Desert-derived actinomycete with micrococcus luteus antagonistic activity

Abstract

The invention belongs to the field of microbial fermentation and natural product development and utilization. The invention discloses a strain of Streptomyces griseoincarnatusTRM 58446-WSY with micrococcus luteus antagonistic activity and an application of the strain of Streptomyces griseoincarnatusTRM 58446-WSY, wherein the strain of Streptomyces griseoincarnatusTRM 58446-WSY is derived from Taxalacaea desert, and the collection number of the strain of Streptomyces griseoincarnatusTRM is GDMCC NO: 65718. The strain has remarkable bacteriostatic activity on micrococcus luteus, and the bacteriostatic active substance obtained through separation has guiding significance in development, prevention and treatment of gram-positive strain infection, can be applied to research and development of antibiotic drugs and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the fields of microbial fermentation and development and utilization of natural products, and particularly relates to a Streptomyces strain from the Taklimakan Desert S. griseoincarnatus TRM 58446-WSY, and a method for extracting 3,4-di-tert-butylphenol with Micrococcus luteus antagonistic activity from its liquid fermentation culture and its application Background Art

[0002] Micrococcus luteus widely exists in external environments such as soil and water, and is also found on the skin of humans and animals. Micrococcus luteus generally does not cause diseases, but it can cause opportunistic infections such as bacteremia, meningitis, endocarditis, etc. when the human body has low immunity or wound infection. Currently, commonly used drugs for treating Micrococcus luteus infections include ceftriaxone, clindamycin, vancomycin, etc. And the problem of antibiotic resistance is one of the greatest threats to global health, food safety and development at present. More and more infections are becoming more difficult to treat because the effectiveness of antibiotics used to treat infections has declined. The research and development of new antibiotic drugs is extremely urgent

[0003] In recent years, researchers' research and development of antibiotic drugs have mainly focused on secondary metabolites produced by microbial fermentation. More than half of the natural antibiotics derived from microorganisms are produced by actinomycetes. Actinomycetes in extreme environments are more likely to metabolize antibiotics with novel structures, high efficacy and low toxicity than those in ordinary environments

[0004] The Taklimakan Desert is located in the Tarim Basin in the southern part of Xinjiang Uygur Autonomous Region, China. Its dry, high-temperature and less-rainy climate has also given birth to a series of unique microbial individuals. Currently, there are few reports on screening Micrococcus luteus antagonistic strains from the Taklimakan Desert and isolating and purifying antibacterial active substances. Therefore, exploring secondary metabolites from the desert is an important way to discover new antibiotics Summary of the Invention

[0005] The technical problem to be solved by the present invention is: to provide a desert-derived actinomycete and its application in the production of an active substance antagonistic to Micrococcus luteus. This antibacterial active substance has significant Micrococcus luteus antagonistic activity and can be applied to the research and development of antibiotic drugs, and has good application prospects

[0006] The technical solution adopted by the present invention to solve the above technical problem is: a Streptomyces griseocarneus S. griseoincarnatusTRM 58446-WSY, deposit number: GDMCC NO: 65718, originated from the Taklimakan Desert, Xinjiang Uygur Autonomous Region, China (40°5'52"N, 84°18'40"E), classified as Streptomyces griseoincarnatus, deposited on January 6, 2025, deposited by Guangdong Microbiological Culture Collection Center (GDMCC), and tested to be alive.

[0007] At the same time, the present invention also provides the use of the above-mentioned actinomycetes in the production of an active substance antagonizing Micrococcus luteus, the active substance is a compound 3,4-di-tert-butylphenol, obtained from Streptomyces S. griseoincarnatus It was isolated from the metabolites of TRM 58446-WSY. The structural formula of the compound is shown below:

[0008] At the same time, the present invention also provides a method for producing the compound 3,4-di-tert-butylphenol, which comprises the following steps: culturing the actinomycetes to obtain a fermentation liquid; and separating and purifying the compound 3,4-di-tert-butylphenol from the fermentation liquid.

[0009] The method of the present invention specifically comprises the following steps: (1) Fermentation production The actinomycetes according to claim 1 are cultured in a seed culture medium TSB at 25-30°C for 2-4 days, inoculated into an M fermentation medium at an inoculum amount of 12.0-15%, cultured at 25-28°C and 180 r / min for 6-8 days, and the cells are collected; (2) Preparation of bacterial extract Add 3 times the volume of methanol to the bacterial cells obtained in step (1), extract on a shaker at 180 r / min overnight, collect the extract, rotary evaporate the methanol, repeat the extraction 3 times to obtain a bacterial extract; (3) Separation of compounds The bacterial extract obtained in step (2) is dissolved in a 60% methanol aqueous solution, filtered and collected, and then dissolved in a solution of dichloromethane: methanol = 3:1, filtered and collected, concentrated, and separated by gel column chromatography, with the eluent being dichloromethane: methanol = 3:1; the components are combined after TLC thin layer chromatography detection; the obtained target fraction is repeatedly dissolved and filtered with dichloromethane, and the filtrate is collected and rotary evaporated to obtain compound 3,4-di-tert-butylphenol.

[0010] Preferably, the culture medium formula and culture conditions of the gray-fleshed Streptomyces described in the present invention are: seed liquid culture medium: 17.0 g of trypticase, 5.0 g of sodium chloride, 3.0 g of soybean papain hydrolyzate, 2.5 g of dipotassium hydrogen phosphate, 2.5 g of glucose dissolved in 1000 mL of water, pH adjusted to 7.2-7.4, 121°C, sterilized for 30 min. The formula and preparation method of the M fermentation medium are: first dissolve 10 g of lactose and 30 g of dextrin and boil until translucent, then weigh 10 g of fermentation fish meal and 5 g of calcium carbonate and dilute to 1000 mL of distilled water, adjust pH to 7.2-7.4, 115°C, and sterilize for 30 min.

[0011] The method for separating the compound 3,4-di-tert-butylphenol from the fermentation broth is specifically as follows: after the fermentation is completed, 8 layers of gauze are used to separate the bacterial cells and the bacterial broth. The obtained bacterial cells are first washed with distilled water to remove the culture medium components, then soaked with 3 times the volume of methanol, ultrasonicated, and the methanol is rotary evaporated to obtain a bacterial cell extract, which is repeated 3 times and the 3 extracts are combined.

[0012] The method for purifying compound 3,4-di-tert-butylphenol from fermentation extract is as follows: the fermentation extract is dissolved and filtered with 60% methanol water, and the filtrate is collected and the methanol water solution is evaporated to obtain extract-1. Extract-1 is dissolved and filtered with an organic solvent (dichloromethane: methanol = 3:1), and the filtrate is collected and the organic solvent is evaporated to obtain extract-2. Extract-2 is dissolved again with an organic solvent (dichloromethane: methanol = 3:1), filtered through a 0.45μm sterile filter membrane, and separated by gel column chromatography, and the same fractions are combined.

[0013] The gel column chromatography conditions are as follows: the gel separation column is Sephadex LH-20, dichloromethane:methanol=3:1, the flow rate is 6-10s / d, 30-40min / 20mL, the column is passed at normal pressure, and the liquid is collected by an automatic collector.

[0014] After gel column chromatography separation, the further purification method is: after combining the same fractions by TLC, rotary evaporate the target fraction, dissolve and filter with dichloromethane, collect the filtrate and rotary evaporate, and repeat this step at least 3 times. The substance obtained after removing the insoluble matter is 3,4-di-tert-butylphenol, and the structural formula is as follows.

[0015]

[0016] The application of the actinomycetes is: the above-mentioned Streptomyces S. griseoincarnatus TRM 58446-WSY or its metabolites are used in any of the following applications.

[0017] (1) Used to inhibit Micrococcus luteus; (2) preparing products for inhibiting Micrococcus luteus; (3) preparing drugs for inhibiting infection with Micrococcus luteus; (4) Preparation of drugs for treating or preventing diseases caused by Gram-positive bacteria.

[0018] The application of the actinomycetes is: the above-mentioned Streptomyces S. griseoincarnatus The metabolite of TRM 58446-WSY, 3,4-di-tert-butylphenol, was verified by the double-layer pour plate method + drug sensitivity sheet embedding method to have a significant antagonistic effect on Micrococcus luteus compared with known homologues.

[0019] The application of the actinomycetes is: the above-mentioned Streptomyces S. griseoincarnatus The metabolite of TRM 58446-WSY, 3,4-di-tert-butylphenol, was verified to have good biocompatibility by cell morphology observation and MTT cytotoxicity assay.

[0020] The present invention has the following beneficial technical effects: The inventor isolated an actinomycete from the extreme environment of the Taklimakan Desert S. griseoincarnatus TRM 58446-WSYSY, obtain the fermentation product by liquid fermentation of Streptomyces TRM 58446-WSYSY, soak the fermentation product in methanol, dissolve and filter it in methanol water, perform gel column chromatography, and repeatedly dissolve and filter it in dichloromethane to obtain 3,4-di-tert-butylphenol, a compound with significant antagonism to Micrococcus luteus. Compared with known homologues, this compound has obvious antibacterial effect and good biocompatibility, and can be used for the development of natural drugs to inhibit related diseases caused by Micrococcus luteus. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 These are the statistical results of cultivable actinomycetes in the Taklimakan Desert.

[0022] Figure 2 These are the results of screening for antagonistic strains of Micrococcus luteus.

[0023] Figure 3 The plate photos and scanning electron microscope photos of Streptomyces griseus TRM 58446-WSY. A: Colony morphology of TRM58446-WSY on Gao's No. 1 plate, B: Microscopic state of spore filaments and spores.

[0024] Figure 4 This is the taxonomic status and phylogenetic tree of Streptomyces griseus TRM 58446-WSY.

[0025] Figure 5The antibacterial spectrum of Streptomyces griseus TRM 58446-WSY is determined. Among them: 1-4 are Micrococcus luteus (M.luteus ATCC 9341), Klebsiella pneumoniae (K.pneumoniae ATCC 11296), Staphylococcus aureus (S.aureus ATCC 29213), and Erwinia rhapontici (E.rhapontici ATCC29283).

[0026] Figure 6 The results of fermentation medium screening.

[0027] Figure 7 This is the nuclear magnetic resonance hydrogen spectrum of 3,4-di-tert-butylphenol.

[0028] Figure 8 This is the carbon nuclear magnetic resonance spectrum of 3,4-di-tert-butylphenol.

[0029] Fig. 9 This is the HSQC spectrum of 3,4-di-tert-butylphenol.

[0030] Fig.10 This is the HSQC-TOCSY spectrum of 3,4-di-tert-butylphenol.

[0031] Fig.11 is the MIC value of 3,4-di-tert-butylphenol against Micrococcus luteus under the microplate method.

[0032] Fig.12 MBC value of 3,4-di-tert-butylphenol against Micrococcus luteus under the microplate method.

[0033] Fig.13 It is the MIC value of 3,4-di-tert-butylphenol against Micrococcus luteus under the double-layer inverted plate method + drug sensitivity sheet embedding method. In the figure: 1-7 are the antagonistic effects of different concentrations of 3,4-di-tert-butylphenol on Micrococcus luteus, which are 60.00 μg / mL, 58.80 μg / mL, 57.62 μg / mL, 56.47 μg / mL, 55.34 μg / mL, 54.24 μg / mL, and 53.15 μg / mL, respectively.

[0034] Fig.14The antagonistic effect of 3,4-di-tert-butylphenol and its homologues on Micrococcus luteus. In the figure: 1-9 are 59.38 μg / mL of 3,4-di-tert-butylphenol, 2,4-dimethyl-6-tert-butylphenol, 4-bromo-2,6-di-tert-butylbenzaldehyde, 4,4'-methylene (N-(sec-butyl)aniline), 4,6-di-tert-butyl-4-methylphenol, 2-phenyl-1,3-dithiolane, 2,4-di-tert-butylphenol, 2,4,6-tri-tert-butylphenol, and 3-(3,5-di-tert-butyl-4-hydroxyphenol) propionate.

[0035] Fig.15 The antagonistic effect of 3,4-di-tert-butylphenol on 8 pathogens. In the figure: 1-8 are Micrococcus luteus (M.luteus ATCC 9341), Shigella flexneri (S.Castellani ATCC 12022), Erwinia rhapontici (E.rhapontici ATCC29283), Acinetobacter baumannii (A.baumannii ATCC 15308), Staphylococcus aureus (S.aureus ATCC 29213), Escherichia coli (E.coli ATCC 25922), Candida albicans (C.Albicans CMCC98001), Klebsiella pneumoniae (K.pneumoniae ATCC 11296); E is a negative control; F is a blank control; G is a positive control.

[0036] Fig.16 The OD of 3,4-di-tert-butylphenol against 8 pathogens 600nm The influence of the value, in the figure: 1-8 are Micrococcus luteus (M.luteus ATCC 9341), Shigella flexneri (S.Castellani ATCC 12022), Erwinia rhapontici (E.rhapontici ATCC29283), Acinetobacter baumannii (A.baumannii ATCC 15308), Staphylococcus aureus (S.aureus ATCC 29213), Escherichia coli (E.coli ATCC 25922), Candida albicans (C.Albicans CMCC98001), Klebsiella pneumoniae (K.pneumoniae ATCC 11296).

[0037] Fig.17 The effect of 3,4-di-tert-butylphenol on the biocompatibility of RAW 264.7 macrophages, where: a is the control group at 0h; b is the experimental group (55.35μg / mL) at 0h; c is the control group at 24h; d is the experimental group (55.34μg / mL) at 24h.

[0038] Fig.18 This is the effect of 3,4-di-tert-butylphenol on the viability of RAW 264.7 macrophages. DETAILED DESCRIPTION

[0039] The present invention is further described in detail below with reference to the accompanying drawings.

[0040] Example 1

[0041] 1. Isolation and purification of cultivable actinomycetes in the Taklimakan Desert Weigh 15 g of soil sample (see Table 1) and place it in a conical flask filled with 150 mL of sterile water to make a soil suspension. Place the soil suspension on a shaker at 27 °C and 200 r / min for 24-48 h, then perform gradient dilution and draw 10 -1 to 10 -5 100 μL of soil suspension was applied to Gao's medium No. 1. Incubate the plate upside down in a 37 ℃ constant temperature incubator for 5-7 days, count the plates and select actinomycetes with different morphology and color for purification to obtain culturable actinomycetes around the Taklimakan Desert.

[0042] The preparation method of Gao's solid medium No. 1 is as follows: dissolve 20 g of soluble starch in an appropriate amount of distilled water and boil until it becomes translucent, then accurately weigh K 2 HPO 4 0.5 g, MgSO 4 7H 2 O 0.5 g, NaCl 0.5 g, FeSO 4 7H 2 Dissolve 0.01 g of O and 20 g of agar powder in 1000 mL of distilled water, adjust the pH to 7.2-7.4, sterilize at 121°C for 30 min, cool to 60°C and pour into plates.

[0043] Experimental results: A total of 414 strains of culturable actinomycetes from the Taklimakan Desert were isolated using Gao's medium No. 1, belonging to 57 different actinomycete taxa (see Figure 1 ), belonging to 6 orders, 7 families and 7 genera, namely, Stenotrophomonas ( Stenotrophomonas ), Micromonas ( Micromonospora ), Nocardia ( Nocardioides ), Streptomyces ( Streptomyces ), Nocardia spp. Nocardiopsis ), Madura ( Actinomadura ) and Amycolatopsis spp. ( Amycolatopsis ), see Table 2.

[0044] Table 1. Sampling site information

[0045] Table 2. Diversity of 414 actinomycetes

[0046] 2. Screening of strains of the present invention Preparation of mixed bacteria plate: inoculate the target bacteria into LB liquid culture medium and culture at 30℃ and 150 r / min for 24 h. 600nm ≥0.5) was inoculated into LB solid plates at a 1% inoculum size to prepare mixed bacterial plates.

[0047] Plate confrontation method: Use a hole puncher to punch a 8.0 mm bacterial cake on the cultured plate, place the bacterial cake upside down on the mixed bacterial plate, and culture it in a 30℃ constant temperature incubator for 2-3 days. Set up 3 parallels for each group and measure the diameter of the inhibition zone.

[0048] Test results: The results of screening for antagonistic strains of Micrococcus luteus are shown in Tables 3 and Figure 2 The target strain TRM 58446-WSY had the largest inhibition zone diameter, which has great research value.

[0049] Table 3. Screening results of antagonistic strains of Micrococcus luteus

[0050] 3. Identification and morphological analysis of the strains of the present invention (1) Identification of target strains The total DNA of the target strain was extracted and 16S rDNA was amplified after agarose gel electrophoresis. The universal primers were: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′). PCR (20 μL) system: 2×Taq Plus PCR Master Mix 10 μL, 27F / 1492R (10 μmol / L) 1 μL each, template DNA 1 μL, ddH 2 O 7 μL. The PCR reaction system was: 94℃ 5 min; 94℃ 45 s, 63℃ 90 s, 72℃ 2min, 30 cycles; 72℃ 5 min. The PCR amplification products were then tested by agarose gel electrophoresis, and after passing the test, they were sent to Sangon Biotech (Xi'an) Co., Ltd. for sequencing. The returned sequencing results were spliced ​​in SeqMen and compared with the EzBioCloud database.

[0051] (2) Morphological analysis of target strains Figure 3The morphology of the target strain after 15 days of culture on Gao's medium No. 1 and the morphology under scanning electron microscope. The color of the plate colony is grayish white; the surface of the colony is not smooth and fluffy; the edge of the colony is irregular. Under scanning electron microscope, the mycelium shows slender irregular branching hyphae, the mature spore hyphae are spiral, and the spores are strip-shaped.

[0052] (3) Construction of homologous evolutionary tree The phylogenetic tree was constructed using the neighbor-joining method for species identification. S. griseoincarnatus The similarity of LMG 19316 reached 100% and clustered on the same branch (see Figure 4 ). Combined with the colony morphology and microstructure of strain TRM 58446-WSY, strain TRM 58446-WSY was identified as Streptomyces griseus ( S. griseoincarnatus .

[0053] Test results: The 16s rDNA information of the target strain TRM 58446-WSY is as follows (SEQ ID No. 1):

[0054] Example 2 1. Determination of the antibacterial spectrum of Streptomyces griseus TRM 58446-WSY The antibacterial spectrum of Streptomyces griseus TRM 58446-WSY was determined according to the plate confrontation method in Example 1.

[0055] Test results: Figure 5 As shown in Figure 2, Streptomyces griseus TRM 58446-WSY was active against four pathogens, namely Micrococcus luteus ( M.luteus ATCC 9341), Klebsiella pneumoniae ( K. pneumoniae ATCC 11296), Staphylococcus aureus ( S. aureus ATCC 29213), Erwinia rhubarb ( E. rhapontici ATCC29283). Among them, the antagonistic activity against Micrococcus luteus was significantly higher than that of other strains.

[0056] Example 3 1. Screening of fermentation medium for target strain Refer to the culture medium commonly used for Streptomyces liquid fermentation that has been published, and the specific formula is shown in Table 4.

[0057] Table 4. Fermentation medium formula

[0058] The fermented broth was separated from the bacterial cell and the bacterial liquid using 8 layers of gauze. Three volumes of methanol were added to the bacterial cell and extracted overnight on a shaker at 150 r / min. The extract was collected and the methanol was evaporated by rotary evaporation to obtain a bacterial cell extract. The bacterial liquid was subjected to liquid-liquid exchange extraction using an equal volume of ethyl acetate. The extract was collected and the ethyl acetate was evaporated by rotary evaporation to obtain a bacterial liquid extract. The bacterial cell extract and the bacterial liquid extract were combined to obtain the fermentation crude extract of TRM 58446-WSY. The crude extract was dissolved in sterile water (containing 10% DMSO) to prepare a 50 mg / mL mother liquor, filtered through a universal 0.25 μm sterile filter membrane for sterilization, and stored at 4°C for later use.

[0059] Activity assay: Using the filter paper method, a dry and sterile 6.0 mm filter paper was immersed in the fermentation crude extract for 30 min, and then removed with sterile tweezers to control the water and attached to the mixed bacteria plate, and cultured in a constant temperature culture medium at 30°C for 2-3 days. Three parallels were set up for each group, and the diameter of the inhibition zone was measured. The negative control was a filter paper treated with sterile water.

[0060] Test results: Different fermentation media have different abilities to stimulate the target strain TRM 58446-WSY to produce antibacterial active substances. The target strain TRM 58446-WSY produced substances that antagonized Micrococcus luteus in B, H, I, L and M media (see Figure 6 Among them, the antibacterial effect of metabolites produced by fermentation in M ​​medium was significantly stronger than that of other strains (P < 0.05), which has great research value.

[0061] Example 4 1. Fermentation of Streptomyces griseus TRM 58446-WSY using the optimal culture medium An appropriate amount of bacterial spores were inoculated from Gao's No. 1 plate into TSB medium, cultured at 28°C for 3 days, and then inoculated into M fermentation medium at a rate of 12.5%, and cultured at 26°C and 180 r / min for 6-10 days. 2. Preparation of 3,4-di-tert-butylphenol using Streptomyces griseus TRM 58446-WSY The fermented liquid was collected and separated from the bacterial body and the liquid with 8 layers of gauze. The obtained bacterial body was first washed with distilled water to remove the culture medium components, then soaked and ultrasonicated with 3 volumes of methanol, and the methanol was rotary evaporated to obtain a bacterial body extract, which was repeated 3 times and the extracts were combined 3 times.

[0062] The method for purifying compound 3,4-di-tert-butylphenol from fermentation extract is as follows: the fermentation extract is dissolved and filtered with 60% methanol water, and the filtrate is collected and the methanol aqueous solution is rotary evaporated to obtain extract-1. Extract-1 is dissolved and filtered with an organic solvent (dichloromethane: methanol = 3:1), and the filtrate is collected and the organic solvent is rotary evaporated to obtain extract-2. Extract-2 is dissolved again with an organic solvent (dichloromethane: methanol = 3:1), filtered through a 0.45μm sterile filter membrane, and then separated by chromatography on a gel column (LH 20), and the same fractions are combined. The target fraction is rotary evaporated, dissolved and filtered with dichloromethane, the filtrate is collected and rotary evaporated, and this step is repeated at least 3 times.

[0063] Test results: The substance was identified as 3,4-di-tert-butylphenol by NMR spectrum analysis. The NMR hydrogen spectrum and carbon spectrum are shown in Figure 7 , Figure 8 HSQC spectra and HSQC-TOCSY spectra are shown in Fig. 9 and Fig.10 The assigned hydrogen and carbon NMR spectrum data of 3,4-di-tert-butylphenol are shown in Table 5.

[0064] Table 5. 3,4-di-tert-butylphenol 1 H and 13 C NMR data (CHCl 3 - d6）

[0065] Note: The signal attribution in Table 5 is based on 1 H. 13 C. 1 H- 1 The analysis results of H COSY, HSQC and HMBC spectra. The multiplicity of hydrogen signals is represented by s (singlet), brs (broad singlet), t (triplet), q (quartet) and m (multiplet).

[0066] Example 5 1. Determination of the antagonistic activity of 3,4-di-tert-butylphenol of Streptomyces griseus TRM 58446-WSY metabolite against Micrococcus luteus The MIC and MBC values ​​were determined using the micropore method. 3,4-di-tert-butylphenol was dissolved in sterile water (containing 10% DMSO), filtered and sterilized with a 0.22 μm sterile filter membrane to prepare a 1 mg / mL stock solution, and then diluted with LB medium to the specified concentrations (60.00, 58.80, 57.62, 56.74, 55.34, 54.24, 53.15, 52.09, 51.05 and 50.02 μg / mL); the suspension of Micrococcus luteus in the logarithmic growth phase was adjusted to 1×10 by McFarland turbidimetry. 6 CFU / mL; 3,4-di-tert-butylphenol of each concentration was mixed with the bacterial solution in an equal volume in a 96-well plate and cultured at 37°C for 24 h. The OD was then measured using an enzyme-labeled instrument. 600nm Value. Use blank LB culture medium as blank control, bacterial suspension without 3,4-di-tert-butylphenol as negative control, and tetracycline (2 mg / mL) as positive control. MIC is the lowest concentration that significantly inhibits the growth of indicator bacteria. Take 100 μL of bacterial suspension above MIC concentration, spread on LB plates respectively, culture at 28℃ for 24 h, observe whether colonies grow, and take the lowest drug concentration for sterile growth as MBC.

[0067] Test results: The antagonistic activity of 3,4-di-tert-butylphenol against Micrococcus luteus is as follows Fig.11 As shown in the figure, when the concentration is higher than 55.34 μg / mL, the growth of Micrococcus luteus is inhibited. When it is lower than 55.34 μg / mL, the inhibitory effect gradually weakens, and Micrococcus luteus shows a growth trend, indicating that 55.34 μg / mL is the MIC of 3,4-di-tert-butylphenol for Micrococcus luteus. The bacterial solution of each well with a concentration above the MIC was taken for subculture. It was found that when the concentration of 3,4-di-tert-butylphenol was 56.47 μg / mL, there was still colony growth, and when the concentration was 57.62 μg / mL, there was no colony growth ( Fig.12 ), indicating that 57.62 μg / mL is the MBC of 3,4-di-tert-butylphenol against Micrococcus luteus.

[0068] Example 6

[0069] 1. MIC value of 3,4-di-tert-butylphenol against Micrococcus luteus using double-layer inverted plate method + drug sensitive tablet embedding method Dilute 3,4-di-tert-butylphenol according to the method in Example 5, and the diluent is methanol. First add 10 μL of the diluent to the dry and sterile filter paper, air-dry at room temperature, then add the diluent and air-dry, repeat three times to accumulate 30 μL of the drug solution on the filter paper. After a layer of mixed bacterial solution is poured and dried and solidified, stick the treated drug-sensitive sheet on the mixed bacterial plate, and then pour another layer of mixed bacterial solution to cover the drug-sensitive sheet. Place in a 37°C constant temperature incubator for 24 hours to observe whether an inhibition zone is produced.

[0070] 2. Determination of the antagonistic effect of 3,4-di-tert-butylphenol and known homologues of Streptomyces griseus TRM 58446-WSY against Micrococcus luteus 3,4-di-tert-butylphenol and its homologues were diluted to 55.34 μg / mL according to the method in Reference Example 5, and the diluent was methanol, and then the above method was followed.

[0071] Test results: Fig.13 As shown in the figure, the MIC value of 3,4-di-tert-butylphenol against Micrococcus luteus was still 55.34 μg / mL under the double-layer inverted plate method + drug sensitivity tablet embedding method, indicating that this method is feasible and effective. Fig.14 The results showed that at a concentration of 55.34 μg / mL, only 3,4-di-tert-butylphenol produced an inhibition zone against Micrococcus luteus, and other homologues had no ability to inhibit Micrococcus luteus.

[0072] Example 7 1. Determination of the antibacterial spectrum of 3,4-di-tert-butylphenol The micropore method was used to prepare bacterial suspensions of 8 indicator bacteria according to the bacterial suspension preparation method described in Example 5. 3,4-di-tert-butylphenol with a prepared concentration of 55.34 μg / mL was added to the bacterial suspension in a volume of 1:1, and cultured at 37°C for 24 h. The OD600nm value was then determined using an enzyme reader, and 10 μL of 1 mg / mL resazurin dye was added, and the color change was observed after incubation at 30°C for 2 h.

[0073] Test results: Resazurin solution can penetrate living cells and be irreversibly reduced to pink. If the culture medium remains blue, it indicates that bacterial growth is restricted. Therefore, the activity of indicator bacteria can be reflected by color changes. Fig.15 As shown in the figure, at a concentration of 55.34 μg / mL, 3,4-di-tert-butylphenol not only had an antagonistic effect on Micrococcus luteus, but also on Erwinia. Fig.16 OD 600nmThe corresponding results can also be seen from the values. The OD of 3,4-di-tert-butylphenol at a concentration of 55.34 μg / mL for Micrococcus luteus and Erwinia 600nm The value remained at a low level (<0.1).

[0074] Example 8

[0075] 1. Cultivation of RAW 264.7 Complete medium: DMEM + 10% fetal bovine serum + 1% blue chain antimycin RAW 264.7 cells with good and stable growth were selected and cultured at 1×10 4 The cells were inoculated at a density of 100 μL / mL in a 96-well plate, with 100 μL per well. The cell density, morphology, and adhesion were observed for 8-12 hours in a 37°C constant temperature incubator, and the culture medium was replaced every 24 hours.

[0076] 2. Gradient dilution of 3,4-di-tert-butylphenol 3,4-di-tert-butylphenol was diluted according to the method in Example 5 to a concentration of 57.62, 56.74, 55.34, and 54.24 μg / mL, and the diluent was complete culture medium. 2. Cell viability assay When the cells have grown to more than 50% of the 96-well plate, add 100 μL of 3,4-di-tert-butylphenol of different concentrations in turn, set up five replicate wells in each group, and continue to culture in a constant temperature incubator at 37°C for 24 hours. Then, remove the supernatant, add 100 μL of 1×PBS for washing, and then add 10% MTT solution and incubate for 4 hours in the dark. After incubation, add 100 μL of dissolving solution and measure the absorbance at 570 nm. Calculate cell activity according to the following formula.

[0077] Cell viability = A 实验组- A 空白组 / A 对照组- A 空白组 ×100% Test results: Fig.17 As shown in the figure, at 0h, the cell growth state of the experimental group and the control group was stable, and the morphology of RAW 264.7 cells was a round semi-adherent state. After 24h, the control group continued to grow and covered 80% of the wells. The growth state of the experimental group cells added with 55.34μg / mL of 3,4-di-tert-butylphenol did not change significantly compared with the control group. This shows that 55.34 μg / mL of 3,4-di-tert-butylphenol has no toxicity to RAW 264.7 cells. Fig.18 It can also be seen that after 24 h of culture, 3,4-di-tert-butylphenol at 55.34 μg / mL had no significant effect on the viability of RAW 264.7 cells compared with the control group ( P>0.05). This indicates that 3,4-di-tert-butylphenol has no toxicity to RAW 264.7 cells and is expected to be used in the treatment of Micrococcus luteus infection in humans.

[0078] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. An actinomycete, characterized in that: It is Streptomyces griseus ( S. griseoincarnatus TRM 58446-WSY), the deposit number is GDMCC NO: 65718.

2. Use of the actinomycete as claimed in claim 1 in the production of the compound 3,4-di-tert-butylphenol, the structural formula of the compound is as follows: 。 3. A method for producing the compound 3,4-di-tert-butylphenol, characterized in that: The method comprises the following steps: culturing the actinomycetes as claimed in claim 1 to obtain fermentation liquid; and separating and purifying the compound 3,4-di-tert-butylphenol from the fermentation liquid.

4. The method according to claim 3 is characterized in that the method specifically comprises the following steps: (1) Fermentation production The actinomycetes according to claim 1 are cultured in a seed culture medium TSB at 25-30°C for 2-4 days, inoculated into an M fermentation medium at an inoculum amount of 12.0-15%, cultured at 25-28°C and 180 r / min for 6-8 days, and the cells are collected; (2) Preparation of bacterial extract Add 3 times the volume of methanol to the bacterial cells obtained in step (1), extract on a shaker at 180 r / min overnight, collect the extract, rotary evaporate the methanol, repeat the extraction 3 times to obtain a bacterial extract; (3) Separation of compounds The bacterial extract obtained in step (2) is dissolved in a 60% methanol aqueous solution, filtered and collected, and then dissolved in a solution of dichloromethane: methanol = 3:1, filtered and collected, concentrated, and separated by gel column chromatography, with the eluent being dichloromethane: methanol = 3:1; the components are combined after TLC thin layer chromatography detection; the obtained target fraction is repeatedly dissolved and filtered with dichloromethane, and the filtrate is collected and rotary evaporated to obtain compound 3,4-di-tert-butylphenol.

5. The method according to claim 4, characterized in that In step (1), the formula of the seed culture medium TSB is: 17.0 g trypticase, 5.0 g sodium chloride, 3.0 g soybean papain hydrolyzate, 2.5 g dipotassium hydrogen phosphate, 2.5 g glucose, 1000 mL distilled water, adjusted to pH = 7.2-7.4; the formula of the M fermentation medium is: 10 g lactose, 30 g dextrin, 10 g fish meal, 5 g calcium carbonate, 1000 mL distilled water, adjusted to pH = 7.2-7.

4.

6. The method according to claim 4, characterized in that In step (1), the actinomycetes according to claim 1 are cultured in a seed culture medium TSB at 28°C for 3 days, then inoculated into an M fermentation medium at an inoculum size of 12.5%, cultured at 26°C and 180 r / min for 7 days, and the bacteria are collected.

7. Use of 3,4-di-tert-butylphenol obtained by the method according to any one of claims 3 to 6 in antagonizing Micrococcus luteus.

8. The use according to claim 7, characterized in that: Preparation of products for inhibiting Micrococcus luteus; or preparation of drugs for inhibiting Micrococcus luteus infection; preparation of drugs for treating or preventing diseases caused by Gram-positive bacteria.

9. Use of the actinomycetes or their metabolites in the preparation of antibacterial drugs according to claim 1, characterized in that: The target bacteria is Micrococcus luteus ( Micrococcus luteus ATCC 9341), Staphylococcus aureus ( Staphylococcus aureus ATCC 29213), Escherichia coli ( Escherichia coli ATCC 25922), Klebsiella pneumoniae ( Klebsiella pneumoniae ATCC 11296), Acinetobacter baumannii ( Acinetobacter baumannii ATCC 15308), Candida albicans ( Canidia Albicans CMCC 98001), Shigella flexneri ( Shigella Castellani ATCC 12022) or Erwinia rhubarb ( Erwinia rhapontici ATCC29283) or more.

10. The use according to any one of claims 7 to 8, characterized in that The 3,4-di-tert-butylphenol is first dissolved with DMSO, preferably in a 37°C water bath to promote full dissolution; and then methanol is used as a diluent to dilute the drug to a desired concentration.

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