AU2020102973A4 - Method for preparing asterric acid through microbial fermentation - Google Patents
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Abstract
The invention discloses a method for preparing asterric acid through microbial
fermentation. In the first aspect, the present invention claims the application of
Pseudogymnoascus pannorum BJZ13 in the preparation of asterric acid. In the second aspect,
the present invention claims a method for preparing asterric acid, which includes the following
steps: fermenting and cultivating Pseudogymnoascus pannorum BJZ13 to obtain asterric acid.
The present invention discloses a new usage of Pseudogymnoascus pannorum BJZ13, namely,
fermentation and cultivation of Pseudogymnoascus pannorum BJZ13, and separation and
purification of asterric acid from the fermentation product. The method for preparing asterric
acid provided by the invention has a simple operation, low cost and a short production cycle,
and has the application potential of industrialized scale production. The invention provides a
new way for resource development of asterric acid drugs.
Description
Method for preparing asterric acid through microbial fermentation
[0001] The invention belongs to the field of biochemistry, and particularly relates to a method for preparing asterric acid through microbial fermentation.
[0002] Asterric acid is a kind of compound containing a diphenyl ether structure, which has the medical activity of resisting bacteria and tumors, as well as the pesticide activity of killing bacteria, killing nematodes and inhibiting the germination of oenanthe javanica seeds. The high purity of asterric acid monomer can be obtained by using microorganism to produce asterric acid so as to avoid the residues of chemical synthesis by-products. In addition, the microorganism is a living body and a synthetic place, and can avoid the waste of resources and the damage to the environment by using the resources of organisms to generate the asterric acid. The microorganism is easy to breed and culture in large quantities, and has the advantages of a simple operation, low cost, a short production period, and application potential for industrial scale production. It is an ideal path to produce asterric acid drugs.
[0003] It is an object of the present invention to provide a method for producing asterric acid through microbial fermentation.
[0004] In a first aspect, the present invention claims the use of Pseudogymnoascus pannorum BJZ13 in the preparation of asterric acid. Pseudogymnoascus pannorum BJZ13 was deposited on March 08, 2018 at the General Microbiological Center of the China Commission for Culture Collection of Microorganisms (CGMCC for short, address: No.
3, Institute of Microbiology, China Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing) with a registration number of CGMCC No. 15389.
[0005] In a second aspect, the present invention claims a process for preparing asterric acid, comprising the steps of: fermenting and culturing Pseudogymnoascus pannorum BJZ13 to obtain asterric acid.
[0006] The fermentation culture specifically comprises the following steps:
[0007] (1) inoculating a single colony of Pseudogymnoascus pannorum BJZ13 to a seed culture medium, and performing shaking culture for 7 days at the temperature of 15 °C and the speed of 120 r/min to obtain a seed solution;
[0008] (2) inoculating the seed solution obtained in the step (1) into a conical flask (4 mL seed solution/flask) filled with a fermentation medium, and carrying out standing at 15 °C for 40 days to obtain a fermentation culture product.
[0009] Preparation method of the seed culture medium: using soluble starch 20g/L, FeCl3 0.00065g/L, MgSO4 0.5 g/L, KH2PO4 0.5 g/L, (NH4)2SO4 0.65 g/L, and the balance of water, adjusting the pH to 7.2-7.4, autoclaving at 121 °C for 20 min, and cooling to a room temperature while adding ampicillin antibiotic and making the concentration become 1pL/mL.
[0010] The preparation method of the fermentation medium: Taking a 500 mL conical flask, add 50 g of rice and 80 mL of distilled water, and autoclaving at 121 °C for 20min.
[0011] The method further comprises the step of subjecting the product of the fermentation culture to an organic phase extraction.
[0012] The organic solvents used in the organic phase extraction are ethyl acetate and dichloromethane.
[0013] The method for extracting the organic phase comprises the following steps of: extracting a fermentation culture product by using ethyl acetate, and then collecting the ethyl acetate phase, and concentrating under reduced pressure to obtain an extract; and extracting the extract by using dichloromethane, and then collecting the dichloromethane phase, and concentrating under reduced pressure to obtain a dichloromethane phase extract, which is also called a crude extract.
[0014] The method for extracting the organic phase comprises the following steps:
[0015] (1) taking a fermentation culture product, adding ethyl acetate, carrying out ultrasonic crushing, and collecting an ethyl acetate phase;
[0016] (2) after the step (1) is completed, adding ethyl acetate to the residue, then carrying out ultrasonic crushing, and collecting an ethyl acetate phase;
[0017] (3) after the completion of step (2), adding ethyl acetate to the residue, then carrying out ultrasonic crushing, and collecting an ethyl acetate phase;
[0018] (4) After completing step (3), adding ethyl acetate to the residue, then carrying out ultrasonic crushing, and collecting the ethyl acetate phase;
[0019] (5) combining and concentrating the ethyl acetate phase obtained in step (2), the ethyl acetate phase obtained in step (3) and the ethyl acetate phase obtained in step (4)under a reduced pressure to obtain an extract;
[0020] (6) extracting the extract obtained in step (5) with dichloromethane, and carrying out pressure-reduced concentration to obtain a dichloromethane phase extract, also called a crude extract.
[0021] The method for extracting an organic phase comprises the following steps:
[0022] (1) after the fermentation culture is finished, adding ethyl acetate to 400 mL in the conical flask, then carrying out ultrasonic crushing for 20 min, and collecting an ethyl acetate phase;
[0023] (2) after completing the step (1), adding ethyl acetate to 400 mL in the conical flask, then carrying out ultrasonic crushing for 20 min, and collecting an ethyl acetate phase;
[0024] (3) after the step (2) is completed, adding ethyl acetate to 400 mL in the conical flask, then carrying out ultrasonic crushing for 20 min, and collecting an ethyl acetate phase;
[0025] (4) after the step (3) is completed, adding ethyl acetate to 400 mL in the conical flask, then carrying out ultrasonic crushing for 20 min, and collecting an ethyl acetate phase;
[0026] (5) combining and concentrating the ethyl acetate phase obtained in step (2), the ethyl acetate phase obtained in step (3) and the ethyl acetate phase obtained in step (4) under a reduced pressure to obtain an extract;
[0027] (6) extracting the extract obtained in step (5) with dichloromethane (after the extract was sufficiently infiltrated with dichloromethane, the extract was allowed to stand at room temperature for 1 hour), and then carrying out pressure-reduced concentration a dichloromethane phase extract, also called as a crude extract.
[0028] The method further comprises the step of obtaining asterric acid from the dichloromethane phase extract.
[0029] Obtaining asterric acid from dichloromethane phase extract comprises the following steps: dissolving dichloromethane phase extract with dichloromethane, then performing normal phase silica gel open column chromatography separation, and then crystallizing.
[0030] The parameters of the normal phase silica gel open column chromatography separation are as follows: the silica gel particle size is 100-200 meshes; the mobile phase at the initial time of elution is dichloromethane, the mobile phase at the end of elution is methanol, and the mobile phase in the elution process is a mixture of dichloromethane and methanol; the flow rate of the mobile phase is 3 mL/min; the elution process lasts for 50h, the volume fraction of dichloromethane in the mobile phase is linearly reduced from 100% to 0%, and the corresponding volume fraction of methanol in the mobile phase is linearly increased from 0% to 100%; and components with the volume fraction of dichloromethane accounting for 98%-94% in parts by volume in the mobile phase are collected.
[0031] The specific steps of crystallization are as follows: taking the liquid collected by normal phase silica gel open column chromatography, concentrating under a reduced pressure (the component cannot be completely dissolved in dichloromethane, so that methanol is dripped into the liquid to make the solution clear and not viscous), then crystallizing, separating out redundant liquid after crystallization, adding dichloromethane/methanol mixed system to completely dissolve the crystal, then recrystallizing, separating out redundant liquid after crystallization, adding dichloromethane/methanol mixed system to completely dissolve the crystal, and then recrystallizing to obtain acicular crystal; and uniformly mixing 10 parts by volume of dichloromethane and 1 part by volume of methanol to obtain the dichloromethane/methanol mixed system.
[0032] The present invention discloses a new usage of Pseudogymnoascus pannorum BJZ13, namely, fermentation and cultivation of Pseudogymnoascus pannorum BJZ13, and separation and purification of asterric acid from the fermentation product. The method for preparing asterric acid provided by the invention has a simple operation, low cost and a short production cycle, and has the application potential of industrialized scale production. The invention provides a new way for resource development of asterric acid drugs.
[0033] Figure 1 shows a result of BLAST sequence alignment.
[0034] Figure 2 is a crystal1H-NMR diagram (hydrogen spectrum; 400 MHz, deuterated acetone).
[0035] Figure 3 is a crystal 13C-NMR diagram (carbon spectrum; 100 MHz, deuterated acetone).
[0036] Figure 4 is a crystalline HSQC spectrum (400/100 MHz, deuterated acetone).
[0037] Figure 5 is a crystalline HMBC spectrum (400/100 MHz, deuterated acetone).
[0038] Figure 6 is a crystalline HR-ESI-MS spectrum.
[0039] Note of preservation
[0040] Strain name: BJZ13
[0041] Latin name: Pseudogymnoascus pannorum
[0042] Preservation institution: China General Microbiological Culture Collection Center
[0043] Name of preservation institution for short: CGMCC
[0044] Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing
[0045] Date of preservation: March 08, 2018
[0046] Accession No. of preservation center: CGMCC No. 15389
[0047] The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods, if not specified. The test materials used in the following examples are purchased from a conventional biochemical reagent store, if not specified. The quantitative tests in the following examples are repeated three times, and average values are taken as the results.
[0048] BILON92-IIDL Ultrasonic Cell Grinder (used for ultrasonic disruption): Shanghai Kehuai Instrumentation Co., Ltd. RE-52AA Rotary Evaporator (used for reduced pressure concentration): Shanghai Yarong Biochemical Instrument Co., Ltd. SHZ-III Circulating Water Vacuum Pump: Shanghai Yarong Biochemical Instrument Co., Ltd. Nuclear Magnetic Resonance apparatus: Bruker Avance III400 MHz, TMS as internal standard. High Performance Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometer: micrOTOF-Q, Bruker Co., Ltd.
[0049] Embodiment 1 Acquisition and Identification of Pseudogymnoascus Pannorum BJZ13
[0050] Collection time of Sanionia: August 8, 2015.
[0051] Site of collection of Sanionia: Ny-Alesund, Svalbard Islands, (Ny-Alesund, 78 054 '01 .242"N, 12 009 '33 .816"E).
[0052] 1. Pretreatment
[0053] The collected Sanionia was washed with sterile water to remove impurities such as silt.
[0054] 2. Surface Disinfection
The pre-treated Sanionia was cut under sterile conditions, sterilized with 75% ethanol for 1 min, and the rinsed for 5 times (1 min each) with sterile water.
[0056] 3. Culture of Symbiotic Bacteria
[0057] The cut Sanionia after surface sterilization was uniformly spread on a culture medium under sterile conditions, and cultured at 15 °C for 5-15 days. The culture medium: soluble starch of 20 g/L, FeCl3 of 0.00065 g/L, MgSO4 of 0.5 g/L, KH2PO4 of 0.5 g/L,(NH4)2SO4 of 0.65 g/L, pH of 7.2-7.4, agar of 2 g/100 mL, was autoclaved at 121 °C for 20 min, and then an ampicillin antibiotic was added at a concentration of1pL/mL.
[0058] 4. Purification of Symbiotic Bacteria
[0059] After culturing for several days in Step 3, under aseptic conditions, the newly grown thallus is gradually picked on a new plate for culturing, the colony is formed after several days, the type of the strain is preliminarily judged according to the difference of the morphology and the color of the colony and the growing time of the colony, and different strains are selected and respectively inoculated on the new plate.
[0060] The above operations were repeated until pure colonies were isolated.
[0061] 5. Identification of Strains
[0062] The purified strain was inoculated on a plate and sent to Beijing Liuhe BGI Co., Ltd. for sequencing identification. The ITS sequence of the purified strain is shown in the Sequence 1 of the sequence table.
[0063] The nucleic acid data in the Blast data of NCBI were subjected to alignment analysis, and the BLAST sequence alignment result is shown in Figure 1. By sequence alignment, the homology between the strain and Pseudogymnoascus pannorum (Geomyces pannorus) was 99%.
[0064] 6. Culture Preservation
[0065] Pseudogymnoascus pannorum BJZ13 was preserved on March 8, 2018 at the China General Microbiological Culture Collection Center (CGMCC for short, address: Institute of Microbiology, China Academy of Sciences, No.3, Yard No.1, Beichen West Road, Chaoyang District, Beijing). The preservation registration numbera is CGMCC No. 15389.
[0066] Embodiment 2 Preparation of Asterric Acid Using Pseudogymnoascus Pannorum BJZ13
[0067] I. culture of Pseudogymnoascus pannorum BJZ13
[0068] 1. A single colony of Pseudogymnoascus pannorum BJZ13 was inoculated into a seed medium, and cultured at 15 DEG C and 120 r/min with shaking of 7 days to obtain a seed solution.
[0069] Preparation method of seed culture medium: soluble starch of 20 g/L, FeCl3 of 0.00065 g/L, MgSO4 of 0.5g/L, KH2PO4 of 0.5g/L, (NH4)2SO4 of 0.65 g/L, and the balance of water, adjusting the pH to 7.2-7.4, autoclaving at 121 °C for 20min, and cooling to a room temperature while adding ampicillin antibiotic and making the concentration become 1 tL/mL.
[0070] 2. The seed solution obtained in Step 1 was inoculated into a conical flask (4 mL seed solution/flask) filled with a fermentation medium, and carrying out standing at 15 °C for 40 days.
[0071] Method for preparing fermentation medium: A 500 mL Erlenmeyer flask was added with 50 g of rice and 80 mL of distilled water, and autoclaved at 121 °C for 20 min.
[0072] II. Preparation of Crude Extract
[0073] 1. After completion of Step 1 (2), ethyl acetate was added to the flask to 400 mL, the mixture was triturated with a glass rod and ultrasonically crushed for 20 min, and the ethyl acetate phase was collected.
[0074] 2. After completion of Step 1, ethyl acetate was added to 400 mL of the flask (containing the residue existing after the ethyl acetate phase was collected in Step 1), then the mixture was ultrasonically crushed for 20 min, and the ethyl acetate phase was collected.
[0075] 3. After completion of Step 2, ethyl acetate was added to the flask (containing the residue existing after the ethyl acetate phase was collected in Step 2) to 400 mL, then the mixture was ultrasonically crushed, and the ethyl acetate phase was collected.
[0076] 4. After completion of Step 3, ethyl acetate was added to 400 mL of the flask (containing the residue existing after the ethyl acetate phase was collected in Step 3), then the mixture was ultrasonically crushed for 20 min, and the ethyl acetate phase was collected.
[0077] 5. The ethyl acetate phase obtained in the step 2, the ethyl acetate phase obtained in the step 3 and the ethyl acetate phase obtained in the step 4 were combined, and then concentrated under a reduced pressure using a rotary evaporator to obtain an extract.
[0078] 6. The extract obtained in the Step 5 was extracted with dichloromethane (after the dichloromethane is fully infiltrated into the extract, standing was carried out at room temperature for 1 hour), and concentrated under a reduced pressure by using a rotary evaporator to obtain a dichloromethane phase extract, also called a crude extract.
[0079] 72.2 g of the crude extract was prepared in Step 2 by per liter of the fermentation system completing Step 1.
[0080] III. Obtaining of Asterric Acid
[0081] 1. The crude extract obtained in Step 2 (6) was dissolved in dichloromethane and subjected to normal phase silica gel open column chromatography separation.
[0082] Silica gel particle size 100-200 mesh.
[0083] The mobile phase at the initial time of elution is dichloromethane, the mobile phase at the end of elution is methanol, and the mobile phase in the elution process is a mixture of dichloromethane and methanol; the flow rate of the mobile phase is 3 mL/min; the elution process lasts for 50h, the volume fraction of dichloromethane in the mobile phase is linearly reduced from 100% to 0%, and the corresponding volume fraction of methanol in the mobile phase is linearly increased from 0% to 100%.
[0084] Components with the volume fraction of dichloromethane accounting for 98%-94% in parts by volume in the mobile phase were collected.
[0085] 2. The liquid collected in Step 1 was taken and then concentrated under reduced pressure using a rotary evaporator. This component was not completely dissolved in dichloromethane, so that a small amount of methanol was added dropwise thereto to make the solution clear and non-viscous.
[0086] 3. After the step 2 is completed, the container is sealed with tin foil paper, dense small holes are drawn out with toothpicks, and the container is put into a fume hood for waiting of subsequent crystallization. After the crystal is separated out, redundant liquid is separated out, and then a dichloromethane/methanol mixed system (obtained by uniformly mixing 10 parts by volume of dichloromethane and 1 part by volume of methanol in the dichloromethane/methanol mixed system) is added, so that the crystallization is completely dissolved.
[0087] 4. After the step 3 is completed: sealing the container with tin foil paper, pricking out dense small holes with toothpicks, placing into a fume hood, recrystallizing, separating out excess liquid after the crystallization is separated out, and adding a dichloromethane/methanol mixed system to completely dissolve the crystallization.
[0088] [5] After the step 4 is completed, the container is sealed with tin foil paper, dense small holes are formed by using toothpicks, and then the small holes are put into a fume hood and recrystallized to obtain acicular crystals.
[0089] 13.2 mg of acicular crystals were obtained per g of crude extract.
[0090] 6. The acicular crystals obtained in Step 5 were dissolved in deuterated acetone for nuclear magnetic identification and HR-ESI-MS identification.
[0091] Parameters for nuclear magnetic identification: 1H-NMR (acetone-d6, 400 MHz), 13C-NMR (acetone-d6, 100 MHz), HSQC (acetone-d6, 400/100 MHz), HMBC (acetone d6, 400/100 MHz). The results of nuclear magnetic identification are shown in Table 1. 1 H-NMR diagram is shown in Figure 2. 13 C-NMR diagram is shown in Figure 3. HSQC diagram is shown in Figure 4. HMBC diagram is shown in Figure 5.
[0092] Parameters identified by HR-ESI-MS: injection voltage of 3500V, temperature of 180 °C, Set Nebulizer 0.6 Bar, Scan Begin 50 m/z, Set End plate offset-500V, Set Dry Gas 6.0 1/min, Scan End 1000 m/z, Set Collision Cell RF 150.Vpp. The HR-ESI-MS diagram is shown in Figure 6.
[0093] The result shows that the structural formula of the acicular crystal obtained in the Step 5 is shown as the formula (I), being asterric acid.
[0094] Table 1 Nuclear magnetic data (delta in ppm and J in Hz) of the crystals
[0095]
Position 'H "C 1 8.99 (H, S) 156 2 7.06 108.7 3 124 4 133 154 6 6.92 (1H, d, J=2.8) 106.2 1' 159.5 2' 5.91 106.0 3' 147.8 4' 6.49 112.3 ' -OH 11.9 164.2 6' 100.7 6' -OH 11.4 C=0 171.5,
[0096]
3' -CH3 2. 16 (s) 21. 1 3 3.82,(3H, s )-CH3 55.8 3.74, (3, s )-OCH 51.9
[0097]
OH 1 6 N .2
H 3CO O O OCH 3 HOC~
A( I). Formula (I).
<110 > Beijing Normal University
<120 > Method for preparing asterric acid through microbial fermentation
<130> GNCYX191084
<160> 1
<170> Patentln version 3 .5
<210> 1
<211> 570
<212> DNA
<213> Pseudogymnoascus pannorum
<400> 1
tttccgtagg gtgacctgcg gaaggatcat tacagtagtc gcccgggttg ccgcaaggcc 60 tcccgggtaa cctaccaccc tttgtttatt acactttgtt gctttggcaa gcctgccctc 120 gggctgctgg ctccggccgg cgagcgcttg ccagaggacc taaactctgt ttgtctatac 180 tgtctgagta ctatataata gttaaaactt tcaacaacgg atctcttggt tctggcatcg 240 atgaagaacg cagcgaaatg cgataagtaa tgtgaattgc agaattcagt gaatcatcga 300 atctttgaac gcacattgcg ccccctggta ttccgggggg catgcctgtc cgagcgtcat 360 tacaaccctc aagctcagct tggtgttggg ccccgccgcc ccggcgggcc ctaaagtcag 420 tggcggtgcc gtccggctcc gagcgtagta attcttctcg ctctggaggt ccggtcgtgt 480 gctcgccagc aacccccaat ttttttcagg ttgacctcgg atcaggtagg gatacccgct 540 gaacttaagc atatcaataa agcggaggaa 570
Claims (10)
1. Application of Pseudogymnoascus. pannorum BJZ13 in the preparation of asterric acid; and an accession number of Pseudogymnoascus pannorum BJZ13 is CGMCC No. 15389.
2. A method for preparing asterric acid, comprising the steps of: fermenting and culturing Pseudogymnoascus pannorum BJZ13 to obtain asterric acid; wherein the accession number of Pseudogymnoascus pannorum BJZ13 is CGMCC No. 15389.
3. The method according to claim 2, further comprising the step of subjecting the product of the fermentation culture to an organic phase extraction.
4. The method according to claim 3, wherein an organic solvent used in the organic phase extraction is ethyl acetate and dichloromethane.
5. The method according to claim 4, wherein the method for extracting an organic phase comprises the steps of: extracting a fermentation culture product with ethyl acetate, collecting the ethyl acetate phase, and concentrating under reduced pressure to obtain an extract; and extracting the extract with dichloromethane, and collecting the dichloromethane phase, and concentrating under reduced pressure to obtain a dichloromethane phase extract.
6. The method according to claim 5, wherein the method for extracting the organic phase comprises the steps of: (1) adding ethyl acetate to a fermentation culture product, then carrying out ultrasonic crushing, and collecting an ethyl acetate phase; (2) after the step (1) is completed, adding ethyl acetate to the residue, then carrying out ultrasonical crushing, and collecting an ethyl acetate phase; (3) after the step (2) is completed, adding ethyl acetate to the residue, then carrying out ultrasonic crushing, and collecting an ethyl acetate phase; (4) after the step (3) is completed, adding ethyl acetate to the residue, then carrying out ultrasonic crushing, and collecting an ethyl acetate phase; (5) concentrating the ethyl acetate phase obtained from the step (2), the ethyl acetate phase obtained from the step (3) and the ethyl acetate phase obtained from the step (4) under a reduced pressure to obtain an extract; and (6) extracting the extract obtained from the step (5) with dichloromethane, and then carrying out pressure-reduced concentration to obtain a dichloromethane phase extract.
7. The method according to claim 5 or 6, further comprising the step of obtaining asterric acid from the dichloromethane phase extract.
8. The method according to claim 7, wherein obtaining the asterric acid from the dichloromethane phase extract comprises the steps of: dissolving the dichloromethane phase extract with dichloromethane, subjecting to normal phase silica gel open column chromatography separation, and crystallizing.
9. The method according to claim 8, wherein the parameters of the normal phase silica gel open column chromatography separation are as follows: the silica gel particle size is 100-200 meshes; the mobile phase at the initial time of elution is dichloromethane, the mobile phase at the end of elution is methanol, and the mobile phase in the elution process is a mixture of dichloromethane and methanol; the flow rate of the mobile phase is 3mL/min; the elution process lasts for 50 h, the volume fraction of dichloromethane in the mobile phase is linearly reduced from 100% to 0%, and the corresponding volume fraction of methanol in the mobile phase is linearly increased from 0% to 100%; and components with the volume fraction of dichloromethane accounting for 98%-94% in parts by volume in the mobile phase are collected.
10. The method according to claim 8, wherein the step of crystallization comprises the steps of: taking the liquid collected by normal phase silica gel open column chromatography separation, concentrating under reduced pressure, crystallizing, separating out excess liquid after crystallization separation, adding a dichloromethane/methanol mixed system to completely dissolve the crystal, recrystallizing, separating out excess liquid after crystallization separation, adding a dichloromethane/methanol mixed system to completely dissolve the crystal, and recrystallizing to obtain acicular crystal, namely the asterric acid; and uniformly mixing 10 parts by volume of dichloromethane and 1 part by volume of methanol to obtain the dichloromethane/methanol mixed system.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108611278A (en) * | 2018-05-02 | 2018-10-02 | 北京师范大学 | A kind of method and its bacterial strain preparing phenazocine by microbial fermentation |
CN108611278B (en) * | 2018-05-02 | 2021-09-28 | 北京师范大学 | Method for preparing phenazocine through microbial fermentation and bacterial strain thereof |
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