Method for preparing sulfur garcinia by using new species of streptomyces deserticola

Abstract

The invention relates to a method for preparing thiogambogic bacteria by utilizing a streptomyces deserticola new strain, which comprises the following steps: (1) activating: inoculating a yeast starch solid culture medium with the streptomyces deserticola new strain Streptomyces sp. HMX112 preserved at-80 DEG C in a streak manner, and culturing at 30 DEG C for 14 days to obtain activated pure culture strain HMX112 spores; (2) seed culture: inoculating activated pure culture strain HMX112 spores into a casein tryptone liquid culture medium, and performing shake cultivation to obtain a first-stage seed solution; (3) fermentation culture: inoculating the first-grade seed liquid into a Gause No.1 liquid fermentation culture medium, and carrying out shake culture to obtain fermentation liquid; (4) fermentation liquor treatment: pre-cooling the fermentation liquor at 4 DEG C, then carrying out solid-liquid separation, and repeatedly extracting and concentrating the obtained supernatant for more than three times by using isometric ethyl acetate to obtain a crude extract; and (5) preparing the sulfur garcinia. The preparation method is simple, and the sample purity is high.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation and extraction and separation of active natural products, and in particular to a method for preparing thioglucosinolates using a new species of Streptomyces desertis. Background Technology

[0002] Thiolutin is a dithiopyrrolidone compound with significant biological activity, originally derived from Streptomyces spp. Streptomyces This compound was isolated from a strain of bacteria. It exhibits multiple biological activities, including broad-spectrum antibacterial activity (especially against Gram-positive bacteria and fungi), potent anti-angiogenic effects, anti-inflammatory properties (achieved through inhibition of pathways such as NF-κB), and function as a molecular tool as an inhibitor of eukaryotic RNA polymerase II / III. These properties make it a promising candidate for applications in antibacterial drug development, antitumor therapy, biopesticides, and basic scientific research.

[0003] Currently, the production of thioclase mainly relies on known Streptomyces strains (such as...) Streptomyces albulus , S. luteosporeus (etc.). However, the existing technology has significant limitations: (1) Production bottleneck - traditional strains have low fermentation efficiency, and the product concentration is difficult to meet the industrial needs, which increases production costs; (2) Process complexity - long fermentation cycle and many by-products (such as structural analogs) make downstream purification difficult, affecting product purity and yield; (3) Insufficient strain stability - some production strains are prone to metabolic drift during subculturing or scale-up, causing yield fluctuations.

[0004] Therefore, the industry needs to discover new high-yield strains—especially those originating from extreme environments (such as desert environments)—in order to overcome yield limitations, optimize product characteristics, and provide a technological foundation for the industry. Against this backdrop, developing new sources of thiogamma-fortified sulfadiazine with high fermentation efficiency and excellent product performance has become a key method for solving industry problems. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a simple and efficient method for preparing thiocrotonin using a new species of Streptomyces desertis.

[0006] To address the above problems, the present invention provides a method for preparing thiophene citrate using a new species of *Streptomyces desertica*, comprising the following steps: (1) Activation: A new species of *Streptomyces desertica* preserved at -80℃ Streptomyces sp. HMX112 was streaked onto yeast starch solid medium and cultured at 30°C for 14 days to obtain activated pure culture strain HMX112 spores; (2) Seed culture: The activated pure culture strain HMX112 spores were inoculated into tryptic soybean broth (TSB) and cultured at 30°C and 160 rpm for 2 days until the OD value was 0.5, thus obtaining the first-grade seed culture. (3) Fermentation culture: The primary seed culture was inoculated at a rate of 10% into 20% of Gaoshi No. 1 liquid fermentation medium, and cultured at a constant temperature of 30°C and shaking at 160 rpm for 7 days to obtain the fermentation broth. (4) Fermentation broth treatment: After the fermentation broth was pre-cooled at 4°C, it was centrifuged at 8000 rpm for 10 min in a low-temperature centrifuge. After solid-liquid separation, the supernatant was repeatedly extracted and concentrated with an equal volume of ethyl acetate more than 3 times to obtain the crude extract. (5) Preparation of thioglucosinolate: a. The crude extract was eluted with methanol and separated into two parts: a bright yellow and a dark red. b. The bright yellow portion is fractionated by normal column chromatography to obtain the target component; c. The target component is further subjected to thin-layer chromatography on a silica gel plate. When the bright yellow target component is located in the third band, it is scraped, eluted, and air-dried to obtain the purified compound Thiolutin.

[0007] The new species of *Streptomyces desertis* in step (1) Streptomyces The accession number for sp. HMX112 is GDMCC No:66755. This strain was deposited on July 24, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Institute of Microbiology, Chinese Academy of Sciences, 510070, China.

[0008] In step (1), the yeast starch solid culture medium is prepared by first adding water to 10g of soluble starch to mix the starch evenly, then adding boiling water to fully gelatinize the starch, then adding 2g of yeast extract and 15-20g of agar, adding distilled water to make up to 1000mL, and sterilizing at 121℃ under high temperature and high pressure for 20 minutes.

[0009] In step (2), the tryptic soy broth (TSB) is prepared by adding 17.0g of tryptone, 3.0g of soybean protein hydrolysate, 5.0g of sodium chloride, 2.5g of dipotassium hydrogen phosphate, and 2.5g of glucose to distilled water, mixing well, adjusting the pH to 7.3±0.2, and bringing the volume to 1000mL. The mixture is then autoclaved at 121℃ for 20min.

[0010] In step (3), the Gao's No. 1 liquid fermentation medium is prepared by first adding water to 20g of soluble starch to mix the starch evenly, then adding 800mL of boiling water to fully gelatinize the starch, then adding 1.0g KNO3, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 0.5g sodium chloride and 0.01g FeSO4·7H2O, mixing evenly, adding distilled water to make up to 1000mL, and then sterilizing at 121℃ under high temperature and high pressure for 20 minutes.

[0011] In step b, the mobile phase used for the normal column chromatography is a mixture of chloroform and acetone in a volume ratio of 5:1.

[0012] The developing solvent used in step c for thin-layer chromatography is a mixture of petroleum ether and acetone in a volume ratio of 9:1.

[0013] The eluent used in step c is a mixture of acetone and methanol in a volume ratio of 5:1.

[0014] Compared with the prior art, the present invention has the following advantages: 1. The fermentation culture medium of this invention is simple, low in cost, and has a short fermentation time.

[0015] 2. The strain used in this invention is a novel species of *Streptomyces desertica*, and the preparation method is simple, resulting in high sample purity. Using the method of this invention, high-yield Thiolutin can be prepared, with a yield as high as 61 mg / L. Attached Figure Description

[0016] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0017] Figure 1 The colony morphology (left) and scanning electron microscope image (right) of the strain HMX112 of this invention are shown.

[0018] Figure 2 This is a flowchart illustrating the isolation process of the active natural products from strain HMX112 of this invention.

[0019] Figure 3 Thiolutin produced by strain HMX112 of this invention 1 H-NMR spectrum.

[0020] Figure 4 Thiolutin produced by strain HMX112 of this invention 13 C-NMR spectrum.

[0021] Figure 5 The nucleocytic-to-cytoplasmic ratio ([M+Na)) of the Thiolutin produced by strain HMX112 of this invention. + ).

[0022] Figure 6 Standard curve of Thiolutin concentration and peak area (a) and Thiolutin production over time (b) under different treatments. DETAILED DESCRIPTION

[0023] As Figure 2 shown, a method for preparing thiolutin by using a new Streptomyces deserti species, comprising the following steps: (1) Activation: The new Streptomyces deserti species sp. HMX112 preserved at -80°C was streaked on a yeast starch solid medium and cultured at 30°C for 14 days to obtain activated pure culture strain HMX112 spores. Streptomyces (2) Seed culture:

[0024] The preservation number of the new Streptomyces deserti species sp. HMX112 is GDMCC No: 66755. The strain has been preserved in the Guangdong Microbial Culture Collection Center on July 24, 2025. The address of the preservation center is: No. 59, Building 5, Institute of Microbiology, Chinese Academy of Sciences, Guangzhou Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, Guangdong, China, with a postcode of 510070. Streptomyces The yeast starch solid medium refers to first adding water to 10 g of soluble starch to mix the starch evenly, then adding boiling water to gelatinize the starch thoroughly, and then adding 2 g of yeast extract, 15-20 g of agar, adding distilled water to 1000 mL, and sterilizing at 121°C high temperature and high pressure for 20 minutes.

[0025] (2) Seed culture:

[0026] The activated pure culture strain HMX112 spores were inoculated in a Tryptic Soy Broth (TSB) liquid medium and cultured at 30°C and 160 rpm on a shaker for 2 days to an OD value of 0.5 to obtain a primary seed liquid. The Tryptic Soy Broth (TSB) liquid medium refers to adding Tryptone 17.0 g, soybean protein hydrolysate 3.0 g, sodium chloride 5.0 g, potassium phosphate dibasic 2.5 g, and glucose 2.5 g in distilled water, mixing evenly and adjusting the pH to 7.3±0.2, then adding distilled water to 1000 mL, and sterilizing at 121°C high pressure steam for 20 min.

[0027] (3) Fermentation culture:

[0028] The primary seed liquid was inoculated in a 20% Gao No. 1 liquid fermentation medium at a 10% inoculation amount, and cultured at 30°C under constant temperature conditions with 160 rpm shaking for 7 days to obtain a fermentation liquid.

[0029] ​Gao's liquid fermentation medium refers to first adding water to 20 g of soluble starch to make the starch uniform, then adding 800 mL of boiling water to make the starch gelatinize completely, then adding 1.0 g of KNO3, 0.5 g of K2HPO4, 0.5 g of MgSO4·7H2O, 0.5 g of sodium chloride and 0.01 g of FeSO4·7H2O, mixing uniformly, adding distilled water to make the volume 1000 mL, and sterilizing at 121 ℃ high temperature and high pressure for 20 minutes to obtain.

[0030] ⑷Fermentation broth treatment: After the fermentation broth is pre-cooled at 4 ℃, it is centrifuged at a speed of 8000 rpm in a low-temperature centrifuge for 10 min, and after solid-liquid separation, the supernatant is repeatedly extracted with an equal volume of ethyl acetate and concentrated for more than 3 times to obtain the crude extract.

[0031] ⑸Preparation of thiolutin: a. The crude extract is eluted with methanol, and divided into two parts of bright yellow and dark red.

[0032] During the collection process, it is found that the solubility of the crude extract is poor, and it is more soluble in chloroform, acetone and DMSO, and solid can be precipitated and separated out in the collection bottle, which can be divided into two parts of bright yellow flocculent and dark red hard block.

[0033] b. The bright yellow part is subjected to forward column chromatography for segmentation, and the target component is obtained. The mobile phase used in forward column chromatography is a mixture of chloroform and acetone in a volume ratio (mL / mL) of 5:1.

[0034] c. The target component is further subjected to thin layer chromatography silica gel plate, and the developing agent is a mixture of petroleum ether and acetone in a volume ratio (mL / mL) of 9:1; when the bright yellow target component is located in the 3rd band, it is scraped off, and then eluted with acetone:methanol (v / v)=5:1, and placed in a ventilated place to dry naturally to form a sheet, and the purified compound thiolutin is obtained.

[0035]

New species of desert Streptomyces sp. HMX112 Streptomyces Morphological characteristics

[0036] Strain HMX112 is a Gram-positive bacterium. During cultivation, it was found to grow well on Gao's No. 1, TSB, and yeast starch media. It produced a yellow pigment on Gao's No. 1 media, and spore growth was optimal on yeast starch media. As shown in the image, the spores are initially white, gradually turning pinkish-brown. Scanning electron microscopy revealed that the spores are cylindrical with a smooth surface.

[0037] [Identification of compounds isolated from strain HMX112] This compound is bright yellow under natural light and exhibits high antibacterial activity.

[0038] High-resolution mass spectrometry ([M+Na]⁺ m / z 250.9922) and carbon nuclear magnetic resonance spectroscopy (…) were used to determine the atomic number of Na atoms. 13 Based on C10 NMR data, its molecular formula was determined to be C8H8N2O2S2, with an unsaturation degree Ω=6. The carbon spectrum showed a total of 8 carbon signals, including 2 ester carbonyl carbons (δ¹⁰). C 166.1, 168.8), 4 aromatic ring carbons (δ C 111.0, 114.8, 132.4, 136.0) and 2 methyl carbons (δ C 22.4, 27.5). Combined with proton spectrum ( 1 H NMR) characteristic signal: δ H 7.34 represents the aromatic proton (H-3) in the thiolopyrrolone ring; the doublet originates from the weak coupling of the sulfur atom; δ H 2.02: The methyl proton of the acetoxy group (-OAc), the singlet indicates no ortho coupling; δ H 3.25 represents the methyl proton of the methoxy group (-OCH3), with a low field shift (δ). C 27.5) This suggests an electron-withdrawing effect from the adjacent thioester group (-S-CO-); δ H 9.98 represents the amide proton (-NH) in the thioester ring, and the broad singlet is characteristic of an active proton. Mass spectrometry isotope analysis showed M+2 / M≈10%, consistent with the theoretical abundance (≈9%) of molecules containing two sulfur atoms, ruling out the possibility of chlorine inclusion. NMR data further support the fused-ring structure of the thiopyrrolidone skeleton, where the sulfur atom connects C-3a (δC 136.0) and C-6a (δC 136.0). C 132.4), forming a rigid planar system. Combined with literature search data comparison and compound characteristics, it was found that this compound is highly consistent with the data of thiolutin, thus confirming that this compound is thiolutin. The comparison results are shown in Table 1. 1 H NMR spectrum as shown Figure 3 As shown, 13 The C NMR spectrum is as follows Figure 4 As shown, the mass spectrum is as followsFigure 5 The results are shown.

[0039] Table 1: Comparison of 1H-NMR and 13C-NMR data of compound Thiolutin

Bacteriostatic application test of S. thiolutin by strain HMX112

[0040] (1) The standard agar diffusion method was used to determine the antibacterial activity of the sample. The specific steps are as follows: ① The pathogenic bacteria were inoculated in LB liquid medium and cultured at 37°C with shaking until the logarithmic phase (about 6h). The McFarland turbidity method was used to adjust the concentration of the bacterial solution to 1×10 8 -1×10 9 CFU / mL. LB medium (Luria-Bertani, LB): yeast extract 5.0g, tryptone 10.0g, sodium chloride 10.0g, pH 7.4, add distilled water to 1000mL, 121℃ high temperature and high pressure sterilization for 20 minutes for standby.

[0041] ② The melted LB agar medium was cooled to 50℃, and the bacterial solution was added at a ratio of 2% (v / v). After mixing, it was poured into a flat plate. LB agar medium: yeast extract 5.0g, tryptone 10.0g, sodium chloride 10.0g, 15~20 g agar, pH 7.4, add distilled water to 1000mL, 121℃ high temperature and high pressure sterilization for 20 minutes for standby.

[0042] ③ Refer to the punching method in the literature (Wang Shiqiang. Punch method for determining the potency of antibiotics [J]. Bulletin of Biology, 2005, (03): 2.), use a 6mm aperture punch to prepare sample holes, and add 200µL of sample to be tested to each hole.

[0043] ④ After 8h of incubation at 37℃, the diameter of the inhibition zone was measured, and three parallel repeats were set for each experiment.

[0044] The results are shown in Table 2. Thiolutin showed significant inhibition effect on 7 common pathogenic strains (gram-positive and gram-negative bacteria), especially on Staphylococcus pasteuri and Staphylococcus epidermidis The inhibition effect on

[0045] Table 2: Results of the antibacterial activity test of thiolutin produced by strain HMX112 on common pathogenic strains 2. The antibacterial activity of the test sample on 4 strains of methicillin-resistant Staphylococcus aureus was tested by micro-broth dilution method (BARBARA D, GARY W, PHILIPPE DUFRESNE, et al. Clinical and Laboratory Standards Institute [Z] / / PA: CLSI. Reference method for broth dilution antifungal susceptibility testing of yeasts. Wayne. 2017).

[0046] In each well of the 96-well plate, bacterial solution and test sample were added respectively, with vancomycin as positive control and sample solvent as negative control. After incubation, the OD 600 value was determined by a microplate reader. The MIC value of the test sample was obtained according to the standard curve established by OD 600 value, and the results are shown in Table 3.

[0047] As shown in Table 3, thiolutin showed significant inhibitory effect on 1 strain of quality control MRSA (ATCC29213) and 3 strains of MRSA (N315, clinical strain 1 and clinical strain 2). The minimum inhibitory concentration (MIC) determined by micro-dilution method was 16 µg / mL (ATCC29213), 16 µg / mL (N315), 8 µg / mL (clinical strain 1) and 16 µg / mL (clinical strain 2), respectively.

[0048] Table 3: Results of the antibacterial activity test of thiolutin produced by strain HMX112 on MRSA strains 6. The antibacterial activity of the test sample on 4 strains of methicillin-resistant Staphylococcus aureus was tested by micro-broth dilution method (BARBARA D, GARY W, PHILIPPE DUFRESNE, et al. Clinical and Laboratory Standards Institute [Z] / / PA: CLSI. Reference method for broth dilution antifungal susceptibility testing of yeasts. Wayne. 2017).

[0049] The 6mm diameter puncher was used to take the fungus cake on the pathogenic fungus species cultured for 5 days, and the fungus cake was transplanted to the center of the solid culture medium containing the sample to be tested, so that the mycelium faced down and contacted the medium, and the solid culture medium with the sample solvent was set as the blank control. The diameter of the colony was measured when the colony diameter reached 5 cm or more, wherein the mycelial growth inhibition rate = (control colony diameter - treatment colony diameter) / control colony diameter. The inhibition effect of thiolutin on various crop pathogenic fungi at a concentration of 50 ppm is shown in Table 4.

[0050] Table 4: Inhibition activity test results of thiolutin produced by strain HMX112 on plant pathogenic fungi Note: 1: Fusarium graminearum (F. graminearum) F.graminearum ), 2: Fusarium oxysporum (F. oxysporum) F.oxysporum ), 3: Fusarium solani (F. solani) F.solani ), 4: Rhizoctonia solani (R. solani) R.solani ), 5: Sclerotinia sclerotiorum (S. sclerotiorum) S.sclerotiorum ), 6: Alternaria alternate (A. alternate) A.tenuissima ), 7: Alternaria solani (A. solani) A.solani ); 8: Botrytis cinerea (B. cinerea) B.cinerea ); 9: Phytophthora capsici (P. capsici) P.capsici ); 10: Guignardia bidwellii (G. bidwellii) C.fructicola ); 11: Valsa mali (V. mali) Valsa mali ).

[0051] As can be seen from Table 4, thiolutin has a significant inhibitory effect on various pathogenic fungi, especially the inhibition rates of F. graminearum Fusarium graminearum ), R. solani Rhizoctonia solani ), S. sclerotiorum Sclerotinia sclerotiorum ), P. capsici Phomopsis capsici ), and V. mali Valsa mali ) reached 100%. In addition, the inhibition rates of thiolutin on F. oxysporum Fusarium oxysporum ), F. solani (F. solani) usarium solan ), A. alternate Alternaria tenuissima ), A. solani Alternaria solani ), and B. cinerea Botrytis cinerea ) were 81.80%, 27.10%, 75.63%, 72.80%, 76.20%, and 82.60%, respectively. The inhibition rate on G. bidwellii Colletotrichum fructicola ) was 52.30%.

[0052] The above tests show that Thiolutin exhibits broad-spectrum antibacterial activity against various pathogenic bacteria, especially Gram-positive bacteria (such as Staphylococcus pasteuri and Staphylococcus epidermidis The inhibitory effect of Thiolutin is significantly better than that of the traditional antibiotic Kanamycin. In addition, Thiolutin also exhibits strong inhibitory activity against MRSA strains, with a minimum inhibitory concentration (MIC) of 8-16 µg / mL. In plant pathogenic fungus inhibition experiments, the inhibition rate of Thiolutin against various crop pathogenic fungi (such as Fusarium graminearum and Rhizoctonia solani and the like) reaches 100%, showing its potential application value in agricultural disease control.

[0053]

Optimization of fermentation conditions for Thiolutin production by strain HMX112

[0054] In summary, the preparation method of Thiolutin provided by the present application adds L-cystine to the fermentation medium, greatly increasing the production, which is 61 mg / L. Compared with existing Thiolutin preparation methods, the preparation process is simple, the strain fermentation level is high, the cost is low, the fermentation period is short, and large-scale fermentation can be used to produce active substances in large quantities, thereby fundamentally solving the shortage of drug sources; the new species of desert Streptomyces Streptomyces flavimicrosus HMX112, the main secondary metabolite of which is Thiolutin, has good antibacterial ability against various pathogenic bacteria, drug-resistant bacteria, and plant pathogenic fungi, and has high application value in various fields of pathogenic prevention and control, and can be used to develop efficient biological pesticides and biological medicines.

Claims

1. A method for preparing thiophene citrate using a new species of *Streptomyces desertica*, comprising the following steps: (1) Activation: A new species of *Streptomyces desertica* preserved at -80℃ Streptomyces sp. HMX112 was streaked onto yeast starch solid medium and cultured at 30°C for 14 days to obtain activated pure culture strain HMX112 spores; (2) Seed culture: The activated pure culture strain HMX112 spores were inoculated into tryptic soybean peptone liquid medium and cultured in a shaker at 30°C and 160 rpm for 2 days until the OD value was 0.5, thus obtaining the first-grade seed liquid; (3) Fermentation culture: The primary seed culture was inoculated at a rate of 10% into 20% of Gaoshi No. 1 liquid fermentation medium, and cultured at a constant temperature of 30°C and shaking at 160 rpm for 7 days to obtain the fermentation broth. (4) Fermentation broth treatment: After the fermentation broth was pre-cooled at 4°C, it was centrifuged at 8000 rpm for 10 min in a low-temperature centrifuge. After solid-liquid separation, the supernatant was repeatedly extracted and concentrated with an equal volume of ethyl acetate more than 3 times to obtain the crude extract. (5) Preparation of thioglucosinolate: a. The crude extract was eluted with methanol and separated into two parts: a bright yellow and a dark red. b. The bright yellow portion is fractionated by normal column chromatography to obtain the target component; c. The target component is further passed through a thin-layer chromatography silica gel plate. When the bright yellow target component is located in the third band, it is scraped, eluted, and air-dried to obtain the purified compound thioclase.

2. The method for preparing thiophene citrate using a new species of *Streptomyces desertica* as described in claim 1, characterized in that: The new species of *Streptomyces desertis* in step (1) Streptomyces sp. HMX112 has the accession number GDMCC No: 66755.

3. The method for preparing thiophene citrate using a new species of *Streptomyces desertica* as described in claim 1, characterized in that: In step (1), the yeast starch solid culture medium is prepared by first adding water to 10g of soluble starch to mix the starch evenly, then adding boiling water to fully gelatinize the starch, then adding 2g of yeast extract and 15-20g of agar, adding distilled water to make up to 1000mL, and sterilizing at 121℃ under high temperature and high pressure for 20 minutes.

4. The method for preparing thiophene citrate using a new species of *Streptomyces desertica* as described in claim 1, characterized in that: In step (2), the tryptone-soybean liquid culture medium is prepared by adding 17.0g of tryptone, 3.0g of soybean protein hydrolysate, 5.0g of sodium chloride, 2.5g of dipotassium hydrogen phosphate and 2.5g of glucose to distilled water, mixing well, adjusting the pH to 7.3±0.2, and bringing the volume to 1000mL. The medium is then sterilized by autoclaving at 121℃ for 20min.

5. The method for preparing thiophene citrate using a new species of *Streptomyces desertica* as described in claim 1, characterized in that: In step (3), the Gao's No. 1 liquid fermentation medium is prepared by first adding water to 20g of soluble starch to mix the starch evenly, then adding 800mL of boiling water to fully gelatinize the starch, then adding 1.0g KNO3, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 0.5g sodium chloride and 0.01g FeSO4·7H2O, mixing evenly, adding distilled water to make up to 1000mL, and then sterilizing at 121℃ under high temperature and high pressure for 20 minutes.

6. The method for preparing thiophene citrate using a new species of *Streptomyces desertica* as described in claim 1, characterized in that: In step b, the mobile phase used for the normal column chromatography is a mixture of chloroform and acetone in a volume ratio of 5:

1.

7. The method for preparing thiophene citrate using a new species of *Streptomyces desertica* as described in claim 1, characterized in that: The developing solvent used in step c for thin-layer chromatography is a mixture of petroleum ether and acetone in a volume ratio of 9:

1.

8. The method for preparing thiophene citrate using a new species of *Streptomyces desertica* as described in claim 1, characterized in that: The eluent used in step c is a mixture of acetone and methanol in a volume ratio of 5:1.

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