Stenotrophomonas sp. and its application in synthesis of acarbose

Abstract

This invention discloses a strain of oligotrophomonas ( Stenotrophomonas Oligotrophozoites A322 and their application in acarbose synthesis. The oligotrophozoite A322 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2025917. Oligotrophozoites A322 can synthesize acarbose using maltose and glucose as carbon sources. The acarbose yield of Oligotrophozoites A322 in a 100 mL shake-flask fermentation system for 1–7 days is 0.4–4 g / L. The acarbose isolated and purified from Oligotrophozoites A322 showed consistent high-performance liquid chromatography (HPLC) retention time and NMR chemical shift with the standard. Activity assays showed that the isolated and purified acarbose sample possessed α-amylase inhibitory activity.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to an oligotrophic bacterium and its application in the synthesis of acarbose. Background Technology

[0002] Acarbose was first isolated by researchers at Bayer AG in Germany from the fermentation broth of *Actinomyces lucifera*, revealing it as a substance with α-glucosidase inhibitory activity. Further structural identification showed that acarbose is a complex oligosaccharide, a pseudotetrasaccharide composed of an acarvityl group and maltose. The acarvityl moiety is an N-glycoside composed of an unsaturated cyclool and 4-amino-4,6-dideoxy-α-D-glucopyranose, which plays a major inhibitory role against glucosidase. After discovering acarbose's α-glucosidase inhibitory activity, Bayer conducted a series of preclinical studies to evaluate its safety and efficacy. In 1986, acarbose was first approved in Germany for the treatment of type 2 diabetes. Subsequently, after large-scale clinical trials, acarbose was approved for marketing by the U.S. Food and Drug Administration (FDA) in 1995, and has gradually been widely used globally for the treatment of type 2 diabetes, now becoming a first-line clinical drug.

[0003] Currently, acarbose cannot be chemically synthesized; industrially, its production through fermentation mainly utilizes actinomycetes. Actinoplanes utahensis ZJB-08196, Actinomycetes Actinoplanes sp. SE50 / 110 was used as the production strain. Wild-type actinomycetes produce low acarbose yields, ranging from 8-10 g / L. Furthermore, the fermentation process generates various acarbose homologues, making subsequent acarbose isolation and purification challenging.

[0004] Therefore, the limited availability of single-strain production and low fermentation yield have hindered further improvements in acarbose fermentation production. To enhance the production performance of the strains, the solution proposed in this application is presented. Summary of the Invention

[0005] In view of this, one of the objectives of this invention is to address the problem of insufficient existing acarbose production strains by providing an oligotrophic monocytogenes strain and its application in the synthesis of acarbose.

[0006] The specific technical solution adopted in this invention is as follows:

[0007] The first aspect of this invention provides an oligotrophomonium strain, wherein the oligotrophomonium is named *Oligotrophomonas* (…). Stenotrophomonas sp. Accession number A322 was deposited on April 27, 2025, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China, 430072, China), with accession number CCTCC NO: M 2025917.

[0008] Furthermore, the 16S rDNA sequence of the oligotrophomonas described above is shown in SEQ ID NO:1.

[0009] The second aspect of this invention provides the application of the above-mentioned oligotrophomonas in the fermentation preparation of acarbose.

[0010] Furthermore, the substrates for fermentation to prepare acarbose are glucose and / or maltose.

[0011] The third aspect of the present invention provides a method for preparing acarbose by fermentation, comprising the following steps: placing the above-described oligotrophomonas in a liquid culture medium for fermentation culture, and then separating and purifying to obtain acarbose.

[0012] Furthermore, the fermentation temperature is 25-40℃; the pH value of the fermentation system is 7.0; the fermentation time is 1-7 days, and maltose may or may not be added daily during the fermentation process.

[0013] Furthermore, the liquid culture medium specifically comprises, per 1L, 16.0-50.0g of maltose, 4.0-20.0g of glucose, 4.0-8.0g of sodium glutamate, 2.5g of peptone, 2.5g of yeast extract, 1.0g of dipotassium hydrogen phosphate, 1.0g of magnesium sulfate heptahydrate, 0.1g of ferrous sulfate heptahydrate, with the remainder being water.

[0014] Specifically, Oligotrophomonas ( Stenotrophomonas Acarbose (sp.) A322 was inoculated into test tubes containing 5 mL of LB liquid medium for activation culture at 30℃ and 220 rpm. After 18 hours of culture, 1% of the culture was inoculated into 500 mL Erlenmeyer flasks containing 100 mL of liquid fermentation medium (pH 7.0) for shake culture. After 1-7 days of culture, the acarbose yield was 0.4-4.0 g / L.

[0015] Furthermore, the method for separating and purifying acarbose includes the following steps: after the fermentation culture is completed, the fermentation broth is centrifuged to obtain the fermentation supernatant; the fermentation supernatant is precipitated with anhydrous ethanol to obtain the precipitate; and the precipitate is separated and purified by cation exchange resin and silica gel plate to obtain acarbose.

[0016] Specifically, the cation exchange resin separation and purification was performed using a strong acid cation exchange resin, 001x7. First, the 001x7 resin was activated. Then, the crude acarbose extract solution was transferred to the activated 001x7 resin column and loaded onto the column. After standing, the eluent was collected fractionally. Next, the eluent was eluted with ultrapure water and 0.03-0.15M hydrochloric acid solution, and the eluent was collected fractionally until the OD of the collected solution reached a certain value. 210Approximately 0, the content and purity of acarbose in each collected solution were determined by HPLC, and the eluent was freeze-dried. The freeze-dried product was further separated and purified by silica gel plate chromatography. The chromatographic development conditions were n-butanol:ethanol:water = 9:7:5, and the development height was 17 cm. The development degree of the sample and standard was observed under 254 nm fluorescence, and the developed band consistent with the standard was scraped from the sample. The scraped silica gel particles were ground uniformly, dissolved in methanol, and sonicated to dissolve the acarbose attached to the silica gel particles into the methanol solution. After filtration through a 0.22 μm filter membrane, the purity and content of acarbose were determined by high-performance liquid chromatography.

[0017] Furthermore, the retention times of fermented acarbose and acarbose standard were consistent in high-performance liquid chromatography (HPLC), and the carbon shifts in the NMR spectra of fermented acarbose and acarbose standard were consistent. The Mass value in cation mode detected by high-resolution mass spectrometry was 646.2577, and elemental analysis showed that the molecular ion peak was C646.2577. 25 H 44 NO 18 This indicates that oligotrophomonas ( Stenotrophomonas The molecular formula of the acarbose sample synthesized by fermentation of sp.) A322 is C 25 H 43 NO 18 .

[0018] Furthermore, by measuring oligotrophomonas ( Stenotrophomonas Acarbose prepared by fermentation of A322 was used to inhibit α-amylase, and the activity of acarbose samples was detected. The results showed that acarbose samples had significant α-amylase inhibitory activity.

[0019] The beneficial effects of this invention are:

[0020] This invention provides a Stenotrophomonas sp. A322 strain capable of fermenting and synthesizing acarbose, and provides a method and application for the fermentation and synthesis of acarbose by this strain, providing a new strain resource for the fermentation production of acarbose. Attached Figure Description

[0021] Figure 1 It is an oligotrophomonas ( Stenotrophomonas A phylogenetic tree of sp.)A322 based on 16S rDNA;

[0022] Figure 2 It is an oligotrophomonas ( Stenotrophomonas Growth and yield curves of acarbose synthesis by fermentation of sp.)A322 in liquid fermentation medium;

[0023] Figure 3 It is an oligotrophomonas ( StenotrophomonasGrowth and yield curves of acarbose synthesis by fed-batch fermentation of sp.) A322 in liquid fermentation medium in shake flask;

[0024] Figure 4 It is an oligotrophomonas ( Stenotrophomonas Liquid chromatograms of acarbose synthesized by fermentation of sp.)A322 after separation and purification, and acarbose standard;

[0025] Figure 5 It is an oligotrophomonas ( Stenotrophomonas Carbon NMR spectra of acarbose synthesized by fermentation of sp.) A322 after separation and purification (right ordinate 1) and acarbose standard (right ordinate 2);

[0026] Figure 6 It is an oligotrophomonas ( Stenotrophomonas High-resolution mass spectrum of acarbose synthesized by fermentation of sp.)A322 after separation and purification;

[0027] Figure 7 It is an oligotrophomonas ( Stenotrophomonas Activity detection curve of acarbose synthesized by fermentation of sp.)A322 after separation and purification. Detailed Implementation

[0028] To explain in detail the technical content, structural features, objectives, and effects of the technical solution, the following description is provided in conjunction with specific embodiments and accompanying drawings.

[0029] The experimental methods used in the following examples are all conventional methods.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] The bacterial strains used in the following examples are all oligotrophomonas ( Stenotrophomonas sp.) A322, the culture medium formulation used in the examples is as follows:

[0032] Each liter of liquid fermentation medium contains 16.0g maltose, 4.0g glucose, 4.0g monosodium glutamate, 2.5g peptone, 2.5g yeast extract, 1.0g dipotassium hydrogen phosphate, 1.0g magnesium sulfate heptahydrate, and 0.1g ferrous sulfate heptahydrate, with water added to a final volume of 1 liter.

[0033] Solid culture medium is based on liquid fermentation medium with 20g of agar added per liter.

[0034] 2216E liquid culture medium contains 5.0g peptone, 1.0g yeast extract, 0.1g ferric citrate, 19.45g sodium chloride, 5.98g magnesium chloride, 3.24g sodium sulfate, 1.8g calcium chloride, 0.55g potassium chloride, 0.16g sodium carbonate, 0.08g potassium bromide, 0.034g strontium chloride, 0.022g boric acid, 0.004g sodium silicate, 0.0024g sodium fluoride, 0.0016g sodium nitrate, and 0.008g disodium hydrogen phosphate per liter. Water is added to a final volume of 1L, and the pH is 7.6 ± 0.2.

[0035] 2216E solid agar medium is based on 2216E liquid medium with 20g of agar added per liter.

[0036] LB medium contains 10.0g peptone, 5.0g yeast extract, and 10.0g sodium chloride per liter, with water added to a final volume of 1 L. The pH value is 7.0. Example 1

[0037] Isolation and Identification of Acarbose-Borose Synthetic Strains A322

[0038] Seawater samples were diluted with sterile water and spread on 2216E solid medium containing 0.1 mg / mL acarbose. After single colonies grew, they were inoculated into 2216E liquid culture containing 0.1 mg / mL acarbose, resulting in a strain with acarbose tolerance, designated A322. The genome of this strain was extracted and whole-genome sequencing was performed. The 16S rDNA of strain A322 was compared with that of the standard strain at https: / / www.ezbiocloud.net / . Stenotrophomonas pavanii The DSM 25135 similarity was 99.93%, indicating that strain A322 is an oligotrophomonad. Stenotrophomonas sp. The strain was named Oligotrophomonas ( ). Stenotrophomonas sp. A322. A phylogenetic tree based on the 16S rDNA sequence of this strain was constructed using Mega 6.0, showing that strain A322 is related to strain... Stenotrophomonas sp. Multiple strains clustered in the same branch, indicating a close phylogenetic relationship. Figure 1 Oligotrophomonas ( Stenotrophomonas sp. A322 was deposited on April 27, 2025, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China, 430072, China), with accession number CCTCC NO: M 2025917.

[0039] Oligotrophomonas ( Stenotrophomonas The 16S rDNA gene sequence of sp.)A322 is shown in the sequence listing, and the specific sequence SEQ ID No. 1 is as follows:

[0040] Example 2

[0041] Oligotrophomonas ( Stenotrophomonas sp. A322 fermentation synthesis of acarbose

[0042] Oligotrophomonas ( Stenotrophomonas sp. A322 was streaked and activated onto LB agar plates and incubated at 30°C for 2 days. A single colony was picked and inoculated into a test tube containing 5 mL of LB liquid medium. The culture was maintained at 30°C and a rotation speed of 220 rpm. After 18 hours of incubation, a 1% inoculum was transferred to a 500 mL Erlenmeyer flask containing 100 mL of liquid fermentation medium (pH 7.0) and cultured on a shaker at 30°C and a shaker speed of 220 rpm. Samples were taken every 6 hours to detect OD. 600 The fermentation supernatant was analyzed by liquid chromatography to determine the acarbose yield, and quantification was performed using an external standard method with acarbose standards. Results showed that in the early stages of fermentation, with increasing fermentation time, the yield of *Oligotrophomonas* (…) increased. Stenotrophomonas sp. The acarbose production of A322 gradually increased, reaching a peak of 0.44 mg / mL at 42 hours. After 42 hours, the yield of Oligotrophomonas (…) decreased with increasing fermentation time. Stenotrophomonas sp. The yield of acarbose decreased with A322, with a minimum yield of 0.18 mg / mL, after 60 hours of fermentation by *Oligotrophomonas*. Stenotrophomonas sp. The yield of A322 acarbose increased with increasing fermentation time. Figure 2 ). Example 3

[0043] Oligotrophomonas ( Stenotrophomonas sp. A322 shake-flask fed-batch fermentation for the synthesis of acarbose

[0044] Oligotrophomonas ( Stenotrophomonas sp.) A322 was streaked and activated onto LB agar plates and incubated at 30°C for 2 days. A single colony was picked and inoculated into a test tube containing 5 mL of LB liquid medium. The culture temperature was 30°C and the rotation speed was 220 rpm. After 18 hours of incubation, a 1% inoculum was transferred to a 500 mL Erlenmeyer flask containing 100 mL of liquid fermentation medium (pH 7.0) and cultured on a shaker at 30°C and a shaking speed of 220 rpm. Maltose was added daily at a rate of 20 g per liter of medium. Samples were taken daily to detect OD. 600 The fermentation supernatant was analyzed by liquid chromatography to determine the acarbose yield, and quantification was performed using an external standard method with acarbose standards. Results showed that with increasing fermentation time, the yield of *Oligotrophomonas* (…) increased. Stenotrophomonas sp.)A322's 0D 600 After a period of growth of 3 days, it entered a plateau phase, and acarbose production gradually increased, reaching a peak of 4.0 mg / mL at 7 days.Figure 3 ). Example 4

[0045] Oligotrophomonas ( Stenotrophomonas sp. Isolation and purification of acarbose synthesized by fermentation of A322

[0046] Oligotrophomonas ( Stenotrophomonas sp. A322 was inoculated into test tubes containing 5 mL of liquid fermentation medium and cultured. The seed culture was then inoculated at 1% into 500 mL shake flasks containing 100 mL of liquid fermentation medium. The culture temperature was 30℃, the shaker speed was 220 rpm, and the culture time was 2 days. The fermentation broth was transferred to centrifuge tubes and centrifuged at 8000 rpm at 4℃ for half an hour. After centrifugation, the supernatant was transferred to a clean ground glass joint round-bottom flask and rotary evaporated at 45℃ and 90 rpm to half the original volume. After rotary evaporation, three volumes of anhydrous ethanol were added, and the mixture was allowed to stand overnight to precipitate. The supernatant was discarded, and the precipitate was freeze-dried under vacuum and weighed. The freeze-dried precipitate was dissolved in ultrapure water, centrifuged at 4000 rpm at 4℃, and after removing insoluble matter, the supernatant was obtained. The pH was adjusted to 6.5 to obtain a crude acarbose extract solution, which was then purified using a strong acid cation exchange resin. First, the strong acid cation exchange resin 001x7 was activated. First, rinse with ultrapure water until no impurities remain, then soak overnight in ultrapure water. Discard the ultrapure water, soak in 4% dilute hydrochloric acid for 4 hours, rinse with ultrapure water, then soak in 4% sodium hydroxide solution for 4 hours, and rinse again with ultrapure water. Repeat this process two to three times. The activated resin is then soaked in ultrapure water for later use. Next, perform strong acid cation exchange resin column purification. Transfer the crude acarbose extract solution to an activated 001x7 resin column, let it stand for two hours, then pass it through the column at a flow rate of 5 mL / min, collecting the eluent in 50 mL fractions until all the crude acarbose extract solution has been eluented. Finally, pass the eluent through the column with ultrapure water at a flow rate of 5 mL / min, collecting the wash solution in 50 mL fractions until the OD of the collected solution reaches a certain level. 210 Approaching 0. Subsequently, the column was passed through dilute hydrochloric acid solutions of concentration gradients of 0.03M, 0.06M, 0.09M, and 0.15M, and the hydrochloric acid eluent was collected fractionally until the OD of the collected solution was close to 0. 210 The concentration was close to 0. After HPLC analysis of the hydrochloric acid gradient eluent, the collected 0.09M hydrochloric acid eluent was subjected to vacuum freeze-drying, yielding a total of 2.9383g of freeze-dried product, which was crude acarbose.

[0047] 100 mg of crude acarbose was further purified by silica gel plate chromatography. The chromatography conditions were n-butanol:ethanol:water = 9:7:5, and the development height was 17 cm. The development degree of the sample and standard was observed under 254 nm fluorescence, and the developed band consistent with the standard was scraped from the sample. The scraped silica gel particles were ground evenly and transferred to a 15 mL centrifuge tube. 3 mL of methanol was added to dissolve the acarbose attached to the silica gel particles into the methanol solution. After sonication, the mixture was allowed to stand and separate into layers. The solution was filtered through a 0.22 μm filter membrane into a new 15 mL centrifuge tube. 100 μL of each sample was reserved for high-performance liquid chromatography (HPLC) detection. The remaining samples were dried under nitrogen to obtain 5.6 mg of high-purity acarbose, with a sample yield of 5.6%. Example 5

[0048] Oligotrophomonas ( Stenotrophomonas sp. High-performance liquid chromatography analysis of acarbose synthesized from A322

[0049] Acarbose samples prepared from silica gel plates were filtered through a 0.22 μm filter membrane and then qualitatively analyzed by high-performance liquid chromatography (HPLC). The HPLC conditions were: Thermo Fisher Hypersil APS-2 NH2 column (250 × 4.6 mm, 5 μm), methanol 75%, phosphate (600 mg potassium dihydrogen phosphate, 279 mg anhydrous disodium hydrogen phosphate, 1 L water, pH 6.9) 25%, column temperature 30℃, and detection wavelength 210 nm. Acarbose samples prepared from silica gel plates were compared with purchased acarbose standards under the same HPLC conditions. The results showed that *Oligotrophomonas* (…) Stenotrophomonas sp. The acarbose synthesized by fermentation of A322, after preparation, had the same retention time in liquid chromatography as the standard, which was 4.85 min. Figure 4 ). Example 6

[0050] Oligotrophomonas ( Stenotrophomonas sp. Nuclear magnetic resonance and mass spectrometry detection of acarbose synthesized from A322

[0051] Oligotrophomonas ( Stenotrophomonas sp. Acarbose synthesized by fermentation of A322 was prepared, and the resulting acarbose sample was dissolved in heavy water for 400 M nuclear magnetic resonance analysis. Simultaneously, standard samples were analyzed. The analysis revealed that the acarbose standard and oligotrophomonas (…) Stenotrophomonas sp. The carbon spectra of acarbose samples synthesized by fermentation of A322 all showed the presence of 34 carbon atoms, and the chemical shifts of the 34 carbon atoms were completely consistent. Figure 5 (Table 1) The peak with a chemical shift of 50 ppm in the carbon NMR spectrum of the acarbose sample is due to residual methanol solvent used during purification, indicating that it is caused by oligotrophomonas (…). Stenotrophomonas sp.Acarbose synthesized by fermentation of A322 has a completely identical structure to the standard acarbose. The molecular formula of acarbose is C322. 25 H 43 NO 18 The molecule contains 25 carbon atoms, and the carbon NMR spectrum shows a chemical shift of 34 carbon atoms. This shift is due to the α and β configurations of the hydroxyl group at the reducing end of acarbose, which is caused by the mutarotation of the sugar in solution. (Oligotrophomonas) Stenotrophomonas sp. High-resolution mass spectrometry (HMS) analysis of acarbose synthesized by fermentation of A322 showed a Mass value of 646.2557 in positive ion mode. Elemental analysis revealed that the molecular ion peak was C. 25 H 44 NO 18 ( Figure 6 ), indicating that oligotrophomonas ( Stenotrophomonas sp. The molecular formula of the acarbose sample synthesized by fermentation (CCTCC NO: M2025917) is C 25 H 43 NO 18 .

[0052] Table 1: Oligotrophomonas ( Stenotrophomonas sp. Chemical shifts in carbon NMR spectra of acarbose synthesized by fermentation of A322 after separation and purification, and acarbose standards.

[0053] Example 7

[0054] Oligotrophomonas ( Stenotrophomonas Activity assay of acarbose synthesized by sp.)A322

[0055] The principle behind acarbose activity detection is based on the fact that 2-chloro-4-nitrophenyl-α-galactosyl-maltoside (Gal-G2-α-CNP), a commonly used reagent for determining amylase activity, can be hydrolyzed by α-amylase into two parts without byproduct formation. The hydrolysis product, 2-chloro-4-nitrophenol (CNP), exhibits a characteristic absorption peak at 398 nm. This process does not require any cofactors. Acarbose competitively binds to amylase, thus hindering the normal binding of amylase to the substrate and inhibiting product formation. Based on this principle, a substrate-enzyme-inhibitor screening model has been established, which serves as the basis for detecting acarbose activity.

[0056] Oligotrophomonas ( Stenotrophomonas sp.Acarbose synthesized by fermentation of A322 was prepared into solutions with a series of concentration gradients from 0.1 mg / mL to 1.0 mg / mL for activity assay. The activity assay reaction system consisted of 40 μL of 0.05 M 2-chloro-4-nitrobenzene-α-galactosyl-maltobiose (Gal-G2-α-CNP) solution, 30 μL of 10 U / mL α-amylase solution, 40 μL of acarbose solution (the blank control was 40 μL of pH 6.5 phosphate buffer), and 90 μL of pH 6.5 phosphate buffer. The reaction was carried out at 37℃ for 50 minutes, and the OD was then measured using a microplate reader. 398 The inhibition rate was calculated. Analysis revealed *Oligotrophomonas* (…). Stenotrophomonas sp. The inhibitory activity of acarbose synthesized by fermentation of A322 increased with increasing acarbose concentration. Stenotrophomonas sp. The inhibitory activity of acarbose synthesized by fermentation of A322 increased with increasing concentration in the range of 0.1 mg / mL to 1.0 mg / mL. The inhibition rate was 22.57% at a concentration of 0.1 mg / mL, and the maximum inhibition rate of 36.92% was achieved at a concentration of 1.0 mg / mL. Figure 7 ).

[0057] Although the above embodiments have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the above descriptions are merely embodiments of the present invention and do not limit the scope of patent protection of the present invention. Any equivalent structural or procedural transformations made using the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A strain of oligotrophomonas ( Stenotrophomonas sp.), characterized in that, The oligotrophomonium was named Oligotrophomonium A322 and was deposited at the China Center for Type Culture Collection on April 27, 2025, with accession number CCTCC NO: M 2025917.

2. The oligotrophomonad strain according to claim 1, characterized in that, The 16S rDNA sequence of the oligotrophomonad is shown in SEQ ID NO:

1.

3. The use of the oligotrophomonas according to claim 1 or 2 in the fermentation preparation of acarbose.

4. The application according to claim 3, characterized in that, The substrates for fermentation preparation of acarbose are glucose and / or maltose.

5. A method for preparing acarbose by fermentation, characterized in that, The process includes the following steps: placing the oligotrophomonas described in claim 1 in a liquid culture medium for fermentation, and then separating and purifying it to obtain acarbose.

6. The method according to claim 5, characterized in that, The fermentation temperature is 25-40℃; the pH value of the fermentation system is 7.0; the fermentation time is 1-7 days, and maltose may or may not be added daily during the fermentation process.

7. The method according to claim 5, characterized in that, The liquid culture medium specifically comprises 16.0-50.0g maltose, 4.0-20.0g glucose, 4.0-8.0g sodium glutamate, 2.5g peptone, 2.5g yeast extract, 1.0g dipotassium hydrogen phosphate, 1.0g magnesium sulfate heptahydrate, and 0.1g ferrous sulfate heptahydrate per 1L of culture medium, with the remainder being water.

8. The method according to claim 5, characterized in that, The method for separating and purifying acarbose includes the following steps: after the fermentation culture is completed, the fermentation broth is centrifuged to obtain the fermentation supernatant, the fermentation supernatant is precipitated with anhydrous ethanol to obtain the precipitate, and the precipitate is separated and purified by cation exchange resin and silica gel plate to obtain acarbose.

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