Application of Burkholderia sp. WJ-02 in conversion of ginsenoside and conversion method of Burkholderia sp. WJ-02

Abstract

The invention belongs to the technical field of microbial bioconversion, and particularly relates to application of Burkholderia sp. WJ-02 in ginsenoside conversion and a conversion method, and the Burkholderia sp. WJ-02 can convert ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2. The Burkholderia sp. WJ-02 is separated from rhizosphere soil of American ginseng, the Burkholderia sp. WJ-02 can efficiently and selectively convert main ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2 by taking American ginseng stem and leaf saponin as a substrate, and conversion target products are concentrated, so that subsequent purification is facilitated.

Description

Technical Field

[0001] This invention belongs to the field of microbial biotransformation technology, specifically relating to the application and transformation method of Burkholderia WJ-02 in the transformation of ginsenosides. Background Technology

[0002] Ginsenosides, the core active ingredients of Panax ginseng plants (Araliaceae family), can be classified into three types based on their aglycone structure: protopanaxadiol (PPD), protopanatriol (PPT), and oleanolic acid (OA). Ginsenoside Rd and the rare ginsenoside F2 both belong to the PPD type, exhibiting a simpler sugar chain structure and better bioavailability.

[0003] Studies have shown that ginsenoside Rd exhibits outstanding performance in anti-tumor activity, cardiovascular protection, and metabolic regulation. It effectively inhibits tumor cell proliferation and metastasis by regulating signaling pathways such as PI3K / Akt and MAPK, and can enhance the sensitivity to chemotherapeutic drugs. In the cardiovascular field, ginsenoside Rd can improve myocardial energy metabolism, reduce oxidative stress and inflammatory responses, and has a protective effect against myocardial ischemia-reperfusion injury. Rare ginsenoside F2, while retaining the core activity of PPD-type saponins, exhibits better bioavailability due to its simpler glycosylation structure, and has unique value in anti-tumor, anti-inflammatory, and metabolic regulation.

[0004] However, rare ginsenosides are present in very low amounts in natural plants, and traditional preparation methods have obvious limitations: heating and hydrolysis can easily destroy the active structure; chemical methods produce complex products and require harsh conditions; and although enzymatic methods offer good selectivity, they are cumbersome and costly. Summary of the Invention

[0005] The purpose of this invention is to provide an application and conversion method of Burkholderia WJ-02 in the conversion of ginsenosides, so as to solve the above-mentioned technical problems.

[0006] To achieve the above-mentioned technical objectives, the technical solution of the present invention is as follows: Application of Burkholderia WJ-02 in the conversion of ginsenosides, wherein Burkholderia WJ-02 converts ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2.

[0007] As a further improvement, the Burkholderia WJ-02 is Burkholderia (… Burkholderia sp.) WJ-02 was deposited at the China Center for Type Culture Collection on October 17, 2025, with accession number CCTCC NO: M20252235.

[0008] This invention also provides a method for converting ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2 using Burkholderia WJ-02, comprising the following steps: S1, strain activation culture; The Burkholderia WJ-02 strain is inoculated into LB liquid culture medium, and is cultured at 20-35 DEG C and pH 6.0-9.0 for 10-24h to obtain a seed liquid; S2, scale-up culture; The seed liquid is inoculated into fresh LB liquid culture medium for continuous culture to obtain a high-density bacterial body culture liquid; S3, fermentation conversion; The filtered ginsenoside from Panax quinquefolium stem and leaf is added to the sterilized LB liquid culture medium, and the high-density bacterial body culture liquid is inoculated, and the mixture is uniformly mixed to obtain a fermentation liquid, which is fermented and converted at 37 DEG C and pH 7.0 for 7d to obtain a ginsenoside conversion liquid.

[0009] As a further improvement, in step S1, the inoculation amount of the Burkholderia WJ-02 strain is 1-5%.

[0010] As a further improvement, in step S2, the inoculation amount of the seed liquid is 1-5%, and the continuous culture conditions are 20-35 DEG C, pH 6.0-9.0, and culture for 10-24h.

[0011] As a further improvement, in step S3, in the fermentation liquid, the concentration of Panax quinquefolium stem and leaf saponin is 1mg / mL, and the concentration of Burkholderia WJ-02 is 8x10 8 CFU / mL.

[0012] As a further improvement, in the ginsenoside conversion liquid, the concentrations of ginsenoside Rb3, ginsenoside Rd and rare ginsenoside F2 are 0.0258mg / mL, 0.0448mg / mL and 0.0113mg / mL, respectively.

[0013] Due to the adoption of the above technical solutions, the beneficial effects of the present application are: The present application provides a kind of application and conversion method of Burkholderia WJ-02 in the conversion of ginsenoside, and a strain of Burkholderia WJ-02 is obtained from the rhizosphere soil of Panax quinquefolium, which can efficiently and selectively use Panax quinquefolium stem and leaf saponin as substrate to convert main ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2, and the target product is concentrated in the conversion process, which is convenient for subsequent purification.

[0014] The obtained Burkholderia strain WJ-02 (strain preservation number CCTCC NO: M20252235) can use crude ginsenoside extract of Panax quinquefolius stems and leaves as a substrate, and can convert the main ginsenoside Rb3 in the fermentation process into the rare ginsenoside F2 with better biological activity. The conversion process has mild conditions, is green and environmentally friendly, has strong specificity, can realize directional conversion, and has certain industrialization potential. This technology not only provides a new scheme for large-scale production of rare ginsenosides, but also promotes the upgrading of Panax quinquefolius processing industry to the field of biological medicine deep processing, provides certain raw material guarantee for drug research and development and mechanism exploration, and can realize the dual goals of efficient resource utilization and industrial value improvement. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 Figure 1 is a colony morphology diagram of Burkholderia WJ-02 in Example 1 of the present application, A is a single colony morphology diagram of the strain on R2A solid culture medium, and B is a single colony morphology diagram of the strain on R2A screening medium; Figure 2 Figure 2 is a gram staining diagram of Burkholderia WJ-02 in Example 1 of the present application; Figure 3 Figure 3 is a phylogenetic tree of Burkholderia WJ-02 in Example 1 of the present application; Figure 4 Figure 4 is a high performance liquid chromatogram of ginsenoside conversion liquid in Example 2 of the present application, A is a high performance liquid chromatogram of Panax quinquefolius stem and leaf saponin, B is a high performance liquid chromatogram of n-butanol extract of Panax quinquefolius stem and leaf saponin, C is a high performance liquid chromatogram of n-butanol extract of ginsenoside conversion liquid, and D is a blank control high performance liquid chromatogram of LB liquid medium; The Burkholderia WJ-02 is Burkholderia sp. WJ-02, which was preserved in China Center for Type Culture Collection on October 17, 2025, with a preservation number of CCTCC NO: M20252235 and a preservation address of Wuhan, China. Burkholderia sp. ) WJ-02, which was preserved in China Center for Type Culture Collection on October 17, 2025, with a preservation number of CCTCC NO: M20252235 and a preservation address of Wuhan, China. DETAILED DESCRIPTION

[0016] The technical solutions of the present application will be described clearly and completely in combination with specific embodiments, but those skilled in the art will understand that the following described embodiments are part of the embodiments of the present application, not all the embodiments, and should not be regarded as limiting the scope of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application. The specific conditions are not specified in the embodiments, and are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be purchased on the market.

[0017] Example 1 Screening and identification of Burkholderia WJ-02.

[0018] Preparation of culture medium: R2A solid medium: 18.12 g of R2A solid medium was dissolved in 1 L of distilled water, autoclaved at 121 ℃ for 20 min, and stored at 4 ℃ for standby; R2A screening medium: 18.12 g of R2A solid medium, 1.00 g of aescin, and 0.50 g of ferric citrate were dissolved in 1 L of distilled water, autoclaved at 121 ℃ for 20 min, and stored at 4 ℃ for standby; LB liquid medium: 10.00 g of tryptone, 5.00 g of yeast extract, and 10.00 g of sodium chloride were dissolved in 1 L of distilled water, autoclaved at 121 ℃ for 20 min, and stored at 4 ℃ for standby.

[0019] Isolation of strains: 1 g of the rhizosphere soil sample of Panax quinquefolius was taken into a 15 mL centrifuge tube, 9 mL of sterile normal saline was added after mixing well, and a 10 -1 soil dilution liquid; 1 mL of the 10 -1 soil dilution liquid was transferred to a centrifuge tube containing 9 mL of sterile normal saline, mixed well by shaking, and a 10 -2 soil dilution liquid was obtained, and the method was used to dilute step by step to 10 -6 , so that 10 -1 , 10 -6 , and 10 gradient soil dilution liquids were obtained; -1 -6 Using the spread plate method, 200 μL of 10

[0020] Screening of strains: The isolated and purified single colonies were picked up using a sterile inoculation loop, inoculated into a 48-well plate containing R2A screening medium, and placed in a 37 ℃ constant temperature incubator for culture for 48 h. The color change of the culture medium in each well was observed regularly, and the strains were systematically numbered for subsequent data recording and analysis; If the strain can secrete β-glucosidase, aescin in the culture medium can be hydrolyzed to aescinogen, which can further react with Fe 3+ ​The reaction generates black precipitate, which makes the medium in the well black. The strain showing this phenomenon is the candidate strain. The strain showing obvious phenomenon is further isolated and purified. After 48h culture, single colony is picked and inoculated in LB liquid medium for overnight culture. The culture liquid is mixed with 60% glycerol at a volume ratio of 1:1 and stored in -80℃ ultra-low temperature refrigerator for standby. The strain with the ability to produce β-glucosidase is reactivated and inoculated into LB liquid medium. After 24h culture at 37℃ and 120r / min, 3mL bacterial liquid is mixed with 5mL water solution of American ginseng stem and leaf saponin (1mg / mL). After 7d continuous culture, equal volume of water-saturated n-butanol solution is added for extraction to terminate the transformation. The transformation ability is verified by high performance liquid chromatography. Finally, saponin transformation strain Burkholderia WJ-02 is screened.

[0021] Culture characteristics: (1) Liquid culture characteristics: The strain grows uniformly turbid in LB liquid medium. The fermentation broth is yellow. After standing, the bacterial precipitate is white.

[0022] (2) Colony characteristics: After purification, Burkholderia WJ-02 is inoculated in R2A solid medium and R2A screening medium. After 18~24h culture at 37℃, the colony morphology is observed. The colony morphology of the strain is shown in Figure 1 , wherein A is the single colony morphology of the strain on R2A solid medium, and B is the single colony morphology of the strain on R2A screening medium.

[0023] As shown in Figure 1 , the colony of the strain on R2A solid medium is round, with neat edge, protrusion, smooth and moist surface, and luster. The texture is slightly mucous, light yellow, uniform in color and without obvious pigment diffusion.

[0024] Bacterial morphology: The gram staining result of the strain is shown in Figure 2 . The strain is gram-negative and rod-shaped. Scanning electron microscope observation shows that the bacterial body is rod-shaped and has linear vein on the surface.

[0025] Physiological and biochemical characteristics: Physiological and biochemical indicators were determined using conventional methods, and the results are as follows: the optimal growth temperature is 25℃, and the optimal growth pH is 7; the gelatin test, arginine (ADH) test, lysine (LDH) test, ornithine (ODC) test, and citric acid (CIT) test are positive; the hydrogen sulfide (H2S) test, urease (URE) test, indole (IND) test, and VP test are negative; the lactose (LAC) test, glucose (GLU) test, and rhamnose (RHA) test are positive; and the mannitol (MAN) test, inositol (INO) test, sorbitol (SOR) test, sucrose (SAC) test, melibiose (MEL) test, amygdalin (AMY) test, arabinose (ARA) test, and oxidase (OX) test are negative.

[0026] Strain identification: 16S rRNA gene sequence homology analysis: 1 mL of Burkholderia WJ-02 strain cultured at 37℃ and 120 r / min for 12 h was centrifuged at 10000 r / min for 2 min, the supernatant was discarded, 100 μL of ddH2O was added to the precipitate, and the mixture was vortexed to prepare a bacterial suspension. The suspension was heated in a boiling water bath at 100℃ for 10 min, and then centrifuged at 10000 r / min for 2 min. The supernatant was used as a PCR template. Using the bacterial supernatant as a template, the 16S rRNA gene was amplified using universal primers 27F and 1492R. The 27F sequence is shown in SEQ ID NO: 1, and the 1492R sequence is shown in SEQ ID NO: 2. After the reaction, the amplified fragment was confirmed by agarose gel electrophoresis. The PCR product was sequenced, and the sequence was aligned using the EzTaxon database (similarity ≥ 98%) and analyzed using MEGA. 11. A phylogenetic tree was constructed using software to further confirm the taxonomic position of the strain. The results showed that the bacterium belonged to the genus Burkholderia (…). Burkholderia sp.), named Burkholderia sp. WJ-02 (Burkholderia WJ-02), its phylogenetic tree is as follows Figure 3 As shown.

[0027] In this embodiment, Burkholderia WJ-02 is Burkholderia ( Burkholderia sp.) WJ-02 was deposited at the China Center for Type Culture Collection (CCTCC) on October 17, 2025, with accession number CCTCC NO: M20252235, and the deposit address is Wuhan University, Wuhan, China.

[0028] Example 2: A method for converting ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2 using Burkholderia WJ-02, comprising the following steps: S1. Microbial strain activation culture; The frozen strain Burkholderia WJ-02 was inoculated into LB liquid medium at a rate of 1% (v / v) and cultured at 25°C and pH 7.0 for 12 h to obtain the seed culture. S2, Expanded cultivation; The seed culture was inoculated into LB liquid medium at a rate of 1% (v / v) and cultured at 25°C and pH 7.0 for 12 h to obtain a high-density cell culture medium, which was used as the bacterial culture medium for subsequent fermentation and transformation. S3, fermentation conversion; Filtered and sterilized American ginseng stem and leaf saponins were added to sterilized LB liquid medium, inoculated with high-density bacterial culture, and mixed thoroughly to obtain a fermentation broth. The concentration of American ginseng stem and leaf saponins in the fermentation broth was 1 mg / mL, and the concentration of Burkholderia WJ-02 was 8 × 10⁻⁶. 8 The ginsenoside conversion solution was obtained by fermenting at 37℃ and pH 7.0 for 7 days with CFU / mL, thus realizing the conversion of the main ginsenoside Rb3 in American ginseng stem and leaf saponins into ginsenoside Rd and rare ginsenoside F2.

[0029] The ginsenoside conversion solution obtained above was analyzed by high performance liquid chromatography under the following conditions: The chromatographic column was a YMC C18 column (250×4.6mm, 5μm), the injection volume was 20μL, the flow rate was 1mL / min, and the detection wavelength was 202nm; the mobile phase was A: water; B: acetonitrile. Gradient elution: 0-5 min, mobile phase B 25%-30%; 5-14 min, mobile phase B 30%-38%; 14-28 min, mobile phase B 38%-38%; 28-30 min, mobile phase B 38%-46%; 30-50 min, mobile phase B 46%-74%; 50-60 min, mobile phase B 74%-100%; 60-65 min, mobile phase B 100%-25%; 65-70 min, mobile phase B 25%-25%.

[0030] like Figure 4 The figures shown are high-performance liquid chromatograms of ginsenoside conversion solutions. Figure A is a high-performance liquid chromatogram of saponins from American ginseng stems and leaves; Figure B is a high-performance liquid chromatogram of the n-butanol extract of saponins from American ginseng stems and leaves; Figure C is a high-performance liquid chromatogram of the n-butanol extract of ginsenoside conversion solutions; and Figure D is a high-performance liquid chromatogram of the blank control in LB liquid culture medium.

[0031] Depend on Figure 4As can be seen, comparing Figure B (before conversion) and Figure C (after conversion), the intensity of the chromatographic peak corresponding to ginsenoside Rb3 at a retention time of about 20 min is significantly reduced; meanwhile, the intensities of the chromatographic peaks at about 21 min and 37 min corresponding to the retention times of ginsenoside Rd and rare ginsenoside F2 standard samples, respectively, are both significantly increased, confirming that ginsenoside Rb3 is effectively converted and successfully generates ginsenoside Rd and rare ginsenoside F2.

[0032] The quantitative analysis results show that, before conversion, the concentrations of ginsenoside Rb3, ginsenoside Rd and rare ginsenoside F2 in the panax quinquefolium saponins are 0.0407 mg / mL, 0.0228 mg / mL and 0.00626 mg / mL, respectively; after conversion, the concentrations of ginsenoside Rb3, ginsenoside Rd and rare ginsenoside F2 in the ginsenoside conversion solution are 0.0258 mg / mL, 0.0448 mg / mL and 0.0113 mg / mL, respectively, and the conversion rate of ginsenoside Rb3 is 36.5%.

[0033] Example 3 A method for converting ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2 by Burkholderia WJ-02, comprising the following steps: S1, activating and culturing the strain; The cryopreserved strain Burkholderia WJ-02 was inoculated into LB liquid medium at an inoculation amount of 5% (v / v) and cultured at 20°C and pH 6.0 for 24 h to obtain a seed solution; S2, expanding the culture; The seed solution was inoculated into LB liquid medium at an inoculation amount of 5% (v / v) and cultured at 20°C and pH 6.0 for 24 h to obtain a high-density bacterial culture solution, which was used as the bacterial solution for subsequent fermentation conversion; S3, fermentation conversion; The filtered and sterilized panax quinquefolium saponins were added to the sterilized LB liquid medium, and the high-density bacterial culture solution was inoculated. After uniform mixing, a fermentation solution was obtained, in which the concentration of panax quinquefolium saponins was 1 mg / mL, and the concentration of Burkholderia WJ-02 was 8×10 8 CFU / mL. The fermentation solution was subjected to fermentation conversion at 37°C and pH 7.0 for 7 d to obtain a ginsenoside conversion solution.

[0034] Example 4 A method for converting ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2 by Burkholderia WJ-02, comprising the following steps: S1, activating and culturing the strain; The cryopreserved strain Burkholderia WJ-02 was inoculated into LB liquid medium at an inoculation amount of 2.5% (v / v) and cultured at 20°C and pH 9.0 for 10 h to obtain a seed solution; S2, scale-up culture; The seed liquid was inoculated into LB liquid medium at an inoculation amount of 2.5% (v / v) and cultured at 20°C and pH 9.0 for 10h to obtain a high-density bacterial culture liquid as a bacterial liquid for subsequent fermentation conversion; S3, fermentation conversion; The filtered and sterilized panax quinquefolium stem and leaf saponin was added to the sterilized LB liquid medium, inoculated with the high-density bacterial culture liquid, and mixed uniformly. The concentration of panax quinquefolium stem and leaf saponin in the fermentation liquid was 1mg / mL, and the concentration of Burkholderia WJ-02 was 8x10 8 CFU / mL. The fermentation conversion was carried out at 37°C and pH 7.0 for 7d to obtain a ginsenoside conversion liquid.

[0035] The method for converting ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2 by Burkholderia WJ-02 according to the embodiment 5 comprises the following steps: S1, activation culture of bacterial strain; The frozen Burkholderia WJ-02 strain was inoculated into LB liquid medium at an inoculation amount of 3% (v / v) and cultured at 30°C and pH 7.5 for 18h to obtain a seed liquid; S2, scale-up culture; The seed liquid was inoculated into LB liquid medium at an inoculation amount of 3% (v / v) and cultured at 30°C and pH 7.5 for 18h to obtain a high-density bacterial culture liquid as a bacterial liquid for subsequent fermentation conversion; S3, fermentation conversion; The panax quinquefolium stem and leaf saponin raw material was dissolved in the culture medium and uniformly mixed with the high-density bacterial culture liquid to obtain a fermentation liquid. The concentration of panax quinquefolium stem and leaf saponin in the fermentation liquid was 1mg / mL, and the concentration of Burkholderia WJ-02 was 8x10 8 CFU / mL. The fermentation conversion was carried out at 37°C and pH 7.0 for 7d to obtain a ginsenoside conversion liquid.

[0036] The specific embodiments of the application described above do not constitute a limitation on the scope of protection of the application. Any various other corresponding changes and modifications made according to the technical concept of the application should be included in the scope of protection of the claims of the application.

Claims

1. Use of Burkholderia WJ-02 in transforming ginsenosides, characterized in that, The Burkholderia WJ-02 converts ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2.

2. The use of B. WJ-02 according to claim 1 in the transformation of ginsenosides, characterized in that, The Burkholderia WJ-02 is Burkholderia ( Burkholderia sp.) WJ-02 was deposited at the China Center for Type Culture Collection on October 17, 2025, with accession number CCTCC NO: M20252235.

3. A method for transformation of ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2 by Burkholderia WJ-02, characterized in that, The method comprises the following steps: S1, activating culture of the strain; The Burkholderia WJ-02 strain is inoculated into LB liquid culture medium, and is cultured at 20-35 DEG C and pH 6.0-9.0 for 10-24 hours to obtain a seed liquid; S2, expanding culture; The seed liquid is inoculated into fresh LB liquid culture medium for further culture to obtain a high-density bacterial body culture liquid; S3, fermentation conversion; The filtered ginsenoside of Panax quinquefolium stem and leaf is added into sterilized LB liquid culture medium, and the high-density bacterial body culture liquid is inoculated, and then the mixture is uniformly mixed to obtain a fermentation liquid; the fermentation liquid is subjected to fermentation conversion at 37 DEG C and pH 7.0 for 7 days to obtain a ginsenoside conversion liquid.

4. The method for converting ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2 by Burkholderia WJ-02 according to claim 3, characterized in that, In step S1, the inoculation amount of the Burkholderia WJ-02 strain is 1-5%.

5. The method for converting ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2 by Burkholderia WJ-02 according to claim 3, characterized in that, In step S2, the inoculation amount of the seed liquid is 1-5%, and the further culture is carried out at 20-35 DEG C and pH 6.0-9.0 for 10-24 hours. 6.The method of claim 3, wherein the Burkholderia WJ-02 transforms ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2, and wherein the method comprises the steps of: (a) culturing the Burkholderia WJ-02 in a culture medium containing ginsenoside Rb3; (b) extracting ginsenoside Rd and rare ginsenoside F2 from the culture medium; and (c) purifying ginsenoside Rd and rare ginsenoside F2. In step S3, the concentration of ginsenoside in the fermentation broth is 1 mg / mL, and the concentration of Burkholderia WJ-02 is 8 x 10 8 CFU / mL.

7. The method for converting ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2 by Burkholderia WJ-02 according to claim 3, characterized in that, In the ginsenoside conversion liquid, the concentrations of ginsenoside Rb3, ginsenoside Rd and rare ginsenoside F2 are 0.0258 mg / mL, 0.0448 mg / mL and 0.0113 mg / mL respectively.

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