Application of burkholderia WJ-02 in transforming ginsenosides and transformation method

Abstract

The application belongs to the technical field of microbial bioconversion, and particularly relates to application of Burkholderia WJ-02 in conversion of ginsenoside and a conversion method, wherein the Burkholderia WJ-02 can convert ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2. The Burkholderia WJ-02 is obtained by being separated from rhizosphere soil of American ginseng, the strain can efficiently and selectively convert ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2 with American ginseng stem and leaf saponin as a substrate, and the conversion target product is 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:

[0007] Application of Burkholderia WJ-02 in the conversion of ginsenosides, wherein Burkholderia WJ-02 converts ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2.

[0008] 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.

[0009] 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:

[0010] S1. Microbial strain activation culture;

[0011] Burkholderia WJ-02 strain was inoculated into LB liquid medium and cultured at 20-35℃ and pH 6.0-9.0 for 10-24 h to obtain seed culture;

[0012] S2, Expanded cultivation;

[0013] The seed culture was inoculated into fresh LB liquid medium and cultured for a further period to obtain a high-density bacterial culture medium.

[0014] S3, fermentation conversion;

[0015] The filtered and sterilized American ginseng stem and leaf saponins were added to sterile LB liquid medium, inoculated with high-density bacterial culture, and mixed evenly to obtain fermentation broth. The fermentation broth was fermented and transformed for 7 days at 37℃ and pH 7.0 to obtain ginsenoside transformation broth.

[0016] As a further improvement, in step S1, the inoculation amount of Burkholderia WJ-02 strain is 1-5%.

[0017] As a further improvement, in step S2, the inoculation amount of the seed liquid is 1-5%, and the conditions for continued cultivation are: culturing at 20-35℃ and pH 6.0-9.0 for 10-24 hours.

[0018] As a further improvement, in step S3, the concentration of ginseng stem and leaf saponins in the fermentation broth is 1 mg / mL, and the concentration of Burkholderia WJ-02 is 8 × 10⁻⁶. 8 CFU / mL.

[0019] As a further improvement, 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.

[0020] Due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows:

[0021] This invention provides the application and transformation method of Burkholderia WJ-02 in the transformation of ginsenosides. A Burkholderia WJ-02 strain was isolated from the rhizosphere soil of American ginseng. This strain can efficiently and selectively use American ginseng stem and leaf saponins as substrates to transform the main ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2. The target products are concentrated in the transformation process, which facilitates subsequent purification.

[0022] The Burkholderia strain WJ-02 (CCTCC NO: M20252235) obtained by this invention can convert the main ginsenoside Rb3 in crude extract of American ginseng stems and leaves into the rarer ginsenoside F2, which has better biological activity, using it as a substrate during fermentation. This conversion process is mild, environmentally friendly, and highly specific, enabling targeted conversion and possessing significant industrialization potential. This technology not only provides a new solution for the large-scale production of rare ginsenosides but also promotes the upgrading of the American ginseng processing industry towards the field of deep processing in biopharmaceuticals, providing a certain raw material guarantee for drug research and mechanism exploration, and achieving the dual goals of efficient resource utilization and enhanced industrial value. Attached Figure Description

[0023] Figure 1 These are colony morphology diagrams of Burkholderia WJ-02 in Example 1 of the present invention. A is a single colony morphology diagram of the strain on R2A solid medium, and B is a single colony morphology diagram of the strain on R2A screening medium.

[0024] Figure 2 This is a Gram staining image of Burkholderia WJ-02 in Example 1 of this invention;

[0025] Figure 3 This is the phylogenetic tree of Burkholderia WJ-02 in Example 1 of this invention;

[0026] Figure 4 These are high-performance liquid chromatograms of ginsenoside conversion solution in Example 2 of the present invention. A is a high-performance liquid chromatogram of American ginseng stem and leaf saponins, B is a high-performance liquid chromatogram of n-butanol extract of American ginseng stem and leaf saponins, C is a high-performance liquid chromatogram of n-butanol extract of ginsenoside conversion solution, and D is a high-performance liquid chromatogram of blank control of LB liquid culture medium.

[0027] Among them, 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. Detailed Implementation

[0028] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0029] Example 1: Screening and identification of Burkholderia WJ-02.

[0030] Preparation of culture medium:

[0031] R2A solid medium: Dissolve 18.12g of R2A solid medium in 1L of distilled water, autoclave at 121℃ for 20min, and store at 4℃ for later use.

[0032] R2A screening medium: Dissolve 18.12g R2A solid medium, 1.00g aescin and 0.50g ferric citrate in 1L distilled water, autoclave at 121℃ for 20min, and store at 4℃ for later use.

[0033] LB liquid medium: Dissolve 10.00g tryptone, 5.00g yeast extract and 10.00g sodium chloride in 1L distilled water, autoclave at 121℃ for 20min, and store at 4℃ for later use.

[0034] Isolation of strains:

[0035] Take 1g of American ginseng rhizosphere soil sample and place it in a 15mL centrifuge tube. Add 9mL of sterile physiological saline and mix thoroughly to prepare 10 -1 Soil dilution solution;

[0036] Take 1 mL of 10 -1 The soil dilution was transferred to a centrifuge tube containing 9 mL of sterile physiological saline, and after vortexing, 10 μL of solution was obtained. -2 Soil dilution solution, diluted stepwise to 10 using this method. -6 Thus, we get 10 -1 ~10 -6 Soil gradient dilution solution;

[0037] Using the coating plate method, 10 samples were taken respectively. -1 ~10 -6Six gradient soil dilutions (200 μL each) were evenly spread on the surface of R2A solid medium, with three replicates for each dilution gradient. The plates were inverted and incubated in a 37°C incubator. Colony morphology was observed periodically. Colonies with different morphologies were randomly selected and streaked onto R2A plates until single colonies were isolated. The cultured plates were then stored at 4°C for later use.

[0038] Strain screening:

[0039] Use a sterile inoculation loop to pick up a single colony that has been isolated and purified, and inoculate it into a 48-well plate containing R2A selection medium. Incubate the plate at 37°C for 48 hours. Observe the color change of the medium in each well regularly, and systematically number the colonies for subsequent data recording and analysis.

[0040] If the strain can secrete β-glucosidase, then aescin in the culture medium can be hydrolyzed into aescin, which in turn reacts with Fe in the culture medium. 3+ The reaction produces a black precipitate, turning the culture medium in the wells black. Strains exhibiting this phenomenon are candidate strains. Strains with obvious phenomena are selected for further isolation and purification. After culturing for 48 hours, single colonies are selected and placed in LB liquid medium and cultured overnight. The culture medium is then mixed with 60% glycerol at a 1:1 volume ratio and stored in an ultra-low temperature freezer at -80℃ for later use.

[0041] The strain capable of producing β-glucosidase was reactivated and inoculated into LB liquid medium. After culturing at 37℃ and 120 rpm for 24 h, 3 mL of the bacterial culture was mixed with 5 mL of American ginseng stem and leaf saponin aqueous solution (1 mg / mL), and cultured for another 7 days. Then, an equal volume of water-saturated n-butanol solution was added for extraction to terminate the transformation. The transformation ability was verified by high performance liquid chromatography, and the saponin-transforming strain Burkholderia WJ-02 was finally screened.

[0042] Culture characteristics:

[0043] (1) Liquid culture characteristics: The strain grows in LB liquid medium with uniform turbidity, the fermentation broth is yellow, and the bacterial cells precipitate as white after standing.

[0044] (2) Colony characteristics: The purified Burkholderia WJ-02 was inoculated into R2A solid medium and R2A selection medium, and its colony morphology was observed after incubation at 37℃ for 18-24 h. The colony morphology of this bacterium is as follows: Figure 1 As shown, A is a single colony morphology diagram of the strain on R2A solid medium, and B is a single colony morphology diagram of the strain on R2A selection medium.

[0045] Depend on Figure 1It can be seen that the colonies of this strain on R2A solid medium are round, with neat and raised edges, smooth, moist and glossy surface, slightly viscous texture, pale yellow color, uniform color and no obvious pigment diffusion.

[0046] Cell morphology:

[0047] The Gram staining results of this bacterium are shown in the figure below. Figure 2 As shown, it is Gram-negative and rod-shaped; scanning electron microscopy results show that its cells are rod-shaped with linear veins on the surface.

[0048] Physiological and biochemical characteristics:

[0049] 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.

[0050] Strain identification:

[0051] 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.

[0052] 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.

[0053] Example 2: A method for converting ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2 using Burkholderia WJ-02, comprising the following steps:

[0054] S1. Microbial strain activation culture;

[0055] 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.

[0056] S2, Expanded cultivation;

[0057] 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.

[0058] S3, fermentation conversion;

[0059] 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.

[0060] The ginsenoside conversion solution obtained above was analyzed by high performance liquid chromatography under the following conditions:

[0061] 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.

[0062] 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%.

[0063] 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.

[0064] Depend on Figure 4 As can be seen from the comparison of Figure B (before conversion) and Figure C (after conversion), the chromatographic peak intensity of ginsenoside Rb3 at a retention time of approximately 20 min is significantly reduced; meanwhile, at approximately 21 min and 37 min, the chromatographic peak intensities corresponding to the retention times of ginsenoside Rd and rare ginsenoside F2 standards, respectively, show a significant increase, confirming that ginsenoside Rb3 was effectively converted and successfully generated into ginsenoside Rd and rare ginsenoside F2.

[0065] Quantitative analysis showed that before transformation, the concentrations of ginsenoside Rb3, ginsenoside Rd, and rare ginsenoside F2 in American ginseng stem and leaf saponins were 0.0407 mg / mL, 0.0228 mg / mL, and 0.00626 mg / mL, respectively. After transformation, the concentrations of ginsenoside Rb3, ginsenoside Rd, and rare ginsenoside F2 in the ginsenoside transformation solution were 0.0258 mg / mL, 0.0448 mg / mL, and 0.0113 mg / mL, respectively, with a transformation rate of ginsenoside Rb3 of 36.5%.

[0066] Example 3: A method for converting ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2 using Burkholderia WJ-02, comprising the following steps:

[0067] S1. Microbial strain activation culture;

[0068] The frozen strain Burkholderia WJ-02 was inoculated into LB liquid medium at a rate of 5% (v / v) and cultured at 20°C and pH 6.0 for 24 h to obtain the seed culture.

[0069] S2, Expanded cultivation;

[0070] The seed culture was inoculated into LB liquid medium at a rate of 5% (v / v) and cultured at 20°C and pH 6.0 for 24 h to obtain a high-density cell culture medium, which was used as the bacterial culture medium for subsequent fermentation and transformation.

[0071] S3, fermentation conversion;

[0072] The filtered and sterilized American ginseng stem and leaf saponins were added to sterile LB liquid medium, inoculated with high-density bacterial culture, and mixed thoroughly to obtain the 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 fermentation at 37℃ and pH 7.0 for 7 days with CFU / mL.

[0073] Example 4: A method for converting ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2 using Burkholderia WJ-02, comprising the following steps:

[0074] S1. Microbial strain activation culture;

[0075] The frozen strain Burkholderia WJ-02 was inoculated into LB liquid medium at an inoculation rate of 2.5% (v / v) and cultured at 20℃ and pH 9.0 for 10 h to obtain seed culture;

[0076] S2, Expanded cultivation;

[0077] The seed culture was inoculated into LB liquid medium at an inoculation rate of 2.5% (v / v) and cultured at 20℃ and pH 9.0 for 10 h to obtain a high-density cell culture medium, which was used as the bacterial culture medium for subsequent fermentation and transformation.

[0078] S3, fermentation conversion;

[0079] The filtered and sterilized American ginseng stem and leaf saponins were added to sterile LB liquid medium, inoculated with high-density bacterial culture, and mixed thoroughly. 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 fermentation at 37℃ and pH 7.0 for 7 days with CFU / mL.

[0080] Example 5: A method for converting ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2 using Burkholderia WJ-02, comprising the following steps:

[0081] S1. Microbial strain activation culture;

[0082] The frozen strain Burkholderia WJ-02 was inoculated into LB liquid medium at a rate of 3% (v / v) and cultured at 30°C and pH 7.5 for 18 h to obtain seed culture.

[0083] S2, Expanded cultivation;

[0084] The seed culture was inoculated into LB liquid medium at an inoculation rate of 3% (v / v) and cultured at 30°C and pH 7.5 for 18 h to obtain a high-density cell culture medium, which was used as the bacterial culture medium for subsequent fermentation and transformation.

[0085] S3, fermentation conversion;

[0086] American ginseng stem and leaf saponin raw materials were dissolved in culture medium and uniformly mixed with high-density bacterial culture solution to obtain 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 fermentation at 37℃ and pH 7.0 for 7 days with CFU / mL.

[0087] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of Burkholderia WJ-02 in the conversion of ginsenosides, characterized in that, Burkholderia ( Burkholderia sp.)WJ-02 transforms ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2; The Burkholderia WJ-02 strain was deposited at the China Center for Type Culture Collection on October 17, 2025, with accession number CCTCC NO: M20252235.

2. A method for converting ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2 using Burkholderia WJ-02, characterized in that, Includes the following steps: S1. Microbial strain activation culture; Burkholderia WJ-02 strain was inoculated into LB liquid medium and cultured at 20-35℃ and pH 6.0-9.0 for 10-24 h to obtain seed culture; S2, Expanded cultivation; The seed culture was inoculated into fresh LB liquid medium and cultured for a further period to obtain a high-density bacterial culture medium. S3, fermentation conversion; The filtered and sterilized American ginseng stem and leaf saponins were added to sterile LB liquid medium, inoculated with high-density bacterial culture, and mixed evenly to obtain fermentation broth. The fermentation broth was fermented and transformed for 7 days at 37℃ and pH 7.0 to obtain ginsenoside conversion broth. The Burkholderia WJ-02 strain was deposited at the China Center for Type Culture Collection on October 17, 2025, with accession number CCTCC NO: M20252235.

3. The method for converting ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2 by Burkholderia WJ-02 according to claim 2, characterized in that, In step S1, the inoculation amount of Burkholderia WJ-02 strain is 1-5%.

4. The method for converting ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2 by Burkholderia WJ-02 according to claim 2, characterized in that, In step S2, the inoculation amount of the seed liquid is 1-5%, and the conditions for continued culture are: culture at 20-35℃ and pH 6.0-9.0 for 10-24 hours.

5. The method for converting ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2 by Burkholderia WJ-02 according to claim 2, characterized in that, In step S3, the concentration of American ginseng stem and leaf saponins in the fermentation broth is 1 mg / mL, and the concentration of Burkholderia WJ-02 is 8 × 10⁻⁶. 8 CFU / mL.

6. The method for converting ginsenoside Rb3 into ginsenoside Rd and rare ginsenoside F2 by Burkholderia WJ-02 according to claim 2, characterized in that, In the ginsenoside conversion solution, 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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