Bacillus pseudomycoides Sys-Gs-005 strain capable of producing ginsenoside Rg2 and application of bacillus pseudomycoides Sys-Gs-005 strain
The fermentation of ginsenoside Rg2 by Bacillus pseudomycosis strain Sys-Gs-005 solves the problems of high production cost and environmental pollution in existing technologies, and realizes efficient and low-cost large-scale fermentation production.
Patent Information
- Application Number
- CN202511842648.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-09
- Publication Date
- 2026-03-06
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Existing technologies are insufficient for the efficient and low-cost production of rare ginsenoside Rg2. Chemical methods have low yields and pollute the environment, while biotransformation methods are limited by poor enzyme stability and high immobilization costs, and there is a lack of microbial fermentation production methods.
Ginsenoside Rg2 was prepared by fermentation using Bacillus pseudomycetamol strain Sys-Gs-005. The optimized fermentation medium and conditions were: 5 g/L yeast extract, 8 g/L peptone, 3 g/L sodium chloride, pH 7.0, 37 ℃, 150 r/min, and fermentation for 7 days.
This method enables large-scale fermentation production of ginsenoside Rg2 with high yield and low cost, avoiding the pollution problems of chemical methods and overcoming the stability and immobilization cost limitations of biotransformation methods.
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Abstract
Description
Technical Field
[0001] This invention relates to a strain of Bacillus pseudomycoides and its uses, which can ferment and prepare ginsenoside Rg2, belonging to the field of microbial technology. Background Technology
[0002] Ginsenosides are the main active medicinal components of ginseng. Due to the different types and contents of ginsenoside monomers, the pharmacological effects of various monomeric saponins differ. Therefore, isolating the effective monomers and clarifying the pharmacological activities of each monomer has become a research hotspot in this field. Currently, more than 60 known ginsenosides have been discovered, each with unique medicinal functions. They are mainly divided into two categories: ① Protopanaxadiol (PPD), including ginsenosides Ra1, Ra2, Rb1, Rb2, Rb3, Rc, Rd, Rg3, and Rh2; ② Protopanatriol-type saponins (PPT), including ginsenosides Re, Rg1, Rg2, Rf, and Rh1.
[0003] The majority of ginsenosides in ginseng are Rb1, Rb2, Rc, Rd, Re, and Rg1 ginsenosides. Ginsenoside Rg2 is a rare ginsenoside, present in low amounts in ginseng, but possesses extremely high physiological and pharmacological activity. Rg2 has a protective effect against acute cardiogenic shock, exhibiting anti-shock, anti-heart failure, anticoagulant, and antithrombotic effects. Its main manifestations include strengthening the myocardium, enhancing myocardial contractility, slowing heart rate, dilating blood vessels, increasing cardiac output and coronary blood flow, rapidly improving myocardial ischemia and hypoxia, and significantly enhancing cardiac function.
[0004] High-content ginsenosides can be converted into ginsenoside Rg2 using chemical methods such as acid and alkaline hydrolysis and biotransformation. However, chemical methods have low yields, produce many byproducts, consume large amounts of organic solvents, and pollute the environment. While biotransformation (enzymatic conversion) offers advantages in regional selectivity, it faces industrialization barriers due to poor enzyme stability and high immobilization costs. Currently, there are no reports in this field regarding the direct production of rare ginsenoside Rg2 using microbial fermentation. Summary of the Invention
[0005] This invention solves the problems in the background art and provides a strain of Bacillus pseudomycetes Sys-Gs-005 that can produce ginsenoside Rg2, which can be fermented to prepare ginsenoside Rg2.
[0006] This application screened a novel bacterial strain, named Sys-Gs-005, which is a Bacillus pseudomycoides. This strain is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, 430072, China, on December 4, 2025, with accession number CCTCC NO: M20252781. The 16S rDNA sequence of this strain is shown in SEQ ID NO.1.
[0007] The aforementioned *Bacillus pseudomyceti* strain Sys-Gs-005 can be used for fermentation to prepare ginsenoside Rg2. The microbial fermentation conditions were: yeast extract 5 g / L, peptone 8 g / L, sodium chloride 3 g / L, pH 7.0; the culture conditions were: 37 ℃, 150 r / min, fermentation for 7 days.
[0008] Compared with existing technologies, the present invention has the following advantages: The *Bacillus pseudomycosis* strain Sys-Gs-005 provided by the present invention can ferment and produce ginsenoside Rg2. The strain provided by the present invention can be cultured on a large scale in a short period of time, with low fermentation cost, and is not limited by time, season, or other conditions, exhibiting significant advantages. Furthermore, the present invention also provides optimized microbial fermentation conditions and culture medium formulations, resulting in a higher yield of ginsenoside Rg2. Attached Figure Description
[0009] Figure 1 The HPLC chromatogram shows the detection of ginsenoside Rg2 in the standard sample.
[0010] Figure 2 This is an HPLC chromatogram showing the detection of ginsenoside Rg2 in the culture medium after fermentation of Bacillus pseudomycosis Sys-Gs-005 strain. Detailed Implementation
[0011] The present invention will now be described in detail with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.
[0012] Isolation and culture of strains
[0013] In this embodiment, the root tissue of fresh wild ginseng collected from Changbai Mountain was surface-sterilized with 75% ethanol for 20 minutes, then washed 3-5 times with sterile water, placed in a pre-sterilized mortar, and ground with 5 mL of sterile water to form a white suspension. 300 μL of the suspension was transferred to each LB solid medium plate, spread evenly with a sterile spreader, and after air-drying, sealed with sealing film. The plates were then incubated upside down in a 37°C incubator for 3-5 days, with timely observation and daily recording. The resulting bacteria were repeatedly purified.
[0014] Meanwhile, wash the stems, remove any necrotic or hardened parts from the surface, and then cut them crosswise into 1cm pieces. 2 The thin slices were first soaked in 75% alcohol for 3-5 minutes, then rinsed 2-3 times with sterile water to remove residual alcohol. They were then soaked in sterile water for about 5 minutes, and finally blotted dry with sterile filter paper. The treated slices were then placed on LB agar plates, 5 slices per plate. The plates were sealed with sealing film and incubated at 37°C for 3-5 days. Observations and records were recorded daily, and the resulting bacteria were repeatedly purified.
[0015] The purified bacteria on the plate were picked and transferred to 1.5 mL EP tubes containing 600 μL LB liquid medium. The tubes were sealed with sealing film and then cultured at 37°C in a shaker at 190 r / min for 1 day. An equal volume of 50% glycerol was added, and the mixture was mixed and stored in an ultra-low temperature freezer at -80°C. At least 3 copies of each strain were stored.
[0016] 10 μL of the preserved bacterial strain was inoculated into 5 mL of LB liquid medium and activated in a shaker at 37°C and 150 rpm for 24 h. 3 mL of the activated bacterial solution was then inoculated into 100 mL of LB liquid medium for fermentation and pre-cultured in a shaker at 37°C and 150 rpm for 7 days. After the above fermentation process, the fermentation broth was collected.
[0017] Strain screening
[0018] By performing HPLC analysis on the ginsenoside Rg2 in the filtered fermentation broth samples of each strain and comparing the results with the data of the standard, it can be preliminarily determined whether the strain has the ability to produce ginsenoside Rg2.
[0019] After the above tests, a strain of *Pseudomonas aeruginosa* producing ginsenoside Rg2 was selected and named strain Sys-Gs-005. HPLC detection is as follows. Figure 2 As shown. The HPLC detection conditions are as follows:
[0020] HPLC detection conditions: The chromatograph was an Agilent 1290 system, with an ODS-C18 reversed-phase column (4.6 mm × 250 mm, 5 μm, Agilent); the mobile phase was acetonitrile:water = 65:35; the standard concentration was 100 μg / mL; the flow rate was 1 mL / min; the UV wavelength was set to 203 nm; and the column temperature was 20℃. The detection results are as follows: Figure 1 As shown. From Figure 1 and Figure 2 The comparison shows that the fermentation broth of the Sys-Gs-005 strain contains ginsenoside Rg2.
[0021] Sequencing and sequence alignment and analysis
[0022] The strain was sent to a biotechnology company for sequencing. The 16S rDNA sequence of this strain is shown in SEQ ID NO.1. The sequencing results were analyzed using BLAST in the NCBI nucleic acid database. BLAST sequence alignment and phylogenetic analysis showed that the 16S rDNA sequence of this strain had a 99% similarity to *Bacillus pseudomycoides* (GeneBank ID: NR_113991.1), confirming it as a bacterium of *Bacillus pseudomycoides*.
[0023] Preservation of strains:
[0024] The strain is deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China 430072, China, on December 4, 2025, with accession number CCTCC NO: M20252781.
[0025] Optimization of fermentation conditions
[0026] The fermentation medium composition of microorganisms must meet the needs of rapid growth and reproduction of the strain, while the optimal fermentation conditions must ensure the accumulation of a large amount of secondary metabolites. This invention preliminarily optimized the fermentation medium composition and culture conditions for strain Sys-Gs-005. The optimized medium formulation is: 5 g / L yeast extract, 8 g / L peptone, 3 g / L sodium chloride, pH 7.0; the culture conditions are: 37 ℃, 150 r / min, fermentation for 7 days.
Claims
1. A Bacillus pseudomycoides. Sys-Gs-005 strain for producing ginsenoside Rg2, the strain has a preservation number of CCTCC NO: M20252781, and a 16S rDNA sequence of the strain is shown as SEQ ID NO.
1.
2. Use of the Bacillus pseudofirmus strain Sys-Gs-005 of claim 1, characterized in that: Ginsenoside Rg2 is prepared by fermentation.
3. Use according to claim 2, characterized in that: The fermentation method is as follows: a fermentation temperature is 37 ℃, a rotation speed is 150 r / min, and fermentation culture is performed for 7 days.
4. Use according to claim 2, characterized in that: A culture medium formula used in the fermentation process is as follows: 5 g / L of yeast powder, 8 g / L of proteose peptone, 3 g / L of sodium chloride, and pH 7.0.