Bacillus firmus Sys-Gs-002 strain for producing ginsenoside Rg1 and application of bacillus firmus Sys-Gs-002 strain
By screening and confirming the Bacillus thymophilus strain Sys-Gs-002, the problem of producing rare ginsenoside Rg1 in existing technologies has been solved, enabling efficient and low-cost large-scale fermentation production and increasing the yield of ginsenoside Rg1.
Patent Information
- Application Number
- CN202511808320.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-03
- Publication Date
- 2026-01-20
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
There is currently no effective microbial fermentation method to directly produce rare ginsenoside Rg1, and artificially cultivated ginseng suffers from quality decline and resource depletion.
A strain of Bacillus thymophilus, Sys-Gs-002, was screened and confirmed. This strain can efficiently convert ginsenosides into rare ginsenoside Rg1 under specific fermentation conditions. The fermentation conditions are: 5 g/L yeast powder, 8 g/L peptone, 3 g/L sodium chloride, pH 7.0, 37 ℃, 150 r/min, and fermentation for 7 days.
This method enables efficient and low-cost large-scale fermentation production of ginsenoside Rg1, solving the problems of resource shortage and quality decline, and providing a high-yield fermentation method.
Smart Images
Figure CN121362696A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to a Cytobacillus firmus strain and use, the strain can ferment to prepare ginsenoside Rg1, belongs to the field of microbial technology. BACKGROUND
[0002] Panax ginseng C. A. Meyer, also known as Dijing, Shengcao and Huangshen, is a perennial herbaceous medicinal plant of the Araliaceae family, and its medicinal parts are roots and rhizomes. It has magical effects such as tonifying primordial qi, strengthening pulse and preventing collapse, tonifying the spleen and lung, generating fluid and nourishing blood, and tranquilizing the mind and benefiting intelligence. The cultivation of Panax ginseng has a history of more than 400 years. In recent years, with the further increase of market demand, artificial domestication and cultivation is the only way to meet market demand. However, in recent decades, due to overexploitation and destruction of its habitat, the number of wild Panax ginseng resources has decreased sharply, and artificial cultivation of Panax ginseng also has problems such as rapid decline in quality and mixed varieties. Therefore, it is particularly important to find a reasonable and effective way to promote the sustainable development of the Panax ginseng industry.
[0003] Ginsenoside is the main active medicinal component of Panax ginseng. Due to the different types and contents of ginsenoside monomers contained, the pharmacological effects of various monomer saponins are different. Therefore, separating effective monomers and clarifying the pharmacological activities of each monomer have become a research hotspot in this field. At present, more than 60 known ginsenosides have been found, and each has a unique medicinal function. They can be mainly divided into two categories: ① protopanax diol (PPD) including ginsenoside Ra1, Ra2, Rb1, Rb2, Rb3, Rc, Rd, Rg3 and Rh2, etc.; ② protopanax triol type saponin (PPT) including ginsenoside Re, Rg1, Rg2, Rf and Rh1, etc.
[0004] Ginsenoside Rg1 is a high-value rare ginsenoside. Ginsenoside Rg1 has good effects on the protection of the central nervous system. Rg1 can delay and reduce the apoptosis of neurons and has a protective effect on neurons. Rg1 has the effect of promoting the proliferation of cortical neural stem cells and the directional differentiation of glial-like cells. Rg1 can also reduce the content of β-amyloid in the brain by increasing the expression of enkephalinase, and has good anti-Alzheimer's disease effect. Rg1 also has good effects on the protection of the cardiovascular system. Rg1 can significantly reduce the incidence of arrhythmia after ischemia / reperfusion by regulating the endogenous mitochondrial apoptosis pathway. Rg1 can also reduce myocardial blood flow and inhibit myocardial structural changes by down-regulating the activity of the RhoA signaling pathway, thereby protecting the myocardium from damage. Rg1 can also play an anti-myocardial ischemia-reperfusion injury role by inhibiting inflammatory reactions and changing myocardial pathological changes.
[0005] Ginsenoside Rg1 is low in ginseng, which can be obtained by bioconversion of ginsenoside Rf. At present, researchers screen microbial strains with high conversion capacity, such as specific types of bacteria, fungi and the like, and use the enzymes produced in the fermentation process to convert ginsenosides. A variety of microorganisms have been confirmed to have the ability to convert ginsenosides into rare ginsenoside Rg1. However, there is currently no direct report on the production of rare ginsenoside Rg1 by microbial fermentation in the field. SUMMARY
[0006] The present application solves the problems in the background art and provides a Cytobacillus firmus Sys-Gs-002 strain capable of producing ginsenoside Rg1, which can be used for fermentation to prepare ginsenoside Rg1.
[0007] A new strain is screened in the present application, which is named Sys-Gs-002 and is a Cytobacillus firmus. The strain is preserved in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with a postal code of 430072, and the preservation date is November 24, 2025, and the preservation number is CCTCC NO: M20252684. The 16S rDNA sequence of the strain is shown in SEQ ID NO. 1.
[0008] The above-mentioned Cytobacillus firmus Sys-Gs-002 strain can be used for fermentation to prepare ginsenoside Rg1. The microbial fermentation conditions are: 5 g / L of yeast powder, 8 g / L of proteose peptone, 3 g / L of sodium chloride, and pH 7.0; the culture conditions are: 37℃, 150 r / min, and fermentation culture for 7 days.
[0009] Compared with the prior art, the present application has the following advantages: the Cytobacillus firmus Sys-Gs-002 strain provided by the present application can ferment to produce ginsenoside Rg1. The strain provided by the present application can be used for large-scale fermentation culture in a short period of time, and the fermentation cost is low, and it is not limited by time domain, season and other conditions, and has obvious advantages. Meanwhile, the present application also provides the optimized microbial fermentation conditions and the formula of the culture medium, and the yield of ginsenoside Rg1 is high. BRIEF DESCRIPTION OF DRAWINGS
[0010] Figure 1 is the HPLC detection chart of ginsenoside Rg1 in the standard sample;
[0011] Figure 2 is the HPLC detection chart of ginsenoside Rg1 in the culture medium after fermentation of the Cytobacillus firmus Sys-Gs-002 strain. DETAILED DESCRIPTION
[0012] The application will be described in detail below with specific examples, but the scope of protection of the application is not limited to the following examples.
[0013] Isolation and culture of strains
[0014] In this embodiment, the root tissue of fresh wild ginseng collected from Changbai Mountain was surface sterilized with 75% ethanol for 20 min, then washed with sterile water for 3-5 times, and then put into a pre-sterilized mortar, 5 mL of sterile water was added to grind into a white suspension. 300 μL of the suspension was taken to each LB solid medium plate, and then evenly coated with a sterile swab. After air-drying, the plate was sealed with a sealing film and incubated at 37°C in an incubator for 3-5 days. The bacteria growing out were observed and recorded in time, and the bacteria growing out were repeatedly purified.
[0015] At the same time, the stems were washed, the necrotic surface hard parts were removed, and then cut into 1 cm 2 thin slices, soaked in 75% alcohol for 3-5 min, washed with sterile water for 2-3 times to remove residual alcohol, and then soaked in sterile water for about 5 min. After taking out, the surface moisture was absorbed with sterile filter paper. The treated small pieces were attached to the LB solid medium plate, 5 pieces were placed on each plate, and then sealed with a sealing film and incubated at 37°C in an incubator for 3-5 days. The bacteria growing out were observed and recorded in time, and the bacteria growing out were repeatedly purified.
[0016] The bacteria purified on the plate were picked into a 1.5 mL EP tube containing 600 μL of LB liquid medium, sealed with a sealing film, and then incubated at 37°C in a 190 r / min shaking incubator for 1 day. Then an equal volume of 50% glycerol was added, mixed well, and stored in a -80°C ultra-low temperature refrigerator. Each strain was stored in at least 3 copies.
[0017] 10 μL of the stored strain was inoculated into 5 mL of LB liquid medium, incubated at 37°C in a 150 r / min shaking incubator for 24 h, and then 3 mL of the activated bacterial solution was inoculated into 100 mL of LB liquid medium for fermentation culture at 37°C in a 150 r / min shaking incubator for 7 days. After the above fermentation culture, the fermentation broth was collected.
[0018] Strain screening
[0019] The HPLC detection of ginsenoside Rg1 in the filtered fermentation broth sample of each strain was performed, and the detection results were compared with the standard product data, so as to preliminarily determine whether the strain had the ability to produce ginsenoside Rg1.
[0020] After the above detection, a strain of Bacillus firmus producing ginsenoside Rg1 was screened, which was named as Sys-Gs-002 strain. The HPLC detection was as follows: Figure 2The HPLC detection conditions are as follows:
[0021] The HPLC detection conditions are as follows: the chromatograph is Agilent 1290 chromatographic system, the ODS-C18 reversed-phase column (4.6 mm x 250 mm, 5 μm, Agilent), the mobile phase is acetonitrile: water = 65:35; the standard concentration is 100 μg / mL, the flow rate is 1 mL / min, the ultraviolet wavelength is set to 203 nm, the column temperature is 20℃, and the detection results are as shown in the following table: Figure 1 The HPLC detection conditions are as follows: Figure 1 and Figure 2 As can be seen from the comparison of the above two tables, the fermentation broth of the strain Sys-Gs-002 after fermentation contains ginsenoside Rg1.
[0022] Sequencing and sequence alignment and analysis
[0023] The strain is sent to a biotechnology company for sequencing, and the 16S rDNA sequence of the strain is shown as SEQ ID NO. 1. The sequencing results are subjected to Blast similarity analysis in the NCBI nucleic acid database, and through Blast sequence alignment and phylogenetic tree analysis, the 16S rDNA sequence of the strain has a similarity of 98% to Cytobacillus firmus (GeneBank number: NR_025842.1) and a similarity of 98% to Cytobacillus firmus (GeneBank number: NR_112635.1), which is determined to be a bacterium of Cytobacillus firmus.
[0024] Preservation of the strain:
[0025] The strain is preserved in the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with a postal code of 430072, and the preservation date is November 24, 2025, and the preservation number is CCTCC NO: M20252684.
[0026] Optimization of fermentation conditions
[0027] The fermentation medium components of microorganisms must be able to meet the needs of rapid growth and reproduction of the strain, and the optimal fermentation culture conditions can ensure the accumulation of secondary metabolites. The fermentation medium components and culture conditions of the strain Sys-Gs-002 are preliminarily optimized, and the optimized medium formula is as follows: yeast powder 5 g / L, peptone 8 g / L, sodium chloride 3 g / L, pH 7.0; the culture conditions are as follows: 37℃, 150 r / min, and fermentation culture for 7 days.
Claims
1. A Cytobacillus firmus. Sys-Gs-002 strain for producing ginsenoside Rg1, the strain has a preservation number of CCTCC NO: M20252684, and a 16S rDNA sequence of the strain is shown as SEQ ID NO.
1.
2. Use of Bacillus firmus strain Sys-Gs-002 according to claim 1, characterized in that: The ginsenoside Rg1 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.