Bacillus cereus Sys-Gs-004 strain for producing ginsenoside Rh2 and application of bacillus cereus Sys-Gs-004 strain
The preparation of ginsenoside Rh2 by fermentation with Bacillus cereus strain Sys-Gs-004 solves the problems of high production cost and limited conditions in the existing technology, and realizes large-scale production with high efficiency and low cost.
Patent Information
- Application Number
- CN202511842380.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-09
- Publication Date
- 2026-02-27
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Existing technologies are insufficient for the efficient and low-cost production of ginsenoside Rh2, and are limited by time and seasonal conditions, making it difficult to meet the needs of large-scale industrial production.
Ginsenoside Rh2 was prepared by fermentation using Bacillus cereus strain Sys-Gs-004. 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 high-yield, low-cost, short-term fermentation production of ginsenoside Rh2, avoiding time and seasonal limitations and making it suitable for large-scale industrial production.
Smart Images
Figure CN121574877A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to a Bacillus cereus strain and use, the strain can ferment to prepare ginsenoside Rh2, belongs to the field of microbial technology. BACKGROUND
[0002] Ginsenoside is the main active medicinal component of ginseng. Due to the different types and contents of ginsenoside monomers contained, the pharmacological effects of various monomer saponins are different, so separating effective monomers and clarifying the pharmacological activities of each monomer have become a research hotspot in the field. At present, more than 60 known ginsenosides have been found, and each has a unique medicinal function, mainly divided into two categories: ① protopanaxadiol (PPD) including ginsenoside Ra1, Ra2, Rb1, Rb2, Rb3, Rc, Rd, Rg3 and Rh2, etc.; ② protopanaxatriol (PPT) including ginsenoside Re, Rg1, Rg2, Rf and Rh1, etc.
[0003] Most of the saponins in ginseng are Rb1, Rb2, Rc, Rd, Re and Rg1. Ginsenoside Rh2 is a very rare ginsenoside in ginseng. Ginsenoside Rh2 is one of the most important anti-tumor active substances in ginseng, which has the effects of inhibiting tumor cell growth, inhibiting telomerase activity of tumor cells, inducing tumor cell apoptosis, anti-tumor cell metastasis, reversing abnormal differentiation of tumor cells, etc., thereby having good anti-tumor and preventing and treating tumor recurrence and metastasis effects. In clinical practice, ginsenoside Rh2 combined with radiotherapy and chemotherapy can enhance the effect of radiotherapy and chemotherapy in cancer treatment. In addition, ginsenoside Rh2 also has a series of physiological functions such as anti-allergy, improving immunity, anti-fatigue and anti-inflammatory.
[0004] At present, there are mainly two ways to produce ginsenoside Rh2. The first way is to use ginsenoside Rh2 produced by acid hydrolysis, alkali hydrolysis, enzyme hydrolysis and microbial fermentation of ginsenoside Rh2. Due to the long growth cycle of ginseng, the harsh growth conditions, the low content of ginsenoside Rh2, the complex extraction process and the low yield of ginsenoside Rh2, the production cost of ginsenoside Rh2 is extremely high, which is difficult to meet the requirements of large-scale industrial production. (2) The precursor of ginsenoside Rh2 is protopanaxadiol. The chemical method for glycosylation modification of ginsenoside Rh2 has the problems of complex synthesis route, high cost, low conversion rate, poor stereoselectivity and generation of a large amount of by-products. In recent years, the chemical method has been rarely used. The above two production methods have their own disadvantages, so seeking other production methods is a research hotspot in the field. At present, there is no related report on the direct microbial fermentation production of rare ginsenoside Rh2. SUMMARY
[0005] The present application solves the problems in the background art and provides a Bacillus cereus Sys-Gs-004 strain capable of producing ginsenoside Rh2. The strain can be used for fermentation to prepare ginsenoside Rh2.
[0006] A new strain is screened in the present application, which is named Sys-Gs-004 and is a Bacillus cereus. The strain is preserved in China Center for Type Culture Collection, Wuhan University, Wuhan, China, with a postal code of 430072, and the preservation date is December 4, 2025, and the preservation number is CCTCC NO: M20252780. The 16S rDNA sequence of the strain is shown as SEQ ID NO. 1.
[0007] The above-mentioned Bacillus cereus Sys-Gs-004 strain can be used for fermentation to prepare ginsenoside Rh2. 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°C, 150 r / min, and fermentation culture for 7 days.
[0008] Compared with the prior art, the present application has the following advantages: the Bacillus cereus Sys-Gs-004 strain provided by the present application can produce ginsenoside Rh2. 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, season and other conditions, which 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 Rh2 is high. BRIEF DESCRIPTION OF DRAWINGS
[0009] Figure 1 HPLC detection chart of ginsenoside Rh2 in the standard sample;
[0010] Figure 2 HPLC detection chart of ginsenoside Rh2 in the fermentation medium of Bacillus cereus Sys-Gs-004 strain. DETAILED DESCRIPTION
[0011] 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.
[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 min, then washed with sterile water for 3-5 times, and 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 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.
[0014] At the same time, the stem was washed, the necrotic surface hard part was removed, and then cut into 1 cm 2 thin pieces. The pieces were first soaked in 75% alcohol for 3-5 min, then washed with sterile water for 2-3 times to remove the 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 pieces were placed on the LB solid medium plate, 5 pieces on each plate, 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] 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. An equal volume of 50% glycerol was added, mixed well, and stored in a -80°C ultra-low temperature refrigerator. Each strain was stored for at least 3 copies.
[0016] 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 3 mL of the activated bacterial liquid was inoculated into 100 mL of LB liquid medium for fermentation culture, and incubated at 37°C in a 150 r / min shaking incubator for 7 d. After the above fermentation culture, the fermentation broth was collected.
[0017] Strain screening
[0018] The fermentation liquid sample of each strain after filtration was detected by HPLC to detect ginsenoside Rh2, and the detection results were compared with the standard data, so as to preliminarily determine whether the strain has the ability to produce ginsenoside Rh2.
[0019] After the above detection, a Bacillus cereus strain producing ginsenoside Rh2 was screened, which was named Sys-Gs-004 strain. The HPLC detection is shown in Figure 2 The HPLC detection conditions are as follows:
[0020] The HPLC detection conditions are as follows: the chromatograph is Agilent 1290 chromatograph system, 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 shown in Figure 1 The comparison of Figure 1 and Figure 2 can be seen that the fermentation liquid of the Sys-Gs-004 strain contains ginsenoside Rh2.
[0021] Sequencing and sequence alignment and analysis
[0022] The strain was 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 were subjected to Blast similarity analysis in the NCBI nucleic acid database, and the Blast sequence alignment and phylogenetic tree analysis showed that the 16S rDNA sequence of the strain had a similarity of 99% with Bacillus cereus (GeneBank number: NR_074540.1), 99% with Bacillus cereus (GeneBank number: NR_115714.1), and 99% with Bacillus cereus (GeneBank number: NR_115526.1), confirming that it is a Bacillus cereus bacteria.
[0023] Preservation of the strain:
[0024] The strain was preserved in China Center for Type Culture Collection, Wuhan University, Wuhan, China, with a post code of 430072, on December 4, 2025, with a preservation number of CCTCC NO: M20252780.
[0025] Optimization of fermentation conditions
[0026] The fermentation medium components of the microorganism 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 Sys-Gs-004 strain are preliminarily optimized, and the optimized medium formula is: yeast powder 5 g / L, peptone 8 g / L, sodium chloride 3 g / L, pH 7.0; the culture conditions are: 37 DEG C, 150 r / min, and fermentation culture for 7 days.
Claims
1. A Bacillus cereus. Sys-Gs-004 strain for producing ginsenoside Rh2, the strain has a preservation number of CCTCC NO: M20252780, and a 16S rDNA sequence of the strain is shown as SEQ ID NO.
1.
2. Use of Bacillus cereus strain Sys-Gs-004 according to claim 1, characterized in that: Ginsenoside Rh2 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.