Achromobacter PGOD518 strain for producing ginsenoside Rg3 and application of Achromobacter PGOD518 strain

Abstract

The invention provides an achromobacter sp. PGOD518 strain for producing ginsenoside Rg3, and the strain is preserved in China Center for Type Culture Collection (address: Wuhan University, Wuhan, China). The strain is preserved in China Center for Type Culture Collection (CCTCC), the postal code is 430072, the preservation date is November 11, 2024, and the preservation number is CCTCC NO: M20242516; the 16SrDNA (deoxyribonucleic acid) sequence of the strain is as shown in SEQ ID NO. 1. The PGOD518 strain can be used for producing ginsenoside Rg3, and the yield is 8.2 g / L. The strain can be used for mass production of ginsenoside Rg3 in a short time, the conversion condition is mild, the yield is high, and the Rg3 can be produced by using a substrate to the maximum extent through fermentation. The method for converting and producing ginsenoside Rg3 by using the strain is low in cost and quick in reaction, and has obvious advantages in a large-scale fermentation production process.

Description

Technical Field

[0001] The invention relates to a colorless bacterium, which can produce ginsenoside Rg3 and belongs to the technical field of microorganisms. Background Art

[0002] Ginseng (Pannax gxinseng CAMeyer), also known as goblin, magic grass, and yellow ginseng, is a perennial herbaceous medicinal plant of the genus Panax in the Araliaceae family. The medicinal parts are the roots and rhizomes. It has magical effects such as replenishing vital energy, restoring pulse and strengthening deficiency, tonifying the spleen and lungs, promoting body fluid and nourishing blood, calming the mind and improving intelligence. Ginseng cultivation has a history of more than 400 years. In recent years, with the further increase in market demand, artificial domestication and cultivation is the only way to meet market demand. However, in recent decades, due to over-exploitation and the destruction of its habitat, the number of wild ginseng resources has sharply decreased. Artificially cultivated ginseng also has problems such as a sharp decline in quality and mixed varieties. Therefore, it is particularly important to find reasonable and effective ways to promote the sustainable development of the ginseng industry.

[0003] Ginsenoside is the main active medicinal ingredient of ginseng. Due to the different types and contents of ginsenoside monomers, the pharmacological effects of various monomer saponins are different. Therefore, isolating the 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 discovered, and each has unique medicinal functions, mainly divided into two categories: ① Protopanaxadiol (PPD) includes ginsenosides Ra1, Ra2, Rb1, Rb2, Rb3, Rc, Rd, Rg3 and Rh2; ② Protopanaxantriol saponins (PPT) include ginsenosides Re, Rg1, Rg2, Rf and Rh1.

[0004] Ginsenoside Rg3 is mainly used to inhibit the production of tumor cells and can be used to prevent the occurrence and recurrence of tumors, including tumor metastasis. Compared with other anticancer drugs, ginsenoside Rg3 has no obvious toxic side effects and can be used for a long time. Ginsenoside Rg3 can inhibit the proliferation and infiltration of tumor cells and induce apoptosis of tumor cells. Therefore, ginsenoside Rg3 is often used in combination with radiotherapy and chemotherapy, which can significantly improve the efficacy of radiotherapy and chemotherapy, while also reducing the side effects of radiotherapy and chemotherapy, and improving the quality of life of patients to a certain extent. It has certain effects on gastric cancer, colon cancer, liver cancer, and non-small cell lung cancer.

[0005] Ginsenoside Rg3 has unique biological activity, but its content in ginseng is extremely low. The existing technology for preparing ginsenoside Rg3 includes microbial fermentation, which converts ginsenosides into rare saponins Rg3 through microbial metabolism. For example, the "Study on the Process of Lactic Acid Bacteria Fermentation to Convert Ginsenoside Rg3" in the journal Food and Fermentation Industry discloses a method of converting ginsenoside Rg3 using lactic acid bacteria, which uses microorganisms such as Lactobacillus and Pediococcus pentosaceus to convert ginsenosides into ginsenoside Rg3. However, there are no reports on other microbial fermentation. Summary of the invention

[0006] The present invention solves the problem in the background technology and provides a strain of Achromobacter PGOD518 producing ginsenoside Rg3. The strain can produce ginsenoside Rg3 with a high fermentation yield of 8.2 g / L.

[0007] The inventors screened a new strain, which was named PGOD518, and is a colorless bacterium (Achromobacter sp.). The strain is deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China; postal code 430072, the deposit date is November 11, 2024, and the deposit number is: CCTCC NO: M20242516; the 16S rDNA sequence of the strain is shown in SEQ ID NO.1.

[0008] The above-mentioned Achromobacter PGOD518 strain can be used for fermentation to prepare ginsenoside Rg3. The microbial fermentation conditions are: culture medium formula: NaCl 12g / L, yeast extract 15g / L, peptone 15g / L, pH 9.0; culture conditions: 31°C, shaking speed 150r / min.

[0009] Compared with the prior art, the present invention has the following advantages: the Achromobacter PGOD518 strain provided by the present invention can produce ginsenoside Rg3 with a yield of 8.2 g / L. The strain can produce ginsenoside Rg3 in large quantities in a short period of time, the conversion conditions are mild, the yield is high, and the utilization of substrate fermentation to produce Rg3 can be maximized. In the reaction of the strain producing ginsenoside Rg3, the target product is clear, other impurities have less interference, and it is easier to prepare ginsenoside Rg3. The cost of strain transformation to produce ginsenoside Rg3 is low, the reaction is fast, and it can have obvious advantages in the process of large-scale fermentation production. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is the first HPLC test chart of ginsenoside Rg3 standard;

[0011] Figure 2This is the first HPLC detection chart of ginsenoside Rg3 in the fermentation broth of Achromobacter PGOD518 strain;

[0012] Figure 3 This is the second HPLC test chart of ginsenoside Rg3 standard;

[0013] Figure 4 This is the second HPLC detection chart of ginsenoside Rg3 in the fermentation broth of Achromobacter PGOD518 strain. DETAILED DESCRIPTION

[0014] The present invention is described in detail below in conjunction with specific embodiments, but the protection scope of the present invention is not limited to the following embodiments.

[0015] Isolation and cultivation of strains

[0016] In this embodiment, the root tissue of fresh wild ginseng collected from Changbai Mountain was surface disinfected with 75% ethanol for 20 minutes, then washed with sterile water for 3-5 times, placed in a pre-sterilized standby mortar, and ground into a white suspension with 5 mL of sterile water. Take 300 μL of the suspension in each LA plate culture medium, spread it evenly with a sterile coating rod, seal it with a sealing film after air drying, and invert it in a 37°C constant temperature incubator for 3-5 days, observe it in time, and take photos and record it on the 1st, 3rd, and 5th days. If the bacteria grow all over the plate, they will no longer be separated and sterilized. Use a toothpick to pick the bacteria grown on the plate and purify them repeatedly on the LB solid culture medium plate until a single colony is obtained.

[0017] At the same time, wash the stems, remove the necrotic surface hard parts, and then cut them into 1cm 2 The thin slices were soaked in 75% alcohol for 3-5 minutes, then washed with sterile water for 2-3 times to remove the residual alcohol, and then soaked in sterile water for about 5 minutes. After taking out, the surface moisture was absorbed with sterilized filter paper. The processed small pieces were pasted on LA solid plates, 5 pieces were placed on each plate, sealed with sealing film, and cultured in a constant temperature incubator at 37℃ for 3-5 days. Observe and record every day, and purify the grown bacteria repeatedly.

[0018] The purified bacteria on the plate were picked into a 1.5 mL EP tube containing 600 μL LB liquid culture medium, sealed with a sealing film, and then cultured in a 37°C constant temperature shaker at 190 r / min for 1 day, and then an equal volume of 50% glycerol was added, mixed and stored in a -80°C ultra-low temperature refrigerator. At least 6 copies of each strain were stored.

[0019] Screening of strains

[0020] Take 10 μL of the preserved strain and inoculate it into 1000 μL LB liquid medium, and activate it in a 37℃ constant temperature shaker at 180 r / min. After 24 hours, take 1 mL of the activated strain and ferment it in LB liquid medium in a triangular flask, with a liquid volume of 100 mL / 250 mL, and culture it in a 37℃, 180 r / min shaker until OD 600 =0.8, and then switched to purple light culture. After 12 h of fermentation culture, the fermentation broth was collected.

[0021] The first test of ginsenoside Rg3 in the fermentation broth samples of each strain was conducted, and its spectrum and data were compared with the standard product data to roughly determine whether the strain has the ability to produce ginsenoside Rg3. Then, based on the standard curve of ginsenoside Rg3, the content of ginsenoside Rg3 in the sample and the yield of ginsenoside Rg3 in the fermentation broth of the strain can be calculated. In order to avoid possible interference from other substances, the applicant conducted a second confirmatory qualitative test on the positive strain fermentation broth obtained in the first test after changing the chromatographic detection conditions, including the detection of the standard product and the detection of the sample.

[0022] After the above tests, a strain of ginseng endophyte that can produce ginsenoside Rg3 was screened and named PGOD518. The yield of ginsenoside Rg3 in the fermentation broth of this strain was 8.2 g / L. The first HPLC test results were as follows: Figure 1 and Figure 2 As shown; the second HPLC test results are as follows Figure 3 and Figure 4 shown.

[0023] Extraction and electrophoresis detection of endophytic bacterial DNA

[0024] Primer sequences for 16S rDNA:

[0025] Upstream primer: 515F (5'-GTGYCAGCMGCCGCGGTAA-3')

[0026] Downstream primer: 806R (5'-GGACTACNVGGGTWTCTAAT-3')

[0027] The 16S rDNA PCR product was purified and recovered using the Sigma NA1020 PCR product recovery kit.

[0028] Ligation of 16S rDNA fragment and cloning vector: Ligate the recovered 16S rDNA fragment with the pMD18-T Vector vector, flick to mix, and then ligate at 16°C for 6 hours or 4°C for 12 hours. Transform competent cells with the ligation product.

[0029] Bacterial liquid PCR-clone detection

[0030] Universal primers were used to perform bacterial solution PCR to determine whether the target fragment was successfully transformed and to screen positive clones.

[0031] Primer sequence: M13R: CAGGAAACAGCTATGACC

[0032] M13F:TGTAAAACGACGGCCAGT

[0033] A conventional amplification reaction system and amplification procedure were used, wherein 2 μL of bacterial solution was used to replace the original 3 μL of template in the reaction system. 10 μL of amplification product was taken for agarose gel electrophoresis detection, and the corresponding positive clones were packaged and preserved.

[0034] Sequencing, sequence alignment and analysis

[0035] The positive clones were sent to a biotechnology company for sequencing, and the 16S rDNA sequence of the strain is shown in SEQ ID NO. 1. The sequencing results were subjected to Blast similarity analysis in the GenBank nucleic acid database. After Blast sequence alignment and phylogenetic tree analysis, the homology of PGOD518 strain with Achromobacter sp. strain S4b60 was 99%, and the homology with Achromobacter sp. MMJ Inhibition was 99%. Combined with the phylogenetic tree, it was determined to be Achromobacter sp.

[0036] Deposit of strains:

[0037] The strain was deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China; postal code 430072, the deposit date is November 11, 2024, and the deposit number is: CCTCC NO: M20242516.

[0038] Optimization of fermentation conditions

[0039] The fermentation medium components of microorganisms must be able to meet the needs of rapid growth and reproduction of the strains, and at the same time, the optimal fermentation culture conditions can ensure the accumulation of a large number of secondary metabolites. The applicant optimized the fermentation medium components of the PGOD518 strain through single-factor and orthogonal experiments. The optimized optimal medium formula is: NaCl 12g / L, yeast extract 15g / L, peptone 15g / L. After determining the culture medium components and corresponding concentrations, the optimal initial pH value, culture temperature and shaker speed for the strain to transform ginsenosides were further optimized, and the optimal culture conditions for the HGS-487 strain were finally determined to be: initial pH 9.0, culture temperature 31°C, and shaker speed 150r / min.

Claims

1. A strain of Achromobacter sp. PGOD518, the deposit number of which is CCTCCNO: M20242516, and the 16S rDNA sequence of which is shown in SEQ ID NO.

1.

2. The use of the Achromobacterium PGOD518 strain according to claim 1, characterized in that: Used for fermentation to prepare ginsenoside Rg3.

3. The use according to claim 2, characterized in that: The fermentation conditions were as follows: culture medium formula: NaCl 12 g / L, yeast extract 15 g / L, peptone 15 g / L, pH 9.0; culture conditions: 31°C, shaking speed 150 r / min.

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