Anomalus sp. For converting ginsenoside Rb1 into ginsenoside Rd and application of Anomalus sp.

Abstract

The invention discloses another strain capable of converting ginsenoside Rb1 into ginsenoside Rd and application of the another strain. The another strain (Alistipes sp.) CMLF01 is preserved in China General Microbiological Culture Collection Center, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, the preservation date is January 25, 2024, and the preservation number is CGMCC NO.41092. The invention further discloses a preparation method of the another strain. The invention provides an application of the other fungus in the conversion of the ginsenoside Rb1 into the ginsenoside Rd. The invention solves the problems of low specificity, low conversion rate and generation of other byproducts in the existing microbial conversion of ginsenoside Rb1. The other strain CMLF01 provided by the invention can efficiently and singly convert ginsenoside Rb1 into ginsenoside Rd, and the conversion rate reaches 95.5%.

Description

Technical Field

[0001] The present invention relates to a strain capable of converting ginsenoside Rb1 into ginsenoside Rd, and in particular to another strain capable of converting ginsenoside Rb1 into ginsenoside Rd and application thereof. Background Art

[0002] Ginseng (Panax ginseng) is a plant of the genus Panax. It is a traditional precious Chinese medicinal material in my country. It has complex chemical components, extensive biological activities, and unique pharmacological effects. With the advancement of modern separation and analysis techniques, the chemical components of ginseng have been further elucidated. Studies have shown that ginsenosides are the main bioactive substances in these isolated components. So far, more than 50 ginsenoside monomers have been isolated and identified. Ginsenosides are composed of aglycones and glycosyls. According to the different parent nucleus skeletons of aglycones, they can be divided into dammarane-type tetracyclic triterpenoid saponins and oleanolic acid-type pentacyclic triterpenoid saponins. Dammarane-type ginsenosides can be divided into protopanaxadiol-type saponins (PPD) and protopanaxatriol-type saponins (PPT) according to the different glycosyl substituents on C-3, C-6, and C-20.

[0003] Ginsenoside Rb1 is a monomeric saponin of protopanaxadiol saponins, which is widely distributed in plants such as ginseng, American ginseng and Panax notoginseng. The isolated ginsenoside Rb1 is as high as about 30%, which is a major saponin in ginseng plants. However, due to the large number of side chain sugar groups and high polarity, it is not easily absorbed by the human gastrointestinal tract, resulting in low biological activity. Ginsenoside Rd has the function of specifically blocking receptor-dependent calcium ion channels, and has unique effects that other monomeric ginsenosides do not have in protecting renal function, regulating immunity, inhibiting HeLa cell growth, inducing COX22 production, and protecting against radiation; in terms of analgesia and neuroprotection, Rd is also stronger than other monomeric ginsenosides, and is a candidate drug with great application prospects.

[0004] The content of ginsenoside Rd varies in different ginseng plants, but the overall content is low, and the content in ginseng is only about 0.1%. Obtaining ginsenoside Rd from natural plants can no longer meet market demand. At present, ginsenoside Rd is mainly prepared by chemical synthesis, chemical transformation and biological transformation. The structure of Rd is relatively complex, and chemical synthesis requires high experimental conditions and is also difficult; acid hydrolysis is a method of chemical transformation. This method is simple to operate, but the hydrolysis specificity is poor, and the sapogenin undergoes structural changes during the hydrolysis process. Therefore, many researchers are looking for microorganisms that can convert ginsenoside Rb1 into ginsenoside Rd, but currently microorganisms lack high specificity, the conversion rate is not high enough, and further transformation will produce other by-products. Therefore, it is of great significance to find microorganisms that can efficiently and specifically convert ginsenoside Rb1 into ginsenoside Rd. Summary of the invention

[0005] The purpose of the present invention is to provide another branch of bacteria for converting ginsenoside Rb1 into ginsenoside Rd and its application, which solves the problems that the existing microorganisms for converting ginsenoside Rb1 have low specificity and conversion rate and produce other by-products. Ginsenoside Rb1 can be efficiently and solely converted into ginsenoside Rd, and the conversion rate reaches 95.5%.

[0006] In order to achieve the above-mentioned purpose, the present invention provides another branch of bacteria that converts ginsenoside Rb1 into ginsenoside Rd. The other branch of bacteria (Alistipes sp.) CMLF01 is preserved in the General Microbiological Center of China Microbiological Culture Collection Administration, the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the preservation date is January 25, 2024, and the preservation number is CGMCC NO.41092.

[0007] The present invention provides an application of the aforementioned fungus in converting ginsenoside Rb1 into ginsenoside Rd.

[0008] Preferably, the bioconversion route for converting ginsenoside Rb1 into ginsenoside Rd is: the other bacterium CMLF01 hydrolyzes the glucose group at the C-20 position of ginsenoside Rb1 to generate ginsenoside Rd.

[0009] Preferably, the method for converting ginsenoside Rb1 into ginsenoside Rd is as follows:

[0010] The bacterial liquid of another branch as described above was inoculated into a brain heart infusion liquid culture medium containing saponin, and cultured statically at 37° C. under an anaerobic gas environment. The bacterial liquid after culture was taken for detection of saponin component conversion.

[0011] Preferably, the preparation method of the brain heart infusion liquid culture medium is: mix peptone, dehydrated calf brain infusion powder, dehydrated buffalo heart infusion powder, sodium chloride, glucose, disodium phosphate and L-cysteine ​​hydrochloride, adjust the pH to 7.2-7.6, sterilize at 101 kPa and 121° C., wait for it to cool after sterilization, and then add hemin and vitamins.

[0012] More preferably, in each liter of brain heart infusion liquid culture medium, the mass ratio of the peptone, dehydrated calf brain extract powder, dehydrated buffalo heart extract powder, sodium chloride, glucose and disodium phosphate is 10.0:12.5:5.0g:5.0:2.0:2.5; the mass of the L-cysteine ​​hydrochloride is 1.0‰ of the mass of the brain heart infusion liquid culture medium, the mass of the hemin chloride is 0.01‰ of the mass of the brain heart infusion liquid culture medium, and the mass of the vitamin K1 is 0.01‰ of the mass of the brain heart infusion liquid culture medium.

[0013] Preferably, the composition of the anaerobic gas environment is 5% CO2, 5% H2 and 90% N2.

[0014] Preferably, the detection is performed by high performance liquid chromatography to analyze the components of saponins in the cultured bacterial liquid.

[0015] Preferably, the cultured bacterial liquid is taken out once every 24 hours and then tested.

[0016] Preferably, during the inoculation, the volume ratio of the bacterial liquid to the BHI culture medium containing 1.5‰ notoginseng total saponins and 1.5‰ ginsenoside Rb1 is 1:10.

[0017] The main influencing factor of the present invention is the activity of the bacteria. On the one hand, the strain has high activity and strong metabolic capacity. If it grows fast, it needs to obtain more energy, so it will absorb its own available energy, so the conversion of ginsenoside Rb1 will be faster; on the other hand, the strain has high activity and better reproduction ability. During the conversion, the concentration of the bacterial solution can be increased to further accelerate the conversion. The bacterial growth cycle has four stages: the delayed period, the logarithmic period, the stable period and the decay period. In the logarithmic period, the strain activity is the best during this period. During this period, the strain grows faster and has stronger reproduction ability, so that ginsenoside Rb1 can be basically converted into ginsenoside Rd in a short time. In the delayed period, the strain starts to grow with low activity during this period. If the bacterial solution of this period is used for the conversion experiment, the conversion effect is poor due to the slow growth of the strain, and the conversion time needs to be extended. In the stable period, the strain activity tends to be stable. If the bacterial solution of this period is used for the conversion experiment, the strain stops growing due to the accumulation of its own metabolites and other reasons, and the metabolic activity is poor, so that the conversion rate does not reach the above value. In the decay phase, the strain begins to die. If the bacterial solution of this phase is used for transformation, the strain may not grow and the experiment cannot be carried out. In summary, the activity of the strain will affect the experimental results. The logarithmic phase of the strain is between 36 and 48 hours of cultivation. The bacterial solution inoculated in the transformation experiment is the bacterial solution of the period. The transformation rate can reach 95.5% 48 hours after inoculation with the strain of this period.

[0018] The present invention discloses another branch of bacteria for converting ginsenoside Rb1 into ginsenoside Rd and its application, which solves the problems of low specificity and low conversion rate of existing microorganisms for converting ginsenoside Rb1 and the generation of other by-products, and has the following advantages:

[0019] Another branch of the fungus CMLF01 of the present invention can efficiently and solely convert ginsenoside Rb1 into ginsenoside Rd, with a conversion rate of 95.5%. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a plate colony morphology diagram of another mycobacterial strain CMLF01 of the present invention.

[0021] Figure 2This is a high performance liquid chromatogram of the fermentation product of Panax notoginseng total saponins fermented by another fungus strain CMLF01 of the present invention for 48 hours.

[0022] Figure 3 This is a high performance liquid chromatogram of the fermentation product of ginsenoside Rb1 fermented by another mycorrhizal strain CMLF01 of the present invention for 48 hours.

[0023] Figure 4 Schematic diagram of the conversion of ginsenoside Rb1 into ginsenoside Rd according to the present invention. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Example 1: Isolation and identification of another fungus CMLF01

[0026] The specific steps are as follows:

[0027] 1. Preparation of 10% (w / v) human feces suspension:

[0028] About 3 g of fresh feces from healthy people who took Panax notoginseng powder for a long time were taken. The fresh feces samples were quickly placed in 30 mL of sterile phosphate buffered saline (PBS, pH 7.2) containing 20% ​​glycerol at a volume ratio of 1:10, vortexed and mixed, and the supernatant was taken after centrifugation to prepare a human fecal bacterial suspension.

[0029] Sterile phosphate buffered saline (PBS, pH 7.2) containing 20% ​​glycerol was prepared as follows:

[0030] Glycerol was sterilized at 101 kPa and 121° C. for 30 min. 6 mL of sterilized glycerol was added to 24 mL of sterile phosphate buffer (PBS, pH 7.2) and mixed to prepare 30 mL of sterile phosphate buffer (PBS, pH 7.2) containing 20% ​​glycerol.

[0031] 2. Configuration of Brucella blood agar solid medium (LKV):

[0032] The amount of each component added to the LKV medium is 10.0g / L of peptone, 2.0g / L of yeast extract powder, 10.0g / L of trypticase peptone, 5.0g / L of sodium chloride, 1.0g / L of glucose, 0.1g / L of sodium bisulfite, 15.0g / L of agar and 1.0‰ of L-cysteine ​​hydrochloride, the pH is adjusted to 6.8-7.2, and sterilized at 101kPa and 121℃ for 15min. After sterilization, add 0.01‰ hemin (0.01‰ hemin means that the mass of hemin is 0.01‰ of LKV medium, the same below), 0.01‰ vitamin K1, 0.1‰ kanamycin, 0.0075‰ vancomycin and 5% sterile defibrinated sheep whole blood. After the LKV plate solidifies, move it to an anaerobic gas environment and place it for 48h for use.

[0033] 3. Isolation of other fungal strains:

[0034] The human fecal bacterial suspension was diluted with sterile phosphate buffer solution (PBS, pH 7.2) containing cysteine ​​hydrochloride (the dilution for isolation was 900 μl of PBS plus 100 μl of bacterial suspension; the gradient dilution was 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 ), 100 μL of bacterial solution from each gradient dilution was evenly spread on LKV solid culture medium and cultured at 37°C in an anaerobic gas environment (5% CO2, 5% H2 and about 90% N2) for 48 h.

[0035] The preparation method of sterile phosphate buffered solution (PBS, pH 7.2) containing cysteine ​​hydrochloride is as follows:

[0036] First, prepare 0.2 M disodium hydrogen phosphate solution and 0.1 M citric acid solution, mix to obtain pH 7.2 phosphate buffer (PBS), and then filter and sterilize through a sterile syringe with a 0.45 um filter to obtain sterile phosphate buffer (PBS, pH 7.2).

[0037] 4. Purification of other bacterial strains:

[0038] Single colonies with different colors, shapes, sizes, etc. were picked from the LKV plate and streaked onto new LKV plates until the purified bacterial strain CMLF01 was obtained.

[0039] 5. Identification of colony morphology of other fungal strains:

[0040] Observe the colony morphology on the plate after 48 h of culture.

[0041] like Figure 1As shown in the figure, the plate colony morphology of another mycobacterium strain CMLF01 of the present invention. Figure 1 It can be seen that the colony as a whole is a relatively regular circle with complete edges, a colony diameter of about 0.5 mm, and a milky white, moist, smooth and translucent surface.

[0042] 6. 16S rDNA analysis of other bacterial strains:

[0043] The genome of strain CMLF01 was extracted, and 16S rDNA was amplified using primers 27F and 1492r (the nucleotide sequence of the universal primer 27F is agagtttgatcctggctcag, and the nucleotide sequence of the universal primer 1492R is ggttaccttgttacgactt). The amplified product was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. After homologous comparison of the results returned by the company on NCBI, it was found that the 16S rDNA of strain CMLF01 and Alistipes shahii had the highest similarity, with a consistency of 99.86%.

[0044] 7. Genome sequencing and classification identification of other fungal strains:

[0045] The strain CMLF01 was sent to Beijing Biomarker Biotechnology Co., Ltd. for genome sequencing. The CMLF01 genome was compared with the public database genome using the online genome hybridization analysis software GGDC (Genome-to-Genome Distance Calculator, https: / / ggdc.dsmz.de / ggdc.php#) and the whole genome average nucleotide similarity (average nucleotide identity, ANI, https: / / www.ezbiocloud.net / tools / ani) analysis tool. The results showed that the DDH value between the CMLF01 genome and the genome of the model strain Alistipes shahiiWAL 8301 was 87.90%, and the ANI value was 98.63%. Based on the 16S rDNA sequencing identification and DDH and ANI genome classification identification results of the strain (the nucleotide sequence of the other branch fungus strain CMLF01 is shown in SEQ ID NO.1), the strain CMLF01 belongs to another branch fungus (Alistipes sp.), and is named Alistipes shahii CMLF01, recorded as CMLF01, and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 25, 2024. The storage address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the storage number is CGMCC NO.41092.

[0046] The 16S DNA nucleotide sequence of the Alternaria strain CMLF01 is as follows:

[0047] gggctaacggggcagctacacatgcagtcgaggggcagcacggtgtagcaatacactggtggcgaccggcgcacgggtg

[0048] cgtaacgcgtatgcaacctacccataacagggggataacactgagaaattggtactaataccccataacatcaggaccggcat

[0049] cggttctggttgaaaactccggtggttatggatgggcatgcgttgtattagctggttggtgaggtaacggctcaccaaggcaac

[0050] gatacatagggggactgagaggttaaccccccacattggtactgagacacggaccaaactcctacgggaggcagcagtgag

[0051] gaatattggtcaatggacgcaagtctgaaccagccatgccgcgtgcaggaagacggctctatgagttgtaaactgcttttgtac

[0052] gagagtaaacgctcttacgtgtaagagcctgaaagtatcgtacgaataaggatcggctaactccgtgccagcagccgcggta

[0053] atacggaggatccaagcgttatccggatttattgggtttaaagggtgcgtaggcggtttgataagttagaggtgaaataccggtg

[0054] cttaacaccggaactgcctctaatactgttgaactagagagtagttgcggtaggcggaatgtatggtgtagcggtgaaatgctta

[0055] gagatcatacagaacaccgattgcgaaggcagcttaccaaactatatctgacgttgaggcacgaaagcgtggggagcaaac

[0056] aggattagataccctggtagtccacgcagtaaacgatgataactcgctgtcggcgatacacagtcggcggctaagcgaaagc

[0057] gataagttatccacctggggagtacgttcgcaagaatgaaactcaaaggaattgacgggggcccgcacaagcggaggaaca

[0058] tgtggtttaattcgatgatacgcgaggaaccttacccgggcttgaaagttactgacgattctggaaacaggatttcccttcgggg

[0059] caggaaactaggtgctgcatggttgtcgtcagctcgtgccgtgaggtgtcgggttaagtcccataacgagcgcaacccctacc

[0060] gttagttgccatcaggtcaagctgggcactctggcgggactgccggtgtaagccgagaggaaggtggggatgacgtcaaat

[0061] cagcacggcccttacgtccggggctacacacgtgttacaatggtaggtacagagggcagctacccagtgatgggatgcgaat

[0062] ctcgaaagcctatctcagttcggatcggaggctgaaacccgcctccgtgaagttggattcgctagtaatcgcgcatcagcca

[0063] tggcgcggtgaatacgttcccgggccttgtacacaccgcccgtcaagccatggaagctgggggtgcctgaagttcgtgaccgcaaggagcgacctagcaatccccgttc。

[0064] Example 2: Transformation of Panax notoginseng saponins and ginsenoside Rb1 by another strain CMLF01

[0065] The strain CMLF01 was mixed with notoginseng saponins or ginsenoside Rb1 for fermentation, and the saponin components were analyzed by HPLC. Figure 4 As shown, the schematic diagram of the present invention for converting ginsenoside Rb1 into ginsenoside Rd, the specific steps are as follows:

[0066] 1. Preparation of brain heart infusion liquid medium (BHI medium):

[0067] The amount of each component added to the culture medium is 10.0 g / L of peptone, 12.5 g / L of dehydrated calf brain extract powder, 5.0 g / L of dehydrated ox heart extract powder, 5.0 g / L of sodium chloride, 2.0 g / L of glucose, 2.5 g / L of disodium hydrogen phosphate and 1‰ of L-cysteine ​​hydrochloride (1‰ of L-cysteine ​​hydrochloride means that the mass of L-cysteine ​​hydrochloride is 1‰ of the culture medium, the same below), the pH is adjusted to 7.2-7.6, sterilized at 101 kPa and 121°C for 30 min, and after the sterilization is completed, it is cooled, and then 0.01‰ of hemin and 10.01‰ of vitamin K are added;

[0068] 2. Fermentation culture:

[0069] A single colony was picked from the LKV solid plate in Example 1 and inoculated (the inoculation volume was 100 μl for each gradient inoculation, the same below) into the BHI medium, and the bacteria were enriched at 37°C for 48 h in an anaerobic gas environment. 3 mL of the bacterial solution was inoculated into 30 mL of the BHI medium containing 1.5‰ total saponins of Panax notoginseng and 1.5‰ ginsenoside Rb1, and the culture was kept at 37°C in an anaerobic gas (5% CO2, 5% H2 and 90% N2) environment. 300 μl was taken after each 24 h of culture to detect the conversion of the saponin components; the BHI fermentation broth without saponin was used as a control.

[0070] 3. High performance liquid chromatography (HPLC) analysis:

[0071] The detailed conditions of HPLC are: YMC-PackODS-AM (250mm×4.6mm, 5μm) chromatographic column, mobile phase water (A)-acetonitrile (B), detection wavelength 203nm; column temperature 40°C, total flow rate 1.0L / min, sample amount 10μL; gradient elution, elution program is 0-25min, 20%B; 25-30min, 20%-25%B; 30-41min, 25%-41%B; 41-55min, 41%-45%B; 55-80min, 45%-74%B; 80-82min, 74%-100%B; 82-92min, 100%B; 92-95min, 100%-20%B; 95-100min, 20%B.

[0072] like Figure 2 As shown in the figure, the high performance liquid chromatogram of the fermentation product of Panax notoginseng total saponins fermented by another strain CMLF01 of the present invention for 48 hours, wherein A is the control result of Panax notoginseng total saponins; B is the result of Panax notoginseng total saponins conversion at 48 hours. Figure 2 It can be seen that the transformation results of Panax notoginseng total saponins are as follows: at the 48th hour, compared with the control group, the ultraviolet absorption peak of ginsenoside Rb1 in the experimental group basically disappeared, and the ultraviolet absorption peak of Rd became larger.

[0073] like Figure 3 As shown in the figure, the high performance liquid chromatogram of the fermentation product of ginsenoside Rb1 fermented by another strain of the present invention, CMLF01, for 48 hours, wherein A is the control result of Rb1; B is the result of Rb1 conversion at 48 hours. Figure 3 It can be seen that the conversion results of ginsenoside Rb1 are as follows: at 48h, compared with the control group, the ultraviolet absorption peak of ginsenoside Rb1 in the experimental group basically disappeared, and a new ultraviolet absorption peak appeared. After comparing with the peak graph of the ginsenoside Rd standard, it was determined that the conversion product was ginsenoside Rd.

[0074] In summary, the fungus strain CMLF01 has a transformation effect on ginsenoside Rb1 in Panax notoginseng total saponins, and the transformation product is ginsenoside Rd, with a conversion rate of 95.5%.

[0075] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A fungus for converting ginsenoside Rb1 into ginsenoside Rd, characterized in that: The other branch of fungus (Alistipes sp.) CMLF01 is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit date is January 25, 2024, and the deposit number is CGMCC NO.41092.

2. Use of the fungus as claimed in claim 1 in converting ginsenoside Rb1 into ginsenoside Rd.

3. The use according to claim 2, characterized in that: The bioconversion route for converting ginsenoside Rb1 into ginsenoside Rd is as follows: the other bacterium CMLF01 hydrolyzes the glucose group at the C-20 position of ginsenoside Rb1 to generate ginsenoside Rd.

4. The use according to claim 3, characterized in that: The method for converting ginsenoside Rb1 into ginsenoside Rd is specifically as follows: The bacterial liquid of another branch as claimed in claim 1 is inoculated into a brain heart infusion liquid culture medium containing saponin, and is cultured statically at 37° C. in an anaerobic gas environment. The cultured bacterial liquid is taken for detection of saponin component conversion.

5. The use according to claim 4, characterized in that: The preparation method of the brain heart infusion liquid culture medium is as follows: peptone, dehydrated calf brain infusion powder, dehydrated ox heart infusion powder, sodium chloride, glucose, disodium phosphate and L-cysteine ​​hydrochloride are mixed, the pH is adjusted to 7.2-7.6, sterilized at 101 kPa and 121° C., cooled after sterilization, and then hemin and vitamins are added.

6. The use according to claim 5, characterized in that: In each liter of brain heart infusion liquid culture medium, the mass ratio of the peptone, dehydrated calf brain extract powder, dehydrated buffalo heart extract powder, sodium chloride, glucose and disodium phosphate is 10.0:12.5:5.0g:5.0:2.0:2.5; the mass of the L-cysteine ​​hydrochloride is 1.0‰ of the mass of the brain heart infusion liquid culture medium, the mass of the hemin chloride is 0.01‰ of the mass of the brain heart infusion liquid culture medium, and the mass of the vitamin K1 is 0.01‰ of the mass of the brain heart infusion liquid culture medium.

7. The use according to claim 4, characterized in that: The composition of the anaerobic gas environment is 5% CO2, 5% H2 and 90% N2.

8. The use according to claim 4, characterized in that: The detection is to use high performance liquid phase to analyze the components of saponin in the bacterial liquid after cultivation.

9. The use according to claim 4, characterized in that: The cultured bacterial liquid is taken out once every 24 hours and then tested.

10. The use according to claim 4, characterized in that: During the inoculation, the volume ratio of the bacterial liquid to the BHI culture medium containing 1.5‰ notoginseng total saponins and 1.5‰ ginsenoside Rb1 was 1:10.

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