CN116286389A - Aspergillus niger and method for removing ginkgolic acid in ginkgo leaves by fermentation method thereof - Google Patents
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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- C12R2001/66—Aspergillus
- C12R2001/685—Aspergillus niger
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Abstract
The invention discloses an aspergillus niger and a method for removing ginkgolic acid in ginkgo leaves by a fermentation method thereof. Aspergillus niger Aspergillus nigerOP811219 strain with the preservation number of CGMCC No.40430. 5g of ginkgo leaf powder, 0.1g of glucose, (NH) 4 ) 2 SO 4 0.15g，MgSO 4 ·7H 2 O0.025g，KH 2 PO 4 0.1g, and adding 3% of Aspergillus niger for fermenting for 3 d. The Aspergillus niger fermentation method removes ginkgolic acid from the production source, and achieves the purpose of eliminating ginkgolic acid on the production line.
Description
Technical Field
The invention relates to aspergillus niger and a method for extracting ginkgolic acid thereof, in particular to a method for removing ginkgolic acid in ginkgo leaves by using aspergillus niger and a fermentation method thereof.
Background
Ginkgo biloba (Ginkgo biloba l.) is a deciduous tree of the genus Ginkgo of the family ginkgaceae, ginkgo biloba is a dry leaf of Ginkgo tree, ginkgo biloba is rich in more than 200 active substances including flavonoids, alkaloids, lactones, organic acids, polysaccharides, catechins and the like, and Ginkgo biloba plays an important role in the treatment of cerebral ischemic stroke, alzheimer's disease and other neurodegenerative diseases. The active ingredients in folium Ginkgo also have effects of resisting hyperuricemia, regulating immunity, resisting bacteria and viruses, treating vitiligo, preventing obesity, resisting ultraviolet injury, and improving tinnitus. The ginkgo leaf extract is prepared by processing ginkgo leaves, has the functions of activating blood circulation to dissipate blood stasis and dredging collaterals, and is used for treating chest stuffiness and pains, stroke, hemiplegia and tongue strength and Chinese tamponade caused by blood stasis and obstruction of collaterals; coronary heart disease stable angina pectoris and cerebral infarction with the above symptoms.
The ginkgo leaf extract contains a certain amount of harmful substances of ginkgolic acid. Ginkgolic acid (GAs for short) is another component of ginkgo with important physiological activity except ginkgo flavone and ginkgolide, and exists in leaves, fruits and exodermis of ginkgo, wherein the content of the exodermis is highest. Ginkgolic acid is a long-chain benzoic acid compound with the same pharmacophore, the length of a side chain is 13-19 carbons, the number of double bonds is 0-3, and the structure of 5 common ginkgolic acid compounds is shown in figure 1. The ginkgolic acid has cytotoxicity, embryotoxicity, sensitization and mutation effects, and can greatly reduce the survival rate of rat liver cells after the concentration of ginkgolic acid reaches a certain level. Therefore, the European and American pharmacopoeia standard is that the content of ginkgolic acid in the ginkgo leaf extract is less than 5ppm, and the Chinese pharmacopoeia standard is that the content of ginkgolic acid in the ginkgo leaf extract is less than 10ppm.
Conventional detoxification methods include physical methods and chemical methods. Cooking, ultrasound and microwaves are the most common physical methods for attenuation of ginkgolic acid in ginkgo leaves. The digestion method has lower attenuation efficiency, and the ginkgolic acid in the ginkgo leaves is not thoroughly removed, so that more residues are left. The disadvantage of removing ginkgolic acid by chemical method is: some drugs may have a greater impact on the active ingredients in the sample; incomplete removal of ginkgolic acid and certain residue; the environment is polluted to a certain extent; the experimental operation cost is higher, and mass production is not facilitated. The solid state fermentation method of the microorganism adopted in the experiment. The microbial solid state fermentation has the following advantages: the equipment is simple and easy to operate; fermentation or optimization conditions are convenient to control; the nutrient components produced by the fermented product are rarely lost, and can be fully utilized.
Disclosure of Invention
The invention aims to: the invention aims to provide a method for removing ginkgolic acid in ginkgo leaves by an Aspergillus niger fermentation method.
The technical scheme is as follows: a strain of Aspergillus niger Aspergillus niger OP811219 with a preservation number of CGMCC No.40430.
The 18S rDNA sequence of Aspergillus niger OP811219 strain is shown in SEQ ID NO. 1.
A method for removing ginkgolic acid in ginkgo leaves by an Aspergillus niger fermentation method comprises the following steps:
(1) Performing expansion culture on Aspergillus niger strains;
(2) Inoculating the seed liquid into fermentation culture medium with folium Ginkgo as substrate, without changing the properties of intermediate materials, and removing ginkgolic acid.
The method for removing ginkgolic acid in ginkgo leaves by using the Aspergillus niger fermentation method comprises the following steps of: 2g of tryptone, 1g of yeast extract and 2g of glucose, dissolving in distilled water, fixing the volume, adding glass beads, and sterilizing by steam; aspergillus niger is inoculated in YPD liquid culture medium and cultured in shaking mode.
The inoculum size of the aspergillus niger is 0% -12%.
The liquid-solid ratio of the fermentation is 0.9-1.8.
The beneficial effects are that: the Aspergillus niger fermentation method of the invention removes ginkgolic acid from the production source, thereby achieving the purpose of removing ginkgolic acid on the production line. The removal rate of ginkgolic acid can reach 90.21% by fermenting with Aspergillus niger for 3d, as shown in figure 5 (a); the inoculum size of Aspergillus niger is 4%, and the removal rate of ginkgolic acid can reach 86.93%, as shown in figure 5 (b); the solid-solid ratio of the fermentation liquid is 1.5, and the removal rate of ginkgolic acid can reach 90.23%.
Drawings
FIG. 1 is a diagram of the structure of ginkgolic acid;
FIG. 2 is a schematic representation of a phylogenetic tree constructed from Aspergillus niger OP811219 and other fungi;
FIG. 3 is a liquid chromatogram of a ginkgolic acid standard;
fig. 4 is a graph showing the front-to-back change of ginkgolic acid in Aspergillus niger OP811219 fermented ginkgo leaf powder: (A) Before fermentation ginkgolic acid content, and (B) ginkgolic acid content after fermentation.
FIG. 5 optimization of conditions for Aspergillus niger Aspergillus niger OP811219 fermented ginkgo leaf: the method comprises the steps of (A) changing the content of ginkgolic acid in different fermentation times, (B) changing the content of ginkgolic acid after fermentation in different inoculum sizes, and (C) changing the content of ginkgolic acid after fermentation in different liquid-solid ratios.
Detailed Description
Example 1
Collecting folium Ginkgo from campus of Huaiyin institute of technology, oven drying at 60deg.C, pulverizing, sieving with 60 mesh sieve, weighing 1g folium Ginkgo powder, spraying sterile distilled water, and standing at 37deg.C for 2d. Taking appropriate amount of solid, culturing in YPD liquid culture medium at 37deg.C and 200rpm for 2d, sequentially and gradually diluting 10 -1 -10 -6 YpD the bacterial solutions with different dilution factors are respectively sucked into YPD solid culture dishes by a sterile micropipette, uniformly coated, sealed and inverted, and cultured in a constant temperature incubator at 37 ℃ for 2d. The isolated microorganisms were streaked on multiple plates and single colonies were picked up for expansion culture in YPD liquid medium, 60% glycerol was stored in-80℃refrigerator. The strains screened are respectively inoculated into fermentation culture medium taking ginkgo leaf as substrate after being activated, and are cultivated for 6d at 37 ℃. Finding out strain 37 ℃ strip for efficiently removing ginkgolic acidThe strain is activated at 200rpm to obtain bacterial liquid, and 18S rDNA sequence determination (sequencing by Shanghai Jie Li You Co., ltd.) is carried out, and the determined sequence result shows that the 18S rDNA fragment of the Aspergillus niger OP811219 strain is 604bp long, and the sequence is shown as SEQ ID NO. 1. The resulting sequences were BLAST aligned through the GenBank database of NCBI and phylogenetic tree was constructed (fig. 2). And determining that the strain is Aspergillus niger by combining the traditional physiological and biochemical characteristic identification and the result of 18S rDNA sequence analysis.
Example 2
HPLC detection of ginkgolic acid:
in the embodiment, the content of ginkgolic acid is measured by HPLC, and the content of ginkgolic acid is measured by HPLC analysis conditions are as follows:
the chromatographic conditions were as follows: agilent Eclipse XDB-C18 (4.6X250 mm,5 μm) column, detection wavelength: 310nm, mobile phase: acetonitrile-1% trichloroacetic acid (75:25), flow rate: 1.0mL/min, column temperature: 40 ℃, sample injection amount: 20. Mu.L.
Mobile phase condition ratio
Mobile phase conditional scale
The liquid phase diagram of the standard under this condition is shown in fig. 2. Ginkgolic acid: the peak time of the ginkgolic acid (C13:0) is 22.224min, the peak time of the ginkgolic acid (C15:1) is 23.173min, the peak time of the hydrogenated ginkgolic acid (C15:0) is 24.657min, the peak time of the heptadecadiene ginkgolic acid (C17:2) is 29.158min, and the peak time of the heptadecene ginkgolic acid (C17:1) is 29.675min. As can be seen from FIG. 2, the five ginkgolic acids have good peak patterns and are completely separated, and can be used for detection of the invention.
Example 3
Weighing 5g of ginkgo leaf powder in each fermentation bottle, sealing, and sterilizing at 115 ℃ for 20min by high-pressure steam. The single factor experiment is adopted, the ginkgolic acid content in the fermented product is taken as a main investigation index, and the fermentation time (0 d, 1d, 2d, 3d, 4d, 5d and 6 d), the inoculum size (0%, 2%, 4%, 6%, 8%, 10% and 12%) and the liquid-solid ratio (0.9, 1.2, 1.5 and 1.8) of fermentation are used for researching the influence on the fermentation effect. Single factor experimental fermentation results: the removal rate of ginkgolic acid can reach 90.21% by fermenting with Aspergillus niger for 3d, as shown in figure 5 (a); the inoculum size of Aspergillus niger is 4%, and the removal rate of ginkgolic acid can reach 86.93%, as shown in figure 5 (b); the solid-solid ratio of the fermentation liquid is 1.5, and the removal rate of ginkgolic acid can reach 90.23 percent, as shown in figure 5 (c).
Claims (6)
1. A strain of Aspergillus niger Aspergillus niger OP811219 with a preservation number of CGMCC No.40430.
2. The 18S rDNA sequence of Aspergillus niger OP811219 strain is shown in SEQ ID NO. 1.
3. A method for removing ginkgolic acid in ginkgo leaves by an Aspergillus niger fermentation method is characterized in that: the method comprises the following steps:
(1) Performing expansion culture on Aspergillus niger strains;
(2) Inoculating the seed liquid into fermentation culture medium with folium Ginkgo as substrate, without changing the properties of intermediate materials, and removing ginkgolic acid.
4. The method for removing ginkgolic acid from ginkgo leaves by aspergillus niger fermentation method according to claim 1, which is characterized in that: the method for expanding culture comprises the following steps: 2g of tryptone, 1g of yeast extract and 2g of glucose, dissolving in distilled water, fixing the volume, adding glass beads, and sterilizing by steam; aspergillus niger is inoculated in YPD liquid culture medium and cultured in shaking mode.
5. The method for removing ginkgolic acid from ginkgo leaves by using an Aspergillus niger fermentation method of claim 4, which is characterized in that: the inoculum size of the aspergillus niger is 0% -12%.
6. The method for removing ginkgolic acid from ginkgo leaves by using an Aspergillus niger fermentation method of claim 4, which is characterized in that: the liquid-solid ratio of the fermentation is 0.9-1.8.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117814265A (en) * | 2024-03-01 | 2024-04-05 | 临沂大学 | Method for fermenting ginkgo exocarp by microorganisms and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117814265A (en) * | 2024-03-01 | 2024-04-05 | 临沂大学 | Method for fermenting ginkgo exocarp by microorganisms and application thereof |
| CN117814265B (en) * | 2024-03-01 | 2024-05-17 | 临沂大学 | Method for fermenting ginkgo exocarp by microorganisms and application thereof |
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