CN1944626A - Microbial bacteria for preparing ginkalide by microbial fermenting and method for preparing ginkalide - Google Patents
Microbial bacteria for preparing ginkalide by microbial fermenting and method for preparing ginkalide Download PDFInfo
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Abstract
The present invention is method of separating and screening fungus from ginkgo bark and fermenting to produce ginkalide. The collected fungus is one kind of fusarium in the preservation number of CGMCC No. 1677. The ginkalide producing process includes the following steps: 1. preparing culture medium comprising carbon source matters glucose, starch, dextrin, etc; nitrogen source matters peptone, peanut cake portions, fish meal, etc; and inorganic salt; 2. activating the fungus seed, first shake flask fermentation to preparing seed, and second shake flask fermentation to obtain the fermented liquid; and 3. post-treating the fermented liquid and extracting and purifying to prepare ginkalide. The present invention provides one new mode of producing ginkalide, and the mode has great product and stable product quality.
Description
Technical field:
The present invention relates to biological pharmacy technical field, is that the method-microbial fermentation of the precious traditional Chinese medicine ingredients-bilobalide of a kind of microbial fermentation preparation prepares the microbial bacteria (Fusarium oxysporum) of bilobalide and prepares the bilobalide method specifically.
Background technology:
Ginkgo (Ginkgo biloba L.) has another name called Gong Sunshu, is one of ancient seeds, is that a section one belongs to a kind of special plant.The geology transition that ginkgo can experience more than 200 ten thousand years still can the few survivors be got off with time brings great changes to the world, and the place of its uniqueness must be arranged.So the plant scholar has done number of research projects to the chemical ingredients and the pharmacological action of ginkgo tissue both at home and abroad to the sixties in last century, now the composition of clear and definite structure is nearly about 70 kinds.Table 1 has been listed partly bioactive material in the ginkgo.(Ginkgo Biloba extract GBE) is extensively applied to medicine, food supplement, makeup etc. to Folium Ginkgo extract at present.Though ginkgo begins plantation in Europe, North America now, China is still the main place of production.2004, China provided 2.4 ten thousand tons of Ginkgo Leaves to the world, general 600,000,000 yuans of sales volume, and the total sales volume of whole world ginkgo agent reaches 5,000,000,000 dollars, and the 98%th, external company produces.
Bioactive main component is arranged in table 1 ginkgo
| Kind | Title | Molecular formula | Tissue-derived | Biological activity |
| Glycosides million | Kaempferol | C15H10O 6 | Ginkgo Leaf | The expansion coronary vasodilator, the activity of inhibition Zinc metallopeptidase Zace1 (ACEZ). |
| Kaempferol-3-O-glycoside | C21H20O 11 | |||
| Kaempferol-3-O-rutinoside | C27H30O 15 | |||
| And flavonoid glycoside | Kaempferol-7-O-glycoside | C21H20O 11 | ||
| Kaempferol-3-glucose-2,6-two rhamnosides | C33H40O 19 | |||
| Quercetin | C15H10O 7 | |||
| Quercetin-3-O-glycoside | C21H20O 12 | |||
| Quercetin-3-O-rhamnoside | C21H20O 11 |
| Quercetin-3-O-rutinoside | C27H30O 16 | |||
| Quercetin-3-glucose-2,6-two rhamnosides | C33H40O 20 | |||
| Isorhamnetol | C16H12O 7 | |||
| Isorhamnetol-3-O-glycoside | C22H22O 12 | |||
| Isorhamnetol-3-O-rutinoside | C28H32O 16 | |||
| Biflavone | Ginkegetin | C32H22O 10 | The Ginkgo Leaf gingko episperm | The cytotoxicity mediation suppresses the NO Synthesis |
| Different ginkegetin | C32H22O 10 | |||
| The demethyl ginkegetin | C31H20O 10 | |||
| Tridemethylsciadopitysin | C30H18O 10 | |||
| The golden larch biflavone | C33H24O 10 | |||
| The 5-methoxyl group removes the first ginkegetin | C32H22O 11 | |||
| Folium Ginkgo terpene lactones | Bilobalide | C15H18O 8 | Ginkgo blade root skin tissue | To the effect of nervus centralis, the effect of anti-platelet activating factor (PAF) |
| Ginkgolide A | C20H24O 9 | |||
| Ginkgolide B | C20H24O 10 | |||
| Ginkalide C | C20H24O 11 | |||
| Bilobalide J | C20H24O 10 | |||
| Bilobalide M | C20H24O 10 | Exist only in the root skin | ||
| Organic acid | The 6-hydroxykynurenic acid | C10H7O4 N | Ginkgo Leaf | The N-methyl-D-aspartate receptor antagonist |
| The alkyl phenolic acid | 4-methoxyl group pyridoxic acid | C9H13O3 N | The ginkgo fruit | Cytotoxin |
| Bilobol | C21H34O | The ginkgo fruit | Antitumor action | |
| Bilobol | C21H34O 2 | The ginkgo fruit | ||
| Ginkgolic acid | C22H34O 3 | The ginkgo fruit | Antitumor action, bacteriostatic action | |
| Hydroginkgolic acid | C22H36O 3 | The ginkgo fruit | Anti-inflammatory, anti-allergic effects, bacteriostatic action | |
| Hydroginkgolinic acid | C21H34O 3 | The ginkgo fruit |
As can be seen from the above table, bilobalide-like is special efficacy platelet activation factor (PAF) antagonist.Platelet activation factor is a kind of Mammals endogenous physiologically active substance, main and allergy, ulcer, asthma, thrombus generate, the allosome of some inflammation, organ transplantation repels, the coronary disease patient is because the morbidity of the myocardial damage that anoxic causes etc. is closely related, therefore bilobalide also is mainly used in above-mentioned treatment of diseases, be mainly used in senile dementia, cardiovascular and cerebrovascular diseases clinically, the allosome repulsion of bronchial asthma and organ transplantation etc.
At present, the activeconstituents in the ginkgo tissue is mainly derived from ginkgo leaf, bark and fruit.Because ginkgo is subjected to the restriction of weather, region, harvest season, the more important thing is that big area felling ginkgo can destroy the eubiosis, be unfavorable for environment protection, therefore must develop other resources and substitute extraction ginkgo active ingredient medicine from ginkgo.
Summary of the invention:
The purpose of this invention is to provide a kind of can the fermentation and produce the microorganism strains (preservation date: on 04 17th, 2006 of bilobalide, depositary institution's title: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), depositary institution address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, deposit number: CGMCC No1677, bacterium classification name: Fusarium oxysporum).This bacterial classification is that the bacterial classification of separating from ginkgo intracutaneous side is Fusarium tricinctum (Fusarfum tricinctum) through the GB-1-3 that screening obtains, and belongs to and partly knows mattress subphylum (Deuteromycotina), hyphomycetes (Hyphomycetes), knurl seat spore order (Tuberculariales), Tuberculariaceae (Tuberculariaceae), Fusarium fungi.。
The method that the present invention also provides a kind of microbial fermentation to prepare bilobalide, comprise original strain activation, shake-flask seed cultivation, fermentation culture and after extract various steps.
Skimmed milk freeze-drying pipe bacterial classification of the present invention is meant a kind of preserving type of producing bacterial classification.It is the germfree defatted milk solution that adds required ratio in cultured slant tube, uses to connect collarium and scrape mycelium gently and break up as far as possible, makes it homodisperse and becomes suspension.With aseptic long dropper with the suspension branch aseptic clean ampoul tube bottom (0.9 milliliter/every) of packing into.In the dry ice alcohol of the ampoul tube that branch is installed between-25 to-40 ℃ after the pre-freeze 1 hour, ampoul tube is put into vacuum then, start vacuum pump and carry out drying, last tube sealing.
Strain inclined plane activation of the present invention is meant the freeze-drying pipe of opening preservation, is uniformly coated on the slant medium behind the picking bacterial classification, cultivates 7 days in 28 ℃.
Shake-flask seed of the present invention is cultivated and is meant the needed carbon source of employing strain growth, nitrogenous source, inorganic salt etc., after sterilization, take an amount of thalline inclined-plane, transferring to seed shakes in the bottle, automatically shaking cultivation on the rotation bottle swingging machine, 200rpm, cultivates and obtained shake-flask seed in 22-26 hour by 28 ℃.
Fermentation of the present invention is meant adopts the needed carbon source of strain growth, nitrogenous source, inorganic salt, defoamer etc., after sterilization, according to certain inoculum size ratio (10-15%), change shake-flask seed over to, automatically shaking cultivation, 220-240rpm, 28 ℃ on the rotation bottle swingging machine, cultivated 140-150 hour, and obtained fermentating metabolism product.
After fermented liquid post-treating method of the present invention refers to that fermented liquid is put bottle, under room temperature, normal pressure, use rotating speed to be centrifugal 30 minutes of the whizzer of 4000rpm, abandon fermented supernatant fluid, collect mycelium, dry to weight for 55 ℃ and reduce by half, with the aqueous suspension mycelium of 10 times of amounts, the sherwood oil that adds 2 times of water yields then, stir and divide bed thickness, collect water, repeat above-mentioned petroleum ether extraction operation once, purpose is to remove the less materials such as lipid acid of polarity, and internally the extraction of Ester influence is less.Add 2N CaOH2 in aqueous phase, regulate PH to 8.0, make free fatty acids salify precipitation (because the free-fat acid calcium salt is a lyophobic dust, the solubleness in water is very little), treat precipitation fully, centrifugal acquisition supernatant liquor 1 and precipitation 1.Precipitation 1 is used the water concussion washing of 2 times of amounts, centrifugal 20 minutes of 3000rpm, and repeat twice, and collect supernatant liquor 2, merge with supernatant liquor 1.The ethyl acetate that adds equal volume amounts then, after extracting 1 time, obtain ester phase 1 and water 1, wherein (experimental result of the present invention shows a kind of adding of water 2N HCl adjusting PH to 4.7, in the PH4.7-6.1 scope, help improving the yield of bilobalide), the ethyl acetate that adds 3 times of amounts is afterwards divided 3 fully extractions, the ester phase 2 of acquisition and ester 1 merging mutually.Volatilize the dry thing of ethyl acetate acquisition extract.Mix silicagel column on the sample, use chloroform-methanol to carry out gradient elution, collect each cut, obtain Ginkgolide A, B, bilobalide.
Advantage of the present invention is:
1, substitutes plant origin, the new resources of ginkgo activeconstituents are provided
The present invention prepares bilobalide for using microbial fermentation first, substituted the ginkgo of plant origin fully, therefore more environmental, do not destroy the eubiosis, and can control its output and quality during the fermentation, not being subjected to the influence of region and changes of seasons, is great advantage of the present invention.
2, do not contain objectionable impurities-ginkgol and ginkgoic acid in the ginkgo leaf extract
Ginkgol (Ginkgols) and ginkgoic acid can cause severe anaphylactic reaction.The reaction of this class belongs to the alkane phenol of urshiol type, is considered to the part in the intensive contactant of occurring in nature.Treat patient with Ginkgo Leaf, existing report numerical example generation cycle penalty comprises anaphylactic shock.Therefore in August, 1999, the medicine for animals council of medicine examination board of European Community issue Semen Ginkgo extrac final report (EMEA/MRL/668/99 final draft) has been made regulation (being lower than 5mg/kg) to ginkgoic acid wherein.EMEA this document emphasizes that this standard is used for the people equally, and this standard is also adopted by north america.Do not find ginkgoic acid in the meta-bolites of microbial fermentation preparation of the present invention.
3, production cost is low, and is pollution-free
Raw materials cost used in the present invention is low, and production technique advanced person is suitable for large-scale industrial production, and production unit can be general with other microniological proudcts.In addition, the discharging of unharmful substance in the production process of this product,, compliance with environmental protection requirements pollution-free, nuisanceless to surrounding environment.
Culture presevation among the present invention:
Preservation date: on 04 17th, 2006.
Depositary institution's title: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC).
Deposit number: CGMCC No1677.
Bacterium classification name: Fusarium oxysporum.
Description of drawings:
Fig. 1 is the production technique schema
Fig. 2 is a fermented liquid aftertreatment technology schema
Embodiment:
Embodiment 1: the microbial strains GB-1-3 feature that is used for the fermentative preparation bilobalide of the present invention.
On the PDA substratum, cultivated two days the cotton-shaped mycelia of growth white cotton, colony edge cilium shape; Cultivated the 3rd day, substrate mycelium is red; Aerial hyphae is a white, and grow prolifically is covered with full ware.Opticmicroscope is observed on the mycelia does not down have chlamydospore, and conidium is divided into two kinds of spores of size, and megaspore is a sickleshaped, and is colourless, 3～5 every, size is 25～48 * 3～4.5 μ m; The sporule oblong, colourless, 1～2 every, size is 12～15 * 2.4～3 μ m.Use the shake-flask seed substratum to obtain mycelium, extract its karyomit(e), use the ITS primer, be masterplate, carry out pcr amplification with the karyomit(e) of this thalline, and its PCR product reclaimed, clones and check order, obtaining sequence is compared in Genbank, analyzed.Simultaneously in conjunction with investigating cultural characteristic, mycelia morphology, determine that this bacterial strain is Fusarium tricinctum (Fusarfum tricinctum), belong to and partly know mattress subphylum (Deuteromycotina), hyphomycetes (Hyphomycetes), knurl seat spore order (Tuberculariales), Tuberculariaceae (Tuberculariaceae), Fusarium fungi.
Microbial fermentation of the present invention prepares the production technique of bilobalide
One, substratum
1, slant medium:
Glucose 10g/L, potato (peeling) 200g/L, agar 15g/L is with distilled water configuration, pH7.2-7.4,121 ℃ of 30min.
Potato is soaked the preparation of juice: get 200g peeling potato, be cut into small pieces, place 1000ml water to boil a hour after-filtration, with cold boiling water filtrate is supplied 1000ml.
2, shake-flask seed substratum
Glucose 20g/L, groundnut meal 3.0g/L, NH
4Cl 3.0g/L, KH
2PO
43.0g/L, MgSO
47H2O 3.0g/L, CaCO
34.0g/L,
NaAC3H
2O 1.0g/L, ordinary water configuration, PH6.0 before the sterilization.
Packing 40ml/250ml triangular flask, 121 ℃ of 30min.
3, fermentation tank culture medium
Glucose 10g/L, groundnut meal 10g/L, soybean cake powder 10g/L,
W-Gum 40g/L, NH4Cl 3g/L, KH
2PO4 3g/L,
NaAC·3H
2O 3g/L， CaCO
3 4g/L， MgSO
4·7H
2O 3g/L。
The ordinary water configuration, PH6.0 before the sterilization, packing 50ml/250ml triangular flask, 121 ℃ of 30min.
Groundnut meal, soybean cake powder, W-Gum require fresh not mouldy and mistake 160 mesh sieves in more than forming.
Two, fermentative production
1, the slant activation of bacterial classification: open bacterial classification skimmed milk freeze-drying pipe, the picking small quantities of powder is uniformly coated on the inclined-plane of the above-mentioned bacterium of going out, cultivated 5-7 days under 28 ℃ of temperature, treat that lawn grows into white aerial mycelium densification, is covered with the inclined-plane, does not have assorted bacterium, refrigerator 4 is preserved.
2, shake-flask seed is cultivated: will activate good slant strains and dig piece 1*1cm2 and be inoculated in each seed culture bottle, isothermal vibration is cultivated under 28 ℃ of temperature, 200rpm, and 22-26 hour, that the microscopy mycelial growth is unfolded, dye was dark, nothing is mixed, and bacterium gets final product.
3, the fermentation: fermentative medium formula as above, after the sterilization, with cultured seed, the transferred species amount according to 10% changes in the fermentation flask, 180-240rpm, 25-28 ℃, incubation time is 140-150 hour.
4, stop fermentation after, under room temperature, normal pressure, use rotating speed to be centrifugal 30 minutes of the whizzer of 4000rpm fermented liquid, abandon fermented supernatant fluid, collect mycelium, dry to weight for 55 ℃ and reduce by half, with the aqueous suspension mycelium of 10 times of amounts, the sherwood oil that adds 2 times of water yields then, stir and divide bed thickness, collect water, repeat above-mentioned petroleum ether extraction operation once, purpose is to remove the less materials such as lipid acid of polarity, and internally the extraction of Ester influence is less.Add 2N CaOH in aqueous phase
2, regulate PH to 8.0, make free fatty acids salify precipitation (because the free-fat acid calcium salt is a lyophobic dust, the solubleness in water is very little), treat precipitation fully, centrifugal acquisition supernatant liquor 1 and precipitation 1.Precipitation 1 is used the water concussion washing of 2 times of amounts, centrifugal 20 minutes of 3000rpm, and repeat twice, and collect supernatant liquor 2, merge with supernatant liquor 1.The ethyl acetate that adds equal volume amounts then, after extracting 1 time, obtain ester phase 1 and water 1, wherein (experimental result of the present invention shows a kind of adding of water 2N HCl adjusting PH to 4.7, in the PH4.7-6.1 scope, help improving the yield of bilobalide), the ethyl acetate that adds 3 times of amounts is afterwards divided 3 fully extractions, the ester phase 2 of acquisition and ester 1 merging mutually.Volatilize the dry thing of ethyl acetate acquisition extract.Mix silicagel column on the sample, use chloroform-methanol to carry out gradient elution, collect each cut, obtain Ginkgolide A, B, bilobalide.
The detection that embodiment 2 microbial fermentations of the present invention prepare bilobalide
With the centrifugal acquisition mycelium of the fermented liquid of bacterial strain GD3-1, after embodiment 1 described extracting process processing, the crude extract dry sample use dissolve with methanol with ethyl acetate is volatilized mutually and afterwards obtained is configured to 7.6mg/ml solution; With after Ginkgolide A, B, C and the bilobalide dissolving, be configured to contain above-mentioned various standard substance concentration the solution that is 0.2mg/ml respectively.With above-mentioned standard solution is contrast, and the method that adopts external standard method and interior addition to combine is analyzed sample.。The retention time of standard substance and each close peak position of sample and peak area relatively see Table 2.The chromatographic peak that overlaps with standard substance GA, GB, GC and BB is arranged in the sample as can be seen from Table 2 really.
The chromatographic data of the material close statistics in table 2 sample with the standard substance retention time
| Standard substance (0.2mg/ml) | GA | GB | GC | BB | |
| Retention time t`R (min) | 16.375 | 18.492 | 8.748 | 7.937 | |
| Peak area Ar | 50708 | 45875 | 58342 | 34699 | |
| Sample (the 7.6mg crude extract/ml) | GA｀ | GB｀ | GC｀ | BB｀ | |
| Retention time tR (min) | 16.668 | 18.15 | 8.542 | 8.005 | |
| Peak area Ar | 3426 | 30169 | 11906 | 66403 | |
| Sample (the 7.6mg crude extract/ml)+standard substance (0.2mg/ml) | |||||
| Retention time tR (min) | 16.59 | 18.775 | 8.757 | 8.173 | |
| Peak area Ar | 53995 | 86291 | 65164 | 96603 |
The one-level mass spectrum of bilobalide composition is identified in embodiment 3 microbial fermentation products of the present invention
With GD 3-1 is the starting strain shake-flask culture, mycelium is handled the ethyl acetate extract that the back obtains according to extraction process, compare with standard substance (Ginkgolide A, B, C and bilobalide), carrying out HPLC under the same conditions analyzes, gather and the approaching chromatographic peak of standard substance retention time, compare with standard substance again after concentrating, carry out mass spectroscopy.The parsing that relatively reaches of standard substance BB, GA and GB mass spectrometry results sees Table 4, BB standard substance and sample mass spectrometry results relatively see Table 5, GA standard substance and sample mass spectrometry results relatively see Table 6, GB standard substance and sample mass spectrometry results relatively see Table 7.
Table 4 standard substance mass spectrometry results relatively reach parsing
| The collection of illustrative plates attribute | BB | GA | GB | Resolve |
| The standard substance molecular weight | 326 | 408 | 424 | M |
| Positive ion is composed (m/z) entirely + | 426.0 833.4 838.7 855.5 | 442.0 463.0 865.7 886.8 | M+NH 4 + M+K + 2M+NH 4 + 2M+Na + 2M+K + |
| Positive ion secondary spectrum (m/z) + | 440.5 425.7 | 456.6 442.5 378.6 | M+CH 3OH+H + M+NH 4 + M-CO 2+H + | |
| Negative ion is composed (m/z) entirely - | 325.0 370.6 650.9 | 406.9 452.8 814.9 | 422.9 458.9 468.6 847.0 | M-1 M+Cl - M+HCOO - 2M-1 |
| Negative ion secondary spectrum (m/z) - | 325.0 306.5 250.9 206.8 163.3 | 406.8 378.6 351.0 | 423.0 394.7 367.0 | M-1 [M-H 3O +] [M-CO-H +] - [M-2CO-H +] - 250.9-CO 2 206.8-CO 2 |
The result of table 5 bilobalide and sample thereof relatively
| The collection of illustrative plates attribute | The BB standard substance | BB ｀ sample | Resolve |
| Negative ion is composed entirely | 325.0 650.9 | 325.0 | M-1 2M-1 |
| 325 negative ion secondarys spectrum | 325.8 325.0 306.5 250.9 206.8 163.3 | 326.3 325.2 306.1 250.9 206.9 163.2 | M-1 [M-H 3O +] - [M-CO-H +] - 250.9-CO 2 206.9-CO 2 |
The result of table 6 GA standard substance and sample thereof relatively
| The collection of illustrative plates attribute | The GA standard substance | GA ｀ sample | Resolve |
| Positive ion is composed entirely | 426.0 833.4 839.7 | 425.9 | M+NH 4 + 2M+NH 4 + 2M+Na + |
| 426 positive ion secondarys spectrum | 441.7 440.5 426.5 425.7 | 441.8 440.6 426.4 425.0 | M+CH 3OH+H + M+NH 4 + |
| Negative ion is composed entirely | 406.9 452.8 811.0 | 452.9 810.9 | M-1 M+HCOO - 2M-1 |
| 453 negative ion secondarys spectrum | 452.5 406.8 378.6 351.0 | 453.1 406.9 378.6 350.9 | M+HCOO - M-1 M-CO-H + M-2CO-H + |
The result of table 7 GB standard substance and sample thereof relatively
| The collection of illustrative plates attribute | The GB standard substance | GB ｀ sample | Resolve |
| Positive ion is composed entirely | 442.0 865.7 886.7 | 442.0 463.4 | M+NH 4 + M+K + 2M+NH 4 + 2M+K + |
| 442.0 positive ion secondary spectrum | 457.7 456.6 442.5 378.6 | 457.6 456.6 442.2 378.7 | M+CH 3OH+H + M+NH 4 + M-CO 2+H + |
| Negative ion is composed entirely | 422.9 468.6 847.0 | 423.0 458.9 | M-1 M+Cl - M+HCOO - 2M-1 |
| 423.0 negative ion secondary spectrum | 424.0 423.0 394.7 367.0 | 424.4 423.0 394.8 366.9 | M-1 M-CO-H + M-2CO-H + |
Above data show, exist in the broth extraction sample with standard substance BB, GA, GB retention time approaching, the material that quasi-molecular ion is identical.
High performance liquid phase/electrospray ionization mass spectrum (HPLC/ESI-MS) Analysis and Identification of Ginkgolide B composition in embodiment 4 microbial fermentation products of the present invention
Through silica gel column chromatography, the chloroform-methanol gradient elution is found to contain and the consistent material of Ginkgolide B (GB) retention time in 96: 4 the effluent liquid of chloroform-methanol with the mycelium acetic acid ethyl ester extract after the GD3-1 fermentation; Hold back this section cut, obtain the sample (GB ｀) of about 50 μ g, use the 4mL dissolve with methanol, compare with the GB standard substance, under identical chromatogram and mass spectrum condition, carry out high performance liquid phase/electrospray ionization mass spectrum (HPLC/ESI-MS) analysis, find both unanimities, further confirmed the existence of Ginkgolide B.
Claims (5)
1, microbial fermentation prepares the microbial bacteria of bilobalide, it is characterized in that: employing be Fusarfum tricinctum, preserving number CGMCC No1677, bacterium classification name: Fusariumoxysporum.
2, a kind of method of cultivation of bacterial classification Fusarfum tricinctum as claimed in claim 1 is characterized in that: the preparation of shake-flask seed: substratum Ph6.0 before the sterilization, dig piece inoculation 1*1cm
2Production bacterial strain inclined-plane lawn, revolution 200rpm, temperature is 25-28 ℃, incubation time is 30-36 hour.
3, the method for cultivation of Fusarfum tricinctum according to claim 2, it is characterized in that: shake flask fermentation technology is: substratum Ph6.0 before the sterilization, according to the inoculum size inoculation of 10-15%, stir revolution 200-240rpm, temperature is 25-28 ℃, and incubation time is 140-148 hour.
4, a kind of method of utilizing microbial fermentation to prepare bilobalide, it is characterized in that: employed production bacterial classification GB-1-3 is a Fusarfum tricinctum, belong to and partly know mattress subphylum, hyphomycetes, knurl seat spore order, Tuberculariaceae, Fusarium fungi, its technology is as follows: carbon source and nitrogenous source are adopted in the nutrition in (1) substratum seed and the fermention medium, wherein carbon source can be glucose, starch, dextrin, maltose, nitrogenous source can be peptone, groundnut meal, soybean cake powder, extractum carnis, fish meal, need to add inorganic salt in addition, NH
4Cl, KH
2PO
4, MgSO
4.7H
2O, CH
3COONa, CaCO
3(2) zymotechnique uses this production bacterial strain, can obtain its meta-bolites---bilobalide by shaking the method for bottle second order fermentation, cultural method comprises: the aftertreatment of the activation of original strain, the preparation of shake-flask seed, shake flask fermentation, fermented liquid, extraction purifying prepare bilobalide.
5, require 4 described a kind of methods of utilizing microbial fermentation to prepare bilobalide according to profit, it is characterized in that: its fermented liquid aftertreatment technology is as follows:
The thalline crude extract adds the sherwood oil of 2 times of amounts with the aqueous suspension of 10 times of amounts, gained feed liquid, extracts 2 times, isolates ether phase and water; Add 2N CaOH in aqueous phase
2, regulate PH to 8.0, make free fatty acids salify precipitation, treat precipitation fully, the centrifugal 20min of 3000rpm obtains supernatant liquor 1 and precipitation 1; Precipitation 1 is used the water concussion washing of 2 times of amounts, centrifugal 20 minutes of 3000rpm, and repeat twice, and obtain precipitation and supernatant liquor 2, collect supernatant liquor 2 and merge with supernatant liquor 1, the ethyl acetate that adds equal volume amounts then, after extracting 1 time, obtain ester phase 1 and water 1, wherein add 2N HCl in the water 1 and regulate PH to 4.7, the ethyl acetate that adds 3 times of amounts is afterwards divided 3 fully extractions, the ester phase 2 of acquisition and ester 1 merging mutually; Volatilize the dry thing of ethyl acetate acquisition extract, mix silicagel column on the sample, use chloroform-methanol to carry out gradient elution, collect each cut, obtain Ginkgolide A, B, bilobalide.
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| CN103451250A (en) * | 2013-09-02 | 2013-12-18 | 张志才 | Application of tremella fuciformis thalli in preparation of ginkgolide B |
| CN103451251A (en) * | 2013-09-02 | 2013-12-18 | 张志才 | Method for preparing ginkgolide B by using stereum hirsutum thalli as catalyst |
| CN103468760A (en) * | 2013-09-02 | 2013-12-25 | 张志才 | Method for preparing bilobalide B by using Stereum hirsutum thalli as catalyst |
| CN108684713A (en) * | 2018-05-12 | 2018-10-23 | 湖南科技学院 | The growth-promoting preparation of lactone B content in a kind of promotion ginkgo leaf |
| CN112646732A (en) * | 2020-12-24 | 2021-04-13 | 湖南科技学院 | Growth promoting preparation capable of simultaneously increasing content of lactone A, C and bilobalide in ginkgo leaf and preparation method and application thereof |
| 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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2006
- 2006-07-26 CN CN 200610103651 patent/CN1944626A/en active Pending
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103450217A (en) * | 2013-09-02 | 2013-12-18 | 张志才 | Application of stereum hirsutum thalli in preparation of ginkgolide B |
| CN103451250A (en) * | 2013-09-02 | 2013-12-18 | 张志才 | Application of tremella fuciformis thalli in preparation of ginkgolide B |
| CN103451251A (en) * | 2013-09-02 | 2013-12-18 | 张志才 | Method for preparing ginkgolide B by using stereum hirsutum thalli as catalyst |
| CN103468760A (en) * | 2013-09-02 | 2013-12-25 | 张志才 | Method for preparing bilobalide B by using Stereum hirsutum thalli as catalyst |
| CN103451250B (en) * | 2013-09-02 | 2015-08-26 | 张志才 | White fungus thalline is preparing the application in Ginkgolide B |
| CN103450217B (en) * | 2013-09-02 | 2015-12-02 | 北京绿科天成生物科技有限公司 | Thick hair Boreostereum vibrans thalline is preparing the application in Ginkgolide B |
| CN103451251B (en) * | 2013-09-02 | 2016-06-01 | 北京绿科天成生物科技有限公司 | Utilize thick hair Boreostereum vibrans thalline as the method for catalyst preparing Ginkgolide B |
| CN103468760B (en) * | 2013-09-02 | 2016-07-06 | 北京绿科天成生物科技有限公司 | Utilize Tremella thalline as the method for catalyst preparing ginkalide B |
| CN108684713A (en) * | 2018-05-12 | 2018-10-23 | 湖南科技学院 | The growth-promoting preparation of lactone B content in a kind of promotion ginkgo leaf |
| CN108684713B (en) * | 2018-05-12 | 2020-09-01 | 湖南科技学院 | Growth-promoting preparation for increasing content of lactone B in ginkgo leaves |
| CN112646732A (en) * | 2020-12-24 | 2021-04-13 | 湖南科技学院 | Growth promoting preparation capable of simultaneously increasing content of lactone A, C and bilobalide in ginkgo leaf and preparation method and application thereof |
| 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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