CN105907810A

CN105907810A - Method for simultaneously preparing ginkgolide B and quercetin from stereum hirsutum

Info

Publication number: CN105907810A
Application number: CN201610391342.XA
Authority: CN
Inventors: 张志才; 李金花; 樊亚娟; 胡坤雅
Original assignee: Jiangsu University
Current assignee: Jiangsu University
Priority date: 2016-06-06
Filing date: 2016-06-06
Publication date: 2016-08-31

Abstract

The invention discloses a method for simultaneously preparing ginkgolide B and quercetin from stereum hirsutum and relates to the technical field of biological engineering. The preparation method includes the steps that fermentation liquor containing ginkgolide B and a mycelium containing quercetin are prepared sequentially through the steps of tube enlarged culture, liquid shake culture, seed tank enlarged culture, solid primary fermentation culture, liquid post-fermentation culture and extraction and separation; ginkgolide B and quercetin crystal are prepared through extraction. Peeled ginkgoes and rice serve as a solid matrix, gingko leaf extracts are added, stereum hirsutum serves as a strain to convert ginkgolide and ginkgetin through solid primary fermentation, then quercetin and ginkgolide B are separated through post-fermentation, large-scale industrial production can be achieved, and ginkgolide B and quercetin in gingkoes can be produced at the same time. The process is simple, the purity of ginkgolide B is 90% or above and the purity of quercetin is 95% or above in the obtained product, and preparation purity is high.

Description

A kind of method utilizing the tough lead fungi of rough bark simultaneously to prepare ginkalide B and Quercetin

Technical field

The present invention relates to technical field of bioengineering, be specifically related to one utilize the tough lead fungi of rough bark prepare simultaneously ginkalide B and The method of Quercetin.

Background technology

At present, research proves that bilobalide is one of active component main in Folium Ginkgo, is the special effective blood of class Platelet activation factor (Platelet activating factor) receptor antagonist.Platelet activating factor (PAF) is by blood A kind of endogenous phosphide that platelet and inflammation tissue secretion produce, is the maximally effective platelet aggregation induction having now been found that Agent, closely related with the Emergence and Development of numerous disease.Bilobalide, as paf receptor specific antagonists, has medicine widely Reason effect.Research find ginkalide B to central nervous system and the protected effect of ischemic injuries, shock, antiallergic, antibacterial Rejection in antiinflammatory action, and anti-organ transplantation.The most also find that ginkalide B can reduce hepatic portal vein pressure, improves System vascular toleration, illustrates that it has certain curative effect to liver cirrhosis.Bilobalide has therapeutical effect for injury of kidney.These are made With all relevant as paf receptor antagonists with ginkalide B, and it be presently believed to be effect the strongest, most have clinical should With native platelets activation factor (PAF) receptor antagonist of prospect.

Quercetin and derivant thereof are that plant kingdom is widely distributed, have the flavone compound of various biological activity. In recent years, domestic and international medical personal is increasing to the research of Quercetin, finds that it has many pharmacologically actives, such as antioxidation And scavenging activated oxygen effect, anti-fibrosis effect, antitumor action, can reduce blood pressure, Ischemic myocardium reperfusion injury, Antiviral, analgesia and antidiarrheal etc., and Quercetin is to human non-toxic, harmless, without carcinogenic, without lethal, without the advantages such as teratogenesis, state Once tried out in the treatment of multiple disease outward, achieved good effect.Quercetin is present in the many flower of plant, leaf, fruit, Many presented in glycoside, such as rutin, quercitrin, hyperin etc., these glycosides generally prepare Quercetin through acid hydrolysis.Few use Biological method prepares Quercetin.

Scholar is had to propose herb fermenting pharmaceutical technology in recent years.So-called herb fermenting pharmaceutical technology is to inherit Chinese medicine Concoct on the basis of learning fermentation method, drawn Microecology achievement, formed in conjunction with the fermentation technique of modern biological project High-tech herbal pharmaceutical new technique, be in terms of Chinese medicine (natural drug) pharmacy find medicine new curative effect.Traditional Chinese medicine Mostly fermentation is to carry out under conditions of natural, and present herb fermenting pharmaceutical technology is to fully absorb Tiny ecosystem in modern age Learn, bionic achievement in research and gradually form.

Commonly used in prior art ginkalide B and Quercetin are utilized respectively simple process for separation and purification obtain Substantial amounts of ginkalide B and Quercetin, separation efficiency is low, it is impossible to is applicable to industrialization large-scale production, and individually purifies Separation method is more backward, and production cost is higher.

Inventor, through further investigation and industrialized production test repeatedly, finds out industrially scalable and produces in Semen Ginkgo simultaneously Ester B and Quercetin technology, no matter the present invention from the mode of production and the product of production of product, is different from conventional patent and literary composition The content of chapter report, a kind of method utilizing the tough lead fungi of rough bark simultaneously to produce ginkalide B and Quercetin.

Summary of the invention

, problem to be solved

For the above-mentioned problems in the prior art, the present invention provides one to utilize the tough lead fungi of rough bark to prepare bilobalide simultaneously B and the method for Quercetin, it adds Folium Ginkgo extract for solid matrix, with the tough lead fungi of rough bark as bacterium with peeling Semen Ginkgo and rice Plant by solid primary fermentation, convert bilobalide and ginkgetin, by after fermentation, make Quercetin and ginkalide B be divided From, industrialization large-scale production can be realized, it is possible to produce ginkalide B and Quercetin simultaneously；Technique is simple, and obtained In product, ginkalide B purity is more than 90%, and Quercetin purity is more than 95%, and the purity produced is higher.

, technical scheme

In order to solve the problems referred to above, the technical solution adopted in the present invention is as follows:

A kind of described method utilizing the tough lead fungi of rough bark simultaneously to prepare ginkalide B and Quercetin, described preparation method is with greatly Rice, peeling Semen Ginkgo and Folium Ginkgo extract are primary raw material, with the tough lead fungi of rough bark as starting strain, sequentially pass through test tube and expand training Support, liquid submerged culture and seed tank amplification culture, solid primary fermentation are cultivated, liquid post-fermentation and culture and the separating step system of extraction Obtain the fermentation liquid containing ginkalide B and the mycelium containing Quercetin；Described fermentation liquid containing ginkalide B and containing Mongolian oak The mycelium of Pi Su prepares ginkalide B and Quercetin crystal respectively through extracting again, and wherein ginkalide B purity reaches 90%, Quercetin purity reaches 95%.

A kind of method utilizing the tough lead fungi of rough bark simultaneously to prepare ginkalide B and Quercetin, is carried out as steps described below:

A1 test tube amplification culture: slant strains in tough for rough bark lead fungi (Stereum hirsutum) is inoculated in potato glucose Culture medium is cultivated, prepares rough bark tough lead fungi test tube slant strain；

A2 liquid submerged culture: tough for the rough bark obtained by step A1 lead fungi test tube slant strain is inoculated into and trains equipped with liquid shaking bottle Support in the shaking flask of base, cultivate, prepare rough bark tough lead fungi liquid shaking bottle strain；

A3 seed tank amplification culture: tough for the rough bark obtained by step A2 lead fungi liquid shaking bottle strain is inoculated into seed tank culture base In cultivate, make rough bark tough lead fungi seed tank strain；

A4 solid primary fermentation is cultivated: be inoculated in solid medium by tough for the rough bark obtained by step A3 lead fungi seed tank strain, And mix homogeneously, carry out fermentation culture, prepare rough bark tough lead fungi solid fermentation thing；

A5 liquid post-fermentation and culture: tough for the rough bark obtained by step A4 lead fungi solid primary fermentation thing is transferred to liquid fermentation tank training Support in base, carry out liquid after fermentation, prepare the fermentation liquid containing ginkalide B and the rough bark tough lead fungi mycelia containing Quercetin Body；

A6 extracts separation: by the tough lead fungi mycelium of the rough bark containing Quercetin obtained by step A5 with containing ginkalide B Fermentation liquid, respectively through extraction, obtains Quercetin and ginkalide B crystal.

Preferably, a kind of method utilizing the tough lead fungi of rough bark simultaneously to prepare ginkalide B and Quercetin, as steps described below Carry out:

B1 test tube amplification culture: slant strains in tough for rough bark lead fungi test tube is cut into 3 × 3 mm fritter strains, inoculates a fritter and arrive In test tube slant culture medium, cultivating 4～15 days for 20～35 DEG C, prepare test tube slant strain, 4 DEG C, this test tube slant saves backup；

B2 liquid submerged culture: the test tube slant strain obtained by step B1 is cut into 3 × 3 mm fritter strains, picking 3～10 Block is inoculated in the 250mL triangular flask equipped with 20～150mL liquid submerged culture bases, triangular flask rotating speed be 50～200 turns/ Point, under conditions of temperature 20～35 DEG C, 18-86h cultivated by shaking table, makes liquid shaking bottle strain；

B3 seed tank amplification culture: the liquid shaking bottle strain obtained by step B2 is inoculated in seed tank amplification culture base, and Described liquid shaking bottle strain and seed tank amplification culture base are the inoculum concentration inoculation of 1～20% by volume, are 20～35 in temperature DEG C, speed of agitator is 50～160 revs/min, is 0.2 at the volume being passed through gas per minute and seed tank amplification culture base volume ratio ～under the conditions of the ventilation of 1.8:1, cultivate 18～96h, make rough bark tough lead fungi seed tank strain；

B4 solid primary fermentation is cultivated: is accessed by tough for the rough bark obtained by step B3 lead fungi seed tank strain and trains equipped with solid primary fermentation Support in the culture bag of base, and described rough bark tough lead fungi seed tank strain is with the culture bag equipped with solid primary fermentation culture medium by volume Than being the inoculum concentration inoculation of 2～10%, in temperature 20～35 DEG C, humidity 80% primary fermentation 20～40 days, wherein every 3～6 days, turn over Bag once, prepares solid primary fermentation thing；

B5 liquid post-fermentation and culture: the solid primary fermentation thing obtained by step B4 is crushed to 20 mesh, joins liquid fermentation tank In culture medium, described liquid fermentation tank culture volume is 5～10 with the weight ratio of solid primary fermentation thing, is 20～43 in temperature DEG C, speed of agitator is 50～160 revs/min, at the volume being passed through gas per minute with liquid fermentation tank culture volume ratio is Fermentation 24～72h under the conditions of the ventilation of 0.2-1.8:1, obtained fermented liquid is under the conditions of 2000～6000 revs/min The centrifugal fermentation liquid prepared containing ginkalide B and the mycelium containing Quercetin；

B6 extracts separation: by the tough lead fungi mycelium of the rough bark containing Quercetin obtained by step A5 with containing ginkalide B Fermentation liquid, respectively through extraction, obtains Quercetin and ginkalide B crystal.

Preferably, liquid submerged culture base described in step B2 is sterilizing 90～360 min under the conditions of 100～130 DEG C Prepare, and containing wheat bran 5～20g, the raw material of rice 5～20g in every liter of liquid submerged culture base.

Preferably, the base of seed tank amplification culture described in step B3 is sterilizing 90～360 under the conditions of 100～130 DEG C Min prepares, and containing wheat bran 5～20g, the raw material of rice 5～20g in every liter of liquid submerged culture base.

Preferably, the culture medium of solid primary fermentation described in step B4 is sterilizing 90～360 under the conditions of 100～130 DEG C Min prepares, and containing peeling Semen Ginkgo 200～400g, Folium Ginkgo extract 30～50g in each kilogram of solid primary fermentation culture medium Raw material with rice 750～570g.

Preferably, the raw material in described solid primary fermentation culture medium is mixed with water in the ratio of 1:0.6～1.5.

Preferably, liquid fermentation medium described in step B5 is sterilizing 30～60 min under the conditions of 100～130 DEG C Prepare, and every liter of liquid fermentation medium contains 5～40g glucoses, 0.5～4g peptone and 0.01～0.1gK₂HPO₄ 。

Preferably, from the tough lead fungi mycelium of the rough bark containing Quercetin, Quercetin is produced described in step A6 or step B6 The method of crystal comprises the steps:

Obtained rough bark tough lead fungi mycelium is dried by C1, is then crushed to 20 mesh, adds 5～20 times of bodies of mycelium weight 60 long-pending～the ethanol of 100%, repeatedly extracts 3～5 times, obtains extracting solution one；

Extracting solution one obtained by step C1 is merged by C2, is then evaporated to the 1/30 of original volume, adds residual volume 5 times Water, under the conditions of 4 DEG C crystallize 3～18 hours, then sucking filtration is dried, and i.e. obtains Quercetin crystal, and Quercetin purity is more than 95%。

Preferably, from the fermentation liquid containing ginkalide B, ginkalide B crystalline substance is produced described in step A6 or step B6 The method of body comprises the steps:

D1 is by the fermentation liquid concentrating under reduced pressure of obtained ginkalide B and is dried and to obtain product；

D2 adds 60～100% alcohol reflux 3～5 times of 5～20 times of weight in step D1 products therefrom, obtains extraction Liquid two；

Extracting solution two obtained by step D2 is merged by D3, is then evaporated to the 1/40 of original volume, ties under the conditions of-20 DEG C Brilliant 3～18 hours；

Product after step D3 is crystallized by D4 carries out sucking filtration, and sucking filtration gained filtering residue ethyl acetate is redissolved, then by ethyl acetate Decompression is concentrated to dryness mutually, then redissolves with dehydrated alcohol, and adds the water of 3～10 times of volumes, precipitation, sucking filtration, and gained filtering residue is dried Being ginkalide B, its purity is more than 90%.

, beneficial effect

Compared to prior art, the invention have the benefit that

(1) present invention is on the basis of drawing herb fermenting pharmaceutical technology, respective in conjunction with modern solid-state fermentation and liquid fermentation Advantage, uses solid-state primary fermentation to convert Semen Ginkgo active component flavone and bilobalide, overcomes substrate (silver during liquid fermentation The active component of Folium Pruni) inhibitory action of lead fungi mycelial growth tough to rough bark, utilize submerged fermentation technology, the tough lead fungi of rough bark simultaneously Ginkalide B is secreted into extracellular and flavone is changed into Quercetin and is accumulated in intracellular, ginkalide B and Quercetin are divided From；

(2) a kind of method utilizing the tough lead fungi of rough bark simultaneously to prepare ginkalide B and Quercetin of the present invention, is not simple The process for separation and purification that utilizes obtain substantial amounts of ginkalide B and Quercetin, but with peeling Semen Ginkgo and rice as solid matrix Add Folium Ginkgo extract, with the tough lead fungi of rough bark for strain by solid primary fermentation, convert bilobalide and ginkgetin, pass through After fermentation, makes Quercetin and ginkalide B be separated, can realize industrialization large-scale production, it is possible to produce Semen Ginkgo simultaneously Lactone B and Quercetin；

(3) a kind of method utilizing the tough lead fungi of rough bark simultaneously to prepare ginkalide B and Quercetin of the present invention, technique letter Single, and in obtained product, ginkalide B purity is more than 90%, Quercetin purity is more than 95%, and the purity produced is higher.

Accompanying drawing explanation

Fig. 1 is a kind of method utilizing the tough lead fungi of rough bark simultaneously to prepare ginkalide B and Quercetin of the present invention Preparation flow schematic diagram.

Detailed description of the invention

Below in conjunction with specific embodiment, the present invention is further described below.

Embodiment 1

As it is shown in figure 1, a kind of method utilizing the tough lead fungi of rough bark to prepare ginkalide B and Quercetin specifically includes following step simultaneously Rapid:

(1) test tube amplification culture: (Stereum hirsutum ATCC 24992 receives purchased from Beijing North and creates connection by tough for rough bark lead fungi Bioteknologisk Institut, lower with) slant strains is cut into 3 × 3 mm fritter strains in test tube, inoculate a fritter and train to test tube slant Supporting in base, cultivate 15 days for 20 DEG C, prepare test tube slant strain, 4 DEG C, this test tube slant saves backup；

(2) liquid submerged culture: the test tube slant strain obtained by step (1) is cut into 3 × 3 mm fritter strains, picking 10 pieces Being inoculated in the 250mL triangular flask equipped with 150mL liquid submerged culture base, triangular flask is 50 revs/min at rotating speed, temperature 35 DEG C Under the conditions of, 18h cultivated by shaking table, makes liquid shaking bottle strain；Described liquid submerged culture base is sterilizing 360 under the conditions of 130 DEG C Min prepares, and containing wheat bran 5g, the raw material of rice 20g in every liter of liquid submerged culture base；

(3) seed tank amplification culture: the liquid shaking bottle strain obtained by step (2) is inoculated in seed tank amplification culture base, And described liquid shaking bottle strain and the inoculum concentration inoculation that seed tank amplification culture base is 1% by volume, it is 35 DEG C in temperature, stirs Mixing rotating speed is 160 revs/min, is the ventilation of 0.2:1 at the volume being passed through gas per minute and seed tank amplification culture base volume ratio Under the conditions of amount, cultivate 96h, make rough bark tough lead fungi seed tank strain；Described seed tank amplification culture base is under the conditions of 130 DEG C Sterilizing 360 min prepares, and containing wheat bran 5g, the raw material of rice 20g in every liter of liquid submerged culture base；

(4) solid primary fermentation is cultivated: access the rough bark tough lead fungi seed tank strain obtained by step (3) equipped with solid primary fermentation In the culture bag of culture medium, and described rough bark tough lead fungi seed tank strain presses body with the culture bag equipped with solid primary fermentation culture medium Long-pending ratio is the inoculum concentration inoculation of 10%, at temperature 20 DEG C, humidity 80% primary fermentation 40 days, wherein every 6 days, turns over bag once, prepares Solid primary fermentation thing；Described solid primary fermentation culture medium is that sterilizing 360 min prepares under the conditions of 130 DEG C, and each kilogram solid Containing peeling Semen Ginkgo 200g, Folium Ginkgo extract 50g and the raw material of rice 750g in body primary fermentation culture medium；Send out before described solid Raw material in ferment culture medium is mixed with water in the ratio of 1:0.6；

(5) liquid post-fermentation and culture: the solid primary fermentation thing obtained by step (4) is crushed to 20 mesh, joins liquid fermentation In tank culture medium, described liquid fermentation tank culture volume is 10 with the weight ratio of solid primary fermentation thing, is 20 DEG C in temperature, stirs Mixing rotating speed is 50 revs/min, at the volume being passed through gas per minute with liquid fermentation tank culture volume than the ventilation for 0.2:1 Under the conditions of ferment 72h, obtained fermented liquid is centrifugal under the conditions of 2000 revs/min prepares the fermentation containing ginkalide B Liquid and the mycelium containing Quercetin；Described liquid fermentation medium is that sterilizing 60 min prepares under the conditions of 130 DEG C, and every liter Liquid fermentation medium contains 5g glucose, 4g peptone and 0.01gK₂HPO₄；

(6) separation is extracted: by the tough lead fungi mycelium of the rough bark containing Quercetin obtained by step (5) with containing ginkalide B Fermentation liquid respectively through extraction, obtain Quercetin and ginkalide B crystal.

It should be noted that and from the tough lead fungi mycelium of the rough bark containing Quercetin, produce Quercetin described in step (6) The method of crystal comprises the steps:

Obtained rough bark tough lead fungi mycelium is dried by A1, is then crushed to 20 mesh, adds 20 times of volumes of mycelium weight 100% ethanol, repeatedly extract 3 times, obtain extracting solution one；

Extracting solution one obtained by step A1 is merged by A2, is then evaporated to the 1/30 of original volume, adds residual volume 5 times Water, under the conditions of 4 DEG C crystallize 3 hours, then sucking filtration is dried, and i.e. obtains Quercetin crystal, and quercetin content is 95%.

In the present embodiment, from the fermentation liquid containing ginkalide B, ginkalide B crystal is produced described in step (6) Method comprise the steps:

B1 is by the fermentation liquid concentrating under reduced pressure of obtained ginkalide B and is dried and to obtain product；

B2 adds 60% alcohol reflux 5 times of 20 times of weight in step B1 products therefrom, obtains extracting solution two；

Extracting solution two obtained by step B2 is merged by B3, is then evaporated to the 1/40 of original volume, ties under the conditions of-20 DEG C Brilliant 18 hours；

Product after step B3 is crystallized by B4 carries out sucking filtration, and sucking filtration gained filtering residue ethyl acetate is redissolved, then by ethyl acetate Decompression is concentrated to dryness mutually, then redissolves with dehydrated alcohol, and adds the water of 3 times of volumes, precipitation, sucking filtration, and gained filtering residue is dried and is Ginkalide B, its content is 90%.

The mensuration of described quercetin content uses high performance liquid chromatography (Qi Ningli etc.: high performance liquid chromatography-two pole Pipe array detection measures rutin and the content of Quercetin in Radix Et Rhizoma Fagopyri Tatarici simultaneously. food science and technology, and 2013,38 (10): 301-304): Intersil ODS-SP C 18 post (150 mm × 4.6 mm, 5 μm), flowing is used (to use phosphorus for methanol-0.5% phosphoric acid solution mutually Acid dihydride potassium regulation p H to 4.5) (40:60), flow velocity: 1.0 m L/min, column temperature: 35 DEG C, sample size: 10 μ L, detects ripple Long 255 nm, using Quercetin standard substance as comparison, according to Quercetin and the corresponding peak area of variable concentrations, carry out recurrence and obtain Quercetin concentration and face, peak relation equation, further according to Quercetin peak area in sample and extension rate and regression equation calculation sample Middle quercetin content.

The mensuration of described ginkalide B content uses High Performance Liquid Chromatography/Mass Spectrometry: be furnished with high pressure binary pump, water this 996 diode array detector and Agilent 1200 series autosampler, chromatographic column BEH C-18 post (2.5 μm, 3.0 × 100) high performance liquid chromatography (HPLC) is used for analyzing bilobalide.With first alcohol and water (7:3) as flowing phase, flow velocity is 0.2 mL/ min carries out eluting.Three grades of quadrupole rod mass spectrum 6400 ion trap mass spectrometers of Agilent, as detector, are born at full scan The mode operation of ion electrospray ionization (ESI) (M/Z500-800).Ion trap mass spectrometer, condition ISO be: ESI, Negative polarity ionizes；Spray voltage: 4.0 kilovolts；Heated capillary temperature: 275 DEG C；Sheath gas (N2 gas) flow velocity: 30 Arb；Auxiliary/ Purge gas (N2 gas)；Flow velocity: 50 Arb.Using ginkalide B standard substance as comparison, according to the ginkalide B of variable concentrations with Corresponding peak area, carries out recurrence and obtains ginkalide B concentration and face, peak relation equation, further according to face, ginkalide B peak in sample Amass and ginkalide B content in extension rate and regression equation calculation sample.

Based on above-mentioned, invention, on the basis of drawing herb fermenting pharmaceutical technology, ferments in conjunction with modern solid-state Advantage respective with liquid fermentation, uses solid-state primary fermentation to convert Semen Ginkgo active component flavone and bilobalide, overcomes liquid The inhibitory action of sweat mesostroma (active component of Folium Ginkgo) lead fungi tough to rough bark mycelial growth, utilizes deep layer to send out simultaneously Ferment technology, ginkalide B is secreted into extracellular and flavone changes into Quercetin and is accumulated in intracellular, by silver by the tough lead fungi of rough bark Fructus Pruni lactone B separates with Quercetin；And the present invention be not simple utilize process for separation and purification obtain substantial amounts of ginkalide B and Quercetin, but add Folium Ginkgo extract with peeling Semen Ginkgo and rice for solid matrix, pass through with the tough lead fungi of rough bark for strain Solid primary fermentation, converts bilobalide and ginkgetin, by after fermentation, makes Quercetin and ginkalide B be separated, can be real Existing industrialization large-scale production, it is possible to simultaneously produce ginkalide B and Quercetin；There is the simple advantage of technique, and gained To product in, ginkalide B purity be more than 90%, Quercetin purity be more than 95%, the purity produced is higher.

Embodiment 2

A kind of method utilizing the tough lead fungi of rough bark simultaneously to prepare ginkalide B and Quercetin specifically includes following steps:

(1) test tube amplification culture: slant strains in tough for rough bark lead fungi test tube is cut into 3 × 3 mm fritter strains, inoculates a fritter In test tube slant culture medium, cultivating 4 days for 35 DEG C, prepare test tube slant strain, 4 DEG C, this test tube slant saves backup；

(2) liquid submerged culture: the test tube slant strain obtained by step (1) is cut into 3 × 3 mm fritter strains, picking 3 pieces Being inoculated in the 250mL triangular flask equipped with 20mL liquid submerged culture base, triangular flask is 200 revs/min at rotating speed, temperature 20 DEG C Under the conditions of, 86h cultivated by shaking table, makes liquid shaking bottle strain；Described liquid submerged culture base is sterilizing 90 under the conditions of 100 DEG C Min prepares, and containing bran 20g, the raw material of rice 5g in every liter of liquid submerged culture base；

(3) seed tank amplification culture: the liquid shaking bottle strain obtained by step (2) is inoculated in seed tank amplification culture base, And described liquid shaking bottle strain and the inoculum concentration inoculation that seed tank amplification culture base is 20% by volume, it is 20 DEG C in temperature, stirs Mixing rotating speed is 50 revs/min, is 0.2～1.8:1 at the volume being passed through gas per minute and seed tank amplification culture base volume ratio Under the conditions of ventilation, cultivate 18h, make rough bark tough lead fungi seed tank strain；Described seed tank amplification culture base is at 100 DEG C of bars Under part, sterilizing 90min prepares, and containing wheat bran 20g, the raw material of rice 5g in every liter of liquid submerged culture base；

(4) solid primary fermentation is cultivated: access the rough bark tough lead fungi seed tank strain obtained by step (3) equipped with solid primary fermentation In the culture bag of culture medium, and described rough bark tough lead fungi seed tank strain presses body with the culture bag equipped with solid primary fermentation culture medium Long-pending ratio is the inoculum concentration inoculation of 2%, at temperature 35 DEG C, humidity 80% primary fermentation 20 days, wherein every 3 days, turns over bag once, prepares solid Body primary fermentation thing；Described solid primary fermentation culture medium is that sterilizing 90min prepares under the conditions of 100 DEG C, and before each kilogram of solid Containing peeling Semen Ginkgo 400g, Folium Ginkgo extract 30g and the raw material of rice 570g in fermentation medium；Described solid primary fermentation is trained The raw material supported in base is mixed with water in the ratio of 1:1.5；

(5) liquid post-fermentation and culture: the solid primary fermentation thing obtained by step (4) is crushed to 20 mesh, joins liquid fermentation In tank culture medium, described liquid fermentation tank culture volume is 5 with the weight ratio of solid primary fermentation thing, is 35 DEG C in temperature, stirs Mixing rotating speed is 160 revs/min, at the volume being passed through gas per minute with liquid fermentation tank culture volume than the ventilation for 1.8:1 Ferment under the conditions of amount 24h, and obtained fermented liquid centrifugal preparing under the conditions of 6000 revs/min contains sending out of ginkalide B Ferment liquid and the mycelium containing Quercetin；Described liquid fermentation medium is that sterilizing 30min prepares under the conditions of 100 DEG C, and often Rise liquid fermentation medium and contain 40g glucose, 0.5g peptone and 0.01gK₂HPO₄；

Obtained rough bark tough lead fungi mycelium is dried by A1, is then crushed to 20 mesh, adds 5 times of volumes of mycelium weight The ethanol of 60%, repeatedly extracts 5 times, obtains extracting solution one；

Extracting solution one obtained by step A1 is merged by A2, is then evaporated to the 1/30 of original volume, adds residual volume 5 times Water, under the conditions of 4 DEG C crystallize 18 hours, then sucking filtration is dried, and i.e. obtains Quercetin crystal, and quercetin content is 99.9%.

B2 adds 60～100% alcohol reflux 3 times of 5 times of weight in step B1 products therefrom, obtains extracting solution two；

Extracting solution two obtained by step B2 is merged by B3, is then evaporated to the 1/40 of original volume, ties under the conditions of-20 DEG C Brilliant 3 hours；

Product after step B3 is crystallized by B4 carries out sucking filtration, and sucking filtration gained filtering residue ethyl acetate is redissolved, then by ethyl acetate Decompression is concentrated to dryness mutually, then redissolves with dehydrated alcohol, and adds the water of 10 times of volumes, precipitation, sucking filtration, and gained filtering residue is dried and is Ginkalide B, its content is 99.8%.

Embodiment 3

(1) test tube amplification culture: slant strains in tough for rough bark lead fungi test tube is cut into 3 × 3 mm fritter strains, inoculates a fritter In test tube slant culture medium, cultivating 10 days for 28 DEG C, prepare test tube slant strain, 4 DEG C, this test tube slant saves backup；

(2) liquid submerged culture: the test tube slant strain obtained by step (1) is cut into 3 × 3 mm fritter strains, picking 7 pieces Being inoculated in the 250mL triangular flask equipped with 80mL liquid submerged culture base, triangular flask is 120 revs/min at rotating speed, temperature 28 DEG C Under the conditions of, 57h cultivated by shaking table, makes liquid shaking bottle strain；Described liquid submerged culture base is sterilizing 260 under the conditions of 120 DEG C Min prepares, and containing wheat bran 13g, the raw material of rice 13g in every liter of liquid submerged culture base；

(3) seed tank amplification culture: the liquid shaking bottle strain obtained by step (2) is inoculated in seed tank amplification culture base, And described liquid shaking bottle strain and the inoculum concentration inoculation that seed tank amplification culture base is 10% by volume, it is 28 DEG C in temperature, stirs Mixing rotating speed is 110 revs/min, is the ventilation of 1:1 at the volume being passed through gas per minute and seed tank amplification culture base volume ratio Under the conditions of, cultivate 57h, make rough bark tough lead fungi seed tank strain；Described seed tank amplification culture base is to go out under the conditions of 120 DEG C Bacterium 260 min prepares, and containing wheat bran 13g, the raw material of rice 13g in every liter of liquid submerged culture base；

(4) solid primary fermentation is cultivated: access the rough bark tough lead fungi seed tank strain obtained by step (3) equipped with solid primary fermentation In the culture bag of culture medium, and described rough bark tough lead fungi seed tank strain presses body with the culture bag equipped with solid primary fermentation culture medium Long-pending ratio is the inoculum concentration inoculation of 6%, at temperature 28 DEG C, humidity 80% primary fermentation 30 days, wherein every 4.5 days, turns over bag once, prepares Solid primary fermentation thing；Described solid primary fermentation culture medium is that sterilizing 260 min prepares under the conditions of 120 DEG C, and each kilogram solid Containing peeling Semen Ginkgo 300g, Folium Ginkgo extract 40g and the raw material of rice 660g in body primary fermentation culture medium；Send out before described solid Raw material in ferment culture medium is mixed with water in the ratio of 1:1；

(5) liquid post-fermentation and culture: the solid primary fermentation thing obtained by step (4) is crushed to 20 mesh, joins liquid fermentation In tank culture medium, described liquid fermentation tank culture volume is 8 with the weight ratio of solid primary fermentation thing, is 27 DEG C in temperature, stirs Mixing rotating speed is 110 revs/min, logical than for 0.2-1.8 of the volume being passed through gas per minute and liquid fermentation tank culture volume Ferment under the conditions of tolerance 48h, and obtained fermented liquid is centrifugal under the conditions of 4000 revs/min to be prepared containing ginkalide B Fermentation liquid and the mycelium containing Quercetin；Described liquid fermentation medium is that sterilizing 45 min prepares under the conditions of 120 DEG C, and Every liter of liquid fermentation medium contains 23g glucose, 2.2g peptone and 0.05gK₂HPO₄；

Obtained rough bark tough lead fungi mycelium is dried by A1, is then crushed to 20 mesh, adds 13 times of volumes of mycelium weight 80% ethanol, repeatedly extract 4 times, obtain extracting solution one；

Extracting solution one obtained by step A1 is merged by A2, is then evaporated to the 1/30 of original volume, adds residual volume 5 times Water, under the conditions of 4 DEG C crystallize 11 hours, then sucking filtration is dried, and i.e. obtains Quercetin crystal, and quercetin content is 98%.

B2 adds 80% alcohol reflux 4 times of 13 times of weight in step B1 products therefrom, obtains extracting solution two；

Extracting solution two obtained by step B2 is merged by B3, is then evaporated to the 1/40 of original volume, ties under the conditions of-20 DEG C Brilliant 11 hours；

Product after step B3 is crystallized by B4 carries out sucking filtration, and sucking filtration gained filtering residue ethyl acetate is redissolved, then by ethyl acetate Decompression is concentrated to dryness mutually, then redissolves with dehydrated alcohol, and adds the water of 7 times of volumes, precipitation, sucking filtration, and gained filtering residue is dried and is Ginkalide B, its content is 95%.

Embodiment 4

(1) test tube amplification culture: slant strains in tough for rough bark lead fungi test tube is cut into 3 × 3 mm fritter strains, inoculates a fritter In test tube slant culture medium, cultivating 12 days for 23 DEG C, prepare test tube slant strain, 4 DEG C, this test tube slant saves backup；

(2) liquid submerged culture: the test tube slant strain obtained by step (1) is cut into 3 × 3 mm fritter strains, picking 8 pieces Being inoculated in the 250mL triangular flask equipped with 30mL liquid submerged culture base, triangular flask is 75 revs/min at rotating speed, temperature 20～35 Under conditions of DEG C, 30h cultivated by shaking table, makes liquid shaking bottle strain；Described liquid submerged culture base is sterilizing under the conditions of 100 DEG C 100 min prepare, and containing wheat bran 8g, the raw material of rice 12g in every liter of liquid submerged culture base；

(3) seed tank amplification culture: the liquid shaking bottle strain obtained by step (2) is inoculated in seed tank amplification culture base, And described liquid shaking bottle strain and the inoculum concentration inoculation that seed tank amplification culture base is 4% by volume, it is 25 DEG C in temperature, stirs Mixing rotating speed is 120 revs/min, is the ventilation of 0.6:1 at the volume being passed through gas per minute and seed tank amplification culture base volume ratio Under the conditions of amount, cultivate 35h, make rough bark tough lead fungi seed tank strain；Described seed tank amplification culture base is under the conditions of 100 DEG C Sterilizing 100 min prepares, and containing wheat bran 8g, the raw material of rice 15g in every liter of liquid submerged culture base；

(4) solid primary fermentation is cultivated: access the rough bark tough lead fungi seed tank strain obtained by step (3) equipped with solid primary fermentation In the culture bag of culture medium, and described rough bark tough lead fungi seed tank strain presses body with the culture bag equipped with solid primary fermentation culture medium Long-pending ratio is the inoculum concentration inoculation of 4%, at temperature 22 DEG C, humidity 80% primary fermentation 25 days, wherein every 3 days, turns over bag once, prepares solid Body primary fermentation thing；Described solid primary fermentation culture medium is that sterilizing 100 min prepares under the conditions of 100 DEG C, and each kilogram of solid Containing peeling Semen Ginkgo 250g, Folium Ginkgo extract 35g and the raw material of rice 715g in primary fermentation culture medium；Described solid primary fermentation Raw material in culture medium is mixed with water in the ratio of 1:0.6；

(5) liquid post-fermentation and culture: the solid primary fermentation thing obtained by step (4) is crushed to 20 mesh, joins liquid fermentation In tank culture medium, described liquid fermentation tank culture volume is 6 with the weight ratio of solid primary fermentation thing, is 24 DEG C in temperature, stirs Mixing rotating speed is 65 revs/min, at the volume being passed through gas per minute with liquid fermentation tank culture volume than the ventilation for 1.5:1 Under the conditions of ferment 60h, obtained fermented liquid is centrifugal under the conditions of 5000 revs/min prepares the fermentation containing ginkalide B Liquid and the mycelium containing Quercetin；Described liquid fermentation medium is that sterilizing 50 min prepares under the conditions of 115 DEG C, and every liter Liquid fermentation medium contains 30g glucose, 3g peptone and 0.09gK₂HPO₄；

Obtained rough bark tough lead fungi mycelium is dried by A1, is then crushed to 20 mesh, adds 18 times of volumes of mycelium weight 95% ethanol, repeatedly extract 3 times, obtain extracting solution one；

Extracting solution one obtained by step A1 is merged by A2, is then evaporated to the 1/30 of original volume, adds residual volume 5 times Water, under the conditions of 4 DEG C crystallize 4 hours, then sucking filtration is dried, and i.e. obtains Quercetin crystal, Quercetin purity be more than 95%.

B2 adds 95% alcohol reflux 4 times of 18 times of weight in step B1 products therefrom, obtains extracting solution two；

Extracting solution two obtained by step B2 is merged by B3, is then evaporated to the 1/40 of original volume, ties under the conditions of-20 DEG C Brilliant 17 hours；

Product after step B3 is crystallized by B4 carries out sucking filtration, and sucking filtration gained filtering residue ethyl acetate is redissolved, then by ethyl acetate Decompression is concentrated to dryness mutually, then redissolves with dehydrated alcohol, and adds the water of 9 times of volumes, precipitation, sucking filtration, and gained filtering residue is dried and is Ginkalide B, its content is 92%.

Embodiment 5

(1) test tube amplification culture: slant strains in tough for rough bark lead fungi test tube is cut into 3 × 3 mm fritter strains, inoculates a fritter In test tube slant culture medium, cultivating 6 days for 32 DEG C, prepare test tube slant strain, 4 DEG C, this test tube slant saves backup；

(2) liquid submerged culture: the test tube slant strain obtained by step (1) is cut into 3 × 3 mm fritter strains, picking 4 pieces Being inoculated in the 250mL triangular flask equipped with 40mL liquid submerged culture base, triangular flask is 160 revs/min at rotating speed, temperature 30 DEG C Under the conditions of, 36h cultivated by shaking table, makes liquid shaking bottle strain；Described liquid submerged culture base is sterilizing 320 under the conditions of 128 DEG C Min prepares, and containing wheat bran 6g, the raw material of rice 20g in every liter of liquid submerged culture base；

(3) seed tank amplification culture: the liquid shaking bottle strain obtained by step (2) is inoculated in seed tank amplification culture base, And described liquid shaking bottle strain and the inoculum concentration inoculation that seed tank amplification culture base is 15% by volume, it is 30 DEG C in temperature, stirs Mixing rotating speed is 160 revs/min, is the ventilation of 0.6:1 at the volume being passed through gas per minute and seed tank amplification culture base volume ratio Under the conditions of amount, cultivate 68h, make rough bark tough lead fungi seed tank strain；Described seed tank amplification culture base is under the conditions of 128 DEG C Sterilizing 320 min prepares, and containing wheat bran 15g, the raw material of rice 7g in every liter of liquid submerged culture base；

(4) solid primary fermentation is cultivated: access the rough bark tough lead fungi seed tank strain obtained by step (3) equipped with solid primary fermentation In the culture bag of culture medium, and described rough bark tough lead fungi seed tank strain presses body with the culture bag equipped with solid primary fermentation culture medium Long-pending ratio is the inoculum concentration inoculation of 6%, at temperature 30 DEG C, humidity 80% primary fermentation 28 days, wherein every 5 days, turns over bag once, prepares solid Body primary fermentation thing；Described solid primary fermentation culture medium is that sterilizing 320 min prepares under the conditions of 128 DEG C, and each kilogram of solid Containing peeling Semen Ginkgo 350g, Folium Ginkgo extract 50g and the raw material of rice 600g in primary fermentation culture medium；Described solid primary fermentation Raw material in culture medium is mixed with water in the ratio of 1:1.2；

(5) liquid post-fermentation and culture: the solid primary fermentation thing obtained by step (4) is crushed to 20 mesh, joins liquid fermentation In tank culture medium, described liquid fermentation tank culture volume is 7 with the weight ratio of solid primary fermentation thing, is 30 DEG C in temperature, stirs Mixing rotating speed is 120 revs/min, at the volume being passed through gas per minute with liquid fermentation tank culture volume than the ventilation for 1.6:1 Ferment under the conditions of amount 58h, and obtained fermented liquid centrifugal preparing under the conditions of 3000 revs/min contains sending out of ginkalide B Ferment liquid and the mycelium containing Quercetin；Described liquid fermentation medium is that sterilizing 60 min prepares under the conditions of 128 DEG C, and often Rise liquid fermentation medium and contain 35g glucose, 0.5g peptone and 0.04gK₂HPO₄；

Obtained rough bark tough lead fungi mycelium is dried by A1, is then crushed to 20 mesh, adds 18 times of volumes of mycelium weight 90% ethanol, repeatedly extract 4 times, obtain extracting solution one；

Extracting solution one obtained by step A1 is merged by A2, is then evaporated to the 1/30 of original volume, adds residual volume 5 times Water, under the conditions of 4 DEG C crystallize 12 hours, then sucking filtration is dried, and i.e. obtains Quercetin crystal, and quercetin content is 96%.

B2 adds 70% alcohol reflux 5 times of 8 times of weight in step B1 products therefrom, obtains extracting solution two；

Extracting solution two obtained by step B2 is merged by B3, is then evaporated to the 1/40 of original volume, ties under the conditions of-20 DEG C Brilliant 6 hours；

Product after step B3 is crystallized by B4 carries out sucking filtration, and sucking filtration gained filtering residue ethyl acetate is redissolved, then by ethyl acetate Decompression is concentrated to dryness mutually, then redissolves with dehydrated alcohol, and adds the water of 4 times of volumes, precipitation, sucking filtration, and gained filtering residue is dried and is Ginkalide B, its content is 92%.

Embodiment 6

(1) test tube amplification culture: slant strains in tough for rough bark lead fungi test tube is cut into 3 × 3 mm fritter strains, inoculates a fritter In test tube slant culture medium, cultivating 5 days for 32 DEG C, prepare test tube slant strain, 4 DEG C, this test tube slant saves backup；

(2) liquid submerged culture: the test tube slant strain obtained by step (1) is cut into 3 × 3 mm fritter strains, picking 4 pieces Being inoculated in the 250mL triangular flask equipped with 20mL liquid submerged culture base, triangular flask is 170 revs/min at rotating speed, temperature 25 DEG C Under the conditions of, 60h cultivated by shaking table, makes liquid shaking bottle strain；Described liquid submerged culture base is sterilizing 250 under the conditions of 110 DEG C Min prepares, and containing wheat bran 16g, the raw material of rice 8g in every liter of liquid submerged culture base；

(3) seed tank amplification culture: the liquid shaking bottle strain obtained by step (2) is inoculated in seed tank amplification culture base, And described liquid shaking bottle strain and the inoculum concentration inoculation that seed tank amplification culture base is 15% by volume, it is 25 DEG C in temperature, stirs Mixing rotating speed is 120 revs/min, is the ventilation of 1.6:1 at the volume being passed through gas per minute and seed tank amplification culture base volume ratio Under the conditions of amount, cultivate 29h, make rough bark tough lead fungi seed tank strain；Described seed tank amplification culture base is under the conditions of 110 DEG C Sterilizing 250 min prepares, and containing wheat bran 14g, the raw material of rice 10g in every liter of liquid submerged culture base；

(4) solid primary fermentation is cultivated: access the rough bark tough lead fungi seed tank strain obtained by step (3) equipped with solid primary fermentation In the culture bag of culture medium, and described rough bark tough lead fungi seed tank strain presses body with the culture bag equipped with solid primary fermentation culture medium Long-pending ratio is the inoculum concentration inoculation of 6%, at temperature 25 DEG C, humidity 80% primary fermentation 26 days, wherein every 5 days, turns over bag once, prepares solid Body primary fermentation thing；Described solid primary fermentation culture medium is that sterilizing 250 min prepares under the conditions of 110 DEG C, and each kilogram of solid Containing peeling Semen Ginkgo 276g, Folium Ginkgo extract 33g and the raw material of rice 691g in primary fermentation culture medium；Described solid primary fermentation Raw material in culture medium is mixed with water in the ratio of 1:0.9；

(5) liquid post-fermentation and culture: the solid primary fermentation thing obtained by step (4) is crushed to 20 mesh, joins liquid fermentation In tank culture medium, described liquid fermentation tank culture volume is 9 with the weight ratio of solid primary fermentation thing, is 25 DEG C in temperature, stirs Mixing rotating speed is 110 revs/min, at the volume being passed through gas per minute with liquid fermentation tank culture volume than the ventilation for 1.4:1 Ferment under the conditions of amount 48h, and obtained fermented liquid centrifugal preparing under the conditions of 3500 revs/min contains sending out of ginkalide B Ferment liquid and the mycelium containing Quercetin；Described liquid fermentation medium is that sterilizing 35 min prepares under the conditions of 110 DEG C, and often Rise liquid fermentation medium and contain 34g glucose, 4g peptone and 0.03gK₂HPO₄；

Obtained rough bark tough lead fungi mycelium is dried by A1, is then crushed to 20 mesh, adds 16 times of volumes of mycelium weight 70% ethanol, repeatedly extract 5 times, obtain extracting solution one；

Extracting solution one obtained by step A1 is merged by A2, is then evaporated to the 1/30 of original volume, adds residual volume 5 times Water, under the conditions of 4 DEG C crystallize 12 hours, then sucking filtration is dried, and i.e. obtains Quercetin crystal, and quercetin content is 98%.

B2 adds 70% alcohol reflux 4 times of 10 times of weight in step B1 products therefrom, obtains extracting solution two；

Extracting solution two obtained by step B2 is merged by B3, is then evaporated to the 1/40 of original volume, ties under the conditions of-20 DEG C Brilliant 8 hours；

Product after step B3 is crystallized by B4 carries out sucking filtration, and sucking filtration gained filtering residue ethyl acetate is redissolved, then by ethyl acetate Decompression is concentrated to dryness mutually, then redissolves with dehydrated alcohol, and adds the water of 7 times of volumes, precipitation, sucking filtration, and gained filtering residue is dried and is Ginkalide B, its content is 94%.

Embodiment 7

(1) test tube amplification culture: slant strains in tough for rough bark lead fungi test tube is cut into 3 × 3 mm fritter strains, inoculates a fritter In test tube slant culture medium, cultivating 13 days for 23 DEG C, prepare test tube slant strain, 4 DEG C, this test tube slant saves backup；

(2) liquid submerged culture: the test tube slant strain obtained by step (1) is cut into 3 × 3 mm fritter strains, picking 10 pieces Being inoculated in the 250mL triangular flask equipped with 130mL liquid submerged culture base, triangular flask is 150 revs/min at rotating speed, temperature 23 DEG C Under conditions of, 36h cultivated by shaking table, makes liquid shaking bottle strain；Described liquid submerged culture base is sterilizing under the conditions of 115 DEG C 260 min prepare, and containing wheat bran 9g, the raw material of rice 20g in every liter of liquid submerged culture base；

(3) seed tank amplification culture: the liquid shaking bottle strain obtained by step (2) is inoculated in seed tank amplification culture base, And described liquid shaking bottle strain and the inoculum concentration inoculation that seed tank amplification culture base is 15% by volume, it is 21 DEG C in temperature, stirs Mixing rotating speed is 120 revs/min, is the ventilation of 0.2:1 at the volume being passed through gas per minute and seed tank amplification culture base volume ratio Under the conditions of amount, cultivate 81h, make rough bark tough lead fungi seed tank strain；Described seed tank amplification culture base is under the conditions of 115 DEG C Sterilizing 260 min prepares, and containing wheat bran 19g, the raw material of rice 18g in every liter of liquid submerged culture base；

(4) solid primary fermentation is cultivated: access the rough bark tough lead fungi seed tank strain obtained by step (3) equipped with solid primary fermentation In the culture bag of culture medium, and described rough bark tough lead fungi seed tank strain presses body with the culture bag equipped with solid primary fermentation culture medium Long-pending ratio is the inoculum concentration inoculation of 4%, at temperature 21 DEG C, humidity 80% primary fermentation 34 days, wherein every 6 days, turns over bag once, prepares solid Body primary fermentation thing；Described solid primary fermentation culture medium is that sterilizing 250 min prepares under the conditions of 110 DEG C, and each kilogram of solid Containing peeling Semen Ginkgo 370g, Folium Ginkgo extract 40g and the raw material of rice 590g in primary fermentation culture medium；Described solid primary fermentation Raw material in culture medium is mixed with water in the ratio of 1:0.7；

(5) liquid post-fermentation and culture: the solid primary fermentation thing obtained by step (4) is crushed to 20 mesh, joins liquid fermentation In tank culture medium, described liquid fermentation tank culture volume is 10 with the weight ratio of solid primary fermentation thing, is 21 DEG C in temperature, stirs Mixing rotating speed is 60 revs/min, at the volume being passed through gas per minute with liquid fermentation tank culture volume than the ventilation for 1.4:1 Under the conditions of ferment 66h, obtained fermented liquid is centrifugal under the conditions of 5700 revs/min prepares the fermentation containing ginkalide B Liquid and the mycelium containing Quercetin；Described liquid fermentation medium is that sterilizing 35 min prepares under the conditions of 110 DEG C, and every liter Liquid fermentation medium contains 35g glucose, 5g peptone and 0.05gK₂HPO₄；

Obtained rough bark tough lead fungi mycelium is dried by A1, is then crushed to 20 mesh, adds 17 times of volumes of mycelium weight 90% ethanol, repeatedly extract 4 times, obtain extracting solution one；

Extracting solution one obtained by step A1 is merged by A2, is then evaporated to the 1/30 of original volume, adds residual volume 5 times Water, under the conditions of 4 DEG C crystallize 18 hours, then sucking filtration is dried, and i.e. obtains Quercetin crystal, and quercetin content is 99.5%.

B2 adds 65% alcohol reflux 5 times of 8 times of weight in step B1 products therefrom, obtains extracting solution two；

Product after step B3 is crystallized by B4 carries out sucking filtration, and sucking filtration gained filtering residue ethyl acetate is redissolved, then by ethyl acetate Decompression is concentrated to dryness mutually, then redissolves with dehydrated alcohol, and adds the water of 5 times of volumes, precipitation, sucking filtration, and gained filtering residue is dried and is Ginkalide B, its content is 99%.

Embodiment 8

(1) test tube amplification culture: slant strains in tough for rough bark lead fungi test tube is cut into 3 × 3 mm fritter strains, inoculates a fritter In test tube slant culture medium, cultivating 10 days for 29 DEG C, prepare test tube slant strain, 4 DEG C, this test tube slant saves backup；

(2) liquid submerged culture: the test tube slant strain obtained by step (1) is cut into 3 × 3 mm fritter strains, picking 8 pieces Being inoculated in the 250mL triangular flask equipped with 60mL liquid submerged culture base, triangular flask is 160 revs/min at rotating speed, temperature 33 DEG C Under the conditions of, 80h cultivated by shaking table, makes liquid shaking bottle strain；Described liquid submerged culture base is sterilizing 300 under the conditions of 120 DEG C Min prepares, and containing wheat bran 19g, the raw material of rice 19g in every liter of liquid submerged culture base；

(3) seed tank amplification culture: the liquid shaking bottle strain obtained by step (2) is inoculated in seed tank amplification culture base, And described liquid shaking bottle strain and the inoculum concentration inoculation that seed tank amplification culture base is 18% by volume, it is 33 DEG C in temperature, stirs Mixing rotating speed is 140 revs/min, is the ventilation of 1.8:1 at the volume being passed through gas per minute and seed tank amplification culture base volume ratio Under the conditions of amount, cultivate 90h, make rough bark tough lead fungi seed tank strain；Described seed tank amplification culture base is under the conditions of 120 DEG C Sterilizing 300 min prepares, and containing wheat bran 18g, the raw material of rice 18g in every liter of liquid submerged culture base；

(4) solid primary fermentation is cultivated: access the rough bark tough lead fungi seed tank strain obtained by step (3) equipped with solid primary fermentation In the culture bag of culture medium, and described rough bark tough lead fungi seed tank strain presses body with the culture bag equipped with solid primary fermentation culture medium Long-pending ratio is the inoculum concentration inoculation of 9%, at temperature 33 DEG C, humidity 80% primary fermentation 37 days, wherein every 6 days, turns over bag once, prepares solid Body primary fermentation thing；Described solid primary fermentation culture medium is that sterilizing 350 min prepares under the conditions of 105 DEG C, and each kilogram of solid Containing peeling Semen Ginkgo 400g, Folium Ginkgo extract 30g and the raw material of rice 570g in primary fermentation culture medium；Described solid primary fermentation Raw material in culture medium is mixed with water in the ratio of 1:1.4；

(5) liquid post-fermentation and culture: the solid primary fermentation thing obtained by step (4) is crushed to 20 mesh, joins liquid fermentation In tank culture medium, described liquid fermentation tank culture volume is 10 with the weight ratio of solid primary fermentation thing, is 33 DEG C in temperature, stirs Mixing rotating speed is 150 revs/min, at the volume being passed through gas per minute with liquid fermentation tank culture volume than the ventilation for 1.8:1 Ferment under the conditions of amount 70h, and obtained fermented liquid centrifugal preparing under the conditions of 5800 revs/min contains sending out of ginkalide B Ferment liquid and the mycelium containing Quercetin；Described liquid fermentation medium is that sterilizing 35 min prepares under the conditions of 105 DEG C, and often Rise liquid fermentation medium and contain 38g glucose, 4g peptone and 0.09g K₂HPO₄；

Obtained rough bark tough lead fungi mycelium is dried by A1, is then crushed to 20 mesh, adds 19 times of volumes of mycelium weight 100% ethanol, repeatedly extract 5 times, obtain extracting solution one；

Extracting solution one obtained by step A1 is merged by A2, is then evaporated to the 1/30 of original volume, adds residual volume 5 times Water, under the conditions of 4 DEG C crystallize 16 hours, then sucking filtration is dried, and i.e. obtains Quercetin crystal, and quercetin content is 99%.

B2 adds 98% alcohol reflux 5 times of 18 times of weight in step B1 products therefrom, obtains extracting solution two；

Product after step B3 is crystallized by B4 carries out sucking filtration, and sucking filtration gained filtering residue ethyl acetate is redissolved, then by ethyl acetate Decompression is concentrated to dryness mutually, then redissolves with dehydrated alcohol, and adds the water of 10 times of volumes, precipitation, sucking filtration, and gained filtering residue is dried and is Ginkalide B, its content is 98%.

Embodiment 9

(1) test tube amplification culture: slant strains in tough for rough bark lead fungi test tube is cut into 3 × 3 mm fritter strains, inoculates a fritter In test tube slant culture medium, cultivating 6 days for 24 DEG C, prepare test tube slant strain, 4 DEG C, this test tube slant saves backup；

(2) liquid submerged culture: the test tube slant strain obtained by step (1) is cut into 3 × 3 mm fritter strains, picking 4 pieces Being inoculated in the 250mL triangular flask equipped with 24mL liquid submerged culture base, triangular flask is 58 revs/min at rotating speed, temperature 24 DEG C Under the conditions of, 20h cultivated by shaking table, makes liquid shaking bottle strain；Described liquid submerged culture base is sterilizing 100 under the conditions of 100 DEG C Min prepares, and containing wheat bran 5g, the raw material of rice 5g in every liter of liquid submerged culture base；

(3) seed tank amplification culture: the liquid shaking bottle strain obtained by step (2) is inoculated in seed tank amplification culture base, And described liquid shaking bottle strain and the inoculum concentration inoculation that seed tank amplification culture base is 3% by volume, it is 24 DEG C in temperature, stirs Mixing rotating speed is 58 revs/min, is the ventilation of 1.8:1 at the volume being passed through gas per minute and seed tank amplification culture base volume ratio Under the conditions of, cultivate 25h, make rough bark tough lead fungi seed tank strain；Described seed tank amplification culture base is to go out under the conditions of 100 DEG C Bacterium 100 min prepares, and containing wheat bran 5g, the raw material of rice 6g in every liter of liquid submerged culture base；

(4) solid primary fermentation is cultivated: access the rough bark tough lead fungi seed tank strain obtained by step (3) equipped with solid primary fermentation In the culture bag of culture medium, and described rough bark tough lead fungi seed tank strain presses body with the culture bag equipped with solid primary fermentation culture medium Long-pending ratio is the inoculum concentration inoculation of 3%, at temperature 24 DEG C, humidity 80% primary fermentation 23 days, wherein every 3 days, turns over bag once, prepares solid Body primary fermentation thing；Described solid primary fermentation culture medium is that sterilizing 150 min prepares under the conditions of 100 DEG C, and each kilogram of solid Containing peeling Semen Ginkgo 220g, Folium Ginkgo extract 33g and the raw material of rice 747g in primary fermentation culture medium；Described solid primary fermentation Raw material in culture medium is mixed with water in the ratio of 1:0.8；

(5) liquid post-fermentation and culture: the solid primary fermentation thing obtained by step (4) is crushed to 20 mesh, joins liquid fermentation In tank culture medium, described liquid fermentation tank culture volume is 6 with the weight ratio of solid primary fermentation thing, is 24 DEG C in temperature, stirs Mixing rotating speed is 55 revs/min, at the volume being passed through gas per minute with liquid fermentation tank culture volume than the ventilation for 1.8:1 Under the conditions of ferment 26h, obtained fermented liquid is centrifugal under the conditions of 2300 revs/min prepares the fermentation containing ginkalide B Liquid and the mycelium containing Quercetin；Described liquid fermentation medium is that sterilizing 35 min prepares under the conditions of 100 DEG C, and every liter Liquid fermentation medium contains 6g glucose, 0.6g peptone and 0.02gK₂HPO₄；

Obtained rough bark tough lead fungi mycelium is dried by A1, is then crushed to 20 mesh, adds 6 times of volumes of mycelium weight The ethanol of 67%, repeatedly extracts 4 times, obtains extracting solution one；

Extracting solution one obtained by step A1 is merged by A2, is then evaporated to the 1/30 of original volume, adds residual volume 5 times Water, under the conditions of 4 DEG C crystallize 5 hours, then sucking filtration is dried, and i.e. obtains Quercetin crystal, and quercetin content is 95%.

B2 adds 65% alcohol reflux 3 times of 6 times of weight in step B1 products therefrom, obtains extracting solution two；

Extracting solution two obtained by step B2 is merged by B3, is then evaporated to the 1/40 of original volume, ties under the conditions of-20 DEG C Brilliant 4 hours；

Product after step B3 is crystallized by B4 carries out sucking filtration, and sucking filtration gained filtering residue ethyl acetate is redissolved, then by ethyl acetate Decompression is concentrated to dryness mutually, then redissolves with dehydrated alcohol, and adds the water of 4 times of volumes, precipitation, sucking filtration, and gained filtering residue is dried and is Ginkalide B, its content is 91%.

Schematically being described the present invention and embodiment thereof above, this description does not has restricted, actual knot Structure is not limited thereto.So, if those of ordinary skill in the art is enlightened by it, without departing from the invention objective In the case of, design the frame mode similar to this technical scheme and embodiment without creative, all should belong to the present invention's Protection domain.

Claims

1. one kind utilizes the method that the tough lead fungi of rough bark prepares ginkalide B and Quercetin simultaneously, it is characterised in that described preparation side Method, with rice, peeling Semen Ginkgo and Folium Ginkgo extract as primary raw material, with the tough lead fungi of rough bark as starting strain, sequentially passes through test tube Amplification culture, liquid submerged culture and seed tank amplification culture, the cultivation of solid primary fermentation, liquid post-fermentation and culture and extraction separate Step prepares the fermentation liquid containing ginkalide B and the mycelium containing Quercetin；The described fermentation liquid containing ginkalide B and Mycelium containing Quercetin prepares ginkalide B and Quercetin crystal respectively through extracting again, and wherein ginkalide B purity reaches To 90%, Quercetin purity reaches 95%.

A kind of method utilizing the tough lead fungi of rough bark simultaneously to prepare ginkalide B and Quercetin the most according to claim 1, its Being characterised by, described preparation method comprises the steps:

A1 test tube amplification culture: slant strains in tough for rough bark lead fungi is inoculated in potato dextrose medium and cultivates, Prepare rough bark tough lead fungi test tube slant strain；

A kind of method utilizing the tough lead fungi of rough bark simultaneously to prepare ginkalide B and Quercetin the most according to claim 2, its Being characterised by, described preparation method is specific as follows:

A kind of method utilizing the tough lead fungi of rough bark simultaneously to prepare ginkalide B and Quercetin the most according to claim 3, its Being characterised by, liquid submerged culture base described in step B2 is that sterilizing 90～360 min prepares under the conditions of 100～130 DEG C, and Containing wheat bran 5～20g in every liter of liquid submerged culture base, the raw material of rice 5～20g.

A kind of method utilizing the tough lead fungi of rough bark simultaneously to prepare ginkalide B and Quercetin the most according to claim 3, its Being characterised by, the base of seed tank amplification culture described in step B3 is that sterilizing 90～360 min prepares under the conditions of 100～130 DEG C, And containing wheat bran 5～20g, the raw material of rice 5～20g in every liter of liquid submerged culture base.

A kind of method utilizing the tough lead fungi of rough bark simultaneously to prepare ginkalide B and Quercetin the most according to claim 3, its Being characterised by, the culture medium of solid primary fermentation described in step B4 is that sterilizing 90～360 min prepares under the conditions of 100～130 DEG C, And containing peeling Semen Ginkgo 200～400g, Folium Ginkgo extract 30～50g and rice in each kilogram of solid primary fermentation culture medium The raw material of 750～570g.

A kind of method utilizing the tough lead fungi of rough bark simultaneously to prepare ginkalide B and Quercetin the most according to claim 6, its Being characterised by, the raw material in described solid primary fermentation culture medium is mixed with water in the ratio of 1:0.6～1.5.

A kind of method utilizing the tough lead fungi of rough bark simultaneously to prepare ginkalide B and Quercetin the most according to claim 3, It is characterized in that, liquid fermentation medium described in step B5 is that sterilizing 30～60 min prepares under the conditions of 100～130 DEG C, And every liter of liquid fermentation medium contains 5～40g glucoses, 0.5～4g peptone and 0.01～0.1gK₂HPO₄ 。

9. according to a kind of side utilizing the tough lead fungi of rough bark to prepare ginkalide B and Quercetin described in Claims 2 or 3 simultaneously Method, it is characterised in that produce Quercetin from the tough lead fungi mycelium of the rough bark containing Quercetin described in step A6 or step B6 The method of crystal comprises the steps:

10. according to a kind of side utilizing the tough lead fungi of rough bark to prepare ginkalide B and Quercetin described in Claims 2 or 3 simultaneously Method, it is characterised in that produce ginkalide B crystal described in step A6 or step B6 from the fermentation liquid containing ginkalide B Method comprise the steps: