CN106399431A - Preparation method of micafungin precursor - Google Patents
Preparation method of micafungin precursor Download PDFInfo
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Abstract
The invention provides a preparation method of micafungin precursor. The preparation method includes steps of producing strain spore suspension liquid through a few amounts of micafungin precursor and cultivating, and adding oxygen carrier in the fermentation liquid during the fermenting process, thus the dissolved oxygen concentration in a fermentation cylinder is improved, and 20-40% of output of the micafungin precursor is improved; performing the acidification and wall breaking treatment on the fermentation liquid, absorbing polar solvent by macroporous resin, analyzing and obtaining desorbed solution of micafungin precursor; crystalizing the desorbed solution and obtaining micafungin precursor powder. The invention can be used for compounding the micafungin precursor, and is simple in operation procedure, stable in method, good in reproducibility, and very suitable for industrial production; meanwhile, the method avoids using acetone and other poisonous solvent and lightens hazard to environment and human body.
Description
Technical field
The present invention relates to biological pharmacy technical field, more particularly, to a kind of preparation method of MFG precursor.
Background technology
In recent years, with the extensive application of extensive pedigree antibiotic and immunodepressant, chemotherapy of tumors, organ transplant and Chinese mugwort
Grow spreading of disease, lead to the crowd that immune system is suppressed to be on the increase, the incidence of disease of deep fungal infection starts to present
Significantly ascendant trend, and the utilization with antifungal drug, the drug resistance of fungi also increasingly stronger so that antifungal drug
Application has the trend of swift and violent increase.Therefore, the medicine of anti-deep fungal infection has become one of study hotspot of anti-infectious agent, day
Benefit causes the concern of people.
Echinocandin is the new antifungal of a class, obvious not with the mechanism of action of current clinical conventional antifungal drug
With, it is β -1, the inhibitor of 3- glucan synthase, act on the cell membrane of fungi.Cell membrane is fungi and mammalian cell
Between maximum difference, so the synthesis of interference cell wall, the integrality of impact cell membrane will lead to fungal cell dead and right
People's fanout free region, therefore side effect are less, and security is higher.
MFG is a kind of water-soluble semi synthesis echinocandin antifungal agent thing, by precursor compound FR901379
(or claiming " WF11899A ") synthesizes after deacylation enzyme hydrolysis and obtains.Micafungin precursor compound FR901379 is sheath stem point
The fermentating metabolism product of mould genus (Coleophoma sp.), is the primary raw material of synthesis MFG, and its structural formula is as follows：
Chinese invention patent, notification number CN102952179B, disclose《A kind of high-purity micafungin precursor compound
The preparation method of FR901379》, technical scheme disclosed in it is essentially the purification circuit to MFG precursor, and it is not taken off
Show how to synthesize MFG precursor, and there is complex operation, the longer problem of syntheti c route.Meanwhile, Chinese invention patent, public
The number of opening CN103848900A, it discloses《A kind of method isolating and purifying WF11899A from zymotic fluid》, it substantially also belongs to
In the purification circuit to MFG precursor and it needs to soak, using the alcohol or ketone of C1～C4, the zymotic fluid containing WF11899A, and
Need to carry out wash-out collection and so as to HPLC tracing detection by reverse chromatograms post.Therefore, there is also and prepare MFG precursor
Complex operation step, complicated process of preparation, and need chemical reagent that these are harmful to using ketone to human body and environment, therefore not
Too suitable industrialization large-scale production.
In view of this it is necessary to be improved to the preparation method of MFG precursor of the prior art, on solving
State problem.
Content of the invention
It is an object of the invention to disclosing a kind of preparation method of MFG precursor, in order to realize MFG precursor
Commercial application, reduces preparation difficulty, reduces preparation process and human body and environment are had undesirable effect.
For achieving the above object, the invention provides a kind of preparation method of MFG precursor, walk including following
Suddenly：
Step (1), take a small amount of MFG precursor producing strains spore suspension, be inoculated on seed culture medium, in 24～28
At DEG C, cultivate 2～4 days；
Step (2), seed culture medium is inoculated in fermentation tank and adds fermentation medium and is fermented, in fermentation tank
Fermentation temperature is 24～28 DEG C, and fermentation time is 10～12 days；
Step (3), when fermentation to 40～60h when add the carrier of oxygen, and during the fermentation when total reducing sugar be less than 2.0g/
Flow feeding culture medium during 100mL；
Step (4), in zymotic fluid add acid make zymotic fluid pH be in 3～4, plate-frame filtering, then add polar solvent
It is stripped concentrating, prepared MFG precursor concentrate；
Step (5), by MFG precursor concentrate through macroporous resin adsorption, polar solvent parses, prepared MFG
Precursor desorbed solution；
Step (6), MFG precursor desorbed solution are crystallized, are dried, and MFG precursor in powder is obtained.
As a further improvement on the present invention, the seed culture medium in described step (1) consists of：Glucose 5～15g/
L, glycerine 5～15g/L, Dried Corn Steep Liquor Powder 10～30g/L, dusty yeast 5～15g/L, tryptone 5～15g/L, sodium chloride 1～
3g/L, potassium dihydrogen phosphate 1～3g/L and calcium carbonate 1～3g/L.
As a further improvement on the present invention, the fermentation medium in described step (2) consists of：Glucose 5～15g/
L, sorbierite 70～90g/L, Dried Corn Steep Liquor Powder 6～10g/L, soybean cake powder 20～30g/L, Triammonium citrate 2～4g/L, seven water
Ferrous sulfate 0.2～0.4g/L, ammonium sulfate 2～4g/L, epsom salt 1～3g/L, calcium carbonate 3～5g/L and dipotassium hydrogen phosphate
1～3g/L.
As a further improvement on the present invention, the carrier of oxygen in described step (3) is selected from n-hexane, n-dodecane or tells
Temperature -80；The additional amount of the carrier of oxygen is the 1.0%～3.0% of fermenter volume.
As a further improvement on the present invention, the supplemented medium in described step (3) consists of：Glucose 0.5～
1.0g/L, Dried Corn Steep Liquor Powder 0.05～0.2g/L, soybean cake powder 0.05～0.2g/L and calcium carbonate 0.01～0.05g/L.
As a further improvement on the present invention, the polar solvent in described step (4) and step (5) is selected from methyl alcohol, ethanol
Or acetonitrile.
As a further improvement on the present invention, the macroreticular resin in described step (5) is selected from HP20 macroreticular resin, D1300
Macroreticular resin or SP207 macroreticular resin.
As a further improvement on the present invention, the crystallization mode in described step (6) is concentration and evaporation crystallization or solvent
Sedimentation crystallization, crystallization temperature is 40～60 DEG C；The used solvent of described solvent sedimentation crystallization be selected from petroleum ether, n-hexane or
Hexamethylene.
As a further improvement on the present invention, the acid added in zymotic fluid in described step (4) is selected from dilute sulfuric acid, dilute
Hydrochloric acid or acetic acid.
As a further improvement on the present invention, the drying mode in described step (6) includes vacuum drying, falling film evaporation is done
Dry, baking temperature is 40～105 DEG C.
Compared with prior art, the invention has the beneficial effects as follows：In the present invention, lead to too small amount of MFG precursor to produce
Raw bacterium spore suspension is simultaneously cultivated, and during fermentation is carried out to zymotic fluid in add the carrier of oxygen, improve fermentation tank in dissolving
Oxygen concentration, improves the yield 20%～40% of MFG precursor；The acidified broken wall treatment of zymotic fluid, and use macroreticular resin
Absorption polar solvent parsing obtains MFG precursor desorbed solution, then obtains MFG precursor powder by desorbed solution by crystallization
End；The present invention can be used for synthesizing MFG precursor, and operation sequence is simple, and method is stable, favorable reproducibility.
Specific embodiment
With reference to each embodiment, the present invention is described in detail, but it should explanation, these embodiments are simultaneously
Non- limitation of the present invention, those of ordinary skill in the art are according in these embodiment institute work energy, method or structure
Equivalent transformation or replacement, belong within protection scope of the present invention.
Unless there is specified otherwise in specification, the component in each embodiment in the present invention, raw material are all pure using analyzing
Rank.In addition, " g " in each embodiment is unit of weight " gram "；" h " is chronomere's " hour "；" ml " be volume unit " in the least
Rise "；" room temperature " is 23 DEG C；“BV/H”：The expression of space flow speed, that is, refer to that in post, the unit interval (h) flows through unit volume resin
Average liquid measure.“HPLC”：High performance liquid chromatography.
Embodiment one：
The preparation method of this MFG precursor comprises the following steps.
Step (1), take a small amount of MFG precursor, that is, FR179642 (that is, WF11899A) producing strains spore suspension, connects
Plant and cultivated on seed culture medium.Each group in seed culture medium is divided into (unit：g/L)：Glucose 15, glycerine 15, beautiful
Rice ＆ peanut milk dry powder 30, dusty yeast 15, tryptone 15, sodium chloride 3, potassium dihydrogen phosphate 3, calcium carbonate 3, balance of water.24 DEG C of cultures 2
My god.
Step (2), seed culture medium is inoculated in fermentation tank and adds fermentation medium and fermented.In fermentation medium
Each group be divided into (unit：g/L)：Glucose 15, sorbierite 90, Dried Corn Steep Liquor Powder 10, soybean cake powder 30, Triammonium citrate 4, seven
Aqueous ferrous sulfate 0.4, ammonium sulfate 4, epsom salt 3, calcium carbonate 5, dipotassium hydrogen phosphate 3, balance of water.Cultivate 10 days for 28 DEG C.
Step (3), when fermentation is carried out to 40h, add n-hexane as the carrier of oxygen.The additional amount of n-hexane is fermentation tank
Long-pending 1.0%.When total sugar amount is less than 2.0g/100mL, proceed by stream plus flow feeding culture medium.In this supplemented medium
Each group be divided into (unit：g/L)：Glucose 0.5, Dried Corn Steep Liquor Powder 0.05, soybean cake powder 0.05, calcium carbonate 0.01, balance of
Water.
Step (4), fermentation are extracted after completing to carry out putting tank, and are specially：Extract and add in zymotic fluid during WF11899A
Acid, making zymotic fluid be acidified to pH is in 3, and after crossing sheet frame, plus methyl alcohol extracting concentrates, prepared MFG precursor concentrate.In this reality
Apply in example, the acid added in zymotic fluid is the dilute sulfuric acid of 10wt% for concentration.
Step (5), by the MFG precursor concentrate macroporous resin adsorption of model HP20.Adsorption flow rate controls
0.5～1.5BV/H, washes after the completion of absorption, is parsed as polar solvent with the methyl alcohol of 60wt%, and parsing flow velocity 1～
2BV/H, merges active principle, to prepare MFG precursor desorbed solution.
Step (6), MFG precursor desorbed solution are crystallized, are dried, and MFG precursor in powder is obtained.At this
In embodiment, crystallization mode crystallizes for concentration and evaporation, and drying mode is dried for concentration and evaporation, 40 DEG C of baking temperature.
MFG precursor obtained by the present embodiment detects through HPLC, normalizes purity 95.5%.
Embodiment two：
The preparation method of this MFG precursor comprises the following steps.
Step (1), take a small amount of MFG precursor, i.e. FR179642 (that is, WF11899A) producing strains spore suspension,
It is inoculated in and cultivated on seed culture medium.Each group in seed culture medium component is divided into (unit：g/L)：Glucose 5, glycerine
5, Dried Corn Steep Liquor Powder 10, dusty yeast 5, tryptone 5, sodium chloride 1, potassium dihydrogen phosphate 1, calcium carbonate 1, balance of water.28 DEG C of trainings
Support 4 days.
Step (2), seed culture medium is inoculated in fermentation tank and adds fermentation medium and fermented.In fermentation medium
Each group be divided into (unit：g/L)：Glucose 5, sorbierite 70, Dried Corn Steep Liquor Powder 6, soybean cake powder 20, Triammonium citrate 2, seven water
Ferrous sulfate 0.2, ammonium sulfate 2, epsom salt 1, calcium carbonate 3, dipotassium hydrogen phosphate 1, balance of water.Cultivate 12 days for 24 DEG C.
When step (3), fermentation are carried out to 60h, add n-dodecane as the carrier of oxygen.The additional amount of n-dodecane is fermentation
The 3.0% of tank volume.When total sugar amount is less than 2.0g/100mL, proceed by flow feeding culture medium.In this supplemented medium
Each group be divided into (unit：g/L)：Glucose 1.0, Dried Corn Steep Liquor Powder 0.2, soybean cake powder 0.2, calcium carbonate 0.05, balance of water.
Step (4), fermentation are extracted after completing to carry out putting tank, and are specially：Extract and first add in zymotic fluid during WF11899A
Acid adding, making zymotic fluid be acidified to pH is in 4, and after crossing sheet frame, plus methyl alcohol extracting concentrates and MFG precursor concentrate is obtained.In this reality
Apply in example, the acid added in zymotic fluid is the watery hydrochloric acid of 10wt% for concentration.
Step (5), by the MFG precursor concentrate macroporous resin adsorption of model D1300.Adsorption flow rate controls
0.5～1.5BV/H, washes after the completion of absorption, is parsed as polar solvent with 60wt% ethanol, parses flow velocity 1～2BV/
H, merges active principle, to prepare MFG precursor desorbed solution.
Step (6), MFG precursor desorbed solution are crystallized, are dried, and MFG precursor in powder is obtained.At this
In embodiment, crystallization mode settles crystallization for solvent, and the used solvent of solvent sedimentation crystallization is petroleum ether, and is specially：Will
Desorbed solution concentrates, plus 15～petroleum ether of 20 times of volume of concentrate.50 DEG C of baking temperature.
MFG precursor obtained by the present embodiment detects through HPLC, normalizes purity 96.1%.
Embodiment three：
The preparation method of this MFG precursor comprises the following steps.
Step (1), take a small amount of MFG precursor, i.e. FR179642 (that is, WF11899A) producing strains spore suspension,
It is inoculated in and cultivated on seed culture medium.Each group in seed culture medium is divided into (unit：g/L)：Glucose 10, glycerine 10,
Dried Corn Steep Liquor Powder 20, dusty yeast 10, tryptone 10, sodium chloride 2, potassium dihydrogen phosphate 2, calcium carbonate 2, balance of water.26 DEG C of trainings
Support 3 days.
Step (2), seed culture medium is inoculated in fermentation tank and adds fermentation medium and fermented.In fermentation medium
Each group be divided into (unit：g/L)：Glucose 10, sorbierite 80, Dried Corn Steep Liquor Powder 8, soybean cake powder 25, Triammonium citrate 3, seven
Aqueous ferrous sulfate 0.3, ammonium sulfate 3, epsom salt 2, calcium carbonate 4, dipotassium hydrogen phosphate 2, balance of water.Cultivate 11 days for 26 DEG C.
Step (3), fermentation are carried out to 50h, add Tween-80 as the carrier of oxygen.The additional amount of Tween-80 is fermentation tank
Long-pending 2.0%.When total sugar amount is less than 2.0g/100mL, proceed by flow feeding culture medium.Each in this supplemented medium
Group is divided into (unit：g/L)：Glucose 0.8, Dried Corn Steep Liquor Powder 0.1, soybean cake powder 0.1, calcium carbonate 0.03, balance of water.
Step (4), fermentation are extracted after completing to carry out putting tank, and are specially：Extract and first add in zymotic fluid during WF11899A
Acid adding, making zymotic fluid be acidified to pH is in 3, and after crossing sheet frame, plus ethanolic extraction concentrates, prepared MFG precursor concentrate.At this
In embodiment, the acid added in zymotic fluid is the acetic acid of 1mol/L.
Step (5), by the MFG precursor concentrate macroporous resin adsorption of model SP207.Adsorption flow rate controls
0.5～1.5BV/H, washes after the completion of absorption, is parsed as polar solvent with the acetonitrile of 60wt%, and parsing flow velocity 1～
2BV/H, merges active principle, to prepare MFG precursor desorbed solution.
Step (6), MFG precursor desorbed solution are crystallized, are dried, and MFG precursor in powdery is obtained.In this reality
Apply in mode, drying mode is dried for concentration and evaporation, 50 DEG C of baking temperature.In the present embodiment, crystallization mode settles for solvent
Crystallization, the used solvent of solvent sedimentation crystallization is n-hexane, and crystallization temperature is 55 DEG C.
MFG precursor obtained by the present embodiment detects through HPLC, normalizes purity 96.3%.
Example IV：
The preparation method of this MFG precursor comprises the following steps.
Step (1), take a small amount of MFG precursor, i.e. FR179642 (that is, WF11899A) producing strains spore suspension,
It is inoculated in and cultivated on seed culture medium.Each group in seed culture medium component is divided into (unit：g/L)：Glucose 15, glycerine
15, Dried Corn Steep Liquor Powder 30, dusty yeast 15, tryptone 15, sodium chloride 3, potassium dihydrogen phosphate 3, calcium carbonate 2, balance of water.24℃
Culture 3 days.
Step (2), seed culture medium is inoculated in fermentation tank and adds fermentation medium and fermented.Fermentation medium group
Each group in point is divided into (unit：g/L)：Glucose 10, sorbierite 80, Dried Corn Steep Liquor Powder 8, soybean cake powder 25, Triammonium citrate
3, ferrous sulfate heptahydrate 0.3, ammonium sulfate 3, epsom salt 2, calcium carbonate 4, dipotassium hydrogen phosphate 2, balance of water.24 DEG C of cultures 12
My god.
Step (3), fermentation are carried out to 50h, add n-hexane as the carrier of oxygen.The additional amount of n-hexane is fermenter volume
2.5%.When total sugar amount is less than 2.0g/100mL, proceed by flow feeding culture medium.Each group in this supplemented medium
It is divided into (unit：g/L)：Glucose 0.5, Dried Corn Steep Liquor Powder 0.2, soybean cake powder 0.2, calcium carbonate 0.05, balance of water.
Step (4), fermentation are extracted after completing to carry out putting tank, and are specially：Extract and first add in zymotic fluid during WF11899A
Acid adding, making zymotic fluid be acidified to pH is in 3, and after crossing sheet frame, plus methyl alcohol extracting concentrates, prepared MFG precursor concentrate.At this
In embodiment, the acid added in zymotic fluid is the watery hydrochloric acid of 8wt% for concentration.
Step (5), by the MFG precursor concentrate macroporous resin adsorption of model SP207.Adsorption flow rate controls
0.5～1.5BV/H, washes after the completion of absorption, is parsed as polar solvent with the methyl alcohol of 60wt%, and parsing flow velocity 1～
2BV/H, merges active principle, to prepare MFG precursor desorbed solution.
Step (6), MFG precursor desorbed solution are concentrated, crystallization, and MFG precursor in powdery is obtained.In this reality
Apply in example, drying mode is dried for falling film evaporation, baking temperature is 105 DEG C.Crystallization mode settles crystallization for solvent.Crystallization temperature
For 45 DEG C.The used solvent of this solvent sedimentation crystallization is hexamethylene.
MFG precursor obtained by the present embodiment detects through HPLC, normalizes purity 95.5%.
Embodiment five：
The preparation method of this MFG precursor comprises the following steps.
Step (1), take a small amount of MFG precursor, i.e. FR179642 (that is, WF11899A) producing strains spore suspension,
It is inoculated in and cultivated on seed culture medium.Each group in seed culture medium is divided into (unit：g/L)：Glucose 8, glycerine 8, beautiful
Rice ＆ peanut milk dry powder 15, dusty yeast 10, tryptone 10, sodium chloride 3, potassium dihydrogen phosphate 3, calcium carbonate 3, balance of water, train at 28 DEG C
Support 2 days.
Step (2), seed culture medium is inoculated in fermentation tank and adds fermentation medium and fermented.In fermentation medium
Each group be divided into (unit：g/L)：Glucose 10, sorbierite 75, Dried Corn Steep Liquor Powder 10, soybean cake powder 30, Triammonium citrate 4, seven
Aqueous ferrous sulfate 0.4, ammonium sulfate 2, epsom salt 1, calcium carbonate 3, dipotassium hydrogen phosphate 1, balance of water, cultivate 12 days for 24 DEG C.
Step (3), when fermentation to 50h when, add Tween-80 as the carrier of oxygen.The additional amount of Tween-80 is fermentation tank
Long-pending 1.2%.When total sugar amount is less than 2.0g/100mL, proceed by flow feeding culture medium.Each in this supplemented medium
Group is divided into (unit：g/L)：Glucose 0.6, Dried Corn Steep Liquor Powder 0.15, soybean cake powder 0.2, calcium carbonate 0.05, balance of water.
Step (4), fermentation are extracted after completing to carry out putting tank, and are specially：Extract and first add in zymotic fluid during WF11899A
Acid adding, making zymotic fluid be acidified to pH is in 4, and after crossing sheet frame, plus ethanolic extraction concentrates, prepared MFG precursor concentrate.At this
In embodiment, the acid added in zymotic fluid is the dilute sulfuric acid of 7wt% for concentration.
Step (5), the MFG precursor concentrate macroporous resin adsorption of model D1300.Adsorption flow rate control 0.5～
1.5BV/H, washes after the completion of absorption, is parsed as polar solvent with the ethanol of 60wt%, parses flow velocity 1～2BV/H, closes
And active principle, to prepare MFG precursor desorbed solution.
Step (6), MFG precursor desorbed solution are crystallized, are dried, and MFG precursor in powdery is obtained.In this reality
Apply in mode, drying mode is vacuum drying, 50 DEG C of baking temperature.Crystallization mode settles crystallization for solvent.Crystallization temperature is 47
℃.The mixture that the used solvent of this solvent sedimentation crystallization is formed by hexamethylene and petroleum ether, and both ratios are any.
MFG precursor obtained by the present embodiment detects through HPLC, normalizes purity 95.8%.
Embodiment six：
The preparation method of this MFG precursor comprises the following steps.
Step (1), take a small amount of MFG precursor, i.e. FR179642 (that is, WF11899A) producing strains spore suspension,
It is inoculated in and cultivated on seed culture medium.Each group in seed culture medium is divided into (unit：g/L)：Glucose 5, glycerine 10, beautiful
Rice ＆ peanut milk dry powder 10, dusty yeast 8, tryptone 5, sodium chloride 1, potassium dihydrogen phosphate 1, calcium carbonate 1, balance of water, cultivate 3 at 26 DEG C
My god.
Step (2), seed culture medium is inoculated in fermentation tank and adds fermentation medium and fermented.In fermentation medium
Each group be divided into (unit：g/L)：Glucose 12, sorbierite 80, Dried Corn Steep Liquor Powder 8, soybean cake powder 20, Triammonium citrate 2, seven
Aqueous ferrous sulfate 0.2, ammonium sulfate 2, epsom salt 1, calcium carbonate 3, dipotassium hydrogen phosphate 1, balance of water, cultivate 12 days for 24 DEG C.
Step (3), when fermentation to 55h when, add n-dodecane as the carrier of oxygen.The additional amount of n-dodecane is fermentation tank
The 2.8% of volume.When total sugar amount is less than 2.0g/100mL, proceed by flow feeding culture medium.In this supplemented medium
Each group is divided into (unit：g/L)：Glucose 0.7, Dried Corn Steep Liquor Powder 0.16, soybean cake powder 0.08, calcium carbonate 0.05, balance of water.
Step (4), fermentation are extracted after completing to carry out putting tank, and are specially：Extract and first add in fermentation tank during WF11899A
Acid adding, making zymotic fluid be acidified to pH is in 4, and after crossing sheet frame, plus ethanolic extraction concentrates, prepared MFG precursor concentrate.
Step (5), by the MFG precursor concentrate macroporous resin adsorption of model HP20.Adsorption flow rate controls 0.5
～1.5BV/H, washes after the completion of absorption, is parsed as polar solvent with the ethanol of 60wt%, parses flow velocity 1～2BV/H,
Merge active principle, to prepare MFG precursor desorbed solution.
Step (6), MFG precursor desorbed solution are crystallized, are dried, and MFG precursor in powder is obtained.At this
In embodiment, crystallization mode settles crystallization for solvent, and MFG precursor desorbed solution is concentrated, and adds the volume of concentrate 15～20
Petroleum ether again, 40 DEG C of sedimentation crystallizations.Drying mode is vacuum drying, 40 DEG C of baking temperature.Vacuum drying vacuum is
0.1MPa.
MFG precursor obtained by the present embodiment detects through HPLC, normalizes purity 96.5%.
The a series of detailed description of those listed above is only for the feasibility embodiment of the present invention specifically
Bright, they simultaneously are not used to limit the scope of the invention, all equivalent implementations made without departing from skill spirit of the present invention
Or change should be included within the scope of the present invention.
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of the spirit or essential attributes of the present invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit requires rather than described above limits, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.
Moreover, it will be appreciated that although this specification is been described by according to embodiment, not each embodiment only wraps
Containing an independent technical scheme, only for clarity, those skilled in the art should for this narrating mode of specification
Using specification as an entirety, the technical scheme in each embodiment can also form those skilled in the art through appropriately combined
Understandable other embodiment.
Claims (10)
1. a kind of preparation method of MFG precursor is it is characterised in that comprise the following steps：
Step (1), take a small amount of MFG precursor producing strains spore suspension, be inoculated on seed culture medium, at 24～28 DEG C,
Culture 2～4 days；
Step (2), seed culture medium is inoculated in fermentation tank and adds fermentation medium and is fermented, the fermentation in fermentation tank
Temperature is 24～28 DEG C, and fermentation time is 10～12 days；
Step (3), when fermentation to 40～60h when add the carrier of oxygen, and during the fermentation when total reducing sugar be less than 2.0g/100mL when
Flow feeding culture medium；
Step (4), in zymotic fluid add acid make zymotic fluid pH be in 3～4, plate-frame filtering, then add polar solvent carry out
Extracting concentrates, prepared MFG precursor concentrate；
Step (5), by MFG precursor concentrate through macroporous resin adsorption, polar solvent parses, prepared MFG precursor
Desorbed solution；
Step (6), MFG precursor desorbed solution are crystallized, are dried, and MFG precursor in powder is obtained.
2. preparation method according to claim 1 is it is characterised in that the seed culture medium in described step (1) consists of：
Glucose 5～15g/L, glycerine 5～15g/L, Dried Corn Steep Liquor Powder 10～30g/L, dusty yeast 5～15g/L, tryptone 5～
15g/L, sodium chloride 1～3g/L, potassium dihydrogen phosphate 1～3g/L and calcium carbonate 1～3g/L.
3. preparation method according to claim 1 is it is characterised in that the fermentation medium in described step (2) consists of：
Glucose 5～15g/L, sorbierite 70～90g/L, Dried Corn Steep Liquor Powder 6～10g/L, soybean cake powder 20～30g/L, citric acid three
Ammonium 2～4g/L, ferrous sulfate heptahydrate 0.2～0.4g/L, ammonium sulfate 2～4g/L, epsom salt 1～3g/L, calcium carbonate 3～
5g/L and dipotassium hydrogen phosphate 1～3g/L.
4. preparation method according to claim 1 it is characterised in that the carrier of oxygen in described step (3) be selected from n-hexane,
N-dodecane or Tween-80；The additional amount of the carrier of oxygen is the 1.0%～3.0% of fermenter volume.
5. preparation method according to claim 1 is it is characterised in that the supplemented medium in described step (3) consists of：
Glucose 0.5～1.0g/L, Dried Corn Steep Liquor Powder 0.05～0.2g/L, soybean cake powder 0.05～0.2g/L and calcium carbonate 0.01～
0.05g/L.
6. preparation method according to claim 1 is it is characterised in that polar solvent in described step (4) and step (5)
Selected from methyl alcohol, ethanol or acetonitrile.
7. preparation method according to claim 1 is it is characterised in that the macroreticular resin in described step (5) is selected from HP20
Macroreticular resin, D1300 macroreticular resin or SP207 macroreticular resin.
8. preparation method according to claim 1 is it is characterised in that the crystallization mode in described step (6) is to concentrate to steam
Send out crystallization or solvent sedimentation crystallization, crystallization temperature is 40～60 DEG C；The used solvent of described solvent sedimentation crystallization is selected from stone
Oily ether, n-hexane or hexamethylene.
9. preparation method according to claim 1 is it is characterised in that added in described step (4) in zymotic fluid
Acid is selected from dilute sulfuric acid, watery hydrochloric acid or acetic acid.
10. preparation method according to claim 1 is it is characterised in that the drying mode in described step (6) includes vacuum
It is dried, falling film evaporation is dried, baking temperature is 40～105 DEG C.
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| CN108753878A (en) * | 2018-05-30 | 2018-11-06 | 博瑞生物医药（苏州）股份有限公司 | The fermentation process of micafen sodium intermediate |
| CN108753881A (en) * | 2018-05-30 | 2018-11-06 | 博瑞生物医药（苏州）股份有限公司 | The preparation method of FR179642 |
| CN108753880A (en) * | 2018-05-30 | 2018-11-06 | 博瑞生物医药（苏州）股份有限公司 | The new preparation method of micafen sodium precursor |
| CN108753877A (en) * | 2018-05-30 | 2018-11-06 | 博瑞生物医药（苏州）股份有限公司 | The fermentation process of micafen sodium intermediate FR901379 |
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| CN116496911A (en) * | 2023-04-23 | 2023-07-28 | 浙江昊清生物科技有限公司 | Ricasfungin intermediate FR901379 high-yield strain and application thereof |
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| CN108753881A (en) * | 2018-05-30 | 2018-11-06 | 博瑞生物医药（苏州）股份有限公司 | The preparation method of FR179642 |
| CN108753880A (en) * | 2018-05-30 | 2018-11-06 | 博瑞生物医药（苏州）股份有限公司 | The new preparation method of micafen sodium precursor |
| CN108753877A (en) * | 2018-05-30 | 2018-11-06 | 博瑞生物医药（苏州）股份有限公司 | The fermentation process of micafen sodium intermediate FR901379 |
| CN108441534B (en) * | 2018-05-30 | 2023-10-03 | 博瑞生物医药（苏州）股份有限公司 | New preparation method of micafungin sodium precursor |
| CN111434675A (en) * | 2019-01-11 | 2020-07-21 | 苏州纳微科技股份有限公司 | Separation and purification method of micafungin mother ring |
| CN116496911A (en) * | 2023-04-23 | 2023-07-28 | 浙江昊清生物科技有限公司 | Ricasfungin intermediate FR901379 high-yield strain and application thereof |
| CN116496911B (en) * | 2023-04-23 | 2023-12-26 | 浙江昊清生物科技有限公司 | Ricasfungin intermediate FR901379 high-yield strain and application thereof |
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