CN106498011A - A kind of preparation method of Gentamicin C1a - Google Patents
A kind of preparation method of Gentamicin C1a Download PDFInfo
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- C12P19/485—Having two saccharide radicals bound through only oxygen to non-adjacent ring carbons of the cyclohexyl radical, e.g. gentamycin, kanamycin, sisomycin, verdamycin, mutamycin, tobramycin, nebramycin, antibiotics 66-40B, 66-40D, XK-62-2, 66-40, G-418, G-52
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Abstract
The invention provides a kind of preparation method of Gentamicin C1a, which is by carrying out microwave irradiation and mutagenesis to gentamicinB producing strains, select the new strains that a Gentamicin C1a is Main Components, microwave irradiation is first carried out, mutagenesis is carried out again, so as to obtain the producing strains of Gentamicin C1a, and fermented and cultured is carried out, the zymotic fluid for obtaining carries out the crude product of isolated Gentamicin C1a by microfiltration membranes, milipore filter, NF membrane., by continuous passage, Gentamicin C1a yield is highly stable for Gentamicin C1a producing strains obtained by the present invention, and has production cost relatively low, is very suitable for commercial application.
Description
Technical field
A kind of the present invention relates to biological pharmacy technical field, more particularly to preparation method of Gentamicin C1a.
Background technology
Gentamicin is a kind of aminoglycoside antibiotics, is mainly used in treating bacterium infection, especially Gram-negative
Microbial infection.Gentamicin can be combined with bacterial ribosome 30s subunits, blocking bacterioprotein synthesis.
《The People's Republic of China's pharmacopeia》Version regulation gentamicin C components include C1, C1a, C2a, C2 within 2000, wherein,
Gentamicin C1a can replace the parent of star as synthesising bacteria anti-reflecting medicine according to rice.The molecular structural formula of Gentamicin C1a is as follows：
Gentamicin C1a is the parent for synthesizing Etimicin, and Etimicin Sulfate one kind that to be China voluntarily develop is high
Effect, low toxicity, the semi-synthetic aminoglycoside antibiotics of anti-drug resistance bacterium, are uniquely to obtain the anti-infective of first class national new drug certificate
Medicine.This product be applied to its sensitive Escherichia coli, Klebsiella pneumoniae, Serratia category, long Bacillus,
All kinds of infection that acinetobacter, Proteus, haemophilus influenzae, pseudomonas aeruginosa and staphylococcus cause.Clinical
Show this product to the preferable curative effect of following infection：Respiratory tract infection：As acute bronchitis, AECB,
Community's pulmonary infection etc..Kidney and urogenital infections：Such as acute pyelonephritis, vesical pyelonephritis or chronic bladder
Scorching acute attack etc..Skin soft-tissue infection includes furuncle, carbuncle, acute cellulitis etc..Wound, perioperatively treatment of infection or
Preventive usage.And having relatively low ear, a renal toxicity adverse reaction rate, it was demonstrated that Etimicin Sulfate is in clinical practice
Efficiently, safe of new generation semi-synthetic aminoglycoside antibiotics.
The conventional preparation method of Gentamicin C1a is isolated in zymotic fluid by micronomicin.The preparation method
There is many technology inferior positions, for example, production technology is longer, complex manufacturing, need to pass through using mixed solvent after resin adsorption
Cross three wash-outs, Gentamicin C1a can be just obtained after parsing.Meanwhile, the ethanol in mixtures of eluents and parsing agent is using distillation
Tower is reclaimed, and is therefore caused production equipment costly, and is needed to consume substantial amounts of steam, so as to cause production cost higher.
In view of this, it is necessary to which the preparation method of Gentamicin C1a of the prior art is improved, above-mentioned to solve
Problem.
Content of the invention
It is an object of the invention to a kind of preparation method of Gentamicin C1a is disclosed, in order to overcome system of the prior art
Production cost existing for Preparation Method is higher, the defect such as cumbersome.
For achieving the above object, the invention provides a kind of preparation method of Gentamicin C1a, including following step
Suddenly：
Step (1), gentamicinB producing strains are carried out microwave irradiation, then mutagenesis is carried out using chemical mutagen
Obtain Gentamicin C1a producing strains；
Step (2), the Gentamicin C1a producing strains for selecting stable and relative high yield, 35～40 DEG C of lower inclined plane cultures 8～
15 days；
Step (3), select maturation strain inclined plane be inoculated in seed culture medium, at 35～40 DEG C, under 200～250rpm
40～60h is cultivated on shaking table；Then, it is inoculated on the primary-seed medium in first class seed pot, at 30～40 DEG C, cultivates 40
～60h；Then, it is inoculated in the secondary seed medium in secondary seed tank, at 30～40 DEG C, cultivates 20～30h；
Step (4), it is inoculated on the fermentation medium in fermentation tank, at 30～40 DEG C, fermentation carried out to 110～
160h, puts tank, obtains zymotic fluid；
Step (5), the zymotic fluid obtained by step (4) remove mycelium and zymotic fluid residue using microfiltration membranes；Micro-filtration is saturating
Cross liquid isolating protein, pigment and polysaccharide is removed through milipore filter；By the ultrafiltration permeate of collection through NF membrane carry out normal temperature desalination,
Enrichment, desalinization liquor is concentrated, dry prepared Gentamicin C1a crude product.
As a further improvement on the present invention, the microwave irradiation in step (1) is specially：Draw 2mL spore liquids to hang
In EP pipes, EP pipes are put in ice bath beaker, 10～60s are processed with 700W, 2450MHz in micro-wave oven, then 20～
6～8h is carried out at 38 DEG C in constant incubator；The chemical mutagen is nitrosoguanidine that concentration is 0.4～1.0mg/mL.
As a further improvement on the present invention, slant medium in step (2) is consisted of：Cornstarch 1.8
～2.2g/L, KNO30.05～0.15g/L, NaCl0.04～0.06g/L, MgSO40.03～0.07g/L, K2HPO40.02～
0.04g/L, 1.8～2.2g/L of wheat flour, 0.09～0.11g/L of calcium carbonate, 1.7～2.3g/L of agar, balance of water.
As a further improvement on the present invention, Shake flask medium in step (3) is consisted of：Starch 1.8～
2.2g/L, 1.8～2.2g/L of soybean cake powder, 0.4～0.6g/L of glucose, 0.3～0.7g/L of corn flour, peptone 0.1～
0.3g/L, KNO30.04～0.06g/L, K2HPO40.01～0.03g/L, 0.3～0.5g/L of calcium carbonate, balance of water.
As a further improvement on the present invention, in step (3), the inoculum concentration of Shake flask medium is 10～15%；
The primary-seed medium is consisted of：0.8～1.2g/L of starch, 1.4～1.6g/L of soybean cake powder, corn flour
0.8～1.2g/L, KNO30.04～0.06g/L, 0.1～0.3g/L of peptone, 0.03～0.05g/L of bubble enemy, calcium carbonate 0.3～
0.5g/L, balance of water, the pH of primary-seed medium is 8.0～8.4；
The secondary seed medium is consisted of：1.8～2.2g/L of starch, 1.4～1.6g/L of soybean cake powder, corn flour
0.4～0.6g/L, 0.4～0.6g/L of glucose, 0.1～0.3g/L of peptone, KNO30.05～0.15g/L, bubble enemy 0.03～
0.05g/L, balance of water, the pH of secondary seed medium is 8.0～8.4.
As a further improvement on the present invention, fermentation medium in step (3) is consisted of：Starch 3.8～
4.2g/L, 3.3～3.7g/L of soybean cake powder, 0.4～0.6g/L of glucose, 1.4～1.6g/L of corn flour, peptone 0.1～
0.3g/L, KNO30.05～0.15g/L, (NH4)2SO40.04～0.06g/L, 0.03～0.05g/L of bubble enemy, calcium carbonate 0.3～
0.5g/L, balance of water, the pH of fermentation medium is 5.0～5.4.
As a further improvement on the present invention, used in step (5), microfiltration membranes remove mycelium and zymotic fluid residue
Operating pressure be 1.0～3.0bar；Used in step (5), milipore filter goes the operation pressure of isolating protein, pigment and polysaccharide
Power is 1.0～6bar；Used in step (5), NF membrane carries out normal temperature desalination, the operating pressure of enrichment is 6.0～10bar.
As a further improvement on the present invention, the microfiltration membranes in step (5) from molecular cut off be 20000～
500000 seperation film；Milipore filter in step (5) is from the seperation film that molecular cut off is 5000～20000；Described
NF membrane in step (5) is from the seperation film that molecular cut off is 200～800.
As a further improvement on the present invention, the membrane material that the microfiltration membranes are adopted is pottery or metal；Milipore filter is adopted
Membrane material is polyethylene, Kynoar or polyvinyl chloride；The membrane material that the NF membrane is adopted is polyvinyl alcohol, sulphur
Change PS membrane or cellulose acetate.
Compared with prior art, the invention has the beneficial effects as follows：In the present invention, by entering to gentamicinB producing strains
Row microwave irradiation and mutagenesis, select the new strains that a Gentamicin C1a is Main Components, first carry out microwave irradiation, then
Mutagenesis is carried out, so as to obtain the producing strains of Gentamicin C1a, and fermented and cultured is carried out, the zymotic fluid for obtaining passes through micro-filtration
Film, milipore filter, NF membrane carry out the crude product of isolated Gentamicin C1a, the Gentamicin C1a producing strains obtained by the present invention
By continuous passage, Gentamicin C1a yield is highly stable, and has production cost relatively low, is very suitable for industry
Change application.
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 structures
Equivalent transformation or replacement, belong within protection scope of the present invention.
Unless there is specified otherwise in specification, the component in each embodiment, raw material in the present invention is pure using analysis
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；" inoculum concentration " is " ratio of nutrient solution volume after moving into the volume of culture medium and being inoculated with "；When " s " is
Between unit " second "；" mg " is unit of weight " milligram "；The Speed unit " rev/min " of " rpm " for Shaking culture；" bar " is pressure
Unit of force " bar ", 1bar=0.1 MPa (Mpa).
Embodiment one：
The preparation method of the Gentamicin C1a is comprised the following steps.
Step (1), gentamicinB (mutant of micromonospora echinospora) producing strains are carried out microwave irradiation, draw 2mL spores
Sub- liquid is suspended from EP pipes, and EP pipes are put in ice bath beaker, in micro-wave oven processes 10s with 700W, 2450MHz；Then utilize
Chemical mutagen carries out mutagenesis, and chemical mutagen is nitrosoguanidine, and mutagens concentration is 0.4mg/mL, at 20 DEG C
Under, the incubated of 6h is carried out in constant incubator.Screening obtains Gentamicin C1a producing strains.
Step (2), the Gentamicin C1a producing strains for selecting stable and relative high yield, carry out inclined-plane in slant medium
Culture.Group in the slant medium is divided into (unit：g/L)：Cornstarch 1.8, KNO30.05, NaCl0.04, MgSO40.03,
K2HPO40.02, wheat flour 1.8, calcium carbonate 0.09, agar 1.7, balance of water.The pH of the slant medium natural (pH7 ±
0.2).At 35 DEG C, cultivate 8 days.
Step (3), select maturation strain inclined plane be inoculated in Shake flask medium, inoculum concentration is 10%, carries out on shaking table
Culture.Group in Shake flask medium is divided into (unit：g/L)：Starch 1.8, soybean cake powder 1.8, glucose 0.4, corn flour 0.3,
Peptone 0.1, KNO30.04, K2HPO40.01, calcium carbonate 0.3, balance of water.The pH of the Shake flask medium natural (pH7 ±
0.2).Shaking flask cultivates 40h at 35 DEG C under 200rpm.Then, be inoculated on the primary-seed medium in first class seed pot after
Continuous culture, inoculum concentration is 10%.
Group in the primary-seed medium is divided into (unit：g/L)：Starch 0.8, soybean cake powder 1.4, corn flour 0.8,
KNO30.04, peptone 0.1, bubble enemy 0.03, calcium carbonate 0.3, balance of water.The pH of primary-seed medium is 8.0.At 30 DEG C
Under, tank pressure is 0.03Mpa, cultivates 40h, continues culture in secondary seed medium of the culture transferring in secondary seed tank.
Group in the secondary seed medium is divided into (unit：g/L)：Starch 1.8, soybean cake powder 1.4, corn flour 0.4, Portugal
Grape sugar 0.4, peptone 0.1, KNO30.05, bubble enemy 0.03, balance of water.The pH of the secondary seed medium is 8.0.Inoculum concentration
For 10%, at 30 DEG C, tank pressure is 0.1Mpa, cultivates 20h.
Step (4), be inoculated in the fermentation medium in fermentation tank ferment, inoculum concentration is 10%.In the fermentation medium
Group be divided into (unit：g/L)：Starch 3.8, soybean cake powder 3.3, glucose 0.4, corn flour 1.4, peptone 0.1,
KNO30.05, (NH4)2SO40.04, bubble enemy 0.035, calcium carbonate 0.3, balance of water.The pH of the fermentation medium is 5.0, fermentation
Carry out at 30 DEG C, fermentation carries out to 110h putting tank.
(5) zymotic fluid obtained by step (4) removes mycelium and zymotic fluid residue using microfiltration membranes, in the behaviour of 1.0bar
Make under pressure, microfiltration membranes collect microfiltration membranes permeate from the ceramic membrane that molecular cut off is 20000.
By microfiltration membranes permeate through milipore filter, isolating protein, pigment and polysaccharide is removed under the conditions of 1.0bar operating pressures
Etc. big molecular impurity, milipore filter collects milipore filter permeate from the polyvinylidene fluoride film that molecular cut off is 5000.
By milipore filter permeate under the conditions of 6.0bar operating pressures through CAM that molecular cut off is 200
(a kind of NF membrane) carries out normal temperature desalination, enrichment, and desalinization liquor is concentrated, dry the solid-state Gentamicin C1a of prepared content 60%
Crude product.
Embodiment two：
The preparation method of the Gentamicin C1a is comprised the following steps.
Step (1), gentamicinB producing strains are carried out microwave irradiation, draw 2mL spore liquids and be suspended from EP pipes, EP is managed
It is put in ice bath beaker, 30s is processed with 700W, 2450MHz in micro-wave oven；Then carry out chemistry using chemical mutagen to lure
Become, chemical mutagen is nitrosoguanidine, and mutagens concentration is 0.7mg/mL, carries out 7h's in constant incubator at 30 DEG C
Incubated.Screening obtains Gentamicin C1a producing strains.
Step (2), the Gentamicin C1a producing strains for selecting stable and relative high yield, carry out inclined-plane culture.Slant medium
In group be divided into (unit：g/L)：Cornstarch 2.0, KNO30.1, NaCl0.05, MgSO40.05, K2HPO40.03, wheat flour
2.0, calcium carbonate 0.10, agar 2.0, balance of water.The pH of the slant medium is natural (pH7 ± 0.2).At 38 DEG C, culture 10
My god；
Step (3), select maturation strain inclined plane be inoculated in Shake flask medium, inoculum concentration is 12%, carries out on shaking table
Culture.Group in the Shake flask medium is divided into (unit：g/L)：Starch 2.0, soybean cake powder 2.0, glucose 0.5, corn flour
0.5, peptone 0.2, KNO30.05, K2HPO40.02, calcium carbonate 0.4, balance of water.Natural (the pH7 of the pH of the Shake flask medium
±0.2).Shaking flask cultivates 50h at 38 DEG C, under 220rpm.Then, be inoculated on the primary-seed medium in first class seed pot after
Continuous culture, inoculum concentration is 12%.
Group in the primary-seed medium is divided into (unit：g/L)：Starch 1.0, soybean cake powder 1.5, corn flour 1.0,
KNO30.05, peptone 0.2, bubble enemy 0.04, calcium carbonate 0.4, balance of water.The pH of the primary-seed medium is 8.2.35
At DEG C, tank pressure is 0.04Mpa, cultivates 50h, continues culture in secondary seed medium of the culture transferring in secondary seed tank.
Group in the secondary seed medium is divided into (unit：g/L)：Starch 2.0, soybean cake powder 1.5, corn flour 0.5, Portugal
Grape sugar 0.5, peptone 0.2, KNO30.1, bubble enemy 0.04, balance of water.The pH of the secondary seed medium is 8.2, inoculum concentration
For 12%.At 35 DEG C, tank pressure is 0.15Mpa, cultivates 25h.
Step (4), be inoculated in the fermentation medium in fermentation tank ferment, inoculum concentration is 12%.In the fermentation medium
Group be divided into (unit：g/L)：Starch 4.0, soybean cake powder 3.5, glucose 0.5, corn flour 1.5, peptone 0.2, KNO30.1,
(NH4)2SO40.05, bubble enemy 0.04, calcium carbonate 0.4, balance of water.The pH of the fermentation medium is 5.2, and fermentation is entered at 35 DEG C
OK, fermentation carries out to 135h putting tank.
Step (5), the zymotic fluid obtained by step (4) remove mycelium and zymotic fluid residue using microfiltration membranes,
Under the operating pressure of 2.0bar, microfiltration membranes collect microfiltration membranes permeate from the ceramic membrane that molecular cut off is 200000.
Microfiltration membranes permeate is removed isolating protein, pigment and polysaccharide etc. under the conditions of 3.0bar operating pressures through milipore filter
Big molecular impurity, milipore filter collect ultrafiltration permeate from the polyethylene film that molecular cut off is 10000.
The milipore filter permeate of collection is gathered through the sulfonation that molecular cut off is 500 under the conditions of 8.0bar operating pressures
Sulfone film (a kind of NF membrane) carries out normal temperature desalination, enrichment, and desalinization liquor is concentrated, dry the solid-state gentamicin of prepared content 72%
C1a crude products.
Embodiment three：
The preparation method of the Gentamicin C1a is comprised the following steps.
Step (1), gentamicinB producing strains are carried out microwave irradiation, draw 2mL spore liquids and be suspended from EP pipes, EP is managed
It is put in ice bath beaker, 60s is processed with 700W, 2450MHz in micro-wave oven；Then carry out chemistry using chemical mutagen to lure
Become, chemical mutagen is nitrosoguanidine, and mutagens concentration is 1.0mg/mL, carries out 8h's in constant incubator at 38 DEG C
Incubated.Screening obtains Gentamicin C1a producing strains.
Step (2), the Gentamicin C1a producing strains for selecting stable and relative high yield, carry out inclined-plane culture, slant medium
In group be divided into (unit：g/L)：Cornstarch 2.2, KNO30.15, NaCl0.06, MgSO40.07, K2HPO40.04, wheat flour
2.2, calcium carbonate 0.11, agar 2.3, balance of water.The pH of the slant medium is natural (pH7 ± 0.2).15 are cultivated at 40 DEG C
My god.
Step (3), select maturation strain inclined plane be inoculated in Shake flask medium, inoculum concentration is 15%, carries out on shaking table
Culture.Group in Shake flask medium is divided into (unit：g/L)：Starch 2.2, soybean cake powder 2.2, glucose 0.6, corn flour 0.7,
Peptone 0.3, KNO30.06, K2HPO40.03, calcium carbonate 0.5, balance of water.The pH of the seed culture medium natural (pH7 ±
0.2).Shaking flask cultivates 60h at 40 DEG C, under 250rpm.Then, it is inoculated on the primary-seed medium in first class seed pot and continues
Culture, inoculum concentration is 15%.
Group in the primary-seed medium is divided into (unit：g/L)：Starch 1.2, soybean cake powder 1.6, corn flour 1.2,
KNO30.06, peptone 0.3, bubble enemy 0.05, calcium carbonate 0.5, balance of water.The pH of the primary-seed medium be 8.0～
8.4.At 40 DEG C, tank pressure is 0.05Mpa, cultivates 60h, and culture transferring continues training in the secondary seed medium of secondary seed tank
Support.
Group in the secondary seed medium is divided into (unit：g/L)：Starch 2.2, soybean cake powder 1.6, corn flour 0.6, Portugal
Grape sugar 0.6, peptone 0.3, KNO30.15, bubble enemy 0.05, balance of water.The pH of the secondary seed medium is 8.4, inoculum concentration
For 15%.At 40 DEG C, tank pressure is 0.2Mpa, cultivates 30h.
Step (4), be inoculated in the fermentation medium in fermentation tank ferment, inoculum concentration is 15%.In the fermentation medium
Group be divided into (unit：g/L)：Starch 4.2, soybean cake powder 3.7, glucose 0.6, corn flour 1.6, peptone 0.3,
KNO30.15, (NH4)2SO40.06, bubble enemy 0.05, calcium carbonate 0.5, balance of water.The pH of the fermentation medium is 5.4.Fermentation
Carry out at 40 DEG C, fermentation carries out to 160h putting tank.
Step (5), the zymotic fluid obtained by step (4) remove mycelium using microfiltration membranes, and zymotic fluid residue, in 3.0bar
Operating pressure under, microfiltration membranes are 400000 microfiltration membranes from molecular cut off, and the membrane material of the microfiltration membranes is metal.Collect micro-
Filter membrane permeate.
Microfiltration membranes permeate is removed isolating protein, pigment and polysaccharide etc. under the conditions of 6.0bar operating pressures through milipore filter
Big molecular impurity, milipore filter collect milipore filter permeate from the pdythene film that molecular cut off is 20000.
By the milipore filter permeate of collection under the conditions of 10bar operating pressures through polyethylene that molecular cut off is 800
Alcohol film (a kind of NF membrane) carries out normal temperature desalination, enrichment, and desalinization liquor is concentrated, dry the solid-state gentamicin of prepared content 65%
C1a crude products.
The a series of detailed description in detail of those listed above is only for feasibility embodiment of the invention specifically
Bright, they are simultaneously not used to limit the scope of the invention, all equivalent implementations that is 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 spirit or essential attributes without departing substantially from 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 is required rather than described above is limited, 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 is only wrapped
Contain an independent technical scheme, this narrating mode of specification is only that those skilled in the art should for clarity
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 (9)
1. a kind of preparation method of Gentamicin C1a, it is characterised in that comprise the following steps：
Step (1), gentamicinB producing strains are carried out microwave irradiation, then mutagenesis is carried out using chemical mutagen and obtained
Gentamicin C1a producing strains；
Step (2), the Gentamicin C1a producing strains for selecting stable and relative high yield, in 35～40 DEG C of lower inclined plane cultures 8～15
My god；
Step (3), select maturation strain inclined plane be inoculated in seed culture medium, at 35～40 DEG C, in shaking table under 200～250rpm
40～60h of upper culture；Then, be inoculated on the primary-seed medium in first class seed pot, at 30～40 DEG C culture 40～
60h；Then, it is inoculated in the secondary seed medium in secondary seed tank, at 30～40 DEG C, cultivates 20～30h；
Step (4), it is inoculated on the fermentation medium in fermentation tank, at 30～40 DEG C, fermentation is carried out to 110～160h, is put
Tank, obtains zymotic fluid；
Step (5), the zymotic fluid obtained by step (4) remove mycelium and zymotic fluid residue using microfiltration membranes；Micro-filtration permeate
Isolating protein, pigment and polysaccharide are removed through milipore filter；The ultrafiltration permeate of collection is carried out normal temperature desalination, richness through NF membrane
Collection, desalinization liquor is concentrated, dry prepared Gentamicin C1a crude product.
2. preparation method according to claim 1, the microwave irradiation in step (1) are specially：Draw 2mL spore liquids
It is suspended from EP pipes, EP pipes is put in ice bath beaker, 10～60s is processed with 700W, 2450MHz in micro-wave oven, then 20
6～8h is carried out at～38 DEG C in constant incubator；The chemical mutagen is nitrosoguanidine that concentration is 0.4～1.0mg/mL.
3. preparation method according to claim 1, the slant medium in step (2) are consisted of：Cornstarch
1.8～2.2g/L, KNO30.05～0.15g/L, NaCl0.04～0.06g/L, MgSO40.03～0.07g/L, K2HPO4
0.02～0.04g/L, 1.8～2.2g/L of wheat flour, 0.09～0.11g/L of calcium carbonate, 1.7～2.3g/L of agar, balance of water.
4. preparation method according to claim 1, the Shake flask medium in step (3) are consisted of：Starch 1.8～
2.2g/L, 1.8～2.2g/L of soybean cake powder, 0.4～0.6g/L of glucose, 0.3～0.7g/L of corn flour, peptone 0.1～
0.3g/L, KNO30.04～0.06g/L, K2HPO40.01～0.03g/L, 0.3～0.5g/L of calcium carbonate, balance of water.
5. preparation method according to claim 1, in step (3), the inoculum concentration of Shake flask medium is 10～15%；
The primary-seed medium is consisted of：0.8～1.2g/L of starch, 1.4～1.6g/L of soybean cake powder, corn flour 0.8
～1.2g/L, KNO30.04～0.06g/L, 0.1～0.3g/L of peptone, 0.03～0.05g/L of bubble enemy, calcium carbonate 0.3～
0.5g/L, balance of water, the pH of primary-seed medium is 8.0～8.4；
The secondary seed medium is consisted of：1.8～2.2g/L of starch, 1.4～1.6g/L of soybean cake powder, corn flour 0.4
～0.6g/L, 0.4～0.6g/L of glucose, 0.1～0.3g/L of peptone, KNO30.05～0.15g/L, bubble enemy 0.03～
0.05g/L, balance of water, the pH of secondary seed medium is 8.0～8.4.
6. preparation method according to claim 1, the fermentation medium in step (3) are consisted of：Starch 3.8～
4.2g/L, 3.3～3.7g/L of soybean cake powder, 0.4～0.6g/L of glucose, 1.4～1.6g/L of corn flour, peptone 0.1～
0.3g/L, KNO30.05～0.15g/L, (NH4)2SO40.04～0.06g/L, 0.03～0.05g/L of bubble enemy, calcium carbonate 0.3
～0.5g/L, balance of water, the pH of fermentation medium is 5.0～5.4.
7. preparation method according to claim 1, used in step (5), microfiltration membranes remove mycelium and zymotic fluid is residual
The operating pressure of base is 1.0～3.0bar；Used in step (5), milipore filter goes the operation of isolating protein, pigment and polysaccharide
Pressure is 1.0～6bar；Used in step (5) NF membrane carry out normal temperature desalination, enrichment operating pressure be 6.0～
10bar.
8. preparation method according to claim 1, the microfiltration membranes in step (5) are 20000 from molecular cut off
～500000 seperation film；Milipore filter in step (5) is from the seperation film that molecular cut off is 5000～20000；Institute
The NF membrane in step (5) is stated from the seperation film that molecular cut off is 200～800.
9. preparation method according to claim 8, the membrane material that the microfiltration membranes are adopted are pottery or metal；Milipore filter
The membrane material for adopting is polyethylene, Kynoar or polyvinyl chloride；The membrane material that the NF membrane is adopted for polyvinyl alcohol,
Sulfonated polysulfone membrane or cellulose acetate.
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| CN107686490A (en) * | 2017-10-25 | 2018-02-13 | 陈磊 | A kind of method for extracting rifamycin B |
| CN110499308A (en) * | 2019-09-23 | 2019-11-26 | 华东理工大学青岛创新研究院 | The method of high flux screening gentamicin superior strain based on ARTP complex mutation technology |
| CN110628850A (en) * | 2019-09-23 | 2019-12-31 | 华东理工大学青岛创新研究院 | Method for semi-continuously producing gentamicin C1a based on control of specific growth rate |
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| CN110499308A (en) * | 2019-09-23 | 2019-11-26 | 华东理工大学青岛创新研究院 | The method of high flux screening gentamicin superior strain based on ARTP complex mutation technology |
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