CN103205479B - A kind of culture medium for being used to produce ECB - Google Patents
A kind of culture medium for being used to produce ECB Download PDFInfo
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- CN103205479B CN103205479B CN201210015967.8A CN201210015967A CN103205479B CN 103205479 B CN103205479 B CN 103205479B CN 201210015967 A CN201210015967 A CN 201210015967A CN 103205479 B CN103205479 B CN 103205479B
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Abstract
The invention discloses a kind of culture medium for being used to produce ECB and ECB (ECB) production method using the culture medium.Described culture medium contains carbon source, organic nitrogen source and inorganic salts, wherein described carbon source includes glucose, starch and grease.The yield for producing ECB using Aspergillus (Aspergillus nidulans) using the culture medium of the present invention is high, and the more existing culture medium of potency improves 3~4 times, reaches 750~900 μ g/ml.
Description
Technical field
The invention belongs to biological technical field, more particularly to a kind of culture medium and use for being used to produce ECB
The ECB production method of the culture medium.
Background technology
In the 1970s, people obtain first naturally in fungi (Aspergillus nidulans) zymotic fluid
Lipopeptide compound ECB (echinocandin B, hereinafter referred to as ECB), research finds that it is β -1,3-D- glucans
The noncompetitive inhibitor of synzyme, mainly acts on Mycotoruloides (Candidas) and Eurotium (Aspergillus).
ECB prevents fungi from synthesizing glucan by suppressing β -1,3-D- glucan synthases in fungus body, so that cell membrane knot
Structure is abnormal, causes cell rupture, and cellular content seepage causes fungi dead.ECB to Cytochrome P450 without effect, it is bad
Reaction is few, with amphotericin B and triazole antifungal agent thing without cross resistance, but due to toxicity, especially hemolytic, and
Fail to be used for clinic.
Using natural products ECB as raw material, the antifungal drug anidulafungin obtained is modified by side chain
(anidulafungin) it is, by Li Lai companies and the joint research and development exploitation of Vicuron Pharmaceuticals companies, to have completed
The III phases are clinical, and in the registration last stage, method of administration is drip-feed.Animal experiment study shows, with antifungal drug Ka Bo
Sweet smell is compared with MFG only, and anidulafungin is stronger to aspergillus fumigatus activity.Oesophagus monilial infection patient is carried out at one
Double blind controling test in, the effective percentage of drip-feed anidulafungin is basically identical with Fluconazole, is 97.2%, and Fluconazole has
Efficiency is 98.8%, and the adverse reaction rate of anidulafungin is low, is 10%, and control group is 13%, therefore is had well
Market prospects.
ECB structural formulas are as follows：
ECB
United States Patent (USP) 4,024,246 is disclosed is fermented using Aspergillus (Aspergillus nidulans) NRRL 8112
Produce ECB process：From slant medium, (catsup 2%, baby oat 2%, agar 2%, water 94%, above-mentioned percentage is equal
Be weight percentage) on take ECB producing strains to be inoculated in containing seed culture medium (glucose 1%, glycerine 1%, Cottonseed Meal 2.5%, carbon
Sour calcium 0.1%, water 95.4%, above-mentioned percentage is weight percentage) in, 25 DEG C of shaken cultivations are inoculated in after 48 hours containing hair
Ferment culture medium (sucrose 2%, maltose 1%, malt extract 1%, molasses 0.5%, corn steep liquor 0.5%, casein hydrolysis
0.5%, water 94.5%, above-mentioned percentage is weight percentage) fermentation tank in, under ventilation condition, 25 DEG C cultivate 5 days, warp
HPLC detection potency is 150~200 μ g/ml.
United States Patent (USP) 4,024,245 is disclosed is fermented using Aspergillus (Aspergillus rugulosus) NRRL 8113
Produce ECB process：From slant medium (dextrin 1%, casein hydrolysis 0.2%, yeast extract 0.1%, beef extract 0.1%,
KCl 0.02%, MgSO4·7H2O 0.02%, FeSO4·7H2O 0.0004%, water 98.5596%) on take ECB producing strains to connect
Plant in containing seed culture medium (sucrose 2.5%, molasses 3.6%, corn steep liquor 0.6%, casein hydrolysis 1%, K2HPO40.2%, water
92.1%, above-mentioned percentage is weight percentage) in, 25 DEG C of shaken cultivations are inoculated in containing fermentation medium (Portugal after 24 hours
Grape sugar 2.5%, starch 1%, peptone 1%, molasses 0.5%, casein hydrolysis 0.4%, MgSO4·7H2O 0.7%,
KCl0.2%, FeSO4·7H2O 0.004%, CaCO30.2%, above-mentioned percentage is weight percentage) fermentation tank in,
Under ventilation condition, 25 DEG C are cultivated 4 days, are 100~200 μ g/ml through HPLC detection potency.
Above-mentioned two invention respectively discloses compound ECB that a kind of bacterium produces and preparation method thereof, but uses both
Method produces ECB, and fermented and cultured products collection efficiency is relatively low, therefore can not all meet industrialized requirement.
Therefore, this area is in the urgent need to a kind of fermentation medium for making tunning ECB yields high.
The content of the invention
Therefore, the technical problem to be solved in the present invention is sent out aiming at current Aspergillus (Aspergillus nidulans)
The not enough of ferment production ECB low yield is suitable for culture Aspergillus (Aspergillus nidulans) there is provided one kind, can
Significantly improve the culture medium of ECB fermentation level.
The technical scheme that the present invention solves above-mentioned technical problem is as follows：
One of technical scheme is：A kind of culture medium for being used to produce ECB, described culture medium contains
Carbon source, organic nitrogen source and inorganic salts, wherein, described carbon source includes glucose, starch and grease.
In the preference of the present invention, described culture medium includes：Glucose 2-10%, starch 2-6% and grease
0.1-2%.In the present invention, described percentage is the mass percent for accounting for culture medium.
It is furthermore preferred that described culture medium includes：Glucose 4-8%, starch 3-5% and grease 0.5-1.5%.
Described starch can be the one or more in conventional various plant amylums and converted starch, such as plant is formed sediment
Powder includes farina, cornstarch, wheaten starch, sweet potato starch, green starch, tapioca, water caltrop starch, lotus root and formed sediment
Powder；Converted starch includes dextrin, maltodextrin etc..One kind preferably in farina, cornstarch and converted starch
Or a variety of, most preferably farina.The preferred natural oil of described grease, more preferably selected from following one or more：Beans
Oil, corn oil, sunflower oil, fish oil and lecithin.
In the preference of the present invention, described culture medium includes：Glucose 4-8%, farina 3-5% and beans
Oily 0.5-1.5%.
In the preference of the present invention, described culture medium includes：Glucose 2-10%, starch 2-6%, organic nitrogen source
1-5%, grease 0.1-2%, inorganic salts 0.04-0.8%.Inorganic salts preferred 0.06-0.5%, more preferably 0.1-0.3%.
In the present invention, described organic nitrogen source is conventional, preferably is selected from following one or more：Fish meal protein peptone, soybean
Peptone, Dried Corn Steep Liquor Powder, peanut meal, soyabean cake, cotton seed meal and dregs of beans.
In the present invention, described inorganic salts are conventional, preferably are selected from following one or more：Divalent magnesium, ferrous iron, monovalence
The inorganic salts of potassium and divalent calcium.It is furthermore preferred that described inorganic salts are selected from following one or more：MgSO4·7H2O、KCl、
FeSO4·7H2O and CaCO3
In the preference of the present invention, described culture medium includes：Glucose 2-10%, starch 2-6%, organic nitrogen source
1-5%, grease 0.1-2%, MgSO4·7H2O 0.01-0.1%, KCl 0.01-0.1%, FeSO4·7H2O 0.001-
0.1% and CaCO30.02-0.5%.
In another preference of the present invention, described culture medium includes：Glucose 2-10%, farina 2-6%,
Organic nitrogen source 1-5%, grease 0.1-2%, MgSO4·7H2O 0.01-0.1%, KCl0.01-0.1%, FeSO4·7H2O
0.001-0.1% and CaCO30.02-0.5%.
In another preference of the present invention, described culture medium includes：Glucose 6%, farina 4%, fish meal
Peptone 2%, soya-bean oil 0.5%, KCl 0.02%, MgSO4·7H2O 0.02%, FeSO4·7H2O 0.004%, CaCO3
0.2%, surplus is water, pH6.5.
In the present invention, the pH value of described culture medium is routine, can be so that naturally, can also adjust, preferably pH4-8 is more excellent
Select pH6-7, most preferably pH6.5.
In another preference of the present invention, during the fermentation with NaOH, NH3Or CaCO3Adjust the pH of the culture medium
Value.
In the present invention, the strain of fermenting and producing ECB is containing ECB in any tunning in this area
Mushroom, preferably Aspergillus (Aspergillus nidulans).Described Aspergillus (Aspergillus nidulans) includes
Any species of (but not limited to) Eurotium, preferably is selected from Aspergillus nidulans ATCC 58396,
Aspergillus nidulans NRRL 8112, Aspergillus nidulans NRRL 8113 etc..For example also include
Aspergillus rugulosus。
The two of technical scheme are：The purposes of described culture medium, the fermenting and producing for ECB.
The three of technical scheme are：A kind of method that ECB is produced using described culture medium, it is described
Method includes：
(1) under conditions of suitable Aspergillus (Aspergillus nidulans) growth, sent out in described culture medium
Ferment culture Aspergillus (Aspergillus nidulans)；With
(2) separation obtains ECB from Aspergillus (Aspergillus nidulans) culture.
Raw material or reagent used in the present invention is commercially available in addition to special instruction.
Compared to prior art, beneficial effects of the present invention are as follows：The present invention provides a kind of for producing ECB
Culture medium, the yield for producing ECB using the fermentation medium of the present invention is high, and the more existing culture medium of potency improves 3~4 times,
Reach 750~900 μ g/ml.
Embodiment
The present inventor is had found during fermentation method production ECB, fermented and cultured by for a long time and widely studying
Base is to influence the key factor of ECB fermentation yield, and the carbon source in conventional ECB fermentation medium is carried out
Improvement, using combination carbon source, can greatly improve and (improve more than 3 times, preferred 4 times of raising) yield of ECB.Base
The present invention is completed in this.
In the present invention, described carbon source refers to the nutrient source of carbon needed for providing microbial nutrition.Nitrogen source refers to energy
The nutrient source of nitrogen needed for enough providing microbial nutrition.Inorganic salts are to exercise to constitute drive member, enzymatic activity composition or maintain
Enzymatic activity, regulation osmotic pressure, pH etc. composition.
The present inventor optimizes to fermentation medium by the following method：
Single factor experiment is done to the carbon source of culture medium, nitrogen source, inorganic salts ingredients and content, these factors is observed and thalline is given birth to
The influence of long and Product formation amount, filters out carbon source, nitrogen source, inorganic salts kind with higher ECB fermentation level respectively
Class.Then two factors interaction experiment and multifactorial experiment are carried out again.The present invention using " Orthogonal Experiment and Design " mathematical method come
Nutrient media components and concentration are determined, and by variance analysis, it is determined that the larger factor of influence.As a result find, carbon is worked as in culture medium
When source includes glucose, starch and grease, ECB fermentation yield is carried higher significantly, and this is and existing spine
The different place of white rhzomorph B fermentation culture medium, and organic nitrogen source and inorganic salts are using in conventional ECB fermentation medium
Corresponding composition and content.These compositions can be substituted with other compositions well known to those skilled in the art, and this area
Technical staff be not difficult determine its content.The present inventors have additionally discovered that, formed sediment when the carbon source in culture medium includes glucose, potato
When powder and grease, for promoting the production of ECB more effective, other starch effects are slightly inferior.
The great discovery of the present inventor is the combination that the carbon source in culture medium is improved to glucose, starch and grease.
Glucose is quickly to utilize carbon source, and grease is to utilize carbon source at a slow speed.Present invention discover that the combination of the short-acting carbon source of the length is white for spine
Rhzomorph B high yield is highly beneficial.Further and again on the basis of this to other components and content, and between these components and carbon source
Combination be optimized, obtain finally a comparison be adapted to Aspergillus ferment ECB factory formula, so as to try to achieve height
Production.
Through the culture medium prescription after present invention optimization, containing enough nutritional ingredients, the need for shake flask test being met,
Shake flask fermentation result successfully can be amplified to fermentation tank again.In one embodiment of the invention, the culture medium and optimization
Culture medium in the past is compared, the yield of ECB reaches 900mg/L, 3.5 times of output increased.And because of culture medium used
Ingredient prices are relatively low, production cost is also greatly reduced.
As a kind of preferred embodiment of the present invention, the composition and content of described culture medium are：Glucose 2-10%,
Farina 2-6%, fish meal protein peptone 1-5%, soya-bean oil 0.1-2%, MgSO4·7H2O0.01-0.1%, KCl 0.01-
0.1%th, FeSO4·7H2O 0.001-0.1%, CaCO30.02-0.5%, surplus is water, pH6-7.
The culture medium of the present invention, which is suitable for any this area, is used for the Aspergillus of fermenting and producing ECB, including but not
It is limited to Aspergillus nidulans.For example, it is also possible to be Aspergillus rugulosus.
Present invention also offers the method for preparing the culture medium, comprise the following steps：By described glucose, starch,
Grease, organic nitrogen source, inorganic salts are dissolved in most of water or are fully mixed into water homogeneous emulsion system in proportion,
PH value is adjusted to required scope, water constant volume is used after mixing, is then sterilized.Preferably, for some kinds of inorganic salts in regulation
Added after pH value, such as CaCO3 is added after adjustment pH value.
It is preferred that, methods described includes：By glucose, starch, grease, KCl, MgSO4·7H2O、FeSO4·
7H2O is dissolved in ninety percent water in proportion, and pH is to 6.5 for regulation, adds CaCO3, it is sufficiently mixed after dissolving, water constant volume is used, after packing
High-temperature sterilization, preferably such as 121 DEG C, 0.1MPa sterilizings 20min.
Main advantages of the present invention are：The present inventor has found in conventional ECB fermentation medium first
Carbon source optimizing is combined as glucose, starch, grease, makes after quick-acting, slow carbon source optimizing combination within limits, is conducive to
The production of ECB, the output increased of ECB is up to as many as 4 times, the life of the raising scope of the yield in medicine intermediate
Thing fermentation arts are extremely significant.
The present invention is further illustrated with embodiment below, but the present invention is not intended to be limited thereto.Do not noted in the following example
The experimental method of bright actual conditions, generally according to normal condition, or according to the condition proposed by manufacturer.Described in embodiment
" room temperature " refer to the temperature of the operation room tested, generally 25 DEG C.
The present invention uses bacterial strain for Aspergillus nidulans ATCC 58396.
The formulation of the fermentation culture method of embodiment 1 and standard potency
1st, the preparation of seed
Inclined-plane culture is inoculated with after Aspergillus nidulans ATCC 58396 in glycerine cryopreservation tube are taken out into activation
Base (agar 1.8%, surplus is water, pH naturally, 121 DEG C of sterilizing 20min for potato 2%, glucose 1%), will be inoculated with strain
Inclined-plane is placed in 28 DEG C of constant incubators, is cultivated 6 days, obtains the spore of maturation.Then by ripe spore inoculating in equipped with
30ml seed culture mediums (glucose 1%, glycerine 1%, Cottonseed Meal 2.5%, calcium carbonate 0.1%, water 95.4%, above-mentioned percentage
Be weight percentage) 250ml shaking flasks in, on 28 DEG C, 220rpm shaking tables cultivate 2 days, obtain maturation seed.
2nd, the preparation of fermentation medium
Fermentation medium is：Glucose 6%, farina 4%, fish meal protein peptone 3%, corn oil 1%, KCl
0.02%th, MgSO4·7H2O 0.02%, FeSO4·7H2O 0.004%, CaCO30.2%, surplus is water, pH6.5.
Preparation steps：Accurately weigh respectively 60g glucose, 40g farinas, 20g fish meal proteins peptone, 5g soya-bean oil,
0.2g KCl、0.2g MgSO4·7H2O、0.04g FeSO4·7H2O, is dissolved in 900ml water, adjusts pH to 6.5, adds 2g
CaCO3, 1000ml is settled to water.Per 250ml shaking flasks packing 40ml fermentation mediums, 121 DEG C, 0.1MPa sterilizings 20min.
3rd, ferment
Obtained seed is equipped with the 250ml shaking flasks of 40ml fermentation mediums with 2% inoculum concentration access, 28 are placed in
DEG C, 220rpm shaking tables continue cultivate 120 hours, take zymotic fluid 3ml, plus 3ml acetone soaks to stay overnight, centrifuging and taking supernatant is entered with HPLC
The measure of row ECB content, is 744mg/L.
The chromatograph that HPLC is used is Waters510 type high performance liquid chromatographs, and chromatographic column is C18 posts, and detector is
The Photodiode Array Detector of Waters 996, mobile phase is methanol: acetonitrile: water (7: 2: 1), and flow velocity is 1.0ml/
Min, sample size is 20 μ l, and column temperature is room temperature.
Embodiment 2-8 difference organic nitrogen sources are investigated
Slant medium and seed culture medium be the same as Example 1.
Fermentation medium：Based on the fermentative medium formula in embodiment 1, the organic nitrogen source point in fermentation medium
Do not replaced by peptone (fish meal), peptone (soybean), cotton seed meal, soyabean cake, dregs of beans, peanut meal, Dried Corn Steep Liquor Powder, content is equal
For 3%, other compositions and content are constant.
The influence of the different organic nitrogen sources of table 1
Other method be the same as Example 1.
Fermentation termination detects product potency with HPLC, the results are shown in Table 1.
As a result show, peptone (fish meal), soy peptone and Dried Corn Steep Liquor Powder effect are preferable, wherein peptone (fish meal)
Effect it is best.
The combination of embodiment 9-15 different carbon sources is investigated
Slant medium and seed culture medium be the same as Example 1.
Fermentation medium：Based on the fermentative medium formula in embodiment 1, the glucose difference in fermentation medium
Replaced by maltodextrin, sucrose, lactose, cornstarch, dextrin, farina, content is 6%, other compositions and content
It is constant.
The influence of the different carbon source of table 2
Other method be the same as Example 1.
Fermentation termination detects product potency with HPLC, the results are shown in Table 2.
As a result show, the effect of glucose and farina is preferable, wherein again optimal with glucose.
Embodiment 16-20 difference natural oils are investigated
Slant medium and seed culture medium be the same as Example 1.
Fermentation medium：Based on the fermentative medium formula in embodiment 1, soya-bean oil therein respectively by corn oil,
Sunflower oil, fish oil, lecithin are replaced, and content is 1%, and other compositions and content are constant.Lecithin is purchased from Shanghai day edible vegetable oil
Fat Co., Ltd：
The influence of the different natural oils of table 3
| Embodiment | Embodiment 16 | Embodiment 17 | Embodiment 18 | Embodiment 19 | Embodiment 20 |
| Component | Soya-bean oil | Corn oil | Sunflower oil | Fish oil | Lecithin |
| Potency mg/L | 768 | 907 | 803 | 628 | 769 |
Other method be the same as Example 1.
Fermentation termination detects product potency with HPLC, the results are shown in Table 3.
As a result show, the effect for adding corn oil is best.
The investigation of embodiment 21-25 fish meal protein peptone contents
Slant medium and seed culture medium be the same as Example 1.
Fermentation medium：Based on the fermentative medium formula in embodiment 1, organic nitrogen source peptone (fish therein
Powder) content replace with 1%, 2%, 3%, 4%, 5% respectively, other compositions and content are constant.
The influence of the fish meal protein peptone content of table 4
| Embodiment | Embodiment 21 | Embodiment 22 | Embodiment 23 | Embodiment 24 | Embodiment 25 |
| Content | 1% | 2% | 3% | 4% | 5% |
| Potency mg/L | 707 | 764 | 900 | 769 | 669 |
Other method be the same as Example 1.
Fermentation termination detects product potency with HPLC, the results are shown in Table 4.
As a result show, the effect for adding 3% fish meal protein peptone is best.
The investigation of embodiment 26-30 glucose contents
Slant medium and seed culture medium be the same as Example 1.
Fermentation medium：Based on the fermentative medium formula in embodiment 1, the content of glucose therein is replaced respectively
2%, 4%, 6%, 8%, 10% is changed to, other compositions and content are constant.
The influence of the glucose content of table 5
| Embodiment | Embodiment 26 | Embodiment 27 | Embodiment 28 | Embodiment 29 | Embodiment 30 |
| Content | 2% | 4% | 6% | 8% | 10% |
| Potency mg/L | 868 | 734 | 896 | 793 | 609 |
Other method be the same as Example 1.
Fermentation termination detects product potency with HPLC, the results are shown in Table 5.
As a result show, the effect for adding 6% glucose is best.
The investigation of embodiment 31-35 farina contents
Slant medium and seed culture medium be the same as Example 1.
Fermentation medium：Based on the fermentative medium formula in embodiment 1, farina content difference therein
2%, 3%, 4%, 5%, 6% is replaced with, other compositions and content are constant.
The influence of the farina content of table 6
| Embodiment | Embodiment 31 | Embodiment 32 | Embodiment 33 | Embodiment 34 | Embodiment 35 |
| Content | 2% | 3% | 4% | 5% | 6% |
| Potency mg/L | 762 | 851 | 896 | 815 | 654 |
Other method be the same as Example 1.
Fermentation termination detects product potency with HPLC, the results are shown in Table 6.
As a result show, the effect for adding 4% farina is best.
The investigation of embodiment 36-40 corn oil contents
Slant medium and seed culture medium be the same as Example 1.
Fermentation medium：Based on the fermentative medium formula in embodiment 1, corn oil content therein is respectively
0.1%th, 0.5%, 1%, 1.5%, 2%, other compositions and content are constant.
The influence of the corn oil content of table 7
| Embodiment | Embodiment 36 | Embodiment 37 | Embodiment 38 | Embodiment 39 | Embodiment 40 |
| Content | 0.1% | 0.5% | 1% | 1.5% | 2% |
| Potency mg/L | 719 | 845 | 908 | 828 | 619 |
Other method be the same as Example 1.
Fermentation termination detects product potency with HPLC, the results are shown in Table 7.
As a result show, the effect for adding 1% corn oil is best.
The investigation of embodiment 41-44 KCl contents
Slant medium and seed culture medium be the same as Example 1.
Fermentation medium：Based on the fermentative medium formula in embodiment 1, KCl therein content is respectively
0.01%th, 0.02%, 0.05%, 0.1%, other compositions and content are constant.
The influence of the KCl contents of table 8
| Embodiment | Embodiment 41 | Embodiment 42 | Embodiment 43 | Embodiment 44 |
| Content | 0.01% | 0.02% | 0.05% | 0.1% |
| Potency mg/L | 819 | 896 | 818 | 748 |
Other method be the same as Example 1.
Fermentation termination detects product potency with HPLC, the results are shown in Table 8.
As a result show, the effect for adding 0.02%KCl is best.
Embodiment 45-48 MgSO4The investigation of content
Slant medium and seed culture medium be the same as Example 1.
Fermentation medium：Based on the fermentative medium formula in embodiment 1, MgSO therein4Content is respectively
0.01%th, 0.02%, 0.05%, 0.1%, other compositions and content are constant.
The MgSO of table 94The influence of content
| Embodiment | Embodiment 45 | Embodiment 46 | Embodiment 47 | Embodiment 48 |
| Content | 0.01% | 0.02% | 0.05% | 0.1% |
| Potency mg/L | 834 | 896 | 850 | 793 |
Other method be the same as Example 1.
Fermentation termination detects product potency with HPLC, the results are shown in Table 9.
As a result show, add 0.02% MgSO4Effect it is best.
Embodiment 49-52 FeSO4The investigation of content
Slant medium and seed culture medium be the same as Example 1.
Fermentation medium：Based on the fermentative medium formula in embodiment 1, FeSO therein4Content is respectively
0.001%th, 0.004%, 0.01%, 0.1%, other compositions and content are constant.
The FeSO of table 104The influence of content
| Embodiment | Embodiment 49 | Embodiment 50 | Embodiment 51 | Embodiment 52 |
| Content | 0.001% | 0.004% | 0.01% | 0.1% |
| Potency mg/L | 768 | 896 | 787 | 743 |
Other method be the same as Example 1.
Fermentation termination detects product potency with HPLC, the results are shown in Table 10.
As a result show, add 0.004%FeSO4Effect it is best.
Embodiment 53-55 CaCO3The investigation of content
Slant medium and seed culture medium be the same as Example 1.
Fermentation medium：Based on the fermentative medium formula in embodiment 1, CaCO therein3Content is respectively
0.02%th, 0.2%, 0.5%, other compositions and content are constant.
The CaCO of table 103The influence of content
| Embodiment | Embodiment 53 | Embodiment 54 | Embodiment 55 |
| Content | 0.02% | 0.2% | 0.5% |
| Potency | 822 | 896 | 886 |
Other method be the same as Example 1.
Fermentation termination detects product potency with HPLC, the results are shown in Table 10.
As a result show, add 0.2% CaCO3Effect it is best.
Embodiment 56-59 pH investigation
Slant medium and seed culture medium be the same as Example 1.
Fermentation medium：Based on the fermentative medium formula in embodiment 1, pH therein is adjusted to 5.5 respectively, 6.5,
7.5th, 8.5, other compositions and content are constant.
The influence of the pH contents of table 11
| Embodiment | Embodiment 56 | Embodiment 57 | Embodiment 58 | Embodiment 59 |
| pH | 5.5 | 6.5 | 7.5 | 8.5 |
| Potency | 522 | 896 | 696 | 386 |
Other method be the same as Example 1.
Fermentation termination detects product potency with HPLC, the results are shown in Table 10.
As a result show, pH is best for 6.5 effect.
Comparative example 1
Using the identical step of embodiment 1, only difference is that used fermentation medium is United States Patent (USP) 4024246
In fermentation medium (sucrose 2%, maltose 1%, malt extract 1%, molasses 0.5%, corn steep liquor 0.5%, digest junket egg
White 0.5%, water 94.5%, above-mentioned percentage is weight percentage) fermented.It is 150~200 μ through HPLC detection potency
g/ml。
It should be understood that after the above of the present invention has been read, those skilled in the art can make various to the present invention
Change or change, these equivalent form of values equally fall within the application appended claims limited range.
Claims (4)
1. a kind of culture medium for being used to produce ECB, described culture medium contains carbon source, organic nitrogen source and inorganic salts, its
It is characterised by, described culture medium is made up of following component：Glucose 2-10%, starch 2-6%, organic nitrogen source 1-5%, grease
0.1-2%, MgSO4·7H2O 0.01-0.1%, KCl 0.01-0.1%, FeSO4·7H2O 0.001-0.1% and CaCO3
0.02-0.5%, surplus is water；The pH value of described culture medium is pH4-8；Described starch is selected from following one or more：
Farina, cornstarch and converted starch；Described grease is selected from following one or more：Soya-bean oil, corn oil, sunflower
Seed oil, fish oil and lecithin；Described organic nitrogen source is selected from following one or more：Fish meal protein peptone, soy peptone, jade
Rice ＆ peanut milk dry powder, peanut meal, soyabean cake, cotton seed meal and dregs of beans；Described percentage is the mass percent for accounting for culture medium.
2. culture medium as claimed in claim 1, it is characterised in that the Aspergillus of ECB described in fermenting and producing
Aspergillus nidulans are Aspergillus nidulans ATCC 58396.
3. the purposes of culture medium as claimed in claim 1, it is characterised in that the fermenting and producing for ECB.
4. the method that the culture medium described in a kind of any one of utilization claim 1~2 produces ECB, it is characterised in that institute
The method stated includes：
(1) under conditions of suitable Aspergillus Aspergillus nidulans growths, in the culture medium described in claim 1
Fermented and cultured Aspergillus Aspergillus nidulans；With
(2) separation obtains ECB from Aspergillus Aspergillus nidulans cultures.
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| CN201210015967.8A CN103205479B (en) | 2012-01-17 | 2012-01-17 | A kind of culture medium for being used to produce ECB |
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| CN103205479B true CN103205479B (en) | 2017-10-27 |
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| CN103555591B (en) * | 2013-10-12 | 2015-06-03 | 浙江工业大学 | Method and bacterial strain for fermentation preparation of Echinocandin B |
| CN103509840B (en) * | 2013-10-12 | 2015-08-05 | 浙江工业大学 | A kind of method improving anidulafungin precursor compound Echinocandin B output |
| CN106497794B (en) * | 2015-09-08 | 2019-09-27 | 浙江海正药业股份有限公司 | One plant of naked born of the same parents' shell of structure nest for producing echinocandin B and its application |
| CN106011204B (en) * | 2016-07-08 | 2020-01-14 | 浙江工业大学 | Method for synthesizing echinocandin B by fermentation method |
| CN111517463A (en) * | 2020-05-08 | 2020-08-11 | 绵津环保科技(上海)有限公司 | Preparation method of sewage biological denitrification composite carbon source |
| CN112646854B (en) * | 2020-12-11 | 2022-10-11 | 浙江工业大学 | Echinocandin B synthetic medium and application |
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| US4024245A (en) * | 1975-10-02 | 1977-05-17 | Eli Lilly And Company | Antibiotic A-30912 and process for production thereof |
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| HUE029147T2 (en) * | 2009-08-14 | 2017-02-28 | Xellia Pharmaceuticals Aps | Separation and/or purification of pneumocandin b0 from c0 |
| CN101993903B (en) * | 2009-08-18 | 2013-05-08 | 上海医药工业研究院 | Fermentation medium for producing ascomycin and fermentation method thereof |
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| US4024245A (en) * | 1975-10-02 | 1977-05-17 | Eli Lilly And Company | Antibiotic A-30912 and process for production thereof |
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| Title |
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| Improving the fermentation medium for echinocandin B production.PartI:sequential statistical experimental design;Cockshott A R et al;《Process Biochemistry》;20011231;647-660 * |
| 棘白菌素类抗真菌药物的结构与微生物合成;滕云等;《中国医药工业杂志》;20101231;第41卷(第10期);776-782 * |
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