CN103205479A - Culture medium used for producing echinocandin B - Google Patents
Culture medium used for producing echinocandin B Download PDFInfo
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- CN103205479A CN103205479A CN2012100159678A CN201210015967A CN103205479A CN 103205479 A CN103205479 A CN 103205479A CN 2012100159678 A CN2012100159678 A CN 2012100159678A CN 201210015967 A CN201210015967 A CN 201210015967A CN 103205479 A CN103205479 A CN 103205479A
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Abstract
The invention discloses a culture medium used for producing echinocandin B (ECB), and a production method employing the ECB. The culture medium comprises a carbon source, an organic nitrogen source, and inorganic salt, wherein the carbon source comprises glucose, starch and fat. With the culture medium provided by the invention, the yield of ECB produced by using aspergillus nidulans is high, and a titer is increased by 3-4 times than that of existing culture media. The titer can reach 750-900mug/ml.
Description
Technical field
The invention belongs to biological technical field, particularly a kind of for the production of the substratum of ECB and the ECB production method that adopts described substratum.
Background technology
The seventies in 20th century, people are by obtaining first neutral grease peptides ECB (echinocandin B in the fermented liquid of fungi (Aspergillus nidulans), hereinafter to be referred as ECB), discover that it is β-1, the noncompetitive inhibitor of 3-D-glucan synthase mainly acts on Candida (Candidas) and Eurotium (Aspergillus).ECB is by suppressing the β-1 in the fungus body, and the 3-D-glucan synthase makes fungi can not synthesize dextran, thereby makes cell wall structure unusual, causes cell rupture, and the entocyte seepage causes fungi death.ECB is, and Cytochrome P450 does not have effect, and untoward reaction is few, does not have cross resistance with amphotericin B and triazole antifungal agent thing, but owing to have toxicity, especially hemolytic, and fail for clinical.
Be raw material with natural product ECB, modifying the antifungal drug anidulafungin (anidulafungin) that obtains by side chain is to come company and the joint research and development exploitation of Vicuron Pharmaceuticals company by gift, it is clinical to have finished the III phase, is in the registration last stage, and route of administration is intravenous drip.Animal experiment study shows that with antifungal drug Caspofungin and Mi Kafen net phase ratio, anidulafungin is stronger to the aspergillus fumigatus activity.In a double blind controling test that oesophagus monilial infection patient is carried out, efficient and the fluconazole basically identical of intravenous drip anidulafungin, be 97.2%, the efficient of fluconazole is 98.8%, and the adverse reaction rate of anidulafungin is low, be 10%, control group is 13%, therefore has good market outlook.
The ECB structural formula is as follows:
ECB
United States Patent (USP) 4,024,246 have announced the process of utilizing aspergillus tubigensis (Aspergillus nidulans) NRRL 8112 fermentative production ECB: from slant medium (tomato-sauce 2%, baby oat 2%, agar 2%, water 94%, above-mentioned per-cent all is weight percentage) on get ECB and produce bacterium and be inoculated in and contain seed culture medium (glucose 1%, glycerine 1%, cottonseed meal 2.5%, calcium carbonate 0.1%, water 95.4%, above-mentioned per-cent all is weight percentage) in, 25 ℃ of shaking culture are inoculated in after 48 hours and contain fermention 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 per-cent all is weight percentage) fermentor tank in, under the ventilation condition, cultivated 5 days for 25 ℃, tiring through the HPLC detection is 150～200 μ g/ml.
United States Patent (USP) 4,024,245 have announced the process of utilizing aspergillus tubigensis (Aspergillus rugulosus) NRRL 8113 fermentative production ECB: from slant medium (dextrin 1%, casein hydrolysis 0.2%, yeast extract paste 0.1%, extractum carnis 0.1%, KCl 0.02%, MgSO
47H
2O 0.02%, FeSO
47H
2O 0.0004%, water 98.5596%) on get ECB and produce bacterium and be inoculated in and contain seed culture medium (sucrose 2.5%, molasses 3.6%, corn steep liquor 0.6%, casein hydrolysis 1%, K
2HPO
40.2%, water 92.1%, above-mentioned per-cent all is weight percentage) in, 25 ℃ of shaking culture are inoculated in after 24 hours and contain fermention medium (glucose 2.5%, starch 1%, peptone 1%, molasses 0.5%, casein hydrolysis 0.4%, MgSO
47H
2O 0.7%, KCl0.2%, FeSO
47H
2O 0.004%, CaCO
30.2%, above-mentioned per-cent all is weight percentage) fermentor tank in, under the ventilation condition, cultivated 4 days for 25 ℃, detect through HPLC that to tire be 100～200 μ g/ml.
Each discloses compd E CB of a kind of bacterium generation and preparation method thereof above-mentioned two inventions, but is to use this two kinds of methods to produce ECB, and fermentation culture product productive rate is on the low side, therefore all can not satisfy industrialization demands.
Therefore, this area presses for a kind of high fermention medium of tunning ECB productive rate that makes.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is exactly at present aspergillus tubigensis (Aspergillus nidulans) deficiency that fermentative production ECB productive rate is low, a kind of aspergillus tubigensis (Aspergillus nidulans) that is suitable for cultivating is provided, can significantly improves the substratum of ECB fermentation level.
The technical scheme that the present invention solves the problems of the technologies described above is as follows:
One of technical scheme of the present invention is: a kind of substratum for the production of ECB, and described substratum contains carbon source, organic nitrogen source and inorganic salt, and wherein, described carbon source comprises glucose, starch and grease.
In a preference of the present invention, described substratum comprises: glucose 2-10%, starch 2-6% and grease 0.1-2%.Among the present invention, described per-cent is the mass percent that accounts for substratum.
Preferred, described substratum comprises: glucose 4-8%, starch 3-5% and grease 0.5-1.5%.
Described starch can be conventional various plant amylums and in the modified starch one or more, comprises yam starch, W-Gum, wheat starch, sweet potato starch, green starch, tapioca (flour), water caltrop starch, Rhizoma Nelumbinis starch such as plant amylum; Modified starch comprises dextrin, maltodextrin etc.Preferably one or more in yam starch, W-Gum and the modified starch, most preferably yam starch.The preferred natural fats and oils of described grease more preferably is selected from following one or more: soya-bean oil, Semen Maydis oil, sunflower seed oil, fish oil and Yelkin TTS.
In a preference of the present invention, described substratum comprises: glucose 4-8%, yam starch 3-5% and soya-bean oil 0.5-1.5%.
In a preference of the present invention, described substratum comprises: glucose 2-10%, starch 2-6%, organic nitrogen source 1-5%, grease 0.1-2%, inorganic salt 0.04-0.8%.The preferred 0.06-0.5% of inorganic salt, more preferably 0.1-0.3%.
Among the present invention, described organic nitrogen source is conventional, preferably from following one or more: fish meal protein peptone, soy peptone, Dried Corn Steep Liquor Powder, peanut meal, soyabean cake, cottonseed meal and dregs of beans.
Among the present invention, described inorganic salt are conventional, preferably from following one or more: the inorganic salt of divalence magnesium, ferrous iron, monovalence potassium and divalent calcium.Preferred, described inorganic salt are selected from following one or more: MgSO
47H
2O, KCl, FeSO
47H
2O and CaCO
3
In a preference of the present invention, described substratum comprises: glucose 2-10%, starch 2-6%, organic nitrogen source 1-5%, grease 0.1-2%, MgSO
47H
2O 0.01-0.1%, KCl 0.01-0.1%, FeSO
47H
2O 0.001-0.1% and CaCO
30.02-0.5%.
In another preference of the present invention, described substratum comprises: glucose 2-10%, yam starch 2-6%, organic nitrogen source 1-5%, grease 0.1-2%, MgSO
47H
2O 0.01-0.1%, KCl0.01-0.1%, FeSO
47H
2O 0.001-0.1% and CaCO
30.02-0.5%.
In another preference of the present invention, described substratum comprises: glucose 6%, yam starch 4%, fish meal protein peptone 2%, soya-bean oil 0.5%, KCl 0.02%, MgSO
47H
2O 0.02%, FeSO
47H
2O 0.004%, CaCO
30.2%, surplus is water, pH6.5.
Among the present invention, the pH value of described substratum is conventional, can be natural, also can regulate preferred pH4-8, more preferably pH6-7, most preferably pH6.5.
In another preference of the present invention, use NaOH, NH during the fermentation
3Or CaCO
3Regulate the pH value of described substratum.
Among the present invention, the bacterial classification of fermentative production ECB is the mushroom that contains ECB in any tunning in this area, preferred aspergillus tubigensis (Aspergillus nidulans).Described aspergillus tubigensis (Aspergillus nidulans) includes, but is not limited to any kind of of Eurotium, preferably from Aspergillus nidulans ATCC 58396, Aspergillus nidulans NRRL 8112, Aspergillus nidulans NRRL 8113 etc.For example also comprise Aspergillus rugulosus.
Two of technical scheme of the present invention is: the purposes of described substratum, and for the fermentative production of ECB.
Three of technical scheme of the present invention is: a kind of method of utilizing described substratum to produce ECB, and described method comprises:
(1) under the condition that is fit to aspergillus tubigensis (Aspergillus nidulans) growth, fermentation culture aspergillus tubigensis in described substratum (Aspergillus nidulans); With
(2) from aspergillus tubigensis (Aspergillus nidulans) culture, separate the acquisition ECB.
The raw material that the present invention is used or reagent except specifying, equal commercially available getting.
Than prior art, beneficial effect of the present invention is as follows: the invention provides a kind of substratum for the production of ECB, adopt fermention medium of the present invention to produce the productive rate height of ECB, the more existing substratum of tiring has improved 3～4 times, reaches 750～900 μ g/ml.
Embodiment
The inventor is through research for a long time and widely, discovery is in fermentative Production ECB process, fermention medium is the key factor that influences the ECB fermentation yield, and the carbon source in the ECB fermention medium of routine is improved, adopt the combination carbon source, can improve the output of (improving more than 3 times 4 times of preferred raisings) ECB greatly.Finished the present invention based on this.
Among the present invention, described carbon source refers to provide the nutrition source of the required carbon of microbial nutrition.Nitrogenous source refers to provide the nutrition source of the required nitrogen element of microbial nutrition.Inorganic salt are to exercise the composition that constitutes thalline composition, enzymic activity composition or keep enzymic activity, adjusting osmotic pressure, pH etc.
The inventor is optimized fermention medium by the following method:
Carbon source, nitrogenous source, inorganic salt composition and content to substratum are done single factor experiment, observe these factors to the influence of thalli growth and product resultant quantity, filter out carbon source, nitrogenous source, inorganic salt kind with higher ECB fermentation level respectively.The mutual test of two factors of carrying out again then and multifactorial experiment.The present invention adopts " orthogonal experimental design " mathematical method to determine nutrient media components and concentration, and by variance analysis, determines the factor that influence is bigger.Found that, in the substratum when carbon source comprises glucose, starch and grease, the ECB fermentation yield is carried higher greatly, this is and the different place of existing ECB fermention medium that corresponding composition and content gets final product in the conventional ECB fermention medium and organic nitrogen source and inorganic salt adopt.These compositions can substitute with other compositions well known to those skilled in the art, and those skilled in the art is not difficult to determine its content.The inventor finds that also when the carbon source in the substratum comprised glucose, yam starch and grease, more effective for the production that promotes ECB, other starch based effect was slightly inferior.
The inventor's great discovery is the carbon source in the substratum is improved to the combination of glucose, starch and grease.Glucose is that to utilize carbon source, grease fast be to utilize carbon source at a slow speed.The present invention finds that the combination of this length effect carbon source is very favourable for the high yield of ECB.Further again again this basis go up other components and content, and the combination between these components and the carbon source optimizes, and obtains a production formula that relatively is fit to aspergillus tubigensis fermentation ECB finally, thereby tries to achieve high yield.
Culture medium prescription after the present invention optimizes contains enough nutritive ingredients, can satisfy the needs of shake flask test, again can success the shake flask fermentation result is amplified to fermentor tank.In one embodiment of the invention, described substratum is compared with the substratum before optimizing, and the output of ECB reaches 900mg/L, and output has improved 3.5 times.And because used substratum ingredient prices is lower, production cost is also significantly reduced.
As a kind of preferred implementation of the present invention, the composition of described substratum and content are: glucose 2-10%, yam starch 2-6%, fish meal protein peptone 1-5%, soya-bean oil 0.1-2%, MgSO
47H
2O0.01-0.1%, KCl 0.01-0.1%, FeSO
47H
2O 0.001-0.1%, CaCO
30.02-0.5%, surplus is water, pH6-7.
Substratum of the present invention is suitable for the aspergillus tubigensis that any this area is used for the fermentative production ECB, includes but not limited to Aspergillus nidulans.For example, can also be Aspergillus rugulosus.
The present invention also provides the method for preparing described substratum, may further comprise the steps: described glucose, starch, grease, organic nitrogen source, inorganic salt are dissolved in most of water in proportion or and water fully be mixed into the emulsion system of homogeneous, regulate the pH value to required scope, water constant volume behind the mixing, sterilization then.Preferable, regulating the adding of pH value back for the inorganic salt of some kind, add in adjustment pH value back such as CaCO3.
As optimal way, described method comprises: with glucose, starch, grease, KCl, MgSO
47H
2O, FeSO
47H
2O is dissolved in ninety percent water in proportion, regulates pH to 6.5, adds CaCO again
3, fully mix dissolving back, the water constant volume, and high-temperature sterilization after the packing, preferable as 121 ℃, the 0.1MPa 20min that sterilizes.
Major advantage of the present invention is: the inventor finds that first the carbon source optimizing in the ECB fermention medium of routine is combined as glucose, starch, grease, after making the combination within limits of quick-acting, slow carbon source optimizing, be conducive to the production of ECB, the output raising of ECB reaches 4 times more than, and the raising scope of this output is very significant in the biological fermentation field of medicine intermediate.
Further specify the present invention with embodiment below, but the present invention is not limited.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer." room temperature " described in the embodiment refers to the temperature of the operation room tested, is generally 25 ℃.
It is Aspergillus nidulans ATCC 58396 that the present invention adopts bacterial strain.
The formulation that embodiment 1 fermentation culture method and standard are tired
1, the preparation of seed
Aspergillus nidulans ATCC 58396 in the frozen pipe of glycerine is taken out activation back inoculation slant medium (potato 2%, glucose 1%, agar 1.8%, surplus is water, the pH nature, 121 ℃ of sterilization 20min), the inclined-plane of inoculation bacterial classification is placed 28 ℃ of constant incubators, cultivated 6 days, and obtained ripe spore.Then with the spore inoculating of maturation in 30ml seed culture medium (glucose 1%, glycerine 1%, cottonseed meal 2.5% are housed, calcium carbonate 0.1%, water 95.4%, above-mentioned per-cent all is weight percentage) 250ml shake in the bottle, cultivated 2 days at 28 ℃, 220rpm shaking table, obtain ripe seed.
2, the preparation of fermention medium
Fermention medium is: glucose 6%, yam starch 4%, fish meal protein peptone 3%, Semen Maydis oil 1%, KCl 0.02%, MgSO
47H
2O 0.02%, FeSO
47H
2O 0.004%, CaCO
30.2%, surplus is water, pH6.5.
Preparation steps: accurately take by weighing 60g glucose, 40g yam starch, 20g fish meal protein peptone, 5g soya-bean oil, 0.2g KCl, 0.2g MgSO respectively
47H
2O, 0.04g FeSO
47H
2O is dissolved in the 900ml water, regulates pH to 6.5, adds 2g CaCO
3, water is settled to 1000ml.Every 250ml shakes a bottle packing 40ml fermention medium, 121 ℃, the 0.1MPa 20min that sterilizes.
3, fermentation
The 250ml that the seed that obtains is equipped with the 40ml fermention medium with 2% inoculum size access shakes in the bottle, place 28 ℃, 220rpm shaking table to continue to cultivate 120 hours, get fermented liquid 3ml, add 3ml acetone soaked overnight, the centrifuging and taking supernatant carries out the ECB Determination on content with HPLC, is 744mg/L.
The chromatographic instrument that HPLC adopts is Waters510 type high performance liquid chromatograph, and chromatographic column is the C18 post, and detector is Waters 996 Photodiode Array Detector, moving phase is methyl alcohol: acetonitrile: water (7: 2: 1), flow velocity is 1.0ml/min, and sample size is 20 μ l, and column temperature is room temperature.
Embodiment 2-8 is different, and organic nitrogen source is investigated
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: based on the fermentative medium formula among the embodiment 1, organic nitrogen source in the fermention medium is replaced by peptone (fish meal), peptone (soybean), cottonseed meal, soyabean cake, dregs of beans, peanut meal, Dried Corn Steep Liquor Powder respectively, content is 3%, and other compositions and content are constant.
The influence of the different organic nitrogen sources of table 1
Additive method is with embodiment 1.
Fermentation termination detects product with HPLC and tires, and the results are shown in Table 1.
The result shows that peptone (fish meal), soy peptone and Dried Corn Steep Liquor Powder effect are preferable, and wherein the effect of peptone (fish meal) is best.
Embodiment 9-15 is different, and the carbon source combination is investigated
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: based on the fermentative medium formula among the embodiment 1, the glucose in the fermention medium is replaced by maltodextrin, sucrose, lactose, W-Gum, dextrin, yam starch respectively, and content is 6%, and other compositions and content are constant.
The influence of the different carbon sources of table 2
Additive method is with embodiment 1.
Fermentation termination detects product with HPLC and tires, and the results are shown in Table 2.
The result shows that the effect of glucose and yam starch is preferable, wherein again with glucose the best.
Embodiment 16-20 is different, and natural fats and oils is investigated
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: based on the fermentative medium formula among the embodiment 1, soya-bean oil is wherein replaced by Semen Maydis oil, sunflower seed oil, fish oil, Yelkin TTS respectively, and content is 1%, and other compositions and content are constant.Yelkin TTS is available from Shanghai day edible vegetable oil fat company limited:
The influence of the different natural fats and oils of table 3
| Embodiment | Embodiment 16 | Embodiment 17 | Embodiment 18 | Embodiment 19 | Embodiment 20 |
| Component | Soya-bean oil | Semen Maydis oil | Sunflower seed oil | Fish oil | Yelkin TTS |
| Mg/L tires | 768 | 907 | 803 | 628 | 769 |
Additive method is with embodiment 1.
Fermentation termination detects product with HPLC and tires, and the results are shown in Table 3.
The result shows that the effect that adds Semen Maydis oil is best.
The investigation of embodiment 21-25 fish meal protein peptone content
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: based on the fermentative medium formula among the embodiment 1, the content of organic nitrogen source peptone (fish meal) wherein replaces with 1%, 2%, 3%, 4%, 5% respectively, and other compositions and content are constant.
The influence of table 4 fish meal protein peptone content
| Embodiment | Embodiment 21 | Embodiment 22 | Embodiment 23 | Embodiment 24 | Embodiment 25 |
| Content | 1％ | 2％ | 3％ | 4％ | 5％ |
| Mg/L tires | 707 | 764 | 900 | 769 | 669 |
Additive method is with embodiment 1.
Fermentation termination detects product with HPLC and tires, and the results are shown in Table 4.
The result shows that the effect that adds 3% fish meal protein peptone is best.
The investigation of embodiment 26-30 glucose content
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: based on the fermentative medium formula among the embodiment 1, the content of glucose wherein replaces with 2%, 4%, 6%, 8%, 10% respectively, and other compositions and content are constant.
The influence of table 5 glucose content
| Embodiment | Embodiment 26 | Embodiment 27 | Embodiment 28 | Embodiment 29 | Embodiment 30 |
| Content | 2％ | 4％ | 6％ | 8％ | 10％ |
| Mg/L tires | 868 | 734 | 896 | 793 | 609 |
Additive method is with embodiment 1.
Fermentation termination detects product with HPLC and tires, and the results are shown in Table 5.
The result shows that the effect that adds 6% glucose is best.
The investigation of embodiment 31-35 yam starch content
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: based on the fermentative medium formula among the embodiment 1, yam starch content wherein replaces with 2%, 3%, 4%, 5%, 6% respectively, and other compositions and content are constant.
The influence of table 6 yam starch content
| Embodiment | Embodiment 31 | Embodiment 32 | Embodiment 33 | Embodiment 34 | Embodiment 35 |
| Content | 2％ | 3％ | 4％ | 5％ | 6％ |
| Mg/L tires | 762 | 851 | 896 | 815 | 654 |
Additive method is with embodiment 1.
Fermentation termination detects product with HPLC and tires, and the results are shown in Table 6.
The result shows that the effect that adds 4% yam starch is best.
The investigation of embodiment 36-40 Semen Maydis oil content
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: based on the fermentative medium formula among the embodiment 1, Semen Maydis oil content wherein is respectively 0.1%, 0.5%, 1%, 1.5%, 2%, and other compositions and content are constant.
The influence of table 7 Semen Maydis oil content
| Embodiment | Embodiment 36 | Embodiment 37 | Embodiment 38 | Embodiment 39 | Embodiment 40 |
| Content | 0.1％ | 0.5％ | 1％ | 1.5％ | 2％ |
| Mg/L tires | 719 | 845 | 908 | 828 | 619 |
Additive method is with embodiment 1.
Fermentation termination detects product with HPLC and tires, and the results are shown in Table 7.
The result shows that the effect that adds 1% Semen Maydis oil is best.
The investigation of embodiment 41-44 KCl content
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: based on the fermentative medium formula among the embodiment 1, the content of KCl wherein is respectively 0.01%, 0.02%, 0.05%, 0.1%, and other compositions and content are constant.
The influence of table 8 KCl content
| Embodiment | Embodiment 41 | Embodiment 42 | Embodiment 43 | Embodiment 44 |
| Content | 0.01％ | 0.02％ | 0.05％ | 0.1％ |
| Mg/L tires | 819 | 896 | 818 | 748 |
Additive method is with embodiment 1.
Fermentation termination detects product with HPLC and tires, and the results are shown in Table 8.
The result shows that the effect that adds 0.02%KCl is best.
Embodiment 45-48 MgSO
4The investigation of content
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: based on the fermentative medium formula among the embodiment 1, MgSO wherein
4Content is respectively 0.01%, 0.02%, 0.05%, 0.1%, and other compositions and content are constant.
Table 9 MgSO
4The influence of content
| Embodiment | Embodiment 45 | Embodiment 46 | Embodiment 47 | Embodiment 48 |
| Content | 0.01％ | 0.02％ | 0.05％ | 0.1％ |
| Mg/L tires | 834 | 896 | 850 | 793 |
Additive method is with embodiment 1.
Fermentation termination detects product with HPLC and tires, and the results are shown in Table 9.
The result shows, adds 0.02% MgSO
4Effect best.
Embodiment 49-52 FeSO
4The investigation of content
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: based on the fermentative medium formula among the embodiment 1, FeSO wherein
4Content is respectively 0.001%, 0.004%, 0.01%, 0.1%, and other compositions and content are constant.
Table 10 FeSO
4The influence of content
| Embodiment | Embodiment 49 | Embodiment 50 | Embodiment 51 | Embodiment 52 |
| Content | 0.001％ | 0.004％ | 0.01％ | 0.1％ |
| Mg/L tires | 768 | 896 | 787 | 743 |
Additive method is with embodiment 1.
Fermentation termination detects product with HPLC and tires, and the results are shown in Table 10.
The result shows, adds 0.004%FeSO
4Effect best.
Embodiment 53-55 CaCO
3The investigation of content
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: based on the fermentative medium formula among the embodiment 1, CaCO wherein
3Content is respectively 0.02%, 0.2%, 0.5%, and other compositions and content are constant.
Table 10 CaCO
3The influence of content
| Embodiment | Embodiment 53 | Embodiment 54 | Embodiment 55 |
| Content | 0.02％ | 0.2％ | 0.5％ |
| Tire | 822 | 896 | 886 |
Additive method is with embodiment 1.
Fermentation termination detects product with HPLC and tires, and the results are shown in Table 10.
The result shows, adds 0.2% CaCO
3Effect best.
The investigation of embodiment 56-59 pH
Slant medium and seed culture medium are with embodiment 1.
Fermention medium: based on the fermentative medium formula among the embodiment 1, pH wherein is adjusted to 5.5,6.5,7.5,8.5 respectively, and other compositions and content are constant.
The influence of table 11 pH content
| Embodiment | Embodiment 56 | Embodiment 57 | Embodiment 58 | Embodiment 59 |
| pH | 5.5 | 6.5 | 7.5 | 8.5 |
| Tire | 522 | 896 | 696 | 386 |
[0148]Additive method is with embodiment 1.
Fermentation termination detects product with HPLC and tires, and the results are shown in Table 10.
The result shows, pH is that 6.5 effect is best.
Comparative Examples 1
Adopt the identical step of embodiment 1, unique difference is that employed fermention medium is the fermention medium (sucrose 2% in the United States Patent (USP) 4024246, maltose 1%, malt extract 1%, molasses 0.5%, corn steep liquor 0.5%, casein hydrolysis 0.5%, water 94.5%, above-mentioned per-cent all is weight percentage) ferment.Tiring through the HPLC detection is 150～200 μ g/ml.
Should be understood that after having read foregoing of the present invention, those skilled in the art can make various changes or modifications the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. substratum for the production of ECB, described substratum contains carbon source, organic nitrogen source and inorganic salt, it is characterized in that, and described carbon source comprises glucose, starch and grease.
2. substratum as claimed in claim 1 is characterized in that, described substratum comprises: glucose 2-10%, starch 2-6% and grease 0.1-2%, described per-cent are the mass percent that accounts for substratum.
3. substratum as claimed in claim 1 is characterized in that, described starch is selected from following one or more: yam starch, W-Gum and modified starch; Described grease is selected from following one or more: soya-bean oil, Semen Maydis oil, sunflower seed oil, fish oil and Yelkin TTS.
4. substratum as claimed in claim 1 is characterized in that, described substratum comprises: glucose 2-10%, starch 2-6%, organic nitrogen source 1-5%, grease 0.1-2% and inorganic salt 0.04-0.8%.
5. substratum as claimed in claim 1 is characterized in that, described organic nitrogen source is selected from following one or more: fish meal protein peptone, soy peptone, Dried Corn Steep Liquor Powder, peanut meal, soyabean cake, cottonseed meal and dregs of beans; Described inorganic salt are selected from following one or more: KCl, MgSO
4, FeSO
4And CaCO
3
6. substratum as claimed in claim 1 is characterized in that, described substratum comprises: glucose 2-10%, starch 2-6%, organic nitrogen source 1-5%, grease 0.1-2%, MgSO
47H
2O0.01-0.1%, KCl 0.01-0.1%, FeSO
47H
2O 0.001-0.1% and CaCO
30.02-0.5%.
7. substratum as claimed in claim 1 is characterized in that, the pH value of described substratum is pH4-8.
8. substratum as claimed in claim 1 is characterized in that, described aspergillus tubigensis (Aspergillus nidulans) is Aspergillus nidulans ATCC 58396.
9. the purposes of substratum as claimed in claim 1 is characterized in that, is used for the fermentative production of ECB.
10. method of utilizing each described substratum of claim 1-8 to produce ECB is characterized in that described method comprises:
(1) under the condition that is fit to aspergillus tubigensis (Aspergillus nidulans) growth, fermentation culture aspergillus tubigensis in the described substratum of claim 1 (Aspergillus nidulans); With
(2) from aspergillus tubigensis (Aspergillus nidulans) culture, separate the acquisition ECB.
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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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| 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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Cited By (6)
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| CN103509840A (en) * | 2013-10-12 | 2014-01-15 | 浙江工业大学 | Method for increasing yield of anidulafungin precursor compound Echinocandin B |
| CN103555591A (en) * | 2013-10-12 | 2014-02-05 | 浙江工业大学 | Method and bacterial strain for fermentation preparation of Echinocandin B |
| CN106011204A (en) * | 2016-07-08 | 2016-10-12 | 浙江工业大学 | Method for synthesizing echinocandin B through fermentation method |
| CN106497794A (en) * | 2015-09-08 | 2017-03-15 | 浙江海正药业股份有限公司 | The naked born of the same parents' shell of structure nest of one plant of product echinocandin B and its application |
| 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 |
| CN112646854A (en) * | 2020-12-11 | 2021-04-13 | 浙江工业大学 | Echinocandin B synthetic medium and application |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103509840A (en) * | 2013-10-12 | 2014-01-15 | 浙江工业大学 | Method for increasing yield of anidulafungin precursor compound Echinocandin B |
| CN103555591A (en) * | 2013-10-12 | 2014-02-05 | 浙江工业大学 | Method and bacterial strain for fermentation preparation of Echinocandin B |
| CN103555591B (en) * | 2013-10-12 | 2015-06-03 | 浙江工业大学 | Method and bacterial strain for fermentation preparation of Echinocandin B |
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| CN106497794A (en) * | 2015-09-08 | 2017-03-15 | 浙江海正药业股份有限公司 | The naked born of the same parents' shell of structure nest of one plant of product echinocandin B and its application |
| 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 |
| CN106011204A (en) * | 2016-07-08 | 2016-10-12 | 浙江工业大学 | Method for synthesizing echinocandin B through fermentation method |
| 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 |
| CN112646854A (en) * | 2020-12-11 | 2021-04-13 | 浙江工业大学 | Echinocandin B synthetic medium and application |
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| CN103205479B (en) | 2017-10-27 |
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