CN101319241A - Solid fermentation method of ansamitocin - Google Patents
Solid fermentation method of ansamitocin Download PDFInfo
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- CN101319241A CN101319241A CNA2008100406880A CN200810040688A CN101319241A CN 101319241 A CN101319241 A CN 101319241A CN A2008100406880 A CNA2008100406880 A CN A2008100406880A CN 200810040688 A CN200810040688 A CN 200810040688A CN 101319241 A CN101319241 A CN 101319241A
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Abstract
The invention discloses a solid fermentation method for ansamitocin. The method comprises the following steps of: step one, seed culture: a thallophytic streak culture medium plate which is freezingly preserved in a saccharose solution and produces the ansamitocin is subject to inversion activation for 2 to 9 days at a temperature of 28 DEG C and then is transferred to a seed culture medium, and a seed liquid is cultured at a temperature of 28 DEG C; and step two, solid fermentation: the seed liquid obtained in the step one is diluted to a concentration between 10<2> and 10<6>CFU/ml and is coated to a fermentation culture medium plate paved with a regenerative cellulose membrane support to be cultured for 7 to 10 days at a temperature of 28 DEG C. Compared with the prior liquid fermentation method, the method has the advantages that: the output of the ansamitocin is more than 2 times higher, and the unit cell content is near 4 times higher; at the same time, the solid fermentation has small energy consumption, the treatment of a sample is simple, thereby contributing to the industrialized preparation of the ansamitocin.
Description
Technical field
The present invention relates to the fermentation process of a kind of biotechnology and technical field, particularly, relate to a kind of solid fermentation method of ansamitocin.
Background technology
Ansamitocin (ansamitocin) is the maytenin derivative in precious orange synnema actinomycetes (Actinosynnema pretiosum) source; its parent nucleus is the Macrocyclic lactams ring of 19-C; C-3 connects the carbochain of different lengths; the side chain of main active ingredient AP-3 is an isobutyryl, can suppress aleukemic leukemia clone and human solid tumor under lower concentration.Because stomach side effect and neurotoxicity, its research also rests on the clinical two-stage.Yet because ansamitocin can be clinical one interim as antibody-toxin conjugate and immune conjugate, the research of each side is still underway.
Solid fermentation is a kind of fermentation that approaches state of nature, and it has many differently with liquid submerged fermentation, produces the product of greater concn under less energy consumption and fermentation time.Compare with liquid fermenting, precious orange synnema actinomycetes can produce under the solid fermentation condition has active ansamitocin glycosides compound.
The prosperity of precious orange synnema actinomycetes mycelia combines closely with substratum when the YMG solid culture, can't directly survey, and therefore can't simply directly characterize the output of ansamitocin with unit volume.Simultaneously, indirect determination method all has certain limitation, O
2And CO
2The metabolism amount can change with cell age; The special biological components content of measuring cell can be because of indefinite, and because the polymorphism of mycelial growth, the thalli growth amount of different batches also is very different, and this same bacterial classification, culture condition be with incubation time and different, the accuracy that detection time is long, the complicated influence of process is measured; Then can be subjected to thalline whether to produce the outer deoxyribonuclease restriction of born of the same parents etc. to the detection of DNA influences.
Find that by prior art documents the wild-type precious orange synnema actinomycetes of having reported at present produces ansamitocin and is mainly liquid fermenting, generally by the rapid technology of the multistep ansamitocin that reclaims and purify.It as the patent No. method of having described a kind of ansamitocin of purifying in the United States Patent (USP) of US6573074, this method is to utilize toluene or dimethylbenzene slightly to carry product and concentrated from fermented liquid, behind petroleum ether precipitation, pass through further separation and purification of various column chromatographies and crystallization again, with ethyl acetate and heptane is the ansamitocin that obtains certain output and purity behind the solvent secondary crystal, yet this method relates to multiple organic solvent and extracting method, brings certain difficulty for large-scale industrial fermentation.Simultaneously, because precious orange synnema actinomycetes mycelium in the liquid culture process is grown in flakes, fermentation whole culture system of middle and later periods is highly viscous state, and a large amount of mycelium adsorbed close are unfavorable for mass transfer at the culture vessel wall, have significantly reduced the product amount.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of solid fermentation method of ansamitocin is provided.Method among the present invention is on the basis of solid-fermented technique, by at the renewable cellulosefilm upholder of agar plate upper berth one deck, making solid fermentation finish can scrape mycelium, collect mycelium quickly and easily, quantitatively the product of unit cell is plain measures, improve the output of ansamitocin simultaneously by the optimum inoculation amount of determining best seed activation time and solid fermentation, method of the present invention helps the optimizing fermentation regulation and control, and suitability for industrialized production is had potential value.
The present invention is achieved by the following technical solutions:
The first step, seed culture: will be in sucrose solution the thalline streak plate of the product ansamitocin of freezing preservation, be inverted activation down for 28 ℃ and be transferred in the seed culture medium after 2-9 days, cultivate this seed liquor down for 28 ℃;
Described culture dish diameter is 10cm, liquid amount 15ml.
The mass percent of sucrose is 10.3% in the described sucrose solution.
Second step, solid fermentation: the seed liquor that obtains in the first step is diluted to 10
2-10
6The concentration of CFU/ml is applied on the fermention medium flat board that is covered with renewable cellulosefilm upholder, cultivates 7 days down for 28 ℃.
Described fermention medium is the YMG substratum, and the weight percent of its moiety is: yeast extract matter 0.4%, maltose 1%, glucose 0.4%, agar powder 1.4% and distilled water 96.8%.
The subsides adding method of described renewable cellulosefilm upholder is as follows: renewable cellulosefilm is cut into the circle that diameter is 10cm, in 121 ℃ the sterilization 20 minutes after, 50 ℃ of oven dry, under the aseptic technique, with tunica fibrosa of tweezers folder, paste the solid plate of going into to have substratum, and guarantee that the tunica fibrosa full extension covers whole flat board.。
The present invention utilizes renewable cellulosefilm upholder to carry out the solid fermentation microorganism collection, and is applied in the solid fermentation production process of ansamitocin.Simultaneously, by the optimum inoculation amount of definite best seed activation time and solid fermentation, and under this top condition, carry out solid fermentation.Method of the present invention with compare with liquid fermenting: the output of ansamitocin exceeds more than 2 times, and unit cell content exceeds nearly 4 times; The solid fermentation energy consumption is little simultaneously, and sample preparation is simple, helps its preparation of industrialization.
Related bacterial strain is precious orange synnema actinomycetes (Actinosynnema pretiosumATCC 31565) among the present invention.
Description of drawings
The ansamitocin output comparison diagram of solid fermentation and liquid fermenting under the different seed activation time conditions of Fig. 1.
Fig. 2 different vaccination density is to the dynamic effects figure of ansamitocin output.
Embodiment
Below in conjunction with accompanying drawing embodiments of the invention are elaborated: present embodiment has provided detailed embodiment and process being to implement under the prerequisite with the technical solution of the present invention, but protection scope of the present invention is not limited to following embodiment.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
The comparison of the ansamitocin output of YMG substratum solid fermentation and the fermentation of YMG medium liquid:
Seed and fermentation culture: seed culture medium is the YMG substratum, the weight percent of its integral part is: yeast extract matter 0.4%, maltose 1%, glucose 0.4%, agar powder 1.5% and distilled water 96.7%, the culture dish diameter is 10cm, liquid amount 15ml.Use is stored in the high density thalline line YMG culture medium flat plate that mass percent is 10.3% sucrose, is inverted activation 9 days for 28 ℃, is inoculated into the shaking in the bottle of 250ml that the 30mlYMG substratum is housed with the inoculation shovel, and 28 ℃, cultivated 2 days for 220 rev/mins, press 10
2The inoculum size of CFU/ml is coated the YMG culture medium flat plate that is covered with renewable cellulosefilm upholder, cultivated 7 days down for 28 ℃, simultaneously be inoculated into shaking in the bottle of 250ml that the 30mlYMG substratum is housed with the inoculation shovel with identical inoculum size, 28 ℃, 220 rev/mins of cultivations 7 days.
Sample preparation and detection method: (1) solid culture is cut into small pieces after finishing, and soaks 2 times with the 15ml ethyl acetate, each 1 hour, merges the ethyl acetate soak solution twice; (2) after liquid culture finished, fermented liquid each 1 hour, merged acetic acid ethyl acetate extract twice with 150 rev/mins of hybrid extractions of isopyknic ethyl acetate 2 times.Above-mentioned acetic acid ethyl fluid adds the dissolve with methanol of 1/10 volume behind 50 ℃ of rotary evaporations, 4 ℃ of preservations are standby behind the 0.22 μ m organic membrane filter.The reverse post of C18 (moving phase: 85% methyl alcohol and 15% water, flow velocity: 0.6ml/min) detect ansamitocin content.
Experimental result: under different seed activation time conditions, the ansamitocin output of two kinds of fermentation modes as shown in Figure 1.Experimental result shows: solid fermentation is under the seed condition of different soak times, and the output of ansamitocin is inoculated the seed of shorter soak time (5 days) all than liquid culture height, and the ansamitocin output of two kinds of fermentation modes differs about 8 times.
The solid-liquid fermentative production ansamitocin that utilizes the more different substratum of renewable cellulosefilm upholder to form:
Seed culture: seed culture medium is the YMG substratum, and the mass percent of its each integral part is: yeast extract matter 0.4%, maltose 1%, glucose 0.4%, agar powder 1.5% and distilled water 96.7%, culture dish diameter are 10cm, liquid amount 15ml.The high density thalline line YMG flat board that 10.3% sucrose is preserved is inverted activation 5 days for 28 ℃, is inoculated into the shaking in the bottle of 250ml that the 30mlYMG substratum is housed with the inoculation shovel, under 28 ℃ with 220 rev/mins rotating speed cultivation 2 days, behind the stepwise dilution, with 10
6The concentration of CFU/ml is coated the YMG flat board that is covered with renewable cellulosefilm upholder, and 28 ℃, cultivated 9 days, the shaking in the bottle of 250ml of 30mlYMG substratum is housed with identical inoculum size inoculation simultaneously, 28 ℃, cultivated 9 days with 220 rev/mins rotating speed under 28 ℃.。
The mass percent of each moiety of fermention medium L that control group uses is: fructose 1%, glucose 0.5%, dextrin 5%, glycerine 2%, yeast extract powder 0.5%, Semen Maydis powder 1%, ammonium chloride 0.5%, lime carbonate 0.5%, dipotassium hydrogen phosphate 0.05%, ferrous sulfate 0.0002%, surplus are distilled water.
Liquid fermenting: the inoculum size with 3.3% is inoculated in the fermention medium L of 60ml, and the rotating speed with 220 rev/mins under 28 ℃ was cultivated 9 days.
Solid fermentation: with 10
6The inoculum size coating of CFU/ml is covered with the above-mentioned fermention medium L flat board (liquid amount 15ml) of RCF regenerated cellulose film upholder, cultivates 9 days down for 28 ℃.
Sample preparation and detection method: after (1) liquid culture finished, fermented liquid was in 5000 rev/mins of centrifugal 10min, and supernatant each 1 hour, merges acetic acid ethyl acetate extract twice with 150 rev/mins of hybrid extractions of isopyknic ethyl acetate 2 times.Above-mentioned acetic acid ethyl fluid adds the dissolve with methanol of 1/10 volume behind 50 ℃ of rotary evaporations, 4 ℃ of preservations are standby behind the 0.22 μ m organic membrane filter.The reverse post of C18 (moving phase: 85% methyl alcohol and 15% water, flow velocity: 0.6ml/min) detect ansamitocin content.The remaining mycelium in centrifugal back is washed 2 times with deionized water, again with 0.1M HCl wash once after, dry to constant weight.(2) after solid culture finishes, take film off, agar is cut into small pieces, soak 2 times with the 15ml ethyl acetate, each 1 hour, merge the ethyl acetate soak solution twice, behind 50 ℃ of rotary evaporations, add the dissolve with methanol of 1/10 volume, 4 ℃ of preservations are standby behind the 0.22 μ m organic membrane filter.The reverse post of C18 (moving phase: 85% methyl alcohol and 15% water, flow velocity: 0.6ml/min) detect ansamitocin content.Thalline on the fiber thin scrapes gently with blade, washes 3 times with deionized water, dries to constant weight for 50 ℃.
Experimental result: solid fermentation 5 days, there is the ansamitocin more than 80% to be secreted in the agar, increase with fermentation time, up to 98% be secreted in the agar, so after solid fermentation finishes, directly with the outer product of ethyl acetate lixiviate born of the same parents.Above-mentioned experimental result shows, ansamitocin is at solid fermentation rate ratio liquid fermenting height, compare with liquid fermenting with the solid of fermention medium, the output of using the YMG substratum to carry out ansamitocin in the solid fermentation exceeds more than 2 times, unit cell content exceeds nearly 4 times, liquid fermenting then detects basically less than product, and the result is as shown in table 1.
The different substratum of table 1 are formed the solid-liquid fermentation relatively
Therefore proof uses the YMG substratum to carry out solid fermentation, can obtain the production peak of ansamitocin.
Utilize renewable cellulosefilm upholder relatively in the solid fermentation different vaccination density to the influence of ansamitocin.
Seed and fermentation culture: the mass percent of seed culture each component is: yeast extract matter 0.4%, maltose 1%, glucose 0.4%, agar powder 1.5% and distilled water 96.7%, culture dish diameter are 10cm, liquid amount 15ml.The high density thalline line YMG flat board that 10.3% sucrose is preserved, be inverted activation after 2 days for 28 ℃, be inoculated in next fritter of inoculation shovel shovel and fill the 30mlYMG substratum and volume is shaking in the bottle of 250ml, under 28 ℃ with 220 rev/mins rotating speed cultivation 2 days, behind the stepwise dilution, with 10
4The concentration of CFU/ml is coated the YMG flat board that is covered with renewable cellulosefilm upholder, 28 ℃, cultivates 10 days.
The subsides adding method of renewable cellulosefilm upholder is as described in the embodiment 1.
Sample preparation and detection method: after (1) solid culture finishes, take film off, agar is cut into small pieces, soak 2 times with the 15ml ethyl acetate, each 1 hour, merge the ethyl acetate soak solution twice, behind 50 ℃ of rotary evaporations, the dissolve with methanol that adds 1/10 volume, 4 ℃ of preservations are standby behind the 0.22 μ m organic membrane filter.The reverse post of C18 (moving phase: 85% methyl alcohol and 15% water, flow velocity: the 0.6ml/min) content of detection ansamitocin.(2) thalline on the fiber thin scrapes gently with blade, washes 3 times with deionized water, dries to constant weight for 50 ℃.
For quantitative initial inoculum size, all adopt the coating inoculation, seed adopts 10 times of dilution methods, and coating 0.1ml is added with the YMG agar plate of tunica fibrosa in subsides; Different seed density is with single enumeration (CFU/ml).
Experimental result as shown in Figure 2, with 10
4The inoculum size of CFU/ml (by 100 times of the seed liquor redilution of above-mentioned cultivation) is carried out solid fermentation, and the unit cell content of ansamitocin reaches the 20mg/g stem cell, ferments after 8 days, and production peak reaches 29mg/L, much larger than the YMG liquid fermenting.Other batch experiment shows that inoculum density is 10
7CFU/ml, the highest ansamitocin content is about the 4mg/g stem cell, illustrates that in the solid fermentation process, inoculum density is a key factor.Collect mycelial method by adding solid support (RCF regenerated cellulose film), the ansamitocin content and the efficient that can compare solid and liquid fermenting effectively, for further illustrating solid and liquid fermenting mechanism provides foundation: from cell concentration, relatively produce plain difference on the one hand, help follow-up genetically engineered operation (as the extraction of DNA and RNA etc.) on the other hand, the solid fermentation sample preparation is simple simultaneously, energy consumption is little, helps the mass preparation ansamitocin; And the seed that the inoculum density of lower level can reduce early stage preparation.
Claims (4)
1, a kind of solid fermentation method of ansamitocin is characterized in that, comprises the steps:
The first step, seed culture: will be in sucrose solution the streak culture base of the thalline of the product ansamitocin of freezing preservation dull and stereotyped, be inverted activation down for 28 ℃ and be transferred in the seed culture medium after 2-9 days, cultivate this seed liquor down for 28 ℃;
Second step, solid fermentation: the seed liquor that obtains in the first step is diluted to 10
2-10
6The concentration of CFU/ml is applied on the fermention medium flat board that is covered with renewable cellulosefilm upholder, cultivates 7-10 days down for 28 ℃.
2, the solid fermentation method of ansamitocin according to claim 1 is characterized in that, described in the first step in the sucrose solution mass percent of sucrose be 10.3%.
3, the solid fermentation method of ansamitocin according to claim 1, it is characterized in that, fermention medium is the YMG substratum described in second step, and the weight percent of its moiety is: yeast extract 0.4%, maltose 1%, glucose 0.4%, agar powder 1.4% and distilled water 96.8%.
4, the solid fermentation method of ansamitocin according to claim 1, it is characterized in that, the subsides adding method of renewable cellulosefilm upholder is described in second step: renewable cellulosefilm is cut into the circle that diameter is 10cm, in 121 ℃ the sterilization 20 minutes after, 50 ℃ of oven dry are under the aseptic technique, with tunica fibrosa of tweezers folder, paste the solid plate of going into to have substratum, and guarantee that the tunica fibrosa full extension covers whole flat board.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102732581A (en) * | 2012-07-18 | 2012-10-17 | 上海交联药物研发有限公司 | Method for highly expressing ansamitocinP-3 |
| CN102731526A (en) * | 2012-05-14 | 2012-10-17 | 福建省微生物研究所 | New ansamitocin compounds and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105039304A (en) * | 2015-08-12 | 2015-11-11 | 四川省农业科学院土壤肥料研究所 | Monospore hybridization method of edible fungus |
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| US7432088B2 (en) * | 2003-05-08 | 2008-10-07 | Immunogen Inc. | Methods for the production of ansamitocins |
| CN1253464C (en) * | 2003-08-13 | 2006-04-26 | 中国科学院昆明植物研究所 | Ansi glycoside compound and its medicinal composition, preparation and use |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102731526A (en) * | 2012-05-14 | 2012-10-17 | 福建省微生物研究所 | New ansamitocin compounds and preparation method thereof |
| CN102732581A (en) * | 2012-07-18 | 2012-10-17 | 上海交联药物研发有限公司 | Method for highly expressing ansamitocinP-3 |
| CN102732581B (en) * | 2012-07-18 | 2014-07-16 | 上海交联药物研发有限公司 | Method for highly expressing ansamitocinP-3 |
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