CN104673677A - Colletotrichum lini strain LJYH20130405-1 for steride fermentation as well as application technology thereof - Google Patents
Colletotrichum lini strain LJYH20130405-1 for steride fermentation as well as application technology thereof Download PDFInfo
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Abstract
The invention discloses a high-yielding colletotrichum lini strain LJYH20130405-1 for steride fermentation as well as an application technology of the strain. A taxonomic status of the strain LJYH20130405-1 is that the sexual generation belongs to the fungi, ascomycota, sordariomycetes, sordariomycetidae, small nectriaceae and glomerella; the strain is preserved in (China General Microbiological Culture Collection Center on June 8th, 2013, with the preservation number of CGMCC 7715. According to the colletotrichum lini strain, 7alpha, 15alpha-dyhydroxyl androstenolone can be produced by using dehydroepiandrosterone (DHEA) as a substrate in a conversion process of producing plant hormones by using steroids as raw materials. The produced 7alpha, 15alpha-dyhydroxyl androstenolone has the characteristics of high conversion rate, high yield, comparatively stable biological properties and the like. According to the colletotrichum lini strain for steride fermentation, the technical support can be provided for improving the production yield of plant steroid hormone using dioscin as a raw material and reducing the cost.
Description
Technical field
The invention belongs to industrial fungi technical field, a kind of high yield steroidal of specific design ferment flax thorn dish spore trichoderma strain and steroidal compounds fermentation utilisation technology.
Background technology
steroidal compounds biological fermentation
Drospirenone (Drospirenone) is aldosterone antagonists, has the effect of anti-mineral Kendall compound activity.It is combined with Ethinylestradiol and forms new and effective oral contraceptive Yasmin.Yasmin(drospirenone adds ethinylestradiol) be the oral contraceptive that French Schering company succeeds in developing, go on the market at US and European.7 α, 15 alpha-dihydroxy-dihydrotestosterones (7 α, 15 α-dihydroxyandrostenolone) are the important intermediate of synthesis drospirenone.Its mode of production has traditional chemical synthesis, semi-synthesis method, biotransformation method three class.Conventional chemical methods synthesis needs nearly ten step reactions, and reaction process needs a large amount of toxic reagent.Semi-synthesis method only carries out the hydroxylation fermentation of a position, and the hydroxylation of another position also needs to use a large amount of toxic reagent and through multiple chemical reaction step.The method of bio-transformation once hydroxylation is directly carried out in two positions by microorganism, and production technique is simple, decreases synthesis step, reduces energy consumption, and significantly cut down the discharge of pollutent.The method of direct bioconversion can reduce production cost, reaches the object of cleanly production.Therefore, 7 α, the production of 15 alpha-dihydroxy-dihydrotestosterones all adopts the mode of fermentable to carry out.
At present, steroidal 7 α can be carried out, the bacterial classification of 15 alpha-dihydroxy-s have gibberella (
gibberella fujikuroi), Colletotrichum lini (
colletotrichum lini) etc.And wherein Colletotrichum lini is industrial widely used bacterial classification.The hydroxylase system of application flax thorn dish spore carries out bio-transformation to dehydroepiandros-sterone (DHEA) substrate and synthesizes 7 α, 15 alpha-dihydroxy-dihydrotestosterones, can adopt direct translation method and indirect reformer method.Experimental result shows, directly transform with the transformation efficiency of indirect reformer all at 60%-70%, but the by product of indirect reformer is few, Isolation and purification yield is high.Direct method for transformation is simple, and suitability for industrialized production still uses direct translation method.In order to improve constantly microbial transformation productive rate, must transforming microorganism, can power improve constantly, bringing larger benefit for producing, the final cost reducing final drug, makes drug price progressively reduce.Therefore, to the protection with the bacterial strain of high yield selected, attention should be strengthened.
flax thorn dish spore classification position and utilize present situation
To be Corda set up in 1831 Colletotrichum, for small cluster shell belong to (
glomerellaspauld. & H. Schrenk) imperfect stage.Its sexual generation be under the jurisdiction of mycota (Fungi), Ascomycota guiding principle (Ascomycota), excrement shell Gammaproteobacteria (Sordariomycetes), excrement shell bacterium subclass (Sordariomycetidae), little from Chi Ke section (Glomerellaceae), small cluster shell belong to.Since Corda sets up Colletotrichum, the kind successively described has exceeded 1000 kinds.After Von Arx arranges, until 1970,23 taxonomical units are accepted.Sutton only admitted 22 kinds afterwards.
The acervulus of flax thorn dish spore produces under the stratum corneum of host plant, and raw or table life under epidermis, dispersion or gathering, irregular pattern type cracking, is had 3 transeptate brown bristles, not branch.Conidiophore is colourless to brown, tool tabula, base portion branch.Conidium is colourless, unit cell, directly, round shape, or slightly in fusiformis, wall is thin, smooth surface, sometimes containing oil droplet, 11 ~ 26 ' 3 ~ 5.5mm.
Under field conditions (factors), flax thorn dish spore is a kind of common phytopathogen, usually colonizes on flax blade, causes anthrax.Cotyledon forms brown and has the round spot of taking turns line; If seedling stem foot is injured, usually complete stool is withered, and tikka and stem spot are all brown; Sometimes can also cause harm and spend and capsule.
On PDA substratum, temperature is comparatively large to flax thorn dish spore growth effect, and this bacterial strain all can grow at 10 ~ 35 DEG C, cultivates lower growth the fastest, be secondly 20 DEG C with 25 DEG C, lower than 5 DEG C or higher than 40 DEG C of mycelia without growth.All can grow at pH 2 ~ 11, optimal pH is 8, and mycelial growth is luxuriant, and colony diameter reaches 6.0cm, and be secondly pH 7, colony diameter reaches 5.1 cm.Under each illumination condition process, mycelia all can grow, and wherein under lasting dark condition, the fastest colony diameter of mycelial growth reaches 2.4cm, and alternation of light and darkness takes second place, and continuous illumination is the poorest, and show that dark is conducive to mycelial growth, illumination then produces restraining effect.PMA and PSA substratum is all conducive to mycelial growth, and wherein optimum medium is PSA, and colony diameter all reaches 6.4 cm, and PMA also can supply nutrition preferably and promote mycelial growth.Mycelia grows comparatively fast on the substratum being carbon source with N.F,USP MANNITOL, sucrose, sorbyl alcohol, starch.On the substratum being carbon source with maltose, fructose, glucose, side of body wood sugar, lactose, growth phase is to slowly.Anthrax bacteria is best to utilizing of peptone, and colony diameter reaches 6.3cm.Extractum carnis takes second place, and the substratum mycelial growth of other various nitrogenous source is slow.
Summary of the invention
One of goal of the invention of the present invention is to provide a kind of steroidal and ferments flax thorn dish spore trichoderma strain, and it produces 7 α, and 15 alpha-dihydroxy-dihydrotestosterones have the features such as transformation efficiency, yield, biological character be more stable.
Two of goal of the invention of the present invention is to provide described steroidal and ferments flax thorn dish spore trichoderma strain at preparation 7 α, the application in 15 alpha-dihydroxy-dihydrotestosterones.
It is being that substrate produces 7 α with DHEA that three of goal of the invention of the present invention is to provide described steroidal flax thorn dish spore trichoderma strain that ferments, the method for 15 alpha-dihydroxy-dihydrotestosterones.
The strain classification status realizing one of the object of the invention is: its sexual generation be under the jurisdiction of mycota (Fungi), Ascomycota guiding principle (Ascomycota), excrement shell Gammaproteobacteria (Sordariomycetes), excrement shell bacterium subclass (Sordariomycetidae), little Nectriaceae (Glomerellaceae), small cluster shell belong to (
glomerellaspauld. & H. Schrenk).This culture presevation is in " China General Microbiological culture presevation administrative center ", and the preservation time is on June 8th, 2013, and preserving number is: CGMCC7715.
Flax thorn dish spore of the present invention mould new strains biological property is as follows.
Strain culturing proterties: after inoculation, culture dish is cultivated about 30 hours at 27 DEG C ~ 28 DEG C, and visible tiny radial growth bacterium colony, cultivates after ripening in 5 days, the general 1.5cm ~ 2.5cm of diameter.Early stage, bacterium colony took center as basic point, and to the radial growth of surrounding, edge is irregular, and middle slightly projection, color is red-brown more deeply, and edge color is more shallow is sorrel, bacterium colony pros and cons solid colour.Bacterium colony surface villous shape in early stage white aerial hyphae, after ripe, bacterium colony surface and edge present black mycelia.The ripe rear surface of bacterium colony is slightly moistening, and bacterium colony matrix is combined with substratum closely, more difficult picking, produces spore like relevant with color, obviously produces spore feature without other.
Strain morphology feature: mycelia nodular grows, clear-cut, alkalescence and acid dyeing node are transparence.Hyphal diameter 3 μm ~ 4 μm.Spore dumbbell shaped, two ends are elliposoidal, and top is point slightly.
Steroidal of the present invention flax thorn dish spore trichoderma strain that ferments can be used for standby 7 α, 15 alpha-dihydroxy-dihydrotestosterones.
Steroidal of the present invention flax thorn dish spore trichoderma strain that ferments is being that substrate produces 7 α with DHEA, and the method for 15 alpha-dihydroxy-dihydrotestosterones is as follows:
1. prepare primary seed solution: original seed brings back to life, and inserts in shaking flask, cultivate under culture temperature on shaking table together with nutrient solution, obtain primary seed solution;
2. prepare secondary seed solution: substratum remains unchanged down, primary seed solution is inserted second-level shake flask according to 20% inoculum size and puts into granulated glass sphere, identical with primary seed solution culture condition, cultivate 24 hours, obtain secondary seed solution;
3. feed intake fermentation: add sterilized water and soya-bean oil in secondary seed solution, and the DMF solution dropping into substrate DHEA according to the charging capacity of 0.8wt% transforms to shaking flask in secondary seed solution, consistent with front step seed culture;
4. result detects: after the fermentation liquor in shaking flask being crossed press filtration-filter cake pulverizing-alcohol reflux extraction-press filtration-concentration-crystallization-filtration-drying program, calculate filter cake raw product yield and transformation efficiency, filtrate adds the ethyl acetate of same volume, quantitative solvent is got after extraction, evaporate into dry, adopt liquid phase process to calculate yield and transformation efficiency;
5. choose production bacterial strain: the bacterial strain of yield and transformation efficiency all >=90% is made on the one hand freeze-drying pipe original seed be stored under 4 DEG C of conditions in refrigerator as original seed for produce later and seed selection strain excellent for subsequent use; Go down to posterity for steroidal fermentative production on the other hand;
5. Secondary Culture: the production bacterial strain access slant medium test tube slant chosen being cultivated, covering with behind inclined-plane namely for cultivating production bacterial classification until bacterium colony;
6. cultivate and produce female kind: the millet boiled is put into triangular flask, then access the spawn culture that goes down to posterity, make bacterial classification cover with substratum;
7. one-level seeding liquid is cultivated: female kind in access seeding tank will be produced, cultivate under growth temperature condition, and after mycelium apparent volume more than 90%, move in second order fermentation tank and cultivate;
8. substrate solution preparation: add DMF by substrate dissolving vessel, drops into DHEA wherein, is substrate solution after being warming up to 125 DEG C after airtight, for subsequent use;
9. cultivate secondary seeding liquid: all moved into by one-level seeding liquid and be equipped with in the fermentor tank of nutrient solution, cultivate under growth temperature condition, detect qualified after, the DMF solution of press-in DHEA ferments;
10. by 7 α, the production technology preparing product of 15 alpha-dihydroxy-dihydrotestosterones.
At production 7 α of the present invention, in the method for 15 alpha-dihydroxy-dihydrotestosterones, more specifically processing condition are as follows:
1. original seed brings back to life is under 27 ~ 30 DEG C of temperature condition, is cultivated 5 ~ 7 days by the original seed preserved on slant medium test tube slant, covers with behind inclined-plane prepare to prepare bacteria suspension until bacterium;
2. primary seed solution inserts in 500ml shaking flask by the nutrient solution 200ml containing bacterial classification, under 27 ~ 30 DEG C of temperature condition, obtain after shaking table cultivates 48h with 200 ~ 220 revs/min, secondary seed solution is that loading amount is reduced to 100ml/500ml shaking flask, primary seed solution is inserted second-level shake flask according to 20% inoculum size, put into 10 granulated glass spherees, with primary seed solution culture condition, cultivate acquisition in 24 hours;
3. Secondary Culture condition is, under 27 ~ 30 DEG C of temperature condition, by production bacterial strain access 30 ' 200 slant medium test tubes chosen, inclined-plane is cultivated 5 ~ 7 days;
4. cultivating and producing female method of planting is that the millet 160g boiled is put into 1000ml triangular flask, then by the bacterial classification after Secondary Culture from test tube slant with under aseptic washing, bacteria suspension is prepared by collecting cells method, and after shaking up by the amount access triangular flask of 2ml every bottle, cultivate 5 days under 27 ~ 30 DEG C of conditions;
5. cultivating one-level seeding liquid is 5 bottles of above-mentioned production mothers are planted access inserted in the seeding tank of 0.7t bacteria culture fluid, cultivate 48h under 27 ~ 30 DEG C of temperature condition after, sampling microscopy is determined not contaminated, and after mycelium apparent volume more than 90%, move in second order fermentation tank and cultivate; Cultivating secondary seeding liquid is insert 2t bacteria culture fluid by the fermentor tank of 6t volume, again the nutrient solution in seeding tank is all moved into this fermentor tank, cultivate 24h under 27 ~ 30 DEG C of temperature condition after, sampling microscopy is determined not contaminated, and mycelium weight in wet base reaches more than 4g/100ml, be pressed into described substrate solution and can start fermentation;
6. secondary seeding hydraulic pressure enters fermenting substrate is be pressed into 1.5 tons of sterilized waters in the fermentor tank filling secondary seeding liquid, then substrate solution is pressed into, cultivate 24h under 27 ~ 30 DEG C of temperature condition after, according to the conversion situation sampled in detection fermentor tank at that time, control fermentation time 30 ~ 48h.
Advantageous Effects of the present invention is: have the features such as transformation efficiency, yield, biological character be more stable, and can be that the plant steroid hormone of raw material is produced and improved output with dioscin, reducing production cost provides technical support.The DHEA solid phase transformation rate that flax thorn dish spore trichoderma strain of the present invention is verified on lab shaker is stabilized in 83 ~ 91%; And 7 α, 15 alpha-dihydroxy-dihydrotestosterone stable yield are 78 ~ 82%.In scale operation (6t fermentor tank), DHEA transformation efficiency is 83 ~ 86%, 7 α, and 15 alpha-dihydroxy-dihydrotestosterone (fine work) yields are 78 ~ 85%.
Accompanying drawing explanation
Fig. 1: flax thorn dish spore mould steroidal compounds fermentable production scheme.
Fig. 2: the mould fermentative production schema of flax thorn dish spore.
Embodiment
Original seed brings back to life: under 27-30 DEG C of temperature condition, the original seed of preservation is cultivated 5-7 days on slant medium test tube slant, covers with behind inclined-plane prepare to prepare bacteria suspension until bacterium.
Prepare bacteria suspension: get 30ml sterilized water and add strain inclined plane, wash lower spore and thalline with cotton swab, suspension is poured in the 250ml triangular flask containing the granulated glass sphere (diameter 2-3mm) of 70-80.By suspension, on shaking table, (200-240 rev/min) shakes broken 0.5h, is filtered by suspension by sterile absorbent cotton, and after being diluted to 10-3 ~ 10-5 multiple, being coated on diameter is on 150mm culture dish.Cultivate 5 days under 27-30 DEG C of temperature condition.
Prepare test tube slant: the culture dish cultivated, select single colony inoculation on test tube (30 ' 200) inclined-plane, cultivate 5 days under 27-30 DEG C of temperature condition.
Preparation primary seed solution: prepare nutrient solution 200ml and insert in 500ml shaking flask, under 27-30 DEG C of temperature condition, after (200-220 rev/min) cultivates 48h on shaking table, namely obtains primary seed solution.
Preparation secondary seed solution: substratum is identical with previous step, and loading amount is reduced to 100ml/500ml shaking flask.Primary seed solution is inserted second-level shake flask according to 20% inoculum size and puts into 10 granulated glass spherees. with primary seed solution culture condition, cultivate 24 hours, namely obtain secondary seed solution.
Feed intake fermentation: add 80ml sterilized water and 0.2% soya-bean oil in secondary seed solution, charging capacity according to 0.8% drops into substrate (the DMF solution of DHEA) and transforms to shaking flask in secondary seed solution, condition and seed culture consistent, transform after 48h and be satisfactory fermented liquid, blanking.
Result detects: by the fermented liquid press filtration of each shaking flask.After getting filter cake pulverizing, add 300ml edible ethanol and be warmed up to boiling point evaporation backflow 2h.Then press filtration, is concentrated into a large amount of crystal and separates out by filtrate, add the rear crystallisation by cooling of a small amount of water dilution.After crystallisate suction filtration is obtained raw product and mother liquor, raw product is dried in 70 DEG C of baking ovens.Take raw product weight, calculate raw product yield and transformation efficiency according to charging capacity.Liquid phase fermented liquid adds same volume ethyl acetate, gets quantitative solvent after extraction, evaporates into dry, adopts liquid phase method to obtain product yield and transformation efficiency.
Choose production bacterial strain: yield and the relatively high bacterial strain (selecting all higher bacterial strain of yield and transformation efficiency in the bacterial strain of yield 90%, transformation efficiency more than 90%) of transformation efficiency are made on the one hand freeze-drying pipe original seed be stored under 4 DEG C of conditions in refrigerator as original seed for produce later and seed selection strain excellent for subsequent use; Go down to posterity for steroidal fermentative production on the other hand.
Secondary Culture: under 27-30 DEG C of temperature condition, cultivating 5-7 days by the production bacterial strain chosen access slant medium test tube (30 ' 200) inclined-plane, covering with behind inclined-plane namely for cultivating production bacterial classification until bacterium colony.
Cultivate and produce female kind: the millet 160g boiled is put into 1000ml triangular flask, then by the bacterial classification after Secondary Culture from test tube slant with under aseptic washing, bacteria suspension is prepared by collecting cells method, and after shaking up by the amount access triangular flask of 2ml every bottle, cultivate 5 days under 27-30 DEG C of condition, make bacterial classification cover with substratum.
Cultivate one-level seeding liquid: 5 bottles are produced bacterial classification access and inserted in the seeding tank (1t volume) of 0.7t nutrient solution (filling a prescription identical with shake flask culture).Cultivate 48h under 27-30 DEG C of temperature condition after, sampling microscopy is determined not have contaminated, and after mycelium apparent volume more than 90%, can move in second order fermentation tank and cultivate.
Prepared by substrate solution: will add the DMF of 62.5L in substrate (DHEA) dissolving vessel (0.1t volume), is dropped into wherein by 32kgDHEA, is substrate solution after being warming up to 125 DEG C after airtight, for subsequent use.
Cultivate secondary seeding liquid: 2t nutrient solution (filling a prescription identical with shake flask culture) will be inserted in the fermentor tank of 6t volume, then the nutrient solution in seeding tank is all moved into this fermentor tank, cultivate 24h under 27-30 DEG C of temperature condition after, sampling microscopy is determined not contaminated, and mycelium weight in wet base reaches more than 4g/100ml, press-in substrate solution can start fermentation.
Throw fermenting substrate: in the fermentor tank filling secondary seed solution, be pressed into 1.5 tons of sterilized waters, then substrate solution is pressed into, after cultivating 24h under the same conditions, the conversion situation according to sampling in detection fermentor tank at that time determines the fermentation termination time, general control fermentation time 30 ~ 48h.
Press filtration: after fermentation reaches end condition, press filtration is carried out to fermented liquid in fermentor tank, get filter cake beat powder after for subsequent use, filtrate collection extract.
Filter cake powder extracts: filter cake powder is dropped into extractor, adds 10 times amount edible ethanols of substrate, and be warming up to backflow 1h, then press filtration concentrates to concentration tank; Squeeze into 10 times amount edible ethanol pump around circuits of substrate again, until filter cake extracts clean (sampling detects) after extracting 3 times.
Filtrate is extracted: filtrate is pressed into high level tank, makes it adsorb from flowing in resin column, and water outlet is after testing without discharge after material.The polymeric adsorbent edible ethanol of 5 times amount carries out wash-out, merges filter cake extracting solution and concentrates.
Raw product concentrates: extracting solution is concentrated into atherosclerotic thing, adds amount of substrate 1 times of process water, stirs and is down to crystallizing at room temperature.
Raw product is centrifugal: above-mentioned solution is inserted centrifuge and dry.Limpid to water outlet with the washing of a small amount of process water, continue centrifugally to ooze to anhydrous.
Highly finished product: raw product is dropped into treatment tank, add the 2-methylpentanone of raw product 30 times of quantity of solvent and the gac temperature rising reflux 1h of 0.1 times, then filtered while hot, be concentrated into a large amount of crystal to separate out, be cooled to 0 ~ 5 DEG C of crystallization, centrifugal, dry, and use appropriate solvent rinse, dry.Re-refine 1 time according to said procedure.After sampling detection is qualified, puts into baking oven (70-80 DEG C) baking 16h and namely obtain 7 α, 15 alpha-dihydroxy-dihydrotestosterone highly finished product.
example:to novel flax thorn dish spore trichoderma strain that the present invention proposes, with regard to its strain cultures, culture condition and shaking table checking (be 7 αs to DHEA microbe conversion, 15 alpha-dihydroxy-dihydrotestosterones) whole process, 5 case verifications are carried out.Its result shows: under substratum and culture condition (shaking speed and culture temperature) do the condition of corresponding variation, flax thorn dish spore trichoderma strain LJYH20130405-1 is to the solid phase transformation rate of DHEA and 7 α, the yield of 15 alpha-dihydroxy-dihydrotestosterones is stabilized between 83 ~ 91% and 78 ~ 82% respectively, illustrates that the mould novel strain of flax thorn dish spore that the present invention proposes has excellent proterties (see table 1); And its this bacterial strain in large-scale industrial scale also has higher transformation efficiency and yield, its DHEA transformation efficiency is 83 ~ 86%, 7 α, and 15 alpha-dihydroxy-dihydrotestosterone (fine work) yields are that 78 ~ 85%(is in table 2).
Table 1 flax thorn dish spore mould fermentation dehydroepiandros-sterone sample result synopsis (shaking table)
。
Table 2 flax thorn dish spore mould fermentation dehydroepiandros-sterone sample result synopsis (6 tons of tanks)
。
Claims (4)
1. a steroidal ferments flax thorn dish spore trichoderma strain, its classification position is: its sexual generation is under the jurisdiction of mycota, Ascomycota guiding principle, excrement shell Gammaproteobacteria, excrement shell bacterium subclass, little Nectriaceae, small cluster shell genus, this culture presevation is in " China General Microbiological culture presevation administrative center ", the preservation time is on June 8th, 2013, and preserving number is: CGMCC7715.
2. steroidal according to claim 1 ferments flax thorn dish spore trichoderma strain at preparation 7 α, the application in 15 alpha-dihydroxy-dihydrotestosterones.
3. steroidal according to claim 1 flax thorn dish spore trichoderma strain that ferments is being that substrate produces 7 α with DHEA, and the method for 15 alpha-dihydroxy-dihydrotestosterones, is characterized in that comprising the following steps:
(1) prepare primary seed solution: original seed brings back to life, and inserts in shaking flask, cultivate under culture temperature on shaking table together with nutrient solution, obtain primary seed solution;
(2) prepare secondary seed solution: substratum remains unchanged down, primary seed solution is inserted second-level shake flask according to 20% inoculum size and puts into granulated glass sphere, identical with primary seed solution culture condition, cultivate 24 hours, obtain secondary seed solution;
(3) feed intake fermentation: add sterilized water and soya-bean oil in secondary seed solution, and the DMF solution dropping into substrate DHEA according to the charging capacity of 0.8wt% transforms to shaking flask in secondary seed solution, consistent with front step seed culture;
(4) result detects: after the fermentation liquor in shaking flask being crossed press filtration-filter cake pulverizing-alcohol reflux extraction-press filtration-concentration-crystallization-filtration-drying program, calculate filter cake raw product yield and transformation efficiency, filtrate adds the ethyl acetate of same volume, quantitative solvent is got after extraction, evaporate into dry, adopt liquid phase process to calculate yield and transformation efficiency;
(5) choose production bacterial strain: the bacterial strain of yield and transformation efficiency all >=90% is made on the one hand freeze-drying pipe original seed be stored under 4 DEG C of conditions in refrigerator as original seed for produce later and seed selection strain excellent for subsequent use; Go down to posterity for steroidal fermentative production on the other hand;
(6) Secondary Culture: the production bacterial strain access slant medium test tube slant chosen being cultivated, covering with behind inclined-plane namely for cultivating production bacterial classification until bacterium colony;
(7) cultivate and produce female kind: the millet boiled is put into triangular flask, then access the spawn culture that goes down to posterity, make bacterial classification cover with substratum;
(8) cultivate one-level seeding liquid: female kind in access seeding tank will be produced, cultivate under growth temperature condition, and after mycelium apparent volume more than 90%, move in second order fermentation tank and cultivate;
9. substrate solution preparation: add DMF by substrate dissolving vessel, drops into DHEA wherein, is substrate solution after being warming up to 125 DEG C after airtight, for subsequent use;
(10) cultivate secondary seeding liquid: all moved into by one-level seeding liquid and be equipped with in the fermentor tank of nutrient solution, cultivate under growth temperature condition, detect qualified after, the DMF solution of press-in DHEA ferments;
(11) by 7 α, the production technology preparing product of 15 alpha-dihydroxy-dihydrotestosterones.
4. be that substrate produces 7 α with DHEA by steroidal according to claim 4 flax thorn dish spore trichoderma strain that ferments, the method for 15 alpha-dihydroxy-dihydrotestosterones, is characterized in that:
1. original seed brings back to life is under 27 ~ 30 DEG C of temperature condition, is cultivated 5 ~ 7 days by the original seed preserved on slant medium test tube slant, covers with behind inclined-plane prepare to prepare bacteria suspension until bacterium;
2. primary seed solution inserts in 500ml shaking flask by the nutrient solution 200ml containing bacterial classification, under 27 ~ 30 DEG C of temperature condition, obtain after shaking table cultivates 48h with 200 ~ 220 revs/min, secondary seed solution is that loading amount is reduced to 100ml/500ml shaking flask, primary seed solution is inserted second-level shake flask according to 20% inoculum size, put into 10 granulated glass spherees, with primary seed solution culture condition, cultivate acquisition in 24 hours;
3. Secondary Culture condition is, under 27 ~ 30 DEG C of temperature condition, by production bacterial strain access 30 ' 200 slant medium test tubes chosen, inclined-plane is cultivated 5 ~ 7 days;
4. cultivating and producing female method of planting is that the millet 160g boiled is put into 1000ml triangular flask, then by the bacterial classification after Secondary Culture from test tube slant with under aseptic washing, bacteria suspension is prepared by collecting cells method, and after shaking up by the amount access triangular flask of 2ml every bottle, cultivate 5 days under 27 ~ 30 DEG C of conditions;
5. cultivating one-level seeding liquid is 5 bottles of above-mentioned production mothers are planted access inserted in the seeding tank of 0.7t bacteria culture fluid, cultivate 48h under 27 ~ 30 DEG C of temperature condition after, sampling microscopy is determined not contaminated, and after mycelium apparent volume more than 90%, move in second order fermentation tank and cultivate; Cultivating secondary seeding liquid is insert 2t bacteria culture fluid by the fermentor tank of 6t volume, again the nutrient solution in seeding tank is all moved into this fermentor tank, cultivate 24h under 27 ~ 30 DEG C of temperature condition after, sampling microscopy is determined not contaminated, and mycelium weight in wet base reaches more than 4g/100ml, be pressed into described substrate solution and can start fermentation;
6. secondary seeding hydraulic pressure enters fermenting substrate is be pressed into 1.5 tons of sterilized waters in the fermentor tank filling secondary seeding liquid, then substrate solution is pressed into, cultivate 24h under 27 ~ 30 DEG C of temperature condition after, according to the conversion situation sampled in detection fermentor tank at that time, control fermentation time 30 ~ 48h.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410136406.2A CN104673677A (en) | 2014-04-08 | 2014-04-08 | Colletotrichum lini strain LJYH20130405-1 for steride fermentation as well as application technology thereof |
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