CN104059944A - Method for preparing ascochlorin by using strains of Cylindrocarpon sp. - Google Patents
Method for preparing ascochlorin by using strains of Cylindrocarpon sp. Download PDFInfo
- Publication number
- CN104059944A CN104059944A CN201410311994.9A CN201410311994A CN104059944A CN 104059944 A CN104059944 A CN 104059944A CN 201410311994 A CN201410311994 A CN 201410311994A CN 104059944 A CN104059944 A CN 104059944A
- Authority
- CN
- China
- Prior art keywords
- ascochlorin
- gram
- ncc3746
- spore
- fermentation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for preparing ascochlorin by using strains of Cylindrocarpon sp. The strains used in the method are strains of the Cylindrocarpon sp. NCC3746 with the preservation number being CGMCCNo.7756. According to the method for preparing the ascochlorin, the fermentation titer unit of the ascochlorin reaches more than 1600 mg/L and is higher than the fermentation titer unit of ascochlorin reported at present, a simple and practical crystallization purification process is developed, the yield is higher than 60%, the product purity is higher than 97.0%, and the method can be used for industrial production of the ascochlorin.
Description
Technical field
The present invention relates to microbial technology field, specifically a kind of method of utilizing the mould genus fungal bacterial strain of post spore to prepare ascochlorin.
Background technology
Ascochlorin is nineteen sixty-eight, first by Japanese scholars Tamura etc. from hyphomycetes ascochyta fungi Testa Viciae fabae two spores (
ascochyta viciae) meta-bolites in separated a kind of isopentene phenolic compound (the Tamura G obtaining; et al. J. Antibiot; 21:539-544; 1968), there is antiviral, antitumor, antibacterium and sedative activity and there is cholesterol and the triglyceride levels of reduction, reduce blood pressure, improving effect (the Hong S of I type and type ii diabetes; et al.; J. Biol Chem, 280 (26): 25202-25209,2005; Tsuruga M, et al., J. Antibiot, 60 (1): 20-26,2007; Sakaguchi K, et al., Biochem Bioph Res Commun, 329 (1): 46-50,2005; Tsuruga M, et al., Apoptosis, 9 (4): 429-435,2004), there is huge application prospect.
Ascochlorin can by Fusarium (
fusariumsp.) (Ralph Henry Evans, et al., US3546073), Nectria (
nectria coccinea) (Andridge, et al., J. Chem. Soc., Perkin Trans. 1,2136-2141,1972), Verticillium (
verticilliumsp.) (Satoshi T, et al., Chem. Pharm. Bull., 42 (4): 953-956,1994), post spore mould (
cylindrocarponsp.) bacterial strain of a plurality of fungi Pseudomonas such as (Mio K, et al., J. Antibiot, 66:23 – 29,2013) produces, but the meta-bolites complicated component of above-mentioned bacterial strains and the fermentation unit of ascochlorin are all very low, are all no more than 50mg/L.As can be seen here, find ascochlorin high efficient strain, improve ascochlorin fermentation yield, develop the technique that is suitable for industrialized preparation ascochlorin and become problem demanding prompt solution.
Summary of the invention
The object of this invention is to provide a kind of method of utilizing the mould genus fungal bacterial strain of post spore to prepare ascochlorin.
The mould genus fungal bacterial strain of post spore that the present invention utilizes, its preservation name is called NCC3746, its Classification And Nomenclature be post spore (
cylindrocarponsp.), China Committee for Culture Collection of Microorganisms of depositary institution common micro-organisms center, preservation address is: No. 3, No. 1, Chaoyang District Beijing North Star West Road institute, preservation date on June 19th, 2013, deposit number is CGMCC No.7756.
The fungal bacterial strain NCC3746 that the present invention utilizes, through macroscopic view and micro-morphology and the analysis of ITS sequence alignment be accredited as the mould genus of post spore (
cylindrocarponsp.).
The mould genus fungal bacterial strain of post spore of the present invention NCC3746 authentication method, for the spore liquid of bacterial strain NCC3746 is coated on slide glass, carries out microscopic morphology observation under 400 power microscopes.By the spore liquid dibbling of bacterial strain NCC3746 in 4 kinds of collective medias: CMA(Corn Meal Agar), PDA(potato dextrose agar), MA(malt meal agar), CzA(Czapek's agar) in, cultivate 14 days at 26 ℃, carry out macroscopic form observation.Compare of analysis through macroscopic view and microscopic morphology observation and ITS sequence, is accredited as the mould genus of post spore by NCC3746.
The mould genus of post spore of the present invention (
cylindrocarponsp.) the separated strain wild strain obtaining in watt room mountain plateau beginning forest soil of Hongya County, fungal bacterial strain NCC3746 Shi Cong Meishan city.
The collecting soil sample method of bacterial strain NCC3746 of the present invention is placed in sterilized paper bag and preserves for scalping the vegetable mould on the top layer of close plant root soil with aseptic shovel, then get apart from the earth's surface degree of depth approximately 30 grams of the soil samples that is 5-10cm.
The separation method of bacterial strain NCC3746 pedotheque of the present invention joins fully vibration in 10mL sterilized water for getting 1 gram of this sample, get subsequently 1mL suspension and join fully vibration in another test tube that 9mL sterilized water is housed, the rest may be inferred carries out gradient dilution until 10
-10concentration.Each concentration is got the two dish of 0.2mL coating PDA, carries out subsequently standing cultivation at 26 ℃, and two dish culture cycle are 3-10 days, and the bacterium colony that picking form is different from two dish is during this period in PDA(Beijing bispin) to cultivate in inclined-plane, the slant culture time is 14 days.By aforesaid method, obtained a fungal strain, by its called after NCC3746.
Adopt bacterial strain NCC3746 of the present invention, production ascochlorin simultaneously and dihydro ascochlorin, more than the fermentation unit of its ascochlorin has reached 1600mg/L, more than the fermentation unit of dihydro ascochlorin has reached 1300mg/L, in crystallisation process, the high ascochlorin preferential crystallization of fermentation unit out, thereby suppress the crystallization of dihydro ascochlorin, it is separated cleverly, ascochlorin yield is more than 60%, product purity reaches more than 97.0%, can be for the suitability for industrialized production of ascochlorin.
The method of utilizing the mould genus fungal bacterial strain of post spore to prepare ascochlorin provided by the present invention, comprises the following steps:
The seed liquor of a, the mould genus of preparative column spore fungi NCC3746:
The slant culture of bacterial strain NCC3746 or spore liquid are inoculated in to seed culture medium, 22-30 ℃, rotating speed 100-220 rpm cultivation 48-72 h acquisition seed liquor; Wherein said seed culture medium makes by the following method: W-Gum 10.0-30.0 gram, glucose 10.0-30.0 gram, 0.5-2.0 gram of hot moulding soybean cake powder, malt meal 3.0-6.0 gram, yeast powder 1.0-3.0 gram, sodium-chlor 0.5-2.0 gram, magnesium sulfate heptahydrate 0.5-2.0 gram, add water and be settled to 1000 mL, pH6.5-7.0,121 ℃ of sterilizing 30min.
The fermented liquid of b, the mould genus of preparative column spore fungi NCC3746:
Above-mentioned seed liquor is inoculated in to fermention medium with the inoculum size of volume percent 3-10 %, and in shaking flask or fermentation cylinder for fermentation, 22-30 ℃ of leavening temperatures, fermentation time 120-180 h, obtain fermented liquid; Wherein said fermention medium makes by the following method: glucose 20.0-40.0 gram, Zulkovsky starch 20.0-40.0 gram, 4.0-20.0 gram of hot moulding soybean cake powder, cottonseed meal 4.0-20.0 gram, potassiumphosphate 1.0-10.0 gram, sodium-chlor 0.5-2.0 gram, magnesium sulfate heptahydrate 0.5-2.0 gram, add tap water and be settled to 1000 mL, pH6.5-7.0,121 ℃ of sterilizing 30 min.
C, by the centrifugal 10-20 min of above-mentioned fermented liquid, abandoning supernatant, obtains mycelium.
D, with aqueous solutions of organic solvent to mycelium lixiviate 2-4 time, each 2-4 hour, discards vat liquor for the first time, vat liquor after merging, concentrating under reduced pressure, removes organic solvent, obtains concentrated solution.
E, concentrated solution add methyl alcohol, dissolve, and obtain solution.
F, by above-mentioned solution left standstill, crystallization, removes by filter mother liquor, obtains ascochlorin crude product.
G, ascochlorin crude product is added in the mixed solvent of methyl alcohol and acetone, dissolve, standing recrystallization, filtering mother liquor, obtains ascochlorin sterling.
Shake flask fermentation in described step b, its rotating speed is 100-220 rpm;
Ferment tank in described step b, fermentation condition for its tank pressure be that 0.05 ± 0.01Mpa, air flow are 10-30 L/min, stirring velocity is 400-500 rpm.
In described steps d, lixiviate is for the first time 30-60% by the concentration expressed in percentage by volume of aqueous solutions of organic solvent, and lixiviate is afterwards 90-100% by the concentration expressed in percentage by volume of aqueous solutions of organic solvent; Described aqueous solutions of organic solvent is aqueous ethanolic solution or aqueous acetone solution.
In described step c, centrifugal rotational speed is 4000 rpm.
In described steps d, concentrating under reduced pressure carries out at 30-50 ℃.
Crystallization in described step f, g, ascochlorin concentration is at 90-130 mg/mL, and temperature is room temperature 10-25 ℃, and crystallization time, between 24-48 h, filters rear with a small amount of organic solvent washing or mother liquor washing leaching cake.
Dissolving in described step e, g, is ultrasonic dissolution or stirring and dissolving, can Ultrasonic Heating or otherwise under stirring state, heat, and Heating temperature is 40-50 ℃.
In described step g, the volume ratio of methyl alcohol and acetone is 1:1 to 3:1.
Above-mentioned optimum condition, it is higher that it obtains ascochlorin output.
The mould genus of center pillar spore of the present invention fungi NCC3746 is wild strain, utilize it to prepare ascochlorin, more than fermentation unit has reached 1600 mg/L, higher than current reported ascochlorin fermentation yield, adopt two main ingredient ascochlorin and dihydro ascochlorin that structure is closely similar in crystallization method separation and purification fermented liquid, ascochlorin total recovery reaches more than 60%, and product purity reaches more than 97.0%, and preparation technology is simple and easy to do, be suitable for suitability for industrialized production.
Accompanying drawing explanation
Fig. 1 is conidial microscopic morphology of bacterial strain.
Fig. 2 is that bacterial strain is at the CMA colonial morphology of upper 14 day.
Fig. 3 is that bacterial strain is at the PDA colonial morphology of upper 14 day.
Fig. 4 is that bacterial strain is at the MA colonial morphology of upper 14 day.
Fig. 5 is that bacterial strain is at the CzA colonial morphology of upper 14 day.
Fig. 6 is that 50L tank fermented liquid HPLC analyzes color atlas.
Fig. 7 is that ascochlorin HPLC analyzes color atlas.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited to this.Experiment material used in following embodiment is industrial raw material if no special instructions; Analyze chromatographic column Inertsil ODS-SP, 5 μ m, 4.6 * 250 mm are purchased from Japanese Shimadzu, and leaching process organic solvent used is purchased from Beijing Chemical Plant, and chromatographic solvent is purchased from Honeywell trade (Shanghai) Co., Ltd..
the Isolation and Identification of embodiment 1, the mould genus bacterial strain of post spore NCC3746
(1), the separation of bacterial strain NCC3746
The separation substratum of fungal bacterial strain NCC3746 is PDA(Beijing bispin);
Get 1 gram of pedotheque (picking up from the soil of Wa Wu mountain, Hongya County, Sichuan Province China province Meishan City virgin forest), put into the 50mL centrifuge tube that 9mL sterilized water is housed, fully vibration.Get 1mL suspension liquid and put into the test tube that 9mL sterilized water is housed, mix.Carry out serial gradient dilution until 10 successively
-10.Under each gradient, get 0.2mL coated plate.The flat board that is coated with is placed at 26 ℃ to be cultivated, and has therefrom obtained a fungal strain, by its called after NCC3746.
(2), the evaluation of bacterial strain NCC3746
The spore liquid of bacterial strain NCC3746 is coated on slide glass, under 400 power microscopes, carried out microscopic morphology observation.By the spore liquid dibbling of bacterial strain NCC3746 in 4 kinds of collective medias: CMA(Corn Meal Agar) PDA(potato dextrose agar) MA(malt meal agar) CzA(Czapek's agar), cultivate 14 days at 26 ℃, carry out macroscopic form observation.
The conidium that bacterial strain NCC3746 produces has 3 or 4 tabulas conventionally, and conidium is cylindrical, and two ends are more blunt; Conidial principal length is 75-95um left and right; The conidium microscopic morphology photo (Fig. 1) of bacterial strain NCC3746.
The morphological specificity of bacterial strain NCC3746 in 4 kinds of collective medias is as follows:
NCC3746 is at CMA(Corn Meal Agar) upper 14 day, colony diameter reaches 47-50mm; Bacterium colony baldness, is grey black; There is sparse flocculence mycelia on top layer; The micro-protuberance of bacterium colony central part, and have maple leaf shape irregular pattern, decorative pattern diameter range is in 30mm left and right; Back is grey black; Produce spore comparatively small amt, spore quantity is about 1.4 ╳ 10
6individual, spore length range is at 45-100um, and width range is at 7.5-10 um.NCC3746 bacterial strain is at the CMA colonial morphology of upper 14 day (Fig. 2).
NCC3746 is at PDA(potato dextrose agar) upper 14 day, colony diameter reaches 47-49mm; There is flocculence mycelia on bacterium colony surface; Bacterium colony central part is purple, and all the other positions are yellowish white; Central part protuberance, scope is about 10mm left and right; Bacterium colony has more regular radial lines; Bacterium colony back central authorities are brown color, and thin out gradually to edge, edge is white in color; Produce spore quantity more, spore quantity is about 2.25 ╳ 10
7individual, spore length range is at 50-100 um, and width range is at 7.5-10 um, and NCC3746 bacterial strain is at the PDA colonial morphology of upper 14 day (Fig. 3).
NCC3746 is at MA(malt meal agar) upper 14 day, colony diameter reaches 42-44mm; Bacterium colony surface is flocculence; Central part protuberance, the about 5-6mm of scope; Ridge bottom is pale blue green, and top is brown; Ridge is coated with the cotton-shaped mycelia of white cotton around, and mycelia is sparse gradually by central authorities to edge; Back central authorities are brown color, and thin out gradually to edge, edge is white in color.Produce spore quantity more, spore quantity is about 4.75 ╳ 10
6individual, spore length range is at 55-85um, and width range is at 7.5-10 um, and NCC3746 bacterial strain is at the MA colonial morphology of upper 14 day (Fig. 4).
NCC3746 is at CzA(Czapek's agar) upper 14 day, colony diameter reaches 48-50mm; Bacterium colony baldness; It is colourless that bacterium colony is; There is sparse cotton-shaped mycelia on top layer; The micro-protuberance of central part; Bacterium colony has 3-4 to enclose irregular concentric annular rill; Bacterium colony back is colourless; Produce spore comparatively small amt, 1.4 ╳ 10
6individual, spore length range is at 50-95 um, and width range is at 5-10 um, and NCC3746 bacterial strain is at the CzA colonial morphology of upper 14 day (Fig. 5).
ITS sequence contains 587bp after measured, measurement result (seeing specification sheets sequence table).Through NCBI comparison, with the mould genus of post spore (
cylindrocarponsp.) sibship is nearest.
Therefore according to macroscopic view and micro-morphology and ITS sequence alignment analytical results, by NCC3746 be accredited as the mould genus of post spore (
cylindrocarponsp.).
the preparation of embodiment 2,50L tank fermentation ascochlorin
(a) the mould genus of preparative column spore fungi NCC3746 seed liquor
The mould genus of post spore fungi NCC3746 is inoculated in to seed culture medium, and 26 ℃, rotating speed 200 rpm are cultivated 72 h and are obtained seed liquor.The preparation method of seed culture medium is: 20.0 grams of W-Gums, 10.0 grams of glucose, 2.0 grams of hot moulding soybean cake powders, 6.0 grams of malt meals, 2.5 grams of yeast powders, 2.0 grams, sodium-chlor, 1.0 grams of magnesium sulfate heptahydrates, add tap water dissolving and be settled to 1000 mL, pH value 6.5-7.0,121 ℃ of sterilizing 30 min.
(b) fermented liquid of the mould genus of preparative column spore fungi NCC3746
Seed liquor is inoculated in to the fermentor tank that 30 L fermention mediums are housed with the inoculum size of volume percent 5%, 26 ℃ of tank temperature, tank pressure 0.05 ± 0.01 Mpa, air flow 30 L/min, 400 rpm stir, fermentation 168 h.The preparation method of fermention medium is: 29.0 grams of glucose, 30.0 grams of Zulkovsky starches, 14.0 grams of cottonseed meals, 14.0 grams of hot moulding soybean cake powders, K
2hPO
43.0 grams, MgSO
47H
20.5 gram of O, 0.5 gram of NaCl, add tap water dissolving and be settled to 1000 mL, pH value 6.5-7.0,121 ℃ of sterilizing 30 min.
(c) after fermentation, carry out HPLC detection (moving phase: 75% CH
3cN, flow velocity: 1.0 mL/min), color atlas is shown in Fig. 6, ascochlorin fermentation unit is 1806 mg/L.By centrifugal 15 min of 27 L fermented liquid 4000 rpm of the mould genus of post spore fungi NCC3746, supernatant discards, and obtains mycelium approximately 3.3 L.
(d) first mycelium is used 10 L 50% aqueous ethanolic solution lixiviate 4 h, and after lixiviate, vat liquor is given up for the first time.Use afterwards 13 L 95% alcohol steeps, lixiviate twice, each 4 h at every turn.Merge vat liquor twice, 40 ℃ are evaporated to without ethanol, obtain concentrated solution approximately 300 mL.Wherein ascochlorin and dihydro ascochlorin content ratio are 11:9.
(e) concentrated solution adds 80mL methyl alcohol, and the 50 ℃ of heating of ultrasonic while obtain viscous solution, wherein ascochlorin concentration approximately 128 mg/mL to fully dissolving in 3-5 minute.
(f) above-mentioned solution at room temperature obtains a large amount of crystalline particles precipitate ascochlorin crystal after standing about 40h, and filtering mother liquor, also by 30 mL washed with methanol, cleans twice, and decompressing and extracting solvent, obtains ascochlorin crude product 39.5 g, HPLC purity assay 81%.
(g) above-mentioned ascochlorin crude product is added in 330mL methyl alcohol and acetone (v:v=2:1) mixing solutions, 50 ℃ of heating in water bath ultrasonic to dissolving, ascochlorin concentration is 96.9 mg/mL, standing 28 h of room temperature after qualitative filter paper filters, obtain ascochlorin crystallization, after filtering mother liquor with twice, 35 mL washed with methanol crystal, with 50 mL sherwood oils, clean once again, finally draining solvent obtains ascochlorin fine work 32.1 g(HPLC analysis of spectra and sees Fig. 7), content 97.5 %, total recovery 63.9%.
Ascochlorin molecular formula is: C
23h
29clO
4, ESI-MS m/z 405.18 [M+H]
+, 403.17 [M-H]
-; UV λ
max(in EtOH): 240,250,292,346nm; 153 ~ 154 ℃ of MP, [α]=-31
o(c, 0.99, MeOH).
1H-NMR(500MHz, CDCl
3) δ(ppm): 0.70(3H, s), 0.81(3H,d, J=6.5Hz), 0.83(3H,d, J=6.5Hz), 1.62 (1H, m), 1.92(3H, s), 1.95(2H, m), 2.35-2.43 (3H, m), 2.60(3H, s), 3.54(2H,d, J=7.5Hz), 5.38(1H,d, J=16.0Hz), 5.52(1H,t, J=7.5Hz), 5.91(1H,d, J=16.0Hz), 6.42(1H, s), 10.14(1H, s), 12.71(1H, s)。
13C-NMR(125MHz, CDCl
3) δ(ppm): 8.9(11′-CH
3), 10.3(6′-CH
3), 12.6(7′-CH
3), 14.5(3′-CH
3), 16.3(6-CH
3), 22.2(C-1′), 31.1(C-8′), 40.8(C-9′), 41.6(C-7′), 48.5(C-6′), 53.6(C-11′), 113.1(C-5), 113.6(C-3), 113.7(C-1), 127.5(C-2′), 133.2(C-4′), 134.1(C-3′), 135.6(C-5′), 137.8(C-6), 156.1(C-4), 162.2(C-2), 193.2(1-CHO), 212.8(C-10′)。
The chemical structural formula of ascochlorin
the preparation of embodiment 3, shake flask fermentation ascochlorin
(a) seed liquor of the mould genus of preparative column spore fungi NCC3746
By the mould genus of post spore (
cylindrocarponsp.) fungi NCC3746 is inoculated in seed culture medium, and 26 ℃, rotating speed 200 rpm are cultivated 72 h and obtained seed liquor.The preparation method of seed culture medium is: 20.0 grams of W-Gums, 20.0 grams of glucose, 2.0 grams of hot moulding soybean cake powders, 4.0 grams of malt meals, 3.0 grams of yeast powders, 2.0 grams, sodium-chlor, 1.0 grams of magnesium sulfate heptahydrates, add tap water dissolving and be settled to 1000 mL, pH value 6.5-7.0,121 ℃ of sterilizing 30 min.
(b) fermented liquid of the mould genus of preparative column spore fungi NCC3746
Seed liquor is inoculated in to the 1000mL shaking flask that 150mL fermention medium is housed with the inoculum size of volume percent 5%, 28.5 ℃, rotating speed 220 rpm, fermentation 144 h.The preparation method of fermention medium is: 29.0 grams of glucose, 30.0 grams of Zulkovsky starches, 14.0 grams of cottonseed meals, 14.0 grams of hot moulding soybean cake powders, K
2hPO
43.0 grams, MgSO
47H
20.5 gram of O, 0.5 gram of NaCl, add tap water dissolving and be settled to 1000 mL, pH value 6.5-7.0,121 ℃ of sterilizing 30 min.
(c) after fermentation, ascochlorin fermentation unit is 2115 mg/L after testing.4L fermented liquid 4000 rpm of the mould genus of post spore fungi NCC3746 are centrifugal, and supernatant discards, and obtains mycelium approximately 500 mL.
(d) first mycelium is used 2 L 35% aqueous acetone solution lixiviate 3 h, and after lixiviate, vat liquor is given up for the first time, uses afterwards 2 L industrial acetone lixiviates, repeats lixiviate three times, each 3 h.Merge three times vat liquor, 50 ℃ are evaporated to without acetone, obtain concentrated solution approximately 50 mL.Wherein the mass content of ascochlorin and dihydro ascochlorin is than being 9:8.
(e) concentrated solution adds the methyl alcohol of 30 mL, and Ultrasonic Heating 2 min are to fully dissolving.Ascochlorin concentration is 105.8 mg/mL.
(f) above-mentioned solution at room temperature standing about 40h obtain solid precipitate ascochlorin crystal, filtering mother liquor also use 15mL washed with methanol, cleaning twice, decompressing and extracting solvent, obtains ascochlorin crude product 6.93g, HPLC purity assay 82%.
(g) ascochlorin crude product is dissolved in 64 mL methyl alcohol and acetone (v:v=3:1) mixing solutions, 45 ℃ of heating in water bath ultrasonic to all dissolving, ascochlorin concentration is 90.6 mg/mL, after filter paper filtering, is statically placed under room temperature.After 24h, obtain ascochlorin crystal, filtering mother liquor, 10 mL washed with methanol twice for crystal, then clean once with 25 mL sherwood oils, drain afterwards solvent, obtain ascochlorin fine work 5.25g, HPLC content 98 %, total recovery 62 %.
the preparation of embodiment 4, shake flask fermentation ascochlorin
(a) seed liquor of the mould genus of preparative column spore fungi NCC3746
By the mould genus of post spore (
cylindrocarponsp.) fungi NCC3746 is inoculated in seed culture medium, and 22 ℃, rotating speed 100 rpm are cultivated 68 h and obtained seed liquor.The preparation method of seed culture medium is: 10.0 grams of W-Gums, 30.0 grams of glucose, 0.5 gram of hot moulding soybean cake powder, 3.0 grams of malt meals, 1.5 grams of yeast powders, 1.0 grams, sodium-chlor, 0.5 gram of magnesium sulfate heptahydrate, add tap water dissolving and be settled to 1000 mL, 6.5,121 ℃ of sterilizing 30 min of pH value.
(b) fermented liquid of the mould genus of preparative column spore fungi NCC3746
Seed liquor is inoculated in to the 250mL shaking flask that 40mL fermention medium is housed with the inoculum size of volume percent 3%, 22 ℃, rotating speed 100 rpm, fermentation 180 h.The preparation method of fermention medium is: 20.0 grams of glucose, 40.0 grams of Zulkovsky starches, 4.0 grams of cottonseed meals, 4.0 grams of hot moulding soybean cake powders, K
2hPO
410.0 grams, MgSO
47H
20.5 gram of O, 2.0 grams of NaCl, add tap water dissolving and be settled to 1000 mL, 6.5,121 ℃ of sterilizing 30 min of pH value.
(c) after fermentation, ascochlorin fermentation unit is 1768 mg/L after testing.2L fermented liquid 4000 rpm of the mould genus of post spore fungi NCC3746 are centrifugal, and supernatant discards, and obtains mycelium approximately 200 mL.
(d) first mycelium is used 1 L 30% aqueous acetone solution lixiviate 4 h, and after lixiviate, vat liquor is given up for the first time.Use afterwards the lixiviate of 1L industrial acetone, lixiviate twice, each 4 h at every turn.Merge vat liquor twice, 30 ℃ are evaporated to without acetone, obtain concentrated solution approximately 30 mL.
(e) concentrated solution adds the methyl alcohol of 6 mL, and Ultrasonic Heating 4min is to fully dissolving.Ascochlorin concentration is 98.2mg/mL.
(f) above-mentioned solution obtains solid precipitate ascochlorin crystal after standing about 24h at 25 ℃ of room temperatures, filtering mother liquor, and solid 10mL washed with methanol, cleans twice, and decompressing and extracting solvent, obtains ascochlorin crude product 3.31g, HPLC purity assay 80%.
(g) ascochlorin crude product is dissolved in 25 mL methyl alcohol and acetone (v:v=1:1) mixing solutions, 40 ℃ of heating in water bath ultrasonic to all dissolving, are statically placed in after filter paper filtering under room temperature, wherein ascochlorin concentration 108.1mg/mL.After 24h, obtain ascochlorin crystal, filtering mother liquor, 8mL washed with methanol twice for crystal, then clean once with 10 mL sherwood oils, drain afterwards solvent, obtain ascochlorin fine work 2.23g, HPLC content 97.9 %, total recovery 63 %.
the preparation of embodiment 5, shake flask fermentation ascochlorin
(a) seed liquor of the mould genus of preparative column spore fungi NCC3746
By the mould genus of post spore (
cylindrocarponsp.) fungi NCC3746 is inoculated in seed culture medium, and 30 ℃, rotating speed 220 rpm are cultivated 48 h and obtained seed liquor.The preparation method of seed culture medium is: 30.0 grams of W-Gums, 10.0 grams of glucose, 1.0 grams of hot moulding soybean cake powders, 5.0 grams of malt meals, 1.0 grams of yeast powders, 2.0 grams, sodium-chlor, 2.0 grams of magnesium sulfate heptahydrates, add tap water dissolving and be settled to 1000 mL, 7.0,121 ℃ of sterilizing 30 min of pH value.
(b) fermented liquid of the mould genus of preparative column spore fungi NCC3746
Seed liquor is inoculated in to the 250mL shaking flask that 40mL fermention medium is housed with the inoculum size of volume percent 10%, 30 ℃, rotating speed 220 rpm, fermentation 120 h.The preparation method of fermention medium is: 40.0 grams of glucose, 20.0 grams of Zulkovsky starches, 20.0 grams of cottonseed meals, 20.0 grams of hot moulding soybean cake powders, K
2hPO
41.0 grams, MgSO
47H
22.0 grams of O, 0.5 gram of NaCl, add tap water dissolving and be settled to 1000 mL, 7.0,121 ℃ of sterilizing 30 min of pH value.
(c) after fermentation, ascochlorin fermentation unit is 1961 mg/L after testing.2L fermented liquid 4000 rpm of the mould genus of post spore fungi NCC3746 are centrifugal, and supernatant discards, and obtains mycelium approximately 200 mL.
(d) first mycelium is used 1 L 55% aqueous ethanolic solution lixiviate 2 h, and after lixiviate, vat liquor is given up for the first time.Use afterwards lixiviate twice, use 1L 95% alcohol steep at every turn, each 2 h.Merge vat liquor twice, 50 ℃ are evaporated to without ethanol, obtain concentrated solution approximately 30 mL.
(e) concentrated solution adds the methyl alcohol of 10 mL, and Ultrasonic Heating 2min is to fully dissolving.Ascochlorin concentration 98mg/mL.
(f) above-mentioned solution obtains solid precipitate ascochlorin crystal after standing about 48h at approximately 10 ℃ of room temperatures, and filtering mother liquor also use 10mL washed with methanol, cleaning twice, and decompressing and extracting solvent, obtains ascochlorin crude product 3.86g, HPLC purity assay 83%.
(g) ascochlorin crude product is dissolved in 30 mL methyl alcohol and acetone (v:v=3:1) mixing solutions, 50 ℃ of heating in water bath ultrasonic to all dissolving, are statically placed in after filter paper filtering under room temperature, wherein ascochlorin concentration 91.4mg/mL.After 24h, obtain ascochlorin crystal, filtering mother liquor, 10 mL washed with methanol twice for crystal, then clean once with 15 mL sherwood oils, drain afterwards solvent, obtain ascochlorin fine work 2.52g, HPLC content 98 %, total recovery 64%.
Claims (8)
1. utilize the mould genus fungal bacterial strain of post spore to prepare a method for ascochlorin, comprise the following steps:
The seed liquor of a, the mould genus of preparative column spore fungi NCC3746:
The slant culture of bacterial strain NCC3746 or spore liquid are inoculated in to seed culture medium, 22-30 ℃, rotating speed 100-220 rpm cultivation 48-72 h acquisition seed liquor;
Wherein said seed culture medium makes by the following method: W-Gum 10.0-30.0 gram, glucose 10.0-30.0 gram, 0.5-2.0 gram of hot moulding soybean cake powder, malt meal 3.0-6.0 gram, yeast powder 1.0-3.0 gram, sodium-chlor 0.5-2.0 gram, magnesium sulfate heptahydrate 0.5-2.0 gram, add water and be settled to 1000 mL, pH6.5-7.0,121 ℃ of sterilizing 30min;
The fermented liquid of b, the mould genus of preparative column spore fungi NCC3746:
Above-mentioned seed liquor is inoculated in to fermention medium with the inoculum size of volume percent 3-10 %, and in shaking flask or fermentation cylinder for fermentation, 22-30 ℃ of leavening temperatures, fermentation time 120-180 h, obtain fermented liquid;
Wherein said fermention medium makes by the following method: glucose 20.0-40.0 gram, Zulkovsky starch 20.0-40.0 gram, 4.0-20.0 gram of hot moulding soybean cake powder, cottonseed meal 4.0-20.0 gram, potassiumphosphate 1.0-10.0 gram, sodium-chlor 0.5-2.0 gram, magnesium sulfate heptahydrate 0.5-2.0 gram, add tap water and be settled to 1000 mL, pH6.5-7.0,121 ℃ of sterilizing 30 min;
C, by the centrifugal 10-20 min of above-mentioned fermented liquid, abandoning supernatant, obtains mycelium;
D, with aqueous solutions of organic solvent to mycelium lixiviate 2-4 time, each 2-4 hour, discards vat liquor for the first time, vat liquor after merging, concentrating under reduced pressure, removes organic solvent, obtains concentrated solution;
E, concentrated solution add methyl alcohol, dissolve, and obtain solution;
F, by above-mentioned solution left standstill, crystallization, after crystal is separated out completely, removes by filter mother liquor, obtains ascochlorin crude product;
G, ascochlorin crude product is added in the mixed solvent of methyl alcohol and acetone and dissolves, standing recrystallization, to be crystallized completely after, filtering mother liquor, obtains ascochlorin sterling;
Wherein the aqueous solutions of organic solvent described in steps d is aqueous ethanolic solution or aqueous acetone solution;
Wherein in step g, the volume ratio of methyl alcohol and acetone is 1:1 to 3:1.
2. method according to claim 1, wherein in steps d, lixiviate is for the first time 30-60% by the concentration expressed in percentage by volume of aqueous solutions of organic solvent, lixiviate is afterwards 90-100% by the concentration expressed in percentage by volume of aqueous solutions of organic solvent.
3. method according to claim 1, wherein the concentrating under reduced pressure described in steps d carries out at 30-50 ℃.
4. method according to claim 1, the wherein crystallization described in step f, g, ascochlorin concentration is at 90-130 mg/mL.
5. method according to claim 1, the wherein crystallization described in step f, g, temperature is room temperature 10-25 ℃, crystallization time is between 24-48 h.
6. method according to claim 1, is wherein dissolved as ultrasonic dissolution or heating for dissolving described in step e, g, and Heating temperature is at 40-50 ℃.
7. method according to claim 1, the shake flask fermentation described in step b wherein, its rotating speed is 100-220 rpm.
8. method according to claim 1, the ferment tank described in step b wherein, its tank pressure is that 0.05 ± 0.01 Mpa, air flow are 10-30 L/min, stirring velocity is 400-500 rpm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410311994.9A CN104059944B (en) | 2014-07-02 | 2014-07-02 | A kind of method utilizing post spore mould genus fungal bacterial strain to prepare ascochlorin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410311994.9A CN104059944B (en) | 2014-07-02 | 2014-07-02 | A kind of method utilizing post spore mould genus fungal bacterial strain to prepare ascochlorin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104059944A true CN104059944A (en) | 2014-09-24 |
| CN104059944B CN104059944B (en) | 2016-08-24 |
Family
ID=51547875
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410311994.9A Active CN104059944B (en) | 2014-07-02 | 2014-07-02 | A kind of method utilizing post spore mould genus fungal bacterial strain to prepare ascochlorin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104059944B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110770347A (en) * | 2017-05-11 | 2020-02-07 | 龟甲万株式会社 | Method for producing isoprenoid, and protein, gene and transformant used for same |
| CN111943828A (en) * | 2020-08-20 | 2020-11-17 | 广西中医药大学 | Ascochylomycete compound and application thereof in preparation of antitumor drugs or dihydroorotate dehydrogenase inhibitor drugs |
-
2014
- 2014-07-02 CN CN201410311994.9A patent/CN104059944B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| M KAWAGUCHI等: "A new ascochlorin derivative from cylindrocarpon sp. fki-4602", 《J. ANTIBIOT》, vol. 66, 21 November 2012 (2012-11-21) * |
| 李业英等: "壳二袍氯素产生菌的鉴定及发酵培养基优化", 《中国抗生素杂志》, vol. 37, no. 5, 31 May 2012 (2012-05-31) * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110770347A (en) * | 2017-05-11 | 2020-02-07 | 龟甲万株式会社 | Method for producing isoprenoid, and protein, gene and transformant used for same |
| CN111943828A (en) * | 2020-08-20 | 2020-11-17 | 广西中医药大学 | Ascochylomycete compound and application thereof in preparation of antitumor drugs or dihydroorotate dehydrogenase inhibitor drugs |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104059944B (en) | 2016-08-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101445784B (en) | Eupenicillium brefeldianum variety ZJB082702 and application thereof in preparation of Brefeldin A by fermentation | |
| CN101085989B (en) | Process for producing tomatine using bacteria fermentation | |
| CN104694401B (en) | Penicillium viridicatum strain and the method that ergosterol peroxide is produced with it | |
| CN106010980B (en) | A kind of endogenetic fungus Brazil class shell roundlet spore bacterial strain and its application | |
| CN110527629A (en) | A kind of brefeldin A in marine fungi source, preparation method and its application in terms of anti-agriculture pathogenic bacteria | |
| CN104726347A (en) | Penicillium vulpinum fungi strain and method for preparing levo 7-hydroxyl butylphthalide employing fungi strain | |
| CN107189949A (en) | Rhizopus oryzae LJH3 and the application in bioconversion Sophoricoside prepares genistein | |
| CN108315265B (en) | Aspergillus versicolor Av-2 strain and application thereof | |
| CN104059944A (en) | Method for preparing ascochlorin by using strains of Cylindrocarpon sp. | |
| CN105779348A (en) | Method for producing Rakicidins compounds by virtue of marine micromonospora fermentation | |
| CN104726345A (en) | Combined strains for highly producing baccatin III and method for producing baccatin III | |
| CN102226152B (en) | Cylindrocarpon didymium and method for preparing ginsenoside Rh2 by using same | |
| CN102161973B (en) | Method for preparing homoharringtonine and special strain | |
| CN104611283A (en) | Recombinational streptomyces lydicus and application thereof | |
| CN106282032B (en) | Penicillium citrinum LB and the application in phillygenol is prepared in bioconversion forsythin | |
| CN110527632B (en) | Endophytic fungus strain for efficiently biotransformation of betulinic acid and application thereof | |
| CN104059858B (en) | One strain post spore mould genus fungal bacterial strain and utilize the method that this bacterial strain prepares dihydro ascochlorin | |
| CN102329829B (en) | Method for converting daidzein into 8-hydroxydaidzein by utilizing penicillium | |
| CN109182142A (en) | Skin falls mould and its application | |
| CN105602856B (en) | Aspergillus niger (Aspergillus niger) An-19 bacterial strain and its purposes and fermentation process of the production for Lovastatin | |
| CN114806888B (en) | Aspergillus versicolor ZJUTE2 and application thereof in preparation of aflatoxin | |
| CN103966289A (en) | Preparation method for malformin C | |
| CN102851216B (en) | Aspergillus fumigatus and its use in microbial preparation of (R,R)-3-phenylglcidol | |
| CN108218884B (en) | Huperzine B derivative and preparation method thereof | |
| CN102703529B (en) | Method for generating betulinic ketone alcohol by catalyzing betulin through rhodotorula mucilaginosa |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |