CN114276392A - Zhongshengmycin F component mother medicine and preparation method thereof - Google Patents
Zhongshengmycin F component mother medicine and preparation method thereof Download PDFInfo
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- VMFXMTJCTSYHCF-HHQUSWFZSA-N [(2r,3r,4s,5r)-5-(hexylamino)-4-hydroxy-2-(hydroxymethyl)-6-[(7-hydroxy-4-oxo-1,3a,5,6,7,7a-hexahydroimidazo[4,5-c]pyridin-2-yl)amino]oxan-3-yl] carbamate Chemical compound CCCCCCN[C@@H]1[C@H](O)[C@@H](OC(N)=O)[C@@H](CO)OC1\N=C\1NC(C(=O)NCC2O)C2N/1 VMFXMTJCTSYHCF-HHQUSWFZSA-N 0.000 title claims abstract description 65
- 239000003814 drug Substances 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 238000000855 fermentation Methods 0.000 claims abstract description 60
- 230000004151 fermentation Effects 0.000 claims abstract description 60
- 239000001963 growth medium Substances 0.000 claims abstract description 31
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- 238000011218 seed culture Methods 0.000 claims abstract description 18
- 238000012360 testing method Methods 0.000 claims abstract description 16
- 238000004519 manufacturing process Methods 0.000 claims abstract description 15
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- 229940079593 drug Drugs 0.000 claims abstract description 10
- 238000001035 drying Methods 0.000 claims abstract description 10
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- 239000002994 raw material Substances 0.000 claims abstract description 9
- 238000005342 ion exchange Methods 0.000 claims abstract description 8
- 241001655322 Streptomycetales Species 0.000 claims abstract description 5
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 54
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 48
- 238000003756 stirring Methods 0.000 claims description 43
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 32
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 28
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 27
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 24
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 24
- 239000011780 sodium chloride Substances 0.000 claims description 24
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- 230000001954 sterilising effect Effects 0.000 claims description 21
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 20
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 20
- 239000004202 carbamide Substances 0.000 claims description 20
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- 239000008399 tap water Substances 0.000 claims description 20
- 235000020679 tap water Nutrition 0.000 claims description 20
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 18
- 229920002261 Corn starch Polymers 0.000 claims description 17
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- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 claims description 15
- 238000012258 culturing Methods 0.000 claims description 13
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 13
- 235000012424 soybean oil Nutrition 0.000 claims description 12
- 239000003549 soybean oil Substances 0.000 claims description 12
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 11
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 11
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- 244000068988 Glycine max Species 0.000 claims description 7
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 5
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 2
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- 240000007124 Brassica oleracea Species 0.000 description 1
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- 241000233679 Peronosporaceae Species 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 241000187389 Streptomyces lavendulae Species 0.000 description 1
- 241001398008 Streptomyces qinlingensis Species 0.000 description 1
- 229930189330 Streptothricin Natural products 0.000 description 1
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- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
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- 230000015572 biosynthetic process Effects 0.000 description 1
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229940126534 drug product Drugs 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
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- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
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- 230000000630 rising effect Effects 0.000 description 1
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- REFMEZARFCPESH-UHFFFAOYSA-M sodium;heptane-1-sulfonate Chemical compound [Na+].CCCCCCCS([O-])(=O)=O REFMEZARFCPESH-UHFFFAOYSA-M 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a zhongshengmycin F component mother medicine and a preparation method thereof, which comprises the following steps: (1) preparing a production test tube slant culture medium, and inoculating streptomycete on a slant for culture; when a single colony grows in the slant culture medium, picking the single colony by using an inoculating needle and carrying out the next culture; (2) preparing a seed culture medium for strain fermentation, and inoculating the single colony obtained in the step (1) for culture to obtain a seed culture solution; (3) preparing a fermentation tank culture medium, and inoculating the seed culture solution obtained in the step (2), wherein the volume ratio of the seed culture solution to the fermentation culture solution is 10-15%; (4) filtering the fermentation liquor, performing ion exchange, eluting, concentrating, decoloring, cooling and drying to obtain the zhongshengmycin mother drug. The method can prepare the mother medicine containing 60.8-78.7wt% of the zhongshengmycin F component, fills the blank of the high-purity zhongshengmycin mother medicine, and provides raw material medicines for the production and development of mixed preparations and pesticide products with the content higher than 12%.
Description
Technical Field
The invention belongs to the field of pesticides, and particularly relates to a zhongshengmycin F component mother drug and a preparation method thereof.
Background
Zhongshengmycin (zhongshengmycin) is also called as paragonin 751 bacterin and streptothricin, and belongs to N-glycoside antibiotics. According to the literature reports, various Streptomyces such as Streptomyces lavendulae var heinenensis n.var, Streptomyces qinlingensis sp. nov can produce the zhongshengmycin. The zhongshengmycin has wide antibacterial spectrum, can resist gram-positive bacteria, gram-negative bacteria, mycobacteria, saccharomycetes, filamentous fungi and the like, and also has a certain killing effect on insects. The sterilization mechanism is that polypeptide synthesis guided by polyU can be inhibited, misreading in protein translation can be caused, and bacteria can not synthesize protein and die. The zhongshengmycin is agriculturally mainly used for preventing and treating bacterial diseases such as rice bacterial leaf blight, cucumber bacterial angular leaf spot, cabbage soft rot, bacillus ulcer and the like, and also can be used for preventing and treating fungal diseases such as apple ring spot, cucumber downy mildew, gray mold, powdery mildew, tomato late blight and the like. The zhongshengmycin has a plurality of effective components, wherein the zhongshengmycin F component is one of the components of the zhongshengmycin with higher pesticide utilization value. The F component structure is as follows (n = 1):
because of its remarkable and direct bactericidal effect, the streptomyces producing the zhongshengmycin is relatively easy to be found and separated in nature. The invention obtains the bacterial strain producing the zhongshengmycin for a plurality of times in the long-term collection and separation of the field bacterial strain. In 2003, a strain of actinomycete is separated from the soil of the great wall of the small wall of the great wall of the small wall of the great wall of the small wall. The product separation and identification show that the bacteriostatic component is mainly Zhongshengmycin C, D, F component, the reported Zhongshengmycin A and X components are not found, and the B, E component content is low. Subsequently, the strain center of Shaanxi Meconlo Biotechnology Limited company carries out long-term continuous single ultraviolet mutagenesis screening to obtain a strain ZS-II-D190515 (identified by Chinese academy of sciences microorganism in 2019, the result is also streptomyces), and then researches on the aspects of culture medium and culture condition optimization, liquid fermentation, product separation identification, activity test and the like are carried out, and the specific characteristics are shown in figures 1-3. With the assistance of related colleges and scientific research units, in the year 2015, a 10M3 fermentation tank is adopted, a deep liquid fermentation test is carried out, a product sample takes acetonitrile, water, sodium heptanesulfonate, disodium hydrogen phosphate and phosphoric acid as mobile phases, a stainless steel column and an ultraviolet detector which take RP-C18 as fillers are used, high performance liquid chromatography separation is carried out on each component in the sample at the wavelength of 200nm, the product is mainly zhongshengmycin F, the components A and X are not found, and the content of other components such as BCDE is low.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides a zhongshengmycin F component mother medicine and a preparation method thereof. The method can prepare the mother medicine containing 60.8-78.7wt% of the zhongshengmycin F component, the content of the product is far higher than that of the 12% zhongshengmycin mother medicine registered by the department of agriculture at present, the product is dissolved in water, insoluble substances are less than 0.2%, the blank of the high-purity zhongshengmycin mother medicine is filled, and raw material medicines are provided for the production and development of mixed preparations and pesticide products with the content higher than 12%.
In order to realize the purpose of the invention, the invention adopts the following technical scheme: a preparation method of a zhongshengmycin F component mother medicine comprises the following steps:
(1) preparing a production test tube slant culture medium, and inoculating streptomycete on a slant for culture; when a single colony grows in the slant culture medium, picking the single colony by using an inoculating needle and carrying out the next culture;
(2) preparing a seed culture medium for strain fermentation, and inoculating the single colony obtained in the step (1) for culture to obtain a seed culture solution;
(3) preparing a fermentation tank culture medium, and inoculating the seed culture solution obtained in the step (2), wherein the volume ratio of the seed culture solution to the fermentation culture solution is 10-15%;
(4) filtering the fermentation liquor, performing ion exchange, eluting, concentrating, decoloring, cooling and drying to obtain the zhongshengmycin mother drug.
More preferably, the preparation method comprises the following steps:
(1) the slant culture medium for producing the test tube adopts corn flour or corn starch as a carbon source, and the using amount is 3-7%; urea is used as a nitrogen source, and the using amount is 1-3%; trace element sodium chloride, the consumption is 0.1-0.2%; the dosage of the trace element magnesium chloride is 0.1 to 0.2 percent; the phosphorus source adopts monopotassium phosphate, and the using amount is 0.2-0.4%; the dosage of the agar is 20 g/L; adjusting pH value to 7.0-7.4 with 2mol/L hydrochloric acid or 2mol/L sodium hydroxide; supplementing the raw materials with tap water, and preparing the raw materials by the following steps:
firstly, sequentially adding sodium chloride, magnesium chloride, potassium dihydrogen phosphate and urea into tap water at the temperature of 40 +/-2 ℃, stirring to fully dissolve, then adding corn starch, and stirring until the mixture is uniform and free of lumps; the tap water is used as 70 percent of the total preparation water consumption, and the preparation process is finished in a water bath;
secondly, using tap water with the same temperature in the first step to fix the volume;
thirdly, placing the solution with the constant volume obtained in the second step at room temperature, and adjusting the solution with 2mol/L hydrochloric acid when the pH value is higher than 7.4 after the temperature is reduced to 20 ℃; if the pH value is lower than 7.0, adjusting the pH value by using 2mol/L sodium hydroxide; then adding agar and stirring uniformly;
fourthly, preparing an inclined plane after the adjusted culture medium is sterilized, and culturing for 72 hours at 37 ℃ until no mixed bacteria fall;
fifthly, inoculating streptomyces on the inclined plane, and culturing for 140-150 hours to grow offwhite lawn;
(2) corn flour or corn starch is adopted as a carbon source in a seed culture medium for strain fermentation, and the using amount is 2-3%; cold-pressed soybean cake powder is used as a nitrogen source, and the using amount is 2-3 percent; adding 0.5-2% of glucose as a quick-acting carbon source; 1-2% of quick-acting nitrogen source urea is added; adding inorganic salts of sodium chloride, potassium dihydrogen phosphate and magnesium chloride, wherein the dosage is 0.1-0.2% of sodium chloride, 0.1-0.2% of potassium dihydrogen phosphate and 0.2-0.4% of magnesium chloride respectively; meanwhile, soybean oil is added as a defoaming agent, and the using amount of the soybean oil is 0.3 to 0.5 percent; sterilizing, wherein the volume percentage of the seed culture medium to the fermentation culture medium is 10-15%, and the ventilation volume during culture is 300L/min; the stirring speed is 120-150rmp, the culture temperature is 28 +/-2 ℃, the pH value is natural, and the culture period is 24-28 h;
(3) the culture medium of the strain fermentation tank adopts corn flour or corn starch as a carbon source, and the using amount is 2-3%; using cold-pressed soybean cake powder as a nitrogen source, wherein the using amount is 2-3%; 1-2% of quick-acting nitrogen source urea is added; adding inorganic salts of sodium chloride, potassium dihydrogen phosphate and magnesium chloride, wherein the dosage of the sodium chloride is 0.1-0.2%, the dosage of the potassium dihydrogen phosphate is 0.1-0.2%, and the dosage of the magnesium chloride is 0.2-0.4%; meanwhile, soybean oil is added as an antifoaming agent with the dosage of 0.1-0.2 percent for disinfection, the percentage of the volume of the seed culture medium to the volume of the fermentation culture medium is 10-15 percent, the ventilation volume is 300L/min during culture, the stirring speed is 120-150rmp, the culture temperature is 28 +/-2 ℃, the pH is natural, the fermentation period is 72-90h, and the pH is 7.2, and then the tank is placed;
(4) when the fermentation broth is put in a tank, the content of the zhongshengmycin F in the fermentation broth is 3-5g/L, and the flow of producing the zhongshengmycin F mother drug by the fermentation broth is as follows:
firstly, adjusting the pH value of fermentation liquor to 3.5 +/-0.3 by using oxalic acid, and then filtering by adopting ultrafiltration;
secondly, the filtrate passes through resin for ion exchange at the speed of 2BV/h, then is eluted by eluent, and the pH value of the collected eluent is adjusted to 3.5;
thirdly, concentrating the eluent under reduced pressure until the concentration is 40 +/-5 g/L;
fourthly, adding active carbon into the concentrated solution in the previous step, stirring and decoloring for 30min at normal temperature, and filtering;
fifthly, concentrating the filtrate at 60 ℃ under reduced pressure until the concentration is 90 +/-5 g/L;
sixthly, reducing the temperature of the concentrated solution to 9 ℃ at a cooling speed of 10 ℃/min, keeping the temperature at 9 ℃ for 10h, and keeping the stirring speed at 50-100 rpm during the period to separate out a large amount of crystals;
seventhly, filtering and drying the mixture until the water content is less than or equal to 5 percent under the conditions of pressure of 0.1Mpa and temperature of 60 ℃, thus obtaining the zhongshengmycin mother medicine with the content of 60-80 percent through detection.
In a preferred embodiment of the invention, the streptomyces is preferably streptomyces produced by Shaanxi Mecono Biotechnology GmbH (the company strain label ZS-II-D190515) as a production strain, the strain is produced by adopting a submerged liquid fermentation technology, and the streptomyces has the characteristic of high proportion of F component in a product, and is particularly suitable for preparing a zhongshengmycin F component mother medicine.
In a preferred embodiment of the present invention, the sterilization in the fourth step of step (1) is sterilization at 121 ℃ for 30 minutes.
In a preferred embodiment of the invention, the sterilization is performed at 121 ℃ for 30 min.
In the preferred embodiment of the present invention, in step (3), when the dissolved oxygen begins to decrease and becomes stable after the fermentation is carried out for 24 + -5 hours, the culture medium of the fermentation tank with the total amount of 1/4 as the formula is needed to be supplemented so as to rapidly supplement the required nutrients for the propagation of the bacterial cells.
In a preferred embodiment of the present invention, in the step (4), in the second step, the resin is a sodium type resin, preferably a 724 resin; the eluent is hydrochloric acid with the concentration of 1mol/L, and the pH value is adjusted by using sodium hydroxide with the concentration of 2 mol/L.
In a preferred embodiment of the present invention, in step (4), the parameters of the reduced pressure concentration used in the third step are: the pressure is-0.1 Mpa, and the concentration temperature is 50 ℃.
In a preferred embodiment of the present invention, in the step (4), the amount of the activated carbon added in the fourth step is 2.0% by mass of the concentrated solution.
In the preferred embodiment of the present invention, in the step (4), the seventh step is filtering by a punching bag centrifuge and drying by a double cone dryer.
Compared with the prior art, the invention has the beneficial technical effects that:
1. the process can reduce energy consumption and the production cost of the zhongshengmycin raw pesticide;
2. the pigment of the zhongshengmycin is obviously reduced after the activated carbon is added, and the product quality is improved;
3. the purity of the zhongshengmycin F component is improved by utilizing the process.
Drawings
The following is further described with reference to the accompanying drawings.
FIG. 1 shows the morphology of ZS-II-D190515 colonies.
FIG. 2 is a liquid chromatogram of ZS-II-D190515 fermentation product.
FIG. 3 is a liquid chromatogram of ZS-II-D190515 fermentation product.
FIG. 4 is a ZS-II-D190515 identification report.
FIG. 5 is a liquid chromatogram of a Zhongshengmycin F parent drug product.
Detailed Description
The following examples are intended to illustrate the present invention in detail and should not be construed as limiting the scope of the present invention in any way.
Example one
The zhongshengmycin F component mother medicine is prepared according to the following method:
weighing 30g of corn starch, 10g of urea, 2g of sodium chloride and 2g of magnesium chloride; 3g of monopotassium phosphate and 200g of agar. Firstly, measuring 700mL of tap water, heating to 40 ℃, putting the tap water into a 40 ℃ water bath kettle, sequentially adding weighed sodium chloride, magnesium chloride, potassium dihydrogen phosphate and urea, stirring uniformly, wherein the color is slightly milky white, then slowly adding corn flour under stirring, and stirring uniformly. The volume is adjusted to 1000mL by using tap water with the temperature of 40 ℃, then the pH value is measured by using a pH meter to be 6.7, and the pH value is adjusted to be 7.2 by using 2mol/L sodium hydroxide; adding agar powder, and stirring. Subpackaging into common test tubes with diameter of 3cm, each test tube contains 15 ml. The subpackaged test tubes are sterilized for 30min at 121 ℃. Taking out from the sterilizing pot, putting out the inclined plane, cooling, putting into a 37 ℃ incubator, culturing for 72h, and observing the growth of the bacteria without impurities. Inoculating streptomycete on the inclined plane. After 145 hours of cultivation, an off-white lawn developed.
Simultaneously weighing 9Kg of corn starch, 9Kg of cold-pressed soybean meal, 6Kg of glucose, 6g of urea, 300g of sodium chloride, 300g of monopotassium phosphate, 300g of magnesium chloride, 600g of magnesium chloride, 900g of soybean oil and 100Kg of water, sequentially adding the materials into a 500L seed tank, and supplementing water to 230L in volume, 121 ℃ and 30 min. Sterilizing, cooling to 30 deg.C, inoculating streptomyces in an amount of 10-15%. The ventilation rate was adjusted to 600L/min. The stirring line speed was 326 m/min. Culturing for 26h, and observing the hypha to form a net shape under a microscope, so that the hypha grows well.
Weighing 240Kg of corn starch; 240kg of cold-pressed soybean cake powder; 120kg of urea; 8kg of sodium chloride, 8kg of monopotassium phosphate, 16kg of magnesium chloride and 16kg of soybean oil 4000L of water. Sequentially adding the above raw materials into water, adding into 15000L fermentation tank to constant volume of 6500L, sterilizing at 121 deg.C for 30min, adding streptomyces when the temperature is reduced to 30 deg.C, inoculating at an inoculum size of 10-15%, and regulating ventilation rate to 600L/min. The stirring line speed was 343-350 m/min (fluctuation). The culture temperature is 28 +/-2 ℃. The pH is natural. Culturing for 22h, finding that dissolved oxygen slowly rises, and 24h rises slowly and becomes stable, supplementing feed liquid with good sterilization effect, 2000L of feed liquid with the same formula as a fermentation tank, about 10000L in volume, and when the fermentation period is 84h, raising the pH to 7.2 and putting the fermentation tank.
The volume of the fermentation liquid is 9100L when the fermentation liquid is placed in a tank, and the content of the zhongshengmycin F in the fermentation liquid is 4.5g/L through liquid chromatography detection. Adding 92Kg of oxalic acid, stirring fully to dissolve, then, putting into an ultrafiltration unit for filtration, adding the water for continuous filtration when the filtrate 3700L is discharged, and diluting for three times in sequence to obtain 17000L of filtrate with the content of the biotins F of 2.09 g/L. Filtrate in 6m3The speed/h was passed through 2 ion exchange columns 734 connected in series, after which all the filtrate was squeezed out with tap water. Then using the previously prepared 6m3Eluting with 1mol/L hydrochloric acid, monitoring concentration by liquid chromatography, and collecting eluate of 6.2m3The content of Zhongshengmycin F is 5.16 g/L. Concentrating under-0.1 Mpa at 60 deg.C to obtain 800L, 39.7 g/L. Then adding 24Kg of activated carbon into the concentrated solution, stirring for 60min, and filtering to obtain 750L of filtrate, wherein the active ingredients adsorbed on the activated carbon and the filtrate are washed with water for the next use. Under the conditions of pressure of-0.1 Mpa and concentration temperature of 60 ℃, the concentration is carried out until the volume is 325L, and the content of the protopectin F in the concentrated solution is 92.3 g/L. And then, the temperature is controlled to be reduced by 10 ℃/min to 9 ℃, a large amount of white precipitate is separated out under the slight stirring of the stirring speed of 60m/min, after the centrifugal filtration by a hanging bag, the mixture is transferred into a double-cone mixer to be dried for 6 hours under the conditions of the pressure of-0.1 Mpa and the concentration temperature of 60 ℃ to obtain 23.4Kg of the zhongshengmycin mother medicine, the content of the zhongshengmycin F is detected to be 60.8 percent, the weight is reduced by 3.7 percent after the drying, the content of the zhongshengmycin F in the mother liquor is 43.8g/L, and the zhongshengmycin F mother medicine is applied to the next crystallization or used for processing a zhongshengmycin F water aqua.
Example two
The zhongshengmycin F component mother medicine is prepared according to the following method:
weighing 70g of corn starch, 30g of urea, 2g of sodium chloride and 4g of magnesium chloride; 3g of monopotassium phosphate and 200g of agar. Firstly, measuring 700mL of tap water, heating to 40 ℃, putting the tap water into a 40 ℃ water bath kettle, sequentially adding weighed sodium chloride, magnesium chloride, potassium dihydrogen phosphate and urea, stirring uniformly, wherein the color is slightly milky white, then slowly adding corn flour under stirring, and stirring uniformly. The volume is adjusted to 1000mL by using tap water with the temperature of 40 ℃, then the pH value is measured by using a pH meter to be 6.5, and the pH value is measured by using 2mol/L to be 7.2; adding agar powder, and stirring. Subpackaging into common test tubes with diameter of 3cm, each test tube contains 15 ml. The subpackaged test tubes are sterilized for 30min at 121 ℃. Taking out from the sterilizing pot, putting out the inclined plane, cooling, putting into a 37 ℃ incubator, culturing for 72h, and observing the growth of the bacteria without impurities. Streptomyces was inoculated on the slant. After 140 hours of culture, gray-white lawn grows and is full.
Simultaneously weighing 9Kg of corn starch, 9Kg of cold-pressed soybean meal, 1.5Kg of glucose, 6g of urea, 300g of sodium chloride, 300g of monopotassium phosphate, 300g of magnesium chloride, 600g of magnesium chloride, 900g of soybean oil and 100Kg of water, sequentially adding the materials into a 500L seeding tank, supplementing water to 232L volume, 121 ℃ and 30 min. Sterilizing, cooling to 30 deg.C, inoculating streptomyces at an inoculum size of 10-15%, and regulating ventilation rate to 600L/min. . The stirring line speed was 317 m/min. Culturing for 25h, and observing the hypha to form a net shape under a microscope, wherein the hypha grows well.
Weighing 240Kg of corn starch; 160kg of cold-pressed soybean cake powder; 160kg of urea; 8kg of sodium chloride, 8kg of monopotassium phosphate, 16kg of magnesium chloride and 16kg of soybean oil 4000L of water. Sequentially adding the above raw materials into water, adding into 15000L fermentation tank to constant volume of 6500L, sterilizing at 121 deg.C for 30min, sterilizing to volume of 7760L, inoculating streptomyces at an inoculation amount of 10-15%, and regulating ventilation rate to 600L/min. The stirring line speed was 345-350 m/min (fluctuation). The culture temperature is 28 +/-2 ℃. The pH is natural. Culturing for 22h, finding that dissolved oxygen slowly rises, and 24h rises slowly and becomes stable, supplementing feed liquid with good sterilization effect, 2000L of feed liquid with the same formula as a fermentation tank, about 10000L in volume, and when the fermentation period is 84h, raising the pH to 7.2 and putting the fermentation tank.
The volume of the fermentation liquid is 9090L when the fermentation liquid is placed in a tank, and the content of the zhongshengmycin F in the fermentation liquid is 4.8g/L through liquid chromatography detection. Adding 91.6Kg oxalic acid, stirring thoroughly to dissolve, then pH3.3, then filtering in ultrafiltration unit, adding water when the liquid is 3750L, filtering, diluting for three times to obtain 18200L filtrate, the content of biotins F in the filtrate is 2.19 g/L. Filtrate in 6m3The speed/h was passed through 2 ion exchange columns 734 connected in series, after which all the filtrate was squeezed out with tap water. Then using the previously prepared 6m3Eluting with 1mol/L hydrochloric acid, monitoring concentration by liquid chromatography, and collecting eluate of 6.2m3The content of Zhongshengmycin F is 5.24 g/L. Concentrating under-0.1 Mpa at 60 deg.C to 820L, the content is 41.6g/L, adding 24.6Kg activated carbon into the concentrated solution, stirring for 60min, and filtering to obtain 770L filtrate, which is washed with water to obtain effective components adsorbed on the activated carbon for reuse. Under the conditions of pressure of-0.1 Mpa and concentration temperature of 60 ℃, the concentration is carried out until the volume is 325L, and the content of the protopectin F in the concentrated solution is 94.3 g/L. At this time, the temperature is controlled to be reduced by 10 ℃/min to 9 ℃, a large amount of white precipitate is separated out under the slight stirring of the stirring speed of 60m/min, after the centrifugal filtration by a hanging bag, the mixture is transferred into a double-cone mixer to be dried for 6 hours under the conditions of the pressure of-0.1 Mpa and the concentration temperature of 60 ℃ to obtain 23.4Kg of the zhongshengmycin mother drug, the content of zhongshengmycin F is detected to be 61.9 percent, and the drying weight is reduced by 4.8. The content of the zhongshengmycin F in the mother liquor is 44.3g/L, and the zhongshengmycin F is applied to the next crystallization or is used for processing a zhongshengmycin F water aqua.
EXAMPLE III
The zhongshengmycin F component mother medicine is prepared according to the following method:
weighing 70g of corn starch, 30g of urea, 2g of sodium chloride and 3g of magnesium chloride; 4g of monopotassium phosphate and 200g of agar. Firstly, measuring 700mL of tap water, heating to 40 ℃, putting the tap water into a 40 ℃ water bath kettle, sequentially adding weighed sodium chloride, magnesium chloride, potassium dihydrogen phosphate and urea, stirring uniformly, wherein the color is slightly milky white, then slowly adding corn flour under stirring, and stirring uniformly. The volume is adjusted to 1000mL by using tap water with the temperature of 40 ℃, then the pH value is measured by using a pH meter to be 6.6, and the pH value is measured by using 2mol/L to be 7.2; adding agar powder, and stirring. Subpackaging into common test tubes with diameter of 3cm, each test tube contains 15 ml. The subpackaged test tubes are sterilized for 30min at 121 ℃. Taking out from the sterilizing pot, putting out the inclined plane, cooling, putting into a 37 ℃ incubator, culturing for 72h, and observing the growth of the bacteria without impurities. Inoculating streptomycete on the inclined plane. After 141 hours of cultivation, an off-white lawn appeared.
Simultaneously weighing 9Kg of corn starch, 9Kg of cold-pressed soybean meal, 6Kg of glucose, 6g of urea, 300g of sodium chloride, 300g of monopotassium phosphate, 450g of magnesium chloride, 600g of magnesium chloride, 900g of soybean oil and 100Kg of water, sequentially adding the materials into a 500L seed tank, and supplementing water to 244L volume, 121 ℃ and 30 min. Sterilizing, cooling to 30 deg.C, inoculating streptomyces at an inoculum size of 10-15%, and regulating ventilation rate to 600L/min. The stirring line speed was 313 m/min. Culturing for 24.4h, and observing hypha to form net under microscope to grow well.
Weighing 240Kg of corn starch; 240kg of cold-pressed soybean cake powder; 160kg of urea; 8kg of sodium chloride, 8kg of monopotassium phosphate, 16kg of magnesium chloride and 16kg of soybean oil 4000L of water. Sequentially adding the above raw materials into water, adding into 15000L fermentation tank to constant volume of 6500L, sterilizing at 121 deg.C for 30min, inoculating streptomyces, and regulating ventilation rate to 600L/min. The stirring line speed was 348 ℃ and 350 m/min (fluctuation). The culture temperature is 28 +/-2 ℃. The pH is natural. Culturing for 25h, finding that the dissolved oxygen slowly rises, and the rising for 26h becomes slow and stable, supplementing the feed liquid which is well sterilized and has the same formula with the fermentation tank with 2000L, the volume is about 10000L, and when the fermentation period is 81h, the pH value rises to 7.2 and the fermentation tank is placed.
The volume of the fermentation tank is 9250L when the fermentation tank is placed, and the content of the zhongshengmycin F in the fermentation liquor is 5.0g/L through liquid chromatography detection. Adding 95Kg oxalic acid, stirring thoroughly to dissolve, then pH3.4, then filtering in ultrafiltration unit, adding water when the liquid goes out of 4050L, filtering continuously, diluting three times in sequence to obtain 18700L filtrate with 2.24g/L of biotins F content in the filtrate. Filtrate in 6m3The speed/h was passed through 2 ion exchange columns 734 connected in series, after which all the filtrate was squeezed out with tap water. Then using the previously prepared 6m3 Eluting with 1mol/L hydrochloric acid, monitoring concentration by liquid chromatography, and collecting eluate of 6.8m3The content of Zhongshengmycin F is 5.04 g/L. Concentrating under-0.1 Mpa at 60 deg.C to obtain 834L concentrate with a content of 41.6g/L, and adding 25.6KStirring activated carbon for 60min, and filtering to obtain 779L filtrate, which is used for the next time after the active ingredients adsorbed on the activated carbon and water are washed. Concentrating under-0.1 Mpa at 60 deg.C to 329L volume, and the concentration liquid contains 94.3g/L of protopectin F. At this time, the temperature is controlled to be reduced by 10 ℃/min to 9 ℃, a large amount of white precipitate is separated out under the slight stirring of the stirring speed of 60m/min, after the centrifugal filtration by a hanging bag, the mixture is transferred into a double-cone mixer to be dried for 6 hours under the conditions of the pressure of-0.1 Mpa and the concentration temperature of 60 ℃, 18.4Kg of the zhongshengmycin mother medicine is obtained, the content of zhongshengmycin F is detected to be 78.7 percent, and the drying weight is reduced by 2.9. The content of the zhongshengmycin F in the mother liquor is 41.7g/L, and the zhongshengmycin F is applied to the next crystallization or is used for processing a zhongshengmycin F water aqua.
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that are not thought of through the inventive work should be included in the scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.
Claims (10)
1. The Zhongshengmycin F component mother medicine is characterized in that the content of the Zhongshengmycin F component is 60.8-78.7 wt%.
2. The process for preparing a zhongshengmycin F component parent drug according to claim 1, which comprises the steps of:
(1) preparing a production test tube slant culture medium, and inoculating streptomycete on a slant for culture; when a single colony grows in the slant culture medium, picking the single colony by using an inoculating needle and carrying out the next culture;
(2) preparing a seed culture medium for strain fermentation, and inoculating the single colony obtained in the step (1) for culture to obtain a seed culture solution;
(3) preparing a fermentation tank culture medium, and inoculating the seed culture solution obtained in the step (2), wherein the volume ratio of the seed culture solution to the fermentation culture solution is 10-15%;
(4) filtering the fermentation liquor, performing ion exchange, eluting, concentrating, decoloring, cooling and drying to obtain the zhongshengmycin mother drug.
3. The production method according to claim 2, characterized by comprising the steps of:
(1) the slant culture medium for producing the test tube adopts corn flour or corn starch as a carbon source, and the using amount is 3-7%; urea is used as a nitrogen source, and the using amount is 1-3%; trace element sodium chloride, the consumption is 0.1-0.2%; the dosage of the trace element magnesium chloride is 0.1 to 0.2 percent; the phosphorus source adopts monopotassium phosphate, and the using amount is 0.2-0.4%; the dosage of the agar is 20 g/L; adjusting pH value to 7.0-7.4 with 2mol/L hydrochloric acid or 2mol/L sodium hydroxide; supplementing the raw materials with tap water, and preparing the raw materials by the following steps:
firstly, sequentially adding sodium chloride, magnesium chloride, potassium dihydrogen phosphate and urea into tap water at the temperature of 40 +/-2 ℃, stirring to fully dissolve, then adding corn starch, and stirring until the mixture is uniform and free of lumps; the tap water is used as 70 percent of the total preparation water consumption, and the preparation process is finished in a water bath;
secondly, using tap water with the same temperature in the first step to fix the volume;
thirdly, placing the solution with the constant volume obtained in the second step at room temperature, and adjusting the solution with 2mol/L hydrochloric acid when the pH value is higher than 7.4 after the temperature is reduced to 20 ℃; if the pH value is lower than 7.0, adjusting the pH value by using 2mol/L sodium hydroxide; then adding agar and stirring uniformly;
fourthly, preparing an inclined plane after the adjusted culture medium is sterilized, and culturing for 72 hours at 37 ℃ until no mixed bacteria fall;
fifthly, inoculating streptomyces on the inclined plane, and culturing for 140-150 hours to grow offwhite lawn;
(2) corn flour or corn starch is adopted as a carbon source in a seed culture medium for strain fermentation, and the using amount is 2-3%; cold-pressed soybean cake powder is used as a nitrogen source, and the using amount is 2-3 percent; adding 0.5-2% of glucose as a quick-acting carbon source; 1-2% of quick-acting nitrogen source urea is added; adding inorganic salts of sodium chloride, potassium dihydrogen phosphate and magnesium chloride, wherein the dosage is 0.1-0.2% of sodium chloride, 0.1-0.2% of potassium dihydrogen phosphate and 0.2-0.4% of magnesium chloride respectively; meanwhile, soybean oil is added as a defoaming agent, and the using amount of the soybean oil is 0.3 to 0.5 percent; sterilizing, wherein the volume percentage of the seed culture medium to the fermentation culture medium is 10-15%, and the ventilation volume during culture is 300L/min; the stirring speed is 120-150rmp, the culture temperature is 28 +/-2 ℃, the pH value is natural, and the culture period is 24-28 h;
(3) the culture medium of the strain fermentation tank adopts corn flour or corn starch as a carbon source, and the using amount is 2-3%; using cold-pressed soybean cake powder as a nitrogen source, wherein the using amount is 2-3%; 1-2% of quick-acting nitrogen source urea is added; adding inorganic salts of sodium chloride, potassium dihydrogen phosphate and magnesium chloride, wherein the dosage of the sodium chloride is 0.1-0.2%, the dosage of the potassium dihydrogen phosphate is 0.1-0.2%, and the dosage of the magnesium chloride is 0.2-0.4%; meanwhile, soybean oil is added as an antifoaming agent with the dosage of 0.1-0.2 percent for disinfection, the percentage of the volume of the seed culture medium to the volume of the fermentation culture medium is 10-15 percent, the ventilation volume is 300L/min during culture, the stirring speed is 120-150rmp, the culture temperature is 28 +/-2 ℃, the pH is natural, the fermentation period is 72-90h, and the pH is 7.2, and then the tank is placed;
(4) when the fermentation broth is put in a tank, the content of the zhongshengmycin F in the fermentation broth is 3-5g/L, and the flow of producing the zhongshengmycin F mother drug by the fermentation broth is as follows:
firstly, adjusting the pH value of fermentation liquor to 3.5 +/-0.3 by using oxalic acid, and then filtering by adopting ultrafiltration;
secondly, the filtrate passes through resin for ion exchange at the speed of 2BV/h, then is eluted by eluent, and the pH value of the collected eluent is adjusted to 3.5;
thirdly, concentrating the eluent under reduced pressure until the concentration is 40 +/-5 g/L;
fourthly, adding active carbon into the concentrated solution in the previous step, stirring and decoloring for 30min at normal temperature, and filtering;
fifthly, concentrating the filtrate at 60 ℃ under reduced pressure until the concentration is 90 +/-5 g/L;
sixthly, reducing the temperature of the concentrated solution to 9 ℃ at a cooling speed of 10 ℃/min, keeping the temperature at 9 ℃ for 10h, and keeping the stirring speed at 50-100 rpm during the period to separate out a large amount of crystals;
seventhly, filtering and drying the mixture until the water content is less than or equal to 5 percent under the conditions of pressure of 0.1Mpa and temperature of 60 ℃, thus obtaining the zhongshengmycin mother medicine with the content of 60-80 percent through detection.
4. The production method according to claim 3, wherein the streptomyces is preferably streptomyces produced by Shaanxi Mecono Biotech limited (strain number ZS-II-D190515) as a production strain.
5. The production method according to claim 3, wherein the sterilization in the fourth step of step (1) is sterilization at 121 ℃ for 30 minutes.
6. The method of claim 3, wherein the sterilization is performed at 121 ℃ for 30 min.
7. The process according to claim 3, wherein in the step (3), when the dissolved oxygen begins to decrease and becomes stable after the fermentation is carried out for 24. + -.5 h, the culture medium of the fermenter containing 1/4 is supplemented to supplement the required nutrients for the growth of the fungus body.
8. The production method according to claim 3, wherein in the step (4), in the second step, the resin is a sodium type resin, preferably a 724 resin; the eluent is hydrochloric acid with the concentration of 1mol/L, and the pH value is adjusted by using sodium hydroxide with the concentration of 2 mol/L.
9. The production method according to claim 3, wherein in the step (4), the parameters of the reduced pressure concentration adopted in the third step are as follows: the pressure is-0.1 Mpa, and the concentration temperature is 50 ℃.
10. The production method according to claim 3, wherein in the step (4), the adding amount of the activated carbon in the fourth step is 2.0 percent of the mass of the concentrated solution; and (4) in the seventh step, filtering by using a punching bag centrifuge, and drying by using a double-cone dryer.
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| CN116649356A (en) * | 2023-07-31 | 2023-08-29 | 中国远大集团有限责任公司 | Pesticide raw material or parent medicine and preparation method and application thereof |
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