CN114107103A - Method for preparing maduramicin ammonium production strain - Google Patents
Method for preparing maduramicin ammonium production strain Download PDFInfo
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- CN114107103A CN114107103A CN202111389154.0A CN202111389154A CN114107103A CN 114107103 A CN114107103 A CN 114107103A CN 202111389154 A CN202111389154 A CN 202111389154A CN 114107103 A CN114107103 A CN 114107103A
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- 238000004519 manufacturing process Methods 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 39
- BXHCJLRTXPHUGH-UHFFFAOYSA-N 2-oxo-1h-pyridine-4-carboxylic acid Chemical compound OC(=O)C=1C=CNC(=O)C=1 BXHCJLRTXPHUGH-UHFFFAOYSA-N 0.000 title claims abstract description 18
- 235000002597 Solanum melongena Nutrition 0.000 claims abstract description 51
- 244000061458 Solanum melongena Species 0.000 claims abstract description 49
- 239000001963 growth medium Substances 0.000 claims abstract description 23
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 14
- 238000000855 fermentation Methods 0.000 claims abstract description 13
- 230000004151 fermentation Effects 0.000 claims abstract description 13
- 239000000843 powder Substances 0.000 claims abstract description 13
- 238000005303 weighing Methods 0.000 claims abstract description 11
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims abstract description 8
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229920001817 Agar Polymers 0.000 claims abstract description 7
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- 235000003891 ferrous sulphate Nutrition 0.000 claims abstract description 7
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims abstract description 7
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims abstract description 7
- 239000011780 sodium chloride Substances 0.000 claims abstract description 7
- 239000008107 starch Substances 0.000 claims abstract description 7
- 235000019698 starch Nutrition 0.000 claims abstract description 7
- 229940041514 candida albicans extract Drugs 0.000 claims abstract description 4
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims abstract description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims abstract description 4
- 235000019341 magnesium sulphate Nutrition 0.000 claims abstract description 4
- 235000010333 potassium nitrate Nutrition 0.000 claims abstract description 4
- 239000004323 potassium nitrate Substances 0.000 claims abstract description 4
- 239000012138 yeast extract Substances 0.000 claims abstract description 4
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 36
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 27
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 18
- 239000000203 mixture Substances 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 14
- 239000008399 tap water Substances 0.000 claims description 12
- 235000020679 tap water Nutrition 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000002655 kraft paper Substances 0.000 claims description 9
- RWVUEZAROXKXRT-VQLSFVLHSA-N maduramicin Chemical compound O1[C@@H](C)[C@H](OC)[C@@H](OC)C[C@H]1O[C@@H]1[C@H]([C@@]2(C)O[C@H](CC2)[C@@]2(C)O[C@]3(O[C@@H]([C@H](C)[C@@H](O)C3)[C@@H](C)[C@H]3[C@@H]([C@@H](OC)[C@H](C)[C@@](O)(CC(O)=O)O3)OC)CC2)O[C@@H]([C@@H]2[C@H](C[C@@H](C)[C@@](C)(O)O2)C)C1 RWVUEZAROXKXRT-VQLSFVLHSA-N 0.000 claims description 9
- 229950006915 maduramicin Drugs 0.000 claims description 9
- 239000002994 raw material Substances 0.000 claims description 9
- 238000005070 sampling Methods 0.000 claims description 9
- 230000036512 infertility Effects 0.000 claims description 7
- 239000000463 material Substances 0.000 claims description 7
- 230000008569 process Effects 0.000 claims description 7
- 229920000742 Cotton Polymers 0.000 claims description 6
- 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 6
- 244000068988 Glycine max Species 0.000 claims description 6
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- 240000008042 Zea mays Species 0.000 claims description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
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- 239000012153 distilled water Substances 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 238000011218 seed culture Methods 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 5
- 239000012297 crystallization seed Substances 0.000 claims description 4
- 238000011068 loading method Methods 0.000 claims description 4
- KKUKTXOBAWVSHC-UHFFFAOYSA-N Dimethylphosphate Chemical compound COP(O)(=O)OC KKUKTXOBAWVSHC-UHFFFAOYSA-N 0.000 claims description 3
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
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- 238000012258 culturing Methods 0.000 claims description 2
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- 238000004659 sterilization and disinfection Methods 0.000 claims description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims 6
- 238000002360 preparation method Methods 0.000 abstract description 9
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12N3/00—Spore forming or isolating processes
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
- C12P17/162—Heterorings having oxygen atoms as the only ring heteroatoms, e.g. Lasalocid
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Abstract
The invention relates to the technical field of microbial fermentation working strain preparation, and discloses a preparation method of a maduramicin ammonium production strain. 2.0% of bran is added into the changed formula, and the rest is unchanged: preparing 1L of slant culture medium: soluble starch: 20g, yeast extract: 2g, magnesium sulfate: 0.5g, sodium chloride: 0.5g, dipotassium hydrogen phosphate: 0.5g, potassium nitrate: 1.0g, ferrous sulfate: 0.01g, weighing agar powder: 20g, separately weighing bran: 1g per eggplant bottle. According to the manufacturing method of the maduramicin ammonium production strain, a large number of spores are produced by changing the inclined plane formula, so that the production level of the original method is improved by 8.98%, the risk of strain contamination is reduced by the strain through the manufacturing method of the maduramicin ammonium production strain, the survival rate of the strain is improved, the cultivation efficiency is improved, and the stability of the strain is improved by reducing the transfer passage.
Description
Technical Field
The invention relates to the technical field of microbial fermentation working strain preparation, in particular to a preparation method of a maduramicin ammonium production strain.
Background
After the production strains or the screened excellent strains are subjected to passage or preservation, the morphological characteristics and the physiological characteristics of some groups gradually decline. In industrial production, strains are usually purified and screened after several passages and storage for a period of time, and excellent strains with high production level are bred as first-generation strains. The stability of the production performance of the strains in industrial production is a very important factor influencing the high and low yield. In production, the first generation of strain is generally subcultured to verify the stability, and the third generation of strain with high stability is used as the production strain. Because actinomadura has limited vitality, it is easy to change or even die during the process of passage and preservation, often causes the degeneration of industrial production strains, and may cause the loss of excellent strains. Therefore, a correct and efficient production method is very important for the preparation of a strain.
The existing preparation method is used for preserving seed liquid at present, because mycelium preservation time is short, purification is required to be carried out every year, a large amount of manpower and material resources are consumed, and actinomycetes of the maduracilla cannot produce abundant spores on the existing solid culture medium and cannot preserve the spores.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a method for preparing maduramicin ammonium production strains, which solves the problems in the background art.
(II) technical scheme
In order to achieve the above purpose, the invention provides the following technical scheme: a method for preparing a strain for producing maduramicin ammonium comprises a spore eggplant bottle inclined plane, wherein the original formula uses a Gao's No. one culture medium which produces a small amount of spores and cannot be used for producing the eggplant bottle inclined plane. 2.0% of bran is added into the changed formula, and the rest is unchanged: preparing 1L of slant culture medium: soluble starch: 20g, yeast extract: 2g, magnesium sulfate: 0.5g, sodium chloride: 0.5g, dipotassium hydrogen phosphate: 0.5g, potassium nitrate: 1.0g, ferrous sulfate: 0.01g, weighing agar powder: 20g, separately weighing bran: weighing each reagent in a container according to 1g per eggplant bottle, distilling a small amount of cold distilled gelatinized starch, then using boiled distilled water to slide off all the reagents, using distilled water to fix the volume to 1L, using 20% sodium hydroxide solution to adjust the pH value to 7.0-7.2, adding 20g of agar powder into the prepared solution, gelatinizing on an electric furnace, pouring 50ml of gelatinized culture medium into the eggplant bottle added with 1g of bran, subpackaging 20 eggplant bottle inclined planes, wrapping by using a cotton plug, gauze and kraft paper, sterilizing at 121 +/-1 ℃ for 30min, manufacturing the eggplant bottle into an inclined plane with an angle of 15 degrees when the inclined plane of the killed eggplant bottle is not solidified, and carrying out blank culture at 37 ℃ for 24 h. The slope of the eggplant bottle is sterilized for standby inoculation.
Preferably, 2 quick-freezing tubes taken out of the refrigerator are connected with a mother bottle according to the original method, the other seed liquid is added on the inclined plane of an empty eggplant bottle, 0.2ml of seed liquid is added on each inclined plane, the mixture is uniformly coated, the mixture is placed in a constant-temperature incubator at the temperature of 32 +/-0.5 ℃ for culturing for 7-8 days, a large amount of off-white spores are generated on the inclined plane of the eggplant bottle at the moment, the cultured inclined plane strain is a cultured inclined plane strain, the cultured inclined plane of the eggplant bottle is wrapped by sterile tinfoil paper, the eggplant bottle is refrigerated for standby at the temperature of 4 ℃, and the refrigerating time of the inclined plane refrigerator is not more than 40 days.
Preferably, the cultured slant strains are inoculated with 0.5cm 2/bottle slant spores to shake flask seeds, the shake flask seeds are inoculated with sterile nutrient broth, meanwhile, the fermentation flask is transferred for culture for 5 days, the strains are detected to be sterile, the titer is more than 8000u/ml, and the batch of slant can be used for seed supply.
Preferably, the production of the seed culture medium comprises preparing 1L of seed culture medium: glucose: 20g, soybean cake powder: 20g, corn steep liquor: 10g, light calcium carbonate: 5g of light calcium carbonate is weighed singly, other raw materials and auxiliary materials are weighed together, a small amount of cold tap water is used for pasting, and then boiled hot water is used for sliding. Then, the volume is determined to be 1L by cold tap water, the pH value is adjusted to be 7.2-7.4 by 20 percent sodium hydroxide solution, finally, light calcium carbonate is added, the mixture is stirred evenly and subpackaged, the seed loading of each bottle is 100ml/750ml triangular bottle, a cotton plug, 2 layers of gauze and kraft paper are used for wrapping, the temperature is 121 +/-1 ℃, the sterilization is carried out for 30min, and the mixture is cooled to the room temperature for standby.
Preferably, the preparation of the fermentation medium comprises the following steps of preparing 1L of fermentation medium: glucose: 40g, soybean cake powder 40g, dimethyl hydrogen phosphate: 0.2g, ferrous sulfate: 0.05g, light calcium carbonate: 5g, corn steep liquor: 5g, sodium chloride: 4g of light calcium carbonate is weighed singly, other raw materials and auxiliary materials are weighed together, a small amount of cold tap water is used for pasting, and then boiled hot water is used for sliding. Then, the volume is determined to be 1L by cold tap water, the pH value is adjusted to be 7.2-7.4 by 20 percent sodium hydroxide solution, finally, light calcium carbonate is added, the mixture is stirred evenly and subpackaged, the seed loading of each bottle is 60ml/500ml triangular bottle, eight layers of gauze and kraft paper are used for packing, the mixture is sterilized at the temperature of 121 +/-1 ℃ for 30min, and the mixture is cooled to the room temperature for standby.
Preferably, the bottle inoculation is that prepared eggplant bottle slope seeds are taken from a refrigerator and are inoculated into a seed bottle filled with a sterile culture medium, the eggplant bottle is cultured for 48 hours, the bottle is closed and the tank is put into the refrigerator, when the eggplant bottle is closed into a large triangular flask of the tank, a small amount of seed liquid is reserved in each bottle of seeds and is put into a sampling bottle, the seed liquid in the sampling bottle is inoculated into 3 bottles for fermentation and tracking, the sterility and the titer of the eggplant bottle are detected after 5 days of culture, meanwhile, the seed liquid in the sampling bottle is inoculated with broth to detect the sterility of the seeds, the aseptic bacteria are detected by picture microscopy, the pH is 7.0 to 8.0, the bacterial concentration is more than 10 percent, and the eggplant bottle can be used for tank putting. The maduramicin produced by the method has 9 batches, the production level is improved by 18 percent, and the average yield is not changed by 75 percent. The content of maduramicin ammonium raw material medicine is more than 91%. All the products are qualified.
Preferably, the new process produces 10 batches of pots, requiring 1 batch of eggplant flask slants and 10 batches of shake flask seeds to be formulated.
(III) advantageous effects
Compared with the prior art, the invention provides a method for preparing maduramicin ammonium production strains, which has the following beneficial effects:
according to the manufacturing method of the maduramicin ammonium production strain, a large number of spores are produced by changing the inclined plane formula, so that the production level of the original method is improved by 8.98%, the risk of strain contamination is reduced by the strain through the manufacturing method of the maduramicin ammonium production strain, the survival rate of the strain is improved, the cultivation efficiency is improved, and the stability of the strain is improved by reducing the transfer passage.
Drawings
FIG. 1 is a comparison graph of the manufacturing method of maduramicin ammonium production strain provided by the invention and the experiment of the existing manufacturing method;
FIG. 2 is a comparison chart of the process of the preparation method of maduramicin ammonium production strain provided by the invention and the existing preparation method.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1-2, the present invention provides a technical solution: the culture medium comprises a spore eggplant bottle inclined plane, the original formula uses a Gao's No. one culture medium, a small amount of spores are generated, and the spore eggplant bottle inclined plane cannot be manufactured for production and use. 2.0% of bran is added into the changed formula, and the rest is unchanged: preparing 1L of slant culture medium: soluble starch: 20g, yeast extract: 2g, magnesium sulfate: 0.5g, sodium chloride: 0.5g, dipotassium hydrogen phosphate: 0.5g, potassium nitrate: 1.0g, ferrous sulfate: 0.01g, weighing agar powder: 20g, separately weighing bran: weighing each reagent in a container according to 1g per eggplant bottle, distilling a small amount of cold distilled gelatinized starch, then using boiled distilled water to slide off all the reagents, using distilled water to fix the volume to 1L, using 20% sodium hydroxide solution to adjust the pH value to 7.0-7.2, adding 20g of agar powder into the prepared solution, gelatinizing on an electric furnace, pouring 50ml of gelatinized culture medium into the eggplant bottle added with 1g of bran, subpackaging 20 eggplant bottle inclined planes, wrapping by using a cotton plug, gauze and kraft paper, sterilizing at 121 +/-1 ℃ for 30min, manufacturing the eggplant bottle into an inclined plane with an angle of 15 degrees when the inclined plane of the killed eggplant bottle is not solidified, and carrying out blank culture at 37 ℃ for 24 h. Aseptic stand-by inoculation is carried out on the slope of eggplant bottle, 2 quick-freezing tubes taken out from a refrigerator are inoculated into a mother bottle according to the original method, the other seed solution is added on the slope of an empty white eggplant bottle, 0.2ml of seed solution is added on each slope, the mixture is uniformly coated and cultured in a constant-temperature incubator at the temperature of 32 +/-0.5 ℃ for 7-8 days, at the moment, a large amount of off-white spores are generated on the slope of the eggplant bottle, the slope is folded, namely the cultured slope strain is wrapped by sterile tinfoil paper, the slope of the cultured eggplant bottle is refrigerated for standby use at 4 ℃ in a refrigerator, the refrigerating time of the slope refrigerator is not more than 40 days, the cultured slope strain is inoculated with 0.5cm2 per bottle of slope spores to shake flask seeds, the shake flask seeds are inoculated with sterile nutrient broth, meanwhile, the culture is transferred to a fermentation bottle for 5 days, the bacteria is detected to be sterile, the titer is more than 8000u/ml, the batch of inclined planes can be used for supplying seeds, the production of a seed culture medium is carried out, and 1L of the seed culture medium is prepared: glucose: 20g, soybean cake powder: 20g, corn steep liquor: 10g, light calcium carbonate: 5g of light calcium carbonate is weighed singly, other raw materials and auxiliary materials are weighed together, a small amount of cold tap water is used for pasting, and then boiled hot water is used for sliding. Then, metering the volume to 1L by using cold tap water, adjusting the pH value to 7.2-7.4 by using 20% sodium hydroxide solution, finally adding light calcium carbonate, uniformly stirring and subpackaging, wrapping each bottle of seeds in a triangular bottle with the volume of 100ml/750ml by using a cotton plug, 2 layers of gauze and kraft paper, sterilizing at the temperature of 121 +/-1 ℃ for 30min, cooling to room temperature for later use, preparing a fermentation medium with the volume of 1L: glucose: 40g, soybean cake powder 40g, dimethyl hydrogen phosphate: 0.2g, ferrous sulfate: 0.05g, light calcium carbonate: 5g, corn steep liquor: 5g, sodium chloride: 4g of light calcium carbonate is weighed singly, other raw materials and auxiliary materials are weighed together, a small amount of cold tap water is used for pasting, and then boiled hot water is used for sliding. Then, the volume is determined to be 1L by cold tap water, the pH value is adjusted to be 7.2-7.4 by 20 percent sodium hydroxide solution, finally, light calcium carbonate is added, the mixture is stirred and split charged, the seed content of each bottle is 60ml/500ml triangular flask, eight layers of gauze and kraft paper are used for wrapping, the mixture is sterilized at the temperature of 121 +/-1 ℃ for 30min, the mixture is cooled to the room temperature for standby, the seeds are inoculated in the seed bottles, one prepared eggplant bottle inclined plane seed is taken from a refrigerator and is inoculated in the seed bottle filled with the sterile culture medium, the culture is carried out for 48h, the bottles are closed and the bottles are put into a tank, when the seeds are closed into a large triangular flask of the tank, a small amount of seed liquid is remained in each bottle of seeds and is put into a sampling bottle, the seed liquid in the sampling bottle is inoculated into 3 bottles for fermentation and tracking, the sterility and the titer are detected after 5 days of culture, meanwhile, the seed liquid of a sampling bottle is inoculated with broth to detect the sterility degree of the seeds, the picture is microscopically free of foreign bacteria, the pH is 7.0-8.0, the bacteria concentration is more than 10%, and the method can be used for feeding into a tank. The maduramicin produced by the method has 9 batches, the production level is improved by 18 percent, and the average yield is not changed by 75 percent. The content of maduramicin ammonium raw material medicine is more than 91%. All are qualified, 10 batches of jars are produced by the new method, 1 batch of eggplant bottle inclined planes and 10 batches of shake flask seeds are required to be prepared.
In conclusion, the manufacturing method of the maduramicin ammonium production strain improves the production level of the original method by 8.98% by changing the formula of the inclined plane, reduces the risk of strain contamination, improves the survival rate of the strain, improves the cultivation efficiency, and improves the stability of the strain by reducing the transfer passage.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A method for preparing maduramicin ammonium production strains comprises a spore eggplant bottle inclined plane, and is characterized in that: the original formula uses a Gao's No. one culture medium, produces a small amount of spores, and cannot be used for producing the inclined plane of an eggplant bottle. 2.0% of bran is added into the changed formula, and the rest is unchanged: preparing 1L of slant culture medium: soluble starch: 20g, yeast extract: 2g, magnesium sulfate: 0.5g, sodium chloride: 0.5g, dipotassium hydrogen phosphate: 0.5g, potassium nitrate: 1.0g, ferrous sulfate: 0.01g, weighing agar powder: 20g, separately weighing bran: weighing each reagent in a container according to 1g per eggplant bottle, distilling a small amount of cold distilled gelatinized starch, then using boiled distilled water to slide off all the reagents, using distilled water to fix the volume to 1L, using 20% sodium hydroxide solution to adjust the pH value to 7.0-7.2, adding 20g of agar powder into the prepared solution, gelatinizing on an electric furnace, pouring 50ml of gelatinized culture medium into the eggplant bottle added with 1g of bran, subpackaging 20 eggplant bottle inclined planes, wrapping by using a cotton plug, gauze and kraft paper, sterilizing at 121 +/-1 ℃ for 30min, manufacturing the eggplant bottle into an inclined plane with an angle of 15 degrees when the inclined plane of the killed eggplant bottle is not solidified, and carrying out blank culture at 37 ℃ for 24 h. The slope of the eggplant bottle is sterilized for standby inoculation.
2. The method for preparing an ammonium maduramicin producer strain according to claim 1, wherein the method comprises the steps of: 2 quick-freezing tubes taken out of a refrigerator are connected with a mother bottle according to the original method, the other seed liquid is added on the inclined plane of an empty white eggplant bottle, 0.2ml of seed liquid is added on each inclined plane, the mixture is uniformly coated, the mixture is placed in a constant-temperature incubator at the temperature of 32 +/-0.5 ℃ for culturing for 7-8 days, a large amount of off-white spores are generated on the inclined plane of the eggplant bottle at the moment, the cultured inclined plane strain is the cultured inclined plane strain, the cultured inclined plane of the eggplant bottle is wrapped by sterile tinfoil paper, the eggplant bottle is refrigerated for standby at the temperature of 4 ℃, and the refrigerating time of the inclined plane refrigerator is not more than 40 days.
3. The method for preparing an ammonium maduramicin producer strain according to claim 2, wherein the method comprises the steps of: the cultured slant strain is inoculated with 0.5cm 2/bottle slant spores to shake flask seeds, the shake flask seeds are inoculated with sterile nutrient broth, and simultaneously the strain is transferred to a fermentation flask for culture for 5 days, the sterility of the strain is detected, and the titer is more than 8000u/ml.
4. The method for preparing an ammonium maduramicin producer strain according to claim 3, wherein the method comprises the steps of: the production of the seed culture medium comprises the following steps of preparing 1L of seed culture medium: glucose: 20g, soybean cake powder: 20g, corn steep liquor: 10g, light calcium carbonate: 5g of light calcium carbonate is weighed singly, other raw materials and auxiliary materials are weighed together, a small amount of cold tap water is used for pasting, and then boiled hot water is used for sliding. Then, the volume is determined to be 1L by cold tap water, the pH value is adjusted to be 7.2-7.4 by 20 percent sodium hydroxide solution, finally, light calcium carbonate is added, the mixture is stirred evenly and subpackaged, the seed loading of each bottle is 100ml/750ml triangular bottle, a cotton plug, 2 layers of gauze and kraft paper are used for wrapping, the temperature is 121 +/-1 ℃, the sterilization is carried out for 30min, and the mixture is cooled to the room temperature for standby.
5. The method for preparing an ammonium maduramicin producer strain according to claim 4, wherein the method comprises the steps of: preparing the fermentation culture medium, namely preparing 1L of the fermentation culture medium: glucose: 40g, soybean cake powder 40g, dimethyl hydrogen phosphate: 0.2g, ferrous sulfate: 0.05g, light calcium carbonate: 5g, corn steep liquor: 5g, sodium chloride: 4g of light calcium carbonate is weighed singly, other raw materials and auxiliary materials are weighed together, a small amount of cold tap water is used for pasting, and then boiled hot water is used for sliding. Then, the volume is determined to be 1L by cold tap water, the pH value is adjusted to be 7.2-7.4 by 20 percent sodium hydroxide solution, finally, light calcium carbonate is added, the mixture is stirred evenly and subpackaged, the seed loading of each bottle is 60ml/500ml triangular bottle, eight layers of gauze and kraft paper are used for packing, the mixture is sterilized at the temperature of 121 +/-1 ℃ for 30min, and the mixture is cooled to the room temperature for standby.
6. The method for preparing an ammonium maduramicin producer strain according to claim 5, wherein the method comprises the steps of: the bottle inoculation is characterized in that prepared eggplant bottle inclined plane seeds are taken from a refrigerator and are inoculated into a seed bottle filled with a sterile culture medium, the eggplant bottle inclined plane seeds are cultured for 48 hours, the bottle is closed and the tank is thrown, when a large triangular bottle of the tank is closed, a small amount of seed liquid is reserved in each bottle of seeds and is put into a sampling bottle, the seed liquid in the sampling bottle is inoculated into 3 bottles for fermentation and tracking, the sterility and the titer of the eggplant bottle are detected after the cultivation for 5 days, meanwhile, the seed liquid in the sampling bottle is inoculated into broth for detecting the sterility of the seeds, the picture microscopic examination shows that the eggplant bottle is free of foreign bacteria, the pH value is 7.0-8.0, and the concentration of the eggplant bottle is greater than 10%, so that the eggplant bottle inclined plane seeds can be used for throwing the tank. The maduramicin produced by the method has 9 batches, the production level is improved by 18 percent, and the average yield is not changed by 75 percent. The content of maduramicin ammonium raw material medicine is more than 91%. All the products are qualified.
7. The method for preparing an ammonium maduramicin producer strain according to claim 6, wherein the method comprises the steps of: the new process produces 10 batches of jars, requiring 1 batch of eggplant flask slopes, 10 batches of shake flask seeds to be formulated.
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| 沈雪亮;: "分批发酵及补料分批发酵工艺生产马杜霉素的研究", 高校化学工程学报, no. 06, 15 December 2008 (2008-12-15), pages 98 - 103 * |
| 赵子刚, 夏黎明: "马杜霉素发酵法生产工艺的研究", 中国兽药杂志, no. 01, 20 January 2004 (2004-01-20), pages 47 - 50 * |
| 高自召, 薛彩琴, 黄玉欣, 周超, 扈留轩, 陈永辉, 王新灵, 薛建敏, 高巧转: "斜面保藏及冷冻保藏对马杜霉素产生菌的影响", 中国兽药杂志, no. 11, 25 November 2003 (2003-11-25), pages 48 * |
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