CN117487872A - Fermentation method of oligomycin A - Google Patents

Fermentation method of oligomycin A Download PDF

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CN117487872A
CN117487872A CN202210884800.9A CN202210884800A CN117487872A CN 117487872 A CN117487872 A CN 117487872A CN 202210884800 A CN202210884800 A CN 202210884800A CN 117487872 A CN117487872 A CN 117487872A
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fermentation
percent
oligomycin
corn
glucose
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侯宏波
周陈锋
陈琳
陈斌
颜林江
张宇
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Hangzhou Zhongmei Huadong Pharmaceutical Co Ltd
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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
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    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
    • C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces
    • C12R2001/545—Streptomyces griseus

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Abstract

The invention discloses a fermentation method of oligomycin A, which is characterized in that quick-acting/slow-release carbon and nitrogen sources in a fermentation medium and various inorganic salts in a feeding process are subjected to DOE experiment design, and proper parameters are optimized. And (3) finishing fermentation optimization control, and guiding to the biosynthesis path of the oligomycin A to obtain the fermentation method of the high-yield oligomycin A. The oligomycin A fermentation method provided by the invention is suitable for laboratory-scale preparation and industrial mass production, improves the fermentation level, and can stably reach more than 3000ug/ml after 13 days of fermentation period.

Description

Fermentation method of oligomycin A
Technical Field
The invention belongs to the technical field of medicines, further belongs to the technical field of fermentation, and particularly relates to a fermentation method of oligomycin A.
Background
The oligomycin antibiotic composition is a class of structurally similar twenty-six membered ring macrolide antibiotics isolated for the first time from Streptomyces diastatochromogenes in 1954 by Smith et al, university of Wesconsin, U.S.A. The biological activity is various, including antifungal activity, antitumor activity, respiratory depression, insecticidal activity, etc. The oligomycin A is mainly derived from streptomyces diastatochromogenes, is an inhibitor of ATP synthase, and inhibits oxidative phosphorylation and all ATP-dependent processes occurring in mitochondrial coupling membranes. Oligomycin a inhibits ATP synthesis by blocking its proton channel (Fo subunit), thereby affecting ADP conversion to ATP by oxidative phosphorylation.
The chemical structural formula of the oligomycin is as follows:
CN200810223500.6, chinese university of agriculture discloses a method for producing oligomycin a and its special strain, its process uses venezuelan streptomycete, and adopts the processes of plate activation, block-cutting inoculation bottle, shaking culture, fermentation, pH 7.0-7.2, and bottle-placing end-point titer is 1461 mug/ml in 9 days (216 hours).
DE3503568A1, bayer discloses a process for the preparation of oligomycin A using Streptomyces griseus, wherein the plate culture medium used is described as comprising yeast extract, malt extract, glucose, agar, etc., and the fermentation medium comprises fermentation medium: skimmed milk, yeast autolysate, dextrin, maltose, etc.
Since the 60 s of the 19 th century, studies on oligomycin have been mainly focused on downstream processes, and there have been literature on sporadic descriptions of designing oligomycin fermentation formulation tests through experiments such as orthogonalization, and formulation optimization has been mainly focused on basic grain material ratios, and has not been designed or introduced for metabolic processes, biological metabolism, or synthetic pathways.
In the microbial fermentation process, the conventional inorganic salt mainly has the following functions: the components forming the cell are used as the components of the enzyme, maintain the activity of the enzyme, maintain the acid-base balance in the organism, regulate the concentration of hydrogen ions, regulate the oxidation-reduction potential, maintain the osmotic pressure of the cell and other factors. Phosphorus plays an extremely important role in bacterial growth, reproduction and metabolic activity.
Phosphorus is an important raw material constituting nucleic acid, phospholipid, many coenzymes and high-energy phosphate compounds, and on the other hand, phosphorus plays an important role in regulating metabolism, and can promote the progress of sugar metabolism and promote the growth of microorganisms. Although potassium is not involved in cellular structural components, it is an activator of many enzymatic actions, potassium also has a regulatory effect on the colloidal state of cell protoplasm and permeability of cell membranes. Manganese plays an important role in the growth and spore production of streptomyces, and when properly adding manganese ions into a culture medium, the generation of spores is promoted, and the increase of the concentration of the manganese ions can cause a series of changes such as imbalance of the component proportion of the culture medium, increase of osmotic pressure and the like, so that the growth state of microorganisms is influenced. Cobalt is one of the elements constituting vitamin B2, vitamin B12 can promote the metabolic rate of one carbon unit of microorganisms, but overfeeding affects the metabolic balance of cells.
Disclosure of Invention
According to the invention, the quick-acting/slow-release carbon and nitrogen sources in the fermentation medium and the inorganic salts in the feeding process are subjected to DOE experiment design, and the proper parameters are optimized. And (3) completing fermentation optimization control and guiding to the oligomycin biosynthesis pathway. The oligomycin A fermentation method provided by the invention is suitable for laboratory-scale preparation and industrial mass production, improves the fermentation level, and can reach more than 3000ug/ml after 13 days of fermentation period.
A fermentation medium for oligomycin a comprising the following components:
fermentation medium composition Mass to volume ratio (W/V)
Quick-acting carbon source 1～8％
Sustained release carbon source 4～15％
Quick-acting nitrogen source 0.2～2.5％
Sustained release nitrogen source 0.5～5％
Inorganic salt 0.01～0.5％
The pH value of the fermentation medium is 6.5-7.5.
The fermentation medium is prepared according to the process formula, water is added into a fermentation tank, and raw materials are added while stirring;
the temperature of the fermentation tank is 10-37 ℃, preferably 25-30 ℃;
the culture time of the fermentation tank is 12-14 days;
the fermentation culture starts flow supplement at 48h after the self-fermentation culture; the flow compensation is calculated according to daily flow compensation;
the daily flow supplement feed formula is as follows:
feed supplement component Mass to volume ratio (W/V)
Carbon source 30～60％
Inorganic salt 0.005～0.01％
As a specific embodiment, the carbon source includes, but is not limited to: glucose, sucrose, fructose, lactose, maltose, glycerol, mannitol, maltodextrin, high maltose flour, molasses, corn flour, potato starch, tapioca starch, corn steep liquor dry powder, glutinous rice flour, soluble starch, yeast powder, etc.
As a specific embodiment, the nitrogen source includes, but is not limited to: ammonium sulfate, urea, malt extract, yeast powder, yeast peptone, soybean peptone, corn gluten meal, corn steep liquor dry powder, soybean protein meal, cotton seed meal, soybean meal, raw soybean meal, silkworm chrysalis meal, beef extract, fish meal, peanut cake meal, soybean cake meal, etc.
As a specific embodiment, the carbon source includes a quick-acting carbon source and a slow-release carbon source.
As a specific embodiment, the nitrogen source includes a quick-acting nitrogen source and a slow-release nitrogen source.
As a specific embodiment, the quick-acting carbon source includes, but is not limited to: glucose, fructose, sucrose, glycerol, mannitol, lactose, maltose, and the like.
As a specific embodiment, the slow release carbon source includes, but is not limited to: corn flour, potato starch, tapioca starch, corn starch, glutinous rice flour, soluble starch, etc.
As a specific embodiment, the quick-acting nitrogen source includes, but is not limited to: ammonium sulfate, urea, malt extract, yeast extract powder, etc.
As a specific embodiment, the slow release nitrogen source includes, but is not limited to: corn gluten meal, soy protein meal, cotton seed meal, soybean cake meal, corn steep liquor dry powder and the like.
As a specific embodiment, the inorganic salts include, but are not limited to: carbonates, sulfates, phosphates, chlorides, and the like.
As a specific embodiment, the inorganic salt is selected from the group consisting of calcium carbonate, dipotassium hydrogen phosphate, potassium chloride, cobalt chloride, magnesium sulfate, zinc sulfate, ferrous sulfate, and manganese sulfate.
The invention also provides a fermentation method of the oligomycin A, which comprises the following steps:
1. shake flask seed culture: comprises the steps of preparing shake flask seeds, split charging, sterilizing, inoculating and culturing to obtain shake flask seed bacterial liquid;
2. seed pot culture: the method comprises the steps of proportioning, sterilizing, inoculating and culturing seed culture to obtain seed bacterial liquid;
3. fermentation culture: comprises the steps of proportioning, sterilizing, inoculating and culturing a fermentation medium to obtain an oligomycin fermentation liquor;
4. and (3) feeding and culturing: starting flow supplement from 48h of the initiation of fermentation culture, wherein the flow supplement is calculated by the daily flow supplement amount;
5. and (5) placing the pot.
As a specific embodiment, the fermentation medium of the present invention is formulated as follows:
fermentation medium composition Mass to volume ratio (W/V)
Glucose 0.5～4％；
Mannitol (mannitol) 0.5～5％；
Corn starch 2～12％；
Corn flour 0.5～5％；
Ammonium sulfate 0.5～2.5％；
Cottonseed fine powder 0.5～5％；
Dipotassium hydrogen phosphate 0.01～0.1％；
Manganese sulfate 0.001～0.05％；
Cobalt chloride 0.001～0.05％；
As a specific implementation scheme, the fermentation culture mode is to mix the components according to the proportion, add water into a fermentation tank, add raw materials while stirring, adjust pH, and hydraulically press seeds into the fermentation tank by using sterile air.
As a specific embodiment, the feed culture method of the invention is as follows: starting flow supplement from 48h of the initiation of fermentation culture, supplementing 30-60% glucose daily, maintaining the residual sugar of a fermentation system at 0.5-1%, and supplementing an inorganic salt mixed solution daily, wherein the inorganic salt mixed solution is favorable for the generation and metabolism balance of the oligomycin A.
As a specific embodiment, the inorganic salt mixture includes dipotassium hydrogen phosphate, manganese sulfate and cobalt chloride.
As a specific embodiment, the content of the daily fluid mineral salt mixture is 0.005-0.01%.
As a specific embodiment, the content of the dipotassium hydrogen phosphate in the stream is 0.005-0.008%, preferably 0.006-0.007%.
As a specific embodiment, the content of the fed-batch manganese sulfate is 0.0001 to 0.0003%, preferably 0.0002 to 0.0003%.
As a specific embodiment, the content of the cobalt chloride in the stream is 0.0001-0.0003%, preferably 0.0001-0.0002%.
The invention provides an oligomycin A fermentation method, which comprises the following steps:
1. fermentation culture: weighing the raw materials, wherein the weight volume ratio of the raw materials is 0.5-4% of glucose, 1-5% of mannitol, 2-11% of corn starch, 0.5-5% of corn powder, 0.5-2.5% of ammonium sulfate, 0.5-5% of cotton seed refined powder, 0.01-0.1% of dipotassium hydrogen phosphate, 0.001-0.05% of manganese sulfate and 0.001-0.005% of cobalt chloride; adding raw materials while stirring, regulating pH, sterilizing, preserving heat and pressure, and culturing in a fermentation tank at a tank temperature of 25-30 ℃;
2. and (3) feeding and culturing: starting flow supplement from 48h of the beginning of fermentation culture, wherein the flow supplement comprises 30-60% of glucose and 0.005-0.01% of inorganic salt mixed liquor, and the inorganic salt mixed liquor comprises dipotassium hydrogen phosphate, manganese sulfate and cobalt chloride;
3. and (3) tank discharge: after 12-14 days of fermentation culture, the fermentation is finished.
The beneficial effects of the invention are as follows:
1. quick-acting/slow-releasing carbon source, quick-acting/slow-releasing nitrogen source type screening and proportion screening in the fermentation culture medium. The primary metabolism of the bacteria is affected to grow, and the secondary metabolism of the carbon-nitrogen source is utilized to generate API, and the initial carbon source in the biological metabolism path is glucose by taking oligomycin A as an example. However, the addition of excess glucose to the basal medium causes a glucose repression effect, and catabolites repress transcription of certain genes encoding the inducible enzyme system, thereby affecting its efficiency of utilization of other carbon sources and inhibiting cell growth. The patent performs type screening on initial carbon and nitrogen sources, and uses Tian Koushi experiment to finish the optimization of the proportion of each component of the fermentation formula.
2. Compared with the prior art, the invention has the advantages that the mixed solution of inorganic salt is introduced into daily flow supplement to form balance with the growth and metabolism of thalli, thereby being beneficial to the biosynthesis of the API. However, some inorganic salts such as cobalt, too much flow can inhibit the growth of the cells, and manganese can promote the formation of spores and thus affect osmotic pressure. The patent obtains the optimal solution in the selection of the components and the total amount of the fluid compensation inorganic salt of the fermentation system through the response surface experiment.
3. According to the fermentation method provided by the invention, through verification of a fermentation system, the fermentation period can reach more than 3000ug/ml stably after being placed in a tank in 12-14 days.
Drawings
FIG. 1-HPLC chart of the fermentation product obtained in example 2
FIG. 2-Mintab Multi-response prediction result graph described in example 5
Strain and preservation method
The oligomycin A strain used in the present invention is Streptomyces griseus (Streptomyces griseus).
The strain is preserved in two forms of glycerol pipe and inclined plane.
Detailed Description
The present invention will be described in further detail with reference to specific examples. The following examples are presented to illustrate the method and core concepts of the present invention and, as such, to those skilled in the art, any possible variations or alterations may be made without departing from the spirit of the present invention. The experimental method without specific conditions noted in the embodiments of the present invention is generally conventional conditions or conditions suggested by the manufacturer of the raw materials or goods; the starting materials and reagents from which they are not identified are typically conventional reagents that are commercially available.
Example 1: fermentation culture (shaking flask systematic experiment)
Fermentation medium formulation (W/V): glucose 2%, mannitol 3%, corn starch 11%, corn flour 1%, ammonium sulfate 1%, cotton seed fine powder 1%, and inorganic salt mixed solution (total dipotassium hydrogen phosphate 0.05%, manganese sulfate 0.0024% and cobalt chloride 0.002%) are added for strain fermentation shake flask systematic test, and the fermentation period is 13 days.
The fermentation titer of oligomycin A obtained by the fermentation method described in example 1 was 1964.6ug/ml.
Example 2: fermentation culture (put quantity production)
Fermentation medium formulation (W/V): 2% of glucose, 3% of mannitol, 5% of corn starch, 2% of corn powder, 1.5% of ammonium sulfate, 3% of cotton seed fine powder, 0.05% of dipotassium hydrogen phosphate, 0.0024% of manganese sulfate and 0.002% of cobalt chloride.
The preparation method comprises the steps of proportioning according to the process formula, adding raw materials while stirring, regulating the pH to 6.5-7.5, controlling the pot temperature and the pot pressure, preserving heat and pressure, sterilizing, hydraulically feeding seeds into a fermentation pot by using sterile air, and heating the fermentation liquor to the pot temperature of 28 ℃.
The daily flow supplement is started from the beginning of fermentation culture, 50% glucose solution is fed daily, and the residual sugar detection of a fermentation system is maintained to be 0.5% -1%; daily flow supplement inorganic salt mixed solution: 0.00643% of dipotassium hydrogen phosphate, 0.00026% of manganese sulfate and 0.00018% of cobalt chloride.
The fermentation period was 13 days, and after 13 days, the tank was placed and examined.
The fermentation titer of oligomycin A obtained by the fermentation method described in example 2 was 3255.0ug/ml.
FIG. 1 is an HPLC chart of the end-point product of the fermentation in this example 2.
Example 3: investigation of fermentation culture Material
Experiment design:
the experiment was intended to screen the selection of carbon and nitrogen sources in the fermentation culture formulation.
Except for carbon and nitrogen sources, the introduced content of the inorganic salt mixed solution (total dipotassium hydrogen phosphate 0.05%, manganese sulfate 0.0024% and cobalt chloride 0.002%) is unchanged.
Experimental protocol:
scheme 1-1: the formula (W/V) of the fermentation medium is 5% of corn starch; 2% of cotton seed refined powder;
scheme 1-2: the formula (W/V) of the fermentation medium is 5% of corn starch; 2% of cotton seed refined powder; 3% of maltose;
scheme 1-3: the formula (W/V) of the fermentation medium is 5% of corn starch; 2% of cotton seed refined powder; mannitol 3%;
schemes 1-4: the formula (W/V) of the fermentation medium is 5% of corn starch; 2% of cotton seed refined powder; glucose 3%;
schemes 1-5: the formula (W/V) of the fermentation medium is 5% of corn starch; 2% of cotton seed refined powder; sucrose 3%;
schemes 1-6: the formula (W/V) of the fermentation medium is 5% of corn starch; 2% of cotton seed refined powder; 3% of potato starch;
schemes 1-7: the formula (W/V) of the fermentation medium is 5% of corn starch; 2% of cotton seed refined powder; glycerol 3%;
schemes 1-8: the formula (W/V) of the fermentation medium is 5% of corn starch; 2% of cotton seed refined powder; corn flour 3%;
schemes 1-9: the formula (W/V) of the fermentation medium is 5% of corn starch; 2% of cotton seed refined powder; 3% of molasses;
schemes 1-10: fermentation medium formula (W/V) corn starch 5%; 2% of cotton seed refined powder; maltodextrin 3%;
schemes 1-11: the formula (W/V) of the fermentation medium is 8% of corn starch; 1% of cotton seed refined powder; 1% of ammonium sulfate;
schemes 1-12: the formula (W/V) of the fermentation medium is 8% of corn starch; 1% of cotton seed refined powder; 1% of raw soybean flour;
schemes 1-13: the formula (W/V) of the fermentation medium is 8% of corn starch; 1% of cotton seed refined powder; urea 1%;
schemes 1-14: the formula (W/V) of the fermentation medium is 8% of corn starch; 2% of yeast powder;
schemes 1-15: the formula (W/V) of the fermentation medium is 8% of corn starch; soybean peptone 2%;
schemes 1-16: the formula (W/V) of the fermentation medium is 8% of corn starch; 2% of cotton seed refined powder;
schemes 1-17: the formula (W/V) of the fermentation medium is 8% of corn starch; 2% of soybean powder;
schemes 1-18: the formula (W/V) of the fermentation medium is 8% of corn starch; 2% of beef extract;
schemes 1-19: the formula (W/V) of the fermentation medium is 8% of corn starch; silkworm chrysalis meal 2%;
schemes 1-20: the formula (W/V) of the fermentation medium is 8% of corn starch; 2% of fish meal;
schemes 1-21: the formula (W/V) of the fermentation medium is 8% of corn starch; corn steep liquor dry powder 2%;
schemes 1-22: the formula (W/V) of the fermentation medium is 8% of corn starch; 2% of peanut cake powder;
experimental results:
conclusion of experiment: it was basically confirmed that the quick-acting carbon source of the present invention may be glucose, mannitol and glycerol; the slow release carbon source can be corn starch, maltodextrin and corn flour; the nitrogen source can be ammonium sulfate, raw soybean powder, cotton seed refined powder, etc.
Basically confirms that the carbon source, the nitrogen source and the composite carbon-nitrogen source of the fermentation tank and the feed can be selected from glucose, mannitol, corn starch, corn steep liquor, ammonium sulfate and cotton seed fine powder.
Example 4: investigation of fermentation culture material proportion based on shake flask experiment
Experiment design: based on the selection of carbon and nitrogen sources in example 3, the reasonable proportions of glucose, mannitol, corn starch, corn meal, ammonium sulfate, cottonseed meal in the fermentation medium were further tested.
In addition to the carbon and nitrogen source, the addition or absence of the inorganic salt component is also a confirmation option.
Scheme 2-1: the formula (W/V) of the fermentation medium is 1% of glucose, 1% of mannitol, 5% of corn starch, 1% of corn meal, 0.5% of ammonium sulfate and 1% of cottonseed meal; inorganic salt mixed solution is not added;
scheme 2-2: the formula (W/V) of the fermentation medium is 1% of glucose, 3% of mannitol, 8% of corn starch, 2% of corn meal, 1% of ammonium sulfate and 2% of cottonseed meal; inorganic salt mixed solution is not added;
scheme 2-3: the formula (W/V) of the fermentation medium is 1% of glucose, 5% of mannitol, 11% of corn starch, 3% of corn meal, 1.5% of ammonium sulfate and 3% of cottonseed meal; inorganic salt mixed solution is not added;
scheme 2-4: the formula (W/V) of the fermentation medium is 2% of glucose, 1% of mannitol, 5% of corn starch, 2% of corn meal, 1% of ammonium sulfate and 3% of cottonseed meal; inorganic salt mixed solution is not added;
scheme 2-5: the formula (W/V) of the fermentation medium is 3% of glucose, 5% of mannitol, 8% of corn starch, 3% of corn meal, 1.5% of ammonium sulfate and 1% of cottonseed meal; inorganic salt mixed solution is not added;
scheme 2-6: the formula (W/V) of the fermentation medium is 2% of glucose, 5% of mannitol, 11% of corn starch, 1% of corn meal, 0.5% of ammonium sulfate and 2% of cotton seed fine powder; inorganic salt mixed solution is not added;
scheme 2-7: the formula (W/V) of the fermentation medium is 3% of glucose, 1% of mannitol, 8% of corn starch, 1% of corn meal, 1.5% of ammonium sulfate and 2% of cotton seed fine powder; inorganic salt mixed solution is not added;
scheme 2-8: the formula (W/V) of the fermentation medium is 3% of glucose, 3% of mannitol, 11% of corn starch, 2% of corn meal, 0.5% of ammonium sulfate and 3% of cotton seed fine powder; inorganic salt mixed solution is not added;
scheme 2-9: the formula (W/V) of the fermentation medium is 3% of glucose, 5% of mannitol, 5% of corn starch, 3% of corn meal, 1% of ammonium sulfate and 1% of cottonseed meal; adding an inorganic salt mixed solution;
scheme 2-10: the formula (W/V) of the fermentation medium is 1% of glucose, 1% of mannitol, 11% of corn starch, 3% of corn meal, 1% of ammonium sulfate and 2% of cottonseed meal; adding an inorganic salt mixed solution;
scheme 2-11: the formula (W/V) of the fermentation medium is 1% of glucose, 3% of mannitol, 5% of corn starch, 1% of corn meal, 1.5% of ammonium sulfate and 3% of cottonseed meal; adding an inorganic salt mixed solution;
scheme 2-12: the formula (W/V) of the fermentation medium is 1% of glucose, 5% of mannitol, 8% of corn starch, 2% of corn meal, 0.5% of ammonium sulfate and 1% of cottonseed meal; adding an inorganic salt mixed solution;
scheme 2-13: the formula (W/V) of the fermentation medium is 2% of glucose, 1% of mannitol, 8% of corn starch, 3% of corn meal, 0.5% of ammonium sulfate and 3% of cotton seed fine powder; adding an inorganic salt mixed solution;
scheme 2-14: the formula (W/V) of the fermentation medium is 2% of glucose, 3% of mannitol, 11% of corn starch, 1% of corn meal, 1% of ammonium sulfate and 1% of cottonseed meal; adding an inorganic salt mixed solution;
scheme 2-15: the formula (W/V) of the fermentation medium is 2% of glucose, 5% of mannitol, 5% of corn starch, 2% of corn meal, 1.5% of ammonium sulfate and 2% of cotton seed fine powder; adding an inorganic salt mixed solution;
schemes 2-16: the formula (W/V) of the fermentation medium is 3% of glucose, 1% of mannitol, 11% of corn starch, 2% of corn meal, 1.5% of ammonium sulfate and 1% of cottonseed meal; adding an inorganic salt mixed solution;
scheme 2-17: the formula (W/V) of the fermentation medium is 3% of glucose, 3% of mannitol, 5% of corn starch, 3% of corn meal, 0.5% of ammonium sulfate and 2% of cotton seed fine powder; adding an inorganic salt mixed solution;
scheme 2-18: the formula (W/V) of the fermentation medium is 3% of glucose, 5% of mannitol, 8% of corn starch, 1% of corn meal, 1% of ammonium sulfate and 3% of cottonseed meal; adding an inorganic salt mixed solution;
experimental results:
conclusion of experiment: experiments show that 2% of glucose, 3% of mannitol, 5% of corn starch, 2% of corn powder, 1.5% of ammonium sulfate and 3% of cottonseed meal, and an inorganic salt mixed solution is needed to be added, so that a relatively good material proportion selection is provided, and a foundation is provided for the follow-up carefully chosen proportion.
Example 5: investigation of fermentation flow-supplementing inorganic salt mixed liquor
Experiment design: based on the selection and the ratio of the carbon-nitrogen source in examples 3 and 4, the proportions of the respective components in the respective inorganic salt mixed liquids to be added were examined.
Experimental results:
conclusion of experiment:
and (3) performing multiple regression analysis according to the obtained data to obtain a multiple quadratic regression equation of the response variable (daily flux supplement of X dipotassium hydrogen phosphate, Y manganese sulfate and Z cobalt chloride) and the response value (oligomycin titer (ug/ml)).
The oligomycin potency (ug/ml) =856+255406x dipotassium hydrogen phosphate+ 9601160Y manganese sulfate+ 7192371Z cobalt chloride-24316335X dipotassium hydrogen phosphate-18252519207Y manganese sulfate-28518323967Z cobalt chloride-67919898X dipotassium hydrogen phosphate, Y manganese sulfate+ 412348616X dipotassium hydrogen phosphate+z cobalt chloride+ 2340896967Y manganese sulfate-Z cobalt chloride.
The significance of the relationship between each response variable (dipotassium hydrogen phosphate, manganese sulfate Y, cobalt chloride Z and daily flux supplement) and the response value (oligomycin potency (ug/ml)), and the overall F value of the design model in the analysis of variance table is 28.81, and the P value is 0.001 (< 0.05%), so the design of the model is significant. And further analyzing each response variable of the dipotassium hydrogen phosphate, the manganese sulfate Y and the cobalt chloride Z, squaring the response variable and performing double-factor interaction on the F value and the P value of the response variable. The model influence among dipotassium hydrogen phosphate, manganese sulfate Y, cobalt chloride Z, dipotassium hydrogen phosphate, manganese sulfate Y and dipotassium hydrogen phosphate Z cobalt chloride is obvious.
Interaction contour maps and surface maps of various response variables (X dipotassium hydrogen phosphate, Y manganese sulfate and Z cobalt chloride daily flow supplement) are analyzed, mintab multi-response prediction is used, and model prediction results are shown in figure 2.
The specific prediction result of the scheme is as follows:
conclusion of experiment: according to the orthogonal experiment and data analysis of the flow compensation inorganic salt mixed solution, the optimization result of the daily flow compensation response surface experiment of dipotassium hydrogen phosphate, manganese sulfate Y and cobalt chloride Z is that dipotassium hydrogen phosphate 0.00643%, manganese sulfate 0.00026% and cobalt chloride 0.00018% are compensated daily.
The above embodiments are only for understanding the method and core idea of the present invention, and do not limit the scope of the present invention. It will be apparent to those skilled in the art that any possible variations or substitutions can be made without departing from the spirit of the invention.

Claims (9)

1. The fermentation method of the oligomycin A comprises fermentation culture and daily flow supplement after 48 hours from the fermentation culture, wherein the daily flow supplement comprises 30-60% of glucose and 0.005-0.01% of inorganic salt mixed solution according to the mass volume ratio; the inorganic salt mixed solution comprises dipotassium hydrogen phosphate, manganese sulfate and cobalt chloride.
2. A fermentation process of oligomycin a according to claim 1, wherein the content of dipotassium hydrogen phosphate in the stream is 0.005-0.008%, preferably 0.006-0.007%;
and/or the content of the flow-compensated manganese sulfate is 0.0001-0.0003%, preferably 0.0002-0.0003%;
and/or the content of the cobalt chloride in the stream is 0.0001-0.0003%, preferably 0.0001-0.0002%.
3. A fermentation process of oligomycin a as claimed in claim 1, wherein the medium of the fermentation culture comprises, in mass to volume ratio: 1 to 8 percent of quick-acting carbon source, 4 to 15 percent of slow-release carbon source, 0.2 to 2.5 percent of quick-acting nitrogen source, 0.5 to 5 percent of slow-release nitrogen source and 0.01 to 0.5 percent of inorganic salt;
and/or the quick-acting carbon source is selected from one or more of glucose, fructose, sucrose, glycerol, mannitol, lactose and maltose;
and/or the slow-release carbon source is selected from one or more of corn flour, potato starch, corn starch, molasses, maltodextrin and soluble starch;
and/or the quick-acting nitrogen source is selected from one or more of ammonium sulfate, urea and yeast powder;
and/or the slow release nitrogen source is selected from one or more of soybean powder, beef extract, cotton seed extract powder, soybean peptone, fish meal, peanut cake powder and corn steep liquor dry powder;
and/or the inorganic salt is selected from carbonate, sulfate, phosphate or chloride, preferably one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, cobalt chloride, manganese sulfate, magnesium sulfate and zinc sulfate.
4. A fermentation process of oligomycin a according to claim 1, wherein the medium of the fermentation culture comprises, in mass to volume ratio: 0.5 to 4 percent of glucose, 0.5 to 5 percent of mannitol, 2 to 12 percent of corn starch, 0.5 to 5 percent of corn meal, 0.5 to 2.5 percent of ammonium sulfate, 0.5 to 5 percent of cotton seed fine powder, 0.01 to 0.1 percent of dipotassium hydrogen phosphate, 0.001 to 0.05 percent of manganese sulfate and 0.001 to 0.05 percent of cobalt chloride.
5. A fermentation process of oligomycin a according to claim 1, wherein the pH of the fermentation medium is 6.5-7.5; and/or the temperature of the fermentation culture is 25-30 ℃; and/or the fermentation culture period is 12-14 days.
6. A fermentation process of oligomycin a according to claim 1, comprising the steps of:
a. fermentation culture: weighing the raw materials, wherein the weight volume ratio of the raw materials is 0.5-4% of glucose, 1-5% of mannitol, 2-11% of corn starch, 0.5-5% of corn powder, 0.5-2.5% of ammonium sulfate, 0.5-5% of cotton seed refined powder, 0.01-0.1% of dipotassium hydrogen phosphate, 0.001-0.05% of manganese sulfate and 0.001-0.005% of cobalt chloride; adding raw materials while stirring, regulating pH, sterilizing, preserving heat and pressure, and culturing in a fermentation tank at a tank temperature of 25-30 ℃;
b. daily current supplement: starting flow compensation from 48h of the beginning of fermentation culture, maintaining residual sugar of a fermentation system by 30-60% of glucose, and carrying out daily flow compensation by 0.005-0.01% of inorganic salt mixed liquor, wherein the inorganic salt mixed liquor comprises dipotassium hydrogen phosphate, manganese sulfate and cobalt chloride;
c. and (3) tank discharge: after 12-14 days of fermentation culture, the fermentation is finished.
7. A fermentation process of oligomycin a according to claim 1, wherein the fermentation medium comprises, by mass, 2% glucose, 3% mannitol, 5% corn starch, 2% corn meal, 1.5% ammonium sulfate, 3% cotton seed meal, 0.05% dipotassium hydrogen phosphate, 0.0024% manganese sulfate, 0.002% cobalt chloride;
and/or 2% of glucose, 5% of mannitol, 5% of corn starch, 2% of corn meal, 1.5% of ammonium sulfate, 2% of cotton seed fine powder, 0.05% of dipotassium hydrogen phosphate, 0.0024% of manganese sulfate and 0.002% of cobalt chloride;
and/or 3% of glucose, 1% of mannitol, 11% of corn starch, 2% of corn meal, 1.5% of ammonium sulfate, 1% of cotton seed fine powder, 0.05% of dipotassium hydrogen phosphate, 0.0024% of manganese sulfate and 0.002% of cobalt chloride;
and/or 3% of glucose, 5% of mannitol, 8% of corn starch, 1% of corn meal, 1% of ammonium sulfate, 3% of cottonseed meal, 0.05% of dipotassium hydrogen phosphate, 0.0024% of manganese sulfate and 0.002% of cobalt chloride.
8. The application of a culture medium in the preparation of oligomycin A comprises the following components in mass volume ratio: 0.5 to 4 percent of glucose, 1 to 5 percent of mannitol, 2 to 11 percent of corn starch, 0.5 to 5 percent of corn meal, 0.5 to 2.5 percent of ammonium sulfate, 0.5 to 5 percent of cotton seed fine powder, 0.01 to 0.1 percent of dipotassium hydrogen phosphate, 0.001 to 0.05 percent of manganese sulfate and 0.001 to 0.005 percent of cobalt chloride.
9. The fermentation method of oligomycin A according to claim 1, wherein the daily fluid mineral salt mixture comprises 0.00643% of dipotassium hydrogen phosphate, 0.00026% of manganese sulfate and 0.00018% of cobalt chloride.
CN202210884800.9A 2022-07-26 2022-07-26 Fermentation method of oligomycin A Pending CN117487872A (en)

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