CN112410390A - Fermentation process of neomycin sulfate - Google Patents
Fermentation process of neomycin sulfate Download PDFInfo
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- CN112410390A CN112410390A CN202011376376.4A CN202011376376A CN112410390A CN 112410390 A CN112410390 A CN 112410390A CN 202011376376 A CN202011376376 A CN 202011376376A CN 112410390 A CN112410390 A CN 112410390A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/46—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical bound to a cyclohexyl radical, e.g. kasugamycin
- C12P19/48—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical bound to a cyclohexyl radical, e.g. kasugamycin the cyclohexyl radical being substituted by two or more nitrogen atoms, e.g. destomycin, neamin
- C12P19/50—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical bound to a cyclohexyl radical, e.g. kasugamycin the cyclohexyl radical being substituted by two or more nitrogen atoms, e.g. destomycin, neamin having two saccharide radicals bound through only oxygen to adjacent ring carbon atoms of the cyclohexyl radical, e.g. ambutyrosin, ribostamycin
- C12P19/52—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical bound to a cyclohexyl radical, e.g. kasugamycin the cyclohexyl radical being substituted by two or more nitrogen atoms, e.g. destomycin, neamin having two saccharide radicals bound through only oxygen to adjacent ring carbon atoms of the cyclohexyl radical, e.g. ambutyrosin, ribostamycin containing three or more saccharide radicals, e.g. neomycin, lividomycin
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- C—CHEMISTRY; METALLURGY
- 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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- C—CHEMISTRY; METALLURGY
- 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
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
Abstract
The invention relates to the technical field of biology, in particular to a fermentation process of neomycin sulfate. A fermentation process of neomycin sulfate at least comprises the following steps: preparing a slant culture medium, inoculating the slant, preparing a spore suspension, culturing in a seeding tank and culturing in a fermentation tank. The invention provides a fermentation method of neomycin sulfate, which comprises the following steps of firstly, compounding and optimizing seed culture medium materials to improve the seed bacterial concentration; secondly, the titer of neomycin sulfate obtained by fermentation is improved by compounding and optimizing materials of a fermentation culture medium; finally, through the compounding and optimization of the supplementary materials, the growth fluctuation caused by sudden overhigh sugar concentration is avoided, and the problem that the synthesis of products is inhibited due to overhigh concentration of inorganic salts of magnesium ions and zinc ions can be solved.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a fermentation process of neomycin sulfate.
Background
In recent years, neomycin has been widely used as a feed additive. The feed additive has a large using amount at home and abroad, and has a wide application range, thereby showing a good prospect. The demand of the domestic and foreign markets for neomycin has a greatly increased trend in recent years. Develops neomycin, improves the production level, and has wide market prospect and obvious economic benefit.
At present, the fermentation level of the neomycin is mainly improved at home and abroad without considering the unit consumption of sugar substances in the fermentation process and the control of the residual sugar concentration during tank discharging, so that the pollutant discharge caused by the neomycin fermentation process is high, wherein the residual sugar concentration during tank discharging can reach more than 3-3.5 percent sometimes. The production process is an extensive process with greatly changed substrate concentration, so that nutrient substances can not be supplied according to needs in the fermentation process, the normal metabolism of microorganisms is disturbed, the production efficiency is low (the concentration of neomycin is low) and the raw materials are wasted (the composition of an initial formula and a feed liquid is unreasonable, the feed is excessive, and the concentration of residual sugar after tank placement is overhigh), so that the production cost is increased, the yield is reduced, and the environmental pollution caused by emissions is also caused. Meanwhile, the abnormal growth of thalli is caused by the impact of high-concentration total sugar and intermittent feeding, the generation of products is influenced due to the extensive control of the concentration of ammonia nitrogen, the increase rate of the concentration of the products in the resistance period is low, and the concentration of the final products is low. The traditional process fully emphasizes the necessity of a large amount of sugar supplement. The starch hydrolysis sugar solution is added in a large amount blindly, so that a process of mass multiplication of thalli still exists for many times in the product synthesis period, and the product synthesis rate is reduced. Meanwhile, because the microorganisms cannot utilize the polysaccharide, a large amount of polysaccharide which cannot be utilized by the microorganisms is accumulated in the bioreactor, and the polysaccharide is discharged along with wastewater, so that environmental pollution and resource waste are caused.
Aiming at the problems, the invention provides a fermentation process of neomycin sulfate by optimizing a seed culture medium, a fermentation culture medium and a sugar supplement material.
Disclosure of Invention
In order to solve the above technical problems, a first aspect of the present invention provides a fermentation process of neomycin sulfate, which at least comprises the following steps:
(1) preparing a slant culture medium: sterilizing slant culture medium, shaking, spreading into slant, culturing at 28 + -0.5 deg.C for 1-5 days, and checking for sterility to obtain slant culture medium;
(2) slant inoculation: dissolving sand spore in sterile water, uniformly coating on slant culture medium, culturing at 28 + -0.5 deg.C for 5-10 days, and storing at 1-8 deg.C to obtain slant spore;
(3) preparing spore suspension: adding glass beads into the bevel spores, and shaking to knock down the spores; adding sterile water, shaking, sealing, and storing at 1-8 deg.C to obtain spore suspension;
(4) seed tank culture: inoculating the spore suspension to a seed culture medium in a seeding tank, then starting stirring, introducing sterile air, and culturing for 30-50h to obtain a culture solution;
(5) culturing in a fermentation tank: and (3) inoculating the culture solution onto a fermentation culture medium in a fermentation tank, starting stirring, introducing sterile air, detecting at regular time, and placing the fermentation tank after fermentation culture for 200-260 h.
As a preferred technical scheme of the invention, in the step (1), the raw materials for preparing the slant culture medium at least comprise the following components in percentage by weight: 0.5-2% of glucose, 0.01-0.5% of beef extract, 0.1-0.5% of peptone, 0.01-0.3% of corn steep liquor, 0.1-1% of sodium chloride, 1-5% of agar and the balance of water.
As a preferred technical scheme of the present invention, in step (4), the raw materials for preparing the seed culture medium at least comprise the following components by weight: 1-4% of glucose, 0.5-2% of corn steep liquor, 0.1-1% of peptone, 0.5-2% of corn starch, 0.5-2% of peanut cake powder, 1-3% of yeast extract, 0.1-1% of soybean oil, 1-2% of calcium carbonate, 0.05-0.3% of ammonium sulfate, 0.05-0.3% of disodium hydrogen phosphate and the balance of water.
As a preferred technical scheme of the invention, the raw materials for preparing the seed culture medium at least comprise the following components in percentage by weight: 1% of glucose, 1.0% of corn steep liquor, 0.3% of peptone, 1% of corn starch, 0.5% of peanut cake powder, 3.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.1% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water.
As a preferred technical solution of the present invention, the preparation method of the seed culture medium comprises the following steps:
(1) adding at least half of water into a seeding tank, then adding glucose, corn steep liquor, peptone, corn starch, peanut cake powder, yeast extract, ammonium sulfate and disodium hydrogen phosphate, uniformly stirring, and adjusting the pH value to 7-8 by using alkali;
(2) adding calcium carbonate, and washing the tank wall with the rest water; then adding soybean oil, and closing the tank cover;
(3) heating the seeding tank to 115-130 ℃, controlling the pressure in the tank to be 0.1-0.15MPa, preserving the temperature for 20-50min, and sterilizing;
(4) and (4) after the sterilization is finished, introducing sterile air into the tank, and cooling the seeding tank to 30-40 ℃ to obtain the product.
As a preferred technical scheme of the invention, in the step (5), the raw materials for preparing the fermentation medium at least comprise the following components in percentage by weight: 1-3% of glucose, 0.05-0.3% of corn steep liquor, 0.5-2% of peptone, 6-8% of long-shaped rice flour, 1.5-3.5% of peanut cake powder, 0.1-1% of soybean cake powder, 0.5-2% of yeast extract, 0.05-0.2% of soybean oil, 0.4-0.5% of sodium chloride, 0.3-0.7% of calcium carbonate, 0.3-0.8% of ammonium sulfate, 0.01-0.05% of amylase, 0.01-0.05% of potassium dihydrogen phosphate, 0.01-0.1% of sodium dihydrogen phosphate and the balance of water.
As a preferable technical scheme of the invention, the preparation raw materials of the fermentation medium at least comprise the following components in percentage by weight: 1% of glucose, 3% of corn steep liquor, 0.5% of peptone, 7% of long-shaped rice flour, 2.8% of peanut cake powder, 0.8% of soybean cake powder, 1% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.4% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water.
As a preferable technical scheme, the preparation method of the fermentation medium comprises the following steps:
(1) adding at least 1/4% water into the fermentation tank, adding indica rice flour and amylase, heating to 55-70 deg.C, and stirring for 10-40 min;
(2) adding soybean cake powder, peanut cake powder, corn steep liquor, yeast extract, glucose, peptone, ammonium sulfate, sodium chloride, calcium carbonate, potassium dihydrogen phosphate and disodium hydrogen phosphate into a fermentation tank, adding the rest water, and stirring uniformly; then adding soybean oil, and closing the tank cover;
(3) heating the fermentation tank to 115 ℃ and 130 ℃, controlling the pressure in the fermentation tank to be 0.1-0.15MPa, and preserving the temperature for 20-50min for sterilization;
(4) and (4) after the sterilization is finished, introducing sterile air into the tank, and cooling the fermentation tank to 30-40 ℃ to obtain the product.
As a preferable technical scheme of the invention, in the step (5), in the fermentation culture process, the weight ratio of reducing sugar is maintained between 0.5 and 3 percent, and the content of ammonia nitrogen is controlled between 4 and 25 mg/mL.
According to a second aspect of the invention, neomycin sulfate is prepared according to the fermentation process of neomycin sulfate.
Advantageous effects
The invention provides a fermentation method of neomycin sulfate, which comprises the following steps of firstly, compounding and optimizing seed culture medium materials to improve the seed bacterial concentration; secondly, the titer of neomycin sulfate obtained by fermentation is improved by compounding and optimizing materials of a fermentation culture medium; finally, through the compounding and optimization of the supplementary materials, the growth fluctuation caused by sudden overhigh sugar concentration is avoided, and the problem that the synthesis of products is inhibited due to overhigh concentration of inorganic salts of magnesium ions and zinc ions can be solved.
Detailed Description
The disclosure may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the examples included therein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.
The term "prepared from …" as used herein is synonymous with "comprising". The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.
The conjunction "consisting of …" excludes any unspecified elements, steps or components. If used in a claim, the phrase is intended to claim as closed, meaning that it does not contain materials other than those described, except for the conventional impurities associated therewith. When the phrase "consisting of …" appears in a clause of the subject matter of the claims rather than immediately after the subject matter, it defines only the elements described in the clause; other elements are not excluded from the claims as a whole.
When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.
The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. "optional" or "any" means that the subsequently described event or events may or may not occur, and that the description includes instances where the event occurs and instances where it does not.
Approximating language, as used herein throughout the specification and claims, is intended to modify a quantity, such that the invention is not limited to the specific quantity, but includes portions that are literally received for modification without substantial change in the basic function to which the invention is related. Accordingly, the use of "about" to modify a numerical value means that the invention is not limited to the precise value. In some instances, the approximating language may correspond to the precision of an instrument for measuring the value. In the present description and claims, range limitations may be combined and/or interchanged, including all sub-ranges contained therein if not otherwise stated.
In order to solve the above technical problems, a first aspect of the present invention provides a fermentation process of neomycin sulfate, which at least comprises the following steps:
(1) preparing a slant culture medium: sterilizing slant culture medium, shaking, spreading into slant, culturing at 28 + -0.5 deg.C for 1-5 days, and checking for sterility to obtain slant culture medium;
(2) slant inoculation: dissolving sand spore in sterile water, uniformly coating on slant culture medium, culturing at 28 + -0.5 deg.C for 5-10 days, and storing at 1-8 deg.C to obtain slant spore;
(3) preparing spore suspension: adding glass beads into the bevel spores, and shaking to knock down the spores; then adding sterile water, shaking, sealing, and storing at 1-8 deg.C to obtain spore suspension;
(4) seed tank culture: inoculating the spore suspension to a seed culture medium in a seeding tank, then starting stirring, introducing sterile air, and culturing for 30-50h to obtain a culture solution;
(5) culturing in a fermentation tank: and (3) inoculating the culture solution onto a fermentation culture medium in a fermentation tank, starting stirring, introducing sterile air, detecting at regular time, and placing the fermentation tank after fermentation culture for 200-260 h.
Preparation of slant culture medium
In the present invention, the following medium was prepared: preparing a slant culture medium: sterilizing the slant culture medium, shaking while hot, spreading into slant, culturing at 28 + -0.5 deg.C for 1-5 days, and checking for sterility to obtain slant culture medium.
In a preferred embodiment, the raw materials for preparing the slant culture medium at least comprise the following components in percentage by weight: 0.5-2% of glucose, 0.01-0.5% of beef extract, 0.1-0.5% of peptone, 0.01-0.3% of corn steep liquor, 0.1-1% of sodium chloride, 1-5% of agar and the balance of water.
In a more preferred embodiment, the raw materials for preparing the slant culture medium at least comprise the following components in percentage by weight: 1% of glucose, 0.25% of beef extract, 0.3% of peptone, 0.18% of corn steep liquor, 0.4% of sodium chloride, 2.6% of agar and the balance of water.
In a preferred embodiment, the preparation method of the slant culture medium comprises the following steps:
(1) mixing glucose, beef extract, peptone, corn steep liquor, sodium chloride, agar and water, adjusting pH to 7-8 with alkali, packaging, and sealing;
(2) sterilizing with steam of 0.10-0.15MPa at 110-130 deg.C for 10-50 min.
In a preferred embodiment, the base, without particular limitation, may be mentioned potassium hydroxide, sodium hydroxide and the like.
In a preferred embodiment, the pH is adjusted to 7.5-7.6.
Slant inoculation
In the invention, slant inoculation is as follows: dissolving sand spore in sterile water, uniformly coating on slant culture medium, culturing at 28 + -0.5 deg.C for 5-10 days, and storing at 1-8 deg.C to obtain slant spore;
in a preferred embodiment, the inoculation culture time is 6 to 8 days.
In a preferred embodiment, the storage temperature is 4 ± 2 ℃.
In a preferred embodiment, the shelf life of the bevel spores is 60 days.
Spore suspension preparation
In the invention, the spore suspension is prepared by the following steps: adding glass beads into the bevel spores, and shaking to knock down the spores; then adding sterile water, shaking, sealing, and storing at 1-8 deg.C to obtain spore suspension.
In a preferred embodiment, the spore suspension is prepared by: adding glass beads into the slant spore in the inoculation chamber, shaking to remove the spore, adding sterile water, shaking, sealing, and storing in a refrigerator at 2-6 deg.C.
In a preferred embodiment, the spore suspension has a shelf life of 24 hours.
Seeding tank culture
In the invention, the seeding tank culture comprises the following steps: inoculating the spore suspension to a seed culture medium in a seeding tank, then starting stirring, introducing sterile air, and culturing for 30-50h to obtain a culture solution.
In a preferred embodiment, the raw materials for preparing the seed culture medium at least comprise the following components in percentage by weight: 1-4% of glucose, 0.5-2% of corn steep liquor, 0.1-1% of peptone, 0.5-2% of corn starch, 0.5-2% of peanut cake powder, 1-3% of yeast extract, 0.1-1% of soybean oil, 1-2% of calcium carbonate, 0.05-0.3% of ammonium sulfate, 0.05-0.3% of disodium hydrogen phosphate and the balance of water.
In a more preferred embodiment, the raw material for preparing the seed culture medium at least comprises the following components in percentage by weight: 1% of glucose, 1.0% of corn steep liquor, 0.3% of peptone, 1% of corn starch, 0.5% of peanut cake powder, 3.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.1% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water.
In a preferred embodiment, the method for preparing the seed culture medium comprises the following steps:
(1) adding at least half of water into a seeding tank, then adding glucose, corn steep liquor, peptone, corn starch, peanut cake powder, yeast extract, ammonium sulfate and disodium hydrogen phosphate, uniformly stirring, and adjusting the pH value to 7-8 by using alkali;
(2) adding calcium carbonate, and washing the tank wall with the rest water; then adding soybean oil, and closing the tank cover;
(3) heating the seeding tank to 115-130 ℃, controlling the pressure in the tank to be 0.1-0.15MPa, preserving the temperature for 20-50min, and sterilizing;
(4) and (4) after the sterilization is finished, introducing sterile air into the tank, and cooling the seeding tank to 30-40 ℃ to obtain the product.
In a more preferred embodiment, the base, without particular limitation, may be mentioned sodium hydroxide, potassium hydroxide and the like.
In a more preferred embodiment, the pH is 7.5 ± 0.1.
In a preferred embodiment, the heating temperature of the seed can during the sterilization process is 121 ± 2 ℃.
In a more preferred embodiment, the pressure inside the seed tank during the sterilization process is 0.11 ± 0.1 MPa.
On the basis of the original culture medium proportion, the invention adopts an orthogonal experiment to optimize the carbon-nitrogen ratio of the culture medium, and takes the bacterial concentration as an index to obtain the optimal level of each material. Through many times of parallel orthogonal experiments, result analysis shows that the primary and secondary sequence of the influence of the seed bacterial concentration is as follows: glucose, yeast extract, ammonium sulfate, corn steep liquor, peanut cake powder, corn starch and peptone. However, the inventors have unexpectedly found that although glucose has the greatest effect on the bacterial concentration, the bacterial concentration can be increased by decreasing the amount of glucose and increasing the amount of yeast extract in the system, and determined that the optimum combination is 1.0% of corn steep liquor, 0.3% of peptone, 3.0% of yeast extract, 0.5% of peanut meal, 1.0% of corn starch, 1.0% of glucose, 0.1% of ammonium sulfate, 1.4% of calcium carbonate, 0.1% of sodium dihydrogen phosphate, 1% of soybean oil and the balance of water. The inventor believes that the possible reasons are that the seed culture medium has relatively less materials, organic sugar and nitrogen source substances which are easy to consume are required to be quickly consumed in the early stage after inoculation, the carbon source substances such as glucose are reduced in the optimized control and adjustment of the seed culture medium by the system, the seed grows more quickly after a small part of organic quick-acting nitrogen source substances such as yeast extract and peptone are added, the continuous growth metabolism is supplied by supplementing materials, the production level is improved, and the production cost is reduced. Tests prove that the seed concentration can reach 36.0% by using the optimal combination to carry out seed shake flask culture. The optimization of the seed culture medium material of the system not only simplifies the operation of batching, improves the batching efficiency, and reduces the risk of errors in batching, which shows that the bacterial concentration can not be reduced by reducing the content of glucose in the raw materials in the formula.
Cultivation in fermenter
In the invention, the fermentation tank culture comprises the following steps: and (3) inoculating the culture solution onto a fermentation culture medium in a fermentation tank, starting stirring, introducing sterile air, detecting at regular time, and placing the fermentation tank after fermentation culture for 200-260 h.
In a preferred embodiment, the raw materials for preparing the fermentation medium at least comprise the following components in percentage by weight: 1-3% of glucose, 0.05-0.3% of corn steep liquor, 0.5-2% of peptone, 6-8% of long-shaped rice flour, 1.5-3.5% of peanut cake powder, 0.1-1% of soybean cake powder, 0.5-2% of yeast extract, 0.05-0.2% of soybean oil, 0.4-0.5% of sodium chloride, 0.3-0.7% of calcium carbonate, 0.3-0.8% of ammonium sulfate, 0.01-0.05% of amylase, 0.01-0.05% of potassium dihydrogen phosphate, 0.01-0.1% of sodium dihydrogen phosphate and the balance of water.
In a preferred embodiment, the raw materials for preparing the fermentation medium at least comprise the following components in percentage by weight: 1% of glucose, 3% of corn steep liquor, 0.5% of peptone, 7% of long-shaped rice flour, 2.8% of peanut cake powder, 0.8% of soybean cake powder, 1% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.4% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water.
In a preferred embodiment, the method for preparing the fermentation medium comprises the following steps:
(1) adding at least 1/4% water into the fermentation tank, adding indica rice flour and amylase, heating to 55-70 deg.C, and stirring for 10-40 min;
(2) adding soybean cake powder, peanut cake powder, corn steep liquor, yeast extract, glucose, peptone, ammonium sulfate, sodium chloride, calcium carbonate, potassium dihydrogen phosphate and disodium hydrogen phosphate into a fermentation tank, adding the rest water, and stirring uniformly; then adding soybean oil, and closing the tank cover;
(3) heating the fermentation tank to 115 ℃ and 130 ℃, controlling the pressure in the fermentation tank to be 0.1-0.15MPa, and preserving the temperature for 20-50min for sterilization;
(4) and (4) after the sterilization is finished, introducing sterile air into the tank, and cooling the fermentation tank to 30-40 ℃ to obtain the product.
In a preferred embodiment, the heating temperature of the fermenter during the sterilization process is 121 ± 2 ℃.
In a more preferred embodiment, the pressure in the fermenter during the sterilization process is 0.11 ± 0.1 MPa.
In the present invention, the method of introducing the culture solution into the fermentation tank is not particularly limited, and the culture solution can be introduced into the fermentation tank by using a pressure difference.
In a preferred embodiment, the flow rate of the sterile gas is 15-30L/min.
In a preferred embodiment, the fermenter pressure is from 0.04 to 0.06 MPa.
In a preferred embodiment, the fermenter temperature is 35. + -. 1 ℃.
In a preferred embodiment, during the fermentation, the pH, viscosity, tank temperature, tank pressure, air flow, stirring speed are recorded every 6 hours; and sampling and detecting the amount of reducing sugar, total sugar and ammonia nitrogen in the tank, and performing aseptic inspection.
In a preferred embodiment, the bio-titer is measured every 24h starting on day 4 of the fermentation.
In the invention, the fermentation yield of the fermentation culture medium is 128% of that of a control group, and the biological potency of the fermentation culture medium is 13058 u/mL. In addition, the inventor unexpectedly finds that a certain amount of soybean oil is added into the raw materials for preparing the culture medium, which is beneficial to defoaming of fermentation liquor in a tank. The inventor believes that the possible reasons are that foam is easily generated in the fermentation production process, the foam can reduce dissolved oxygen in fermentation liquor, normal metabolism of hyphae is affected, the charging coefficient of a fermentation tank is reduced, the tank discharge volume is reduced, and the tank batch yield is reduced. Therefore, good foam control is an important link for improving the fermentation level. Normally, fermentation often uses antifoams to eliminate foam. However, antifoaming agents are expensive and too much can have adverse effects on fermentation. In the invention, two components of corn steep liquor and soybean oil are directly added, the oil content is high, and the organic carbon/nitrogen source is provided, and simultaneously a certain amount of vegetable oil can be provided, so that the fermentation liquor plays a role in foam inhibition, and the tank placing volume is effectively increased.
In the invention, in the fermentation culture process, the weight ratio of reducing sugar is maintained between 0.5 and 3 percent, and the content of ammonia nitrogen is controlled between 4 and 25 mg/mL.
In a preferred embodiment, according to the utilization condition of reducing sugar and ammonia nitrogen, a certain amount of sugar and ammonia nitrogen need to be supplemented in time, namely sugar supplement and nitrogen supplement, so as to keep the amount of the reducing sugar and the ammonia nitrogen within the process requirement range, and sugar supplement is stopped 6-20h before the tank is placed.
In a preferred embodiment, the sugar feed comprises, in weight percent, at least the following components: 15-25% of indica rice flour, 15-35% of glucose, 0.01-0.1% of zinc sulfate, 0.05-0.5% of magnesium sulfate and water to balance.
In a more preferred embodiment, the sugar feed comprises, in weight percent, at least the following components: 20% of long-shaped rice flour, 20% of glucose, 0.05% of zinc sulfate, 0.2% of magnesium sulfate and water to balance.
In a preferred embodiment, the nitrogen feed is ammonium sulfate.
In a more preferred embodiment, the feeding is performed as fed-batch; the first feeding is 60-100 hours after fermentation, and the sugar feeding is stopped 6-20 hours before tank placing.
The inventor thinks that the traditional fermentation process adopts starch polysaccharide and glucose as carbon source supplement, and the starch polysaccharide has the advantages of low price, but the high-concentration feed liquid can increase the operation difficulty, has high viscosity and is difficult to sterilize thoroughly; glucose is used as a quick-acting carbon source, and pure glucose feeding can cause pH fluctuation and influence the metabolism of thalli. The invention adopts a composite material feeding mode to better solve the technical problems, and particularly, the feeding is started 80 hours after the fermentation is started, so that the fermentation and the synthesis of neomycin can be remarkably promoted. The inventor believes that the possible reason is that the feeding materials can rapidly act on an enzyme system of a neomycin synthesis pathway after fermentation for 80 hours, and the synthesis from neomycin C to neomycin B can be rapidly realized; if the addition is advanced, the hypha growth is affected, and the titer is reduced; the effect of the nutrient is reduced by adding the nutrient after the delay, and the conversion rate of the neomycin C to the neomycin B is reduced, so that the effect cannot be achieved. Meanwhile, the fermentation medium has a nutrient condition for good seed growth, if the fermentation medium is directly added into the culture medium, the components of the early-stage culture medium are changed, the rapid growth of the early-stage seeds during fermentation is not facilitated, the growth and metabolism of the early-stage seeds are adversely affected, and the fermentation titer is low, so that the fed-batch method is required, and otherwise, the titer cannot be improved. Secondly, the amount of the feeding material per flow controlled by the invention is 150-250mL, preferably 200mL, the thallus metabolism can be influenced when the feeding material is excessively added, and the conversion rate can be reduced when the feeding material is excessively added, so that the action effect can not be achieved.
The inventor also finds that magnesium ions can improve the tolerance of aminoglycoside antibiotic producing bacteria to antibiotics produced by the bacteria and improve the yield of the antibiotics, but magnesium ions can form precipitates in an alkaline solution, so that loss is easily caused by directly adding the magnesium ions in ingredients; zinc ions are essential trace elements for fermentation of streptomyces, and can promote hypha growth and antibiotic synthesis at low concentration, and inhibit antibiotic synthesis at high concentration. Therefore, the feeding mode of the invention adopts a small amount of feeding for a plurality of times, can avoid the growth fluctuation caused by sudden overhigh sugar concentration, and can reduce the overhigh concentration of inorganic salts of magnesium ions and zinc ions to inhibit the synthesis of products. In addition, the dosage proportion of fed-batch substances must be controlled, the change of the dosage proportion can affect the titer of the fermentation liquor and the contents of neomycin B and C, and the dosage is more than that, firstly, the saturated titer of nutrient substances can not be improved, secondly, the inhibition and repression effects can be caused, and the titer of the fermentation liquor can not be improved but can be reduced; the dosage is small, the titer of the fermentation liquor can not be improved or the improvement range is very small, so that the strict control of the dosage proportion is very important.
The present invention will be specifically described below by way of examples. It should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and that the insubstantial modifications and adaptations of the present invention by those skilled in the art based on the above disclosure are still within the scope of the present invention.
In addition, the starting materials used are all commercially available, unless otherwise specified.
Examples
Example 1
Example 1 provides a fermentation process for neomycin sulfate, comprising at least the following steps:
(1) preparing a slant culture medium: sterilizing the slant culture medium, shaking, spreading into slant, culturing at 28 + -0.5 deg.C for 3 days, and checking sterility to obtain slant culture medium;
the preparation raw materials of the slant culture medium comprise the following components in percentage by weight: 1% of glucose, 0.25% of beef extract, 0.3% of peptone, 0.18% of corn steep liquor, 0.4% of sodium chloride, 2.6% of agar and the balance of water;
the preparation method of the slant culture medium comprises the following steps:
(a) mixing glucose, beef extract, peptone, corn steep liquor, sodium chloride, agar and water, adjusting pH to 7.5 with sodium hydroxide, packaging, and sealing;
(b) sterilizing with 0.15MPa steam at 120 deg.C for 30 min;
(2) slant inoculation: dissolving sand spore in sterile water, uniformly coating on slant culture medium, culturing at 28 + -0.5 deg.C for 7 days, and storing at 4 deg.C to obtain slant spore;
(3) preparing spore suspension: adding glass beads into the bevel spores, and shaking to knock down the spores; then adding sterile water, shaking, sealing, and storing at 1-8 deg.C to obtain spore suspension;
the spore suspension is prepared by the following steps: taking slant spores, adding glass beads into an inoculation chamber, shaking to remove the spores, adding a proper amount of sterile water, shaking uniformly, sealing, and storing in a refrigerator at 4 ℃ for later use;
(4) seed tank culture: inoculating the spore suspension to a seed culture medium in a seeding tank, then starting stirring, introducing sterile air, and culturing for 40h to obtain a culture solution;
the preparation raw materials of the seed culture medium at least comprise the following components in percentage by weight: 1% of glucose, 1.0% of corn steep liquor, 0.3% of peptone, 1% of corn starch, 0.5% of peanut cake powder, 3.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.1% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water;
the preparation method of the seed culture medium comprises the following steps:
(a) adding at least half of water into a seeding tank, then adding glucose, corn steep liquor, peptone, corn starch, peanut cake powder, yeast extract, ammonium sulfate and disodium hydrogen phosphate, uniformly stirring, and adjusting the pH value to 7.5 +/-0.1 by using sodium hydroxide;
(b) adding calcium carbonate, and washing the tank wall with the rest water; then adding soybean oil, and closing the tank cover;
(c) heating the seeding tank to 122 ℃, controlling the pressure in the tank to be 0.12MPa, preserving the temperature for 30min, and sterilizing;
(d) after the sterilization is finished, introducing sterile air into the tank, and cooling the seeding tank to 350 ℃ to obtain the product;
(5) culturing in a fermentation tank: inoculating the culture solution onto fermentation medium in a fermentation tank, stirring, introducing sterile air, detecting at regular time, fermenting for 250 hr, and placing into the tank;
the preparation raw materials of the fermentation medium comprise the following components in percentage by weight: 1% of glucose, 3% of corn steep liquor, 0.5% of peptone, 7% of long-shaped rice flour, 2.8% of peanut cake powder, 0.8% of soybean cake powder, 1% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.4% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water;
the preparation method of the fermentation medium comprises the following steps:
(1) adding at least 1/4% water into the fermentation tank, adding indica rice flour and amylase, heating to 60 deg.C, and stirring for 20 min;
(2) adding soybean cake powder, peanut cake powder, corn steep liquor, yeast extract, glucose, peptone, ammonium sulfate, sodium chloride, calcium carbonate, potassium dihydrogen phosphate and disodium hydrogen phosphate into a fermentation tank, adding the rest water, and stirring uniformly; then adding soybean oil, and closing the tank cover;
(3) heating the fermentation tank to 122 ℃, controlling the pressure in the fermentation tank to be 0.12MPa, preserving the temperature for 30min, and sterilizing;
(4) after the sterilization is finished, introducing sterile air into the tank, and cooling the fermentation tank to 35 ℃ to obtain the product;
in the fermentation culture process, the weight ratio of reducing sugar is maintained between 0.5 and 3 percent, and the content of ammonia nitrogen is controlled between 4 and 25 mg/mL; according to the utilization conditions of reducing sugar and ammonia nitrogen, a certain amount of sugar and ammonia nitrogen are required to be supplemented in time, namely sugar supplementing and nitrogen supplementing, so as to keep the amount of the reducing sugar and the ammonia nitrogen within the process requirement range, and sugar supplementing is stopped 6-20 hours before the tank is placed; the feeding is carried out in a fed-batch mode; the first feeding is 80 hours after fermentation;
the sugar supplement comprises the following components in percentage by weight: 20% of long-shaped rice flour, 20% of glucose, 0.05% of zinc sulfate, 0.2% of magnesium sulfate and water to make up the balance; the nitrogen supplement is an ammonium sulfate solution.
Example 2
Example 2 differs from example 1 in that the raw materials for preparing the seed culture medium comprise at least the following components in percentage by weight: 1% of glucose, 0.5% of corn steep liquor, 0.3% of peptone, 0.5% of corn starch, 0.5% of peanut cake powder, 1.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.05% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water.
Example 3
Example 3 differs from example 1 in that the raw materials for preparing the seed culture medium comprise at least the following components in percentage by weight: 2% of glucose, 0.5% of corn steep liquor, 0.5% of peptone, 1.0% of corn starch, 1.0% of peanut cake powder, 2.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.1% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water.
Example 4
Example 4 differs from example 1 in that the raw materials for preparing the seed culture medium comprise at least the following components in percentage by weight: 3% of glucose, 0.5% of corn steep liquor, 0.7% of peptone, 1.5% of corn starch, 1.5% of peanut cake powder, 3.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.15% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water.
Example 5
Example 5 differs from example 1 in that the raw materials for preparing the seed culture medium comprise at least the following components in percentage by weight: 3% of glucose, 1.0% of corn steep liquor, 0.3% of peptone, 1.0% of corn starch, 1.0% of peanut cake powder, 1.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.15% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water.
Example 6
Example 6 differs from example 1 in that the raw materials for preparing the seed culture medium comprise at least the following components in percentage by weight: 1.0% of glucose, 1.0% of corn steep liquor, 0.5% of peptone, 1.5% of corn starch, 1.5% of peanut cake powder, 2.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.05% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water.
Example 7
Example 7 differs from example 1 in that the raw materials for preparing the seed culture medium comprise at least the following components in percentage by weight: 2.0% of glucose, 1.0% of corn steep liquor, 0.7% of peptone, 0.5% of corn starch, 0.5% of peanut cake powder, 3.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.1% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water.
Example 8
Example 8 differs from example 1 in that the raw materials for preparing the seed culture medium comprise at least the following components in percentage by weight: 2.0% of glucose, 1.5% of corn steep liquor, 0.3% of peptone, 1.5% of corn starch, 0.5% of peanut cake powder, 2.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.15% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water.
Example 9
Example 9 differs from example 1 in that the raw materials for preparing the seed culture medium comprise at least the following components in percentage by weight: 3.0% of glucose, 1.5% of corn steep liquor, 0.3% of peptone, 0.5% of corn starch, 1.0% of peanut cake powder, 3.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.05% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water.
Example 10
Example 10 differs from example 1 in that the raw materials for preparing the seed culture medium comprise at least the following components in percentage by weight: 1.0% of glucose, 1.5% of corn steep liquor, 0.7% of peptone, 1.0% of corn starch, 1.5% of peanut cake powder, 1.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.1% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water.
Example 11
Example 11 differs from example 1 in that the raw materials for preparing the seed culture medium comprise at least the following components in percentage by weight: 2.0% of glucose, 0.5% of corn steep liquor, 0.3% of peptone, 1.0% of corn starch, 1.5% of peanut cake powder, 3.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.05% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water.
Example 12
Example 12 differs from example 1 in that the raw materials for preparing the seed culture medium comprise at least the following components in percentage by weight: 3.0% of glucose, 0.5% of corn steep liquor, 0.5% of peptone, 1.5% of corn starch, 0.5% of peanut cake powder, 1.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.1% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water.
Example 13
Example 13 differs from example 1 in that the raw materials for preparing the seed culture medium comprise at least the following components in percentage by weight: 1.0% of glucose, 0.5% of corn steep liquor, 0.7% of peptone, 0.5% of corn starch, 1.0% of peanut cake powder, 2.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.15% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water.
Example 14
Example 14 differs from example 1 in that the raw materials for preparing the seed culture medium comprise at least the following components in percentage by weight: 3.0% of glucose, 1.0% of corn steep liquor, 0.3% of peptone, 0.5% of corn starch, 1.5% of peanut cake powder, 2.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.1% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water.
Example 15
Example 15 differs from example 1 in that the raw materials for preparing the seed culture medium comprise at least the following components in percentage by weight: 2.0% of glucose, 1.5% of corn steep liquor, 0.3% of peptone, 1.5% of corn starch, 0.5% of peanut cake powder, 2.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.15% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water.
Example 16
Example 16 differs from example 1 in that the raw materials for preparing the seed culture medium comprise at least the following components in percentage by weight: 2.0% of glucose, 1.0% of corn steep liquor, 0.7% of peptone, 1.5% of corn starch, 1.0% of peanut cake powder, 1.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.05% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water.
Example 17
Example 17 differs from example 1 in that the raw materials for preparing the seed culture medium comprise at least the following components in percentage by weight: 1.0% of glucose, 1.5% of corn steep liquor, 0.3% of peptone, 1.5% of corn starch, 1.0% of peanut cake powder, 3.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.1% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water.
Example 18
Example 18 differs from example 1 in that the raw materials for preparing the seed culture medium comprise at least the following components in percentage by weight: 1.0% of glucose, 1.5% of corn steep liquor, 0.3% of peptone, 1.5% of corn starch, 1.0% of peanut cake powder, 3.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.1% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water.
Example 19
Example 19 differs from example 1 in that the raw materials for preparing the seed culture medium comprise at least the following components in percentage by weight: 3.0% of glucose, 1.5% of corn steep liquor, 0.7% of peptone, 1.0% of corn starch, 0.5% of peanut cake powder, 2.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.05% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water.
Example 20
Example 20 differs from example 1 in that the raw materials for the preparation of the fermentation medium comprise, in weight percent, the following components: 1% of glucose, 0.1% of corn steep liquor, 0.5% of peptone, 7% of long-shaped rice flour, 2.4% of peanut cake powder, 0.3% of soybean cake powder, 3% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.4% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water.
Example 21
Example 21 differs from example 1 in that the raw materials for the preparation of the fermentation medium comprise, in weight percent, the following components: 2% of glucose, 0.1% of corn steep liquor, 1% of peptone, 7% of long-shaped rice flour, 2.8% of peanut cake powder, 0.5% of soybean cake powder, 4% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.6% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water.
Example 22
Example 22 differs from example 1 in that the raw materials for the preparation of the fermentation medium comprise, in weight percent, the following components: 3% of glucose, 0.1% of corn steep liquor, 1.5% of peptone, 7% of long-shaped rice flour, 3.2% of peanut cake powder, 0.8% of soybean cake powder, 5% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.8% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water.
Example 23
Example 23 differs from example 1 in that the starting materials for the preparation of the fermentation medium comprise, in weight percent, the following components: 3% of glucose, 0.2% of corn steep liquor, 0.5% of peptone, 7% of long-shaped rice flour, 2.4% of peanut cake powder, 0.5% of soybean cake powder, 3% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.8% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water.
Example 24
Example 24 differs from example 1 in that the raw materials for the preparation of the fermentation medium comprise, in weight percent, the following components: 1% of glucose, 0.2% of corn steep liquor, 1% of peptone, 7% of long-shaped rice flour, 2.8% of peanut cake powder, 0.8% of soybean cake powder, 4% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.4% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water.
Example 25
Example 25 differs from example 1 in that the raw materials for the preparation of the fermentation medium comprise, in weight percent, the following components: 2% of glucose, 0.2% of corn steep liquor, 1.5% of peptone, 7% of long-shaped rice flour, 3.2% of peanut cake powder, 0.3% of soybean cake powder, 5% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.6% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water.
Example 26
Example 26 differs from example 1 in that the starting materials for the preparation of the fermentation medium comprise, in weight percent: 2% of glucose, 0.3% of corn steep liquor, 0.5% of peptone, 7% of long-shaped rice flour, 2.4% of peanut cake powder, 0.8% of soybean cake powder, 3% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.8% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water.
Example 27
Example 27 differs from example 1 in that the starting materials for the preparation of the fermentation medium comprise, in weight percent: 3% of glucose, 0.3% of corn steep liquor, 1% of peptone, 7% of long-shaped rice flour, 2.8% of peanut cake powder, 0.3% of soybean cake powder, 4% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.4% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water.
Example 28
Example 28 differs from example 1 in that the starting materials for the preparation of the fermentation medium comprise, in weight percent: 1% of glucose, 0.3% of corn steep liquor, 1.5% of peptone, 7% of long-shaped rice flour, 3.2% of peanut cake powder, 0.5% of soybean cake powder, 5% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.6% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water.
Example 29
Example 29 differs from example 1 in that the starting materials for the preparation of the fermentation medium comprise, in weight percent: 2% of glucose, 0.1% of corn steep liquor, 0.5% of peptone, 7% of long-shaped rice flour, 3.2% of peanut cake powder, 0.5% of soybean cake powder, 3% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.4% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water.
Example 30
Example 30 differs from example 1 in that the raw materials for the preparation of the fermentation medium comprise, in weight percent, the following components: 3% of glucose, 0.1% of corn steep liquor, 1% of peptone, 7% of long-shaped rice flour, 2.4% of peanut cake powder, 0.8% of soybean cake powder, 4% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.6% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water.
Example 31
Example 31 differs from example 1 in that the raw materials for the preparation of the fermentation medium comprise, in weight percent, the following components: 1% of glucose, 0.1% of corn steep liquor, 1.5% of peptone, 7% of long-shaped rice flour, 2.8% of peanut cake powder, 0.3% of soybean cake powder, 5% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.8% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water.
Example 32
Example 32 differs from example 1 in that the starting materials for the preparation of the fermentation medium comprise, in weight percent: 3% of glucose, 0.2% of corn steep liquor, 0.5% of peptone, 7% of long-shaped rice flour, 3.2% of peanut cake powder, 0.3% of soybean cake powder, 3% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.6% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water.
Example 33
Example 33 differs from example 1 in that the starting materials for the preparation of the fermentation medium comprise, in weight percent: 1% of glucose, 0.2% of corn steep liquor, 1% of peptone, 7% of long-shaped rice flour, 2.4% of peanut cake powder, 0.5% of soybean cake powder, 4% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.8% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water.
Example 34
Example 34 differs from example 1 in that the starting materials for the preparation of the fermentation medium comprise, in weight percent, the following components: 2% of glucose, 0.2% of corn steep liquor, 1.5% of peptone, 7% of long-shaped rice flour, 2.8% of peanut cake powder, 0.8% of soybean cake powder, 5% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.4% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water.
Example 35
Example 35 differs from example 1 in that the starting materials for the preparation of the fermentation medium comprise, in weight percent: 1% of glucose, 0.3% of corn steep liquor, 0.5% of peptone, 7% of long-shaped rice flour, 2.8% of peanut cake powder, 0.8% of soybean cake powder, 3% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.6% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water.
Example 36
Example 36 differs from example 1 in that the raw materials for the preparation of the fermentation medium comprise, in weight percent, the following components: 2% of glucose, 0.3% of corn steep liquor, 1% of peptone, 7% of long-shaped rice flour, 3.2% of peanut cake powder, 0.3% of soybean cake powder, 4% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.8% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water.
Example 37
Example 37 differs from example 1 in that the starting materials for the preparation of the fermentation medium comprise, in weight percent: 3% of glucose, 0.3% of corn steep liquor, 1.5% of peptone, 7% of long-shaped rice flour, 2.4% of peanut cake powder, 0.5% of soybean cake powder, 5% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.4% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water.
Example 38
Example 38 differs from example 1 in that the sugar feed comprises, in weight percent, the following components: 20% of long-shaped rice flour, 20% of corn starch and the balance of water.
Example 39
Example 39 differs from example 1 in that the sugar feed comprises, in weight percent, the following components: glucose 40% and water to make up the balance.
Performance testing
1. Bacterial concentration test
The principle is as follows: the growth state of the microorganism is reflected by detecting the amount of hyphae contained in a certain amount of culture solution.
The method comprises the following steps: about 30mL of the sample was placed in a 50mL graduated cylinder, a drop of antifoam was added, and the sample volume (V) was recorded. Placing in a centrifuge, centrifuging at 5000rpm for 5 minutes, removing the centrifuge tube, transferring the supernatant to a 50mL measuring cylinder, and recording the volume (V)1)。
And (3) calculating: concentration of fungus (V-V)1)/V*100％；
V: the volume of the culture solution to be measured, Ml;
V1: volume of supernatant, mL.
Examples 1-19 tested the inoculum concentration of the broth after the seeding tank culture.
2. Potency assay (bioassay)
And (3) detecting bacteria: staphylococcus aureus [ CMCC (B) 26003 ].
Preparing bacterial liquid: the slant of the tube was used temporarily, and the lawn was washed with 5ml of 0.9% sterile sodium chloride solution.
Preparation of double dish plate: preparing 110mL of bottom layer culture medium antibiotic verification culture medium II and 60mL of upper layer, shaking up, and sterilizing for 30 minutes at 115 ℃. Pouring the lower layer on the sterilized glass plate while the lower layer is hot after sterilization, and cooling. And (3) cooling the upper culture medium in a water bath to 48-50 ℃, adding 0.1mL of slant bacterial suspension of the staphylococcus aureus, uniformly mixing, immediately pouring onto a glass plate, and uniformly spreading the glass plate on the whole plate. And uniformly placing a sterile dry oxford cup on the cooled upper culture medium. Each plate has six rows and seven columns, and totally has 42 oxford cups.
Intermediate: the fermentation broth was acidified with oxalic acid to pH 3, centrifuged, and the supernatant was diluted to 10u/ml (dT1) and 20u/ml (dT2) with 3% NaCl in pH 7.8 phosphate buffer according to the estimated titer.
And (3) diluting the standard: 20 to 30mg of the standard was weighed out precisely, and diluted to 10u/ml (dS1) and 20u/ml (dS2) with a phosphate buffer solution containing 3% NaCl at pH 7.8.
Dish dropping, culturing and result calculation: respectively adding dS2, dT2, dS1 and dT1 into an oxford cup, covering a cover plate, and culturing in an incubator at 35-37 ℃ for 14-16 hours.
Measuring the bacteriostatic zone: and (3) respectively measuring the diameters of the dS2, dT2, dS1 and dT1 by using a bacteriostatic circle measuring system, wherein the diameters are accurate to 0.001mm, and only the diameters of the middle twenty bacteriostatic circles are measured to eliminate the edge effect by counting one circle around.
Calculating relative titer: the relative potency is the diameter of the bacteriostatic circle of the test sample/the diameter of the bacteriostatic circle of the reference sample x 100%.
And (3) titer calculation: dS2 and dT2 estimated titer (AT) are respectively input into computer calculation software, and R value, PT value and FL% are obtained after calculation. The R value should be between 0.9 and 1.1, and the FL% should not be more than 5%. Otherwise, the AT should be adjusted and then re-checked.
Examples 1, 20-37 the relative titer of the fermentation broth after fermentor culture was calculated. Examples 1, 38-39 calculate the titer of the fermentation broth after the tank discharge. The results of the examples are shown in table 1; "/" indicates that the item was not tested.
TABLE 1 results of the Performance test of examples
The foregoing examples are merely illustrative and serve to explain some of the features of the method of the present invention. The appended claims are intended to claim as broad a scope as is contemplated, and the examples presented herein are merely illustrative of selected implementations in accordance with all possible combinations of examples. Accordingly, it is applicants' intention that the appended claims are not to be limited by the choice of examples illustrating features of the invention. Also, where numerical ranges are used in the claims, subranges therein are included, and variations in these ranges are also to be construed as possible being covered by the appended claims.
Claims (10)
1. A fermentation process of neomycin sulfate is characterized by at least comprising the following steps:
(1) preparing a slant culture medium: sterilizing slant culture medium, shaking, spreading into slant, culturing at 28 + -0.5 deg.C for 1-5 days, and checking for sterility to obtain slant culture medium;
(2) slant inoculation: dissolving sand spore in sterile water, uniformly coating on slant culture medium, culturing at 28 + -0.5 deg.C for 5-10 days, and storing at 1-8 deg.C to obtain slant spore;
(3) preparing spore suspension: adding glass beads into the bevel spores, and shaking to knock down the spores; adding sterile water, shaking, sealing, and storing at 1-8 deg.C to obtain spore suspension;
(4) seed tank culture: inoculating the spore suspension to a seed culture medium in a seeding tank, then starting stirring, introducing sterile air, and culturing for 30-50h to obtain a culture solution;
(5) culturing in a fermentation tank: and (3) inoculating the culture solution onto a fermentation culture medium in a fermentation tank, starting stirring, introducing sterile air, detecting at regular time, and placing the fermentation tank after fermentation culture for 200-260 h.
2. The fermentation process of neomycin sulfate according to claim 1, wherein in step (1), the raw materials for preparing the slant culture medium at least comprise the following components in percentage by weight: 0.5-2% of glucose, 0.01-0.5% of beef extract, 0.1-0.5% of peptone, 0.01-0.3% of corn steep liquor, 0.1-1% of sodium chloride, 1-5% of agar and the balance of water.
3. The fermentation process of neomycin sulfate according to claim 1, wherein in step (4), the raw materials for preparing the seed culture medium at least comprise the following components in percentage by weight: 1-4% of glucose, 0.5-2% of corn steep liquor, 0.1-1% of peptone, 0.5-2% of corn starch, 0.5-2% of peanut cake powder, 1-3% of yeast extract, 0.1-1% of soybean oil, 1-2% of calcium carbonate, 0.05-0.3% of ammonium sulfate, 0.05-0.3% of disodium hydrogen phosphate and the balance of water.
4. The fermentation process of neomycin sulfate according to claim 3, wherein the raw materials for preparing said seed culture medium comprise, in weight percent, at least the following components: 1% of glucose, 1.0% of corn steep liquor, 0.3% of peptone, 1% of corn starch, 0.5% of peanut cake powder, 3.0% of yeast extract, 1% of soybean oil, 1.4% of calcium carbonate, 0.1% of ammonium sulfate, 0.1% of disodium hydrogen phosphate and the balance of water.
5. The fermentation process of neomycin sulfate according to claim 3, wherein said method for preparing a seed culture medium comprises the following steps:
(1) adding at least half of water into a seeding tank, then adding glucose, corn steep liquor, peptone, corn starch, peanut cake powder, yeast extract, ammonium sulfate and disodium hydrogen phosphate, uniformly stirring, and adjusting the pH value to 7-8 by using alkali;
(2) adding calcium carbonate, and washing the tank wall with the rest water; then adding soybean oil, and closing the tank cover;
(3) heating the seeding tank to 115-130 ℃, controlling the pressure in the tank to be 0.1-0.15MPa, preserving the temperature for 20-50min, and sterilizing;
(4) and (4) after the sterilization is finished, introducing sterile air into the tank, and cooling the seeding tank to 30-40 ℃ to obtain the product.
6. The fermentation process of neomycin sulfate according to claim 1, wherein in step (5), the raw materials for preparing the fermentation medium at least comprise the following components in percentage by weight: 1-3% of glucose, 0.05-0.3% of corn steep liquor, 0.5-2% of peptone, 6-8% of long-shaped rice flour, 1.5-3.5% of peanut cake powder, 0.1-1% of soybean cake powder, 0.5-2% of yeast extract, 0.05-0.2% of soybean oil, 0.4-0.5% of sodium chloride, 0.3-0.7% of calcium carbonate, 0.3-0.8% of ammonium sulfate, 0.01-0.05% of amylase, 0.01-0.05% of potassium dihydrogen phosphate, 0.01-0.1% of sodium dihydrogen phosphate and the balance of water.
7. The fermentation process of neomycin sulfate according to claim 6, wherein the raw materials for preparing the fermentation medium comprise at least the following components in percentage by weight: 1% of glucose, 3% of corn steep liquor, 0.5% of peptone, 7% of long-shaped rice flour, 2.8% of peanut cake powder, 0.8% of soybean cake powder, 1% of yeast extract, 0.2% of soybean oil, 0.45% of sodium chloride, 0.5% of calcium carbonate, 0.4% of ammonium sulfate, 0.03% of amylase, 0.03% of potassium dihydrogen phosphate, 0.05% of sodium dihydrogen phosphate and the balance of water.
8. The fermentation process of neomycin sulfate according to claim 6, wherein said fermentation medium is prepared by a process comprising the steps of:
(1) adding at least 1/4% water into the fermentation tank, adding indica rice flour and amylase, heating to 55-70 deg.C, and stirring for 10-40 min;
(2) adding soybean cake powder, peanut cake powder, corn steep liquor, yeast extract, glucose, peptone, ammonium sulfate, sodium chloride, calcium carbonate, potassium dihydrogen phosphate and disodium hydrogen phosphate into a fermentation tank, adding the rest water, and stirring uniformly; then adding soybean oil, and closing the tank cover;
(3) heating the fermentation tank to 115 ℃ and 130 ℃, controlling the pressure in the fermentation tank to be 0.1-0.15MPa, and preserving the temperature for 20-50min for sterilization;
(4) and (4) after the sterilization is finished, introducing sterile air into the tank, and cooling the fermentation tank to 30-40 ℃ to obtain the product.
9. The fermentation process of neomycin sulfate according to claim 1, wherein in step (5), the weight ratio of reducing sugars is maintained between 0.5 and 3% and the content of ammonia nitrogen is controlled between 4 and 25mg/mL during the fermentation culture.
10. Neomycin sulfate, characterized in that it is obtained by a fermentation process of neomycin sulfate according to any of claims 1-9.
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