CN112795487A

CN112795487A - Fermentation medium and fermentation method for producing fusidic acid

Info

Publication number: CN112795487A
Application number: CN201911108883.7A
Authority: CN
Inventors: 范萍; 方丽纳; 张薇; 方一民; 胡坚; 张菊芳
Original assignee: Hangzhou Zhongmei Huadong Pharmaceutical Co Ltd
Current assignee: Hangzhou Zhongmei Huadong Pharmaceutical Co Ltd
Priority date: 2019-11-13
Filing date: 2019-11-13
Publication date: 2021-05-14
Anticipated expiration: 2039-11-13
Also published as: CN112795487B

Abstract

The invention provides a fermentation medium for producing fusidic acid and a method for producing fusidic acid by adopting the fermentation medium to ferment fusarium oxysporum, wherein the fermentation medium comprises a growth factor, preferably trisodium citrate. The fermentation medium and the fermentation method obviously improve the titer of fusidic acid produced by utilizing the fusarium oxysporum, particularly the addition of the trisodium citrate serving as a growth factor, and finally improve the fermentation titer to 7019 mu g/mL.

Description

Fermentation medium and fermentation method for producing fusidic acid

Technical Field

The invention relates to the technical field of biological pharmacy, in particular to a fermentation medium and a fermentation method for producing fusidic acid.

Background

Fusidic acid is also known as fusidic acid, brown mycin or steromycin. The action mechanism of the medicine is to inhibit the biosynthesis of bacterial protein, can be used for treating various infections caused by drug-resistant staphylococcus aureus, such as septicemia, pneumonia, endocarditis, osteomyelitis, skin soft tissue infection and the like, and has good safety, low toxicity and rare anaphylactic reaction. The first time the us came into the market in 6 months in 1984.

The chemical name of fusidic acid is trans-16 alpha-carboxyl-3 beta, 11 beta-dihydroxy-4 beta, 8 beta, 14 alpha-trimethyl-18-demethyl-5 beta, 10 alpha-cholest- (17Z) -17(20), 24-diene-21-acid hemihydrate, and the molecular formula is C₃₁H₄₈O₆The fusidic acid is usually prepared into sodium salt clinically, and the structural formula of the fusidic acid is shown as a formula I.

Fusidic acid is produced by the fungus Alternaria sphaerica (Fusidium coccineum) or certain Cephalosporium spp. Chinese patent application CN101812498A discloses a fermentation production method of fusidic acid, which discloses the following process steps: (1) preparing seed solution from Thielavia (Fusidumcoccineum); (2) sterilizing the fermentation medium, wherein dissolved oxygen DO is corrected to be 0% in the sterilization process; (3) inoculating the seed solution prepared in the step (1) into a sterilized fermentation medium according to the inoculation amount of 10% (v/v) to ferment for 5-8 days to produce fusidic acid; wherein, in the inoculation process, the culture temperature of the fermentation medium is 25-28 ℃, the pH value is controlled to be 6.0-8.0, and the dissolved oxygen DO is corrected to be 100%; in the fermentation process, before 72 hours, the air flow is 0.6-0.8 vvm, the stirring speed is 260-340 rpm, and the dissolved oxygen DO is controlled at 50-80%; after 72 hours, the air flow is 0.2-0.4 vvm, the stirring speed is 340-360 rpm, and the dissolved oxygen DO is controlled at 30-40%; meanwhile, when the concentration of residual sugar is lower than 5g/L in the fermentation process, feeding 80-100 g/L of glucose, controlling the concentration of residual sugar in the fermentation tank within the range of 4-6 g/L, wherein the whole fermentation process is 6 days, and finally measuring the titer of the fusidic acid to be 3027U/mL; at the same time, the embodiment of the application disclosesThe formula of the fermentation medium is as follows: 20g/L of cane sugar, 5g/L of glycerin, 20g/L of cottonseed cake powder, 1g/L of soybean meal, (NH)₄)₂SO₄ 1g/L，KH₂PO₄ 0.2g/L，MgSO₄·7H₂O 0.01g/L，FeCl₃·6H₂O0.01 g/L, pH7.0. The application produces the fusarium oxysporum through multi-stage fermentation by culturing seeds, preparing and inoculating before inoculating, multi-stage dissolved oxygen control and fed-batch fermentation stages, so that the fermentation titer of a secondary metabolite of the strain is improved to 3027U/mL.

Chinese patent application CN101818186A discloses a culture medium for producing fusidic acid by fermentation, disclosing that the fermentation medium contains the following components in concentration: 20g/L of cane sugar, 5g/L of glycerin, 20g/L of cottonseed cake powder, 1g/L of soybean meal, (NH)₄)₂SO₄ 1g/L，KH₂PO₄ 0.2g/L，MgSO₄·7H₂O 0.01g/L，FeCl₃·6H₂O0.01 g/L, solvent is water, pH 7.8. The application selects cheap cottonseed cake meal as an organic nitrogen source, reduces the cost of a culture medium, integrates fermentation process conditions, improves the conversion rate of the fusidic acid, ensures that the whole fermentation culture time is 144 hours, the conversion rate of the fusidic acid reaches 52.7 percent, and does not relate to a material supplementing process in the fermentation process.

Chinese patent application CN109468233A discloses a fusidic acid high-producing strain and a breeding method and application thereof, the name of the fusidic acid high-producing strain is Fusarium coccineum NJWW 0520(Fusidium coccineum NJWW 0520), the invention application carries out stress directed domestication on a fusidic acid producing strain by continuously utilizing DMSO (dimethyl sulfoxide) for a long time, and the fusidic acid high-producing strain is bred. The yield of the fusidic acid produced by the domesticated strain through fermentation is 4932 mu g/mL, is improved by 53.88 percent compared with the yield (3205 mu g/mL) of the original strain, and the application discloses the composition of a fermentation culture medium: 10.0 percent of sucrose, 0.5 percent of gluten powder, 2.0 percent of yeast powder, 1.0 percent of corn steep liquor, 0.5 percent of soybean cake powder and KH₂PO₄ 0.1％，MgSO₄0.05 percent, 0.3 percent of light calcium carbonate and pH 5.9 +/-0.1.

However, the above-mentioned culture medium has the following technical problems: although the above patent application improves the titer of fusidic acid to some extent by changing the composition of the fermentation medium and controlling the process conditions during fermentation, the operation is complicated for industrial production and the fermentation yield is still low. In order to further improve the potency of fusidic acid and better meet the needs of industrial production, more fermentation media and fermentation processes which can replace the prior art need to be found to obtain higher potency.

Disclosure of Invention

The invention provides a fermentation medium for producing fusidic acid, which comprises growth factors.

Preferably, the growth factor is selected from one or more of sodium acetate, sodium propionate, citric acid, trisodium citrate or triammonium citrate. Further preferably, the growth factor is one or a combination of more than two of citric acid, trisodium citrate and triammonium citrate.

Still more preferably, the growth factor is citric acid, trisodium citrate or triammonium citrate.

In one embodiment of the present invention, the growth factor is trisodium citrate.

Preferably, the fermentation medium contains 0.1-0.6g of growth factor per 100 mL.

Further preferably, the fermentation medium contains 0.2-0.6g of growth factor per 100 mL.

In one embodiment of the present invention, the fermentation medium contains 0.4-0.6g/100mL of growth factor.

In one embodiment of the present invention, the fermentation medium contains 0.2-0.4g/100mL of growth factor.

Preferably, the fermentation medium comprises 0.05-0.3g/100mL of inorganic salts. Further preferably, the fermentation medium comprises 0.1-0.3g/100mL of inorganic salts.

In one embodiment of the invention, the fermentation medium comprises 0.2-0.3g/100mL of inorganic salts.

Further preferably, the inorganic salt is selected from one or a combination of more than two of magnesium sulfate, ferrous sulfate, calcium carbonate, zinc sulfate, sodium chloride, potassium chloride and manganese sulfate. Still more preferably, the inorganic salt is selected from one or a combination of magnesium sulfate and calcium carbonate. Most preferably, the inorganic salt is calcium carbonate.

In one embodiment of the invention, the fermentation medium comprises 0.2-0.3g/100mL of calcium carbonate, which is proved to contribute to accumulation of target products, and the fermentation titer is improved by about 40% after 7 days, because the calcium carbonate is added into the medium to neutralize acidic substances generated by glucose metabolism in time, the pH in the optimal strain production range of 5-7 in the whole fermentation process is maintained, and simultaneously, Ca is added into the medium²⁺Has certain regulation effect on the permeability of fungal cell membranes.

Preferably, the weight ratio of the inorganic salt to the growth factor in the fermentation medium is 1:6 to 3: 2.

Further preferably, the weight ratio of the inorganic salt to the growth factor in the fermentation medium is 1:4 to 3: 4.

Preferably, the fermentation medium also comprises a carbon source, a nitrogen source, an antifoaming agent and water which are normally used in the fermentative production of fusidic acid.

The carbon source is used for synthesizing carbon-containing substances and skeletons thereof of the thalli in microbial fermentation and providing energy for microbial metabolism, and the type and concentration of the carbon source are key factors for strain growth and formation of target products.

In one embodiment of the invention, the glucose tolerance of the fusarium is initially researched, and comparison experiments are carried out on various carbon sources, so that different carbon sources make different contributions to the good growth of strains and the generation of anti-carbon sources. The specific scheme is as follows: glucose, sucrose, glycerol, maltose, dextrin, potato starch, soybean oil and corn starch with the same concentration are respectively used as carbon sources, and other components such as nitrogen sources and inorganic salts are unchanged to form different fermentation formulas. The same seed source is inoculated into a fermentation shake flask, the flask is placed for 3, 5 and 7 days of growth for detecting parameters, and other carbon sources can be used for the growth of thalli except that the soybean oil is not suitable for the growth of strains.

Therefore, the carbon source of the present invention includes, but is not limited to, one or a combination of two or more of glucose, sucrose, glycerol, maltose, dextrin, potato starch, and corn starch.

In one embodiment of the present invention, the carbon source is glucose.

The nitrogen source is essential for microbial growth and product synthesis. Is mainly used for synthesizing thallus cell substances (amino acid, protein, nucleic acid and the like) and nitrogen-containing metabolites. The type and concentration of the fermentation nitrogen source are the key factors influencing the fermentation titer.

The nitrogen source of the present invention includes an organic nitrogen source and/or an inorganic nitrogen source; the inorganic nitrogen source includes, but is not limited to, various ammonium salts, nitrates, or aqueous ammonia, etc. The organic nitrogen source includes, but is not limited to, soybean cake powder, cotton seed cake powder, corn steep liquor, yeast powder, silkworm chrysalis powder, peptone or bran, etc.

The defoaming agent of the present invention includes, but is not limited to, foamed enemy.

In one embodiment of the present invention, the fermentation medium comprises a carbon source, a nitrogen source, a growth factor, inorganic salts, a foam and water.

In one embodiment of the invention, the fermentation medium comprises the following components: 8-12g/100mL of glucose, 3-4g/100mL of yeast powder, 0.1-0.3g/100mL of calcium carbonate, 0.2-0.6g/100mL of trisodium citrate, 0.2g/100mL of molinate and the balance of water.

In one embodiment of the invention, the fermentation medium comprises the following components: 10g/100mL of glucose, 3.5g/100mL of Angel yeast powder, 0.2g/100mL of calcium carbonate, 0.4g/100mL of trisodium citrate, 0.2g/100mL of natural killer and the balance of water.

Preferably, the species producing fusidic acid is Thielavia SIIA 06-05-201.

The invention also provides a fermentation method for producing fusidic acid, which comprises seed culture and fermentation culture, wherein the fermentation culture adopts the fermentation culture medium to culture.

Preferably, the seed culture medium used for the seed culture can be any culture medium which can be used for germination, growth and mass propagation of mycelia by spores and enables the mycelia to grow robustly and become strong 'seeds'. Further preferably, the seed medium contains a carbon source, a nitrogen source, inorganic salts, antifoaming agents, and the like.

In one embodiment of the invention, the composition of the seed culture medium comprises 2g/100mL of glucose, 1g/100mL of soybean meal, 1g/100mL of solid corn steep liquor powder, 0.2g/100mL of calcium carbonate, 0.05g/100mL of natural killer and the balance of water.

Preferably, the seed culture is first cultured in a shake flask, followed by inoculation into a seed tank culture.

The shake flask culture method comprises the following steps:

preparing spore suspension, inoculating into shake flask containing seed culture medium, culturing at 25-30 deg.C for 66-80 hr to obtain shake flask seed culture solution; and inoculating the shake flask seed culture solution into a seed tank filled with a seed culture medium for culture at the temperature of 25-30 ℃ for 44-52h to obtain the seed tank culture solution.

Preferably, the culture temperature in the shake flask culture is 27 +/-1 ℃.

Preferably, the spore suspension is inoculated into the shake flask culture medium in an inoculum size of 10⁵-10⁷one/mL, preferably, the inoculation amount is 10⁶one/mL.

Preferably, the seed culture liquid in the shake flask is inoculated into a seed tank filled with a seed culture medium for culture in an inoculation amount of 0.1-0.5% (V/V), and more preferably, the seed culture liquid in the shake flask is inoculated into the seed tank filled with the seed culture medium for culture in an inoculation amount of 0.3% (V/V).

Preferably, the air flow rate in the seeding tank for culture is controlled between 0.8vvm and 1.0 vvm.

In one embodiment of the present invention, the shake flask cultivation step comprises:

washing spores of Fusarium oxysporum with sterile water from the inclined plane to obtain spore suspension; then, the spore suspension was inoculated to a shakerIn the bottle culture medium, the inoculation amount is 10⁶Culturing at 27 deg.C for 66-80 hr under shaking condition to obtain seed culture solution, inoculating the seed culture solution into seed tank containing seed culture medium at 0.3% (V/V) for culturing at 27 deg.C with air flow rate of 0.8-1.0vvm under stirring and aeration for 44-52 hr to obtain seed tank culture solution. Wherein, the culture mediums in the seeding tank and the shaking bottle are seed culture mediums.

The fermentation culture method comprises the following steps: and transferring the culture solution of the seeding tank into a fermentation tank for fermentation culture, wherein the fermentation temperature is 25-30 ℃, and the fermentation period is 7-11 days.

Preferably, the seeding tank culture solution is transferred into the fermentation tank for fermentation culture in an inoculation amount of 5-15% (V/V), and more preferably, the seeding tank culture solution is transferred into the fermentation tank for fermentation culture in an inoculation amount of 10% (V/V).

Preferably, the culture temperature in the fermentation culture is 27 +/-1 ℃.

Preferably, the pressure of the fermentation culture medium is 0.04-0.05 Mpa.

Preferably, the initial air flow rate in the fermentation culture is 0.8-1vvm, and the air flow rate is increased to 1.3-1.5vvm at the beginning of the middle fermentation period. More preferably, the initial dissolved oxygen in the fermentation culture is controlled to be more than 25%, and the dissolved oxygen is maintained to be more than 30% at the beginning of the middle period of the fermentation.

In one embodiment of the present invention, the fermentation culturing step comprises:

cooling the seed tank culture solution, transferring into a fermentation tank with 10% (V/V) inoculum size for fermentation culture at 27 + -1 deg.C under 0.04-0.05Mpa at initial stirring speed of 30Hz and initial air flow rate of 0.8-1vvm, controlling dissolved oxygen to be more than 25%, increasing air flow rate to 1.3-1.5vvm in the middle stage of fermentation to maintain dissolved oxygen at more than 30%, and fermenting for 9-11 days.

In one embodiment of the present invention, the fermentation method comprises the following steps:

washing from PDA slant with sterile waterPreparing spores of Neurospora exigua into spore suspension, inoculating into shake flask containing seed culture medium, and inoculating with inoculum size of 10⁶Performing shake culture in a shaker at 27 deg.C and 200r/min for 66-80 hr to obtain shake flask seed culture solution;

inoculating the shake flask seed culture solution into a seed tank filled with a seed culture medium for culture at the inoculation amount of 0.3% (V/V), wherein the culture temperature is 27 ℃, the air flow is controlled between 0.8-1.0vvm, the stirring and the ventilation are carried out, and the culture time is 44-52h, so as to obtain a seed tank culture solution;

cooling the seed tank culture solution, transferring the cooled seed tank culture solution into a fermentation tank with the inoculation amount of 10% (V/V) for fermentation culture, controlling the fermentation temperature to be 27 +/-1 ℃, the tank pressure to be 0.04-0.05Mpa, the initial stirring speed to be 30Hz, the initial air flow to be 0.8-1vvm, controlling the dissolved oxygen to be more than 25%, starting to improve the air flow to be 1.3-1.5vvm in the middle stage of fermentation, and keeping the dissolved oxygen at more than 30% for 7-11 days.

Preferably, the spores of the fusarium oxysporum are washed on the PDA slant and obtained by culturing the strains of the fusarium oxysporum on the PDA slant.

Preferably, the fermentation method further comprises a feeding step of adding a feeding medium to the fermentation medium on the 4 th day and/or the 6 th day after the start of the fermentation culture, respectively, and continuing the culture.

Further preferably, the feed medium comprises a carbon source and/or a nitrogen source.

In one embodiment of the invention, the feed medium is glucose and/or ammonium sulfate.

In one embodiment of the present invention, the feed medium is glucose and ammonium sulfate.

Preferably, the concentrations of the carbon source and the nitrogen source in the feed medium are 3g/100mL and 0.075g/100mL, respectively, as the concentration of the components in the fermentation medium.

Preferably, the seed medium and the fermentation medium are sterilized before being inoculated and the feed medium is fed to the fermentation medium, and are cooled to the culture temperature (25-30 ℃) after sterilization

Then inoculation or feeding operation is carried out. Further preferably, the sterilization is preferably performed for 30min at the temperature of 120-123 ℃.

The pH value in the whole fermentation process is controlled to be 5.5-8.0, the pH value is slowly increased to about 7.8 along with the consumption of a nitrogen source in the early stage, glucose and ammonium sulfate are supplemented in the middle stage, the pH value is slightly decreased to about 6.5, the carbon source is basically consumed in the later stage of fermentation, the pH value is increased again, and the end point of fermentation is reached.

The invention also provides fusidic acid prepared by the fermentation method.

The invention also provides application of fusidic acid prepared by the fermentation method in antibiosis and treatment of septicemia, pneumonia, endocarditis, osteomyelitis and skin soft tissue infection. Preferably, the antimicrobial is resistant to a major proportion of gram positive bacteria and a minor proportion of gram negative bacteria. More preferably, the antibiotic is staphylococcus aureus, etc.

The invention also provides the use of inorganic salts and citrate in the preparation of a culture medium for the fermentative production of fusidic acid.

Preferably, the citric acid or the salt thereof is selected from one or the combination of citric acid, trisodium citrate or triammonium citrate; the inorganic salt is selected from one or the combination of magnesium sulfate, ferrous sulfate, calcium carbonate, zinc sulfate or manganese sulfate.

Further preferably, the citric acid or the salt thereof is trisodium citrate, and the inorganic salt is calcium carbonate.

The PDA is a Potato Dextrose Agar (Medium), which is a short name for Potato Dextrose Agar culture Medium. The culture medium is a solid culture medium or a semi-synthetic culture medium, and is mainly used for preservation, activation, passage, amplification culture and the like of strains.

The fermentation medium and the fermentation method improve the titer of fusidic acid produced by utilizing the fermentation of the fusarium oxysporum, particularly the addition of growth factors and the application of a feeding process, finally can improve the fermentation titer to 7019 mu g/mL, and prolong the fermentation time. Meanwhile, the inventor verifies through a shake flask, a pilot plant test and a fermentation tank that the production capacity is stable and the fermentation titer is high, so that the method is suitable for industrial mass production and has high productivity.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1: graph of the effect of different yeast powder concentrations on the titer of fusidic acid produced by fermentation, wherein the ordinate is the 7 balance average titer.

FIG. 2: and (3) a curve chart of change of glucose, bacterial concentration and pH in the whole fermentation process.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Each component in the medium formulation in the following examples is a commercially available product.

Example 1

Firstly, the formula of the fermentation medium is determined, and relatively appropriate carbon source, nitrogen source and inorganic salt are selected.

The method is characterized in that the growth of strains and the influence of production resistance are inspected on several commonly used nitrogen sources, and yeast powder is selected as the nitrogen source of the implementation based on the analysis of the viscosity of fermentation liquor, the oxygen transfer process, the growth condition of the strains and the production condition of target products. When the yeast powder is used as a fermentation nitrogen source, the growth speed of the thalli is proper, the bacteria concentration in the whole fermentation process is always kept in a lower state, the fermentation liquid is dilute, the oxygen consumption requirement of the thalli in the fermentation process is met, and the formation of a target product is facilitated. Meanwhile, the microbial concentration is low, and the target product is an intracellular product, so that the microbial concentration is low, the fermentation liquor is dilute, and the later-stage extraction is facilitated. Since the concentration of nitrogen source is also the key to influence the fermentation titer, the results of the concentration gradient investigation of yeast powder are shown in FIG. 1. Meanwhile, attempts to add an inorganic nitrogen source to the fermentation medium showed that the addition of the inorganic nitrogen source had an inhibitory effect on the formation of the target product.

The tolerance of the fusidium to glucose is preliminarily researched, 4%, 6%, 8%, 10% and 12% of glucose is respectively added into a fermentation culture medium for culture, the same conditions (fermentation nitrogen source, inorganic salt and strain source) are adopted, and the fermentation is respectively carried out for 3, 5 and 7 days for bottle placing detection, wherein detection indexes mainly relate to parameters such as thallus concentration, pH, residual glucose, titer and the like.

And (3) displaying a detection result: the concentration of the thalli reaches more than 20 percent in 3 days, the concentration of the thalli in the formulas of 8 percent, 10 percent and 12 percent of glucose is slightly low, the titer is slightly low, and the formation of the target product is slightly slow. By the 5 th day of fermentation, the bacterial concentration of each formula is about 25%, the fermentation titer of the glucose formula with 6-12% is obviously higher than that of the glucose formula with 4%, and according to the detection value of the residual glucose, the glucose is basically consumed by fermenting the glucose formula with 4% for 5 days, so that the requirement of the strain on fast resistance production cannot be maintained. By the 7 th day of fermentation, the positive correlation phenomenon of the potency and the glucose concentration is obvious, but the potency is slightly lower than that of a 10% glucose formula in the 7 th day of 12% glucose, high sugar has slight inhibition effect, and specific potency data are shown in table 1.

TABLE 1 Effect of glucose on growth and resistance of bacteria

The inorganic salt of this example is selected as calcium carbonate, the concentration of calcium carbonate is determined here, other formulations of the fermentation medium are selected as 10g/100mL of glucose, 3.5g/100mL of Angel yeast powder (YP100), 0.2g/100mL of kyotoxin, no material is added in the fermentation process, the temperature is controlled at 27 + -1 ℃ in the fermentation process with 10% (V/V) of seed transfer amount, the pot pressure is controlled at 0.04-0.05Mpa, the stirring speed is 30Hz, the initial air flow is 0.8-1vvm, the dissolved oxygen is controlled to be more than 25% in the whole process, the dissolved oxygen starts to decrease in the middle stage of fermentation, the dissolved oxygen is maintained by increasing the air flow or increasing the stirring speed, the air flow is increased to 1.3-1.5vvm, the dissolved oxygen can be increased to 35%, and the dissolved oxygen is maintained at more than 30% in the later stage. The fermentation culture time is 7 days. The results of the measurements of the effect of different concentrations of calcium carbonate on the fermentation titer are shown in Table 2.

TABLE 2 results of measurements of the influence of calcium carbonate of different concentrations on the fermentation titer

Calcium carbonate concentration	0	0.1	0.2	0.3
					7 days of potency	2045	3247	3526	3502

In summary, it was determined that the fermentation medium formulations, as well as the seed medium formulations and the feed medium formulations of this example are shown in Table 3.

TABLE 3 formulation of each medium in this example

Firstly, preparing a culture medium: the media were prepared according to the concentration composition of table 3, as follows:

seed medium in shake flasks (500 mL): 2g of glucose, 1g of soybean meal, 1g of solid corn steep liquor, 0.2g of calcium carbonate and 0.05g of molinate, wherein the total volume of the components is 100mL by tap water, and the pH value is natural.

Seed medium in seeding tank (500L): 6kg of glucose, 3kg of soybean meal, 3kg of solid corn steep liquor, 600g of calcium carbonate and 150g of natural pH, wherein the amount of the solid corn steep liquor is supplemented to 300L by tap water.

Fermentation medium in fermentor (3500L): 150kg of glucose, 52.5kg of Angel yeast powder YP10052, 3kg of calcium carbonate, 6kg of trisodium citrate and 3kg of natural pH, wherein the balance is 1500L by using tap water.

Feed medium in feed tank (500L): glucose 90kg and ammonium sulfate 2.25kg, and 200L of tap water was replenished.

II, determination of material feeding process

In order to improve the yield, prolong the resistance period and increase the accumulation of the target product, the method of supplementing carbon source, nitrogen source and the like in the middle stage of fermentation is adopted, and the supplementing time, the supplementing content and the supplementing method are determined in the embodiment.

1. Supplementing carbon source

And (3) feeding materials for the first time in 3, 4 and 5 days of fermentation culture, placing bottles in 7 and 8 days of fermentation to detect the content of the target product, and finding that the feeding effect in 4 days is better than that in 3 days and 5 days. Referring to the residual glucose content in the fermentation broth in 3, 4 and 5 days under the condition of no supplementation, the residual glucose content in 3 days is about 6%, the residual glucose content in 4 days is about 4.5% and the residual glucose content in 5 days is about 2%, so that the glucose consumption in the later period can be judged to be fast, and the glucose can be supplemented when the glucose content in the fermentation broth is lower than 5%. And then respectively performing second-grade material supplement on 5 days, 6 days and 7 days, finally determining the second-grade material supplement on the 6 th day of fermentation, and then inspecting the supplement amount of each grade, wherein the supplement amount is appropriate by taking the final titer as an index and matching with factors such as convenience of actual operation and the like, and 3% glucose is supplemented each time.

2. Nitrogen source

The supplement of organic nitrogen source in the later stage of fermentation may result in excessive growth of thallus and is not favorable for the accumulation of secondary metabolic target product, so the supplement amount must be controlled. On the basis of supplementing 3% of glucose, 0.2-0.5% of yeast powder and 0.05-0.1% of ammonium sulfate are respectively added, so that certain effects are achieved, particularly the supplementing effects of 3% of glucose and 0.075% of ammonium sulfate are remarkable, and the titer reaches 6000-doped 7000 mu g/mL after fermentation for 9-11 days. The supplementation of ammonium sulfate provides a small amount of nitrogen source on one hand, and on the other hand, the ammonium sulfate is used as a quick-acting nitrogen source, which is metabolized by the bacteria to generate acidic substances, so that the fermentation pH can be effectively adjusted, the pH is prevented from being always higher than 7.5, and the catabolism of the bacteria to glucose is enhanced, and the bacteria enter a rapid resistance production period.

Finally, the feeding process is determined as follows: feeding was performed on day 4 and day 6 of fermentation with 3% glucose and 0.075% ammonium sulfate. Under this process, the shake flask fermentation level reached 7000. mu.g/mL.

Thirdly, fermentation process:

taking out the Fusarium species for slant culture, wherein the slant culture medium is PDA culture medium, washing off Fusarium spore with sterile water from PDA slant, making into spore suspension, inoculating into 500mL shake flask containing 100mL seed culture medium, and inoculating with the amount of 10⁶Performing shake culture for 77h in a shaking table at 27 ℃ and 200r/min to obtain shake flask seed culture solution; then inoculating the strain into a 500L seeding tank filled with 300L seed culture medium for culture at the temperature of 27 ℃, controlling the air flow between 0.8vvm and 1.0vvm, stirring and ventilating for 46h to obtain a seeding tank culture solution, wherein the inoculation amount of 0.3% (V/V) is used for inoculation; cleaning a fermentation tank, filling a temperature probe and a pH probe, pouring the fermentation medium into a 3500L fermentation tank, sterilizing at 120-123 ℃ for 30min, correcting dissolved oxygen DO to 0% in the sterilization process, cooling to the strain culture temperature, transferring the culture solution of the seeding tank into the fermentation tank in a 10% (V/V) seed transferring amount, controlling the temperature to be 27 +/-1 ℃ in the fermentation process, controlling the tank pressure to be 0.04-0.05MPa, controlling the stirring speed to be 30Hz, controlling the initial air flow to be 0.8-1vvm, controlling the dissolved oxygen to be more than 25% in the whole process, controlling the dissolved oxygen to be reduced in the middle stage of fermentation, maintaining the dissolved oxygen by increasing the air flow or accelerating the stirring speed, increasing the air flow to be 1.3-1.5vvm, increasing the dissolved oxygen to be 35%, and maintaining the dissolved oxygen to be more than 30% in the later stage.

Adding 100L of feed media into the fermentation tank for continuous culture at the fourth and sixth days after the start of the fermentation culture, wherein the concentrations of glucose and ammonium sulfate in the feed media are respectively 3g/100mL and 0.075g/100mL, and the concentration components are concentration components in the fermentation media; the feed medium was solubilized to 200L with water because: the first desirable supplement volume is smaller, so that the effect on thalli and other materials is smaller when the first desirable supplement volume is supplemented into a fermentation tank; the second glucose has an upper solubility limit, which is generally about 50% suitable. Therefore, the glucose concentration is 45% and the ammonium sulfate can be completely dissolved when the selection is made to dissolve up to 200L. The supplementary material can supplement fermentation carbon source and a small amount of nitrogen source, simultaneously can regulate and control fermentation metabolism pH, the pH is gradually increased at the initial stage of fermentation, the fermentation pH begins to be in a descending trend through the supplement of glucose and ammonium sulfate, the pH is maintained between 5.7 and 8.2 in the whole fermentation process, and the fermentation is grown for 11 days.

Finally, fusidic acid titer was determined to be 7019. mu.g/mL. The glucose consumption curve in the fermentation process is shown in figure 2, the glucose consumption in the whole process is basically in positive correlation with the increasing rate of the fermentation titer, the titer daily increasing rate in the early stage of fermentation is 500-.

Example 2

TABLE 4 formulation of each medium in this example

Firstly, preparing a culture medium: the media were prepared according to the concentration composition of table 4, as follows:

fermentation medium in fermentor (3500L): 120kg of glucose, YP 10045 kg of Angel yeast powder, 1.5kg of calcium carbonate, 6kg of trisodium citrate and 3kg of natural pH, wherein the weight of the mixture is supplemented to 1500L by tap water.

The other media were the same as in example 1.

Secondly, fermentation process:

the procedure of example 1 was repeated, except that the fermentation medium formulation was prepared as shown in Table 4.

Finally, fusidic acid titer 6827. mu.g/mL was measured.

Example 3

TABLE 5 formulation of each medium in this example

Firstly, preparing a culture medium: the media were prepared according to the concentration composition of table 5, as follows:

fermentation medium in fermentor (3500L): 180kg of glucose, 60kg of Angel yeast powder (YP100), 4.5kg of calcium carbonate, 6kg of trisodium citrate and 3kg of natural pH, wherein the weight of the mixture is 1500L by supplementing tap water.

The other media were the same as in example 1.

Secondly, fermentation process:

the procedure of example 1 was repeated, except that the fermentation medium formulation was prepared as shown in Table 5.

Finally, the fusidic acid titer was measured to be 6927. mu.g/mL.

Example 4

TABLE 6 formulation of each medium in this example

Firstly, preparing a culture medium: each medium was prepared according to the concentration composition shown in Table 6.

Fermentation medium in fermentor (3500L): 150kg of glucose, 52.5kg of Angel yeast powder YP10052, 3kg of calcium carbonate, 3kg of trisodium citrate and 3kg of natural pH, wherein the balance is 1500L by using tap water.

The other media were the same as in example 1.

Secondly, fermentation process:

the procedure of example 1 was repeated, except that the fermentation medium formulation was prepared as shown in Table 6.

Finally, fusidic acid titer was found to be 6457 μ g/mL.

Example 5

TABLE 7 formulation of each medium in this example

Firstly, preparing a culture medium: each medium was prepared according to the concentration composition shown in Table 7.

Fermentation medium in fermentor (3500L): 150kg of glucose, 52.5kg of Angel yeast powder YP10052, 3kg of calcium carbonate, 9kg of trisodium citrate and 3kg of natural pH, wherein the balance is 1500L by using tap water.

The other media were the same as in example 1.

Secondly, fermentation process:

the procedure of example 1 was repeated, except that the fermentation medium formulation was prepared as shown in Table 7.

Finally fusidic acid titer was found to be 6328 μ g/mL.

Comparative example 1

TABLE 8 formulation of each medium in this comparative example

Firstly, preparing a culture medium: each medium was prepared according to the concentration composition shown in Table 8.

Fermentation medium in fermentor (3500L): 150kg of glucose, 52.5kg of Angel yeast powder YP10052, 3kg of calcium carbonate and 3kg of natural pH, wherein the balance is 1500L by using tap water.

The other media were the same as in example 1.

Secondly, fermentation process:

the procedure of example 1 was repeated, except that the fermentation medium formulation was prepared in accordance with Table 8.

Finally the fusidic acid titer was measured to be 5412. mu.g/mL.

Comparative example 2

TABLE 9 formulation of each medium in this comparative example

Firstly, preparing a culture medium: each medium was prepared according to the concentration composition shown in Table 9.

Fermentation medium in fermentor (3500L): 150kg of glucose, 52.5kg of Angel yeast powder YP10052, 3kg of calcium carbonate, 1.5kg of trisodium citrate and 3kg of natural pH, wherein the weight of the mixture is supplemented to 1500L by tap water.

The other media were the same as in example 1.

Secondly, fermentation process:

the procedure of example 1 was repeated, except that the fermentation medium formulation was prepared in accordance with Table 9.

Finally the fusidic acid titer was measured to be 5684. mu.g/mL.

Comparative example 3

TABLE 10 formulation of each medium in this comparative example

Firstly, preparing a culture medium: each medium was prepared according to the concentration composition shown in Table 10.

Fermentation medium in fermentor (3500L): 150kg of glucose, 52.5kg of Angel yeast powder YP10052, 3kg of calcium carbonate, 12kg of trisodium citrate and 3kg of natural pH, wherein the balance is 1500L by using tap water.

The other media were the same as in example 1.

Secondly, fermentation process:

the procedure of example 1 was repeated, except that the fermentation medium formulation was prepared as shown in Table 10.

Finally, fusidic acid titer was determined to be 5212. mu.g/mL.

Comparative example 4

TABLE 11 respective media formulations in this comparative example

Fermentation medium formula	Same as example 1
		Seed culture medium	Same as example 1
Supplementary culture medium	Is free of
		Material supplementing process	Non-supplementary material

Firstly, preparing a culture medium: each medium was prepared according to the concentration composition shown in Table 11.

No supplement medium.

The other media were the same as in example 1.

Secondly, fermentation process:

taking out the Fusarium species for slant culture, wherein the slant culture medium is PDA culture medium, washing off Fusarium spore with sterile water from PDA slant, making into spore suspension, inoculating into 500mL shake flask containing 100mL seed culture medium, and inoculating with the amount of 10⁶Performing shake culture for 77h in a shaking table at 27 ℃ and 200r/min to obtain shake flask seed culture solution; then inoculating the strain into a 500L seeding tank filled with 300L seed culture medium for culture at 27 ℃, controlling the air flow between 0.8vvm and 1.0vvm, stirring and ventilating for 46h to obtain a seeding tank culture solution; cleaning fermentation tank, charging temperature and pH probe, pouring the fermentation medium into 3500L fermentation tank, sterilizing at 120-123 deg.C for 30min,correcting dissolved oxygen DO to be 0% in the sterilization process, cooling the seed tank, transferring the seed tank culture solution into a fermentation tank in a 10% (V/V) seed transferring amount after cooling, controlling the temperature to be 27 +/-1 ℃ in the fermentation process, controlling the tank pressure to be 0.04-0.05MPa, controlling the stirring speed to be 30Hz, controlling the initial air flow to be 0.8-1vvm, controlling the dissolved oxygen to be more than 25% in the whole process, starting to reduce the dissolved oxygen in the middle fermentation period, maintaining the dissolved oxygen by improving the air flow or accelerating the stirring speed at the moment, improving the air flow to be 1.3-1.5vvm, increasing the dissolved oxygen to be 35%, and maintaining the dissolved oxygen to be more than 30% in the later period. The fermentation culture time is 7 days.

Finally, fusidic acid titer 4215 μ g/mL was measured.

Comparative example 5

TABLE 12 formulation of each medium in this comparative example

Fermentation medium formula	Same as example 1
		Seed culture medium	Same as example 1
Supplementary culture medium	3g/100mL, 2g/100mL or 4g/100mL of glucose, tap water.
		Material supplementing process	Feeding the fermentation tank on the fourth and sixth days of fermentation.

Firstly, preparing a culture medium: each medium was prepared according to the concentration composition shown in Table 12

Feed medium in feed tank (500L): glucose (90 kg) was supplemented to 200L with tap water.

Feed medium in feed tank (500L): 60kg of glucose was supplemented to 200L with tap water.

Feed medium in feed tank (500L): 120kg of glucose was supplemented to 200L with tap water.

The other media were the same as in example 1.

Secondly, fermentation process:

taking out the Fusarium species for slant culture, wherein the slant culture medium is PDA culture medium, washing off Fusarium spore with sterile water from PDA slant, making into spore suspension, inoculating into 500mL shake flask containing 100mL seed culture medium, and inoculating with the amount of 10⁶Performing shake culture for 77h in a shaking table at 27 ℃ and 200r/min to obtain shake flask seed culture solution; then inoculating the strain into a 500L seeding tank filled with 300L seed culture medium for culture at 27 ℃, controlling the air flow between 0.8vvm and 1.0vvm, stirring and ventilating for 46h to obtain a seeding tank culture solution; cleaning a fermentation tank, filling a temperature probe and a pH probe, pouring the fermentation medium into a 3500L fermentation tank, sterilizing for 30min at 120-123 ℃, correcting dissolved oxygen DO to be 0% in the sterilization process, cooling the fermentation tank, transferring the culture solution of the seeding tank into the fermentation tank in a 10% (V/V) seed transferring amount after cooling, controlling the temperature to be 27 +/-1 ℃ in the fermentation process, controlling the tank pressure to be 0.04-0.05MPa, controlling the stirring speed to be 30Hz, controlling the initial air flow to be 0.8-1vvm, controlling the dissolved oxygen to be more than 25% in the whole process, beginning to reduce the dissolved oxygen in the middle stage of fermentation, maintaining the dissolved oxygen by increasing the air flow or accelerating the stirring speed, increasing the air flow to 1.3-1.5vvm, increasing the dissolved oxygen to 35%, and maintaining the dissolved oxygen at the later stage to be more than 30%.

And respectively adding 100L of feed culture medium into the fermentation tank for continuous culture on the fourth day and the sixth day after the start of the fermentation culture, wherein the glucose concentration in the feed culture medium is 3g/100mL, 2g/100mL or 4g/100mL, and the concentration component is the concentration component in the fermentation culture medium, and the fermentation is grown for 11 days.

When the concentration of glucose in the fed medium was 3g/100mL, the final fusidic acid titer was found to be 6412. mu.g/mL. The fusidic acid titer was finally measured to be 6058 μ g/mL when the concentration of glucose in the feed medium was 2g/100mL, and 6250 μ g/mL when the concentration of glucose in the feed medium was 5g/100 mL.

In summary, based on the differences of the fermentation media and feeding processes used in examples 1-5 and comparative examples 1-5, the following conclusions can be drawn:

1. regarding the fermentation medium:

under the same conditions of other culture media and fermentation processes, the fermentation media of examples 1-5 are all added with trisodium citrate as a growth factor, and the fermentation titers of examples 1-5 are 7019. mu.g/mL, 6827. mu.g/mL, 6927. mu.g/mL, 6457. mu.g/mL and 6328. mu.g/mL in this order.

In comparative example 1, trisodium citrate was not added, and the final fermentation titer was 5412. mu.g/mL, so that, comparing examples 1-5 with comparative example 1, the addition of trisodium citrate, a growth factor, at a certain concentration, to the fermentation medium was effective in improving the fermentation titer.

In comparative example 2, the concentration component of trisodium citrate added is 0.1g/100mL, the final fusidic acid titer is 5684 mug/mL, in comparative example 3, the concentration component of trisodium citrate added is 0.8g/100mL, and the final fusidic acid titer is 5212 mug/mL, so that the titer of the fermentation is improved to a certain extent after the growth factor trisodium citrate is added compared with comparative example 2, and the fermentation titer is obviously affected by the different concentrations of the growth factor trisodium citrate added in examples 1-5 compared with comparative example 2 and comparative example 3, and has a certain rule that the fermentation titer is reduced after the growth factor trisodium citrate is added, and the concentration component is preferably controlled within the range of 0.2-0.6g/100mL in a certain range, less than this concentration range, the fermentation titer cannot reach the maximum yet, and above this range, the fermentation titer is also lowered.

2. Regarding the fermentation process:

example 1 was fed to the fermentor on the fourth and sixth days of fermentation with 3g/100mL glucose and 0.075g/100mL ammonium sulfate, and the final fermentation titer of example 1 was 7019. mu.g/mL.

In the fermentation process of comparative example 4, no feeding process was used, and finally the fusidic acid titer was measured to be 4215 μ g/mL, therefore, it can be seen that feeding 3g/100mL of glucose and 0.075g/100mL of ammonium sulfate to the fermentation tank on the fourth and sixth days of fermentation effectively increased the fermentation titer of fusidic acid when comparing example 1 with comparative example 4.

In the fermentation process of comparative example 5, the fermentation tank was fed with glucose at the fourth and sixth days of fermentation, the feed was 3g/100mL, and the final fusidic acid titer was 6412. mu.g/mL, and although the titer was improved as compared with comparative example 4 without feeding, the final titer was significantly lower than example 1 in which glucose at 3g/100mL and ammonium sulfate at 0.075g/100mL were both fed, because only glucose at 3g/100mL was fed and no ammonium sulfate at 0.075g/100mL was fed.

Example 6

Examples 1-5 verify that the addition of trisodium citrate to the fermentation medium has a good effect on the improvement of the fermentation titer, and the examples mainly verify the influence of other growth factors on the fermentation titer.

Specifically, the method comprises the steps of taking 10g/100mL of glucose, YP1003.5g/100 mL of yeast powder, 0.2g/100mL of calcium carbonate, 0.2g/100mL of natural sodium citrate and 0.2g/100mL of natural sodium citrate as basic fermentation culture media, adding different contents of sodium acetate, sodium propionate, citric acid, triammonium citrate or trisodium citrate into the basic fermentation culture media respectively, and fermenting by using a fermentation process of a comparative example 4 (the result is shown in Table 13), wherein the seed culture media are also the same as the comparative example 4. TABLE 13 influence of the addition of different growth factors to the fermentation broth on the fermentation titer

As shown in Table 13, the addition of different concentrations of growth factors to the fermentation medium has a significant impact on the titer of fusidic acid produced by fermentation. In particular, the fermentation medium added with citric acid, trisodium citrate and triammonium citrate has the most obvious improvement on the fermentation titer. More surprisingly, the fermentation medium added with trisodium citrate is adopted for fermentation, in the fermentation process without feeding, the titer can be improved by 15% only by the single action of the trisodium citrate, and the trisodium citrate is monitored in the whole fermentation process, so that the trisodium citrate has an obvious positive regulation and control action in the whole fermentation process.

Example 7

In the embodiment, different proportions of trisodium citrate and calcium carbonate are used as variables, the contents of other components in the fermentation medium are 10g/100mL of glucose, YP1003.5g/100 mL of yeast powder and 0.2g/100mL of natural pH, the influence of different proportions of growth factors and inorganic salts on the fermentation titer is examined, and fermentation is carried out by the fermentation process of comparative example 4 (the result is shown in Table 14), wherein the seed culture medium is also the same as the comparative example 4.

TABLE 14 influence of different ratios of growth factors to inorganic salts on fermentation titers

As can be seen from Table 14, the different ratios of calcium carbonate to trisodium citrate in the fermentation medium significantly affected the fermentation titer, especially when the calcium carbonate to trisodium citrate were 1:4 (0.1 g/100mL of calcium carbonate, 0.4g/100mL of trisodium citrate), 1:3 (0.2 g/100mL of calcium carbonate, 0.6g/100mL of trisodium citrate), 1:2 (0.2 g/100mL of calcium carbonate, 0.4g/100mL of trisodium citrate), 3:4 (0.3 g/100mL of calcium carbonate, 0.4g/100mL of trisodium citrate), and 1:1 (0.2 g/100mL of calcium carbonate, 0.2g/100mL of trisodium citrate), the highest aging titer was observed when the calcium carbonate to trisodium citrate was 1:2 (0.2 g/100mL of calcium carbonate, 0.4g/100mL of trisodium citrate).

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Claims

1. A fermentation medium for the production of fusidic acid, wherein the fermentation medium comprises a growth factor selected from one or a combination of more than two of sodium acetate, sodium propionate, citric acid, trisodium citrate or triammonium citrate; preferably, the growth factor is one or a combination of more than two of citric acid, trisodium citrate or triammonium citrate.

2. The fermentation medium of claim 1, wherein the fermentation medium comprises 0.1-0.6g/100mL growth factor.

3. The fermentation medium of claim 1 or 2, wherein the fermentation medium comprises 0.05-0.3g/100mL of inorganic salts; preferably, the inorganic salt is selected from one or a combination of more than two of magnesium sulfate, ferrous sulfate, calcium carbonate, zinc sulfate or manganese sulfate; further preferably, the inorganic salt is selected from one or a combination of magnesium sulfate and calcium carbonate; most preferably, the inorganic salt is calcium carbonate.

4. The fermentation medium of claim 3, wherein the weight ratio of inorganic salts to growth factors in the fermentation medium is 1:6 to 3: 2; preferably, the weight ratio of the inorganic salt to the growth factor in the fermentation medium is 1:4 to 3: 4.

5. The fermentation medium of any one of claims 1, 2 and 4, wherein the fermentation medium comprises the following components: 8-12g/100mL of glucose, 3-4g/100mL of yeast powder, 0.1-0.3g/100mL of calcium carbonate, 0.2-0.6g/100mL of trisodium citrate and 0.2g/100mL of molinate.

6. The fermentation medium of any one of claims 1, 2 and 4, wherein the fusidic acid-producing species is Thielavia SIIA 06-05-201.

7. A fermentation process for the production of fusidic acid comprising a seed culture, a fermentation culture wherein the fermentation medium comprises a growth factor selected from one or a combination of two or more of sodium acetate, sodium propionate, citric acid, trisodium citrate or triammonium citrate.

8. The fermentation process of claim 7, wherein the seed culture comprises the steps of:

preparing spore suspension, inoculating into shake flask containing seed culture medium, culturing at 25-30 deg.C for 66-80 hr to obtain shake flask seed culture solution;

and inoculating the shake flask seed culture solution into a seed tank filled with a seed culture medium for culture at the temperature of 25-30 ℃ for 44-52h to obtain the seed tank culture solution.

9. The fermentation process of claim 8, wherein the fermentation culture comprises the steps of: and transferring the culture solution of the seeding tank into a fermentation tank for fermentation culture, wherein the fermentation temperature is 25-30 ℃, and the fermentation period is 7-11 days.

10. The fermentation method according to any one of claims 7 to 9, further comprising a feeding step of adding a feed medium to the fermentation medium at the 4 th and/or 6 th day from the start of the fermentation culture, respectively, and continuing the culture.

11. Use of an inorganic salt and a citrate salt in the preparation of a fermentation production medium for fusidic acid, wherein the citric acid or salt thereof is selected from one or a combination of citric acid, trisodium citrate or triammonium citrate; the inorganic salt is selected from one or the combination of magnesium sulfate, ferrous sulfate, calcium carbonate, zinc sulfate or manganese sulfate.

12. Use according to claim 11, characterized in that the citric acid or salt thereof is trisodium citrate and the inorganic salt is calcium carbonate.