CN112795487B

CN112795487B - Fermentation medium and fermentation method for producing fusidic acid

Info

Publication number: CN112795487B
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: 2023-10-20
Anticipated expiration: 2039-11-13
Also published as: CN112795487A

Abstract

The invention provides a fermentation medium for producing fusidic acid and a method for producing fusidic acid by fermenting fusarium by using the fermentation medium, 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 production by utilizing the fusarium, especially the addition of the growth factor trisodium citrate, and finally the fermentation titer can be improved to 7019 mug/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 for producing fusidic acid and a fermentation method.

Background

Fusidic acid is also known as fusidic acid, brown mycin or steromycin. The mechanism of action is to inhibit the biosynthesis of bacterial proteins, and 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 simultaneously has good safety, low toxicity and rare anaphylactic reaction. The first time us was marketed in 6 months of 1984.

Fusidic acid is named as trans-16α -carboxyl-3β,11β -dihydroxy-4β,8β,14α -trimethyl-18-desmethyl-5β,10α -cholest- (17Z) -17 (20), 24-diene-21-acid hemihydrate, and has a molecular formula of C ₃₁ H ₄₈ O ₆ The sodium salt is usually prepared in clinical application, and the structural formula of the fusidic acid is shown as a formula I.

Fusidic acid is produced by a ball shuttle in fungiNeurospora (Fusidium coccineum) or some ceftiella (Cephalosporium spp). Chinese patent application CN101812498A discloses a method for producing fusidic acid by fermentation, which comprises the following steps: (1) Preparing seed solution by using Fusidium (Fusidumcoccineum); (2) Sterilizing the fermentation culture medium, wherein the DO of the dissolved oxygen 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 an inoculum size of 10% (v/v) for fermenting for 5-8 days to produce fusidic acid; wherein, in the inoculation process, the culture temperature of the fermentation culture medium is 25-28 ℃, the pH 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 72h, 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, 80-100 g/L glucose is fed in, the concentration of residual sugar in a fermentation tank is controlled to be in the range of 4-6 g/L, the whole fermentation process is carried out for 6 days, and finally the titer of fusidic acid is measured to be 3027U/mL; meanwhile, the examples of the application disclose the formulation of the fermentation medium as follows: 20g/L sucrose, 5g/L glycerin, 20g/L cottonseed meal, 1g/L soybean meal, (NH) ₄ ) ₂ SO ₄ 1g/L，KH ₂ PO ₄ 0.2g/L，MgSO ₄ ·7H ₂ O 0.01g/L，FeCl ₃ ·6H ₂ O0.01 g/L, pH7.0. The application produces the fusarium by multi-stage fermentation through seed culture, pre-inoculation preparation and inoculation, multi-stage dissolved oxygen control and fed-batch fermentation stages, so that the fermentation titer of the secondary metabolite of the strain is improved to 3027U/mL.

Chinese patent application CN101818186a discloses a medium for producing fusidic acid by fermentation, which comprises the following components in concentration: 20g/L sucrose, 5g/L glycerin, 20g/L cottonseed meal, 1g/L soybean meal, (NH) ₄ ) ₂ SO ₄ 1g/L，KH ₂ PO ₄ 0.2g/L，MgSO ₄ ·7H ₂ O 0.01g/L，FeCl ₃ ·6H ₂ O0.01 g/L, water as solvent, pH 7.8. The application selects the low-price cottonseed cake powder as the organic nitrogen source,the cost of the culture medium is reduced, the fermentation process conditions are synthesized, the conversion rate of the fusidic acid is improved, the whole fermentation culture time is 144 hours, the conversion rate of the fusidic acid reaches 52.7%, and the feeding process is not involved in the fermentation process.

Chinese patent application CN109468233A discloses a fusidic acid high-yield strain named as Fusarium oxysporum NJWW 0520 (Fusidium coccineum NJWW 0520), and a breeding method and application thereof. The output of the domesticated strain for producing fusidic acid by fermentation is up to 4932 mug/mL, which is 53.88% higher than that of the original strain (3205 mug/mL), and the application discloses the composition of a fermentation culture medium: 10.0% of sucrose, 0.5% of gluten powder, 2.0% of yeast powder, 1.0% of corn steep liquor, 0.5% of soybean cake powder and KH ₂ PO ₄ 0.1％，MgSO ₄ 0.05%, light calcium carbonate 0.3% and pH 5.9.+ -. 0.1.

However, the above culture medium has the following technical problems: although the above patent application improves the potency 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 and the fermentation yield is still low for industrial production. To further increase the potency of fusidic acid, better meet the needs of industrial production, more alternative fermentation media and fermentation processes need to be searched for in order to obtain higher potency.

Disclosure of Invention

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

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

Still more preferably, the growth factor is citric acid, trisodium citrate, or tri-ammonium citrate.

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

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

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

In one embodiment of the invention, the fermentation medium comprises growth factors of 0.4-0.6g/100mL.

In one embodiment of the invention, the fermentation medium comprises growth factors of 0.2-0.4g/100mL.

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

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 more 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 a specific embodiment of the invention, the fermentation medium comprises 0.2-0.3g/100mL of calcium carbonate, and the calcium carbonate contributes to the accumulation of target products, the fermentation titer is improved by about 40% in 7 days, because the acid substances generated by glucose metabolism can be timely neutralized by adding the calcium carbonate into the medium, the pH value of the whole fermentation process is maintained in the optimal strain resistance range of 5-7, and meanwhile Ca ²⁺ Has certain regulating effect on fungal cell membrane permeability.

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

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

Preferably, the fermentation medium further comprises a carbon source, a nitrogen source, an antifoaming agent and water which are commonly used for producing fusidic acid by fermentation.

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

In a specific embodiment of the invention, first, preliminary study on glucose tolerance of the fusarium is carried out, and comparison experiments are carried out on various carbon sources, so that different carbon sources are proved to make different contributions to good growth of strains and production 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 used as carbon sources, and other components such as nitrogen sources, inorganic salts and the like are unchanged to form different fermentation formulas. The same seed source is connected into a fermentation shake flask, and the fermentation shake flask grows for 3, 5 and 7 days, and the fermentation shake flask is used for detecting parameters, so that the soybean oil is not suitable for strain growth, and other carbon sources can be used for thallus growth.

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 or corn starch.

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

The nitrogen source is necessary for microbial growth and product synthesis. The method is mainly used for synthesizing cell substances (amino acids, proteins, nucleic acids and the like) of the thalli and nitrogen-containing metabolites. The type and concentration of the fermentation nitrogen source are key to influencing the fermentation titer.

The nitrogen source comprises an organic nitrogen source and/or an inorganic nitrogen source; the inorganic nitrogen source includes, but is not limited to, various ammonium salts, nitrates, ammonia water, 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.

Defoamers described herein include, but are not limited to, dichlord.

In one embodiment of the invention, the fermentation medium comprises a carbon source, a nitrogen source, a growth factor, an inorganic salt, bufomide and water.

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

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 dichlord and the balance of water.

Preferably, the species producing fusidic acid is fusiform S II A06-05-201.

The invention also provides a fermentation method for producing fusidic acid, which comprises seed culture and fermentation culture, wherein the fermentation culture is carried out by adopting the fermentation culture medium.

Preferably, the seed culture medium used for the seed culture can be any culture medium which is used for germination, growth and mass propagation of mycelium and enables the mycelium to grow robustly and become a strong 'seed'. Further preferably, the seed medium comprises a carbon source, a nitrogen source, an inorganic salt, an antifoaming agent, and the like.

In one specific 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 enemy and the balance of water.

Preferably, the seed culture is first cultured in shake flasks and then inoculated into seed pots for culture.

The shake flask culture step provided by the invention comprises the following steps:

preparing spore suspension and inoculating into shake flask containing seed culture medium, culturing at 25-30deg.C for 66-80 hr to obtain shake flask seed culture solution; and (3) inoculating the shake flask seed culture solution into a seed tank filled with a seed culture medium for culture, wherein the culture temperature is 25-30 ℃ and the culture time is 44-52h, and obtaining 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 medium at an inoculum size of 10 ⁵ -10 ⁷ The seed amount is 10 per mL, preferably ⁶ And each mL.

Preferably, the shake flask seed culture solution is inoculated into a seed tank filled with a seed culture medium at an inoculum size of 0.1-0.5% (V/V) for cultivation, and more preferably, the shake flask seed culture solution is inoculated into a seed tank filled with a seed culture medium at an inoculum size of 0.3% (V/V) for cultivation.

Preferably, the air in the culture in the seed tank is controlled between 0.8 and 1.0 vvm.

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

washing spores of the fusarium from the inclined plane by using sterile water to prepare spore suspension; the spore suspension was then inoculated into shake flask medium at an inoculum size of 10 ⁶ Shaking culture is carried out for 66-80h at the temperature of 27 ℃ under the condition of per mL, shake flask seed culture solution is obtained, the shake flask seed culture solution is inoculated into a seed tank filled with seed culture medium for culture at the inoculation amount of 0.3% (V/V), the culture temperature is 27 ℃, the air flow is controlled between 0.8-1.0vvm, stirring and ventilation are carried out, and the culture time is 44-52h, so that the seed tank culture solution is obtained. Wherein, the culture mediums in the seed tank and the shake flask are seed culture mediums.

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

Preferably, the seed tank culture solution is transferred into the fermentation tank for fermentation culture at an inoculum size of 5-15% (V/V), more preferably, the seed tank culture solution is transferred into the fermentation tank for fermentation culture at an inoculum size of 10% (V/V).

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

Preferably, the tank pressure in the fermentation culture is 0.04-0.05Mpa.

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 in the middle period of fermentation. More preferably, the dissolved oxygen is controlled to be more than 25% initially in the fermentation culture, and the dissolved oxygen is maintained to be more than 30% at the beginning of the middle fermentation period.

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

cooling the culture solution of the seed tank, transferring the cooled culture solution into a fermentation tank for fermentation culture at an inoculation amount of 10% (V/V), wherein the fermentation temperature is 27+/-1 ℃, the tank pressure is 0.04-0.05Mpa, the initial stirring speed is 30Hz, the initial air flow is 0.8-1vvm, the dissolved oxygen is controlled to be more than 25%, the air flow is increased to 1.3-1.5vvm at the middle stage of fermentation, the dissolved oxygen is maintained to be more than 30%, and the fermentation period is 9-11 days.

In one embodiment of the invention, the fermentation process comprises the steps of:

washing spores of Thielavia from PDA slant with sterile water to obtain spore suspension, inoculating into shake flask containing seed culture medium, and inoculating with 10% amount ⁶ Shake culturing at 27deg.C in shaking table with rotation speed of 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 according to the inoculation amount of 0.3% (V/V), culturing at 27 ℃, controlling the air flow rate between 0.8 and 1.0vvm, stirring and ventilating, and culturing for 44 to 52 hours to obtain the seed tank culture solution;

cooling the seed tank culture solution, transferring the cooled seed tank culture solution into a fermentation tank for fermentation culture at an inoculum size of 10% (V/V), wherein the fermentation temperature is 27+/-1 ℃, the tank pressure is 0.04-0.05Mpa, the initial stirring speed is 30Hz, the initial air flow is 0.8-1vvm, the dissolved oxygen is controlled to be more than 25%, the air flow is increased to 1.3-1.5vvm at the beginning of the middle fermentation period, the dissolved oxygen is maintained to be more than 30%, and the fermentation period is 7-11 days.

Preferably, the spores of the fusarium on the PDA inclined plane are obtained by culturing the strain of the fusarium on the PDA inclined plane.

Preferably, the fermentation method further comprises a feeding step, wherein the feeding step is to add a feeding culture medium to the fermentation culture medium for continuous culture on the 4 th day and/or the 6 th day of the fermentation 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 invention, the feed medium is glucose and ammonium sulfate.

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

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

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

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

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

The invention also provides application of the fusidic acid prepared by the fermentation method in resisting bacteria, treating septicemia, pneumonia, endocarditis, osteomyelitis and skin soft tissue infection. Preferably, the antibacterial is against a major part of gram-positive bacteria and a minor part of gram-negative bacteria. Further preferably, the antibacterial agent is an antibacterial agent against staphylococcus, staphylococcus aureus, etc.

The invention also provides the application of the inorganic salt and the citrate in preparing culture medium for producing fusidic acid by fermentation.

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

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

The PDA is Potato Dextrose Agar (Medium), namely potato dextrose agar culture Medium for short. The culture medium is a solid culture medium and a semisynthetic culture medium, and is mainly used for preserving, activating, passaging, amplifying and culturing strains and the like.

The fermentation medium and the fermentation method improve the titer of fusidic acid produced by utilizing the fermentation of the fusarium, particularly the addition of the growth factors and the application of a feeding process, and finally can improve the fermentation titer to 7019 mug/mL and prolong the fermentation time. Meanwhile, the inventor passes shake flask, small test, pilot test and fermentation tank verification, and the production capacity is stable, the fermentation titer is high, and the method is suitable for industrial mass production and has higher productivity.

Drawings

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

fig. 1: results of the effect of different yeast powder concentrations on the potency of the fermentative production of fusidic acid, wherein the ordinate is the average potency over 7 days.

Fig. 2: and in the whole fermentation process, glucose, bacteria concentration and pH change are plotted.

Detailed Description

The technical solutions of 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 apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. Each component in the medium formulation in the following examples is commercially available.

Example 1

First, the formulation of the fermentation medium is determined, and a relatively suitable carbon source, nitrogen source and inorganic salt are selected.

The method is characterized in that common nitrogen sources are subjected to strain growth and resistance effect investigation, and yeast powder is selected as the nitrogen source of the implementation based on fermentation broth viscosity, oxygen transfer process, strain growth condition and production condition analysis of target products. When the yeast powder is used as a fermentation nitrogen source, the growth speed of thalli is proper, the concentration of the thalli is always kept in a lower state in the whole fermentation process, the fermentation liquid is dilute, the oxygen consumption requirement of the thalli in the fermentation process is met, and the formation of target products is facilitated. Meanwhile, the bacteria concentration is low, and as the target product is an intracellular product, the bacteria concentration is low, and the fermentation liquor is thin, thereby being beneficial to the later extraction. Since the concentration of the nitrogen source is also a key to influence the fermentation titer, concentration gradient investigation was performed on the yeast powder, and the result is shown in fig. 1. Meanwhile, an attempt to add an inorganic nitrogen source to a fermentation medium shows that the addition of the inorganic nitrogen source has an inhibitory effect on the formation of the objective product.

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

The detection result shows that: the concentration of the thalli reaches more than 20% in 3 days, the concentration of the thalli in the glucose formulations of 8%, 10% and 12% is slightly low, the titer is slightly low, and the formation start 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 a 6-12% glucose formula is obviously higher than that of a 4% glucose formula, and meanwhile, the 4% glucose formula is fermented for 5 days according to the residual glucose detection value, so that the glucose is basically consumed, and the quick production and resistance requirements of the bacterial 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 of 12% glucose in 7 days is slightly lower than that of a 10% glucose formula, and the high sugar has slight inhibition effect, and the specific potency data are shown in Table 1.

TABLE 1 Effect of glucose on cell growth and resistance

The concentration of the inorganic salt in this example was determined by selecting calcium carbonate, the other formulation of the fermentation medium was glucose 10g/100mL, angel yeast powder (YP 100) 3.5g/100mL, papaver 0.2g/100mL, the fermentation process was not fed with feed, the temperature during the fermentation was controlled at 27.+ -. 1 ℃ at 10% (V/V) seed transfer, the pot pressure was controlled at 0.04-0.05MPa, the stirring rate was 30Hz, the initial air flow was 0.8-1vvm, the whole process required to control dissolved oxygen to be greater than 25%, the dissolved oxygen began to decrease during the middle of the fermentation, the dissolved oxygen was maintained by increasing the air flow to 1.3-1.5vvm, the dissolved oxygen was allowed to rise to 35%, and the later period was always maintained at 30% or more. The fermentation culture time is 7 days. The results of the detection of the effect of different concentrations of calcium carbonate on the fermentation titer are shown in Table 2.

TABLE 2 results of detection of the Effect of calcium carbonate at different concentrations on fermentation titers

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

In summary, the fermentation medium formulation, as well as the seed medium formulation and the feed medium formulation, in this example were determined as shown in Table 3.

TABLE 3 Medium formulations in this example

1. Preparing a culture medium: the respective media were prepared according to the concentration components of table 3, specifically as follows:

seed medium in shake flasks (500 mL): glucose 2g, soybean meal 1g, solid corn steep liquor 1g, calcium carbonate 0.2g and dichlord 0.05g, and the pH is natural after the mixture is complemented to 100mL with tap water.

Seed culture medium in seed pot (500L): glucose 6kg, soybean meal 3kg, solid corn steep liquor 3kg, calcium carbonate 600g and dichlord 150g, and the pH is natural after the mixture is supplemented to 300L with tap water.

Fermentation medium in fermentors (3500L): 150kg of glucose, 100.5 kg of Angel Yeast powder YP, 3kg of calcium carbonate, 6kg of trisodium citrate and 3kg of dichlord are supplemented to 1500L by tap water, and the pH is natural.

Feed medium in feed tank (500L): glucose 90kg, ammonium sulfate 2.25kg, was supplemented to 200L with tap water.

2. Determination of the feed Process

In order to improve the yield, prolong the yield-resistance period and increase the accumulation amount of target products, the method of supplementing carbon sources, nitrogen sources and the like in the middle fermentation period is adopted, and the feeding time, the feeding content and the feeding mode are determined in the embodiment.

1. Supplementing carbon source

The first feeding is carried out in 3 days, 4 days and 5 days of fermentation culture respectively, and the target product content is detected after 7 days and 8 days of fermentation, and the feeding effect of 4 days is found to be superior to that of 3 days and 5 days. And referring to the residual glucose content in the fermentation liquid for 3, 4 and 5 days under the condition of no feeding, the residual glucose content is about 6% in 3 days, about 4.5% in 4 days and about 2% in 5 days, the later glucose consumption can be judged to be faster, and the glucose can be fed in when the glucose content in the fermentation liquid is lower than 5%. And then respectively carrying out second-grade feeding on 5 days, 6 days and 7 days, finally determining that the second-grade material is fed into the fermentation on the 6 th day, and then examining the feeding amount of each grade, wherein the feeding amount of each grade is proper by taking the final potency as an index and matching with factors such as convenience of actual operation and the like.

2. Nitrogen source

The organic nitrogen source is fed in the later fermentation period, so that the microbial cells can grow excessively and are unfavorable for accumulation of target secondary metabolic products, and the feeding amount must be controlled. On the basis of supplementing 3% glucose, 0.2-0.5% yeast powder and 0.05-0.1% ammonium sulfate are added respectively, so that the effects are certain, especially the supplementing effect of 3% glucose and 0.075% ammonium sulfate is obvious, and the titer reaches 6000-7000 mug/mL after 9-11 days of fermentation. On the one hand, the ammonium sulfate is supplemented with a small amount of nitrogen source, on the other hand, the ammonium sulfate is taken as a quick-acting nitrogen source, and is metabolized by thalli to generate acidic substances, so that the fermentation pH can be effectively regulated, the pH is prevented from being always higher than 7.5, meanwhile, the catabolism of strains on glucose is enhanced, and the quick-acting period is entered.

The final material supplementing process is as follows: the feed was fed on days 4 and 6 of the fermentation with 3% glucose and 0.075% ammonium sulphate. Under this process, the shake flask fermentation level reached 7000. Mu.g/mL.

3. The fermentation process comprises the following steps:

taking out the streptomyces species for slant culture, wherein the slant culture medium is PDA culture medium, washing spores of the streptomyces from the PDA slant with sterile water after the slant culture is completed, preparing spore suspension, and inoculating into 500mL shake flask filled with 100mL seed culture medium, wherein the inoculating amount is 10 ⁶ Shake culturing in shaking table at 27deg.C and rotation speed of 200r/min for 77 hr/mL to obtain shake flask seed culture solution; then inoculating into 500L seed tank containing 300L seed culture medium at 27deg.C under 0.3% (V/V) inoculum size, and air culturingControlling the flow to be between 0.8 and 1.0vvm, stirring and ventilating, and culturing for 46 hours to obtain a seed tank culture solution; cleaning a fermentation tank, filling a temperature and 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 to the strain culture temperature, transferring the seed tank culture solution into the fermentation tank in a seed transfer amount of 10% (V/V), 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, starting the 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 start to fall in the middle of the fermentation, and maintaining the dissolved oxygen by improving the air flow or accelerating the stirring speed, wherein the air flow is improved to be 1.3-1.5vvm, the dissolved oxygen can be increased to be 35%, and the later period is always maintained to be more than 30%.

Adding 100L of feed medium to the fermentation tank for continuous culture on the fourth day and the sixth day of fermentation culture, wherein the concentration of glucose and ammonium sulfate in the feed medium is 3g/100mL and 0.075g/100mL respectively, and the concentration components are the concentration components in the fermentation medium; the feed medium was dissolved to 200L with water because: the first hope is to supplement the volume smaller, so that the supplementing to the fermentation tank has smaller influence on thalli and other materials; the second glucose has an upper solubility limit, and is preferably about 50%. Therefore, the concentration of glucose was 45L at this time, and ammonium sulfate was completely dissolved. The fed material can supplement fermentation carbon source and a small amount of nitrogen source, and can regulate and control fermentation metabolism pH, the pH at the initial stage of fermentation gradually rises, the fermentation pH starts to be in a descending trend through the feeding of glucose and ammonium sulfate, and the pH is maintained between 5.7 and 8.2 in the whole fermentation process, and the fermentation grows for 11 days.

The final measured fusidic acid titer was 7019 μg/mL. The consumption curve of glucose in the fermentation process is shown in figure 2, the consumption of glucose in the whole process is basically positively correlated with the increasing rate of the fermentation titer, the daily increasing rate of the titer in the early stage of fermentation is 500-700 units, the daily increasing rate of the titer in the middle and later stages of fermentation is 800-1200 units, the fermentation liquor in the whole process is thinner, the concentration of bacteria in the early stage is about 20%, the oxygen transfer in the fermentation process is facilitated, the concentration of bacteria is increased in the middle stage by two times of feeding, the concentration of bacteria is still controlled within 35%, and the fermentation liquor still maintains a thinner state, so that the process of later extraction is facilitated.

Example 2

TABLE 4 Medium formulations in this example

1. Preparing a culture medium: the respective media were prepared according to the concentration components of table 4, specifically as follows:

fermentation medium in fermentors (3500L): 120kg of glucose, 100 kg of Angel yeast powder YP, 1.5kg of calcium carbonate, 6kg of trisodium citrate and 3kg of dichlord, and the mixture was supplemented with 1500L of tap water to a natural pH.

Other media were as in example 1.

2. The fermentation process comprises the following steps:

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

The final fusidic acid titer was measured to be 6827 μg/mL.

Example 3

TABLE 5 Medium formulations in this example

1. Preparing a culture medium: the respective media were prepared according to the concentration components of table 5, specifically as follows:

fermentation medium in fermentors (3500L): 180kg of glucose, 60kg of Angel yeast powder (YP 100), 4.5kg of calcium carbonate, 6kg of trisodium citrate and 3kg of dichlord, and the pH is natural after supplementing 1500L with tap water.

Other media were as in example 1.

2. The fermentation process comprises the following steps:

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

The final fusidic acid titer was found to be 6927 μg/mL.

Example 4

TABLE 6 Medium formulations in this example

1. Preparing a culture medium: each medium was prepared according to the concentration components of Table 6.

Fermentation medium in fermentors (3500L): 150kg of glucose, 100.5 kg of Angel Yeast powder YP, 3kg of calcium carbonate, 3kg of trisodium citrate and 3kg of dichlord are supplemented to 1500L by tap water, and the pH is natural.

Other media were as in example 1.

2. The fermentation process comprises the following steps:

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

The final fusidic acid titer was 6457 μg/mL.

Example 5

TABLE 7 Medium formulations in this example

1. Preparing a culture medium: each medium was prepared according to the concentration components of Table 7.

Fermentation medium in fermentors (3500L): 150kg of glucose, 100.5 kg of Angel Yeast powder YP, 3kg of calcium carbonate, 9kg of trisodium citrate and 3kg of dichlord are supplemented to 1500L by tap water, and the pH is natural.

Other media were as in example 1.

2. The fermentation process comprises the following steps:

example 1 was repeated except that the fermentation medium was prepared in accordance with Table 7.

The final fusidic acid titer was determined to be 6328 μg/mL.

Comparative example 1

Table 8 Medium formulations for this comparative example

1. Preparing a culture medium: each medium was prepared according to the concentration components of Table 8.

Fermentation medium in fermentors (3500L): 150kg of glucose, 100.5 kg of Angel Yeast powder YP, 3kg of calcium carbonate and 3kg of dichlord are supplemented to 1500L by tap water, and the pH is natural.

Other media were as in example 1.

2. The fermentation process comprises the following steps:

example 1 was repeated except that the fermentation medium was prepared as shown in Table 8.

The final fusidic acid titer was determined to be 5412 μg/mL.

Comparative example 2

Table 9 Medium formulations for this comparative example

1. Preparing a culture medium: each medium was prepared according to the concentration components of Table 9.

Fermentation medium in fermentors (3500L): 150kg of glucose, 100.5 kg of Angel Yeast powder YP, 3kg of calcium carbonate, 1.5kg of trisodium citrate and 3kg of dichlord are supplemented to 1500L by tap water, and the pH is natural.

Other media were as in example 1.

2. The fermentation process comprises the following steps:

example 1 was repeated except that the fermentation medium was prepared as shown in Table 9.

The final fusidic acid titer was determined to be 5684 μg/mL.

Comparative example 3

Table 10 Medium formulations for the present comparative example

1. Preparing a culture medium: each medium was prepared according to the concentration components of Table 10.

Fermentation medium in fermentors (3500L): 150kg of glucose, 100.5 kg of Angel Yeast powder YP, 3kg of calcium carbonate, 12kg of trisodium citrate and 3kg of dichlord are supplemented to 1500L by tap water, and the pH is natural.

Other media were as in example 1.

2. The fermentation process comprises the following steps:

example 1 was repeated except that the fermentation medium was prepared in accordance with Table 10.

The final fusidic acid titer was 5212 μg/mL.

Comparative example 4

Table 11 Medium formulations of the present comparative examples

Fermentation medium formula	Same as in example 1
		Seed culture medium	Same as in example 1
Feed medium	Without any means for
		Feed supplement process	Non-supplementing material

1. Preparing a culture medium: each medium was prepared according to the concentration components of Table 11.

No feed medium.

Other media were as in example 1.

2. The fermentation process comprises the following steps:

taking out the streptomyces species for slant culture, wherein the slant culture medium is PDA culture medium, washing spores of the streptomyces from the PDA slant with sterile water after the slant culture is completed, preparing spore suspension, and inoculating into 500mL shake flask filled with 100mL seed culture medium, wherein the inoculating amount is 10 ⁶ Shake culturing in shaking table at 27deg.C and rotation speed of 200r/min for 77 hr/mL to obtain shake flask seed culture solution; inoculating into a 500L seed tank filled with 300L seed culture medium at a seed culture rate of 0.3% (V/V) for culturing at 27deg.C under air flow rate of 0.8-1.0vvm for 46 hr; cleaning a fermentation tank, filling a temperature and 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 seed tank culture solution into the fermentation tank in a 10% (V/V) 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, starting the 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 start to fall in the middle of fermentation, and maintaining the dissolved oxygen by improving the air flow or accelerating the stirring speed, wherein the air flow is improved to be 1.3-1.5vvm, the dissolved oxygen can be increased to be 35%, and the later period is always maintained to be more than 30%. The fermentation culture time is 7 days.

The final measured fusidic acid titer was 4215 μg/mL.

Comparative example 5

Table 12 the medium formulations of this comparative example

Fermentation medium formula	Same as in example 1
		Seed culture medium	Same as in example 1
Feed medium	Glucose 3g/100mL, glucose 2g/100mL or glucose 4g/100mL, tap water.
		Feed supplement process	The fermenter was fed on the fourth and sixth days of fermentation.

1. Preparing a culture medium: the media were prepared according to the concentration components of Table 12

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

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

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

Other media were as in example 1.

2. The fermentation process comprises the following steps:

taking out the streptomyces species for slant culture, wherein the slant culture medium is PDA culture medium, washing spores of the streptomyces from the PDA slant with sterile water after the slant culture is completed, preparing spore suspension, and inoculating into 500mL shake flask filled with 100mL seed culture medium, wherein the inoculating amount is 10 ⁶ Shake culturing in shaking table at 27deg.C and rotation speed of 200r/min for 77 hr/mL to obtain shake flask seed culture solution; inoculating into 500L seed tank containing 300L seed culture medium at 27deg.C under stirring and aeration for 46 hr at air flow rate of 0.8-1.0vvmTo a seed tank culture solution; cleaning a fermentation tank, filling a temperature and PH probe, pouring the fermentation culture 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 seed tank culture solution into the fermentation tank in a seed transfer amount of 10% (V/V) after cooling, controlling the temperature at 27+/-1 ℃ in the fermentation process, controlling the tank pressure to be 0.04-0.05MPa, controlling the stirring speed to be 30Hz, starting the 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 start to fall in the middle of fermentation, and maintaining the dissolved oxygen by improving the air flow or accelerating the stirring speed, wherein the air flow is improved to be 1.3-1.5vvm, the dissolved oxygen can be increased to be 35%, and the later period is always maintained to be more than 30%.

And (3) respectively adding 100L of feed medium to the fermentation tank for continuous culture in the fourth day and the sixth day of the fermentation culture, wherein the glucose concentration in the feed medium is 3g/100mL, 2g/100mL or 4g/100mL, and the concentration components are the concentration components in the fermentation medium, so that the fermentation growth is carried out for 11 days.

When the concentration of glucose in the feed medium was 3g/100mL, the final fusidic acid titer was 6412. Mu.g/mL. The final measured fusidic acid titer was 6058 μg/mL when the concentration of glucose in the feed medium was 2g/100mL, and 6250 μg/mL when the concentration of glucose in the feed medium was 5g/100 mL.

In summary, according to the differences of the fermentation media and the feeding processes used in examples 1 to 5 and comparative examples 1 to 5, the following conclusions were drawn:

1. regarding the fermentation medium:

under the same conditions of other culture mediums and fermentation processes, the fermentation culture mediums in examples 1-5 are added with the growth factor trisodium citrate, and the final measured fermentation titers in examples 1-5 are 7019 mug/mL, 6827 mug/mL, 6927 mug/mL, 6457 mug/mL and 6328 mug/mL in sequence.

Since trisodium citrate is not added in comparative example 1 and the final fermentation titer is 5412. Mu.g/mL, comparing examples 1-5 with comparative example 1, it is known that the fermentation titer can be effectively improved by adding a certain concentration of trisodium citrate as a growth factor to the fermentation medium.

The concentration component of trisodium citrate added in comparative example 2 is 0.1g/100mL, the final measured value of trisodium citrate added in comparative example 3 is 5684 mug/100 mL, the final measured value of trisodium citrate added in comparative example 3 is 0.8g/100mL, therefore, the concentration component is preferably controlled within the range of 0.2-0.6g/100mL, the fermentation titer is not highest in comparison with comparative example 2, the concentration of trisodium citrate added in examples 1-5 is different from that in comparison with comparative example 2 and comparative example 3, the effect on the fermentation titer is remarkable, and the fermentation titer is regular in a certain range, namely, the fermentation titer is reduced after the addition of trisodium citrate is increased, so that the concentration component is preferably controlled within the range of 0.2-0.6g/100mL, the fermentation titer is not highest in comparison with comparative example 2, and the fermentation titer is reduced in comparison with comparative example 3.

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 example 1 had a final fermentation titer of 7019. Mu.g/mL.

In the fermentation process of comparative example 4, there was no feeding process, and thus, the final measured fusidic acid titer was 4215. Mu.g/mL, and thus, example 1 was compared with comparative example 4, and it was found that feeding glucose in an amount of 3g/100mL and ammonium sulfate in an amount of 0.075g/100mL to the fermenter at the fourth and sixth days of fermentation was effective in improving the fusidic acid fermentation titer.

In the fermentation process of comparative example 5, the fermenter was fed with glucose of 3g/100mL on the fourth and sixth days of fermentation, and the final valance of fusidic acid was 6412. Mu.g/mL, which was significantly lower than in example 1 in which glucose of 3g/100mL and ammonium sulfate of 0.075g/100mL were fed at the same time, although the valance was improved as compared with comparative example 4 in which no feed was fed.

Example 6

Examples 1-5 demonstrate that the addition of trisodium citrate to the fermentation medium has a good effect on the improvement of fermentation titer, and this example mainly demonstrates the effect of other growth factors on fermentation titer.

Specifically, the fermentation medium is based on 10g/100mL glucose, 100.5 g/100mL yeast powder YP, 0.2g/100mL calcium carbonate, 0.2g/100mL bufomide and natural pH, and after sodium acetate, sodium propionate, citric acid, tri-ammonium citrate or tri-sodium citrate with different contents are respectively added into the basic fermentation medium, the fermentation process of comparative example 4 is carried out (the result is shown in Table 13), wherein the seed culture medium is also the same as that of comparative example 4. TABLE 13 influence of addition of different growth factors to fermentation Medium on fermentation titers

According to Table 13, the addition of different concentrations of growth factors to the fermentation medium has a great influence on the potency of the fermentation to fusidic acid. Especially, the fermentation medium added with citric acid, trisodium citrate and triammonium citrate has the most obvious improvement on fermentation titer. More surprisingly, the fermentation is carried out by adopting a fermentation medium added with trisodium citrate, 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 obvious positive regulation and control effect in the whole fermentation process.

Example 7

In the embodiment, different proportions of trisodium citrate and calcium carbonate are used as variables, the content of other components of the fermentation medium is 10g/100mL of glucose, 100.5 g/100mL of yeast powder YP, 0.2g/100mL of dichlord and natural pH are adopted to examine the influence condition of different proportions of growth factors and inorganic salts on fermentation titer, and the fermentation process of comparative example 4 is used for fermentation (the result is shown in Table 14), wherein the seed culture medium is the same as that of 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 had a significant effect on the fermentation titer, especially when the ratio of calcium carbonate to trisodium citrate was 1:4 (calcium carbonate 0.1g/100mL, trisodium citrate 0.4g/100 mL), 1:3 (calcium carbonate 0.2g/100mL, trisodium citrate 0.6g/100 mL), 1:2 (calcium carbonate 0.2g/100mL, trisodium citrate 0.4g/100 mL), 3:4 (calcium carbonate 0.3g/100mL, trisodium citrate 0.4g/100 mL), and 1:1 (calcium carbonate 0.2g/100mL, trisodium citrate 0.2g/100 mL), the titer was highest when the ratio was 1:2 (calcium carbonate 0.2g/100mL, trisodium citrate 0.4g/100 mL).

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Claims

1. A fermentation medium for producing fusidic acid from fusarium, comprising the following components: glucose 8-12g/100mL, yeast powder 3-4g/100mL, inorganic salt 0.1-0.3g/100mL, growth factor 0.2-0.6g/100mL, defoamer, wherein,

the growth factor is selected from one or more than two of citric acid, trisodium citrate or tri-ammonium citrate;

the inorganic salt is calcium carbonate.

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

3. The fermentation medium of claim 2, wherein the weight ratio of inorganic salts to growth factors in the fermentation medium is from 1:4 to 3:4.

4. The fermentation medium of claim 1, wherein the antifoaming agent comprises a foam enemy.

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

6. The fermentation medium of any one of claims 1-5, wherein the species producing fusidic acid is fusidic acid S II A06-05-201.

7. A fermentation method for producing fusidic acid by using fusarium, which is characterized by comprising seed culture and fermentation culture, wherein a fermentation medium in the fermentation culture is the fermentation medium of claim 1.

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

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

and (3) inoculating the shake flask seed culture solution into a seed tank filled with a seed culture medium for culture, wherein the culture temperature is 25-30 ℃ and the culture time is 44-52h, and obtaining the seed tank culture solution.

9. The fermentation process of claim 7, wherein the fermentation culture comprises the steps of: transferring the seed tank culture solution into a fermentation tank for fermentation culture at 25-30deg.C for 7-11 days.

10. The fermentation process of claim 9, further comprising a feeding step of continuously adding a feeding medium to the fermentation medium at the 4 th and/or 6 th day of the start of the fermentation culture, respectively.