阅读说明：本技术 一种发酵罐生产两性霉素b的分批补料发酵方法 (Fed-batch fermentation method for producing amphotericin B in fermentation tank ) 是由 柳志强 张博 郭一平 杨晓章 党万利 吴哲明 黄良刚 周俊平 郑裕国 于 2021-09-16 设计创作，主要内容包括：本发明涉及一种发酵罐生产两性霉素B的分批补料发酵方法。本发明分阶段发酵调控策略能够提供结节链霉菌一个最佳的菌体生长和产物合成的外部环境,有利于两性霉素B的积累；采用本发明发酵方法,两性霉素B在温度在31℃,发酵溶氧维持在20％,发酵液残糖控制在0.5～1％时,最终两性霉素B在50吨发酵罐中产量为14g/l左右。(The invention relates to a fed-batch fermentation method for producing amphotericin B in a fermentation tank. The staged fermentation regulation strategy can provide an optimal external environment for growth of the streptomyces tubercle and synthesis of the product, and is beneficial to accumulation of amphotericin B; by adopting the fermentation method, the yield of the amphotericin B in a 50-ton fermentation tank is about 14g/l finally when the temperature of the amphotericin B is 31 ℃, the fermentation dissolved oxygen is maintained at 20 percent, and the residual sugar in the fermentation liquor is controlled at 0.5-1 percent.)

1. A fed-batch fermentation process for producing amphotericin B in a fermentor, said process comprising: inoculating streptomyces tuberculatus to a seed culture medium to obtain a seed solution; inoculating the seed liquid to a fermentation culture medium, and carrying out fermentation culture, wherein the total fermentation time is 120 +/-36 hours, and the parameters in the fermentation process are controlled as follows:

(1) controlling the pH value of the fermentation system to be maintained at 6.5-7.8 until the fermentation is finished;

(2) fermenting and culturing for 0-36h, maintaining the temperature at 31 +/-2 ℃, reducing the temperature to 26 +/-2 ℃ when fermenting for 36h, and maintaining the temperature until the fermentation is finished;

(3) in the early stage of fermentation, the thalli grow under the condition of natural DO, and when the DO value of a fermentation system is reduced to 20% -30%, the DO value is controlled to be maintained to be more than 20% until the fermentation is finished;

(4) controlling the glucose concentration in the fermentation medium, feeding materials at a constant speed when the glucose concentration is reduced to 1-2% in the fermentation process, and controlling the glucose content to be 0.5-1% until the fermentation is finished.

2. The method of claim 1, wherein the Streptomyces nodosus is Streptomyces nodosus CCTCC NO. M2017426.

3. The method of claim 1, wherein the fermenter capacity is 500L to 10000L.

4. The method of claim 1, wherein said seed medium consists of: 10-30 g/L of peptone, 5-20 g/L of yeast extract, 1-10 g/L of sodium chloride, 5-20 g/L of glucose, 0.5-2 g/L of calcium carbonate, water as a solvent and pH of 7.0-7.2.

5. The method of claim 1, wherein the fermentation medium consists of: 80-100 g/L of glucose, 30-50 g/L of soybean meal, 5-10 g/L of sodium citrate, 0.5-5 g/L of magnesium sulfate, 0.05-1 g/L of manganese sulfate, 0.05-1 g/L of calcium chloride, 5-10 g/L of calcium carbonate, 20000.5-5 g/L of PPG, water as a solvent and natural pH.

6. The method of claim 1, wherein the pH is controlled during the fermentation with 30. + -. 2% strength by mass of aqueous ammonia.

(I) technical field

The invention relates to a fed-batch fermentation method for producing amphotericin B in a fermentation tank.

(II) background of the invention

Amphotericin B (AmB) is an important antifungal antibiotic and has been the first choice for deep fungal infections since the time of 1966. The pure product of the AmB is yellow or orange, and the solubility of the AmB in a water phase and most of organic phases is extremely low due to the asymmetric distribution of hydrophilic groups and hydrophobic groups in the molecular structure and the characteristic of acid-base property. AmB has wide antifungal spectrum and strong activity, has strong antifungal effect on most deep fungi such as candida, cryptococcus, mucor, aspergillus, histoplasma, coccidioidomycosis and the like, can effectively inhibit candida and is used for treating leishmaniasis and the like, so that amphotericin B and derivatives thereof are still widely applied.

At present, the industrial production of amphotericin B mainly adopts a microbial fermentation method, and the level of antibiotics produced by microbial fermentation is related to the genetic characteristics of strains, fermentation medium components, fermentation conditions and fermentation process control. The former has mutation breeding and molecular modification means, and the latter obtains the most suitable fermentation medium formula and fermentation culture condition mainly through optimization. At present, the development of amphotericin B is limited by the problems of low yield and slow development of industrial production of amphotericin B, and conditions for expanding culture of amphotericin B are optimized, and especially the optimization of fermentation process conditions of a large fermentation tank has very important significance for industrial production.

Disclosure of the invention

The invention aims to provide a fermentation method for improving the synthetic amount of Escherichia coli L-cysteine.

The technical method adopted by the invention is as follows:

a fed-batch fermentation process for producing amphotericin B in a fermentor, said process comprising: inoculating streptomyces tuberculatus to a seed culture medium to obtain a seed solution; inoculating the seed liquid to a fermentation culture medium, and carrying out fermentation culture, wherein the total fermentation time is 120 +/-36 hours, and the parameters in the fermentation process are controlled as follows:

(1) controlling the pH value of the fermentation system to be maintained at 6.5-7.8 until the fermentation is finished;

(2) fermenting and culturing for 0-36h, maintaining the temperature at 31 +/-2 ℃, reducing the temperature to 26 +/-2 ℃ when fermenting for 36h, and maintaining the temperature until the fermentation is finished;

(3) in the early stage of fermentation, the thalli grow under the condition of natural DO, and when the DO value of a fermentation system is reduced to 20% -30%, the DO value is controlled to be maintained to be more than 20% until the fermentation is finished;

controlling the concentration of glucose in the fermentation medium, feeding materials at a constant speed when the concentration is reduced to 1-2% in the fermentation process, and controlling the content of glucose to be 0.5-1% until the fermentation is finished. And detecting the residual sugar concentration in real time every 12h, and supplementing liquid sugar according to the residual sugar concentration.

According to the invention, the fermentation process of amphotericin B in the fermentation tank is optimized, so that the possible problems of amphotericin B in the process of enlarged culture can be known, and the feed supplement can relieve the repression of substrate inhibition, product feedback inhibition and catabolite; the influence caused by the mass growth of cells due to excessive feeding at one time in batch fermentation can be avoided, and the rheological property of fermentation is improved; can be used as a means of controlling cell quality to increase the proportion of germinated spores. The fed-batch regulation of the glucose avoids the problems that the concentration of the glucose is not uniform in the whole fermentation process, and a large amount of glucose is supplemented at one time, which is not beneficial to the growth and metabolism of the thalli. In addition, high sugar content can generate acidic substances, such as lactic acid and the like, which causes the growth of thalli and the reduction of pH; the low sugar content and insufficient nutrition cause the growth of thalli. The thalli is not long or grows slowly, so that the normal metabolic pathway in the biosynthesis process of amphotericin B can be broken, pH change is caused, more complex byproducts are finally formed, and the separation and purification at the later stage are not facilitated. The feeding control method can ensure the relatively stable concentration of glucose in the fermentation liquor, so that each stage of thallus growth can keep the optimal production state, and the yield of amphotericin B is effectively improved.

Preferably, the streptomyces nodorusis streptomyces nodoruscctcc No. m 2017426, disclosed in CN 110564718A.

Specifically, the capacity of the fermentation tank is 500L-10000L.

The seed culture medium comprises the following components: 10-30 g/L of peptone, 5-20 g/L of yeast extract, 1-10 g/L of sodium chloride, 5-20 g/L of glucose, 0.5-2 g/L of calcium carbonate, water as a solvent and pH of 7.0-7.2.

The fermentation medium comprises the following components: 80-100 g/L of glucose, 30-50 g/L of soybean meal, 5-10 g/L of sodium citrate, 0.5-5 g/L of magnesium sulfate, 0.05-1 g/L of manganese sulfate, 0.05-1 g/L of calcium chloride, 5-10 g/L of calcium carbonate, 20000.5-5 g/L of PPG, water as a solvent and natural pH.

Specifically, ammonia water with mass concentration of 30 +/-2% is used for controlling the pH value in the fermentation process.

The invention has the following beneficial effects: according to the invention, an optimal external environment for growth of the streptomyces tubercle and synthesis of the product can be provided through a staged fermentation regulation strategy, so that the accumulation of amphotericin B is facilitated; meanwhile, the feeding regulation and control avoids the problems that nutrient substances are consumed in the fermentation process and necessary supplement cannot be obtained in the later period through feeding of the feeding culture medium and the exogenous compound. Meanwhile, unnecessary byproducts in the fermentation process are reduced by controlling dissolved oxygen, residual sugar and PH, so that the yield of amphotericin B can be increased. According to the method of the present invention, an amphotericin B yield of 14g/L could be obtained in a 50 ton fermentor.

(IV) description of the drawings

FIG. 1 is a fermentation curve of a 5 ton fermentor at a fermentation temperature of 30 ℃ showing the yield of AMB and the ratio of AMA to AMB.

FIG. 2 is a fermentation curve at a fermentation temperature of 26 ℃ showing AMB production and the ratio of AMA to AMB.

FIG. 3 is a fermentation curve for a 5 ton fermentor without ph control showing AMB production, the ratio of AMA to AMB and the trend of ph.

FIG. 4 is a fermentation curve of a 5 ton fermentor with controlled dissolved oxygen of 10% or more, showing the AMB yield, the ratio of AMA to AMB and the trend of the dissolved oxygen.

FIG. 5 is a fermentation curve of a 5 ton fermentor with controlled dissolved oxygen of 20% or more, showing the AMB yield, the ratio of AMA to AMB and the trend of the dissolved oxygen.

FIG. 6 is a fermentation curve of a 5 ton fermentor with controlled dissolved oxygen of 30% or more, showing the AMB yield, the ratio of AMA to AMB and the trend of the dissolved oxygen.

FIG. 7 is a fermentation curve of a 5 ton fermentor with a residual sugar control of 2.0% or more, showing the AMB yield, the ratio of AMA to AMB and the trend of the residual sugar.

FIG. 8 is a fermentation curve of a 5 ton fermentor with residual sugar controlled at 0.5-1%, showing AMB yield, AMA to AMB ratio and the trend of residual sugar.

FIG. 9 is a graph showing the temperature, dissolved oxygen, ph and residual sugar profiles of fermentation in a 50 ton fermentor, showing the temperature, dissolved oxygen, ph and residual sugar profiles during fermentation.

FIG. 10 is the yield of the 50 ton fermentor from the fermentation curve of FIG. 9 showing the yield of AMB, the ratio of AMA to AMB.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

comparative example:

the strain used in the embodiment of the invention is Streptomyces nodosus ZDB 2016050(CCTCC No: M2017426) and comes from the China center for type culture collection.

The primary and secondary seed culture media comprise the following components: 15g/L of peptone, 10g/L of yeast extract, 5g/L of sodium chloride, 10g/L of glucose, 1g/L of calcium carbonate, distilled water as solvent, 7.0 pH value adjusted by NaOH, and 30min of sterilization at 115 ℃.

The fermentation medium comprises the following components: 100g/L glucose, 50g/L soybean meal, 10g/L sodium citrate, 5g/L magnesium sulfate, 1g/L manganese sulfate, 1g/L anhydrous calcium chloride, 10g/L calcium carbonate, PPG 20005 g/L, distilled water as a solvent, natural pH value and steam sterilization.

In the fermentation process, ammonia water is added to adjust the pH value, and the mass percentage concentration of the ammonia water is 30%.

A supplemented medium: liquid sugar (30% glucose solution, 300g/L)

The fermentation production method of amphotericin B comprises the following steps:

(1) streptomyces nodosus ZJB 50 seed culture:

taking a ring belt gray spore colony from a GYM slant culture medium, inoculating the ring belt gray spore colony into a bottle of 250mL triangular flask containing 50mL seed culture medium, culturing at 26 ℃ and at a shaking table rotating speed of 200r/min for 2 days to obtain a first-grade seed solution; after 2 days, transferring the strain to a 500mL triangular flask containing 100mL seed culture medium with the inoculation amount of 2%, culturing at 26 ℃ at the rotation speed of a shaking table of 200r/min for 2 days to obtain a secondary seed solution.

(2) Streptomyces nodosus ZJB 50 fermentation culture:

inoculating the secondary seed liquid into a 5000L fermentation tank at an inoculation amount of 10%, wherein the initial fermentation liquid is 3000L, and the fermentation liquid is 1500m at 31 deg.C and 100r/min³Culturing for 5 days under the condition of ventilation volume per hour, feeding materials at a constant speed of 1.5g/L/h after the glucose concentration is reduced to 20g/L in the fermentation, and detecting the glucose concentration in real time every 12 hours. The mycelium quantity is obviously increased, the PMV is more than 50% when the mycelium is placed in a pot, the shape of the mycelium is straight, and the mycelium is thick and strong, which proves that the mycelium is rapidly and vigorously produced at the temperature. The fermentation is finished when the fermentation culture lasts for 96 hours, sampling is carried out to detect the AMB content and AMA, the result is shown in figure 1, the AMB content is 9.43g/L and the amphotericin A/B content is 1.1 percent by using a high performance liquid chromatography, the titer is slowly increased after the fermentation lasts for 60 hours, and the titer is not increased basically when the fermentation lasts for about 90 hours. At this temperature, growth of Streptomyces nodularis was demonstrated to be appropriate and the mycelium growth was rapid, but higher temperatures were detrimental to the production of the amphotericin B product at later stages of fermentation.

Example 1:

the primary and secondary seed culture media comprise the following components: 15g/L of peptone, 10g/L of yeast extract, 5g/L of sodium chloride, 10g/L of glucose, 1g/L of calcium carbonate, distilled water as solvent, 7.0 pH value adjusted by NaOH, and 30min of sterilization at 115 ℃.

The fermentation medium comprises the following components: 100g/L glucose, 50g/L soybean meal, 10g/L sodium citrate, 5g/L magnesium sulfate, 1g/L manganese sulfate, 1g/L anhydrous calcium chloride, 10g/L calcium carbonate, PPG 20005 g/L, distilled water as a solvent, natural pH value and steam sterilization.

In the fermentation process, ammonia water is added to adjust the pH value, and the mass percentage concentration of the ammonia water is 30%.

A supplemented medium: liquid sugar (30% glucose solution, 300g/L)

The fermentation production method of amphotericin B comprises the following steps:

(1) streptomyces nodosus ZJB 50 seed culture:

taking a ring belt gray spore colony from a GYM slant culture medium, inoculating the ring belt gray spore colony into a bottle of 250mL triangular flask containing 50mL seed culture medium, culturing at 26 ℃ and at a shaking table rotating speed of 200r/min for 2 days to obtain a first-grade seed solution; after 2 days, transferring the strain to a 500mL triangular flask containing 100mL seed culture medium with the inoculation amount of 2%, culturing at 26 ℃ at the rotation speed of a shaking table of 200r/min for 2 days to obtain a secondary seed solution.

(2) Streptomyces nodosus ZJB 50 fermentation culture:

inoculating the secondary seed liquid into a 5000L fermentation tank at an inoculation amount of 10%, wherein the initial fermentation liquid is 3000L, and the fermentation liquid is 1500m at 26 deg.C and 100r/min³The culture was carried out for 5 days under the condition of ventilation amount per hour, and the glucose concentration was measured in real time every 12 hours in the feeding manner with reference to the control example. The mycelium amount is obviously reduced, PMV is 39% when the mycelium is placed in a pot, the shape of the mycelium is bent, the mycelium is thin and short, and the growth of the mycelium is slow at the temperature. When the fermentation is finished when the fermentation is cultured for 96 hours, sampling is carried out to detect the AMB content and AMA, and the result is shown in figure 2, the AMB content is 8.21g/L and the amphotericin A/B content is 0.59 percent by using a high performance liquid chromatography, compared with a control group, the by-product is reduced, but the yield is also reduced by 13 percent. The streptomyces nodorum mycelium grows slowly in the whole fermentation process becauseThe slow growth of the mycelium eventually leads to a significant decrease in the amount of amphotericin B produced during fermentation. The mycelium is finer and shorter than normal, and the mycelium begins to break at the later stage of fermentation. It was demonstrated that at this temperature, growth of S.nodularis is not suitable, and that amphotericin B production is significantly lower due to poor mycelium development.

Example 2:

the primary and secondary seed culture media comprise the following components: 15g/L of peptone, 10g/L of yeast extract, 5g/L of sodium chloride, 10g/L of glucose, 1g/L of calcium carbonate, distilled water as solvent, 7.0 pH value adjusted by NaOH, and 30min of sterilization at 115 ℃.

The fermentation medium comprises the following components: 100g/L glucose, 50g/L soybean meal, 10g/L sodium citrate, 5g/L magnesium sulfate, 1g/L manganese sulfate, 1g/L anhydrous calcium chloride, 10g/L calcium carbonate, PPG 20005 g/L, distilled water as a solvent, natural pH value and steam sterilization.

In the fermentation process, ammonia water is added to adjust the pH value, and the mass percentage concentration of the ammonia water is 30%.

A supplemented medium: liquid sugar (30% glucose solution, 300g/L)

The fermentation production method of amphotericin B by a staged temperature regulation strategy comprises the following steps:

(1) streptomyces nodosus ZJB 50 seed culture:

taking a ring belt gray spore colony from a GYM slant culture medium, inoculating the ring belt gray spore colony into a bottle of 250mL triangular flask containing 50mL seed culture medium, culturing at 26 ℃ and at a shaking table rotating speed of 200r/min for 2 days to obtain a first-grade seed solution; after 2 days, transferring the strain to a 500mL triangular flask containing 100mL seed culture medium with the inoculation amount of 2%, culturing at 26 ℃ at the rotation speed of a shaking table of 200r/min for 2 days to obtain a secondary seed solution.

(2) Streptomyces nodosus ZJB 50 fermentation culture:

inoculating the secondary seed liquid into a 5000L fermentation tank with 10% inoculum size, wherein the initial fermentation liquid is 3000L, the initial fermentation condition is 100r/min, and 1500m³Per hour ventilation, feed mode reference control, glucose concentration was measured in real time every 12 hours. In the hairThe early stage of fermentation is 0-36h, the temperature is controlled to be 31 ℃, and the fermentation temperature is controlled to be 26 ℃ after the late stage of fermentation is 36h until the end of fermentation. The ph was not controlled for natural fermentation. And (3) ending fermentation at the 90 th hour of fermentation culture, sampling and detecting the AMB content and AMA, wherein the results are shown in figure 3, the AMB content is 10.2g/L, the amphotericin A/B is 6.3 percent and the final ph is 3.6 measured by high performance liquid chromatography, and the AMB yield is improved by 8 percent compared with the control example.

Example 3:

the primary and secondary seed culture media comprise the following components: 15g/L of peptone, 10g/L of yeast extract, 5g/L of sodium chloride, 10g/L of glucose, 1g/L of calcium carbonate, distilled water as solvent, 7.0 pH value adjusted by NaOH, and 30min of sterilization at 115 ℃.

The fermentation medium comprises the following components: 100g/L glucose, 50g/L soybean meal, 10g/L sodium citrate, 5g/L magnesium sulfate, 1g/L manganese sulfate, 1g/L anhydrous calcium chloride, 10g/L calcium carbonate, PPG 20005 g/L, distilled water as a solvent, natural pH value and steam sterilization.

In the fermentation process, ammonia water is added to adjust the pH value, and the mass percentage concentration of the ammonia water is 30%.

A supplemented medium: liquid sugar (30% glucose solution, 300g/L)

The fermentation production method of amphotericin B comprises the following steps:

(1) streptomyces nodosus ZJB 50 seed culture:

taking a ring belt gray spore colony from a GYM slant culture medium, inoculating the ring belt gray spore colony into a bottle of 250mL triangular flask containing 50mL seed culture medium, culturing at 26 ℃ and at a shaking table rotating speed of 200r/min for 2 days to obtain a first-grade seed solution; after 2 days, transferring the strain to a 500mL triangular flask containing 100mL seed culture medium with the inoculation amount of 2%, culturing at 26 ℃ at the rotation speed of a shaking table of 200r/min for 2 days to obtain a secondary seed solution.

(2) Streptomyces nodosus ZJB 50 fermentation culture:

inoculating the secondary seed liquid into a 5000L fermentation tank at an inoculation amount of 10%, wherein the initial fermentation liquid is 3000L, the initial fermentation condition is 100r/min, and the glucose concentration is detected in real time every 12h by referring to the control example in a feeding manner. Controlling the temperature to be 31 ℃ in the early stage of fermentation for 0-36h, and controlling the fermentation temperature to be 26 ℃ in the later stage of fermentation for 36h till the end of fermentation. Controlling aeration quantity in the fermentation process to adjust dissolved oxygen to be more than or equal to 10 percent, ending the fermentation when the fermentation culture lasts for 116 hours, sampling and detecting the AMB content and AMA, wherein the result is shown in figure 4, the AMB content is 12.35g/L, the amphotericin A/B is 4.2 percent and the AMB yield is improved by 31 percent compared with the control example which are measured by high performance liquid chromatography.

Example 4:

the primary and secondary seed culture media comprise the following components: 15g/L of peptone, 10g/L of yeast extract, 5g/L of sodium chloride, 10g/L of glucose, 1g/L of calcium carbonate, distilled water as solvent, 7.0 pH value adjusted by NaOH, and 30min of sterilization at 115 ℃.

The fermentation medium comprises the following components: 100g/L glucose, 50g/L soybean meal, 10g/L sodium citrate, 5g/L magnesium sulfate, 1g/L manganese sulfate, 1g/L anhydrous calcium chloride, 10g/L calcium carbonate, PPG 20005 g/L, distilled water as a solvent, natural pH value and steam sterilization.

In the fermentation process, ammonia water is added to adjust the pH value, and the mass percentage concentration of the ammonia water is 30%.

A supplemented medium: liquid sugar (30% glucose solution, 300g/L)

The fermentation production method of amphotericin B comprises the following steps:

(1) streptomyces nodosus ZJB 50 seed culture:

taking a ring belt gray spore colony from a GYM slant culture medium, inoculating the ring belt gray spore colony into a bottle of 250mL triangular flask containing 50mL seed culture medium, culturing at 26 ℃ and at a shaking table rotating speed of 200r/min for 2 days to obtain a first-grade seed solution; after 2 days, transferring the strain to a 500mL triangular flask containing 100mL seed culture medium with the inoculation amount of 2%, culturing at 26 ℃ at the rotation speed of a shaking table of 200r/min for 2 days to obtain a secondary seed solution.

(2) Streptomyces nodosus ZJB 50 fermentation culture:

inoculating the secondary seed liquid into a 5000L fermentation tank by 10% of inoculation amount, wherein the initial fermentation liquid is 3000L, the initial fermentation condition is 100r/min, feeding at a constant speed of 1.5g/L/h after the glucose concentration is reduced to 20g/L in the fermentation, and detecting the glucose concentration in real time every 12 h. Controlling the temperature to be 31 ℃ in the early stage of fermentation for 0-36h, and controlling the fermentation temperature to be 26 ℃ in the later stage of fermentation for 36h till the end of fermentation. Controlling aeration quantity in the fermentation process to adjust dissolved oxygen to be more than or equal to 20 percent, ending the fermentation when the fermentation culture lasts for 120 hours, sampling and detecting the AMB content and AMA, wherein the result is shown in figure 5, the AMB content is 13.89g/L, the amphotericin A/B is 3.2 percent and the AMB yield is improved by 42 percent compared with the control example by using a high performance liquid chromatography.

Example 5:

the primary and secondary seed culture media comprise the following components: 15g/L of peptone, 10g/L of yeast extract, 5g/L of sodium chloride, 10g/L of glucose, 1g/L of calcium carbonate, distilled water as solvent, 7.0 pH value adjusted by NaOH, and 30min of sterilization at 115 ℃.

The fermentation medium comprises the following components: 100g/L glucose, 50g/L soybean meal, 10g/L sodium citrate, 5g/L magnesium sulfate, 1g/L manganese sulfate, 1g/L anhydrous calcium chloride, 10g/L calcium carbonate, PPG 20005 g/L, distilled water as a solvent, natural pH value and steam sterilization.

In the fermentation process, ammonia water is added to adjust the pH value, and the mass percentage concentration of the ammonia water is 30%.

A supplemented medium: liquid sugar (30% glucose solution, 300g/L)

The fermentation production method of amphotericin B comprises the following steps:

(1) streptomyces nodosus ZJB 50 seed culture:

taking a ring belt gray spore colony from a GYM slant culture medium, inoculating the ring belt gray spore colony into a bottle of 250mL triangular flask containing 50mL seed culture medium, culturing at 26 ℃ and at a shaking table rotating speed of 200r/min for 2 days to obtain a first-grade seed solution; after 2 days, transferring the strain to a 500mL triangular flask containing 100mL seed culture medium with the inoculation amount of 2%, culturing at 26 ℃ at the rotation speed of a shaking table of 200r/min for 2 days to obtain a secondary seed solution.

(2) Streptomyces nodosus ZJB 50 fermentation culture:

inoculating the secondary seed liquid into a 5000L fermentation tank at an inoculation amount of 10%, wherein the initial fermentation liquid is 3000L, the initial fermentation condition is 100r/min, and the glucose concentration is detected in real time every 12h by referring to the control example in a feeding manner. Controlling the temperature to be 31 ℃ in the early stage of fermentation for 0-36h, and controlling the fermentation temperature to be 26 ℃ in the later stage of fermentation for 36h till the end of fermentation. Controlling the ventilation amount and supplementing materials in the fermentation process to jointly adjust the dissolved oxygen to be more than or equal to 30%, ending the fermentation when the fermentation culture lasts to 120h, sampling and detecting the AMB content and AMA, and obtaining the result as shown in figure 6, wherein the AMB content is 13.71g/L and the amphotericin A/B is 2.9% by high performance liquid chromatography, compared with the comparative example 5, the yield is not obviously changed, and the ventilation amount needs to be greatly improved when the dissolved oxygen is controlled to be more than 30 at the later stage of the fermentation, so that the energy is greatly wasted.

Example 6:

the primary and secondary seed culture media comprise the following components: 15g/L of peptone, 10g/L of yeast extract, 5g/L of sodium chloride, 10g/L of glucose, 1g/L of calcium carbonate, distilled water as solvent, 7.0 pH value adjusted by NaOH, and 30min of sterilization at 115 ℃.

The fermentation medium comprises the following components: 100g/L glucose, 50g/L soybean meal, 10g/L sodium citrate, 5g/L magnesium sulfate, 1g/L manganese sulfate, 1g/L anhydrous calcium chloride, 10g/L calcium carbonate, PPG 20005 g/L, distilled water as a solvent, natural pH value and steam sterilization.

In the fermentation process, ammonia water is added to adjust the pH value, and the mass percentage concentration of the ammonia water is 30%.

A supplemented medium: liquid sugar (30% glucose solution, 300g/L)

The fermentation production method of amphotericin B comprises the following steps:

(1) streptomyces nodosus ZJB 50 seed culture:

taking a ring belt gray spore colony from a GYM slant culture medium, inoculating the ring belt gray spore colony into a bottle of 250mL triangular flask containing 50mL seed culture medium, culturing at 26 ℃ and at a shaking table rotating speed of 200r/min for 2 days to obtain a first-grade seed solution; after 2 days, transferring the strain to a 500mL triangular flask containing 100mL seed culture medium with the inoculation amount of 2%, culturing at 26 ℃ at the rotation speed of a shaking table of 200r/min for 2 days to obtain a secondary seed solution.

(2) Streptomyces nodosus ZJB 50 fermentation culture:

inoculating the secondary seed liquid into a 5000L fermentation tank with 10% inoculum size, wherein the initial fermentation liquid is 3000L, the initial fermentation condition is 100r/min, and 1500m³Ventilation in/h. Controlling the temperature to be 31 ℃ in the early stage of fermentation for 0-36h, and controlling the fermentation temperature to be 26 ℃ in the later stage of fermentation for 36h till the end of fermentation. The feeding rate is controlled according to the concentration of glucose in the fermentation liquor, and the glucose amount in the fermentation liquor is controlled to be more than or equal to 2.0 percent. When the fermentation is finished when the fermentation culture time reaches 116h, sampling and detecting the AMB content and AMA, wherein the AMB content is 11.6g/L and the amphotericin A/B content is 6% by using high performance liquid chromatography as shown in figure 7. The fermentation process controls the residual sugar content of the fermentation liquor to be more than or equal to 2.0 percent, the titer of the amphotericin B is slowly produced in the later period of fermentation, the amphotericin A is rapidly grown, and the fermentation liquor is very viscous.

Example 7:

the primary and secondary seed culture media comprise the following components: 15g/L of peptone, 10g/L of yeast extract, 5g/L of sodium chloride, 10g/L of glucose, 1g/L of calcium carbonate, distilled water as solvent, 7.0 pH value adjusted by NaOH, and 30min of sterilization at 115 ℃.

The fermentation medium comprises the following components: 100g/L glucose, 50g/L soybean meal, 10g/L sodium citrate, 5g/L magnesium sulfate, 1g/L manganese sulfate, 1g/L anhydrous calcium chloride, 10g/L calcium carbonate, PPG 20005 g/L, distilled water as a solvent, natural pH value and steam sterilization.

In the fermentation process, ammonia water is added to adjust the pH value, and the mass percentage concentration of the ammonia water is 30%.

A supplemented medium: liquid sugar (30% glucose solution, 300g/L)

The fermentation production method of amphotericin B comprises the following steps:

(1) streptomyces nodosus ZJB 50 seed culture:

taking a ring belt gray spore colony from a GYM slant culture medium, inoculating the ring belt gray spore colony into a bottle of 250mL triangular flask containing 50mL seed culture medium, culturing at 26 ℃ and at a shaking table rotating speed of 200r/min for 2 days to obtain a first-grade seed solution; after 2 days, transferring the strain to a 500mL triangular flask containing 100mL seed culture medium with the inoculation amount of 2%, culturing at 26 ℃ at the rotation speed of a shaking table of 200r/min for 2 days to obtain a secondary seed solution.

(2) Streptomyces nodosus ZJB 50 fermentation culture:

inoculating the secondary seed liquid into a 5000L fermentation tank with 10% inoculum size, wherein the initial fermentation liquid is 3000L, the initial fermentation condition is 100r/min, and 1500m³Ventilation in/h. Controlling the temperature to be 31 ℃ in the early stage of fermentation for 0-36h, and controlling the fermentation temperature to be 26 ℃ in the later stage of fermentation for 36h till the end of fermentation. The feeding rate is controlled according to the concentration of glucose in the fermentation liquor, and the glucose amount in the fermentation liquor is controlled to be 0.5-1%. The fermentation was terminated when the fermentation was cultured for 124 hours, and the AMB content and AMA were sampled, and the results are shown in FIG. 8, where the AMB content was 12.5g/L and amphotericin A/B was 2.1% by HPLC, the yield was improved as compared with example 7, and the amount of by-products was small. The residual sugar content of the fermentation liquor is reduced, the fermentation time is prolonged, the fermentation titer is slowly grown, because the sugar supplement amount is reduced and the sugar concentration is lower in the fermentation process, the whole fermentation environment is better, and the amphotericin A is less generated.

Example 8:

the primary and secondary seed culture media comprise the following components: 15g/L of peptone, 10g/L of yeast extract, 5g/L of sodium chloride, 10g/L of glucose, 1g/L of calcium carbonate, distilled water as solvent, 7.0 pH value adjusted by NaOH, and 30min of sterilization at 115 ℃.

The fermentation medium comprises the following components: 100g/L glucose, 50g/L soybean meal, 10g/L sodium citrate, 5g/L magnesium sulfate, 1g/L manganese sulfate, 1g/L anhydrous calcium chloride, 10g/L calcium carbonate, PPG 20005 g/L, distilled water as a solvent, natural pH value and steam sterilization.

In the fermentation process, ammonia water is added to adjust the pH value, and the mass percentage concentration of the ammonia water is 30%.

A supplemented medium: liquid sugar (30% glucose solution, 300g/L)

The fermentation production method of amphotericin B comprises the following steps:

(1) streptomyces nodosus ZJB 50 seed culture:

taking a ring belt gray spore colony from a GYM slant culture medium, inoculating the ring belt gray spore colony into a bottle of 250mL triangular flask containing 50mL seed culture medium, culturing at 26 ℃ and at a shaking table rotating speed of 200r/min for 2 days to obtain a first-grade seed solution; after 2 days, transferring the strain to a 500mL triangular flask containing 100mL seed culture medium with the inoculation amount of 2%, culturing at 26 ℃ at the rotation speed of a shaking table of 200r/min for 2 days to obtain a secondary seed solution.

(2) Streptomyces nodosus ZJB 50 fermentation culture:

inoculating the secondary seed liquid into a 50000L fermentation tank by 10 percent of inoculation amount, wherein the initial fermentation liquid is 35000L, the initial fermentation condition is 100r/min, the fermentation time is 0-24 h, and the ventilation volume is 1000m³H, controlling the ventilation quantity to be 1500m from 24h to the end of fermentation³And/h, specifically controlling dissolved oxygen through rotating speed and material distribution, controlling the temperature to be 31 ℃ in the early stage of fermentation for 0-36 hours, and controlling the fermentation temperature to be 26 ℃ in the later stage of fermentation for 36 hours till the end of fermentation. The feeding rate is controlled according to the concentration of glucose in the fermentation liquor, and the glucose amount in the fermentation liquor is controlled to be 0.5-1%. The fermentation is finished when the fermentation culture lasts for 120h, and the pH, residual sugar, dissolved oxygen and temperature change trends in the fermentation process are shown in FIG. 9. The AMB content and AMA were sampled and detected, and the results are shown in FIG. 10, the AMB content was 13.996g/L and amphotericin A/B was 2.2% by HPLC, which is a 48.3% increase in yield over the control.