阅读说明：本技术 一种氢化可的松的生产发酵工艺 (Production fermentation process of hydrocortisone ) 是由 孙琼芳 付林 徐明琴 冯蕾 朱云 王霜 刘威 田玉林 于 2019-12-09 设计创作，主要内容包括：本发明公开了一种氢化可的松的生产发酵工艺,包括如下步骤：S1：菌种传代：选取蓝色犁头霉,培养温度为28℃摄氏度,培养时间为7～8天,至长出灰色的气生菌丝；S2：菌种扩大培养：将步骤(1)的菌种接入已灭菌的种子罐,进行一级培养；S3：发酵培养：在无菌条件下,将步骤S3中培养出的种子液接入发酵罐,发酵罐内放置有第二培养基；发酵过程中,检测发酵液的PH值小于3.8、DO值大于20％、PMV大于10％、还原糖的含量在1.0～2.0％之间、游离氨基氮的含量40～150mg/ml之间、游离磷酸根的含量20～500ug/ml之间；S4：底物转化。本发明较大程度地提高了氢化可的松生产的效率,降低了生产的综合成本。(The invention discloses a production fermentation process of hydrocortisone, which comprises the following steps: s1: and (3) strain passage: selecting Absidia coerulea, culturing at 28 ℃ for 7-8 days until gray aerial hyphae grow out; s2: and (3) strain amplification culture: inoculating the strain obtained in the step (1) into a sterilized seeding tank for primary culture; s3: fermentation culture: under the aseptic condition, the seed solution cultured in the step S3 is inoculated into a fermentation tank, and a second culture medium is placed in the fermentation tank; in the fermentation process, detecting that the pH value of the fermentation liquor is less than 3.8, the DO value is more than 20%, the PMV is more than 10%, the content of reducing sugar is 1.0-2.0%, the content of free amino nitrogen is 40-150 mg/ml, and the content of free phosphate radical is 20-500 ug/ml; s4: and (4) substrate conversion. The invention improves the production efficiency of hydrocortisone to a great extent and reduces the comprehensive cost of production.)

1. A production fermentation process of hydrocortisone is characterized by comprising the following steps:

s1: and (3) strain passage: selecting absidia coerulea, carrying out slant passage on the absidia coerulea, wherein a culture medium is a PDA culture medium, the culture temperature is 28 ℃, and the culture time is 7-8 days until gray aerial hyphae grow out;

s2: and (3) strain amplification culture: inoculating the strain obtained in the step (1) into a sterilized seed tank, placing a first culture medium in the seed tank, performing primary culture, controlling the fermentation temperature to be 28 +/-0.5 ℃, stirring at 0-500 rpm, and culturing at 0.02-0.08 MPa for 10-24 hours;

s3: fermentation culture:

under the aseptic condition, the seed solution cultured in the step S3 is inoculated into a fermentation tank, a second culture medium is placed in the fermentation tank, the fermentation temperature is controlled to be 28 +/-0.5 ℃, the stirring speed is 0-500 rpm, the pressure is 0.02-0.08 MPa, and the culture is carried out for 8-24 hours;

in the fermentation process, the PH value of the fermentation liquor is detected to be less than 3.8, the DO value is detected to be more than 20%, the PMV is detected to be more than 10%, the content of reducing sugar is 1.0-2.0%, the content of free amino nitrogen is 40-150 mg/ml, the content of free phosphate radical is 20-500 ug/ml, hyphae under an optical microscope with 400 times of color depth, multiple and thick branches are formed, most sporangium germinates, and part of hypha slightly expands and is transparent;

s4: substrate conversion: when the microbial culture meets the requirement, the pH value of the fermentation liquor is adjusted to be 5.4-5.8, the temperature is controlled to be 26 +/-0.5 ℃, the stirring is controlled to be 100-300 rpm, the air flow is controlled to be 20-400L/min, the tank pressure is controlled to be 0-0.02 Mpa, after the system is stabilized, an R.S.A solution which is completely dissolved by a solution of water and ethanol in a ratio of 1:4 is added, the ratio of the weight of the R.S.A to the volume of the fermentation liquor is (w/v) to 2.5g/L, after a substrate is added, the aeration ratio is adjusted to be 1: 0-0.5, the stirring rotation speed is 100-500 rpm, the temperature is controlled to be 28 +/-0.5 ℃, the conversion reaction is carried out for 15-25 hours under the condition, and the content of the R.S.A (HPLC) is less than 1%, and the tank is placed.

2. The production and fermentation process of hydrocortisone as claimed in claim 1, which comprises the following steps: in step S2, the first medium formulation, in percent: 0.8-1.5% of glucose, 0.5-1.6% of corn steep liquor, 0.1-1.2% of yeast extract, 0.1-1.0% of ammonium sulfate, 0.01-0.1% of magnesium sulfate, 0.01-0.1% of soybean oil and the balance of water; the pH value is 6.1-6.5.

3. The production and fermentation process of hydrocortisone as claimed in claim 1, which comprises the following steps: in step S3, the second medium formulation, in percent: 0.5-1.5% of glucose, 0.5-1.5% of corn steep liquor, 0.1-0.6% of yeast extract, 0.1-1.0% of ammonium sulfate, 0.01-0.1% of magnesium sulfate, 0.01-0.1% of soybean oil and the balance of water; the pH value is 6.1-6.5.

4. A fermentation process for the production of hydrocortisone according to claim 2 or 3, wherein the fermentation process comprises the following steps: the sterilization temperature of the culture medium is 118-125 ℃, and the time is 30 minutes.

Technical Field

The invention relates to the technical field of improvement of a hydrocortisone production process, in particular to a production fermentation process of hydrocortisone.

Background

Since Absidia coerulea (Absidiacoerulea) is adopted as a bacterial strain for industrial production of hydrocortisone in the early 60 s in China, remarkable economic benefit is created. However, the biotransformation process has the problems of more byproducts and low hydrocortisone yield. To increase the conversion of the product, many researchers have conducted extensive research including first stage mutagenesis or fermentation process improvement and second stage substrate conversion. Currently, the substrate conversion in the second stage mainly comprises the following six methods: (1) a one-step conversion method; (2) resting cell transformation; (3) a co-transformation method; (4) immobilized cell transformation technology; (5) cell-free system transformation; (6) protoplast transformation technique.

Disclosure of Invention

The invention provides a production fermentation process of hydrocortisone, which aims to solve the problems in the background art.

The invention provides a production fermentation process of hydrocortisone, which comprises the following steps:

s1: and (3) strain passage: selecting absidia coerulea, carrying out slant passage on the absidia coerulea, wherein a culture medium is a PDA culture medium, the culture temperature is 28 ℃, and the culture time is 7-8 days until gray aerial hyphae grow out;

s2: and (3) strain amplification culture: inoculating the strain obtained in the step (1) into a sterilized seed tank, placing a first culture medium in the seed tank, performing primary culture, controlling the fermentation temperature to be 28 +/-0.5 ℃, stirring at 0-500 rpm, and culturing at 0.02-0.08 MPa for 10-24 hours;

s3: fermentation culture:

under the aseptic condition, the seed solution cultured in the step S3 is inoculated into a fermentation tank, a second culture medium is placed in the fermentation tank, the fermentation temperature is controlled to be 28 +/-0.5 ℃, the stirring speed is 0-500 rpm, the pressure is 0.02-0.08 MPa, and the culture is carried out for 8-24 hours;

in the fermentation process, the PH value of the fermentation liquor is detected to be less than 3.8, the DO value is detected to be more than 20%, the PMV is detected to be more than 10%, the content of reducing sugar is 1.0-2.0%, the content of free amino nitrogen is 40-150 mg/ml, the content of free phosphate radical is 20-500 ug/ml, hyphae under an optical microscope with 400 times of color depth, multiple and thick branches are formed, most sporangium germinates, and part of hypha slightly expands and is transparent;

s4: substrate conversion: when the microbial culture meets the requirement, the pH value of the fermentation liquor is adjusted to be 5.4-5.8, the temperature is controlled to be 26 +/-0.5 ℃, the stirring is controlled to be 100-300 rpm, the air flow is controlled to be 20-400L/min, the tank pressure is controlled to be 0-0.02 Mpa, after the system is stabilized, an R.S.A solution which is completely dissolved by a solution of water and ethanol in a ratio of 1:4 is added, the ratio of the weight of the R.S.A to the volume of the fermentation liquor is (w/v) to 2.5g/L, after a substrate is added, the aeration ratio is adjusted to be 1: 0-0.5, the stirring rotation speed is 100-500 rpm, the temperature is controlled to be 28 +/-0.5 ℃, the conversion reaction is carried out for 15-25 hours under the condition, and the content of the R.S.A (HPLC) is less than 1%, and the tank is placed.

Preferably, in step S2, the first medium formulation comprises, in percent: 0.8-1.5% of glucose, 0.5-1.6% of corn steep liquor, 0.1-1.2% of yeast extract, 0.1-1.0% of ammonium sulfate, 0.01-0.1% of magnesium sulfate, 0.01-0.1% of soybean oil and the balance of water; the pH value is 6.1-6.5.

Preferably, in step S3, the second medium formulation comprises, in percent: 0.5-1.5% of glucose, 0.5-1.5% of corn steep liquor, 0.1-0.6% of yeast extract, 0.1-1.0% of ammonium sulfate, 0.01-0.1% of magnesium sulfate, 0.01-0.1% of soybean oil and the balance of water; the pH value is 6.1-6.5.

Preferably, the sterilization temperature of the culture medium is 118-125 ℃, and the time is 30 minutes.

The production fermentation process of hydrocortisone provided by the invention has the beneficial effects that: according to the invention, the concentration of nitrogen sources and enzyme active factors in a fermentation formula is optimized, the model of the stirring paddle is adjusted, and the stirring rotating speed and the air flow are optimally regulated and controlled in the fermentation process, so that the speed of the hydrocortisone biotransformation reaction process is greatly increased on the premise of ensuring the yield and quality of the hydrocortisone product; under the condition that the concentration of the substrate RSA is not lower than 2.5g/L (RSA weight/total volume of fermentation liquor), after the biotransformation reaction is carried out for 18-24 hours, the residual quantity of the RSA is less than 1 percent (HPLC), the proportion of hydrocortisone is more than 70 percent, and the proportion of hydrocortisone on the maximum byproduct table is less than 12 percent; the determination of the transfer timing, the starting timing of substrate conversion and the optimal timing of tank release of the microorganism culture improves the production efficiency of hydrocortisone to a great extent and reduces the comprehensive cost of production.

Drawings

FIG. 1 is an HPLC chart of the objective product hydrocortisone and the main byproduct hydrocortisone in the fermentation broth of the present invention.

FIG. 2 is a comparison graph of the content and ratio of the target product hydrocortisone and the main by-product hydrocortisone in the fermentation broth.

Detailed Description

The invention is further illustrated by the following examples.