CN112543811B - Production method of arachidonic acid - Google Patents

Production method of arachidonic acid Download PDF

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CN112543811B
CN112543811B CN202080002953.5A CN202080002953A CN112543811B CN 112543811 B CN112543811 B CN 112543811B CN 202080002953 A CN202080002953 A CN 202080002953A CN 112543811 B CN112543811 B CN 112543811B
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王霄凤
陈必钦
严必能
刘子睿
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INNER MONGOLIA KINGDOMWAY PHARMACEUTICAL CO Ltd
Xiamen Kingdomway Group Co
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Abstract

The invention belongs to the field of fermentation engineering, and relates to a production method of arachidonic acid, which comprises the steps of sequentially carrying out strain activation, seed expansion culture and fermentation culture on Mortierella alpina or mutant strains thereof, and in the fermentation culture process, monitoring OUR on line and controlling the OUR value to be 10-100 mmol/L.h, and/or after the 100 th h of fermentation culture, monitoring ORP in fermentation liquor on line and controlling the ORP value to be 50-150 mv. The method provided by the invention can stably improve the fermentation production level, so that the content of arachidonic acid in the obtained fermentation liquid is obviously improved.

Description

Production method of arachidonic acid
Technical Field
The invention belongs to the field of fermentation engineering, relates to a method for producing arachidonic acid by utilizing Mortierella alpina or mutant strains thereof, and particularly relates to a method for producing arachidonic acid by utilizing metabolic parameters OUR and/or ORP to regulate fermentation process.
Background
Arachidonic acid (abbreviated as AA or ARA) is an omega-6 polyunsaturated fatty acid which is all-cis-5, 8,11, 14-eicosatetraenoic acid containing four carbon-carbon double bonds and one carbon-oxygen double bond and is a higher unsaturated fatty acid. Arachidonic acid plays an important role as a phospholipid-bound structural lipid in blood, liver, muscle and other organ systems. In addition, arachidonic acid is a biologically active substance of many circulating eicosanoic acid derivatives, such as prostaglandin E2 (PGE 2), prostacyclin (PGI 2), thromboxane A2 (TXA 2), leukotrienes, and direct precursors of C4 (LTC 4). These bioactive substances have important regulatory effects on lipoprotein metabolism, haemorheology, vascular elasticity, leukocyte function, platelet activation, etc. Arachidonic acid is an important substance for the development of brain and optic nerve of human body, and has important effects in improving intelligence and enhancing visual acuity. Arachidonic acid has a series of physiological activities such as esterifying cholesterol, increasing vascular elasticity, reducing blood viscosity, and regulating blood cell function. Arachidonic acid has important effects in preventing cardiovascular diseases, diabetes, tumor, etc. High purity arachidonic acid is a direct precursor of eicosa-derived compounds such as prostaglandins (prostamanns), thromboxane (thrombioxanes) and leukotrienes (leukotrienes), which have important roles in the cardiovascular and immune systems of humans.
Currently, arachidonic acid is produced mainly by microbial fermentation. The existing fermentation is mainly improved in two directions of strain breeding and process control. Conventional process control is mainly regulated and controlled by single process parameters such as pH value, dissolved Oxygen (DO), temperature, feed, etc. Although the regulation and control method plays a certain role, as a plurality of interference factors of the fermentation process, such as the differences of strains, equipment, environment and the like, can generate larger interference on process analysis and regulation, the regulation and control method of a single factor is difficult to accurately reflect and control the whole fermentation process, the comprehensive analysis of the fermentation dynamic process is not needed fundamentally, the reproducibility is poor, the fluctuation of the fermentation level is larger, and the content of arachidonic acid in the obtained fermentation product is lower.
Disclosure of Invention
The invention aims to overcome the defects of large fluctuation of fermentation level and lower content of arachidonic acid in the obtained fermentation liquid when the fermentation process of the arachidonic acid is controlled by adopting single parameters such as pH value, DO, temperature, feeding and the like in the prior art, and provides a novel production method of the arachidonic acid.
Specifically, the present invention provides a method for producing arachidonic acid, which comprises subjecting Mortierella alpina or a mutant strain thereof to strain activation, seed expansion culture and fermentation culture in this order, and during fermentation culture, monitoring the oxygen consumption rate (Oxygen Uptake Rate, hereinafter abbreviated as OUR) on line and controlling the OUR value to 10 to 100 mmol/L.h, and/or, after 100h of fermentation culture, monitoring the oxidation-reduction potential (Oxidation Reduction Potential, hereinafter abbreviated as ORP) in the fermentation broth on line and controlling the ORP value to 50 to 150mv.
Further, the OUR is controlled in six stages, and the control manner is as follows: 0 to 20 hours is controlled to be 10 to 40 mmol/L.h, 20 to 55 hours is controlled to be 20 to 80 mmol/L.h, 55 to 80 hours is controlled to be 25 to 100 mmol/L.h, 80 to 120 hours is controlled to be 20 to 90 mmol/L.h, 120 to 160 hours is controlled to be 15 to 70 mmol/L.h, and 160 to fermentation end is controlled to be 15 to 60 mmol/L.h.
Further, the OUR is controlled in nine stages, and the control manner is as follows: 0 to 20h is controlled to be 10 to 35 mmol/L.h, 20 to 40h is controlled to be 20 to 70 mmol/L.h, 40 to 55h is controlled to be 25 to 80 mmol/L.h, 55 to 80h is controlled to be 25 to 100 mmol/L.h, 80 to 100h is controlled to be 25 to 90 mmol/L.h, 100 to 120h is controlled to be 20 to 80 mmol/L.h, 120 to 140h is controlled to be 20 to 70 mmol/L.h, 140 to 160h is controlled to be 20 to 65 mmol/L.h, and 160 to the end of fermentation is controlled to be 15 to 60 mmol/L.h.
Further, the ORP is controlled in three stages and in the following manner: 100-120 h, 50-100 mv, 120-160 h, 60-130 mv and 70-150 mv after fermentation.
Further, the ORP is controlled in four stages and in the following manner: 100-120 h is controlled at 50-100 mv, 120-140 h is controlled at 60-120 mv, 140-160 h is controlled at 65-130 mv, and 160-fermentation end is controlled at 70-140 mv.
Further, the OUR is controlled by adjusting at least one of ventilation, rotational speed, and tank pressure.
Further, the ORP is controlled by adjusting at least one of ventilation, rotational speed, tank pressure, temperature, and pH.
Further, the production method of arachidonic acid provided by the invention comprises the following steps:
(1) Strain activation: inoculating Mortierella alpina strain or mutant strain thereof into a shake flask containing sterilized strain activation medium, and culturing at 25-32deg.C and 100-200 rpm for 40-48 hr to obtain shake flask seed solution;
(2) Seed expansion culture: inoculating the shake flask seed liquid into a seed tank filled with the sterilized seed culture medium at an inoculum size of 0.4-1%, culturing for 30-35 h at a temperature of 25-32 ℃ under a ventilation rate of 1-2 vvm and a tank pressure of 0.02-0.05 MPa, optionally inoculating into the seed tank filled with the sterilized seed culture medium at an inoculum size of 1-3%, culturing for 20-25 h at a temperature of 25-32 ℃ under a ventilation rate of 1-2 vvm and a tank pressure of 0.02-0.05 MPa, and optionally repeatedly culturing for 1-3 times to obtain the seed liquid;
(3) Fermentation culture: inoculating 1% -3% of seed liquid into a fermentation tank filled with a sterilized fermentation medium, fermenting and culturing for 150-168 h under the conditions of 20-30 ℃ of temperature, 1-2 vvm of ventilation volume, 0.02-0.05 MPa of tank pressure and 0-100 rpm of stirring rotation speed, monitoring OUR on line and controlling the value of OUR in a preset range in the fermentation and culturing process, and/or monitoring ORP in the fermentation liquid on line and controlling the value of ORP in a preset range after 100h of fermentation and culturing process, and controlling the pH value of a fermentation system in the whole fermentation process to be 5-7.5.
After intensive research, the inventor of the invention discovers that in the process of producing arachidonic acid by using Mortierella alpina or mutant strains thereof, on one hand, the OUR value in the whole fermentation culture process can well reflect the growth process of thalli, and the fermentation level can be stably improved by precisely controlling the value of the OUR value, so that the content of arachidonic acid in fermentation liquor is obviously improved; on the other hand, the formation of arachidonic acid in the fermentation process is accompanied by a large amount of dehydrogenation reaction, the ORP value is precisely controlled after the 100h of fermentation, the oxidability of the fermentation liquid can be adjusted, the effect of dehydrogenase is facilitated, the fermentation level is stably improved, the formation of arachidonic acid is promoted, and the percentage of the arachidonic acid in total oil is obviously improved.
Detailed Description
In the invention, the OUR needs to be controlled to be 10-100 mmol/L.h in the whole fermentation process.
In some embodiments of the invention, OUR is controlled in six stages (i.e., 0-20 h, 20-55 h, 55-80 h, 80-120 h, 120-160 h, 160-end of fermentation) and in the following manner: the 0 to 20 hours are controlled to 10 to 40 mmol/L.h, and may be, for example, 10, 15, 20, 25, 30, 35, 40 mmol/L.h, etc.; the 20 to 55 hours are controlled to 20 to 80 mmol/L.h, and for example, may be 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 mmol/L.h, etc.; 55 to 80 hours are controlled to 25 to 100 mmol/L.h, and may be, for example, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mmol/L.h, etc.; 80 to 120 hours is controlled to 20 to 90 mmol/L.h, and for example, may be 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 mmol/L.h, etc.; 120 to 160 hours is controlled to 15 to 70 mmol/L.h, and for example, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 mmol/L.h, etc. can be used; 160 to the end of fermentation is controlled to 15 to 60 mmol/L.h, and may be, for example, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 mmol/L.h, etc. 0 to 20h include 1h, 2h … … h and 19h, 20h total 20h,20 to 55h include 21h, 22h … … h and 54h, 55h total 35h,55 to 80h include 56h, 57h … … h and 79h, 80 to 25h total 25h,80 to 120h include 81h, 82h … … h 119h and 120h total 40h,120 to 160h include 121h, 122h … … h 159h and 160h total 40h,160h to end fermentation include 161h and 162h … … end fermentation. In the present invention, the definition of the time period related to the phased control is the same as the definition, and the detailed description is omitted here.
In some embodiments of the invention, OUR is controlled in nine stages (i.e., 0-20 h, 20-40 h, 40-55 h, 55-80 h, 80-100 h, 100-120 h, 120-140 h, 140-160 h, 160-end of fermentation) and in the following manner: the 0 to 20 hours are controlled to 10 to 35 mmol/L.h, and for example, 10, 15, 20, 25, 30, 35 mmol/L.h, etc. can be used; the 20 to 40 hours are controlled to 20 to 70 mmol/L.h, and for example, may be 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 mmol/L.h, etc.; the 40 to 55 hours are controlled to 25 to 80 mmol/L.h, and may be, for example, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 mmol/L.h, etc.; 55 to 80 hours are controlled to 25 to 100 mmol/L.h, and may be, for example, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mmol/L.h, etc.; 80 to 100 hours is controlled to 25 to 90 mmol/L.h, and may be, for example, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 mmol/L.h, etc.; the 100 to 120 hours are controlled to 20 to 80 mmol/L.h, and for example, may be 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 mmol/L.h, etc.; 120 to 140 hours is controlled to 20 to 70 mmol/L.h, and for example, may be 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 mmol/L.h, etc.; 140 to 160 hours to 20 to 65 mmol/L.h, for example, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 mmol/L.h, etc.; the concentration of 160 to the end of fermentation is controlled to 15 to 60 mmol/L.h, and may be, for example, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 mmol/L.h, etc.
In the present invention, the OUR is calculated according to the following formula:
F in : ventilation (L/h or m) 3 /h)；
V: volume of fermentation broth (L or m) 3 )；
Co 2in : oxygen concentration (%) in intake air;
C idle in : concentration (%) of inert gas in the intake air;
Co 2out : oxygen concentration (%) in the exhaust gas of the fermentation broth;
Cco 2out : carbon dioxide concentration (%) in the effluent gas of the fermentation broth.
Wherein Co is 2in 、C Idle in 、Co 2out 、Cco 2out Monitoring was performed in real time by mass spectrometry.
In the present invention, the ORP needs to be controlled at 50-150 mv after the 100 th hour of fermentation culture.
In some embodiments of the invention, ORP is controlled in three stages (i.e., 100-120 h, 120-160 h, 160-end of fermentation) and in the following manner: the time of 100 to 120 hours is controlled to be 50 to 100mv, for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mv and the like; 120 to 160 hours is controlled to 60 to 130mv, for example, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130mv, etc.; the fermentation completion is controlled to 70 to 150mv, and may be, for example, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150mv, etc.
In some embodiments of the invention, ORP is controlled in four stages (i.e., 100-120 h, 120-140 h, 140-160 h, 160-end of fermentation) and in the following manner: the time of 100 to 120 hours is controlled to be 50 to 100mv, for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mv and the like; the 120-140 h is controlled to be 60-120 mv, for example, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120mv and the like; 140 to 160 hours is controlled to 65 to 130mv, and for example, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130mv, etc. are possible; the fermentation completion is controlled to 70 to 140mv, and may be, for example, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140mv, etc.
In the present invention, the ORP is measured by an oxidation-reduction potential electrode.
In one embodiment of the invention, the OUR is controlled by adjusting at least one of ventilation, rotational speed, and tank pressure. When OUR exceeds a predetermined range, then it is desirable to decrease OUR by adjusting at least one of ventilation, rotational speed, and tank pressure downward; when OUR is below the predetermined range, then it is desirable to increase OUR by up-regulating at least one of ventilation, rotational speed, and tank pressure.
In one embodiment of the present invention, the ORP is controlled by adjusting at least one of ventilation, rotational speed, tank pressure, temperature, and pH. When the ORP exceeds a predetermined range, then it is necessary to reduce the ORP by either adjusting at least one of ventilation, rotational speed and tank pressure down or adjusting the temperature and/or pH up; when the ORP is below the predetermined range, then it is necessary to increase the ORP by adjusting at least one of the ventilation, the rotational speed and the tank pressure up or by adjusting the temperature and/or the pH down.
In the present invention, the mortierella alpina and mutant strains thereof can be commercially obtained, and specifically can be selected from strains with the preservation numbers of CCTCC No. m2012073, CCTCC No. m2013392, CCTCC No. m 2015121 or ATCC No. 42430.
The production process of the arachidonic acid comprises strain activation, seed expansion culture and fermentation culture, wherein the seed expansion culture generally comprises primary seed culture and secondary seed culture. The strain activation medium, the seed medium and the fermentation medium used in each stage may be those used in the art for culturing Mortierella alpina strain or its mutant strain, and are known to those skilled in the art, and are not described herein.
In one embodiment, the method for producing arachidonic acid comprises the steps of:
(1) Strain activation: inoculating Mortierella alpina strain or mutant strain thereof into a shake flask containing sterilized strain activation medium, and culturing at 25-32deg.C and 100-200 rpm for 40-48 hr to obtain shake flask seed solution;
(2) Seed expansion culture: inoculating the shake flask seed liquid into a seed tank filled with the sterilized seed culture medium at an inoculum size of 0.4-1%, culturing for 30-35 h at a temperature of 25-32 ℃ under a ventilation rate of 1-2 vvm and a tank pressure of 0.02-0.05 MPa, optionally inoculating into the seed tank filled with the sterilized seed culture medium at an inoculum size of 1-3%, culturing for 20-25 h at a temperature of 25-32 ℃ under a ventilation rate of 1-2 vvm and a tank pressure of 0.02-0.05 MPa, and optionally repeatedly culturing for 1-3 times to obtain the seed liquid;
(3) Fermentation culture: inoculating 1% -3% of seed liquid into a fermentation tank filled with a sterilized fermentation medium, fermenting and culturing for 150-168 h under the conditions of 20-30 ℃ of temperature, 1-2 vvm of ventilation volume, 0.02-0.05 MPa of tank pressure and 0-100 rpm of stirring rotation speed, monitoring OUR on line and controlling the value of OUR in a preset range in the fermentation and culturing process, and/or monitoring ORP in the fermentation liquid on line and controlling the value of ORP in a preset range after 100h of fermentation and culturing process, and controlling the pH value of a fermentation system in the whole fermentation process to be 5-7.5. Wherein the agent for adjusting the pH value comprises but is not limited to: sodium hydroxide solution, potassium hydroxide solution, ammonia water, citric acid, etc. In addition, the fermenter volume used for the fermentation culture may be 0.5L to 500m 3 。
The following examples further illustrate the invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In the following examples and comparative examples, the strains and media used, if not specified, were as follows:
(1) The strain used was Mortierella alpina Mortierella alpine (ATCC No. 42430);
(2) The strain activation medium used was formulated as follows: 200g of potato (peeled), 20g of glucose, 5g of yeast extract, 1000mL of distilled water and natural pH;
(3) The seed culture medium used was as follows: 200g of potato (peeled), 20g of glucose, 1000mL of distilled water and natural pH;
(4) The fermentation medium used was as follows: glucose 22g/L, yeast powder 12g/L, peptone 8g/L, sodium chloride 15g/L, ammonium sulfate 6g/L、KH 2 PO 4 5g/L、CuSO 4 ·5H 2 O 1.5μg/L、MnSO 4 2μg/L、ZnSO 4 ·7H 2 O3 mug/L, pH value is 6-7.
In the following examples and comparative examples, the measurement methods of the respective parameters were carried out in the following manner:
(1) The method for measuring the biomass comprises the following steps: and (3) taking a proper amount of fermentation liquor in a weighing bottle, drying for 4 hours in an electrothermal constant-temperature drying oven at 105 ℃, then putting the fermentation liquor into a dryer, cooling to room temperature, weighing, and subtracting the net weight of the weighing bottle from the weight to obtain the dry weight of the fermentation liquor, wherein the dry weight of the fermentation liquor divided by the volume of the fermentation liquor is biomass, and the unit g/L.
(2) The method for measuring the crude oil yield comprises the following steps: adding 2 times of concentrated hydrochloric acid into a certain volume of fermentation liquor, stirring at a constant temperature of 70 ℃ for 50min until thalli are completely digested, adding a proper amount of normal hexane, standing for layering, taking an upper organic phase into a eggplant type bottle by using a dropper, continuously extracting for 6 times until the upper organic phase is colorless, removing the normal hexane by rotary evaporation in a water bath at 80 ℃, then placing the eggplant type bottle into an electric heating constant temperature drying oven at 105 ℃ for drying for 1h, then placing the bottle into a dryer for cooling to room temperature and weighing, wherein the dry weight of crude oil is obtained by subtracting the dry weight of the eggplant type bottle, and the dry weight of the crude oil is obtained by dividing the volume of the fermentation liquor by the unit g/L.
(3) The method for measuring the ARA mass percent comprises the following steps: the mass percent of ARA in the crude oil is measured by adopting a gas chromatography method.
Example 1:100L fermentation tank process control
(1) Strain activation: the Mortierella alpina slope preservation strain is inoculated into a 2L shake flask filled with 400mL sterilized strain activation medium, and is cultured for 48 hours at the temperature of 28 ℃ and the rotating speed of 150rpm, so that the strain activation culture is completed, and shake flask seed liquid is obtained.
(2) Seed expansion culture: inoculating the shake flask seed liquid into a seed tank filled with the sterilized seed culture medium according to the inoculation amount of 0.4%, culturing at 28 ℃ and aeration rate of 1vvm, and culturing at 0.04MPa for 35h to obtain the seed liquid.
(3) Fermentation culture: inoculating the seed solution of the seed tank into a fermentation tank filled with a sterilized fermentation medium according to the inoculation amount of 3%, and performing initial culture conditions: the culture temperature is 28 ℃, the aeration rate is 1.0vvm, the tank pressure is 0.04MPa, the rotating speed is 70rpm, and the fermentation culture is carried out for 168 hours, thus obtaining the fermentation broth. During the fermentation culture, OUR is monitored online and its value is controlled within the following ranges:
1) Fermenting and culturing for 0-20 h, controlling OUR at 10-40 mmol/L.h,
2) Fermenting and culturing for 20-55 h, controlling OUR at 20-80 mmol/L.h,
3) The OUR is controlled to be 25-100 mmol/L.h in the fermentation culture for 55-80 h,
4) The OUR is controlled to be 20-90 mmol/L.h in the fermentation culture for 80-120 h,
5) The OUR is controlled to be 15-70 mmol/L.h in the fermentation culture for 120-160 h,
6) The OUR is controlled to be 15-60 mmol/L.h in the fermentation culture for 160-168 h.
The value of OUR is controlled by adjusting at least one of ventilation, tank pressure, and rotational speed. When the OUR is below the control range, up-regulating at least one of ventilation, tank pressure, and rotational speed; when the OUR is above the control range, at least one of the air flow, tank pressure, and rotational speed is adjusted downward to control the OUR within the desired range. And culturing for 168 hours to stop fermentation, wherein the biomass, the oil yield and the mass percent of arachidonic acid in the fermentation liquid are respectively 45g/L, 26.7g/L and 67 percent according to detection.
Example 2:100m 3 Fermentation tank process control
(1) Strain activation: the Mortierella alpina slope preservation strain is inoculated into a 2L shake flask filled with 400mL sterilized strain activation medium, and is cultured for 48 hours at the temperature of 28 ℃ and the rotating speed of 150rpm, so that the strain activation culture is completed, and shake flask seed liquid is obtained.
(2) Primary seed culture: inoculating the shake flask seed liquid into a first-stage seed tank filled with a sterilized seed culture medium according to the inoculation amount of 0.4%, culturing at 28 ℃ and aeration rate of 1vvm, and culturing for 35h under the pressure of 0.04MPa to complete the expansion culture of the first-stage seeds to obtain the first-stage seed liquid.
(3) Secondary seed culture: and (3) inoculating the first-stage seed liquid into a second-stage seed tank filled with the sterilized seed culture medium according to the inoculation amount of 3%, wherein the culture temperature is 28 ℃, the aeration rate is 1vvm, the tank pressure is 0.04MPa, and the second-stage seed liquid is obtained after the expansion culture of the second-stage seeds.
(4) Fermentation culture: inoculating the seed solution of the secondary seed tank into a fermentation tank filled with a sterilized fermentation medium according to the inoculation amount of 3%, and performing initial culture conditions: the culture temperature is 28 ℃, the aeration rate is 1.0vvm, the tank pressure is 0.04MPa, the rotating speed is 50rpm, and the fermentation culture is carried out for 168 hours, thus obtaining the fermentation broth. During the fermentation culture, OUR is monitored online and its value is controlled within the following ranges:
1) The OUR is controlled to be 10-35 mmol/L.h for 0-20 h,
2) The OUR is controlled to be 20-70 mmol/L.h for 20-40 h,
3) 40-55 h, controlling OUR at 25-80 mmol/L.h,
4) The OUR is controlled to be 25-100 mmol/L.h for 55-80 h,
5) The OUR is controlled to be 25-90 mmol/L.h for 80-100 h,
6) The OUR is controlled to be 20-80 mmol/L.h for 100-120 h,
7) The OUR is controlled to be 20-70 mmol/L.h for 120-140 h,
8) The OUR is controlled to be 20-65 mmol/L.h for 140-160 h,
9) The OUR is controlled to be 15-60 mmol/L.h in 160-168 h.
The value of OUR is controlled by adjusting at least one of ventilation, tank pressure, and rotational speed. When the OUR is below the control range, up-regulating at least one of ventilation, tank pressure, and rotational speed; when the OUR is above the control range, at least one of the air flow, tank pressure, and rotational speed is adjusted downward to control the OUR within the desired range. The fermentation is stopped after 168 hours of culture, and the biomass, the oil yield and the mass percent of arachidonic acid in the fermentation liquid are respectively 48.5g/L, 30.1g/L and 66.8 percent according to detection.
Example 3:300m 3 Fermentation tank process control
(1) Strain activation: the Mortierella alpina slope preservation strain is inoculated into a 2L shake flask filled with 400mL sterilized strain activation medium, and is cultured for 48 hours at the temperature of 28 ℃ and the rotating speed of 150rpm, so that the strain activation culture is completed, and shake flask seed liquid is obtained.
(2) Primary seed culture: inoculating the shake flask seed liquid into a first-stage seed tank filled with a sterilized seed culture medium according to the inoculation amount of 0.4%, culturing at 28 ℃ and aeration rate of 1vvm, and culturing for 35h under the pressure of 0.04MPa to complete the expansion culture of the first-stage seeds to obtain the first-stage seed liquid.
(3) Secondary seed culture: and (3) inoculating the first-stage seed liquid into a second-stage seed tank filled with the sterilized seed culture medium according to the inoculation amount of 3%, wherein the culture temperature is 28 ℃, the aeration rate is 1vvm, the tank pressure is 0.04MPa, and the second-stage seed liquid is obtained after the expansion culture of the second-stage seeds.
(4) Fermentation culture: inoculating the seed solution of the secondary seed tank into a fermentation tank filled with a sterilized fermentation medium according to the inoculation amount of 3%, and performing initial culture conditions: the culture temperature is 28 ℃, the aeration rate is 1.0vvm, the tank pressure is 0.04MPa, the rotating speed is 30rpm, and the fermentation culture is carried out for 168 hours, thus obtaining the fermentation broth. During the fermentation culture, OUR is monitored online and its value is controlled within the following ranges:
1) The OUR is controlled to be 10-35 mmol/L.h for 0-20 h,
2) The OUR is controlled to be 20-70 mmol/L.h for 20-40 h,
3) 40-55 h, controlling OUR at 25-80 mmol/L.h,
4) The OUR is controlled to be 25-100 mmol/L.h for 55-80 h,
5) The OUR is controlled to be 25-90 mmol/L.h for 80-100 h,
6) The OUR is controlled to be 20-80 mmol/L.h for 100-120 h,
7) The OUR is controlled to be 20-70 mmol/L.h for 120-140 h,
8) The OUR is controlled to be 20-65 mmol/L.h for 140-160 h,
9) The OUR is controlled to be 15-60 mmol/L.h in 160-168 h.
The value of OUR is controlled by adjusting at least one of ventilation, tank pressure, and rotational speed. When the OUR is below the control range, up-regulating at least one of ventilation, tank pressure, and rotational speed; when the OUR is above the control range, at least one of the air flow, tank pressure, and rotational speed is adjusted downward to control the OUR within the desired range. Culturing for 168h to stop fermentation, and detecting that the biomass, the oil yield and the mass percentage of arachidonic acid in the fermentation liquid are respectively 48.7g/L, 29.6g/L and 67.2 percent.
Example 1-1:100m 3 Fermentation tank process control
Arachidonic acid was produced as in example 2, except that OUR was monitored online and controlled within the following limits during the fermentation culture:
1) The OUR is controlled to be 10-35 mmol/L.h for 0-20 h,
2) The OUR is controlled to be 20-70 mmol/L.h for 20-40 h,
3) 40-55 h, controlling OUR at 25-80 mmol/L.h,
4) The OUR is controlled to be 25-100 mmol/L.h for 55-80 h,
5) The OUR is controlled to be 25-90 mmol/L.h for 80-100 h,
6) The OUR is controlled to be 20-80 mmol/L.h for 100-120 h,
7) The OUR is controlled to be 70-80 mmol/L.h for 120-140 h,
8) The OUR is controlled to be 10 to 20 mmol/L.h for 140 to 160 hours,
9) The OUR is controlled to be 15-60 mmol/L.h in 160-168 h.
The fermentation is stopped after 168 hours of culture, and the biomass, the oil yield and the mass percent of arachidonic acid in the fermentation liquid are respectively 46.2g/L, 27.1g/L and 66.5 percent according to detection. Compared with example 2, the partial interval is not controlled in the OUR preferable range, and the biomass, the oil yield and the mass percentage of the arachidonic acid in the fermentation liquid are all lower than those in example 2.
Comparative example 1
Arachidonic acid was produced as in example 1-1, except that OUR was monitored online and its value controlled within the following ranges during the fermentation culture:
1) The OUR is controlled to be 5-35 mmol/L.h for 0-20 h,
2) The OUR is controlled to be 20-70 mmol/L.h for 20-40 h,
3) 40-55 h, controlling OUR at 25-80 mmol/L.h,
4) The OUR is controlled to be 25-120 mmol/L.h for 55-80 h,
5) The OUR is controlled to be 25-90 mmol/L.h for 80-100 h,
6) The OUR is controlled to be 20-80 mmol/L.h for 100-120 h,
7) The OUR is controlled to be 70-80 mmol/L.h for 120-140 h,
8) The OUR is controlled to be 10 to 20 mmol/L.h for 140 to 160 hours,
9) The OUR is controlled to be 15-60 mmol/L.h in 160-168 h.
And culturing for 168 hours to stop fermentation, wherein the mass percentages of biomass, oil yield and arachidonic acid in the fermentation liquid are respectively 43.2g/L, 24.8g/L and 57.8% through detection. In comparison with example 1-1, the OUR value falls outside the scope of the present invention in the partial interval, and the biomass, the oil yield, and the mass percentage of arachidonic acid in the fermentation broth are all lower than in example 1-1.
Example 4:100m 3 Fermentation tank process control
(1) Strain activation: the Mortierella alpina slope preservation strain is inoculated into a 2L shake flask filled with 400mL sterilized strain activation medium, and is cultured for 48 hours at the temperature of 28 ℃ and the rotating speed of 150rpm, so that the strain activation culture is completed, and shake flask seed liquid is obtained.
(2) Primary seed culture: inoculating the shake flask seed liquid into a first-stage seed tank filled with a sterilized seed culture medium according to the inoculation amount of 0.4%, culturing at 28 ℃ and aeration rate of 1vvm, and culturing for 35h under the pressure of 0.04MPa to complete the expansion culture of the first-stage seeds to obtain the first-stage seed liquid.
(3) Secondary seed culture: and (3) inoculating the first-stage seed liquid into a second-stage seed tank filled with the sterilized seed culture medium according to the inoculation amount of 3%, wherein the culture temperature is 28 ℃, the aeration rate is 1vvm, the tank pressure is 0.04MPa, and the second-stage seed liquid is obtained after the expansion culture of the second-stage seeds.
(4) Fermentation culture: inoculating the seed solution of the secondary seed tank into a fermentation tank filled with a sterilized fermentation medium according to the inoculation amount of 3%, and performing initial culture conditions: the culture temperature is 28 ℃, the aeration rate is 1.0vvm, the tank pressure is 0.04MPa, the rotating speed is 45rpm, and the fermentation culture is carried out for 168 hours, thus obtaining the fermentation broth. After 100h of fermentation culture, ORP in the broth was monitored online and its value was controlled within the following range:
1) The ORP is controlled to be 50-100 mv in 100-120 h,
2) The ORP is controlled to be 60-130 mv within 120-160 h,
3) The ORP is controlled to be 70-150 mv in 160-168 h.
The value of ORP is controlled by adjusting at least one of ventilation, tank pressure, rotational speed, temperature, and pH. When the ORP is below the control range, at least one of aeration rate, tank pressure and rotational speed is adjusted up, or fermentation temperature and/or pH is adjusted down; when the ORP is above the control range, at least one of aeration rate, tank pressure and rotational speed is adjusted down, or fermentation temperature and/or pH is adjusted up to control the ORP within the desired range. And culturing for 168 hours to stop fermentation, wherein the mass percentages of biomass, oil yield and arachidonic acid in the fermentation liquid are respectively 43.5g/L, 25.2g/L and 68.3 percent according to detection.
Example 5:100m 3 Fermentation tank process control
(1) Strain activation: the Mortierella alpina slope preservation strain is inoculated into a 2L shake flask filled with 400mL sterilized strain activation medium, and is cultured for 48 hours at the temperature of 28 ℃ and the rotating speed of 150rpm, so that the strain activation culture is completed, and shake flask seed liquid is obtained.
(2) Primary seed culture: inoculating the shake flask seed liquid into a first-stage seed tank filled with a sterilized seed culture medium according to the inoculation amount of 0.4%, culturing at 28 ℃ and aeration rate of 1vvm, and culturing for 35h under the pressure of 0.04MPa to complete the expansion culture of the first-stage seeds to obtain the first-stage seed liquid.
(3) Secondary seed culture: and (3) inoculating the first-stage seed liquid into a second-stage seed tank filled with the sterilized seed culture medium according to the inoculation amount of 3%, wherein the culture temperature is 28 ℃, the aeration rate is 1vvm, the tank pressure is 0.04MPa, and the second-stage seed liquid is obtained after the expansion culture of the second-stage seeds.
(4) Fermentation culture: inoculating the seed solution of the secondary seed tank into a fermentation tank filled with a sterilized fermentation medium according to the inoculation amount of 3%, and performing initial culture conditions: the culture temperature is 28 ℃, the aeration rate is 1.0vvm, the tank pressure is 0.04MPa, the rotating speed is 45rpm, and the fermentation culture is carried out for 168 hours. After 100h of fermentation culture, ORP in the broth was monitored online and its value was controlled within the following range:
1) The ORP is controlled to be 50-100 mv in 100-120 h,
2) The ORP is controlled to be 60-120 mv within 120-140 h,
3) The ORP is controlled to be 65-130 mv within 140-160 h,
4) The ORP is controlled to be 70-140 mv in 160-168 h.
The value of ORP is controlled by adjusting at least one of ventilation, tank pressure, rotational speed, temperature, and pH. When the ORP is below the control range, at least one of aeration rate, tank pressure and rotational speed is adjusted up, or fermentation temperature and/or pH is adjusted down; when the ORP is above the control range, at least one of aeration rate, tank pressure and rotational speed is adjusted down, or fermentation temperature and/or pH is adjusted up to control the ORP within the desired range. And culturing for 168 hours to stop fermentation, wherein the mass percentages of biomass, oil yield and arachidonic acid in the fermentation liquid are respectively 44.1g/L, 25.8g/L and 68.6% through detection.
Comparative example 2:100m 3 Fermentation tank process control
Arachidonic acid was produced as in example 5, except that after 100h of culture, ORP in the broth was monitored on line and controlled to the following values:
1) The ORP is controlled to be 50-100 mv in 100-120 h,
2) The ORP is controlled to be 60-120 mv within 120-140 h,
3) The ORP is controlled to be 40-65 mv within 140-160 h,
4) The ORP is controlled to be 35-60 mv within 160-168 hours.
And culturing for 168 hours to stop fermentation, wherein the mass percentages of biomass, oil yield and arachidonic acid in the fermentation liquor are respectively 43.1g/L, 23.6g/L and 58.4 percent according to detection. In comparison with example 5, ORP values falling outside the scope of the present invention in the partial interval range, biomass, oil yield, and mass percent of arachidonic acid in the fermentation broth were all lower than in example 5.
Example 6:100m 3 Fermentation tank process control
(1) Strain activation: the Mortierella alpina slope preservation strain is inoculated into a 2L shake flask filled with 400mL sterilized strain activation medium, and is cultured for 48 hours at the temperature of 28 ℃ and the rotating speed of 150rpm, so that the strain activation culture is completed, and shake flask seed liquid is obtained.
(2) Primary seed culture: inoculating the shake flask seed liquid into a first-stage seed tank filled with a sterilized seed culture medium according to the inoculation amount of 0.4%, culturing at 28 ℃ and aeration rate of 1vvm, and culturing for 35h under the pressure of 0.04MPa to complete the expansion culture of the first-stage seeds to obtain the first-stage seed liquid.
(3) Secondary seed culture: and (3) inoculating the first-stage seed liquid into a second-stage seed tank filled with the sterilized seed culture medium according to the inoculation amount of 3%, wherein the culture temperature is 28 ℃, the aeration rate is 1vvm, the tank pressure is 0.04MPa, and the second-stage seed liquid is obtained after the expansion culture of the second-stage seeds.
(4) Fermentation culture: inoculating the seed solution of the secondary seed tank into a fermentation tank filled with a sterilized fermentation medium according to the inoculation amount of 3%, and performing initial culture conditions: the culture temperature is 28 ℃, the aeration rate is 1.0vvm, the tank pressure is 0.04MPa, the rotating speed is 45rpm, and the fermentation culture is carried out for 168 hours. During the fermentation culture, OUR is monitored on line and its value is controlled within the following range, and after 100 hours of fermentation culture, ORP in the fermentation broth is monitored on line and its value is controlled within the following range:
1) The OUR is controlled to be 10-35 mmol/L.h for 0-20 h,
2) The OUR is controlled to be 20-70 mmol/L.h for 20-40 h,
3) 40-55 h, controlling OUR at 25-80 mmol/L.h,
4) The OUR is controlled to be 25-100 mmol/L.h for 55-80 h,
5) The OUR is controlled to be 25-90 mmol/L.h for 80-100 h,
6) The OUR is controlled to be 20-80 mmol/L.h for 100-120 h, the ORP is controlled to be 50-100 mv,
7) The OUR is controlled to be 20-70 mmol/L.h for 120-140 h, the ORP is controlled to be 60-120 mv,
8) The OUR is controlled to be 20-65 mmol/L.h for 140-160 h, the ORP is controlled to be 65-130 mv,
9) The OUR is controlled to be 15-60 mmol/L.h in 160-168 h, and the ORP is controlled to be 70-140 mv.
The value of OUR is controlled by adjusting at least one of ventilation, tank pressure, and rotational speed. When the OUR is below the control range, up-regulating at least one of ventilation, tank pressure, and rotational speed; when the OUR is above the control range, at least one of the air flow, tank pressure, and rotational speed is adjusted downward to control the OUR within the desired range. The value of ORP is controlled by adjusting at least one of ventilation, tank pressure, rotational speed, temperature, and pH. When the ORP is below the control range, at least one of aeration rate, tank pressure and rotational speed is adjusted up, or fermentation temperature and/or pH is adjusted down; when the ORP is above the control range, at least one of aeration rate, tank pressure and rotational speed is adjusted down, or fermentation temperature and/or pH is adjusted up to control the ORP within the desired range. And culturing for 168 hours to stop fermentation, wherein the mass percentages of biomass, oil yield and arachidonic acid in the fermentation liquid are respectively 50.2g/L, 31.3g/L and 69.2 percent through detection.
Example 7:100m 3 Fermentation tank process control
(1) Strain activation: the Mortierella alpina slope preservation strain is inoculated into a 2L shake flask filled with 400mL sterilized strain activation medium, and is cultured for 48 hours at the temperature of 28 ℃ and the rotating speed of 150rpm, so that the strain activation culture is completed, and shake flask seed liquid is obtained.
(2) Primary seed culture: inoculating the shake flask seed liquid into a first-stage seed tank filled with a sterilized seed culture medium according to the inoculation amount of 0.4%, culturing at 28 ℃ and aeration rate of 1vvm, and culturing for 35h under the pressure of 0.04MPa to complete the expansion culture of the first-stage seeds to obtain the first-stage seed liquid.
(3) Secondary seed culture: and (3) inoculating the first-stage seed liquid into a second-stage seed tank filled with the sterilized seed culture medium according to the inoculation amount of 3%, wherein the culture temperature is 28 ℃, the aeration rate is 1vvm, the tank pressure is 0.04MPa, and the second-stage seed liquid is obtained after the expansion culture of the second-stage seeds.
(4) Fermentation culture: inoculating the seed solution of the secondary seed tank into a fermentation tank filled with a sterilized fermentation medium according to the inoculation amount of 3%, and performing initial culture conditions: the culture temperature is 28 ℃, the aeration rate is 1.0vvm, the tank pressure is 0.04MPa, the rotating speed is 45rpm, and the fermentation culture is carried out for 168 hours. During the fermentation culture, OUR is monitored on line and its value is controlled within the following range, and after 100 hours of fermentation culture, ORP in the fermentation broth is monitored on line and its value is controlled within the following range:
1) The OUR is controlled to be 10-40 mmol/L.h for 0-20 h,
2) The OUR is controlled to be 20-80 mmol/L.h for 20-55 h,
3) The OUR is controlled to be 25-100 mmol/L.h for 55-80 h,
4) The OUR is controlled to be 20-90 mmol/L.h for 80-120 h, the ORP is controlled to be 50-100 mv for 100-120 h,
5) The OUR is controlled to be 15-70 mmol/L.h for 120-160 h, the ORP is controlled to be 60-130 mv,
6) The OUR is controlled to be 15-60 mmol/L.h in 160-168 h, and the ORP is controlled to be 70-150 mv.
The value of OUR is controlled by adjusting at least one of ventilation, tank pressure, and rotational speed. When the OUR is below the control range, up-regulating at least one of ventilation, tank pressure, and rotational speed; when the OUR is above the control range, at least one of the air flow, tank pressure, and rotational speed is adjusted downward to control the OUR within the desired range. The value of ORP is controlled by adjusting at least one of ventilation, tank pressure, rotational speed, temperature, and pH. When the ORP is below the control range, at least one of aeration rate, tank pressure and rotational speed is adjusted up, or fermentation temperature and/or pH is adjusted down; when the ORP is above the control range, at least one of aeration rate, tank pressure and rotational speed is adjusted down, or fermentation temperature and/or pH is adjusted up to control the ORP within the desired range. And culturing for 168 hours to stop fermentation, wherein the mass percentages of biomass, oil yield and arachidonic acid in the fermentation liquid are respectively 47.6g/L, 30.1g/L and 67.6% through detection.
Example 8:100m 3 Fermentation tank process control
(1) Strain activation: the Mortierella alpina slope preservation strain is inoculated into a 2L shake flask filled with 400mL sterilized strain activation medium, and is cultured for 48 hours at the temperature of 28 ℃ and the rotating speed of 150rpm, so that the strain activation culture is completed, and shake flask seed liquid is obtained.
(2) Primary seed culture: inoculating the shake flask seed liquid into a first-stage seed tank filled with a sterilized seed culture medium according to the inoculation amount of 0.4%, culturing at 28 ℃ and aeration rate of 1vvm, and culturing for 35h under the pressure of 0.04MPa to complete the expansion culture of the first-stage seeds to obtain the first-stage seed liquid.
(3) Secondary seed culture: and (3) inoculating the first-stage seed liquid into a second-stage seed tank filled with the sterilized seed culture medium according to the inoculation amount of 3%, wherein the culture temperature is 28 ℃, the aeration rate is 1vvm, the tank pressure is 0.04MPa, and the second-stage seed liquid is obtained after the expansion culture of the second-stage seeds.
(4) Fermentation culture: inoculating the seed solution of the secondary seed tank into a fermentation tank filled with a sterilized fermentation medium according to the inoculation amount of 3%, and performing initial culture conditions: the culture temperature is 28 ℃, the aeration rate is 1.0vvm, the tank pressure is 0.04MPa, the rotating speed is 45rpm, and the fermentation culture is carried out for 168 hours.
The oxygen consumption rate OUR and the oxidation-reduction potential ORP are not controlled in stages during fermentation. The oxygen consumption rate OUR is controlled to be 10-100 mmol/L.h in the fermentation process, the ORP is controlled to be 50-150 mv after the 100 th hour of fermentation, the OUR is controlled to be in the range by adjusting at least one of the ventilation, the rotating speed and the tank pressure, and the ORP is controlled to be in the range by adjusting at least one of the ventilation, the rotating speed, the tank pressure, the temperature and the pH value. And culturing for 168 hours to stop fermentation, wherein the mass percentages of biomass, oil yield and arachidonic acid in the fermentation liquid are respectively 44.5g/L, 25.2g/L and 62.1 percent according to detection.
Examples 9 to 11: production of arachidonic acid by fermentation of different strains
Examples 9 to 11, the arachidonic acid was produced by fermentation using different production strains, and the specific conditions and fermentation results are shown in Table 1.
Table 1: fermentation levels of different arachidonic acid producing strains
Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives, and variations may be made in the above embodiments by those skilled in the art without departing from the spirit and principles of the invention.

Claims (6)

1. The production method of the arachidonic acid is characterized by comprising the steps of sequentially carrying out strain activation, seed expansion culture and fermentation culture on Mortierella alpina, and in the fermentation culture process, monitoring OUR on line and controlling the OUR value in six stages, wherein the control mode is as follows: 0-20 h is controlled to be 10-40 mmol/L.h, 20-55 h is controlled to be 20-80 mmol/L.h, 55-80 h is controlled to be 25-100 mmol/L.h, 80-120 h is controlled to be 20-90 mmol/L.h, 120-160 h is controlled to be 15-70 mmol/L.h, and 160-fermentation end is controlled to be 15-60 mmol/L.h; and/or, after 100h of fermentation culture, monitoring ORP in the fermentation broth on line and controlling the ORP value in three stages in the following manner: 100-120 hours, 50-100 mv, 120-160 hours, 60-130 mv and 160 hours, and 70-150 mv after fermentation.
2. The method for producing arachidonic acid according to claim 1, wherein the OUR is controlled in nine stages in the following manner:
controlling the time of 0-20 h to be 10-35 mmol/L.h,
controlling the time of 20-40 h to be 20-70 mmol/L.h,
40 to 55 hours is controlled to be 25 to 80 mmol/L.h,
55 to 80 hours is controlled to be 25 to 100 mmol/L.h,
80-100 h is controlled to be 25-90 mmol/L.h,
100 to 120 hours is controlled to be 20 to 80 mmol/L.h,
120-140 h is controlled to be 20-70 mmol/L.h,
140-160 h is controlled to be 20-65 mmol/L.h,
160h to the end of fermentation, and 15-60 mmol/L.h.
3. The method for producing arachidonic acid according to claim 1, characterized in that the ORP is controlled in four stages and in the following manner:
controlling the time to be 50-100 mv within 100-120 hours,
120-140 h is controlled at 60-120 mv,
140 to 160 hours is controlled to 65 to 130mv,
and controlling the fermentation time to be 160h to 70-140 mv.
4. The method for producing arachidonic acid according to any one of claims 1 to 3, wherein the OUR is controlled by adjusting at least one of ventilation, rotation speed, and tank pressure.
5. The method for producing arachidonic acid according to any one of claims 1 to 3, wherein the ORP is controlled by adjusting at least one of ventilation, rotation speed, tank pressure, temperature, and pH.
6. A method for producing arachidonic acid according to any one of claims 1 to 3, characterized in that it comprises the steps of:
(1) Strain activation: inoculating Mortierella alpina strains into shake flasks containing sterilized strain activation culture medium, and culturing at 25-32 ℃ and 100-200 rpm for 40-48 hours to complete strain activation culture to obtain shake flask seed liquid;
(2) Seed expansion culture: inoculating the shake flask seed liquid into a seed tank filled with the sterilized seed culture medium at an inoculum size of 0.4% -1%, culturing for 30-35 h at a temperature of 25-32 ℃ and a ventilation rate of 1-2 vvm under a tank pressure of 0.02-0.05 MPa, inoculating the shake flask seed liquid into the seed tank filled with the sterilized seed culture medium at an inoculum size of 1% -3%, culturing for 20-25 h at a temperature of 25-32 ℃ and a ventilation rate of 1-2 vvm under a tank pressure of 0.02-0.05 MPa, and optionally repeatedly culturing for 1-3 times to obtain the seed liquid;
(3) Fermentation culture: inoculating 1% -3% of seed liquid into a fermentation tank filled with a sterilized fermentation medium, fermenting and culturing for 150-168 h under the conditions of 20-30 ℃ of temperature, 1-2 vvm of ventilation, 0.02-0.05 MPa of tank pressure and 0-100 rpm of stirring rotation speed, monitoring OUR on line and controlling the value of OUR in a preset range in the fermentation and culturing process, and/or monitoring ORP in the fermentation liquid on line and controlling the value of ORP in a preset range after 100h of fermentation and culturing process, and controlling the pH value of a fermentation system in the whole fermentation process to be 5-7.5.
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