CN112543811A - Production method of arachidonic acid - Google Patents

Production method of arachidonic acid Download PDF

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CN112543811A
CN112543811A CN202080002953.5A CN202080002953A CN112543811A CN 112543811 A CN112543811 A CN 112543811A CN 202080002953 A CN202080002953 A CN 202080002953A CN 112543811 A CN112543811 A CN 112543811A
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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 amplification culture and fermentation culture on mortierella alpina or mutant strains thereof, and in the process of the fermentation culture, monitoring OUR on line and controlling the OUR numerical value to be 10-100 mmol/L.h, and/or after the 100 th hour of the fermentation culture, monitoring ORP in fermentation liquor on line and controlling the ORP numerical value to be 50-150 mv. The method provided by the invention can stably improve the fermentation production level, and obviously improve the content of arachidonic acid in the obtained fermentation liquor.

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 fermentation of mortierella alpina or mutant strains thereof, and particularly relates to a method for producing arachidonic acid by utilizing metabolic parameter OUR and/or ORP to regulate and control the fermentation process.
Background
Arachidonic acid (AA or ARA for short) is an omega-6 polyunsaturated fatty acid, is all-cis-5, 8,11, 14-eicosatetraenoic acid, contains 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. Furthermore, arachidonic acid is a biologically active substance of many circulating eicosanoic acid derivatives, such as prostaglandin E2(PGE2), prostacyclin (PGI2), thromboxane a2(TXA2), leukotrienes, and direct precursors of C4(LTC 4). These bioactive substances have important regulatory effects on lipid protein metabolism, hemorheology, vascular elasticity, leukocyte function, platelet activation, and the like. Arachidonic acid is an important substance for the development of human brain and optic nerve, and has important effects on improving intelligence and enhancing visual acuity. Arachidonic acid has a series of physiological activities such as esterifying cholesterol, increasing blood vessel elasticity, reducing blood viscosity, regulating blood cell function and the like. Arachidonic acid has important effects on preventing cardiovascular diseases, diabetes, tumors, and the like. High purity arachidonic acid is a direct precursor for synthesizing eicosane derivatives such as prostaglandins (prostaglandins), thromboxanes (thromboxanes) and leukotrienes (leucotrienes), and the bioactive substances have very important functions on the cardiovascular system and the immune system of a human body.
At present, arachidonic acid is mainly produced by a microbial fermentation method. The existing fermentation is mainly improved through two directions of strain breeding and process control. Conventional process control is mainly regulated and controlled by single process parameters such as pH, Dissolved Oxygen (DO), temperature, feed, etc. Although the regulation and control method plays a certain role, the process analysis and regulation and control are greatly interfered by more interference factors in the fermentation process, such as the difference of strains, equipment, environment and the like, the regulation and control method with a single factor is difficult to accurately reflect and control the whole fermentation process, the comprehensive analysis of the dynamic fermentation process is lacked fundamentally, the reproducibility is poor, the fermentation level fluctuation is large, and the arachidonic acid content in the obtained fermentation product is low.
Disclosure of Invention
The invention aims to overcome the defects that the fermentation level fluctuation is large and the content of arachidonic acid in the obtained fermentation liquid is low when the fermentation process of the arachidonic acid is controlled by adopting single parameters such as pH value, DO, temperature, supplementary materials and the like in the prior art, and provides a novel production method of the arachidonic acid.
Specifically, the invention provides a production method of arachidonic acid, which comprises the steps of sequentially carrying out strain activation, seed amplification culture and fermentation culture on mortierella alpina or mutant strains thereof, and in the process of fermentation culture, monitoring an Oxygen consumption Rate (OUR) on line and controlling the OUR value to be 10-100 mmol/L.h, and/or monitoring an Oxidation Reduction Potential (ORP) in fermentation liquor on line and controlling the ORP value to be 50-150 mv after 100h of fermentation culture.
Further, the OUR is controlled in six stages in the following control mode: the concentration is controlled to be 10-40 mmol/L.h for 0-20 h, 20-80 mmol/L.h for 20-55 h, 25-100 mmol/L.h for 55-80 h, 20-90 mmol/L.h for 80-120 h, 15-70 mmol/L.h for 120-160 h, and 15-60 mmol/L.h for 160-end of fermentation.
Further, the OUR is controlled in nine stages in the following manner: the concentration is controlled to be 10-35 mmol/L.h for 0-20 h, 20-70 mmol/L.h for 20-40 h, 25-80 mmol/L.h for 40-55 h, 25-100 mmol/L.h for 55-80 h, 25-90 mmol/L.h for 80-100 h, 20-80 mmol/L.h for 100-120 h, 20-70 mmol/L.h for 120-140 h, 20-65 mmol/L.h for 140-160 h, and 15-60 mmol/L.h for 160-160 h.
Further, the ORP is controlled in three stages in the following manner: the fermentation time is controlled to be 50-100 mv for 100-120 h, 60-130 mv for 120-160 h, and 70-150 mv for 160-fermentation end.
Further, the ORP is controlled in four stages in the following manner: the fermentation time is controlled to be 50-100 mv for 100-120 h, 60-120 mv for 120-140 h, 65-130 mv for 140-160 h, and 70-140 mv for 160-fermentation end.
Further, the OUR is controlled by adjusting at least one of ventilation, rotational speed, and canister pressure.
Further, the ORP is controlled by adjusting at least one of ventilation, rotational speed, tank pressure, temperature, and pH.
Further, the present invention provides a method for producing arachidonic acid, comprising the steps of:
(1) strain activation: inoculating the mortierella alpina strain or the mutant strain thereof into a shake flask filled with a sterilized strain activation culture medium, and culturing for 40-48 h under the conditions that the temperature is 25-32 ℃ and the rotating speed is 100-200 rpm, so as to complete strain activation culture and obtain shake flask seed liquid;
(2) seed amplification culture: inoculating the shake flask seed solution into a seed tank filled with a sterilized seed culture medium in an inoculation amount of 0.4-1%, culturing for 30-35 h under the conditions of a temperature of 25-32 ℃, an air throughput of 1-2 vvm and a tank pressure of 0.02-0.05 MPa, optionally inoculating the shake flask seed solution into the seed tank filled with the sterilized seed culture medium in an inoculation amount of 1-3%, culturing for 20-25 h under the conditions of a temperature of 25-32 ℃, an air throughput of 1-2 vvm and a tank pressure of 0.02-0.05 MPa, and optionally repeatedly culturing for 1-3 times to obtain a seed solution;
(3) fermentation culture: inoculating 1-3% of seed liquid into a fermentation tank filled with a sterilized fermentation medium, performing fermentation culture for 150-168 h under the conditions of temperature of 20-30 ℃, ventilation volume of 1-2 vvm, tank pressure of 0.02-0.05 MPa and stirring speed of 0-100 rpm, monitoring OUR on line and controlling the numerical value within a preset range in the fermentation culture process, and/or monitoring ORP in fermentation liquor on line and controlling the numerical value within the preset range after the 100h of the fermentation culture, and controlling the pH value of a fermentation system within 5-7.5 in the whole fermentation process.
After intensive research, the inventor of the invention discovers that in the process of producing arachidonic acid by adopting 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 accurately controlling the numerical value, so that the content of the arachidonic acid in fermentation liquor is obviously improved; on the other hand, the formation of arachidonic acid is accompanied with a large amount of dehydrogenation reaction in the fermentation process, the ORP value is accurately controlled after the 100 th hour of the fermentation, the oxidability of the fermentation liquor can be adjusted, the action of dehydrogenase is facilitated, the fermentation level is stably improved, the formation of arachidonic acid is promoted, and the percentage of arachidonic acid in the 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 present 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 the control method is as follows: 0-20 h is controlled to be 10-40 mmol/L.h, for example, 10, 15, 20, 25, 30, 35, 40 mmol/L.h; 20-55 h is controlled to be 20-80 mmol/L.h, for example, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 mmol/L.h; 55-80 h is controlled to be 25-100 mmol/L.h, for example, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mmol/L.h; the concentration of the organic solvent is controlled to be 20 to 90 mmol/L.h, for example, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 mmol/L.h; 120-160 h is controlled to be 15-70 mmol/L.h, for example, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 mmol/L.h; the concentration of 160 to 60 mmol/L.h 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. It is noted that 0 to 20 hours includes 20 hours of the 1 st hour, the 19 th hour and the 20 th hour … …, 20 to 55 hours includes 35 hours of the 21 st hour, the 22 nd hour … … th hour and the 55 th hour, 55 to 80 hours includes 25 hours of the 56 th hour, the 57 th hour … … th hour and the 80 th hour, 80 to 120 hours includes 40 hours of the 81 th hour, the 82 th hour … … th hour and the 120 th hour, 120 to 160 hours includes 40 hours of the 121 th hour, the 122 th hour … … th 159 hour and the 160 th hour, 160 hours includes the 161 th hour and the 162 th … … hour after the fermentation is finished. In the present invention, the time period definition related to the staged control is the same, and is not described herein again.
In some embodiments of the present 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 the control method is as follows: 0-20 h is controlled to be 10-35 mmol/L.h, for example, 10, 15, 20, 25, 30, 35 mmol/L.h; the concentration of the organic solvent is controlled to be 20 to 70 mmol/L.h, for example, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 mmol/L.h; 40-55 h is controlled to be 25-80 mmol/L.h, for example, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 mmol/L.h; 55-80 h is controlled to be 25-100 mmol/L.h, for example, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mmol/L.h; the concentration of the organic solvent is controlled to be 25 to 90 mmol/L.h, for example, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 mmol/L.h; the concentration of the organic solvent is controlled to be 20 to 80 mmol/L.h for 100 to 120h, and may be, for example, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 mmol/L.h; 120-140 h is controlled to be 20-70 mmol/L.h, for example, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 mmol/L.h; 140-160 h is controlled to be 20-65 mmol/L.h, for example, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 mmol/L.h; 160 to 60 mmol/L.h at the end of fermentation, for example, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 mmol/L.h, etc.
In the invention, the OUR is calculated according to the following formula:
Figure BDA0002796419680000051
Fin: ventilation (L/h or m)3/h)；
V: volume of fermentation broth (L or m)3)；
Co2in: oxygen concentration (%) in intake air;
Cidle in: inert gas concentration (%) in intake air;
Co2out: the oxygen concentration (%) in the exhaust gas discharged from the fermentation broth;
Cco2out: carbon dioxide concentration (%) in the exhaust gas discharged from the fermentation broth.
Wherein Co2in、CIdle in、Co2out、Cco2outReal-time monitoring by mass spectrometer.
In the invention, the ORP needs to be controlled to be 50-150 mv after the 100 th hour of fermentation culture.
In some embodiments of the invention, the ORP is controlled in three stages (i.e., 100-120 h, 120-160 h, 160-fermentation end), and the control is as follows: the amount of 100-120 h is controlled to be 50-100 mv, for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mv, etc.; 120-160 h is controlled at 60-130 mv, for example, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130mv, etc.; the fermentation completion range from 160 to 150mv is controlled, 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 present invention, the ORP is controlled in four stages (i.e., 100 to 120 hours, 120 to 140 hours, 140 to 160 hours, 160 to the end of fermentation), and the control method is as follows: the amount of 100-120 h is controlled to be 50-100 mv, for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mv, etc.; 120-140 h is controlled at 60-120 mv, for example, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120mv, etc.; 140-160 h is controlled at 65-130 mv, for example, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130mv, etc.; the fermentation completion range from 160 to 140mv is controlled, 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 canister pressure. When OUR exceeds a predetermined range, then it is necessary to reduce OUR by down-regulating at least one of aeration, rotational speed, and tank pressure; when the OUR is below a predetermined range, it is then necessary to increase the OUR by up-regulating at least one of the aeration, rotational speed, and tank pressure.
In one embodiment of the 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 the ORP needs to be reduced by adjusting at least one of the ventilation, rotation speed and tank pressure down or adjusting the temperature and/or pH up; when the ORP is below a predetermined range, it is then necessary to increase the ORP by adjusting at least one of the ventilation, rotation speed and tank pressure up or by adjusting the temperature and/or pH down.
In the invention, the mortierella alpina and the mutant strain thereof can be obtained commercially, and can be specifically selected from strains with the preservation numbers of CCTCC No. M2012073, CCTCC No. M2013392, CCTCC No. M2015421 or ATCC No. 42430.
The production process of the arachidonic acid comprises strain activation, seed amplification culture and fermentation culture, wherein the seed amplification 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 a strain activation medium, a seed medium and a fermentation medium for culturing mortierella alpina strain or a mutant strain thereof in the field, which can be known by those skilled in the art and are not described herein again.
In one embodiment, the method for producing arachidonic acid comprises the steps of:
(1) strain activation: inoculating the mortierella alpina strain or the mutant strain thereof into a shake flask filled with a sterilized strain activation culture medium, and culturing for 40-48 h under the conditions that the temperature is 25-32 ℃ and the rotating speed is 100-200 rpm, so as to complete strain activation culture and obtain shake flask seed liquid;
(2) seed amplification culture: inoculating the shake flask seed solution into a seed tank filled with a sterilized seed culture medium in an inoculation amount of 0.4-1%, culturing for 30-35 h under the conditions of a temperature of 25-32 ℃, an air throughput of 1-2 vvm and a tank pressure of 0.02-0.05 MPa, optionally inoculating the shake flask seed solution into the seed tank filled with the sterilized seed culture medium in an inoculation amount of 1-3%, culturing for 20-25 h under the conditions of a temperature of 25-32 ℃, an air throughput of 1-2 vvm and a tank pressure of 0.02-0.05 MPa, and optionally repeatedly culturing for 1-3 times to obtain a seed solution;
(3) fermentation culture: inoculating 1-3% of seed liquid into a fermentation tank filled with a sterilized fermentation medium, performing fermentation culture for 150-168 h under the conditions of temperature of 20-30 ℃, ventilation volume of 1-2 vvm, tank pressure of 0.02-0.05 MPa and stirring speed of 0-100 rpm, monitoring OUR on line and controlling the numerical value within a preset range in the fermentation culture process, and/or monitoring ORP in fermentation liquor on line and controlling the numerical value within the preset range after the 100h of the fermentation culture, and controlling the pH value of a fermentation system within 5-7.5 in the whole fermentation process. Among the agents that adjust the pH include, but are not limited to: sodium hydroxide solution, potassium hydroxide solution, ammonia water, citric acid, and the like. In addition, the fermentation tank used for the fermentation culture may have a volume of 0.5L to 500m3。
The following examples further illustrate the present invention but are not to be construed as limiting thereof. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
In the following examples and comparative examples, the strains and media used, if not otherwise specified, were as follows:
(1) the strain used was Mortierella alpina alpine (ATCC No. 42430);
(2) the formula of the strain activation medium is as follows: 200g of potato (peeled), 20g of glucose, 5g of yeast extract and 1000mL of distilled water, wherein the pH value is natural;
(3) the formula of the seed culture medium is as follows: 200g of potato (peeled), 20g of glucose and 1000mL of distilled water, wherein the pH value is natural;
(4) the formula of the fermentation medium is as follows: 22g/L glucose, 12g/L yeast powder, 8g/L peptone, 15g/L sodium chloride and 6g/L, KH ammonium sulfate2PO4 5g/L、CuSO4·5H2O 1.5μg/L、MnSO4 2μg/L、ZnSO4·7H2O3 mug/L and pH value of 6-7.
In the following examples and comparative examples, the measurement of each parameter was carried out in the following manner:
(1) method for determining biomass: taking a proper amount of fermentation liquor in a weighing bottle, drying in an electrothermal constant-temperature drying oven at 105 ℃ for 4h, then putting in 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 is divided by the volume of the fermentation liquor to obtain the biomass in unit g/L.
(2) The method for measuring the yield of the crude oil comprises the following steps: taking a certain volume of fermentation liquor, adding concentrated hydrochloric acid with the volume being 2 times that of the fermentation liquor, stirring for 50min at a constant temperature of 70 ℃ until thalli are completely digested, adding a proper amount of normal hexane, standing for layering, taking an upper organic phase into an eggplant-shaped bottle by using a dropper, continuously extracting for 6 times until the upper organic phase is colorless, removing the normal hexane through rotary evaporation in a water bath at 80 ℃, then placing the eggplant-shaped bottle into an electrothermal constant-temperature drying oven at 105 ℃ for drying for 1h, then placing the eggplant-shaped bottle into a dryer for cooling to room temperature and weighing, wherein the dry weight of crude oil is the dry weight of crude oil by subtracting the net weight of the eggplant-shaped bottle, and the dry weight of crude oil is the yield.
(3) The method for measuring the mass percentage of the ARA comprises the following steps: the content of ARA in crude oil is measured by gas chromatography in unit percent.
Example 1: process control for 100L fermentation tank
(1) Strain activation: inoculating the Mortierella alpina slant preservation strain into a 2L shake flask containing 400mL of sterilized strain activation medium, culturing at 28 deg.C and 150rpm for 48h to complete strain activation culture, and obtaining shake flask seed solution.
(2) Seed amplification culture: and inoculating the shake flask seed solution into a seed tank filled with the sterilized seed culture medium according to the inoculation amount of 0.4%, culturing at 28 ℃, ventilating 1vvm and tank pressure of 0.04MPa for 35h, and completing seed amplification culture to obtain the seed solution.
(3) Fermentation culture: inoculating the seed solution in the seed tank into a fermentation tank filled with a sterilized fermentation medium according to the inoculation amount of 3 percent, wherein the initial culture conditions are as follows: the culture temperature is 28 ℃, the ventilation volume is 1.0vvm, the tank pressure is 0.04MPa, the rotating speed is 70rpm, and the fermentation culture is carried out for 168 hours to obtain the fermentation liquor. During the fermentation culture process, OUR is monitored on line and controlled within the following range:
1) OUR is controlled to be 10-40 mmol/L.h in fermentation culture for 0-20 h,
2) OUR is controlled to be 20-80 mmol/L.h in fermentation culture for 20-55 h,
3) OUR is controlled to be 25-100 mmol/L.h in 55-80 h of fermentation culture,
4) OUR is controlled to be 20-90 mmol/L.h in fermentation culture for 80-120 h,
5) OUR is controlled to be 15-70 mmol/L.h within 120-160 h of fermentation culture,
6) OUR is controlled to be 15-60 mmol/L.h in fermentation culture for 160-168 h.
The OUR value is controlled by adjusting at least one of ventilation, canister pressure, and rotational speed. When the OUR is lower than the control range, at least one of the ventilation volume, the tank pressure and the rotating speed is adjusted upwards; when the OUR is above the control range, at least one of the aeration, canister pressure, and rotational speed is adjusted downward to control the OUR within a desired range. Culturing for 168h, stopping fermentation, and detecting that the biomass, the oil yield and the mass percentage of the arachidonic acid in the fermentation liquor are 45g/L, 26.7g/L and 67 percent respectively.
Example 2: 100m3Fermentation tank process control
(1) Strain activation: inoculating the Mortierella alpina slant preservation strain into a 2L shake flask containing 400mL of sterilized strain activation medium, culturing at 28 deg.C and 150rpm for 48h to complete strain activation culture, and obtaining shake flask seed solution.
(2) First-order seed culture: and inoculating the shake flask seed solution into a primary seed tank filled with the sterilized seed culture medium according to the inoculation amount of 0.4%, culturing at 28 ℃, ventilating 1vvm and tank pressure of 0.04MPa for 35h to complete primary seed amplification culture, and obtaining the primary seed solution.
(3) Secondary seed culture: and (3) inoculating the primary seed solution into a secondary seed tank filled with a sterilized seed culture medium according to the inoculation amount of 3%, culturing at 28 ℃, ventilating 1vvm and tank pressure of 0.04MPa for 24h, and completing secondary seed amplification culture to obtain a secondary seed solution.
(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 percent, wherein the initial culture conditions are as follows: the culture temperature is 28 ℃, the ventilation volume is 1.0vvm, the tank pressure is 0.04MPa, the rotating speed is 50rpm, and the fermentation culture is carried out for 168 hours to obtain the fermentation liquor. During the fermentation culture process, OUR is monitored on line and controlled within the following range:
1) OUR is controlled to be 10 to 35 mmol/L.h within 0 to 20h,
2) OUR is controlled to be 20-70 mmol/L.h within 20-40 h,
3) OUR is controlled to be 25-80 mmol/L.h within 40-55 h,
4) OUR is controlled to be 25-100 mmol/L.h within 55-80 h,
5) OUR is controlled to be 25-90 mmol/L.h within 80-100 h,
6) OUR is controlled to be 20-80 mmol/L.h within 100-120 h,
7) OUR is controlled to be 20-70 mmol/L.h within 120-140 h,
8) OUR is controlled to be 20 to 65 mmol/L.h within 140 to 160h,
9) OUR is controlled to be 15-60 mmol/L.h within 160-168 h.
The OUR value is controlled by adjusting at least one of ventilation, canister pressure, and rotational speed. When the OUR is lower than the control range, at least one of the ventilation volume, the tank pressure and the rotating speed is adjusted upwards; when the OUR is above the control range, at least one of the aeration, canister pressure, and rotational speed is adjusted downward to control the OUR within a desired range. Culturing for 168h, stopping fermentation, and detecting that the biomass, the oil yield and the mass percentage content of the arachidonic acid in the fermentation liquor are 48.5g/L, 30.1g/L and 66.8% respectively.
Example 3: 300m3Fermentation tank process control
(1) Strain activation: inoculating the Mortierella alpina slant preservation strain into a 2L shake flask containing 400mL of sterilized strain activation medium, culturing at 28 deg.C and 150rpm for 48h to complete strain activation culture, and obtaining shake flask seed solution.
(2) First-order seed culture: and inoculating the shake flask seed solution into a primary seed tank filled with the sterilized seed culture medium according to the inoculation amount of 0.4%, culturing at 28 ℃, ventilating 1vvm and tank pressure of 0.04MPa for 35h to complete primary seed amplification culture, and obtaining the primary seed solution.
(3) Secondary seed culture: and (3) inoculating the primary seed solution into a secondary seed tank filled with a sterilized seed culture medium according to the inoculation amount of 3%, culturing at 28 ℃, ventilating 1vvm and tank pressure of 0.04MPa for 24h, and completing secondary seed amplification culture to obtain a secondary seed solution.
(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 percent, wherein the initial culture conditions are as follows: the culture temperature is 28 ℃, the ventilation volume is 1.0vvm, the tank pressure is 0.04MPa, the rotating speed is 30rpm, and the fermentation culture is carried out for 168 hours to obtain the fermentation liquor. During the fermentation culture process, OUR is monitored on line and controlled within the following range:
1) OUR is controlled to be 10 to 35 mmol/L.h within 0 to 20h,
2) OUR is controlled to be 20-70 mmol/L.h within 20-40 h,
3) OUR is controlled to be 25-80 mmol/L.h within 40-55 h,
4) OUR is controlled to be 25-100 mmol/L.h within 55-80 h,
5) OUR is controlled to be 25-90 mmol/L.h within 80-100 h,
6) OUR is controlled to be 20-80 mmol/L.h within 100-120 h,
7) OUR is controlled to be 20-70 mmol/L.h within 120-140 h,
8) OUR is controlled to be 20 to 65 mmol/L.h within 140 to 160h,
9) OUR is controlled to be 15-60 mmol/L.h within 160-168 h.
The OUR value is controlled by adjusting at least one of ventilation, canister pressure, and rotational speed. When the OUR is lower than the control range, at least one of the ventilation volume, the tank pressure and the rotating speed is adjusted upwards; when the OUR is above the control range, at least one of the aeration, canister pressure, and rotational speed is adjusted downward to control the OUR within a desired range. Culturing for 168h, stopping fermentation, and detecting that the biomass, the oil yield and the mass percentage content of the arachidonic acid in the fermentation liquor are 48.7g/L, 29.6g/L and 67.2 percent respectively.
Example 1-1: 100m3Fermentation tank process control
Arachidonic acid was produced according to the procedure of example 2, except that during the fermentation culture, OUR was monitored on-line and controlled to the following range:
1) OUR is controlled to be 10 to 35 mmol/L.h within 0 to 20h,
2) OUR is controlled to be 20-70 mmol/L.h within 20-40 h,
3) OUR is controlled to be 25-80 mmol/L.h within 40-55 h,
4) OUR is controlled to be 25-100 mmol/L.h within 55-80 h,
5) OUR is controlled to be 25-90 mmol/L.h within 80-100 h,
6) OUR is controlled to be 20-80 mmol/L.h within 100-120 h,
7) OUR is controlled to be 70-80 mmol/L.h within 120-140 h,
8) OUR is controlled to be 10 to 20 mmol/L.h within 140 to 160h,
9) OUR is controlled to be 15-60 mmol/L.h within 160-168 h.
The fermentation is stopped after 168 hours of culture, and the biomass, the oil yield and the mass percentage content of the arachidonic acid in the fermentation liquor are respectively 46.2g/L, 27.1g/L and 66.5 percent by detection. Compared with the example 2, part of the interval is not controlled in the OUR preferred range, and the biomass, the oil yield and the arachidonic acid mass percentage content in the fermentation liquor are all lower than those in the example 2.
Comparative example 1
Arachidonic acid was produced according to the method of example 1-1, except that during the fermentation culture, OUR was monitored on-line and controlled to the following range:
1) OUR is controlled to be 5-35 mmol/L.h within 0-20 h,
2) OUR is controlled to be 20-70 mmol/L.h within 20-40 h,
3) OUR is controlled to be 25-80 mmol/L.h within 40-55 h,
4) OUR is controlled to be 25-120 mmol/L.h within 55-80 h,
5) OUR is controlled to be 25-90 mmol/L.h within 80-100 h,
6) OUR is controlled to be 20-80 mmol/L.h within 100-120 h,
7) OUR is controlled to be 70-80 mmol/L.h within 120-140 h,
8) OUR is controlled to be 10 to 20 mmol/L.h within 140 to 160h,
9) OUR is controlled to be 15-60 mmol/L.h within 160-168 h.
And (3) culturing for 168h, and stopping fermentation, wherein the biomass, the oil yield and the mass percentage content of the arachidonic acid in the fermentation liquid are respectively 43.2g/L, 24.8g/L and 57.8% by detection. Compared with example 1-1, OUR value falls outside the scope of the present invention in part of the interval, and biomass, oil yield, and arachidonic acid content are all lower than in example 1-1.
Example 4: 100m3Fermentation tank process control
(1) Strain activation: inoculating the Mortierella alpina slant preservation strain into a 2L shake flask containing 400mL of sterilized strain activation medium, culturing at 28 deg.C and 150rpm for 48h to complete strain activation culture, and obtaining shake flask seed solution.
(2) First-order seed culture: and inoculating the shake flask seed solution into a primary seed tank filled with the sterilized seed culture medium according to the inoculation amount of 0.4%, culturing at 28 ℃, ventilating 1vvm and tank pressure of 0.04MPa for 35h to complete primary seed amplification culture, and obtaining the primary seed solution.
(3) Secondary seed culture: and (3) inoculating the primary seed solution into a secondary seed tank filled with a sterilized seed culture medium according to the inoculation amount of 3%, culturing at 28 ℃, ventilating 1vvm and tank pressure of 0.04MPa for 24h, and completing secondary seed amplification culture to obtain a secondary seed solution.
(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 percent, wherein the initial culture conditions are as follows: the culture temperature is 28 ℃, the ventilation volume is 1.0vvm, the tank pressure is 0.04MPa, the rotating speed is 45rpm, and the fermentation culture is carried out for 168 hours to obtain the fermentation liquor. After 100h of fermentation culture, the ORP in the fermentation broth was monitored on-line and controlled to the following range:
1) controlling ORP at 50-100 mv for 100-120 h,
2) controlling ORP at 60-130 mv within 120-160 h,
3) controlling ORP at 70-150 mv within 160-168 h.
The ORP value is controlled by adjusting at least one of aeration, tank pressure, rotational speed, temperature, and pH. When the ORP is lower than the control range, at least one of ventilation, tank pressure and rotating speed is adjusted upwards, or fermentation temperature and/or pH value is adjusted downwards; when the ORP is above the control range, at least one of aeration, tank pressure and rotational speed is adjusted down, or fermentation temperature and/or pH is adjusted up, to control ORP within the desired range. And (3) culturing for 168h, and stopping fermentation, wherein the biomass, the oil yield and the mass percentage content of the arachidonic acid in the fermentation liquid are respectively 43.5g/L, 25.2g/L and 68.3% by detection.
Example 5: 100m3Fermentation tank process control
(1) Strain activation: inoculating the Mortierella alpina slant preservation strain into a 2L shake flask containing 400mL of sterilized strain activation medium, culturing at 28 deg.C and 150rpm for 48h to complete strain activation culture, and obtaining shake flask seed solution.
(2) First-order seed culture: and inoculating the shake flask seed solution into a primary seed tank filled with the sterilized seed culture medium according to the inoculation amount of 0.4%, culturing at 28 ℃, ventilating 1vvm and tank pressure of 0.04MPa for 35h to complete primary seed amplification culture, and obtaining the primary seed solution.
(3) Secondary seed culture: and (3) inoculating the primary seed solution into a secondary seed tank filled with a sterilized seed culture medium according to the inoculation amount of 3%, culturing at 28 ℃, ventilating 1vvm and tank pressure of 0.04MPa for 24h, and completing secondary seed amplification culture to obtain a secondary seed solution.
(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 percent, wherein the initial culture conditions are as follows: the culture temperature is 28 ℃, the ventilation volume is 1.0vvm, the tank pressure is 0.04MPa, the rotation speed is 45rpm, and the fermentation culture is 168 hours. After 100h of fermentation culture, the ORP in the fermentation broth was monitored on-line and controlled to the following range:
1) controlling ORP at 50-100 mv for 100-120 h,
2) controlling ORP at 60-120 mv for 120-140 h,
3) controlling ORP at 65-130 mv for 140-160 h,
4) controlling ORP at 70-140 mv within 160-168 h.
The ORP value is controlled by adjusting at least one of aeration, tank pressure, rotational speed, temperature, and pH. When the ORP is lower than the control range, at least one of ventilation, tank pressure and rotating speed is adjusted upwards, or fermentation temperature and/or pH value is adjusted downwards; when the ORP is above the control range, at least one of aeration, tank pressure and rotational speed is adjusted down, or fermentation temperature and/or pH is adjusted up, to control ORP within the desired range. And (3) culturing for 168h, and stopping fermentation, wherein the biomass, the oil yield and the mass percentage content of the arachidonic acid in the fermentation liquid are respectively 44.1g/L, 25.8g/L and 68.6% by detection.
Comparative example 2: 100m3Fermentation tank process control
Arachidonic acid was produced according to the method of example 5, except that after 100 hours of culture, ORP in the fermentation broth was monitored on-line and controlled to the following range:
1) controlling ORP at 50-100 mv for 100-120 h,
2) controlling ORP at 60-120 mv for 120-140 h,
3) controlling ORP at 40-65 mv for 140-160 h,
4) controlling the ORP to be 35-60 mv within 160-168 hours.
And (3) stopping fermentation after culturing for 168h, wherein the biomass, the oil yield and the mass percentage content of the arachidonic acid in the fermentation liquid are respectively 43.1g/L, 23.6g/L and 58.4 percent by detection. Compared with example 5, the ORP value falls outside the scope of the invention in part of the interval, and the biomass, the oil yield and the arachidonic acid content in mass percent in the fermentation liquor are all lower than those in example 5.
Example 6: 100m3Fermentation tank process control
(1) Strain activation: inoculating the Mortierella alpina slant preservation strain into a 2L shake flask containing 400mL of sterilized strain activation medium, culturing at 28 deg.C and 150rpm for 48h to complete strain activation culture, and obtaining shake flask seed solution.
(2) First-order seed culture: and inoculating the shake flask seed solution into a primary seed tank filled with the sterilized seed culture medium according to the inoculation amount of 0.4%, culturing at 28 ℃, ventilating 1vvm and tank pressure of 0.04MPa for 35h to complete primary seed amplification culture, and obtaining the primary seed solution.
(3) Secondary seed culture: and (3) inoculating the primary seed solution into a secondary seed tank filled with a sterilized seed culture medium according to the inoculation amount of 3%, culturing at 28 ℃, ventilating 1vvm and tank pressure of 0.04MPa for 24h, and completing secondary seed amplification culture to obtain a secondary seed solution.
(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 percent, wherein the initial culture conditions are as follows: the culture temperature is 28 ℃, the ventilation volume is 1.0vvm, the tank pressure is 0.04MPa, the rotation speed is 45rpm, and the fermentation culture is 168 hours. During the fermentation culture, OUR is monitored on-line and controlled to be within the following range, and after 100h of fermentation culture, ORP in the fermentation broth is monitored on-line and controlled to be within the following range:
1) OUR is controlled to be 10 to 35 mmol/L.h within 0 to 20h,
2) OUR is controlled to be 20-70 mmol/L.h within 20-40 h,
3) OUR is controlled to be 25-80 mmol/L.h within 40-55 h,
4) OUR is controlled to be 25-100 mmol/L.h within 55-80 h,
5) OUR is controlled to be 25-90 mmol/L.h within 80-100 h,
6) OUR is controlled to be 20-80 mmol/L.h for 100-120 h, ORP is controlled to be 50-100 mv,
7) OUR is controlled to be 20-70 mmol/L.h within 120-140 h, ORP is controlled to be 60-120 mv,
8) OUR is controlled to be 20-65 mmol/L.h within 140-160 h, ORP is controlled to be 65-130 mv,
9) OUR is controlled to be 15-60 mmol/L.h within 160-168 h, and ORP is controlled to be 70-140 mv.
The OUR value is controlled by adjusting at least one of ventilation, canister pressure, and rotational speed. When the OUR is lower than the control range, at least one of the ventilation volume, the tank pressure and the rotating speed is adjusted upwards; when the OUR is above the control range, at least one of the aeration, canister pressure, and rotational speed is adjusted downward to control the OUR within a desired range. The ORP value is controlled by adjusting at least one of aeration, tank pressure, rotational speed, temperature, and pH. When the ORP is lower than the control range, at least one of ventilation, tank pressure and rotating speed is adjusted upwards, or fermentation temperature and/or pH value is adjusted downwards; when the ORP is above the control range, at least one of aeration, tank pressure and rotational speed is adjusted down, or fermentation temperature and/or pH is adjusted up, to control ORP within the desired range. And (3) culturing for 168h, and stopping fermentation, wherein the biomass, the oil yield and the mass percentage content of the arachidonic acid in the fermentation liquor are respectively 50.2g/L, 31.3g/L and 69.2% by detection.
Example 7: 100m3Fermentation tank process control
(1) Strain activation: inoculating the Mortierella alpina slant preservation strain into a 2L shake flask containing 400mL of sterilized strain activation medium, culturing at 28 deg.C and 150rpm for 48h to complete strain activation culture, and obtaining shake flask seed solution.
(2) First-order seed culture: and inoculating the shake flask seed solution into a primary seed tank filled with the sterilized seed culture medium according to the inoculation amount of 0.4%, culturing at 28 ℃, ventilating 1vvm and tank pressure of 0.04MPa for 35h to complete primary seed amplification culture, and obtaining the primary seed solution.
(3) Secondary seed culture: and (3) inoculating the primary seed solution into a secondary seed tank filled with a sterilized seed culture medium according to the inoculation amount of 3%, culturing at 28 ℃, ventilating 1vvm and tank pressure of 0.04MPa for 24h, and completing secondary seed amplification culture to obtain a secondary seed solution.
(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 percent, wherein the initial culture conditions are as follows: the culture temperature is 28 ℃, the ventilation volume is 1.0vvm, the tank pressure is 0.04MPa, the rotation speed is 45rpm, and the fermentation culture is 168 hours. During the fermentation culture, OUR is monitored on-line and controlled to be within the following range, and after 100h of fermentation culture, ORP in the fermentation broth is monitored on-line and controlled to be within the following range:
1) OUR is controlled to be 10 to 40 mmol/L.h within 0 to 20h,
2) OUR is controlled to be 20-80 mmol/L.h within 20-55 h,
3) OUR is controlled to be 25-100 mmol/L.h within 55-80 h,
4) OUR is controlled to be 20-90 mmol/L.h within 80-120 h, ORP is controlled to be 50-100 mv within 100-120 h,
5) OUR is controlled to be 15-70 mmol/L.h within 120-160 h, ORP is controlled to be 60-130 mv,
6) OUR is controlled to be 15-60 mmol/L.h within 160-168 h, and ORP is controlled to be 70-150 mv.
The OUR value is controlled by adjusting at least one of ventilation, canister pressure, and rotational speed. When the OUR is lower than the control range, at least one of the ventilation volume, the tank pressure and the rotating speed is adjusted upwards; when the OUR is above the control range, at least one of the aeration, canister pressure, and rotational speed is adjusted downward to control the OUR within a desired range. The ORP value is controlled by adjusting at least one of aeration, tank pressure, rotational speed, temperature, and pH. When the ORP is lower than the control range, at least one of ventilation, tank pressure and rotating speed is adjusted upwards, or fermentation temperature and/or pH value is adjusted downwards; when the ORP is above the control range, at least one of aeration, tank pressure and rotational speed is adjusted down, or fermentation temperature and/or pH is adjusted up, to control ORP within the desired range. And (3) culturing for 168h, and stopping fermentation, wherein the biomass, the oil yield and the mass percentage content of the arachidonic acid in the fermentation liquor are respectively 47.6g/L, 30.1g/L and 67.6 percent through detection.
Example 8: 100m3Fermentation tank process control
(1) Strain activation: inoculating the Mortierella alpina slant preservation strain into a 2L shake flask containing 400mL of sterilized strain activation medium, culturing at 28 deg.C and 150rpm for 48h to complete strain activation culture, and obtaining shake flask seed solution.
(2) First-order seed culture: and inoculating the shake flask seed solution into a primary seed tank filled with the sterilized seed culture medium according to the inoculation amount of 0.4%, culturing at 28 ℃, ventilating 1vvm and tank pressure of 0.04MPa for 35h to complete primary seed amplification culture, and obtaining the primary seed solution.
(3) Secondary seed culture: and (3) inoculating the primary seed solution into a secondary seed tank filled with a sterilized seed culture medium according to the inoculation amount of 3%, culturing at 28 ℃, ventilating 1vvm and tank pressure of 0.04MPa for 24h, and completing secondary seed amplification culture to obtain a secondary seed solution.
(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 percent, wherein the initial culture conditions are as follows: the culture temperature is 28 ℃, the ventilation volume is 1.0vvm, the tank pressure is 0.04MPa, the rotation speed is 45rpm, and the fermentation culture is 168 hours.
The oxygen consumption rate OUR and the oxidation-reduction potential ORP are not controlled in stages in the fermentation process. During the fermentation process, the oxygen consumption rate OUR is controlled to be 10-100 mmol/L.h, after the 100 th hour of the fermentation, the ORP is controlled to be 50-150 mv, during the process, the OUR is controlled to be in a range by adjusting at least one of the ventilation volume, the rotation speed and the tank pressure, and the ORP is controlled to be in the range by adjusting at least one of the ventilation volume, the rotation speed, the tank pressure, the temperature and the pH value. And (3) stopping fermentation after culturing for 168h, wherein the biomass, the oil yield and the mass percentage content of the arachidonic acid in the fermentation liquid are respectively 44.5g/L, 25.2g/L and 62.1 percent by detection.
Examples 9 to 11: production of arachidonic acid by fermentation of different strains
Examples 9 to 11, 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
Figure BDA0002796419680000181
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.

Claims (6)

1. A production method of arachidonic acid is characterized in that the method comprises the steps of sequentially carrying out strain activation, seed amplification 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 in six stages in a control mode as follows: controlling the concentration of the active components in the solution to be 10-40 mmol/L.h for 0-20 h, 20-80 mmol/L.h for 20-55 h, 25-100 mmol/L.h for 55-80 h, 20-90 mmol/L.h for 80-120 h, 15-70 mmol/L.h for 120-160 h, and 15-60 mmol/L.h for 160-end of fermentation; and/or, after the 100 th hour of the fermentation culture, monitoring the ORP in the fermentation liquor on line and controlling the ORP value in three stages in the following way: the fermentation time is controlled to be 50-100 mv for 100-120 h, 60-130 mv for 120-160 h, and 70-150 mv for 160-fermentation end.
2. A process for producing arachidonic acid according to claim 1 wherein the OUR is controlled in nine stages in the following manner:
controlling the concentration of the solution in a range of 10 to 35 mmol/L.h for 0 to 20h,
controlling the concentration of the solution in 20-40 h to 20-70 mmol/L.h,
controlling the concentration of the solution to be 25-80 mmol/L.h within 40-55 h,
the time is controlled to be 25 to 100 mmol/L.h within 55 to 80h,
controlling the concentration of the solution to be 25-90 mmol/L.h within 80-100 h,
controlling the concentration of the solution in 20-80 mmol/L.h for 100-120 h,
controlling the concentration of the solution to be 20-70 mmol/L.h for 120-140 h,
the concentration of the catalyst is controlled to be 20 to 65 mmol/L.h within 140 to 160h,
and controlling the concentration of the fermentation liquor between 160 and 60 mmol/L.h after the fermentation is finished.
3. A process for producing arachidonic acid according to claim 1, characterized in that the ORP is controlled in four stages in the following manner:
the time is controlled to be 50-100 mv for 100-120 h,
120-140 hours is controlled to be 60-120 mv,
140 to 160 hours are controlled to be 65 to 130mv,
the fermentation time is controlled to be between 160 and 140mv after the fermentation is finished.
4. A process for producing arachidonic acid according to any of claims 1-3, wherein the OUR is controlled by adjusting at least one of the aeration rate, the rotation speed and the tank pressure.
5. A method for producing arachidonic acid according to any of claims 1-3, characterized in that the ORP is controlled by adjusting at least one of the aeration rate, the rotation speed, the tank pressure, the temperature and the pH.
6. A process for producing arachidonic acid according to any of claims 1 to 3, characterized in that it comprises the following steps:
(1) strain activation: inoculating the mortierella alpina strain or the mutant strain thereof into a shake flask filled with a sterilized strain activation culture medium, and culturing for 40-48 h under the conditions that the temperature is 25-32 ℃ and the rotating speed is 100-200 rpm, so as to complete strain activation culture and obtain shake flask seed liquid;
(2) seed amplification culture: inoculating the shake flask seed solution into a seed tank filled with a sterilized seed culture medium in an inoculation amount of 0.4-1%, culturing for 30-35 h under the conditions of a temperature of 25-32 ℃, an air throughput of 1-2 vvm and a tank pressure of 0.02-0.05 MPa, optionally inoculating the shake flask seed solution into the seed tank filled with the sterilized seed culture medium in an inoculation amount of 1-3%, culturing for 20-25 h under the conditions of a temperature of 25-32 ℃, an air throughput of 1-2 vvm and a tank pressure of 0.02-0.05 MPa, and optionally repeatedly culturing for 1-3 times to obtain a seed solution;
(3) fermentation culture: inoculating 1-3% of seed liquid into a fermentation tank filled with a sterilized fermentation medium, performing fermentation culture for 150-168 h under the conditions of temperature of 20-30 ℃, ventilation volume of 1-2 vvm, tank pressure of 0.02-0.05 MPa and stirring speed of 0-100 rpm, monitoring OUR on line and controlling the numerical value within a preset range in the fermentation culture process, and/or monitoring ORP in fermentation liquor on line and controlling the numerical value within the preset range after the 100h of the fermentation culture, and controlling the pH value of a fermentation system within 5-7.5 in the whole fermentation process.
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