WO2022104673A1

WO2022104673A1 - Method for producing arachidonic acid

Info

Publication number: WO2022104673A1
Application number: PCT/CN2020/130322
Authority: WO
Inventors: 王霄凤; 陈必钦; 严必能; 刘子睿
Original assignee: 内蒙古金达威药业有限公司; 厦门金达威集团股份有限公司
Priority date: 2020-11-20
Filing date: 2020-11-20
Publication date: 2022-05-27
Also published as: CN112543811A; CN112543811B

Abstract

The invention belongs to the field of fermentation engineering, and relates to a method for producing arachidonic acid. The method comprises: successively subjecting Mortierella alpine or mutant strains thereof to strain activation, seed amplification culture and fermentation culture, wherein OUR is monitored online, and the OUR value is controlled at 10-100 mmol/L.h during the fermentation culture, and/or after 100 h of fermentation culture, ORP is monitored in fermentation liquid online, and the ORP value is controlled to be within the range of 50-150 mv. By adopting the method, the fermentation production level can be stably improved, such that the content of the arachidonic acid in the obtained fermentation solution is obviously improved.

Description

A kind of production method of arachidonic acid

technical field

The invention belongs to the field of fermentation engineering, and relates to a method for producing arachidonic acid by utilizing Mortierella alpina or its mutant strain by fermentation, in particular to a method for producing arachidonic acid by utilizing metabolic parameters OUR and/or ORP to regulate and control the fermentation process. method.

Background technique

Arachidonic acid (AA or ARA for short) is an omega-6 polyunsaturated fatty acid that is all-cis-5,8,11,14-eicosatetraenoic acid, which contains four carbon-carbon double bonds and A carbon-oxygen double bond is a higher unsaturated fatty acid. Arachidonic acid plays an important role as a phospholipid-bound structural lipid in the blood, liver, muscle and other organ systems. In addition, arachidonic acid is the biologically active substance of many circulating eicosanoid derivatives, such as prostaglandin E2 (PGE2), prostacyclin (PGI2), thromboxane A2 (TXA2), leukotrienes and C4 (LTC4 ) direct precursor. These biologically active substances have important regulatory effects on lipid and protein metabolism, blood rheology, vascular elasticity, leukocyte function and platelet activation. Arachidonic acid is an important substance for the development of the human brain and optic nerve, and plays an important role in improving intelligence and enhancing visual acuity. Arachidonic acid has a series of physiological activities such as esterification of cholesterol, increase of blood vessel elasticity, reduction of blood viscosity and regulation of blood cell function. Arachidonic acid has important effects on the prevention of cardiovascular disease, diabetes and tumors. High-purity arachidonic acid is the direct precursor for the synthesis of prostaglandins, thromboxanes, and leukotrienes and other icosyl derivatives. These biologically active substances have effects on the human cardiovascular system and immune system. The system plays a very important role.

At present, arachidonic acid is mainly produced by microbial fermentation. The existing fermentation is mainly improved through strain selection and process control. Conventional process control is mainly adjusted and controlled by a single process parameter such as pH value, dissolved oxygen (DO), temperature, and feeding. Although this control method also plays a certain role, due to the many interference factors in the fermentation process, such as differences in bacterial species, equipment, and environment, the process analysis and control will be greatly interfered. It is difficult to accurately reflect and control the entire fermentation process, lack of a fundamental comprehensive analysis of the fermentation dynamic process, poor reproducibility, large fluctuations in the fermentation level, and low arachidonic acid content in the resulting fermentation product.

SUMMARY OF THE INVENTION

The object of the present invention is to overcome the prior art and adopt single parameters such as pH value, DO, temperature, feeding to control the arachidonic acid fermentation process, and there is a large fluctuation in the fermentation level, and the arachidonic acid content in the obtained fermentation broth is relatively high. Therefore, a new method for producing arachidonic acid is provided, which can stably improve the fermentation production level and significantly increase the content of arachidonic acid in the obtained fermentation broth.

Specifically, the present invention provides a method for producing arachidonic acid, the method comprising sequentially performing strain activation, seed expansion culture and fermentation culture on Mortierella alpina or a mutant thereof, and during the fermentation culture process, online monitoring Oxygen consumption rate (Oxygen Uptake Rate, the abbreviation OUR is used below) and the value of OUR is controlled at 10-100 mmol/L h, and/or, after the 100th hour of fermentation culture, online monitoring of the redox potential (Oxidation potential) in the fermentation broth. Reduction Potential, the abbreviation ORP is used below) and the ORP value is controlled at 50-150mv.

Further, the OUR is controlled in six stages, and the control method is as follows: 0～20h is controlled at 10～40mmol/L·h, 20～55h is controlled at 20～80mmol/L·h, 55～80h is controlled at 25～80 mmol/L·h 100mmol/L·h, 80～120h controlled at 20～90mmol/L·h, 120～160h controlled at 15～70mmol/L·h, 160～end of fermentation controlled at 15～60mmol/L·h.

Further, the OUR is controlled in nine stages, and the control method is as follows: 0～20h is controlled at 10～35mmol/L·h, 20～40h is controlled at 20～70mmol/L·h, 40～55h is controlled at 25～ 80mmol/L·h, 55～80h at 25～100mmol/L·h, 80～100h at 25～90mmol/L·h, 100～120h at 20～80mmol/L·h, 120～140h at 20～80mmol/L·h 20～70mmol/L·h, 140～160h controlled at 20～65mmol/L·h, 160～fermentation end controlled at 15～60mmol/L·h.

Further, the ORP is controlled in three stages, and the control method is as follows: 100-120 h is controlled at 50-100 mv, 120-160 h is controlled at 60-130 mv, and 160-fermentation end is controlled at 70-150 mv.

Further, the ORP is controlled in four stages, and the control method is as follows: 100-120h is controlled at 50-100mv, 120-140h is controlled at 60-120mv, 140-160h is controlled at 65-130mv, 160-fermentation end is controlled at 60-130mv 70～140mv.

Further, the OUR is controlled by adjusting at least one of ventilation volume, rotational speed and tank pressure.

Further, the ORP is controlled by adjusting at least one of ventilation rate, rotational speed, tank pressure, temperature and pH value.

Further, the production method of arachidonic acid provided by the invention may further comprise the steps:

(1) Strain activation: insert the Mortierella alpina strain or its mutant strain into the shake flask containing the sterilized strain activation medium, and cultivate for 40 to 48 hours at a temperature of 25 to 32 °C and a rotation speed of 100 to 200 rpm. , to complete the activation and cultivation of strains to obtain shake flask seed liquid;

(2) Seed expansion culture: insert the shake flask seed liquid into the seed tank containing the sterilized seed medium at an inoculation amount of 0.4% to 1%, at a temperature of 25 to 32 ° C, aeration volume of 1 to 2 vvm, and the tank Under the condition of pressure of 0.02-0.05MPa, cultivate for 30-35h, and optionally insert 1%-3% of the inoculum into the seed tank containing the sterilized seed medium. Cultivate for 20-25h under the conditions of ~2vvm and tank pressure of 0.02-0.05MPa, optionally repeat the cultivation 1-3 times to obtain seed liquid;

(3) Fermentation culture: insert the seed liquid into a fermenter with a sterilized fermentation medium at an inoculation amount of 1% to 3%, at a temperature of 20 to 30° C., a ventilation volume of 1 to 2 vvm, and a tank pressure of 0.02 to 0.02 Fermentation and culture under the conditions of 0.05MPa and 0-100rpm stirring speed for 150-168h, during the fermentation and culture process, monitor OUR online and control its value within a predetermined range, and/or, after the 100th hour of fermentation and culture, online monitoring The ORP in the fermentation broth is controlled within a predetermined range, and the pH value of the fermentation system is controlled at 5-7.5 during the entire fermentation process.

After in-depth research, the inventors of the present invention found that in the process of using Mortierella alpina or its mutant to produce arachidonic acid, on the one hand, the OUR value in the whole fermentation and culture process can well reflect the growth process of the bacterial cells, Through precise control of its value, the fermentation level can be stably improved, so that the content of arachidonic acid in the fermentation broth is significantly increased; on the other hand, the formation of arachidonic acid in the fermentation process is accompanied by a large number of dehydrogenation reactions, Precise control of the ORP value after the 100th hour of fermentation can adjust the oxidative properties of the fermentation broth, help the action of dehydrogenase, stably improve the fermentation level, and promote the formation of arachidonic acid, so that arachidonic acid accounts for the total oil and fat. percentage has increased significantly.

Detailed ways

In the present invention, the OUR needs to be controlled at 10-100 mmol/L·h during the entire fermentation process.

In some embodiments of the present invention, OUR is controlled in six stages (ie, 0-20h, 20-55h, 55-80h, 80-120h, 120-160h, and 160-fermentation end), and the control method is as follows: 0~20h is controlled at 10~40mmol/L·h, for example, it can be 10, 15, 20, 25, 30, 35, 40mmol/L·h, etc.; 20~55h is controlled at 20~80mmol/L·h, for example , can be 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80mmol/L·h, etc.; 55-80h can be controlled at 25-100mmol/L·h, for example, can 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mmol/L·h, etc.; 80～120h controlled at 20～90mmol/L·h , for example, it can be 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 mmol/L h, etc.; 120 to 160 h is controlled at 15 to 70 mmol/L h, for example, can be 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 mmol/L h, etc.; 160 to the end of fermentation is controlled at 15 to 60 mmol/L h, For example, it can be 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 mmol/L·h, or the like. It should be noted that 0~20h includes 1h, 2h... 19h, 20h total 20h, 20~55h includes 21h, 22h... 54h, 55h total 35h, 55~80h includes 56h, 57h...79h, 80h total 25h, 80~120h includes 81h, 82h...119h, 120h total 40h, 120~160h includes 121h, 122h...159h, 160h total 40h, 160h to the end of fermentation, including 161h, 162h...the end of fermentation. In the present invention, the definition of the time period related to the staged control is the same, and will not be repeated here.

In some embodiments of the present invention, there are nine stages (ie 0-20h, 20-40h, 40-55h, 55-80h, 80-100h, 100-120h, 120-140h, 140-160h, 160-fermentation End) Control OUR, and the control method is as follows: 0~20h is controlled at 10~35mmol/L·h, for example, it can be 10, 15, 20, 25, 30, 35mmol/L·h, etc.; 20~40h control At 20～70mmol/L·h, for example, it can be 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70mmol/L·h, etc.; 40～55h can be controlled at 25～80mmol/L h, for example, can be 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 mmol/L h, etc.; 55 to 80 h is controlled at 25 to 100 mmol/L h, for example , can be 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mmol/L h, etc.; 80 ~ 100h control at 25 ~ 90 mmol / L h, for example, can be 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 mmol/L h, etc.; 100-120 h is controlled at 20-80 mmol/L h, for example, can be 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 mmol/L h, etc.; 120 to 140 h is controlled at 20 to 70 mmol/L h , for example, it can be 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70mmol/L·h, etc.; 140-160h can be controlled at 20-65mmol/L·h, for example, it can be 20 , 25, 30, 35, 40, 45, 50, 55, 60, 65 mmol/L h, etc.; 160 to the end of fermentation, control at 15 to 60 mmol/L h, for example, it can be 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 volume (L/h or m ³ /h);

V: volume of fermentation broth (L or m ³ );

Co _2in : oxygen concentration in intake air (%);

C _{inert in} : the concentration of inert gas in the intake gas (%);

Co _2out : oxygen concentration (%) in the exhaust gas discharged from the fermentation broth;

Cco _2out : carbon dioxide concentration (%) in the exhaust gas of the fermentation broth.

Among them, Co _2in , C _{inert in} , Co _2out , and Cco _2out are monitored in real time by mass spectrometer.

In the present invention, the ORP needs to be controlled at 50-150 mv after the 100th hour of fermentation.

In some embodiments of the present invention, ORP is controlled in three stages (ie, 100-120h, 120-160h, and 160-fermentation end), and the control method is as follows: 100-120h is controlled at 50-100mv, for example, it can be 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mv, etc; , 95, 100, 105, 110, 115, 120, 125, 130mv, etc; 115, 120, 125, 130, 135, 140, 145, 150mv, etc.

In some embodiments of the present invention, the ORP is controlled in four stages (ie, 100-120h, 120-140h, 140-160h, and 160-fermentation end), and the control method is as follows: 100-120h is controlled at 50-100mv , for example, it can be 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mv, etc; , 85, 90, 95, 100, 105, 110, 115, 120mv, etc; , 115, 120, 125, 130mv, etc; 135, 140mv, etc.

In the present invention, the ORP is measured by a redox potential electrode.

In a specific embodiment of the present invention, the OUR is controlled by adjusting at least one of ventilation volume, rotational speed and tank pressure. When the OUR exceeds the predetermined range, it is necessary to reduce the OUR by reducing at least one of the ventilation volume, the rotation speed and the tank pressure; when the OUR is lower than the predetermined range, it is necessary to increase at least one of the ventilation volume, the rotation speed and the tank pressure. to improve OUR.

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 the predetermined range, it is necessary to reduce the ORP by reducing at least one of the ventilation volume, rotational speed and tank pressure, or increasing the temperature and/or pH value; when the ORP is lower than the predetermined range, it is necessary to increase the ventilation volume, At least one of rotational speed and tank pressure can either decrease temperature and/or pH to increase ORP.

In the present invention, the Mortierella alpina and its mutant strains can be obtained commercially, and can be specifically selected from the strains whose deposit numbers are CCTCC No.M2012073, CCTCC No.M2013392, CCTCC No.M2015421 or ATCC No.42430.

The production process of arachidonic acid includes strain activation, seed expansion culture and fermentation culture, wherein the seed expansion culture generally includes primary seed culture and secondary seed culture. Wherein, the bacterial strain activation medium, seed medium and fermentation medium adopted in each stage can be the strain activation medium, seed medium and fermentation medium for culturing Mortierella alpine strain or its mutant strain in this area. Those skilled in the art can all know, and will not be repeated here.

In a specific embodiment, the production method of described arachidonic acid comprises the following steps:

(3) Fermentation culture: insert the seed liquid into a fermenter with a sterilized fermentation medium at an inoculation amount of 1% to 3%, at a temperature of 20 to 30° C., a ventilation volume of 1 to 2 vvm, and a tank pressure of 0.02 to 0.02 Fermentation and culture under the conditions of 0.05MPa and 0-100rpm stirring speed for 150-168h, during the fermentation and culture process, monitor OUR online and control its value within a predetermined range, and/or, after the 100th hour of fermentation and culture, online monitoring The ORP in the fermentation broth is controlled within a predetermined range, and the pH value of the fermentation system is controlled at 5-7.5 during the entire fermentation process. Wherein, the reagent for adjusting the pH value includes but is not limited to: sodium hydroxide solution, potassium hydroxide solution, ammonia water, citric acid and the like. In addition, the volume of the fermentor used for the fermentation culture may be 0.5 L to 500 m ³ .

The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Modifications or substitutions made to the methods, steps or conditions of the present invention without departing from the spirit and essence of the present invention all belong to the scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.

In the following examples and comparative examples, unless otherwise specified, the strains and media used are as follows:

(1) the used bacterial strain is Mortierella alpine (ATCC No.42430);

(2) the used strain activation medium formula is as follows: potato (peeled) 200g, glucose 20g, yeast extract 5g, distilled water 1000mL, pH is natural;

(3) the used seed culture medium formula is as follows: potato (peeled) 200g, glucose 20g, distilled water 1000mL, pH is natural;

(4) The used fermentation medium formula is 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 1.5 μg/L, MnSO ₄ 2 μg/L, ZnSO ₄ ·7H ₂ O 3 μg/L, pH 6-7.

In the following examples and comparative examples, the measuring methods of each parameter are all carried out in the following manner:

(1) Determination method of biomass: take an appropriate amount of fermentation broth in a weighing bottle, dry it in an electric heating constant temperature drying oven at 105 °C for 4 hours, then put it in a desiccator to cool to room temperature and weigh, and subtract the net weight of the weighing bottle from the weight. It is the dry weight of the fermentation broth, and the dry weight of the fermentation broth divided by the volume of the fermentation broth is the biomass, in g/L.

(2) Determination method of crude oil yield: take a certain volume of fermentation broth, add 2 times the volume of concentrated hydrochloric acid, stir at a constant temperature for 50 min at 70 ° C until the bacterial cells are completely digested, add an appropriate amount of n-hexane, let stand for stratification, and use a dropper to take The upper organic phase was placed in an eggplant-shaped bottle, and extracted continuously for 6 times until the upper organic phase was colorless. The n-hexane was removed by rotary evaporation in a water bath at 80 °C. Put it in a desiccator to cool to room temperature and weigh it. The weight minus the net weight of the eggplant bottle is the dry weight of crude oil, and the dry weight of crude oil divided by the volume of fermentation broth is the yield of crude oil, in g/L.

(3) Determination method of ARA mass percentage content: The mass percentage content of ARA in crude oil and fat was measured by gas chromatography, unit %.

Embodiment 1: 100L fermentation tank process technology control

(1) Strain activation: The slanted strain of Mortierella alpina was placed in a 2L shake flask containing 400 mL of sterilized strain activation medium, and cultured at a temperature of 28°C at a speed of 150 rpm for 48 hours to complete the activation of the strain. Get the shake flask seed solution.

(2) Seed expansion culture: insert the shake flask seed liquid into the seed tank containing the sterilized seed medium according to the inoculation amount of 0.4%. The seed expansion is completed to obtain seed liquid.

(3) Fermentation culture: insert the seed liquid of the seed tank into the fermentation tank equipped with the sterilized fermentation medium according to the inoculation amount of 3%, the initial culture conditions: the culture temperature is 28°C, the ventilation rate is 1.0vvm, and the tank pressure is 0.04 MPa, rotating speed 70rpm, fermented and cultured for 168h to obtain fermentation broth. During the fermentation process, OUR is monitored online and its value is controlled within the following ranges:

1) Control OUR at 10-40mmol/L·h during 0-20h of fermentation and culture,

2) Control OUR at 20-80 mmol/L·h during 20-55 h of fermentation and culture,

3) Control OUR at 25-100 mmol/L·h during 55-80 h of fermentation and culture,

4) Control OUR at 20-90 mmol/L·h during 80-120 h of fermentation and culture,

5) Control OUR at 15-70 mmol/L·h during 120-160 h of fermentation and culture,

6) Control OUR at 15-60 mmol/L·h during 160-168 h of fermentation.

The value of the OUR is controlled by adjusting at least one of ventilation volume, tank pressure and rotational speed. When OUR is lower than the control range, at least one of ventilation volume, tank pressure and rotation speed is increased; when OUR is higher than the control range, at least one of ventilation volume, tank pressure and rotation speed is decreased to control OUR within the desired range. within the required range. After culturing for 168 hours, the fermentation was stopped. After testing, the biomass, oil yield and arachidonic acid mass percentage in the fermentation broth were 45g/L, 26.7g/L and 67%, respectively.

Embodiment 2: 100m ³ fermentor process technology control

(2) First-class seed cultivation: according to the inoculation amount of 0.4%, the shake flask seed liquid is inserted into the first-class seed tank containing the sterilized seed medium, the cultivation temperature is 28°C, the ventilation volume is 1vvm, and the tank pressure is 0.04MPa, After culturing for 35 hours, the expanded cultivation of the first-class seeds was completed, and the first-class seed liquid was obtained.

(3) Secondary seed cultivation: insert the primary seed liquid into the secondary seed tank containing the sterilized seed medium according to the inoculation amount of 3%, the cultivation temperature is 28°C, the ventilation volume is 1vvm, and the tank pressure is 0.04MPa, After culturing for 24 hours, the expanded cultivation of secondary seeds was completed, and secondary seed liquid was obtained.

(4) Fermentation culture: insert the seed liquid of the secondary seed tank into the fermentation tank equipped with the sterilized fermentation medium according to the inoculum amount of 3%, the initial culture conditions: the culture temperature is 28° C. The pressure was 0.04MPa, the rotation speed was 50rpm, and the fermentation was carried out for 168h to obtain a fermentation broth. During the fermentation process, OUR is monitored online and its value is controlled within the following ranges:

1) 0～20h control OUR at 10～35mmol/L·h,

2) 20～40h control OUR at 20～70mmol/L·h,

3) 40～55h control OUR at 25～80mmol/L·h,

4) 55～80h control OUR at 25～100mmol/L·h,

5) 80～100h control OUR at 25～90mmol/L·h,

6) 100～120h control OUR at 20～80mmol/L·h,

7) 120～140h control OUR at 20～70mmol/L·h,

8) 140～160h control OUR at 20～65mmol/L·h,

9) 160～168h control OUR at 15～60mmol/L·h.

The value of the OUR is controlled by adjusting at least one of ventilation volume, tank pressure and rotational speed. When OUR is lower than the control range, at least one of ventilation volume, tank pressure and rotation speed is increased; when OUR is higher than the control range, at least one of ventilation volume, tank pressure and rotation speed is decreased to control OUR within the desired range. within the required range. After culturing for 168 hours, the fermentation was stopped. After testing, the biomass, oil yield and arachidonic acid mass percentage in the fermentation broth were 48.5g/L, 30.1g/L and 66.8%, respectively.

Embodiment 3: 300m ³ fermentor process technology control

(4) Fermentation culture: insert the seed liquid of the secondary seed tank into the fermentation tank equipped with the sterilized fermentation medium according to the inoculum amount of 3%, the initial culture conditions: the culture temperature is 28° C. The pressure was 0.04MPa, the rotation speed was 30rpm, and the fermentation was carried out for 168h to obtain a fermentation broth. During the fermentation process, OUR is monitored online and its value is controlled within the following ranges:

1) 0～20h control OUR at 10～35mmol/L·h,

2) 20～40h control OUR at 20～70mmol/L·h,

3) 40～55h control OUR at 25～80mmol/L·h,

4) 55～80h control OUR at 25～100mmol/L·h,

5) 80～100h control OUR at 25～90mmol/L·h,

6) 100～120h control OUR at 20～80mmol/L·h,

7) 120～140h control OUR at 20～70mmol/L·h,

8) 140～160h control OUR at 20～65mmol/L·h,

9) 160～168h control OUR at 15～60mmol/L·h.

The value of the OUR is controlled by adjusting at least one of ventilation volume, tank pressure and rotational speed. When OUR is lower than the control range, at least one of ventilation volume, tank pressure and rotation speed is increased; when OUR is higher than the control range, at least one of ventilation volume, tank pressure and rotation speed is decreased to control OUR within the desired range. within the required range. After culturing for 168 hours, the fermentation was stopped. After testing, the biomass, oil yield and arachidonic acid mass percentage in the fermentation broth were 48.7g/L, 29.6g/L and 67.2%, respectively.

Embodiment 1-1: ^100m3 fermentor process technology control

According to the method for embodiment 2, arachidonic acid is produced, and the difference is that during the fermentation and cultivation process, OUR is monitored online and its numerical value is controlled within the following range:

1) 0～20h control OUR at 10～35mmol/L·h,

2) 20～40h control OUR at 20～70mmol/L·h,

3) 40～55h control OUR at 25～80mmol/L·h,

4) 55～80h control OUR at 25～100mmol/L·h,

5) 80～100h control OUR at 25～90mmol/L·h,

6) 100～120h control OUR at 20～80mmol/L·h,

7) 120～140h control OUR at 70～80mmol/L·h,

8) 140～160h control OUR at 10～20mmol/L·h,

9) 160～168h control OUR at 15～60mmol/L·h.

After culturing for 168 hours, the fermentation was stopped. After testing, the biomass, oil yield and arachidonic acid mass percentage in the fermentation broth were 46.2g/L, 27.1g/L and 66.5%, respectively. Compared with Example 2, some sections were not controlled within the preferred range of OUR, and the biomass, oil yield and arachidonic acid mass percentage in the fermentation broth were all lower than those in Example 2.

Comparative Example 1

Produce arachidonic acid according to the method of embodiment 1-1, the difference is, in the fermentation culture process, monitor OUR online and control its value within the following scope:

1) 0～20h control OUR at 5～35mmol/L·h,

2) 20～40h control OUR at 20～70mmol/L·h,

3) 40～55h control OUR at 25～80mmol/L·h,

4) 55～80h control OUR at 25～120mmol/L·h,

5) 80～100h control OUR at 25～90mmol/L·h,

6) 100～120h control OUR at 20～80mmol/L·h,

7) 120～140h control OUR at 70～80mmol/L·h,

8) 140～160h control OUR at 10～20mmol/L·h,

9) 160～168h control OUR at 15～60mmol/L·h.

After culturing for 168 hours, the fermentation was stopped. After testing, the biomass, oil yield and arachidonic acid mass percentage in the fermentation broth were 43.2g/L, 24.8g/L and 57.8%, respectively. Compared with Example 1-1, the value of OUR is outside the scope of the present invention in some intervals, and the biomass, oil yield and arachidonic acid mass percentage in the fermentation broth are all lower than those in Example 1-1.

Embodiment ⁴ : 100m3 fermentor process technology control

(1) Strain activation: The slant preservation strain of Mortierella alpina was placed in a 2L shake flask containing 400mL of sterilized strain activation medium, and cultured at a temperature of 28°C for 48h at a rotation speed of 150rpm to complete the strain activation culture. Get the shake flask seed solution.

(4) Fermentation culture: insert the seed liquid of the secondary seed tank into the fermentation tank equipped with the sterilized fermentation medium according to the inoculum amount of 3%, the initial culture conditions: the culture temperature is 28°C, the ventilation rate is 1.0vvm, the tank The pressure was 0.04MPa, the rotation speed was 45rpm, and the fermentation was cultured for 168h to obtain a fermentation broth. After 100h of fermentation, the ORP in the fermentation broth was monitored online and its value was controlled within the following range:

1) 100～120h control ORP at 50～100mv,

2) 120～160h control ORP at 60～130mv,

3) 160～168h control ORP at 70～150mv.

The value of the ORP is controlled by adjusting at least one of ventilation, tank pressure, rotational speed, temperature and pH. When the ORP is lower than the control range, at least one of the ventilation volume, tank pressure and rotation speed is increased, or the fermentation temperature and/or pH value is decreased; when the ORP is higher than the control range, the ventilation volume, tank pressure and rotation speed are decreased. At least one, or increase fermentation temperature and/or pH to control ORP within the desired range. After culturing for 168 hours, the fermentation was stopped. After testing, the biomass, oil yield and arachidonic acid mass percentage in the fermentation broth were 43.5g/L, 25.2g/L and 68.3%, respectively.

Embodiment 5: 100m ³ fermentor process technology control

(4) Fermentation culture: insert the seed liquid of the secondary seed tank into the fermentation tank equipped with the sterilized fermentation medium according to the inoculum amount of 3%, the initial culture conditions: the culture temperature is 28°C, the ventilation rate is 1.0vvm, the tank The pressure was 0.04MPa, the rotation speed was 45rpm, and the fermentation was cultured for 168h. After 100h of fermentation, the ORP in the fermentation broth was monitored online and its value was controlled within the following range:

1) 100～120h control ORP at 50～100mv,

2) 120～140h control ORP at 60～120mv,

3) 140～160h control ORP at 65～130mv,

4) 160～168h control ORP at 70～140mv.

The value of the ORP is controlled by adjusting at least one of ventilation, tank pressure, rotational speed, temperature and pH. When the ORP is lower than the control range, at least one of the ventilation volume, tank pressure and rotation speed is increased, or the fermentation temperature and/or pH value is decreased; when the ORP is higher than the control range, the ventilation volume, tank pressure and rotation speed are decreased. At least one, or increase fermentation temperature and/or pH to control ORP within the desired range. After culturing for 168 hours, the fermentation was stopped. After testing, the biomass, oil yield and arachidonic acid mass percentage in the fermentation broth were 44.1g/L, 25.8g/L and 68.6%, respectively.

Comparative example 2: Process control of ^100m3 fermentation tank

Arachidonic acid was produced according to the method of Example 5, except that after culturing for 100h, the ORP in the fermentation broth was monitored online and its value was controlled within the following range:

1) 100～120h control ORP at 50～100mv,

2) 120～140h control ORP at 60～120mv,

3) 140～160h control ORP at 40～65mv,

4) 160～168h control ORP at 35～60mv.

After culturing for 168 hours, the fermentation was stopped. After testing, the biomass, oil yield and arachidonic acid mass percentage in the fermentation broth were 43.1g/L, 23.6g/L and 58.4%, respectively. Compared with Example 5, the ORP value is outside the scope of the present invention in some intervals, and the biomass, oil yield and arachidonic acid mass percentage in the fermentation broth are all lower than those in Example 5.

Embodiment ⁶ : 100m3 fermentor process technology control

(4) Fermentation culture: insert the seed liquid of the secondary seed tank into the fermentation tank equipped with the sterilized fermentation medium according to the inoculum amount of 3%, the initial culture conditions: the culture temperature is 28° C. The pressure was 0.04MPa, the rotation speed was 45rpm, and the fermentation was cultured for 168h. During the fermentation and culture process, OUR was monitored online and its value was controlled within the following range, and after 100h of fermentation and culture, the ORP in the fermentation broth was monitored online and its value was controlled within the following range:

1) 0～20h control OUR at 10～35mmol/L·h,

2) 20～40h control OUR at 20～70mmol/L·h,

3) 40～55h control OUR at 25～80mmol/L·h,

4) 55～80h control OUR at 25～100mmol/L·h,

5) 80～100h control OUR at 25～90mmol/L·h,

6) 100～120h control OUR at 20～80mmol/L·h, control ORP at 50～100mv,

7) 120～140h control OUR at 20～70mmol/L·h, control ORP at 60～120mv,

8) 140～160h control OUR at 20～65mmol/L·h, control ORP at 65～130mv,

9) During 160-168 h, control OUR at 15-60 mmol/L·h, and control ORP at 70-140 mv.

The value of the OUR is controlled by adjusting at least one of ventilation volume, tank pressure and rotational speed. When OUR is lower than the control range, at least one of ventilation volume, tank pressure and rotation speed is increased; when OUR is higher than the control range, at least one of ventilation volume, tank pressure and rotation speed is decreased to control OUR within the desired range. within the required range. The value of the ORP is controlled by adjusting at least one of ventilation, tank pressure, rotational speed, temperature and pH. When the ORP is lower than the control range, at least one of the ventilation volume, tank pressure and rotation speed is increased, or the fermentation temperature and/or pH value is decreased; when the ORP is higher than the control range, the ventilation volume, tank pressure and rotation speed are decreased. At least one, or increase fermentation temperature and/or pH to control ORP within the desired range. After culturing for 168 hours, the fermentation was stopped. After testing, the biomass, oil yield and arachidonic acid mass percentage in the fermentation broth were 50.2g/L, 31.3g/L and 69.2%, respectively.

Embodiment 7: 100m ³ fermentor process technology control

1) 0～20h control OUR at 10～40mmol/L·h,

2) 20～55h control OUR at 20～80mmol/L·h,

3) 55～80h control OUR at 25～100mmol/L·h,

4) 80～120h control OUR at 20～90mmol/L·h, 100～120h control ORP at 50～100mv,

5) 120～160h control OUR at 15～70mmol/L·h, control ORP at 60～130mv,

6) During 160-168h, control OUR at 15-60mmol/L·h, and control ORP at 70-150mv.

The value of the OUR is controlled by adjusting at least one of ventilation volume, tank pressure and rotational speed. When OUR is lower than the control range, at least one of ventilation volume, tank pressure and rotation speed is increased; when OUR is higher than the control range, at least one of ventilation volume, tank pressure and rotation speed is decreased to control OUR within the desired range. within the required range. The value of the ORP is controlled by adjusting at least one of ventilation, tank pressure, rotational speed, temperature, and pH. When the ORP is lower than the control range, at least one of the ventilation volume, tank pressure and rotation speed is increased, or the fermentation temperature and/or pH value is decreased; when the ORP is higher than the control range, the ventilation volume, tank pressure and rotation speed are decreased. At least one, or increase fermentation temperature and/or pH to control ORP within the desired range. After culturing for 168 hours, the fermentation was stopped. After testing, the biomass, oil yield and arachidonic acid mass percentage in the fermentation broth were 47.6g/L, 30.1g/L and 67.6%, respectively.

Embodiment ⁸ : 100m3 fermentor process technology control

(4) Fermentation culture: insert the seed liquid of the secondary seed tank into the fermentation tank equipped with the sterilized fermentation medium according to the inoculum amount of 3%, the initial culture conditions: the culture temperature is 28° C. The pressure was 0.04MPa, the rotation speed was 45rpm, and the fermentation was cultured for 168h.

No staged control of oxygen consumption rate OUR and redox potential ORP was performed during fermentation. During the fermentation process, the oxygen consumption rate OUR was controlled at 10-100 mmol/L·h, and the ORP was controlled at 50-150 mv after the 100th hour of fermentation. During the process, OUR was controlled within the range by adjusting at least one of the ventilation rate, rotation speed and tank pressure. The ORP is controlled within the range by adjusting at least one of ventilation, rotational speed, tank pressure, temperature, and pH. After culturing for 168 hours, the fermentation was stopped. After testing, the biomass, oil yield and arachidonic acid mass percentage in the fermentation broth were 44.5g/L, 25.2g/L and 62.1%, respectively.

Examples 9-11: Fermentation of different strains to produce arachidonic acid

Embodiments 9-11 adopt different production strains to ferment and produce arachidonic acid, and the specific conditions and fermentation results are shown in Table 1.

Table 1: Fermentation levels of different arachidonic acid producing strains

Although the embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Variations, modifications, substitutions, and alterations to the above-described embodiments are possible within the scope of the present invention without departing from the scope of the present invention.

Claims

A method for producing arachidonic acid, characterized in that the method comprises sequentially performing strain activation, seed expansion culture and fermentation culture on Mortierella alpina or a mutant thereof, and in the process of fermentation culture, online monitoring of OUR and The OUR value is controlled in six stages, and the control method is as follows: 0~20h is controlled at 10~40mmol/L·h, 20~55h is controlled at 20~80mmol/L·h, 55~80h is controlled at 25~100mmol/L·h h, 20-90 mmol/L·h for 80-120 h, 15-70 mmol/L·h for 120-160 h, 15-60 mmol/L·h for 160-to the end of fermentation; and/or, in the fermentation culture After the 100th hour, the ORP in the fermentation broth was monitored online and the ORP value was controlled in three stages. 70～150mv.
The production method of arachidonic acid according to claim 1, is characterized in that, described OUR is controlled in nine stages, and control mode is as follows:

0～20h is controlled at 10～35mmol/L·h,

20～40h controlled at 20～70mmol/L·h,

40～55h controlled at 25～80mmol/L·h,

55～80h controlled at 25～100mmol/L·h,

80～100h controlled at 25～90mmol/L·h,

100～120h controlled at 20～80mmol/L·h,

120～140h controlled at 20～70mmol/L·h,

140～160h controlled at 20～65mmol/L·h,

From 160 to the end of fermentation, control at 15 to 60 mmol/L·h.
The production method of arachidonic acid according to claim 1, is characterized in that, described ORP is controlled in four stages, and control mode is as follows:

100～120h control at 50～100mv,

120～140h control at 60～120mv,

140～160h controlled at 65～130mv,

160～The end of fermentation is controlled at 70～140mv.
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 rate, rotational speed and tank pressure.
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, rotational speed, tank pressure, temperature and pH value.
The production method of arachidonic acid according to any one of claims 1 to 3, wherein the method comprises the following steps:

(1) Strain activation: insert the Mortierella alpina strain or its mutant strain into the shake flask containing the sterilized strain activation medium, and cultivate for 40 to 48 hours at a temperature of 25 to 32 °C and a rotation speed of 100 to 200 rpm. , to complete the activation and cultivation of strains to obtain shake flask seed liquid;

(2) Seed expansion culture: insert the shake flask seed liquid into the seed tank containing the sterilized seed medium at an inoculation amount of 0.4% to 1%, at a temperature of 25 to 32 ° C, aeration volume of 1 to 2 vvm, and the tank Under the condition of pressure of 0.02-0.05MPa, cultivate for 30-35h, and optionally insert 1%-3% of the inoculum into the seed tank containing the sterilized seed medium. Cultivate for 20-25h under the conditions of ~2vvm and tank pressure of 0.02-0.05MPa, optionally repeat the cultivation 1-3 times to obtain seed liquid;

(3) Fermentation culture: insert the seed liquid into a fermenter with a sterilized fermentation medium at an inoculation amount of 1% to 3%, at a temperature of 20 to 30° C., a ventilation volume of 1 to 2 vvm, and a tank pressure of 0.02 to 0.02 Fermentation and culture under the conditions of 0.05MPa and 0-100rpm stirring speed for 150-168h, during the fermentation and culture process, monitor OUR online and control its value within a predetermined range, and/or, after the 100th hour of fermentation and culture, online monitoring The ORP in the fermentation broth is controlled within a predetermined range, and the pH value of the fermentation system is controlled at 5-7.5 during the entire fermentation process.