CN104278107B - Method for producing arachidonic acid oil by fermenting mortierella alpina based on dissolved oxygen regulation - Google Patents

Abstract

The invention relates to a method for producing arachidonic acid oil by regulating Mortierella alpine fermentation based on dissolved oxygen. The Mortierella alpina strain was amplified step by step through the test tube slant medium and the shake flask medium, and the seed liquid was prepared, and then inoculated in the fermenter for fermentation culture. The whole fermentation process was divided into the early stage of fermentation, the middle stage of fermentation and the late stage of fermentation3 The initial setting of rotation speed is 0rpm, and the initial setting of dissolved oxygen is 100%. In the early stage of fermentation, the dissolved oxygen is stabilized at 40%-70%; in the middle stage of fermentation, the dissolved oxygen is controlled at 20%-40%. ; In the later stage of fermentation, the dissolved oxygen is controlled at 40%‑70%. At the end of fermentation, the dry weight of Mortierella alpina cells, the oil content, the percentage of arachidonic acid in the total oil, and the unit yield of arachidonic acid can reach 55g/L, 54%, 65%, and 20.628g/L respectively. L, the production intensity of arachidonic acid reaches 2.292g/(L*d).

Description

A method for producing arachidonic acid oil by regulating the fermentation of Mortierella alpina based on dissolved oxygen

technical field

The invention relates to a method for producing arachidonic acid oil by regulating Mortierella alpine fermentation based on dissolved oxygen, and belongs to the technical field of microbial fermentation.

Background technique

Since the 1990s, people have paid more and more attention to healthy diet and nutrition, and long-chain polyunsaturated fatty acids have gradually come into people's sight. Long-chain polyunsaturated fatty acids mainly include arachidonic acid (ARA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). These fatty acids not only play an important role in the composition of membrane phospholipids, but also can be used as precursors for the synthesis of signal molecules eicosanoids (including prostaglandins, thromboxanes and leukotrienes, etc.), and are widely used in Pharmaceuticals and cosmetics, especially in the field of infant food and nutritional products.

At present, the process of producing arachidonic acid oil by microbial fermentation has entered the stage of industrial production. However, fermentation production also faces many problems, such as long fermentation period, low glucose conversion rate, low cell biomass, low oil content, high content of saturated and monounsaturated fatty acids, especially the difficulty of cell morphology during fermentation. control etc.

Fermentation tanks used in traditional production mainly include mechanical stirring type and air lift type. The mechanical agitation fermenter has high power and high energy consumption. At the same time, the shear force of the blade is relatively large, which has a great impact on the shape of the bacteria. It is easy to break the bacteria into pieces and take the shape of broken feathers. On the other hand, the air-lift fermenter uses sterile air as the driving force to roll the fermentation liquid without shear force, but the biomass of the bacteria grows to a certain extent in the later stage of fermentation, the density of the fermentation liquid is high, and the viscosity is high. The effect of gas propulsion on the tumbling and mixing of the fermentation broth is significantly weakened, the dissolved oxygen is not easy to control, and the shape of the bacteria is also difficult to control. However, the morphology of the bacteria has a direct impact on indicators such as the biomass of the bacteria, the oil content and the percentage of arachidonic acid.

At present, the general level of arachidonic acid oil produced by Mortierella alpina fermentation is: the cycle is 10-16 days, the biomass is 22-37g/L, and arachidonic acid accounts for 33-60% of the total oil content. Among them, the group with the highest reported arachidonic acid content has a period of 12.1 days, a biomass of 38.3g/L, an oil content of 26.4g/L, and arachidonic acid accounting for 75% of the total oil. However, the production intensity of arachidonic acid is basically 0.3-1.45g/(L*d), and the highest reported net content of arachidonic acid is only 18g/L. In view of the problems of long fermentation cycle, easy to cause bacterial contamination, and low production intensity, there is room for further improvement. In addition, the currently reported experiments with high net content of arachidonic acid are basically carried out at the level of 250mL shake flasks, 1L, 5L, and 20L small fermentation tanks, and there are few reports of good examples in industrial scale-up.

Therefore, in view of the disadvantages of the existing mechanically stirred fermenters and airlift fermenters on the influence of the bacterial morphology, for the fermentation of Mortierella alpina to produce arachidonic acid oil, the cycle is long, extremely aerobic, and the viscosity increases significantly in the later stage of fermentation. The fermentation system optimizes the appropriate high-density fermentation process conditions, and provides a new process suitable for the accumulation of Mortierella alpine bacteria to produce arachidonic acid oil, which increases the biomass of the bacteria and the content of arachidonic acid oil, and improves the quality of peanuts. Tetraenoic acid production intensity, and it is necessary to put it into industrialized large-scale production.

Contents of the invention

In view of the above-mentioned problems in the prior art, the purpose of the present invention is to propose a method based on dissolved oxygen regulation Mortierella alpina fermentation to produce arachidonic acid oil, which is aimed at the shape of the bacteria, combined with mechanical stirring fermenter and gas The advantages of the ascending fermenter, through the regulation of the process, the fermentation capacity is improved.

In order to achieve the above object, the technical scheme adopted in the present invention is:

A method based on dissolved oxygen regulation of Mortierella alpina fermentation to produce arachidonic acid oil. The Mortierella alpina strain is gradually amplified and cultivated on a test tube slope and a shaker flask to prepare seed liquid, and then inoculated in a fermenter for fermentation and cultivation , in the fermentation process, with the goal of controlling the fermentation morphology of Mortierella alpina, the fermentation process is regulated in stages with the fermentation dissolved oxygen parameters as the index, and the entire fermentation process is divided into three stages: the early stage of fermentation, the middle stage of fermentation and the late stage of fermentation ; In the early stage of fermentation, when the biomass of the bacteria grows vigorously, the initial setting of the rotating speed is 0rpm, and the initial setting of the dissolved oxygen is 100%. As the concentration of the bacteria increases, the dissolved oxygen will show a downward trend. Increase 5rpm until 50rpm, so that the dissolved oxygen can be stabilized at 40%-70% after the drop; in the middle stage of fermentation, the oil accumulation stage, the dissolved oxygen is controlled at 20%-40%; in the late stage of fermentation, arachidonic acid biosynthesis stage, the dissolved oxygen is controlled at 40%-70%.

The early stage of fermentation is the period of 0-72 hours of fermentation.

The dissolved oxygen control in the early stage of fermentation is realized by air volume and speed control, the air volume is controlled at 1-3VVM (VVM: the air volume per unit volume per minute), and the speed is controlled at 0-50 rpm.

The middle stage of the fermentation is the period of 73-120 hours of fermentation.

The dissolved oxygen control in the mid-fermentation stage is realized by air volume and rotational speed control, the air volume is controlled at 0.5-1.5VVM, and the rotational speed is controlled at 50-200 rpm.

The late stage of fermentation is the period of 121-216 hours of fermentation.

The dissolved oxygen control in the late stage of fermentation is realized through the control of air volume and rotational speed, the air volume is controlled at 1-3VVM, and the rotational speed is controlled at 100-200 rpm.

The control of dissolved oxygen in the early stage of fermentation is realized by air volume and speed control, the air volume is controlled at 1-2VVM, the speed is controlled at 0-50 rpm, and the dissolved oxygen is controlled between 40%-55%.

The dissolved oxygen control in the middle stage of fermentation is realized by air volume and speed control, the air volume is controlled at 0.8-1.2VVM, the speed is controlled at 80-150 rpm, and the dissolved oxygen is controlled between 20%-35%.

The control of dissolved oxygen in the late stage of fermentation is realized by air volume and speed control, the air volume is controlled at 1.5-2VVM, the speed is controlled at 150-200 rpm, and the dissolved oxygen is controlled at 40%-50%.

The Mortierella alpina strain used in the present invention is Mortierella alpina strain R807 (CCTCC M 2012118)

The control principle of the present invention is as follows: the fermentation step is divided into three stages (biomass accumulation stage, oil accumulation stage, and arachidonic acid biosynthesis stage), and different regulation processes are adopted for different stages. In the biomass accumulation stage, The seed liquid has just been put into the fermenter, and the vigor of the bacteria is low and fragile, and it is not resistant to shearing. The air-lift tank culture method is adopted, with high dissolved oxygen and low shear, which is conducive to the accumulation of Mortierella alpina cells. When entering the stage of oil accumulation and the biomass reaches a certain level, a single aeration method is not enough to create a tumbling effect for the fermentation broth. With an appropriate stirring speed, it can make a good contribution to the gas-liquid mixing effect of the fermentation broth. In the biosynthesis stage of arachidonic acid in the later stage of fermentation, appropriately reducing the rotation speed and increasing the air volume is beneficial to the Δ9 desaturase, Δ12 desaturase, Δ6 desaturase and Δ5 desaturase to play a role, making the arachidonic acid content rises.

Beneficial effects of the present invention:

(1) In view of the problems that the bacteria are fragile in the early stage and the density is too large in the later stage, combined with the better dissolved oxygen in the airlift tank and the blades of the mechanical stirring tank, it can provide advantages such as shearing. Through the regulation of the process, the fermentation capacity was improved. The unit yield of arachidonic acid in the 7.5L tank reached 20.628g/L fermentation broth, and the production intensity of arachidonic acid reached 2.291g/(L*d).

(2) The process involved in the present invention is easy to operate and is beneficial to industrial production. At present, the trial operation has been completed at the level of 7m³ and 25m³ fermentation tanks, and good benefits have been obtained.

detailed description

The present invention will be described in detail below in combination with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

The culture medium adopted in the following examples is as follows:

Test tube slant medium (PDA slant medium): 200g of fresh peeled potatoes, boiled for 30 minutes, filtered through 4 layers of gauze to remove solids, added 20g of glucose, 20g of agar, distilled water to 1L, natural pH, extinguished at 121°C Bacteria 30min.

Seed medium: glucose 30g/L, yeast extract 6g/L, KH ₂ PO ₄ 3g/L, pH natural, sterilized at 121°C for 30min.

Fermentation medium: glucose 80g/L, yeast extract 11g/L, KH ₂ PO ₄ 3.8g/L, NaNO ₃ 3.4g/L, MgSO ₄ 7H ₂ O 0.5g/L, natural pH, sterilized at 121°C for 30min .

The strain used in the examples is: Mortierella alpina strain R807 (CCTCC M 2012118), and other similar strains can also be used.

Example 1 Fermentation of Mortierella alpina in a 7.5L tank to produce arachidonic acid oil based on dissolved oxygen control strategy

1. Activation of strains and preparation of seed solution: Mortierella alpina strain R807 (CCTCC M 2012118) was selected as the starting strain, and the preserved strains were inserted into PDA slant medium, and placed in an incubator at 25°C Cultivate for 10 days, transfer to a 500mL grooved bottle containing 100mL seed medium and culture in a constant temperature shaker, the inoculum size is 10% (v/v, 10mL), the culture conditions are 120rpm, the temperature is 25°C, and the culture time is 1 -2 days.

2. Inoculate the seed liquid obtained in step 1 into a 7.5L tank (New Brunswick Scientific, USA), the liquid volume is 5L, the inoculum volume is 10% (v/v, 500mL), and the culture temperature is controlled at 28°C for 0-120h , 121-216h controlled at 18°C,

At the beginning of fermentation, the seed liquid has just been put into the fermenter, there is a period of adaptation, the initial air volume is 2VVM, the initial stirring speed is 50 rpm, the initial setting of dissolved oxygen is 100%, and the period of 0-72 hours of fermentation is the early stage of fermentation. In the vigorous growth stage of bacterial biomass, with the increase of bacterial concentration, the dissolved oxygen will continue to decrease. After the dissolved oxygen is controlled by the air volume and speed control, it can be stabilized at 40%-70%. The air volume is controlled at 1-2VVM, and the speed The initial setting is 0rpm, and the speed is increased by 5rpm every 4 hours until it reaches 50rpm; during the 73-120 hour period of fermentation, it is the middle stage of fermentation, and this stage is the stage of oil accumulation. The dissolved oxygen is controlled at 20 by air volume and speed control. %-40%, the air volume is controlled at 1-1.5VVM, and the speed is controlled at 150-200 rpm; during the 121-216 hour period of fermentation, it is the late stage of fermentation, which is the biosynthesis stage of arachidonic acid, and dissolved oxygen Control at 40%-70%, air volume control at 1-2VVM, speed control at 150-200 rpm. The following table takes the control conditions of the boundary points of different stages as an example, see Table 1.

Table 1

Note: When the inoculation was just completed, due to the low biomass of the bacteria, it was not easy to detect, so no samples were taken to detect the biomass, oil content and percentage of arachidonic acid at 0 hours.

At the beginning of fermentation, the seed liquid has just been connected to the fermenter, and there is a period of adaptation. The initial air volume is 2VVM, the stirring speed is initially 50 rpm, and the dissolved oxygen is initially set at 100%. After the bacterial concentration grows, the dissolved oxygen continues to decrease. Around 40%;

When the fermentation culture reaches 72 hours, the growth of bacterial biomass slows down, and at this time the intracellular oil begins to be synthesized, the air volume is controlled at 1VVM, the stirring speed is controlled at 150 rpm, and the dissolved oxygen is controlled at 20%;

After fermentation and cultivation for 120 hours, the concentration of bacteria in the tank is too large at this time, and the viscosity of the fermentation liquid rises obviously. About 50%;

When the fermentation culture reaches 216 hours, the fermentation ends, and samples are taken to detect the biomass, oil content and arachidonic acid percentage content at the end point. Mortierella alpine cell dry weight, oil content, percentage of arachidonic acid, and unit yield of arachidonic acid can reach 56.1g/L, 53.9%, 68.22%, 20.628g/L, respectively, and the production of arachidonic acid The strength reaches 2.292g/(L*d).

Example 2. Arachidonic acid produced by fermenting Mortierella alpina in a 7m³ tank based on the dissolved oxygen regulation strategy

1. Activation of strains and preparation of seed solution: Mortierella alpina strain R807 (CCTCC M 2012118) was selected as the starting strain, and the preserved strains were inserted into PDA slant medium, and placed in an incubator at 25°C Cultivate for 10 days, transfer to a 500mL grooved bottle containing 100mL seed medium and culture in a constant temperature shaker, the inoculum size is 10% (v/v, 10mL), the culture conditions are 120rpm, the temperature is 25°C, and the culture time is 1 -2 days, prepare a first-grade seed solution. Then, according to the inoculation amount of 10% (v/v), the primary seed solution was inserted into a 100L seed tank, the cultivation conditions were 200 rpm, the temperature was 25°C, and the cultivation time was 1 day to prepare the secondary seed solution. Next, all the prepared secondary seed liquids were put into a 3000L seed tank, the cultivation conditions were 100 rpm, the temperature was 25° C., and the cultivation time was 1 day to prepare the tertiary seed liquids.

2. Put all the tertiary seed liquid obtained in step 1 into a 7m³ fermenter, control the culture temperature at 28°C for 0-120h, and 18°C for 121-216h, and see Table 2 for the control of other parameters. The height-to-diameter ratio of the fermenter used is 3:1, manufactured and installed by Nanjing Huike Bioengineering Equipment Co., Ltd., used and managed by Jiangsu Tiankai Biotechnology Co., Ltd., the liquid volume is 5m³, and the fermentation period is 0-72 hours. In the early stage of fermentation, the biomass of the bacteria grows vigorously. With the increase of the concentration of the bacteria, the dissolved oxygen continues to decrease. After the air volume and speed control are used to control the dissolved oxygen, the drop can be stabilized at 40%-45%, and the air volume is controlled at 1- 2VVM, the speed is initially set to 0rpm, and the speed is increased by 5rpm every 4 hours until it reaches 50rpm; during the 73-120 hour period of fermentation, it is the middle stage of fermentation, and this stage is the stage of oil accumulation. Dissolved oxygen is controlled by air volume and speed. Control at 20%-35%, air volume control at 0.8-1.2VVM, speed control at 80-150 rpm; during the 121-216 hours of fermentation, it is the late stage of fermentation, and this stage is the biosynthesis stage of arachidonic acid. Control the dissolved oxygen at 40%-50%, the air volume at 1.5-2VVM, and the speed at 150-200 rpm. The following table takes the control conditions of the boundary points of different stages as an example, and the control conditions are shown in Table 2.

Table 2

Note: When the inoculation was just completed, due to the low biomass of the bacteria, it was not easy to detect, so no samples were taken to detect the biomass, oil content and percentage of arachidonic acid at 0 hours.

At the beginning of fermentation, the seed liquid has just been connected to the fermenter, and there is a period of adaptation. The initial air volume is 1.5VVM, and the stirring speed is initially 0. Every four hours, adjust the frequency converter to increase the speed by 5 revolutions until it reaches 50 revolutions per minute. The initial oxygen setting is 100%. After the growth of the bacteria concentration, the dissolved oxygen will continue to decrease, which can be controlled at about 45%;

When the fermentation culture reaches 72 hours, the growth of bacterial biomass slows down, and at this time, the intracellular oil begins to be synthesized, the air volume is controlled at 1VVM, the stirring speed is controlled at 100 rpm, and the dissolved oxygen is controlled at 20%;

After 120 hours of fermentation and cultivation, the concentration of bacteria in the tank is too large at this time, and the viscosity of the fermentation liquid rises significantly. Control the speed at 160 rpm and the air volume at 1.7VVM to control the gas-liquid mixing effect in the fermentation tank and control the dissolved oxygen. Around 40%;

When the fermentation culture reaches 216 hours, the fermentation ends, and samples are taken to detect the biomass, oil content and arachidonic acid percentage content at the end point. Mortierella alpine cell dry weight, oil content, percentage of arachidonic acid, and unit yield of arachidonic acid can reach 53.08g/L, 57.05%, 62.37%, 18.887g/L, respectively, and the production of arachidonic acid The strength reaches 2.099g/(L*d).

Example 3. Arachidonic acid produced by fermenting Mortierella alpina in a 25m³ tank based on the dissolved oxygen regulation strategy

1. Activation of strains and preparation of seed solution: Mortierella alpina strain R807 (CCTCC M 2012118) was selected as the starting strain, and the preserved strains were inserted into PDA slant medium, and placed in an incubator at 25°C Cultivate for 10 days, transfer to a 500mL grooved bottle containing 100mL seed medium and culture in a constant temperature shaker, the inoculum size is 10% (v/v, 10mL), the culture conditions are 120rpm, the temperature is 25°C, and the culture time is 1 -2 days, prepare a first-grade seed solution. Then, according to the inoculation amount of 10% (v/v), the primary seed solution was inserted into a 150L seed tank, the cultivation conditions were 200rpm, the temperature was 25°C, and the cultivation time was 1 day to prepare the secondary seed solution. Next, all the prepared secondary seed liquids were put into a 5000L seed tank, the cultivation conditions were 100 rpm, the temperature was 25° C., and the cultivation time was 1 day to prepare the tertiary seed liquids.

2. Put all the three-stage seed liquid obtained in step 1 into a 25m³ fermenter, control the culture temperature at 28°C for 0-120h, and control at 18°C for 121-216h. The period of 0-72 hours of fermentation is the early stage of fermentation. In the vigorous growth stage of bacterial biomass, with the increase of bacterial concentration, the dissolved oxygen will continue to decrease. After the dissolved oxygen is controlled by air volume and speed control, it can be stabilized at 40%-55%. The air volume is controlled at 1-2VVM, and the speed The initial setting is 0rpm, and the speed is increased by 5rpm every 4 hours until it reaches 50rpm; during the 73-120 hour period of fermentation, it is the middle stage of fermentation, and this stage is the stage of oil accumulation. The dissolved oxygen is controlled at 20 by air volume and speed control. %-35%, the air volume is controlled at 0.8-1.2VVM, and the speed is controlled at 80-150 rpm; during the 121-216 hour period of fermentation, it is the late stage of fermentation, which is the biosynthesis stage of arachidonic acid, and dissolved oxygen Control at 40%-50%, air volume control at 1.5-2VVM, speed control at 150-200 rpm. The following table takes the control conditions of the boundary points of different stages as an example, and the control of the parameter process is shown in Table 3. The height-to-diameter ratio of the fermenter used is 2.5:1. It is manufactured and installed by Chaoyang Light Chemical Equipment Factory in Yiyang City, Hunan Province. It is used and managed by Jiangsu Tiankai Biotechnology Co., Ltd. The liquid volume is 21m³. The control of the fermentation process is shown in Table 3:

table 3

Note: When the inoculation was just completed, due to the low biomass of the bacteria, it was not easy to detect, so no samples were taken to detect the biomass, oil content and percentage of arachidonic acid at 0 hours.

At the beginning of fermentation, the seed liquid has just been connected to the fermenter, and there is a period of adaptation. The initial air volume is 1.5VVM, and the stirring speed is initially 0. Every four hours, adjust the frequency converter to increase the speed by 5 revolutions until it reaches 50 revolutions per minute. The initial oxygen setting is 100%. After the bacterial concentration grows, the dissolved oxygen continues to decrease, which can be controlled at about 60%;

When the fermentation culture reaches 72 hours, the growth of bacterial biomass slows down, at this time, the intracellular oil begins to synthesize, the air volume is controlled at 1VVM, the stirring speed is controlled at 80 rpm, and the dissolved oxygen can be controlled at 35%;

After fermentation and cultivation for 120 hours, the concentration of bacteria in the tank is too large at this time, and the viscosity of the fermentation liquid rises significantly. Control the speed at 150 rpm and the air volume at 1.7VVM to control the gas-liquid mixing effect in the fermentation tank and control the dissolved oxygen. Around 40%;

When the fermentation culture reaches 216 hours, the fermentation ends, and samples are taken to detect the biomass, oil content and arachidonic acid percentage content at the end point. Mortierella alpine cell dry weight, oil content, percentage of arachidonic acid, and unit yield of arachidonic acid can reach 49g/L, 50.42%, 63.86%, and 15.777g/L respectively, and the production intensity of arachidonic acid It reaches 1.753g/(L*d).

As can be seen from the above examples, the method for producing arachidonic acid oil based on dissolved oxygen regulation and control Mortierella alpina fermentation of the present invention can make biomass, oil content, arachidonic acid percentage content, and arachidonic acid content The output per unit of acid and production intensity have been greatly improved.

Claims (4)

1. A method based on dissolved oxygen regulation and control of Mortierella alpina fermentation to produce arachidonic acid oil, the Mortierella alpina strain is gradually enlarged and cultivated through the test tube slant medium and the shake flask medium, the seed liquid is prepared, and then inoculated in Fermentation culture is carried out in a fermenter, which is characterized in that: during the fermentation process, the fermentation process is controlled in stages with the goal of controlling the fermentation form of Mortierella alpina, and the fermentation dissolved oxygen parameter is used as an index, and the entire fermentation process is divided into fermentation processes. There are three stages: the early stage, the middle stage of fermentation and the late stage of fermentation; the speed is initially set at 0rpm, and the dissolved oxygen is initially set at 100%. As the concentration of bacteria increases, the dissolved oxygen will show a downward trend. The operation method of air volume is to stabilize the dissolved oxygen at 40%-70% in the early stage of fermentation; in the middle stage of fermentation, control the dissolved oxygen in 20%-40%; in the late stage of fermentation, control the dissolved oxygen in 40%-70% %; the early stage of fermentation is during the 0-72 hours of fermentation; the dissolved oxygen control in the early stage of fermentation is realized by air volume and speed control, the air volume is controlled at 1-3VVM, and the speed is controlled at 0-50 rpm; the fermentation The mid-stage is the period of 73-120 hours of fermentation; the dissolved oxygen control in the mid-fermentation is realized by air volume and speed control, the air volume is controlled at 0.5-1.5VVM, and the speed is controlled at 50-200 rpm; During the period of 121-216 hours; the dissolved oxygen control in the late stage of fermentation is realized through air volume and speed control, the air volume is controlled at 1-3VVM, and the speed is controlled at 100-200 rpm. 2. the method for regulating Mortierella alpine fermentation based on dissolved oxygen according to claim 1 to produce arachidonic acid oil, is characterized in that: the dissolved oxygen control in the early stage of fermentation is realized by air volume and speed control, and the air volume is controlled at 1 -2VVM, the speed is controlled at 0-50 rpm, and the dissolved oxygen is controlled between 40%-55%. 3. the method for regulating Mortierella alpine fermentation based on dissolved oxygen according to claim 1 to produce arachidonic acid oil, characterized in that: the control of dissolved oxygen in the middle stage of fermentation is realized by air volume and speed control, and the air volume is controlled at 0.8 -1.2VVM, the speed is controlled at 80-150 rpm, and the dissolved oxygen is controlled between 20%-35%. 4. the method for regulating Mortierella alpine fermentation based on dissolved oxygen according to claim 1 to produce arachidonic acid oil, characterized in that: the control of dissolved oxygen in the later stage of fermentation is realized by air volume and speed control, and the air volume is controlled at 1.5 -2VVM, the speed is controlled at 150-200 rpm, and the dissolved oxygen is controlled at 40%-50%.