CN106987528B

CN106987528B - Bacterium for producing docosahexaenoic acid and application thereof

Info

Publication number: CN106987528B
Application number: CN201710398286.7A
Authority: CN
Inventors: 陈礼毅; 钟惠昌; 陈水荣
Original assignee: Xiamen Huison Biotech Co ltd
Current assignee: Xiamen Huison Biotech Co ltd
Priority date: 2017-05-31
Filing date: 2017-05-31
Publication date: 2020-05-05
Anticipated expiration: 2037-05-31
Also published as: CN106987528A

Abstract

The invention discloses a bacterium for producing docosahexaenoic acid and application thereof. The bacteria for producing the docosahexaenoic acid provided by the invention is Schizochytrium limacinum (Schizochytrium limacinum) HS01, and the preservation number of the bacteria in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 13746. Experiments prove that schizochytrium limacinum HS01 is fermented and cultured to obtain fermentation liquor; the DHA in the fermentation liquor accounts for 45.0-60.0% of the oil content. Therefore, the invention has important application value.

Description

Bacterium for producing docosahexaenoic acid and application thereof

Technical Field

The invention relates to the industrial fields of industrial microorganisms, food and feed industries, in particular to a bacterium for producing docosahexaenoic acid and application thereof.

Background

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid essential to the human body. In terms of molecular structure, DHA is a straight-chain fatty acid having 22 carbon atoms and 6 double bonds, and belongs to OMEGA-3 series fatty acids (OMEGA-3) because the 1 st double bond is present at the 3 rd carbon atom on the methyl end of the fatty acid chain. DHA has important physiological functions such as promoting development of the nervous system, improving retinal function, improving vision, preventing cardiovascular disease, treating cardiovascular disease, anti-inflammation, and suppressing allergic reactions, among others. Because the DHA content in daily diet is often insufficient, the addition of DHA to foods or milk powder is of great significance to humans, especially infants and the elderly.

At present, there are two main methods for producing DHA: one is a conventional DHA source, i.e., extracted from tissues of marine fishes (mainly including salmon, mackerel, salmon and sardine), but the quality of fish oil obtained by extraction is affected by the variety, season and place of fishing of the fishes, and the quality of fish oil is also affected by the increasingly serious shortage of fish resources and environmental pollution; the other is a novel DHA production method, namely DHA is produced by fermenting marine microorganisms, and the method has the advantages of short period, no objective condition influence, no fishy smell and the like, and has wide prospect. The marine microorganism used for fermentation production of DHA is mainly Schizochytrium (Schizochytrium), but the current Schizochytrium used for fermentation production of DHA cannot further improve yield and reduce cost due to the limitation of technical indexes such as self total fatty acid content, DHA content, growth rate and the like.

Disclosure of Invention

The technical problem to be solved by the invention is how to produce docosahexaenoic acid.

In order to solve the technical problems, the invention firstly provides a bacterium for producing docosahexaenoic acid.

The bacteria for producing the docosahexaenoic acid provided by the invention is Schizochytrium (Schizochytrium limacinum) HS01, and the preservation number of the bacteria in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 13746.

In order to solve the technical problems, the invention also provides a microbial inoculum, which contains the Schizochytrium limacinum HS01CGMCC No. 13746.

The Schizochytrium limacinum HS01CGMCC No.13746 or the application of the microbial inoculum in producing docosahexaenoic acid also belong to the protection scope of the invention.

In order to solve the above technical problems, the present invention also provides a method for producing docosahexaenoic acid, which comprises the step of fermenting and culturing the schizochytrium limacinum HS01CGMCC No.13746 to obtain docosahexaenoic acid.

In the above method, the fermentation medium solute and its solubility used in the fermentation culture may be: 20-120 g/L (such as 20-60 g/L, 60-120 g/L, 20g/L, 60g/L or 120g/L) of glucose, 5-15 g/L (such as 5-10 g/L, 10-15 g/L, 5g/L, 10g/L or 15g/L) of glutamic acid or sodium glutamate, 3-15 g/L (such as 3-10 g/L, 10-15 g/L, 3g/L, 10g/L or 15g/L) of corn steep liquor dry powder, NaSO₄5-24 g/L (e.g., 5-14 g/L, 14-24 g/L, 5g/L, 14g/L or 24g/L), KCl 0.1-1.0 g/L (e.g., 0.1-0.5 g/L, 0.5-1.0 g/L, 0.1g/L, 0.5g/L or 1.0g/L), MgSO₄1.0-3.0 g/L (e.g., 1.0-2.0 g/L, 2.0-3.0 g/L, 1.0g/L, 2.0g/L, or 3.0g/L), K₂SO₄0.3-1.5 g/L (e.g., 0.3-1.0 g/L, 1.0-1.5 g/L, 0.3g/L, 1.0g/L, or 1.5 g%L)、KH₂PO₄0.5-1.5 g/L (e.g., 0.5-1.0 g/L, 1.0-1.5 g/L, 0.5g/L, 1.0g/L or 1.5g/L), (NH)₄)₂SO₄0.5-1.5 g/L (e.g., 0.5-1.0 g/L, 1.0-1.5 g/L, 0.5g/L, 1.0g/L, or 1.5g/L), CaCl₂0.1-1.0 g/L (e.g., 0.1-0.5 g/L, 0.5-1.0 g/L, 0.1g/L, 0.5g/L, or 1.0 g/L); the solute may be water; the pH value can be 5.0-6.5 (such as 5.0, 6.0 or 6.5). The fermentation medium may specifically be: mixing glucose 60g, glutamic acid or sodium glutamate 10g, corn steep liquor dry powder 10g, and NaSO₄14g、KCl0.5g、MgSO₄2.0g、K₂SO₄1.0g、KH₂PO₄1.0g、(NH₄)₂SO₄1.0g and CaCl₂0.5g of this solution was dissolved in 1L of distilled water, and the pH was adjusted to 6.0.

In the above process, the initial biomass in the fermentation culture may be 1.0X 10⁸～2.5×10⁸cfu/mL (e.g., 1.0X 10)⁸～1.5×10⁸cfu/mL、1.5×10⁸～2.5×10⁸cfu/mL、1.0×10⁸cfu/mL、1.5×10⁸cfu/mL or 2.5X 10⁸cfu/mL)。

In the above process, the initial biomass in the fermentation culture may be 5.0X 10⁸～3.0×10⁹cfu/mL (e.g., 5.0X 10)⁸～1.0×10⁹cfu/mL、1.0×10⁹～3.0×10⁹cfu/mL、5.0×10⁸cfu/mL、1.0×10⁹cfu/mL or 3.0X 10⁹cfu/mL)。

In the above method, the fermentation culture inoculum size may be 3-10% (e.g., 3-5%, 5-10%, 3%, 5%, or 10%).

In the above method, the fermentation culture may be carried out under a culture condition of 22-28 ℃ (e.g., 22-25 ℃, 25-28 ℃, 22 ℃, 25 ℃ or 28 ℃) for 72-120 hours (e.g., 72-100 hours, 100-120 hours, 72 hours, 100 hours or 120 hours) and a dissolved oxygen concentration of 5-80% (e.g., 5-50%, 50-80%, 5%, 50% or 80%).

In the above method, said "fermenting and culturing said schizochytrium limacinum (schizochytrium limacinum) HS01CGMCC No. 13746" may further comprise preparing a primary seed solution and/or preparing a secondary seed solution and/or preparing a primary fermentation seed solution and/or preparing a secondary fermentation seed solution;

the steps for preparing the first-order seed solution may be as follows: culturing the Schizochytrium limacinum HS01CGMCC No.13746 in a shake flask to obtain a first-level seed solution;

the steps for preparing the secondary seed liquid may be as follows: shake flask culture first seed liquid, get second seed liquid;

the steps for preparing the fermented primary seed liquid may be as follows: fermenting and culturing the secondary seed liquid to obtain a fermented primary seed liquid;

the steps for preparing the fermentation secondary seed liquid can be as follows: fermenting the primary seed liquid by fermentation culture to obtain a secondary seed liquid by fermentation.

In the first-stage seed liquid preparation and the second-stage seed liquid preparation, the solute and the solubility of the shake flask culture medium used for shake flask culture can be as follows: 10-90 g/L (such as 10-50 g/L, 50-90 g/L, 10g/L, 50g/L or 90g/L) of glucose, 5-25 g/L (such as 5-15 g/L, 15-25 g/L, 5g/L, 15g/L or 25g/L) of yeast powder; the solute may be water; the pH is natural. The 'shaking culture' is carried out under the culture conditions of 22-28 ℃ (such as 22-25 ℃, 25-28 ℃, 22 ℃, 25 ℃ or 28 ℃), 150-250 rpm/min (such as 150-200 rpm/min, 200-250 rpm/min, 150rpm/min, 200rpm/min or 250rpm/min) and 24-48 h (such as 24-36 h, 36-48 h, 24h, 36h or 48 h). The inoculation amount of the shake flask culture is 3-10% (such as 3-5%, 5-10%, 3%, 5% or 10%). The shake flask culture medium may specifically be: dissolving glucose 50g and yeast powder 15g in 1L distilled water, and adjusting pH to natural value.

In the preparation of the first-stage fermentation seed liquid and the preparation of the second-stage fermentation seed liquid, the solutes and the solubilities of a seed culture medium used in the fermentation culture can be as follows: 20-100 g/L (such as 20-60 g/L, 60-120 g/L, 20g/L, 60g/L or 120g/L) of glucose, 5-15 g/L (such as 5-10 g/L, 10-15 g/L, 5g/L, 10g/L or 15g/L) of yeast powder, NaSO₄5-24 g/L (e.g., 5-10 g/L, 10-24 g/L, 5g/L, 10g/L or 24g/L), KCl 0.1-1.0 g/L (e.g., 0.1-0.5 g/L, 0.5-1.0 g/L, 0.1g/L, 0.5g/L or 1.0g/L), MgSO₄1.0-3.0 g/L (e.g., 1.0-2.0 g/L, 2.0-3.0 g/L, 1.0g/L, 2.0g/L, or 3.0g/L), K₂SO₄0.3～1.5g/L (e.g. 0.3-1.0 g/L, 1.0-1.5 g/L, 0.3g/L, 1.0g/L or 1.5g/L), KH₂Po₄0.5-1.5 g/L (e.g., 0.5-1.0 g/L, 1.0-1.5 g/L, 0.5g/L, 1.0g/L or 1.5g/L), (NH)₄)₂SO₄0.5-1.5 g/L (e.g., 0.5-1.0 g/L, 1.0-1.5 g/L, 0.5g/L, 1.0g/L, or 1.5g/L), CaCl₂0.1-1.0 g/L (e.g., 0.1-0.5 g/L, 0.5-1.0 g/L, 0.1g/L, 0.5g/L, or 1.0 g/L); the solute may be water; the pH value can be 5.0-6.5 (such as 5.0, 6.0 or 6.5). The culture condition of the fermentation culture is 22-28 ℃ (such as 22-25 ℃, 25-28 ℃, 22 ℃, 25 ℃ or 28 ℃) for 24-48 h (such as 24-36 h, 36-48 h, 24h, 36h or 48h), and the dissolved oxygen concentration is 10-80% (such as 10-50%, 50-80%, 10%, 50% or 80%). The inoculation amount of the fermentation culture is 3-10% (such as 3-5%, 5-10%, 3%, 5% or 10%). The seed culture medium can be specifically: mixing glucose 60g, yeast powder 10g, and NaSO₄10g、KCl 0.5g、MgSO₄2.0g、K₂SO₄1.0g、KH₂Po₄1.0g、(NH₄)₂SO₄1.0g and CaCl₂0.5g of this solution was dissolved in 1L of distilled water, and the pH was adjusted to 6.0.

Fermenting and culturing Schizochytrium limacinum HS01 to obtain fermentation liquid. The result shows that the DHA in the fermentation liquor accounts for 45.0-60.0% of the grease content. Therefore, the DHA can be produced by using Schizochytrium limacinum HS01 provided by the invention, and the DHA has important application value.

Drawings

FIG. 1 shows the colony morphology characteristics of Schizochytrium HS 01.

FIG. 2 shows morphological characteristics of Schizochytrium HS 01.

Deposit description

The strain name is as follows: schizochytrium limacinum (Fr.) Kuntze

Latin name: schizochytrium limacinum

The strain number is as follows: HS01

The preservation organization: china general microbiological culture Collection center

The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)

Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district

The preservation date is as follows: 3 and 10 months in 2017

Registration number of the preservation center: CGMCC No.13746

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The media used in the following examples are as follows:

wort agar medium: dissolving 150g of malt extract powder in 1L of mixed solution (prepared by mixing 1 volume of natural seawater and 1 volume of distilled water), wherein the pH value is natural; agar powder was then added to a concentration of 15g/100mL to obtain a medium.

Screening a liquid culture medium: 50g of glucose and 15g of yeast powder are dissolved in 1L of mixed solution (formed by mixing 1 volume of natural seawater and 1 volume of distilled water), and the pH value is natural.

Screening a solid culture medium: agar powder was added to the screening liquid medium to a concentration of 15g/100mL to obtain a medium.

Screening the plates: the screened solid medium at about 55 ℃ is poured into a petri dish and cooled to obtain a solid plate.

Shake flask culture medium: dissolving glucose 50g and yeast powder 15g in 1L distilled water, and adjusting pH to natural value.

Seed culture medium: 60g of glucose, 10g of yeast powder and NaSO₄10g、KCl 0.5g、MgSO₄2.0g、K₂SO₄1.0g、KH₂Po₄1.0g、(NH₄)₂SO₄1.0g and CaCl₂0.5g of this solution was dissolved in 1L of distilled water, and the pH was adjusted to 6.0.

Fermentation medium: mixing glucose 60g, glutamic acid or sodium glutamate 10g, corn steep liquor dry powder 10g, and NaSO₄14g、KCl 0.5g、MgSO₄2.0g、K₂SO₄1.0g、KH₂PO₄1.0g、(NH₄)₂SO₄1.0g and CaCl₂0.5g of this solution was dissolved in 1L of distilled water, and the pH was adjusted to 6.0.

The corn steep liquor dry powder is a product of Beijing Sorlebao science and technology Limited, and the product catalog number is FA 0010.

The yeast powder is a product of Angel Yeast GmbH, and the product catalog number is LMO 2.

Example 1 isolation, identification and preservation of Schizochytrium limacinum HS01CGMCC No.13746

Separation of Schizochytrium HS01

1. The inventor collects schizochytrium limacinum from a plurality of mangrove forests in Yuanxiao county of Zhangzhou city in Fujian province, and mixes the schizochytrium limacinum and the mangrove forests to obtain mixed liquid; inoculating 0.5mL of the mixed solution to 5mL of a screening liquid culture medium, and then culturing at 25 ℃ and 200rpm/min for 2d to obtain a culture solution.

2. And (3) uniformly coating the culture bacterial liquid obtained in the step (1) on a screening plate, and performing static culture at 25 ℃ for 2d to generate a single bacterial colony.

3. After the step 2 is completed, single colonies are respectively selected and inoculated to 5mL of fermentation culture medium, and then cultured for 2d at 25 ℃ and 200rpm/min to obtain culture bacteria liquid.

4. And (4) centrifuging the culture solution obtained in the step (3) at 4 ℃ and 2000rpm for 5min, and collecting thalli.

5. Taking 1.0-2.0 g of the thalli to a measuring cylinder with a plug (the specification is 100mL), firstly adding 15mL of HCl aqueous solution with the concentration of 8.3mol/L, covering a cover, and hydrolyzing in a water bath at 70-80 ℃ for 50-60 min (during the hydrolysis, placing the measuring cylinder with the plug on a vortex mixer for 1 time every 10 min); after cooling to room temperature, firstly adding 10mL of 95% (v/v) ethanol aqueous solution, sufficiently and uniformly shaking, then adding 20mL of anhydrous ether, sufficiently shaking for extraction for 1-2 min, finally adding 20mL of petroleum ether, sufficiently shaking for extraction for 1-2 min, standing for layering, placing the upper organic phase in a glass weighing dish (dried and weighed), placing the glass weighing dish on a boiling water bath in a ventilation cabinet to sufficiently evaporate the organic phase completely (sufficiently and completely volatilize completely), and obtaining the liquid phase as the grease.

6. And (3) taking the grease extracted in the step (5), detecting the DHA content according to the national standard of GB 26400-2011 food safety, and detecting the composition and the content of the fatty acid according to the method of AOAC 996.06.

And selecting strains with higher DHA content, and repeatedly purifying for 24 times. The screened schizochytrium limacinum strain is named as schizochytrium limacinum HS 01.

And (3) inoculating the schizochytrium HS01 monoclonal to a fermentation medium for continuous passage of 12 generations, and detecting the DHA content according to the steps. The result shows that the stability of DHA produced by schizochytrium HS01 is good.

II, identification of Schizochytrium HS01

1. Morphological identification

The schizochytrium HS01 is inoculated on a wort agar culture medium, dark culture is carried out at 25 ℃, the morphology of a bacterial colony is observed after 5 days, and the morphological characteristics of the bacterial body are analyzed and observed by a high-resolution transmission electron microscope.

The results of the experiment are shown in FIGS. 1 and 2. The result shows that the colony diameter of the schizochytrium HS01 is 2-4.3 mm, the schizochytrium HS01 is white (light orange at the later stage), and the edge is irregular; the thallus is proliferated in a fission mode, the cell wall is thin, spherical, colorless or light orange, transparent and 4.5-15.5 mu m in size, and zoospores and an exoplasmic reticulum are not seen.

2. 18s rDNA sequence homology analysis

The partial sequence of the 18s rDNA of the schizochytrium HS01 is shown as the sequence 1 in the sequence table.

The partial sequence of the 18s rDNA of the schizochytrium HS01 is shown as the sequence 2 in the sequence table.

Combining the above identification results, the Schizochytrium HS01 is Schizochytrium (Schizochytrium limacinum).

Third, preservation of Schizochytrium HS01

Schizochytrium limacinum HS01 has been deposited in China general microbiological culture Collection center (CGMCC, address: No. 3, Xilu No.1, Beijing area, Chaoyang area, China) in 2017, 03 and 10 days, and the deposit number is CGMCC No. 13746. The schizochytrium limacinum HS01 is called schizochytrium limacinum HS01CGMCC No.13746, abbreviated as schizochytrium limacinum HS 01.

Example 2 fermentation of Schizochytrium limacinum HS01 to produce DHA

Production of DHA by fermentation of Schizochytrium limacinum HS01

1. The method comprises the steps of inoculating a schizochytrium limacinum HS01 monoclonal to a shake flask (with the specification of 10mL) filled with 2mL of shake flask culture medium, and culturing at 22-28 ℃ and 150-250 rpm/min for 24-48 h to obtain a first-stage seed solution.

2. Taking the first-stage seed solution, inoculating the first-stage seed solution into a shake flask (the shake flask specification is 1L) filled with 250mL of shake flask culture medium in an inoculation amount of 3-10% (v/v), and culturing at 22-28 ℃ at 150-250 rpm/min for 24-48 h to obtain a second-stage seed solution.

3. Inoculating the second-stage seed solution into a fermentation tank (5L in the specification of the fermentation tank) filled with 3L of seed culture medium in an inoculation amount of 3-10% (v/v), and culturing at 22-28 ℃ for 24-48 h (10-80% of dissolved oxygen) to obtain a first-stage fermentation seed solution.

4. Inoculating the first-stage fermentation seed liquid with 3-10% (v/v) of inoculum size to a fermentation tank (100L fermentation tank specification; initial biomass is 1.0 × 10 after inoculation)⁸～2.5×10⁸cfu/mL) at 22-28 ℃ for 72-120 h (dissolved oxygen of 5-80%) to obtain a fermentation broth. The fermentation broth contains DHA.

Secondly, analysis of fatty acid component in fermentation liquor

According to the method in the step one of the embodiment 1, 5, the grease of the fermentation liquid is extracted, then the DHA content is detected according to the national standard for food safety GB 26400-2011, and the composition and the content of the fatty acid are detected according to the method of AOAC 996.06.

The results are shown in Table 1. The result shows that the DHA accounts for 45.0-60.0% of the grease.

TABLE 1

Third, separation and quality identification of DHA in fermentation liquor

1. Taking the fermentation liquor obtained in the first step, and sequentially performing cell wall breaking of schizochytrium limacinum and extraction of crude DHA algae oil (the method for performing cell wall breaking of schizochytrium limacinum and extraction of crude DHA algae oil is described in chinese patent application CN 101817738B).

2. The DHA algal oil crude oil extracted in step 1 is refined (the refining method is described in chinese patent document CN 103865642B).

The quality indexes of the DHA algae oil crude oil after refining are shown in Table 2.

TABLE 2

Example 3 production of DHA by Large-Scale fermentation of Schizochytrium limacinum HS01

1. Inoculating single schizochytrium limacinum HS01 colony to a shake flask (250 mL in shake flask specification) filled with 20mL of shake flask culture medium, and culturing at 22-28 ℃ and 150-250 rpm/min for 24-48 h to obtain a first-stage seed solution.

2. Taking the first-stage seed solution, inoculating the first-stage seed solution into a shake flask (2L in shake flask specification) filled with 250mL of shake flask culture medium in an inoculation amount of 3-10% (v/v), and culturing at 22-28 ℃ and 150-250 rpm/min for 24-48 h to obtain a second-stage seed solution.

3. Inoculating the secondary seed solution into a fermentation tank (the specification of the fermentation tank is 1000L) filled with 500L of seed culture medium in an inoculation amount of 3-10% (v/v), and culturing at 22-28 ℃ for 24-48 h (the dissolved oxygen is 10-80%) to obtain a primary fermentation seed solution with the biomass of 15-30 g/L.

4. Inoculating the first-stage fermentation seed liquid into a fermentation tank (the specification of the fermentation tank is 8000-10000L) filled with 5000L of seed culture medium in an inoculation amount of 5-15% (v/v), and culturing at 22-28 ℃ for 24-48 h (the dissolved oxygen is 10-80%) to obtain a second-stage fermentation seed liquid with the biomass of 15-30 g/L.

5. Inoculating the fermentation secondary seed liquid with an inoculation amount of 5-15% (v/v) to 30000L of the fermentation mediumFermenter for fermentation medium (fermenter size 75000L; initial biomass after inoculation is 5.0X 10⁸～3.0×10⁹cfu/mL) at 22-28 ℃ for 72-120 h (dissolved oxygen of 5-80%) to obtain a fermentation broth. The fermentation broth contains DHA.

The fatty acid content of the fermentation broth was analyzed according to the method of step two in example 2. The result shows that the DHA in the fermentation liquor accounts for 35.0-60.0% of the oil content.

Therefore, the DHA produced by using Schizochytrium limacinum HS01 provided by the invention has higher production value.

<110> Xiamen Huishi Biometrics Ltd

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Claims

1. Schizochytrium limacinum (Schizochytrium limacinum) HS01, wherein the preservation number of the Schizochytrium limacinum in the China general microbiological culture Collection center is CGMCC No. 13746.

2. A microbial inoculum, which is characterized in that: the microbial inoculum contains Schizochytrium limacinum (Schizoochytrium limacinum) HS01CGMCC No.13746 according to claim 1.

3. Use of the schizochytrium limacinum HS01CGMCC No.13746 or the microbial inoculum of claim 2 of claim 1 for the production of docosahexaenoic acid.

4. A method for producing docosahexaenoic acid, comprising the step of fermentatively culturing the Schizochytrium limacinum HS01CGMCC No.13746 of claim 1 to obtain docosahexaenoic acid;

the method specifically comprises the following steps:

1) preparing a first-stage seed liquid, a second-stage seed liquid, a fermented first-stage seed liquid and a fermented second-stage seed liquid;

the first-stage seed liquid is prepared by the following steps: culturing the schizochytrium limacinum HS01CGMCC No.13746 in a shake flask to obtain a first-level seed solution;

the steps for preparing the secondary seed liquid are as follows: shake flask culture first seed liquid, get second seed liquid;

the steps for preparing the fermentation primary seed liquid are as follows: fermenting and culturing the secondary seed liquid to obtain a fermented primary seed liquid;

the preparation method of the fermentation secondary seed liquid comprises the following steps: fermenting the primary seed liquid by fermentation culture to obtain a secondary seed liquid by fermentation.

2) Taking 5-15% by volume of the secondary fermentation seed liquid to a fermentation tank filled with 30000L of fermentation medium, and performing fermentation culture to obtain docosahexaenoic acid;

the culture medium solute and the solubility used in the fermentation culture are as follows: 20-120 g/L glucose, 5-15 g/L glutamic acid or sodium glutamate and 3-15 g/L, NaSO g/L corn steep liquor dry powder₄5～24g/L、KCl 0.1～1.0g/L、MgSO₄1.0～3.0g/L、K₂SO₄0.3～1.5g/L、KH₂PO₄0.5～1.5g/L、（NH₄）₂SO₄0.5～1.5g/L、CaCl₂0.1-1.0 g/L; the solute is water; pH5.0-6.5;

the fermentation culture condition is that the culture is carried out for 72-120 h at 22-28 ℃, and the dissolved oxygen concentration is 5-80%;

the initial biomass in the fermentation culture is 5.0 x 10⁸～3.0×10⁹cfu/mL；

The volume ratio of the inoculum size of shake flask culture in the preparation of the secondary seed liquid is 3-10%;

the volume ratio of the inoculum size of the fermentation culture in the preparation of the fermentation secondary seed liquid is 5-15%.