CN106636235B

CN106636235B - Method for producing DHA (docosahexaenoic acid) by microbial fermentation

Info

Publication number: CN106636235B
Application number: CN201611270522.9A
Authority: CN
Inventors: 王炳荣; 文昌; 陈必钦; 张斌; 韩雯; 庞毅; 李丹; 詹光煌
Original assignee: INNER MONGOLIA KINGDOMWAY PHARMACEUTICAL CO Ltd; Xiamen Kingdomway Group Co
Current assignee: INNER MONGOLIA KINGDOMWAY PHARMACEUTICAL CO Ltd; Xiamen Kingdomway Group Co
Priority date: 2016-12-30
Filing date: 2016-12-30
Publication date: 2020-08-04
Anticipated expiration: 2036-12-30
Also published as: CN109777606A; CN109777607A; CN109777607B; WO2018120574A1; CN109777606B; CN106636235A

Abstract

The invention belongs to the field of fermentation engineering, relates to a method for producing DHA (docosahexaenoic acid) by microbial fermentation, and particularly relates to a method for producing DHA-containing mixed grease by industrial fermentation of schizochytrium limacinum strains. More specifically, the present invention relates to a method for culturing a microorganism for the production of DHA, wherein: controlling the dissolved oxygen saturation at 5% -10% from 36-60 hours of the fermenter culture, and/or not adding any nitrogen source from 36-60 hours of the fermenter culture. The method for preparing DHA has high yield and high purity, and is beneficial to large-scale industrial production of DHA.

Description

Method for producing DHA (docosahexaenoic acid) by microbial fermentation

Technical Field

The invention belongs to the field of fermentation engineering, relates to a method for producing DHA (docosahexaenoic acid) by microbial fermentation, and particularly relates to a method for producing DHA-containing mixed grease by industrial fermentation of schizochytrium limacinum strains.

Background

DHA, the full name docosahexaenoic acid (DHA), is a polyunsaturated fatty acid. The human body itself is difficult to synthesize and must be ingested from the outside. DHA is one of essential fatty acids, has important physiological regulation function and health care function, can cause a series of diseases including growth retardation, skin abnormality, scale, sterility, dysnoesia and the like when lacking, and has special prevention and treatment effects on cardiovascular diseases. The related research also shows that DHA can act on various tissues and cells, has the effects of inhibiting inflammation and immunity, including reducing the generation of inflammatory factors, inhibiting the proliferation of lymphocytes and the like, and also has multiple effects of preventing senile dementia, neurological diseases and the like.

Current commercial sources of DHA are mainly fish oil and microalgae. DHA extracted from traditional deep sea fish oil is unstable due to the influence of fish varieties, seasons and geographical positions, the content of cholesterol and other unsaturated fatty acids is high, the length and the unsaturation degree of fatty acid chains are different greatly, and the problems of limited DHA yield, low content, difficult separation and purification, high cost and the like are caused. With the increasing shortage of fish oil raw material sources, it is difficult to realize the wide application of DHA, a high value-added product, in the industries of food, medicine and the like. The DHA produced by the microbial fermentation method can overcome the defects of the traditional fish oil extraction, can be used for mass production of DHA, continuously meets the requirements of people, has wide application prospect, and is well concerned by scholars at home and abroad. The microbial fermentation method adopts oil-producing microorganisms such as fungi and microalgae to ferment and produce DHA-containing algae oil, and the essential oil with high DHA content is obtained through refining and extraction. DHA-producing strains approved by the national ministry of health include Schizochytrium sp, Ukenia amoeboida, and Crypthecodinium cohnii.

The market share of DHA produced by a microbial fermentation method rapidly rises year by year, has a tendency of replacing fish oil DHA, improves the production technology and quality of microalgae DHA, and has a wide market prospect in the process of microalgae DHA.

The patent with publication number CN103882072A discloses a method for producing docosahexaenoic acid by utilizing Schizochytrium, and the highest disclosed yield is 61.2 g/L of dry cell weight, 55.07% of DHA content and 22.17 g/L of DHA content, the patent with publication number CN101812484A discloses a method for producing DHA by fermenting Schizochytrium through high-density culture, the disclosed yields are 150 g/L of dry cell weight and 26-30 g/L of DHA, which is the highest production level of DHA produced by adopting Schizochytrium sp reported at present, although the productivity of DHA is greatly improved compared with the previous research, the production cost is greatly reduced, the unit yield is improved, and the method for producing DHA through microbial fermentation can be greatly popularized and is still insufficient.

The existing methods for extracting DHA from schizochytrium limacinum fermentation liquor mainly comprise three methods, namely a centrifugal method, an organic solvent extraction method and a supercritical extraction method. Centrifugation as disclosed in patent publication No. CN101817738B, discloses a method for extracting DHA from algal and fungal cells by breaking the walls: separating and collecting cells of the fermented microalgae or fungus fermentation liquor by a separation system, adjusting the pH of the bacterial sludge to 2.0-4.0 by acid, controlling the temperature of the bacterial sludge to 10-20 ℃, adding an antioxidant into the bacterial sludge, and performing high-pressure homogenization and wall breaking by a high-pressure homogenizer; and adding water into the bacterial sludge after wall breaking, stirring, and separating the feed liquid by a three-phase separator to obtain DHA grease. The invention adopts physical wall breaking and physical extraction methods, has simple process and high cell wall breaking efficiency, can effectively protect the biological activity of substances in algae and fungus cells by low-temperature and antioxidant treatment of bacterial sludge, and is green, nontoxic and residue-free. However, the oil layer after centrifugation is poor in quality, contains impurities such as water, culture medium components and cell debris besides oil and fat, and is not beneficial to subsequent refining, and the waste water layer after centrifugation contains a large amount of mushroom dregs, so that COD is high, and the treatment is difficult or the treatment cost is extremely high. An organic solvent extraction method, for example, a patent with publication number of CN101824363B discloses a method for extracting docosahexaenoic acid oil: after the fermentation liquor containing docosahexaenoic acid is broken by an enzyme method, organic solvent is adopted to carry out first-stage water separation to enrich thalli, and then organic solvent is adopted to carry out second-stage extraction to obtain crude oil. The method is simple to operate, equipment investment is low, but the method uses an organic solvent for extraction, the final product may have solvent residues, and potential safety hazards such as flammability and explosiveness exist in the extraction process. Supercritical extraction method for example, patent with publication number CN102181320B discloses an extraction method of biological fermentation DHA algae oil, comprising the following steps: a) drying the solid obtained after solid-liquid separation of the microalgae fermentation liquor to obtain dry thalli; b) extracting the dried thalli by using supercritical carbon dioxide as an extracting agent to obtain carbon dioxide fluid; c) and carrying out reduced pressure separation on the carbon dioxide fluid to obtain DHA algae oil. Experiments show that the DHA algae oil obtained by the method provided by the invention has a DHA content of more than 40%, the highest extraction yield of only 85.23%, ethanol is required to be added as an auxiliary extraction agent, certain safety risk exists, the supercritical equipment is expensive, and the extraction yield is not high.

In the prior art, the DHA crude oil is refined by adopting a chemical refining technology, and the DHA essential oil is obtained by degumming, alkali refining, decoloring and deodorizing the DHA crude oil. The process technology inevitably has some problems, such as: alkali refining in order to meet the requirement of low acid value control, excessive alkali is usually added, and partial triglyceride is inevitably saponified; high COD wastewater generated by alkali refining pollutes the environment; the alkali refining needs long high-temperature treatment time, and the peroxide value and the anisidine value of the product are easily increased; high deodorization temperature, long deodorization time, easy generation of trans-fatty acid and the like.

At present, a new DHA production process needs to be developed.

Disclosure of Invention

The present inventors have made intensive studies and creative efforts to obtain a method for culturing microorganisms for producing DHA. The inventors have surprisingly found that the cultivation process allows to significantly increase biomass and DHA yield. Further, the present inventors have also found a method for extracting DHA crude oil, which can improve the extraction yield of DHA crude oil. Further, the inventor also finds a method for purifying DHA crude oil, which can improve various technical indexes and purification yield of DHA product oil. The invention obviously improves the yield of DHA-containing crude oil, the yield of DHA and the productivity of DHA. The following invention is thus provided:

one aspect of the present invention relates to a method for culturing a microorganism for the production of DHA (docosahexaenoic acid), wherein:

controlling the dissolved oxygen saturation (DO) at 5% -10% (e.g., 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%) from 36-60 hours of fermentor culture (preferably, until the end of fermentation); and/or

From 36 to 60 hours of the fermentor culture (preferably, until the end of fermentation), the nitrogen source is not added any more or the amount of the nitrogen source added is reduced by 50% or more (for example, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more).

In some embodiments of the invention, the method of culturing, wherein:

controlling the dissolved oxygen at 5% -10% and/or starting from 40-56 hours (e.g., 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, or 56 hours), 44-52 hours, 46-50 hours, or 48 hours of fermentor culture

From 40-56 hours (e.g., 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, or 56 hours), 44-52 hours, 46-50 hours, or 48 hours of fermentor culture, no more nitrogen source is added.

In some embodiments of the invention, the culture method wherein the dissolved oxygen saturation is 30% -50% (e.g., 30% -45%, 35% -45%, 40% -50%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%) before controlling the dissolved oxygen saturation to 5% -10%.

In some embodiments of the invention, the culture method, wherein the fermentation broth has a pH of 6.0 to 7.0.

In some embodiments of the invention, the culture method wherein the glucose concentration in the fermentation broth is maintained at 1-5 g/L (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 g/L).

In some embodiments of the present invention, the nitrogen source is ammonia water, preferably 25% to 45% ammonia water, more preferably 35% to 45% or 38% to 42% ammonia water, and particularly preferably 40% ammonia water.

In some embodiments of the invention, the method for culturing, wherein the fermentor culture further comprises the step of conducting a split culture 12-36 hours (e.g., 16-32 hours, 18-30 hours, 20-28 hours, 22-26 hours, 24 hours) from the beginning of the fermentor culture; for example, the fermenter culture is carried out in two or more fermenters.

In some embodiments of the present invention, the cultivation method further comprises the steps of inoculation and seed expansion culture before the fermenter culture;

preferably, the seed expansion culture comprises a primary seed expansion culture and a secondary seed expansion culture;

preferably, the primary seed expansion culture comprises the following steps:

inoculating the shake flask seed solution into a first-stage seed tank filled with a sterilized culture medium according to the inoculation amount of 0.4-1%, wherein the culture temperature is 25-32 ℃, the ventilation amount is 1-2 vvm, the tank pressure is 0.02-0.05 MPa, the stirring speed is 50-100 rpm, and the first-stage seed is cultured for 30-35 h to complete the first-stage seed expanded culture;

preferably, the secondary seed expansion culture comprises the following steps:

inoculating the seed liquid of the first-stage seed tank into a second-stage seed tank filled with a sterilized culture medium according to the inoculation amount of 1-3%, wherein the culture temperature is 25-32 ℃, the ventilation amount is 1-2 vvm, the tank pressure is 0.02-0.05 MPa, the stirring speed is 50-100 rpm, and the second-stage seed is cultured for 20-25 h to complete the second-stage seed expanded culture.

In some embodiments of the present invention, the culturing method further comprises the step of performing activation culture before inoculation and before expansion culture; preferably, the temperature of the activation culture is 25-32 ℃, and the time is 20-25 h.

In some embodiments of the invention, the cultivation method, wherein the fermenter cultivation comprises the steps of:

inoculating the seed liquid of the secondary seed tank into a fermentation tank filled with the sterilized culture medium according to the inoculation amount of 1-3%, wherein the culture temperature is 25-32 ℃, the ventilation amount is 1-2 vvm, the tank pressure is 0.02-0.05 MPa, and the stirring speed is 50-100 rpm, so as to culture in the fermentation tank.

In some embodiments of the invention, the culturing method, wherein the microorganism used to produce DHA is schizochytrium sp; preferably, the schizochytrium is selected from the strains with the preservation number of CGMCC No.6843, ATCCNO.20888, ATCC No.20889, ATCC No.28209 or ATCC MYA-1381.

In a specific embodiment of the present invention, the cultivation method comprises the following steps:

1) inoculating Schizochytrium sp slant preserved strain into 2L shake flask containing 400m L seed culture medium, and culturing at 25-32 deg.C at 200rpm for 20-25 h to complete strain activation culture;

2) inoculating the shake flask seed solution into a first-stage seed tank filled with a sterilized seed culture medium according to the inoculation amount of 0.4-1%, wherein the culture temperature is 25-32 ℃, the ventilation amount is 1-2 vvm, the tank pressure is 0.02-0.05 MPa, the stirring speed is 50-100 rpm, and the first-stage seed is cultured for 30-35 h to complete the first-stage seed expanded culture;

3) inoculating the seed solution of the first-stage seed tank into a second-stage seed tank filled with a sterilized seed culture medium according to the inoculation amount of 1-3%, wherein the culture temperature is 25-32 ℃, the ventilation volume is 1-2 vvm, the tank pressure is 0.02-0.05 MPa, the stirring rotation speed is 50-100 rpm, and the culture is carried out for 20-25 h, so as to complete the second-stage seed expanded culture;

4) inoculating the seed solution of the secondary seed tank into a fermentation tank filled with a sterilized fermentation medium according to the inoculation amount of 1-3%, wherein the culture temperature is 25-32 ℃, the ventilation amount is 1-2 vvm, the tank pressure is 0.02-0.05 MPa, and the stirring speed is 50-100 rpm, so as to culture in the fermentation tank;

5) DO is controlled to be 30% -50% before 48h of fermentation and 5% -10% after 48h by a dissolved oxygen control strategy, the DO is used as a control index in the process, the ventilation volume, the tank pressure and the stirring speed are adjusted, and a sterilized fresh culture medium or sterile water is appropriately supplemented;

6) before 48h of fermentation, 40% ammonia water is fed in to control the pH of the fermentation liquor to be 6.0-7.0, the pH of the fermentation liquor can be controlled to be stable while the nitrogen source is supplemented by the fed ammonia water, and after 48h, the feeding of the ammonia water is stopped, nitrogen-deficient culture is carried out, and the pH is not controlled any more;

7) fermenting for 24h, dividing fermentation liquor in a fermentation tank into two parts according to the volume, continuously culturing one half of the fermentation liquor in a main tank, transferring the other half of the fermentation liquor to an auxiliary tank with sterile pressure maintaining through a pressure difference method, respectively supplementing a proper amount of sterilized fresh culture medium or sterile water into the main tank and the auxiliary tank, and correspondingly adjusting the ventilation quantity, the tank pressure and the stirring rotation speed of the main tank and the auxiliary tank according to a dissolved oxygen regulation strategy after the separated culture;

8) in the fermentation process, the glucose concentration is continuously reduced along with the growth of the thalli, and a carbon source is fed-batch to maintain the sugar point (glucose concentration) in the fermentation liquor at 1-5 g/L;

9) fermenting and culturing for 84-108 h, terminating fermentation, placing in a tank, determining that the biomass in the fermentation liquor reaches 180 g/L, the crude oil content in the biomass reaches 45-60%, the DHA accounts for 40-55% of the total oil content, and the maximum DHA yield can reach 44.9 g/L and can reach 11.2 g/(L d).

The formulation of the seed culture medium used in the above steps 1) to 3) and the formulation of the fermentation medium used in the above step 4) are conventional formulations known to those skilled in the art. For example, the seed medium formulation is: 3% of glucose, 1% of peptone, 0.5% of yeast powder, 2% of sea crystal and natural pH. The formula of the fermentation medium is as follows: 12% of glucose, 1% of peptone, 0.5% of yeast powder, 2% of sea crystal and 6.5% of pH (researches on DHA production process of schizochytrium limacinum and breeding of high-yield strains, Wangshengqiang, etc., Master academic paper of Jiangnan university, 2013, p 13-14).

The method comprises the following steps of i) mixing crude glycerol and deionized water in a volume ratio of 1:4, ii) adjusting the pH to about 6.5 with hydrochloric acid, iii) separating and removing precipitated substances at a rotating speed of 5000rpm, wherein the insoluble saponin substances in the crude glycerol can be reduced after dilution, and in the step ii), soluble saponin substances in the crude glycerol are converted into insoluble free fatty acids, and in the step iii), the precipitated substances comprise free fatty acid solids and insoluble heavy metal impurities.

In step 8) above, the sugar point (concentration of glucose) is determined using methods known to those skilled in the art, such as biosensor determination.

In the fermentation process, DO is controlled to be 30% -50% in 48h before fermentation and 5% -10% after fermentation for 48h by adopting a dissolved oxygen control strategy, and in the process, the DO is used as a control index, the ventilation volume, the tank pressure and the stirring speed are adjusted, and a sterilized fresh culture medium or sterile water is appropriately supplemented.

And (3) feeding 40% ammonia water before fermenting for 48h in the fermentation process to control the pH of the fermentation liquor to be 6.0-7.0, controlling the pH of the fermentation liquor to be stable while feeding a nitrogen source, stopping feeding the ammonia water after 48h, and carrying out nitrogen deficiency culture, wherein the pH is not controlled any more.

And in the fermentation process, the fermentation liquor in the fermentation tank is divided into two parts for culture after fermentation is carried out for 24 hours, half of the fermentation liquor is left in the main tank for continuous culture, the other half of the fermentation liquor is transferred into an auxiliary tank for culture under sterile pressure maintaining through a pressure difference method, a proper amount of sterilized fresh culture medium or sterile water is respectively supplemented into the main tank and the auxiliary tank, and the ventilation volume, the tank pressure and the stirring speed of the main tank and the auxiliary tank are correspondingly adjusted according to the dissolved oxygen regulation strategy after the sub-tank culture.

The carbon source is fed during the fermentation process to maintain the sugar point (glucose concentration) in the fermentation liquor at 1-5 g/L.

The carbon source in the fermentation medium comprises one or more of glucose, corn steep liquor powder, molasses, glycerol and starch; the nitrogen source comprises one or more of soybean flour, yeast extract, peptone, ammonia water, sodium nitrate, sodium glutamate, and ammonium sulfate.

The microelements added into the fermentation medium comprise alanine, glutamic acid, lysine, calcium pantothenate, biotin, and vitamin B₁Microorganism B₆Microorganism B₁₂And one or more of vitamin K, wherein when the vitamin K is one of the vitamin K and the vitamin K, the addition amount is 0.001% -0.01%; when the amount of the components is more than one, the addition amount of any one component is 0.001-0.005%.

The inorganic salt added into the fermentation medium comprises one or more of magnesium sulfate, potassium chloride, sodium chloride, calcium chloride, potassium dihydrogen phosphate and dipotassium hydrogen phosphate.

The species Schizochytrium (Schizochytrium sp.) used in the above fermentation is not particularly limited, and may be any of various species classified into Schizochytrium, specifically, but not limited to Schizochytrium No. CGMCC No.6843, Schizochytrium No. ATCC No.20888, Schizochytrium No. ATCC No.20889, Schizochytrium No. ATCC No.28209, Schizochytrium limacinum Honda ethokochiatca-1381, and the source thereof is not particularly limited, and may be obtained from a fermentation institute or a microorganism depository organization such as ATCC, cgmccc, CCTCC, or may be Schizochytrium (Schizochytrium sp.) strain obtained from a natural environment by a known screening method.

Another aspect of the invention relates to a microbial fermentation broth obtained by the cultivation process according to any one of the invention. Preferably, the microbial fermentation broth is a schizochytrium fermentation broth.

Still another aspect of the present invention relates to a method for extracting DHA crude oil, comprising the steps of:

1) dehydrating fermentation liquor of microorganisms for producing DHA;

2) flexibly squeezing the product obtained in the step 1) to obtain DHA crude oil.

In some embodiments of the invention, the extraction method is described, wherein, in step 1), the fermentation broth is a schizochytrium limacinum fermentation broth; preferably, the fermentation broth is a microbial fermentation broth of the invention.

In some embodiments of the invention, the extraction method, wherein, in step 1), the dehydration treatment is selected from any one, two or three of the following:

centrifugation, first stage flexible pressing, drying such as spray drying;

preferably, the dehydration treatment comprises centrifugation and first-stage flexible pressing in sequence, or comprises centrifugation and spray drying in sequence;

preferably, the spray pressure of the spray drying is 4-8 MPa, the air inlet temperature is 160-220 ℃, and the air outlet temperature is 80-120 ℃.

In some embodiments of the invention, in the extraction method, in step 1), the first-stage flexible pressing adopts a gradual pressurization mode, the set pressure is 20-40 MPa, the pressurization time is 1-6 h, and the pressure is maintained for 1-4 h after the set pressure is reached.

In some embodiments of the invention, in the extraction method, in the step 2), the flexible pressing is performed in a step-by-step pressurizing mode, the set pressure is 50-150 MPa, the pressurizing time is 1-6 h, and the pressure is maintained for 1-4 h after the set pressure is reached.

In a specific embodiment of the present invention, the extraction method comprises the following steps:

1) material distribution: and conveying a certain amount of DHA fermentation liquor to the material distribution cavity by using a material distributor, returning the material distributor to the initial position, and waiting for material distribution in the next step.

2) Primary flexible squeezing: and (3) adopting a gradual pressurization mode, wherein the pressure range of the primary flexible pressing is 20-40 MPa, the pressurization time is 1-6 h, the set pressure is reached, the pressure is maintained for 1-4 h until no water drops flow out basically, and after the pressure is maintained for 1-4 h, the material is descended into the heavy pressure cavity, and the material is pushed into the secondary flexible pressing position.

3) Secondary flexible squeezing: and (3) adopting a gradual pressurization mode, keeping the final pressure of the pressing at 50-150 MPa for 1-6 h until the set pressure is reached, maintaining the pressure for 1-4 h until no oil drops flow out basically, collecting the pressed DHA crude oil, releasing the pressure, removing a secondary pressing cage, and separating the microalgae meal from the filter cloth.

The DHA fermentation liquor can be directly subjected to primary flexible squeezing, or a centrifugal method can be adopted to remove a part of water, so that the solid content of the fermentation liquor is increased, and then the primary flexible squeezing is performed, so that the squeezing time can be shortened and the production capacity can be improved.

The fermentation liquor centrifugation method can be carried out by adopting one of a horizontal spiral centrifuge, a disc centrifuge and a tubular centrifuge.

The method for extracting the DHA crude oil can also adopt the following modes: and (3) spray-drying the DHA fermentation liquor to obtain microalgae powder (namely dried thalli which are subjected to drying and moisture removal, and are called as microalgae powder, wherein the appearance of the dried thalli is granular powder), and performing flexible pressing on the microalgae powder (the condition is equal to that of the two-stage flexible pressing) to obtain the DHA crude oil.

The spray drying pressure is 4-8 MPa, the air inlet temperature is 160-220 ℃, the air outlet temperature is 80-120 ℃, and the moisture content of the DHA microalgae powder is controlled within 10%.

The DHA fermentation liquor can be directly subjected to spray drying, or a centrifugal method is adopted to remove a part of water before the spray drying, so that the solid content of the fermentation liquor is increased, and then the spray drying is carried out, thereby improving the productivity and saving the energy consumption.

The flexible pressing comprises the following steps:

(1) material distribution: and conveying a certain amount of DHA microalgal powder to the distribution cavity by using a distributor, returning the distributor to the initial position, and waiting for next distribution.

(2) And (2) performing flexible pressing, namely adopting a gradual pressing mode, keeping the pressing final pressure at 50-150 MPa for 1-6 h until the set pressure is reached, maintaining the pressure for 1-4 h until no oil drops flow out basically, collecting the pressed DHA crude oil, releasing the pressure, removing a secondary pressing cage, and separating the microalgae dregs from the filter cloth.

Still another aspect of the present invention relates to a DHA raw oil obtained by any of the extraction methods of the present invention.

Yet another aspect of the present invention relates to a method for purifying DHA crude oil, comprising the steps of hydrating, decolorizing, and molecular distilling DHA crude oil; preferably, the DHA crude oil is a DHA crude oil of the invention.

In some embodiments of the invention, the purification method, wherein the hydrating comprises the steps of:

heating DHA crude oil to 70-85 deg.C, adding 75-90 deg.C water according to the ratio of 1kg crude oil to 50-150 g water, stirring for 10-60 min at stirring speed of 30-90 r/min, standing for 1-6 h, and removing lower layer phospholipid to obtain the hydrated oil.

In some embodiments of the invention, the purification method, wherein the decolorizing comprises the steps of:

heating the hydration product to 90-110 ℃, controlling the vacuum degree to be less than or equal to-0.07 MPa, dehydrating in vacuum for 0.5-2 h, then cooling to 60-80 ℃, adding a decolorizing agent (such as activated carbon accounting for 1-3% of the weight of the hydration oil and activated clay accounting for 2-4%) and stirring for 0.5-1 h, stopping stirring, and filtering to remove the decolorizing agent to obtain the decolorized oil.

In some embodiments of the invention, the purification process, wherein the molecular distillation is a three-stage molecular distillation;

preferably, the molecular distillation comprises the steps of:

performing three-stage molecular distillation on the decolorized oil, controlling the first-stage vacuum degree to be less than or equal to 100Pa and the temperature to be 150-200 ℃, and removing the light components of the first stage; the obtained first heavy component enters a second-stage molecular distillation, the second-stage vacuum degree is controlled to be less than or equal to 50Pa, the temperature is controlled to be 180-220 ℃, and second-stage light components are removed; and performing third-stage molecular distillation on the obtained second heavy component, controlling the third-stage vacuum degree to be less than or equal to 5Pa and the temperature to be 200-250 ℃, and removing a third-stage light component to obtain a third heavy component, namely DHA finished oil.

Preferably, the molecular distillation is repeated 1 or more times.

In a specific embodiment of the present invention, the purification method comprises the following steps:

and refining the DHA crude oil through the steps of hydration, decoloration, molecular distillation and the like to finally obtain DHA product oil. The refining method comprises the following specific steps:

1) hydration: heating DHA crude oil to 70-85 deg.C, adding water of 75-90 deg.C according to the proportion of adding 50-150 g water into 1kg crude oil, stirring for 10-60 min at stirring speed of 30-90 r/min, standing for 1-6 h, and removing lower layer phospholipid by layering to obtain the hydrated oil.

2) And (3) decoloring: transferring the hydrated oil into a decoloring pot, heating to 90-110 ℃, controlling the vacuum degree to be less than or equal to-0.07 MPa, dehydrating for 0.5-2 h in vacuum, then cooling to 60-80 ℃, adding decoloring agents (activated carbon accounting for 1-3% of the weight of the hydrated oil and activated clay accounting for 2-4% of the weight of the hydrated oil), stirring and decoloring for 0.5-1 h, stopping stirring, and filtering to remove the decoloring agents to obtain the decolored oil.

3) Molecular distillation: and (3) performing three-stage molecular distillation on the decolorized oil, controlling the first-stage vacuum degree to be less than or equal to 100Pa and the temperature to be 150-200 ℃, removing the first-stage light components, controlling the second-stage vacuum degree to be less than or equal to 50Pa and the temperature to be 180-220 ℃, removing the second-stage light components, allowing the heavy components to enter the third-stage molecular distillation, controlling the third-stage vacuum degree to be less than or equal to 5Pa and the temperature to be 200-250 ℃, removing the third-stage light components, and collecting the heavy components to obtain the molecular distillation oil. The number of times of molecular distillation is controlled for 1-3 times until the acid value and the odor meet the standard requirements. And after the molecular distillation is finished, cooling to 20-40 ℃, adding an antioxidant, and packaging to obtain the DHA product oil.

In the step 1), preferably, the water is purified water.

Yet another aspect of the present invention relates to a DHA product oil obtained by any of the purification methods described herein.

Yet another aspect of the invention relates to a method for producing DHA or a DHA-containing product (e.g., DHA finished oil), comprising:

method for culturing a microorganism producing DHA according to any one of the above aspects,

Method for extracting DHA crude oil, and/or method for extracting DHA crude oil

The method for purifying the DHA raw oil in any one of the inventions.

In one embodiment of the present invention, the production process is as shown in FIG. 1.

In another embodiment of the present invention, the production method is as shown in fig. 2.

In the present invention,

the term "flexible press" refers to a high-pressure press mode in which a predetermined pressure is gradually reached by performing a press-dwell-press cycle using program control of P L C (programmable logic controller).

The term "purified water" refers to water for pharmaceutical use which is obtained by subjecting drinking water to distillation, ion exchange, reverse osmosis or other suitable method, and does not contain any additives. In one embodiment of the invention, the purified water is according to the regulations of the "Chinese pharmacopoeia".

The term "crude DHA oil" refers to a primary oil obtained from DHA fermentation broth without refining.

The term "DHA product oil" refers to essential oil obtained by refining DHA crude oil.

The term "fermentor culture" refers to a fermentation culture performed in a fermentor for the production of a product of interest following seed expansion.

The invention has the beneficial effects that:

the invention provides a method for producing docosahexaenoic acid grease through industrial fermentation, which can produce environment-friendly high-quality DHA grease with low cost and high yield.

The invention has at least one of the following technical effects:

(1) the technological indexes of the invention are obviously superior to those of the existing technological indexes, the obtained DHA grease has high yield and high purity, and is beneficial to large-scale industrial production of DHA, the addition of the crude glycerol also reduces the DHA fermentation production cost, and the market competitiveness of DHA fermentation production is greatly improved.

(2) According to the invention, the DHA crude oil is prepared by adopting a flexible squeezing process, an organic solvent is not required for extraction, the whole production process route does not need to use the organic solvent, the final product does not have solvent residue, and on one hand, the obtained product is green and healthy and has good product quality, and on the other hand, the production workshop is safe and environment-friendly, so that the method is a green clean production process.

(3) The aqueous solution removed by the primary flexible pressing of the fermentation liquor basically contains no bacterial residues, has low COD (chemical oxygen demand) ratio and is easy for biochemical treatment, and after the secondary flexible pressing is carried out to obtain crude oil, the remaining microalgae meal also contains a small amount of grease and a large amount of protein, so that the microalgae meal can be used as a feed additive, and is economic and environment-friendly.

(4) The invention adopts a molecular distillation one-step process to replace the traditional two-step processes of deacidification and deodorization. Molecular distillation can rapidly remove a large amount of free fatty acid and odor on the basis of keeping the physiological activity of the substance. Compared with the traditional alkali-refining deacidification method, the molecular distillation deacidification method has the advantages that the molecular distillation deacidification process is simple, the risk of excessive alkali-refining is reduced, the loss of neutral oil taken away from soapstock is reduced, the deacidification yield is obviously improved, the deacidification process is carried out under the high vacuum condition for a short time, the risk of increasing peroxide value and anisidine value caused by long heating time of alkali-refining deacidification is avoided, and the product stability is good; compared with the traditional steam distillation deodorization process, the molecular distillation deodorization time is short, the vacuum degree is high, the generation of trans-fatty acid is reduced, the deodorization effect of odor substances is good, and the product has no fishy smell.

(5) The production process avoids the use of organic solvent, avoids the cost of solvent consumption and solvent recovery, has low COD in the sewage, is easy to treat, has high refining yield, and greatly reduces the production cost.

Drawings

FIG. 1: the process flow of producing the DHA product oil in one embodiment of the invention is schematic.

FIG. 2: the process flow of producing DHA product oil according to another embodiment of the present invention is schematically illustrated.

FIG. 3: schizochytrium sp.CGMCC No.6843 is adopted to cultivate 100m in different modes³Fermentation in a fermenter to produce DHA.

Detailed Description

The present invention will be described in detail below with reference to specific examples. However, those skilled in the art will readily appreciate that the specific material ratios, process conditions and results thereof described in the examples are illustrative only and should not be taken as limiting the invention as detailed in the claims.

The method for measuring or calculating the physical quantity or index according to the present invention is carried out by the following method, unless otherwise specified:

the biomass measuring method includes placing a proper amount of fermentation liquor in a flat weighing bottle, drying in an electrothermal constant-temperature drying oven at 105 ℃ for 4h, placing in a dryer, cooling to room temperature, weighing, subtracting the weight of the weighing bottle, and dividing by the volume of the fermentation liquor to obtain a value, namely the biomass, with the unit g/L.

The method for measuring the yield of the crude oil comprises the steps of taking a certain volume of fermentation liquor, adding concentrated hydrochloric acid with the volume being 2 times that of the fermentation liquor, stirring for 50min at a constant temperature of 70 ℃ until thalli are completely digested, adding a proper amount of n-hexane, standing for layering, taking an upper layer organic phase into an eggplant-shaped bottle by a dropper, continuously extracting for 5-8 times until the upper layer organic phase is colorless, removing the n-hexane through water bath rotary evaporation at 80 ℃, then placing the eggplant-shaped bottle into an electrothermal constant-temperature drying oven at 105 ℃ for drying for 1h, placing the eggplant-shaped bottle into a dryer for cooling to room temperature, weighing, subtracting the weight of the eggplant-shaped bottle, and then dividing by the volume of the fermentation liquor to obtain a.

The DHA yield is obtained by multiplying the DHA content of the crude oil and fat measured by gas chromatography by the crude oil and fat yield, and is unit g/L.

The DHA productivity is a value obtained by dividing the DHA production by the fermentation period (days) in g/(L. d).

The fatty acid component analysis method and the DHA product oil detection method in the invention are based on GB26400-2011 food safety national standard food additive docosahexaenoic acid grease (fermentation method).

The crude oil extraction yield is calculated in a mode that the yield is × 100 percent, wherein the yield is the weight g of the crude oil/(the volume of fermentation liquor L× of the crude oil yield g/L of the fermentation liquor).

Fermentation culture

Example 1 is an original culture mode (no dissolved oxygen regulation strategy, nitrogen source regulation strategy and split-tank culture strategy are adopted); example 2 is the use of dissolved oxygen regulation strategy; example 3 is the use of nitrogen source regulation strategy; example 4 is a split-tank culture strategy; examples 5 to 13 were carried out by simultaneously employing a dissolved oxygen control strategy, a nitrogen source control strategy and a split-tank culture strategy.

In the following examples 1 to 13, if not otherwise specified, the seed medium formulation used was: 3% of glucose, 1% of peptone, 0.5% of yeast powder, 2% of sea crystal and natural pH (the balance being water). The formula of the fermentation medium is as follows: 12% of glucose, 1% of peptone, 0.5% of yeast powder and 2% of sea crystal (the balance being water).

Example 1: original culture mode (not adopting dissolved oxygen regulation strategy, nitrogen source regulation strategy and split-tank culture strategy) pair ³Influence of fermentation in 100m fermenter to produce DHA

Inoculating Schizochytrium sp (ATCC 20888), Schizochytrium limacinum Honda et Yokochi ATCCMA-1381) and Schizochytrium sp (CGMCCCNo.6843) slant preserved strains into a 2L shake flask filled with 400m L culture medium, culturing at 25 ℃ and 200rpm for 24h, completing strain activation culture, inoculating the shake flask seed solution into a primary seed tank filled with a sterilized culture medium according to an inoculation amount of 0.4%, culturing at 28 ℃, aeration rate of 1vvm, tank pressure of 0.02MPa and stirring rotation rate of 50rpm for 30h, completing primary seed expansion culture, inoculating the seed solution in the primary seed tank into a secondary seed tank filled with the sterilized culture medium according to an inoculation amount of 3%, culturing at 28 ℃, aeration rate of 1vvm, tank pressure of 0.02MPa and stirring rotation rate of 75rpm for 24h, and inoculating the sterilized seed solution into a secondary seed tank according to an inoculation amount of 3%.

In the fermentation process, the culture temperature is 28 ℃, the ventilation volume is 1vvm, the tank pressure is 0.02MPa, the stirring speed is 75rpm, a carbon source containing 30 percent of pretreated crude glycerol is fed in a flow mode, the glucose concentration is controlled to be 5 g/L, a nitrogen source is fed in a flow mode, fermentation culture is carried out, and the glucose concentration, the pH value, the thallus biomass, the crude oil yield and the DHA yield change of fermentation liquor are detected in the fermentation process.

And (3) terminating the fermentation after culturing for 96h, wherein the following table 1 is a table for respectively measuring the biomass, the crude oil yield, the DHA yield and the DHA productivity of the three strains after culturing in the original culture mode, and the following table 2 is a table for analyzing the gas phase of the fatty acid composition of the mixed oil obtained after fermentation. The biomass, crude oil yield and DHA yield of CGMCC No.6843 are also shown in FIG. 3.

Table 1: fermentation results of different strains in original culture mode

Table 2: 100m³The fatty acid composition of the mixed oil obtained after fermentation in the original culture mode of a fermentation tank

As can be seen from tables 1 and 2, the three strains have larger difference in yield and fatty acid composition in the original culture mode, wherein each index of the schizochytrium (Schizochytrium sp.CGMCC No.6843) is better than that of the other two strains, so that the schizochytrium (Schizochytrium sp.CGMCC No.6843) is used as the starting strain to optimize different culture modes.

³Example 2: influence of dissolved oxygen regulation strategy on DHA fermentation production in 100m fermentation tank

Inoculating a 2L shake flask filled with 400m L culture medium into a schizochytrium sp.CGMCC No.6843 slant preservation strain, culturing at 25 ℃ for 24h at the rotation speed of 200rpm to complete strain activation culture, inoculating a shake flask seed solution into a primary seed tank filled with the sterilized culture medium according to the inoculation amount of 0.4%, culturing at 28 ℃, the aeration amount of 1vvm, the tank pressure of 0.02MPa and the stirring rotation speed of 50rpm for 30h to complete primary seed expansion culture, inoculating the seed solution in the primary seed tank into a secondary seed tank filled with the sterilized culture medium according to the inoculation amount of 3%, culturing at 28 ℃, the aeration amount of 1vvm, the tank pressure of 0.02MPa and the stirring rotation speed of 75rpm for 24h to complete secondary seed expansion culture, and inoculating the seed solution in the secondary seed tank into a fermentation tank filled with the sterilized culture medium according to the inoculation amount of 3%.

The culture temperature in the fermentation process is 28 ℃, the ventilation volume is 1-2 vvm, the tank pressure is 0.02-0.05 MPa, the stirring speed is 50-100 rpm, a carbon source containing 30% of pretreated crude glycerol is fed, the glucose concentration is controlled at 5 g/L, a nitrogen source is fed in the feeding process to carry out fermentation culture, DO in 48 hours before the fermentation is controlled at 40%, DO in 48 hours after the fermentation is controlled at 8%, the ventilation volume, the tank pressure and the stirring speed are adjusted by taking the DO as a control index in the process, and a sterilized fresh culture medium or sterile water is properly added, and the glucose concentration, the pH, the thallus biomass, the crude oil yield and the DHA yield of the fermentation liquid are detected in the fermentation process.

After 96h of culture, the fermentation is stopped, the biomass in the fermentation liquor is measured to be 125 g/L, the yield of crude oil and fat is measured to be 56.3 g/L, the yield is 29.3 g/L, the productivity is 7.3 g/(L d), as shown in figure 3, the tank-placing volume of the fermentation liquor is 78m³The yield of DHA-containing crude oil in the whole batch of fermentation is 4391.4kg, which is 0.67 times higher than that in the original culture mode. The following table 3 shows the gas phase analysis results of the fatty acid composition of the mixed oil obtained after fermentation:

table 3: 100m³Composition of mixed oil fatty acid obtained after fermentation by using dissolved oxygen regulation strategy of fermentation tank

³Example 3: influence of nitrogen source regulation strategy on DHA fermentation production in 100m fermentation tank

Inoculating a 2L shake flask filled with 400m L culture medium into a schizochytrium sp.CGMCC No.6843 slant preservation strain, culturing at 25 ℃ for 24h at the rotation speed of 200rpm to complete strain activation culture, inoculating a shake flask seed solution into a primary seed tank filled with the sterilized culture medium according to the inoculation amount of 0.4%, culturing at 28 ℃, the ventilation amount of 1vvm, the tank pressure of 0.02MPa and the stirring rotation speed of 50rpm for culturing for 30h to complete primary seed expansion culture, inoculating the seed solution in the primary seed tank into a secondary seed tank filled with the sterilized culture medium according to the inoculation amount of 3%, culturing at 28 ℃, the ventilation amount of 1vvm, the tank pressure of 0.02MPa and the stirring rotation speed of 75rpm for culturing for 24h to complete secondary seed expansion culture, and inoculating the seed solution in the secondary seed tank into a fermentation tank filled with the sterilized culture medium according to the inoculation amount of 3%.

The culture temperature in the fermentation process is 28 ℃, the ventilation volume is 1vvm, the tank pressure is 0.02MPa, the stirring speed is 75rpm, a carbon source containing 30 percent of pretreated crude glycerol is fed in a flowing mode, the glucose concentration is controlled to be 5 g/L, the fermentation culture is carried out, in the fermentation process, before 48 hours of fermentation, 40 percent of ammonia water is fed in a flowing mode to control the pH of the fermentation liquor to be 6.5, the pH of the fermentation liquor can be controlled to be stable while the nitrogen source is supplemented by the fed-in ammonia water, after 48 hours, the ammonia water is stopped being fed in a flowing mode to carry out nitrogen deficiency culture, the pH is not controlled, and the glucose concentration, the pH, the thallus biomass, the crude oil yield and.

After 96h of culture, the fermentation is stopped, the biomass in the fermentation liquor is determined to be 118 g/L, the yield of crude oil is determined to be 64.2 g/L, the yield is 32.6 g/L, the productivity is 8.2 g/(L d), as shown in figure 3, the tank-placing volume of the fermentation liquor is 78m³The yield of DHA-containing crude oil in the whole batch of fermentation is 5007.6kg, which is 0.91 times higher than that in the original culture mode. The following table 4 shows the gas phase analysis results of the fatty acid composition of the mixed oil obtained after fermentation:

table 4: 100m³Mixed grease fatty acid composition obtained after fermentation by nitrogen source regulation strategy of fermentation tank

Fatty acid composition	Content%
		C12:0	0.48
C14:0	4.96
		C16:0	17.12
C16:1	1.26
		C18:0	1.88
C18:1	1.67
		C20:4	6.23
C20:5	1.25
		C22:5	14.35
C22:6	50.8

³Example 4: influence of split-tank culture strategy on DHA fermentation production in 100m fermentation tank

In the fermentation process, the culture temperature is 28 ℃, the ventilation volume is 1vvm, the tank pressure is 0.02MPa, the stirring speed is 75rpm, a carbon source containing 30 percent of pretreated crude glycerol is fed in a flowing mode, the glucose concentration is controlled to be 5 g/L, a nitrogen source is fed in a flowing mode, fermentation culture is carried out, fermentation is carried out for 24 hours, fermentation liquor in a fermentation tank is divided into two parts according to the volume, one half of the fermentation liquor is left in a main tank for continuous culture, the other half of the fermentation liquor is transferred to an auxiliary tank for sterile pressure maintaining for culture through a pressure difference method, a proper amount of sterilized fresh culture medium or sterile water is respectively fed in the main tank and the auxiliary tank, and the glucose concentration, the pH value, the thallus biomass, the crude oil yield and the DHA yield of.

After 96h of culture, the fermentation is stopped, the biomass in the fermentation liquor is determined to be 73 g/L, the yield of crude oil is determined to be 36.3 g/L, the yield is 16.4 g/L, the productivity is 4.1 g/(L. d), as shown in figure 3, the tank placing volume of the fermentation liquor is 132m³The yield of DHA-containing crude oil in the whole batch of fermentation is 4791.6kg, which is 0.83 times higher than that in the original culture mode, and although the indexes such as tank-releasing biomass, crude oil yield, DHA yield and the like are not much different from those in the original culture mode, the tank-releasing volume of the fermentation liquid is 1.74 times of that in the original culture mode due to the adoption of the split-tank culture, so that the yield in the whole batch of fermentation is 0.83 times higher than that in the original culture mode. The method has important significance for industrial large-scale production of the DHA-containing grease, can greatly save the cost and improve the market competitiveness of DHA production. The following table 5 shows the gas phase analysis results of the fatty acid composition of the mixed oil obtained after fermentation:

table 5: 100m³Fatty acid composition of mixed oil obtained after fermentation in fermentation tank

Fatty acid composition	Content%
		C12:0	0.58
C14:0	4.88
		C16:0	20.21
C16:1	1.91
		C18:0	1.54
C18:1	1.22
		C20:4	6.13
C20:5	1.25
		C22:5	17.08
C22:6	45.2

³Example 5: fission of 100m fermentation tank by adopting dissolved oxygen regulation strategy, nitrogen source regulation strategy and split-tank culture strategy Influence of fermentation of Chytridiomycetes (Schizochytrium sp.CGMCC No.6843) on production of DHA

The fermentation process comprises the steps of culturing at the temperature of 28 ℃, the ventilation amount of 1-2 vvm, the tank pressure of 0.02-0.05 MPa, the stirring speed of 50-100 rpm, feeding a carbon source containing 30% of pretreated crude glycerol, controlling the glucose concentration to be 5 g/L, carrying out fermentation culture, controlling DO to be 40% before 48h of fermentation and controlling DO to be 8% after 48h of fermentation by adopting an oxygen-dissolved regulation strategy, adjusting the ventilation amount, the tank pressure and the stirring speed by taking DO as a control index, properly supplementing a sterilized fresh culture medium or sterile water, feeding 40% of ammonia water before 48h of fermentation to control the pH of a fermentation liquid to be about 6.5, feeding ammonia water to supplement a nitrogen source while controlling the pH of the fermentation liquid to be stable, stopping feeding ammonia water after 48h of the fermentation liquid, carrying out nitrogen-deficient culture, not controlling the pH to be not to be controlled any more than 24h, dividing the fermentation liquid in a fermentation tank into two parts by volume, keeping half of the fermentation liquid in the main tank, continuously culturing the fermentation liquid by a pressure difference method, transferring the fermentation liquid to an aseptic secondary tank, respectively adding ammonia water to adjust the fresh culture medium or the fermentation liquid, and slightly adjusting the fermentation liquid yield and the fermentation liquid concentration and the fermentation liquid by the fermentation medium, the fermentation water concentration and the fermentation tank after the fermentation and the fermentation process of the fermentation.

After 96h of culture, the fermentation is stopped, the biomass in the fermentation liquor is determined to be 145 g/L, the yield of crude oil is determined to be 82.6 g/L, the yield is 44.9 g/L, the productivity is 11.2 g/(L. d), as shown in figure 3, the tank placing volume of the fermentation liquor is 130m³The yield of DHA-containing crude oil in the whole batch of fermentation is 10738kg, which is 3.10 times higher than that in the original culture mode. Meanwhile, by adopting a dissolved oxygen regulation strategy, a nitrogen source regulation strategy and a split-tank culture strategy, the DHA-containing grease can be produced at high yield, and the obtained DHA grease has high DHA content and strong market competitiveness. Table 6 below shows the results after fermentationObtaining a gas phase analysis result of the fatty acid composition of the mixed oil:

table 6: 100m³Fatty acid composition of mixed oil obtained after fermentation in fermentation tank

Fatty acid composition	Content%
		C12:0	0.45
C14:0	4.86
		C16:0	14.22
C16:1	1.68
		C18:0	1.72
C18:1	1.59
		C20:4	6.36
C20:5	1.54
		C22:5	13.28
C22:6	54.3

³Example 6: fission of 100m fermentation tank by adopting dissolved oxygen regulation strategy, nitrogen source regulation strategy and split-tank culture strategy Effect of fermentation of Chytridiomycetes (Schizochytrium sp.ATCC 20888) on DHA production

The method comprises the following steps of culturing schizochytrium sp (ATCC 20888) according to the culture method of example 5, terminating fermentation after culturing for 96 hours, determining that the biomass in fermentation liquor is 65 g/L, the yield of crude oil is 15.2 g/L and is 6.5 g/L, and the yield is increased by 2.49 times compared with the yield of the original culture method, and simultaneously adopting a dissolved oxygen regulation strategy, a nitrogen source regulation strategy and a pot culture strategy, not only can the DHA-containing oil be produced, but also the DHA content in the obtained DHA oil is high, and the DHA oil has strong market competitiveness, and the following table 7 shows the gas phase analysis result of the fatty acid composition of the mixed oil obtained after fermentation:

table 7: 100m³Fatty acid composition of mixed oil obtained after fermentation in fermentation tank

³Example 7: fission of 100m fermentation tank by adopting dissolved oxygen regulation strategy, nitrogen source regulation strategy and split-tank culture strategy Production of DHA ghost by fermentation of Chytridium limacinum Honda et Yokochi ATCC MYA-1381 Sound box

The method comprises the following steps of culturing Schizochytrium limacinum Honda et Yokochi ATCC MYA-1381) in a culture mode of example 5, terminating fermentation after culturing for 96 hours, determining that the biomass in fermentation liquor is 105 g/L, the yield of crude oil is 37 g/L and is 17.2 g/L, and the yield is improved by 2.81 times compared with that of an original culture mode, and simultaneously adopting a dissolved oxygen regulation strategy, a nitrogen source regulation strategy and a tank separation culture strategy, not only can high-yield DHA-containing oil be produced, but also the DHA content of the obtained DHA oil is high, and the obtained DHA oil has strong market competitiveness, and the following table 8 is a gas phase analysis result of the fatty acid composition of the mixed oil obtained after fermentation:

table 8: 100m³Fatty acid composition of mixed oil obtained after fermentation in fermentation tank

Fatty acid composition	Content%
		C12:0	0.79
C14:0	5.48
		C16:0	20.63
C16:1	1.25
		C18:0	1.96
C18:1	1.12
		C20:4	7.95
C20:5	2.87
		C22:5	11.45
C22:6	46.5

³Example 8: fission of 100m fermentation tank by adopting dissolved oxygen regulation strategy, nitrogen source regulation strategy and split-tank culture strategy Influence of fermentation of Chytridiomycetes (Schizochytrium sp.CGMCC No.6843) on production of DHA

The fermentation process comprises the steps of culturing at the temperature of 28 ℃, the ventilation amount of 1-2 vvm, the tank pressure of 0.02-0.05 MPa and the stirring speed of 50-100 rpm, feeding a carbon source containing 20% of pretreated crude glycerol, controlling the glucose concentration to be 1 g/L, carrying out fermentation culture, controlling DO to be 30% before 36h fermentation and controlling DO to be 5% after 36h fermentation by adopting an oxygen-dissolved regulation strategy, adjusting the ventilation amount, the tank pressure and the stirring speed by taking DO as a control index, properly supplementing a sterilized fresh culture medium or sterile water, feeding 25% of ammonia water before 36h fermentation in the fermentation process to control the pH of the fermentation liquor to be about 6, feeding ammonia water to supplement a nitrogen source while controlling the pH of the fermentation liquor to be stable, stopping feeding ammonia water after 36h, carrying out nitrogen-deficient culture, not controlling the pH, fermenting to 12h, dividing the fermentation liquor in a fermentation tank into two parts by volume, keeping half of the fermentation liquor for continuous culture, transferring the other half of the fermentation liquor to an aseptic secondary tank by a pressure difference method, maintaining the culture, respectively adding an appropriate amount of fresh culture medium or a fermentation tank after sterilization, and adjusting the fermentation liquor by the fermentation medium, the fermentation liquor, and the fermentation process of fermentation liquor, and the fermentation liquor.

After culturing for 96h, terminating the fermentation, determining that the biomass in the fermentation liquor is 140 g/L, the yield of crude oil is 78.5 g/L, the yield is 40.4 g/L, the productivity is 10.1 g/(L d), and the following table 9 shows the gas phase analysis result of the fatty acid composition of the mixed oil obtained after fermentation:

table 9: 100m³Fatty acid composition of mixed oil obtained after fermentation in fermentation tank

Fatty acid composition	Content%
		C12:0	0.42
C14:0	4.87
		C16:0	15.07
C16:1	1.65
		C18:0	1.7
C18:1	1.58
		C20:4	6.35
C20:5	1.62
		C22:5	15.24
C22:6	51.5

³Example 9: fission of 100m fermentation tank by adopting dissolved oxygen regulation strategy, nitrogen source regulation strategy and split-tank culture strategy Influence of fermentation of Chytridiomycetes (Schizochytrium sp.CGMCC No.6843) on production of DHA

The fermentation process comprises the steps of culturing at the temperature of 28 ℃, the ventilation amount of 1-2 vvm, the tank pressure of 0.02-0.05 MPa and the stirring speed of 50-100 rpm, feeding a carbon source containing 20% of pretreated crude glycerol, controlling the glucose concentration to be 5 g/L, carrying out fermentation culture, controlling DO to be 50% before 60h of fermentation and DO to be 10% after 60h of fermentation by adopting an oxygen-dissolved regulation strategy, adjusting the ventilation amount, the tank pressure and the stirring speed by taking DO as a control index, properly supplementing a sterilized fresh culture medium or sterile water, feeding 45% of ammonia water before 60h of fermentation in the fermentation process to control the pH of the fermentation liquid to be about 7, feeding ammonia water to supplement a nitrogen source while controlling the pH of the fermentation liquid to be stable, stopping feeding ammonia water after 60h, carrying out nitrogen-deficient culture, not controlling the pH, fermenting to 36h, dividing the fermentation liquid in a fermentation tank into two parts by volume, keeping half of the fermentation liquid for continuous culture, transferring the other half of the fermentation liquid to an aseptic secondary tank by a pressure difference method, maintaining the culture, respectively adding an appropriate amount of fresh culture medium or a fresh culture tank after the sterilization, and adjusting the fermentation liquid by the fermentation medium, the fermentation liquid, and the fermentation process of fermentation, and the fermentation liquid, and the fermentation.

After culturing for 96h, terminating the fermentation, determining that the biomass in the fermentation liquor is 138 g/L, the yield of crude oil is 78.6 g/L, the yield is 39.6 g/L, and the productivity is 9.9 g/(L. d). The following table 10 is the gas phase analysis result of the fatty acid composition of the mixed oil obtained after fermentation:

table 10: 100m³Fatty acid composition of mixed oil obtained after fermentation in fermentation tank

Fatty acid composition	Content%
		C12:0	0.41
C14:0	4.88
		C16:0	15.32
C16:1	1.64
		C18:0	1.71
C18:1	1.6
		C20:4	6.38
C20:5	1.58
		C22:5	16.08
C22:6	50.4

³Example 10: fission of 100m fermentation tank by adopting dissolved oxygen regulation strategy, nitrogen source regulation strategy and split-tank culture strategy Influence of fermentation of Chytridiomycetes (Schizochytrium sp.CGMCC No.6843) on production of DHA

The fermentation process comprises the steps of culturing at the temperature of 28 ℃, the ventilation amount of 1-2 vvm, the tank pressure of 0.02-0.05 MPa and the stirring speed of 50-100 rpm, feeding a carbon source containing 20% of pretreated crude glycerol, controlling the glucose concentration to be 2 g/L, carrying out fermentation culture, controlling DO to be 35% before 40h of fermentation and controlling DO to be 6% after 40h of fermentation by adopting an oxygen-dissolved regulation strategy, adjusting the ventilation amount, the tank pressure and the stirring speed by taking DO as a control index, and properly supplementing a sterilized fresh culture medium or sterile water, feeding 38% of ammonia water before 40h of fermentation to control the pH of the fermentation liquor to be about 6.5, feeding the ammonia water to supplement a nitrogen source while controlling the pH of the fermentation liquor to be stable, stopping feeding the ammonia water after 40h of the fermentation, carrying out nitrogen-deficient culture, not controlling the pH to be not to be controlled any more, dividing the fermentation liquor in a fermentation tank into two parts by volume for culture, keeping half of the fermentation liquor in a main tank for continuous culture, transferring the other half of the fermentation liquor to an aseptic secondary tank by a pressure difference method for culture, respectively adding an appropriate amount of ammonia water, adjusting the fresh culture medium or the fermentation tank, and adjusting the fermentation liquor, the fermentation liquor by the fermentation medium, and the fermentation liquor by adjusting the fermentation medium, the fermentation and the fermentation water concentration and the fermentation process of the fermentation.

After culturing for 96h, terminating the fermentation, determining that the biomass in the fermentation liquor is 139 g/L, the yield of crude oil is 78.2 g/L, the yield is 38.0 g/L, and the productivity is 9.5 g/(L d), and the following table 11 shows the gas phase analysis results of the fatty acid composition of the mixed oil obtained after fermentation:

table 11: 100m³Fatty acid composition of mixed oil obtained after fermentation in fermentation tank

³Example 11: fission of 100m fermentation tank by adopting dissolved oxygen regulation strategy, nitrogen source regulation strategy and split-tank culture strategy Fermentation production of chytriumsp.CGMCC No.6843Effect of DHA

The fermentation process comprises the steps of culturing at the temperature of 28 ℃, the ventilation volume of 1-2 vvm, the tank pressure of 0.02-0.05 MPa, the stirring speed of 50-100 rpm, feeding a carbon source containing 40% of pretreated crude glycerol, controlling the glucose concentration to be 4 g/L, carrying out fermentation culture, controlling DO to be 45% before 56h of fermentation and controlling DO to be 9% after 56h of fermentation by adopting an oxygen-dissolved regulation strategy, adjusting the ventilation volume, the tank pressure and the stirring speed by taking DO as a control index, properly supplementing a sterilized fresh culture medium or sterile water, feeding 42% of ammonia water before 56h of fermentation to control the pH of the fermentation liquor to be about 7 during the fermentation process, feeding the ammonia water to supplement a nitrogen source while controlling the pH of the fermentation liquor to be stable, stopping feeding the ammonia water after 56h, carrying out nitrogen-deficient culture, not controlling the pH, fermenting to 28h, dividing the fermentation liquor in a fermentation tank into two parts by volume, keeping half of the fermentation liquor for continuous culture, transferring the other half of the fermentation liquor to an aseptic secondary tank by a pressure difference method, respectively supplementing the fresh culture medium or the fermentation tank after the sterilization, and adjusting the fermentation liquor by appropriate amount, the fermentation medium, the fermentation liquor, and the fermentation process of fermentation liquor, and the.

After culturing for 96h, terminating the fermentation, determining that the biomass in the fermentation liquor is 140 g/L, the yield of crude oil is 80.8 g/L, the yield is 42.0 g/L, the productivity is 10.5 g/(L d), and the following table 12 shows the gas phase analysis result of the fatty acid composition of the mixed oil obtained after fermentation:

table 12: 100m³Fatty acid composition of mixed oil obtained after fermentation in fermentation tank

Fatty acid composition	Content%
		C12:0	0.44
C14:0	4.86
		C16:0	14.61
C16:1	1.67
		C18:0	1.7
C18:1	1.58
		C20:4	6.38
C20:5	1.64
		C22:5	15.12
C22:6	52

³Example 12: fission of 100m fermentation tank by adopting dissolved oxygen regulation strategy, nitrogen source regulation strategy and split-tank culture strategy Influence of fermentation of Chytridiomycetes (Schizochytrium sp.CGMCC No.6843) on production of DHA

The fermentation process comprises the steps of culturing at the temperature of 28 ℃, the ventilation amount of 1-2 vvm, the tank pressure of 0.02-0.05 MPa, the stirring speed of 50-100 rpm, feeding a carbon source containing 40% of pretreated crude glycerol, controlling the glucose concentration to be 3 g/L, carrying out fermentation culture, controlling DO to be 38% before 44h fermentation and 7% after 44h fermentation by adopting an oxygen-dissolved regulation strategy, adjusting the ventilation amount, the tank pressure and the stirring speed by taking DO as control indexes, and properly supplementing a sterilized fresh culture medium or sterile water, feeding 40% of ammonia water before 44h fermentation in the fermentation process to control the pH of the fermentation liquor to be about 6.5, feeding ammonia water to supplement a nitrogen source while controlling the pH of the fermentation liquor to be stable, stopping feeding ammonia water after 44h, carrying out nitrogen-deficient culture, not controlling the pH, fermenting to 22h, dividing the fermentation liquor in a fermentation tank into two parts by volume, keeping half of the fermentation liquor in the main tank for continuous culture, transferring the other half of the fermentation liquor to an aseptic secondary tank by a pressure difference method, maintaining the culture, respectively adding an appropriate amount of ammonia water into the fresh culture medium after 44h, adjusting the fermentation tank, and the fermentation liquor, and the fermentation medium, and the fermentation liquor, and slightly adjusting the fermentation liquor yield and the fermentation process of the fermentation medium, the fermentation liquor, and the fermentation.

After culturing for 96h, terminating the fermentation, determining that the biomass in the fermentation liquor is 141 g/L, the yield of crude oil is 81.2 g/L, the yield is 42.4 g/L, the productivity is 10.6 g/(L d), and the following table 13 is the gas phase analysis result of the fatty acid composition of the mixed oil obtained after fermentation:

table 13: 100m³Fatty acid composition of mixed oil obtained after fermentation in fermentation tank

³Example 13: fission of 100m fermentation tank by adopting dissolved oxygen regulation strategy, nitrogen source regulation strategy and split-tank culture strategy Influence of fermentation of Chytridiomycetes (Schizochytrium sp.CGMCC No.6843) on production of DHA

The fermentation process comprises the steps of culturing at the temperature of 28 ℃, the ventilation volume of 1-2 vvm, the tank pressure of 0.02-0.05 MPa, the stirring speed of 50-100 rpm, feeding a carbon source containing 40% of pretreated crude glycerol, controlling the glucose concentration to be 5 g/L, carrying out fermentation culture, controlling DO to be 42% before 52h fermentation and 8% after 52h fermentation by adopting an oxygen-dissolved regulation strategy, adjusting the ventilation volume, the tank pressure and the stirring speed by taking DO as control indexes, properly supplementing a sterilized fresh culture medium or sterile water, feeding 40% of ammonia water before 52h fermentation in the fermentation process to control the pH of the fermentation liquor to be about 6.5, feeding ammonia water to supplement a nitrogen source while controlling the pH of the fermentation liquor to be stable, stopping feeding ammonia water after 52h, carrying out nitrogen-deficient culture, not controlling the pH, fermenting to 26h, dividing the fermentation liquor in a fermentation tank into two parts by volume, keeping half of the fermentation liquor in the main tank, continuously culturing, transferring the other half of the fermentation liquor to an aseptic secondary tank by a pressure difference method, respectively adding ammonia water into the fresh culture tank, adjusting the fermentation liquor after the fermentation medium, the fermentation tank, the fermentation medium, the fermentation liquor, and the fermentation tank, and slightly adjusting the fermentation liquor yield and the fermentation liquor concentration, the fermentation liquor, the fermentation medium, the fermentation water, the fermentation liquor.

After culturing for 96h, terminating the fermentation, determining that the biomass in the fermentation liquor is 142 g/L, the crude oil yield is 80.2 g/L and the yield is 41.2 g/L, and the productivity is 10.3 g/(L. d). The following table 14 is the gas phase analysis result of the fatty acid composition of the mixed oil obtained after fermentation:

table 14: 100m³Fatty acid composition of mixed oil obtained after fermentation in fermentation tank

Fatty acid composition	Content%
		C12:0	0.45
C14:0	4.86
		C16:0	15.06
C16:1	1.65
		C18:0	1.72
C18:1	1.56
		C20:4	6.35
C20:5	1.59
		C22:5	15.36
C22:6	51.4

Extracting crude oil

Example 14

Taking the DHA fermentation liquor 100L obtained in the example 5, heating and inactivating, conveying the fermentation liquor to a cloth cavity by a distributor, wrapping the cloth, performing primary pressing after the cloth is distributed, maintaining the pressure within a set 2h to a set pressure of 30MPa in a gradual pressurization mode for 2h until no water drops flow out basically, removing a primary pressing cage, replacing a secondary pressing cage, pushing the secondary pressing cage into a secondary pressing position, performing secondary pressing, maintaining the pressure within a set 2h to a set pressure of 100MPa in a gradual pressurization mode for 2h until no oil drops flow out basically, collecting the pressed DHA crude oil to obtain 6.8kg of DHA crude oil, removing the secondary pressing cage from the fermentation liquor until the crude oil yield reaches 82.3%, and separating the pressed microalgae meal from the filter cloth.

Example 15

Taking the DHA fermentation liquor 100L obtained in the example 5, heating and inactivating, conveying the fermentation liquor to a cloth cavity by a distributor, wrapping cloth, performing primary pressing after the cloth is distributed, maintaining the pressure within 5 hours to 40MPa in a step-by-step pressurizing manner until no water drops flow out basically, removing a primary pressing cage, replacing a secondary pressing cage, pushing the secondary pressing cage into a secondary pressing position, performing secondary pressing, maintaining the pressure within 5 hours to 150MPa in a step-by-step pressurizing manner until no oil drops flow out basically, collecting pressed DHA crude oil to obtain 7.3kg of DHA crude oil, removing the secondary pressing cage until the yield of the crude oil is 88.4%, and separating the pressed microalgae meal from filter cloth.

Example 16

Taking 150L DHA fermentation broth obtained in example 5, heating for inactivation, centrifuging by a butterfly centrifuge, removing centrifuged light liquid to obtain 95L concentrated fermentation broth, conveying the concentrated fermentation broth into a material distribution cavity by a material distributor, distributing and wrapping, performing primary pressing after distributing, performing primary pressing in a gradual pressurization manner until the set pressure of 40MPa is reached within 5 hours, maintaining the pressure for 4 hours until no water drops flow out basically, removing a primary pressing cage, replacing the primary pressing cage with a secondary pressing cage, pushing the secondary pressing cage into a secondary pressing position, performing secondary pressing in a gradual pressurization manner until the set pressure of 150MPa is reached within 5 hours, maintaining the pressure for 4 hours until no oil drops flow out basically, collecting the pressed DHA crude oil to obtain 10.8kg DHA crude oil, obtaining the fermentation broth until the crude oil yield is 87.2%, removing the secondary pressing cage, and separating the pressed microalgae cake from filter cloth.

Example 17

Heating and inactivating the DHA fermentation liquor 300L obtained in the example 5, adding the DHA fermentation liquor into a spray dryer, setting the spray pressure to be 5MPa, the air inlet temperature to be 180 ℃, the air outlet temperature to be 80 ℃, performing spray drying to obtain powder, conveying the DHA microalgae powder to a cloth cavity, wrapping the cloth, performing flexible squeezing and squeezing after the cloth is finished, maintaining the pressure to be 100MPa within 2 hours by adopting a gradual pressurizing mode, keeping the pressure for 2 hours until no oil drops flow out basically, collecting the squeezed DHA crude oil to obtain 22.1kg DHA crude oil, and obtaining the fermentation liquor with the crude oil yield of 89.2%.

Example 18

Heating and inactivating the DHA fermentation broth 300L obtained in example 5, adding the DHA fermentation broth into a spray dryer, setting the spray pressure to be 6MPa, the air inlet temperature to be 200 ℃, the air outlet temperature to be 100 ℃, performing spray drying to obtain powder, conveying the DHA microalgae powder 51.0kg to a cloth cavity, wrapping the cloth, performing flexible squeezing and squeezing after the cloth is finished, maintaining the pressure to be 150MPa within 4h by adopting a gradual pressurizing mode, keeping the pressure for 4h until no oil drops flow out basically, collecting the squeezed DHA crude oil to obtain 22.8kg of DHA crude oil, and obtaining the crude oil yield of the fermentation broth to be 92.0%.

Example 19

Taking the DHA fermentation broth 300L obtained in example 5, heating for inactivation, centrifuging by a butterfly centrifuge, removing centrifuged light liquid to obtain 200L concentrated fermentation broth, adding into a spray dryer, setting a spray pressure of 8MPa, an air inlet temperature of 220 ℃, an air outlet temperature of 110 ℃, spray drying into powder to obtain 47.5kg of DHA microalgal powder, conveying the DHA microalgal powder into a cloth cavity, carrying out cloth wrapping, carrying out flexible squeezing and squeezing after cloth is finished, keeping the pressure for 4h until the set 150MPa pressure is reached within the set 4h by adopting a gradual pressurizing mode, keeping the pressure for 4h until no oil drops flow out basically, collecting squeezed DHA crude oil, obtaining 23.3kg of DHA crude oil altogether, and obtaining the fermentation broth until the crude oil yield is 94.0%.

Comparative example 1

Taking 300L of the DHA fermentation broth obtained in example 5, heating and inactivating, centrifuging by a butterfly centrifuge, removing centrifuged light liquid to obtain 200L concentrated fermentation broth, adding into a spray dryer, setting the spray pressure to be 8MPa, the air inlet temperature to be 220 ℃, the air outlet temperature to be 110 ℃, and spray drying to obtain powder, preheating 47.8 kg. double-screw press to 80 ℃, adding DHA microalga powder into the double-screw press to extract oil, so as to obtain 11.6kg of DHA crude oil, wherein the fermentation broth has a crude oil yield of 46.81%.

Refining of crude oil

Example 20

10kg of DHA crude oil obtained in examples 14, 15 and 16 are combined and refined according to the steps of hydration, decolorization and molecular distillation.

Hydration: heating 10kg of DHA crude oil to 75 ℃, adding 1kg of purified water with the temperature of 80 ℃, stirring for 30min at the stirring speed of 30 r/min, standing for 2h, and removing the lower layer to obtain 9.75kg of hydrated oil.

And (3) decoloring: heating the hydrated oil to 100 ℃, controlling the vacuum degree to be-0.075 MPa, dehydrating in vacuum for 1h, then cooling to 70 ℃, adding a decolorizing agent (191g of activated carbon and 286g of activated clay), stirring and decolorizing for 0.5h, stopping stirring, and filtering to remove the decolorizing agent to obtain 9.30kg of decolorized oil.

Molecular distillation: and (3) performing three-stage molecular distillation on the decolorized oil, controlling the first-stage vacuum degree to be about 90Pa and the temperature to be 160 ℃, removing the light components of the first stage, controlling the second-stage vacuum degree to be about 40Pa and the temperature to be 200 ℃, removing the light components of the second stage, performing third-stage molecular distillation on the heavy components, controlling the third-stage vacuum degree to be about 3Pa and the temperature to be 220 ℃, removing the light components of the third stage, collecting the heavy components, performing deodorization, cooling to 30 ℃, adding an antioxidant, and packaging to obtain 9.10kg of DHA product oil, wherein the inspection result is shown in Table 15.

Table 15: DHA product oil inspection result

Example 21

Hydration: heating 10kg of DHA crude oil to 85 ℃, adding 1kg of purified water with the temperature of 90 ℃, stirring for 15min at the stirring speed of 90 r/min, standing for 4h, and removing the lower layer to obtain 9.70kg of hydrated oil.

And (3) decoloring: heating the hydrated oil to 110 ℃, controlling the vacuum degree to be-0.075 MPa, carrying out vacuum dehydration for 0.5h, then cooling to 80 ℃, adding a decoloring agent (192g of activated carbon and 288g of activated clay), stirring and decoloring for 1h, stopping stirring, and filtering to remove the decoloring agent to obtain 9.31kg of decolored oil.

Molecular distillation: and (3) performing three-stage molecular distillation on the decolorized oil, controlling the first-stage vacuum degree to be about 90Pa and the temperature to be 200 ℃, removing the light components of the first stage, controlling the heavy components to be about 3Pa and the temperature to be 250 ℃, removing the light components of the third stage, collecting the heavy components, cooling to 30 ℃ after the molecular distillation is finished, adding an antioxidant, and packaging to obtain 9.03kg of DHA product oil, wherein the inspection result is shown in Table 16.

Table 16: DHA product oil inspection result

Example 22

10kg of the DHA raw oil obtained by combining examples 17, 18 and 19 was refined by the refining method of example 20 to obtain 9.13kg of DHA product oil, and the test results are shown in Table 17.

Table 17: DHA product oil inspection result

Example 23

10kg of the DHA raw oil obtained by combining examples 17, 18 and 19 was refined by the refining method of example 21 to obtain 9.08kg of DHA product oil, and the test results are shown in Table 18.

Table 18: DHA product oil inspection result

Comparative example 2

10kg of the DHA raw oil obtained by combining the DHA in the examples 17, 18 and 19 was refined by the conventional refining method, i.e., hydration, alkali refining, decolorization and deodorization.

Hydration: heating 10kg of DHA crude oil to 85 ℃, adding 1kg of purified water with the temperature of 90 ℃, stirring for 15min at the stirring speed of 90 r/min, standing for 4h, and removing the lower layer to obtain 9.71kg of hydrated oil.

And (2) alkali refining, namely, keeping the temperature of the hydrated oil at 75 ℃, adding NaOH solution with the concentration of 1L (mass fraction) for stirring for 30min at the stirring speed of 30 r/min, standing for 4h, separating soapstock to obtain alkali refined oil, spraying purified water with the weight of 10% of the oil at 80 ℃ for washing under the stirring condition of the alkali refined oil, controlling the water adding time to be 10-30 min, standing for 2h after the water is added, separating a water layer, and repeatedly washing for 2 times to obtain 8.92kg of the alkali refined oil.

And (3) decoloring: heating the alkali refined oil to 110 ℃, controlling the vacuum degree to be-0.075 MPa, dehydrating in vacuum for 0.5h, then cooling to 80 ℃, adding a decolorizing agent (186g of activated carbon and 280g of activated clay), stirring and decolorizing for 1h, stopping stirring, and filtering to remove the decolorizing agent to obtain 8.49kg of decolorized oil.

Deodorizing: transferring the decolorized oil into a deodorizing pot, introducing steam for deodorization, controlling the deodorizing temperature at 185 ℃, controlling the vacuum degree within 600Pa, deodorizing for 2h, stopping introducing steam after deodorization, cooling to 30 ℃, adding an antioxidant, and packaging to obtain 8.31kg of DHA product oil, wherein the detection results are shown in Table 19.

Table 19: DHA product oil inspection result

Claims

1. A method of culturing a microorganism for the production of DHA, wherein:

controlling the dissolved oxygen saturation at 5% -10% from 36-60 hours of the fermentation tank culture; and

starting from 36-60 hours of the fermentation tank culture, no nitrogen source is added or the adding amount of the nitrogen source is reduced by more than 50%;

wherein the content of the first and second substances,

the fermenter culture further comprises a step of performing split-tank culture 12-36 hours after the start of the fermenter culture; and is

The microorganism for producing DHA is Schizochytrium limacinum.

2. The culture method according to claim 1, wherein:

controlling the dissolved oxygen at 5% -10% from 40-56 hours of the fermenter culture, and/or

From 40 to 56 hours of the fermenter culture, no further nitrogen source was added.

3. The culture method according to claim 1, wherein:

dissolved oxygen was controlled at 5% -10% starting from 44-52 hours of fermentor culture.

4. The culture method according to claim 1, wherein:

dissolved oxygen was controlled at 5% -10% starting from 46-50 hours of fermentor culture.

5. The culture method according to claim 1, wherein:

dissolved oxygen was controlled at 5% -10% starting from 48 hours of fermentor culture.

6. The culture method according to claim 1, wherein:

from 44 to 52 hours of the fermenter culture, no further nitrogen source was added.

7. The culture method according to claim 1, wherein:

from 46 to 50 hours of the fermenter culture, no further nitrogen source was added.

8. The culture method according to claim 1, wherein:

from 48 hours of the fermenter culture, no further nitrogen source was added.

9. The culture method according to claim 1, wherein the dissolved oxygen saturation is 30% -50% before controlling the dissolved oxygen saturation to be 5% -10%.

10. The culture method according to claim 1, wherein the pH of the fermentation broth is 6.0 to 7.0.

11. The culture method according to claim 10, wherein the concentration of glucose in the fermentation broth is maintained at 1-5 g/L.

12. The culture method according to claim 1, wherein the nitrogen source is ammonia.

13. The culture method according to claim 12, wherein the ammonia water is 25% to 45% ammonia water.

14. The culture method according to claim 12, wherein the ammonia water is 35% to 45% ammonia water.

15. The culture method according to claim 12, wherein the ammonia water is 40% ammonia water.

16. The culture method according to claim 1, wherein the fermenter culture further comprises a step of performing the split culture at 24 hours from the start of the fermenter culture.

17. The culture method according to claim 1, wherein the split-tank culture is a fermentor culture divided into two or more fermentors.

18. The method according to claim 1, further comprising the steps of inoculation and seed expansion before the fermenter culture.

19. The method according to claim 18, wherein the seed expansion culture comprises a primary seed expansion culture and a secondary seed expansion culture.

20. The method of claim 19, wherein the expanding culture of the primary seed comprises the steps of:

inoculating the shake flask seed solution into a first-stage seed tank filled with a sterilized culture medium according to the inoculation amount of 0.4-1%, culturing at 25-32 deg.C, ventilation amount of 1-2 vvm, tank pressure of 0.02-0.05 MPa, stirring at 50-100 rpm, and culturing for 30-35 h to complete first-stage seed amplification culture.

21. The method of claim 19, wherein the secondary seed expansion culture comprises the steps of:

22. The culture method according to claim 18, further comprising the step of performing activation culture before the inoculation and expansion culture.

23. The method of claim 22, wherein the activation culture is at a temperature of 25 ℃ to 32 ℃ for a period of 20 to 25 hours.

24. The cultivation method according to claim 18, wherein the fermenter cultivation comprises the steps of:

25. The culture method according to claim 1, wherein the schizochytrium is selected from the strains with a preservation number of CGMCCNo.6843, ATCC 20888, ATCC 20889, ATCC 28209 or ATCC MYA-1381.

26. A method for producing DHA or a DHA-containing product using a microorganism, comprising:

a method according to any one of claims 1 to 25 for culturing a microorganism for the production of DHA.

27. The method of claim 26, wherein the DHA-containing product is a DHA product oil.