DHA (docosahexaenoic acid) production strain and application thereof

Abstract

The invention discloses a DHA (docosahexaenoic acid) production strain and application thereof, and belongs to the technical field of microorganisms. According to the present invention, by using the ARTP mutagenesis technology, the screening procedures such as shake flask adaptive screening, plate pre-screening, shake flask primary screening and shake flask secondary screening are respectively utilized to superpose dominant mutation, such that the strain capable of producing DHA at the high yield by using the inorganic nitrogen source is obtained through screening, the strain has good passage stability, and the DHA yield can be maintained at more than 54 mg / L after the passage is performed for more than 5 times; the strain can maintain high DHA fermentation capability in a culture medium in which inorganic nitrogen source nitrate is used for replacing part of organic nitrogen sources, and is suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to a DHA-producing strain and its application, belonging to the field of microbial technology. Background Art

[0002] Docosahexaenoic acid (DHA) is an ω-3 polyunsaturated fatty acid, which plays an important role in preventing cardiovascular diseases, inhibiting cancer, maintaining the health of mothers and infants, and the development and function of the brain and eyes. At present, DHA mainly comes from fish oil and algal oil. Due to the complex purification process of fish oil, large consumption of marine resources, and marine pollution, the demand for DHA algal oil is gradually increasing. Schizochytrium limacinum is considered to be the most promising microorganism for DHA production and is also an important strain for industrial production of DHA. However, one of the most outstanding problems is to reduce the cost of heterotrophic culture and increase the yield of DHA in algal oil. Economically, the cost of obtaining DHA from microbial strains is still 1-2 times higher than that from fish, and fish oil resources are very scarce. Developing new DHA microbial resources has become a research hotspot in recent years. In order to reduce production costs, current research mainly focuses on mutagenesis breeding, fermentation process optimization, lipid extraction optimization, and gene editing.

[0003] Ben Zhao et al. developed a new strategy for ARTP mutants combining stepwise malonic acid and zeocin resistance screening in Schizochytrium limacinum. Finally, the DHA yield of the mutant strain mz-17 increased by 1.8 times. However, the nitrogen source used for the fermentation of this strain is yeast extract, which is expensive and not suitable for industrial production.

[0004] Min Lian et al. screened through N-methyl-N-nitro-N-nitrosoguanidine and ultraviolet mutagenesis using a plate containing 0.06 g / L iodoacetic acid and 0.1 g / L malonic acid simultaneously. Compared with the parental strain, the lipid accumulation of the mutant strain increased by 34.84%, and the proportion of DHA in total fatty acids increased by 38.88%.

[0005] Xin Sun et al. obtained a high-lipid-yielding Desmodesmus sp. strain b15 through the method of combining malonic acid resistance screening and ARTP mutagenesis. Compared with the screening method only by ARTP mutagenesis, malonic acid improved the screening efficiency for targeting oil-rich algae. Compared with the control strain, the triglyceride and total lipid contents increased by 48.98% and 114.99% respectively.

[0006] Although the DHA yield in the prior art has been greatly improved compared with previous studies, it is still far from meeting the requirements for large-scale industrial production at a lower production cost. Summary of the Invention

[0007] To reduce the production cost of DHA, the present invention uses Schizochytrium with high DHA production and efficient utilization of sodium nitrate as a secondary screening target. After ARTP mutagenesis, screening is carried out on a malonic acid plate containing an inorganic nitrogen source of sodium nitrate, combined with a simple and highly operable adaptive evolution program, and considering economic benefits comprehensively, strains that can utilize inorganic nitrogen sources and produce high yields of DHA are screened.

[0008] The present invention provides a strain of Schizochytrium limacinum ZD-NHU-11, with a preservation number of CGMCC No. 41559 and a preservation date of October 28, 2024.

[0009] The present invention also provides a method for screening the Schizochytrium, including:

[0010] (1) Performing ARTP mutagenesis on the initial strain of Schizochytrium and culturing it overnight in an activation medium;

[0011] (2) Transferring the bacterial liquid obtained from the overnight culture to a screening medium containing malonic acid for adaptive evolution to obtain an adaptively evolved strain;

[0012] (3) Selecting strains with relatively high cell biomass and DHA content after adaptive evolution, and performing primary screening and re-screening in shake flasks to obtain Schizochytrium with high DHA production.

[0013] In one embodiment, in step (2), the adaptive evolution uses malonic acid as a stress factor, first performing adaptive culture in a shake flask, and then screening those with better growth for pre-screening on a malonic acid plate. During the adaptive evolution process, the concentration of malonic acid is maintained at MIC 50 , and the concentration range of the used malonic acid is between 1.5 g / L and 2.0 g / L. By gradually increasing the concentration of malonic acid, the adaptability of cells to malonic acid is gradually improved.

[0014] In one embodiment, the screening medium contains: 11 g / L of glucose monohydrate, 1.5 g / L of yeast extract, 1.5 g / L of sodium nitrate, 14.5 g / L of sodium chloride, 6 g / L of magnesium sulfate heptahydrate, 16 g / L of agar, and malonic acid at a corresponding concentration.

[0015] In one embodiment, steps (1) to (2) can be repeated multiple times.

[0016] The present invention also provides a method for producing DHA, using the Schizochytrium limacinum ZD-NHU-11 for fermentation.

[0017] In one embodiment, the method includes:

[0018] (A) Seed culture of Schizochytrium limacinum CGMCC No.41559 is carried out in a seed culture medium to obtain a seed solution.

[0019] (B) The seed solution prepared in step (A) is inoculated into a fermentation medium for fermentation culture to obtain a fermentation broth.

[0020] In one embodiment, the seed culture is carried out at 26 - 30 °C and 160 - 180 rpm for 20 - 24 h.

[0021] In one embodiment, when the pH of the seed culture medium > 6 or OD 650 = 5, the seed solution is inoculated into the fermentation medium.

[0022] In one embodiment, the fermentation culture is carried out at 26 - 30 °C and 150 - 180 rpm for 96 - 144 h.

[0023] In one embodiment, the nitrogen source of the fermentation medium includes but is not limited to sodium nitrate.

[0024] In one embodiment, the nitrogen source of the fermentation medium is sodium nitrate and yeast extract.

[0025] In one embodiment, the fermentation medium contains: glucose, sodium glutamate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, sodium chloride, sodium sulfate, ammonium sulfate, potassium chloride, nickel sulfate monohydrate, copper sulfate pentahydrate, sodium molybdate dihydrate, manganese chloride tetrahydrate, cobalt chloride hexahydrate, zinc sulfate, ferrous sulfate heptahydrate.

[0026] In one embodiment, the mass ratio of the sodium nitrate to the yeast extract is (6:4) - (9:1).

[0027] In one embodiment, glucose is fed during the fermentation process every 8 - 12 h.

[0028] The present invention also provides the application of the Schizochytrium or the method in the preparation of DHA or products containing DHA.

[0029] Beneficial effects: Combining with the ARTP mutagenesis technology, regarding the large-capacity mutant library as a population, and respectively using four screening procedures of shake-flask adaptive screening, plate pre-screening, shake-flask primary screening, and shake-flask re-screening to continuously stack advantageous mutations, a strain capable of producing high-yield DHA using inorganic nitrogen source was screened. This strain has good passage stability and can still maintain the DHA production above 54 mg / L after being passaged more than 5 times. This strain can also maintain a relatively high DHA fermentation ability in the medium where part of the organic nitrogen source is replaced by the inorganic nitrogen source nitrate. Since the market price of yeast extract is 20 - 25 yuan / kg and the market price of sodium nitrate is about 2 yuan / kg, the above characteristics of the strain will greatly reduce the cost of industrial fermentation for preparing DHA.

[0030] Biological material preservation

[0031] Schizochytrium limacinum ZD-NHU-11, classified and named as Schizochytrium limacinum, was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on October 28, 2024. The deposit number is CGMCC No. 41559, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Description of the drawings

[0032] Figure 1 It is the ARTP lethality curve diagram shown in Example 1.

[0033] Figure 2 It is the data diagram of DHA content of the better strains in the shake-flask primary screening at different evolutionary stages.

[0034] Figure 3 It is the shake-flask stability screening diagram of the initial strain and the mutagenized strain. Detailed implementation manners

[0035] Culture medium:

[0036] Plate culture medium: Glucose monohydrate 11 g / L, yeast extract 1.5 g / L, sodium nitrate 1.5 g / L, sodium chloride 14.5 g / L, magnesium sulfate heptahydrate 6 g / L, agar 16 g / L.

[0037] Activation culture medium: Glucose monohydrate 44 g / L, sodium glutamate 30 g / L, yeast extract 6 g / L, potassium dihydrogen phosphate 6 g / L, magnesium sulfate heptahydrate 8 g / L, sodium chloride 20 g / L, calcium chloride 0.3 g / L.

[0038] Screening medium containing malonic acid: Glucose monohydrate 11 g / L, yeast extract 1.5 g / L, sodium nitrate 1.5 g / L, sodium chloride 14.5 g / L, magnesium sulfate heptahydrate 6 g / L, agar 16 g / L. Add malonic acid as needed.

[0039] Adaptive evolution medium: Glucose monohydrate 11 g / L, yeast extract 1.5 g / L, sodium nitrate 1.5 g / L, potassium dihydrogen phosphate 6 g / L, magnesium sulfate heptahydrate 8 g / L, sodium chloride 20 g / L, calcium chloride 0.17 g / L. Add malonic acid as needed.

[0040] Flask fermentation medium: Glucose monohydrate 55 g / L, sodium glutamate 30 g / L, yeast extract 8 g / L, sodium nitrate 2 g / L, potassium dihydrogen phosphate 1.0 g / L, magnesium sulfate heptahydrate 12 g / L, sodium chloride 2.0 g / L, sodium sulfate 10 g / L, ammonium sulfate 1 g / L, potassium chloride 0.8 g / L, nickel sulfate monohydrate 2 g / L, copper sulfate pentahydrate 2 g / L, sodium molybdate dihydrate 0.04 g / L, manganese chloride tetrahydrate 3 g / L, cobalt chloride hexahydrate 0.04 g / L, zinc sulfate 3 g / L, ferrous sulfate heptahydrate 10 g / L.

[0041] Detection method:

[0042] Cell density: Detected using an ultraviolet spectrophotometer at a wavelength of 650 nm. Appropriately dilute the sample, with a measurement range of 0.2 - 0.8. Multiply the dilution factor by the measured value during calculation.

[0043] Determination of total lipids: Accurately weigh approximately 1 g (mL) of the above-mentioned freeze-dried cells into a 50 mL centrifuge tube, add 10 mL of concentrated hydrochloric acid and 5 mL of deionized water, heat in a water bath at 70 - 80 °C for about 1 h, and shake once every 10 min during this period. After cooling, add 4 mL of absolute ethanol and 10 mL of n-hexane, shake well, centrifuge at 3000 rpm for 2 min to extract the oil. Collect the upper n-hexane layer in a pre-weighed rotary flask, repeat 3 times, and perform rotary evaporation in a vacuum water bath at 65 °C for 10 min until the n-hexane is completely evaporated. Weigh the total weight of the rotary flask to obtain the total oil weight.

[0044] Determination of DHA content: Weigh approximately 0.05 g of the extracted oil into a centrifuge tube, add 0.5 mol·L -12 mL of NaOH-methanol solution was taken, shaken evenly, and reacted in a water bath at 65 °C for 1 h. After cooling to room temperature for 5 min, 2 mL of 25% boron trifluoride-ether solution was added, mixed evenly, esterified in a water bath at 65 °C for 20 min, 2 mL of saturated NaCl solution was added, and DHA methyl ester was extracted with n-hexane, and then gas chromatography analysis was carried out with a gas chromatograph, and the detector was a hydrogen flame detector. The specific GC conditions were set as follows: the inlet temperature was 250 °C, the injection volume was 1 μL, and splitless injection was used; the detector temperature was 250 °C, N2 was used as the carrier gas, after maintaining the initial column temperature for 2 min, the temperature was raised to 180 °C at a rate of 10 °C·min -1 and then the temperature was raised to 240 °C at a rate of 4 °C·min -1 and maintained for 8 min. Qualitative analysis of DHA was carried out by comparing the retention time with a 40-component FAME mixed standard; quantitative analysis of DHA was determined by comparing the peak areas of DHA and the added internal standard C19:0 to determine the percentage of DHA in the total oil content.

[0045] Example 1 Establishment of ARTP mutagenesis method

[0046] First, the cryopreservation tube of the initial strain Schizochytrium NHU-SL01 (disclosed in the patent application document with the publication number CN118562902A) was thawed naturally at room temperature. 1 mL of the bacterial liquid in the cryopreservation tube was inoculated into a 250 mL unbaffled straight-mouth activation flask containing 50 mL of activation medium, and cultured on a shaker at 28 °C and 170 rpm with an amplitude of 50 mm for about 22 h. When pH>6 or OD 650 was about 5, the cells were collected by centrifugation at 5000 rpm for 3 min. The cells were washed twice with sterile 5% glycerol, and then the cell OD 650 was diluted between 0.6 and 0.7, and mutagenesis was carried out using the ARTP breeding system. The mutagenesis steps were as follows:

[0047] 10 μL of the diluted bacterial suspension was taken and evenly spread on a sterile slide. In this invention, the helium flow rate and input power were controlled at 10.0 L / min and 120 W respectively, and the irradiation distance was kept at 2 mm. The treatment times were set as 0 s, 5 s, 10 s, 15 s, 20 s, 25 s, 30 s and 35 s in turn. After the treatment was completed, the slide was placed in an EP tube containing sterile 5% glycerol, shaken and mixed for 2-3 s until the cells were fully eluted, appropriately diluted and spread on a plate, and cultured at 28 °C for 2-3 d.

[0048] During the mutagenesis breeding process, in order to effectively mutate and screen mutants, the lethality rate was controlled at a relatively high level. As Figure 1As shown, with the increase of treatment time, the lethality gradually increases. When the treatment time is 5s and 10s, the lethality does not exceed 20%. When the treatment time is extended to 15s, the lethality increases sharply, reaching 67%. When the treatment time is increased to 20s, the lethality increases significantly again, reaching 90%. When the treatment time is 25s and 30s, the lethality exceeds 95%. When the treatment time is 35s, the lethality reaches 100%. It is inferred that before processing 10s, the cell membrane protects cells from death to a certain extent. When this limit is exceeded, the cell death rate increases sharply. When the treatment time is extended to 20s, the self-repair mechanism of the cell is triggered. At this time, the surviving cells provide a very favorable mutation rate. Therefore, a treatment time of 20 seconds was selected and used for subsequent ARTP mutagenesis.

[0049] Example 2 Malonic Acid Screening Concentration Optimization

[0050] Using enzyme inhibitors in related metabolic pathways as screening factors can make the screening process more selective and efficient. Malonic acid is used as an inhibitor of succinate dehydrogenase, resulting in inhibition of normal cell growth. In order to determine the sensitivity of Schizochytrium to malonate, plates containing different malonate concentrations were prepared to test the lethal concentration range of malonate. The specific implementation steps are as follows:

[0051] Take 1 mL of the frozen tube bacterial solution and inoculate it into a 250 mL straight-mouth activation shake flask without a barrier containing 50 mL of activation medium. Incubate on a shaker with an amplitude of 50 mm and 170 rpm at 28°C for about 22 h. When pH>6 or OD 650 When the pH was ≈5, the cells were collected by centrifugation at 5000 rpm for 3 min. The cells were washed twice with distilled water and resuspended in 0.2 mol / L PBS buffer solution at pH 7.0.

[0052] Press 10 respectively 2 -10 5 100 μL of the bacterial solution was diluted in a gradient manner and spread on the plate culture medium containing 1.0, 1.5, 2.0, 2.5, 3.0, and 4.0 g / L of malonic acid. Single colonies were grown at 28°C for 2-3 days and counted.

[0053] As shown in Table 1, with the increase of malonate concentration, the lethality rate increased rapidly. When the malonate concentration was 2.0 g / L, the lethality rate increased to 100%, and when the malonate concentration was 1.5 g / L, the lethality rate was around 50%, which can be used as a reference for adaptive evolution experiments. In order to facilitate the subsequent single colony selection and verification, 10 3 -10 4 Dilution multiple is preferred.

[0054] Table 1 Colony counts at different malonic acid concentrations

[0055]

[0056] Example 3 Adaptive Evolution

[0057] Adaptive evolution can simulate the natural evolution of microorganisms under environmental stress and ultimately obtain the desired phenotypes, which is a powerful method for microbial mutation breeding. After ARTP mutagenesis, the cells are first cultured overnight in the activation medium and then transferred to the screening medium containing malonic acid for adaptive evolution. The specific implementation steps are as follows:

[0058] Transfer 1 mL of the mixed bacterial suspension after ARTP mutagenesis eluted with 5% glycerol into a 250 mL baffled straight-mouth activation flask containing 50 mL of the activation medium, and culture at 28 °C and 170 rpm for 12 - 14 h. Pipette 2 mL of the bacterial solution and transfer it into a 1 L baffled straight-mouth enlarged flask containing 200 mL of the adaptive evolution medium for adaptive culture.

[0059] According to the results of Example 2, once the malonic acid concentration exceeds the tolerance of the strain, it will cause serious damage to the strain and further lead to cell death. Generally, we select the malonic acid concentration of about MIC 50 (defined as the minimum inhibitory concentration of malonic acid required to reduce the growth of Schizochytrium by 50%) as the screening concentration. Therefore, we select 1.5 g / L as the concentration for the first round of malonic acid tolerance evolution. Culture the enlarged flask at 28 °C and 170 rpm for 24 h to complete the first generation of evolution. Then transfer it to a new enlarged flask with an inoculation amount of 1% and keep the malonic acid concentration unchanged, and continue to culture under the same conditions, which is the second generation of evolution. And so on, depending on the specific growth situation, each round generally undergoes 6 - 10 generations of evolution.

[0060] According to the evolution requirements, multiple evolution processes are carried out in parallel for each round of evolution until there is a certain improvement in the cell growth trend, or select the process with the best growth among multiple processes, take out its bacterial solution and spread it on the malonic acid screening plate, and culture at 28 °C for 2 - 3 d to grow single colonies. The malonic acid concentration of the screening plate is generally 5% higher than the malonic acid concentration at the corresponding adaptive evolution level. Pick the single colonies that grow faster and larger from the plate for a new round of ARTP mutagenesis evolution. The malonic acid concentration for each round of adaptive evolution increases step by step with an increment of 0.1 g / L. Finally, the malonic acid concentration increases to 2.0 g / L, and the cells can grow nearly half normally, and a few tiny single colonies can be seen on the plate medium with 2.1 g / L malonic acid. When further attempting to culture in the adaptive evolution medium with 2.1 g / L malonic acid, the cells cannot grow effectively and always maintain at a low concentration level.

[0061] Example 4 Shake Flask Screening

[0062] Single colonies on plates at different evolutionary stages were picked for shake flask fermentation verification. For single colonies with good growth, they were inoculated into a 50 ml / 250 mL straight-mouth unbaffled activation shake flask and cultured on a shaker at 28 °C with an amplitude of 50 mm and 170 rpm for about 22 h. When pH > 6 or OD 650 = 5, 2 mL of the activated flask seed liquid was inoculated into a 250 mL fermentation shake flask containing 40 mL of fermentation medium and cultured on a shaker at 28 °C with an amplitude of 50 mm and 160 rpm for 120 h. After fermentation, the DHA content was detected. During the fermentation process, the glucose concentration in the culture system was maintained at 20 - 40 g / L by supplementing a glucose solution with a concentration of 500 g / L. The specific glucose supplementation method was: 4.6 mL of the glucose solution with a concentration of 500 g / L was supplemented at fermentation times 0 h, 10 h, 20 h, 30 h, 40 h, 50 h, 60 h, 70 h, 80 h, 90 h, 100 h, and 110 h respectively.

[0063] As Figure 2 shown, the DHA content data of the better strains initially screened by shake flask at different evolutionary stages were sorted out. It can be seen that among these more than 370 mutant strains, with the progress of adaptive evolution, the DHA content in shake flask fermentation gradually increased, but the overall evolution process was relatively gentle. In the later stage of evolution, there was a small obvious rising period in the DHA content, and it became more difficult to maintain a higher level later. At the later stage of evolution, the DHA content showed a slight fluctuation and then tended to be stable. This indicates that the ability of cells to adapt to malonic acid was enhanced, but further enhancing adaptability was restricted. Finally, a mutant strain ZD-NHU-11 was obtained through adaptive evolution, and its DHA content increased by 44.12% compared with the initial strain, reaching 55.98 mg / L.

[0064] Example 5 Shake Flask Re-screening

[0065] Take the cryopreservation tube of the mutant strain ZD-NHU-11 screened by shake flask in Example 4. After natural thawing at room temperature, 1 mL of the bacterial liquid was taken and inoculated into a 1 L unbaffled straight-mouth activation shake flask containing 200 mL of activation medium. Using the initial strain NHU-SL01 as a control (CK), it was cultured on a shaker at 28 °C with an amplitude of 50 mm and 170 rpm for about 22 h. When pH > 6 or OD 650 = 5, 2 mL of the activated seed liquid was inoculated into a 250 mL fermentation shake flask containing 40 mL of fermentation medium and cultured on a shaker at 28 °C with 170 rpm for 120 h, and the DHA content was detected. During the fermentation process, glucose was still supplemented according to the method described in Example 4.

[0066] Subculture experiment: 1 mL of bacterial solution was taken from the last batch of fermentation shake flasks (ZD-NHU-11, NHU-SL01) that had been cultured for 120 h and inoculated into 1 L straight-mouth activation shake flasks without baffles containing 200 mL of activation medium. The culture was carried out on a shaker at 28°C and 170 rpm for 22 h. When pH>6 or OD 650 =5, take 2mL of the activated bottle seed solution and inoculate it into the fermentation shake flask, and culture it on a shaker at 28°C, 170rpm and an amplitude of 50mm for 120h. Figure 3 As shown, strain ZD-NHU-11 exhibited good stability in succession, and the DHA production was maintained above 54 mg / L; compared with the control bacteria (CK), it always maintained an advantage in DHA production and had good genetic stability.

[0067] Example 6 Effect of sodium nitrate on biomass and DHA content of Schizochytrium

[0068] In order to further verify the mutant strain's ability to utilize sodium nitrate, the total content of yeast extract and sodium nitrate in the fermentation medium was controlled to be 10 g / L, and the mass ratio of the two was changed according to Table 2. The specific verification process is as follows:

[0069] Take the control bacteria and ZD-NHU-11 cryopreserved tubes, thaw them naturally at room temperature, and then draw 1 mL of the bacterial solution to inoculate into a 1L straight-mouth activation shake flask without a barrier containing 200 mL of activation medium. Incubate on a shaker with an amplitude of 50 mm at 28°C and 170 rpm for about 22 hours. When the pH is > 6 or the OD 650 When the activation temperature was about ≈5, 2 mL of the activated bottle seed solution was inoculated into fermentation shake flasks filled with different fermentation mediums and cultured on a shaker at 28°C, 170 rpm and an amplitude of 50 mm for 120 h. After the fermentation was completed, the biomass and DHA content were detected. During the fermentation process, glucose was still added according to the method described in Example 4.

[0070] Table 2 Biomass and DHA content in Schizochytrium fermentation broth after fermentation under different sodium nitrate ratios

[0071]

[0072] As shown in Table 2, with the increase of sodium nitrate ratio, the biomass and DHA content of the control strain (CK) decreased significantly, and the biomass and DHA production of the mutant strain ZD-NHU-11 also gradually decreased. When only sodium nitrate was used as the nitrogen source in the culture medium, the biomass and DHA content of the mutant strain ZD-NHU-11 were significantly improved compared with the initial strain, which were 90.69% and 103.03% higher, respectively. This shows that compared with the control strain, the mutant strain ZD-NHU-11 can use sodium nitrate more efficiently.

[0073] Although the present invention has been disclosed above in preferred embodiments, it is not intended to limit the present invention. Anyone skilled in this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Schizochytrium limacinum ZD-NHU-11, deposit number is CGMCC No.41559, and deposit date is October 28, 2024.

2. A method for preparing DHA, characterized in that: The method comprises fermenting the Schizochytrium sp. ZD-NHU-11 according to claim 1.

3. The method according to claim 2, characterized in that include: (A) culturing the Schizochytrium in a seed culture medium to obtain a seed solution; (B) inoculating the seed solution prepared in step (A) into a fermentation medium for fermentation to obtain a fermentation solution.

4. The method according to claim 3, characterized in that The seed culture is carried out at 26-30° C. and 160-180 rpm for 20-24 hours.

5. The method according to claim 3, characterized in that: The fermentation culture is carried out at 26-30° C. and 150-180 rpm for 96-144 hours.

6. The method according to any one of claims 2 to 5, characterized in that: During the fermentation process, glucose was fed every 8 to 12 hours.

7. The method according to any one of claims 2 to 6, characterized in that: The nitrogen source of the fermentation medium included sodium nitrate.

8. The method according to claim 7, characterized in that The nitrogen sources of the fermentation medium are sodium nitrate and yeast extract.

9. The method according to claim 8, characterized in that The mass ratio of the sodium nitrate to the yeast extract is (6:4) to (9:1).

10. Use of the Schizochytrium according to claim 1 or the method according to any one of claims 2 to 9 in the preparation of DHA or a product containing DHA.

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