Production method for improving DHA content in schizochytrium limacinum algae powder and application and method

Abstract

The invention belongs to the technical field of bioengineering, and discloses a production method and application for improving DHA content in schizochytrium limacinum algae powder, and the method comprises the following steps: using schizochytrium limacinum to carry out ARTP mutagenesis treatment to obtain a schizochytrium limacinum mutant strain with DHA% greater than or equal to 55%, carrying out fermentation regulation to obtain a fermentation broth, and then using a spray drying method to obtain the schizochytrium limacinum algae powder with high DHA content. According to the method, the DHA content in schizochytrium limacinum can be remarkably increased by combining a simple mutagenesis technology with fermentation regulation and control, and then spray drying is performed to obtain the DHA algae powder, so that the production cost is reduced, economic benefits are achieved, and an effective solution is expected to be provided for the problems that the schizochytrium limacinum algae powder is applied to the feed industry, the interest rate of the DHA production industry is low and the like.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular to a production method and application for increasing the DHA content in Schizochytrium algae powder, specifically involving ARTP mutagenesis technology, small molecule regulation strategy and nitrogen restriction strategy in the production method of increasing the DHA content in Schizochytrium algae powder. Background Technology

[0002] Docosahexaenoic acid (DHA) is an important long-chain polyunsaturated fatty acid. Due to its functions such as promoting brain development, preventing cardiovascular disease, and anti-cancer properties, it is widely used in the food, health product, and feed additive industries. The Chinese market is projected to reach 45 billion yuan by 2025. The traditional source of DHA is deep-sea fish oil; however, natural extraction methods are easily affected by marine pollution and environmental problems, making them unsustainable.

[0003] Schizochytrium is a microalga isolated from seawater, possessing advantages such as rapid growth, short fermentation cycle, and high biomass. It can be used to produce polyunsaturated fatty acids such as EPA, DPA, and DHA. Furthermore, Schizochytrium is rich in acetyl-CoA and NADPH precursors, and is considered a potential FDA-approved, edible strain for industrial DHA production. Currently, DHA derived from Schizochytrium is widely used in food and health products. However, my country's DHA production industry still faces challenges such as low DHA content and susceptibility to oxidation, as well as low oil yield, resulting in low production efficiency and limiting its application in animal feed. Therefore, improving the efficiency and quality of DHA production from Schizochytrium remains a challenge for further development in this field.

[0004] Chinese patent publication CN110885816A describes the use of ARTP to mutagenesis of microalgae, resulting in the screening of a genetically stable mutant strain with an oil yield of 343 mg / L. Chinese patent publication CN116179619A describes the synergistic use of two synthetic plant growth regulators, diethylaminoethanol hexanoate (DA-6) and 2,4-dichloronaphthyloxyacetic acid (2,4-D), to promote the DHA content and yield in Schizochytrium to 58.32% and 5.17 g / L, respectively. Chinese patent publication CN117821532A describes the addition of six amino acids (leucine, lysine, tryptophan, tyrosine, isoleucine, phenylalanine, and leucine) to the fermentation medium of Schizochytrium, promoting a DHA content of 55%.

[0005] In summary, current technologies mostly employ single methods, such as mutagenesis, addition of small molecule antioxidants, and fermentation condition control, and still suffer from various drawbacks such as low yield, high cost, and insufficient oxidative stability. This limits the comprehensive optimization of the oil yield and DHA content of Schizochytrium. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a production method and application for increasing the DHA content in Schizochytrium algae powder.

[0007] The technical solution adopted by this invention to solve its technical problem is: A method for increasing the lipid and DHA content of Schizochytrium, wherein the method utilizes ambient pressure room temperature plasma ARTP mutagenesis technology assisted by small molecule regulation and nitrogen-limiting strategies to increase the lipid and DHA content of Schizochytrium.

[0008] Furthermore, the *Schizochytridactylum* was mutagenized using an ARTP mutagen, and Nile Red staining was used to initially screen *Schizochytridactylum* mutants with high oil content (≥50 g / L). These high-oil-content mutants were then rescreened using gas chromatography to identify fatty acid profiles, resulting in mutants with high DHA content (≥55%). The DHA% ≥55% mutants were further enhanced using a small molecule regulation strategy combined with a nitrogen-limiting strategy, ultimately achieving an oil yield ≥65 g / L and DHA (%TFA) ≥60% in the *Schizochytridactylum* mutants. Alternatively, the Schizochytrium fungus is ATCC 20888.

[0009] Furthermore, the steps for obtaining the mutant strain with DHA% ≥ 55% are as follows: (1) Select wild-type fissuriyces growing in the logarithmic phase and prepare a bacterial suspension using 2% PBS solution; (2) Place the bacterial suspension in an ARTP mutagen for mutagenesis. The power supply voltage input is 120V, the current is 1A, the mutagenesis distance is 2 mm, the air flow rate is 7 L / min, and the mutagenesis treatment time is 30 s. The lethality rate is above 95% as a reference. (3) Transfer the mutagenized strain to a protective solution containing 10% (v / v) glycerol and Schizochytrium seed culture medium at a volume ratio of 1:1, and stabilize for 3-4 hours; (3) Dilute the mutagenized strain after stabilization treatment to 1×10⁻⁶. 7 The mixture was evenly spread on a solid culture medium and incubated at 30 °C for 48 h. (4) The mutant strains grown on the solid culture medium were transferred to the primary seed culture medium and cultured for 48 h to activate them. Then, the primary seed was used to continue to subculture to the tertiary seed using the seed culture medium. The inoculum amount for each subculture was 2% (v / v), the temperature was 30℃, and the rotation speed was 170 rpm for 24 h. Then, the tertiary seed cultured to the logarithmic phase was inoculated into the fermentation medium for fermentation culture. The inoculum amount was 2% (v / v), and the culture was carried out at 30℃ and 170 rpm for 120 h to obtain the fermentation broth. At the end of fermentation, the mutant strains with high oil content (≥50 g / L) were initially screened by Nile red staining. (5) Using gas chromatography, mutant strains with high DHA content (i.e., DHA%≥55%) were further screened.

[0010] Furthermore, (5) the specific steps for further screening using gas chromatography to obtain mutant strains with high DHA content, i.e., DHA%≥55%, are as follows: 1) Adjust the pH of the fermentation broth after fermentation in step (4) to 10-12, and add alkaline protease with a final mass concentration of 0.6%. 2) The Schizochytrium cells were completely digested by alkaline protease at 50℃ for 6 h until microscopic examination. 3) Add 95% ethanol to the fermentation liquid obtained in step (4) at a volume ratio of 1:1 to precipitate the protein; 4) Add an equal volume of n-hexane to the fermentation broth, mix well, let stand and wait for the layers to separate, remove the upper layer of n-hexane and oil mixture, and extract with n-hexane 2-3 times until the upper liquid is colorless and transparent. 5) Extract the obtained liquid by rotary evaporation at 50°C. Weigh the bottle before rotary evaporation. Evaporate until the condensate no longer drips. After rotary evaporation, dry in an oven until constant weight and weigh. 6) Ethylating the oil obtained in step 5): First, pipette 0.5-1 ml of potassium hydroxide-ethanol solution into a 2 ml centrifuge tube, add 40 µl of the liquid dried to constant weight in step 5), mix well, then add it to a 20 ml volumetric flask, add 2 ml of potassium hydroxide-ethanol solution to the volumetric flask, mix well, and then incubate at 65°C for 20 min, and cool to room temperature; next, add 2 ml of boron trifluoride ether, i.e., a mixture of boron trifluoride and ether in a volume ratio of 3:7, mix well, and then incubate at 65°C for 7 min; finally, add 2 ml of saturated potassium chloride and shake well, add 3 ml of n-hexane, let stand until the layers separate, pipette the supernatant organic phase and filter it through a microporous membrane to remove impurities, and then detect it using a gas chromatograph; Alternatively, the seed culture medium can be formulated as follows: glucose 60 g / L, yeast extract 5 g / L, monosodium glutamate 20 g / L, Na2SO4 10 g / L, CaCl2 0.5 g / L, KH2PO4 4 g / L, KCl 2 g / L, MgSO4•7H2O 5 g / L, vitamin B1 5 mg / L, vitamin B6 5 mg / L, vitamin B12 2 mg / L, and trace elements ZnSO4•7H2O 2 mg / L, FeCl3 10 mg / L, CuSO4 4 mg / L, MnCl2 8 mg / L, with water as the solvent. The solid culture medium comprises: 50 g / L glucose, 5 g / L yeast extract, 20 g / L monosodium glutamate, 10 g / L Na₂SO₄, 0.5 g / L CaCl₂, 4 g / L KH₂PO₄, 2 g / L KCl, 5 g / L MgSO₄•7H₂O, 5 mg / L vitamin B1, 5 mg / L vitamin B6, 2 mg / L vitamin B12, trace elements namely ZnSO₄•7H₂O 2 mg / L, FeCl₃ 10 mg / L, CuSO₄ 4 mg / L, MnCl₂ 8 mg / L, 20 g / L agar powder, and water as the solvent. The fermentation medium is formulated as follows: glucose 80 g / L, yeast extract 5 g / L, sodium glutamate 20 g / L, (NH4)2SO4 6 g / L, Na2SO4 15 g / L, CaCl2 1 g / L, KCl 1.5 g / L, KH2PO4 4 g / L, MgSO4•7H2O 5 g / L, vitamin B1 5 mg / L, vitamin B6 5 mg / L, vitamin B12 2 mg / L, and trace elements ZnSO4•7H2O 2 mg / L, FeCl3 10 mg / L, CuSO4 4 mg / L, and MnCl2 8 mg / L, with water as the solvent.

[0011] Furthermore, the specific steps of combining the small molecule regulation strategy with the nitrogen-limiting strategy include: (1) The mutant strain of Schizochytrium with DHA%≥55% was inoculated from the cryopreserved glycerol tube into the seed culture medium for activation, and the activated strain was transferred into the seed culture medium and cultured for 24h to obtain the first-level seed. (2) The primary seeds obtained in step (1) are inoculated into seed culture medium and cultured for 24 hours to obtain secondary seeds; (3) The secondary seeds obtained in step (2) are inoculated into the seed culture medium and cultured for 24 hours to obtain tertiary seeds; (4) The third-level seeds in step (3) are transferred to a fermentation medium with added tocopherol and reduced nitrogen source for fermentation culture to obtain DHA; wherein, the final concentration of added tocopherol in the fermentation medium is 0-1.5 g / L, and the final concentration of added sodium glutamate is 0-40 g / L, and the values ​​are not 0.

[0012] Further, the seed cultivation method in steps (1) to (3) is as follows: the inoculation amount of the primary or secondary seeds of the Schizochytrium is 2% (v / v, volume percentage), the cultivation temperature is 30 ℃, the rotation speed is 170 rpm, and the cultivation time is 24 h; Alternatively, in step (4), the tertiary seeds obtained in step (3) are inoculated into a fermentation medium with added tocopherol and reduced monosodium glutamate for fermentation culture. The inoculation amount is 2% (v / v, volume percentage), the temperature is 30 ℃, and the rotation speed is 170 rpm for 120 h. Alternatively, the seed culture medium in steps (1) to (3) may include a carbon source, a nitrogen source, inorganic salt ions, vitamins and trace elements; Alternatively, the fermentation medium in step (4) may include a carbon source, a nitrogen source (including monosodium glutamate), inorganic salt ions, vitamins, and trace elements.

[0013] Furthermore, in step (4), the final concentration of tocopherol added to the fermentation medium is 1 g / L, and the final concentration of sodium glutamate added is 10 g / L.

[0014] Furthermore, the seed culture medium is formulated as follows: glucose 60 g / L, yeast extract 5 g / L, monosodium glutamate 20 g / L, Na2SO4 10 g / L, CaCl2 0.5 g / L, KH2PO4 4 g / L, KCl 2 g / L, MgSO4•7H2O 5 g / L, vitamin B1 5 mg / L, vitamin B6 5 mg / L, vitamin B12 2 mg / L, and trace elements namely ZnSO4•7H2O 2 mg / L, FeCl3 10 mg / L, CuSO4 4 mg / L, and MnCl2 8 mg / L, with water as the solvent; The fermentation medium is formulated as follows: glucose 80 g / L, yeast extract 5 g / L, tocopherol, monosodium glutamate, (NH4)2SO4 6 g / L, Na2SO4 15 g / L, CaCl2 1 g / L, KCl 1.5 g / L, KH2PO4 4 g / L, MgSO4•7H2O 5 g / L, vitamin B1 5 mg / L, vitamin B6 5 mg / L, vitamin B12 2 mg / L, and trace elements ZnSO4•7H2O 2 mg / L, FeCl3 10 mg / L, CuSO4 4 mg / L, and MnCl2 8 mg / L, with water as the solvent.

[0015] The above-described method is used to increase the DHA content in Schizochytrium.

[0016] A production method for increasing the DHA content in Schizochytrium algae powder using the method described above is characterized by: using Schizochytrium to perform ARTP mutagenesis to obtain a Schizochytrium mutant strain with DHA%≥55%, and performing fermentation regulation to obtain a fermentation broth, followed by using spray drying to obtain Schizochytrium algae powder with high DHA content. The inlet temperature during drying is 180℃, and the outlet temperature is 80℃.

[0017] The advantages and positive effects of this invention are as follows: 1. This invention is a method for increasing the DHA content in Schizochytrium using mutagenesis technology, small molecule regulation strategy, and nitrogen-limiting strategy. Specifically, a mutant strain of Schizochytrium with increased oil and DHA content is obtained by using ambient pressure room temperature plasma (ARTP) mutagenesis technology. Subsequently, by adding tocopherol and reducing the concentration of monosodium glutamate, the DHA content and oil yield can be further increased. Finally, high-DHA algal powder can be obtained by spray drying at the end of fermentation. This method is a way to promote the increase of DHA and oil content in Schizochytrium. The method is simple, low-cost, and easy to apply to the feed industry.

[0018] 2. This invention rapidly obtains mutant strains rich in lipids using ARTP mutagenesis combined with Nile Red staining. Gas chromatography is then used to further identify mutant strains with high DHA content. Subsequently, this invention adds antioxidants such as tocopherol to the Schizochytrium fermentation system, which can not only alleviate oxidative stress within the Schizochytrium cells but also potentially address the problem of lipid oxidation. Furthermore, reducing the concentration of monosodium glutamate in the fermentation system may promote more acetyl-CoA flow to the lipid synthesis pathway, thereby increasing lipid yield. Using the above methods, a Schizochytrium strain with a lipid content of 69 g / L was finally obtained, representing a 43.75% increase compared to the control. The DHA yield was approximately 80.04% higher than the control, and high-DHA algal powder was obtained using spray drying. The obtained algal powder has a lower acid value and a peroxide value <0.06 under the same conditions, exhibiting superior quality. This invention, utilizing mutagenesis technology combined with a simple fermentation control strategy, not only saves production costs and is economically beneficial but also alleviates the problem of lipid oxidation, making it applicable to the feed production field.

[0019] 3. This invention utilizes ambient pressure room temperature plasma (ARTP) mutagenesis technology assisted by Nile Red staining and gas chromatography detection to screen for DHA-rich mutant strains. Subsequently, a small molecule regulation strategy combined with a nitrogen-limiting strategy is used to further increase the oil and DHA content of Schizochytrium, resulting in a significant increase in DHA yield in the Schizochytrium fermentation broth. The product is then spray-dried to obtain the DHA algal powder. This invention is expected to provide an effective solution for applying Schizochytrium algal powder in the feed industry and addressing the problem of low profit margins in DHA production.

[0020] 4. To address the issues of low DHA content, easy oxidation, and low oil yield in Schizochytrium, this invention provides a method for further increasing the oil and DHA content of Schizochytrium using ambient pressure room temperature plasma (ARTP) mutagenesis technology assisted by small molecule regulation and nitrogen-limiting strategies. After spray drying, high-DHA algal powder can be obtained. This method is a way to promote the increase of DHA and oil content in Schizochytrium. The method is simple, low-cost, and easy to apply to the feed industry.

[0021] 5. The method of the present invention can significantly increase the DHA content and oil yield in the Schizochytrium mutant strain by adding tocopherol and reducing the sodium glutamate concentration in the fermentation medium, thereby increasing the DHA production in Schizochytrium.

[0022] 6. This invention utilizes ambient pressure room temperature plasma (ARTP) mutagenesis assisted by Nile Red staining and gas chromatography to screen for high-DHA-yielding Schizochytrium mutant strains. Subsequently, tocopherol is added to the Schizochytrium fermentation medium to alleviate oxidative stress during fermentation. Furthermore, a nitrogen-limiting strategy allows Schizochytrium to quickly enter the lipid accumulation phase, further increasing both lipid and DHA content. Finally, spray-drying the high-DHA algal powder yields algal powder with more stable and superior lipid quality. This invention, through simple mutagenesis combined with fermentation regulation, significantly increases DHA content in Schizochytrium. Subsequent spray drying yields the DHA algal powder, reducing production costs and offering economic benefits. It is expected to provide an effective solution to the low profit margins in DHA production and the application of Schizochytrium algal powder in the feed industry. Detailed Implementation

[0023] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0024] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0025] A method for increasing the lipid and DHA content of Schizochytrium, wherein the method utilizes ambient pressure room temperature plasma ARTP mutagenesis technology assisted by small molecule regulation and nitrogen-limiting strategies to increase the lipid and DHA content of Schizochytrium.

[0026] Preferably, the *Schizochytridactylum* is mutagenized using an ARTP mutagen, and Nile Red staining is used to initially screen *Schizochytridactylum* mutants with high oil content (≥50 g / L). These high-oil-content mutants are then rescreened using gas chromatography for fatty acid profiling to identify mutants with high DHA content, resulting in mutant strains with DHA content ≥55%. The DHA% ≥55% mutant strains are further enhanced using a small molecule regulation strategy combined with a nitrogen-limiting strategy, ultimately achieving an oil yield ≥65 g / L and DHA (%TFA) ≥60% in the *Schizochytridactylum* mutant strains. Alternatively, the Schizochytrium fungus is ATCC 20888.

[0027] Preferably, the steps for obtaining the mutant strain with DHA% ≥ 55% are as follows: (1) Select wild-type fissuriyces growing in the logarithmic phase and prepare a bacterial suspension using 2% PBS solution; (2) Place the bacterial suspension in an ARTP mutagen for mutagenesis. The power supply voltage input is 120V, the current is 1A, the mutagenesis distance is 2 mm, the air flow rate is 7 L / min, and the mutagenesis treatment time is 30 s. The lethality rate is above 95% as a reference. (3) Transfer the mutagenized strain to a protective solution containing 10% (v / v) glycerol and Schizochytrium seed culture medium at a volume ratio of 1:1, and stabilize for 3-4 hours; (3) Dilute the mutagenized strain after stabilization treatment to 1×10⁻⁶. 7 The mixture was evenly spread on a solid culture medium and incubated at 30 °C for 48 h. (4) The mutant strains grown on the solid culture medium were transferred to the primary seed culture medium and cultured for 48 h to activate them. Then, the primary seed was used to continue to subculture to the tertiary seed using the seed culture medium. The inoculum amount for each subculture was 2% (v / v), the temperature was 30℃, and the rotation speed was 170 rpm for 24 h. Then, the tertiary seed cultured to the logarithmic phase was inoculated into the fermentation medium for fermentation culture. The inoculum amount was 2% (v / v), and the culture was carried out at 30℃ and 170 rpm for 120 h to obtain the fermentation broth. At the end of fermentation, the mutant strains with high oil content (≥50 g / L) were initially screened by Nile red staining. (5) Using gas chromatography, mutant strains with high DHA content (i.e., DHA%≥55%) were further screened.

[0028] Preferably, (5) the specific steps for further screening using gas chromatography to obtain mutant strains with high DHA content, i.e., DHA%≥55%, are as follows: 1) Adjust the pH of the fermentation broth after fermentation in step (4) to 10-12, and add alkaline protease with a final mass concentration of 0.6%. 2) The Schizochytrium cells were completely digested by alkaline protease at 50℃ for 6 h until microscopic examination. 3) Add 95% ethanol to the fermentation liquid obtained in step (4) at a volume ratio of 1:1 to precipitate the protein; 4) Add an equal volume of n-hexane to the fermentation broth, mix well, let stand and wait for the layers to separate, remove the upper layer of n-hexane and oil mixture, and extract with n-hexane 2-3 times until the upper liquid is colorless and transparent. 5) Extract the obtained liquid by rotary evaporation at 50°C. Weigh the bottle before rotary evaporation. Evaporate until the condensate no longer drips. After rotary evaporation, dry in an oven until constant weight and weigh. 6) Ethylating the oil obtained in step 5): First, pipette 0.5-1 ml of potassium hydroxide-ethanol solution into a 2 ml centrifuge tube, add 40 µl of the liquid dried to constant weight in step 5), mix well, then add it to a 20 ml volumetric flask, add 2 ml of potassium hydroxide-ethanol solution to the volumetric flask, mix well, and then incubate at 65°C for 20 min, and cool to room temperature; next, add 2 ml of boron trifluoride ether, i.e., a mixture of boron trifluoride and ether in a volume ratio of 3:7, mix well, and then incubate at 65°C for 7 min; finally, add 2 ml of saturated potassium chloride and shake well, add 3 ml of n-hexane, let stand until the layers separate, pipette the supernatant organic phase and filter it through a microporous membrane to remove impurities, and then detect it using a gas chromatograph; Alternatively, the seed culture medium can be formulated as follows: glucose 60 g / L, yeast extract 5 g / L, monosodium glutamate 20 g / L, Na2SO4 10 g / L, CaCl2 0.5 g / L, KH2PO4 4 g / L, KCl 2 g / L, MgSO4•7H2O 5 g / L, vitamin B1 5 mg / L, vitamin B6 5 mg / L, vitamin B12 2 mg / L, and trace elements ZnSO4•7H2O 2 mg / L, FeCl3 10 mg / L, CuSO4 4 mg / L, MnCl2 8 mg / L, with water as the solvent. The solid culture medium comprises: 50 g / L glucose, 5 g / L yeast extract, 20 g / L monosodium glutamate, 10 g / L Na₂SO₄, 0.5 g / L CaCl₂, 4 g / L KH₂PO₄, 2 g / L KCl, 5 g / L MgSO₄•7H₂O, 5 mg / L vitamin B1, 5 mg / L vitamin B6, 2 mg / L vitamin B12, trace elements namely ZnSO₄•7H₂O 2 mg / L, FeCl₃ 10 mg / L, CuSO₄ 4 mg / L, MnCl₂ 8 mg / L, 20 g / L agar powder, and water as the solvent. The fermentation medium is formulated as follows: glucose 80 g / L, yeast extract 5 g / L, sodium glutamate 20 g / L, (NH4)2SO4 6 g / L, Na2SO4 15 g / L, CaCl2 1 g / L, KCl 1.5 g / L, KH2PO4 4 g / L, MgSO4•7H2O 5 g / L, vitamin B1 5 mg / L, vitamin B6 5 mg / L, vitamin B12 2 mg / L, and trace elements ZnSO4•7H2O 2 mg / L, FeCl3 10 mg / L, CuSO4 4 mg / L, and MnCl2 8 mg / L, with water as the solvent.

[0029] Preferably, the specific steps of the small molecule regulation strategy combined with the nitrogen-limiting strategy include: (1) The mutant strain of Schizochytrium with DHA%≥55% was inoculated from the cryopreserved glycerol tube into the seed culture medium for activation, and the activated strain was transferred into the seed culture medium and cultured for 24h to obtain the first-level seed. (2) The primary seeds obtained in step (1) are inoculated into seed culture medium and cultured for 24 hours to obtain secondary seeds; (3) The secondary seeds obtained in step (2) are inoculated into the seed culture medium and cultured for 24 hours to obtain tertiary seeds; (4) The third-level seeds in step (3) are transferred to a fermentation medium with added tocopherol and reduced nitrogen source for fermentation culture to obtain DHA; wherein, the final concentration of added tocopherol in the fermentation medium is 0-1.5 g / L, and the final concentration of added sodium glutamate is 0-40 g / L, and the values ​​are not 0.

[0030] Preferably, the seed culture method in steps (1) to (3) is as follows: the inoculation amount of the primary or secondary seeds of the Schizochytrium is 2% (v / v, volume percentage), the culture temperature is 30 ℃, the rotation speed is 170 rpm, and the culture time is 24 h; Alternatively, in step (4), the tertiary seeds obtained in step (3) are inoculated into a fermentation medium with added tocopherol and reduced monosodium glutamate for fermentation culture. The inoculation amount is 2% (v / v, volume percentage), the temperature is 30 ℃, and the rotation speed is 170 rpm for 120 h. Alternatively, the seed culture medium in steps (1) to (3) may include a carbon source, a nitrogen source, inorganic salt ions, vitamins and trace elements; Alternatively, the fermentation medium in step (4) may include a carbon source, a nitrogen source (including monosodium glutamate), inorganic salt ions, vitamins, and trace elements.

[0031] Preferably, in step (4), the final concentration of tocopherol added to the fermentation medium is 1 g / L, and the final concentration of sodium glutamate added is 10 g / L.

[0032] Preferably, the seed culture medium is formulated as follows: glucose 60 g / L, yeast extract 5 g / L, monosodium glutamate 20 g / L, Na2SO4 10 g / L, CaCl2 0.5 g / L, KH2PO4 4 g / L, KCl 2 g / L, MgSO4•7H2O 5 g / L, vitamin B1 5 mg / L, vitamin B6 5 mg / L, vitamin B12 2 mg / L, and trace elements namely ZnSO4•7H2O 2 mg / L, FeCl3 10 mg / L, CuSO4 4 mg / L, and MnCl2 8 mg / L, with water as the solvent; The fermentation medium is formulated as follows: glucose 80 g / L, yeast extract 5 g / L, tocopherol, monosodium glutamate, (NH4)2SO4 6 g / L, Na2SO4 15 g / L, CaCl2 1 g / L, KCl 1.5 g / L, KH2PO4 4 g / L, MgSO4•7H2O 5 g / L, vitamin B1 5 mg / L, vitamin B6 5 mg / L, vitamin B12 2 mg / L, and trace elements ZnSO4•7H2O 2 mg / L, FeCl3 10 mg / L, CuSO4 4 mg / L, and MnCl2 8 mg / L, with water as the solvent.

[0033] The above-described method is used to increase the DHA content in Schizochytrium.

[0034] A production method for increasing the DHA content in Schizochytrium algae powder using the method described above is characterized by: using Schizochytrium to perform ARTP mutagenesis to obtain a Schizochytrium mutant strain with DHA%≥55%, and performing fermentation regulation to obtain a fermentation broth, followed by using spray drying to obtain Schizochytrium algae powder with high DHA content. The inlet temperature during drying is 180℃, and the outlet temperature is 80℃.

[0035] Specifically, the relevant preparation and testing methods are as follows: Implementation Case 1 In this embodiment, Schizochytrium ATCC-20888 (purchased from the American Type Culture Collection (ATCC) and currently stored in 20% (v / v, volume percentage) glycerol tubes) was used.

[0036] A method for generating Schizochytrium mutant strains with high lipid and DHA content using ARTP mutagenesis includes the following steps: (1) Select wild-type Schizochytrium fungi cultured in seed culture medium to the logarithmic growth phase ( Schizochytrium sp. ATCC 20888), a bacterial suspension was prepared using 0.01 mol / L PBS solution with a mass concentration of 2% (w / v); (2) The bacterial suspension was placed in an ARTP mutagen for mutagenesis. Specifically, the power supply voltage was set to 120V, the current to 1A, the mutagenesis distance to 2 mm, the air flow rate to 7 L / min, and the mutagenesis treatment times were 0s, 10s, 20s, 30s, 40s, 50s, 60s, and 70s. By adjusting the different mutagenesis times, the optimal mutagenesis time was determined to be 30s, with a lethality rate of over 95% as a reference. The lethality rate % = (number of unmutated colonies - number of mutagenated colonies) / number of unmutated colonies × 100%; (3) Transfer the mutagenized strain to a protective solution containing 10% (v / v) glycerol and Schizochytrium seed culture medium at a volume ratio of 1:1, and stabilize for 3-4 hours; (3) Dilute the mutagenized strain after stabilization treatment to 1×10⁻⁶. 7 The mixture was evenly spread on a solid culture medium and incubated at 30 °C for 48 h. The solid culture medium comprises: 50 g / L glucose, 5 g / L yeast extract, 20 g / L sodium glutamate, 10 g / L Na2SO4, 0.5 g / L CaCl2, 4 g / L KH2PO4, 2 g / L KCl, 5 g / L MgSO4•7H2O, 5 mg / L vitamin B1, 5 mg / L vitamin B6, 2 mg / L vitamin B12, trace elements (2 mg / L ZnSO4•7H2O, 10 mg / L FeCl3, 4 mg / L CuSO4, 8 mg / L MnCl2), 20 g / L agar powder, and water as the solvent.

[0037] (4) The mutant strains grown on solid culture medium were transferred to primary seed culture medium and cultured for 48 h for activation. Then, the primary seed was passaged to tertiary seed using seed culture medium. The inoculum size for each passage was 2% (v / v), the temperature was 30℃, and the rotation speed was 170 rpm for 24 h. Subsequently, the tertiary seed cultured to the logarithmic growth phase was inoculated into fermentation medium for fermentation culture. The inoculum size was 2% (v / v), and the culture was carried out at 30℃ and 170 rpm for 120 h to obtain fermentation broth. At the end of fermentation, the fluorescence value per unit cell was determined by Nile red staining to obtain mutant strains with high oil content (i.e., oil content ≥50 g / L). Specifically, Nile red was used to stain the strains, and the fluorescence value was measured by a fluorescence spectrophotometer. The excitation wavelength was 480 nm, and the emission wavelength was 500-600 nm. The OD value of Schizochytrium was measured by a spectrophotometer at a detection wavelength of 600 nm. The fluorescence value per unit cell = fluorescence value / OD 600In this embodiment, the genetic stability considered is based on whether the difference between the growth rate and relative fluorescence value of Schizochytrium is significant.

[0038] The seed culture medium is formulated as follows: glucose 60 g / L, yeast extract 5 g / L, monosodium glutamate 20 g / L, Na2SO4 10 g / L, CaCl2 0.5 g / L, KH2PO4 4 g / L, KCl 2 g / L, MgSO4•7H2O 5 g / L, vitamin B1 5 mg / L, vitamin B6 5 mg / L, vitamin B12 2 mg / L, and trace elements (ZnSO4•7H2O 2 mg / L, FeCl3 10 mg / L, CuSO4 4 mg / L, MnCl2 8 mg / L).

[0039] The fermentation medium is formulated as follows: glucose 80 g / L, yeast extract 5 g / L, sodium glutamate 20 g / L, (NH4)2SO4 6 g / L, Na2SO4 15 g / L, CaCl2 1 g / L, KCl 1.5 g / L, KH2PO4 4 g / L, MgSO4•7H2O 5 g / L, vitamin B1 5 mg / L, vitamin B6 5 mg / L, vitamin B12 2 mg / L, and trace elements (ZnSO4•7H2O 2 mg / L, FeCl3 10 mg / L, CuSO4 4 mg / L, MnCl2 8 mg / L).

[0040] (5) Using gas chromatography, mutant strains with high DHA content (DHA%≥55%) were further screened. The pH of the fermentation broth after fermentation was completed was adjusted to 10-12, and alkaline protease with a final mass concentration of 0.6% (w / v) was added. (6) The Schizochytrium cells were completely digested by alkaline protease at 50℃ for 6 h until microscopic examination. (7) Add 95% ethanol (volume concentration) to the enzyme hydrolysate from step (6) in a ratio of 1:1 to the volume of the fermentation broth obtained in step (4) to precipitate the protein. (8) Add an equal volume of n-hexane to the fermentation broth and mix well. Let stand and wait for the layers to separate. Take off the upper layer of n-hexane and oil mixture and extract with n-hexane 2-3 times until the upper liquid is colorless and transparent. (9) Extract the obtained liquid by rotary evaporation at 50°C. Weigh the bottle before rotary evaporation. Evaporate until the condensate no longer drips. After rotary evaporation, dry in an oven until constant weight and weigh. (10) Ethylate the oil obtained in step (9): First, take 0.5-1 ml of 1 mol / L potassium hydroxide-ethanol solution into a 2 ml centrifuge tube, add 40 µl (the liquid obtained in step (9) dried to constant weight) and mix well. Then add it to a 20 ml volumetric flask, add 2 ml of 1 mol / L potassium hydroxide-ethanol solution to the volumetric flask, mix well, and then heat in a water bath at 65°C for 20 min and cool to room temperature. Next, add 2 ml of boron trifluoride ether (a mixture of boron trifluoride and ether in a volume ratio of 3:7), mix well, and then heat in a water bath at 65°C for 7 min. Finally, add 2 ml of saturated potassium chloride and shake well. Add 3 ml of n-hexane and let stand until the layers separate. Take the supernatant organic phase and filter it with a microporous membrane to remove impurities. Then, use a gas chromatograph to detect it.

[0041] (11) The fermentation results of a batch of mutant strains obtained in step (10) were statistically analyzed. The results are shown in Table 1. The best Schizochytrium mutant strain obtained an oil yield of 60 g / L, which is 1.25 times that of the wild-type strain. The DHA content was 58.97%, which is 17.19% higher than that of the control.

[0042] Table 1

[0043] Implementation Case 2 The method for adding tocopherol to the fermentation medium of Schizochytrium includes the following steps: 1. The *Schizochytrium* mutant strain from Example 1 (total oil content 60 g / L, DHA (%TFA) 58.97%) was inoculated into 50 mL of seed culture medium at an inoculation volume of 2% (v / v). The culture was incubated at 30 °C and 170 rpm for approximately 24 h to obtain primary seeds. 2% (v / v) of the seed culture medium containing the primary seeds was added to 50 mL of seed culture medium and incubated at 30 °C and 170 rpm for approximately 24 h to obtain secondary seeds. 2% (v / v) of the seed culture medium containing the secondary seeds was added to 200 mL of seed culture medium and incubated at 30 °C and 170 rpm to obtain tertiary seed culture in the logarithmic growth phase. The formulation of the seed culture medium is the same as that described in Example 1.

[0044] 2. The tertiary seed culture, cultured to the logarithmic growth phase, was inoculated at a rate of 5% (v / v) into the fermentation medium described in Example 1. The difference was that a gradient of tocopherols (0 g / L, 0.25 g / L, 0.5 g / L, 0.75 g / L, 1 g / L, and 1.5 g / L) was added to the fermentation medium. The culture was then shake-cultured at 30℃ and 170 rpm for 120 h, after which the total lipid content and DHA percentage were measured. The results are shown in Table 2.

[0045] Table 2

[0046] As shown in Table 2, after adding different concentrations of tocopherol, the total lipid content and DHA proportion significantly increased with the addition of 0-1.5 g / L of tocopherol in the fermentation medium, with the optimal concentration being 1 g / L (Table 2). The results were as follows: the lipid yield of the Schizochytrium mutant strain increased from 60 g / L to 63.1 g / L, and the DHA proportion increased from 58.97% to 60.35%. Calculations showed that the DHA yield increased from 35.38 g / L to 38.08 g / L, which is 1.08 times that without tocopherol. It is evident that the amount of tocopherol added in this invention is crucial; both excessively low and high concentrations are ineffective, and even minute changes can lead to significant increases or decreases in various indicators. Furthermore, after adding tocopherol, the mutant strain achieved a 1.31-fold increase in total lipid content compared to the wild-type strain, and the proportion of DHA in total fatty acids (TFA) increased by 19.93%. This indicates that the mutant strain itself had a significantly higher total lipid content and DHA production than the wild-type strain, and the addition of tocopherol to the fermentation medium resulted in even higher total lipid content and DHA production.

[0047] Implementation Case 3 A method for reducing monosodium glutamate in Schizochytrium fermentation medium includes the following steps: 1. The *Schizochytrium* mutant strain from Example 1 (total oil content 60 g / L, DHA (%TFA) 58.97%) was inoculated into 50 mL of seed culture medium at an inoculation volume of 2% (v / v). The culture was incubated at 30°C and 170 rpm for approximately 24 hours to obtain primary seeds. 2% (v / v) of the seed culture medium containing the primary seeds was added to 50 mL of seed culture medium and incubated at 30°C and 170 rpm for approximately 24 hours to obtain secondary seeds. 2% (v / v) of the seed culture medium containing the secondary seeds was added to 200 mL of seed culture medium and incubated at 30°C and 170 rpm to obtain tertiary seed culture in the logarithmic growth phase. The formulation of the seed culture medium is the same as that described in Example 1.

[0048] 2. The tertiary seed culture, cultured to the logarithmic growth phase, was inoculated at a rate of 5% (v / v) into the fermentation medium described in Case 1. The difference was that the sodium glutamate (MSG) concentration in the fermentation medium was adjusted according to a specific concentration gradient (i.e., 0 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L). The culture was then in shake flasks at 30℃ and 170 rpm for 120 h, at which point the fermentation was stopped, and the total lipid content and DHA percentage were measured. The results are shown in Table 3.

[0049] Table 3

[0050] As shown in Table 3, after adding different concentrations of monosodium glutamate (MSG), the total lipid content and DHA proportion in the fermentation medium were significantly increased with the addition of 0-40 g / L MSG, with the optimal concentration being 10 g / L (Table 3). The results were as follows: the lipid yield of the Schizochytrium mutant strain increased from 60 g / L to 65.7 g / L, and the DHA proportion increased from 58.97% to 62.18%. Calculations showed that the DHA yield increased from 35.38 g / L to 40.85 g / L, which is 1.15 times that of the control. In this invention, the concentration of MSG has a significant impact on the DHA and lipid content. Both excessively low and high concentrations can lead to poor fermentation results in lipid and DHA content; even slight changes can result in significant increases or decreases in various indicators. Furthermore, after adjusting the nitrogen source concentration, this mutant strain achieved a total lipid content 1.37 times higher than the wild-type strain, and the proportion of DHA in total fatty acids (TFA) increased by 23.57%. This indicates that the mutant strain itself had a significantly higher total lipid content and DHA production than the wild-type strain, and that reducing the sodium glutamate concentration in its fermentation medium resulted in even higher total lipid content and DHA production.

[0051] Implementation Case 4 Based on Implementation Case 2 and Implementation Case 3, the method for adding tocopherol and reducing monosodium glutamate in the fermentation medium of Schizochytrium includes the following steps: 1. The *Schizochytrium* mutant strain from Example 1 (total oil content 60 g / L, DHA (%TFA) 58.97%) was inoculated into 50 mL of seed culture medium at an inoculation volume of 2% (v / v). The culture was incubated at 30°C and 170 rpm for approximately 24 hours to obtain primary seeds. 2% (v / v) of the seed culture medium containing the primary seeds was added to 50 mL of seed culture medium and incubated at 30°C and 170 rpm for approximately 24 hours to obtain secondary seeds. 2% (v / v) of the seed culture medium containing the secondary seeds was added to 200 mL of seed culture medium and incubated at 30°C and 170 rpm to obtain tertiary seed culture in the logarithmic growth phase. The formulation of the seed culture medium is the same as that described in Example 1.

[0052] 2. The tertiary seed culture, cultured to the logarithmic growth phase, was inoculated at a rate of 5% (v / v) into a fermentation medium with a tocopherol concentration of 1 g / L and a monosodium glutamate concentration of 10 g / L (except for the addition of tocopherol and the change in the monosodium glutamate concentration, the other components of the medium were the same as those in Case 1). The medium was then shake-cultured at 30℃ and 170 rpm for 120 h, after which the fermentation was stopped, and the total lipid content and DHA percentage were measured. The results are shown in Table 4.

[0053] Table 4

[0054] As shown in Table 4, after adding 1 g / L tocopherol and 10 g / L monosodium glutamate to the fermentation medium of the Schizochytrium mutant strain, the total lipid content and DHA ratio of the Schizochytrium mutant strain were further increased. The results were as follows: the lipid yield of the Schizochytrium mutant strain increased from the initial 58.97 g / L to 69 g / L, and the DHA ratio increased from 58.97% to 63.01%. Calculations showed that the DHA yield increased from 35.38 g / L to 43.48 g / L, an increase of 22.89% compared to the control. In this invention, the combined addition of tocopherol and modification of the monosodium glutamate concentration further improved the DHA and lipid content, indicating that adjusting the concentrations of tocopherol and monosodium glutamate has a synergistic effect on promoting the total lipid and DHA content of this Schizochytrium mutant strain. Furthermore, after simultaneously adding tocopherol and adjusting the nitrogen source concentration in the fermentation medium, the mutant strain achieved a total lipid content 1.44 times higher than the wild-type strain, and the proportion of DHA in total fatty acids (TFA) increased by 25.22%. This indicates that the mutant strain itself already showed a significant increase in total lipid content and DHA production compared to the wild-type strain, and adding 1 g / L tocopherol and reducing the sodium glutamate concentration in the fermentation medium resulted in even higher total lipid content and DHA production.

[0055] Meanwhile, by comparing Examples 2 to 4, it can be seen that the 1 g / L tocopherol and 10 g / L sodium glutamate added to the fermentation culture medium in the method of the present invention have a synergistic effect, which can synergistically improve the DHA content and oil content of the prepared product.

[0056] Implementation Case 5 The fermentation broth obtained by the preparation method in Case 4 was spray-dried to produce powder. The inlet temperature during drying was 180℃ and the outlet temperature was 80℃, resulting in Schizochytrium algal powder. The control group consisted of wild-type Schizochytrium algal powder. The acid value and peroxide value of the oil in the algal powder were detected using a kit according to the instructions. The results are shown in Table 4.

[0057] Table 4

[0058] The results are shown in Table 4. In Case 4, the oil content in the spray-dried algal powder was not only higher than that of the wild-type Schizochytrium strain, but the DHA (%TFA) content was also further improved. In addition, by comparing the acid value and peroxide value of the two strains, it can be found that the oil in the spray-dried algal powder is more stable under the same storage conditions and has a more outstanding quality performance, especially in terms of acid value.

[0059] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for increasing the lipid content and DHA content of a Schizochytrium sp., characterized by: The method is to improve the oil content and DHA content of Schizochytrium sp. by using atmospheric room temperature plasma (ARTP) mutagenesis technology assisted small molecule regulation strategy and nitrogen limitation strategy.

2. The method of claim 1, wherein: The Schizochytrium sp. is mutagenized by using an ARTP mutagenizer, and the high-oil-content Schizochytrium sp. mutant strain with an oil content of ≥50 g / L is preliminarily screened by using Nile red staining method. The high-oil-content Schizochytrium sp. mutant strain with an oil content of ≥50 g / L is further screened by using a gas chromatograph to identify the DHA content of the Schizochytrium sp. mutant strain, and the mutant strain with a high DHA content, i.e. DHA%≥55%, is screened. The mutant strain with DHA%≥55% is further improved in DHA content and oil content by using a small molecule regulation strategy combined with a nitrogen limitation strategy. Finally, the oil yield of the Schizochytrium sp. mutant strain is ≥65 g / L, and DHA (%TFA) is ≥60%. Alternatively, the Schizochytrium sp. is ATCC 20888.

3. The method of claim 2, wherein: The mutant strain with DHA%≥55% is obtained by the following steps: (1) The wild-type Schizochytrium sp. in the logarithmic growth phase is selected, and a bacterial suspension is prepared by using a PBS solution with a mass concentration of 2%; (2) The bacterial suspension is placed in the ARTP mutagenizer for mutagenesis. The power supply voltage is 120 V, the current is 1 A, the mutagenesis distance is 2 mm, the air flow is 7 L / min, and the mutagenesis treatment time is 30 s, with a lethal rate of more than 95% as a reference; (3) The mutagenized strain is transferred to a protective solution prepared by mixing 10% (v / v) glycerol and Schizochytrium sp. seed culture medium at a volume ratio of 1:1, and is stabilized for 3-4 hours; (3) The concentration of the mutagenized strain after stable treatment is diluted to 1 x 10 7 It is uniformly coated in solid culture medium and cultured in a 30 °C incubator for 48 h; (4) The mutagenized strain grown on the solid culture medium is transferred to the first-stage seed culture medium for culture for 48 h for activation. Then, the first-stage seed is further subcultured to the third-stage seed by using the seed culture medium. The inoculation amount of each subculture is 2% (v / v), the temperature is 30°C, and the rotation speed is 170 rpm, and each culture is performed for 24 h. Then, the third-stage seed in the logarithmic growth phase is inoculated into the fermentation medium for fermentation culture. The inoculation amount is 2% (v / v), the temperature is 30°C, and the rotation speed is 170 rpm, and the culture is performed for 120 h to obtain a fermentation broth. At the end of the fermentation, the mutant strain with a high oil content, i.e. ≥50 g / L, is preliminarily screened by using Nile red staining method; (5) The mutant strain with a high DHA content, i.e. DHA%≥55%, is further screened by using gas chromatography.

4. The method of claim 3, wherein: The specific steps for further screening the mutant strain with a high DHA content, i.e. DHA%≥55%, by using gas chromatography are as follows: 1) The pH of the fermentation broth obtained in step (4) is adjusted to 10-12, and 0.6% alkaline protease is added; 2) The alkaline protease is used for enzymolysis at 50°C for 6 h until the Schizochytrium sp. is completely enzymolyzed by microscopic observation; 3) 95% ethanol is added at a volume ratio of 1:1 to the fermentation broth obtained in step (4) to precipitate the protein; 4) An equal volume of n-hexane is added and mixed, and the mixture is allowed to stand to be stratified. The upper layer of n-hexane and oil mixture is sucked and extracted with n-hexane for 2-3 times until the upper layer of liquid is colorless and transparent. 5) The obtained liquid is subjected to rotary evaporation extraction at 50°C, the bottle is weighed before rotary evaporation, rotary evaporation is performed until no water drops from the condenser, and the bottle is placed in an oven for drying to constant weight after rotary evaporation; 6) The oil obtained in step 5) is subjected to ethyl esterification: first, 0.5-1 ml of potassium hydroxide-ethanol solution is sucked into a 2 ml centrifuge tube, 40 μl of the liquid dried to constant weight in step 5) is added and mixed, then it is added to a 20 ml volumetric flask, 2 ml of potassium hydroxide-ethanol solution is added to the volumetric flask, mixed, and then placed in a 65°C water bath for 20 min and cooled to room temperature; secondly, 2 ml of boron trifluoride diethyl ether, i.e. a mixture of boron trifluoride and diethyl ether in a volume ratio of 3:7, is added, mixed, and then placed in a 65°C water bath for 7 min; finally, 2 ml of saturated potassium chloride is added and shaken, 3 ml of n-hexane is added and allowed to stand until the layers separate, the supernatant organic phase is sucked and filtered using a microporous membrane to remove impurities, and then detected using a gas chromatograph; Alternatively, the formula of the seed culture medium is: glucose 60 g / L, yeast extract 5 g / L, sodium glutamate 20 g / L, Na2SO4 10 g / L, CaCl2 0.5 g / L, KH2PO4 4 g / L, KCl 2 g / L, MgSO4•7H2O 5 g / L, vitamin B1 5 mg / L, vitamin B6 5 mg / L, vitamin B12 2 mg / L, trace elements, i.e. ZnSO4•7H2O 2 mg / L, FeCl3 10 mg / L, CuSO4 4 mg / L, MnCl2 8 mg / L, and the solvent is water; The components of the solid culture medium include: glucose 50 g / L, yeast extract 5 g / L, sodium glutamate 20 g / L, Na2SO4 10 g / L, CaCl2 0.5 g / L, KH2PO4 4 g / L, KCl 2 g / L, MgSO4•7H2O 5 g / L, vitamin B1 5 mg / L, vitamin B6 5 mg / L, vitamin B12 2 mg / L, trace elements, i.e. ZnSO4•7H2O 2 mg / L, FeCl3 10 mg / L, CuSO4 4 mg / L, MnCl2 8 mg / L, agar powder 20 g / L, and the solvent is water The formula of the fermentation medium is: glucose 80 g / L, yeast extract 5 g / L, sodium glutamate 20 g / L, (NH4)2SO4 6 g / L, Na2SO4 15 g / L, CaCl2 1 g / L, KCl 1.5 g / L, KH2PO4 4 g / L, MgSO4·7H2O 5 g / L, vitamin B1 5 mg / L, vitamin B6 5 mg / L, vitamin B12 2 mg / L, trace elements, namely ZnSO4·7H2O 2 mg / L, FeCl3 10 mg / L, CuSO4 4 mg / L, MnCl2 8 mg / L, and the solvent is water.

5. The method according to any one of claims 2 to 4, characterized in that: The specific steps of the small molecule regulation strategy combined with the nitrogen limitation strategy include: (1) A Schizochytrium sp. mutant strain with DHA≥55% is activated from a glycerol tube of low-temperature storage in a seed culture medium and then transferred into the seed culture medium for 24 h to obtain a first-level seed; (2) The first-level seed obtained in step (1) is inoculated into the seed culture medium for 24 h to obtain a second-level seed; (3) The second-level seed obtained in step (2) is inoculated into the seed culture medium for 24 h to obtain a third-level seed; (4) The third-level seed in step (3) is transferred into a fermentation medium added with tocopherol and reduced nitrogen source for fermentation to obtain DHA; wherein the final concentration of tocopherol added in the fermentation medium is 0-1.5 g / L, the final concentration of sodium glutamate added is 0-40 g / L, and neither of them is 0.

6. The method of claim 5, wherein: The seed culture method in steps (1)-(3) is as follows: the inoculation amount of the Schizochytrium sp. first-level seed or second-level seed is 2% (v / v, volume percentage), the culture temperature is 30°C, the rotation speed is 170 rpm, and the culture time is 24 h; Alternatively, the third-level seed obtained in step (3) is inoculated into a fermentation medium added with tocopherol and reduced sodium glutamate in step (4) for fermentation culture, the inoculation amount is 2% (v / v, volume percentage), the temperature is 30°C, and the rotation speed is 170 rpm for 120 h of culture; Alternatively, the seed culture medium in steps (1)-(3) comprises a carbon source, a nitrogen source, inorganic salt ions, vitamins and trace elements; Alternatively, the fermentation medium in step (4) comprises a carbon source, a nitrogen source (including sodium glutamate), inorganic salt ions, vitamins and trace elements.

7. The method of claim 5, wherein: The final concentration of tocopherol added in the fermentation medium in step (4) is 1 g / L, and the final concentration of sodium glutamate added is 10 g / L.

8. The method of claim 5, wherein: The formula of the seed culture medium is: glucose 60 g / L, yeast extract 5 g / L, sodium glutamate 20 g / L, Na2SO4 10 g / L, CaCl2 0.5 g / L, KH2PO4 4 g / L, KCl 2 g / L, MgSO4·7H2O 5 g / L, vitamin B1 5 mg / L, vitamin B6 5 mg / L, vitamin B12 2 mg / L, trace elements, namely ZnSO4·7H2O 2 mg / L, FeCl3 10 mg / L, CuSO4 4 mg / L, MnCl2 8 mg / L, and water as a solvent; The formula of the fermentation medium is: glucose 80 g / L, yeast extract 5 g / L, tocopherol, sodium glutamate, (NH4)2SO4 6 g / L, Na2SO4 15 g / L, CaCl2 1 g / L, KCl 1.5 g / L, KH2PO4 4 g / L, MgSO4·7H2O 5 g / L, vitamin B1 5 mg / L, vitamin B6 5 mg / L, vitamin B12 2 mg / L, trace elements, namely ZnSO4·7H2O 2 mg / L, FeCl3 10 mg / L, CuSO4 4 mg / L, MnCl2 8 mg / L, and water as a solvent.

9. Use of the method according to any one of claims 1 to 8 for increasing the content of DHA in Schizochytrium sp.

10. A production method for increasing the content of DHA in a Schizochytrium sp. powder by the method according to any one of claims 1 to 8, characterized by: The method comprises the following steps: using Schizochytrium sp. to perform ARTP mutagenesis treatment to obtain a Schizochytrium sp. mutant strain with DHA%≥55%, and performing fermentation regulation to obtain a fermentation liquor, and then using a spray drying method to obtain Schizochytrium sp. algal powder with high DHA content; In the drying process, the inlet temperature is 180°C, and the outlet temperature is 80°C.

Images