Composite high-yield DHA and EPA schizochytrium limacinum and application thereof

Abstract

The invention discloses a schizochytrium limacinum for composite high yield of DHA and EPA and application of the schizochytrium limacinum. The schizochytrium sp. HG-EPA20 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on October 11, 2024, the preservation number is CGMCC No.41545, and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The schizochytrium limacinum can realize the combined high yield of DHA and EPA; after 228 hours of fermentation culture, the biomass finally obtained in a 3L fermentation tank can reach 224.7 g / L; the grease yield reaches 68.5 g / L; the ratio of the DHA is 38.62%; and the EPA accounts for 3.42% of the total weight.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to a Schizochytrium strain with high yields of DHA and EPA in combination and its application. Background Art

[0002] Polyunsaturated fatty acids (PUFAs) are essential for the human body and are divided into two types: omega-3 and omega-6 (Sijtsma, L., et al., Biotechnological production and applications of the ω-3 polyunsaturated fatty acid docosahexaenoic acid. Applied Microbiology and Biotechnology, 2004. 64(2): p. 146-153.). Docosahexaenoic acid (DHA, 22:6n-3) and eicosapentaenoic acid (EPA, 20:5n-3) belong to omega-3 long-chain polyunsaturated fatty acids. Moreover, the intake of DHA and EPA is beneficial for hypertension (Bercea, C.I., et al., Omega-3 polyunsaturated fatty acids and hypertension: a review of vasodilatory mechanisms of docosahexaenoic acid and eicosapentaenoic acid. British Journal of Pharmacology, 2021. 178(4): p. 860-877.) and the prevention of chronic kidney disease (Ong, K.L., et al., Association of omega 3 polyunsaturated fatty acids with incident chronic kidney disease: pooled analysis of 19 cohorts. Bmj, 2023.).In addition, supplementing DHA during pregnancy can enhance the immune function of infants (Byun, M.J., et al., Omega-3 polyunsaturated fatty acids modify glucose metabolism in THP-1 monocytes, 2024.) and promote brain and optic nerve development (Wang, Q., et al., The Effect of Supplementation of Long-Chain Polyunsaturated Fatty Acids During Lactation on Neurodevelopmental Outcomes of Preterm Infant From Infancy to School Age: A Systematic Review and Meta-analysis. Pediatric Neurology, 2016. 59:54-61.).

[0003] Currently, fish oil and microalgae are the main sources of DHA and EPA. However, due to strict fishing and catch quota regulations, production is restricted and the yield is low (Khan, I., et al., Omega-3 long-chain polyunsaturated fatty acids: Metabolism and health implications, Progress in Lipid Research, 2023, 92.), unable to meet market demand. The synthesis of polyunsaturated fatty acids by oil-producing microorganisms can achieve the green, environmentally friendly, and efficient production of polyunsaturated fatty acids, which is a new trend in sustainable development. Schizochytrium sp. is a single-celled marine fungus belonging to the Thraustochytrid family (Huang Y S, et al., γ-linolenic acid: recent advances in biotechnology and clinical applications. European Journal of Lipid Science and Technology, 2002, 104: 706-706). It has industrial potential due to its high biomass and DHA content and its advantage of being tolerant to agitation; because of its relatively fast growth rate, the fatty acids can account for up to 70% of the cell dry weight and 90% of them exist in the form of triglycerides, which are easily absorbed by the human body. It has been used for commercial production in the United States, and the Ministry of Health of China also approved the DHA produced by Schizochytrium sp. for infant food in 2012. In addition, Schizochytrium sp. can also produce many high-value-added products such as EPA (Brito C, et al., Corema album spp: Edible wild crowberries with a high content in minerals and organic acids. Food Chemistry, 2021, 345: 128732-128740.). Therefore, the large-scale production of DHA and EPA using Schizochytrium sp. has broad prospects.

[0004] In the related art, for example, in Chinese invention patent CN 114907988 B, through two-stage temperature-variable fermentation, after 144 hours of fermentation, the dry cell weight reached 98.9 g / L, 40.43 g / L of oil was obtained, the DHA yield was 22.2 g / L, and the proportion in total fatty acids was 55%. In Chinese invention patent application CN 118755584 A, through ultraviolet mutagenesis and selective culture screening, a mutant Schizochytrium sp. with high EPA yield was obtained. After 120 h of fermentation in a 5 L fermenter, the biomass could reach 76.48 g / L, the oil could reach 50.03 g / L, the average DHA content was 46.02%, and the average EPA content was 10.11%. Another example is Chinese invention patent CN109913513 B. After high-sugar domestication of Schizochytrium sp., fed-batch fermentation culture of the 20th-generation 150 g / L domesticated strain was carried out in a 3.5 L fermenter, and the biomass and total oil could reach 261.3 g / L and 94.6 g / L, which were increased by 105% and 15.5% compared with the original strain. Among them, the proportion of DHA in total oil was 38.68%, the yield was 34.89 g / L, the proportion of EPA was 1.91%, and the yield was 1.74 g / L.

[0005] However, it is difficult for the above-mentioned Schizochytrium sp. to achieve the combined and efficient synthesis of DHA and EPA, especially the low proportion of EPA in the oil. Summary of the Invention

[0006] To solve the above problems, the present invention provides a Schizochytrium sp. with high combined yield of DHA and EPA and its application, which can achieve the combined and efficient synthesis of DHA and EPA.

[0007] In the first aspect of the present invention, a Schizochytrium sp. with high combined yield of DHA and EPA is proposed. Schizochytrium sp. HG-EPA20 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on October 11, 2024, with the deposit number CGMCC No. 41545 and the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] According to a Schizochytrium sp. with high yield of DHA and EPA of the present invention, when the Schizochytrium sp. HG-EPA20 is inoculated into a fermenter and cultured for 228 h, the biomass can reach 224.7 g / L; the oil yield can reach 68.5 g / L; the DHA proportion reaches 38.62%; the EPA proportion reaches 3.42%. The production efficiency of DHA and EPA is significantly improved, and the strain quality is superior to the prior art.

[0009] Optionally, the Schizochytrium sp. HG-EPA20 is obtained through domestication.

[0010] In a second aspect, the present invention provides the use of the above-mentioned Schizochytrium sp. in the production of DHA and EPA. After activating the Schizochytrium sp. HG-EPA20, it can be transferred to a fermentation medium for cultivation.

[0011] Specifically, it includes the following steps:

[0012] (1) Strain activation

[0013] Take out the Schizochytrium sp. HG-EPA20 stored at -80°C from the glycerol tube, thaw it at room temperature, and inoculate it in a 100 mL conical flask in a laminar flow hood at an inoculation amount of 5% (v / v). Cultivate it at 28°C and 200 rpm for 36 h;

[0014] (2) Seed culture

[0015] Inoculate the activated strain into a new seed medium at an inoculation amount of 5% (v / v), and cultivate it at 28°C and 200 rpm for 36 h to obtain a secondary seed solution;

[0016] (3) Fermentation tank fermentation culture

[0017] Inoculate the secondary seed culture solution into a 3 L fermentation tank containing 2 L of fermentation medium by flame inoculation method at an inoculation amount of 10%. At the same time, dissolve 5 g / L of methionine, trace elements, calcium pantothenate, and vitamins into the dispensed glucose and add it. Set the rotation speed at 250 rpm, the aeration rate at 2 vvm, and control the pH at 6.5 with ammonia water and citric acid. During the fermentation process, supplement glucose by pulse feeding. The addition of the nitrogen source is achieved by adding ammonia water to control the pH. Add 100 mL of inorganic salt components at 72 h, and add 5 g / L of methionine again at 120 h of fermentation.

[0018] This application uses the strain after being domesticated under specific conditions, combines optimized fermentation culture conditions, and appropriately adds amino acids to the medium as key precursors and nutritional promoting factors for cell metabolism, effectively promoting the accumulation of EPA. The efficiency of the optimized strain in producing DHA and EPA is improved, and the stability of the culture process is enhanced, effectively solving the problems existing in the early patent solutions.

[0019] Optionally, the pulse feeding method is to set a peristaltic pump to run at 10 rpm, supplement 9 mL of solution per minute, and set the pump to run for 1 minute and stop for 35 minutes, that is, run 20 cycles every 12 h.

[0020] Optionally, the fermentation medium formulation is 120 g / L glucose, 10 g / L solid corn steep liquor, 10 g / L sodium glutamate, inorganic salt components, trace elements, 3.2 mg / L calcium pantothenate, 9.5 mg / L VB1, and 0.15 mg / L VB12; the trace elements, calcium pantothenate, VB1, and VB12 are all added after filtration and sterilization before inoculation.

[0021] Further, the trace element components are 10 mg / L FeSO4·7H2O, 0.34 g / L CaCl2·2H2O, 1.5 g / L KH2PO4, 3 mg / L MnCl2·4H2O, 3 mg / L ZnSO4·7H2O, 2.1 g / L MgSO4·7H2O, 2 mg / L NiSO4·6H2O, 6 mg / L CuSO4·5H2O, 0.04 mg / L CoCl2·6H2O, 0.04 mg / L Na2MoO4·2H2O, and the rest is water.

[0022] Further, the inorganic salt components are 12 g / L Na2SO4, 4.2 g / L MgSO4·7H2O, 0.5 g / L KCl, 1 g / L KH2PO4, 0.65 g / L K2SO4, 1 g / L (NH4)2SO4, 0.17 g / L CaCl2·2H2O, and the rest is water.

[0023] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. Description of the Drawings

[0024] Figure 1 It is a morphological diagram of the Schizochytrium sp. HG-EPA20 colony under an electron microscope in the embodiment of the present invention;

[0025] Figure 2 It is a morphological diagram of the Schizochytrium sp. HG-EPA20 strain on a solid plate in the embodiment of the present invention;

[0026] Figure 3 It is the relative value of TAG / PL during fermentation after adding methionine at 72 h of fermentation of Schizochytrium sp. HG-EPA20 in the embodiment of the present invention. Detailed Embodiments

[0027] The technical solution of the present invention will be described below through specific specific examples. It should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combined steps or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Moreover, unless otherwise specified, the numbers of each method step are only convenient tools for identifying each method step, rather than limiting the arrangement order of each method step or the scope in which the present invention can be implemented. The change or adjustment of their relative relationship, without substantial change in the technical content, should also be regarded as the scope in which the present invention can be implemented.

[0028] To better understand the above technical solution, the exemplary embodiments of the present invention will be described in more detail below. Although the exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0029] The test materials used in the present invention are all ordinary commercially available products and can be purchased in the market.

[0030] Determination of biomass: Take 1 mL of the bacterial solution into a pre-weighed centrifuge tube, centrifuge at 9000 rpm for 5 min, discard the supernatant, wash with deionized water, centrifuge at 9000 rpm for 5 min, discard the supernatant, repeat the washing several times, and then freeze-dry the bacterial cells for 24 h and weigh. The difference between the final weight of the centrifuge tube and the initial weight of the centrifuge tube divided by the volume is the biomass.

[0031] Extraction of DHA and EPA in the fermentation broth: Accurately pipette 4 mL of the fermentation broth into a 15 mL centrifuge tube according to the fermentation volume, and then add an equal volume of concentrated hydrochloric acid and mix well. Place the centrifuge tube in a magnetic stirring water bath at 65 °C for about 1 h, and add a rotor at the bottom of the centrifuge tube before the water bath to ensure complete digestion. After cooling to room temperature, add 5 mL of n-hexane, mix well, and centrifuge at 5800 rpm for 1 min in a centrifuge. Pipette the organic phase into a pre-weighed 50 mL centrifuge tube, and continue to use n-hexane. Repeat this operation about 3 - 5 times until the organic phase becomes colorless. Then, remove the organic phase from the 50 mL centrifuge tube containing the organic phase by nitrogen blowing, and place it in an oven at 65 °C to dry for about 1 h. Take out the centrifuge tube and wait until it cools to room temperature and weigh. The difference between the weight of the centrifuge tube after drying and the initial weight is the weight of the oil.

[0032] After weighing, add 5 mL of 0.5 M KOH-CH3OH solution to the centrifuge tube containing oil and place it in a water bath at 65 °C for about 20 min until the oil droplets are completely dissolved. Then immediately add 5 mL of 30% boron trifluoride etherate solution to it and continue to water bath for about 30 min to fully convert fatty acids into fatty acid methyl esters with lower boiling points. After taking it out and waiting for it to cool to room temperature, add 5 mL of chromatographic grade n-hexane to it and shake well. Continue to add about 3 mL of saturated brine, shake well and let it stand for layering. Then add anhydrous sodium sulfate for dehydration, suck the supernatant with a 2 mL syringe, filter impurities through a 0.22 μm organic filter membrane and inject it into a brown chromatographic vial for gas chromatography analysis.

[0033] Determination of DHA and EPA contents in the fermentation broth by gas chromatography: Use a gas chromatograph equipped with an Agilent HP-88 chromatographic column to analyze each fatty acid component in the sample. The injection volume is set to 1 μL, and it is washed 3 times before and after injection. The split ratio is set to 5:1, and the flow rate of nitrogen as the carrier gas is set to 20 mL / min. Temperature programming: The initial temperature is 140 °C, maintained for 5 min, then heated at a rate of 4 °C per minute to 210 °C and held for 31.5 min. Then heated at a rate of 10 °C per minute to 230 °C and held for 3 min. Finally, heated at a rate of 10 °C per minute to 240 °C and held for 10 min. The control mode adopts constant pressure, the column pressure is set to 25 psi, and the detector temperature is set to 260 °C.

[0034] The seed medium is 150 g / L glucose, 10 g / L yeast extract and inorganic salt components, the pH is adjusted to 6.5, and it is sterilized at 121 °C for 20 min. The inorganic salt components are: 12 g / L Na2SO4, 4.2 g / L MgSO4·7H2O, 0.5 g / L KCl, 1 g / L KH2PO4, 0.65 g / L K2SO4, 1 g / L (NH4)2SO4, 0.17 g / L CaCl2·2H2O, and the rest is water.

[0035] The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0036] Example 1

[0037] (1) Inoculate Schizochytrium sp. ATCC-1381 (purchased from the American Type Culture Collection (ATCC)) stored in a glycerol tube into the seed medium at an inoculation amount of 5% (v / v) to obtain a primary seed solution. The culture conditions are 28 °C, 220 rpm, and cultured for 36 h.

[0038] (2) Inoculate the above primary seeds into the secondary seed medium at an inoculation amount of 5% (v / v) to obtain the secondary seed liquid. The culture conditions are 28 °C and 220 rpm for 36 h.

[0039] (3) Prepare six different glucose concentration gradients in conical flasks. Glucose is separated from the sugar-free seed medium and sterilized at 115 °C under high-pressure steam for 15 min. Then, add glucose solution to the sugar-free seed medium in a laminar flow hood so that the final glucose concentrations are 50 g / L, 100 g / L, 150 g / L, and 180 g / L, respectively. Inoculate 5% (v / v) of the bacterial solution and place it in a shaker at 200 rpm and 28 °C for culture. One generation is 48 h, and continuous subculture is carried out. The sugar concentration gradient is increased every 10 generations.

[0040] Compared with the original strain, the biomass of the strain slightly decreases at 10 generations, but significantly increases at a glucose concentration of 150 g / L. Finally, a genetically stable domesticated strain, namely Schizochytrium sp. HG-EPA20, is obtained.

[0041] Schizochytrium sp. HG-EPA20 was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on October 11, 2024, with the deposit number CGMCC No. 41545 and the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0042] Schizochytrium sp. HG-EPA20 is a marine heterotrophic microorganism with single-celled and spherical cells. Its morphology under the microscope is as Figure 1 shown, and the colony morphology on the solid plate is as Figure 2 shown. The overall colony shows an opaque and light yellow spherical shape.

[0043] Example 2 Optimization of the nitrogen source in the fermentation medium

[0044] This example provides an application of Schizochytrium sp. in the preparation of DHA and EPA, and the strain used is Schizochytrium sp. HG-EPA20 in Example 1.

[0045] The specific steps of this example are as follows:

[0046] (1) Strain activation

[0047] The Schizochytrium sp. HG-EPA20 stored at -80°C was taken out from the glycerol tube. After thawing at room temperature, it was inoculated in a 100 mL conical flask in a laminar flow hood at an inoculation amount of 5% (v / v), and cultured at 28°C and 200 rpm for 36 h.

[0048] (2) Seed culture

[0049] The activated strain was inoculated into a new seed medium at an inoculation amount of 5% (v / v), and cultured at 28°C and 200 rpm for 36 h to obtain a secondary seed solution.

[0050] (3) Fermentation culture

[0051] Four nitrogen sources, namely sodium glutamate, solid corn steep liquor, yeast extract, and peptone, were selected for fermentation culture. The carbon source was 120 g / L glucose. These four nitrogen sources were combined into 5 different fermentation media, as shown in Table 1.

[0052] The fermentation results are shown in Table 2. Under the condition of the same nitrogen source concentration (10 g / L), nitrogen source A had the largest biomass and the largest oil content. However, in terms of the fatty acid composition, it can be seen that the proportion of EPA reached the maximum of 0.74% in the nitrogen source B medium, and at the same time, it also had the largest EPA yield of 178.3 mg / L. Considering the proportion and yield of EPA, sodium glutamate and solid corn steep liquor were selected as the nitrogen sources for the fermentation medium in the following experiments.

[0053] Table 1 Different nitrogen source combinations

[0054]

[0055]

[0056] Table 2 Effects of different nitrogen sources on the cell growth and oil synthesis of Schizochytrium sp. HG-EPA20

[0057]

[0058] Example 3 Effect of initial C / N of the medium on the synthesis of EPA by Schizochytrium sp. HG-EPA20

[0059] This example provides an application of Schizochytrium sp. in the preparation of DHA and EPA. The strain used is Schizochytrium sp. HG-EPA20 in Example 1, and the nitrogen sources used are sodium glutamate and solid corn steep liquor in Example 2.

[0060] The specific steps of this example are as follows:

[0061] (1) Strain activation

[0062] Take Schizochytrium sp. HG-EPA20 stored at -80°C out of the glycerol tube. After thawing at room temperature, inoculate it into a 100 mL conical flask in a laminar flow hood with an inoculum size of 5% (v / v), and culture it at 28°C and 200 rpm for 36 h.

[0063] (2) Seed culture

[0064] Inoculate the activated strain into a new seed medium with an inoculum size of 5% (v / v), and culture it at 28°C and 200 rpm for 36 h to obtain the secondary seed liquid.

[0065] (3) Fermentation culture

[0066] In this example, glucose concentrations of 60, 90, and 120 g / L were respectively selected as the carbon source, and the concentrations of sodium glutamate and solid corn steep liquor were the same, which were 5, 10, 20, and 30 g / L respectively as the nitrogen source for fermentation culture. The ratio of glucose concentration / (solid corn steep liquor concentration + sodium glutamate concentration) was used as C / N for research and analysis. The results after 144 h of fermentation are shown in Table 3. When C / N was 6 (glucose 120 g / L + glutamic acid 10 g / L + solid corn steep liquor 10 g / L), the EPA proportion was 1.08%, and the EPA yield reached 255.7 mg / L (shake flask level), which was 38.4% higher than that of the control group.

[0067] Table 3 Effects of different initial C / N on the cell growth and lipid synthesis of Schizochytrium sp. HG-EPA20

[0068]

[0069] Example 4 Fermentation tank culture of Schizochytrium sp. HG-EPA20 for combined production of DHA and EPA

[0070] (1) Strain activation

[0071] Take Schizochytrium sp. HG-EPA20 stored at -80°C out of the glycerol tube. After thawing at room temperature, inoculate it into a 100 mL conical flask in a laminar flow hood with an inoculum size of 5% (v / v), and culture it at 28°C and 200 rpm for 36 h.

[0072] (2) Seed culture

[0073] The activated strain was inoculated into a new seed medium at an inoculum size of 5% (v / v) and cultured at 28 °C and 200 rpm for 36 h to obtain a secondary seed solution.

[0074] (3) Fermentation culture in a fermenter

[0075] The secondary seed culture solution was inoculated into a 3 L fermenter containing 2 L of fermentation medium by the flame inoculation method at an inoculum size of 10%. At the same time, 5 g / L of methionine, trace elements, calcium pantothenate, and vitamins were dissolved in the dispensed glucose and added. The rotation speed was set at 250 rpm, the aeration rate was 2 vvm, and the pH was controlled at 6.5 with ammonia water and citric acid. During the fermentation process, glucose was supplemented by pulse feeding, and the addition of nitrogen source was achieved by adding ammonia water to control the pH. 100 mL of inorganic salt components were added at 72 h, and 5 g / L of methionine was added again at 120 h of fermentation. The fermentation medium formula was 120 g / L glucose, 10 g / L solid corn steep liquor, 10 g / L sodium glutamate, inorganic salt components, trace elements, 3.2 mg / L calcium pantothenate, 9.5 mg / L VB1, and 0.15 mg / L VB12. Trace elements, calcium pantothenate, VB1, and VB12 were all added after filtration and sterilization before inoculation. The trace element components were 10 mg / L FeSO4·7H2O, 0.34 g / L CaCl2·2H2O, 1.5 g / L KH2PO4, 3 mg / L MnCl2·4H2O, 3 mg / L ZnSO4·7H2O, 2.1 g / L MgSO4·7H2O, 2 mg / L NiSO4·6H2O, 6 mg / L CuSO4·5H2O, 0.04 mg / L CoCl2·6H2O, 0.04 mg / L Na2MoO4·2H2O, and the rest was water. The inorganic salt components were 12 g / L Na2SO4, 4.2 g / L MgSO4·7H2O, 0.5 g / L KCl, 1 g / L KH2PO4, 0.65 g / L K2SO4, 1 g / L (NH4)2SO4, 0.17 g / L CaCl2·2H2O, and the rest was water.

[0076] As shown in Table 4, after culturing for 228 h, the biomass could reach 224.7 g / L; the oil yield could reach 68.5 g / L; the DHA proportion was 38.62%; the EPA proportion reached 3.42%.

[0077] Table 4 Fatty acid composition of Schizochytrium sp. HG-EPA20 in a 3 L fermenter

[0078]

[0079] To determine whether the addition of methionine affects the ratio of triglycerides to phospholipids and thus the content of EPA, the relative content of the two was expressed as RV = TAG / PL. As Figure 3 shown, from 96 h to 144 h, the RV value of the original strain became larger and larger. This indicates that over time, the proportion of triglycerides in the total oil became larger and larger. In contrast, after the addition of methionine, the RV decreased compared to the control group, and the RV value decreased slowly over time. At 144 h, the RV values of the control group and the addition group were 34.37 and 5.43, respectively, a decrease of approximately 84.2%. This indicates that the addition of Met can increase the proportion of phospholipids in the total oil.

[0080] The determination results of fatty acids are shown in Table 5. Compared with the control group, the fatty acid composition in phospholipids differed greatly before and after the addition of Met. At 144 h, the saturated fatty acids C16:0 and C18:0 decreased from 59.20% and 12.92% to 51.65% and 10.77%, respectively, with a decrease of 12.7% and 16.6%. Among the unsaturated fatty acids, EPA, DPA, and DHA were 4.72%, 6.77%, and 24.30%, respectively, with an increase of 68.6%, 23.1%, and 31.2%.

[0081] Table 5 Fatty acid proportion in phospholipids of Schizochytrium sp. HG-EPA20 after methionine addition at 72 h

[0082]

[0083] In summary, according to the embodiments of the present invention, by adding methionine, the content of cell phospholipids can be increased, and the fatty acid composition in phospholipids can be changed. The proportion of polyunsaturated fatty acids contained in phospholipids has been significantly increased, especially the proportion of EPA. The finally obtained Schizochytrium sp. HG-EPA20 maintains a relatively high level of 38.62% in the DHA proportion through fermentation culture, and the EPA proportion can also be increased to 3.42%. It has significant advantages over the existing Schizochytrium sp.

[0084] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples", etc., mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms should not be understood as necessarily referring to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine the different embodiments or examples described in this specification.

[0085] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A Schizochytrium strain with high yields of both DHA and EPA, characterized in that, Schizochytrium sp. HG-EPA20 was deposited at the China General Microbiological Culture Collection Center on October 11, 2024. The deposit number is CGMCC No. 41545, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. The Schizochytrium for producing high yields of DHA and EPA as claimed in claim 1, characterized in that, The Schizochytrium sp. HG-EPA20 was obtained through domestication.

3. Use of the Schizochytrium sp. according to claim 1 or 2 in the production of DHA and EPA, characterized in that, After activating the Schizochytrium sp. HG-EPA20, transfer it to a fermentation medium for cultivation.

4. The application according to claim 3, characterized in that, It includes the following steps: (1) Strain activation Take out Schizochytrium sp. HG-EPA20 stored at -80°C from the glycerol tube, thaw it at room temperature, and inoculate it in a 100 mL conical flask in a laminar flow hood at an inoculation amount of 5% (v / v). Cultivate it at 28°C and 200 rpm for 36 h. (2) Seed culture Inoculate the activated strain into a new seed medium at an inoculation amount of 5% (v / v), and cultivate it at 28°C and 200 rpm for 36 h to obtain a secondary seed liquid. (3) Fermentation tank fermentation culture Inoculate the secondary seed culture broth into a 3 L fermenter containing 2 L of fermentation medium by flame inoculation at an inoculation amount of 10%. At the same time, dissolve 5 g / L of methionine, trace elements, calcium pantothenate, and vitamins into the dispensed glucose and add it. Set the rotation speed at 250 rpm, the aeration rate at 2 vvm, and control the pH at 6.5 with ammonia water and citric acid. During the fermentation process, supplement glucose by pulse feeding. The addition of nitrogen source is achieved by adding ammonia water to control the pH. Add 100 mL of inorganic salt components at 72 h and add 5 g / L of methionine again at 120 h of fermentation.

5. The application according to claim 4, characterized in that, The pulse feeding method is to set a peristaltic pump to run at 10 rpm, supplement 9 mL of solution per minute, and set the pump to run for 1 minute and stop for 35 minutes.

6. The application according to claim 4, characterized in that, The fermentation medium formula is 120 g / L glucose, 10 g / L solid corn steep liquor, 10 g / L sodium glutamate, inorganic salt components, trace elements, 3.2 mg / L calcium pantothenate, 9.5 mg / L VB1, and 0.15 mg / L VB12. Trace elements, calcium pantothenate, VB1, and VB12 are all added after filtration sterilization before inoculation.

7. The application according to claim 6, characterized in that, The inorganic salt components are 12 g / L Na2SO4, 4.2 g / L MgSO4·7H2O, 0.5 g / L KCl, 1 g / L KH2PO4, 0.65 g / L K2SO4, 1 g / L (NH4)2SO4, 0.17 g / L CaCl2·2H2O, and the rest is water.

8. The application according to claim 6, characterized in that, The trace element components are 10 mg / L FeSO4·7H2O, 0.34 g / L CaCl2·2H2O, 1.5 g / L KH2PO4, 3 mg / L MnCl2·4H2O, 3 mg / L ZnSO4·7H2O, 2.1 g / L MgSO4·7H2O, 2 mg / L NiSO4·6H2O, 6 mg / L CuSO4·5H2O, 0.04 mg / L CoCl2·6H2O, 0.04 mg / L Na2MoO4·2H2O, and the rest is water.

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