Schizochytrium sp. with high eicosapentaenoic acid production and application thereof

Abstract

This invention belongs to the fields of biomedicine, marine microbiology, and microbial fermentation, and discloses a high-EPA-producing Schizochytrium fungus and its applications. The Schizochytrium fungus KDW-306 provided by this invention has the accession number CGMCC No. 42323. This Schizochytrium fungus KDW-306 exhibits high biomass, high oil yield, high EPA titer, and high DHA titer, excellent EPA and DHA synthesis capabilities, and excellent production stability and genetic stability through generations, thus well meeting the needs of large-scale industrial production of EPA and DHA.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine, marine microbiology, and microbial fermentation, and particularly relates to a high-EPA-producing Schizochytrium and its applications. Background Technology

[0002] Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are two important ω-3 long-chain polyunsaturated fatty acids (ω-3 LC-PUFAs). Numerous studies have shown that EPA and DHA can promote brain development and have physiological effects in preventing and treating cardiovascular diseases, inflammation, hypertension, and cancer, playing a crucial role in the prevention and treatment of human diseases.

[0003] Extraction is the traditional method for producing ω-3 LC-PUFA, specifically referring to the extraction of ω-3 LC-PUFA from deep-sea fish oil. However, this method is cumbersome, has a persistent odor, and is affected by factors such as severe marine pollution and overfishing, and has been gradually replaced by microbial fermentation. Various microorganisms in the ocean, including microalgae, marine bacteria, and fungi, have the ability to produce ω-3 LC-PUFA; among them, Schizochytrium (… Schizochytrium sp Schizochytrium is a fungus belonging to the family Schizochytriaceae. It is characterized by simple cultivation conditions, rapid growth, and a short fermentation cycle. The fungus is rich in lipids, with total lipids accounting for 40%–70% of its dry weight. Of this, DHA accounts for approximately 20%–60%, and it also contains a certain amount of EPA. It is currently the most widely used strain for industrial DHA production and a potential producer of EPA. However, most wild-type Schizochytrium produce extremely low EPA yields. Therefore, methods such as mutagenesis, stress, and genetic modification are typically needed to enhance its EPA synthesis capacity, enabling it to better adapt to the demands of large-scale industrial EPA production. Currently, research on screening wild-type strains to obtain high-EPA-producing strains is limited, restricting the application of Schizochytrium in EPA production.

[0004] Chinese patent application CN118755584A discloses a high-EPA-producing Schizochytrium and its applications. This high-yielding Schizochytrium was obtained through UV mutagenesis and selective culture screening. Under normal nutrient conditions, it can synthesize large amounts of EPA and DHA, with EPA content reaching 10.11% and DHA content reaching 46.02%. However, it suffers from reduced biomass, resulting in an oil yield of only 50.03 g / L, an EPA titer of 5.06 g / L, and a DHA titer of 23.03 g / L. Chinese patent application CN119307531A introduces elongated desaturase genes from *Schizochytrium* and *Fusarium* into Schizochytrium, obtaining a high-EPA-producing engineered strain. This engineered strain achieves an oil content of 74.25 g / L and an EPA content of 7.8 g / L. However, the potential safety risks associated with mutagenesis and the introduction of exogenous genes reduce consumer acceptance of the product.

[0005] Therefore, obtaining a Schizochytrium strain with high biosafety and excellent EPA and DHA production capacity and stability is of great significance for realizing large-scale industrial production of EPA. Summary of the Invention

[0006] The primary objective of this invention is to provide a Schizochytrium fungus that produces high levels of EPA. Schizochytrium sp. KDW-306. This Schizochytrium KDW-306 was obtained by screening and culturing biological samples obtained through pine pollen fishing. It has excellent EPA and DHA production capacity and stability, and has excellent application prospects in realizing low-cost, large-scale industrial production of EPA and DHA.

[0007] A second objective of this invention is to provide a microbial agent.

[0008] A third objective of this invention is to provide the application of the above-mentioned Schizochytrium KDW-306 or its agent in the preparation of EPA and / or DHA.

[0009] The fourth objective of this invention is to provide a method for preparing EPA and / or DHA.

[0010] Specifically, the accession number of the Schizochytrium KDW-306 provided by this invention is CGMCC No.42323.

[0011] Furthermore, the selection method for the Schizochytrium KDW-306 includes: taking the original strain obtained by pine pollen fishing and inoculating it on a selective solid medium for screening culture to obtain a single colony; taking the single colony for fermentation culture to obtain a fermentation broth; and selecting the Schizochytrium KDW-306 according to the biomass and EPA yield of the fermentation broth; wherein the selective solid medium includes 1 mg / L to 10 mg / L of terbinafine.

[0012] Furthermore, the selective solid culture medium comprises 3 mg / L to 7 mg / L terbinafine, 1 g / L to 10 g / L glucose, 1 g / L to 5 g / L yeast extract, 1 g / L to 10 g / L peptone, 10 g / L to 20 g / L sea salt, and 15 g / L to 25 g / L agar, and the pH value of the selective solid culture medium is 6 to 7.

[0013] Furthermore, the screening culture is conducted at a temperature of 25℃~30℃ for a time of 36h~60h.

[0014] Furthermore, the fermentation culture temperature is 25℃~30℃, the rotation speed is 180r / min~220r / min, and the time is 36h~144h.

[0015] The microbial agent provided by this invention includes the above-mentioned Schizochytrium KDW-306.

[0016] Furthermore, the microbial agent includes one or more of the following: live cells, dead cells, and spores of Schizochytrium KDW-306.

[0017] This invention provides the application of the above-mentioned Schizochytrium KDW-306 or its agent in the preparation of EPA and / or DHA.

[0018] The method for preparing EPA and / or DHA provided by the present invention includes: S1, taking the above-mentioned Schizochytrium KDW-306 or bacterial agent and performing activation culture and seed expansion culture in sequence to obtain seed culture solution; S2, taking the seed culture solution and inoculating it into fermentation medium for fermentation culture to obtain fermentation culture solution; wherein, the fermentation culture solution contains EPA and / or DHA.

[0019] Further, in step S1, the solid activation culture medium used in the activation culture includes 1 g / L to 10 g / L of glucose, 1 g / L to 10 g / L of peptone, 1 g / L to 5 g / L of yeast extract, 10 g / L to 20 g / L of sea salt and 15 g / L to 25 g / L of agar, and the pH value of the solid activation culture medium is 6 to 7.

[0020] Furthermore, in step S1, the activation culture temperature is 25℃~30℃, and the time is 36h~60h.

[0021] Further, in step S1, the seed culture medium used for the seed expansion culture includes 10 g / L to 20 g / L of glucose, 1 g / L to 5 g / L of peptone, 5 g / L to 15 g / L of yeast extract and 20 g / L to 40 g / L of sea salt, and the pH value of the seed culture medium is 6 to 7. Further, in step S1, the temperature for seed expansion culture is 25℃~30℃, the rotation speed is 180r / min~220r / min, and the time is 36h~60h.

[0022] Further, in step S2, the fermentation medium comprises 80 g / L to 120 g / L glucose, 5 g / L to 20 g / L yeast extract, 5 g / L to 10 g / L monosodium glutamate, 6 g / L to 12 g / L sodium sulfate, 2 g / L to 4 g / L magnesium sulfate, 4 g / L to 8 g / L ammonium sulfate, 0.5 g / L to 2 g / L potassium chloride, 0.1 g / L to 0.2 g / L calcium chloride, 0.5 g / L to 1 g / L potassium sulfate, 1 g / L to 2 g / L potassium dihydrogen phosphate, 0.02 mg / L to 0.05 mg / L sodium molybdate dihydrate, and 2 mg / L... The fermentation medium contains 5 mg / L manganese chloride tetrahydrate, 0.02 mg / L to 0.08 mg / L cobalt chloride hexahydrate, 1 mg / L to 5 mg / L copper sulfate pentahydrate, 1 mg / L to 3 mg / L nickel sulfate hexahydrate, 1 mg / L to 5 mg / L zinc sulfate heptahydrate, 5 mg / L to 15 mg / L ferric sulfate heptahydrate, 2 mg / L to 5 mg / L calcium pantothenate, 5 mg / L to 20 mg / L vitamin B1, 4 mg / L to 10 mg / L vitamin B6, and 0.1 mg / L to 0.5 mg / L vitamin B12, and the pH of the fermentation medium is 6 to 7.

[0023] Further, in step S2, the fermentation culture is a batch culture, and the temperature of the batch culture is 25℃~30℃, the rotation speed is 180r / min~220r / min, and the time is 36h~144h.

[0024] Furthermore, the fermentation culture is a fed-batch fermentation culture, which includes adjusting the glucose concentration of the culture medium to 10 g / L to 15 g / L during the culture process; the temperature of the fed-batch fermentation culture is 25℃ to 30℃, the pH value is 6 to 7, the DO value is 25% to 35%, the rotation speed is 300 r / min to 500 r / min, the aeration rate is 1 vvm to 3 vvm, and the time is 60 h to 240 h.

[0025] Biological Preservation: The Schizochytrium provided by this invention ( Schizochytrium sp. KDW-306, deposited on November 18, 2025, with accession number CGMCC No.42323, deposited at the China General Microbiological Culture Collection Center, located at No.3, No.1 Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0026] Figure 1 This is a micrograph of the cell of Schizochytrium KDW-306 provided in Example 1 of this invention.

[0027] Figure 2 This is a diagram showing the experimental results of the passage stability test of Schizochytrium KDW-306 provided in Example 3 of this invention. Detailed Implementation

[0028] This invention provides a high-EPA-producing Schizochytrium fungus ( Schizochytrium sp. KDW-306, with accession number CGMCC No. 42323 and accession date November 18, 2025, was obtained from decaying leaves collected from the mangrove forest in Haicang Bay, Haicang District, Xiamen City. Oil-producing and / or EPA-producing strains were captured and enriched using a pine pollen fishing method to obtain biological material for subsequent screening and culture. Based on the oil, EPA, and DHA production capabilities of each strain in the biological material, original strains with good potential for EPA and DHA production were screened. Finally, the original strains were screened and cultured using terbinafine, ultimately yielding a Schizochytrium strain with high biomass, oil yield, EPA titer, and DHA titer. This Schizochytrium strain exhibits excellent EPA and DHA synthesis capabilities, high production stability, and high genetic stability, effectively meeting the needs of large-scale industrial production of EPA and DHA.

[0029] In this invention, the selection method of the Schizochytrium KDW-306 specifically includes: taking the original strain obtained by pine pollen fishing and inoculating it on a selective solid culture medium for screening culture to obtain a single colony; taking the single colony for fermentation culture to obtain a fermentation broth; and selecting the Schizochytrium KDW-306 according to the biomass and EPA yield of the fermentation broth.

[0030] In this invention, the introduction of terbinafine during the selection and cultivation process can inhibit the MVA pathway of Schizochytrium, causing more acetyl-CoA and NADPH to flow to the unsaturated fatty acid synthesis pathway, thereby establishing screening pressure to eliminate strains sensitive to terbinafine and enrich strains containing the corresponding metabolic compensation pathway. This achieves a high hit rate in screening high-EPA-producing Schizochytrium, significantly reducing screening costs and shortening the breeding cycle. It has excellent application prospects in achieving high-throughput improvement of EPA industrial strains.

[0031] In this invention, the pine pollen fishing method refers to a screening technique that uses pine pollen to separate specific microorganisms from natural water bodies. This is a commonly used technique in the prior art. Those skilled in the art can make adaptive selections and adjustments according to actual needs. This invention does not impose any particular limitations on it.

[0032] In this invention, the selective solid culture medium specifically refers to a functional culture medium prepared by adding terbinafine to a Schizochytrium solid culture medium. The Schizochytrium solid culture medium is a conventionally used technique for Schizochytrium and can be any of the existing options; this invention does not impose any particular limitation. More specifically, the concentration of terbinafine in the selective solid culture medium is 1 mg / L to 10 mg / L, specifically 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 8 mg / L, 10 mg / L, or any value between them.

[0033] In some specific embodiments, the selective solid culture medium preferably includes: terbinafine at a concentration of 3 mg / L to 7 mg / L, specifically 3 mg / L, 3.5 mg / L, 4 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, 7 mg / L, or any value between them; glucose at a concentration of 1 g / L to 10 g / L, specifically 1 g / L, 2.5 g / L, 5 g / L, 8 g / L, 10 g / L, or any value between them; yeast extract at a concentration of 1 g / L to 5 g / L, specifically 1 g / L, 1.2 g / L, 2 g / L, 2.5 g / L, 3 g / L, 4 g / L, 5 g / L, or any value between them; and a concentration of 1 g / L... The selective solid culture medium contains peptone at a concentration of ~10 g / L, specifically 1 g / L, 2 g / L, 3 g / L, 5 g / L, 8 g / L, 10 g / L, or any value between them; sea salt crystals at a concentration of 10 g / L to 20 g / L, specifically 10 g / L, 12 g / L, 14 g / L, 15 g / L, 18 g / L, 20 g / L, or any value between them; and agar at a concentration of 15 g / L to 25 g / L, specifically 15 g / L, 18 g / L, 20 g / L, 23 g / L, 25 g / L, or any value between them; and the pH value of the selective solid culture medium is preferably 6 to 7, specifically 6, 6.2, 6.3, 6.5, 6.8, 7, or any value between them.

[0034] In this invention, the screening culture refers to the process of using terbinafine to achieve targeted enrichment of strains with metabolic compensation pathways in biological samples. The key to achieving enrichment lies in the introduction of terbinafine into the culture system, while other reagents and conditions can be various existing options, and this invention does not impose any particular limitations on them.

[0035] In some specific embodiments, the screening culture conditions specifically include a temperature preferably of 25℃~30℃, specifically 25℃, 26℃, 27℃, 28℃, 29℃, 30℃ or any value between them; and a time preferably of 36h~60h, specifically 36h, 38h, 40h, 48h, 50h, 54h, 58h, 60h or any value between them.

[0036] In this invention, the fermentation culture refers to the process of synthesizing, transforming and accumulating EPA using the metabolic activities of strains obtained through screening and culture. Various existing options are available, and this invention does not impose any particular limitation on them.

[0037] In some specific embodiments, the fermentation culture conditions specifically include a temperature preferably of 25℃~30℃, specifically 25℃, 26℃, 27.5℃, 28℃, 29℃, 30℃ or any value between them; a rotation speed preferably of 180r / min~220r / min, specifically 180r / min, 195r / min, 200r / min, 210r / min, 220r / min or any value between them; and a time preferably of 36h~144h, specifically 36h, 48h, 60h, 72h, 120h, 144h or any value between them.

[0038] In this invention, the method for testing and obtaining the biomass and EPA yield of the fermentation culture can be any of the existing options, limited to those that can obtain the biomass and EPA yield of the fermentation culture, and this invention does not impose any particular limitation.

[0039] The fungal agent provided by this invention includes the aforementioned Schizochytrium KDW-306. More specifically, the fungal agent contains one or more of the following: live cells, dead cells, and spores of the Schizochytrium KDW-306.

[0040] In this invention, the dosage form of the microbial agent can be any of the existing options, specifically including, but not limited to, liquid and / or solid microbial agents. More specifically, depending on the intended use, the microbial agent can be formulated into different dosage forms by adding appropriate excipients and other components. The added excipients are a conventional technique in the art and can be of various types; this invention does not impose any particular limitation on them.

[0041] This invention provides the application of the above-mentioned Schizochytrium KDW-306 or its agent in the preparation of EPA and / or DHA.

[0042] This invention provides a method for preparing EPA and / or DHA. The method specifically includes: S1, sequentially activating and seed culture of the above-mentioned Schizochytrium KDW-306 or its inoculum to obtain a seed culture solution; S2, inoculating the seed culture solution into a fermentation medium for fermentation culture to obtain a fermentation broth.

[0043] In this invention, step S1, the activation culture specifically refers to the process of activating Schizochytrium KDW-306 using a solid activation culture medium to restore its metabolic activity and proliferation capacity; the solid activation culture medium can be any of the existing options, and this invention does not impose any particular limitation on it.

[0044] In some specific embodiments, in step S1, the solid activation culture medium specifically includes: glucose at a concentration preferably from 1 g / L to 10 g / L, specifically 1 g / L, 3 g / L, 5 g / L, 8 g / L, 10 g / L, or any value between them; peptone at a concentration preferably from 1 g / L to 10 g / L, specifically 1 g / L, 2.5 g / L, 5 g / L, 7.5 g / L, 10 g / L, or any value between them; and yeast extract at a concentration preferably from 1 g / L to 5 g / L, specifically 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, or any value between them. L or any value therebetween; the concentration of sea salt crystals is preferably 10 g / L to 20 g / L, specifically 10 g / L, 12 g / L, 14 g / L, 16 g / L, 18 g / L, 20 g / L or any value therebetween; and the concentration of agar is preferably 15 g / L to 25 g / L, specifically 15 g / L, 18 g / L, 20 g / L, 23 g / L, 25 g / L or any value therebetween; and the pH value of the solid activated culture medium is preferably 6 to 7, specifically 6, 6.1, 6.3, 6.5, 6.8, 7 or any value therebetween.

[0045] In some specific embodiments, in step S1, the activation culture conditions include a temperature preferably of 25℃~30℃, specifically 25℃, 26℃, 27℃, 29℃, 30℃ or any value between them; and a time preferably of 36h~60h, specifically 36h, 38h, 40h, 45h, 50h, 60h or any value between them.

[0046] In this invention, in step S1, the seed expansion culture specifically refers to the process of using a seed culture medium to provide nutrition and a growth environment for Schizochytrium KDW-306, so as to promote rapid cell proliferation, maintain high activity and stability, and thus obtain a seed liquid that meets the fermentation requirements; the seed culture medium can be any of the existing options, and this invention does not impose any particular limitation on it.

[0047] In some specific embodiments, in step S1, the seed culture medium specifically includes: glucose at a concentration preferably of 10 g / L to 20 g / L, specifically 10 g / L, 12 g / L, 15 g / L, 18 g / L, 20 g / L, or any value between them; peptone at a concentration preferably of 1 g / L to 5 g / L, specifically 1 g / L, 1.5 g / L, 2 g / L, 2.4 g / L, 2.8 g / L, 3 g / L, 4 g / L, 5 g / L, or any value between them; and yeast extract at a concentration preferably of 5 g / L to 15 g / L, specifically 5 g... / L, 8g / L, 10g / L, 12g / L, 15g / L or any value between them; and, the concentration of sea salt crystals is preferably 20g / L to 40g / L, specifically 20g / L, 23g / L, 24g / L, 26g / L, 28g / L, 30g / L, 32g / L, 35g / L, 38g / L, 40g / L or any value between them; and the pH value of the seed culture medium is preferably 6 to 7, specifically 6, 6.1, 6.2, 6.4, 6.48, 6.5, 6.52, 6.7, 6.8, 7 or any value between them.

[0048] In some specific embodiments, in step S1, the conditions for seed expansion culture specifically include a temperature preferably of 25℃~30℃, specifically 25℃, 26℃, 28℃, 29℃, 30℃ or any value between them; a rotation speed preferably of 180r / min~220r / min, specifically 180r / min, 185r / min, 190r / min, 200r / min, 210r / min, 220r / min or any value between them; and a time preferably of 36h~60h, specifically 36h, 38h, 40h, 45h, 50h, 60h or any value between them.

[0049] In this invention, in step S2, the fermentation medium refers to a special culture medium that provides a unique nutritional and metabolic environment for Schizochytrium KDW-306 during the fermentation culture stage, so as to efficiently synthesize and accumulate EPA and DHA. Various existing options can be used, and this invention does not impose any particular limitation on it.

[0050] In some specific embodiments, in step S2, the fermentation culture medium specifically includes: glucose with a concentration preferably between 80 g / L and 120 g / L, specifically 80 g / L, 90 g / L, 100 g / L, 110 g / L, 120 g / L, or any value between them; yeast extract with a concentration preferably between 5 g / L and 20 g / L, specifically 5 g / L, 8 g / L, 10 g / L, 12 g / L, 15 g / L, 18 g / L, 20 g / L, or any value between them; and monosodium glutamate with a concentration preferably between 5 g / L and 10 g / L, specifically 5 g / L, 7 g / L, 9 g / L, 10 g / L, or... Any value between these; the preferred concentration is sodium sulfate of 6 g / L to 12 g / L, specifically 6 g / L, 7 g / L, 8 g / L, 10 g / L, 12 g / L, or any value between these; the preferred concentration is magnesium sulfate of 2 g / L to 4 g / L, specifically 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, or any value between these; the preferred concentration is ammonium sulfate of 4 g / L to 8 g / L, specifically 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, or any value between these; the preferred concentration is potassium chloride of 0.5 g / L to 2 g / L, specifically 0.5 g / L... The concentrations are preferably 0.1 g / L to 0.2 g / L of calcium chloride, specifically 0.1 g / L, 0.15 g / L, 0.18 g / L, 0.2 g / L, or any value between them; the concentrations are preferably 0.5 g / L to 1 g / L of potassium sulfate, specifically 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, or any value between them; and the concentrations are preferably 1 g / L to 2 g / L of potassium dihydrogen phosphate, specifically 1 g / L, 1.3 g / L, 1.5 g / L, 1.8 g / L. The concentrations are preferably 0.02 mg / L to 0.05 mg / L of sodium molybdate dihydrate, specifically 0.02 mg / L, 0.03 mg / L, 0.04 mg / L, 0.05 mg / L, or any value between them; the concentrations are preferably 2 mg / L to 5 mg / L of manganese chloride tetrahydrate, specifically 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, or any value between them; the concentrations are preferably 0.02 mg / L to 0.08 mg / L of cobalt chloride hexahydrate, specifically 0.02 mg / L, 0.04 mg / L, 0.06 mg / L, 0.08 mg / L, or 0.08 mg / L.The concentrations are preferably 0.8 mg / L or any value between them; copper sulfate pentahydrate with a concentration of 1 mg / L to 5 mg / L, specifically 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L or any value between them; nickel sulfate hexahydrate with a concentration of 1 mg / L to 3 mg / L, specifically 1 mg / L, 1.5 mg / L, 2 mg / L, 3 mg / L or any value between them; zinc sulfate heptahydrate with a concentration of 1 mg / L to 5 mg / L, specifically 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L or any value between them; ferric sulfate heptahydrate with a concentration of 5 mg / L to 15 mg / L, specifically 5 mg / L, 8 mg / L, 10 mg / L, 12 mg / L, 15 mg / L or any value between them; and calcium pantothenate with a concentration of 2 mg / L to 5 mg / L, specifically 2 mg / L... The concentrations are preferably 5 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, or any value between them; the concentration of vitamin B1 is preferably 5 mg / L to 20 mg / L, specifically 5 mg / L, 7 mg / L, 9 mg / L, 10 mg / L, 12 mg / L, 15 mg / L, 20 mg / L, or any value between them; the concentration of vitamin B6 is preferably 4 mg / L to 10 mg / L, specifically 4 mg / L, 6 mg / L, 8 mg / L, 10 mg / L, or any value between them; and the concentration of vitamin B12 is preferably 0.1 mg / L to 0.5 mg / L, specifically 0.1 mg / L, 0.3 mg / L, 0.5 mg / L, or any value between them; and the pH of the fermentation medium is preferably 6 to 7, specifically 6, 6.1, 6.2, 6.4, 6.48, 6.5, 6.52, 6.7, 6.8, 7, or any value between them.

[0051] In this invention, step S2, the fermentation culture refers to the process of synthesizing, transforming, and accumulating EPA and DHA using the metabolic activity of Schizochytrium KDW-306. Various existing options are possible, and this invention does not impose any particular limitation on them. More specifically, depending on the timing, method, and continuity of the addition of nutrients such as carbon sources, nitrogen sources, and precursors, specific examples of the fermentation culture include, but are not limited to, batch culture and / or fed-batch fermentation culture.

[0052] In some specific embodiments, in step S2, the fermentation culture is preferably a batch culture. The conditions for the batch culture specifically include a temperature preferably of 25℃~30℃, specifically 25℃, 26℃, 27℃, 28℃, 29℃, 30℃ or any value between them; a rotation speed preferably of 180r / min~220r / min, specifically 180r / min, 190r / min, 200r / min, 210r / min, 220r / min or any value between them; and a time preferably of 36h~144h, specifically 36h, 48h, 60h, 72h, 120h, 144h or any value between them.

[0053] In some specific embodiments, in step S2, the fermentation culture is preferably a fed-batch fermentation culture. The conditions for the fed-batch fermentation culture specifically include: controlling the glucose concentration of the culture medium to 10 g / L~15 g / L during the culture process, specifically 10 g / L, 12 g / L, 14 g / L, 15 g / L, or any value between them; the temperature is preferably 25℃~30℃, specifically 25℃, 26℃, 28℃, 29℃, 30℃, or any value between them; the pH value is preferably 6~7, specifically 6, 6.3, 6.5, 6.8, 7, or any value between them; the DO value is preferably 25%~35%, specifically 25%, 27%, 29%, 30%, 32%, 35%, or any value between them. The preferred values ​​are: rotational speed (300 r / min to 500 r / min), specifically 300 r / min, 320 r / min, 350 r / min, 380 r / min, 400 r / min, 420 r / min, 450 r / min, 480 r / min, 500 r / min or any value between them; ventilation rate (VVM) is preferably 1 vvm to 3 vvm, specifically 1 vvm, 1.3 vvm, 1.5 vvm, 1.8 vvm, 2 vvm, 2.5 vvm, 3 vvm or any value between them; and duration (VVM) is preferably 60 h to 240 h, specifically 60 h, 72 h, 84 h, 96 h, 120 h, 180 h, 240 h or any value between them.

[0054] In this invention, in step S2, the obtained fermentation culture broth includes EPA and / or DHA. The method for obtaining EPA and / or DHA from the fermentation culture broth is a conventional technical means used in the existing production of EPA and DHA, and can be any of the existing options. This invention does not impose any particular limitation on it.

[0055] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0056] The culture media and their formulations involved in the following examples and comparative examples specifically include: (1) The solid activated culture medium consists of 5 g / L glucose, 2 g / L yeast extract, 5 g / L peptone, 15 g / L sea salt crystals and 20 g / L agar, with pH=6.5.

[0057] (2) The seed culture medium consists of 15 g / L glucose, 2.5 g / L peptone, 10 g / L yeast extract and 30 g / L sea salt, pH=6.5.

[0058] (3) Selective solid culture medium includes 5 g / L glucose, 2 g / L yeast extract, 5 g / L peptone, 15 g / L sea salt, 20 g / L agar and 5 mg / L terbinafine, pH=6.5.

[0059] (4) Fermentation medium I includes 120 g / L glucose, 8 g / L yeast extract, 8 g / L corn steep liquor powder and 20 g / L sea salt, pH=6.5.

[0060] (5) Fermentation medium II consists of 100 g / L glucose, 15 g / L yeast extract, 8 g / L monosodium glutamate, 10 g / L sodium sulfate, 3 g / L magnesium sulfate, 6 g / L ammonium sulfate, 1 g / L potassium chloride, 0.1 g / L calcium chloride, 0.8 g / L potassium sulfate, 1.5 g / L potassium dihydrogen phosphate, 0.03 mg / L sodium molybdate dihydrate, 4 mg / L manganese chloride tetrahydrate, 0.06 mg / L cobalt chloride hexahydrate, 3 mg / L copper sulfate pentahydrate, 2 mg / L nickel sulfate hexahydrate, 3 mg / L zinc sulfate heptahydrate, 10 mg / L ferric sulfate heptahydrate, 4 mg / L calcium pantothenate, 12 mg / L vitamin B1, 8 mg / L vitamin B6 and 0.3 mg / L vitamin B12, pH=6.5.

[0061] Example 1 This embodiment illustrates the acquisition and preservation of a high-EPA-producing schistocytic fungus, KDW-306, including the following steps: 1. Collection and separation of biological samples (1) Collect decaying leaves from the mangrove forest in Haicang Bay, Haicang District, Xiamen City, Fujian Province. Wash the soil off the decaying leaves with sterile seawater and cut the decaying leaves into small pieces with a side length of 1cm.

[0062] (2) After washing the small fragments with sterile seawater containing 1 g / L streptomycin and 1 g / L penicillin, the small fragments were inoculated onto solid activation medium at a rate of 4 tablets / 1 culture medium. A small amount of sterile pine pollen was evenly sprinkled on the solid activation medium, and 5 mL of sterile seawater was added. The medium was then incubated upright at 28°C for 7 days.

[0063] (3) Pick pine pollen with culture adhering to it from the solid activated medium and streak it onto a new solid activated medium. Incubate at 28°C until a single colony grows.

[0064] (4) Select a single colony with good growth and repeat step (3) until there are no contaminants in the plate and no contaminants under microscopic observation to obtain a biological sample. Store the solid activated culture with the biological sample at 4°C.

[0065] 2. Identification of biological samples and determination of oil-producing capacity (1) Identification of biological samples: A total of 12 strains were isolated from the biological samples. They were sent to a biological company for whole-genome sequencing identification. The results are shown in Table 1.

[0066] Table 1.

[0067] (2) Determination of oil production capacity: i. Select a single colony from each solid activation medium and streak it onto the solid activation medium. After incubating at 28℃ for 48h, select 8-10 healthy single colonies and inoculate them into a shake flask containing 50mL of seed medium. Incubate at 28℃ and 200r / min for 48h to obtain the activated bacterial solution. ii. Take 1% of the activated bacterial solution and inoculate it into 50 mL of fermentation medium I. Incubate at 28℃ and 200 r / min for 72 h to obtain the fermentation broth. iii. The fermentation broth was freeze-dried at -4℃ for 24 hours to obtain freeze-dried cells; the freeze-dried cells were then ground and added to a chloroform-methanol mixture at a ratio of 1g:15mL (V 氯仿 :V 甲醇 The mixture was extracted with a ratio of 2:1 for 24 hours. Chloroform and methanol were removed by rotary evaporation. The oil mass was weighed and the oil content (in g / L) and oil amount (in %) were calculated according to the following formula. Oil content = Oil mass / Fermentation broth volume Oil content = (Oil mass / Freeze-dried bacterial cell mass) × 100% iv. Using EPA methyl ester and DHA methyl ester as standards, the oil obtained by rotary evaporation in step iii was subjected to methyl esterification, and the contents (in %) and titers (in g / L) of EPA and DHA were determined by gas chromatography according to the following formula. EPA content = (EPA mass / freeze-dried bacterial cell mass) × 100% DHA content = (DHA mass / freeze-dried bacterial cell mass) × 100% EPA titer = EPA mass / fermentation broth volume DHA titer = DHA mass / fermentation broth volume Furthermore, the test reagents and conditions used for each strain were kept consistent, and the results are shown in Table 2.

[0068] Table 2.

[0069] As shown in Table 2, strain 2 has an oil content of 2.19 g / L, an EPA content of 2.28%, an EPA titer of 49.93 mg / L, a DHA content of 15.98%, and a DHA titer of 349.96 mg / L, which are higher than the other 11 strains. It has good potential for producing EPA and DHA. Therefore, strain 2 was selected as the original strain for subsequent screening and culture.

[0070] 3. Screening and culture of schistocytrium fungi that produce high levels of EPA (1) Straw strain 2 was streaked onto a solid activation medium and cultured at 28°C for 72 hours until single colonies grew. 8-10 well-grown single colonies were picked and inoculated into 50 mL of seed medium and cultured at 28°C and 200 r / min for 48 hours to obtain activated bacterial solution.

[0071] (2) Dilute the activated bacterial solution 10% using seed culture medium. 6 After dilution, the culture was spread onto a selective solid medium and screened at 28°C for 48 hours until single colonies grew.

[0072] (3) Pick each single colony on the selective solid medium and inoculate it into 50 mL of fermentation medium I. Incubate at 28 °C and 200 r / min for 72 h to obtain fermentation culture.

[0073] (4) Centrifuge the fermentation culture at 12000 rpm for 10 min, collect the bottom sludge, dry it at 60℃ to constant weight, weigh it to obtain the sludge mass, and calculate the biomass (unit: g / L) according to the following formula.

[0074] Biomass = Mass of mycelium sludge / Volume of fermentation broth (5) The oil content, EPA and DHA content of each fermentation culture medium were measured using the method provided in step “2. Identification of biological samples and determination of oil production capacity”.

[0075] Table 3.

[0076] A total of 500 single colonies grew on selective solid culture medium. Among them, 20 strains, accounting for 4%, exhibited rapid growth, high biomass, and high EPA production. The biomass and EPA and DHA production capacity of each strain are shown in Table 3. The screening results show that the high EPA-producing strains had a high success rate, indicating that the method provided in this embodiment can achieve highly efficient screening of high-EPA-producing Schizochytrid fungi.

[0077] As shown in Table 3, strain 15 possesses excellent biomass, oil content, EPA titer, and DHA titer, demonstrating promising application prospects for the efficient and safe production of EPA. Strain 15 was named *Schizochytrium*. Schizochytrium sp. )KDW-306.

[0078] 4. Preservation of Schizochytrium KDW-306 Schizochytrium ( Schizochytrium sp. KDW-306 was deposited with the China General Microbiological Culture Collection Center (CGMCC) on November 18, 2025, with accession number CGMCC No. 42323.

[0079] Figure 1 These are photographs of the fungal cells of *Schizochytrium* KDW-306, obtained using an Olympus CX-23 optical microscope (20x eyepiece, 40x objective). Figure 1 It is known that Schizochytrium KDW-306 is spherical or ellipsoidal in shape, with a diameter of 5μm~15μm.

[0080] Example 2 This embodiment illustrates the EPA and DHA production capacity of the Schizochytrium KDW-306 provided in Example 1, specifically including: 1. Shake flask fermentation culture (1) Preparation of seed culture medium: Schizochytrium KDW-306 was inoculated on solid activated medium and cultured at 28℃ for 48h until single colonies grew. Single colonies of Schizochytrium KDW-306 were picked and inoculated into 50mL of seed culture medium and cultured at 28℃ and 200r / min for 120h to obtain shake flask fermentation culture medium.

[0081] (2) The biomass, oil content, and EPA and DHA content in the shake flask fermentation broth were tested according to the method provided in “3. Screening and culture of high EPA-producing Schizochytrium” in Example 1. The results are shown in Table 4.

[0082] Table 4.

[0083] As shown in Table 4, the Schizochytrium KDW-306 provided by this invention has excellent EPA and DHA production capacity under shake-flask fermentation culture conditions.

[0084] 2.5L fermenter fed-batch fermentation culture (1) Preparation of seed culture medium: Take Schizochytrium KDW-306 and inoculate it on solid activated medium. Incubate at 28℃ for 48h until single colonies grow. Select well-grown single colonies and inoculate them into 50mL of seed medium. Incubate at 28℃ and 200r / min for 48h to obtain seed culture medium.

[0085] (2) Fermentation culture: The seed culture solution was inoculated into 5L of fermentation medium II at an inoculation rate of 10% (v / v). Fermentation culture was carried out for 120h at a temperature of 28±0.1℃, pH of 6.5±0.02, DO of 30±5%, rotation speed of 400r / min and aeration rate of 1.5vvm to obtain the fermentation culture solution. During the fermentation culture, the glucose concentration was sampled and tested regularly. When the glucose concentration was less than 10g / L, 60% glucose solution was added to maintain the glucose concentration in the culture solution at 10g / L~15g / L.

[0086] (3) The biomass, oil content and EPA and DHA content in the fermentation culture were tested according to the method provided in “3. Screening and culture of high EPA-producing Schizochytrium” in Example 1. The results are shown in Table 5.

[0087] Table 5.

[0088] As shown in Table 5, the Schizochytrium KDW-306 provided by this invention has excellent EPA and DHA production capacity under fed-batch fermentation culture conditions in a 5L fermenter.

[0089] 3. Feed-and-cure fermentation culture in a 50L fermenter (1) Preparation of seed culture medium: Take Schizochytrium KDW-306 and inoculate it on solid activated medium. Incubate at 28℃ for 48h until single colonies grow. Select well-grown single colonies and inoculate them into 50mL of seed medium. Incubate at 28℃ and 200r / min for 48h to obtain seed culture medium.

[0090] (2) Fermentation culture: The seed culture solution was inoculated into 50L of fermentation medium II at an inoculation rate of 10% (v / v). Fermentation culture was carried out for 120h at a temperature of 28±0.1℃, pH of 6.5±0.02, DO of 30±5%, rotation speed of 400r / min and aeration rate of 1.5vvm to obtain the fermentation culture solution. During the fermentation culture, the glucose concentration was sampled and tested regularly. When the glucose concentration was less than 10g / L, 60% glucose solution was added to maintain the glucose concentration in the culture solution at 10g / L~15g / L.

[0091] (3) The biomass, oil content and EPA and DHA content in the fermentation culture were tested according to the method provided in “3. Screening and culture of high EPA-producing Schizochytrium” in Example 1. The results are shown in Table 6.

[0092] Table 6.

[0093] As shown in Table 6, the Schizochytrium KDW-306 provided by this invention has excellent EPA and DHA production capacity under fed-batch fermentation culture conditions in a 50L fermenter; and the consistency of the results of the 6 parallel replicate experiments is high, indicating that it has excellent production stability.

[0094] As shown in Tables 4-6, the Schizochytrium KDW-306 provided by this invention has excellent and stable EPA and DHA production capacity under both small-scale and large-scale fermentation culture conditions, and has excellent application prospects in realizing low-cost, large-scale industrial production of EPA and DHA.

[0095] Example 3 This embodiment is used to illustrate the passage stability of the Schizochytrium KDW-306 provided in Example 1, specifically including: (1) Preparation of seed culture medium: A single colony of Schizochytrium KDW-306 provided in Example 1 was picked and inoculated into 50 mL of seed culture medium and cultured at 28℃ and 200 r / min for 48 h to obtain seed culture medium, which was recorded as generation 0.

[0096] (2) Subculture of Schizochytrium KDW-306: The seed culture solution was inoculated into a new seed culture medium at an inoculation rate of 10% (v / v) and cultured at 28℃ and 200r / min for 48h to complete the subculture. The above operation was repeated 10 times to complete the continuous subculture.

[0097] (3) Testing of the subculture medium: After each subculture, the obtained subculture medium was fermented according to the method provided in "1. Shake flask fermentation conditions" in Example 2, and the biomass, lipid content, EPA and DHA content of the obtained fermentation medium were measured. The results are as follows: Figure 2 As shown.

[0098] Depend on Figure 2 The results show that, during 10 subcultures, the biomass of Schizochytrium KDW-306 in the fermentation culture medium was 22 g / L to 23 g / L, the lipid content was 10 g / L to 11 g / L, the EPA content was 9% to 10%, and the DHA content was 36% to 40%, indicating good genetic stability and practical application value, which can meet the needs of industrial production.

[0099] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A high-EPA-producing Schizochytrium fungus ( Schizochytrium sp. KDW-306, characterized in that, The accession number of the Schizochytrium KDW-306 is CGMCC No. 42323.

2. The high-EPA-producing Schizochytrium KDW-306 according to claim 1, characterized in that, The selection method for Schizochytrium KDW-306 includes: inoculating the original strain obtained by pine pollen fishing onto a selective solid medium for screening culture to obtain single colonies; fermenting the single colonies to obtain fermentation broth; and screening Schizochytrium KDW-306 based on the biomass and EPA yield of the fermentation broth; wherein the selective solid medium includes 1 mg / L to 10 mg / L of terbinafine.

3. The high-EPA-producing Schizochytrium KDW-306 according to claim 2, characterized in that, The selective solid culture medium comprises 3 mg / L to 7 mg / L terbinafine, 1 g / L to 10 g / L glucose, 1 g / L to 5 g / L yeast extract, 1 g / L to 10 g / L peptone, 10 g / L to 20 g / L sea salt, and 15 g / L to 25 g / L agar, and the pH value of the selective solid culture medium is 6 to 7; Optionally, the screening culture is conducted at a temperature of 25℃~30℃ for a time of 36h~60h; Optionally, the fermentation culture temperature is 25℃~30℃, the rotation speed is 180r / min~220r / min, and the time is 36h~144h.

4. A microbial agent, characterized in that, The microbial agent includes the Schizochytrium KDW-306 as described in claim 1.

5. The microbial agent according to claim 5, characterized in that, The inoculum includes one or more of the following: live cells, dead cells, and spores of Schizochytrium KDW-306.

6. The use of the Schizochytrium KDW-306 of claim 1 or the microbial agent of claim 4 in the preparation of EPA and / or DHA.

7. A method for preparing EPA and / or DHA, characterized in that, The preparation method includes: S1, taking the Schizochytrium KDW-306 of claim 1 or the fungal agent of claim 4 and performing activation culture and seed expansion culture in sequence to obtain seed culture solution; S2, taking the seed culture solution and inoculating it into a fermentation medium for fermentation culture to obtain fermentation culture solution; wherein, the fermentation culture solution contains EPA and / or DHA.

8. The method for preparing EPA and / or DHA according to claim 7, characterized in that, In step S1, the solid activation culture medium used in the activation culture includes 1 g / L to 10 g / L of glucose, 1 g / L to 10 g / L of peptone, 1 g / L to 5 g / L of yeast extract, 10 g / L to 20 g / L of sea salt and 15 g / L to 25 g / L of agar, and the pH value of the solid activation culture medium is 6 to 7. Optionally, the activation culture is carried out at a temperature of 25°C to 30°C for a time of 36 to 60 hours. Optionally, the seed culture medium used for the seed expansion culture includes 10 g / L to 20 g / L of glucose, 1 g / L to 5 g / L of peptone, 5 g / L to 15 g / L of yeast extract and 20 g / L to 40 g / L of sea salt, and the pH value of the seed culture medium is 6 to 7. Optionally, the seed culture is carried out at a temperature of 25℃~30℃ for 36h~60h.

9. The method for preparing EPA and / or DHA according to claim 7, characterized in that, In step S2, the fermentation medium comprises 80 g / L to 120 g / L glucose, 5 g / L to 20 g / L yeast extract, 5 g / L to 10 g / L monosodium glutamate, 6 g / L to 12 g / L sodium sulfate, 2 g / L to 4 g / L magnesium sulfate, 4 g / L to 8 g / L ammonium sulfate, 0.5 g / L to 2 g / L potassium chloride, 0.1 g / L to 0.2 g / L calcium chloride, 0.5 g / L to 1 g / L potassium sulfate, 1 g / L to 2 g / L potassium dihydrogen phosphate, 0.02 mg / L to 0.05 mg / L sodium molybdate dihydrate, and 2 mg / L to 5 mg / L sodium chloride. The fermentation medium contains g / L manganese chloride tetrahydrate, 0.02 mg / L to 0.08 mg / L cobalt chloride hexahydrate, 1 mg / L to 5 mg / L copper sulfate pentahydrate, 1 mg / L to 3 mg / L nickel sulfate hexahydrate, 1 mg / L to 5 mg / L zinc sulfate heptahydrate, 5 mg / L to 15 mg / L ferric sulfate heptahydrate, 2 mg / L to 5 mg / L calcium pantothenate, 5 mg / L to 20 mg / L vitamin B1, 4 mg / L to 10 mg / L vitamin B6, and 0.1 mg / L to 0.5 mg / L vitamin B12, and the pH of the fermentation medium is 6 to 7.

10. The method for preparing EPA and / or DHA according to claim 7, characterized in that, In step S2, the fermentation culture is a batch culture, and the temperature of the batch culture is 25℃~30℃, the rotation speed is 180r / min~220r / min, and the time is 36h~144h. Optionally, the fermentation culture is a fed-batch fermentation culture, which includes adjusting the glucose concentration of the culture medium to 10 g / L to 15 g / L during the culture process; the temperature of the fed-batch fermentation culture is 25℃ to 30℃, the pH value is 6 to 7, the DO value is 25% to 35%, the rotation speed is 300 r / min to 500 r / min, the aeration rate is 1 vvm to 3 vvm, and the time is 60 h to 240 h.

Images