A high-oil high-dha schizochytrium sp. engineering strain and a culture and algal oil extraction method

Abstract

The application relates to a high-oil high-DHA schizochytrium sp. engineering strain and a culture and algal oil extraction method. The engineering strain is obtained by compound mutagenesis and multi-generation directional domestication of schizochytrium sp., has a fat content of greater than or equal to 35%, a DHA content of greater than or equal to 48%, stable properties after continuous generations of 8 to 12, the characteristics of temperature resistance, light resistance, pollution resistance, memory growth rate, a biomass of greater than or equal to 1.5 g / L, and the like. From primary shake flask activation expansion fermentation to secondary fermentation tank expansion fermentation, to tertiary oil enrichment fermentation, in the step-by-step amplification process, key parameters such as temperature, pH, CO2 supply, dissolved oxygen content and the like are accurately controlled in different stages, and in the late culture stage, nitrogen reduction induction is combined with weak light regulation to direct promote oil and DHA enrichment, so that the amplification effect problem that the biomass and DHA content are greatly reduced after amplification from small-scale test to industrial large tank in the traditional process is effectively solved.

Description

Technical Field

[0001] This invention relates to the fields of microbial breeding and natural oil extraction technology, specifically to an engineered strain of *Schizochytrium schizochytii* with high oil and high DHA content, and a method for its cultivation and oil extraction. Background Technology

[0002] Docosahexaenoic acid (DHA) is an essential omega-3 polyunsaturated fatty acid for the human body. It plays a vital role in promoting brain and vision development in infants and young children, preventing cardiovascular and cerebrovascular diseases, and possessing anti-inflammatory and anti-tumor properties. It is widely used in infant formula, health products, and the pharmaceutical industry. Currently, the main industrial source of DHA is marine microalgae such as Schizochytrium, which are high in DHA, virtually EPA-free, safe, and of stable quality, making them a recognized high-quality source of DHA.

[0003] Currently, the wild-type *Schizochytrium* strains, conventionally induced mutant strains, and supporting production processes used in domestic and international industrial production have several shortcomings, specifically: First, traditional algal strains generally have an oil content below 30% and a DHA content below 45%, exhibiting either high oil content and low DHA content or high DHA content and low oil content. These strains have slow growth rates, poor stress resistance, weak stability, are prone to contamination during large-scale fermentation, and show significant batch-to-batch variations. After continuous subculturing, traits rapidly degrade, and batch production indicators fluctuate greatly, making them unsuitable for long-term continuous industrial production. Second, conventional mutagenesis methods often employ single UV or chemical mutagenesis, resulting in low mutation rates and low probabilities of positive mutations, making it difficult to simultaneously achieve the dual goals of high oil content and high DHA. Third, traditional fermentation lacks a scale-up gradient culture system, resulting in a lack of precise control over key parameters such as temperature, pH, carbon dioxide supply, stirring speed, aeration rate, and dissolved oxygen. The absence of a targeted oil induction strategy leads to low oil and DHA enrichment efficiency in algal cells, and a significant fermentation scale-up effect; after scaling up from small-scale processes to large industrial tanks, biomass and DHA content decrease dramatically. In addition, traditional cell wall breaking methods are mostly simple mechanical breaking, which can easily cause excessive damage to algal cells and lead to DHA oxidation and deterioration. Single extraction methods have drawbacks such as solvent residue, high extraction cost, low extraction rate, lack of low temperature and light protection control throughout the extraction process, and high DHA oxidation loss rate.

[0004] Currently, there is no publicly available, domestically or internationally, high-oil, high-DHA algal oil strain and cultivation and extraction method for Schizochytrium algae that can be stably genetically derived, has an oil content of ≥35%, DHA content of ≥48%, exhibits rapid growth, is pollution-resistant, and is suitable for large-scale fermentation. Existing technologies cannot simultaneously address the four core challenges of high oil content, high DHA content, stability, and industrial compatibility, thus hindering the large-scale development of the high-quality DHA algal oil industry. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a high-oil, high-DHA Schizochytrium engineered strain and a method for its cultivation and oil extraction. Through a combination of ultraviolet light and compound mutagenesis with multiple generations of targeted domestication, a genetically stable Schizochytrium engineered strain with high oil and DHA content is obtained. A three-stage gradient precision fermentation process solves the fermentation scale-up problem, and a compound cell wall-breaking extraction process reduces DHA oxidation loss and increases the extraction rate. Ultimately, this achieves the goals of high quality, high yield, low cost, and easy industrial-scale production, thus overcoming the deficiencies of existing technologies.

[0006] The technical solution of this invention is achieved as follows: a high-oil, high-DHA Schizochytrium engineered strain, which is obtained from Schizochytrium through compound mutagenesis and multiple generations of directional domestication, has an oil content ≥35%, a DHA content ≥48%, and stable traits after 8 to 12 generations of continuous subculturing. It has the characteristics of being temperature-resistant, light-resistant, pollution-resistant, and having a fast growth rate with a biomass ≥1.5g / L.

[0007] A method for culturing the high-oil, high-DHA Schizochytrium engineered strain as described above, comprising the following steps: S1. Algal strain isolation and screening: Microalgae are isolated from natural freshwater and seawater bodies, and after initial screening by oil staining and secondary screening by DHA content, algal strains with oil content ≥35%, DHA content ≥48% and biomass ≥1.2g / L are selected. S2. Combined mutagenesis: The algal strains were treated with a combination of ultraviolet mutagenesis and EMS chemical mutagenesis, with the ultraviolet mutagenesis duration being 30 to 60 seconds and the EMS mutagenesis concentration being 0.1% to 0.3%. S3. Targeted domestication: The algal strains after compound mutagenesis are subjected to 8 to 12 generations of continuous targeted domestication, with a domestication cycle of 4 to 6 months. The domestication direction is to improve the lipid synthesis capacity, DHA enrichment capacity, temperature tolerance, light tolerance and pollution tolerance, and finally obtain the engineered strain of Schizochytrium.

[0008] A method for extracting algal oil from the high-oil, high-DHA Schizochytrium engineered strain as described above, comprising: subjecting the engineered strain to three-stage gradient fermentation culture, algal cell collection and concentration, compound cell wall disruption, and compound algal oil extraction, ultimately yielding crude DHA oil.

[0009] The three-stage gradient fermentation culture includes the following steps: S1. Primary shake-flask activation and expansion fermentation: Using glucose, yeast powder, inorganic salts, and sterile water as the culture medium, the culture temperature is 25℃ to 30℃, the pH value is 7.0 to 8.5, the light-dark cycle is 12h light / 12h dark, the light intensity is 2000 lux to 3000 lux, the shaker speed is 150 rpm to 200 rpm, the inoculum size is 10% to 15%, and the culture period is 7 to 10 days; culture until the algal solution OD680 is ≥0.8 and the biomass is ≥0.5g / L, the pH fluctuation during the culture process is ≤0.5, and aseptic operation is carried out throughout the process; S2. Secondary fermentation tank expansion fermentation: The algal solution after activation and expansion fermentation in the primary shake flask is transferred to a 5L fermenter for fermentation culture. The culture temperature is 26℃ to 28℃, the pH value is 7.2 to 8.2, and 1% to 5% CO2 mixed with air is introduced. The stirring speed is 100 rpm to 300 rpm, the aeration rate is 0.5 vv to 2.0 vvm, and the culture period is 7 days. During the culture process, the dissolved oxygen (DO) is controlled to be ≥4 mg / L, and the culture is continued until the algal biomass is ≥1.0 g / L, the oil content is ≥25%, and the DHA content is ≥45%. S3. Tertiary Lipid Enrichment Fermentation: The algal broth from the secondary fermentation tank is transferred to a 50L fermenter. The cultivation temperature is 26℃ to 28℃, the pH is 7.5 to 8.5, and 2% to 4% CO2 mixed air is introduced. The stirring speed is 150 rpm to 250 rpm, the aeration rate is 1.0 vvm to 1.5 vvm, and the cultivation period is 7 to 10 days. In the later stage of cultivation, lipid synthesis is induced by nitrogen reduction and low light regulation is adopted. Positive pressure is maintained in the tank and sterilization is achieved by 0.22μm air filtration. Cultivation continues until the lipid content is ≥35%, the DHA content is ≥48%, and the biomass is ≥1.5g / L.

[0010] The algal collection and concentration steps are as follows: the algal liquid after three-stage gradient fermentation culture is centrifuged at 8000rpm to 10000rpm for 10 to 15 minutes to obtain concentrated algal mud. The water content of the algal mud is controlled at 80% to 85%, and it is stored in a low-temperature and light-proof environment at 4℃. It is then put into the next process within 24 hours.

[0011] The composite cell wall disruption process employs a combined enzymatic hydrolysis and high-pressure homogenization process, achieving a cell wall disruption rate ≥95%. First, the concentrated algal sludge obtained from the algal collection and concentration step undergoes enzymatic hydrolysis. During this process, the pH is adjusted to 10.5 to 11.0, the temperature is controlled at 50℃ to 55℃, and 0.4% (by mass) of cellulase and pectinase are added. The hydrolysis time is 5 to 6 hours. Then, the hydrolyzed material is placed in a high-pressure homogenizer, with the homogenization pressure set to 1000 bar to 1500 bar, and the entire process is conducted in the dark.

[0012] The algal oil composite extraction involves organic solvent pre-extraction combined with supercritical CO2 purification. First, a mixed solvent of n-hexane and ethanol is used, with a material-to-solvent ratio of 1:5 to 10. The extraction temperature is 40°C to 50°C, and the extraction time is 2 hours to obtain a pre-extracted product. Then, the pre-extracted product is placed in a supercritical extraction device, with an extraction pressure of 30 MPa to 40 MPa, a temperature of 40°C to 50°C, and an extraction time of 1 to 2 hours. During the extraction process, the moisture content of the material is controlled to be ≤5%, and the final extraction rate is ≥90%, with a DHA retention rate of ≥98%.

[0013] The crude DHA oil specifications are as follows: DHA content ≥48%, acid value ≤1.0 mg / g, peroxide value ≤2.0 meq / kg, and moisture content ≤0.5%.

[0014] The present invention has the following positive effects: 1. The high-oil, high-DHA Schizochytridium engineered strain described in this invention is obtained through compound mutagenesis combined with multi-generation directional domestication cultivation, which solves the technical problems of traditional strains being high in oil and low in DHA or high in DHA and low in oil. The oil content can reach more than 35%, and the DHA content is stable at more than 48%. The traits do not degenerate after 8 to 12 generations of continuous subculturing. It also has a fast growth rate, strong resistance to temperature, light and pollution, and a biomass of more than 1.5 g / L, which is suitable for the needs of long-term continuous industrial production and solves the problem of large batch fluctuations in industrial production from the root.

[0015] 2. The algal oil extraction method of the high-oil, high-DHA Schizochytrium engineered strain described in this invention adopts a three-stage gradient precision fermentation system, from the first-stage shake flask activation and expansion fermentation to the second-stage fermenter expansion fermentation, and then to the third-stage oil enrichment fermentation. During the step-by-step scale-up process, key parameters such as temperature, pH, CO2 supply, and dissolved oxygen are precisely controlled at different stages. In the later stage of cultivation, nitrogen reduction induction combined with weak light regulation is used to directionally promote the enrichment of oil and DHA, which effectively solves the scale-up effect problem of significant decrease in biomass and DHA content after scale-up from small-scale to large-scale industrial tanks in traditional processes. After fermentation, the production indicators can be stably achieved as follows: oil ≥35%, DHA ≥48%, and biomass ≥1.5g / L.

[0016] 3. The algal oil extraction method of the high-oil, high-DHA Schizochytrium engineered strain described in this invention adopts an enzymatic hydrolysis combined with high-pressure homogenization coupled cell wall disruption process. Compared with the traditional simple mechanical cell wall disruption, it ensures a cell wall disruption rate of over 95% and avoids the problem of DHA oxidation and deterioration caused by excessive crushing. Subsequently, an organic solvent pre-extraction combined with supercritical CO2 refining process is used. The entire process is strictly controlled by low temperature and light protection. The final extraction rate can reach over 90%, the DHA retention rate is ≥98%, the obtained DHA crude oil acid value is ≤1.0mg / g, peroxide value is ≤2.0meq / kg, moisture content is ≤0.5%, and there is no chemical solvent residue. The product quality is far superior to the product obtained by traditional extraction processes. At the same time, it reduces extraction costs and water treatment burden, and has both economic and environmental advantages.

[0017] 4. This invention solves the problems of existing technologies by covering the entire chain from strain cultivation and fermentation culture to oil extraction. It obtains a high-oil, high-DHA Schizochytrium engineered strain that can be stably inherited and adapted for large-scale production, realizing high-efficiency and low-cost mass production of high-quality DHA algal oil. It can effectively promote the large-scale development of the high-quality DHA algal oil industry and has high industrial application value and market prospects.

[0018] 5. This invention uses only physical mutagenesis, chemical mutagenesis and natural domestication methods, without introducing exogenous genes, so it has a high safety factor. At the same time, the whole process route is simple, has good repeatability and is easy to scale up, and can be produced on a large scale. The overall process is highly compatible and has outstanding comprehensive benefits. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the framework for the cultivation method of the high-oil, high-DHA Schizochytrium sarcodactylon engineered strain of the present invention.

[0020] Figure 2 This is a schematic diagram of the algal oil extraction method of the high-oil, high-DHA Schizochytrium engineered strain of the present invention. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1: As Figure 1 As shown, this embodiment provides a high-oil, high-DHA Schizochytrium engineered strain, which is obtained from Schizochytrium through compound mutagenesis and multiple generations of directional domestication. Its oil content is ≥35%, DHA content is ≥48%, and its traits are stable after 8 to 12 generations of continuous subculturing. It has the characteristics of being temperature resistant, light resistant, pollution resistant, and having a fast growth rate. Its biomass is ≥1.5g / L.

[0023] Example 2: As Figure 1 As shown in this embodiment, a method for cultivating a high-oil, high-DHA *Schizochytridica* engineered strain as described in this embodiment is provided. First, algal strain isolation and screening are performed by collecting surface water samples rich in organic matter from natural water bodies such as seas and freshwater lakes. After initial screening with oil staining and secondary screening with DHA content, an algal strain with good growth characteristics, a basic oil content of approximately 35%, a DHA content of approximately 48%, and a biomass ≥1.2 g / L is selected. This strain is then processed into a bacterial suspension for mutagenesis. Next, UV mutagenesis is performed under a 20W UV lamp for 50 seconds. After irradiation, the algal solution is immediately wrapped in sterile black paper to protect it from light and transferred to subsequent chemical mutagenesis. Sterile EMS solution is added to the UV-mutated algal solution to a final concentration of 0.2% (v / v). After terminating the mutagenesis reaction and removing residual EMS, continuous subculturing is performed for approximately 10 generations over a 5-month acclimatization period. After about 10 generations of targeted domestication, a strain of *Schizochytrium* with the best overall performance was obtained through screening. The trait stability of this strain was verified, and the results showed that the main trait indicators, such as biomass, lipid content, and DHA content, fluctuated by less than 5%, indicating good genetic stability.

[0024] Example 3: As Figure 2 As shown, this embodiment provides a method for extracting algal oil from the high-oil, high-DHA Schizochytrium engineered strain described in Example 1. First, the engineered strain undergoes a three-stage gradient fermentation culture. The specific steps are as follows: Primary shake-flask activation and expansion fermentation: Using glucose, yeast powder, inorganic salts and sterile water as the culture medium, the culture temperature is 25℃, the pH value is 7, the light-dark cycle is 12h light / 12h dark, the light intensity is 2000 lux, the shaker speed is 150 rpm, the inoculum size is 10%, and the culture period is 7 days; culture until the algal solution OD680≥0.8 and the biomass≥0.5g / L, the pH fluctuation during the culture process is ≤0.5, and the entire process is carried out under aseptic conditions.

[0025] Secondary fermenter expansion fermentation: The algal solution after activation and expansion fermentation in the primary shake flask was transferred to a 5L fermenter for fermentation and culture. The culture temperature was 27℃, the pH value was 8, and 2% CO2 mixed with air was introduced. The stirring speed was 200 rpm, the aeration rate was 1.0 vvm, and the culture period was 7 days. During the culture process, the dissolved oxygen (DO) was controlled to be ≥4 mg / L, and the culture was continued until the algal biomass was ≥1.0 g / L, the oil content was ≥25%, and the DHA content was ≥45%.

[0026] Three-stage lipid enrichment fermentation: The algal broth from the secondary fermenter was transferred to a 50L fermenter. The culture temperature was 28℃, the pH was 8.5, and 4% CO2 mixed with air was introduced. The stirring speed was 250 rpm, the aeration rate was 1.5 vvm, and the culture period was 10 days. In the later stage of culture, lipid synthesis was induced by nitrogen reduction and weak light regulation was used. Positive pressure was maintained in the tank and sterilization was carried out by 0.22μm air filtration. The culture continued until the lipid content was ≥35%, the DHA content was ≥48%, and the biomass was ≥1.5g / L.

[0027] The fermentation broth after the three-stage fermentation was placed in a high-speed centrifuge and centrifuged at 9000 rpm for 12 minutes at 4°C. The supernatant was discarded, and the wet algal sludge was collected. The algal sludge was quickly washed once with distilled water and then centrifuged again. The final algal sludge had a moisture content of approximately 82%. The wet algal sludge was immediately transferred to a pre-cooled, sealed, light-proof container and temporarily stored at 4°C in the dark, and then subjected to subsequent cell wall disruption treatment within 18 hours.

[0028] The obtained wet algal sludge was weighed and suspended in sterile water. The pH of the system was adjusted to 10.8, and the mixture was heated to 52°C in a water bath. A complex enzyme preparation of cellulase and pectinase at a mass ratio of 1:1 (0.4% w / w) was added to the system for enzymatic hydrolysis for 5.5 hours. After hydrolysis, the mixture was cooled and then pumped into a high-pressure homogenizer under completely dark conditions. The homogenization pressure was set to 1200 bar, and two cycles were performed. Sampling and testing showed that the algal cell wall disruption rate reached 96.5%. The homogenized algal slurry was then ready for use.

[0029] Homogenized algal slurry was mixed with a mixture of n-hexane and ethanol at a ratio of 1:6 (m / v). The mixture was then transferred to a constant-temperature extraction tank equipped with a reflux condenser. The extraction temperature was set at 45°C, and the mixture was kept at this temperature and stirred for 2 hours. After extraction, centrifugation was performed to obtain a crude algal oil extract rich in DHA. The crude algal oil extract was then transferred to a supercritical CO2 extraction device. The extraction pressure was set at 35 MPa, the extraction temperature at 45°C, and the extraction time at 1.5 hours, ultimately yielding crude DHA oil. The final DHA algal oil was tested, and the results were as follows: DHA content 49.5%, acid value 0.8 mg, peroxide value 1.6 meq / kg, and moisture content 0.25%.

[0030] The process parameters, process control range, and acceptance criteria for each stage of this invention are shown in the table below:

[0031] Comparative Example 1: This comparative example is a traditional single-mutation algal strain and process control. Commercially available wild strains of Schizochytrium were selected. After single-mutation under ultraviolet light, the algae were cultured in a conventional manner. Fermentation, cell wall disruption, and extraction all adopted traditional industry processes.

[0032] Its oil content was 27.1%, and its DHA content was 43.2%. After five generations of continuous subculturing, its properties significantly deteriorated. After fermentation scale-up, the biomass decreased to 0.92 g / L, and the oil and DHA contents decreased significantly. Pure mechanical cell disruption was used, with a cell disruption rate of 81.2%, a DHA oxidation loss of 3.8%, and an oil extraction rate of 82.7%. The acid value was 1.85 mg / g, and the peroxide value was 3.62 meq / kg. The overall product quality and yield were significantly lower than those of the embodiments of this invention.

[0033] Verification through the above specific embodiments shows that the high-oil, high-DHA Schizochytridium tumefaciens engineered strain and its supporting cultivation method and algal oil extraction method provided by this invention can successfully and stably produce high-quality algal oil with an oil content ≥35% and a DHA content ≥48%. The entire process parameters are controllable and have good repeatability, and the various indicators of the obtained product meet the requirements of high-quality DHA raw materials such as infant formula.

[0034] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A high-oil, high-DHA Schizochytridium strain, characterized in that: It was obtained from Schizochytrium through complex mutagenesis and multiple generations of directional domestication. Its oil content is ≥35%, DHA content is ≥48%, and its traits are stable after 8 to 12 generations of continuous subculturing. It has the characteristics of being heat-resistant, light-resistant, pollution-resistant, and having a fast growth rate with a biomass of ≥1.5g / L.

2. The method for culturing the high-oil, high-DHA Schizochytridium sarcodactylon engineered strain according to claim 1, characterized in that, The method includes the following steps: S1. Algal strain isolation and screening: Microalgae are isolated from natural freshwater and seawater bodies, and after initial screening by oil staining and secondary screening by DHA content, algal strains with oil content ≥35%, DHA content ≥48% and biomass ≥1.2g / L are selected. S2. Combined mutagenesis: The algal strains were treated with a combination of ultraviolet mutagenesis and EMS chemical mutagenesis, with the ultraviolet mutagenesis duration being 30 to 60 seconds and the EMS mutagenesis concentration being 0.1% to 0.3%. S3. Targeted domestication: The algal strains after compound mutagenesis are subjected to 8 to 12 generations of continuous targeted domestication, with a domestication cycle of 4 to 6 months. The domestication direction is to improve the lipid synthesis capacity, DHA enrichment capacity, temperature tolerance, light tolerance and pollution tolerance, and finally obtain the engineered strain of Schizochytrium.

3. A method for extracting algal oil from the high-oil, high-DHA *Schizochytrium* engineered strain as described in claim 1, characterized in that, The method is as follows: the engineered strain is subjected to three-stage gradient fermentation culture, algal cell collection and concentration, compound cell wall disruption and compound extraction of algal oil, and finally DHA crude oil is obtained.

4. The method for extracting algal oil from the high-oil, high-DHA *Schizochytrium* engineered strain according to claim 3, characterized in that, The three-stage gradient fermentation culture includes the following steps: S1. Primary shake-flask activation and expansion fermentation: Using glucose, yeast powder, inorganic salts, and sterile water as the culture medium, the culture temperature is 25℃ to 30℃, the pH value is 7.0 to 8.5, the light-dark cycle is 12h light / 12h dark, the light intensity is 2000 lux to 3000 lux, the shaker speed is 150 rpm to 200 rpm, the inoculum size is 10% to 15%, and the culture period is 7 to 10 days; culture until the algal solution OD680 is ≥0.8 and the biomass is ≥0.5g / L, the pH fluctuation during the culture process is ≤0.5, and aseptic operation is carried out throughout the process; S2. Secondary fermentation tank expansion fermentation: The algal solution after activation and expansion fermentation in the primary shake flask is transferred to a 5L fermenter for fermentation culture. The culture temperature is 26℃ to 28℃, the pH value is 7.2 to 8.2, and 1% to 5% CO2 mixed with air is introduced. The stirring speed is 100 rpm to 300 rpm, the aeration rate is 0.5 vv to 2.0 vvm, and the culture period is 7 days. During the culture process, the dissolved oxygen (DO) is controlled to be ≥4 mg / L, and the culture is continued until the algal biomass is ≥1.0 g / L, the oil content is ≥25%, and the DHA content is ≥45%. S3. Tertiary Lipid Enrichment Fermentation: The algal broth from the secondary fermentation tank is transferred to a 50L fermenter. The cultivation temperature is 26℃ to 28℃, the pH is 7.5 to 8.5, and 2% to 4% CO2 mixed air is introduced. The stirring speed is 150 rpm to 250 rpm, the aeration rate is 1.0 vvm to 1.5 vvm, and the cultivation period is 7 to 10 days. In the later stage of cultivation, lipid synthesis is induced by nitrogen reduction and low light regulation is adopted. Positive pressure is maintained in the tank and sterilization is achieved by 0.22μm air filtration. Cultivation continues until the lipid content is ≥35%, the DHA content is ≥48%, and the biomass is ≥1.5g / L.

5. The method for extracting algal oil from the high-oil, high-DHA *Schizochytrium* engineered strain according to claim 3, characterized in that, The algal collection and concentration steps are as follows: the algal liquid after three-stage gradient fermentation culture is centrifuged at 8000rpm to 10000rpm for 10min to 15min to obtain concentrated algal mud. The water content of the algal mud is controlled at 80% to 85%, and it is stored in a low-temperature and light-proof environment at 4℃. It is then put into the next process within 24h.

6. The method for extracting algal oil from the high-oil, high-DHA Schizochytrium engineered strain according to claim 3, characterized in that: The composite cell wall disruption process employs a combined enzymatic hydrolysis and high-pressure homogenization process, achieving a cell wall disruption rate ≥95%. First, the concentrated algal sludge obtained from the algal collection and concentration step undergoes enzymatic hydrolysis. During this process, the pH is adjusted to 10.5 to 11.0, the temperature is controlled at 50℃ to 55℃, and 0.4% (by mass) of cellulase and pectinase are added. The hydrolysis time is 5 to 6 hours. Then, the hydrolyzed material is placed in a high-pressure homogenizer, with the homogenization pressure set to 1000 bar to 1500 bar, and the entire process is conducted in the dark.

7. The method for extracting algal oil from the high-oil, high-DHA *Schizochytrium* engineered strain according to claim 3, characterized in that: The algal oil composite extraction involves organic solvent pre-extraction combined with supercritical CO2 purification. First, a mixed solvent of n-hexane and ethanol is used, with a material-to-solvent ratio of 1:5 to 10. The extraction temperature is 40°C to 50°C, and the extraction time is 2 hours to obtain a pre-extracted product. Then, the pre-extracted product is placed in a supercritical extraction device, with an extraction pressure of 30 MPa to 40 MPa, a temperature of 40°C to 50°C, and an extraction time of 1 to 2 hours. During the extraction process, the moisture content of the material is controlled to be ≤5%, and the final extraction rate is ≥90%, with a DHA retention rate of ≥98%.

8. The method for extracting algal oil from the high-oil, high-DHA *Schizochytrium* engineered strain according to claim 3, characterized in that, The crude DHA oil specifications are as follows: DHA content ≥48%, acid value ≤1.0 mg / g, peroxide value ≤2.0 meq / kg, and moisture content ≤0.5%.

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