A method, product, and application for regulating DHA content, DHA / DPA ratio, and sn-2 DHA proportion in Schizochytrium fermentation products.

Abstract

The present application belongs to the technical field of bioengineering and food engineering, and discloses a method for improving the content of DHA, the ratio of DHA / DPA and the proportion of sn-2 DHA in the fermentation product of Schizochytrium sp., a product and application, wherein the oil-producing Schizochytrium sp. is subjected to fed-batch fermentation culture, and shikonin is added during the fermentation culture. The present application provides a fermentation strategy for producing high-quality DHA algal oil by using Schizochytrium sp., wherein shikonin is added to the fermentation medium of Schizochytrium sp., so that the Schizochytrium sp. obtains lipids with high DHA ratio, high DHA / DPA ratio and high sn-2 DHA proportion. The obtained Schizochytrium sp. lipids not only have a significantly improved total DHA content compared with traditional fermentation strategies, but also have a significantly improved DHA / DPA ratio and sn-2 DHA proportion.

Description

Technical Field

[0001] This invention belongs to the fields of bioengineering technology and food engineering technology, and in particular, it relates to a method, product and application for regulating the DHA content, DHA / DPA ratio and sn-2 DHA proportion in Schizochytrium fermentation products. Background Technology

[0002] Docosahexaenoic acid (DHA, 22:6, n-3) is an important polyunsaturated fatty acid that has been extensively studied for its beneficial health effects. DHA is crucial for the growth and functional maintenance of the infant brain and retina, and also plays a preventive role in cardiovascular and inflammatory diseases in adults. Algal oil is one source of n-3 polyunsaturated fatty acids, rather than fish oil or marine mammal oil. Among microalgae, Schizochytrium is a well-studied oleogenous marine microalga with a high lipid content (35%–84% of biomass), of which neutral lipids account for approximately 97% of the total lipids. Neutral lipids are mainly composed of triglycerides (TAG), accounting for approximately 94% of the lipids.

[0003] Commercial algal oils are primarily composed of triglycerides (TAGs, approximately 97%) and contain DHA, which accounts for about 45% of the total fatty acids. Algal oils derived from Schizochytrium have played a significant role in the food market, including bread, nutrition bars, dairy beverages, fish feed, poultry and cattle (DHA-rich milk), margarine, and infant formula.

[0004] Furthermore, existing research indicates that the distribution within the TAG structure affects its absorption efficiency and nutritional value. Fatty acids in natural oils undergo esterification at three stereospecific numbered (sn) sites on the glycerol backbone, exhibiting different distribution characteristics. Studies have found that DHA located at the sn-2 position of TAGs is more readily absorbed by intestinal epithelial cells, thus enhancing its bioavailability. However, positional analysis of highly unsaturated fatty acids (PUFAs) in oils remains a challenge in lipid research. Recent studies have shown that DHA in human milk fat is primarily esterified at the sn-2 position of the TAG, a finding that has attracted attention, and algal oil has been added to infant formula to supplement DHA. However, most DHA supplementation recommendations focus on total intake rather than its positional distribution. Given that the storage form of DHA significantly impacts its pharmacological and nutritional value for human brain development and maintenance, achieving efficient and specific production of sn-2 DHA is of great significance for improving the dietary health of Chinese residents.

[0005] Currently, there are many reports on the production of DHA-rich oils from *Schizochytrium*, but the production process suffers from several problems, including low DHA content, high DPA content, low sn-2 DHA content, and a lack of application testing. Chinese patent publication CN120060388A, by adding inositol and betaine to the fermentation broth, can promote DHA synthesis and reduce the DPA content in *Schizochytrium*, achieving a DHA content of 51.2%. Chinese patent publication CN114525312A, by using a fermentation medium without additional sodium or chloride ions, achieved a DHA content of 50% in *Schizochytrium*. Chinese patent publication CN119351635A, by controlling the pH of the fermentation broth through staged acid or alkali supplementation during *Schizochytrium* fermentation, ultimately obtained oils with a sn-2 DHA content as high as 60.4%. Chinese patent publication CN119061084A describes a calcium ion-induced regulation strategy combined with nitrogen-limited induced fermentation to achieve sn-2 DHA and carotenoid content of 57.3% and 78.3 μg / g, respectively, in lipids. However, existing research on the production or application of DHA lipids using Schizochytrium is relatively limited, with related strategies focusing only on one or two of the lipids, DHA, DHA / DPA, sn-2 DHA, or their applications, without a comprehensive discussion.

[0006] Therefore, it is urgent to obtain a fermentation method that can produce high-quality DHA algal oil more efficiently (i.e., high DHA ratio, high DHA / DPA ratio, and high sn-2DHA content) and that performs well in the application of Schizochytrium products in the food industry. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method, product, and application for regulating the DHA content, DHA / DPA ratio, and sn-2 DHA proportion in Schizochytrium fermentation products.

[0008] The technical solution adopted by this invention to solve its technical problem is: A method for regulating the DHA content, DHA / DPA ratio, and sn-2 DHA percentage in the fermentation products of Schizochytrium oleiferum, wherein the method involves feeding the oil-producing Schizochytrium oleiferum into a batch fermentation culture, and adding shikonin during the fermentation culture process.

[0009] Furthermore, the concentration of the added shikonin is between 1 μg / L and 50 μg / L, and the addition time of the shikonin is between 6h and 18h. Alternatively, the oil-producing schistocytrium includes Schistocytrium. Schizochytriumsp. HX-308, Schizochytrium ATCC 20888 and Schizochytrium ATCC PRA-276.

[0010] Furthermore, it includes the following steps: Single colonies of *Schizochytrium* were selected and inoculated into seed culture medium. After incubation at 28°C and 180 rpm for 24 h, they were used as first-generation seed cultures. 1 mL of first-generation seed culture was inoculated into 50 mL of seed culture medium and incubated at 28°C and 180 rpm for 24 h to obtain second-generation seed cultures. 1 mL of second-generation seed culture was inoculated into 50 mL of seed culture medium and incubated at 28°C and 180 rpm for 24 h to obtain third-generation seed cultures, which were used as inoculum for fermentation. Fermentation culture: 10 mL of third-generation seeds were inoculated into 90 mL of fermentation medium. 20 μg / L of shikonin was added after 18 h of fermentation. The culture was carried out at 28℃ and 180 r / min in a shaker. The glucose concentration in the fermentation medium was measured every 12 h. The glucose concentration in the fermentation medium was maintained above 50 g / L by supplementing with 800 g / L of glucose mother liquor. Fermentation was completed after 120 h. The oil was collected to obtain the fermentation product of Schizochytrium.

[0011] Furthermore, the DHA content in the Schizochytrium fermentation product was 57.1%, the Sn-2 DHA content was 69.2%, and the DHA / DPA ratio was 10.8; the lipid yield of Schizochytrium increased by 49.1% compared with the original fermentation conditions. Alternatively, the concentration of shikonin added is 20 μg / L, and the addition time is 18h.

[0012] The fermentation product of Schizochytrium was prepared using the control method described above.

[0013] Algal powder prepared using the fermentation products of Schizochytrium as described above.

[0014] The method for preparing algae powder as described above includes the following steps: The fermentation product of Schizochytrium was spray-dried using an LPG-10 spray dryer. The fermentation broth injection rate was set to 8 L / hour, and the inlet air temperature was set to 180℃ to ensure rapid evaporation of moisture and avoid polyunsaturated fatty acids in Schizochytrium. The outlet air temperature was controlled at 80℃ to obtain algal powder.

[0015] The application of algae powder in food preparation as described above.

[0016] Furthermore, the food is a food with high DHA content and high sn-2 DHA content, including milk, fish oil, goat milk and eggs.

[0017] Furthermore, when the algae powder was applied to dairy cows, at a rate of 6g algae powder / kg feed, after 30 days, the DHA content in the milk reached and remained stable at 350mg / kg, and the sn-2 DHA content remained stable at 250mg / kg. After feeding rainbow trout with the algae powder continuously for 60 days, at a rate of 100g algae powder / kg feed, the DHA content in the fish oil reached 7.7 mg / kg, and the sn-2 DHA content reached 5.2 mg / kg. After feeding goats with the algae powder continuously for 45 days, at a rate of 5g algae powder / kg feed, the DHA content in goat milk reached and remained stable at 500mg / kg, and the sn-2 DHA content remained stable at 320mg / kg. After feeding laying hens with the algae powder continuously for 13 days, at a rate of 100g algae powder / kg chicken feed, the DHA content in the eggs reached and remained stable at 310mg / kg.

[0018] The advantages and positive effects of this invention are as follows: 1. This invention provides a fermentation strategy for producing high-quality DHA algal oil using Schizochytrium. By adding shikonin to the Schizochytrium fermentation medium, the Schizochytrium produces lipids with high DHA content, high DHA / DPA ratio, and high sn-2 DHA content. The obtained Schizochytrium lipids not only have a significantly higher total DHA content than traditional fermentation strategies, but also show a significantly higher DHA / DPA ratio and sn-2 DHA content. Furthermore, applying this regulation method to the preparation of Schizochytrium lipids and algal powder makes the obtained Schizochytrium lipids and algal powder more suitable for application in the food industry.

[0019] 2. In this invention, the fermentation of Schizochytrium HX-308 induced by Shikonin increases the total DHA content in lipids from 45.7% to 57.1%, the proportion of DHA at the sn-2 position from 42.6% to 69.2%, and the DHA / DPA ratio from 2.8 to 10.8. This strategy simultaneously optimizes the three-dimensional indicators of content, position, and purity, breaking through the bottleneck of microalgal oil bioavailability.

[0020] 3. In this invention, the lipid yield of Schizochytrium HX-308 induced by shikonin was increased from 65.2 g / L to 97.2 g / L, an increase of 49.1%. This strategy also increased the lipid yield of other Schizochytrium strains by 38.2%-59.8%, and the trace amount of shikonin did not lead to a significant increase in cost.

[0021] 4. This invention uses empirical evidence from four major economic animals—dairy cows, rainbow trout, goats, and laying hens—to demonstrate the core value of this technology in transforming into functional foods.

[0022] 5. This invention has been verified to be effective with three strains: Schizochytrium sp. HX-308 (CCTCC M 209059), ATCC 20888, and ATCCPRA-276. The process is compatible with mainstream commercial strains, avoiding the narrowing of the authorized scope caused by limiting to a single strain. It can cover 90% of the industrial production strains of Schizochytrium worldwide (giants such as Cargill and DSM use ATCC strains).

[0023] 6. The method of this invention can promote the synthesis of DHA and the selective storage of sn-2 DHA during the growth of Schizochytrium, thereby improving the quality of DHA in Schizochytrium oil and algal powder. The fermentation strategy of this invention increases the proportion of sn-2 DHA in Schizochytrium oil to 69.2%, which can simulate the structure of breast milk (human milk sn-2 DHA ≈ 70%). This technology breaks through the limitation of existing technologies that only focus on the "total amount of DHA", and can help related DHA products seize the high-end market of infant formula and special medical foods. Attached Figure Description

[0024] Figure 1 This is a graph showing the changes in lipid production of Schizochytrium HX-308 under different addition times of 10 μg / L Shikonin in this invention; Figure 2 This is a graph showing the change in lipid production of Schizochytrium HX-308 under different addition amounts at the optimal addition time of shikonin in this invention. Figure 3 This is a diagram showing the optimized addition time of the present invention under the condition of confirming the optimal amount of shikonin added to Schizochytrium HX-308. Figure 4 This is a comparison chart of the fatty acid ratio of oil in Schizochytrium HX-308 under the optimal addition time and amount of shikonin and the fatty acid ratio of oil in normal fermentation. Figure 5 This is a comparison chart of the proportion of fatty acids at the sn-2 position of the oil in *Schizochytrium HX-308* under the optimal addition time and amount of shikonin, and the proportion of fatty acids at the sn-2 position of the oil in normal fermentation.

[0025] The Schizochytrium sp. HX-308 strain used in this invention is a strain already existing in the art. This strain has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC No. M209059, as disclosed in Chinese patent publication CN116478835A. It is currently stored at -80 °C in 50% (v / v) glycerol. Detailed Implementation

[0026] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0027] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0028] A method for regulating the DHA content, DHA / DPA ratio, and sn-2 DHA percentage in the fermentation products of Schizochytrium oleiferum, wherein the method involves feeding the oil-producing Schizochytrium oleiferum into a batch fermentation culture, and adding shikonin during the fermentation culture process.

[0029] Furthermore, the concentration of the added shikonin is between 1 μg / L and 50 μg / L, and the addition time of the shikonin is between 6h and 18h. Alternatively, the oil-producing schistocytrium includes Schistocytrium. Schizochytrium sp. HX-308, Schizochytrium ATCC 20888 and Schizochytrium ATCC PRA-276.

[0030] Furthermore, it includes the following steps: Single colonies of *Schizochytrium* were selected and inoculated into seed culture medium. After incubation at 28°C and 180 rpm for 24 h, they were used as first-generation seed cultures. 1 mL of first-generation seed culture was inoculated into 50 mL of seed culture medium and incubated at 28°C and 180 rpm for 24 h to obtain second-generation seed cultures. 1 mL of second-generation seed culture was inoculated into 50 mL of seed culture medium and incubated at 28°C and 180 rpm for 24 h to obtain third-generation seed cultures, which were used as inoculum for fermentation. Fermentation culture: 10 mL of third-generation seeds were inoculated into 90 mL of fermentation medium. 20 μg / L of shikonin was added after 18 h of fermentation. The culture was carried out at 28℃ and 180 r / min in a shaker. The glucose concentration in the fermentation medium was measured every 12 h. The glucose concentration in the fermentation medium was maintained above 50 g / L by supplementing with 800 g / L of glucose mother liquor. Fermentation was completed after 120 h. The oil was collected to obtain the fermentation product of Schizochytrium.

[0031] Furthermore, the DHA content in the Schizochytrium fermentation product was 57.1%, the Sn-2 DHA content was 69.2%, and the DHA / DPA ratio was 10.8; the lipid yield of Schizochytrium increased by 49.1% compared with the original fermentation conditions. Alternatively, the concentration of shikonin added is 20 μg / L, and the addition time is 18h.

[0032] The fermentation product of Schizochytrium was prepared using the control method described above.

[0033] Algal powder prepared using the fermentation products of Schizochytrium as described above.

[0034] The method for preparing algae powder as described above includes the following steps: The fermentation product of Schizochytrium was spray-dried using an LPG-10 spray dryer. The fermentation broth injection rate was set to 8 L / hour, and the inlet air temperature was set to 180℃ to ensure rapid evaporation of moisture and avoid polyunsaturated fatty acids in Schizochytrium. The outlet air temperature was controlled at 80℃ to obtain algal powder.

[0035] The application of algae powder in food preparation as described above.

[0036] Furthermore, the food is a food with high DHA content and high sn-2 DHA content, including milk, fish oil, goat milk and eggs.

[0037] Furthermore, when the algae powder was applied to dairy cows, at a rate of 6g algae powder / kg feed, after 30 days, the DHA content in the milk reached and remained stable at 350mg / kg, and the sn-2 DHA content remained stable at 250mg / kg. After feeding rainbow trout with the algae powder continuously for 60 days, at a rate of 100g algae powder / kg feed, the DHA content in the fish oil reached 7.7 mg / kg, and the sn-2 DHA content reached 5.2 mg / kg. After feeding goats with the algae powder continuously for 45 days, at a rate of 5g algae powder / kg feed, the DHA content in goat milk reached and remained stable at 500mg / kg, and the sn-2 DHA content remained stable at 320mg / kg. After feeding laying hens with the algae powder continuously for 13 days, at a rate of 100g algae powder / kg chicken feed, the DHA content in the eggs reached and remained stable at 310mg / kg.

[0038] Specifically, the relevant preparation and testing methods are as follows: In the following examples, Schizochytrium Schizochytrium sp. HX-308 is currently deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 209059, and has been published in Chinese patent publication document CN101575584A; Schizochytrium Schizochytrium sp. was purchased from the American Type Culture Collection (ATCC20888); it is also disclosed in Chinese patent publication CN120888620A.

[0039] Schizochytrium Schizochytriumsp. was purchased from the American Type Culture Collection (ATCCPRA-276); it is also disclosed in Chinese patent publication CN120888620A.

[0040] Unless otherwise specified, room temperature refers to 25±5℃.

[0041] The method for determining biomass is as follows: Take 1 mL of the fermentation broth that has just finished fermentation, centrifuge at 4000 r / min for 5 min, discard the supernatant, add 1 mL of 2% NaCl solution to wash once, then wash once with 1 mL of distilled water, dry at 105℃ to constant weight, and finally weigh and calculate.

[0042] The culture media used in the following examples are as follows: The plate culture medium had a pH of 5.6 and included: 50 g / L glucose, 5 g / L yeast extract, 8 g / L sodium sulfate, 4 g / L magnesium sulfate, 8 g / L ammonium sulfate, 2 g / L potassium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 10 g / L monosodium glutamate, 2 mg / L zinc sulfate heptahydrate, 0.04 mg / L cobalt chloride hexahydrate, 4 mg / L copper sulfate pentahydrate, 2 mg / L nickel sulfate hexahydrate, 10 mg / L ferric sulfate heptahydrate, 4 mg / L manganese chloride tetrahydrate, 0.04 mg / L sodium molybdate dihydrate, 20 g / L agar powder, 10 mg / L vitamin B6, and 0.1 mg / L vitamin B12, with water as the solvent. The culture medium was sterilized using an autoclave at 115°C for 30 minutes.

[0043] The seed culture medium for the seed culture solution had a pH of 5.6 and included: 50 g / L glucose, 5 g / L yeast extract, 8 g / L sodium sulfate, 4 g / L magnesium sulfate, 8 g / L ammonium sulfate, 2 g / L potassium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 10 g / L monosodium glutamate, 2 mg / L zinc sulfate heptahydrate, 0.04 mg / L cobalt chloride hexahydrate, 4 mg / L copper sulfate pentahydrate, 2 mg / L nickel sulfate hexahydrate, 10 mg / L ferric sulfate heptahydrate, 4 mg / L manganese chloride tetrahydrate, 0.04 mg / L sodium molybdate dihydrate, 10 mg / L vitamin B6, and 0.1 mg / L vitamin B12, with water as the solvent. The culture medium was sterilized using an autoclave at 115°C for 30 minutes.

[0044] The fermentation medium had a pH of 5.6 and consisted of: 80 g / L glucose, 5 g / L yeast extract, 8 g / L sodium sulfate, 4 g / L magnesium sulfate, 8 g / L ammonium sulfate, 2 g / L potassium chloride, 0.6 g / L potassium sulfate, 1 g / L potassium dihydrogen phosphate, 10 g / L monosodium glutamate, 2 mg / L zinc sulfate heptahydrate, 0.04 mg / L cobalt chloride hexahydrate, 4 mg / L copper sulfate pentahydrate, 2 mg / L nickel sulfate hexahydrate, 10 mg / L ferric sulfate heptahydrate, 4 mg / L manganese chloride tetrahydrate, 0.04 mg / L sodium molybdate dihydrate, 10 mg / L vitamin B6, and 0.1 mg / L vitamin B12, with water as the solvent. The medium was sterilized using an autoclave at 115°C for 30 minutes.

[0045] Example 1. Determination of the dosage and timing of addition of shikonin. Selecting Schizochytrium Schizochytrium A single colony of sp. HX-308 was inoculated into a 250 mL Erlenmeyer flask (containing 50 mL of seed culture medium) and cultured at 28°C and 180 rpm for 24 h to obtain the first-generation seed culture. 1 mL of the first-generation seed culture was inoculated into a 250 mL Erlenmeyer flask (containing 50 mL of seed culture medium) and cultured at 28°C and 180 rpm for 24 h to obtain the second-generation seed culture. 1 mL of the second-generation seed culture was inoculated into a 250 mL Erlenmeyer flask (containing 50 mL of seed culture medium) and cultured at 28°C and 180 rpm for 24 h to obtain the third-generation seed culture, which was used as the inoculum for fermentation.

[0046] 1. Shake-flask fermentation culture: Take 10 mL of tertiary seed culture and inoculate it into a 250 mL Erlenmeyer flask (containing 90 mL of fermentation medium). Add 10 μg / L of shikonin at 0 h / 6 h / 12 ​​h / 18 / 24 / 30 / 36 / 42 h of fermentation. Culture in a shaker at 28℃ and 180 r / min. Take a sample every 12 h to detect the glucose concentration in the fermentation medium. Supplement with glucose stock solution (800 g / L) to maintain the glucose concentration in the fermentation medium above 50 g / L. Fermentation ends after 120 h, and the oil is collected.

[0047] 2. Collect the bacterial cells and extract the oil, including the following steps: (1) Collect 50 mL of fermentation broth after fermentation culture, add NaOH solution to adjust pH to 10-12, add 150-300 mg of cell wall breaking enzyme, and shake at 40-60℃ and 100-200 r / min for 5-15 h; (2) Cool to room temperature and add 50 mL of anhydrous ethanol to inactivate the cell wall-breaking enzyme; (3) Add 50 mL of n-hexane to extract the oil: After extraction, the organic phase was allowed to stand for 5 hours. Once the upper and lower layers were clearly separated, the upper organic phase was collected. The upper organic phase was then removed and placed in a rotary evaporator flask. The organic phase was then evaporated by rotary evaporation at 45°C in a water bath and at 120 r / min. Once no more organic phase was evaporated, the rotary evaporator flask was removed and dried in a 60°C oven until its weight no longer changed. The oil was then weighed to obtain the oil. 3. After determining the optimal addition time of shikonin, the optimal addition amount of shikonin was optimized. At the optimal addition time, 1, 2, 5, 10, 20, 30, and 50 μg / L of shikonin were added to the fermentation medium, and the mixture was cultured at 28℃ and 180 r / min for 120 h. The oil was collected and the fatty acid composition was measured.

[0048] 4. After determining the optimal dosage of shikonin, the addition time was further optimized by incubating the product in a shaker at 28℃ and 180r / min for 120h, collecting the oil, and determining the optimal conditions for adding shikonin.

[0049] 5. Determine the sn-2 DHA content in oils, including the following steps: (1) Removal of phospholipids: ① Place about 10 mL of the oil obtained from fermentation into a bottle, seal it, and heat it in a water bath to about 90°C. Then filter / squeeze to remove insoluble matter and obtain the sample.

[0050] ② Weigh 2g of the sample obtained in the previous step into a clean beaker, heat it to 80℃, add 2.0mL of ultrapure water, and stir thoroughly to hydrate it.

[0051] ③ Centrifuge in centrifuge tubes at 12,000 rpm for 5 minutes. The upper layer is glycerides, the middle layer is phospholipids, and the lower layer is aqueous phase. Try to collect the upper glycerides A.

[0052] (2) Preparation of 2-glycerol monoester ① Add 1 g of upper glycerol ester A and 4 g of anhydrous ethanol to the reaction vessel, and then add 0.03 g (3%, w / w, mass concentration) of lipase to start the alcoholysis reaction. The reaction temperature is 23℃, and the mixture is stirred continuously at 350 r / min for 4 h.

[0053] ② Centrifuge the reaction mixture at 12,000 rpm for 5 minutes, then filter it through an organic filter membrane to remove the lipase.

[0054] ③ Dissolve the product in 15 mL of n-hexane, then add 6 mL of 75% ethanol-water solution, and transfer the mixture to a separation funnel to allow it to stand and separate into layers.

[0055] ④ After the two layers are formed, the upper layer containing esters and unreacted triglyceride residues is removed, and the extract layer containing 2-MAG, i.e. the lower organic phase, is retained.

[0056] ⑤ Add 10 mL of a mixed solution of ethanol and n-hexane (V(ethanol):V(n-hexane) = 90:10 (volume ratio)) to the lower organic phase, centrifuge at 3500 r / min for 1 min, and collect the upper n-hexane phase containing 2-MAG for gas phase detection to analyze the DHA and sn-2 DHA content.

[0057] (3) Methylation The sample to be analyzed by gas chromatography is methylated using the following steps: ① Add 1 ml of 1M potassium hydroxide methanol solution to a 1.5 ml centrifuge tube, add 20 μl of grease and mix well. Then add the mixture to a 20 ml volumetric flask, and add 2 ml of 1M potassium hydroxide methanol solution to the volumetric flask (rinse the 1.5 ml centrifuge tube and draw as much grease as possible into the volumetric flask). Mix well and incubate in a 65°C water bath for 17 min, then cool to room temperature. ② Add 2 ml of boron trifluoride-ether mixture (boron trifluoride: ether = 3:7, volume ratio), mix well, and then incubate in a water bath at 65°C for 7 min; ③ Add 2 ml of saturated potassium chloride solution and shake well, then add 3 ml of n-hexane (chromatographic grade) and let stand to allow the layers to separate. ④ Pour into a small centrifuge tube, take the upper n-hexane phase and pass it through a microporous membrane to remove impurities; ⑤ Seal and store before gas chromatography.

[0058] 5. Gas phase detection and analysis of fatty acids, the specific procedure is as follows: Analysis was performed using a GC-2010 (Shimadzu, Japan) gas chromatography system equipped with a DB-23 capillary column (60 m). A 0.22 mm column and a flame ionization detector (FID) were used. Nitrogen was used as the carrier gas. The injection volume was 1 μL, and the injection temperature was 250 °C. The column temperature was increased from 100 °C to 200 °C at a rate of 25 °C / min, then increased to 230 °C at a rate of 4 °C / min and held for 9 min. The FID detector temperature was 280 °C. Different fatty acid compositions were identified by comparison with Sigma standards (related external standards, Sigma, USA). The content of individual fatty acids was calculated from the peak area on the chromatogram using non-endogenous fatty acids (C19:0) as internal standards.

[0059] The results of the time-based filtering for adding Zicrolimus are as follows: Figure 1 As shown. Although adding shikonin at 0h will cause the fissuri fungus to... SchizochytriumWhile lipid production in *Schizochytrium sp.* HX-308 decreased, the addition of shikonin at other time points promoted lipid accumulation in *Schizochytrium sp.*. Specifically, adding shikonin between 6 and 18 hours gradually increased lipid production, but adding it after 18 hours resulted in lower lipid production compared to adding it at 18 hours. Ultimately, adding 10 μg / L shikonin at 18 hours yielded 91.3 g / L of lipids, while adding 10 μg / L at 12 hours and 24 hours yielded 84.7 g / L and 87.6 g / L of lipids, respectively. The control group without shikonin only yielded 65.2 g / L of lipids. Therefore, adding 10 μg / L shikonin at the optimal 18 hours increased lipid production in *Schizochytrium sp.* by 40.0%.

[0060] The screening results of the amount of zikonin added are as follows: Figure 2 As shown in the figure, the addition of all concentrations of shikonin increased lipid production in *Schizochytrium*. Specifically, the addition of 1-20 μg / L shikonin over 18 hours gradually increased lipid production, but above 20 μg / L, lipid production decreased compared to 20 μg / L. Ultimately, adding 20 μg / L shikonin at 18 hours yielded 97.2 g / L of lipids, while adding 10 μg / L and 30 μg / L yielded 91.3 g / L and 92.7 g / L, respectively. Furthermore, adding 10 μg / L shikonin at 18 hours yielded 91.3 g / L of lipids, while only adding 20 μg / L achieved 93.9% of the expected lipid production. Therefore, adding the optimal amount of 20 μg / L shikonin at the optimal 18h yields the highest lipid production, and the lipid production obtained is 49.1% higher than that obtained by normal fermentation of Schizochytrium.

[0061] Furthermore, since the addition time of shikonin was determined at a concentration of 10 μg / L, we further confirmed that maintaining the shikonin addition level at 20 μg / L and comparing the fermentation results with shikonin addition times of 12 h and 24 h were also considered. The comparison results are as follows: Figure 3As shown, when the amount of shikonin added is 20 μg / L, adding it at 12 h yields 91.5 g / L of lipids, adding it at 18 h yields 97.2 g / L of lipids, and adding it at 24 h yields 93.7 g / L of lipids. The lipid yields obtained by adding 20 μg / L shikonin at 12 h and 24 h are only 94.1% and 96.4% of the lipid yield obtained by adding shikonin at 18 h, respectively. Therefore, 18 h is the optimal time for adding shikonin. In summary, by determining the optimal time for adding shikonin to be 18 h and the optimal amount to be 20 μg / L, the lipid yield of Schizochytrium under the optimal conditions was increased by 49.1% compared to the original fermentation conditions, confirming the correctness of our fermentation strategy. It can also be seen that the two conditions of 18 h and 20 μg / L shikonin in the method of this invention have a synergistic effect, which can synergistically improve the lipid yield in the prepared Schizochytrium oil.

[0062] Furthermore, we analyzed the fermentation performance of each experimental group, and the results are shown in Table 1. Under normal fermentation conditions, the *Schizochytrium* oil contained 45.7% DHA, with a sn-2 DHA content of 42.6% and a DHA / DPA ratio of 2.8. After 18 hours of fermentation with 10 μg / L shikonin, the *Schizochytrium* oil contained 52.1% DHA, with a sn-2 DHA content of 60.4% and a DHA / DPA ratio of 8.4. After 18 hours of fermentation with 20 μg / L shikonin, the *Schizochytrium* oil contained 57.1% DHA, with a sn-2 DHA content of 69.2% and a DHA / DPA ratio of 10.8. After 12 hours of fermentation with 20 μg / L shikonin, the *Schizochytrium* oil contained 53.3% DHA, with a sn-2 DHA content of 59.7% and a DHA / DPA ratio of 8.9. After 24 hours of fermentation with 20 μg / L shikonin, the *Schizochytrium* oil contained 54.2% DHA, with a sn-2 DHA content of 45.7% and a sn-2 DHA ratio of 42.6%. The DHA content was 62.4%, and the DHA / DPA ratio was 8.1. Therefore, adding the optimal 20 μg / L shikonin at the optimal 18h not only yielded the highest lipid yield but also resulted in the highest DHA and sn-2 DHA content in the oil, with a significantly higher DHA / DPA ratio than other groups. Therefore, adding the optimal 20 μg / L shikonin at the optimal 18h for Schizochytrium fermentation resulted in oil of higher quality than other experimental groups.

[0063] It can also be seen that the two conditions of 18h and 20 μg / L shikonin in the method of the present invention have a synergistic effect, which can synergistically improve the DHA content, sn-2 DHA content and DHA / DPA value in the prepared Schizochytrium oil.

[0064] Table 1. Performance differences among the optimized fermentation groups of *Schizochytrium* with added shikonin.

[0065] Fatty acid analysis results under the optimal dosage and addition time conditions of shikonin are as follows: Figure 4 As shown in Table 2, under the optimal dosage and time of shikonin addition, the DHA content in the *Schizochytrium* oil was 57.1%, and the DPA content was 4.9%, while in the group without shikonin addition, the DHA content was 45.7%, and the DPA content was as high as 16.5%. This indicates that under the optimal dosage and time of shikonin addition, not only was the oil yield of *Schizochytrium* increased, but the DHA content in the oil was also significantly increased, and the DPA content was significantly decreased, indicating that the quality of *Schizochytrium* oil was significantly improved. Furthermore, under the optimal dosage and time of shikonin addition, the C14:0 content in the *Schizochytrium* oil was 3.1%, while the C16:0 content was as high as 27.3%, while in the group without shikonin addition, the C14:0 content was as high as 14.1%, and the C16:0 content was 20.5%. This indicates that the fatty acid profile of *Schizochytrium* oil was significantly altered, proving that shikonin has a comprehensive effect on *Schizochytrium* oil. Furthermore, the DHA / DPA ratio in the oil after fermentation optimization with the addition of shikonin was 10.8, while the DHA / DPA ratio in the unoptimized fermentation group was only 2.8. This indicates that the oil obtained through shikonin-optimized fermentation has a higher omega-3 DHA / omega-6 DPA ratio, better meeting the market demand for omega-3 oils.

[0066] Table 2. Differences in fermentation performance of Schizochytrium before and after optimization with the addition of shikonin.

[0067] The results of the analysis of the sn-2 fatty acid under the optimal addition amount and time conditions of shikonin are as follows: Figure 5As shown in Table 1, under the optimal dosage and time of shikonin addition, the sn-2 position DHA content in *Schizochytrium* oil was 69.2%, and the DPA content was 6.1%, while in the group without shikonin addition, the sn-2 position DHA content was 42.6%, and the DPA content was as high as 18.3%. This indicates that under the optimal dosage and time of shikonin addition, not only was the DHA content of *Schizochytrium* oil increased, but the sn-2 DHA content was also significantly increased. Consistent with the DPA content, the sn-2 DPA content was also significantly decreased, indicating a significant improvement in the quality of *Schizochytrium* oil. Furthermore, under the optimal dosage and time of shikonin addition, the sn-2 position C14:0 content in *Schizochytrium* oil was 4.5%, and the C16:0 content was 15.8%, while in the group without shikonin addition, the sn-2 position C14:0 content was as high as 13.2%, and the C16:0 content was as high as 21.3%. This indicates that the fatty acid profile at the sn-2 position in the oil of Schizochytrium is also significantly altered, proving that the effect of shikonin on the oil of Schizochytrium is comprehensive. However, overall, the accumulation of sn-2 DHA content is more significant under the condition of shikonin addition.

[0068] In summary, under the optimal addition amount and time of shikonin, the lipid production of Schizochytrium HX-308 was significantly increased, and the DHA content, DHA / DPA ratio and sn-2 DHA content in the oil were also significantly increased, thus improving the quality of Schizochytrium oil.

[0069] Example 2. Fermentation of Schizochytrium ATCC 20888 under optimal conditions for the addition of shikonin. The specific fermentation steps for Schizochytrium ATCC 20888 are the same as in Example 1 (adding the optimal amount of 20 μg / L Shikonin at the optimal 18h).

[0070] The method for obtaining oil through fermentation and related detection is the same as in Example 1.

[0071] Table 3. Fermentation performance of Schizochytrium ATCC 20888 under optimal conditions for the addition of shikonin.

[0072] The fermentation results are shown in Table 3. Before fermentation optimization, the proportions of C14:0 in the oil from *Schizochytridacna ATCC 20888* were 12.3%, C16:0 was 19.3%, DPA was 19.2%, and DHA was 45.2%, with a DHA / DPA ratio of only 2.4. After fermentation optimization, the proportions of C14:0 in the oil were only 5.3%, while the proportion of C16:0 was as high as 24.7%. The proportion of DPA decreased to 5.2%, and the proportion of DHA increased to 52.7%, with a DHA / DPA ratio as high as 10.1. This indicates that under the optimal addition amount and time of shikonin, not only was the DHA content in the oil significantly increased, but the DPA content was also significantly decreased. Furthermore, the omega-3 DHA / omega-6 DPA ratio in the oil was higher, better meeting the market demand for omega-3 oils. This demonstrates that the quality of *Schizochytridacna ATCC 20888* oil was significantly improved. Furthermore, before fermentation optimization, the sn-2 position DHA content in the oil from *Schizochytrium ATCC 20888* was 43.1%, while after optimization, it increased to 66.3%. This indicates that under the optimal addition amount and time of shikonin, not only was the DHA content in *Schizochytrium ATCC 20888* oil increased, but the sn-2 DHA content was also significantly increased. Consistent with DPA content, the sn-2 DPA content was also significantly reduced, indicating a significant improvement in the quality of *Schizochytrium ATCC 20888* oil. Moreover, before fermentation optimization, the oil yield from *Schizochytrium ATCC 20888* was 64.1 g / L, while after optimization, it increased to 88.6 g / L, representing a 38.2% increase.

[0073] In summary, under the optimal addition amount and time of shikonin, the lipid production of Schizochytrium ATCC 20888 was significantly increased, and the DHA content, DHA / DPA ratio and sn-2 DHA content in the oil were also significantly increased. This indicates that the shikonin fermentation conditions are suitable for the DHA oil fermentation of Schizochytrium ATCC 20888.

[0074] Example 3. Fermentation of Schizochytrium ATCC PRA-276 under optimal conditions for the addition of shikonin. The specific fermentation steps for Schizochytrium ATCC PRA-276 are the same as in Example 1 (adding the optimal amount of 20 μg / L Shikonin at the optimal 18h).

[0075] The method for obtaining oil through fermentation and related detection is the same as in Example 1.

[0076] Table 4. Fermentation performance of Schizochytrium ATCC PRA-276 under optimal conditions for the addition of shikonin.

[0077] The fermentation results are shown in Table 4. Before fermentation optimization with *Schizochytrium ATCC PRA-276*, the proportion of C14:0 in the oil was 10.7%, C16:0 was 21.5%, DPA was 17.9%, and DHA was 42.6%, with a DHA / DPA ratio of only 2.4. After fermentation optimization with *Schizochytrium ATCC PRA-276*, the proportion of C14:0 in the oil was only 4.1%, the proportion of C16:0 was as high as 25.8%, the proportion of DPA decreased to 6.2%, the proportion of DHA increased to 55.9%, and the DHA / DPA ratio reached 9.0. This indicates that under the optimal addition amount and time of shikonin, not only was the DHA content in the oil significantly increased, but the DPA content was also significantly decreased, and the omega-3 DHA / omega-6 DPA ratio in the oil was higher, better meeting the market demand for omega-3 oils. This shows that the quality of *Schizochytrium ATCC PRA-276* oil was significantly improved. Furthermore, before fermentation optimization, the sn-2 position DHA content in the oil from *Schizochytrium ATCC PRA-276* was 40.3%, while after optimization, it increased to 68.1%. This indicates that under the optimal addition amount and time of shikonin, not only was the DHA content in *Schizochytrium ATCC PRA-276* oil increased, but the sn-2 DHA content was also significantly increased. Consistent with DPA content, the sn-2 DPA content was also significantly decreased, indicating a significant improvement in the quality of *Schizochytrium ATCC PRA-276* oil. Moreover, before fermentation optimization, the oil yield from *Schizochytrium ATCC PRA-276* was 57.2 g / L, while after optimization, it increased to 91.4 g / L, representing a 59.8% increase in oil yield.

[0078] In summary, under the optimal addition amount and time of shikonin, the lipid production of Schizochytrium ATCC PRA-276 was significantly increased, and the DHA content, DHA / DPA ratio and sn-2 DHA content in the oil were also significantly increased. This indicates that the shikonin fermentation conditions are suitable for the DHA oil fermentation of Schizochytrium ATCC PRA-276.

[0079] Example 4. Feeding Experiment with Dairy Cows The *Schizochytridactylogyrus* fermentation broths obtained before and after fermentation optimization (i.e., normal fermentation) were used in Example 1. Optimized fermentation conditions involved adding 20 μg / L of shikonin to *Schizochytridactylogyrus* HX-308 fermentation culture for 18 hours, incubating at 28°C and 180 rpm on a shaker, and sampling the glucose concentration in the fermentation medium every 12 hours. A glucose stock solution (800 g / L) was added to maintain the glucose concentration above 50 g / L. Fermentation was completed after 120 hours, and the fermentation broth was recovered. Normal fermentation conditions involved incubating the *Schizochytridactylogyrus* HX-308 strain fermentation broth at 28°C and 180 rpm on a shaker, sampling the glucose concentration in the fermentation medium every 12 hours, and adding a glucose stock solution (800 g / L) to maintain the glucose concentration above 50 g / L. Fermentation was completed after 120 hours, and the fermentation broth was recovered. 100L of each sample was spray-dried using an LPG-10 spray dryer. The fermentation broth injection rate was set to 8L / hour. The inlet air temperature was set to 180℃ during spray drying to ensure rapid evaporation of moisture and avoid polyunsaturated fatty acids in the fissicryte. The outlet air temperature was controlled at 80℃ to obtain algal powder.

[0080] 20.2 kg and 13.4 kg of algal powder were obtained respectively. The two types of algal powder were mixed with dairy cow feed at a ratio of 6 g algal powder / kg feed, and fed to dairy cows continuously. The milk was collected and compared with the milk produced by dairy cows fed with normal feed continuously. The contents of DHA and sn-2 DHA in the milk were detected. The results are shown in Table 5.

[0081] Table 5. Changes in DHA and sn-2DHA in milk from dairy cows fed with algae powder obtained from normal fermentation and optimized fermentation with shikonin.

[0082] In Table 5, the algae powder in "Added algae powder" refers to the algae powder prepared from the Schizochytrium fermentation broth before fermentation optimization, while the algae powder in "Added optimized fermented algae powder" refers to the algae powder prepared from the Schizochytrium fermentation broth after fermentation optimization.

[0083] Table 5 shows that when dairy cows were fed a normal diet without Schizochytrium algae powder on day 1, the DHA content in the milk reached approximately 11 mg / kg, and the sn-2 DHA content was approximately 4 mg / kg; while when dairy cows were fed a normal algae powder diet on day 1, the DHA content in the milk was approximately 15 mg / kg, and the sn-2 DHA content was approximately 6 mg / kg; when dairy cows were fed an optimized algae powder diet on day 1, the DHA content in the milk was approximately 16 mg / kg, and the sn-2 DHA content was approximately 7 mg / kg. After 15 days of feeding dairy cows with regular feed, the DHA content in milk reached approximately 12 mg / kg, and the sn-2 DHA content was approximately 5 mg / kg. In contrast, cows fed with normal algae powder had a DHA content of approximately 188 mg / kg and a sn-2 DHA content of approximately 102 mg / kg. Cows fed with the optimized algae powder had a DHA content of approximately 243 mg / kg and a sn-2 DHA content of approximately 176 mg / kg. Finally, after 30 days of continuous feeding dairy cows with the fermented and optimized Schizochytrium algae powder corresponding to Example 1, the DHA content in milk reached and remained stable at approximately 350 mg / kg, and the sn-2 DHA content remained stable at approximately 250 mg / kg. In contrast, cows fed with normal algae powder required 60 days of feeding to reach and maintain a DHA content of approximately 300 mg / kg and a sn-2 DHA content of approximately 140 mg / kg. Therefore, dairy cows fed with Schizochytrium algae powder obtained through zikonin fermentation are more likely to accumulate DHA and sn-2 DHA in their milk, giving them a greater advantage in producing DHA-rich milk. DHA is an essential Omega-3 fatty acid, while sn-2 DHA is known as a "brain express" in its utilization pathway, playing a crucial role in the normal development of the brain and retina. In addition to DHA, DHA-rich milk also contains abundant vitamins, proteins, fats, calcium, iron, phosphorus, and other nutrients, all of which are essential for maintaining normal physiological functions. Therefore, producing high-quality DHA-rich milk (high in both DHA and sn-2 DHA) better meets the growth needs of children.

[0084] Example 5. Experiment on feeding freshwater rainbow trout The algae powder obtained in Example 4 was used to further feed freshwater rainbow trout. The two types of algae powder were mixed with rainbow trout feed at a ratio of 100g algae powder / kg feed. Thirty rainbow trout fry of similar age, size, and development were selected and divided into three groups. Each group was fed the same amount of normal feed, feed supplemented with algae powder, and feed supplemented with optimized fermented algae powder, for 60 consecutive days. The DHA and sn-2 DHA content in the fish oil was measured each time. Two rainbow trout were randomly selected from each test group for testing, and the average value was taken. The results are shown in Table 6.

[0085] Table 6. Changes in DHA and sn-2 DHA in fish oil from rainbow trout fed with Schizochytrium algae powder obtained before and after normal culture medium optimization.

[0086] In Table 6, the algae powder in "Added algae powder" refers to the algae powder prepared from the Schizochytrium fermentation broth before fermentation optimization, while the algae powder in "Added optimized fermented algae powder" refers to the algae powder prepared from the Schizochytrium fermentation broth after fermentation optimization.

[0087] The results in Table 6 show that after feeding rainbow trout with a diet containing Schizochytrium algae powder obtained in Example 4 for 15 consecutive days, the DHA content in the fish oil reached 0.9 mg / kg and the sn-2 DHA content reached 0.5 mg / kg; after feeding for 30 days, the DHA content in the fish oil reached 2.1 mg / kg and the sn-2 DHA content reached 1.4 mg / kg; after feeding for 60 days, the DHA content in the fish oil reached 7.7 mg / kg and the sn-2 DHA content reached 5.2 mg / kg. When rainbow trout are fed with normal algae powder, after 15 days of feeding, the DHA content in their fish oil is only 0.7 mg / kg, and the sn-2 DHA content is only 0.4 mg / kg; after 30 days of feeding, the DHA content is only 1.2 mg / kg, and the sn-2 DHA content is only 0.8 mg / kg; after 60 days of feeding, the DHA content is only 2.1 mg / kg, and the sn-2 DHA content is only 1.2 mg / kg. Therefore, rainbow trout fed with Schizochytrium algae powder obtained through zikonin fermentation are more likely to accumulate DHA and sn-2 DHA in their fish oil, giving them a greater advantage in improving the quality of rainbow trout. DHA is an essential Omega-3 fatty acid for the growth and development of rainbow trout. It cannot be synthesized by rainbow trout themselves and must be obtained from their food. DHA can promote the development of the nervous and visual systems in juvenile fish, improving the survival rate and health of fry. Moreover, as an edible fish, rainbow trout's core nutritional selling point is DHA, and supplementing with DHA algae powder can significantly increase the DHA content in the fish, allowing consumers to obtain more nutrients beneficial to the brain and cardiovascular system.

[0088] Example 6. Feeding experiment with dairy goats The algae powder obtained in Example 4 was further used in experiments feeding dairy goats. The two types of algae powder were mixed with dairy goat feed at a ratio of 5g algae powder / kg feed, and the goats were fed continuously. Goat milk was collected and compared with the milk produced by cows and goats fed with normal feed. The content of DHA and sn-2 DHA in the milk was detected. The results are shown in Table 7.

[0089] Table 7. Changes in DHA and sn-2 DHA in goat milk fed with Schizochytrium algae powder obtained before and after normal culture medium optimization.

[0090] In Table 7, "algae powder" refers to the algae powder prepared from the Schizochytrium fermentation broth before fermentation optimization, while "algae powder" refers to the algae powder prepared from the Schizochytrium fermentation broth after fermentation optimization.

[0091] The results in Table 7 show that after feeding goats with a diet containing the Schizochytrium algae powder obtained in Example 4 for 15 consecutive days, the DHA content in goat milk reached and remained stable at around 169 mg / kg, and the sn-2 DHA content remained stable at around 86 mg / kg; after 30 days of feeding, the DHA content in goat milk reached and remained stable at around 317 mg / kg, and the sn-2 DHA content remained stable at around 223 mg / kg; after 45 days of feeding, the DHA content in goat milk reached and remained stable at around 514 mg / kg, and the sn-2 DHA content remained stable at around 327 mg / kg; after 60 days of feeding, the DHA content in goat milk reached and remained stable at around 521 mg / kg, and the sn-2 DHA content remained stable at around 352 mg / kg. When goats are fed with normal algae powder, after 15 days, the DHA content in goat milk reaches and stabilizes at around 140 mg / kg, and the sn-2 DHA content stabilizes at around 60 mg / kg; after 30 days, the DHA content reaches and stabilizes at around 272 mg / kg, and the sn-2 DHA content stabilizes at around 146 mg / kg; after 45 days, the DHA content only reaches and stabilizes at around 410 mg / kg, and the sn-2 DHA content only stabilizes at around 242 mg / kg; after 60 days, the DHA content reaches and stabilizes at around 410 mg / kg, and the sn-2 DHA content stabilizes at around 241 mg / kg. Goat milk obtained from goats fed with ordinary feed does not contain DHA.

[0092] Therefore, goats fed with Schizochytrium algae powder obtained through zikonin fermentation are more likely to accumulate DHA and sn-2 DHA in their milk, giving them a greater advantage in producing DHA-rich goat milk. Natural raw goat milk contains almost no direct form of DHA and can only be inefficiently converted through precursors such as linoleic acid and linolenic acid. When dairy goats are fed DHA algae powder, this DHA accumulates in the fat globules of the milk. However, the algae powder obtained through fermentation optimization not only has a high DHA content but also a high sn-2 DHA content, resulting in the DHA in goat milk being mainly distributed at the sn-2 position. This form of DHA is more easily absorbed by the human body and can directly affect brain and retinal development. Compared to goat milk with artificially added DHA, this natural DHA goat milk is more recognized for its safety and naturalness, allowing it to fetch a higher price and increase farming profits.

[0093] Example 7. Feeding experiment with laying hens The algae powder obtained in Example 4 was further used in the feeding experiment on laying hens. The two types of algae powder were mixed with chicken feed at a ratio of 100g algae powder / kg chicken feed, and the chickens were fed continuously. Eggs were collected and compared with eggs laid by chickens fed with normal feed. The DHA content in the eggs was detected, and the results are shown in Table 8.

[0094] Table 8. Changes in DHA in eggs from laying hens fed with Schizochytrium algae powder obtained before and after normal culture medium optimization.

[0095] In Table 8, the algae powder in "Added algae powder" refers to the algae powder prepared from the Schizochytrium fermentation broth before fermentation optimization, while the algae powder in "Added optimized fermented algae powder" refers to the algae powder prepared from the Schizochytrium fermentation broth after fermentation optimization.

[0096] Table 8 shows that after feeding laying hens with the fermented and optimized Schizochytrium algae powder corresponding to Example 1 for 7 days, the DHA content in eggs reached and remained stable at around 269 mg / kg; after feeding the laying hens for 13 days, the DHA content in eggs reached and remained stable at around 310 mg / kg. In contrast, when laying hens were fed with normal algae powder, after 7 days the DHA content in eggs only reached and remained stable at around 202 mg / kg; after 13 days the DHA content only reached and remained stable at around 240 mg / kg. Therefore, laying hens fed with Schizochytrium algae powder obtained through zikonin fermentation are more likely to accumulate DHA in their eggs, giving them an advantage in producing DHA-rich eggs. The study found that this DHA accumulates in the egg yolk, mainly in the form of phospholipid DHA, which is more easily absorbed and utilized by the human body than the triglyceride DHA in fish oil, making it especially suitable for the elderly, children, and pregnant women.

[0097] In summary, this invention provides a fermentation control strategy for producing high-quality DHA from Schizochytrium by adding shikonin, and the application of Schizochytrium products in the functional food field. Firstly, this strategy, applied to Schizochytrium HX-308, can increase oil yield by 49.1%, with a DHA content as high as 57.1%, a sn-2 DHA proportion as high as 69.2%, and a DHA / DPA ratio increased to 10.8. Furthermore, this fermentation strategy also has a positive impact on the production of high-quality DHA algal oil from Schizochytrium ATCC 20888 and Schizochytrium ATCC PRA-276. Chinese patent publication CN120060388A increased the DHA / DPA ratio of Schizochytrium to 5.1 by adding inositol and betaine; Chinese patent publication CN119351635A controlled the pH of the fermentation broth during the Schizochytrium fermentation process by supplementing acid or alkali in stages, resulting in an oil with a sn-2 DHA content as high as 60.4%; Chinese patent publication CN119061084A achieved sn-2 DHA and carotenoid content in lipids of 57.3% and 78.3 μg / g, respectively, through a calcium ion-induced regulation strategy combined with nitrogen-limited induced fermentation. However, these strategies did not significantly improve the quality of Schizochytrium algal oil as the present invention, and the strategy of the present invention has been proven to be universal for different Schizochytrium species.

[0098] Furthermore, when this strategy was applied to regulate the fermentation of Schizochytrium HX-308 to obtain algal powder, and it was used in dairy cow feed, the DHA content in the milk reached and stabilized at around 350 mg / kg after 30 days, and the sn-2 DHA content stabilized at around 250 mg / kg. This is significantly higher than the DHA content in existing DHA-rich milk, indicating that the high sn-2 DHA content is very beneficial for DHA absorption. After feeding rainbow trout with this algal powder for 60 days, the DHA content in the fish oil reached 7.7 mg / kg, and the sn-2 DHA content reached 5.2 mg / kg. After feeding goats with this algal powder for 45 days, the DHA content in goat milk reached and stabilized at around 500 mg / kg, and the sn-2 DHA content stabilized at around 320 mg / kg. After feeding laying hens with this algal powder for 13 days, the DHA content in eggs reached and stabilized at around 310 mg / kg. These data are more widely used and have a more prominent effect than existing Chinese patent publications CN119351635A, CN116622514A and other published data.

[0099] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for regulating the DHA content, DHA / DPA ratio, and sn-2 DHA proportion in Schizochytrium fermentation products, characterized in that: The method involves feeding-in-batch fermentation of oil-producing Schizochytrium, with the addition of shikonin during the fermentation process.

2. The control method according to claim 1, characterized in that: The concentration of shikonin added is between 1 μg / L and 50 μg / L, and the addition time of shikonin is between 6h and 18h. Alternatively, the oil-producing schistocytrium includes Schistocytrium. Schizochytrium sp. HX-308, Schizochytrium ATCC20888 and Schizochytrium ATCC PRA-276.

3. The control method according to claim 1 or 2, characterized in that: The steps include the following: Single colonies of *Schizochytrium* were selected and inoculated into seed culture medium. After incubation at 28°C and 180 rpm for 24 h, they were used as first-generation seed cultures. 1 mL of first-generation seed culture was inoculated into 50 mL of seed culture medium and incubated at 28°C and 180 rpm for 24 h to obtain second-generation seed cultures. 1 mL of second-generation seed culture was inoculated into 50 mL of seed culture medium and incubated at 28°C and 180 rpm for 24 h to obtain third-generation seed cultures, which were used as inoculum for fermentation. Fermentation culture: 10 mL of third-generation seeds were inoculated into 90 mL of fermentation medium. 20 μg / L of shikonin was added after 18 h of fermentation. The culture was carried out at 28℃ and 180 r / min in a shaker. The glucose concentration in the fermentation medium was measured every 12 h. The glucose concentration in the fermentation medium was maintained above 50 g / L by supplementing with 800 g / L of glucose mother liquor. Fermentation was completed after 120 h. The oil was collected to obtain the fermentation product of Schizochytrium.

4. The control method according to claim 1, characterized in that: The fermentation product of *Schizochytrium* contained 57.1% DHA, 69.2% Sn-2 DHA, and a DHA / DPA ratio of 10.8; the lipid yield in the fermentation product was increased by 49.1% compared with the original fermentation conditions. Alternatively, the concentration of shikonin added is 20 μg / L, and the addition time is 18h.

5. The Schizochytrium fermentation product prepared by the control method according to any one of claims 1 to 4.

6. Algal powder prepared using the fermentation product of Schizochytrium as described in claim 5.

7. The method for preparing algae powder as described in claim 6, characterized in that: The steps include the following: The fermentation product of Schizochytrium was spray-dried. The inlet air temperature was set to 180℃ and the outlet air temperature was controlled at 80℃ to obtain algal powder.

8. The application of the algae powder as described in claim 6 in food preparation.

9. The application according to claim 8, characterized in that: The food products include milk, fish oil, goat milk, and eggs.

10. The application according to claim 9, characterized in that: When algae powder was applied to dairy cows, at a rate of 6g algae powder / kg feed, after 30 days, the DHA content in milk reached and remained stable at 350mg / kg, and the sn-2 DHA content remained stable at 250mg / kg. After feeding rainbow trout with algae powder continuously for 60 days, at a rate of 100g algae powder / kg feed, the DHA content in fish oil reached 7.7mg / kg, and the sn-2 DHA content reached 5.2mg / kg. After feeding goats with algae powder continuously for 45 days, at a rate of 5g algae powder / kg feed, the DHA content in goat milk reached and remained stable at 500mg / kg, and the sn-2 DHA content remained stable at 320mg / kg. After feeding laying hens with algae powder continuously for 13 days, at a rate of 100g algae powder / kg chicken feed, the DHA content in eggs reached and remained stable at 310mg / kg.

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