A fermentation method and application for enhancing the DHA production capacity of Schizochytrium phospholipids.

Abstract

This invention belongs to the field of microbial fermentation and food engineering technology, and discloses a fermentation method and application for enhancing the DHA production capacity of *Schizochytrium* phospholipids. *Schizochytrium* is fermented for 32-60 hours, with optimized addition of organic nitrogen sources and phosphates during the fermentation process. The seed culture medium formulation for *Schizochytrium* remains unchanged; only the fermentation medium formulation is modified. This invention provides a fermentation strategy to enhance the DHA production capacity of *Schizochytrium* phospholipids by optimizing the organic nitrogen sources and phosphates in the fermentation medium, enabling *Schizochytrium* to obtain a higher content of DHA phospholipids. The obtained *Schizochytrium* oil not only has a significantly higher total DHA content than traditional fermentation strategies, but also a significantly higher proportion of DHA phospholipids. Applying this method to the preparation of *Schizochytrium* algal powder yields *Schizochytrium* algal powder rich in DHA phospholipids, which is more beneficial for its application in aquatic feed.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation and food engineering technology, and in particular to a fermentation method and application for enhancing the DHA production capacity of Schizochytrium phospholipids. Background Technology

[0002] Phospholipid DHA is a complex lipid molecule formed by the combination of docosahexaenoic acid (DHA) and phospholipids (such as phosphatidylcholine and phosphatidylethanolamine). Unlike common fish oil (triglyceride-type DHA), phospholipid DHA is naturally found in marine organisms (such as krill, fish eggs / brains) and breast milk, possessing unique biological activities and physiological functions. Phospholipid DHA is amphiphilic (hydrophilic + hydrophobic groups), forming micelles for direct absorption through the intestinal wall without pancreatic enzyme hydrolysis (triglyceride-type DHA requires bile emulsification). Phospholipid DHA in the placenta and breast milk is the main source of DHA for fetuses / infants, accounting for 70% of brain lipid DHA. Phospholipid DHA inhibits hepatic lipid synthesis enzymes, protecting the cardiovascular system with superior effects compared to fish oil. Retinal photoreceptor cells are rich in DHA phospholipids (such as phosphatidylserine), and supplementation improves dark adaptation ability by 17%. Therefore, phospholipid DHA has superior bioavailability and tissue targeting, and is gradually replacing traditional fish oil as the new standard for neurological health supplements.

[0003] Schizochytrium ( Schizochytrium As a star strain in microbial DHA production, *Schizochytrium sp.* has demonstrated disruptive advantages in industrial applications, becoming the core source of global algal oil DHA (accounting for over 80% of commercial production). Furthermore, compared to obtaining fish oil DHA through fishing, algal oil DHA obtained through *Schizochytrium sp.* fermentation has advantages such as lower production costs, shorter production cycles, independence from the natural environment, smaller land area requirements, and stronger sustainability. Moreover, replacing fish oil DHA with 1 ton of *Schizochytrium sp.* DHA can reduce approximately 200 tons of fishery byproducts and 800 tons of CO2 emissions. Therefore, utilizing *Schizochytrium sp.* to produce phospholipid DHA is a feasible strategy.

[0004] However, existing research on Schizochytrium often focuses on improving the production of DHA, which involves almost no phospholipids. Chinese patent application CN104894176A uses iodoacetamide as a regulator added to the fermentation medium of Schizochytrium, effectively adjusting the DPA / DHA ratio in the oil from 0.2 to 0.39, resulting in a 69% increase in DPA production. Chinese patent application CN120060388A, by adding inositol and betaine to the fermentation broth, promotes DHA synthesis and reduces the proportion of DPA in Schizochytrium, achieving a DHA content of 51.15%. Chinese patent application CN114525312A achieves a DHA content of 50% in Schizochytrium by using a fermentation medium without additional sodium or chloride ions. Chinese patent application CN114703238A addresses the low efficiency of Schizochytrium in utilizing both glucose and glycerol by controlling the timing of glycerol supplementation, achieving a DHA content of 46.28% in the oil. However, there is almost no research on how to utilize Schizochytrium to produce phospholipid DHA. Therefore, it is of great significance to provide a fermentation method and application that enhances the phospholipid DHA production capacity of Schizochytrium. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a fermentation method and application that enhances the DHA production capacity of Schizochytrium phospholipids.

[0006] The technical solution adopted by this invention to solve its technical problem is:

[0007] A fermentation method for enhancing the DHA production capacity of Schizochytrium phospholipids includes the following steps:

[0008] Schizochytrium was fermented for 32-60 hours, and the addition of organic nitrogen source and phosphate was optimized during the fermentation process. The seed culture medium formula of Schizochytrium was not changed, only the fermentation culture medium formula of Schizochytrium was changed.

[0009] The organic nitrogen source is at least one of soybean meal powder, yeast extract, corn steep liquor powder, beef extract, and trypsin, and the phosphate is at least one of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

[0010] The final concentration of the organic nitrogen source is between 1 g / L and 10 g / L, and the final concentration of the phosphate is between 0.2 g / L and 2 g / L. These concentrations refer to the final concentrations of the organic nitrogen source and phosphate in the fermentation medium.

[0011] Furthermore, the organic nitrogen source is soybean meal powder, and its final concentration is 4 g / L.

[0012] Furthermore, the phosphate is sodium dihydrogen phosphate, and its final concentration is 1 g / L.

[0013] Furthermore, the specific steps are as follows:

[0014] (1) Take out 100 μL of the preserved Schizochytrium spp. and inoculate it into the seed culture medium at a 2% inoculation rate. Do not adjust the pH and culture at 28℃ and 230 rpm for 48 h.

[0015] (2) Inoculate 1% into seed culture medium, do not adjust pH, and incubate at 28℃ and 170rpm for 24h. OD at 24h 600 >3. And microscopic examination showed no bacterial contamination before inoculation of the next generation;

[0016] (3) 1% inoculum was inoculated into the seed culture medium, without adjusting the pH, and cultured at 28°C and 170 rpm for 24 h. OD at 24 h was... 600 >5. If microscopic examination shows no bacterial contamination, then inoculate the next generation;

[0017] (4) Inoculate 1% of the inoculum into the fermentation medium, do not adjust the pH, start fermentation at 28° and 170 rpm, and ferment for 48 hours to obtain the product.

[0018] Furthermore, the Schizochytrium includes Schizochytrium HX-308, Schizochytrium ATCC 20888, or Schizochytrium ATCC PRA-276.

[0019] Furthermore, the seed culture medium for the Schizochytrium fungus comprises: 40-60 g / L glucose, 4-6 g / L yeast extract, 5-8 g / L sodium sulfate, 2-4 g / L magnesium sulfate, 4-8 g / L ammonium sulfate, 1-2 g / L potassium chloride, 0.5-1 g / L potassium sulfate, 0.5-2 g / L potassium dihydrogen phosphate, 8-12 g / L monosodium glutamate, 1-5 mg / L zinc sulfate heptahydrate, 0.01-0.1 mg / L cobalt chloride hexahydrate, 2-6 mg / L copper sulfate pentahydrate, 1-2 mg / L nickel sulfate hexahydrate, 8-15 mg / L ferric sulfate heptahydrate, 3-5 mg / L manganese chloride tetrahydrate, 0.04 mg / L sodium molybdate dihydrate, with water as the solvent;

[0020] The fermentation medium for the Schizochytrium fungus comprises: glucose 60-100 g / L, sodium sulfate 5-12 g / L, organic nitrogen source 1-10 g / L, phosphate 0.2-2 g / L, magnesium sulfate 2-4 g / L, ammonium sulfate 4-8 g / L, potassium chloride 1-2 g / L, potassium sulfate 0.5-1 g / L, monosodium glutamate 15-20 g / L, zinc sulfate heptahydrate 1-5 mg / L, cobalt chloride hexahydrate 0.01-0.1 mg / L, copper sulfate pentahydrate 2-6 mg / L, nickel sulfate hexahydrate 1-2 mg / L, ferric sulfate heptahydrate 8-15 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.04 mg / L, vitamin B6 4-10 mg / L, vitamin B12 0.1-0.5 mg / L, and water as the solvent.

[0021] The fermentation method described above is applied in the preparation of algae powder for aquatic feed.

[0022] A Schizochytrium algae powder for aquatic feed is prepared by the following steps:

[0023] The fermentation broth obtained by the fermentation method described above is then dried and powdered.

[0024] A method for obtaining oil from a fermentation broth obtained by the fermentation method described above includes the following steps:

[0025] (1) After adjusting the pH to 10-13 by adding NaOH solution to the fermentation broth, add 0.01-0.5% of the cell wall breaking enzyme by mass of the fermentation broth, and shake at 100-200 r / min for 5-10 h at 40-60℃;

[0026] (2) Cool to room temperature and add an equal volume of anhydrous ethanol to the fermentation broth to inactivate the cell wall breaking enzyme;

[0027] (3) Add n-hexane for extraction and collect the upper organic phase;

[0028] (4) Repeat step 3) several times, combine the organic phases, evaporate the solvent, and obtain lipids, which are oils.

[0029] Furthermore, the lipids are separated to obtain phospholipids and glycerides, and the specific steps are as follows:

[0030] (1) Place the homogeneous lipid in a bottle and seal it. Heat it in a water bath to 90°C and then filter it to remove insoluble matter.

[0031] (2) Weigh the lipids, heat them to 80°C, add ultrapure water, the ratio of lipids to ultrapure water g:mL is 25:2, stir thoroughly to hydrate them;

[0032] (3) Centrifuge the precipitate in centrifuge tubes. The upper layer is glycerol, the middle layer is phospholipid, and the lower layer is aqueous phase. Collect the upper layer of glycerol and the middle layer of phospholipid separately and calculate the yield of glycerol and phospholipid.

[0033] Alternatively, the analytical method for obtaining fatty acids in phospholipids and glycerides may include the following steps:

[0034] (1) Add 1 ml of potassium hydroxide methanol solution to a 1.5 ml centrifuge tube, add 20 μl of oil to it and mix well, then add it to a 20 ml volumetric flask, add 2 ml of 1 M potassium hydroxide-methanol solution to the volumetric flask, rinse the 1.5 ml centrifuge tube, draw the oil into the volumetric flask, mix well, and then heat in a 65 °C water bath for 17 min, and cool to room temperature;

[0035] (2) Add 2 ml of boron trifluoride ether. The boron trifluoride ether is obtained by mixing boron trifluoride and ether in a volume ratio of 3:7. After mixing, heat in a water bath at 65°C for 7 min.

[0036] (3) Add 2 ml of saturated potassium chloride and shake well, then add 3 ml of n-hexane and let stand to allow the layers to separate.

[0037] (4) Remove impurities from the upper n-hexane phase by passing it through a microporous membrane;

[0038] (5) Seal and store immediately for gas chromatography.

[0039] The advantages and positive effects of this invention are as follows:

[0040] 1. This invention provides a fermentation strategy to enhance the DHA production capacity of Schizochytrium phospholipids. By optimizing the organic nitrogen source and phosphate in the Schizochytrium fermentation medium, Schizochytrium phospholipids can obtain a higher content of DHA. The obtained Schizochytrium oil not only has a significantly higher total DHA content than traditional fermentation strategies, but also a significantly higher proportion of DHA from phospholipids. Furthermore, applying this regulation method to the preparation of Schizochytrium algal powder yields Schizochytrium algal powder rich in DHA from phospholipids, which is more conducive to its application in the field of aquatic feed.

[0041] 2. The present invention adopts a fermentation optimization strategy to produce phospholipid DHA, which does not involve genetic engineering or the addition of exogenous chemical regulators. Therefore, the strategy of the present invention meets the production requirements of food-grade phospholipid DHA.

[0042] 3. The fermentation optimization strategy adopted in this invention has universal applicability to the Schizochytrium genus and has a significant promoting effect on the production of DHA phospholipids from multiple Schizochytrium genus species. Therefore, this strategy is suitable for the universal production of DHA phospholipids from the Schizochytrium genus.

[0043] 4. Currently, research on the production of phospholipid DHA using microorganisms is still in its infancy, and there are no related patent reports in China, especially regarding the production of phospholipid DHA using Schizochytrium. Therefore, this invention fills the gap in related research.

[0044] 5. The bacterial powder obtained by direct spray drying of the fermentation broth obtained by the method of the present invention is called Schizochytrium algae powder or DHA algae powder (hereinafter collectively referred to as algae powder), which can be applied in the field of aquatic feed. Attached Figure Description

[0045] Figure 1 This is a graph showing the phospholipid production of Schizochytrium HX-308 under different organic nitrogen source conditions in this invention;

[0046] Figure 2 The graph shows the phospholipid yield of HX-308 under different concentrations of soybean meal nitrogen source conditions in this invention.

[0047] Figure 3 This is a graph showing the phospholipid yield of HX-308 under different phosphate conditions in this invention;

[0048] Figure 4 This is a graph showing the phospholipid yield of HX-308 under different concentrations of sodium dihydrogen phosphate in this invention;

[0049] Figure 5 This is a graph showing the yields of phospholipids, glycerides, and total lipids of HX-308 under the optimal soybean meal concentration and sodium dihydrogen phosphate concentration conditions in this invention.

[0050] Figure 6 This is a graph showing the yields of phospholipids, glycerides, and total lipids of ATCC 20888 under the optimal soybean meal concentration and sodium dihydrogen phosphate concentration conditions in this invention.

[0051] Figure 7 This is a graph showing the production of phospholipids, glycerides, and total lipids of Schizochytrium ATCC PRA-276 under the optimal soybean meal concentration and sodium dihydrogen phosphate concentration conditions in this invention. Detailed Implementation

[0052] 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.

[0053] 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.

[0054] Specifically, the relevant preparation and testing methods are as follows:

[0055] 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 patent application CN101575584A;

[0056] Schizochytrium Schizochytrium sp. ATCC 20888 was purchased from the American Type Culture Collection, with the catalog number ATCC 20888;

[0057] Schizochytrium Schizochytrium sp. ATCC PRA-276 was purchased from the American Collection of Type Cultures (ATCC PRA-276). The culture media used in the following examples are as follows:

[0058] The seed culture medium for Schizochytrium includes: 40 g / L glucose, 4 g / L yeast extract, 5 g / L sodium sulfate, 2 g / L magnesium sulfate, 4 g / L ammonium sulfate, 1 g / L potassium chloride, 0.5 g / L potassium sulfate, 0.5 g / L potassium dihydrogen phosphate, 12 g / L monosodium glutamate, 5 mg / L zinc sulfate heptahydrate, 0.01 mg / L cobalt chloride hexahydrate, 2 mg / L copper sulfate pentahydrate, 1 mg / L nickel sulfate hexahydrate, 8 mg / L ferric sulfate heptahydrate, 3 mg / L manganese chloride tetrahydrate, and 0.04 mg / L sodium molybdate dihydrate.

[0059] The fermentation medium for Schizochytrium includes: glucose 80 g / L, sodium sulfate 5 g / L, organic nitrogen source 3 g / L, phosphate 0.8 g / L, magnesium sulfate 4 g / L, ammonium sulfate 4 g / L, potassium chloride 1 g / L, potassium sulfate 0.5 g / L, monosodium glutamate 20 g / L, zinc sulfate heptahydrate 5 mg / L, cobalt chloride hexahydrate 0.01 mg / L, copper sulfate pentahydrate 2 mg / L, nickel sulfate hexahydrate 1 mg / L, ferric sulfate heptahydrate 8 mg / L, manganese chloride tetrahydrate 3 mg / L, sodium molybdate dihydrate 0.04 mg / L, vitamin B6 10 mg / L, and vitamin B12 0.1 mg / L.

[0060] Example 1. Screening and concentration optimization of organic nitrogen sources for fermentation by Schizochytrium HX-308

[0061] The specific fermentation steps of Schizochytrium HX-308 are as follows (i.e., the steps of a fermentation method to enhance the DHA production capacity of Schizochytrium phospholipids):

[0062] 1. Take 100 μL of the preserved bacterial culture from the cryopreservation tube and inoculate it into a test tube (test tube generation 1) (5 mL seed culture medium, without pH adjustment), and incubate at 28℃ and 230 rpm for 48 h;

[0063] 2. Inoculate 1% of the culture medium into 250mL baffled shake flasks (50mL seed culture medium, pH unadjusted), and incubate at 28℃ and 170rpm for 24h (2 generations in shake flasks). OD at 24h... 600 >3. If microscopic examination shows no contamination, the next generation can be inoculated;

[0064] 3. Inoculate 1% of the culture medium into a 250mL baffled shake flask (50mL seed culture medium, pH unadjusted), and incubate at 28℃ and 170rpm for 24h (3 generations in shake flasks). OD at 24h... 600 >5. If microscopic examination shows no bacterial contamination, the next generation can be inoculated;

[0065] 4. Inoculate 1% of the culture medium into a 1000mL baffled shaker flask (100mL of fermentation medium, pH not adjusted), and start fermentation at 28℃ and 170rpm.

[0066] The fermentation medium is the same as the aforementioned Schizochytrium fermentation medium, except that the initial concentration of the organic nitrogen source is 3 g / L, and the organic nitrogen source is at least one of soybean meal, yeast extract, corn steep liquor powder, beef extract, and trypsin, with a concentration between 1 g / L and 10 g / L. Potassium dihydrogen phosphate is tentatively selected at 0.8 g / L.

[0067] Furthermore, the method for obtaining oil through fermentation culture includes the following steps:

[0068] 1. The entire cycle can reach 60 hours, for example, it can be 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 52 hours, 56 hours, or 60 hours. After the fermentation culture is completed, add NaOH solution to the fermentation broth to adjust the pH to 10-13, then add 0.01-0.5% of the fermentation broth mass of cell wall-breaking enzyme, and shake at 100-200 rpm for 5-10 hours at 40-60℃.

[0069] 2. Cool to room temperature, then add an equal volume of anhydrous ethanol to the fermentation broth to inactivate the cell wall-breaking enzyme;

[0070] 3. Add n-hexane for extraction, and collect the upper organic phase;

[0071] 4. Repeat step 3) several times, combine the organic phases, evaporate the solvent, and obtain lipids.

[0072] Furthermore, the method for separating phospholipids and glycerides from lipids obtained through fermentation culture includes the following steps:

[0073] 1. Place the homogeneous lipids in a bottle, seal it, and heat it in a water bath to about 90°C before filtering to remove insoluble matter.

[0074] 2. Weigh 25g of lipids into a clean beaker, heat to 80℃, add 2mL of ultrapure water, and stir thoroughly to hydrate it.

[0075] 3. Centrifuge the pellet in centrifuge tubes. The upper layer consists of glycerides, the middle layer of phospholipids, and the lower layer of aqueous phase. Collect the upper layer of glycerides and the middle layer of phospholipids separately, and calculate the yield of glycerides and phospholipids.

[0076] Fermentation results of different organic nitrogen sources, such as Figure 1 As shown. Fermentation was carried out for 48 hours with the addition of 3 g / L organic nitrogen source and ensuring that other fermentation medium components were identical. Lipids were extracted and phospholipid yields were calculated. Ultimately, 2.5 g / L of phospholipids were obtained when the organic nitrogen source was soybean meal; 1.1 g / L when the organic nitrogen source was corn steep liquor powder; 1.5 g / L when the organic nitrogen source was beef extract; and 0.8 g / L when the organic nitrogen source was trypsin. However, using traditional yeast extract as the organic nitrogen source only yielded 0.9 g / L of phospholipids after fermentation. Therefore, soybean meal was chosen as the organic nitrogen source.

[0077] Figure 2 Fermentation of *Schizochytrium* HX-308 under different concentrations of soybean meal nitrogen source (1 g / L to 10 g / L), with other fermentation medium components remaining the same, was conducted for 48 hours. The highest phospholipid yield (3.5 g / L) was obtained at a soybean meal concentration of 4 g / L. The yields were: 1.6 g / L at 1 g / L; 2.1 g / L at 2 g / L; 2.5 g / L at 3 g / L; 3.3 g / L at 5 g / L; 3.2 g / L at 6 g / L; and 2.5 g / L at 7 g / L. When the soybean meal concentration was 8 g / L, the yield of phospholipids was 2.1 g / L. At 9 g / L, the yield was 1.8 g / L. At 10 g / L, the yield was 1.9 g / L. Further increases in soybean meal concentration did not lead to an increase in phospholipid yield. Therefore, 4 g / L soybean meal was chosen as the organic nitrogen source.

[0078] Example 2: Screening and concentration optimization of phosphate fermentation by Schizochytrium HX-308

[0079] The specific fermentation steps for Schizochytrium HX-308 are the same as in Example 1.

[0080] The fermentation medium is the same as the fermentation medium for Schizochytrium mentioned above, except that the initial phosphate concentration is 0.8 g / L, and the phosphate is at least one of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate. The phosphate concentration is between 0.2 g / L and 2 g / L. The organic nitrogen source is tentatively selected as 3 g / L yeast extract.

[0081] The method for obtaining oil through fermentation culture is the same as in Example 1.

[0082] The method for separating the lipids obtained from fermentation culture to obtain phospholipids and glycerides is the same as in Example 1.

[0083] Fermentation results of different organic nitrogen sources, such as Figure 3 As shown in the figure. Fermentation was carried out for 48 hours with 0.8 g / L phosphate added and other fermentation medium components kept constant. Lipids were extracted and phospholipid yield was calculated. Ultimately, 2.2 g / L of phospholipids were obtained when sodium dihydrogen phosphate was used; 0.8 g / L when dipotassium hydrogen phosphate was used; and 1.3 g / L when disodium hydrogen phosphate was used. However, when potassium dihydrogen phosphate was used as the fermentation phosphate, only 0.9 g / L of phospholipids were obtained after fermentation. Therefore, sodium dihydrogen phosphate was chosen as the organic nitrogen source.

[0084] Figure 4 Fermentation of *Schizochytrium* HX-308 under different concentrations of sodium dihydrogen phosphate (SDP) was conducted for 48 hours, ensuring that other fermentation medium components remained the same. The highest phospholipid yield (2.7 g / L) was obtained when the SDP concentration was 1 g / L. The yields were: 0.8 g / L at 0.2 g / L, 1.3 g / L at 0.4 g / L, 1.5 g / L at 0.6 g / L, 2.2 g / L at 0.8 g / L, 2.4 g / L at 1.2 g / L, 2.3 g / L at 1.4 g / L, and 2 g / L at 1.6 g / L. When the sodium dihydrogen phosphate concentration is 1.8 g / L, it results in 1.8 g / L phospholipids. When the sodium dihydrogen phosphate concentration is 2 g / L, it results in 1.7 g / L phospholipids. Therefore, 1 g / L sodium dihydrogen phosphate is chosen as the phosphate.

[0085] Example 3: Fermentation of Schizochytrium HX-308 under optimal organic nitrogen source and phosphate conditions

[0086] The specific fermentation steps for Schizochytrium HX-308 are the same as in Example 1.

[0087] The fermentation medium is the same as the fermentation medium for Schizochytrium mentioned above, except that the phosphate is 1 g / L sodium dihydrogen phosphate and the organic nitrogen source is 4 g / L soybean meal.

[0088] The method for obtaining oil through fermentation culture is the same as in Example 1.

[0089] The method for separating the lipids obtained from fermentation culture to obtain phospholipids and glycerides is the same as in Example 1.

[0090] Furthermore, the method for analyzing fatty acids in phospholipids and glycerides includes the following steps:

[0091] ① Add 1 ml of 1M potassium hydroxide-methanol solution to a 1.5 ml centrifuge tube, add 20 μl of oil and mix well, then add it to a 20 ml volumetric flask, and then add 2 ml of 1M potassium hydroxide-methanol solution to the volumetric flask (rinse the 1.5 ml centrifuge tube and draw as much oil as possible into the volumetric flask), mix well, and then incubate in a 65℃ water bath for 17 min, and cool to room temperature;

[0092] ② Add 2 ml of boron trifluoride ether (i.e., a mixture of boron trifluoride and ether in a volume ratio of 3:7), mix well, and then heat in a water bath at 65°C for 7 min.

[0093] ③ Add 2 ml of saturated potassium chloride and shake well, then add 3 ml of n-hexane (chromatographic grade) and let stand to separate the layers;

[0094] ④ Pour into a small centrifuge tube, take the upper n-hexane phase and pass it through a microporous membrane to remove impurities;

[0095] ⑤ Seal and store before gas chromatography.

[0096] Figure 5 Schizochytrium HX-308 was fermented for 48 h under the following conditions: before optimization, 3 g / L yeast extract and 0.8 g / L potassium dihydrogen phosphate; after optimization, 4 g / L soybean meal and 1 g / L sodium dihydrogen phosphate, while ensuring other fermentation medium components remained the same. Lipids were extracted and phospholipid yields were calculated. Before optimization, Schizochytrium HX-308 yielded 0.9 g / L phospholipids, 13.3 g / L glycerides, and 14.2 g / L lipids. After optimization, Schizochytrium HX-308 yielded 4.7 g / L phospholipids, 8.8 g / L glycerides, and 13.5 g / L lipids.

[0097] In addition, we also tested the optimal organic and inorganic nitrogen sources separately using *Schizochytrium* HX-308 fermentation culture medium (the fermentation medium was the same as the *Schizochytrium* fermentation medium mentioned above, except that it contained 3 g / L yeast extract and 1 g / L sodium dihydrogen phosphate, 4 g / L soybean meal and 0.8 g / L potassium dihydrogen phosphate). The results are shown in Table 1. When 3 g / L yeast extract and 1 g / L sodium dihydrogen phosphate were added as nitrogen sources, only 1.1 g / L phospholipids were obtained; when 4 g / L soybean meal and 0.8 g / L potassium dihydrogen phosphate were added as nitrogen sources, only 2.3 g / L phospholipids were obtained. Therefore, 4 g / L soybean meal and 1 g / L sodium dihydrogen phosphate are the optimal nitrogen source combination. It can also be seen that 4 g / L soybean meal and 1 g / L sodium dihydrogen phosphate have a synergistic effect in the method of this invention, which can synergistically increase the yield of the prepared phospholipids.

[0098] Table 1. Phospholipid production of Schizochytrium HX-308 under different combinations of organic and inorganic nitrogen sources.

[0099]

[0100] The final fatty acid distributions of phospholipids and lipids in *Schizochytrium* HX-308 before and after fermentation optimization are shown in Table 2. Before optimization, the fatty acid distribution in *Schizochytrium* phospholipids was mainly 8.5% C14:0, 20.4% C14:0, 19.3% DPA, and 44.8% DHA; while the fatty acid distribution in lipids was mainly 12.3% C14:0, 18.9% C14:0, 18.2% DPA, and 47.8% DHA. After optimization, the fatty acid distribution in *Schizochytrium* phospholipids was mainly 8.3% C14:0, 18.2% C14:0, 16.8% DPA, and 52.2% DHA; while the fatty acid distribution in lipids was mainly 10.6% C14:0, 19.7% C14:0, 16.8% DPA, and 48.2% DHA.

[0101] Table 2 Fatty acid distribution of phospholipids and lipids before and after fermentation optimization of Schizochytrium HX-308

[0102]

[0103] Ultimately, through optimization of the fermentation medium, the phospholipid yield of *Schizochytrium* increased from 0.9 g / L to 4.7 g / L, a 4.2-fold increase. Furthermore, the DHA content in the phospholipids increased from 44.8% to 52.2%, a 16.5% increase. Therefore, this fermentation strategy significantly improved the DHA yield of *Schizochytrium* HX-308 phospholipids and laid the foundation for the industrial-scale production of DHA from phospholipids.

[0104] Example 4: Fermentation of Schizochytrium ATCC 20888 under optimal organic nitrogen source and phosphate conditions

[0105] The specific fermentation steps for Schizochytrium ATCC 20888 are the same as in Example 1.

[0106] The fermentation medium is the same as the fermentation medium for Schizochytrium mentioned above, except that the phosphate is 1 g / L sodium dihydrogen phosphate and the organic nitrogen source is 4 g / L soybean meal.

[0107] The method for obtaining oil through fermentation culture is the same as in Example 1.

[0108] The method for separating the lipids obtained from fermentation culture to obtain phospholipids and glycerides is the same as in Example 1.

[0109] Furthermore, the analytical method for obtaining fatty acids in phospholipids and glycerides is the same as in Example 3.

[0110] Figure 6 The *Schizochytridacna ATCC* 20888 was fermented for 48 h under the following conditions: before optimization, 3 g / L yeast extract and 0.8 g / L potassium dihydrogen phosphate; after optimization, 4 g / L soybean meal and 1 g / L sodium dihydrogen phosphate, while ensuring other fermentation medium components remained the same. Lipids were extracted and phospholipid yields were calculated. Before optimization, *Schizochytridacna ATCC* 20888 yielded 0.8 g / L phospholipids, 12.5 g / L glycerols, and 13.3 g / L lipids. After optimization, *Schizochytridacna ATCC* 20888 yielded 4.2 g / L phospholipids, 8.2 g / L glycerols, and 12.4 g / L lipids.

[0111] In addition, we also tested the optimal organic and inorganic nitrogen sources separately, performing fermentation tests on *Schizochytridum ATCC20888* (the fermentation medium was the same as the *Schizochytridum* fermentation medium mentioned above, the only difference being: 3 g / L yeast extract and 1 g / L sodium dihydrogen phosphate, 4 g / L soybean meal and 0.8 g / L potassium dihydrogen phosphate). The results are shown in Table 3. When 3 g / L yeast extract and 1 g / L sodium dihydrogen phosphate were added as nitrogen sources, only 1.3 g / L phospholipids were obtained; when 4 g / L soybean meal and 0.8 g / L potassium dihydrogen phosphate were added as nitrogen sources, only 2.4 g / L phospholipids were obtained. Therefore, 4 g / L soybean meal and 1 g / L sodium dihydrogen phosphate are the optimal nitrogen source combination. It can also be seen that 4 g / L soybean meal and 1 g / L sodium dihydrogen phosphate have a synergistic effect in the method of this invention, which can synergistically increase the yield of the prepared phospholipids.

[0112] Table 3. Phospholipid production of Schizochytrium ATCC 20888 under different combinations of organic and inorganic nitrogen sources.

[0113]

[0114] The final fatty acid distribution of phospholipids and lipids in *Schizochytrium ATCC 20888* before and after fermentation optimization is shown in Table 4. Before optimization, the fatty acid distribution in *Schizochytrium ATCC 20888* phospholipids was mainly 9.5% C14:0, 20.3% C14:0, 19.8% DPA, and 43.3% DHA; while the fatty acid distribution in lipids was mainly 12.3% C14:0, 19.3% C14:0, 19.2% DPA, and 45.2% DHA. After optimization, the fatty acid distribution in *Schizochytrium ATCC 20888* phospholipids was mainly 8.1% C14:0, 17.6% C14:0, 17.6% DPA, and 51.6% DHA; while the fatty acid distribution in lipids was mainly 11.9% C14:0, 19.4% C14:0, 15.9% DPA, and 46.5% DHA.

[0115] Table 4. Fatty acid distribution of phospholipids and lipids in Schizochytrium ATCC 20888 before and after fermentation optimization.

[0116]

[0117] Therefore, this fermentation strategy significantly increased the phospholipid DHA yield of Schizochytrium ATCC 20888 and laid the foundation for the industrial production of phospholipid DHA.

[0118] Example 5. Fermentation of Schizochytrium ATCC PRA-276 under optimal organic nitrogen source and phosphate conditions.

[0119] The specific fermentation steps for Schizochytrium ATCC PRA-276 are the same as in Example 1.

[0120] The fermentation medium is the same as the fermentation medium for Schizochytrium mentioned above, except that the phosphate is 1 g / L sodium dihydrogen phosphate and the organic nitrogen source is 4 g / L soybean meal.

[0121] The method for obtaining oil through fermentation culture is the same as in Example 1.

[0122] The method for separating the lipids obtained from fermentation culture to obtain phospholipids and glycerides is the same as in Example 1.

[0123] Furthermore, the analytical method for obtaining fatty acids in phospholipids and glycerides is the same as in Example 3.

[0124] Figure 7The *Schizochytridacna ATCC PRA-276* strain was fermented for 48 h under the following conditions: before optimization, 3 g / L yeast extract and 0.8 g / L potassium dihydrogen phosphate; after optimization, 4 g / L soybean meal and 1 g / L sodium dihydrogen phosphate, while ensuring other fermentation medium components remained the same. Lipids were extracted and phospholipid yields were calculated. Before optimization, *Schizochytridacna ATCC PRA-276* yielded 0.6 g / L phospholipids, 11.3 g / L glycerols, and 11.9 g / L lipids. After optimization, *Schizochytridacna ATCC PRA-276* yielded 3.9 g / L phospholipids, 7.8 g / L glycerols, and 11.7 g / L lipids.

[0125] In addition, we also tested the optimal organic and inorganic nitrogen sources separately, performing fermentation tests on *Schizochytrium ATCC PRA-276* (the fermentation medium was the same as the *Schizochytrium* fermentation medium described above, except that it consisted of 3 g / L yeast extract and 1 g / L sodium dihydrogen phosphate, 4 g / L soybean meal and 0.8 g / L potassium dihydrogen phosphate). The results are shown in Table 5. When 3 g / L yeast extract and 1 g / L sodium dihydrogen phosphate were added as nitrogen sources, only 1.4 g / L phospholipids were obtained; when 4 g / L soybean meal and 0.8 g / L potassium dihydrogen phosphate were added as nitrogen sources, only 2.1 g / L phospholipids were obtained. Therefore, 4 g / L soybean meal and 1 g / L sodium dihydrogen phosphate are the optimal nitrogen source combination. It can also be seen that 4 g / L soybean meal and 1 g / L sodium dihydrogen phosphate have a synergistic effect in the method of this invention, which can synergistically increase the yield of the prepared phospholipids.

[0126] Table 5. Phospholipid production of Schizochytrium ATCC PRA-276 under different combinations of organic and inorganic nitrogen sources.

[0127]

[0128] The final fatty acid distribution of phospholipids and lipids in *Schizochytrium ATCC PRA-276* before and after fermentation optimization is shown in Table 6. Before optimization, the fatty acid distribution in *Schizochytrium ATCC PRA-276* phospholipids was mainly 10.7% C14:0, 21.5% C14:0, 17.9% DPA, and 42.6% DHA; while the fatty acid distribution in lipids was mainly 11.1% C14:0, 18.9% C14:0, 20.2% DPA, and 45.6% DHA. After optimization, the fatty acid distribution in *Schizochytrium ATCC PRA-276* phospholipids was mainly 8.5% C14:0, 17.9% C14:0, 16.9% DPA, and 51.8% DHA; while the fatty acid distribution in lipids was mainly 11.4% C14:0, 19.9% ​​C14:0, 15.4% DPA, and 47.2% DHA.

[0129] Table 6 Fatty acid distribution of phospholipids and lipids before and after fermentation optimization of Schizochytrium ATCC PRA-276

[0130]

[0131] Therefore, this fermentation strategy significantly increased the phospholipid DHA yield of Schizochytrium ATCC PRA-276 and laid the foundation for the industrial production of phospholipid DHA.

[0132] Therefore, this fermentation strategy is universal for Schizochytrium and can significantly increase the yield of phospholipid DHA.

[0133] Meanwhile, by comparing Examples 3 to 6, it can be seen that 4 g / L soybean meal powder and 1 g / L sodium dihydrogen phosphate have a synergistic effect in the method of the present invention, which can synergistically increase the yield of the prepared phospholipids.

[0134] Example 6: Experiment on feeding freshwater rainbow trout

[0135] The *Schizochytrium* fermentation broth obtained before and after optimization of the fermentation medium corresponding to Example 3 was spray-dried to obtain algal powder. The fermentation broth was then dried to form powder at an inlet temperature of 180℃ and an outlet temperature of 100℃. The two types of algal powder were mixed with rainbow trout feed at a ratio of 100g algal 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 containing algal powder obtained before nitrogen source optimization, and feed containing algal powder obtained after nitrogen source optimization, for 60 consecutive days. The DHA content in the fish oil was measured each time, with two rainbow trout randomly selected from each test group for testing, and the average value was taken. The results are shown in Table 7.

[0136] Table 7. Changes in DHA content in fish oil from rainbow trout fed with Schizochytrium algae powder obtained before and after normal culture medium optimization.

[0137]

[0138] The results in Table 7 show that when rainbow trout were fed with algal powder feed obtained by spray drying the Schizochytrium fermentation broth after optimization of the fermentation medium for 30 consecutive days, the DHA content in the fish oil exceeded 2%, and exceeded 7% after 60 days of feeding.

[0139] The method for determining the polyunsaturated fatty acid composition in DHA algae powder / fish oil samples is as follows:

[0140] 1. Take 10 mg of fish oil for fatty acid methyl esterification, add it to an EP tube containing 1 mL of 1 M potassium hydroxide-methanol solution, and shake for 6 h at 20 °C and 1000 rpm.

[0141] 2. Add 50 μL of concentrated sulfuric acid to terminate the reaction, and add 1 mL of n-hexane to extract by shaking at 20 °C and 1000 rpm for 0.5 h.

[0142] 3. The extract phase was loaded into a liquid chromatography vial for gas chromatography analysis: A GC-2030 (Shimadzu, Japan) gas chromatography system was used, equipped with a DB-23 capillary column (60m * 0.22mm) and a flame ionization detector (FID). Nitrogen was used as the carrier gas, with an injection volume of 1μL and an injection temperature of 250℃. The column temperature was increased from 100℃ to 200℃ at a rate of 25℃ / min, then increased to 230℃ at a rate of 4℃ / min and held for 9 min. The FID detector temperature was 280℃.

[0143] By comparing with Sigma standards (related external standards, Sigma, USA), the composition of different polyunsaturated fatty acids was identified. Using non-endogenous fatty acids (C19:0) as internal standards, the content of individual polyunsaturated fatty acids was calculated from the peak area on the chromatogram.

[0144] This invention utilizes optimized fermentation medium and controlled fermentation of *Schizochytrium* HX-308 to obtain 4.7 g / L phospholipids within 48 hours, with a DHA content of 52.2%. This phospholipid-DHA strategy is currently unreported, and it also achieved significant fermentation results in *Schizochytrium* ATCC 20888 and *Schizochytrium* ATCC PRA-276. Furthermore, the *Schizochytrium* algal powder containing this high-yield phospholipid-DHA was used in a feeding experiment with rainbow trout. After 30 days of continuous feeding to rainbow trout, the DHA content in the fish oil exceeded 2%, and after 60 days, it exceeded 7%. We hypothesize that phospholipids are a major structural component of cell membranes, and DHA carried by phospholipids can be directly integrated into the intestinal cell membrane of rainbow trout. Compared to triglyceride-type DHA, which requires pancreatic lipase to break down into free fatty acids before absorption, phospholipid-type DHA has a shorter absorption pathway and higher efficiency. This is the main reason why rainbow trout fed with algal powder obtained by regulating the fermentation medium and controlling the Schizochytrium HX-308 are more likely to accumulate DHA.

[0145] In summary, this invention provides a fermentation method and application for increasing the yield of Schizochytrium phospholipid DHA, which can promote the production of Schizochytrium phospholipid DHA. It also provides a method for applying Schizochytrium algal powder and demonstrates its application in aquatic feed with high phospholipid DHA content.

[0146] Our research found that the production of phospholipid DHA using microorganisms is currently a blank area, with no related patent reports in China, especially regarding the production of phospholipid DHA using Schizochytrium. Therefore, this study fills a gap in this research.

[0147] 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 fermentation method for enhancing the DHA production capacity of a Schizochytrium sp. phospholipid, characterized by: It comprises the following steps: S. schizochytrium is fermented for 32-60 hours, and the addition of organic nitrogen source and phosphate is optimized during the fermentation; the seed culture medium formula for culturing S. schizochytrium is not changed, only the fermentation medium formula is changed; The final concentration of the added organic nitrogen source is 4 g / L, and the final concentration of the added phosphate is 1 g / L, which refers to the final concentration of the organic nitrogen source and the phosphate in the fermentation medium; The organic nitrogen source is soybean meal; The phosphate is sodium dihydrogen phosphate; The specific steps are as follows: (1) Take out 100 μL of the preserved S. schizochytrium liquid and inoculate it into the seed culture medium at an inoculation amount of 2%, without adjusting pH, and cultivate it at 28°C and 230 rpm for 48 hours; (2) Inoculate in seed culture medium at 1% inoculum, without adjusting pH, cultivate at 28°C and 170 rpm for 24 h, OD 600 >3 and no contamination is found by microscope to inoculate the next generation; (3) 1% inoculation amount inoculated in seed culture medium, no pH adjustment, 28°C and 170 rpm culture for 24 h, OD 600 >5 and no staining under microscopy, inoculate the next generation; (4) Inoculate it into the fermentation medium at an inoculation amount of 1%, without adjusting pH, start fermentation at 28°C and 170 rpm, and ferment for 48 hours to obtain the product; The S. schizochytrium is S. schizochytrium HX-308, whose preservation number is CCTCC M 209059, S. schizochytrium ATCC 20888, or S. schizochytrium ATCC PRA-276; The components of the seed culture medium for the S. schizochytrium include: glucose 40-60 g / L, yeast extract powder 4-6 g / L, sodium sulfate 5-8 g / L, magnesium sulfate 2-4 g / L, ammonium sulfate 4-8 g / L, potassium chloride 1-2 g / L, potassium sulfate 0.5-1 g / L, potassium dihydrogen phosphate 0.5-2 g / L, sodium glutamate 8-12 g / L, zinc sulfate heptahydrate 1-5 mg / L, cobalt chloride hexahydrate 0.01-0.1 mg / L, copper sulfate pentahydrate 2-6 mg / L, nickel sulfate hexahydrate 1-2 mg / L, iron sulfate heptahydrate 8-15 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.04 mg / L, and water as the solvent; The components of the fermentation medium for the S. schizochytrium include: glucose 60-100 g / L, sodium sulfate 5-12 g / L, organic nitrogen source 1-10 g / L, phosphate 0.2-2 g / L, magnesium sulfate 2-4 g / L, ammonium sulfate 4-8 g / L, potassium chloride 1-2 g / L, potassium sulfate 0.5-1 g / L, sodium glutamate 15-20 g / L, zinc sulfate heptahydrate 1-5 mg / L, cobalt chloride hexahydrate 0.01-0.1 mg / L, copper sulfate pentahydrate 2-6 mg / L, nickel sulfate hexahydrate 1-2 mg / L, iron sulfate heptahydrate 8-15 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.04 mg / L, vitamin B6 4-10 mg / L, vitamin B12 0.1-0.5 mg / L, and water as the solvent.

2. The use of the fermentation method according to claim 1 in the preparation of algal powder for aquatic feed.

3. A Schizochytrium sp. algal meal for use in aquaculture feed, characterized in that: The preparation steps are as follows: Dry the fermentation broth obtained by the fermentation method according to claim 1 to obtain the algal powder.

4. A method of obtaining oil from the fermentation broth obtained from the fermentation process as claimed in claim 1, characterized in that: It comprises the following steps: 1) After adding NaOH solution to adjust pH 10-13 in the fermentation broth, add 0.01-0.5% of the fermentation broth mass of cell wall breaking enzyme, 40~60℃, 100~200r / min oscillation for 5~10h; 2) Cool to room temperature, add anhydrous ethanol to inactivate the cell wall breaking enzyme; 3) Add n-hexane to extract, collect the upper organic phase; 4) Repeat step 3) several times, combine the organic phase, and evaporate the solvent to obtain the lipid, which is oil.

5. The method of claim 4, wherein: The lipid is separated to obtain phospholipid and glycerolipid, and the specific steps are as follows: S1 Put the uniform lipid in a bottle, seal, and filter when heated to 90℃ in a water bath to remove insoluble substances; S2 Weigh the lipid, heat to 80℃, add ultrapure water, and the ratio of lipid to ultrapure water is 25:2 g:mL, fully stir to hydrate; S3 Centrifugal tube centrifugal precipitation, upper glycerolipid, middle phospholipid, and lower water phase, respectively collect the upper glycerolipid and middle phospholipid, and calculate the yield of glycerolipid and phospholipid.

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