Fermentation process and application of algal oil containing specific DHA distribution characteristic

Abstract

The invention discloses a fermentation process and application of algal oil containing specific DHA distribution characteristics, and belongs to the technical field of microbial fermentation and functional algal oil preparation. According to the method, sodium citrate participating in tricarboxylic acid circulation is utilized to supplement and enhance central carbon metabolic flux, and more sufficient acetyl coenzyme A and precursor substances are provided for fatty acid synthesis, so that the fatty acid synthesis flux is up-regulated; the phytosterol is used as a cell membrane fluidity regulator, so that the activity of the Sn-2 site acyltransferase in the schizochytrium limacinum is favorably activated, and the DHA is directionally guided to be combined to the Sn-2 site of the TAG. According to the invention, the two substances are synergistically added to a schizochytrium limacinum fermentation system, so that the three targets that the total DHA content gt is 40%, the ratio of Sn-2DHA to the total DHA gt is 50% and the specific triglyceride molecule content reaches the standard can be simultaneously realized accidentally, and the problem that the DHA content and distribution are difficult to regulate and control synchronously in the prior art is effectively solved.

Description

Technical Field

[0001] This invention relates to the intersection of microbial fermentation and functional algal oil preparation, specifically providing an algal oil fermentation process and application containing specific DHA distribution characteristics. Background Technology

[0002] Docosahexaenoic acid (DHA), a core component of ω-3 polyunsaturated fatty acids (ω-3 PUFAs), is a crucial nutrient for the growth of brain and retinal nerve cells. Its bioavailability depends not only on its total content but also, and more importantly, on its position within triglyceride (TAG) molecules, directly determining absorption efficiency. Human digestive enzymes (such as pancreatic lipase) exhibit position-specific hydrolysis of TAGs, preferentially targeting Sn-1 / Sn-3 fatty acids, which require esterification for absorption. Sn-2 fatty acids, however, enter the bloodstream directly as monoacylglycerols (MAGs), with an absorption rate 30%-50% higher than Sn-1 / Sn-3 fatty acids. For infants and young children, Sn-2 DHA can reduce calcium soap formation and lower the risk of constipation. Therefore, the core quality indicator for high-end infant formula algal oil has been upgraded from "total DHA content" to "Sn-2 DHA percentage."

[0003] Currently, the main sources of DHA include fish oil and algal oil. Fish oil DHA is susceptible to marine pollution (such as heavy metals and polychlorinated biphenyls) and ecological fishing restrictions; moreover, the Sn-2 position DHA content in natural fish oil is only 20%-35%, which cannot meet the needs of high-end formulations; traditional algal oils (Crypthecodinium cohnii, Schizochytrium, and Ulkenia amoeboida) have become mainstream due to their stable quality and mild odor. The commercially available product DSM "life's DHA™" algal oil has a total DHA content of 40% and a Sn-2 position DHA content of 30%. However, existing technologies still suffer from two major flaws: Firstly, while prioritizing the total DHA content of the product, they neglect its distribution characteristics. Existing patents often aim to increase the total DHA content by optimizing the carbon-nitrogen ratio of the fermentation medium (WO2019218839A1) or constructing genetically engineered strains (CN116814447A), but they fail to address the regulation of the Sn-2 position DHA proportion and triglyceride molecule composition, resulting in low bioavailability of algal oil. Secondly, the additives used in existing processes are functionally singular and cannot achieve synergistic regulation of DHA positional distribution. Currently, existing technologies mostly employ single nutrients (such as amino acids or plant growth regulators) or genetic engineering methods. The former can increase the total DHA proportion but cannot directionally regulate the targeted enrichment of Sn-2 positions; the latter faces difficulties such as high construction costs, difficulty in scaling up the process, and the potential for food safety controversies. Summary of the Invention

[0004] [Technical Issues] To overcome the shortcomings of existing Schizochytrium fermentation processes, which "emphasize total DHA content but neglect distribution characteristics" and "have limited functionality of additives," this paper proposes an algal oil fermentation technology that is simple, cost-controllable, and capable of targeted regulation of DHA distribution, thereby producing algal oils with specific DHA distribution characteristics that meet the needs of high-end formulated foods.

[0005] [Technical Solution] Sodium citrate, as a natural organic acid salt, can promote fatty acid synthesis by supplementing acetyl-CoA precursor (pyruvate); phytosterols, as cell membrane fluidity regulators, can activate the activity of "Sn-2 acyltransferase" in Schizochytrium, guiding DHA to bind to the Sn-2 position of TAG. This invention is the first to synergistically add both to a Schizochytrium fermentation system, simultaneously achieving three major objectives: total DHA content >40%, Sn-2 DHA proportion >50% of total DHA, and specific triglyceride molecule content meeting standards, filling a gap in existing technologies for DHA distribution regulation.

[0006] The first objective of this invention is to provide a fermentation medium for increasing the DHA content and the proportion of sn-2 DHA in the oil fermentation broth of Schizochytrium. The fermentation medium comprises: anhydrous glucose: 80-90 g / L, monosodium glutamate: 6-10 g / L, yeast extract: 8-12 g / L, Na₂SO₄: 8-14 g / L, MgSO₄·7H₂O: 1-3 g / L, NaCl: 3-5 g / L, KCl: 2-4 g / L, KH₂PO₄: 3-5 g / L, K₂HPO₄: 2-4 g / L, sodium citrate: 1.5-3.0 g / L, and phytosterols: 0.2-0.5 g / L; and vitamin additions: VB1: 0.004-0.008 g / L, VB6: 0.001-0.004 g / L, VB… 12 : 0.004-0.008g / L.

[0007] The second objective of this invention is to provide a method for increasing the DHA content and the proportion of sn-2 DHA in the fermentation broth of Schizochytrium esculentum, wherein the method involves adding sodium citrate and phytosterols to the fermentation medium.

[0008] In one embodiment of the present invention, the phytosterols include β-sitosterol, stigmasterol, or campesterol.

[0009] Preferably, the phytosterol is β-sitosterol.

[0010] In one embodiment of the present invention, the amount of sodium citrate added is 1.5-3.0 g / L.

[0011] In one embodiment of the present invention, the amount of phytosterol added is 0.2-0.5 g / L.

[0012] In one embodiment of the present invention, the method steps are as follows: S1: Inoculate Schizochytrium into seed culture medium and collect seed liquid activated to the logarithmic growth phase; S2: Inoculate the seed culture into a fermentation medium containing sodium citrate and phytosterols, and ferment for 120-140 hours.

[0013] In one embodiment of the present invention, in S1, frozen Schizochytrium is inoculated into the seed culture medium at a volume ratio of 2-6%.

[0014] In one embodiment of the present invention, in S1, the culture is shaken and cultured at 26-32℃ and 160-190rpm for 18-26h, and the culture is repeated 2-4 times.

[0015] Preferably, the passage is repeated 3 times.

[0016] In one embodiment of the present invention, in step S2, the seed liquid is inoculated into the fermentation medium at an inoculation rate of 7-11% by volume.

[0017] In one embodiment of the present invention, in S2, the culture is shaken at 26-32°C and 160-190 rpm.

[0018] In one embodiment of the present invention, the Schizochytrium includes Schizochytrium sp. ATCC 20888 or Schizochytrium sp. HX-308.

[0019] Preferably, the Schizochytrium is Schizochytrium sp. ATCC 20888.

[0020] In one embodiment of the invention, the phytosterols are emulsified with Tween-80.

[0021] In one embodiment of the present invention, the final concentration of Tween-80 is 0.1 g / L.

[0022] In one embodiment of the present invention, in step S2, when the glucose concentration is below 8-12 g / L, a sterile glucose solution is added to restore the glucose concentration in the fermentation broth to 45-55 g / L.

[0023] A third objective of this invention is to provide the application of the fermentation medium in increasing the DHA content and the proportion of Sn-2 DHA in the products of Schizochytrium, wherein the application is to use the fermentation medium for the fermentation culture of Schizochytrium.

[0024] In one embodiment of the present invention, during the fermentation process, when the glucose concentration is below 8-12 g / L, glucose is added to control the glucose concentration at 45-55 g / L.

[0025] A fourth objective of this invention is to provide the application of the method or the fermentation medium in the preparation of algal oil for infant formula and algal oil for DHA health products.

[0026] Beneficial effects The DHA distribution characteristics were significantly optimized: the total DHA content of the prepared algal oil was >40%, the proportion of Sn-2 position DHA in the total DHA was >50%, and the contents of the three core triglyceride molecules (16:0-22:6-16:0, 16:0-22:6-22:6, 22:6-22:6-22:6) were stable at 17%-22%, 18%-23%, and 7%-12%, respectively, which meets the requirements of infant formula for highly absorbable DHA; Synergistic addition is superior to single regulation: Sodium citrate (carbon metabolism regulation) and phytosterol (acyltransferase activation) work synergistically, increasing the proportion of DHA at the Sn-2 position by 5%-8% compared to adding sodium citrate alone (42%-47% of DHA at the Sn-2 position) or phytosterol alone (45%-49% of DHA at the Sn-2 position); Highly economical process: Sodium citrate and phytosterols are widely available and require small amounts, making them easier to industrialize than recombinant strain construction (cost reduction of more than 60%) and special amino acid mixtures (cost reduction of more than 40%). Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to these embodiments.

[0028] Terms and Definitions Phytosterols: Unless otherwise stated, "phytosterols" in this specification, also known as plant sterols, belong to the category of plant steroidal compounds. "Phytosterols" refers to steroidal compounds having a cyclopentane-polyhydrophenanthrene skeleton, with a methyl or ethyl substitution at position 24, and their saturated forms (steranols). The phytosterols are preferably derived from non-animal sources, such as β-sitosterol, stigmasterol, campesterol, brassosterol, sitosterol, campesterol, and their pharmaceutically or food-acceptable esterified derivatives (such as fatty acid esters, ferulic acid esters, and p-coumaric acid esters). The phytosterols may exist in free, alkylated, or esterified forms (such as phytosterol esters formed with fatty acids) and may be derived from natural extracts or synthetic sources.

[0029] Sodium citrate: Unless otherwise stated, "sodium citrate" in this specification refers to the sodium salt obtained by neutralizing citric acid with sodium hydroxide or sodium carbonate, including anhydrous sodium citrate (Na3C6H5O7), sodium citrate dihydrate (Na3C6H5O7·2H2O), and any equivalent soluble derivative that releases citrate ions (such as disodium hydrogen citrate, sodium dihydrogen citrate). This term does not include citrate salts formed with metal ions other than sodium.

[0030] DHA: Unless otherwise stated, "DHA (docosahexaenoic acid)" in this specification refers to an n-3 polyunsaturated fatty acid with 22 carbon atoms and 6 cis double bonds, systematically named all-cis-docosa-4,7,10,13,16,19-hexaenoic acid, abbreviated as 22:6(n-3). Triacylglycerol (TAG) is a triester composed of one molecule of glycerol (propane-1,2,3-triol) and three molecules of fatty acid through ester bonds. The three carbon atoms of the glycerol skeleton are defined sequentially using stereospecific numbering (sn): sn-1 position: the position where the hydroxyl group at the C-1 position of glycerol is esterified; sn-2 position: the position where the hydroxyl group at the C-2 (central) position of glycerol is esterified; sn-3 position: the position where the hydroxyl group at the C-3 position of glycerol is esterified. Sn-2 DHA specifically refers to the structural form in which DHA is linked to the Sn-2 position of the TAG molecule via an ester bond; it is represented by sn-1,3-diacyl-sn-2-DHA-glycerol ester (i.e., sn-2 DHA-TAG), and also includes Sn-2-DHA-monoacylglycerol (sn-2 DHA-MAG) formed after its digestion. In this patent, the content of Sn-2 DHA is expressed as "the percentage of Sn-2 DHA in the total mass of DHA".

[0031] Experimental materials and equipment involved in the following embodiments Strain: Schizochytrium sp. ATCC 20888; Reagents: Food-grade sodium citrate, phytosterols (β-sitosterol content ≥95%), Tween-80, anhydrous glucose, and chromatographically pure n-hexane; Equipment: Shaking table, ultra-high performance liquid chromatography-triple quadrupole-time-of-flight mass spectrometer, gas chromatograph.

[0032] Example 1 1. Seed activation: *Schizochytrium* was inoculated into seed culture medium at a 3% (v / v) inoculum and cultured at 28℃ and 170 rpm for 24 hours, followed by 3 subcultures. The seed culture medium composition (g / L) was: anhydrous glucose 15 g / L, monosodium glutamate 6 g / L, yeast extract 8 g / L, Na₂SO₄ 8 g / L, MgSO₄·7H₂O 1 g / L, NaCl 3 g / L, KCl 2 g / L, KH₂PO₄ 3 g / L, K₂HPO₄ 2 g / L; vitamin supplementation: VB1 0.004 g / L, VB6 0.001 g / L, VB… 12 0.004 g / L.

[0033] 2. Fermentation Culture: Inoculate the basal fermentation medium at a 10% (v / v) inoculation rate and incubate at 28℃ and 170 rpm for 130 hours. Control the pH to 6.5-7.5 with NaOH solution. Add glucose every 15 hours (to 50 g / L if glucose falls below 10 g / L). The composition of the basal fermentation medium is as follows: anhydrous glucose 80 g / L, monosodium glutamate 6 g / L, yeast extract 8 g / L, Na2SO4 8 g / L, MgSO4·7H2O 1 g / L, NaCl 3 g / L, KCl 2 g / L, KH2PO4 3 g / L, K2HPO4 2 g / L, sodium citrate 2 g / L, and phytosterols 0.3 g / L. Vitamin supplementation: VB1 0.004 g / L, VB6 0.001 g / L, VB... 12 0.004 g / L, of which phytosterols need to be emulsified with Tween-80 in advance to ensure uniform dispersion.

[0034] 3. Collection of bacterial cells and extraction of oil esters: Algal oil was obtained using a process of "enzymatic crushing of fermentation broth - hexane extraction - acidification and degumming". (1) Add NaOH solution to the collected fermentation broth to adjust the pH to 10-13, add 0.01-0.2% of cell wall breaking enzyme by weight of fermentation broth, shake at 100-200 r / min for 5-15 h at 40-60℃; (2) Cool to room temperature and add an equal volume of anhydrous ethanol to the fermentation broth to inactivate the cell wall-breaking enzyme; (3) Extract the oil by adding an equal volume of n-hexane to the fermentation broth: Let the extracted organic phase stand for 5 hours. After the upper and lower layers are clearly separated, collect the upper organic phase. Place the collected organic phase into a rotary evaporator flask and evaporate the organic phase by rotary evaporation at 45°C water bath and 120 r / min. When the organic phase no longer evaporates, remove the rotary evaporator flask and dry it in a 60°C oven until the weight no longer changes. Weigh the oil to obtain the oil.

[0035] 4. Gas-phase analysis of fatty acids: Total DHA content was determined by gas chromatography (GC), the composition of fatty acids at the 2-position of algal oil was determined by enzymatic alcoholysis combined with GC, and the composition of triglyceride molecules was analyzed by ultra-high performance liquid chromatography-triple quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS). For detailed methods, please refer to: Li Feng. Analysis, preparation and physicochemical properties of triglycerides with high sn-2-position DHA structure [D]. Wuxi, Jiangnan University, 2023. The results are shown in Table 1. The total DHA content in the algal oil prepared using the method of Example 1 is close to 45%, and the proportion of Sn-2 site DHA in the total DHA is as high as 52.3%, which is far higher than that of existing commercially available products.

[0036] Table 1 Fatty acid content Table 2 Comparison of Example 1 and Existing Technology Example 2 1. Seed activation: *Schizochytrium* was inoculated into seed culture medium at a 3% (v / v) inoculum and cultured at 28℃ and 170 rpm for 24 hours, followed by 3 subcultures. The seed culture medium composition (g / L) was: anhydrous glucose 15 g / L, monosodium glutamate 6 g / L, yeast extract 8 g / L, Na₂SO₄ 8 g / L, MgSO₄·7H₂O 1 g / L, NaCl 3 g / L, KCl 2 g / L, KH₂PO₄ 3 g / L, K₂HPO₄ 2 g / L; vitamin supplementation: VB1 0.004 g / L, VB6 0.001 g / L, VB… 12 0.004 g / L.

[0037] 2. Fermentation Culture: Inoculate the basal fermentation medium at a 10% (v / v) inoculation rate and incubate at 28℃ and 170 rpm for 130 hours, adding glucose every 15 hours (to 50 g / L if glucose falls below 10 g / L). The basal fermentation medium consists of: anhydrous glucose 80 g / L, monosodium glutamate 6 g / L, yeast extract 8 g / L, Na₂SO₄ 8 g / L, MgSO₄·7H₂O 1 g / L, NaCl 3 g / L, KCl 2 g / L, KH₂PO₄ 3 g / L, K₂HPO₄ 2 g / L, sodium citrate 3 g / L, and phytosterols 0.3 g / L. Vitamin supplementation includes: VB1 0.004 g / L, VB6 0.001 g / L, and VB… 12 0.004 g / L, of which phytosterols need to be emulsified with Tween-80 in advance to ensure uniform dispersion.

[0038] Example 3 1. Seed activation: *Schizochytrium* was inoculated into seed culture medium at a 3% (v / v) inoculum and cultured at 28℃ and 170 rpm for 24 hours, followed by 3 subcultures. The seed culture medium composition (g / L) was: anhydrous glucose 15 g / L, monosodium glutamate 6 g / L, yeast extract 8 g / L, Na₂SO₄ 8 g / L, MgSO₄·7H₂O 1 g / L, NaCl 3 g / L, KCl 2 g / L, KH₂PO₄ 3 g / L, K₂HPO₄ 2 g / L; vitamin supplementation: VB1 0.004 g / L, VB6 0.001 g / L, VB… 12 0.004 g / L.

[0039] 2. Fermentation Culture: Inoculate the basal fermentation medium at a 10% (v / v) inoculation rate and incubate at 28℃ and 170 rpm for 130 hours, adding glucose every 15 hours (to 50 g / L if glucose falls below 10 g / L). The basal fermentation medium consists of: anhydrous glucose 80 g / L, monosodium glutamate 6 g / L, yeast extract 8 g / L, Na₂SO₄ 8 g / L, MgSO₄·7H₂O 1 g / L, NaCl 3 g / L, KCl 2 g / L, KH₂PO₄ 3 g / L, K₂HPO₄ 2 g / L, sodium citrate 2 g / L, and phytosterols 0.2 g / L. Vitamin supplementation includes: VB1 0.004 g / L, VB6 0.001 g / L, and VB… 12 0.004 g / L, of which phytosterols need to be emulsified with Tween-80 in advance to ensure uniform dispersion.

[0040] Example 4 1. Seed activation: *Schizochytrium* was inoculated into seed culture medium at a 3% (v / v) inoculum and cultured at 28℃ and 170 rpm for 24 hours, followed by 3 subcultures. The seed culture medium composition (g / L) was: anhydrous glucose 15 g / L, monosodium glutamate 6 g / L, yeast extract 8 g / L, Na₂SO₄ 8 g / L, MgSO₄·7H₂O 1 g / L, NaCl 3 g / L, KCl 2 g / L, KH₂PO₄ 3 g / L, K₂HPO₄ 2 g / L; vitamin supplementation: VB1 0.004 g / L, VB6 0.001 g / L, VB… 12 0.004 g / L.

[0041] 2. Fermentation Culture: Inoculate the basal fermentation medium at a 10% (v / v) inoculation rate and incubate at 28℃ and 170 rpm for 130 hours, adding glucose every 15 hours (to 50 g / L if glucose falls below 10 g / L). The basal fermentation medium consists of: anhydrous glucose 80 g / L, monosodium glutamate 6 g / L, yeast extract 8 g / L, Na₂SO₄ 8 g / L, MgSO₄·7H₂O 1 g / L, NaCl 3 g / L, KCl 2 g / L, KH₂PO₄ 3 g / L, K₂HPO₄ 2 g / L, sodium citrate 2 g / L, and phytosterols 0.5 g / L. Vitamin supplementation includes: VB1 0.004 g / L, VB6 0.001 g / L, and VB… 12 0.004 g / L, of which phytosterols need to be emulsified with Tween-80 in advance to ensure uniform dispersion.

[0042] Example 5 1. Seed activation: *Schizochytrium* was inoculated into seed culture medium at a 3% (v / v) inoculum and cultured at 28℃ and 170 rpm for 24 hours, followed by 3 subcultures. The seed culture medium composition (g / L) was: anhydrous glucose 15 g / L, monosodium glutamate 6 g / L, yeast extract 8 g / L, Na₂SO₄ 8 g / L, MgSO₄·7H₂O 1 g / L, NaCl 3 g / L, KCl 2 g / L, KH₂PO₄ 3 g / L, K₂HPO₄ 2 g / L; vitamin supplementation: VB1 0.004 g / L, VB6 0.001 g / L, VB… 12 0.004 g / L.

[0043] 2. Fermentation Culture: Inoculate the basal fermentation medium at a 10% (v / v) inoculation rate and incubate at 28℃ and 170 rpm for 130 hours, adding glucose every 15 hours (to 50 g / L if glucose falls below 10 g / L). The basal fermentation medium consists of: anhydrous glucose 80 g / L, monosodium glutamate 6 g / L, yeast extract 8 g / L, Na₂SO₄ 8 g / L, MgSO₄·7H₂O 1 g / L, NaCl 3 g / L, KCl 2 g / L, KH₂PO₄ 3 g / L, K₂HPO₄ 2 g / L, sodium citrate 1.5 g / L, and phytosterols 0.3 g / L. Vitamin supplementation includes: VB1 0.004 g / L, VB6 0.001 g / L, and VB… 12 0.004 g / L, of which phytosterols need to be emulsified with Tween-80 in advance to ensure uniform dispersion.

[0044] Comparative Example 1 1. Seed activation: *Schizochytrium* was inoculated into seed culture medium at a 3% (v / v) inoculum and cultured at 28℃ and 170 rpm for 24 hours, followed by 3 subcultures. The seed culture medium composition (g / L) was: anhydrous glucose 15 g / L, monosodium glutamate 6 g / L, yeast extract 8 g / L, Na₂SO₄ 8 g / L, MgSO₄·7H₂O 1 g / L, NaCl 3 g / L, KCl 2 g / L, KH₂PO₄ 3 g / L, K₂HPO₄ 2 g / L; vitamin supplementation: VB1 0.004 g / L, VB6 0.001 g / L, VB… 12 0.004 g / L.

[0045] 2. Fermentation Culture: Inoculate the basal fermentation medium at a 10% (v / v) inoculation rate and incubate at 28℃ and 170 rpm for 130 hours, adding glucose every 15 hours (to 50 g / L if glucose falls below 10 g / L). The composition of the basal fermentation medium is as follows: anhydrous glucose 80 g / L, monosodium glutamate 6 g / L, yeast extract 8 g / L, Na₂SO₄ 8 g / L, MgSO₄·7H₂O 1 g / L, NaCl 3 g / L, KCl 2 g / L, KH₂PO₄ 3 g / L, K₂HPO₄ 2 g / L; Vitamin supplementation: VB1 0.004 g / L, VB6 0.001 g / L, VB… 12 0.004 g / L, of which phytosterols need to be emulsified with Tween-80 in advance to ensure uniform dispersion.

[0046] The test results showed that the total DHA content of algal oil was 37.2%, Sn-2DHA accounted for 39.5% of the total DHA, 16:0-22:6-16:0 content was 14.3%, 16:0-22:6-22:6 content was 12.1%, and 22:6-22:6-22:6 content was 6.8% (not up to standard).

[0047] Comparative Example 2 1. Sodium citrate only group Sodium citrate (final concentration 2.0 g / L) was added to the basic fermentation medium, and the other conditions were the same as in Example 1; the test results were as follows: total DHA content was 40.8%, Sn-2DHA accounted for 46.2% of total DHA, 16:0-22:6-16:0 content was 16.5%, 16:0-22:6-22:6 content was 17.3%, and 22:6-22:6-22:6 content was 6.7% (some indicators did not meet the standards).

[0048] 2. Phytosterol-only group Phytosterols (final concentration 0.3 g / L, emulsified at Tween-80) were added to the basic fermentation medium, and the other conditions were the same as in Example 1; Test results: Total DHA content was 39.5%, Sn-2DHA accounted for 48.8% of total DHA, 16:0-22:6-16:0 content was 15.8%, 16:0-22:6-22:6 content was 18.1%, and 22:6-22:6-22:6 content was 7.2% (total DHA did not meet the standard).

[0049] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for increasing the DHA content and the proportion of DHA at the sn-2 position in the oil fermentation broth of Schizochytrium, characterized in that, The method involves adding sodium citrate and phytosterols to the fermentation medium.

2. The method according to claim 1, characterized in that, The phytosterols include β-sitosterol, stigmasterol, or campesterol.

3. The method according to claim 1 or 2, characterized in that, The phytosterol is β-sitosterol.

4. The method according to claim 1, characterized in that, The amount of sodium citrate added is 1.5-3.0 g / L, and the amount of phytosterol added is 0.2-0.5 g / L.

5. The method according to claim 1, characterized in that, The steps of the method are as follows: S1: Inoculate Schizochytrium into seed culture medium and collect seed liquid activated to the logarithmic growth phase; S2: Inoculate the seed culture of S1 into a fermentation medium containing sodium citrate and phytosterols, and ferment for 120-140 hours.

6. The method according to claim 5, characterized in that, In S1, Schizochytrium is inoculated into the seed culture medium at a volume ratio of 2-6%, and cultured with shaking at 26-32℃ and 160-190rpm for 18-26h, and the culture is repeated 2-4 times.

7. The method according to claim 5, characterized in that, In S2, the seed liquid is inoculated into the fermentation medium at an inoculation rate of 7-11% by volume and cultured under shaking conditions at 26-32℃ and 160-190rpm.

8. The method according to any one of claims 1 to 7, characterized in that, The Schizochytrium species include Schizochytrium sp. ATCC 20888 or Schizochytrium sp. HX-308.

9. The method according to claim 5, characterized in that, The phytosterols were emulsified with Tween-80.

10. The method according to claim 5, characterized in that, In S2, when the glucose concentration is below 8-12 g / L, a sterile glucose solution is added to restore the glucose concentration in the fermentation broth to 45-55 g / L.

11. The method according to claim 5, characterized in that, In S1, the seed culture medium comprises: anhydrous glucose 15-25 g / L, sodium glutamate 6-10 g / L, yeast extract 8-12 g / L, Na2SO4 8-14 g / L, MgSO4·7H2O 1-3 g / L, (NH4)2SO4 3-5 g / L, NaCl 3-5 g / L, KCl 2-4 g / L, KH2PO4 3-5 g / L, K2HPO4 2-4 g / L, VB1 0.004-0.008 g / L, VB6 0.001-0.004 g / L, VB... 12 0.004-0.008 g / L.

12. A fermentation medium for increasing the DHA content and the proportion of DHA at the sn-2 position in the oil fermentation broth of Schizochytrium, characterized in that, The fermentation medium comprises: anhydrous glucose: 80-90 g / L, monosodium glutamate: 6-10 g / L, yeast extract: 8-12 g / L, Na₂SO₄: 8-14 g / L, MgSO₄·7H₂O: 1-3 g / L, NaCl: 3-5 g / L, KCl: 2-4 g / L, KH₂PO₄: 3-5 g / L, K₂HPO₄: 2-4 g / L, sodium citrate: 1.5-3.0 g / L, and phytosterols: 0.2-0.5 g / L; vitamin additions: VB1: 0.004-0.008 g / L, VB6: 0.001-0.004 g / L, VB… 12 : 0.004-0.008g / L.

13. The fermentation medium according to claim 12, characterized in that, The phytosterols include β-sitosterol, stigmasterol, or campesterol.

14. The application of the fermentation medium according to claim 12 or 13 in increasing the DHA content and the proportion of Sn-2 DHA in Schizochytrium products, characterized in that, The application is the fermentation culture of Schizochytrium using the fermentation medium described in claim 12 or 13.

15. The application of the method according to any one of claims 1-1 or the fermentation medium according to claim 12 or 13 in the preparation of algal oil for infant formula and algal oil for DHA health products.

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