Method for co-producing squalene and n-3 fatty acid by using single cells

Abstract

The invention relates to the technical field of microbial fermentation, and particularly discloses a method for co-producing squalene and n-3 fatty acid by using single cells, which comprises the following steps: feeding exogenous factors at different proliferation and growth stages by using 15-35% of inoculum size in combination with single-cell algae, regulating the pH value of a fermentation medium by using substances such as succinic acid and ammonia water, and the like. The amount of microbial oil obtained through fermentation and EPA, n-3DPA, DHA and squalene in the microbial oil can be remarkably increased, the amount of the microbial oil obtained through fermentation can be increased to 46% or above, the n-3DPA in the microbial oil can be increased to 4.7% or above, the DHA can be increased to 43% or above, especially the content of squalene can be increased to 18.6% or above, and squalene is widely used for resisting tumors and inflammation, preventing cardiovascular diseases and the like. The application in the fields of medicine, nutrition, beauty makeup and the like is wide.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and specifically discloses a method for co-producing squalene and n-3 fatty acids by single cells. Background Art

[0002] Squalene (C30H50) is an open-chain triterpenoid compound with six all-trans double bonds. Its chemical name is 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-triacontanehexaenoic acid. The pure product is a colorless or light yellow oily liquid and is insoluble in water. In 1906, squalene was first discovered in the extract of shark liver and got its name accordingly, also known as "fish liver terpene". Deep-sea fish are one of the main traditional sources of squalene, such as salmon, sardines, sharks, etc. Among them, the content of squalene in deep-sea sharks, an important traditional source, can reach about 15-60%. In addition to shark liver, squalene also exists in various plants such as olives, amaranth, camellia oleifera, and rice. Among them, the content of squalene in olive oil can reach 1.16%, and the content is generally low. In addition, squalene widely exists in higher mammals, especially in human sebum, with a content of up to 12%. Research shows that squalene has a positive effect on oxidative stress caused by ultraviolet rays and also has various physiological functions such as anti-tumor, anti-inflammatory, and prevention of cardiovascular diseases. It is mainly used in the fields of medicine, nutrition, beauty, etc. However, due to the problems of declining quality and reduced production of squalene from shark liver, and at the same time, its acquisition process also involves many drawbacks such as environmental pollution, ecological balance destruction, and marine resource limitations, it is of great practical significance to find sustainable alternative resources.

[0003] n-3 fatty acids are a class of long-chain fatty acids containing multiple double bonds, usually referring to fatty acids with a carbon chain length of more than 18 carbon atoms, at least containing 3 double bonds and the first double bond at the methyl end located at the third carbon atom. Eicosatrienoic acid, arachidonic acid (C20:4N-3), eicosapentaenoic acid (EPA), docosapentaenoic acid (n-3DPA), and docosahexaenoic acid (DHA) all belong to n-3 polyunsaturated fatty acids and have important physiological functions in the human body, such as preventing atherosclerotic heart disease, anti-cancer, protecting eyes, anti-allergy, anti-thrombosis, etc. However, the human body does not have the ability to synthesize n-3 fatty acids de novo, so it is necessary to ingest fatty acids from the outside to supplement the required amount. Deep-sea fish oil is currently the main source for humans to obtain n-3 polyunsaturated fatty acids. It is particularly urgent to develop sustainable alternative resources to meet the growing market demand.

[0004] Microalgae are primary producers in nature and the initial source of bioactive substances such as EPA and DHA, with great development potential. Among them, Schizochytrium sp is a heterotrophic single-celled microorganism that aggregates in nutrient-rich seawater and mangrove sediments. It contains rich active substances such as proteins, fatty acids, amino acids, sterols, squalene, and carotenoids. Its fermentation cycle is short and the process requirements are simple, and it is mostly used for commercial production of DHA, with a maximum yield of up to 200 g / L. Given the advantages of Schizochytrium sp in the production of polyunsaturated fatty acids, it is considered a potential sustainable resource for co-producing squalene and n-3 fatty acids, with broad application prospects. et al. used Aurantiochytrium sp. 18W-13a to ferment and produce squalene, and the final yield reached 1.29 g / L. Schütte et al. adjusted the dissolved oxygen and feeding strategies to improve the squalene content in Schizochytrium sp. S31. The results showed that the squalene content increased to 39.67 mg / g under the fermentation process of oxygen limitation and fed-batch, which was much higher than the control group. It can be seen that using microalgae to ferment and produce squalene and co-producing n-3 fatty acids has broad market prospects. Summary of the Invention

[0005] To solve the above problems, the first aspect of the present invention proposes a method for co-producing squalene and n-3 fatty acids by single cells, including: Inoculating the Schizochytrium sp seed liquid into the fermentation medium for fermentation. The fermentation process is divided into different fermentation periods according to time. Among them, the pH value of the fermentation medium is controlled in some fermentation periods, the dissolved oxygen level of the fermentation medium is controlled in some periods, ornithine and sarcosine are added to the fermentation medium in some fermentation periods, choline phosphate is added to the fermentation medium in some fermentation periods, and sodium phytate and potassium sodium tartrate are added to the operation medium in some fermentation periods.

[0006] The fermentation periods are divided into cases of time coincidence, partial time coincidence, or time non-coincidence.

[0007] In some specific embodiments of the method for co-producing squalene and n-3 fatty acids by single cells in the first aspect, the fermentation period for adding ornithine and sarcosine to the fermentation medium is from 75 h to the end of fermentation.

[0008] In some specific embodiments of the method for co-producing squalene and n-3 fatty acids by single cells in some first aspects, after the operation of adding ornithine to the fermentation medium, the concentration of ornithine in the fermentation medium is not less than 0.1 g / L. In some preferred embodiments of some first aspects, the concentration of ornithine in the fermentation medium is 2.5 - 3 g / L. In some embodiments, the concentration of ornithine in the fermentation medium is 2.75 g / L.

[0009] In some specific embodiments of the method for co-producing squalene and n-3 fatty acids by single cells in some first aspects, after the operation of adding sarcosine to the fermentation medium, the concentration of sarcosine in the fermentation medium is not less than 0.01 g / L. In some preferred embodiments of some first aspects, the concentration of sarcosine in the fermentation medium is 0.02 - 0.055 g / L. In some embodiments, the concentration of sarcosine in the fermentation medium is 0.055 g / L.

[0010] In some specific embodiments of the method for co-producing squalene and n-3 fatty acids by single cells in some first aspects, the fermentation period for the operation of adding choline phosphate to the fermentation medium is from 95 h to the end of fermentation.

[0011] In some specific embodiments of the method for co-producing squalene and n-3 fatty acids by single cells in some first aspects, the fermentation period for the operation of adding sodium phytate and sodium potassium tartrate to the fermentation medium is from 110 h to the end of fermentation.

[0012] In some specific embodiments of the method for co-producing squalene and n-3 fatty acids by single cells in some first aspects, after the operation of adding sodium phytate and sodium potassium tartrate to the fermentation medium, the concentration of sodium phytate in the fermentation medium is not less than 0.1 g / L. In some preferred embodiments of some first aspects, the concentration of sodium phytate in the fermentation medium is 0.5 - 1.5 g / L. In some embodiments, the concentration of sodium phytate in the fermentation medium is 1.0 g / L.

[0013] In some specific embodiments of the method for co-producing squalene and n-3 fatty acids by single cells in some first aspects, the concentration of sodium potassium tartrate in the fermentation medium is not less than 0.1 g / L. In some preferred embodiments of some first aspects, the concentration of sodium potassium tartrate in the fermentation medium is 0.5 - 2.5 g / L. In some embodiments, the concentration of sodium potassium tartrate in the fermentation medium is 1.5 g / L.

[0014] In some specific embodiments of the method for co-producing squalene and n-3 fatty acids by single cells in some first aspects, the fermentation periods for controlling the pH and dissolved oxygen of the fermentation medium are 0 to 70 h, 70 to 90 h, and 90 to the end of fermentation.

[0015] In some specific embodiments of the method for co-producing squalene and n-3 fatty acids by single cells in some first aspects, when the fermentation period is 0 to 70 h, the pH of the fermentation medium is adjusted to 5.0 - 6.0. In some embodiments, when the fermentation period is 0 to 70 h, the pH of the fermentation medium is adjusted to 5.5.

[0016] In some specific embodiments of the method for co-producing squalene and n-3 fatty acids by single cells in some first aspects, when the fermentation period is 70 to 90 h, the pH of the fermentation medium is adjusted to 6.0 - 7.0. In some embodiments, when the fermentation period is 70 to 90 h, the pH of the fermentation medium is adjusted to 6.5.

[0017] In some specific embodiments of the method for co-producing squalene and n-3 fatty acids by single cells in some first aspects, when the fermentation period is from 90 h to the end of fermentation, the pH of the fermentation medium is adjusted to 7.0 - 7.5. In some embodiments, when the fermentation period is from 90 h to the end of fermentation, the pH of the fermentation medium is adjusted to 7.25.

[0018] In some specific embodiments of the method for co-producing squalene and n-3 fatty acids by single cells in some first aspects, when the fermentation period is 0 to 70 h, the dissolved oxygen of the fermentation medium is controlled at 90 - 100%. In some embodiments, when the fermentation period is 0 to 70 h, the dissolved oxygen of the fermentation medium is controlled at 95%.

[0019] In some specific embodiments of the method for co-producing squalene and n-3 fatty acids by single cells in some first aspects, when the fermentation period is 70 to 90 h, the dissolved oxygen of the fermentation medium is controlled at 50 - 80%. In some embodiments, when the fermentation period is 70 to 90 h, the dissolved oxygen of the fermentation medium is controlled at 65%.

[0020] In some specific embodiments of the method for co-producing squalene and n-3 fatty acids by single cells in some first aspects, when the fermentation period is from 90 h to the end of fermentation, the dissolved oxygen of the fermentation medium is controlled at 1 - 5%. In some embodiments, when the fermentation period is from 90 h to the end of fermentation, the dissolved oxygen of the fermentation medium is controlled at 3%.

[0021] In some specific embodiments of the method for co-producing squalene and n-3 fatty acids by single cells in some first aspects, each 1 L of the fermentation medium comprises 10 - 30 g of glucose, 5 - 20 g of yeast extract, 4.0 - 5.0 g of ammonium sulfate, 10 - 20 g of anhydrous sodium sulfate, 4.0 - 5.0 g of magnesium sulfate, 6.0 - 10 g of potassium dihydrogen phosphate, 0.1 - 0.5 g of potassium sulfate, and 0.2 - 0.5 g of anhydrous calcium chloride.

[0022] In some specific embodiments of the method for co-producing squalene and n-3 fatty acids by single cells in some first aspects, the inoculation volume ratio of the seed liquid is 15 - 35%.

[0023] In some specific embodiments of the method for co-producing squalene and n-3 fatty acids from single cells in the first aspect, the pH is controlled by adding a mixture of succinic acid, citric acid, and malic acid to the fermentation medium, or by adding ammonia water to the fermentation medium.

[0024] In the second aspect of the present invention, a microbial oil obtained by fermentation in the first aspect is provided. The microbial oil contains at least 43% DHA, at least 6.8% EPA, at least 4.4% n-3 DPA, and at least 18% squalene.

[0025] In the seed culture step of the present invention, the glycerol tube Schizochytrium strain used is Schizochytrium HS08; preservation information: preserved in the China General Microbiological Culture Collection Center (CGMCC), with the preservation number CGMCC No. 40902; All the medicaments used in the present invention are purchased from open and legal markets and are not further purified.

[0026] In the present invention, room temperature is 5 - 40 °C in some embodiments, 10 - 35 °C in some embodiments, 15 - 30 °C in some embodiments, and 25 °C in some embodiments.

[0027] In the present invention, the inoculation amount refers to the ratio of the volume of the transferred seed liquid to the volume of the medium after inoculation.

[0028] In the present invention, "g / L" is relative to the volume of the liquid in the medium or fermenter. For example, "adding a mixed solution of 1.0 g / L sodium phytate + 2.0 g / L sodium potassium tartrate" means adding sodium phytate to the fermentation medium at a concentration of 1.0 g / L and adding sodium potassium tartrate to the fermentation medium at a concentration of 2.0 g / L.

[0029] Advantages of the present invention: The present invention adopts a method of controlling the dissolved oxygen and pH in the fermentation broth in stages, in combination with the staged feeding of choline phosphate and sodium phytate, and the addition of ornithine, sarcosine, etc. at different fermentation periods, and uses substances such as succinic acid and ammonia water to adjust the pH of the fermentation medium, significantly increasing the amount of microbial oil obtained by fermentation, as well as EPA, n-3 DPA, DHA, and squalene in the microbial oil. The amount of microbial oil obtained by fermentation can be increased to more than 46%, the content of squalene in the microbial oil weight can be increased to more than 18.6%, n-3 DPA can be increased to more than 4.7%, and DHA can be increased to more than 43%. Specific embodiments

[0030] In order to enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to further elaborate on the present invention.

[0031] Preparation of Seed Liquid Composition of the first-stage medium: glucose 40 - 60, yeast extract powder 10 - 20, anhydrous sodium sulfate 15 - 20, anhydrous calcium chloride 0.1 - 0.2, potassium chloride 0.3 - 0.6, potassium dihydrogen phosphate 0.5 - 1.5, ammonium sulfate 2.0 - 4.0, zinc sulfate 1.0 - 4.0, magnesium sulfate 2.0 - 6.0, potassium sulfate 0.5 - 1.5; unit: g / L.

[0032] Composition of the second-stage medium: glucose 60 - 100, yeast extract powder 10 - 15, anhydrous sodium sulfate 10 - 15, anhydrous calcium chloride 0.1 - 0.2, potassium chloride 0.3 - 0.6, potassium dihydrogen phosphate 0.5 - 1.5, ammonium sulfate 2.0 - 4.0, zinc sulfate 1.0 - 4.0, magnesium sulfate 2.0 - 6.0, potassium sulfate 0.5 - 1.5; unit: g / L.

[0033] Steps for preparing the seed liquid: Take out a glycerol tube of Schizochytrium sp. strain from a -80 °C refrigerator and put it into the first-stage seed medium, and culture it at 28 °C and 180 r / min for 48 h to obtain the first-stage Schizochytrium sp. seed liquid; Inoculate the first-stage seed liquid into the second-stage seed medium at an inoculation amount of 10%, and culture it at 28 °C and 180 r / min for 24 h to obtain the second-stage Schizochytrium sp. seed liquid; Composition of the fermenter medium: glucose 10 - 30, yeast extract 5 - 20, ammonium sulfate 4.0 - 5.0, anhydrous sodium sulfate 10 - 20, magnesium sulfate 4.0 - 5.0, potassium dihydrogen phosphate 6.0 - 10, potassium sulfate 0.1 - 0.5 g, anhydrous calcium chloride 0.2 - 0.5; unit: g / L.

[0034] Comparative Example 1 Inoculate the second-stage Schizochytrium sp. seed liquid into an 8-ton tank containing the fermenter medium at an inoculation amount of 15 - 35%, and control the dissolved oxygen throughout the fermentation process to be above 90%; control the pH throughout the fermentation process to be 5.0 - 6.0, and use ammonia water and 50% malic acid to adjust the pH. After fermentation for 120 h, obtain the fermentation broth, and detect the oil content in the fermentation broth, the squalene content, EPA content, n-3 DPA content, and DHA content in the microbial algal oil.

[0035] Comparative Example 2 Inoculate the Schizochytrium sp. secondary seed liquid into an 8-ton tank containing the fermentation medium at an inoculation amount of 15 - 35%. Control the dissolved oxygen at 80% - 100% before 90 h of fermentation and at 10 - 20% after 90 h; control the pH at 5.0 - 6.0, and adjust the pH using ammonia water and 50% malic acid. After 120 h of fermentation, obtain the fermentation broth, and detect the oil content in the fermentation broth, the squalene content, EPA content, n-3DPA content, and DHA content in the microbial algal oil.

[0036] Comparative Example 3 Inoculate the Schizochytrium sp. secondary seed liquid into an 8-ton tank containing the fermentation medium at an inoculation amount of 15 - 35%. Control the dissolved oxygen in the fermentation above 90%; control the pH at 5.0 - 6.0, and adjust the pH using 40% succinic acid + 50% citric acid and ammonia water. After 120 h of fermentation, obtain the fermentation broth, and detect the oil content in the fermentation broth, the squalene content, EPA content, n-3DPA content, and DHA content in the microbial algal oil.

[0037] Comparative Example 4 Inoculate the Schizochytrium sp. secondary seed liquid into an 8-ton tank containing the fermentation medium at an inoculation amount of 15 - 35%. Control the dissolved oxygen in the fermentation above 90%; control the pH at 5.0 - 6.0 from 0 to 70 h and at 6.0 - 7.0 after 70 h, and adjust the pH using 40% succinic acid + 50% citric acid and ammonia water. After 120 h of fermentation, obtain the fermentation broth, and detect the oil content in the fermentation broth, the squalene content, EPA content, n-3DPA content, and DHA content in the microbial algal oil.

[0038] Example 1 Inoculate the Schizochytrium sp. secondary seed liquid into an 8-ton tank containing the fermentation medium at an inoculation amount of 15 - 35%. Control the dissolved oxygen at 80% - 100% before 90 h of fermentation and at 10 - 20% after 90 h; control the pH at 5.0 - 6.0 from 0 to 70 h and at 6.0 - 7.0 after 70 h, and adjust the pH using 40% succinic acid + 50% citric acid and ammonia water; Start feeding choline phosphate at 75 h to make its concentration in the fermentation medium 0.05 g / L. After 120 h of fermentation, obtain the fermentation broth, and detect the oil content in the fermentation broth, the squalene content, EPA content, n-3DPA content, and DHA content in the microbial algal oil.

[0039] Example 2 Inoculate the Schizochytrium sp. secondary seed liquid into an 8-ton tank containing the fermenter medium at an inoculation amount of 15 - 35%. Control the dissolved oxygen at 80% - 100% before 90 h of fermentation and at 10% - 20% after 90 h; control the pH at 5.0 - 6.0 from 0 to 70 h and at 6.0 - 7.0 after 70 h, and adjust the pH using 40% succinic acid + 50% citric acid and ammonia water.

[0040] Start feeding sodium phytate at 75 h to make its concentration in the fermentation medium 1 g / L, and at the same time feed potassium sodium tartrate to make its concentration in the fermentation medium 1.5 g / L. After 156 h of fermentation, obtain the fermentation broth and detect the oil content in the fermentation broth, the squalene content, EPA content, n - 3 DPA content, and DHA content in the microbial algal oil.

[0041] Example 3 Inoculate the Schizochytrium sp. secondary seed liquid into an 8-ton tank containing the fermenter medium at an inoculation amount of 15 - 35%. Control the dissolved oxygen at 80% - 100% before 90 h of fermentation and at 10% - 20% after 90 h; control the pH at 5.0 - 6.0 from 0 to 70 h and at 6.0 - 7.0 after 70 h, and adjust the pH using 40% succinic acid + 50% citric acid and ammonia water.

[0042] Start supplementing ornithine at 75 h to make its concentration in the fermentation medium 3.0 g / L, and at the same time supplement sarcosine to make its concentration in the fermentation medium 0.065 g / L. After 156 h of fermentation, obtain the fermentation broth and detect the oil content in the fermentation broth, the squalene content, EPA content, n - 3 DPA content, and DHA content in the microbial algal oil.

[0043] Example 4 Inoculate the Schizochytrium sp. secondary seed liquid into an 8-ton tank containing the fermenter medium at an inoculation amount of 15 - 35%. Control the dissolved oxygen at 80% - 100% before 90 h of fermentation and at 10% - 20% after 90 h; control the pH at 5.0 - 6.0 from 0 to 70 h and at 6.0 - 7.0 after 70 h, and adjust the pH using 40% succinic acid + 50% citric acid and ammonia water.

[0044] Start feeding ornithine at 75 h to make its concentration in the fermentation medium 3.0 g / L, and at the same time feed sarcosine to make its concentration in the fermentation medium 0.055 g / L. Start supplementing sodium phytate at 110 h to make its concentration in the fermentation medium 1.0 g / L, and at the same time start supplementing potassium sodium tartrate to make its concentration in the fermentation medium 2.0 g / L. After 156 h of fermentation, obtain the fermentation broth and detect the oil content in the fermentation broth, the squalene content, EPA content, n - 3 DPA content, and DHA content in the microbial algal oil.

[0045] Example 5 Inoculate the Schizochytrium sp. secondary seed liquid into an 8-ton tank containing the fermentation medium at an inoculation amount of 15% - 35%. Control the dissolved oxygen at 90% - 100% before 70 h of fermentation, at 50% - 80% from 70 h to 90 h, and at 1% - 5% after 90 h. Adjust the pH using 40% succinic acid + 50% malic acid and ammonia water. Control the pH at 5.0 - 6.0 from 0 h to 70 h, at 6.0 - 7.0 from 70 h to 90 h, and at 7.0 - 7.5 from 90 h to the end of fermentation.

[0046] Start feeding ornithine at 75 h to make its concentration in the fermentation medium 3.0 g / L, and at the same time feed sarcosine to make its concentration in the fermentation medium 0.055 g / L. Feed choline phosphate at 95 h to make its concentration in the fermentation medium 0.05 g / L. Start adding sodium phytate at 110 h to make its concentration in the fermentation medium 1.0 g / L, and at the same time add potassium sodium tartrate to make its concentration in the fermentation medium 2.0 g / L. After 156 h of fermentation, obtain the fermentation broth, and detect the oil content in the fermentation broth, the squalene content, EPA content, n - 3DPA content, and DHA content in the microbial algal oil.

[0047] Example 6 Inoculate the Schizochytrium sp. secondary seed liquid into an 8-ton tank containing the fermentation medium at an inoculation amount of 15% - 35%. Control the dissolved oxygen at 90% - 100% before 70 h of fermentation, at 50% - 80% from 70 h to 90 h, and at 1% - 5% after 90 h. Adjust the pH using 40% succinic acid + 40% citric acid + 50% malic acid and ammonia water. Control the pH at 5.0 - 6.0 from 0 h to 70 h, at 6.0 - 7.0 from 70 h to 90 h, and at 7.0 - 7.5 from 90 h to the end of fermentation. Start feeding ornithine at 75 h to make its concentration in the fermentation medium 3.0 g / L, and at the same time feed sarcosine to make its concentration in the fermentation medium 0.055 g / L. Feed choline phosphate at 95 h to make its concentration in the fermentation medium 0.05 g / L. Start adding sodium phytate at 110 h to make its concentration in the fermentation medium 1.0 g / L, and at the same time add potassium sodium tartrate to make its concentration in the fermentation medium 2.0 g / L. After 156 h of fermentation, obtain the fermentation broth, and detect the oil content in the fermentation broth, the squalene content, EPA content, n - 3DPA content, and DHA content in the microbial algal oil.

[0048] Table 1 shows the squalene, total oil, DHA content, and DHA content in Comparative Examples 1 - 4 and Examples 1 - 6.

[0049] It can be seen from the comparison between Comparative Examples 1-1, 1-2 and Comparative Examples 1-3, 1-4 that the stepwise combined control of dissolved oxygen and pH in the fermentation broth can, to a certain extent, increase the squalene content and DHA content. When a single exogenous factor was added dropwise, it was found that the addition of choline phosphate could significantly increase the oil content, and the addition of the amino acid mixture could significantly increase the squalene content. Subsequently, by adding different exogenous substances and mixtures dropwise in stages, the yields of oil, squalene, EPA, n-3 DPA, and DHA in the bacterial cells were finally increased significantly at the same time.

[0050] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for co-producing squalene and n-3 fatty acids in a single cell, comprising: The Schizochytrium seed liquid is inoculated into a fermentation medium for fermentation, and the fermentation process is divided into different fermentation time periods, characterized in that, in part of the fermentation time period, the pH value of the fermentation medium is controlled, in part of the time period, the dissolved oxygen level of the fermentation medium is controlled, in part of the fermentation time period, ornithine and sarcosine are added to the fermentation medium, in part of the fermentation time period, choline phosphate is added to the fermentation medium, and in part of the fermentation time period, sodium phytate and potassium sodium tartrate are added to the operation medium.

2. The method for co-producing squalene and n-3 fatty acids in a single cell according to claim 1, characterized in that: The fermentation period for adding ornithine and sarcosine to the fermentation medium is 75 hours to the end of fermentation, and / or the fermentation period for adding phosphorylcholine to the fermentation medium is 95 hours to the end of fermentation, and / or the fermentation period for adding sodium phytate and potassium sodium tartrate to the fermentation medium is 110 hours to the end of fermentation.

3. The method for co-producing squalene and n-3 fatty acids in a single cell according to claim 2, characterized in that: After adding ornithine and sarcosine to the fermentation medium, the concentration of ornithine in the fermentation medium is not less than 0.1 g / L, preferably 2.5-3 g / L, and / or the concentration of sarcosine in the fermentation medium is not less than 0.01 g / L, preferably 0.02-0.055 g / L.

4. The method for co-producing squalene and n-3 fatty acids in a single cell according to claim 5, characterized in that: After adding sodium phytate and sodium potassium tartrate to the fermentation medium, the concentration of sodium phytate in the fermentation medium is not less than 0.1 g / L, preferably 0.5-1.5 g / L, and / or the concentration of potassium sodium tartrate in the fermentation medium is not less than 0.1 g / L, preferably 0.5-2.5 g / L.

5. The method for co-producing squalene and n-3 fatty acids in a single cell according to any one of claims 1 to 4, characterized in that: The fermentation periods for controlling the pH and dissolved oxygen of the fermentation medium are 0 to 70 h, 70 to 90 h, and 90 to the end of fermentation.

6. The method for co-producing squalene and n-3 fatty acids in a single cell according to any one of claims 1 to 5, characterized in that: When the fermentation period is 0 to 70 hours, the pH of the fermentation medium is adjusted to 5.0-6.0, and / or, when the fermentation period is 70 to 90 hours, the pH of the fermentation medium is adjusted to 6.0-7.0, and / or, when the fermentation period is 90 hours to the end of fermentation, the pH of the fermentation medium is adjusted to 7.0-7.5, and / or, when the fermentation period is 0 to 70 hours, the dissolved oxygen of the fermentation medium is controlled to 90-100%, and / or, when the fermentation period is 70 to 90 hours, the dissolved oxygen of the fermentation medium is controlled to 50-80%, and / or, when the fermentation period is 90 hours to the end of fermentation, the dissolved oxygen of the fermentation medium is controlled to 1-5%.

7. The method for co-producing squalene and n-3 fatty acids in a single cell according to any one of claims 1 to 6, characterized in that: The pH is controlled by adding a mixture of succinic acid, citric acid and malic acid into the fermentation medium, or adding ammonia water into the fermentation medium.

8. The method for co-producing squalene and n-3 fatty acids in a single cell according to any one of claims 1 to 7, characterized in that: Each 1L of the fermentation medium includes 10-30g of glucose, 5-20g of yeast extract, 4.0-5.0g of ammonium sulfate, 10-20g of anhydrous sodium sulfate, 4.0-5.0g of magnesium sulfate, 6.0-10g of potassium dihydrogen phosphate, 0.1-0.5g of potassium sulfate, and 0.2-0.5g of anhydrous calcium chloride.

9. The method for co-producing squalene and n-3 fatty acids in a single cell according to any one of claims 1 to 8, characterized in that: The inoculation volume ratio of the seed solution is 15-35%.

10. A microbial oil obtained by fermenting the arbitrary method of claim 1 to 9, wherein the microbial oil contains at least 43% DHA, and / or contains at least 6.8% EPA, and / or contains at least 4.4% n-3 DPA, and / or contains at least 18% squalene.

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