Method for promoting oleaginous microorganisms to quickly accumulate functional grease by using abscisic acid and application

Abstract

The invention belongs to the technical field of microbial fermentation and food engineering, and discloses a method for promoting oleaginous microorganisms to quickly accumulate functional grease by using abscisic acid and application of the method. According to the method, the oleaginous microorganisms are subjected to fermentation culture, an exogenous compound abscisic acid is added in the fermentation culture process, and the functional grease is obtained. The final concentration of the exogenous compound added in the fermentation culture process of the abscisic acid is 0.5-3 mM. The abscisic acid can be applied to promoting the fermentation of the oleaginous microorganisms to quickly accumulate the functional grease. As an important plant hormone, abscisic acid is added as a regulator to promote the production of grease from microorganisms, which is a green production mode. And the abscisic acid has poor thermal stability, can be degraded in the oil extraction process, and does not affect the quality of the grease.

Description

Technical Field

[0001] This invention belongs to the fields of microbial fermentation technology and food engineering technology, and in particular, it is a method and application of using abscisic acid to promote the rapid accumulation of functional oils by oil-producing microorganisms. Background Technology

[0002] Long-chain polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), eicosapentaenoic acid (ARA), and eicosapentaenoic acid (EPA), are commonly found in deep-sea fish, seal oil, and certain plants. They are an indispensable class of unsaturated fatty acids and play an important role in human health. In recent years, deep-sea fish have become the main source of oils containing long-chain polyunsaturated fatty acids for humans. However, fish oil yield and quality are easily affected by environmental and seasonal factors, and it has drawbacks such as a fishy smell, susceptibility to oxidation and contamination, and high processing costs. Chemical synthesis methods suffer from cumbersome synthesis steps, low yields, and severe environmental pollution. Therefore, further developing strategies for the rapid and stable production of long-chain polyunsaturated fatty acid oils is of great significance.

[0003] Oil-producing microorganisms are a type of microorganism with a rapid growth rate and rich in polyunsaturated fatty acids. Their growth is not affected by the seasons, and they can accumulate oils accounting for nearly 70% of their biomass. Therefore, they have long been regarded as an ideal strategy for achieving the industrial production of long-chain polyunsaturated fatty acids. Consequently, strategies to regulate and promote microbial oil production through fermentation have been widely applied in the industrial fermentation production of functional oils.

[0004] Chinese patent publication CN104894176A describes the use of iodoacetamide as a regulatory factor in the fermentation medium of *Schizochytrium*, effectively adjusting the DPA / DHA ratio in *Schizochytrium* oil from 0.2 to 0.39 and increasing DPA production by 69%. Chinese patent application CN101979623A demonstrates that adding one or more exogenous regulatory factors, acetic acid, citric acid, and simvastatin, can promote DHA synthesis in *Schizochytrium*, increasing the final DHA yield from an initial 35.51% to 45%. Chinese patent application CN114525312A achieves a DHA content of 50% in *Schizochytrium* through fermentation culture without additional sodium or chloride ion supplementation. 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.

[0005] However, existing fermentation strategies often have limitations, and the added chemical regulators often fail to meet food safety requirements. Furthermore, existing regulations typically only work on single oil-producing microorganisms, failing to achieve universal applicability in improving microbial oil production capacity. Therefore, adding abscisic acid to increase the yield of oils fermented by different microorganisms and enhance the content of polyunsaturated fatty acids in the oils is highly significant. Summary of the Invention

[0006] The technical solution adopted by this invention to solve its technical problem is: Application of abscisic acid in promoting the rapid accumulation of functional oils through the fermentation of oil-producing microorganisms.

[0007] Furthermore, the oil-producing microorganism is selected from Schizochytrium (… Schizochytrium sp.) HX-308, Schizochytrium ( Schizochytrium sp.) ATCC 20888, Alpine Flocculation ( Mortierella alpina ACCC 31952, Alpine Morphyra ( Mortierella alpina ACCC 32468, Alpine Morphyra ( Mortierella alpina ACCC 31578, Chlorella ( Chlorella ATCC 50258, Chlorella ( Chlorella ) Chlorella At least one of ATCC 11469; Alternatively, the final concentration of the exogenous compound added during the fermentation process of the abscisic acid is 0.5-3 mM; Alternatively, the functional oils may include docosahexaenoic acid, eicosapentaenoic acid, or eicosapentaenoic acid.

[0008] A method for promoting the rapid accumulation of functional oils by oleogenous microorganisms using abscisic acid, wherein the oleogenous microorganisms are fermented and cultured, and exogenous compound abscisic acid is added during the fermentation process.

[0009] Furthermore, the oil-producing microorganism is selected from at least one of Schizochytrium, Alpine Monascus, or Chlorella; Alternatively, the final concentration of abscisic acid added during the fermentation process is 0.5-3 mM; Alternatively, the functional oils may include docosahexaenoic acid, eicosapentaenoic acid, or eicosapentaenoic acid.

[0010] Furthermore, the oil-producing microorganism is selected from Schizochytrium (… Schizochytrium sp.) HX-308, Schizochytrium ( Schizochytrium sp.) ATCC 20888, Alpine Flocculation ( Mortierella alpina ACCC 31952, Alpine Morphyra ( Mortierella alpinaACCC 32468, Alpine Morphyra ( Mortierella alpina ACCC 31578, Chlorella ( Chlorella ATCC 50258, Chlorella ( Chlorella ) Chlorella At least one of ATCC 11469; When the oil-producing microorganism is Schizochytrium ( Schizochytrium When using sp.) HX-308, abscisic acid was added at a final concentration of 1.8 mM at 0 h of fermentation culture, and fermentation continued for 120 h. When the oil-producing microorganism is Mortierella alpina ACCC 31952, abscisic acid is added at a final concentration of 1.5 mM at 0 h of fermentation culture and fermented for 96 days. When the oil-producing microorganism is Chlorella ATCC 50258, abscisic acid is added at a final concentration of 1.2 mM at 0 h of fermentation culture and fermented for 168 h.

[0011] Furthermore, it includes the following steps: The seed culture of oil-producing microorganisms was obtained by culturing the microbial strains. The seed culture was then inoculated into a fermentation medium and fermented to obtain a fermentation broth. At 0 h of fermentation, the exogenous compound abscisic acid was added.

[0012] Furthermore, the fermentation medium can provide the nutrients required for the growth and proliferation of the oil-producing microorganism. When the oil-producing microorganism is *Schizochytrium*, the fermentation medium comprises: glucose 60-100 g / L, yeast extract 5-15 g / L, sodium sulfate 5-12 g / L, magnesium sulfate 2-4 g / L, ammonium sulfate 4-8 g / L, potassium chloride 1-2 g / L, calcium chloride 0.1-0.2 g / L, potassium sulfate 0.5-1 g / L, and phosphate. Potassium dihydrogen 0.5-2 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, ferrous sulfate heptahydrate 8-15 mg / L, calcium pantothenate 2-4 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.02-0.06 mg / L, vitamin B6 4-10 mg / L, vitamin B12 0.1-0.5 mg / L, pH 6.0-6.5; When the oil-producing microorganism is *Morchella alpineensis*, the fermentation medium comprises: glucose 60-100 g / L, yeast extract 8-15 g / L, sodium sulfate 3-12 g / L, potassium sulfate 2-4 g / L, ammonium sulfate 4-8 g / L, potassium chloride 1-2 g / L, calcium chloride 0.1-0.2 g / L, potassium dihydrogen phosphate 5-20 g / L, magnesium sulfate 2-10 g / L, and a pH of 5.0-6.5. When the oil-producing microorganism is Chlorella, the fermentation medium comprises: sodium sulfate 0.1-4 g / L, potassium dihydrogen phosphate 0.2-4 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, ferrous sulfate heptahydrate 8-15 mg / L, calcium pantothenate 2-4 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.02-0.06 mg / L, vitamin B1 0.02-0.4 mg / L, vitamin B6 0.04-0.5 mg / L, and vitamin B12 0.01-0.2 mg / L; Alternatively, the seed culture of the oil-producing microorganism is obtained by inoculating the microbial strain into a seed culture medium, which provides the nutrients required for the growth and proliferation of the oil-producing microorganism. When the oil-producing microorganism is a Schizochytrium fungus, the seed culture medium comprises: glucose 40-60 g / L, yeast extract 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, calcium chloride 0.1-0.2 g / L, and sulfur... 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, ferrous sulfate heptahydrate 8-15 mg / L, calcium pantothenate 2-4 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.02-0.06 mg / L, pH 4.0-6.5; When the oil-producing microorganism is *Morchella alpineensis*, the seed culture medium comprises: 20-40 g / L glucose, 4-8 g / L yeast extract, 2-8 g / L sodium sulfate, 0.5-4 g / L potassium dihydrogen phosphate, 0.1-5 g / L magnesium sulfate heptahydrate, and a pH of 4.0-6.5.

[0013] Furthermore, when the oil-producing microorganism is *Schizochytrium*, the seed culture conditions shall at least include: an inoculum size of 0.5-2% by volume, a temperature of 20-30°C, a rotation speed of 150-250 rpm, and a time of 18-30 h; the fermentation culture conditions shall at least include: an inoculum size of 2-20% by volume, a temperature of 20-30°C, and a rotation speed of 150-550 rpm; when the oil-producing microorganism is *Morchella alpineensis*, the seed culture conditions shall at least include: an inoculum size of 0.5-2% by volume, a temperature of 10-30°C, and a rotation speed of 150-250 rpm. The fermentation time is 2-4 days; the fermentation conditions include at least the following: inoculum size of 2-20% by volume, temperature of 20-30℃, and rotation speed of 100-450 rpm; when the oil-producing microorganism is Chlorella, the fermentation conditions include at least the following: inoculum size of 0.5-10% by volume, temperature of 20-30℃; the fermentation process includes: after continuous light treatment at a light intensity of 3000-5000 lux for 20-28 hours, shaking culture is performed every 10-15 hours at an intensity of 50-250 rpm for 1-30 minutes.

[0014] Furthermore, the specific steps are as follows: S1. The seed culture medium formula is as follows: glucose 50g / L, yeast extract 5g / L, sodium sulfate 6g / L, magnesium sulfate 3g / L, ammonium sulfate 6g / L, potassium chloride 1.5g / L, calcium chloride 0.15g / L, potassium sulfate 0.8g / L, potassium dihydrogen phosphate 1.5g / L, monosodium glutamate 10g / L, zinc sulfate heptahydrate 3mg / L, cobalt chloride hexahydrate 0.05mg / L, copper sulfate pentahydrate 4mg / L, nickel sulfate hexahydrate 1.5mg / L, ferrous sulfate heptahydrate 12mg / L, calcium pantothenate 3mg / L, manganese chloride tetrahydrate 4mg / L, sodium molybdate dihydrate 0.04mg / L, pH 5. Sterilize at 121℃ for 20 minutes before use. The fermentation medium formula is as follows: glucose 80g / L, yeast extract 10g / L, sodium sulfate 8g / L, magnesium sulfate 3g / L, ammonium sulfate 6g / L, potassium chloride 1.5g / L, calcium chloride 0.15g / L, potassium sulfate 0.8g / L, potassium dihydrogen phosphate 1.5g / L, monosodium glutamate 18g / L, zinc sulfate heptahydrate 3mg / L, cobalt chloride hexahydrate 0.05mg / L, copper sulfate pentahydrate 4mg / L, nickel sulfate hexahydrate 1.5mg / L, ferrous sulfate heptahydrate 12mg / L, calcium pantothenate 3mg / L, manganese chloride tetrahydrate 4mg / L, sodium molybdate dihydrate 0.04mg / L, vitamin B6 7mg / L, vitamin B12 0.3mg / L, pH 6.0. It is sterilized at 121℃ for 20 minutes before use. S2. The Schizochytrium sp. HX-308 strain stored at -80℃ was inoculated into seed culture medium at a volume percentage of 1% and cultured at 25℃ and 180 rpm for 24 h to obtain primary seed culture. The primary seed culture was then inoculated into seed culture medium at a volume percentage of 1% and cultured at 25℃ and 180 rpm for 24 h to obtain secondary seed culture. The secondary seed culture was then inoculated into seed culture medium at a volume percentage of 1% and cultured at 25℃ and 180 rpm for 24 h to obtain tertiary seed culture. S3. The third-stage seed culture was inoculated into the fermentation medium at a volume percentage of 10%, and fermented at 25℃ and 200 rpm for 120 h. After fermentation began, 1.8 mmol / L of abscisic acid was added to the fermentation broth. The results showed that the oil yield increased from 29.4 g / L to 50.1 g / L. The fermentation broth was obtained after fermentation. S4. Add the cell wall breaking enzyme to the fermentation broth at a dosage of 3 g / L, and enzymatically hydrolyze it for 10 h at pH 12, rotation speed of 150 rpm and temperature of 50℃ to obtain the cell wall breaking liquid. After cooling the cell wall breaking liquid, mix it with ethanol at a volume ratio of 1:1, and then mix it with n-hexane for extraction to obtain the n-hexane phase. Remove the n-hexane phase by rotary evaporation to obtain microbial oil. Alternatively, the specific steps are as follows: S1. The formula for seed culture medium is: glucose 30g / L, yeast extract 6g / L, sodium sulfate 6g / L, potassium dihydrogen phosphate 1.5g / L, magnesium sulfate heptahydrate 3g / L, pH 5, sterilized at 121℃ for 20min before use. The fermentation medium formula is as follows: glucose 80g / L, yeast extract 12g / L, sodium sulfate 8g / L, potassium sulfate 3g / L, ammonium sulfate 6g / L, potassium chloride 1.5g / L, calcium chloride 0.15g / L, potassium dihydrogen phosphate 10g / L, magnesium sulfate heptahydrate 6g / L, pH 6.0, sterilized at 121℃ for 20min before use; S2. The mycelium of Mortierella alpina ACCC 31952 stored at -80℃ was inoculated into seed culture medium and cultured for 3 days at 25℃ and 180 rpm to obtain seed liquid. S3. The seed culture was inoculated into the fermentation medium at a volume percentage of 10%, and fermented at 25℃ and 200 rpm for 96 days. After the start of fermentation, 1.5 mmol / L of abscisic acid was added to the fermentation broth. The results showed that the oil yield increased from 12.6 g / L to 20.8 g / L. The fermentation broth was obtained after the fermentation was completed. S4. Add the cell wall breaking enzyme to the fermentation broth at a dosage of 3 g / L, and enzymatically hydrolyze it for 10 h at pH 12, rotation speed of 150 rpm and temperature of 50℃ to obtain the cell wall breaking liquid. After cooling the cell wall breaking liquid, mix it with ethanol at a volume ratio of 1:1, and then mix it with n-hexane for extraction to obtain the n-hexane phase. Remove the n-hexane phase by rotary evaporation to obtain microbial oil. Alternatively, the specific steps are as follows: S1. The fermentation medium formula is as follows: sodium sulfate 2g / L, potassium dihydrogen phosphate 2g / L, zinc sulfate heptahydrate 3mg / L, cobalt chloride hexahydrate 0.05mg / L, copper sulfate pentahydrate 4mg / L, nickel sulfate hexahydrate 1.5mg / L, ferrous sulfate heptahydrate 12mg / L, calcium pantothenate 3mg / L, manganese chloride tetrahydrate 4mg / L, sodium molybdate dihydrate 0.04mg / L, vitamin B1 0.3mg / L, vitamin B6 0.3mg / L, vitamin B12 0.1mg / L. Sterilize at 121℃ for 20min before use. S2. Chlorella ATCC 50258 stored at -80℃ was inoculated into the fermentation medium at a volume percentage of 5%. Under the condition of 25℃, it was first treated with continuous light intensity of 4000 lux for 24 h, and then the shake flask was shaken every 12 h. The shaking intensity of each shaking culture was 150 rpm and the shaking time was 20 min, for a total of 168 h. After the fermentation started, 1.2 mmol / L of abscisic acid was added to the fermentation broth. The results showed that the oil yield increased from 1.1 g / L to 1.9 g / L. The fermentation broth was obtained after the fermentation was completed. S3. Add the cell wall breaking enzyme to the fermentation broth at a dosage of 3 g / L. Enzymatically hydrolyze for 10 h at pH 12, rotation speed of 150 rpm and temperature of 50℃ to obtain the cell wall breaking liquid. After cooling the cell wall breaking liquid, mix it with ethanol at a volume ratio of 1:1, and then mix it with n-hexane for extraction to obtain the n-hexane phase. Remove the n-hexane phase by rotary evaporation to obtain microbial oil.

[0015] The above-described method is applied to increasing the yield of functional oils.

[0016] The advantages and positive effects of this invention are as follows: 1. This invention proposes for the first time a method to increase the content of polyunsaturated fatty acids during microbial fermentation by adding a novel exogenous compound, abscisic acid (ABA). Compared with the control group without ABA, the oil content of Schizochytrium abscisic acid-added strains was significantly increased, as were the contents of DHA-producing oil and β-carotene. Compared with the control, the β-carotene content increased by 47% with the addition of 1.8 g / L ABA. Specifically, the oil yield increased by 47.61%, 55.1%, and 70.4% with the addition of 1.2 g / L, 1.5 g / L, and 1.8 g / L ABA, respectively, with an average increase of approximately 57.7% in oil content. The addition of ABA not only increases the oil content during fermentation but also promotes the growth of Schizochytrium abscisic acid and the accumulation of DHA.

[0017] 2. Compared with the control group without abscisic acid, the addition of 1.5 mM abscisic acid increased the oil content of *Morchella alpina* and the content of ARA-producing oil. Compared with the control, the addition of 1.8 g / L abscisic acid increased the ARA oil content by 79.2%, with the addition of 1.5 g / L abscisic acid increasing the oil yield by 65%. Appropriate addition of abscisic acid not only increases the oil content during fermentation but also promotes the growth of *Morchella alpina* and the accumulation of ARA.

[0018] 3. Compared with the control group without abscisic acid, the oil content of Chlorella with 1.2 mM abscisic acid was increased, as were the contents of DHA-producing and EPA-producing oils. Compared with the control group, the DHA-producing oil content of Chlorella with 1.5 mM abscisic acid was significantly increased, and the EPA-producing oil content increased by 17% with 2 mM abscisic acid. The addition of abscisic acid not only increased the oil content during fermentation but also promoted the growth of Chlorella.

[0019] 4. In the experiment of fermenting and producing oil from Schizochytrium, the inventors found that adding abscisic acid during fermentation would increase the yield of oil, and the β-carotene content would increase significantly within a specific concentration range, rising by 47%. Moreover, abscisic acid is inexpensive and readily available. This method saves manpower, material resources and costs, and greatly improves the efficiency of oil production, making it suitable for industrial production.

[0020] 5. The strategy of this invention has been proven effective in a variety of oil-producing microorganisms, including Schizochytrium, Alpine Monascus, and Chlorella, and has positive significance for the production of functional polyunsaturated fatty acids (DHA, EPA, ARA), showing the general applicability of the treatment.

[0021] 6. Abscisic acid (ABA), as an important plant hormone, is used as a regulator to promote microbial oil production, representing a green production method. Furthermore, ABA has poor thermal stability and degrades during oil extraction, without affecting the quality of the oil. Attached Figure Description

[0022] Figure 1 A comparison of the fermentation results of Schizochytrium with 1.8 mM abscisic acid in this invention and the control. Figure 2 This is a comparison chart showing the fermentation results of adding 1.8 mM abscisic acid at different times after the start of Schizochytrium fermentation in this invention. Figure 3 This is a comparison chart of the fermentation results of *Morchella alpina* with 1.5 mM abscisic acid added and the control in this invention; Figure 4 This is a comparison chart of the fermentation results of adding 1.5 mM abscisic acid at different times after the start of fermentation of *Morchella alpina* in this invention; Figure 5 A comparison of the fermentation results of Chlorella with 1.2 mM abscisic acid in this invention and the control. Figure 6 This is a comparison chart showing the fermentation results of adding 1.2 mM abscisic acid at different times after the start of Chlorella fermentation in this invention. Detailed Implementation

[0023] 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.

[0024] 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.

[0025] Application of abscisic acid in promoting the rapid accumulation of functional oils through the fermentation of oil-producing microorganisms.

[0026] Preferably, the oil-producing microorganism is selected from Schizochytrium (Schizochytrium). Schizochytrium sp.) HX-308, Schizochytrium ( Schizochytrium sp.) ATCC 20888, Alpine Flocculation ( Mortierella alpina ACCC 31952, Alpine Morphyra ( Mortierella alpina ACCC 32468, Alpine Morphyra ( Mortierella alpina ACCC 31578, Chlorella ( Chlorella ATCC 50258, Chlorella ( Chlorella ) Chlorella At least one of ATCC 11469; Alternatively, the final concentration of the exogenous compound added during the fermentation process of the abscisic acid is 0.5-3 mM; Alternatively, the functional oils may include docosahexaenoic acid, eicosapentaenoic acid, or eicosapentaenoic acid.

[0027] A method for promoting the rapid accumulation of functional oils by oleogenous microorganisms using abscisic acid, wherein the oleogenous microorganisms are fermented and cultured, and exogenous compound abscisic acid is added during the fermentation process.

[0028] Preferably, the oil-producing microorganism is selected from at least one of Schizochytrium, Morphozoa alpinea, or Chlorella. Alternatively, the final concentration of abscisic acid added during the fermentation process is 0.5-3 mM; Alternatively, the functional oils may include docosahexaenoic acid, eicosapentaenoic acid, or eicosapentaenoic acid.

[0029] Preferably, the oil-producing microorganism is selected from Schizochytrium (Schizochytrium). Schizochytrium sp.) HX-308, Schizochytrium ( Schizochytrium sp.) ATCC 20888, Alpine Flocculation ( Mortierella alpina ACCC 31952, Alpine Morphyra ( Mortierella alpina ACCC 32468, Alpine Morphyra ( Mortierella alpina ACCC 31578, Chlorella ( Chlorella ATCC 50258, Chlorella ( Chlorella ) Chlorella At least one of ATCC 11469; When the oil-producing microorganism is Schizochytrium ( Schizochytrium When using sp.) HX-308, abscisic acid was added at a final concentration of 1.8 mM at 0 h of fermentation culture, and fermentation continued for 120 h. When the oil-producing microorganism is Mortierella alpina ACCC 31952, abscisic acid is added at a final concentration of 1.5 mM at 0 h of fermentation culture and fermented for 96 days. When the oil-producing microorganism is Chlorella ATCC 50258, abscisic acid is added at a final concentration of 1.2 mM at 0 h of fermentation culture and fermented for 168 h.

[0030] Preferably, the steps include: The seed culture of oil-producing microorganisms was obtained by culturing the microbial strains. The seed culture was then inoculated into a fermentation medium and fermented to obtain a fermentation broth. At 0 h of fermentation, the exogenous compound abscisic acid was added.

[0031] Preferably, the fermentation medium can provide the nutrients required for the growth and proliferation of the oil-producing microorganism. When the oil-producing microorganism is a Schizochytrium, the fermentation medium comprises: glucose 60-100 g / L, yeast extract 5-15 g / L, sodium sulfate 5-12 g / L, magnesium sulfate 2-4 g / L, ammonium sulfate 4-8 g / L, potassium chloride 1-2 g / L, calcium chloride 0.1-0.2 g / L, potassium sulfate 0.5-1 g / L, and diphosphate phosphate. Potassium hydrogen 0.5-2 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, ferrous sulfate heptahydrate 8-15 mg / L, calcium pantothenate 2-4 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.02-0.06 mg / L, vitamin B6 4-10 mg / L, vitamin B12 0.1-0.5 mg / L, pH 6.0-6.5; When the oil-producing microorganism is *Morchella alpineensis*, the fermentation medium comprises: glucose 60-100 g / L, yeast extract 8-15 g / L, sodium sulfate 3-12 g / L, potassium sulfate 2-4 g / L, ammonium sulfate 4-8 g / L, potassium chloride 1-2 g / L, calcium chloride 0.1-0.2 g / L, potassium dihydrogen phosphate 5-20 g / L, magnesium sulfate 2-10 g / L, and a pH of 5.0-6.5. When the oil-producing microorganism is Chlorella, the fermentation medium comprises: sodium sulfate 0.1-4 g / L, potassium dihydrogen phosphate 0.2-4 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, ferrous sulfate heptahydrate 8-15 mg / L, calcium pantothenate 2-4 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.02-0.06 mg / L, vitamin B1 0.02-0.4 mg / L, vitamin B6 0.04-0.5 mg / L, and vitamin B12 0.01-0.2 mg / L; According to the present invention, the seed culture of the oil-producing microorganism can be obtained by activating the oil-producing microorganism strain on a plate and then diluting it into a liquid culture medium, or by inoculating the oil-producing microorganism strain into a liquid seed culture medium and culturing it. Preferably, the preparation process of the seed culture of the oil-producing microorganism includes: inoculating the oil-producing microorganism strain into a seed culture medium for at least one seed culture. More preferably, the preparation process of the seed culture of the oil-producing microorganism includes: inoculating the oil-producing microorganism strain into a seed culture medium for 1-3 seed cultures.

[0032] Alternatively, the seed culture of the oil-producing microorganism is obtained by inoculating the microbial strain into a seed culture medium, which provides the nutrients required for the growth and proliferation of the oil-producing microorganism. When the oil-producing microorganism is a Schizochytrium fungus, the seed culture medium comprises: glucose 40-60 g / L, yeast extract 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, calcium chloride 0.1-0.2 g / L, and sulfur... 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, ferrous sulfate heptahydrate 8-15 mg / L, calcium pantothenate 2-4 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.02-0.06 mg / L, pH 4.0-6.5; When the oil-producing microorganism is *Morchella alpineensis*, the seed culture medium comprises: 20-40 g / L glucose, 4-8 g / L yeast extract, 2-8 g / L sodium sulfate, 0.5-4 g / L potassium dihydrogen phosphate, 0.1-5 g / L magnesium sulfate heptahydrate, and a pH of 4.0-6.5.

[0033] Preferably, when the oil-producing microorganism is *Schizochytrium*, the seed culture conditions include at least: an inoculum size of 0.5-2% by volume, a temperature of 20-30°C, a rotation speed of 150-250 rpm, and a time of 18-30 h; the fermentation culture conditions include at least: an inoculum size of 2-20% by volume, a temperature of 20-30°C, and a rotation speed of 150-550 rpm; when the oil-producing microorganism is *Morchella alpineensis*, the seed culture conditions include at least: an inoculum size of 0.5-2% by volume, a temperature of 10-30°C, and a rotation speed of 150-250 rpm. The fermentation time is 2-4 days; the fermentation conditions include at least the following: inoculum size of 2-20% by volume, temperature of 20-30℃, and rotation speed of 100-450 rpm; when the oil-producing microorganism is Chlorella, the fermentation conditions include at least the following: inoculum size of 0.5-10% by volume, temperature of 20-30℃; the fermentation process includes: after continuous light treatment at a light intensity of 3000-5000 lux for 20-28 hours, shaking culture is performed every 10-15 hours at an intensity of 50-250 rpm for 1-30 minutes.

[0034] Preferably, the specific steps are as follows: S1. The seed culture medium formula is as follows: glucose 50g / L, yeast extract 5g / L, sodium sulfate 6g / L, magnesium sulfate 3g / L, ammonium sulfate 6g / L, potassium chloride 1.5g / L, calcium chloride 0.15g / L, potassium sulfate 0.8g / L, potassium dihydrogen phosphate 1.5g / L, monosodium glutamate 10g / L, zinc sulfate heptahydrate 3mg / L, cobalt chloride hexahydrate 0.05mg / L, copper sulfate pentahydrate 4mg / L, nickel sulfate hexahydrate 1.5mg / L, ferrous sulfate heptahydrate 12mg / L, calcium pantothenate 3mg / L, manganese chloride tetrahydrate 4mg / L, sodium molybdate dihydrate 0.04mg / L, pH 5. Sterilize at 121℃ for 20 minutes before use. The fermentation medium formula is as follows: glucose 80g / L, yeast extract 10g / L, sodium sulfate 8g / L, magnesium sulfate 3g / L, ammonium sulfate 6g / L, potassium chloride 1.5g / L, calcium chloride 0.15g / L, potassium sulfate 0.8g / L, potassium dihydrogen phosphate 1.5g / L, monosodium glutamate 18g / L, zinc sulfate heptahydrate 3mg / L, cobalt chloride hexahydrate 0.05mg / L, copper sulfate pentahydrate 4mg / L, nickel sulfate hexahydrate 1.5mg / L, ferrous sulfate heptahydrate 12mg / L, calcium pantothenate 3mg / L, manganese chloride tetrahydrate 4mg / L, sodium molybdate dihydrate 0.04mg / L, vitamin B6 7mg / L, vitamin B12 0.3mg / L, pH 6.0. It is sterilized at 121℃ for 20 minutes before use. S2. The Schizochytrium sp. HX-308 strain stored at -80℃ was inoculated into seed culture medium at a volume percentage of 1% and cultured at 25℃ and 180 rpm for 24 h to obtain primary seed culture. The primary seed culture was then inoculated into seed culture medium at a volume percentage of 1% and cultured at 25℃ and 180 rpm for 24 h to obtain secondary seed culture. The secondary seed culture was then inoculated into seed culture medium at a volume percentage of 1% and cultured at 25℃ and 180 rpm for 24 h to obtain tertiary seed culture. S3. The third-stage seed culture was inoculated into the fermentation medium at a volume percentage of 10%, and fermented at 25℃ and 200 rpm for 120 h. After fermentation began, 1.8 mmol / L of abscisic acid was added to the fermentation broth. The results showed that the oil yield increased from 29.4 g / L to 50.1 g / L. The fermentation broth was obtained after fermentation. S4. Add the cell wall breaking enzyme to the fermentation broth at a dosage of 3 g / L, and enzymatically hydrolyze it for 10 h at pH 12, rotation speed of 150 rpm and temperature of 50℃ to obtain the cell wall breaking liquid. After cooling the cell wall breaking liquid, mix it with ethanol at a volume ratio of 1:1, and then mix it with n-hexane for extraction to obtain the n-hexane phase. Remove the n-hexane phase by rotary evaporation to obtain microbial oil. Alternatively, the specific steps are as follows: S1. The formula for seed culture medium is: glucose 30g / L, yeast extract 6g / L, sodium sulfate 6g / L, potassium dihydrogen phosphate 1.5g / L, magnesium sulfate heptahydrate 3g / L, pH 5, sterilized at 121℃ for 20min before use. The fermentation medium formula is as follows: glucose 80g / L, yeast extract 12g / L, sodium sulfate 8g / L, potassium sulfate 3g / L, ammonium sulfate 6g / L, potassium chloride 1.5g / L, calcium chloride 0.15g / L, potassium dihydrogen phosphate 10g / L, magnesium sulfate heptahydrate 6g / L, pH 6.0, sterilized at 121℃ for 20min before use; S2. The mycelium of Mortierella alpina ACCC 31952 stored at -80℃ was inoculated into seed culture medium and cultured for 3 days at 25℃ and 180 rpm to obtain seed liquid. S3. The seed culture was inoculated into the fermentation medium at a volume percentage of 10%, and fermented at 25℃ and 200 rpm for 96 days. After the start of fermentation, 1.5 mmol / L of abscisic acid was added to the fermentation broth. The results showed that the oil yield increased from 12.6 g / L to 20.8 g / L. The fermentation broth was obtained after the fermentation was completed. S4. Add the cell wall breaking enzyme to the fermentation broth at a dosage of 3 g / L, and enzymatically hydrolyze it for 10 h at pH 12, rotation speed of 150 rpm and temperature of 50℃ to obtain the cell wall breaking liquid. After cooling the cell wall breaking liquid, mix it with ethanol at a volume ratio of 1:1, and then mix it with n-hexane for extraction to obtain the n-hexane phase. Remove the n-hexane phase by rotary evaporation to obtain microbial oil. Alternatively, the specific steps are as follows: S1. The fermentation medium formula is as follows: sodium sulfate 2g / L, potassium dihydrogen phosphate 2g / L, zinc sulfate heptahydrate 3mg / L, cobalt chloride hexahydrate 0.05mg / L, copper sulfate pentahydrate 4mg / L, nickel sulfate hexahydrate 1.5mg / L, ferrous sulfate heptahydrate 12mg / L, calcium pantothenate 3mg / L, manganese chloride tetrahydrate 4mg / L, sodium molybdate dihydrate 0.04mg / L, vitamin B1 0.3mg / L, vitamin B6 0.3mg / L, vitamin B12 0.1mg / L. Sterilize at 121℃ for 20min before use. S2. Chlorella ATCC 50258 stored at -80℃ was inoculated into the fermentation medium at a volume percentage of 5%. Under the condition of 25℃, it was first treated with continuous light intensity of 4000 lux for 24 h, and then the shake flask was shaken every 12 h. The shaking intensity of each shaking culture was 150 rpm and the shaking time was 20 min, for a total of 168 h. After the fermentation started, 1.2 mmol / L of abscisic acid was added to the fermentation broth. The results showed that the oil yield increased from 1.1 g / L to 1.9 g / L. The fermentation broth was obtained after the fermentation was completed. S3. Add the cell wall breaking enzyme to the fermentation broth at a dosage of 3 g / L. Enzymatically hydrolyze for 10 h at pH 12, rotation speed of 150 rpm and temperature of 50℃ to obtain the cell wall breaking liquid. After cooling the cell wall breaking liquid, mix it with ethanol at a volume ratio of 1:1, and then mix it with n-hexane for extraction to obtain the n-hexane phase. Remove the n-hexane phase by rotary evaporation to obtain microbial oil.

[0035] The above-described method is applied to increasing the yield of functional oils.

[0036] In this invention, the fermentation culture of *Schizochytrium* and *Morchella alpineensis* can be carried out by shaking culture in shake flasks or by stirring culture in fermentation tanks (e.g., 5L or 50L fermentation tanks). Specifically, the shaking culture conditions for *Schizochytrium* are 20-30℃ and 150-250 rpm; the fermentation tank culture conditions for *Schizochytrium* are 20-30℃ and 150-550 rpm, with timely monitoring of glucose content to ensure that the carbon source concentration in the fermentation broth remains above 20 g / L; the shaking culture conditions for *Morchella alpineensis* are 20-30℃ and 150-250 rpm; and the fermentation tank culture conditions for *Schizochytrium* are 20-30℃ and 100-450 rpm, with timely monitoring of glucose content to ensure that the carbon source concentration in the fermentation broth remains above 20 g / L.

[0037] According to a particularly preferred embodiment of the present invention, a method for preparing microbial oil is provided, the method comprising: S1. Inoculate the oil-producing microorganisms into the seed culture medium at an inoculation rate of 0.5-2% by volume, and culture them for 18-30 hours at a temperature of 20-30℃ and a rotation speed of 150-200 rpm to obtain the primary seed culture. Take the primary seed culture and inoculate it into the seed culture medium at an inoculation rate of 0.5-2% by volume, and culture it for 18-30 hours at a temperature of 20-30℃ and a rotation speed of 150-200 rpm to obtain the secondary seed culture. Take the secondary seed culture and inoculate it into the seed culture medium at an inoculation rate of 0.5-2% by volume, and culture it for 18-30 hours at a temperature of 20-30℃ and a rotation speed of 150-200 rpm to obtain the tertiary seed culture. S2. Inoculate the tertiary seed culture into the fermentation medium at an inoculation rate of 2-20% by volume. Ferment at a temperature of 20-30℃ and a rotation speed of 150-550rpm for 96-168h. After the fermentation starts, add abscisic acid to the fermentation broth so that the amount of abscisic acid added during the fermentation stage is 0.5-3mM. After the fermentation is completed, the fermentation broth is obtained. S3. Add the cell wall disrupting enzyme to the fermentation broth at a dosage of 0.1-5 g / L, and enzymatically hydrolyze it for 5-15 h at a pH of 10-14, a rotation speed of 100-200 rpm, and a temperature of 40-60℃ to obtain the cell wall disrupting liquid. After cooling the cell wall disrupting liquid, mix it with ethanol at a volume ratio of 1:0.8-1.2, and then mix it with n-hexane for extraction to obtain the n-hexane phase. Remove the n-hexane phase by rotary evaporation to obtain microbial oil.

[0038] The microbial oil prepared by the method provided in the above preferred embodiment has a higher DHA content.

[0039] Specifically, the relevant preparation and testing methods are as follows: In the following examples, 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 disclosed in Chinese patent publication CN101575584A. Schizochytrium Schizochytrium sp. Purchased from the American Type Culture Collection, catalog number ATCC20888; Mortierella alpina was purchased from the China Agricultural Microbial Culture Collection Center, with the number ACCC 31952; alpine spores Mortierella alpina Purchased from the China Agricultural Microbial Culture Collection Center, catalog number ACCC 32468; alpine spores Mortierella alpina Purchased from the China Agricultural Microbial Culture Collection Center, catalog number ACCC 31578 (all related alpine spores were purchased from the China Agricultural Microbial Culture Collection Center and are also disclosed in Chinese patent application CN116622514A). Chlorella Chlorella Purchased from the American Type Culture Collection, catalog number ATCC 50258; Chlorella Chlorella Purchased from the American Type Culture Collection, catalog number ATCC 11469; Unless otherwise specified, room temperature refers to 25±5℃.

[0040] Biomass determination: Take 1 mL of the fermentation broth that has just finished fermentation, centrifuge at 4000 r / min for 5 min, discard the supernatant, add 1 mL of 2% NaCl solution to wash once, then wash once with 1 mL of distilled water, dry at 105℃ to constant weight, and finally weigh and calculate.

[0041] Example 1 A method for promoting the rapid accumulation of functional oils by oleogen-producing microorganisms using abscisic acid includes the following steps: S1. The seed culture medium formula is as follows: glucose 50g / L, yeast extract 5g / L, sodium sulfate 6g / L, magnesium sulfate 3g / L, ammonium sulfate 6g / L, potassium chloride 1.5g / L, calcium chloride 0.15g / L, potassium sulfate 0.8g / L, potassium dihydrogen phosphate 1.5g / L, monosodium glutamate 10g / L, zinc sulfate heptahydrate 3mg / L, cobalt chloride hexahydrate 0.05mg / L, copper sulfate pentahydrate 4mg / L, nickel sulfate hexahydrate 1.5mg / L, ferrous sulfate heptahydrate 12mg / L, calcium pantothenate 3mg / L, manganese chloride tetrahydrate 4mg / L, sodium molybdate dihydrate 0.04mg / L, pH 5. Sterilize at 121℃ for 20 minutes before use. The fermentation medium formula is as follows: glucose 80g / L, yeast extract 10g / L, sodium sulfate 8g / L, magnesium sulfate 3g / L, ammonium sulfate 6g / L, potassium chloride 1.5g / L, calcium chloride 0.15g / L, potassium sulfate 0.8g / L, potassium dihydrogen phosphate 1.5g / L, monosodium glutamate 18g / L, zinc sulfate heptahydrate 3mg / L, cobalt chloride hexahydrate 0.05mg / L, copper sulfate pentahydrate 4mg / L, nickel sulfate hexahydrate 1.5mg / L, ferrous sulfate heptahydrate 12mg / L, calcium pantothenate 3mg / L, manganese chloride tetrahydrate 4mg / L, sodium molybdate dihydrate 0.04mg / L, vitamin B6 7mg / L, vitamin B12 0.3mg / L, pH 6.0. It is sterilized at 121℃ for 20 minutes before use. S2. The Schizochytrium sp. HX-308 strain stored at -80℃ was inoculated into seed culture medium at a volume percentage of 1% and cultured at 25℃ and 180 rpm for 24 h to obtain primary seed culture. The primary seed culture was then inoculated into seed culture medium at a volume percentage of 1% and cultured at 25℃ and 180 rpm for 24 h to obtain secondary seed culture. The secondary seed culture was then inoculated into seed culture medium at a volume percentage of 1% and cultured at 25℃ and 180 rpm for 24 h to obtain tertiary seed culture. S3. The third-stage seed culture was inoculated into the fermentation medium at a volume percentage of 10%, and fermented at 25℃ and 200 rpm for 120 h. Abscisic acid (Abscisic Acid) of 0.5-3 mmol / L was added to the fermentation broth after fermentation began, with an optimal concentration of 1.8 mmol / L. Results showed that the oil yield increased from 29.4 g / L to 50.1 g / L. The fermentation broth was obtained after fermentation. S4. Add the cell-wall-breaking enzyme at a dosage of 3 g / L to the fermentation broth. Enzymatic hydrolysis is carried out for 10 hours at pH 12, a rotation speed of 150 rpm, and a temperature of 50℃ to obtain a cell-wall-breaking solution. After cooling, the cell-wall-breaking solution is mixed with ethanol at a volume ratio of 1:1, and then mixed with n-hexane for extraction to obtain the n-hexane phase. The n-hexane phase is then removed by rotary evaporation to obtain microbial oil. After rotary evaporation, the oil is dried in an oven to constant weight and weighed to obtain the oil. S5. Esterification of the oil: ① Add 1 ml of 1 mol / L potassium hydroxide-methanol solution to a 1.5 ml centrifuge tube, add 20 μl of oil (the liquid after constant weight in step S4) and mix well. Then add it to a 20 ml volumetric flask, and add 2 ml of 1 mol / L potassium hydroxide-methanol solution to the volumetric flask (rinse the 1.5 ml centrifuge tube to draw as much oil as possible into the volumetric flask), mix well, and incubate at 65°C for 17 min, then cool to room temperature; ② Add 2 ml of boron trifluoride-ether mixture (boron trifluoride: ether = 3:7, volume ratio), mix well, and incubate at 65°C for 7 min; ③ Add 2 ml of saturated potassium chloride and shake well, add 3 ml of n-hexane (chromatographic grade), and allow to stand for layering; S6. After separation, pour the upper layer of liquid into a small centrifuge tube and pass it through a microporous membrane to remove impurities. After sample preparation, perform gas chromatography (GC) analysis and calculate the content of lipids and DHA. GC analysis method: Refer to the chromatographic conditions for the detection of docosahexaenoic acid (DHA) in Chen Lizhu's "Analysis Method for the Production of DHA by Feed-Stick Culture of Schizochytrium": Select a capillary column (60m x 0.32nm x 15um). Use programmed temperature ramping: initial temperature 200℃, hold for 2 min, then ramp to 240℃ at 10℃ / min, hold for 40 min. Column pressure 200kPa, inlet temperature 250℃, detector temperature 280℃. Quantify using the internal standard method, with eicosanoic acid as the internal standard.

[0042] S7 β - Carotene content detection: Take 5 mL of freshly fermented broth (i.e., the fermentation broth obtained in step S3), centrifuge, add an equal volume of methanol to the fermentation broth for several soaking extractions, combine the methanol extracts, remove methanol using a rotary vacuum evaporator, add CCl4 to dissolve the concentrate, filter through a 0.22 μm organic filter membrane, and set aside for later use. Determination was performed using high performance liquid chromatography (HPLC). β- Carotene content. Chromatographic column: Amethyst C18-H column (4.6 x 150 mm, 5 μm), detection wavelength 4750 nm, column temperature room temperature, mobile phase flow rate 1.0 mL / min, gradient elution, preparation. β -Carotene standard sample was used to obtain the standard curve equation. The elution procedure is as follows:

[0043] Other indicators were measured using conventional methods available in the prior art.

[0044] Example 2 Using the method described in Example 1, abscisic acid was added to the fermentation medium at a certain concentration gradient (0.5 mmol / L, 1 mmol / L, 1.2 mol / L, 1.5 mol / L, 1.8 mol / L, 2 mol / L, 2.5 mol / L, 3 mol / L). Fermentation was stopped after 120 h, and various indicators were measured. The results are shown in Table 1.

[0045] Table 1

[0046]

[0047] As shown in Table 1, without the addition of abscisic acid (Schizochytrium control), *Schizochytrium* accumulated a biomass of 62.2 g / L, an oil yield of 29.4 g / L, and DHA content and yield of 42.5% and 12.5 g / L, respectively, with a β-carotene yield of 35 μg / g. After adding different concentrations of exogenous abscisic acid, the cell biomass increased, and all groups with added abscisic acid showed increased oil yield, DHA content, DHA yield, and β-carotene yield. Among the various treatments, the addition of 1.8 mM abscisic acid-containing *Schizochytrium* resulted in the highest oil content, effectively increasing the DHA content of the oil produced by *Schizochytrium* fermentation. The DHA content increased from 42.5% to 51.3%; biomass increased from 62.2 g / L to 86 g / L; oil yield increased from 29.4 g / L to 50.1 g / L; DHA yield increased from 12.5 g / L to 25.7 g / L; and β-carotene yield increased from 35 μg / g to 52.4 μg / g. Overall, DHA yield increased by over 20.7%, total oil yield increased by over 70%, and β-carotene yield also showed a significant increase. The amount of abscisic acid added is crucial in this invention; both excessively low and excessively high concentrations are ineffective, and even slight variations can lead to a significant decrease in various indicators.

[0048] Example 3 Using the method described in Example 1, 1.8 mM abscisic acid was added to the fermentation medium, and oil yield was measured at 0 h, 24 h, 48 h, 72 h, 96 h, and 120 h of fermentation. The results are shown below. Figure 1 .

[0049] The results showed that adding 1.8 mM abscisic acid significantly increased the final oil yield compared to the original strain, and required less fermentation time to produce the same amount of oil. Specifically, the group with abscisic acid added at 0 h of fermentation accumulated a high oil yield of 28.8 g / L at 72 h and 50.1 g / L at 120 h. In contrast, the control group without abscisic acid accumulated only 13.6 g / L of oil at 72 h and only 29.4 g / L at 120 h.

[0050] Furthermore, the addition time of abscisic acid was optimized, and tests were conducted at 0h, 12h, 24h, 36h, 48h, 60h, and 72h, with the addition of 1.8mM abscisic acid. The results are as follows... Figure 2 As shown, the oil yields obtained by adding 1.8 mM abscisic acid at 0h, 12h, 24h, 36h, 48h, 60h, and 72h were 50.1 g / L, 48.9 g / L, 45.6 g / L, 40.6 g / L, 36.2 g / L, 35.1 g / L, and 32.2 g / L, respectively. The oil yield decreased with delayed abscisic acid addition, because abscisic acid accelerates the growth of oil-producing microorganisms, and its effect is shortened the later it is added. Therefore, adding abscisic acid at 0h of fermentation is beneficial for the accumulation of oil by oil-producing microorganisms. It can also be seen that the amount and timing of abscisic acid addition in the method of this invention have a synergistic effect, jointly increasing the yield of the prepared oil. In particular, during the fermentation of Schizochytrium sp. HX-308, the addition of abscisic acid at a final concentration of 1.8 mM and the addition of abscisic acid at 0 h of fermentation showed a significant synergistic effect, which could significantly increase the yield of the prepared oil.

[0051] Example 4 A method for promoting the rapid accumulation of functional oils by oleogen-producing microorganisms using abscisic acid includes the following steps: S1. The formula for seed culture medium is: glucose 30g / L, yeast extract 6g / L, sodium sulfate 6g / L, potassium dihydrogen phosphate 1.5g / L, magnesium sulfate heptahydrate 3g / L, pH 5, sterilized at 121℃ for 20min before use. The fermentation medium formula is as follows: glucose 80g / L, yeast extract 12g / L, sodium sulfate 8g / L, potassium sulfate 3g / L, ammonium sulfate 6g / L, potassium chloride 1.5g / L, calcium chloride 0.15g / L, potassium dihydrogen phosphate 10g / L, magnesium sulfate heptahydrate 6g / L, pH 6.0, sterilized at 121℃ for 20min before use; S2. A small amount of mycelium of Mortierella alpina ACCC 31952, stored at -80℃, was inoculated into seed culture medium and cultured for 3 days at 25℃ and 180 rpm to obtain seed liquid. S3. The seed culture was inoculated into the fermentation medium at a volume percentage of 10%, and fermented for 96 days at 25℃ and 200 rpm. Abscisic acid (Abscisic Acid) of 0.5-3 mmol / L was added to the fermentation broth after fermentation began, with an optimal concentration of 1.5 mmol / L. Results showed that the oil yield increased from 12.6 g / L to 20.8 g / L. The fermentation broth was obtained after fermentation. S4. Add the cell wall breaking enzyme to the fermentation broth at a dosage of 3 g / L. Enzymatically hydrolyze for 10 h at pH 12, rotation speed of 150 rpm and temperature of 50℃ to obtain the cell wall breaking liquid. After cooling the cell wall breaking liquid, mix it with ethanol at a volume ratio of 1:1, and then mix it with n-hexane for extraction to obtain the n-hexane phase. Remove the n-hexane phase by rotary evaporation to obtain microbial oil.

[0052] Example 5 Using the method described in Example 4, abscisic acid was added to the fermentation medium at a certain concentration gradient (0.5 mmol / L, 1 mmol / L, 1.2 mol / L, 1.5 mol / L, 1.8 mol / L, 2 mol / L, 2.5 mol / L, 3 mol / L). Fermentation was stopped after 96 hours, and various indicators were measured. The results are shown in Table 2.

[0053] Table 2

[0054] As shown in Table 2, in the group without abscisic acid (ABA), *Morchella alpina* fermentation yielded 24 g / L of biomass and 12.6 g / L of oil, with an ARA content as high as 24%. After adding different concentrations of exogenous ABA, the biomass, oil content, and ARA content of *Morchella alpina* increased in almost all groups. The group with 1.5 mM ABA showed the highest biomass and oil content, reaching 42 g / L and 20.8 g / L, respectively. Furthermore, the addition of 1.5 mM ABA effectively increased the ARA content of the oil produced by *Morchella alpina* fermentation, reaching as high as 43%, which is more than 79% higher than the control group without ABA. The amount of ABA added in this invention is crucial; both excessively low and excessively high concentrations are ineffective, and even minute changes can lead to significant variations in various indicators.

[0055] Furthermore, the addition time of abscisic acid was optimized, and tests were conducted at 0h, 8h, 16h, 24h, 32h, 40h, and 48h with an addition of 1.5mM abscisic acid. The results are as follows: Figure 4 As shown, the oil yields obtained by adding 1.5 mM abscisic acid at 0h, 8h, 16h, 24h, 32h, 40h, and 48h were 20.8 g / L, 20.2 g / L, 19.5 g / L, 17.5 g / L, 15.7 g / L, 15.1 g / L, and 13.8 g / L, respectively. The oil yield decreased with delayed abscisic acid addition, because abscisic acid accelerates the growth of oil-producing microorganisms, and its effect is shortened the later it is added. Therefore, adding abscisic acid at 0h of fermentation is beneficial for the accumulation of oil by oil-producing microorganisms. It can also be seen that the amount and timing of abscisic acid addition in the method of this invention have a synergistic effect, jointly increasing the yield of the prepared oil. In particular, during the fermentation of Mortierella alpina ACCC 31952 strain, the addition of abscisic acid at a final concentration of 1.5 mM and the addition of abscisic acid at 0 h of fermentation showed a significant synergistic effect, which could significantly increase the yield of the prepared oil.

[0056] Example 6 Using the method described in Example 4, 1.5 mM abscisic acid was added to the fermentation medium, and oil yield was measured at 0 h, 24 h, 48 h, 72 h, and 96 h of fermentation. The results are shown below. Figure 3 .

[0057] The results showed that adding 1.5 mM abscisic acid significantly increased the final oil yield compared to the original strain, and required less fermentation time to produce the same amount of oil. Specifically, the group with added abscisic acid at 0 h of fermentation accumulated a high oil yield of 10.7 g / L at 48 h and 20.8 g / L at 96 h. In contrast, the control group without added abscisic acid accumulated only 4.8 g / L of oil at 72 h and only 12.6 g / L at 120 h.

[0058] Example 7 A method for promoting the rapid accumulation of functional oils by oleogen-producing microorganisms using abscisic acid includes the following steps: S1. The fermentation medium formula is as follows: sodium sulfate 2g / L, potassium dihydrogen phosphate 2g / L, zinc sulfate heptahydrate 3mg / L, cobalt chloride hexahydrate 0.05mg / L, copper sulfate pentahydrate 4mg / L, nickel sulfate hexahydrate 1.5mg / L, ferrous sulfate heptahydrate 12mg / L, calcium pantothenate 3mg / L, manganese chloride tetrahydrate 4mg / L, sodium molybdate dihydrate 0.04mg / L, vitamin B1 0.3mg / L, vitamin B6 0.3mg / L, vitamin B12 0.1mg / L. Sterilize at 121℃ for 20min before use. S2. Chlorella ATCC 50258, stored at -80℃, was inoculated into fermentation medium at a volume percentage of 5%. Under 25℃, the medium was first treated with continuous light at 4000 lux for 24 hours. Then, the flasks were shaken every 12 hours at a shaking intensity of 150 rpm for 20 minutes, for a total of 168 hours. Abscisic acid (0.5-3 mmol / L) was added to the fermentation broth after fermentation began, with an optimal concentration of 1.2 mmol / L. Results showed that the oil yield increased from 1.1 g / L to 1.9 g / L. The fermentation broth was obtained after fermentation. S3. Add the cell wall breaking enzyme to the fermentation broth at a dosage of 3 g / L. Enzymatically hydrolyze for 10 h at pH 12, rotation speed of 150 rpm and temperature of 50℃ to obtain the cell wall breaking liquid. After cooling the cell wall breaking liquid, mix it with ethanol at a volume ratio of 1:1, and then mix it with n-hexane for extraction to obtain the n-hexane phase. Remove the n-hexane phase by rotary evaporation to obtain microbial oil.

[0059] Example 8 Using the method described in Example 7, abscisic acid was added to the fermentation medium at a certain concentration gradient (0.5 mmol / L, 1 mmol / L, 1.2 mol / L, 1.5 mol / L, 1.8 mol / L, 2 mol / L, 2.5 mol / L, 3 mol / L). Fermentation was stopped after 168 hours, and various indicators were measured. The results are shown in Table 3.

[0060] Table 3

[0061]

[0062] As shown in Table 3, in the group without added abscisic acid, fermented Chlorella yielded 4.2 g / L biomass and 1.1 g / L oil, with a cell oil content of 26.2%. The EPA and DHA contents in the oil were 24% and 12%, respectively. Adding different concentrations of exogenous abscisic acid increased cell biomass and effectively improved the content of DHA and EPA oil produced by Chlorella fermentation. Furthermore, Chlorella with 1.2 mM abscisic acid showed the highest oil yield, reaching 1.9 g / L, more than 72% higher than the control group without abscisic acid; Chlorella with 2 mM abscisic acid showed the highest EPA content, reaching 28%, more than 17% higher than the control group without abscisic acid; and Chlorella with 1.5 mM abscisic acid showed the highest DHA content, reaching 25%, more than 108% higher than the control group without abscisic acid. The amount of abscisic acid added is crucial in this invention; both excessively low and high concentrations are ineffective, and even minute changes can lead to significant variations in various indicators.

[0063] Example 9 Using the method described in Example 7, 1.2 mM abscisic acid was added to the fermentation medium, and oil yield was measured at fermentation times of 0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h. The results are shown below. Figure 5 .

[0064] The results showed that adding 1.2 mM abscisic acid significantly increased the final oil yield compared to the original strain, and the fermentation time required to produce the same amount of oil was less.

[0065] Furthermore, the addition time of abscisic acid was optimized, and tests were conducted at 0h, 12h, 24h, 36h, 48h, 60h, and 72h, with the addition of 1.2mM abscisic acid. The results are as follows... Figure 6As shown, the oil yields obtained by adding 1.2 mM abscisic acid at 0h, 12h, 24h, 36h, 48h, 60h, and 72h were 1.9 g / L, 1.8 g / L, 1.7 g / L, 1.5 g / L, 1.4 g / L, 1.3 g / L, and 1.2 g / L, respectively. The amount of oil obtained decreased with the later the abscisic acid was added. This is because abscisic acid accelerates the growth of oil-producing microorganisms, and the later the abscisic acid is added, the shorter its effective period. Therefore, adding abscisic acid at 0h of fermentation is beneficial for the accumulation of oil by oil-producing microorganisms. It can also be seen that the amount and timing of abscisic acid addition in the method of this invention have a synergistic effect, jointly increasing the yield of the prepared oil. In particular, during the fermentation of Chlorella ATCC 50258, the addition of abscisic acid at a final concentration of 1.2 mM and the addition of abscisic acid at 0 h of fermentation showed a significant synergistic effect, which could significantly increase the yield of the prepared oil.

[0066] Application publication number CN108342349A showed that adding abscisic acid (ABA) to *Phaeodactylum tricornutum* significantly increased triglyceride production at an ABA concentration of 2 mg / L, but the total triglyceride production was less than 0.03 g / L. Application publication number CN107446875A showed that adding ABA to *Phaeodactylum tricornutum* resulted in a biomass of 0.183 g / L at an ABA concentration of 20 mg / L. Application publication number CN104726508A showed that adding 20 mg / L ABA to *Cryptodinium kowti* increased lipid accumulation by 11.1%. However, these cases of lipid accumulation using ABA did not have the significant impact on oil-producing microbial fermentation as our strategy, and it also promoted the accumulation of DHA, EPA, ARA, and β-carotene. This is partly due to the difference in ABA concentration, and partly because our optimization of the ABA concentration and addition time was more specific. Furthermore, our strategy is not limited to a single oil-producing microorganism, but includes a variety of oil-producing microorganisms such as Schizochytrium, Alpine Sporozoa, and Chlorella, making its application more extensive.

[0067] 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. Application of abscisic acid in promoting the rapid accumulation of functional oils by oil-producing microorganisms.

2. The application according to claim 1, characterized in that: The oil-producing microorganisms are selected from Schizochytrium (… Schizochytrium sp.) HX-308, Schizochytrium ( Schizochytrium sp.) ATCC 20888, Alpine Flocculation ( Mortierella alpina ACCC 31952, Alpine Morphyra ( Mortierella alpina ACCC 32468, Alpine Morphyra ( Mortierella alpina ACCC 31578, Chlorella ( Chlorella ATCC 50258, Chlorella ( Chlorella ) Chlorella At least one of ATCC 11469; Alternatively, the final concentration of the exogenous compound added during the fermentation process of the abscisic acid is 0.5-3 mM; Alternatively, the functional oils may include docosahexaenoic acid, eicosapentaenoic acid, or eicosapentaenoic acid.

3. A method for promoting the rapid accumulation of functional oils by oleogen-producing microorganisms using abscisic acid, characterized in that: The method involves fermenting oil-producing microorganisms and adding the exogenous compound abscisic acid during the fermentation process.

4. The method according to claim 3, characterized in that: The oil-producing microorganism is selected from at least one of Schizochytrium, Morphyra hygrostis, or Chlorella. Alternatively, the final concentration of abscisic acid added during the fermentation process is 0.5-3 mM; Alternatively, the functional oils may include docosahexaenoic acid, eicosapentaenoic acid, or eicosapentaenoic acid.

5. The method according to claim 4, characterized in that: The oil-producing microorganisms are selected from Schizochytrium (… Schizochytrium sp.) HX-308, Schizochytrium ( Schizochytrium sp.) ATCC 20888, Alpine Flocculation ( Mortierella alpina ACCC 31952, Alpine Morphyra ( Mortierella alpina ACCC 32468, Alpine Morphyra ( Mortierella alpina ACCC 31578, Chlorella ( Chlorella ATCC 50258, Chlorella ( Chlorella ) Chlorella At least one of ATCC 11469; When the oil-producing microorganism is Schizochytrium ( Schizochytrium When using sp.) HX-308, abscisic acid was added at a final concentration of 1.8 mM at 0 h of fermentation culture, and fermentation continued for 120 h. When the oil-producing microorganism is Mortierella alpina ACCC 31952, abscisic acid is added at a final concentration of 1.5 mM at 0 h of fermentation culture and fermented for 96 days. When the oil-producing microorganism is Chlorella ATCC 50258, abscisic acid is added at a final concentration of 1.2 mM at 0 h of fermentation culture and fermented for 168 h.

6. The method according to claim 3, characterized in that: Includes the following steps: The seed culture of oil-producing microorganisms was obtained by culturing the microbial strains. The seed culture was then inoculated into a fermentation medium and fermented to obtain a fermentation broth. At 0 h of fermentation, the exogenous compound abscisic acid was added.

7. The method according to claim 6, characterized in that: The fermentation medium provides the nutrients required for the growth and proliferation of the oil-producing microorganism. When the oil-producing microorganism is *Schizochytrium*, the fermentation medium comprises: glucose 60-100 g / L, yeast extract 5-15 g / L, sodium sulfate 5-12 g / L, magnesium sulfate 2-4 g / L, ammonium sulfate 4-8 g / L, potassium chloride 1-2 g / L, calcium chloride 0.1-0.2 g / L, potassium sulfate 0.5-1 g / L, and potassium dihydrogen phosphate. 0.5-2 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, ferrous sulfate heptahydrate 8-15 mg / L, calcium pantothenate 2-4 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.02-0.06 mg / L, vitamin B6 4-10 mg / L, vitamin B12 0.1-0.5 mg / L, pH 6.0-6.5; When the oil-producing microorganism is *Morchella alpineensis*, the fermentation medium comprises: glucose 60-100 g / L, yeast extract 8-15 g / L, sodium sulfate 3-12 g / L, potassium sulfate 2-4 g / L, ammonium sulfate 4-8 g / L, potassium chloride 1-2 g / L, calcium chloride 0.1-0.2 g / L, potassium dihydrogen phosphate 5-20 g / L, magnesium sulfate 2-10 g / L, and a pH of 5.0-6.

5. When the oil-producing microorganism is Chlorella, the fermentation medium comprises: sodium sulfate 0.1-4 g / L, potassium dihydrogen phosphate 0.2-4 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, ferrous sulfate heptahydrate 8-15 mg / L, calcium pantothenate 2-4 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.02-0.06 mg / L, vitamin B1 0.02-0.4 mg / L, vitamin B6 0.04-0.5 mg / L, and vitamin B12 0.01-0.2 mg / L; Alternatively, the seed culture of the oil-producing microorganism is obtained by inoculating the microbial strain into a seed culture medium, which provides the nutrients required for the growth and proliferation of the oil-producing microorganism. When the oil-producing microorganism is a Schizochytrium fungus, the seed culture medium comprises: glucose 40-60 g / L, yeast extract 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, calcium chloride 0.1-0.2 g / L, and sulfur... 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, ferrous sulfate heptahydrate 8-15 mg / L, calcium pantothenate 2-4 mg / L, manganese chloride tetrahydrate 3-5 mg / L, sodium molybdate dihydrate 0.02-0.06 mg / L, pH 4.0-6.5; When the oil-producing microorganism is *Morchella alpineensis*, the seed culture medium comprises: 20-40 g / L glucose, 4-8 g / L yeast extract, 2-8 g / L sodium sulfate, 0.5-4 g / L potassium dihydrogen phosphate, 0.1-5 g / L magnesium sulfate heptahydrate, and a pH of 4.0-6.

5.

8. The method according to claim 6, characterized in that: When the oil-producing microorganism is *Schizochytrium*, the seed culture conditions shall at least include: inoculum size of 0.5-2% by volume, temperature of 20-30℃, rotation speed of 150-250 rpm, and time of 18-30 h; the fermentation culture conditions shall at least include: inoculum size of 2-20% by volume, temperature of 20-30℃, and rotation speed of 150-550 rpm; when the oil-producing microorganism is *Morchella alpineensis*, the seed culture conditions shall at least include: inoculum size of 0.5-2% by volume, temperature of 10-30℃, rotation speed of 150-250 rpm, and time of 18-30 h. The fermentation period is 2-4 days; the fermentation conditions include at least the following: inoculum size of 2-20% by volume, temperature of 20-30℃, and rotation speed of 100-450 rpm; when the oil-producing microorganism is Chlorella, the fermentation conditions include at least the following: inoculum size of 0.5-10% by volume, temperature of 20-30℃; the fermentation process includes: continuous light treatment at a light intensity of 3000-5000 lux for 20-28 hours, followed by shaking culture at a vibration intensity of 50-250 rpm for 1-30 minutes every 10-15 hours.

9. The method according to claim 3, characterized in that: The specific steps are as follows: S1. The seed culture medium formula is as follows: glucose 50g / L, yeast extract 5g / L, sodium sulfate 6g / L, magnesium sulfate 3g / L, ammonium sulfate 6g / L, potassium chloride 1.5g / L, calcium chloride 0.15g / L, potassium sulfate 0.8g / L, potassium dihydrogen phosphate 1.5g / L, monosodium glutamate 10g / L, zinc sulfate heptahydrate 3mg / L, cobalt chloride hexahydrate 0.05mg / L, copper sulfate pentahydrate 4mg / L, nickel sulfate hexahydrate 1.5mg / L, ferrous sulfate heptahydrate 12mg / L, calcium pantothenate 3mg / L, manganese chloride tetrahydrate 4mg / L, sodium molybdate dihydrate 0.04mg / L, pH 5. Sterilize at 121℃ for 20 minutes before use. The fermentation medium formula is as follows: glucose 80g / L, yeast extract 10g / L, sodium sulfate 8g / L, magnesium sulfate 3g / L, ammonium sulfate 6g / L, potassium chloride 1.5g / L, calcium chloride 0.15g / L, potassium sulfate 0.8g / L, potassium dihydrogen phosphate 1.5g / L, monosodium glutamate 18g / L, zinc sulfate heptahydrate 3mg / L, cobalt chloride hexahydrate 0.05mg / L, copper sulfate pentahydrate 4mg / L, nickel sulfate hexahydrate 1.5mg / L, ferrous sulfate heptahydrate 12mg / L, calcium pantothenate 3mg / L, manganese chloride tetrahydrate 4mg / L, sodium molybdate dihydrate 0.04mg / L, vitamin B6 7mg / L, vitamin B12 0.3mg / L, pH 6.

0. It is sterilized at 121℃ for 20 minutes before use. S2. The Schizochytrium sp. HX-308 strain stored at -80℃ was inoculated into seed culture medium at a volume percentage of 1% and cultured at 25℃ and 180 rpm for 24 h to obtain primary seed culture. The primary seed culture was then inoculated into seed culture medium at a volume percentage of 1% and cultured at 25℃ and 180 rpm for 24 h to obtain secondary seed culture. The secondary seed culture was then inoculated into seed culture medium at a volume percentage of 1% and cultured at 25℃ and 180 rpm for 24 h to obtain tertiary seed culture. S3. The third-stage seed culture was inoculated into the fermentation medium at a volume percentage of 10%, and fermented at 25℃ and 200 rpm for 120 h. After the fermentation started, 1.8 mmol / L of abscisic acid was added to the fermentation broth. The results showed that the oil yield increased from 29.4 g / L to 50.1 g / L. The fermentation broth was obtained after the fermentation was completed. S4. Add the cell wall breaking enzyme to the fermentation broth at a dosage of 3 g / L, and enzymatically hydrolyze it for 10 h at pH 12, rotation speed of 150 rpm and temperature of 50℃ to obtain the cell wall breaking liquid. After cooling the cell wall breaking liquid, mix it with ethanol at a volume ratio of 1:1, and then mix it with n-hexane for extraction to obtain the n-hexane phase. Remove the n-hexane phase by rotary evaporation to obtain microbial oil. Alternatively, the specific steps are as follows: S1. The formula for seed culture medium is: glucose 30g / L, yeast extract 6g / L, sodium sulfate 6g / L, potassium dihydrogen phosphate 1.5g / L, magnesium sulfate heptahydrate 3g / L, pH 5, sterilized at 121℃ for 20min before use. The fermentation medium formula is as follows: glucose 80g / L, yeast extract 12g / L, sodium sulfate 8g / L, potassium sulfate 3g / L, ammonium sulfate 6g / L, potassium chloride 1.5g / L, calcium chloride 0.15g / L, potassium dihydrogen phosphate 10g / L, magnesium sulfate heptahydrate 6g / L, pH 6.0, sterilized at 121℃ for 20min before use; S2. The mycelium of Mortierella alpina ACCC 31952 stored at -80℃ was inoculated into seed culture medium and cultured for 3 days at 25℃ and 180 rpm to obtain seed liquid. S3. The seed culture was inoculated into the fermentation medium at a volume percentage of 10%, and fermented at 25℃ and 200 rpm for 96 days. After the start of fermentation, 1.5 mmol / L of abscisic acid was added to the fermentation broth. The results showed that the oil yield increased from 12.6 g / L to 20.8 g / L. The fermentation broth was obtained after the fermentation was completed. S4. Add the cell wall breaking enzyme to the fermentation broth at a dosage of 3 g / L, and enzymatically hydrolyze it for 10 h at pH 12, rotation speed of 150 rpm and temperature of 50℃ to obtain the cell wall breaking liquid. After cooling the cell wall breaking liquid, mix it with ethanol at a volume ratio of 1:1, and then mix it with n-hexane for extraction to obtain the n-hexane phase. Remove the n-hexane phase by rotary evaporation to obtain microbial oil. Alternatively, the specific steps are as follows: S1. The fermentation medium formula is as follows: sodium sulfate 2g / L, potassium dihydrogen phosphate 2g / L, zinc sulfate heptahydrate 3mg / L, cobalt chloride hexahydrate 0.05mg / L, copper sulfate pentahydrate 4mg / L, nickel sulfate hexahydrate 1.5mg / L, ferrous sulfate heptahydrate 12mg / L, calcium pantothenate 3mg / L, manganese chloride tetrahydrate 4mg / L, sodium molybdate dihydrate 0.04mg / L, vitamin B1 0.3mg / L, vitamin B6 0.3mg / L, vitamin B12 0.1mg / L. Sterilize at 121℃ for 20min before use. S2. Chlorella ATCC 50258 stored at -80℃ was inoculated into the fermentation medium at a volume percentage of 5%. Under the condition of 25℃, it was first treated with continuous light intensity of 4000 lux for 24 h, and then the shake flask was shaken every 12 h. The shaking intensity of each shaking culture was 150 rpm and the shaking time was 20 min, for a total of 168 h. After the fermentation started, 1.2 mmol / L of abscisic acid was added to the fermentation broth. The results showed that the oil yield increased from 1.1 g / L to 1.9 g / L. The fermentation broth was obtained after the fermentation was completed. S3. Add the cell wall breaking enzyme to the fermentation broth at a dosage of 3 g / L. Enzymatically hydrolyze for 10 h at pH 12, rotation speed of 150 rpm and temperature of 50℃ to obtain the cell wall breaking liquid. After cooling the cell wall breaking liquid, mix it with ethanol at a volume ratio of 1:1, and then mix it with n-hexane for extraction to obtain the n-hexane phase. Remove the n-hexane phase by rotary evaporation to obtain microbial oil.

10. The application of the method according to any one of claims 3 to 8 in increasing the yield of functional oils.

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