Sn-2 ARA oil produced by fermentation of Mortierella alpina mutant bacteria and preparation method thereof

Abstract

The present invention belongs to the technical field of microbial oil and fat nutritional products, and relates to a Sn-2 ARA oil produced by fermentation of a Mortierella alpine mutant strain and a preparation method thereof. The Mortierella alpine mutant strain is Mortierella alpine A1.0, and its deposit number is CCTCC: M20222092. The Sn-2 ARA content in the ARA oil produced by the present invention can be as high as 55.1%, and the process flow is simple, suitable for industrial-scale production, reducing environmental pollution and other problems, filling the gap in pure natural Sn-2 ARA oil.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial oil nutritional products, and in particular relates to a Sn-2 ARA oil produced by fermentation of a mutant strain of Mortierella alpina and a preparation method thereof. Background Art

[0002] ARA (AA), scientifically known as eicosatetraenoic acid, also known as arachidonic acid, is a long-chain polyunsaturated fatty acid from the Omega-6 family. ARA plays a crucial role as a phospholipid-bound structural lipid in the blood, liver, muscles, and other organ systems. Furthermore, ARA is a direct precursor of many bioactive circulating eicosanoic acid derivatives, such as prostaglandin E2 (PGE2), prostacyclin (PGI2), thromboxane A2 (TXA2), and leukotrienes C4 (LTC4). These bioactive substances play important regulatory roles in lipid and protein metabolism, hemorheology, vascular elasticity, leukocyte function, and platelet activation. Because the body's ability to synthesize ARA is low during infancy, ARA is considered an essential fatty acid during this period. On the one hand, providing a certain amount of ARA in the diet of infants and young children during this prime period of physical development can benefit their physical development. On the other hand, ARA deficiency can have serious adverse effects on the development of human tissues and organs, particularly the brain and nervous system.

[0003] Since infants and young children cannot synthesize ARA in their bodies and must obtain it externally, fermentation to produce high-content ARA oils has become an industrialized production method. ARA oils obtained through fermentation processes are triglyceride-type oils, and the structures of triglyceride oils are divided into Sn-1, Sn-2, and Sn-3 fatty acids. Scientific research shows that specific pancreatic lipases in the human body selectively hydrolyze Sn-1 and Sn-3 fatty acids to produce Sn-2 fatty acid monoglycerides and free fatty acids. Sn-2 fatty acid monoglycerides can be absorbed by the small intestinal mucosa and metabolized into triglycerides and phospholipids. The free fatty acids hydrolyzed from the Sn-1 and Sn-3 positions are partially absorbed and partially oxidized and decomposed into energy. Therefore, Sn-2 ARA is more easily absorbed by the human body than Sn-1 and Sn-3 ARA. Therefore, increasing the content of Sn-2 ARA in microbial oils through microbial fermentation processes, thereby enhancing the body's absorption and utilization of ARA, has become an urgent problem that needs to be solved.

[0004] Currently, the fermentation strain for arachidonic acid oil is Mortierella alpina. However, existing Mortierella alpina strains have low oil conversion yields, which has led to high costs for industrialized ARA production. Furthermore, literature reports that using glucose, yeast extract, glutamic acid and other raw materials as culture medium components does not effectively increase relative yield. As far as current technology is concerned, corn steep liquor has not yet been applied as a culture medium component in Mortierella alpina fermentation technology. Due to the many uncertainties in the microbial fermentation process, it is still unknown whether Mortierella alpina can grow normally in corn steep liquor fermentation medium. Summary of the Invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a Sn-2 ARA oil produced by fermentation of a mutant strain of Mortierella alpina and a preparation method thereof, thereby improving the absorption rate of ARA oil in the human body and effectively reducing production costs.

[0006] In order to achieve the above technical objectives, the inventors have screened out a high-ARA-yielding alpine fungus from the soil in a high-salinity area through a large number of experimental studies and unremitting efforts. They also achieved the industrial production of Sn-2 ARA oil through laboratory mutagenesis and domestication, as well as seed culture, fermentation culture, and oil extraction.

[0007] Specifically, the inventors screened out a high-ARA-producing Mortierella alpina strain from the soil in a high-salinity area, and induced and domesticated the strain according to the following method:

[0008] (1) Take OD 660 To prepare 20 mL of bacterial suspension with a concentration of 0.6-0.8, add 0.8 mL of prepared EMS stock solution, place in an incubator at 26-28°C and 120 rpm for mutagenesis for 40-60 minutes, draw some of the mutagenized bacterial liquid and add it to the 5% sodium thiosulfate solution prepared in advance to react for 5 minutes, wash with PBS 2-3 times, then dilute to an appropriate concentration and apply to the selection culture medium.

[0009] (2) Select a 30W UV lamp and irradiate for 30 minutes in advance, and take the OD 660 20 mL of bacterial suspension with a pH of 0.6 to 0.8 was placed in a flat-bottomed glass culture dish and stirred with a magnetic stirrer. The linear distance between the UV lamp and the culture dish was 30 cm. The bacterial suspension irradiated for 10 to 30 seconds was appropriately diluted and spread on the inhibitory solid culture medium and cultured in the dark.

[0010] (3) Strain selection: The larger strains on the inhibition plate are selected as candidate strains for fermentation culture (the fermentation container can be a microtiter plate or a conical flask, and the fermentation volume can be minimized to improve the mutagenesis efficiency).

[0011] (4) Drawing of the standard curve for determining oil content using vanillin phosphate: Release the fermentation broth (calculate the oil content in advance); dilute the broth several times (to keep the oil content within 0-10 g / L) and measure the OD 515 .

[0012] (5) Vanillin phosphate reaction method for rapid determination of bacterial oil content: Take 0.5 mL of uniform fermentation liquid, add 1.5 mL of sterile water to wash, centrifuge at 8000 r / min for 5 minutes, and repeat twice. After dilution with 2 mL of sterile water, shake evenly, take 0.1 mL of bacterial suspension and place it in a 25 mL stoppered test tube (distilled water is the control sample), add 2 mL of 98% H2SO4, boil in boiling water bath for 10 minutes, and then bathe in room temperature water bath for 5 minutes. Take out the stoppered test tube and add 5 mL of vanillin phosphate solution, shake evenly, bathe in 37°C water bath for 15 minutes, and then bathe in room temperature water bath for 10 minutes, measure its OD value at 515 nm, and set the OD value to 0. 515 The strains with larger values ​​and the strains with high ARA content at the Sn-2 position in the strains analyzed by ARA structure were selected as high-yield strains and named Mortierella alpine A1.0. They were deposited in the China Type Culture Collection of Wuhan University on December 28, 2022, with the deposit number CCTCC: M20222092.

[0013] In addition, the present invention provides a microbial oil rich in Sn-2 ARA, which is fermented by a mutant of Mortierella alpine. The mutant of Mortierella alpine is Mortierella alpine A1.0, and its deposit number is CCTCC: M20222092.

[0014] Furthermore, the present invention provides a method for preparing a microbial oil rich in Sn-2 ARA, which comprises culturing a mutant strain of Mortierella alpine through seed culture and fermentation culture to obtain a microbial oil rich in Sn-2 ARA, wherein the mutant strain of Mortierella alpine is Mortierella alpine A1.0, and its deposit number is CCTCC: M20222092.

[0015] Further preferably, the preparation method of the microbial oil rich in Sn-2 ARA as described above, wherein the seed culture is to make the shake flask seed liquid of the alpine morphology after activation and inoculate it into the seed culture medium for culture; the culture medium composition of the seed culture is: glucose 47-53g / L, sodium glutamate 2.5-3.5g / L, corn steep liquor 9-11g / L, magnesium sulfate 2.5-3.5g / L, potassium dihydrogen phosphate 0.15-0.25g / L, sodium chloride 1.2-1.6g / L. In a most preferred embodiment of the present invention, the culture medium composition of the seed culture is: glucose 50g / L, sodium glutamate 3g / L, corn steep liquor 10g / L, magnesium sulfate 3g / L, potassium dihydrogen phosphate 0.2g / L, sodium chloride 1.4g / L. It should be noted that the solid concentration of the corn steep liquor used in the present invention is 45-50%, and the amino nitrogen content is 4.0-4.5%.

[0016] Still further preferably, in the method for preparing microbial oils rich in Sn-2 ARA as described above, the culture conditions of the seed culture are: stirring speed 80-120 r / min, ventilation volume 0.6-1.0 m 3 / min, temperature 27~29℃.

[0017] Further preferably, in the method for preparing a microbial oil rich in Sn-2 ARA as described above, the fermentation culture medium composition is: glucose 55-63g / L / L, sodium glutamate 4.5-5.5g / L, corn steep liquor 9-11g / L, magnesium sulfate 4.5-5.5g / L, potassium dihydrogen phosphate 0.15-0.25g / L, sodium chloride 1.2-1.6g / L. In a most preferred embodiment of the present invention, the fermentation culture medium composition is: glucose 60g / L, sodium glutamate 5g / L, corn steep liquor 10g / L, magnesium sulfate 5g / L, potassium dihydrogen phosphate 0.2g / L; sodium chloride 1.4g / L.

[0018] Still further preferably, the method for preparing microbial oils rich in Sn-2 ARA as described above, wherein the culture conditions of the fermentation culture are: stirring speed 50-80 r / min, culture temperature 27-29 ° C, ventilation volume 700-800 m 3 / h, the fermentation cycle is 110 to 120 hours, the pH of the fermentation liquid is controlled at 6.0 to 7.0 by intermittently adding ammonia water, the glucose concentration is controlled at 50 g / L to 63 g / L in the early stage of fermentation, and glucose solution is used for sugar supplementation when the sugar concentration is lower than 50 g / L. The sugar supplementation is stopped in the late stage of fermentation until the glucose is completely consumed.

[0019] Further preferably, the method for preparing microbial oil rich in Sn-2 ARA as described above further comprises extracting and separating the fermentation product to obtain microbial oil, the steps being: filtering the fermentation broth, drying it, and then soaking it in food-grade n-butane for 8 to 16 hours, and finally separating the solvent from the oil, recovering the solvent, and obtaining microbial oil.

[0020] Still further preferably, in the method for preparing the microbial oil rich in Sn-2 ARA as described above, the ARA content in the microbial oil is not less than 60%, and the proportion of Sn-2 ARA in the ARA is not less than 55%.

[0021] Compared with the prior art, the present invention adopts a Mortierella alpina fermentation method to produce high-content ARA oil and high-content Sn-2 position ARA oil, which has the following advantages and significant improvements:

[0022] (1) The Sn-2 ARA grease produced by the present invention has a simple process flow, is suitable for industrial-scale production, reduces environmental pollution and other problems, and fills the gap in pure natural Sn-2 ARA grease.

[0023] (2) The content of Sn-2 ARA in the ARA oil produced by the present invention can be as high as 55.1%.

[0024] (3) The average ARA content in the ARA oil produced by the present invention is as high as 60%, and the oil has good stability and nutritional value.

[0025] (4) The present invention shortens the fermentation cycle and reduces production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the morphology of Mortierella alpina under a microscope;

[0027] Figure 2 This is the gas chromatogram of the fatty acid composition of Sn-2 ARA oil;

[0028] Figure 3 This is the gas chromatogram of the fatty acid composition of ARA oil. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below through specific embodiments. Where specific technical operating steps or conditions are not specified in the examples, they were performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional products.

[0030] Example 1 Mutagenesis of Sn-2 ARA Oil Production Strains

[0031] (1) A high-yield ARA Mortierella alpina was screened from the soil in a high-salinity area, and the OD 660 Add 0.8 mL of the prepared EMS solution to 20 mL of a 0.7% suspension of Mortierella alpina. Place in an incubator at 26-28°C and 120 rpm for mutagenesis for 40-60 minutes. Add a portion of the mutagenized bacterial suspension to a previously prepared 5% sodium thiosulfate solution and incubate for 5 minutes. Wash with PBS 2-3 times. Dilute to an appropriate concentration and apply to selective culture medium.

[0032] (2) Select a 30W UV lamp and irradiate for 30 minutes in advance, and take the OD 660 Place 20 mL of a 0.7% bacterial suspension into a flat-bottomed glass culture dish and stir with a magnetic stirrer. The linear distance between the UV lamp and the culture dish is 30 cm. Take the bacterial suspension irradiated for 10 to 30 seconds, dilute it appropriately, and spread it on the inhibitory solid culture medium. Culture in the dark.

[0033] (3) Strain selection: The larger strains on the inhibition plate are selected as candidate strains for fermentation culture (the fermentation container can be a microtiter plate or a conical flask, and the fermentation volume can be minimized to improve the mutagenesis efficiency).

[0034] (4) Drawing of the standard curve for determining oil content by vanillin phosphate: The fermentation broth was released and diluted several times (to keep the oil content within 0-10 g / L) to measure the OD 515 .

[0035] (5) Vanillin phosphate reaction method for rapid determination of bacterial oil content: Take 0.5 mL of uniform fermentation liquid, add 1.5 mL of sterile water to wash, centrifuge at 8000 r / min for 5 minutes, and repeat twice. After dilution with 2 mL of sterile water, shake evenly, take 0.1 mL of bacterial suspension and place it in a 25 mL stoppered test tube (distilled water is the control sample), add 2 mL of 98% H2SO4, boil in boiling water bath for 10 minutes, and then bathe in room temperature water bath for 5 minutes. Take out the stoppered test tube and add 5 mL of vanillin phosphate solution, shake evenly, bathe in 37°C water bath for 15 minutes, and then bathe in room temperature water bath for 10 minutes, measure its OD value at 515 nm, and set the OD value to 0. 515 The strains with larger values ​​and the strains with high ARA content at the Sn-2 position in the strains analyzed by ARA structure were preserved as high-yield strains.

[0036] Example 2Sn-2 ARA Grease Production

[0037] 1. Preparation of seed culture medium: glucose 50g / L, sodium glutamate 3g / L, corn steep liquor 10g / L, magnesium sulfate 3g / L, potassium dihydrogen phosphate 0.2g / L, sodium chloride 1.4g / L.

[0038] 2. Preparation of fermentation medium: glucose 60g / L, monosodium glutamate 5g / L, corn steep liquor 10g / L, magnesium sulfate 5g / L, potassium dihydrogen phosphate 0.2g / L; sodium chloride 1.4g / L.

[0039] 3. Seed culture: The seed culture solution of Mortierella alpina strain A1.0 (CCTCC: M20222092) of the present invention was added to the primary fermentation seed tank at an inoculation ratio of 10%, with a stirring speed of 110 r / min and a ventilation volume of 0.9 m 3 / min, and the culture temperature was 28.5℃, and the first-level seed culture was carried out to allow the bacteria to grow to the logarithmic phase to obtain the first-level seed liquid.

[0040] 4. Fermentation culture: Inoculate the first-grade seed liquid into the fermentation tank at a ratio of 10% for fermentation culture. During the culture process, the stirring speed is 70r / min, the culture temperature is 28.5℃, and the ventilation volume is controlled at 750m 3 / h, the pH of the fermentation liquid is controlled at 6.5 by intermittently adding ammonia water. In the early stage of the fermentation process, the glucose concentration is controlled at 50g / L-63g / L. When the sugar concentration is lower than 50g / L, 50-60% glucose solution is used for sugar supplementation. After fermentation for 90h, the sugar supplementation is stopped until the glucose is completely consumed. The fermentation cycle is 120h.

[0041] 5. Oil extraction: After the fermentation culture is completed, the fermentation liquid is filtered and dried, and then soaked in food-grade n-butane for 12 hours. Finally, the solvent is separated from the oil, and the solvent is recovered to obtain ARA oil.

[0042] Comparative Example 1Sn-2 ARA Grease Production (Culture Medium Formula is Different from Example 2)

[0043] 1. Seed culture medium preparation: glucose 50g / L, yeast extract 20g / L, magnesium sulfate 3g / L, potassium dihydrogen phosphate 0.2g / L, sodium chloride 1.4g / L.

[0044] 2. Preparation of fermentation medium: glucose 45 g / L, peptone 10 g / L, corn steep liquor powder 10 g / L, magnesium sulfate 3 g / L, potassium dihydrogen phosphate 0.2 g / L, sodium chloride 1.4 g / L.

[0045] 3. Seed culture: The seed culture solution of Mortierella alpina strain A1.0 (CCTCC: M20222092) of the present invention was added to the primary fermentation seed tank at an inoculation ratio of 10%, with a stirring speed of 110 r / min and a ventilation volume of 0.9 m 3 / min, and the culture temperature was 28.5℃, and the first-level seed culture was carried out to allow the bacteria to grow to the logarithmic phase to obtain the first-level seed liquid.

[0046] 4. Fermentation culture: Inoculate the first-grade seed liquid into the fermentation tank at a ratio of 10% for fermentation culture. During the culture process, the stirring speed is 70r / min, the culture temperature is 28.5℃, and the ventilation volume is controlled at 750m 3 / h, the pH of the fermentation liquid is controlled at 6.5 by intermittently adding ammonia water. In the early stage of the fermentation process, the glucose concentration is controlled at 50g / L-63g / L. When the sugar concentration is lower than 50g / L, 50-60% glucose solution is used for sugar supplementation. After fermentation for 90h, the sugar supplementation is stopped until the glucose is completely consumed. The fermentation cycle is 120h.

[0047] 5. Oil extraction: After the fermentation culture is completed, the fermentation liquid is filtered, dried, and then soaked in food-grade n-butane for 12 hours. Finally, the solvent is separated from the oil and the solvent is recovered to obtain ARA oil.

[0048] Comparative Example 2Sn-2 ARA Grease Production (Fermentation Process Parameters Different from Example 2)

[0049] 1. Preparation of seed culture medium: glucose 50g / L, sodium glutamate 3g / L, corn steep liquor 10g / L, magnesium sulfate 3g / L, potassium dihydrogen phosphate 0.2g / L, sodium chloride 1.4g / L.

[0050] 2. Preparation of fermentation medium: glucose 60g / L, monosodium glutamate 5g / L, corn steep liquor 10g / L, magnesium sulfate 5g / L, potassium dihydrogen phosphate 0.2g / L; sodium chloride 1.4g / L.

[0051] 3. Seed culture: The shake flask seed culture solution of the alpine fungus species A1.0 (CCTCC: M20222092) of the present invention is added to a primary fermentation seed tank at an inoculation ratio of 10%, the stirring speed is 180 r / min, the ventilation volume is controlled at 0.65 vvm, the culture temperature is 29°C, and the pH is 7.5. The primary seed culture is carried out until the Sn-2 position ARA strain grows to the logarithmic phase to obtain a primary seed solution.

[0052] 4. Fermentation culture: The first-level seed liquid is inoculated into the fermentation tank at a ratio of 10% for fermentation culture. During the culture process, the stirring speed is 90r / min, the culture temperature is 29°C, the ventilation volume is controlled at 0.9vvm, and the pH of the fermentation liquid is controlled at 7.5 by intermittent addition of ammonia water. In the early stage of the fermentation process, the glucose concentration is controlled at 50g / L-63g / L. When the sugar concentration is lower than 50g / L, 50-60% glucose solution is used for sugar supplementation. After fermentation for 90h, the flow addition of sugar is stopped until the glucose is completely consumed. The fermentation cycle is 120h.

[0053] 5. Oil extraction: After the fermentation culture is completed, the fermentation liquid is filtered, dried, and then soaked in food-grade n-butane for 12 hours. Finally, the solvent is separated from the oil and the solvent is recovered to obtain ARA oil.

[0054] Example 3 Comparison of fermentation results with different treatments

[0055] Following the steps of Example 2, the fermentation strains of Mortierella alpina A1.0 (CCTCC: M20222092) before and after mutagenesis in Example 1 were used to obtain the corresponding ARA oils. The Sn-2ARA content, ARA content, and total oil content of the products were tested.

[0056] According to the steps of Comparative Example 1, the mutagenized Mortierella alpina strain CCTCC: M20222092 was used as the fermentation strain, and a culture medium different from the fermentation formula of Example 2 was used as the raw material and the same fermentation control parameters were used for fermentation culture to obtain the corresponding ARA oil, and its Sn-2ARA content, ARA content, and total oil were detected.

[0057] According to the steps of Comparative Example 2, the mutagenized Mortierella alpina strain CCTCC: M20222092 was used as the fermentation strain, and the culture medium with the same fermentation formula was used as the raw material, but the fermentation process control parameters different from those in Example 2 were selected for fermentation and culture to obtain the corresponding ARA oil, and its Sn-2ARA content, ARA content, and total oil were detected.

[0058] Total oil: refers to the weight of total fat in every 100g of fermented dry bacteria.

[0059] ARA content: refers to the weight of ARA in every 100g of oil.

[0060] Sn-2 ARA content: refers to the ratio of Sn-2ARA content to ARA content.

[0061] The ARA content in the present invention can be determined by referring to the method of GB5009.168.

[0062] The test method for the Sn-2 position ARA content in the present invention is as follows:

[0063] 1) Weigh 0.1g of sample into a 10mL centrifuge tube, add approximately 30mg of lipase (porcine pancreatic lipase) and 2mL of 1mol / L tris(hydroxymethyl)aminomethane buffer solution, and shake evenly. Then, add 0.5mL of 1‰ sodium cholate solution and 0.2mL of calcium chloride solution. Cover with the stopper and shake carefully. Immediately place the centrifuge tube in a 40°C water bath and shake for 1 minute. Remove the centrifuge tube from the water bath and shake vigorously at 40°C for 2 minutes. Immediately add 1mL of hydrochloric acid and 1mL of dichloromethane, shake vigorously on a shaker, and centrifuge. Transfer the organic layer with a syringe and filter through a 0.45μm filter membrane into a liquid chromatography sample for later use.

[0064] 2) Liquid chromatography: Mobile phase: acetonitrile (containing 0.1% glacial acetic acid): dichloromethane (50:50); 4.6 mm × 250 mm, 5 μm amino column; column temperature: 40°C; flow rate: 1 ml / min; evaporative light scattering detector (ELSD); offset tube: 70°C; nebulizer pressure: 0.3 MPa.

[0065] 3) Separation of monoglycerides using a chromatographic column: Inject 40 μl of monoglyceride standard into a liquid chromatograph to determine the retention time of monoglycerides. Then inject 40 μl of sample into the liquid chromatograph and collect the monoglyceride fractions according to the retention time of monoglycerides.

[0066] 4) The collected monoglycerides were blown dry with a nitrogen blower, dissolved in 1 ml of n-hexane, and then 0.2 ml of a 2 mol / L potassium hydroxide methanol solution was added. The mixture was shaken vigorously for 2 min, allowed to stand for separation, and the n-hexane layer was transferred to a gas chromatography sample bottle for later use.

[0067] 5) Fatty acid determination was performed according to GB5009.168 method.

[0068] The assay method of total oil in the present invention is as follows:

[0069] 1) Take a certain amount of fermentation liquid, filter, dry, and crush for later use;

[0070] 2) Weigh 4-5 g (accurate to 0.001 g) of the crushed powder into a filter paper tube, and then seal the filter paper tightly to prevent the powder from leaking out.

[0071] 3) Place the filter paper tube into the extraction chamber of the direct drop fat extractor, connect a 250ml flat-bottom flask with constant weight below, add an appropriate amount (about 30ml) of petroleum ether to the flat-bottom flask, and then connect the flat-bottom flask to the condenser to keep it vertical.

[0072] 4) After reflux extraction for 2 h, the petroleum ether in the flat-bottom flask was desolventized and recovered, and the oil was then dried at a constant temperature of 105°C for 2 h and weighed.

[0073] 5) The absolute difference between the weights before and after weighing the bottle divided by the sample volume equals the total oil content in the bacteria.

[0074] Table 1 Comparison of fermentation results of strains before and after mutagenesis As can be seen from the test results in Table 1, the content of Sn-2ARA produced by the mutagenized strain is significantly higher than that before mutagenization. In addition, although the mutagenized strain was also used, the Sn-2ARA content in the ARA oil prepared in Example 2 was greatly increased. The reason may be that the corn steep liquor with an appropriate solid content not only contains amino nitrogen, but also contains other nutrients that are conducive to the growth of Mortierella alpina, and the high ventilation volume during the production process creates a good oxidizing environment for the microorganisms, promoting their accelerated metabolism and fat accumulation, while promoting the enrichment of Sn-2ARA.

[0075] The above is only a preferred specific implementation method of the present invention, but the scope of protection of the present invention is not limited to this. Any technician familiar with this technical field, within the technical scope disclosed by the present invention, can make equivalent substitutions or changes based on the technical solution and inventive concept of the invention, which should be covered by the scope of protection of the present invention.

Claims

1. A method for preparing a microbial oil rich in Sn-2 ARA, characterized in that: The method comprises the steps of: subjecting a Mortierella alpina mutant to seed culture and fermentation culture to obtain a microbial oil rich in Sn-2 ARA; wherein the Mortierella alpina mutant is Mortierella alpina ( Mortierella alpine ) A1.0, its deposit number is CCTCC: M20222092.

2. The method for preparing a microbial oil rich in Sn-2 ARA according to claim 1, wherein The seed culture comprises the following steps: activating the Mortierella alpina mutant bacteria to prepare a shake flask seed solution, inoculating the solution into a seed culture medium for culture; and the culture medium for the seed culture comprises the following components: 47-53 g / L glucose, 2.5-3.5 g / L sodium glutamate, 9-11 g / L corn steep liquor, 2.5-3.5 g / L magnesium sulfate, 0.15-0.25 g / L potassium dihydrogen phosphate, and 1.2-1.6 g / L sodium chloride.

3. The method for preparing a microbial oil rich in Sn-2 ARA according to claim 2, wherein: The culture conditions of the seed culture are: stirring speed 80~120r / min, ventilation volume 0.6~1.0m 3 / min, temperature 27~29℃.

4. The method for preparing a microbial oil rich in Sn-2 ARA according to claim 1, wherein The fermentation culture medium comprises: 55-63 g / L / L glucose, 4.5-5.5 g / L sodium glutamate, 9-11 g / L corn steep liquor, 4.5-5.5 g / L magnesium sulfate, 0.15-0.25 g / L potassium dihydrogen phosphate, and 1.2-1.6 g / L sodium chloride.

5. The method for preparing the microbial oil rich in Sn-2 ARA according to claim 4, characterized in that: The fermentation culture conditions are as follows: stirring speed 50-80 r / min, culture temperature 27-29°C, ventilation volume 700-800 m 3 / h, the fermentation cycle is 110~120h, the pH of the fermentation liquid is controlled at 6.0~7.0 by intermittent addition of ammonia water, the glucose concentration is controlled at 50g / L~63g / L in the early stage of fermentation, and glucose solution is used for sugar supplementation when the sugar concentration is lower than 50g / L. The sugar supplementation is stopped in the late stage of fermentation until the glucose is completely consumed.

6. The method for preparing the microbial oil rich in Sn-2 ARA according to claim 1, characterized in that: The method also includes extracting and separating the fermentation product to obtain microbial oil, the steps of: filtering the fermentation liquid, drying it, then soaking it in food-grade n-butane for 8 to 16 hours, and finally separating the solvent from the oil, recovering the solvent, and obtaining the microbial oil.

7. The method for preparing the microbial oil rich in Sn-2 ARA according to claim 6, characterized in that: The ARA content in the microbial oil is not less than 60%, and the proportion of Sn-2 position ARA in the ARA is not less than 55%.

8. A Mortierella alpina mutagen, which is Mortierella alpina ( Mortierella alpine ) A1.0, its deposit number is CCTCC: M20222092.

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