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    • 专利摘要:
    The present disclosure relates to microbial technology, and provides a microbial oil containing ARA at an sn—2 position, a preparation method and uses therefor. A weight percentage of ARA at the sn—2 position of triglyceride in the microbial oil is not less than 23%. The method for producing the microbial oil is carried by inoculating Martierella alpina strains into a fermentation medium to for fermentation. The Martierella alpina strains is assigned accession No. 607344. The ARA at the sn—2 position of triglyceride in the microbial oil is far more than the ARA at the sn—1 and sn—3 position of triglyceride, which improves the absorption of the ARA in the microbial oil in the human body and effectively facilitates the utilization of ARA in the human body.
    公开号:NL2026989A
    申请号:NL2026989
    申请日:2020-11-26
    公开日:2021-09-02
    发明作者:Wang Shenjian；Liang Yun；Cao Sheng
    申请人:Qu hanpeng；
    IPC主号:
    专利说明:

    MICROBIAL OIL CONTAINING ARA AT SN-2 POSITION ANDPREPARATION METHOD AND USES THEREFOR
    TECHNICAL FIELD The present disclosure relates to microbial technology, and more particularly to a microbial oil containing ARA at an sn-2 position, and a preparation method and uses therefor.
    BACKGROUND Lipids, as essential nutrients for the human body, act as indispensable raw materials for building cells and tissues and supply energy for the body, and they can also provide fat-soluble vitamins. However, lipids need to be digested and absorbed in the intestines first and then play roles in the human body. Lipases from the pancreas and small intestines and cholate in the bile participate in the digestion and absorption of the lipids under an alkaline environment formed by bicarbonate secretion of the pancreas and bile. The lipid absorption efficiency depends on the types of enzymes involved in digestion and the action mechanisms thereof, and the structures of the lipid. A large number of studies have shown that the structures of the lipid largely influence the absorption rate of lipid in the human body. The lipids of triglyceride are classified into fatty acids at the sn-1, sn-2 or sn-2 position according to their binding positions on the glycerol. Pancreatic lipase, as a primary lipase enzyme, attaches a water-oil interface to hydrolyze dietary fat molecules. Meanwhile, pancreatic lipase only breaks down specific ester bonds at the sn-1 and sn-3 positions. Hydrolyzed by pancreatic lipase, fat molecules with triglyceride structure are converted into free fatty acids and monoglycerides. In the conversion, the free fatty acids are formed from the fatty acids at the sn-1 and sn-3 positions and hard to penetrate bile salt micelle and absorb, and the free fatty acids are then combined with calcium and magnesium ions in the intestine to form intestine soap salts and wasted, whereas the monoglycerides are formed from the fatty acids at the sn-3 position and easy to penetrate bile salt micelle and absorb. Therefore, the absorption rate of fatty acids at the sn-2 position in the human body is higher than that of the fatty acids at the sn-1 and sn-3 positions.
    As consumers become more aware of the health impact of fatty acids, microbial oils, as primary resources of fatty acids, have been largely adopted in infant food and nutraceuticals, and their nutritional benefits are increasingly recognized by the public. In addition, people are more and more concerned about the absorption rate of polyunsaturated fatty acids that are functional compositions in microbial oils, for example, arachidonic acid (ARA) and docosahexaenoic acid (DHA). 90% of fatty acids in microbial oils are triglycerides; however, in the existing microbial oils, the fatty acids at the sn-2 position of triglyceride are far less than those at the sn-1 and sn-3 positions. Most of fatty acids are formed into soap salts and wasted, which decreases the benefits of the microbial oils.
    SUMMARY The object of the present disclosure is to provide a microbial oil containing ARA at an sn-2 position, and a preparation method and uses therefor, in order to solve the problem that fatty acids are hard to absorb by the human body for there are too many fatty acids at the sn-1 and sn-3 positions of triglyceride in those microbial oils. The microbial oil provided herein is produced by a fermentation using Mortierella alpina, and a weight percentage of the ARA at the sn-2 position of triglyceride in the microbial oil is not less than 23%, improving absorption of ARA in the human body.
    In order to realize the above-mentioned object, the present disclosure provides a microbial oil containing ARA at the sn-2 position, and a weight percentage of the ARA at the sn-2 position of triglyceride in the microbial oil is not less than 23%, and a weight percentage of triglyceride ARA in the microbial oil is not less than 38%.
    The present disclosure further provides a method for producing the microbial oil mentioned above, comprising: inoculating Mortierella alpina strains into a fermentation medium for fermentation; wherein the Mortierella alpina strains is assigned accession No. 60734 In some embodiments, the fermentation medium comprises a carbon source and a nitrogen source; and a fermentation is performed at 27-31°C for 4-8 days under a ventilation at 0.5-1.1 vvm, wherein a carbon-to-nitrogen ratio is (3-18):1, and an inoculation volume ratio is 5-10%. In some embodiments, the method comprises: activating the Mortierella alpina strains; culturing Mortierella alpina strains for a propagation to obtain an inoculum; and inoculating the inoculum into the fermentation medium for the fermentation.
    In some embodiments, the activating and propagating process comprises: a) inoculating the Mortierella alpina strains into a potato dextrose agar (PDA) slant medium, culturing the Mortierella alpina strains until a formation and a maturity of spores, and performing an elution with sterile water to obtain a spore suspension; b)inoculating the spore suspension obtained in step a)into a seed culture medium in a shake flask for a propagation to obtain an inoculum; and c)inoculating the inoculum obtained in step b)into a primary seed tank, culturing the inoculum for 1-2 days. and then inoculating the inoculum into a secondary tank and culturing the inoculum for 1-2 days.
    In some embodiments, the step a)comprises: inoculating the Mortierella alpina strains into a PDA slant medium in a test tube, and culturing the Mortierella alpina strains at 25-30°C for 5-7 days for the formation of the spores; inoculating the spores into a PDA slant medium in an eggplant-shaped bottle, and culturing the spores at 25-30°C for 4-6 days for the maturity of the spores; and removing mycelium and the spores on the medium PDA slant for preparing the spore suspension with sterile water.
    In some embodiments, the step b)comprises: inoculating the spore suspension obtained in step a)into the seed culture medium in the shake flask; and culturing the spore suspension at 25-30°C for 5-8 days at a speed of 180-200 r/min, wherein in the seed culture medium, a concentration of the carbon source is at 50- 70 g/L, a concentration of the nitrogen source is at 15-25 g/L and a pH is at 6.5-7.2. In some embodiments, the step c)comprises:
    culturing the inoculum in the primary seed tank at 27-31°C for 1-2 days, wherein a ventilation is (.5-0.8 vvm and a carbon-to-nitrogen ratio is (3-8): 1; and culturing the inoculum in the secondary seed tank at 27-31°C for 1-2 days, wherein a ventilation is 0.5-0.8 vvm and a carbon-to-nitrogen ratio is {3-8):1.
    In some embodiments, the carbon source is selected from the group consisting of glucose, starch and a combination thereof, and the nitrogen source is selected from the group consisting of peptone, yeast powder, yeast extract, a cornstarch slurry and a combination thereof.
    In some embodiments, the method further comprises: extracting an oil product from a resulting product of the fermentation.
    The present disclosure further provides a triglyceride microbial oil produced according to any one of the methods mentioned above, in the microbial oil, a weight percentage of ARA at the sn-2 position of triglyceride is not less than 23% and a weight percentage of triglyceride ARA is not less than 38%.
    The present disclosure provides a use of the microbial oil produced according to any one of the methods mentioned above in food, wherein the food is preferably infant formulas, nutraceuticals or health food.
    According to the technical solutions mentioned above, the present disclosure, by a fermentation using Mortierella alpina strains, produces a microbial oil containing ARA, wherein the ARA at the sn-2 position of triglyceride in the microbial oil is far more than the ARA at the sn-1 and sn-3 position of triglyceride, which improves absorption of the ARA in the microbial oil in the human body and effectively facilitates the utilization of ARA in the human body.
    The features and beneficial effects will be further described in detail below with reference to the embodiments.
    Deposit of microorganisms The Mortierella alpina strains of present disclosure assigned GDMCC No. 60734 was deposited on August 8, 2019 with Guangdong Microbial Culture Collection Center (GDMCC, Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China).
    DETAILED DESCRIPTION OF EMBODIMENTS It should be noted that endpoints and values within ranges disclosed herein are only exemplary, and are intended to include any values close to these values. Any possible 5 combination of numerical values within the range to form one or more new ranges should be considered to be expressly disclosed in this disclosure.
    The present disclosure provides a microbial oil containing ARA at an sn-2 position, and a weight percentage of the ARA at the sn-2 position of triglyceride in the microbial oil is not less than 23%.
    The present disclosure further provides a method for producing the microbial oil mentioned above. Mortierella alpina strains are inoculated into a fermentation medium for fermentation, and the Mortierella alpina strains is assigned accession No. 60734.
    A microbial oil containing ARA are produced by the fermentation using the Mortierella alpina strains. The fermentation, without special requirements, is for a propagation of the Mortierella alpina strains.
    The fermentation conditions, including temperature, time, ventilation, carbon- nitrogen ratio, are all generally used and have no special requirements. In order to increase a yield of the fermentation, in some embodiments, the fermentation medium includes a carbon source and a nitrogen source, and the fermentation is performed at 27-31°C for 4- 8 days under a ventilation at 0.5-1.1 vvm, wherein a carbon-to-nitrogen ratio is (3-18):1, and an inoculation volume ratio 1s 5-10%.
    During the fermentation, the nitrogen source is added into the fermentation medium as need at one time, whereas beside the addition at an initial, the further addition of the carbon source is according to adjustments of carbon-to-nitrogen ratios. When the fermentation is close to a terminal, no carbon source is supplied into the medium so that the residual carbohydrate source is reduced to 0). In this way, an auxotroph condition is formed by adjusting the carbon-to-nitrogen ratios to improve the oil production of the Mortierella alpina strains In order to increase a yield of the fermentation, the Mortierella alpina strains are activated and cultured for a propagation to obtain an inoculum, and then the inoculum is inoculated into the fermentation medium for the fermentation.
    In some embodiments, the activating and propagating process includes the following steps.
    a) The Mortierella alpina strains are inoculated into a potato dextrose agar (PDA) slant medium, and cultured until a formation and a maturity of spores to obtain a spore suspension.
    b) The spore suspension obtained in step a) is inoculated into a seed culture medium in a shake flask for a propagation to obtain an inoculum.
    c) The inoculum obtained in step b) is inoculated into a primary seed tank and cultured for 1-2 days. Then the inoculum is inoculated into a secondary tank and cultured for 1-2 days.
    Preferably, in the step a), the Mortierella alpina strains are inoculated into a PDA slant medium in a test tube, and cultured the at 25-30°C for 5-7 days for the formation of the spores. Then the spores are inoculated into a PDA slant medium in an eggplant-shaped bottle, and cultured at 25-30°C for 4-6 days for the maturity of the spores. Mycelium and the spores on the medium PDA slant are removed for preparing the spore suspension with sterile water.
    Preferably, in the step b), the spore suspension obtained in step a)is inoculated into the seed culture medium in the shake flask; and cultured at 25-30°C for 3-6 days at a speed of 180-200 r/min, wherein in the seed culture medium, a concentration of the carbon source is at 50-70 g/L, a concentration of the nitrogen source is at 15-25 g/L and a pH is at 6.8-7.2. Specifically, 2-3 eggplant-shaped bottles of the spore suspension are inoculated into one shake flask, and storage capacities of the eggplant-shaped bottle and the flask are both 500 mL.
    Specifically, in the stepc), the inoculum is cultured in the primary seed tank at 27- 31°C for 1-2 days, wherein a ventilation is 0.5-0.8 vvm and a carbon-to-nitrogen ratio is (3-8):1, and then the inoculum is cultured in the secondary seed tank at 27-31°C for 1-2 days, wherein a ventilation is 0.5-0.8 vvm and a carbon-to-nitrogen ratio is (3-8):1.
    In some embodiments, the carbon source is glucose, starch or any substrate that can be supplied as carbon sources, and the nitrogen source is peptone, yeast powder, a cornstarch slurry or substrate that can supply nitrogen sources. Preferably, the carbon source is selected from the group consisting of glucose, sucrose and a combination thereof, and the nitrogen source is selected from the group consisting of peptone, yeast powder, yeast extract, a cornstarch slurry and a combination thereof.
    The present disclosure may further process the above-mentioned resulting product of the fermentation to obtain a microbial oil. The process, without special requirements, is for extracting the microbial oil from the resulting product of the fermentation. In order to improve the production of the microbial oil, during the processing, walls of the resulting product of the fermentation are broken, and an oil product is extracted from the resulting product. The present disclosure provides a microbial oil produced by any one of the methods mentioned above, and a weight percentage of ARA at the sn-2 position of triglyceride in the microbial oil is not less than 23%. The present disclosure further provides a use of the microbial oil produced by any one of the methods mentioned above the in food, and the food is preferably infant formulas, nutraceuticals or health food. The present disclosure will be further described in detail below accompanying with the embodiments. In the embodiments, a content of ARA and a fatty acid composition in a microbial oil are detected according to GB 26401-2011 and Standard Test Method for Determination of Fatty Acids in Food, GB5009. 168-2016, respectively. A weight percentage of the ARA at the sn-2 position of triglyceride in the microbial oil is detected according to Animal and Vegetable Fats and Oils—Determination of the Composition of Fatty Acids in the 2-position of the Triglyceride Molecules, GB/T24984- 2010/I1SO6800:1997. A weight percentage of the ARA at the sn-1 and sn-3 positions of triglyceride is calculated according to an 1,3-random-2-random distribution theory based on the analyses of all fatty acids in the sample and the fatty acids at sn-2 position. The absorption rate of ARA in the human body is detected by an efficacy trial, where male and female volunteers are required to take in the microbial oil produced by the method of the present disclosure and the microbial oil produced by ordinary Mortierella alpina strains, and then have their blood drawn to determine a content of the ARA at the sn-2 position in the blood to calculate the absorption rate of ARA. Mortierella alpina strains are provided by China Center of Industrial Culture Collection, and the accession number is CICC 11092s. The glucose, starch, yeast powder, yeast extract, peptone, a cornstarch slurry, potatoes and agar are all commercially available.
    A formula of the PDA medium in the embodiments herein is: 200 g of potatoes, 20 g of sucrose, 15-17 g of agar and 1000 mL of water.
    Examples 1-3 describe the method for producing microbial oil by a fermentation using Mortierella alpine.
    Example 1 a) Ordinary Mortierella alpine strains and Mortierella alpine strains used herein (Accession No. 60734) were inoculated into a PDA slant medium in a test tube, respectively, cultured at 27°C for 5 days. The spores were inoculated into a PDA slant medium in an eggplant-shaped bottle, cultured at 27°C for 4 days to obtain mature spores.
    The mycelium and the spores on the medium were removed for preparing the spore suspension with 20 mL of sterile water.
    b) The spore suspension obtained in step a) was inoculated into the seed culture medium in the shake flask to obtain an inoculum. The spore suspension in each eggplant- shaped bottle was inoculated into 2-3 shake flasks, and cultured on a shaker at pH 7.2 and 27°C for 4 days at a speed of 180 r/min. The carbon source and the nitrogen source in the medium were 50 g/L of glucose and 15 g/L of yeast powder, respectively.
    c) 1 Lof the inoculum obtained in step b)was inoculated in a 1 m primary seed tank containing 500 L of a medium, cultured at pH 7.2 and 29°C for 2 days at a speed of 180 r/min, under a ventilation of 0.65 vvm. The carbon source and nitrogen source in the medium were 30 g/L of glucose and 15 g/L of yeast extract, respectively.
    d)500 L of the inoculum obtained in step c) was inoculated in a 10 m secondary seed tank containing 7 m’ of a medium, cultured at pH 7.2 and 29°C for 2 days at a speed of 120 r/min, under a ventilation of 0.65 vvm. The carbon source and nitrogen source in the medium were 30 g/L of glucose and 15 g/L of yeast extract, respectively. The inoculum was inoculated into a fermentor when a concentration of the mycelium was
    >3.5%.
    e) 2 m’ of the inoculum was inoculated into the 45 m’ fermentor containing 22 m’ of the fermentation medium, cultured at pH 7.5 and 29°C for 6 days at a speed of 90 r/min, under a ventilation of 0.9 vvm to obtain a fermentation solution. The carbon source and nitrogen source in the medium fermentation were 30 g/L of sucrose and 15 g/L of yeast extract, respectively. A carbon-nitrogen ratio was (8-16):1, and was controlled by adding a sterile glucose solution (250 g/L) during the fermentation.
    f) The fermentation solution obtained in step e) was separated to obtain a wet cell mass. Then the wet cell mass was dried to be a dried cell mass, and an oil was extracted from the dried cell mass with hexane. The solid phase separated after extraction was transferred to an extraction vessel for repeated extraction 4-7 times, and the residual oil in the residue after extractions was controlled to be less than 7%. A weight ratio of the solvent to the dried cell mass during extraction was 1.5:1. The mixed oil obtained by filtration and separation after each extraction was desolventized to obtain a microbial oil. g) The microbial oil obtained in step f) was detected to obtain a content of ARA, a fatty acid composition and a weight percentage of ARA at the sn-2 position of triglyceride in the microbial oil. The results were shown in Table 1.
    Table 1 Parameters of the microbial oils in Example | Ordinary Mortierella Mortierella alpina alpina strains used herein Palmitic acid (C16:0), g/100g Stearic acid (C18:0), g/100g 6.825 Linoleic acid (C18:2), g/100g Weight percentage of ARA at the sn- aw we
    21.97 32.65 2 position of triglyceride, % =
    56.15 52.8 and sn-2 positions of triglyceride, %
    Example 2 a) Ordinary Mortierella alpine strains and Mortierella alpine strains used herein {Accession No. 60734) were inoculated into a PDA slant medium in a test tube, respectively, cultured at 28°C for 6 days.
    The spores were inoculated into a PDA slant medium in an eggplant-shaped bottle, cultured at 28°C for 5 days to obtain mature spores.
    The mycelium and the spores on the medium were removed for preparing the spore suspension with 20 mL of sterile water. b) The spore suspension obtained in step a) was inoculated into the seed culture medium in the shake flask to obtain an inoculum.
    The spore suspension in each eggplant- shaped bottle was inoculated into 2-3 shake flasks, and cultured on a shaker at pH 7.0 and 28°C for 6 days at a speed of 200 r/min.
    The carbon source in the medium was 60 g/L of glucose, and the nitrogen source was 10 g/L yeast powder and 10 g/L of peptone. c) 200 mL of the inoculum obtained in step b)was inoculated in a 5 L seed tank containing 3 L of a medium, cultured at pH 7.2 and 28°C for 1 day at a speed of 200 r/min, under a ventilation of 0.6 vvm.
    The carbon source and the nitrogen source in the medium were 35 g/L of glucose and 20 g/L of yeast powder, respectively. d) 1.5 L of the inoculum obtained in step c) was inoculated into the 30 L fermentor containing 15 L of the fermentation medium, cultured at pH 8 and 28°C for 7 days at a speed of 220 r/min, under a ventilation of 0.8 vvm to obtain a fermentation solution.
    The carbon source in the medium fermentation was 50 g/L of glucose and 10 g/L starch, and nitrogen source was 20 g/L of yeast extract.
    A carbon-nitrogen ratio was (5-12):1, and was controlled by adding a sterile glucose solution (250 g/L) during the fermentation. e) The fermentation solution obtained in step d)was separated to obtain a wet cell mass.
    Then the wet cell mass was dried to be a dried cell mass, and an oil was extracted with hexane from the dried cell mass.
    The solid phase separated after extraction was transferred to an extraction vessel for repeated extraction 4-7 times, and the residual oil in the residue after extractions was controlled to be less than 7%. A weight ratio of the solvent to the dries cell mass during extraction was 1.5:1. The mixed oil obtained by filtration and separation after each extraction was desolventized to obtain a microbial oil. f) The microbial oil obtained in step e) was detected to obtain a content of ARA, a fatty acid composition and a weight percentage of ARA at the sn-2 position of triglyceride in the microbial oil. The results were shown in Table 2. Table 2 Parameters of the microbial oils in Example 2 Ordinary Mortierella Mortierella alpina alpina strains used herein Stearic acid (C18:0), g/100g Oleic acid (C18:1), g/100g Weight percentage of ARA at sn-2 own
    20.97 32.17 position of triglyceride, % Weight percentage of ARA at sn-1 me
    57.83 514 and sn-2 positions of triglyceride, % Example 3 1) Ordinary Mortierella alpine strains and Mortierella alpine strains used herein {Accession No. 60734) were inoculated into a PDA slant medium in a test tube, respectively, cultured at 29°C for 7 days. The spores were inoculated into a PDA slant medium in an eggplant-shaped bottle, cultured at 29°C for 6 days to obtain mature spores. The mycelium and the spores on the medium were removed for preparing the spore suspension with 20 mL of sterile water. b)The spore suspension obtained in step a) was inoculated into the seed culture medium in the shake flask to obtain an inoculum. The spore suspension in each eggplant- shaped bottle was inoculated into 2-3 shake flasks, and cultured on a shaker at pH 7.2 and 29°C for 6 days at a speed of 190 r/min. The carbon source in the medium was 60 g/L of glucose and 10 g/L starch, and the nitrogen source was 25 g/L yeast powder.
    c) 300 mL of the inoculum obtained in step b) was inoculated in a 10 L seed tank containing 6 L of a medium, cultured at pH 6.8 and 30°C for 2 days at a speed of 220 r/min, under a ventilation of 0.75 vvm. The carbon source and the nitrogen source in the medium were 40 g/L of glucose and 25 g/L of yeast powder, respectively.
    d) 2.75 L of the inoculum obtained in step ¢) was inoculated into the 100 L fermentor containing 55 L of the fermentation medium, cultured at pH 7 and 30°C for 5 days at a speed of 200 r/min, under a ventilation of 0.85 vvm to obtain a fermentation solution. The carbon source in the medium fermentation was 45 g/L of glucose, and the nitrogen source was 10 g/L of the cornstarch slurry and 10 g/L of peptone. A carbon-nitrogen ratio was (3-10):1, and was controlled by adding a sterile glucose solution (250 g/L) during the fermentation.
    e) The fermentation solution obtained in step d) was separated to obtain a wet cell mass. Then the wet cell mass was dried to be a dried cell mass, and an oil was extracted with hexane from the dried cell mass. The solid phase separated after extraction was transferred to an extraction vessel for repeated extraction 4-7 times, and the residual oil in the residue after extractions was controlled to be less than 7%. A weight ratio of the solvent to the dried cell mass during extraction was 1.5:1. The mixed oil obtained by filtration and separation after each extraction was desolventized to obtain a microbial oil.
    f) The microbial oil obtained in step e) was detected to obtain a content of ARA, a fatty acid composition of and a weight percentage of ARA at the sn-2 position of triglyceride in the microbial oil. The results were shown in Table 3.
    Table 3 Parameters of the microbial oils in Example 3 Ordinary Mortierella Mortierella alpina alpina strains used herein Stearic acid (C18:0), g/100g
    Linoleic acid (C18:2), g/100g 6.871 6.009 Weight percentage of ARA at sn-2
    20.15 31.98 position of triglyceride, % Weight percentage of ARA at sn-1
    56.91 52.31 and sn-2 positions of triglyceride, % The above-mentioned embodiments are only preferred embodiments, and not intend to limit the scope of the present invention. It should be noted that variations and modifications can be made without departing from the spirit of the invention should fall in the scope of the present invention.
    权利要求:
    Claims (12)
    [1]
    A microbial oil comprising ARA at an sn-2 position, characterized in that in the microbial oil, a weight percentage of ARA at the sn-2 position of triglyceride is not less than 23%.
    [2]
    A method for producing the microbial oil according to claim 1, characterized by : inoculating Mortierella alpina strains into a fermentation medium for fermentation; where the Mortierella alpina strains are given accession number 60734.
    [3]
    Process according to claim 2, characterized in that the fermentation medium comprises a carbon source and a nitrogen source; and a fermentation is carried out at 27-31°C for 4-8 days under a ventilation of 0.5-1.1 VVM, wherein a carbon to nitrogen ratio is (3-18)::1, and an inoculation volume ratio is 5-10%.
    [4]
    A method according to claim 2 or 3, characterized in that the method comprises: activating the Mortierella alpina strains; culturing Mortierella alpina strains for propagation to obtain an inoculum; and inoculating the inoculum into the fermentation medium for the fermentation.
    [5]
    Method according to claim 5, characterized in that the activation and the propagation process comprises: a) inoculating the Mortierella alpina strains in a potato dextrose agar (PDA) slant medium, culturing the Mortierella alpina strains to form and spore maturity, and performing an elution with sterile water to obtain a spore suspension; b) inoculating the spore suspension obtained in step a) into a seed culture medium in a shaker flask for propagation to obtain an inoculum; and c) inoculating inoculum obtained in step b) into a primary seed vessel, culturing the inoculum for 1-2 days, and then inoculating the inoculum into a secondary vessel and culturing the inoculum for 1-2 days.
    [6]
    Method according to claim 5, characterized in that step a) comprises: inoculating the Mortierella alpina strains in a PDA slant medium in a test tube, and culturing the Mortierella alpina strains at 25-30°C for 5-7 days for the formation of the spores; inoculating the spores in a PDA slant medium in an aubergine-shaped flask, and culturing the spores at 25-30°C for 4-6 days for spore maturity; and removing mycelium and the spores on the medium PDA ramp to prepare the spore suspension with sterile water.
    [7]
    A method according to claim 5 or 6, characterized in that step b) comprises: inoculating the spore suspension obtained in step a) into the culture medium in the shaker flask, and; culturing the spore suspension at 25-30°C for 5-8 days at a rate of 180-200 rpm, wherein in the culture medium the concentration of the carbon source is 50-70 g/L, and the concentration of the nitrogen source 15-25 g/l and the pH is 6.5-7.2.
    [8]
    Method according to any one of claims 5-7, characterized in that step ¢) comprises: culturing the inoculum in the primary seed vessel for 1-2 days at 27-31°C, the ventilation being 0.5-0 .8 vvm and a carbon-to-nitrogen ratio is (3-8):1; and culturing the inoculum in the secondary seed vessel at 27-31°C for 1-2 days, wherein a ventilation is 0.5-0.8 vvm and a carbon-to-nitrogen ratio is (3-8):1.
    [9]
    A method according to claim 3, 7 or 8, characterized in that the carbon source is selected from a group consisting of; glucose, starch and a combination thereof; and the nitrogen source is selected from a group consisting of; peptone, yeast powder, yeast extract, a cornstarch suspension and a combination thereof.
    [10]
    A method according to any one of claims 2-9, characterized in that the method further comprises:
    extracting an oil product from a resulting product of the fermentation
    [11]
    A microbial oil produced by the method according to any one of claims 2-10, characterized in that a weight percentage of ARA at the sn-2 position of triglyceride in the microbial oil is not less than 23%, and a weight percentage of triglyceride ARA in the microbial oil is not less than 38%.
    [12]
    Use of the microbial oil according to claim 11 in food, wherein the food is preferably infant formula, nutraceuticals or health food
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    同族专利:
    公开号 | 公开日
    WO2021104164A1|2021-06-03|
    CN110747239B|2021-06-22|
    AU2020277167A1|2021-06-10|
    CN110747239A|2020-02-04|
    US20210340582A1|2021-11-04|
    AU2020277167B2|2022-02-03|
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    申请号 | 申请日 | 专利标题
    CN201911175789.3A|CN110747239B|2019-11-26|2019-11-26|Microbial oil rich in Sn-2 ARA and preparation method and application thereof|
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