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WO2018152984A1 - 一种利用微生物发酵生产ara的方法 - Google Patents

一种利用微生物发酵生产ara的方法 Download PDF

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Publication number
WO2018152984A1
WO2018152984A1 PCT/CN2017/087749 CN2017087749W WO2018152984A1 WO 2018152984 A1 WO2018152984 A1 WO 2018152984A1 CN 2017087749 W CN2017087749 W CN 2017087749W WO 2018152984 A1 WO2018152984 A1 WO 2018152984A1
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WIPO (PCT)
Prior art keywords
culture
ara
fermentation
oil
temperature
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PCT/CN2017/087749
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English (en)
French (fr)
Inventor
文昌
陈必钦
王炳荣
王跃飞
蒋四富
徐鲁明
吴轶
Original Assignee
内蒙古金达威药业有限公司
厦门金达威集团股份有限公司
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Publication of WO2018152984A1 publication Critical patent/WO2018152984A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/02—Pretreatment
    • C11B1/025—Pretreatment by enzymes or microorganisms, living or dead
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/06—Production of fats or fatty oils from raw materials by pressing
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/001—Refining fats or fatty oils by a combination of two or more of the means hereafter
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/008—Refining fats or fatty oils by filtration, e.g. including ultra filtration, dialysis
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/10—Refining fats or fatty oils by adsorption
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/12—Refining fats or fatty oils by distillation
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B3/00—Refining fats or fatty oils
    • C11B3/16—Refining fats or fatty oils by mechanical means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone

Definitions

  • the invention belongs to the field of fermentation engineering, and relates to a method for producing ARA by microbial fermentation, in particular to a method for producing arachidonic acid (ARA) mixed oil by industrial fermentation of a strain of Mortierella alpina.
  • ARA arachidonic acid
  • ARA also known as arachidonic acid, the full name aesthetic acid (cis-5, 8, 11, 14-arachidonic acid, ARA), is a polyunsaturated fatty acid of the omega-6 series.
  • ARA is an essential essential fatty acid in the human body, and it is also the most abundant and widely distributed polyunsaturated fatty acid in the human body.
  • ARA plays an important role as a phospholipid-bound structural lipid in the blood, liver, muscle and other organ systems, and ARA is a direct precursor of many circulating eicosanic acid derivatives. These bioactive substances have important regulatory effects on lipid protein metabolism, hemorheology, vascular elasticity, leukocyte function, and platelet activation.
  • ARA lowering blood fat, lowering blood pressure and lowering blood cholesterol are stronger than linoleic acid and linolenic acid; at the same time, it can alleviate arrhythmia caused by strontium chloride and aconitine, and its effect is also strong.
  • strontium chloride and aconitine arrhythmia caused by strontium chloride and aconitine, and its effect is also strong.
  • ARA has important nutritional, health and medical functions.
  • ARA is mainly produced by microbial fermentation.
  • the publication No. CN105112466A discloses a method for preparing arachidonic acid by adding a product promoter, wherein the shake flask of the disclosed product promoter is used to detect the ARA content in the fermentation liquid after the culture is 5.41 g/L. The yield increased by 45.1% compared to the control.
  • the patent publication No. CN102925502A discloses an industrial process for producing arachidonic acid oil by using Mortierella alpina, which discloses a yield of arachidonic acid having a unit yield of 10 g/L of fermentation broth.
  • CN104278107A discloses a method for controlling the production of arachidonic acid oil by Mortierella alpina based on dissolved oxygen, and the disclosed yield is 25m 3 fermenter fermenting the dry weight and oil content of Mortierella alpina cells.
  • the content of arachidonic acid in total fat and oil, and the unit yield of arachidonic acid can reach 55g/L, 54%, 65%, 20.628g/L, respectively, and the production intensity of arachidonic acid reaches 1.753g/(L ⁇ d) This is also the highest level of production currently reported for the large-scale industrial production of ARA using Mortierella alpina.
  • Centrifugation as disclosed in the publication No. CN1282745C, discloses a method for obtaining oil from a microbial cell, the oil comprising one or more unsaturated fatty acids, the method comprising: (a) lysing the cell wall of the microbial cell to release Oil is extracted; and (b) the oil is separated from the cell wall fragments formed in at least part of (a) by centrifugation.
  • the quality of the oil layer after centrifugation of the invention is poor.
  • the organic solvent extraction method discloses a method for preparing an arachidonic acid oil, wherein the oil extraction method comprises: collecting, drying and grinding the aged bacteria, using petroleum ether. After extraction with ethanol for 12 hours, petroleum ether and ethanol were removed by distillation under reduced pressure to obtain an arachidonic acid oil.
  • the method uses an organic solvent for extraction, and the final product may have solvent residues, and the extraction process may be flammable and explosive.
  • the supercritical extraction method as disclosed in the publication No. CN101579019A, discloses a method for extracting polyunsaturated fatty acid oil by CO 2 supercritical extraction method, the steps of which are as follows: 1 pulverization and sieving; 2 heating extraction; 3 liquid CO 2 Inject into the extraction vessel, pressurize; 4 extract the oil after collecting.
  • supercritical equipment is expensive and the extraction yield is not high.
  • the extraction rate of the invention is only 50%.
  • ARA essential oil is obtained by degumming, alkali refining, decolorizing and deodorizing.
  • alkali refining usually adds excessive alkali, and some triglycerides are inevitably saponified; high COD wastewater produced by alkali refining will pollute the environment; Alkali refining requires long time of high temperature treatment, which is easy to cause the product's peroxide value and anisidine value to increase; the deodorization temperature is high, and the long time is easy to produce trans fatty acids.
  • the inventors have obtained a method of cultivating microorganisms for producing ARA through intensive research and creative labor.
  • the inventors have surprisingly found that the culture method can greatly increase biomass and ARA yield.
  • the inventors have also found a method of extracting ARA hair oil, which can improve the extraction yield and quality of ARA hair oil.
  • the inventors have also found a method for purifying ARA hair oil, which can enhance various technical indexes and purification yields of ARA product oil.
  • the present invention significantly improves the crude oil production, ARA yield, and ARA productivity of ARA. The following invention is thus provided:
  • One aspect of the invention relates to a method of cultivating a microorganism for producing ARA, wherein:
  • the fermentation temperature is maintained at 20-25 ° C from the fermentation tank for 100-140 hours (eg 21-25 ° C, 20-24 ° C, 21-23 ° C, 22-25 ° C, 22-24 ° C, 23-25 °C, 20 ° C, 21 ° C, 22 ° C, 23 ° C, 24 ° C or 25 ° C); preferably, until the end of the fermentation;
  • stop ventilation or reduce ventilation by 50% or more (for example, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85%) Above, 90% or more or 95% or more); preferably, until the end of the fermentation.
  • the culture method wherein:
  • the fermentation temperature is controlled at 20-25 ° C (for example, 21-25 ° C, 20-24 ° C, 21-23 ° C, 22-25 ° C, 22-24 ° C, 23-25 ° C, 20 ° C, 21 ° C, 22 ° C, 23 °C, 24°C or 25°C), and/or
  • the ventilation is stopped or the ventilation is reduced by 50% or more (for example, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more).
  • the culture method wherein the fermentation temperature is 25-30 ° C (for example, 26-30 ° C, 25-29 ° C, 26-28) before the fermentation temperature is maintained at 20-25 ° C. °C, 27-30 ° C, 26-29 ° C, 28-30 ° C, 25 ° C, 26 ° C, 27 ° C, 28 ° C, 29 ° C or 30 ° C).
  • the fermentation temperature is 25-30 ° C (for example, 26-30 ° C, 25-29 ° C, 26-28) before the fermentation temperature is maintained at 20-25 ° C. °C, 27-30 ° C, 26-29 ° C, 28-30 ° C, 25 ° C, 26 ° C, 27 ° C, 28 ° C, 29 ° C or 30 ° C).
  • the culture method wherein the pH of the fermentation broth is 6.0-7.0;
  • the concentration of glucose in the fermentation broth is maintained at 1-5 g/L (eg 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 g/L) before the aeration is stopped or the ventilation is reduced by more than 50%. .
  • the culture method wherein the concentration of glucose in the fermentation broth is 1-2 g/L while or after stopping the ventilation or reducing the ventilation volume by 50% or more;
  • the fermentation temperature is 20-25 ° C while or after stopping the ventilation or reducing the ventilation by more than 50%;
  • the stirring is stopped while or after the ventilation is stopped or the ventilation is reduced by 50% or more;
  • the above three conditions namely glucose concentration, fermentation temperature, and stirring are stopped, until the end of fermentation;
  • the fermentation broth is filtered off and the appropriate amount of water or fresh fermentation medium is added before the aeration is stopped or the aeration is reduced by more than 50%.
  • the culture method wherein the fermenter culture further comprises 36-60 hours (eg, 40-56 hours, 42-54 hours, 44-52) of initiation of fermentation in the fermentor.
  • the hour, 46-50 hours, 46, 47, 48, 49 or 50 hours) the step of performing the tank culture; for example, splitting into two or more fermentors for fermentor culture.
  • the culture method further comprises the steps of inoculating and seed expanding culture before the fermenter is cultured;
  • the seed expansion culture comprises a primary seed expansion culture and a secondary seed expansion culture
  • the primary seed expansion culture comprises the following steps:
  • the shake flask seed liquid is connected to the first-stage seed tank containing the sterilized medium, the culture temperature is 25-32 ° C, the aeration amount is 1-2 vvm, the tank pressure is 0.02-0.05 MPa, After culturing for 30-35 hours, complete the first-stage seed expansion culture;
  • the secondary seed expansion culture comprises the following steps:
  • the seed liquid of the primary seed tank is connected to the secondary seed tank containing the sterilized medium according to the inoculation amount of 1%-3%, the culture temperature is 25-32 ° C, the ventilation volume is 1-2 vvm, the tank pressure is 0.02- 0.05MPa, cultured for 20-25h, complete secondary seed expansion culture.
  • the culture method further comprises the step of performing activating culture before inoculation and seed expansion culture; preferably, the temperature of the activation culture is 25-32 ° C, and the stirring speed is 100- 200 rpm, time is 40-48h.
  • the culture method wherein the fermentor culture comprises the steps of:
  • the seed liquid of the secondary seed tank is connected to the fermenter containing the sterilized medium according to the inoculation amount of 1%-3%, the culture temperature is 20-30 ° C, the aeration amount is 1-2 vvm, and the tank pressure is 0.02-0.05 MPa.
  • the fermentation tank culture was carried out at a stirring speed of 0-50 rpm.
  • the culture method, wherein the microorganism for producing ARA is Mortierella alpina or a mutant strain thereof; and the Mortierella alpina may be a strain of Mortierella alpina known in the art.
  • the height Mortierella alpina is selected from the strains having the accession numbers CCTCC No. M2012073, CCTCC No. M2013392, CCTCC No. M2015421 and ATCC No. 42430.
  • the culture method comprises the following steps:
  • Mortierella alpine slant-preserved strain was inserted into a 2L shake flask containing 400 mL of medium, and cultured at 150-32 ° C for 150-48 h at 150 rpm to complete the activated culture of the strain;
  • the shake flask seed liquid is connected to the first-stage seed tank containing the sterilized medium, the culture temperature is 25-32 ° C, the ventilation volume is 1-2 vvm, the tank pressure is 0.02-0.05. MPa, culture for 30-35h, complete the first-stage seed expansion culture;
  • the seed liquid of the primary seed tank is connected to the secondary seed tank containing the sterilized medium, the culture temperature is 25-32 ° C, the ventilation volume is 1-2 vvm, the tank pressure 0.02-0.05MPa, cultured for 20-25h, complete secondary seed expansion culture;
  • the seed liquid of the secondary seed tank is connected to the fermenter containing the sterilized medium, the culture temperature is 20-30 ° C, the ventilation volume is 1-2 vvm, the tank pressure is 0.02- Fermentation culture at 0.05 MPa, stirring speed 0-50 rpm;
  • glucose concentration decreases with the growth of the bacteria, and the carbon source is added to maintain the sugar point (glucose concentration) in the fermentation liquid at 1-5 g/L;
  • Fermentation culture for 8-10d the fermentation is stopped, the main and auxiliary tanks are filtered through the discharge pipeline screen, and the obtained bacteria are re-added to the same volume of water once, the bacteria are suspended after a little stirring, the stirring is stopped, and the ventilation is stopped. Maintain the tank temperature of 20-25 ° C, add carbon source to control the main and auxiliary tank sugar points at 1-2g / L, let stand 1-2d;
  • step 8) can stimulate the conversion of short-chain and low-carbon fatty acids in the ARA by the ARA, and increase the ARA content in the cells;
  • the sugar spot (concentration of glucose) is determined by a method known to those skilled in the art, for example. Biosensor measurement.
  • the culture method wherein the fermentation medium carbon source used is added with 20% to 40% (mass of crude glycerin/mass of carbon source ⁇ 100%) of pretreated biodiesel By-product crude glycerin.
  • Biodiesel is a renewable energy source made from renewable oils such as vegetable oils and animal fats. It can produce biodiesel and by-product glycerol after transesterification.
  • the pretreatment of crude glycerol involves adjusting the pH to acidity, dilution, hydrolysis, and separation.
  • the pretreatment of the crude glycerol comprises the steps of: i) mixing the crude glycerol with deionized water at a ratio of 1:4 (volume ratio); ii) adjusting the pH to about 6.5 with hydrochloric acid; iii) The precipitated material was separated and removed at a speed of 5000 rpm.
  • step i) the viscosity can be lowered after dilution; in step ii), the soluble saponin in the crude glycerol is converted into an insoluble free fatty solid material; in step iii), the precipitated substance includes free Fatty acid solids and insoluble heavy metal impurities.
  • the above seed culture medium can be cultured in the field by the existing strain Mortierella alpine seed culture medium.
  • the above fermentation medium can be cultured in the art by cultivating an existing strain of Mortierella alpine fermentation medium.
  • the carbon source in the above fermentation medium includes one or more of glucose, corn syrup powder, molasses, glycerin and starch; the nitrogen source includes soybean powder, yeast powder, peptone, ammonia water, sodium nitrate, sodium glutamate, ammonium sulfate One or more of them.
  • the trace elements added in the above fermentation medium include one or more of alanine, glutamic acid, lysine, calcium pantothenate, biotin, vitamin B 1 , microorganism B 6 , microorganism B 12 , and vitamin K.
  • the addition amount is 0.001% to 0.01%; when it is plural, any one component is added in an amount of 0.001% to 0.005%.
  • the inorganic salt added to the above fermentation medium includes one or more of magnesium sulfate, potassium chloride, sodium chloride, calcium chloride, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate.
  • Another aspect of the invention relates to a fermentation culture of a microorganism obtained by the culture method according to any one of the inventions.
  • a further aspect of the invention relates to a method of extracting ARA hair oil, comprising the steps of:
  • step 2) The product of step 1) is subjected to a flexible press to obtain ARA hair oil.
  • the extraction method wherein, in step 1), the fermentation culture
  • the material is a Mortierella alpina fermentation culture; preferably, the fermentation culture is a fermentation culture of the invention.
  • the dehydrating treatment is selected from any one, two or three of the following:
  • the dehydration treatment comprises, in order, centrifugation and a first stage flexible press, or sequentially comprises plate and frame filtration and air flow drying;
  • the airflow is dried at an inlet air temperature of 110-150 ° C, an outlet air temperature of 30-70 ° C, and a moisture content of less than 10%.
  • the first stage flexible press adopts a stepwise pressurization mode
  • the set pressure is 20-40 MPa
  • the pressurization time is 1-6 h. , after reaching the set pressure, keep pressure for 1-4h.
  • the flexible press adopts a stepwise pressurization mode, the set pressure is 50-150 MPa, and the pressurization time is 1-6 h. After constant pressure, keep pressure for 1-4h.
  • the extraction method includes the following steps:
  • Cloth A certain amount of the fermentation culture is transported to the cloth cavity by the distributor, and the distributor returns to the initial position, and the fabric is to be replaced.
  • First-stage flexible press adopts step-by-step pressurization method.
  • the pressure range of the first-stage flexible press is 20-40MPa, the pressurizing time is about 1-6h, the set pressure is reached, and the pressure is kept for 1-4h until no water droplets flow out. After the end, the material is lowered into the heavy pressure chamber and the material is pushed into the secondary flexible press position.
  • Two-stage flexible press adopting stepwise pressurization method, the final pressure of the press is 50-150MPa, the pressurizing time is about 1-6h, the set pressure is reached, the pressure is kept for 1-4h until the oil-free dripping is basically collected, and the press is collected.
  • the ARA hair oil is released, the pressure is removed, the secondary pressing cage is removed, and the slag is separated from the filter cloth.
  • the ARA fermentation broth can be directly subjected to a first-stage flexible pressing, or a centrifugal method can be used to remove a part of the water, and the solid content of the fermentation broth can be increased, and then a first-stage flexible pressing can be performed, which can shorten the pressing time and increase the production capacity.
  • the fermentation liquid centrifugation method may be carried out by one of a horizontal spiral centrifuge, a disc centrifuge, and a tubular centrifuge.
  • the extraction method comprises the following steps: the fermentation culture is subjected to plate and frame filtration, airflow drying to obtain a bacterial powder, and the bacterial powder is subjected to flexible pressing to obtain ARA hair oil.
  • the above-mentioned plate frame filtering method is as follows: after the fermentation liquid is fed, the filter is filtered until the filter press is fully opened and the feed pressure reaches 0.6-1.0 MPa, and the air is blown after the filtration is completed, the blowing pressure is 0.3-0.6 MPa, and the blowing time is 1 -3h.
  • the airflow drying inlet air temperature is 110-150 ° C
  • the outlet air temperature is 30-70 ° C
  • the ARA bacterial powder moisture is controlled within 10%.
  • a further aspect of the invention relates to an ARA hair oil produced by the extraction method of any of the inventions.
  • a further aspect of the invention relates to a method of purifying ARA hair oil comprising the steps of hydrating, decolorizing and molecularly distilling ARA hair oil; preferably, the ARA hair oil is the ARA hair oil of the invention.
  • the purification method wherein the hydrating comprises the steps of:
  • the purification method, wherein the discoloration comprises the following steps:
  • the hydration product is heated to 90-110 ° C, the controlled vacuum is ⁇ -0.07 MPa, vacuum dehydrated for 0.5-2 h, then cooled to 60-80 ° C, and a decolorizing agent (for example, hydration oil weight 1% - 3% activated carbon and 2%-4% activated clay), stirring for 0.5-1 h, stirring was stopped, and the decolorizing agent was removed by filtration to obtain a decolorizing oil.
  • a decolorizing agent for example, hydration oil weight 1% - 3% activated carbon and 2%-4% activated clay
  • the purification method wherein the molecular distillation is a tertiary molecular distillation
  • the molecular distillation comprises the following steps:
  • the decolorized oil enters the tertiary molecular distillation, and the first stage vacuum degree is controlled to be ⁇ 100 Pa and the temperature is 150-200 ° C to remove the light component of the first stage; the obtained first heavy component enters the second stage molecular distillation to control the secondary vacuum Degree ⁇ 50Pa, temperature 180-220 ° C, remove the second-level light component; the second heavy component obtained enters the third-stage molecular distillation, controls the third-stage vacuum ⁇ 5Pa, temperature 200-250 ° C, removes the third level Light component, the third heavy component is obtained, which is ARA product oil.
  • the molecular distillation is repeated one or more times (e.g., 2, 3, 4 or 5 times).
  • the purification method comprises the following steps:
  • ARA hair oil is heated to 70-85 ° C, and added with 75-90 ° C purified water in the proportion of 1 kg of hair oil added to 50-150 g of purified water, stirred for 10-60 min, stirring speed 30-90 rev / min, static Set 1-6h, layering off the lower layer of phospholipids to obtain hydrated oil;
  • Decolorization Hydration oil is transferred into the decolorizing pot, heated to 90-110 ° C, controlled vacuum ⁇ -0.07 MPa, vacuum dehydrated 0.5-2 h, then cooled to 60-80 ° C, added decolorizing agent (hydration oil weight 1%) - 3% activated carbon and 2% - 4% activated clay), stirring and decolorizing for 0.5-1 h, stirring is stopped, and the decolorizing agent is removed by filtration to obtain a decolorizing oil;
  • the decolorized oil enters the tertiary molecular distillation, controls the first stage vacuum degree ⁇ 100Pa, the temperature is 150-200°C, removes the light components of the first stage, and the heavy components enter the second stage molecular distillation to control the secondary vacuum.
  • Degree ⁇ 50Pa temperature 180-220 ° C, remove the second-level light components, heavy components into the third-stage molecular distillation, control the third-stage vacuum ⁇ 5Pa, temperature 200-250 ° C, remove the third-level light components,
  • the heavy components were collected to obtain a molecular distillation oil.
  • the molecular distillation number is controlled 1-3 times, and the acid value and odor meet the standard requirements.
  • the temperature is lowered to 20-40 ° C, and an antioxidant is added and packaged to obtain an ARA product oil.
  • a further aspect of the invention relates to an ARA product oil obtained by the purification method of any of the inventions.
  • a further aspect of the invention relates to a method for producing ARA or a product containing ARA (for example, ARA finished oil) using a microorganism, comprising:
  • a method for extracting ARA hair oil according to any one of the present invention, and/or
  • a method of purifying ARA hair oil according to any one of the inventions.
  • the term "flexible press” refers to a high pressure press that uses a PLC (Programmable Logic Controller) program control to perform a pressurization-pressure-pressurization cycle to gradually reach a predetermined pressure.
  • PLC Programmable Logic Controller
  • purified water refers to pharmaceutically acceptable water obtained by distillation, ion exchange, reverse osmosis or other suitable methods, without any additives.
  • the purified water is in accordance with the provisions of the Chinese Pharmacopoeia.
  • ARA hair oil means a primary oil obtained from an ARA fermentation broth without undergoing refining.
  • ARA finished oil refers to an essential oil obtained by refining ARA wool.
  • fertilizer culture refers to a fermentation culture for producing a desired product, which is carried out in a fermenter after the seed is expanded and cultured.
  • ventilation when referring to “ventilation” or “ventilation amount”, unless otherwise specified, it means the amount of air or air introduced.
  • the technical indexes of the invention are obviously superior to the existing technical indexes, and the obtained ARA oil has high yield and high purity, which is favorable for large-scale industrial production of ARA, and the addition of crude glycerin also reduces the production cost of ARA fermentation. Greatly improved the market competitiveness of ARA fermentation production.
  • the invention adopts the flexible pressing process to prepare ARA hair oil, and does not need to use organic solvent for extraction.
  • the whole production process route does not need to use organic solvent, and the final product does not have solvent residue.
  • the obtained product is green and healthy, and the product quality is good.
  • the production workshop is safe and environmentally friendly, and it is a green clean production process.
  • the aqueous solution of the fermentation culture which is removed by the first-stage flexible pressing is basically free of slag, the COD is relatively low, and it is easy to be biochemically treated.
  • the second-stage flexible pressing obtains the oil, the remaining slag also contains a small amount of oil and a large amount of oil.
  • the protein can be used as a feed additive and is economical and environmentally friendly.
  • the invention adopts a molecular distillation one-step process to replace the traditional two steps of deacidification and deodorization.
  • Molecular distillation can quickly remove a large amount of free fatty acids and odors based on the physiological activity of the retained materials.
  • the molecular distillation deacidification process is simple, the risk of excessive alkali refining is reduced, the loss of neutral oil from the soap foot is reduced, the deacidification yield is obviously improved, and the deacidification process is under high vacuum conditions.
  • the process is carried out under a short period of time, avoiding the risk of excessive oxidation and de-acidification, causing an increase in peroxide value and anisidine value, and good product stability; compared with the conventional steam distillation deodorization process, the molecular distillation deodorization time is short, and the vacuum is high. The production of trans fatty acids is reduced, and the odorous substance is removed well, and the product has no astringency.
  • the production process avoids the use of organic solvents, avoids the cost of solvent consumption and solvent recovery, and the low COD of the sewage is easy to handle, and the refining yield is high, thereby greatly reducing the production cost.
  • Figure 1 is a schematic view showing the process flow for producing ARA product oil according to an embodiment of the present invention.
  • FIG. 2 is a schematic view showing a process flow for producing an ARA product oil according to another embodiment of the present invention.
  • Figure 3 Results of the disclosed production of ARA by Mortierella alpine Mortierella alpine in a 100 m 3 fermentor under different culture modes.
  • Method for measuring biomass Take appropriate amount of fermentation culture (fermentation liquid) in a flat weighing bottle, dry it in a 105 °C electric oven, and then dry it in a desiccator to room temperature, weigh it, and weigh it.
  • the weight of the bottle, divided by the volume of the fermentation broth, is the biomass, in g/L.
  • Determination method of crude oil production Take a certain volume of fermentation culture (fermentation liquid), add 2 times volume of concentrated hydrochloric acid, stir at 70 °C for 50 min to complete digestion of the cells, add appropriate amount of n-hexane, let stand for stratification, use drops Pipe the upper organic phase into the eggplant bottle and extract it continuously for 5-8 times until the upper organic phase is colorless. The n-hexane is removed by rotary evaporation in a water bath at 80 ° C, and then the eggplant bottle is placed in a 105 ° C electric oven. Dry for 1h, put into a desiccator to cool to room temperature, weigh, subtract the weight of the eggplant bottle, and then divide by the volume of the fermentation broth, the value obtained is the crude oil production, g/L.
  • ARA yield The content of ARA in the crude oil and fat was measured by gas chromatography, multiplied by the crude oil production, in g/L.
  • ARA productivity The value obtained by dividing the ARA yield by the fermentation cycle (days) in g/(L ⁇ d).
  • the method for analyzing fatty acid composition and the method for detecting ARA product oil in the present invention are all carried out according to GB 26401-2011 arachidonic acid oil (fermentation method).
  • Examples 1-5 relate to the production of ARA by fermentation with a 100 m 3 fermentor.
  • the strain used was Mortierella alpine (ATCC No. 42430);
  • the seed medium used is: potato (peeled) 200g, glucose 20g, agar 15-20g, distilled water 1000ml, natural pH;
  • the fermentation medium used was: glucose 22g/L, yeast powder 12g/L, peptone 8g/L, sodium chloride 15g/L, ammonium sulfate 6g/L, KH 2 PO 4 5g/L, CuSO 4 ⁇ 5H 2 O 1.5 ⁇ g/L, MnSO 4 2 ⁇ g/L, ZnSO 4 ⁇ 7H 2 O 3 ⁇ g/L, pH 6-7.
  • the Mortierella alpine slant-preserved strain was inserted into a 2 L shake flask containing 400 mL of medium, and cultured at a temperature of 28 ° C for 45 hours at 150 rpm to complete the activated culture of the strain. According to the inoculation amount of 0.4%, the shake flask seed solution was connected to the first-stage seed tank containing the sterilized medium, and cultured for 30 hours under the conditions of culture temperature of 28 ° C, aeration rate of 1 vvm and tank pressure of 0.04 MPa. Seeds are expanded and cultured.
  • the seed solution of the primary seed tank was connected to the secondary seed tank containing the sterilized medium according to the inoculation amount of 3%, and cultured for 24 hours under the conditions of culture temperature of 28 ° C, aeration of 1 vvm, and tank pressure of 0.04 MPa. Complete secondary seed expansion culture.
  • the seed solution of the secondary seed tank was connected to a fermentor containing the sterilized medium according to a 3% inoculum.
  • the fermentation temperature of the fermentation process was 28 ° C, the aeration rate was 1 vvm, the tank pressure was 0.04 MPa, the stirring speed was 50 rpm, and a carbon source containing 30% of the pretreated crude glycerin was added thereto, and the glucose concentration was controlled at about 5 g/L to carry out fermentation culture.
  • the glucose concentration, pH, bacterial biomass, crude oil production and ARA yield of the fermentation broth were measured.
  • the fermentation tank has a tank volume of 83 m 3 , and the whole batch fermentation contains ARA crude oil with a yield of 1784.5 kg.
  • Table 1 shows the results of gas phase analysis of the fatty acid composition of the mixed fat obtained after fermentation.
  • the Mortierella alpine slant-preserved strain was inserted into a 2 L shake flask containing 400 mL of medium, and cultured at a temperature of 28 ° C for 45 hours at 150 rpm to complete the activated culture of the strain. According to the inoculation amount of 0.4%, the shake flask seed solution was connected to the first-stage seed tank containing the sterilized medium, and cultured for 30 hours under the conditions of culture temperature of 28 ° C, aeration rate of 1 vvm and tank pressure of 0.04 MPa. Seeds are expanded and cultured.
  • the seed solution of the primary seed tank was connected to the secondary seed tank containing the sterilized medium according to the inoculation amount of 3%, and cultured for 24 hours under the conditions of culture temperature of 28 ° C, aeration of 1 vvm, and tank pressure of 0.04 MPa. Complete secondary seed expansion culture.
  • the seed solution of the secondary seed tank was connected to a fermentor containing the sterilized medium according to a 3% inoculum.
  • the aeration rate of the fermentation process was 1vvm, the tank pressure was 0.04MPa, the stirring speed was 50rpm, and the tank temperature before 120h was controlled at 28 ⁇ 1°C, 120h and the tank temperature was controlled at 22 ⁇ 1°C, and the flow was 30% pretreated.
  • the carbon source of the crude glycerin is controlled to have a glucose concentration of about 5 g/L and is subjected to fermentation culture. During the fermentation process, the glucose concentration, pH, bacterial biomass, crude oil production and ARA yield of the fermentation broth were measured.
  • the fermentation tank had a tank volume of 82 m 3 , and the whole batch fermentation containing ARA crude oil yield was 2378 kg, which was 0.33 times higher than that of Example 1.
  • Table 2 below shows the results of gas phase analysis of the fatty acid composition of the mixed fat obtained after fermentation.
  • the Mortierella alpine slant-preserved strain was inserted into a 2 L shake flask containing 400 mL of medium, and cultured at a temperature of 28 ° C for 45 hours at 150 rpm to complete the activated culture of the strain. According to the inoculation amount of 0.4%, the shake flask seed solution was connected to the first-stage seed tank containing the sterilized medium, and cultured for 30 hours under the conditions of culture temperature of 28 ° C, aeration rate of 1 vvm and tank pressure of 0.04 MPa. Seeds are expanded and cultured.
  • the seed solution of the primary seed tank was connected to the secondary seed tank containing the sterilized medium according to the inoculation amount of 3%, and cultured for 24 hours under the conditions of culture temperature of 28 ° C, aeration of 1 vvm, and tank pressure of 0.04 MPa. Complete secondary seed expansion culture.
  • the seed solution of the secondary seed tank was connected to a fermentor containing the sterilized medium according to a 3% inoculum.
  • the fermentation temperature of the fermentation process was 28 ° C, the aeration rate was 1 vvm, the tank pressure was 0.04 MPa, the stirring speed was 50 rpm, and a carbon source containing 30% of the pretreated crude glycerin was added thereto, and the glucose concentration was controlled at 5 g/L to carry out fermentation culture. Fermentation to 48h in the fermentation process, the fermentation broth in the fermenter is divided into two cultures, half of the fermentation broth is left in the main tank to continue the cultivation, and the other half of the fermentation broth is transferred to the aseptically held sub-tank by differential pressure method.
  • the appropriate amount of fresh medium or sterile water was added to the auxiliary tank, and the main and auxiliary tank ventilation, tank pressure and stirring speed were adjusted slightly after the tank was cultured. During the fermentation process, the glucose concentration, pH, bacterial biomass, crude oil production and ARA yield of the fermentation broth were measured.
  • the fermentation tank has a tank volume of 145m 3 , and the whole batch fermentation contains ARA crude oil output of 3197.25kg, which is 0.79 times higher than the yield of Example 1, although the canned biomass, crude oil yield, ARA yield and other indicators and original culture methods.
  • the Mortierella alpine slant-preserved strain was inserted into a 2 L shake flask containing 400 mL of medium, and cultured at a temperature of 28 ° C for 45 hours at 150 rpm to complete the activated culture of the strain. According to the inoculation amount of 0.4%, the shake flask seed solution was connected to the first-stage seed tank containing the sterilized medium, and cultured for 30 hours under the conditions of culture temperature of 28 ° C, aeration rate of 1 vvm and tank pressure of 0.04 MPa. Seeds are expanded and cultured.
  • the seed solution of the primary seed tank was connected to the secondary seed tank containing the sterilized medium according to the inoculation amount of 3%, and cultured for 24 hours under the conditions of culture temperature of 28 ° C, aeration of 1 vvm, and tank pressure of 0.04 MPa. Complete secondary seed expansion culture.
  • the seed solution of the secondary seed tank was connected to a fermentor containing the sterilized medium according to a 3% inoculum.
  • the fermentation temperature of the fermentation process was 28 ° C, the aeration rate was 1 vvm, the tank pressure was 0.04 MPa, the stirring speed was 50 rpm, and a carbon source containing 30% of the pretreated crude glycerin was added thereto, and the glucose concentration was controlled at 5 g/L to carry out fermentation culture.
  • the fermentation was stopped, the fermentation broth was filtered through the discharge line screen, and an equal volume of water was added again.
  • the bacteria were suspended after a little stirring, the stirring was stopped, the aeration was stopped, and the carbon source was controlled to control the sugar spot at 2 g.
  • the biomass in the fermentation broth was determined to be 52 g/L, the crude oil production was 31.2 g/L, the ARA yield was 16.5 g/L, and the ARA productivity was 1.5 g/(L ⁇ d), as shown in FIG.
  • the fermentation tank had a tank volume of 83 m 3 , and the whole batch fermentation containing ARA crude oil yield was 2589.6 kg, which was 0.45 times higher than that of Example 1.
  • Table 4 below shows the results of gas phase analysis of the fatty acid composition of the mixed fat obtained after fermentation.
  • the Mortierella alpine slant-preserved strain was inserted into a 2 L shake flask containing 400 mL of medium, and cultured at a temperature of 25 ° C for 24 h at 200 rpm to complete the activated culture of the strain.
  • the shake flask seed solution was inserted into the first-stage seed tank containing the sterilized medium according to the inoculation amount of 0.4%, and cultured for 30 hours under the conditions of culture temperature of 28 ° C, aeration rate of 1 vvm, tank pressure of 0.02 MPa, and stirring speed of 50 rpm. , complete the first-level seed expansion training.
  • the seed liquid of the primary seed tank was connected to the secondary seed tank containing the sterilized medium according to the inoculation amount of 3%, and the culture temperature was 28 ° C, the aeration rate was 1 vvm, the tank pressure was 0.02 MPa, and the stirring speed was 75 rpm.
  • the culture was carried out for 24 hours to complete the secondary seed expansion culture.
  • the seed solution of the secondary seed tank was connected to a fermentor containing the sterilized medium according to a 3% inoculum.
  • the aeration rate of the fermentation process was 1vvm, the tank pressure was 0.04MPa, the stirring speed was 50rpm, and the tank temperature before 120h was controlled at 28 ⁇ 1°C, 120h and the tank temperature was controlled at 22 ⁇ 1°C, and the flow was 30% pretreated.
  • the carbon source of the crude glycerin is controlled to have a glucose concentration of about 5 g/L and is subjected to fermentation culture. Fermentation to 48h in the fermentation process, the fermentation broth in the fermenter is divided into two cultures, half of the fermentation broth is left in the main tank to continue the cultivation, and the other half of the fermentation broth is transferred to the aseptically held sub-tank by differential pressure method. Add appropriate amount of fresh medium or sterile water to the auxiliary tank.
  • the main and auxiliary tank ventilation, tank pressure and stirring speed were slightly adjusted. After culturing for 9 days, the fermentation was stopped. The main and auxiliary tanks were filtered through the discharge line screen to remove the fermentation broth. The same volume of water was added again. The bacteria were suspended after a little stirring, the agitation was stopped, the aeration was stopped, and the tank temperature was maintained at 22 ⁇ . At 1 °C, the carbon source was controlled to control the sugar content of the main and auxiliary tanks at 2g/L, and it was allowed to stand for 2d, which stimulated the conversion of short-chain and low-carbon fatty acids in the ARA by the algae, and increased the ARA content in the cells. During the fermentation process, the glucose concentration, pH, bacterial biomass, crude oil production and ARA yield of the fermentation broth were measured.
  • the biomass in the fermentation broth was determined to be 54 g/L, the crude oil production was 34.02 g/L, the ARA yield was 20.1 g/L, and the ARA productivity was 1.82 g/(L ⁇ d), as shown in FIG.
  • the fermentation tank had a tank volume of 146 m 3 , and the whole batch fermentation containing ARA crude oil yield was 4696.92 kg, which was 1.78 times higher than that of Example 1.
  • Table 5 below shows the results of gas phase analysis of the fatty acid composition of the mixed fat obtained after fermentation.
  • Examples 6-10 and Comparative Example 1 below relate to the extraction of ARA oil
  • the fermentation broth was transported to the cloth cavity by a distributor to carry out the cloth wrapping, and after the cloth was finished, the first-stage pressing was performed, and the stepwise pressing method was adopted.
  • the set pressure of 30MPa is reached, and the pressure is kept for 2h until basically no water droplets flow out.
  • Use the stepwise pressurization method to reach the set pressure of 100MPa within the set 2h, and keep the pressure for 2h to the basic.
  • the fermentation broth was transported to the cloth cavity by a distributor to carry out the cloth wrapping, and after the cloth was finished, the first-stage pressing was performed, and the stepwise pressing method was adopted.
  • the set pressure of 40 MPa was reached within 5 hours of the set pressure, and the pressure was kept for 4 hours until substantially no water droplets flowed out.
  • Use the stepwise pressurization method to reach the set pressure of 150MPa within the set 5h, and keep the pressure for 4h to the basic.
  • 300 L of the ARA fermentation broth obtained in Example 5 was subjected to heat inactivation treatment, and then centrifuged in a butterfly centrifuge to remove the centrifugal light liquid to obtain 145 L of the concentrated fermentation broth, and the concentrated fermentation broth was transported to the cloth by a distributor.
  • the fabric is wrapped, and after the fabric is finished, the first-stage pressing is carried out, and the pressure of 40 MPa is reached within the set 5h by the stepwise pressing method, and the water is kept for 4 hours until substantially no water droplets flow out.
  • stepwise pressurization method uses the stepwise pressurization method to reach the set pressure of 150MPa within the set 5h, and keep the pressure for 4h to the basic. No oil droplets were discharged, and the pressed ARA hair oil was collected, and a total of 9.4 kg of ARA hair oil was obtained, and the yield of the fermentation liquid to the hair oil was 92.1%.
  • the secondary press cage is removed and the pressed residue is separated from the filter cloth.
  • the flexible pressing is carried out, and the pressure of 100 MPa is set within 2 hours within the set pressure, and the pressure is kept for 2 hours until there is no oil drop.
  • the crushed ARA hair oil was collected, and a total of 31.8 kg of ARA hair oil was obtained, and the fermentation liquid to the hair oil yield was 93.5%.
  • the flexible pressing is carried out, and the pressure of 150 MPa is set within 4 hours within the set pressure, and the pressure is kept for 4 hours until there is no oil drop.
  • the crushed ARA hair oil was collected, and a total of 32.5 kg of ARA hair oil was obtained, and the fermentation liquid to the hair oil yield was 95.5%.
  • the twin-screw press was preheated to 80 ° C, and the ARA powder was added to a twin-screw press to extract oil, and 18.2 kg of ARA hair oil was obtained, and the yield of the fermentation liquid to the hair oil was 53.5%.
  • Decolorization The hydration oil is heated to 100 ° C, the vacuum degree is controlled to -0.075 MPa, vacuum dehydration is carried out for 1 h, then the temperature is lowered to 70 ° C, decoloring agent (191 g activated carbon and 286 g of activated clay) is added, and the mixture is decolorized for 0.5 h, the stirring is stopped, and the decolorization is removed by filtration.
  • the agent obtained 9.33 kg of decolorizing oil.
  • the decolorized oil enters the tertiary molecular distillation, controls the first stage vacuum degree of about 90Pa, the temperature of 160 ° C, removes the light components of the first stage, the heavy components enter the second stage molecular distillation, and controls the secondary vacuum degree of about 40 Pa,
  • the temperature is 200 ° C
  • the second-stage light component is removed
  • the heavy component enters the third-stage molecular distillation
  • the third-stage vacuum is controlled to about 3 Pa
  • the temperature is 220 ° C
  • the third-level light component is removed
  • the heavy component is collected
  • the deodorization is completed
  • the temperature is lowered.
  • To 30 ° C add antioxidants and package to obtain 9.13 kg of ARA refined oil. The test results are shown in Table 6.
  • Decolorization The hydration oil is heated to 110 ° C, the vacuum degree is controlled to -0.075 MPa, vacuum dehydrated for 0.5 h, then the temperature is lowered to 80 ° C, decoloring agent (192 g activated carbon and 288 g of activated clay) is added, and the mixture is decolorized for 1 h, the stirring is stopped, and the decolorization is removed by filtration. The agent obtained 9.39 kg of decolorizing oil.
  • the decolorized oil enters the tertiary molecular distillation, controls the first stage vacuum degree of about 90Pa, the temperature of 200 ° C, removes the light components of the first stage, the heavy components enter the second stage molecular distillation, and controls the secondary vacuum degree of about 40 Pa,
  • the temperature is 220 ° C
  • the second-stage light component is removed
  • the heavy component enters the third-stage molecular distillation
  • the third-stage vacuum is controlled to about 3 Pa
  • the temperature is 250 ° C
  • the third-level light component is removed
  • the heavy component is collected, and the molecular distillation is completed.
  • the temperature was lowered to 30 ° C, antioxidants were added, and packaging was carried out to obtain 9.08 kg of ARA refined oil.
  • the test results are shown in Table 7.
  • Alkali refining hydrated oil is kept at 75 °C, 1L concentration of 10% (mass fraction) NaOH solution is added, stirred for 30min, stirring speed is 30rev/min, and it is allowed to stand for 4h. The soap residue is separated to obtain alkali refining and alkali refining and stirring. Under the circumstances, 10% of the oil with a weight of 10% was sprayed with water, and the water addition time was controlled at 10-30 min. After the completion of the water, the mixture was allowed to stand for 2 h, and the water layer was separated and washed twice to obtain 9.10 kg of alkali refinery.
  • Decolorization The alkali refining oil is heated to 110 ° C, the vacuum degree is controlled to -0.075 MPa, vacuum dehydration is 0.5 h, then the temperature is lowered to 80 ° C, decoloring agent (182 g activated carbon and 273 g of activated clay) is added, and the mixture is decolorized for 1 h, the stirring is stopped, and the decolorizing agent is removed by filtration. , 8.65 kg of decolorized oil was obtained.
  • Deodorization The decolorizing oil is transferred into the deodorizing pot, deodorized by steam, the deodorizing temperature is controlled at 185 ° C, the vacuum degree is controlled within 600 Pa, the deodorizing time is 2 h, the deodorization is completed, the steam is stopped, the temperature is lowered to 20-40 ° C, and the antioxidant is added. Packing, get 8.41kg of ARA refined oil, the test results are shown in Table 10.

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Abstract

一种利用高山被孢霉菌株工业化发酵生产含花生四烯酸混合油脂的方法，从发酵罐培养的100-140小时开始，将发酵温度控制在20-25℃；和/或，从发酵罐培养的第8-10天开始，停止通气或者将通气量减少50％以上。

Description

一种利用微生物发酵生产ARA的方法 技术领域
本发明属于发酵工程领域，涉及一种利用微生物发酵生产ARA的方法，具体地，涉及利用高山被孢霉菌株工业化发酵生产含花生四烯酸(ARA)混合油脂的方法。
背景技术
ARA，又称花生四烯酸，全名二十碳四烯酸(cis-5，8，11，14-arachidonic acid，ARA)，是ω-6系列的一种多元不饱和脂肪酸。ARA是一种人体重要的必需脂肪酸，也是人体中含量最高、分布最广的一种多不饱和脂肪酸。ARA在血液、肝脏、肌肉和其他器官系统中作为磷脂结合的结构脂类起重要作用，ARA是许多循环二十烷酸衍生物的直接前体。这些生物活性物质对脂质蛋白的代谢、血液流变学、血管弹性、白细胞功能和血小板激活等具有重要的调节作用。此外，有研究表明，ARA降血脂、降血压和降血胆固醇的效果均强于亚油酸和亚麻酸；同时能缓解氯化钡、乌头碱等引起的心律不齐，其作用效果也强于亚油酸和亚麻酸。因此，ARA具有重要的营养、保健和医疗功能。
目前，ARA主要由微生物发酵法生产。公开号为CN105112466A的专利公开了一种添加产物促进剂发酵制备花生四烯酸的方法，其所公开的添加产物促进剂的摇瓶在培养结束后检测发酵液中ARA含量为5.41g/L，产量较对照提高了45.1％。公开号为CN102925502A的专利公开了一种利用高山被孢霉生产花生四烯酸油脂的工业方法，其所公开的产量为花生四烯酸的单位产量达到10g/L发酵液。公开号为CN104278107A的专利公开了一种基于溶氧调控高山被孢霉发酵产花生四烯酸油脂的方法，其所公开的产量为25m3发酵罐发酵高山被孢霉细胞干重、油脂含量、花生四烯酸占总油脂的百分含量、花生四烯酸的单位产量分别可以达到55g/L、54％、65％、20.628g/L，花生四烯酸生产强度达到1.753g/(L·d)，这也是目前报道的采用高山被孢霉大规模工业化生产ARA的最高生产水平。虽然其ARA生产率较之前的研究有了较大提高，但对于利用高山被孢霉进行工业化生产花生四烯酸，大大降低其生产成本，提高单位产量，使微生物发酵工业化生产ARA的方法能够得到大力推广和普及使用还是远远不够的。
现有从高山被孢霉发酵培养物中提取ARA的方法主要有三种，一是离心法，二是有机溶剂萃取法，三是超临界萃取法。离心法如公告号为CN1282745C的专利公开了一种从微生物细胞中获得油的方法，所述的油包含一种或多种不饱和脂肪酸，该方法包括：(a)裂解微生物细胞的细胞壁以释放出油来；和(b)通过离心从至少部分(a)中所形成的细胞壁碎片中分离油。但该发明离心后的油层品质较差，除含油脂外，还含有水分、培养基成分和细胞碎片，不利于后续的精炼，另外离心后的废水层含有大量菌渣，COD很高，难以处理或处理成本极高。有机溶剂萃取法如公告号为CN101109015B的专利公开了一种花生四烯酸油脂的制备方法，其中所述的油脂提取方法为：将老化后的菌体收集、烘干、磨碎，用石油醚和乙醇萃取12小时，再将石油醚和乙醇通过减压蒸馏除去即得到花生四烯酸油脂。但该方法使用到有机溶剂进行萃取，最终产品可能会有溶剂残留，且萃取过程存在易燃易爆等安全隐患。超临界萃取方法如公开号为CN101579019A的专利公开了一种用CO2超临界萃取法提取多不饱和脂肪酸油脂的方法，其步骤如下：①粉碎过筛；②加温萃取；③将液态CO2注入萃取釜中，加压；④萃取完毕收集油脂。但超临界设备价格昂贵，萃取收率也不高，该发明萃取出油率最高只有50％。
现有技术中ARA毛油的精炼多采用化学精炼技术，ARA毛油经过脱胶、碱炼、脱色、脱臭后得到ARA精油。该工艺技术不可避免地存在一些问题，如：碱炼为了达到控制酸价低的要求，通常都会加入过量碱，部分甘油三酯不可避免会被皂化；碱炼产生的高COD废水会污染环境；碱炼需要高温处理时间长，容易造成产品过氧化值、茴香胺值升高；脱臭温度高、时间长易产生反式脂肪酸等缺点。
目前，尚需要开发新的ARA生产工艺。
发明内容
本发明人经过深入的研究和创造性的劳动，得到了一种培养用于生产ARA的微生物的方法。本发明人惊奇地发现，所述培养方法能够大幅度地提高生物量及ARA产量。进一步地，本发明人还发现了一种提取ARA毛油的方法，所述提取方法能够提高ARA毛油的提取收率和品质。进一步地，本发明人还发现了一种纯化ARA毛油的方法，其能够提升ARA成品油的各项技术指标和纯化收率。本发明显著地提高了含ARA的粗油脂产量、ARA产量以及ARA生产率。由此提供了下述发明：
本发明的一个方面涉及一种培养用于生产ARA的微生物的方法，其中：
从发酵罐培养的100-140小时开始，将发酵温度保持在20-25℃(例如21-25℃、20-24℃、21-23℃、22-25℃、22-24℃、23-25℃、20℃、21℃、22℃、23℃、24℃或25℃)；优选地，直至发酵结束；
和/或
从发酵罐培养的第8-10天开始，停止通气或者将通气量减少50％以上(例如55％以上、60％以上、65％以上、70％以上、75％以上、80％以上、85％以上、90％以上或95％以上)；优选地，直至发酵结束。
在本发明的一个实施方案中，所述的培养方法，其中：
从发酵罐培养的105-135小时、110-130小时、115-125小时(例如115、116、117、118、119、120、121、122、123、124或125小时)或者120小时开始，将发酵温度控制在20-25℃(例如21-25℃、20-24℃、21-23℃、22-25℃、22-24℃、23-25℃、20℃、21℃、22℃、23℃、24℃或25℃)，和/或
从发酵罐培养的第8天、第9天或者第10天开始，停止通气或者将通气量减少50％以上(例如55％以上、60％以上、65％以上、70％以上、75％以上、80％以上、85％以上、90％以上或95％以上)。
在本发明的一个实施方案中，所述的培养方法，其中，在保持发酵温度为20-25℃之前，发酵温度为25-30℃(例如26-30℃、25-29℃、26-28℃、27-30℃、26-29℃、28-30℃、25℃、26℃、27℃、28℃、29℃或30℃)。
在本发明的一个实施方案中，所述的培养方法，其中，所述发酵液的pH为6.0-7.0；
优选地，在停止通气或者将通气量减少50％以上之前，发酵液中葡萄糖的浓度保持1-5g/L(例如1、1.5、2、2.5、3、3.5、4、4.5或5g/L)。
在本发明的一个实施方案中，所述的培养方法，其中，在停止通气或者将通气量减少50％以上的同时或之后，发酵液中葡萄糖的浓度1-2g/L；
优选地，在停止通气或者将通气量减少50％以上的同时或之后，发酵温度20-25℃；
优选地，在停止通气或者将通气量减少50％以上的同时或之后，停止搅拌；
优选地，将上述3个条件即葡萄糖浓度、发酵温度、停止搅拌，保持至发酵结束；
优选地，在停止通气或者将通气量减少50％以上之前，滤去发酵液，并加入适量水或新鲜的发酵培养基。
在本发明的一个实施方案中，所述的培养方法，其中，所述发酵罐培养还包括在发酵罐培养起始的36-60小时(例如40-56小时、42-54小时、44-52小时、46-50小时、46、47、48、49或50小时)，进行分罐培养的步骤；例如，分为两个或更多个发酵罐进行发酵罐培养。
在本发明的一个实施方案中，所述的培养方法，其在发酵罐培养之前，还包括接种和种子扩大培养的步骤；
优选地，所述种子扩大培养包括一级种子扩大培养和二级种子扩大培养；
优选地，所述一级种子扩大培养包括下述步骤：
按照0.4％-1％的接种量将摇瓶种子液接入装有灭菌后培养基的一级种子罐中，培养温度25-32℃，通气量1-2vvm，罐压0.02-0.05MPa，培养30-35h，完成一级种子扩大培养；
优选地，所述二级种子扩大培养包括下述步骤：
按照1％-3％的接种量将一级种子罐的种子液接入装有灭菌后培养基的二级种子罐中，培养温度25-32℃，通气量1-2vvm，罐压0.02-0.05MPa，培养20-25h，完成二级种子扩大培养。
在本发明的一个实施方案中，所述的培养方法，还包括在接种和种子扩大培养之前，进行活化培养的步骤；优选地，所述活化培养的温度为25-32℃，搅拌转速100-200rpm，时间为40-48h。
在本发明的一个实施方案中，所述的培养方法，其中，所述发酵罐培养包括下述步骤：
按照1％-3％的接种量将二级种子罐的种子液接入装有灭菌后培养基的发酵罐中，培养温度20-30℃，通气量1-2vvm，罐压0.02-0.05MPa，搅拌转速0-50rpm，进行发酵罐培养。
在本发明的一个实施方案中，所述的培养方法，其中用于生产ARA的微生物为高山被孢霉或其突变菌株；所述高山被孢霉可以是本领域已知的高山被孢霉，例如，所述高 山被孢霉选自保藏号为CCTCC No.M2012073、CCTCC No.M2013392、CCTCC No.M2015421和ATCC No.42430的菌株。
在本发明一个具体的实施方式中，所述的培养方法，包括下述步骤：
1)将高山被孢霉(Mortierella alpine)斜面保藏菌株接入装有400mL培养基的2L摇瓶，在25-32℃的温度下以150rpm的转速培养40-48h，完成菌株活化培养；
2)按照0.4％-1％的接种量将摇瓶种子液接入装有灭菌后培养基的一级种子罐中，培养温度25-32℃，通气量1-2vvm，罐压0.02-0.05MPa，培养30-35h，完成一级种子扩大培养；
3)按照1％-3％的接种量将一级种子罐的种子液接入装有灭菌后培养基的二级种子罐中，培养温度25-32℃，通气量1-2vvm，罐压0.02-0.05MPa，培养20-25h，完成二级种子扩大培养；
4)按照1％-3％的接种量将二级种子罐的种子液接入装有灭菌后培养基的发酵罐中，培养温度20-30℃，通气量1-2vvm，罐压0.02-0.05MPa，搅拌转速0-50rpm，进行发酵培养；
5)将120h前的罐温控制在25-30℃、120h及之后的罐温控制在20-25℃；
6)发酵至48h将发酵罐中发酵液一分为二培养，一半发酵液留在主罐中继续培养，另一半发酵液通过压差法转移至无菌保压的副罐中培养，主副罐中分别补入适量灭菌后新鲜培养基或无菌水，分罐培养后主副罐通气量、罐压及搅拌转速均稍作调整；
7)发酵过程中葡萄糖浓度随菌体生长不断下降，流加碳源将发酵液中糖点(葡萄糖浓度)维持在1-5g/L；
8)发酵培养8-10d，中止发酵，主副罐均通过放料管路筛网过滤，得到的菌体重新加入等体积一次水，稍开搅拌将菌体重悬后，停搅拌，停止通气，维持罐温20-25℃，流加碳源控制主副罐糖点在1-2g/L，静置1-2d；
不拘于理论的限制，步骤8)可以刺激高山被孢霉菌体中短链及低碳脂肪酸往ARA转化积累，提高菌体中ARA含量；
9)放罐，测定发酵液中生物量达50-60g/L，粗油脂含量达50％-65％，ARA占总油脂含量为55％-60％，ARA产量最高可达20.1g/L，ARA产率最高可达1.83g/(L·d)。
上述步骤7)中，糖点(葡萄糖的浓度)采用本领域技术人员知悉的方法测定，例如 生物传感仪测定。
在本发明的一个实施方案中，所述的培养方法，其中所用的发酵培养基碳源中添加20％-40％(粗甘油的质量/碳源的质量×100％)预处理后的生物柴油副产物粗甘油。生物柴油是以植物油和动物油脂等可再生油脂为原料制成的可再生能源，经过转酯反应后可生成生物柴油及副产物甘油。粗甘油的预处理过程包括调节pH值至酸性、稀释、水解、分离。在本发明的一个实施方案中，粗甘油的预处理包括下述步骤：i)将粗甘油与去离子水以1∶4(体积比)混合；ii)以盐酸调节pH至6.5左右；iii)以5000rpm的转速分离去除沉淀物质。不拘于理论的限制：步骤i)中，稀释后可以降低粘度；步骤ii)中，将粗甘油中可溶性的皂角类物质转化为不溶的游离脂肪酸固体物质；步骤iii)中，沉淀物质包括游离脂肪酸固体和不溶解的重金属杂质。
上述种子培养基可以采用本领域培养已有的菌种高山被孢霉(Mortierella alpine)种子培养基。上述发酵培养基可以采用本领域培养已有的菌种高山被孢霉(Mortierella alpine)发酵培养基。
上述发酵培养基中碳源包括葡萄糖、玉米浆粉、糖蜜、甘油和淀粉中的一种或多种；氮源包括大豆粉、酵母粉、蛋白胨、氨水、硝酸钠、谷氨酸钠、硫酸铵中的一种或多种。
上述发酵培养基中添加的微量元素包括丙氨酸、谷氨酸、赖氨酸、泛酸钙、生物素、维生素B1、微生物B6、微生物B12、维生素K中的一种或多种，当为其中一种时，添加量为0.001％-0.01％；当为其中多种时，任意一种组分的添加量为0.001％-0.005％。
上述发酵培养基中添加的无机盐包括硫酸镁、氯化钾、氯化钠、氯化钙、磷酸二氢钾、磷酸氢二钾中的一种或多种。
本发明的另一方面涉及一种微生物的发酵培养物，其由本发明中任一项所述的培养方法得到。
本发明的再一方面涉及一种提取ARA毛油的方法，包括如下步骤：
1)将用于生产ARA的微生物的发酵培养物进行脱水处理；
2)将步骤1)的产物进行柔性压榨，得到ARA毛油。
在本发明的一个实施方案中，所述的提取方法，其中，步骤1)中，所述发酵培养 物为高山被孢霉发酵培养物；优选地，所述发酵培养物为本发明的发酵培养物。
在本发明的一个实施方案中，所述的提取方法，其中，步骤1)中，所述脱水处理选自如下的任意一种、两种或三种：
离心或板框过滤、第一级柔性压榨、干燥例如气流干燥；
优选地，所述脱水处理依次包括离心和第一级柔性压榨，或者依次包括板框过滤和气流干燥；
优选地，所述气流干燥的进风温度110-150℃，出风温度30-70℃，至水分含量小于10％。
在本发明的一个实施方案中，所述的提取方法，其中，步骤1)中，所述第一级柔性压榨采用逐步加压方式，设定压力为20-40MPa，加压时间为1-6h，到达设定压力后保压1-4h。
在本发明的一个实施方案中，所述的提取方法，其中，步骤2)中，所述柔性压榨采用逐步加压方式，设定压力为50-150MPa，加压时间为1-6h，到达设定压力后保压1-4h。
在本发明一个具体的实施方式中，所述的提取方法，包括下述步骤：
1)布料：将一定量的发酵培养物用布料器运送至布料腔中，布料器回到初始位置，待下一步布料。
2)一级柔性压榨：采用逐步加压方式，一级柔性压榨的压力范围为20-40MPa，加压时间约为1-6h，到达设定压力，保压1-4h至基本无水滴流出，结束后，待物料下降到重压腔中，将物料推入二级柔性压榨位置。
3)二级柔性压榨：采用逐步加压方式，压榨的最终压力为50-150MPa，加压时间大约为1-6h，到达设定压力，保压1-4h至基本无油滴流出，收集压榨出来的ARA毛油，卸压，去掉二级压榨笼，将菌渣与滤布进行分离。
上述ARA发酵液可直接进行一级柔性压榨，也可以先采用离心方法除去一部分水分，提高发酵液的含固量后再进行一级柔性压榨，可缩短压榨时间和提高生产能力。
上述发酵液离心方法可采用卧式螺旋离心机、碟式离心机、管式离心机中的一种进行。
在本发明另一个具体的实施方式中，所述的提取方法，包括下述步骤：发酵培养物经板框过滤、气流干燥得到菌粉，菌粉进行柔性压榨，得到ARA毛油。
上述板框过滤方式为：发酵液进料后，过滤至压滤机滤嘴全开且进料压力达到0.6-1.0MPa，过滤完毕后吹气，吹气压力0.3-0.6MPa，吹气时间1-3h。
上述气流干燥进风温度110-150℃，出风温度30-70℃，ARA菌粉水分控制在10％以内。
本发明的再一方面涉及一种ARA毛油，其由本发明中任一项所述的提取方法制得。
本发明的再一方面涉及一种纯化ARA毛油的方法，包括将ARA毛油进行水化、脱色和分子蒸馏的步骤；优选地，所述ARA毛油为本发明的ARA毛油。
在本发明的一个实施方案中，所述的纯化方法，其中，所述水化包括如下步骤：
将ARA毛油加热至70-85℃，按1kg毛油加入50-150g水的比例加入75-90℃水，搅拌10-60min，搅拌速度30-90转/min，静置1-6h，分层去掉下层磷脂，得到水化油。
在本发明的一个实施方案中，所述的纯化方法，其中，所述脱色包括如下步骤：
将水化产物升温至90-110℃，控制真空度≤-0.07MPa，真空脱水0.5-2h，然后降温至60-80℃，加入脱色剂(例如水化油重量1％-3％的活性炭和2％-4％活性白土)，搅拌0.5-1h，停止搅拌，过滤去除脱色剂，得到脱色油。
在本发明的一个实施方案中，所述的纯化方法，其中，所述分子蒸馏为三级分子蒸馏；
优选地，所述分子蒸馏包括如下步骤：
将脱色油进入三级分子蒸馏，控制第一级真空度≤100Pa、温度150-200℃，去除第一级的轻组分；得到的第一重组分进入第二级分子蒸馏，控制二级真空度≤50Pa、温度180-220℃，去除第二级轻组分；得到的第二重组分进入第三级分子蒸馏，控制第三级真空度≤5Pa、温度200-250℃，去除第三级轻组分，得到第三重组分，为ARA成品油。
优选地，重复分子蒸馏1次或多次(例如2、3、4或5次)。
在本发明一个具体的实施方式中，所述的纯化方法，包括下述步骤：
1)水化：ARA毛油加热至70-85℃，按1kg毛油加入50-150g纯化水的比例加入75-90℃纯化水，搅拌10-60min，搅拌速度30-90转/min，静置1-6h，分层去掉下层磷脂，得到水化油；
2)脱色：水化油移入脱色锅，升温至90-110℃，控制真空度≤-0.07MPa，真空脱水0.5-2h，然后降温至60-80℃，加入脱色剂(水化油重量1％-3％的活性炭和2％-4％活性白土)，搅拌脱色0.5-1h，停止搅拌，过滤去除脱色剂，得到脱色油；
3)分子蒸馏：脱色油进入三级分子蒸馏，控制第一级真空度≤100Pa、温度150-200℃，去除第一级的轻组分，重组分进入第二级分子蒸馏，控制二级真空度≤50Pa、温度180-220℃，去除第二级轻组分，重组分进入第三级分子蒸馏，控制第三级真空度≤5Pa、温度200-250℃，去除第三级轻组分，收集重组分，得到分子蒸馏油。分子蒸馏遍数控制1-3遍，至酸价、气味符合标准要求。分子蒸馏完毕，降温至20-40℃，添加抗氧化剂，包装，得到ARA成品油。
本发明的再一方面涉及一种ARA成品油，其由本发明中任一项所述的纯化方法制得。
本发明的再一方面涉及一种利用微生物生产ARA或含有ARA的产品(例如ARA成品油)的方法，包括：
本发明任一项所述的培养用于生产ARA的微生物的方法、
本发明任一项所述的提取ARA毛油的方法、和/或
本发明任一项所述的纯化ARA毛油的方法。
在本发明的一个实施方式中，生产ARA成品油的工艺流程如图1所示。
在本发明的另一个实施方式中，生产ARA成品油的工艺流程如图2所示。
本发明中，
术语“柔性压榨”是指采用PLC(可编程逻辑控制器)程序控制来进行加压-保压-加压循环，逐步达到预定压力的一种高压压榨方式。
术语“纯化水”是指饮用水经蒸馏法、离子交换法、反渗透法或其他适宜的方法制得的供药用的水，不含任何添加剂。在本发明的一个实施方案中，所述纯化水参照《中国药典》的规定。
术语“ARA毛油”是指，从ARA发酵液中制取、没经过精炼加工的初级油。
术语“ARA成品油”是指，ARA毛油经过精炼加工得到的精油。
术语“发酵罐培养”是指种子扩大培养之后，在发酵罐中进行的用于生产目的产物的发酵培养。
本发明中，当提及“通气”或“通气量”时，如果没有特别说明，是指通入空气或者通入空气的量。
发明的有益效果
本发明具有下述技术效果中的至少一种：
(1)本发明的工艺技术指标均明显优于现有的工艺技术指标，所得ARA油脂产量高、纯度高，有利于ARA的大规模工业化生产，粗甘油的添加也降低了ARA发酵生产成本，大大提高了ARA发酵生产的市场竞争力。
(2)本发明采用柔性压榨工艺制备ARA毛油，不需要使用有机溶剂进行提取，整条生产工艺路线无需使用有机溶剂，最终产品不会有溶剂残留，一方面所得产品绿色健康，产品质量好，另一方面生产车间安全环保，是一个绿色的清洁生产工艺。
(3)发酵培养物经一级柔性压榨除去的水溶液，基本不含菌渣，COD比较低，容易生化处理，二级柔性压榨得到毛油后，剩下的菌渣还含有少量的油脂和大量的蛋白，可以作为饲料添加剂使用，经济环保。
(4)本发明采用分子蒸馏一步工艺替代传统脱酸、脱臭两步工艺。分子蒸馏能够在保留物质生理活性的基础上快速去除大量的游离脂肪酸和臭味。对比传统的碱炼脱酸方法，分子蒸馏脱酸工艺简单，降低了过度碱炼的风险，减少从皂脚带走中性油脂的损失，脱酸收率明显提高，脱酸过程在高真空条件下进行且时间短，避免了碱炼脱酸受热时间长造成过氧化值、茴香胺值升高的风险，产品稳定性好；对比传统水蒸气蒸馏脱臭工艺，分子蒸馏脱臭时间短，真空度高，减少了反式脂肪酸的产生，且臭味物质脱除效果好，产品无腥味。
(5)生产过程避免了有机溶剂的使用，避免了溶剂消耗和溶剂回收的成本，污水COD低容易处理，精炼收率高，从而大大降低了生产成本。
附图说明
图1：本发明一个实施方式的生产ARA成品油的工艺流程示意图。
图2：本发明另一个实施方式的生产ARA成品油的工艺流程示意图。
图3：所公开的高山被孢霉Mortierella alpine在不同培养方式下100m3发酵罐发酵生产ARA的结果。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
关于本发明涉及的物理量或者指标的测定方法或者计算方法，如果没有特别说明，按照下面所述的方法进行：
生物量的测定方法：取适量发酵培养物(发酵液)置于扁形称量瓶中，于105℃电热恒温干燥箱中干燥4h后，放入干燥器冷却至室温，称重，减去称量瓶重量，再除以发酵液体积，所得数值即为生物量，单位g/L。
粗油脂产量的测定方法：取一定体积发酵培养物(发酵液)，加入2倍体积的浓盐酸，70℃下恒温搅拌50min至菌体完全消化，加入适量正己烷，静置分层，用滴管取上层有机相至茄型瓶中，连续萃取5-8次，直至上层有机相为无色，通过80℃水浴旋转蒸发除去正己烷，然后将茄型瓶置于105℃电热恒温干燥箱中干燥1h，放入干燥器冷却至室温，称重，减去茄型瓶重量，再除以发酵液体积，所得数值即为粗油脂产量，单位g/L。
ARA产量：以气相色谱法测得粗油脂中ARA的含量，乘以粗油脂产量所得，单位g/L。
ARA生产率：ARA产量除以发酵周期(天数)所得数值，单位g/(L·d)。
脂肪酸组成分析的方法和本发明中ARA成品油检测方法均依据GB 26401-2011花生四烯酸油脂(发酵法)进行。
毛油提取收率计算方式：收率＝毛油重量g/(发酵液体积L×发酵液粗油脂产量g/L)×100％。
以下的实施例1-5涉及用100m3发酵罐发酵生产ARA。
下面的实施例1-5中，如果没有特别说明：
所用的菌株为高山被孢霉Mortierella alpine(ATCC No.42430)；
所用种子培养基配方为：马铃薯(去皮)200g、葡萄糖20g、琼脂15-20g、蒸馏水1000ml，自然pH；
所用发酵培养基配方为：葡萄糖22g/L，酵母粉12g/L，蛋白胨8g/L，氯化钠15g/L，硫酸铵6g/L，KH2PO4 5g/L，CuSO4·5H2O 1.5μg/L，MnSO4 2μg/L，ZnSO4·7H2O 3μg/L，pH 6-7。
实施例1
将高山被孢霉(Mortierella alpine)斜面保藏菌株接入装有400mL培养基的2L摇瓶，在28℃的温度下以150rpm的转速培养48h，完成菌株活化培养。按照0.4％的接种量将摇瓶种子液接入装有灭菌后培养基的一级种子罐中，在培养温度28℃、通气量1vvm、罐压0.04MPa的条件下培养30h，完成一级种子扩大培养。按照3％的接种量将一级种子罐的种子液接入装有灭菌后培养基的二级种子罐中，在培养温度28℃、通气量1vvm、罐压0.04MPa的条件下培养24h，完成二级种子扩大培养。按照3％的接种量将二级种子罐的种子液接入装有灭菌后培养基的发酵罐中。
发酵过程的培养温度28℃、通气量1vvm、罐压0.04MPa、搅拌转速50rpm，流加含30％预处理后粗甘油的碳源，控制葡萄糖浓度在5g/L左右，进行发酵培养。发酵过程中检测发酵液葡萄糖浓度、pH、菌体生物量、粗油脂产量及ARA产量变化。
培养9d后终止发酵，测定发酵液中生物量为43g/L，粗油脂产量为21.5g/L，ARA产量为10.1g/L，ARA生产率为1.12g/(L·d)，如图3。发酵液放罐体积为83m3，整批发酵含ARA粗油脂产量为1784.5kg。表1为发酵后所得混合油脂脂肪酸组成气相分析结果。
表1：实施例1所得混合油脂脂肪酸组成
脂肪酸组成 含量％
C14：0 0.27
C15：0 0.12
C16：0 17.63
C17：0 0.46
C18：0 19.13
C18：1 5.34
C18：2 4.61
C18：3 4.47
C20：0 0.99
C20：4 46.98
实施例2
将高山被孢霉(Mortierella alpine)斜面保藏菌株接入装有400mL培养基的2L摇瓶，在28℃的温度下以150rpm的转速培养48h，完成菌株活化培养。按照0.4％的接种量将摇瓶种子液接入装有灭菌后培养基的一级种子罐中，在培养温度28℃、通气量1vvm、罐压0.04MPa的条件下培养30h，完成一级种子扩大培养。按照3％的接种量将一级种子罐的种子液接入装有灭菌后培养基的二级种子罐中，在培养温度28℃、通气量1vvm、罐压0.04MPa的条件下培养24h，完成二级种子扩大培养。按照3％的接种量将二级种子罐的种子液接入装有灭菌后培养基的发酵罐中。
发酵过程的通气量1vvm、罐压0.04MPa、搅拌转速50rpm，将120h前的罐温控制在28±1℃、120h及之后的罐温控制在22±1℃，流加含30％预处理后粗甘油的碳源，控制葡萄糖浓度在5g/L左右，进行发酵培养。发酵过程中检测发酵液葡萄糖浓度、pH、菌体生物量、粗油脂产量及ARA产量变化。
培养9d后终止发酵，测定发酵液中生物量为50g/L，粗油脂产量为29g/L，ARA产量为15.1g/L，ARA生产率为1.68g/(L·d)，如图3。发酵液放罐体积为82m3，整批发酵含ARA粗油脂产量为2378kg，比实施例1的产量提高了0.33倍。以下表2为发酵后所得混合油脂脂肪酸组成气相分析结果。
表2：实施例2所得混合油脂脂肪酸组成
脂肪酸组成 含量％
C14：0 0.43
C15：0 0.16
C16：0 15.21
C17：0 0.56
C18：0 16.89
C18：1 5.07
C18：2 4.51
C18：3 4.12
C20：0 0.98
C20：4 52.07
实施例3
将高山被孢霉(Mortierella alpine)斜面保藏菌株接入装有400mL培养基的2L摇瓶，在28℃的温度下以150rpm的转速培养48h，完成菌株活化培养。按照0.4％的接种量将摇瓶种子液接入装有灭菌后培养基的一级种子罐中，在培养温度28℃、通气量1vvm、罐压0.04MPa的条件下培养30h，完成一级种子扩大培养。按照3％的接种量将一级种子罐的种子液接入装有灭菌后培养基的二级种子罐中，在培养温度28℃、通气量1vvm、罐压0.04MPa的条件下培养24h，完成二级种子扩大培养。按照3％的接种量将二级种子罐的种子液接入装有灭菌后培养基的发酵罐中。
发酵过程的培养温度28℃、通气量1vvm、罐压0.04MPa、搅拌转速50rpm，流加含30％预处理后粗甘油的碳源，控制葡萄糖浓度在5g/L，进行发酵培养。发酵过程中发酵至48h将发酵罐中发酵液一分为二培养，一半发酵液留在主罐中继续培养，另一半发酵液通过压差法转移至无菌保压的副罐中培养，主副罐中分别补入适量灭菌后新鲜培养基或无菌水，分罐培养后主副罐通气量、罐压及搅拌转速均稍作调整。发酵过程中检测发酵液葡萄糖浓度、pH、菌体生物量、粗油脂产量及ARA产量变化。
培养9d后终止发酵，测定发酵液中生物量为45g/L，粗油脂产量为22.05g/L，ARA产量为10.6g/L，ARA生产率为1.18g/(L·d)，如图3。发酵液放罐体积为145m3，整批发酵含ARA粗油脂产量为3197.25kg，比实施例1的产量提高了0.79倍，虽然放罐生物量、粗油脂产量、ARA产量等指标与原始培养方式比较差异不大，但是由于采用分罐培养，发酵液放罐体积为原始培养方式的1.75倍，所以整批发酵产量较原始培养方式提高了0.79倍。这对于工业化大规模生产含ARA油脂具有重要意义，可以极大的节约成本，提高ARA生产的市场竞争力。以下表3为发酵后所得混合油脂脂肪酸组成气相分析结果。
表3：实施例3所得混合油脂脂肪酸组成
脂肪酸组成 含量％
C14：0 0.48
C15：0 0.17
C16：0 17.12
C17：0 0.52
C18：0 18.54
C18：1 5.28
C18：2 4.25
C18：3 4.36
C20：0 1.21
C20：4 48.07
实施例4
将高山被孢霉(Mortierella alpine)斜面保藏菌株接入装有400mL培养基的2L摇瓶，在28℃的温度下以150rpm的转速培养48h，完成菌株活化培养。按照0.4％的接种量将摇瓶种子液接入装有灭菌后培养基的一级种子罐中，在培养温度28℃、通气量1vvm、罐压0.04MPa的条件下培养30h，完成一级种子扩大培养。按照3％的接种量将一级种子罐的种子液接入装有灭菌后培养基的二级种子罐中，在培养温度28℃、通气量1vvm、罐压0.04MPa的条件下培养24h，完成二级种子扩大培养。按照3％的接种量将二级种子罐的种子液接入装有灭菌后培养基的发酵罐中。
发酵过程的培养温度28℃、通气量1vvm、罐压0.04MPa、搅拌转速50rpm，流加含30％预处理后粗甘油的碳源，控制葡萄糖浓度在5g/L，进行发酵培养。培养9d后，中止发酵，通过放料管路筛网滤去发酵液，重新加入等体积一次水，稍开搅拌将菌体重悬后，停搅拌，停止通气，流加碳源控制糖点在2g/L左右，静置2d，刺激高山被孢霉菌体中短链及低碳脂肪酸往ARA转化积累，提高菌体中ARA含量。发酵过程中检测发酵液葡萄糖浓度、pH、菌体生物量、粗油脂产量及ARA产量变化。
培养11d后，测定发酵液中生物量为52g/L，粗油脂产量为31.2g/L，ARA产量为16.5g/L，ARA生产率为1.5g/(L·d)，如图3。发酵液放罐体积为83m3，整批发酵 含ARA粗油脂产量为2589.6kg，比实施例1的产量提高了0.45倍。以下表4为发酵后所得混合油脂脂肪酸组成气相分析结果。
表4：实施例4所得混合油脂的脂肪酸组成
脂肪酸组成 含量％
C14：0 0.58
C15：0 0.18
C16：0 15.21
C17：0 0.76
C18：0 14.94
C18：1 5.29
C18：2 4.45
C18：3 4.15
C20：0 1.56
C20：4 52.88
实施例5
将高山被孢霉(Mortierella alpine)斜面保藏菌株接入装有400mL培养基的2L摇瓶，在25℃的温度下以200rpm的转速培养24h，完成菌株活化培养。按照0.4％的接种量将摇瓶种子液接入装有灭菌后培养基的一级种子罐中，在培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速50rpm的条件下培养30h，完成一级种子扩大培养。按照3％的接种量将一级种子罐的种子液接入装有灭菌后培养基的二级种子罐中，在培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速75rpm的条件下培养24h，完成二级种子扩大培养。按照3％的接种量将二级种子罐的种子液接入装有灭菌后培养基的发酵罐中。
发酵过程的通气量1vvm、罐压0.04MPa、搅拌转速50rpm，将120h前的罐温控制在28±1℃、120h及之后的罐温控制在22±1℃，流加含30％预处理后粗甘油的碳源，控制葡萄糖浓度在5g/L左右，进行发酵培养。发酵过程中发酵至48h将发酵罐中发酵液一分为二培养，一半发酵液留在主罐中继续培养，另一半发酵液通过压差法转移至无菌保压的副罐中培养，主副罐中分别补入适量灭菌后新鲜培养基或无菌水， 分罐培养后主副罐通气量、罐压及搅拌转速均稍作调整。培养9d后，中止发酵，主副罐均通过放料管路筛网滤去发酵液，重新加入等体积一次水，稍开搅拌将菌体重悬后，停搅拌，停止通气，维持罐温22±1℃，流加碳源控制主副罐糖点在2g/L左右，静置2d，刺激高山被孢霉菌体中短链及低碳脂肪酸往ARA转化积累，提高菌体中ARA含量。发酵过程中检测发酵液葡萄糖浓度、pH、菌体生物量、粗油脂产量及ARA产量变化。
培养11d后，测定发酵液中生物量为54g/L，粗油脂产量为34.02g/L，ARA产量为20.1g/L，ARA生产率为1.82g/(L·d)，如图3。发酵液放罐体积为146m3，整批发酵含ARA粗油脂产量为4966.92kg，比实施例1的产量提高了1.78倍。以下表5为发酵后所得混合油脂脂肪酸组成气相分析结果。
表5：实施例5所得混合油脂脂肪酸组成
脂肪酸组成 含量％
C14：0 0.45
C15：0 0.11
C16：0 14.22
C17：0 0.45
C18：0 10.46
C18：1 5.28
C18：2 4.16
C18：3 4.37
C20：0 1.42
C20：4 59.08
以下的实施例6-10和对比例1涉及提取ARA毛油
实施例6
取实施例5得到的ARA发酵液200L，经加热灭活处理后，用布料器将发酵液运至布料腔中，进行布料包裹，布料完毕后，进行一级压榨，采用逐步加压的方式，在 设定的2h内达到设定的30MPa压力，保压2h到基本没有水滴流出。去掉一级压榨笼，换上二级压榨笼，推入二级压榨处，进行二级压榨，采用逐步加压的方式，在设定的2h内达到设定的100MPa压力，保压2h到基本没有油滴流出，收集压榨出来的ARA毛油，共得到ARA毛油6.0kg，发酵液至毛油收率88.2％。去掉二级压榨笼，将压完的菌渣与滤布进行分离。
实施例7
取实施例5得到的ARA发酵液200L，经加热灭活处理后，用布料器将发酵液运至布料腔中，进行布料包裹，布料完毕后，进行一级压榨，采用逐步加压的方式，在设定的5h内达到设定的40MPa压力，保压4h到基本没有水滴流出。去掉一级压榨笼，换上二级压榨笼，推入二级压榨处，进行二级压榨，采用逐步加压的方式，在设定的5h内达到设定的150MPa压力，保压4h到基本没有油滴流出，收集压榨出来的ARA毛油，共得到ARA毛油6.2kg，发酵液至毛油收率91.1％。去掉二级压榨笼，将压完的菌渣与滤布进行分离。
实施例8
取实施例5得到的ARA发酵液300L，经加热灭活处理后，采用蝶式离心机离心，去除离心轻液，得到145L浓缩后的发酵液，用布料器将浓缩后的发酵液运至布料腔中，进行布料包裹，布料完毕后，进行一级压榨，采用逐步加压的方式，在设定的5h内达到设定的40MPa压力，保压4h到基本没有水滴流出。去掉一级压榨笼，换上二级压榨笼，推入二级压榨处，进行二级压榨，采用逐步加压的方式，在设定的5h内达到设定的150MPa压力，保压4h到基本没有油滴流出，收集压榨出来的ARA毛油，共得到ARA毛油9.4kg，发酵液至毛油收率92.1％。去掉二级压榨笼，将压完的菌渣与滤布进行分离。
实施例9
取实施例5得到的ARA发酵液1000L，经加热灭活处理后，进入板框压滤机进行过滤，过滤至进料压力达到0.7MPa，过滤完毕后，用压缩空气进行吹气，吹气压力0.4MPa，吹气2h，吹气完毕后，拆卸板框，得到滤泥。设定气流干燥进风温度120℃， 出风温度40℃，滤泥进入气流干燥，得到ARA菌粉58.1kg。ARA菌粉运至布料腔中，进行布料包裹，布料完毕后，进行柔性压榨，采用逐步加压的方式，在设定的2h内达到设定的100MPa压力，保压2h到基本没有油滴流出，收集压榨出来的ARA毛油，共得到ARA毛油31.8kg，发酵液至毛油收率93.5％。
实施例10
取实施例5得到的ARA发酵液1000L，经加热灭活处理后，进入板框压滤机进行过滤，过滤至进料压力达到1.0MPa，进料完毕后，用压缩空气进行吹气，吹气压力0.6MPa，吹气1h，吹气完毕后，拆卸板框，得到滤泥。设定气流干燥进风温度140℃，出风温度50℃，滤泥进入气流干燥，得到ARA菌粉55.8kg。ARA菌粉运至布料腔中，进行布料包裹，布料完毕后，进行柔性压榨，采用逐步加压的方式，在设定的4h内达到设定的150MPa压力，保压4h到基本没有油滴流出，收集压榨出来的ARA毛油，共得到ARA毛油32.5kg，发酵液至毛油收率95.5％。
对比例1
取实施例5得到的ARA发酵液1000L，经加热灭活处理后，进入板框压滤机进行过滤，过滤至进料压力达到1.0MPa，进料完毕后，用压缩空气进行吹气，吹气压力0.6MPa，吹气1h，吹气完毕后，拆卸板框，得到滤泥。设定气流干燥进风温度140℃，出风温度50℃，滤泥进入气流干燥，得到ARA菌粉55.6kg。双螺杆压榨机先预热至80℃，将ARA菌粉加入双螺杆压榨机中榨油，得到ARA毛油18.2kg，发酵液至毛油收率53.5％。
可见，本发明的提取ARA毛油的方法的毛油收率显著高于现有方法。
以下的实施例11-14和对比例2涉及纯化(精炼)ARA毛油
实施例11
取10kg实施例6、7、8合并所得的ARA毛油，按水化、脱色、分子蒸馏步骤进行精炼。
水化：10kg ARA毛油，升温至至75℃，加入1kg温度为80℃的纯化水，搅拌 30min，搅拌速度30转/min，静置2h，分去下层，得到水化油9.76kg。
脱色：水化油升温至100℃，控制真空度-0.075MPa，真空脱水1h，然后降温至70℃，加入脱色剂(191g活性炭和286g活性白土)，搅拌脱色0.5h，停止搅拌，过滤去除脱色剂，得到脱色油9.33kg。
分子蒸馏：脱色油进入三级分子蒸馏，控制第一级真空度90Pa左右、温度160℃，去除第一级的轻组分，重组分进入第二级分子蒸馏，控制二级真空度40Pa左右、温度200℃，去除第二级轻组分，重组分进入第三级分子蒸馏，控制第三级真空度3Pa左右、温度220℃，去除第三级轻组分，收集重组分，脱臭完毕，降温至30℃，添加抗氧化剂，包装，得到ARA成品油9.13kg，检验结果见表6。
表6：ARA成品油检验结果
Figure PCTCN2017087749-appb-000001
实施例12
取10kg实施例6、7、8合并所得的ARA毛油，按水化、脱色、分子蒸馏步骤进行精炼。
水化：10kg ARA毛油，升温至至85℃，加入1kg温度为90℃的纯化水，搅拌15min，搅拌速度90转/min，静置4h，分去下层，得到水化油9.81kg。
脱色：水化油升温至110℃，控制真空度-0.075MPa，真空脱水0.5h，然后降温至80℃，加入脱色剂(192g活性炭和288g活性白土)，搅拌脱色1h，停止搅拌，过滤去除脱色剂，得到脱色油9.39kg。
分子蒸馏：脱色油进入三级分子蒸馏，控制第一级真空度90Pa左右、温度200℃，去除第一级的轻组分，重组分进入第二级分子蒸馏，控制二级真空度40Pa左右、温度220℃，去除第二级轻组分，重组分进入第三级分子蒸馏，控制第三级真空度3Pa左右、温度250℃，去除第三级轻组分，收集重组分，分子蒸馏完毕，降温至30℃，添加抗氧化剂，包装，得到ARA成品油9.08kg，检验结果见表7。
表7：ARA成品油检验结果
Figure PCTCN2017087749-appb-000002
Figure PCTCN2017087749-appb-000003
实施例13
取10kg实施例9、10合并所得的ARA毛油，按实施例11的精炼方法进行精炼，得到ARA成品油9.20kg，检验结果见表8。
表8：ARA成品油检验结果
Figure PCTCN2017087749-appb-000004
实施例14
取10kg实施例9、10合并所得的ARA毛油，按实施例12的精炼方法进行精炼，得到ARA成品油9.13kg，检验结果见表9。
表9：ARA成品油检验结果
Figure PCTCN2017087749-appb-000005
对比例2
取10kg实施例9、10合并所得的ARA毛油，按传统精炼方法即水化、碱炼、脱色、脱臭步骤进行精炼。
水化：10kg ARA毛油，升温至至85℃，加入1kg温度为90℃的纯化水，搅拌15min，搅拌速度90转/min，静置4h，分去下层，得到水化油9.80kg。
碱炼：水化油保温在75℃，加入1L浓度10％(质量分数)的NaOH溶液，搅拌30min，搅拌速度30转/min，静置4h，分离掉皂脚，得到碱炼油，碱炼油搅拌情况下喷洒入油重10％的80℃纯化水进行水洗，加水时间控制在10-30min，加完水后采用静置2h，分离水层，重复洗涤2次，得到碱炼油9.10kg。
脱色：碱炼油升温至110℃，控制真空度-0.075MPa，真空脱水0.5h，然后降温至80℃，加入脱色剂(182g活性炭和273g活性白土)，搅拌脱色1h，停止搅拌，过滤去除脱色剂，得到脱色油8.65kg。
脱臭：脱色油移入脱臭锅，通水蒸气进行脱臭，脱臭温度控制在185℃，真空度控制在600Pa以内，脱臭时间2h，脱臭完毕，停止通蒸汽，降温至20-40℃，添加抗氧化剂，包装，得到ARA成品油8.41kg，检测结果见表10。
表10：ARA成品油检验结果
Figure PCTCN2017087749-appb-000006
Figure PCTCN2017087749-appb-000007
结果表明，本发明的纯化ARA毛油的方法能够更好地去除过氧化物和不皂化物，避免反式脂肪酸的产生，酸价也更低。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (23)

  1. 一种培养用于生产ARA的微生物的方法，其中：
    从发酵罐培养的100-140小时开始，发酵温度为20-25℃；优选地，直至发酵结束；
    和/或
    从发酵罐培养的第8-10天开始，停止通气或者将通气量减少50％以上；优选地，直至发酵结束。
  2. 根据权利要求1所述的培养方法，其中：
    从发酵罐培养的105-135小时、110-130小时、115-125小时或者120小时开始，发酵温度为20-25℃，和/或
    从发酵罐培养的第8天、第9天或者第10天开始，停止通气或者将通气量减少50％以上。
  3. 根据权利要求1或2所述的培养方法，其中，在发酵温度为20-25℃之前，发酵温度为25-30℃。
  4. 根据权利要求1至3中任一权利要求所述的培养方法，其中，所述发酵液的pH为6.0-7.0；
    优选地，在停止通气或者将通气量减少50％以上之前，发酵液中葡萄糖的浓度为1-5g/L。
  5. 根据权利要求1至4中任一权利要求所述的培养方法，其中，在停止通气或者将通气量减少50％以上的同时或之后，发酵液中葡萄糖的浓度为1-2g/L；
    优选地，在停止通气或者将通气量减少50％以上的同时或之后，发酵温度为20-25℃；
    优选地，在停止通气或者将通气量减少50％以上的同时或之后，停止搅拌；
    优选地，在停止通气或者将通气量减少50％以上之前，滤去发酵液，并加入适量 水或新鲜的发酵培养基。
  6. 根据权利要求1至5中任一权利要求所述的培养方法，其中，所述发酵罐培养还包括在发酵罐培养起始的36-60小时(例如48小时)，进行分罐培养的步骤；例如，分为两个或更多个发酵罐进行发酵罐培养。
  7. 根据权利要求1至6中任一权利要求所述的培养方法，其在发酵罐培养之前，还包括接种和种子扩大培养的步骤；
    优选地，所述种子扩大培养包括一级种子扩大培养和二级种子扩大培养；
    优选地，所述一级种子扩大培养包括下述步骤：
    按照0.4％-1％的接种量将摇瓶种子液接入装有灭菌后培养基的一级种子罐中，培养温度25-32℃，通气量1-2vvm，罐压0.02-0.05MPa，培养30-35h，完成一级种子扩大培养；
    优选地，所述二级种子扩大培养包括下述步骤：
    按照1％-3％的接种量将一级种子罐的种子液接入装有灭菌后培养基的二级种子罐中，培养温度25-32℃，通气量1-2vvm，罐压0.02-0.05MPa，培养20-25h，完成二级种子扩大培养。
  8. 根据权利要求7所述的培养方法，还包括在接种和种子扩大培养之前，进行活化培养的步骤；优选地，所述活化培养的温度为25-32℃，搅拌转速100-200rpm，时间为40-48h。
  9. 根据权利要求7或8所述的培养方法，其中，所述发酵罐培养包括下述步骤：
    按照1％-3％的接种量将二级种子罐的种子液接入装有灭菌后培养基的发酵罐中，培养温度20-30℃，通气量1-2vvm，罐压0.02-0.05MPa，搅拌转速0-50rpm，进行发酵罐培养。
  10. 根据权利要求1至9中任一权利要求所述的培养方法，其中用于生产ARA的微生物为高山被孢霉或其突变菌株；优选地，所述高山被孢霉选自保藏号为CCTCC No. M2012073、CCTCC No.M2013392、CCTCC No.M2015421和ATCC No.42430的菌株。
  11. 一种微生物的发酵培养物，其由权利要求1至10中任一权利要求所述的培养方法得到。
  12. 一种提取ARA毛油的方法，包括如下步骤：
    1)将用于生产ARA的微生物的发酵培养物进行脱水处理；
    2)将步骤1)的产物进行柔性压榨，得到ARA毛油。
  13. 根据权利要求12所述的提取方法，其中，步骤1)中，所述发酵培养物为高山被孢霉发酵培养物；优选地，所述发酵培养物为权利要求11所述的发酵培养物。
  14. 根据权利要求12所述的提取方法，其中，步骤1)中，所述脱水处理选自如下的任意一种、两种或三种：
    离心或板框过滤、第一级柔性压榨、干燥例如气流干燥；
    优选地，所述脱水处理依次包括离心和第一级柔性压榨，或者依次包括板框过滤和气流干燥；
    优选地，所述气流干燥的进风温度110-150℃，出风温度30-70℃，至水分含量小于10％。
  15. 根据权利要求12所述的提取方法，其中，步骤1)中，所述第一级柔性压榨采用逐步加压方式，设定压力为20-40MPa，加压时间为1-6h，到达设定压力后保压1-4h。
  16. 根据权利要求12所述的提取方法，其中，步骤2)中，所述柔性压榨采用逐步加压方式，设定压力为50-150MPa，加压时间为1-6h，到达设定压力后保压1-4h。
  17. 一种ARA毛油，其由权利要求12至16中任一权利要求所述的提取方法制得。
  18. 一种纯化ARA毛油的方法，包括将ARA毛油进行水化、脱色和分子蒸馏的步骤；优选地，所述ARA毛油为权利要求17所述的ARA毛油。
  19. 根据权利要求18所述的纯化方法，其中，所述水化包括如下步骤：
    将ARA毛油加热至70-85℃，按1kg毛油加入50-150g水的比例加入75-90℃水，搅拌10-60min，搅拌速度30-90转/min，静置1-6h，分层去掉下层磷脂，得到水化油。
  20. 根据权利要求18所述的纯化方法，其中，所述脱色包括如下步骤：
    将水化产物升温至90-110℃，控制真空度≤-0.07MPa，真空脱水0.5-2h，然后降温至60-80℃，加入脱色剂(例如水化油重量1％-3％的活性炭和2％-4％活性白土)，搅拌0.5-1h，停止搅拌，过滤去除脱色剂，得到脱色油。
  21. 根据权利要求18所述的纯化方法，其中，所述分子蒸馏为三级分子蒸馏；
    优选地，所述分子蒸馏包括如下步骤：
    将脱色油进入三级分子蒸馏，控制第一级真空度≤100Pa、温度150-200℃，去除第一级的轻组分；得到的第一重组分进入第二级分子蒸馏，控制二级真空度≤50Pa、温度180-220℃，去除第二级轻组分；得到的第二重组分进入第三级分子蒸馏，控制第三级真空度≤5Pa、温度200-250℃，去除第三级轻组分，得到第三重组分，为ARA成品油。
    优选地，重复分子蒸馏1次或多次。
  22. 一种ARA成品油，其由权利要求18至21中任一权利要求所述的纯化方法制得。
  23. 一种利用微生物生产ARA或含有ARA的产品(例如ARA成品油)的方法，包括：
    权利要求1至10中任一权利要求所述的培养用于生产ARA的微生物的方法、
    权利要求12至16中任一权利要求所述的提取ARA毛油的方法、和/或
    权利要求18至21中任一权利要求所述的纯化ARA毛油的方法。
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