WO2018120574A1 - 一种利用微生物发酵生产dha的方法 - Google Patents

一种利用微生物发酵生产dha的方法 Download PDF

Info

Publication number
WO2018120574A1
WO2018120574A1 PCT/CN2017/083461 CN2017083461W WO2018120574A1 WO 2018120574 A1 WO2018120574 A1 WO 2018120574A1 CN 2017083461 W CN2017083461 W CN 2017083461W WO 2018120574 A1 WO2018120574 A1 WO 2018120574A1
Authority
WO
WIPO (PCT)
Prior art keywords
culture
dha
fermentation
oil
hours
Prior art date
Application number
PCT/CN2017/083461
Other languages
English (en)
French (fr)
Inventor
王炳荣
文昌
陈必钦
张斌
韩雯
庞毅
李丹
詹光煌
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
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 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 filed Critical 内蒙古金达威药业有限公司
Publication of WO2018120574A1 publication Critical patent/WO2018120574A1/zh

Links

Images

Classifications

    • 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
    • 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
    • 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
    • C12P7/6434—Docosahexenoic acids [DHA]

Definitions

  • the invention belongs to the field of fermentation engineering, and relates to a method for producing DHA by microbial fermentation, in particular to a method for industrially fermenting a compound containing docosahexahydrate (DHA) by using a Schizochytrium strain.
  • DHA docosahexahydrate
  • DHA the full name doc-4,7,10,13,16,19-docosahexaenoic acid, DHA
  • DHA is a polyunsaturated fatty acid.
  • the human body is difficult to synthesize itself and must be taken from the outside world.
  • DHA is one of the essential fatty acids in the human body. It has important physiological regulation functions and health care functions. When it is lacking, it will cause a series of diseases, including growth retardation, skin abnormalities, scales, infertility, mental retardation, etc. Special preventive and therapeutic effects. Studies have also shown that DHA can act on many different types of tissues and cells, inhibit inflammation and immune function, including reducing the production of inflammatory factors, inhibit lymphocyte proliferation, etc. DHA also has multiple effects in preventing Alzheimer's disease and neurological diseases. .
  • DHA The current commercial sources of DHA are mainly fish oil and microalgae.
  • DHA extracted from traditional deep-sea fish oil is unstable due to the variety, season and geographical location of fish, and the content of cholesterol and other unsaturated fatty acids is high.
  • the difference in length and degree of unsaturation of fatty acid chains is large, resulting in limited production and content of DHA. It is not high, it is difficult to separate and purify, and the cost is high.
  • With the growing shortage of fish oil raw materials it is difficult to achieve the widespread use of DHA, a high value-added product in the food and pharmaceutical industries.
  • the production of DHA by microbial fermentation can overcome the defects of traditional fish oil extraction, can be used for mass production of DHA, continuously meet people's needs, has broad application prospects, and has attracted the attention of scholars at home and abroad.
  • the microbial fermentation method uses fermented microorganisms such as fungi and microalgae to produce DHA-containing algae oil, and refined to obtain essential oil with high DHA content.
  • DHA-producing strains approved by the Ministry of Health include Schizochytrium sp., Ulkenia amoeboida, and Crypthecodinium cohnii.
  • the publication No. CN103882072A discloses a method for producing docosahexaenoic acid by using Schizochytrium, and the highest yield disclosed is a cell dry weight of 61.2 g/L, a DHA content of 55.07%, and a DHA yield of 22.17 g. /L.
  • the publication No. CN101812484A discloses a method for fermentative production of DHA by high-density culture of Schizochytrium, which discloses a dry cell weight of 120-150 g/L and a DHA yield of 26-30 g/L, which is also reported. Mining The highest production level of DHA was produced by Schizochytrium sp.
  • Centrifugation such as the publication No. CN101817738B, discloses a method for extracting DHA from algae and fungal cells by separating the microalgae or fungal fermentation broth after fermentation by a separation system to collect cells, and adjusting the pH of the sludge with acid.
  • the invention adopts physical wall breaking and physical extraction methods, has simple process, high cell breakage, low temperature treatment of bacteria sludge and antioxidant treatment, can effectively protect the biological activity of algae and fungal cells, and the product is green and non-toxic. Residual. However, the quality of the oil layer after centrifugation of the invention is poor.
  • the organic solvent extraction method discloses a method for extracting docosahexaenoic acid oil: the fermentation liquid containing docosahexaenoic acid is subjected to enzymatic breaking, and then an organic solvent is used first. The first stage water is divided, the cells are enriched, and the organic solvent is used for secondary extraction to obtain a crude oil.
  • the method is simple in operation and low in equipment investment, but the method uses organic solvent for extraction, and the final product may have solvent residue, and the extraction process has safety hazards such as flammability and explosion.
  • the supercritical extraction method as disclosed in the publication No. CN102181320B, discloses a method for extracting bio-fermented DHA algae oil, comprising the following steps: a) drying the solid matter obtained by solid-liquid separation of the microalgae fermentation liquid to obtain a dried bacterial cell; b) extracting the dried cells with supercritical carbon dioxide as an extracting agent to obtain a carbon dioxide fluid; c) separating the carbon dioxide fluid under reduced pressure to obtain DHA algae oil.
  • the refining of DHA hair oil is mostly carried out by chemical refining technology, and the DHA hair oil is degummed, alkali refining, decolorized and deodorized to obtain DHA essential oil.
  • 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 conducted intensive research and creative labor to obtain a method of cultivating microorganisms for producing DHA.
  • the inventors have surprisingly found that the culture method can greatly increase biomass and DHA production. Further, the inventors have also found a method of extracting DHA hair oil, which can improve the extraction yield of DHA hair oil. Further, the inventors have also found a method for purifying DHA hair oil, which can improve various technical indexes and purification yields of DHA refined oil.
  • the present invention significantly improves the crude oil production, DHA production, and DHA productivity of DHA. The following invention is thus provided:
  • One aspect of the invention relates to a method of cultivating a microorganism for producing DHA (docosahexaenoic acid), wherein:
  • the dissolved oxygen saturation (DO) is controlled at 5%-10% (eg 5%, 5.5%, 6%, 6.5%, 7%) , 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%); and/or
  • the nitrogen source is no longer added or the amount of nitrogen source added 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:
  • 40-56 hours of culture from the fermenter eg 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 or 56 hours
  • 44-52 Start at 46-50 hours or 48 hours, control dissolved oxygen at 5%-10%, and/or
  • the culture method wherein the dissolved oxygen saturation is 30%-50% (for example, 30%-45%, 35) before controlling the dissolved oxygen saturation to be 5%-10%. %-45%, 40%-45%, 40%-50%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44% or 45%) .
  • the culture method wherein the pH of the fermentation broth is 6.0- 7.0.
  • the culture method wherein the concentration of glucose in the fermentation broth is maintained at 1-5 g/L (for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 g/L) ).
  • the culture method wherein the nitrogen source is ammonia water, preferably 25%-45% ammonia water, more preferably 35%-45% or 38%-42% ammonia water. Particularly preferred is 40% aqueous ammonia.
  • the culture method wherein the fermenter culture further comprises 12-36 hours (eg, 16-32 hours, 18-30 hours, 20-28) of the start of the fermentation tank culture.
  • the hour, 22-26 hours, 24 hours) the step of performing the tank culture; for example, the fermenter culture is carried out by dividing into two or more fermentors.
  • 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°C-32°C, the ventilation volume is 1-2vvm, the tank pressure is 0.02-0.05MPa. , the stirring speed is 50-100 rpm, and the culture is carried out for 30-35 hours to 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 ° C - 32 ° C, the ventilation volume is 1-2 vvm, the tank pressure is 0.02. -0.05 MPa, stirring speed 50-100 rpm, culture 20-25h, complete secondary seed expansion culture.
  • the culture method further comprises the step of activating the culture before the inoculation and before the expanding the culture; preferably, the temperature of the activated culture is 25° C. to 32° C., and the time is 20 -25h.
  • the culture method, wherein the fermentor culture comprises the following steps:
  • the seed liquid of the secondary seed tank is connected to the fermenter containing the sterilized medium, the culture temperature is 25°C-32°C, the ventilation volume is 1-2vvm, and the tank pressure is 0.02-0.05. Fermentor culture was carried out at MPa, stirring speed of 50-100 rpm.
  • the culture method wherein the microorganism for producing DHA is Schizochytrium sp.; preferably, the Schizochytrium is selected from the group of CGMCC No. 6843 , ATCC No. 20888, ATCC No. 20889, ATCC No. 28209 or ATCC MYA-1381 strain.
  • the culture method comprises the following steps:
  • the Schizochytrium sp. strained strain was inserted into a 2 L shake flask containing 400 mL of seed culture medium, and cultured at a temperature of 25 ° C to 32 ° C at a rotation speed of 200 rpm for 20-25 h to complete the activated culture of the strain;
  • the shake flask seed solution is connected to the first-stage seed tank containing the sterilized seed culture medium, the culture temperature is 25 ° C - 32 ° C, the ventilation volume is 1-2 vvm, the tank pressure is 0.02 - 0.05MPa, stirring speed 50-100rpm, culture 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 seed culture medium after sterilization, the culture temperature is 25 ° C -32 ° C, the ventilation volume is 1-2 vvm, The tank pressure is 0.02-0.05 MPa, the stirring speed is 50-100 rpm, and the culture is carried out for 20-25 hours to complete the secondary seed expansion culture;
  • the seed liquid of the secondary seed tank is connected to the fermenter containing the fermentation medium after sterilization, the culture temperature is 25 ° C -32 ° C, the ventilation volume is 1-2 vvm, the tank pressure Fermentation tank culture is carried out at 0.02-0.05 MPa and stirring speed of 50-100 rpm;
  • the fermenter is cultured for 24 hours, the fermentation broth in the fermenter is divided into two by volume, half of the fermentation broth is left in the main tank to continue the culture, and the other half of the fermentation broth is transferred to the aseptically held auxiliary tank by differential pressure method.
  • the main and auxiliary tanks are respectively filled with appropriate amount of fresh medium or sterile water after sterilization, and the main and auxiliary tank ventilation, tank pressure and stirring speed are adjusted according to step 5);
  • the 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;
  • the formulation of the seed medium used in the above steps 1) to 3) and the formulation of the fermentation medium used in the above step 4) are conventional formulations known to those skilled in the art.
  • the seed medium formula is: glucose 3%, peptone 1%, yeast powder 0.5%, sea crystal 2%, pH natural.
  • Fermentation medium formula glucose 12%, peptone 1%, yeast powder 0.5%, sea crystal 2%, pH 6.5 (process research of DHA produced by Schizochytrium and high-yield strain selection, Wang Shenqiang, etc., Jiangnan University Master thesis , 2013, p13-14).
  • the above-mentioned fermentation medium carbon source is added with 20% to 40% (mass of crude glycerin / mass of carbon source ⁇ 100%) of the 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 sugar spot (concentration of glucose) is measured by a method known to those skilled in the art, for example, by a biosensor.
  • the DO of the first 48 h of the fermenter culture was controlled at 30%-50%, and the DO was controlled at 5%-10% after the fermentation tank was cultured for 48 h.
  • DO was used as a control index to adjust the aeration volume and the tank pressure. And stir the rotation speed, and properly add fresh medium or sterile water after sterilization.
  • the fermenter is cultured for 24 hours, and the fermentation liquid in the fermenter is divided into two by volume, half of the fermentation liquid is left in the main tank to continue the cultivation, and the other half of the fermentation liquid is transferred to the aseptic pressure by the differential pressure method.
  • the main and auxiliary tanks are filled with appropriate amount of fresh medium or sterile water after sterilization. After the tank is cultured, the main and auxiliary tanks are ventilated, the tank pressure and the stirring speed are all in the first 48 hours of the fermenter culture. After adjusting for 48 hours with the fermenter, adjust accordingly.
  • the carbon source is added during the above fermentation to maintain the sugar point (glucose concentration) in the fermentation liquid at 1-5 g/L.
  • 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 extract, peptone, ammonia water, sodium nitrate, sodium glutamate, sulfuric acid One or more of ammonium Kind.
  • 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.
  • the strain Schizochytrium sp. used in the above fermentation may be various fungi classified into the genus Schizochytrium, specifically including but not limited to Schizochytrium sp. CGMCC No. 6843, Schizochytrium sp. ATCC No. 20888, Schizochytrium sp. ATCC No. 20889, Schizochytrium sp. ATCC No. 28209, Schizochytrium limacinum Honda et Yokochi ATCCMYA-1381, and the source thereof is also not particularly limited, and may be from a fermentation institute or a microorganism such as ATCC, CGMCC, CCTCC, or the like.
  • the Schizochytrium sp. strain obtained from a natural environment by a well-known screening method can also be obtained by the depository authority.
  • Another aspect of the invention relates to a microbial fermentation broth obtained by the culture method according to any one of the inventions.
  • the microbial fermentation broth is a Schizochytrium fermentation broth.
  • a further aspect of the invention relates to a method of extracting DHA hair oil, comprising the steps of:
  • step 2) The product of step 1) is subjected to a flexible press to obtain DHA hair oil.
  • the extraction method wherein, in the step 1), the fermentation liquid is a Schizochytrium fermentation broth; preferably, the fermentation liquid is the microorganism fermentation broth of the present invention.
  • the extraction method wherein, in the step 1), the dehydration 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 centrifugation and spray drying;
  • the spray drying spray pressure is 4-8 MPa
  • the inlet air temperature is 160 ° C - 220 ° C
  • the outlet air temperature is 80 ° C - 120 ° C.
  • 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, the pressurization time is 1-6 h, and the setting is reached. After constant pressure, keep pressure for 1-4h.
  • the extraction method comprises the following steps:
  • Cloth A certain amount of DHA fermentation broth is transported to the cloth cavity by the distributor, and the hopper returns to the initial position, waiting for the next fabric.
  • 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 1-6h, the set pressure is reached, the pressure is maintained for 1-4h, and basically no water droplets flow out, ending After that, the material is lowered into the heavy pressure chamber and the material is pushed into the secondary flexible press position.
  • Two-stage flexible press adopt the stepwise pressurization method, the final pressure of the press is 50-150MPa, the pressurizing time is 1-6h, the set pressure is reached, the pressure is maintained for 1-4h until the oil-free drip is basically discharged, and the press is collected. DHA oil, pressure relief, remove the secondary press cage, and separate the microalgae from the filter cloth.
  • the above DHA fermentation liquid can be directly subjected to one-stage flexible pressing, or a part of water can be removed by centrifugation first, and the solid content of the fermentation liquid 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 DHA hair oil extraction method can also be adopted as follows: DHA fermentation liquid is spray-dried to obtain microalgae powder (refers to the dried bacteria body obtained after the bacteria are dried and removed, and the appearance is a granular powder, which is called microalgae powder. ), the microalgae powder is subjected to a flexible press (conditions equivalent to the previous two-stage flexible press) to obtain DHA hair oil.
  • microalgae powder refers to the dried bacteria body obtained after the bacteria are dried and removed, and the appearance is a granular powder, which is called microalgae powder.
  • the microalgae powder is subjected to a flexible press (conditions equivalent to the previous two-stage flexible press) to obtain DHA hair oil.
  • the spray drying spray pressure is 4-8 MPa
  • the inlet air temperature is 160 ° C-220 ° C
  • the outlet air temperature is 80 ° C - 120 ° C
  • the DHA microalgae powder moisture is controlled within 10%.
  • the DHA fermentation broth can be directly spray-dried, or a part of the water can be removed by centrifugation before the spray drying, and the solid content of the fermentation broth can be increased and then spray-dried to increase the productivity and save energy.
  • the fermentation liquid centrifugation method may be carried out by one of a horizontal spiral centrifuge, a disc centrifuge, and a tubular centrifuge.
  • the above flexible press comprises the following steps:
  • Cloth A certain amount of DHA microalgae powder is transported to the cloth cavity by the distributor, and the distributor returns to the initial position, waiting for the next fabric.
  • a further aspect of the invention relates to a DHA hair oil produced by the extraction method of any of the inventions.
  • a further aspect of the invention relates to a method of purifying DHA hair oil comprising the steps of hydrating, decolorizing and molecularly distilling DHA hair oil; preferably, the DHA hair oil is a DHA hair oil of the invention.
  • the purification method wherein the hydrating comprises the steps of:
  • DHA hair oil is heated to 70 ° C -85 ° C, according to the ratio of 1 kg of wool added 50-150g water, 75 ° C -90 ° C water, stirring 10-60min, stirring speed 30-90 rev / min, standing 1-6h
  • the layer of phospholipids is removed in layers to obtain a hydrated oil.
  • the purification method, wherein the discoloration comprises the following steps:
  • the hydration product is heated to 90 ° C -110 ° C, the control vacuum degree ⁇ -0.07MPa, vacuum dehydration 0.5-2h, then cooled to 60 ° C -80 ° C, adding decolorizing agent (such as 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.
  • decolorizing agent such as 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 ° C to 200 ° C to remove the light component of the first stage; the first heavy component obtained enters the second stage molecular distillation, and the second stage is controlled. Vacuum degree ⁇ 50Pa, temperature 180°C-220°C, removing the second-stage light component; the second heavy component obtained enters the third-stage molecular distillation, and controls the third-stage vacuum degree ⁇ 5Pa, temperature 200°C-250°C, and removes The third grade light component, the third heavy component, is the DHA refined oil.
  • the molecular distillation is repeated one or more times.
  • the purification method comprises the steps of:
  • DHA hair oil is refined by hydration, decolorization, molecular distillation and other steps to finally obtain DHA refined oil.
  • the specific steps for refining are as follows:
  • DHA oil is heated to 70 ° C -85 ° C, added to the ratio of 50 kg to 150 g of 1 kg of wool oil added to 75 ° C -90 ° C water, stirring 10-60min, stirring speed 30-90 rev / min, standing 1-6h, layering off the lower layer of phospholipids, to obtain hydration oil.
  • Decolorization Hydration oil is transferred into the decolorizing pot, heated to 90 ° C -110 ° C, controlled vacuum ⁇ -0.07 MPa, vacuum dehydrated 0.5-2 h, then cooled to 60 ° C -80 ° C, added decolorizing agent (hydrated oil weight 1%-3% activated carbon and 2%-4% activated clay), decolorization by stirring for 0.5-1 h, stirring is stopped, and the decolorizing agent is removed by filtration to obtain a decolorizing oil.
  • decolorizing agent hydrated oil weight 1%-3% activated carbon and 2%-4% activated clay
  • the decolorized oil enters the tertiary molecular distillation, controls the first stage vacuum degree ⁇ 100Pa, the temperature 150°C-200°C, removes the light components of the first stage, and the heavy components enter the second stage molecular distillation to control the second stage.
  • heavy component enters the third-stage molecular distillation, control the third-stage vacuum ⁇ 5Pa, temperature 200°C-250°C, remove the third-level light
  • the components are collected and the heavy components are 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 ° C - 40 ° C, and the antioxidant is added and packaged to obtain DHA refined oil.
  • the water is purified water.
  • a further aspect of the invention relates to a DHA product oil obtained by the purification method of any of the inventions.
  • a further aspect of the invention relates to a method of producing DHA or a product containing DHA, such as a DHA refined oil, comprising:
  • a method for cultivating a microorganism for producing DHA according to any one of the present invention is to cultivating a microorganism for producing DHA according to any one of the present invention.
  • a method for extracting DHA hair oil according to any one of the present invention, and/or
  • a method of purifying DHA hair oil according to any one of the inventions.
  • the production method is illustrated in FIG.
  • the production method is as shown in FIG.
  • 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 referred to the "Chinese Pharmacopoeia” Provisions.
  • DHA hair oil refers to a primary oil that has been prepared from a DHA fermentation broth without undergoing refining.
  • DHA refined oil means an essential oil obtained by refining DHA 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.
  • the invention provides a method for industrially producing docosahexaenoic acid oil, which can produce green high quality DHA grease with low cost and high output.
  • the technical indexes of the invention are obviously superior to the existing technical indexes, and the obtained DHA oil has high yield and high purity, which is favorable for large-scale industrial production of DHA, and the addition of crude glycerin also reduces the production cost of DHA fermentation. Significantly increased the market competitiveness of DHA fermentation production.
  • the invention adopts the flexible pressing process to prepare DHA 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 removed by the first-stage flexible pressing of the fermentation broth is basically free of microbial residue, the COD is relatively low, and it is easy to be biochemically treated.
  • the second-stage flexible pressing obtains the oil, the remaining microalgae also contain 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 DHA refined oil according to an embodiment of the present invention.
  • FIG. 2 is a schematic view showing a process flow for producing DHA refined oil according to another embodiment of the present invention.
  • Figure 3 Results of DHA production by fermentation of a 100 m 3 fermentor in different culture modes using Schizochytrium sp. CGMCC No. 6843.
  • the biomass is determined by placing an appropriate amount of the fermentation broth in a flat weighing bottle, drying it in an electric oven at 105 ° C for 4 hours, then placing it in a desiccator to cool to room temperature, weighing it, subtracting the weight of the weighing bottle, and then Divided by the volume of the fermentation broth, the resulting value is biomass, in g/L.
  • Determination method of crude oil production take a certain volume of fermentation broth, 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, take the upper organic phase with a dropper
  • To the eggplant bottle extract continuously for 5-8 times until the upper organic phase is colorless, remove the n-hexane by rotary evaporation in a water bath at 80 ° C, then place the eggplant bottle in a 105 ° C electric heating oven for 1 h, put in The dryer is cooled to room temperature, weighed, the weight of the eggplant bottle is subtracted, and the volume of the fermentation broth is divided, and the obtained value is the crude oil production in g/L.
  • the DHA yield is: the content of DHA in the crude oil and fat by gas chromatography, multiplied by the crude oil production, in g/L.
  • the DHA productivity is the value obtained by dividing the DHA yield by the fermentation cycle (days) in g/(L ⁇ d).
  • the method for analyzing fatty acid composition and the method for detecting DHA refined oil in the present invention are based on GB26400-2011 national standard for food safety, food additive docosahexaenoic acid oil (fermentation method).
  • the seed medium formulations used were: glucose 3%, peptone 1%, yeast powder 0.5%, sea crystal 2%, and pH natural (the rest being water).
  • the fermentation medium formula is: glucose 12%, peptone 1%, yeast powder 0.5%, sea crystal 2% (the rest is water).
  • the Schizochytrium sp. ATCC 20888, Schizochytrium limacinum Hyundai et Yokochi ATCCMYA-1381, and Schizochytrium sp. CGMCC No. 6843 slope-preserved strains were respectively inserted into 400 mL of medium.
  • the 2L shake flask was cultured at a temperature of 25 ° C at a rotation speed of 200 rpm for 24 hours to complete the activated culture of the strain.
  • the shake flask seed liquid was connected to the first-stage seed tank containing the sterilized medium, and the culture temperature was 28 ° C, the aeration amount was 1 vvm, the tank pressure was 0.02 MPa, and the stirring speed was 50 rpm for 30 hours to complete the first stage. Seeds are expanded and cultured.
  • 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 amount was 1 vvm, the tank pressure was 0.02 MPa, and the stirring speed was 75 rpm for 24 hours.
  • 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 process was carried out at a temperature of 28 ° C, aeration of 1 vvm, a pressure of 0.02 MPa, a stirring speed of 75 rpm, a carbon source containing 30% of pretreated crude glycerol, a glucose concentration of 5 g/L, and a nitrogen source. Fermentation culture. During the fermentation process, the glucose concentration, pH, bacterial biomass, crude oil production and DHA yield of the fermentation broth were measured.
  • Table 1 shows the biomass, crude oil production, DHA production and DHA productivity of the three strains cultured in the original culture mode.
  • Table 2 shows the mixed fatty acid composition of the gas obtained after fermentation. Analysis results. The biomass, crude oil production and DHA production of CGMCC No.6843 are also shown in Figure 3.
  • Table 2 The composition of the mixed fats and fats obtained after fermentation in the original culture mode of the 100m 3 fermenter
  • the Schizochytrium sp. (CGMCC No. 6843) 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 liquid was connected to the first-stage seed tank containing the sterilized medium, and the culture temperature was 28 ° C, the aeration amount was 1 vvm, the tank pressure was 0.02 MPa, and the stirring speed was 50 rpm for 30 hours to complete the first stage. Seeds are expanded and cultured.
  • 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 amount was 1 vvm, the tank pressure was 0.02 MPa, and the stirring speed was 75 rpm for 24 hours.
  • Complete secondary seed expansion culture was connected to a fermentor containing the sterilized medium according to a 3% inoculum.
  • the fermentation process has a culture temperature of 28 ° C, aeration volume of 1-2 vvm, a tank pressure of 0.02-0.05 MPa, a stirring speed of 50-100 rpm, and a carbon source containing 30% of the pretreated crude glycerol, and a glucose concentration of 5 g/L.
  • the nitrogen source is supplemented and fermented.
  • the DO was controlled at 40% before fermentation and the DO was controlled at 8% after 48 hours of fermentation.
  • DO was used as the control index, the aeration volume, tank pressure and stirring speed were adjusted, and the fresh medium or sterilized after sterilization was appropriately added. water.
  • the glucose concentration, pH, bacterial biomass, crude oil production and DHA yield of the fermentation broth were measured.
  • the fermentation tank had a tank volume of 78 m 3 , and the whole batch fermentation containing DHA crude oil yield was 4391.4 kg, which was 0.67 times higher than the original culture method.
  • Table 3 below shows the results of gas phase analysis of the fatty acid composition of the mixed fat obtained after fermentation:
  • Table 3 100m 3 fatty acids, oils and fats obtained after mixing composition fermentor
  • the Schizochytrium sp. (CGMCC No. 6843) 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 connected to the first-stage seed tank containing the sterilized medium, 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 50 rpm for 30 hours to complete the first stage. Seeds are expanded and cultured.
  • 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 amount was 1 vvm, the tank pressure was 0.02 MPa, and the stirring speed was 75 rpm for 24 hours.
  • Complete secondary seed expansion culture was connected to a fermentor containing the sterilized medium according to a 3% inoculum.
  • the fermentation process was carried out at a culture temperature of 28 ° C, aeration rate of 1 vvm, a tank pressure of 0.02 MPa, a stirring speed of 75 rpm, and a carbon source containing 30% of the pretreated crude glycerol, and a glucose concentration of 5 g/L to carry out fermentation culture.
  • 40% ammonia water was added before fermentation for 48 hours to control the pH of the fermentation broth at 6.5.
  • the pH of the fermentation broth could be controlled while adding ammonia water to supplement the nitrogen source. After 48 hours, the ammonia flow was stopped and the nitrogen-free culture was carried out. The pH was no longer used. control. Detection of fermentation broth during fermentation Changes in glucose concentration, pH, bacterial biomass, crude oil production, and DHA production.
  • the fermentation was terminated after 96 hours of culture, and the biomass in the fermentation broth was 118 g/L, the crude oil production was 64.2 g/L, the DHA yield was 32.6 g/L, and the DHA productivity was 8.2 g/(L ⁇ d), as shown in FIG.
  • the fermentation tank has a tank volume of 78 m 3 , and the whole batch fermentation contains DHA crude oil with a yield of 5007.6 kg, which is 0.91 times higher than the original culture method.
  • Table 4 below shows the results of gas phase analysis of the fatty acid composition of the mixed fat obtained after fermentation:
  • Table 4 Fatty acid composition of mixed fats and oils obtained after fermentation in a 100m 3 fermentor
  • the Schizochytrium sp. (CGMCC No. 6843) 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 liquid was connected to the first-stage seed tank containing the sterilized medium, and the culture temperature was 28 ° C, the aeration amount was 1 vvm, the tank pressure was 0.02 MPa, and the stirring speed was 50 rpm for 30 hours to complete the first stage. Seeds are expanded and cultured.
  • 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 amount was 1 vvm, the tank pressure was 0.02 MPa, and the stirring speed was 75 rpm for 24 hours.
  • Complete secondary seed expansion culture was connected to a fermentor containing the sterilized medium according to a 3% inoculum.
  • the fermentation process was carried out at a temperature of 28 ° C, aeration of 1 vvm, a pressure of 0.02 MPa, a stirring speed of 75 rpm, a carbon source containing 30% of pretreated crude glycerol, a glucose concentration of 5 g/L, and a nitrogen source. Fermentation culture. Fermentation to 24h, the fermentation broth in the fermenter is divided into two by volume, half of the fermentation broth is left in the main tank to continue cultivation, and the other The semi-fermented solution is transferred to a sterile holding sub-tank by a differential pressure method, and an appropriate amount of fresh medium or sterile water is separately added to the main and auxiliary tanks. During the fermentation process, the glucose concentration, pH, bacterial biomass, crude oil production and DHA yield of the fermentation broth were measured.
  • the Schizochytrium sp. (CGMCC No. 6843) 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 liquid was connected to the first-stage seed tank containing the sterilized medium, and the culture temperature was 28 ° C, the aeration amount was 1 vvm, the tank pressure was 0.02 MPa, and the stirring speed was 50 rpm for 30 hours to complete the first stage. Seeds are expanded and cultured.
  • 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, aeration of 1vvm, canister pressure of 0.02MPa, stirring speed of 75rpm for 24h, 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 process has a culture temperature of 28 ° C, aeration volume of 1-2 vvm, a tank pressure of 0.02-0.05 MPa, a stirring speed of 50-100 rpm, a carbon source containing 30% of the pretreated crude glycerin, and a glucose concentration of 5 g/L to carry out fermentation. to cultivate.
  • the DO was controlled at 40% before fermentation for 48 hours, and the DO was controlled at 8% after 48 hours of fermentation. During the process, DO was used as the control index to adjust the aeration volume, tank pressure and stirring speed, and appropriately added to the fresh medium or sterilized after sterilization. water. In the fermentation process, 40% ammonia water was added before fermentation for 48 hours to control the pH of the fermentation broth to about 6.5.
  • the pH of the fermentation broth could be controlled while adding ammonia water to supplement the nitrogen source. After 48 hours, the ammonia solution was stopped and the nitrogen deficiency culture was carried out. The pH was no longer used. Control. Fermentation to 24h, the fermentation broth in the fermenter is divided into two by volume, 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 tank, and the main and auxiliary tank ventilation, tank pressure and stirring speed were adjusted according to the conditions before and after 48 h. During the fermentation process, the glucose concentration, pH, bacterial biomass, crude oil production and DHA yield of the fermentation broth were measured.
  • the fermentation was terminated after 96 hours of culture, and the biomass in the fermentation broth was determined to be 145 g/L, the crude oil production was 82.6 g/L, the DHA yield was 44.9 g/L, and the DHA productivity was 11.2 g/(L ⁇ d), as shown in FIG.
  • the fermentation tank has a tank volume of 130m 3 , and the whole batch fermentation contains DHA crude oil with a yield of 10738kg, which is 3.10 times higher than the original culture method. It can not only produce high DHA-containing oil, but also has high DHA content in the obtained DHA oil. Market competitiveness. Table 6 below shows the results of gas phase analysis of the fatty acid composition of the mixed fat obtained after fermentation:
  • the Schizochytrium sp. (Schizochytrium sp. ATCC 20888) was cultured in the manner of Example 5, and the fermentation was terminated after 96 hours of culture.
  • the biomass in the fermentation broth was determined to be 65 g/L, the crude oil production was 15.2 g/L, and the DHA yield was 6.5g / L, the ratio of the original culture method increased by 2.49 times, not only can produce high DHA-containing oil, but also the DHA content of the obtained DHA oil is high, and has a strong market competitiveness.
  • Table 7 below shows the gas phase analysis results of the fatty acid composition of the mixed fat obtained after fermentation:
  • Table 7 Fatty acid composition of mixed fats obtained after fermentation in a 100m 3 fermentor
  • the Schizochytrium limacinum Hyundai et Yokochi ATCC MYA-1381 was cultured according to the culture method of Example 5. After 96 hours of culture, the fermentation was terminated, and the biomass in the fermentation broth was determined to be 105 g/L, and the crude oil production was 37 g/L.
  • the yield of DHA is 17.2g/L, which is 2.81 times higher than that of the original culture method. It can not only produce high-yield DHA-containing fat, but also has high DHA content in DHA oil and has strong market competitiveness.
  • Table 8 below shows the results of gas phase analysis of the fatty acid composition of the mixed fat obtained after fermentation:
  • Table 8 Fatty acid composition of mixed fats and oils obtained after fermentation in a 100m 3 fermentor
  • the Schizochytrium sp. (CGMCC No. 6843) 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 liquid was connected to the first-stage seed tank containing the sterilized medium, and the culture temperature was 28 ° C, the aeration amount was 1 vvm, the tank pressure was 0.02 MPa, and the stirring speed was 50 rpm for 30 hours to complete the first stage. Seeds are expanded and cultured.
  • 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 amount was 1 vvm, the tank pressure was 0.02 MPa, and the stirring speed was 75 rpm for 24 hours.
  • Complete secondary seed expansion culture was connected to a fermentor containing the sterilized medium according to a 3% inoculum.
  • the fermentation process has a culture temperature of 28 ° C, aeration volume of 1-2 vvm, a tank pressure of 0.02-0.05 MPa, a stirring speed of 50-100 rpm, a carbon source containing 20% of the pretreated crude glycerin, and a glucose concentration of 1 g/L to carry out fermentation. to cultivate.
  • the DO was controlled at 30% before fermentation for 36h, and the DO was controlled at 5% after fermentation for 36h.
  • DO was used as the control index to adjust the aeration volume, tank pressure and stirring speed, and properly added to the fresh medium or sterilized after sterilization. water.
  • 25% ammonia water was added before fermentation for 36h to control the pH of the fermentation broth to about 6.
  • the pH of the fermentation broth could be controlled while adding ammonia water to supplement the nitrogen source. After 36h, the ammonia solution was stopped and the nitrogen deficiency culture was carried out. Control. Fermentation to 12h, the fermentation broth in the fermenter is divided into two by volume, half of the fermentation broth is left in the main tank to continue the culture, and the other half of the fermentation broth is transferred to the aseptically held sub-tank by differential pressure method. The tank was filled with appropriate amount of fresh medium or sterile water after sterilization. After the tank was cultured, the main and auxiliary tank ventilation, tank pressure and stirring speed were adjusted according to the conditions before and after 36 h. During the fermentation process, the glucose concentration, pH, bacterial biomass, crude oil production and DHA yield of the fermentation broth were measured.
  • the Schizochytrium sp. (CGMCC No. 6843) 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 liquid was connected to the first-stage seed tank containing the sterilized medium, and the culture temperature was 28 ° C, the aeration amount was 1 vvm, the tank pressure was 0.02 MPa, and the stirring speed was 50 rpm for 30 hours to complete the first stage. Seeds are expanded and cultured.
  • 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 amount was 1 vvm, the tank pressure was 0.02 MPa, and the stirring speed was 75 rpm for 24 hours.
  • Complete secondary seed expansion culture was connected to a fermentor containing the sterilized medium according to a 3% inoculum.
  • the fermentation process has a culture temperature of 28 ° C, aeration volume of 1-2 vvm, a tank pressure of 0.02-0.05 MPa, a stirring speed of 50-100 rpm, a carbon source containing 20% of the pretreated crude glycerin, and a glucose concentration of 5 g/L for fermentation. to cultivate.
  • the DO was controlled at 50% before fermentation for 60 hours, and the DO was controlled at 10% after 60 hours of fermentation.
  • DO was used as the control index, and the aeration, tank pressure and stirring speed were adjusted, and the fresh medium or sterilized after sterilization was appropriately added. water. In the fermentation process, 45% of ammonia water was added before fermentation for 60 hours to control the pH of the fermentation broth to about 7.
  • the pH of the fermentation broth could be controlled while adding ammonia water to supplement the nitrogen source. After 60 hours, the ammonia solution was stopped and the nitrogen deficiency culture was carried out. The pH was no longer used. Control. Fermentation to 36h will be sent in the fermenter The fermentation broth is divided into two by volume, half of the fermentation broth is left in the main tank to continue the culture, and the other half of the fermentation broth is transferred to the aseptically held sub-tank by differential pressure method, and the main and auxiliary tanks are respectively filled with appropriate amount. Fresh medium or sterile water after the bacteria, the main and auxiliary tank ventilation, tank pressure and stirring speed after the tank culture were adjusted according to the conditions before and after the above 60h. During the fermentation process, the glucose concentration, pH, bacterial biomass, crude oil production and DHA yield of the fermentation broth were measured.
  • the Schizochytrium sp. (CGMCC No. 6843) 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 liquid was connected to the first-stage seed tank containing the sterilized medium, and the culture temperature was 28 ° C, the aeration amount was 1 vvm, the tank pressure was 0.02 MPa, and the stirring speed was 50 rpm for 30 hours to complete the first stage. Seeds are expanded and cultured.
  • 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 amount was 1 vvm, the tank pressure was 0.02 MPa, and the stirring speed was 75 rpm for 24 hours.
  • Complete secondary seed expansion culture was connected to a fermentor containing the sterilized medium according to a 3% inoculum.
  • the DO was controlled at 35% before fermentation for 40h, and the DO was controlled at 6% after fermentation for 40h. During the process, DO was used as the control index to adjust the aeration volume, tank pressure and stirring speed, and properly added to the fresh medium or sterilized after sterilization. water. In the fermentation process, 38% ammonia water was added before fermentation for 40h to control the pH of the fermentation broth to about 6.5.
  • the pH of the fermentation broth could be controlled while adding ammonia water to supplement the nitrogen source. After 40h, the ammonia solution was stopped and the nitrogen deficiency culture was carried out. The pH was no longer used. Control. Fermentation to 20h, the fermentation broth in the fermenter is divided into two by volume, half of the fermentation broth is left in the main tank to continue the culture, 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 tank, and the aeration, tank pressure and stirring speed of the main and auxiliary tanks were adjusted according to the conditions before and after 40 h. During the fermentation process, the glucose concentration, pH, bacterial biomass, crude oil production and DHA yield of the fermentation broth were measured.
  • Table 11 Fatty acid composition of mixed fats obtained after fermentation in 100m 3 fermentor
  • the Schizochytrium sp. (CGMCC No. 6843) 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 connected to the first-stage seed tank containing the sterilized medium according to the inoculation amount of 0.4%, and the culture temperature was 28 ° C.
  • the gas volume was 1vvm
  • the tank pressure was 0.02 MPa
  • the stirring speed was 50 rpm for 30 hours to complete the first-stage seed expansion culture.
  • 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 amount was 1 vvm, the tank pressure was 0.02 MPa, and the stirring speed was 75 rpm for 24 hours.
  • Complete secondary seed expansion culture was connected to a fermentor containing the sterilized medium according to a 3% inoculum.
  • the fermentation process has a culture temperature of 28 ° C, aeration volume of 1-2 vvm, a tank pressure of 0.02-0.05 MPa, a stirring speed of 50-100 rpm, a carbon source containing 40% of the pretreated crude glycerin, and a glucose concentration of 4 g/L for fermentation. to cultivate.
  • the DO was controlled at 45% before fermentation for 56h, and the DO was controlled at 9% after fermentation for 56h.
  • DO was used as the control index to adjust the aeration volume, tank pressure and stirring speed, and properly added to the fresh medium or sterilized after sterilization. water.
  • 56% ammonia water was added before fermentation for 56h to control the pH of the fermentation broth to about 7.
  • the pH of the fermentation broth could be controlled while adding ammonia water to supplement the nitrogen source. After 56h, the ammonia solution was stopped and the nitrogen deficiency culture was carried out. The pH was no longer used. Control. Fermentation to 28h, the fermentation broth in the fermenter is divided into two by volume, half of the fermentation broth is left in the main tank to continue the culture, and the other half of the fermentation broth is transferred to the aseptically held sub-tank by differential pressure method. The tank was filled with appropriate amount of fresh medium or sterile water after sterilization. The main and auxiliary tank ventilation, tank pressure and stirring speed were adjusted according to the conditions before and after 56 h. During the fermentation process, the glucose concentration, pH, bacterial biomass, crude oil production and DHA yield of the fermentation broth were measured.
  • Table 12 Fatty acid composition of mixed fats obtained after fermentation in 100m 3 fermentor
  • the Schizochytrium sp. (CGMCC No. 6843) 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 liquid was connected to the first-stage seed tank containing the sterilized medium, and the culture temperature was 28 ° C, the aeration amount was 1 vvm, the tank pressure was 0.02 MPa, and the stirring speed was 50 rpm for 30 hours to complete the first stage. Seeds are expanded and cultured.
  • 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 amount was 1 vvm, the tank pressure was 0.02 MPa, and the stirring speed was 75 rpm for 24 hours.
  • Complete secondary seed expansion culture was connected to a fermentor containing the sterilized medium according to a 3% inoculum.
  • the fermentation process has a culture temperature of 28 ° C, aeration volume of 1-2 vvm, a tank pressure of 0.02-0.05 MPa, a stirring speed of 50-100 rpm, a carbon source containing 40% of the pretreated crude glycerin, and a glucose concentration of 3 g/L for fermentation. to cultivate.
  • the DO was controlled at 38% before fermentation for 44h, and the DO was controlled at 7% after fermentation for 44h.
  • DO was used as the control index to adjust the aeration volume, tank pressure and stirring speed, and appropriately added to the fresh medium or sterilized after sterilization. water.
  • 40% ammonia water was added before fermentation for 44h to control the pH of the fermentation broth to about 6.5.
  • the pH of the fermentation broth could be controlled while adding ammonia water to supplement the nitrogen source. After 44h, the ammonia solution was stopped and the nitrogen deficiency culture was carried out. The pH was no longer used. Control. Fermentation to 22h, the fermentation broth in the fermenter is divided into two by volume, half of the fermentation broth is left in the main tank to continue the culture, and the other half of the fermentation broth is transferred to the aseptically held sub-tank by differential pressure method. The tank was filled with appropriate amount of fresh medium or sterile water after sterilization. The main and auxiliary tank ventilation, tank pressure and stirring speed were adjusted according to the conditions before and after 44 h. During the fermentation process, the glucose concentration, pH, bacterial biomass, crude oil production and DHA yield of the fermentation broth were measured.
  • Table 13 Fatty acid composition of mixed fats obtained after fermentation in 100m 3 fermentor
  • the Schizochytrium sp. (CGMCC No. 6843) 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 liquid was connected to the first-stage seed tank containing the sterilized medium, and the culture temperature was 28 ° C, the aeration amount was 1 vvm, the tank pressure was 0.02 MPa, and the stirring speed was 50 rpm for 30 hours to complete the first stage. Seeds are expanded and cultured.
  • 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 amount was 1 vvm, the tank pressure was 0.02 MPa, and the stirring speed was 75 rpm for 24 hours.
  • Complete secondary seed expansion culture was connected to a fermentor containing the sterilized medium according to a 3% inoculum.
  • the fermentation process has a culture temperature of 28 ° C, aeration volume of 1-2 vvm, a tank pressure of 0.02-0.05 MPa, a stirring speed of 50-100 rpm, a carbon source containing 40% of the pretreated crude glycerin, and a glucose concentration of 5 g/L to conduct fermentation. to cultivate.
  • the DO was controlled at 42% before fermentation for 52 hours, and the DO was controlled at 8% after fermentation for 52 hours.
  • DO was used as the control index to adjust the aeration volume, tank pressure and stirring speed, and appropriately added to the fresh medium or sterilized after sterilization. water.
  • 40% ammonia water was added before fermentation for 52h to control the pH of the fermentation broth to about 6.5.
  • the pH of the fermentation broth could be controlled while adding ammonia water to supplement the nitrogen source. After 52h, the ammonia solution was stopped and the nitrogen deficiency culture was carried out. The pH was no longer used. Control. Fermentation to 26h, the fermentation broth in the fermenter is divided into two by volume, half of the fermentation broth is left in the main tank to continue the culture, and the other half of the fermentation broth is transferred to the aseptically held sub-tank by differential pressure method. The tank was filled with appropriate amount of fresh medium or sterile water after sterilization. The main and auxiliary tank ventilation, tank pressure and stirring speed were adjusted according to the conditions before and after 52 hours. During the fermentation process, the glucose concentration, pH, bacterial biomass, crude oil production and DHA yield of the fermentation broth were measured.
  • Table 14 Fatty acid composition of mixed fats obtained after fermentation in 100m 3 fermentor
  • Example 5 100 L of the DHA fermentation broth obtained in Example 5 was heated and inactivated, and then the fermentation broth was transported to the cloth cavity by a distributor to carry out the cloth wrapping. After the cloth was finished, the first-stage pressing was performed, and the stepwise pressing method was adopted. The set pressure of 30 MPa was reached within the set 2 h, and the pressure was kept for 2 h until substantially no water droplets flowed out. Remove the first-stage squeeze cage, replace it with the second-stage press cage, push it into the second-stage press, and carry out the second-stage press. 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.
  • Example 5 100 L of the DHA fermentation broth obtained in Example 5 was heated and inactivated, and then the fermentation broth was transported to the cloth cavity by a distributor to carry out the cloth wrapping. 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. Remove the first-stage press cage, replace it with the second-stage press cage, push it into the second-stage press, and carry out the second-stage press. 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.
  • Example 5 150 L of the DHA fermentation broth obtained in Example 5 was subjected to heat inactivation treatment, and then centrifuged by a butterfly centrifuge to remove the centrifugal light liquid to obtain 95 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.
  • Example 5 300 L of the DHA fermentation broth obtained in Example 5 was heated and inactivated, and then added to a spray dryer to set a spray pressure of 5 MPa, an inlet air temperature of 180 ° C, an outlet air temperature of 80 ° C, and spray-dried into a powder to obtain DHA micro Algal flour 52.5kg.
  • DHA microalgae powder is transported into the cloth cavity, and the fabric is wrapped. After the fabric is finished, the flexible press is pressed, and the pressure is gradually increased to reach the set pressure of 100 MPa within 2 hours, and the pressure is kept for 2 hours until there is no oil.
  • the DHA oil was pressed out and the pressed DHA hair oil was collected, and a total of 22.1 kg of DHA hair oil was obtained, and the fermentation liquid to hair oil yield was 89.2%.
  • Example 5 300 L of the DHA fermentation broth obtained in Example 5 was heated and inactivated, and then added to a spray dryer to set a spray pressure of 6 MPa, an inlet air temperature of 200 ° C, an outlet air temperature of 100 ° C, and spray-dried into a powder to obtain DHA micro Algal flour 51.0kg.
  • DHA microalgae powder is transported into the cloth cavity, and the fabric is wrapped. After the fabric is finished, the flexible press is pressed, and the pressure is gradually increased to reach the set pressure of 150 MPa within 4 hours, and the pressure is kept for 4 hours until there is no oil.
  • the DHA oil was pressed out and the pressed DHA hair oil was collected, and a total of 22.8 kg of DHA hair oil was obtained, and the fermentation liquid to the hair oil yield was 92.0%.
  • Example 5 300 L of the DHA 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, thereby obtaining 200 L of the concentrated fermentation liquid, and added to a spray dryer to set a spray pressure of 8 MPa.
  • the inlet air temperature was 220 ° C
  • the outlet air temperature was 110 ° C
  • spray-dried into a powder to obtain 47.5 kg of DHA microalgal flour.
  • DHA microalgae powder is transported into the cloth cavity to wrap the fabric. After the fabric is finished, the flexible press is pressed and the pressure is gradually applied.
  • the set pressure of 150MPa was reached, and the pressure was kept for 4h until almost no oil droplets flowed out.
  • the DHA oil extracted from the press was collected, and 23.3kg of DHA hair oil was obtained, and the fermentation liquid to hair oil yield was 94.0%.
  • 300 L of the DHA 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, thereby obtaining 200 L of the concentrated fermentation liquid, and added to a spray dryer to set a spray pressure of 8 MPa.
  • the inlet air temperature was 220 ° C
  • the outlet air temperature was 110 ° C
  • the twin-screw press was preheated to 80 ° C, and the DHA microalgae powder was added to a twin-screw press to obtain 11.6 kg of DHA wool, and the fermentation liquid to the oil yield was 46.81%.
  • 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 9.30 kg of decolorized 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 9.10kg of DHA refined oil. The test results are shown in Table 15.
  • 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 9.31 kg of decolorized 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, Temperature 220 ° C, remove the second-level light components, heavy components into the third-stage molecular distillation, control the third-stage vacuum of about 3Pa, temperature 250 ° C, remove the third-level light components, collect heavy components, molecular distillation is completed, cooling To 30 ° C, add antioxidants, and package to obtain 9.03 kg of DHA refined oil.
  • Table 16 The test results are shown in Table 16.
  • 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 aqueous layer was separated and washed twice to obtain a base refining oil of 8.92 kg.
  • 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 (186 g activated carbon and 280 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. , 8.49kg 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 30 ° C, the antioxidant is added, and the packaging is performed.
  • the DHA refined oil was obtained in 8.31 kg, and the test results are shown in Table 19.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

本发明属于发酵工程领域，公开了一种利用微生物发酵生产二十二碳六稀酸(DHA)的方法，具体公开了利用裂殖壶菌菌株工业化发酵生产含DHA混合油脂的方法。

Description

一种利用微生物发酵生产DHA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DHA的方法，具体地，涉及利用裂殖壶菌菌株工业化发酵生产含二十二碳六稀酸(DHA)混合油脂的方法。
背景技术
DHA，全名二十二碳六烯酸(cis-4，7，10，13，16，19-docosahexaenoic acid，DHA)，是一种多元不饱和脂肪酸。人体自身难以合成，必须从外界摄取。DHA属于人体必需脂肪酸之一，有重要的生理调节功能和保健作用，缺乏时会引发一系列病症，包括生长发育迟缓、皮肤异常、鳞屑、不育、智力障碍等，另外还对心血管疾病有特殊的预防和治疗效果。有关研究还表明，DHA能作用于许多不同类型的组织和细胞，具有抑制炎症及免疫作用，包括减少炎症因子的产生、抑制淋巴细胞增殖等，DHA还具有预防老年痴呆、神经性疾病等多重功效。
目前DHA的商业来源主要是鱼油和微藻。传统深海鱼油提取的DHA受鱼的品种、季节及地理位置的影响而不稳定，且胆固醇和其他不饱和脂肪酸含量高，脂肪酸链的长度和不饱和度差异均较大，导致DHA产量有限、含量不高、分离纯化困难，成本较高等问题。随着鱼油原料来源日渐紧缺，难以实现DHA这种高附加值产品在食品和医药等行业中的广泛应用。微生物发酵法生产DHA可克服传统鱼油提取的缺陷，可用于大量生产DHA，不断满足人们的需求，具有广阔的应用前景，备受国内外学者关注。微生物发酵法采用真菌及微藻等产油微生物发酵生产含DHA藻油，经精制提取得DHA含量高的精油。国家卫生部许可的DHA生产菌种包括裂殖壶菌(Schizochytrium sp.)、吾肯氏壶藻(Ulkenia amoeboida)和寇氏隐甲藻(Crypthecodinium cohnii)。
微生物发酵法生产DHA的市场份额在逐年快速上升，有取代鱼油DHA的趋势，提高微藻DHA生产技术及品质，进军微藻DHA市场前景广阔。
公开号为CN103882072A的专利公开了一种利用裂殖壶菌生产二十二碳六烯酸的方法，其所公开的最高产量为细胞干重61.2g/L、DHA含量55.07％、DHA产量22.17g/L。公开号为CN101812484A的专利公开了一种高密度培养裂殖壶菌发酵生产DHA的方法，其所公开的产量为细胞干重120-150g/L、DHA产量26-30g/L，这也是目前报道的采 用裂殖壶菌(Schizochytrium sp.)生产DHA的最高生产水平。虽然其DHA生产率较之前的研究有了较大提高，但对于利用微藻进行工业化生产二十二碳六烯酸，大大降低其生产成本，提高单位产量，使微生物发酵产DHA的方法能够得到大力推广和普及使用还是远远不够的。
现有从裂殖壶菌发酵液中提取DHA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。离心法如公开号为CN101817738B的专利公开了一种从藻类和真菌细胞中破壁提取DHA的方法：将发酵结束后的微藻或真菌发酵液通过分离系统分离收集细胞，用酸调节菌泥pH 2.0-4.0，然后控制菌泥温度在10℃-20℃，在菌泥中加入抗氧化剂后通过高压均质机进行高压均质破壁；将破壁后的菌泥加入水，搅拌后将料液通过三相分离机分离得到DHA油脂。该发明采用物理的破壁和物理提取方法，工艺简单，细胞破壁高效，对菌泥的低温和抗氧化剂处理，可有效的保护藻类和真菌细胞内物质的生物活性，且产品绿色无毒无残留。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等杂质，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开号为CN101824363B的专利公开了一种提取二十二碳六烯酸油脂的方法：将含有二十二碳六烯酸的发酵液经酶法破壁后，先采用有机溶剂进行一级分水，对菌体进行富集，再用有机溶剂进行二级萃取，得到粗油。该方法操作简单，设备投入低，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CN102181320B的专利公开了一种生物发酵DHA藻油的提取方法，包括以下步骤：a)将微藻发酵液固液分离后得到的固体物干燥，得到干燥菌体；b)以超临界二氧化碳为萃取剂对所述干燥菌体进行萃取，得到二氧化碳流体；c)对所述二氧化碳流体进行减压分离，得到DHA藻油。实验表明，采用本发明提供的方法得到的DHA藻油中，DHA的含量大于40％，提取收率最高仅为85.23％，且需要加入乙醇作为助萃取剂，存在一定的安全风险，同时超临界设备价格昂贵，萃取收率也不高。
现有技术中DHA毛油的精炼多采用化学精炼技术，DHA毛油经过脱胶、碱炼、脱色、脱臭后得到DHA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DHA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DHA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DHA产量。进一步地，本发明人还发现了一种提取DHA毛油的方法，所述提取方法能够提高DHA毛油的提取收率。进一步地，本发明人还发现了一种纯化DHA毛油的方法，其能够提升DHA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DHA的粗油脂产量、DHA产量及DHA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DHA(二十二碳六烯酸)的微生物的方法，其中：
从发酵罐培养的36-60小时开始(优选地，直至发酵结束)，将溶解氧饱和度(DO)控制在5％-10％(例如5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％或10％)；和/或
从发酵罐培养的36-60小时开始(优选地，直至发酵结束)，不再加入氮源或者将氮源的加入量减少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)。
在本发明的一些实施方式中，所述的培养方法，其中：
从发酵罐培养的40-56小时(例如40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55或56小时)、44-52小时、46-50小时或者48小时开始，将溶解氧控制在5％-10％，和/或
从发酵罐培养的40-56小时(例如40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55或56小时)、44-52小时、46-50小时或者48小时开始，不再加入氮源。
在本发明的一些实施方式中，所述的培养方法，其中，在控制溶解氧饱和度为5％-10％之前，溶解氧饱和度为30％-50％(例如30％-45％、35％-45％、40％-45％、40％-50％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％或45％)。
在本发明的一些实施方式中，所述的培养方法，其中，所述发酵液的pH为6.0- 7.0。
在本发明的一些实施方式中，所述的培养方法，其中，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)。
在本发明的一些实施方式中，所述的培养方法，其中，所述氮源为氨水，优选为25％-45％的氨水，更优选为35％-45％或38％-42％的氨水，特别优选为40％的氨水。
在本发明的一些实施方式中，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12-36小时(例如16-32小时、18-30小时、20-28小时、22-26小时、24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。
在本发明的一些实施方式中，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，搅拌转速50-100rpm，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，搅拌转速50-100rpm，培养20-25h，完成二级种子扩大培养。
在本发明的一些实施方式中，所述的培养方法，还包括在接种之前和扩大培养之前，进行活化培养的步骤；优选地，所述活化培养的温度为25℃-32℃，时间为20-25h。
在本发明的一些实施方式中，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25℃-32℃，通气量1-2vvm，罐压0.02-0.05MPa，搅拌转速50-100rpm，进行发酵罐培养。
在本发明的一些实施方式中，所述的培养方法，其中用于生产DHA的微生物为裂殖壶菌(Schizochytriumsp.)；优选地，所述裂殖壶菌选自保藏号为CGMCC No.6843、ATCC No.20888、ATCC No.20889、ATCC No.28209或ATCC MYA-1381的菌株。
在本发明一个具体的实施方案中，所述的培养方法，包括下述步骤：
1)将裂殖壶菌(Schizochytriumsp.)斜面保藏菌株接入装有400mL种子培养基的2L摇瓶，在25℃-32℃的温度下以200rpm的转速培养20-25h，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种子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，搅拌转速50-100rpm，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种子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，搅拌转速50-100rpm，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发酵培养基的发酵罐中，培养温度25℃-32℃，通气量1-2vvm，罐压0.02-0.05MPa，搅拌转速50-100rpm，进行发酵罐培养；
5)将发酵罐培养的前48h的DO控制在30％-50％，48h及之后DO控制在5％-10％，过程中以DO为控制指标，调整通气量、罐压及搅拌转速，并适当补入灭菌后新鲜培养基或无菌水；
6)发酵罐培养的前48h，流加40％的氨水控制发酵液pH在6.0-7.0，流加氨水补充氮源的同时可以控制发酵液pH稳定，48h后停止流加氨水，进行缺氮培养，pH不再作控制；
7)发酵罐培养至24h，将发酵罐中发酵液按体积一分为二培养，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、罐压及搅拌转速均按步骤5)作相应调整；
8)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；
9)发酵培养84-108h，终止发酵，放罐，测定发酵液中生物量达120-180g/L，生物量中粗油脂含量达45％-60％，DHA占总油脂含量为40％-55％，DHA产量最高可达 44.9g/L，DHA产率最高可达11.2g/(L·d)。
上述步骤1)至步骤3)中所用的种子培养基的配方以及上述的步骤4)中所用的发酵培养基的配方，为本领域技术人员知悉的常规配方。例如，种子培养基配方为：葡萄糖3％，蛋白胨1％，酵母粉0.5％，海水晶2％，pH自然。发酵培养基配方为：葡萄糖12％，蛋白胨1％，酵母粉0.5％，海水晶2％，pH 6.5(裂殖壶菌产DHA的工艺研究及高产菌株选育，王申强等，江南大学硕士学位论文，2013年，p13-14)。
上述发酵培养基碳源中添加了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。
上述步骤8)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。
上述发酵过程中，将发酵罐培养的前48h的DO控制在30％-50％、发酵罐培养48h后DO控制在5％-10％，过程中以DO为控制指标，调整通气量、罐压及搅拌转速，并适当补入灭菌后新鲜培养基或无菌水。
上述发酵过程中，发酵罐培养的前48h流加40％的氨水控制发酵液pH在6.0-7.0，流加氨水补充氮源的同时可以控制发酵液pH稳定，48h后停止流加氨水，进行缺氮培养，pH不再作控制。
上述发酵过程中，发酵罐培养至24h，将发酵罐中发酵液按体积一分为二培养，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、罐压及搅拌转速均按前述的发酵罐培养的前48h和发酵罐培养48h后作相应调整。
上述发酵过程中流加碳源将发酵液中糖点(葡萄糖浓度)维持在1-5g/L。
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、酵母提取物、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。
对上述发酵所用菌种裂殖壶菌(Schizochytriumsp.)没有特殊的限制，可以是归类于裂殖壶菌属的各种菌类，具体来说，包括但不限于Schizochytriumsp.CGMCC No.6843、Schizochytriumsp.ATCC No.20888、Schizochytriumsp.ATCC No.20889、Schizochytriumsp.ATCC No.28209、Schizochytrium limacinum Honda et YokochiATCCMYA-1381，对其来源也无特殊限制，可以从发酵研究所或ATCC、CGMCC、CCTCC等微生物保藏寄存机关获得，还可以使用从自然环境中通过公知的筛选方法获得的裂殖壶菌(Schizochytriumsp.)菌株。
本发明的另一方面涉及一种微生物发酵液，其由本发明中任一项所述的培养方法得到。优选地，所述微生物发酵液为裂殖壶菌发酵液。
本发明的再一方面涉及一种提取DHA毛油的方法，包括如下步骤：
1)将用于生产DHA的微生物的发酵液进行脱水处理；
2)将步骤1)的产物进行柔性压榨，得到DHA毛油。
在本发明的一些实施方式中，所述的提取方法，其中，步骤1)中，所述发酵液为裂殖壶菌发酵液；优选地，所述发酵液为本发明的微生物发酵液。
在本发明的一些实施方式中，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、第一级柔性压榨、干燥例如喷雾干燥；
优选地，所述脱水处理依次包括离心和第一级柔性压榨，或者依次包括离心和喷雾干燥；
优选地，所述喷雾干燥的喷雾压力为4-8MPa，进风温度160℃-220℃，出风温度80℃-120℃。
在本发明的一些实施方式中，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。
在本发明的一些实施方式中，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案中，所述的提取方法，包括下述步骤：
1)布料：将一定量的DHA发酵液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DHA毛油，卸压，去掉二级压榨笼，将微藻粕与滤布进行分离。
上述DHA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。
提取DHA毛油方法也可以采用如下方式：DHA发酵液经喷雾干燥得到微藻粉(是指菌体经干燥去除水分后，得到的干菌体，外观为颗粒状粉末，称之为微藻粉)，微藻粉进行柔性压榨(条件等同于前面的二级柔性压榨)，得到DHA毛油。
上述喷雾干燥喷雾压力为4-8MPa，进风温度160℃-220℃，出风温度80℃-120℃，DHA微藻粉水分控制在10％以内。
上述DHA发酵液可直接进行喷雾干燥，也可以喷雾干燥前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喷雾干燥，可提高产能和节约能耗。
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。
上述柔性压榨包括以下步骤：
(1)布料：将一定量的DHA微藻粉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)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DHA毛油，卸压，去掉二级压榨笼，将微藻粕与滤布进行分离。
本发明的再一方面涉及一种DHA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DHA毛油的方法，包括将DHA毛油进行水化、脱色和分子蒸馏的步骤；优选地，所述DHA毛油为本发明的DHA毛油。
在本发明的一些实施方式中，所述的纯化方法，其中，所述水化包括如下步骤：
将DHA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。
在本发明的一些实施方式中，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。
在本发明的一些实施方式中，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DHA成品油。
优选地，重复分子蒸馏1次或多次。
在本发明一个具体的实施方案中，所述的纯化方法，包括下述步骤：
DHA毛油经水化、脱色、分子蒸馏等步骤进行精炼，最终得到DHA成品油。精炼具体步骤如下：
1)水化：DHA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。
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DHA成品油。
上述步骤1)中，优选地，所述水为纯化水。
本发明的再一方面涉及一种DHA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生产DHA或含有DHA的产品(例如DHA成品油)的方法，包括：
本发明中任一项所述的培养用于生产DHA的微生物的方法、
本发明中任一项所述的提取DHA毛油的方法、和/或
本发明中任一项所述的纯化DHA毛油的方法。
在本发明的一个实施方式中，所述生产方法的如图1所示。
在本发明的另一个实施方式中，所述生产方法的如图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“DHA毛油”是指，从DHA发酵液中制取、没经过精炼加工的初级油。
术语“DHA成品油”是指，DHA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。
本发明的有益效果：
本发明提供了一种工业化发酵生产二十二碳六烯酸油脂的方法，该方法能够低成本、高产量生产绿色环保的高品质DHA油脂。
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DHA油脂产量高、纯度高，有利于DHA的大规模工业化生产，粗甘油的添加也降低了DHA发酵生产成本，大幅提高了DHA发酵生产的市场竞争力。
(2)本发明采用柔性压榨工艺制备DHA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)发酵液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，剩下的微藻粕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DHA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DHA成品油的工艺流程示意图。
图3：采用裂殖壶菌Schizochytriumsp.CGMCC No.6843在不同培养方式下100m3发酵罐发酵生产DHA的结果。
具体实施方式
下面通过具体实施例对本发明作详细说明。然而，本领域的技术人员容易理解，实施例所描述的具体的物料配比、工艺条件及其结果仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。
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：
生物量的测定方法为：取适量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。
DHA产量为：以气相色谱法测得粗油脂中DHA的含量，乘以粗油脂产量所得，单位g/L。
DHA生产率为：DHA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DHA成品油检测方法依据GB26400-2011食品安全国家标准食品添加剂二十二碳六烯酸油脂(发酵法)。
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％。
发酵培养
下面的实施例1-13中，如果没有特别说明，所用种子培养基配方为：葡萄糖3％，蛋白胨1％，酵母粉0.5％，海水晶2％，pH自然(其余为水)。发酵培养基配方为：葡萄糖12％，蛋白胨1％，酵母粉0.5％，海水晶2％(其余为水)。
实施例1
分别将裂殖壶菌(Schizochytriumsp.ATCC 20888)、裂殖壶菌(Schizochytrium limacinum Honda et YokochiATCCMYA-1381)、裂殖壶菌(Schizochytriumsp.CGMCC No.6843)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，培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速50rpm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，培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速75rpm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。
发酵过程培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速75rpm，流加含30％预处理后粗甘油的碳源，控制葡萄糖浓度在5g/L，流加补入氮源，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DHA产量变化。
培养96h后终止发酵，下表1为分别测定所述三株菌在原始培养模式下培养后的生物量、粗油脂产量、DHA产量及DHA生产率，下表2为发酵后所得混合油脂脂肪酸组成气相分析结果。CGMCC No.6843的生物量、粗油脂产量、DHA产量亦如图3所示。
表1：不同菌株在原始培养模式下的发酵结果
Figure PCTCN2017083461-appb-000001
表2：100m3发酵罐原始培养方式发酵后所得混合油脂脂肪酸组成
Figure PCTCN2017083461-appb-000002
Figure PCTCN2017083461-appb-000003
通过表1及表2可见，三株菌在原始培养方式下产量及脂肪酸组成均差异较大，其中裂殖壶菌(Schizochytriumsp.CGMCC No.6843)各项指标均优于另外两株菌，因此以裂殖壶菌(Schizochytriumsp.CGMCC No.6843)为出发菌株来进行不同培养方式的优化。
实施例2
将裂殖壶菌(Schizochytriumsp.CGMCC No.6843)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，培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速50rpm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，培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速75rpm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。
发酵过程培养温度28℃、通气量1-2vvm、罐压0.02-0.05MPa、搅拌转速50-100rpm，流加含30％预处理后粗甘油的碳源，控制葡萄糖浓度在5g/L，流加补入氮源，进行发酵培养。将发酵前48h DO控制在40％、发酵48h后DO控制在8％，过程中以DO为控制指标，调整通气量、罐压及搅拌转速，并适当补入灭菌后新鲜培养基或无菌水。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DHA产量变化。
培养96h后终止发酵，测定发酵液中生物量为125g/L，粗油脂产量为56.3g/L，DHA产量为29.3g/L，DHA生产率为7.3g/(L·d)，如图3。发酵液放罐体积为78m3，整批发酵含DHA粗油脂产量为4391.4kg，比原始培养方式产量提高了0.67倍。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3：100m3发酵罐发酵后所得混合油脂脂肪酸组成
脂肪酸组成 含量％
C12：0 0.43
C14：0 5.04
C16：0 15.21
C16：1 1.76
C18：0 1.68
C18：1 1.52
C20：4 6.49
C20：5 1.05
C22：5 14.72
C22：6 52.1
实施例3
将裂殖壶菌(Schizochytriumsp.CGMCC No.6843)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，培养温度28℃、通气量1vvm，罐压0.02MPa、搅拌转速50rpm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，培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速75rpm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。
发酵过程培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速75rpm，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48h前流加40％的氨水控制发酵液pH在6.5，流加氨水补充氮源的同时可以控制发酵液pH稳定，48h后停止流加氨水，进行缺氮培养，pH不再作控制。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DHA产量变化。
培养96h后终止发酵，测定发酵液中生物量为118g/L，粗油脂产量为64.2g/L，DHA产量为32.6g/L，DHA生产率为8.2g/(L·d)，如图3。发酵液放罐体积为78m3，整批发酵含DHA粗油脂产量为5007.6kg，比原始培养方式产量提高了0.91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4：100m3发酵罐发酵后所得混合油脂脂肪酸组成
脂肪酸组成 含量％
C12：0 0.48
C14：0 4.96
C16：0 17.12
C16：1 1.26
C18：0 1.88
C18：1 1.67
C20：4 6.23
C20：5 1.25
C22：5 14.35
C22：6 50.8
实施例4
将裂殖壶菌(Schizochytriumsp.CGMCC No.6843)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，培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速50rpm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，培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速75rpm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。
发酵过程培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速75rpm，流加含30％预处理后粗甘油的碳源，控制葡萄糖浓度在5g/L，流加补入氮源，进行发酵培养。发酵至24h将发酵罐中发酵液按体积一分为二培养，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。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DHA产量变化。
培养96h后终止发酵，测定发酵液中生物量为73g/L，粗油脂产量为36.3g/L，DHA产量为16.4g/L，DHA生产率为4.1g/(L·d)，如图3。发酵液放罐体积为132m3，整批发酵含DHA粗油脂产量为4791.6kg，比原始培养方式产量提高了0.83倍，虽然放罐生物量、粗油脂产量、DHA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1.74倍，所以整批发酵产量较原始培养方式提高了0.83倍。这对于工业化大规模生产含DHA油脂具有重要意义，可以极大的节约成本，提高DHA生产的市场竞争力。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5：100m3发酵罐发酵后所得混合油脂脂肪酸组成
脂肪酸组成 含量％
C12：0 0.58
C14：0 4.88
C16：0 20.21
C16：1 1.91
C18：0 1.54
C18：1 1.22
C20：4 6.13
C20：5 1.25
C22：5 17.08
C22：6 45.2
实施例5
将裂殖壶菌(Schizochytriumsp.CGMCC No.6843)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，培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速50rpm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，培养温度 28℃、通气量1vvm、罐压0.02MPa、搅拌转速75rpm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。
发酵过程培养温度28℃、通气量1-2vvm、罐压0.02-0.05MPa、搅拌转速50-100rpm，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。将发酵48h前DO控制在40％、发酵48h后DO控制在8％，过程中以DO为控制指标，调整通气量、罐压及搅拌转速，并适当补入灭菌后新鲜培养基或无菌水。发酵过程中发酵48h前流加40％的氨水控制发酵液pH在6.5左右，流加氨水补充氮源的同时可以控制发酵液pH稳定，48h后停止流加氨水，进行缺氮培养，pH不再作控制。发酵至24h将发酵罐中发酵液按体积一分为二培养，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、罐压及搅拌转速均按前述的48h之前和之后的条件作相应调整。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DHA产量变化。
培养96h后终止发酵，测定发酵液中生物量为145g/L，粗油脂产量为82.6g/L，DHA产量为44.9g/L，DHA生产率为11.2g/(L·d)，如图3。发酵液放罐体积为130m3，整批发酵含DHA粗油脂产量为10738kg，比原始培养方式产量提高了3.10倍，不仅可以高产含DHA的油脂，而且所得DHA油脂中DHA的含量高，具有强势的市场竞争力。以下表6为发酵后所得混合油脂脂肪酸组成气相分析结果：
表6：100m3发酵罐发酵后所得混合油脂脂肪酸组成
脂肪酸组成 含量％
C12：0 0.45
C14：0 4.86
C16：0 14.22
C16：1 1.68
C18：0 1.72
C18：1 1.59
C20：4 6.36
C20：5 1.54
C22：5 13.28
C22：6 54.3
实施例6
将裂殖壶菌(Schizochytriumsp.ATCC 20888)按实施例5的培养方式进行培养，培养96h后终止发酵，测定发酵液中生物量为65g/L，粗油脂产量为15.2g/L，DHA产量为6.5g/L，比原始培养方式产量提高了2.49倍，不仅可以高产含DHA的油脂，而且所得DHA油脂中DHA的含量高，具有强势的市场竞争力。以下表7为发酵后所得混合油脂脂肪酸组成气相分析结果：
表7：100m3发酵罐发酵后所得混合油脂脂肪酸组成
脂肪酸组成 含量％
C12：0 0.56
C14：0 4.89
C16：0 22.16
C16：1 1.52
C18：0 1.28
C18：1 1.26
C20：4 7.85
C20：5 2.56
C22：5 15.12
C22：6 42.8
实施例7
将裂殖壶菌(Schizochytrium limacinum Honda et Yokochi ATCC MYA-1381)按实施例5的培养方式进行培养，培养96h后终止发酵，测定发酵液中生物量为105g/L，粗油脂产量为37g/L，DHA产量为17.2g/L，比原始培养方式产量提高了2.81倍，不仅可以高产含DHA的油脂，而且所得DHA油脂中DHA的含量高，具有强势的市场竞争力。以下表8为发酵后所得混合油脂脂肪酸组成气相分析结果：
表8：100m3发酵罐发酵后所得混合油脂脂肪酸组成
脂肪酸组成 含量％
C12：0 0.79
C14：0 5.48
C16：0 20.63
C16：1 1.25
C18：0 1.96
C18：1 1.12
C20：4 7.95
C20：5 2.87
C22：5 11.45
C22：6 46.5
实施例8
将裂殖壶菌(Schizochytriumsp.CGMCC No.6843)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，培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速50rpm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，培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速75rpm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。
发酵过程培养温度28℃、通气量1-2vvm、罐压0.02-0.05MPa、搅拌转速50-100rpm，流加含20％预处理后粗甘油的碳源，控制葡萄糖浓度在1g/L，进行发酵培养。将发酵36h前DO控制在30％、发酵36h后DO控制在5％，过程中以DO为控制指标，调整通气量、罐压及搅拌转速，并适当补入灭菌后新鲜培养基或无菌水。发酵过程中发酵36h前流加25％的氨水控制发酵液pH在6左右，流加氨水补充氮源的同时可以控制发酵液pH稳定，36h后停止流加氨水，进行缺氮培养，pH不再作控制。发酵至12h将发酵罐中发酵液按体积一分为二培养，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、罐压及搅拌转速均按前述的36h之前和之后的条件作相应调整。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DHA产量变化。
培养96h后终止发酵，测定发酵液中生物量为140g/L，粗油脂产量为78.5g/L，DHA 产量为40.4g/L，DHA生产率为10.1g/(L·d)。以下表9为发酵后所得混合油脂脂肪酸组成气相分析结果：
表9：100m3发酵罐发酵后所得混合油脂脂肪酸组成
脂肪酸组成 含量％
C12：0 0.42
C14：0 4.87
C16：0 15.07
C16：1 1.65
C18：0 1.7
C18：1 1.58
C20：4 6.35
C20：5 1.62
C22：5 15.24
C22：6 51.5
实施例9
将裂殖壶菌(Schizochytriumsp.CGMCC No.6843)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，培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速50rpm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，培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速75rpm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。
发酵过程培养温度28℃、通气量1-2vvm、罐压0.02-0.05MPa、搅拌转速50-100rpm，流加含20％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。将发酵60h前DO控制在50％、发酵60h后DO控制在10％，过程中以DO为控制指标，调整通气量、罐压及搅拌转速，并适当补入灭菌后新鲜培养基或无菌水。发酵过程中发酵60h前流加45％的氨水控制发酵液pH在7左右，流加氨水补充氮源的同时可以控制发酵液pH稳定，60h后停止流加氨水，进行缺氮培养，pH不再作控制。发酵至36h将发酵罐中发 酵液按体积一分为二培养，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、罐压及搅拌转速均按前述的60h之前和之后的条件作相应调整。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DHA产量变化。
培养96h后终止发酵，测定发酵液中生物量为138g/L，粗油脂产量为78.6g/L，DHA产量为39.6g/L，DHA生产率为9.9g/(L·d)。以下表10为发酵后所得混合油脂脂肪酸组成气相分析结果：
表10：100m3发酵罐发酵后所得混合油脂脂肪酸组成
脂肪酸组成 含量％
C12：0 0.41
C14：0 4.88
C16：0 15.32
C16：1 1.64
C18：0 1.71
C18：1 1.6
C20：4 6.38
C20：5 1.58
C22：5 16.08
C22：6 50.4
实施例10
将裂殖壶菌(Schizochytriumsp.CGMCC No.6843)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，培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速50rpm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，培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速75rpm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。
发酵过程培养温度28℃、通气量1-2vvm、罐压0.02-0.05MPa、搅拌转速50-100 rpm，流加含20％预处理后粗甘油的碳源，控制葡萄糖浓度在2g/L，进行发酵培养。将发酵40h前DO控制在35％、发酵40h后DO控制在6％，过程中以DO为控制指标，调整通气量、罐压及搅拌转速，并适当补入灭菌后新鲜培养基或无菌水。发酵过程中发酵40h前流加38％的氨水控制发酵液pH在6.5左右，流加氨水补充氮源的同时可以控制发酵液pH稳定，40h后停止流加氨水，进行缺氮培养，pH不再作控制。发酵至20h将发酵罐中发酵液按体积一分为二培养，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、罐压及搅拌转速均按前述的40h之前和之后的条件作相应调整。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DHA产量变化。
培养96h后终止发酵，测定发酵液中生物量为139g/L，粗油脂产量为78.2g/L，DHA产量为38.0g/L，DHA生产率为9.5g/(L·d)。以下表11为发酵后所得混合油脂脂肪酸组成气相分析结果：
表11：100m3发酵罐发酵后所得混合油脂脂肪酸组成
脂肪酸组成 含量％
C12：0 0.43
C14：0 4.89
C16：0 16.34
C16：1 1.63
C18：0 1.71
C18：1 1.57
C20：4 6.39
C20：5 1.59
C22：5 16.85
C22：6 48.6
实施例11
将裂殖壶菌(Schizochytriumsp.CGMCC No.6843)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，培养温度28℃、通 气量1vvm、罐压0.02MPa、搅拌转速50rpm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，培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速75rpm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。
发酵过程培养温度28℃、通气量1-2vvm、罐压0.02-0.05MPa、搅拌转速50-100rpm，流加含40％预处理后粗甘油的碳源，控制葡萄糖浓度在4g/L，进行发酵培养。将发酵56h前DO控制在45％、发酵56h后DO控制在9％，过程中以DO为控制指标，调整通气量、罐压及搅拌转速，并适当补入灭菌后新鲜培养基或无菌水。发酵过程中发酵56h前流加42％的氨水控制发酵液pH在7左右，流加氨水补充氮源的同时可以控制发酵液pH稳定，56h后停止流加氨水，进行缺氮培养，pH不再作控制。发酵至28h将发酵罐中发酵液按体积一分为二培养，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、罐压及搅拌转速均按前述的56h之前和之后的条件作相应调整。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DHA产量变化。
培养96h后终止发酵，测定发酵液中生物量为140g/L，粗油脂产量为80.8g/L，DHA产量为42.0g/L，DHA生产率为10.5g/(L·d)。以下表12为发酵后所得混合油脂脂肪酸组成气相分析结果：
表12：100m3发酵罐发酵后所得混合油脂脂肪酸组成
脂肪酸组成 含量％
C12：0 0.44
C14：0 4.86
C16：0 14.61
C16：1 1.67
C18：0 1.7
C18：1 1.58
C20：4 6.38
C20：5 1.64
C22：5 15.12
C22：6 52
实施例12
将裂殖壶菌(Schizochytriumsp.CGMCC No.6843)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，培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速50rpm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，培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速75rpm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。
发酵过程培养温度28℃、通气量1-2vvm、罐压0.02-0.05MPa、搅拌转速50-100rpm，流加含40％预处理后粗甘油的碳源，控制葡萄糖浓度在3g/L，进行发酵培养。将发酵44h前DO控制在38％、发酵44h后DO控制在7％，过程中以DO为控制指标，调整通气量、罐压及搅拌转速，并适当补入灭菌后新鲜培养基或无菌水。发酵过程中发酵44h前流加40％的氨水控制发酵液pH在6.5左右，流加氨水补充氮源的同时可以控制发酵液pH稳定，44h后停止流加氨水，进行缺氮培养，pH不再作控制。发酵至22h将发酵罐中发酵液按体积一分为二培养，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、罐压及搅拌转速均按前述的44h之前和之后的条件作相应调整。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DHA产量变化。
培养96h后终止发酵，测定发酵液中生物量为141g/L，粗油脂产量为81.2g/L，DHA产量为42.4g/L，DHA生产率为10.6g/(L·d)。以下表13为发酵后所得混合油脂脂肪酸组成气相分析结果：
表13：100m3发酵罐发酵后所得混合油脂脂肪酸组成
脂肪酸组成 含量％
C12：0 0.45
C14：0 4.85
C16：0 15.34
C16：1 1.68
C18：0 1.72
C18：1 1.54
C20：4 6.37
C20：5 1.57
C22：5 14.28
C22：6 52.2
实施例13
将裂殖壶菌(Schizochytriumsp.CGMCC No.6843)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，培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速50rpm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，培养温度28℃、通气量1vvm、罐压0.02MPa、搅拌转速75rpm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。
发酵过程培养温度28℃、通气量1-2vvm、罐压0.02-0.05MPa、搅拌转速50-100rpm，流加含40％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。将发酵52h前DO控制在42％、发酵52h后DO控制在8％，过程中以DO为控制指标，调整通气量、罐压及搅拌转速，并适当补入灭菌后新鲜培养基或无菌水。发酵过程中发酵52h前流加40％的氨水控制发酵液pH在6.5左右，流加氨水补充氮源的同时可以控制发酵液pH稳定，52h后停止流加氨水，进行缺氮培养，pH不再作控制。发酵至26h将发酵罐中发酵液按体积一分为二培养，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、罐压及搅拌转速均按前述的52h之前和之后的条件作相应调整。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DHA产量变化。
培养96h后终止发酵，测定发酵液中生物量为142g/L，粗油脂产量为80.2g/L，DHA产量为41.2g/L，DHA生产率为10.3g/(L·d)。以下表14为发酵后所得混合油脂脂肪酸组成气相分析结果：
表14：100m3发酵罐发酵后所得混合油脂脂肪酸组成
脂肪酸组成 含量％
C12：0 0.45
C14：0 4.86
C16：0 15.06
C16：1 1.65
C18：0 1.72
C18：1 1.56
C20：4 6.35
C20：5 1.59
C22：5 15.36
C22：6 51.4
提取毛油
实施例14
取实施例5得到的DHA发酵液100L，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DHA毛油，共得到DHA毛油6.8kg，发酵液至毛油收率82.3％。去掉二级压榨笼，将压完的微藻粕与滤布进行分离。
实施例15
取实施例5得到的DHA发酵液100L，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DHA毛油，共得到DHA毛油7.3kg，发酵液至毛油收率88.4％。去掉二级压榨笼，将压完的微藻粕与滤布进行分离。
实施例16
取实施例5得到的DHA发酵液150L，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95L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DHA毛油，共得到DHA毛油10.8kg，发酵液至毛油收率87.2％。去掉二级压榨笼，将压完的微藻粕与滤布进行分离。
实施例17
取实施例5得到的DHA发酵液300L，经加热灭活处理后，加入喷雾干燥器中，设定喷雾压力5MPa，进风温度180℃，出风温度80℃，喷雾干燥成粉末，得到DHA微藻粉52.5kg。DHA微藻粉运至布料腔中，进行布料包裹，布料完毕后，进行柔性压榨压榨，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DHA毛油，共得到DHA毛油22.1kg，发酵液至毛油收率89.2％。
实施例18
取实施例5得到的DHA发酵液300L，经加热灭活处理后，加入喷雾干燥器中，设定喷雾压力6MPa，进风温度200℃，出风温度100℃，喷雾干燥成粉末，得到DHA微藻粉51.0kg。DHA微藻粉运至布料腔中，进行布料包裹，布料完毕后，进行柔性压榨压榨，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DHA毛油，共得到DHA毛油22.8kg，发酵液至毛油收率92.0％。
实施例19
取实施例5得到的DHA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200L浓缩后的发酵液，加入喷雾干燥器中，设定喷雾压力8MPa，进风温度220℃，出风温度110℃，喷雾干燥成粉末，得到DHA微藻粉47.5kg。DHA微藻粉运至布料腔中，进行布料包裹，布料完毕后，进行柔性压榨压榨，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DHA毛油，共得到DHA毛油23.3kg，发酵液至毛油收率94.0％。
对比例1
取实施例5得到的DHA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200L浓缩后的发酵液，加入喷雾干燥器中，设定喷雾压力8MPa，进风温度220℃，出风温度110℃，喷雾干燥成粉末，得到DHA微藻粉47.8kg。双螺杆压榨机先预热至80℃，将DHA微藻粉加入双螺杆压榨机中榨油，得到DHA毛油11.6kg，发酵液至毛油收率46.81％。
毛油精炼
实施例20
取10kg实施例14、15、16合并所得的DHA毛油，按水化、脱色、分子蒸馏步骤进行精炼。
水化：10kg DHA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9.75kg。
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9.30kg。
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DHA成品油9.10kg，检验结果见表15。
表15：DHA成品油检验结果
Figure PCTCN2017083461-appb-000004
Figure PCTCN2017083461-appb-000005
实施例21
取10kg实施例14、15、16合并所得的DHA毛油，按水化、脱色、分子蒸馏步骤进行精炼。
水化：10kg DHA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9.70kg。
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9.31kg。
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DHA成品油9.03kg，检验结果见表16。
表16：DHA成品油检验结果
Figure PCTCN2017083461-appb-000006
实施例22
取10kg实施例17、18、19合并所得的DHA毛油，按实施例20的精炼方法进行精炼，得到DHA成品油9.13kg，检验结果见表17。
表17：DHA成品油检验结果
Figure PCTCN2017083461-appb-000007
实施例23
取10kg实施例17、18、19合并所得的DHA毛油，按实施例21的精炼方法进行精炼，得到DHA成品油9.08kg，检验结果见表18。
表18：DHA成品油检验结果
Figure PCTCN2017083461-appb-000008
Figure PCTCN2017083461-appb-000009
对比例2
取10kg实施例17、18、19合并所得的DHA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 DHA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9.71kg。
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8.92kg。
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(186g活性炭和280g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8.49kg。
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30℃，添加抗氧化剂，包装，得到DHA成品油8.31kg，检测结果见表19。
表19：DHA成品油检验结果
Figure PCTCN2017083461-appb-000010

Claims (23)

  1. 一种培养用于生产DHA的微生物的方法，其中：
    从发酵罐培养的36-60小时开始，将溶解氧饱和度控制在5％-10％；和/或
    从发酵罐培养的36-60小时开始，不再加入氮源或者将氮源的加入量减少50％以上。
  2. 根据权利要求1所述的培养方法，其中：
    从发酵罐培养的40-56小时、44-52小时、46-50小时或者48小时开始，将溶解氧控制在5％-10％，和/或
    从发酵罐培养的40-56小时、44-52小时、46-50小时或者48小时开始，不再加入氮源。
  3. 根据权利要求1或2所述的培养方法，其中，在控制溶解氧饱和度为5％-10％之前，溶解氧饱和度为30％-50％。
  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，发酵液中葡萄糖的浓度保持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，所述氮源为氨水，优选为25％-45％的氨水，更优选为35％-45％的氨水，特别优选为40％的氨水。
  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12-36小时(例如24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，搅拌转速50-100rpm，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，搅拌转速50-100rpm，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，还包括在接种之和扩大培养之前，进行活化培养的步骤；优选地，所述活化培养的温度为25℃-32℃，时间为20-25h。
  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25℃-32℃，通气量1-2vvm，罐压0.02-0.05MPa，搅拌转速50-100rpm，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DHA的微生物为裂殖壶菌；优选地，所述裂殖壶菌选自保藏号为CGMCC No.6843、ATCC No.20888、ATCC No.20889、ATCC No.28209或ATCC MYA-1381的菌株。
  11. 一种微生物的发酵液，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DHA毛油的方法，包括如下步骤：
    1)将用于生产DHA的微生物的发酵液进行脱水处理；
    2)将步骤1)的产物进行柔性压榨，得到DHA毛油。
  13. 根据权利要求12所述的提取方法，其中，步骤1)中，所述发酵液为裂殖壶菌 发酵液；优选地，所述发酵液为权利要求11所述的发酵液。
  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、第一级柔性压榨、干燥例如喷雾干燥；
    优选地，所述脱水处理依次包括离心和第一级柔性压榨，或者依次包括离心和喷雾干燥；
    优选地，所述喷雾干燥的喷雾压力为4-8MPa，进风温度160℃-220℃，出风温度80℃-120℃。
  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DHA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DHA毛油的方法，包括将DHA毛油进行水化、脱色和分子蒸馏的步骤；优选地，所述DHA毛油为权利要求17所述的DHA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DHA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DHA成品油。
    优选地，重复分子蒸馏1次或多次。
  22. 一种DHA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DHA或含有DHA的产品(例如DHA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DHA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DHA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DHA毛油的方法。
PCT/CN2017/083461 2016-12-30 2017-05-08 一种利用微生物发酵生产dha的方法 WO2018120574A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201611270522.9 2016-12-30
CN201611270522.9A CN106636235B (zh) 2016-12-30 2016-12-30 一种利用微生物发酵生产dha的方法

Publications (1)

Publication Number Publication Date
WO2018120574A1 true WO2018120574A1 (zh) 2018-07-05

Family

ID=58838534

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/083461 WO2018120574A1 (zh) 2016-12-30 2017-05-08 一种利用微生物发酵生产dha的方法

Country Status (2)

Country Link
CN (3) CN109777607B (zh)
WO (1) WO2018120574A1 (zh)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020081637A1 (en) * 2018-10-17 2020-04-23 Archer Daniels Midland Company Enzyme addition to fermentation broth for the reduction of oligosaccharides introduced through sterilized dextrose solution
CN111363730A (zh) * 2020-03-23 2020-07-03 焦作健康元生物制品有限公司 一种用于生产4-aa的酮还原酶制备方法
CN112646749A (zh) * 2021-01-15 2021-04-13 驻马店华中正大有限公司 一种利用氨水调节培养基pH的金霉素种子生产方法
CN113004971A (zh) * 2021-03-15 2021-06-22 东北农业大学 一种萌发调控结合酶法制备功能性粉末油脂的方法
CN113444569A (zh) * 2021-06-29 2021-09-28 江西尊创新能源有限公司 一种废弃动植物油精制的方法
CN115340950A (zh) * 2022-09-09 2022-11-15 漯河市田润宝生物科技有限公司 一种营养促生长的微生物发酵工艺

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110317672A (zh) * 2019-08-02 2019-10-11 重庆市三品功能食品研究院有限公司 一种从虹鳟鱼鱼脂肪中制备鱼油的方法
CN111011621B (zh) * 2019-12-30 2023-04-07 内蒙古金达威药业有限公司 一种裂壶藻粉的制备方法
CN111378699A (zh) * 2019-12-31 2020-07-07 嘉必优生物技术（武汉）股份有限公司 一种裂殖壶菌发酵产dha的方法
CN114164125A (zh) * 2021-12-28 2022-03-11 湖南农业大学 一种培养高油脂含量裂殖壶藻的发酵工艺

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101519678A (zh) * 2000-01-28 2009-09-02 马泰克生物科学公司 通过在发酵罐中高密度培养真核微生物来增加含有多烯脂肪酸的脂质的产生
CN101543695A (zh) * 2009-03-26 2009-09-30 南京泰分利分离技术有限公司 柔性压榨法处理发酵液的工艺
CN101585759A (zh) * 2009-07-08 2009-11-25 内蒙古金达威药业有限公司 从双鞭甲藻发酵液中提取dha不饱和脂肪酸的方法
CN101638361A (zh) * 2009-05-06 2010-02-03 厦门金达威集团股份有限公司 一种从裂殖壶菌提取并精制富含二十二碳六烯酸的方法
WO2014101857A1 (zh) * 2012-12-31 2014-07-03 厦门金达威集团股份有限公司 一种固料培养裂殖壶菌液体发酵生产dha的方法
CN105349588A (zh) * 2015-12-02 2016-02-24 福建师范大学 利用裂殖壶菌生产二十二碳六烯酸的方法

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101519676B (zh) * 2009-04-03 2011-09-14 湖北福星生物科技有限公司 用裂殖壶菌发酵生产二十二碳六烯酸的方法
SG10201401472YA (en) * 2009-04-14 2014-08-28 Solazyme Inc Methods Of Microbial Oil Extraction And Separation
CN101560440B (zh) * 2009-06-02 2012-01-11 北京化工大学 一种微生物油脂的制备方法
CN102100260B (zh) * 2010-12-03 2013-07-31 滨州学院 酵母油脂及其制备方法和应用
CN102492544B (zh) * 2011-12-07 2013-09-25 湖北福星生物科技有限公司 干法制取dha微藻油的方法
FR2991337B1 (fr) * 2012-05-29 2016-09-02 Roquette Freres Procede continu d'enrichissement en esters ethyliques de dha d'une huile produite par des microalgues
CN104263770A (zh) * 2014-09-04 2015-01-07 青岛海智源生命科技有限公司 裂壶藻半连续阶段式发酵、絮凝板框干法制取dha的方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101519678A (zh) * 2000-01-28 2009-09-02 马泰克生物科学公司 通过在发酵罐中高密度培养真核微生物来增加含有多烯脂肪酸的脂质的产生
CN101543695A (zh) * 2009-03-26 2009-09-30 南京泰分利分离技术有限公司 柔性压榨法处理发酵液的工艺
CN101638361A (zh) * 2009-05-06 2010-02-03 厦门金达威集团股份有限公司 一种从裂殖壶菌提取并精制富含二十二碳六烯酸的方法
CN101585759A (zh) * 2009-07-08 2009-11-25 内蒙古金达威药业有限公司 从双鞭甲藻发酵液中提取dha不饱和脂肪酸的方法
WO2014101857A1 (zh) * 2012-12-31 2014-07-03 厦门金达威集团股份有限公司 一种固料培养裂殖壶菌液体发酵生产dha的方法
CN105349588A (zh) * 2015-12-02 2016-02-24 福建师范大学 利用裂殖壶菌生产二十二碳六烯酸的方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHI, Z.Y. ET AL.: "Study of a Two-stage Growth of DHA-producing Marine Algae Schizochytrium limacinum SR21 with Shifting Dissolved Oxygen Level", APPL MICROBIOL BIOTECHNOL, vol. 81, no. 6, 21 October 2008 (2008-10-21), pages 1141 - 1148, XP019654222, Retrieved from the Internet <URL:doi:10.1007/s00253-008-1740-7> *
WEI, PING ET AL. ET AL.: "Research Progress on DHA Production by Schizochytrium", SCIENCE AND TECHNOLOGY OF FOOD INDUSTRY, 1 October 2010 (2010-10-01), pages 400 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020081637A1 (en) * 2018-10-17 2020-04-23 Archer Daniels Midland Company Enzyme addition to fermentation broth for the reduction of oligosaccharides introduced through sterilized dextrose solution
US20230242949A1 (en) * 2018-10-17 2023-08-03 Archer Daniels Midland Company Enzyme addition to omega 3 fermentation broth for the reduction of oligosaccharides through sterilized dextrose solution
US11946093B2 (en) * 2018-10-17 2024-04-02 Archer-Daniels-Midland Company Enzyme addition to omega 3 fermentation broth for the reduction of oligosaccharides through sterilized dextrose solution
CN111363730A (zh) * 2020-03-23 2020-07-03 焦作健康元生物制品有限公司 一种用于生产4-aa的酮还原酶制备方法
CN112646749A (zh) * 2021-01-15 2021-04-13 驻马店华中正大有限公司 一种利用氨水调节培养基pH的金霉素种子生产方法
CN113004971A (zh) * 2021-03-15 2021-06-22 东北农业大学 一种萌发调控结合酶法制备功能性粉末油脂的方法
CN113444569A (zh) * 2021-06-29 2021-09-28 江西尊创新能源有限公司 一种废弃动植物油精制的方法
CN115340950A (zh) * 2022-09-09 2022-11-15 漯河市田润宝生物科技有限公司 一种营养促生长的微生物发酵工艺

Also Published As

Publication number Publication date
CN109777606B (zh) 2022-09-23
CN109777607A (zh) 2019-05-21
CN106636235B (zh) 2020-08-04
CN109777606A (zh) 2019-05-21
CN109777607B (zh) 2022-06-10
CN106636235A (zh) 2017-05-10

Similar Documents

Publication Publication Date Title
WO2018120574A1 (zh) 一种利用微生物发酵生产dha的方法
WO2018152984A1 (zh) 一种利用微生物发酵生产ara的方法
CN102746947B (zh) 一种分离、纯化裂壶藻油中dha和饱和脂肪酸的方法
WO2017166831A1 (zh) 一种ω-7脂肪酸合成物及培养黄丝藻生产该合成物的方法与应用
CN101092344B (zh) 一种用分子蒸馏技术从元宝枫油中提取神经酸的方法
JP6118904B2 (ja) 微生物（単細胞真菌モルチエレラ・アルピナ（Ｍｏｒｔｉｅｒｅｌｌａａｌｐｉｎａ））のアラキドン酸が富化された油の製造方法
CN105925627B (zh) 微生物油及其制备方法
US20150159116A1 (en) Method for continuously enriching an oil produced by microalgae with ethyl esters of dha
CN104212848B (zh) 以生物柴油副产物粗甘油为碳源的微生物油脂制备方法
CN102796675A (zh) 一种粘红酵母油脂基因工程菌及其构建方法和应用
CN110747239B (zh) 富含Sn-2位ARA的微生物油脂及其制备方法和应用
CN100408543C (zh) 一种联产脂肪酸酯、甘油和植物粗蛋白的工艺
CN106520852A (zh) 一种利用淡水微藻生产含神经酸微藻油脂的生产方法
CN102352400B (zh) 微生物发酵植物油脂脱臭馏出物生产植物甾醇的方法
CN109266698B (zh) 被孢霉属微生物油脂中脂肪酸组合物成分调整的方法
WO2019218839A1 (zh) Dha微生物油脂中脂肪酸组合物成分调整的方法
CN114032259B (zh) 一种酵母菌的高密度发酵及十六碳烯酸提取方法
CN106318984B (zh) 一种产油微生物制脂肪酸甘油脂的方法及其甘油脂和应用
CN105779518A (zh) 一种高稳定性dha藻油的制备方法
CN107083280B (zh) 一种利用微生物纤维分离微生物油脂的方法
CN104263770A (zh) 裂壶藻半连续阶段式发酵、絮凝板框干法制取dha的方法
CN105331670B (zh) 裂壶藻与寇氏隐甲藻混合发酵的方法
CN105713936B (zh) 微生物油的制备方法
CN110295086A (zh) 一种从沙棘果油中提纯棕榈油酸的方法
CN115353528B (zh) 一种粉末磷脂的绿色生产新工艺

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17886069

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17886069

Country of ref document: EP

Kind code of ref document: A1