CN106636235A - Method for producing DHA (Docosahexaenoic Acid) by utilizing microorganism fermentation - Google Patents
Method for producing DHA (Docosahexaenoic Acid) by utilizing microorganism fermentation Download PDFInfo
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- CN106636235A CN106636235A CN201611270522.9A CN201611270522A CN106636235A CN 106636235 A CN106636235 A CN 106636235A CN 201611270522 A CN201611270522 A CN 201611270522A CN 106636235 A CN106636235 A CN 106636235A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
- C11B1/06—Production of fats or fatty oils from raw materials by pressing
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- 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]
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
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- Oil, Petroleum & Natural Gas (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
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- 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
The invention belongs to the field of fermentation engineering, relates to a method for producing DHA (Docosahexaenoic Acid) by utilizing microorganism fermentation and in particular relates to a method for producing mixed oil containing the DHA by utilizing industrial fermentation of schizochytrium strains. More specifically, the invention relates a method for cultivating microorganisms for producing the DHA, wherein the dissolved oxygen saturation is controlled to be 5 percent to 10 percent from 36h to 60h of a culture process of a fermentation tank, and/or a nitrogen source is not added any more from 36h to 60h of the culture process of the fermentation tank. When the method provided by the invention is used for preparing the DHA, the yield is high and the purity is high; large-scale industrial production of the DHA is facilitated.
Description
Technical field
The invention belongs to field of fermentation engineering, is related to a kind of method that utilization fermentable produces DHA, specifically, relate to
And using the method for the schizochytrium limacinum bacterial strain industrial fermentation production diluted acid containing 22 carbon six (DHA) compound lard.
Background technology
DHA, full name DHA (cis-4,7,10,13,16,19-docosahexaenoic acid, DHA),
It is a kind of polybasic unsaturated fatty acid.Human body itself is difficult to synthesize, it is necessary to from extraneous intake.DHA belongs to essential fatty acid
One of, there is important physiological regulation function and health-care effect, can cause a series of illnesss during shortage, including growth retardation,
Skin abnormality, the scales of skin that peel off, infertility, dysnoesia etc., also there is in addition special prevention and treatment effect to angiocardiopathy.It is relevant to grind
Study carefully it is also shown that DHA can act on many different types of tissues and cell, with suppressing inflammation and immunization, including reduction
Generation, suppression lymphopoiesis of inflammatory factor etc., DHA also has the multi-efficiencies such as prevention senile dementia, neurogenic disease.
At present the commercial source of DHA is mainly fish oil and microalgae.Kind, seasons of the DHA that traditional deep sea fish oil is extracted by fish
Section and the impact in geographical position and it is unstable, and cholesterol and other unsaturated fatty acid contents are high, the length of fatty acid chain and
Degree of unsaturation difference is larger, causes that DHA limits throughputs, content be not high, isolates and purifies difficulty, the problems such as relatively costly.With
Fish oil raw material sources are day by day in short supply, it is difficult to realize this high value added products of DHA extensively should in the industries such as food and medicine
With.DHA Production by Microorganism Fermentation can overcome the defect that traditional fish oil is extracted, and can be used to produce DHA in a large number, constantly meet people
Demand, have broad application prospects, enjoy Chinese scholars to pay close attention to.Microbe fermentation method adopts the oil-producing such as fungi and microalgae
Fermentable production contains DHA algal oil, and Jing is refined and extracted to obtain the high essential oil of DHA content.The DHA production bacterium of health ministry license
Kind include schizochytrium limacinum (Schizochytrium sp.), my Ken Shi kettle algaes (Ulkenia amoeboida) and the hidden dinoflagellates of Kou Shi
(Crypthecodinium cohnii)。
The market share of DHA Production by Microorganism Fermentation is improved in rapid increase year by year, the trend of substituted fish oil DHA
Microalgae DHA production technology and quality, march microalgae DHA wide market.
The patent of Publication No. CN103882072A discloses a kind of utilization schizochytrium limacinum production DHA
Method, the maximum output disclosed in it is dry cell weight 61.2g/L, DHA content 55.07%, DHA yield 22.17g/L.It is open
Number a kind of method of producing DHA by Schizochytrium in high-density culture through fermentation is disclosed for the patent of CN101812484A, disclosed in it
Yield be dry cell weight 120-150g/L, DHA yield 26-30g/L, this is also the employing schizochytrium limacinum of current report
(Schizochytrium sp.) produces the highest level of production of DHA.Although its DHA productivity ratio relatively before research have larger
Improve, but for industrialized production DHA is carried out using microalgae, substantially reduce its production cost, improve unit and produce
Amount, enables the method that fermentable produces DHA to be widelyd popularize and popularizes to use or far from being enough.
The method of DHA is extracted in the existing liquid from schizochytrium limacinum fermentation mainly three kinds, and one is centrifugal process, and two is organic solvent
Extraction, three is supercritical extraction.The patent of centrifugal process such as Publication No. CN101817738B disclose it is a kind of from algae and
The method of extraction DHA in fungal cell：Microalgae or fungal fermented filtrate after fermentation ends is separated by piece-rate system and is received
Collection cell, with acid bacterium mud pH 2.0-4.0 is adjusted, and then controls bacterium mud temperature at 10 DEG C -20 DEG C, and antioxygen is added in bacterium mud
High-pressure homogeneous broken wall is carried out by high pressure homogenizer after agent；Bacterium mud after broken wall is added into water, feed liquid is passed through into three after stirring
The isolated DHA grease of phase separation machine.The invention adopts the broken wall and physical extracting method of physics, process is simple, breaking-wall cell
Efficiently, to bacterium mud low temperature and antioxidant is processed, and can be effectively protected the biologically active of algae and fungi intracellular matter, and
Product Green nontoxic residue-free.But the oil reservoir inferior quality after the invention centrifugation, in addition to containing grease, also containing moisture, culture medium
The impurity such as composition and cell fragment, is unfavorable for follow-up refining, and the waste water layer after being centrifuged in addition contains a large amount of bacteria residues, and COD is very high,
It is difficult to process or processing cost is high.The patent of organic solvent extractionprocess such as Publication No. CN101824363B discloses one kind and carries
The method for taking docosahexaenoic acid grease：By the zymotic fluid containing DHA Jing after enzymatic shell-broken, first adopting has
Machine solvent carries out a fraction water, and thalline is enriched with, then two grades of extractions are carried out with organic solvent, obtains crude oil.The method is grasped
Make simple, equipment investment is low, but the method uses organic solvent to be extracted, and final products might have dissolvent residual, and
There is the potential safety hazard such as inflammable and explosive in extraction process.The patent of supercritical extraction method such as Publication No. CN102181320B is disclosed
A kind of extracting method of biofermentation DHA algal oil, comprises the following steps：A) by consolidating for obtaining after the separation of Microalgae fermentation solid and liquid
Body thing is dried, and obtains being dried thalline；B) thalline that is dried is extracted by extractant of supercritical carbon dioxide, obtains two
Carbon oxide fluid；C) decompression separation is carried out to the CO 2 fluid, obtains DHA algal oil.Experiment is shown, is carried using the present invention
For the DHA algal oil that obtains of method in, the content of DHA is more than 40%, and extract yield is only up to 85.23%, and needs to add
There is certain security risk in ethanol, while overcritical equipment price is expensive, extraction yield is not also high as extractant is helped.
In prior art the refining of DHA crude oils more adopt chemical refining technology, DHA crude oils through degumming, alkali refining, decolouring,
DHA essential oils are obtained after deodorization.The technology is inevitably present some problems, such as：Alkali refining is low in order to reach control acid value
Requirement, generally can all add excess base, the ester of partial glycerol three to be inevitably saponified；The high-COD waste water meeting that alkali refining is produced
Pollution environment；Alkali refining needs the high-temperature process time long, easily causes product peroxide value, anisidine value and raises；Deodorization temperature height,
The shortcomings of time length is also easy to produce trans-fatty acid.
At present, still needing will develop new DHA production technologies.
The content of the invention
The present inventor has obtained a kind of micro- life cultivated for producing DHA through in-depth study and performing creative labour
The method of thing.Surprisingly, it was found that the cultural method can be significantly increased biomass and DHA yield.Enter one
Step ground, the present inventors have additionally discovered that a kind of method of extraction DHA crude oils, the extracting method can improve the extraction of DHA crude oils
Yield.Further, the present inventors have additionally discovered that a kind of method of purifying DHA crude oils, it can lift the items of DHA product oils
Technical indicator and purifying yield.Invention significantly increases the thick grease yield containing DHA, DHA yield and DHA productivity ratio.By
This provides following inventions：
One aspect of the present invention is related to one kind and cultivates side for producing the microorganism of DHA (DHA)
Method, wherein：
(preferably, until fermentation ends) from the beginning of the 36-60 hours of fermentation tank culture, by saturation dissolved oxygen (DO)
Control is at 5%-10% (such as 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10%)；
And/or
(preferably, until fermentation ends) from the beginning of the 36-60 hours of fermentation tank culture, do not add nitrogen source or incite somebody to action
The addition of nitrogen source reduce more than 50% (such as more than 55%, more than 60%, more than 65%, more than 70%, more than 75%,
More than 80%, more than 85%, more than 90% or more than 95%).
In certain embodiments of the present invention, described cultural method, wherein：
From the 40-56 hours of fermentation tank culture (such as 40,41,42,43,44,45,46,47,48,49,50,51,52,
53rd, 54,55 or 56 hours), 44-52 hours, 46-50 hours or start for 48 hours, dissolved oxygen is controlled in 5%-
10%, and/or
From the 40-56 hours of fermentation tank culture (such as 40,41,42,43,44,45,46,47,48,49,50,51,52,
53rd, 54,55 or 56 hours), 44-52 hours, 46-50 hours or start for 48 hours, do not add nitrogen source.
In certain embodiments of the present invention, described cultural method, wherein, it is saturation dissolved oxygen is controlled
Before 5%-10%, saturation dissolved oxygen be 30%-50% (such as 30%-45%, 35%-45%, 40%-45%,
40%-50%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44% or 45%).
In certain embodiments of the present invention, described cultural method, wherein, the pH of the zymotic fluid is 6.0-
7.0。
In certain embodiments of the present invention, described cultural method, wherein, the concentration of glucose keeps in zymotic fluid
1-5g/L (such as 1,1.5,2,2.5,3,3.5,4,4.5 or 5g/L).
In certain embodiments of the present invention, described cultural method, wherein, the nitrogen source is ammoniacal liquor, preferably
The ammoniacal liquor of the ammoniacal liquor of 25%-45%, more preferably 35%-45% or 38%-42%, particularly preferably 40% ammoniacal liquor.
In certain embodiments of the present invention, described cultural method, wherein, the fermentation tank culture is additionally included in be sent out
12-36 hours (such as 16-32 hours, 18-30 hours, 20-28 hours, 22-26 hours, 24 little of fermentation tank culture starting
When), the step of carry out point tank culture；For example, being divided into two or more fermentation tanks carries out fermentation tank culture.
In certain embodiments of the present invention, described cultural method, its before fermentation tank culture, also including inoculation
The step of with seed Amplification Culture；
Preferably, the seed Amplification Culture includes first order seed Amplification Culture and secondary seed Amplification Culture；
Preferably, the first order seed Amplification Culture comprises the steps：
Inoculum concentration according to 0.4%-1% accesses shake-flask seed liquid in the first class seed pot equipped with sterilizing wild Oryza species,
25 DEG C -32 DEG C of cultivation temperature, throughput 1-2vvm, tank pressure 0.02-0.05MPa, speed of agitator 50-100rpm, culture
30-35h, completes first order seed Amplification Culture；
Preferably, the secondary seed Amplification Culture comprises the steps：
The seed liquor of first class seed pot is accessed two grades of kinds equipped with sterilizing wild Oryza species by the inoculum concentration according to 1%-3%
In sub- tank, 25 DEG C -32 DEG C of cultivation temperature, throughput 1-2vvm, tank pressure 0.02-0.05MPa, speed of agitator 50-100rpm,
Culture 20-25h, completes secondary seed Amplification Culture.
In certain embodiments of the present invention, described cultural method, be additionally included in inoculation before and Amplification Culture it
Before, the step of carry out activation culture；Preferably, the temperature of the activation culture is 25 DEG C -32 DEG C, and the time is 20-25h.
In certain embodiments of the present invention, described cultural method, wherein, the fermentation tank culture includes following steps
Suddenly：
The seed liquor of secondary seed tank is accessed the fermentation tank equipped with sterilizing wild Oryza species by the inoculum concentration according to 1%-3%
In, 25 DEG C -32 DEG C of cultivation temperature, throughput 1-2vvm, tank pressure 0.02-0.05MPa, speed of agitator 50-100rpm are carried out
Fermentation tank culture.
In certain embodiments of the present invention, described cultural method, wherein the microorganism for producing DHA is fragmentation
Chytrid (Schizochytriumsp.)；Preferably, the schizochytrium limacinum is CGMCC No.6843, ATCC selected from preserving number
The bacterial strain of No.20888, ATCC No.20889, ATCC No.28209 or ATCC MYA-1381.
In a specific embodiment of the invention, described cultural method comprises the steps：
1) schizochytrium limacinum (Schizochytriumsp.) slant preservation bacterial strain is accessed equipped with 400mL seed culture mediums
2L shaking flasks, with the rotating speed culture 20-25h of 200rpm at a temperature of 25 DEG C -32 DEG C, complete bacterial strain activation culture；
2) shake-flask seed liquid is accessed into the first order seed equipped with seed culture medium after sterilizing according to the inoculum concentration of 0.4-1%
In tank, 25 DEG C -32 DEG C of cultivation temperature, throughput 1-2vvm, tank pressure 0.02-0.05MPa, speed of agitator 50-100rpm, training
Foster 30-35h, completes first order seed Amplification Culture；
3) seed liquor of first class seed pot is accessed equipped with seed culture medium after sterilizing according to the inoculum concentration of 1%-3%
In secondary seed tank, 25 DEG C -32 DEG C of cultivation temperature, throughput 1-2vvm, tank pressure 0.02-0.05MPa, speed of agitator 50-
100rpm, cultivates 20-25h, completes secondary seed Amplification Culture；
4) seed liquor of secondary seed tank is accessed equipped with sterilizing after fermentation culture medium according to the inoculum concentration of 1%-3%
In fermentation tank, 25 DEG C -32 DEG C of cultivation temperature, throughput 1-2vvm, tank pressure 0.02-0.05MPa, speed of agitator 50-
100rpm, carries out fermentation tank culture；
5) DO controls before fermentation 48h are existed in 30%-50%, 48h and DO controls afterwards by Dissolved oxygen regulation strategy
5%-10%, during with DO as Con trolling index, adjustment throughput, tank pressure and speed of agitator, and suitably fill into fresh after sterilizing
Culture medium or sterilized water；
6) the ammoniacal liquor control zymotic fluid pH for Jia 40% is flowed before fermentation 48h in 6.0-7.0, Feeding ammonia water supplements the same of nitrogen source
When can control zymotic fluid pH stablize, Feeding ammonia water is stopped after 48h, carry out nitrogen stress culture, pH does not remake control；
7) ferment to 24h and fermentation cylinder for fermentation liquid is divided into two culture by volume, half zymotic fluid stays in main tank relaying
Continuous culture, second half zymotic fluid is transferred in the assistant tank of aseptic pressurize by pressure differential method and is cultivated, and is filled into respectively in major-minor tank appropriate
Fresh culture or sterilized water after sterilizing, major-minor tank throughput, tank pressure and speed of agitator press Dissolved oxygen regulation plan after point tank culture
Slightly it is adjusted accordingly；
8) concentration of glucose constantly declines with thalli growth in sweat, and sugar in zymotic fluid is selected (grape by stream plus carbon source
Sugared concentration) maintain 1-5g/L；
9) fermented and cultured 84-108h, terminates fermentation, puts tank, determines biomass in zymotic fluid and reaches 120-180g/L, biological
Thick fat content reaches 45%-60% in amount, and it is 40%-55% that DHA accounts for total fat content, and DHA yield reaches as high as 44.9g/
L, DHA yield reaches as high as 11.2g/ (Ld).
Above-mentioned steps 1) to step 3) in seed culture medium used formula and above-mentioned step 4) in used send out
The formula of ferment culture medium, is conventional formulation that those skilled in the art know.For example, seed culture based formulas are：Glucose
3%, peptone 1%, dusty yeast 0.5%, sea crystal 2%, pH natures.Fermentative medium formula is：Glucose 12%, peptone
1%, dusty yeast 0.5%, sea crystal 2%, (schizochytrium limacinum produces the technical study of DHA and superior strain seed selection, Wang Shenqiang to pH 6.5
Deng, Southern Yangtze University's master thesis, 2013, p13-14).
20%-40% (quality × 100% of the quality/carbon source of crude glycerine) is with the addition of in above-mentioned fermentation medium carbon source
Pretreated biological diesel oil byproduct crude glycerine.Biodiesel is with the renewable grease such as vegetable oil and animal fat as raw material
Made by regenerative resource, biodiesel and by-product glycerin can be generated after transesterification reaction.The preprocessing process of crude glycerine
Including regulation pH value to acid, dilution, hydrolysis, separate.In one embodiment of the invention, the pretreatment of crude glycerine includes
Following step：I) by crude glycerine and deionized water with 1:4 (volume ratios) mix；Ii) with salt acid for adjusting pH to 6.5 or so；iii)
Separated with the rotating speed of 5000rpm and remove deposit.It is not limited to the restriction of theory：In step i), viscosity can be reduced after dilution；
Step ii) in, saponin material soluble in crude glycerine is converted into insoluble free fatty solid matter；Step iii)
In, deposit includes free fatty solid and undissolved beavy metal impurity.
Above-mentioned steps 8) in, sugared point (concentration of glucose) is determined using the method that those skilled in the art know, for example
Bio-sensing instrument is determined.
48h DO controls are in 30%-50%, fermentation 48h before being fermented using Dissolved oxygen regulation strategy in above-mentioned sweat
Afterwards DO is controlled in 5%-10%, during with DO as Con trolling index, adjustment throughput, tank pressure and speed of agitator, and suitably fill into
Fresh culture or sterilized water after sterilizing.
The ammoniacal liquor for Jia 40% is flowed in above-mentioned sweat before fermentation 48h and controls zymotic fluid pH in 6.0-7.0, Feeding ammonia water
Zymotic fluid pH being controlled while supplementing nitrogen source to stablize, Feeding ammonia water being stopped after 48h, carry out nitrogen stress culture, pH does not remake control
System.
Ferment in above-mentioned sweat to 24h and fermentation cylinder for fermentation liquid is divided into two culture by volume, half zymotic fluid
Stay in main tank and continue to cultivate, second half zymotic fluid is transferred in the assistant tank of aseptic pressurize by pressure differential method and is cultivated, in major-minor tank
Fresh culture or sterilized water after appropriate sterilizing is filled into respectively, and major-minor tank throughput, tank pressure and speed of agitator are equal after point tank culture
It is adjusted accordingly by Dissolved oxygen regulation strategy.
Sugared point (concentration of glucose) in zymotic fluid is maintained 1-5g/L by stream plus carbon source in above-mentioned sweat.
Carbon source includes the one kind or many in glucose, corn starch, molasses, glycerine and starch in above-mentioned fermentation medium
Kind；Nitrogen source includes the one kind or many in soy meal, yeast extract, peptone, ammoniacal liquor, sodium nitrate, sodium glutamate, ammonium sulfate
Kind.
In above-mentioned fermentation medium add trace element include alanine, glutamic acid, lysine, calcium pantothenate, biotin,
Cobastab1, microorganism B6, microorganism B12, one or more in vitamin K, when for one of which when, addition is
0.001%-0.01%；When for it is many of when, the addition of any one component is 0.001%-0.005%.
The inorganic salts added in above-mentioned fermentation medium include magnesium sulfate, potassium chloride, sodium chloride, calcium chloride, biphosphate
One or more in potassium, dipotassium hydrogen phosphate.
Can return to bacterial classification schizochytrium limacinum (Schizochytriumsp.) used by above-mentioned fermentation without special restriction
Class in the various mushrooms of Schizochytrium, specifically, including but not limited to Schizochytriumsp.CGMCC No.6843,
Schizochytriumsp.ATCC No.20888、Schizochytriumsp.ATCC No.20889、
Schizochytriumsp.ATCC No.28209、Schizochytrium limacinum Honda et
YokochiATCCMYA-1381, originates also without specifically limited to it, can be from fermentation research institute or ATCC, CGMCC, CCTCC etc.
Microbial preservation deposit office obtains, and can also use the schizochytrium limacinum obtained by known screening technique from natural environment
(Schizochytriumsp.) bacterial strain.
Another aspect of the present invention is related to a kind of microbial fermentation solution, its cultural method by any one of the present invention
Obtain.Preferably, the microbial fermentation solution is schizochytrium limacinum fermentation liquid.
Another aspect of the invention is related to a kind of method of extraction DHA crude oils, comprises the steps：
1) zymotic fluid of the microorganism for producing DHA is carried out into processed；
2) by step 1) product carry out flexible squeezing, obtain DHA crude oils.
In certain embodiments of the present invention, described extracting method, wherein, step 1) in, the zymotic fluid is to split
Grow chytrid zymotic fluid；Preferably, the zymotic fluid is the microbial fermentation solution of the present invention.
In certain embodiments of the present invention, described extracting method, wherein, step 1) in, the processed choosing
From any one following, two or three：
Centrifugation, the flexible squeezing of the first order, drying are for example spray-dried；
Preferably, the processed includes successively centrifugation and the flexible squeezing of the first order, or includes centrifugation successively and spray
Mist is dried；
Preferably, the atomisation pressure of the spray drying be 4-8MPa, 160 DEG C -220 DEG C of EAT, leaving air temp
80 DEG C -120 DEG C.
In certain embodiments of the present invention, described extracting method, wherein, step 1) in, the first order is flexible
Squeezing sets pressure as 20-40MPa using progressively pressuring method, and pressing time is 1-6h, reaches pressurize after setting pressure
1-4h.
In certain embodiments of the present invention, described extracting method, wherein, step 2) in, the flexible squeezing is adopted
With progressively pressuring method, pressure is set as 50-150MPa, pressing time is 1-6h, reach pressurize 1-4h after setting pressure.
In a specific embodiment of the invention, described extracting method comprises the steps：
1) cloth：A certain amount of DHA zymotic fluids distributing device is transported in distribution cavity, distributing device returns to initial position,
Treat next step cloth.
2) the flexible squeezing of one-level：Using progressively pressuring method, the pressure limit of the flexible squeezing of one-level is 20-40MPa, plus
The pressure time is 1-6h, reaches setting pressure, and pressurize 1-4h to substantially anhydrous drip goes out, and after terminating, treats that material drops to weight
In chamber, material is pushed into two grades of flexible squeezing positions.
3) two grades of flexible squeezings：Using progressively pressuring method, the final pressure of squeezing is 50-150MPa, and pressing time is
1-6h, reaches setting pressure, and pressurize 1-4h to basic oil-free drip goes out, and collection squeezes out the DHA crude oils for coming, and release is removed
Two grades of squeezing cages, the microalgae dregs of rice are separated with filter cloth.
Above-mentioned DHA zymotic fluids can directly carry out the flexible squeezing of one-level, it is also possible to first remove a part of water using centrifugal method
Point, carry out the flexible squeezing of one-level after the solid content for improving zymotic fluid again, the squeezing time can be shortened and production capacity is improved.
Above-mentioned zymotic fluid centrifugal method can adopt the one kind in horizontal spiral centrifuge, disk centrifugal separator, tube centrifuge
Carry out.
Extracting DHA crude oil methods can also be in the following way：The spray-dried microalgae powder that obtains of DHA zymotic fluids (is referred to
Thalline drying removes the dry mycelium for after moisture removal, obtaining, and outward appearance is particulate powder, referred to as microalgae powder), microalgae powder carries out soft
Property squeezing (condition is equal to above two grades flexible squeezings), obtain DHA crude oils.
Above-mentioned Spray Drying Spray pressure be 4-8MPa, 160 DEG C -220 DEG C of EAT, 80 DEG C -120 of leaving air temp
DEG C, DHA microalgae powders moisture is controlled within 10%.
Above-mentioned DHA zymotic fluids can be directly spray-dried, it is also possible to first using centrifugal method removing one before being spray-dried
Part moisture, is spray-dried again after the solid content for improving zymotic fluid, can improve production capacity and saving energy consumption.
Above-mentioned zymotic fluid centrifugal method can adopt the one kind in horizontal spiral centrifuge, disk centrifugal separator, tube centrifuge
Carry out.
Above-mentioned flexible squeezing is comprised the following steps：
(1) cloth：A certain amount of DHA microalgae powders distributing device is transported in distribution cavity, distributing device returns to initial position,
Treat next step cloth.
(2) flexible squeezing, using progressively pressuring method, the final pressure of squeezing is 50-150MPa, and pressing time is 1-
6h, reaches setting pressure, and pressurize 1-4h to basic oil-free drip goes out, and collection squeezes out the DHA crude oils for coming, and release removes two grades
Squeezing cage, the microalgae dregs of rice are separated with filter cloth.
Another aspect of the invention is related to a kind of DHA crude oils, and its extracting method by any one of the present invention is obtained.
Another aspect of the invention is related to a kind of method of purifying DHA crude oils, and including by DHA crude oils aquation, decolouring are carried out
And the step of molecular clock；Preferably, the DHA crude oils are the DHA crude oils of the present invention.
In certain embodiments of the present invention, described purification process, wherein, the aquation comprises the steps：
DHA crude oils are heated into 70 DEG C -85 DEG C, add the ratio of 50-150g water to add 75 DEG C -90 DEG C in 1kg crude oils
Water, stirs 10-60min, and mixing speed 30-90 turns/min, stands 1-6h, and lower floor's phosphatide is removed in layering, obtains aquation oil.
In certain embodiments of the present invention, described purification process, wherein, the decolouring comprises the steps：
Hydrated product is warming up into 90 DEG C -110 DEG C, vacuum≤- 0.07MPa, vacuum dehydration 0.5-2h is controlled, then
60 DEG C -80 DEG C are cooled to, decolorising agent (such as activated carbon of aquation weight of oil 1%-3% and 2-4% atlapulgites) is added,
Stirring 0.5-1h, stops stirring, filters and removes decolorising agent, obtains bleached oil.
In certain embodiments of the present invention, described purification process, wherein, the molecular clock is the steaming of three-level molecule
Evaporate；
Preferably, the molecular clock comprises the steps：
Bleached oil is entered into three-level molecular clock, control first order vacuum≤100Pa, 150 DEG C -200 DEG C of temperature are gone
Except the light component of the first order；The first heavy constituent for obtaining enters second level molecular clock, control secondary vacuum degree≤50Pa, temperature
180 DEG C -220 DEG C, remove second level light component；The second heavy constituent for obtaining enters third level molecular clock, and the control third level is true
200 DEG C -250 DEG C of reciprocal of duty cycle≤5Pa, temperature, removes third level light component, obtains the 3rd heavy constituent, is DHA product oils.
Preferably, molecular clock 1 time or multiple is repeated.
In a specific embodiment of the invention, described purification process comprises the steps：
The steps such as DHA crude oil Jing aquations, decolouring, molecular clock are refined, and finally give DHA product oils.Refining is concrete
Step is as follows：
1) aquation：DHA crude oils are heated to 70 DEG C -85 DEG C, add the ratio of 50-150g water to add 75 in 1kg crude oils
DEG C -90 DEG C of water, stir 10-60min, and mixing speed 30-90 turns/min, stand 1-6h, and layering is removed lower floor's phosphatide, obtained
Aquation oil.
2) decolourize：Aquation oil moves into Decolouring pot, is warming up to 90 DEG C -110 DEG C, controls vacuum≤- 0.07MPa, and vacuum takes off
Water 0.5-2h, is then cooled to 60 DEG C -80 DEG C, adds the decolorising agent (activated carbon and 2%- of aquation weight of oil 1%-3%
4% atlapulgite), decolouring 0.5-1h is stirred, stop stirring, filter and remove decolorising agent, obtain bleached oil.
3) molecular clock：Bleached oil enter three-level molecular clock, control first order vacuum≤100Pa, 150 DEG C of temperature-
200 DEG C, the light component of the first order is removed, heavy constituent enters second level molecular clock, control secondary vacuum degree≤50Pa, temperature
180 DEG C -220 DEG C, remove second level light component, heavy constituent enter third level molecular clock, control third level vacuum≤5Pa,
200 DEG C -250 DEG C of temperature, removes third level light component, collects heavy constituent, obtains molecular clock oil.Molecular clock pass is controlled
1-3 time, meet standard to acid value, smell and require.Molecular clock is finished, and is cooled to 20 DEG C -40 DEG C, adds antioxidant, bag
Dress, obtains DHA product oils.
Above-mentioned steps 1) in, it is preferable that the water is purified water.
Another aspect of the invention is related to a kind of DHA product oils, its purification process system by any one of the present invention
.
Another aspect of the invention is related to a kind of method for producing DHA or the product (such as DHA product oils) containing DHA,
Including：
Any one of the present invention culture for produce DHA microorganism method,
The method of the extraction DHA crude oils any one of the present invention, and/or
The method of the purifying DHA crude oils any one of the present invention.
In an embodiment of the invention, the production method is as shown in Figure 1.
In yet another embodiment of the present invention, the production method is as shown in Figure 2.
In the present invention,
Term " flexibility squeezing " refers to using PLC (programmable logic controller (PLC)) programme-control carry out-pressurize-of pressurizeing
Pressurized circulation, progressively reaches a kind of high-pressure compressing mode of predetermined pressure.
Term " purified water " refers to the drinking water Jing way of distillations, ion-exchange, hyperfiltration or other suitable method systems
The water of the hyoscine for obtaining, without any additive.In one embodiment of the invention, the purified water reference《Middle traditional Chinese medicines
Allusion quotation》Regulation.
Term " DHA crude oils " refers to, produces from DHA zymotic fluids, do not pass through the primary oil that refining is processed.
Term " DHA product oils " refers to that DHA crude oils process the essential oil for obtaining through refining.
Term " fermentation tank culture " referred to after seed Amplification Culture, carry out in fermentation tank for producing purpose product
Fermented and cultured.
Beneficial effects of the present invention：
The invention provides a kind of method that industrial fermentation produces docosahexaenoic acid grease, the method can it is low into
Originally, high yield produces the high-quality DHA grease of environmental protection.
The present invention have the following technical effect that at least one：
(1) technical indicator of the invention is significantly better than that existing technical indicator, gained DHA grease yield
High, purity is high, and the addition for being conducive to the large-scale industrial production of DHA, crude glycerine also reduces DHA fermenting and producing costs, greatly
Width improves the market competitiveness of DHA fermenting and producings.
(2) present invention prepares DHA crude oils using flexible squeezing process, it is not necessary to extracted using organic solvent, whole piece
Without using organic solvent, final products do not have dissolvent residual to production process route, and one side products obtained therefrom green health is produced
Quality is good, another aspect workshop safety and environmental protection, is the process for cleanly preparing of a green.
(3) aqueous solution that the flexible squeezing of zymotic fluid Jing one-levels is removed, is substantially free of bacteria residue, COD than relatively low, easily at biochemistry
Reason, two grades of flexible squeezings are obtained after crude oil, and the remaining microalgae dregs of rice also contain a small amount of grease and substantial amounts of albumen, can be used as feeding
Feed additives are used, economic and environment-friendly.
(4) present invention substitutes traditional depickling, deodorization two-step process using the step process of molecular clock one.Molecular clock can be
Retain and quickly remove substantial amounts of free fatty and stink on the basis of material physiologically active.The traditional alkali-refining deacidification side of contrast
Method, molecular clock deacidifying process is simple, reduces the risk of excessive alkali refining, and the loss of neutral grease is taken away in reduction from soap stock, takes off
Sour yield is significantly improved, deacidification is carried out in high vacuum conditions and the time is short, it is to avoid alkali-refining deacidification heated time length is made
Into peroxide value, the elevated risk of anisidine value, product stability is good；The traditional steam distillation deodorizing technology of contrast, molecule steams
Evaporate deodorization time short, vacuum is high, reduces the generation of trans-fatty acid, and stink substance removal effect is good, and product is without fishy smell.
(5) production process avoids the use of organic solvent, it is to avoid the cost of solvent consumption and solvent recovery, sewage
COD is low to be easily processed, and high income is refined, so as to greatly reduce production cost.
Description of the drawings
Fig. 1：The process flow diagram of the production DHA product oils of one embodiment of the present invention.
Fig. 2：The process flow diagram of the production DHA product oils of another embodiment of the invention.
Fig. 3：Using schizochytrium limacinum Schizochytriumsp.CGMCC No.6843 under different training methods 100m3Send out
Fermenting pot produces the result of DHA.
Specific embodiment
The present invention is elaborated below by specific embodiment.However, as it will be easily appreciated by one skilled in the art that real
Apply the specific material proportion described by example, process conditions and its result and be merely to illustrate the present invention, and also will not should not limit
The present invention described in detail in claims processed.
With regard to the assay method or computational methods of physical quantity according to the present invention either index, if especially not saying
Bright, method as described below is carried out：
The assay method of biomass is：Take appropriate zymotic fluid to be placed in flat measuring cup, in 105 DEG C of Constant Temp. Ovens
In be dried after 4h, be put into drier and be cooled to room temperature, weigh, deduct measuring cup weight, then divided by fermentating liquid volume, institute's value
As biomass, unit g/L.
The assay method of thick grease yield：Certain volume zymotic fluid is taken, the concentrated hydrochloric acid of 2 times of volumes, constant temperature at 70 DEG C is added
Stirring 50min digests completely to thalline, adds appropriate n-hexane, stratification, and upper organic phase is taken into eggplant type bottle with dropper,
Continuous extraction 5-8 time, until upper organic phase is colourless, removes n-hexane, then by eggplant type by 80 DEG C of water-bath rotary evaporations
Bottle is placed in 105 DEG C of Constant Temp. Ovens and is dried 1h, is put into drier and is cooled to room temperature, weighs, and deducts eggplant type bottle weight, then
Divided by fermentating liquid volume, institute's value is thick grease yield, unit g/L.
DHA yield is：With the content that gas chromatography measures DHA in crude oil fat, thick grease yield gained, unit g/ are multiplied by
L。
DHA productivity ratio is：DHA yield is divided by fermentation period (number of days) institute value, unit g/ (Ld).
DHA product oils detection method is according to GB26400-2011 food peace in the method and the present invention of fatty acid compositional analysis
National family's standard food additive docosahexaenoic acid grease (fermentation method).
Crude oil extract yield calculation：Yield=crude oil weight g/ (fermentating liquid volume L × thick grease yield g/ of zymotic fluid
L) × 100%.
Fermented and cultured
Embodiment 1 is original training method (not adopting Dissolved oxygen regulation strategy, nitrogen Regulation strategy and point tank training strategy)；
Embodiment 2 is using Dissolved oxygen regulation strategy；Embodiment 3 is using nitrogen Regulation strategy；Embodiment 4 is using a point tank culture plan
Slightly；Embodiment 5-13 is for simultaneously using Dissolved oxygen regulation strategy, nitrogen Regulation strategy and point tank training strategy.
In example 1 below -13, if not otherwise specified, seed culture based formulas used are：Glucose 3%,
Peptone 1%, dusty yeast 0.5%, sea crystal 2%, pH is natural (remaining is water).Fermentative medium formula is：Glucose 12%,
Peptone 1%, dusty yeast 0.5%, sea crystal 2% (remaining is water).
Embodiment 1：Original training method (not adopting Dissolved oxygen regulation strategy, nitrogen Regulation strategy and point tank training strategy) is right
100m
3
Ferment tank produces the impact of DHA
Respectively by schizochytrium limacinum (Schizochytriumsp.ATCC 20888), schizochytrium limacinum (Schizochytrium
Limacinum Honda et YokochiATCCMYA-1381), schizochytrium limacinum (Schizochytriumsp.CGMCC
No.6843) slant preservation bacterial strain accesses the 2L shaking flasks equipped with 400mL culture mediums, with the rotating speed of 200rpm at a temperature of 25 DEG C
Culture 24h, completes bacterial strain activation culture.Shake-flask seed liquid is accessed equipped with sterilizing wild Oryza species according to 0.4% inoculum concentration
In first class seed pot, 28 DEG C of cultivation temperature, throughput 1vvm, tank pressure 0.02MPa, speed of agitator 50rpm culture 30h complete one
Level seed Amplification Culture.The seed liquor of first class seed pot is accessed into two grades equipped with sterilizing wild Oryza species according to 3% inoculum concentration
In seeding tank, 28 DEG C of cultivation temperature, throughput 1vvm, tank pressure 0.02MPa, speed of agitator 75rpm culture 24h complete two grades of kinds
Sub- Amplification Culture.The seed liquor of secondary seed tank is accessed in the fermentation tank equipped with sterilizing wild Oryza species according to 3% inoculum concentration.
28 DEG C of sweat cultivation temperature, throughput 1vvm, tank pressure 0.02MPa, speed of agitator 75rpm, stream adds and contains 30%
The carbon source of crude glycerine after pretreatment, control concentration of glucose flows plus fills into nitrogen source in 5g/L, carries out fermented and cultured.Sweat
Middle detection zymotic fluid concentration of glucose, pH, Fungal biodiversity, thick grease yield and DHA change of production.
Terminate fermentation after culture 96h, table 1 below is to determine the life after three plants of bacterium are cultivated under original training mode respectively
Thing amount, thick grease yield, DHA yield and DHA productivity ratio, table 2 below is gained compound lard aliphatic acid composition gas phase point after fermentation
Analysis result.The biomass of CGMCC No.6843, thick grease yield, DHA yield are as also shown in Figure 3.
Table 1：Fermentation results of the different strains under original training mode
Table 2：100m3Gained compound lard aliphatic acid composition after the original training method fermentation of fermentation tank
Visible by table 1 and table 2, three plants of bacterium yield and aliphatic acid compositions under original training method differ greatly, its
Middle schizochytrium limacinum (Schizochytriumsp.CGMCC No.6843) indices are superior to other two plants of bacterium, therefore with fragmentation
Chytrid (Schizochytriumsp.CGMCC No.6843) carries out the optimization of different training methods for starting strain.
Embodiment 2：Using Dissolved oxygen regulation strategy to 100m
3
Ferment tank produces the impact of DHA
Schizochytrium limacinum (Schizochytriumsp.CGMCC No.6843) slant preservation bacterial strain is accessed equipped with 400mL trainings
The 2L shaking flasks of foster base, with the rotating speed culture 24h of 200rpm at a temperature of 25 DEG C, complete bacterial strain activation culture.According to 0.4%
Inoculum concentration by shake-flask seed liquid access equipped with sterilizing wild Oryza species first class seed pot in, 28 DEG C of cultivation temperature, throughput 1vvm,
Tank pressure 0.02MPa, speed of agitator 50rpm cultivate 30h, complete first order seed Amplification Culture.According to 3% inoculum concentration by one-level kind
The seed liquor of sub- tank is accessed in the secondary seed tank equipped with sterilizing wild Oryza species, 28 DEG C of cultivation temperature, throughput 1vvm, tank pressure
0.02MPa, speed of agitator 75rpm cultivate 24h, complete secondary seed Amplification Culture.According to 3% inoculum concentration by secondary seed tank
Seed liquor access equipped with sterilizing wild Oryza species fermentation tank in.
28 DEG C of sweat cultivation temperature, throughput 1-2vvm, tank pressure 0.02-0.05MPa, speed of agitator 50-
100rpm, flows plus the carbon source containing crude glycerine after 30% pretreatment, and control concentration of glucose flows plus fill into nitrogen source, carries out in 5g/L
Fermented and cultured.48h DO controls DO after 40%, fermentation 48h is controlled 8% before being fermented using Dissolved oxygen regulation strategy, during
With DO as Con trolling index, adjustment throughput, tank pressure and speed of agitator, and suitably fill into fresh culture or sterilized water after sterilizing.
Zymotic fluid concentration of glucose, pH, Fungal biodiversity, thick grease yield and DHA change of production are detected in sweat.
Terminate fermentation after culture 96h, it is 125g/L to determine biomass in zymotic fluid, and thick grease yield is 56.3g/L, DHA
Yield is 29.3g/L, and DHA productivity ratio is 7.3g/ (Ld), such as Fig. 3.Zymotic fluid puts tank volume for 78m3, by the gross fermentation is containing DHA
Thick grease yield is 4391.4kg, 0.67 times than original training method output increased.Table 3 below is gained miscella after fermentation
Fat aliphatic acid constitutes gas phase analysis result：
Table 3：100m3Gained compound lard aliphatic acid composition after the fermentation of fermentation tank Dissolved oxygen regulation strategy
Embodiment 3：Using nitrogen Regulation strategy to 100m
3
Ferment tank produces the impact of DHA
Schizochytrium limacinum (Schizochytriumsp.CGMCC No.6843) slant preservation bacterial strain is accessed equipped with 400mL trainings
The 2L shaking flasks of foster base, with the rotating speed culture 24h of 200rpm at a temperature of 25 DEG C, complete bacterial strain activation culture.According to 0.4%
Inoculum concentration accesses shake-flask seed liquid in the first class seed pot equipped with sterilizing wild Oryza species, 28 DEG C of cultivation temperature, throughput 1vvm,
Tank pressure 0.02MPa, speed of agitator 50rpm cultivate 30h, complete first order seed Amplification Culture.According to 3% inoculum concentration by one-level kind
The seed liquor of sub- tank is accessed in the secondary seed tank equipped with sterilizing wild Oryza species, 28 DEG C of cultivation temperature, throughput 1vvm, tank pressure
0.02MPa, speed of agitator 75rpm cultivate 24h, complete secondary seed Amplification Culture.According to 3% inoculum concentration by secondary seed tank
Seed liquor access equipped with sterilizing wild Oryza species fermentation tank in.
28 DEG C of sweat cultivation temperature, throughput 1vvm, tank pressure 0.02MPa, speed of agitator 75rpm, stream adds and contains 30%
The carbon source of crude glycerine after pretreatment, control concentration of glucose carries out fermented and cultured in 5g/L.Flow before fermentation 48h in sweat
6.5, Feeding ammonia water can control zymotic fluid pH and stablize the ammoniacal liquor control zymotic fluid pH for plus 40% while supplementing nitrogen source, 48h
After stop Feeding ammonia water, carry out nitrogen stress culture, pH does not remake control.Zymotic fluid concentration of glucose, pH, bacterium are detected in sweat
Body biomass, thick grease yield and DHA change of production.
Terminate fermentation after culture 96h, it is 118g/L to determine biomass in zymotic fluid, and thick grease yield is 64.2g/L, DHA
Yield is 32.6g/L, and DHA productivity ratio is 8.2g/ (Ld), such as Fig. 3.Zymotic fluid puts tank volume for 78m3, by the gross fermentation is containing DHA
Thick grease yield is 5007.6kg, 0.91 times than original training method output increased.Table 4 below is gained miscella after fermentation
Fat aliphatic acid constitutes gas phase analysis result：
Table 4：100m3Gained compound lard aliphatic acid composition after the fermentation of fermentation tank nitrogen Regulation strategy
| Aliphatic acid is constituted | Content % |
| 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 |
Embodiment 4：Using point tank training strategy to 100m
3
Ferment tank produces the impact of DHA
Schizochytrium limacinum (Schizochytriumsp.CGMCC No.6843) slant preservation bacterial strain is accessed equipped with 400mL trainings
The 2L shaking flasks of foster base, with the rotating speed culture 24h of 200rpm at a temperature of 25 DEG C, complete bacterial strain activation culture.According to 0.4%
Inoculum concentration by shake-flask seed liquid access equipped with sterilizing wild Oryza species first class seed pot in, 28 DEG C of cultivation temperature, throughput 1vvm,
Tank pressure 0.02MPa, speed of agitator 50rpm cultivate 30h, complete first order seed Amplification Culture.According to 3% inoculum concentration by one-level kind
The seed liquor of sub- tank is accessed in the secondary seed tank equipped with sterilizing wild Oryza species, 28 DEG C of cultivation temperature, throughput 1vvm, tank pressure
0.02MPa, speed of agitator 75rpm cultivate 24h, complete secondary seed Amplification Culture.According to 3% inoculum concentration by secondary seed tank
Seed liquor access equipped with sterilizing wild Oryza species fermentation tank in.
28 DEG C of sweat cultivation temperature, throughput 1vvm, tank pressure 0.02MPa, speed of agitator 75rpm, stream adds and contains 30%
The carbon source of crude glycerine after pretreatment, control concentration of glucose flows plus fills into nitrogen source in 5g/L, carries out fermented and cultured.Ferment to 24h
Fermentation cylinder for fermentation liquid is divided into two culture by volume, half zymotic fluid stays in main tank and continues to cultivate, second half zymotic fluid
It is transferred in the assistant tank of aseptic pressurize by pressure differential method and is cultivated, fills into fresh culture or nothing after appropriate sterilizing in major-minor tank respectively
Bacterium water.Zymotic fluid concentration of glucose, pH, Fungal biodiversity, thick grease yield and DHA change of production are detected in sweat.
Terminate fermentation after culture 96h, it is 73g/L to determine biomass in zymotic fluid, and thick grease yield is 36.3g/L, and DHA is produced
Measure as 16.4g/L, DHA productivity ratio is 4.1g/ (Ld), such as Fig. 3.Zymotic fluid puts tank volume for 132m3, by the gross fermentation is containing DHA
Thick grease yield is 4791.6kg, 0.83 times than original training method output increased, although put tank biomass, crude oil fat and produce
The indexs such as amount, DHA yield with original training method comparing difference less, but divide a tank culture, zymotic fluid to put tank body due to adopting
Product is 1.74 times of original training method, so by the gross the more original training method of fermentation yield improves 0.83 times.This is for work
Industryization large-scale production is significant containing DHA grease, can be greatly cost-effective, improves the market competition of DHA productions
Power.Table 5 below is gained compound lard aliphatic acid composition gas phase analysis result after fermentation：
Table 5：100m3Gained compound lard aliphatic acid composition after ferment tank
| Aliphatic acid is constituted | Content % |
| 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 |
Embodiment 5：Using Dissolved oxygen regulation strategy, nitrogen Regulation strategy and point tank training strategy to 100m
3
Fermentation tank fragmentation
The impact of chytrid (Schizochytriumsp.CGMCC No.6843) fermenting and producing DHA
Schizochytrium limacinum (Schizochytriumsp.CGMCC No.6843) slant preservation bacterial strain is accessed equipped with 400mL trainings
The 2L shaking flasks of foster base, with the rotating speed culture 24h of 200rpm at a temperature of 25 DEG C, complete bacterial strain activation culture.According to 0.4%
Inoculum concentration by shake-flask seed liquid access equipped with sterilizing wild Oryza species first class seed pot in, 28 DEG C of cultivation temperature, throughput 1vvm,
Tank pressure 0.02MPa, speed of agitator 50rpm cultivate 30h, complete first order seed Amplification Culture.According to 3% inoculum concentration by one-level kind
The seed liquor of sub- tank is accessed in the secondary seed tank equipped with sterilizing wild Oryza species, 28 DEG C of cultivation temperature, throughput 1vvm, tank pressure
0.02MPa, speed of agitator 75rpm cultivate 24h, complete secondary seed Amplification Culture.According to 3% inoculum concentration by secondary seed tank
Seed liquor access equipped with sterilizing wild Oryza species fermentation tank in.
28 DEG C of sweat cultivation temperature, throughput 1-2vvm, tank pressure 0.02-0.05MPa, speed of agitator 50-
100rpm, stream plus the carbon source containing crude glycerine after 30% pretreatment, control concentration of glucose carries out fermented and cultured in 5g/L.Using
Dissolved oxygen regulation strategy will ferment, and 40%, after fermentation 48h, DO is controlled 8% for DO controls before 48h, during refer to by control of DO
Mark, adjustment throughput, tank pressure and speed of agitator, and suitably fill into fresh culture or sterilized water after sterilizing.Send out in sweat
The ammoniacal liquor control zymotic fluid pH for Jia 40% is flowed before ferment 48h 6.5 or so, Feeding ammonia water can control fermentation while supplementing nitrogen source
Liquid pH stablizes, and Feeding ammonia water is stopped after 48h, carries out nitrogen stress culture, and pH does not remake control.Ferment fermentation cylinder for fermentation to 24h
Liquid is divided into two culture by volume, and half zymotic fluid stays in main tank and continues to cultivate, and second half zymotic fluid is shifted by pressure differential method
Cultivate into the assistant tank of aseptic pressurize, fill into fresh culture or sterilized water after appropriate sterilizing, point tank culture in major-minor tank respectively
Afterwards major-minor tank throughput, tank pressure and speed of agitator are adjusted accordingly by Dissolved oxygen regulation strategy.Zymotic fluid is detected in sweat
Concentration of glucose, pH, Fungal biodiversity, thick grease yield and DHA change of production.
Terminate fermentation after culture 96h, it is 145g/L to determine biomass in zymotic fluid, and thick grease yield is 82.6g/L, DHA
Yield is 44.9g/L, and DHA productivity ratio is 11.2g/ (Ld), such as Fig. 3.Zymotic fluid puts tank volume for 130m3, ferment contain by the gross
The thick grease yields of DHA are 10738kg, 3.10 times than original training method output increased.Dissolved oxygen regulation strategy, nitrogen are adopted simultaneously
Source regulating strategy and point tank training strategy not only can grease of the high yield containing DHA, and in gained DHA grease DHA content it is high,
With the surging market competitiveness.Table 6 below is gained compound lard aliphatic acid composition gas phase analysis result after fermentation：
Table 6：100m3Gained compound lard aliphatic acid composition after ferment tank
| Aliphatic acid is constituted | Content % |
| 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 |
Embodiment 6：Using Dissolved oxygen regulation strategy, nitrogen Regulation strategy and point tank training strategy to 100m
3
Fermentation tank fragmentation
The impact of chytrid (Schizochytriumsp.ATCC 20888) fermenting and producing DHA
Schizochytrium limacinum (Schizochytriumsp.ATCC 20888) is cultivated by the training method of embodiment 5, is trained
Terminate fermentation after foster 96h, it is 65g/L to determine biomass in zymotic fluid, and thick grease yield is 15.2g/L, and DHA yield is 6.5g/
L, 2.49 times than original training method output increased.Simultaneously using Dissolved oxygen regulation strategy, nitrogen Regulation strategy and point tank culture
Strategy not only can grease of the high yield containing DHA, and in gained DHA grease DHA content it is high, with surging market competition
Power.Table 7 below is gained compound lard aliphatic acid composition gas phase analysis result after fermentation：
Table 7：100m3Gained compound lard aliphatic acid composition after ferment tank
Embodiment 7：Using Dissolved oxygen regulation strategy, nitrogen Regulation strategy and point tank training strategy to 100m
3
Fermentation tank fragmentation
The shadow of chytrid (Schizochytrium limacinum Honda et Yokochi ATCC MYA-1381) fermenting and producing DHA
Ring
Schizochytrium limacinum (Schizochytrium limacinum Honda et Yokochi ATCC MYA-1381) is applied
The training method of example 5 is cultivated, culture 96h after terminate fermentation, determine zymotic fluid in biomass be 105g/L, thick grease yield
For 37g/L, DHA yield is 17.2g/L, 2.81 times than original training method output increased.Simultaneously using Dissolved oxygen regulation strategy,
Nitrogen Regulation strategy and point tank training strategy not only can grease of the high yield containing DHA, and in gained DHA grease DHA content
Height, with the surging market competitiveness.Table 8 below is gained compound lard aliphatic acid composition gas phase analysis result after fermentation：
Table 8：100m3Gained compound lard aliphatic acid composition after ferment tank
| Aliphatic acid is constituted | Content % |
| 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 |
Embodiment 8：Using Dissolved oxygen regulation strategy, nitrogen Regulation strategy and point tank training strategy to 100m
3
Fermentation tank fragmentation
The impact of chytrid (Schizochytriumsp.CGMCC No.6843) fermenting and producing DHA
Schizochytrium limacinum (Schizochytriumsp.CGMCC No.6843) slant preservation bacterial strain is accessed equipped with 400mL trainings
The 2L shaking flasks of foster base, with the rotating speed culture 24h of 200rpm at a temperature of 25 DEG C, complete bacterial strain activation culture.According to 0.4%
Inoculum concentration by shake-flask seed liquid access equipped with sterilizing wild Oryza species first class seed pot in, 28 DEG C of cultivation temperature, throughput 1vvm,
Tank pressure 0.02MPa, speed of agitator 50rpm cultivate 30h, complete first order seed Amplification Culture.According to 3% inoculum concentration by one-level kind
The seed liquor of sub- tank is accessed in the secondary seed tank equipped with sterilizing wild Oryza species, 28 DEG C of cultivation temperature, throughput 1vvm, tank pressure
0.02MPa, speed of agitator 75rpm cultivate 24h, complete secondary seed Amplification Culture.According to 3% inoculum concentration by secondary seed tank
Seed liquor access equipped with sterilizing wild Oryza species fermentation tank in.
28 DEG C of sweat cultivation temperature, throughput 1-2vvm, tank pressure 0.02-0.05MPa, speed of agitator 50-
100rpm, stream plus the carbon source containing crude glycerine after 20% pretreatment, control concentration of glucose carries out fermented and cultured in 1g/L.Using
Dissolved oxygen regulation strategy will ferment, and 30%, after fermentation 36h, DO is controlled 5% for DO controls before 36h, during refer to by control of DO
Mark, adjustment throughput, tank pressure and speed of agitator, and suitably fill into fresh culture or sterilized water after sterilizing.Send out in sweat
The ammoniacal liquor control zymotic fluid pH for Jia 25% is flowed before ferment 36h 6 or so, Feeding ammonia water can control zymotic fluid while supplementing nitrogen source
PH stablizes, and Feeding ammonia water is stopped after 36h, carries out nitrogen stress culture, and pH does not remake control.Ferment fermentation cylinder for fermentation liquid to 12h
It is divided into two culture by volume, half zymotic fluid stays in main tank and continues to cultivate, and second half zymotic fluid is transferred to by pressure differential method
Cultivate in the assistant tank of aseptic pressurize, fresh culture or sterilized water after appropriate sterilizing is filled into major-minor tank respectively, after point tank culture
Major-minor tank throughput, tank pressure and speed of agitator are adjusted accordingly by Dissolved oxygen regulation strategy.Zymotic fluid Portugal is detected in sweat
Grape sugar concentration, pH, Fungal biodiversity, thick grease yield and DHA change of production.
Terminate fermentation after culture 96h, it is 140g/L to determine biomass in zymotic fluid, and thick grease yield is 78.5g/L, DHA
Yield is 40.4g/L, and DHA productivity ratio is 10.1g/ (Ld).Table 9 below is gained compound lard aliphatic acid composition gas after fermentation
Facies analysis result：
Table 9：100m3Gained compound lard aliphatic acid composition after ferment tank
| Aliphatic acid is constituted | Content % |
| 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 |
Embodiment 9：Using Dissolved oxygen regulation strategy, nitrogen Regulation strategy and point tank training strategy to 100m
3
Fermentation tank fragmentation
The impact of chytrid (Schizochytriumsp.CGMCC No.6843) fermenting and producing DHA
Schizochytrium limacinum (Schizochytriumsp.CGMCC No.6843) slant preservation bacterial strain is accessed equipped with 400mL trainings
The 2L shaking flasks of foster base, with the rotating speed culture 24h of 200rpm at a temperature of 25 DEG C, complete bacterial strain activation culture.According to 0.4%
Inoculum concentration by shake-flask seed liquid access equipped with sterilizing wild Oryza species first class seed pot in, 28 DEG C of cultivation temperature, throughput 1vvm,
Tank pressure 0.02MPa, speed of agitator 50rpm cultivate 30h, complete first order seed Amplification Culture.According to 3% inoculum concentration by one-level kind
The seed liquor of sub- tank is accessed in the secondary seed tank equipped with sterilizing wild Oryza species, 28 DEG C of cultivation temperature, throughput 1vvm, tank pressure
0.02MPa, speed of agitator 75rpm cultivate 24h, complete secondary seed Amplification Culture.According to 3% inoculum concentration by secondary seed tank
Seed liquor access equipped with sterilizing wild Oryza species fermentation tank in.
28 DEG C of sweat cultivation temperature, throughput 1-2vvm, tank pressure 0.02-0.05MPa, speed of agitator 50-
100rpm, stream plus the carbon source containing crude glycerine after 20% pretreatment, control concentration of glucose carries out fermented and cultured in 5g/L.Using
Dissolved oxygen regulation strategy will ferment, and 50%, after fermentation 60h, DO is controlled 10% for DO controls before 60h, during refer to by control of DO
Mark, adjustment throughput, tank pressure and speed of agitator, and suitably fill into fresh culture or sterilized water after sterilizing.Send out in sweat
The ammoniacal liquor control zymotic fluid pH for Jia 45% is flowed before ferment 60h 7 or so, Feeding ammonia water can control zymotic fluid while supplementing nitrogen source
PH stablizes, and Feeding ammonia water is stopped after 60h, carries out nitrogen stress culture, and pH does not remake control.Ferment fermentation cylinder for fermentation liquid to 36h
It is divided into two culture by volume, half zymotic fluid stays in main tank and continues to cultivate, and second half zymotic fluid is transferred to by pressure differential method
Cultivate in the assistant tank of aseptic pressurize, fresh culture or sterilized water after appropriate sterilizing is filled into major-minor tank respectively, after point tank culture
Major-minor tank throughput, tank pressure and speed of agitator are adjusted accordingly by Dissolved oxygen regulation strategy.Zymotic fluid Portugal is detected in sweat
Grape sugar concentration, pH, Fungal biodiversity, thick grease yield and DHA change of production.
Terminate fermentation after culture 96h, it is 138g/L to determine biomass in zymotic fluid, and thick grease yield is 78.6g/L, DHA
Yield is 39.6g/L, and DHA productivity ratio is 9.9g/ (Ld).Table 10 below is gained compound lard aliphatic acid composition gas after fermentation
Facies analysis result：
Table 10：100m3Gained compound lard aliphatic acid composition after ferment tank
| Aliphatic acid is constituted | Content % |
| 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 |
Embodiment 10：Using Dissolved oxygen regulation strategy, nitrogen Regulation strategy and point tank training strategy to 100m
3
Fermentation tank fragmentation
The impact of chytrid (Schizochytriumsp.CGMCC No.6843) fermenting and producing DHA
Schizochytrium limacinum (Schizochytriumsp.CGMCC No.6843) slant preservation bacterial strain is accessed equipped with 400mL trainings
The 2L shaking flasks of foster base, with the rotating speed culture 24h of 200rpm at a temperature of 25 DEG C, complete bacterial strain activation culture.According to 0.4%
Inoculum concentration by shake-flask seed liquid access equipped with sterilizing wild Oryza species first class seed pot in, 28 DEG C of cultivation temperature, throughput 1vvm,
Tank pressure 0.02MPa, speed of agitator 50rpm cultivate 30h, complete first order seed Amplification Culture.According to 3% inoculum concentration by one-level kind
The seed liquor of sub- tank is accessed in the secondary seed tank equipped with sterilizing wild Oryza species, 28 DEG C of cultivation temperature, throughput 1vvm, tank pressure
0.02MPa, speed of agitator 75rpm cultivate 24h, complete secondary seed Amplification Culture.According to 3% inoculum concentration by secondary seed tank
Seed liquor access equipped with sterilizing wild Oryza species fermentation tank in.
28 DEG C of sweat cultivation temperature, throughput 1-2vvm, tank pressure 0.02-0.05MPa, speed of agitator 50-
100rpm, stream plus the carbon source containing crude glycerine after 20% pretreatment, control concentration of glucose carries out fermented and cultured in 2g/L.Using
Dissolved oxygen regulation strategy will ferment, and 35%, after fermentation 40h, DO is controlled 6% for DO controls before 40h, during refer to by control of DO
Mark, adjustment throughput, tank pressure and speed of agitator, and suitably fill into fresh culture or sterilized water after sterilizing.Send out in sweat
The ammoniacal liquor control zymotic fluid pH for Jia 38% is flowed before ferment 40h 6.5 or so, Feeding ammonia water can control fermentation while supplementing nitrogen source
Liquid pH stablizes, and Feeding ammonia water is stopped after 40h, carries out nitrogen stress culture, and pH does not remake control.Ferment fermentation cylinder for fermentation to 20h
Liquid is divided into two culture by volume, and half zymotic fluid stays in main tank and continues to cultivate, and second half zymotic fluid is shifted by pressure differential method
Cultivate into the assistant tank of aseptic pressurize, fill into fresh culture or sterilized water after appropriate sterilizing, point tank culture in major-minor tank respectively
Afterwards major-minor tank throughput, tank pressure and speed of agitator are adjusted accordingly by Dissolved oxygen regulation strategy.Zymotic fluid is detected in sweat
Concentration of glucose, pH, Fungal biodiversity, thick grease yield and DHA change of production.
Terminate fermentation after culture 96h, it is 139g/L to determine biomass in zymotic fluid, and thick grease yield is 78.2g/L, DHA
Yield is 38.0g/L, and DHA productivity ratio is 9.5g/ (Ld).Table 11 below is gained compound lard aliphatic acid composition gas after fermentation
Facies analysis result：
Table 11：100m3Gained compound lard aliphatic acid composition after ferment tank
Embodiment 11：Using Dissolved oxygen regulation strategy, nitrogen Regulation strategy and point tank training strategy to 100m
3
Fermentation tank fragmentation
The impact of chytrid (Schizochytriumsp.CGMCC No.6843) fermenting and producing DHA
Schizochytrium limacinum (Schizochytriumsp.CGMCC No.6843) slant preservation bacterial strain is accessed equipped with 400mL trainings
The 2L shaking flasks of foster base, with the rotating speed culture 24h of 200rpm at a temperature of 25 DEG C, complete bacterial strain activation culture.According to 0.4%
Inoculum concentration by shake-flask seed liquid access equipped with sterilizing wild Oryza species first class seed pot in, 28 DEG C of cultivation temperature, throughput 1vvm,
Tank pressure 0.02MPa, speed of agitator 50rpm cultivate 30h, complete first order seed Amplification Culture.According to 3% inoculum concentration by one-level kind
The seed liquor of sub- tank is accessed in the secondary seed tank equipped with sterilizing wild Oryza species, 28 DEG C of cultivation temperature, throughput 1vvm, tank pressure
0.02MPa, speed of agitator 75rpm cultivate 24h, complete secondary seed Amplification Culture.According to 3% inoculum concentration by secondary seed tank
Seed liquor access equipped with sterilizing wild Oryza species fermentation tank in.
28 DEG C of sweat cultivation temperature, throughput 1-2vvm, tank pressure 0.02-0.05MPa, speed of agitator 50-
100rpm, stream plus the carbon source containing crude glycerine after 40% pretreatment, control concentration of glucose carries out fermented and cultured in 4g/L.Using
Dissolved oxygen regulation strategy will ferment, and 45%, after fermentation 56h, DO is controlled 9% for DO controls before 56h, during refer to by control of DO
Mark, adjustment throughput, tank pressure and speed of agitator, and suitably fill into fresh culture or sterilized water after sterilizing.Send out in sweat
The ammoniacal liquor control zymotic fluid pH for Jia 42% is flowed before ferment 56h 7 or so, Feeding ammonia water can control zymotic fluid while supplementing nitrogen source
PH stablizes, and Feeding ammonia water is stopped after 56h, carries out nitrogen stress culture, and pH does not remake control.Ferment fermentation cylinder for fermentation liquid to 28h
It is divided into two culture by volume, half zymotic fluid stays in main tank and continues to cultivate, and second half zymotic fluid is transferred to by pressure differential method
Cultivate in the assistant tank of aseptic pressurize, fresh culture or sterilized water after appropriate sterilizing is filled into major-minor tank respectively, after point tank culture
Major-minor tank throughput, tank pressure and speed of agitator are adjusted accordingly by Dissolved oxygen regulation strategy.Zymotic fluid Portugal is detected in sweat
Grape sugar concentration, pH, Fungal biodiversity, thick grease yield and DHA change of production.
Terminate fermentation after culture 96h, it is 140g/L to determine biomass in zymotic fluid, and thick grease yield is 80.8g/L, DHA
Yield is 42.0g/L, and DHA productivity ratio is 10.5g/ (Ld).Table 12 below is gained compound lard aliphatic acid composition after fermentation
Gas phase analysis result：
Table 12：100m3Gained compound lard aliphatic acid composition after ferment tank
| Aliphatic acid is constituted | Content % |
| 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 |
Embodiment 12：Using Dissolved oxygen regulation strategy, nitrogen Regulation strategy and point tank training strategy to 100m
3
Fermentation tank fragmentation
The impact of chytrid (Schizochytriumsp.CGMCC No.6843) fermenting and producing DHA
Schizochytrium limacinum (Schizochytriumsp.CGMCC No.6843) slant preservation bacterial strain is accessed equipped with 400mL trainings
The 2L shaking flasks of foster base, with the rotating speed culture 24h of 200rpm at a temperature of 25 DEG C, complete bacterial strain activation culture.According to 0.4%
Inoculum concentration by shake-flask seed liquid access equipped with sterilizing wild Oryza species first class seed pot in, 28 DEG C of cultivation temperature, throughput 1vvm,
Tank pressure 0.02MPa, speed of agitator 50rpm cultivate 30h, complete first order seed Amplification Culture.According to 3% inoculum concentration by one-level kind
The seed liquor of sub- tank is accessed in the secondary seed tank equipped with sterilizing wild Oryza species, 28 DEG C of cultivation temperature, throughput 1vvm, tank pressure
0.02MPa, speed of agitator 75rpm cultivate 24h, complete secondary seed Amplification Culture.According to 3% inoculum concentration by secondary seed tank
Seed liquor access equipped with sterilizing wild Oryza species fermentation tank in.
28 DEG C of sweat cultivation temperature, throughput 1-2vvm, tank pressure 0.02-0.05MPa, speed of agitator 50-
100rpm, stream plus the carbon source containing crude glycerine after 40% pretreatment, control concentration of glucose carries out fermented and cultured in 3g/L.Using
Dissolved oxygen regulation strategy will ferment, and 38%, after fermentation 44h, DO is controlled 7% for DO controls before 44h, during refer to by control of DO
Mark, adjustment throughput, tank pressure and speed of agitator, and suitably fill into fresh culture or sterilized water after sterilizing.Send out in sweat
The ammoniacal liquor control zymotic fluid pH for Jia 40% is flowed before ferment 44h 6.5 or so, Feeding ammonia water can control fermentation while supplementing nitrogen source
Liquid pH stablizes, and Feeding ammonia water is stopped after 44h, carries out nitrogen stress culture, and pH does not remake control.Ferment fermentation cylinder for fermentation to 22h
Liquid is divided into two culture by volume, and half zymotic fluid stays in main tank and continues to cultivate, and second half zymotic fluid is shifted by pressure differential method
Cultivate into the assistant tank of aseptic pressurize, fill into fresh culture or sterilized water after appropriate sterilizing, point tank culture in major-minor tank respectively
Afterwards major-minor tank throughput, tank pressure and speed of agitator are adjusted accordingly by Dissolved oxygen regulation strategy.Zymotic fluid is detected in sweat
Concentration of glucose, pH, Fungal biodiversity, thick grease yield and DHA change of production.
Terminate fermentation after culture 96h, it is 141g/L to determine biomass in zymotic fluid, and thick grease yield is 81.2g/L, DHA
Yield is 42.4g/L, and DHA productivity ratio is 10.6g/ (Ld).Table 13 below is gained compound lard aliphatic acid composition after fermentation
Gas phase analysis result：
Table 13：100m3Gained compound lard aliphatic acid composition after ferment tank
Embodiment 13：Using Dissolved oxygen regulation strategy, nitrogen Regulation strategy and point tank training strategy to 100m
3
Fermentation tank fragmentation
The impact of chytrid (Schizochytriumsp.CGMCC No.6843) fermenting and producing DHA
Schizochytrium limacinum (Schizochytriumsp.CGMCC No.6843) slant preservation bacterial strain is accessed equipped with 400mL trainings
The 2L shaking flasks of foster base, with the rotating speed culture 24h of 200rpm at a temperature of 25 DEG C, complete bacterial strain activation culture.According to 0.4%
Inoculum concentration by shake-flask seed liquid access equipped with sterilizing wild Oryza species first class seed pot in, 28 DEG C of cultivation temperature, throughput 1vvm,
Tank pressure 0.02MPa, speed of agitator 50rpm cultivate 30h, complete first order seed Amplification Culture.According to 3% inoculum concentration by one-level kind
The seed liquor of sub- tank is accessed in the secondary seed tank equipped with sterilizing wild Oryza species, 28 DEG C of cultivation temperature, throughput 1vvm, tank pressure
0.02MPa, speed of agitator 75rpm cultivate 24h, complete secondary seed Amplification Culture.According to 3% inoculum concentration by secondary seed tank
Seed liquor access equipped with sterilizing wild Oryza species fermentation tank in.
28 DEG C of sweat cultivation temperature, throughput 1-2vvm, tank pressure 0.02-0.05MPa, speed of agitator 50-
100rpm, stream plus the carbon source containing crude glycerine after 40% pretreatment, control concentration of glucose carries out fermented and cultured in 5g/L.Using
Dissolved oxygen regulation strategy will ferment, and 42%, after fermentation 52h, DO is controlled 8% for DO controls before 52h, during refer to by control of DO
Mark, adjustment throughput, tank pressure and speed of agitator, and suitably fill into fresh culture or sterilized water after sterilizing.Send out in sweat
The ammoniacal liquor control zymotic fluid pH for Jia 40% is flowed before ferment 52h 6.5 or so, Feeding ammonia water can control fermentation while supplementing nitrogen source
Liquid pH stablizes, and Feeding ammonia water is stopped after 52h, carries out nitrogen stress culture, and pH does not remake control.Ferment fermentation cylinder for fermentation to 26h
Liquid is divided into two culture by volume, and half zymotic fluid stays in main tank and continues to cultivate, and second half zymotic fluid is shifted by pressure differential method
Cultivate into the assistant tank of aseptic pressurize, fill into fresh culture or sterilized water after appropriate sterilizing, point tank culture in major-minor tank respectively
Afterwards major-minor tank throughput, tank pressure and speed of agitator are adjusted accordingly by Dissolved oxygen regulation strategy.Zymotic fluid is detected in sweat
Concentration of glucose, pH, Fungal biodiversity, thick grease yield and DHA change of production.
Terminate fermentation after culture 96h, it is 142g/L to determine biomass in zymotic fluid, and thick grease yield is 80.2g/L, DHA
Yield is 41.2g/L, and DHA productivity ratio is 10.3g/ (Ld).Table 14 below is gained compound lard aliphatic acid composition after fermentation
Gas phase analysis result：
Table 14：100m3Gained compound lard aliphatic acid composition after ferment tank
| Aliphatic acid is constituted | Content % |
| 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 |
Extract crude oil
Embodiment 14
The DHA zymotic fluid 100L that Example 5 is obtained, after heated inactivation treatment, cloth are transported to distributing device by zymotic fluid
In material chamber, cloth parcel is carried out, after cloth is finished, carry out one-level squeezing, by the way of progressively pressurizeing, reached in the 2h of setting
To the 30MPa pressure of setting, pressurize 2h is to basic without water droplet outflow.Remove one-level squeezing cage, change two grades of squeezing cages, push
At two grades of squeezings, two grades of squeezings are carried out, by the way of progressively pressurizeing, the 100MPa pressure of setting are reached in the 2h of setting,
Pressurize 2h flows out to basic without oil droplet, and collection squeezes out the DHA crude oils for coming, and DHA crude oil 6.8kg are obtained, and zymotic fluid is to hair
Oil yield 82.3%.Remove two grades of squeezing cages, the microalgae dregs of rice pressed are separated with filter cloth.
Embodiment 15
The DHA zymotic fluid 100L that Example 5 is obtained, after heated inactivation treatment, cloth are transported to distributing device by zymotic fluid
In material chamber, cloth parcel is carried out, after cloth is finished, carry out one-level squeezing, by the way of progressively pressurizeing, reached in the 5h of setting
To the 40MPa pressure of setting, pressurize 4h is to basic without water droplet outflow.Remove one-level squeezing cage, change two grades of squeezing cages, push
At two grades of squeezings, two grades of squeezings are carried out, by the way of progressively pressurizeing, the 150MPa pressure of setting are reached in the 5h of setting,
Pressurize 4h flows out to basic without oil droplet, and collection squeezes out the DHA crude oils for coming, and DHA crude oil 7.3kg are obtained, and zymotic fluid is to hair
Oil yield 88.4%.Remove two grades of squeezing cages, the microalgae dregs of rice pressed are separated with filter cloth.
Embodiment 16
The DHA zymotic fluid 150L that Example 5 is obtained, after heated inactivation treatment, are centrifuged using butterfly centrifugal machine, are removed
Centrifugation light liquid, obtains the zymotic fluid after 95L concentrations, and the zymotic fluid after concentration is transported in distribution cavity with distributing device, carries out cloth
Parcel, after cloth is finished, carries out one-level squeezing, by the way of progressively pressurizeing, the 40MPa pressures of setting is reached in the 5h of setting
Power, pressurize 4h flows out to basic without water droplet.Remove one-level squeezing cage, change two grades of squeezing cages, push at two grades of squeezings, carry out
Two grades of squeezings, using progressively pressurizeing by the way of, reach the 150MPa pressure of setting in the 5h for setting, and pressurize 4h to not having substantially
Oil droplet flows out, and collection squeezes out the DHA crude oils for coming, and DHA crude oil 10.8kg are obtained, and zymotic fluid is to crude oil yield 87.2%.Go
Fall two grades of squeezing cages, the microalgae dregs of rice pressed are separated with filter cloth.
Embodiment 17
The DHA zymotic fluid 300L that Example 5 is obtained, after heated inactivation treatment, in adding spray dryer, setting spray
Mist pressure 5MPa, 180 DEG C of EAT, 80 DEG C of leaving air temp is spray dried to powder, obtains DHA microalgae powder 52.5kg.DHA
Microalgae powder is transported in distribution cavity, carries out cloth parcel, after cloth is finished, flexible squeezing squeezing is carried out, using the side progressively pressurizeed
Formula, reaches the 100MPa pressure of setting in the 2h of setting, and pressurize 2h flows out to basic without oil droplet, and collection squeezes out what is come
DHA crude oils, are obtained DHA crude oil 22.1kg, and zymotic fluid is to crude oil yield 89.2%.
Embodiment 18
The DHA zymotic fluid 300L that Example 5 is obtained, after heated inactivation treatment, in adding spray dryer, setting spray
Mist pressure 6MPa, 200 DEG C of EAT, 100 DEG C of leaving air temp is spray dried to powder, obtains DHA microalgae powder 51.0kg.DHA
Microalgae powder is transported in distribution cavity, carries out cloth parcel, after cloth is finished, flexible squeezing squeezing is carried out, using the side progressively pressurizeed
Formula, reaches the 150MPa pressure of setting in the 4h of setting, and pressurize 4h flows out to basic without oil droplet, and collection squeezes out what is come
DHA crude oils, are obtained DHA crude oil 22.8kg, and zymotic fluid is to crude oil yield 92.0%.
Embodiment 19
The DHA zymotic fluid 300L that Example 5 is obtained, after heated inactivation treatment, are centrifuged using butterfly centrifugal machine, are removed
Centrifugation light liquid, obtains the zymotic fluid after 200L concentrations, in adding spray dryer, sets atomisation pressure 8MPa, EAT 220
DEG C, 110 DEG C of leaving air temp is spray dried to powder, obtains DHA microalgae powder 47.5kg.DHA microalgae powders are transported in distribution cavity, are entered
Row cloth is wrapped up, and after cloth is finished, carries out flexible squeezing squeezing, by the way of progressively pressurizeing, is reached in the 4h of setting and is set
Fixed 150MPa pressure, pressurize 4h flows out to basic without oil droplet, and collection squeezes out the DHA crude oils for coming, and DHA crude oils are obtained
23.3kg, zymotic fluid is to crude oil yield 94.0%.
Comparative example 1
The DHA zymotic fluid 300L that Example 5 is obtained, after heated inactivation treatment, are centrifuged using butterfly centrifugal machine, are removed
Centrifugation light liquid, obtains the zymotic fluid after 200L concentrations, in adding spray dryer, sets atomisation pressure 8MPa, EAT 220
DEG C, 110 DEG C of leaving air temp is spray dried to powder, obtains DHA microalgae powder 47.8kg.Twin-screw squeezer is first preheated to 80 DEG C,
DHA microalgae powders are added in twin-screw squeezer and is extracted oil, obtain DHA crude oil 11.6kg, zymotic fluid is to crude oil yield 46.81%.
Crude oil is refined
Embodiment 20
The DHA crude oils obtained by the merging of 10kg embodiments 14,15,16 are taken, by aquation, decolouring, molecular clock step essence is carried out
Refining.
Aquation：10kg DHA crude oils, are warming up to 75 DEG C, add the purified water that 1kg temperature is 80 DEG C, stir 30min, stir
30 turns/min of speed is mixed, 2h is stood, point sub-cloud obtains aquation oil 9.75kg.
Decolourize：Aquation oil is warming up to 100 DEG C, controls vacuum -0.075MPa, vacuum dehydration 1h, is then cooled to 70 DEG C,
Decolorising agent (191g activated carbons and 286g atlapulgites) is added, decolouring 0.5h is stirred, stops stirring, filtered and remove decolorising agent, obtained
To bleached oil 9.30kg.
Molecular clock：Bleached oil enters three-level molecular clock, control first order vacuum 90Pa or so, 160 DEG C of temperature, goes
Except the light component of the first order, heavy constituent enters second level molecular clock, control secondary vacuum degree 40Pa or so, 200 DEG C of temperature, goes
Except second level light component, heavy constituent enters third level molecular clock, control third level vacuum 3Pa or so, 220 DEG C of temperature, goes
Except third level light component, heavy constituent is collected, deodorization is finished, be cooled to 30 DEG C, add antioxidant, packaging obtains DHA product oils
9.10kg, assay is shown in Table 15.
Table 15：DHA product oil assays
Embodiment 21
The DHA crude oils obtained by the merging of 10kg embodiments 14,15,16 are taken, by aquation, decolouring, molecular clock step essence is carried out
Refining.
Aquation：10kg DHA crude oils, are warming up to 85 DEG C, add the purified water that 1kg temperature is 90 DEG C, stir 15min, stir
90 turns/min of speed is mixed, 4h is stood, point sub-cloud obtains aquation oil 9.70kg.
Decolourize：Aquation oil is warming up to 110 DEG C, controls vacuum -0.075MPa, vacuum dehydration 0.5h, is then cooled to 80
DEG C, decolorising agent (192g activated carbons and 288g atlapulgites) is added, decolouring 1h is stirred, stop stirring, filter and remove decolorising agent, obtain
To bleached oil 9.31kg.
Molecular clock：Bleached oil enters three-level molecular clock, control first order vacuum 90Pa or so, 200 DEG C of temperature, goes
Except the light component of the first order, heavy constituent enters second level molecular clock, control secondary vacuum degree 40Pa or so, 220 DEG C of temperature, goes
Except second level light component, heavy constituent enters third level molecular clock, control third level vacuum 3Pa or so, 250 DEG C of temperature, goes
Except third level light component, collect heavy constituent, molecular clock is finished, be cooled to 30 DEG C, add antioxidant, packaging, obtain DHA into
Product oil 9.03kg, assay is shown in Table 16.
Table 16：DHA product oil assays
Embodiment 22
The DHA crude oils obtained by the merging of 10kg embodiments 17,18,19 are taken, is refined by the method for refining of embodiment 20, obtained
To DHA product oil 9.13kg, assay is shown in Table 17.
Table 17：DHA product oil assays
Embodiment 23
The DHA crude oils obtained by the merging of 10kg embodiments 17,18,19 are taken, is refined by the method for refining of embodiment 21, obtained
To DHA product oil 9.08kg, assay is shown in Table 18.
Table 18：DHA product oil assays
Comparative example 2
Take 10kg embodiments 17,18,19 merge obtained by DHA crude oils, by traditional method of refining be aquation, alkali refining, decolouring,
Deodorising step is refined.
Aquation：10kg DHA crude oils, are warming up to 85 DEG C, add the purified water that 1kg temperature is 90 DEG C, stir 15min, stir
90 turns/min of speed is mixed, 4h is stood, point sub-cloud obtains aquation oil 9.71kg.
Alkali refining：Aquation oil thermal insulating adds the NaOH solution of 1L concentration 10% (mass fraction) at 75 DEG C, stirs 30min, stirs
30 turns/min of speed is mixed, 4h is stood, soap stock is separated, alkali refining oil is obtained, under alkali refining oil stirring state oily weight 10% is sprayed into
80 DEG C of purified waters are washed, and the time control that adds water is added using 2h is stood after water in 10-30min, and separate aqueous layer, repetition is washed
Wash 2 times, obtain alkali refining oil 8.92kg.
Decolourize：Alkali refining oil is warming up to 110 DEG C, controls vacuum -0.075MPa, vacuum dehydration 0.5h, is then cooled to 80
DEG C, decolorising agent (186g activated carbons and 280g atlapulgites) is added, decolouring 1h is stirred, stop stirring, filter and remove decolorising agent, obtain
To bleached oil 8.49kg.
Deodorization：Bleached oil moves into odor removing pot, and water flowing steam carries out deodorization, deodorization temperature control at 185 DEG C, vacuum degree control
Within 600Pa, deodorization time 2h, deodorization is finished, and stops logical steam, is cooled to 30 DEG C, adds antioxidant, and packaging is obtained
DHA product oil 8.31kg, testing result is shown in Table 19.
Table 19：DHA product oil assays
Claims (23)
1. a kind of method cultivated for producing the microorganism of DHA, wherein：
From the beginning of the 36-60 hours of fermentation tank culture, saturation dissolved oxygen is controlled in 5%-10%；And/or
From the beginning of the 36-60 hours of fermentation tank culture, do not add nitrogen source or the addition of nitrogen source is reduced into more than 50%.
2. cultural method according to claim 1, wherein：
40-56 hours, 44-52 hours, 46-50 hours from fermentation tank culture or from the beginning of 48 hours, by dissolved oxygen control
In 5%-10%, and/or
40-56 hours, 44-52 hours, 46-50 hours from fermentation tank culture or from the beginning of 48 hours, do not add nitrogen
Source.
3. cultural method according to claim 1 and 2, wherein, before saturation dissolved oxygen is controlled for 5%-10%,
Saturation dissolved oxygen is 30%-50%.
4. the cultural method according to any claim in claims 1 to 3, wherein, the pH of the zymotic fluid is 6.0-
7.0；Preferably, the concentration of glucose keeps 1-5g/L in zymotic fluid.
5. the cultural method according to any claim in Claims 1-4, wherein, the nitrogen source is ammoniacal liquor, preferably
The ammoniacal liquor of the ammoniacal liquor of 25%-45%, more preferably 35%-45%, particularly preferably 40% ammoniacal liquor.
6. the cultural method according to any claim in claim 1 to 5, wherein, the fermentation tank culture also includes
In the 12-36 hours (such as 24 hours) of fermentation tank culture starting, the step of carry out point tank culture；For example, it is divided into two or more
Multiple fermentation tanks carry out fermentation tank culture.
7. the cultural method according to any claim in claim 1 to 6, it also included before fermentation tank culture
The step of inoculation and seed Amplification Culture；
Preferably, the seed Amplification Culture includes first order seed Amplification Culture and secondary seed Amplification Culture；
Preferably, the first order seed Amplification Culture comprises the steps：
Inoculum concentration according to 0.4%-1% accesses shake-flask seed liquid in the first class seed pot equipped with sterilizing wild Oryza species, culture
25 DEG C -32 DEG C of temperature, throughput 1-2vvm, tank pressure 0.02-0.05MPa, speed of agitator 50-100rpm cultivate 30-
35h, completes first order seed Amplification Culture；
Preferably, the secondary seed Amplification Culture comprises the steps：
The seed liquor of first class seed pot is accessed the secondary seed tank equipped with sterilizing wild Oryza species by the inoculum concentration according to 1%-3%
In, 25 DEG C -32 DEG C of cultivation temperature, throughput 1-2vvm, tank pressure 0.02-0.05MPa, speed of agitator 50-100rpm, culture
20-25h, completes secondary seed Amplification Culture.
8. cultural method according to claim 7, before being additionally included in inoculation sum Amplification Culture, carries out activation culture
Step；Preferably, the temperature of the activation culture is 25 DEG C -32 DEG C, and the time is 20-25h.
9. the cultural method according to claim 7 or 8, wherein, the fermentation tank culture comprises the steps：
Inoculum concentration according to 1%-3% accesses the seed liquor of secondary seed tank in the fermentation tank equipped with sterilizing wild Oryza species, training
25 DEG C -32 DEG C of foster temperature, throughput 1-2vvm, tank pressure 0.02-0.05MPa, speed of agitator 50-100rpm, is fermented
Tank culture.
10. the cultural method according to any claim in claim 1 to 9, wherein the microorganism for producing DHA is
Schizochytrium limacinum；Preferably, the schizochytrium limacinum is CGMCC No.6843, ATCC No.20888, ATCC selected from preserving number
The bacterial strain of No.20889, ATCC No.28209 or ATCC MYA-1381.
A kind of 11. zymotic fluids of microorganism, its cultural method by described in any claim in claim 1 to 10 is obtained.
A kind of 12. methods of extraction DHA crude oils, comprise the steps：
1) zymotic fluid of the microorganism for producing DHA is carried out into processed；
2) by step 1) product carry out flexible squeezing, obtain DHA crude oils.
13. extracting methods according to claim 12, wherein, step 1) in, the zymotic fluid is schizochytrium limacinum fermentation liquid；
Preferably, the zymotic fluid is the zymotic fluid described in claim 11.
14. extracting methods according to claim 12, wherein, step 1) in, the processed is selected from following any
It is a kind of, two or three：
Centrifugation, the flexible squeezing of the first order, drying are for example spray-dried；
Preferably, the processed includes successively centrifugation and the flexible squeezing of the first order, or includes centrifugation successively and spray dry
It is dry；
Preferably, the atomisation pressure of the spray drying be 4-8MPa, 160 DEG C -220 DEG C of EAT, leaving air temp 80
DEG C -120 DEG C.
15. extracting methods according to claim 12, wherein, step 1) in, the flexible squeezing of the first order is using progressively
Pressuring method, sets pressure as 20-40MPa, and pressing time is 1-6h, reaches pressurize 1-4h after setting pressure.
16. extracting methods according to claim 12, wherein, step 2) in, the flexible squeezing is using progressively pressurization side
Formula, sets pressure as 50-150MPa, and pressing time is 1-6h, reaches pressurize 1-4h after setting pressure.
A kind of 17. DHA crude oils, its extracting method by described in any claim in claim 12 to 16 is obtained.
A kind of 18. methods of purifying DHA crude oils, including carrying out aquation, decolourize and the step of molecular clock DHA crude oils；It is preferred that
Ground, the DHA crude oils are the DHA crude oils described in claim 17.
19. purification process according to claim 18, wherein, the aquation comprises the steps：
DHA crude oils are heated into 70 DEG C -85 DEG C, add the ratio of 50-150g water to add 75 DEG C of -90 DEG C of water in 1kg crude oils,
Stirring 10-60min, mixing speed 30-90 turns/min, stands 1-6h, and lower floor's phosphatide is removed in layering, obtains aquation oil.
20. purification process according to claim 18, wherein, the decolouring comprises the steps：
Hydrated product is warming up into 90 DEG C -110 DEG C, vacuum≤- 0.07MPa, vacuum dehydration 0.5-2h is controlled, is then lowered the temperature
To 60 DEG C -80 DEG C, decolorising agent (such as activated carbon of aquation weight of oil 1%-3% and 2%-4% atlapulgites) is added, stirred
0.5-1h is mixed, stops stirring, filtered and remove decolorising agent, obtain bleached oil.
21. purification process according to claim 18, wherein, the molecular clock is three-level molecular clock；
Preferably, the molecular clock comprises the steps：
Bleached oil is entered into three-level molecular clock, control first order vacuum≤100Pa, 150 DEG C -200 DEG C of temperature remove the
The light component of one-level；The first heavy constituent for obtaining enters second level molecular clock, control secondary vacuum degree≤50Pa, temperature 180
DEG C -220 DEG C, remove second level light component；The second heavy constituent for obtaining enters third level molecular clock, controls third level vacuum
200 DEG C -250 DEG C of degree≤5Pa, temperature, removes third level light component, obtains the 3rd heavy constituent, is DHA product oils.
Preferably, molecular clock 1 time or multiple is repeated.
A kind of 22. DHA product oils, its purification process by described in any claim in claim 18 to 21 is obtained.
The method of a kind of 23. utilization micro-organisms DHA or the product (such as DHA product oils) containing DHA, including：
In claim 1 to 10 described in any claim culture for produce DHA microorganism method,
The method of the extraction DHA crude oils in claim 12 to 16 described in any claim, and/or
The method of the purifying DHA crude oils in claim 18 to 21 described in any claim.
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| CN201611270522.9A CN106636235B (en) | 2016-12-30 | 2016-12-30 | Method for producing DHA (docosahexaenoic acid) by microbial fermentation |
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