CN104450810A - Method of preparing arachidonic acid - Google Patents
Method of preparing arachidonic acid Download PDFInfo
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- CN104450810A CN104450810A CN201410800525.3A CN201410800525A CN104450810A CN 104450810 A CN104450810 A CN 104450810A CN 201410800525 A CN201410800525 A CN 201410800525A CN 104450810 A CN104450810 A CN 104450810A
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Abstract
The invention discloses a method of preparing arachidonic acid, and relates to arachidonic acid. The method comprises the following steps: optimizing the culture condition of producing ARA from mortierella alpine, fermenting in a basal culture medium in batches, fermenting in an optimization culture medium in batches by taking yeast powder and corn steep liquor as nitrogen sources, then carrying out high-sugar acclimation on the mortierella alpine through a fermentation tank enlargement experiment carried out by taking the yeast powder as the nitrogen source, fermenting the acclimated strains in a mixed nitrogen source optimization culture medium in batches within a capacity of 2L, filling the strains in the mixed nitrogen source optimization culture medium in a fermentation tank with the capacity of 2L, feeding 600g/L glucose when the concentration of the glucose is reduced to 10g/L to control residual glucose at 10g/L, culturing for 168h and then starting to harvest dry cells, oil and the ARA.
Description
Technical field
The present invention relates to arachidonic acid, especially relate to one and prepare arachidonic method.
Background technology
Arachidonic acid (Arachidonic Acid, be called for short ARA, namely 5,8,11,14-eicosatetraenoic acid) belong to the serial long chain polyunsaturated fatty acids of ω-6, owing to not possessing corresponding enzyme system in human body to synthesize specific unsaturated double-bond, therefore being considered to essential fatty acid, is that many 20 carbon derivatives are as PGE
2(PGE
2), prostacyclin (PGI
2), thromboxane A
2(TXA
2), leukotriene Leukotirene B
4(LTB
4) and C
4(LTC
4) direct precursor.ARA also has different kinds of ions channeling in control agent, controls membrane passage, maintains many important physiological functions such as body water-retentivity.Research simultaneously shows, arachidonic acid has very important meaning to the brain development of baby and the perfect of sight function, arachidonic acid also has significant effect (Roger et al.Biotechnology Bioengineering to the prevention of the diseases such as hypertension, hyperlipidemia, diabetes, virus infection and treatment, 2010,9 (20): 245-257).Therefore, arachidonic acid is a kind of nutrition-fortifying agent, is again the important healthcare products of the mankind.ARA plays as essential fatty acid the effect that can not be substituted, and has a extensive future.
ARA main source has plant, animal tissues, fish oil and microorganism and microalgae etc., but ARA content in animal tissues is low, originate also limited, and fermentable rule opens new way for producing ARA, it has not by starting material and weather restriction, growth cycle is short, culture process is simple, ARA content high, has become the focus of research both at home and abroad in recent years.Carry out the microbe research producing polyunsaturated fatty acid (PFUAs) in the world and mainly concentrate on the following aspects: (1) produces the isolation and screening of PFUAs bacterial classification; (2) optimization of culture condition and zymotechnique; (3) research (comprising batch, feed supplement, continuous culture process etc.) of training method; (4) separation and purification of PFUAs and Structural Identification.At present, people only find zygomycetes be compare have researching value a guiding principle, and general good mortierella, thinks that it produces the most potential bacterial classification of ARA, EPA, DHA.At present, the national leavened prod of ARA that successively come out such as Japan, the U.S., the domestic starting of the research in this field is late, though produce ARA aspect to fermentable to have done some researchs, but it is on the low side also to there is biomass, the problems such as fat content is lower, still have certain distance from large-scale industrial production.
The open one of Chinese patent CN101613724 utilizes Mortierella alpina mycelium to prepare arachidonic method, includes following step: first in constant-temperature table by the deep drainpipe of Mortierella alpina inoculum size; Transfer again in the vessel that fermention medium is housed, fermentation liquid is obtained after cultivation, wherein growth medium is or/and the nitrogenous source of fermention medium replaces the conventional nitrogenous source of part with the Mortierella alpine mould dregs of rice, and it is 10% ~ 90% that the nitrogenous source in the Mortierella alpine mould dregs of rice substitutes than by percentage to the quality; Fermentation liquid after filtration, the mould organism of collecting, through washing, dry, pulverizing, adopt non-polar solvent to carry out lixiviate or squeezing, results grease, the grease of results obtains target product through processing further, mould organism is remaining solid material after non-polar solvent lixiviate, be the Mortierella alpine mould dregs of rice that can be recycled, this invention utilizes the waste produced in Mortierella alpina fermentative production arachidonic acid process to obtain recycle, avoids the discharge of environmental pollutant.
Summary of the invention
One is the object of the present invention is to provide to prepare arachidonic method.
The present invention includes following steps:
1) be optimized the culture condition that Mortierella alpina produces ARA by shake flat experiment, the method for described optimization is equipped with 50mL basic medium (glucose 40g/L for getting the access of 4mL seed liquor; Yeast powder 5g/L; MgSO
4-7H
2o 0.1g/L; KH
2pO
40.5g/L; CaCO
30.05g/L; ZnSO
4-7H
2o 0.1g/L; PH 6.0) 250mL triangular flask, 28 DEG C, 150rpm cultivates 5 ~ 6d, be optimized substratum: glucose 60g/L; Yeast powder or corn steep liquor 10g/L; L-glutamic acid 1.0g/L; MgSO
4-7H
2o0.1g/L; KH
2pO
42.0g/L; CaCO
30.05g/L; ZnSO
4-7H
2o 0.1g/L; Mixed trace elements 1mL/L; Mixed vitamin 1mL/L; PH is adjusted to 6.0;
2) high sugar domestication is carried out to Mortierella alpina and in mixed nitrogen Optimal Medium, 2L batch fermentation is carried out to the bacterial classification after domestication, mixed nitrogen Optimal Medium is loaded in 2L fermentor tank, when being down to 10g/L when glucose concn, stream adds 600g/L glucose, make residual sugar control at 10g/L, start to gather in the crops dry mycelium, grease and ARA after cultivating 168h.
In step 1) in, described mixed trace elements can comprise FeCl
36H
2o, H
3bO
3, MnCl
24H
2o, CoCl
26H
2o, CuSO
45H
2o, NiSO
46H
2o etc.; Described mixed vitamin can comprise vitamins B
12, vitamin H, vitamins B
1, calcium pantothenate etc.
In step 2) in, the concrete grammar of described 2L batch fermentation can be: first adopt solid medium domestication, (sugar degree is respectively 2% bacterial classification to be coated step by step the high sugar flat board of gradient, 5%, 7%, 10% and 15%) cultivate, (sugar degree is respectively 3% to the high sugar of two kinds of gradients containing different nitrogen sources to select the strain transfer that can tolerate 10% high glucose concentration flat board after solid domestication, 4%, 5% and 6%) tame in liquid nutrient medium, then to the bacterial classification after this domestication, use step 2) described in mixed nitrogen Optimal Medium carry out batch fermentation, described dry mycelium can obtain 58g/L, and grease can obtain 28g/L, and ARA can obtain 16g/L.
Described Mortierella alpina can inoculate appropriate mortierella in the shaking flask being loaded with substratum, is placed on shaking table and cultivates, and rotating speed is 100 ~ 300rpm, is 150 ~ 200rpm preferably.
The moiety of the substratum that the present invention relates to, such as carbon source, nitrogenous source can be different.Carbon source can use sucrose, maltose, dextrin, starch etc., and glucose is the carbon source be comparatively suitable for, and source is simple, and cheap, usage quantity is 30 ~ 80g/L.Shake-flask seed is cultivated, generally usage quantity is reduced to 10 ~ 30g/L, consumption can be selected according to the time of cultivating and required biomass density.Specifically suitable consumption is selected, and those skilled in the art can by the kind of relating operation determination carbon source and suitable amounts.Meanwhile, visual microbial growth situation, selects disposablely add whole carbon source or fill in batches.Nitrogenous source can select at least one in yeast powder, corn steep liquor, peptone, soyflour etc., yeast powder or corn steep liquor are considered to reasonable nitrogenous source, add concentration at 5 ~ 15g/L, those skilled in the art can determine kind and the consumption of nitrogenous source by related experiment operation.
Can the single nitrogenous source of choice for use or mixed nitrogen, better result uses the suitable nitrogen source that mixed nitrogen (corn steep liquor, yeast powder) is fermentor cultivation, and the mass ratio of the two is larger for the accumulation impact of grease, the statistical methods such as response surface analysis can be utilized to optimize the combination of carbon nitrogen source further, and good two kinds of nitrogenous source ratios are 3:8 (w/w).
Temperature will start to produce EPA in a large number lower than 20 DEG C of Mortierella alpinas, and fostering requirement obtains the ARA of high level, and lower simultaneously as far as possible or avoid producing EPA, culture temperature remains on 25 ~ 30 DEG C usually.Fermentable produces PUFAs, and its production peak is generally in logarithmic phase later stage or early stage stationary phase, therefore will obtain the biomass of maximum possible and the content of total fatty acids within the shortest time, and in general, the time of the suitableeest results ARA is at 5th ~ 6 days.
The pH scope of fungal growth is comparatively wide, more prefers to and grows in meta-acid environment, and pH all can normal growth between 4.0 to 8.0, and find in implementation process of the present invention, initial pH 6.0 ~ 6.5, in culturing process, pH is conducive to the accumulation of ARA between 7 ~ 8; Be conducive to the accumulation of biomass between pH 4 ~ 5, in culturing process can adjustment pH level stage by stage to adapt to required growing environment.
Inoculum size can at 2% to 14% (V/V), inoculum size is low, biomass does not reach required level, inoculum size is high, will suppress the continued growth of thalline to a certain extent at biomass accumulation, in shake-flask culture, inoculation controls at 3% ~ 5% (V/V), and in fermentor cultivation, inoculum size controls obtain good result at 8% ~ 10% (V/V).
In substratum, carbon-nitrogen ratio affects the accumulation of fungal oil, and carbon-nitrogen ratio, between 10: 1 to 60: 1, is preferably 40: 1 to 50: 1, sterilizing can be selected in culturing process before disposable add or cultivate start after add in batches.
Microbial growth needs the somatomedins such as VITAMIN, and when being deficient in vitamin in culture environment, the content of intracellular rna, DNA and protein can decline.Add vitamins B
1, vitamins B
12, vitamin H and calcium pantothenate be conducive to the accumulation of ARA most, its consumption is respectively between 50 ~ 150mg/L, 300 ~ 600 μ g/L, 400 ~ 600 μ g/L and 80 ~ 150mg/L.L-glutamic acid can improve the activity of G6PDH in cell, for the growth of mortierella cell provides nucleic acid raw material, thus is closely related with arachidonic synthesis, and the L-glutamic acid adding 0.5 ~ 2g/L is in the medium conducive to the accumulation of ARA.
In order to the stability in the sugared ability of the consumption improving Mortierella alpina and preservation process, can carry out high sugar domestication to bacterial classification and cultivate, found that after domestication, the consumption sugar ability of bacterial classification and oil-producing capacity obviously promote, and the content of ARA is multiple growth.
Fermentation culture process can adopt batch fermentation or fed-batch fermentation, batch fermentation requires that nutritive substance is disposable and adds, nutrient inventory is too high may extend lag phase in the growth of cultivating Antifungi in early stage, fermentation period extends thereupon, and nutrient inventory is too low, the phase cannot meet the nutritional supplementation of fungal growth needs after incubation.Those skilled in the art, can operate by series of experiments the functional leaves thing content determining to be suitable for; In fed-batch fermentation culturing process, must detect the nutritive substance in nutrient solution, particularly when glucose concn is less than 5g/L, need to supplement glucose in time, this needs sterilizing respectively., in implementation process, may there is foaming phenomena in above-mentioned two kinds of training methods, this does not wish to occur, can select between sterilizing, to add proper quantity of defoaming agent or when foaming phenomena occurs, in time add defoamer and prevent excess foam.
According to the fermentation condition of above-mentioned optimization Mortierella alpina, higher biomass and total fat content can be obtained, can often 12h or 24h sampling and measuring biomass, grease and ARA content, or cultivate and terminate harvesting biomass mensuration.
Can gather in the crops biological plastid by multiple method, such as filtration, vacuum filtration, centrifugal, membrane sepn, flocculation sediment etc., general, alternative costs low and the filtration of handled easily or vacuum filtration harvesting biomass body.After results, can select to adopt dry mycelium or wet thallus to carry out the extraction of grease, in this implementation process, water content is dried to thalline and is less than 4%, now, use alcohols, alkane such as the oil and grease extractings such as normal hexane, normal heptane such as acetone, ethanol, Virahol to be comparatively be suitable for, but normal hexane is best.Dried thalline adds normal hexane, carries out grinding, homogenate or subtraction wall-breaking abstraction, and the extraction liquid obtained carries out centrifugation and gets upper strata, evaporation removing organic solvent wherein, obtains total grease i.e. thick oil.
The present invention compared with prior art has the following advantages and effect: raw material is easily purchased, moderate, substratum preparation is convenient, zymotechnique is easy, arachidonic acid synthesis is stable, grease yield is 16 ~ 28g/L, wherein arachidonic acid yield is at 6.5 ~ 16g/L, solves arachidonic fermentation production efficiency and the problem yielded poorly, is applicable to suitability for industrialized production.
Accompanying drawing explanation
Fig. 1 is the fermentation results of Mortierella alpina in basic medium.
The fermentation results of Fig. 2 Mortierella alpina in the substratum that with the addition of VITAMIN, amino acid and trace element.
Fig. 3 Mortierella alpina with the addition of the fermentation results of mixed nitrogen in embodiment 3 substratum.
Fermentation results in Mortierella alpina after Fig. 4 domestication substratum in example 4.
Fig. 1 ~ 4 are respectively different culture media in embodiment 2 ~ 5 affects result to the biomass of Mortierella alpina, total grease and ARA output.In Fig. 1 ~ 4, X-coordinate is incubation time (h), left ordinate zou is glucose consumption situation Glucose (■, g/L), right ordinate zou is respectively biomass Biomass (●, g/L), total grease Total fatty acid (★, and ARA output (▲, g/L) g/L).
Embodiment
Following examples are used for further illustrating the present invention.
Embodiment 1. utilizes Mortierella alpina to produce ARA
In a 250mL shaking flask, cultivate Mortierella alpina, carrier fluid amount is the activation medium of 50mL, 150rpm, 28 DEG C, activates 3 days.Again by the bacterial strain access basic medium after activation, the shaking flask of liquid amount 250mL loads the basic medium of 50mL, inoculum size 10% (v/v), cultivates 5 ~ 6d in 28 DEG C of reciprocal shaker, rotating speed 150rpm, 28 DEG C, gather in the crops after cultivating 5d, suction filtration harvesting biomass, by its freeze-day with constant temperature to constant weight, grind oil and grease extracting with normal hexane, after the organic appearance agent of evaporation removing, obtain total grease 5g/L.
Activation medium composition (g/L):
Basic medium composition (g/L):
Embodiment 2. utilizes basic medium to produce ARA at fermentor cultivation Mortierella alpina
2L fermentor cultivation, liquid amount 1.5L basic medium (as embodiment 1), initial glucose concentration is decided to be 60 ~ 65g/L, independent sterilizing, leavening temperature maintains 28 DEG C, initial mixing speed 150 ~ 200rpm, under the condition of air flow 1vvm, batch fermentation cultivation is carried out to Mortierella alpine mould, by pH regulator to 6.5 ~ 7.0 before sterilizing, initial pH after sterilizing is 6.0 ~ 6.5, after cultivating 12h, dissolved oxygen starts rapid decline, grow into logarithmic phase, after cultivating 48h, growth starts to enter stationary phase, in whole growth cycle, initial pH value is 6.0 ~ 6.5, in process, pH value first raises rear reduction and raises, along with the arrival of stationary phase, last pH value is stabilized between 7.0 ~ 7.9, dissolved oxygen declines rapidly along with the increase of biomass, maintains dissolved oxygen 0.5% ~ 10% at logarithmic phase.
Between incubation period, the analysis of biomass and lipid acid is carried out in every 12h sampling.Be cultured to 168h, start to gather in the crops thalline, adopt the method for embodiment 1, obtain dry mycelium 18g/L, total grease 7g/L, ARA 2.7g/L, the glucose concn in fermented liquid is down to 15g/L from initial 60g/L, and concrete outcome as shown in Figure 1.
Embodiment 3. amino acid, VITAMIN and trace element are on the impact of Mortierella alpina biomass and ARA fat content
In the basic medium of embodiment 2, with the addition of somatomedin (VITAMIN, amino acid), and trace element improves output.
The somatomedin added comprises:
VITAMIN is made into 500 × concentrated solution, and use 0.2 μm of aseptic filter membrane filtration sterilization, and place 4 DEG C of Refrigerator stores, directly add in basic medium before to be seeded, addition is 2mL/L.L-glutamic acid adds when preparing basic medium before sterilization, with substratum together sterilizing.
The trace element added comprises (g/L):
Trace element is made into 1000 × concentrated solution (namely adding 1mL microelement concentrate in 1L substratum), is adjusted to pH 7.Use 0.2 μm of aseptic filter membrane filtration sterilization, and place 4 DEG C of Refrigerator stores, directly add in basic medium before to be seeded.
In order to prepare inoculation thalline, the seed of use is seeded in 3 shaking flasks containing 50mL activation medium, 28 DEG C, after 150rpm cultivates 3 days, the form that thalline presents in fermented liquid is spherical, and diameter is 2 ~ 4mm, or loose mycelial congeries.Be seeded to the 2L fermentor tank that carrier fluid amount is 1.5L, inoculum size 10% (V/V), if the density that seed grows in shaking flask is lower, then suitable when being seeded to fermentor tank increase inoculum size.Substratum in fermentor tank, for the addition of the Optimal Medium of somatomedin (VITAMIN, amino acid) and trace element, carries out batch fermentation cultivation.Wherein initial glucose concentration is 60 ~ 65g/L, separately sterilizing, 120 DEG C of sterilizing 30min.
The parameter of fermentor tank is as follows: temperature: 28 DEG C; Ventilation: 0.5 ~ 1vvm; PH: maintain about 6.0 with 25% ammoniacal liquor; Initial mixing speed: 150rpm.
12h, dissolved oxygen starts rapid decline, and pH also starts to decline, and thalli growth enters logarithmic phase, and rotating speed is adjusted to 200rpm, after this starts in 1 ~ 2h to produce foam, adds proper quantity of defoaming agent.After this rotating speed is adjusted to 250rpm.
During inoculation, the thalline in fermentor tank is diameter is the coccoid of 1 ~ 2mm, and outer rim is smooth, be cultured to 12h to start, spheroid starts to become large, to 24h, spheroid form is more clear, continues to become large, and 48h starts cell density in tank and increases, to 72h, spheroid outer rim seems fluffy, and occurs the loose mycelium that similar feather is cotton-shaped, this may start due to larger spherical thallus to disintegrate, to 120h, the diameter of ball, between 2 ~ 4mm, defines many loose mycelial congeries simultaneously.Every 12h, fermentor tank is sampled to the analysis carrying out biomass and lipid acid, 168h secondary fermentation terminates, and in tank, the final volume of fermented liquid is 1.2L.Obtain dry mycelium 21g/L, total grease 9g/L, ARA 3.8g/L, the glucose concn in fermented liquid is down to 13g/L from initial 65g/L, and concrete outcome as shown in Figure 2.
Embodiment 4. mixed nitrogen is on the impact of Mortierella alpina Mortierella alpina biomass and ARA fat content
The experiment of this group improves output further by using mixed nitrogen, and process is substantially the same manner as Example 3, and moiety and the ratio of the mixed nitrogen of interpolation are obtained by series of experiments.
The mixed nitrogen added comprises (g/L):
Corn steep liquor 3
Yeast powder 7
Fermentor tank is at 11h, and dissolved oxygen starts rapid decline, starts to produce a small amount of foam at about 14h, controls by manually adding defoamer.To 24h, thalline is the spherical of diameter 1 ~ 2mm, and to 48h, thalline is the spherical of diameter 2 ~ 3mm, and outer rim is smooth, and to 72h, start to occur the cotton-shaped mycelial congeries of loose feather, spheroid outer rim is loose.Every 12h, fermentor tank is sampled to the analysis carrying out biomass and lipid acid, gather in the crops after 168h, obtain dry mycelium 36g/L, total grease 16g/L, ARA6.5g/L, the glucose concn in fermented liquid is down to 5g/L from initial 60g/L, and concrete outcome as shown in Figure 3.
Embodiment 5. utilizes the Mortierella alpina after domestication to produce ARA
For making Mortierella alpina in fermentation culture process, more abundant to the absorption of nutritive substance, reduce the waste of raw material, improve bacterial classification and consume sugared ability, bacterial classification is tamed, high step by step sugar mainly through solid medium and liquid nutrient medium is tamed, and improve the consumption sugar ability of thalline, and the bacterial classification after domestication is all rendered as the loose spheres of the outer rim band thorn-like being conducive to oil and fat accumulation.Utilize the bacterial classification after this domestication, use the optimization wild Oryza species described in embodiment 4 to carry out Fed batch fementation, and when glucose concn is down to about 10g/L, stream adds 600g/L glucose, makes residual sugar control at about 10g/L, all the other operating process are identical with enforcement 4.
In fermenting process with 25% ammoniacal liquor regulable control pH 6.0 ~ 6.5.
12h, thalline starts growth, thalline is the bead of outer rim thorn-like, diameter 1 ~ 2mm, afterwards along with thalline raised growth, be rendered as the loose spherical of outer rim thorn-like, diameter 2 ~ 5mm, now pH starts to decline, this is that carbohydrate metabolism first produces the organic acids such as ketone acid, then be utilized and rise gradually again, now, pH is allowed to be down to 4.5, to 72h, pH slowly can go up along with thalline enters stationary phase, now thalline is mainly the loose spherical of outer rim thorn-like, secondly be the cotton-shaped mycelial congeries of feather, to 98h, pH is slowly risen between 6.5 ~ 7.5, but not higher than 8.0.Every 12h, fermentor tank is sampled to the analysis carrying out biomass and lipid acid,
Start results after cultivating 168h, obtain dry mycelium 58g/L, total grease 28g/L, ARA 16g/L, glucose concn during fermentation ends in fermented liquid goes to zero.According to the batch fermentation result display that the present embodiment carries out, ARA output is existing comparatively before to be increased substantially, and concrete outcome as shown in Figure 4.
Claims (5)
1. prepare an arachidonic method, it is characterized in that comprising the following steps:
1) by shake flat experiment, the culture condition that Mortierella alpina produces ARA is optimized, the method of described optimization is get the 250mL triangular flask that 50mL basic medium is equipped with in the access of 4mL seed liquor, 28 DEG C, 150rpm cultivates 5 ~ 6d, be optimized substratum: glucose 60g/L; Yeast powder or corn steep liquor 10g/L; L-glutamic acid 1.0g/L; MgSO
4-7H
2o0.1g/L; KH
2pO
42.0g/L; CaCO
30.05g/L; ZnSO
4-7H
2o 0.1g/L; Mixed trace elements 1mL/L; Mixed vitamin 1mL/L; PH is adjusted to 6.0; Described basic medium consist of glucose 40g/L; Yeast powder 5g/L; MgSO
4-7H
2o 0.1g/L; KH
2pO
40.5g/L; CaCO
30.05g/L; ZnSO
4-7H
2o 0.1g/L; PH 6.0;
2) high sugar domestication is carried out to Mortierella alpina and in mixed nitrogen Optimal Medium, 2L batch fermentation is carried out to the bacterial classification after domestication, mixed nitrogen Optimal Medium is loaded in 2L fermentor tank, when being down to 10g/L when glucose concn, stream adds 600g/L glucose, make residual sugar control at 10g/L, start to gather in the crops dry mycelium, grease and ARA after cultivating 168h.
2. one prepares arachidonic method as claimed in claim 1, it is characterized in that in step 1) in, described mixed trace elements comprises FeCl
36H
2o, H
3bO
3, MnCl
24H
2o, CoCl
26H
2o, CuSO
45H
2o, NiSO
46H
2o.
3. one prepares arachidonic method as claimed in claim 1, it is characterized in that in step 1) in, described mixed vitamin comprises vitamins B
12, vitamin H, vitamins B
1, calcium pantothenate.
4. one prepares arachidonic method as claimed in claim 1, it is characterized in that in step 2) in, the concrete grammar of described 2L batch fermentation is: first adopt solid medium domestication, bacterial classification is coated step by step the high sugared slat chain conveyor of gradient, select the strain transfer that can tolerate 10% high glucose concentration flat board after solid domestication to tame to two kinds containing in the gradient high sugar liquors substratum of different nitrogen sources, then to the bacterial classification after this domestication, use step 2) described in mixed nitrogen Optimal Medium carry out batch fermentation.
5. one prepares arachidonic method as claimed in claim 4, it is characterized in that the sugar degree of the high sugar flat board of described gradient is respectively 2%, 5%, 7%, 10% and 15%; The sugar degree of the high sugar of described two kinds of gradients containing different nitrogen sources is respectively 3%, 4%, 5% and 6%.
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| CN112625999A (en) * | 2020-12-30 | 2021-04-09 | 嘉必优生物技术（武汉）股份有限公司 | Method for domesticating filamentous fungi through continuous fermentation |
| WO2024131744A1 (en) * | 2022-12-19 | 2024-06-27 | 嘉必优生物技术（武汉）股份有限公司 | Method for reducing content of chloropropanol in arachidonic acid oil |
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| CN110129384A (en) * | 2018-02-08 | 2019-08-16 | 浙江海正药业股份有限公司 | A kind of arachidonic preparation method |
| CN110129384B (en) * | 2018-02-08 | 2022-07-01 | 浙江海正药业股份有限公司 | Preparation method of arachidonic acid |
| CN112625999A (en) * | 2020-12-30 | 2021-04-09 | 嘉必优生物技术（武汉）股份有限公司 | Method for domesticating filamentous fungi through continuous fermentation |
| CN112625999B (en) * | 2020-12-30 | 2022-08-09 | 嘉必优生物技术（武汉）股份有限公司 | Method for domesticating filamentous fungi through continuous fermentation |
| WO2024131744A1 (en) * | 2022-12-19 | 2024-06-27 | 嘉必优生物技术（武汉）股份有限公司 | Method for reducing content of chloropropanol in arachidonic acid oil |
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