CN1323904A - Fermentation prepn. of arachidonic acid - Google Patents
Fermentation prepn. of arachidonic acid Download PDFInfo
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- CN1323904A CN1323904A CN 00114562 CN00114562A CN1323904A CN 1323904 A CN1323904 A CN 1323904A CN 00114562 CN00114562 CN 00114562 CN 00114562 A CN00114562 A CN 00114562A CN 1323904 A CN1323904 A CN 1323904A
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Abstract
A fermentation method for preparing arachidonic acid includes the processes and steps as: (A). for seed expansion culture: the first is to prepare the bean sprouts juice, the second is to prepare the formula of seed substratum, the third is to prepare the seed cultuivation conditions and the fourth is to carry out the seed expansion culture; (B). for fermentation culture: the first is to prepare corn saccharified liquid, the second is to build up fermentation substratum, the third is to prepare the fermentation conditions and the fourth is to carry out the shaking culture or fermentation tank cultivation. After fermentation, the process of extracting, filtering, thallus' collecting, drying, smashing, petroleum ethers extracting will be carried out for obtaining the final product of arachidonic acid.
Description
The present invention relates to field of fermentation engineering, more specifically relate to a kind of arachidonic acid-fermentation method for formulating.
Research work around polyunsaturated fatty acid (PuFAs) fermentation since the eighties makes significant progress.At present, U.S. Starge company, Japanese bright dipping petroleum chemistry company etc. are with gamma-linolenic acid (GLA) launch products of fermentative production, the GLA grease of fermentative production is compared with Oenothera oil, not only have GLA content height, stable yield, suitability for industrialized production and be not subjected to advantages such as natural condition restriction, and its lubricant component is more near breast milk.Except that GLA, other PuFAs fermentation still is in the experimental phase, is the research focus of fermentation industry and medicine, foodstuffs industry.The present domestic GLA microorganism fermentation process research aspect that also mainly rests on, then emphasis is transferred on the study on the industrialization of arachidonic acid (AA), timnodonic acid (EPA), docosahexenoic acid (DHA) abroad, but up to the present, the external report that does not still have GLA PuFAs microbial fermentation launch products in addition, but the experimental study of this respect has bigger progress, especially walk up front with Japan and Canada, U.S. oil chemistry association also very pays close attention to the progress of production and the application of PuFAs.The microbe research of carrying out PuFAs in the world mainly concentrates on the following aspects: the strain separating and the seed selection of 1, producing PuFAs; 2, the optimization of culture condition and zymotechnique; 3, the research of training method (comprising batch formula, feed supplement, continuous culture process etc.); 4, the separation and purification of PuFAs and structure are identified.People only find that zygomycetes is a unique promising guiding principle at present, and generally good mortierella, think that it is to produce arachidonic acid, EPA, the best bacterial classification of DHA.Domestic aspect, research GLA and obtain certain progress Shanghai plant physiology institute of the Chinese Academy of Sciences, Nankai University, biotechnology institute of Fujian Normal University and life science institute of Shandong University arranged, but now the main source of GLA is still and extracts from plants such as root of Redsepal Eveningprimrose, utilizes microbiological industry production GLA not appear in the newspapers as yet.South China Science ﹠ Engineering University has reported arachidonic fermentation research.Its seed culture medium is: glucose 30g/1000mL, yeast extract paste 2g/1000mL, peptone 3g/1000mL, K
2HPO
40.5g/1000mL, CaCl
2.2H
2O 0.2g/1000mL, MgSO
4.7H
2O 0.3g/1000mL, pH6.0.Its fermention medium is: glucose 80g/1000ml,, yeast extract paste 0.5g/1000mL, KNO
340g/1000mL, K
2HPO
43.0g/1000mL, CaCl
2.2H
2O 0.1g/1000ml, MgSO
4.7H
2O 0.5g/1000mL, FeCl
26H
2O 0.015g/1000mL, ZnSO
47H
2O 0.0075g/1000mL, CuSO
45H
2O 0.0005g/1000mL, pH6.0.Its seed liquor is prepared as follows: adopt the 5ml sterilized water slant strains to be washed in the 250ml triangular flask that the 50ml seed culture medium is housed, shake-flask culture is 4 days under 25 ℃, 180rpm.Its shake flask fermentation cultural method is as follows: get the 2ml seed liquor in the 250ml triangular flask that the 50ml fermention medium is housed, 25 ℃, shake-flask culture is 7 days under the 180rpm.Present domestic research still is in the shake flat experiment stage, does not carry out arachidonic fermentor tank amplification culture as yet, and the highest yield that arachidonic acid shake flask fermentation system is joined is 0.827g/1000mL.
The object of the present invention is to provide a kind of arachidonic acid-fermentation method for formulating, technology is easy, and system is filled a prescription just, and cost is low, the problem that has solved arachidonic fermentation production efficiency and yielded poorly.
In order to achieve the above object, the present invention by the following technical solutions, on the basis that culture medium prescription and arachidonic acid fermentation condition are tested repeatedly, a large amount of synthetic substratum of arachidonic acid and fermentation condition have been found, its culture medium cost is cheap, draw materials easily, operating process is simple, and its fermentation method for formulating is as follows:
1, seed enlarged culturing:
(1) preparation of bean sprouts juice: fresh soybean sprout 400-600g, add water 800-1200mL and boiled 0.5-1.0 hour, take out bean sprouts juice, standby.
(2) seed culture based formulas: adopt bean sprouts juice 800-1200mL, glucose 40-60g/1000mL, K
2HPO
40.5-1.0g/1000mL, pH 6.0-6.5.
(3) seed culture condition: culture temperature 10-20 ℃, shaking speed is 110-150rpm, cultivates 3-5 days.
(4) seed enlarged culturing: shake bottled 100ml seed culture medium by 500ml, adopt the common mortierella preservation bacterial classification of yield peanut tetraenoic acid then, after slant activation, add the 8-10ml sterilized water, wash spore, make spore suspension.Add the 8-10ml spore suspension by the 100ml seed culture medium, carry out the seed enlarged culturing by the seed culture condition in (3) again, obtain the inoculum that the arachidonic acid fermentative production is used.
2, fermentation culture
(1) Semen Maydis powder saccharification liquid preparation: Semen Maydis powder 180-220g adds water 800-1200mL, boils expansion, adds α-Dian Fenmei 4-6g and saccharifying enzyme 1-3g, and 40-60 ℃, be incubated 10-14 hour, filter filtrate for later use.
(2) fermention medium: Semen Maydis powder saccharification liquid 800-1200mL, yeast powder 12-18g/1000mL, extractum carnis 3-6g/1000mL, NaNO
31-5g/1000mL, K
2HPO
40.5-1.0g/1000mL, pH 6.0-6.5.
(3) ferment tank culture condition: leavening temperature 10-18 ℃, mixing speed 150-250rpm, air flow 120-180VVM cultivated 10-20 days.Or shake flask fermentation culture condition: leavening temperature is 10-18 ℃, and shaking speed is 110-150rpm, cultivates 10-20 days.
(4) fermentor cultivation: by the 65-75ml fermention medium of packing in every 100mL tank volume, adopt the above-mentioned seed liquor for preparing to insert the 15-25ml seed liquor by the 100ml fermention medium, it is 10-18 ℃ by leavening temperature again, mixing speed is 150-250rpm, air flow 120-180VVM, cultivate incubation time 10-20 days.Or shake flask fermentation is cultivated: shake the bottled 20ml of going into fermention medium by 100ml, add the above-mentioned seed liquor for preparing of 15-25ml again by leavening temperature 10-18 ℃ by the 100ml fermention medium then, shaking speed 110-150rpm cultivated 10-20 days.
After the fermentation ends, suction filtration is collected thalline and is dried to constant weight, pulverizing sherwood oil extracting grease for 40-60 ℃.Arachidonic acid content in the gas chromatography determination grease gets the pure product of arachidonic acid through the separation and purification grease again.
The present invention compared with prior art, have the following advantages and effect: raw material is easily purchased, and is cheap, the arachidonic acid synthesizing stable, output is at 1.35-1.55g/1000ml, fermenting process is simple, and is easy and simple to handle, is fit to suitability for industrialized production.
Embodiment 1:
1, seed enlarged culturing
(1) preparation of bean sprouts juice: fresh soybean sprout 500g, add water 1000mL, boiled 1 hour, take out and soak juice, standby.
(2) seed culture based formulas: bean sprouts juice 1000mL, glucose 50g/1000mL, K
2HPO
41g/1000mL, pH6.0.
(3) seed culture condition: culture temperature is 18 ℃, and to shake bottle rotating speed be 120rpm, cultivated 3 days.
(4) seed enlarged culturing: shake bottled 100ml seed culture medium by 500ml, after adopting the mortierella slant preservation actication of culture of common yield peanut tetraenoic acid then, wash spore with the 10ml sterilized water, adding the access of 10ml spore suspension by every 100ml seed culture medium shakes in the bottle, and the seed culture condition of pressing in (3) is carried out the seed enlarged culturing, the inoculum that acquisition arachidonic acid fermentative production is used.
2, fermentation culture:
(1) preparation of Semen Maydis powder saccharification liquid: Semen Maydis powder 200g adds deionized water 1000mL, boils expansion, adds α-Dian Fenmei 5g and saccharifying enzyme 2g, and 60 ℃, be incubated 12 hours, filter filtrate for later use.
(2) fermention medium: Semen Maydis powder saccharification liquid 1000mL, yeast powder 15g/1000mL, extractum carnis 5g/1000mL, NaNO
33g/1000mL, K
2HPO
41g/1000mL, pH6.0.
(3) fermentation culture conditions: leavening temperature is 15 ℃, and shaking bottle rotating speed is 120rpm, cultivates 15 days.
(4) arachidonic acid fermentation culture: shake bottled 400ml fermention medium by 2000ml, adopt a large amount of mortierella seed liquor that seed enlarged culturing system is joined to add the 20ml seed liquor then and join in the fermention medium, carry out arachidonic fermentation culture by the fermentation culture conditions in above-mentioned (3) again by every 100ml fermention medium.After the fermentation ends, suction filtration is collected thalline and is dried to constant weight, pulverizing for 40-60 ℃, adopts petroleum ether solvent to extract grease; Arachidonic acid content in the gas chromatography determination grease gets the pure product of arachidonic acid through the separation and purification grease again.
Embodiment 2:
1, seed enlarged culturing
(1) preparation of bean sprouts juice: fresh soybean sprout 400g, add water 1000mL, boiled 1 hour, take out and soak juice, standby.
(2) seed culture based formulas: bean sprouts juice 1000mL, glucose 60g/1000mL, K
2HPO
41g/1000mL, pH6.0.
(3) seed culture condition: culture temperature is 20 ℃, and shaking bottle rotating speed is 120rpm, cultivates 3 days.
(4) seed enlarged culturing: shake bottled 100ml seed culture medium by 500ml, adopt the common mortierella slant preservation bacterial classification of yield peanut tetraenoic acid then, after slant activation, add the 8ml sterilized water, wash spore, make spore suspension.Add the 8ml spore suspension by every 100ml seed culture medium, carry out the seed enlarged culturing by the seed culture condition of (3) again, obtain the inoculum that the arachidonic acid fermentative production is used.
2, fermentation culture:
(1) preparation of Semen Maydis powder saccharification liquid: Semen Maydis powder 180g adds deionized water 1000mL, boils expansion, adds α-Dian Fenmei 4g and saccharifying enzyme 2g, and 60 ℃, be incubated 12 hours, filter filtrate for later use.
(2) fermention medium: Semen Maydis powder saccharification liquid 1000mL, yeast powder 12g/1000mL, extractum carnis 4.5g/1000mL, NaNO
33g/1000mL, K
2HPO
41g/1000mL, pH6.0.
(3) fermentation culture conditions: leavening temperature is 12 ℃, and shaking bottle rotating speed is 150rpm, cultivates 15 days.
(4) arachidonic acid fermentation culture: shake bottled 400ml fermention medium by 2000ml, a large amount of mortierella seed liquor that seed enlarged culturing system is joined join in the fermention medium by every 100ml fermention medium adding 18ml seed liquor then, carry out arachidonic fermentation culture by the fermentation culture conditions in above-mentioned (3) again.After the fermentation ends, suction filtration is collected thalline, dry to constant weight, pulverizing to 55 ℃, and petroleum ether extraction, arachidonic acid content in the gas chromatography determination grease gets the pure product of arachidonic acid through the separation and purification grease again.
Embodiment 3:
1, seed enlarged culturing
(1) preparation of bean sprouts juice: fresh soybean sprout 600g, add water 1000mL, boiled 1 hour, take out and soak juice, standby.
(2) seed culture based formulas: bean sprouts juice 1000mL, glucose 40g/1000mL, K
2HPO
40.5g/1000mL, pH6.2.
(3) seed culture condition: culture temperature is 20 ℃, and shaking bottle rotating speed is 140rpm, cultivates 4 days.
(4) seed enlarged culturing: shake bottled 100ml seed culture medium by 500ml, adopt the common mortierella slant preservation bacterial classification of yield peanut tetraenoic acid then, after slant activation, add the 10ml sterilized water, wash spore, make spore suspension.Add the 10ml spore suspension by every 100ml seed culture medium, carry out the seed enlarged culturing by the seed culture condition of (3) again, obtain the inoculum that the arachidonic acid fermentative production is used.
2, fermentation culture:
(1) preparation of Semen Maydis powder saccharification liquid: Semen Maydis powder 190g adds deionized water 1000mL, boils expansion, adds α-Dian Fenmei 5.5g and saccharifying enzyme 1.5g, and 60 ℃, be incubated 14 hours, filter filtrate for later use.
(2) fermention medium: Semen Maydis powder saccharification liquid 1000mL, yeast powder 10g/1000mL, extractum carnis 5.5g/1000mL, NaNO
32g/1000mL, K
2HPO
41g/1000mL, pH6.4.
(3) fermentation culture conditions: leavening temperature is 17 ℃, and shaking bottle rotating speed is 130rpm, cultivates 14 days.
(4) arachidonic acid fermentation culture: shake bottled 400ml fermention medium by 2000ml, a large amount of mortierella seed liquor that seed enlarged culturing system is joined join in the fermention medium by every 100ml fermention medium adding 20ml seed liquor then, carry out arachidonic fermentation culture by the fermentation culture conditions in above-mentioned (3) again.After the fermentation ends, suction filtration is collected thalline, dry to constant weight, pulverizing to 50 ℃, and petroleum ether extraction, arachidonic acid content in the gas chromatography determination grease gets the pure product of arachidonic acid through the separation and purification grease again.
Embodiment 4:
1, seed enlarged culturing
(1) preparation of bean sprouts juice: fresh soybean sprout 450g, add water 1000mL, boiled 1 hour, take out and soak juice, standby.
(2) seed culture based formulas: bean sprouts juice 1000mL, glucose 45g/1000mL, K
2HPO
40.8g/1000mL, pH6.5.
(3) seed culture condition: culture temperature is 16 ℃, and shaking bottle rotating speed is 130rpm, cultivates 3 days.
(4) seed enlarged culturing: shake bottled 100ml seed culture medium by 500ml, adopt the common mortierella slant preservation bacterial classification of yield peanut tetraenoic acid then, after slant activation, add the 10ml sterilized water.Wash spore, make spore suspension.Add the 8ml spore suspension by every 100ml seed culture medium.Carry out the seed enlarged culturing by the seed culture condition of (3) again, obtain the inoculum that the arachidonic acid fermentative production is used.
2, fermentation culture:
(1) preparation of Semen Maydis powder saccharification liquid: Semen Maydis powder 210g adds deionized water 1100mL, boils expansion, adds α-Dian Fenmei 4.5g and saccharifying enzyme 2.5g, and 50 ℃, be incubated 14 hours, filter filtrate for later use.
(2) fermention medium: Semen Maydis powder saccharification liquid 1000mL, yeast powder 16g/1000mL, extractum carnis 5g/1000mL, NaNO
34g/1000mL, K
2HPO
40.6g/1000mL, pH6.0.
(3) fermentation culture conditions: leavening temperature is 14 ℃, and mixing speed is 200rpm, and air flow is 140VVM, cultivates 16 days.
Arachidonic acid fermentation culture in (4) 15 liters of fermentor tanks: by the 15000ml mechanical agitation type canned 10500ml fermention medium that ferments, adopt a large amount of mortierella seed liquor that seed enlarged culturing system is joined to add the 25ml seed liquor then and join in the fermention medium, carry out arachidonic fermentation culture by the fermentation culture conditions in above-mentioned (3) again by every 100ml fermention medium.After the fermentation ends, suction filtration is collected thalline, dry to constant weight, pulverizing to 60 ℃, and petroleum ether extraction, arachidonic acid content in the gas chromatography determination grease gets the pure product of arachidonic acid through the separation and purification grease again.
Embodiment 5:
1, seed enlarged culturing
(1) preparation of bean sprouts juice: fresh soybean sprout 550g, add water 1000mL, boiled 1 hour, take out and soak juice, standby.
(2) seed culture based formulas: bean sprouts juice 1000mL, glucose 55g/1000mL, K
2HPO
41g/1000mL, pH6.5.
(3) seed culture condition: culture temperature is 17 ℃, and shaking bottle rotating speed is 130rpm, cultivates 4 days.
(4) seed enlarged culturing: shake bottled 100ml seed culture medium by 500ml, adopt the common mortierella slant preservation bacterial classification of yield peanut tetraenoic acid then, after slant activation, add the 10ml sterilized water, wash spore, make spore suspension.Add the 10ml spore suspension by every 100ml seed culture medium, carry out the seed enlarged culturing by the seed culture condition of (3) again, obtain the inoculum that the arachidonic acid fermentative production is used.
2, fermentation culture:
(1) preparation of Semen Maydis powder saccharification liquid: Semen Maydis powder 200g adds deionized water 1000mL, boils expansion, adds α-Dian Fenmei 6g and saccharifying enzyme 2g, and 55 ℃, be incubated 14 hours, filter filtrate for later use.
(2) fermention medium: Semen Maydis powder saccharification liquid 1000mL, yeast powder 18g/1000mL, extractum carnis 3.5g/1000mL, NaNO
32g/1000mL, K
2HPO
41g/1000mL, pH6.4.
(3) fermentation culture conditions: leavening temperature is 12 ℃, and mixing speed is 250rpm, and air flow is 150VVM, cultivates 14 days.
Arachidonic acid fermentation culture in (4) 15 liters of fermentor tanks: by the 15000ml mechanical agitation type fermentor tank 11250ml fermention medium of packing into, a large amount of mortierella seed liquor that seed enlarged culturing system is joined join in the fermention medium by every 100ml fermention medium adding 18ml seed liquor then, carry out arachidonic fermentation culture by the fermentation culture conditions in above-mentioned (3) again.After the fermentation ends, suction filtration is collected thalline, dry to constant weight, pulverizing to 55 ℃, and petroleum ether extraction, arachidonic acid content in the gas chromatography determination grease gets the pure product of arachidonic acid through the separation and purification grease again.
Claims (1)
1, it is as follows that a kind of arachidonic acid-fermentation method for formulating, its system are joined step:
A, seed enlarged culturing:
The preparation of a, bean sprouts juice, fresh soybean sprout 400-600g adds water 800-1200ml and boiled 0.5-1.0 hour, takes out bean sprouts juice, and is standby;
B, seed culture based formulas, bean sprouts juice 800-1200mL, glucose 40-60g/1000mL, K
2HPO
40.5-1.0g/1000mL, pH6.0-6.5;
C, seed culture condition, culture temperature is 10-20 ℃, shaking bottle rotating speed is 110-150rpm, cultivates 3-5 days;
D, seed enlarged culturing: shake bottled 100ml seed culture medium by 500ml, adopt the mortierella slant preservation bacterial classification of common yield peanut tetraenoic acid, after slant activation, use the 8-10ml sterilized water, wash spore, make spore suspension, by every 100ml seed culture medium, add the 8-10ml spore suspension, and carry out the seed enlarged culturing by the seed culture condition in (c);
B, fermentation culture:
The preparation of a, Semen Maydis powder saccharification liquid: Semen Maydis powder 180-220g adds water 800-1200mL, boils expansion, adds α-Dian Fenmei 4-6g and saccharifying enzyme 1-3g, and 40-60 ℃, be incubated 10-14 hour, filter filtrate for later use;
B, fermention medium: Semen Maydis powder saccharification liquid 800-1200mL, yeast powder 12-18g/1000mL, extractum carnis 3-6g/1000mL, NaNO
31-5g/1000mL, K
2HPO
40.5-1.0g/1000mL, pH6.0-6.5;
C, ferment tank culture condition: leavening temperature is 10-18 ℃, and mixing speed is 150-250rpm, and air flow 120-180VVM cultivated 10-20 days, or the shake flask fermentation culture condition: leavening temperature 10-18 ℃, shake a bottle rotating speed 110-150rpm, and cultivated 10-20 days;
D, fermentor cultivation: by every 100ml tank volume 65-75ml fermention medium of packing into, insert the 15-25m1 seed liquor by every 100ml fermention medium, leavening temperature is 10-18 ℃, and mixing speed is 150-250rpm, air flow 120-180VVM, incubation time 10-20 days, or shake flask fermentation is cultivated: shake the bottled 20ml of going into fermention medium by 100ml, press the 100ml fermention medium and add the 15-25ml seed liquor, leavening temperature is 10-18 ℃, shaking bottle rotating speed is 110-150rpm, cultivates 10-20 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00114562 CN1323904A (en) | 2000-05-12 | 2000-05-12 | Fermentation prepn. of arachidonic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00114562 CN1323904A (en) | 2000-05-12 | 2000-05-12 | Fermentation prepn. of arachidonic acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1323904A true CN1323904A (en) | 2001-11-28 |
Family
ID=4584225
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|---|---|---|---|
| CN 00114562 Pending CN1323904A (en) | 2000-05-12 | 2000-05-12 | Fermentation prepn. of arachidonic acid |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2003235950B2 (en) * | 2002-04-26 | 2008-12-11 | Suntory Holdings Limited | Process for producing highly unsaturated fatty acid-containing lipid |
| CN101153298B (en) * | 2007-09-07 | 2010-11-24 | 武汉麦可得生物技术有限公司 | Method for ferment production of arachidonic acid grease with low-content nervonic acid and EPA |
| EP2333094A1 (en) | 2005-02-08 | 2011-06-15 | Nippon Suisan Kaisha, Ltd. | Production of polyunsaturated fatty acids using novel cell treatment method |
| US8241868B2 (en) | 2005-02-08 | 2012-08-14 | Nippon Suisan Kaisha, Ltd. | Production of polyunsaturated fatty acids using cell treatment method |
-
2000
- 2000-05-12 CN CN 00114562 patent/CN1323904A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2003235950B2 (en) * | 2002-04-26 | 2008-12-11 | Suntory Holdings Limited | Process for producing highly unsaturated fatty acid-containing lipid |
| AU2003235950B8 (en) * | 2002-04-26 | 2009-08-06 | Suntory Holdings Limited | Process for producing highly unsaturated fatty acid-containing lipid |
| US7863024B2 (en) | 2002-04-26 | 2011-01-04 | Suntory Holdings Limited | Process for producing highly unsaturated fatty acid-containing lipid |
| EP2333094A1 (en) | 2005-02-08 | 2011-06-15 | Nippon Suisan Kaisha, Ltd. | Production of polyunsaturated fatty acids using novel cell treatment method |
| US8241868B2 (en) | 2005-02-08 | 2012-08-14 | Nippon Suisan Kaisha, Ltd. | Production of polyunsaturated fatty acids using cell treatment method |
| CN101153298B (en) * | 2007-09-07 | 2010-11-24 | 武汉麦可得生物技术有限公司 | Method for ferment production of arachidonic acid grease with low-content nervonic acid and EPA |
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