CN104651423A - Method for fermentation production of arachidonic acid by adopting two-stage pH control - Google Patents
Method for fermentation production of arachidonic acid by adopting two-stage pH control Download PDFInfo
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- CN104651423A CN104651423A CN201510045230.4A CN201510045230A CN104651423A CN 104651423 A CN104651423 A CN 104651423A CN 201510045230 A CN201510045230 A CN 201510045230A CN 104651423 A CN104651423 A CN 104651423A
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- 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
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Abstract
The invention discloses a method for fermentation production of arachidonic acid by adopting two-stage pH control. According to the method, mortierella alpine is taken as a production strain; a two-stage pH control strategy is adopted; the pH of a fermentation solution is regulated to be 4 to 6 in an early stage of fermentation; after the strain is transformed from a growth type to a grease accumulation type, the pH of the fermentation solution is regulated to 5 to 8 for fermentation culture. By adopting the method disclosed by the invention, the yield of the arachidonic acid can be remarkably increased.
Description
Technical field
The invention belongs to bioengineering field, relate to fermentation processes, particularly a kind of two benches pH control strategy, is applied to arachidonic fermentative production.
Background technology
Arachidonic acid (Arachidonic acid, C20:4 are called for short ARA), belongs to the serial polyunsaturated fatty acid of ω-6 (PUFAs), has very important effect for the physiological activity maintaining human normal.It is not only extensively present in mammiferous histoorgan as a kind of very important struetural lipid, and be the important precursor of human prostate's element synthesis, there is biological activity and important trophism widely, be widely used at food, medicine and cosmetic field.In addition, arachidonic acid to the nerve of baby and physiological growth essential, to grow and amphiblestroid perfect in shape and function significant, be considered to the essential nutrients of mankind's early development.The Ministry of Health, official approval in 1994, can add arachidonic acid in dispensed food for baby, and within 1999, official approval arachidonic acid is as novel nourishing reinforcer.At present, arachidonic acid, by internal authority agency recommends such as the World Health Organization, adds in infant formula powder as nutritional supplementation.Arachidonic traditional source is fish oil, yolk, animal tissues etc. mainly, and its shortcoming is that content is low and output unstable, is difficult to the heavy demand meeting society.And microorganism has the features such as fast growth, training method is simple, lubricant component is single, therefore fermentable is utilized to be the arachidonic main flow means of industrial production.Mortierella alpina is a kind of filamentous fungus of genus mortierella, and the grease contained by it and arachidonic acid content are all higher, therefore utilizes Mortierella alpina fermentation yield peanut tetraenoic acid to have very large using value.
Chinese scholars utilizes the ferment research of yield peanut tetraenoic acid of Mortierella alpina mainly to concentrate on two aspects: the control of fermentation condition and the seed selection of bacterial strain and molecular modification.But how on earth improve arachidonic acid yield also urgently to study.
Summary of the invention
The object of this part is some aspects of general introduction embodiments of the invention and briefly introduces some better embodiment.May do in the specification digest and denomination of invention of this part and the application a little simplify or omit with avoid making this part, specification digest and denomination of invention object fuzzy, and this simplification or omit and can not be used for limiting the scope of the invention.
In view of Problems existing in the arachidonic method of above-mentioned and/or existing control fermentative production, propose the present invention.
Therefore, an object of the present invention is to solve deficiency of the prior art, the invention provides a kind of two benches pH that adopts and control the arachidonic method of fermentative production.
For solving the problems of the technologies described above, according to an aspect of the present invention, the invention provides following technical scheme: a kind of two benches pH that adopts controls the arachidonic method of fermentative production, it is produce bacterial strain with Mortierella alpina, adopt two benches pH control strategy, earlier fermentation regulates fermented liquid pH to be 4-6, regulates fermented liquid pH to be that 5-8 carries out fermentation culture after bacterial strain is converted to oil and fat accumulation type by growth form.
A kind of preferred version of the arachidonic method of fermentative production is controlled as employing two benches pH of the present invention, wherein: take Mortierella alpina as starting strain, be seeded to activated inclined plane, cultivate after 7-8 days for 25 DEG C, spore suspension is prepared into the mycelium on transfering loop scraping inclined-plane and spore after adding sterilized water, spore suspension is inoculated into seed culture medium, 25 DEG C, and 200rpm cultivates 36-40h; Be seeded in the fermentor tank that fermention medium is housed by the inoculum size of 10-15% and ferment, fermentation condition is: 25 DEG C, mixing speed 250rpm, and air flow quantity is 1.0-1.2m
3/ m
3fermented liquid/min.
A kind of preferred version of the arachidonic method of fermentative production is controlled, wherein: using NaOH/HCl, ammoniacal liquor, ammonia, ammonium nitrate or ammonium hydroxide to control Preliminary fermentation pH is in earlier stage 4-6 as employing two benches pH of the present invention; Treat that fermentation is to grease Rapid Accumulation period phase, uses NaOH/HCl, ammoniacal liquor, ammonia, ammonium nitrate or ammonium hydroxide regulating and controlling pH to 5-8, maintain always and be stable at this pH and last till fermentation ends.
Utilize the present invention can significantly improve arachidonic output.By two stages control pH respectively, finally make arachidonic acid yield reach 8.2g/L, improve 32%.Meanwhile, two benches pH controls the utilization ratio that improve glucose and nitrogenous source, can shorten fermentation period, reduces production cost.It is simple, economical, efficient that two benches pH provided by the present invention controls fermentation strategies, do not need to increase extra fermentation equipment, the zymotechnique relevant according to the adjustment of bacterial metabolism characteristic, realize arachidonic Rapid Accumulation, have important industrial application value, produce microbial oil to other produce oil fungi fermentations has certain directive significance simultaneously.
Embodiment
For enabling above-mentioned purpose of the present invention, feature and advantage become apparent more, are described in detail below by the specific embodiment of the present invention.
Set forth a lot of detail in the following description so that fully understand the present invention, but the present invention can also adopt other to be different from alternate manner described here to implement, those skilled in the art can when without prejudice to doing similar popularization when intension of the present invention, therefore the present invention is by the restriction of following public specific embodiment.
The method for producing bacterial strain with Mortierella alpina (Mortierela alpina), adopts the fermentation strategies of two benches control pH respectively, improves arachidonic production efficiency.Fermenting, first stage (0-48h) control pH is 4-6, makes somatic cells fast breeding, is 5-8, makes it be in best arachidonic acid synthesis environment at fermentation subordinate phase (after 48h) control pH.Concrete grammar is as follows:
(1) activation of bacterial strain: Mortierella alpine trichoderma strain after 7-8 days in slant culture activation, is added the sterilized water of certain volume, scrapes mycelia and spore with transfering loop, obtain the spore suspension of Mortierella alpina after concussion is filtered;
(2) seed culture: by Mortierella alpina spore suspension (10
8individual/mL) be inoculated in seed culture medium, inoculum size is every 50mL culture medium inoculated 1mL spore suspension, and 25 DEG C, 200rpm cultivates 36-40h, obtains seed culture fluid;
(3) fermentation culture: seed culture fluid is inoculated into the fermentor tank that fermention medium is housed and ferments.Design parameter is: liquid amount 60%, inoculum size 15%, temperature 25 DEG C, air flow 1.0-1.2m
3/ m
3fermented liquid/min, mixing speed is 250rpm.
(4) pH controls: using NaOH/HCl, ammoniacal liquor, ammonia, ammonium nitrate or ammonium hydroxide to control Preliminary fermentation pH is in earlier stage 4-6; Treat that fermentation is to grease Rapid Accumulation period phase (48h), uses NaOH/HCl, ammoniacal liquor, ammonia, ammonium nitrate or ammonium hydroxide regulating and controlling pH to 5-8, maintenance is always stable at this pH and lasts till fermentation ends.
(5) extraction of grease: after fermentation ends, carries out vacuum filtration by fermented liquid, and wet thallus is constant weight at 60 DEG C.After mycelium after oven dry is ground to uniform powder, utilize organic solvent (normal hexane, chloroform/methanol) to carry out the extraction of grease, obtain containing the arachidonic bacterium oil of high density.
Above-mentioned bacterial strains activation medium is PDA substratum: potato 200g, glucose 20g, agar 20g, water 1000g, pH nature.
Above-mentioned seed culture based component is (g/L): glucose 30, yeast extract paste 20, potassium primary phosphate 0.1;
Above-mentioned fermentation medium components is (g/L): glucose 80, yeast extract paste 30, potassium primary phosphate 0.1, magnesium sulfate heptahydrate 2.
The method of above-mentioned acquisition spore suspension is specially: what the mycelium scraped and spore mixed solution are aseptically transferred to sterilising treatment crosses is covered with in the 250mL Erlenmeyer flask of granulated glass sphere (3-4mm), and granulated glass sphere thickness is at about 0.5mm.25 DEG C, at shaking table concussion 20min under 200rpm condition, then filter with 4 layers of aseptic lens wiping paper, finely dispersed spore suspension can be obtained.
The correlation parameter related in aforesaid method measures:
The mensuration of biomass: by fermented liquid vacuum filtration, distilled water wash 3 times, is placed in 60 DEG C of oven dry and spends the night, weigh.
Grease extraction: by dry mycelium grind into powder, with concentrated hydrochloric acid 75 DEG C of broken wall 1h, divides with normal hexane and extracts thick bacterium oil for 3 times, finally dry up solvent with nitrogen, weigh.
Determination of fatty acid: methyl esterification of fatty acid: get thalline grease 0.1g, adds 0.5M NaOH-methanol solution 2mL, 65 DEG C of water-bath 30min.2mL boron trifluoride-methanol solution (volume ratio is 1:3) is added, 70 DEG C of water-bath concussion 5min after cooling room temperature.Add 2mL normal hexane vibration layering, add the saturated NaCl solution of 1mL, Aspirate supernatant adds anhydrous Na
2sO
4centrifugal laggard promoting the circulation of qi facies analysis.
Gas-chromatography working conditions: gas chromatographic analysis adopts GC2014 (Japanese Shimadzu), chromatographic column: P/N053187 (30m × 0.32mm, 0.22 μm); Fid detector, purge flow rate 3.0mL/min; Injection port and detector temperature are 250 DEG C; Shunting mode sample introduction 1 μ L, splitting ratio 50:1, carrier gas is nitrogen.
Comparative examples 1 maintains fermented liquid pH=4-6 and accumulates arachidonic acid
With Mortierella alpina (Mortierella alpina) for producing bacterial strain, being seeded to PDA inclined-plane, under 25 DEG C of conditions, activating 7 days.Cover with after mycelia and spore until inclined-plane, with mycelia under the aseptic washing of 15mL, and make spore suspension, be inoculated in seed culture medium, 25 DEG C, 200rpm cultivates 36h; Inoculum size by 15% is seeded in fermentor tank ferments, and fermentation condition is: liquid amount 60%, inoculum size 15%, temperature 25 DEG C, air flow 1.0-1.2m
3/ m
3fermented liquid/min, mixing speed is 250rpm, and the pH using NaOH/HCl, ammoniacal liquor, ammonia, ammonium nitrate or ammonium hydroxide to control whole fermenting process is 4-6, and fermentation period is 8 days.Biomass, fat content and arachidonic acid content is measured after fermentation ends.Wherein culture medium prescription is as follows:
Seed culture based component is (g/L): glucose 30, yeast extract paste 20, potassium primary phosphate 0.1;
Fermentation medium components is (g/L): glucose 80, yeast extract paste 30, potassium primary phosphate 0.1, magnesium sulfate heptahydrate 2.
Comparative examples 2 maintains fermented liquid pH=5-8 and accumulates arachidonic acid
Fermentation process, condition are except following variation, and all the other are with comparative examples 1: the pH that fermenting process NaOH/HCl, ammoniacal liquor, ammonia, ammonium nitrate or ammonium hydroxide control whole fermenting process is 5-8.Biomass, fat content and arachidonic acid content is measured after fermentation ends.
Comparative examples 3 accumulates arachidonic acid under maintaining fermented liquid pH natural condition
Fermentation process, condition are except following variation, and all the other are with comparative examples 1: whole fermenting process does not regulate pH, and only initial pH regulator is to 4-6.Biomass, fat content and arachidonic acid content is measured after fermentation ends.
Embodiment 1 two benches pH control strategy accumulation arachidonic acid
Fermentation process, condition are except following variation, and all the other are with comparative examples 1: using NaOH/HCl, ammoniacal liquor, ammonia, ammonium nitrate or ammonium hydroxide to control Preliminary fermentation pH is in earlier stage 4-6; Treat that fermentation is to grease Rapid Accumulation period phase (48h), uses NaOH/HCl, ammoniacal liquor, ammonia, ammonium nitrate or ammonium hydroxide regulating and controlling pH to 5-8, maintenance is always stable at this pH and lasts till fermentation ends.Biomass, fat content and arachidonic acid content is measured after fermentation ends.
Wherein the measuring method of above each example biomass is: by fermented liquid vacuum filtration, distilled water wash 3 times, wet thallus is placed in 60 DEG C of baking ovens oven dry and spends the night, weigh.The calculation formula of biomass is:
Grease extraction: the dry mycelium taking certain mass, by dry mycelium grind into powder, with concentrated hydrochloric acid 75 DEG C of broken wall 1h, divide with 2mL normal hexane and extract thick bacterium oil for 3 times, finally dry up solvent with nitrogen, weigh, obtain the relative content of grease:
Determination of fatty acid: methyl esterification of fatty acid: get thalline grease 0.1g, adds 0.5M NaOH-methanol solution 2mL, 65 DEG C of water-bath 30min.2mL boron trifluoride-methanol solution (volume ratio is 1:3) is added, 70 DEG C of water-bath concussion 5min after cooling room temperature.Add 2mL normal hexane vibration layering, add the saturated NaCl solution of 1mL, Aspirate supernatant adds anhydrous Na
2sO
4centrifugal laggard row vapor detection is analyzed, and utilizes area normalization method to obtain arachidonic relative content, and calculates final arachidonic acid yield:
Arachidonic acid yield (g/L)=biomass × grease relative content × arachidonic acid relative content
Gas-chromatography working conditions: gas chromatographic analysis adopts GC2014 (Japanese Shimadzu), chromatographic column: P/N053187 (30m × 0.32mm, 0.22 μm); Fid detector, purge flow rate 3.0mL/min; Injection port and detector temperature are 250 DEG C; Shunting mode sample introduction 1 μ L, splitting ratio 50:1, carrier gas is nitrogen.
Concentration of residual glucose measures: adopt 3,5-dinitrosalicylic Acid Colorimetry to measure.
Two benches pH control strategy is compared with control group:
(1) in comparative examples 1 (constant pH 4-6), arachidonic acid yield is 6.4g/L, in comparative examples 2 (constant pH 5-8), arachidonic acid yield is 7.4g/L, in comparative examples 3 (pH nature), arachidonic acid yield is 6.2g/L, in embodiment 1 (two benches pH controls), arachidonic acid output increased is to 8.2g/L, does not improve 28.1%, 10.8% and 32.3% than contrast component;
(2) in comparative examples 1 (constant pH 4-6), glucose utilization rate is 87.5%, glucose utilization rate 91.2% in comparative examples 2 (constant pH 5-8), in comparative examples 3 (pH nature), glucose utilization rate is 85.3%, embodiment 1 (two benches pH controls) glucose utilization rate brings up to 100%, does not improve 14.3%, 9.6% and 17.2% than contrast component.
It should be noted that, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.
Claims (3)
1. one kind adopts two benches pH to control the arachidonic method of fermentative production, it is characterized in that: be produce bacterial strain with Mortierella alpina, adopt two benches pH control strategy, earlier fermentation regulates fermented liquid pH to be 4-6, regulates fermented liquid pH to be that 5-8 carries out fermentation culture after bacterial strain is converted to oil and fat accumulation type by growth form.
2. method according to claim 1, it is characterized in that: take Mortierella alpina as starting strain, be seeded to activated inclined plane, cultivate after 7-8 days for 25 DEG C, spore suspension is prepared into the mycelium on transfering loop scraping inclined-plane and spore after adding sterilized water, spore suspension is inoculated into seed culture medium, 25 DEG C, and 200rpm cultivates 36-40h; Be seeded in the fermentor tank that fermention medium is housed by the inoculum size of 10-15% and ferment, fermentation condition is: 25 DEG C, mixing speed 250rpm, and air flow quantity is 1.0-1.2m
3/ m
3fermented liquid/min.
3. method according to claim 1, is characterized in that: using NaOH/HCl, ammoniacal liquor, ammonia, ammonium nitrate or ammonium hydroxide to control Preliminary fermentation pH is in earlier stage 4-6; Treat that fermentation is to grease Rapid Accumulation period phase, uses NaOH/HCl, ammoniacal liquor, ammonia, ammonium nitrate or ammonium hydroxide regulating and controlling pH to 5-8, maintain always and be stable at this pH and last till fermentation ends.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105907691A (en) * | 2016-07-13 | 2016-08-31 | 四川大学 | Method for shortening high-salt fermentation period of lactic acid bacteria by controlling pH of two phases |
| 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102925502A (en) * | 2011-08-10 | 2013-02-13 | 嘉必优生物工程（湖北）有限公司 | Industry method for producing arachidonic acid grease by using mortierella alpine |
| CN103667068A (en) * | 2012-09-14 | 2014-03-26 | 罗盖特兄弟公司 | Arachidonic acid-rich oil produced by microorganism (unicellular fungal mortierella alpina) and preparation process thereof |
-
2015
- 2015-01-28 CN CN201510045230.4A patent/CN104651423A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102925502A (en) * | 2011-08-10 | 2013-02-13 | 嘉必优生物工程（湖北）有限公司 | Industry method for producing arachidonic acid grease by using mortierella alpine |
| CN103667068A (en) * | 2012-09-14 | 2014-03-26 | 罗盖特兄弟公司 | Arachidonic acid-rich oil produced by microorganism (unicellular fungal mortierella alpina) and preparation process thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105907691A (en) * | 2016-07-13 | 2016-08-31 | 四川大学 | Method for shortening high-salt fermentation period of lactic acid bacteria by controlling pH of two phases |
| CN105907691B (en) * | 2016-07-13 | 2019-09-27 | 四川大学 | A kind of method that the lactic acid bacteria high salt fermentation period is shortened in two stages pH control |
| 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 |
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Application publication date: 20150527 |