CN106244468A - A kind of regulate and control Mortierella alpina fermentation producing arachidonic acid preparation method - Google Patents
A kind of regulate and control Mortierella alpina fermentation producing arachidonic acid preparation method Download PDFInfo
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Abstract
A kind of regulate and control Mortierella alpina fermentation producing arachidonic acid preparation method, relate to arachidonic acid.Mortierella alpina (Mortierella alpina) LU166, deposit number: CGMCC No.12764.Preparation method: Mortierella alpina (Mortierella alpina) LU166 is cultivated and seed culture medium activation through plating medium, obtains seed liquor；Seed liquor is inoculated in fermentation medium, adds organic acid, after fermentation stability mycelium synthesizes, add organic acid and nitrogen source, promote that oils and fats accumulates and arachidonic conversion in a large number, obtain arachidonic acid.Promote cell and the accumulation of total oils and fats, improve Biomass, effectively facilitated the oils and fats conversion to ARA, improve ARA and account for the ratio of oils and fats；Improve the yield of total oils and fats, improve the yield of ARA.
Description
Technical field
The present invention relates to arachidonic acid, especially relate to a kind of regulate and control Mortierella alpina fermentation producing arachidonic acid system
Preparation Method.
Background technology
Arachidonic acid (Arachidonic acid, ARA) the most full ARACHIDONIC ACID, molecule
Formula is C20H32O2(Dedyukhina E G.et al.2011), for the senior polyunsaturated fatty acid of ω-6, has promotion infant
The multiple important physiological functions such as brain development, regulation nerve conduction, reduction cholesterol, at diet nutritional, medicine, cosmetics etc.
Multi-field be widely used (Njie-Mbye Y F.et al.2013).The traditional source of ARA is from animal livers, bathypelagic fish
Oil, evening primrose seed etc. extract (Kusumoto A.et al.2007).But due in animal tissue ARA content limited and
Having abnormal flavour, plant growing cycle is very long, it is difficult to meet the market demand, utilizes Production by Microorganism Fermentation ARA to have the cycle short, no
By advantages such as region are limited, become the focus (Wu W.et al.2015) of current research.
The common microorganism that can synthesize ARA mainly includes part antibacterial, yeast, microalgae and most filamentous fungi
(Dyal S D.et al.2005).Industrial conventional strain is concentrated mainly on Phycomycetes: Mortierella, Rhizopus, and ear is mould
Belong to, mucor and Thamnidium etc..Wherein, Mortierella alpina lipid accumulation is up to about the 40% of dry cell weight, and ARA content also may be used
Reach more than the 50% of PUFAs, become industrial main bacterial strain (Li X.et al.2015).Mortierella alpina is as life
The microorganism that product uses, by the safety evaluation of United States Department of Agriculture (USDA), is to produce long-chain polyunsaturated fatty acid up to now
Microorganism in one of the minority strain with formal safety evaluation (GRAS) (Streekstra H.et al.2015；
Nisha A.et al.2009)。
The research producing ARA currently, with respect to microbial method is concentrated mainly on screening strain excellent, the biosynthesis of ARA, training
Support in the exploration of base and the optimization of condition of culture and industrialization large-scale production process.Degerming strain itself is to microbial metabolic products
Impact outside, the composition of culture medium to its growth and product accumulation also have a great impact, as optimum carbon source, nitrogen source, carbon-nitrogen ratio,
Cofactor and the interpolation etc. of precursor.Currently, existing in a large number about the researchs of the basal nutrient compositions such as carbon nitrogen source, and cofactor and front
The achievement in research that body thing adds is less, also has the result of study of a large amount of difference.Metabolism due to Mortierella alpina fermenting and producing ARA
Process is complex, becomes the metabolic regulation of product process also to become one of difficult point.
The oil synthesis approach of Mortierella alpina fermenting and producing ARA tends to be considered FAS approach (Ward O P.et
al.2005).During the biosynthetic metabolism of ARA, the synthesis essence of polyunsaturated fatty acid is the continuous prolongation of carbochain
The common effect of the enzymatic reaction (NADPH is reducing power) with oxidative dehydrogenation.First it is combined in fatty acid synthase by acetyl-CoA
Under the enzymatic catalysis of body, go through acetyl grouptransfer, malonyl-group-transfer, be condensed, reduce, be dehydrated, the circulation such as to restore anti-
Should, generate C16:0；Then being added carbon by C16:0 through extending enzyme, (NADPH is electronics to introduce double bond through fatty acid desaturase dehydrogenation
Donor, molecular oxygen is electron acceptor), synthesizing a series of polyunsaturated fatty acid, ARA is as the terminal unsaturated lipid of ω-6 series
Fat acid thus produces.Research is thought, in M.alpina thalline, synthesizes particularly polyunsaturated fatty acid dehydrogenation for fatty acid
The NADPH of journey is mainly produced the process generation of acetone acid under the decarboxylation of malate dehydrogenase by malic acid, it is seen that malic acid
The activity of enzyme most important in fatty acid synthesis process (Wynn J P.et al.2001).Kendrick(Kendrick
A.et al.1992) etc. research think that malic acid can stimulate the activity of malate dehydrogenase；Lan (Lan W Z.et al.2002) etc.
Result of study is analyzed MSG and be have stimulated phosphopentose pathway (PPP), improves glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) vigor,
Producing more NADPH, the synthesis for oils and fats provides reducing power, promotes the synthesis of ARA.The present invention is based on this metabolic pathway
And the achievement in research of forefathers, by the interpolation of malic acid and MSG, its sweat is regulated and controled, to improving fermentation productivity.
Summary of the invention
The first object of the present invention is to provide Mortierella alpina (Mortierella alpina) LU166.
The second object of the present invention be to provide a kind of regulate and control Mortierella alpina fermentation producing arachidonic acid preparation method.
Described Mortierella alpina (Mortierella alpina) LU166, was preserved in China on 07 05th, 2016
Microbiological Culture Collection administration committee common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 China
Institute of microbiology of academy of science, postcode: 100101, preservation center registers on the books numbering: CGMCC No.12764.
The preparation method of described regulation and control Mortierella alpina fermentation producing arachidonic acid, comprises the following steps:
1) Mortierella alpina (Mortierella alpina) LU166 is cultivated and seed culture medium through plating medium
Activation, obtains seed liquor；
2) by step 1) seed liquor that obtains is inoculated in fermentation medium, adds organic acid, treat fermentation stability mycelium
Add organic acid and nitrogen source after synthesis, promote that oils and fats accumulates and arachidonic conversion in a large number, obtain arachidonic acid
(ARA)。
In step 1) in, the composition of described plating medium can be: Rhizoma Solani tuber osi 200g/L, glucose 20g/L, agar 20g/
L；The composition of described seed culture medium can be: glucose 30g/L, yeast powder 4g/L, KH2PO42g/L, MgSO4·7H2O
0.3g/L, pH are adjusted to 6.0.
In step 2) in, the composition of described fermentation medium can be: glucose 50g/L, yeast powder 8g/L, Semen Maydis pulp 3g/
L, KH2PO42g/L, glutamic acid 1g/L, MgSO4·7H2O 0.1g/L, ZnSO4·7H2O 0.1g/L, CaCO30.05g/L,
1000 × trace element 1mL；PH is adjusted to 6.0；The composition of 1000 × trace element can be: MnCl2·4H2O 0.8g/L,
H3BO30.5g/L, FeCl3·6H2O 0.2g/L, NiSO4·6H2O 0.05g/L, CoCl2·6H2O 0.005g/L, CuSO4·
5H2O0.002g/L, places 4 DEG C of preservations；The incubation time of described fermentation stability mycelium synthesis can be 120h；
Described organic acid can use malic acid, and the mass percentage concentration of described malic acid can be 0.025%.
Described nitrogen source can use organic nitrogen source, described organic nitrogen source can use NaNO3、KNO3, in sodium glutamate (MSG) etc.
One, the mass percentage concentration of described sodium glutamate (MSG) can be 0.05%.
Principle of adjustment and control of the present invention is as follows: the initial stage adds malic acid and mainly improves Biomass, and ARA synthesis is had certain pressing down
System, and initial stage and the compound malic acid that adds of stable phase are except further increasing Biomass, the synthesis to oils and fats and ARA also has one
Fixed facilitation, especially promotes the oils and fats conversion to ARA so that ARA accounts for the ratio of oils and fats and significantly improves.Contrast only exists
The experiment initial stage adds malic acid and initial stage and adds the malic acid stable composition phase and add the Biomass increase rate of MSG it appeared that MSG
Add inconspicuous to the raising effect of Biomass, and from the change of production of oils and fats and ARA it will be seen that stable phase MSG's adds
Add the synthesis to both and all have obvious facilitation, especially the raising effect highly significant to total oil.Adding plan above
On the basis of Lve, (adding malic acid at the fermentation initial stage, the stable composition phase adds Fructus Mali pumilae to the compound interpolation strategy that the present invention finally determines
Acid and MSG) ferment effect best, under this strategy, Biomass, oils and fats and ARA yield have obtained the increase of amplitude peak, yield
Respectively reach 24.5g/L, 12.4g/L and 6.2g/L, compare matched group and be respectively increased 21.21%, 50.67% and 43.47%.
Accompanying drawing explanation
Fig. 1 is to add malic acid the fermentation initial stage to Mortierella alpina (Mortierella alpina) LU166 fermented cells
Dry weight, total oil and the impact of ARA yield.
Fig. 2 is to add malic acid oil total to Mortierella alpina (Mortierella alpina) LU166 fermentation the fermentation initial stage
The impact of lubricant component.
Fig. 3 is the fermentation initial stage and stable phase adds malic acid for twice to Mortierella alpina (Mortierella alpina)
LU166 fermented cells dry weight, total oil and the impact of ARA yield.
Fig. 4 is the fermentation initial stage and stable phase adds malic acid for twice to Mortierella alpina (Mortierella alpina)
LU166 ferments oils and fats, the impact of ARA content.
Fig. 5 is to add the malic acid stable composition phase initial stage to add different nitrogen sources to Mortierella alpina (Mortierella
Alpina) LU166 fermented cells dry weight, total oil and the impact of ARA yield.
Fig. 6 is to add the malic acid stable composition phase initial stage to add different nitrogen sources to Mortierella alpina (Mortierella
Alpina) LU166 fermentation oils and fats, the impact of ARA content.
Fig. 7 is that stream adds malic acid compound interpolation MSG to Mortierella alpina (Mortierella alpina) LU166 fermentation carefully
Born of the same parents' dry weight, total oil and the impact of ARA yield.
Fig. 8 is that stream adds the compound MSG that adds of malic acid to Mortierella alpina (Mortierella alpina) LU166 ferment oil
Fat, the impact of ARA content.
Detailed description of the invention
Following example are used for further illustrating the present invention.
Embodiment 1. is tested the initial stage and is added what Mortierella alpina (Mortierella alpina) LU166 was fermented by malic acid
Impact
Mortierella alpina (Mortierella alpina) LU166, after PDA plate activates, makes spore suspension, connects
Cultivating in 250mL shaking flask, carrier fluid amount is 50mL, 150rpm, 28 DEG C, activates 2 days, turns secondary seed, by the bacterial strain after activation
Accessing 250mL shake-flask culture, carrier fluid amount is 50mL, adds 0.025% malic acid in basal fermentation medium, 28 DEG C,
150rpm cultivates.Result is as illustrated in fig. 1 and 2.
As seen from Figure 1, promote Biomass and total grease yield adding malic acid in the fermentation starting stage, but necessarily
The yield of ARA is inhibited in degree.Analyze visible in conjunction with Fig. 2, compared with matched group, in experimental group, ARA accounts for the ratio of total oil relatively
Low, and the accounting of C16:0, C18:1 and C18:2 is higher accordingly, although the interpolation of this explanation malic acid promotes FAS approach
Synthetic ester oil, but do not improve the effective of satisfied fatty acid and monounsaturated fatty acid downstream polyunsaturated fatty acid ARA
Convert.
Embodiment 2. tests initial stage and the compound malic acid that adds of stable phase to Mortierella alpina (Mortierella
Alpina) impact of LU166 fermentation
Add malic acid in view of the initial stage and be primarily to facilitate cell and the accumulation of total oil, do not have at stable phase enough
Malic acid stimulates the activity of malate dehydrogenase to provide enough reducing power NADPH as reaction substrate, and therefore, experiment considers to add in the early stage
On the basis of adding malic acid, add the malic acid of 0.025% again at stable phase, to improving the conversion ratio of ARA.Result such as Fig. 3
Shown in 4.
From Fig. 3 and 4, again add malic acid at stable phase and not only increase Fungal biodiversity and grease yield, ARA
Yield and content have also been obtained certain lifting, although total oil content has and somewhat reduces, but ARA accounts for proportion and the ARA of cell
The ratio weight average accounting for total oil has risen.Visible it be properly added malic acid at stable phase and be conducive to satisfied fatty acid and single unsaturated lipid
Fat acid is to the conversion of ARA, and this interpolation being likely due to stable phase malic acid again have activated the activity of malate dehydrogenase, " turns hydrogen
Enzyme circulates " flux increase, the NADPH of generation now can effectively participate in the desat process of fatty acid, promotes turning of ARA
Change.
Embodiment 3. is tested the initial stage and is added malic acid and stable phase interpolation nitrogen source to Mortierella alpina (Mortierella
Alpina) impact of LU166 fermentation
The interpolation in a small amount of nitrogen source can activate the activity of malate dehydrogenase, releases the restriction of NADPH, promotes the conversion of ARA.This
Test and be combined three kinds of different nitrogen source NaNO of interpolation in the cytotostatic phase on the basis of the experiment initial stage adds malic acid3、KNO3With
Sodium glutamate (MSG) (adds concentration and is all 0.05%), investigates it to Mortierella alpina (Mortierella alpina) LU166
The conversion impact of middle ARA.Result is as illustrated in Figures 5 and 6.
By Fig. 5 and 6 it can be seen that add the compound nitrogen source of adding of stable phase on the basis of malic acid at Initial stage of culture and not only carry
The yield of high Biomass, total oils and fats and ARA improves total oil and ARA content in cell the most to a certain extent and is adding
Different nitrogen sources in, the MSG synthetic effect to promoting ARA is the most obvious, NaNO3Take second place, and KNO3Effect is minimum.MSG effect is
It is good, it may be possible to because MSG, as organic nitrogen source, is converted into NH4 +In hgher efficiency, NH4 +It is efficiently absorbed as nitrogen source, excites
The activity of malate dehydrogenase, and MSG can be effectively transformed as α-ketoglutaric acid, promotes that TCA circulation generates acetyl-CoA, for
Fatty acid synthesis provides more precursors.
Embodiment 4. is tested the initial stage and is added malic acid and compound malic acid and the nitrogen source of adding of stable phase to Mortierella alpina
The impact of (Mortierella alpina) LU166 fermentation
On the basis of embodiment 2 and 3, experiment considers to add malic acid at the experiment initial stage further, is combined at stable phase
Adding malic acid and MSG, investigating the fermentation on Mortierella alpina (Mortierella alpina) LU166 affects, to entering one
Step improves the flux of malate dehydrogenase decarboxylic reaction, it is provided that more NADPH reducing power, thus improves the synthesis of ARA.Result such as figure
Shown in 7 and 8.
As seen from Figure 7, it is combined at stable phase and adds malic acid and MSG and further increase the yield of ARA.See in conjunction with Fig. 8
Going out, in experimental group, ARA accounts for the ratio of total oils and fats and does not improve, it is seen that the raising of ARA yield derives from the raising of grease yield.Right
Than above strategy, obtaining optimal ferment effect under the interpolation strategy of embodiment 4, Biomass, oils and fats and ARA yield have obtained maximum
The increase of amplitude, yield respectively reaches 24.5g/L, 12.4g/L and 6.2g/L, compare matched group be respectively increased 21.21%,
50.67% and 43.47%.
Claims (10)
1. Mortierella alpina (Mortierella alpina) LU166, was preserved in China Microbiological on 07 05th, 2016
Culture presevation administration committee common micro-organisms center, preservation center registers on the books numbering: CGMCC No.12764.
2. the preparation method regulating and controlling Mortierella alpina fermentation producing arachidonic acid, it is characterised in that comprise the following steps:
1) Mortierella alpina as claimed in claim 1 (Mortierella alpina) LU166 is cultivated through plating medium
Activate with seed culture medium, obtain seed liquor；
2) by step 1) seed liquor that obtains is inoculated in fermentation medium, adds organic acid, treat that fermentation stability mycelium synthesizes
After add organic acid and nitrogen source, promote that oils and fats accumulates and arachidonic conversion in a large number, obtain arachidonic acid (ARA).
The most as claimed in claim 2 a kind of regulate and control Mortierella alpina fermentation producing arachidonic acid preparation method, it is characterised in that
In step 1) in, consisting of of described plating medium: Rhizoma Solani tuber osi 200g/L, glucose 20g/L, agar 20g/L.
The most as claimed in claim 2 a kind of regulate and control Mortierella alpina fermentation producing arachidonic acid preparation method, it is characterised in that
In step 1) in, consisting of of described seed culture medium: glucose 30g/L, yeast powder 4g/L, KH2PO42g/L, MgSO4·
7H2O 0.3g/L, pH are adjusted to 6.0.
The most as claimed in claim 2 a kind of regulate and control Mortierella alpina fermentation producing arachidonic acid preparation method, it is characterised in that
In step 2) in, consisting of of described fermentation medium: glucose 50g/L, yeast powder 8g/L, Semen Maydis pulp 3g/L, KH2PO4 2g/
L, glutamic acid 1g/L, MgSO4·7H2O 0.1g/L, ZnSO4·7H2O 0.1g/L, CaCO30.05g/L, 1000 × trace element
1mL；PH is adjusted to 6.0；Consisting of of 1000 × trace element: MnCl2·4H2O 0.8g/L, H3BO30.5g/L, FeCl3·
6H2O 0.2g/L, NiSO4·6H2O 0.05g/L, CoCl2·6H2O 0.005g/L, CuSO4·5H2O 0.002g/L, places 4
DEG C preserve.
The most as claimed in claim 2 a kind of regulate and control Mortierella alpina fermentation producing arachidonic acid preparation method, it is characterised in that
In step 2) in, the incubation time of described fermentation stability mycelium synthesis is 120h.
The most as claimed in claim 2 a kind of regulate and control Mortierella alpina fermentation producing arachidonic acid preparation method, it is characterised in that
In step 2) in, described organic acid uses malic acid.
The most as claimed in claim 7 a kind of regulate and control Mortierella alpina fermentation producing arachidonic acid preparation method, it is characterised in that
The mass percentage concentration of described malic acid is 0.025%.
The most as claimed in claim 2 a kind of regulate and control Mortierella alpina fermentation producing arachidonic acid preparation method, it is characterised in that
In step 2) in, described nitrogen source uses organic nitrogen source.
The most as claimed in claim 9 a kind of regulate and control Mortierella alpina fermentation producing arachidonic acid preparation method, it is characterised in that
Described organic nitrogen source uses NaNO3、KNO3, one in sodium glutamate, the mass percentage concentration of described sodium glutamate can be
0.05%.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108517338A (en) * | 2018-03-15 | 2018-09-11 | 南京工业大学 | Method for producing arachidonic acid oil by fermenting mortierella alpina based on active oxygen regulation |
| CN108823109A (en) * | 2018-07-24 | 2018-11-16 | 江苏远大仙乐药业有限公司 | A kind of Mortierella alpine trichoderma strain of high-yield peanut tetraene acid lipid and its application |
| RU2716106C1 (en) * | 2019-05-13 | 2020-03-05 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Уфимский государственный нефтяной технический университет" | Method of producing polyunsaturated fatty acids with high content of arachidonic acid in lipids of aerial mycelium of fungus mortierella alpina (versions) |
| CN112625912A (en) * | 2020-09-09 | 2021-04-09 | 中国科学院微生物研究所 | Mortierella alpina strain XY05201 and application thereof |
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| CN101109015A (en) * | 2007-07-09 | 2008-01-23 | 南京工业大学 | Preparation method of arachidonic acid grease |
| CN103451247A (en) * | 2013-09-13 | 2013-12-18 | 厦门大学 | Preparation method of arachidonic acid |
| CN104278107A (en) * | 2014-10-20 | 2015-01-14 | 南京工业大学 | Method for producing arachidonic acid oil by fermenting mortierella alpina based on dissolved oxygen regulation |
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| CN101109015A (en) * | 2007-07-09 | 2008-01-23 | 南京工业大学 | Preparation method of arachidonic acid grease |
| CN103451247A (en) * | 2013-09-13 | 2013-12-18 | 厦门大学 | Preparation method of arachidonic acid |
| CN104278107A (en) * | 2014-10-20 | 2015-01-14 | 南京工业大学 | Method for producing arachidonic acid oil by fermenting mortierella alpina based on dissolved oxygen regulation |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108517338A (en) * | 2018-03-15 | 2018-09-11 | 南京工业大学 | Method for producing arachidonic acid oil by fermenting mortierella alpina based on active oxygen regulation |
| CN108517338B (en) * | 2018-03-15 | 2021-07-23 | 南京工业大学 | Method for producing arachidonic acid oil by fermenting mortierella alpina based on active oxygen regulation |
| CN108823109A (en) * | 2018-07-24 | 2018-11-16 | 江苏远大仙乐药业有限公司 | A kind of Mortierella alpine trichoderma strain of high-yield peanut tetraene acid lipid and its application |
| RU2716106C1 (en) * | 2019-05-13 | 2020-03-05 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Уфимский государственный нефтяной технический университет" | Method of producing polyunsaturated fatty acids with high content of arachidonic acid in lipids of aerial mycelium of fungus mortierella alpina (versions) |
| CN112625912A (en) * | 2020-09-09 | 2021-04-09 | 中国科学院微生物研究所 | Mortierella alpina strain XY05201 and application thereof |
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