CN106636236A - Fermentation medium for promoting mortierella alpina to produce EPA (eicosapentaenoic acid) and application thereof - Google Patents
Fermentation medium for promoting mortierella alpina to produce EPA (eicosapentaenoic acid) and application thereof Download PDFInfo
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Abstract
The invention relates to a fermentation medium for promoting mortierella alpina to produce EPA (eicosapentaenoic acid). The fermentation medium for promoting the mortierella alpina to produce the EPA consists of 50g/L of starch, 30-70g/L of soybean meal extract, 0-10g/L of potassium nitrate, 1g/L of phosphate, 0.25g/L of magnesium sulfate heptahydrate and the balance of water, and PH (potential of hydrogen) value of the fermentation medium is 6.0-8.0. The fermentation medium for promoting the mortierella alpina to produce the EPA achieves the purpose of substantially improving EPA yield of the mortierella alpina CCFM695 under normal temperature environment, enables the EPA yield to reach 1.7g/L, and increases the yield by about 3 times of that under original conditions.
Description
【Technical field】
The present invention relates to culture medium, more particularly to a kind of fermentation medium containing soybean meal extractive, and the culture
Application of the base in Mortierella alpina product EPA is promoted.
【Background technology】
EPA (all-cis formula -5,8,11, l4,17- eicosapentaenoic acid) be a kind of ω -3 series long-chain polyunsaturated fat
Acid, with special physiological functions such as anticoagulation, anticancer, anti-inflammatory, anti-cardiovascular disease, reducing blood lipid, in Medicines and Health Product, food and feeding
Material industry has more application.The biogenetic derivation of EPA mainly includes that marine fishes and fermentable are produced, fermentable
Method production EPA abundant raw materials, cycle is short, low price, the little, weatherproof of floor space etc., it is more and more concerned.At present
It is the New Harvest of E.I.Du Pont Company's report by the commercially produced product of Production by Microorganism Fermentation EPATMEPAoil andSalmon, bacterial strain uses therefor is Yarrowia lipolytica gene engineering bacteria.Mortierella alpina fat content enriches, at present
It has been that arachidonic acid (AA) commercially produces bacterium.Wild type M Mortierella need at low temperature synthesize EPA, and yield compared with
It is low.Chinese invention patent application CN2016101289357 discloses the restructuring Mortierella alpina MA- that a plant height produces EPA
OPaFADS17-3 (it is equally named as CCFM695), the bacterial strain is originated by the overexpression in wild type Mortierella alpine enzyme
In the rotten mould omega-fatty acid desaturase of melon and fruit, the high mountain quilt of successful expression candidate's omega-fatty acid desaturase is finally given
The mould recombinant bacterial strain of spore.According to records, the recombinant bacterium is inoculated in Broth culture mediums, in 28 DEG C of 200rpm shaking table cultures 7d, is measured
Its EPA (C20:5) yield reaches 617.1mg/L, accounts for the 18.7% of total fat (TFA), and shows AA expeditiously
EPA is catalytically conveted to, conversion ratio reaches 49.9%.Further to improve its EPA yield, the present invention is carried out to its culture medium composition
Optimization.
【The content of the invention】
The purpose of the present invention overcomes prior art defect, by optimizing fermentation medium on the premise of fermentation costs are reduced
Compositing formula, further improve EPA yield of the Mortierella alpina CCFM695 under normal temperature condition.
To achieve these goals, the present invention provides application of the soybean meal extractive in Mortierella alpina product EPA is promoted.
Further, the present invention provides soybean meal extractive in Mortierella alpina fermentation medium as the application of nitrogen source.
In the present invention, the preparation method of soybean meal extractive is：By weight, 1 part of dregs of beans is taken, 5 parts of water are added, is boiled simultaneously
Keep boiling 10-30min；Products therefrom is filtered with double gauze, discards filter residue, and gained filtrate is cooled to room temperature, as described
Soybean meal extractive.
Preferably, concentration of the soybean meal extractive in Mortierella alpina fermentation medium is 10-70g/L.
The present invention also provides a kind of fermentation medium, and the fermentation medium composition is as follows：
The pH value of the fermentation medium is 6.0-8.0；
The one kind of the phosphate in potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate and disodium hydrogen phosphate.
In the present invention, the starch is cornstarch, soluble starch or farina.
The present invention also provides the application that above-mentioned fermentation medium produces the culture medium of EPA as Mortierella alpina.
Technical scheme is explained in detailed below.
The present invention is with Mortierella alpina MA-oPaFADS17- disclosed in Chinese invention patent application CN 2016101289357
3 (it is equally named as CCFM695), are used as object.The bacterial strain is from rotten mould ω -3 desaturations of melon and fruit by overexpression
The restructuring Mortierella alpina of enzyme oPaFADS17 genes, the bacterial strain is preserved in Chinese microorganism strain guarantor on October 12nd, 2015
Administration committee's common micro-organisms center is hidden, deposit number is CGMCCNo.11394.
Historically, the bacterial strain be with Broth culture mediums as fermentation medium, consisting of：20g/L glucose, 5g/L
Yeast extract, 1g/L potassium dihydrogen phosphates, 0.25g/L epsom salts, 10g/L potassium nitrate, balance of water, pH 6.0.
The present invention is the composition by such as successive optimization fermentation medium, it is intended to further improve Mortierella alpina
The EPA yield of CCFM695.
Optimization process is as follows：
A, actication of culture
Mortierella alpina CCFM695 is inoculated into activation medium with 1% inoculum concentration, under the rotating speed of 28 DEG C of 200rpm
Activation 36h, activated for two generations；
Activation medium is consisted of：20g/L glucose, 5g/L yeast extracts (Yeast extract), 10g/L nitre
Sour potassium, 1g/L potassium dihydrogen phosphates, 0.25g/L bitter salts, using the 1M HCl and 1M NaOH aqueous solution by its pH adjust to
6.0, then the 20min that sterilizes at 115 DEG C of temperature, obtain described activation medium.
The impact of B, concentration of glucose to EPA yield
The Mortierella alpina CCFM695 in two generations of activation is inoculated into concentration of glucose by 1% inoculum concentration and is respectively 40g/
In the fermentation medium of L, 50g/L and 60g/L, then under the room temperature condition of 28 DEG C of temperature, with the rotating speed of 200rpm 7 are fermented
My god, each concentration do 3 it is parallel, respectively determine gained tunning aliphatic acid composition, confirm carbon source (i.e. glucose) conjunction
Reason concentration.
Fermentation medium is consisted of：Glucose (variable concentrations), 5g/L yeast extracts (Yeast extract),
10g/L potassium nitrate, 1g/L potassium dihydrogen phosphates, 0.25g/L bitter salts, using the 1M HCl and 1M NaOH aqueous solution by its
PH is adjusted to 6.0, then the 20min that sterilizes at 115 DEG C of temperature, obtains described fermentation medium.
The optimization of C, carbon source
Confirm carbon source (i.e. glucose) reasonable concentration after, respectively with cornstarch, soluble starch, farina and
Their mixture, and glycerine, used as carbon source, the glucose in replacement fermentation medium, other condition of culture divide with step B
Not Ce Ding gained tunning aliphatic acid composition, confirm carbon source reasonable selection.
The optimization of D, organic nitrogen source
Confirm carbon source reasonable concentration and it is specifically chosen after, respectively with 10g/L, 30g/L, 50g/L, 70g/L dregs of beans extraction
Thing substitutes the yeast extract (Yeast extract) in fermentation medium, the same step of other condition of culture as organic nitrogen source
B, determines respectively the aliphatic acid composition of gained tunning, confirms the reasonable selection of organic nitrogen source.
Wherein, the preparation method of the soybean meal extractive is：Dregs of beans is weighed appropriate, by dregs of beans and water weight ratio 1：5 difference
Boil and keep the 20min that seethes with excitement, products therefrom to be filtered with double gauze after adding water, discard filter residue, gained filtrate is cooled to room temperature,
As described soybean meal extractive.
In the present invention, dregs of beans should be understood to the byproduct obtained after soybean extracting bean oil, and it is in irregular fragment shape,
Color is light yellow to light brown, and taste has roasting soybean fragrance.Dregs of beans is the product that acquisition can be bought on market, for example in
The soybean products of grain storage Zhenjiang grain and oil Co., Ltd sale.
E, inorganic nitrogen-sourced optimization
After confirming carbon source and organic nitrogen source, using potassium nitrate as the inorganic nitrogen-sourced of fermentation medium, addition is respectively provided with dense
Degree is set to 0g/L, 5g/L, 10g/L, and other condition of culture determine respectively the aliphatic acid composition of gained tunning, really with step B
Recognize inorganic nitrogen-sourced reasonable selection.
The optimization of F, pH value
The pH value for adjusting fermentation medium by hydrochloric acid and/or sodium hydroxide solution is set to 5,6,7,8,9, cultivates bar
Part determines respectively the aliphatic acid composition of gained tunning with step B, confirms the reasonable selection of pH value.
Through above-mentioned test and results contrast, the present invention is surprised to find that can by reasonable selection carbon source and organic nitrogen source
EPA yield of the Mortierella alpina CCFM695 under normal temperature environment is greatly improved, the 617.1mg/L recorded by prior art is improved
It is the 275.5% of prior art to 1.7g/L.And selected carbon nitrogen source is glucose and dregs of beans, the two market price compared with
It is low and be readily available, can effectively reduce the production cost of fermentation medium.
【Specific embodiment】
The present invention is will be better understood that by following embodiments.
In the present invention, if no special instructions, it is weight percentage for representing " % " of concentration, or " part " is attached most importance to
Amount part.
The culture device that the present invention is used is the shaken cultivation case (model of Shanghai Zhi Chu Instrument Ltd. sale:ZQZY-
70B)。
Embodiment 1：The determination of concentration of glucose in fermentation medium
Mortierella alpina CCFM695 is inoculated into activation medium with 1% inoculum concentration, in 28 DEG C of 200rpm speed conditions
Lower activation 36h, activated for two generations；
Activation medium is consisted of：20g/L glucose, 5g/L yeast extracts (Yeast extract), 10g/L nitre
Sour potassium, 1g/L potassium dihydrogen phosphates, 0.25g/L bitter salts, are adjusted the pH of its aqueous solution using 1M HCl and 1M NaOH
To 6.0, then the 20min that sterilizes at 115 DEG C of temperature, obtain described activation medium.
In Broth culture mediums (20g/L glucose, 5g/L yeast extracts, 1g/L biphosphates that prior art is recorded
Potassium, 0.25g/L epsom salts, 10g/L potassium nitrate, balance of water, pH 6.0.) on the basis of, investigate and improve concentration of glucose
Whether be conducive to Mortierella alpina grow and improve aliphatic acid yield, so by concentration of glucose therein be set to 40g/L,
50g/L, 60g/L (other components in culture medium are constant), by experimental strain Mortierella alpina MA-oPaFADS17 in 28 DEG C of bars
Normal temperature fermentation 7 days under part, content of fatty acid in determination sample.
Described determination of fatty acid method bibliography：
Wang L,Chen W,Feng Y,et al.Genome characterization of the oleaginous
fungusMortierella alpina.Plos One,2011,6(12):e28319.
The different initial glucose concentrations of table 1 ferment to Mortierella alpina CCFM695 and produce the impact of EPA
Note：Data are represented with mean+SD in table, and using the Tukey HSD methods in SPSS work property point is carried out
Analysis.
As a result show, CCFM695 bacterial strains can accumulate substantial amounts of EPA under normal temperature condition, its result is recorded with prior art
It is consistent.Compared with the data that CN 2016101289357 is recorded, properly increase concentration of glucose and be conducive to TFA (TFA)
With the raising of EPA yield, the increase rate is less than expection.Wherein, too high or too low concentration of glucose is all unfavorable for EPA yield
Raising, the concentration of glucose optimum of the 50g/L bacterial strain ferments product EPA in shaking flask.
Embodiment 2：The determination of carbon source kind
Trained with the fermentation of the cornstarch of 50g/L, soluble starch, farina or glycerine alternate embodiment 1 respectively
Glucose in foster base, also sets up the group of glucose and each 25g of soluble starch, and other compositions and condition are constant, by experimental bacteria
Strain Mortierella alpina CCFM695 normal temperature fermentation 7 days under the conditions of 28 DEG C, content of fatty acid in determination sample.
The different carbon source species of table 2 is fermented to Mortierella alpina CCFM695 and produces the impact of EPA
Note：Data are represented with mean+SD in table, and using the Tukey HSD methods in SPSS work property point is carried out
Analysis.
As a result show, Mortierella alpina CCFM695 can carry out fermentation and produce fat using various starch as substrate in various degree,
Wherein best results of cornstarch, EPA yield is up to 1237mg/L and TFA (TFA) content highest, significantly excellent
In other groups, and the effect of soluble starch is also significantly greater than prior art and reaches 935mg/L, and this is primarily due to corn shallow lake
The reason for powder or soluble starch can significantly improve the biomass of thalline.But during using soluble starch as carbon source, hence it is evident that
See that its total fatty acid content is relatively low, this is likely due to soluble starch and is conducive to the raising of biomass and is unfavorable for aliphatic acid
Accumulation, therefore its resultant effect be not so good as cornstarch.And the mixture of farina, glucose and soluble starch and
When glycerine is as carbon source, EPA yield is then without significant changes.
Embodiment 3：The determination of organic nitrogen source
In the medium on the premise of glucose of the carbon source for 50g/L, organic nitrogen source yeast extract is contrasted
(Yeastextract, expensive) produces the impact of aliphatic acid, wherein yeast with soybean meal extractive (cheap) to Mortierella alpina
Extract concentration in the medium is 5g/L, and soybean meal extractive concentration in the medium is 50g/L, and other are constituted and condition
It is constant, by experimental strain Mortierella alpina CCFM695 under the conditions of 28 DEG C normal temperature fermentation 7 days, content of fatty acid in determination sample.
The different organic nitrogen sources of table 3 ferment to Mortierella alpina CCFM695 and produce the impact of EPA
Note：Data are represented with mean+SD in table, and using the Tukey HSD methods in SPSS work property point is carried out
Analysis.
Further the different of setting soybean meal extractive add concentration, respectively 10g/L, 30g/L, 50g/L and 70g/L, its
He constitutes and condition is constant, by experimental strain Mortierella alpina CCFM695 under the conditions of 28 DEG C normal temperature fermentation 7 days, determination sample
Middle content of fatty acid.
The different soybean meal extractive concentration of table 4 are fermented to Mortierella alpina CCFM695 and produce the impact of EPA
Note：Data are represented with mean+SD in table, and using the Tukey HSD methods in SPSS work property point is carried out
Analysis.
As a result show, compare the yeast extract of costliness, when soybean meal extractive is used as organic nitrogen source, not only price is low
Honest and clean, it has been unexpectedly discovered that it has significant facilitation to thalli growth, EPA yield first mate improves, up to 1551mg/L.
Additionally, when with yeast extract as organic nitrogen source, the yield ratio about 1 of AA and EPA:1, show that AA is converted into
The conversion ratio of EPA about 50% (conversion ratio=EPA yield/AA+EPA total amounts * 100%), and organic nitrogen source is made with soybean meal extractive
When, apparently higher than AA yield, about 2 times, conversion ratio reaches 61.4% to the yield of EPA, shows that soybean meal extractive may advantageously facilitate
Catalyzed conversion of the Mortierella alpina from AA to EPA, is more suitable for the industrialized production of EPA.
Central, soybean meal extractive concentration shows obvious yield increasing effect when being 30-70g/L, wherein with 50g/L effects
Most preferably, EPA yield highest.
Embodiment 4：The determination of inorganic nitrogen-sourced concentration
Make in the case that carbon source, 5g/L yeast extracts make nitrogen source, to arrange inorganic in fermentation medium in 50g/L glucose
The different addition concentration of nitrogen source potassium nitrate, are set to 0g/L, 5g/L, 10g/L, and other condition of culture are constant, by experimental strain
Mortierella alpina CCFM695 normal temperature fermentation 7 days under the conditions of 28 DEG C, content of fatty acid in determination sample.
The different nitric acid potassium concns of table 5 ferment to Mortierella alpina CCFM695 and produce the impact of EPA
Note：Data are represented with mean+SD in table, and using the Tukey HSD methods in SPSS work property point is carried out
Analysis.
Consistent with teaching in prior art, nitrogen source is limited and is conducive to Mortierella alpina to produce fat unanimously, different by adjusting
Inorganic nitrogen-sourced concentration it is found that when add in culture medium low concentration it is inorganic nitrogen-sourced when, the yield of EPA can be properly increased.
Embodiment 5：The determination of pH value
Investigate impact of the Medium's PH Value to bacterial strain EPA yield.1M hydrochloric acid and 1M sodium hydroxide solutions are used respectively by Broth
The pH value of basal medium is adjusted to 5,6,7,8,9, and other conditions are constant, by experimental strain Mortierella alpina CCFM695 at 28 DEG C
Under the conditions of normal temperature fermentation 7 days, content of fatty acid in determination sample.
The different pH value of table 8 are fermented to Mortierella alpina CCFM695 and produce the impact of EPA
Note：Data are represented with mean+SD in table, and using the Tukey HSD methods in SPSS work property point is carried out
Analysis.
From list data as can be seen that there is during pH6-8 the fermentation different from bacterial strain to produce fat, yield is shown as higher than existing
The record of technology, wherein, effect is optimum during pH=6, EPA yield highests.
Embodiment 6：Orthogonal optimization test
Carbon source, organic nitrogen source, inorganic nitrogen-sourced three are synthesized on EPA affects three larger factors to carry out the water of three factor three
Flat orthogonal experiment, design table (schedule) is as follows：
Table 9 affects the factor and level of EPA synthesis
The Orthogonal experiment results of table 10SPSS designs
The variance analysis of the Orthogonal experiment results of table 11
Note：K values represent the corresponding EPA content of certain level of A, B, C
RjIt is worth for kmax-kmin
May determine that according to extreme value R, RA>RB>RC, so impact of the carbon source kind to EPA is maximum, organic nitrogen source dregs of beans
The concentration of extract is taken second place, inorganic nitrogen-sourced KNO3The impact of concentration is minimum in three.By the size for comparing k values, can obtain
Go out, optimum fermentation process is A1B2C2, i.e., carbon source be glucose, dregs of beans concentration 50g/L, KNO3Concentration 5g/L, with this understanding,
EPA yield reaches as high as 1733.5mg/L.Than improve 2.9 times under initial condition (600mg/L).
In sum, the present invention passes through the carbon source and organic nitrogen source in reasonable selection fermentation medium, with glucose or shallow lake
As carbon source and using soybean meal extractive as organic nitrogen source, realization greatly improves Mortierella alpina to powder (particularly cornstarch)
EPA yield of the CCFM695 under normal temperature environment, to 1.7g/L, is the 275.5% of prior art.And the market of selected raw material
Price is relatively low and is readily available, and can effectively reduce the production cost of fermentation medium.
Claims (8)
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Cited By (2)
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| WO2018130204A1 (en) * | 2017-01-13 | 2018-07-19 | 江南大学 | Preparation of mortierella alpina ccfum698 thalli and application thereof to feed additives |
| CN108651755A (en) * | 2018-05-08 | 2018-10-16 | 江南大学 | A kind of application of high yield EPA Mortierella alpinas in egg feedstuff |
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| CN104087512A (en) * | 2014-05-23 | 2014-10-08 | 中国科学院青岛生物能源与过程研究所 | Mortierella alpine producing polyunsaturated fatty acids and application thereof |
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| WO2018130204A1 (en) * | 2017-01-13 | 2018-07-19 | 江南大学 | Preparation of mortierella alpina ccfum698 thalli and application thereof to feed additives |
| US11266167B2 (en) | 2017-01-13 | 2022-03-08 | Jiangnan University | Preparation of Mortierella alpina CCFM698 thalli and application thereof in feed additive |
| CN108651755A (en) * | 2018-05-08 | 2018-10-16 | 江南大学 | A kind of application of high yield EPA Mortierella alpinas in egg feedstuff |
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| CN106636236B (en) | 2020-10-23 |
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