CN105132480B - The method for inducing chlorella vulgaris ZF algae strain efficient accumulation EPA - Google Patents
The method for inducing chlorella vulgaris ZF algae strain efficient accumulation EPA Download PDFInfo
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Abstract
The present invention provides the method for induction chlorella vulgaris ZF algae strain efficient accumulation EPA a kind of, it is characterized in that using following steps: 1) preparing algae solution: under conditions of 23 DEG C ± 2 DEG C, 1900 ± 100lx of light intensity and Light To Dark Ratio is 12h/12h, logarithmic growth phase is arrived within BG11 culture medium culture chlorella vulgaris ZF algae strain cell 28 days using 1/4 times, the algae solution for plant growth regulator induction is obtained, the BG11 nutritive salt of 1 times of concentration is added within every 14 days during culture；2) accumulate EPA: logarithmic growth phase algae solution 200mL is placed in 300mL triangular pyramidal bottle, then plant growth regulator 6-BA is added, concentration of the plant growth regulator 6-BA in algae solution is 1 ± 0.05mg/L, induction processing 14 ± 1 days is carried out in 23 DEG C ± 2 DEG C, processing stage manually shakes algae daily and is no less than 3 times, adjacent algae time interval of shaking realized accumulation of the EPA in algae solution not less than 2 hours.The invention is simple and feasible, low in cost, can significantly accumulate EPA content in a short time.
Description
Technical field
The present invention provides the method for induction chlorella vulgaris ZF algae strain efficient accumulation EPA a kind of, belongs to field of biotechnology.
Background technique
Chlorella (Chlorella) is a kind of common aquatic unicellular alga, is a weight in green alga chlorella section
Belong to, ecologicaI distribution is extensive, is distributed in seawater and fresh water, at present includes about 10 kinds.Contain in chlorella cells
Protein (the 50% of dry weight) abundant, essential amino acid, polysaccharide, lipid (result of fatty acid accumulation), chlorophyll, carrot
Element and vitamin etc., are widely used in health food, aquaculture (feed addictive), cosmetics, medicine and other fields,
Supply falls short of demand for domestic and international market.Compared with the countries such as the U.S., Japan, Israel, the chlorella in China industrializes large-scale culture
Level relatively lags behind.Chlorella vulgaris ZF algae strain is a kind of spherical unicellular fresh water algae, is usually grown in the water of eutrophication
In body, but can raised growth breeding under culture conditions.Nutritive salt (nitrogen, phosphorus etc.), illumination, pH value, temperature, microelement
(iron, manganese, selenium etc.), ammonium hydrogen carbonate, etc. factors can influence chlorella growth.
Eicosapentaenoic acid (eicosapentaenoic acid, EPA), is a kind of n-3 polyunsaturated fatty acid
(polyunsaturated fatty acid, PUFA), i.e. the 1st double bond appear in the 3rd of carbochain methyl end.EPA is being sought
It supports and medically all there is important physiological activity, can promote brain development, improve cerebral function, improve memory, to prevention and treatment
Heart disease, artery sclerosis, cancer, rheumathritis, asthma and diabetes etc. also have positive effect.At present mainly from fish oil
It obtains, but the product fishy smell in fish oil source is larger, quality is not high, and yield is unstable.Recently pass through microalgae extraction purification DHA, EPA
Have become the hot spot studied both at home and abroad.Plant growth regulator is generated in plant to plant growth and development regulation work
Chemical substance can be divided into five major class: auxins, cytokinin, gibberellin class, abscisic acid and ethylene, and this
Five major class growth regulators are in algae it has been found that plant growth regulator has apparent promote to the growth and development of plant
It acts on and is widely used in agricultural production.Plant growth regulator as a plant growth regulators, have it is easy to operate,
Many advantages, such as inductivity is high, speed is fast, low cost, damage are light, pollution-free, high safety.Microalgae is to carry out plant growth tune
The excellent material of agent induction is saved, structure is simple, life cycle is short, light sensitivity is strong, metabolic process is easily affected by environment, also holds
Easily it is detected.
Currently, the related patents technology of some purifications, the method and process for preparing EPA has been disclosed both at home and abroad, it is most of
It is for technique improvements such as some purifications, separation.As Shandong Inst. of Marine Medicinal Sci. discloses one kind from marine growth
Alkali separation prepares the method (CN88101811) of eicosapentaenoic acid, docosahexaenoic acid or its esters, feature in grease
It is first to make fat saponification with the ethanol solution of metal hydroxides, then molten with the low carbon numbers straight chain alcohol such as the ethyl alcohol of urea or methanol
The obtained unsaturated fatty acid of liquid processing.Directly to the alcoholic solution of resulting high unsaturated fatty acid after removing urea inclusion
Inorganic acid is added to carry out Catalytic esterification.In the technique of the invention, alkali metal crystallisation and urea inclusion method are improved, and
It is combined, the oil-soluble impurities such as saturated fatty acid and the cholesterol in degreasing is removed with preceding method, it is low not with the removing of rear method
Thus EPA, DHA or its ester derivative of the non-isomerization of high-content is made in saturated fatty acid.Lv Weixue etc. provide it is a kind of from
The method (CN201110106175) of EPA and DHA is extracted in seaweed.Its step: seaweed raw material is crushed, and hydrochloric acid hydrolysis is added,
After colloid mill grinding homogenate, successively into ultrafiltration, nanofiltration I, nanofiltration II is crossed, ethyl alcohol is extracted.As Harbin patented technology develops public affairs
Department provides a kind of using fish oil as the method for waste eicosapentaenoic acid, docosahexaenoic acid and its esters
(CN88106038), EPA, DHA content are high in the product of this method, and product yield is high.Inner Mongolia Kingdomway Pharmaceutical Co., Ltd.
It is provided with Xiamen Jindawei Group Co., Ltd and a kind of extracts DHA unsaturated fatty acid from dino flagellate fermentation liquor
Method (CN200910159368), it is characterized in that: first by the hydrochloric acid solution tune of dino flagellate fermentation liquor mass concentration 10-30%
Section pH value is water-soluble for the noresidue monomer cationic polyacrylamide of 0.1-0.5% with fermentating liquid volume 2-5%, concentration to 3-6
Liquid is flocculant flocculation, and plate-frame filtering obtains filter cake, the shell-broken liquid broken wall of 5-10 times of weight of filter cake, uses high speed centrifugation after broken wall
Machine realizes that slag, water phase, oily phase three phase separation, substantially oil-free phase after repeating 2-5 times merge oil mutually and be after washing to contain DHA
Crude oil, DHA crude oil enter the continuous molecular distillation of Pyatyi, and the heavy constituent of prime is directly entered rear class and is distilled, absolute pressure
0.5-5Pa, 120-140 DEG C of temperature, final the unsaturated fatty acid for containing DH.Hubei Fuxing Biological Technology Co., Ltd. discloses
A kind of side with the hidden dinoflagellate of Kou Shi (Crypthecodinium cohnii) industrial fermentation production docosahexaenoic acid grease
Method (CN200910061833).This method provides a kind of hidden dinoflagellate of Kou Shi (Crypthecodinium cohnii) original strain
For the method that strain, industrial fermentation produce docosahexaenoic acid grease, this method being capable of low cost, mass production high-content
Triglyceride type DHA grease.Above-mentioned related purification, the related patents technology for the method and process for preparing DHA are although advanced, but
It is not directed to the content of plant growth regulator induction chlorella vulgaris accumulation EPA, and technical matters is relative complex, and costly,
This undoubtedly increases the difficulty of production cost and Technique Popularizing.
Summary of the invention
The object of the present invention is to provide it is a kind of it is easy to operate, speed is fast, low cost, pollution-free, high safety induction are general
The method of logical chlorella ZF algae strain efficient accumulation EPA.Its specific technical solution are as follows:
A method of passing through induction chlorella vulgaris ZF algae strain efficient accumulation EPA, it is characterised in that use following steps:
1) it prepares algae solution: under conditions of 23 DEG C ± 2 DEG C, 1900 ± 100lx of light intensity and Light To Dark Ratio are 12h/12h, using
1/4 times is arrived logarithmic growth phase in BG11 culture medium culture chlorella vulgaris ZF algae strain cell 28 days, is obtained and is used for plant growth regulating
The algae solution of agent induction, adds the BG11 nutritive salt of 1 times of concentration for every 14 days during culture；
2) accumulate EPA: logarithmic growth phase algae solution 200mL is placed in 300mL triangular pyramidal bottle, and 6-BA is then added and swashs
Element, concentration of the plant growth regulator 6-BA in algae solution are 1 ± 0.05mg/L, and induction processing 14 ± 1 is carried out in 23 DEG C ± 2 DEG C
It, processing stage manually shakes algae daily and is no less than 3 times, and adjacent algae time interval of shaking realized EPA in algae solution not less than 2 hours
Accumulation.
Compared with prior art, the present invention its advantage is that:
1, simple and easy, raw material chlorella vulgaris ZF algae strain cell is easy to cultivate, and the period is short, at low cost.
2, the intracellular EPA content of chlorella vulgaris ZF algae strain can be significantly improved, the results showed that, frustule after induction processing
EPA content is 2.18-the 2.30% of frustule dry weight, is 8.15-8.59 times of cellular control unit respectively.
Specific embodiment
Embodiment 1, using following steps:
1) it prepares algae solution: under conditions of 23 DEG C, light intensity 1800lx and Light To Dark Ratio are 12h/12h, being trained using 1/4 times of BG11
It supports the strain of base culture chlorella vulgaris ZF algae and arrives logarithmic growth phase in cell 28 days, obtain the algae for plant growth regulator induction
Liquid adds the BG11 nutritive salt of 1 times of concentration for every 14 days during culture；
2) accumulate EPA: logarithmic growth phase algae solution 200mL is placed in 300mL triangular pyramidal bottle, and Beijing Suo Lai is then added
The plant growth regulator 6-BA of precious Science and Technology Ltd.'s production, makes concentration of the plant growth regulator 6-BA in algae solution
1mg/L carries out induction processing 14 days in 23 DEG C, and processing stage manually shakes algae daily and is no less than 3 times, adjacent to shake algae time interval not
Less than 2 hours, accumulation of the EPA in algae solution is realized.
Embodiment 2, using following steps:
1) it prepares algae solution: under conditions of 24 DEG C, light intensity 2000lx and Light To Dark Ratio are 12h/12h, being trained using 1/4 times of BG11
It supports the strain of base culture chlorella vulgaris ZF algae and arrives logarithmic growth phase in cell 28 days, obtain the algae for plant growth regulator induction
Liquid adds the BG11 nutritive salt of 1 times of concentration for every 14 days during culture；
2) accumulate EPA: logarithmic growth phase algae solution 200mL is placed in 300mL triangular pyramidal bottle, and Beijing Suo Lai is then added
The plant growth regulator 6-BA of precious Science and Technology Ltd.'s production, makes concentration of the plant growth regulator 6-BA in algae solution
0.95mg/L carries out induction processing 15 days in 24 DEG C, and processing stage manually shakes algae daily and is no less than 3 times, adjacent to shake between the algae time
Every being not less than 2 hours, accumulation of the EPA in algae solution is realized.
Experiment detection:
1) be enriched with frustule: 8000-9000rpm, 4 DEG C centrifugation 5-8 minutes, obtain bath mud, then by algal gel in -40 DEG C of freezings
Dry algae powder.
2) it extracts grease: the algae powder after freeze-drying being extracted to get grease using soxhlet extraction.
3) to grease esterification: 3ml 0.4mol/L KOH/ methanol, 60 DEG C of water being added first into the total grease extracted
Bathe 1h；14%BF is added after cooling3·CH3OH solution 3ml, 60 DEG C of water-bath 1h；20 μ L 10mg/mL nonadecanoic acids are added after cooling down again
Methyl esters, 1ml n-hexane (chromatographically pure) and 1ml are saturated NaCl, and stratification after anhydrous sodium sulfate is added, on 1ml syringe extracts
Layer organic phase, is stored in 1.5mL EP after filtering.
4) EPA content is analyzed: it is analyzed using chromatography of gases, chromatographic column is the anti-oxidant crosslinking quartz capillary column of HP-FFAP,
Specification is 30m × 0.25mm × 0.3m；Injector temperature is 260 DEG C, split ratio 50: 1；Carrier gas is high-purity N 2, and column flow is
1mL/min, total flow 55mL/min, pressure 114Kpa, linear velocity 29.6cm/s, column temperature is by 160 DEG C of speed liters with 2 DEG C/min
To 230 DEG C, holding 3min to appearance is finished；Fid detector, temperature are 230 DEG C, and sample volume 1uL obtains chromatography of gases figure.Meter
Calculate result are as follows: 1 gained EPA content of embodiment is the 2.18% of frustule dry weight, is 8.15 times of cellular control unit, embodiment 2
Gained EPA content is the 2.30% of frustule dry weight, is 8.59 times of cellular control unit.
Claims (1)
1. a kind of method of induction chlorella vulgaris ZF algae strain efficient accumulation EPA, it is characterised in that use following steps:
1) algae solution is prepared: under conditions of 23 DEG C ± 2 DEG C, 1900 ± 100lx of light intensity and Light To Dark Ratio are 12h/12h, using 1/4 times
It arrives logarithmic growth phase within BG11 culture medium culture chlorella vulgaris ZF algae strain cell 28 days, obtains and induced for plant growth regulator
Algae solution, the BG11 nutritive salt of every 14 days plus 1 times of concentration during culture；
2) accumulate EPA: logarithmic growth phase algae solution 200mL is placed in 300mL triangular pyramidal bottle, and plant growth regulating is then added
The concentration of agent 6-BA, plant growth regulator 6-BA in algae solution is 1 ± 0.05mg/L, carries out induction processing in 23 DEG C ± 2 DEG C
14 ± 1 days, processing stage manually shook algae daily and is no less than 3 times, and adjacent algae time interval of shaking realized EPA in algae not less than 2 hours
Accumulation in liquid.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102281756A (en) * | 2009-01-13 | 2011-12-14 | α-J研究有限合伙公司 | Use of plant growth regulators to enhance algae growth |
| CN103305560A (en) * | 2013-06-06 | 2013-09-18 | 山东理工大学 | Method for inducing fresh water chlorella to fast accumulate grease through plant hormone jasmonic acid |
| WO2014081963A2 (en) * | 2012-11-21 | 2014-05-30 | Nair, Ramesh | Engineering plants to produce farnesene and other terpenoids |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102281756A (en) * | 2009-01-13 | 2011-12-14 | α-J研究有限合伙公司 | Use of plant growth regulators to enhance algae growth |
| WO2014081963A2 (en) * | 2012-11-21 | 2014-05-30 | Nair, Ramesh | Engineering plants to produce farnesene and other terpenoids |
| CN103305560A (en) * | 2013-06-06 | 2013-09-18 | 山东理工大学 | Method for inducing fresh water chlorella to fast accumulate grease through plant hormone jasmonic acid |
Non-Patent Citations (2)
| Title |
|---|
| Enhancement of microalgae growth and fatty acid content under the influence of phytohormones;El-Sayed Salama,et al.;《Bioresource Technology》;20141130;第172卷;第97–103页 |
| 蹄形藻和单针藻生长与油脂合成影响因素的研究;徐阳;《优秀硕士学位论文全文数据库 工程科技Ⅰ辑》;20140215(第2期);第6页第3-8段，第46-49页3.3.10部分以及表3.20，表3.21 |
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