CN105132484B - The method for inducing chlorella vulgaris ZF algae strain efficient accumulation DHA - Google Patents
The method for inducing chlorella vulgaris ZF algae strain efficient accumulation DHA Download PDFInfo
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Abstract
The present invention provides the method for induction chlorella vulgaris ZF algae strain efficient accumulation DHA 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 DHA: 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 0.01 ± 0.001mg/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 DHA in algae solution not less than 2 hours.The invention is simple and feasible, low in cost, can significantly accumulate DHA content in a short time.
Description
Technical field
The present invention provides the method for induction chlorella vulgaris ZF algae strain efficient accumulation DHA 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.
All-cis formula-7,10,13,16,19-docosahexaenoic acids (docosahexaenoic cid, abbreviation DHA), custom
It is the polyunsaturated fatty acid that a kind of pair of human body has extremely important effect at docosapentaenoic acid is in omega-3 unsaturated fatty acid family
Important member, dynamic and people itself, which cannot synthesize, to be taken in from the external world.DHA has important physiological action: with antitumor immune
It is related, while having the effects that cerebrum tonifying brain tonic, improve eyesight, is reducing blood lipid, blood pressure lowering, antiatherosclerosis, anti-inflammatory, have very big
Application value.DHA is mainly obtained from fish oil at present, but the product fishy smell in fish oil source is larger, and quality is not high, and yield is unstable
It is fixed.Recently the hot spot studied both at home and abroad is had become by microalgae extraction purification DHA, EPA.Plant growth regulator is plant
The chemical substance that regulating and controlling effect is played to plant growth and development of interior generation, can be divided into five major class: auxins, the basic element of cell division
Class, gibberellin class, abscisic acid and ethylene, and this five major class growth regulator is equal it has been found that plant growth regulating in algae
Agent has apparent facilitation to the growth and development of plant and is widely used in agricultural production.Plant growth regulator conduct
One plant growth regulators, with easy to operate, inductivity is high, speed is fast, low cost, damages light, pollution-free, high safety
Many advantages, such as.Microalgae is the excellent material for carrying out growth regulator induction, structure is simple, life cycle is short, light sensitivity is strong,
Metabolic process is easily affected by environment, is also easy to be detected.
Currently, the relevant technologies of some purifications, the method and process for preparing DHA have been disclosed in foreign countries, most of is needle
To technique improvements such as some purifications, separation.As Shandong Inst. of Marine Medicinal Sci. discloses one kind from marine growth grease
Middle separation prepares the method (CN88101811) of eicosapentaenoic acid, docosahexaenoic acid or its esters, is sending bright work elsewhere
In skill, alkali metal crystallisation and urea inclusion method are improved, and is combined, removed with preceding method full in degreasing
The non-isomerization of high-content is thus made with the low unsaturated fatty acid of rear method removing with oil-soluble impurities such as fatty acid and cholesterol
EPA, DHA or its ester derivative.A kind of method that EPA and DHA is extracted from seaweed of Lv Weixue invention
(CN201110106175), manufacture craft, which is followed successively by, crushes seaweed raw material, and hydrochloric acid hydrolysis is added, grinds and is homogenized through colloid mill
Afterwards, successively into ultrafiltration, nanofiltration I, nanofiltration II is crossed, ethyl alcohol is extracted.As Harbin Patent Technique Development Corp. provides one kind with fish
Oil is the method (CN88106038) of waste eicosapentaenoic acid, docosahexaenoic acid and its esters, this method product
Middle EPA, DHA content are high, and product yield is high.Inner Mongolia Kingdomway Pharmaceutical Co., Ltd. and the limited public affairs of Xiamen Jin Dawei group share
Department provides a kind of method (CN200910159368) that DHA unsaturated fatty acid is extracted from dino flagellate fermentation liquor, special
Sign is: the hydrochloric acid solution of dino flagellate fermentation liquor mass concentration 10-30% first being adjusted pH value to 3-6, with fermentating liquid volume
The noresidue monomer cationic polyacrylamide aqueous solution that 2-5%, concentration are 0.1-0.5% is flocculant flocculation, plate-frame filtering
Obtain filter cake, the shell-broken liquid broken wall of 5-10 times of weight of filter cake realizes slag, water phase, oily phase three-phase with supercentrifuge after broken wall
Separation, substantially oil-free phase after repeating 2-5 times merge oil mutually and are after washing crude oil containing DHA, and DHA crude oil enters five cascades
Continuous molecular distillation, the heavy constituent of prime are directly entered rear class and are distilled, absolute pressure 0.5-5Pa, and 120-140 DEG C of temperature, most
The unsaturated fatty acid of DHA must be contained eventually.Hubei Fuxing Biological Technology Co., Ltd. discloses a kind of hidden dinoflagellate of Kou Shi
The method of (Crypthecodinium cohnii) industrial fermentation production docosahexaenoic acid grease
(CN200910061833).This method provides a kind of is with the hidden dinoflagellate of Kou Shi (Crypthecodinium cohnii) original strain
Strain, the method that industrial fermentation produces docosahexaenoic acid grease, this method can be inexpensive, 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 not
It is related to the content of plant growth regulator induction chlorella vulgaris accumulation DHA, and technical matters is relative complex, and costly, this
Undoubtedly increase 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 DHA.Its specific technical solution are as follows:
A method of passing through induction chlorella vulgaris ZF algae strain efficient accumulation DHA, 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 DHA: logarithmic growth phase algae solution 200mL is placed in 300mL triangular pyramidal bottle, and plant growth is then added
Concentration of regulator 6-BA, the 6-BA element in algae solution is 0.01 ± 0.001mg/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 DHA 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 DHA content of chlorella vulgaris ZF algae strain can be significantly improved, the results showed that, after induction processing, DHA content
It is 1.22-the 1.29% of frustule dry weight, is 7.21-7.62 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 DHA: 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
0.01mg/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 between the algae time
Every being not less than 2 hours, accumulation of the DHA 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 DHA: 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
0.011mg/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 DHA 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) DHA 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 DHA content of embodiment is frustule dry weight 1.22%, is 7.21 times of cellular control unit, 2 institute of embodiment
1.29% that DHA content is frustule dry weight is obtained, is 7.62 times of cellular control unit.
Claims (1)
1. a kind of method of induction chlorella vulgaris ZF algae strain efficient accumulation DHA, 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 DHA: 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 0.01 ± 0.001mg/L, is induced in 23 DEG C ± 2 DEG C
Processing 14 ± 1 days, processing stage manually shakes algae daily and is no less than 3 times, and adjacent algae time interval of shaking realized DHA not less than 2 hours
Accumulation in algae solution.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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;《中国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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