CN105132481A - Method for inducing chlorella vulgaris ZF algal strains to efficiently accumulate linolenic acid - Google Patents
Method for inducing chlorella vulgaris ZF algal strains to efficiently accumulate linolenic acid Download PDFInfo
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- CN105132481A CN105132481A CN201510609109.XA CN201510609109A CN105132481A CN 105132481 A CN105132481 A CN 105132481A CN 201510609109 A CN201510609109 A CN 201510609109A CN 105132481 A CN105132481 A CN 105132481A
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Abstract
The invention provides a method for inducing chlorella vulgaris ZF algal strains to efficiently accumulate linolenic acid. The method is characterized by adopting the following steps : 1) preparation of algae solution : under the conditions of the temperature being 23+/-2 DEG C, the light intensity being 1900+/-1001 x and the ratio of light to dark being 12h/12h, a 1/4 BG11 culture medium is adopted for culturing chlorella vulgaris ZF algal strain cells from 28 days to an exponential phase, an algae solution used for plant growth regulator induction is obtained, and BG11 nutritive salt with twice concentration is added once every 14 days during the culture period; 2) accumulation of linolenic acid: 200ml of the algae solution in the exponential phase is taken and placed into a 300ml conical flask, then a plant growth regulator 6-BA is added, the concentration of the plant growth regulator 6-BA in the algae solution is 1+/-0.05 mg/L, induction treatment is performed at 23+/-2 DEG C for 14+/-1 days, manual shaking of the algae solution is not less than three times every day in the treatment stage, time intervals between adjacent shaking of the algae solution are not less than 2 hours, and the accumulation of linolenic acid in the algae solution is realized. The method is simple and feasible, the cost is low, and the accumulation of the linolenic acid content can be remarkably improved in a short term.
Description
Technical field
The invention provides a kind of linolenic method of induction Chlorella vulgaris ZF algae strain efficient accumulation, belong to biological technical field.
Background technology
Chlorella (Chlorella) is a kind of common aquatic unicellular algae, is an important genus in green alga chlorella section, and ecologicaI distribution is extensive, all has distribution, comprise about 10 kinds at present in seawater and fresh water.Containing rich in protein (50% of dry weight), indispensable amino acid, polysaccharide, lipid (result of lipid acid accumulation), chlorophyll, carotene and VITAMIN etc. in chlorella cells, be widely used in protective foods, aquaculture (fodder additives), makeup, medicine and other fields, supply falls short of demand in domestic and international market.Compared with the countries such as the U.S., Japan, Israel, the chlorella industrialization large scale culturing level of China relatively lags behind.The strain of Chlorella vulgaris ZF algae is a kind of spherical unicellular fresh water algae, and growth is in the water body of eutrophication usually, but can raised growth breeding under culture conditions.Nutritive salt (nitrogen, phosphorus etc.), illumination, pH value, temperature, trace element (iron, manganese, selenium etc.), bicarbonate of ammonia, etc. factor all can affect chlorella growth.
Conjugate linolenic acid (conjugaedlinolenicacid is called for short CLN) refers to that a class has the punicic acid of three conjugated double bonds, and occurring in nature is mainly present in tung oil tree, Semen Granati, bitter melon seed etc. with the form of plant seed oils.There is different positional isomerss due to the difference of position of double bond in CLN, as 9,11,13 1 CLN and 8,10,12 1 CLN etc., often organize isomer again because the geometric configuration of double bond exists different geometrical isomers, as c, and c, c-CLN and c, c, t-CLN etc.Naturally occurring mainly three kind 8,10,12 1 CLN isomer and four kind 9,11,13 1 CLN isomer.CLN does not cause the attention of people at the discovery initial stage, in recent years along with its multiple beneficial physiological function is proved, become the focus of Chinese scholars research gradually, be described as " the novel nourishing factor of 21 century ", CLN has the physiologically active be highly profitable, comprise anticancer, reducing blood-fat, raising immunizing power, fat-reducing, hypoglycemic, improve immunizing power etc.Micro-algae is the excellent material carrying out plant-growth regulator induction, and its structure is simple, life cycle is short, photosensitivity is strong, metabolic process is very easily affected by environment, is also easily detected.
At present, disclosed the correlation technique of some linolenic method techniques of purifying, prepare both at home and abroad, great majority are for technique improvements such as some purification, preparations.As Northeast Forestry University discloses a kind of production method (CN1344706A) of alpha-linolenic acid, it is characterized in that with camplotheca acuminata fatty-acid ethyl ester for raw material, obtain alpha-linolenic acid through camplotheca acuminata fatty-acid ethyl ester → complexing Silver Nitrate → organic solvent extraction → recycling design → washing.This invention belongs to the production method of alpha-linolenic acid in plant seed oils, particularly relates to a kind of method adopting the purification of alpha-fiber crops acid from camptotheca seed fatty-acid ethyl ester of Silver Nitrate complexing technology.The Hou Xianglin of Shanxi Inst. of Coal Chemistry, Chinese Academy of Sciences etc. provide a kind of method (CN1366064A) of preparing alpha-linolenic acid by integrated supercritical reaction and separation.This invention be adopt be rich in linolenic vegetables oil supercritical co mutually in be that catalyst is hydrolyzed with lipase, realized the separation of inhomogeneity lipid acid in conjunction with the method that rectifying column is separated by supercritical extraction simultaneously.The method need use high-pressure reactor and relate to the problems such as thermodynamic (al) balance, and operation inconvenience, processing condition require higher.Inner Mongolia Kingdomway Pharmaceutical Co., Ltd. and Xiamen Jindawei Group Co., Ltd provide a kind of processing method (CN200910159368) extracting linolenic oil from Kiwifruit, it is characterized in that: supercritical CO
2extraction process carries out in extraction kettle, and extraction temperature is 30 DEG C-45 DEG C, extracting pressure is 25 ~ 35Mpa; The method relates to extraction agent distil process, and conditional request is stricter.Nanjing University discloses a kind of technique (CN1448383A) from algae extraction, preparation, purifying gamma-linolenic acid methyl esters.Its step: after algae powder being added the homogenate of entrainment agent methyl alcohol, at 20-50MPa, 20-60 DEG C, uses CO
2carry out supercritical extraction as solvent, after concentrated, obtain total lipid, the total lipid obtained is dissolved in 12 times of its volumes containing chloracetyl 5% methyl alcohol in, under nitrogen atmosphere protection, 80 DEG C of back flow reaction are after 2 hours, be cooled to room temperature, add 0.3% sodium carbonate solution of two volumes, washing, centrifuging and taking upper strata oil reservoir, concentrate and obtain total fatty acids methyl esters and reclaim methyl alcohol, total methyl esters is under condition of high vacuum degree 6-10Pa, and 160 DEG C of molecular distillations, collect overhead product, the overhead product of gained is added the acetone that triploid is long-pending, successively at 4 DEG C,-10 DEG C, equilibrium crystallization 24 hours at-30 DEG C, each crystallization and filtration removes crystal, finally at-30 DEG C, crystallization also crosses elimination precipitate in 24 hours again, in fatty acid methyl ester (liter) after concentration and recovery acetone: urea (kilogram): the ratio of methyl alcohol (kilogram)=1: 2-4: 8-10 adds urea and methyl alcohol, 80 DEG C are refluxed 1 hour under nitrogen protection, slowly cool to room temperature, place to filter for 24 hours and decrystallize, under zero degree environment, placement is again filtered after 24 hours and is decrystallized, after adding equal-volume water, with the petroleum ether extraction gamma-linolenic acid methyl esters of two volumes, urea clathration crystallisation process is repeated after concentration and recovery sherwood oil, after adding isopyknic water again, with the petroleum ether extraction gamma-linolenic acid methyl esters of two volumes, Distillation recovery sherwood oil, obtain gamma-linolenic acid methyl esters, by the gamma-linolenic acid methyl esters that obtains in high vacuum, 100-150 DEG C of fractional distillation, obtains gamma-linolenic acid methyl esters sterling.The method requires that pressure is higher, and relate to the technology such as molecular distillation, poly-talented requirement is higher, is not easy to operation.The Hu Defu etc. of Beijing University of Beijing Kerma (unit of kinetic energy) Technical Development Center provides a kind of preparation method (CN1317477A) of high-purity alpha-linolenic acid, oleum lini or perilla oil is characterized in be dissolved in saponification reaction in aqueous ethanolic solution, obtain mixed fatty acid, then urea clathration, slow cooling and evaporation concentration, obtain alpha-linolenic acid, the alpha-linolenic acid ester that purity is 95 ~ 99.9% is obtained successively again through esterification, column chromatography for separation, finally by saponification reaction, namely molecular distillation obtains the colourless alpha-linolenic acid that purity is 95 ~ 99.9%.Zhu Tiebao etc. disclose a kind of method (CN1235956A) of purification & isolation alpha-linolenic acid, it is included in seed adds separation promoter and antioxidant, in inert gas atmosphere, extract fat, free fatty acids is generated again through hydrolysis, in free fatty acids, add tensio-active agent, separation promoter and antioxidant in nitrogen atmosphere, carry out three times and be separated, obtain the alpha-linolenic acid of high density; In the alpha-linolenic acid of high density, add antioxidant again and carry out complete esterification in nitrogen atmosphere, the ester of generation carries out purifying and namely obtains product.As Changcheng Fubang Biological Engineering Co., Ltd., Beijing provides a kind of processing method (CN1414080A) extracting alpha-linolenic acid with membrane separation technique from vegetables oil, comprise vegetable oil raw materials pre-treatment, separation, aftertreatment and purification.The mixed fatty acid of pre-treatment and separation promoter, anti-oxidant activity agent, tensio-active agent mix and obtain mixed solution, mixed solution is separated at I cold bottom film filter and obtains I liquid, I liquid is separated at II freezing mask strainer and obtains II liquid, II liquid is separated at III cold bottom film filter and obtains III liquid, III liquid is separated at IV cold bottom film filter and obtains IV liquid, completes separation.The heating of IV liquid obtains lipid acid, and lipid acid film filter separating-purifying, multipole molecular sieves have been purified aftertreatment and purification.Through pre-treatment, separation, aftertreatment and purification, obtain C 18 unsaturated fatty acid product.Although the Patents technology of above-mentioned relevant purification, the linolenic method technique of preparation D is advanced, but all do not relate to plant-growth regulator induction Chlorella vulgaris and accumulate linolenic content, and technical matters relative complex, and costly, this adds the difficulty of production cost and technology popularization undoubtedly.
Summary of the invention
The object of this invention is to provide a kind of simple to operate, speed is fast, cost is low, free of contamination by the linolenic method of induction chlorella ZF algae strain efficient accumulation.Its concrete technical scheme is:
A kind of by the linolenic method of induction Chlorella vulgaris ZF algae strain efficient accumulation, it is characterized in that adopting following steps:
1) prepare algae liquid: 23 DEG C ± 2 DEG C, under light intensity 1900 ± 100lx and Light To Dark Ratio be the condition of 12h/12h, adopt 1/4 times of BG11 culture medium culturing Chlorella vulgaris ZF algae strain cell 28 days to logarithmic phase, obtain the algae liquid being used for plant-growth regulator induction, between incubation period every 14 days, add the BG11 nutritive salt of 1 times of concentration;
2) linolenic acid is accumulated: the vegetative period algae liquid 200mL of taking the logarithm is placed in 300mL triangular pyramidal bottle, then plant-growth regulator 6-BA is added, the concentration of plant-growth regulator 6-BA in algae liquid is 1 ± 0.05mg/L, induction process is carried out 14 ± 1 days in 23 DEG C ± 2 DEG C, treatment stage every day manually shake algae and be no less than 3 times, adjacently shake the algae timed interval and be not less than 2 hours, realize the accumulation of linolenic acid in algae liquid.
Compared with prior art, its advantage is in the present invention:
1, simple, starting material Chlorella vulgaris ZF algae strain cell is easy to cultivate, and the cycle is short, and cost is low.
2, can significantly improve linolenic acid content in Chlorella vulgaris ZF algae strain cell, result shows, after induction process, linolenic acid content is 93.92-94.99% of total fatty acids in frustule, is 5.41-5.47 times of control group frustule respectively.
Embodiment
Embodiment 1, adopts following steps:
1) prepare algae liquid: 23 DEG C, under light intensity 1800lx and Light To Dark Ratio be the condition of 12h/12h, 1/4 times of BG11 culture medium culturing Chlorella vulgaris ZF algae strain cell is adopted to arrive logarithmic phase in 28 days, obtain the algae liquid being used for plant-growth regulator induction, between incubation period every 14 days, add the BG11 nutritive salt of 1 times of concentration;
2) linolenic acid is accumulated: the vegetative period algae liquid 200mL of taking the logarithm is placed in 300mL triangular pyramidal bottle, then the plant-growth regulator 6-BA that Beijing Suo Laibao Science and Technology Ltd. produces is added, the concentration of plant-growth regulator 6-BA in algae liquid is made to be 1.00mg/L, induction process is carried out 14 days in 23 DEG C, treatment stage every day manually shake algae and be no less than 3 times, adjacently shake the algae timed interval and be not less than 2 hours, realize the accumulation of linolenic acid in algae liquid.
Embodiment 2, adopts following steps:
1) prepare algae liquid: 24 DEG C, under light intensity 2000lx and Light To Dark Ratio be the condition of 12h/12h, 1/4 times of BG11 culture medium culturing Chlorella vulgaris ZF algae strain cell is adopted to arrive logarithmic phase in 28 days, obtain the algae liquid being used for plant-growth regulator induction, between incubation period every 14 days, add the BG11 nutritive salt of 1 times of concentration;
2) linolenic acid is accumulated: the vegetative period algae liquid 200mL of taking the logarithm is placed in 300mL triangular pyramidal bottle, then the plant-growth regulator 6-BA that Beijing Suo Laibao Science and Technology Ltd. produces is added, the concentration of plant-growth regulator 6-BA in algae liquid is made to be 0.95mg/L, induction process is carried out 15 days in 24 DEG C, treatment stage every day manually shake algae and be no less than 3 times, adjacently shake the algae timed interval and be not less than 2 hours, realize the accumulation of linolenic acid in algae liquid.
Experiment detects:
1) enrichment frustule: 8000-9000rpm, 4 DEG C of centrifugal 5-8 minute, obtain bath mud, then by algae mud at-40 DEG C of lyophilize algae powders.
2) grease is extracted: utilize soxhlet extraction to extract the algae powder after lyophilize, obtain grease.
3) to grease esterification: first add 3ml0.4mol/LKOH/ methyl alcohol in the total grease extracted, 60 DEG C of water-bath 1h; 14%BF is added after cooling
3cH
3oH solution 3ml, 60 DEG C of water-bath 1h; Add 20 μ L10mg/mL methyl nonadecanoate, 1ml normal hexane (chromatographically pure) and the saturated NaCl of 1ml after cooling again, add stratification after anhydrous sodium sulphate, 1ml syringe extraction upper organic phase, is stored in 1.5mLEP after filtration.
4) linolenic acid content analysis: adopt chromatography of gases analysis, chromatographic column is the anti-oxidant crosslinked quartz capillary column of HP-FFAP, and specification is 30m × 0.25mm × 0.3m; Injector temperature is 260 DEG C, and splitting ratio is 50: 1; Carrier gas is high-purity N
2, post flow is 1mL/min, total flux 55mL/min, pressure 114Kpa, linear velocity 29.6cm/s, and column temperature rises to 230 DEG C by 160 DEG C with the speed of 2 DEG C/min, keeps 3min complete to going out peak; Fid detector, temperature is 230 DEG C, and sample size 1uL obtains chromatography of gases figure.Calculation result is: embodiment 1 gained linolenic acid content is 93.92% of the total fat acid of frustule, is 5.41 times of control group frustule, and embodiment 2 gained linolenic acid content is 93.99% of the total fat acid of frustule, is 5.47 times of control group frustule.
Claims (1)
1. induce the linolenic method of Chlorella vulgaris ZF algae strain efficient accumulation, it is characterized in that adopting following steps:
1) prepare algae liquid: 23 DEG C ± 2 DEG C, under light intensity 1900 ± 100lx and Light To Dark Ratio be the condition of 12h/12h, adopt 1/4 times of BG11 culture medium culturing Chlorella vulgaris ZF algae strain cell 28 days to logarithmic phase, obtain the algae liquid being used for plant-growth regulator induction, between incubation period every 14 days, add the BG11 nutritive salt of 1 times of concentration;
2) linolenic acid is accumulated: the vegetative period algae liquid 200mL of taking the logarithm is placed in 300mL triangular pyramidal bottle, then plant-growth regulator 6-BA is added, the concentration of plant-growth regulator 6-BA in algae liquid is 1 ± 0.05mg/L, induction process is carried out 14 ± 1 days in 23 DEG C ± 2 DEG C, treatment stage every day manually shake algae and be no less than 3 times, adjacently shake the algae timed interval and be not less than 2 hours, realize the accumulation of linolenic acid in algae 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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2015
- 2015-09-23 CN CN201510609109.XA patent/CN105132481A/en not_active Withdrawn
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 |
|---|
| EL-SAYED SALAMA,ET AL.: "Enhancement of microalgae growth and fatty acid content under the influence of phytohormones", 《BIORESOURCE TECHNOLOGY》 * |
| 徐阳: "蹄形藻和单针藻生长与油脂合成影响因素的研究", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
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