CN1865242A - Method for extracting lutein from chlorella cell - Google Patents
Method for extracting lutein from chlorella cell Download PDFInfo
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- CN1865242A CN1865242A CN 200610011994 CN200610011994A CN1865242A CN 1865242 A CN1865242 A CN 1865242A CN 200610011994 CN200610011994 CN 200610011994 CN 200610011994 A CN200610011994 A CN 200610011994A CN 1865242 A CN1865242 A CN 1865242A
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- xenthophylls
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- chlorella
- octane
- methyl alcohol
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Abstract
This invention relates to a method for extracting lutein from Chlorella pyrenoidosa cells, belonging to the biotechnology field. It uses a mixed solvent of polar solvent methanol and non-polar solvent octane to extract lutein from Chlorella pyrenoidosa cells, wherein: centrifuge to collect the cultured Chlorella pyrenoidosa cells with 50-500ml centrifuge tubes, decant the supernatant and add in 5.0-50ml mixed solvent at carbon and oxygen atom ratio of 10:1-40:1, put the centrifuge tube on the ice and then disintegrate by ultrasonic, disintegrating time 10-30min, interval time 2-5sec between every 5-10sec ultrasonic, output power of the cell ultrasonic bucker 200-600W, after disintegration centrifuge at 5000-15000rpmat 4-8Deg C for 5-20min. It can obtain large amount of lutein extracted from the Chlorella pyrenoidosa cells from the extracting solvent supernatant octane phase.
Description
Technical field
The invention belongs to biological technical field, a kind of method of extracting xenthophylls from chlorella cells particularly is provided, adopt a kind of polar organic solvent methyl alcohol and a kind of nonpolar reagent octane of small molecular weight to mix as extraction agent, can from chlorella cells, extract xenthophylls efficiently, be worth and prospect adopting chlorella to produce to have important use aspect the important life active compound xenthophylls.
Background technology
Xenthophylls belongs to a kind of asymmetric dihydroxyl carotenoid, is the main moiety of macular pigment among the human eye retina.Discover that at present xenthophylls mainly contains the pharmaceutical value of following three aspects: prevent that 1) the elderly from looking macular degeneration; 2) has antitumous effect; 3) cardiovascular and cerebrovascular diseases there is preventive and therapeutic effect.In addition, xenthophylls can be widely used in the industries such as food color, makeup and medicine as the pure natural pigment of high value.At present price and the price of gold of xenthophylls are suitable substantially, and the laudatory title of food gold is arranged, and its domestic market year demand reaches 100,000 tons, but turnout 6 kilotons only do not satisfy the needs of domestic and international market far away, so have the vast market potentiality.
At present, the chemical synthesising technology of xenthophylls is not achieved success as yet, therefore can only mainly extract to purify from plants such as Flower of Aztec Marigold to obtain.The xenthophylls production technique is at first with Flower of Aztec Marigold ensiling, squeezing, drying and granulating, adopts normal hexane extraction and distillation back to obtain then.In addition, Chinese scholars compares deep research aspect the organic reagent kind of extracting xenthophylls from Flower of Aztec Marigold, aspect single organic solvent extraction xenthophylls, finds that hexanaphthene, normal hexane, sherwood oil, acetone and extraction using alcohol effect are better.Aspect mixed organic solvents extraction xenthophylls, find that normal hexane-acetone-methyl alcohol, sherwood oil-ethanol and normal hexane-extraction using alcohol efficient are higher.
Because the Flower of Aztec Marigold poor growth, therefore study fast growth and other high living species of lutein content and replace Flower of Aztec Marigold and become a very important research topic as the Biological resources of producing xenthophylls.We discover that lutein content is suitable with the lutein content in the Flower of Aztec Marigold substantially in the chlorella cells, particularly we adopt the technology of heterotrophic fermentation method cultivation superelevation cell concn chlorella ripe, can fast culture go out a large amount of chlorella biomasss.Therefore adopting chlorella is fully possible as a kind of Biological resources of producing xenthophylls to substitute Flower of Aztec Marigold.
There are differences because can produce the cell size and the structure of the different vegetation types of xenthophylls, the type of solvent of therefore therefrom extracting xenthophylls also should be different.Though Chinese scholars has been carried out a large amount of research aspect the optimizing and controlling method of extracting xenthophylls from Flower of Aztec Marigold, in that how the research report that goes out aspect the xenthophylls of high efficiency extraction is rare from chlorella.At present, mainly contain ethanol, acetone and normal hexane etc. at the reagent that from chlorella cells, extracts xenthophylls, the Chinese scholar discovery is compared with single solvents such as methyl alcohol, ethanol, sherwood oil, acetone, ether and normal hexanes, adopts methyl alcohol-methylene dichloride (volume ratio 2: 1) mixed solvent can reach better extraction xenthophylls effect.
Successfully filtering out and also to cultivate on the basis of controlling acquisition superelevation cell concn chlorella by the heterotrophic growth chlorella vulgaris by optimizing, we compare system and comprehensively research the single polar solvent of selection, non-polar solvent and the nonpolar mixed solvent of polarity extract xenthophylls from chlorella cells aspect, find to adopt polarity and nonpolar mixed solvent high efficiency extraction to go out xenthophylls from frustule, this has established very important basis for further adopting chlorella to produce xenthophylls.
Summary of the invention
The object of the present invention is to provide a kind of method of from chlorella cells, extracting xenthophylls, realized that from chlorella cells high efficiency extraction goes out the solvent of xenthophylls, for the industrialization production of adopting cultivating chlorella by heterotrophic nutrition fermentation to produce xenthophylls lays the foundation.
1, polar solvent extracts the effect of xenthophylls from chlorella cells: with the cultured chlorella cells of the centrifugal results of 50～500ml centrifuge tube, each 5.0～50ml methyl alcohol of difference after the evacuation supernatant liquor, ethanol, ethyl acetate and methylene dichloride, on the cytoclasis instrument, carry out the fragmentation of ultrasonic wave frustule after centrifuge tube is inserted into rubble ice, condition is broken 10～30 minutes time, every ultrasonic 5～10 seconds, 2～5 seconds at interval, circulation, cytoclasis instrument output rating is 200～600W, after the frustule fragmentation at 5000～15000 rev/mins, 4～8 ℃ of temperature, low-temperature centrifugation 5～20 minutes, extract the solvent supernatant liquor and directly on high pressure liquid chromatography, measure xenthophylls concentration, to determine the effect of xenthophylls in the opposed polarity solvent extraction frustule.Result of study shows, in the different solvents of test, methyl alcohol xenthophylls concentration in the supernatant liquor when extracting solvent is the highest, and the efficient of extracting xenthophylls is best.
2, non-polar solvent extracts the effect of xenthophylls from chlorella cells: adopt and use the same method, tested non-polar solvent benzene, sherwood oil, normal hexane, hexanaphthene, octane and decane respectively and extracted the effect of xenthophylls, it is best to find that octane extracts the effect of xenthophylls from chlorella cells.
3, mixed solvent extracts the effect of xenthophylls from chlorella: adopt and use the same method, tested methyl alcohol respectively and made up the effect of extracting xenthophylls in different carbon atoms and Sauerstoffatom number ratio respectively with the octane mix reagent, best methyl alcohol and methylene dichloride (volume ratio 2: 1, the two all is that polar solvent can the mix fully) mixed solvent of xenthophylls effect that extract from chlorella cells that has increased present bibliographical information simultaneously compares.In methyl alcohol and octane mixed solvent, because methyl alcohol is polar solvent, and octane is a non-polar solvent, and is therefore immiscible when the two mixes.Because the density of methyl alcohol is slightly larger than octane, therefore pass through the centrifugal back of cytoclasis methyl alcohol in lower floor, and octane is on the upper strata.The supernatant liquor of getting this 2 phase respectively carries out the xenthophylls concentration determination, find under different ratios methyl alcohol and octane mix reagent extraction conditions, xenthophylls only be distributed in octane mutually in, and do not detect the existence of xenthophylls in mutually at methyl alcohol.Discover that further mixed solvent carbon atom and Sauerstoffatom number compare from 10: 1 to 40: 1 in the scope, no matter be the xenthophylls concentration of octane in mutually, still extract the xenthophylls total amount all apparently higher than adopting methyl alcohol and the level of methylene dichloride (volume ratio 2: 1) as xenthophylls extraction solvent.Therefore adopt methyl alcohol and octane mixed solvent, the two mixes from 10: 1 by 40: 1 according to carbon atom in the mixed solvent and Sauerstoffatom ratio, be the mixed solvent of high efficiency extraction xenthophylls from chlorella cells, have important use to be worth at the industrialization producer mask that utilizes chlorella to produce xenthophylls.
Principle of work
Adopting the opposed polarity solvent from chlorella, to extract in the experiment of xenthophylls, finding that the methyl alcohol effect is best, the easiest frustule that enters of methyl alcohol of small molecular weight is being described, easily the xenthophylls in the frustule is being extracted.From chlorella, extract in the experiment of xenthophylls at molecular weight non-polar solvent from small to large, find that the octane effect is best, illustrate that octane helps the xenthophylls in the chlorella cells is dissolved and extracts most.Extracting the best reason of xenthophylls effect for methyl alcohol and octane mixed solvent from chlorella is: 1, at first the molecular structure of xenthophylls had both comprised the nonpolar carbochain that contains the two keys in 17 intervals, also comprised on 2 phenyl ring, respectively having 1 polar component hydroxyl, therefore made xenthophylls can be dissolved in nonpolar or polar organic solvent.But in general, the whole molecule of xenthophylls is more prone to nonpolar structure, therefore cause at methyl alcohol and octane during as immiscible mixed extraction solvent, xenthophylls almost all is assigned to the octane phase, and does not detect the existence of xenthophylls in mutually at methyl alcohol.2, when methyl alcohol and octane extracted xenthophylls as mixed solvent from chlorella cells, two kinds of immiscible solvents were brought into play different effects separately.The methanol molecules amount is little, enters frustule easily, therefore can and extract the dissolving of the xenthophylls in the frustule.But compare with methyl alcohol, xenthophylls is easier to be dissolved in the octane, therefore the xenthophylls that extracts from frustule be assigned to immediately methyl alcohol and the octane mixed solvent octane mutually in.Therefore methyl alcohol mainly is responsible for the xenthophylls in the frustule is extracted, and octane mainly is responsible for the dissolving of xenthophylls, be methyl alcohol and octane mixed solvent can be from chlorella cells high efficiency extraction go out the mechanism of xenthophylls.3, carbon atom in the xenthophylls molecule and Sauerstoffatom number ratio are 20: 1, methyl alcohol and octane according to carbon atom number and Sauerstoffatom number ratio when mixing in 10: 1 to 40: 1, the effect of extracting xenthophylls from chlorella cells is best, has illustrated that the carbon atom of mixed extraction solvent and the ratio and being in similar proportion of carbon atom in the xenthophylls molecule and Sauerstoffatom of Sauerstoffatom can obtain better to extract the xenthophylls effect.
Advantage of the present invention
Adopt a kind of small molecular weight polar solvent methyl alcohol and a kind of non-polar solvent octane, mix in the scope than from 10: 1 to 40: 1 as xenthophylls extraction solvent according to carbon atom and Sauerstoffatom number, can be from chlorella cells more effective xenthophylls that extracts, at the industrialization producer mask that adopts chlorella to produce xenthophylls significant values is arranged.
Description of drawings
Fig. 1 extracts the effect of xenthophylls for the present invention adopts the opposed polarity solvent from chlorella.X-coordinate is the polar solvent kind, and ordinate zou is the xenthophylls concentration mg/L in the non-polar solvent.Show among the figure that in the opposed polarity solvent of test, methyl alcohol xenthophylls concentration in the supernatant liquor when extracting solvent is the highest, the efficient of extracting xenthophylls is best.
Different non-polar solvents extract the effect of xenthophylls to Fig. 2 from chlorella for the present invention adopts.X-coordinate is the non-polar solvent kind, and ordinate zou is the xenthophylls concentration mg/L in the non-polar solvent.Show among the figure that in the different non-polar solvents of test, octane xenthophylls concentration in the supernatant liquor when extracting solvent is the highest, the efficient of extracting xenthophylls is best.
Fig. 3 extracts the effect of xenthophylls for the present invention adopts methyl alcohol and octane mixed solvent from chlorella.X-coordinate be methyl alcohol, octane, methyl alcohol with octane by different carbon atoms and Sauerstoffatom mixed and methyl alcohol and methylene dichloride mixed solvent (volume ratio 2: 1), ordinate zou is to extract xenthophylls total amount μ g in xenthophylls concentration mg/L in the solvent or the solvent.Show among the figure, methyl alcohol compares from 10: 1 to 40: 1 as mixed solvent according to carbon atom and Sauerstoffatom number with octane, no matter be the xenthophylls concentration of octane in mutually, still extract the xenthophylls total amount all apparently higher than adopting methyl alcohol and the level of methylene dichloride (volume ratio 2: 1) as xenthophylls extraction solvent.
Embodiment
1, chlorella cells experiment material: the present invention test used algae kind be a strain we oneself screening can unglazed chlorella-USTB01 (Chlorella sp) according to heterotrophic growth, cgmcc No1448, preservation date: on August 25th, 2005.After cultivating in batches through 5 days heterotrophism, by centrifugal (10000 rev/mins, 10 minutes) gather in the crops frustule in the 50ml centrifuge tube, after removing supernatant liquor, the frustule that falls to centrifuge tube bottom is suspended again with 50mmol phosphate buffer soln (pH7.0), and recentrifuge removes supernatant liquor, repeat 2 cleaning frustules after, recentrifuge results frustule, making the frustule dry weight that is retained in each centrifuge tube bottom all is 35mg.
2, from chlorella cells, extract xenthophylls: in centrifuge tube, add respectively 5.0ml methyl alcohol and octane according to carbon atom and Sauerstoffatom ratio from 10: 1 to 40: 1 behind the mixed solvent, centrifuge tube is inserted on the inherent cytoclasis instrument of rubble ice carries out the fragmentation of ultrasonic wave frustule, condition is broken (the ultrasonic 10 seconds 24 minutes time, 2 seconds at interval, circulation), broken instrument output rating is 600W.
3, the xenthophylls in the extraction agent is measured: prepare xenthophylls concentration with methyl alcohol-methylene dichloride (2: 1) before the experiment and be respectively 4,8,12,16 and 20mgL
-1Standardized solution, on high pressure liquid chromatography, measure successively, obtain one-variable linear regression equation (y=4.0408x-0.8169, the R of xenthophylls peak height (y) and xenthophylls concentration (x)
2=0.9987).Experiment is got supernatant liquor after by centrifugal extraction agent, directly measures xenthophylls concentration on HPLC.Condition determination is: high performance liquid chromatograph (Tianjin, island LC-10ATvp pump, SPD-M10Avp diode-array detector), Zorbax SB-C
18(separator column of 4.6mm * 25cm), moving phase is acetonitrile-methyl alcohol-ethyl acetate (volume ratio 45: 10: 45), flow velocity is 1 ml/min, sample size 20 μ L, the detection wavelength is 477nm.
4, adopt chlorella to produce xenthophylls: to adopt polar solvent methyl alcohol to mix than from 10: 1 to 40: 1 as extracting solvent according to carbon atom and Sauerstoffatom with the non-polar solvent octane, can from chlorella cells, high efficiency extraction go out xenthophylls, established very important basis for the industrialization production of adopting chlorella to produce xenthophylls, had important use and be worth.
Claims (1)
1, a kind of method of extracting xenthophylls from chlorella cells is characterized in that: take polarity and nonpolar mixed solvent to extract xenthophylls from chlorella; Extracting xenthophylls technology from chlorella cells is: with the cultured chlorella cells of the centrifugal results of 50～500ml centrifuge tube, add 5.0～50.0ml mixing liquid solvent after the evacuation supernatant liquor, on the cytoclasis instrument, carry out the fragmentation of ultrasonic wave frustule after centrifuge tube is inserted into rubble ice, condition is broken 10～30 minutes time, every ultrasonic 5～10 seconds, 2～5 seconds at interval, circulation, cytoclasis instrument output rating is 200～600W, after the frustule fragmentation at 5000～15000 rev/mins, 4～8 ℃ of temperature, low-temperature centrifugation 5～20 minutes extracts the xenthophylls that the middle mutually acquisition of solvent supernatant liquor octane extracts from chlorella cells; Described mixing liquid solvent is a polar solvent methyl alcohol, the non-polar solvent octane; The two mixes from 10: 1 by 40: 1 according to carbon atom in the mixed solvent and Sauerstoffatom ratio.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2006100119942A CN100372834C (en) | 2006-05-25 | 2006-05-25 | Method for extracting lutein from chlorella cell |
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| CNB2006100119942A CN100372834C (en) | 2006-05-25 | 2006-05-25 | Method for extracting lutein from chlorella cell |
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| CN100372834C CN100372834C (en) | 2008-03-05 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100445378C (en) * | 2006-12-27 | 2008-12-24 | 天津商学院 | Ultrasonic method for breaking up micro alga cells |
| CN102206539A (en) * | 2010-03-31 | 2011-10-05 | 中国科学院青岛生物能源与过程研究所 | Method for extracting grease from wet microalgae mud by using subcritical alcohols |
| CN102976992A (en) * | 2012-12-14 | 2013-03-20 | 国家海洋局第三海洋研究所 | Method for extracting lutein from chlorella |
| CN103045626A (en) * | 2012-12-20 | 2013-04-17 | 国家海洋局第三海洋研究所 | High-yielding lutein transgenic chlorella and preparation thereof |
| CN103567042A (en) * | 2013-11-19 | 2014-02-12 | 北京缘草堂生物科技开发有限公司 | Ultrasonic positioning wall-breaking method for Chinese herbal medicine |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1464877A (en) * | 2001-05-18 | 2003-12-31 | 阿迪索法国两合公司 | Process for the extraction of esters of xanthophyll and of carotenoids |
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2006
- 2006-05-25 CN CNB2006100119942A patent/CN100372834C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100445378C (en) * | 2006-12-27 | 2008-12-24 | 天津商学院 | Ultrasonic method for breaking up micro alga cells |
| CN102206539A (en) * | 2010-03-31 | 2011-10-05 | 中国科学院青岛生物能源与过程研究所 | Method for extracting grease from wet microalgae mud by using subcritical alcohols |
| CN102206539B (en) * | 2010-03-31 | 2012-11-07 | 中国科学院青岛生物能源与过程研究所 | Method for extracting grease from wet microalgae mud by using subcritical alcohols |
| CN102976992A (en) * | 2012-12-14 | 2013-03-20 | 国家海洋局第三海洋研究所 | Method for extracting lutein from chlorella |
| CN103045626A (en) * | 2012-12-20 | 2013-04-17 | 国家海洋局第三海洋研究所 | High-yielding lutein transgenic chlorella and preparation thereof |
| CN103045626B (en) * | 2012-12-20 | 2014-11-05 | 国家海洋局第三海洋研究所 | High-yielding lutein transgenic chlorella and preparation thereof |
| CN103567042A (en) * | 2013-11-19 | 2014-02-12 | 北京缘草堂生物科技开发有限公司 | Ultrasonic positioning wall-breaking method for Chinese herbal medicine |
| CN103567042B (en) * | 2013-11-19 | 2015-11-25 | 北京缘草堂生物科技开发有限公司 | A kind of Chinese medicine ultrasonic wave location wall-breaking method |
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