CN104529852A - Method for extracting astaxanthin from haematococcus pluvialis - Google Patents
Method for extracting astaxanthin from haematococcus pluvialis Download PDFInfo
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- CN104529852A CN104529852A CN201410748498.XA CN201410748498A CN104529852A CN 104529852 A CN104529852 A CN 104529852A CN 201410748498 A CN201410748498 A CN 201410748498A CN 104529852 A CN104529852 A CN 104529852A
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Abstract
A disclosed method for extracting astaxanthin from haematococcus pluvialis comprises algae strain culture and pretreatment, mixing with glacial acetic acid and processing in an ice bath, a cell wall breaking processing combining homogenizing-machine homogenizing and liquid-nitrogen low-temperature processing, CO2 supercritical extraction of astaxanthin, and the like. According to the method, glacial acetic acid can relatively rapidly penetrate into the surface layer of haematococcus pluvialis cells under the ice bath condition; algae cells are crushed under effects of stirring centrifugal force, extrusion force and shearing force during homogenizing, and destroy on glycoprotein in cell wall structure by glacial acetic acid at the cell surface is accelerated because of the friction force among materials; and also, intervention of ice-bath glacial acetic acid, algae cell temperature is prevented from rising during homogenizing, and the physical and chemical activities of astaxanthin are furthest protected. By employing the wall breaking process, the cell wall breaking rate is up to 97.7%, the extraction rate of the astaxanthin extractive reaches 37%, the extraction rate of astaxanthin reaches 35.05%, and the purity of astaxanthin in the product is extremely high and reaches 95.25%.
Description
Technical field
The present invention relates to a kind of method extracting astaxanthin from Haematocoocus Pluvialls, belong to technical field of biological culture.
Background technology
Haematocoocus Pluvialls is the unicell green alga of one way of life in fresh water, and can accumulate a large amount of carotenoid (can reach 2%-5% of dry weight) under given conditions, wherein more than 80% is astaxanthin and ester class D thereof.The life cycle of Haematocoocus Pluvialls is mainly divided into vegetative cell and chlamydospore, when it is generally acknowledged environmental benefits, forms trophozooid; When environment is unfavorable, form chlamydospore and a large amount of accumulation astaxanthin.Astaxanthin (Astaxanthin) can be used as fat-soluble pigment, has gorgeous redness and strong antioxidant property; In the food industry, can not only effectively play fresh-keeping, protect look, protect taste, the effect such as to guarantee the quality, and can be multiclass food color, the color and luster aesthetic feeling of increase food; Experimentation on animals shows that astaxanthin has Tumor suppression to occur, strengthens many-sided physiological actions such as immunologic function in addition, and therefore astaxanthin is widely used in functional food and medicine etc.Utilize organic solvent directly can extract astaxanthin from haematococcus pluvialis cell, but the chlamydospore due to Haematocoocus Pluvialls has tough and tensile cell walls, if directly utilizing to cause its bioavailability to reduce, and chlamydospore structure hinders organic solvent and enters in cell and extract astaxanthin, thus Astaxanthin extraction rate is lower.Therefore first must carry out the broken wall treatment of spore state cell before Astaxanthin extraction, to destroy the structure of the cellularstructure of Haematocoocus Pluvialls, particularly its cell walls, and the wall-breaking method of Haematocoocus Pluvialls for the extraction yield of astaxanthin and quality most important.
At present, the wall-breaking method of Haematocoocus Pluvialls mainly contains physical mechanical wall-breaking method, enzymolysis process and chemical method: enzyme solution is minimum for the destruction of astaxanthin, but sporoderm-broken rate is lower and add the difficulty of later separation, is not suitable for large-scale industrial production; Chemical process comparison system, but in its process, employ organic solvent, and not only cost is higher and not environmentally, can not reach the green standard of foodstuff production; The environmental protection of physical mechanical wall-breaking method, is separated simple, be applicable to large-scale industrial production, but at present, the sporoderm-broken rate of cell is lower, is generally 70%, and reaches serious, the cost intensive of wall-breaking method power consumption of 90%.
The physical mechanical wall-breaking method of Haematocoocus Pluvialls mainly comprises high pressure homogenization method, supersonic method, polishing, homogenate method, freeze thawing freezing process and microwave and cracks method, Ou Yangqin (University of Fuzhou's journal, 2005,33(1): 111-115) etc. find after research, the broken wall of the most applicable Haematocoocus Pluvialls of high pressure homogenization method, can reach the sporoderm-broken rate of 92.9%, but the extraction yield of astaxanthin is unsatisfactory is only 28.0 μ g/mg(dry cell weights); And Zhou Xiangchi (Oceanologia et Limnologia Sinica, 2006,37(5): 424-428) etc. research shows, homogenate method and supersonic method for follow-up Astaxanthin extraction be separated impact, and directly grind the activity reducing astaxanthin in broken wall process, add liquid nitrogen cryogenic milling process pollution-free and remain the physiologically active of astaxanthin to greatest extent, but liquid nitrogen cryogenics polishing is only applicable to research department and is not suitable for large-scale industrial production.Patent CN101691348A adopts SCF-CO 2 extracting directly Haematocoocus Pluvialls dry powder, this technique environmental protection and less on the impact of the physiologically active of astaxanthin, but the method equipment used is expensive, and cost is very high, the technology for broken wall that thing the is special and extraction yield of astaxanthin is low.
At present, be badly in need of wanting a kind of environmental protection, with low cost, on astaxanthin physiologically active without impact and the high method of Astaxanthin extraction rate.
Summary of the invention
Technical problem to be solved by this invention is to provide that a kind of sporoderm-broken rate is high, environmental protection is nuisanceless, cost is low, be applicable to the method for the extraction astaxanthin of large-scale industrialized production, by the method, broken wall is carried out to Haematocoocus Pluvialls, minimum on the impact of the physiologically active of astaxanthin, the extraction yield of astaxanthin is high and meet the green standard of foodstuff production.
In order to solve the problems of the technologies described above, the present invention adopts following technical scheme:
From Haematocoocus Pluvialls, extract a method for astaxanthin, comprise the following steps:
(1) algal species cultivation and pre-treatment: algae kind through BBM substratum 18-20 DEG C, cultivate 10d under 3000-4000Lx condition, abandoning supernatant after centrifugal 10-15min under 300-500r/min, adds nitrogen stress substratum and continues to cultivate under 5000-6000Lx in precipitation; To cultivate after 7d centrifugal 10-15min under 3000-3500r/min, removal supernatant liquor adds 20% methyl alcohol and 5% potassium hydroxide mixed solution processes 10-20min removal chlorophyll at 60-65 DEG C, and finally under 300-500r/min condition, centrifugal 5-10min abandons supernatant liquor and obtains spore state frustule suspension;
(2) cell wall breaking: the spore state frustule suspension of gained in step 1 is mixed with Glacial acetic acid, ice bath 10-20min at-4-0 DEG C, then in above-mentioned mixed solution, add deionized water and be placed in refiner homogenate 5-10min and obtain algae slurries, after leaving standstill 5-10min add liquid nitrogen mixing in algae slurries after, obtain broken wall marine alga liquid;
(3) Astaxanthin extraction: the broken wall marine alga liquid of gained in step 3 is concentrated, be dried to powder after be loaded in supercritical extraction unit, the temperature and pressure of separation reactor I is 30-40 DEG C, 5-8MPa, the temperature and pressure of separation reactor I I is 30-40 DEG C, 20-25MPa, CO
2flow velocity is 10-15L/h, and extraction time is obtain astaxanthin extract after 0.5-1h.
Further, in step 2, the volume ratio of described spore state frustule suspension and Glacial acetic acid, deionized water is 1:2-5:1-3.
Further, in step 2, the concentration of described Glacial acetic acid is 0.3%-1%.
Further, in step 2, the volume that adds of described liquid nitrogen is add 2-5mL liquid nitrogen in 100mL algae slurries.
Further, in step 3, the temperature and pressure of described separation reactor I is 35 DEG C, the temperature and pressure of 7MPa, separation reactor I I is 40 DEG C, 22MPa, CO
2flow velocity is 12L/h, and extraction time is 0.5h.
The present invention compared with prior art, has following beneficial effect:
Under condition of ice bath, Glacial acetic acid can penetrate into the top layer of haematococcus pluvialis cell faster; In homogenization process, the stirring centrifugal force utilizing water screw to apply, squeeze, shearing force carry out fragmentation to alginic cell, and the frictional force between material accelerates the Glacial acetic acid of cell surface to the destruction of glycoprotein in cell wall structure; Meanwhile, the intervention due to the acetic acid of ice bath avoids the rising of temperature in homogenization process, and the physics and chemistry farthest protecting astaxanthin is active, and the astaxanthin extract of gained is garnet thick substances.Wall-breaking method of the present invention, the sporoderm-broken rate of cell is up to 97.7%, and the extraction rate reached of astaxanthin extract is to 37%, and the extraction rate reached of astaxanthin is to 35.05%, and in product, astaxanthin very high purity reaches 95.25%.
Embodiment
Set forth beneficial effect of the present invention further by the following examples:
Embodiment 1:
From Haematocoocus Pluvialls, extract a method for astaxanthin, comprise the following steps:
(1) algal species cultivation and pre-treatment: algae kind through BBM substratum 18 DEG C, cultivate 10d, abandoning supernatant after centrifugal 10min under 450r/min under 3000-4000Lx condition, in precipitation, add nitrogen stress substratum under 5000-6000Lx, continue cultivation; Cultivate and under 3000r/min, to remove supernatant liquor after 7d after centrifugal 10min add 20% methyl alcohol and 5% potassium hydroxide mixed solution and at 60-65 DEG C, process 15min remove chlorophyll, finally under 400r/min condition, centrifugal 5min abandons supernatant liquor and obtains spore state frustule suspension;
(2) cell wall breaking: the spore state frustule suspension of gained in step 1 is mixed with 1% Glacial acetic acid, ice bath 15min at-4-0 DEG C, then in above-mentioned mixed solution, add deionized water and be placed in refiner homogenate 5min and obtain algae slurries, the volume ratio of spore state frustule suspension and Glacial acetic acid, deionized water is 1:2:1; Add in algae slurries after liquid nitrogen leaves standstill 5min and obtain broken wall marine alga liquid, in 100mL algae slurries, add 2mL liquid nitrogen;
(3) Astaxanthin extraction: the broken wall marine alga liquid of gained in step 3 is concentrated, be dried to powder after be loaded in supercritical extraction unit, the temperature and pressure of separation reactor I is 35 DEG C, 7MPa, the temperature and pressure of separation reactor I I is 40 DEG C, 22MPa, CO2 flow velocity is 12L/h, and extraction time is obtain astaxanthin extract after 0.5h.
Embodiment 2:
Operating process is identical with embodiment 1, and difference is in step 2, and the concentration of Glacial acetic acid is 0.3%.
Embodiment 3:
Operating process is identical with embodiment 1, and difference is in step 2, and the concentration of Glacial acetic acid is 0.6%.
Embodiment 4:
Operating process is identical with embodiment 3, and difference is in step 2, and the volume ratio of spore state frustule suspension and Glacial acetic acid, deionized water is 1:5:2.
Embodiment 5:
Operating process is identical with embodiment 3, and difference is in step 2, and the volume ratio of spore state frustule suspension and Glacial acetic acid, deionized water is 1:3:2.
Embodiment 6:
Operating process is identical with embodiment 5, and difference is in step 2, and the volume that adds of liquid nitrogen is add 5mL liquid nitrogen in 100mL algae slurries.
Embodiment 7:
Operating process is identical with embodiment 5, and difference is in step 2, and the volume that adds of liquid nitrogen is add 5mL liquid nitrogen in 100mL algae slurries.
Simultaneous test:
Cytoclastic spore state frustule suspension will do not carried out and be designated as control group 0, by only using the Glacial acetic acid of 0.6% (spore state frustule suspension and Glacial acetic acid volume ratio are 1:3), broken wall treatment is carried out to spore state frustule suspension and be designated as control group 1, only will carry out homogenate broken wall treatment to spore state frustule suspension and be designated as control group 2, only will add liquid nitrogen broken wall treatment to spore state frustule suspension and be designated as control group 3, embodiment 1-7 is designated as experimental group 1-7 simultaneously, measure following index, experimental result is as shown in table 1:
Dry cell weight calculates: the spore state frustule suspension 100ml getting gained in step 1, is placed in 80 DEG C of baking ovens and dries 24h, weigh after cooling.
Calculate astaxanthin extract yield (100%)=astaxanthin extract weight/dry cell weight × 100%.
Calculate content astaxanthin in product: accurately take 25mg astaxanthin pure sample product, 25 mL are settled to acetone solution, accurately pipette out 0.5 respectively, 1.0,1.5,2.0,2.5,3.0mL, 50mL is settled to by acetone diluted, survey light absorption value in 490nm place, do with acetone the typical curve that blank group draws sample; Take astaxanthin extract 10mg, with acetone diluted and and be settled to 25 mL, survey light absorption value at 490nm place, light absorption value is brought in typical curve and release liquid concentration C to be measured, astaxanthin weight=(C × 25 mL/10mg × 100%) × astaxanthin extract weight in product.
Calculate astaxanthin weight/dry cell weight × 100% in astaxanthin yield (100%)=product.
The product yield of control group and embodiment is as shown in table 1:
| Group | Dry cell weight mg (100ml) | Astaxanthin extract weight mg | Astaxanthin extract yield (%) | Astaxanthin weight mg | Astaxanthin yield (100%) |
| Control group 0 | 399.7 | 3 | 0.75 | 1.7 | 0.42 |
| Control group 1 | 400.8 | 28 | 6.99 | 20 | 5.0 |
| Control group 2 | 392.9 | 15 | 3.82 | 10.2 | 2.5 |
| Control group 3 | 399.3 | 10 | 2.5 | 7 | 1.75 |
| Embodiment 1 | 399.4 | 120.8 | 30.25 | 99.9 | 25.0 |
| Embodiment 2 | 400.4 | 128.1 | 31.99 | 100.8 | 25.17 |
| Embodiment 3 | 400.9 | 128.9 | 32.15 | 118.2 | 29.48 |
| Embodiment 4 | 399.1 | 130.1 | 32.60 | 120.9 | 30.29 |
| Embodiment 5 | 398.7 | 136.7 | 34.29 | 130.2 | 32.66 |
| Embodiment 6 | 401.2 | 140.2 | 34.95 | 132.5 | 33.02 |
| Embodiment 7 | 400.0 | 148.1 | 37 | 140.2 | 35.05 |
From table 1 data of the present invention, the method that Glacial acetic acid ice bath of the present invention, refiner homogenate and liquid nitrogen cryogenics process combine carries out broken wall treatment to Haematocoocus Pluvialls, the extraction yield of its astaxanthin is apparently higher than control group, the concentration of astaxanthin in product can be calculated simultaneously, in embodiment 1-7, astaxanthin concentration is respectively 82.7%, 78.69%, 91.70%, 92.93%, 95.25%, 94.51%, 94.66%, the very high concentrations of product, and control group 0-3 is respectively 56.67%, 71.42%, 68% and 70%.
Above-mentioned example just for technical conceive of the present invention and technical characterstic are described, can not limit the scope of the invention with this.The equivalent transformation that all essence according to the present invention is done or modification, all should be encompassed within protection scope of the present invention.
Claims (5)
1. from Haematocoocus Pluvialls, extract a method for astaxanthin, it is characterized in that, comprise the following steps:
(1) algal species cultivation and pre-treatment: algae kind through BBM substratum 18-20 DEG C, cultivate 10d under 3000-4000Lx condition, abandoning supernatant after centrifugal 10-15min under 300-500r/min, adds nitrogen stress substratum and continues to cultivate under 5000-6000Lx in precipitation; To cultivate after 7d centrifugal 10-15min under 3000-3500r/min, remove supernatant liquor afterwards, the mixed solution adding 20% methyl alcohol and 5% potassium hydroxide in precipitation processes 10-20min and removes chlorophyll at 60-65 DEG C, and finally under 300-500r/min condition, centrifugal 5-10min abandons supernatant liquor and obtains spore state frustule suspension;
(2) cell wall breaking: the spore state frustule suspension of gained in step 1 is mixed with Glacial acetic acid, ice bath 10-20min at-4-0 DEG C, then in above-mentioned mixed solution, add deionized water and be placed in refiner homogenate 5-10min and obtain algae slurries, after leaving standstill 5-10min add liquid nitrogen mixing in algae slurries after, obtain broken wall marine alga liquid;
(3) Astaxanthin extraction: the broken wall marine alga liquid of gained in step 3 is concentrated, be dried to powder after be loaded in supercritical extraction unit, the temperature and pressure of separation reactor I is 30-40 DEG C, 5-8MPa, the temperature and pressure of separation reactor I I is 30-40 DEG C, 20-25MPa, CO
2flow velocity is 10-15L/h, and extraction time is 0.5-1h.
2. method according to claim 1, is characterized in that, in step 2, the volume ratio of described spore state frustule suspension and Glacial acetic acid, deionized water is 1:2-5:1-3.
3. method according to claim 1, is characterized in that, in step 2, the concentration of described Glacial acetic acid is 0.3%-1%.
4. method according to claim 1, is characterized in that, in step 2, the volume that adds of described liquid nitrogen is add 2-5mL liquid nitrogen in 100mL algae slurries.
5. method according to claim 1, is characterized in that, in step 3, the temperature and pressure of described separation reactor I is 35 DEG C, the temperature and pressure of 7MPa, separation reactor I I is 40 DEG C, 22MPa, CO
2flow velocity is 12L/h, and extraction time is 0.5h.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106867899A (en) * | 2015-11-12 | 2017-06-20 | 现代自动车株式会社 | Microalgae photochemistry culture device |
| CN107053898A (en) * | 2016-12-28 | 2017-08-18 | 浙江彭胜文教用品股份有限公司 | A kind of super smooth coloured plastic pencil and its production technology |
| CN107484829A (en) * | 2017-08-10 | 2017-12-19 | 山东阳春羊奶乳业有限公司 | A kind of DHA algal oil goat milk piece and preparation method thereof |
| CN113214998A (en) * | 2021-06-16 | 2021-08-06 | 江苏格局生物医药科技有限公司 | Preparation method of natural astaxanthin |
| CN115161200A (en) * | 2021-04-02 | 2022-10-11 | 中科技术物理青岛研究院 | Green and efficient haematococcus pluvialis wall breaking method |
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| CN102337215A (en) * | 2011-10-20 | 2012-02-01 | 烟台华融生物科技有限公司 | Methods for culturing haematococcus pluvialis and producing astaxanthin |
| CN103232375A (en) * | 2013-04-03 | 2013-08-07 | 大连医诺生物有限公司 | Novel high-efficiency extraction process for astaxanthin in Haematococcus pluvialis |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101381337A (en) * | 2007-09-03 | 2009-03-11 | 陈锦猜 | Astaxanthin extraction method |
| CN102337215A (en) * | 2011-10-20 | 2012-02-01 | 烟台华融生物科技有限公司 | Methods for culturing haematococcus pluvialis and producing astaxanthin |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106867899A (en) * | 2015-11-12 | 2017-06-20 | 现代自动车株式会社 | Microalgae photochemistry culture device |
| CN107053898A (en) * | 2016-12-28 | 2017-08-18 | 浙江彭胜文教用品股份有限公司 | A kind of super smooth coloured plastic pencil and its production technology |
| CN107484829A (en) * | 2017-08-10 | 2017-12-19 | 山东阳春羊奶乳业有限公司 | A kind of DHA algal oil goat milk piece and preparation method thereof |
| CN115161200A (en) * | 2021-04-02 | 2022-10-11 | 中科技术物理青岛研究院 | Green and efficient haematococcus pluvialis wall breaking method |
| CN113214998A (en) * | 2021-06-16 | 2021-08-06 | 江苏格局生物医药科技有限公司 | Preparation method of natural astaxanthin |
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