CN107235881B - The supercritical carbon dioxide extraction method of astaxanthin in transgenic engineering tamato fruit - Google Patents
The supercritical carbon dioxide extraction method of astaxanthin in transgenic engineering tamato fruit Download PDFInfo
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- CN107235881B CN107235881B CN201710452369.XA CN201710452369A CN107235881B CN 107235881 B CN107235881 B CN 107235881B CN 201710452369 A CN201710452369 A CN 201710452369A CN 107235881 B CN107235881 B CN 107235881B
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- JEBFVOLFMLUKLF-IFPLVEIFSA-N Astaxanthin Natural products CC(=C/C=C/C(=C/C=C/C1=C(C)C(=O)C(O)CC1(C)C)/C)C=CC=C(/C)C=CC=C(/C)C=CC2=C(C)C(=O)C(O)CC2(C)C JEBFVOLFMLUKLF-IFPLVEIFSA-N 0.000 title claims abstract description 84
- 235000013793 astaxanthin Nutrition 0.000 title claims abstract description 84
- 239000001168 astaxanthin Substances 0.000 title claims abstract description 84
- MQZIGYBFDRPAKN-ZWAPEEGVSA-N astaxanthin Chemical compound C([C@H](O)C(=O)C=1C)C(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)C(=O)[C@@H](O)CC1(C)C MQZIGYBFDRPAKN-ZWAPEEGVSA-N 0.000 title claims abstract description 84
- 229940022405 astaxanthin Drugs 0.000 title claims abstract description 84
- 238000000034 method Methods 0.000 title claims abstract description 42
- 235000013399 edible fruits Nutrition 0.000 title claims abstract description 32
- 230000009261 transgenic effect Effects 0.000 title claims abstract description 32
- 238000003815 supercritical carbon dioxide extraction Methods 0.000 title claims abstract description 24
- 238000000605 extraction Methods 0.000 claims abstract description 64
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims abstract description 29
- 235000007688 Lycopersicon esculentum Nutrition 0.000 claims abstract description 22
- UPYKUZBSLRQECL-UKMVMLAPSA-N Lycopene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1C(=C)CCCC1(C)C)C=CC=C(/C)C=CC2C(=C)CCCC2(C)C UPYKUZBSLRQECL-UKMVMLAPSA-N 0.000 claims abstract description 18
- JEVVKJMRZMXFBT-XWDZUXABSA-N Lycophyll Natural products OC/C(=C/CC/C(=C\C=C\C(=C/C=C/C(=C\C=C\C=C(/C=C/C=C(\C=C\C=C(/CC/C=C(/CO)\C)\C)/C)\C)/C)\C)/C)/C JEVVKJMRZMXFBT-XWDZUXABSA-N 0.000 claims abstract description 18
- 235000021466 carotenoid Nutrition 0.000 claims abstract description 18
- 235000012661 lycopene Nutrition 0.000 claims abstract description 18
- 239000001751 lycopene Substances 0.000 claims abstract description 18
- OAIJSZIZWZSQBC-GYZMGTAESA-N lycopene Chemical compound CC(C)=CCC\C(C)=C\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C=C(/C)CCC=C(C)C OAIJSZIZWZSQBC-GYZMGTAESA-N 0.000 claims abstract description 18
- 229960004999 lycopene Drugs 0.000 claims abstract description 18
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- ZCIHMQAPACOQHT-ZGMPDRQDSA-N trans-isorenieratene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/c1c(C)ccc(C)c1C)C=CC=C(/C)C=Cc2c(C)ccc(C)c2C ZCIHMQAPACOQHT-ZGMPDRQDSA-N 0.000 claims abstract description 18
- 150000001747 carotenoids Chemical class 0.000 claims abstract description 17
- 239000001569 carbon dioxide Substances 0.000 claims abstract description 14
- 229910002092 carbon dioxide Inorganic materials 0.000 claims abstract description 14
- 238000002156 mixing Methods 0.000 claims abstract description 11
- 239000000843 powder Substances 0.000 claims abstract description 11
- 230000001105 regulatory effect Effects 0.000 claims abstract description 9
- 241000227653 Lycopersicon Species 0.000 claims abstract 5
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 33
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 30
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- ONTQJDKFANPPKK-UHFFFAOYSA-L chembl3185981 Chemical compound [Na+].[Na+].CC1=CC(C)=C(S([O-])(=O)=O)C=C1N=NC1=CC(S([O-])(=O)=O)=C(C=CC=C2)C2=C1O ONTQJDKFANPPKK-UHFFFAOYSA-L 0.000 claims description 2
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- 238000011084 recovery Methods 0.000 abstract description 31
- 239000000463 material Substances 0.000 abstract description 5
- 230000000050 nutritive effect Effects 0.000 abstract description 2
- 240000003768 Solanum lycopersicum Species 0.000 description 19
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 11
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- HDLNSTQYXPTXMC-UHFFFAOYSA-N Astaxanthin-diacetat Natural products O=C1C(OC(=O)C)CC(C)(C)C(C=CC(C)=CC=CC(C)=CC=CC=C(C)C=CC=C(C)C=CC=2C(CC(C(=O)C=2C)OC(C)=O)(C)C)=C1C HDLNSTQYXPTXMC-UHFFFAOYSA-N 0.000 description 3
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C403/00—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone
- C07C403/24—Derivatives of cyclohexane or of a cyclohexene or of cyclohexadiene, having a side-chain containing an acyclic unsaturated part of at least four carbon atoms, this part being directly attached to the cyclohexane or cyclohexene or cyclohexadiene rings, e.g. vitamin A, beta-carotene, beta-ionone having side-chains substituted by six-membered non-aromatic rings, e.g. beta-carotene
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C7/00—Purification; Separation; Use of additives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C7/00—Purification; Separation; Use of additives
- C07C7/10—Purification; Separation; Use of additives by extraction, i.e. purification or separation of liquid hydrocarbons with the aid of liquids
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Water Supply & Treatment (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The supercritical carbon dioxide extraction method of astaxanthin in transgenic engineering tamato fruit, it is characterized in that this method includes taking the astaxanthin engineering tamato fruit dry powder being sieved in advance, regulating system temperature and pressure is to setting value, after the temperature and pressure of kettle to be extracted reach setting value and stablize, the static extracting of 1h is carried out；Constant-flux pump is opened after static extracting, entrainer is transported into equipment with 1.0g/min, and the fluid-mixing of entrainer and carbon dioxide gas enters extraction kettle, starts dynamic extraction.This method materials safety, recovery rate is high, is very suitable to the extraction of transgenic engineering tomato astaxanthin.In addition, the carotenoid such as lycopene and bata-carotene in transgenic engineering tomato containing high nutritive value, under the optimum extraction condition of astaxanthin, lycopene 82%, bata-carotene recovery rate is 89%.Astaxanthin, which is extracted, for transgenic engineering tomato industrialization has established technical foundation.
Description
Technical field:
The invention belongs to field of biotechnology, and in particular, to astaxanthin is super in a kind of transgenic engineering tamato fruit
Critical carbon dioxide fluid extraction process, while the overcritical dioxy of carotenoid in transgenic engineering tamato fruit being also provided
Change carbon fluid extraction process.
Background technique:
Astaxanthin is the strongest bioactive substance of antioxidant activity in nature, with the hair of plant gene engineering technology
Exhibition is produced astaxanthin using plant especially industrial crops as bioreactor and has become the hot spot studied both at home and abroad, astaxanthin
It is the unique keto-acid carotenoid of molecular structure, is the strongest bioactive substance of antioxidant activity in nature, there is anti-spoke
Penetrate, anti-aging, the antitumor and prevention diseases such as angiocarpy the effect of, be applied to cosmetics, health care product and aquaculture etc. just
Face.Commercialized natural astaxanthin is mainly derived from haematococcus pluvialis, which is photoautotrophy green alga, environmentally sensitive, growth
Slowly, it is difficult to realize the growth of highdensity cell, this is can not to break through its yield bottleneck both at home and abroad at present and astaxanthin price is high
Your the main reason for.Due to a lack of carotenoid assimilation enzyme (BKT) without synthesizing astaxanthin, the BKT for expressing external source may make plant
Plant especially industrial crops become efficient production of astaxanthin factory.Huang Junchao etc. will be sieved by technique for gene engineering in green alga
Beta carotene and luteole in plant cell can be catalyzed and be transferred at the assimilation enzyme and '-hydroxylase gene of astaxanthin by selecting and being separated to
In wild-type tomatoes plant, a kind of transgenic engineering tomato rich in astaxanthin, astaxanthin in ripening fruits are obtained for the first time
Content be up to 16.1mg/g, overcome plant and be difficult to accumulate the problem of high-content astaxanthin, with such transgenic engineering tomato
There is great commercial promise as bioreactor production astaxanthin.
Astaxanthin is more sensitive to light, temperature, and in extracting process, the several factors such as light, heat, oxygen can promote or accelerate
Astaxanthin is degraded；And traditional organic solvent extraction can not be completely removed because of residue, and cause harm to the human body, it breaks
Bad body immune system causes the generation of the diseases such as cancer.Supercritical carbon dioxide extraction is a kind of novel extraction skill
Art has extraction and isolated double action, and extraction process material is without phase-change thus energy saving obvious, and process flow is simple, extraction
Take high-efficient, organic solvent-free residual, good product quality, non-environmental-pollution.Herein to supercritical carbon dioxide extraction skill
The technique that art extracts astaxanthin from astaxanthin engineering tomato has done preliminary discussion, while to having high exploitation value in extract
Lycopene, the bata-carotene of value are analyzed, to have established technology using transgenic engineering tomato industrialization production astaxanthin
Basis.It is mentioned from transgenic engineering tomato currently, there are no in the prior art using supercritical carbon dioxide extraction technology
Take the report of the method for astaxanthin.Also without the supercritical carbon dioxide fluid of carotenoid in transgenic engineering tamato fruit
The report of extracting process.
Summary of the invention:
It is an object of the invention to places in view of the shortcomings of the prior art, provide a kind of using supercritical carbon dioxide
The method that fluid extraction technology extracts astaxanthin from transgenic engineering tomato, while providing from transgenic engineering tamato fruit
The supercritical carbon dioxide extraction method of carotenoid.
In order to realize above-mentioned purpose of the invention, the present invention provides the following technical solutions:
The supercritical carbon dioxide extraction method of astaxanthin in transgenic engineering tamato fruit, this method are pre- including taking
The astaxanthin engineering tamato fruit dry powder being first sieved, regulating system temperature and pressure to setting value, the temperature and pressure of kettle to be extracted
After power reaches setting value and stablizes, the static extracting of 1h is carried out；Constant-flux pump is opened after static extracting, will be pressed from both sides with 1.0g/min
Band agent is transported into equipment, and the fluid-mixing of entrainer and carbon dioxide gas enters extraction kettle, starts dynamic extraction, so
After be saponified.
The supercritical carbon dioxide extraction method of astaxanthin, this method in transgenic engineering tamato fruit as mentioned
Astaxanthin ester is further completely converted into free state astaxanthin using saponification means, by the extract liquor of astaxanthin engineering tomato into
Row saponification, takes 1ml extracting solution, is concentrated and is done using concentrating instrument, is dissolved in 1ml ether, and 1ml KOH MeOH is added and mixes, 0 DEG C of dark
15min is reacted in environment, 2ml 10%NaCl is added and mixes, and 2000g is centrifuged 2min, removes water phase, is washed with 2ml 10%NaCl
Twice, it is dissolved in 1ml acetone soln, loading measures pigment content after suction filtration.
The supercritical carbon dioxide extraction method of astaxanthin, takes in advance in transgenic engineering tamato fruit as mentioned
The tamato fruit dry powder of sieving, to 50 DEG C, extracting pressure 35MPa, the temperature and pressure of kettle to be extracted reaches regulating system temperature
Setting value and after stablizing, carries out the static extracting of 1h；Constant-flux pump is opened after static extracting, and dehydrated alcohol is transported to and is set
In standby, the fluid-mixing of dehydrated alcohol and carbon dioxide gas enters extraction kettle, starts dynamic extraction, and dynamic extraction time is
2h。
Carotenoid supercritical carbon dioxide extraction method in transgenic engineering tamato fruit, this method include
Take the tamato fruit dry powder being sieved in advance, regulating system temperature is to 50 DEG C, extracting pressure 35MPa, the temperature and pressure of kettle to be extracted
After power reaches setting value and stablizes, the static extracting of 1h is carried out；Constant-flux pump is opened after static extracting, and dehydrated alcohol is conveyed
To into equipment, the fluid-mixing of dehydrated alcohol and carbon dioxide gas enters extraction kettle, starts dynamic extraction, dynamic extraction
Time is 2h.
Carotenoid supercritical carbon dioxide extraction method in transgenic engineering tamato fruit as mentioned, should
Method further quantitative determines carotenoid content, and the carotenoid is astaxanthin, lycopene, β carrot
Element, method for quantitatively determining use: using acetone after accurately weighing each 5mg mixing of astaxanthin, lycopene, bata-carotene standard items
100ml is dissolved and be settled to, 1ml, 5ml are taken, 10ml mixed dissolution liquid is settled to 50ml with acetone respectively, 1ug/ml is made,
5ug/ml, 10ug/ml standard items aggregate sample make standard curve using UPLC loading, calculate astaxanthin, lycopene, β Hu trailing plants
The content of Bu Su, the UPLC method are as follows: flow velocity: 1ml/min；Sample volume: 5ul；0-1.0min: water: 20%, acetonitrile:
60%, isopropanol: 5%, methanol: 15%；1.00-2.00min: water: 0%, acetonitrile: 80%, isopropanol: 5%, methanol: 15%；
2.00-8.00min: water: 0%, acetonitrile: 80%, isopropanol: 5%, methanol: 15%；
The supercritical carbon dioxide extraction method of lycopene in transgenic engineering tamato fruit, this method include taking
The tamato fruit dry powder being sieved in advance, regulating system temperature is to 50 DEG C, extracting pressure 35MPa, the temperature and pressure of kettle to be extracted
After reaching setting value and stablizing, the static extracting of 1h is carried out；Constant-flux pump is opened after static extracting, and dehydrated alcohol is transported to
Into equipment, the fluid-mixing of dehydrated alcohol and carbon dioxide gas enters extraction kettle, starts dynamic extraction, when dynamic extraction
Between be 2h.
The supercritical carbon dioxide extraction method of bata-carotene in transgenic engineering tamato fruit, this method include taking
The tamato fruit dry powder being sieved in advance, regulating system temperature is to 50 DEG C, extracting pressure
35MPa carries out the static extracting of 1h after the temperature and pressure of kettle to be extracted reach setting value and stablize；Static state extraction
Constant-flux pump is opened after taking, and dehydrated alcohol is transported into equipment, the mixed flow of dehydrated alcohol and carbon dioxide gas
Body enters extraction kettle, starts dynamic extraction, dynamic extraction time 2h.
The present invention is to extract astaxanthin from transgenic engineering tomato using supercritical carbon dioxide extraction technology.?
In invention, 50 DEG C of extraction temperature, extracting pressure 35MPa, in the case where extracting 2h, the recovery rate of astaxanthin is up to 95%, this side
Method materials safety, recovery rate is high, is very suitable to the extraction of transgenic engineering tomato astaxanthin.In addition, in transgenic engineering tomato
The carotenoid such as lycopene and bata-carotene containing high nutritive value, under the optimum extraction condition of astaxanthin, tomato red
Plain recovery rate is 82%, and bata-carotene recovery rate is 89%.This research is that transgenic engineering tomato industrialization extraction astaxanthin is established
Technical foundation is determined.
Detailed description of the invention:
Influence of Fig. 1 difference entrainer to astaxanthin recovery rate；
Influence of Fig. 2 extraction temperature to astaxanthin recovery rate；
Influence of Fig. 3 extracting pressure to astaxanthin recovery rate；
Influence of Fig. 4 extraction time to astaxanthin recovery rate；
Fig. 5 transgenic engineering tamato fruit carotenoid chromatogram.
Specific embodiment:
With reference to the accompanying drawing, essentiality content of the invention is further illustrated with the embodiment of the present invention, but not with
This limits the present invention.
Embodiment 1:
Material and method
Material and facility:
Dehydrated alcohol, 75% ethyl alcohol, ethyl acetate, acetone, n-hexane are that domestic analysis is pure；Isopropanol, acetonitrile, methanol
For German Fisher chromatographically pure；Supercritical carbon dioxide extraction device, U.S. Waters (TharSFE)；Ultra high efficiency liquid phase
Chromatograph, Anjelen Sci. & Tech. Inc, the U.S., 1290Infinity；Chromatographic column, Anjelen Sci. & Tech. Inc, the U.S.,
Eclipse plus C18 RRHD 1.8μm；Rotary Evaporators, Tokyo is physical and chemical, EYELE N-1100；Assay balance, Beijing match
More Li Si instrument systems Co., Ltd, TP-213.
Tamato fruit dry powder: (yellow pretty tide etc. (Huang JC etc., 2013) pass through gene engineering method to astaxanthin engineering tomato
To screen and be separated in green alga can be catalyzed in plant cell beta carotene and luteole into the assimilation enzyme and hydroxylation of astaxanthin
Enzyme gene is transferred in wild-type tomatoes plant (cv.UC82B), and obtained transgene tomato is (on May 14th, 2014 in China
Microbiological Culture Collection administration committee common micro-organisms center preservation, deposit number are as follows: CGMCC No 9223, classification naming:
Red No. 1 transgene tomato of shrimp.) F4 tomato dry powder, be sieved 200 mesh.
Method:
Supercritical carbon dioxide extraction method: the astaxanthin engineering tamato fruit dry powder being sieved in advance is taken every time
200g, regulating system temperature and pressure to setting value after the temperature and pressure of kettle to be extracted reach setting value and stablize, carry out 1h
Static extracting；Constant-flux pump is opened after static extracting, and entrainer is transported into equipment with 1.0g/min, entrainer
Enter extraction kettle with the fluid-mixing of carbon dioxide gas, starts dynamic extraction.This research will to extracting pressure, extraction temperature,
Entrainer, extraction time are investigated, and determine optimum extraction condition according to the recovery rate of astaxanthin, when investigating a parameter,
It is constant to set other parameters.
Astaxanthin method for saponification: astaxanthin a part in Transgenic tomato fruit is existed in the form of astaxanthin ester,
Therefore it needs that astaxanthin ester is completely converted into free state astaxanthin using saponification means.Specific method is: taking 1ml overcritical two
Astaxanthin extract liquor obtained by carbon oxide fluid extracting process is concentrated using concentrating instrument and is done, and 1ml ether is dissolved in, and 1ml KOH is added
MeOH is mixed, and 15min is reacted in 0 DEG C of dark surrounds, and 2ml 10%NaCl is added and mixes, and 2000g is centrifuged 2min, removes water phase,
It is washed twice with 2ml 10%NaCl, is dissolved in 1ml acetone soln, loading measures pigment content after suction filtration.
Astaxanthin, lycopene, the quantitative approach of bata-carotene are as follows: accurately weighing astaxanthin, lycopene, β carrot
With acetone solution and it is settled to 100ml after each 5mg mixing of plain standard items, takes 1ml, 5ml, 10ml mixed dissolution liquid is distinguished with acetone
It is settled to 50ml, 1ug/ml, 5ug/ml is made, 10ug/ml standard items aggregate sample makes standard curve, meter using UPLC loading
Calculate the content of astaxanthin, lycopene, bata-carotene.UPLC method: flow velocity: 1ml/min；Sample volume: 5ul；0-1.0min:
Water: 20%, acetonitrile: 60%, isopropanol: 5%, methanol: 15%；1.00-2.00min: water: 0%, acetonitrile: 80%, isopropanol:
5%, methanol: 15%；2.00-8.00min: water: 0%, acetonitrile: 80%, isopropanol: 5%, methanol: 15%；
As a result with analysis
Influence of the different entrainers to astaxanthin recovery rate:
During supercritical carbon dioxide extraction, when investigating the influence of entrainer, other parameters are selected are as follows: extraction
35 DEG C of temperature, extracting pressure 35MPa, extraction time 3h.Respectively with 50% ethyl alcohol, 75% ethyl alcohol, dehydrated alcohol, acetone, acetic acid
Ethyl ester is tested as entrainer.Astaxanthin extraction rate is measured, as a result such as Fig. 1.
As seen from Figure 1, when using dehydrated alcohol, acetone, ethyl acetate as entrainer, Astaxanthin extraction rate compared with
It is high.This explanation, in the supercritical state, the polarity of CO 2 fluid is very small, and the addition of entrainer, especially highly polar
Entrainer, be conducive to astaxanthin and be dissolved in supercritical carbon dioxide fluid.But acetone, ethyl acetate are organic solvents, residual
Make things difficult for completely remove, and dehydrated alcohol is not only safe, and at low cost, recovery rate is relatively high, therefore can choose anhydrous
Ethyl alcohol is as best entrainer.
Influence of the extraction temperature to astaxanthin recovery rate:
During supercritical carbon dioxide extraction, when investigating the influence of temperature, other parameters are selected are as follows: extraction pressure
Power 35MPa, extraction time 3h, entrainer dehydrated alcohol, at 30 DEG C of this experiment condition set temperature, 35 DEG C, 40 DEG C, 45 DEG C, 50
DEG C, 55 DEG C, 60 DEG C.Astaxanthin extraction rate is measured, as a result such as Fig. 2.
As seen from Figure 2, the recovery rate of astaxanthin is increased with the raising of temperature, and temperature reaches maximum when being 50 DEG C,
Later, recovery rate is reduced with the raising of temperature.This may be since when temperature increases, vapour pressure increases, and faces astaxanthin super
Solubility in boundary's CO 2 fluid increases, but when temperature continues to increase since free state astaxanthin holds under high temperature environment
Vulnerable to thermal decomposition so as to cause recovery rate reduction.Therefore, the optimum temperature of supercritical carbon dioxide extraction astaxanthin is 50
℃。
Influence of the extracting pressure to astaxanthin recovery rate:
During supercritical carbon dioxide extraction, when investigating the influence of pressure, other experiment parameters are selected are as follows: temperature
Degree 50 DEG C, entrainer dehydrated alcohol, extraction time 3h, this experiment condition set 10 DEG C of pressure, 15 DEG C, 20 DEG C, 25 DEG C, 30
DEG C, 35 DEG C, 40 DEG C.Astaxanthin extraction rate is measured, as a result such as Fig. 3.
As seen from Figure 3, the recovery rate of astaxanthin increases with increasing for extracting pressure, when pressure 35MPa, extracts
Rate is 90%, when pressure 40MPa, recovery rate 92%.This explanation increases when pressure, and fluid density increases, and extract is caused to exist
Solubility in supercritical carbon dioxide fluid improves, and causes to extract the increase of Astaxanthin extraction rate.It is more advantageous although pressure is higher
In the raising of recovery rate, but since experimental facilities limits, and in production application, the excessively high of pressure will lead to equipment life drop
It is low, increase operation cost, therefore use 35MPa for best extracting pressure.
Influence of the extraction time to astaxanthin recovery rate:
During supercritical carbon dioxide extraction, when investigating the influence of extraction time, other experiment parameters are selected
Are as follows: 50 DEG C of extraction temperature, extracting pressure 35MPa, entrainer is dehydrated alcohol；1.0h, 1.5h, 2.0h, 2.5h are extracted respectively,
3.0h, 3.5h, 4.0h.Astaxanthin extraction rate is measured, as a result such as Fig. 4.
As seen from Figure 4, the recovery rate of astaxanthin increases with the increase of extraction time, and recovery rate reaches when 2.5h
Maximum, in 4h, recovery rate sharply declines, this may be due to thermal sensitivity astaxanthin in long-time in 50 DEG C of extraction temperature,
Under conditions of 35MPa, caused by astaxanthin is thermally decomposed.Therefore 2.5h is optimum extraction time.
Carotenoid content comparative analysis in tamato fruit:
During supercritical carbon dioxide extraction, the carotenoid content and when recovery rate in investigating fruit, if
Determine the optimum extraction condition that experiment parameter is astaxanthin: 50 DEG C of extraction temperature, extracting pressure 35MPa, dehydrated alcohol extracts 2h, point
It Ji Suan not astaxanthin, lycopene, the content of bata-carotene and recovery rate.
From Fig. 5, table 1 as can be seen that not only containing astaxanthin in transgenic engineering tamato fruit, there are also lycopene and β
Carrotene.The recovery rate of astaxanthin is 95%, and content is 1.90mg/g dry weight, and lycopene 82%, content is
0.74mg/g dry weight, the recovery rate of bata-carotene are 89%, and content is 0.85mg/g dry weight.
1 transgenic engineering tamato fruit carotenoid content of table
Best-of-breed technology scheme of the invention:
Take the tamato fruit dry powder 200g being sieved in advance, regulating system temperature is to 50 DEG C, and extracting pressure 35MPa is to be extracted
After the temperature and pressure of kettle reach setting value and stablize, the static extracting of 1h is carried out；Constant-flux pump is opened after static extracting, it will
Dehydrated alcohol is transported into equipment, and the fluid-mixing of dehydrated alcohol and carbon dioxide gas enters extraction kettle, starts dynamic
Extraction, dynamic extraction time 2h, the recovery rate of astaxanthin are 95%.While extracting astaxanthin under this extraction conditions, this
Invention also obtains lycopene, the bata-carotene of high value simultaneously, and recovery rate is respectively 82% and 89%.
Claims (1)
1. the carotenoid supercritical carbon dioxide extraction method in transgenic engineering tamato fruit, it is characterised in that should
Method includes taking the tamato fruit dry powder being sieved in advance, regulating system temperature to 50 DEG C, extracting pressure 35MPa, kettle to be extracted
After temperature and pressure reach setting value and stablizes, the static extracting of 1h is carried out；Constant-flux pump is opened after static extracting, it will be anhydrous
Ethyl alcohol is transported into equipment, and the fluid-mixing of dehydrated alcohol and carbon dioxide gas enters extraction kettle, starts dynamic extraction,
Dynamic extraction time is 2h, and the transgenic engineering tomato deposit number is CGMCC No 9223, classification naming: shrimp red No. 1
Transgene tomato, on May 14th, 2014 in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation；It should
Method further quantitative determines carotenoid content, and the carotenoid is astaxanthin, lycopene, β carrot
Element, method for quantitatively determining use: using acetone after accurately weighing each 5mg mixing of astaxanthin, lycopene, bata-carotene standard items
100ml is dissolved and be settled to, 1ml, 5ml are taken, 10ml mixed dissolution liquid is settled to 50ml with acetone respectively, 1ug/ml is made,
5ug/ml, 10ug/ml standard items aggregate sample make standard curve using UPLC loading, calculate astaxanthin, lycopene, β Hu trailing plants
The content of Bu Su, the UPLC method are as follows: flow velocity: 1ml/min；Sample volume: 5ul；Column temperature: 60 DEG C；Eluent gradient variation:
0-1.0min: water: 20%, acetonitrile: 60%, isopropanol: 5%, methanol: 15%；1.00-2.00min: water: 0%, acetonitrile: 80%, isopropyl
Alcohol: 5%, methanol: 15%；2.00-8.00min: water: 0%, acetonitrile: 80%, isopropanol: 5%, methanol: 15%.
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| CN1334328A (en) * | 2001-07-25 | 2002-02-06 | 新疆大学 | Process for extracting prolycopene oil resin with supercritical fluid |
| CN1364832A (en) * | 2002-02-07 | 2002-08-21 | 山东大学 | Method for extracting and separating lycopene from tamato |
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| CN1334328A (en) * | 2001-07-25 | 2002-02-06 | 新疆大学 | Process for extracting prolycopene oil resin with supercritical fluid |
| CN1364832A (en) * | 2002-02-07 | 2002-08-21 | 山东大学 | Method for extracting and separating lycopene from tamato |
| CN101148631A (en) * | 2007-11-09 | 2008-03-26 | 天津商业大学 | Method for preparing lycopene-enriched vegetable oil |
| CN102783650A (en) * | 2012-09-04 | 2012-11-21 | 郭威 | Nutritional supplement of compound collagen and processing method thereof |
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