CN110452189A - A kind of total extracting method of Phaeodactylum tricornutum fucoxanthine and polyunsaturated fatty acid - Google Patents
A kind of total extracting method of Phaeodactylum tricornutum fucoxanthine and polyunsaturated fatty acid Download PDFInfo
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- CN110452189A CN110452189A CN201910695756.5A CN201910695756A CN110452189A CN 110452189 A CN110452189 A CN 110452189A CN 201910695756 A CN201910695756 A CN 201910695756A CN 110452189 A CN110452189 A CN 110452189A
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Abstract
The present invention relates to the total extracting method of a kind of Phaeodactylum tricornutum fucoxanthine and polyunsaturated fatty acid, includes the following steps: that (1) takes wet algal gel, add ethyl alcohol, stirring is extracted, the mixed liquor of wet algal gel and ethyl alcohol is obtained, is separated by solid-liquid separation, obtains preextraction liquid and algal biscuit 1；(2) algal biscuit 1 is mixed with ethyl alcohol, n-hexane, stirring extracts and obtains Two Liquid Phases mixed liquor, is separated by solid-liquid separation, obtains Two Liquid Phases extracting solution and filter cake 2；(3) preextraction liquid is mixed with Two Liquid Phases extracting solution, mixed liquor, layered processing is made, ethyl alcohol phase and n-hexane phase are obtained, the ethyl alcohol is mutually dried, fucoxanthine extract is made, the n-hexane is mutually dried, polyunsaturated fatty acid extract is made.Fucoxanthine and polyunsaturated fatty acid can effectively be extracted using method provided by the present invention altogether, Phaeodactylum tricornutum is enable to make the best use of everything.
Description
Technical field
The present invention relates to the total extracting methods of a kind of Phaeodactylum tricornutum fucoxanthine and polyunsaturated fatty acid, belong to biological work
Journey technical field.
Background technique
Fucoxanthine (Fucoxanthin) also known as fucoxanthin or brown alga flavine, belong to the oxygen-containing derivative of carotenoid
Object is common in brown alga and diatom.Fucoxanthine exercises the function of catching photopigment in alga cells.Fucoxanthine and chlorophyll
A and Chlorofucsin composition fucoxanthine-Chlorophyll Protein Complexes (Fucoxanthin-Chlorophyll Protein, FCP),
It is photosynthetical system element important in diatom.Fucoxanthine has proved to be a kind of safely and effectively dietary supplements, has anti-
Obesity, anti-diabetic, anti-oxidant, a variety of physiological activity [1] such as anti-inflammatory, antitumor.
Fucoxanthine is a kind of fat-soluble pigment, is insoluble in the higher solvent of water isopolarity in neutral conditions, is soluble in
Nonpolar organic solvent [2].Different according to the source of fucoxanthine at present, the conventional extraction of fucoxanthine predominantly has
Solvent extraction method and supercritical extraction.Since organic solvent is at the extraction to substrate without selectivity, organic solvent
Extraction method is suitable for the unified of multi-products and extracts, and this method is also most common extracting method during pigment extracts.This method tool
There is simple and easy, of less demanding to experiment condition advantage.It is demonstrated experimentally that fucoxanthine most preferably mentions in microalgae Phaeodactylum tricornutum
Taking solvent is ethyl alcohol, and optimum extraction temperature is 40 DEG C, and best solid-liquid ratio is 1:6 (mass volume ratio), and extraction time is no more than 12
Hour [3].In large-scale segasso sea band, also solvent extraction method can be used to extract fucoxanthine in kelp.
Supercritical extract extraction method is with CO2Target product is analysed by changing pressure and temperature as separation fluid
Out, to achieve the purpose that isolate and purify, this method can carry out at room temperature, while reduce oxidation again, therefore can
To keep the activity of natural materials.Armando etc. [4] is to extract raw material with thallus laminariae, is obtained using supercritical extraction technique
The optimal extraction process of fucoxanthine selects 40 DEG C to be used as optimum extraction temperature, when pressure is 40Mpa, extraction 3 hours, most
The rate of recovery for obtaining fucoxanthine in thallus laminariae eventually is 80%.Tang Jie [5] selects ethyl alcohol for entrainer, using supercritical CO2Extraction
It takes technology extraction from kelp base to prepare fucoxanthine, finally obtains the extract containing 5~20% fucoxanthines.
In addition, microwave extraction method, ultrasonic extraction, enzyme formulation etc. are also common extracting method, these are novel to be mentioned
It takes method to have improve extraction efficiency, shorten extraction time and the advantages such as environmental pollution is small, and is more and more studied
Person utilizes.
Eicosapentaenoic acid (Eicosapentaenoic acid, EPA, C20H30O2) it is the how unsaturated containing five double bonds
Fatty acid (polyunsaturated fatty acid, PUFA), belongs to omega-fatty acid.EPA is primarily present the dynamic plant of Yu Haiyang
It, can not de novo formation omega-fatty acid in human body in object.Prevention and treatment of the EPA to developing infant brain and adult disease of cardiovascular system
With good effect, while also there is anti-tampon, prevent coagulating platelets, vasodilator reduces cholesterol and blood pressure etc.
It acts on [6].It is mainly extracted from deep sea fish oil currently used for the natural EPA of food and health care product, the ratio that EPA accounts for fish oil is reachable
To 20%~30% [7].However, the yield of EPA is influenced by fishing type, the fishing season and place in fish oil；With me
State's health care product and the Fast Growth in high level food additives market, need the EPA in a variety of sources to meet the needs of market.
Marine microalgae plays an important role in marine ecosystems, and as primary producer, they are marine organisms
Important foodstuffs source.Marine microalgae contains a large amount of long-chain unsaturated fatty acid into the cell, and wherein EPA content is abundant.Microalgae
PUFAs in grease has the characteristics that composition is simple, cholesterol is low, without fishlike smell.Therefore, the insatiable hungers such as microalgae production EPA are utilized
It is had a vast market foreground with fatty acid.
Microalgae grease is present on cell membrane or is stored in into the cell as energy matter, and the grease on cell membrane contains
Amount will not change during micro algae growth, and furthermore polyunsaturated fatty acid is more common in ergastic substances.Since microalgae is thin
Born of the same parents have cell wall, to efficiently extract microalgae grease, cell wall damage need to be released to grease intracellular or use penetrability
Good extractant.The extraction of microalgae grease mainly uses traditional solvent extraction method and soxhlet extraction at present, in recent years, some
Novel microalgae grease extractive technique especially supercritical fluid extraction, ethyl alcohol multigelation method, subcritical solvent extraction etc.
To more and more extensive concern [8].
Marine microalgae is many kinds of, wherein Phaeodactylum tricornutum due to its growth rate it is fast, the features such as fat content is high, is by people
It is of interest.Its growth rate can reach 0.38g/L/d, and fat content can reach 48% or more, EPA yield and can reach after harvest
36mg/L/d produces source as new EPA in recent years.And as typical diatom, fucoxanthine content 15.42~
Between 16.51mg/g [9].
In current report, is extracted about microalgae pigment and a kind of substance is mainly individually extracted in fatty acid extraction, three
In the brown algae of angle, two kinds of products market value all with higher is converted into respective sterling and calculates, and about 2000 yuan of the value of EPA/
Kilogram, about 12000~16000 yuan/kilogram of the value of fucoxanthine, the value ratio of two kinds of products is about 1:(6~8).EPA contains
Amount accounts for about the 5~8% of dry cell weight, and fucoxanthine content accounts for about the 1.5~2.5% of dry cell weight.Therefore Unit Weight
Microalgae biomass in, EPA: the value of the product of fucoxanthine is than being about 1:1.8~1:2.5.If only mentioned during the extraction process
A kind of high value added product of fucoxanthine is taken, then the EPA for accounting for microalgae value 28%~36% will be wasted.How to pass through
Ji effectively extracts two kinds of products simultaneously, is to reduce production cost, the effective way that realized value is broken through.
In the technical literature of existing microalgae product development, the technical side that pigment and fatty acid product extract altogether is not retrieved
Case.Paper document " technical study that n-hexane-water-ethyl alcohol double liquid phase system extracts tea seed oil and Tea Saponin " (Lv Qi,
2017-05-01, " Institutes Of Technology Of Jiangxi ") in, tea seed oil and Tea Saponin are extracted using n-hexane-ethyl alcohol double liquid phase system
Process flow, but fucoxanthine and EPA from document tea seed oil and Tea Saponin physicochemical property it is different, therefore, in document
Flow and method be not particularly suited for the extraction of fucoxanthine and unsaturated fatty acid in Phaeodactylum tricornutum.
The present invention is intended to provide in a kind of Phaeodactylum tricornutum pigment fucoxanthine and polyunsaturated fatty acid EPA total extraction
Method, the purpose is to be comprehensively utilized to the Phaeodactylum tricornutum of culture, so that reaching reduces the mesh that toxigenic capacity improves the output value
's.
Summary of the invention
In view of the above-mentioned deficiencies in the prior art, it is an object of the present invention to provide a kind of Phaeodactylum tricornutum fucoxanthine and more insatiable hungers
With the total extracting method of fatty acid.
Technical solution of the present invention is as follows:
A kind of total extracting method of Phaeodactylum tricornutum fucoxanthine and polyunsaturated fatty acid, includes the following steps:
(1) preextraction: taking water content is the wet algal gel within 85%, adds ethyl alcohol, the quality volume of wet algal gel and ethyl alcohol
Than for 1:(1~4), unit g/ml, stirring is extracted 3~12 hours, and the mixed liquor of wet algal gel and ethyl alcohol is obtained, and is separated by solid-liquid separation, is obtained
To preextraction liquid and algal biscuit 1；
(2) n-hexane-ethanol solution Two Liquid Phases extracts: by algal biscuit 1 made from step (1) according to algal biscuit 1: ethyl alcohol: just oneself
Alkane=(1.5~2.5): (0.8~1): 1 mass volume ratio mixing, unit g/ml/ml, stirring are extracted 3~12 hours, are obtained
Two Liquid Phases mixed liquor is separated by solid-liquid separation gained Two Liquid Phases mixed liquor, obtains Two Liquid Phases extracting solution and filter cake 2；
(3) it separates drying: preextraction liquid made from step (1) is mixed with Two Liquid Phases extracting solution made from step (2), make
Mixed liquor is obtained, layered processing obtains ethyl alcohol phase and n-hexane phase, the ethyl alcohol is mutually dried, and fucoxanthine is made, right
The n-hexane is mutually dried, and polyunsaturated fatty acid is made.
Preferred according to the present invention, in the step (1), wet algal gel is selected from Cyclotella (Cyclotella), diamond shape algae
Belong to (Nitzschia), Navicula (Navicula), barrel mast Trentepohlia (Cylindrotheca) or brown algae category
(Phaeodactylum) wet algal gel；It is further preferred that the brown algae category (Phaeodactylum) is Phaeodactylum tricornutum.
Preferred according to the present invention, in the step (1) or step (2), ethyl alcohol is dehydrated alcohol.
It is preferred according to the present invention, in the step (1) or step (2), stir extraction conditions are as follows: 45 DEG C of Extracting temperature,
It is protected from light.
Preferred according to the present invention, in the step (1), the wet algal gel water content is 80%, wet algal gel and ethyl alcohol
Mass volume ratio is 1:3, unit g/ml；
It is preferred according to the present invention, it in the step (1) or step (2), is separated by solid-liquid separation to filter, filters filter cloth hole used
Diameter is 8~13 μm.
According to the present invention it is further preferred that the filter cloth aperture is 10 μm, material is polypropylene material.
It is preferred according to the present invention, in the step (2), the optimum quality volume ratio of filter cake 1 and extracting solution are as follows: filter cake 1:
Ethyl alcohol: n-hexane=2:1:1.
Preferred according to the present invention, in the step (3), layered shaping is that 5~20% volume basis are pressed into mixed liquor
Than KCl solution is added, the concentration of KCl solution is 0.5~1.5g/L, stratification.
Preferred according to the present invention, in the step (3), ethyl alcohol is mutually dry, and steps are as follows: ethyl alcohol phase and chloroform are pressed body
Product is mixed than 1:1, KCl solution then is added by 5~20% percents by volume, the concentration of KCl solution is 0.5~1.5g/L, quiet
Set layering, take chloroform phase, through rotary evaporation it is dry to get.
Preferred according to the present invention, in the step (3), it is that rotary evaporation is dry that n-hexane is mutually dry.
It is preferred according to the present invention, it further include the step of HPLC detection ethyl alcohol phase and n-hexane phase constituent in the step (3)
Suddenly, the step of carrying out esterification to the ingredient in ethyl alcohol phase and n-hexane phase carries out gas chromatographic analysis to esterification product
Step.
The product of the present invention that extracts altogether is mentioned including but not limited to the polyunsaturated fatty acid and fucoxanthine of diatom
The grease taken includes the metabolite that all fatty acid and sterol etc. are soluble in non-polar organic solvent；The pigment of extraction includes
Chlorophyll and carotenoid, lutein class etc. are soluble in the metabolite of polar organic solvent.
Phaeodactylum tricornutum fucoxanthine and polyunsaturated fatty acid of the present invention are total to extracting method, and fucoxanthine obtains
Yield > 85% of rate > 95%, EPA.
Technical solution provided by the present invention has the following beneficial effects:
1. can effectively extract fucoxanthine and polyunsaturated fatty acid altogether using method provided by the present invention, make three
Angle brown algae can make the best use of everything；
2. being operated using the present invention, agents useful for same is easily obtained, and Yi Huishou can be effectively controlled cost；And the present invention
The extraction system of use compares with the extraction system reported at present, and solid-liquid ratio still with higher ensure that extraction process
Economy.
3. the present invention is extracted using wet algal gel, the cost allowance and fucoxanthine of algae powder drying process are effectively prevented
The loss of isoreactivity substance.
It, also can be by the chlorophyll a in pigment from pigment 4. the present invention is separating pigment and while polyunsaturated fatty acid
In separate, to improve pigment quality it is helpful.
Detailed description of the invention
High performance liquid chromatography detection result picture after the extraction of Fig. 1 embodiment 1；
(A) is alcohol layer high-efficient liquid phase chromatogram in figure；(B) n-hexane layer high-efficient liquid phase chromatogram；
Gas chromatographic detection result picture after the extraction of Fig. 2 embodiment 1；
(A) is alcohol layer gas chromatogram in figure；(B) be positive hexane layer gas chromatogram；
Fucoxanthine and EPA yield histogram in Fig. 3 embodiment 2 under different Extracting temperatures；
Fucoxanthine and EPA yield histogram in Fig. 4 embodiment 3 under different solvents ratio；
Fucoxanthine and EPA yield histogram in Fig. 5 embodiment 4 under different extraction times；
Fucoxanthine and EPA yield histogram are directly extracted using preextraction and Two Liquid Phases in Fig. 6 comparative example 1.
Using other organic solvents to tomographic results after the total extraction of wet algae progress fucoxanthine and EPA in Fig. 7 comparative example 2
Photo；
Wherein: figure A, pigment exhibition layer analysis TLC analyze result photo；Scheme B, grease exhibition layer analysis TLC analyzes result photo.
Specific embodiment
Technical solution of the present invention is further elaborated below with reference to embodiment and Figure of description, but the present invention is protected
It is without being limited thereto to protect range.
Biological material source
Phaeodactylum tricornutum (Phaeodactylum tricornutum) is purchased from Chinese Marine University's microalge library, number
MACC/B228。
Detection method
1. the measurement of fucoxanthine:
The measurement of fucoxanthine content uses Hitachi's high performance liquid chromatograph detection system in the pigment extracted, and selection is anti-
Phase Agilent C18Chromatographic column (2.7 μm, 100 × 4.6mm).Detection wavelength is 445nm, flow rate of mobile phase 1mL/min, sample introduction
Amount: 5 μ L.Mobile phase is that acetonitrile and water carry out gradient elution.Acetonitrile ratio rises to 100% by 80% in 8min, maintains this
After ratio elutes 3min, acetonitrile ratio is linearly down to 80% in 5min, acetonitrile concentration is then risen to 100% again and is maintained
15 minutes.
The measurement of 2.EPA
By the algae oil after drying, 1.5mL n-hexane and 4.5mL 2%H is added2SO4(being dissolved in methanol), 85 DEG C of esterifications are anti-
It answers 3 hours, adds 0.9%KCl solution 2mL after cooling, fully shake.5 minutes are stood after concussion, takes upper organic phase in centrifuge tube
In it is spare.If EPA concentration is low, revolving instrument concentration can be used.200 μ L are finally taken to analyze for gas-chromatography (GC).
It is formed using the fatty acid methyl ester in Agilent 7890A gas chromatography analysis sample, chromatographic column is HP-5 (30m
×320μm×0.25μm).Gas chromatographic analysis program is as follows: injector temperature: 250 DEG C, sampling volume: 1 μ L；Carrier gas uses
High-purity nitrogen；Temperature program setting: then 120 DEG C of maintenance 5min rise to 240 DEG C with 3.5 DEG C/min, keep 10min.
In the present invention, the algae that each embodiment is selected is Phaeodactylum tricornutum.
Embodiment 1:
A kind of total extracting method of Phaeodactylum tricornutum fucoxanthine and polyunsaturated fatty acid, includes the following steps:
It weighs on a small quantity 1. the wet algal gel of harvest is taken, after calculated weight, wet algal gel is put into 105 DEG C of baking ovens, place
12 hours, the weight after to be dried, after calculating drying.Weight difference according to front and back calculates the water content of wet algal gel.
2. dehydrated alcohol, wet algal gel and nothing are added into wet algal gel in the case that the water content of wet algal gel is no more than 85%
The mass volume ratio of water-ethanol is 1:3, unit g/ml.
3. mixed solution is stirred at 45 DEG C using automatic stirrer, whole process is protected from light operation.
4. being separated solid algae-residue and extracting solution using Suction filtration device after stirring is extracted 3 hours, filters and use poly- third
Preextraction liquid and algal biscuit 1 is made in alkene filter membrane, 10 μm of aperture.
5. filtered algal biscuit 1 is weighed, and according to algal biscuit: ethyl alcohol: the mass volume ratio of n-hexane is the ratio of 2:1:1
Mixing.
6. collecting extract with step 3-4.
7. extract made from preextraction liquid made from step 4 and step 6 is mixed, and the KCl solution of 5% volume is added
(mass concentration 0.9%).It is layered ethyl alcohol phase mutually with n-hexane, uses liquid distributing device separating alcohol phase and n-hexane phase.
8. ethyl alcohol phase is mutually dried with n-hexane using Rotary Evaporators.
Preferred scheme: it in step 8, since alcohol layer contains most water, is not easy during rotary evaporation
It makes it dry, therefore adds chloroform according to 1:1 volume ratio to the alcohol layer containing pigment, mass concentration 0.9%KCL solution is added to make
It is layered, and chloroform rotary evaporation is dry.
9. using the ingredient of HPLC detection ethyl alcohol phase and n-hexane phase.
10. ingredient obtained in ethyl alcohol phase and n-hexane phase is carried out esterification respectively.
11. the esterification product that step 10 is obtained carries out gas chromatographic analysis.
Through detecting, this method, which is extracted, can will mainly contain the pigment separated of fucoxanthine to alcohol layer, 90~95%
Fucoxanthine is extracted to alcohol layer, and the fucoxanthine for only losing about 5~7% is extracted into hexane layer (Fig. 1).
Oil extraction to hexane layer, 80~85% EPA can be extracted to hexane layer by this method, only lose about 10~
12% EPA is extracted to alcohol layer (Fig. 2).
Embodiment 2
Embodiment 2 is essentially identical with embodiment 1, and difference is extraction temperature used in step 3 and step 6 point
It Wei not be 24 DEG C, 37 DEG C, 45 DEG C and 60 DEG C.
Through detecting, the recovery rate for extracting fucoxanthine at different temperatures is different, wherein under the conditions of 45 DEG C, extraction efficiency
Highest.There is also differences for the extraction efficiency of EPA under different temperatures, wherein EPA extraction efficiency is most under the conditions of 37 DEG C and 45 DEG C
It is high.In summary, 45 DEG C of optimum temperatures (Fig. 3) as extraction.
Embodiment 3
Embodiment 3 is essentially identical with embodiment 1, and difference is ethyl alcohol used in step 5 and n-hexane ratio
Difference is extracted, respectively when 1 mass of algal biscuit that step 5 obtains is 1 using the ethyl alcohol and n-hexane of different volumes ratio
Ethyl alcohol: n-hexane=1:3；Ethyl alcohol: n-hexane=1:2；Ethyl alcohol: n-hexane=1:1；Ethyl alcohol: n-hexane=1:0.5.
Through detecting, different solvent ratios can also be impacted extraction efficiency, mainly cause shadow to the extraction of grease
It rings, in ethyl alcohol: under conditions of n-hexane=1:2 or 1:3, the extraction efficiency highest of grease uses second based on the considerations of economy
Alcohol: n-hexane=1:2 condition is as best grease extraction conditions (Fig. 4).
Embodiment 4
Embodiment 4 is essentially identical with embodiment 1, and difference is extraction time used in step 4 and step 6 point
It Wei not 1h, 3h, 6h, 9h and 12h.
Through detecting, extraction time has a certain impact to extraction efficiency, after extracting 3h, the extraction of fucoxanthine and EPA
Rate variation is no longer significant, therefore selective extraction 3h is as optimum extraction time (Fig. 5).
Comparative example 1
It is total to extracting method as described in Example 1, (is mentioned in advance without using ethyl alcohol the difference is that omitting step 2~4
It takes).
Through detecting, in the experimental group for not carrying out preextraction, the recovery rate of cytochromes reduces 14%, and grease mentions
Rate is taken to reduce 37% (Fig. 6).This result is that due to water presence, result in the higher water-ethanol phase of polarity and formed with cell
Homogenate, and the lower n-hexane of polarity generates layering with it, can not be sufficiently mixed with components such as the higher film rouge of polarity, causes
Extraction efficiency is low.
In microalgae process, by algal gel it is dry dry algae powder be made be used for the cost of Downstream processing usually account for and be entirely processed into
This 30% or more, and usually with the loss of product, therefore, drying process is avoided to will be helpful to reduce cost raising production
Rate.But the moisture in wet algal gel can affect to downstream extraction, therefore the application is mentioned by the way that the pre- of ethyl alcohol is arranged
Step is taken, the recovery rate of wet algal gel is substantially increased.
Comparative example 2
It is total to extracting method as described in Example 1, the difference is that organic solvent similar in other polarity has been used to make
To extract reagent, ethyl alcohol is substituted using methanol, is extracted using normal heptane substitution n-hexane, comparative analysis extraction efficiency.It is right
In ratio, four groups of combinations are set, are respectively as follows: 1, n-hexane and ethyl alcohol carry out total extraction；2, normal heptane and ethyl alcohol carry out total extraction；
3, normal heptane and methanol carry out total extraction；4, n-hexane and methanol carry out total extraction.The oil layer and pigment that four kinds of combinations are extracted
Layer carries out TLC analysis respectively.
TLC is divided into pigment exhibition layer analysis and grease exhibition two aspects of layer analysis.Pigment opens up layer analysis and uses exhibition layer liquid:
N-hexane: acetone=6:4 (volume ratio)；Grease opens up layer analysis and uses exhibition layer liquid: n-hexane: ether: acetic acid=70:30:1 (body
Product ratio).Pigment exhibition layer analysis by open up layer after color and color spot size be compared；It is smoked using iodine vapor that grease opens up layer analysis
It steams post analysis color spot size and is compared.
It is detected, the oil layer being obtained by extraction, the separation through thin-layer chromatography exhibition layer liquid, it can be clearly seen that the rock in group 1
Algae Huang matter content is minimum, illustrates that oil layer will not be split into using the combination pigment, can preferably realize two kinds of components
Separation.The uvea being obtained by extraction, the separation through thin-layer chromatography exhibition layer liquid, it can be clearly seen that the fucoxanthine content in group 1
Highest, and the trace of grease is also least significant, when this explanation carries out total extract using n-hexane and ethyl alcohol, for fucoxanthine
It is best with the extraction efficiency of grease, and separating effect is best.It can be said that bright n-hexane used in embodiment 1 and ethyl alcohol
Combination has significant difference in total extraction process, the separating effect of fucoxanthine is significantly better than other similar solvent.
Grease opens up layer analysis and fumigates post analysis color spot size using iodine vapor, finds separation of four kinds of combinations for neutral fats
Effect is all very significant.Comprehensive grease and pigment separated effect analysis, can be real when be total to and extract using n-hexane and ethyl alcohol
The separation of existing fucoxanthine and polyunsaturated fatty acid, and separating effect is significantly better than the Two Liquid Phases of similar polar solvent composition
Extraction system, this has greatly exceeded the expectation of those skilled in the art.
The above is only the Optimizing Suggestions in presently preferred embodiments of the present invention and implementation process, not to this hair
The bright limitation done in any form, therefore all contents without departing from technical solution of the present invention, according to the technical essence of the invention to
Any simple modification, equivalent change and modification that upper embodiment is done, all of which are still within the scope of the technical scheme of the invention.
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Determination of Fucoxanthin in Diatom[J].Marine Drugs,2018,16(1):33.
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tricornutum[J].Separation Science&Technology,2014,49(3):410-415.
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Claims (10)
1. the total extracting method of a kind of Phaeodactylum tricornutum fucoxanthine and polyunsaturated fatty acid, which is characterized in that including as follows
Step:
(1) preextraction: taking water content is the wet algal gel within 85%, adds ethyl alcohol, and the mass volume ratio of wet algal gel and ethyl alcohol is
1:(1~4), unit g/ml, stirring is extracted 3~12 hours, and the mixed liquor of wet algal gel and ethyl alcohol is obtained, and is separated by solid-liquid separation, is obtained pre-
Extracting solution and algal biscuit 1；
(2) n-hexane-ethanol solution Two Liquid Phases extract: by algal biscuit 1 made from step (1) according to algal biscuit 1: ethyl alcohol: and n-hexane=
(1.5~2.5): (0.8~1): 1 mass volume ratio mixing, unit g/ml/ml, stirring are extracted 3~12 hours, and biliquid is obtained
Liquid is mixed, gained Two Liquid Phases mixed liquor is separated by solid-liquid separation, Two Liquid Phases extracting solution and filter cake 2 are obtained；
(3) it separates drying: preextraction liquid made from step (1) is mixed with Two Liquid Phases extracting solution made from step (2), be made mixed
Liquid is closed, layered processing obtains ethyl alcohol phase and n-hexane phase, the ethyl alcohol is mutually dried, and fucoxanthine is made, to described
N-hexane is mutually dried, and polyunsaturated fatty acid is made.
2. being total to extracting method as described in claim 1, which is characterized in that in the step (1), wet algal gel is selected from Cyclotella
(Cyclotella), Nitzschia (Nitzschia), Navicula (Navicula), barrel mast Trentepohlia (Cylindrotheca) or
The wet algal gel of brown algae category (Phaeodactylum)；It is further preferred that the brown algae category (Phaeodactylum) is three
Angle brown algae.
3. being total to extracting method as described in claim 1, which is characterized in that in the step (1) or step (2), ethyl alcohol is nothing
Water-ethanol；
Preferably, in the step (1) or step (2), extraction conditions are stirred are as follows: 45 DEG C of Extracting temperature, be protected from light.
4. being total to extracting method as described in claim 1, which is characterized in that in the step (1), the wet algal gel water content is
80%, the mass volume ratio of wet algal gel and ethyl alcohol is 1:3, unit g/ml.
5. being total to extracting method as described in claim 1, which is characterized in that in the step (1) or step (2), be separated by solid-liquid separation
To filter, filtering filter cloth aperture used is 8~13 μm；
It is further preferred that the filter cloth aperture is 10 μm, material is polypropylene material.
6. being total to extracting method as described in claim 1, which is characterized in that in the step (2), filter cake 1 and extracting solution are most
High quality volume ratio are as follows: filter cake 1: ethyl alcohol: n-hexane=2:1:1.
7. being total to extracting method as described in claim 1, which is characterized in that in the step (3), layered shaping is to mixed liquor
In by 5~20% percents by volume KCl solution is added, the concentration of KCl solution is 0.5~1.5g/L, stratification.
8. being total to extracting method as described in claim 1, which is characterized in that in the step (3), ethyl alcohol phase drying steps are such as
Under: ethyl alcohol is want that 1:1 is mixed by volume with chloroform, KCl solution then is added by 5~20% percents by volume, KCl solution
Concentration is 0.5~1.5g/L, and stratification takes chloroform phase, through rotary evaporation it is dry to get.
9. being total to extracting method as described in claim 1, which is characterized in that in the step (3), n-hexane is mutually dry for rotation
Evaporation drying.
10. being total to extracting method as described in claim 1, which is characterized in that further include HPLC detection second in the step (3)
The step of alcohol phase and n-hexane phase constituent, in ethyl alcohol phase and n-hexane phase ingredient carry out esterification the step of, to esterification
Product carries out the step of gas chromatographic analysis.
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