CN111187524B - Method for extracting carotenoid from waste algae oil - Google Patents
Method for extracting carotenoid from waste algae oil Download PDFInfo
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- CN111187524B CN111187524B CN202010035864.2A CN202010035864A CN111187524B CN 111187524 B CN111187524 B CN 111187524B CN 202010035864 A CN202010035864 A CN 202010035864A CN 111187524 B CN111187524 B CN 111187524B
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources
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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
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B67/00—Influencing the physical, e.g. the dyeing or printing properties of dyestuffs without chemical reactions, e.g. by treating with solvents grinding or grinding assistants, coating of pigments or dyes; Process features in the making of dyestuff preparations; Dyestuff preparations of a special physical nature, e.g. tablets, films
- C09B67/0096—Purification; Precipitation; Filtration
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2601/00—Systems containing only non-condensed rings
- C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
- C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated
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Abstract
The invention discloses a method for extracting carotenoid from waste algae oil, belonging to the field of extraction of carotenoid. The method comprises the following steps: firstly, adding a certain volume of mixed organic solvent into waste algae oil, diluting and dissolving the waste algae oil to obtain a diluent, keeping the diluent in a weak alkali environment, and adding an oxidation protective agent into the diluent; then, the diluted solution is subjected to gel chromatography to remove impurities, and a crude pigment solution is obtained; and finally, rotationally evaporating the pigment crude liquid, redissolving the pigment crude liquid by using a certain amount of mixed organic solvent, carrying out chromatographic separation by using a chromatographic column, collecting carotenoid mixed liquid, and further rotationally evaporating to remove the solvent to obtain dry powder. On one hand, the invention extracts the carotenoid from the waste algae oil, thereby changing waste into valuable; on the other hand, by improving the extraction method, the carotenoid beneficial components in the waste algae oil can be fully extracted, and the microalgae value is maximized.
Description
Technical Field
The invention relates to the field of extraction of carotenoid, in particular to a method for extracting carotenoid from waste algae oil.
Background
Carotenoids (Carotenoids) are a generic term for a class of natural pigment compounds. It is an isoprenoid polymer containing 40 carbons, i.e., a tetraterpene compound. Carotenoids are fairly stable within the cell, but are extracted within the cell and are susceptible to destabilization by oxygen, light, temperature and acid. The carotenoid has strong reducibility due to the structure of rich conjugated double bonds, and has the effects of resisting oxidation, regulating immunity, resisting cancer and delaying aging; meanwhile, the carotenoid compound is also a precursor of vitamin A and can protect vision to a certain extent, so that the carotenoid compound is widely applied to the fields of food, medicines, cosmetics, feed additives and the like and has important development and utilization values. Generally, carotenoids appear from bright yellow to dark red in color, with bright colors for ease of extraction, isolation and purification. Carotenoids are ubiquitous in plants, algae and some photosynthetic bacteria, and are not synthesized in animals, but are available in the diet. To date, there are more than about 750 carotenoids known in nature, of which more than 200 are found in algal cells and are concentrated in very limited algae. Functional carotenoids found in algae include lutein, violaxanthin, astaxanthin, diatoxanthin, diadinoxanthin, fucoxanthin, zeaxanthin, antheraflavin, and the like.
Microalgae are lower plants that produce energy by photoautotrophy and have a variety of biological functional components; the device has simple structure and easy control of culture conditions, can realize large-scale stable high-yield culture, and is widely applied to the development of energy and biological functional products. Microalgae are important sources of carotenoids such as astaxanthin, fucoxanthin, zeaxanthin and the like.
Related researches on extracting carotenoids from microalgae also exist in the prior art, for example, application No. 201410790236.X discloses a process for extracting lutein from marine microalgae, which comprises the steps of carrying out molecular biological modification on high-yield lutein marine microalgae by using a genetic engineering technology to obtain high-quality lutein-producing algal seeds, separating and purifying lutein in the microalgae by using column chromatography, and refining a lutein product by using a recrystallization technology; application No. 201811623577.2 discloses an industrial process for the extraction of high content of beta-carotene from dunaliella salina, comprising the steps of: (1) adding a nonpolar solvent into the dunaliella salina powder for extraction to obtain an extracting solution; (2) concentrating the extracting solution, standing for crystallization to obtain a crystal layer and a solvent layer; (3) washing the crystal layer, and drying to obtain trans-beta-carotene crystals; (4) concentrating the solvent layer to remove solvent to obtain dunaliella salina oil, and removing impurities from the dunaliella salina oil with supercritical fluid to obtain cis-form beta-carotene soft extract.
Although the prior art mentioned above has made some progress in extracting carotenoids from microalgae, it still has the technical defect of low extraction efficiency.
Disclosure of Invention
The invention aims to provide a method for extracting carotenoid from waste algae oil, which can change waste into valuable by extracting the carotenoid from the waste algae oil on one hand; on the other hand, by improving the extraction method, the carotenoid beneficial components in the waste algae oil can be fully extracted, and the microalgae value is maximized.
The technical scheme is as follows:
a method for extracting carotenoid from waste algae oil sequentially comprises the following steps:
a. adding a certain volume of mixed organic solvent into the waste algae oil, diluting and dissolving the waste algae oil to obtain a diluent, keeping the diluent in a weak alkali environment, and adding an oxidation protective agent into the diluent;
b. subjecting the diluent to gel chromatography to remove impurities to obtain a pigment crude solution;
c. performing rotary evaporation on the crude pigment liquid, redissolving the crude pigment liquid by using a certain amount of mixed organic solvent, performing chromatographic separation by using a chromatographic column, collecting carotenoid mixed liquid, and further performing rotary evaporation to remove the solvent to obtain dry powder;
the waste algae oil is the residue of oil-rich microalgae after extraction of DHA and EPA.
In a preferable embodiment of the invention, in the step a, the mixed organic solvent is ethyl acetate-cyclohexane or ethyl acetate-n-hexane, the volume ratio of ethyl acetate to cyclohexane to ethyl acetate to n-hexane is 1: 1-1.5, and the volume ratio of the waste algal oil to the mixed organic solvent is 1: 3-5.
As another preferred embodiment of the present invention, calcium carbonate particles are added to the diluent in an amount to maintain a weak alkaline environment, and the oxidation protective agent is vitamin C or tea polyphenols.
Further, subjecting the diluent to gel chromatography, wherein the specification of a gel purification column adopted in the gel process is 600mm in height multiplied by 40mm in inner diameter, the sample amount of each time is 20-50 mL, an elution solvent is ethyl acetate-cyclohexane or ethyl acetate-n-hexane, the volume ratio of ethyl acetate to cyclohexane and ethyl acetate to n-hexane is 1: 1-1.5, the flow rate is controlled by adopting a decompression mode to be 8-12 mL/min, the first 90mL fraction is discarded, 90-160 mL fraction is collected, and the gel column is washed by 160-250 mL.
Further, in the step c, the filler used by the chromatographic column is silica gel, diatomite or alumina, and the specification of the filler is 150-200 meshes; the mixed organic solvent is ethanol-petroleum ether, wherein the volume ratio of ethanol to petroleum ether is 1: 1-1.5, the elution solution required in the chromatographic separation process is acetone-petroleum ether, and the volume ratio of acetone to petroleum ether is 1: 8-9.
Further, the diluent and the elution solution are processed in an ice bath for 15-30 min before use.
Furthermore, the glass tubes of the gel purification column adopted in the gel process and the chromatography column adopted in the chromatography process are both made of brown materials or are processed in a dark place by tinfoil paper.
Compared with the prior art, the invention has the following beneficial technical effects:
(1) the invention separates fat-soluble pigment from grease by using gel chromatography, effectively removes various impurities in the grease, and has the characteristics of simple separation method and high separation efficiency.
(2) The method effectively avoids the carotenoid from deteriorating in the separation and purification process by adding the antioxidant, the acid-base regulator, low-temperature and light-resistant treatment and other means, and ensures good biological activity and effectiveness of subsequent use.
(3) The invention can recycle various organic solvents by rotary evaporation, thereby avoiding environmental pollution and effectively reducing production cost.
(4) The invention provides a simple and effective separation method for extracting carotenoid from waste algae oil, and performs primary separation condition optimization, thereby being beneficial to further research of an extraction process and providing basic conditions for industrialization.
Detailed Description
The invention provides a method for extracting carotenoid from waste algae oil, and in order to make the advantages and technical scheme of the invention clearer and clearer, the invention is described in detail with reference to specific examples.
The raw materials selected by the invention can be purchased from commercial sources.
In the following examples, SX-3,200 to 400mesh or the same standard was used for the gel, and the gel column standard was 600mm (height) by 40mm (inner diameter). The sample amount is 20-50 mL each time, the elution solvent is ethyl acetate-cyclohexane or n-hexane (1: 1-1.5, v/v), the flow rate is controlled by a decompression mode to be 8-12 mL/min, the first 90mL of fractions are discarded, 90-160 mL of fractions are collected, and 160-250 mL of gel columns are washed.
The glass tube of the gel purification column is made of brown materials or is processed in a dark place by using tin foil paper.
The chromatographic column filler used in the chromatography is silica gel, diatomite or alumina, and the specification of the filler is 150-200 meshes.
The glass tube of the chromatographic column is made of brown material or is processed in a dark place by tinfoil paper.
The waste algae oil mentioned in the invention is residual algae oil after DHA and EPA extraction of oil-rich microalgae, and is rich in various algae pigments, organic phosphorus, heavy metals and the like.
Example 1:
the method for extracting carotenoid from waste algae oil comprises the following steps:
in the first step, 10mL of waste algae oil is weighed, 30mL of ice-cooled ethyl acetate-cyclohexane (1:1, v/v) mixed solution is added, 0.5g of vitamin C and CaCO are added32.0g, and uniformly stirring;
step two, transferring 40mL of the diluent obtained in the step one to the upper end of a gel column, using an ice-cooled ethyl acetate-cyclohexane (1:1, v/v) mixed solution as an elution solution, controlling the flow rate by adopting a decompression mode to be 8mL/min, discarding the previous 90mL fraction, collecting 90-160 mL fraction, and washing the gel column by 160-250 mL to obtain a crude pigment solution;
and thirdly, rotationally evaporating the crude pigment liquid at 30 ℃ in vacuum, redissolving the crude pigment liquid by using 5mL of ethanol-petroleum ether (1:1, v/v), carrying out chromatographic separation by using an alumina chromatographic column, collecting carotenoid mixed liquid timely according to the movement of an observed pigment strip, rotationally evaporating the mixed liquid to remove most of the solvent, and then carrying out freeze drying to obtain dry powder.
Example 2:
the method for extracting carotenoid from waste algae oil comprises the following steps:
in the first step, 10mL of waste algae oil is weighed, and ethyl acetate-n-hexane (1) which is subjected to ice bath is added into the waste algae oil1.5, v/v) 50mL of mixed solution, 0.5g of vitamin C and CaCO32.0g, and uniformly stirring;
step two, transferring 40mL of the diluent obtained in the step one to the upper end of a gel column, using an ice-cooled ethyl acetate-cyclohexane (1:1.5, v/v) mixed solution as an elution solution, controlling the flow rate by adopting a decompression mode to be 8mL/min, discarding the first 90mL of fraction, collecting 90-160 mL of fraction, and washing the gel column by 160-250 mL to obtain a crude pigment solution;
and thirdly, rotationally evaporating the crude pigment liquid at 30 ℃ in vacuum, redissolving the crude pigment liquid by using 5mL of ethanol-petroleum ether (1:1, v/v), carrying out chromatographic separation by using an alumina chromatographic column, collecting carotenoid mixed liquid timely according to the movement of an observed pigment strip, rotationally evaporating the mixed liquid to remove most of the solvent, and then carrying out freeze drying to obtain dry powder.
Example 3:
the method for extracting carotenoid from waste algae oil comprises the following steps:
in the first step, 10mL of waste algae oil is weighed, 20mL of ice-cooled ethyl acetate-cyclohexane (1:1.5, v/v) mixed solution is added, 0.5g of vitamin C and CaCO are added32.0g, and uniformly stirring;
transferring 40mL of the diluent obtained in the first step to the upper end of a gel column, using an ice-bath ethyl acetate-n-hexane (1:1.5, v/v) mixed solution as an elution solution, controlling the flow rate by adopting a decompression mode to be 8mL/min, discarding the first 90mL of fraction, collecting 90-160 mL of fraction, and washing the gel column by 160-250 mL to obtain a crude pigment solution;
and thirdly, rotationally evaporating the crude pigment liquid at 30 ℃ in vacuum, redissolving the crude pigment liquid by using 5mL of ethanol-petroleum ether (1:1, v/v), carrying out chromatographic separation by using an alumina chromatographic column, collecting carotenoid mixed liquid timely according to the movement of an observed pigment strip, rotationally evaporating the mixed liquid to remove most of the solvent, and then carrying out freeze drying to obtain dry powder.
The parts which are not described in the invention can be realized by taking the prior art as reference.
It should be noted that: any equivalents, or obvious variations thereof, which may occur to those skilled in the art and which are commensurate with the teachings of this disclosure, are intended to be within the scope of this invention.
Claims (5)
1. A method for extracting carotenoid from waste algae oil is characterized by comprising the following steps: the method sequentially comprises the following steps:
a. adding a certain volume of mixed organic solvent into the waste algae oil, diluting and dissolving the waste algae oil to obtain a diluent, keeping the diluent in a weak alkali environment, and adding an oxidation protective agent into the diluent;
b. subjecting the diluent to gel chromatography to remove impurities to obtain a pigment crude solution;
c. performing rotary evaporation on the crude pigment liquid, redissolving the crude pigment liquid by using a certain amount of mixed organic solvent, performing chromatographic separation by using a chromatographic column, collecting carotenoid mixed liquid, and further performing rotary evaporation to remove the solvent to obtain dry powder;
the waste algae oil is the residual algae oil after DHA and EPA extraction of oil-rich microalgae, and is rich in various algae pigments, organic phosphorus and heavy metals;
in the step a, the mixed organic solvent is ethyl acetate-cyclohexane or ethyl acetate-n-hexane, the volume ratio of ethyl acetate to cyclohexane to ethyl acetate to n-hexane is 1: 1-1.5, and the volume ratio of the waste algae oil to the mixed organic solvent is 1: 3-5;
and in the step b, subjecting the diluent to gel chromatography, wherein the specification of a gel purification column adopted in the gel process is 600mm in height multiplied by 40mm in inner diameter, the sample amount of each time is 20-50 mL, the elution solvent is ethyl acetate-cyclohexane or ethyl acetate-n-hexane, the volume ratio of ethyl acetate to cyclohexane to ethyl acetate to n-hexane is 1: 1-1.5, the flow rate is controlled by adopting a decompression mode to be 8-12 mL/min, the first 90mL fraction is discarded, 90-160 mL fraction is collected, and the gel column is washed by 160-250 mL.
2. The method for extracting carotenoid from waste algae oil according to claim 1, wherein the carotenoid is: in the step a, a certain amount of calcium carbonate particles are added into the diluent to maintain a weak alkali environment, and the oxidation protective agent is vitamin C or tea polyphenol.
3. The method for extracting carotenoid from waste algae oil according to claim 1, wherein the carotenoid is: in the step c, the filler used by the chromatographic column is silica gel, diatomite or alumina, and the specification of the filler is 150-200 meshes; the mixed organic solvent is ethanol-petroleum ether, wherein the volume ratio of ethanol to petroleum ether is 1: 1-1.5, the elution solution required in the chromatographic separation process is acetone-petroleum ether, and the volume ratio of acetone to petroleum ether is 1: 8-9.
4. The method of claim 3, wherein the carotenoid is extracted from the waste algae oil by: the diluent and the elution solution are processed in an ice bath for 15-30 min before use.
5. The method of claim 3, wherein the carotenoid is extracted from the waste algae oil by: the glass tubes of the gel purification column adopted in the gel process and the chromatography column adopted in the chromatography process are both made of brown materials or are processed in a dark place by using tinfoil paper.
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| US8591912B1 (en) * | 2013-02-28 | 2013-11-26 | Kiran L. Kadam | Algae extraction process |
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| US20020082459A1 (en) * | 1997-05-28 | 2002-06-27 | Bailey David T. | High purity beta-carotene and process for obtaining same |
| CN1771966B (en) * | 2005-10-28 | 2010-04-28 | 浙江大学宁波理工学院 | Application of natural beta-carotene oil and beta-carotene crystal |
| CN101445565A (en) * | 2008-12-24 | 2009-06-03 | 江南大学 | Separation and purification method for Dunaliella salina polysaccharide |
| CN106478554A (en) * | 2016-10-17 | 2017-03-08 | 青岛博恩高科生物技术有限公司 | A kind of method extracting fucoxanthine and Sargassum polysaccharides comprehensive from Sargassum |
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