CN113817336A - Method for efficiently extracting carotenoid from dunaliella salina by DMSO (dimethyl sulfoxide) - Google Patents
Method for efficiently extracting carotenoid from dunaliella salina by DMSO (dimethyl sulfoxide) Download PDFInfo
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- CN113817336A CN113817336A CN202111101347.1A CN202111101347A CN113817336A CN 113817336 A CN113817336 A CN 113817336A CN 202111101347 A CN202111101347 A CN 202111101347A CN 113817336 A CN113817336 A CN 113817336A
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 title claims abstract description 48
- 235000021466 carotenoid Nutrition 0.000 title claims abstract description 41
- 150000001747 carotenoids Chemical class 0.000 title claims abstract description 41
- 241000195633 Dunaliella salina Species 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 18
- 241000195493 Cryptophyta Species 0.000 claims abstract description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 10
- 239000011780 sodium chloride Substances 0.000 claims abstract description 9
- 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 4
- 150000001746 carotenes Chemical class 0.000 claims abstract description 4
- 235000005473 carotenes Nutrition 0.000 claims abstract description 4
- NCYCYZXNIZJOKI-UHFFFAOYSA-N vitamin A aldehyde Natural products O=CC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-UHFFFAOYSA-N 0.000 claims abstract description 4
- 238000002386 leaching Methods 0.000 claims description 9
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- 239000002184 metal Substances 0.000 claims description 5
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- 238000000605 extraction Methods 0.000 abstract description 74
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- 230000004044 response Effects 0.000 abstract description 10
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- 235000012162 pavlova Nutrition 0.000 abstract description 3
- 230000000975 bioactive effect Effects 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 238000013461 design Methods 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
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- 238000004519 manufacturing process Methods 0.000 description 4
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- 239000003960 organic solvent Substances 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- OENHQHLEOONYIE-UKMVMLAPSA-N all-trans beta-carotene Natural products CC=1CCCC(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)CCCC1(C)C OENHQHLEOONYIE-UKMVMLAPSA-N 0.000 description 3
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- TUPZEYHYWIEDIH-WAIFQNFQSA-N beta-carotene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CCCC1(C)C)C=CC=C(/C)C=CC2=CCCCC2(C)C TUPZEYHYWIEDIH-WAIFQNFQSA-N 0.000 description 3
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- OENHQHLEOONYIE-JLTXGRSLSA-N β-Carotene Chemical compound CC=1CCCC(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C OENHQHLEOONYIE-JLTXGRSLSA-N 0.000 description 3
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- 229930002868 chlorophyll a Natural products 0.000 description 1
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
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- 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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- 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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Abstract
The invention discloses a method for efficiently extracting saline algae carotene by DMSO (dimethyl sulfoxide), belonging to the field of extraction of bioactive substances. The theoretical value of the optimal extraction condition of the dunaliella salina carotenoid obtained through the response surface is that when the extraction time is 11.35min, the temperature is 57.157 ℃, and the extraction reagent amount is 410 mu L, the predicted value of the carotenoid extraction rate is highest and can reach 0.576 per thousand, the reliability of the optimized condition is verified through experiments, and a certain theoretical basis is provided for the extraction and utilization of the carotenoid in the pavlova in the future; the experimental result shows that the extraction rate of the carotenoid is 0.517 per thousand, which is 31.55 percent higher than that of the control DMSO (0.393 per thousand), the invention reduces the dosage of the DMSO and improves the extraction rate of the carotenoid, thereby saving the cost and effectively protecting the environment.
Description
Technical Field
The invention relates to the field of extraction of bioactive substances, and particularly relates to a method for efficiently extracting saline algae carotene by DMSO.
Background
The Dunaliella bardawil (Dunaliella parva) is unicellular Dunaliella salina, can normally grow in a culture medium containing 0.05-5M NaCl, and has strong anti-pollution capability in outdoor large-scale culture. It has the following advantages: photoautotrophic property, strong stress resistance and simple culture condition; no cell wall, which is beneficial to genetic transformation; is rich in carotenoid and glycerol, and has high nutritive value.
At present, domestic carotenoids are mostly imported, and domestic produced natural carotenoids are extracted from fruits and vegetables, such as carrots, corns, potatoes and the like, but the raw materials have low effective components and high production cost. In contrast, extraction and utilization of carotenoids by using dunaliella salina as a raw material have many significant advantages. For example, dunaliella salina grows relatively rapidly, is easy to culture, has high yield, is less influenced by seasons, has strong adaptability, can grow and propagate in sewage, and the like. Therefore, dunaliella salina is the first alga which is considered to have potential commercial value and is used for producing beta-carotene. To date, Dunaliella salina has been utilized in large scale production of natural beta-carotene in several countries, such as the United states, Australia, Israel, and the like. In China, the phenomenon that a large amount of dunaliella salina is cultivated to extract beta-carotene by utilizing the advantages of local environment also exists, such as a Gilan salt farm of inner Mongolia.
The research and development of the carotenoid in the dunaliella salina are enhanced, the source of the natural carotenoid can be expanded, the demand quantity which is increased year by year in each field in the current society is met, the full utilization of the algae seed value can be realized, and considerable economic benefit is brought. At present, scholars at home and abroad carry out relevant research on the optimization of the extraction process of carotenoid in dunaliella salina, and report and summarize a plurality of methods and technologies related to the extraction of carotenoid. The production and the carotenoid acquisition by utilizing the dunaliella salina need to be carried out through the following three processes: culturing dunaliella salina, collecting algal bodies and extracting and purifying carotenoid. Wherein, the collection of the algae and the extraction of the carotenoid have decisive effects on the production cost. The common methods for extracting carotenoids from dunaliella salina include: organic solvent extraction, in-situ extraction, supercritical carbon dioxide extraction and the like, but no method which can really realize economic, environment-friendly and high-efficiency extraction exists so far. In comparison, the organic solvent extraction method is most convenient to operate, influence factors are easy to control, required instruments and equipment are few, cost is low, and safety of the whole process is high. Therefore, the extraction steps can be continuously perfected and simplified by optimizing various extraction factors of the organic solvent extraction method, and the development and utilization of the baffea duff algae and the commercial production of the carotenoid can be effectively promoted.
Disclosure of Invention
According to the method, the Dunaliella bardawil is used as a raw material, response surface analysis tests of a five-factor level are selected, the influence of interaction among factors on the extraction of the carotenoid in the Dunaliella bardawil is evaluated, and the relation between the extraction rate and the extraction factors is established through regression analysis, so that the extraction conditions of the carotenoid in the Dunaliella bardawil are optimized.
The invention aims to provide a method for efficiently extracting dunaliella salina carotenoid by DMSO, which comprises the following steps:
a. centrifuging the shaken dunaliella salina culture solution in a centrifuge tube, and removing supernatant; repeatedly centrifuging, and removing the supernatant; then, in the algae mud, according to the volume ratio of the saline algae culture solution to DMSO of 1 mL: adding DMSO into 900 mu L of 410-;
b. uniformly mixing the algae mud block and DMSO, and leaching in a constant-temperature metal bath at 40-60 ℃ for 10-20 min;
c. centrifuging after leaching, and taking supernatant as the extracted saline algae carotene solution.
Preferably, the volume ratio of the saline algae culture solution to DMSO in the step a is 1 mL: 410 mu L of the solution;
preferably, the leaching in step b is leaching in a thermostatted metal bath at 57 ℃ for 12 min.
Compared with the prior art, the invention has the following beneficial effects: the method optimizes the extraction conditions of the carotenoids in the pavlova, and provides a certain theoretical basis for the extraction and utilization of the carotenoids in the pavlova in the future. The invention can reduce the dosage of DMSO, improve the extraction rate of carotenoid, save cost and effectively protect environment.
Drawings
FIG. 1 shows the extraction rate of carotenoids from Dunaliella salina with different extraction reagents.
FIG. 2 is a graph comparing the predicted response value and the actual response value of the DMSO-extracted Dunaliella salina carotenoid under different conditions.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
The test methods in the following examples are all conventional test methods unless otherwise specified, and the test reagents and consumables described in the following examples are all available from conventional biochemical reagents company, unless otherwise specified.
EXAMPLE 1 determination of optimal extraction reagent
Dimethyl sulfoxide (DMSO), 95% ethanol, ethyl acetate, acetone, absolute ethanol, petroleum ether were selected as extraction reagents according to the solubility of carotenoids and literature consulted. Taking out 1mL of shaken up Dunaliella bardawil culture solution, centrifuging at 12000rpm for 5min, and discarding the supernatant; repeatedly centrifuging at the same rotation speed for 1min, and discarding the supernatant; then, 1mL of the above 6 extraction reagents are added to the algal mud respectively, and the mixture is placed on a vortex mixer to be shaken until the algal mud pieces are fully mixed and dissolved in the extraction solution. After mixing uniformly, the mixture is put into a constant temperature metal bath at 60 ℃ for leaching for 20min, and then the mixture is centrifuged at 12000rpm for 2 min. And finally, 300 mu L of supernatant is absorbed into an enzyme label plate to serve as detection liquid, an Epoch 2T microplate spectrophotometer is used for detecting light absorption values of the detection liquid under the three wavelengths of 480nm, 649nm and 665nm, and the extraction effects of different extraction reagents are researched. Three replicates of each group were performed. The carotenoid extraction rate was calculated according to the following formula.
chla＝12.47×A665-3.62×A649
chlb＝25.06×A649-6.5×A665
C＝(1000×A480-1.29×Chla-53.78×Chlb)/220
In the formula: chla is the concentration of chlorophyll a; chlb is the concentration of chlorophyll b; c is the content of carotenoid in the sample, mu g/mL; y is the extraction rate of carotenoid,% o; v is the volume of the extraction reagent, mu L; m is the dry weight of dunaliella salina in the sample, mg.
As a result, as shown in FIG. 1, carotenoids were soluble in most organic solvents, but different extraction reagents had different extraction effects on carotenoids in Dunaliella bardawil. In the six extraction reagents selected in the experiment, the extraction effects of DMSO, 95% ethanol, absolute ethanol, acetone, ethyl acetate and petroleum ether are sequentially from large to small, wherein the extraction effect of DMSO is the best, 95% ethanol is the second, and the extraction effect of petroleum ether is the worst. Therefore, DMSO was chosen as the extraction reagent in later experiments that optimized other extraction conditions.
Example 2 response surface optimization test design
On the basis of primarily exploring an optimal extraction reagent and referring to relevant reference document data, a Design-Expert 10.0.4 analysis software is utilized, according to a Central Composite Design (CCD) experiment principle, the extraction rate of carotenoid in Dunaliella bardawil is determined as a response value, the extraction time (A), the extraction temperature (B) and the extraction reagent amount (C) are used as influence factors, experiment Design and condition optimization are carried out within a certain numerical range, and the optimal extraction rate is finally obtained.
According to the experimental design, 5 levels of the three factors of the extraction time (A), the extraction temperature (B) and the amount of the extraction reagent (C) are studied, and the levels of the experimental factors are specifically set as shown in Table 1. The following experimental protocol was developed using a CCD design, with a total of 20 groups of three replicates per group, as detailed in Table 2.
TABLE 1 setting of various factors and levels in CCD
Table 2 design of the experimental protocol
According to the scheme designed by the principle of Central Complex Design (CCD), 1mL of the dunaliella salina culture solution is respectively added for experiments, and after relevant experiments are carried out according to the experimental steps of example 1, the results of the carotenoid extraction rate under different extraction conditions are finally calculated and obtained, and are shown in Table 3. Regression fitting analysis was performed on the data obtained in table 3 using Design Expert, and the regression equation of the response variable extraction time (a), temperature (B), and extraction reagent amount (C) versus the extraction rate (Y) was obtained as:
Y＝54.09-1.12A+4.43B-0.74C+5.32AB+4.68AC-2.71BC-1.57A2-5.54B2-1.86C2+3.94ABC-3.34A2B+0.53A2C-3.43AB2
the actual extraction rate in table 3 was analyzed by Diagnostics using Design-Expert to obtain the relationship between the actual extraction rate and the predicted value, as shown in fig. 2. In this figure, the closer the test point is to the straight line, the more reliable the actual extraction rate value is. According to the analysis of the graph, most of the test points are distributed near the theoretical line, which shows that the experimental value of the test is not much different from the theoretical value, and the experimental value and the theoretical value have better correlation.
TABLE 3 CCD analysis results
TABLE 4 analysis of variance of response surface
Note: as indicated by (p <0.05) for significance, and (p <0.01) for extreme significance.
As can be seen from table 4, the F value of this model is 20.34, and the p value is 0.0007<0.001, which shows that the correlation between the dependent variable (extraction rate) and the independent variable (extraction factor) is extremely different in the regression model, and that the subsequent optimization design can be performed using this model of the response surface.
Example 3 validation experiment
Through analysis of the model and prediction of optimal extraction conditions of the carotenoid, the predicted value of the carotenoid extraction rate is the highest and can reach 0.576 per thousand when the extraction time is 11.35min, the temperature is 57.157 ℃ and the extraction reagent amount is 410 mu L. In order to verify the accuracy of the regression model in the response surface method for optimizing the extraction conditions, the predicted optimal extraction conditions are reasonably and simply adjusted, namely the extraction time is changed to 12min, the temperature is 57 ℃, the extraction reagent amount is 410 mu L, 3 times of parallel verification tests are set, the extraction rate of the carotenoid is 0.517 per thousand, the extraction rate is 31.55 percent higher than that of a reference DMSO (0.393 per thousand), and the verification value of the test is not much different from the extraction rate value predicted by the regression model. The experimental results are combined, so that the model is real and reliable, and has certain reference significance for practical operation extraction of the carotenoids.
Claims (3)
- The method for efficiently extracting the carotenoid from the dunaliella salina by using DMSO (dimethyl sulfoxide) is characterized by comprising the following steps of:a. centrifuging the shaken dunaliella salina culture solution in a centrifuge tube, and removing supernatant; repeatedly centrifuging, and removing the supernatant; then, in the algae mud, according to the volume ratio of the saline algae culture solution to DMSO of 1 mL: adding DMSO into 900 mu L of 410-;b. uniformly mixing the algae mud block and DMSO, and leaching in a constant-temperature metal bath at 40-60 ℃ for 10-20 min;c. centrifuging after leaching, and taking supernatant as the extracted saline algae carotene solution.
- 2. The method of claim 1, wherein the volume ratio of the saline algae culture solution to DMSO in the step a is 1 mL: 410 μ L.
- 3. A process according to claim 1, characterized in that the leaching in step b is a leaching in a thermostatted metal bath at 57 ℃ for 12 min.
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| CN108949888A (en) * | 2018-06-28 | 2018-12-07 | 华南理工大学 | A method of promoting Du Shi algae accumulation carotenoid and beta carotene using alpha, beta-lonone |
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| CN112501234A (en) * | 2020-12-15 | 2021-03-16 | 华南理工大学 | Method for producing carotenoid by treating Dunaliella tertiolecta with creatinine |
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- 2021-09-18 CN CN202111101347.1A patent/CN113817336A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050203321A1 (en) * | 2002-05-08 | 2005-09-15 | Hejazi Mohammand A. | Process for obtaining carotenoids from natural sources |
| JP2006070114A (en) * | 2004-08-31 | 2006-03-16 | Fuji Chem Ind Co Ltd | Method for preparing carotenoid-containing extract |
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| CN112143770A (en) * | 2020-09-04 | 2020-12-29 | 清华苏州环境创新研究院 | Rhodotorula benthica and application thereof in production of beta-carotene by taking straws as raw material |
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Application publication date: 20211221 |