CN113817335A - Organic extraction reagent for efficiently extracting carotenoid from dunaliella salina and use method thereof - Google Patents
Organic extraction reagent for efficiently extracting carotenoid from dunaliella salina and use method thereof Download PDFInfo
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- CN113817335A CN113817335A CN202111101316.6A CN202111101316A CN113817335A CN 113817335 A CN113817335 A CN 113817335A CN 202111101316 A CN202111101316 A CN 202111101316A CN 113817335 A CN113817335 A CN 113817335A
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Abstract
The invention discloses an organic extraction reagent for efficiently extracting dunaliella salina carotenoid and a use method thereof, belonging to the field of extraction of bioactive substances. The invention determines the organic extraction reagent mixed solvent (DMSO: 95% ethanol) with good effect through single factor test, and then obtains the best extraction condition of the dunaliella salina carotenoid through the response surface: the extraction time is 20min, the temperature is 40 ℃, the proportion of mixed solvents (DMSO: 95% ethanol) is 3.64:1, and experiments prove that the extraction rate of the saline algae carotene is 0.0464%, which is 18.07% higher than that of the equivalent single solvent DMSO (0.0393%).
Description
Technical Field
The invention relates to the field of extraction of bioactive substances, in particular to an organic extraction reagent for efficiently extracting dunaliella salina carotenoid and a using method thereof.
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.
Carotenoids are a class of pure natural pigments that are widely distributed in various higher animals and plants and in certain microorganisms, and have a variety of important biological roles in the organism. Research shows that the carotenoid has the functions of immunoregulation, strengthening the resistance of the organism, resisting cancer and aging, and is a natural antioxidant with physiological activity in the organism. In recent years, due to the good biological effect of the carotenoid, natural carotenoid is often applied to the industries of food, cultivation, medicine, cosmetics and the like to prepare functional products such as various health-care foods, beverages, feeds, functional medicines, various anti-wrinkle anti-aging cosmetics and the like, and has potential value and research prospect.
Carotenoid extraction techniques have become a focus of research. At present, many documents have already provided methods for extracting carotenoids, mainly including enzymatic medium extraction, supercritical fluid extraction, ultrasonic extraction, organic solvent extraction, and the like. Since the carotenoid belongs to a fat-soluble pigment, the pigment can be dissolved out by using a chemical solvent capable of rapidly dissolving the lipid, so that the aim of extracting the pigment is further fulfilled. The invention adopts an organic solvent extraction method, and the experimental principle is clear. Common organic solvents include absolute ethanol, ethyl acetate, 95% ethanol, n-hexane, petroleum ether, methanol solution, acetone, etc.
Disclosure of Invention
The invention determines the organic extraction reagent mixed solvent (DMSO: 95% ethanol) with good effect through single factor test; the response surface software is used for designing an experimental scheme, so that the extraction condition of the mixed solvent is optimized; the reliability of the optimized conditions was verified by experiments.
The first purpose of the invention is to provide an organic extraction reagent for efficiently extracting the carotene of the saltalgae, wherein the organic extraction reagent is a mixed solvent of DMSO and 95% ethanol, and the weight ratio of DMSO: the volume ratio of 95% ethanol is 0.3-6.3: 1, specifically, DMSO: the volume ratio of 95% ethanol can be 0.3:1, 1.5:1, 3.25:1, 3.64:1, 5:1, 6.3: 1;
preferably, the ratio of DMSO: the volume ratio of 95% ethanol is 3.64: 1.
The second purpose of the invention is to provide a method for efficiently extracting the carotene from the salt algae, which is characterized by comprising the following steps:
a. centrifuging the salted algae culture solution, and removing the supernatant to obtain algae cell precipitate;
b. uniformly suspending the algal cell precipitate in a mixed solvent of DMSO and 95% ethanol for extraction, wherein the extraction time is 10-20min, and the extraction temperature is 40-60 ℃, and the ratio of DMSO: the volume ratio of 95% ethanol is 0.3-6.3: 1;
c. centrifuging after extraction is finished, wherein the supernatant is the extracted saline algae carotene solution;
preferably, the DMSO: the volume ratio of 95% ethanol is 3.64: 1;
preferably, the extraction time in step b is 20 min;
preferably, the extraction temperature in step b is 40 ℃.
Compared with the prior art, the invention has the following beneficial effects: the price of the used mixed extracting reagent is lower, the extracting temperature is obviously lower than 55 ℃ of the original technology, the extracting rate of the carotenoid is improved, the cost can be effectively saved, and the environment can be protected.
Drawings
FIG. 1 shows the results of a single solvent extraction of salt algae carotene.
FIG. 2 is a graph comparing the predicted response value and the actual response value of the extraction efficiency of the mixed solvent (DMSO: 95% ethanol) under different conditions.
The specific implementation mode is as follows:
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 Single factor assay to determine optimal extraction of organic reagents
1mL of shaken dunaliella salina culture solution is respectively measured and placed in 21 centrifuge tubes of 1.5mL, each 3 centrifuge tubes are divided into 7 groups, and 1mL of different extraction solvents, namely petroleum ether, ethyl acetate, 95% ethanol, n-ethane, absolute ethanol, dimethyl sulfoxide (DMSO) and acetone, are respectively added into the centrifuge tubes. The influence of different organic extraction solvents on the extraction rate was examined at an extraction temperature of 60 ℃ for 20 min. When the light absorption value is measured, different organic solvents are respectively used as controls, and each group of experiments is repeated for 3 times. The detailed extraction steps are as follows: centrifuging 1mL of algae solution at 12000rpm for 2min, and discarding the supernatant; suspending the algae cell sediment by 1mL of extraction solvent uniformly for extraction, wherein the time is set by the test; centrifuging at 12000rpm for 2min after extraction, placing 300 μ L of the extractive solution into 96-well microporous plate, measuring absorbance at 665nm, 649nm and 480nm, and adding corresponding extraction reagent without algae extraction solution into microporous plate as control. Measuring the absorbance of Dunaliella salina leaching solution at specific wavelength (665nm, 649nm, 480nm) with a microplate spectrophotometer, calculating the carotenoid content in each experiment, and mixing the two extraction solvents with good extraction effect to obtain a mixed extraction solvent. 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.
Experiments are carried out according to a single solvent extraction test method, and the experimental results (figure 1) show that different organic solvent extractants have different affinities for carotenoid and different influences on the extraction rate. The highest carotenoid content was extracted with dimethyl sulfoxide (DMSO), 95% ethanol and n-hexane as the minimum. It is demonstrated that the carotenoid in Dunaliella salina can be dissolved in these 7 solvents, and the content of carotenoid extracted is different due to different dissolving capacity. According to the extraction results of different organic solvents, the DMSO and the 95% ethanol have the best extraction effect, and the DMSO and the 95% ethanol are selected to be prepared into a mixed solvent extractant according to different proportions.
Example 2 optimization of extraction protocol for Mixed solvent (DMSO: 95% ethanol) in response to surface method
On the premise that a mixed solvent is determined in a single solvent extraction test, the influence of the extraction time (A), the extraction temperature (B) and the DMSO: 95% ethanol mixed solvent ratio (C) on the carotenoid extraction result is examined. The levels of experimental influencing factors were determined by prior investigation and reading of the reference (table 1), and then these influencing factors were further optimized by CCD design according to the level settings, and data were collected experimentally.
The experimental design scheme is provided by CCD center combination design, the influence of 3 factors of extraction time (A), extraction temperature (B) and mixed solvent ratio (C) on the extraction rate is considered, and all the factors and level settings are listed in Table 1.
TABLE 1 various factors and level settings in CCD design
The Design of the test is completed by using Design Expert software (Design Expert 10.0.4.0), and the following experimental scheme is designed by using CCD, wherein each group is repeated for 3 times, and the experimental scheme is shown in Table 2.
TABLE 2 CCD design experiment scheme
According to the experimental group setting, mixed solvents of DMSO and 95% ethanol in different mixing ratios are prepared according to the volume ratio of the solutions, and the mixed solvents and the 95% ethanol are uniformly mixed according to the ratio and then are filled into a reagent bottle for later use. The volume of the mixed solvent added in the experiment is 1mL, the volume of the saline algae culture solution is 1mL, and the specific experimental steps refer to example 1.
According to the test scheme optimized by the test method and the response surface method, after the experiments of each group are completed, the carotenoid extraction rate is obtained by excel calculation, and the results are input into Design Expert software (Design-Expert 10.0.4.0) for further data analysis and are listed in table 3.
TABLE 3 CCD design test results
Polynomial model fitting and regression analysis are carried out on the saline algae carotenoid extraction test result data by utilizing design expert software, the actual response value of the extraction rate in the table 3 is processed, and a polynomial regression equation with the actual carotenoid extraction rate (Y) as a dependent variable and the time (A), the temperature (B) and the mixed solvent ratio (C) as independent variables is obtained:
Y＝34.81+0.012A+B+0.5C-0.25AB+0.038AC+0.15BC-1.07A2+1.75B2-1.34C2-0.063ABC-4.89A2B+0.14A2C+0.26AB2
the experimental data of Table 3 were analyzed by Diagnostics using Design-Expert 10.0.4.0 software, and the results are shown in FIG. 2. Fig. 2 shows the relationship between the theoretical value and the actual response value, and the straight line in the figure indicates that the theoretical value is equal to the experimental value. As shown in the figure, most of the experimental values are distributed on both sides of a straight line, which indicates that the experimental values are not much different from the theoretical values, which indicates that the correlation between the theoretical extraction rate and the actual extraction rate is obvious and the degree of coincidence is high. Table 4 shows that the model is extremely remarkable, and the simulation result of the invention is ideal.
TABLE 4 analysis of variance of response surface
Note: (p <0.05) for significance, (< 0.01) for extreme significance, -this term has no data.
Example 3 validation experiment
The theoretical values of the optimal extraction conditions for the dunaliella salina carotenoids, which have been obtained by the response surface analysis method, are extraction time of 20min, temperature of 40 ℃, and mixed solvent ratio (DMSO: 95% ethanol) of 3.64: 1. Under this experimental condition, the theoretically optimal extraction rate of carotenoids was 0.04%. In order to further verify whether the selected model can reflect the actual situation, a sexual verification experiment is carried out according to predicted experimental conditions, 3 times of repetition is set, the extraction rate of the dunaliella salina carotenoid is finally measured to be 0.0464%, the extraction rate is improved by 18.07% compared with the extraction rate (0.0393%) of an equivalent amount of single solvent DMSO, under the condition that errors are allowed, the value is not greatly different from the predicted value, and the selected regression model is proved to be effective for predicting the influence of three factors on the extraction rate.
Claims (6)
1. An organic extraction reagent for efficiently extracting the carotenoid of the dunaliella salina is characterized in that the organic extraction reagent is a mixed solvent of DMSO and 95% ethanol, wherein the weight ratio of DMSO: the volume ratio of the 95% ethanol is 0.3-6.3: 1.
2. The organic extraction reagent according to claim 1, wherein the ratio of DMSO: the volume ratio of 95% ethanol is 3.64: 1.
3. A method for efficiently extracting carotene from salt algae is characterized by comprising the following steps:
a. centrifuging the salted algae culture solution, and removing the supernatant to obtain algae cell precipitate;
b. uniformly suspending the algal cell precipitate in a mixed solvent of DMSO and 95% ethanol for extraction, wherein the extraction time is 10-20min, and the extraction temperature is 40-60 ℃, and the ratio of DMSO: the volume ratio of 95% ethanol is 0.3-6.3: 1;
c. centrifuging after extraction, and collecting supernatant as extractive salt algae carotene solution.
4. The method according to claim 3, wherein the DMSO in step b: the volume ratio of 95% ethanol is 3.64: 1.
5. The method of claim 3, wherein the extraction time in step b is 20 min.
6. The method of claim 3, wherein the extraction temperature in step b is 40 ℃.
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