CN112778240A - A method for simultaneously extracting feed additive rich in DMPT and fucoxanthin from Chrysophyta - Google Patents
A method for simultaneously extracting feed additive rich in DMPT and fucoxanthin from Chrysophyta Download PDFInfo
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Abstract
The invention provides a method for simultaneously extracting a feed additive rich in DMPT and a feed additive rich in fucoxanthin from golden algae, belonging to the technical field of material extraction. According to the invention, 60-75% ethanol water solution is used as an extractant, DMPT and fucoxanthin in algae powder can be greatly extracted, the obtained extracting solution passes through a D101 macroporous resin column, fucoxanthin and the like can be adsorbed on the resin column, DMPT flows out of the adsorption column along with the effluent, and the obtained effluent is subjected to rotary evaporation to obtain the feed additive rich in DMPT; and (3) selecting a proper eluent to elute the fucoxanthin adsorbed on the resin column, and performing rotary evaporation on the eluent to obtain the feed additive rich in the fucoxanthin. The bait additive obtained by the invention has high DMPT content, improves the quality of bait, obtains the bait additive rich in fucoxanthin, and improves the utilization rate of the raw material golden algae.
Description
Technical Field
The invention relates to the technical field of substance extraction, in particular to a method for simultaneously extracting a feed additive rich in DMPT and a feed additive rich in fucoxanthin from golden algae.
Background
The golden algae belongs to the phylum of golden algae, the class of Prymycetes, the order of Hemicellales and the family of Isodinoflagellates, is rich in various nutrients such as polysaccharide, carotene, fucoxanthin (fucoxanthin) and high-energy lipid substances, unsaturated fatty acid and the like, has small size due to no cell wall, is high in production and propagation speed and easy to culture, and is an important economic bait algae for marine culture at home and abroad.
Dimethyl-betapropiothionine (DMPT) is present in many phytoplankton, macroalgae and molluscs symbiotic with algae, and is a representative substance for algae and plankton to convert inorganic sulfur into organic molecules. The research reports that DMPT has a healing effect on the stomach injury of experimental mice and can be used as a natural food additive, but the research focus of DMPT at present is the promotion effect on the feeding behavior and the growth of aquatic animals. DMPT has been reported at home and abroad to have good food calling effect on freshwater and seawater animals, and obvious food calling and growth promoting effects can be achieved by adding low-concentration DMPT in bait. At present, the commercial DMPT used as the aquatic product phagostimulant is a chemically synthesized product, and the extraction of natural DMPT from marine microalgae is beneficial to ensuring the safety of the product.
However, there are few studies and reports on DMPT extracted from seaweeds, and the DMPT used in current aquatic feeds is a chemical synthetic product, and some of the DMPT are even other compounds with similar chemical structures; this leads to differences in the effects of food calling and growth promotion, and even safety issues. Meanwhile, the simultaneous extraction of a DMPT-rich bait additive and a fucoxanthin-rich bait additive from seaweeds has not been reported.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for simultaneously extracting a DMPT-rich bait additive and a fucoxanthin-rich bait additive from chrysophyceae. The method provided by the invention can simultaneously obtain the feed additive rich in DMPT and the feed additive rich in fucoxanthin.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for simultaneously extracting a feed additive rich in DMPT and a feed additive rich in fucoxanthin from golden algae, which comprises the following steps:
separating solid from liquid of the algae culture solution in the logarithmic growth phase, collecting algae mud, and freeze-drying to obtain algae powder;
mixing the algae powder with an extracting agent, and carrying out ultrasonic extraction to obtain an extracting solution; the extractant is an ethanol water solution with the volume concentration of 60-75%;
passing the extract through a D101 macroporous resin column to obtain an effluent liquid and an adsorption column;
the effluent liquid is subjected to first concentration to obtain the bait additive rich in DMPT;
eluting the adsorption column to obtain an eluent;
performing second concentration on the eluent to obtain the feed additive rich in fucoxanthin;
the elution comprises the following steps: performing first elution on the adsorption column by using a first eluent to obtain a first eluent and an elution column; performing second elution on the elution column by using a second eluent to obtain an eluent; the first eluent is an ethanol water solution with the volume concentration of 60-70%, and the second eluent is an ethanol water solution with the volume concentration of 90%.
Preferably, the dosage ratio of the algae powder to the extractant is 1 g: (60-100) mL.
Preferably, the initial temperature of the ultrasonic extraction is 20-25 ℃, and the time is 40-45 min.
Preferably, the ultrasonic extraction further comprises: centrifuging the obtained extraction system to obtain an extracting solution; the rotation speed of the centrifugation is 4000r/min, and the time is 12-20 min.
Preferably, the ratio of the volume of the extracting solution passing through the D101 macroporous resin column to the volume of the D101 macroporous resin column is (50-75): 1.
preferably, the flow rate of the extracting solution passing through the D101 macroporous resin column is 3-6 mL/min.
Preferably, the first and second concentration modes are independently rotary evaporation.
Preferably, the dosage of the first eluent is 100-150% of the volume of the D101 macroporous resin column; the dosage of the second eluent is 400-600% of the volume of the D101 macroporous resin column.
Preferably, the mass concentration of DMPT in the feed additive rich in DMPT is 17-30%, and the mass concentration of fucoxanthin in the feed additive rich in fucoxanthin is 15-20%.
The invention provides a method for simultaneously extracting a feed additive rich in DMPT and a feed additive rich in fucoxanthin from golden algae, which comprises the following steps: separating solid from liquid of the algae culture solution in the logarithmic growth phase, collecting algae mud, and freeze-drying to obtain algae powder; mixing the algae powder with an extracting agent, and carrying out ultrasonic extraction to obtain an extracting solution; the extractant is an ethanol water solution with the volume concentration of 60-75%; passing the extract through a D101 macroporous resin column to obtain an effluent liquid and an adsorption column; the effluent liquid is subjected to first concentration to obtain the bait additive rich in DMPT; eluting the adsorption column to obtain an eluent; performing second concentration on the eluent to obtain the feed additive rich in fucoxanthin; the elution comprises the following steps: performing first elution on the adsorption column by using a first eluent to obtain a first eluent and an elution column; performing second elution on the elution column by using a second eluent to obtain an eluent; the first eluent is an ethanol water solution with the volume concentration of 60-70%, and the second eluent is an ethanol water solution with the volume concentration of 90%. The invention adopts ethanol water solution as an extractant, which can greatly extract DMPT and fucoxanthin in algae powder, the obtained extracting solution passes through a D101 macroporous resin column, the fucoxanthin can be adsorbed on the resin column, and the DMPT flows out of the adsorption column along with the effluent liquid; and concentrating the obtained effluent to obtain the feed additive rich in DMPT. And performing first elution on the adsorption column by using a first eluent to remove impurities with poor adhesive force, performing second elution on the elution column by using a second eluent to elute the fucoxanthin, and concentrating to obtain the bait additive rich in the fucoxanthin. The content of DMPT and fucoxanthin in the bait additive obtained by the invention is high, and the quality of the bait additive is improved.
Drawings
FIG. 1 is a photograph of an extract obtained by extracting chrysophytes with an aqueous solution of ethanol having different volume concentrations;
FIG. 2 is a response surface diagram showing the interaction between the concentration of the ethanol aqueous solution and the extraction time on the extraction amount of fucoxanthin;
FIG. 3 is a response surface diagram of the interaction effect of the concentration of the ethanol water solution and the extraction temperature on the extraction amount of the fucoxanthin;
FIG. 4 is a response surface diagram showing the interaction effect of the concentration of the ethanol aqueous solution and the feed liquid ratio on the extraction amount of fucoxanthin;
FIG. 5 is a response surface graph showing the interaction of extraction time and temperature on the amount of fucoxanthin extracted;
FIG. 6 is a graph of the response of the extraction time and feed liquid ratio versus the interactive effect on the amount of fucoxanthin extracted;
FIG. 7 is a graph of the response surface of the interaction of the extraction temperature and feed liquid ratio on the fucoxanthin extraction amount;
FIG. 8 is a response surface plot of the interactive effect of ethanol concentration and extraction time on DMPT extraction;
FIG. 9 is a response surface plot of the interactive effect of ethanol concentration and extraction temperature on DMPT extraction;
FIG. 10 is a response surface graph showing the interaction effect of ethanol aqueous solution concentration and feed solution ratio on DMPT extraction amount;
FIG. 11 is a response surface plot of the interaction of extraction time and temperature on DMPT extraction volume;
FIG. 12 is a response surface plot of the interaction of extraction time and feed solution ratio versus DMPT extraction volume;
FIG. 13 is a graph of the response of the extraction temperature and feed solution ratio versus DMPT extraction.
Detailed Description
The invention provides a method for simultaneously extracting a feed additive rich in DMPT and a feed additive rich in fucoxanthin from golden algae, which comprises the following steps:
separating solid from liquid of the algae culture solution in the logarithmic growth phase, collecting algae mud, and freeze-drying to obtain algae powder;
mixing the algae powder with an extracting agent, and carrying out ultrasonic extraction to obtain an extracting solution;
passing the extract through a D101 macroporous resin column to obtain an effluent liquid and an adsorption column;
the effluent liquid is subjected to first concentration to obtain the bait additive rich in DMPT;
eluting the adsorption column to obtain an eluent;
performing second concentration on the eluent to obtain the feed additive rich in fucoxanthin;
the invention separates the solid and liquid of the algae culture solution in logarithmic growth phase, collects the algae mud and carries out freeze drying to obtain the algae powder.
The invention does not specifically limit the acquisition mode of the algae culture solution in the logarithmic growth phase, and the skilled person can select the culture solution according to the actual situation. In the present invention, the algae in the algae culture solution is preferably chrysophyceae. In the invention, the solid-liquid separation mode is preferably centrifugation, the rotation speed of the centrifugation is preferably 6000r/min, and the time is preferably 10-15 min; the temperature of the centrifugation is preferably 10 ℃. In the present invention, the parameters of the freeze-drying preferably include: freezing at-18 deg.C for 24h, and drying in a freeze dryer.
After the algae powder is obtained, the algae powder and an extracting agent are mixed and subjected to ultrasonic extraction to obtain an extracting solution.
In the invention, the extracting agent is preferably ethanol water solution with volume concentration of 60-75%; the using amount ratio of the algae powder to the extracting agent is preferably 1 g: (60-100) mL. In the invention, the starting temperature of ultrasonic extraction is preferably 20-25 ℃, and the time is preferably 40-45 min. In the present invention, after the ultrasonic extraction, it is preferable to further include centrifuging the obtained ultrasonic extraction system to obtain an extract; the rotation speed of the centrifugation is preferably 4000r/min, and the time is preferably 12-20 min.
The method takes 60-75% ethanol water solution as an extracting agent, and can greatly extract DMPT and fucoxanthin in the algae powder by extracting under the ultrasonic condition.
After the extracting solution is obtained, the extracting solution is processed by a D101 macroporous resin column to obtain an effluent liquid and an adsorption column.
In the invention, the ratio of the volume of the extracting solution passing through the D101 macroporous resin column to the volume of the D101 macroporous resin column is preferably (50-75): 1. in the specific embodiment of the invention, the inner diameter of the D101 macroporous resin column is preferably 1.6cm, the height of the column bed is preferably 20cm, and the average pore diameter is preferably 25-28 nm; when the inner diameter of the D101 macroporous resin column is 1.6cm, the height of the column bed is 20cm, and the average pore diameter is 25-28 nm, the volume of the extracting solution passing through the D101 macroporous resin column is preferably 2000-3000 mL. In the invention, the flow rate of the extracting solution passing through the D101 macroporous resin column is preferably 3-6 mL/min, and more preferably 4-5 mL/min.
In the invention, the extract passes through a D101 macroporous resin column, because the D101 macroporous resin is a nonpolar polymer adsorbent, DMPT is a polar compound and is not adsorbed by the resin, and fucoxanthin is a fat-soluble pigment and can be adsorbed by nonpolar resin; therefore, the fucoxanthin in the extracting solution is adsorbed on the D101 macroporous resin column by passing the extracting solution through the D101 macroporous resin column, and the DMPT does not adsorb on the D101 macroporous resin column and flows out of the D101 macroporous resin column in the form of an effluent liquid.
After obtaining the effluent, the invention firstly concentrates the effluent to obtain the bait additive rich in DMPT. In the present invention, the first concentration mode is preferably rotary evaporation, the temperature of the rotary evaporation is preferably 60 ℃, the time of the rotary evaporation is not particularly limited, and the first concentration mode can be set by a person skilled in the art according to actual conditions.
After the adsorption column is obtained, the invention elutes the adsorption column to obtain the eluent. In the present invention, the elution preferably comprises the steps of: performing first elution on the adsorption column by using a first eluent to obtain a first eluent and an elution column; performing second elution on the elution column by using a second eluent to obtain an eluent; the first eluent is an ethanol water solution with the volume concentration of 60-70%, and the second eluent is an ethanol water solution with the volume concentration of 90%. In the invention, the dosage of the first eluent is 100-150% of the volume of the D101 macroporous resin column; the dosage of the second eluent is 400-600% of the volume of the D101 macroporous resin column. In the present invention, the eluate obtained in the second elution needs to discard the initially obtained solution, and the discarded amount is not particularly limited, and may be selected by one skilled in the art according to actual situations. In the present invention, the first elution can remove some non-target substances (for example, water-soluble components contained in the extraction liquid and some more polar pigments) adsorbed on the adsorption column; the second elution can elute the fucoxanthin adsorbed on the adsorption column, and the first elution and the second elution can improve the purity of the fucoxanthin.
After the eluent is obtained, the second concentration is carried out on the eluent to obtain the bait additive rich in the fucoxanthin. In the invention, the second concentration mode is preferably rotary evaporation, and the temperature of the rotary evaporation is preferably 50 ℃; the time of the rotary evaporation is not specifically and newly stabilized, and the skilled person can select the time according to the actual situation. In the invention, the mass concentration of the fucoxanthin in the crude extract rich in the fucoxanthin is 15-20%.
The bait additive obtained by the method provided by the invention has high DMPT and fucoxanthin contents, and when the bait additive is applied to baits, the addition amount is small, the addition amount of other nutrient elements such as betaine and the like can be increased, and the quality of the baits is improved.
The method for simultaneously extracting DMPT-rich and fucoxanthin-rich bait additives from chrysophyceae according to the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.
Example 1
Centrifuging the golden algae culture solution in logarithmic growth phase at 6000r/min for 10min, collecting algae mud, freeze drying at-18 deg.C for 24h, and drying in freeze dryer to obtain algae powder;
mixing 1g of algae powder with 100mL of 70% ethanol aqueous solution, performing ultrasonic treatment at 20 ℃ for 45min, centrifuging at the rotation speed of 4000rpm for 15min, and collecting supernatant as an extracting solution;
adding 100mL of deionized water into 100mL of extracting solution, and passing through a D101 macroporous resin column at the flow rate of 5mL/min, wherein the inner diameter of the D101 macroporous resin column is 1.6cm, the height of a column bed is 20cm, and the volume of the column is 40mL to obtain an effluent liquid;
carrying out rotary evaporation on the effluent at 60 ℃ to obtain the feed additive rich in DMPT;
eluting and adsorbing the D101 macroporous resin column by adopting 60mL ethanol water solution with volume concentration of 70 percent, and discarding the elution solution; and then continuously eluting and adsorbing the D101 macroporous resin column by using 300mL of ethanol water solution with the volume concentration of 90%, discarding the first 40mL of elution solution, collecting 240mL of elution solution flowing out after the elution solution is used as eluent, and performing rotary evaporation on the eluent at 50 ℃ to obtain the fucoxanthin crude extract.
1. The fucoxanthin content of the resulting fucoxanthin-enriched bait additive was tested using the following liquid chromatography conditions:
a chromatographic column: develosil C30-UG-5, 250mm × 4.6mm (i.d.), 5 μm;
mobile phase: methanol-acetonitrile solution (49: 51, V/V); flow rate: 1.0 mL/min;
detection wavelength: 450 nm;
column temperature: 30 ℃;
sample introduction volume: 20 mu L of the solution;
establishment of fucoxanthin standard curve
Accurately weighing 0.01g (accurate to 0.0001g) fucoxanthin standard substance, dissolving with methanol, diluting to 100mL, preparing into standard stock solution with concentration of 100 μ g/mL, preparing, and storing in 4 deg.C refrigerator in dark place with shelf life of no more than 3 months.
Diluting with methanol to obtain a series of standard solutions of 0.020mg/L, 0.100mg/L, 0.400mg/L, 1.00mg/L, 5.00mg/L and 10.0mg/L at the time of clinical application, sampling 20 μ L, performing linear regression on the measured fucoxanthin chromatographic peak area and the corresponding concentration, and obtaining a standard curve regression equation of Y-1.7725X-0.0633, wherein X is the concentration (mg/L) of fucoxanthin in the measured solution, and Y is the fucoxanthin chromatographic peak area.
The content of fucoxanthin in the obtained fucoxanthin crude extract is 15-20%;
2. the DMPT content of the DMPT-rich bait additive was determined according to the following conditions:
headspace gas chromatography determination parameters of DMPT:
a chromatographic column: DB-624, 0.32mm × 30m, liquid film thickness 1.8 μm;
carrier gas: high-purity nitrogen; flow rate: 1.0 mL/min;
sample inlet temperature: 120 ℃;
column temperature: 50 ℃;
a detector: FID, temperature 150 ℃.
Headspace sample introduction: the split ratio is 20: 1, sample injection time of 0.5min, equilibration time of 7min, and temperature of 50 ℃.
Establishment of DMPT standard curve
Accurately weighing 0.01g (accurate to 0.0001g) of DMPT standard substance, dissolving with water, diluting to 10mL, preparing into standard stock solution with concentration of 1mg/mL, and storing in refrigerator at 4 deg.C. Diluting the solution into a series of standard solutions of 1mg/L, 5mg/L, 10mg/L, 25mg/L and 50mg/L by using water for clinical use, injecting samples according to the measurement parameters of the headspace gas chromatography of DMPT, performing linear regression on the measured DMPT chromatographic peak area and the corresponding concentration, and obtaining a standard curve regression equation as follows: Y4.3458X +3.3896, where X is the mass of DMPT in the headspace (μ g) and Y is the chromatographic peak area for DMPT.
The mass content of DMPT in the feed additive rich in DMPT is 17-30%.
2.1 measurement of DMPT in extract
And (3) putting 0.2mL of the extracting solution into a 20mL headspace bottle, adding 2mL of 5mol/L NaOH solution, immediately sealing to obtain a solution to be detected, and measuring by adopting headspace gas chromatography measurement parameters of DMPT to obtain the content of DMPT in the extracting solution of 112 mg/L.
2.2 determination of DMPT in algal powder
Putting 0.05g of golden algae powder into a 20mL headspace bottle, adding 2mL of 5mol/L NaOH solution, immediately sealing, carrying out ultrasonic treatment for 10min, standing overnight to obtain a solution to be detected, and measuring by adopting headspace gas chromatography measurement parameters of DMPT to obtain the content of DMPT in the algae powder of 13.2 mg/g.
Example 2
The only difference from example 1 is that the extractant is an aqueous ethanol solution with a concentration of 60% by volume.
Example 3
The only difference from example 1 is that the extractant is a 75% strength by volume aqueous ethanol solution.
Comparative example 1
The only difference from example 1 is that the extractant is a 30% strength by volume aqueous ethanol solution.
Comparative example 2
The only difference from example 1 is that the extractant is an aqueous ethanol solution with a volume concentration of 45%.
Comparative example 3
The only difference from example 1 is that the extractant is a 90% strength by volume aqueous ethanol solution.
Table 1 shows the contents of DMPT and fucoxanthin in the bait additives obtained in examples 2 to 3 and comparative examples 1 to 3.
TABLE 1 extraction of target substance by ethanol aqueous solution of different volume concentration
Note: the results are shown as the average value, n is 3
As can be seen from table 1: when the volume concentration of the ethanol water solution is below 45%, the extraction amount of two target substances is low, and especially the fucoxanthin content is low; for fucoxanthin, the extraction amounts of 60% and 75% ethanol water solutions are basically not different, and the color of the 90% ethanol water solution extracting solution is too dark, which indicates that the pigment in the extracting solution is too much and is not measured on a machine for protecting an instrument. For DMPT, 75% aqueous ethanol extraction was the greatest.
FIG. 1 is a photograph of an extract obtained by extracting chrysophytes with an aqueous solution of ethanol having different volume concentrations; the volume concentration of the ethanol aqueous solution is 30%, 45%, 60%, 75% and 90% from left to right.
As can be seen from fig. 1: the extract obtained from 60% ethanol water solution contains less green pigment. Although the green pigment has no interference on the determination of the fucoxanthin, the reduction of impurities can protect an analysis system and is beneficial to the subsequent separation and purification of the fucoxanthin, so that the extractant selects a lower-concentration ethanol aqueous solution, namely the volume concentration of the ethanol aqueous solution is 60%.
As a result, it was found that: the extract obtained by 30% ethanol aqueous solution is turbid and difficult to filter; the extraction effect of 45% ethanol aqueous solution is slightly better; the extract obtained from ethanol water solution with volume concentration of above 60% is easy to filter.
Extraction process of fucoxanthin and DMPT
A4-factor and 3-level response surface experiment is designed by taking the liquid-material ratio, the ultrasonic extraction temperature, the ultrasonic extraction time and the concentration of an ethanol aqueous solution as investigation factors and the extraction amount of fucoxanthin and DMPT in an extracting solution as an experimental index according to a Box-Behnken combined experimental design principle, and the result is shown in Table 2.
TABLE 2 response surface design and test results for fucoxanthin and DMPT extraction
And (3) performing multivariate regression fitting analysis on the experimental results in the table 2 by using Design Expert 8.0.6.1 software, establishing a secondary response model, and obtaining a multivariate quadratic regression model by using the fucoxanthin extraction amount as an optimization index: y1 ═ 95.43352+2.97577A-0.051250B-0.023292C +0.11494D +0.003577788AB-0.00220000AC-0.000220833AD-0.00166667BC + 0.0020379170 BD +0.00020812500CD-0.021046A2-0.00430185B2+0.00289583C2-0.00104 792D2. Taking the DMPT extraction amount as an optimization index to obtain a multiple quadratic regression model: y2 ═ 5.20643 +0.16803A-0.15986B-0.20305C +0.10592D +0.00390333A +0.00220250 AC-0.00221917 AD-0.00349000BC +0.00216792 BD-0.000184375 CD-0.00137047A2– 0.00144491B2+0.00215380C2。
In order to test the effectiveness of the regression equation, the regression equation is subjected to variance analysis and significance test, and the results are shown in tables 3 and 4.
TABLE 3 analysis of fucoxanthin extraction variance
Note: p <0.0100 indicates that the index is extremely significant; p <0.0500 indicates that the index is significant; p >0.0500 indicates that the index is not significant.
As can be seen from table 3, the regression equation is highly significant (p < 0.01), the misfit term is not significant (p: 0.1249 > 0.05), and the correlation coefficient of the model is high (R ═ 0.1249 ≧ 0.05)2＝88.51％)、R2Adj 77.02%. The regression equation is good in fitting degree and small in experimental error, and the fucoxanthin extraction amount can be predicted according to the fitting degree. As can be seen from Table 3, the A, D effect was significant (p < 0.05), with the A effect being very significant (p < 0.01). This indicates that 2 factors have a direct relationship with the amount of fucoxanthin extracted, which is the main limiting factor, and that small changes will cause significant changes in the amount of fucoxanthin extracted. The order of the extracted influencing factors which can be obtained by comparing the magnitudes of the p values is as follows: concentration of ethanol aqueous solution>Liquid to feed ratio>Temperature of extraction>And (4) extracting time.
Response surface and contour maps of the interaction effect between the two factors are obtained through Design-Expert 8.0.6.1 software, and the results are shown in fig. 2-7. According to the design principle and analysis of the response surface experiment, the larger the curvature of the three-dimensional response surface graph is, the more obvious the interaction effect between the two factors is, and therefore the influence of the interaction between the factors on the extraction amount of the fucoxanthin can be effectively reflected.
Comparing fig. 2 to fig. 7, it can be seen that the interaction between the concentration of the extractant ethanol aqueous solution and the ultrasonic extraction time, and the concentration of the extractant ethanol aqueous solution and the feed-liquid ratio has a significant effect on the extraction of fucoxanthin, and the interaction between the other two factors has a insignificant effect on the extraction of fucoxanthin.
TABLE 4 DMPT extraction analysis of variance results
Note: p <0.0100 indicates that the index is extremely significant; p <0.0500 indicates that the index is significant; p >0.0500 indicates that the index is not significant.
As can be seen from table 4, the regression equation is significant (p ═ 0.0335 < 0.05), the mismatching term is not significant (p ═ 0.4245 > 0.05), and the correlation coefficient of the equation is high (R ═ 0.4245 > 0.05)2＝70.21％)、R2Adj 44.39%. The regression model is good in fitting degree and small in experimental error, and the DMPT extraction amount can be predicted accordingly. As can be seen from Table 4, A, D had a significant effect (p < 0.05). This indicates that 2 factors have a direct relationship with DMPT extraction and are the major limiting factors, with small changes causing significant changes in extraction. The order of the influence factors extracted by the DMPT from the comparison of the p values is: concentration of ethanol aqueous solution>Liquid to feed ratio>Temperature of extraction>And (4) extracting time.
Response surface and contour plots of the interaction effect between the two factors are obtained through Design-Expert 8.0.6.1 software, and the results are shown in fig. 8-13. According to the design principle and analysis of the response surface experiment, the larger the curvature of the three-dimensional response surface graph is, the more obvious the interaction effect between the two factors is, and therefore the influence of the interaction between the factors on the extraction amount of the DMPT can be effectively reflected.
Comparing fig. 8 to fig. 13, it can be seen that the interaction between the concentration of the extractant ethanol aqueous solution and the ultrasonic extraction temperature, the ultrasonic extraction temperature and the feed-liquid ratio has a significant effect on the DMPT extraction, and the interaction between the other two factors has a insignificant effect on the DMPT extraction.
Comparative example 4
The difference from the example 1 is that the D101 macroporous resin column is replaced by AB-8 macroporous adsorption resin, and the effluent of the column passing contains pigment such as fucoxanthin which is not adsorbed, so that the DMPT concentrate has low content and dark color.
Comparative example 5
The difference from example 1 is that the first eluent is 75% ethanol, and fucoxanthin is eluted with loss.
Comparative example 6
The difference from the example 1 is that the second eluent is ethanol, and green pigment such as chlorophyll adsorbed on the column is eluted together, so that the fucoxanthin content in the crude extract is low.
Comparative example 7
The difference from example 1 is that the volume of the collected eluate exceeds 6 times of the column volume, because the fucoxanthin concentration in the eluate at the later stage is low and other impurities continue to elute. In addition, the volume of the collected eluate is increased, the concentration time is prolonged, and partial degradation loss of fucoxanthin is caused, so that the fucoxanthin content in the crude extract is low.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A method for simultaneously extracting a feed additive rich in DMPT and a feed additive rich in fucoxanthin from golden algae is characterized by comprising the following steps:
separating solid from liquid of the algae culture solution in the logarithmic growth phase, collecting algae mud, and freeze-drying to obtain algae powder;
mixing the algae powder with an extracting agent, and carrying out ultrasonic extraction to obtain an extracting solution; the extractant is an ethanol water solution with the volume concentration of 60-75%;
passing the extract through a D101 macroporous resin column to obtain an effluent liquid and an adsorption column;
the effluent liquid is subjected to first concentration to obtain the bait additive rich in DMPT;
eluting the adsorption column to obtain an eluent;
performing second concentration on the eluent to obtain the feed additive rich in fucoxanthin;
the elution comprises the following steps: performing first elution on the adsorption column by using a first eluent to obtain a first eluent and an elution column; performing second elution on the elution column by using a second eluent to obtain an eluent; the first eluent is an ethanol water solution with the volume concentration of 60-70%, and the second eluent is an ethanol water solution with the volume concentration of 90%.
2. The method according to claim 1, wherein the ratio of the algae meal to the extractant is 1 g: (60-100) mL.
3. The method according to claim 1 or 2, wherein the ultrasonic extraction is carried out at an initial temperature of 20-25 ℃ for 40-45 min.
4. The method of claim 3, further comprising, after the ultrasonic extraction: centrifuging the obtained extraction system to obtain an extracting solution; the rotation speed of the centrifugation is 4000r/min, and the time is 12-20 min.
5. The method according to claim 1, wherein the ratio of the volume of the extracting solution passing through the D101 macroporous resin column to the volume of the D101 macroporous resin column is (50-75): 1.
6. the method according to claim 1 or 5, wherein the flow rate of the extracting solution passing through the D101 macroporous resin column is 3-6 mL/min.
7. The method of claim 1, wherein the first and second concentrations are independently rotary evaporation.
8. The method according to claim 1, wherein the amount of the first eluent is 100-150% of the volume of the D101 macroporous resin column; the dosage of the second eluent is 400-600% of the volume of the D101 macroporous resin column.
9. The method according to claim 1, wherein the mass concentration of DMPT in the DMPT-rich bait additive is 17-30%, and the mass concentration of fucoxanthin in the fucoxanthin-rich bait additive is 15-20%.
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