CN113150045A - Method for efficiently extracting and purifying anthocyanin of purple corn cob - Google Patents
Method for efficiently extracting and purifying anthocyanin of purple corn cob Download PDFInfo
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Abstract
The invention discloses a method for efficiently extracting and purifying purple corn cob anthocyanin, which comprises the following steps: (1) pretreating raw materials; (2) and (3) circulating column chromatography extraction: loading the pretreated raw material into a column, adding an extraction reagent for elution, collecting eluent, directly using the eluent with the column volume of 1-1.5 times for subsequent resin adsorption, using the eluent with the column volume of 1-1.5 times for column chromatography extraction of the swollen purple corn cob material of the second batch, and using the eluent with the column volume of 1-1.5 times for column chromatography extraction of the swollen purple corn cob material of the third batch; by analogy, the first eluent with the column volume of 1-1.5 times of that of each batch of the imbibed purple corn axial material is directly used for the subsequent resin adsorption, and the second eluent with the column volume of 1-1.5 times of that of the second eluent and the third eluent with the column volume of 1-1.5 times of that of the subsequent batch are used for the column chromatography extraction of the subsequent batch; (3) separating with macroporous adsorption resin (4) and carrying out post-treatment. The method is simple, low in material cost and high in anthocyanin extraction efficiency, and is suitable for industrial production.
Description
Technical Field
The invention belongs to the technical field of anthocyanin extraction, and particularly relates to a method for efficiently extracting and purifying anthocyanin of purple corn stalks.
Background
Anthocyanins are compounds formed by combining anthocyanidins and sugars, and are widely present in cell sap of flowers, fruits, stems, leaves and root organs of plants, so that the plants are in different colors from red, purple red to blue and the like. It is a natural pigment, safe and nontoxic, has various health care functions for human body, can remove free radicals in the body, resist tumors, cancers and inflammation, inhibit lipid peroxidation and platelet aggregation, prevent diabetes, reduce weight and protect eyesight, and has been applied to the industries of food, health care products, cosmetics, medicines and the like.
The purple corn cob is rich in anthocyanin, however, the existing soaking extraction method needs a large amount of used extraction reagents, has low extraction efficiency and is not suitable for industrial production, so that how to provide the extraction and purification method of the purple corn cob anthocyanin, which has low extraction reagent amount and high extraction efficiency, is a problem to be solved in the field.
Disclosure of Invention
The invention discloses a method for efficiently extracting and purifying purple corn cob anthocyanin, which is suitable for industrial production.
In order to achieve the purpose, the invention adopts the following technical scheme:
the method for efficiently extracting and purifying the purple corn cob anthocyanin comprises the following steps:
(1) pretreatment of raw materials
Cleaning purple corn cob, drying, pulverizing, adding extraction reagent, and soaking to obtain imbibition purple corn cob material;
(2) circulating column chromatography extraction
Loading the swollen purple corn cob material in the step (1) into a column, adding an extraction reagent for elution, collecting eluent, directly using the eluent with the column volume of 1-1.5 times as large as that of the first eluent for subsequent resin adsorption, using the eluent with the column volume of 1-1.5 times as large as that of the second eluent for column chromatography extraction of the swollen purple corn cob material of the second batch, and using the eluent with the column volume of 1-1.5 times as large as that of the third eluent for column chromatography extraction of the swollen purple corn cob material of the third batch;
by analogy, the first eluent with the column volume of 1-1.5 times of that of each batch of the imbibed purple corn axial material is directly used for the subsequent resin adsorption, and the second eluent with the column volume of 1-1.5 times of that of the second eluent and the third eluent with the column volume of 1-1.5 times of that of the subsequent batch are used for the column chromatography extraction of the subsequent batch;
(3) separation by macroporous adsorbent resin
Loading the eluent obtained in the step (2) into a macroporous adsorption resin column, eluting by using distilled water after all eluent flows into the column, and then eluting by using acidic ethanol to obtain anthocyanin eluent;
(4) post-separation treatment
And standing the anthocyanin eluent, filtering or centrifuging, removing precipitates, concentrating and drying to obtain the anthocyanin.
The method comprises the steps of drying and crushing the purple corn cob before extraction, which is beneficial to flowing of a subsequent column chromatography extraction reagent, and soaking the purple corn cob in the extraction reagent to ensure that the purple corn cob is fully imbibed and the extraction efficiency of the purple corn cob can be improved. The eluent is collected in sections and is reused, the extraction rate of anthocyanin is ensured to reach more than 99 percent while the dosage of the extraction reagent is reduced, and the method is suitable for industrial continuous production. Separating with macroporous adsorbent resin, refining, and drying to obtain anthocyanin powder with high anthocyanin recovery rate.
Preferably, in the step (1),
selecting ripe, dried and threshed purple corn cobs, washing surface impurities with clear water, drying at 40-45 ℃ until the water content is 2-5%, and crushing to 40-60 meshes.
If the purple corn cob is crushed to be too fine, the flow of an extraction reagent in the subsequent extraction process is influenced, so that the extraction column pressure is increased; if the purple corn cob is crushed too coarsely, the extraction reagent is difficult to soak, the pretreatment time is long, and the industrial application is not facilitated.
Preferably, in the step (1),
the extraction reagent is ethanol water solution with volume fraction of 40-60%, and pH is 3.0-5.0;
the ratio of the purple corn cob to the extracting reagent material liquid is 1 (2.5-5) g/mL; the soaking time is at least 1 h.
Preferably, in the step (2),
loading the imbibed purple corn cob material and the extraction reagent for soaking into the column; the height of the purple corn shaft material which is imbibed in the column is 10-15 times of the diameter of the column;
when the extraction reagent is added for elution, the liquid level in the column is always higher than the purple corn axis.
Preferably, in the step (3),
the macroporous adsorption resin is S-8 macroporous adsorption resin, and is pretreated before use as follows:
sequentially soaking the macroporous adsorption resin in 95% alcohol, 0.1mol/L hydrochloric acid and 0.1mol/L NaOH for 12h, loading into a chromatographic column, wherein the height of the resin in the column is 10-15 times of the diameter of the column, and fully washing with distilled water until the effluent is no longer turbid.
Preferably, during the sample adding process in the step (3),
loading the sample at a flow rate of 1.5BV/h, wherein the loading volume is not more than 4.5 times of the column volume;
the effluent liquid of the first column volume is distilled water and is used for the pretreatment of macroporous adsorption resin of the next batch;
the effluent from the second column volume is 40-60% ethanol water solution, and is used for the next batch of circulating column chromatography extraction.
Preferably, in the step (3),
the acidic ethanol is an ethanol water solution with the volume fraction of 80 percent, and the pH value is 1.0; the dosage of the acidic ethanol is 2 to 3 times of the column volume, and the elution flow rate is 1.5 BV/h.
The ethanol water solution with the volume fraction of 80 percent is used for macroporous adsorption resin elution, and the subsequent direct standing can remove the residual polysaccharide and protein impurities in the eluent, thereby simplifying the purification process.
Preferably, in the step (4),
sealing anthocyanin eluate, keeping out of the sun, standing at 4 deg.C for 12 hr, and filtering or centrifuging; concentrating the filtrate or supernatant under reduced pressure at 40-45 deg.C to remove ethanol, and vacuum freeze drying or spray drying to obtain anthocyanin.
The reduced pressure concentration temperature is not higher than 45 ℃, otherwise, the degradation rate of anthocyanin is remarkably accelerated, and the loss is more; the ethanol is not easy to be completely removed by reduced pressure distillation at the reduced pressure concentration temperature of less than 40 ℃, and the residual ethanol brings difficulty to the next drying.
Preferably, the vacuum freeze drying is pre-cooling for 2h at-20 ℃, and then freeze drying for 12h at-80 ℃;
the temperature of an air inlet of the spray drying is 145 ℃, and the feeding flow rate is 10 mL/min.
In conclusion, the method is simple, low in material cost and high in anthocyanin extraction efficiency, and is suitable for industrial production.
Drawings
FIG. 1 shows the results of the experiment in example 2;
FIG. 2 shows the results of the experiment in example 3;
FIG. 3 shows the results of the experiment in example 4;
FIG. 4 shows the results of the experiment in example 5;
FIG. 5 shows the results of the experiment in example 6;
FIG. 6 shows the results of the experiment in example 7.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The process of the method for extracting the purple corn cob anthocyanin by the high-efficiency circulating column chromatography comprises the following steps:
(1) pretreatment of raw materials
Selecting ripe, dried and threshed purple corn cobs, washing surface impurities with clear water, drying at 45 ℃ for 12h until the water content is 2-5%, and mechanically shearing and crushing to 40 meshes.
Adding an extraction solvent into the crushed purple corn cobs according to the material-liquid ratio of the dry weight of the purple corn cobs to the extraction solvent (ethanol water solution with volume fraction of 50% and concentrated hydrochloric acid for adjusting the pH value to 4) of 1:2.5g/mL, and soaking for 1h at room temperature to ensure that the purple corn cobs are fully swelled.
(2) Circulating column chromatography extraction
Filling the fully imbibed purple corn shafts obtained in the step (1) and an extraction reagent used for soaking into a column; the height of the purple corn shaft material which is imbibed in the column is 15 times of the diameter of the column;
eluting anthocyanin from purple corn cob in the chromatographic column by adopting an ethanol water solution (pH 4.0) with the volume fraction of 50% at room temperature by adopting a column chromatography elution method, wherein the liquid level in the column is always higher than the purple corn cob during elution, and the elution flow rate is 1.5 BV/h. The eluent is separately collected by taking the column volume as a unit, the first eluent with the column volume of 1.5 times is directly used for the subsequent resin adsorption, the second eluent with the column volume of 1.5 times is used for the column chromatography extraction of the second swollen purple corn axial material, and the third eluent with the column volume of 1.5 times is used for the column chromatography extraction of the third swollen purple corn axial material; the first 1.5 times of column volume eluent of the second swollen purple corn cob material is directly used for the subsequent resin adsorption, the second 1.5 times of column volume eluent is used for the column chromatography extraction of the third swollen purple corn cob material, and the third 1.5 times of column volume eluent is used for the column chromatography extraction of the fourth swollen purple corn cob material; by analogy, the first 1.5 times of column volume eluent of each batch of the imbibed purple corn shaft material is directly used for the subsequent resin adsorption, and the second and the third 1.5 times of column volume eluents are used for the column chromatography extraction of the subsequent batch; through detection and extraction by circulating column chromatography, the extraction rate of anthocyanin in the purple corn cobs reaches 99.05 percent.
(3) Separation by macroporous adsorbent resin
Taking S-8 macroporous adsorption resin, and carrying out pretreatment:
the macroporous absorption resin is respectively soaked for 12 hours by using 95 percent of alcohol, 0.1mol/L hydrochloric acid and 0.1mol/L NaOH in sequence, then is loaded into a chromatographic column, the height of the resin in the chromatographic column is 15 times of the diameter of the chromatographic column, and then is fully washed by using distilled water at the flow rate of 1.5BV/h until the effluent is not turbid any more.
Adding the eluent obtained in the step (2) into the pretreated macroporous adsorption resin column at the flow rate of 1.5BV/h, wherein the sample adding volume is not more than 4.5 times of the volume of the macroporous adsorption resin column, and no air bubbles or faults are generated in the macroporous adsorption resin column during sample adding; and during sample addition, effluent liquid in the column is separately collected by taking the column volume as a unit, the volume of the 1 st column is mainly distilled water and is used for pretreatment of the macroporous adsorption resin of the next batch, and the effluent liquid of the 2 nd to 5.5 th column volumes is mainly 50% ethanol and can be directly used for cyclic column chromatography extraction of the next batch.
And (3) after the eluent obtained in the step (2) completely flows into the column, 2BV of distilled water is used for passing through the resin column at the flow rate of 2BV/h, most of impurities are washed away, and anthocyanin is not lost.
Eluting anthocyanin from the macroporous adsorption resin column by using 3BV of ethanol water solution (pH is 1.0) with volume fraction of 80 percent, eluting at the flow rate of 1.5BV/h, and collecting anthocyanin eluent. In the elution process, anthocyanin moves downwards in a black-purple strip and can be used as a visual index in chromatographic elution. Through detection, the recovery rate of anthocyanin can reach 84.75 percent after the separation of macroporous absorption resin.
(4) Post-separation treatment
Sealing the collected anthocyanin eluate, keeping away from light, placing in a low-temperature refrigerator at 4 deg.C for 12 hr, filtering under reduced pressure or centrifuging to remove precipitate, and concentrating the filtrate or supernatant under reduced pressure at 45 deg.C to remove ethanol.
Pre-cooling the concentrated solution after decompression concentration at-20 deg.C for 2 hr, and vacuum freeze-drying at-80 deg.C for 12 hr to obtain mauve powder; or spray drying at 145 deg.C of air inlet and 10mL/min of feed flow rate to obtain mauve powder.
Tests show that the purity of the anthocyanin powder prepared by the two drying ways can reach more than 86 percent, and the water content is only 3-4.5 percent. Wherein, the vacuum freeze drying consumes long time, but the degradation rate of anthocyanin is lower and is only 3.92 percent; the spray drying process is short in time consumption, can be continuously processed in large batches, and is suitable for industrial production, but because the high-temperature stability of the anthocyanin is poor, the degradation rate of the anthocyanin in the spray drying process reaches 13.57%.
Example 2
The ethanol concentrations of the extraction reagents used in the steps (1) and (2) are verified on the basis of the example 1, the ethanol concentrations are respectively set to be 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100%, and the results of 1-time column volume elution show that the extraction rate of the solvent to the purple corn axial anthocyanin tends to increase and then decrease along with the increase of the ethanol volume fraction, and the extraction rate is the highest when the ethanol volume fraction is 50%.
Example 3
The pH of the extraction reagent used in steps (1) and (2) was confirmed based on example 1, and was set to 1.0, 2.0, 3.0, 4.0, and 5.0, respectively, and the column volume was 1-fold eluted, and as a result, as shown in fig. 2, the extraction yield was the highest when pH4 and a 50% ethanol aqueous solution were used as the extraction solvent.
Example 4
The column diameter-height ratio and the elution volume in the step (2) are verified on the basis of the example 1, and the result is shown in fig. 3, when the ratio of the column diameter to the column bed height is 1:15, only 1 column volume of extraction reagent is needed to enable the extraction rate to reach more than 85%, and the extraction effect is remarkably superior to that of the groups with the ratio of the diameter to the height of 1:10 and 1: 20.
Example 5
The anthocyanin concentration of the first batch of imbibed purple corn cob material eluent is detected on the basis of the example 1, the result is shown in figure 4, the anthocyanin concentration measured by collecting 0.2BV eluent at each point in the figure is rapidly dissolved out along with the increase of the elution volume, the dissolution rate of the anthocyanin in the purple corn cob is gradually slowed down when the elution volume reaches 1.5BV, and the complete extraction of the anthocyanin is realized when the elution volume is 4 BV. Therefore, if the purple corn cob anthocyanin is extracted by adopting column chromatography, the complete extraction of the anthocyanin can be realized only by eluting the eluent with 4 times of column volume. If the extraction is carried out by adopting the circulating column chromatography, the extraction solution with the volume of 1.5 times of the column volume is selected for circulating extraction, and the complete extraction of anthocyanin can be realized when the circulating times reach 3 times.
Example 6
The method of example 1 is used for cyclic column chromatography extraction, and the first 1.5 column volumes of eluents of the three consecutive batches of the imbibed purple corn cob materials are detected, and the result is shown in fig. 5, in the cyclic column chromatography extraction process, the concentration of anthocyanin in the eluents is basically kept in the range of 1.00-1.45mg/mL, the fluctuation range is far smaller than that of the simple column chromatography extraction, and the smaller concentration fluctuation is beneficial to the quality control in the industrial production process.
Example 7
On the basis of example 1, the amount of extraction reagents used for soaking, extracting and eluting a first batch of imbibed purple corn cob materials is adjusted, and a control group for direct soaking and extraction (without column chromatography) is arranged, so that the result is shown in fig. 6, the amount of solvent used in the extraction process can be effectively reduced by adopting column chromatography extraction, the extraction rate can reach more than 90% by adopting column chromatography extraction when the material-liquid ratio is 1:5(g: mL), and if the common solvent is adopted for soaking and extraction, the extraction rate can reach more than 90% only when the material-liquid ratio is 1:20(g: mL).
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to the above-described embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (9)
1. The method for efficiently extracting and purifying purple corn cob anthocyanin is characterized by comprising the following steps:
(1) pretreatment of raw materials
Cleaning purple corn cob, drying, pulverizing, adding extraction reagent, and soaking to obtain imbibition purple corn cob material;
(2) circulating column chromatography extraction
Loading the swollen purple corn cob material in the step (1) into a column, adding an extraction reagent for elution, collecting eluent, directly using the eluent with the column volume of 1-1.5 times as large as that of the first eluent for subsequent resin adsorption, using the eluent with the column volume of 1-1.5 times as large as that of the second eluent for column chromatography extraction of the swollen purple corn cob material of the second batch, and using the eluent with the column volume of 1-1.5 times as large as that of the third eluent for column chromatography extraction of the swollen purple corn cob material of the third batch;
by analogy, the first eluent with the column volume of 1-1.5 times of that of each batch of the imbibed purple corn axial material is directly used for the subsequent resin adsorption, and the second eluent with the column volume of 1-1.5 times of that of the second eluent and the third eluent with the column volume of 1-1.5 times of that of the subsequent batch are used for the column chromatography extraction of the subsequent batch;
(3) separation by macroporous adsorbent resin
Loading the eluent obtained in the step (2) into a macroporous adsorption resin column, eluting by using distilled water after all eluent flows into the column, and then eluting by using acidic ethanol to obtain anthocyanin eluent;
(4) post-separation treatment
And standing the anthocyanin eluent, filtering or centrifuging, removing precipitates, concentrating and drying to obtain the anthocyanin.
2. The method for efficiently extracting and purifying purple corn cob anthocyanin according to claim 1,
in the step (1), the step (c),
selecting ripe, dried and threshed purple corn cobs, washing surface impurities with clear water, drying at 40-45 ℃ until the water content is 2-5%, and crushing to 40-60 meshes.
3. The method for efficiently extracting and purifying purple corn cob anthocyanin according to claim 1,
in the step (1), the step (c),
the extraction reagent is ethanol water solution with volume fraction of 40-60%, and pH is 3.0-5.0;
the ratio of the purple corn cob to the extracting reagent material liquid is 1 (2.5-5) g/mL; the soaking time is at least 1 h.
4. The method for efficiently extracting and purifying purple corn cob anthocyanin according to claim 1,
in the step (2),
loading the imbibed purple corn cob material and the extraction reagent for soaking into the column; the height of the purple corn shaft material which is imbibed in the column is 10-15 times of the diameter of the column;
when the extraction reagent is added for elution, the liquid level in the column is always higher than the purple corn axis.
5. The method for efficiently extracting and purifying purple corn cob anthocyanin according to claim 1,
in the step (3), the step (c),
the macroporous adsorption resin is S-8 macroporous adsorption resin, and is pretreated before use as follows:
sequentially soaking the macroporous adsorption resin in 95% alcohol, 0.1mol/L hydrochloric acid and 0.1mol/L NaOH for 12h, loading into a chromatographic column, wherein the height of the resin in the column is 10-15 times of the diameter of the column, and fully washing with distilled water until the effluent is no longer turbid.
6. The method for efficiently extracting and purifying purple corn cob anthocyanin according to claim 5,
in the process of adding the sample in the step (3),
loading the sample at a flow rate of 1.5BV/h, wherein the loading volume is not more than 4.5 times of the column volume;
the effluent liquid of the first column volume is distilled water and is used for the pretreatment of macroporous adsorption resin of the next batch;
the effluent from the second column volume is 40-60% ethanol water solution, and is used for the next batch of circulating column chromatography extraction.
7. The method for efficiently extracting and purifying purple corn cob anthocyanin according to claim 1,
in the step (3), the step (c),
the acidic ethanol is an ethanol water solution with the volume fraction of 80 percent, and the pH value is 1.0; the dosage of the acidic ethanol is 2 to 3 times of the column volume, and the elution flow rate is 1.5 BV/h.
8. The method for efficiently extracting and purifying purple corn cob anthocyanin according to claim 1,
in the step (4), the step (c),
sealing anthocyanin eluate, keeping out of the sun, standing at 4 deg.C for 12 hr, and filtering or centrifuging; concentrating the filtrate or supernatant under reduced pressure at 40-45 deg.C to remove ethanol, and vacuum freeze drying or spray drying to obtain anthocyanin.
9. The method for efficiently extracting and purifying purple corn cob anthocyanin according to claim 8,
vacuum freeze drying at-20 deg.C for 2 hr, and freeze drying at-80 deg.C for 12 hr;
the temperature of an air inlet of the spray drying is 145 ℃, and the feeding flow rate is 10 mL/min.
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