阅读说明：本技术 一种可食用刺葡萄愈伤组织细胞花色苷提取纯化的方法 (Method for extracting and purifying anthocyanin of edible vitis davidii callus cells ) 是由 赖呈纯 张静 赖恭梯 潘红 黄贤贵 高慧颖 王�琦 于 2021-06-18 设计创作，主要内容包括：本发明提供一种可食用刺葡萄愈伤组织细胞花色苷提取纯化的方法,首次以刺葡萄愈伤组织细胞为原料提取纯化可食用花色苷,依次通过刺葡萄愈伤组织细胞的获取、刺葡萄愈伤组织细胞的预处理、制备花色苷提取液、花色苷提取液过滤、花色苷提取液浓缩、花色苷浓缩液检测、花色苷纯化和洗脱、及纯化花色苷获取等步骤获取可食用花色苷。本发明具有对刺葡萄愈伤组织细胞花色苷提取效果好、纯度高的显著特征,提取剂采用食品级乙醇与食品级柠檬酸作为混合提取溶剂,溶剂使用量小,无有毒物质残留,具备作为安全性食用花色苷生产的条件,同时采用刺葡萄愈伤组织细胞作为天然花色苷提取材料,该材料具有周年、全天候生产的特性、产量稳定、人为可控性等特点。(The invention provides a method for extracting and purifying anthocyanin from calluses of edible vitis amurensis, which comprises the steps of extracting and purifying the edible anthocyanin by taking the calluses of the vitis amurensis as raw materials for the first time, and sequentially obtaining the edible anthocyanin through the steps of obtaining the calluses of the vitis amurensis, pretreating the calluses of the vitis amurensis, preparing anthocyanin extracting solution, filtering the anthocyanin extracting solution, concentrating the anthocyanin extracting solution, detecting the anthocyanin concentrating solution, purifying and eluting the anthocyanin, obtaining the purified anthocyanin and the like. The method has the remarkable characteristics of good effect of extracting anthocyanin from the calluses of the vitis amurensis, high purity, small solvent usage amount and no toxic substance residue, adopts the food grade ethanol and the food grade citric acid as the mixed extraction solvent for the extracting agent, has the condition for producing safe edible anthocyanin, adopts the calluses of the vitis amurensis as the natural anthocyanin extraction material, and has the characteristics of annual and all-weather production, stable yield, artificial controllability and the like.)

1. A method for extracting and purifying anthocyanin from calluses of edible Vitis davidii is characterized by comprising the following steps: comprises the following steps:

(1) obtaining calluses of the vitis davidii: culturing the calli cells of the red amur grape in a solid culture set for 30-35 days by using a plant tissue culture technology to obtain fully mature calli cells of the amur grape rich in anthocyanin;

(2) pretreatment of vitis davidii callus cells: taking the vitis davidii callus cells obtained in the step (1) out of the culture bottle, removing the culture medium, collecting the vitis davidii callus cells, and drying by using a freeze drying method or a low-temperature vacuum drying method to obtain a dry vitis davidii callus cell product; the product can be stored in a refrigerator at 4 deg.C for a short time, or frozen at-40 deg.C or-80 deg.C for a long time;

(3) preparing an anthocyanin extracting solution: taking the dry vitis davidii callus cell product obtained in the step (2), putting the dry vitis davidii callus cell product into a wall breaking and homogenizing machine, adding 70-75% of frozen and acidified food grade ethanol according to the dosage of 18ml:1g of extracting agent-material ratio of the volume of the extracting liquid to the weight of the dry vitis davidii callus cell product, and homogenizing; placing the homogenized extraction mixed solution into an ultrasonic instrument, and extracting anthocyanin in the calluses of the vitis amurensis for 50-60 min at the temperature of 35-45 ℃ and the power of 300-400W; after the ultrasonic treatment is finished, centrifuging for 10-15 min at the rotation speed of 5000-7000 rpm at 4 ℃ and taking the supernatant; adding proper amount of extractant into the precipitate again according to the above operation to extract anthocyanin, and repeating for 2 times; finally, mixing the supernatants to obtain the vitis amurensis callus cell anthocyanin extracting solution;

(4) filtering an anthocyanin extracting solution: vacuum filtering the vitis davidii callus cell anthocyanin extracting solution obtained in the step (3) through a 0.22-micron organic filter membrane, and obtaining pure vitis davidii callus cell anthocyanin extracting solution after filtering;

(5) concentrating anthocyanin extracting solution: transferring the pure vitis davidii callus cell anthocyanin extracting solution obtained in the step (4) into a rotary evaporation bottle, carrying out vacuum evaporation concentration in a water bath at 35-45 ℃ in the absence of light, and separating ethanol from the anthocyanin extracting solution to obtain vitis davidii callus cell anthocyanin concentrated solution;

(6) detecting an anthocyanin concentrated solution: detecting the mass concentration of the anthocyanin concentrated solution;

(7) anthocyanin purification and elution: according to the mass concentration of the anthocyanin concentrated solution detected in the step (6), diluting the concentration of the anthocyanin concentrated solution to 0.8-1.0 mg/mL by using acidified purified water, and adsorbing and eluting the diluted anthocyanin diluted solution by using pretreated weak-polarity or non-polarity high-molecular porous microspheres to obtain purified eluent containing the vitis davidii callus cell anthocyanin;

(8) obtaining purified anthocyanin: transferring the purified anthocyanin eluent obtained in the step (7) into a rotary evaporation bottle, carrying out vacuum evaporation concentration in a water bath at the temperature of 35-45 ℃ in the dark until ethanol can not be evaporated; and pre-freezing the obtained evaporation concentrated solution, transferring the evaporation concentrated solution into a freeze dryer, and freeze-drying for 48-60 hours at the temperature of minus 45-50 ℃ and the vacuum degree of 15-25 Pa to obtain the purified anthocyanin.

2. The method for extracting and purifying anthocyanin from the callus cells of the edible vitis amurensis as claimed in claim 1, wherein the anthocyanin extraction and purification method comprises the following steps: the solid culture medium is an MS solid culture medium added with 1.0mg/mL2,4-D, 30g/L sugar and 6g/L agar powder.

3. The method for extracting and purifying anthocyanin from the callus cells of the edible vitis amurensis as claimed in claim 1, wherein the anthocyanin extraction and purification method comprises the following steps: in the step (2), the vitis davidii callus cell freeze-drying specifically comprises the following steps: rapidly freezing the collected vitis davidii callus cells for 5-6 min by using liquid nitrogen, transferring the vitis davidii callus cells into a freeze-drying machine, carrying out freeze-drying for 72-80 h at the temperature of-45-50 ℃ and the vacuum degree of 15-25 Pa, and taking out to obtain the dry vitis davidii callus cells.

4. The method for extracting and purifying anthocyanin from the callus cells of the edible vitis amurensis as claimed in claim 1, wherein the anthocyanin extraction and purification method comprises the following steps: in the step (2), the low-temperature vacuum drying of the vitis davidii callus cells comprises the following specific steps: collecting the calluses of the vitis amurensis, drying the calluses of the vitis amurensis by adopting a low-temperature vacuum dryer, setting the temperature to be 35 ℃ and the vacuum degree to be 20-35 Pa, and drying the calluses of the vitis amurensis to constant weight to obtain a dry calluses of the vitis amurensis.

5. The method for extracting and purifying anthocyanin from the callus cells of the edible vitis amurensis as claimed in claim 1, wherein the anthocyanin extraction and purification method comprises the following steps: in the step (3), the preparation method of the food grade ethanol with 70% -75% of frozen acidification comprises the following steps: firstly, preparing a 70-75% food grade ethanol solution according to the volume ratio of purified water to food grade ethanol of 2.5-3: 7-7.5; then preparing 70-75% food grade ethanol solution containing 3-5% citric acid according to the mass volume (w/v) ratio of 3-5: 100, and then placing the solution in a refrigerator at the temperature of-20 ℃ for freezing to obtain the frozen acidified food grade ethanol.

6. The method for extracting and purifying anthocyanin from the callus cells of the edible vitis amurensis as claimed in claim 1, wherein the anthocyanin extraction and purification method comprises the following steps: in the step (3), in the three-time extraction process of anthocyanin, the extraction agent-material ratio is as follows: during the first extraction, the ratio of the volume of the extracting agent to the mass of the calluses of the vitis amurensis is 18mL:1 g; performing secondary extraction on the centrifuged precipitate, wherein the ratio of the volume of the extracting agent to the mass of the calluses of the vitis amurensis is 14mL:1 g; and performing third extraction on the sediment after the centrifugation again, wherein the ratio of the volume of the extracting agent to the mass of the calluses of the vitis amurensis is 10mL to 1 g.

7. The method for extracting and purifying anthocyanin from the callus cells of the edible vitis amurensis as claimed in claim 1, wherein the anthocyanin extraction and purification method comprises the following steps: the anthocyanin concentration detection method in the step (6) is as follows: accurately weighing 50mg of cornflower-3-glucoside standard substance, and dissolving in 1mL of fixed solvent to obtain 50mg/mL of standard solution;

the standard solution was diluted to 50. mu.g/mL and 2-fold diluted to obtain 6 concentration gradients of 50. mu.g/mL, 25. mu.g/mL, 12.5. mu.g/mL, 6.25. mu.g/mL, 3.125. mu.g/mL and 1.5625. mu.g/mL. Adjusting 0 with a fixed solvent as a blank, measuring light absorption values at wavelengths of 530nm, 620nm and 650nm in an enzyme labeling instrument, and using Greey formula OD_λ＝(OD₅₃₀-OD₆₂₀)-0.1(OD₆₅₀-OD₆₂₀) Accurately calculating the light absorption value of the anthocyanin standard substance with each concentration, and drawing a standard curve;

diluting the vitis davidii callus cell anthocyanin concentrated solution to a certain multiple, and determining the light absorption value of the anthocyanin extracting solution according to the determination step established by anthocyanin marker yeast; calculating the mass concentration of anthocyanin in the sample through a cornflower-3-glucoside standard curve;

an anthocyanin content calculation formula: (μ g/g DW) ═ cxv/wxtx dilution factor; wherein, C: obtaining the mass concentration (mu g/mL) of anthocyanin according to the standard koji; v: total reaction volume (mL); w: cell dry weight (g).

8. The method for extracting and purifying anthocyanin from the callus cells of the edible vitis amurensis as claimed in claim 1, wherein the anthocyanin extraction and purification method comprises the following steps: in the step (7), the preparation process of the acidified purified water is as follows: and (3) taking purified water, and adjusting the pH value of the purified water by using a proper amount of citric acid to ensure that the pH value of the purified water is 2.0-3.0.

9. The method for extracting and purifying anthocyanin from the callus cells of the edible vitis amurensis as claimed in claim 1, wherein the anthocyanin extraction and purification method comprises the following steps: in the step (7), the method for pretreating the weak-polarity or non-polarity polymer porous microspheres comprises the following steps: soaking the weak-polarity or non-polarity high-molecular porous microspheres in absolute ethyl alcohol for 24-36 h, fully swelling, filling columns by a wet method, washing with purified water, and washing until no alcohol smell exists; then washing with 0.05-0.1 mol/L hydrochloric acid until the effluent is acidic, soaking for 2-4 h, and washing with ultrapure water until the effluent is neutral; and finally, washing the mixture by using 1.0-1.5 mol/L sodium hydroxide solution until the effluent is alkaline, soaking the mixture for 2-4 hours, and finally washing the mixture by using purified water until the mixture is neutral.

10. The method for extracting and purifying anthocyanin from the callus cells of the edible vitis amurensis as claimed in claim 1, wherein the anthocyanin extraction and purification method comprises the following steps: in the step (7), the adsorption and elution steps are as follows: pumping a diluent of the vitis davidii callus anthocyanin extracting solution into the filled weak-polarity or non-polarity polymer porous microspheres by using a constant flow pump, wherein the flow rate is set to be 3-4 mL per minute; stopping pumping when the diluent is adsorbed to 30-40% of the column body, standing for 30-40 min to ensure that the diluent is fully adsorbed, washing 3-4 column volumes with acidified pure water to fully remove impurities such as sugar, desorbing with 75-80% acidified food grade ethanol containing 3-5% citric acid, wherein the desorption flow rate is set to be 5-6 mL per minute; when the effluent had a red color, the eluate was collected until the color disappeared.

[ technical field ] A method for producing a semiconductor device

The invention belongs to the technical field of extraction and purification of natural plant bioactive substances, and particularly relates to a method for extracting and purifying anthocyanin from an edible vitis davidii callus cell.

[ background of the invention ]

The anthocyanin is a natural edible pigment, and has the characteristics of safety and no toxicity. Although anthocyanins are widely present in natural plants, the dependence on natural plants for extracting anthocyanins has limitations. At present, the extraction of anthocyanin substances of grapes is mainly from grape skin residues in the wine industry, but the grape skin residues have the defects of high sugar content, high protein content, more impurities, high non-toxic separation cost, long time consumption and the like, and the grape skin residues are limited by conditions such as market, climate and the like, so that the development space is limited. With the development of the food industry and the attention of people on food safety, the demand of anthocyanin in the industries of food processing, health products and the like is increasing day by day, and the original source approach is not enough to meet the market demand. Therefore, under the condition of low carbon economy, the natural secondary substances obtained by culturing the vitis amurensis cells have higher feasibility and large-scale production potential. At present, main reports of anthocyanin extraction and purification are concentrated on grape skin residues and the like, and reports of extraction, separation and purification of vitis davidii cell anthocyanin are not seen.

[ summary of the invention ]

The technical problem to be solved by the invention is to provide a method for extracting and purifying anthocyanin from calluses of edible vitis amurensis, the method has good extraction effect and high purity on the anthocyanin from the calluses of the vitis amurensis, an extracting agent adopts food-grade ethanol and food-grade citric acid as a mixed extraction solvent, the usage amount of the solvent is small, no toxic substance is left, and the method has the production condition of safe edible anthocyanin.

The invention is realized by the following steps:

a method for extracting and purifying anthocyanin from calluses of edible Vitis davidii comprises the following steps:

(1) obtaining calluses of the vitis davidii: culturing the calli cells of the red amur grape in a solid culture set for 30-35 days by using a plant tissue culture technology to obtain fully mature calli cells of the amur grape rich in anthocyanin;

(2) pretreatment of vitis davidii callus cells: taking the vitis davidii callus cells obtained in the step (1) out of the culture bottle, removing the culture medium, collecting the vitis davidii callus cells, and drying by using a freeze drying method or a low-temperature vacuum drying method to obtain a dry vitis davidii callus cell product; the product can be stored in a refrigerator at 4 deg.C for a short time, or frozen at-40 deg.C or-80 deg.C for a long time;

(3) preparing an anthocyanin extracting solution: taking the dry vitis davidii callus cell product obtained in the step (2), putting the dry vitis davidii callus cell product into a wall breaking and homogenizing machine, adding 70-75% of frozen and acidified food grade ethanol according to the dosage of 18ml:1g of extracting agent-material ratio of the volume of the extracting liquid to the weight of the dry vitis davidii callus cell product, and homogenizing; placing the homogenized extraction mixed solution into an ultrasonic instrument, and extracting anthocyanin in the calluses of the vitis amurensis for 50-60 min at the temperature of 35-45 ℃ and the power of 300-400W; after the ultrasonic treatment is finished, centrifuging for 10-15 min at the rotation speed of 5000-7000 rpm at 4 ℃ and taking the supernatant; adding proper amount of extractant into the precipitate again according to the above operation to extract anthocyanin, and repeating for 2 times; finally, mixing the supernatants to obtain the vitis amurensis callus cell anthocyanin extracting solution;

(4) filtering an anthocyanin extracting solution: vacuum filtering the vitis davidii callus cell anthocyanin extracting solution obtained in the step (3) through a 0.22-micron organic filter membrane, and obtaining pure vitis davidii callus cell anthocyanin extracting solution after filtering;

(5) concentrating anthocyanin extracting solution: transferring the pure vitis davidii callus cell anthocyanin extracting solution obtained in the step (4) into a rotary evaporation bottle, carrying out vacuum evaporation concentration in a water bath at 35-45 ℃ in the absence of light, and separating ethanol from the anthocyanin extracting solution to obtain vitis davidii callus cell anthocyanin concentrated solution;

(6) detecting an anthocyanin concentrated solution: detecting the mass concentration of the anthocyanin concentrated solution;

(7) anthocyanin purification and elution: according to the mass concentration of the anthocyanin concentrated solution detected in the step (6), diluting the concentration of the anthocyanin concentrated solution to 0.8-1.0 mg/mL by using acidified purified water, and adsorbing and eluting the diluted anthocyanin diluted solution by using pretreated weak-polarity or non-polarity high-molecular porous microspheres to obtain purified eluent containing the vitis davidii callus cell anthocyanin;

(8) obtaining purified anthocyanin: transferring the purified anthocyanin eluent obtained in the step (7) into a rotary evaporation bottle, carrying out vacuum evaporation concentration in a water bath at the temperature of 35-45 ℃ in the dark until ethanol can not be evaporated; and pre-freezing the obtained evaporation concentrated solution, transferring the evaporation concentrated solution into a freeze dryer, and freeze-drying for 48-60 hours at the temperature of minus 45-50 ℃ and the vacuum degree of 15-25 Pa to obtain the purified anthocyanin.

Furthermore, the solid culture medium is MS solid culture medium added with 1.0mg/mL2,4-D, 30g/L sugar and 6g/L agar powder.

Further, in the step (2), the vitis davidii callus cells are freeze-dried by the following specific steps: rapidly freezing the collected vitis davidii callus cells for 5-6 min by using liquid nitrogen, transferring the vitis davidii callus cells into a freeze-drying machine, carrying out freeze-drying for 72-80 h at the temperature of-45-50 ℃ and the vacuum degree of 15-25 Pa, and taking out to obtain the dry vitis davidii callus cells.

Further, in the step (2), the low-temperature vacuum drying of the vitis davidii callus cells comprises the following specific steps: collecting the calluses of the vitis amurensis, drying the calluses of the vitis amurensis by adopting a low-temperature vacuum dryer, setting the temperature to be 35 ℃ and the vacuum degree to be 20-35 Pa, and drying the calluses of the vitis amurensis to constant weight to obtain a dry calluses of the vitis amurensis.

Further, in the step (3), the preparation method of the food grade ethanol with 70% -75% of frozen acidification comprises the following steps: firstly, preparing a 70-75% food grade ethanol solution according to the volume ratio of purified water to food grade ethanol of 2.5-3: 7-7.5; then preparing a 70-75% food grade ethanol solution containing 3-5% citric acid according to the mass volume fraction ratio of 3-5: 100(w/v), and then placing the solution in a refrigerator at the temperature of-20 ℃ for freezing to obtain the frozen acidified food grade ethanol.

Further, in the step (3), in the three-time extraction process of anthocyanin, the ratio of the extracting agents is as follows: during the first extraction, the ratio of the volume of the extracting agent to the mass of the calluses of the vitis amurensis is 18mL:1 g; performing secondary extraction on the centrifuged precipitate, wherein the ratio of the volume of the extracting agent to the mass of the calluses of the vitis amurensis is 14mL:1 g; and performing third extraction on the sediment after the centrifugation again, wherein the ratio of the volume of the extracting agent to the mass of the calluses of the vitis amurensis is 10mL to 1 g.

Further, the anthocyanin concentration detection method in the step (6) is as follows: accurately weighing 50mg of cornflower-3-glucoside standard substance, and dissolving in 1mL of fixed solvent to obtain 50mg/mL of standard solution;

the standard solution was diluted to 50. mu.g/mL and 2-fold diluted to obtain 6 concentration gradients of 50. mu.g/mL, 25. mu.g/mL, 12.5. mu.g/mL, 6.25. mu.g/mL, 3.125. mu.g/mL and 1.5625. mu.g/mL. Adjusting 0 with a fixed solvent as a blank, measuring light absorption values at wavelengths of 530nm, 620nm and 650nm in an enzyme labeling instrument, and using Greey formula OD_λ＝(OD₅₃₀-OD₆₂₀)-0.1(OD₆₅₀-OD₆₂₀) Accurately calculating the light absorption value of the anthocyanin standard substance with each concentration, and drawing a standard curve;

diluting the vitis davidii callus cell anthocyanin concentrated solution to a certain multiple, and determining the light absorption value of the anthocyanin extracting solution according to the determination step established by anthocyanin marker yeast; calculating the mass concentration of anthocyanin in the sample through a cornflower-3-glucoside standard curve;

an anthocyanin content calculation formula: (μ g/gDW) ═ cxv/wxtx dilution factor; wherein, C: obtaining the mass concentration (mu g/mL) of anthocyanin according to the standard koji; v: total reaction volume (mL); w: cell dry weight (g).

Further, in the step (7), the acidified purified water is prepared as follows: and (3) taking purified water, and adjusting the pH value of the purified water by using a proper amount of citric acid to ensure that the pH value of the purified water is 2.0-3.0.

Further, in the step (7), the method of the pretreated weakly polar or non-polar polymer porous microspheres includes the following steps: soaking the weak-polarity or non-polarity high-molecular porous microspheres in absolute ethyl alcohol for 24-36 h, fully swelling, filling columns by a wet method, washing with purified water, and washing until no alcohol smell exists; then washing with 0.05-0.1 mol/L hydrochloric acid until the effluent is acidic, soaking for 2-4 h, and washing with ultrapure water until the effluent is neutral; and finally, washing the mixture by using 1.0-1.5 mol/L sodium hydroxide solution until the effluent is alkaline, soaking the mixture for 2-4 hours, and finally washing the mixture by using purified water until the mixture is neutral.

Further, in the step (7), the adsorption and elution steps are as follows: pumping a diluent of the vitis davidii callus anthocyanin extracting solution into the filled weak-polarity or non-polarity polymer porous microspheres by using a constant flow pump, wherein the flow rate is set to be 3-4 mL per minute; stopping pumping when the diluent is adsorbed to 30-40% of the column body, standing for 30-40 min to ensure that the diluent is fully adsorbed, washing 3-4 column volumes with acidified pure water to fully remove impurities such as sugar, desorbing with 75-80% acidified food grade ethanol containing 3-5% citric acid, wherein the desorption flow rate is set to be 5-6 mL per minute; when the effluent had a red color, the eluate was collected until the color disappeared.

The invention has the following advantages:

the calli cells of the red spine grape adopted by the invention have good stability, vigorous growth and high anthocyanin yield; the method has high extraction efficiency and high purity of the vitis davidii callus cell anthocyanin, the extracting agent adopts food-grade ethanol and food-grade citric acid as a mixed extraction solvent, the usage amount of the solvent is small, no toxic substance is left, the method has the production condition of safe edible anthocyanin, and the method has wide application prospect in the industries of food, medicine, cosmetics and the like.

The method is different from the traditional method for extracting and purifying anthocyanin by using the vitis amurensis peels, and has the advantages that impurities such as sugar, protein and the like are less than the peels, anthocyanin substances are easy to separate, and the purity is high; the method is rapid and convenient, short in extraction time, high in purification yield, simple in extraction equipment and low in cost; the obtained purified anthocyanin extract has strong oxidation resistance; the extracting agent adopts food grade ethanol and food grade citric acid as mixed extracting solvent, the using amount of the solvent is small, no toxic substance residue exists, the extracting agent has the production condition of safe edible anthocyanin, and the extracting agent has wide application prospect in the industries of food, medicine, cosmetics and the like.

[ detailed description ] embodiments

The technical solution of the present invention will be clearly and completely described with reference to the following detailed description. 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. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1:

1. culture and acquisition of vitis davidii callus cells

By utilizing a plant tissue culture technology, culturing the calli cells of the red amur grape in an MS solid culture medium added with 1.0mg/mL2,4-D, 30g/L sugar and 6g/L agar powder for 30-35 days to obtain fully mature calli cells of the amur grape rich in anthocyanin. The calluses of the vitis davidii have high water content and need to be prepared into dry products to facilitate the extraction of anthocyanin.

2. Pretreatment of vitis davidii callus cells

And taking the obtained vitis davidii callus cells out of the culture bottle, removing the culture medium, and collecting to obtain a vitis davidii callus cell fresh sample. And (3) rapidly freezing the collected vitis davidii callus cells for 5-6 min by using liquid nitrogen, transferring the vitis davidii callus cells into a freeze dryer, carrying out freeze drying for 72h at the temperature of-45-50 ℃ and the vacuum degree of 15Pa, and taking out to obtain a vitis davidii callus cell dry product. The product can be stored in refrigerator at 4 deg.C for short time, or frozen at-40 deg.C or-80 deg.C for long time.

3. Frozen acidified ethanol preparation

Firstly, preparing 70-75% of food grade ethanol solution (the volume ratio of purified water to ethanol is 2.5-3: 7-7.5), then preparing 70-75% of food grade ethanol solution containing 3-5% of citric acid according to the mass volume fraction ratio of 3:100, and then placing the solution in a refrigerator at the temperature of-20 ℃ for freezing to obtain the frozen acidified food grade ethanol. The ethanol and citric acid are food grade products, as follows.

4. Preparation of anthocyanin extract

Taking dry vitis davidii callus cells, putting the dry vitis davidii callus cells into a wall breaking and homogenizing machine, adding 70-75% of frozen and acidified food grade ethanol according to the extractant-material ratio of 18:1 (volume of extracting solution (mL): weight (g)) and homogenizing; placing the homogenized extraction mixed solution in an ultrasonic instrument, and extracting anthocyanin in the calluses of the Vitis davidii for 50min at 35 ℃ and 400W of power; centrifuging at 4 deg.C and 7000rpm for 12min after the ultrasound is finished, and collecting the supernatant; adding proper amount of extractant into the precipitate again according to the above operation to extract anthocyanin, repeating for 2 times, and specifically performing the following steps: performing secondary extraction on the centrifuged precipitate, wherein the ratio of the volume of the extracting agent to the mass of the calluses of the vitis amurensis is 14: 1; and performing third extraction on the sediment after the centrifugation again, wherein the ratio of the volume of the extracting agent to the mass of the calluses of the vitis amurensis is 10: 1. And finally, mixing the supernatants to obtain the calli cell anthocyanin extracting solution of the vitis davidii. The method can not only fully extract anthocyanin in the calluses of the vitis amurensis, but also greatly save the extraction cost.

5. Filtration of anthocyanin extract

The vitis davidii callus cell anthocyanin extracting solution is subjected to vacuum filtration through a 0.22 mu m organic filter membrane to remove substances such as cell disruption residues, macromolecular proteins and the like, reduce the influence on later-stage purification and increase the purity of purified anthocyanin. And (4) after suction filtration, obtaining a pure vitis davidii callus cell anthocyanin extracting solution.

6. Concentration of anthocyanin extract

And (4) transferring the pure vitis davidii callus cell anthocyanin extracting solution obtained in the step (4) into a rotary evaporation bottle, carrying out vacuum evaporation concentration in a water bath at 35 ℃ in the dark, and separating ethanol from the anthocyanin extracting solution to obtain the vitis davidii callus cell anthocyanin concentrated solution.

7. Detection of anthocyanin content in anthocyanin concentrated solution

(1) Drawing an anthocyanin standard curve

Accurately weighing 50mg of cornflower-3-glucoside standard substance, and dissolving in 1mL of cosolvent to obtain 50mg/mL of standard solution. The standard solution was diluted to 50. mu.g/mL and 2-fold diluted to obtain 6 concentration gradients of 50. mu.g/mL, 25. mu.g/mL, 12.5. mu.g/mL, 6.25. mu.g/mL, 3.125. mu.g/mL and 1.5625. mu.g/mL. Adjusting 0 with a fixed solvent as a blank, measuring light absorption values at wavelengths of 530nm, 620nm and 650nm in an enzyme labeling instrument, and using Greey formula OD_λ＝(OD₅₃₀-OD₆₂₀)-0.1(OD₆₅₀-OD₆₂₀) And accurately calculating the light absorption value of the anthocyanin standard substance with each concentration, and drawing a standard curve.

(2) Anthocyanin content detection

Diluting the vitis davidii callus cell anthocyanin concentrated solution to a certain multiple, and determining the light absorption value of the anthocyanin extracting solution according to the determination steps established by anthocyanin marker yeast. And calculating the mass concentration of the anthocyanin in the sample through a cornflower-3-glucoside standard curve. The anthocyanin content calculation formula (μ g/gDW) is cxv/wx dilution factor. C: obtaining the mass concentration (mu g/mL) of anthocyanin according to the standard koji; v: total reaction volume (mL); w: cell dry weight (g).

8. Anthocyanin purification and elution

(1) Preparation of acidified pure water

And (3) taking purified water, and adjusting the pH value of the purified water by using a proper amount of citric acid to ensure that the pH value of the purified water is 2.0-3.0.

(2) Pretreatment of weak-polarity or non-polarity high-molecular porous microspheres

And (3) soaking the weak-polarity or non-polarity high-molecular porous microspheres in absolute ethyl alcohol for 24-36 h, fully swelling, filling the microspheres into a column by a wet method, washing the column by purified water, and washing the column until no alcohol smell exists. Then washing with 0.05-0.1 mol/L hydrochloric acid until the effluent is acidic, soaking for 2-4 h, and washing with ultrapure water until the effluent is neutral; and finally, washing the mixture by using 1.0-1.5 mol/L sodium hydroxide solution until the effluent is alkaline, soaking the mixture for 2-4 hours, and finally washing the mixture by using purified water until the mixture is neutral.

(2) Purification and elution

And (3) diluting the concentration of the anthocyanin concentrated solution to 0.8-1.0 mg/mL by using acidified purified water according to the mass concentration of the anthocyanin concentrated solution. Pumping a diluent of the vitis davidii callus cell anthocyanin extracting solution into the filled weak-polarity or non-polarity polymer porous microspheres by using a constant flow pump, wherein the flow rate is set to be 3-4 mL per minute; and stopping pumping when the diluent is adsorbed to 30-40% of the column body, standing for 30-40 min to ensure that the diluent is fully adsorbed, washing 3-4 column volumes with acidified pure water, and fully removing impurities such as sugar. And desorbing with 75-80% acidified food grade ethanol (containing 3-5% citric acid) at a desorption flow rate of 5-6 mL/min. When the effluent had a red color, the eluate was collected until the color disappeared.

9. Obtaining dry product of purified anthocyanin

Transferring the obtained purified anthocyanin eluent into a rotary evaporation bottle, carrying out vacuum evaporation concentration in a water bath at 35 ℃ in the dark until ethanol can not be evaporated; and pre-freezing the obtained evaporation concentrated solution, transferring into a freeze dryer, and freeze-drying for 48 hours at the temperature of minus 45 ℃ and under the vacuum degree of 15PaPa to obtain the purified anthocyanin. The extraction rate of the vitis davidii callus cell purified anthocyanin is 1.8 percent, and the purity is 95.4 percent.

10. Antioxidant capacity of purified anthocyanin dry product

The anthocyanin of the Vitis davidii prepared by the method has strong oxidation resistance which is 176.89U/g, is far higher than that of like products, and is 15-20 times of Vc oxidation resistance.

Example 2:

1. culture and acquisition of vitis davidii callus cells

By utilizing a plant tissue culture technology, culturing the calli cells of the red amur grape in an MS solid culture medium added with 1.0mg/mL2,4-D, 30g/L sugar and 6g/L agar powder for 30-35 days to obtain fully mature calli cells of the amur grape rich in anthocyanin. The calluses of the vitis davidii have high water content and need to be prepared into dry products to facilitate the extraction of anthocyanin.

2. Pretreatment of vitis davidii callus cells

And taking the obtained vitis davidii callus cells out of the culture bottle, removing the culture medium, and collecting to obtain a vitis davidii callus cell fresh sample. Collecting the vitis davidii callus cells, drying the vitis davidii callus cells by adopting a low-temperature vacuum dryer, setting the temperature at 45 ℃ and the vacuum degree at 35Pa, and drying the vitis davidii callus cells to constant weight to obtain a vitis davidii callus cell dry product. The product can be stored in refrigerator at 4 deg.C for short time, or frozen at-40 deg.C or-80 deg.C for long time.

3. Frozen acidified ethanol preparation

Firstly, preparing 70-75% of food grade ethanol solution (the volume ratio of purified water to ethanol is 2.5-3: 7-7.5), then preparing 70-75% of food grade ethanol solution containing 5% of citric acid according to the mass volume ratio of 5:100, and then placing the solution in a refrigerator at the temperature of-20 ℃ for freezing to obtain the frozen and acidified food grade ethanol. The ethanol and citric acid are food grade products, as follows.

4. Preparation of anthocyanin extract

Taking dry vitis davidii callus cells, putting the dry vitis davidii callus cells into a wall breaking and homogenizing machine, adding 70-75% of frozen and acidified food grade ethanol according to the extractant-material ratio of 18:1 (volume of extracting solution (mL): weight (g)) and homogenizing; placing the homogenized extraction mixed solution in an ultrasonic instrument, and extracting anthocyanin in the calluses of the Vitis davidii for 60min at 45 ℃ and 300W of power; centrifuging at 4 deg.C and 5000rpm for 10min after the ultrasound is finished, and collecting supernatant; adding proper amount of extractant into the precipitate again according to the above operation to extract anthocyanin, repeating for 2 times, and specifically performing the following steps: performing secondary extraction on the centrifuged precipitate, wherein the ratio of the volume of the extracting agent to the mass of the calluses of the vitis amurensis is 14: 1; and performing third extraction on the sediment after the centrifugation again, wherein the ratio of the volume of the extracting agent to the mass of the calluses of the vitis amurensis is 10: 1. And finally, mixing the supernatants to obtain the calli cell anthocyanin extracting solution of the vitis davidii. The method can not only fully extract anthocyanin in the calluses of the vitis amurensis, but also greatly save the extraction cost.

5. Filtration of anthocyanin extract

The vitis davidii callus cell anthocyanin extracting solution is subjected to vacuum filtration through a 0.22 mu m organic filter membrane to remove substances such as cell disruption residues, macromolecular proteins and the like, reduce the influence on later-stage purification and increase the purity of purified anthocyanin. And (4) after suction filtration, obtaining a pure vitis davidii callus cell anthocyanin extracting solution.

6. Concentration of anthocyanin extract

And (4) transferring the pure vitis davidii callus cell anthocyanin extracting solution obtained in the step (4) into a rotary evaporation bottle, carrying out vacuum evaporation concentration in a water bath at 45 ℃ in the dark, and separating ethanol from the anthocyanin extracting solution to obtain the vitis davidii callus cell anthocyanin concentrated solution.

7. Detection of anthocyanin content in anthocyanin concentrated solution

(1) Drawing an anthocyanin standard curve

Accurately weighing 50mg of cornflower-3-glucoside standard substance, and dissolving in 1mL of cosolvent to obtain 50mg/mL of standard solution. The standard solution was diluted to 50. mu.g/mL and 2-fold diluted to obtain 6 concentration gradients of 50. mu.g/mL, 25. mu.g/mL, 12.5. mu.g/mL, 6.25. mu.g/mL, 3.125. mu.g/mL and 1.5625. mu.g/mL. Adjusting 0 with a fixed solvent as a blank, measuring light absorption values at wavelengths of 530nm, 620nm and 650nm in an enzyme labeling instrument, and using Greey formula OD_λ＝(OD₅₃₀-OD₆₂₀)-0.1(OD₆₅₀-OD₆₂₀) And accurately calculating the light absorption value of the anthocyanin standard substance with each concentration, and drawing a standard curve.

(2) Anthocyanin content detection

Diluting the vitis davidii callus cell anthocyanin concentrated solution to a certain multiple, and determining the light absorption value of the anthocyanin extracting solution according to the determination steps established by anthocyanin marker yeast. And calculating the mass concentration of the anthocyanin in the sample through a cornflower-3-glucoside standard curve. The anthocyanin content calculation formula (μ g/gDW) is cxv/wx dilution factor. C: obtaining the mass concentration (mu g/mL) of anthocyanin according to the standard koji; v: total reaction volume (mL); w: cell dry weight (g).

8. Anthocyanin purification and elution

(1) Preparation of acidified pure water

And (3) taking purified water, and adjusting the pH value of the purified water by using a proper amount of citric acid to ensure that the pH value of the purified water is 2.0-3.0.

(2) Pretreatment of weak-polarity or non-polarity high-molecular porous microspheres

And (3) soaking the weak-polarity or non-polarity high-molecular porous microspheres in absolute ethyl alcohol for 24-36 h, fully swelling, filling the microspheres into a column by a wet method, washing the column by purified water, and washing the column until no alcohol smell exists. Then washing with 0.05-0.1 mol/L hydrochloric acid until the effluent is acidic, soaking for 2-4 h, and washing with ultrapure water until the effluent is neutral; and finally, washing the mixture by using 1.0-1.5 mol/L sodium hydroxide solution until the effluent is alkaline, soaking the mixture for 2-4 hours, and finally washing the mixture by using purified water until the mixture is neutral.

(2) Purification and elution

And (3) diluting the concentration of the anthocyanin concentrated solution to 0.8-1.0 mg/mL by using acidified purified water according to the mass concentration of the anthocyanin concentrated solution. Pumping a diluent of the vitis davidii callus cell anthocyanin extracting solution into the filled weak-polarity or non-polarity polymer porous microspheres by using a constant flow pump, wherein the flow rate is set to be 3-4 mL per minute; and stopping pumping when the diluent is adsorbed to 30-40% of the column body, standing for 30-40 min to ensure that the diluent is fully adsorbed, washing 3-4 column volumes with acidified pure water, and fully removing impurities such as sugar. And desorbing with 75-80% acidified food grade ethanol (containing 3-5% citric acid) at a desorption flow rate of 5-6 mL/min. When the effluent had a red color, the eluate was collected until the color disappeared.

9. Obtaining dry product of purified anthocyanin

Transferring the obtained purified anthocyanin eluent into a rotary evaporation bottle, carrying out vacuum evaporation concentration in a water bath at 45 ℃ in a dark place until ethanol can not be evaporated; and pre-freezing the obtained evaporation concentrated solution, transferring the evaporation concentrated solution into a freeze dryer, and freeze-drying for 48-60 hours at the temperature of minus 45-50 ℃ and the vacuum degree of 15-25 Pa to obtain the purified anthocyanin. The extraction rate of the vitis davidii callus cell purified anthocyanin is 1.2%, and the purity is 93.8%.

10. Antioxidant capacity of purified anthocyanin dry product

The anthocyanin of the Vitis davidii prepared by the method has strong oxidation resistance which is 150.55U/g, is far higher than that of like products, and is 15-20 times of Vc oxidation resistance.

Although specific embodiments of the invention have been described above, it will be understood by those skilled in the art that the specific embodiments described are illustrative only and are not limiting upon the scope of the invention, and that equivalent modifications and variations can be made by those skilled in the art without departing from the spirit of the invention, which is to be limited only by the appended claims.