阅读说明：本技术 一种从植物中提取花色苷的方法 (Method for extracting anthocyanin from plant ) 是由 李丽梅 马艳芳 丁秋艳 鲁恒 李云龙 于 2021-10-14 设计创作，主要内容包括：本发明属于天然产物有效成份的分离提取技术领域,具体涉及一种从植物中提取花色苷的方法；包括如下步骤：S1、回流提取；S2、减压浓缩；S3、糖基化反应；S4、一次过滤：将稀释的发酵液依次经过孔径为150～250目和5～15μm的过滤装置进行过滤,得到粗滤液；S5、二次过滤：将粗滤液用超滤膜及反渗透膜过滤,得到精滤液；S6、喷雾干燥：采用喷雾干燥方法将液体形式的花色苷色素产品制备成粉状形式的产品。该制备方法不仅可以改善花色苷色素产品的溶解性,而且还可提高产品收率。(The invention belongs to the technical field of separation and extraction of active ingredients of natural products, and particularly relates to a method for extracting anthocyanin from plants; the method comprises the following steps: s1, reflux extraction; s2, concentrating under reduced pressure; s3, glycosylation reaction; s4, primary filtration: filtering the diluted fermentation liquor by a filtering device with the aperture of 150-250 meshes and the diameter of 5-15 mu m in sequence to obtain crude filtrate; s5, secondary filtration: filtering the crude filtrate with ultrafiltration membrane and reverse osmosis membrane to obtain fine filtrate; s6, spray drying: the anthocyanin pigment product in liquid form is prepared into a product in powder form by a spray drying method. The preparation method can not only improve the solubility of anthocyanin pigment product, but also improve the product yield.)

1. A method for extracting anthocyanin from plants is characterized in that: the method comprises the following steps:

s1, reflux extraction: carrying out reflux extraction on anthocyanin plant raw materials by using an acidic alcohol solution with the pH value of 3.0-5.0, and collecting an extracting solution;

s2, concentration under reduced pressure: concentrating the collected extracting solution at 40-60 ℃ under reduced pressure until the Baume degree is 4-7 degrees, and obtaining a concentrated solution;

s3, glycosylation reaction: adding glycosyltransferase and a sugar donor into the collected concentrated solution, placing the concentrated solution into a constant-temperature fermentation tank, stirring and reacting for 20-30 hours, collecting fermentation liquor, adding 2-3 times of hot water, stirring and uniformly mixing, and standing at normal temperature for 24-30 hours to obtain diluted fermentation liquor;

s4, primary filtration: filtering the diluted fermentation liquor by a filtering device with the aperture of 150-250 meshes and the diameter of 5-15 mu m in sequence to obtain crude filtrate;

s5, secondary filtration: filtering the crude filtrate with ultrafiltration membrane and reverse osmosis membrane to obtain fine filtrate;

s6, spray drying: the anthocyanin pigment product in liquid form is prepared into a product in powder form by a spray drying method.

2. The method for extracting anthocyanin from plant as claimed in claim 1, wherein the anthocyanin extraction step comprises the following steps: in step S1, the anthocyanin plant material is pulverized to 20-40 mesh before reflux extraction, and the anthocyanin plant is one or more of purple sweet potato, purple cabbage, red heart radish and red rice.

3. The method for extracting anthocyanin from plant as claimed in claim 1, wherein the anthocyanin extraction step comprises the following steps: in step S1, the amount of acidic alcohol solution used in the reflux extraction is 10-15 times of the weight of the anthocyanin raw material, the extraction is carried out at 40-60 ℃ for 50-60 minutes, the acidic alcohol solution is prepared by mixing acid and 15-45 vol% alcohol according to the volume ratio of 1: 1000-5: 1000, and in step S1, the pH adjusting process is as follows: and slowly adding an acid into the alcohol solution while stirring to detect the change condition of the pH value of the solution until the pH value reaches 3.0-5.0, wherein the acid is one of citric acid, hydrochloric acid and phosphoric acid.

4. The method for extracting anthocyanin from plant as claimed in claim 1, wherein the anthocyanin extraction step comprises the following steps: in step S2, the pressure of reduced pressure concentration is set to be 6000-8000 Pa, and the concentration is carried out for 3-5 hours under the condition to reach the required Baume degree of 4-7 degrees, so that the concentrated solution is obtained.

5. The method for extracting anthocyanin from plant as claimed in claim 1, wherein the anthocyanin extraction step comprises the following steps: in step S3, the glycosyltransferase is at least one of transglucosidase, a-glucosidase, β -glucosidase, lactase, and β -galactosidase, the sugar donor is at least one of maltose, glucose, lactose, and galactose, the glycosyltransferase is added in an amount of 0.1 to 0.5 per mill of the initial raw material amount, the sugar donor is added in an amount of 5.0 to 10.0 per mill of the initial raw material amount, the mixture is fermented in a fermentation tank at a temperature of 40 to 55 ℃ for 20 to 30 hours, the fermentation process needs to be continuously stirred, and 2 to 3 times of hot water at a temperature of 75 to 90 ℃ is added to the fermentation broth after the reaction is completed.

6. The method for extracting anthocyanin from plant as claimed in claim 1, wherein the anthocyanin extraction step comprises the following steps: in step S4, the filtering device may be a filtering screen, a filtering bag, a filtering net, a filtering plate, etc., and the filtering aperture of the filtering device is preferably 150-250 mesh and 5-15 μm.

7. The method for extracting anthocyanin from plant as claimed in claim 1, wherein the anthocyanin extraction step comprises the following steps: in step S5, a polypropylene hollow fiber ultrafiltration membrane is adopted, and the particle size of the ultrafiltration membrane is preferably 0.01-0.02 μm.

8. The method for extracting anthocyanin from plant as claimed in claim 1, wherein the anthocyanin extraction step comprises the following steps: in step S6, a spray dryer is used for drying, wherein the air inlet temperature is 160-180 ℃ and the air outlet temperature is 65-80 ℃ under the spray drying condition.

Technical Field

The invention belongs to the technical field of separation and extraction of active ingredients of natural products, and particularly relates to a method for extracting anthocyanin from plants.

Background

Glycosylation is an important late-stage modification reaction in the biosynthesis process of natural products, and many natural products exist in the form of glycoside. Glycosylation not only increases the water solubility of natural products and thus their bioavailability, but also has an extremely important role in the recognition of natural products from targets of action, even though their biological activity and selectivity often depend on the glycosyl groups contained, and thus glycosylation often has a decisive influence on the pharmaceutical potential of natural products. Glycosylation modification has abundant diversity, and the diversity mainly comes from the difference of glycosyl molecular species and sugar chain length, and the difference of glycosidic bond type, glycosylation site and spatial configuration. The glycosylation modification of the diversification in the nature makes the use of genetic engineering or combined biosynthesis technology to reasonably modify the glycosylation process of natural products become an important means and an effective way for creating novel active substances and improving the activity or the drug effect.

The extraction method adopted by the existing anthocyanin pigment has low yield, and the obtained anthocyanin pigment product has poor quality, which greatly influences the application of the product.

Disclosure of Invention

The invention aims to provide a method for extracting anthocyanin from plants, which can improve the solubility of anthocyanin pigment products and can also improve the product yield.

In order to solve the technical problems, the invention adopts the following technical scheme:

a method for extracting anthocyanin from plants comprises the following steps:

s1, reflux extraction: carrying out reflux extraction on anthocyanin plant raw materials by using an acidic alcohol solution with the pH value of 3.0-5.0, and collecting an extracting solution;

s2, concentration under reduced pressure: concentrating the collected extracting solution at 40-60 ℃ under reduced pressure until the Baume degree is 4-7 degrees, and obtaining a concentrated solution;

s3, glycosylation reaction: adding glycosyltransferase and a sugar donor into the collected concentrated solution, placing the concentrated solution into a constant-temperature fermentation tank, stirring and reacting for 20-30 hours, collecting fermentation liquor, adding 2-3 times of hot water, stirring and uniformly mixing, and standing at normal temperature for 24-30 hours to obtain diluted fermentation liquor;

s4, primary filtration: filtering the diluted fermentation liquor by a filtering device with the aperture of 150-250 meshes and the diameter of 5-15 mu m in sequence to obtain crude filtrate;

s5, secondary filtration: filtering the crude filtrate with ultrafiltration membrane and reverse osmosis membrane to obtain fine filtrate;

s6, spray drying: the anthocyanin pigment product in liquid form is prepared into a product in powder form by a spray drying method.

Further, in step S1, the anthocyanin plant material is pulverized to 20-40 mesh before reflux extraction, and the anthocyanin plant is one or more of purple sweet potato, purple cabbage, red heart radish and red rice.

Further, in step S1, the amount of the acidic alcohol solution used in the reflux extraction is 10-15 times of the weight of the anthocyanin raw material, the extraction is carried out at 40-60 ℃ for 50-60 minutes, the acidic alcohol solution is prepared by mixing acid and 15-45 vol% alcohol according to the volume ratio of 1: 1000-5: 1000, and in step S1, the pH adjusting process is as follows: and slowly adding an acid into the alcohol solution while stirring to detect the change condition of the pH value of the solution until the pH value reaches 3.0-5.0, wherein the acid is one of citric acid, hydrochloric acid and phosphoric acid.

Further, in step S2, the pressure of reduced pressure concentration is set to be 6000-8000 Pa, and the concentration is carried out for 3-5 hours under the condition to reach the required Baume degree of 4-7 degrees, so that the concentrated solution is obtained.

Further, in step S3, the glycosyltransferase is at least one of transglucosidase, a-glucosidase, β -glucosidase, lactase, and β -galactosidase, the glycosyl donor is at least one of maltose, glucose, lactose, and galactose, the glycosyltransferase is added in an amount of 0.1 to 0.5 per mill of the initial raw material amount, the glycosyl donor is added in an amount of 5.0 to 10.0 per mill of the initial raw material amount, the mixture is fermented in a fermentation tank at a temperature of 40 to 55 ℃ for 20 to 30 hours, the fermentation process needs to be continuously stirred, and 2 to 3 times of hot water at a temperature of 75 to 90 ℃ is added to the fermentation broth after the reaction is completed.

Further, in step S4, the filtering device may be a filtering screen, a filtering bag, a filtering net, a filtering plate, or other common filtering devices, and the filtering apertures of the filtering devices are preferably 150-250 mesh and 5-15 μm in sequence.

Further, in step S5, a polypropylene hollow fiber ultrafiltration membrane is used, and the particle size of the ultrafiltration membrane is preferably 0.01 to 0.02 μm.

Further, in step S6, a spray dryer is used for drying, wherein the air inlet temperature is 160 to 180 ℃ and the air outlet temperature is 65 to 80 ℃ under the spray drying condition.

Compared with the prior art, the invention has at least one of the following beneficial effects:

reflux-extracting anthocyanin plants with acidic alcohol solution, concentrating the extract under reduced pressure, adding glycosyltransferase and glycosyl donor, placing the mixture into a fermentation tank, stirring at constant temperature for reaction, collecting the reaction solution, diluting and inactivating the reaction solution with hot water, standing the mixture at normal temperature after stirring uniformly, performing coarse filtration, purifying and concentrating the obtained coarse filtrate with an ultrafiltration membrane and a reverse osmosis membrane to obtain anthocyanin pigment product liquid, and preparing the liquid product into a powdery product by adopting a spray drying method. The preparation method can not only improve the solubility of anthocyanin pigment product, but also improve the product yield.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the following embodiments further describe the present invention in detail. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1

1000 kg of purple sweet potato as a raw material was washed, pulverized to 40 mesh, and then extracted under reflux with 15000 kg of acidic alcohol solution (citric acid and 30 vol% alcohol) of pH3.5 at 55 ℃ for 50 minutes to obtain 15400 kg of extract. Concentrating the extractive solution at 60 deg.C under reduced pressure to obtainTo 6000 kg of concentrate. 200 g (0.2 per mill of the initial raw material) of transglucosidase and 5 kg (5 per mill of the initial raw material) of maltose are added into the concentrated solution, then the concentrated solution is placed into a constant-temperature fermentation tank at 55 ℃ for stirring reaction for 24 hours, 12000 kg of hot water at 85 ℃ is added into the collected fermentation liquor, the mixture is stirred and mixed evenly, and then the mixture is placed for 26 hours at normal temperature, so that 18000 kg of diluted fermentation liquor is obtained. The diluted fermentation broth was sequentially filtered through a filter sieve of 250 mesh and a filter bag of 15 μm to obtain 17700 kg of crude filtrate. Filtering the coarse filtrate with hollow fiber ultrafiltration membrane with filtering pore diameter of 0.02 μm to obtain refined filtrate 15000. Filtering the fine filtrate with reverse osmosis membrane with molecular weight cutoff of 1500 to obtain 47 kgThe purple sweet potato pigment liquid is easy to dissolve and good in stability. Re-spray-drying the pigment liquid4.2 kg of purple sweet potato pigment powder product.

Example 2

1000 kg of purple cabbage was used as a raw material, washed, crushed to 30 mesh, extracted under reflux with 15000 kg of acidic alcohol solution (phosphoric acid and 25 vol% alcohol) of pH3.5, and extracted at 50 ℃ for 50 minutes to obtain 15500 kg of extract. The extract was concentrated under reduced pressure at 60 ℃ to obtain 5700 kg of a concentrated solution. Adding 100 g (0.1 per mill of the initial raw material) of alpha-glucosidase and 6 kg (6 per mill of the initial raw material) of glucose into the concentrated solution, placing the concentrated solution into a constant-temperature fermentation tank at 50 ℃ for stirring reaction for 28 hours, collecting fermentation liquor, adding 11500 kg of hot water at 85 ℃, stirring and mixing uniformly, standing the mixture at normal temperature for 28 hours, and obtaining 17000 kg of diluted fermentation liquor. The diluted fermentation broth was sequentially filtered through a filter sieve of 200 mesh and a filter bag of 10 μm to obtain 16800 kg of crude filtrate. And (3) filtering the coarse filtrate by using a hollow fiber ultrafiltration membrane with the filtering pore diameter of 0.01 mu m to obtain 16600 kg of fine filtrate. Filtering the fine filtrate with reverse osmosis membrane with molecular weight cutoff of 1200 to obtain 40 kgThe purple cabbage pigment liquid is easy to dissolve and good in stability. Re-spray-drying the pigment liquid2.4 kg of purple cabbage pigment powder product.

Example 3

1000 kg of red-heart radish as a raw material was washed, crushed to 30 mesh, and subjected to reflux extraction with 15000 kg of an acidic alcohol solution (composed of phosphoric acid and 25 vol% alcohol) having a pH of 3.0 at 50 ℃ for 60 minutes to give 15800 kg of an extract. The extract was concentrated under reduced pressure at 55 ℃ to obtain 5750 kg of a concentrated solution. Adding 100 g of beta-glucosidase, 50 g of lactase (0.15 per thousand of the initial raw material), 4 kg of glucose and 1 kg of lactose (5 per thousand of the initial raw material) into the concentrated solution, placing the concentrated solution into a constant-temperature fermentation tank at 50 ℃, stirring and reacting for 30 hours, collecting fermentation liquor, adding 11600 kg of hot water at 85 ℃, stirring and uniformly mixing, and standing for 30 hours at normal temperature to obtain 17000 kg of diluted fermentation liquor. The diluted fermentation broth was sequentially filtered through a filter sieve having a pore size of 200 mesh and a filter bag having a pore size of 10 μm to obtain 16650 kg of crude filtrate. And (3) filtering the coarse filtrate by using a hollow fiber ultrafiltration membrane with the filtering pore diameter of 0.01 mu m to obtain 16600 kg of fine filtrate. Filtering the fine filtrate with reverse osmosis membrane with molecular weight cutoff of 1200 to obtain 43 kgThe liquid is easy to dissolve and has good stability. Re-spray-drying the pigment liquid3.4 kg of the radish red pigment powder product.

Example 4

1000 kg of red rice is used as a raw material, washed and crushed into 20 meshes, 10000 kg of acidic alcohol solution (composed of hydrochloric acid and 35 vol% alcohol) with pH of 3.0 is used for carrying out reflux extraction on the crushed red rice, and the extraction is carried out for 50 minutes at 45 ℃ to obtain 9900 kg of extracting solution. Will be provided withThe extract was concentrated under reduced pressure at 50 ℃ to give 5000 kg of a concentrated solution. Adding 300 g of beta-galactosidase (0.3 per mill of the initial raw material) and 6 kg of galactose (6 per mill of the initial raw material) into the concentrated solution, placing the concentrated solution into a constant-temperature fermentation tank at 45 ℃, stirring and reacting for 35 hours, collecting the fermentation solution, adding 10000 kg of hot water at 85 ℃, stirring and uniformly mixing, and standing at normal temperature for 30 hours to obtain 15000 kg of diluted fermentation solution. Filtering the diluted fermentation liquor by a filter sieve with the aperture of 250 meshes and a filter bag with the aperture of 15 mu m in sequence to obtain 14800 kg of coarse filtrate. The crude filtrate was filtered with a hollow fiber ultrafiltration membrane having a filtration pore size of 0.02 μm to obtain 14000 kg of a fine filtrate. Filtering the fine filtrate with reverse osmosis membrane with molecular weight cutoff of 1500 to obtain 185 kgThe red rice pigment liquid is easy to dissolve and good in stability. Re-spray-drying the pigment liquid8.7 kg of the radish red pigment powder product.

Comparative example 1

1000 kg of purple sweet potato as a raw material was washed, pulverized to 40 mesh, and then extracted under reflux with 15000 kg of acidic alcohol solution (citric acid and 30 vol% alcohol) of pH3.5 at 55 ℃ for 50 minutes to obtain 15500 kg of extract. The extract was concentrated under reduced pressure at 60 ℃ to give 7000 kg of the concentrate. The concentrated solution was sequentially filtered through a filter sieve having a pore size of 250 mesh and a filter bag having a pore size of 15 μm to obtain 6900 kg of a crude filtrate. And filtering the coarse filtrate with hollow fiber ultrafiltration membrane with filtering pore diameter of 0.02 μm to obtain fine filtrate 6700. Filtering the fine filtrate with reverse osmosis membrane with molecular weight cutoff of 1500 to obtain 50 kgPurple sweet potato pigment liquid. Re-spray-drying the pigment liquid 4.2 kg of purple sweet potato pigment powder product.

Comparative example 2

1000 kg of purple cabbage was used as a raw material, washed, crushed to 30 mesh, and then extracted under reflux with 15000 kg of an acidic alcohol solution (consisting of phosphoric acid and 25 vol% alcohol) having a pH of 3.5 at 50 ℃ for 50 minutes to obtain 15600 kg of an extract. The extract was concentrated under reduced pressure at 60 ℃ to give 7000 kg of the concentrate. The concentrated solution was filtered through a 200-mesh filter sieve and a 10 μm filter bag in this order to obtain 6850 kg of crude filtrate. The crude filtrate was filtered with a hollow fiber ultrafiltration membrane having a filtration pore size of 0.01 μm to obtain 6500 kg of the fine filtrate. Filtering the fine filtrate with reverse osmosis membrane with molecular weight cutoff of 1200 to obtain 50 kgPurple cabbage pigment liquid. Re-spray-drying the pigment liquid2.3 kg of purple cabbage pigment powder product.

Comparative example 3

1000 kg of red-heart radish as a raw material was washed, crushed to 30 mesh, and subjected to reflux extraction with 15000 kg of an acidic alcohol solution (composed of phosphoric acid and 25 vol% alcohol) having a pH of 3.0 at 50 ℃ for 60 minutes to obtain 15700 kg of an extract. The extract was concentrated under reduced pressure at 55 ℃ to give 7400 kg of a concentrated solution. The concentrated solution was filtered through a 200 mesh filter sieve and a 10 μm filter bag in this order to obtain 7300 kg of crude filtrate. And filtering the coarse filtrate by using a hollow fiber ultrafiltration membrane with the filtering pore diameter of 0.01 mu m to obtain 7000 kg of fine filtrate. Filtering the fine filtrate with reverse osmosis membrane with molecular weight cutoff of 1200 to obtain 50 kgAnd (4) liquid of radish red pigment. Re-spray-drying the pigment liquid 3.3 kg of the radish red pigment powder product.

Comparative example 4

1000 kg of red rice is used as a raw material, washed and crushed into 20 meshes, 10000 kg of acidic alcohol solution (composed of hydrochloric acid and 35 vol% alcohol) with pH of 3.0 is used for carrying out reflux extraction on the crushed red rice, and the extraction is carried out for 50 minutes at 45 ℃ to obtain 9900 kg of extracting solution. Concentrating the extractive solution at 50 deg.C under reduced pressure to obtain 5000 kg of concentrated solution. The concentrated solution was sequentially filtered through a filter sieve having a pore size of 250 mesh and a filter bag having a pore size of 15 μm to obtain 4900 kg of a crude filtrate. The crude filtrate was filtered with a hollow fiber ultrafiltration membrane having a filtration pore size of 0.02 μm to obtain 4750 kg of the fine filtrate. Filtering the fine filtrate with reverse osmosis membrane with molecular weight cutoff of 1500 to obtain 186 kgRed rice pigment liquid. Re-spray-drying the pigment liquid8.5 kg of the radish red pigment powder product.

Carrying out experimental analysis;

1. and (3) judging the solubility:

the anthocyanin pigment powders obtained in examples 1 to 4 and comparative examples 1 to 4 were diluted with a buffer solution having a pH of 3.0 and a pH of 5.5And 2, 5 and 10, taking 1000 kg of the same amount of diluent for centrifugal treatment, and weighing the mass of the precipitate after centrifugation.

Note: firstly, the solubility of red rice red is poor under the acidic condition; ② and- "indicate no precipitate.

As can be seen from the above table, the present invention can not only improve the solubility of anthocyanin pigment products.

2. Yield of anthocyanin pigment powder

Note: the anthocyanin pigment obtained by adopting the biological enzyme reaction has insignificant wave crest deviation.

As can be seen from the above table, the process of the present invention can improve the product yield.

Although the invention has been described herein with reference to a number of illustrative embodiments thereof, it should be understood that numerous other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure. More specifically, various variations and modifications are possible in the component parts and/or arrangements of the subject combination arrangement within the scope of the disclosure and claims of this application. In addition to variations and modifications in the component parts and/or arrangements, other uses will also be apparent to those skilled in the art.