METHOD FOR EFFICIENT EXTRACTION OF PLANT ANTHOCYANIDIN

Abstract

The present invention relates to the technical field of bioengineering and provides a method for efficient extraction of plant anthocyanidin. The method comprises steps of: hydrolyzing anthocyanin; using one of sodium bicarbonate or potassium bicarbonate NaHCO.sub.3/KHCO.sub.3 to neutralize an aqueous solution obtained after the anthocyanin hydrolysis to pH 7; using a saturated aqueous solution of sodium chloride or potassium chloride NaCl/Kcl to perform salting out for suspended separation of a conjugate of an anthocyanidin sodium salt or potassium salt and a fatty acid or fatty acid ester; allowing aqueous hydrochloric acid to react with the anthocyanidin sodium salt or potassium salt; and filtering and separating an anthocyanidin aqueous solution. The present invention is applied to the extraction of natural plant anthocyanidin.

Claims

1. A method for efficient extraction of plant anthocyanidin, comprising: 1). hydrolyzing a natural plant anthocyanin aqueous solution with β-glucosidase, preventing oxidation by using L-cysteine, protecting the activity of β-glucosidase; wherein the hydrolysis is carried out at a temperature of 45-50° C. for 24-48 hours with pH of 4.5-5;an aqueous solution containing anthocyanidins/anthocyanins/glucose is obtained, and the aqueous solution contains oil-soluble fatty acid or fatty acid impurities which are dispersed in an acidic aqueous solution and have a large total surface area and an absorption surface area and have a relative density of less than 1; the fatty acid or fatty acid impurities refer to oleic acid or/and palmitic acid esters, an appropriate amount of oleic acid or/and palmitic acid esters are added to an anthocyanin aqueous solution with low oleic acid or/and palmitic acid content to perform suspended separation of an anthocyanidin sodium salt or potassium salt in an anthocyanin/glucose aqueous solution in the following process 3); 2) using one of sodium bicarbonate or potassium bicarbonate NaHCO.sub.3/KHCO.sub.3 to neutralize the pH of an aqueous anthocyanin solution obtained in step 1) to 6.8-7, and allowing anthocyanidins to produce sodium salts or potassium salts thereof; 3) adding the above 2) to sodium chloride or potassium chloride NaCl/Kcl to formulate saline saturated aqueous solution of an aqueous solution containing anthocyanidin sodium salts or potassium salts/anthocyanins/glucose in 2); the formulation method is as follows: in order to save energy, when the temperature is in the range of 25-50° C., the saturated solubility of sodium chloride or potassium chloride Nacl/Kcl in water at a certain temperature is used as a reference value, and sodium chloride or potassium chloride Nacl/Kcl at the reference value is added to and is dissolved in the aqueous solution containing anthocyanidins/anthocyanins/glucose, allowing the aqueous solution to stand for 1 to 3 minutes, to precipitate and separate the excess sodium chloride or potassium chloride immediately; an aqueous solution containing anthocyanidin sodium salts or potassium salts/anthocyanins/glucose is taken out and allowed to sit for 3 to 8 hours, such that the fatty acid or fatty acid esters entrap, absorb or physically bind to a water-insoluble conjugate of an anthocyanidin sodium salt or potassium salt, and the conjugate is a solid water-insoluble matter due to its relative density is less than the relative density of a saturated aqueous solution of sodium chloride or potassium chloride NaCl/Kcl, then a densely suspended separation of the conjugate is achieved; the conjugate is then separated from the aqueous solution of anthocyanins and glucose and sodium chloride or potassium chloride; then a conjugate of an anthocyanidin sodium salt or potassium salt and a fatty acid or fatty acid ester is taken out in layers, the moisture in the conjugate is filtered, and water-soluble anthocyanin, glucose and liquid oleic acid esters are also filtered; finally, a conjugate of an anthocyanidin sodium salt or potassium salt and palmitic acid ester; 4) allowing slightly excess aqueous hydrochloric acid to react with the anthocyanidin sodium salt or potassium salt in palmitic acid ester in step 3) by stirring at room temperature, and strong acid generates weak acid, and an aqueous hydrochloric acid solution of hydrogen H reduced anthocyanidin and sodium chloride or potassium chloride is produced, and anthocyanidins are completely dissolved in the aqueous hydrochloric acid solution; the palmitic acid ester is insoluble in the aqueous hydrochloric acid Hcl solution, and it is not reacted with the aqueous hydrochloric acid Hcl solution; sodium chloride or potassium chloride does not react with the aqueous hydrochloric acid Hcl solution; sodium chloride or potassium chloride does not react with the anthocyanidin,weak acid anthocyanidin cannot generate strong acid hydrochloric acid; the anthocyanidin-containing aqueous hydrochloric acid Hcl solution is more acidic, and the pH value of the aqueous solution is below 2; people are not allowed to take it directly; the present invention selects and utilizes important properties that the suspending agent palmitic acid or/and oleic acid ester is insoluble in and does not react with the aqueous hydrochloric acid Hcl solution, the anthocyanidin sodium salt or potassium salt is reduced to an aqueous hydrochloric acid solution of anthocyanidin; 5) filtering the aqueous hydrochloric acid solution of anthocyanidins containing the palmitic acid ester in the above 4) with a compact acid-resistant industrial filter cloth under pressure or vacuum decompression at room temperature to separate the palmitic acid ester; since the palmitic acid ester is a palmitic acid solid, an amorphous substance, it can not be filtered with a filter screen; the aqueous hydrochloric acid Hcl solution of anthocyanidins is obtained; the aqueous hydrochloric acid solution containing anthocyanidins is neutralized by using an aqueous solution of sodium bicarbonate or potassium bicarbonate NaHCO.sub.3/KHCO.sub.3, so that its acidic pH is adjusted to 3.5-4; when the aqueous solution containing anthocyanidins is neutralized by using the aqueous solution of sodium bicarbonate or potassium bicarbonate NaHCO.sub.3/KHCO.sub.3, the aqueous solution of sodium bicarbonate or potassium bicarbonate NaHCO.sub.3/KHCO.sub.3 must be added slowly for several times and it must be stirred quickly at a stirring speed of 300-500 rpm, so that sodium bicarbonate or potassium bicarbonate is evenly distributed in the aqueous hydrochloric acid solution containing anthocyanidins; an appropriate amount of ascorbic acid (i.e., vitamin C) is added to the aqueous hydrochloric acid solution containing anthocyanidins and having a pH of 3.5-4, after the pH value of the aqueous hydrochloric acid solution containing anthocyanidins is adjusted to pH 1-2, the aqueous hydrochloric acid solution containing anthocyanidins and having a pH of 1-2 is neutralized to pH of 3.4-4 by using the aqueous solution of sodium bicarbonate or potassium bicarbonate NaHCO.sub.3/KHCO.sub.3; hydrochloric acid is a strong acid, and ascorbic acid (etc., vitamin C) is a weak acid; here, the competitive reaction of ascorbic acid (etc., vitamin C) with hydrochloric acid greatly reduces the amount of hydrochloric acid in the aqueous hydrochloric acid Hcl solution of anthocyanidins, so that most hydrochloric acid is replaced with ascorbic acid (etc., vitamin C); even if there is a small amount of water-soluble ascorbic acid, that is, the sodium or potassium salt of vitamin C, human body can consume the substance; anthocyanidins are slightly dissolved in an acidic aqueous solution; red ascorbic acid of anthocyanin, namely vitamin C aqueous solution, is obtained; 6) if there is a small amount of anthocyanidin sodium salt or potassium salt products in the above step (5), allowing the ascorbic acid containing anthocyanidins above, that is, the vitamin C aqueous solution, to stand at room temperature for 24 to 72 hours, so that the small amount of anthocyanidin sodium salt or potassium salt products are aggregates, precipitates and separates, or filtered with a compact acid-resistant industrial filter cloth under pressure or vacuum decompression; the small amount of anthocyanidin sodium salt or potassium salt products are reacted with the aqueous hydrochloric acid Hcl solution for repeated uses; red ascorbic acid of anthocyanin, namely vitamin C aqueous solution, is obtained; reactions are all conducted at room temperature under atmospheric conditions, unless otherwise stated in the present invention.

2. The method for efficient extraction of plant anthocyanidin of claim 1, wherein a method for producing solid samples for the detection of the anthocyanidin products according to the present invention comprises steps of: (1) taking out the conjugate of an anthocyanidin sodium salt or potassium salt and a fatty acid or fatty acid ester in step 3) in the above-mentioned technical solution 1, filtering the moisture in the conjugate, wherein water-soluble anthocyanins and glucose are also filtered; then edible soft water is added to the conjugate of an anthocyanidin sodium salt or potassium salt and the fatty acid or fatty acid ester, wherein a volume of the edible soft water is 10 to 20 times that of the conjugate, and water consumption is greatly reduced when compared with the aqueous solution obtained after the anthocyanin hydrolysis; an appropriate amount of sodium chloride or potassium chloride NaCl/Kcl is added at a temperature of 0-5° C. to reach a concentration of a saturated aqueous solution thereof, so that the relative density of the aqueous solution is greater than 1; then the aqueous solution stands at a temperature of 0-5° C. for 8-24 hours, so as to enable solid particles of the fatty acid or fatty acid ester to collide ad aggregate, and to greatly reduce a total surface area and an absorption surface area of the fatty acid or fatty acid ester, and a densely suspended separation of the fatty acid or fatty acid ester in a form of fatty solid is achieved, and solid particles of the anthocyanidin sodium salt or potassium salts also collide, aggregate, precipitate and separate from each other; the relative density of anthocyanidin sodium salts or potassium salts is greater than 18, which is far more greater than the relative density of the saturated aqueous solution of sodium chloride or potassium chloride NaCl/Kcl; 2) filtering and taking out the anthocyanidin sodium salts or potassium salts in layers in the above-mentioned step 1), making the anthocyanidin sodium salts or potassium salts reach with an excess of 10-30% (by weight) aqueous hydrochloric acid Hcl solution under continuous stirring operation, wherein the stirring speed is 300-500 rpm, so that the anthocyanidin sodium salts or potassium salts are fully reacted with the aqueous hydrochloric acid Hcl solution, and an aqueous solution of hydrogen-reduced anthocyanidin precipitate and sodium chloride or potassium chloride is produced; a mass-to-liquid ratio of the anthocyanidin sodium salt or potassium salt to the aqueous hydrochloric acid Hcl solution is in a range of 10 to 20; the anthocyanidin precipitate is removed by filtering or chromatography technique, hydrochloric acid in the anthocyanidin precipitate is washed with a small amount of edible water at a temperature of 1-25° C., to precipitate ad separate the anthocyanidin, and an anthocyanidin solid is obtained; then the anthocyanidin solid is subjected to vacuum freeze dehydrating and drying, so as to obtain an anthocyanidin solid product having the same chemical structure as natural anthocyanidins; the aqueous hydrochloric acid Hcl solution containing a small amount of anthocyanidins is recycled for further use; the relative density of the anthocyanidin is 1.8.

Description

DETAILED DESCRIPTION

Example 1

[0057] 1). Hydrolyzing a natural black rice aqueous anthocyanin solution with β-glucosidase, preventing oxidation by using L-cysteine, protecting the activity of β-glucosidase; wherein the hydrolysis is carried out at a temperature of 45-50° C. for 24-48 hours with pH of 4.5-5; an aqueous solution containing anthocyanidins/anthocyanins/glucose is obtained, and the aqueous solution contains oil-soluble fatty acid or fatty acid impurities which are dispersed in an acidic aqueous solution and have a large total surface area and an absorption surface area and have a relative density of less than 1; the fatty acid or fatty acid impurities refer to oleic acid or/and palmitic acid esters, an appropriate amount of oleic acid or/and palmitic acid esters are added to an anthocyanin aqueous solution with low oleic acid or/and palmitic acid content to perform suspended separation of an anthocyanidin sodium salt or potassium salt in an anthocyanin/glucose aqueous solution in the following step 3);

[0058] 2) using sodium bicarbonate NaHCO.sub.3 to neutralize an aqueous solution obtained after the anthocyanin hydrolysis to 6.8-7, and allowing anthocyanidins to produce sodium salt or potassium salts thereof;

[0059] 3) adding sodium chloride NaCl to the above-mentioned 2) to formulate saline saturated aqueous solution of an aqueous solution containing anthocyanidin sodium salt or potassium salts/anthocyanins/glucose in step 2); the formulation method is as follows: in order to save energy, when the temperature is in the range of 25-50° C., the saturated solubility of sodium chloride Nacl in water at a certain temperature is used as a reference value, and sodium chloride Nacl at the reference value is added to and is dissolved in the aqueous solution containing anthocyanidins/anthocyanins/glucose, allowing the aqueous solution to stand for 1 to 3 minutes, to precipitate and separate the excess sodium chloride immediately; an aqueous solution containing anthocyanidin sodium salt/anthocyanin/glucose is taken out and allowed to sit for 3 to 8 hours, such that the fatty acid or fatty acid esters entrap, absorb or physically bind to a water-insoluble conjugate of an anthocyanidin sodium salt, and the conjugate is a solid water-insoluble matter due to its relative density is less than the relative density of a saturated aqueous solution of sodium chloride NaCl, then a densely suspended separation of the conjugate is achieved; the conjugate is then separated from the aqueous solution of anthocyanin and glucose and sodium chloride; then a conjugate of an anthocyanidin sodium salt and a fatty acid or fatty acid ester is taken out in layers, the moisture in the conjugate is filtered, and water-soluble anthocyanins, glucose and liquid oleic acid esters are also filtered; finally, a conjugate of an anthocyanidin sodium salt and palmitic acid ester;

[0060] 4) allowing slightly excess aqueous hydrochloric acid solution to react with the anthocyanidin sodium salt or potassium salt in palmitic acid ester in step 3) by stirring at room temperature, and strong acid generates weak acid, and an aqueous hydrochloric acid solution of hydrogen H reduced anthocyanidin and sodium chloride is produced, and the anthocyanidin is completely dissolved in the aqueous hydrochloric acid solution; the palmitic acid ester is insoluble in the aqueous hydrochloric acid Hcl solution, and it is not reacted with the aqueous hydrochloric acid Hcl solution; sodium chloride does not react with the aqueous hydrochloric acid Hcl solution; sodium chloride does not react with anthocyanidins, weak acid anthocyanidin cannot generate strong acid hydrochloric acid; the anthocyanidin-containing aqueous hydrochloric acid Hcl solution is more acidic, and the pH value of the aqueous solution is below 2; people are not allowed to take it directly; the present invention selects and utilizes important properties that the suspending agent palmitic acid or/and oleic acid ester is insoluble in and does not react with the aqueous hydrochloric acid Hcl solution, the anthocyanidin sodium salt is reduced to an aqueous hydrochloric acid solution of anthocyanidins, otherwise it makes no sense to enable the aqueous hydrochloric acid Hcl solution to react with the anthocyanidin sodium salt in the palmitic acid ester in step 3) as mentioned above by stirring;

[0061] 5) filtering the aqueous hydrochloric acid solution of anthocyanidins containing the palmitic acid ester in the above 4) with a compact acid-resistant industrial filter cloth under pressure or vacuum decompression at room temperature to separate the palmitic acid ester; since the palmitic acid ester is a palmitic acid solid, an amorphous substance, it can not be filtered with a filter screen; the aqueous hydrochloric acid Hcl solution of anthocyanidins is obtained; the aqueous hydrochloric acid solution containing anthocyanidins is neutralized by using an aqueous solution of sodium bicarbonate NaHCO.sub.3, so that its acidic pH is adjusted to 3.5-4; when the aqueous solution containing anthocyanidins is neutralized by using the aqueous solution of sodium bicarbonate NaHCO.sub.3, the aqueous solution of sodium bicarbonate NaHCO.sub.3 must be added slowly for several times and it must be stirred quickly at a stirring speed of 300-500 rpm, so that sodium bicarbonate is evenly distributed in the aqueous hydrochloric acid solution containing anthocyanidins; an appropriate amount of ascorbic acid (i.e., vitamin C) is added to the aqueous hydrochloric acid solution containing anthocyanidins and having a pH of 3.5-4, after the pH value of the aqueous hydrochloric acid solution containing anthocyanidins is adjusted to pH 1-2, the aqueous hydrochloric acid solution containing anthocyanidins and having a pH of 1-2 is neutralized to pH of 3.4-4 by using the aqueous solution of sodium bicarbonate NaHCO.sub.3; hydrochloric acid is a strong acid, and ascorbic acid (etc., vitamin C) is a weak acid; here, the competitive reaction of ascorbic acid (etc., vitamin C) with hydrochloric acid greatly reduces the amount of hydrochloric acid in the aqueous hydrochloric acid Hcl solution of anthocyanidins, so that most hydrochloric acid is replaced with ascorbic acid (etc., vitamin C); even if there is a small amount of water-soluble ascorbic acid, that is, the sodium or potassium salt of vitamin C, human body can consume the substance; anthocyanidins are slightly dissolved in an acidic aqueous solution; red ascorbic acid of anthocyanin, namely vitamin C aqueous solution, is obtained;

[0062] 6) if there is a small amount of anthocyanidin sodium salt products in the above step (5), allowing the ascorbic acid containing the anthocyanidins above, that is, the vitamin C aqueous solution, to stand at room temperature for 24 hours, so that the small amount of anthocyanidin sodium salt products are aggregated, precipitated and separated, or filtered with a compact acid-resistant industrial filter cloth under pressure or vacuum decompression; the small amount of anthocyanidin sodium salt products are reacted with the aqueous hydrochloric acid Hcl solution for repeated uses; red ascorbic acid of anthocyanin, namely vitamin C aqueous solution, is obtained.

Example 2

[0063] 1). Hydrolyzing a natural plant anthocyanin aqueous solution with β-glucosidase, preventing oxidation by using L-cysteine, protecting the activity of β-glucosidase; wherein the hydrolysis is carried out at a temperature of 45-50° C. for 24-48 hours with pH of 4.5-5; an aqueous solution containing anthocyanidins/anthocyanins/glucose is obtained, and the aqueous solution contains oil-soluble fatty acid or fatty acid impurities which are dispersed in an acidic aqueous solution and have a large total surface area and an absorption surface area and have a relative density of less than 1; the fatty acid or fatty acid impurities refer to oleic acid or/and palmitic acid esters, an appropriate amount of oleic acid or/and palmitic acid esters are added to an anthocyanin aqueous solution with low oleic acid or/and palmitic acid content to perform suspended separation of an anthocyanidin sodium salt or potassium salt in an anthocyanin/glucose aqueous solution in the following step 3);

[0064] 2) using potassium bicarbonate KHCO.sub.3to neutralize an aqueous solution obtained after the anthocyanin hydrolysis in the step 1) to 6.8-7, and allowing the anthocyanidins to produce potassium salts thereof;

[0065] 3) adding potassium chloride Kcl to the above-mentioned 2) to formulate saline saturated aqueous solution of an aqueous solution containing anthocyanidin potassium salts/anthocyanins/glucose in step 2); the formulation method is as follows: in order to save energy, when the temperature is in the range of 25-50° C., the saturated solubility of potassium chloride Kcl in water at a certain temperature is used as a reference value, and potassium chloride Kcl at the reference value is added to and is dissolved in the aqueous solution containing anthocyanidin potassium salts/anthocyanins/glucose, allowing the aqueous solution to stand for 1 to 3 minutes, to precipitate and separate the excess potassium chloride immediately; an aqueous solution containing anthocyanidin sodium salts or potassium salts/anthocyanins/glucose is taken out and allowed to sit for 3 hours, such that the fatty acid or fatty acid esters entrap, absorb or physically bind to a water-insoluble conjugate of an anthocyanidin potassium salt, and the conjugate is a solid water-insoluble matter due to its relative density is less than the relative density of a saturated aqueous solution of potassium chloride Kcl, then a densely suspended separation of the conjugate is achieved; the conjugate is then separated from the aqueous solution of anthocyanins and glucose and potassium chloride; then a conjugate of an anthocyanidin potassium salt and a fatty acid or fatty acid ester is taken out in layers, the moisture in the conjugate is filtered, and water-soluble anthocyanin, glucose and liquid oleic acid esters are also filtered; finally, a conjugate of an anthocyanidin potassium salt and palmitic acid ester;

[0066] 4) allowing slightly excess aqueous hydrochloric acid solution to react with the anthocyanidin potassium salt in palmitic acid ester in step 3) by stirring at room temperature, and strong acid generates weak acid, and a hydrochloric acid aqueous solution of hydrogen H reduced anthocyanidins and potassium chloride is produced, and the anthocyanidin is completely dissolved in the aqueous hydrochloric acid solution; the palmitic acid ester is insoluble in the aqueous hydrochloric acid Hcl solution, and it is not reacted with the hydrochloric acid Hcl aqueous potassium chloride does not react with the aqueous hydrochloric acid Hcl solution; potassium chloride does not react with the anthocyanidin, weak acid anthocyanidin cannot generate strong acid hydrochloric acid; the anthocyanidin-containing aqueous hydrochloric acid Hcl solution is more acidic, and the pH value of the aqueous solution is below 2; people are not allowed to take it directly; the present invention selects and utilizes important properties that the suspending agent palmitic acid or/and oleic acid ester is insoluble in and does not react with the aqueous hydrochloric acid Hcl solution, the anthocyanidin potassium salt is reduced to an aqueous hydrochloric acid solution of anthocyanidins, otherwise it makes no sense to enable the aqueous hydrochloric acid Hcl solution to react with the anthocyanidin potassium salt in the palmitic acid ester in step 3) as mentioned above by stirring;

[0067] 5) filtering the hydrochloric acid aqueous solution of anthocyanidins containing the palmitic acid ester in the above 4) with a compact acid-resistant industrial filter cloth under pressure or vacuum decompression at room temperature to separate the palmitic acid ester; since the palmitic acid ester is a palmitic acid solid, an amorphous substance, it can not be filtered with a filter screen; the aqueous hydrochloric acid Hcl solution of anthocyanidin is obtained; the aqueous hydrochloric acid solution containing the anthocyanidin is neutralized by using an aqueous solution of potassium bicarbonate KHCO.sub.3, so that its acidic pH is adjusted to 3.5-4; when the aqueous solution containing anthocyanidins is neutralized by using the aqueous solution of potassium bicarbonate KHCO.sub.3, the aqueous solution of potassium bicarbonate KHCO.sub.3 must be added slowly for several times and it must be stirred quickly at a stirring speed of 300-500 rpm, so that potassium bicarbonate is evenly distributed in the aqueous hydrochloric acid solution containing anthocyanidins; an appropriate amount of ascorbic acid (i.e., vitamin C) is added to the aqueous hydrochloric acid solution containing the anthocyanidin and having a pH of 3.5-4, after the pH value of the aqueous hydrochloric acid solution containing anthocyanidins is adjusted to pH 1-2, the aqueous hydrochloric acid solution containing anthocyanidins and having a pH of 1-2 is neutralized to pH of 3.4-4 by using the aqueous solution of potassium bicarbonate KHCO.sub.3; hydrochloric acid is a strong acid, and ascorbic acid (etc., vitamin C) is a weak acid; here, the competitive reaction of ascorbic acid (etc., vitamin C) with hydrochloric acid greatly reduces the amount of hydrochloric acid in the aqueous hydrochloric acid Hcl solution of anthocyanidins, so that most hydrochloric acid is replaced with ascorbic acid (etc., vitamin C); even if there is a small amount of water-soluble ascorbic acid, that is, potassium salt of vitamin C, human body can consume the substance; anthocyanidins are slightly dissolved in an acidic aqueous solution; red ascorbic acid of anthocyanins, namely vitamin C aqueous solution, is obtained;

[0068] 6) if there is a small amount of anthocyanidin potassium salt products in the above step (5), allowing the ascorbic acid containing anthocyanidins above, that is, the vitamin C aqueous solution, to stand at room temperature for 72 hours, so that the small amount of anthocyanidin potassium salt products are aggregated, precipitated and separated, or filtered with a compact acid-resistant industrial filter cloth under pressure or vacuum decompression; the small amount of potassium salt products are reacted with the aqueous hydrochloric acid Hcl solution for repeated uses; red ascorbic acid of anthocyanin, namely vitamin C aqueous solution, is obtained.

[0069] Each specific numerical value in the data range of the present invention can be implemented according to the principle of the invention, and the data range includes each specific numerical value therein, which can be independently selected and implemented by those skilled in the art according to the principle of the invention. In order to save the length of the description, each specific numerical value in the data range will not be repeated in the present invention. Reactions are all conducted at room temperature under atmospheric conditions, unless otherwise stated in the present invention.