WO2018205846A1

WO2018205846A1 - Method for extracting highly-effective natural plant anthocyanidin

Info

Publication number: WO2018205846A1
Application number: PCT/CN2018/084667
Authority: WO
Inventors: 耿兆翔
Original assignee: 耿兆翔
Priority date: 2017-05-11
Filing date: 2018-04-26
Publication date: 2018-11-15
Also published as: CN108864027A

Abstract

A method for extracting highly-effective natural plant anthocyanidin, which relates to the technical field of bioengineering. The method comprises the steps of: hydrolyzing anthocyanin; by using sodium bicarbonate or potassium bicarbonate NaHCO3/KHCO3, neutralizing a water solution obtained after the anthocyanin is hydrolyzed, so as to adjust the PH of the obtained solution to PH7; then, by means of suspended separation, salting out a combination between an anthocyanidin sodium salt or potassium salt and a fatty acid or a fatty acid ester by using a saturated water solution of the sodium chloride or potassium chloride Nacl/Kcl; letting them sitting at a low temperature, so as to separate the fatty acid or the fatty acid ester; carrying out a reaction between the hydrochloric acid and the anthocyanidin sodium salt or potassium salt; and depositing to recover the anthocyanidin. The method is used for extracting the natural plant anthocyanidin.

Description

TECHNICAL FIELD The present invention relates to the field of bioengineering technology, and in particular to a method for extracting high-efficiency natural plant anthocyanins. BACKGROUND OF THE INVENTION The separation and extraction of natural plant anthocyanins has been a worldwide problem, and there has been no effective high-yield separation and extraction technology. Prior art, Chinese invention patent application, application number 201210516999.6, application date 2012.11.25, application publication number CN103834702A, application publication date 2014.06.04, invention creation name: a method for extracting cationic anthocyanin aglycon; using anionic surface The active agent reacts with the cationic anthocyanin under acidic (pH 3.5) conditions; according to the prior knowledge, the anthocyanin molecular structure (C ₆ -C ₃ -C ₆ ) in the intermediate C ₃ ring on the oxygen atom The chlorine cl atom can dissociate the chloride ion cl_ under the more acidic conditions of the aqueous solution, and the anthocyanin is cationic; the anthocyanin is under such acidic conditions (pH 3.5), the anionic surfactant first or simultaneously It also reacts with hydrogen ions in an aqueous acid solution to synthesize water-insoluble mercapto acids/fatty acids, and the anionic surfactant is too much lost; using anionic surfactants and cations (dissociation of d in anthocyanin molecules) anthocyanins a compound salt compound, because the anthocyanin molecular structure (eg, cornflower chrysanthemum, etc.) mostly contains 5 hydroxyl groups, soluble in acidic aqueous solution, and anthocyanin and anionic surface The salt compound of the active agent is acid-soluble, and it is difficult to separate the anthocyanin from a large amount of water. Thus, most of the anthocyanins hydrolyzed by β-glucosidase are dissolved in an acidic hydrolyzed aqueous solution; It is serious that the salt compound of anthocyanin and anionic surfactant is simultaneously ionized in an acidic hydrolyzed aqueous solution, and the anionic group in the anionic surfactant is detached from the anthocyanin molecule, and the anthocyanin containing five hydroxyl groups. Unable to extract; Experiments prove that the above patent application method can only extract anthocyanins (eg, peony anthocyanins) containing 4 hydroxyl groups, resulting in extremely valuable waste of anthocyanin resources; severely restricting the yield of anthocyanin extraction.

The biological activity of the anthocyanins of the four hydroxyl groups is not as strong as that of the anthocyanins of the five hydroxyl groups.

The extraction method of anthocyanins requires substantial improvement, innovation and fundamental breakthroughs.

Replacement page (Article 26) SUMMARY OF THE INVENTION - Principles of the Invention -

1. Anthocyanins are aglycones in anthocyanins and do not contain glycosyl groups. Anthocyanins cannot be reacted with a strong alkali sodium hydroxide or potassium hydroxide NaOH/KOH solution with a pH greater than pH 7.5, otherwise the molecular structure of the anthocyanins will change to become a colorless chalcone. The relative density of sodium chloride is 2.165, and the relative density of potassium chloride is 1.98. Relative density units: gram / cubic centimeter or milliliter, the same below.

2. The hydrogen atom on the hydroxyl group bound by the glycosidic bond in the anthocyanin molecule dissociates the water-soluble H+ ion in the aqueous solution of PH4-7.5, and is itself a water-soluble anion group. Natural plants do not contain free anthocyanin molecules, but exist as anthocyanins, an anthocyanins.

In the prior art, an aqueous solution of anthocyanins is hydrolyzed by β-glucosidase at a temperature of 40 ° C to 50 ° C and a pH of 4.5 to 5 to form sugars and free anthocyanin molecules.

PH4-7.5 when in an aqueous solution, a sodium bicarbonate or potassium bicarbonate NaHC0 _₃ KHC0 ₃ in acidic aqueous solution and the hydrolysis of anthocyanins, sodium or potassium bicarbonate NaHC0 _₃ / KHC0 ₃ also reacted with anthocyanins, The sodium or potassium salt of anthocyanin is produced and carbon dioxide C0 _{2 is} released.

3. Anthocyanin of the present invention refers to an aglycone in an anthocyanin glycoside (i.e., anthocyanin), which does not contain a glycosyl group, i.e., an active aglycon.

4. Anthocyanins are only slightly soluble in acidic aqueous solutions. Anthocyanins produce a colorless chalcone in an aqueous alkaline solution of pH 7.5 or higher, and the structure changes.

5. In the flavonoid structure of the anthocyanin molecular structure (C ₆ -C ₃ -C ₆ ), the chlorine cl atom on the 0 atom in the middle C ₃ ring is only solved when the pH of the aqueous solution is less than or equal to PH3.5. Chloride cl_ ions are separated; chlorine cl- ions cannot be dissociated at pH 4 -7.5.

6. Principle of salting out of anthocyanin/anthocyanin sodium or potassium salt and separation of anthocyanin/glucose aqueous solution: In the enzymatic hydrolysis of β-glucosidase in anthocyanin aqueous solution, anthocyanins cannot be completely hydrolyzed. Anthocyanins require separation of anthocyanins/glucose from anthocyanins and separation of anthocyanins from its 1000-fold or more mass of water.

Anthocyanins contain highly water-soluble sugar groups (eg, glucosyl, galactosyl), plus 3-5 hydrophilic hydroxyl groups (-ΟΗ) contained in their aglycone (ie, anthocyanins) structure. , so anthocyanin

2

Replacement page (Article 26) It has strong hydrophilicity and water solubility; while anthocyanin contains only 4-6 hydrophilic hydroxyl groups, and anthocyanins containing 4-5 hydrophilic hydroxyl groups (-OH) are mainly available in nature. The hydrophilicity of anthocyanins and anthocyanins is very different. The water solubility (hydrophilicity) of anthocyanins/glucose is much higher than the hydrophilicity of anthocyanins. The hydrophilicity of anthocyanins is greater than the hydrophilicity of anthocyanin sodium or potassium salts, because anthocyanin sodium or potassium salts (weak acid salts) contain only 3-5 hydrophilic hydroxyl groups (-OH), One less hydrophilic hydroxyl group (-OH).

Sodium chloride/potassium chloride Nacl Kcl destroys the hydrogen bond or polar attraction between the hydroxyl group on the anthocyanin molecule and the water molecule to reduce the water solubility of anthocyanin and anthocyanin sodium or potassium salt; The solubility of sodium chloride/potassium chloride in NaCl/Kcl water increases. At pH 6.8-7, the hydrophilic anthocyanin and anthocyanin sodium or potassium salt will gradually separate from water and become water insoluble aggregates. Precipitated, and separated from the aqueous solution of anthocyanin/glucose.

The hydrophilicity of anthocyanin/glucose is greater than the hydrophilicity of sodium chloride or potassium chloride NaCl/Kcl, and the hydrophilicity of sodium chloride or potassium chloride NaCl/Kcl is much greater than anthocyanin and anthocyanin sodium or Hydrophilicity of potassium salt; Glucose is more soluble in water than sodium chloride or potassium chloride, NaCl, and it is more hydrophilic; so glucose and anthocyanin in water can reduce sodium chloride or potassium chloride Nacl/Kcl Saturated solubility. Since the water solubility (hydrophilicity) of anthocyanin/glucose is much greater than the hydrophilicity of anthocyanins and anthocyanin sodium or potassium salts, an extreme contrast is formed; anthocyanins/glucose in sodium chloride or chlorination Can not be aggregated and separated in potassium NaCl/Kcl aqueous solution, so that anthocyanin sodium or potassium salt is combined with anthocyanin/glucose in saturated aqueous solution of sodium chloride or potassium chloride at pH 6.8-7 and 25°C-50°C. The aqueous solution is separated. The hydrophilicity of glucose is stronger than that of sodium chloride or potassium chloride, Nacl/Kcl and anthocyanin sodium or potassium salt. Saturated aqueous solution of sodium chloride or potassium chloride Nacl/Kcl cannot precipitate and precipitate glucose.

The aqueous solution of anthocyanin hydrolyzed with anthocyanin and anthocyanin sodium or potassium salt is at a temperature between 25 ° C and 50 ° C and keeps the aqueous solution neutral; saturated with sodium chloride or potassium chloride NaCl/Kcl An aqueous solution of an aqueous solution (containing anthocyanins and glucose) containing anthocyanins hydrolyzed by anthocyanins and anthocyanin sodium or potassium salts, which can cause anthocyanins and anthocyanin sodium or potassium salts from anthocyanins Aggregation and precipitation in a hydrolyzed aqueous solution; anthocyanin and anthocyanin sodium or potassium salt when saturated with sodium chloride or potassium chloride NaCl/Kcl in an aqueous solution of anthocyanin hydrolyzed at 25 ° C to 50 ° C The salting out is best precipitated and precipitated from the aqueous solution of anthocyanin, and the anthocyanin sodium or potassium salt can be completely separated. 0 ° C -25 ° C low temperature can be accelerated

3

Replacement page (Article 26) The anthocyanin sodium or potassium salt is precipitated and precipitated from an aqueous solution of anthocyanin. Anthocyanins and anthocyanin sodium or potassium salts are insoluble in a saturated aqueous solution of sodium chloride or potassium chloride NaCl/Kcl in an anthocyanin/glucose aqueous solution.

7. The relative density of oil-soluble fatty acids or fatty acid esters (such as oleic acid, palmitic acid) in natural plants is less than 1 (water is 1, g / cm3 or ml). Rumic acid freezing point (°C): 13.2, palmitic acid melting point (°C): 63.1. Naturally anthocyanin-containing plants (or fruits) mainly contain palmitic acid and oleic acid (such as black rice), of which palmitic acid is the most abundant, and palmitic acid content is 5-10 times or more of the anthocyanin content after anthocyanin hydrolysis.

The invention utilizes the acid acid of palmitic acid or/and oleic acid to suspend the sodium or potassium anthocyanin from the anthocyanin/glucose aqueous solution; and further uses an aqueous solution of sodium chloride or potassium chloride NaCl/Kcl to increase the relative ratio of the aqueous solution. Density, and then stand at 0 ° C -5 ° C for 8-24 hours, so that the fatty acid or fatty acid lipids are suspended in a solid oily form, anthocyanin sodium or potassium salt precipitates, with anthocyanin sodium or The potassium salt is separated to obtain an anthocyanin sodium or potassium salt. Adding a combination of sodium or potassium anthocyanin and fatty acid or fatty acid fat to soft water of 10-20 times its volume, which greatly reduces the amount of water compared with the aqueous solution of anthocyanin, and makes solid or fatty acid solid particles (relative The density is less than 1) The collision is combined into a whole solid oil, which is densely suspended in solid oil at 0 °C-5 °C; the particles of sodium or potassium anthocyanin (relative density greater than 1.8) are also insoluble. The neutral saline solution collides with each other and aggregates and precipitates.

Palmitic acid, also known as palmitic acid, is known as palmitic acid. Widely found in nature, almost all oils and fats have varying levels of palmitic acid components. Molecular formula: C ₁₆ H ₃₂ 0 ₂ , Structural formula: CH ₃ (CH ₂ ) ₁₄ COOH. It is a white pearlescent scale that is present as a glyceride in many oils and fats. Do not dissolve in water, slightly soluble in cold alcohol and petroleum ether, soluble in hot ethanol, ether and chloroform. Density (g/mL, 25/4 °C): 0. 8527; Relative density (25 ° C, 4 ° C): 0.841480. Oleic acid, oleic acid, together with other fatty acids, is present in all animal and vegetable oils in the form of glycerides. Relative density

(g/mL, 25/4 ° C): 0.8935, relative density (25 ° C, 4 ° C): 0.845590, relative density (g/mL, 90/4 ° C): 0.8429.

8. The combination of anthocyanin sodium or potassium salt and fatty acid or fatty acid ester, the main component is fatty acid or fatty acid ester (oleic acid or / and palmitic acid acid ester), so the relative density of the combination is not more than

4

Replacement page (Article 26) 1.1; The relative density of sodium or potassium chloride NaCl/Kcl saturated aqueous solution containing anthocyanin/glucose is greater than 1.1, reaching 1.2; therefore, the combination of anthocyanin sodium or potassium salt with fatty acid or fatty acid can be included The anthocyanin/glucose is suspended in a sodium chloride or potassium chloride NaCl/Kcl saturated aqueous solution.

9. 10%-30% aqueous solution of HCI and anthocyanin sodium or potassium salt are stirred and metathesized, and the principle of precipitation and recovery of anthocyanins: mass ratio of anthocyanin sodium or potassium salt to hydrochloric acid Hcl aqueous solution For 1 to 10-20, with 300-500 rpm shear agitation, continuously change the shape and size of the anthocyanin sodium or potassium salt particles, so that the anthocyanin sodium or potassium salt and hydrochloric acid Hcl are sufficient The reaction is contacted to form an aqueous solution of hydrogen chloride-reduced anthocyanin and sodium chloride or potassium chloride NaCl/Kcl containing hydrochloric acid. Since anthocyanin is only slightly soluble in aqueous solution of HCI, the water solubility of hydrochloric acid and sodium chloride or potassium chloride is much greater than the water solubility of anthocyanin. Hydrochloric acid and sodium chloride or potassium chloride also inhibit anthocyanin in hydrochloric acid. Dissolved in an aqueous solution. Sodium chloride or potassium chloride cannot react with anthocyanins because weak acids (hydroxy-OH on the anthocyanin molecule) do not form strong acid acids; the reaction can be carried out thoroughly and completely. An absolute excess of anthocyanins can only accumulate and precipitate due to their relative density (1.8) being much greater than the relative density of hydrochloric acid and sodium chloride or potassium chloride solution (less than 1.2).

10. The isoelectric point (ΡΙ) of β-glucosidase is mostly in the acidic range, so this property can be used to adjust the enthalpy of the acid aqueous solution after anthocyanin hydrolysis to the isoelectric point of the enzyme, so that β-glucoside Enzymatic precipitation recovery and reuse, a specific β-glucosidase isoelectric point (ΡΙ) can be measured and calculated according to the prior art; the isoelectric point (ΡΙ) is higher than the acid aqueous solution after the anthocyanin hydrolysis , using sodium bicarbonate or potassium bicarbonate NaHC0 ₃ /KHC0 ₃ to neutralize the pH of the aqueous acid solution to the isoelectric point; when the isoelectric point (PI) is lower than the pH of the aqueous acid solution after the anthocyanin hydrolysis, use The aqueous solution of HCI hydrochloride adjusts the enthalpy of the aqueous acid solution to the isoelectric point.

11. According to the prior knowledge, L-cysteine is a reducing agent, and its role in the hydrolysis of anthocyanins is to prevent the active center of β-glucosidase from being oxidized and to restore the activity of β-glucosidase; Cystine itself is oxidized to a disulfide bond of L-cystine, L-cystine is slightly soluble in water, soluble in dilute aqueous acid solution, and the isoelectric point (ΡΙ) of L-cystine is 4.6, which can be adjusted. The aqueous acid solution after hydrolysis of the anthocyanin is decanted to the isoelectric point (ΡΙ) of L-cystine, and the L-cystine is precipitated and separated. The β-glucosidase enzymatic hydrolysate of anthocyanins is anthocyanin and glucose, but glucose has an aldehyde group, which is oxidizing and reducing.

Replacement page (Article 20) Sex, can oxidize the active center of β-glucosidase, so glucose is a typical inhibitor of enzymatic hydrolysis of β-glucosidase; thus adding the reducing agent L-cysteine to prevent the active center of β-glucosidase It is oxidized and restores the activity of β-glucosidase.

Cystine is also known as L-cystine, 3,3'-dithiodialanine, etc., CAS number is 56-89-3, and the chemical formula is C ₆ H ₁₂ N ₂ 0 ₄ S ₂ . It was discovered by Wollaston from bladder stones in 1810. In 1832, Berzelius named it cystine, a sulfur-containing amino acid that was present in small amounts in proteins and contained in keratin such as hair and fingers. It can also be obtained by a synthetic method. Used in medicine, food, cosmetics and other industries.

L-cystine, water solubility: 0. 112g / L (25 ° C), white flaky crystal or crystalline powder; very slightly soluble in water; insoluble in ethanol and other organic solvents, soluble in dilute acid; The isoelectric point is pI4.6.

12. Activated carbon can adsorb anthocyanins in anthocyanin aqueous solution and has decolorization effect.

13. Sodium chloride Nad water solubility (g / 100 g water): 0 ° C: 35.7 g, 10 ° C: 35.8 g, 20 ° C: 36. Og, 30 ° ° C: 36. 3g, 40 °C : 36.6g, 50 °C : 37. Og, 60 °C: 37.3g, 70 °C: 37.8g, 80 °C: 38.4g, 90 °C: 39. Og, 100 °C: 39. 8g.

14, potassium chloride Kcl water solubility (g / 100 grams of water):

0°C 10°C 20 °C 30°C 40 °C 60 °C 70 °C 80 °C 90 °C 100°C 28g 31.2g 34.2g 37.2g 40. lg 45.8g 48.8g 51.3g 53.9g 56.3g .

Technical solution of the invention

1. (1) The natural plant anthocyanin aqueous solution is hydrolyzed by β-glucosidase, and the activity of β-glucosidase is protected by L-cysteine anti-oxidation; hydrolysis temperature 45 ° C -50 ° C, pH 4.5 -5 , time 24-48 hours; obtaining an aqueous solution containing anthocyanin/anthocyanin/glucose, containing an oil-soluble fatty acid or fatty acid ester impurity having a relative density of less than 1 dispersed in an acidic aqueous solution; fatty acid or fatty acid impurity impurity means Acidic acid of oleic acid or/and palmitic acid, an aqueous solution of anthocyanin with low content of oleic acid or/and palmitic acid should be added with an appropriate amount of acid acid of oleic acid or/and palmitic acid to enable completeness in (3) below. Suspension separation of anthocyanin sodium or potassium salt in an aqueous solution of anthocyanin;

(2) neutralizing the anthocyanin aqueous solution of the above (1) with sodium hydrogencarbonate or potassium hydrogencarbonate NaHC0 ₃ /KHC0 ₃ to pH 6.8-7, anthocyanin to form its sodium or potassium salt;

6

Replacement page (Article 26) (3) adding the above (2) sodium chloride or potassium chloride Nacl Kcl to prepare a salt saturated aqueous solution of the above (2) aqueous solution containing anthocyanin sodium or potassium salt/anthocyanin/glucose; In order to save energy, at 25 °C -50 °C, the saturation solubility of sodium chloride or potassium chloride NaCl in a certain temperature is used as a reference value, and then the value of sodium chloride or Potassium chloride Nacl/Kcl is dissolved in this aqueous solution containing anthocyanin/anthocyanin/glucose, and allowed to stand for 1-3 minutes to precipitate and separate excess sodium chloride or potassium chloride; then extract anthocyanin/ An aqueous solution of anthocyanin/glucose/sodium chloride or potassium chloride; a mixture of 3-8 hours, which is a mixture of fatty acid or fatty acid impurities, which is adsorbed and physically bound to water-insoluble anthocyanin sodium or potassium. The relative density is less than the relative density of the saturated aqueous solution of sodium chloride or potassium chloride NaCl/Kcl, and is separated by solid water insoluble matter; and separated from the aqueous solution of anthocyanin and glucose and sodium chloride or potassium chloride;

(4) taking out the mixed combination of sodium or potassium anthocyanin or fatty acid or fatty acid ester of (3) above, and filtering the water; and combining the sodium or potassium salt of anthocyanin with fatty acid or fatty acid Add 10-20 times of edible soft water, add appropriate amount of sodium chloride or potassium chloride NaCl/Kcl at 0 °C -5 °C or reach the concentration of saturated aqueous solution, so that the relative density of the aqueous solution is greater than 1; It is allowed to stand at 0 °C - 5 °C for 8-24 hours, so that the fatty acid or fatty acid ester is densely suspended and separated in solid oil form. At this time, the anthocyanin sodium salt or potassium salt is mostly aggregated and precipitated and separated; anthocyanin sodium salt Or the relative density of the potassium salt is greater than 1.8, which is much larger than the relative density of the saturated aqueous solution of sodium chloride or potassium chloride NaCl/Kcl; the fatty acid or fatty acid ester is partially recycled in the above (1) if necessary;

(5), at 25 ° C -50 ° C, the above or (4) anthocyanin sodium or potassium salt is removed by filtration or layering; and a slight excess of 10%-30% aqueous solution of HCI and The anthocyanin sodium or potassium salt is stirred and stirred at a rate of 300-500 rpm to fully react the anthocyanin sodium or potassium salt with the aqueous solution of HCI to form an anthocyanin precipitate and sodium chloride reduced by hydrogen H. Or potassium chloride; the mass ratio of anthocyanin sodium or potassium salt to HCI aqueous solution is 1 to 10-20; filtered or layered to remove anthocyanin precipitate, used at 1 °C -25 °C A small amount of edible water is used to wash the hydrochloric acid, and the anthocyanin is precipitated and separated to obtain an anthocyanin solid; and then dehydrated and dried under vacuum to obtain an anthocyanin product having the same chemical structure as natural anthocyanin; use. Anthocyanins are only slightly soluble in aqueous HCI solution, and sodium chloride or potassium chloride Nacl/Kcl further reduces the solubility of anthocyanins in aqueous HHCl solution.

Replacement page (Article 26) In the prior art, the chlorine cl atom on the oxygen atom in the middle C ₃ ring in the molecular structure of anthocyanins is easily replaced by an ethanol group to form an ethanol-based anthocyanin.

2. The natural structure of the anthocyanin product is dissolved in a hydrochloric acid and ethanol aqueous solution having a pH of 2.5 to 3.5, a reaction temperature of 50 ° C, an ethanol volume concentration of 50% to 95%, and a ratio of material to liquid of 1: 10-20. The chlorine ion on the anthocyanin molecule is replaced by an ethanol group to form a salt-type ethanol-based anthocyanin; and then the ethanol is recovered by evaporation under reduced pressure at a pressure of not more than 100 Pa and a temperature of 40 ° C to 60 ° C; The insoluble ethanol-based anthocyanins are aggregated and precipitated in the remaining aqueous hydrochloric acid solution; layered and filtered to obtain an ethanol-based anthocyanin solid containing a small amount of water; vacuum dehydration and drying; and an ethanol-based anthocyanin product is obtained.

3. The isoelectric point PI of β-glucosidase is mostly in the acidic range, so this property is used to adjust the enthalpy of the acid aqueous solution after anthocyanin hydrolysis to the isoelectric point of the enzyme to recover the β-glucosidase precipitation. Reuse; a specific β-glucosidase isoelectric point is measured and calculated according to the prior art; when the isoelectric point is higher than the acid aqueous solution after the anthocyanin hydrolysis, sodium bicarbonate or carbonic acid is used. The potassium hydroxide NaHC0 ₃ /KHC0 _{3 is} neutralized, and the pH of the acid aqueous solution is adjusted to the isoelectric point; when the isoelectric point PI is lower than the pH value of the aqueous solution of the anthocyanin hydrolyzed, the pH of the aqueous acid solution is adjusted with the aqueous solution of HCI hydrochloric acid to The isoelectric point; the isoelectric point PI of L-cystine is 4.6, and the pH of the aqueous acid solution after hydrolysis of the anthocyanin is adjusted to the isoelectric point PI of L-cystine to precipitate and separate the L-cystine.

4, instead of with KHC0 ₃ Kcl NaHC0 ₃ / Nacl, containing an aqueous solution Kcl / KHC0 ₃ as fertilizer or crop recovered solid was dried Kcl / KHC0 _3.

5. The aqueous solution of anthocyanin-containing saturated sodium chloride or potassium chloride NaCl/Kcl in the above technical schemes 1 and (3) is vacuum-dried at 70 ° C -100 ° C; the continuous recovery during the drying process is due to dehydration. Excessive precipitation of sodium chloride or potassium chloride, NaCl/Kcl, sodium chloride or potassium chloride, NaCl/Kcl, recycling, anthocyanins containing a small amount of sodium chloride or potassium chloride, NaCl/Kcl and glucose, and recycling; The above aqueous solution of the anthocyanin-containing saturated sodium chloride or potassium chloride NaCl/Kcl aqueous solution in the above technical scheme 1, (3) is electrolyzed by a cation exchange membrane, and the sodium hydroxide or potassium hydroxide NaOH/KOH aqueous solution and the aqueous solution of HCI are recovered or Chlorine gas, non-electrolyte anthocyanin and glucose aqueous solution recycling; or the above technical scheme 1,

(3) Saturated potassium chloride containing anthocyanins and glucose in KCL aqueous solution adsorbed by activated carbon

8

Replacement page (Article 26) The anthocyanins are decolorized, and the anthocyanins are separated by precipitation, and the saturated potassium chloride KCl aqueous solution containing glucose is recycled or directly used for agricultural fertilizers.

6. Using sodium hydroxide or potassium hydroxide NaOH KOH to partially replace sodium bicarbonate or potassium hydrogencarbonate NaHC0 ₃ /KHC0 _{3 to} neutralize the aqueous solution of anthocyanin, and hydrolyze anthocyanin with an appropriate amount of PH9-14 NaOH/KOH aqueous solution. The pH of the aqueous solution is adjusted to pH 5.5-6, slowly adding NaOH/KOH aqueous solution and stirring rapidly at a speed of 300-500 rpm or more, so that NaOH KOH is evenly distributed in the aqueous solution of anthocyanin hydrolysis; The pH of the aqueous solution of NaHC0 ₃ /KHCO^ anthocyanin hydrolyzed is adjusted to PH7;

An aqueous solution in which an aqueous solution of anthocyanin is hydrolyzed by an aqueous solution of sodium carbonate or potassium carbonate in an aqueous solution of potassium carbonate or potassium carbonate in an amount of sodium carbonate or potassium carbonate in place of sodium hydrogencarbonate or potassium hydrogencarbonate NaHC0 ₃ /KHC0 ₃ . The pH value is adjusted to pH 5.5-6, and the sodium carbonate or potassium carbonate aqueous solution is slowly added while stirring rapidly, and the rotation speed is 300-500 rpm or more, so that the sodium carbonate or potassium carbonate is uniformly distributed in the aqueous solution of anthocyanin hydrolysis; Further, an appropriate amount of sodium hydrogencarbonate or potassium hydrogencarbonate NaHC0 ₃ /KHC0 _{3 was} added to adjust the pH of the anthocyanin-hydrolyzed aqueous solution to pH 7.

Aspect of the present invention 1, the hydrolysis of anthocyanins, after the hydrolysis with sodium bicarbonate and anthocyanins or potassium bicarbonate NaHC0 _₃ / KHC0 ₃ solution to PH7, again with sodium or potassium chloride Nacl / Kcl saturated Aqueous solution salting-off suspension separation of anthocyanin sodium or potassium salt with fatty acid or fatty acid ester, separation of fatty acid or fatty acid ester at low temperature, reaction of hydrochloric acid with anthocyanin sodium or potassium salt to precipitate anthocyanin Combination is the core technology of the present invention, that is, the necessary technical features of innovation.

Beneficial effects:

1. The invention greatly improves the industrial production yield of anthocyanins, and can extract all anthocyanins after hydrolysis of β-glucosidase, including anthocyanins and 6 hydroxyl groups containing 4 hydroxyl groups and 5 hydroxyl groups. Anthocyanins.

2. Avoiding the waste of anthocyanin resources and providing a strong health material support for the health of the people.

3. It does not need to dry a large amount of water, which greatly saves energy, greatly shortens the drying time of anthocyanin products, and greatly improves the production efficiency of industrial production of anthocyanins.

9

Replacement page (Article 26) Preferred technical solution of the invention:

(1) The black plant anthocyanin aqueous solution of natural plant is hydrolyzed by β-glucosidase, and the activity of β-glucosidase is protected by oxidation of L cysteine; hydrolysis temperature is 45 ° C -50 ° C, PH 4. 5-5, time ₂₄ - ₄₈ hours; obtaining an aqueous solution containing anthocyanin/anthocyanin/glucose, containing an oil-soluble fatty acid or fatty acid ester impurity having a relative density of less than 1 dispersed in an acidic aqueous solution; fatty acid or fatty acid impurity impurity is Refers to the acid acid of oleic acid or/and palmitic acid. For the aqueous solution of anthocyanin with low content of oleic acid or/and palmitic acid, an appropriate amount of acid acid of oleic acid or/and palmitic acid is added to enable the following (3). Completely suspending the anthocyanin sodium salt in an aqueous solution of anthocyanin;

(2) neutralizing the anthocyanin aqueous solution of the above (1) with sodium hydrogencarbonate NaHC0 ₃ to pH 6.8-7, anthocyanin to form its sodium salt;

(3) Adding the above (2) to sodium chloride, to prepare a salt saturated aqueous solution of the above (2) aqueous solution containing anthocyanin sodium/anthocyanin/glucose; the specific preparation method, in order to save energy, at 25 ° At C-50 °C, the saturation solubility of sodium chloride in a certain temperature in water is taken as the reference value, and the sodium chloride NaCl of this value is added to the aqueous solution containing anthocyanin/anidin/glucose. Dissolve, let stand for 1-3 minutes, so that the excess sodium chloride is precipitated and separated immediately; then take out the aqueous solution containing anthocyanin / anthocyanin / glucose / sodium chloride; let stand for 3-8 hours, make fatty acid or fatty acid fat The mixed conjugate of impurity-trapped, adsorbed, physically bound water-insoluble anthocyanin sodium salt is densely suspended in solid water insoluble matter because its relative density is less than the relative density of saturated aqueous solution of sodium chloride NaCl; and with anthocyanins and glucose and Separation of aqueous solution of sodium chloride;

(4) taking out the mixed combination of the anthocyanin sodium salt of the above (3) and the fatty acid or fatty acid ester, and filtering the water therein; adding the combination of the anthocyanin sodium salt and the fatty acid or fatty acid fat to the volume of 10- 20 times of soft water, add appropriate amount of sodium chloride NaCl at 0 °C -5 °C or reach its saturated aqueous solution concentration, so that the relative density of the aqueous solution is greater than 1; then stand at 0 °C -5 °C 8 -24 hours, the fatty acid or fatty acid ester is densely suspended and separated in solid oil form. At this time, the anthocyanin sodium salt is mostly aggregated and precipitated. The relative density of the anthocyanin sodium salt is more than 1.8, which is much larger than the saturated aqueous solution of sodium chloride and NaCl. Relative density; fatty acid or fatty acid ester is partially recycled in the above (a) if necessary;

10

Replacement page (Article 20) (5), at 25 ° C -50 ° C, the above (4) anthocyanin sodium salt is removed by filtration or layering; and a slight excess of 10%-30% hydrochloric acid aqueous solution of HCI and anthocyanin is used. The sodium salt is stirred and reacted at a stirring speed of 300-500 rpm to fully react the anthocyanin sodium salt with an aqueous solution of HCI to form an anthocyanin precipitate and sodium chloride reduced by hydrogen H; anthocyanin sodium salt and hydrochloric acid Hcl The mass ratio of the aqueous solution is 1 to 10-20; the anthocyanin precipitate is removed by filtration or layering, and the hydrochloric acid is washed with a small amount of edible water at 1 ° C to 25 ° C to precipitate the anthocyanin to obtain a flower. The phthalocyanine solid is further dehydrated and dried under vacuum to obtain an anthocyanin product having the same chemical structure as natural anthocyanin; and an aqueous solution of hydrochloric acid containing a trace amount of anthocyanin is recycled.

DETAILED DESCRIPTION OF THE INVENTION - Example 1

(1) hydrolyzing the aqueous solution of black rice anthocyanin in natural plants with β-glucosidase, protecting the activity of β-glucosidase with L-cysteine; hydrolysis temperature 50 ° C, PH 5, time 24 hours; Obtaining an aqueous solution containing anthocyanin/anthocyanin/glucose, containing an oil-soluble fatty acid or fatty acid ester impurity having a relative density of less than 1 dispersed in an acidic aqueous solution; fatty acid or fatty acid lipid impurity means an acid of oleic acid or/and palmitic acid For the anthocyanin aqueous solution with low oleic acid or/and palmitic acid content, an appropriate amount of acid acid of oleic acid or/and palmitic acid is added to enable complete suspension of the anthocyanin solution in the aqueous solution of anthocyanin in the following (3). Sodium salt

(2) neutralizing the anthocyanin aqueous solution of the above (1) with sodium hydrogencarbonate NaHC0 ₃ to pH 7, and the anthocyanin is formed into a sodium salt thereof;

(3) Adding the above (2) to sodium chloride, to prepare a salt saturated aqueous solution of the above (2) aqueous solution containing anthocyanin sodium/anthocyanin/glucose; the specific preparation method, in order to save energy, at 25 ° At C, the saturation solubility of sodium chloride in water at this temperature is used as a reference value, and the sodium chloride NaCl of this value is added to the aqueous solution containing anthocyanin/anidin/glucose to dissolve, and 1- 3 minutes, the excess sodium chloride was immediately precipitated and separated; then the aqueous solution containing anthocyanin / anthocyanin / glucose / sodium chloride was taken out; allowed to stand for 8 hours, the fatty acid or fatty acid impurities were entrapped, adsorbed, physically bound to water The mixed conjugate of the insoluble anthocyanin sodium salt is densely suspended in solid water insoluble matter because its relative density is less than the relative density of the saturated aqueous solution of sodium chloride NaCl; and is separated from the aqueous solution of anthocyanin and glucose and sodium chloride;

11

Replacement page (Article 26) (4) taking out the mixed combination of the anthocyanin sodium salt of the above (3) and the fatty acid or fatty acid ester, filtering the water therein; adding the combination of the anthocyanin sodium salt and the fatty acid or fatty acid fat to the volume 10 times For soft water, add an appropriate amount of sodium chloride at 5 °C or reach a saturated aqueous solution concentration to make the relative density of the aqueous solution greater than 1; then let stand at 5 °C for 24 hours to make the fatty acid or fatty acid fat solid. Intensive suspension separation, at this time, anthocyanin sodium salt is mostly aggregated and precipitated; the relative density of anthocyanin sodium salt is greater than 1.8, which is much larger than the relative density of saturated aqueous solution of sodium chloride Nad; fatty acid or fatty acid fat is necessary in the above ( a) partial recycling;

(5), at 50 ° C, the above (4) anthocyanin sodium salt is removed by filtration or layering; and a slight excess of 10% hydrochloric acid aqueous solution of HCI and anthocyanin sodium salt are stirred and reacted at a stirring speed. 500 rpm, the anthocyanin sodium salt and the aqueous solution of HCI are fully reacted to form an anthocyanin precipitate reduced by hydrogen H and sodium chloride; the mass ratio of the anthocyanin sodium salt to the aqueous solution of HCI is 1 ratio 10; filtering or layering out the anthocyanin precipitate, washing the hydrochloric acid with a small amount of edible water at 1 ° C -25 ° C, separating and separating the anthocyanins to obtain the anthocyanin solid; vacuum dehydration and drying The anthocyanin product has the same chemical structure as the natural anthocyanin; the aqueous solution containing trace anthocyanin is recycled.

Example 2:

(1) hydrolyzing the natural plant anthocyanin aqueous solution with β-glucosidase, protecting against the activity of β-glucosidase by using L-cysteine; hydrolysis temperature 50 ° C, PH 5, time 24 hours; An aqueous solution of anthocyanin/anthocyanin/glucose containing an oil-soluble fatty acid or fatty acid ester impurity having a relative density of less than 1 dispersed in an acidic aqueous solution; a fatty acid or a fatty acid lipid impurity means an acid acid of oleic acid or/and palmitic acid, An aqueous solution of anthocyanin with low oleic acid or/and palmitic acid content is added with an appropriate amount of acid acid of oleic acid or/and palmitic acid to enable complete suspension of the anthocyanin potassium salt in the aqueous solution of anthocyanin in the following (3). ;

(2) neutralizing the anthocyanin aqueous solution of the above (1) with potassium bicarbonate KHC0 ₃ to pH 7, and the anthocyanin forms its potassium salt;

(3) Adding the above (2) potassium chloride KCl to prepare a salt saturated aqueous solution of the above (2) aqueous solution containing anthocyanin potassium salt/anthocyanin/glucose; specific preparation method, in order to save energy, at 25 ° C, the saturation solubility of potassium chloride KCl in water at this temperature is the reference value, and then

12

Replacement page (Article 26) Potassium chloride KCl of this value is added to the aqueous solution containing anthocyanin/anthocyanin/glucose, and allowed to stand for 1-3 minutes to precipitate and separate excess potassium chloride immediately; then extract anthocyanin/anthocyanin/ An aqueous solution of glucose/potassium chloride; a mixed conjugate of an anthocyanin potassium salt that binds fatty acid or fatty acid ester impurities, adsorbed, and physically binds water insoluble for 3-8 hours because its relative density is less than that of a saturated aqueous solution of potassium chloride KCl The relative density of the solid water insoluble matter is densely suspended and separated; and separated from the aqueous solution of anthocyanins and glucose and potassium chloride;

(4) taking out the mixed combination of the anthocyanin potassium or potassium salt of the above (3) with a fatty acid or a fatty acid ester, and filtering the water therein; adding the combination of the anthocyanin potassium salt and the fatty acid or fatty acid ester thereto 10 times the volume of soft water, add appropriate potassium chloride KCl at 5 °C or reach its saturated aqueous solution concentration, so that the relative density of the aqueous solution is greater than 1; then stand at °C for 8 hours, so that fatty acids or fatty acid lipids Solid oily and intensive suspension separation, at this time, anthocyanin potassium salt is mostly aggregated and precipitated; the relative density of anthocyanin potassium salt is greater than 1.8, which is much larger than the relative density of saturated aqueous solution of potassium chloride Kcl; fatty acid or fatty acid fat is necessary Partial recycling in (a) above;

(S), at 25 ° C, filter or layering out the above (4) anthocyanin potassium salt; and then use a slight excess of 30% hydrochloric acid aqueous solution of HCI and anthocyanin potassium salt to stir the reaction, stirring speed 300-500 rpm, the anthocyanin potassium salt and the aqueous solution of HCI are fully reacted to form an anthocyanin precipitate and potassium chloride reduced by hydrogen H; the mass ratio of the anthocyanin potassium salt to the aqueous solution of HCI is 1 to 10; filtering or layering out the anthocyanin precipitate, washing the hydrochloric acid with a small amount of edible water at C-25'C, separating and separating the anthocyanin to obtain an anthocyanin solid; The anthocyanin product has the same chemical structure as the natural anthocyanin; the aqueous solution containing trace anthocyanin is recycled.

The specific points 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 number of points, which is independently selected by those skilled in the art according to the inventive principle. To save the space of the specification, the present invention does not unnecessarily repeat and describe various specific points in the data range.

13

Replacement page (Article 20)

Claims

O 2018/205846 et TM? PCT/CN2018/084667 The method for extracting high-efficiency natural plant anthocyanins, characterized in that:

1. (1) The natural plant anthocyanin aqueous solution is hydrolyzed by β-glucosidase, and the activity of β-glucosidase is protected by the anti-oxidation of L-cysteine; hydrolysis temperature 45 ° C -50 ° C, pH 4.5 -5 , time 24-48 hours; obtaining an aqueous solution containing anthocyanin/anthocyanin/glucose, containing an oil-soluble fatty acid or fatty acid ester impurity having a relative density of less than 1 dispersed in an acidic aqueous solution; fatty acid or fatty acid impurity impurity means Acidic acid of oleic acid or/and palmitic acid, an aqueous solution of anthocyanin with low content of oleic acid or/and palmitic acid should be added with an appropriate amount of acid acid of oleic acid or/and palmitic acid to enable completeness in (3) below. Suspension separation of anthocyanin sodium or potassium salt in an aqueous solution of anthocyanin;

(2) neutralizing the anthocyanin aqueous solution of the above (1) with sodium hydrogencarbonate or potassium hydrogencarbonate NaHC0 ₃ KHC0 ₃ to pH 6.8-7, anthocyanin to form its sodium or potassium salt;

(3) Adding the above (2) sodium chloride or potassium chloride Nacl/Kcl to prepare a salt saturated aqueous solution of the above (2) aqueous solution containing anthocyanin sodium or potassium salt/anthocyanin/glucose; Method, in order to save energy, at 25 ° C -50 ° C, the saturation solubility of sodium chloride or potassium chloride NaCl / Kcl in water at a certain temperature is used as a reference value, and then the value of chlorination Sodium or potassium chloride NaCl/Kcl is added to the aqueous solution containing anthocyanin/anthocyanin/glucose, and allowed to stand for 1-3 minutes to precipitate and separate excess sodium chloride or potassium chloride; An aqueous solution of anthocyanin/glucoside/sodium chloride or potassium chloride; allowed to stand for 3-8 hours to bind fatty acid or fatty acid impurities, adsorb, and physically bind water-insoluble anthocyanin sodium or potassium salt. The solid water insoluble matter is densely suspended and separated because the relative density is less than the relative density of the saturated aqueous solution of sodium chloride or potassium chloride NaCl/Kcl; and separated from the aqueous solution of anthocyanin and glucose and sodium chloride or potassium chloride;

(4) taking out the mixed combination of sodium or potassium anthocyanin or fatty acid or fatty acid ester of (3) above, and filtering the water; and combining the sodium or potassium salt of anthocyanin with fatty acid or fatty acid Add 10-20 times of edible soft water, add appropriate amount of sodium chloride or potassium chloride NaCl/Kcl at 0 °C -5 °C or reach the concentration of saturated aqueous solution, so that the relative density of the aqueous solution is greater than 1; It is allowed to stand at 0 °C -5 °C for 8-24 hours, so that the fatty acid or fatty acid ester is densely suspended and separated in solid oil form. At this time, the anthocyanin sodium salt or potassium salt is mostly aggregated and precipitated and separated; fatty acid or fatty acid ester is necessary. Partially recycled in (a) above;

14

Replacement page (Article 26) (E), at 25 ° C -50 ° C, filter or layered to remove the anthocyanin sodium or potassium salt of (4) above; and then use a slight excess of 10%-30% aqueous solution of HCI and The anthocyanin sodium or potassium salt is stirred and stirred at a rate of 300-500 rpm to fully react the anthocyanin sodium or potassium salt with the aqueous solution of HCI to form an anthocyanin precipitate and sodium chloride reduced by hydrogen H. Or potassium chloride; the mass ratio of anthocyanin sodium or potassium salt to HCI aqueous solution is 1 to 10-20; filtered or layered to remove anthocyanin precipitate, eat with a small amount at rC-25 °C The hydrochloric acid is washed with water, and the anthocyanin is precipitated and separated to obtain an anthocyanin solid; and then dehydrated and dried under vacuum to obtain an anthocyanin product having the same chemical structure as that of natural anthocyanin; and an aqueous solution of hydrochloric acid containing a trace amount of anthocyanin is recycled.

2. The method for extracting an efficient natural plant anthocyanin according to claim 1, wherein: the natural structure anthocyanin product is dissolved in a pH of 2.5 to 3.5, a reaction temperature of 50 ° C, and an ethanol volume concentration of 50% - 95%, the ratio of material to liquid 1: 10-20 hydrochloric acid and ethanol, so that the chlorine ion on the anthocyanin molecule of the natural structure is replaced by the ethanol group to form a salt-type ethanol-based anthocyanin; and the pressure is not more than 100Pa. Evaporating ethanol under reduced pressure at a temperature of 40 ° C -60 ° C; agglomerating and separating the acid-insoluble ethanol - based anthocyanins in the remaining aqueous hydrochloric acid; layering and filtering to obtain an ethanol-based flower containing a small amount of water Anthraquinone solid; vacuum dehydration drying; ethanol-based anthocyanin product.

3. The method for extracting high-efficiency natural plant anthocyanins according to claim 1, wherein: the isoelectric point PI of the β-glucosidase is mostly in the acidic range, so the acid after hydrolysis of the anthocyanin is utilized by using this property. The enthalpy of the aqueous solution is adjusted to the isoelectric point of the enzyme to recover and reuse the β-glucosidase precipitate; the specific isoelectric point of a specific β-glucosidase is measured and calculated according to the prior art; an aqueous solution of an acid ΡΗ value, adjusted to the above anthocyanin hydrolysis with sodium or potassium hydrogen carbonate and the aqueous acid PH value of NaHC0 _₃ / KHC0 ₃ to the isoelectric point; below the isoelectric point PI hydrolysis of anthocyanins After the pH of the aqueous acid solution, the pH of the aqueous acid solution was adjusted to the isoelectric point with an aqueous solution of HCI; the isoelectric point PI of L-cystine was 4.6, and the pH of the aqueous solution of the anthocyanin was adjusted to L- The isoelectric point PI of cystine precipitates and separates L-cystine.

The method of extracting a natural plant efficiency anthocyanin claim wherein: substituting _{_{KHC0 3 / Kcl NaHC0 3 / Nacl}} , aqueous Kcl / KHC0 ₃ containing as a fertilizer or crop recovered solid was dried Kcl / KHC0 _3.

15

Replacement page (Article 26) The method for extracting high-efficiency natural plant anthocyanins according to claim 1, wherein: the aqueous solution containing anthocyanin or the potassium chloride NaCl/Kcl solution in the above technical scheme 1, (3) is Vacuum dehydration and drying at 70 ° C-100 ° C; continuous recovery of sodium chloride or potassium chloride NaClClCl due to dehydration during the drying process, sodium chloride or potassium chloride NaCl/Kcl recycling, containing a small amount of chlorination Recovery of anthocyanins from sodium or potassium chloride, NaCl/Kcl and glucose; or electrolysis of an aqueous solution of anthocyanin-containing saturated sodium chloride or potassium chloride Nacl Kcl in the above technical schemes 1, (3) with a cation exchange membrane Recycling sodium hydroxide or potassium hydroxide NaOH/KOH aqueous solution and hydrochloric acid Hcl aqueous solution or chlorine gas, non-electrolyte anthocyanin and glucose aqueous solution recycling; or the above technical scheme 1,

(3) The saturated potassium chloride KCl aqueous solution containing anthocyanins and glucose is decolorized by activated carbon, and the anthocyanins are separated and precipitated, and the saturated potassium chloride KCl aqueous solution containing glucose is recycled or reused or directly used in agriculture. fertilizer.

6, according to claim 1, method of efficiently extracting a natural plant anthocyanin wherein: in place of sodium or potassium bicarbonate NaHC0 _₃ KHC0 ₃ and anthocyanin with sodium or potassium hydroxide NaOH / KOH section For the hydrolysis of the aqueous solution, the pH of the anthocyanin-hydrolyzed aqueous solution is adjusted to pH 5.5-6 with an appropriate amount of PH9-14 NaOH/KOH aqueous solution, and the NaOH/KOH aqueous solution is slowly added while rapidly stirring at a speed of 300-500 rpm. or above the NaOH / KOH in aqueous anthocyanin distribution hydrolyzed; adding an appropriate amount of NaHC0 _₃ KHC0 ₃ to adjust the PH value of the aqueous solution of anthocyanin hydrolyzed to PH7;

A method of partially neutralizing an aqueous solution of anthocyanin by using sodium carbonate/potassium carbonate in place of sodium hydrogencarbonate or potassium hydrogencarbonate NaHC0 ₃ KHC0 ₃ , and hydrolyzing an aqueous solution of anthocyanin with an appropriate amount of an aqueous solution of sodium carbonate or potassium carbonate of pH 9-10 Adjust the pH to PH5.5-6, slowly add sodium carbonate or potassium carbonate aqueous solution and stir at the same time, rotate at 300-500 rpm or above, so that sodium carbonate or potassium carbonate is evenly distributed in the aqueous solution of anthocyanin hydrolysis; The pH of the anthocyanin-hydrolyzed aqueous solution was adjusted to pH 7 by adding an appropriate amount of sodium hydrogencarbonate or potassium hydrogencarbonate NaHC0 ₃ KHC0 ₃ .

16

Replacement page (Article 26)