Preparation method of high-purity anthocyanin

Abstract

The invention discloses a preparation method of high-purity anthocyanin, which comprises the following steps: taking mulberry residue as a raw material, adding an acidified ethanol aqueous solution, standing for 2 hours at a low temperature of 4 DEG C, carrying out suction filtration to collect filtrate, and carrying out vacuum concentration to obtain concentrated anthocyanin crude extract; enabling the crude extract to flow through a D001 cationic resin column for adsorption, firstly washing away polysaccharide substances by using an acidic solution, then washing away flavonoid substances by using an acidified ethanol aqueous solution, finally eluting adsorbed anthocyanin by using an acidified ethanol salt-containing solution, and concentrating the collected anthocyanin salt-containing solution under reduced pressure to obtain an anthocyanin salt-containing solution; removing salt in the anthocyanin salt-containing solution by a resin method or a dialysis method, and freeze-drying to obtain anthocyanin with the purity of at least 95%. According to the preparation method, polysaccharide substances and flavonoid substances in the crude extract can be efficiently removed, anthocyanin is enriched, high-purity anthocyanin is obtained through desalting and freeze drying, no toxic reagent is used in the whole process, the product is non-toxic and harmless, and waste liquid does not pollute the environment.

Description

Technical Field

[0001] The invention belongs to the field of separation and purification, and relates to a method for preparing anthocyanidins, in particular to a method for preparing high-purity anthocyanidins. Background Art

[0002] Anthocyanins, as a natural plant pigment, are not only widely available but also possess good biological activity. In recent years, research on anthocyanins has gradually deepened, and the demand for higher-purity anthocyanins has also increased. However, the current anthocyanins on the market are priced high, and most of them use a lot of toxic reagents, making them difficult to use at a high level, mainly because the purification cost of anthocyanins is low.

[0003] Currently, fruits and vegetables containing anthocyanins on the market include mulberries, Sanhua plums, blueberries, purple sweet potatoes, black beans, etc. Mulberries are a relatively common fruit containing anthocyanins, which contain glucose, vitamins, amino acids, mineral elements, etc. Currently, in addition to being prepared into juice, the by-product of mulberries on the market, mulberry residue, is produced in large quantities and is often discarded as waste. Studies have shown that mulberry residue contains a large amount of anthocyanins, and the anthocyanins in it are relatively stable. Extracting and purifying anthocyanins from it can turn waste into treasure, improve the utilization rate of mulberry residue, and achieve sustainable resource utilization.

[0004] Currently, methods for purifying anthocyanins include macroporous resins, membrane separation, gel chromatography, and high-speed countercurrent chromatography. However, these methods all have drawbacks such as low purification rates, high costs, and the use of a large number of toxic reagents, which are associated with limitations. Meanwhile, cationic resins can also be used for anthocyanin purification, but these also have drawbacks such as the use of toxic reagents and limited sample loading. Summary of the Invention

[0005] In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, the object of the present invention is to provide a method for preparing high-purity anthocyanins, which is non-toxic and harmless, green and environmentally friendly, and has high efficiency and high product purity.

[0006] The method for preparing high-purity anthocyanidins of the present invention comprises the following steps: A. Add acidified ethanol and water to mulberry residue, let stand at 4°C for 2 hours, collect the filtrate by suction, and concentrate under reduced pressure to obtain a concentrated mulberry residue anthocyanin crude extract. B. Pass the crude extract obtained in step A through a D001 cationic resin column for adsorption. First, wash away the polysaccharide with an acidic solution, then wash away the flavonoids with an acidified ethanol aqueous solution, and finally elute the adsorbed anthocyanidins with an acidified ethanol saline solution. The collected anthocyanidin saline solution is concentrated under reduced pressure to obtain an anthocyanidin saline solution. C. removing salt from the anthocyanin salt solution by a resin method or a dialysis method, and collecting anthocyanin purified samples by freeze-drying to obtain anthocyanin with a purity of at least 95%.

[0007] According to a further feature of the method for preparing high-purity anthocyanins of the present invention, in step A, the acidified ethanol aqueous solution is an ethanol aqueous solution acidified with citric acid, wherein the volume fraction of ethanol is 60% and the volume fraction of citric acid is 1%.

[0008] According to a further feature of the method for preparing high-purity anthocyanins of the present invention, in step B, the acidic solution is a citric acid solution with a volume fraction of 1%, and its amount is at least three times the volume of the D001 cationic adsorption resin column.

[0009] According to a further feature of the method for preparing high-purity anthocyanins according to the present invention, in step B, the acidified ethanol aqueous solution is an ethanol aqueous solution acidified with citric acid, wherein the volume fraction of ethanol is 60% and the volume fraction of citric acid is 1%; the amount of the acidified ethanol aqueous solution is at least three times the volume of the D001 cationic adsorption resin column.

[0010] According to a further feature of the method for preparing high-purity anthocyanins according to the present invention, in step B, the acidified ethanol saline solution is an ethanol saline solution acidified with citric acid, wherein the volume fraction of ethanol is 50%, the volume fraction of citric acid is 1%, and the molar concentration of sodium chloride is 0.25 mol / L.

[0011] According to a further feature of the method for preparing high-purity anthocyanins of the present invention, in step C, the resin method is to add the anthocyanin salt solution obtained in step B to a pretreated resin for adsorption, slowly add water for elution, and measure the soluble salt content in the water with a salinity meter. When the soluble content remains constant, the water elution is stopped, and acidified ethanol is added to elute the anthocyanins adsorbed on the macroporous resin. The collected anthocyanin purified solution is concentrated under reduced pressure to obtain a salt-free anthocyanin solution.

[0012] Preferably, the resin is AB-8 or D101 macroporous resin; the acidified ethanol solution is anhydrous ethanol acidified with citric acid, wherein the volume fraction of citric acid is 1%.

[0013] According to a further feature of the method for preparing high-purity anthocyanins of the present invention, in step C, the dialysis method is to add the anthocyanin salt solution obtained in step B to a dialysis bag, stir it magnetically at low temperature, change the water every 12 hours, and measure the soluble salt content in the water with a salinity meter. When the soluble content remains constant, stop the dialysis, and freeze-dry to collect the purified anthocyanin sample.

[0014] Preferably, the cut-off capacity of the dialysis bag is 3000Da.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) This invention uses mulberry residue, a byproduct of mulberry juice processing, as raw material and achieves efficient resource conversion through innovative technology. Compared with traditional single processing methods, it not only significantly reduces application costs but also develops a breakthrough deep processing and utilization path for mulberry residue.

[0016] (2) The preparation method described in the present invention can efficiently remove polysaccharides and flavonoids from the crude extract, enrich anthocyanins, elute to obtain a high-purity anthocyanin salt solution, and then obtain high-purity anthocyanins through desalination and freeze-drying.

[0017] (3) D001 strong acid cationic resin is used for purification and preparation. No toxic reagents are used throughout the process. The product is non-toxic and harmless. The waste liquid will not pollute the environment. The main ethanol used can be recycled.

[0018] (4) In the process of desalination, the method of removing salt by dialysis can achieve the same effect as the resin method of desalination. However, compared with the resin desalination method which requires reduced pressure concentration, the purified mulberry anthocyanin solution after dialysis to remove salt can be directly freeze-dried. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the liquid chromatogram of mulberry residue anthocyanin D113 resin at 280 nm and 520 nm.

[0020] Figure 2 This is the liquid chromatogram of mulberry residue anthocyanin D152 resin at 280 nm and 520 nm.

[0021] Figure 3 This is the liquid chromatogram of anthocyanin from mulberry residue SCX resin at 280 nm and 520 nm.

[0022] Figure 4 Shows the change in sugar content of mulberry residue anthocyanin D001 resin when using acidified aqueous solution.

[0023] Figure 5 This is the liquid chromatogram at 280 nm of the amount of flavonoids eluted from mulberry residue anthocyanin D001 resin using acidified ethanol.

[0024] Figure 6 This is the liquid chromatogram of mulberry residue anthocyanin D001 resin using acidified ethanol aqueous solution with different salt concentrations at 280nm.

[0025] Figure 7 The following are the liquid chromatograms of mulberry anthocyanins without and without desalination at 280 nm.

[0026] Figure 8 This is a diagram of the screening factors for using macroporous resin in the process of removing salt from mulberry anthocyanin purification solution.

[0027] Figure 9 This is a comparison chart of two methods for desalting anthocyanins in dialysis bags. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below with reference to the examples and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the experimental methods used in the present invention are conventional methods; the materials and reagents used are all commercially available.

[0029] Experimental reagents: Strong cationic resin D001, Beijing Solebow Co., Ltd.; formic acid (chromatographic grade), Tianjin Kemeiou Chemical Reagent Co., Ltd.; acetonitrile (chromatographic grade), Merck KGaA, Germany.

[0030] The entire extraction, impurity removal and concentration process involved in the examples were carried out under light-proof conditions. The chemical reagents used in the operation steps involved in the examples were all analytically pure.

[0031] Experimental instruments and equipment: glass column, Guangzhou Puzhi Co., Ltd.; dialysis bag (cut-off volume 3000Da), Shanghai Yuanye Biotechnology Co., Ltd.; C18 (2.1×100mm, 1.8um) chromatographic column, Agel-Phenome Company; high-performance liquid chromatograph, Thermo Fisher Scientific.

[0032] Example 1: Extraction of anthocyanins from mulberry residue Preparation of mulberry residue: mulberries purchased from the market are used, crushed and filtered using a wall breaking machine to obtain the mulberry residue.

[0033] Acidified ethanol aqueous solution was added to the mulberry residue, and the mixture was placed in a 4°C refrigerator for low temperature standing for 2 hours. Then, the crude anthocyanin extract of the mulberry residue was collected by extraction, and the extract was concentrated under reduced pressure to obtain a concentrated solution.

[0034] This example adopts a three-extraction method, specifically: 467.5 g of mulberry residue was weighed, 9.35 L of 1% citric acid 60% ethanol aqueous solution was added for the first extraction, and the first extraction residue was obtained after filtration; 390.0 g of mulberry residue anthocyanins were weighed, 3.9 L of 1% citric acid 60% ethanol aqueous solution was added for the second extraction, and the second extraction residue was obtained after filtration; 390.0 g of mulberry residue anthocyanins were weighed, 2 L of 1% citric acid 60% ethanol aqueous solution was added for the third extraction, and the third extraction residue was obtained after filtration. The three anthocyanin crude extract solutions were concentrated under reduced pressure at 42° C. until there was no alcohol taste, to obtain 6 L of anthocyanin concentrate.

[0035] Example 2: Purification of anthocyanins from mulberry residue Screening and pretreatment of cationic resins: Select more common cationic resins for comparative experiments.

[0036] The anthocyanin concentrate obtained in Example 1 was purified using two weakly acidic cationic resins, D152 and D113. The HPLC spectrum is shown in FIG. Figure 1 and Figure 2 As shown in the figure, the results show that the purification effect of these two resins is not ideal. Anthocyanin molecules are difficult to effectively adsorb on these resins, which may be because the exchange capacity of the resin is reduced under acidic conditions, and hydrogen ions are not easily dissociated, thus affecting the resin's adsorption capacity. Weakly acidic cationic resins can only perform their exchange function more effectively in a moderately alkaline environment, but under such conditions, anthocyanins may be degraded. Therefore, these two resins did not show good adsorption effect.

[0037] The results of purifying the anthocyanin concentrate obtained in Example 1 using SCX strong acid cationic resin are as follows: Figure 3 As shown, the purification effect is good. Both peaks appearing in the HPLC spectrum represent anthocyanins. However, SCX resin uses toxic reagents such as trifluoroacetic acid during the activation process. Trifluoroacetic acid is highly corrosive and irritating, posing a potential hazard to human health, which may limit its application in production. In addition, SCX resin is packaged in a relatively small column, requiring a vacuum pump to expel the liquid faster. Therefore, the sample volume is small, typically 100 mL.

[0038] D001 Strong Acid Cationic Resin achieves the same purification results as SCX resin, without the use of toxic reagents, making it suitable for production applications. D001 resin can be installed in large columns, eliminating the need for a vacuum pump. The flow rate can be freely controlled using a valve below, allowing for larger sample loads, up to approximately 400 mL.

[0039] Pretreatment method for D001 cationic resin: After loading the resin into the column, rinse with deionized water until the outflow or backwash water is colorless and has minimal foam. First, slowly flow hydrochloric acid solution through the resin at a dosage of 3 times the resin volume and a flow rate of 1.5 times the resin volume / hour (discharge twice the volume before, recover twice the volume after), rinse with water, and the pH of the effluent is around 2-4. Then, flow sodium hydroxide solution through the resin at the same dosage and flow rate as in step 1 (discharge once the volume before, recover twice the volume after), rinse with water to a pH of around 10-12. Then, convert the resin to the hydrogen form with 4% hydrochloric acid at a dosage of 3 times the resin volume and the same flow rate as in step 1 (discharge twice the volume before, recover twice the volume before, and prepare fresh water for recovery). Finally, after the hydrochloric acid solution has flowed through the resin, rinse with deionized water until the pH of the effluent is around 7, and it is ready for use.

[0040] The concentrated solution obtained in Example 1 is added to the D001 cationic resin. When the concentrated solution is completely adsorbed in the column, the polysaccharide substances are first removed by washing with an acidic solution, and then the flavonoid substances are removed by washing with acidified ethanol. Finally, the anthocyanins adsorbed on the cationic resin are eluted with a saline solution of acidified ethanol. Finally, the collected anthocyanin saline solution is concentrated under reduced pressure to obtain an anthocyanin saline solution with a purity of more than 95%.

[0041] In this embodiment, the collected concentrate was loaded in an amount of about 800 mL, and the elution volumes of the eluted polysaccharides, flavonoids, and anthocyanins were screened according to the HPLC chart. Figure 4 、 Figure 5 and Figure 6 The volume of the acidified aqueous solution elution is 3.2 L, the volume of the acidified ethanol aqueous solution elution is 3.2 L, and then the anthocyanin is eluted with the acidified ethanol aqueous solution containing salt. The specific volume of the eluted anthocyanin is based on visual observation. When the color of the eluted effluent becomes significantly lighter, the sample collection can be stopped. The above-obtained anthocyanin salt-containing purified solution is concentrated under reduced pressure at 42°C until there is no alcohol taste.

[0042] Example 3: Desalination of anthocyanin salt-containing purified solution Resin desalination or dialysis desalination can be used.

[0043] Resin method: First pre-treat the resin, screen it according to the adsorption rate and resolution rate, and select the most suitable resin model for desalting operation. Add an appropriate amount of mulberry anthocyanin salt-containing purified solution to the resin, add water at a slower rate to elute, and use a salinity meter to measure the soluble salt content in the water. When the soluble content remains constant, the water elution can be stopped. In the subsequent process, acidified ethanol is added to elute the anthocyanin adsorbed on the macroporous resin. The collected anthocyanin purified solution is concentrated under reduced pressure to obtain a salt-free anthocyanin solution. Finally, the anthocyanin purified sample is freeze-dried and the anthocyanin chromatogram is detected by HPLC ( Figure 7 ), the purity reached 95% by summing the peak areas at 280 nm.

[0044] In this example, four resin pretreatments were selected, and the resins were subjected to static adsorption and desorption experiments respectively. In the static adsorption experiment, the volume ratio of the resin mass to the anthocyanin crude extract was 1:50 (g / mL), and the mixture was shaken at an ambient temperature of 30°C and a rotation speed of 100 rpm for 4 hours. 1 mL was collected every hour and placed in a -80°C environment for subsequent liquid phase detection of anthocyanin content. In the static desorption experiment, the volume ratio of the resin mass to the acidified ethanol solution was 1:50, and the ambient conditions remained unchanged. 1 mL was collected every hour and placed in a -80°C environment for subsequent liquid phase detection of anthocyanin content. Figure 8As shown in the figure, the adsorption effect of XAD-7 resin is poor, and the desorption effect of XDA-6 is not as good as the other three resins. Considering the adsorption effect and desorption effect, AB-8 and D101 resins perform better in terms of adsorption rate and desorption rate, and can both be used for desalination.

[0045] Resin desalination experiment: The salt-containing mulberry anthocyanin solution collected above was eluted with AB-8 or D101 resin, with a sample volume of about 200 mL and a water elution volume of 600 mL to remove salt. The soluble salt content was measured with a salinity meter until it was less than 100 ppm. Then, the volume of acidified ethanol aqueous solution added was 600 mL, and the salt-free mulberry anthocyanin purified solution was collected and concentrated under reduced pressure at 42°C until there was no alcohol taste.

[0046] Dialysis method: Boil the dialysis bag and use it directly.

[0047] This example compares the use of flowing water combined with magnetic stirring with the use of magnetic stirring alone (see Figure 9 ), using flowing water combined with magnetic stirring method, the water flow rate is 20mL / min, 10mL of the solution to be desalted is added to the dialysis bag, and the dialysis bag is placed in 250mL of pure water. After 7.5h, the liquid in the dialysis bag is measured by a salinity meter and shows 195ppm. After using only a magnetic stirrer, the water is changed every 2.5h. At this time, the liquid in the dialysis bag is measured by a salinity meter and shows 40ppm. Under the same time, magnetic stirring combined with mid-way water change is a relatively fast desalination method, and the water consumption is also less at this time. The above-collected salt-containing mulberry anthocyanin purified solution is placed in a dialysis bag, and the water is changed every 12 hours. The soluble salt content is measured with a salinity meter until it is less than 100ppm. The dialysis can be stopped and freeze-dried to obtain the anthocyanin purified product.

[0048] All the above purity determinations were performed using HPLC. A C18 reversed-phase chromatography column (2.1x100 mm, 1.8 um) was used, the column temperature was 30°C, mobile phase A was 100% acetonitrile, mobile phase B was 2% formic acid in water, the single injection volume was 15.0 uL, the flow rate was 1.0 mL / min, the detection wavelength was 520 nm, and the gradient elution program was: 0-34 min, 6%-30% A; 34-35 min, 30%-95% A; 35-40 min, 95% A; 40-41 min, 95%-6% A; 41-45 min, 6% A.

Claims

1. A method for preparing high-purity anthocyanidins, characterized in that: The following steps are involved: A. Add acidified ethanol and water to mulberry residue, let stand at 4°C for 2 hours, collect the filtrate by suction, and concentrate under reduced pressure to obtain a concentrated mulberry residue anthocyanin crude extract. B. Pass the crude extract obtained in step A through a D001 cationic resin column for adsorption, first wash away the polysaccharide with an acidic solution, then wash away the flavonoids with an acidified ethanol aqueous solution, and finally elute the adsorbed anthocyanidins with an acidified ethanol saline solution. The collected anthocyanidin saline solution is concentrated under reduced pressure to obtain an anthocyanidin saline solution; C. removing salt from the anthocyanin salt solution by a resin method or a dialysis method, and collecting anthocyanin purified samples by freeze-drying to obtain anthocyanin with a purity of at least 95%.

2. The method for preparing high-purity anthocyanidins according to claim 1, wherein: In step A, the acidified ethanol aqueous solution is an ethanol aqueous solution acidified with citric acid, wherein the volume fraction of ethanol is 60% and the volume fraction of citric acid is 1%.

3. The method for preparing high-purity anthocyanidins according to claim 1, wherein: In step B, the acidic solution is a citric acid solution with a volume fraction of 1%, and its amount is at least three times the volume of the D001 cationic adsorption resin column.

4. The method for preparing high-purity anthocyanidins according to claim 1, wherein: In step B, the acidified ethanol aqueous solution is an ethanol aqueous solution acidified with citric acid, wherein the volume fraction of ethanol is 60% and the volume fraction of citric acid is 1%; and the amount of the acidified ethanol aqueous solution used is at least three times the volume of the D001 cationic adsorption resin column.

5. The method for preparing high-purity anthocyanidins according to claim 1, wherein: In step B, the acidified ethanol saline solution is a citric acid acidified ethanol saline solution, wherein the volume fraction of ethanol is 50%, the volume fraction of citric acid is 1%, and the molar concentration of sodium chloride is 0.25 mol / L.

6. The method for preparing high-purity anthocyanidins according to claim 1, wherein: In step C, the resin method is to add the anthocyanin salt solution obtained in step B to the pretreated resin for adsorption, slowly add water for elution, and measure the soluble salt content in the water with a salinity meter. When the soluble content remains constant, the water elution is stopped, and acidified ethanol is added to elute the anthocyanin adsorbed on the macroporous resin. The collected anthocyanin purified solution is concentrated under reduced pressure to obtain a salt-free anthocyanin solution.

7. The method for preparing high-purity anthocyanidins according to claim 6, wherein: The resin is AB-8 or D101 macroporous resin; the acidified ethanol solution is anhydrous ethanol acidified with citric acid, wherein the volume fraction of citric acid is 1%.

8. The method for preparing high-purity anthocyanidins according to claim 1, wherein: In step C, the dialysis method is to add the anthocyanin salt solution obtained in step B into a dialysis bag, stir it with low-temperature magnetic force, change the water every 12 hours, and measure the soluble salt content in the water with a salinity meter. When the soluble content remains constant, stop dialysis, and freeze-dry to collect the anthocyanin purified sample.

9. The method for preparing high-purity anthocyanidins according to claim 8, wherein: The cut-off capacity of the dialysis bag is 3000Da.

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