Purification method and application of plant source high-purity betacyanin

Abstract

The invention provides a purification method of plant-derived high-purity betacyanin, which comprises the following steps: firstly, mashing plant raw materials, filtering to obtain juice, standing, and taking supernate; secondly, adding the supernate into a cation exchange resin column, firstly eluting with water, and then eluting with an aqueous solution of inorganic salt to obtain a first eluent; thirdly, adding the first eluent into a glass column filled with ODS filler, firstly eluting with water, and then eluting with an ethanol solution to obtain a second eluent; and finally, carrying out drying treatment on the second eluent to obtain the betacyanin. The purification method provided by the invention has specificity on the betacyanin, and can realize high-purity extraction of the betacyanin and effective removal of impurities. The method is widely applicable to various edible plants and non-edible materials containing the betacyanin, material sources for purifying the betacyanin are expanded, comprehensive utilization of resources is facilitated, and the method has wide popularization and application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural pigment purification, and particularly relates to a method for purifying high-purity betacyanin from plant sources, and also relates to the application of the above purification method in the purification of betacyanin from plant sources. Background Art

[0002] Betacyanin is a red pigment present in plants such as Caryophyllaceae, Chenopodiaceae, Amaranthaceae, Phytolacca acinosa, etc. This type of pigment has characteristics such as non-toxicity and bright color, and also has health care effects such as antioxidant properties. It can be used as a colorant for foods (such as ice cream, red wine, cocktails, etc.) and cosmetics (such as lipsticks, rouges, etc.). Currently, the red pigment extract from beets has been approved by the United States and the European Union for use as a food colorant. However, plant-derived extracts containing betacyanin often contain impurities unrelated to the pigment, and these impurities may be toxic. For example, phytolaccasaponin in the red pigment extract of Phytolacca americana berries can cause diarrhea when mixed in; the red pigment extract of pitaya contains other small molecules, impurities such as polysaccharides and proteins, and mixing them will affect the edible taste. Therefore, a suitable process is needed to remove these toxic or taste-affecting impurities.

[0003] Currently, the existing methods for purifying plant-derived betacyanin mainly include the solvent method, macroporous resin method, and ion resin method. CN 101429347A and CN 111480828A use styrene-based macroporous resins to purify betacyanin from red beets and Suaeda salsa respectively. This method utilizes the different adsorption properties of macroporous resins due to their low polarity towards organic molecules, and does not have specificity, so it cannot remove the mixed impurities. CN 108624082A purifies betacyanin from Phytolacca acinosa using the ion exchange resin method, but the resin used is an anion resin, which does not have the ability to specifically adsorb betacyanin, and the elution solvent contains hydrochloric acid solution, which is likely to cause the decomposition of betacyanin and may also introduce strongly acidic impurities. In summary, the existing methods cannot effectively remove the impurities mixed in betacyanin, so high-purity betacyanin cannot be obtained.

[0004] Based on this, providing a method for obtaining high-purity red pigment from plant extracts containing betacyanin to effectively remove irrelevant impurities and improve the purity of the product betacyanin is a technical problem that urgently needs to be solved. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a method for specifically purifying betacyanin contained in plant sources to effectively remove irrelevant impurities and ensure the purity of the product betacyanin.

[0006] The second purpose of the present invention is to provide the application of a method for purifying high-purity betacyanin from plant sources.

[0007] One of the technical solutions adopted to achieve the purpose of the present invention is to provide a method for purifying high-purity betacyanin from plants, which includes the following steps:

[0008] S1. Mash the plant raw materials and filter to obtain juice, and take the supernatant after standing;

[0009] S2. Add the supernatant into a cation exchange resin column, first elute with water, and then elute with an aqueous solution of inorganic salt with a mass concentration of 1% - 10% to obtain a first eluate;

[0010] S3. Add the first eluate into a glass column filled with ODS packing material, first elute with water, and then elute with an ethanol solution with a volume concentration of 20% - 100% to obtain a second eluate;

[0011] S4. Perform drying treatment on the second eluate to obtain betacyanin.

[0012] The general idea of the method for purifying high-purity betacyanin from plants provided by the present invention is as follows:

[0013] In view of the problems in the prior art that the purity of the prepared betacyanin is not high and it is mixed with other components, which restricts the safety or taste of betacyanin as a food additive and health product, the present invention provides a specific method for purifying betacyanin, removing the impurities mixed in the betacyanin extract, obtaining high-purity plant-derived betacyanin, and improving its safety and edibility.

[0014] In the present invention, the idea of "cation exchange resin + inorganic salt eluent + ODS packing material treatment" is adopted. First, the cation exchange resin is used to specifically adsorb betacyanin, excluding the co-adsorption of most impurities; secondly, an aqueous solution of inorganic salt with a mass concentration of 1% - 10% is used to elute the adsorbed betacyanin, avoiding the use of strong acidic solutions such as hydrochloric acid, and preventing the destruction of betacyanin by strong acids, as well as the damage to operators and equipment; finally, the ODS packing material (Octadecylsilyl) is used to further remove impurities from the betacyanin eluate and specifically remove inorganic salts, and then elute with an ethanol solution with a volume concentration of 20% - 100%. Finally, a betacyanin product with a pigment purity greater than 97% is obtained.

[0015] Among them, the aqueous solution of inorganic salts can provide cations, which combine with the functional groups in the cation exchange resin, thereby replacing and eluting the betacyanin substances adsorbed in the cation exchange resin. For the aqueous solution of inorganic salts, if its concentration is too low, the elution ability is limited, which will cause the volume of the eluate to be too large, and the betacyanin elution band will be elongated and difficult to collect; if its concentration is too high, the salt content in the eluate will be too high, increasing the pressure of eluting salts in the next ODS step and also increasing unnecessary purification costs. In summary, the present invention sets the mass concentration of the aqueous solution of inorganic salts to 1% - 10%. Further, during the elution process of the ethanol solution, similar problems will occur if the concentration of the ethanol solution is too low or too high. Therefore, the present invention uses an ethanol solution with a volume concentration of 20% - 100%. Preferably, the mass concentration of the aqueous solution of inorganic salts is 2%, and the volume concentration of the ethanol solution is 20%.

[0016] Further, in step S1, the plant raw material includes one or more combinations of pitaya, beet tubers, and pokeberries.

[0017] Preferably, for pokeberries, in step S1, it further includes: adding an ethanol solution with a volume concentration of 70% - 100% to the filtered juice, fully mixing to make the final volume concentration of the ethanol solution in the juice 60% - 80%, standing for 1 - 5 h to precipitate impurities, and then taking the supernatant for subsequent operations. Pokeberries belong to non-edible plant raw materials (the contained phytolaccasaponin has certain toxicity). Using a high-concentration ethanol solution to pretreat the juice can remove toxic small-molecule compounds, and combined with subsequent treatment steps, effective impurity removal can be achieved to ensure the purity of betacyanin.

[0018] Further, in step S2, the cation exchange resin column is filled with a cation exchange resin; the functional groups of the cation exchange resin include one or more combinations of carboxyl group, sulfonic acid group, sulfate group, and phenolic hydroxyl group.

[0019] Preferably, the model of the cation exchange resin is selected from one of D113, D204, 001*7H, Zheng Guang D001FC, Amberlite IRA-67, Dowex 66, Dowex 50, Amberlite IRA-400, Dowex1X8, Amberlite IRA-402, Dowex M419. More preferably, the D113 cation exchange resin is used, which has a weak acidic group, can specifically adsorb the imino positive ion of betacyanin, and has the characteristic of not adsorbing other irrelevant impurities.

[0020] Further, in step S2, the volume-mass ratio of the supernatant to the cation exchange resin is 1:(2-10) mL / g; the water consumption for water elution and the volume ratio of the supernatant is (2-20):1.

[0021] Further, in step S2, in the aqueous solution of the inorganic salt, the inorganic salt is selected from one or a combination of more of sodium chloride, sodium bromide, sodium iodide, potassium chloride, potassium bromide, potassium iodide, magnesium chloride, magnesium bromide, sodium sulfate, potassium sulfate, magnesium sulfate, sodium nitrate, potassium nitrate, and magnesium nitrate.

[0022] Preferably, the aqueous solution of the inorganic salt is an aqueous solution of sodium chloride, which has advantages such as non-toxicity, wide raw material sources, and low price.

[0023] Further, in step S2, the volume ratio of the amount of the aqueous solution of the inorganic salt used to the volume of the supernatant is (2-20):1.

[0024] Preferably, in step S2, the part with a relatively strong red color in the solution obtained after elution is collected to obtain a first eluate.

[0025] Further, in step S3, the volume-mass ratio of the first eluate to the ODS packing is 1:(2-10) mL / g.

[0026] Further, in step S3, the water consumption for water elution and the volume ratio of the first eluate is (2-20):1; the volume ratio of the ethanol solution with a volume concentration of 20%-100% used to the volume of the first eluate is (2-20):1.

[0027] Preferably, in the elution processes of step S2 and step S3, the amount of each eluate used is 3-4 column bed volumes. Specifically, considering the differences in the content of betacyanin in different plant raw materials, during the elution process, it can be flexibly adjusted by observing the color of the resin column or silica gel column, and elution can be stopped until the red color band disappears.

[0028] Preferably, in step S3, the part with a relatively strong red color in the solution obtained after elution is collected to obtain a second eluate.

[0029] Further, in step S4, the drying treatment includes freeze-drying or spray-drying. Preferably, the drying treatment is carried out by freeze-drying. The temperature of freeze-drying is -40 to -10°C, and the time is 1 to 8 h. The finally obtained product is in the form of a solid powder.

[0030] The technical solution adopted to achieve the second object of the present invention is: to provide an application of the purification method according to the first object of the present invention.

[0031] Further, the purification method is used to purify betacyanin in plant raw materials.

[0032] In the present invention, the plant raw materials include both edible plants such as pitaya and beet, and non-edible materials such as pokeberry berries. The purification method provided by the present invention has good impurity removal effect and high purity of the product pigment, expanding the material source for purifying betacyanin. The high-purity betacyanin purified by the present invention can be used as an additive for food and health products, has good compatibility with other additives, is not easily decomposed, is beneficial to maintaining the stability of the product, and prolongs the storage period of the product.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] (1) The present invention provides a method for purifying high-purity betacyanin from plants, adopting the idea of combining "cation exchange resin + salt eluent + ODS packing treatment". First, the cation exchange resin is used to specifically adsorb betacyanin, excluding the co-adsorption of most impurities; secondly, a neutral inorganic salt solution is used to elute the adsorbed betacyanin, avoiding the destruction of betacyanin by strong acids and the damage to operators and equipment; finally, the ODS packing is used to further remove impurities and salts from the betacyanin eluent, and finally a betacyanin product with a pigment purity greater than 97% is obtained.

[0035] (2) The present invention provides a method for purifying high-purity betacyanin from plants, using an aqueous solution of a neutral inorganic salt as the eluent, avoiding the use of strong acid solutions such as hydrochloric acid. The existing purification scheme using hydrochloric acid as the eluent will cause the pH of the product to be acidic. Using acidic betacyanin as a food additive will not only affect the compatibility of the additive, but also have an adverse effect on the taste of the food. The purification conditions of the present invention are mild, the product purity is high, the pH value of the product is 6.5 - 6.8, the prepared betacyanin has good compatibility with other additives, is not easily decomposed, is beneficial to maintaining the stability of the product, and prolongs the storage period of the product.

[0036] (3) The present invention provides a method for purifying high-purity betacyanin from plants, which is specific for betacyanin, and is not only applicable to extracting betacyanin from edible plants such as pitaya and beet, but also applicable to obtaining high-purity betacyanin from non-edible materials such as pokeberry berries, expanding the material source for purifying betacyanin, and being beneficial to the comprehensive utilization of resources. Description of the Drawings

[0037] Figure 1 It is a process schematic diagram of a method for purifying high-purity betacyanin from plants provided by an embodiment of the present invention;

[0038] Figure 2 It is the chemical structural formula of betacyanin, where R1 and R2 are different glycosyl substitutions;

[0039] Figure 3 FIG. 3 is the HPLC chromatogram of the crude extract of betacyanin extracted from pokeberry fruits in Example 3 of the present invention; (a) is the chromatogram at 220 nm; (b) is the chromatogram at 520 nm.

[0040] Figure 4 FIG. 4 is the HPLC chromatogram of the purified extract of betacyanin extracted from pokeberry fruits in Example 3 of the present invention; (a) is the chromatogram at 220 nm; (b) is the chromatogram at 520 nm. Detailed Description of the Invention

[0041] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the scope of protection of the present invention.

[0042] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments may be combined with each other.

[0043] Figure 1 FIG. 1 is a schematic flow chart of the purification method of high-purity betacyanin from plants provided by the present invention. This purification method utilizes the characteristics that cation exchange resin can specifically adsorb betacyanin in the extract and can be eluted by sodium chloride aqueous solution to remove most of the impurities. Subsequently, ODS packing can specifically remove sodium chloride, thereby obtaining high-purity betacyanin, which can be used as an additive for food and health care.

[0044] The chemical structure of betacyanin is as Figure 2 shown. It has a characteristic iminium cation in its structure, which can specifically bind to the anionic group of the cation exchange resin.

[0045] The present invention will be further described below in conjunction with specific embodiments, but it is not limited to the present invention.

[0046] Example 1

[0047] The pitaya was mashed and filtered. After the juice was allowed to stand, the supernatant was taken. 10 mL of the red supernatant was added to a cation exchange resin column filled with 70 g of D113. After washing with 160 mL of water, it was washed with 160 mL of sodium chloride with a mass concentration of 2%. The eluate in the concentrated red part was collected. 20 mL of the red eluate was loaded onto a glass column filled with 50 g of ODS packing. After washing with 120 mL of water, it was eluted with 120 mL of ethanol solution with a volume concentration of 20%. The eluate in the concentrated red part was collected and freeze-dried to obtain a red solid. The pigment purity was determined by HPLC normalization method to be over 98.3%.

[0048] Comparative Example 1

[0049] The pitaya was mashed, filtered, and after the juice was allowed to stand, the supernatant was taken. 10 mL of the red supernatant was added to a cation exchange resin column filled with 70 g of D113. After washing with 160 mL of water, it was washed with 160 mL of hydrochloric acid with a mass concentration of 2%, and the eluate in the concentrated red part was collected. 15 mL of the red eluate was loaded onto a glass column filled with 30 g of ODS packing material. After washing with 120 mL of water, it was eluted with 120 mL of an ethanol solution with a volume concentration of 20%, and the eluate in the concentrated red part was collected and freeze-dried to obtain a red solid. The pigment purity determined by HPLC normalization method was less than 95%.

[0050] In this comparative example, a hydrochloric acid solution with a mass concentration of 2% was used as the eluent. In addition to the pigment purity of the product being lower than that of Example 2, the pH of the product was 3.5, showing acidity. Using the betacyanin prepared in this comparative example as an additive not only affects the taste of the food, but also has poor compatibility with other additives, which is not conducive to the stable storage of the product.

[0051] Example 2

[0052] The beet tubers were mashed, filtered, and after the juice was allowed to stand, the supernatant was taken. 100 mL of the red supernatant was added to a cation exchange resin column filled with 450 g of 001*7H. After washing with 1000 mL of water, it was washed with 1000 mL of sodium sulfate with a mass concentration of 2%, and the eluate in the concentrated red part was collected. 150 mL of the red eluate was loaded onto a glass column filled with 300 g of ODS packing material. After washing with 500 mL of water, it was eluted with 1000 mL of an ethanol solution with a volume concentration of 20%, and the eluate in the concentrated red part was collected and freeze-dried to obtain a red solid. The pigment purity determined by HPLC normalization method was 97.2%.

[0053] Comparative Example 2

[0054] The beet tubers were mashed, filtered, and after the juice was allowed to stand, the supernatant was taken. 100 mL of the red supernatant was added to a cation exchange resin column filled with 500 g of 001*7H. After washing with 1000 mL of water, it was washed with 600 mL of sodium chloride with a mass concentration of 20%, and the eluate in the concentrated red part was collected. 50 mL of the red eluate was loaded onto a glass column filled with 300 g of ODS packing material. After washing with 800 mL of water, it was eluted with 1000 mL of an ethanol solution with a volume concentration of 20%, and the eluate in the concentrated red part was collected and freeze-dried to obtain a red solid.

[0055] In this comparative example, the sodium chloride concentration was relatively high (mass concentration of 20%), resulting in some incompletely removed sodium chloride being mixed into the product, and the absolute pigment content of the product was less than 90%.

[0056] Example 3

[0057] The pokeberry berries are mashed, and water is added to the mashed berries for extraction according to a mass ratio of 1:1. After filtration, a 70% ethanol solution by volume is added for precipitation, and then it is allowed to stand for 5 h. The supernatant is taken as the material for the next step. 100 mL of the supernatant is added to 200 g of carboxyl cation exchange resin. After rinsing with 500 mL of water, it is rinsed with 500 mL of 2% sodium chloride by mass concentration, and the eluate in the red concentrated part is collected. 60 mL of the red eluate is loaded onto a glass column filled with 400 g of ODS packing. After washing with 1000 mL of water, it is eluted with 1000 mL of 20% ethanol solution by volume, and the eluate in the red concentrated part is collected and freeze-dried to obtain a red solid. The pigment purity is determined by HPLC normalization method to be 98.6%.

[0058] Figure 3 and Figure 4 are the HPLC chromatograms of the extracts before and after purification of betacyanin purified from pokeberry berries in Example 3, respectively.

[0059] From Figure 3 and Figure 4 the comparison of the chromatograms before and after purification, it can be seen that before purification, in addition to the red pigments with retention times of 9.1, 10.6, 11.6, 12.4, and 13.4 minutes, there are also a large number of other impurities at the 220 nm chromatogram. However, from the chromatogram after purification, it can be seen that only the red pigments with retention times of 9.1, 10.6, 11.6, 12.4, and 13.4 minutes exist in the chromatogram, and there are basically no impurities at the 220 nm chromatogram (the solvent front is at 1.2 minutes). This proves that the purification effect of the purification method of high-purity plant-derived betacyanin provided by the present invention is good.

[0060] Furthermore, combined with Figure 4 the HPLC chromatogram where there are no obvious peaks at 220 nm, no peaks at 520 nm, and the impurity peaks with later elution times, it can be proved that the betacyanin product purified from pokeberry berries in Example 3 basically does not contain toxic phytolaccasaponin.

[0061] The above are only the preferred embodiments of the present invention, and do not limit the implementation manners and protection scope of the present invention accordingly. For those skilled in the art, it should be able to realize that all the equivalent replacements and obvious changes made by using the content of the specification of the present invention should be included in the protection scope of the present invention.

Claims

1. A purification method for high-purity betacyanin from plants, characterized in that, It includes the following steps: S1. Mash and filter the plant raw materials to obtain juice, and take the supernatant after standing; S2. Add the supernatant into a cation exchange resin column, first elute with water, and then elute with an aqueous solution of inorganic salt with a mass concentration of 1% - 10% to obtain a first eluate; S3. Add the first eluate into a glass column filled with ODS packing, first elute with water, and then elute with an ethanol solution with a volume concentration of 20% - 100% to obtain a second eluate; S4. Perform a drying treatment on the second eluate to obtain betacyanin.

2. The purification method according to claim 1, wherein, In step S1, the plant raw materials include one or a combination of more than one of pitaya, beet tuber, and pokeberry; 3. The purification method according to claim 1, wherein In step S2, the cation exchange resin column is filled with a cation exchange resin; the functional groups of the cation exchange resin include one or a combination of more than one of carboxyl group, sulfonic acid group, sulfuric acid group, and phenolic hydroxyl group; 4. The purification method according to claim 1, characterized in that, In step S2, the volume-mass ratio of the supernatant to the cation exchange resin is 1:(2 - 10) mL / g; the water consumption for water elution and the volume ratio of the supernatant is (2 - 20):1; 5. The purification method according to claim 1, characterized in that In step S2, in the aqueous solution of the inorganic salt, the inorganic salt is selected from one or a combination of more than one of sodium chloride, sodium bromide, sodium iodide, potassium chloride, potassium bromide, potassium iodide, magnesium chloride, magnesium bromide, sodium sulfate, potassium sulfate, magnesium sulfate, sodium nitrate, potassium nitrate, and magnesium nitrate; 6. The purification method according to claim 1, wherein In step S2, the volume ratio of the amount of the aqueous solution of the inorganic salt to the volume of the supernatant is (2 - 20):1; 7. The purification method according to claim 1, characterized in that, In step S3, the volume-mass ratio of the first eluate to the ODS packing is 1:(2 - 10) mL / g; 8. The purification method according to claim 1, wherein In step S3, the water consumption for water elution and the volume ratio of the first eluate is (2 - 20):1; the volume ratio of the amount of the ethanol solution with a volume concentration of 20% - 100% to the volume of the first eluate is (2 - 20):1; 9. The purification method according to claim 1, wherein In step S4, the drying treatment includes freeze-drying or spray-drying; 10. Use of the purification method according to any one of claims 1-9, characterized in that, Use the above purification method to purify betacyanin in plant raw materials.

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