Method for separating high-purity chlorogenic acid and neochlorogenic acid from stevia rebaudiana chlorogenic acid product based on membrane coupling extraction crystallization

Abstract

The invention discloses a method for separating high-purity chlorogenic acid and neochlorogenic acid from a stevia rebaudiana chlorogenic acid product based on membrane coupling extraction crystallization. The method comprises the following steps: preparing a first to-be-treated solution by taking the stevia rebaudiana chlorogenic acid product as a raw material; treating the first to-be-treated liquid through an ultrafiltration membrane and a nanofiltration membrane in sequence to obtain a trapped fluid II, concentrating the trapped fluid II, and adjusting the pH value to obtain a second to-be-treated liquid; extracting the second to-be-treated liquid with an ethyl acetate-n-butyl alcohol saturated aqueous solution to obtain a raffinate phase I and an extract phase I; adjusting the pH of the raffinate phase I to obtain a third to-be-treated solution; extracting the third to-be-treated liquid with an ethyl acetate-n-butyl alcohol saturated aqueous solution to obtain a raffinate phase II and an extract phase II; drying the extract phase I, crystallizing, pulping and purifying to obtain high-purity chlorogenic acid; and drying the extract phase II, crystallizing, pulping and purifying to obtain the high-purity neochlorogenic acid. According to the method, membrane separation, solvent extraction and fractional crystallization are coupled, and high-efficiency and high-selectivity separation and purification of chlorogenic acid and neochlorogenic acid in a stevia rebaudiana chlorogenic acid product are realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of natural product extraction, and in particular to a method for separating high-purity chlorogenic acid and neochlorogenic acid from a steviol glycoside product based on membrane coupling extraction crystallization. BACKGROUND

[0002] Chlorogenic acid and neochlorogenic acid are phenolic acid compounds with important biological activities in stevia, and have wide application value in the fields of food, medicine and health care products. Due to the fact that they are structural isomers, the molecular structures are highly similar, and the physical and chemical properties are close, it is challenging to efficiently separate and simultaneously obtain high-purity chlorogenic acid and neochlorogenic acid from stevia crude extract.

[0003] At present, the separation and purification of chlorogenic acid compounds in industry mainly relies on the following methods: 1. Macroporous resin adsorption method: the selectivity of the resin is limited, it is difficult to effectively separate chlorogenic acid and neochlorogenic acid, the product purity is usually less than 90%, and the solvent consumption is large in the elution process, and the regeneration wastewater treatment cost is high; 2. Organic solvent fractional precipitation method: the separation efficiency is low, the yield is unstable, the target substance is easy to be lost, and the residual solvent in the product is difficult to completely remove; 3. High performance liquid chromatography method: although this method can realize high-purity separation, the equipment investment is large, the processing capacity is small, and the operation cost is high, which is difficult to meet the demand of large-scale production.

[0004] In view of the above problems, it is urgent to develop an efficient and low-cost separation process to separate chlorogenic acid and neochlorogenic acid from stevia chlorogenic acid product. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a method for separating high-purity chlorogenic acid and neochlorogenic acid from a stevia chlorogenic acid product based on membrane coupling extraction crystallization, which couples membrane separation, solvent extraction and step crystallization, and realizes efficient and high-selectivity separation and purification of chlorogenic acid and neochlorogenic acid with very similar structures in stevia chlorogenic acid product by effectively adjusting specific conditions, and the waste liquid in the separation process is effectively used, which significantly improves the resource utilization rate, reduces the cost and environmental burden.

[0006] To solve the above technical problems, the technical scheme of the present application is:

[0007] A method for separating high-purity chlorogenic acid and neochlorogenic acid from a stevia chlorogenic acid product based on membrane coupling extraction crystallization, comprising the following steps:

[0008] (1) Dissolve the stevia chlorogenic acid product in purified water, adjust the pH of the solution to obtain a first liquid to be treated;

[0009] (2) Concentrating the first liquid to be treated by using ultrafiltration membrane, and repeatedly taking the liquid with purified water, and combining the permeate of each time to obtain permeate I, and collecting the retentate I;

[0010] (3) Concentrating the permeate I of step (2) by using nanofiltration membrane to obtain permeate II and retentate II, and continuing to concentrate the retentate II, and adjusting the pH of the concentrated solution to obtain the second liquid to be treated;

[0011] (4) Extracting the second liquid to be treated by using ethyl acetate-n-butanol saturated aqueous solution for multiple times, collecting the raffinate I, and combining the extraction phases to obtain extraction phase I; and adjusting the pH of the raffinate I to obtain the third liquid to be treated;

[0012] (5) Extracting the third liquid to be treated by using ethyl acetate-n-butanol saturated aqueous solution for multiple times, and collecting the raffinate II, and combining the extraction phases II;

[0013] (6) After the extraction phase I is dried, it is dissolved in ethyl acetate saturated aqueous solution, and stirring and crystallization are performed, after which filtration is performed to obtain filter cake I and filtrate I, the filter cake I is dried and then treated by beating in purified water, and then filtration is performed to obtain filter cake II and filtrate II, and the filter cake II is dried to obtain high-purity chlorogenic acid;

[0014] (7) After the extraction phase II is dried, it is dissolved in acetone solution, high-content neochlorogenic acid crystal seeds are added, stirring and crystallization are performed, and then filtration is performed to obtain filter cake III and filtrate III, the filter cake III is dried and then mixed with purified water, and beating treatment is performed, and then filtration is performed to obtain filter cake IV and filtrate IV, and the filter cake IV is dried to obtain high-purity neochlorogenic acid.

[0015] Preferably, in step (1), the mass ratio of the stevia chlorogenic acid product to purified water is 1:(10-30); the pH of the first liquid to be treated is 3.0-3.5.

[0016] Preferably, in step (2), the molecular weight cut-off of the ultrafiltration membrane is 2000-3000 Da, the concentration conditions are: pressure 0.8-1.0 MPa, temperature 30-50℃, and the solid content of the first liquid to be treated concentrated by using the ultrafiltration membrane is 15-18wt%.

[0017] Preferably, in step (2), when the liquid is taken with purified water, the amount of purified water added is 1 / 2 of the volume of the material, and the liquid is taken 3-5 times.

[0018] Preferably, in step (3), the molecular weight cut-off of the nanofiltration membrane is 400-800 Da, the concentration conditions are: pressure 2.0-3.5 MPa, temperature 30-50℃; the retentate is concentrated to a solid content of 20-30wt%; and the pH of the second liquid to be treated is 2.0-2.5.

[0019] Preferably, in step (4), the volume ratio of the saturated ethyl acetate-n-butanol aqueous solution to the third liquid to be treated is (1-3):1, and the volume ratio of ethyl acetate to n-butanol in the saturated ethyl acetate-n-butanol aqueous solution is (8.5-9.0):1.

[0020] Preferably, in step (4), the pH of the third liquid to be treated is 1.3-1.5.

[0021] Preferably, in step (5), the volume ratio of the saturated ethyl acetate-n-butanol aqueous solution to the third liquid to be treated is (2-3):1, and the volume ratio of ethyl acetate to n-butanol in the saturated ethyl acetate-n-butanol aqueous solution is (7.0-7.5):1.

[0022] Preferably, in step (5), the raffinate is combined into the retentate in step (2), and the sweetener polyphenol is dried.

[0023] Preferably, in step (6), the addition amount of the saturated ethyl acetate aqueous solution is 2-3 times the mass of the dried extraction phase one, and the crystallization is carried out at room temperature for 16-24h.

[0024] Preferably, in step (6), the addition amount of the purified water is 2-3 times the mass of the dried filter cake, and the beating treatment is carried out at a temperature of 30-40℃ for 1-3h.

[0025] Preferably, in step (7), the concentration of the acetone solution is 95-98% v / v, the addition amount of the acetone solution is 1.5-3.0 times the mass of the dried extraction phase two, the addition amount of the high-content neochlorogenic acid seed crystal is 1.0-2.0% of the mass of the dried extraction phase two, and the crystallization is carried out at room temperature for 8-12h.

[0026] Preferably, in step (7), the addition amount of the purified water is 2.0-3.0 times the mass of the dried filter cake three, and the beating treatment is carried out at a temperature of 35-45℃ for 1-3h.

[0027] Preferably, the filtrate one and the filtrate three are combined into the permeate one in step (2), the filtrate two is combined into the extraction phase one, and the filtrate four is combined into the extraction phase two.

[0028] The high-content neochlorogenic acid seed crystal can be selected from the un-dried filter cake three of the previous batch in the production process or commercially available neochlorogenic acid with a purity of ≥98%, such as neochlorogenic acid with a purity of 99% from Shanghai Yongheng Biotechnology Co., Ltd., batch number: 103460.

[0029] Thanks to the above technical solutions, the present application has the following advantages:

[0030] The application provides a method for separating chlorogenic acid and neochlorogenic acid from a stevia rebaudiana chlorogenic acid product, which is carried out by a membrane coupling extraction crystallization method, macromolecular chlorogenic acids such as iso-chlorogenic acid A, iso-chlorogenic acid B and iso-chlorogenic acid C and macromolecular impurities such as proteins and polysaccharides in the stevia rebaudiana chlorogenic acid product are removed by an ultrafiltration membrane, small molecular impurities and salts are removed by a nanofiltration membrane, and effective grading separation and concentration of chlorogenic acid, neochlorogenic acid and impurities are initially realized, so that the efficiency and selectivity of subsequent treatment are greatly improved.

[0031] The application adopts a certain proportion of ethyl acetate-n-butanol saturated aqueous solution for multiple and step-by-step extraction, according to the polarity difference between chlorogenic acid and neochlorogenic acid, the two are effectively separated by using different ethyl acetate-n-butanol saturated aqueous solutions, and subsequent crystallization treatment is facilitated.

[0032] The application uses ethyl acetate saturated aqueous solution as a crystallization solvent of chlorogenic acid, uses a specific concentration of acetone solution as a crystallization solvent of neochlorogenic acid, and adds crystal seeds for induction, and high-purity chlorogenic acid and neochlorogenic acid are prepared by using the solubility and crystallinity difference of chlorogenic acid and neochlorogenic acid in different solvent systems.

[0033] In order to further improve the purity of chlorogenic acid and neochlorogenic acid, the application also mixes and beats the filter cake after crystallization and purified water, further removes the impurities co-crystallized or adsorbed, and the purity of the prepared neochlorogenic acid and chlorogenic acid reaches more than 98%.

[0034] In the crystallization process, the filtrate one and the filtrate three are combined to the ultrafiltration membrane treatment process, the filtrate two is recovered to the extraction phase one, and the filtrate four is recovered to the extraction phase two, so that the target product and the solvent are recovered to the maximum extent, and the total yield of the product is improved.

[0035] In the extraction process, the raffinate phase two and the retained liquid one in the ultrafiltration membrane treatment process are combined and dried to obtain a byproduct stevia rebaudiana polyphenol, improve the resource utilization rate, and reduce waste. DETAILED DESCRIPTION

[0036] In order to more clearly understand the above-mentioned purposes, features and advantages of the application, the schemes of the application will be further described below. It should be noted that the embodiments of the application and the features in the embodiments can be combined with each other without conflict.

[0037] In the following description, many specific details are set forth in order to provide a thorough understanding of the application, but the application can also be implemented in other ways different from those described herein; obviously, the embodiments in the description are only some embodiments of the application, not all embodiments.

[0038] For a further understanding of the present application, preferred embodiments thereof will be described in detail below with reference to the following examples, but it is to be understood that the description is merely by way of illustration and is not intended to limit the present application as claimed.

[0039] The steviol glucuronide product in the present application is a by-product from the extraction process of stevia, which specifically includes the following steps:

[0040] S1: Stevia is extracted with 15-35% v / v ethanol solution (the mass ratio of ethanol solution to stevia is 8-15:1), the extraction temperature is 25-40℃, after the extraction is completed, filtration is performed, the filter residue is added with ethanol solution for secondary extraction, and the extraction is repeated for 3-5 times, the extraction solutions of multiple times are combined, and the extraction solution is obtained;

[0041] S2, the extraction solution is de-alcoholized by using a 200-400 Da nanofiltration membrane, and the pH of the cut-off solution is adjusted to 3-3.5, then weakly polar macroporous adsorption resin is used for adsorption (when adsorbing, the feed amount is 1 ml: (1-1.2) g of the ratio of the volume of the resin to the weight of stevia, and the flow rate of the feed liquid is 1-2 BV / h of the volume of the resin), after the adsorption is completed, pure water is used for elution (when eluting, the amount of the feed liquid is 1.5-3.5 BV of the volume of the resin, and the flow rate of the feed liquid is 1-2 BV / h of the volume of the resin); after the water elution is completed, an ethanol solution with a concentration of 60-80% v / v is used for elution, and the amount of the ethanol solution used for elution is 2-3 BV of the volume of the resin, and the flow rate of the feed liquid is 1-2 BV / h of the volume of the resin; the elution solution is dried to obtain the steviol glucuronide product.

[0042] The preparation method of the steviol glucuronide product in the following examples and comparative examples specifically includes the following steps:

[0043] 10 kg of stevia is extracted with 120 L of 25% v / v ethanol at 30℃ for four times, and the filtrates of multiple times are combined to obtain the extraction solution;

[0044] The extraction solution is de-alcoholized by using a 200 Da nanofiltration membrane, the pH of the cut-off solution is adjusted to 3.2, 9 L of weakly polar macroporous adsorption resin is used for adsorption, the flow rate of the feed liquid during adsorption is 1.5 BV / h, after the adsorption is completed, 2 BV of pure water of the volume of the resin is used for elution, the flow rate of the feed liquid during elution is 1.5 BV / h, then 3 BV of 70% v / v ethanol solution of the volume of the resin is used for elution, the flow rate of the feed liquid during elution is 1.5 BV / h, and the elution solution is dried to obtain the steviol glucuronide product.

[0045] Example 1

[0046] A method for separating high-purity chlorogenic acid and neochlorogenic acid from steviol glucuronide product based on membrane coupling extraction crystallization, which includes the following steps:

[0047] (1) 500 g of stevia green acid product (total content of chlorogenic acid and its isomers is 45.8 wt%, content of neochlorogenic acid is 6.3 wt%, content of chlorogenic acid is 14.1 wt%) is dissolved in 5 kg of purified water, and the pH of the solution is adjusted to 3.5 to obtain a first to-be-treated solution;

[0048] (2) The first to-be-treated solution is concentrated to a solid content of 15 wt% by using an ultrafiltration membrane (molecular weight cut-off is 2000 Da) under the concentration conditions of a pressure of 0.8 MPa and a temperature of 30°C, and then 1 / 2 of the volume of the remaining material is added to the concentrated solution, and this process is repeated 5 times to obtain a permeate one and a retentate one;

[0049] (3) The permeate one of step (2) is concentrated by using a nanofiltration membrane (molecular weight cut-off is 400 Da) under the concentration conditions of a pressure of 2.0 MPa and a temperature of 30°C to obtain a permeate two and a retentate two, and the retentate two is concentrated to a solid content of 20 wt% and the pH of the concentrated solution is adjusted to 2.0 to obtain a second to-be-treated solution;

[0050] (4) The second to-be-treated solution is extracted 3 times with ethyl acetate-n-butanol saturated aqueous solution (the volume ratio of ethyl acetate to n-butanol is 8.5:1), and the volume ratio of the ethyl acetate-n-butanol saturated aqueous solution to the first to-be-treated solution is controlled to be 1:1 during the extraction, and a raffinate phase one is collected and combined with the extraction phase one; and the pH of the raffinate phase one is adjusted to 1.3 to obtain a third to-be-treated solution;

[0051] (5) The third to-be-treated solution is extracted 3 times with ethyl acetate-n-butanol saturated aqueous solution (the volume ratio of ethyl acetate to n-butanol is 7.0:1), and a raffinate phase two is collected and combined with the extraction phase two; the volume ratio of the ethyl acetate-n-butanol saturated aqueous solution to the third to-be-treated solution is 2:1 during the extraction; and the raffinate phase two is combined with the retentate one of step (2) to obtain stevia polyphenols after drying;

[0052] (6) The extraction phase one of step (4) is dissolved in ethyl acetate saturated aqueous solution (the amount of ethyl acetate saturated aqueous solution added is 2 times the mass of the dried extraction phase one) after drying, and is stirred at room temperature for 16 h for crystallization treatment, and then filtered to obtain a filter cake one and a filtrate one, and the filter cake one is dried and then dissolved in purified water (the amount of purified water added is 2 times the mass of the dried filter cake) for beating, the temperature is 30°C, and the time is 1 h, and then filtered to obtain a filter cake two and a filtrate two, and the filter cake two is dried to obtain high-purity chlorogenic acid; and the filtrate one is combined with the permeate one of step (2), and the filtrate two is combined with the extraction phase one;

[0053] (7) The extraction phase two of step (5) is dissolved in an acetone solution with a concentration of 95% v / v (the added amount of acetone solution is 1.5 times the mass of the dried extraction phase two), high content neochlorogenic acid crystal seeds are added (the added amount is 1.0% of the mass of the dried extraction phase two, and the filter cake three of the previous batch is used as the crystal seeds), and the mixture is stirred at room temperature for 12 hours, then filtered to obtain the filter cake three and the filtrate three, the filter cake three is dried and mixed with purified water (the added amount of purified water is 2.0 times the mass of the dried filter cake three), and the mixture is treated by beating at 35°C for 1 hour, then filtered to obtain the filter cake four and the filtrate four, and the filter cake four is dried to obtain high purity neochlorogenic acid; the filtrate three is combined with the permeate one of step (2); and the filtrate four is combined with the extraction phase two.

[0054] Example 2

[0055] A method for separating high purity chlorogenic acid and neochlorogenic acid from a stevia rebaudiana chlorogenic acid product based on membrane coupled extraction crystallization, comprising the following steps:

[0056] (1) 500 g of a stevia rebaudiana chlorogenic acid product (total content of chlorogenic acid and its isomers is 45.8 wt%, content of neochlorogenic acid is 6.3 wt%, and content of chlorogenic acid is 14.1 wt%) is dissolved in 15 kg of purified water, and the pH of the solution is adjusted to 3.5 to obtain a first to-be-processed solution;

[0057] (2) The first to-be-processed solution is concentrated to a solid content of 18 wt% by using an ultrafiltration membrane (molecular weight cut-off is 3000 Da) under the following concentration conditions: pressure is 1.0 MPa and temperature is 50°C, then 1 / 2 of the volume of the remaining material is added to the solution, and the process is repeated 3 times, the permeate is combined to obtain a permeate one, and the retentate is collected as a retentate one;

[0058] (3) The permeate one of step (2) is concentrated by using a nanofiltration membrane (molecular weight cut-off is 800 Da) under the following concentration conditions: pressure is 3.5 MPa and temperature is 50°C, and the permeate two and the retentate two are collected, the retentate two is concentrated to a solid content of 30 wt%, and the pH of the concentrated solution is adjusted to 2.5 to obtain a second to-be-processed solution;

[0059] (4) The second to-be-processed solution is extracted 4 times with an ethyl acetate-n-butanol saturated aqueous solution (the volume ratio of ethyl acetate to n-butanol is 9.0:1), the volume ratio of the ethyl acetate-n-butanol saturated aqueous solution to the first to-be-processed solution is controlled to be 3:1 during the extraction, the raffinate one is collected, and the extraction phase one is combined; and the pH of the raffinate one is adjusted to 1.5 to obtain a third to-be-processed solution;

[0060] (5) the third to-be-treated liquid is extracted 5 times with ethyl acetate-n-butanol saturated aqueous solution (the volume ratio of ethyl acetate to n-butanol is 7.5:1), and the second raffinate phase is collected and combined with the second extract phase; the volume ratio of ethyl acetate-n-butanol saturated aqueous solution to the third to-be-treated liquid is 3:1 during extraction; the second raffinate phase is combined with the cut liquid one in step (2), and dried to obtain stevia polyphenols;

[0061] (6) the first extract phase after drying in step (4) is dissolved in ethyl acetate saturated aqueous solution (the amount of ethyl acetate saturated aqueous solution added is 3 times the mass of the dried first extract phase), and stirred at room temperature for 24 h for crystallization treatment; after crystallization is completed, filtration is performed to obtain a filter cake one and a filtrate one; the filter cake one is dried and then dissolved in purified water (the amount of purified water added is 3 times the mass of the dried filter cake two); the temperature is 40°C, and the time is 3 h; then filtration is performed to obtain a filter cake two and a filtrate two; the filter cake two is dried to obtain high-purity chlorogenic acid; and the filtrate one is combined with the permeate one in step (2), and the filtrate two is combined with the first extract phase;

[0062] (7) the second extract phase after drying in step (5) is dissolved in a 98% v / v acetone solution (the amount of acetone solution added is 3.0 times the mass of the dried second extract phase); high-content neochlorogenic acid crystal seeds are added (the amount of addition is 2.0% of the mass of the dried second extract phase, and the filter cake three of the previous batch is used as the crystal seeds); stirring is performed at room temperature for 8 h for crystallization treatment; then filtration is performed to obtain a filter cake three and a filtrate three; the filter cake three is dried and then mixed with purified water (the amount of purified water added is 3.0 times the mass of the dried filter cake three); the temperature is 45°C, and the time is 3 h for beating treatment; then filtration is performed to obtain a filter cake four and a filtrate four; the filter cake four is dried to obtain high-purity neochlorogenic acid; the filtrate three is combined with the permeate one in step (2); and the filtrate four is combined with the second extract phase.

[0063] Example 3

[0064] A method for separating high-purity chlorogenic acid and neochlorogenic acid from stevia chlorogenic acid products based on membrane coupling extraction crystallization, comprising the following steps:

[0065] (1) 500 g of stevia chlorogenic acid products (the total content of chlorogenic acid and its isomers is 45.8 wt%, the content of neochlorogenic acid is 6.3 wt%, and the content of chlorogenic acid is 14.1 wt%) is dissolved in 10 kg of purified water, and the pH of the solution is adjusted to 3.2 to obtain a first to-be-treated liquid;

[0066] (2) the first to-be-treated liquid is concentrated to a solid content of 16 wt% by using an ultrafiltration membrane (the molecular weight cut-off is 2500 Da); the concentration conditions are a pressure of 0.9 MPa and a temperature of 42°C; then 1 / 2 of the remaining material volume of purified water is added for material transfer; this is repeated for 4 times; the permeate is combined to obtain a permeate one, and the cut liquid one is collected;

[0067] (3) Concentrating the permeate 1 of step (2) by using nanofiltration membrane (molecular weight cut-off 600 Da) under the condition of pressure 3.0 MPa and temperature 40℃, collecting the permeate 2 and the retentate 2, concentrating the retentate 2 to solid content 25 wt%, and adjusting the pH of the concentrated solution to 2.3, to obtain the second liquid to be treated;

[0068] (4) Extracting the second liquid to be treated 4 times by using ethyl acetate-n-butanol saturated aqueous solution (volume ratio of ethyl acetate to n-butanol 8.8:1), and controlling the volume ratio of ethyl acetate-n-butanol saturated aqueous solution to the first liquid to be treated to be 2:1, collecting the raffinate phase 1 and combining the extract phase 1; and adjusting the pH of the raffinate phase 1 to 1.4, to obtain the third liquid to be treated;

[0069] (5) Extracting the third liquid to be treated 4 times by using ethyl acetate-n-butanol saturated aqueous solution (volume ratio of ethyl acetate to n-butanol 7.3:1), collecting the raffinate phase 2 and combining the extract phase 2; the volume ratio of ethyl acetate-n-butanol saturated aqueous solution to the third liquid to be treated is 3:1 during extraction; and the raffinate phase 2 is combined into the retentate 1 of step (2), and dried to obtain stevia polyphenols;

[0070] (6) After drying the extract phase 1 of step (4), dissolving it in ethyl acetate saturated aqueous solution (the amount of ethyl acetate saturated aqueous solution added is 2.5 times the mass of the dried extract phase 1), stirring at room temperature for 20 h, filtering after crystallization, to obtain the filter cake 1 and the filtrate 1, drying the filter cake 1, dissolving it in purified water (the amount of purified water added is 2.5 times the mass of the dried filter cake), and beating at a temperature of 35℃ for 2 h, then filtering, to obtain the filter cake 2 and the filtrate 2, drying the filter cake 2, to obtain high-purity chlorogenic acid; and combining the filtrate 1 into the permeate 1 of step (2), and combining the filtrate 2 into the extract phase 1;

[0071] (7) After drying the extract phase 2 of step (5), dissolving it in 96% v / v acetone solution (the amount of acetone solution added is 2.0 times the mass of the dried extract phase 2), adding high-content neochlorogenic acid crystal seeds (the amount of crystal seeds added is 1.5 wt% of the mass of the dried extract phase 2, and the crystal seeds are derived from neochlorogenic acid with batch number 103460 and purity 99% from Shanghai Yongheng Biological Technology Co., Ltd.), stirring at room temperature for 10 h, then filtering, to obtain the filter cake 3 and the filtrate 3, drying the filter cake 3, mixing it with purified water (the amount of purified water added is 2.5 times the mass of the dried filter cake 3), beating at 40℃ for 2 h, filtering, to obtain the filter cake 4 and the filtrate 4, and drying the filter cake 4, to obtain high-purity neochlorogenic acid; combining the filtrate 3 into the permeate 1 of step (2); and combining the filtrate 4 into the extract phase 2.

[0072] Comparative Example 1

[0073] The present comparative example differs from Example 3 in that in step (1), the pH of the first liquid to be treated is 2.0, and the other operations are the same as in Example 3.

[0074] Comparative Example 2

[0075] The present comparative example differs from Example 3 in that in step (1), the pH of the first liquid to be treated is 4.0, and the other operations are the same as in Example 3.

[0076] Comparative Example 3

[0077] The present comparative example differs from Example 3 in that in step (3), the pH of the second liquid to be treated is 1.5, and the other operations are the same as in Example 3.

[0078] Comparative Example 4

[0079] The present comparative example differs from Example 3 in that in step (3), the pH of the second liquid to be treated is 3.0, and the other operations are the same as in Example 3.

[0080] Comparative Example 5

[0081] The present comparative example differs from Example 3 in that in step (4), the volume ratio of ethyl acetate to n-butanol in the ethyl acetate-n-butanol saturated aqueous solution is 10.0:1, and the other operations are the same as in Example 3.

[0082] Comparative Example 6

[0083] The present comparative example differs from Example 3 in that in step (4), the volume ratio of ethyl acetate to n-butanol in the ethyl acetate-n-butanol saturated aqueous solution is 8.0:1, and the other operations are the same as in Example 3.

[0084] Comparative Example 7

[0085] The present comparative example differs from Example 3 in that in step (4), the pH of the third liquid to be treated is 1.0, and the other operations are the same as in Example 3.

[0086] Comparative Example 8

[0087] The present comparative example differs from Example 3 in that in step (4), the pH of the third liquid to be treated is 2.0, and the other operations are the same as in Example 3.

[0088] Comparative Example 9

[0089] The present comparative example differs from Example 3 in that in step (5), the volume ratio of ethyl acetate to n-butanol in the ethyl acetate-n-butanol saturated aqueous solution is 8.0:1, and the other operations are the same as in Example 3.

[0090] Comparative Example 10

[0091] The present comparative example differs from Example 3 in that in step (5), the volume ratio of ethyl acetate to n-butanol in the ethyl acetate-n-butanol saturated aqueous solution is 6.5:1, and other operations are the same as in Example 3.

[0092] Comparative Example 11

[0093] The present comparative example differs from Example 3 in that in step (6), the amount of ethyl acetate saturated aqueous solution added is 5 times the mass of the dried extraction phase. Other operations are the same as in Example 3.

[0094] Comparative Example 12

[0095] The present comparative example differs from Example 3 in that in step (6), the temperature of the beating treatment is 20℃, and other operations are the same as in Example 3.

[0096] Comparative Example 13

[0097] The present comparative example differs from Example 3 in that in step (6), the temperature of the beating treatment is 55℃, and other operations are the same as in Example 3.

[0098] Comparative Example 14

[0099] The present comparative example differs from Example 3 in that in step (6), the concentration of the acetone solution is 92% v / v, and other operations are the same as in Example 3.

[0100] Comparative Example 15

[0101] The present comparative example differs from Example 3 in that in step (7), no neochlorogenic acid seed crystals are added, and other operations are the same as in Example 3.

[0102] The yields, the purities and the yields of neochlorogenic acid and chlorogenic acid in the above examples and comparative examples are shown in Table 1.

[0103] Table 1

[0104]

[0105]

[0106] From the above test results, it can be seen that the present application uses stevia chlorogenic acid product as raw material, and realizes high-efficiency separation of chlorogenic acid and neochlorogenic acid by coupling technologies of multi-stage membrane separation, solvent gradient extraction and directional crystallization, and optimizing the conditions of each stage.

[0107] In Comparative Example 1 and Comparative Example 2, the pH of the first liquid to be treated is too low or too high, and the purity and yield of chlorogenic acid and neochlorogenic acid are affected. Mainly, the pH of the first liquid to be treated is not properly controlled, which not only destroys the structure of the target molecules, but also changes the structure of the impurity molecules, affecting the separation effect.

[0108] In order to improve the extraction separation effect, the pH of the second to-be-treated liquid needs to be reasonably adjusted. In Comparative Example 3, the pH of the second to-be-treated liquid is too low, and the yield of chlorogenic acid and neochlorogenic acid decreases significantly. In Comparative Example 4, the pH of the second to-be-treated liquid is too high, and the yield of neochlorogenic acid is significantly affected. Because excessive acidification will reduce the solubility of the target molecule in the second to-be-treated liquid, affecting the extraction selectivity, and alkaline enhancement will increase the solubility of the target molecule in the second to-be-treated liquid, reducing the extraction rate of the extraction solvent.

[0109] The selection of the extraction solvent is also extremely critical. In Comparative Example 5, during the first extraction, a high proportion of ethyl acetate will reduce the solubility of polar components, thereby significantly reducing the yield of chlorogenic acid and neochlorogenic acid. In Comparative Example 6 or Comparative Example 10, a low proportion of ethyl acetate will reduce the selectivity of chlorogenic acid or neochlorogenic acid, so the yield of chlorogenic acid in Comparative Example 6 decreases significantly, and the yield of neochlorogenic acid in Comparative Example 10 decreases significantly.

[0110] The pH before the second extraction is also critical. In Comparative Example 7, the pH of the third to-be-treated liquid is too low, which will destroy the structure of neochlorogenic acid, and thus the yield thereof decreases. In Comparative Example 8, the pH of the third to-be-treated liquid is too high, and the solubility of neochlorogenic acid in the raffinate phase increases, affecting the extraction of neochlorogenic acid, and thus the yield thereof decreases.

[0111] The principles and implementation modes of the present application are described by applying specific examples in the present text, and the above description of the examples is only used to help understand the method of the present application and its core idea, including the best mode, and also enables any person skilled in the art to practice the present application, including manufacturing and using any device or system, and implementing any combined method. It should be noted that, for those skilled in the art, some improvements and modifications can be made to the present application without departing from the principles of the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application. The scope of protection of the present application is defined by the claims, and can include other embodiments that can be thought of by those skilled in the art. If these other embodiments have structural elements similar to the literal expression of the claims, or if they include equivalent structural elements that are not substantially different from the literal expression of the claims, then these other embodiments should also be included in the scope of the claims.

Claims

1. A method for separating high purity chlorogenic acid and neochlorogenic acid from a steviol glycoside product based on membrane coupled extraction crystallization, characterized in that, The method comprises the following steps: (1) dissolving a stevia green acid product in purified water, adjusting the pH of the solution to obtain a first to-be-treated solution; (2) concentrating the first to-be-treated solution by using an ultrafiltration membrane, and using purified water to carry the material for multiple times, and combining the permeate of multiple times to obtain permeate I, and collecting the retentate I; (3) concentrating the permeate I of step (2) by using a nanofiltration membrane, collecting the permeate II and the retentate II, continuously concentrating the retentate II, and adjusting the pH of the concentrated solution to obtain a second to-be-treated solution; (4) extracting the second to-be-treated solution multiple times by using an ethyl acetate-n-butanol saturated aqueous solution, collecting the raffinate phase I, and combining the extraction phases to obtain the extraction phase I; and adjusting the pH of the raffinate phase I to obtain a third to-be-treated solution; (5) extracting the third to-be-treated solution multiple times by using an ethyl acetate-n-butanol saturated aqueous solution, and collecting the raffinate phase II, and combining the extraction phase II; (6) after the extraction phase I is dried, it is dissolved in an ethyl acetate saturated aqueous solution, stirring and crystallizing treatment, filtering after the crystallization is completed to obtain the filter cake I and the filtrate I, and after the filter cake I is dried, it is treated by beating in purified water, and then filtered to obtain the filter cake II and the filtrate II, and the filter cake II is dried to obtain high-purity green acid; (7) after the extraction phase II is dried, it is dissolved in an acetone solution, high-content neogreen acid crystal seeds are added, stirring and crystallizing, and then filtering to obtain the filter cake III and the filtrate III, and after the filter cake III is dried, it is mixed with purified water, and treated by beating, and then filtered to obtain the filter cake IV and the filtrate IV, and the filter cake IV is dried to obtain high-purity neogreen acid.

2. The method for separating high-purity chlorogenic acid and neochlorogenic acid from steviolbioside product based on membrane coupling extraction crystallization according to claim 1, characterized in that, In step (1), the mass ratio of the stevia green acid product to the purified water is 1:(10-30); the pH of the first to-be-treated solution is 3.0-3.

5.

3. The method for separating high-purity chlorogenic acid and neochlorogenic acid from steviolbioside product based on membrane coupling extraction crystallization according to claim 1, characterized in that, In step (2), the molecular weight cut-off of the ultrafiltration membrane is 2000-3000 Da, the concentration conditions are as follows: the pressure is 0.8-1.0 MPa, the temperature is 30-50 ℃, the solid content of the first to-be-treated solution concentrated by using the ultrafiltration membrane is 15-18 wt%, and when the purified water is used to carry the material, the addition amount of the purified water is 1 / 2 of the volume of the material, and the material is carried for 3-5 times.

4. The method for separating high-purity chlorogenic acid and neochlorogenic acid from steviolbioside product based on membrane coupling extraction crystallization according to claim 1, characterized in that, In step (3), the molecular weight cut-off of the nanofiltration membrane is 400-800 Da, the concentration conditions are as follows: the pressure is 2.0-3.5 MPa, the temperature is 30-50 ℃, the retentate is concentrated to a solid content of 20-30 wt%, and the pH of the second to-be-treated solution is 2.0-2.

5.

5. The method for separating high-purity chlorogenic acid and neochlorogenic acid from steviolbioside product based on membrane coupling extraction crystallization according to claim 1, characterized in that, In step (4), the volume ratio of the ethyl acetate-n-butanol saturated aqueous solution to the second to-be-treated solution is (1-3):1 during extraction, and the volume ratio of ethyl acetate to n-butanol in the ethyl acetate-n-butanol saturated aqueous solution is (8.5-9.0):

1.

6. The method for separating high-purity chlorogenic acid and neochlorogenic acid from steviolbioside product based on membrane coupling extraction crystallization according to claim 1, characterized in that, In step (4), the pH of the third to-be-treated solution is 1.3-1.5, the volume ratio of the ethyl acetate-n-butanol saturated aqueous solution to the third to-be-treated solution is (2-3):1 during extraction, and the volume ratio of ethyl acetate to n-butanol in the ethyl acetate-n-butanol saturated aqueous solution is (7.0-7.5):

1.

7. The method for separating high-purity chlorogenic acid and neochlorogenic acid from steviolbioside product based on membrane coupling extraction crystallization according to claim 1, characterized in that, In step (5), the raffinate phase II is combined with the retentate I of step (2), and dried to obtain stevia polyphenol.

8. The method for separating high-purity chlorogenic acid and neochlorogenic acid from steviolbioside product based on membrane coupling extraction crystallization according to claim 1, characterized in that, In step (6), the adding amount of ethyl acetate saturated aqueous solution is 2-3 times of the mass of the dried extraction phase one, and the stirring crystallization is carried out at room temperature for 16-24 h; the adding amount of purified water is 2-3 times of the mass of the dried filter cake; the temperature of the beating treatment is 30-40℃, and the time is 1-3 h.

9. The method for separating high-purity chlorogenic acid and neochlorogenic acid from steviolbioside product based on membrane coupling extraction crystallization according to claim 1, characterized in that, In step (7), the concentration of the acetone solution is 95-98% v / v, the adding amount of the acetone solution is 1.5-3.0 times of the mass of the dried extraction phase two, and the stirring crystallization is carried out at room temperature for 8-12 h.

10. The method for separating high-purity chlorogenic acid and neochlorogenic acid from steviolbioside product based on film-coupled extraction crystallization according to claim 1, characterized in that, In step (7), the adding amount of purified water is 2.0-3.0 times of the mass of the dried filter cake three, and the temperature of the beating treatment is 35-45℃, and the time is 1-3 h.

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