Preparation method of stevia polyphenol

Abstract

The invention discloses a preparation method of stevia rebaudiana polyphenol, which comprises the following steps: adsorbing stevia rebaudiana leach liquor through medium-polarity macroporous resin, desorbing the adsorbed resin by using an ethanol solution to obtain a desorbed solution, concentrating the desorbed solution to obtain a concentrated solution, adding an acid solution into the concentrated solution to adjust the pH value to 1-1.5, settling and separating to obtain a precipitate, and drying to obtain the stevia rebaudiana polyphenol. And adding water into the precipitate, adding a sodium hydroxide solution to adjust the pH value to 5-6, redissolving the precipitate to obtain a redissolved solution, enabling the redissolved solution to pass through a nanofiltration membrane with the molecular weight cutoff of 2000-3000Da to obtain a trapped fluid, adding the acid solution into the trapped fluid to adjust the pH value to 1-1.5, and carrying out settling separation to obtain the stevia polyphenol precipitate. According to the method, after the stevia polyphenol solution is separated from the stevia rebaudiana leaching solution, the acid solution is added into the solution to adjust the pH value to be strongly acidic, so that the stevia polyphenol is settled and is separated from other impurities, and then the high-purity stevia polyphenol is prepared in a manner of adding alkali for redissolving and adding acid for resettling.

Description

Technical Field

[0001] The invention relates to the technical field of organic matter extraction, and in particular to a method for preparing stevia polyphenols. Background Art

[0002] Stevia polyphenols are naturally occurring compounds in plant foods, including chlorogenic acid, flavonoids, flavonols, flavanols, isoflavones and proanthocyanidins. At present, at least 30 stevia polyphenols with clear structures have been reported. Stevia polyphenols are mostly water-soluble polyphenol compounds with good and stable free radical scavenging ability, anti-inflammatory, diuretic, antihypertensive, hypoglycemic, anti-tumor and antioxidant properties. In addition, they also have the effect of lowering blood pressure and blood lipids.

[0003] Stevia contains a certain amount of stevioside polyphenols. Currently, the main use of stevia is to extract steviol glycosides, and most of the stevioside polyphenols contained in it are wasted. In the prior art, when extracting steviol glycosides, iron salts and calcium salts are usually used for complexation and impurity removal. This method will incorporate stevioside polyphenols into the waste residue and also produce a large amount of organic and inorganic mixed solid waste.

[0004] Therefore, it is urgent to establish a technical solution in the prior art for extracting stevioside from stevia and preparing high-purity stevia polyphenols at the same time, so that the stevia polyphenols in stevia can be effectively recovered and the comprehensive utilization value of stevia can be further improved. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for preparing stevia polyphenols, which overcomes the technical problem that stevia polyphenols cannot be recovered during the process of extracting steviol glycosides from stevia in the prior art.

[0006] In order to achieve the above object, the present invention provides a method for preparing stevia polyphenols, comprising the following steps:

[0007] The stevia extract is adsorbed by a medium polarity macroporous resin;

[0008] The adsorbed resin is analyzed with an ethanol solution to obtain an analytical solution;

[0009] Concentrating the analytical solution to obtain a concentrated solution;

[0010] Adding an acid solution to the concentrated solution to adjust the pH to 1-1.5, and performing sedimentation separation to obtain a precipitate;

[0011] Adding water to the precipitate, and adding sodium hydroxide solution to adjust the pH to 5-6 to redissolve the precipitate to obtain a reconstituted solution;

[0012] Passing the reconstituted solution through a nanofiltration membrane with a molecular weight cutoff of 2000-3000Da to obtain a retentate;

[0013] The acid solution is added to the retentate to adjust the pH to 1-1.5, and the stevia polyphenol precipitate is obtained by sedimentation separation.

[0014] The preparation method of stevioside provided by the present invention firstly adsorbs the stevioside extract through a medium polar macroporous resin, and the stevioside is adsorbed on the resin, and the steviol glycoside is retained in the solution and discharged with the effluent, and the effluent is used to prepare steviol glycoside; therefore, the preparation of stevioside in the present invention can be prepared on the basis of the preparation process of steviol glycoside, so that the production process can be optimized, and there is no need to set up a separate production line; after the resin adsorbing stevioside is analyzed and concentrated by an alcohol solution, an acid solution is added to the concentrated solution to adjust the pH to a strong acidity, and under a strong acid condition, the stevioside will settle in the form of a precipitate, thereby separating from other impurities and improving the purity of the stevioside product; after settling, alkali is added for redissolution, and acidification and purification are performed again, and finally a high-purity stevioside product is obtained. Therefore, the present invention effectively separates stevioside from impurities by acidifying the stevioside concentrate, and separates high-purity stevioside on the basis of the process for separating and preparing steviol glycoside from stevioside, further improving the economic value and comprehensive utilization value of stevioside. According to detection, the content of polyphenols in the stevia polyphenol product prepared by the method of the present invention can reach more than 95%. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Described is the HPLC spectrum of stevia polyphenols obtained in Example 1 DETAILED DESCRIPTION

[0016] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0017] The present invention provides a method for preparing stevia polyphenols, comprising the following steps:

[0018] The stevia extract is adsorbed by a medium polarity macroporous resin;

[0019] The adsorbed resin is analyzed with an ethanol solution to obtain an analytical solution;

[0020] Concentrating the analytical solution to obtain a concentrated solution;

[0021] Adding an acid solution to the concentrated solution to adjust the pH to 1-1.5, and performing sedimentation separation to obtain a precipitate;

[0022] Adding water to the precipitate, and adding sodium hydroxide solution to adjust the pH to 5-6 to redissolve the precipitate to obtain a reconstituted solution;

[0023] Passing the reconstituted solution through a nanofiltration membrane with a molecular weight cutoff of 2000-3000Da to obtain a retentate;

[0024] The acid solution is added to the retentate to adjust the pH to 1-1.5, and the stevia polyphenol precipitate is obtained by sedimentation separation.

[0025] The present invention firstly adsorbs the stevia extract through a medium polarity macroporous resin. In the present invention, the stevia extract is prepared by extracting stevia in an ethanol solution; preferably, the volume concentration of the ethanol solution is 20-40% (v / v); the mass ratio of stevia to the ethanol solution is preferably 1: (8-12), the extraction time is preferably 1-3h, the number of extractions is preferably 3 times, and after the extraction is completed, the extracts are combined to obtain the stevia extract; after the extraction is completed, the present invention preferably filters the extract to remove impurities in the extract, and the filtration is preferably plate-frame filtration. The present invention has no special limitation on the pore size of the filter cloth for the plate-frame filtration, as long as the impurities can be filtered out.

[0026] In the present invention, the medium polarity macroporous resin is preferably a polyamide resin, and LX-8 or 900E can be more preferably selected; the feed amount of the stevia extract is preferably 1-2BV, and the flow rate is preferably 1-2BV / h. The present invention has no special limitation on the feeding method of the extract, such as pumping feeding; the present invention has no special limitation on the carrier of the resin, such as a resin column or a glass column. The use of the medium polarity macroporous resin in the present invention for adsorption can effectively adsorb the stevioside in the extract onto the resin, while steviol glycosides and other components are retained in the effluent, so the effluent can be further separated and steviosides can be prepared, that is, the present invention can be produced as a by-product in the process of preparing steviosides, and has certain advantages in the process flow.

[0027] After adsorption, the present invention uses an ethanol solution to resolve the adsorbed resin to obtain a resolution solution. In the present invention, the volume concentration of the ethanol solution is preferably 60-80% (v / v); the amount of the ethanol solution is preferably 2-3BV of the volume of the resin. The purpose of the resolution is to replace the substance adsorbed on the resin with a resolution agent, and replace the substance to be recovered into the resolution solution. Through the resolution, the stevia polyphenols originally adsorbed on the resin are replaced into the resolution solution.

[0028] After obtaining the analytical solution, the present invention concentrates the analytical solution to obtain a concentrated solution. In the present invention, concentrating the analytical solution to obtain a concentrated solution preferably includes: passing the analytical solution through a 800-1000Da nanofiltration membrane for dealcoholization and concentration to obtain a concentrated solution; the solid content of the concentrated solution is 10-12wt%.

[0029] After obtaining the concentrated solution, the present invention adds an acid solution to the concentrated solution to adjust the pH to 1-1.5, and obtains a precipitate by sedimentation separation. In the present invention, the acid solution is preferably a hydrochloric acid solution with a concentration of 5-30% (m / v) or a citric acid solution with a concentration of 10-30% (m / v); the sedimentation time is preferably 0.5-1h. The role of adding acid in this step is to utilize the characteristics that stevioside polyphenols have extremely low solubility in an acidic environment and are relatively stable in nature, but are easily soluble in weakly acidic and alkaline solutions and are unstable in nature; the stevioside polyphenols in the concentrated solution are precipitated into the precipitate under strong acidic conditions, so that the stevioside polyphenols are separated from other impurities and the purity of the stevioside polyphenols is improved.

[0030] After obtaining the sediment, the present invention adds water to the sediment, and adds sodium hydroxide solution to adjust the pH to 5-6 to redissolve the sediment to obtain a reconstituted solution. In the present invention, the amount of water added to the sediment is preferably 2-5BV of the volume of the sediment; the mass concentration of the sodium hydroxide solution is preferably 5-30wt%. The function of adding water to the sediment in the present invention is to provide a solvent to ensure that the stevia polyphenols can be completely dissolved (redissolved) into the solution after the subsequent pH adjustment; the purpose of adding the sodium hydroxide solution is to adjust the pH of the solution and promote the reconstitution of the stevia polyphenols.

[0031] After obtaining the reconstituted solution, the present invention passes the reconstituted solution through a nanofiltration membrane with a molecular weight cutoff of 2000-3000Da to obtain a retentate. In the present invention, when the acid solution is added for precipitation, large molecular weight impurities will be precipitated together with stevia polyphenols, and when the alkaline solution is added for pH adjustment, some salts will also be generated. The function of passing the reconstituted solution through the nanofiltration membrane is to use the nanofiltration membrane to filter out the large molecular impurities and salts obtained above, and stevia polyphenols will not pass through the nanofiltration membrane and be retained in the retentate, and the purity is also improved.

[0032] After obtaining the retentate, the present invention adds the acid solution to the retentate to adjust the pH to 1-1.5, and separates the stevia polyphenol precipitate by sedimentation. In the present invention, the effect of adding the acid solution for sedimentation is the same as the aforementioned sedimentation effect, so it is not repeatedly limited; the secondary sedimentation here is also to convert the stevia polyphenol into a solid again, which is convenient for subsequent processing and sale.

[0033] Furthermore, after obtaining the stevia polyphenol precipitate, the method further comprises: filtering the stevia polyphenol precipitate with filter paper to obtain a filter cake; and rinsing the filter cake with water to obtain high-purity stevia polyphenol. In the present invention, the pore size of the filter paper is preferably 300-500um; and the amount of water used in the rinsing is preferably 1-3BV of the volume of the filter cake.

[0034] In order to better illustrate the technical solution of the present invention, the present invention also provides the following specific examples. It should be understood that the raw materials used in the following examples are all commercially available raw materials unless otherwise specified.

[0035] In particular, the main components of three batches of stevia extracts are listed in Table 1 below, and the extracts were all provided by Zhucheng Haotian Pharmaceutical Co., Ltd.

[0036] Table 11-3 Main components of stevia extract

[0037] batch Moisture / % Stevia polyphenols / % Stevioside / % 1 94.7 0.9 3.0 2 94.8 0.9 3.1 3 94.6 0.9 3.2

[0038] Example 1

[0039] S1. 1.2 L of stevia extract from batch 1 was adsorbed by 0.8 L of medium polarity macroporous resin (resin model 900E, flow rate 1.5 BV / h);

[0040] S2. The adsorbed resin was analyzed with 2 L of 70% (v / v) ethanol solution to obtain 1.8 L of analytical solution;

[0041] S3. The analytical solution was dealcoholized and concentrated through an 800Da nanofiltration membrane to obtain 0.5L of a concentrate having a solid content of 10%;

[0042] S4. Add 20% (m / v) hydrochloric acid solution to the concentrate to adjust the pH to 1.25, and separate by sedimentation to obtain 20 g of sediment;

[0043] S5. 0.5 L of water was added to the sediment, and 20 wt % sodium hydroxide solution was added to adjust the pH to 5.5 to redissolve the sediment to obtain 0.6 L of the reconstituted solution;

[0044] S6. The reconstituted solution was passed through a nanofiltration membrane with a molecular weight cutoff of 2500Da to obtain 0.5L of retentate;

[0045] S7. Add 20% (m / v) citric acid solution to the retentate to adjust the pH to 1.2, and separate by sedimentation to obtain 10 g of stevia polyphenol sediment.

[0046] The results of the test showed that the purity of stevioside in the stevioside sediment was 95%.

[0047] Example 2

[0048] S1. 0.8 L of stevia extract from batch 2 was adsorbed by 0.8 L of medium polarity macroporous resin (resin model LX-8, flow rate 1 BV / h);

[0049] S2. The adsorbed resin was analyzed with 1.6 L of 60% (v / v) ethanol solution to obtain 1.4 L of analytical solution;

[0050] S3. The analytical solution was de-alcoholized and concentrated through a 900Da nanofiltration membrane to obtain 0.4L of a concentrated solution having a solid content of 9%;

[0051] S4. A 10% (m / v) hydrochloric acid solution was added to the concentrate to adjust the pH to 1.5, and 13 g of sediment was obtained by sedimentation separation;

[0052] S5. 0.4 L of water was added to the sediment, and 5 wt % sodium hydroxide solution was added to adjust the pH to 5 to redissolve the sediment to obtain 0.5 L of the reconstituted solution;

[0053] S6. The reconstituted solution was passed through a nanofiltration membrane with a molecular weight cutoff of 2000Da to obtain 0.4L of retentate;

[0054] S7. Add 30% (m / v) citric acid solution to the retentate to adjust the pH to 1, and separate by sedimentation to obtain 7 g of stevia polyphenol sediment.

[0055] The results of the test showed that the purity of stevioside in the stevioside sediment was 94.8%.

[0056] Example 3

[0057] S1. 1.6 L of stevia extract from batch 3 was adsorbed by 0.8 L of medium polarity macroporous resin (resin model 900E, flow rate 2 BV / h);

[0058] S2. The adsorbed resin was analyzed with 2.4 L of 80% (v / v) ethanol solution to obtain 2.1 L of analytical solution;

[0059] S3. The analytical solution was dealcoholized and concentrated through a 1000Da nanofiltration membrane to obtain 0.7L of a concentrate having a solid content of 12%;

[0060] S4. A 30% (m / v) citric acid solution was added to the concentrate to adjust the pH to 1, and 40 g of sediment was obtained by sedimentation separation;

[0061] S5. 0.6 L of water was added to the sediment, and 30 wt % sodium hydroxide solution was added to adjust the pH to 6 to redissolve the sediment to obtain 0.7 L of the reconstituted solution;

[0062] S6. The reconstituted solution was passed through a nanofiltration membrane with a molecular weight cutoff of 3000Da to obtain 0.5L of retentate;

[0063] S7. Add 30% (m / v) hydrochloric acid solution to the retentate to adjust the pH to 1.5, and separate by sedimentation to obtain 20 g of stevia polyphenol precipitate.

[0064] The results of the test showed that the purity of stevioside in the stevioside sediment was 95.5%.

[0065] Comparative Example 1

[0066] The step S4 in Example 1, "adding 20% ​​(m / v) hydrochloric acid solution to the concentrated solution to adjust the pH to 1.25" is adjusted to "adding 20% ​​(m / v) hydrochloric acid solution to the concentrated solution to adjust the pH to 1.75", and other conditions remain unchanged.

[0067] After testing, it was found that 8 g of stevia polyphenol precipitate was obtained, and the purity of stevia polyphenol in the stevia polyphenol precipitate was 94.2%. This was because the precipitation of stevia polyphenol was incomplete after the pH was increased, resulting in a decrease in the final yield and purity.

[0068] Comparative Example 2

[0069] The step S6 in Example 2, "passing the reconstituted solution through a nanofiltration membrane with a molecular weight cutoff of 2000 Da" was adjusted to "passing the reconstituted solution through a nanofiltration membrane with a molecular weight cutoff of 1000 Da", and other conditions remained unchanged.

[0070] After testing, it was found that 10 g of stevia polyphenol precipitate was obtained, and the purity of stevia polyphenol in the stevia polyphenol precipitate was 93%. This is because some impurities cannot pass through after the molecular weight cut-off of the nanofiltration membrane becomes smaller, and are retained in the retentate, resulting in an increase in the final yield of stevia polyphenol precipitate, but a decrease in purity.

[0071] Comparative Example 3

[0072] Steps S4 and S5 in Example 3 are removed, that is, the nanofiltration membrane filtration operation is directly performed after the concentrated solution is obtained, and other conditions remain unchanged.

[0073] After testing, it was found that 24 g of stevia polyphenol precipitate was finally obtained, and the purity of stevia polyphenol in the stevia polyphenol precipitate was 79.6%. This was because after the precipitation and impurity removal process in step S4 was eliminated, the impurities in the solution were not effectively separated from the stevia polyphenol, resulting in a high level of impurities in the final product.

[0074] Test Case

[0075] The purity of stevia polyphenols was determined by high performance liquid chromatography

[0076] UV detector detection wavelength: 327nm;

[0077] Chromatographic column: C18, 4.6×250mm;

[0078] Column temperature: 25°C;

[0079] Mobile phase: acetonitrile-water-formic acid gradient elution, flow rate 1.0mL / min. Injection volume 10μL;

[0080] Mobile phase A: acetonitrile;

[0081] Mobile phase B: acetonitrile-water-formic acid (100:900:1, mL / mL);

[0082] The gradient operation conditions are shown in Table 2:

[0083] Time / min Mobile phase A% Mobile phase B% 0-15 0 100 15-15.01 0→10 100→90 15-25 10→20 90→80 25-25.01 20→5 80→95 25.01-30 5→0 95→100 30-35 0 100

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing stevia polyphenols, characterized in that: The following steps are involved: The stevia extract is adsorbed by a medium polarity macroporous resin; The adsorbed resin is analyzed with an ethanol solution to obtain an analytical solution; Concentrating the analytical solution to obtain a concentrated solution; Adding an acid solution to the concentrated solution to adjust the pH to 1-1.5, and performing sedimentation separation to obtain a precipitate; Adding water to the precipitate, and adding sodium hydroxide solution to adjust the pH to 5-6 to redissolve the precipitate to obtain a reconstituted solution; Passing the reconstituted solution through a nanofiltration membrane with a molecular weight cutoff of 2000-3000Da to obtain a retentate; The acid solution is added to the retentate to adjust the pH to 1-1.5, and the stevia polyphenol precipitate is obtained by sedimentation separation.

2. The method according to claim 1, characterized in that: The medium polar macroporous resin is a polyamide resin; and / or, The feed amount of the stevia extract is 1-2 BV, and the flow rate is 1-2 BV / h.

3. The method according to claim 1, characterized in that The volume concentration of the ethanol solution is 60-80% (v / v); and / or, The amount of the ethanol solution used is 2-3 BV of the resin volume.

4. The method according to claim 1, characterized in that The analytical solution is concentrated to obtain a concentrated solution, comprising: The analytical solution is passed through a 800-1000Da nanofiltration membrane for dealcoholization and concentration to obtain a concentrated solution; and / or, The solid content of the concentrated liquid is 10-12 wt %.

5. The method according to claim 1, characterized in that The acid solution is a hydrochloric acid solution with a mass fraction of 5-30% (m / v) or a citric acid solution with a mass fraction of 10-30% (m / v); and / or, The sedimentation time is 0.5-1h.

6. The method according to claim 1, characterized in that The amount of water added to the sediment is 2-5 BV of the volume of the sediment; and / or, The mass concentration of the sodium hydroxide solution is 5-30wt%.

7. The method according to claim 1, characterized in that After obtaining the stevia polyphenol precipitate, it also includes: The stevia polyphenol precipitate is filtered through filter paper to obtain a filter cake; The filter cake is rinsed with water to obtain high-purity stevia polyphenols.

8. The method according to claim 7, characterized in that The pore size of the filter paper is 300-500um; and / or, The amount of water used in the rinsing is 1-3 BV of the volume of the filter cake.

9. The method according to claim 1, characterized in that: The stevia extract is prepared by extracting stevia in an ethanol solution.

10. The method according to claim 9, characterized in that The volume concentration of the ethanol solution is 20-40% (v / v).

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