CN117701661A - Production and preparation process of enzyme modified isoquercitrin - Google Patents

Abstract

The invention relates to the technical field of preparation of enzyme-modified isoquercetin, and discloses a production and preparation process of enzyme-modified isoquercetin, which includes the following steps: S1: Preparation of raw materials: isoquercetin and dextrin substances are used as the Raw materials, prepare enzyme-modified isoquercetin through enzymatic conversion method; S2: Remove oligosaccharides: dilute the raw materials in S1, pass the diluted solution through an ion exchange resin column, use alcohol to analyze the fully adsorbed resin column, and then pass it through Remove the alcohols in the analytical solution by distillation under reduced pressure. S3: collect enzyme-modified isoquercetin: separate the aqueous solution after removing alcohol from S2 by chromatography to extract the enzyme-modified isoquercetin and collect it; S4: concentrate: collect S3 The collected materials containing high enzyme-modified isoquercetin are concentrated; S5: drying: the concentrated materials in S4 are spray-dried; the present invention uses ion exchange resin and chromatographic separation method to carry out enzyme-modified isoquercetin. Isolation and purification can increase the purity of enzyme-modified isoquercetin to more than 90%, thereby making the product more stable.

Description

A production and preparation process of enzyme-modified isoquercetin

Technical field

The present invention relates to the technical field of preparation of enzyme-modified isoquercetin, specifically a production and preparation process of enzyme-modified isoquercetin.

Background technique

Isoquercetin is a flavonoid compound that is very rare in nature but has significant antioxidant, anti-tumor and other biological activities. It exists in the flowers of Malvaceae plant grass and the leaves of Apocynaceae plant kenaf, but Its water solubility is very poor, which also affects its application. In order to improve its water solubility, people add hydrophilic groups to isoquercetin through enzyme modification, thereby increasing its water solubility. Currently, most enzyme modifications on the market The content of isoquercetin (EMIQ) is 50-60%. The impurities of this enzyme-modified isoquercetin are mainly isoquercetin and some oligosaccharides. These impurities will affect the stability of the EMIQ aqueous solution. At the same time This makes EMIQ's finished products more likely to absorb moisture and agglomerate. To solve these problems, the present invention is proposed.

Contents of the invention

The purpose of the present invention is to provide a production and preparation process of enzyme-modified isoquercetin to solve the problems raised in the above background technology: most enzyme-modified isoquercetin (EMIQ) currently on the market has a content of 53-53 82%. The impurities in this enzyme-modified isoquercetin are mainly isoquercetin and some oligosaccharides. These impurities will affect the stability of the EMIQ aqueous solution and make the finished EMIQ product more likely to absorb moisture and agglomerate.

In order to achieve the above object, the present invention provides the following technical solution: a production and preparation process of enzyme-modified isoquercetin, including the following steps:

S1: Preparation of raw materials: Using isoquercetin and dextrins as raw materials, enzyme-modified isoquercetin is prepared through enzyme conversion method. The content of enzyme-modified isoquercetin in the prepared reaction liquid solid material is 40- 60wt%;

S2: Remove oligosaccharides: dilute the raw materials in S1, pass the diluted liquid through an ion exchange resin column, analyze the fully adsorbed resin column with alcohol, and then remove the alcohols in the analyzed liquid through reduced pressure distillation. The content of enzyme-modified isoquercetin in solid matter is 53-82wt%;

S3: Collect enzyme-modified isoquercetin: The aqueous solution after removing alcohol in S2 is separated by chromatography to extract enzyme-modified isoquercetin and collected;

S4: Concentration: Concentrate the materials containing high enzyme-modified isoquercetin collected in S3;

S5: Drying: Spray dry the concentrated material in S4.

By adopting the above technical solution, this application uses isoquercetin and dextrin-like substances as raw materials to prepare enzyme-modified isoquercetin through an enzyme conversion method. The content of enzyme-modified isoquercetin in the prepared reaction liquid solid material It is 40-60wt%. At this time, the content of the main product (enzyme-modified isoquercetin) is low and the content of impurities (isoquercetin and oligosaccharides) is high. The obtained raw materials are diluted to obtain more It is good to adsorb through the ion exchange resin column. The enzyme-modified isoquercetin is adsorbed in the ion exchange resin column, and a small amount of impurities are also adsorbed in the ion exchange resin column. Use alcohol to adsorb the completely ion exchange resin column. Analysis is carried out, and the enzyme-modified isoquercetin is decomposed by alcohol. At the same time, the analysis liquid contains a small amount of impurities. The alcohols in the analysis liquid are removed by vacuum distillation. At this time, the solid enzyme-modified isoquercetin in the obtained liquid is The content of cortin is 53-82wt%; the aqueous solution after removing alcohol is separated by chromatography to extract the enzyme-modified isoquercetin and collected. At this time, the purity of the enzyme-modified isoquercetin is increased to more than 90wt%. Therefore, ion is used The separation and purification of enzyme-modified isoquercetin by exchange resin and chromatographic separation can increase the purity of enzyme-modified isoquercetin to more than 90wt%, thereby making the product more stable and the quality of the obtained product higher. It is stable and has better water solubility, which is mainly reflected in the rapid dissolution rate of the product in aqueous solution.

Preferably, the enzyme is one or a combination of glucosyltransferase, pullulanase or beta-amylase.

By adopting the above technical solution, it is preferably one or a combination of glucosyltransferase, pullulanase or beta-amylase. Dextrin is composed of multiple glucose structures connected and is cleaved by the above-mentioned biological enzymes. function, the dextrin is cut into glucose units with different degrees of polymerization, and then these glucose units with different degrees of polymerization are connected to the hydroxyl groups of isoquercetin through biological enzymes, and a single biological enzyme conversion method is used to prepare enzyme-modified isoquercetin. At this time, a single biological enzyme can not only connect these glucose groups to form enzyme-modified isoquercetin, but at the same time, a single biological enzyme can also cut off the glucose connected to the enzyme-modified isoquercetin, thereby making the enzyme conversion reaction If the time is too long, the purity of the enzyme-modified isoquercetin obtained will be low. This application uses a composite enzyme to reduce the enzyme conversion reaction time and at the same time improve the purity of the enzyme-modified isoquercetin obtained.

Preferably, the enzyme is glucosyltransferase, pullulanase and beta-amylase.

By adopting the above technical solution, the enzymes are preferably glucosyltransferase, pullulanase and beta-amylase. By using composite enzymes, the enzyme conversion reaction time is reduced and the obtained enzyme-modified isoquercetin is improved. purity.

Preferably, the raw materials in S2 are diluted to an aqueous solution with a solid content of 10-15 wt%, and the speed of the diluted solution passing through the ion exchange resin column is 0.1-0.5 times the column/hour speed.

By adopting the above technical solution, the concentration of raw material dilution and the speed at which the diluent passes through the ion exchange resin column are optimized, so that the ion exchange resin column can better adsorb the diluent.

Preferably, the alcohol mentioned in S2 is methanol or ethanol, and the range of the alcohol is 60-80wt%.

By adopting the above technical solution, the alcohol is preferably methanol or ethanol. Methanol or ethanol has good solubility for enzyme-modified isoquercetin. However, it is difficult to dissolve impurities (isoquercetin and oligosaccharides). The performance is poor, which helps to resolve the enzyme-modified isoquercetin in the ion exchange resin column, and further optimizes the concentration range of alcohol to 60-80vol%, further improving the complete ion exchange of alcohol for adsorption. Analytical performance of resin columns.

Preferably, the specific method of S3 is: dilute the aqueous solution after removing alcohol in S2 to a concentration of 5-10wt%, and carry out chromatographic separation and feed. The chromatographic separation system consists of 8 chromatographic columns connected in series to form a closed system connected end to end. The diameter-to-height ratio of each chromatographic column is 1:10, the feed rate is 1-2 times the column/hour, the feed volume for chromatographic separation is 5-8 times the column, pure water is used as the flushing liquid, and the enzyme is modified The movement speed of isoquercetin in the mobile phase is faster than that of isoquercetin. The effluent is collected at the outlet of the last chromatographic column of the chromatographic separation system. It is collected after the enzyme-modified isoquercetin is found to flow out. The enzyme-modified isoquercetin Isoquercetin is measured by a UV spectrophotometer. Change the collection bottle every half hour, collect continuously for 6-8 hours, and measure the components in each collection bottle.

Preferably, the stationary phase used in the chromatographic separation system is Rohm and Haas chromatographic separation resin, and the matrix is cross-linked polystyrene.

Preferably, the specific operation method of S4 is: mix the material liquid in the collection bottle whose solid content of enzyme-modified isoquercetin in S3 is more than 90%wt%, and vacuum concentrate to a material concentration of 40wt%- 60wt%.

Preferably, the spray inlet temperature is 130°C-140°C.

Preferably, it also includes S6: recycling: reusing the oligosaccharides flowing out in S2 and the remaining isoquercetin in S3 as raw materials for preparing enzyme-modified isoquercetin.

Compared with the prior art, the beneficial effects of the present invention are:

The present invention uses ion exchange resin and chromatographic separation method to separate and purify enzyme-modified isoquercetin, which can increase the purity of enzyme-modified isoquercetin to more than 90wt%, thereby making the product more stable and obtaining The product quality is more stable and the water solubility is better, which is mainly reflected in the rapid dissolution rate of the product in the aqueous solution, while conventional products require vigorous stirring for a long time to be completely dissolved. Many conventional products are dissolved after being left for a long time, and the foreign substances contained in them are Quercetin will precipitate, but the product of the present invention has a very low isoquercetin content and is relatively stable even if it is placed for a long time. The content of sugar in the finished product of the process of the present invention is extremely low, making the powder less likely to absorb moisture even if it is placed for a long time. , and conventional finished products contain a certain amount of sugar substances, which makes them easy to absorb moisture and agglomerate after being left for a long time.

Description of the drawings

Figure 1 is a flow chart of the production and preparation process of enzyme-modified isoquercetin in the present invention.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

Please refer to Figure 1. The present invention provides a production and preparation process of enzyme-modified isoquercetin, which includes the following steps:

S1: Preparation of raw materials: Use isoquercetin and dextrins as raw materials, add enzyme to the raw materials, and prepare enzyme-modified isoquercetin through enzyme conversion method. The enzyme-modified isoquercetin in the prepared reaction liquid solid material Quercetin content is 40-60wt%;

S2: Remove oligosaccharides: Dilute the raw materials in S1 to a 10-15wt% aqueous solution, and pass the diluted solution through the ion exchange resin column. The speed of the diluted solution passing through the ion exchange resin column is 0.1-0.5 times the column. The fully adsorbed resin column is analyzed with 60-80wt% alcohol at a speed of 53-82wt%;

S3: Collect enzyme-modified isoquercetin: Dilute the aqueous solution after removing alcohol in S2 to a concentration of 5-10wt%, and carry out chromatographic separation and feed. The chromatographic separation system consists of 8 chromatographic columns connected in series to form a closed end-to-end connection. System, the diameter-to-height ratio of each chromatographic column is 1:10, the feed rate is 1-2 times the column/hour, the feed volume for chromatographic separation is 5-8 times the column, pure water is used as the flushing liquid, and the enzyme Modified isoquercetin moves faster than isoquercetin in the mobile phase. The effluent is collected at the outlet of the last chromatographic column of the chromatographic separation system. Collection begins after the enzyme-modified isoquercetin is found to flow out. Enzyme-modified isoquercetin is measured by UV spectrophotometer. Change the collection bottle every half hour, collect continuously for 6-8 hours, and measure the components in each collection bottle, and collect the enzyme modification in the solution in the bottle. The content of isoquercetin in solid matter is more than 90%wt;

S4: Concentration: Concentrate the materials containing high enzyme-modified isoquercetin collected in S3 to a concentration of 40wt%-60wt%;

S5: Drying: Spray dry the concentrated material in S4, and the spray inlet temperature is 130°C-140°C;

S6: Recycling: Reuse the oligosaccharides flowing out of S2 and the remaining isoquercetin in S3 as raw materials for preparing enzyme-modified isoquercetin.

Example

Example 1

S1: Preparation of raw materials: Use 1kg of isoquercetin and 1kg of dextrins as raw materials, add 5ml of glucosyltransferase, 3ml of pullulanase and 3ml of beta-amylase to the above raw materials. The enzyme-modified isoquercetin is prepared by the conversion method. The content of the enzyme-modified isoquercetin in the prepared reaction liquid solid material is 60wt%, isoquercetin is 20wt%, and oligosaccharide substances are 20wt%;

S2: Remove oligosaccharides: dilute the raw materials in S1 to a 13wt% aqueous solution, and pass the diluted solution through the ion exchange resin column. The speed of the diluted solution passing through the ion exchange resin column is 0.3 times the column/hour. Speed, use 70wt% ethanol to analyze the fully adsorbed resin column, and then remove the ethanol in the analytical solution through vacuum distillation. The content of the solid enzyme-modified isoquercetin in the obtained liquid is 82wt%, and the isoquercetin content is 82wt%. 15wt%, oligosaccharides 3wt%;

S3: Collect enzyme-modified isoquercetin: dilute the aqueous solution after removing alcohol in S2 to a concentration of 8wt%, and carry out chromatographic separation and feed. The chromatographic separation system consists of 8 chromatographic columns connected in series to form a closed system connected end to end. The diameter-to-height ratio of each chromatographic column is 1:10, the feed rate is 2 times the column/hour, the feed volume for chromatographic separation is 7 times the column, pure water is used as the flushing liquid, and enzyme-modified isoquercetin It moves faster than isoquercetin in the mobile phase. The effluent is collected at the outlet of the last chromatographic column of the chromatographic separation system. It is collected after the enzyme-modified isoquercetin is found to flow out. The enzyme-modified isoquercetin The peptide is measured by UV spectrophotometer. Change the collection bottle every half hour, collect continuously for 8 hours, and measure the components in each collection bottle. The enzyme-modified isoquercetin in the solution in the collection bottle accounts for 10% of the solid matter. Content is 95wt%.

As shown in Table 1, the difference between Examples 1 to 10 lies in the enzyme used in step S1.

Comparative ratio

Comparative example 1

The difference between this comparative example and Example 1 is that the order of steps 2 and 3 is reversed, chromatographic separation is performed first, and then adsorption is performed through an ion exchange resin column.

Comparative example 2

The difference between this comparative example and Example 1 is that step S3 is eliminated and concentrated and dried directly.

Comparative example 3

The difference between this comparative example and Example 1 is that pullulanase is replaced by rhamnase.

Test Methods

Use a UV spectrophotometer to determine the content of the substances in the liquid collected in each step:

1. Prepare the sample: Dissolve the substance to be measured in an appropriate solvent to make a solution of a certain concentration;

2. Draw a standard curve: Configure standard solutions with different concentrations, measure their absorbance on a UV spectrophotometer, and draw the relationship curve between absorbance and concentration;

3. Determine the absorbance of the sample: Place the solution to be measured in the sample cell of the UV spectrophotometer and measure its absorbance;

4. Calculate the sample concentration based on the standard curve: Compare the measured absorbance with the standard curve to determine the concentration of the solution to be tested;

It should be noted that when using a UV spectrophotometer for measurement, you need to choose a suitable wavelength and a suitable solvent, and pay attention to the stability of the solution. At the same time, in order to improve the accuracy of the measurement, you can perform multiple measurements and average them. value.

Record the reaction time of the enzyme conversion method through a table: record the reaction time and the corresponding conversion rate. As the reaction time changes, the corresponding conversion rate tends to balance, and the inflection point of the conversion rate is the reaction time of the enzyme conversion method.

The content of enzyme-modified isoquercetin in the solid material and the reaction time of the enzyme conversion method in the steps corresponding to Examples 1 to 10 and Comparative Examples 1 to 2 are as shown in Table 2.

Table 2 The reaction time of step S1 and the content of enzyme-modified isoquercetin in the solid material in each step of the reaction

Combining Examples 1 to 10 and Comparative Example 3 and Table 2, it can be seen that the content of enzyme-modified isoquercetin in the solid material in the corresponding steps in Example 1 is greater than that in Examples 2 to 10. In step S3, the content of enzyme-modified isoquercetin in the solid material is as high as 95wt%, and the reaction time is also the shortest (24h), indicating that the complex formed by glucosyltransferase combined with the other two enzymes pullulanase and beta-amylase Enzyme is more effective than a single enzyme or a combination of two other enzymes. At the same time, the three enzymes have a synergistic effect, which can increase the conversion rate of the enzyme conversion reaction and shorten the reaction time.

Combining Example 1 and Comparative Example 1 and Table 2, it can be seen that after changing the order of step S2 and step S3 in Example 1, the purity of enzyme-modified isoquercetin obtained in Example 1 is greater than that in Comparative Example 1. , the order of steps S2 and S3 in the instructions affects the purity of the enzyme-modified isoquercetin product.

Combining Example 1 and Comparative Example 2 and Table 2, it can be seen that the purity of the enzyme-modified isoquercetin product can be further improved through chromatographic separation technology, and the purity of the enzyme-modified isoquercetin product can be increased to 95wt %.

Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art will understand that various changes, modifications, and substitutions can be made to these embodiments without departing from the principles and spirit of the invention. and modifications, the scope of the invention is defined by the appended claims and their equivalents.

Claims (8)

1. The production and preparation process of the enzyme modified isoquercitrin is characterized by comprising the following steps:

s1: the preparation method comprises the following steps: taking isoquercetin and dextrin substances as raw materials, and preparing enzyme modified isoquercetin by an enzyme conversion method;

s2: removing oligosaccharide: diluting the raw materials in the step S1, passing the diluted solution through an ion exchange resin column, analyzing the resin column completely adsorbed by alcohol, and removing the alcohol in the analysis solution by reduced pressure distillation;

s3: collecting enzyme modified isoquercetin: separating and extracting enzyme modified isoquercitrin from the aqueous solution after removing the alcohol in the step S2 by chromatography and collecting the enzyme modified isoquercitrin;

s4: concentrating: concentrating the material containing the enzyme modified isoquercitrin Pi Sugao collected in the step S3;

s5: and (3) drying: and (3) carrying out spray drying on the concentrated material in the step S4.

2. The process for producing and preparing the enzyme-modified isoquercitrin according to claim 1, wherein the raw material in S2 is diluted to a 10-15wt% aqueous solution, and the speed of passing the diluted solution through the ion exchange resin column is 0.1-0.5 times the column speed per hour.

3. The process for producing enzymatically modified isoquercitrin of claim 1 wherein said alcohol in S2 is methanol or ethanol and said alcohol is present in a concentration range of 60-80vol%.

4. The production and preparation process of the enzyme modified isoquercitrin, according to claim 1, characterized in that the specific method of S3 is as follows: diluting the aqueous solution after alcohol removal in S2 to 5-10wt% concentration, carrying out chromatographic separation feeding, connecting 8 chromatographic columns in series by a chromatographic separation system to form a closed system connected end to end, wherein the diameter-to-height ratio of each chromatographic column is 1:10, the feeding speed is 1-2 times of column/hour, the feeding amount of chromatographic separation is 5-8 times of column/hour, taking purified water as flushing liquid, collecting effluent at the outlet of the last chromatographic column of the chromatographic separation system, starting collecting after the enzyme-modified isoquercetin is found, measuring the enzyme-modified isoquercetin by an ultraviolet spectrophotometer, changing one collecting bottle every half hour, continuously collecting for 6-8 hours, and measuring the components in each collecting bottle.

5. The process for preparing enzymatically modified isoquercitrin in accordance with claim 4, wherein said stationary phase used in said chromatographic separation system is Rogowski chromatographic resin and said matrix is crosslinked polystyrene.

6. The production and preparation process of the enzyme modified isoquercitrin, according to claim 1, characterized in that the specific operation method of S4 is as follows: uniformly mixing the material liquid in the collecting bottle with the enzyme modified isoquercitrin in S3 accounting for more than 90 percent by weight of solid substance, and concentrating in vacuum until the concentration of the material is 40 to 60 percent by weight.

7. The process for producing and preparing enzyme-modified isoquercitrin in accordance with claim 1, wherein said spray inlet temperature is 130 ℃ to 140 ℃.

8. The process for producing and preparing the enzyme-modified isoquercitrin of claim 1, further comprising S6: recycling: the oligosaccharides eluted in S2 and the isoquercitrin remaining in S3 are reused as raw materials for preparing the enzyme-modified isoquercitrin.