阅读说明：本技术 一种同时制备l-鼠李糖和异槲皮素的方法 (Method for simultaneously preparing L-rhamnose and isoquercetin ) 是由 郑璞 王德庆 吴丹 陈鹏程 于 2020-04-23 设计创作，主要内容包括：本发明公开了一种同时制备L-鼠李糖和异槲皮素的方法,属于生物技术领域。该方法利以低共溶液DES为介质转化芦丁同时制备L-鼠李糖和异槲皮素。经条件优化后,130g/L芦丁能够完全转化；并利用双水相体系提取,获得了高纯度的异槲皮素和L-鼠李糖,且可重复利用DES。DES经过4次回收后再利用,获得的异槲皮素纯度仍有92.28％。(The invention discloses a method for simultaneously preparing L-rhamnose and isoquercetin, which belongs to the technical field of biology, wherein a low co-solution DES is used as a medium to convert rutin and simultaneously prepare L-rhamnose and isoquercetin, rutin can be completely converted at 130 g/L after condition optimization, isoquercetin and L-rhamnose with high purity can be obtained by using a two-aqueous-phase system for extraction, DES can be repeatedly utilized and recycled after 4 times of recovery, and the obtained isoquercetin still has 92.28% purity.)

1. A method for simultaneously preparing L-rhamnose and isoquercetin, which is characterized by comprising the following steps:

(1) reacting in a eutectic solvent DES by using rutin as a substrate and α -L-rhamnosidase as a catalyst to obtain a reaction solution;

(2) diluting the reaction solution in the step (1), performing suction filtration, and respectively collecting filtrate and insoluble substances during suction filtration;

(3) washing the insoluble substance in the step (2), then carrying out suction filtration, and carrying out vacuum drying on the insoluble substance obtained by suction filtration to obtain a crude isoquercitrin product;

decolorizing the filtrate obtained in the step (2), adding inorganic salt to form a double aqueous phase, and collecting an upper phase solution and a lower phase solution respectively;

(4) and (4) concentrating the lower phase solution obtained in the step (3) by rotary evaporation, adding ethanol, collecting precipitate, and drying the precipitate in vacuum to obtain L-rhamnose.

2. The method as claimed in claim 1, wherein the DES is prepared by mixing a certain amount of hydrogen bond acceptor and hydrogen bond donor at a molar ratio of (1-3) to (1-3), and stirring at 60-80 deg.C to form a uniform transparent liquid, which is DES.

3. The method of claim 1, wherein the amount of α -L-rhamnosidase in step (1) in the reaction system is 4-12U/m L.

4. The method according to claim 1, wherein the DES concentration in step (1) is 10-50%.

5. The method of claim 1, wherein the reaction time of step (1) is at least 30 min; the addition amount of the deionized water in the step (2) is 2 times of that of the reaction solution.

6. The method according to claim 1, wherein the inorganic salt in step (3) is K₂HPO₄。

7. The method of claim 1, wherein reusing the DES specifically comprises: and (3) decoloring the filtrate obtained in the step (2), adding inorganic salt into the decolored solution to form a double aqueous phase, and taking the upper phase for reuse in the step (1).

8. The method of claim 7, wherein the concentration of the DES solution recycled to step (1) is adjusted to 10-50%; or not adjusting the concentration of the DES solution used in the step (1) and properly prolonging the reaction time of the step (1).

9. A method for improving the conversion rate of L-rhamnose and isoquercitrin is characterized in that rutin is used as a substrate, α -L-rhamnosidase is used as a catalyst, and the reaction is carried out in DES.

10. Use of the process of any one of claims 1 to 8, or the process of claim 9, in the preparation of L-rhamnose and isoquercitrin in the fields of medicine, food, agriculture.

Technical Field

The invention discloses a method for simultaneously preparing L-rhamnose and isoquercetin, belonging to the technical field of biology.

Background

L rhamnose is a rare sugar, can be used for producing essence and spice, is edible, can be used as intermediate of medicine and synthetic cardiotonic medicine, can react with other substances to form flavor substance, can be used as sweetener, can be used for preventing and treating crop diseases, can be used as intestinal tract penetration test agent, and has obvious anticancer effect.

Isoquercitrin (isoquercitrin), also called isoquercitrin, has one less rhamnoside group than rutin, has extremely low solubility in water (95 mg/L), is widely present in flowers, leaves, fruits and the like of plants, but has very small extraction amount from the flowers, the leaves, the fruits and the like.

A method for preparing L-rhamnose mainly comprises the steps of preparing flavonoid substances through acid hydrolysis, and a patent with publication number CN102952108A discloses a method for preparing quercetin and rhamnose by using sophora flower bud, wherein the quercetin is obtained by hydrolyzing isoquercitrin to remove terminal glucose groups, and the method is completed by the following steps of A, taking a proper amount of sophora flower buds, crushing, extracting rutin by using saturated and clear lime water, B, preparing quercetin by using the rutin extracted in the step A through hydrochloric acid hydrolysis, C, preparing a rhamnose crude product by fermenting the waste liquid in the step B, D, preparing a rhamnose fine product by using glucose in the rhamnose crude product in the step C through methanol separation.

The existing method for producing isoquercetin mainly comprises the preparation of rutin by hydrolyzing rutin with rhamnosidase, redundant quercetin can be generated when the isoquercetin is prepared by hydrolyzing rutin with acid, and the cost of the step of separating isoquercetin from quercetin is increased. The patent with publication number CN1483825A discloses a method for preparing isoquercetin and quercetin by hydrolyzing rutin with an enzyme method, which comprises the following reaction steps: taking enzyme capable of hydrolyzing rutin glycosyl; mixing enzyme, rutin, buffer solution and ethanol, and reacting at 15-70 deg.C and pH 4-8 for 2-24 hr, wherein the concentration of rutin is 0.1-10% and the concentration of ethanol is 0-35%; extracting to obtain mixture of isoquercetin and quercetin, and separating isoquercetin and quercetin by silica gel column chromatography. The method has long reaction time and low rutin concentration, and the obtained product is a mixture of isoquercetin and quercetin, and the pure isoquercetin can be obtained only by further purification, thus increasing the production cost.

A large amount of acid is used in the process of producing L-rhamnose, equipment is corroded, the risk of production operation is increased, glucose is generated in the hydrolysis process, rhamnose needs to be extracted after the glucose is consumed, and an extraction process is added.

Disclosure of Invention

The invention mainly aims to solve the technical defects of the prior production of rhamnose and isoquercitrin and provides a novel pollution-free and easily-controlled method for preparing rhamnose and isoquercitrin by hydrolyzing rutin with rhamnosidase.

The invention provides a method for preparing DES, which comprises the steps of mixing a certain amount of hydrogen bond acceptors and hydrogen bond donors according to the molar ratio of (1-3) to (1-3), and stirring at 60-80 ℃ until uniform and transparent liquid is formed, wherein the transparent liquid is DES.

In one embodiment of the invention, the hydrogen bond acceptor is choline chloride or betaine, and the hydrogen bond donor is urea, glycerol, ethylene glycol, 1, 2-propylene glycol, citric acid, D, L-malic acid, lactic acid, formic acid, acetic acid, acetamide or sulfur.

In one embodiment of the invention, the hydrogen bond acceptor is preferably choline chloride and the hydrogen bond donor is preferably urea.

The invention provides a method for simultaneously preparing L-rhamnose and isoquercetin, which is characterized by comprising the following steps:

(1) dissolving rutin with DES as solvent, and reacting the dissolved rutin under the action of α -L-rhamnosidase;

(2) adding deionized water into the reaction solution after the reaction, then carrying out suction filtration on the reaction solution, and respectively collecting filtrate and insoluble substances during the suction filtration;

(3) washing the insoluble substance with deionized water, performing suction filtration, and performing vacuum drying on the insoluble substance obtained by suction filtration to obtain a crude isoquercitrin product;

(4) decolorizing the filtrate obtained in the step (2), adding inorganic salt to form a double aqueous phase, and collecting an upper phase solution and a lower phase solution respectively;

(5) and (3) concentrating the lower phase solution by rotary evaporation, adding ethanol, collecting precipitate, and drying the precipitate in vacuum to obtain L-rhamnose crude product.

In one embodiment of the invention, the DES concentration in the step (1) is 30-50% (w/w), and the amount of α -L-rhamnosidase in the reaction system is 4-12U/m L.

In one embodiment of the invention, the reaction time in step (1) is at least 45 min.

In one embodiment of the present invention, the amount of deionized water added in step (2) is 2 times the amount of the reaction solution.

In one embodiment of the present invention, the inorganic salt in the step (4) is K₂HPO₄。

The invention provides a method for improving the conversion rate of L-rhamnose and isoquercitrin, which takes prepared DES as a solvent, dissolves rutin and reacts under the action of α -L-rhamnosidase.

In one embodiment of the invention, the α -L-rhamnosidase is added in an amount of 5-7U/m L.

In one embodiment of the present invention, the concentration of the DES is 30-50% (w/w).

In one embodiment of the invention, the reaction time is at least 45 min.

The invention provides a DES (data encryption standard) recycling method, which comprises the steps of decoloring filtrate obtained by preparing isoquercitrin by using activated carbon to obtain decolored liquid, concentrating the decolored liquid, and adding K₂HPO₄Forming a double aqueous phase, repeatedly using DES in the upper phase for rutin conversion, and using the lower phase solution for preparing rhamnose.

In one embodiment of the invention, a new DES solution is added into DES in the upper phase to make the concentration of DES 30-50% (w/w), and the new DES solution is applied to the method for preparing L-rhamnose and isoquercetin, or the DES in the upper phase is applied to the method for preparing L-rhamnose and isoquercetin, and the reaction time in the step (1) is prolonged and is 0.5-2 h.

In one embodiment of the present invention, the decoloring conditions are: adjusting the pH of the filtrate to 1.5-2.5 by using 5-7M HCl, adding activated carbon with the final concentration of 1.0-2.0% (w/v), decoloring at 60-80 ℃ for 30-50 min at 100-140 r/min, and filtering by using filter paper to remove the activated carbon to obtain a decolored solution.

The invention provides application of the method for improving the conversion rate of L-rhamnose and isoquercetin in preparation of L-rhamnose and isoquercetin in the fields of medicine, food and agriculture.

The invention provides application of the method for simultaneously preparing L-rhamnose and isoquercetin in preparation of L-rhamnose and isoquercetin in the fields of medicine, food and agriculture.

The invention provides application of the DES recycling method in preparation of L-rhamnose and isoquercetin in the fields of medicine, food and agriculture.

The method has the beneficial effects that the L-rhamnose and isoquercitrin are prepared by using a eutectic solvent (DES) medium, the DES is used as a medium to convert rutin and simultaneously prepare L-rhamnose and isoquercitrin, under an optimized condition, 130 g/L of rutin can be completely converted, a double-aqueous-phase system is used for extraction, the isoquercitrin and L-rhamnose with high purity are obtained, the DES can be recycled, and the obtained isoquercitrin still has the purity of 92.28% after 4 times of recycling.

Drawings

FIG. 1 is a diagram of the preparation of L-rhamnose and isoquercitin simultaneously by a eutectic solvent medium.

FIG. 2 is a diagram of the selection of two aqueous phase salts.

FIG. 3 is the HP L C spectrum of the product obtained by preparation, isoquercetin, (a) as isoquercetin standard and (b) as obtained isoquercetin product.

FIG. 4 is an HP L C map of L-rhamnose, (a) L-rhamnose standard and (b) L-rhamnose product obtained from the preparation.

Detailed Description

The enzyme activity is determined by adding 50 μ L pNPR and 50 μ L enzyme solution into 400 μ L citric acid buffer solution (50mM, pH 5.0), adding 1M L1M Na immediately after 1min in 50 deg.C thermostatic water bath₂CO₃The reaction was stopped and developed and the absorbance was measured at 400 nm.

Definition of enzyme activity: the amount of enzyme required to produce 1. mu. mol of p-nitrophenol per minute under the above conditions was defined as one unit of enzyme activity.