阅读说明：本技术 一种莱鲍迪苷d的制备方法及其产品和应用 (Preparation method of rebaudioside D, and product and application thereof ) 是由 李丽 聂在建 程保华 李德春 于 2019-10-30 设计创作，主要内容包括：本发明提供了一种莱鲍迪苷D的制备方法及其产品和应用,所述制备方法包括以甜菊苷为原料,以乙酸乙酯和β-环糊精为糖基供体,在复合酶的催化下发生转化反应,生成莱鲍迪苷D。本发明采用廉价易得的乙酸乙酯和β-环糊精作为糖基供体,替换成本高昂的UDP-葡萄糖,在复合酶的催化作用下生成莱鲍迪苷D,该反应为制备莱鲍迪苷D开辟了新的途径,反应的产物得率高,制备工艺简单,适用于工业化生产,具有广阔的应用前景。(The invention provides a preparation method of rebaudioside D, a product and application thereof. According to the invention, ethyl acetate and beta-cyclodextrin which are cheap and easy to obtain are used as glycosyl donors to replace UDP-glucose with high cost, rebaudioside D is generated under the catalytic action of the complex enzyme, the reaction opens up a new way for preparing rebaudioside D, the product yield of the reaction is high, the preparation process is simple, and the method is suitable for industrial production and has wide application prospect.)

1. The preparation method of rebaudioside D is characterized by taking stevioside as a raw material, taking ethyl acetate and beta-cyclodextrin as glycosyl donors, and carrying out conversion reaction under the catalysis of complex enzyme to generate rebaudioside D.

2. The method of claim 1, wherein the stevioside has an initial reaction concentration of 0.1 to 3 g/L.

3. The preparation method according to claim 1 or 2, wherein the mass ratio of ethyl acetate to stevioside is (40-200): 1;

preferably, the mass ratio of the beta-cyclodextrin to the stevioside is (30-100): 1.

4. The method of any one of claims 1-3, wherein the complex enzyme comprises an acyltransferase and a glycosyltransferase;

preferably, the acyltransferase comprises a palmitoyl transferase;

preferably, the glycosyltransferase comprises a cyclodextrin glycosyltransferase.

5. The method according to claim 4, wherein the cyclodextrin glycosyltransferase is added to the reaction system in an amount of 25000-50000U/L;

preferably, the addition amount of the palmitoyl transferase in the reaction system is 50000-100000U/L.

6. The method according to any one of claims 1 to 5, wherein the conversion reaction is carried out in an aqueous system in a buffer at pH 6-8.

7. The method according to any one of claims 1 to 6, wherein the reaction temperature of the conversion reaction is 20 to 50 ℃; preferably 35-40 ℃;

preferably, the reaction time of the conversion reaction is 1 to 24 hours, preferably 16 to 20 hours.

8. The method according to any one of claims 1 to 7, comprising in particular the steps of: taking stevioside as a raw material, taking ethyl acetate and cyclodextrin as glycosyl donors, taking a complex enzyme consisting of palmitoyl transferase and cyclodextrin glycosyl transferase as a tool enzyme, and carrying out biotransformation in a buffer liquid water phase system with the pH value of 6-8, wherein the reaction temperature is 20-50 ℃, and the reaction time is 1-24h, so as to obtain a water solution containing rebaudioside D; optionally, after the aqueous solution is separated and purified, high-purity rebaudioside D is obtained;

wherein the initial concentration of stevioside is 0.1-3g/L, the mass ratio of ethyl acetate to stevioside is (40-200):1, the mass ratio of beta-cyclodextrin to stevioside is (30-100):1, and the addition amount of cyclodextrin glycosyltransferase in the complex enzyme is 25000-50000U/L; the addition amount of the palmitoyl transferase is 50000-100000U/L.

9. A rebaudioside D composition or rebaudioside D purified product obtained by the preparation method of any one of claims 1-8.

10. Use of the rebaudioside D composition or rebaudioside D purified product of claim 9 in the preparation of a sweetener, a food product, a beverage, an oral hygiene product, or a medicament.

Technical Field

The invention belongs to the technical field of biocatalytic conversion, relates to a preparation method of rebaudioside D, a product and application thereof, and particularly relates to a preparation method of rebaudioside D taking ethyl acetate and beta-cyclodextrin as glycosyl donors, a product and application thereof.

Background

Steviosides (stevia glycosides) are natural sweeteners extracted from the leaves of stevia Rebaudiana Bertoni of Compositae herbs. Is a mixture of various glycosides, and different glycosides have large differences in taste quality. Stevioside has high sweetness, low calorie, high stability, potential curative effects of resisting hyperglycemia, hypertension, inflammation, tumor, diarrhea and the like, and effects of regulating immunity and the like, and has attracted extensive attention as a sweetener for related research and economy in food and beverage. High purity stevia products have been officially approved as food additives in the United states and the European Union.

In recent years, over ten glycosides have been isolated from stevia rebaudiana. Rebaudioside D (Rebaudianoside D, Reb D) is the most potential stevioside, has high sweetness which is about 300 times that of cane sugar and 350 times that of other stevioside, has pure sweetness and better mouthfeel which is close to cane sugar, has no sweetness and bitterness and licorice peculiar smell, has good stability, and is an ideal natural high-power sweetener product. In 2013, the FDA in the united states approved the use of a stevia product extracted from stevia leaves containing 50% rebaudioside D content and recognized its general safety (GRAS).

The rebaudioside D content in stevia leaves is very low (less than 5%), a large amount of stevia raw materials are needed for producing rebaudioside D by adopting an extraction method, the process for enriching rebaudioside D is complicated, column passing, desalting, decoloring and recrystallizing are needed for many times after extraction, a large amount of waste water is generated in the production process, the production cost is high, and the method is not suitable for industrial large-scale production.

CN107446874A discloses a method for producing rebaudioside D by using recombinant escherichia coli, which comprises the steps of constructing the recombinant escherichia coli by using a biological method, introducing an eugt11 gene into the escherichia coli for induction expression to obtain recombinant protease, and catalyzing Rebaudioside A (RA) to be converted into Rebaudioside D (RD), wherein the method relates to strain modification, and the stability of the method needs to be improved.

CN104726523A discloses a method for preparing rebaudioside M by an enzymatic method, which utilizes tomato UDP-glycosyltransferase and potato sucrose synthase to produce rebaudioside M from rebaudioside a and sucrose as raw materials. According to the method, high-yield recombinant strains of UDP-glucosyltransferase and sucrose synthase are obtained by using a genetic engineering technology, then a crude product of the biological enzyme is collected and directly subjected to catalytic reaction, UDP or UDP-glucose is not added in a reaction system, rebaudioside A and sucrose are used as raw materials, the UDP-glucose is catalyzed by the sucrose synthase to efficiently circulate, and the rebaudioside A is catalyzed by the tomato UDP-glucosyltransferase to generate rebaudioside M. This method is not suitable for rebaudioside D production.

The prior method for synthesizing rebaudioside D by a biological enzyme method needs to add expensive UDP-glucose as a substrate, and takes stevioside or rebaudioside A as the substrate to catalytically generate rebaudioside D under the action of UDP-glucosyltransferase. But due to the extremely high selling price of UDP-glucose, the feasibility of industrially preparing the rebaudioside D is almost completely limited, the economy is poor, and the market competitiveness is lacked.

Therefore, the method for preparing rebaudioside D economically has important significance and wide market prospect.

Disclosure of Invention

The invention adopts cheap and easily-obtained ethyl acetate and beta-cyclodextrin as glycosyl donors to replace UDP-glucose with high cost, and generates the rebaudioside D under the catalysis of complex enzyme, and the reaction opens up a new way for preparing the rebaudioside D, has high product yield and simple preparation process, is suitable for industrial production, and has wide application prospect.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a method for preparing rebaudioside D, which comprises the steps of taking stevioside as a raw material, taking ethyl acetate and beta-cyclodextrin as glycosyl donors, and carrying out conversion reaction under the catalysis of complex enzyme to generate rebaudioside D.

In the invention, the inventor unexpectedly finds that ethyl acetate and beta-cyclodextrin which are cheap and easy to obtain can be used as glycosyl donors, stevioside is used as a substrate raw material, rebaudioside D is produced by reaction under the catalysis of complex enzyme, UDP-glucose with high cost in the prior art is replaced, ethyl acetate is usually used as an extractant or an organic solvent, but the ethyl acetate and beta-cyclodextrin are used together to be used as the glycosyl donors of stevioside, so that the whole preparation process is simpler and more efficient, the yield of rebaudioside D is improved, and a foundation is laid for industrial production. It should be noted that, during the process of converting stevioside into rebaudioside D, rebaudioside a is converted first, and then rebaudioside D is converted, so rebaudioside a can be used as a substrate material, and the same technical means as above are adopted to prepare rebaudioside D.

Preferably, the stevioside has an initial reaction concentration of 0.1 to 3g/L, and may be, for example, 0.1g/L, 0.2g/L, 0.3g/L, 0.4g/L, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1g/L, 1.1g/L, 1.2g/L, 1.3g/L, 1.4g/L, 1.5g/L, 1.6g/L, 1.7g/L, 1.8g/L, 1.9g/L, 2g/L, 2.1g/L, 2.2g/L, 2.3g/L, 2.4g/L, 2.5g/L, 2.6g/L, 2.7g/L, 2.8g/L, 2.9g/L, or 3 g/L.

Preferably, the mass ratio of ethyl acetate to stevioside is (40-200):1, and may be, for example, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, 100:1, 105:1, 110:1, 115:1, 120:1, 125:1, 130:1, 135:1, 140:1, 145:1, 150:1, 155:1, 160:1, 165:1, 170:1, 175:1, 180:1, 185:1, 190:1, 195:1, or 200: 1.

In the invention, by adjusting the dosage ratio of ethyl acetate to stevioside, the conversion rate of rebaudioside D is reduced when the ratio of ethyl acetate to stevioside is lower than 40:1 or higher than 200:1, which may be related to that low-concentration ethyl acetate cannot meet the requirement of enzymatic reaction, and high-concentration ethyl acetate influences the enzyme activity in the system.

Preferably, the mass ratio of the beta-cyclodextrin to the stevioside is (30-100: 1), and can be 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1, for example.

In the invention, by adjusting the mass ratio of the beta-cyclodextrin to the stevioside, the fact that the conversion rate of rebaudioside D is reduced when the ratio of the beta-cyclodextrin to the stevioside is lower than 30:1 or higher than 100:1 is found, which may be related to the fact that the low-concentration beta-cyclodextrin cannot meet the requirement of enzymatic reaction, and the embedding effect of the high-concentration beta-cyclodextrin influences the enzymatic reaction in a system.

Preferably, the complex enzyme comprises an acyltransferase and a glycosyltransferase.

Preferably, the acyltransferase comprises a palmitoyl transferase.

Preferably, the glycosyltransferase comprises a cyclodextrin glycosyltransferase.

According to the invention, the combination of palmitoyl transferase and cyclodextrin glycosyltransferase is used as the complex enzyme, and the dosage of the two enzymes is adjusted, so that the yield of rebaudioside D converted from stevioside can be further improved, the stevioside is mainly converted into rebaudioside D, and the yield of rebaudioside D is reduced when the ratio of the stevioside to the rebaudioside D exceeds the range of the complex enzyme.

Preferably, the addition amount of the cyclodextrin glycosyltransferase in the reaction system is 25000-50000U/L, and can be 25000U/L, 30000U/L, 35000U/L, 40000U/L, 45000U/L or 50000U/L, etc.

In the present invention, when the addition amount of cyclodextrin glycosyltransferase is less than 25000U/L, the conversion rate of rebaudioside D is reduced, and when the addition amount is more than 50000U/L, a large amount of by-products are produced, and the conversion rate of rebaudioside D is also reduced.

Preferably, the palmitoyl transferase is added in an amount of 50000-100000U/L, such as 50000U/L, 60000U/L, 70000U/L, 80000U/L, 90000U/L or 100000U/L in the reaction system.

In the present invention, when the amount of palmitoyltransferase added is less than 50000U/L, the conversion rate of rebaudioside D is lowered, and when the amount of palmitoyltransferase added is more than 100000U/L, a large amount of by-products are produced, and the conversion rate of rebaudioside D is also lowered.

Preferably, the conversion reaction is carried out in an aqueous system in a buffer at pH 6-8 (e.g., pH 6, pH 7, pH 8, etc.).

Preferably, the temperature of the conversion reaction is 20-50 ℃, for example, 20 ℃, 22 ℃, 25 ℃, 28 ℃, 30 ℃, 32 ℃, 35 ℃, 38 ℃, 40 ℃, 42 ℃, 45 ℃, 48 ℃ or 50 ℃, preferably 35-40 ℃.

Preferably, the conversion reaction time is 1 to 24 hours, for example, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or 24 hours, preferably 16 to 20 hours.

As a preferred technical scheme of the invention, the preparation method specifically comprises the following steps:

taking stevioside as a raw material, taking ethyl acetate and cyclodextrin as glycosyl donors, taking a complex enzyme consisting of palmitoyl transferase and cyclodextrin glycosyl transferase as a tool enzyme, and carrying out biotransformation in a buffer liquid water phase system with the pH value of 6-8, wherein the reaction temperature is 20-50 ℃, and the reaction time is 1-24h, so as to obtain a water solution containing rebaudioside D; optionally, after the aqueous solution is separated and purified, high-purity rebaudioside D is obtained;

wherein the initial concentration of stevioside is 0.1-3g/L, the mass ratio of ethyl acetate to stevioside is (40-200):1, the mass ratio of beta-cyclodextrin to stevioside is (30-100):1, and the addition amount of cyclodextrin glycosyltransferase in the complex enzyme is 25000-50000U/L; the addition amount of the palmitoyl transferase is 50000-100000U/L.

In the invention, the aqueous solution with the main product rebaudioside D is prepared by the method, and the rebaudioside D with high purity is obtained by optionally carrying out separation and purification, wherein the separation and purification modes are typically but not limited to crystallization, membrane concentration, resin separation and the like.

The enzyme catalysis method using stevioside as a substrate can only obtain a mixture of a plurality of glycosides, a certain specific glycoside is taken as a main product by regulating and controlling reaction conditions and the types of used enzymes, and rebaudioside D is mainly generated in the reaction by regulating and controlling the types and the proportional relation of raw materials, glycosyl donors and complex enzymes, so that the yield of the rebaudioside D is remarkably improved.

In a second aspect, the present invention provides a rebaudioside D composition or a rebaudioside D purified product obtained by the preparation method of the first aspect.

In a third aspect, the present invention provides the use of a rebaudioside D composition or rebaudioside D purified product of the second aspect in the preparation of a sweetener, a food product, a beverage, a dental hygiene product, or a medicament.

Compared with the prior art, the invention has the following beneficial effects:

(1) the rebaudioside D preparation method provided by the invention creatively adopts cheap and easily-obtained ethyl acetate and beta-cyclodextrin as a compound glycosyl donor to replace expensive UDP-glucose, so as to realize the preparation of rebaudioside D;

(2) according to the invention, palmitoyl transferase and cyclodextrin glycosyltransferase are used as a complex enzyme, under the condition of the mixture ratio of the enzyme and the existence of ethyl acetate in the proportion, glucose on beta-cyclodextrin can be added to the corresponding site of stevioside, so that the stevioside is converted into rebaudioside D;

(3) the preparation method provided by the invention is simple and efficient, is suitable for industrial scale production, and improves the yield of rebaudioside D.

Detailed Description

To further illustrate the technical means and effects of the present invention, the following embodiments further illustrate the technical solutions of the present invention, but the present invention is not limited to the scope of the embodiments.

The rebaudioside D conversion referred to in the examples below is the ratio of the mass of rebaudioside D produced to the mass of stevioside.