阅读说明：本技术 一种莱鲍迪苷e的制备方法及其产品和应用 (Preparation method of rebaudioside E, 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E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E。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E,该反应为制备莱鲍迪苷E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-E, 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 E is generated under the catalytic action of the complex enzyme, the reaction opens up a new way for preparing rebaudioside E, 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 E is characterized in that stevioside is used as a raw material, ethyl acetate and beta-cyclodextrin are used as glycosyl donors, and conversion reaction is carried out under the catalysis of acyltransferase and glycosyltransferase to generate rebaudioside E.

2. The method of claim 1, wherein the stevioside is initially reacted at a concentration of 1-300 g/L.

3. The production method according to claim 1 or 2, wherein the mass ratio of ethyl acetate to stevioside is (0.4-10): 1;

preferably, the mass ratio of the beta-cyclodextrin to the stevioside is (3-10): 1.

4. The production method according to any one of claims 1 to 3, wherein the acyltransferase comprises palmitoyltransferase;

preferably, the glycosyltransferase comprises a cyclodextrin glycosyltransferase.

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

preferably, the addition amount of the acyltransferase 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 a buffer solution having a pH of 6 to 8 using an aqueous system.

7. The method according to any one of claims 1 to 6, wherein the temperature of the conversion reaction is 25 to 40 ℃; preferably 35-40 ℃;

preferably, the conversion reaction time is between 1 and 24h, preferably between 16 and 20 h.

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 E; optionally, after the aqueous solution is separated and purified, high-purity rebaudioside E is obtained;

wherein the initial concentration of stevioside is 1-300g/L, the mass ratio of ethyl acetate to stevioside is (0.4-10):1, the mass ratio of beta-cyclodextrin to stevioside is (3-10):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 E composition or rebaudioside E purified product obtained by the preparation method of any one of claims 1-8.

10. Use of the rebaudioside E composition or rebaudioside E 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 field of biocatalytic conversion, relates to a preparation method and application of rebaudioside E, and particularly relates to a preparation method and application of rebaudioside E taking ethyl acetate and beta-cyclodextrin as glycosyl donors.

Background

Stevia sugar, the sweetest sweetener known at present, is 250-450 times as sweet as sucrose, while the calorie is 1/300 of sucrose, and is slightly astringent. Besides, stevia sugar has been receiving more and more attention because it has an auxiliary pharmacological action on hypertension, obesity, diabetes and the like. Stevioside is a generic term for stevioside, and more than 35 stevioside compounds, including stevioside and rebaudioside A, B, C, D, E, M, etc., have been isolated and identified from stevia rebaudiana. The stevioside structure takes diterpene steviol as a framework around one, and unequal glucose groups are respectively connected with a hydroxyl at the C13 position and a carboxyl at the C19 position, wherein carbon-carbon double bonds between C16 and C17 on the framework are pharmacological groups for providing sweet taste and functions of stevioside.

The sweetness of different stevioside is different, the taste of rebaudioside E has no aftertaste, the sweetness is similar to that of cane sugar, a glucose group is added on the side chain of C19 position on the skeleton of the stevioside, the content of the rebaudioside E in the dry leaves of the stevia is very small, the rebaudioside E is directly separated from the stevioside by the conventional physical means, the difficulty is large, and the yield is extremely low. In addition, the process for enriching rebaudioside E is complex, column passing, desalting, decoloring and recrystallizing are needed for multiple times after extraction, a large amount of wastewater is generated in the production process, the production cost is high, and the process is not suitable for industrial mass production.

CN106795547A discloses a method for synthesizing rebaudioside E from rebaudioside KA or rubusoside, the substrate being selected from the group consisting of sucrose, Uridine Diphosphate (UDP) and uridine diphosphate glucose (UDP-glucose), the UDP-glycosyltransferase being selected from the group consisting of HV1, EUGT11 and UDP-glycosyltransferase fusion enzymes, and incubating the reaction mixture for a time sufficient to produce rebaudioside E. However, the method still relies on UDP-glucose as a glycosyl donor, and has high synthesis cost and poor economy.

CN109750072A discloses a method for preparing rebaudioside E by an enzymatic method, which utilizes tomato-derived UDP-glycosyltransferase and potato-derived sucrose synthase, takes stevioside as a raw material to carry out a one-step glycosylation reaction to produce rebaudioside E, and simultaneously adopts a site-directed mutagenesis technology to modify the UDP-glycosyltransferase to further improve the yield of the rebaudioside E. According to the method, UDP-glucose and UDP are not required to be added, UDP-glucose obtained by decomposing UDP in the crude extract and cane sugar additionally added under the action of sucrose synthase is used as a raw material for glycosylation, a double-enzyme circulation reaction system is established, and rebaudioside E is effectively produced by catalyzing stevioside.

The method for synthesizing rebaudioside E by using a biological enzyme method usually needs to add expensive UDP-glucose as a substrate, stevioside or rebaudioside A is used as the substrate to catalyze and generate rebaudioside E under the action of UDP-glucosyltransferase, and due to the extremely high selling price of UDP-glucose, the feasibility of industrially preparing rebaudioside E is almost completely limited, so that the method is poor in economy and lacks of market competitiveness. Or the process steps are complicated, the cost is high, and the market competitiveness is also lacked. Therefore, it would be of great interest to develop a cost effective and simple process for preparing rebaudioside E.

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 rebaudioside E under the catalysis of acyltransferase and glycosyltransferase.

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 E, which comprises taking stevioside as a raw material, taking ethyl acetate and beta-cyclodextrin as glycosyl donors, and carrying out conversion reaction under the catalysis of acyltransferase and glycosyltransferase to generate rebaudioside E.

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

Preferably, the stevioside has an initial reaction concentration of 1-300g/L, and may be, for example, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, 30g/L, 35g/L, 40g/L, 45g/L, 50g/L, 55g/L, 60g/L, 65g/L, 70g/L, 75g/L, 80g/L, 85g/L, 90g/L, 95g/L, 100g/L, 110g/L, 120g/L, 130g/L, 140g/L, 150g/L, 160g/L, 170g/L, 180g/L, 190g/L, 200g/L, 210g/L, 220g/L, 230g/L, 240g/L, 250g/L, 260g/L, 270g/L, 280g/L, 290g/L, or 300 g/L.

Preferably, the mass ratio of ethyl acetate to stevioside is (0.4-10):1, and may be, for example, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10: 1.

In the invention, by adjusting the dosage ratio of ethyl acetate to stevioside, the conversion rate of rebaudioside E 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 (3-10):1, and can be, for example, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1, 9:1, 9.5:1 or 10: 1.

In the invention, by adjusting the mass ratio of the beta-cyclodextrin to the stevioside, the fact that the conversion rate of the rebaudioside E 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 acyltransferase comprises a palmitoyl transferase.

Preferably, the glycosyltransferase comprises a cyclodextrin glycosyltransferase.

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

Preferably, the glycosyltransferase is added in an amount of 25000-50000U/L in the reaction system, such as 25000U/L, 30000U/L, 35000U/L, 40000U/L, 45000U/L or 50000U/L.

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

Preferably, the addition amount of the acyltransferase in the reaction system is 50000-100000U/L, such as 50000U/L, 60000U/L, 70000U/L, 80000U/L, 90000U/L or 100000U/L.

In the present invention, when the addition amount of acyltransferase is less than 50000U/L, the conversion rate of rebaudioside E is reduced, and when the addition amount is more than 100000U/L, a large amount of by-products are produced, and the conversion rate of rebaudioside E is also reduced.

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 deg.c.

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 preferable 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 catalytic enzyme, and carrying out biotransformation in a buffer solution water phase system with the pH value of 6-8 at the reaction temperature of 20-50 ℃ for 1-24h to obtain a main product, namely a rebaudioside-E water solution; optionally, after the aqueous solution is separated and purified, high-purity rebaudioside E is obtained;

wherein the initial concentration of stevioside is 1-300g/L, the mass ratio of ethyl acetate to stevioside is (0.4-10):1, the mass ratio of beta-cyclodextrin to stevioside is (3-10):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 main product of the aqueous solution of rebaudioside E is prepared by the method of the invention, and the rebaudioside E with high purity can be 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 rebaudioside E is mainly generated by reaction by regulating and controlling the types and the proportional relation of raw materials, glycosyl donors and complex enzyme, so that the yield of the rebaudioside E is obviously improved.

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

In a third aspect, the present invention provides the use of a rebaudioside E composition or rebaudioside E 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 E preparation method provided by the invention creatively adopts cheap and easily-obtained ethyl acetate and beta-cyclodextrin as a composite glycosyl donor to replace expensive UDP-glucose, so as to realize the preparation of rebaudioside E;

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

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

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