WO2020001516A1 - Stevioside derivative rebaudioside a1g, preparation, purification and application thereof - Google Patents

Abstract

Rebaudioside A1G, a preparation method therefor, a purification method therefor and a use thereof in food. The preparation method comprises: providing rebaudioside A and a glucose-based donor, and producing rebaudioside A1G by means of the catalysis of cyclodextrin glycosyltransferase and amylase.

Description

Stevioside derivative rebaudioside A1G, its preparation, purification and application Technical field

The present disclosure belongs to the fields of biotechnology and food chemical technology. Specifically, the present disclosure relates to a novel steviol glycoside derivative rebaudioside A1G, a preparation method, a purification method, and an application thereof.

Background technique

Stevia glycosides are a series of glycosides extracted from the leaves of the stevia plant Stevia rebaudiana (Bertoni) and are natural sweeteners. Stevia (Stevioside, abbreviated as Stv) and Rebaudioside A (abbreviated as RA, also known as stevioside A) are the major contents in the leaves of the natural plant stevia.

The molecular structure of rebaudioside A is shown below. It is a tetracyclic diterpenoid glycoside substance containing 20 carbon atoms. It is connected by a diterpene core at the C19 position with a glucosyl group and 3 at the C13 position. Glucose is formed:

[Corrected under Rule 91. 16.07.2019]

.

Studies have shown that rebaudioside A is 200-300 times sweeter than sucrose and has only 1/300 the calories of sucrose. It is stable to acids, alkalis and heat. It is not easy to deteriorate after long-term storage. There will be browning and it is not easy to cause dental caries. China has detailed its use as a food additive in the national standards of "GB8270-2014 National Food Safety Standard" and "GB2760-2014 National Food Safety Standard". In 2009, the US Food and Drug Administration FDA also recognized Rebaudioside A as a "GRAS (Generally Recognized as Safe)." As a natural substitute for sucrose, steviol glycosides can not only reduce costs, but also meet the requirements for food and beverages to gradually develop low sugar and low calorie. It has a broad application prospect and is a very ideal green sweetener with multiple uses.

Although stevioside has many advantages, its main component rebaudioside A has a severe bitter aftertaste, which has hindered its wide application in food and other fields. Currently, stevia glycosides can be improved by bioenzymatic conversion. It has been reported that glucosyltransferase can use UDP glucose as a glucosyl donor to add a glucosyl group to the main component of stevioside. However, the glycosyl donor UDP glucose used in this method is expensive and expensive (Biocatalysis: Industrial, Perspective, Royal, Society of Chemistry, 2017, pp199). It has also been reported that cyclodextrin glycosyltransferase (CGT) can be used to modify steviol glycosides, but the catalytic efficiency and transglycosylation site of CGTase from different biological sources are different, and the resulting enzyme catalytic products are complex and diverse (Simple and efficient enzymatic transglycosylation of stevioside by beta-cyclodextrin glucano transferase from Bacillus firmus. Biotechnol Lett. 2009 (Sep; 31 (9): 1415-1420). In addition, only the single-catalyzed product of CGT is a mixture of differently glycosylated derivatives, which results in uneven taste, and the molecular structure and polarity of these derivatives are very similar, which is difficult to separate and purify. It is difficult to obtain a single effective product of high purity. This also puts forward higher requirements for increasing the concentration of effective products and for innovation and optimization of production processes.

Therefore, there is an urgent need in the art to develop a rebaudioside A derivative product with improved taste, an efficient and high-purity production method thereof, and an application thereof.

Summary of the invention

Provided herein is a method for producing a novel stevia derivative by a double-enzyme catalysis method and a product thereof, which has greatly improved raw material taste. In addition, a purification method and a purified product of rebaudioside A1G are also provided herein, thereby further improving the quality of the product. Based on this, this article provides a low-cost and short production cycle to produce high-quality bioenzymatic rebaudioside A1G, a stevioside derivative, and its wide application.

In some aspects of the present disclosure, a compound rebaudioside A1G is provided that is represented by the following structure:

In some aspects of the present disclosure, a method for preparing rebaudioside A1G is provided, wherein the structural formula of the rebaudioside A1G is as shown above, and the method includes the steps:

(1) providing rebaudioside A and a glucose-based donor;

(2) Cytodextrin glycosyltransferase and amylase catalyze the production of rebaudioside A1G as described above.

In some embodiments, the rebaudioside A is one or more selected from the group consisting of rebaudioside A present in natural plants, extracted rebaudioside A, and synthetic rebaudioside A. Glycoside A.

In some embodiments, the glucosyl donor is one or more selected from the group consisting of: starch, such as soluble starch; dextrin; maltodextrin; α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin; maltose.

In some embodiments, the cyclodextrin glycosyltransferase is selected from the group consisting of α-cyclodextrin glycosyltransferase, β-cyclodextrin glycosyltransferase, and γ-cyclodextrin glycosyltransferase.

In some embodiments, the amylase is one or more selected from the group consisting of saccharifying enzyme, alpha-amylase, beta-amylase, gamma-amylase.

In some embodiments, the amount of the glycosyltransferase is 0.1 to 30 kNU / L, for example, 0.5 to 20 kNU / L, 1 to 15 kNU / L, or 5000 to 50,000 U / mL, such as 10,000 to 40,000 U / mL, 15000 to 35000U / mL. In some embodiments, the amount of the glycosyltransferase is 5 to 200 kNU / kg rebaudioside A, such as 10 to 150 kNU / kg.

In some embodiments, the amount of the amylase is 30-300 U / mL, for example, 50-250 U / mL, 80-220 U / mL. In some embodiments, the amount of the amylase is 300-3000 U / g rebaudioside A, such as 800-2200 U / g rebaudioside A.

In some embodiments, the one or more enzymes used are immobilized enzymes.

In some embodiments, the initial concentration of rebaudioside A is 5 to 200 g / L, such as 8 to 150 g / L, 10 g to 120 g / L.

In some embodiments, the initial concentration of the glucose-based donor is 10-800 g / L, such as 20-700 g / L, 30-600 g / L, 30-300 g / L.

In some embodiments, step (2) is performed in an aqueous phase system, for example, in water (such as pure water, distilled water, ultrapure water, pH 6).

In some embodiments, the reaction temperature of step (2) is 35-90 ° C, such as 40-90 ° C, 45-85 ° C, 50-70 ° C, 45-85 ° C.

In some embodiments, the reaction time of step (2) is 0.5 to 72 hours, such as 1 to 48 hours, 1.5 to 36 hours, and 5 to 20 hours.

In some embodiments, the method further comprises one or more steps selected from the group consisting of terminating the enzyme reaction after completion of the enzyme reaction, such as denaturing the enzyme by boiling (such as boiling at 100 ° C for 5 minutes); isolating glycosyl transfer The product of the enzyme's catalytic reaction is used for the reaction catalyzed by amylase; the reaction solution after the catalytic reaction of glycosyltransferase is directly used for the reaction catalyzed by amylase; rebaudioside A, which is consumed in production, Glucosyl donor, glycosyltransferase and / or amylase; separating, purifying, salting, optically resolving, identifying and / or packaging the rebaudioside A obtained in step (2); and / or, Unused rebaudioside A, glucosyl donor, glycosyltransferase and / or amylase are recycled to the next round of reactions.

In some aspects of the present disclosure, there is provided a method for purifying the compound rebaudioside A1G (RA1G) shown in the following structure,

The method includes:

(a) optionally, pre-purifying the RA1G-containing raw material;

(b) the first crystallization and solid-liquid separation of the raw material containing RA1G with or without pretreatment using a methanol aqueous solution as a solvent;

(c) taking the solid phase obtained from the previous crystallization, dissolving it with a methanol aqueous solution as a solvent, and performing a second crystallization under appropriate conditions to obtain a purified product;

(d) Optionally, the purified product obtained from the previous crystallization may be repeatedly crystallized and purified one or more times or further purified by other purification methods;

(e) Optionally, the remaining liquid phase in the crystallization purification step is recycled into the preparation process of the RA1G-containing raw material.

In some embodiments, the raw material of the RA1G is obtained using the preparation method herein.

In some embodiments, the RA1G-containing raw material is prepared by an enzymatic method, which includes: (1) providing rebaudioside A and a glucosyl donor; (2) using a cyclodextrin glycosyltransferase And amylase catalysis to produce rebaudioside A1G.

In some embodiments, the enzymatic method comprises one or more conditions selected from the group consisting of:

The rebaudioside A is one or more selected from the group consisting of rebaudioside A present in natural plants, extracted rebaudioside A, and synthetic rebaudioside A;

The glucose-based donor is one or more selected from the group consisting of starch, such as soluble starch; dextrin; maltodextrin; α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin; maltose;

The cyclodextrin glycosyltransferase is selected from the group consisting of α-cyclodextrin glycosyltransferase, β-cyclodextrin glycosyltransferase, and γ-cyclodextrin glycosyltransferase;

The amylase is one or more selected from the group consisting of saccharifying enzyme, α-amylase, β-amylase;

The amount of the cyclodextrin glycosyltransferase is 0.1 to 30 kNU / L, for example, 0.5 to 20 kNU / L, 1 to 15 kNU / L, or 5000 to 50000 U / mL, such as 10,000 to 40,000 U / mL, 15000 to 35000 U / mL; And / or the amount of the amylase is 30-300U / mL, for example, 50-250U / mL, 80-220U / mL; and / or the enzyme is an immobilized enzyme;

One or more enzymes used are immobilized enzymes;

The starting concentration of the rebaudioside A is 5 to 200 g / L, such as 8 to 150 g / L, 10 g to 120 g / L; the initial concentration of the glucose-based donor is 10 to 800 g / L, such as 20 ～ 700g / L, 30 ～ 600g / L, 30 ～ 300g / L;

Step (2) is performed in an aqueous phase system, for example, in water (such as pure water, distilled water, ultrapure water, pH 6);

The reaction temperature in step (2) is 35 to 90 ° C, such as 40 to 90 ° C, 45 to 85 ° C, 50 to 70 ° C, 45 to 85 ° C; and / or

The reaction time of step (2) is 0.5 to 72 hours, such as 1 to 48 hours, 1.5 to 36 hours, and 5 to 20 hours; and / or

One or more steps selected from the group consisting of terminating the enzyme reaction after completion of the enzyme reaction, such as denaturing the enzyme by boiling (such as boiling at 100 ° C for 5 minutes); isolating the reaction product of the glycosyltransferase catalysis reaction for use in amylase Catalyzed reaction; directly take the reaction solution after catalyzed reaction with glycosyltransferase for amylase-catalyzed reaction; supplement rebaudioside A, glucosyl donor, glycosyltransferase and / Or amylase; separating, purifying, salting, optically resolving, identifying, and / or packaging the rebaudioside A obtained in step (2); and / or, unused rebaudioside A, glucose The base donor, glycosyltransferase and / or amylase are recycled to the next round of reactions.

In some embodiments, the pre-purification pretreatment of step (a) comprises one or more treatments selected from the group consisting of filtration, adsorption and elution, concentration and drying. Other methods such as solvent precipitation, microporous membrane filtration, etc. can also be used to remove small molecular impurities (such as residual glucose).

In some embodiments, the pre-processing is performed by one or more of the following processes:

(a1) optionally, filtering the raw material containing rebaudioside A1G, for example, using a filter plate, filter paper, and filter element for filtering, preferably using a fine filter plate, and more preferably a fine filter plate having a pore size of 5 to 10 μm;

(a2) Optionally, a macroporous resin is used to adsorb and elute the raw material containing rebaudioside A1G, for example:

Use styrene or acrylate macroporous adsorption resin for adsorption, for example, macroporous adsorption resin with pore diameter of 6-15nm and specific surface area of 600-1300㎡ / g; the following adsorption conditions can be used: injection solution concentration 0.5- 20%, pH 4-8, injection flow rate 0.5-5BV / h;

Wash the macroporous resin with adsorbed raw materials with water (such as 2-10 times the resin volume of water), for example, with purified water, with a flow rate of 0.5-5 BV / h;

Ethanol eluent is used to elute the substance adsorbed on the resin. For example, 30-90% (v / v) (preferably ≥60%) ethanol eluent can be used. The volume is ≥1.5 times, such as 1.5-4. Double the bed volume, the elution solvent is ethanol aqueous solution, no need to adjust pH (about 6), elution flow rate is 0.5-2BV / h;

(a3) the raw materials, filtered, and / or adsorbed and eluted products can be optionally concentrated and / or dried, such as spray drying, vacuum drying, for example, at -0.06 to 0.09 MPa, 60 to 85 ° C The ethanol eluate is concentrated under conditions, and spray-dried at, for example, an air temperature of 65 to 90 ° C.

In some embodiments, the first crystallization of step (b) includes one or more processes selected from the group consisting of:

(b1) dissolving the raw material in solid form with the solvent;

(b2) performing the first crystallization;

(b3) solid-liquid separation of the first crystallization product to obtain a solid phase for further purification or use as a purified product.

In some embodiments, the first crystallization includes one or more conditions selected from the group consisting of:

Methanol aqueous solution is used as a solvent to dissolve the solid raw materials. For example, a concentration of 80 to 99% (v / v) is used, such as a concentration ≥90%, such as a 95% methanol aqueous solution is used as the solvent; The ratio is 1: 2 ~ 5, if the volume of the solvent is 3 ~ 5 times, for example 3 times the weight of the dry product;

The crystallization temperature is 15-30 ° C, such as room temperature, such as 20-25 ° C, such as 25 ° C;

The crystallization time is 10 to 40 hours, such as 15 to 30 hours, such as 20 to 24 hours;

The stirring speed during the crystallization process is 10 to 60 rpm, such as 20 to 50 rpm, such as 30 to 45 rpm;

Solid-liquid separation of the first crystallization product by means of filtration (such as suction filtration) and / or centrifugation;

Optionally, the crystals are washed, the detergent is a 60 to 90% (v / v) methanol aqueous solution, the volume ratio of the wet weight of the crystals to the detergent is preferably 1: 0.5 to 2, and the crystal washing time is 10 to 30 minutes;

Optionally, the resulting solid phase is dried.

In some embodiments, the second crystallization of step (c) includes one or more processes selected from the group consisting of:

(c1) dissolving the solid phase obtained by the first crystallization with the solvent;

(c2) performing a second crystallization;

(c3) Solid-liquid separation of the second crystallization product to obtain a solid phase for further purification or use as a purified product.

In some embodiments, the second crystallization includes one or more conditions selected from the group consisting of:

The methanol phase aqueous solution is used as a solvent to dissolve the solid phase of the first crystallization purification product. For example, a concentration of 50 to 90% (v / v), such as a concentration of 60 to 80%, such as a 65% methanol aqueous solution is used as the solvent; The mass-volume ratio to the solid phase may be 1: 1.5 to 5, such as the solvent volume is 1.5 to 3 times the weight of the dry product, such as 2 to 2.5 times; optionally, the concentration of the methanol aqueous solution used in step (c) is low The concentration of the methanol aqueous solution used in step (b);

The second crystallization temperature is 20-35 ° C, such as room temperature, such as 20-25 ° C, such as 20 ° C;

The second crystallization time is 10 to 40 hours, such as 15 to 30 hours, such as 20 to 24 hours;

The stirring speed during the crystallization process is 10 to 60 rpm, such as 20 to 50 rpm, such as 10 to 30 rpm;

Solid-liquid separation of the second crystallization product by means of filtration (such as suction filtration) and / or centrifugation;

Optionally, the obtained solid phase is dried, for example, spray-dried at an air temperature of 65-90 ° C.

In some embodiments, the further purification mode of step (d) is selected from one or more of the following group: crystallization purification, preparative HPLC purification.

In some embodiments, recycling of step (e) includes one or more treatments selected from the group consisting of:

Recycling the liquid phase of a certain crystallization (such as the first crystallization or the second crystallization), the mixture of liquid phases obtained from multiple crystallizations (such as the mixture of the first and second crystallization liquid phases), and the liquid obtained from multiple batches of crystallization Mixtures of phases (e.g. mixtures of liquid phases obtained from multiple first and / or second crystallization steps);

The liquid phase circulation obtained in the purification process is used in the dual-enzyme process herein;

Before recycling, the liquid phase can be mixed, concentrated, and dried.

The RA1G-containing raw material produced after recycling is used in the purification method herein.

In another aspect of the present disclosure, there is provided a composition comprising: (i) rebaudioside A1G of the present disclosure, and / or, rebaudioside obtained by the preparation and / or purification method of the present disclosure Glycoside A1G; and (ii) pharmaceutically, food science, health science or daily chemically acceptable carriers, excipients and / or excipients; (iii) optionally, other sweeteners Or flavoring agents, such as mogroside, acesulfame, aspartame, sucralose, sodium saccharin, xylitol, sorbitol, erythritol, sucrose, fructose, glucose, maltose, citric acid, apple Acid, tartaric acid, lactic acid, glycine, alanine, serine.

In another aspect of the present disclosure, there is provided a rebaudioside A1G of the present disclosure, or a rebaudioside A1G obtained by the preparation and / or purification method of the present disclosure, or an application of a composition of the present disclosure, which Used as a sweetener, flavoring and / or taste-masking agent, such as in the preparation of food, beverages, tobacco products, condiments, household chemicals, pharmaceutical ingredients, nutritional health products, oral hygiene products and / or cosmetics .

In another aspect of the present disclosure, there is provided a product comprising rebaudioside A1G of the present disclosure, and / or, rebaudioside A1G obtained by the method of the present disclosure, and / or, a combination of the present disclosure Thing.

In some embodiments, the product is selected from the group consisting of food, beverages, tobacco products, condiments, household chemicals, pharmaceutical ingredients, nutritional health products, oral hygiene products, and / or cosmetics.

In another aspect of the present disclosure, there is provided a packaged product comprising: Rebaudioside A1G of the present disclosure, and / or, rebaudioside A1G obtained by the preparation and / or purification method of the present disclosure; As well as packaging and / or containers.

In some embodiments, the package and / or container may be selected from: flexible packages or containers, such as bags (such as paper bags, plastic bags, preferably sealed bags) and bottles (such as plastic bottles); rigid packages or containers , Such as glass containers, metal containers, ceramic containers, etc.

Those skilled in the art can make any combination of the above technical solutions and technical features without departing from the inventive concept and protection scope of the present disclosure. Other aspects of the disclosure will be apparent to those skilled in the art from the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure is further described below with reference to the accompanying drawings, wherein these displays are only for illustrating the embodiments of the disclosure, and are not intended to limit the scope of the disclosure.

Figures 1A and 1B: High-performance liquid chromatograms of rebaudioside A1G product (Figure 1A) and purified rebaudioside A1G catalyzed by a dual-enzyme method (Figure 1B).

Figure 2: A schematic diagram of an exemplary process for the dual-enzyme catalysis of rebaudioside A1G (for example, soluble starch can be used as a glucose-based donor).

Figure 3: High-resolution mass spectrum of Rebaudioside A1G.

Figure 4: Nuclear magnetic resonance proton spectrum of acetylated rebaudioside A1G.

Figure 5: NMR carbon spectrum of acetylated rebaudioside A1G.

Fig. 6: Two-dimensional COSY spectrum of acetylated rebaudioside A1G.

Figure 7: Two-dimensional HSQC spectrum of acetylated rebaudioside A1G.

Figure 8: Two-dimensional NMR NMR spectrum of acetylated rebaudioside A1G.

9A. High performance liquid chromatogram of the first reaction product modified by the double enzyme method;

Figure 9B. High performance liquid chromatogram of the second-step reaction product modified by the double-enzyme method.

Figure 10. High performance liquid chromatogram of the solid phase composition after the first crystallization.

Figure 11. High performance liquid chromatogram of liquid components after the first crystallization.

Figure 12. High performance liquid chromatogram of the solid phase components after the second crystallization.

Figure 13. High performance liquid chromatogram of the liquid phase components after the second crystallization.

Figure 14. High performance liquid chromatogram of the solid phase composition after the third crystallization.

Figure 15. High performance liquid chromatogram of the liquid phase components after the third crystallization.

Figure 16. High performance liquid chromatogram of the product obtained by recycling the crystalline liquid phase into the enzyme modification process.

detailed description

The present disclosure provides a new rebaudioside A derivative which is connected to a glucose group via an α-1,4 bond on the C19-linked glucose of the rebaudioside A diterpene core. Named Rebaudioside A1G (ie RA1G). The present disclosure also provides a method for preparing the derivative. The method uses Rebaudioside A as a raw material and is carried out by two-step enzyme catalysis. Compared with the raw material rebaudioside A, the rebaudioside A1G of the present disclosure has improved sweetness and improved mouthfeel. In addition, the rebaudioside A1G of the present disclosure also has the characteristics of low production cost, short production cycle, and environmental protection in the production process.

As used herein, the term "dual-enzyme method" refers to a method using Rebaudioside A and a glucosyl donor as raw materials to perform a catalytic reaction using two enzymes to obtain Rebaudioside A1G. Specific descriptions of the reaction raw materials, enzymes, reaction conditions, reaction products, etc. used in the dual enzyme method can be found below.

The present disclosure also relates to a purification method of a novel rebaudioside A derivative, rebaudioside A1G (ie, RA1G).

It should be understood that the purification method of the present disclosure may be applicable not only to a raw material containing rebaudioside R1G obtained by an enzyme-catalyzed preparation method as described herein, but also to a raw material containing rebaudioside R1G obtained by other methods . Similarly, the remaining liquid phase in the crystallization method of the present disclosure can also be recycled into the production of rebaudioside R1G, and is not limited to a specific preparation method.

The purification method of the present disclosure mainly includes: (a) optionally, pre-purifying pre-purification of RA1G-containing raw materials; (b) using an appropriate solvent and under appropriate conditions, the RA1G-containing The raw material is subjected to the first crystallization and solid-liquid separation; (c) taking the solid phase obtained from the previous crystallization, dissolving with an appropriate solvent, and under appropriate conditions, performing a second crystallization to obtain a purified product; (d) optionally , Repeating the crystallization and purification one or more times to obtain the purified product from the previous crystallization; (e) optionally, recycling the remaining liquid phase in the crystallization purification step to the RA1G preparation process.

For example, some embodiments of the present disclosure provide a method for increasing the content of rebaudioside A1G in a modified product of rebaudioside A enzyme. The steps are as follows: The product is filtered through a fine plate and frame, the macroporous resin is separated, the single-effect evaporator is concentrated, the concentrated solution is spray-dried, and the solvent is added for crystallization; the above crystallization solution is subjected to solid-liquid separation, and the solid phase is dissolved again in the solvent for secondary crystallization The crystallization solution was subjected to solid-liquid separation again, dissolved in solid phase and purified water, and spray-dried to obtain a purified enzyme-modified product. The liquid phase of the two crystallizations is mixed and used as the raw material for the enzyme-catalyzed modification of rebaudioside A to perform the enzyme modification reaction. The product in this step can be recycled into filtration → separation → concentration → spray drying → crystallization → secondary crystallization Process. The secondary crystallization cycle production process can greatly increase the content of rebaudioside A1G, a new product with a better taste among the enzyme-catalyzed modified products of rebaudioside A, and the content of rebaudioside A1G in the final product. More than 70%; through the recycling process, the utilization rate of production raw materials can be further improved, and production costs and waste emissions can be greatly reduced.

As used herein, the terms "rebaudioside A" and "RA" are used interchangeably and each refers to a compound represented by the following structural formula.

As used herein, the terms "rebaudioside A1G" and "RA1G" are used interchangeably, and both mean that the glucose linked to the C19 position of the diterpene core of rebaudioside A is connected again through an α-1,4 bond A glucosyl rebaudioside A derivative (the structure of which is shown below).

As used herein, the terms "rebaudioside A2G" and "RA2G" are used interchangeably, and both refer to a rebaudioside A derivative in which two glucose are linked to rebaudioside A.

As used herein, the terms "rebaudioside A3G" and "RA3G" are used interchangeably, and both refer to a rebaudioside A derivative in which 3 glucose is linked to rebaudioside A.

As used herein, the terms "rebaudioside D" and "RD" are used interchangeably, and both refer to a glucose linked to the C19 position of the rebaudioside A diterpene core by a β-1,2 bond Rebaudioside A derivative.

[Corrected under Rule 91. 16.07.2019]

As used herein, "containing," "having," or "including" includes "including," "consisting essentially of," "consisting essentially of," and "consisting of;" "Consisting", "consisting essentially of" and "consisting of" belong to the subordinate concepts of "containing", "having" or "including".

All numerical ranges provided herein are intended to clearly include all numerical values that fall between the endpoints of the range and the numerical ranges between them. The features mentioned in the present disclosure or the features mentioned in the embodiments may be combined. All features disclosed in this specification can be used in combination with any composition form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal, or similar purpose. Unless otherwise stated, the disclosed features are only general examples of equal or similar features.

Reaction substrate

The main reaction raw materials that can be used in the present disclosure to produce rebaudioside A1G in a dual-enzyme method are rebaudioside A and a glucose-based donor.

The rebaudioside A raw material used for the double-enzyme production of rebaudioside A1G can be rebaudioside A from various sources. Rebaudioside A raw materials that can be used include, but are not limited to, rebaudioside A extracted from natural plants and used directly in the method of the present disclosure, for example, using stevia leaves as raw materials, through extraction, impurity removal, decolorization, Obtained by processes such as drying; commercially available rebaudioside A; synthetic rebaudioside A, for example, synthesized by microbial fermentation (such as recombinant Pichia, recombinant Saccharomyces cerevisiae, recombinant E. coli). Rebaudioside A in the state of powder, crystal, solution and the like can be used in the reaction system of the present disclosure.

The glucosyl donor used in the dual-enzyme production of rebaudioside A1G can be a substrate capable of being used as a substrate for a glycosyltransferase (such as cyclodextrin glycosyltransferase, CGT) to transfer the sugar molecules contained therein through an enzymatic reaction. Any polysaccharides and / or oligosaccharides onto the receptor. Glucose-based donors useful in the present disclosure include, but are not limited to: starch (preferably soluble starch), β-cyclodextrin, α-cyclodextrin or γ-cyclodextrin, maltodextrin, maltose.

Enzyme

There are two types of enzymes used in the present disclosure for producing rebaudioside A1G, which are enzymes that catalyze transglycosyl and catalytic hydrolysis reactions, preferably glycosyltransferases (such as cyclodextrin glycosyltransferase) and amylase.

Glycosyltransferases are a class of enzymes that catalyze the activation of sugars to different receptor molecules, such as rebaudioside A of the present disclosure. Cyclodextrin glycosyltransferase (CGTase) is a multifunctional enzyme capable of catalyzing different reactions, which can transfer a glycosyl on a glucose-based donor to an acceptor (rebaudioside A in the present disclosure) on. For the characteristics, preparation and application of cyclodextrin glycosyltransferase, please refer to Wu Jing et al., "Preparation and Application of Cyclodextrin Glucosyltransferase" (2011, Chemical Industry Press). Cyclodextrin glycosyltransferase (CGT) can be an enzyme from various sources, such as a commercially available CGT enzyme, a genetically engineered CGT enzyme, and the like.

Amylase is an enzyme that hydrolyzes starch and glycogen. Amylases useful in the disclosed methods include, but are not limited to, saccharifying enzymes, alpha-amylases, beta-amylases. Glycosylase, also known as Glucoamylase (EC 3.2.1.3), can hydrolyze starch from non-reducing ends to α-1,4 and α-1,6 glucoside bonds to produce glucose. Hydrolyzed dextrin and glycogen release the β-D-glucose at the non-reducing end.

These two enzymes used in the present disclosure are commercially available, for example, purchased from Japan Amano Enzyme Co., Ltd., Novozymes (China) Biotechnology Co., Ltd., Jiangxi Baiying Biotechnology Co., Ltd., etc .; also by microbial fermentation Obtained as long as it has the required catalytic activity. An enzyme preparation with high enzyme activity and high stability is preferably used, and an enzyme in an immobilized form may also be used.

Enzymatic reaction

The enzymatic reaction of the present disclosure is performed in an aqueous phase system. Rebaudioside A is used as the acceptor substrate, and soluble starch, β-cyclodextrin or α-cyclodextrin, and maltose are used as the glucose-based donor substrate. Materials, under the catalysis of cyclodextrin glycosyltransferase (CGT), a transglycosyl reaction is performed to generate a mixed system of rebaudioside A derivatives, and the hydrolysis of each component in the mixed system is catalyzed by amylase (such as saccharifying enzyme) By reaction, a new type of rebaudioside A derivative with higher uniformity, that is, rebaudioside A1G is finally obtained.

The two raw materials of the present disclosure can be dissolved in water (such as pure water, distilled water, ultrapure water, etc.) to form an aqueous phase system. The initial concentration of the rebaudioside A raw material in the reaction system may be 5 to 200 g / L, such as 8 to 150 g / L, 10 g to 120 g / L. The initial concentration of the raw material of the glucose-based donor substrate in the reaction system may be 10 to 800 g / L, for example, 20 to 700 g / L, 30 to 600 g / L, and 30 to 300 g / L. Generally, the ratio of the rebaudioside A raw material to the glucose-based donor raw material may be 1: 2 to 4 by weight.

A cyclodextrin glycosyltransferase (CGT) was added to the aqueous system to catalyze the transglycosylation reaction to produce a series of transglycosylated rebaudioside A derivatives. The final concentration of the CGTase in the reaction system may be 0.1 to 30 kNU / L, such as 0.5 to 20 kNU / L, 1 to 15 kNU / L, or 5000 to 50,000 U / mL, such as 10,000 to 40,000 U / mL, 15000 to 35000 U / mL. The content of the CGTase in the reaction system may be 5 to 200 kNU / kg rebaudioside A, for example, 10 to 150 kNU / kg rebaudioside A. Depending on the production of the reaction product, the reaction temperature of the CGTase can be set within the range of 35 to 90 ° C, such as 40 to 90 ° C, 45 to 85 ° C, and 50 to 70 ° C. Depending on the specific enzyme used and industrial cost, etc. Adjust this. The pH of the CGT enzyme reaction system can be set to about the optimum pH of the enzyme, for example, pH 4 to 7, pH 4.5 to 6.5, and pH 5 to 6, which can be adjusted according to the specific enzyme used. The CGT enzyme reaction time can be adjusted according to the reaction progress, for example, the reaction is 0.5 to 72 hours, 1 to 48 hours, 1.5 to 36 hours, and 5 to 20 hours.

After the GCT enzyme reaction is completed, the enzyme reaction can be terminated in various ways (for example, a simpler way is to denature the enzyme by boiling (such as boiling at 100 ° C for 5 minutes) to terminate the reaction). Optionally, the resulting reaction product is centrifuged and the supernatant is separated for use in the next reaction. The obtained reaction product can also be directly used in the next reaction without isolation and purification.

Amylase (such as saccharifying enzyme) is added to the GCT enzyme reaction product to catalytically decompose the components in the rebaudioside A derivative mixed system into glucose at the C19 position of the core of the rebaudioside A diterpene through α-1, The 4-bond is linked to a glucosyl rebaudioside A1G.

In some embodiments, the amount of the amylase is 30-300 U / mL, for example, 50-250 U / mL, 80-220 U / mL. In some embodiments, the amount of the amylase is 300-3000 U / g rebaudioside A, such as 800-2200 U / g rebaudioside A.

Depending on the production of the reaction product, the reaction temperature of the amylase can be set within the range of 35-90 ° C, such as 40-90 ° C, 45-85 ° C, 50-70 ° C, according to the specific enzyme used and industrial cost, etc. Adjust this. The pH of the amylase reaction system can be set to about the optimum pH of the enzyme, for example, pH 4 to 7, pH 4.5 to 7, and pH 5 to 7, which can be adjusted according to the specific enzyme used. The amylase reaction time can be adjusted according to the reaction progress, for example, the reaction is 0.5 to 72 hours, 1 to 48 hours, 1.5 to 36 hours, and 2 to 10 hours.

After the amylase reaction is completed, the enzyme reaction can be terminated in various ways (for example, a simpler way is to denature the enzyme by boiling (such as boiling at 100 ° C for 5 minutes) to terminate the reaction). The obtained reaction product can be further separated, dried, purified, identified and other steps to obtain the desired rebaudioside A1G.

For example, the reaction supernatant and the precipitate can be separated by centrifugation, such as 12000 rpm, centrifugation for 5 minutes, and the like. For example, the reaction products can be separated by chromatography, such as by HPLC. An optional HPLC instrument and conditions are: Agilent 1200HPLC system Phenomenex Luna 5μm C18 (2) 4.6mm × 250mm chromatographic column, and the mobile phase is acetonitrile-sodium dihydrogen phosphate aqueous solution (pH 2.6). According to the present disclosure, the single sample loading amount for the above further separation is 5 μL, the flow rate is 1.0 mL / min, the mobile phase is an acetonitrile: sodium dihydrogen phosphate aqueous solution (pH 2.6) volume ratio 68:32, and the ultraviolet detection wavelength is 210 nm. For example, the obtained product can be dried by a lyophilization method. For example, the product obtained can be further purified by crystallization.

Those skilled in the art should also understand that the above reaction can be performed using an immobilized enzyme system, and an immobilized CGT enzyme and / or an immobilized amylase can be used.

The product obtained by the dual enzyme method of the present disclosure can be characterized by high-resolution mass spectrometry, nuclear magnetic resonance, and other methods to determine that the obtained product is glucose at the C19 position of the rebaudioside A diterpene core through α-1,4 The glucosyl group is connected to the bond, and the rebaudioside A1G is obtained.

In addition, the unused rebaudioside A, glucosyl donor, glycosyltransferase and / or amylase can be recycled to the next round of the reaction to save costs and increase yield.

Exemplary preparation method

An exemplary method of the present disclosure is described below, and it should be understood that the exemplary method is only used to illustrate the present disclosure and not to limit the scope of the present disclosure. Those skilled in the art can make appropriate modifications and changes to the present disclosure, and these modifications and changes are all within the scope of the present disclosure.

An exemplary method of the present disclosure provides a method for preparing a stevia derivative rebaudioside A1G from rebaudioside A by a bioenzymatic method, including the following steps:

(1) In an aqueous system, rebaudioside A is used as the acceptor substrate, and the initial reaction concentration is 10 g to 120 g / L. Soluble starch, β-cyclodextrin or α-cyclodextrin, and maltose are used as Glucose donor substrate, with an initial reaction concentration of 30-300 g / L, and a transglycosyl reaction under the catalysis of α-cyclodextrin glycosyltransferase (CGT) to produce a series of rebaudioside A derivatives;

(2) The reaction system prepared in step (1) is reacted in a water bath at 45-85 ° C for 1-48 hours, the reaction is stopped by boiling, centrifuged, and the supernatant is taken;

(3) adding the saccharifying enzyme to the supernatant obtained in step (2), and using all the components in the mixed system in step (2) as a substrate to perform a hydrolysis reaction;

(4) The reaction system prepared in step (3) is reacted in a water bath at 45-85 ° C for 1-48 hours, the reaction is stopped by boiling, centrifuged, and the supernatant is taken;

(5) The supernatant liquid obtained in step (4) is separated and dried to obtain a rebaudioside A1G, a derivative of rebaudioside A.

In some examples, the aqueous system in step (1) is distilled pure water, pH 6.0.

In some examples, the α-cyclodextrin glycosyltransferase in step (1) can be purchased from Jiangxi Baiying Biotechnology Co., Ltd., Novozymes (China) Biotechnology Co., Ltd. and Japan Amano Enzyme Co., Ltd., The final concentration is 0.05 ～ 2g / L.

In some examples, the glucose donor substrate in step (1) is soluble starch, dextrin, and maltose

In some examples, the reaction condition in the step (2) is a 60 ° C water bath, and the reaction time is 15 hours.

In some examples, the boiling termination reaction condition in step (2) is boiling at 100 ° C. for 5 minutes.

In some examples, the centrifugation speed in the step (2) is 12000 rpm, and the time is 5 minutes.

In some examples, the saccharifying enzyme in step (3) may be purchased from Shaanxi Senfu Natural Products Co., Ltd. and Shanghai Yuanye Biotechnology Co., Ltd. with a final concentration of 0.5-20 g / L.

In some examples, the reaction condition in the step (4) is a 60 ° C water bath, and the reaction time is 3 hours.

In some examples, the boiling termination reaction condition in step (4) is boiling at 100 ° C. for 5 minutes.

In some examples, the centrifugation speed in step (4) is 12000 rpm and the time is 5 minutes.

In some examples, the separation in step (5) uses an Agilent 1200 HPLC system Phenomenex Luna 5 μm C18 (2) 4.6 mm × 250 mm chromatographic column, and the mobile phase is an acetonitrile-sodium dihydrogen phosphate aqueous solution (pH 2.6).

According to the present disclosure, in some examples, the single loading of the above-mentioned further separation is 5 μL, the flow rate is 1.0 mL / min, and the mobile phase is acetonitrile: sodium dihydrogen phosphate aqueous solution (pH 2.6) volume ratio 68:32, UV The detection wavelength was 210 nm.

In some examples, the drying in step (5) is freeze-drying.

Crystallization and purification method of modified product of rebaudioside A

The rebaudioside A1G in the rebaudioside A modified product can be purified by a crystallization method (such as primary crystallization, secondary crystallization, triple crystallization, quaternary crystallization, etc., preferably secondary crystallization).

A. Pretreatment before purification

Prior to crystallization purification, the RA1G-containing raw materials can be pre-treated optionally through filtration, adsorption and elution, drying, concentration and other steps to reduce, for example, the interference of the pollutants on the crystallization purification and / or the relative enrichment of the crystalline materials In RA1G.

In some embodiments, the feedstock comprising Rebaudioside A1G is filtered. For example, the mixture is filtered through a filter (such as a filter plate, filter paper, filter element, etc.). In some examples, filtration is performed using a filter plate, such as a fine filter plate, and a fine filter plate with a pore size of 5-10 μm is preferred.

In some embodiments, a macroporous resin is used to adsorb and elute the rebaudioside A1G-containing material. This step can separate some small molecular impurities in the raw material, such as free glucose. The macroporous resin used may be a styrenic or acrylate macroporous adsorption resin. For example, a large-pore adsorption resin with a pore diameter of 6-15nm and a specific surface area of 600-1300㎡ / g can be used. The concentration of the injection solution can be 0.5-20%, and the pH is 4-8. The injection flow rate can be 0.5-5BV / h.

The macroporous resin to which the raw material is adsorbed can be washed with a large amount of water (preferably purified water) to ensure that small molecules in the raw material are washed away or basically washed away. For example, a volume ≥ 2 times, such as 2 to 10 times the bed volume (Ie, resin volume). The washing flow rate can be 0.5-5BV / h. Then, the substance adsorbed on the resin can be eluted with an ethanol eluent. For example, 30 to 90% (v / v) (preferably ≥60%) of an ethanol eluent (pH about 6, the elution flow rate can be 0.5-2 BV / h), the volume is ≥1.5 times, such as 1.5 to 4 times the bed volume.

In some embodiments, the starting materials or pretreated products that have been filtered or pretreated steps such as adsorption and elution can optionally be concentrated and / or dried. For example, the conditions of -0.06 to 0.09 MPa and 60 to 85 ° C can be used to concentrate the ethanol eluent as described above. For example, the product may be dried by spray drying, vacuum drying, or the like.

In some embodiments, the feedstock is dried to obtain a solid phase feedstock.

It should be understood that the pre-processing steps may be added, subtracted, improved or modified without departing from the scope of the present invention. For example, a solvent precipitation method, a microporous membrane filtration method, or the like can be used to remove small molecule impurities (such as residual glucose).

B. First crystallization purification

The first crystallization purification can be performed on the raw materials with or without pretreatment. Generally, the raw materials in solid form can be dissolved by using an aqueous methanol solution as a solvent. For example, a methanol aqueous solution having a concentration of 80 to 99% (v / v) (such as a concentration ≥90%, such as 95%) can be used as a solvent. The mass-volume ratio of the solvent to the solid phase (that is, the dry product) may be 1: 2 to 5, for example, the solvent volume is 3 to 5 times, for example, 3 times the weight of the dry product.

The raw material methanol solution is crystallized at a crystallization temperature of 15 to 30 ° C, for example, room temperature, for example, 20 to 25 ° C, for example, 25 ° C. The crystallization time is usually 10 to 40 hours, such as 15 to 30 hours, such as 20 to 24 hours. Stirring can be performed during the crystallization process, and the rotation speed is usually 10 to 60 rpm, such as 20 to 50 rpm, such as 30 to 45 rpm.

For example, in some embodiments, the following first crystallization conditions are used: the solvent is 3 times the volume of a 95% (V / V) methanol aqueous solution, the crystallization temperature is 25 ° C., the time is 20 hours, and the stirring speed is 30 rpm.

The first crystallization product can be subjected to solid-liquid separation, for example, by filtration (such as suction filtration) and / or centrifugation. Optionally, the resulting solid phase is dried. The solid phase obtained can be used for further crystallization and purification; the liquid phase can be recycled to the production of raw materials.

C. Second crystallization purification

After obtaining the solid phase of the first crystalline purified product, it can be dissolved and purified by the second crystallization.

An aqueous methanol solution can be used as a solvent to dissolve the solid product of the first crystallization purification. For example, a methanol aqueous solution having a concentration of 50 to 90% (v / v) (eg, a concentration of 60 to 80%, such as 65%) can be used as a solvent. In some embodiments, the concentration of the second crystallization solvent is lower than the concentration of the first crystallization solvent.

The mass-volume ratio of the solvent to the solid phase (that is, the dry product) may be 1: 1.5 to 5, for example, the solvent volume is 1.5 to 3 times the weight of the dry product, such as 2 to 2.5 times. The raw material methanol solution is crystallized at a crystallization temperature of 20 to 35 ° C, such as room temperature, such as 20 to 25 ° C, such as 20 ° C. The crystallization time is usually 10 to 40 hours, such as 15 to 30 hours, such as 20 to 24 hours. Stirring can be performed during the crystallization process, and the rotation speed is usually 10 to 60 rpm, such as 20 to 50 rpm, such as 10 to 30 rpm.

For example, in some embodiments, the following secondary crystallization conditions are used: the solvent is a 65% (V / V) methanol aqueous solution, the crystallization temperature is 20 ° C., the time is 15 hours, and the stirring speed is 15 rpm.

The second crystallization product may be subjected to solid-liquid separation, for example, by filtration (such as suction filtration) and / or centrifugation. Among them, the obtained solid phase can be used for further crystallization purification or post-treatment as the final product of crystallization purification; the liquid phase can be recycled to the production of raw materials.

For example, optionally, the crystallization may be performed after the solid phase of the second crystallization product is isolated. For example, a 60 to 90% (v / v) methanol aqueous solution can be used as a detergent for crystal washing, and the volume ratio of the wet weight of the crystal to the detergent is 1: 0.5 to 2 for washing. The crystal washing time can be 10 to 30 min. .

For example, optionally, after the solid phase of the second crystallization product is separated, it can be dissolved in water (preferably pure water) so that the volume ratio of the wet weight of the crystal to water is 1: 0.5-1, and then Spray-dried at 65-90 ° C to obtain a purified product with RA1G content of more than 70%.

D. Further purification

After the second crystallization purification, the second crystallization purification method can be basically used to further purify the second crystallization purification product. For example, the third, fourth, or more crystallization purification steps may be performed as needed.

The second crystallization purification product of the present invention may be further purified by other purification methods according to needs, for example, by a method such as preparative HPLC.

E. Recycling of purified product liquid phase

The liquid phase recycle obtained in the purification process of the present invention can be used in the production of rebaudioside A derivatives.

For example, a single crystallization liquid phase (such as the first crystallization or a second crystallization), a mixture of liquid phases obtained from multiple crystallizations (such as a mixture of the first and second crystallization liquid phases), A mixture of liquid phases obtained from batch crystallization (e.g., a mixture of liquid phases obtained from multiple batches of the first and / or second crystallization steps).

For example, the liquid phase recycling obtained in the purification process of the present invention can be used in the production of a rebaudioside A derivative by a dual enzyme method.

Before recycling, the liquid phase can be mixed, concentrated, and dried. For example, two crystallized liquid phases can be mixed. For example, the liquid phase can be concentrated under the conditions of -0.06 to 0.09 MPa and 60 to 85 ° C, preferably to a solid content of 30 to 60%. For example, the liquid phase can be dried after being mixed and / or concentrated, such as by spray drying, etc., and the dried material can be recycled to participate in the catalytic reaction as an enzyme-modified raw material.

The RA1G-containing product obtained by using the circulating liquid phase may be subjected to crystallization purification again to obtain high-purity RA1G.

Application of Rebaudioside A1G and related products

The rebaudioside A1G of the present disclosure has many advantages such as high sweetness, good taste, green health, etc., and thus can be widely used in foods, beverages, drugs, health products, tobacco products, seasonings, daily chemical products, oral cavity Hygiene products, cosmetics and other fields.

Rebaudioside A1G can be provided in various forms as required, such as dry powder, crystals, solutions, compositions, and the like. For example, the rebaudioside A1G of the present disclosure can be made into a package that is convenient for storage, transportation, and use. For example, the rebaudioside A1G of the present disclosure may be combined with an acceptable excipient or excipient to form a composition of the present disclosure. The composition of the present disclosure comprises an effective amount of rebaudioside A1G, and may optionally include acceptable excipients or excipients such as water, food additives, food excipients, pharmaceutical excipients, and the like. In one embodiment, the food additive may be selected from, but not limited to, flavors, fragrances, emulsifiers, antioxidants, and food coloring.

Rebaudioside A1G of the present disclosure can be compounded with other sweeteners or flavoring agents to further improve its taste or to meet the desired taste requirements, such as other sweeteners or flavoring agents for compounding, including but not Limited to: Mogroside, Acesulfame, Aspartame, Sucralose, Sodium Saccharin, Xylitol, Sorbitol, Erythritol, Sucrose, Fructose, Glucose, Maltose, Citric Acid, Malic Acid, Tartaric Acid, Lactic acid, glycine, alanine, serine.

As used herein, the term "acceptable" ingredient is a substance that is suitable for use in humans and / or animals without or without excessive adverse side effects such as toxicity, irritation, and allergies, that is, a reasonable benefit / risk ratio. As used herein, the term "effective amount" refers to an amount that can produce a desired sweetness, flavor, and / or taste-masking effect and is acceptable to humans and / or animals.

The composition of the present disclosure can be formulated into usable dosage forms such as powders, granules, suspoemulsions, water emulsions, creams, microcapsules, and the like. One of ordinary skill in the art can select its dosage form and application form according to the needs of the specific application.

The rebaudioside A1G or the rebaudioside A1G composition of the present disclosure can be used in various products that require sweetness or flavor or masking. The rebaudioside A1G or rebaudioside A1G composition may be added in an amount of, for example, 0 to 0.064% or 0% to 0.085% based on the weight of the product. In some applications, the product is liquid. Based on the total volume of the product, the concentration of the rebaudioside A1G or rebaudioside A1G composition can be, for example, much lower than the concentration of sucrose used. 0 to 0.56 g / L or 0 to 0.84 g / L.

Those skilled in the art can also make appropriate adjustments to the amount, time or manner of addition of rebaudioside A1G or its composition according to specific needs to obtain the best effect.

The methods and products of the present disclosure have one or more of the following excellent effects:

(1) The present disclosure uses a cyclodextrin glycosyltransferase-saccharifying enzyme dual enzyme system to prepare a new steviol glycoside rebaudioside A1G (RA1G), and also provides a purification method thereof. The structure of this derivative has not been reported in the world. It has the characteristics of significantly better taste than rebaudioside A, and provides a product with great potential for the development and application of multifunctional sweeteners such as steviol glycosides;

(2) The dual enzyme system of the present disclosure, preferably an industrial / commercial enzyme preparation, is low in cost and stable in quality. At the end of the double-enzyme reaction, the content of the target product can reach more than 50%. No separation and purification step is required in the middle of the two-step reaction. The final reaction solution can be separated and purified to obtain a rebaudioside A derivative product. Simple preparation process, high conversion efficiency, low production cost, short cycle and easy industrialization;

(3) The purification method of the present disclosure can greatly increase the content of rebaudioside A1G (for example, the content of rebaudioside A1G in the modified steviol glycoside product of rebaudioside A obtained by the preparation method of the present disclosure can be increased from 40 5% to 50% is increased to more than 70%, and a new product with a rebaudioside A1G content of more than 70% is obtained, which significantly improves the overall quality of the raw material; and the liquid phase generated by the crystallization in the purification method of the present disclosure can be Recycling, continue to be used for production, solvent can be recycled after distillation, achieving better economic benefits and lower waste liquid discharge. As a result, the production process of the present disclosure has lower energy consumption, simple operation, easy scale and continuous化 生产。 Production.

(4) In the present disclosure, rebaudioside A, the main component of natural steviol glycosides, is used as a glycosyl acceptor substrate, and edible starch, dextrin, or maltose is used as a glycosyl donor substrate. Diglycoside A derivative product, the production process is green and safe, which greatly improves the competitiveness of the product. The products obtained by this disclosure have important application value in industries such as food and beverage.

Examples

The disclosure is further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present disclosure and not to limit the scope of the present disclosure. Those skilled in the art can make appropriate modifications and changes to the present disclosure, and these modifications and changes are all within the scope of the present disclosure.

In the following examples, the experimental methods without specific conditions can be adopted by conventional methods in the art or according to the conditions suggested by the supplier. Unless otherwise stated, percentages and parts are by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equal to those described can be applied to the methods of the present disclosure. The preferred implementation methods and materials described herein are for demonstration purposes only.

Example I. Preparation and characterization of rebaudioside A1G

Example I.1: Preparation of a steviol glycoside derivative using rebaudioside A and soluble starch

Accurately weigh 2g of rebaudioside A (purchased from Haotian Pharmaceutical Co., Ltd., RA97) and 2g of soluble starch (purchased from Tianjin Comeo Chemical Reagent Co., Ltd., 03108003), and dissolve them in a single distillation. Cyclodextrin glycosyltransferase (purchased from Novozymes (China) Biotechnology Co., Ltd., Toruzyme 3.0L, ACN00216, 3kNU / mL) in pure water was added to 1kNU to configure a 40mL, pH6.0 reaction system. The reaction system was placed in a constant temperature shaker at 60 ° C to start the reaction, shaken at 150 rpm for 15 hours, and boiled at 100 ° C to terminate the reaction. Centrifuge at 12000g for 5min, take the supernatant as a sample, use HPLC (Agilent 1200HPLC, mobile phase is acetonitrile-sodium dihydrogen phosphate aqueous solution (pH 2.6), flow rate 1.0mL / min) with Phenomenex Luna 5μm C18 (2) 4.6mm × 250mm Column and UV detector detect (210nm) the formation of derivatives.

Example I.2: The use of saccharifying enzymes to catalyze stevioside derivatives to prepare high-level new rebaudioside A derivative

Take 20 mL of the supernatant after the reaction in Example I.1, and add accurately weighed saccharifying enzyme (purchased from Shaanxi Senfu Biotechnology Co., Ltd., batch number 0108011, 150,000 U / g) 4000 U, and start the reaction in a constant temperature shaker at 60 ° C. The reaction was stopped by shaking at 150 rpm for 3 h and boiling at 100 ° C. Centrifuge at 12000g for 5min, take the supernatant as a sample, use HPLC (DAC50 high pressure preparative chromatography, mobile phase is acetonitrile: aqueous solution = 29: 71, flow rate is 70mL / min) with 10μm C18 (500g) 50mm × 500mm column and UV detector (210nm). The results are shown in Figure 1. The retention time of RA1G was about 6.0 min. The product of the double-enzyme-catalyzed reaction was taken and separated using the above HPLC conditions, and the components within a time range of 5.85-6.25 min were collected, lyophilized, and purified RA1G. As determined by HPLC (see conditions above), the purity of the purified RA1G was 95%.

Example I.3: Rebaudioside A and β-cyclodextrin as raw materials for preparation of rebaudioside by double enzyme modification A1G

Add 100L of purified water to the reaction kettle (Wuxi Hongqi Pressure Vessel Manufacturing Co., Ltd., 500KG emulsification kettle, equipment code 21701098220170001). Weighed 6Kg rebaudioside A (Haotian Pharmaceutical Co., Ltd., RA97) and 6Kg β-cyclodextrin (Qufu Tianli Pharmaceutical Excipients Co., Ltd., 170805), put them into the reaction kettle, and dissolved by heating. Cyclodextrin glycosyltransferase (purchased from Novozymes (China) Biotechnology Co., Ltd., Toruzyme 3.0L, ACN00216, 3kNU / mL) 600 kNU required for the first step of enzyme modification was added. The temperature of the feed liquid was maintained at 60 ° C., the stirring speed was 30 rpm / min, and the reaction was performed for 24 hours. The reaction was stopped by boiling at 100 ° C. The reaction solution was detected by HPLC (the spectrum is shown in Fig. 9A). The specific detection conditions are as follows: The HPLC used is Thermo U3000, the mobile phase is acetonitrile-sodium dihydrogen phosphate aqueous solution (pH 2.6), the flow rate is 1.0 mL / min, and a Thermo C184.6mm × 250mm (5um) column and a UV detector are used for detection ( 210nm).

Then, add the saccharifying enzyme (purchased from Shaanxi Senfu Biotechnology Co., Ltd., batch number 01008011, 150,000 U / g) required for the second step reaction to the above reaction solution, 15 × 10 ⁶ U, 60 ° C., stirring speed 20 rpm / min, reaction 2h, boiling at 100 ° C to terminate the reaction. The reaction liquid was subjected to HPLC to detect the formation of derivatives, and the detection conditions were as described above (the spectrum is shown in FIG. 9B).

The content of each main component in the product during the above preparation process is shown in Table I.1 below. In the table, RA2G and RA3G represent rebaudioside A derivatives in which two or three glucoses are linked to rebaudioside A, respectively.

Table I.1. Product analysis results of rebaudioside A double-enzyme modified 100L reaction system

Component	RA (%)	RA1G (%)	RA2G (%)	RA3G (%)
The first step of enzyme modification	16.02	11.78	13.20	10.03
Enzymatic modification of the second step product	37.03	47.10	4.05	2.95

The product from the second step of enzyme modification was taken and separated and purified using HPLC under the same conditions as in Example 1.2.

Example I.4: Structural identification of novel rebaudioside A derivatives

The structure of the purified rebaudioside A derivative RA1G obtained from Example I.2 and Example 1.3 was analyzed by mass spectrometry and nuclear magnetic resonance technology. Mass spectrometry was performed using Shimadzu HPLC coupled ion trap time-of-flight mass spectrometry (LCMS-IT-TOF). Data were collected in negative ion mode. The mobile phase was acetonitrile: water (68:32), and the flow rate was 1 ml / min. The rate is 10,000 half-height full width. NMR uses Bruker DRXAvance 600MHz spectrometer (Switzerland) to collect data, ¹ H spectrum detection frequency is 600 MHz, ¹³ C spectrum is 150 MHz, both detection temperatures are 25 ° C.

This derivative had a mass-to-charge ratio of 1127.4595 for rebaudioside A plus 1 glucose (see Figure 3). The derivative was fully acetylated, and the derivative was identified as the C19 position of the rebaudioside A diterpene core based on hydrogen, carbon, and two-dimensional nuclear magnetic resonance spectroscopy (see the spectra of Figs. 4 to 8). A glucosyl group is connected to the glucosyl group through an α-1,4 bond, so it is named rebaudioside A1G.

Example I.5: Study on the amount of substrate for transglycosylation reaction in the first step of the dual enzyme method

According to a mass ratio of 1: 1, 10 to 100 mg of rebaudioside A and soluble starch (source: same as in Example I.1) or α-cyclodextrin (Biotech Bioengineering (Shanghai) Co., Ltd. 10016-20-3) or β-cyclodextrin (Qufu Tianli Pharmaceutical Excipients Co., Ltd., 170805), mixed and dissolved in pure water in a single distillation, and added cyclodextrin glycosyltransferase (same as in Example I.1 ) 0.025kNU, configured as a 1mL reaction system, and experimented. The experimental system of each group was placed in a constant temperature shaker at 60 ° C to start the reaction, shaken at 150 rpm for 10 hours, and boiled at 100 ° C to stop the reaction. Centrifuge at 12000g for 5min, take the supernatant as a sample, and use HPLC with Phenomenex Luna 5μm C18 (2) 4.6mm × 250mm column and UV detector to detect the generation of derivatives. The conversion of Rebaudioside A was calculated using the following formula:

Rebaudioside A conversion (%) = (initial RA concentration-RA concentration at the end of the reaction) / initial RA concentration × 100%

The results showed that the conversion rate of rebaudioside A reached more than 60% under all test conditions, of which 82.5 mg β-dextrin donor and 82.5 mg rebaudioside A achieved the highest conversion rate (80%).

Example I.6: Study on the Selection of Enzyme Varieties and Enzyme Amount

The substrate combination (82.5 mg β-dextrin donor + 82.5 mg rebaudioside A) with higher conversion rate in Example I.5 was selected, and cyclodextrin glycosyltransferases purchased from different companies were added to prepare the final enzyme. For reaction systems with different concentrations, the final enzyme concentrations were 10,000 to 50000 U / mL of Jiangxi Baiying Biotechnology Co., Ltd. (batch number 15112514, 400,000 U / mL) and Amano Enzyme Co., Ltd. (CGTAmano, 300,000 U / mL) of Novozymes ( China) Biotechnology Co., Ltd. enzyme (Toruzyme 3.0L, ACN00216, 3kNU / mL) 1-20kNU / L.

The experimental system of each group started the reaction at a constant temperature shaker at 60 ° C, shaken at 150 rpm for 10 hours, and boiled at 100 ° C to terminate the reaction. Centrifuge at 12000g for 5min, take the supernatant as a sample, and use HPLC with a Phenomenex Luna 5μm C18 (2) 4.6mm × 250mm column and a UV detector to detect the generation of derivatives. The conversion rate of rebaudioside A was calculated as before, and the results showed that under all test conditions using Novozymes and Japanese Amanoase, the conversion rate of rebaudioside A reached more than 70%, of which 7.5kNU / L was used Novozymes Toruzyme 3.0L cyclodextrin glycosyltransferase achieved a high conversion rate of 87%.

According to the above enzyme with the highest conversion rate of Rebaudioside A (that is, 7.5kNU / L Novozymes Toruzyme 3.0L cyclodextrin glycosyltransferase, the conversion rate is 87%), take 1 mL of the supernatant sample and add them to different companies Saccharifying enzymes: Shaanxi Senfu Natural Products Co., Ltd. (Cat. No. 01008011, enzyme activity 150,000U / g), or Shanghai Yuanye Biotechnology Co., Ltd. (Cat. No. 9032-08-0, enzyme activity 10000U / ml), or Shanghai Yuanye Bio Technology Co., Ltd. α-amylase (article number 9000-90-2, enzyme activity 12000U / g) or β-amylase (article number 9000-91-3, enzyme activity 50,000U / g), the amount of enzyme added is 100-200U.

The experimental systems of each group were started to react at a constant temperature shaker at 60 ° C, shaken at 150 rpm for 5 hours, and boiled at 100 ° C to terminate the reaction. Centrifuge at 12000g for 5min, take the supernatant as a sample, and use HPLC with a Phenomenex Luna 5μm C18 (2) 4.6mm × 250mm column and UV detector (210nm) to detect the formation of derivatives.

Rebaudioside A1G yield (%) = RA1G concentration at the end of the reaction / RA initial concentration × 100%.

The results show that the conversion of a mixture of rebaudioside A glycosylated derivatives to a single derivative rebaudioside A1G, and the production of rebaudioside A1G can be achieved by using saccharifying enzyme, α-amylase or β-amylase. The rate is 10 to 53%, of which the yield of saccharifying enzyme is 35% to 53%, and the highest yield of 53% is achieved by using 180U saccharifying enzyme of Shaanxi Senfu Natural Company.

The saccharifying enzyme with the highest yield of rebaudioside A1G was selected at 180 U / mL for subsequent optimization.

Example I.7: Study of reaction time and reaction temperature

Using the first transglycosylation reaction system in Example I.6, the catalytic reaction was performed at a reaction time of 1 to 6 h at 50 to 80 ° C. Sampling was performed at intervals. After boiling, the samples were centrifuged and stored on ice. The supernatant sample was subjected to HPLC analysis. The results showed that under all test conditions, the conversion rate of rebaudioside A reached more than 60%. Among them, the reaction time at 70 ° C. was 5 hours, and the conversion rate of rebaudioside A was the highest (87.5%).

Using the second step saccharifying enzyme reaction system in Example I.6, the catalytic reaction was performed at a temperature of 50 to 80 ° C. within a reaction time of 1 h to 6 h. Samples were taken every hour. After the samples were boiled, they were centrifuged and stored on ice. The supernatant sample was subjected to HPLC analysis. The results showed that under all test conditions, the yield of RA1G was above 30%, and the RA1G yield was the highest (53%) at a reaction time of 60 ° C for 3 hours.

Example I.8: Sensory Evaluation Experiment of Rebaudioside A1G

The steviol glycoside raw materials used in the sensory evaluation experiment, wherein RA1G was prepared by the HPLC method of Example I. 2 and the other test products were purchased from Haotian Pharmaceutical Co., Ltd., where the purity of RA1G was 95% and the purity of rebaudioside A (RA) was 97%, Rebaudioside D (RD) purity was 95%.

The above steviol glycoside raw materials are dissolved in purified water according to different mixing ratios (Table I.2) to prepare a sample solution of 360 to 560 ppm. (In order to achieve the purpose of sugar replacement, the industry uses sensory evaluation and comparison of food additives with the same sweetness. ), Take 10mL sample solution into 30mL disposable drinking cups, sensory evaluation (blind evaluation) by 8 trained and experienced sensory personnel, the evaluation result is the average of the scores given by the sensory personnel.

In the evaluation, the sweetness is based on a 10% sucrose aqueous solution (10g / 100ml) as a standard, based on 10 points (sweetness is 10% with 10% sucrose, 9% with 9% sucrose, and so on) No sweetness at all is 0 points.

The bitterness was scored on a scale of 10 points for being very bitter and 0 points for being completely unaware of bitterness.

Comprehensive evaluation Based on the overall taste, 0-100 points are given, 100 points represent the taste of 9% sucrose, and bitterness, astringency, and other miscellaneous tastes are deducted. Miscellaneous tastes refer to other bad tastes other than sweet, bitter, and astringent , Such as alcohol, plastic, metal, licorice, chemical and other bad tastes. When tasting, ensure that the sweetness of each sample is basically the same, and compare their other tastes, such as bitterness and off-flavor, in addition to sweetness.

It can be known from Table I.2 that the sweetness of the 560ppm RA1G solution is similar to that of the 7% sucrose solution, and the post-bitterness and other unpleasant tastes are much lower than RA, and the taste is obviously superior to the raw material rebaudioside A.

Table I.2. Sensory evaluation results of rebaudioside A1G

II. Purification of Rebaudioside A1G and determination of the properties of the purified product

Example II.1: Preparation and identification of rebaudioside A double enzyme modified product rebaudioside A1G

Rebaudioside A modified product was prepared by double enzyme method.

Specifically, the modified product of rebaudioside A was prepared by the method described in Example I.3, and the spectra during the preparation are shown in FIGS. 9A and 9B, respectively. The content of each main component in the product during the preparation process is shown in Table II.1 (data is the same as Table I.1, and repeated here for convenience).

Table II.1. Product analysis results of rebaudioside A double enzyme modified 100L reaction system

Component	RA (%)	RA1G (%)	RA2G (%)	RA3G (%)
The first step of enzyme modification	16.02	11.78	13.20	10.03
Enzymatic modification of the second step product	37.03	47.10	4.05	2.95

Using the method described in Example I.4, the mass spectrum (Figure 3) and nuclear magnetic resonance spectrum of the product RA1G were obtained (Figure 4: Nuclear magnetic hydrogen spectrum, Figure 5: Nuclear magnetic carbon spectrum). The results showed that RA1G is a glucose group at the C19 position of the core of the rebaudioside A diterpene, and a glucose group is connected by an α-1,4 bond.

Example II.2. First Crystallization of Rebaudioside A Enzyme Modified Product

Take 100 L of the rebaudioside A enzyme modified product (that is, the reaction system after the reaction was terminated by boiling in Example II.1) and separate it with a precision filter plate with a pore size of 5 to 10 μm (Shenyang Great Wall Filter Paper Co., Ltd., product number 1001). Then, 100L of macroporous resin (Lan Xiao Technology New Materials Co., Ltd., LX-28; new resin needs to be pretreated as follows: 200L 85% ethanol solution was washed, and then washed with purified water until the effluent is alcohol-free, flow rate 100L / h) Adsorption for 2 hours. The resin adsorbed with the sample was first washed with 300 L of pure water to remove small molecules such as glucose mixed in the product, and then eluted with 200 L of 60% (v / v) ethanol. The eluate was collected, -0.07 MPa, 75 ° C concentrate. After concentrating to 50% of solid content, it was dried in a spray drier, and the air temperature was 75 ° C to obtain 7.5 kg of dry product. Add 3 times the volume of the dry product to a 95% (V / V) methanol aqueous solution, control the temperature to 25 ° C, stir at 30 rpm, and crystallize for 20 hours. The crystallization mixture was subjected to suction filtration using a Buchner funnel to obtain a solid phase and a liquid phase of the first crystallization.

The obtained solid phase was dissolved in double distilled water and analyzed by HPLC according to the method described above. The spectrum is shown in Fig. 10. The content of rebaudioside A1G in the solid phase of the first crystallization product was measured to be 61.41%. Weight 7.5 Kg (Table II.2). The obtained liquid phase was also tested by HPLC (the detection conditions are as described in Example I.1) (the spectrum is shown in Figure 11), and the content of rebaudioside A1G in the liquid phase of the first crystallization product was measured to be 32.01 % (Table II.2).

Table II.2. Analysis results of the first crystallization product of the rebaudioside A enzyme modified product

Component	RA	RA1G	RA2G	RA3G
First crystallized solid phase	28.00	61.41	2.49	1.83
First crystalline liquid phase	44.37	32.01	10.54	3.86

Example II.3. Recrystallization of Rebaudioside A Enzyme Modified Product

7 Kg of the first crystallized wet product (ie, the un-dried solid phase obtained in Example II.2) was dissolved by heating in 15 L of a 65% (V / V) methanol aqueous solution. The temperature of the solution water bath was controlled at 20 ° C, the stirring speed was 15 rpm, and the time was 15 hours for crystallization. The crystallization mixture was suction filtered with a Buchner funnel to obtain a second crystalline solid phase and a liquid phase.

After dissolving the obtained solid phase and ultrapure water, the liquid phase was detected by HPLC (the detection conditions are as described in Example I.1) (the spectrum is shown in Figure 12). %, Wet weight 5 Kg (Table II.3).

The second crystalline solid phase wet product was dissolved by adding 2.5 L of purified water, and spray-dried at 75 ° C. to obtain a final product of 2.25 Kg. The liquid phase obtained by the filtration was also subjected to HPLC detection (the detection conditions are as described in Example I.1) (the spectrum is shown in FIG. 13). The content of rebaudioside A1G in the liquid phase of the second crystallization product was measured as 46.33% (Table II.3).

Table II.3. Product analysis results of the second crystallization of the rebaudioside A enzyme modified product

Component	RA	RA1G	RA2G	RA3G
Second crystallization solid phase	15.25	72.55	3.64	0.81
Second crystallization liquid phase	41.59	46.33	1.61	2.24

Example II.4. Third Crystallization of Rebaudioside A Enzyme Modified Product

Take 2Kg of the second crystallized dry product to heat and dissolve in 10L, 65% (V / V) methanol aqueous solution. The temperature of the solution is controlled at 20 ° C, the stirring speed is 15rpm, and the time is 10 hours. The crystallization mixture was suction filtered with a Buchner funnel to obtain a third crystalline solid phase and a liquid phase.

After the obtained solid-phase ultrapure water was dissolved, the liquid phase was detected by HPLC (the detection conditions are as described in Example I.1) (the spectrum is shown in Figure 14). The content of rebaudioside A1G was measured to be 85.30%. It weighs 3 Kg (Table II.4). The third crystalline solid phase wet product was dissolved with 1.5 L of purified water, and spray-dried at 75 ° C to obtain 1.5 Kg of the final product.

The liquid phase obtained by filtration was also tested by HPLC (the detection conditions are as described in Example I.1) (the spectrum is shown in Figure 15). The content of rebaudioside A1G in the liquid phase of the third crystallization product was measured as 46.83% (Table II.4).

Table II.4. Product analysis results of the third crystallization of the rebaudioside A enzyme modified product

Component	RA	RA1G	RA2G	RA3G
Third crystalline solid phase	10.25	85.30	2.33	0.52
The third crystalline liquid phase	25.20	46.83	6.00	1.36

Example II.5. The crystalline liquid phase is recycled into the enzyme modification reaction

The liquid phases obtained in each crystallization in Examples II.2 to II.4 are mixed and concentrated, and the concentration conditions are -0.07 MPa, 75 ° C, and concentrated to 50% of solid content, dried in a spray dryer, and the air outlet temperature is 75 ° C. Get 5.8Kg of dry product.

The dry product is recycled into the enzyme modification reaction process: 50 g of cyclodextrin glucosyltransferase (source and article number are the same as in Example I.1), 3 Kg of β-cyclodextrin (source is the same as in Example I.1), purified 70L of water was reacted at 60 ° C for 24h, and the reaction was stopped by boiling at 100 ° C. A water bath at 60 ° C. was added with 20 g of saccharifying enzyme (source and article number are the same as in Example I.1), and the reaction was carried out for 2 h. The reaction was stopped by boiling at 100 ° C.

The enzyme-modified product obtained as described above was taken and separated by a precision filter plate having a pore size of 5 to 10 μm, and then adsorbed by 100 L of macroporous resin for 2 hours. The adsorption resin was first washed with 300 L of pure water to remove small molecules such as glucose mixed in the product, and then eluted with 200 L of 60% (v / v) ethanol. The eluate was collected, -0.07 MPa, and concentrated at 75 ° C. After concentrating to 50% of solid content, it was dried in a spray drier, and the air temperature was 75 ° C to obtain 5.5Kg of an enzyme-modified product. After the product was dissolved in ultrapure water, it was detected by HPLC in liquid phase (the detection conditions were as described in Example I.1), and the spectrum is shown in Figure 16. The content of the new product rebaudioside A1G was measured to be 47.81% (Table II.5).

From this result, it can be known that the use of a crystalline liquid phase as a raw material for the enzyme modification reaction can also effectively produce the desired RA1G product, thereby saving production costs.

Table II.5. Analysis results of crystallization of the liquid phase circulating into the enzyme modification reaction product

Component	RA	RA1G	RA2G	RA3G
Enzyme modified product	37.81	47.81	3.18	4.0

Example II.6: Sensory Evaluation Experiment of Rebaudioside A1G

The stevioside raw materials used in the sensory evaluation experiment, where RA1G is prepared as in Examples II.2, II.3, and II.4, and the rest of the test products are from Haotian Pharmaceutical Co., Ltd., the purity of rebaudioside A (RA) is 97%, Rebaudioside D (RD) purity was 95%.

In order to achieve the purpose of sugar replacement, the industry uses sensory evaluation and comparison of food additives under the same sweetness. The stevioside raw materials were dissolved in purified water according to different mixing ratios (Table II.6) to prepare 360-560ppm sample solutions. 10mL sample solutions were taken in 30mL disposable drinking cups, which were trained and experienced by 8 people. The sensory staff performed sensory evaluation (blind evaluation), and the evaluation result was the average of the scores given by the sensory staff.

In the evaluation, the sweetness is based on a 10% sucrose aqueous solution (10g / 100ml) as a standard, based on 10 points (sweetness is 10% with 10% sucrose, 9% with 9% sucrose, and so on) No sweetness at all is 0 points.

The bitterness was scored on a scale of 10 points for being very bitter and 0 points for being completely unaware of bitterness.

Comprehensive evaluation Based on the overall taste, 0-100 points are given, 100 points represent the taste of 9% sucrose, and bitterness, astringency, and other miscellaneous tastes are deducted. Miscellaneous tastes refer to other bad tastes other than sweet, bitter, and astringent. , Such as alcohol, plastic, metal, licorice, chemical and other bad tastes. When tasting, ensure that the sweetness of each sample is basically the same, and compare their other tastes, such as bitterness and off-flavor, in addition to sweetness.

It can be known from Table II.6 that the 600 ppm RA1G solution with a purity of 72.5% and the 560 ppm RA1G solution with a purity of 85.30% are close to each other. The sweetness is similar to the 7% sucrose solution. The post-bitterness and other unpleasant tastes are much lower than RA. The taste is obviously superior to the raw material RA; the RA1G solution with a purity of 660ppm and 61.44% is worse than the high-purity RA1G solution, but also better than the raw material rebaudioside A.

Although the purity of RA1G secondary crystallization and tertiary crystallization products is different, there is little difference between the sensory evaluation results of the two. Therefore, in consideration of the production cost problem, a secondary crystallization cycle process may be adopted to increase the content of rebaudioside A1G in the modified product of rebaudioside A enzyme in practical production applications.

Table II.6. Sensory evaluation results of Rebaudioside A1G

All documents mentioned in this disclosure are incorporated by reference in this application, as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present disclosure, those skilled in the art can make various changes or modifications to the present disclosure, and these equivalent forms also fall within the scope defined by the claims appended to this application.

Claims (22)

1. The compound rebaudioside A1G is shown in the following structure:

   
2. A method for preparing rebaudioside A1G, wherein the structural formula of rebaudioside A1G is as follows:

   

   The method includes steps:

   (1) providing rebaudioside A and a glucose-based donor;

   (2) Rebaudioside A1G is catalyzed by cyclodextrin glycosyltransferase and amylase.
3. The method according to claim 2, wherein the rebaudioside A is one or more selected from the group consisting of rebaudioside A present in natural plants and extracted rebaudioside A A, synthetic rebaudioside A; and / or

   The glucosyl donor is one or more selected from the group consisting of starch; dextrin; maltodextrin; α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin; and maltose.
4. The method according to claim 2, wherein the cyclodextrin glycosyltransferase is selected from the group consisting of α-cyclodextrin glycosyltransferase, β-cyclodextrin glycosyltransferase, and γ-cyclodextrin glycosyltransferase ;and / or

   The amylase is one or more selected from the group consisting of saccharifying enzyme, α-amylase, β-amylase.
5. The method according to any one of claims 2 to 4, wherein the amount of the cyclodextrin glycosyltransferase is 0.1 to 30 kNU / L, or 5000 to 50000 U / mL; and / or the amylase The dosage is 30 to 300 U / mL; and / or the enzyme is an immobilized enzyme.
6. The method according to claim 2, wherein the initial concentration of the rebaudioside A is 5 to 200 g / L; the initial concentration of the glucose-based donor is 10 to 800 g / L.
7. The method according to claim 2, wherein the reaction conditions in step (2) are one or more selected from the group consisting of:

   (a) step (2) is performed in an aqueous phase system;

   (b) the reaction temperature of step (2) is 35-90 ° C; and / or

   (c) The reaction time in step (2) is 0.5 to 72 hours.
8. The method of claim 2, further comprising one or more steps selected from the group consisting of:

   Stop the enzyme reaction after the enzyme reaction is completed;

   Isolate the catalytic reaction product of glycosyltransferase for amylase-catalyzed reaction;

   Directly take the reaction solution after the catalytic reaction of glycosyltransferase for the reaction catalyzed by amylase;

   Replenishing rebaudioside A, glucosyl donor, glycosyltransferase and / or amylase consumed in production;

   Separating, purifying, salting, optically resolving, identifying, and / or packaging the rebaudioside A obtained in step (2); and / or

   Unused rebaudioside A, glucosyl donor, glycosyltransferase and / or amylase were recycled to the next round of reactions.
9. A method for purifying the compound rebaudioside A1G (RA1G) shown in the following structure,

   

   The method includes:

   (a) optionally, pre-purifying the RA1G-containing raw material;

   (b) the first crystallization and solid-liquid separation of the raw material containing RA1G with or without pretreatment using a methanol aqueous solution as a solvent;

   (c) taking the solid phase obtained from the previous crystallization, dissolving it with a methanol aqueous solution as a solvent, and performing a second crystallization under appropriate conditions to obtain a purified product;

   (d) Optionally, the purified product obtained from the previous crystallization may be repeatedly crystallized and purified one or more times or further purified by other purification methods;

   (e) Optionally, the remaining liquid phase in the crystallization purification step is recycled into the preparation process of the RA1G-containing raw material.
10. The method according to claim 9, wherein the raw material containing RA1G is obtained by the method according to any one of claims 1-8.
11. The method of claim 9, wherein the pre-purification pretreatment of step (a) comprises one or more processes selected from the group consisting of filtration, adsorption and elution, concentration and drying, and removal of small molecule impurities.
12. The method of claim 11, wherein the pre-processing is performed by one or more of the following processes:

    (a1) optionally filtering the raw material comprising rebaudioside A1G;

    (a2) Optionally, use a macroporous resin to adsorb and elute the rebaudioside A1G-containing material, for example:

    Adsorption with styrene or acrylate macroporous adsorption resin;

    Wash the macroporous resin with adsorbed raw materials with water;

    Use ethanol eluent to elute the substance adsorbed on the resin;

    (a3) The starting materials, filtered, and / or adsorbed and eluted products can optionally be concentrated and / or dried.
13. The method of claim 9, wherein the first crystallization of step (b) comprises one or more processes selected from the group consisting of:

    (b1) dissolving the raw material in solid form with the solvent;

    (b2) performing the first crystallization;

    (b3) solid-liquid separation of the first crystallization product to obtain a solid phase for further purification or use as a purified product.
14. The method of claim 13, wherein the first crystallization comprises one or more conditions selected from the group consisting of:

    A methanol aqueous solution is used as a solvent to dissolve the raw materials in solid form; the mass-volume ratio of the solvent to the raw materials in solid form is 1: 2 to 5;

    Crystallization temperature is 15 ～ 30 ℃;

    The crystallization time is 10 to 40 hours;

    The stirring speed during the crystallization process is 10 to 60 rpm;

    Solid-liquid separation of the first crystallization product by means of filtration and / or centrifugation;

    Optionally, the crystals are washed, the detergent is a 60 to 90% (v / v) methanol aqueous solution, the volume ratio of the wet weight of the crystals to the detergent is preferably 1: 0.5 to 2, and the crystal washing time is 10 to 30 minutes;

    Optionally, the resulting solid phase is dried.
15. The method of claim 9, wherein the second crystallization of step (c) comprises one or more processes selected from the group consisting of:

    (c1) dissolving the solid phase obtained by the first crystallization with the solvent;

    (c2) performing a second crystallization;

    (c3) Solid-liquid separation of the second crystallization product to obtain a solid phase for further purification or use as a purified product.
16. The method of claim 15, wherein the second crystallization comprises one or more conditions selected from the group consisting of:

    The solid phase of the first crystallization purification product is dissolved by using a methanol aqueous solution as a solvent; the mass-volume ratio of the solvent to the solid phase may be 1: 1.5 to 5;

    The concentration of the aqueous methanol solution used in step (c) is lower than the concentration of the aqueous methanol solution used in step (b);

    The second crystallization temperature is 20-35 ° C;

    The second crystallization time is 10 to 40 hours;

    The stirring speed during the crystallization process is 10 to 60 rpm;

    Solid-liquid separation of the second crystallization product by means of filtration and / or centrifugation;

    Optionally, the resulting solid phase is dried.
17. The method according to claim 9, wherein the further purification method of step (d) is selected from one or more of the following group: crystallization purification, preparative HPLC purification.
18. The method of claim 9, wherein the recycling of step (e) comprises one or more processes selected from the group consisting of:

    Recycling the liquid phase of a single crystallization, the mixture of liquid phases obtained from multiple crystallizations, and the mixture of liquid phases obtained from multiple batches of crystallization;

    Using the liquid phase circulation obtained in the purification process in the dual-enzyme process of claim 2;

    Before recycling, the liquid phase can be mixed, concentrated, and dried.

    The RA1G-containing raw material produced after recycling is used in the purification method according to claim 1.
19. A composition comprising:

    (i) rebaudioside A1G according to claim 1, rebaudioside A1G obtained by a method according to any one of claims 2 to 8, and / or by any one of claims 9 to 18 Rebaudioside A1G obtained by the method described in the item; and

    (ii) pharmaceutically, food, health food or daily chemically acceptable carriers, excipients and / or excipients;

    (iii) optionally, other sweeteners or flavoring agents, such as mogroside, acesulfame, aspartame, sucralose, sodium saccharin, xylitol, sorbitol, erythritol, Sucrose, fructose, glucose, maltose, citric acid, malic acid, tartaric acid, lactic acid, glycine, alanine, serine.
20. Rebaudioside A1G according to claim 1, rebaudioside A1G obtained by the method according to any one of claims 2 to 8, and rebaudioside A1G obtained by the method according to any one of claims 9 to 18. Rebaudioside A1G and / or a composition according to claim 19 for use as a sweetener, flavoring and / or taste-masking agent, for example for the preparation of food, beverages, tobacco products, flavoring Products, daily chemical products, pharmaceutical ingredients, nutritional health products, oral hygiene products and / or cosmetics.
21. A product comprising: rebaudioside A1G according to claim 1, rebaudioside A1G obtained by the method according to any one of claims 2 to 8, and Rebaudioside A1G obtained by the method of any one and / or the composition according to claim 19;

    Preferably, the product is selected from the group consisting of food, beverages, tobacco products, condiments, daily chemical products, pharmaceutical ingredients, nutritional health products, oral hygiene products and / or cosmetics.
22. A packaged product comprising:

    Rebaudioside A1G according to claim 1, rebaudioside A1G obtained by the method according to any one of claims 2 to 8, and rebaudioside A1G obtained by the method according to any one of claims 9 to 18. Rebaudioside A1G and / or the composition according to claim 19; and

    Packages and / or containers, for example the packages and / or containers may be selected from the group consisting of flexible packages or containers, such as bags (such as paper bags, plastic bags, preferably sealed bags) and bottles (such as plastic bottles); rigid Packaging or container, such as glass container, metal container, ceramic container, etc.

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