Crystallization method for increasing the content of rebaudioside A1G, its product and use

Abstract

This text relates to a crystallization method for increasing the content of rebaudioside A1G, its products and uses. In this text, a method of multiple crystallizations (such as twice, three times, etc.) is adopted to purify the stevioside rebaudioside A (RA) derivative RA1G, and high-purity RA1G with improved taste is obtained. Moreover, the crystallization liquid phase can also be recycled for further production, saving production costs. The method of the present invention is simple to operate, the production process is green and environmentally friendly, with low cost, short cycle, high conversion efficiency, and the obtained product has a good taste, and has important application value in industries such as food and beverage.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biotechnology and food chemical engineering. Specifically, the present invention relates to a crystallization method for increasing the content of Rebaudioside A1G in the enzyme-modified product of Rebaudioside A, and its products and uses. Background Art

[0002] Steviol glycosides are a series of glycoside substances extracted from the leaves of the Compositae herb Stevia rebaudiana Bertoni. Among them, the main components in natural products are stevioside (abbreviated as Stv) and rebaudioside A (abbreviated as RA, also known as steviolbioside A).

[0003] The molecular structure of rebaudioside A is shown as follows. It is a tetracyclic diterpenoid glycoside substance containing 20 carbon atoms, which is formed by connecting a glucosyl group at the C19 position of the diterpene core and 3 glucosyl groups at the C13 position:

[0004]

[0005] Research shows that the sweetness of rebaudioside A is 200-300 times that of sucrose, and the calorie is only 1 / 300 of sucrose. It is very stable to acids, alkalis and heat, is not easy to deteriorate during long-term storage, will not have browning phenomenon after heat treatment when added to food, and is not easy to cause dental caries. In China, its use as a food additive is described in detail 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)" level. As a natural substitute for sucrose, steviol glycosides can not only reduce costs, but also meet the requirements of the gradual development of food and beverages towards low sugar and low calorie. They are an ideal type of green sweeteners with multiple uses.

[0006] However, rebaudioside A has a serious bitter aftertaste, which affects its application in food. To improve its taste, in the prior art, a method of biocatalytic modification is used, with rebaudioside A as the receptor substrate, adding different amounts and different bond types of glucose, galactose, etc. at the C19 and C13 positions of its diterpene core to obtain an enzyme-modified product with improved taste. The products of the enzymatic modification of rebaudioside A are usually a mixture of glycosylated products with different sites and different amounts, which results in problems such as complex product components and uneven taste. Moreover, since the components in the product mixture have the same parent nucleus, their molecular structures and polarities are very similar, and it is very difficult to separate and purify them by conventional means. To increase the concentration of the effective product, higher requirements are put forward for the innovation and optimization of the production process.

[0007] Therefore, further developing steviol glycoside derivative products with high purity and improved taste and providing their production processes to increase the content of the effective product have become the development trend of the industrial production of steviol glycosides. Summary of the Invention

[0008] This article precisely provides a purification method and a purified product of a novel steviol glycoside derivative (i.e., rebaudioside A1G) to improve the taste of the raw material and produce high-quality steviol glycoside derivative R1G at a low cost and in a short production cycle.

[0009] In some aspects of the present invention, a method for purifying a compound rebaudioside A1G (RA1G) having the following structure is provided.

[0010]

[0011] The method includes:

[0012] (a) Optionally, perform pre-treatment before purification on the raw material containing RA1G.

[0013] (b) Using an aqueous methanol solution as a solvent, perform first crystallization on the raw material containing RA1G with or without pre-treatment and perform solid-liquid separation.

[0014] (c) Take the solid phase obtained from the previous crystallization, dissolve it using an aqueous methanol solution as a solvent, and under appropriate conditions, perform second crystallization to obtain the purified product.

[0015] (d) Optionally, repeat the crystallization purification on the purified product obtained from the previous crystallization once or multiple times or perform further purification using other purification methods.

[0016] (e) Optionally, recycle the liquid phase remaining in the crystallization purification step to the preparation process of the raw material containing RA1G.

[0017] In some embodiments, the raw material containing RA1G is prepared by an enzyme-catalyzed method, and the enzyme-catalyzed method includes: (1) providing rebaudioside A and a glucosyl donor; (2) producing rebaudioside A1G through the catalysis of cyclodextrin glycosyltransferase and amylase.

[0018] In some embodiments, the enzyme-catalyzed method includes one or more conditions selected from the following group:

[0019] The rebaudioside A is one or more selected from the following group: rebaudioside A existing in natural plants, extracted rebaudioside A, and synthetic rebaudioside A;

[0020] The glucosyl donor is one or more selected from the following group: starch, such as soluble starch; dextrin; maltodextrin; α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin; maltose;

[0021] The cyclodextrin glycosyltransferase is selected from: α-cyclodextrin glycosyltransferase, β-cyclodextrin glycosyltransferase, and γ-cyclodextrin glycosyltransferase;

[0022] The amylase is one or more selected from the following group: glucoamylase, α-amylase, β-amylase;

[0023] The dosage of the cyclodextrin glycosyltransferase is 0.1 - 30 kNU / L, such as 0.5 - 20 kNU / L, 1 - 15 kNU / L, or 5000 - 50000 U / mL, such as 10000 - 40000 U / mL, 15000 - 35000 U / mL; and / or the dosage of the amylase is 30 - 300 U / mL, such as 50 - 250 U / mL, 80 - 220 U / mL; and / or the enzyme is an immobilized enzyme;

[0024] The initial concentration of the rebaudioside A is 5 - 200 g / L, such as 8 - 150 g / L, 10 - 120 g / L; the initial concentration of the glucosyl donor is 10 - 800 g / L, such as 20 - 700 g / L, 30 - 600 g / L, 30 - 300 g / L;

[0025] Step (2) is carried out in an aqueous phase system, for example, in water (such as pure water, distilled water, ultrapure water, pH 6).

[0026] The reaction temperature of step (2) is 35 - 90 °C, such as 40 - 90 °C, 45 - 85 °C, 50 - 70 °C, 45 - 85 °C; and / or

[0027] The reaction time of step (2) is 0.5 - 72 hours, such as 1 - 48 hours, 1.5 - 36 hours, 5 - 20 hours.

[0028] In some embodiments, the pre - treatment before purification in step (a) includes one or more treatments selected from the group consisting of filtration, adsorption and elution, concentration and drying. Other methods such as solvent precipitation method, microfiltration membrane filtration method, etc. can also be used to remove small - molecule impurities (such as residual glucose).

[0029] In some embodiments, the pre - treatment is carried out by one or more of the following treatments:

[0030] (a1) Optionally, filter the raw material containing rebaudioside A1G, for example, filter it using filter plates, filter paper, or filter cartridges, preferably using a fine filter plate, more preferably a fine filter plate with a pore size of 5 - 10 μm;

[0031] (a2) Optionally, adsorb the raw material containing rebaudioside A1G using macroporous resin and elute it, for example:

[0032] Adsorb using styrene - type or acrylate - type macroporous adsorption resins, such as macroporous adsorption resins with a pore size of 6 - 15 nm and a specific surface area of 600 - 1300 ㎡ / g; the following adsorption conditions can be used: the concentration of the sample injection solution is 0.5 - 20%, pH is 4 - 8, and the sample injection flow rate is 0.5 - 5 BV / h;

[0033] Wash the macroporous resin adsorbed with the raw material with water (such as 2 - 10 times the resin volume of water), for example, wash it with purified water, and the flow rate is 0.5 - 5 BV / h;

[0034] Elute the substances adsorbed on the resin with an ethanol eluent, such as an ethanol eluent with a concentration of 30 - 90% (v / v) (preferably ≥60%), with a volume of ≥1.5 times, such as 1.5 - 4 times the column bed volume. The elution solvent is an ethanol aqueous solution, without adjusting the pH (about 6), and the elution flow rate is 0.5 - 2 BV / h;

[0035] (a3) Optionally, concentrate and / or dry the raw material, the filtered product, and / or the product after adsorption and elution, such as spray drying, vacuum drying. For example, concentrate the ethanol eluate under the conditions of - 0.06 - 0.09 MPa and 60 - 85 °C, and perform spray drying at an outlet air temperature of 65 - 90 °C.

[0036] In some embodiments, the first crystallization in step (b) includes one or more treatments selected from the group consisting of:

[0037] (b1) Dissolve the raw material in solid form with the solvent;

[0038] (b2) Perform the first crystallization;

[0039] (b3) Separate the solid - liquid of the first crystallization product to obtain the solid phase for the next - step purification or as the purified product.

[0040] In some embodiments, the first crystallization includes one or more conditions selected from the following group:

[0041] Use an aqueous methanol solution as the solvent to dissolve the raw material in solid form. For example, use an aqueous methanol solution with a concentration of 80-99% (v / v), such as a concentration ≥90%, for example 95% as the solvent; the mass-volume ratio of the solvent to the raw material in solid form is 1:2-5, such as the solvent volume is 3-5 times the weight of the dry product, for example 3 times;

[0042] The crystallization temperature is 15-30°C, for example room temperature, for example 20-25°C, for example 25°C;

[0043] The crystallization time is 10-40 hours, for example 15-30 hours, for example 20-24 hours;

[0044] The stirring speed during the crystallization process is 10-60 rpm, for example 20-50 rpm, for example 30-45 rpm;

[0045] Solid-liquid separation of the first crystallization product is carried out by filtration (such as suction filtration) and / or centrifugation, etc.;

[0046] Optionally, the crystals are washed. The detergent is an aqueous methanol solution of 60-90% (v / v). The volume ratio of the wet weight of the crystals to the detergent is preferably 1:0.5-2, and the crystal washing time is 10-30 min;

[0047] Optionally, the obtained solid phase is dried.

[0048] In some embodiments, the second crystallization in step (c) includes one or more treatments selected from the following group:

[0049] (c1) Dissolve the solid phase obtained from the first crystallization with the said solvent;

[0050] (c2) Conduct the second crystallization;

[0051] (c3) Carry out solid-liquid separation on the second crystallization product to obtain a solid phase for the next purification or used as a purified product.

[0052] In some embodiments, the second crystallization includes one or more conditions selected from the following group:

[0053] The solid phase of the first crystallization and purification product is dissolved using an aqueous methanol solution as the solvent. For example, an aqueous methanol solution with a concentration of 50-90% (v / v), such as a concentration of 60-80%, for example 65%, is used as the solvent; the mass-volume ratio of this solvent to the solid phase can be 1:1.5-5, such as the solvent volume being 1.5-3 times the weight of the dry product, for example 2-2.5 times; optionally, 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).

[0054] The temperature for the second crystallization is 20-35 °C, for example room temperature, for example 20-25 °C, for example 20 °C;

[0055] The time for the second crystallization is 10-40 hours, for example 15-30 hours, for example 20-24 hours;

[0056] The stirring speed during the crystallization process is 10-60 rpm, for example 20-50 rpm, for example 10-30 rpm;

[0057] Solid-liquid separation of the second crystallization product is carried out by filtration (such as suction filtration) and / or centrifugation, etc.;

[0058] Optionally, the obtained solid phase is dried, for example by spray drying at an air outlet temperature of 65-90 °C.

[0059] In some embodiments, the further purification method in step (d) is selected from one or more of the following groups: crystallization purification, preparative HPLC purification.

[0060] In some embodiments, the recycling in step (e) includes one or more treatments selected from the following groups:

[0061] Recycling the liquid phase of a certain crystallization (such as the first crystallization or the second crystallization), a mixture of the liquid phases obtained from multiple crystallizations (such as a mixture of the liquid phases of the first and second crystallizations), a mixture of the liquid phases obtained from multiple batches of crystallizations (such as a mixture of the liquid phases obtained from multiple batches through the first and / or second crystallization steps);

[0062] Recycling the liquid phase obtained during the purification process to the double-enzyme process described in claim 2;

[0063] Before recycling, pretreatment such as mixing, concentration, drying, etc. can be carried out on the liquid phase;

[0064] Using the raw material containing RA1G produced after recycling in the purification method described in claim 1.

[0065] In some aspects of the present invention, a composition is provided, which comprises:

[0066] (i) Rebaudioside A1G obtained by the method as described above; and

[0067] (ii) pharmaceutically, food, health product or daily chemical acceptable carriers, excipients and / or adjuvants;

[0068] (iii) Optionally, other sweeteners or flavoring agents, such as mogroside, acesulfame potassium, aspartame, sucralose, sodium saccharin, xylitol, sorbitol, erythritol, sucrose, fructose, glucose, maltose, citric acid, malic acid, tartaric acid, lactic acid, glycine, alanine, serine.

[0069] In some aspects of the present invention, there is provided Rebaudioside A1G obtained by the method of the present invention or a composition thereof, which is used as a sweetening agent, flavoring agent and / or taste masking agent, for example, for preparing foods, beverages, tobacco products, condiments, daily chemical products, pharmaceutical components, nutritional and health products, oral hygiene products and / or cosmetics.

[0070] In some aspects of the present invention, there is provided a product comprising: Rebaudioside A1G obtained by the method of the present invention or a composition thereof.

[0071] Preferably, the product is selected from the group consisting of: foods, beverages, tobacco products, condiments, daily chemical products, pharmaceutical components, nutritional and health products, oral hygiene products and / or cosmetics.

[0072] In some aspects of the present invention, there is provided a package comprising:

[0073] Rebaudioside A1G obtained by the method of the present invention or a composition thereof; and

[0074] a packaging and / or container, for example, the packaging and / or container can be selected from the group consisting of: flexible packaging or containers, such as bags (such as paper bags, plastic bags, preferably sealed bags) and bottles (such as plastic bottles); rigid packaging or containers, such as glass containers, metal containers, ceramic containers, etc.

[0075] Those skilled in the art can make any combination of the foregoing technical solutions and technical features without departing from the inventive concept and protection scope of the present invention. Other aspects of the present invention will be apparent to those skilled in the art from the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] The present invention will be further described below in conjunction with the drawings, where these illustrations are only for illustrating the embodiments of the present invention and not for limiting the scope of the present invention.

[0077] Figure 1 . Schematic diagram of the process for preparing Rebaudioside A1G by double enzyme method.

[0078] Figure 2A . HPLC chromatogram of the product of the first step of double-enzyme modification;

[0079] Figure 2B . HPLC chromatogram of the product of the second step of double-enzyme modification.

[0080] Figure 3A . Mass spectrum of RA1G;

[0081] Figure 3B . 1H NMR spectrum of RA1G;

[0082] Figure 3C . 13C NMR spectrum of RA1G.

[0083] Figure 4 . HPLC chromatogram of the solid-phase component after the first crystallization.

[0084] Figure 5 . HPLC chromatogram of the liquid-phase component after the first crystallization.

[0085] Figure 6 . HPLC chromatogram of the solid-phase component after the second crystallization.

[0086] Figure 7 . HPLC chromatogram of the liquid-phase component after the second crystallization.

[0087] Figure 8 . HPLC chromatogram of the solid-phase component after the third crystallization.

[0088] Figure 9 . HPLC chromatogram of the liquid-phase component after the third crystallization.

[0089] Figure 10 . HPLC chromatogram of the product obtained by recycling the crystallization liquid phase into the enzyme modification process. Detailed implementation manners

[0090] The present invention relates to a purification method of a novel rebaudioside A derivative, rebaudioside A1G (i.e., RA1G). RA1G is a novel rebaudioside A derivative developed by the applicant in the early stage (see the patent application filed on the same day, with the invention name "Preparation and Application of Stevioside Derivative Rebaudioside A1G by Double Enzyme Method", hereinafter referred to as the "same-day application", the full text of which is incorporated herein by reference). It has a glucose group linked by an α-1,4 bond to the glucose linked to the C19 position of the diterpene core of rebaudioside A, so it is named rebaudioside A1G. The preparation method and characterization of RA1G are described in detail in the same-day application. Taking rebaudioside A as the raw material, it is carried out through two-step enzyme catalysis. Compared with the raw material rebaudioside A, the novel rebaudioside A1G has increased sweetness and improved taste.

[0091] It should be understood that the purification method of the present invention is not only applicable to the raw materials containing rebaudioside R1G obtained by the enzyme-catalyzed modification preparation method described below or as described in the same-day application, but also includes the raw materials containing rebaudioside R1G obtained by other methods. Similarly, the remaining liquid phase in the crystallization method of the present invention can also be recycled to the production of rebaudioside R1G, and is not limited to a specific preparation method.

[0092] The purification method of the present invention mainly includes: (a) Optionally, pre-treat the raw material containing RA1G before purification; (b) Use an appropriate solvent and under appropriate conditions, perform the first crystallization on the raw material containing RA1G with or without pre-treatment and carry out solid-liquid separation; (c) Take the solid phase obtained from the previous crystallization, dissolve it with an appropriate solvent, and under appropriate conditions, perform the second crystallization to obtain the purified product; (d) Optionally, repeat the crystallization purification one or more times for the purified product obtained from the previous crystallization; (e) Optionally, recycle the remaining liquid phase in the crystallization purification step to the RA1G preparation process.

[0093] For example, in some embodiments of the present invention, a method for improving the production of rebaudioside A1G in the enzyme-modified product of rebaudioside A is provided, and the steps are as follows: The enzyme-catalyzed modified product of rebaudioside A is filtered through a fine plate and frame, separated by macroporous resin, concentrated by a single-effect evaporator, the concentrated solution is spray-dried, and a solvent is added for crystallization; the above crystallization solution is subjected to solid-liquid separation, the solid phase is redissolved in the solvent for secondary crystallization, the secondary crystallization solution is subjected to solid-liquid separation again, the solid phase is dissolved in purified water and spray-dried to obtain a refined enzyme-modified product. The liquid phases of the two crystallizations are mixed and reused as the raw material for the enzyme-catalyzed modification of rebaudioside A for the enzyme modification reaction. The product of this step can be recycled into the process flow of filtration → separation → concentration → spray drying → crystallization → secondary crystallization again. This secondary crystallization cycle production process can greatly increase the content of a novel product rebaudioside A1G with a better taste in the enzyme-catalyzed modified product of rebaudioside A, and the content of rebaudioside A1G in the final product reaches more than 70%; through the recycling process, the utilization rate of production raw materials can be further improved, and the production cost and waste discharge can be greatly reduced.

[0094] As used herein, the terms "rebaudioside A" and "RA" are used interchangeably and both refer to the compound shown by the following structural formula.

[0095] As used herein, the terms "rebaudioside A1G" and "RA1G" are used interchangeably and both refer to a rebaudioside A derivative in which a glucose group is further linked by an α-1,4 bond to the glucose linked to the diterpene core C19 position of rebaudioside A (the structure is shown below).

[0096] As used herein, the terms "rebaudioside A2G" and "RA2G" are used interchangeably and both refer to a rebaudioside A derivative in which 2 glucose groups are further linked to rebaudioside A.

[0097] As used herein, the terms "rebaudioside A3G" and "RA3G" are used interchangeably and both refer to a rebaudioside A derivative in which 3 glucose groups are further linked to rebaudioside A.

[0098] As used herein, the terms "rebaudioside D" and "RD" are used interchangeably and both refer to a rebaudioside A derivative in which a glucose group is linked by a β-1,2 bond to the glucose linked to the diterpene core C19 position of rebaudioside A.

[0099]

[0100] All numerical ranges provided herein are intended to clearly include all numerical values falling between the range endpoints and the numerical ranges therebetween. The features mentioned in the present invention or the features mentioned in the embodiments can 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 with any alternative feature that provides the same, equivalent or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.

[0101] As used herein, "comprising", "having" or "including" includes "containing", "consisting essentially of", "consisting substantially of", and "consisting of"; "consisting essentially of", "consisting substantially of" and "consisting of" are subordinate concepts of "comprising", "having" or "including".

[0102] Enzymatic Catalytic Modification of Rebaudioside A

[0103] As used herein, the term "two - enzyme method" refers to a method in which rebaudioside A and a glucosyl donor are used as raw materials, and two enzymes are used for catalytic reaction to obtain rebaudioside A1G. Specific descriptions of the reaction raw materials, enzymes, reaction conditions, reaction products, etc. used in the two - enzyme method can be found below. For more details, see the application filed on the same day.

[0104] The rebaudioside A raw material for the production of rebaudioside A1G by the two - enzyme method can be rebaudioside A from various sources. The selectable rebaudioside A raw materials include but are not limited to: rebaudioside A extracted from natural plants and directly used in the method of the present invention, for example, using stevia leaves as raw materials and obtaining it through processes such as extraction, impurity removal, decolorization, and drying; commercially available rebaudioside A; synthetic rebaudioside A, for example, synthesized by microbial fermentation (such as recombinant Pichia pastoris, recombinant Saccharomyces cerevisiae, recombinant Escherichia coli). Rebaudioside A in the form of powder, crystal, solution, etc. can be used in the reaction system of the present invention.

[0105] The glucosyl donor for the production of rebaudioside A1G by the two - enzyme method can be any polysaccharide and / or oligosaccharide that can serve as a substrate for a glycosyltransferase (such as cyclodextrin glycosyltransferase, CGT) and transfer the sugar molecules contained therein to a receptor through an enzymatic reaction. The glucosyl donors that can be used in the present invention include but are not limited to: starch (preferably soluble starch), β - cyclodextrin, α - cyclodextrin or γ - cyclodextrin, maltodextrin, maltose.

[0106] There are two types of enzymes for the production of rebaudioside A1G, namely enzymes that catalyze transglycosylation and hydrolysis reactions, preferably glycosyltransferases (such as cyclodextrin glycosyltransferase) and amylases.

[0107] Cyclodextrin glycosyltransferase (CGTase) is a multifunctional enzyme that can catalyze different reactions. It can transfer the glycosyl group from a glucose donor to a receptor (Rebaudioside A in the present invention). For the properties, preparation, and applications of cyclodextrin glycosyltransferase, reference can be made to Wu Jing et al., "Preparation and Application of Cyclodextrin Glucosyltransferase" (Chemical Industry Press, 2011). The cyclodextrin glycosyltransferase (CGT) can be an enzyme from various sources, such as commercially available CGTase, CGTase produced by genetic engineering, etc. The amylases that can be used in the double-enzyme method include but are not limited to: glucoamylase, α-amylase, and β-amylase. Glucoamylase, also known as glucose amylase (Glucoamylase, EC 3.2.1.3), can hydrolyze the α-1,4 and α-1,6 glucosidic bonds at the non-reducing end of starch to produce glucose. At the same time, this enzyme can also hydrolyze the non-reducing ends of dextrin and glycogen to release β-D-glucose.

[0108] Both of these two enzymes are commercially available, for example, purchased from Amano Enzyme Inc. of Japan, Novozymes (China) Biotechnology Co., Ltd., Jiangxi Baiying Biotechnology Co., Ltd., etc.; they can also be obtained by means such as microbial fermentation as long as they have the required catalytic activity. It is preferred to use enzyme preparations with high enzyme activity and high stability, and immobilized enzymes can also be used.

[0109] The enzymatic reaction for preparing Rebaudioside A1G is carried out in an aqueous phase system. Using Rebaudioside A as the receptor substrate and soluble starch, β-cyclodextrin or α-cyclodextrin, maltose, etc. as the glucose donor substrates, a transglycosylation reaction is carried out under the catalysis of cyclodextrin glycosyltransferase (CGT) to generate a mixed system of Rebaudioside A derivatives. Then, through the hydrolysis reaction catalyzed by amylase (such as glucoamylase) on the components in the mixed system, a novel Rebaudioside A derivative with higher homogeneity, namely Rebaudioside A1G, is finally obtained.

[0110] The two raw materials of the present invention 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 can be 5 - 200 g / L, for example, 8 - 150 g / L, 10 - 120 g / L. The initial concentration of the glucose donor substrate raw material in the reaction system can be 10 - 800 g / L, for example, 20 - 700 g / L, 30 - 600 g / L, 30 - 300 g / L. Usually, the weight ratio of the Rebaudioside A raw material to the glucose donor raw material used can be 1:2 - 4.

[0111] In an aqueous phase system, cyclodextrin glycosyltransferase (CGT) is added to catalyze the transglycosylation reaction to generate a mixed system of a series of transglycosylated rebaudioside A derivatives. The final concentration of CGT enzyme in the reaction system can be 0.1 - 30 kNU / L, such as 0.5 - 20 kNU / L, 1 - 15 kNU / L, or 5000 - 50000 U / mL, such as 10000 - 40000 U / mL, 15000 - 35000 U / mL. The content of CGT enzyme in the reaction system can be 5 - 200 kNU / kg of rebaudioside A, such as 10 - 150 kNU / kg of rebaudioside A. According to the generation of the reaction product, the reaction temperature of CGT enzyme can be set within the range of 35 - 90 °C, such as 40 - 90 °C, 45 - 85 °C, 50 - 70 °C, and this can be adjusted according to the specific enzyme used and industrial costs, etc. The pH of the CGT enzyme reaction system can be set around the optimal pH of this enzyme, such as pH 4 - 7, pH 4.5 - 6.5, pH 5 - 6, and this can be adjusted according to the specific enzyme used. The reaction time of CGT enzyme can be adjusted according to the reaction process, such as reacting for 0.5 - 72 hours, 1 - 48 hours, 1.5 - 36 hours, 5 - 20 hours.

[0112] After the GCT enzyme reaction is completed, the enzyme reaction can be terminated by various methods (for example, a relatively simple method is to denature the enzyme by boiling (such as boiling at 100 °C for 5 minutes) to terminate the reaction). Optionally, the obtained 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 separation and purification.

[0113] Amylase (such as glucoamylase) is added to the GCT enzyme reaction product to catalytically decompose the components in the rebaudioside A derivative mixed system into rebaudioside A1G in which a glucose group is linked to the C19 position of the rebaudioside A diterpene core through an α-1,4 bond. In some embodiments, the dosage of the amylase is 30 - 300 U / mL, such as 50 - 250 U / mL, 80 - 220 U / mL. In some embodiments, the dosage of the amylase is 300 - 3000 U / g of rebaudioside A, such as 800 - 2200 U / g of rebaudioside A.

[0114] According to the generation 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, and can be adjusted according to the specific enzyme used and industrial costs, etc. The pH of the amylase reaction system can be set around the optimal pH of the enzyme, such as pH 4-7, pH 4.5-7, pH 5-7, and can be adjusted according to the specific enzyme used. The reaction time of the amylase can be adjusted according to the reaction process, such as reacting for 0.5-72 hours, 1-48 hours, 1.5-36 hours, 2-10 hours.

[0115] After the amylase reaction is completed, the enzyme reaction can be terminated by various methods (for example, a relatively simple method 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, identified, etc. to obtain Rebaudioside A1G.

[0116] For example, the reaction supernatant and precipitate can be separated by centrifugation, such as at 12000 rpm for 5 minutes, etc. For example, the reaction product can be separated by chromatography, such as using HPLC. An optional HPLC instrument and conditions are: Agilent 1200 HPLC 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 invention, 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 acetonitrile:sodium dihydrogen phosphate aqueous solution (pH 2.6) with a volume ratio of 68:32, and the ultraviolet detection wavelength is 210 nm. For example, the obtained product can be dried by freeze-drying method.

[0117] Those of ordinary skill in the art should also understand that the above reaction can be carried out using an immobilized enzyme system, and an immobilized CGT enzyme and / or an immobilized amylase can be used.

[0118] The product obtained by the double-enzyme method of the present invention is characterized by high-resolution mass spectrometry, nuclear magnetic resonance, etc., and it is determined that the glucose at the C19 position of the diterpene core of Rebaudioside A is linked to a glucosyl group through an α-1,4 bond, that is, the obtained product is Rebaudioside A1G.

[0119] Exemplary Preparation Method

[0120] An exemplary method for preparing Rebaudioside A1G is described as follows. It should be understood that this exemplary method is only used to illustrate one case of the preparation of the raw materials for the purification method of the present invention and is not used to limit the scope of the present invention. Those skilled in the art can make appropriate modifications, changes, and selections to the preparation method, and these modifications, changes, and selections are all within the scope of the present invention.

[0121] An exemplary method for preparing the steviol glycoside derivative rebaudioside RA1G from rebaudioside A by a bioenzymatic method, comprising the following steps:

[0122] (1) In an aqueous phase system, using rebaudioside A as the acceptor substrate with an initial reaction concentration of 10 g to 120 g / L, and using soluble starch, β-cyclodextrin or α-cyclodextrin, maltose as the glucose donor substrate with an initial reaction concentration of 30 to 300 g / L, a transglycosylation reaction is carried out under the catalytic action of α-cyclodextrin glycosyltransferase (CGT) to generate a series of derivatives of rebaudioside A; (2) The reaction system prepared in step (1) is reacted in a water bath at 45 to 85 °C for 1 to 48 hours, the reaction is terminated by boiling, centrifuged, and the supernatant is taken; (3) To the supernatant obtained in step (2), glucoamylase is added, and hydrolysis reaction is carried out using all the components in the mixed system in step (2) as the substrate; (4) The reaction system prepared in step (3) is reacted in a water bath at 45 to 85 °C for 1 to 48 hours, the reaction is terminated by boiling, centrifuged, and the supernatant is taken; (5) The supernatant obtained in step (4) is separated and dried to obtain a derivative of rebaudioside A, rebaudioside A1G.

[0123] In one example, the aqueous phase system in step (1) is distilled pure water with a pH of 6.0.

[0124] In one example, the α-cyclodextrin glycosyltransferase in step (1) can be purchased from Jiangxi Baiying Biotechnology Co., Ltd., Novozymes (China) Biotechnology Co., Ltd. and Amano Enzyme Co., Ltd. Japan, and the final concentration is 0.05 to 2 g / L.

[0125] In one example, the glucose donor substrate in step (1) is soluble starch, dextrin, maltose

[0126] In one example, the reaction conditions in step (2) are a water bath at 60 °C for 15 hours.

[0127] In one example, the boiling termination reaction condition in step (2) is boiling at 100 °C for 5 minutes.

[0128] In one example, the centrifugation speed in step (2) is 12000 rpm and the time is 5 minutes.

[0129] In one example, the glucoamylase in step (3) can be purchased from Shaanxi Senfu Natural Products Co., Ltd. and Shanghai Yuanye Biotechnology Co., Ltd., and the final concentration is 0.5 to 20 g / L

[0130] In one example, the reaction conditions in step (4) are a water bath at 60 °C for 3 hours

[0131] In one example, the boiling termination reaction condition in step (4) is boiling at 100 °C for 5 minutes.

[0132] In one example, the centrifugation speed in step (4) is 12,000 rpm and the time is 5 minutes.

[0133] In one example, the separation in step (5) uses an Agilent 1200 HPLC system, a Phenomenex Luna 5μm C18(2) 4.6mm×250mm chromatographic column, and the mobile phase is acetonitrile - sodium dihydrogen phosphate aqueous solution (pH 2.6).

[0134] In a further example according to the present invention, the single sample loading amount for the further separation above is 5 μL, the flow rate is 1.0 mL / min, the mobile phase is acetonitrile:sodium dihydrogen phosphate aqueous solution (pH 2.6) with a volume ratio of 68:32, and the ultraviolet detection wavelength is 210 nm.

[0135] In one example, the drying in step (5) is freeze-drying.

[0136] Crystallization and Purification Method for Modified Product of Rebaudioside A

[0137] The purification of rebaudioside A1G in the rebaudioside A modified product can be carried out by crystallization methods (such as primary crystallization, secondary crystallization, tertiary crystallization, quaternary crystallization, etc., preferably secondary crystallization).

[0138] A. Pretreatment before purification

[0139] Before crystallization purification, optionally, the raw material containing RA1G is pretreated through steps such as filtration, adsorption and elution, drying, concentration, etc., to, for example, reduce the interference of contaminants on crystallization purification and / or relatively enrich RA1G in the crystallization raw material.

[0140] In some embodiments, the raw material containing 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, a filter plate is used for filtration, for example, a fine filter plate, preferably a fine filter plate with a pore size of 5 - 10 μm.

[0141] In some embodiments, macroporous resin is used to adsorb the raw material containing rebaudioside A1G and elute it. This step can separate some small molecule impurities in the raw material, such as free glucose, etc. The macroporous resin used can be styrene-based or acrylate-based macroporous adsorption resin. For example, a macroporous adsorption resin with a pore size of 6 - 15 nm and a specific surface area of 600 - 1300 ㎡ / g can be used. The concentration of the sample injection solution can be 0.5 - 20%, and the pH is 4 - 8. The sample injection flow rate can be 0.5 - 5 BV / h.

[0142] The macroporous resin adsorbed with the raw material 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 substantially washed away. For example, water with a volume ≥ 2 times, such as 2 - 10 times the column bed volume (i.e., the resin volume) can be used for washing. The washing flow rate can be 0.5 - 5 BV / h. Then, an ethanol eluent can be used to elute the substances adsorbed on the resin. For example, an ethanol eluent with a concentration of 30 - 90% (v / v) (preferably ≥ 60%) (pH about 6, and the elution flow rate can be 0.5 - 2 BV / h) can be used, and the volume is ≥ 1.5 times, such as 1.5 - 4 times the column bed volume.

[0143] In some embodiments, optionally, the raw material or the pretreatment product after filtration or adsorption and elution and other pretreatment steps can be concentrated and / or dried. For example, the ethanol eluate as described above can be concentrated under the conditions of -0.06 to 0.09 MPa and 60 - 85°C. For example, the product can be dried by spray drying, vacuum drying, etc.

[0144] In some embodiments, the raw material is dried to obtain a solid-phase raw material.

[0145] It should be understood that the pretreatment steps can be increased, decreased, improved, or modified without departing from the concept and protection scope of the present invention. For example, the solvent precipitation method, microfiltration membrane filtration method, etc. can be used to remove small molecule impurities (such as residual glucose).

[0146] B. First crystallization purification

[0147] The raw material, with or without pretreatment, can be subjected to first crystallization purification. Generally, for the raw material in solid form, an aqueous methanol solution can be used as the solvent to dissolve it. For example, an aqueous methanol solution with a concentration of 80 - 99% (v / v) (such as a concentration ≥ 90%, for example 95%) can be used as the solvent. The mass-volume ratio of this solvent to the solid phase (i.e., the dry product) can be 1:2 - 5, such as the solvent volume is 3 - 5 times the weight of the dry product, for example 3 times.

[0148] The methanol solution of the raw material is crystallized at a crystallization temperature of 15 - 30°C, for example, at room temperature, for example, 20 - 25°C, for example, 25°C. The crystallization time is usually 10 - 40 hours, for example, 15 - 30 hours, for example, 20 - 24 hours. Stirring can be carried out during the crystallization process, and the rotation speed is usually 10 - 60 rpm, for example, 20 - 50 rpm, for example, 30 - 45 rpm.

[0149] For example, in some embodiments, the following first crystallization conditions are adopted: the solvent is 3 times the volume of 95% (V / V) aqueous methanol solution, the crystallization temperature is 25°C, the time is 20 hours, and the stirring speed is 30 rpm.

[0150] The solid-liquid separation of the first crystallization product can be carried out, for example, by filtration (such as suction filtration) and / or centrifugation. Optionally, the obtained solid phase is dried. Among them, the obtained solid phase can be used for further crystallization purification; the liquid phase can be recycled to the raw material production.

[0151] C. Second crystallization purification

[0152] After obtaining the solid phase of the first crystallization purification product, it can be dissolved and subjected to second crystallization purification.

[0153] The solid phase of the first crystallization purification product can be dissolved using an aqueous methanol solution as the solvent. For example, an aqueous methanol solution with a concentration of 50-90% (v / v) (such as a concentration of 60-80%, for example 65%) can be used as the solvent. In some embodiments, the concentration of the second crystallization solvent is lower than that of the first crystallization solvent.

[0154] The mass-volume ratio of the solvent to the solid phase (i.e., the dry product) can be 1:1.5-5, such as the solvent volume being 1.5-3 times the weight of the dry product, for example 2-2.5 times. The methanol solution of the raw material is crystallized at a crystallization temperature of 20-35°C, such as room temperature, for example 20-25°C, for example 20°C. The crystallization time is usually 10-40 hours, for example 15-30 hours, for example 20-24 hours. Stirring can be carried out during the crystallization process, and the rotation speed is usually 10-60 rpm, for example 20-50 rpm, for example 10-30 rpm.

[0155] For example, in some embodiments, the following second crystallization conditions are adopted: the solvent is an aqueous methanol solution of 65% (V / V), the crystallization temperature is 20°C, the crystallization time is 15 hours, and the stirring speed is 15 rpm.

[0156] The solid-liquid separation of the second crystallization product can be carried out, 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 raw material production.

[0157] For example, optionally, crystal washing can be carried out after separating the solid phase of the second crystallization product. For example, an aqueous methanol solution of 60-90% (v / v) can be used as the detergent during crystal washing, and the washing is carried out at a volume ratio of crystal wet weight to detergent of 1:0.5-2, and the crystal washing time can be 10-30 min.

[0158] For example, optionally, after separating the solid phase of the second crystallization product, it can be dissolved in water (preferably pure water) such that the volume ratio of crystal wet weight to water is 1:0.5-1, and then spray-dried at 65-90°C to obtain a purified product with a RA1G content of more than 70%.

[0159] D. Further purification

[0160] After the second crystallization purification, the product of the second crystallization purification can be further purified substantially according to the method of the second crystallization purification. For example, according to needs, the third, fourth or more crystallization purification steps can be carried out.

[0161] According to needs, other purification methods can also be used to further purify the product of the second crystallization purification of the present invention, such as using preparative HPLC and other methods.

[0162] E. Recycling of the liquid phase of the purified product

[0163] The liquid phase obtained in the purification process of the present invention can be recycled for the production of rebaudioside A derivatives.

[0164] For example, the liquid phase of a certain crystallization (such as the first crystallization or the second crystallization), a mixture of the liquid phases obtained from multiple crystallizations (such as a mixture of the liquid phases of the first and second crystallizations), or a mixture of the liquid phases obtained from multiple batches of crystallizations (such as a mixture of the liquid phases obtained from multiple batches through the first and / or second crystallization steps) can be recycled.

[0165] For example, the liquid phase obtained in the purification process of the present invention can be recycled for the production of rebaudioside A derivatives by the double enzyme method.

[0166] Before recycling, pretreatment such as mixing, concentration, and drying can be carried out on the liquid phase. For example, the liquid phases of two crystallizations 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 concentrated to a solid content of 30 to 60%. For example, the liquid phase can be dried after mixing and / or concentration, such as by spray drying, etc., and the dried material can be used as an enzyme modification raw material to participate in the catalytic reaction in a recycled manner.

[0167] The product containing RA1G obtained by using the recycled liquid phase can be crystallized and purified again to obtain high-purity RA1G.

[0168] Application of Rebaudioside A1G and Related Products

[0169] The rebaudioside A1G of the present invention has various advantages such as high purity, high sweetness, good taste, green and healthy, etc., and thus can be widely used in various fields such as food, beverage, medicine, health care products, tobacco products, condiments, daily chemical products, oral hygiene products, cosmetics, etc.

[0170] Rebaudioside A1G can be provided in various forms as needed, such as in the form of dry powder, crystals, solutions, compositions, etc. For example, the rebaudioside A1G of the present invention can be made into a packaging that is convenient for storage, transportation, and use. For example, the rebaudioside A1G of the present invention can be combined with acceptable excipients or vehicles to form the composition of the present invention. The composition of the present invention contains an effective amount of rebaudioside A1G and may optionally contain acceptable excipients or vehicles such as water, food additives, food excipients, pharmaceutical excipients, etc. In one embodiment, the food additives can be selected from, but not limited to: flavors, emulsifiers, antioxidants, food colors.

[0171] The rebaudioside A1G of the present invention can be compounded with other sweeteners or flavoring agents to further improve its taste or meet the required taste requirements. For example, other sweeteners or flavoring agents used for compounding include, but are not limited to: mogroside, acesulfame potassium, aspartame, sucralose, sodium saccharin, xylitol, sorbitol, erythritol, sucrose, fructose, glucose, maltose, citric acid, malic acid, tartaric acid, lactic acid, glycine, alanine, serine.

[0172] As used herein, the term "acceptable" ingredients are substances that are suitable for humans and / or animals without or with no excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, substances with a reasonable benefit / risk ratio. As used herein, the term "effective amount" refers to an amount that can produce the desired sweetening, flavoring, and / or taste masking effect and is acceptable to humans and / or animals.

[0173] The composition of the present invention can be formulated into available dosage forms such as powders, granules, suspension emulsions, water emulsion concentrates, emulsifiable concentrates, microcapsules, etc. Those of ordinary skill in the art can select the dosage form and administration form according to the specific application needs.

[0174] The rebaudioside A1G or the rebaudioside A1G composition of the present invention can be applied in various products that require sweetening, flavoring, or taste masking. Calculated based on the weight of the product, the addition amount of rebaudioside A1G or the rebaudioside A1G composition can be, for example, 0 - 0.064% or 0% - 0.085%. In some applications, the product is liquid, and calculated based on the total volume of the product, the concentration of rebaudioside A1G or the rebaudioside A1G composition can be, for example, much lower than the usage concentration of sucrose, for example, it can be 0 - 0.56 g / L or 0 - 0.84 g / L.

[0175] Those skilled in the art can also appropriately adjust the addition amount, addition time, addition method, etc. of rebaudioside A1G or its composition according to specific needs to obtain the best effect.

[0176] Beneficial Effects

[0177] The method and product of the present invention have one or more of the following excellent effects:

[0178] (1) The method disclosed herein can be used for the purification of the novel steviol glycoside derivative rebaudioside A1G (RA1G), the structure of which has not been reported worldwide. It has the characteristic that its taste is significantly better than that of rebaudioside A, providing a highly potential method and product for the development and application of such multifunctional sweeteners as steviol glycosides;

[0179] (2) The method disclosed herein can increase the content of rebaudioside A1G, which is only 40% - 50% in the modified steviol glycoside product of rebaudioside A, to more than 70%, and then obtain a new product with the content of rebaudioside A1G reaching more than 70%. This product significantly improves the overall quality of the raw material;

[0180] (3) The liquid phase generated by crystallization in the present invention can be recycled and continuously used for production. The solvent can be distilled and recycled, achieving good economic benefits and low waste liquid discharge; moreover, the production process of the present invention has low energy consumption, simple operation, and is easy to scale up and continuous production.

[0181] Examples

[0182] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. Those skilled in the art can make appropriate modifications and changes to the present invention, and these modifications and changes are within the scope of the present invention. For example, for those skilled in the art, various changes or modifications to the purification and crystallization conditions in the embodiments or examples also belong to the protection scope of the present invention without departing from the essence and scope of the present invention.

[0183] For the experimental methods without specific conditions noted in the following examples, conventional methods in the art or the conditions recommended by suppliers can be used. Unless otherwise stated, percentages and parts are calculated 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 equivalent to the described content can be applied to the method of the present invention. The preferred implementation methods and materials described herein are only for demonstration purposes.

[0184] Example 1: Preparation and Identification of Rebaudioside A1G, the Product of Double-Enzyme Modification of Rebaudioside A

[0185] According to Figure 1 the brief process shown, the modified product of rebaudioside A is prepared by a double - enzyme method. Specifically:

[0186] Add 100 L of purified water to the reactor (Wuxi Hongqi Pressure Vessel Manufacturing Co., Ltd., 500 KG emulsifying pot, equipment code 21701098220170001). Weigh 6 Kg of Rebaudioside A (Haotian Pharmaceutical Co., Ltd., RA97) and 6 Kg of β-cyclodextrin (Qufu Tianli Pharmaceutical Excipients Co., Ltd., 170805), put them into the reactor, and heat to dissolve. Add 600 kNU of cyclodextrin glycosyltransferase (purchased from Novozymes (China) Biotechnology Co., Ltd., Toruzyme 3.0L, ACN00216, 3 kNU / mL) required for the first step of enzyme modification. Maintain the temperature of the feed liquid at 60 °C, stir at a speed of 30 rpm / min, and react for 24 h. Terminate the reaction by boiling at 100 °C. The reaction solution was detected by HPLC (the chromatogram is as shown in Figure 2A shown). 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 it is detected with a Thermo C18 4.6 mm × 250 mm (5 μm) column and an ultraviolet detector (210 nm).

[0187] Then, add 15 × 10 6 U of glucoamylase (purchased from Shaanxi Senfu Biotechnology Co., Ltd., batch number 01080011, 150000 U / g) required for the second step of enzyme modification to the above reaction solution, react at 60 °C, stir at a speed of 20 rpm / min for 2 h, and terminate the reaction by boiling at 100 °C. The derivative formation of the reaction solution was detected by HPLC, and the detection conditions are as described above. The chromatogram is as shown in Figure 2B shown.

[0188] During the above preparation process, the contents of the main components in the product are as shown in Table 1 below.

[0189] Table 1. Analysis results of products in a 100 L reaction system for the double-enzyme modification of Rebaudioside A

[0190] Component RA(%) RA1G(%) RA2G(%) RA3G(%) Product of the First Step of Enzymatic Modification 16.02 11.78 13.20 10.03 Product of the Second Step of Enzymatic Modification 37.03 47.10 4.05 2.95

[0191] Take the product of the second step of enzyme modification and separate and purify it using HPLC (DAC50 high-pressure preparative chromatography). The mobile phase is acetonitrile: aqueous solution = 29:71, the flow rate is 70 mL / min), and it is detected with a 10 μm C18 (500 g) 50 mm × 500 mm column and an ultraviolet detector (210 nm).

[0192] The purified product obtained was analyzed by mass spectrometry and nuclear magnetic resonance spectroscopy to determine its structure. For mass spectrometry, a Shimadzu high-performance liquid chromatograph coupled with an ion trap time-of-flight mass spectrometer (LCMS-IT-TOF) was used. Data were collected in the negative ion mode. The mobile phase was acetonitrile:water (68:32), the flow rate was 1 ml / min, and the resolution was 10000 full width at half maximum. For nuclear magnetic resonance detection, a Bruker DRX Avance 600 MHz spectrometer (Switzerland) was used to collect data. The detection frequency for the 1H spectrum was 600 MHz, and for the 13C spectrum was 150 MHz. The detection temperature for both was 25 °C.

[0193] The mass spectrum of the obtained product RA1G ( Figure 3A ) and nuclear magnetic resonance spectrum ( Figure 3B : 1H nuclear magnetic resonance spectrum, Figure 3C : 13C nuclear magnetic resonance spectrum) results showed that: In RA1G, a glucose group was linked to the C19 position of the glucose group of the di terpene core of rebaudioside A through an α-1,4 bond.

[0194] Example 2. First Crystallization of Enzymatically Modified Product of Rebaudioside A

[0195] 100 L of the enzyme-modified product of rebaudioside A (i.e., the reaction system after boiling to terminate the reaction in Example 1) was separated using a precision filter plate with a pore size of 5 - 10 μm (Shenyang Great Wall Filter Paper Co., Ltd., product number 1001), and then adsorbed by 100 L of macroporous resin (Lanxiao Technology New Materials Co., Ltd., LX-28; the new resin needs to be pretreated as follows: washed with 200 L of 85% ethanol solution, and then washed with purified water until the effluent had no alcohol smell, with a flow rate of 100 L / h) for 2 hours. The resin adsorbed with the sample was first washed with 300 L of pure water to wash away small molecules such as glucose mixed in the product, and then eluted with 200 L of 60% (v / v) ethanol. The eluate was collected and concentrated at -0.07 MPa and 75 °C. After concentrating to a solid content of 50%, it was dried in a spray dryer at an outlet air temperature of 75 °C to obtain 7.5 Kg of dry product. 95% (V / V) methanol aqueous solution with a volume three times that of the dry product weight was added, the temperature was controlled at 25 °C, the stirring speed was 30 rpm, and the crystallization time was 20 hours. The crystallization mixture was filtered by a Buchner funnel to obtain the solid phase and liquid phase of the first crystallization.

[0196] The obtained solid phase was dissolved in double-distilled water and analyzed by HPLC. The detection conditions were as described in Example 1. The chromatogram was as shown in Figure 4 . The content of rebaudioside A1G in the solid phase of the first crystallization product was measured to be 61.41%, with a wet weight of 7.5 Kg (Table 2). For the obtained liquid phase, HPLC detection was also carried out (detection conditions as described in Example 1) (the chromatogram was as shown in Figure 5 ), and the content of rebaudioside A1G in the liquid phase of the first crystallization product was measured to be 32.01% (Table 2).

[0197] Table 2. Analysis Results of the First Crystallization Product of the Enzyme-Modified Product of Rebaudioside A

[0198] Component RA RA1G RA2G RA3G Solid Phase of the First Crystallization 28.00 61.41 2.49 1.83 Liquid Phase of the First Crystallization 44.37 32.01 10.54 3.86

[0199] Example 3. Second Crystallization of Enzymatically Modified Product of Rebaudioside A

[0200] Take 7 Kg of the wet product of the first crystallization (i.e., the undried solid phase obtained in Example 2) and dissolve it by heating in 15 L of a 65% (V / V) methanol aqueous solution. Control the water bath temperature of the solution at 20 °C, the stirring speed at 15 rpm, and crystallize for 15 hours. Filter the crystallization mixture with a Buchner funnel to obtain the second crystallization solid phase and liquid phase.

[0201] After dissolving the obtained solid phase in ultrapure water, detect it by HPLC liquid phase (the detection conditions are as described in Example 1) (the spectrum is as Figure 6 shown), and the content of the new product rebaudioside A1G is measured to be 72.55%, with a wet weight of 5 Kg (Table 3).

[0202] Dissolve 2.5 L of purified water in the wet product of the second crystallization solid phase and spray-dry it at 75 °C to obtain 2.25 Kg of the final product. For the liquid phase obtained by filtration, also perform HPLC detection (the detection conditions are as described in Example 1) (the spectrum is as Figure 7 shown), and the content of rebaudioside A1G in the liquid phase of the second crystallization product is measured to be 46.33% (Table 3).

[0203] Table 3. Product Analysis Results of the Second Crystallization of the Enzyme-Modified Product of Rebaudioside A

[0204] Component RA RA1G RA2G RA3G Solid Phase of the Second Crystallization 15.25 72.55 3.64 0.81 Liquid Phase of the Second Crystallization 41.59 46.33 1.61 2.24

[0205] Example 4. Third Crystallization of Enzymatically Modified Product of Rebaudioside A

[0206] Take 2 Kg of the dry product of the second crystallization and dissolve it by heating in 10 L of a 65% (V / V) methanol aqueous solution. Control the temperature of the solution in the water bath at 20 °C, the stirring speed at 15 rpm, and crystallize for 10 hours. Filter the crystallization mixture with a Buchner funnel to obtain the third crystallization solid phase and liquid phase.

[0207] After dissolving the obtained solid phase in ultrapure water, detect it by HPLC liquid phase (the detection conditions are as described in Example 1) (the spectrum is as Figure 8 shown), and the content of rebaudioside A1G is measured to be 85.30%, with a wet weight of 3 Kg (Table 4). Dissolve 1.5 L of purified water in the wet product of the third crystallization solid phase and spray-dry it at 75 °C to obtain 1.5 Kg of the final product.

[0208] For the liquid phase obtained by filtration, also perform HPLC detection (the detection conditions are as described in Example 1) (the spectrum is asFigure 9 As shown in the figure, the content of rebaudioside A1G in the liquid phase of the third crystallization product was measured to be 46.83% (Table 4).

[0209] Table 4. Product analysis results of the third crystallization of the enzyme-modified product of rebaudioside A

[0210] Component RA RA1G RA2G RA3G Solid Phase of the Third Crystallization 10.25 85.30 2.33 0.52 Liquid Phase of the Third Crystallization 25.20 46.83 6.00 1.36

[0211] Example 5. Recycling of Crystallized Liquid Phase into Enzymatic Modification Reaction Process

[0212] The liquid phases obtained from each crystallization in Examples 2 to 4 were mixed and concentrated under the conditions of -0.07 MPa and 75 °C until the solid content reached 50%, then fed into a spray dryer for drying at an outlet air temperature of 75 °C, and 5.8 Kg of dry product was obtained.

[0213] The dry product was recycled into the enzyme modification reaction process: 50 g of cyclodextrin glycosyltransferase (the same source and catalog number as in Example 1), 3 Kg of β-cyclodextrin (the same source as in Example 1), and 70 L of purified water were added, and the reaction was carried out at 60 °C for 24 h, and the reaction was terminated by boiling at 100 °C. In a 60 °C water bath, 20 g of glucoamylase (the same source and catalog number as in Example 1) was added, and the reaction was carried out for 2 h, and the reaction was terminated by boiling at 100 °C.

[0214] The enzyme-modified product obtained above was taken, separated by a precision filter plate with a pore size of 5 - 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 wash away small molecules such as glucose mixed in the product, and then eluted with 200 L of 60% (v / v) ethanol. The eluate was collected and concentrated under the conditions of -0.07 MPa and 75 °C. After concentrating to a solid content of 50%, it was fed into a spray dryer for drying at an outlet air temperature of 75 °C, and 5.5 Kg of the enzyme-modified product was obtained. After the product was dissolved in ultrapure water, it was detected by HPLC liquid phase (the detection conditions were as described in Example 1), and the spectrum was as Figure 10 shown. The content of the new product rebaudioside A1G was measured to be 47.81% (Table 5).

[0215] From this result, it can be seen that using the crystallization liquid phase as the raw material for the enzyme modification reaction can also effectively produce the required RA1G product, thus saving production costs.

[0216] Table 5. Product analysis results of the crystallization liquid phase recycled into the enzyme modification reaction

[0217] Component RA RA1G RA2G RA3G Enzymatically Modified Product 37.81 47.81 3.18 4.0

[0218] Example 6: Sensory Evaluation Experiment of Rebaudioside A1G

[0219] The steviol glycoside raw materials used in the sensory evaluation experiment, among which RA1G was prepared as in Examples 2, 3, and 4, and the rest of the test products were all from Haotian Pharmaceutical Co., Ltd. Among them, the purity of rebaudioside A (RA) was 97%, and the purity of rebaudioside D (RD) was 95%.

[0220] In order to achieve the purpose of using sugar substitutes, the food industry conducts sensory evaluation and comparison of food additives at the same sweetness. The steviol glycoside raw materials were dissolved in pure water according to different ratios (Table 6) to prepare 360 - 560 ppm sample solutions. 10 mL of the sample solution was taken into a 30 mL disposable drinking cup respectively, and sensory evaluation (blind evaluation) was carried out by 8 trained and experienced sensory personnel. The evaluation result was the average value of the scores given by the sensory personnel.

[0221] In the evaluation, the sweetness was based on a 10% sucrose aqueous solution (10 g / 100 ml) by mass fraction, scored on a 10 - point scale (the sweetness of the same as 10% sucrose was 10 points, the same as 9% sucrose was 9 points, and so on), and 0 points represented that no sweetness could be detected at all.

[0222] The bitterness was scored according to the standard that very bitter was 10 points and no bitterness could be detected at all was 0 points.

[0223] The comprehensive evaluation gave 0 - 100 points according to the overall taste. 100 points represented the taste of 9% sucrose. The appearance of bitterness, astringency and other miscellaneous tastes were deduction items. Miscellaneous tastes refer to other bad tastes other than sweet, bitter and astringent tastes, such as alcohol taste, plastic taste, metallic taste, licorice taste, chemical taste and other bad tastes. When tasting, ensure that the sweetness of each sample is basically the same, and compare their other tastes except sweetness, such as bitterness, miscellaneous tastes, etc.

[0224] As can be seen from Table 6, the 600 ppm RA1G solution with a purity of 72.55% and the 560 ppm RA1G solution with a purity of 85.30% had similar comprehensive evaluations. Their sweetness was similar to that of a 7% sucrose solution, and the after - bitterness and other bad tastes were much lower than those of RA. Their taste was significantly superior to the raw material RA; the taste of the 660 ppm RA1G solution with a purity of 61.44% was worse than that of the high - purity RA1G solution, but still better than the raw material rebaudioside A.

[0225] Although the product purities of the second - crystallization and third - crystallization of RA1G were different, the difference in their sensory evaluation results was very small. Therefore, considering the production cost issue, the second - crystallization recycling process can be adopted in actual production applications to increase the content of rebaudioside A1G in the enzyme - modified product of rebaudioside A.

[0226] Table 6. Sensory evaluation results of rebaudioside A1G

[0227]

[0228] All documents mentioned in this invention are cited herein as references, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for purifying the compound rebaudioside A1G (RA1G) shown in the following structure, The method comprises: (a) Optionally, performing pre - treatment before purification on the raw material containing RA1G; (b) Using an aqueous methanol solution as a solvent, performing first crystallization on the raw material containing RA1G with or without pre - treatment and performing solid - liquid separation; (c) Taking the solid phase obtained from the previous crystallization, dissolving it with an aqueous methanol solution as a solvent, and performing second crystallization under appropriate conditions to obtain the purified product; (d) Optionally, repeating the crystallization purification one or more times on the purified product obtained from the previous crystallization or performing further purification by other purification methods; (e) Optionally, recycling the remaining liquid phase in the crystallization purification step to the preparation process of the raw material containing RA1G, wherein the content of RA1G in the raw material is 40 - 50%, and wherein the raw material containing RA1G is prepared by an enzyme - catalyzed method, and the enzyme - catalyzed method comprises: (1) Providing rebaudioside A and a glucosyl donor; (2) Catalyzing to produce rebaudioside A1G by cyclodextrin glycosyltransferase and amylase.

2. The method according to claim 1, wherein, the enzyme - catalyzed method comprises one or more conditions selected from the following groups: The rebaudioside A is one or more selected from the following groups: rebaudioside A existing in natural plants, extracted rebaudioside A, synthetic rebaudioside A; The glucosyl donor is one or more selected from the following groups: starch; dextrin; maltose; The cyclodextrin glycosyltransferase is selected from: α - cyclodextrin glycosyltransferase, β - cyclodextrin glycosyltransferase, and γ - cyclodextrin glycosyltransferase; The amylase is one or more selected from the following groups: glucoamylase, α - amylase, β - amylase; The dosage of the cyclodextrin glycosyltransferase is 0.1 - 30 kNU / L, or 5000 - 50000 U / mL; and / or the dosage of the amylase is 30 - 300 U / mL; and / or the enzyme is an immobilized enzyme; The initial concentration of rebaudioside A is 5 - 200 g / L; the initial concentration of the glucosyl donor is 10 - 800 g / L; Step (2) is carried out in an aqueous phase system; The reaction temperature of step (2) is 35 - 90 °C; and / or The reaction time of step (2) is 0.5 - 72 hours.

3. The method according to claim 2, wherein, the dextrin is selected from: maltodextrin, α - cyclodextrin, β - cyclodextrin, and γ - cyclodextrin.

4. The method according to claim 2, wherein, the starch is soluble starch.

5. The method according to claim 2, wherein, the dosage of the cyclodextrin glycosyltransferase is 0.5 - 20 kNU / L.

6. The method according to claim 2, wherein, the dosage of the cyclodextrin glycosyltransferase is 1 - 15 kNU / L.

7. The method according to claim 2, wherein, the dosage of the cyclodextrin glycosyltransferase is 10000 - 40000 U / mL.

8. The method according to claim 2, wherein, the dosage of the cyclodextrin glycosyltransferase is 15000 - 35000 U / mL.

9. The method according to claim 2, Among them, the dosage of the amylase is 50-250 U / mL.

10. The method according to claim 2, wherein, the dosage of the amylase is 80-220 U / mL.

11. The method according to claim 2, wherein, the initial concentration of rebaudioside A is 8-150 g / L.

12. The method according to claim 2, wherein, the initial concentration of rebaudioside A is 10 g-120 g / L.

13. The method according to claim 2, wherein, the initial concentration of the glucosyl donor is 20-700 g / L.

14. The method according to claim 2, wherein, the initial concentration of the glucosyl donor is 30-600 g / L.

15. The method according to claim 2, wherein, the initial concentration of the glucosyl donor is 30-300 g / L.

16. The method according to claim 2, wherein, the aqueous phase system is water.

17. The method according to claim 2, wherein, the aqueous phase system is pure water, distilled water or ultrapure water with a pH of 6.

18. The method according to claim 2, wherein, the reaction temperature of step (2) is 40-90 °C.

19. The method according to claim 2, wherein, the reaction temperature of step (2) is 45-85 °C.

20. The method according to claim 2, wherein, the reaction temperature of step (2) is 50-70 °C.

21. The method according to claim 2, wherein, the reaction time of step (2) is 1-48 hours.

22. The method according to claim 2, wherein, the reaction time of step (2) is 1.5-36 hours.

23. The method according to claim 2, wherein, the reaction time of step (2) is 5-20 hours.

24. The method according to claim 1, wherein, the pre-purification pretreatment in step (a) includes one or more treatments selected from the following group: filtration, adsorption and elution, concentration and drying.

25. The method according to claim 1, wherein, the pre-purification pretreatment in step (a) uses a method selected from the following group to remove small molecule impurities: solvent precipitation method and microfiltration membrane filtration method.

26. The method according to claim 25, wherein, the small molecule impurity is residual glucose.

27. The method according to any one of claims 24-26, wherein, the pretreatment is carried out through the following one or more treatments: (a1) Optionally, filter the raw material containing rebaudioside A1G; (a2) Optionally, adsorb the raw material containing rebaudioside A1G with macroporous resin and elute it; Wash the macroporous resin adsorbed with the raw material with water; Elute the substance adsorbed on the resin with an ethanol eluent; (a3) Optionally concentrate and / or dry the raw material, the product after filtration, and / or the product after adsorption and elution.

28. The method according to claim 27, wherein, filtering is carried out using a filter plate, filter paper or filter element.

29. The method according to claim 27, wherein, filtering is carried out using a fine filter plate.

30. The method according to claim 27, wherein, filtering is carried out using a fine filter plate with a pore size of 5-10 μm.

31. The method according to claim 27, wherein, styrene-based or acrylate macroporous adsorption resins are used for adsorption.

32. The method according to claim 31, wherein, the macroporous adsorption resin has a pore size of 6 - 15 nm and a specific surface area of 600 - 1300 m² / g.

33. The method according to claim 27, wherein, the macroporous resin adsorbed with the raw material is washed with 2 - 10 times the resin volume of water.

34. The method according to claim 27, wherein, the macroporous resin adsorbed with the raw material is washed with washing water at a flow rate of 0.5 - 5 BV / h.

35. The method according to claim 27, wherein, an ethanol eluent of 30 - 90% (v / v) is used.

36. The method according to claim 27, wherein, an ethanol eluent of ≥ 60% (v / v) is used.

37. The method according to claim 27, wherein, the volume of the ethanol eluent is ≥ 1.5 times the column bed volume.

38. The method according to claim 27, wherein, the volume of the ethanol eluent is 1.5 - 4 times the column bed volume.

39. The method according to claim 27, wherein, spray drying or vacuum drying is used for the concentration and / or drying.

40. The method according to claim 27, wherein, the ethanol eluate is concentrated under the conditions of - 0.06 - 0.09 MPa and 60 - 85 °C.

41. The method according to claim 27, wherein, spray drying is carried out at an outlet air temperature of 65 - 90 °C.

42. The method according to claim 1, wherein, the first crystallization in step (b) includes one or more treatments selected from the following group: (b1) dissolving the raw material in solid form with the solvent; (b2) carrying out the first crystallization; (b3) performing solid-liquid separation on the first crystallization product to obtain the solid phase for the next purification or used as the purified product.

43. The method according to claim 42, wherein, the first crystallization includes one or more conditions selected from the following group: using an aqueous methanol solution as the solvent to dissolve the raw material in solid form; the mass-volume ratio of the solvent to the raw material in solid form is 1:2 - 5; the crystallization temperature is 15 - 30 °C; the crystallization time is 10 - 40 hours; the stirring speed during the crystallization process is 10 - 60 rpm; solid-liquid separation of the first crystallization product is carried out by filtration and / or centrifugation; optionally, the crystals are washed, and the washing agent is an aqueous methanol solution of 60 - 90% (v / v); optionally, the obtained solid phase is dried.

44. The method according to claim 43, wherein, an aqueous methanol solution with a concentration of 80 - 99% (v / v) is used as the solvent to dissolve the raw material in solid form.

45. The method according to claim 43, wherein, an aqueous methanol solution with a concentration of ≥ 90% (v / v) is used as the solvent to dissolve the raw material in solid form.

46. The method according to claim 43, wherein, an aqueous methanol solution with a concentration of 95% (v / v) is used as the solvent to dissolve the raw material in solid form.

47. The method according to claim 43, wherein, the solvent volume is 3 - 5 times the weight of the dry product.

48. The method according to claim 43, wherein, the volume of the solvent is 3 times the weight of the dry product.

49. The method according to claim 43, wherein, the crystallization temperature is room temperature.

50. The method according to claim 43, wherein, the crystallization temperature is 20 - 25 °C.

51. The method according to claim 43, wherein, the crystallization temperature is 25 °C.

52. The method according to claim 43, wherein, the crystallization time is 15 - 30 hours.

53. The method according to claim 43, wherein, the crystallization time is 20 - 24 hours.

54. The method according to claim 43, wherein, the stirring speed during crystallization is 20 - 50 rpm.

55. The method according to claim 43, wherein, the stirring speed during crystallization is 30 - 45 rpm.

56. The method according to claim 43, wherein, the filtration is suction filtration.

57. The method according to claim 43, wherein, the volume ratio of the wet weight of the crystal to the detergent is 1∶0.5 - 2, and the crystal washing time is 10 - 30 min.

58. The method according to claim 1, wherein, the second crystallization in step (c) includes one or more treatments selected from the following group: (c1) dissolving the solid phase obtained from the first crystallization with the solvent; (c2) performing the second crystallization; (c3) performing solid-liquid separation on the second crystallization product to obtain the solid phase for the next purification or as the purified product.

59. The method according to claim 58, wherein, the second crystallization includes one or more conditions selected from the following group: dissolving the solid phase of the purified product from the first crystallization with an aqueous methanol solution as the solvent; 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 - 40 hours; the stirring speed during crystallization is 10 - 60 rpm; performing solid-liquid separation on the second crystallization product by filtration and / or centrifugation; optionally, drying the obtained solid phase.

60. The method according to claim 59, wherein, an aqueous methanol solution with a concentration of 50 - 90% (v / v) is used as the solvent.

61. The method according to claim 59, wherein, an aqueous methanol solution with a concentration of 60 - 80% (v / v) is used as the solvent.

62. The method according to claim 59, wherein, an aqueous methanol solution with a concentration of 65% (v / v) is used as the solvent.

63. The method according to claim 59, wherein, the mass-volume ratio of the solvent to the solid phase is 1:1.5 - 5.

64. The method according to claim 59, wherein, the volume of the solvent is 1.5 - 3 times the weight of the dry product.

65. The method according to claim 59, wherein, the volume of the solvent is 2 - 2.5 times the weight of the dry product.

66. The method according to claim 59, wherein, the second crystallization temperature is room temperature.

67. The method according to claim 59, wherein, the second crystallization temperature is 20 - 25 °C.

68. The method according to claim 59, wherein, the second crystallization temperature is 20 °C.

69. The method according to claim 59, wherein, the second crystallization time is 15 to 30 hours.

70. The method according to claim 59, wherein, the second crystallization time is 20 to 24 hours.

71. The method according to claim 59, wherein, the stirring speed during the crystallization process is 20 to 50 rpm.

72. The method according to claim 59, wherein, the stirring speed during the crystallization process is 10 to 30 rpm.

73. The method according to claim 59, wherein, the filtration is suction filtration.

74. The method according to claim 59, wherein, the obtained solid phase is spray-dried at an air outlet temperature of 65 to 90 °C.

75. The method according to claim 1, wherein, the further purification method in step (d) is selected from one or more of the following groups: crystallization purification, preparative HPLC purification.

76. The method according to claim 1, wherein, the recycling in step (e) includes one or more treatments selected from the following groups: recycling the liquid phase of a certain crystallization, a mixture of the liquid phases obtained from multiple crystallizations, a mixture of the liquid phases obtained from multiple batches of crystallizations; recycling the liquid phase obtained during the purification process to the double-enzyme method process according to claim 2; before recycling, performing pretreatment on the liquid phase such as mixing, concentration, and drying; using the raw material containing RA1G produced after recycling in the purification method according to claim 1.

77. The method according to claim 76, wherein, recycle the liquid phase of the first crystallization, the second crystallization, and the mixture of the liquid phases of the first and second crystallizations.

78. The method according to claim 76, wherein, recycle the mixture of the liquid phases obtained from multiple batches through the first and / or second crystallization steps.

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