Two-enzyme preparation of steviol glycoside derivative rebaudioside a1g and applications thereof

Abstract

The application relates to a double-enzyme method for preparing a steviol glycoside derivative, rebaudioside A1G, and application thereof. In the application, rebaudioside A (RA) is used as a receptor substrate, glucose is used as a donor substrate, and a double-enzyme method is used to prepare a steviol glycoside derivative with a novel structure, RA1G. The application obtains a novel steviol glycoside derivative with improved taste through a biological enzyme catalysis method, and has the advantages of simple operation, green and environment-friendly production process, low cost, short cycle, high conversion efficiency, easy purification, good taste of the obtained product, and important application value in the food and beverage industries.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology and food chemical technology. Specifically, the present application relates to a steviol glycoside derivative rebaudioside A1G prepared by a biological enzyme method, a preparation method thereof, and application. BACKGROUND

[0002] Steviol glycoside is a series of glycosides extracted from the leaves of the herbaceous plant Stevia rebaudiana Bertoni, and is a natural sweetener. The main content in the leaves of the natural plant Stevia rebaudiana Bertoni is stevioside (abbreviated as Stv) and rebaudioside A (abbreviated as RA, also known as steviolbioside A). The molecular structure of rebaudioside A is as follows, which is a tetranor-diterpene glycoside containing 20 carbon atoms, and is formed by connecting a glucosyl at C19 of a diterpene core and 3 glucosyls at C13:

[0003]

[0004] Studies have shown that the sweetness of rebaudioside A is 200-300 times that of sucrose, and the heat is only 1 / 300 of sucrose. It is stable to acid, alkali and heat, and is not easy to deteriorate during long-term storage. It will not cause browning phenomenon after being added to food and subjected to heat treatment, and is not easy to cause dental caries. In the national standards of GB8270-2014 Food Safety National Standard and GB2760-2014 Food Safety National Standard, its use as a food additive is specified in detail. In 2009, the U.S. 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 food and beverage gradually developing towards low sugar and low heat. It has a broad application prospect and is an ideal green sweetener with multiple purposes.

[0005] Although steviol glycosides have many advantages, the main component of steviol glycosides, rebaudioside A, has a serious bitter aftertaste, which hinders its wide application in the field of food and other fields. At present, the improvement of the taste of steviol glycosides can be realized by biological enzyme conversion. It has been reported that the main component of steviol glycosides can be added with glucose group by using UDP glucose as glucose group donor by glucose group transferase. However, the sugar group donor UDP glucose used in this method is expensive, and the cost is high (Biocatalysis: An Industrial Perspective, Royal Society of Chemistry, 2017, pp199). It has also been reported that steviol glycosides can be modified by using cyclodextrin glucosyltransferase (CGT), but the catalytic efficiency and transglycosylation sites of CGT enzymes from different biological sources are different, and the enzyme catalyzed products are complex and diverse (Simple and efficient enzymatic transglycosylation of stevioside by beta-cyclodextrin glucanotransferase from Bacillus firmus. Biotechnol Lett. 2009 Sep; 31 (9): 1415-1420). In addition, the single enzyme catalyzed product of CGT is a mixture of glycosylated derivatives of different degrees, which causes the non-uniformity of the taste, and the molecular structure and polarity of these derivatives are very similar, which is difficult to separate and purify, and it is difficult to obtain high-purity single effective product.

[0006] Therefore, there is an urgent need in the art to develop a rebaudioside A derivative product with improved taste and a method for efficient production thereof. SUMMARY

[0007] In order to solve the above problems, the present application provides a method for producing a new steviol glycoside derivative by double enzyme catalysis and the product thereof, so as to improve the taste of the raw material, and to produce high-quality biological enzyme prepared steviol glycoside derivatives with low cost and short production cycle.

[0008] In a first aspect of the present application, a compound rebaudioside A1G is provided, as shown in the following structure:

[0009]

[0010] In another aspect of the present application, a method for preparing rebaudioside A1G is provided, wherein the structural formula of the rebaudioside A1G is shown above, and the method comprises the steps of:

[0011] (1) providing rebaudioside A and glucose group donor;

[0012] (2) producing Rebaudioside AlG having the above structure by catalysis of a cyclodextrin glycosyltransferase and an amylase.

[0013] In some embodiments, the Rebaudioside A is one or more selected from the group consisting of Rebaudioside A present in a natural plant, extracted Rebaudioside A, and synthetic Rebaudioside A.

[0014] In some embodiments, the glucosyl donor is one or more selected from the group consisting of starch, such as soluble starch; dextrin; maltodextrin; a-cyclodextrin, β- cyclodextrin, γ-cyclodextrin; and maltose.

[0015] In some embodiments, the cyclodextrin glycosyltransferase is selected from the group consisting of a-cyclodextrin glycosyltransferase, β-cyclodextrin glycosyltransferase, and γ- cyclodextrin glycosyltransferase.

[0016] In some embodiments, the amylase is one or more selected from the group consisting of saccharifying enzyme, a-amylase, β-amylase, and γ-amylase.

[0017] In some embodiments, the glycosyltransferase is used in an amount of 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. In some embodiments, the glycosyltransferase is used in an amount of 5-200 kNU / kg Rebaudioside A, such as 10-150 kNU / kg.

[0018] In some embodiments, the amylase is used in an amount of 30-300 U / mL, such as 50-250 U / mL, 80-220 U / mL. In some embodiments, the amylase is used in an amount of 300-3000 U / g Rebaudioside A, such as 800-2200 U / g Rebaudioside A.

[0019] In some embodiments, one or more of the enzymes used is an immobilized enzyme.

[0020] In some embodiments, the starting concentration of Rebaudioside A is 5-200 g / L, such as 8-150 g / L, 10-120 g / L.

[0021] In some embodiments, the starting concentration of the glucosyl donor is 10-800 g / L, such as 20-700 g / L, 30-600 g / L, 30-300 g / L.

[0022] In some embodiments, step (2) is performed in an aqueous phase system, such as in water (e.g., pure water, distilled water, ultrapure water, pH 6).

[0023] 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.

[0024] In some embodiments, the reaction time of step (2) is 0.5-72 hours, such as 1-48 hours, 1.5-36 hours, 5-20 hours.

[0025] In some embodiments, the method further comprises one or more steps selected from the group consisting of: terminating the enzymatic reaction after the enzymatic reaction is completed, for example, by boiling (e.g., boiling at 100°C for 5 minutes) to denature the enzyme; separating the catalytic reaction product of the glycosyltransferase for use in the amylase-catalyzed reaction; directly taking the reaction solution after the catalytic reaction of the glycosyltransferase for use in the amylase-catalyzed reaction; supplementing the rebaudioside A, the glucosyl donor, the glycosyltransferase, and / or the amylase consumed in the production; isolating, purifying, salifying, optically resolving, identifying, and / or packaging the rebaudioside A obtained in step (2); and / or, recycling the unused rebaudioside A, the glucosyl donor, the glycosyltransferase, and / or the amylase to the next round of reaction.

[0026] In another aspect of the present application, there is provided a composition comprising: (i) the rebaudioside A1G of the present application, and / or, the rebaudioside A1G obtained by the method of the present application; and (ii) a pharmaceutically, a food scientifically, a nutraceutically, or a daily chemical acceptable carrier, excipient, and / or adjuvant; (iii) optionally, other sweeteners or flavoring agents, such as mogrosides, acesulfame, aspartame, sucralose, sodium saccharin, xylitol, sorbitol, erythritol, sucrose, fructose, glucose, maltose, citric acid, malic acid, tartaric acid, lactic acid, glycine, alanine, serine.

[0027] In another aspect of the present application, there is provided the use of the rebaudioside A1G of the present application, or, the rebaudioside A1G obtained by the method of the present application, or, the composition of the present application, as a sweetener, a flavoring agent, and / or a taste masking agent, for example, in the preparation of food, beverage, tobacco product, condiment, daily chemical product, pharmaceutical component, nutraceutical product, oral hygiene product, and / or cosmetic product.

[0028] In another aspect of the present application, there is provided a product comprising the rebaudioside A1G of the present application, and / or, the rebaudioside A1G obtained by the method of the present application, and / or, the composition of the present application.

[0029] In some embodiments, the product is selected from the group consisting of food, beverage, tobacco product, condiment, daily chemical product, pharmaceutical component, nutraceutical product, oral hygiene product, and / or cosmetic product.

[0030] In another aspect of the present application, there is provided a package comprising: the rebaudioside A1G of the present application, and / or the rebaudioside A1G obtained by the method of the present application; and a package and / or container.

[0031] In some embodiments, the package and / or container can be selected from the group consisting of: flexible packages or containers, such as bags (e.g. paper bags, plastic bags, preferably sealed bags) and bottles (e.g. plastic bottles); rigid packages or containers, such as glass containers, metal containers, ceramic containers, and the like.

[0032] Those skilled in the art can make any combinations of the technical solutions and technical features described above without departing from the inventive concept and protection scope of the present application. Other aspects of the present application are obvious to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF DRAWINGS

[0033] The present application will be further described with reference to the drawings, which are presented herein for illustration purposes only and are not intended to limit the scope of the present application.

[0034] Figure 1 : High performance liquid chromatogram of the rebaudioside A1G product (a) and the purified rebaudioside A1G (b) catalyzed by the double-enzyme method.

[0035] Figure 2 : Exemplary flow diagram of the double-enzyme method for catalyzing the production of rebaudioside A1G (using soluble starch as the glucose donor, and CGT and glucoamylase for catalysis as an example).

[0036] Figure 3 : High resolution mass spectrum of rebaudioside A1G.

[0037] Figure 4 : Nuclear magnetic resonance hydrogen spectrum of acetylated rebaudioside A1G.

[0038] Figure 5 : Nuclear magnetic resonance carbon spectrum of acetylated rebaudioside A1G.

[0039] Figure 6 : Nuclear magnetic resonance two-dimensional COSY spectrum of acetylated rebaudioside A1G.

[0040] Figure 7 : Nuclear magnetic resonance two-dimensional HSQC spectrum of acetylated rebaudioside A1G.

[0041] Figure 8 : Nuclear magnetic resonance two-dimensional HMBC spectrum of acetylated rebaudioside A1G. DETAILED DESCRIPTION

[0042] The present application provides a novel rebaudioside A derivative, which is named rebaudioside A1G (i.e. RA1G) because a glucose group is connected to the glucose group at C19 of the diterpene core of rebaudioside A through an α-1,4 bond. The present application also provides a preparation method of the derivative, which is prepared from rebaudioside A through two-step enzyme catalysis. Compared with the raw material rebaudioside A, the rebaudioside A1G of the present application has improved sweetness and taste. Moreover, the rebaudioside A1G of the present application has the characteristics of low production cost, short production cycle, and green production process.

[0043] As used herein, the term "two-enzyme method" refers to a method of using two enzymes to catalyze a reaction to obtain rebaudioside A1G, using rebaudioside A and a glucosyl donor as raw materials. The specific description of the reaction raw materials, enzymes, reaction conditions, and reaction products used in the two-enzyme method can be found below.

[0044] As used herein, "containing", "having", or "including" includes "comprising", "consisting essentially of", "consisting essentially of", and "consisting of"; "consisting essentially of", "consisting essentially of", and "consisting of" are sub-concepts of "containing", "having", or "including".

[0045] All numerical ranges provided herein are intended to clearly include all numbers and ranges between the endpoints. The features or embodiments mentioned in the present application can be combined. All features disclosed in the specification can be used in any combination, and each feature disclosed in the specification can be replaced by 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.

[0046] Reaction substrate

[0047] The main reaction raw materials used in the two-enzyme method for producing rebaudioside A1G in the present application are rebaudioside A and a glucosyl donor.

[0048] The rebaudioside A raw material used in the two-enzyme method for producing rebaudioside A1G can be rebaudioside A from various sources. The rebaudioside A raw material that can be used includes but is not limited to: rebaudioside A extracted from natural plants and directly used in the method of the present application, such as using stevia leaves as raw material, through processes such as extraction, impurity removal, decolorization, and drying; commercially available rebaudioside A; synthetic rebaudioside A, such as synthesized through microbial fermentation (such as recombinant Pichia pastoris, recombinant Saccharomyces cerevisiae, and recombinant Escherichia coli). The rebaudioside A in the form of powder, crystal, solution, etc. can be used in the reaction system of the present application.

[0049] The glucosyl donor for the production of Rebaudioside A1G by the two-enzyme method can be any polysaccharide and / or oligosaccharide that can be used as a substrate of a glycosyltransferase (such as a cyclodextrin glycosyltransferase, CGT) to transfer the sugar molecules contained therein to an acceptor by an enzymatic reaction. The glucosyl donor that can be used in the present application includes, but is not limited to, starch (preferably soluble starch), β-cyclodextrin, α-cyclodextrin or γ-cyclodextrin, maltodextrin, and maltose.

[0050] Enzyme

[0051] In the present application, the enzymes used for the production of Rebaudioside A1G are of two types, i.e., enzymes catalyzing transglycosylation and hydrolysis reactions, preferably glycosyltransferase (such as cyclodextrin glycosyltransferase) and amylase.

[0052] Glycosyltransferase is a class of enzymes that catalyze the attachment of activated sugars to different acceptor molecules (such as Rebaudioside A in the present application). Cyclodextrin glycosyltransferase (CGTase) is a multifunctional enzyme that can catalyze different reactions, which can transfer the sugar group of a glucosyl donor to an acceptor (in the present application, Rebaudioside A). For the properties, preparation and application of cyclodextrin glycosyltransferase, please refer to Wu Jing et al., Preparation and Application of Cyclodextrin Glycosyltransferase (2011, Chemical Industry Press). Cyclodextrin glycosyltransferase (CGT) can be enzymes of various origins, such as commercially available CGTase, genetically engineered CGTase, etc.

[0053] Amylase is a class of enzymes that hydrolyze starch and glycogen. The amylases that can be used in the method of the present application 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 starch from the non-reducing end to produce glucose by hydrolyzing α-1,4 and α-1,6 glucosidic bonds, and the enzyme can also hydrolyze the non-reducing end of dextrin and glycogen to release β-D-glucose.

[0054] The two enzymes used in the present application can be commercially available, such as from Tianno Enzyme Preparation Co., Ltd., Novozymes (China) Biotechnology Co., Ltd., and Jiangxi Baiying Biotechnology Co., Ltd., etc.; or can be obtained by microbial fermentation, etc., as long as they have the required catalytic activity. Preferably, enzyme preparations with high enzyme activity and high stability are used, and immobilized enzymes can also be used.

[0055] Enzymatic reaction

[0056] The enzymatic reaction of the present application is carried out in an aqueous phase system, with rebaudioside A as the acceptor substrate, soluble starch, β-cyclodextrin or a-cyclodextrin, maltose, etc. as the glucose donor substrate, and under the catalysis of cyclodextrin glycosyltransferase (CGT) to carry out transglycosylation reaction to generate a mixed system of rebaudioside A derivatives, and then through the catalysis of amylase (such as saccharifying enzyme) to hydrolyze each component in the mixed system to finally obtain a novel rebaudioside A derivative with higher uniformity, i.e. rebaudioside AlG.

[0057] The two raw materials of the present application can be dissolved in water (such as pure water, distilled water, ultrapure water, etc.) to form an aqueous phase system. The initial concentration of rebaudioside A raw material in the reaction system can be 5-200 g / L, such as 8-150 g / L, 10-120 g / L. The initial concentration of glucose donor substrate raw material in the reaction system can be 10-800 g / L, such as 20-700 g / L, 30-600 g / L, 30-300 g / L. Generally, the ratio of rebaudioside A raw material to glucose donor raw material can be 1:2-4 by weight.

[0058] Cyclodextrin glycosyltransferase (CGT) is added to the aqueous phase system 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 rebaudioside A, such as 10-150 kNU / kg rebaudioside A. According to the generation of reaction products, the reaction temperature of CGT enzyme can be set in the range of 35-90°C, such as 40-90°C, 45-85°C, 50-70°C, which can be adjusted according to the specific enzyme used and industrial cost, etc. The pH of the CGT enzyme reaction system can be set around the optimum pH of the enzyme, such as pH 4-7, pH 4.5-6.5, pH 5-6, which can be adjusted according to the specific enzyme used. The time of CGT enzyme reaction can be adjusted according to the reaction progress, such as 0.5-72 hours, 1-48 hours, 1.5-36 hours, 5-20 hours.

[0059] After the GCT enzyme reaction is completed, the enzyme reaction can be terminated by various ways (such as a relatively simple way is to denature the enzyme by boiling (such as boiling at 100°C for 5 minutes) to terminate the reaction). Alternatively, the obtained reaction product is centrifuged and the supernatant is separated for the next step reaction. The obtained reaction product can also be directly used for the next step reaction without separation and purification.

[0060] A starch enzyme (e.g., amylase) is added to the GCT enzyme reaction product to catalytically break down the components in the rebaudioside A derivative mixture to rebaudioside AlG, which has a glucose group attached to the C19 position of the rebaudioside A diterpene core via an alpha-1,4 linkage.

[0061] In some embodiments, the amylase is used at a concentration of 30-300 U / mL, e.g., 50-250 U / mL, 80-220 U / mL. In some embodiments, the amylase is used at a concentration of 300-3000 U / g rebaudioside A, e.g., 800-2200 U / g rebaudioside A.

[0062] The temperature of the amylase reaction can be set to a range of 35-90 °C, e.g., 40-90 °C, 45-85 °C, 50-70 °C, depending on the specific enzyme used and other factors such as cost. The pH of the amylase reaction can be set to the optimal pH of the enzyme, e.g., pH 4-7, pH 4.5-7, pH 5-7, depending on the specific enzyme used. The time of the amylase reaction can be adjusted based on the progress of the reaction, e.g., 0.5-72 hours, 1-48 hours, 1.5-36 hours, 2-10 hours.

[0063] The amylase reaction can be stopped by various means (e.g., by denaturing the enzyme by boiling (e.g., 100 °C for 5 minutes) to stop the reaction). The resulting reaction product can be further separated, dried, purified, characterized, etc. to obtain the desired rebaudioside AlG.

[0064] For example, the reaction supernatant and precipitate can be separated by centrifugation, e.g., 12000 rpm for 5 minutes. For example, the reaction product can be separated by chromatography, e.g., HPLC. An example HPLC instrument and conditions are: Agilent 1200 HPLC system Phenomenex Luna 5 μιη C18(2) 4.6 mm x 250 mm column, mobile phase of acetonitrile-sodium dihydrogen phosphate aqueous solution (pH 2.6). According to the present application, the further separation can be performed at a single sample loading of 5 μΐ^, a flow rate of 1.0 mL / min, a mobile phase of acetonitrile: sodium dihydrogen phosphate aqueous solution (pH 2.6) at a volume ratio of 68:32, and a UV detection wavelength of 210 nm. The resulting product can be dried, e.g., by lyophilization. The resulting product can be further purified, e.g., by crystallization.

[0065] It will also be understood by one of ordinary skill in the art that the above reactions can be performed using an immobilized enzyme system, e.g., immobilized CGTase and / or immobilized amylase.

[0066] The product obtained by the double-enzyme method of the present application can be characterized by high-resolution mass spectrometry, nuclear magnetic resonance, etc. to determine that the glucose at the C19 position of the diterpene core of rebaudioside A is connected by an a-1, 4 bond, i.e., the obtained product is rebaudioside A1G.

[0067] In addition, the unspent rebaudioside A, glucosyl donor, glycosyltransferase and / or amylase in the reaction can be recycled to the next round of reaction to save costs and improve yield.

[0068] Exemplary preparation method

[0069] An exemplary method of the present application is described as follows, and it should be understood that the exemplary method is only used to illustrate the present application and is not used to limit the scope of the present application. Those skilled in the art can make appropriate modifications and changes to the present application, and these modifications and changes are within the scope of the present application.

[0070] An exemplary method of the present application provides a method for preparing a steviol glycoside derivative rebaudioside A1G from rebaudioside A by a biological enzyme method, comprising the following steps:

[0071] (1) In an aqueous phase system, rebaudioside A is used as an acceptor substrate with an initial reaction concentration of 10 g to 120 g / L, soluble starch, β-cyclodextrin or a-cyclodextrin, or maltose is used as a glucose donor substrate with an initial reaction concentration of 30 g to 300 g / L, and a transglycosylation reaction is carried out under the catalysis of a-cyclodextrin glycosyltransferase (CGT) to generate a series of rebaudioside A derivatives;

[0072] (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, centrifugation is performed, and the supernatant is taken;

[0073] (3) The supernatant obtained in step (2) is added with saccharifying enzyme, and all the components in the mixed system in step (2) are used as substrates to carry out a hydrolysis reaction;

[0074] (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, centrifugation is performed, and the supernatant is taken;

[0075] (5) The supernatant prepared in step (4) is separated and dried to prepare a rebaudioside A derivative rebaudioside A1G.

[0076] In an example, the aqueous phase system in step (1) is distilled pure water with a pH of 6.0.

[0077] In one example, the alpha-cyclodextrin glycosyltransferase in step (1) can be purchased from Jiangxi Baiyin Biotechnology Co., Ltd., Novozymes (China) Biotechnology Co., Ltd. and Japan Amo Enzyme Products Co., Ltd., with a final concentration of 0.05-2 g / L.

[0078] In one example, the glucose donor substrate in step (1) is soluble starch, dextrin, maltose

[0079] In one example, the reaction condition in step (2) is a 60°C water bath, and the reaction time is 15 hours.

[0080] In one example, the boiling termination reaction condition in step (2) is boiling at 100°C for 5 minutes.

[0081] In one example, the centrifugal speed in step (2) is 12000 rpm for 5 minutes.

[0082] In one example, the glucoamylase in step (3) can be purchased from Shaanxi Senfer Natural Products Co., Ltd. and Shanghai Yuan Ye Biotechnology Co., Ltd., with a final concentration of 0.5-20 g / L

[0083] In one example, the reaction condition in step (4) is a 60°C water bath, and the reaction time is 3 hours

[0084] In one example, the boiling termination reaction condition in step (4) is boiling at 100°C for 5 minutes.

[0085] In one example, the centrifugal speed in step (4) is 12000 rpm for 5 minutes.

[0086] In one example, the separation in step (5) uses an Agilent 1200 HPLC system Phenomenex Luna 5 μm C18(2) 4.6 mm x 250 mm column, with a mobile phase of acetonitrile-sodium dihydrogen phosphate aqueous solution (pH 2.6).

[0087] According to the present application, in a further example, the further separation described above has a single sample loading amount of 5 μL, a flow rate of 1.0 mL / min, a mobile phase of acetonitrile: sodium dihydrogen phosphate aqueous solution (pH 2.6) at a volume ratio of 68:32, and a UV detection wavelength of 210 nm.

[0088] In one example, the drying in step (5) is freeze-drying.

[0089] Application of rebaudioside A1G and related products

[0090] The rebaudioside A1G of the present application has high sweetness, good taste, green and healthy, and many other advantages, and 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 and the like.

[0091] The rebaudioside A1G of the present application can be provided in various forms as needed, such as dry powder, crystal, solution, composition and the like. For example, the rebaudioside A1G of the present application can be made into a package for easy storage, transportation and use. For example, the rebaudioside A1G of the present application can be combined with acceptable adjuvants or excipients to form a composition of the present application. The composition of the present application comprises an effective amount of rebaudioside A1G, and can optionally comprise water, food additives, food adjuvants, pharmaceutical adjuvants and the like acceptable adjuvants or excipients. In one embodiment, the food additives can be selected from, but not limited to, essence and flavor, emulsifier, antioxidant, food colorant.

[0092] The rebaudioside A1G of the present application can be compounded with other sweeteners or flavoring agents to further improve its taste or achieve the required taste requirements, for example, other sweeteners or flavoring agents used for compounding include, but are 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.

[0093] As used herein, the term "acceptable" ingredient is a material that is suitable for use with humans and / or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio, i.e., that are acceptable at the dosage and concentration employed. As used herein, the term "effective amount" means an amount that is effective to produce the desired sweetening, flavoring and / or taste-masking effect and is acceptable to humans and / or animals.

[0094] The composition of the present application can be formulated into powder, granule, suspoemulsion, emulsion, emulsifiable concentrate, microcapsule and other useful dosage forms. The dosage form and administration form thereof can be selected by those of ordinary skill in the art according to the needs of specific applications.

[0095] The rebaudioside A1G or rebaudioside A1G composition of the present application can be applied in various products requiring sweetness or flavoring or taste masking. The rebaudioside A1G or rebaudioside A1G composition can be added in an amount of, for example, 0-0.064% or 0-0.085% by weight of the product. In some applications, the product is liquid, and the concentration of the rebaudioside A1G or rebaudioside A1G composition can be, for example, much lower than the concentration of sucrose used, for example, 0-0.56 g / L or 0-0.84 g / L based on the total volume of the product.

[0096] The skilled in the art can also adjust the adding amount, adding time or adding method of rebaudioside A1G or its composition according to specific needs to obtain the best effect.

[0097] Beneficial effects

[0098] The method and product of the present application have one or more of the following excellent effects:

[0099] (1) The present application adopts a double enzyme system of cyclodextrin glycosyltransferase-glucoamylase to prepare a novel steviol glycoside derivative rebaudioside A1G (RA1G). The structure of the derivative has not been reported worldwide, and the derivative has the characteristics of obviously better taste than rebaudioside A, which provides a product with great potential for the development and application of steviol glycoside, a multifunctional sweetener.

[0100] (2) The double enzyme system of the present application, preferably an industrial / commercial enzyme preparation, has low cost and stable quality. The content of the target product can reach more than 50% at the end of the double enzyme reaction, and the intermediate purification step can be omitted in the two-step reaction. The derivative product of rebaudioside A can be obtained by separating and purifying the final reaction liquid. The preparation process is simple, the conversion efficiency is high, and the production cost is low, the cycle is short, and it is easy to industrialize.

[0101] (3) The present application uses rebaudioside A, the main component of natural steviol glycoside, as a glycosyl acceptor substrate, and edible starch, dextrin or maltose as a glycosyl donor substrate to prepare a rebaudioside A derivative product by using a biological enzyme method. The production process is green and safe, which greatly improves the competitiveness of the product. The product obtained by the present application has important application value in the food, beverage and other industries.

[0102] Examples

[0103] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. Those skilled in the art can make appropriate modifications and changes to the present application, and these modifications and changes are within the scope of the present application.

[0104] The experimental methods in the following examples without specific conditions can use conventional methods in the art or according to the conditions suggested by the suppliers. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as understood by those skilled in the art. In addition, any method and material similar or equivalent to those described can be applied to the method of the present application. The preferred implementation methods and materials described herein are only for demonstration.

[0105] Example 1: Preparation of steviol glycoside derivatives using rebaudioside A and soluble starch

[0106] Dissolve 2 g of rebaudioside A (purchased from Haotian Pharmaceutical Co., Ltd., RA97) and 2 g of soluble starch (purchased from Tianjin Kermel Chemical Reagent Co., Ltd., 03010803) in single distilled water, add 1 kNU of cyclodextrin glycosyltransferase (purchased from Novozymes (China) Biotechnology Co., Ltd., Toruzyme 3.0L, ACN00216, 3 kNU / mL), and prepare a reaction system of 40 mL with pH 6.0. Start the reaction by placing the reaction system in a 60°C constant temperature shaker, oscillate at 150 rpm for 15 h, and terminate the reaction by boiling at 100°C. Centrifuge at 12000 g for 5 min, take the supernatant as a sample, and use HPLC (Agilent 1200 HPLC, mobile phase: acetonitrile-sodium dihydrogen phosphate aqueous solution (pH 2.6), flow rate: 1.0 mL / min) to detect the generation of derivatives by using a Phenomenex Luna 5 μm C18(2) 4.6 mm x 250 mm column and a UV detector (210 nm).

[0107] Example 2: Preparation of high-content novel rebaudioside A derivatives using glycosylase to catalyze steviol glycoside derivatives

[0108] Take 20 mL of the supernatant after the reaction in Example 1, add 4000 U of accurately weighed saccharifying enzyme (purchased from Shaanxi Senfor Biotechnology Co., Ltd., batch number 01080011, 150000 U / g), start the reaction in a 60°C constant temperature shaker, oscillate at 150 rpm for 3 h, terminate the reaction by boiling at 100°C. Centrifuge at 12000 g for 5 min, take the supernatant as a sample, and use HPLC (DAC50 high-pressure preparative chromatography, mobile phase: acetonitrile: water solution = 29:71, flow rate: 70 mL / min) to detect the generation of derivatives by using a 10 μm C18(500 g) 50 mm x 500 mm column and a UV detector (210 nm), and the results are shown in Figure 1 The retention time of RA1G is about 6.0 min. Take the double-enzyme catalytic reaction product, separate it by using the above HPLC conditions, and collect the components in the time range of 5.85-6.25 min, and freeze-dry to obtain purified RA1G. Detect the purity of the purified RA1G by HPLC (conditions are shown above), and the purity of the purified RA1G is 95%.

[0109] Example 3: Preparation of rebaudioside A1G by double-enzyme method using rebaudioside A and β-cyclodextrin as raw materials

[0110] A 100 L reactor (Wuxi Hongqi Pressure Vessel Manufacturing Co., Ltd., 500 KG emulsifying pot, equipment code 21701098220170001) was charged with 100 L purified water. 6 Kg of rebaudioside A (Haotian Pharmaceutical Co., Ltd., RA97) and 6 Kg of β-cyclodextrin (Qufu Tianli Pharmaceutical Auxiliary Material Co., Ltd., 170805) were weighed and added to the reactor and heated to dissolve. 600 kNU of cyclodextrin glycosyltransferase (purchased from Novozymes (China) Biotechnology Co., Ltd., Toruzyme 3.0 L, ACN00216, 3 kNU / mL) required for the first step of enzyme modification was added. The temperature of the material liquid was maintained at 60°C, the stirring speed was 30 rpm / min, and the reaction was carried out for 24 h. The reaction was terminated by boiling at 100°C. The reaction liquid was detected by HPLC. The specific detection conditions were as follows: the HPLC used was Thermo U3000, the mobile phase was acetonitrile-sodium dihydrogen phosphate aqueous solution (pH 2.6), the flow rate was 1.0 mL / min, and Thermo C18 4.6 mm x 250 mm (5 um) column and ultraviolet detector detection (210 nm) were used.

[0111] Then, 15 x 10 6 U of saccharifying enzyme (purchased from Shaanxi Senfe Biotechnology Co., Ltd., batch number 01080011, 150000 U / g) required for the second step of enzyme modification was added to the above reaction liquid, 60°C, stirring speed 20 rpm / min, reaction 2 h, 100°C boiling to terminate the reaction. The reaction liquid was detected by HPLC to detect the generation of derivatives, and the detection conditions were as described above.

[0112] The contents of the main components in the product in the above preparation process are shown in Table 1. In the table, RA2G and RA3G represent rebaudioside A derivatives with 2 or 3 glucose molecules attached to rebaudioside A, respectively.

[0113] Table 1. Product analysis results in 100 L reaction system of rebaudioside A double enzyme modification

[0114]

[0115]

[0116] The product of the second step of enzyme modification was separated and purified by HPLC, and the conditions were as in Example 2.

[0117] Example 4: Structural identification of novel rebaudioside A derivatives

[0118] The structure of purified rebaudioside A derivative RA1G obtained in Example 2 and Example 3 was analyzed by mass spectrometry and nuclear magnetic resonance techniques. Mass spectrometry used a Shimadzu LCMS-IT-TOF coupled with high performance liquid chromatography, data was collected in negative ion mode, mobile phase was acetonitrile: water (68:32), flow rate was 1 ml / min, resolution was 10000 full width at half maximum. Nuclear magnetic resonance used a Bruker DRX Avance 600MHz spectrometer (Switzerland) to collect data, 1 H spectrum detected at a frequency of 600MHz, 13 C spectrum was 150MHz, both detection temperatures were 25°C.

[0119] The mass to charge ratio of this derivative was 1127.4595 which is rebaudioside A plus one glucose (see Figure 3 ). This derivative was fully acetylated and based on hydrogen, carbon and two-dimensional nuclear magnetic resonance spectral analysis (see Figures 4-8 spectra), it was determined that this derivative was a glucose group attached to the C19 position of the diterpene core of rebaudioside A through an alpha-1,4 linkage, so it was named rebaudioside A1G.

[0120] Example 5: Study on the addition amount of the first-step transglycosylation reaction substrate in the double-enzyme method

[0121] According to the mass ratio 1:1, 10-100 mg of rebaudioside A and soluble starch (from the same source as Example 1) or alpha-cyclodextrin (Shanghai Shenguo Bioengineering Co., Ltd., item number 10016-20-3) or beta-cyclodextrin (Qufu Tianli Pharmaceutical Auxiliary Co., Ltd., 170805) were weighed respectively and dissolved in single distilled pure water, 0.025 kNU of cyclodextrin glycosyltransferase (same as Example 1) was added to configure 1 mL of reaction system, and the experiment was carried out. Each group of experimental systems was placed in a 60°C constant temperature shaker to start the reaction, oscillated at 150 rpm for 10h, and the reaction was terminated by boiling at 100°C. Centrifuged at 12000g for 5min, and the supernatant was taken as the sample, and HPLC was used to detect the generation of derivatives with Phenomenex Luna 5μm C18(2)4.6mm×250mm column and ultraviolet detector. The conversion rate of rebaudioside A was calculated using the following formula:

[0122] Rebaudioside A conversion rate (%) = (initial concentration of RA - RA concentration at the end of the reaction) / initial concentration of RA x 100%

[0123] The results show that under all test conditions, the conversion rate of rebaudioside A reaches more than 60%, among which 82.5mg of beta-dextrin donor and 82.5mg of rebaudioside A obtained the highest conversion rate (80%).

[0124] Example 6: Study on enzyme variety selection and enzyme amount

[0125] The substrate combination with higher conversion rate in Example 5 (82.5 mg of β-dextrin donor + 82.5 mg of rebaudioside A) was selected, and different enzyme concentrations of cyclodextrin glycosyltransferase purchased from different companies were added to prepare reaction systems, and the final enzyme concentrations were 10,000-50,000 U / mL of Jiangxi Baiying Biotechnology Co., Ltd. (batch number 15112514, 400,000 U / mL) and Japan Amano Enzyme Products Co., Ltd. (CGTA manno, 300,000 U / mL) 10000-50000 U / mL, and 1-20 kNU / L of Novozymes (China) Biotechnology Co., Ltd. enzyme (Toruzyme 3.0L, ACN00216, 3kNU / mL).

[0126] Each group of experimental systems was started at 60°C constant temperature shaker, 150 rpm oscillation for 10h, 100°C boiling to terminate the reaction. 12000g centrifugation for 5min, take the supernatant as sample, and use HPLC with Phenomenex Luna 5μm C18(2)4.6mm×250mm column and ultraviolet detector to detect the generation of derivatives. As before, the conversion rate of rebaudioside A was calculated, and the results showed that the conversion rate of rebaudioside A reached more than 70% under all test conditions using Novozymes and Japan Amano enzymes, and a higher conversion rate of 87% was obtained when 7.5kNU / L of Novozymes Toruzyme 3.0L cyclodextrin glycosyltransferase was used.

[0127] According to the highest rebaudioside A conversion rate of the enzyme (i.e. 7.5kNU / L of Novozymes Toruzyme 3.0L cyclodextrin glycosyltransferase, conversion rate of 87%), 1mL of supernatant sample was taken, and different companies' glucoamylases were added: Shaanxi Senfer Natural Products Co., Ltd. (item number 01080011, enzyme activity 150,000 U / g), or Shanghai Yuan Ye Biotechnology Co., Ltd. (item number 9032-08-0, enzyme activity 10,000 U / ml), or Shanghai Yuan Ye Biotechnology Co., Ltd. alpha-amylase (item number 9000-90-2, enzyme activity 12,000 U / g) or beta-amylase (item number 9000-91-3, enzyme activity 50,000 U / g), and the enzyme amount was 100-200 U.

[0128] Each group of experimental systems was started at 60°C constant temperature shaker, 150 rpm oscillation for 5h, 100°C boiling to terminate the reaction. 12000g centrifugation for 5min, take the supernatant as sample, and use HPLC with Phenomenex Luna 5μm C18(2)4.6mm×250mm column and ultraviolet detector (210nm) to detect the generation of derivatives.

[0129] Rebaddi glycoside A1G yield (%) = RA1G concentration at reaction termination / initial RA concentration × 100%.

[0130] The results showed that the conversion of a mixture of rebaudioside A glycosylated derivatives to a single derivative rebaudioside A1G could be achieved using saccharifying enzymes, α-amylases, or β-amylases. The yield of rebaudioside A1G ranged from 10% to 53%, with the yield using saccharifying enzymes ranging from 35% to 53%. The highest yield of 53% was achieved using 180U of saccharifying enzyme from Shaanxi Senfu Natural Products Co., Ltd.

[0131] The amount of saccharifying enzyme that yielded the highest yield of rebaudioside A1G was selected at 180 U / mL for further optimization.

[0132] Example 7: Study on reaction time and reaction temperature

[0133] The transglycosylation reaction system from the first step of Example 6 was used, and the catalytic reaction was carried out at 50–80°C for 1–6 hours. Samples were taken at intervals, and after boiling to terminate the reaction, the samples were centrifuged and stored on ice. The supernatant samples were analyzed by HPLC. The results showed that the conversion rate of rebaudioside A reached over 60% under all test conditions, with the highest conversion rate (87.5%) observed at 70°C for 5 hours.

[0134] The saccharifying enzyme reaction system from the second step of Example 6 was used, and the catalytic reaction was carried out at 50–80°C for 1–6 hours. Samples were taken hourly, and after boiling to terminate the reaction, the samples were centrifuged and stored on ice. The supernatant samples were analyzed by HPLC. The results showed that the yield of RA1G was above 30% under all test conditions, with the highest yield (53%) observed at 60°C for 3 hours.

[0135] Example 8: Sensory evaluation experiment of rebaudioside A1G

[0136] The stevioside raw materials used in the sensory evaluation experiment included RA1G, which was prepared by HPLC as described in Example 2, and the remaining test samples were purchased from Haotian Pharmaceutical Co., Ltd. The purity of RA1G was 95%, the purity of rebaudioside A (RA) was 97%, and the purity of rebaudioside D (RD) was 95%.

[0137] The above steviol glycoside raw materials were dissolved in purified water according to different ratios (Table 2) to prepare sample solutions of 360-560 ppm (in order to achieve the purpose of sugar substitute use, the industry uses the same sweetness to compare the sensory evaluation of food additives). 10 mL of the sample solution was taken into 30 mL disposable taste cups and sensory evaluation (blind evaluation) was carried out by 8 trained and experienced sensory personnel. The evaluation result was the average score given by the sensory personnel.

[0138] In the evaluation, the sweetness was evaluated by 10% sucrose solution (10 g / 100 ml) as the standard, and the score was 10 points (the sweetness was the same as 10% sucrose, 9% sucrose was 9 points, and so on), and the complete tasteless was 0 point.

[0139] The bitterness was evaluated by very bitter as 10 points, and the complete tasteless was 0 point.

[0140] The comprehensive evaluation was given 0-100 points according to the overall taste, 100 points represented the taste of 9% sucrose, and the bitter taste, astringency and other flavors were the deduction items, the flavors were the other bad tastes such as alcohol, plastic, metal, liquorice, chemical and other bad tastes. The sweetness of each sample was basically the same, and the other tastes such as bitterness and flavors were compared.

[0141] From table 2, the sweetness of 560 ppm RA1G solution was similar to 7% sucrose solution, the aftertaste and other bad tastes were far lower than RA, and the taste was obviously superior to the raw material rebaudioside A.

[0142] Table 2. Sensory evaluation results of rebaudioside A1G

[0143]

[0144]

[0145] All the documents mentioned in the present application are cited as references in the present application, as if each document is cited as a reference individually. In addition, it should be understood that various modifications or changes can be made to the present application by those skilled in the art after reading the above teaching of the present application, and these equivalent forms also fall within the scope of the appended claims of the present application.

Claims

1. A method for preparing a rebaudioside A derivative product, wherein, The product contains more than 50% of the compound Rebaudioside A1G with the following structure: The method includes the following steps: (1) Provides rebaudine A and a glucose donor, wherein the glucose donor is soluble starch, and wherein the initial concentration of rebaudine A is 8-150 g / L; and the initial concentration of the glucose donor is 30-600 g / L; (2) Rebaudioside A1G is produced by catalysis of cyclodextrin glycosyltransferase and amylase, wherein the cyclodextrin glycosyltransferase is Toruzyme 3.0L, the amylase is a saccharifying enzyme, and the amount of cyclodextrin glycosyltransferase is 0.1-30kNU / L, and the amount of amylase is 30-300U / mL.

2. The method as described in claim 1, characterized in that, The rebaudioside A is selected from one or more of the following groups: extracted rebaudioside A, synthetic rebaudioside A.

3. The method as described in claim 1, characterized in that, The enzyme is an immobilized enzyme.

4. The method as described in claim 1, characterized in that, The amount of cyclodextrin glycosyltransferase used is 0.5–20 kNU / L.

5. The method as described in claim 1, characterized in that, The amount of cyclodextrin glycosyltransferase used is 1–15 kNU / L.

6. The method as described in claim 1, characterized in that, The amount of cyclodextrin glycosyltransferase used is 5000-50000 U / mL.

7. The method as described in claim 1, characterized in that, The amount of cyclodextrin glycosyltransferase used is 10,000–40,000 U / mL.

8. The method as described in claim 1, characterized in that, The amount of cyclodextrin glycosyltransferase used is 15,000–35,000 U / mL.

9. The method as described in claim 1, characterized in that, The amount of amylase used is 50–250 U / mL.

10. The method as described in claim 1, characterized in that, The amount of amylase used is 80–220 U / mL.

11. The method as described in claim 1, characterized in that, The initial concentration of the rebaudioside A is 10 g to 120 g / L.

12. The method as described in claim 1, characterized in that, The initial concentration of the glucose donor is 30–300 g / L.

13. The method as described in claim 1, characterized in that, The reaction conditions for step (2) are selected from one or more of the following groups: (a) Step (2) is carried out in an aqueous system; (b) The reaction temperature in step (2) is 35–90 °C; and / or (c) The reaction time for step (2) is 0.5 to 72 hours.

14. The method as described in claim 13, characterized in that, The aqueous phase system is water.

15. The method as described in claim 13, characterized in that, The aqueous phase system is pure water.

16. The method as described in claim 13, characterized in that, The aqueous phase system is distilled water or ultrapure water.

17. The method as described in claim 14, characterized in that, The pH of the water in step (2) is 6.

18. The method as described in claim 13, characterized in that, The reaction temperature is 40–90℃.

19. The method as described in claim 13, characterized in that, The reaction temperature is 45–85℃.

20. The method as described in claim 13, characterized in that, The reaction temperature is 50–70℃.

21. The method as described in claim 13, characterized in that, The reaction time is 1 to 48 hours.

22. The method as described in claim 13, characterized in that, The reaction time is 1.5 to 36 hours.

23. The method as described in claim 13, characterized in that, The reaction time is 5 to 20 hours.

24. The method as described in claim 1, characterized in that, The method further includes one or more steps selected from the group consisting of: (a) Terminate the enzyme reaction after it is completed to denature the enzyme; (b) Separate the catalytic reaction products of glycosyltransferases for use in amylase-catalyzed reactions; or, The reaction solution after the catalytic reaction of glycosyltransferase is directly used for the reaction catalyzed by amylase; (c) Replenish rebaudioside A, glucosyl donor, glycosyltransferase and / or amylase consumed in production; (d) Separate, purify, salt, optically resolve, identify and / or package the rebaudioside A1G obtained in step (2); and / or (e) Recycle any unused rebaudioside A, glucosyl donor, glycosyltransferase and / or amylase into the next round of reaction.

25. The method of claim 24, wherein, The enzyme reaction is terminated by boiling.

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