Method for producing glucosylstevioside using mother liquor sugar and method for separating and purifying glucosylstevioside to recover glucose

Abstract

The present invention discloses a method for producing glucosylstevioside using mother sugar and a method for separating and purifying glucosylstevioside to recover glucose, which belongs to the technical field of food additives. [Solution] The present invention provides a method for producing glucosylstevioside using mother liquor sugar, which produces glucosylstevioside using mother liquor sugar as a raw material, thereby enriching the utilization routes of mother liquor sugar and filling the technical gap in the production of glucosylstevioside using mother liquor sugar as a raw material. The produced product also has the advantages of good texture and a high total glycoside content. The present invention provides a method for purifying glucosylstevioside, which recovers and utilizes dextrin, reduces waste liquid discharge, significantly reduces production energy consumption and costs, and improves the yield of high-purity glucosylstevioside. The present invention also provides a method for separating and purifying glucosylstevioside to recover glucose, which is simple and easy to operate, not only produces high-purity glucosylstevioside, but also recovers and utilizes unreacted substrate, thereby reducing costs.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to a Chinese patent filed with the China Patent Office on December 29, 2023, bearing application number 202311848635.2, entitled "Method for producing glucosylstevioside using mother sugar," as well as to a Chinese patent filed with the China Patent Office on July 18, 2023, bearing application number 202310879842.8, entitled "Method for purifying glucosylstevioside," and to a Chinese patent filed with the China Patent Office on September 25, 2023, bearing application number 202311239776.4, entitled "Method for separating and purifying glucosylstevioside and recovering glucose," the contents of which are incorporated herein by reference in their entireties.

[0002] The present invention relates to the technical field of food additives, and in particular to a method for producing glucosylstevioside using mother sugar and a method for separating and purifying glucosylstevioside to recover glucose. [Background technology]

[0003] Stevioside is a natural sweetener extracted from the Asteraceae herbaceous plant Stevia rebaudiana. It has the functional characteristics of high sweetness and low calorie content. Its sweetness is 100 to 300 times that of sucrose, but its calorie content is only 1 / 300 of that of sucrose. Stevioside has both sweet and bitter tastes, and the taste qualities of both tastes are related to the number of glycosyl groups attached to its main moiety and the type of glycosidic bond between the glycosyl groups.

[0004] Stevioside mother liquor (MLS) is produced by recovering high-purity rebauside A (RA) and stevioside (ST) through a solution leaching process, followed by spray-drying the resulting crystal mother liquor to produce a powdered solid. Stevioside mother liquor is generally sold as a low-cost sweetener. Another approach is to separate steviosides (e.g., RA, STV, RC, etc.) from the mother liquor using a resin method (e.g., mixed resin chromatography, modified resin chromatography, etc.) and then further utilize the separated steviosides. However, the total glycoside content in the mother liquor is low, making it unsuitable for steviose products. Furthermore, the mother liquor contains residual polyphenols and flavonoids, which not only increase the bitterness of the mother liquor but also affect glycoside recovery.

[0005] Glucosylstevioside is produced by mixing stevioside with dextrin and glucosylating stevioside through an enzyme catalysis, i.e., by covalently bonding one or more glucosyl groups to stevioside, and then evaporating and concentrating and spray-drying the resulting glucosylstevioside, which is a modified product with improved taste and reduced bitterness, and has been approved for use in the United States, China, and other countries. In the actual production process, the DE value of dextrin is related to its hydrolysis degree, and the more dextrin and high molecular weight polysaccharides are added, the greater the viscosity of the product, and the lower the conversion efficiency of glucose groups.

[0006] Based on the above problems, how to use the mother liquor sugar to produce glucosylstevioside with high efficiency has become a problem to be solved.

[0007] Glucosylstevioside is a flavoring agent that is widely used in the food and beverage industry. During the industrial production process, glucosylstevioside needs to be purified to meet the requirements of high purity and purity.

[0008] Currently, the commonly used method for purifying glucosylstevioside is ion exchange chromatography. Ion exchange chromatography is a separation method based on the interaction between ions in a sample and a stationary phase. In an ion exchange column, ionic functional groups are attached to the stationary phase, allowing it to selectively adsorb and release target components in a sample. The commonly used ion exchange resin in the glucosylstevioside purification process is a strong cation exchange resin. The concentrated solution is loaded onto a strong cation exchange resin column, and under appropriate elution conditions, glucosylstevioside is adsorbed onto the resin, while other unrelated components are eluted. Finally, by adjusting the elution conditions, the glucosylstevioside adsorbed on the resin can be eluted to obtain a pure product.

[0009] In the prior art, a large amount of auxiliary material dextrin is produced in the production and purification process of glucosylstevioside, and the subsequent waste liquid needs to be treated and then discharged, which increases production energy consumption and costs.

[0010] The conversion process of glucosylstevioside is not a simple chemical reaction (A+B=C). Depending on the properties of the enzyme used, the sugar chain and glycosyl ligand may be combined in a non-single form. Glucosylstevioside with different grafting numbers appears in the reaction system. Furthermore, the reaction conversion process is difficult to control, and the reaction degree cannot reach 100%. As a result, the purity of the produced glucosylstevioside is low, so it must be separated and purified.

[0011] The patent application number 202010447914.8 provides a method for separating and purifying glucosylstevioside, specifically, the material produced in the previous step is subjected to ion exchange resin to remove cations in the material, and the material is then subjected to 0.5 to 1 m 3 The material was pumped into the simulated moving bed at a rate of 0.3-2 m / h, the temperature was controlled at 25-40°C, and the time was controlled at 1-2.5 h. The total time was calculated based on the material supply rate. The corresponding material was fractionated stepwise according to the time period of 1:3:1. The material supply rate was controlled at 0.3-2 m / s. 3 / h, the material supply temperature is controlled at 20-35°C, and the supply time is controlled at 0.5-3 hours. The material obtained in 30-90 minutes is pumped into a single-effect concentrator, where the pressure is controlled at -0.6--0.9 MPa and the temperature at 45-60°C, and concentrated until the sugar concentration reaches 50% or more. The concentrated material is filtered and then sent to a concentrated sugar solution tank for spray drying. In the above application, the material is decolorized with a resin and then separated using a simulated moving bed, which allows the production of high-purity glucosylstevioside, but the equipment requirements are high and the cost is high. Summary of the Invention [Problem to be solved by the invention]

[0012] In response to the shortcomings of the prior art, a method for producing glucosylstevioside using mother sugar is provided. The present invention enriches the utilization routes of mother sugar and fills the technical gap in producing glucosylstevioside using mother sugar. Glucosylstevioside produced from mother sugar has a good texture, a high yield, and a high total glycoside content.

[0013] Provided is a method for purifying glucosylstevioside, which optimizes and improves the glucosylstevioside purification process, effectively recovering and utilizing the auxiliary material dextrin, reducing the discharge of waste liquid, and significantly reducing the energy consumption and cost of production, as well as improving the yield of high-purity glucosylstevioside.

[0014] Provided is a method for separating and purifying glucosylstevioside to recover glucose. This method involves dissolving glucosylstevioside, then concentrating, nanofiltration, flocculating, settling, and cooling to crystallize, thereby obtaining high-purity glucosylstevioside and recovering glucose at low cost. [Means for solving the problem]

[0015] The method for producing glucosylstevioside using mother sugar is as follows: (1) mixing mother liquor sugar, maltodextrin and water, stirring until the maltodextrin is dissolved, and adding an enzyme to perform enzymatic hydrolysis to obtain an enzymatic hydrolyzed solution; (2) adding activated carbon to the enzymatic decomposition solution obtained in step (1) to remove impurities to obtain an impurity-removed solution, subjecting the impurity-removed solution to enzyme inactivation treatment, filtering the solution, and placing the obtained filtrate in a macroporous adsorption resin for adsorption treatment; (3) performing separation using an acid-alcohol solution having a pH of 1 to 3 composed of an ethanol solution and a hydrochloric acid solution to obtain a separated liquid; (4) adjusting the pH of the separated liquid to 5 to 6, and then concentrating the separated liquid using a nanofiltration membrane to obtain a concentrated liquid; (5) adding a texture modifier to the concentrated liquid, and then spray-drying the liquid to obtain glucosyl stevioside.

[0016] As an improved technical solution, in the step (1), the mass ratio of the mother liquor sugar, maltodextrin and water is 1:(1-3):(5-10).

[0017] In an improved technical solution, in step (1), the enzyme is a mixture of glucosyltransferase and amylase, and the glucosyltransferase is α-cyclodextrin glucosyltransferase. Specifically, when added, the glucosyltransferase is added in the form of a glucosyltransferase solution, the concentration of the glucosyltransferase solution is 0.8-1.2 wt%, and the amylase is added in the form of solid amylase, the concentration of the solid amylase is 0.4-0.6 wt%.

[0018] Furthermore, and / or the amount of glucosyltransferase solution added per 100 g of mother liquor sugar is 0.5 to 2 ml, and the amount of solid amylase added per 100 g of mother liquor sugar is 0.1 to 1 g.

[0019] As an improved technical solution, in the step (1), the enzymatic decomposition conditions are an enzymatic decomposition temperature of 60 to 80°C, a pH of 5.5 to 6.0, and an enzymatic decomposition time of 36 to 48 hours.

[0020] As an improved technical solution, in the step (2), the amount of activated carbon added is 1 to 3% of the mass of the enzymatic decomposition liquid, and the impurity removal time is 1 to 6 hours.

[0021] As an improved technical solution, in step (2), the supply volume of the filtrate when adsorbed by the macroporous adsorption resin is 30-60% of the volume of the macroporous adsorption resin, and the supply rate of the filtrate is 0.5-2 BV / h.

[0022] An improved technical solution further includes washing the macroporous adsorption resin with pure water before separating the macroporous adsorption resin after adsorption, wherein the amount of pure water used during washing is 1 to 3 BV of the volume of the macroporous adsorption resin, and the flow rate of the pure water is 1 to 3 BV / h.

[0023] As an improved technical solution, in step (3), the concentration of the ethanol solution is 40-60 v / v%, the concentration of the hydrochloric acid solution is 0.3-0.5 wt%, and during separation, the volume of the acid-alcohol solution is 2-4 BV of the volume of the macroporous adsorption resin after adsorption, and the flow rate of the acid-alcohol solution is 1-3 BV / h.

[0024] In the improved technical solution, in step (4), the molecular weight cutoff of the nanofiltration membrane is 400-500 Da.

[0025] As an improved technical solution, in step (5), the texture modifier is a composition of erythritol, sodium alginate, and valine, the mass ratio of the three is 2:(2-4):(5-8), and the amount of the texture modifier added is 0.1-0.5% of the mass of the product in the concentrated liquid. [Effects of the Invention]

[0026] (1) The characteristic feature of stevioside mother syrup is that it has a low total glycoside content and a distinct bitter taste due to the presence of residual polyphenols and flavonoid impurities. This invention is innovative in that it uses mother syrup as a raw material, supplemented with maltodextrin as a glucose substrate, and uses glucosyltransferase and amylase for dual catalysis to obtain glucosylstevioside, thereby reducing the bitterness of the mother syrup, and further removing impurities with activated carbon to eliminate the bitterness and further improve the texture of glucosylstevioside. The glucosylstevioside is then adsorbed onto a macroporous adsorption resin to concentrate and separate the glucosylstevioside. The adsorbed resin is separated using an acid-alcohol solution, and the pH of the solution is adjusted to 5-6 to ensure stability of the glycosides in the subsequent concentration process and improve yield and quality. A texture modifier is then added to further modify the glucosylstevioside and adjust its texture. Finally, the glucosylstevioside is spray-dried to obtain a directly marketable glucosylstevioside product with a good texture and a high total glycoside content.

[0027] (2) The present invention solves the problems of low glucosyltransferase conversion rate due to the low degree of dextrin hydrolysis and the high viscosity of the product caused by the high content of dextrin and high molecular weight polysaccharides, which are achieved by supplementing conventional glucosyltransferase with amylase. By combining glucosyltransferase and amylase, the hydrolysis of dextrin and the transfer of glucosyl groups are accelerated, improving the glucosyl group conversion rate.

[0028] (3) In the present invention, separation is performed using a mixture of hydrochloric acid and ethanol solutions, which provides a good separation effect, prevents the loss of part of the product, and significantly improves the product yield. Furthermore, in the present invention, by further washing the resin with pure water before separation, the raw material liquid remaining on the resin that has not been adsorbed is not only re-adsorbed by the resin, but also effectively removes monosaccharides and oligosaccharides produced during the transfer process, thereby further improving the separation effect and product yield.

[0029] (4) The present invention converts stevioside mother syrup directly into marketable glucosylstevioside products through a rational process. Compared with the prior art, the present invention makes full use of the mother syrup, enriches the utilization routes of the mother syrup, and fills the technical gap in producing glucosylstevioside from the mother syrup. <Means for solving the problem>

[0030] The method for purifying glucosylstevioside is as follows: (1) dissolving glucosylstevioside crude product in ethanol solution, then adsorbing the resulting glucosylstevioside ethanol solution with macroporous adsorption resin, collecting the effluent, then separating it using an acid aqueous solution and a high-purity ethanol solution in sequence, and collecting the acid aqueous separation liquid and the high-alcohol separation liquid; (2) concentrating the mixture of the effluent and the acid water separated liquid obtained in step (1), adding α-1,4 glucose hydrolase to the concentrate, raising the temperature to cause a hydrolysis reaction, and after completion of the hydrolysis, adjusting the pH of the reaction system to 3 to inactivate the α-1,4 glucose hydrolase, thereby obtaining a hydrolyzed liquid; (3) adding stevioside to the hydrolyzed solution, then adjusting the pH of the solution to 6.0, adding glucosyltransferase, and reacting at elevated temperature; after the reaction is completed, increasing the temperature to inactivate the glucosyltransferase to obtain a reaction solution; concentrating the reaction solution to a solid content of 40-50 wt%; and then spray-drying the concentrate to obtain high-purity glucosylstevioside A. (4) concentrating the high-alcohol separated liquid to a solid content of 40 to 50 wt %, and spray-drying the concentrate to obtain high-purity glucosylstevioside B.

[0031] As the above solution, preferably, in the step (1), the concentration of the ethanol solution is 5 to 15 wt %, and the concentration of glucosylstevioside in the glucosylstevioside ethanol solution is 30 to 50 g / L.

[0032] As a solution to the above problem, preferably, in the step (1), the flow rate of the glucosylstevioside ethanol solution during the adsorption treatment is 0.25 BV / h to 1 BV / h.

[0033] As a solution to the above problem, preferably, in step (1), the aqueous acid solution is a hydrochloric acid solution having a concentration of 0.04 to 0.06 wt %, and the high-purity ethanol solution has a concentration of 70 to 80 wt %.

[0034] As the above solution, preferably, in the step (1), during separation, the acid aqueous solution has a volume of 2 to 3 BV and a flow rate of 2 to 3 BV / h, and the high-purity ethanol solution has a volume of 2 to 3 BV and a flow rate of 2 to 3 BV / h.

[0035] As the above solution, preferably, in step (1), the volume of the effluent is 1 to 2 BV of the volume of the macroporous adsorption resin, and the volume of the acid water separated liquid is 2 to 3 BV of the volume of the macroporous adsorption resin.

[0036] As a solution to the above problem, preferably, in the step (2), the concentration factor during the concentration treatment is 3 to 5 times.

[0037] As a solution to the above problem, preferably, in the step (2), 0.03 to 0.1 ml of the α-1,4 glucose hydrolase is used per 100 g of the glucosylstevioside crude product.

[0038] As a solution to the above problem, preferably, in step (2), the temperature of the hydrolysis reaction is 40 to 50° C., and the time of the hydrolysis reaction is 1 to 3 hours.

[0039] As the above solution, preferably, in the step (3), the stevioside is stevioside STV, and 0.3 to 0.5 ml of the glucosyltransferase is used per 100 g of glucosylstevioside crude product.

[0040] As the above solution, preferably, in the step (3), the temperature of the temperature-raised reaction is 70 to 80°C, the time is 6 to 10 hours, and the temperature of the temperature-raised inactivation is 95°C. <Effects of the invention>

[0041] In this invention, the glucosylstevioside crude product is first dissolved in an ethanol solution, then adsorbed using a macroporous adsorption resin, and then separated using an acid solution and a high-purity ethanol solution, respectively. The macroporous adsorption resin is a highly efficient adsorption material with a large specific surface area and strong adsorption capacity, which can effectively adsorb the target substance and has good selectivity over other impurities. The action of the macroporous adsorption resin can separate glucosylstevioside from its impurities, improving its purity. After adsorption using the macroporous adsorption resin, the acid solution can separate the dextrin adsorbed as impurities in the macroporous adsorption resin, and the high-purity ethanol solution can desorb glucosylstevioside from the adsorption resin, further improving the purity of glucosylstevioside and removing remaining impurities.

[0042] In addition, the present invention involves mixing the effluent after adsorption by the macroporous adsorption resin with the acid-water separated liquid, adding α-1,4 glucose hydrolase to cause a hydrolysis reaction, and then mixing and reacting with stevioside to obtain high-purity glucose stevioside. In the present invention, the recovered dextrin is reused to produce high-purity glucosyl stevioside, thereby reducing wastewater discharge, reducing energy consumption and costs in the production of glucosyl stevioside, and improving the yield of high-purity glucosyl stevioside.

[0043] The method provided by the present invention is relatively simple in operation and can achieve adsorption separation of glucosylstevioside without requiring complicated equipment and supporting conditions. The macroporous adsorption resin has high adsorption capacity and regeneration ability, and is recyclable. This not only improves the efficiency of the purification process but also reduces costs. <Means for solving the problem>

[0044] The method for isolating and purifying glucosylstevioside and recovering glucose is as follows: (1) adding the glucosylstevioside crude product to purified water and dissolving it again to obtain a raw material solution; (2) subjecting the raw material liquid to nanofiltration concentration treatment and collecting a membrane fraction liquid and a membrane permeate liquid; (3) removing the solvent from the membrane fraction and the membrane permeate, respectively, to obtain intermediate products A and B; (4) mixing the intermediate product A with methanol to precipitate, and then filtering by vacuum suction, collecting the filter cake and the filtrate, and drying the filtrate to obtain high-purity glucosylstevioside; (5) mixing the intermediate product B and the filter cake, adding the purified water, stirring until the intermediate product B and the filter cake are dissolved, adding α-1,4-glucose hydrolase to carry out enzymatic decomposition treatment, and after the enzymatic decomposition is completed, heating the raw material liquid to inactivate the enzyme, thereby obtaining a glucose-containing solution; (6) concentrating the glucose-containing solution at a high temperature to obtain an extract-like intermediate product C, gradually lowering the temperature of the extract-like intermediate product C to crystallize it, and finally filtering it by vacuum suction to recover the filtrate for subsequent production, wherein the filter cake is high-purity glucose.

[0045] As a preferred technical solution, in the step (1), the solid content of the raw material liquid is 1 wt % to 5 wt %.

[0046] As a preferred technical solution, in the step (2), the molecular weight cutoff of the nanofiltration membrane during nanofiltration concentration is 500 Da to 800 Da, and the operating pressure during nanofiltration concentration is 3.5 to 5 bar.

[0047] As a preferred technical solution, in the step (3), the solvent in the membrane permeate and membrane fraction is removed under conditions of a temperature of 80° C. and a pressure of −0.1 MPa.

[0048] As a preferred technical solution, in the step (4), the concentration of the methanol is 95 wt% to 99 wt%, and the mass ratio of the intermediate product A to the methanol is 1:(5 to 10).

[0049] As a preferred technical solution, in the step (4), the mixing and settling time is 2 hours to 8 hours.

[0050] As a preferred technical solution, in step (5), the amount of the α-1,4-glucose hydrolase added is 0.2% to 0.3% of the total weight of the intermediate product B and the filter cake.

[0051] As a preferred technical solution, in step (5), the enzymatic decomposition treatment is carried out at a temperature of 35°C to 45°C, at a pH of 4.0 to 4.5, for 5 to 7 hours, and the raw material liquid is heated to 90°C to inactivate the enzyme.

[0052] As a preferred technical solution, in the step (6), the temperature of the high-temperature concentration treatment is 80°C to 90°C, and the solid content of the extract-like intermediate product C is 70wt% to 75wt%.

[0053] As a preferred technical solution, in the step (6), the temperature is gradually decreased to 20°C at a rate of 10°C / h to 15°C / h, and the temperature is maintained for 5 to 7 hours for crystallization. <Effects of the invention>

[0054] In the present invention, the crude glucosylstevioside product is dissolved in water again, and then concentrated and pre-purified using a nanofiltration membrane. The relative molecular masses of the unreacted rebauside A, glucose, and the product glucosylstevioside in the raw solution vary greatly, and a nanofiltration membrane with a molecular mass of 500 Da to 800 Da can be used to separate glucosylstevioside to a large extent. The membrane fraction solution is then concentrated to remove the solvent, and then methanol is added for re-purification to obtain high-purity glucosylstevioside.

[0055] In the present invention, the solvent is removed from the membrane permeate collected in the nanofiltration process, and the mixture is mixed with the filter cake from the coagulation and sedimentation process, and α-1,4-glucose hydrolase is added to carry out an enzymatic hydrolysis reaction. The mixture is then concentrated under appropriate conditions, and the temperature is lowered to crystallize the mixture, thereby obtaining highly pure glucose.

[0056] As described above, the present invention has a simple process and is easy to operate, and realizes the purification of glucosylstevioside. Not only can it obtain high-purity glucosylstevioside products, but it can also recover and reuse unreacted substrates to a large extent, thereby reducing production costs. DETAILED DESCRIPTION OF THE INVENTION

[0057] The present invention will be further described below in conjunction with examples. Note that these examples are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0058] The present invention provides a method for producing glucosylstevioside using a mother liquor sugar, (1) mixing mother liquor sugar, maltodextrin and water, stirring until the maltodextrin is dissolved, and adding an enzyme to perform enzymatic hydrolysis to obtain an enzymatic hydrolyzed solution; (2) adding activated carbon to the enzymatic decomposition solution obtained in step (1) to remove impurities to obtain an impurity-removed solution, subjecting the impurity-removed solution to enzyme inactivation treatment, filtering the solution, and placing the obtained filtrate in a macroporous adsorption resin for adsorption treatment; (3) performing separation using an acid-alcohol solution having a pH of 1 to 3 composed of an ethanol solution and a hydrochloric acid solution to obtain a separated liquid; (4) adjusting the pH of the separated liquid to 5 to 6, and then concentrating the separated liquid using a nanofiltration membrane to obtain a concentrated liquid; (5) adding a texture modifier to the concentrated liquid, and then spray-drying the liquid to obtain glucosyl stevioside.

[0059] The source of the mother liquor sugar in the present invention is a crystal mother liquor obtained by infusing stevia leaves with a solution. Generally, the total glycoside content of the mother liquor sugar is 60 to 80%, of which RA is 20 to 40%, STV is 10 to 25%, RC is 10 to 20%, and other glycosides are 5 to 20%.

[0060] In the present invention, first, mother liquor sugar, maltodextrin and water are mixed and dissolved, and then an enzyme is added to carry out an enzymatic hydrolysis reaction to obtain an enzymatic hydrolyzed solution.

[0061] In the present invention, the mass ratio of the mother liquor sugar, maltodextrin, and water is preferably 1:(1-3):(5-10). The present invention does not require a specific source of maltodextrin, and commercially available products may be used. The DE value of the maltodextrin in the present invention is preferably 5-10. The present invention does not require any specific water, and preferably uses purified water. The present invention further includes stirring during the mixing and dissolving process, and the stirring speed is preferably 10-100 rpm.

[0062] In the present invention, the enzymatic hydrolysis reaction is preferably initiated after the mother sugar and maltodextrin are completely dissolved. The enzyme is preferably a mixture of glucosyltransferase and amylase, and the glucosyltransferase is preferably α-cyclodextrin glucosyltransferase. The lower the DE value of maltodextrin, the lower the degree of dextrin hydrolysis, resulting in increased amounts of dextrin and large molecular weight polysaccharides, and a higher viscosity of the product. However, in the present invention, the combined addition of glucosyltransferase and amylase can accelerate the hydrolysis of dextrin and the transfer of glucose groups, thereby improving the conversion rate.

[0063] Specifically, in the present invention, the glucosyltransferase is preferably added in the form of a glucosyltransferase solution, the concentration of the glucosyltransferase solution is preferably 0.8 to 1.2 wt%, more preferably 1 wt%, and the activity of the glucosyltransferase is preferably 100 U / g. The amylase is preferably added in the form of a solid amylase, the concentration of the solid amylase is preferably 0.4 to 0.6 wt%, more preferably 0.5 wt%, and the activity of the amylase is preferably 1000 to 3000 U / g.

[0064] In the present invention, the amount of glucosyltransferase solution added per 100 g of mother sugar is preferably 0.5 to 2 ml, and the amount of the solid amylase added per 100 g of mother sugar is preferably 0.1 to 1 g.

[0065] Furthermore, in the present invention, the conditions for carrying out the enzymatic hydrolysis reaction are preferably a reaction temperature of 60 to 80°C, a pH of 5.5 to 6.0, and a reaction time of 36 to 48 hours. Furthermore, the present invention does not particularly require a reagent for adjusting the pH, and preferably employs a potassium hydroxide solution or a sodium hydroxide solution. The present invention does not particularly limit the concentrations of the potassium hydroxide solution and sodium hydroxide solution. For example, the concentration of the potassium hydroxide solution may be 1 to 10 v / v%, and the concentration of the sodium hydroxide solution may be 1 to 10 v / v%.

[0066] In the present invention, after enzymatic hydrolysis is completed, activated carbon is added to the enzymatic hydrolysis solution to remove impurities. The activated carbon's adsorption properties can effectively adsorb impurities such as flavonoids, saponins, and alkaloids present in the enzymatic hydrolysis solution. The activated carbon also has a certain deodorizing effect, eliminating the bitterness of some of the glucosyl stevioside. Furthermore, the amount of activated carbon added is preferably 1-3% of the mass of the enzymatic hydrolysis solution, and the impurity removal time is preferably 1-6 hours. The present invention does not require a specific pore size for the activated carbon, and it can be, for example, 2-50 nm. The present invention does not require a specific source of activated carbon; commercially available activated carbon may be used.

[0067] In the present invention, after removing impurities with activated carbon to obtain an impurity-removed liquid, the impurity-removed liquid is subjected to an enzyme inactivation treatment and then filtered to obtain a filtrate. Furthermore, during the enzyme inactivation treatment, high-temperature inactivation is preferably employed, and the inactivation temperature is preferably 100 to 120°C. The present invention does not particularly limit the inactivation time, and generally, the enzyme is already inactivated by the time the temperature reaches 100 to 120°C. Furthermore, the filtration is preferably performed using a plate and frame filter, with the pore size of the filter cloth preferably being 30 to 50 μm and the operating pressure preferably being 0.5 to 2.5 MPa.

[0068] In the present invention, the purpose of performing enzyme inactivation on the impurity removal solution is to stop the enzymatic decomposition reaction, prevent the enzyme from affecting the final product, and ensure the quality of the product. In the present invention, the first purpose of performing filtration using a plate frame is to filter out the activated carbon after adsorption, and the second purpose is to filter out the inactivated enzyme.

[0069] In the present invention, the obtained filtrate is adsorbed by a macroporous adsorption resin. The type of macroporous adsorption resin is not particularly limited, and is preferably one of SD-9, AB-8, LX-T81, LX-T83, and LX-T28. The source of the resin is not particularly limited, and a commercially available product may be used. The supply method is not particularly limited, and for example, a pump may be used. The support of the macroporous adsorption resin is not particularly limited, and for example, a resin column may be used. Specifically, during the liquid supply, the amount of the filtrate supplied is preferably 30 to 60% of the volume of the macroporous adsorption resin, and the supply rate of the filtrate is preferably 0.5 to 2 BV / h. In the present invention, the effluent adsorbed by the macroporous adsorption resin contains a certain amount of dextrin and glucose, which can be further recovered and utilized to produce other valuable by-products.

[0070] Furthermore, after the filtrate is adsorbed by the macroporous adsorption resin, the macroporous adsorption resin is washed with pure water, and the amount of pure water used is preferably 1 to 3 BV of the volume of the macroporous adsorption resin, and the flow rate of the pure water is 1 to 3 BV / h. In the present invention, the purpose of washing with pure water is to adsorb the filtrate remaining in the resin column by the macroporous adsorption resin, thereby achieving as much complete adsorption of the filtrate as possible and improving the adsorption rate.

[0071] In the present invention, after adsorption by the macroporous adsorption resin, the adsorbed macroporous adsorption resin is separated, and the pH of the effluent is adjusted to 5-6 to obtain a separated solution. Specifically, the separating agent used in the separation is an acid-alcohol solution. The method for preparing the acid-alcohol solution involves adding 0.3-0.5 wt% hydrochloric acid to a 40-60 v / v% ethanol solution and adjusting the pH to 1-3 to obtain the acid-alcohol solution. The amount of the acid-alcohol solution used in the separation is preferably 2-4 BV per volume of the macroporous adsorption resin after adsorption, and the flow rate of the acid-alcohol solution is preferably 1-3 BV / h. In the present invention, the pH adjuster is not particularly limited; any pH adjuster commonly used in this field can be used. In the present invention, the purpose of adjusting the pH of the effluent after separation to 5-6 is to prevent the destruction of glycosides due to excessive acidity during the subsequent concentration process.

[0072] In the present invention, the separated liquid is concentrated using a nanofiltration membrane to obtain a concentrate. The molecular weight cutoff of the nanofiltration membrane is preferably 400 to 500 Da, and the solid content of the concentrate is 30 to 50 wt %.

[0073] After obtaining a concentrate, the present invention adds a texture modifier to the concentrate and spray-dries the mixture to obtain a glucosyl stevioside product. Specifically, the texture modifier is a composition of erythritol, sodium alginate, and valine, with the mass ratio of the three being preferably 2:(2-4):(5-8), more preferably 2:3:5. The amount of texture modifier added is preferably 0.1-0.5% of the mass of the product in the concentrate. The texture modifier used in the present invention is preferably added in solid form. In the present invention, the purpose of adding the texture modifier is to adjust the texture of the product and reduce its bitterness or astringency. In the present invention, the blowing temperature for the spray drying is 180-200°C, and the blowing temperature is 80-100°C.

[0074] To further explain the present invention, the following examples are provided to explain in detail the method for producing glucosylstevioside using the mother saccharide provided by the present invention, but these examples should not be construed as limiting the scope of protection of the present invention. It should be noted that the raw materials used in the following examples and comparative examples are all commercially available raw materials unless otherwise specified. The main components of the stevioside mother saccharide in the following examples are as shown in Table 1, and the stevioside mother saccharide lots were all provided by Zhucheng Haotian Pharmaceutical Co., Ltd.

[0075] [Table 1] Example 1

[0076] S1: 100 g of mother sugar from lot 1 and 150 g of maltodextrin with a DE value of 8 were added to 700 mL of water and stirred at 50 rpm until the maltodextrin was completely dissolved. Then, 0.5 mL of α-cyclodextrin glucosyltransferase (activity: 100 U / g) and 0.1 g of amylase (activity: 1000-3000 U / g) were added and the mixture was subjected to an enzymatic hydrolysis reaction at a temperature of 70°C and a pH of 5.8 for 42 hours to obtain 850 mL of enzymatic hydrolysis solution. S2: 10 g of activated carbon with a pore size of 30 nm was added to 850 mL of the enzymatic decomposition solution, and after removing impurities for 3 hours, 830 mL of impurity-removed solution was obtained. S3: The impurity removal solution was heated to 110°C to perform enzyme inactivation treatment, and then filtered using a plate frame filter with a filter cloth pore size of 40 μm under an operating pressure of 2.0 MPa to obtain 815 mL of filtrate. S4: 815 mL of filtrate was fed to a resin column packed with 2 L of macroporous adsorption resin (SD-9) for adsorption treatment, and the feed rate was controlled at 1 BV / h. S5: 2L of resin after adsorption is separated with an acid-alcohol solution. The amount of the acid-alcohol solution is 4BV of the volume of the macroporous adsorption resin after adsorption, and the flow rate of the acid-alcohol solution is 2BV / h. Then, the pH of the effluent is adjusted to 5 to obtain 7.8L of separated liquid. The acid-alcohol solution is a solution composed of 40v / v% ethanol solution and 0.5wt% hydrochloric acid solution, with a pH of 2. S6: 7.8 L of the separated solution was concentrated using a nanofiltration membrane with a molecular weight cutoff of 400 Da. The nanofiltration fraction was collected to obtain 350 ml of a concentrated solution with a solid content of 40 wt%. S7: 0.14 g of texture modifiers (erythritol 0.028 g, sodium alginate 0.042 g, valine 0.07 g) were added to 350 ml of the concentrate to modify it, and the mixture was spray-dried to obtain 113 g of glucosylstevioside product. The air temperature during spray-drying was 190°C and the outlet temperature was 90°C. Example 2

[0077] S1: 100 g of mother sugar from lot 2 and 100 g of maltodextrin with a DE value of 5 were added to 500 mL of water and stirred at 10 rpm until the maltodextrin was completely dissolved. Then, 1 mL of α-cyclodextrin glucosyltransferase (activity: 100 U / g) and 0.3 g of amylase (activity: 1000-3000 U / g) were added and reacted at 80°C and pH 5.5 for 48 hours to obtain 750 mL of enzymatic hydrolysis solution. S2: 15 g of activated carbon with a pore size of 2 nm was added to 750 mL of the enzyme decomposition solution, and after removing impurities for 6 hours, 730 mL of impurity-removed solution was obtained. S3: The impurity removal solution was heated to 120°C and subjected to enzyme inactivation treatment, and then filtered using a plate frame filter with a filter cloth pore size of 50 μm under an operating pressure of 0.5 MPa to obtain 720 mL of filtrate. S4: 720 mL of filtrate was fed to a resin column packed with 2.4 L of macroporous adsorption resin (AB-8) for adsorption treatment, and the feed rate was controlled at 0.5 BV / h. S5: 2.4L of resin after adsorption is separated with an acid-alcohol solution, the amount of the acid-alcohol solution is 3BV of the volume of the macroporous adsorption resin after adsorption, and the flow rate of the acid-alcohol solution is 1BV / h. Then, the pH of the effluent is adjusted to 5.5 to obtain 7L of separated liquid, of which the acid-alcohol solution is a solution composed of 50v / v% ethanol solution and 0.5wt% hydrochloric acid solution, with a pH of 2; S6: 7 L of the separated solution was concentrated using a nanofiltration membrane with a molecular weight cutoff of 450 Da. The nanofiltration fraction was collected to obtain 430 ml of a concentrated solution with a solid content of 35 wt%. S7: 0.45 g of texture modifiers (erythritol 0.09 g, sodium alginate 0.135 g, valine 0.225 g) were added to 430 ml of the concentrate, and the mixture was spray-dried to obtain 126 g of glucosylstevioside product. The air temperature during spray-drying was 200°C and the outlet temperature was 80°C. Example 3

[0078] S1: 100 g of mother sugar from lot 3 and 200 g of maltodextrin with a DE value of 10 were added to 800 mL of water and stirred at 100 rpm until the maltodextrin was completely dissolved. Then, 1.5 mL of α-cyclodextrin glucosyltransferase (activity: 100 U / g) and 0.7 g of amylase (activity: 1000-3000 U / g) were added and reacted at 60°C and pH 6.0 for 36 hours to obtain 1200 mL of enzymatic hydrolysis solution. S2: 20 g of activated carbon with a pore size of 50 nm was added to 1200 mL of the enzymatic decomposition solution, and after removing impurities for 1 hour, 1900 mL of impurity-removed solution was obtained. S3: The impurity removal solution was heated to 100°C to perform enzyme inactivation treatment, and then filtered using a plate frame filter with a filter cloth pore size of 30 μm under an operating pressure of 2.5 MPa to obtain 1800 mL of filtrate. S4: 1800 mL of filtrate was fed to a resin column packed with 3 L of macroporous adsorption resin (T81) for adsorption treatment, and the feed flow rate was controlled at 2 BV / h. S5: 3L of resin after adsorption is separated with an acid-alcohol solution, the amount of the acid-alcohol solution is 2BV of the volume of the macroporous adsorption resin after adsorption, and the flow rate of the acid-alcohol solution is 3BV / h. Then, the pH of the effluent is adjusted to 6 to obtain 6L of separated liquid, of which the acid-alcohol solution is a solution composed of 60v / v% ethanol solution and 0.5wt% hydrochloric acid solution, with a pH of 2; S6: 6 L of the separated solution was concentrated using a nanofiltration membrane with a molecular weight cutoff of 500 Da. The nanofiltration fraction was collected to obtain 300 ml of a concentrated solution with a solid content of 50 wt%. S7: 0.75 g of texture modifiers (erythritol 0.15 g, sodium alginate 0.225 g, valine 0.375 g) were added to 300 ml of the concentrate to modify it, and then spray-dried to obtain 131 g of glucosylstevioside product. The air temperature during spray-drying was 180°C and the outlet temperature was 100°C. Example 4

[0079] S1: 100 g of mother sugar from lot 4 and 250 g of maltodextrin with a DE value of 6 were added to 1000 mL of water and stirred at 80 rpm until the maltodextrin was completely dissolved. Then, 2 mL of α-cyclodextrin glucosyltransferase (enzyme concentration 0.15%, activity 100 U / g) and 1 g of amylase (activity 1000-3000 U / g) were added and reacted at 70°C and pH 5.6 for 40 hours to obtain 850 mL of enzymatic hydrolysis solution. S2: 25 g of activated carbon with a pore size of 20 nm was added to 850 mL of the enzymatic decomposition solution, and after removing impurities for 2 hours, 830 mL of impurity-removed solution was obtained. S3: The impurity removal solution was heated to 100°C to perform enzyme inactivation treatment, and then filtered using a plate frame filter with a filter cloth pore size of 40 μm under an operating pressure of 1.0 MPa to obtain 815 mL of filtrate. S4: 815 mL of filtrate was supplied to a resin column packed with 1.5 L of macroporous adsorption resin (T83) for adsorption treatment, the flow rate was 1.5 BV / h, and then the macroporous adsorption resin after adsorption was washed with 2 BV of pure water, the flow rate was 2 BV / h, S5: 1.5L of washed resin is separated with an acid-alcohol solution. The amount of the acid-alcohol solution is 4BV of the volume of the macroporous adsorption resin after adsorption, and the flow rate of the acid-alcohol solution is 1.5BV / h. Then, the pH of the effluent is adjusted to 5.5 to obtain 6L of separated liquid. The acid-alcohol solution is a solution composed of 60v / v% ethanol solution and 0.5wt% hydrochloric acid solution, with a pH of 2; S6: 6 L of the separated solution was concentrated using a nanofiltration membrane with a molecular weight cutoff of 500 Da. The nanofiltration fraction was collected to obtain 400 ml of a concentrated solution with a solid content of 40 wt%. S7: 0.8 g of texture modifiers (erythritol 0.16 g, sodium alginate 0.24 g, valine 0.4 g) were added to 400 ml of the concentrate, and the mixture was spray-dried to obtain 145 g of glucosylstevioside product. The air temperature during spray-drying was 180°C and the outlet temperature was 90°C. Comparative Example 1

[0080] In step S2 of this comparative example, 2 L of macroporous adsorption resin SD-300 was used instead of activated carbon, and the other conditions were the same as those of Example 1. Comparative Example 2

[0081] In contrast to Example 1, in this comparative example, first, impurities are removed using activated carbon, and then the enzymatic decomposition reaction is carried out. The specific impurity removal conditions, enzymatic decomposition reaction conditions, and other conditions are the same as those in Example 1. Comparative Example 3

[0082] In contrast to Example 1, in S5 of this comparative example, separation was first performed using a hydrochloric acid solution with a pH of 2, and then using an ethanol solution with a concentration of 40 v / v%, and the two effluents were combined, and the other conditions were the same as in Example 1. Comparative Example 4

[0083] In contrast to Example 1, in S5 of this comparative example, separation was first performed using an ethanol solution with a concentration of 40 v / v%, and then using a hydrochloric acid solution with a pH of 2, and the two effluents were combined, and the other conditions were the same as in Example 1. Comparative Example 5

[0084] In contrast to Example 1, in S5 of this comparative example, the pH of the effluent was adjusted to 9 to obtain a separated liquid, and the other conditions were the same as those in Example 1. Comparative Example 6

[0085] In contrast to Example 2, in this comparative example, the enzyme inactivation treatment in step S3 was omitted, and the other conditions were the same as in Example 2. Comparative Example 7

[0086] In this comparative example, the macroporous adsorption resin (T81) in step S4 was replaced with an equal amount of T-28 resin, and the other conditions were the same as those in Example 3. Comparative Example 8

[0087] In contrast to Example 4, in this comparative example, the texture modifier in step S7 was replaced with an equal amount of sodium citrate, and the other conditions were the same as in Example 4.

[0088] 1. Product quality, purity and texture testing The method for measuring the content of glucosylstevioside in Examples 1 to 4 and Comparative Examples 1 to 8 of the present invention was carried out in accordance with the Chinese national standard GB2760-2014. During the texture test, the collected samples were diluted to 500 ppm, and the test conditions were a temperature of 29°C and humidity of 50% RH. Specifically, a sensory evaluation group was formed by selecting 10 expert sensory evaluators based on the requirements of GB / T 16291.2-2010. During the evaluation, the sensory evaluators were required to restrict their diet within 1 hour prior to the start of the evaluation experiment, particularly for foods that would have a significant impact on taste. The sensory evaluators scored the bitterness of the samples on a 10-point scale, and the final score was the average. The evaluation standard was a 5 wt% sucrose solution. The test results are shown in Table 2.

[0089] [Table 2]

[0090] As can be seen from the test results in Table 2, compared to the comparative example, the present invention uses mother sugar as a raw material and optimizes the conditions to produce glucosylstevioside, which not only has a high yield but also a high total glycoside content and an excellent product flavor.

[0091] 2. Sensory evaluation experiment of sweeteners In the present invention, four sets of control experiments were conducted to measure the difference in texture between the glucosylstevioside produced by the present invention and the stevioside in the original mother sugar, and the collected samples were diluted to 500 ppm. This is mainly because the sweetness of stevioside-based products is high, so dilution is necessary for distinguishing and evaluating them. The specific examples are as follows:

[0092] Experiment 1: 500 ppm mother sugar (total glycosides 68%) Experiment 2: 500 ppm glucosylstevioside obtained in Example 1, Experiment 3: 500 ppm mother sugar (80% total glycosides), Experiment 4: 500 ppm glucosylstevioside prepared in Example 4, The evaluation standard was a 5 wt% sucrose solution. Test conditions: Temperature 29°C, humidity 50RH%, Exam date: May 16, 2023.

[0093] According to the provisions of GB / T16291.2-2010, 10 expert sensory evaluators were selected to form a sensory evaluation group.

[0094] Specifically, during the evaluation, the sensory evaluators were restricted from eating within 1 hour before the start of the evaluation experiment, especially from foods that would have a serious impact on taste.

[0095] The sensory evaluators comprehensively scored the sweetness of the sweeteners based on indicators such as sweetness intensity, speed of sweetness, aftertaste, bitterness, astringency, off-flavors, and overall preference, with speed of sweetness representing how quickly the sweetness was felt and aftertaste representing how long the sweetness lasted. Except for overall preference, which was given a percentile (5 wt% sucrose solution was given a perfect score of 100), all other indicators were given a score of 10, and the final score was calculated as the average. The results of the texture evaluation are shown in Table 3 below, with higher sensory scores indicating more pronounced texture characteristics.

[0096] [Table 3]

[0097] As can be seen from the measurement results in Table 3, the glucosylstevioside produced by the present invention has improved texture in all aspects compared to the original mother syrup, especially in terms of sweetness and speed of sweetening, and at the same time, the bitterness and astringency of the raw material mother syrup are significantly reduced.

[0098] In the following examples, the total stevioside glycoside (TSG) content in 100 g of glucosylstevioside crude product was 71 wt%, the glucosylstevioside (GSG) content was 59 wt%, and the dextrin content was 28 wt%.

[0099] Qualitative analysis of the product: The rearrangement product was analyzed using a liquid chromatograph quadrupole time-of-flight mass spectrometer. The detection conditions were an ACQUITY UPLC BEH HILIC amino column, column temperature 30°C, gradient elution from acetonitrile:water = 80:20 (2 min) to 50:50 (30 min) (v / v), sample injection volume 1 μL, sample injection concentration 5 mg / mL, flow rate 0.3 mL / min, and mass spectrometry conditions were a collision voltage of 6 eV, ionization method electrospray ionization (ESI), negative ion detection mode, and molecular weight range 200 to 2000.

[0100] Quantitative analysis method of the product: In accordance with GB2760-2014, the analytical detection method for glucosylstevioside in Supplementary Document No. 8 of the National Health and Family Planning Commission. Example 5

[0101] S1: 100g of glucosylstevioside crude product is dissolved in 2L of 10wt% ethanol solution, then adsorbed with macroporous adsorption resin, the liquid flow rate is controlled at 0.5BV / h during adsorption, and the effluent is collected. Then, it is separated with 0.05wt% hydrochloric acid solution, the flow rate of the hydrochloric acid solution is controlled at 2BV / h, and the volume is controlled at 2BV, and the acid-water separated liquid is collected. Preferably, it is separated with 70wt% ethanol solution, the flow rate of the ethanol solution is controlled at 2BV / h, and the volume is controlled at 2BV, and the high-alcohol separated liquid is collected. S2: The effluent and the acid water separated liquid were mixed and concentrated to obtain 500 ml of concentrated solution. 0.05 ml of α-1,4 glucose hydrolase was added to the concentrated solution, the temperature was raised to 45°C, and hydrolysis was carried out for 2 hours. After that, the pH of the reaction system was adjusted to 3, and the α-1,4 glucose hydrolase was inactivated to obtain a hydrolyzed solution. S3: 30g of stevioside STV was added to 500ml of hydrolysis solution, the pH of the solution was adjusted to 6.0, the temperature was raised to 75℃, 0.3ml of glucosyltransferase was added and reacted for 8 hours, after the reaction was completed, the temperature was raised to 95℃ to inactivate the glucosyltransferase, and the reaction solution was obtained. The reaction solution was concentrated to a solid content of 50wt%, and then spray-dried to obtain 60g of high-purity glucosylstevioside A. S4: The high-alcohol separated liquid was concentrated to a solid content of 50 wt% and spray-dried to obtain 69 g of highly pure glucosylstevioside B.

[0102] The results of the detection were that the total stevioside glycoside (TSG) content in high-purity glucosylstevioside A was 93 wt%, the glucosylstevioside (GSG) content was 77 wt%, and the dextrin content was 5.8 wt%, and the total stevioside glycoside (TSG) content in high-purity glucosylstevioside B was 99 wt%, the glucosylstevioside (GSG) content was 92 wt%, and the dextrin content was 0.2 wt%. Example 6

[0103] S1: 100g of glucosylstevioside crude product is dissolved in 3L of 10wt% ethanol solution, then adsorbed with macroporous adsorption resin, the liquid flow rate is controlled at 0.75BV / h during adsorption, and the effluent is collected. Then, it is separated with 0.05wt% hydrochloric acid solution, the flow rate of the hydrochloric acid solution is controlled at 2BV / h, and the volume is controlled at 2BV, and the acid-water separated liquid is collected. Preferably, it is separated with 80wt% ethanol solution, the flow rate of the ethanol solution is controlled at 3BV / h, and the volume is controlled at 3BV, and the high-alcohol separated liquid is collected. S2: The effluent and the acid water separated liquid were mixed and concentrated to obtain 500 ml of concentrated solution. 0.05 ml of α-1,4 glucose hydrolase was added to the concentrated solution, the temperature was raised to 50°C, and hydrolysis was carried out for 2 hours. After that, the pH of the reaction system was adjusted to 3, and the α-1,4 glucose hydrolase was inactivated to obtain a hydrolyzed solution. S3: 30g of stevioside STV was added to 500ml of hydrolysis solution, the pH of the solution was adjusted to 6.0, the temperature was raised to 75℃, 0.4ml of glucosyltransferase was added and reacted for 7.5 hours, after the reaction was completed, the temperature was raised to 95℃ to inactivate the glucosyltransferase, and the reaction solution was obtained. The reaction solution was concentrated to a solid content of 40wt%, and then spray-dried to obtain 61g of high-purity glucosylstevioside A. S4: The high-alcohol separated liquid was concentrated to a solid content of 40 wt%, and spray-dried to obtain 68 g of high-purity glucosylstevioside B.

[0104] The results of the detection were that the total stevioside glycoside (TSG) content in high-purity glucosylstevioside A was 93.5 wt%, the glucosylstevioside (GSG) content was 79.5 wt%, and the dextrin content was 5.7 wt%, and the total stevioside glycoside (TSG) content in high-purity glucosylstevioside B was 99 wt%, the glucosylstevioside (GSG) content was 93 wt%, and the dextrin content was 0.2 wt%. Example 7

[0105] S1: 100g of glucosylstevioside crude product is dissolved in 2.5L of 10wt% ethanol solution, then adsorbed on macroporous adsorption resin, the liquid flow rate is controlled at 0.8BV / h during adsorption, and the effluent is collected. Then, separation is performed with 0.05wt% hydrochloric acid solution, the flow rate of the hydrochloric acid solution is controlled at 3BV / h, and the volume is controlled at 3BV, and the acid-water separation liquid is collected. Preferably, separation is performed with 75wt% ethanol solution, the flow rate of the ethanol solution is controlled at 2BV / h, and the volume is controlled at 2BV, and the high-alcohol separation liquid is collected. S2: The effluent and the acid water separated liquid were mixed and concentrated to obtain 500 ml of concentrated solution. 0.05 ml of α-1,4 glucose hydrolase was added to the concentrated solution, the temperature was raised to 45°C, and hydrolysis was carried out for 2 hours. After that, the pH of the reaction system was adjusted to 3, and the α-1,4 glucose hydrolase was inactivated to obtain a hydrolyzed solution. S3: 30 g of stevioside STV was added to 500 ml of hydrolysis solution, the pH of the solution was adjusted to 6.0, the temperature was raised to 75°C, 0.5 ml of glucosyltransferase was added and reacted for 8 hours, after the reaction was completed, the temperature was raised to 95°C to inactivate the glucosyltransferase, and the reaction solution was obtained. The reaction solution was concentrated to a solid content of 45 wt%, and then spray-dried to obtain 61 g of high-purity glucosylstevioside A. S4: The high-alcohol separated liquid was concentrated to a solid content of 45 wt %, and spray-dried to obtain 68.5 g of highly pure glucosylstevioside B.

[0106] The results of the detection were that the total stevioside glycoside (TSG) content in high-purity glucosylstevioside A was 93 wt%, the glucosylstevioside (GSG) content was 78 wt%, and the dextrin content was 5.6 wt%. The total stevioside glycoside (TSG) content in high-purity glucosylstevioside B was 99.5 wt%, the glucosylstevioside (GSG) content was 93 wt%, and the dextrin content was 0.15 wt%.

[0107] The method for calculating the yield of glucosylstevioside in the following examples is as follows: Yield (%) = (actual amount of target product produced / theoretical amount of target product produced) x 100%.

[0108] In the examples below, GSG is glucosylstevioside and TSG is a generic term for all steviosides. Example 8

[0109] S1: 100 g of the glucosylstevioside crude product obtained by the reaction (TSG 85 wt%, GSG 79 wt%, dextrin 14.5 wt%) was taken, 5 L of purified water was added, and the mixture was stirred at room temperature and atmospheric pressure until the glucosylstevioside crude product was dissolved, to obtain a raw material solution with a solid content of 2 wt%. S2: The raw material liquid produced above is concentrated using a nanofiltration membrane with a molecular weight cutoff of 800 Da at an operating pressure of 4 bar, and the membrane fraction and membrane permeate are collected. S3: Using a rotary evaporator, the solvent in the membrane fraction solution and the membrane permeate solution obtained in step S2 was removed under conditions of 80 ° C and a pressure of -0.1 MPa, respectively, to obtain 85 g of intermediate product A and 15 g of intermediate product B. S4: Take 85g of intermediate product A and add 850ml of 99wt% methanol to a beaker. Place in a magnetic stirrer. Slowly add intermediate product A and continue stirring until completely dissolved. Start timing and allow to settle for 5 hours until the reaction is complete. Then, vacuum filter and collect the filter cake for use. Remove the solvent from the filtrate to obtain 83.3g of high-purity glucosylstevioside product. The glucosylstevioside content of the product is 92.3wt% (i.e., product purity), the total glycoside content is 98wt%, and the dextrin content is less than 0.5wt%. The yield of glucosylstevioside is 97.3%. S5: Intermediate product B and the filter cake obtained in S4 were combined to obtain 16.7 g of a solid sample. The solid sample was mixed with purified water in a mass ratio of 1:10 to dissolve, and 0.2% of the weight of the solid sample was added with α-1,4-glucose hydrolase. The enzyme was hydrolyzed at 40°C and pH 4.5 for 6 hours. After the enzyme hydrolysis was completed, the reaction solution was heated to 90°C to inactivate the enzyme, and a glucose-containing solution was obtained. S6: The glucose-containing solution was concentrated at 80°C to an extract-like intermediate product C with a solid content of 75 wt%. The extract-like intermediate product C was then cooled to 20°C and kept at that temperature, during which time crystals gradually precipitated. The mixture was left to stand for 6 hours, and then vacuum filtered to recover the filtrate. The filter cake was high-purity glucose, with a glucose purity of 80%. Example 9

[0110] S1: 100 g of the glucosylstevioside crude product obtained by the reaction (TSG 90 wt%, GSG 83.6 wt%, dextrin 10 wt%) was taken, 10 L of purified water was added, and the mixture was stirred at room temperature and atmospheric pressure until the glucosylstevioside crude product was dissolved, to obtain a raw material solution with a solid content of 1 wt%. S2: The raw material liquid produced above is concentrated using a nanofiltration membrane with a molecular weight cutoff of 800 Da at an operating pressure of 4 bar, and the membrane fraction and membrane permeate are collected. S3: Using a rotary evaporator, the solvent in the membrane fraction solution and the membrane permeate solution obtained in step S2 was removed under conditions of 80 ° C and a pressure of -0.1 MPa, respectively, to obtain 92 g of intermediate product A and 8 g of intermediate product B. S4: Take 92g of intermediate product A and add 850ml of 99wt% methanol to a beaker. Place in a magnetic stirrer. Slowly add intermediate product A and continue stirring until completely dissolved. Start timing and allow to settle for 8 hours until the reaction is complete. Then, vacuum filter and collect the filter cake for use. Remove the solvent from the filtrate to obtain 86g of high-purity glucosylstevioside product. The total glycoside content of the product is 98.4wt%, the glucosylstevioside content is 92.9wt%, and the dextrin content is less than 0.6wt%. The yield of glucosylstevioside is 95.6%. S5: Intermediate product B and the filter cake obtained in S4 were combined to obtain 12.5 g of a solid sample. The solid sample was mixed with purified water in a mass ratio of 1:10 to dissolve, and 0.3% of the weight of the solid sample was added with α-1,4-glucose hydrolase. The enzyme was hydrolyzed at 40°C and pH 4.5 for 7 hours. After the enzyme hydrolysis was completed, the reaction solution was heated to 90°C to inactivate the enzyme, and a glucose-containing solution was obtained. S6: The glucose-containing solution was concentrated at 90°C to produce an extract-like intermediate product C with a solid content of 70 wt%. The extract-like intermediate product C was then cooled to 20°C and kept at that temperature, during which time crystals gradually precipitated. The mixture was then left to stand for 7 hours, and then vacuum filtered to recover the filtrate. The filter cake was high-purity glucose with a purity of 83%. Example 10

[0111] S1: 100 g of the glucosylstevioside crude product obtained by the reaction (TSG 85 wt%, GSG 79 wt%, dextrin 14.5 wt%) was taken, 5 L of purified water was added, and the mixture was stirred at room temperature and atmospheric pressure until the glucosylstevioside crude product was dissolved, to obtain a raw material solution with a solid content of 2 wt%. S2: The raw material liquid produced above is concentrated using a nanofiltration membrane with a molecular weight cutoff of 700 Da at an operating pressure of 4 bar, and the membrane fraction and membrane permeate are collected. S3: Using a rotary evaporator, the solvent in the membrane fraction solution and the membrane permeate solution obtained in step S2 was removed under conditions of 80 ° C and a pressure of -0.1 MPa, respectively, to obtain 83 g of intermediate product A and 16.3 g of intermediate product B. S4: Take 83g of intermediate product A and add 850ml of 99wt% methanol to a beaker. Place in a magnetic stirrer. Slowly add intermediate product A and continue stirring until completely dissolved. Start timing and allow to settle for 5 hours until the reaction is complete. Then, vacuum filter and collect the filter cake for use. Remove the solvent from the filtrate to obtain 82.9g of high-purity glucosylstevioside product. The total glycoside content in the product was 98wt%, the glucosylstevioside content was 93.1wt%, and the dextrin content was less than 0.5wt%. The yield of glucosylstevioside was 97.7%. S5: Intermediate product B and the filter cake obtained in S4 were combined to obtain 17 g of a solid sample. The solid sample was mixed with purified water in a mass ratio of 1:10 to dissolve, and 0.2% of the weight of the solid sample was added with α-1,4-glucose hydrolase. The enzyme was hydrolyzed at 40°C and pH 4.5 for 6 hours. After the enzyme hydrolysis was completed, the reaction solution was heated to 90°C to inactivate the enzyme, and a glucose-containing solution was obtained. S6: The glucose-containing solution was concentrated at 80°C to an extract-like intermediate product C with a solid content of 75 wt%. The extract-like intermediate product C was then cooled to 20°C and kept at that temperature, during which time crystals gradually precipitated. The mixture was left to stand for 6 hours, and then vacuum filtered to recover the filtrate. The filter cake was high-purity glucose, with a glucose purity of 83%. Example 11

[0112] S1: 100 g of the glucosylstevioside crude product obtained by the reaction (TSG 85 wt%, GSG 79 wt%, dextrin 14.5 wt%) was taken, 5 L of purified water was added, and the mixture was stirred at room temperature and atmospheric pressure until the glucosylstevioside crude product was dissolved, to obtain a raw material solution with a solid content of 2 wt%. S2: The raw material liquid produced above is concentrated using a nanofiltration membrane with a molecular weight cutoff of 600 Da at an operating pressure of 4 bar, and the membrane fraction and membrane permeate are collected. S3: Using a rotary evaporator, the solvent in the membrane fraction solution and the membrane permeate solution obtained in step S2 was removed under conditions of 80°C and a pressure of -0.1 MPa, respectively, to obtain 83.9 g of intermediate product A and 15.5 g of intermediate product B. S4: Take 83.9g of intermediate product A and add 850ml of 99wt% methanol to a beaker. Place in a magnetic stirrer. Slowly add intermediate product A and continue stirring until completely dissolved. Start timing and allow to settle for 5 hours until the reaction is complete. Then, vacuum filter and collect the filter cake for use. Remove the solvent from the filtrate to obtain 80g of high-purity glucosylstevioside product, of which the total glycoside content is 99wt%, the glucosylstevioside content is 94.2wt%, and the dextrin content is less than 0.3wt%. The yield of glucosylstevioside is 95.4%. S5: Intermediate product B and the filter cake obtained in S4 were combined to obtain 16 g of a solid sample. The solid sample was mixed with purified water in a mass ratio of 1:10 to dissolve, and 0.2% of the weight of the solid sample was added with α-1,4-glucose hydrolase. The enzyme was hydrolyzed at 40°C and pH 4.5 for 6 hours. After the enzyme hydrolysis was completed, the reaction solution was heated to 90°C to inactivate the enzyme, and a glucose-containing solution was obtained. S6: The glucose-containing solution was concentrated at 80°C to an extract-like intermediate product C with a solid content of 75 wt%. The extract-like intermediate product C was then cooled to 20°C and kept at that temperature, during which time crystals gradually precipitated. The mixture was left to stand for 6 hours, and then vacuum filtered to recover the filtrate. The filter cake was high-purity glucose, with a glucose purity of 82%. Example 12

[0113] S1: 100 g of the glucosylstevioside crude product obtained by the reaction (TSG 85 wt%, GSG 79 wt%, dextrin 14.5 wt%) was taken, 5 L of purified water was added, and the mixture was stirred at room temperature and atmospheric pressure until the glucosylstevioside crude product was dissolved, to obtain a raw material solution with a solid content of 2 wt%. S2: The raw material liquid produced above is concentrated using a nanofiltration membrane with a molecular weight cutoff of 500 Da at an operating pressure of 4 bar, and the membrane fraction and membrane permeate are collected. S3: Using a rotary evaporator, the solvent in the membrane fraction solution and the membrane permeate solution obtained in step S2 was removed under conditions of 80 ° C and a pressure of -0.1 MPa, respectively, to obtain 88.6 g of intermediate product A and 11 g of intermediate product B. S4: Take 88g of intermediate product A and add 850ml of 99wt% methanol to a beaker. Place in a magnetic stirrer. Slowly add intermediate product A and continue stirring until completely dissolved. Start timing and allow to settle for 5 hours until the reaction is complete. Then, vacuum filter and collect the filter cake for use. Remove the solvent from the filtrate to obtain 81.9g of high-purity glucosylstevioside product. The total glycoside content in the product was 97.3wt%, the glucosylstevioside content was 91.2wt%, and the dextrin content was less than 1.0wt%. The yield of glucosylstevioside was 94.5%. S5: Intermediate product B and the filter cake obtained in S4 were combined to obtain 17 g of a solid sample. The solid sample was mixed with purified water in a mass ratio of 1:10 to dissolve, and 0.2% of the weight of the solid sample was added with α-1,4-glucose hydrolase. The enzyme was hydrolyzed at 40°C and pH 4.5 for 6 hours. After the enzyme hydrolysis was completed, the reaction solution was heated to 90°C to inactivate the enzyme, and a glucose-containing solution was obtained. S6: The glucose-containing solution was concentrated at 80°C to an extract-like intermediate product C with a solid content of 75 wt%. The extract-like intermediate product C was then cooled to 20°C and kept at that temperature, during which time crystals gradually precipitated. The mixture was left to stand for 6 hours, and then vacuum filtered to recover the filtrate. The filter cake was high-purity glucose, with a glucose purity of 85%. Example 13

[0114] S1: 100 g of the glucosylstevioside crude product obtained by the reaction (TSG 85 wt%, GSG 79 wt%, dextrin 14.5 wt%) was taken, 5 L of purified water was added, and the mixture was stirred at room temperature and atmospheric pressure until the glucosylstevioside crude product was dissolved, to obtain a raw material solution with a solid content of 2 wt%. S2: The raw material liquid produced above is concentrated using a nanofiltration membrane with a molecular weight cutoff of 800 Da at an operating pressure of 4 bar, and the membrane fraction and membrane permeate are collected. S3: Using a rotary evaporator, the solvent in the membrane fraction solution and the membrane permeate solution obtained in step S2 was removed under conditions of 80 ° C and a pressure of -0.1 MPa, respectively, to obtain 84.6 g of intermediate product A and 15 g of intermediate product B. S4: Take 84.6g of intermediate product A and add 850ml of 99wt% methanol to a beaker. Place in a magnetic stirrer. Slowly add intermediate product A and continue stirring until completely dissolved. Start timing and allow to settle for 5 hours until the reaction is complete. Then, vacuum filter and collect the filter cake for use. Remove the solvent from the filtrate to obtain 83.5g of high-purity glucosylstevioside product. The total glycoside content in the product was 97.8wt%, the glucosylstevioside content was 92.9wt%, and the dextrin content was less than 0.5wt%. The yield of glucosylstevioside was 98.2%. S5: Intermediate product B and the filter cake obtained in S4 were combined to obtain 16.5 g of a solid sample. The solid sample was mixed with purified water in a mass ratio of 1:10 to dissolve, and 0.2% of the weight of the solid sample was added with α-1,4-glucose hydrolase. The enzyme was hydrolyzed at 45°C and pH 4.5 for 8 hours. After the enzyme hydrolysis was completed, the reaction solution was heated to 90°C to inactivate the enzyme, and a glucose-containing solution was obtained. S6: The glucose-containing solution was concentrated at 80°C to an extract-like intermediate product C with a solid content of 75 wt%. The extract-like intermediate product C was then cooled to 20°C and kept at that temperature, during which time crystals gradually precipitated. The mixture was left to stand for 6 hours, and then vacuum filtered to recover the filtrate. The filter cake was high-purity glucose, with a glucose purity of 84.3%. Example 14

[0115] S1: 100 g of the glucosylstevioside crude product obtained by the reaction (TSG 85 wt%, GSG 79 wt%, dextrin 14.5 wt%) was taken, 5 L of purified water was added, and the mixture was stirred at room temperature and atmospheric pressure until the glucosylstevioside crude product was dissolved, to obtain a raw material solution with a solid content of 2 wt%. S2: The raw material liquid produced above is concentrated using a nanofiltration membrane with a molecular weight cutoff of 800 Da at an operating pressure of 4 bar, and the membrane fraction and membrane permeate are collected. S3: Using a rotary evaporator, the solvent in the membrane fraction solution and the membrane permeate solution obtained in step S2 was removed under conditions of 80 ° C and a pressure of -0.1 MPa, respectively, to obtain 84.8 g of intermediate product A and 14.9 g of intermediate product B. S4: Take 84g of intermediate product A and add 850ml of 99wt% methanol to a beaker. Place in a magnetic stirrer. Slowly add intermediate product A and continue stirring until completely dissolved. Start timing and allow to settle for 5 hours until the reaction is complete. Then, vacuum filter and collect the filter cake for use. Remove the solvent from the filtrate to obtain 82.5g of high-purity glucosylstevioside product. The total glycoside content in the product is 98.1wt%, the glucosylstevioside content is 92.9wt%, and the dextrin content is less than 0.7wt%. The yield of glucosylstevioside is 97%. S5: Intermediate product B and the filter cake obtained in S4 were combined to obtain 17 g of a solid sample. The solid sample was mixed with purified water in a mass ratio of 1:10 to dissolve, and 0.2% of the weight of the solid sample was added with α-1,4-glucose hydrolase. The enzyme was hydrolyzed at 35°C and pH 4.5 for 12 hours. After the enzyme hydrolysis was completed, the reaction solution was heated to 90°C to inactivate the enzyme, and a glucose-containing solution was obtained. S6: The glucose-containing solution was concentrated at 80°C to an extract-like intermediate product C with a solid content of 75 wt%. The extract-like intermediate product C was then cooled to 20°C and kept at that temperature, during which time crystals gradually precipitated. The mixture was left to stand for 8 hours, and then vacuum filtered to recover the filtrate. The filter cake was high-purity glucose, with a glucose purity of 83.6%. Example 15

[0116] S1: 100 g of the glucosylstevioside crude product obtained by the reaction (TSG 85 wt%, GSG 79 wt%, dextrin 14.5 wt%) was taken, 5 L of purified water was added, and the mixture was stirred at room temperature and atmospheric pressure until the glucosylstevioside crude product was dissolved, to obtain a raw material solution with a solid content of 2 wt%. S2: The raw material liquid produced above is concentrated using a nanofiltration membrane with a molecular weight cutoff of 800 Da at an operating pressure of 4 bar, and the membrane fraction and membrane permeate are collected. S3: Using a rotary evaporator, the solvent in the membrane fraction solution and the membrane permeate solution obtained in step S2 was removed under conditions of 80°C and a pressure of -0.1 MPa, respectively, to obtain 86.1 g of intermediate product A and 13.8 g of intermediate product B. S4: Take 86g of intermediate product A and add 850ml of 99wt% methanol to a beaker. Place in a magnetic stirrer. Slowly add intermediate product A and continue stirring until completely dissolved. Start timing and allow to settle for 5 hours until the reaction is complete. Then, vacuum filter and collect the filter cake for use. Remove the solvent from the filtrate to obtain 84g of high-purity glucosylstevioside product. The total glycoside content in the product is 98.5wt%, the glucosylstevioside content is 93.1wt%, and the dextrin content is less than 0.6wt%. The yield of glucosylstevioside is 99%. S5: Intermediate product B and the filter cake obtained in S4 were combined to obtain 15 g of a solid sample. The solid sample was mixed with purified water in a mass ratio of 1:10 to dissolve, and 0.2% of the weight of the solid sample was added with α-1,4-glucose hydrolase. The enzyme was hydrolyzed at 30°C and pH 4 for 6 hours. After the enzyme hydrolysis was completed, the reaction solution was heated to 90°C to inactivate the enzyme, and a glucose-containing solution was obtained. S6: The glucose-containing solution was concentrated at 80°C to an extract-like intermediate product C with a solid content of 75 wt%. The extract-like intermediate product C was then cooled to 20°C and kept at that temperature, during which time crystals gradually precipitated. The mixture was left to stand for 6 hours, and then vacuum filtered to recover the filtrate. The filter cake was high-purity glucose, with a glucose purity of 80%.

[0117] From the above, the purity of the glucose produced in the examples of the present application was high at 85%, the total glycoside content of the produced product was high at 99 wt%, the glucosylstevioside content was high at 94.2 wt%, and the yield of glucosylstevioside was high at 99%.

[0118] Furthermore, after reading the content of the present invention described herein, those skilled in the art will understand that various changes or modifications can be made to the present invention, and that equivalents thereof are also within the scope defined by the claims of this application.

Claims

1. (1) mixing mother liquor sugar, maltodextrin, and water, stirring until the maltodextrin is dissolved, and then adding an enzyme to perform enzymatic hydrolysis to obtain an enzymatic hydrolyzed solution; (2) adding activated carbon to the enzymatic decomposition solution obtained in step (1) to remove impurities, thereby obtaining an impurity-removed solution; subjecting the impurity-removed solution to enzyme inactivation treatment, filtering the solution, and introducing the resulting filtrate into a macroporous adsorption resin for adsorption treatment; (3) performing separation using an acid-alcohol solution having a pH of 1 to 3 composed of an ethanol solution and a hydrochloric acid solution to obtain a separated liquid; (4) adjusting the pH of the separated liquid to 5-6, and then concentrating the separated liquid using a nanofiltration membrane to obtain a concentrated liquid; (5) A method for producing glucosylstevioside using mother liquor sugar, comprising the steps of adding a texture modifier to the concentrated liquid and then obtaining glucosylstevioside by spray drying.

2. 2. The method for producing glucosylstevioside using a mother liquor sugar according to claim 1, wherein in step (1), the mass ratio of the mother liquor sugar, the maltodextrin, and the water is 1:(1-3):(5-10).

3. In the step (1), the enzyme is a mixture of glucosyltransferase and amylase, the glucosyltransferase is α-cyclodextrin glucosyltransferase, the glucosyltransferase is added in the form of a glucosyltransferase solution, the concentration of the glucosyltransferase solution is 0.8 to 1.2 wt %, the amylase is added in the form of a solid amylase, and the concentration of the solid amylase is 0.4 to 0.6 wt %, and / or the amount of glucosyltransferase solution added per 100 g of mother liquor sugar is 0.5 to 2 ml, and the amount of solid amylase added per 100 g of mother liquor sugar is 0.1 to 1 g.

4. The method for producing glucosylstevioside using mother liquor sugar according to claim 1, wherein in step (1), the enzymatic degradation conditions are an enzymatic degradation temperature of 60 to 80°C, a pH of 5.5 to 6.0, and an enzymatic degradation time of 36 to 48 hours.

5. 2. The method for producing glucosylstevioside using mother liquor sugar according to claim 1, wherein in step (2), the amount of activated carbon added is 1 to 3% of the mass of the enzymatic hydrolyzed solution, and the impurity removal time is 1 to 6 hours.

6. 2. The method for producing glucosylstevioside using mother liquor sugar according to claim 1, wherein in step (2), the supply volume of the filtrate when adsorbed onto the macroporous adsorption resin is 30 to 60% of the volume of the macroporous adsorption resin, and the supply rate of the filtrate is 0.5 to 2 BV / h.

7. 7. The method for producing glucosylstevioside using mother liquor sugar according to claim 6, further comprising washing the macroporous adsorption resin with pure water before separating the macroporous adsorption resin after adsorption, wherein the amount of pure water used during washing is 1 to 3 BV of the volume of the macroporous adsorption resin, and the flow rate of the pure water is 1 to 3 BV / h.

8. 2. The method for producing glucosylstevioside using mother liquor sugar according to claim 1, wherein in step (3), the concentration of the ethanol solution is 40-60 v / v%, the concentration of the hydrochloric acid solution is 0.3-0.5 wt%, the volume of the acid-alcohol solution during separation is 2-4 BV of the volume of the macroporous adsorption resin after adsorption, and the flow rate of the acid-alcohol solution is 1-3 BV / h.

9. 2. The method for producing glucosylstevioside using mother liquor sugar according to claim 1, wherein in step (4), the molecular weight cutoff of the nanofiltration membrane is 400-500 Da.

10. 2. The method for producing glucosylstevioside using mother liquor sugar according to claim 1, wherein in step (5), the texture modifier is a composition of erythritol, sodium alginate, and valine, the mass ratio of the three is 2:(2-4):(5-8), and the amount of the texture modifier added is 0.1-0.5% of the mass of the product in the concentrated liquid.

11. (1) dissolving glucosylstevioside crude product in ethanol solution, then adsorbing the resulting glucosylstevioside ethanol solution with macroporous adsorption resin, collecting the effluent, then separating it with an acidic aqueous solution and a high-purity ethanol solution in sequence, and collecting the acidic aqueous separation liquid and the high-alcohol separation liquid; (2) concentrating the mixture of the effluent and the acid water separated liquid obtained in the step (1), adding α-1,4 glucose hydrolase to the concentrate, raising the temperature to cause a hydrolysis reaction, and adjusting the pH of the reaction system to 3 after completion of the hydrolysis to inactivate the α-1,4 glucose hydrolase, thereby obtaining a hydrolyzed liquid; (3) adding stevioside to the hydrolyzed solution, then adjusting the pH of the solution to 6.0, adding glucosyltransferase, and reacting at elevated temperature; after the reaction is completed, increasing the temperature to inactivate the glucosyltransferase to obtain a reaction solution; concentrating the reaction solution to a solid content of 40-50 wt %; and then spray-drying the concentrate to obtain high-purity glucosylstevioside A. (4) concentrating the high-alcohol separated liquid to a solid content of 40 to 50 wt %, and then spray-drying the concentrated liquid to obtain high-purity glucosylstevioside B.

12. 12. The method for purifying glucosylstevioside according to claim 11, wherein in step (1), the concentration of the ethanol solution is 5 to 15 wt %, and the concentration of glucosylstevioside in the glucosylstevioside ethanol solution is 30 to 50 g / L.

13. The method for purifying glucosylstevioside according to claim 11, characterized in that in step (1), the flow rate of the glucosylstevioside ethanol solution during the adsorption treatment is 0.25 BV / h to 1 BV / h.

14. 12. The method for purifying glucosylstevioside according to claim 11, wherein in step (1), the aqueous acid solution is a hydrochloric acid solution with a concentration of 0.04-0.06 wt %, the high-purity ethanol solution has a concentration of 70-80 wt %, and during separation, the aqueous acid solution has a volume of 2-3 BV and a flow rate of 2-3 BV / h, and the high-purity ethanol solution has a volume of 2-3 BV and a flow rate of 2-3 BV / h.

15. The method for purifying glucosylstevioside according to claim 11, characterized in that in step (1), the volume of the effluent is 1 to 2 BV of the volume of the macroporous adsorption resin, and the volume of the acid water separation liquid is 2 to 3 BV of the volume of the macroporous adsorption resin.

16. The method for purifying glucosyl stevioside according to claim 11, characterized in that in step (2), the concentration factor during the concentration treatment is 3 to 5 times.

17. 12. The method for purifying glucosylstevioside according to claim 11, wherein in step (2), 0.03 to 0.1 ml of the α-1,4 glucose hydrolase is used per 100 g of crude glucosylstevioside.

18. The method for purifying glucosyl stevioside according to claim 11, characterized in that in step (2), the temperature of the hydrolysis reaction is 40 to 50°C and the time of the hydrolysis reaction is 1 to 3 hours.

19. The method for purifying glucosylstevioside according to claim 11, wherein in step (3), the stevioside is stevioside STV, and 0.3 to 0.5 ml of the glucosyltransferase is used per 100 g of glucosylstevioside crude product.

20. The method for purifying glucosyl stevioside according to claim 11, characterized in that in step (3), the temperature-raised reaction is carried out at a temperature of 70 to 80°C for 6 to 10 hours, and the temperature-raised inactivation is 95°C.

21. (1) adding the glucosylstevioside crude product to purified water and dissolving it again to obtain a raw material solution; (2) subjecting the raw material liquid to nanofiltration concentration treatment and collecting a membrane fraction liquid and a membrane permeate liquid; (3) removing the solvent from the membrane fraction and the membrane permeate, respectively, to obtain intermediate products A and B; (4) mixing the intermediate product A with methanol to precipitate, and then vacuum suction filtering to collect the filter cake and filtrate, and drying the filtrate to obtain high-purity glucosylstevioside; (5) mixing the intermediate product B and the filter cake, adding the purified water, stirring until the intermediate product B and the filter cake are dissolved, adding α-1,4-glucose hydrolase to carry out enzymatic decomposition treatment, and after the enzymatic decomposition is completed, heating the raw material liquid to inactivate the enzyme, thereby obtaining a glucose-containing solution; (6) A method for recovering glucose by separating and purifying glucosylstevioside, comprising the steps of concentrating the glucose-containing solution at a high temperature to obtain an extract-like intermediate product C, gradually lowering the temperature of the extract-like intermediate product C to crystallize it, and finally filtering it by vacuum suction, recovering the filtrate and using it in subsequent production, wherein the filter cake is high-purity glucose.

22. The method for separating and purifying glucosylstevioside to recover glucose according to claim 21, characterized in that in step (1), the solid content of the raw material liquid is 1 wt% to 5 wt%.

23. 22. The method for separating and purifying glucosylstevioside to recover glucose according to claim 21, wherein in step (2), the molecular weight cutoff of the nanofiltration membrane during nanofiltration concentration is 500 Da to 800 Da, and the operating pressure during nanofiltration concentration is 3.5 to 5 bar.

24. The method for separating and purifying glucosylstevioside to recover glucose according to claim 21, characterized in that in step (3), the solvent in the membrane permeate and the membrane fraction is removed under conditions of a temperature of 80°C and a pressure of -0.1 MPa.

25. 22. The method for separating and purifying glucosylstevioside to recover glucose according to claim 21, wherein in step (4), the concentration of the methanol is 95 wt% to 99 wt%, and the mass ratio of the intermediate product A to the methanol is 1:(5 to 10).

26. The method for separating and purifying glucosylstevioside to recover glucose according to claim 21, characterized in that in step (4), the mixing and sedimentation time is 2 to 8 hours.

27. 22. The method for separating and purifying glucosylstevioside to recover glucose according to claim 21, wherein in step (5), the amount of α-1,4-glucose hydrolase added is 0.2% to 0.3% of the total weight of the intermediate product B and the filter cake.

28. The method for separating and purifying glucosylstevioside to recover glucose according to claim 21, characterized in that in step (5), the enzymatic decomposition treatment is carried out at a temperature of 35°C to 45°C, at a pH of 4.0 to 4.5, for 5 to 7 hours, and by heating the raw material solution to 90°C to inactivate the enzyme.

29. 22. The method for separating and purifying glucosylstevioside to recover glucose according to claim 21, wherein in step (6), the temperature of the high-temperature concentration treatment is 80°C to 90°C, and the solid content of the extract-like intermediate product C is 70 wt% to 75 wt%.

30. The method for separating and purifying glucosylstevioside and recovering glucose according to claim 21, characterized in that in step (6), the temperature is gradually lowered to 20°C at a rate of 10°C / h to 15°C / h, and the temperature is maintained for 5 to 7 hours to allow crystallization.

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