Method for preparing glucosyl stevioside using mother liquor sugar, separating and purifying glucosyl stevioside and recovering glucose

Abstract

The present invention belongs to the technical field of food additives. A method for preparing glucosyl stevioside using a mother liquor sugar, separating and purifying the glucosyl stevioside and recovering glucose. Provided is a method for preparing glucosyl stevioside using a mother liquor sugar. By taking a mother liquor sugar as a raw material to prepare glucosyl stevioside, the route of utilization of mother liquor sugars is enriched, the technical gap of preparing glucosyl stevioside using a mother liquor sugar as a raw material is filled, and the prepared product has the advantages of good mouthfeel and high total glycoside content. Provided is a method for purifying glucosyl stevioside, wherein dextrin is recycled, which reduces the discharge of waste liquid, greatly reduces the energy consumption and cost of production, and improves the yield of glucosyl stevioside with high purity. Further provided is a method for separating and purifying glucosyl stevioside and recovering glucose, which method is simple in terms of process and easy to operate, and can not only obtain glucosyl stevioside with high purity, but also recycle unreacted substrates so as to reduce the cost.

Description

Method for preparing glucose-based steviol glycosides from mother liquor sugar, separating and purifying glucose-based steviol glycosides, and recovering glucose

[0001] This application claims the priority of the Chinese patent application number 202311848635.2, filed with the Patent Office of China on December 29, 2023, and entitled “A method for preparing glucosyl stevioside using mother liquor sugar”, the priority of the Chinese patent application number 202310879842.8, filed with the Patent Office of China on July 18, 2023, and entitled “A method for purifying glucosyl stevioside”, and the priority of the Chinese patent application number 202311239776.4, filed with the Patent Office of China on September 25, 2023, and entitled “A method for separating and purifying glucosyl stevioside and recovering glucose”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to the technical field of food additives, and in particular to a method for preparing glucose-based steviol glycosides by utilizing mother liquor sugar, separating and purifying glucose-based steviol glycosides, and recovering glucose. Background Art

[0003] Stevioside is a natural sweetener extracted from the herbaceous plant Stevia rebaudiana. It boasts high sweetness and low calories, with a sweetness 100 to 300 times greater than sucrose and only 1 / 300 of its calories. Stevioside has both sweet and bitter tastes, and the quality of both flavors is related to the number of sugar groups attached to the main component and the type of glycosidic bonds between them.

[0004] Stevioside mother liquor sugar (MLS) is a powdered solid obtained by spray drying the remaining crystallized mother liquor after high-purity rebaudioside A (RA) and stevioside (ST) have been recovered through solution extraction. Generally, steviol glycoside mother liquor sugar is sold as a low-cost sweetener. Another approach involves separating the steviol glycosides (e.g., RA, STV, RC, etc.) from the mother liquor sugar using resin methods (e.g., mixed resin chromatography, modified resin chromatography, etc.), and then further utilizing the separated steviol glycosides. However, the total glycoside content in the mother liquor sugar is low, making it unqualified as a stevioside product. Furthermore, the presence of residual polyphenols and flavonoids not only increases the bitterness of the mother liquor sugar but also hinders the recovery of glycosides.

[0005] Glucosyl steviol glycosides are obtained by mixing steviol glycosides with dextrin and enzymatically glucosylating the steviol glycosides. This involves covalently attaching one or more glucosyl groups to the steviol glycosides, followed by evaporation, concentration, and spray drying. These modified products offer improved flavor and reduced bitterness, and have been approved for use in countries such as the United States and China. In actual production, the DE value of dextrin is related to its degree of hydrolysis. Furthermore, the higher the content of dextrin and macromolecular polysaccharides, the higher the product viscosity, leading to low glucosyl conversion efficiency.

[0006] Based on the above problems, how to use mother liquor sugar to efficiently prepare glucose-based steviol glycosides has become an urgent problem to be solved.

[0007] Glucosyl steviol glycoside is a flavoring and fragrance widely used in the food and beverage industry. During industrial production, Glucosyl steviol glycoside needs to be purified to achieve high purity and purity requirements.

[0008] Currently, the most commonly used method for purifying glucosyl steviol glycosides is through ion exchange chromatography. Ion exchange chromatography is a separation method based on the interaction between ions in the sample and the stationary phase. In an ion exchange chromatography column, the stationary phase carries ionic functional groups that can selectively adsorb and release target components in the sample. Strong cation exchange resins are commonly used in the purification of glucosyl steviol glycosides. The concentrated solution is added to the strong cation exchange resin column. Under appropriate elution conditions, the glucosyl steviol glycosides are adsorbed onto the resin, while other unrelated components are eluted. Finally, by adjusting the elution conditions, the glucosyl steviol glycosides adsorbed on the resin can be eluted to obtain a relatively pure product.

[0009] In the prior art, a large amount of auxiliary material dextrin is generated during the preparation and purification process of glucosyl steviol glycosides. The subsequent waste liquid needs to be treated before it can be discharged, which increases production energy consumption and costs.

[0010] The conversion process of glucosyl steviol glycosides is not a simple chemical reaction (A+B=C). Due to the properties of the enzymes used, it will present a variety of combinations of sugar chains and glycosyl ligands. Glucosyl steviol glycosides with different grafting numbers appear in the reaction system. In addition, the reaction conversion process is difficult to control, and the reaction degree cannot reach 100%. As a result, the purity of the obtained glucosyl steviol glycosides is low, so they need to be separated and purified.

[0011] Patent application number 202010447914.8 provides a method for separating and purifying glucosyl steviol glycosides, specifically: the material generated in the previous process is subjected to ion exchange resin to remove cations in the material; the material is 0.5-1m 3 / h pumped into the simulated moving bed, controlled temperature 25-40℃, time 1-2.5h; calculated total time according to feeding speed, intercepted corresponding materials in sections according to 1:3:1 time period, controlled feeding speed 0.3-2m 3 / h, temperature 20-35°C, feeding time 0.5-3h; the material obtained after 30-90 minutes is pumped into a single-effect concentrator, the pressure is controlled at -0.6 to -0.9 MPa, the temperature is 45°C-60°C, and it is concentrated to a sugar concentration of over 50%. The concentrated material is filtered and sent to a concentrated sugar liquid tank for spray drying. In the above application, the material is decolorized with resin and then separated using a simulated moving bed. Although high-purity glucosyl steviol glycosides can be obtained, the equipment requirements are high and the cost is high.

[0012] Summary of the Invention

[0013] The technical problem to be solved by the present invention is: in view of the shortcomings of the existing technology, a preparation method of glucose-based steviol glycosides using mother liquor sugar is provided. The present invention enriches the ways to utilize mother liquor sugar and fills the technical gap in preparing glucose-based steviol glycosides using mother liquor sugar. The glucose-based steviol glycosides prepared from mother liquor sugar have good taste, high yield and high total glycoside content.

[0014] Provided is a method for purifying glucosyl steviol glycosides. The method optimizes and improves the purification process of glucosyl steviol glycosides and effectively recycles the auxiliary material dextrin, thereby reducing the discharge of waste liquid, greatly lowering the energy consumption and cost of production, and improving the yield of high-purity glucosyl steviol glycosides.

[0015] Provided is a method for separating and purifying glucose-based steviol glycosides and recovering glucose. According to the method, glucose-based steviol glycosides are dissolved, and then concentrated, nanofiltered, flocculated and settled, and cooled and crystallized to obtain high-purity glucose-based steviol glycosides, while glucose recovery is achieved at low cost.

[0016] In order to solve the above technical problems, the technical solution of the present invention is:

[0017] A method for preparing glucosyl steviol glycoside using mother liquor sugar comprises the following steps:

[0018] (1) mixing mother liquor sugar, maltodextrin and water and stirring until the solid is dissolved, and adding enzyme for enzymolysis to obtain enzymolysis solution;

[0019] (2) adding activated carbon to the enzymatic hydrolyzate obtained in step (1) to remove impurities, obtaining a decontaminated liquid, performing enzyme inactivation treatment on the decontaminated liquid, and then filtering the decontaminated liquid, and the obtained filtrate enters a macroporous adsorption resin for adsorption treatment;

[0020] (3) performing decomposition using an acid-alcohol solution with a pH of 1 to 3 consisting of an ethanol solution and a hydrochloric acid solution to obtain a decomposition solution;

[0021] (4) adjusting the pH of the analytical solution to 5-6, and then concentrating the solution using a nanofiltration membrane to obtain a concentrated solution;

[0022] (5) adding a taste modifier to the concentrated solution, and then spray drying to obtain a glucose-based stevioside product.

[0023] As an improved technical solution, in step (1), the mass ratio of the mother liquor sugar, maltodextrin and water is 1: (1-3): (5-10).

[0024] As an improved technical solution, in step (1), the enzyme is a mixture of glucosyltransferase and amylase, the glucosyltransferase is α-cyclodextrin glucosyltransferase, and when added, the glucosyltransferase is added in the form of a glucosyltransferase solution, and 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%.

[0025] Furthermore, the amount of glucosyltransferase solution added to 100 g of mother sugar solution is 0.5 to 2 ml, and the amount of solid amylase added to 100 g of mother sugar solution is 0.1 to 1 g.

[0026] As an improved technical solution, in step (1), the enzymatic hydrolysis conditions are: enzymatic hydrolysis temperature is 60-80°C, pH is 5.5-6.0, and enzymatic hydrolysis time is 36-48h.

[0027] As an improved technical solution, in step (2), the amount of activated carbon added is 1 to 3% of the mass of the enzymatic hydrolyzate, and the impurity removal time is 1 to 6 hours.

[0028] As an improved technical solution, in step (2), when macroporous adsorption resin is used for adsorption, the feed volume of the filtrate is 30-60% of the volume of the macroporous adsorption resin, and the feed rate is 0.5-2 BV / h.

[0029] As an improved technical solution, before analyzing the adsorbed macroporous adsorption resin, it also includes: washing the macroporous adsorption resin with pure water, 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 is 1 to 3 BV / h.

[0030] 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 analysis, the volume of the acid-alcohol solution is 2-4 BV of the volume of the macroporous adsorption resin, and the flow rate is 1-3 BV / h.

[0031] As an improved technical solution, in step (4), the molecular weight cut-off of the nanofiltration membrane is 400 to 500 Da.

[0032] As an improved technical solution, in step (5), the taste modifier is a composition of erythritol, sodium alginate and valine, and the mass ratio of the three is 2: (2-4): (5-8); the amount of the taste modifier added is 0.1-0.5% of the weight of the product in the concentrate.

[0033] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0034] (1) Stevioside mother liquor sugar is characterized by a low total glycoside content and a distinct bitter taste due to the presence of residual polyphenols and flavonoid impurities. The present invention pioneered the use of mother liquor sugar as a raw material, supplemented with maltodextrin as a glucose substrate, and obtained glucose-based stevioside by dual catalysis using glucosyltransferase and amylase, thereby reducing the bitterness of the mother liquor sugar. Activated carbon was then used for impurity removal and deodorization, further improving the taste of the glucose-based stevioside and removing impurities. Glucoside was then enriched and separated by adsorption on a macroporous adsorption resin. The adsorbed resin was analyzed with an acid-alcohol solution and the pH of the solution was adjusted to 5-6, thereby ensuring the stability of the glycoside substances in the subsequent concentration process and improving the yield and quality. Glucoside was further modified by adding a taste modifier to adjust its taste, and finally, after spray drying, a glucose-based stevioside product with a good taste and high total glycoside content that can be directly marketed was obtained.

[0035] (2) The present invention adds amylase to the existing glucosyltransferase to solve the problem of low glucosyl conversion rate caused by low hydrolysis degree of dextrin, large amount of dextrin and macromolecular polysaccharides, and high product viscosity; by the combined use of glucosyltransferase and amylase, the hydrolysis of dextrin and the transglycoside of glucosyl are accelerated, thereby improving the glucosyl conversion rate.

[0036] (3) The present invention uses a mixture of hydrochloric acid solution and ethanol solution for analysis, which has a good analysis effect and does not cause partial product loss, thereby greatly improving the product yield. In addition, the present invention also uses pure water to wash the resin before analysis, which not only allows the residual unadsorbed liquid in the resin to re-enter the resin for adsorption, but also effectively removes the monosaccharides and oligosaccharides produced during the transglycoside process, further improving the analysis effect and product yield.

[0037] (4) The present invention directly converts stevioside mother liquor sugar into commercially available glucose-based stevioside products through a reasonable process. Compared with the existing technology, the present invention makes more complete use of mother liquor sugar, enriches the ways to utilize mother liquor sugar, and fills the technical gap in preparing glucose-based stevioside from mother liquor sugar.

[0038] In order to solve the above technical problems, the technical solution of the present invention is:

[0039] A method for purifying glucosyl steviol glycosides comprises the following steps:

[0040] (1) dissolving crude glucosyl stevioside in an ethanol solution, then subjecting the obtained glucosyl stevioside ethanol solution to adsorption treatment with a macroporous adsorption resin, and collecting the effluent; then successively performing decomposition with an acid aqueous solution and a high-purity ethanol solution, and collecting the acid aqueous decomposition solution and the high-purity ethanol decomposition solution;

[0041] (2) concentrating the mixture of the effluent obtained in step (1) and the acid water analysis solution, adding α-1,4-glucose hydrolase to the concentrate, heating the solution for hydrolysis, adjusting the pH of the reaction system to 3 after the hydrolysis is completed, inactivating the enzyme, and obtaining a hydrolyzed solution;

[0042] (3) adding steviol glycoside to the hydrolyzate, adjusting the pH of the solution to 6.0, adding glucosyltransferase, and heating the reaction. After the reaction is completed, the temperature is further increased to inactivate the reaction to obtain a reaction solution, concentrating the reaction solution to a solid content of 40-50 wt%, and then spray drying the concentrated solution to obtain high-purity glucosyl steviol glycoside A;

[0043] (4) The high alcohol analytical solution is concentrated to a solid content of 40-50 wt%, and then the concentrated solution is spray-dried to obtain high-purity glucosyl steviol glycoside B.

[0044] As a preferred embodiment of the above technical solution, in step (1), the concentration of the ethanol solution is 5-15 wt%; the concentration of glucose stevioside in the glucose stevioside ethanol solution is 30-50 g / L.

[0045] As a preferred embodiment of the above technical solution, in step (1), during the adsorption treatment, the flow rate of the glucose-syl steviol glycoside ethanol solution is 0.25BV / h-1BV / h.

[0046] As a preferred embodiment of the above technical solution, in step (1), the acid aqueous solution is a hydrochloric acid solution with a concentration of 0.04-0.06 wt %; and the concentration of the high-purity ethanol solution is 70-80 wt %.

[0047] As a preferred embodiment of the above technical solution, in step (1), during analysis, the volume of the acid aqueous solution is 2-3BV, and the flow rate is 2-3BV / h; the volume of the high-purity ethanol solution is 2-3BV, and the flow rate is 2-3BV / h.

[0048] As a preferred embodiment of the above technical solution, in step (1), the volume of the effluent is 1-2BV of the resin volume, and the volume of the acid water analysis solution is 2-3BV of the resin volume.

[0049] As a preferred embodiment of the above technical solution, in step (2), the concentration multiple during the concentration treatment is 3-5 times.

[0050] As a preferred embodiment of the above technical solution, in step (2), 0.03-0.1 ml of α-1,4-glucose hydrolase is used for every 100 g of crude glucosyl steviol glycoside.

[0051] As a preferred embodiment of the above technical solution, in step (2), the temperature of the hydrolysis reaction is 40-50° C., and the time of the hydrolysis reaction is 1-3 h.

[0052] As a preferred embodiment of the above technical solution, in step (3), the steviol glycoside is steviol glycoside STV; and 0.3-0.5 ml of glucosyltransferase is used for every 100 g of crude glucosyl steviol glycoside.

[0053] As a preferred embodiment of the above technical solution, in step (3), the temperature of the temperature-raising reaction is 70-80°C, and the time is 6-10 hours; the temperature of the temperature-raising inactivation is 95°C.

[0054] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0055] The present invention first dissolves crude glucosyl stevioside in an ethanol solution, then subjecting it to adsorption treatment with a macroporous adsorption resin. It is then subjected to desorption treatment using an acid aqueous solution and a high-purity ethanol solution in sequence. The macroporous adsorption resin is a highly efficient adsorption material with a large specific surface area and strong adsorption capacity, effectively adsorbing the target substance while exhibiting good selectivity for other impurities. Through the action of the macroporous adsorption resin, the glucosyl stevioside can be separated from the impurities therein, thereby improving its purity. After adsorption treatment with the macroporous adsorption resin, the acid aqueous solution can desorb the dextrin adsorbed within the macroporous adsorption resin, while the high-purity ethanol solution can desorb the glucosyl stevioside from the adsorption resin, thereby improving the purity of the glucosyl stevioside and removing any remaining impurities.

[0056] In addition, the present invention further comprises mixing the effluent after adsorption by the macroporous adsorption resin and the acid water analysis liquid, adding α-1,4-glucose hydrolase to carry out a hydrolysis reaction, and then mixing with steviol glycoside to react, so as to obtain high-purity glucose steviol glycoside; the dextrin recovered by the present invention is reused to prepare high-purity glucose-based steviol glycoside, which not only reduces waste liquid discharge, reduces the production energy consumption and cost of glucose-based steviol glycoside, but also improves the yield of high-purity glucose-based steviol glycoside.

[0057] The method provided by the present invention is relatively simple to operate and does not require complex equipment or supporting conditions to achieve the adsorption separation of glucosyl steviol glycosides. The macroporous adsorption resin has a high adsorption capacity and regeneration ability, allowing for recycling. This not only improves the efficiency of the purification process but also reduces costs.

[0058] In order to solve the above technical problems, the technical solution of the present invention is:

[0059] A method for separating and purifying glucosyl steviol glycosides and recovering glucose comprises the following steps:

[0060] (1) adding purified water to dissolve the crude glucosyl stevioside to prepare a feed solution;

[0061] (2) subjecting the feed liquid to nanofiltration and concentration treatment, collecting the membrane interception liquid and the membrane permeate;

[0062] (3) Removing the solvent from the membrane interception liquid and the membrane permeate respectively to obtain intermediate product A and intermediate product B;

[0063] (4) The intermediate product A and methanol are mixed and precipitated, and then vacuum filtered to collect the filter cake and filtrate, and the filtrate is dried to obtain high-purity glucosyl stevioside;

[0064] (5) The intermediate product B and the filter cake are mixed and purified water is added, stirred until the solid is dissolved, and α-1,4-glucose hydrolase is added for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the feed liquid is heated to inactivate the enzyme to prepare a glucose solution;

[0065] (6) The glucose solution is concentrated at high temperature to obtain an extract-like intermediate product C, which is then slowly cooled and crystallized, and finally vacuum filtered. The filtrate is recovered and retained for subsequent production applications, and the filter cake is high-purity glucose.

[0066] As a preferred technical solution, in step (1), the solid content of the feed liquid is 1wt%-5wt%.

[0067] As a preferred technical solution, in step (2), when nanofiltration concentration is performed, the molecular weight cutoff of the nanofiltration membrane is 500Da-800Da, and the operating pressure during nanofiltration concentration is 3.5-5 bar.

[0068] As a preferred technical solution, in step (3), the solvent in the membrane permeate and the membrane interception liquid is removed under the conditions of a temperature of 80° C. and a pressure of −0.1 MPa.

[0069] As a preferred technical solution, in step (4), the concentration of methanol is 95wt%-99wt%, and the mass ratio of the intermediate product A to methanol is 1:(5-10).

[0070] As a preferred technical solution, in step (4), the mixing and sedimentation time is 2h-8h.

[0071] As a preferred technical solution, in step (5), the amount of α-1,4-glucose hydrolase added is 0.2%-0.3% of the total weight of the intermediate product B and the filter cake.

[0072] As a preferred technical solution, in step (5), the temperature of the enzymatic hydrolysis treatment is 35°C-45°C, the pH is 4.0-4.5, and the time is 5h-7h; the feed liquid is heated to 90°C to inactivate the enzyme.

[0073] As a preferred technical solution, in step (6), the temperature of the high-temperature concentration treatment is 80°C-90°C, and the solid content of the extract-like intermediate product C is 70wt%-75wt%.

[0074] As a preferred technical solution, in step (6), the slow cooling rate is 10°C / h-15°C / h, and the temperature is lowered to 20°C and kept warm for crystallization for 5h-7h.

[0075] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0076] The present invention re-dissolves the crude glucosyl stevioside in water, and then uses a nanofiltration membrane to concentrate and perform preliminary purification. The relative molecular masses of unreacted rebaudioside A and glucose in the feed liquid and the product glucosyl stevioside are very different. The glucosyl stevioside can be separated to a large extent by using a 500Da-800Da nanofiltration membrane. The membrane intercepted liquid is then concentrated to remove the solvent, and methanol is added for re-purification to obtain high-purity glucosyl stevioside.

[0077] The invention removes the solvent from the membrane permeate collected in the nanofiltration process, mixes it with the filter cake in the flocculation precipitation process, adds α-1,4-glucose hydrolase to carry out enzymatic hydrolysis, and then concentrates and cools down the solution to crystallize it under appropriate conditions to obtain high-purity glucose.

[0078] In summary, the process of the present invention is simple and easy to operate. It not only achieves the purification of glucosyl steviol glycosides to obtain high-purity glucosyl steviol glycoside products, but also recovers unreacted substrates to a large extent, which can be reused, thereby reducing production costs. DETAILED DESCRIPTION

[0079] The present invention will be further described below with reference to the following examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0080] The present invention provides a method for preparing glucose-based steviol glycosides by using mother liquor sugar, comprising the following steps:

[0081] (1) mixing mother liquor sugar, maltodextrin and water and stirring until the solid is dissolved, and adding enzyme for enzymolysis to obtain enzymolysis solution;

[0082] (2) adding activated carbon to the enzymatic hydrolyzate obtained in step (1) to remove impurities, obtaining a decontaminated liquid, performing enzyme inactivation treatment on the decontaminated liquid, and then filtering the decontaminated liquid, and the obtained filtrate enters a macroporous adsorption resin for adsorption treatment;

[0083] (3) performing decomposition using an acid-alcohol solution with a pH of 1 to 3 consisting of an ethanol solution and a hydrochloric acid solution to obtain a decomposition solution;

[0084] (4) adjusting the pH of the analytical solution to 5-6, and then concentrating the solution using a nanofiltration membrane to obtain a concentrated solution;

[0085] (5) adding a taste modifier to the concentrated solution, and then spray drying to obtain a glucose-based stevioside product.

[0086] The mother liquor sugar in the present invention is derived from the crystallization mother liquor obtained by solution extraction of stevia leaves. Generally speaking, the total glycoside content thereof is 60-80%, including 20-40% RA, 10-25% STV, 10-20% RC, and 5-20% other glycosides.

[0087] The invention firstly mixes and dissolves mother liquor sugar, maltodextrin and water, and then adds enzyme to carry out enzymolysis reaction to obtain enzymolysis solution.

[0088] 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 has no particular requirements for the source of maltodextrin; commercially available products can be used. The DE value of the maltodextrin in the present invention is preferably 5-10. The present invention has no particular requirements for water; purified water is preferably used. During the mixing and dissolving process, the present invention also includes stirring, and the stirring rate is preferably 10-100 rpm.

[0089] In the present invention, the enzymatic hydrolysis reaction preferably begins after the mother liquor sugar and maltodextrin are completely dissolved. Furthermore, the enzyme is preferably a mixture of glucosyltransferase and amylase, with the glucosyltransferase preferably being α-cyclodextrin glucosyltransferase. The lower the DE value of maltodextrin, the lower the degree of dextrin hydrolysis, the more dextrin and macromolecular polysaccharides are present, and the higher the product viscosity. The combined addition of glucosyltransferase and amylase in the present invention can accelerate the hydrolysis of dextrin and the transglycosylation of glucosyl groups, thereby improving the conversion rate.

[0090] When specifically added, the glucosyltransferase in the present invention is preferably added in the form of a glucosyltransferase solution, the concentration of the glucosyltransferase solution is preferably 0.8-1.2 wt%, further 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 0.4-0.6 wt%, further preferably 0.5 wt%, and the activity of the amylase is preferably 1000-3000 U / g.

[0091] In the present invention, the amount of the glucosyltransferase solution added is preferably 0.5 to 2 ml per 100 g of mother sugar solution, and the amount of the solid amylase added is preferably 0.1 to 1 g per 100 g of mother sugar solution.

[0092] Furthermore, the conditions for the enzymatic hydrolysis reaction of the present invention 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. In addition, the present invention has no special requirements for the reagent for adjusting the pH, and preferably uses a potassium hydroxide solution or a sodium hydroxide solution; the present invention has no special restrictions on the concentrations of the potassium hydroxide solution and the sodium hydroxide solution, for example, the concentration of the potassium hydroxide solution can be 1 to 10 v / v%, and the concentration of the sodium hydroxide solution can be 1 to 10 v / v%.

[0093] In the present invention, after enzymatic hydrolysis is completed, activated carbon is added to the hydrolyzed liquid for impurity removal. The activated carbon's adsorption properties can effectively adsorb impurities such as flavonoids, saponins, and alkaloids present in the hydrolyzed liquid. Furthermore, the activated carbon also has a certain deodorizing effect, removing some of the bitterness of glucosyl steviol glycosides. Furthermore, the amount of activated carbon added is preferably 1-3% of the mass of the hydrolyzed liquid, and the impurity removal time is preferably 1-6 hours. The present invention has no particular requirements for the pore size of the activated carbon, which can, for example, be 2-50 nm. The present invention has no particular requirements for the source of the activated carbon, and commercially available products can be used.

[0094] In the present invention, after removing impurities with activated carbon to obtain a cleaned liquid, the cleaned liquid is subjected to an enzyme inactivation treatment and filtered to obtain a filtrate. Furthermore, the enzyme inactivation treatment is preferably performed at a high temperature, preferably 100-120°C. The present invention does not particularly limit the inactivation time; generally, the enzyme is inactivated when the temperature reaches 100-120°C. Furthermore, the filtration is preferably performed using plate and frame filtration, the filter cloth preferably has a pore size of 30-50 μm, and the operating pressure is preferably 0.5-2.5 MPa.

[0095] The purpose of the present invention to deactivate the enzyme in the impurity removal liquid is to stop the reaction, prevent the enzyme from affecting the final product, and ensure product quality. The purpose of the present invention to use plate and frame filtration is to filter out the activated carbon after adsorption and to filter out the inactivated enzyme.

[0096] In the present invention, the obtained filtrate is adsorbed by a macroporous adsorption resin. The present invention has no special restrictions on the type of the macroporous adsorption resin, and is preferably one of SD-9, AB-8, LX-T81, LX-T83, and LX-T28; the present invention has no special requirements on the source of the resin, and commercially available products can be used; the present invention has no special restrictions on the feeding method, for example, pumping can be used for feeding; the present invention has no special requirements on the carrier of the macroporous adsorption resin, for example, a resin column can be used. Specifically, when feeding the liquid, the feed amount of the filtrate is preferably 30-60% of the volume of the macroporous adsorption resin, and the feed rate is preferably 0.5-2BV / h. The effluent after adsorption by the macroporous adsorption resin of the present invention contains a certain amount of dextrin and glucose, which can be further recycled to prepare other valuable by-products.

[0097] Furthermore, after the filtrate is adsorbed by the macroporous adsorption resin, the macroporous adsorption resin is washed with pure water. Preferably, the amount of pure water used is 1 to 3 BV of the volume of the macroporous adsorption resin, and the flow rate is preferably 1 to 3 BV / h. The purpose of using pure water for washing in the present invention is to allow the filtrate remaining in the resin column to be adsorbed by the macroporous adsorption resin, thereby achieving full adsorption of the filtrate as much as possible and improving the adsorption rate.

[0098] In the present invention, after adsorption by the macroporous adsorption resin, the present invention desorbs the adsorbed macroporous adsorption resin and adjusts the pH of the effluent to 5-6 to obtain a desorbed solution. Specifically, the desorbent used in the desorption is an acid-alcohol solution, and the preparation method of the acid-alcohol solution includes: adding hydrochloric acid with a concentration of 0.3-0.5wt% to an ethanol solution with a concentration of 40-60v / v%, and adjusting the pH to 1-3, thereby obtaining an acid-alcohol solution. During desorption, the amount of the acid-alcohol solution is preferably 2-4BV of the volume of the macroporous adsorption resin, and the flow rate is preferably 1-3BV / h. The present invention does not specifically limit the pH regulator, and the pH regulator commonly used in the art can be used; the purpose of adjusting the pH of the effluent to 5-6 after desorption in the present invention is to avoid the destruction of glycosides due to excessive acidity during the subsequent concentration process.

[0099] In the present invention, the analytical solution is concentrated through a nanofiltration membrane to obtain a concentrated solution; the molecular weight cut-off of the nanofiltration membrane is preferably 400 to 500 Da; and the solid content of the concentrated solution is 30 to 50 wt%.

[0100] After obtaining the concentrated solution, the present invention adds a taste modifier to the concentrated solution, and spray-dries the solution to obtain a glucosyl stevioside product. Specifically, the taste modifier is a composition of erythritol, sodium alginate and valine, and the mass ratio of the three is preferably 2: (2-4): (5-8), and further preferably 2:3:5; the amount of the taste modifier added is preferably 0.1-0.5% of the mass of the product in the concentrated solution. The taste modifier used in the present invention is preferably added in solid form; the purpose of adding the taste modifier in the present invention is to adjust the taste of the product and reduce the bitterness of the product. In the present invention, the inlet air temperature of the spray drying is 180-200°C, and the outlet air temperature is 80-100°C.

[0101] To further illustrate the present invention, the following detailed description of a method for preparing glucosyl steviol glycosides from mother liquor sugars is provided in conjunction with examples. However, these examples should not be construed as limiting the scope of the present invention. In particular, unless otherwise specified, the raw materials used in the following examples and comparative examples are commercially available. The main components of the steviol glycoside mother liquor sugars in the following examples are shown in Table 1. This batch of steviol glycoside mother liquor sugars was provided by Zhucheng Haotian Pharmaceutical Co., Ltd.

[0102] Table 1 Stevioside composition of mother liquors from batches 1 to 4

[0103] Example 1

[0104] S1: 100 g of the mother liquor sugar from batch 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 solids were completely dissolved. Then, 0.5 mL of α-cyclodextrin glucosyltransferase (activity of 100 U / g) and 0.1 g of amylase (activity of 1000-3000 U / g) were added. The mixture was enzymatically hydrolyzed at 70°C and pH 5.8 for 42 h to obtain 850 mL of enzymatic hydrolyzate.

[0105] S2: Add 10 g of activated carbon with a pore size of 30 nm to 850 mL of enzymatic hydrolyzate and remove impurities for 3 h to obtain 830 mL of impurity-removed liquid;

[0106] S3: The impurity removal liquid was heated to 110°C for enzyme inactivation and then filtered through a plate and frame filter with a filter cloth pore size of 40 μm at an operating pressure of 2.0 MPa to obtain 815 mL of filtrate;

[0107] S4: 815 mL of the filtrate was fed into a resin column filled with 2 L of macroporous adsorption resin (SD-9) for adsorption treatment at a feed rate of 1 BV / h;

[0108] S5: Desorbing the adsorbed 2 L of resin with an acid-alcohol solution at a flow rate of 2 BV / h, wherein the amount of the acid-alcohol solution is 4 BV of the volume of the macroporous adsorption resin, and the pH of the effluent is adjusted to 5 to obtain 7.8 L of desorption solution; wherein the acid-alcohol solution is a solution having a pH of 2 composed of a mixture of 40 v / v% ethanol solution and 0.5 wt% hydrochloric acid solution;

[0109] S6: 7.8 L of the analytical solution was concentrated through a nanofiltration membrane with a molecular weight cutoff of 400 Da, and the nanofiltration retentate was collected to obtain 350 ml of a concentrated solution with a solid content of 40 wt%;

[0110] S7: 0.14 g of taste modifier (0.028 g of erythritol, 0.042 g of sodium alginate, and 0.07 g of valine) was added to 350 ml of the concentrate for modification, and 113 g of glucose-based stevioside product was obtained by spray drying. The inlet air temperature during spray drying was 190°C and the outlet air temperature was 90°C.

[0111] Example 2

[0112] S1: Take 100 g of the mother liquor sugar from batch 2 and 100 g of maltodextrin with a DE value of 5, add them to 500 mL of water, and stir at 10 rpm until the solids are completely dissolved. Then, add 1 ml of α-cyclodextrin glucosyltransferase (activity of 100 U / g) and 0.3 g of amylase (activity of 1000-3000 U / g), and react at 80°C and pH 5.5 for 48 h to obtain 750 mL of enzymatic hydrolyzate;

[0113] S2: Add 15 g of activated carbon with a pore size of 2 nm to 750 mL of enzymatic hydrolyzate and remove impurities for 6 h to obtain 730 mL of impurity-removed liquid;

[0114] S3: The impurity removal liquid was heated to 120°C for enzyme inactivation, and then filtered through a plate and frame filter with a filter cloth pore size of 50 μm at an operating pressure of 0.5 MPa to obtain 720 mL of filtrate;

[0115] S4: 720 mL of the filtrate was fed into a resin column filled with 2.4 L of macroporous adsorption resin (AB-8) for adsorption treatment at a feed rate of 0.5 BV / h;

[0116] S5: Desorbing the adsorbed 2.4 L of resin with an acid-alcohol solution at a flow rate of 1 BV / h, adjusting the pH of the effluent to 5.5 to obtain 7 L of desorption solution; wherein the acid-alcohol solution is a solution with a pH of 2 composed of a 50 v / v% ethanol solution and a 0.5 wt% ethanol solution;

[0117] S6: 7 L of the analytical solution was concentrated through a nanofiltration membrane with a molecular weight cutoff of 450 Da, and the nanofiltration retentate was collected to obtain 430 ml of a concentrated solution with a solid content of 35 wt%;

[0118] S7: 0.45 g of taste modifier (0.09 g of erythritol, 0.135 g of sodium alginate, and 0.225 g of valine) was added to 430 ml of the concentrate for modification, and 126 g of glucose-based stevioside product was obtained by spray drying. The inlet air temperature during spray drying was 200°C, and the outlet air temperature was 80°C.

[0119] Example 3

[0120] S1: 100 g of the mother liquor sugar from batch 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 solids were completely dissolved. Then, 1.5 mL of α-cyclodextrin glucosyltransferase (activity of 100 U / g) and 0.7 g of amylase (activity of 1000-3000 U / g) were added. The mixture was reacted at 60°C and pH 6.0 for 36 h to obtain 1200 mL of enzymatic hydrolyzate.

[0121] S2: Add 20 g of activated carbon with a pore size of 50 nm to 1200 mL of enzymatic hydrolyzate and remove impurities for 1 hour to obtain 1900 mL of impurity-removed liquid;

[0122] S3: The impurity removal liquid was heated to 100°C for enzyme inactivation, and then filtered through a plate and frame filter with a filter cloth pore size of 30 μm at an operating pressure of 2.5 MPa to obtain 1800 mL of filtrate;

[0123] S4: 1800 mL of the filtrate was fed into a resin column filled with 3 L of macroporous adsorption resin (T81) for adsorption treatment. The feed flow rate was controlled at 2 BV / h.

[0124] S5: Desorbing the adsorbed 3 L of resin with an acid-alcohol solution at a flow rate of 3 BV / h, wherein the amount of the acid-alcohol solution is 2 BV of the volume of the macroporous adsorption resin, and the pH of the effluent is adjusted to 6 to obtain 6 L of desorption solution; wherein the acid-alcohol solution is a solution with a pH of 2 composed of a 60 v / v% ethanol solution and a 0.5 wt% hydrochloric acid solution;

[0125] S6: 6 L of the analytical solution was concentrated through a nanofiltration membrane with a molecular weight cutoff of 500 Da, and the nanofiltration intercept was collected to obtain 300 ml of a concentrated solution with a solid content of 50 wt%;

[0126] S7: 0.75 g of taste modifier (0.15 g of erythritol, 0.225 g of sodium alginate, and 0.375 g of valine) was added to 300 ml of the concentrate for modification, and 131 g of glucose-based stevioside product was obtained by spray drying. The inlet air temperature during spray drying was 180°C, and the outlet air temperature was 100°C.

[0127] Example 4

[0128] S1: 100 g of the mother liquor sugar from batch 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 solids were 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. The mixture was reacted at 70°C and pH 5.6 for 40 h to obtain 850 mL of enzymatic hydrolyzate.

[0129] S2: Add 25 g of activated carbon with a pore size of 20 nm to 850 mL of enzymatic hydrolyzate and remove impurities for 2 h to obtain 830 mL of impurity-removed liquid;

[0130] S3: The impurity removal liquid was heated to 100°C for enzyme inactivation, and then filtered through a plate and frame filter with a filter cloth pore size of 40 μm at an operating pressure of 1.0 MPa to obtain 815 mL of filtrate;

[0131] S4: 815 mL of the filtrate was fed into a resin column filled with 1.5 L of macroporous adsorption resin (T83) for adsorption treatment at a flow rate of 1.5 BV / h; the adsorbed macroporous adsorption resin was then washed with 2 BV of pure water at a flow rate of 2 BV / h;

[0132] S5: Desorbing 1.5 L of washed resin with an acid-alcohol solution at a flow rate of 1.5 BV / h, using 4 BV of the macroporous adsorption resin volume, and adjusting the pH of the effluent to 5.5 to obtain 6 L of desorption solution; wherein the acid-alcohol solution is a solution with a pH of 2 composed of a 60 v / v% ethanol solution and a 0.5 wt% hydrochloric acid solution;

[0133] S6: 6 L of the analytical solution was concentrated through a nanofiltration membrane with a molecular weight cutoff of 500 Da, and the nanofiltration intercept was collected to obtain 400 ml of a concentrated solution with a solid content of 40 wt%;

[0134] S7: 0.8 g of taste modifier (0.16 g of erythritol, 0.24 g of sodium alginate, and 0.4 g of valine) was added to 400 ml of the concentrate for modification, and 145 g of glucose-based stevioside product was obtained by spray drying. The inlet air temperature during spray drying was 180° C. and the outlet air temperature was 90° C.

[0135] Comparative Example 1

[0136] Compared with Example 1, in step S2 of this comparative example, 2 L of macroporous adsorption resin SD-300 was used to replace the activated carbon, and other conditions were the same as those in Example 1.

[0137] Comparative Example 2

[0138] Compared with Example 1, this comparative example first uses activated carbon for impurity removal and then performs enzymatic hydrolysis. The specific impurity removal conditions, enzymatic hydrolysis reaction conditions and other conditions are the same as those in Example 1.

[0139] Comparative Example 3

[0140] Compared with Example 1, in this comparative example S5, hydrochloric acid solution with a pH of 2 was first used for decomposition, and then ethanol solution with a concentration of 40 v / v% was used for decomposition, and the two effluents were combined. Other conditions were the same as those in Example 1.

[0141] Comparative Example 4

[0142] Compared with Example 1, in this comparative example S5, an ethanol solution with a concentration of 40 v / v% is first used for analysis, and then a hydrochloric acid solution with a pH of 2 is used for analysis, and the two effluents are combined. Other conditions are the same as those in Example 1.

[0143] Comparative Example 5

[0144] Compared with Example 1, in this comparative example S5, the pH of the effluent was adjusted to 9 to obtain a solution, and the other conditions were the same as those in Example 1.

[0145] Comparative Example 6

[0146] Compared with Example 2, the enzyme inactivation treatment in step S3 was omitted in this comparative example, and other conditions were the same as those in Example 2.

[0147] Comparative Example 7

[0148] Compared with Example 3, in this comparative example, the macroporous adsorption resin (T81) in step S4 is replaced by an equal amount of T-28 resin, and other conditions are the same as those in Example 3.

[0149] Comparative Example 8

[0150] Compared with Example 4, the taste modifier in step S7 of this comparative example was replaced by an equal amount of sodium citrate, and other conditions were the same as those of Example 4.

[0151] 1. Product quality, purity and taste testing

[0152] The method for determining the content of glucosyl steviol glycosides in Examples 1-4 and Comparative Examples 1-8 of the present invention was carried out with reference to the national standard GB 2760-2014;

[0153] For taste testing, each sample was diluted to 500 ppm under the following conditions: temperature 29°C, humidity 50%. Specifically, a sensory evaluation panel of 10 professional sensory evaluators was selected according to GB / T 16291.2-2010. The evaluators were required to restrict their diet for 1 hour prior to the start of the experiment, particularly those that could significantly affect taste. The evaluators scored the bitterness of the samples on a 10-point scale, with the final scores averaged. The evaluation standard was a 5wt% sucrose solution. The test results are shown in Table 2.

[0154] Table 2

[0155] It can be seen from the test results in Table 2 that, compared with the comparative example, the present invention uses mother liquor sugar as raw material and optimizes the conditions to produce glucose-syl steviol glycosides with not only high yield but also high total glycoside content and excellent product taste.

[0156] 2. Sweetener sensory evaluation experiment

[0157] The present invention set up four sets of comparative experiments to test the taste difference between the glucose-based steviol glycosides prepared by the present invention and the steviol glycosides in the original mother liquor sugar. The samples were diluted to 500ppm. This is mainly because the sweetness of steviol glycoside products is relatively high, and dilution is required for differentiation and evaluation. The details are as follows:

[0158] Experiment 1: 500 ppm mother liquor sugar (total glycosides 68%);

[0159] Experiment 2: 500 ppm of glucosyl steviol glycoside prepared in Example 1;

[0160] Experiment 3: 500 ppm mother liquor sugar (total glycosides 80%);

[0161] Experiment 4: 500 ppm of glucosyl steviol glycoside prepared in Example 4;

[0162] The evaluation standard was a 5 wt% sucrose solution.

[0163] Test conditions: temperature 29°C, humidity 50RH%;

[0164] Test time: May 16, 2023.

[0165] According to the provisions of GB / T 16291.2-2010, 10 professional sensory evaluators were selected to form a sensory evaluation team.

[0166] During the specific evaluation, sensory evaluators restricted their diet within 1 hour before the start of the evaluation experiment, especially limiting the consumption of foods that can seriously affect the taste.

[0167] Sensory evaluators scored the sweeteners based on a comprehensive range of indicators, including sweetness, onset of sweetness, aftertaste, bitterness, astringency, off-flavors, and overall preference. Onset of sweetness describes how quickly the sweetness is perceived, while aftertaste describes how long the sweetness lasts. All scores were scored on a 10-point scale, with the exception of overall preference, which was rated on a 100-point scale (a 5wt% sucrose solution had a full score of 100). The final score was averaged. The taste evaluation results are shown in Table 3 below, where higher values ​​for each sensory component indicate a more pronounced taste characteristic.

[0168] Table 3

[0169] From the test results in Table 3, it can be seen that the glucose-syl steviol glycoside prepared by the present invention has improved taste in all aspects compared with the original mother liquor sugar, especially in sweetness and sweetening speed, while significantly reducing the bitterness and astringency in the raw mother liquor sugar.

[0170] In the following examples, the content of total stevioside (TSG) in 100 g of crude glucosyl steviol glycoside is 71 wt %, the content of glucosyl steviol glycoside (GSG) is 59 wt %, and the content of dextrin is 28 wt %.

[0171] Qualitative analysis method of the product: The transglycoside products were qualitatively analyzed by liquid chromatography-tandem quadrupole time-of-flight mass spectrometry. The detection conditions were as follows: ACQUITY UPLC BEH HILIC amino column, column temperature at 30°C, gradient elution at acetonitrile:water = 80:20, (2 min)-50:50 (30 min) (v / v), injection volume 1 μL, injection concentration 5 mg / mL, flow rate 0.3 mL / min; mass spectrometry conditions were collision voltage of 6 eV; ionization mode electrospray ionization (ESI), negative ion detection mode, molecular weight range: 200-2000.

[0172] Quantitative analysis method of the product: Based on the analytical detection method for glucosyl steviol glycosides in the supplementary document No. 8 of GB2760-2014 of the National Health and Family Planning Commission.

[0173] Example 5

[0174] S1: 100g of crude glucosylstevioside was dissolved in 2L of 10wt% ethanol solution, and then adsorbed using a macroporous adsorption resin. During adsorption, the liquid flow rate was controlled at 0.5BV / h. The effluent was collected and then analyzed with a 0.05wt% hydrochloric acid aqueous solution. The flow rate of the hydrochloric acid aqueous solution was controlled at 2BV / h and the volume was 2BV. The acid solution was collected. Preferably, a 70wt% ethanol solution was used for analysis. The flow rate of the ethanol solution was controlled at 2BV / h and the volume was 2BV. The high alcohol solution was collected.

[0175] S2: The effluent and the acid solution 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, and the temperature was raised to 45°C for hydrolysis for 2 h. The pH of the reaction system was then adjusted to 3 to inactivate the enzyme to obtain a hydrolyzed solution.

[0176] S3: Add 30 g of stevioside STV to 500 ml of the hydrolyzate, adjust the solution pH to 6.0, raise the temperature to 75° C., add 0.3 ml of glucosyltransferase, and react for 8 hours. After the reaction, raise the temperature to 95° C. to inactivate the reaction solution to obtain a reaction solution; concentrate the reaction solution to a solid content of 50 wt %, and then spray dry the solution to obtain 60 g of high-purity glucosylstevioside A;

[0177] S4: The high alcohol analytical solution was concentrated to a solid content of 50 wt%, and then spray-dried to obtain 69 g of high-purity glucosyl steviol glycoside B.

[0178] After testing, the content of total steviol glycosides (TSG) in high-purity glucose-based steviol glycoside A was 93wt%, the content of glucose-based steviol glycosides (GSG) was 77wt%, and the content of dextrin was 5.8wt%; the content of total steviol glycosides (TSG) in high-purity glucose-based steviol glycoside B was 99wt%, the content of glucose-based steviol glycosides (GSG) was 92wt%, and the content of dextrin was 0.2wt%.

[0179] Example 6

[0180] S1: 100g of crude glucosylstevioside was dissolved in 3L of 10wt% ethanol solution, and then adsorbed using a macroporous adsorption resin. During adsorption, the liquid flow rate was controlled at 0.75BV / h. The effluent was collected and then analyzed with a 0.05wt% hydrochloric acid aqueous solution. The flow rate of the hydrochloric acid aqueous solution was controlled at 2BV / h and the volume was 2BV. The acid solution was collected. Preferably, an 80wt% ethanol solution was used for analysis. The flow rate of the ethanol solution was controlled at 3BV / h and the volume was 3BV. The high alcohol solution was collected.

[0181] S2: The effluent and the acid solution 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, and the temperature was raised to 50°C for hydrolysis for 2 h. The pH of the reaction system was then adjusted to 3 to inactivate the enzyme to obtain a hydrolyzed solution.

[0182] S3: Add 30 g of stevioside STV to 500 ml of the hydrolyzate, adjust the solution pH to 6.0, raise the temperature to 75° C., add 0.4 ml of glucosyltransferase, and react for 7.5 hours. After the reaction, raise the temperature to 95° C. to inactivate the reaction solution to obtain a reaction solution; concentrate the reaction solution to a solid content of 40 wt %, and then spray dry it to obtain 61 g of high-purity glucosylstevioside A;

[0183] S4: The high alcohol analytical solution was concentrated to a solid content of 40 wt%, and then spray-dried to obtain 68 g of high-purity glucosyl steviol glycoside B.

[0184] After testing, the content of total steviol glycosides (TSG) in high-purity glucose-based steviol glycoside A was 93.5wt%, the content of glucose-based steviol glycosides (GSG) was 79.5wt%, and the content of dextrin was 5.7wt%; the content of total steviol glycosides (TSG) in high-purity glucose-based steviol glycoside B was 99wt%, the content of glucose-based steviol glycosides (GSG) was 93wt%, and the content of dextrin was 0.2wt%.

[0185] Example 7

[0186] S1: 100g of crude glucosylstevioside was dissolved in 2.5L of 10wt% ethanol solution, and then adsorbed using a macroporous adsorption resin. During adsorption, the liquid flow rate was controlled at 0.8BV / h. The effluent was collected and then analyzed with a 0.05wt% hydrochloric acid aqueous solution. The flow rate of the hydrochloric acid aqueous solution was controlled at 3BV / h and the volume was 3BV. The acid solution was collected. Preferably, a 75wt% ethanol solution was used for analysis. The flow rate of the ethanol solution was controlled at 2BV / h and the volume was 2BV. The high alcohol solution was collected.

[0187] S2: The effluent and the acid solution 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, and the temperature was raised to 45°C for hydrolysis for 2 h. The pH of the reaction system was then adjusted to 3 to inactivate the enzyme to obtain a hydrolyzed solution.

[0188] S3: Add 30 g of stevioside STV to 500 ml of the hydrolyzate, adjust the solution pH to 6.0, raise the temperature to 75° C., add 0.5 ml of glucosyltransferase, and react for 8 hours. After the reaction, raise the temperature to 95° C. to inactivate the reaction solution to obtain a reaction solution; concentrate the reaction solution to a solid content of 45 wt %, and then spray dry it to obtain 61 g of high-purity glucosylstevioside A;

[0189] S4: The high alcohol analytical solution was concentrated to a solid content of 45 wt %, and then spray-dried to obtain 68.5 g of high-purity glucosyl steviol glycoside B.

[0190] After testing, the content of total steviol glycosides (TSG) in high-purity glucose-based steviol glycoside A was 93wt%, the content of glucose-based steviol glycosides (GSG) was 78wt%, and the content of dextrin was 5.6wt%; the content of total steviol glycosides (TSG) in high-purity glucose-based steviol glycoside B was 99.5wt%, the content of glucose-based steviol glycosides (GSG) was 93wt%, and the content of dextrin was 0.15wt%.

[0191] The yield calculation method of glucosyl steviol glycoside in the following examples is as follows:

[0192] Yield (%) = (actual amount of target product produced / theoretical amount of target product produced) × 100%.

[0193] In the following examples, GSG refers to glucosyl steviol glycoside, and TSG refers to the general term for all steviol glycosides.

[0194] Example 8

[0195] S1: Take 100g of crude glucosyl steviol glycosides (TSG 85wt%, GSG 79wt%, dextrin 14.5wt%) obtained by the reaction, add 5L of purified water, and stir at room temperature and pressure until the solids are dissolved to prepare a feed solution with a solid content of 2wt%;

[0196] S2: The prepared feed liquid is concentrated through a nanofiltration membrane with a molecular weight cut-off of 800 Da at an operating pressure of 4 bar, and the membrane cut-off liquid and the membrane permeate are collected;

[0197] S3: Using a rotary evaporator, the solvent in the membrane intercept liquid and the membrane permeate obtained in step S2 was removed separately at 80°C and a pressure of -0.1 MPa to obtain 85g of intermediate product A and 15g of intermediate product B;

[0198] S4: Take 85g of intermediate product A, add 850ml of 99wt% methanol to a beaker and place it on a magnetic stirrer, slowly add intermediate product A and continue stirring. Start timing after complete dissolution, let it settle for 5h, and the reaction is completed; then vacuum filter, recover the filter cake for standby use, remove the solvent from the filtrate to obtain 83.3g of high-purity glucosyl steviol glycoside product, the content of glucosyl steviol glycoside in the product is 92.3wt% (i.e., product purity), the total glycoside content is 98wt%, the dextrin content is <0.5wt%, and the yield of glucosyl steviol glycoside is 97.3%;

[0199] 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 and purified water were mixed and dissolved in a mass ratio of 1:10. α-1,4-glucose hydrolase was added at a concentration of 0.2% by weight of the solid sample. Enzymatic hydrolysis was carried out at 40°C and pH 4.5 for 6 h. After the enzymatic hydrolysis was completed, the reaction solution was heated to 90°C to inactivate the enzyme to obtain a glucose-containing solution.

[0200] S6: Concentrate the glucose solution at 80°C to an extract-like intermediate product C with a solid content of 75wt%; cool the extract-like intermediate product C to 20°C and keep it warm, at which time crystals slowly precipitate. Let it stand for 6 hours, vacuum filter, and recover the filtrate. The filter cake is high-purity glucose with a purity of 80%.

[0201] Example 9

[0202] S1: Take 100g of crude glucosyl steviol glycosides (TSG 90wt%, GSG 83.6wt%, dextrin 10wt%) obtained by the reaction, add 10L of purified water, and stir at room temperature and pressure until the solids are dissolved to prepare a feed solution with a solid content of 1wt%;

[0203] S2: The prepared feed liquid is concentrated through a nanofiltration membrane with a molecular weight cut-off of 800 Da at an operating pressure of 4 bar, and the membrane cut-off liquid and the membrane permeate are collected;

[0204] S3: Using a rotary evaporator, the solvent in the membrane intercept liquid and the membrane permeate obtained in step S2 was removed separately at 80°C and a pressure of -0.1 MPa to obtain 92g of intermediate product A and 8g of intermediate product B;

[0205] S4: Take 92g of intermediate product A, add 850ml of 99wt% methanol in a beaker and place it on a magnetic stirrer, slowly add intermediate product A and continue stirring. Start timing after complete dissolution, let it settle for 8h, and the reaction is complete; then vacuum filter, recover the filter cake for standby use, remove the solvent from the filtrate to obtain 86g of high-purity glucosyl steviol glycoside product, the total glycoside content of the product is 98.4wt%, the glucosyl steviol glycoside content is 92.9wt%; the dextrin content is less than 0.6wt%, and the glucosyl steviol glycoside yield is 95.6%;

[0206] S5: Combine the intermediate product B and the filter cake obtained in S4 to obtain 12.5 g of a solid sample, mix and dissolve the solid sample with purified water at a mass ratio of 1:10, add 0.3% of α-1,4-glucose hydrolase by weight of the solid sample, and perform enzymatic hydrolysis at 40°C and pH 4.5 for 7 h. After the enzymatic hydrolysis is completed, heat the reaction solution to 90°C to inactivate the enzyme to obtain a glucose-containing solution;

[0207] S6: Concentrate the glucose solution at 90°C to an extract-like intermediate product C with a solid content of 70wt%; cool the extract-like intermediate product C to 20°C and keep it warm, at which time crystals slowly precipitate. Let it stand for 7 hours, vacuum filter, and recover the filtrate. The filter cake is high-purity glucose with a purity of 83%.

[0208] Example 10

[0209] S1: Take 100g of crude glucosyl steviol glycosides (TSG 85wt%, GSG 79wt%, dextrin 14.5wt%) obtained by the reaction, add 5L of purified water, and stir at room temperature and pressure until the solids are dissolved to prepare a feed solution with a solid content of 2wt%;

[0210] S2: The prepared feed liquid is concentrated through a nanofiltration membrane with a molecular weight cut-off of 700 Da at an operating pressure of 4 bar, and the membrane cut-off liquid and the membrane permeate are collected;

[0211] S3: Using a rotary evaporator, the solvent in the membrane intercept liquid and the membrane permeate obtained in step S2 was removed separately at 80°C and a pressure of -0.1 MPa to obtain 83 g of intermediate product A and 16.3 g of intermediate product B;

[0212] S4: Take 83g of intermediate product A, add 850ml of 99wt% methanol in a beaker and place it on a magnetic stirrer, slowly add intermediate product A and continue stirring. Start timing after complete dissolution, let it settle for 5h, and the reaction is completed; then vacuum filter, recover the filter cake for standby use, remove the solvent from the filtrate to obtain 82.9g of high-purity glucosyl steviol glycoside product, the total glycoside content of the product is 98wt%, the glucosyl steviol glycoside content is 93.1wt%, the dextrin content is less than 0.5wt%, and the glucosyl steviol glycoside yield is 97.7%;

[0213] S5: Intermediate product B and the filter cake obtained in S4 were combined to obtain 17 g of a solid sample. The solid sample and purified water were mixed and dissolved in a mass ratio of 1:10. α-1,4-glucose hydrolase was added at a concentration of 0.2% by weight of the solid sample. Enzymatic hydrolysis was carried out at 40°C and pH 4.5 for 6 h. After the enzymatic hydrolysis was completed, the reaction solution was heated to 90°C to inactivate the enzyme to obtain a glucose-containing solution.

[0214] S6: Concentrate the glucose solution at 80°C to an extract-like intermediate product C with a solid content of 75wt%; cool the extract-like intermediate product C to 20°C and keep it warm, at which time crystals slowly precipitate. Let it stand for 6 hours, vacuum filter, and recover the filtrate. The filter cake is high-purity glucose with a glucose purity of 83%.

[0215] Example 11

[0216] S1: Take 100g of crude glucosyl steviol glycosides (TSG 85wt%, GSG 79wt%, dextrin 14.5wt%) obtained by the reaction, add 5L of purified water, and stir at room temperature and pressure until the solids are dissolved to prepare a feed solution with a solid content of 2wt%;

[0217] S2: The prepared feed liquid is concentrated through a nanofiltration membrane with a molecular weight cut-off of 600 Da at an operating pressure of 4 bar, and the membrane cut-off liquid and the membrane permeate are collected;

[0218] S3: Using a rotary evaporator, the solvent in the membrane intercept liquid and the membrane permeate obtained in step S2 was removed separately at 80°C and a pressure of -0.1 MPa to obtain 83.9 g of intermediate product A and 15.5 g of intermediate product B;

[0219] S4: Take 83.9g of intermediate product A, add 850ml of 99wt% methanol to a beaker and place it on a magnetic stirrer, slowly add intermediate product A and continue stirring. Start timing after complete dissolution, let it settle for 5h, and the reaction is completed; then vacuum filter, recover the filter cake for standby use, remove the solvent from the filtrate to obtain 80g of high-purity glucosyl steviol glycoside product, the total glycoside content of the product is 99wt%, the glucosyl steviol glycoside content is 94.2wt%, the dextrin content is less than 0.3wt%, and the glucosyl steviol glycoside yield is 95.4wt%;

[0220] S5: Combine the intermediate product B and the filter cake obtained in S4 to obtain 16 g of a solid sample, mix and dissolve the solid sample with purified water at a mass ratio of 1:10, add 0.2% of α-1,4-glucose hydrolase by weight of the solid sample, and perform enzymatic hydrolysis at 40°C and pH 4.5 for 6 h. After the enzymatic hydrolysis is completed, heat the reaction solution to 90°C to inactivate the enzyme to obtain a glucose-containing solution;

[0221] S6: Concentrate the glucose solution at 80°C to an extract-like intermediate product C with a solid content of 75wt%; cool the extract-like intermediate product C to 20°C and keep it warm, at which time crystals slowly precipitate. Let it stand for 6 hours, vacuum filter, and recover the filtrate. The filter cake is high-purity glucose with a glucose purity of 82%.

[0222] Example 12

[0223] S1: Take 100g of crude glucosyl steviol glycosides (TSG 85wt%, GSG 79wt%, dextrin 14.5wt%) obtained by the reaction, add 5L of purified water, and stir at room temperature and pressure until the solids are dissolved to prepare a feed solution with a solid content of 2wt%;

[0224] S2: The prepared feed liquid is concentrated through a nanofiltration membrane with a molecular weight cut-off of 500 Da at an operating pressure of 4 bar, and the membrane cut-off liquid and the membrane permeate are collected;

[0225] S3: Using a rotary evaporator, the solvent in the membrane intercept liquid and the membrane permeate obtained in step S2 was removed separately at 80°C and a pressure of -0.1 MPa to obtain 88.6 g of intermediate product A and 11 g of intermediate product B;

[0226] S4: Take 88g of intermediate product A, add 850ml of 99wt% methanol in a beaker and place it on a magnetic stirrer, slowly add intermediate product A and continue stirring. Start timing after complete dissolution, let it settle for 5h, and the reaction is completed; then vacuum filter, recover the filter cake for standby use, remove the solvent from the filtrate to obtain 81.9g of high-purity glucosyl steviol glycoside product, the total glycoside content of the product is 97.3wt%, the glucosyl steviol glycoside content is 91.2wt%, the dextrin content is less than 1.0wt%, and the glucosyl steviol glycoside yield is 94.5%;

[0227] S5: Combine the intermediate product B and the filter cake obtained in S4 to obtain 17 g of a solid sample, mix and dissolve the solid sample with purified water at a mass ratio of 1:10, add 0.2% of α-1,4-glucose hydrolase by weight of the solid sample, and perform enzymatic hydrolysis at 40°C and pH 4.5 for 6 h. After the enzymatic hydrolysis is completed, heat the reaction solution to 90°C to inactivate the enzyme to obtain a glucose-containing solution;

[0228] S6: Concentrate the glucose solution at 80°C to an extract-like intermediate product C with a solid content of 75wt%; cool the extract-like intermediate product C to 20°C and keep it warm, at which time crystals slowly precipitate. Let it stand for 6 hours, vacuum filter, and recover the filtrate. The filter cake is high-purity glucose with a purity of 85%.

[0229] Example 13

[0230] S1: Take 100g of crude glucosyl steviol glycosides (TSG 85wt%, GSG 79wt%, dextrin 14.5wt%) obtained by the reaction, add 5L of purified water, and stir at room temperature and pressure until the solids are dissolved to prepare a feed solution with a solid content of 2wt%;

[0231] S2: The prepared feed liquid is concentrated through a nanofiltration membrane with a molecular weight cut-off of 800 Da at an operating pressure of 4 bar, and the membrane cut-off liquid and the membrane permeate are collected;

[0232] S3: Using a rotary evaporator, the solvent in the membrane intercept liquid and the membrane permeate obtained in step S2 was removed separately at 80°C and a pressure of -0.1 MPa to obtain 84.6 g of intermediate product A and 15 g of intermediate product B;

[0233] S4: Take 84.6g of intermediate product A, add 850ml of 99wt% methanol in a beaker and place it on a magnetic stirrer, slowly add intermediate product A and continue stirring. Start timing after complete dissolution, let it settle for 5h, and the reaction is completed; then vacuum filter, recover the filter cake for standby use, remove the solvent from the filtrate to obtain 83.5g of high-purity glucosyl steviol glycoside product, the total glycoside content of the product is 97.8wt%, the glucosyl steviol glycoside content is 92.9wt%, the dextrin content is less than 0.5wt%, and the glucosyl steviol glycoside yield is 98.2%;

[0234] 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 and purified water were mixed and dissolved in a mass ratio of 1:10. α-1,4-glucose hydrolase was added at a concentration of 0.2% by weight of the solid sample. Enzymatic hydrolysis was carried out at 45°C and pH 4.5 for 8 h. After the enzymatic hydrolysis was completed, the reaction solution was heated to 90°C to inactivate the enzyme to obtain a glucose-containing solution.

[0235] S6: Concentrate the glucose solution at 80°C to an extract-like intermediate product C with a solid content of 75wt%; cool the extract-like intermediate product C to 20°C and keep it warm, at which time crystals slowly precipitate. Let it stand for 6 hours, vacuum filter, and recover the filtrate. The filter cake is high-purity glucose with a glucose purity of 84.3%.

[0236] Example 14

[0237] S1: Take 100g of crude glucosyl steviol glycosides (TSG 85wt%, GSG 79wt%, dextrin 14.5wt%) obtained by the reaction, add 5L of purified water, and stir at room temperature and pressure until the solids are dissolved to prepare a feed solution with a solid content of 2%;

[0238] S2: The prepared feed liquid is concentrated through a nanofiltration membrane with a molecular weight cut-off of 800 Da at an operating pressure of 4 bar, and the membrane cut-off liquid and the membrane permeate are collected;

[0239] S3: Using a rotary evaporator, the solvent in the membrane intercept liquid and the membrane permeate obtained in step S2 was removed separately at 80°C and a pressure of -0.1 MPa to obtain 84.8 g of intermediate product A and 14.9 g of intermediate product B;

[0240] S4: Take 84g of intermediate product A, add 850ml of 99wt% methanol in a beaker and place it on a magnetic stirrer, slowly add intermediate product A and continue stirring. Start timing after complete dissolution, let it settle for 5h, and the reaction is completed; then vacuum filter, recover the filter cake for standby use, remove the solvent from the filtrate to obtain 82.5g of high-purity glucosyl steviol glycoside product, the total glycoside content of the product is 98.1wt%, the glucosyl steviol glycoside content is 92.9wt%, the dextrin content is less than 0.7wt%, and the glucosyl steviol glycoside yield is 97%;

[0241] S5: Combine the intermediate product B and the filter cake obtained in S4 to obtain 17 g of a solid sample, mix and dissolve the solid sample with purified water at a mass ratio of 1:10, add 0.2% of α-1,4-glucose hydrolase by weight of the solid sample, and perform enzymatic hydrolysis at 35°C and pH 4.5 for 12 h. After the enzymatic hydrolysis is completed, heat the reaction solution to 90°C to inactivate the enzyme to obtain a glucose-containing solution;

[0242] S6: Concentrate the glucose solution at 80°C to an extract-like intermediate product C with a solid content of 75wt%; cool the extract-like intermediate product C to 20°C and keep it warm, at which time crystals slowly precipitate. Let it stand for 8 hours, vacuum filter, and recover the filtrate. The filter cake is high-purity glucose with a purity of 83.6%.

[0243] Example 15

[0244] S1: Take 100g of crude glucosyl steviol glycosides (TSG 85wt%, GSG 79wt%, dextrin 14.5wt%) obtained by the reaction, add 5L of purified water, and stir at room temperature and pressure until the solids are dissolved to prepare a feed solution with a solid content of 2wt%;

[0245] S2: The prepared feed liquid is concentrated through a nanofiltration membrane with a molecular weight cut-off of 800 Da at an operating pressure of 4 bar, and the membrane cut-off liquid and the membrane permeate are collected;

[0246] S3: Using a rotary evaporator, the solvent in the membrane intercept liquid and the membrane permeate obtained in step S2 was removed separately at 80°C and a pressure of -0.1 MPa to obtain 86.1 g of intermediate product A and 13.8 g of intermediate product B;

[0247] S4: Take 86g of intermediate product A, add 850ml of 99wt% methanol in a beaker and place it on a magnetic stirrer, slowly add intermediate product A and continue stirring. Start timing after complete dissolution, let it settle for 5h, and the reaction is completed; then vacuum filter, recover the filter cake for standby use, remove the solvent from the filtrate to obtain 84g of high-purity glucosyl steviol glycoside product, the total glycoside content of the product is 98.5wt%, the glucosyl steviol glycoside content is 93.1wt%, the dextrin content is less than 0.6wt%, and the yield of glucosyl steviol glycoside is 99%;

[0248] S5: Combine the intermediate product B and the filter cake obtained in S4 to obtain 15 g of a solid sample, mix and dissolve the solid sample with purified water at a mass ratio of 1:10, add 0.2% of α-1,4-glucose hydrolase by weight of the solid sample, and perform enzymatic hydrolysis at 30°C and pH 4 for 6 h. After the enzymatic hydrolysis is completed, heat the reaction solution to 90°C to inactivate the enzyme to obtain a glucose-containing solution;

[0249] S6: Concentrate the glucose solution at 80°C to an extract-like intermediate product C with a solid content of 75wt%; cool the extract-like intermediate product C to 20°C and keep it warm, at which time crystals slowly precipitate. Let it stand for 6 hours, vacuum filter, and recover the filtrate. The filter cake is high-purity glucose with a purity of 80%.

[0250] In summary, the purity of the glucose prepared in the examples of the present application is as high as 85%, the total glycoside content of the prepared product is as high as 99wt%, the content of glucose styrene rebaudioside is as high as 94.2wt%, and the yield of glucose styrene rebaudioside is as high as 99%.

[0251] In addition, it should be understood that after reading the contents of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for preparing glucosyl steviol glycoside using mother liquor sugar, characterized in that: The following steps are involved: (1) mixing mother liquid sugar, maltodextrin and water and stirring until the solid is dissolved, and adding enzyme for enzymolysis to obtain enzymolysis solution; (2) adding activated carbon to the enzymatic hydrolyzate obtained in step (1) to remove impurities, thereby obtaining a decontaminated liquid, performing an enzyme deactivation treatment on the decontaminated liquid, and then filtering the decontaminated liquid, and then passing the obtained filtrate into a macroporous adsorption resin for adsorption treatment; (3) using an acid-alcohol solution with a pH of 1 to 3 composed of an ethanol solution and a hydrochloric acid solution for analysis to obtain an analysis solution; (4) adjusting the pH of the analytical solution to 5-6, and then concentrating the solution using a nanofiltration membrane to obtain a concentrated solution; (5) adding a taste modifier to the concentrated solution, and then spray drying to obtain a glucose-based stevioside product.

2. The method for preparing glucosyl steviol glycoside using mother liquor sugar according to claim 1, characterized in that: In step (1), the mass ratio of the mother liquor sugar, maltodextrin and water is 1:(1-3):(5-10).

3. The method for preparing glucosyl steviol glycoside using mother liquor sugar according to claim 1, characterized in that: In 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 0.8-1.2 wt%, and the amylase is added in the form of a solid amylase, the concentration of the solid amylase is 0.4-0.6 wt%; And / or the amount of glucosyltransferase solution added to 100 g of mother liquid sugar is 0.5-2 ml, and the amount of solid amylase added to 100 g of mother liquid sugar is 0.1-1 g.

4. The method for preparing glucosyl steviol glycoside by using mother liquor sugar according to claim 1, characterized in that: In step (1), the enzymatic hydrolysis conditions are: enzymatic hydrolysis temperature is 60-80°C, pH is 5.5-6.0, and enzymatic hydrolysis time is 36-48h.

5. The method for preparing glucosyl steviol glycoside by using mother liquor sugar according to claim 1, characterized in that: In step (2), the amount of activated carbon added is 1 to 3% of the mass of the enzymatic hydrolyzate, and the impurity removal time is 1 to 6 hours.

6. The method for preparing glucosyl steviol glycoside by using mother liquor sugar according to claim 1, characterized in that: In step (2), when the macroporous adsorption resin is used for adsorption, the feed volume of the filtrate is 30-60% of the volume of the macroporous adsorption resin, and the feed rate is 0.5-2 BV / h.

7. The method for preparing glucosyl steviol glycosides by using mother liquor sugar according to claim 6, characterized in that: Before analyzing the adsorbed macroporous adsorption resin, the method further includes: washing the macroporous adsorption resin with pure water, 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 is 1 to 3 BV / h.

8. The method for preparing glucosyl steviol glycoside by using mother liquor sugar according to claim 1, characterized in that: In step (3), the concentration of the ethanol solution is 40-60 v / v%, and the concentration of the hydrochloric acid solution is 0.3-0.5 wt%. During analysis, the volume of the acid-alcohol solution is 2-4 BV of the volume of the macroporous adsorption resin, and the flow rate is 1-3 BV / h.

9. The method for preparing glucosyl steviol glycoside by using mother liquor sugar according to claim 1, characterized in that: In step (4), the molecular weight cut-off of the nanofiltration membrane is 400 to 500 Da.

10. The method for preparing glucosyl steviol glycoside using mother liquor sugar according to claim 1, characterized in that: In step (5), the taste modifier is a composition of erythritol, sodium alginate and valine, and the mass ratio of the three is 2: (2-4): (5-8); the added amount of the taste modifier is 0.1-0.5% of the mass of the product in the concentrated solution.

11. A method for purifying glucosyl steviol glycoside, characterized in that: The following steps are involved: (1) dissolving crude glucosyl stevioside in an ethanol solution, then subjecting the obtained glucosyl stevioside ethanol solution to adsorption treatment with a macroporous adsorption resin, and collecting the effluent; Then, an acid-water solution and a high-purity ethanol solution are used for analysis in sequence, and the acid-water analysis solution and the high-purity ethanol analysis solution are collected; (2) The mixed solution of the effluent obtained in step (1) and the acid water analysis solution is concentrated, and then α-1,4-glucose hydrolase is added to the concentrated solution to increase the temperature for hydrolysis reaction. After the hydrolysis is completed, the pH of the reaction system is adjusted. to 3, inactivating the enzyme to obtain a hydrolyzate; (3) adding steviol glycoside to the hydrolyzate, adjusting the pH of the solution to 6.0, adding glucosyl transferase, heating the reaction, and further heating the reaction to inactivate the reaction after the reaction is completed to obtain a reaction solution, concentrating the reaction solution to a solid content of 40-50 wt%, and then spray drying the concentrated solution to obtain high-purity glucosyl steviol glycoside A; (4) The high alcohol analysis solution is concentrated to a solid content of 40-50 wt %, and then the concentrated solution is spray-dried to obtain high-purity glucosyl stevioside B.

12. The method for purifying glucosyl steviol glycoside according to claim 11, characterized in that: In step (1), the concentration of the ethanol solution is 5-15wt%; the concentration of the glucosyl steviol glycoside in the glucosyl steviol glycoside ethanol solution is 30-50g / L.

13. The method for purifying glucosyl steviol glycoside according to claim 11, characterized in that: In step (1), during the adsorption treatment, the flow rate of the glucose-syl stevioside ethanol solution is 0.25BV / h-1BV / h.

14. The method for purifying glucosyl steviol glycoside according to claim 11, characterized in that: In step (1), the acid aqueous solution is a hydrochloric acid solution with a concentration of 0.04-0.06wt%, and the concentration of the high-purity ethanol solution is 70-80wt%; during analysis, the volume of the acid aqueous solution is 2-3BV, the flow rate is 2-3BV / h, and the volume of the high-purity ethanol solution is 2-3BV, and the flow rate is 2-3BV / h.

15. The method for purifying glucosyl steviol glycoside according to claim 11, characterized in that: In step (1), the volume of the effluent is 1-2 BV of the resin volume, and the volume of the acid-water analysis solution is 2-3 BV of the resin volume.

16. The method for purifying glucosyl steviol glycoside according to claim 11, characterized in that: In step (2), the concentration multiple during the concentration treatment is 3-5 times.

17. The method for purifying glucosyl steviol glycoside according to claim 11, characterized in that: In step (2), 0.03-0.1 ml of α-1,4-glucose hydrolase is used for every 100 g of crude glucosyl steviol glycoside.

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

19. The method for purifying glucosyl steviol glycoside according to claim 11, characterized in that: In step (3), the steviol glycoside is steviol glycoside STV; and 0.3-0.5 ml of glucosyltransferase is used for every 100 g of crude glucosyl steviol glycoside.

20. The method for purifying glucosyl steviol glycoside according to claim 11, characterized in that: In step (3), the temperature of the temperature-raising reaction is 70-80°C, and the time is 6-10h; the temperature of the temperature-raising inactivation is 95°C.

21. A method for separating and purifying glucosyl steviol glycosides and recovering glucose, characterized in that: The following steps are involved: (1) adding purified water to the crude product of glucosyl stevioside to dissolve it again to obtain a feed solution; (2) subjecting the feed liquid to nanofiltration concentration treatment, and collecting the membrane interception liquid and the membrane permeate; (3) Removing the solvent from the membrane interception liquid and the membrane permeate liquid respectively to obtain intermediate product A and intermediate product B; (4) mixing the intermediate product A and methanol and settling them, then vacuum filtering them, collecting the filter cake and the filtrate, and drying the filtrate to obtain high-purity glucosyl stevioside; (5) mixing the intermediate product B and the filter cake, adding purified water, stirring until the solid is dissolved, adding α-1,4-glucose hydrolase for enzymatic hydrolysis, and heating the feed liquid to inactivate the enzyme after the enzymatic hydrolysis to obtain a glucose-containing solution; (6) The glucose solution is concentrated at high temperature to obtain an extract-like intermediate product C, the extract-like intermediate product C is slowly cooled and crystallized, and finally vacuum filtered, and the filtrate is recovered for subsequent production and application, and the filter cake is high-purity glucose.

22. A method for separating and purifying glucosyl steviol glycosides and recovering glucose according to claim 21, characterized in that: In step (1), the solid content of the feed liquid is 1wt%-5wt%.

23. A method for separating and purifying glucosyl steviol glycosides and recovering glucose according to claim 21, characterized in that: In step (2), when nanofiltration concentration is performed, the molecular weight cutoff of the nanofiltration membrane is 500Da-800Da, and the operating pressure during nanofiltration concentration is 3.5-5bar.

24. A method for separating and purifying glucosyl steviol glycosides and recovering glucose according to claim 21, characterized in that: In step (3), the solvent in the membrane permeate and the membrane interception liquid is removed under the conditions of a temperature of 80° C. and a pressure of −0.1 MPa.

25. The method for separating and purifying glucosyl steviol glycoside and recovering glucose according to claim 21, characterized in that: In step (4), the concentration of methanol is 95wt%-99wt%, and the mass ratio of the intermediate product A to methanol is 1:(5-10).

26. A method for separating and purifying glucosyl steviol glycosides and recovering glucose according to claim 21, characterized in that: In step (4), the mixing and settling time is 2h-8h.

27. A method for separating and purifying glucosyl steviol glycosides and recovering glucose according to claim 21, characterized in that: In step (5), the added amount of the α-1,4-glucose hydrolase is 0.2%-0.3% of the total weight of the intermediate product B and the filter cake.

28. The method for separating and purifying glucosyl steviol glycoside and recovering glucose according to claim 21, characterized in that: In step (5), the temperature of the enzymatic hydrolysis treatment is 35°C-45°C, the pH is 4.0-4.5, and the time is 5h-7h; the feed liquid is heated to 90°C to inactivate the enzyme.

29. The method for separating and purifying glucosyl steviol glycoside and recovering glucose according to claim 21, characterized in that: In step (6), the temperature of the high-temperature concentration treatment is 80°C-90°C, and the solid content of the extract-like intermediate product C is 70wt%-75wt%.

30. The method for separating and purifying glucosyl steviol glycoside and recovering glucose according to claim 21, characterized in that: In step (6), the slow cooling rate is 10°C / h-15°C / h, and the temperature is lowered to 20°C and kept for crystallization for 5h-7h.

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