Method for preparing glucosyl stevioside through multi-enzyme combination

Abstract

The invention discloses a method for preparing glucosyl stevioside by multi-enzyme combination, and relates to the technical field of glucosyl stevioside production, rebaudioside A or stevioside is taken as a raw material, cyclodextrin is taken as an auxiliary material, cyclodextrin glucoside transferase is added for glycosylation reaction, a glucosyl stevioside solution I is cooled, and a glucosyl stevioside solution II is obtained; the preparation method comprises the following steps: firstly, preparing rebaudioside A or stevioside STV, then adding alpha-amylase for hydrolysis, then adding sucrose, UGT glycosyl transferase, sucrose synthase and uridine diphosphate, continuously converting unreacted rebaudioside A or stevioside STV into rebaudioside M, after the reaction is finished, performing inactivation, series resin column adsorption and ethanol desorption, and performing dealcoholization, concentration and drying on a desorption solution to obtain the glucosyl stevioside. The quality of the product is improved, the cost is reduced, and the taste of the product is integrally improved.

Description

Technical Field

[0001] The present invention relates to the technical field of steviol glycoside production, and particularly relates to a method for preparing glucosyl steviol glycoside by multi-enzyme combination. Background Art

[0002] For the glucosyl steviol glycoside obtained by glucosylating rebaudioside A or stevioside with cyclodextrin glycosyltransferase as the raw material, due to the conversion efficiency of beta-cyclodextrin glycosidase to the substrate and the high content of monoglycoside in the raw material, it is difficult for the unreacted glycoside rebaudioside A or stevioside to reach a limit of less than 4%, resulting in the product not meeting the standard. Currently, there are mainly several methods to reduce the substrate residue in the conversion of high-content raw materials as substrates. To a certain extent, they reduce the substrate residue, but the reduction of substrate residue is limited and cannot truly achieve the purpose of reducing the residue. At the same time, to a certain extent, they change the nature and application of the product itself. For example, the first method is to increase the addition amount of dextrin. Although it increases the conversion rate of the substrate and reduces the unreacted glycoside to a certain extent, this method requires a large amount of dextrin added, and it is also difficult to reduce the unreacted glycoside to make rebaudioside A or stevioside reach a limit of less than 4%. At the same time, the excessive dextrin also reduces the sweetness and taste properties of the product. The second method can dilute the unreacted glycoside by compounding steviol glycosides, but there is a large amount of other steviol glycosides added in the compounding method, which causes great changes to the properties of the product and there are also problems with unqualified indicators such as the total glycosides of the product, and the product still cannot meet the quality standards. The third method can be separated by resin. This method has the disadvantages of low yield, increased product waste, and high degree of glycosylation of the product, and also greatly changes the nature of the product itself, restricting the application of glucosyl steviol glycoside products using such raw materials as substrates. Summary of the Invention

[0003] The technical problem to be solved by the present invention is: aiming at the deficiencies existing in the prior art, to provide a method for preparing glucosyl steviol glycoside by multi-enzyme combination, so as to reduce the unreacted glycoside rebaudioside A or stevioside, and at the same time convert it into glucosyl steviol glycoside with good taste and high quality.

[0004] To solve the above technical problem, the technical solution of the present invention is:

[0005] A method for preparing glucosyl steviol glycoside by multi-enzyme combination, comprising the following steps:

[0006] A: Using rebaudioside A or stevioside as the raw material, using beta-cyclodextrin as the auxiliary material, adding cyclodextrin glucosyltransferase for glycosylation reaction to obtain glucosyl steviol glycoside solution I;

[0007] B: The glucosyl stevioside solution I is cooled to 45 - 50 °C, and α - amylase is added for hydrolysis to control the glycosylation chain length. After heating to inactivate the enzyme and then cooling to 30 °C, a glucosyl stevioside solution II with a low glycosylation chain length is obtained;

[0008] C: Using the reaction product in the glucosyl stevioside solution II as the substrate, sucrose, UGT glycosyltransferase, sucrose synthase, and uridine diphosphate glucose are added. The initial reaction temperature is 35 °C, and it is increased by 1 °C every 30 min until the reaction temperature reaches 40 °C, so as to continue to convert the remaining unreacted rebaudioside A or stevioside in the solution into rebaudioside M. After heating to inactivate the enzyme and then cooling to 40 °C, it is filtered to obtain a glucosyl stevioside solution III;

[0009] D: The glucosyl stevioside solution III is passed through two series - connected resin columns to adsorb related steviosides and simultaneously remove residual dextrin and sucrose. After washing with water, the purified glucosyl stevioside solutions IV and V are respectively eluted from the first column and the second column;

[0010] E: The above - mentioned glucosyl stevioside solutions IV and V are de - alcoholized, concentrated, and dried to obtain glucosyl stevioside.

[0011] Preferably, in step A, the total glycosides in rebaudioside A and stevioside > 95%, and the content of rebaudioside A or stevioside ≥ 95%.

[0012] Preferably, in step A, the reaction is first carried out at 75 °C for 6 - 8 h with a stirring rate of 200 - 300 rpm, and then the temperature is raised to 80 °C and the reaction continues for 6 - 16 h with a stirring rate of 100 - 150 rpm;

[0013] The concentration of the stevioside raw material in the glucosyl stevioside solution I is 10 - 20% w / v, the addition amount of beta - cyclodextrin is 1 - 2 times the mass of the stevioside, and the amount of cyclodextrin glucanotransferase is 1 - 2% w / w of the added raw material amount.

[0014] Preferably, in step B, the addition amount of α - amylase is 10 U / ml of enzyme activity, the hydrolysis time is 6 - 8 h, the inactivation temperature is 95 - 100 °C, and the inactivation time is 10 - 20 min.

[0015] Preferably, the UGT glycosyltransferase in step C is a mixed enzyme of UGT91C1 (N4 protein encoded by the UGT91C1 mutant gene from Chinese patent CN119265152B) and UGT76G4 (protein encoded by the UGT76G4 gene from Chinese patent CN115094074B), and the sucrose synthase is the sucrose synthase AtSuSy from Chinese patent CN119265152B, where the enzyme activity ratio of UGT91C1 to UGT76G4 is 1 - 1.5:1.

[0016] Preferably, in step C, the sucrose is at a system concentration of 50 mM, uridine diphosphate glucose is 1 - 2 mM, the UGT glycosyltransferase and sucrose synthase each account for 10 - 15 U / ml of the system, and the conversion time is 2 - 4 h.

[0017] Preferably, in step C, 1 mM NaOH is used to adjust the system pH to 7 - 8 during the reaction process.

[0018] Preferably, in step C, the inactivation temperature is 95 - 100 °C and the inactivation time is 10 - 20 min.

[0019] Preferably, in step D, the tandem resin column is macroporous adsorption resin T28, the resin amount is 20 times the volume of the raw material, the water wash is 2 BV of the resin amount, and the water temperature is controlled at 40 °C.

[0020] Preferably, in step D, first, 1 - 2 BV of 20% ethanol is used for preliminary elution to remove residual sucrose and part of the dextrin; then, 1 - 3 BV of 60% ethanol is used for elution to obtain purified glucosyl stevioside solutions IV and V, and the elution rate is 1 - 3 BV / h for both.

[0021] Due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows:

[0022] 1. By using the multi - enzyme method, the residue of unreacted glycosides in glucosyl stevioside is reduced, making the product meet the quality standards. The multi - enzyme method increases the conversion rate of the substrate and has a high conversion efficiency.

[0023] 2. The combined use of multiple enzymes further converts the substrate residue into rebaudioside M with a better taste, improving the taste of the product.

[0024] 3. After treatment with α - amylase, the proportion of low - substituted glucosyl stevioside is increased, which can increase the sweetness and pre - sweetness. By adding glycosyltransferase, the residual substrate is converted into stevioside with a relatively better taste, improving the overall taste of the product.

[0025] 4. By adopting a segmented temperature control strategy, the reaction is promoted by temperature change, improving the yield of glucosyl stevioside. By controlling the temperature of the feed liquid during resin adsorption and the water - washed feed liquid, a good separation, purification, and impurity removal effect is achieved. Specific Embodiments

[0026] The present invention will be further described below in conjunction with the embodiments.

[0027] Example 1

[0028] A method for preparing glucosyl stevioside by a multi - enzyme combination, comprising the following steps:

[0029] A: Using 50 g of total glycosides with 97.7% rebaudioside A (the content of rebaudioside A is 96.3%) as the raw material, add it to 500 ml of water, heat and stir to dissolve. Using beta-cyclodextrin, which is 1 times the mass of stevioside, as the auxiliary material, add cyclodextrin glucanotransferase in an amount of 1% w / w of rebaudioside A, react at 75 °C for 6 h first, with a stirring rate of 200 rpm, then raise the temperature to 80 °C and continue to react for 6 h, with a stirring rate of 100 rpm, to obtain glucose-based stevioside solution I with a rebaudioside A concentration of 20% w / v;

[0030] B: Cool the glucose-based stevioside solution I to 45 °C, add α-amylase with an enzyme activity of 10 U / ml for hydrolysis for 6 h, control the proportion of glucose 1-3 in the glycosylation chain length to be 60%, inactivate at 95 °C for 10 min, and then cool to 30 °C to obtain glucose-based stevioside solution II with a low glycosylation chain length;

[0031] C: Using the reaction product in the glucose-based stevioside solution II as the substrate, add sucrose with a system concentration of 50 mM, UGT glycosyltransferase at 10 U / ml of the system, sucrose synthase at 10 U / ml of the system, and 1 mM uridine diphosphate glucose. The initial reaction temperature is 35 °C, and it is increased by 1 °C every 30 min until the reaction temperature reaches 40 °C. During the reaction, add 1 mM NaOH to adjust the system pH to 7, and continue to convert the unreacted rebaudioside A in the solution into rebaudioside M. Inactivate at 95 °C for 10 min, filter the inactivated conversion solution, and then cool to 40 °C to obtain glucose-based stevioside solution III. The enzyme activity ratio of UGT91C1 to UGT76G4 is 1:1. The UGT glycosyltransferase is a mixed enzyme of UGT91C1 (N4 protein encoded by the UGT91C1 mutant gene from Chinese patent CN119265152B) and UGT76G4 (protein encoded by the UGT76G4 gene from Chinese patent CN115094074B), and the sucrose synthase is from the sucrose synthase AtSuSy in Chinese patent CN119265152B;

[0032] D: First, pass the glucose-based stevioside solution III through an activated carbon column for decolorization, then adsorb the relevant steviosides and remove the residual dextrin and sucrose through 2 series-connected 500 ml macroporous adsorption resin columns T28, then wash with 2000 ml of water, control the water temperature at 40 °C. Finally, the first column and the second column are first preliminarily eluted with 1000 ml of 20% ethanol to remove the residual sucrose and part of the dextrin; then elute with 1000 ml of 60% ethanol respectively, and after elution, wash the resin with 1000 ml of water. Collect the eluates and the respective water top solutions of the first column and the second column to obtain purified glucose-based stevioside solutions IV and V of the first column and the second column. The elution rate of the first column and the second column is both 1 BV / h;

[0033] E: The above glucosyl steviol glycoside solutions IV and V are subjected to alcohol removal, concentration, and drying at 80°C and -0.1 Mpa to obtain glucosyl steviol glycoside. Among them, the weight of glucosyl steviol glycoside IV is 43.2 g, GSG is 83.2%, TSG is 96.3%, rebaudioside M is 2.7%, and rebaudioside A is 2.2%; among them, the weight of glucosyl steviol glycoside V is 28.3 g, GSG is 79.4%, TSG is 95.8, rebaudioside M is 7.5%, and rebaudioside A is 0.6%.

[0034] Example 2

[0035] A method for preparing glucosyl steviol glycoside by a multi-enzyme combination, comprising the following steps:

[0036] A: Using 50 g of total glycoside 98.8% rebaudioside A (the content of rebaudioside A is 97.9%) as the raw material, adding it to 500 ml of water and heating and stirring to dissolve. Using beta-cyclodextrin 1.5 times the mass of steviol glycoside as the auxiliary material, adding cyclodextrin glucosyltransferase at 1.5% w / w of the rebaudioside A amount and reacting at 75°C for 7 h with a stirring rate of 250 rpm, then raising the temperature to 80°C and continuing to react for 10 h with a stirring rate of 120 rpm to obtain glucosyl steviol glycoside solution I with a rebaudioside A concentration of 25% w / v;

[0037] B: Cooling the glucosyl steviol glycoside solution I to 48°C, adding α-amylase with an enzyme activity of 10 U / ml and hydrolyzing for 7 h, controlling the proportion of glucose 1-3 in the glycosylation chain length to be 65%, inactivating at 98°C for 15 min, and then cooling to 30°C to obtain a low-glycosylation-chain-length glucosyl steviol glycoside solution II;

[0038] C: Using the reaction product in the glucosyl stevioside solution II as the substrate, add sucrose with a system concentration of 50 mM, UGT glycosyltransferase at 12 U / ml in the system, sucrose synthase at 13 U / ml in the system, and 1 mM uridine diphosphate glucose. The initial reaction temperature is 35 °C, and it is increased by 1 °C every 30 min until the reaction temperature reaches 40 °C. During the reaction process, 1 mM NaOH is added to adjust the system pH to 8, and the unreacted rebaudioside A remaining in the solution is continuously converted into rebaudioside M. It is inactivated at 98 °C for 15 min. After filtering the inactivated conversion solution and cooling it to 40 °C, glucosyl stevioside solution III is obtained. The enzyme activity ratio of UGT91C1 to UGT76G4 is 1.2:1. The UGT glycosyltransferase is a mixed enzyme of UGT91C1 (N4 protein encoded by the UGT91C1 mutant gene from Chinese Patent CN119265152B) and UGT76G4 (protein encoded by the UGT76G4 gene from Chinese Patent CN115094074B), and the sucrose synthase is the sucrose synthase AtSuSy from Chinese Patent CN119265152B.

[0039] D: The glucosyl stevioside solution III first enters an activated carbon column for decolorization, then passes through two 500 ml tandem macroporous adsorption resin columns T28 to adsorb relevant steviosides and simultaneously remove residual dextrin and sucrose. Then it is washed with 2000 ml of water, controlling the water temperature at 40 °C. Finally, the first column and the second column are first preliminarily eluted with 1500 ml of 20% ethanol to remove residual sucrose and part of the dextrin; then after eluting with 2000 ml of 60% ethanol respectively, the resin is rinsed with 1000 ml of water. The eluates and their respective water rinsing liquids of the first column and the second column are collected respectively to obtain purified glucosyl stevioside solutions IV and V of the first column and the second column. The elution rate of the first column and the second column is both 2 BV / h.

[0040] E: The above glucosyl stevioside solutions IV and V are subjected to alcohol removal, concentration, and drying at 80 °C and -0.1 Mpa to obtain glucosyl stevioside. Among them, the weight of glucosyl stevioside IV is 44.2 g, GSG is 82.1%, TSG is 95.2%, rebaudioside M is 2.5%, and rebaudioside A is 2.1%. Among them, the weight of glucosyl stevioside V is 27.4 g, GSG is 78.6%, TSG is 96.1%, rebaudioside M is 6.4%, and rebaudioside A is 0.9%.

[0041] Example 3

[0042] A method for preparing glucosyl stevioside by a multi-enzyme combination, comprising the following steps:

[0043] A: Using 50 g of total glycosides with 97.6% stevioside (the content of stevioside is 95.5%) as the raw material, add it to 500 ml of water, heat and stir to dissolve. Using beta-cyclodextrin twice the mass of stevioside as the auxiliary material, add cyclodextrin glucanotransferase at 2% w / w of the stevioside amount and react at 75 °C for 8 h with a stirring rate of 300 rpm. Then raise the temperature to 80 °C and continue to react for 16 h with a stirring rate of 150 rpm to obtain glucose-based stevioside solution I with a stevioside concentration of 30% w / v;

[0044] B: Cool the glucose-based stevioside solution I to 50 °C, add α-amylase with an enzyme activity of 10 U / ml and hydrolyze for 8 h. Control the proportion of glycosylated chain lengths glucose 1-3 to be 60%. Inactivate at 100 °C for 20 min, and then cool to 30 °C to obtain glucose-based stevioside solution II with a low glycosylated chain length;

[0045] C: Using the reaction product in the glucose-based stevioside solution II as the substrate, add sucrose with a system concentration of 50 mM, UGT glycosyltransferase at 15 U / ml of the system, sucrose synthase at 15 U / ml of the system, and 2 mM uridine diphosphate glucose. The initial reaction temperature is 35 °C, and it is increased by 1 °C every 30 min until the reaction temperature reaches 40 °C. During the reaction, add 1 mM NaOH to adjust the system pH to 8, and continue to convert the remaining unreacted stevioside in the solution into rebaudioside M. Inactivate at 100 °C for 20 min. After filtering the inactivated conversion solution and cooling to 40 °C, obtain glucose-based stevioside solution III, where the enzyme activity ratio of UGT91C1 to UGT76G4 is 1.5:1. The UGT glycosyltransferase is a mixed enzyme of UGT91C1 (N4 protein encoded by the UGT91C1 mutant gene from Chinese Patent CN119265152B) and UGT76G4 (protein encoded by the UGT76G4 gene from Chinese Patent CN115094074B), and the sucrose synthase is from sucrose synthase AtSuSy in Chinese Patent CN119265152B.

[0046] D: First, pass the glucose-based stevioside solution III through an activated carbon column for decolorization, and then adsorb the relevant stevioside glycosides and remove the residual dextrin and sucrose through two 500 ml tandem macroporous adsorption resin columns T28. Then wash with 2000 ml of water, control the water temperature at 40 °C. Finally, the first column and the second column are first preliminarily eluted with 2000 ml of 20% ethanol to remove the residual sucrose and part of the dextrin; then elute with 3000 ml of 60% ethanol respectively. After the elution is completed, wash the resin with 1000 ml of water, and collect the eluates and water top solutions of the first and second columns respectively to obtain purified glucose-based stevioside solutions IV and V of the first and second columns, where the elution rate of the first column and the second column is 3 BV / h.

[0047] E: The above glucosyl steviol glycoside solutions Ⅳ and Ⅴ are subjected to alcohol removal, concentration, and drying at 80°C and -0.1 Mpa to obtain glucosyl steviol glycoside. Among them, the weight of glucosyl steviol glycoside Ⅳ is 48.8 g, GSG is 81.6%, TSG is 96.2%, rebaudioside M is 1.3%, and stevioside is 2.4%. The weight of glucosyl steviol glycoside Ⅴ is 26.2 g, GSG is 76.3%, TSG is 95.8%, rebaudioside M is 7.7%, and stevioside is 0.3%.

[0048] Example 4

[0049] A method for preparing glucosyl steviol glycoside by a multi-enzyme combination, comprising the following steps:

[0050] A: Using 50 g of stevioside with a total glycoside of 96.8% (the content of stevioside is 95.3%) as the raw material, using beta-cyclodextrin 1.5 times the mass of steviol glycoside as the auxiliary material, adding 2% w / w of cyclodextrin glucosyltransferase and reacting at 75°C for 7 h first, with a stirring rate of 200 rpm, then raising the temperature to 80°C and continuing to react for 15 h, with a stirring rate of 140 rpm, to obtain glucosyl steviol glycoside solution Ⅰ with a stevioside concentration of 25% w / v;

[0051] B: The glucosyl steviol glycoside solution I is cooled to 42°C, added with α-amylase with an enzyme activity of 10 U / ml for hydrolysis for 7 h, controlling the proportion of glycosylated chain lengths glucose 1-3 to be 50%, inactivated at 100°C for 20 min, and then cooled to 30°C to obtain a low-glycosylated chain length glucosyl steviol glycoside solution Ⅱ;

[0052] C: Using the reaction product in the glucosyl steviol glycoside solution Ⅱ as the substrate, adding 50 mM sucrose in the system, 15 U / ml of UGT glycosyltransferase in the system, 15 U / ml of sucrose synthase in the system, and 2 mM uridine diphosphate glucose. The initial reaction temperature is 30°C, and the temperature is increased by 1°C every 30 min until the reaction temperature reaches 40°C. During the reaction, 1 mM NaOH is added to adjust the system pH to 8, and the remaining unreacted stevioside in the solution is continuously converted into rebaudioside M. It is inactivated at 100°C for 18 min, and after filtering the inactivated conversion solution, it is cooled to 40°C to obtain glucosyl steviol glycoside solution Ⅲ, where the enzyme activity ratio of UGT91C1 to UGT76G4 is 1.4:1. The UGT glycosyltransferase is a mixed enzyme of UGT91C1 (N4 protein encoded by the UGT91C1 mutant gene from Chinese Patent CN119265152B) and UGT76G4 (protein encoded by the UGT76G4 gene from Chinese Patent CN115094074B), and the sucrose synthase is from the sucrose synthase AtSuSy in Chinese Patent CN119265152B;

[0053] D: The glucosyl stevioside solution III is first passed through an activated carbon column for decolorization, and then adsorbed by two 500-ml macroporous adsorption resin columns T28 in series to adsorb the relevant steviosides and simultaneously remove residual dextrin and sucrose. Then it is washed with 2000 ml of water at a water temperature of 40°C. Finally, the first column and the second column are each preliminarily eluted with 1800 ml of 20% ethanol to remove residual sucrose and part of the dextrin. After elution is completed with 2500 ml of 60% ethanol respectively, the resin is rinsed with 1000 ml of water. The eluates and the respective water rinsing solutions of the first and second columns are collected separately to obtain the purified glucosyl stevioside solutions IV and V of the first and second columns. The elution rate of both the first column and the second column is 2 BV / h.

[0054] E: The above-mentioned glucosyl stevioside solutions IV and V are subjected to alcohol removal, concentration, and drying at 80°C and -0.1 Mpa to obtain glucosyl stevioside. Among them, the weight of glucosyl stevioside IV is 47.2 g, GSG is 80.5%, TSG is 95.3%, rebaudioside M is 2.1%, and stevioside is 1.2%. Among them, the weight of glucosyl stevioside V is 31.3 g, GSG is 76.2%, TSG is 95.2%, rebaudioside M is 5.7%, and stevioside is 0.7%.

[0055] Comparative Example 1

[0056] Using the same methods described in steps A, B, and E of Example 1, the glucosyl stevioside solution III is first passed through an activated carbon column for decolorization, and then adsorbed by two 500-ml macroporous adsorption resin columns T28 in series (the resin amount is 20 times the volume of the raw material) to adsorb the relevant steviosides and simultaneously remove residual dextrin and sucrose. Then it is washed with 2000 ml of water. Finally, the first column and the second column are each preliminarily eluted with 1000 ml of 20% ethanol to remove residual sucrose and part of the dextrin. After elution with 1000 ml of 60% ethanol, the purified glucosyl stevioside solutions IV and V are obtained. The resin is rinsed with 1000 ml of water respectively. The eluates and the respective water rinsing solutions of the first and second columns are collected separately to obtain the purified glucosyl stevioside solutions IV and V of the first and second columns. The elution rate of both the first column and the second column is 1 BV / h.

[0057] Finally, after drying, a product with a weight of 73.5 g, TSG of 96.2%, GSG of 80.3%, and rebaudioside A of 7.2% is obtained.

[0058] Comparative Example 2

[0059] Using the methods described in steps A, B, and E of Example 3, the glucosyl stevioside solution III is first passed through an activated carbon column for decolorization, and then adsorbed on two 500-ml tandem macroporous adsorption resin columns T28 (the resin amount is 20 times the volume of the raw material) to adsorb the relevant steviosides and remove residual dextrin and sucrose at the same time. Then it is washed with 2000 ml of water. Finally, the first column and the second column are each preliminarily eluted with 2000 ml of 20% ethanol to remove residual sucrose and part of the dextrin; then eluted with 3000 ml of 60% ethanol to obtain purified glucosyl stevioside solutions IV and V. The resins are each rinsed with 1000 ml of water, and the eluates and the respective water rinsing solutions of the first column and the second column are collected respectively to obtain purified glucosyl stevioside solutions IV and V of the first column and the second column. The elution rate of both the first column and the second column is 3 BV / h.

[0060] Finally, it is dried to obtain 75.3 g in weight, with 96.3% TSG, 79.8% GSG, and 8.2% stevioside.

[0061] Select 10 people for taste evaluation. Compare the tastes of the 2 samples in Example 1 with the 1 sample in Comparative Example 1, and compare and evaluate the tastes of the 2 samples in Example 3 with the 1 sample in Comparative Example 2. Each person in the two groups of comparative samples selects from the dimensions of sweetness, bitterness, and sweetening speed. Each person selects 2 samples with good comprehensive taste in each group. The test results are as follows:

[0062]

[0063] It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for preparing glucosyl steviol glycoside by a multi-enzyme combination, characterized in that It includes the following steps: A: Using rebaudioside A or stevioside as the raw material, beta-cyclodextrin as the auxiliary material, and adding cyclodextrin glycosyltransferase to carry out glycosylation reaction to obtain glucosyl steviol glycoside solution I; B: Cooling the glucosyl steviol glycoside solution I to 45 - 50 °C, adding α-amylase for hydrolysis, controlling the glycosylation chain length, heating to inactivate and then cooling to 30 °C to obtain glucosyl steviol glycoside solution II with a low glycosylation chain length; C: Using the reaction product in the glucosyl steviol glycoside solution II as the substrate, adding sucrose, UGT glycosyltransferase, sucrose synthase and uridine diphosphate glucose, the initial reaction temperature is 35 °C, increasing by 1 °C every 30 min until the reaction temperature reaches 40 °C, continuously converting the unreacted rebaudioside A or stevioside remaining in the solution into rebaudioside M, heating to inactivate and then cooling to 40 °C to obtain glucosyl steviol glycoside solution III; D: Passing the glucosyl steviol glycoside solution III through 2 series-connected resin columns to adsorb relevant steviol glycosides and simultaneously removing residual dextrin and sucrose, washing with water, and separately eluting the first column and the second column to obtain purified glucosyl steviol glycoside solutions IV and V; E: Removing alcohol, concentrating and drying the above glucosyl steviol glycoside solutions IV and V to obtain glucosyl steviol glycoside.

2. The method for preparing glucosyl steviol glycoside by a multi-enzyme combination according to claim 1, characterized in that: In step A, the total glycosides in rebaudioside A and stevioside > 95%, and the content of rebaudioside A or stevioside ≥ 95%.

3. The method for preparing glucosyl steviol glycoside by a multi-enzyme combination according to claim 1, wherein: In step A, first react at 75 °C for 6 - 8 h with a stirring rate of 200 - 300 rpm, and then heat up to 80 °C and continue to react for 6 - 16 h with a stirring rate of 100 - 150 rpm; The concentration of steviol glycoside raw material in the glucosyl steviol glycoside solution I is 10 - 20% w / v, the addition amount of beta-cyclodextrin is 1 - 2 times the mass of steviol glycoside, and the amount of cyclodextrin glycosyltransferase is 1 - 2% w / w of the added raw material amount.

4. The method for preparing glucosylated steviol glycoside by a multi-enzyme combination according to claim 1, characterized in that: In step B, the addition amount of α-amylase is 10 U / ml unit enzyme activity, the hydrolysis time is 6 - 8 h, the inactivation temperature is 95 - 100 °C, and the inactivation time is 10 - 20 min.

5. The method for preparing glucosyl steviol glycoside by a multi-enzyme combination according to claim 1, characterized in that: The UGT glycosyltransferase in step C is a mixed enzyme of UGT91C1 and UGT76G4, and the enzyme activity ratio of UGT91C1 to UGT76G4 is 1 - 1.5:

1.

6. The method for preparing glucosyl stevioside by a multi-enzyme combination according to claim 1, wherein: In step C, the concentration of sucrose in the system is 50 mM, uridine diphosphate glucose is 1 - 2 mM, the UGT glycosyltransferase and sucrose synthase respectively account for 10 - 15 U / ml of the system, and the conversion time is 2 - 4 h.

7. The method for preparing glucosyl steviol glycoside by using a multi-enzyme combination according to claim 1, wherein: In step C, 1 mM NaOH is used to adjust the system pH to 7 - 8 during the reaction process.

8. The method for preparing glucosyl steviol glycoside by a multi-enzyme combination according to claim 1, wherein: In step C, the inactivation temperature is 95 - 100 °C, and the inactivation time is 10 - 20 min.

9. The method for preparing glucosyl steviol glycoside by using a multi-enzyme combination according to claim 1, characterized in that: Before the glucosyl steviol glycoside solution III passes through 2 series-connected resin columns to adsorb relevant steviol glycosides in step D, it first enters an activated carbon column; The series-connected resin column is macroporous adsorption resin T28, the resin amount is 20 times the volume of the raw material, the water washing is 2 BV resin amount, and the water temperature is controlled at 40 °C.

10. The method for preparing glucosyl steviol glycoside by a multi-enzyme combination according to claim 1, characterized in that: In step D, first, 1-2 BV of 20% ethanol is used for preliminary desorption to remove residual sucrose and part of dextrin; then, 1-3 BV of 60% ethanol is used for desorption to obtain purified glucosyl stevioside solutions Ⅳ and Ⅴ, and the desorption rate is 1-3 BV / h for both.

Images