Method for selectively removing rebaudioside M in stevioside and application and product thereof

Abstract

The invention belongs to the field of refining of natural sweeteners, and particularly relates to a method for selectively removing rebaudioside M in stevioside, application of the method and a product. According to the method, beta-1, 2 glucosidase is creatively adopted as a biocatalyst, the Reb M component in stevioside is degraded in a targeted mode through the specific hydrolysis effect of beta-1, 2 glucosidase, and an efficient and high-selectivity Reb M removal technical system is established for the first time in combination with an integrated process of ultrafiltration separation and macroporous adsorption resin purification. The method has the outstanding advantages of mild reaction conditions, high selectivity, high product recovery rate and the like. The invention provides a brand new solution for precise and customized production of stevioside, and has wide application prospects in the fields of healthy food and beverage additives.

Description

Technical Field

[0001] This invention belongs to the field of natural sweetener refining, specifically relating to a method for selectively removing rebaudioside M from steviol glycosides, its application, and products. Background Technology

[0002] Steviol glycosides are derived from the stevia plant (Steviol glycosides) of the Asteraceae family. Stevia Rebaudiana Steviosides, a natural sweetener extracted from Bertoni leaves, are 200-300 times sweeter than sucrose and participate almost entirely in human metabolism. They are widely used in the food, beverage, and pharmaceutical industries as a sucrose substitute. The main active components of steviol glycosides include Reb A, Reb D, and Reb M, with Reb M attracting particular attention due to its highest sweetness (approximately 350 times that of sucrose) and its sweetness profile being closer to that of sucrose. However, in certain applications (such as low-sweetness product development or compound sweetener formulation), it is necessary to selectively reduce the Reb M content to adjust product flavor or cost.

[0003] Currently, international research focuses on purification methods for various single components in stevia. While methods for separating Reb exist, their effectiveness is not ideal. For example, chromatographic separation methods, such as ion exchange resins or simulated moving bed chromatography, can separate Reb M, but suffer from problems like difficult resin regeneration, long operation cycles, and low yields (<70%), and also incur high equipment investment costs. Solvent crystallization methods rely on organic solvents (such as methanol and ethanol) for recrystallization, easily leading to solvent residues that do not meet food-grade safety standards, and have insufficient recovery rates for high-purity Reb A / D. Membrane separation technology, while capable of partially removing impurities, exhibits poor selectivity for Reb M and suffers from significant membrane fouling, limiting its industrial application. Existing enzymatic research primarily focuses on glycosylation modification to synthesize high-sweetness glycosides, but selective removal technologies for Reb M are still underdeveloped. For example, some literature reports the use of α-amylase or cellulase to treat steviol glycosides, but their hydrolysis efficiency for Reb M is low (<30%), and may destroy other glycoside structures (such as the β-1,3 glycosidic bonds of Reb A / D).

[0004] Patent CN107760743A discloses an enzymatic method for preparing n-butyl-β-D-glucosidase. This method prepares a β-glucosidase catalyst, which is then applied to a 1-alkyl-3-methylimidazolium-based ionic liquid microaqueous system to catalyze the reaction of β-D-glucose and n-butanol to synthesize n-butyl-β-D-glucosidase. However, this patent does not address how to selectively remove specific glycosides.

[0005] Patent CN117487871A discloses a method for preparing glucosylstevioside. This method uses a low-alcohol mixture (1-10% by volume) as the reaction matrix, steviol glycoside as the glycosyl acceptor, dextrin as the glycosyl donor, and cyclodextrin glucoside transferase as the catalyst. The mixture is then reacted at a temperature of 60-85°C. However, this patent does not address how to selectively remove specific glycosides.

[0006] Therefore, there is an urgent need to develop an efficient, green, and economical enzymatic reaction method to achieve highly selective removal of Reb M, thereby meeting the demand for customized production of steviol glycosides. Summary of the Invention

[0007] To address the aforementioned shortcomings, this invention provides a highly efficient and selective Reb M removal method using β-1,2-glucosidase as the core catalyst. This enzyme possesses advantages such as high substrate specificity and mild reaction conditions, and is expected to become a key catalyst for achieving this goal.

[0008] The technical solution of this invention is as follows: On the one hand, the present invention provides a method for selectively removing rebaudioside M from steviol glycosides, comprising the following steps: S1. Pretreatment: Dissolve steviol glycosides in buffer solution, keep warm at 45-55℃ for more than 60 minutes, adjust pH to 4.5-5.5, and obtain reaction solution 1; S2. Enzymatic hydrolysis: Add 0.8%-1.5% w / w β-1,2-glucosidase to reaction solution 1, and hydrolyze at 45-55℃ for 2-6 hours to obtain reaction solution 2. S3. Inactivation and separation: The enzyme activity in reaction solution 2 is inactivated by high temperature, and the solution is passed through an ultrafiltration membrane to obtain the filtrate; S4. Elution and purification: The filtrate is passed through a non-polar styrene-based macroporous adsorption resin and eluted sequentially with ethanol at volume concentrations of 20%, 40%, and 70% to obtain the eluent. S5. Concentrate and dry the eluent to obtain the final product.

[0009] Specifically, the buffer solution in step S1 is a phosphate or citrate buffer solution with a pH of 4.5-5.5, such as pH values ​​of 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, and any point within that range.

[0010] Preferably, the buffer solution in step S1 is a citrate buffer solution with a pH of 5.0-5.5, such as pH values ​​of 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, and any point within that range.

[0011] Preferably, in some embodiments, the temperature in step S1 is 45°C. Specifically, the temperature mentioned in step S1 can be any value within the range of 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, and 55.

[0012] Preferably, in some embodiments, the temperature in step S1 is 50°C.

[0013] Specifically, the pH adjustment in step S1 can be any value within the range of 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5.

[0014] Preferably, in some embodiments, step S1 involves adjusting the pH to 4.5.

[0015] Preferably, in some embodiments, step S1 involves adjusting the pH to 5.0.

[0016] Preferably, in some embodiments, step S1 involves adjusting the pH to 5.5.

[0017] Specifically, the amount of β-1,2-glucosidase added in step S2 can be 0.8% w / w, 0.9% w / w, 1.0% w / w, 1.1% w / w, 1.2% w / w, 1.3% w / w, 1.4% w / w, 1.5% w / w, or any value within that range.

[0018] Preferably, in some embodiments, the amount of β-1,2-glucosidase added in step S2 is 0.8% w / w.

[0019] In some embodiments, the amount of β-1,2-glucosidase added in step S2 is 1.1% w / w.

[0020] In some embodiments, the amount of β-1,2-glucosidase added in step S2 is 1.5% w / w.

[0021] Specifically, the enzyme activity of β-1,2-glucosidase in step S2 is 1000 U / g.

[0022] Specifically, the temperature mentioned in step S2 can be any value within the range of 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, and 55.

[0023] In some embodiments, the temperature in step S2 is 45°C. In some embodiments, the temperature in step S2 is 50°C.

[0024] In some embodiments, the temperature in step S2 is 55°C.

[0025] Specifically, the enzymatic hydrolysis time in step S2 can be 5-6 hours.

[0026] Specifically, the enzymatic hydrolysis time in step S2 can be any value within the range of 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, or 2h.

[0027] Preferably, in some embodiments, the enzymatic hydrolysis time in step S2 is 2 hours.

[0028] Preferably, in some embodiments, the enzymatic hydrolysis time in step S2 is 4 hours.

[0029] Preferably, in some embodiments, the enzymatic hydrolysis time in step S2 is 5 hours.

[0030] Preferably, in some embodiments, the enzymatic hydrolysis time in step S2 is 6 hours.

[0031] Specifically, the molecular weight cutoff of the ultrafiltration membrane in step S3 can be 1-3 kDa, such as 1 kDa, 2 kDa, 3 kDa, or any value within that range.

[0032] Preferably, the molecular weight cutoff of the ultrafiltration membrane in step S3 is 2-3 kDa.

[0033] Preferably, the molecular weight cutoff of the ultrafiltration membrane in step S3 is 3 kDa.

[0034] Specifically, the operating pressure of the ultrafiltration membrane in step S3 can be 0.2-0.3 MPa.

[0035] Preferably, the operating pressure of the ultrafiltration membrane in step S3 is 0.3 MPa.

[0036] Specifically, the ultrafiltration membrane in step S3 is made of polyethersulfone (PES) or regenerated cellulose.

[0037] Specifically, the non-polar styrene-based macroporous adsorption resin in step S4 is selected from HPD-800A, XAD-4, D101 or Amberlite XAD-1180.

[0038] Preferably, the non-polar styrene-based macroporous adsorption resin in step S4 is HPD-800A.

[0039] Specifically, the concentration conditions in step S5 are: concentration at 50-60℃ and vacuum degree -0.08MPa until the solid content is ≥30%; the drying is spray drying; the conditions for spray drying are: inlet air temperature: 180±5℃, outlet air temperature: 80±5℃, atomization pressure: 0.2-0.4MPa, and finished product moisture content ≤5%.

[0040] In another aspect, the present invention provides a sweetener obtained by the aforementioned method.

[0041] In another aspect, the present invention provides the application of the aforementioned sweetener in the preparation of products containing sweetener.

[0042] Specifically, the products include, but are not limited to: food, beverages, pharmaceuticals, health products, or daily chemical products.

[0043] The beneficial effects of this invention are as follows: This invention utilizes β-1,2-glucosidase as the core catalyst, combined with ultrafiltration and resin purification processes, to achieve, for the first time, highly efficient and selective removal of Reb M. Compared to existing technologies, this method represents a significant breakthrough in selectivity, cost, and environmental friendliness, providing a new approach for the customized production of steviol glycosides. Detailed Implementation

[0044] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0045] Example 1 A method for the bioenzymatic separation of rebaudioside M from steviol glycosides, the specific implementation steps of which are as follows: (1) Raw material pretreatment: Take 100g of crude steviol glycoside with 15% (w / w) Reb M, dissolve it in 0.1M citrate buffer (pH5.2) to a final concentration of 8% (w / v), make up to 5L of reaction solution, heat treat the reaction solution (keep warm at 50℃ for 60 minutes), and adjust the pH to 5.0; (2) Enzymatic hydrolysis: Add 1.2% (w / w) β-1,2-glucosidase (Wuhan AmyJet Technology Co., Ltd., shipment number: QAB-XYL01, enzyme activity is 1000U / g) to the adjusted reaction solution and continue to react in a 50℃ water bath with shaking (150rpm) for 5 hours. (3) Inactivation and separation: After the enzymatic hydrolysis reaction is completed, the reaction solution is rapidly heated to inactivate the enzyme, and the enzyme is inactivated by boiling water bath for 15 minutes. Then, it is filtered through a 3kDa ultrafiltration membrane (operating pressure 0.3MPa). The filtered supernatant is collected and prepared for resin column filtration. (4) Elution and purification: Before loading the resin column, the resin is pretreated: Styrene-based macroporous adsorption resin is soaked in 95% ethanol (v / v) for 24 hours and then packed into the column with a column volume of 5 BV; loading (the loading amount is within 70%-80% of the maximum adsorption capacity) and elution: loading flow rate: 3 BV / h; elution program: elute with 20% (v / v) ethanol for 2 BV to remove impurities; after elution, elute with 40% (v / v) ethanol for 3 BV and collect the target glycoside (Reb A / D); elute with 70% (v / v) ethanol for 1 BV and recover the residual components.

[0046] (5) Concentrate and dry Vacuum concentration: The eluent is concentrated at 50-60℃ and a vacuum of -0.08 MPa to a solid content ≥30%; Spray drying: Inlet air temperature: 180±5℃; Outlet air temperature: 80±5℃; Atomization pressure: 0.2-0.4MPa; The moisture content of the finished product is ≤5%, the eluent is concentrated to a solid content of 30%, and the final product yield is 88.2% after spray drying.

[0047] The experimental data are shown in Table 1: Table 1

[0048] The experimental data were detected using high-performance liquid chromatography (HPLC).

[0049] Standard product: Stevioside, Putian Tongchuang Biotechnology Co., Ltd., model: STX-395095.

[0050] The parameters for high performance liquid chromatography are as follows: Column: Amino column; Brand: SynHend; Model: HD-NH2 120A; 4.6mm*250mm; 5μm particles; Temperature: 30℃. isocratic mobile phase: 20% buffer solution, 80% acetonitrile; Flow rate: 1.5 mL / min; Injection volume: 12 μL; Detection wavelength: UV210nm (4nmbw), reference: 260nm (120nmbw); Running time: 60 minutes; Sampling temperature: room temperature.

[0051] Example 2 A method for the bioenzymatic separation of rebaudioside M from steviol glycosides, the specific implementation steps of which are as follows: (1) Raw material pretreatment: Take 200g of crude steviol glycoside with Reb M content of 15% (w / w), dissolve it in 0.2M citrate buffer (pH5.2) to a final concentration of 7% (w / v), make up to 10L of reaction solution, heat treat the reaction solution (keep warm at 45℃ for 60 minutes), and adjust the pH to 4.5.

[0052] (2) Enzymatic hydrolysis: Add 1.5% (w / w) of substrate mass β-1,2-glucosidase (Wuhan AmyJet Technology Co., Ltd., shipment number: QAB-XYL01, enzyme activity is 1000U / g) to the adjusted reaction solution and continue to react in a 55℃ water bath with shaking (150rpm) for 6 hours.

[0053] (3) Inactivation and separation: After the enzymatic hydrolysis reaction is completed, the reaction solution is rapidly heated to inactivate the enzyme, and then the enzyme is inactivated by boiling water bath for 10 minutes. After that, it is filtered through a 3kDa ultrafiltration membrane (operating pressure 0.3MPa), and the filtered supernatant is collected for resin column filtration.

[0054] (4) Elution and purification: Before loading the resin column, the resin was pretreated: styrene-based macroporous adsorption resin (HPD-800A) was soaked in 95% (v / v) ethanol for 24 hours and then packed into the column with a column volume of 15 BV; loading (reaction liquid volume 10 L) and elution: loading flow rate: 3 BV / h; elution program: elute with 20% (v / v) ethanol for 5 BV to remove impurities; after elution, elute with 40% (v / v) ethanol for 5 BV and collect the target glycoside (Reb A / D); elute with 70% (v / v) ethanol for 2 BV and recover the residual components.

[0055] (5) Concentrate and dry Vacuum concentration: The eluent is concentrated at 50-60℃ and a vacuum of -0.08 MPa to a solid content ≥30%; Spray drying: Inlet air temperature: 180±5℃; Outlet air temperature: 80±5℃; Atomization pressure: 0.2-0.4 MPa; The moisture content of the finished product is ≤5%, the eluent is concentrated to a solid content of 30%, and the final product yield is 89.9% by spray drying.

[0056] The experimental data are shown in Table 2: Table 2

[0057] 1. Conventional methods Standard experimental protocol: ion exchange chromatography, the specific steps are as follows: (1) Crude steviol glycosides: 10.0g (Reb M content 15.2%), fixed volume 5L, temperature 45℃, pH 4.5, keep warm for 6 hours, and filter the reaction solution through a membrane after the reaction is completed.

[0058] (2) Treatment of the resin column (strong acid cation exchange resin 001×7 type): The resin was soaked in 5% (w / w) HCl for 24 hours, washed with water until neutral, and sample loading: 10g of raw material was dissolved in 100mL of water and loaded at a flow rate of 1BV / h (column volume 50mL); eluted with 0.1M NaCl gradient and the target fraction was collected; desalting and drying: the eluent was desalted by activated carbon and freeze-dried to obtain the final product.

[0059] The experimental data are shown in Table 3: Table 3

[0060] 2. The effect of solution pH The differences from Example 1 and the test results are shown in Tables 4 and 5, respectively: Table 4

[0061] Table 5 Results

[0062] According to experimental data, the enzyme activity retention rate was the highest (95.1%) and the catalytic rate was the largest at pH 5.0. The enzyme activity decreased significantly at pH <4.5 or >5.5. The comprehensive data showed that when the pH was between 4.5 and 5.5, the RebM removal rate was >80% and the total glycoside recovery rate was >87%.

[0063] 3. Effect of water bath temperature on enzymatic hydrolysis The differences from Example 1 and the test results are shown in Tables 6 and 7, respectively: Table 6

[0064] Table 7 Results

[0065] The enzyme hydrolysis efficiency is highest at 50℃ (removal rate 85.2%), but the enzyme half-life decreases significantly at temperatures above 55℃ (6.5-21 h). Based on the experimental data, the RebM removal rate is highest at 45-55℃, and the enzyme half-life time is relatively sufficient to meet production requirements.

[0066] 4. Effect of the amount of β-1,2-glucosidase added The differences from Example 1 and the test results are shown in Tables 8 and 9, respectively: Table 8

[0067] Table 9 Results

[0068] In summary, when enzyme addition is between 0.8% and 1.5%, the RebM removal rate is >80%, and the enzyme activity residue is <0.01%, making this range the most economical.

[0069] 5. Effect of enzymatic hydrolysis time The differences from Example 1 and the test results are shown in Tables 10 and 11, respectively: Table 10

[0070] Table 11 Results

[0071] 6. The impact of the elution process The differences from Example 1 and the test results are shown in Tables 12 and 13, respectively: Table 12

[0072] Table 13 Results

[0073] The results of Comparative Example 10 show that lower concentrations of ethanol cannot effectively elute highly polar hydrolysis products (free glucose, steviol monoglucoside), leaving some products in the resin and affecting subsequent fractionation. The RebA / D recovery rate decreased significantly, and an additional 2-4 BV of ethanol is required to improve the recovery rate.

[0074] The results of Comparative Example 11 show that when using higher concentrations of ethanol, the target fraction will decrease due to the higher ethanol concentration when eluting strongly hydrophobic impurities. Furthermore, the increased use of high-concentration ethanol leads to a rise in the COD value of the production wastewater, significantly increasing treatment costs.

Claims

1. A method for selectively removing rebaudioside M from steviol glycosides, characterized in that, Includes the following steps: S1. Pretreatment: Dissolve steviol glycosides in buffer solution, keep warm at 45-55℃ for more than 60 minutes, adjust pH to 4.5-5.5, and obtain reaction solution 1; S2. Enzymatic hydrolysis: Add 0.8%-1.5% w / w β-1,2-glucosidase to reaction solution 1, and hydrolyze at 45-55℃ for 2-6 hours to obtain reaction solution 2. S3. Inactivation and separation: The enzyme activity in reaction solution 2 is inactivated by high temperature, and the solution is passed through an ultrafiltration membrane to obtain the filtrate; S4. Elution and purification: The filtrate is passed through a non-polar styrene-based macroporous adsorption resin and eluted sequentially with ethanol at volume concentrations of 20%, 40%, and 70% to obtain the eluent. S5. Concentrate and dry the eluent to obtain the final product.

2. The method according to claim 1, characterized in that, The buffer solution in step S1 is a phosphate or citrate buffer solution with a pH of 4.5-5.

5.

3. The method according to claim 2, characterized in that, The buffer solution described in step S1 is a citrate buffer solution with a pH of 5.0-5.

5.

4. The method according to claim 1, characterized in that, The temperature mentioned in step S1 is 45°C or 50°C.

5. The method according to claim 1, characterized in that, Step S1 involves adjusting the pH to 4.5, 5.0, or 5.

5.

6. The method according to claim 1, characterized in that, The amount of β-1,2-glucosidase added in step S2 is 0.8% w / w, 1.1% w / w, or 1.5% w / w.

7. The method according to claim 6, characterized in that, The enzyme activity of β-1,2-glucosidase described in step S2 is 1000 U / g.

8. The method according to claim 1, characterized in that, The temperature mentioned in step S2 is 45°C, 50°C or 55°C.

9. The method according to claim 1, characterized in that, The enzymatic hydrolysis time in step S2 is 2h, 4h, 5h or 6h.

10. The method according to claim 1, characterized in that, The ultrafiltration membrane in step S3 has a molecular weight cutoff of 1-3 kDa, preferably 2-3 kDa, and more preferably 3 kDa; the operating pressure of the ultrafiltration membrane is 0.2-0.3 MPa, preferably 0.3 MPa.

11. The method according to claim 10, characterized in that, The ultrafiltration membrane in step S3 is made of polyethersulfone (PES) or regenerated cellulose.

12. The method according to claim 1, characterized in that, The non-polar styrene-based macroporous adsorption resin in step S4 is selected from HPD-800A, XAD-4, D101 or Amberlite XAD-1180, preferably HPD-800A.

13. The method according to claim 1, characterized in that, The concentration conditions in step S5 are: concentration at 50-60℃ and vacuum degree -0.08MPa until the solid content is ≥30%; the drying is spray drying; the conditions for spray drying are: inlet air temperature: 180±5℃, outlet air temperature: 80±5℃, atomization pressure: 0.2-0.4MPa, and finished product moisture content ≤5%.

14. A sweetener, characterized in that, The sweetener is obtained by the method according to any one of claims 1-13.

15. The use of the sweetener of claim 14 in the preparation of a product containing the sweetener.

16. The application according to claim 15, characterized in that, The products mentioned are food, beverages, pharmaceuticals, health products, or daily chemical products.

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