Synthetic method of rebaudioside M
By using the process flow of filtration, resin decolorization, ultrafiltration, concentration and crystallization in the preparation of rebaudioside M, the problems of high preparation cost, high industrialization difficulty and low yield in the prior art are solved, and high purity and High yield production improves product quality and efficiency.
Patent Information
- Application Number
- CN202411271642.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-11
- Publication Date
- 2024-12-13
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
In the prior art, the preparation cost of rebaudioside M is high, industrialized and has low yields, making it difficult to achieve high purity and high conversion rate production.
Using filtration, resin decolorization, two ultrafiltration, concentration and crystallization, the purity and yield of rebaudioside M are improved by adjusting pH, temperature and the use of additives, including the use of recombinant E. coli fermentation broth and bottom The reaction of the substance system was then subjected to filtration, decolorization, ultrafiltration, concentration and crystallization, and finally crystallization was carried out by the combination of ethanol and seed crystals.
The maximum purity of rebaudioside M was achieved at 98.5% and the yield reached 93.8%, solving the problems of incomplete removal of product pigments and low yields in the prior art, and improving the quality and efficiency of the product.
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Figure CN119120626A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and particularly relates to a legal method for preparing rebaudioside M, and also comprises a method for separating and purifying the rebaudioside M after synthesis. Background Art
[0002] Rebaudioside M is a component of stevioside, accounting for about 0.15% to 0.2% of the total stevioside. It has the best taste and is closest to the taste of sucrose without any bad aftertaste. It is praised by industry insiders as the "gold of steviosides". The sweetness of Rebaudioside M is about 300 times that of sucrose, making it an ideal sucrose substitute.
[0003] A document discloses a method for synthesizing rebaudioside M by catalysis of recombinant yeast, which uses rebaudioside A as a substrate, and in the presence of sucrose and sodium citrate, uses recombinant yeast-expressed enzyme as a catalyst to convert rebaudioside A into rebaudioside M, which is then concentrated, adsorbed on a column, and crystallized to obtain rebaudioside M. However, in this method, the substrate concentration is low, the preparation cost is high, and industrialization is difficult.
[0004] There are also documents that disclose that rebaudioside is produced by host cells during fermentation. After fermentation, the fermentation broth is centrifuged to remove host cells and other dense debris, and then the clarified fermentation broth is diluted with water, and the pH is adjusted to 10 by adding NaOH, and then the clarified fermentation broth is ultrafiltered using a 20kDa molecular weight cutoff to separate large solutes from small steviol glycosides. Rebaudioside M is then crystallized from the solution through nanofiltration to form an acidic slurry, and then the acidic slurry is subjected to a first filter press and washed with acid, and the acid-washed material is subjected to a second filter press, resuspended in water, and spray-dried to form the final purified rebaudioside M. Summary of the invention
[0005] In order to solve the above technical problems, the present invention provides a production process of rebaudioside with a purity of up to 98.5% and a yield of up to 93.8%.
[0006] The synthetic method of rebaudioside M comprises the following steps: (1) Preparation of substrate system In every 1L of water, add the following raw materials in the following proportions: 300-400g of rebaudioside A, 140-150g of glucose, 2.5-4g of dipotassium hydrogen phosphate, and 1250-1500g of sucrose; heat to 90-95°C, stir and dissolve, and when the liquid becomes transparent, cool to 38°C to form a substrate system; (2) Take the following raw materials: add 0.1-1 g of surfactant to every 1 L of recombinant Escherichia coli fermentation broth, stir, transfer to the substrate system to form a reaction system, dilute the reaction system to 5 L with water, react, and obtain a reaction solution; Among them, the volume ratio of E. coli fermentation broth to substrate system is: 0.5~3:1; The reaction solution is filtered, decolorized, ultrafiltered, concentrated, crystallized, and dried to obtain rebaudioside M solid.
[0007] Preferably, in (1), before the reaction, the initial pH value of the reaction system is adjusted to 8.4-8.6, the temperature is controlled to 35-40° C., and the stirring speed is 150-300 rpm.
[0008] Preferably, in (2), the specific steps are: S1 filtration: take the reaction solution, add water, the volume ratio of water to reaction solution is 0.4~2:1, heat to 60~90℃, add activated carbon, heat treat for 30~60min, hot filter at 60~90℃, top wash the filter cake with hot water, combine all the filtrates to obtain rebaudioside M filtrate; S2 decolorizes the Rebaudioside M filtrate in S1 through a macroporous resin to obtain a Rebaudioside M decolorized solution; S3 filters the rebaudioside M decolorized solution in S2 through an ultrafiltration membrane twice to obtain an ultrafiltrate; S4 vacuum rotary evaporation concentrates the ultrafiltrate in S3 to obtain a concentrated solution; S5: adding ethanol to the concentrated solution in S4, followed by cooling, growing crystals, and secondary cooling crystallization to obtain crystalline rebaudioside M; S6 was filtered to collect the crystals, top washed with cold water, dried, and then collected and dried using a double cone vacuum.
[0009] Preferably, in S1, the reaction liquid is heated to 70°C, activated carbon is added for heat treatment for 50 minutes, the amount of activated carbon is 2.5% of the weight of the reaction liquid, and then filtered through a plate and frame, and the liquid temperature is controlled at 60-65°C during filtration; and then top washed with hot water with a volume of 0.5 times the volume of the liquid, and the hot water temperature is 80°C.
[0010] Preferably, in S3, ultrafiltration is performed using ultrafiltration membranes of 10000Da and 1000Da in sequence to cut off materials with a molecular weight of 1000-10000Da.
[0011] Preferably, in S4, the mass concentration of soluble solids in the concentrated solution is 18-23%.
[0012] Preferably, in S5, the volume concentration of ethanol is 95%, the temperature is 70°C, and the amount used is the same as the volume of the concentrate; The concentrated solution was cooled to 60°C, and seed crystals accounting for 0.1% of the volume of the concentrated solution were added. The crystal growth time was 3-5 hours, and the stirring speed was 50 rpm. Then, the temperature was reduced for a second time to 15°C for crystallization at a rate of 2-4°C / h and maintained for 3 hours.
[0013] The beneficial effects of the present invention are: The present invention improves the process and adopts the preparation process of filtration, resin decolorization, two ultrafiltrations, concentration, crystallization and drying, thereby solving the low yield problem of preparing rebaudioside M in the prior art. According to HPLC detection, the rebaudioside M produced by the present invention has the following characteristics: (1) High conversion rate, up to 96.9%; (2) High purity, the total content can reach 98.5%; (3) High yield, up to 93.8%. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 This is the HPLC spectrum of rebaudioside M detected after it was synthesized in Example 1; Figure 2 This is the HPLC spectrum of rebaudioside M detected after it was synthesized in Example 2; Figure 3 This is the HPLC spectrum of rebaudioside M detected after it was synthesized in Example 3; Figure 4 This is the HPLC spectrum of the finished product of rebaudioside M after separation and purification in Example 4; Figure 5 This is the HPLC spectrum of the finished product of rebaudioside M after separation and purification in Example 5; Figure 6 This is the HPLC spectrum of the finished product of rebaudioside M after separation and purification in Example 8; Figure 7 This is the HPLC spectrum of the finished product of rebaudioside M after separation and purification in Comparative Example 1. DETAILED DESCRIPTION
[0015] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.
[0016] Embodiment 1-3 is the synthesis method of rebaudioside M, and the specific steps are as follows: Example 1 The synthesis method of rebaudioside M comprises the following steps: (1) Add 300 g of rebaudioside A, 150 g of glucose, 2.5 g of dipotassium hydrogen phosphate, 1250 g of sucrose, and 1000 mL of water into the reactor; heat to 90-95 °C, stir and dissolve, and when the liquid becomes transparent, cool to 38 °C to form a substrate system; (2) Take 1000 mL of the recombinant E. coli fermentation broth, add 0.5 g of Tween 80, stir for 30 to 60 min, transfer to the substrate system in (1), and adjust the reaction system to 5 L with water; (3) The pH value of the reaction system in (2) was adjusted to about 8.5 with 30% sodium hydroxide, the temperature was controlled at 35-38° C., the stirring speed was 200 rpm, and the reaction was carried out to obtain a reaction solution containing a crude product of rebaudioside M.
[0017] The crude product of rebaudioside M was tested by high performance liquid chromatography to evaluate the conversion rate. The conditions for high performance liquid chromatography testing were as follows: Chromatographic column: Welch Ultimate XB-C18 250×4.6mm, 5μm Injection volume: 20µL; column temperature: 30°C; flow rate: 1.0mL / min; wavelength: 205nm Mobile phase: A water, B acetonitrile, gradient elution 0min 15min 18min 19min 30min A 80 53 53 80 80 B 20 47 47 20 20 The detection conditions in the following examples are the same as above.
[0018] According to the analysis and calculation of the liquid phase detection results, after 36 hours of reaction, rebaudioside A was converted into rebaudioside M with a conversion rate of 87.6% and no by-products were generated.
[0019] Example 2 The synthesis method of rebaudioside M comprises the following steps: (1) Add 400 g of rebaudioside A, 150 g of glucose, 4 g of dipotassium hydrogen phosphate, 1500 g of sucrose, and 1000 mL of water into the reactor; heat to 90-95 °C, stir and dissolve, and when the liquid becomes transparent, cool to 38 °C to form a substrate system; (2) Take 2000 mL of the recombinant E. coli fermentation broth, add 0.5 g of Tween 80, stir for 30-60 min, transfer to the above substrate system, and adjust the reaction system to 5 L with water; (3) adjusting the initial pH value to 8.5 with 30% sodium hydroxide, controlling the temperature at 35-38° C., stirring at 200 rpm, and reacting to obtain a reaction solution containing a crude product of rebaudioside M.
[0020] After 36 hours of reaction, rebaudioside A was almost completely converted into rebaudioside M, with a conversion rate of about 96.9% and no by-products were generated.
[0021] Example 3 The synthesis method of rebaudioside M comprises the following steps: (1) Add 400 g of rebaudioside A, 150 g of glucose, 4 g of dipotassium hydrogen phosphate, 1500 g of sucrose, and 1000 mL of water into the reactor; heat to 90-95 °C, stir and dissolve, and when the liquid becomes transparent, cool to 38 °C to form a substrate system; (2) Take 3000 mL of the recombinant E. coli fermentation broth, add 0.5 g of Tween 80, stir for 30-60 min, transfer to the above substrate system, and adjust the reaction system to 5 L with water; (3) adjusting the initial pH value to 8.5 with 30% sodium hydroxide, controlling the temperature at 35-38° C., stirring at 200 rpm, and reacting to obtain a reaction solution containing a crude product of rebaudioside M.
[0022] After 36 hours of reaction, rebaudioside A was almost completely converted into rebaudioside M, with a conversion rate of up to 96.6% and no by-products generated.
[0023] Example 4-9 is a method for purifying rebaudioside M, and the specific steps are as follows: Example 4 The method for separating and purifying the crude product of rebaudioside M comprises the following steps: (1) Filtration: Prepare 50 liters of reaction solution according to the method in Example 2, add 20 liters of water, raise the temperature to 60°C, add 1.25 kg of activated carbon, heat treat for 50 min, perform hot filtration, and then top wash with 80°C hot water 0.5 times the volume of the feed solution, combine all the filtrates to obtain the rebaudioside M filtrate; (2) Decolorizing the rebaudioside M filtrate with a macroporous resin to obtain a colorless and transparent rebaudioside M decolorized solution; during decolorization, the filtrate temperature was maintained at 50-55°C and the feed rate was 2-3 BV; (3) Ultrafiltration treatment of the decolorized liquid: first, filter it with a 10,000Da ultrafiltration membrane to obtain a permeate; then, filter the permeate through a 1,000Da ultrafiltration membrane to obtain a retentate; (4) The retentate is concentrated by vacuum rotary evaporation, and the retentate of rebaudioside M is concentrated by using an evaporator at a concentration temperature of 70 to 75° C. and a pressure of -0.08 to -0.1 MPa to obtain a concentrated solution of rebaudioside M having a soluble solid mass concentration of 20 wt %; (5) Add 95% ethanol at 70°C to the concentrated solution of rebaudioside M in an amount equal to the volume of the concentrated solution, cool the solution to 60°C, add seed crystals accounting for 0.1% of the volume of the concentrated solution, grow the crystals for 3 to 5 hours, stir at 50 rpm, cool at a rate of 3°C / h, cool to 15°C, and maintain for 3 hours; (6) Filter the crystallization liquid, wash it with cold water, and dry the collected crystals with hot air at a temperature of 105°C and a vacuum degree of -0.08 to -0.1 MPa. After drying, crush the crystals to obtain the finished product of rebaudioside M.
[0024] The obtained finished product Rebaudioside M was weighed, and the product weight was 3.74 kg, and the yield was 72.5%. The product was tested by HPLC, and the testing method and conditions were the same as those in Example 1, and the content of Rebaudioside M (on a dry basis) was 97.5%, white powder, moisture content was 1.98%, and ash content was 0.56%.
[0025] Example 5 The method for separating and purifying the crude product of rebaudioside M comprises the following steps: (1) Filtration: Prepare 50 liters of reaction solution according to the method in Example 2, add 50 liters of water, raise the temperature to 70°C, add 1.25 kg of activated carbon, heat treat for 50 min, perform hot filtration, and then top wash with 80°C hot water 0.5 times the volume of the feed solution, combine all the filtrates to obtain the rebaudioside M filtrate; (2) Decolorizing the Rebaudioside M filtrate through a macroporous resin to obtain a colorless and transparent Rebaudioside M decolorized solution. During the decolorization, the temperature of the Rebaudioside M filtrate is 50-55° C. and the feed rate is 2-3 BV; (3) Ultrafiltration of the decolorized liquid, first using a 10,000Da ultrafiltration membrane to obtain a permeate; then filtering the permeate through a 1,000Da ultrafiltration membrane to obtain a retentate; (4) The retentate was concentrated by vacuum rotary evaporation, and the retentate of rebaudioside M was concentrated by using an evaporator at a concentration temperature of 70 to 75° C. and a pressure of -0.06 to -0.1 MPa to obtain a concentrated solution of rebaudioside M having a soluble solid mass concentration of 23 wt%; (5) Add 95% ethanol at 70°C to the Rebaudioside M concentrate in an amount equal to the volume of the concentrate, cool the solution to 60°C, add seed crystals accounting for 0.1% of the volume of the concentrate, grow the crystals for 3 to 5 hours, stir at 50 rpm, cool at a rate of 3°C / h, cool to 15°C, and maintain for 3 hours; (6) The crystallization liquid is filtered and top washed with cold water. The collected crystals are dried with hot air at a temperature of 105°C and a vacuum degree of -0.08 to -0.1 MPa. After drying, the crystals are crushed to obtain the finished product of rebaudioside M.
[0026] The obtained product, rebaudioside M, was weighed to 4.83 kg, with a yield of 93.8%. The product was tested by HPLC, and the content of rebaudioside M (on a dry basis) was 98.5%, white powder, with a moisture content of 1.95% and an ash content of 0.50%.
[0027] Example 6 The method for separating and purifying the crude product of rebaudioside M comprises the following steps: The difference from Example 5 is that step (1) is specifically as follows: 50 liters of reaction solution is prepared according to the method in Example 2, 100 liters of water is added, the temperature is raised to 70° C., 1.25 kg of activated carbon is added, heat treatment is performed for 50 min, hot filtration is performed, and top washing is performed with 0.5 times the volume of 80° C. hot water, and all filtrates are combined to obtain rebaudioside M filtrate. The other steps are the same as those in Example 5.
[0028] The obtained product, rebaudioside M, was weighed to 4.82 kg, with a yield of 93.4%. The product was tested by HPLC, and the content of rebaudioside M (on a dry basis) was 98.4%, white powder, with a moisture content of 1.90% and an ash content of 0.55%.
[0029] Example 7 The difference from Example 5 is that the resin decolorization in step (2) is not performed, and the filtrate in step (1) is directly subjected to ultrafiltration in step (3).
[0030] The obtained product, rebaudioside M, was weighed to be 4.85 kg, with a yield of 93.9%. The product was tested by HPLC, and the content of rebaudioside M (on a dry basis) was 98.2%, and the product was a light yellow powder with a moisture content of 1.94% and an ash content of 0.51%.
[0031] Example 8 The difference from Example 5 is that the ultrafiltration in step (3) is not performed, and the decolorized liquid in step (2) is directly concentrated and crystallized.
[0032] The obtained product, rebaudioside M, was weighed to have a weight of 4.94 kg and a yield of 95.7%. The product was tested by HPLC, and the content of rebaudioside M (on a dry basis) was 93.0%, the product was white powder, the moisture content was 1.93%, and the ash content was 1.03%.
[0033] Example 9 The difference from Example 5 is that the concentrated solution in step (4) is directly spray-dried, with an inlet air temperature of 180-200°C and an outlet air temperature of 80-100°C to obtain a solid powder of rebaudioside M.
[0034] The obtained product, rebaudioside M, was weighed to be 4.12 kg, with a yield of 79.8%. The product was tested by HPLC, and the content of rebaudioside M (on a dry basis) was 93.2%, and the product was a light yellow powder with a moisture content of 1.84% and an ash content of 0.53%.
[0035] Comparative Example 1 Rebaudioside M was separated and purified according to the method of patent: CN 115697077A: According to the method in Example 2, 5 liters of reaction solution was prepared, and water was added to dissolve the precipitated rebaudioside M; the clear liquid was collected by centrifugation, and the pH of the clear liquid was adjusted to pH=10 with NaOH, and then the clear liquid was ultrafiltered using a 20kDa ultrafiltration membrane, and the filtrate was adjusted to pH=4 with citric acid, and nanofiltration was performed using a 300 to 500Da filter. Rebaudioside M was concentrated by nanofiltration, cooled and crystallized, and crystals were collected by filter pressing, washed with acid water, and filtered twice. The collected solid was resuspended in water and spray-dried to obtain the finished product of rebaudioside M.
[0036] The obtained product, rebaudioside M, was weighed, and the product weight was 361 g, with a yield of 69.9%. The product was tested by HPLC, and the content of rebaudioside M (on a dry basis) was 93.4%, and the product was a light yellow powder with a moisture content of 1.90% and an ash content of 1.25%.
[0037] Table 1 Rebaudioside M finished product test content color Moisture content Ash Product yield Example 4 97.5% White 1.98% 0.56% 72.5% Example 5 98.5% White 1.95% 0.50% 93.8% Example 6 98.4% White 1.90% 0.55% 93.4% Example 7 98.2% Slightly yellow 1.94% 0.51% 93.9% Example 8 93.0% White 1.93% 1.03% 95.7% Example 9 93.2% Slightly yellow 1.84% 0.53% 79.8% Comparative Example 1 93.4% Light yellow 1.90% 1.25% 69.9% According to the data results in the above table, compared with Comparative Example 1, the embodiment of the present invention adopts a combined treatment of filtration + resin decolorization + ultrafiltration + concentration + crystallization, which solves the problems of incomplete pigment removal, low content and low yield of the product obtained in the prior art; the content of rebaudioside M in Example 5 is (on a dry basis) 98.5%, and the yield reaches 93.8%.
[0038] The steps of plate and frame filtration, resin decolorization, ultrafiltration, concentration, crystallization, etc. all have the function of material separation and purification in this field; it can be seen from Examples 7 and 8 that without decolorization and ultrafiltration, the impurity and color problems cannot be solved by crystallization alone; it can be seen from Example 9 that the concentrated solution is directly spray-dried, and the collected product yield is low and the product quality is poor; the crystallization process of the present invention adopts the crystallization method of ethanol + seed + cooling, which can avoid the embedding of impurities and pigments and remove some alcohol-soluble impurities. When the resin decolorization + ultrafiltration + crystallization are combined, the purity of the obtained product is improved and the yield is also significantly improved.
Claims
1. A method for synthesizing rebaudioside M, characterized in that: The steps include: (1) Preparation of substrate system In every 1L of water, add the following raw materials in the following proportions: 300-400g of rebaudioside A, 140-150g of glucose, 2.5-4g of dipotassium hydrogen phosphate, and 1250-1500g of sucrose; heat to 90-95°C, stir to dissolve, and when the liquid becomes transparent, cool to 38°C to form a substrate system; (2) Take the following raw materials: add 0.1-1 g of surfactant to every 1 L of recombinant Escherichia coli fermentation broth, stir, transfer to the substrate system to form a reaction system, dilute the reaction system to 5 L with water, react, and obtain a reaction solution; The volume ratio of E. coli fermentation broth to substrate system is: 0.5~3:1; The reaction solution is filtered, decolorized, ultrafiltered, concentrated, crystallized, and dried to obtain rebaudioside M solid.
2. The method for synthesizing rebaudioside M according to claim 1, characterized in that: In (1), before the reaction, the initial pH value of the reaction system was adjusted to 8.4-8.6, the temperature was controlled to 35-40°C, and the stirring speed was 150-300 rpm.
3. The method for synthesizing rebaudioside M according to claim 1, characterized in that: In (2), the specific steps are: S1 filtration: take the reaction solution, add water, the volume ratio of water to reaction solution is 0.4~2:1, heat to 60~90℃, add activated carbon, heat treat for 30~60min, hot filter at 60~90℃, top wash the filter cake with hot water, combine all the filtrates to obtain rebaudioside M filtrate; S2 decolorizes the Rebaudioside M filtrate in S1 through a macroporous resin to obtain a Rebaudioside M decolorized solution; S3 filters the rebaudioside M decolorized solution in S2 through an ultrafiltration membrane twice to obtain an ultrafiltrate; S4 vacuum rotary evaporation concentrates the ultrafiltrate in S3 to obtain a concentrated solution; S5: adding ethanol to the concentrated solution in S4, followed by cooling, growing crystals, and secondary cooling crystallization to obtain crystalline rebaudioside M; S6 was filtered to collect the crystals, top washed with cold water, dried, and then the crystals were collected and dried.
4. The method for synthesizing rebaudioside M according to claim 3, characterized in that: In S1, the reaction liquid is heated to 70°C, and activated carbon is added for heat treatment for 50 minutes. The amount of activated carbon is 2.5% of the weight of the reaction liquid. Then it is filtered through a plate and frame. The temperature of the liquid is controlled at 60-65°C during filtration. Then, 0.5 times the volume of hot water is used for top washing. The temperature of the hot water is 80°C.
5. The method for synthesizing rebaudioside M according to claim 3, characterized in that: In S3, ultrafiltration is performed using 10000Da and 1000Da ultrafiltration membranes in sequence to cut off materials with a molecular weight of 1000-10000Da.
6. The method for synthesizing rebaudioside M according to claim 3, characterized in that: In S4, the mass concentration of soluble solids in the concentrate is 18~23%.
7. The method for synthesizing rebaudioside M according to claim 3, characterized in that: In S5, the volume concentration of ethanol is 95%, the temperature is 70°C, and the amount used is the same as the volume of the concentrated solution; the concentrated solution is cooled to 60°C, and seed crystals accounting for 0.1% of the volume of the concentrated solution are added, the crystal growth time is 3~5h, and the stirring speed is 50rpm; then the temperature is lowered to 15°C for crystallization at a rate of 2~4°C / h for a second time and maintained for 3h.