Beta-cyclodextrin as well as preparation method and application of beta-cyclodextrin by taking sucrose as substrate
By using sucrose as a substrate and employing multi-enzyme reaction and purification technology to prepare β-cyclodextrin, the problems of low production efficiency and high cost caused by starch substrates have been solved. This has enabled the preparation of high-purity β-cyclodextrin with high recovery rate, which is suitable for food, pharmaceutical and cosmetic fields.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGDONG ZHUMEI BIOMEDICAL TECH CO LTD
- Filing Date
- 2024-11-08
- Publication Date
- 2026-05-12
AI Technical Summary
In the existing technology for preparing β-cyclodextrin using starch as a substrate, there are problems such as low starch utilization, low production efficiency, high cost and poor product uniformity. In particular, due to the unclear molecular structure and wide molecular weight distribution of starch, it is difficult to improve the production efficiency and purity of β-cyclodextrin.
β-Cyclodextrin was prepared by multi-enzyme reaction using sucrose as a substrate. The process was simplified and the reaction efficiency was improved by using sucrose glucosyltransferase mutant and cyclodextrin glucosyltransferase mutant, combined with Licole thermostable amylase. High-purity β-cyclodextrin was obtained by decolorization with activated carbon and purification by hexane crystallization.
This method enables the efficient preparation of β-cyclodextrin with a purity of over 98% and a recovery rate of over 90%. It simplifies the production process, reduces costs, is suitable for large-scale industrial production, and ensures product uniformity and safety.
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Figure CN122012652A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of cyclodextrin production technology, specifically relating to a β-cyclodextrin and its preparation method and application using sucrose as a substrate. Background Technology
[0002] Cyclodextrins (CDs) are cyclic oligosaccharides formed by D-glucanopyranose linked by α-1,4-glycosidic bonds, typically containing 6-12 glucose units. Common cyclodextrins include α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, composed of 6, 7, and 8 glucose units, respectively. The glucose units in the cyclodextrin molecule have a chair-like conformation, meaning they cannot rotate freely, resulting in a cone-shaped cyclic molecule, essentially a hollow cylinder. The cyclodextrin lumen is lipophilic, while the external structure is hydrophilic. This allows CDs to incorporate hydrophobic guest substances or groups suitable for their size and shape, thereby altering the physical and chemical properties of the guest molecule, enhancing its solubility and stability, and improving its usability.
[0003] β-Cyclodextrins (β-CD) are cyclic polymers composed of seven glucose units linked by α-1,4-glycosidic bonds. They have a hollow cylindrical three-dimensional structure with a hydrophobic inner cavity and a hydrophilic outer cavity, which can accommodate hydrophobic guest molecules with suitable shape and size. They are non-toxic or have very low toxicity and are safe and harmless. They are substances permitted for addition under the food additive standard (GB2760-2014). They can be used to improve the defects of hydrophobic guest molecules such as organic acids, fatty acids, aromatics and polar compounds, which are volatile and have poor stability. They are widely used in food, materials, medicine and other fields and have high economic value.
[0004] Currently, the industrial production of cyclodextrins is entirely based on enzymatic methods, using starch as a raw material and catalyzing the production of cyclodextrins by cyclodextrin glycosyltransferase (CGTase). Because β-CD has the lowest solubility in water compared to the other two types of cyclodextrins, it can be easily obtained through stepwise crystallization. While α-CD and γ-CD are difficult to separate and purify industrially, β-CD is the most widely used, accounting for over 90% of cyclodextrin production.
[0005] The industrial production of β-cyclodextrin typically involves the action of β-cyclodextrin glycosyltransferase (β-CGT enzyme) on starch. β-CGT enzyme is a member of the α-amylase 13 family (GH13) and can simultaneously utilize hydrolysis, disproportionation, cyclization, and coupling functions to catalyze the formation of various products from starch, such as cyclodextrins, oligosaccharides, and various dextrin-like substances. However, CGT enzyme is superficially an exoenzyme that degrades starch molecules from the non-reducing ends and cannot cross the branching point (α-1,6 glycosidic bond). Since starch substrates typically contain 75%–80% amylopectin, this significantly reduces the utilization rate of starch substrate and the production efficiency of β-cyclodextrin during cyclodextrin production.
[0006] Chinese patent CN104762345A, filed on March 19, 2015, discloses a process for preparing β-cyclodextrin, using pullulanase or isoamylase in a synergistic reaction with CGT enzyme to produce β-cyclodextrin. However, this method requires pH adjustment, and the pretreatment and enzyme reaction processes involve high temperatures and long reaction times, which are not conducive to reducing energy consumption and improving efficiency in β-cyclodextrin production. Chinese patent CN116656759A, filed on May 25, 2023, provides a method for preparing β-cyclodextrin, using dextrin debranching enzyme to hydrolyze α-1,6 glycosidic bonds during the cyclization reaction, thereby removing the restriction of the branch point on β-CGT enzyme and improving the efficiency of β-cyclodextrin preparation. However, this method still uses starch as a substrate to prepare β-cyclodextrin, and starch, due to its large molecular weight, needs to be pretreated to reduce its molecular weight, which increases production costs and process complexity.
[0007] Current methods for preparing β-cyclodextrin using starch as a substrate suffer from drawbacks, including starch's variable degree of polymerization and wide molecular weight distribution, which may affect the homogeneity of the final product, and the need for necessary pretreatment (such as enzymatic hydrolysis) which may increase costs. Therefore, there is an urgent need to develop a method for preparing β-cyclodextrin with relatively small molecules, well-defined and consistent molecular structures, simple preparation steps, and high reaction efficiency and yield, in order to promote the industrial production of β-cyclodextrin. Summary of the Invention
[0008] To address the problems existing in the prior art, this invention provides a β-cyclodextrin and its preparation method and application using sucrose as a substrate. The preparation method for producing β-cyclodextrin using a multi-enzyme reaction not only yields β-cyclodextrin with good purity and recovery rate, but also eliminates the need for the addition of metal ions. The preparation process is simple and easy to control, enabling the efficient preparation of β-cyclodextrin.
[0009] The technical solution of the present invention is as follows:
[0010] One objective of this invention is to provide a method for preparing β-cyclodextrin using sucrose as a substrate, comprising the following steps:
[0011] (1) Using sucrose as raw material, add sodium citrate buffer to prepare sucrose solution, and adjust the temperature and pH of the solution after stirring;
[0012] (2) Add sucrose glucosyltransferase mutant and cyclodextrin glucosyltransferase mutant to the sucrose solution prepared in step (1) and react. After the reaction is completed, transfer and heat the liquid, and add Licolai high-temperature resistant amylase at a volume ratio of 300:1 and continue stirring.
[0013] (3) Add activated carbon to the liquid and stir thoroughly. After decolorization and filtration, add n-hexane to the filtrate and crystallize at room temperature.
[0014] (4) The β-cyclodextrin was obtained by evaporation to remove n-hexane, filtration to remove oligosaccharides, and vacuum drying.
[0015] Further, in step (1), the concentration of sodium citrate buffer is 0.1M, the final concentration of the sucrose solution is 250g / L, and the stirring conditions are stirring at 350rpm until the temperature of the liquid reaches 45℃.
[0016] Furthermore, in step (1), the temperature of the feed solution is maintained at 45°C, and the pH value is adjusted to 6.5–7.5.
[0017] Further, in step (2), the volume ratio of sucrose solution to sucrose glucosyltransferase mutant and cyclodextrin glucosyltransferase mutant is 15:1-25:1, the reaction time is 2-3h, the heating condition is heating at 90℃ for 15min, and the stirring condition is stirring at 90℃ for 12-15h.
[0018] Furthermore, the sucrose glucosyltransferase mutant is derived from the wild-type sucrose glucosyltransferase by mutating alanine at position 179 to lysine, with the amino acid sequence shown in SEQ ID No:1 and the nucleotide sequence shown in SEQ ID No:2.
[0019] Furthermore, the cyclodextrin glucosyltransferase mutant is derived from the wild-type cyclodextrin glucosyltransferase by mutating tyrosine at position 328 to tryptophan, with the amino acid sequence shown in SEQ ID No:3 and the nucleotide sequence shown in SEQ ID No:4.
[0020] Furthermore, in step (3), the mass of activated carbon is 0.2% of the mass of the liquid material, the stirring conditions are stirring at 60°C for 1 hour, and the volume ratio of the liquid material to n-hexane is 1:1.5.
[0021] Furthermore, in step (4), the evaporation conditions are: evaporation at 60°C, rotation speed of 100 rpm, and vacuum degree of 335 mbar for 2 to 6 hours; and the vacuum drying conditions are: vacuum drying at 50°C for 24 hours.
[0022] The second objective of this invention is to provide a β-cyclodextrin.
[0023] The third objective of this invention is to provide an application of β-cyclodextrin in the preparation of food, pharmaceuticals or cosmetics.
[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0025] 1. This invention is the first to propose using sucrose as a substrate for the preparation of β-cyclodextrin. Compared to the existing technology that typically uses starch as a substrate, sucrose has a well-defined and consistent molecular structure, consisting of a glucose unit and a fructose unit linked by a glycosidic bond. This allows for easier control of reaction conditions during the preparation of β-cyclodextrin to obtain the desired product. Furthermore, due to the relatively small size and good solubility of sucrose molecules, compared to complex polysaccharides such as starch, it not only eliminates the need for pre-decomposition or modification but also improves reaction efficiency, thus enabling the efficient preparation of β-cyclodextrin.
[0026] 2. This invention provides a method for preparing β-cyclodextrin using sucrose as a substrate. Sucrose typically exists in a high-purity form and can serve as a high-quality starting substrate for β-cyclodextrin synthesis, helping to ensure the purity and quality of the final β-cyclodextrin product. Furthermore, this invention removes impurities such as dextran by adding thermostable amylase, eliminating the possibility of impurity peaks in subsequent liquid chromatography that could affect product purity detection. Due to the purity of sucrose itself and the controllability of the reaction conditions, the final β-cyclodextrin obtained has high purity and few impurities. This invention achieves a β-cyclodextrin recovery rate of over 90% after purification, and the purity of the β-cyclodextrin in the final product is over 98%.
[0027] 3. This invention innovatively combines a multi-enzyme reaction with a sucrose substrate for the efficient production of β-cyclodextrin. Sucrose does not require pre-decomposition or modification, simplifying the production process, reducing production costs, and providing a relatively low-cost and stable supply as a raw material. The operation is simple, the reaction cycle is short, and it is suitable for large-scale industrial production. Furthermore, due to the clear structure and high purity of sucrose, the final β-cyclodextrin product exhibits excellent homogeneity and is free of organic solvent residues, making it suitable for applications in food, pharmaceuticals, cosmetics, and other fields with stringent product safety requirements. Attached Figure Description
[0028] Figure 1 This is a schematic diagram showing the results of preparing β-cyclodextrin using sucrose of different concentrations as substrates via a one-pot reaction method in the performance test of this invention.
[0029] Figure 2 This is a schematic diagram showing the results of preparing β-cyclodextrin using a two-step strategy with different concentrations of sucrose as substrate in the performance test of this invention.
[0030] Figure 3 This is a schematic diagram showing the effect of different buffer solutions on the concentration of β-cyclodextrin during the performance test of this invention;
[0031] Figure 4 This is a schematic diagram showing the concentration results of β-cyclodextrin prepared from different concentrations of sucrose in the performance test of this invention;
[0032] Figure 5 This is a schematic diagram showing the concentration results of β-cyclodextrin prepared under different enzyme amounts when the sucrose concentration was 250 g / L during the performance test of this invention.
[0033] Figure 6 This is a schematic diagram showing the concentration results of β-cyclodextrin prepared under different sucrose concentrations and different enzyme amounts during the performance test of this invention;
[0034] Figure 7 This is a schematic diagram comparing the liquid phase results of β-cyclodextrin purity in the feed solution before and after purification following the multi-enzyme reaction in the performance test of this invention. Detailed Implementation
[0035] The present invention will be further described below with reference to preferred embodiments. The endpoints and any values of the ranges disclosed in the present invention are not limited to the precise ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of the various ranges, the endpoint values of the various ranges and individual point values, and individual point values can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed herein.
[0036] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.
[0037] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0038] Example 1
[0039] This embodiment provides a method for preparing β-cyclodextrin using sucrose as a substrate, including the following steps:
[0040] (1) Using sucrose as raw material, add sodium citrate buffer solution with a concentration of 0.1M to prepare a sucrose solution with a concentration of 250g / L. Stir at 350rpm until the temperature of the solution is 45℃, and maintain the temperature of the solution at 45℃. Adjust the pH value to 6.5.
[0041] (2) Add 2 mL of sucrose glucosyltransferase mutant with amino acid sequence as shown in SEQ ID No:1 and 100 μL of cyclodextrin glucosyltransferase mutant with amino acid sequence as shown in SEQ ID No:3 to the sucrose solution obtained in step (1) and react for 2 h. After the reaction is completed, transfer the solution and heat it at 90 °C for 15 min. Add Licolai thermoresistant amylase and stir continuously at 90 °C for 12 h.
[0042] (3) Add 0.2% activated carbon to the feed solution, stir thoroughly at 60°C for 1 hour, decolorize and filter, add n-hexane to the filtrate and crystallize at room temperature. The mass ratio of feed solution to n-hexane is 1:1.5.
[0043] (4) The β-cyclodextrin was obtained by evaporation to remove n-hexane, filtration to remove oligosaccharides, and vacuum drying.
[0044] Example 2
[0045] This embodiment provides a method for preparing β-cyclodextrin using sucrose as a substrate, including the following steps:
[0046] (1) Using sucrose as raw material, add sodium citrate buffer solution with a concentration of 0.1M to prepare a sucrose solution with a concentration of 250g / L. Stir at 350rpm until the temperature of the solution is 45℃, and maintain the temperature of the solution at 45℃. Adjust the pH value to 7.
[0047] (2) Add 2 mL of sucrose glucosyltransferase mutant with amino acid sequence as shown in SEQ ID No:1 and 100 μL of cyclodextrin glucosyltransferase mutant with amino acid sequence as shown in SEQ ID No:3 to the sucrose solution obtained in step (1) and react for 2.5 h. After the reaction is completed, transfer the solution and heat it at 90 °C for 15 min. Add Licolai thermoresistant amylase and stir continuously at 90 °C for 14 h.
[0048] (3) Add 0.2% activated carbon to the feed solution, stir thoroughly at 60°C for 1 hour, decolorize and filter, add n-hexane to the filtrate and crystallize at room temperature. The mass ratio of feed solution to n-hexane is 1:1.5.
[0049] (4) The β-cyclodextrin was obtained by evaporation to remove n-hexane, filtration to remove oligosaccharides, and vacuum drying.
[0050] Example 3
[0051] This embodiment provides a method for preparing β-cyclodextrin using sucrose as a substrate, including the following steps:
[0052] (1) Using sucrose as raw material, add 0.1M sodium citrate buffer to prepare a sucrose solution with a concentration of 250g / L. Stir at 350rpm until the temperature of the solution is 45℃, and maintain the temperature of the solution at 45℃. Adjust the pH value to 7.5.
[0053] (2) Add 2 mL of sucrose glucosyltransferase mutant with amino acid sequence as shown in SEQ ID No:1 and 100 μL of cyclodextrin glucosyltransferase mutant with amino acid sequence as shown in SEQ ID No:3 to the sucrose solution obtained in step (1) and react for 3 h. After the reaction is completed, transfer the solution and heat it at 90 °C for 15 min. Add Licolai thermoresistant amylase and stir continuously at 90 °C for 15 h.
[0054] (3) Add 0.2% activated carbon to the feed solution, stir thoroughly at 60°C for 1 hour, decolorize and filter, add n-hexane to the filtrate and crystallize at room temperature. The mass ratio of feed solution to n-hexane is 1:1.5.
[0055] (4) The β-cyclodextrin was obtained by evaporation to remove n-hexane, filtration to remove oligosaccharides, and vacuum drying.
[0056] Performance testing
[0057] 1. One-pot reaction method test
[0058] The test procedure was the same as in Example 2, except that the sucrose glucosyltransferase mutant was added first and reacted for 1 hour, followed by the addition of cyclodextrin glucosyltransferase mutant and reacted for 1 hour.
[0059] Experimental results are as follows Figure 1 As shown, when the sucrose solution concentration was 200 g / L, the concentration of β-cyclodextrin obtained was higher than that of other concentrations.
[0060] 2. Two-step strategy test
[0061] The test procedure was the same as in Example 2, except that sucrose glucosyltransferase mutant and cyclodextrin glucosyltransferase mutant were added at the same time and reacted for 2 hours.
[0062] Experimental results are as follows Figure 2 As shown, when the sucrose solution concentration is 200 g / L, the concentration of β-cyclodextrin obtained is higher than that of other concentrations. Furthermore, when the substrate concentration is the same, the concentration of β-cyclodextrin obtained by the one-pot reaction method is higher than that obtained by the two-step strategy method.
[0063] 3. Buffer concentration test
[0064] β-cyclodextrin was prepared by using sucrose as raw material and preparing a sucrose solution with a final concentration of 200 g / L using different buffer solutions. The remaining steps and parameters were the same as in Example 2.
[0065] Experimental results are as follows Figure 3 As shown, the most suitable buffer for the substrate is 0.1M sodium citrate, and the concentration of β-cyclodextrin prepared using it as a substrate can reach 22.26 g / L.
[0066] 4. Sucrose solution concentration test
[0067] β-cyclodextrin was prepared by using sucrose as raw material and preparing sucrose solutions of different final concentrations using sodium citrate buffer solution. The remaining steps and parameters were the same as in Example 2.
[0068] Experimental results are as follows Figure 4 As shown, the optimal sucrose solution concentration for the substrate is 250 g / L, and the concentration of the prepared β-cyclodextrin can reach 18.8 g / L when the reaction time is 2 h.
[0069] 5. Multi-enzyme ratio test
[0070] Using a sucrose solution with a final concentration of 250 g / L as a substrate, different amounts of sucrose glucosyltransferase mutant and cyclodextrin glucosyltransferase mutant were added respectively, and the reaction time was adjusted to prepare β-cyclodextrin. The remaining steps and parameters were the same as in Example 2.
[0071] Experimental results are as follows Figure 5 As shown, when the substrate is a sucrose solution with a concentration of 250 g / L, the enzyme ratio of the added sucrose glucosyltransferase mutant to the cyclodextrin glucosyltransferase mutant is 25:1, and the highest concentration of β-cyclodextrin obtained can reach 22.3 g / L.
[0072] 6. Liquid phase analysis of the feed solution before and after purification
[0073] A schematic diagram comparing the β-cyclodextrin purity of the feed solution before and after purification by the multi-enzyme reaction is shown below. Figure 7 As shown in the figure, the method of the present invention removes impurities such as dextran by adding thermostable amylase, effectively overcoming the problem of impurity peaks appearing in the subsequent liquid phase and affecting the detection of product purity. After purification, the recovery rate of β-cyclodextrin can reach more than 90%, and the purity of β-cyclodextrin in the finished product is more than 98%.
[0074] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. The terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes the element.
[0075] The above are merely preferred embodiments of this application and are not intended to limit this application in any way. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the technical solution of this application. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of this application without departing from the content of the technical solution of this application shall still fall within the scope of the technical solution of this application.
Claims
1. A method for preparing β-cyclodextrin using sucrose as a substrate, characterized in that, Includes the following steps: (1) Using sucrose as raw material, add sodium citrate buffer to prepare sucrose solution, and adjust the temperature and pH of the solution after stirring; (2) Add sucrose glucosyltransferase mutant and cyclodextrin glucosyltransferase mutant to the sucrose solution prepared in step (1) and react. After the reaction is completed, transfer and heat the liquid, and add Licolai high-temperature resistant amylase at a volume ratio of 300:1 and continue stirring. (3) Add activated carbon to the liquid and stir thoroughly. After decolorization and filtration, add n-hexane to the filtrate and crystallize at room temperature. (4) The β-cyclodextrin was obtained by evaporation to remove n-hexane, filtration to remove oligosaccharides, and vacuum drying.
2. The method for preparing β-cyclodextrin using sucrose as a substrate according to claim 1, characterized in that, In step (1), the concentration of sodium citrate buffer is 0.1M, the final concentration of the sucrose solution is 250g / L, and the stirring conditions are stirring at 350rpm until the temperature of the liquid reaches 45℃.
3. The method for preparing β-cyclodextrin using sucrose as a substrate according to claim 1, characterized in that, In step (1), the temperature of the feed solution is maintained at 45°C, and the pH value is adjusted to 6.5-7.
5.
4. The method for preparing β-cyclodextrin using sucrose as a substrate according to claim 3, characterized in that, In step (2), the volume ratio of sucrose solution to sucrose glucosyltransferase mutant and cyclodextrin glucosyltransferase mutant is 15:1-25:1, the reaction time is 2-3h, the heating condition is heating at 90℃ for 15min, and the stirring condition is stirring at 90℃ for 12-15h.
5. The method for preparing β-cyclodextrin using sucrose as a substrate according to claim 4, characterized in that, The sucrose glucosyltransferase mutant is derived from the wild-type sucrose glucosyltransferase by mutating alanine at position 179 to lysine. The amino acid sequence is shown in SEQ ID No:1, and the nucleotide sequence is shown in SEQ ID No:
2.
6. The method for preparing β-cyclodextrin using sucrose as a substrate according to claim 4, characterized in that, The cyclodextrin glucosyltransferase mutant is derived from the wild-type cyclodextrin glucosyltransferase by mutating tyrosine at position 328 to tryptophan. The amino acid sequence is shown in SEQ ID No:3, and the nucleotide sequence is shown in SEQ ID No:
4.
7. The method for preparing β-cyclodextrin using sucrose as a substrate according to claim 3, characterized in that, In step (3), the mass of activated carbon is 0.2% of the mass of the liquid material, the stirring conditions are stirring at 60°C for 1 hour, and the volume ratio of the liquid material to n-hexane is 1:1.
5.
8. The method for preparing β-cyclodextrin using sucrose as a substrate according to claim 3, characterized in that, In step (4), the evaporation conditions are evaporation at 60°C, rotation speed of 100 rpm, and vacuum degree of 335 mbar for 2 to 6 hours; the vacuum drying conditions are vacuum drying at 50°C for 24 hours.
9. A β-cyclodextrin prepared by the method according to any one of claims 1-8.
10. The use of a β-cyclodextrin prepared by any one of claims 1-8 in the preparation of food, pharmaceuticals or cosmetics.