A heterogeneous enzymatic synthesis of branched beta-cyclodextrin

Abstract

This invention provides a heterogeneous enzymatic synthesis method for branched-β-cyclodextrin, relating to the field of cyclodextrin derivative synthesis. The method includes: preparing a substrate syrup containing maltose, β-cyclodextrin, and a chelating agent; adding pullulanase to the substrate syrup to induce an enzymatic hydrolysis reaction; and inactivating the enzyme after the hydrolysis reaction to obtain a syrup containing branched-β-cyclodextrin. The addition of the chelating agent disrupts the hydrolyzed layer of pullulanase and β-cyclodextrin, forming a low water activity system. This causes the previously dissolved β-cyclodextrin in the syrup to become supersaturated and crystallize, precipitating out in large quantities as regular polygonal crystals. Simultaneously, pullulanase precipitates and accumulates on the surface of the β-cyclodextrin crystals, forming a heterogeneous enzyme-catalyzed reaction system. This method significantly improves upon the problems of low substrate conversion, long catalytic reaction time, high product impurity content, and high synthesis cost associated with homogeneous enzyme catalysis methods.

Description

Technical Field

[0001] This invention relates to the field of cyclodextrin derivative synthesis, and more specifically to a heterogeneous enzymatic synthesis method for branched β-cyclodextrin. Background Technology

[0002] β-Cyclodextrin is a cyclic oligosaccharide with seven glucose units. It has a truncated pyramidal structure that is hydrophobic on the inside and hydrophilic on the outside. It can form inclusion complexes with a variety of hydrophobic molecules. In the pharmaceutical field, it is used as a pharmaceutical excipient. In the food field, it is used for encapsulation and stabilization of flavor substances. In the cosmetic field, it is used for sustained release of fragrance components. In the chemical and materials fields, it is applied to chemical analysis, molecular recognition, sensor development, etc.

[0003] The solubility of β-cyclodextrin in aqueous solution at room temperature (approximately 25°C) is only about 1.85% (w / v), severely limiting its direct use in high-concentration drug delivery, food additives, or cosmetics. By using pullulanase hydrolysis in a reverse catalytic reaction, disaccharide molecules are grafted onto the hydroxyl groups of the glucose units of β-cyclodextrin to generate branched β-cyclodextrin, significantly improving water solubility and steric hindrance, thus expanding its application scenarios at high concentrations.

[0004] In the industrial production of branched-β-cyclodextrin, a homogeneous enzymatic catalytic process is typically employed. This involves heating and dissolving maltose and β-cyclodextrin to form a homogeneous substrate syrup. Pullulanase is then added to this homogeneous system, and branched-β-cyclodextrin is prepared through retrosynthetic action of pullulanase. This homogeneous catalytic process requires a high-concentration homogeneous substrate solution of disaccharides and β-cyclodextrin. However, the low solubility of β-cyclodextrin is further amplified in this high-concentration syrup, resulting in extremely low levels of free β-cyclodextrin molecules in the solution. This leads to a severely insufficient substrate concentration for pullulanase kinetically distributed in the high-concentration homogeneous system, making it difficult to improve reaction rate and conversion. Furthermore, at the end of the reaction, the percentage of the target product in the solids is low, requiring the separation of a high proportion of disaccharides and β-cyclodextrin, resulting in extremely high enzymatic costs and hindering large-scale adoption.

[0005] For example, non-patent literature 1, "Synthesis of Maltosyl (α1→6)cyclodextrins through the reverse reaction of thermostable Bacillus acidopullulyticus" The patent document "Pululanase" describes an enzymatic synthesis and structural identification of β-cyclodextrin under optimal reaction conditions. The calculated mass percentages of maltose, β-cyclodextrin, and maltodextrin in the final product are 82.9–83.0%, 7.5–7.8%, and 9.2–9.6%, respectively. Non-patent literature 2, "Enzymatic Synthesis and Structural Identification of Maltodextrin (α-1→6)," describes an enzymatic synthesis and structural identification of β-cyclodextrin under optimal reaction conditions. The calculated mass percentages of maltose, β-cyclodextrin, and maltodextrin in the final product are 74.8%, 9.5%, and 15.6% (with a molar ratio of maltose to β-cyclodextrin of 12:1 in the substrate) or 80.1%, 7.6%, and 12.4% (with a molar ratio of maltose to β-cyclodextrin of 16:1 in the substrate).

[0006] Overcoming the shortcomings of existing homogeneous enzyme catalysis methods for branched β-cyclodextrin, such as low β-cyclodextrin conversion rate, high product impurity content, low enzyme reaction efficiency, and high cost, has become a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] Therefore, the purpose of this invention is to propose a new method for synthesizing branched-β-cyclodextrin, which effectively improves the conversion rate of β-cyclodextrin, reduces the impurity content of the product, enhances enzyme reaction efficiency, and controls production costs, thereby providing a heterogeneous enzyme synthesis method for branched-β-cyclodextrin.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a heterogeneous enzyme synthesis method for branched β-cyclodextrin, comprising the following steps: S1. Prepare a substrate syrup containing maltose, β-cyclodextrin and a chelating agent; S2. Pullulanase is added to the substrate syrup to initiate an enzymatic hydrolysis reaction. S3. After the enzymatic hydrolysis reaction is completed, the enzyme is inactivated to obtain a syrup containing branched-β-cyclodextrin.

[0010] Further, in step S1, the chelating agent is selected from at least one of disodium ethylenediaminetetraacetate, trisodium nitrotriacetate, trisodium dicarboxymethylalanine, sodium citrate, and sodium gluconate.

[0011] Further, in step S1, the concentration of the substrate syrup is Bx40~Bx80 (i.e., the mass concentration of solids in the substrate syrup is 40%~80%), preferably Bx70~Bx80, and more preferably Bx80.

[0012] Further, in step S1, the mass of the maltose is 70% to 90% of the solid mass of the substrate syrup, preferably 75% to 85%, and more preferably 80%.

[0013] Further, in step S1, the mass of the β-cyclodextrin is 10% to 30% of the solid mass of the substrate syrup, preferably 15% to 25%, and more preferably 20%.

[0014] Further, in step S1, the chelating agent is added in the form of a chelating agent solution, and the mass concentration of the chelating agent solution is 50~200 ppm, preferably 120~170 ppm, and more preferably 150 ppm.

[0015] Further, in step S1, the mass ratio of the chelating agent to β-cyclodextrin is 1:700~3000, preferably 1:800~1200, and more preferably 1:1000.

[0016] Further, in step S1, the step of preparing the substrate syrup includes: dissolving maltose in water to obtain a maltose solution; adding a chelating agent solution to the maltose solution and fully dispersing and hydrating it to obtain a hydrated solution; adding β-cyclodextrin to the hydrated solution and mixing it evenly to obtain the substrate syrup.

[0017] Furthermore, in step S1, the process of preparing the substrate syrup is carried out under heating conditions of 50~65 ℃, preferably under heating conditions of 55~60 ℃, and more preferably under heating conditions of 60 ℃.

[0018] Further, in step S2, the amount of pullulanase used is 100~300 U / g β-cyclodextrin, preferably 100~200 U / g β-cyclodextrin, more preferably 100 U / g β-cyclodextrin.

[0019] Further, in step S2, the pullulanase is added in the form of an enzyme solution with an enzyme activity of 1000~2000 U / mL, preferably 1000~1500 U / mL, and more preferably 1000 U / mL.

[0020] Further, in step S2, the enzymatic hydrolysis reaction conditions are: temperature 50~65 ℃, stirring speed 100~300 rpm, and time 64~120 h; preferably, the enzymatic hydrolysis reaction conditions are: temperature 55~60 ℃, stirring speed 120~150 rpm, and time 64~96 h; more preferably, the enzymatic hydrolysis reaction conditions are: temperature 60 ℃, stirring speed 120 rpm, and time 96 h.

[0021] Furthermore, in step S3, the enzymatic hydrolysis reaction ends when the reaction solution becomes clear and transparent.

[0022] Further, in step S3, the enzyme inactivation conditions are: 95~100 ℃ for 15~30 min; preferably, the enzyme inactivation conditions are: 95 ℃ for 15 min.

[0023] Furthermore, in step S3, the conversion rate of the β-cyclodextrin is ≥70%, preferably 74%~77%.

[0024] Secondly, the present invention provides a syrup containing branched-β-cyclodextrin, obtained by the heterogeneous enzymatic synthesis method of the branched-β-cyclodextrin.

[0025] Furthermore, the mass ratio of β-cyclodextrin in the syrup solids is ≤10%, and the mass ratio of branched-β-cyclodextrin in the syrup solids is ≥27%.

[0026] Thirdly, the present invention provides a syrup containing branched-β-cyclodextrin obtained by the heterogeneous enzymatic synthesis method of the branched-β-cyclodextrin, or the use of the branched-β-cyclodextrin-containing syrup in the preparation of food, pharmaceuticals or cosmetics.

[0027] The technical solution of this invention has the following advantages: This invention innovatively provides a heterogeneous enzyme synthesis method for branched-β-cyclodextrin. In the preparation of substrate syrup, a chelating agent is added to a substrate containing maltose, β-cyclodextrin, and a chelating agent, and then pullulanase is added to carry out an enzymatic hydrolysis reaction. The addition of the chelating agent disrupted the aqueous solubilization layer of pullulanase and β-cyclodextrin, forming a low water activity system. This caused the previously dissolved β-cyclodextrin in the syrup to become supersaturated and crystallize, precipitating out in large quantities as regular polygonal crystals. Simultaneously, pullulanase precipitated and accumulated on the surface of the β-cyclodextrin crystals, forming a heterogeneous enzyme-catalyzed reaction system. (In the heterogeneous enzyme-catalyzed system with chelating agent, as the reaction proceeded, it was observed that β-cyclodextrin changed from the original powder particles to large, regular, polygonal crystals, and then the crystal size gradually decreased, accompanied by the large-scale synthesis of products. In the homogeneous catalytic system, the β-cyclodextrin particles did not undergo this morphological change. Theoretically, the appearance of large crystals indicates a decrease in liquid concentration and an acceleration of the reaction rate. It is speculated that pullulanase synchronously precipitated onto the concentrated liquid layer on the crystal surface, thereby accelerating the reaction.)

[0028] The β-cyclodextrin concentrate layer on the crystal surface contains a high concentration of β-cyclodextrin, providing the high concentration of substrate required for pullulanase reaction. Together with maltose in the substrate syrup, under the action of pullulanase enriched on the crystal surface, the enzyme kinetics are rapidly converted towards the product direction, synthesizing branched β-cyclodextrin. This effectively improves the β-cyclodextrin conversion rate, reduces the product impurity content, enhances enzyme reaction efficiency, and controls production costs. It significantly improves the problems of low substrate conversion rate, long catalytic reaction time, high product impurity content, and high synthesis cost caused by homogeneous enzyme catalysis methods. Attached Figure Description

[0029] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0030] Figure 1 These are images of the β-cyclodextrin commercial powder used in the embodiments and comparative examples of this invention, observed under a microscope. Figure 2 This is a microscopic image of the β-cyclodextrin crystals formed in Example 1 of this invention. Figure 3 These are images of the reaction solution observed under a microscope after the enzymatic hydrolysis reaction in Example 1 of this invention has been carried out for 43 h and 64 h; Figure 4 This is a high-performance liquid chromatography (HPLC) chromatogram obtained after dilution of the final syrup from the enzymatic hydrolysis reaction in Example 1 of this invention after 96 hours of enzymatic hydrolysis. Detailed Implementation

[0031] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0032] Raw material source: Maltose: Purity ≥90%, sourced from Baolingbao Biotechnology Co., Ltd.

[0033] β-Cyclodextrin powder: food additive grade, purity 98.0%–101.0%, sourced from Henan Mengzhou Huaxing Biochemical Co., Ltd.; observed under a microscope at 10×10 magnification, such as… Figure 1 As shown, the β-cyclodextrin commercial powder is in the form of irregular powder particles with an irregular halo on the surface.

[0034] Disodium ethylenediaminetetraacetic acid (EDTA): analytical grade, sourced from Sinopharm Chemical Reagent Co., Ltd.

[0035] Pullulan enzyme solution: enzyme activity 1000 U / mL, sourced from Ningxia Xiasheng Industrial Group Co., Ltd.

[0036] Instrument source: Thermostatic heating magnetic stirrer: Product model DF-101Z, from Shanghai Lichen Instrument Technology Co., Ltd.

[0037] Electronic balance: Product model HZK-6102, sourced from Huazhi Fujian Electronic Technology Co., Ltd.

[0038] Optical microscope: Baoshide brand.

[0039] High-performance liquid chromatograph: Thermo Fisher Scientific, model U3000, equipped with an evaporative light scattering detector.

[0040] Amino chromatography column: Inertsil NH2 4.6×250 mm 5 μm, GL Sciences.

[0041] Where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in this field. All raw materials or instruments used are commercially available conventional products, including but not limited to those used in the embodiments of this application.

[0042] Example 1 This embodiment provides a heterogeneous enzyme synthesis method for branched β-cyclodextrin, the specific steps of which are as follows: S1. Dissolve maltose in water at 60 °C and stir at 100 rpm for 30 min at this temperature to obtain a maltose solution. Add a 500 ppm EDTA disodium solution (prepared by dissolving EDTA disodium in water) to the maltose solution, maintain the temperature at 60 °C, and stir at 100 rpm for 15 min at this temperature to fully disperse and hydrate, obtaining a hydrated solution. Add β-cyclodextrin commercial powder to the hydrated solution, maintain the temperature at 60 °C, and stir at 100 rpm for 15 min at this temperature to obtain a substrate syrup. The concentration of the substrate syrup is Bx80, the mass of maltose is 80% of the solid mass of the substrate syrup, the mass of β-cyclodextrin is 20% of the solid mass of the substrate syrup, and the mass ratio of EDTA disodium to β-cyclodextrin is 1:1000. Observe the system state: Due to its low water activity, β-cyclodextrin cannot dissolve in the hot syrup, and a large amount is suspended or precipitated in the reactor substrate, making the syrup appear as a milky white crystalline paste; observe under a microscope at 10×10 magnification, as follows... Figure 2 As shown, a large number of regular polygonal crystals (i.e., β-cyclodextrin crystals) overlap and fill the field of view; S2. Add pullulanase solution (addition amount based on 100 U / g β-cyclodextrin) to the substrate syrup, heat in a 60 ℃ water bath jacket while stirring at 120 rpm to induce enzymatic hydrolysis. Observation of the system state: After 64–120 h of reaction, the reaction solution became clear and transparent, with occasional small amounts of fine white particles. Under a microscope at 10×10 magnification, only a small number of small crystals were visible. Microscopic images of the reaction solution at 43 h and 64 h are shown below. Figure 3 As shown, as the reaction time progresses, the size of the large crystal particles continuously decreases, and the number of particles also decreases.

[0043] S3. Take the reaction solutions after 64 h and 96 h of reaction respectively as the final syrup.

[0044] Comparative Example 1 This comparative example provides a homogeneous enzyme synthesis method for branched β-cyclodextrin, the specific steps of which are as follows: S1. Dissolve maltose in water at 60 °C and stir at 100 rpm for 30 min at this temperature to obtain a maltose solution. Add β-cyclodextrin commercial powder to the maltose solution, maintain the temperature at 60 °C, and stir at 100 rpm for 15 min at this temperature to obtain a substrate syrup. The concentration of the substrate syrup is Bx80, the mass of maltose is 80% of the solid mass of the substrate syrup, and the mass of β-cyclodextrin is 20% of the solid mass of the substrate syrup. S2. Add pullulanase solution (added at 100 U / g β-cyclodextrin) to the substrate syrup, heat in a 60 °C water bath jacket while stirring at 120 rpm to induce an enzymatic hydrolysis reaction. Observation of the system state: When the reaction proceeded for 64-120 h, the reaction solution became clear and transparent, with a small number of white fine particles occasionally seen; when observed under a microscope at a magnification of 10×10, very few tiny crystals were seen, and microspheres were also seen (presumably co-crystals of maltose and other components). S3. Take the reaction solution after 64 h of reaction as the final syrup.

[0045] Comparative Example 2 This comparative example provides a heterogeneous enzyme synthesis method for branched β-cyclodextrin, the specific steps of which are as follows: S1. Prepare a 6% (w / w) crystal solution by adding water to β-cyclodextrin commercial powder. Heat the solution at 60 °C until the crystals disappear and the β-cyclodextrin is completely dissolved, obtaining a β-cyclodextrin solution. Slowly cool the β-cyclodextrin solution at room temperature. Large columnar β-cyclodextrin crystals precipitate from the solution. Decant the supernatant and keep the bottom β-cyclodextrin crystals for later use. Dissolve maltose in water at 60 °C and stir at 100 rpm for 30 min at this temperature to obtain a maltose solution. Add β-cyclodextrin crystals to the maltose solution, maintain the temperature at 60 °C, and stir at 100 rpm for 15 min at this temperature to obtain a substrate syrup. The concentration of the substrate syrup is Bx80, the mass of maltose is 80% of the solid mass of the substrate syrup, and the mass of β-cyclodextrin is 20% of the solid mass of the substrate syrup. S2. Add pullulanase solution to the substrate syrup (at a rate of 100 U / g). (β-cyclodextrin), heated in a water bath at 60 ℃ with stirring at 120 rpm, to induce enzymatic hydrolysis; Observe the state of the system: When the reaction proceeds for 64~120 h, the reaction solution changes from a turbid slurry to a slightly clear one; when observed under a microscope at a magnification of 10×10, a large number of irregular crystals can be seen. S3. Take the reaction solution after 64 h of reaction as the final syrup.

[0046] Experimental Example: Determination of Syrup Component Content I. Experimental Methods The component content in the substrate syrup and final syrup collected in Example 1 and Comparative Examples 1-2 was determined by high-performance liquid chromatography.

[0047] 1. Instrument conditions Instrumentation: High-performance liquid chromatograph with evaporative light scattering detector; Chromatographic column: Amino chromatography column; Mobile phase: Acetonitrile:water = 62:38 (v / v); Flow rate: 1.0 mL / min; Injection volume: 10 μL; Column temperature: 30 ℃.

[0048] 2. Qualitative and quantitative methods The substrate syrups from Example 1, Comparative Example 1, and Comparative Example 2 were diluted to Bx2, filtered through a 0.45 μm filter, and injected to analyze the content of each component in the substrate syrup. The final sugar syrups from Example 1, Comparative Example 1, and Comparative Example 2 were diluted to Bx2, inactivated at 95 °C for 15 min, filtered through a 0.45 μm filter, and then injected to analyze the content of each component in the final sugar syrup.

[0049] The high-performance liquid chromatography (HPLC) chromatogram of the final syrup obtained after dilution following 96 hours of enzymatic hydrolysis in Example 1 is shown below. Figure 4 As shown in Table 1, the component identification information is determined based on the peak positions in the chromatogram.

[0050] Table 1. Components and corresponding peak times

[0051] The mass percentage of each component in the solids was calculated using the area normalization method, and the β-cyclodextrin conversion rate was calculated using the following formula:

[0052] II. Experimental Results Table 2 Results of Syrup Component Content Determination

[0053] Note: In the table This indicates that the concentration of this component in the sample is high, exceeding the detector's upper limit.

[0054] As shown in Table 2, for the heterogeneous enzyme synthesis method provided in Example 1, the mass percentage of β-cyclodextrin in the final syrup solids is ≤10% (9.14%~9.28%), the mass percentage of branched-β-cyclodextrin in the final syrup solids is ≥27% (27.50%~29.42%), and the β-cyclodextrin conversion rate is ≥70% (74%~76.3%). However, for the homogeneous enzyme synthesis method provided in Comparative Example 1 and the heterogeneous enzyme synthesis method provided in Comparative Example 2 (without adding chelating agents), the β-cyclodextrin conversion rate is lower than that of the heterogeneous enzyme synthesis method provided in Example 1 (with adding chelating agents). This demonstrates that adding chelating agents to the substrate syrup is beneficial for the conversion of β-cyclodextrin, increases the content of branched-β-cyclodextrin in the syrup, improves enzyme reaction efficiency, and reduces the cost of synthesizing branched-β-cyclodextrin. It should be noted that the glucose in the system was introduced by the raw material maltose powder. Commercially available maltose powder has a purity of 90%, and the solids contain glucose. During the reaction, the mass percentage of glucose did not change significantly, proving that glucose did not participate in enzyme catalysis. Obviously, the above embodiments are merely examples for clear illustration and are not intended to limit the implementation. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A heterogeneous enzyme synthesis method for branched-β-cyclodextrin, characterized in that, Includes the following steps: S1. Prepare a substrate syrup containing maltose, β-cyclodextrin and a chelating agent; S2. Pullulanase is added to the substrate syrup to initiate an enzymatic hydrolysis reaction. S3. After the enzymatic hydrolysis reaction is completed, the enzyme is inactivated to obtain a syrup containing branched-β-cyclodextrin.

2. The heterogeneous enzyme synthesis method for branched-β-cyclodextrin according to claim 1, characterized in that, Step S1 satisfies at least one of the following conditions: (1) The chelating agent is selected from at least one of disodium ethylenediaminetetraacetate, trisodium aziridinetriacetate, trisodium dicarboxymethylalanine, sodium citrate and sodium gluconate; (2) The concentration of the substrate syrup is Bx40~Bx80; (3) The mass of the maltose is 70% to 90% of the solid mass of the substrate syrup; (4) The mass of the β-cyclodextrin is 10% to 30% of the solid mass of the substrate syrup; (5) The chelating agent is added in the form of a chelating agent solution, and the mass concentration of the chelating agent solution is 50~200 ppm; (6) The mass ratio of the chelating agent to β-cyclodextrin is 1:700~3000; (7) The step of preparing the substrate syrup includes: dissolving maltose in water to obtain a maltose solution; adding a chelating agent solution to the maltose solution and fully dispersing and hydrating it to obtain a hydrated solution; adding β-cyclodextrin to the hydrated solution and mixing it evenly to obtain the substrate syrup; (8) The process of preparing the substrate syrup is carried out under heating conditions of 50~65 °C.

3. The heterogeneous enzyme synthesis method for branched-β-cyclodextrin according to claim 1, characterized in that, Step S1 satisfies at least one of the following conditions: (1) The concentration of the substrate syrup is Bx80%; (2) The mass of the maltose is 80% of the solid mass of the substrate syrup; (3) The mass of the β-cyclodextrin is 20% of the solid mass of the substrate syrup; (4) The chelating agent is added in the form of a chelating agent solution, and the mass concentration of the chelating agent solution is 500 ppm; (5) The mass ratio of the chelating agent to β-cyclodextrin is 1:1000; (6) The preparation of the substrate syrup is carried out under heating conditions of 60 °C.

4. The heterogeneous enzyme synthesis method for branched-β-cyclodextrin according to claim 1, characterized in that, Step S2 satisfies at least one of the following conditions: (1) The amount of pullulanase used is 100~300 U / g β-cyclodextrin; (2) The pullulanase is added in the form of an enzyme solution with an enzyme activity of 1000~2000 U / mL; (3) The enzymatic hydrolysis reaction conditions are: temperature 50~65 ℃, stirring speed 100~200 rpm, and time 64~120 h.

5. The heterogeneous enzyme synthesis method for branched-β-cyclodextrin according to claim 1, characterized in that, Step S2 satisfies at least one of the following conditions: (1) The amount of pullulanase used is 100 U / g β-cyclodextrin; (2) The pullulanase is added in the form of an enzyme solution with an enzyme activity of 1000 U / mL. (3) The enzymatic hydrolysis reaction conditions were: temperature 60 ℃, stirring speed 120 rpm, and time 96 h.

6. The heterogeneous enzyme synthesis method for branched-β-cyclodextrin according to claim 1, characterized in that, Step S3 satisfies at least one of the following conditions: (1) The enzymatic hydrolysis reaction ends when the reaction solution becomes clear and transparent; (2) The enzyme inactivation conditions are: 95~100 ℃ for 15~30 min; (3) The conversion rate of the β-cyclodextrin is ≥70%.

7. The heterogeneous enzyme synthesis method for branched-β-cyclodextrin according to claim 1, characterized in that, Step S3 satisfies at least one of the following conditions: (1) The enzyme inactivation conditions are: 95 ℃ for 15 min; (2) The conversion rate of the β-cyclodextrin is 74%~77%.

8. A syrup containing branched-β-cyclodextrin, characterized in that, It is obtained by the heterogeneous enzyme synthesis method of branched-β-cyclodextrin as described in any one of claims 1 to 7.

9. The syrup containing branched-β-cyclodextrin according to claim 8, characterized in that, The mass ratio of β-cyclodextrin in syrup solids is ≤10%, and the mass ratio of branched-β-cyclodextrin in syrup solids is ≥27%.

10. A syrup containing branched-β-cyclodextrin obtained by the heterogeneous enzymatic synthesis method of branched-β-cyclodextrin according to any one of claims 1 to 7, or the use of the syrup containing branched-β-cyclodextrin according to claim 8 or 9 in the preparation of food, pharmaceuticals or cosmetics.

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