A method for preparing gamma-cyclodextrin by biological enzyme
By using phenyl-substituted borate and ethanol as adjuvants in the production of γ-cyclodextrin, combined with γ-cyclodextrin glucosyltransferase, the problem of low yield of γ-cyclodextrin was solved, and a highly efficient preparation and simple separation process of γ-cyclodextrin was achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIANGNAN UNIV
- Filing Date
- 2023-11-30
- Publication Date
- 2026-06-26
Smart Images
Figure CN117551714B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a process for improving the production efficiency of γ-cyclodextrin by utilizing the synergistic effect of phenyl-substituted borate and ethanol, belonging to the field of cyclodextrin production technology. Background Technology
[0002] Cyclodextrins are a class of cyclic oligomers composed of D-glucanopyranoside units linked by α-1,4-glycosidic bonds. Their spatial structure is a hollow cylindrical shape, thus possessing both a hydrophilic surface and a hydrophobic cavity structure. They are promising encapsulation materials with broad applications and have been used in pharmaceuticals, food, cosmetics, and materials science. Cyclodextrins can be classified according to the number of glucose units they comprise; the most common types, composed of 6, 7, and 8 glucose units, are named α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, respectively.
[0003] Compared to α-cyclodextrin and β-cyclodextrin, γ-cyclodextrin has the best solubility and the largest cavity. It can be almost completely broken down by α-amylase in vivo and is rapidly degraded, absorbed, and excreted in the gastrointestinal tract. Therefore, γ-cyclodextrin has the lowest toxicity, and there is currently no minimum daily intake limit. It has broad application prospects in the food and pharmaceutical fields, mainly due to: 1) Due to its unique cavity structure and superior solubility, γ-cyclodextrin is an ideal inclusion carrier. Its large cavity structure can encapsulate large molecules that α-cyclodextrin and β-cyclodextrin cannot, and it is widely used for the inclusion of drugs and food additives with poor solubility. 2) Because γ-cyclodextrin can be rapidly metabolized and excreted in vivo, as an inclusion carrier, it can significantly improve the bioavailability and stability of guest molecules, achieving sustained release. It is often used as a stabilizer for drugs, flavorings, sweeteners, or colorings to reduce their irritation or mask bitterness. 3) The unique spatial structure of γ-cyclodextrin makes it a widely studied organometallic framework material. Organometallic framework materials with γ-cyclodextrin as the framework can be used for gas adsorption, separation and purification of substances, and capture of highly active intermediates, showing broad application prospects as a novel material. 4) Through structural modification and alteration of γ-cyclodextrin, it can be widely used in the synthesis of materials, producing a series of high-value-added derivative materials that can be used in high-tech fields such as liquid crystals and electronics.
[0004] However, the current production of γ-cyclodextrin faces two main problems: first, the lack of γ-cyclodextrin glucosyltransferases with high product specificity, stability, and catalytic efficiency; and second, the lack of mature and efficient γ-cyclodextrin production processes and separation and purification technologies. Therefore, the yield of γ-cyclodextrin prepared by enzymatic methods is low, thus limiting the industrial production of γ-cyclodextrin. Summary of the Invention
[0005] To address the technical problems of low conversion rate, low product proportion, and difficult separation of γ-cyclodextrin, this invention provides a method for producing γ-cyclodextrin using phenyl-substituted borate as a production aid, thereby improving the conversion rate and product proportion of γ-cyclodextrin.
[0006] The first technical solution provided by this invention is a method for preparing γ-cyclodextrin, specifically the following technical solution:
[0007] A method for preparing γ-cyclodextrin, wherein an ethanol solution of phenyl-substituted borate is added as a production aid to a catalytic reaction system for producing γ-cyclodextrin; in the catalytic reaction system, the substrate is starch or a starch derivative liquefaction product, the catalyst is γ-cyclodextrin glucosyltransferase, and the phenyl-substituted borate is a salt formed by replacing the hydroxyl group on the boric acid molecule with a phenyl group.
[0008] In one embodiment of the present invention, the following steps are included:
[0009] S1, an aqueous solution of starch or starch derivative undergoes a liquefaction reaction to obtain a liquefied product;
[0010] S2, γ-cyclodextrin glucosyltransferase is added to the liquefied product, and an ethanol solution of phenyl-substituted borate is added simultaneously to carry out a catalytic reaction; the final concentration of the phenyl-substituted borate in the catalytic reaction system is 0.5-3 g / 100 mL;
[0011] S3 was diluted with water and heated to inactivate the enzyme, yielding a reaction product containing γ-cyclodextrin.
[0012] In one embodiment of the present invention, the specific steps of the liquefaction reaction in step S1 are as follows:
[0013] S11, prepare starch or starch derivatives into an aqueous solution with a concentration of 5% to 20% w / w; the concentration of the aqueous solution of starch or starch derivatives is 5 to 20 g / 100 g water;
[0014] S12, adjust the pH of the aqueous solution from step S11 to 8.0–10.0 using sodium hydroxide solution;
[0015] S13, add γ-cyclodextrin glucosyltransferase, and simultaneously heat to 90℃ for heat preservation to end the liquefaction reaction and obtain the liquefied product.
[0016] In one embodiment of the present invention, the starch or starch derivative may be any one of soluble starch, potato starch, corn starch, cassava starch, wheat starch, rice starch, millet starch or maltodextrin, preferably cassava starch.
[0017] In one embodiment of the present invention, in step S13, the amount of γ-cyclodextrin glucosyltransferase added is 1-5 U / g dry starch, the heating rate is 3-5℃ / min, and the holding time is 20-30min.
[0018] In one embodiment of the present invention, in step S2, the amount of γ-cyclodextrin glucosyltransferase added is 2-10 U / g dry starch.
[0019] In one embodiment of the present invention, in step S2, the amount of ethanol added to the ethanol solution of the phenyl-substituted borate should be 5% to 15% of the volume of the catalytic reaction system.
[0020] In one embodiment of the present invention, in step S2, the catalytic reaction temperature is 45-60°C and the reaction time is 6-10 h.
[0021] In one embodiment of the present invention, in step S2, the phenyl-substituted borate may be a monophenyl-substituted borate, a diphenyl-substituted borate, a triphenyl-substituted borate, a tetraphenyl-substituted borate, or a phenyl-substituted borate containing other modifications.
[0022] In one embodiment of the present invention, in step S2, the phenyl-substituted borate can be a sodium salt, potassium salt, ammonium salt, or other salt.
[0023] In one embodiment of the present invention, in step S3, the dilution factor with water is 5 to 10 times, and the method for inactivating the enzyme is to heat the catalytic reaction system to above 90°C and keep it at that temperature for 20 to 30 minutes.
[0024] The second technical solution provided by the present invention is a method for preparing γ-cyclodextrin product. The product prepared by the method described in the first technical solution is filtered, concentrated and dried to obtain γ-cyclodextrin product.
[0025] The third technical solution provided by this invention is the application of the above method in the preparation of products containing γ-cyclodextrin.
[0026] Beneficial effects
[0027] (1) This invention provides a new enzymatic process for producing γ-cyclodextrin, using γ-cyclodextrin glucosyltransferase derived from Bacillus sp. G-825-6STB17 as a catalyst. The cyclodextrin products are only β-cyclodextrin and γ-cyclodextrin, with a simple product composition that facilitates subsequent separation. Under the highest conversion rate, the conversion rate of γ-cyclodextrin can reach 54.14%, and the product ratio can reach 83.07%.
[0028] (2) This invention provides a novel strategy for the production of γ-cyclodextrin, namely, adding phenyl-substituted borate and ethanol as production aids. Compared with the absence of production aids, the yield of γ-cyclodextrin is increased by 5.6 times, and the product proportion is increased by 35%. Phenyl-substituted borate can encapsulate cyclodextrin, promoting the forward reaction; ethanol can change the solubility of cyclodextrin in the reaction system, promoting the precipitation of cyclodextrin, driving the forward reaction, and improving the overall conversion rate of cyclodextrin; the addition of production aids is also believed to change the conformation of the enzyme used in production, thereby improving the catalytic efficiency of the enzyme. In addition, these salts are easily decomposed at high temperatures, so they can be separated from the product simply by heating, which is simple to operate and greatly reduces production costs. Attached Figure Description
[0029] Figure 1 HPLC chromatogram of the product when 10% (w / w) cassava starch is used as substrate. Detailed Implementation
[0030] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.
[0031] The materials involved in the following embodiments are as follows:
[0032] The γ-cyclodextrin glucosyltransferase is derived from Bacillus sp. G-825-6STB17. A genetically engineered Bacillus subtilis strain containing the gene sequence of this enzyme has been successfully constructed, as detailed in the patent publication number CN114958701A, and will not be elaborated here.
[0033] The detection methods involved in the following embodiments are as follows:
[0034] (1) Methods for determining γ-cyclization activity
[0035] γ-cyclization activity was determined using the bromocresol green (BCG) method. 0.1 mL of appropriately diluted enzyme solution was added to a centrifuge tube containing 0.9 mL of 1% maltodextrin (DE=4) prepared in 20 mM Tris-HCl buffer (pH 9.0). After preheating for 5 min, the reaction was carried out at 55 °C for 10 min. The reaction was terminated by adding 100 μL of 1.0 M hydrochloric acid solution. Subsequently, 2 mL of 0.2 M citrate buffer (pH 4.2) and 100 μL of 5 mM bromocresol green (BCG) solution were added. The reaction was allowed to develop at room temperature for 20 min, and the absorbance was measured at 630 nm. An inactivated enzyme solution was used as a blank.
[0036] One enzyme activity unit is defined as the amount of enzyme required to generate 1 μmol of γ-cyclodextrin per minute under the above conditions.
[0037] (2) Methods for analyzing reaction products
[0038] The detection method for cyclodextrins employed high-performance liquid chromatography (HPLC): the reaction solution was boiled in a water bath for 30 min to inactivate the enzyme, centrifuged (10,000 × g, 10 min), and the supernatant was filtered through a 0.22 μm aqueous filter membrane before HPLC analysis of the product. HPLC conditions were: Waters 600 HPLC system (equipped with a differential refractive index detector), Hypersil GOLD column. TM Amino HPLC (4.6 mm × 250 mm), column temperature 30 °C, mobile phase 70% (v / v) acetonitrile aqueous solution, flow rate 1 mL / min.
[0039] (3) Calculation method for product proportion:
[0040] Cyclodextrin yield = Peak area of the corresponding cyclodextrin / Peak area of the corresponding cyclodextrin standard * Standard concentration
[0041] The product percentage of γ-cyclodextrin = γ-cyclodextrin yield / sum of the yields of the three cyclodextrins.
[0042] Example 1:
[0043] Using cassava starch as a substrate, γ-cyclodextrin was produced using γ-cyclodextrin glucosyltransferase derived from Bacillus sp. G-825-6STB17 (the amount added was based on the enzyme activity required for the cyclization reaction). The specific steps are as follows:
[0044] S1, an aqueous solution of starch or a starch derivative undergoes a liquefaction reaction to obtain a liquefied product, as detailed below:
[0045] S11, add 20g of tapioca starch to 200g of water to prepare a 10% (w / w) tapioca starch solution, preheat it in a 55℃ water bath for 15min, and stir at 200-300rpm to obtain an aqueous solution;
[0046] S12, and adjust the pH of the aqueous solution in step S11 to 9.0 using sodium hydroxide solution;
[0047] S13, add γ-cyclodextrin glucosyltransferase enzyme solution to the system in step S12, the amount added is 2U / g starch, and heat to 90℃ at a rate of 3-5℃ / min for liquefaction, and liquefaction time is 20min.
[0048] S2, after liquefaction is complete, cool to 55℃, and add γ-cyclodextrin glucosyltransferase enzyme solution again at a rate of 2 U / g starch to carry out the cyclization reaction. At the same time, add potassium tetraphenylborate solution dissolved in ethanol. In the catalytic reaction system, the amount of potassium tetraphenylborate added is 1g / 100mL, and the amount of ethanol added is 5% (v / v). That is, in a 200mL catalytic reaction system, 2g of potassium tetraphenylborate is dissolved in 10mL of ethanol and then added to the catalytic reaction system. The reaction time is 10h.
[0049] S3, after the reaction is complete, dilute with water 5 times and heat to 90℃ to inactivate the enzyme until the solution is clear and transparent. Take a sample and analyze the reaction product by HPLC. The final results show that the conversion rate of γ-cyclodextrin in the product can reach 54.14% and the product proportion can reach 83.07%.
[0050] S4, add a chelating agent to the reaction solution to remove metal ions, and adsorb impurities in the reaction solution through resin to obtain cyclodextrin mother liquor;
[0051] S5, the cyclodextrin mother liquor was further concentrated, crystallized, and dried to obtain γ-cyclodextrin.
[0052] Example 2:
[0053] Using cassava starch as a substrate, γ-cyclodextrin was produced using γ-cyclodextrin glucosyltransferase derived from Bacillus sp. G-825-6STB17 (the amount added was based on the enzyme activity required for the cyclization reaction). The specific steps are as follows:
[0054] S1, an aqueous solution of starch or a starch derivative undergoes a liquefaction reaction to obtain a liquefied product, as detailed below:
[0055] S11, prepare a 10% (w / w) cassava starch solution and preheat it in a 55℃ water bath for 15 minutes, with the stirring head rotating at 200-300 rpm to obtain an aqueous solution;
[0056] S12, and adjust the pH of the aqueous solution from step S11 to 9.0 using sodium hydroxide solution;
[0057] S13, add γ-cyclodextrin glucosyltransferase enzyme solution to the system in step S12, the amount of which is 2U / g starch, and liquefy it by heating to 90℃ at 3-5℃ / min for 20min.
[0058] S2, after liquefaction is complete, cool to 55℃, add γ-cyclodextrin glucosyltransferase enzyme solution again, the amount added is 2U / g starch, carry out cyclization reaction, and at the same time add potassium tetraphenylborate solution dissolved in ethanol, wherein the amount of potassium tetraphenylborate added is 0.5g / 100mL (0.5% w / v), the amount of ethanol added is 5% (v / v), and the reaction time is 10h;
[0059] S3, after the reaction is complete, dilute with water 5 times and heat to 90℃ to inactivate the enzyme until the solution is clear and transparent. Take a sample and analyze the reaction product by HPLC. The final results show that the conversion rate of γ-cyclodextrin in the product can reach 18.53% and the product proportion can reach 60.70%.
[0060] S4, add a chelating agent to the reaction solution to remove metal ions, and adsorb impurities in the reaction solution through resin to obtain cyclodextrin mother liquor;
[0061] S5, the cyclodextrin mother liquor was further concentrated, crystallized, and dried to obtain γ-cyclodextrin.
[0062] Example 3:
[0063] Using cassava starch as a substrate, γ-cyclodextrin was produced using γ-cyclodextrin glucosyltransferase derived from Bacillus sp. G-825-6STB17 (the amount added was based on the enzyme activity required for the cyclization reaction). The specific steps are as follows:
[0064] S1, an aqueous solution of starch or a starch derivative undergoes a liquefaction reaction to obtain a liquefied product, as detailed below:
[0065] S11, add 20g of tapioca starch to 200g of water to prepare a 10% (w / w) tapioca starch solution. Preheat the solution in a 55℃ water bath for 15 minutes with the stirring head rotating at 200-300 rpm to obtain an aqueous solution.
[0066] S12, and adjust the pH of the aqueous solution in step S11 to 9.0 with sodium hydroxide solution;
[0067] S13, add γ-cyclodextrin glucosyltransferase enzyme solution, the amount of addition is 2U / g starch, and liquefy at 3~5℃ / min to 90℃, liquefaction time is 20min;
[0068] S2, after liquefaction is complete, cool to 55℃, add γ-cyclodextrin glucosyltransferase enzyme solution again, the amount added is 2U / g starch, carry out cyclization reaction, and at the same time add potassium tetraphenylborate solution dissolved in ethanol, the amount of potassium tetraphenylborate added is 3g / 100mL (3% w / v), the amount of ethanol added is 5% (v / v), and the reaction time is 10h;
[0069] S3, after the reaction is complete, dilute with water 5 times and heat to 90℃ to inactivate the enzyme until the solution is clear and transparent. Take a sample and analyze the reaction product by HPLC. The final results show that the conversion rate of γ-cyclodextrin in the product can reach 51.58% and the product proportion can reach 80.12%.
[0070] S4, add a chelating agent to the reaction solution to remove metal ions, and adsorb impurities in the reaction solution through resin to obtain cyclodextrin mother liquor;
[0071] S5, the cyclodextrin mother liquor was further concentrated, crystallized, and dried to obtain γ-cyclodextrin.
[0072] The γ-cyclodextrins obtained in the above examples can all be applied in fields with strict product safety requirements, such as food, medicine, and cosmetics.
[0073] Comparative Example 1:
[0074] The specific implementation method is the same as in Example 1, except that no production aids are added to the catalytic reaction system, and the total conversion rate of cyclodextrin is only 18.07%, indicating that the addition of production aids is beneficial to improving the overall conversion rate of cyclodextrin and significantly improving the substrate utilization rate.
[0075] Comparative Example 2:
[0076] The specific steps are as follows:
[0077] S1, an aqueous solution of starch or a starch derivative undergoes a liquefaction reaction to obtain a liquefied product, as detailed below:
[0078] S11, prepare a 10% (w / w) cassava starch solution and preheat it in a 55℃ water bath for 15 minutes, with the stirring head rotating at 200-300 rpm to obtain an aqueous solution;
[0079] S12, and adjust the pH of the aqueous solution from step S11 to 9.0 using sodium hydroxide solution;
[0080] S13, add γ-cyclodextrin glucosyltransferase enzyme solution, the amount added is 2U / g starch, and liquefy at a rate of 3-5℃ / min to 90℃, liquefaction time is 20min;
[0081] S2, after liquefaction is complete, cool down to 50℃, add γ-cyclodextrin glucosyltransferase enzyme solution again, the amount added is 2U / g starch, and at the same time add 10% (v / v) ethanol to carry out cyclization reaction, the reaction time is 10h;
[0082] S3. After the reaction was completed, the temperature was raised to 90℃ to inactivate the enzyme until the solution became clear and transparent. Samples were taken and the reaction products were analyzed by HPLC. The final results showed that the total conversion rate of cyclodextrin in the product reached 32.75%, and the proportion of γ-cyclodextrin product reached 52.17%. This indicates that the addition of ethanol may have changed the polar environment of the solution, resulting in a decrease in the solubility of cyclodextrin, which promoted the forward reaction and thus increased the total conversion rate of cyclodextrin.
[0083] Comparative Example 3:
[0084] The specific implementation method is the same as Comparative Example 2, except that the amount of ethanol added in step S2 is 30% (v / v), and the total conversion rate of cyclodextrin is only 17.72%, which is slightly lower than the reaction system without the addition of ethanol. This indicates that the amount of ethanol added is too large, which changes the reaction system environment too much, affects the conformation of the enzyme, and will hinder the catalytic efficiency of the enzyme in the reaction system.
[0085] Comparative Example 4:
[0086] The specific implementation method is the same as in Example 1, except that phenyl-substituted borate solid is directly added in step S2. The conversion rate of γ-cyclodextrin is 50.49%, which is slightly lower than in Example 1. This may be because the addition of a small amount of ethanol changes the distribution of solute in the solution, reduces the solubility of the inclusion complex formed by cyclodextrin, and promotes the forward reaction.
[0087] Comparative Example 5:
[0088] The specific implementation method is the same as in Example 1, except that in step S2, steam distillation is used to remove phenyl borate from the reaction solution. The results showed that the conversion rate of γ-cyclodextrin was 48.55%, which was slightly lower than in Example 1. Moreover, almost all of the cyclodextrin was distributed in the supernatant. Therefore, it is possible to increase the amount of inclusion complex dissolved by directly adding water for dilution and then directly heating to promote the dissociation of the inclusion complex.
[0089] Comparative Example 6:
[0090] The specific implementation method is the same as in Example 1, except that potassium borate is used instead of phenyl borate in step S2. The results showed that the total conversion rate of cyclodextrin was 17.72%, indicating that the addition of potassium borate did not have a significant impact on the production of cyclodextrin.
[0091] The chelating groups of boric acid compounds can bond with sugars. Phenyl-substituted borates have a higher molecular size and better fit with the cavity of γ-cyclodextrin because the hydroxyl groups on the boric acid molecule are replaced by benzene rings with larger spatial structures, making it easier to form stable inclusion complexes. Therefore, this application selects phenyl-substituted borates for inclusion to improve its stability, solubility and bioavailability. Furthermore, phenyl-substituted borates are used as production aids in the production of γ-cyclodextrin to promote the forward reaction and increase the yield of γ-cyclodextrin.
[0092] Ethanol, as a good organic solvent, can significantly improve the solubility of phenyl-substituted borates, thereby increasing the utilization rate of production auxiliaries. Furthermore, the addition of ethanol can alter the solubility of cyclodextrin in the reaction system, leading to its precipitation and further promoting the forward reaction. In addition, the addition of ethanol and phenyl-substituted borates changes the original polar environment of the solution, which can also induce conformational changes in some cyclodextrin glucosyltransferases, potentially improving the enzyme's catalytic efficiency.
[0093] In summary, a strategy of adding phenyl-substituted borate and ethanol as γ-cyclodextrin production aids was adopted and applied to the production of γ-cyclodextrin to improve the yield and proportion of γ-cyclodextrin.
[0094] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A method for preparing γ-cyclodextrin, characterized in that, The method involves adding an ethanol solution of phenyl-substituted borate as a production aid to a catalytic reaction system for the production of γ-cyclodextrin. In the catalytic reaction system, the substrate is a starch liquefaction product or a starch derivative liquefaction product, the catalyst is γ-cyclodextrin glucosyltransferase, the final concentration of the phenyl-substituted borate in the catalytic reaction system is 1-3 g / 100 mL, the amount of γ-cyclodextrin glucosyltransferase added is 2 U / g dry starch, and the amount of ethanol added is 5% of the volume of the catalytic reaction system.
2. The method according to claim 1, characterized in that, Includes the following steps: S1, an aqueous solution of starch or starch derivative undergoes a liquefaction reaction to obtain a liquefied product; S2, γ-cyclodextrin glucosyltransferase is added to the liquefied product, and an ethanol solution of phenyl-substituted borate is added simultaneously to carry out the catalytic reaction; S3 was diluted with water and heated to inactivate the enzyme, yielding a reaction product containing γ-cyclodextrin.
3. The method according to claim 2, characterized in that, In step S1, the specific steps of the liquefaction reaction are as follows: S11, prepare starch or starch derivatives into an aqueous solution with a concentration of 5%~20% w / w; S12, adjust the pH of the aqueous solution from step S11 to 8.0~10.0 using sodium hydroxide solution; S13, add γ-cyclodextrin glucosyltransferase, and simultaneously heat to above 90°C and keep warm to end the liquefaction reaction and obtain the liquefied product.
4. The method according to any one of claims 1-3, characterized in that, The starch is any one of soluble starch, potato starch, corn starch, cassava starch, wheat starch, rice starch, and millet starch, and the starch derivative is maltodextrin.
5. The method according to claim 3, characterized in that, In step S13, the heating rate is 3~5°C / min, and the holding time is 20~30 min.
6. The method according to claim 2, characterized in that, In step S2, the temperature of the catalytic reaction is 45~60°C, and the reaction time is 6~10 h.
7. The method according to claim 2, characterized in that, In step S3, the dilution factor with water is 5 to 10 times, and the enzyme inactivation method is to heat the catalytic reaction system to above 90°C and keep it at that temperature for 20 to 30 minutes.
8. A method for preparing a γ-cyclodextrin product, characterized in that, The product prepared by the method according to any one of claims 1 to 7 is filtered, concentrated and dried to obtain the γ-cyclodextrin product.
9. The use of the method according to any one of claims 1 to 7 or the method according to claim 8 in the preparation of a product containing γ-cyclodextrin.