KR0136363B1 - Preparation process of cyclodextrin - Google Patents

Abstract

The present invention has a temperature range of 10 ℃ higher than the gelatinization initiation temperature at 30 ℃ lower than the gelatinization initiation temperature of the starch selected as a substrate without pretreatment of the substrate starch in the modified starch having a particulate starch or ordinary starch While maintaining, the cyclodextrin glucanotransferase (Cyclodextrin glucanotransferase) is reacted to produce a cyclodextrin mixture, and the resulting cyclodextrin mixture is centrifuged, filtered or the like to remove unreacted starch. A method for producing a high content cyclodextrin.

Description

Method for preparing cyclodextrin

In the present invention, cyclodextrin (Cd) is produced in a cyclodextrin by producing a cyclodextrin by reacting it with a reaction temperature of 10 ° C. higher than the initiation temperature at 30 ° C. below the initiation temperature of the selected substrate starch without pretreatment. By greatly increasing the amount of cyclodextrin produced per unit enzyme titer, and by removing the unreacted starch relates to a method for producing cyclodextrin of high purity products.

Cyclodextrin is a polymer of glucose first discovered by Billier in 1891 and is a cyclic oligosaccharide in which at least six glucose forms alpha-1,4-glucoside linkage. Particularly, those consisting of 6, 7, and 8 glucose are called alpha-cyclodextrin (α-Cyclodextrin), beta-cyclodextrin (β-Cyclodextrin), and gamma-cyclodextrin (γ-Cyclodextrin), respectively. Cyclodextrin is a useful material used for the purpose of stabilizing volatiles, eliminating bitumen, solubilizing insoluble substances, stabilizing color cans, etc., because it has a pupil that can contain hydrophobic substances. Cyclodextrin production method is prepared by reacting the cyclodextrin glucanotransferase (EC 2.4.1.19) or its producing microorganisms with the starch cyclodextrin synthase. In the conventional cyclodextrin production method, the starch solution is liquefied using liquefied enzyme and high temperature heat, and then cyclodextrin-glucanotransferase, which is a cyclodextrin generating enzyme, is used to produce cyclodextrin. However, this method produces a large amount of by-products such as glucose, maltose and oligosaccharides in addition to cyclodextrin, and has the following disadvantages. First, the production rate of cyclodextrin is lowered. Second, the reaction product should be purified to produce high purity cyclodextrin. Third, if dextrins with carbonyls (aldehyde groups or ketone groups) other than cyclodextrins are present in the aqueous solution, these components later cause amino carbonyl reactions and impair the quality of the final product. Fourth, its use is limited because by-products other than cyclodextrins have a sweet taste. Therefore, there is a need for a method of efficiently producing a product with less by-products other than cyclodextrin. In Japanese Patent Laid-Open No. 46-2380, a method of treating substrate starch DE under 15 has been proposed. In Japanese Patent Laid-Open No. 52-25043, cyclodextrin glucanotransferase is used as a liquefied enzyme. A method of increasing the production rate of dextrin has been proposed. The main method is to liquefy the substrate within the range of minimizing the production of by-products, but some by-products are generated before the cyclodextrin production reaction, and when using cyclodextrin glucanotransferase as a liquefied enzyme, Dextrin production is very low. Therefore, methods for converting starch into a form capable of reacting with cyclodextrin glucanotransferase have been proposed by physical methods that do not degrade starch liquor, which is a substrate. Korean Patent Publication No. 92-937 uses crushing friction instead of liquefaction, and Japanese Patent Publication No. Hei 1-6829595 uses starch extrusion to convert the starch liquor into a form that the enzyme can use. Has been proposed. However, these methods require time to pretreat the starch and require the introduction of a new device. In Japanese Patent Laid-Open No. 2-227088, a method of producing cyclodextrin by swelling starch with steam or hot water has been proposed to overcome the problems of the above methods. However, this method heats the starch prior to preparing the cyclodextrin, which increases the viscosity of the starch, which is a substrate, so that high concentration of starch cannot be used, and the production time of the cyclodextrin was shortened by a simple heat treatment method. In order to solve this problem, Japanese Laid-Open Patent Publication No. 3-160896 adds raw starch to an aqueous alcohol solution of 60 to 80%, and then dissolves and reacts again at high temperature and high pressure. And a separate facility is required.

In order to solve the above problems, the inventors of the present invention, the pre-applied invention Republic of Korea Patent Application No. 93-20680 the temperature of the range of possible non-gelatinized starch liquid, that is, the temperature lower than 30 ℃ lower than the start temperature of gelatinization starch The amount of cyclodextrin produced per unit enzyme titer by heat treatment at 10 ℃ higher than the initiation temperature at, followed by the production of cyclodextrin at a constant reaction temperature, followed by removal of unreacted starch by simple physical methods such as centrifugation or filtration. The method of producing cyclodextrin of high yield and high purity has been disclosed. However, in this preparation method, since the heat treatment must be performed before the enzyme acts on the starch, the processing time is required, and the viscosity of the substrate is increased by completing the pretreatment before the reaction, so that it is difficult to apply at a substrate concentration of 15% or more. Therefore, in the present invention, the reaction is not completely gelatinized depending on the type of starch and the temperature at which the cyclodextrin synthase can be converted into a form capable of reacting, i.e., 30 ° C lower than the initiation temperature of the substrate starch, and 10 ° C higher than the initiation temperature. By reacting cyclodextrin glucanotransferase directly with starch without pretreatment at temperature, the produced reaction solution is removed by simple physical methods such as centrifugation or filtration to remove unreacted starch, resulting in low by-product content and high cyclodextrin content. In addition, the present inventors have improved the simpler method of obtaining cyclodextrin with high yield of cyclodextrin per unit enzyme titer and reacting at high concentration of substrate. Accordingly, an object of the present invention is a starch solution in which the substrate selected from modified starch or ordinary starch having a particulate form is dissolved at a temperature lower than 30 ° C. below the gelatinization initiation temperature of the starch liquid selected as the substrate without pretreatment of the substrate starch. While maintaining the temperature range, the cyclodextrin glucanotransferase is reacted with a cyclodextrin mixture to produce a cyclodextrin mixture, and the resulting cyclodextrin mixture is centrifuged to remove unreacted starch by physical means such as filtration. It is to provide a method for producing a high content cyclodextrin.

Hereinafter, the present invention will be described in more detail. Cyclodextrin in the present invention refers to alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin or mixtures thereof. By-products in the present invention are other products produced from starch except cyclodextrin are glucose, maltose, Oligosaccharides.

The effects of cyclodextrin production according to the present invention are as follows. First, it does not require pretreatment of starch, so it is simple and saves money. Second, production time is short because starch is not pretreated. Third, the conventional method is to liquefy the starch using the alpha amylase liquefied enzyme to produce cyclodextrin, so that during the liquefaction reaction starch is decomposed by alpha amylase to produce a lot of dispersions glucose, maltose, oligosaccharides. However, in the present invention, since the liquefied enzyme, alpha amylase, is not used unlike the conventional method, the production of by-products is reduced. Fourth, since the starch, which is an unreacted substrate, can be easily separated through centrifugation or filtration, the purification process is simple since the reaction solution is clear and clean. Fourth, as a feature of the present invention, the ratio of cyclodextrin in the reaction solution is increased by increasing the proportion of cyclodextrin in the product, thereby increasing the content of cyclodextrin. Fifth, the separated unreacted starch can be used to prepare starch and starch sugar for high value-added new applications by removing heat-soluble and soluble parts of starch. Sixth, the conventional method uses a large amount of enzyme to increase the ratio of cyclodextrin in the product, but in the present invention, a high amount of cyclodextrin is produced even with a small amount of enzyme, so that the amount of cyclodextrin per unit enzyme is greatly increased. do. Seventh, the conventional method is difficult to apply at a high concentration of the substrate concentration of the substrate after the pre-treatment of the viscosity of the substrate, but the present invention can be produced cyclodextrin in a high concentration of the substrate in the pre-treatment process. As the enzyme used in the present invention, a cyclodextrin glucarotransferase derived from Bacillus macerans was used. Cyclodextrin glucanotransferase from Bacillus circulans was used. Cyclodextrin glucanotransferases derived from Bacillus macerans are marketed under the name Cyclodexrin Glucanotr ansf erase by Amano Pharmaceutical Co., LTD. Cyclodextrin glucanotransferases derived from Lance (Bacillus circulans) were cultured by Bacillus circulans (Bacillus circulans. ATTCC 21783), centrifuged bacteria and used to powder the supernatant. The enzymes usable in the present invention may be any as cyclodextrin glucanotransferases that maintain activity at a reaction temperature determined according to the gelatinization start temperature of the selected starch.

The most important factor in the present invention is the temperature of the reaction from 30 ° C lower than the initiation temperature of the starch selected as the substrate among the various starches to 10 ° C higher than the initiation temperature, preferably 25 ° C Reacts at temperatures up to Starch used as a substrate was used as corn starch, waxy corn starch, potato starch, sweet potato starch, wheat starch. In the present invention, all starches such as modified starch and general starch are available. In the present invention, the method of separating the unreacted substrate may use centrifugation or filtration that can be used when the solid material is mixed in the liquid phase. In the present invention, the method used to separate the unreacted solids was treated with a centrifugal force of 4000 g for 10 minutes, or a filter cloth made of polyester material was used. Any method can be easily used if the solid unreacted starch and the liquid product are easily separated by a centrifugal separation method having a centrifugal force capable of separating the product or a filtration using an industrially available filter cloth.

Example 1

Make 200 mL of 10% whole milk powder with buffer pH 6.0. After checking the pH, cyclodextrin glucanotransferase derived from Bacillus macerans is added in an amount of 0.25% (V / W, 36.3 U / g) relative to the dry matter. After 24 hours of reaction at 55 ° C, unreacted solid starch is removed by centrifugation at 8000 rpm for 15 minutes using an IC Model Centra-4 Centrifuger and a 820 rotor (CAT 820 Rotor). The enzyme in the supernatant is inactivated and the content of cyclodextrin is quantified by high performance liquid chromatography and phenolphthalein coloring. Hereinafter, one unit of enzyme refers to the amount of enzyme that reacts with 0.2% soluble starch and decreases the iodine absorbance of the substrate by 10% per minute. %, Which indicates the amount of enzyme to be reacted, refers to a volume per mL of substrate (mL / 100 g).

Comparative Example 1

Amanosa's proposed method of producing cyclodextrin is 1.67% of ethanol starch milk in 200mL of liquefaction and 1.67% of enzyme in liquefaction and 3.33% of enzymes reacts with starch to produce cyclodextrin. To produce. The reaction temperature is 60 ℃ and the other reaction conditions are the same pH 6.0 as in Example 1. It is a 24 hour reaction. As shown in Table 1, in the case of Example 1, the purity of the cyclodextrin was 482% higher than that of Comparative Example 1, which is a general cyclodextrin production method, and the purity of the cyclodextrin was 1.1% improved.

TABLE 1

Comparison of cyclodextrin production and yield per enzyme titer

In Table 1 CD represents cyclodextrin.

Example 2

Make 10% wheat and potato starch 200mL with buffer pH 6.0. After checking the pH, the amount of cyclodextrin glucanotransferase derived from Bacillus macerans is added in an amount of 0.833% (121 U / g) relative to the dry matter. After reaction at 60 ° C. for 24 hours, unreacted solid starch is removed by centrifugation at 8000 rpm for 15 minutes using an IC Model Centra-4 Centrifuger and a 820 rotor (CAT 820 Rotor). The enzyme in the supernatant is inactivated and the content of cyclodextrin is quantified by high performance liquid chromatography and phenolphthalein coloring. The detailed results are shown in Table 2.

TABLE 2

Comparison of cyclodextrin production and yield per enzyme titer

In Table 2 CD represents cyclodextrin.

Example 3

In the same manner as in Example 2, the enzyme was treated with corn starch to form a cyclodextrin mixed solution, and the unreacted solid starch was removed with a filter cloth made of polyester, and the produced cyclodextrin was quantitated by high-performance liquid chromatography and phenolphthalein. do. The detailed results are shown in Table 3.

TABLE 3

Comparison of cyclodextrin production and yield per enzyme titer

In Table 3 CD represents cyclodextrin.

Claims (2)

The starch solution in which the substrate selected from denatured starch or ordinary starch having granular shape is dissolved is selected as a substrate without pretreatment of the substrate starch while maintaining a temperature range of 10 ° C. higher than the gelatinization start temperature of 30 ° C. below the gelatinization start temperature of the starch solution. And a cyclodextrin glucanotransferase to produce a cyclodextrin mixture, and the resulting cyclodextrin mixture to remove unreacted starch by physical methods such as centrifugation and filtration. Method for preparing dextrin The method for producing a high content cyclodextrin according to claim 1, wherein the starch solution selected as a substrate is reacted with cyclodextrin glucanotransferase in a temperature range of 25 ° C lower than the initiation temperature to the initiation temperature.