KR101340970B1 - Preparation method of cyclodextrin in liquid form - Google Patents

Abstract

The present invention relates a method for manufacturing liquid cyclodextrin comprising a step for enzyme reaction by adding cyclodextrin synthetase, alpha-amylase, and pullulanase to a starch-derived raw material. The cyclodextrin, which is manufactured by the manufacturing method according to the present invention, is liquid and has no cloudiness when being stored at room temperature for a long time.

Description

Preparation method of liquid cyclodextrin {PREPARATION METHOD OF CYCLODEXTRIN IN LIQUID FORM}

The present invention relates to a process for the preparation of liquid cyclodextrins.

Cyclodextrins are known to contain a variety of chemicals by acting as host molecules. In particular, alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin contain cavities, and the size of these cavities differs depending on the type of cyclodextrin, thereby enclosing guest molecules of various sizes. You can. Therefore, many studies are being conducted on the inclusion effects of cyclodextrins and fields such as food, medicine, and pesticides using these effects. In particular, they are widely used for encapsulating the flavor of food and maintaining it for a long time or for improving the quality of food by encapsulating bitter or other odors.

However, high-purity alpha-cyclodextrins, beta-cyclodextrins and gamma-cyclodextrins are rarely used in the food field because of their high price, and are mainly used in medicine and pesticides. On the other hand, in the food field, cyclodextrin is mainly used in the form of a mixture, which is about 18 to 21% by weight of cyclodextrin, about 78 to 81% by weight of non-cyclic polysaccharides in which four or more glucose molecules are combined, and the like. Mixtures comprising the components. At this time, about 20% of the cyclodextrin consists of alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin.

Currently, the cyclodextrins used in food are mostly in powder form, and the liquid cyclodextrins are not sold because the liquid form of cyclodextrins causes haze phenomenon that causes the components of the product to become cloudy after prolonged storage. . The clouding phenomenon is an irreversible reaction that cannot be restored to its original state, and the liquid cyclodextrin in which clouding occurs has a bad taste of a grassy or starchy system that is not felt in the original product.

However, the cyclodextrin in powder form is not only a problem of scattering (powder blowing) when used by a consumer, but also hassle that the cyclodextrin in powder form is dissolved again in water to be prepared and used as a liquid in the required concentration.

Therefore, the inventors of the present invention, while studying the liquid cyclodextrin without the clouding phenomenon, found that the liquid cyclodextrin prepared by adding certain enzymes simultaneously to the starch-derived raw material can be stably stored for a long time without clouding phenomenon and completed the present invention.

It is an object of the present invention to provide a method for preparing a liquid cyclodextrin that can be stably stored for a long time without clouding.

In order to achieve the above object, the present invention provides a method for preparing a liquid cyclodextrin comprising the step of adding cyclodextrin synthase, alpha-amylase and pullulanase to the starch-derived raw material.

The liquid cyclodextrin prepared by the production method of the present invention can be stably stored for a long time without clouding phenomenon.

The present invention relates to a method for preparing a liquid cyclodextrin comprising the step of enzymatic reaction by adding cyclodextrin synthase, alpha-amylase and pullulanase to starch-derived raw materials.

The present invention also relates to

Enzymatic reaction by adding cyclodextrin synthase, alpha-amylase and pullulanase to starch-derived raw materials; and

It relates to a method for producing a liquid cyclodextrin comprising the step of filtering, purifying and concentrating the enzyme reaction solution.

Starch derived raw materials

The starch-derived raw material of the present invention is starch or maltodextrin (non-cyclic polysaccharide in which 10 to 30 glucose molecules are bound). The starch may be corn starch, potato starch, sweet potato starch, tapioca starch, waxycorn starch, sago starch and the like, but is not particularly limited.

When starch is used as the starch-derived raw material of the present invention, the starch slurry can be liquefied to add an enzyme. When maltodextrin is used as the starch-derived raw material of the present invention, maltodextrin can be dissolved in water to add an enzyme.

The starch-derived raw material is preferably having a concentration of 0.5 to 80% on the basis of anhydride when considering the efficiency of the enzyme reaction, but is not limited thereto.

enzyme

Enzymes of the invention are cyclodextrin synthetase, alpha-amylase and pullulanase. For reference, beta-amylase can reduce the content of macromolecules in the starch-derived raw materials by converting them into glucose and maltose. The use of beta-amylase instead of alpha-amylase is not effective in achieving the object of the present invention because the glucose and maltose produced thereby interfere with the cyclodextrin synthesis reaction of the cyclodextrin synthase.

In the present invention, the cyclodextrin synthase, alpha-amylase and pullulanase are simultaneously added to starch-derived raw materials to perform enzymatic reactions together. That is, the enzymes are not added sequentially.

In the case of the addition of pullulanase, pullulanase decomposes amylopectin of alpha-1,6 bonds of amylopectin and maltodextrin and amylopectin in starch into amylose of alpha-1,4 bond, which is broken down by short chain Is made of low molecular weight. Cyclodextrin synthase can make cyclodextrins using the short chain sugars and amylose. In this case, since the decomposition rate of polymer components such as maltodextrin is increased, the transparency of the enzyme reaction solution itself is increased, and the occurrence of turbidity is greatly reduced.

If no pullulanase is used, the alpha-1,6 bond will remain and the alpha-amylase will not be broken down. Therefore, even when the liquid cyclodextrin is prepared, the glucose molecular chains are combined with each other during the storage period of the enzyme reaction solution by the alpha 1,6 binding of maltodextrin and amylopectin in the starch, resulting in the clouding phenomenon. This will happen.

The cyclodextrin synthase is preferably cycloglucosyltransferase (CGTase (Cycloglucosyltrans-ferase)). The alpha-amylase or beta-amylase is preferably heat resistant and mesophilic.

If the starch-derived raw material is treated with cyclodextrin synthase to undergo primary saccharification and then treated with alpha-amylase to undergo secondary saccharification, this sequential treatment takes a lot of time and money. In addition, the present invention is to prevent the turbidity phenomenon by decomposing even components that could not be resolved by conventional methods, such as components containing alpha 1, 6 bonds by the action of pullulanase, alpha-amylase and cyclodextrin synthase at the same time Because of the characteristics, enzymes should not be added sequentially.

The enzyme is added in an amount of 0.001 to 3.0 parts by weight based on 100 parts by weight of the solid content of the starch-derived raw material. The enzyme is added to the starch-derived raw material to perform an enzymatic reaction, and the enzymatic reaction may be performed at a temperature of pH 2.0 to 8.0 and 10 to 100 ° C.

Enzyme Reaction

The enzyme reaction solution of the present invention is a product obtained by reacting an enzyme with a starch-derived raw material, and is a sugar solution containing sugar. The enzyme reaction solution of the present invention includes cyclodextrins and acyclic polysaccharides in which four or more glucose molecules are bound. In addition, the enzyme reaction solution of the present invention may further comprise glucose or maltose. The enzyme reaction solution may be filtered, the enzyme reaction solution may be more colorless and transparent, and the ionic substance may be removed and then concentrated to a desired concentration to prepare a liquid cyclodextrin. The filtration, color improvement of the enzyme reaction solution, ionic material removal and concentration can be carried out using methods commonly used in the art. For example, the filtration may be performed using perlite, diatomaceous earth, and the like, and the color of the enzyme reaction solution may be colorless and transparent using powdered activated carbon or granular activated carbon, and the removal of the ionic substance may be performed by removing the ion exchange resin. It can be performed using, but is not limited thereto.

Liquid Cyclodextrin

The liquid cyclodextrin of the present invention is not powdered, but is stored, distributed and sold in the liquid form of the enzyme reaction solution (sugar solution). The liquid cyclodextrin of the present invention is not observed visually when stored at room temperature for 600 days, preferably when stored at room temperature for 700 days is not observed with the naked eye, most preferably, stored at room temperature for 800 days No clouding is observed visually. That is, the liquid cyclodextrin of the present invention can be stored for a long time without clouding.

Liquid cyclodextrins of the invention include alpha-cyclodextrin and gamma-cyclodextrin. The liquid cyclodextrin of the present invention does not contain beta-cyclodextrin or even if it contains beta-cyclodextrin relative to 100 parts by weight of the total cyclodextrin (total sum of alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin). It is included in less than 50 parts by weight, preferably less than 40 parts by weight, more preferably less than 30 parts by weight, even more preferably less than 20 parts by weight, most preferably less than 10 parts by weight. In addition, even if beta-cyclodextrin is included in the liquid cyclodextrin of the present invention, less than 10% by weight, preferably less than 8% by weight, more preferably less than 6% by weight of the total sugar is included.

When more than 50 parts by weight of beta-cyclodextrin in the cyclodextrin, the turbidity occurs not only due to the intimacy of acyclic polysaccharides in which four or more glucose molecules are bound, especially maltodextrin and beta-cyclodextrin, but also beta-cyclodextrin. Due to the crystallinity of dextrins, there is a risk of crystallization during storage and distribution of the finished product.

The enzyme reaction solution of the present invention contains less than 55% by weight, preferably less than 50% by weight of acyclic polysaccharides having four or more glucose molecules in the total sugars. In addition, the enzyme reaction solution of the present invention contains more than 23% by weight of cyclodextrin in the total sugar, preferably 23 to 35% by weight, more preferably 25 to 30% by weight.

The liquid cyclodextrin of the present invention is contained in a liquid composition such as an enzyme reaction solution. In addition to the cyclodextrin, the liquid composition may further include acyclic polysaccharides, maltodextrins, glucose or maltose to which four or more glucose molecules are bound.

The liquid cyclodextrin of the present invention is for food addition. Food may be prepared by adding the liquid cyclodextrin of the present invention. The kind of the food is not particularly limited.

BRIEF DESCRIPTION OF THE DRAWINGS The advantages and features of the present invention, and the manner of achieving them, will be apparent from and elucidated with reference to the embodiments described hereinafter in conjunction with the accompanying drawings. It should be understood, however, that the invention is not limited to the disclosed embodiments, but is capable of many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, To fully disclose the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

≪ Materials and methods >

Commercial corn starch and maltodextrin were purchased and used for testing.

Enzyme reaction solution analysis

Cyclodextrin content in the enzyme reaction solution was analyzed using high performance liquid chromatography (HPLC). HPLC operating conditions are as follows.

HPLC column: Carbohydrate column (Aminex HPX-42A)

Length 300 mm x 7.8 mm

- Detector: RID (Refractive Index Detector)

-Operating temperature: injection temperature 85 ℃, detection temperature 45 ℃

Column oven temperature: 25-100 ℃

-Solvent: Ultrapure water (Specific conductivity: 0.05 uS / cm)

Flow rate: 0.6 mL / min

- Run time: 25 minutes

Injection volume: 20 μL

The analysis method is as follows. First, the concentration of the sample solution to be analyzed for cyclodextrin content is adjusted to 2.5-3.0% using ultrapure water. At this time, a handbrix meter is used to analyze the sample concentration. After adjusting the concentration of the sample solution, remove foreign substances and enzyme bodies contained in the sample solution using a 0.2-0.45um filter. At this time, the purpose of removing foreign substances and enzyme bodies from the sample solution is to extend the life of the HPLC column and obtain more accurate analysis results. Thereafter, the sample solution from which the foreign substances are removed is injected into HPLC to analyze the content of the enzyme reaction solution (sugar solution) and the cyclodextrin contained therein.

Cyclodextrin in the enzyme reaction solution was quantified as follows. Samples in an amount equivalent to 0.5 g as cyclodextrin were precisely measured, and water was added thereto to make 50 ml. 20 ml of the solution was taken, heated for 10 minutes in a water bath, cooled, and 2 ml of glucoamylase solution (10 IU / ml) was added thereto, and the mixture was reacted in a 40 ° C. water bath for 1 hour. The reaction solution was heated in a water bath for 10 minutes, filtered, cooled to room temperature, and then diluted to 25 ml with water as a test solution.

Separately, the standards of alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin were each dried, and then 0.1 g of each was precisely measured and water was added thereto to prepare a solution of 20 ml. The solutions were used as mixed standard solutions.

10 L of the mixed standard solution and the test solution were respectively injected into the high-speed liquid chromatograph under the following operating conditions, and the peak areas of alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin and the mixed standard solution of the test solution. The peak areas of alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin were obtained, respectively, and the contents of the three components were calculated according to the following Equations 1 to 3, and the sum was the cyclodextrin content (Equation 4).

<Formula 1>

% Of alpha-cyclodextrin = {(concentration of alpha-cyclodextrin standard solution (ppm) X 50 X 25) / (sample amount (g) X 20)} X {(alpha-cyclodextrin of test solution Peak area) / (peak area of alpha-cyclodextrin of mixed standard solution)} X 100 / 1,000,000.

<Formula 2>

% Of beta-cyclodextrin = {(concentration of beta-cyclodextrin standard solution (ppm) X 50 X 25) / (sample amount (g) X 20)} X {(beta-cyclodextrin of test solution Peak area) / (peak area of beta-cyclodextrin of mixed standard solution)} X 100 / 1,000,000.

<Formula 3>

Gamma-cyclodextrin content (%) = {(concentration of gamma-cyclodextrin standard solution (ppm) X 50 X 25) / (sample amount (g) X 20)} X {(gamma-cyclodextrin of test solution Peak area) / (peak area of gamma-cyclodextrin of mixed standard solution)} X 100 / 1,000,000.

<Equation 4>

Cyclodextrin content (%) = Alpha-cyclodextrin content (%) + Beta-cyclodextrin content (%) + Gamma-cyclodextrin content (%)

&Lt; Example 1 >

Commercial corn starch was prepared and starch boomer (starch concentration unit / Be ') was diluted to 10-20 Be' using process water. Thereafter, the temperature of the dilution solution was increased to 105-108 ° C, and the enzyme reaction was performed by adding 0.15% by weight of CGTase and pullulanase (used in the same amount) and 0.13% by weight of heat-resistant and mesophilic alpha-amylase relative to the solid content of the dilution solution. It was. The enzyme reaction was carried out for about 48 hours, at which time the pH of the enzyme reaction solution was pH 5.5-6.0. Thereafter, the enzyme reaction was stopped by inactivating the enzyme by lowering the pH of the enzyme reaction solution to pH 3.5 using hydrochloric acid. The enzyme reaction solution of which the enzyme reaction was completed was filtered using diatomaceous earth, and the color of the reaction solution was made colorless and transparent using powdered activated carbon. Thereafter, an ionic substance present in the enzyme reaction solution was removed using an ion exchange resin, and then concentrated to a desired concentration to prepare an enzyme reaction solution (sugar solution). The enzyme reaction solution is liquid and contains cyclindextrin.

<Example 2>

Maltodextrin having a reducing equivalent value of 20 or less was dissolved in water at 35-38%, 55-65 ° C. When maltodextrin was completely dissolved, the enzyme reaction was carried out by adding 0.3% by weight of a mixture of CGTase, heat-resistant alpha-amylase, and flulanase (each enzyme in the mixture was included in the same amount) to maltodextrin solids. The enzymatic reaction was carried out for 24 hours, at which time pH 5.5-6.0 was maintained and the pH was adjusted using hydrochloric acid and caustic soda (NaOH). Thereafter, the enzyme reaction was stopped by inactivating the enzyme by lowering the pH of the enzyme reaction solution to pH 3.5 using hydrochloric acid. The enzyme reaction solution of which the enzyme reaction was completed was filtered using diatomaceous earth, and the color of the reaction solution was made colorless and transparent using powdered activated carbon. Thereafter, an ionic substance present in the enzyme reaction solution was removed using an ion exchange resin, and then concentrated to a desired concentration to prepare an enzyme reaction solution (sugar solution). The enzyme reaction solution is liquid and contains cyclindextrin.

&Lt; Comparative Example 1 &

Commercial corn starch was prepared and starch boomer (starch concentration unit / Be ') was diluted to 10-20 Be' using process water. Thereafter, the temperature of the diluted solution was increased to 105-108 ° C., and 0.15% by weight of CGTase was added to the solid content of the diluted solution, followed by enzymatic reaction for 24 hours (primary saccharification). Thereafter, the enzyme reaction was stopped by inactivating the enzyme by lowering the pH of the enzyme reaction solution to 3.5. After adjusting the pH to 5.5-6.0 using hydrochloric acid and caustic soda, the enzyme reaction was performed for 24 hours by adding 0.13% by weight of heat resistant and mesophilic alpha-amylase to the solids of the diluent (secondary glycosylation). Thereafter, the enzyme reaction was stopped by inactivating the enzyme by lowering the pH of the enzyme reaction solution to pH 3.5 using hydrochloric acid. The enzyme reaction solution of which the enzyme reaction was completed was filtered using diatomaceous earth, and the color of the reaction solution was made colorless and transparent using powdered activated carbon. Thereafter, an ionic substance present in the enzyme reaction solution was removed using an ion exchange resin, and then concentrated to a desired concentration to prepare an enzyme reaction solution (sugar solution). The enzyme reaction solution is liquid and contains cyclindextrin.

Comparative Example 2

Maltodextrin having a reducing equivalent value of 20 or less was dissolved in water at 35-38%, 55-65 ° C. When maltodextrin was completely dissolved, enzyme reaction was carried out for 12 hours by adding 0.15% by weight of CGTase to maltodextrin solids (primary saccharification). Thereafter, the enzyme reaction was stopped by inactivating the enzyme by lowering the pH of the enzyme reaction solution to 3.5. After adjusting to pH 5.5-6.0 using hydrochloric acid and caustic soda, the enzyme reaction was performed for 12 hours by adding 0.15% by weight of heat resistant and mesophilic alpha-amylase to maltodextrin solids (secondary glycosylation). Thereafter, the enzyme reaction was stopped by inactivating the enzyme by lowering the pH of the enzyme reaction solution to pH 3.5 using hydrochloric acid. The enzyme reaction solution of which the enzyme reaction was completed was filtered using diatomaceous earth, and the color of the reaction solution was made colorless and transparent using powdered activated carbon. Thereafter, an ionic substance present in the enzyme reaction solution was removed using an ion exchange resin, and then concentrated to a desired concentration to prepare an enzyme reaction solution (sugar solution). The enzyme reaction solution is liquid and contains cyclindextrin.

&Lt; Comparative Example 3 &

Maltodextrin having a reducing equivalent value of 20 or less was dissolved in water at 35-38%, 55-65 ° C. When the maltodextrin was completely dissolved, the enzyme reaction was carried out by adding 0.3 wt% of the mixture of CGTase and the heat-resistant alpha-amylase (the respective enzymes in the mixture were included in the same amount) to the maltodextrin solids. The enzymatic reaction was carried out for 24 hours, at which time pH 5.5-6.0 was maintained and the pH was adjusted using hydrochloric acid and caustic soda (NaOH). Thereafter, the enzyme reaction was stopped by inactivating the enzyme by lowering the pH of the enzyme reaction solution to pH 3.5 using hydrochloric acid. The enzyme reaction solution of which the enzyme reaction was completed was filtered using diatomaceous earth, and the color of the reaction solution was made colorless and transparent using powdered activated carbon. Thereafter, an ionic substance present in the enzyme reaction solution was removed using an ion exchange resin, and then concentrated to a desired concentration to prepare an enzyme reaction solution (sugar solution). The enzyme reaction solution is liquid and contains cyclindextrin.

Experimental Example 1 Composition of Enzyme Reaction Solution

The compositions of the enzyme reaction solutions of Examples 1 and 2 and Comparative Examples 1 to 3 were analyzed using HPLC. As a result, Examples 1 and 2 had a higher content of cyclodextrin in the total sugar than the comparative examples. In addition, Examples 1 and 2 were found to have a significantly low content of beta-cyclodextrin to the total cyclodextrin, and also a low content of acyclic polysaccharides having four or more glucose molecules bound thereto. That is, Example 1 was about 3.7 parts by weight of beta-cyclodextrin relative to 100 parts by weight of total cyclodextrin, and Example 2 was about 1.85 parts by weight based on 100 parts by weight of total cyclodextrin (Table 1). In addition, the examples are expected to have low or no white turbidity due to the low content of acyclic polysaccharides having four or more glucose molecules bound thereto.

Sugar composition
(sugar profile / DB%) Example 1 Example 2 Comparative Example 1 Comparative Example 2 Comparative Example 3 Alpha-cyclodextrin 8.5 11 3 2.5 2.1 Beta-cyclodextrin One 0.7 10.2 10.4 13.6 Gamma-cyclodextrin 17.5 16.3 7.1 5 6.4 Total-cyclodextrin 27 28 20.3 17.9 22.1 Glucose 10.9 9 3 2 2.7 Maltose 16.1 15.8 13.7 9.4 10.2 * G4 < 46 47.2 63 70.7 65 Total 100 100 100 100 100

* G4 <: non-cyclic polysaccharide in which four or more glucose molecules are combined

Content Unit: Anhydrous Basis (DB%): Weight% based on Dry Basis

The figures are averages of three replicates.

Experimental Example 2 Stability in Long-Term Storage

After storing the enzyme reaction solution of Examples 1 and 2 and Comparative Examples 1 to 3 for 100 days at room temperature, the presence or absence of clouding was observed visually over time.

Day Example 1 Example 2 Comparative Example 1 Comparative Example 2 Comparative Example 3 100 days - - - ○ ○ 200 days - - ○ ○ ◎ 400 days - - ○ ◎ ◎ 600 days - - ◎ ◎ ◎ 800 days - - ◎ ◎ ◎

-: No turbidity occurs.

○: somewhat cloudy but generally transparent

◎: heavy turbidity

Claims (16)

A method for producing a liquid cyclodextrin comprising enzymatic reaction by adding cyclodextrin synthase, alpha-amylase and pullulanase to starch or maltodextrin.
The method of claim 1,
The cyclodextrin synthase, alpha-amylase and pullulanase are added simultaneously.
The method of claim 1,
Said cyclodextrin synthetase is cycloglucosyl transferase.
delete The method of claim 1,
The liquid cyclodextrin is not powdered, the manufacturing method characterized in that it is stored in a liquid form.
The method of claim 1,
The liquid cyclodextrin has a beta-cyclodextrin is less than 50 parts by weight based on 100 parts by weight of cyclodextrin.
The method of claim 1,
The liquid cyclodextrin has a beta-cyclodextrin is less than 30 parts by weight based on 100 parts by weight of cyclodextrin.
The method of claim 1,
The liquid cyclodextrin is a manufacturing method characterized in that no whitening phenomenon is observed visually when stored at room temperature for 400 days.
delete The method of claim 1,
Wherein said liquid cyclodextrin comprises alpha-cyclodextrin and gamma-cyclodextrin.
Enzymatic reaction by adding cyclodextrin synthase, alpha-amylase and pullulanase to starch or maltodextrin; and
Method for producing a liquid cyclodextrin comprising the step of filtering and concentrating the enzyme reaction solution.
12. The method of claim 11,
The enzyme reaction solution is characterized in that it comprises a cyclodextrin and maltodextrin.
12. The method of claim 11,
The enzyme reaction solution is characterized in that less than 55% by weight of the non-cyclic polysaccharides in which four or more glucose molecules are combined in the total sugar.
12. The method of claim 11,
The enzyme reaction solution is characterized in that the cyclodextrin in the total sugar contains 23% by weight or more.
12. The method of claim 11,
The enzyme reaction solution is characterized in that the beta-cyclodextrin is less than 50 parts by weight based on 100 parts by weight of cyclodextrin.
12. The method of claim 11,
The enzyme reaction solution is characterized in that less than 10% by weight of beta-cyclodextrin in the total sugar.