KR102711483B1 - Method for the production of modified starch with wild typed cyclodextrin glucanotransferase and rice cake therefrom - Google Patents

Abstract

The present invention relates to producing enzymatically modified starch by hydrolyzing starch with wild-type cyclodextrin glucanotransferase, and to using the same in starch-based foods such as rice cakes. According to the present invention, low-molecular-weight (10 ³ -10 ⁴ Da) modified starch can be produced without producing CD by using wild-type cyclodextrin glucanotransferase (CGTase), and this can be effectively used in the food and starch material industries.

Description

Method for the production of modified starch with wild typed cyclodextrin glucanotransferase and rice cake therefrom

The present invention relates to a method for producing modified starch using wild-type cyclodextrin glucanotransferase, and more specifically, to producing enzymatically modified starch by hydrolyzing starch with wild-type cyclodextrin glucanotransferase, and using the same in starchy foods such as rice cakes.

Starch is a crystalline storage carbohydrate of plants, and is the main component of various types of cereals and paper, and is the main energy source for living organisms including humans. Due to its safety for the human body, starch is utilized in various industries as well as the food industry, and physical, chemical, and bioconversion techniques are being used to change the properties of starch.

Korean Patent Registration No. 10-0868329 (registration date: November 5, 2008) discloses a method for producing a highly branched amylopectin cluster and a highly branched amylose using an enzyme. According to this document, a method is disclosed for effectively producing a branched amylopectin cluster, a highly branched amylose, or a branched oligosaccharide from starch, in which alpha-glucanotransferase or a branching enzyme hydrolyzes linking chains between amylopectin clusters present in starch to produce amylopectin clusters, while the branching enzyme attaches branched side chains to amylose to produce branched amylose, and maltogenic amylase is treated to cleave the long side chains of the amylopectin clusters and branched amylose produced thereby to create short side chains, while simultaneously transferring a sugar to the side chains by alpha-1,6-linkage.

In addition, Korean Patent Registration No. 10-1532025 (registration date June 22, 2015) describes a method for producing an amylopectin cluster having a molecular weight of 10 ⁴ to 10 ⁵ Da by treating a novel cyclodextrin glucanotransferase (CGTase). According to this document, an amylopectin cluster having a low molecular weight can be produced without producing cyclodextrin (CD) by using a mutant CGTase in which three amino acids are artificially modified.

The present invention relates to a method for producing enzymatically modified starch by hydrolyzing starch with cyclodextrin glucanotransferase and to using the same in starchy foods such as rice cakes. According to the present invention, starch is partially hydrolyzed with cyclodextrin glucanotransferase to produce starch with modified properties, and the product suitability of instant rice cakes is modified by adding the same to rice cakes.

The present invention provides a method for producing modified starch, characterized by including the steps of: (a) dissolving starch in a buffer and gelatinizing it; (b) placing the starch gelatinized in step (a) in a constant temperature stirrer and preheating it; and (c) adding cyclodextrin glucanotransferase and inducing a reaction after the preheating in step (b).

In the method for manufacturing modified starch of the present invention, it is preferable that the method for manufacturing modified starch further includes, after the step (c), a step (d) of boiling the reaction solution to stop the enzymatic reaction; and after the step (d), a step (e) of freeze-drying the liquid modified starch.

In the method for manufacturing modified starch of the present invention, the modified starch may preferably have a size of 1×10 ³ to 1×10 ⁴ Da.

The present invention provides a rice cake manufactured by adding modified starch having a size of 1×10 ³ to 1×10 ⁴ Da manufactured by the method of the present invention to rice flour, adding water, kneading, and steaming.

In the rice cake of the present invention, it is preferable that the rice cake is manufactured by mixing 0.1 to 30 wt% of modified starch and 70 to 99.9 wt% of rice flour based on the weight sum of the modified starch and rice flour.

According to the present invention, low-molecular-weight modified starch can be effectively produced using wild-type cyclodextrin glucanotransferase (CGTase). In previous studies, 4-α-GTase or mutant CGTase was used to produce amylopectin clusters having a molecular weight of 10 ⁴ -10 ⁵ Da. However, since these are not commercial enzymes, their industrial utility is very low. However, when the enzyme reaction conditions developed in the present invention are applied, low-molecular-weight (10 ³ -10 ⁴ Da) modified starch can be produced without producing CD using general wild-type CGTase. In addition, this modified starch can be effectively utilized in the food and starch material industries.

Figure 1 shows the results of analyzing the change in molecular weight of starch according to the time period of cyclodextrin glucanotransferase enzyme treatment.
Figure 2 shows the SEC analysis results of starch 4 hours after cyclodextrin glucanotransferase enzyme treatment.
Figure 3 shows the results of the analysis of small sugars in the enzyme reaction solution treated with cyclodextrin glucanotransferase by time zone.

The present invention relates to a method for producing enzymatically modified starch by hydrolyzing starch with cyclodextrin glucanotransferase and to using the same in starchy foods such as rice cakes. The existing invention (No. 10-1532025) can produce amylopectin clusters without effectively producing CD in a short period of time, but has limitations in industrial use because it requires the use of a mutant enzyme. However, in the present invention, starch is partially hydrolyzed with wild-type cyclodextrin glucanotransferase to produce starch with modified properties, and can be added to rice cakes to modify the product properties of instant rice cakes.

Based on this, the present invention provides a method for producing modified starch, characterized by including the steps of: (a) dissolving starch in a buffer and gelatinizing it; (b) placing the starch gelatinized in step (a) in a constant temperature stirrer and preheating it; and (c) adding cyclodextrin glucanotransferase and inducing a reaction after the preheating in step (b).

Hereinafter, the present invention will be described in detail in each step.

<Step (a): Luxury>

This step is a process of dissolving starch in a buffer and gelatinizing it. Various starches derived from cereals and paper can be used as the starch, but corn starch is preferably used. Any buffer having a pH of 5.0 to 7.0 (preferably 6.0) can be used, but sodium acetate buffer (sodium acetate buffer, 50 mM, pH 6.0) is preferably used. It is preferable to add 10% (w/v) of starch (preferably corn starch) to the buffer (preferably sodium acetate buffer) and then gelatinize it. Gelatinization can be performed using a conventional method.

<Step (b): Warm-up>

This step is a process of putting the starch gelatinized in the above step (a) into a constant temperature stirrer and preheating it. It is recommended that the preheating be performed in a constant temperature stirrer at 50 to 70°C (preferably 60°C) for 4 to 6 minutes (preferably 5 minutes). It was confirmed through the present invention that preheating makes the enzyme reaction even and increases the reproducibility of the enzyme reaction.

<Step (c): Enzyme reaction>

This step is a process of adding cyclodextrin glucanotransferase and inducing a reaction after the preheating in the above step (b). It is recommended to add 0.640 to 0.650 U/mg substrate (preferably 0.645 U/mg substrate) of cyclodextrin glucanotransferase. At this time, 1 unit (U) is defined as the amount of enzyme that hydrolyzes 1 mol of glycosidic bond in 1 minute.

It is preferable to use 'Toruzyme 300L' manufactured by Novozyme as the cyclodextrin glucanotransferase enzyme. This enzyme is an alpha-cyclodextrin (α-CD) producing enzyme. In the present invention, by using an extremely small amount, it is possible to selectively hydrolyze a portion of starch without producing α-CD. Due to this, in the present invention, it is possible to produce modified starch having a size of 1×10 ³ to 1×10 ⁴ Da without producing α-CD.

At this stage, it is recommended to perform the enzymatic reaction at 55 to 65°C (preferably 60°C), preferably for 0.5 to 4 hours.

<Step (d): End of enzyme reaction>

This step is the process of boiling the reaction solution after the above step (c) to stop the enzyme reaction. By boiling the reaction solution for 10 to 30 minutes (preferably 20 minutes), the enzyme is inactivated and the reaction is stopped.

<Step (e): Freeze-drying>

This step is a process of freeze-drying the liquid modified starch after the above step (d). The modified starch of the present invention can be recovered in powder form through freeze-drying. Freeze-drying can be performed using a conventional method.

Through the above steps, the present invention was able to manufacture modified starch having a size of 1×10 ³ to 1×10 ⁴ Da, and it was confirmed that almost no disaccharides were detected even after 4 hours of reaction. The fact that no disaccharides were produced is a result confirming that the enzymatic reaction is focused on endo-type hydrolysis of starch and does not produce disaccharides including CD.

The present invention develops the starch hydrolysis reaction conditions of wild-type CGTase and confirms that a low-molecular-weight modified starch of 10 ³ to 10 ⁴ Da can be produced without producing a by-product CD. The low-molecular-weight modified starch produced in the present invention can be utilized as a starch food additive. As a preferred example, the present invention provides rice cake manufactured by adding the modified starch having a size of 1×10 ³ to 1×10 ⁴ Da manufactured by the method of the present invention to rice flour, adding water, kneading, and steaming. The rice cake manufactured including the modified starch of the present invention is refrigerated and dried to a moisture content of 40% or less and stored. When boiled in water for 5 minutes at the time of consumption, the rice cake exhibits the characteristics of a fast gelatinization rate compared to general rice cakes, a rapid decrease in hardness of about 20%, and thus retains its original chewiness. These characteristics are utilized in easy-to-cook foods, providing convenience by allowing people to easily cook and eat tteokbokki and other foods using only boiling water, even when there is no microwave.

In the rice cake of the present invention, it is preferable that the rice cake is manufactured by mixing 0.1 to 30 wt% of modified starch and 70 to 99.9 wt% of rice flour based on the weight sum of the modified starch and rice flour. In addition, it is preferable to add water to the 'modified starch and rice flour mixture' in an amount of about 50 to 60 wt% relative to the weight of the 'modified starch and rice flour mixture'. At this time, it is preferable to sprinkle the water. The rice cake of the present invention can be manufactured by steaming the dough.

Hereinafter, the contents of the present invention will be described in more detail through the following examples and experimental examples. However, the scope of the rights of the present invention is not limited to the following examples and experimental examples, but includes modifications of technical ideas equivalent thereto.

[Example 1: Production of low-molecular-weight modified starch using wild-type CGTase]

Corn starch (10%, w/v) was dissolved in sodium acetate buffer (50 mM, pH 6.0) and gelatinized. The gelatinized starch solution was preheated in a 60°C constant temperature shaker for 5 minutes, and cyclodextrin glucanotransferase (0.645 U/mg substrate) was added. After stirring at 60°C for a predetermined time (0.5, 1, 2, 3, and 4 hours respectively), the enzymatic reaction was stopped by boiling for 20 minutes. The liquid modified starch manufactured by the above method was freeze-dried at -40°C to manufacture a powder form. At this time, the measurement of the enzyme hydrolysis activity was defined as the amount of enzyme that decomposes 5.26 g of starch per hour according to the standard method for measuring α-amylase of Novozyme.

[Experimental Example 1: Measurement of molecular weight of modified starch manufactured in Example 1]

The enzyme-treated starch of Example 1 was sampled by time zone and analyzed by SEC (Size exclusion chromatography). First, the 10% concentration analysis sample was completely dissolved, diluted to 5 mg/mL with a mobile phase solvent, filtered, and used as an analysis sample. The flow rate was 0.5 ml/min, and the composition of the mobile phase was changed by time zone using 0.5% lithium bromide (TCI, Japan) and 50% dimethyl sulfoxide (JUNSEI, Japan), and the measurement was performed by a differential refractometer (RI detector). The results were calculated using Pullulan molecular weight standards (Shodex pullulan standard P-82, Showa Denko K.K., Tokyo, Japan; P-5, Mw=6.3 kDa; P-10, Mw=9.8; P-20, Mw=22 kDa; P50, Mw=49.4 kDa; P-100, Mw=106 kDa; P-200, Mw=201 kDa; P-400, Mw=334 kDa; P-800, Mw=642 kDa) and Dextran standard (Mw=6100 KDa).

Figure 1 shows the results of analysis of changes in the molecular weight of starch by enzyme treatment time. It can be seen that the position of the peak changes as the enzyme reaction time passes, and this change indicates that amylopectin is hydrolyzed into amylopectin cluster units by the enzyme cyclodextrin glucanotransferase (CGTase) reaction. In addition, it can be seen that the molecular weight decreases as hydrolysis occurs.

Figure 2 shows the SEC analysis results of starch 4 hours after enzyme treatment. It was confirmed that the produced modified starch had an average molecular weight of approximately 5×10 ³ Da.

[Experimental Example 2: Analysis of the small sugars in the reaction product of Example 1]

High-performance anion exchange chromatography (HPAEC) was used to analyze the amount of disaccharides produced after the enzymatic reaction of Example 1. A pretreatment process of the reactants is required before use. First, water equivalent to 5 times the amount of enzyme-treated starch was added and mixed well (vortexting). The well-mixed starch solution was centrifuged to collect the supernatant. The recovered supernatant was diluted 10-fold with water and filtered through a 0.45 μm disposable syringe filter (Advantec, Dublin, CA, USA). 20 μL of the analyte was injected into the column (CarboPac PA1 column, 4 × 250 mm; Dionex) and analyzed at a flow rate of 1 mL/min. The solvents used for the analysis were solvent A (150 mM NaOH) and solvent B (600 mM NaOH), and the content was adjusted by time period. The solvent content by time period was 0-10 min, 10-30% B; The analysis conditions were applied as follows: 10-16 min, 30-40% B; 16-27 min, 40-50% B; 27-32 min, 50-100% B; 32-42 min, 100-10% B.

Figure 3 shows the results of the analysis of small sugars in the enzyme reaction solution by time period. Although the enzyme reaction time was 4 hours, it was confirmed that the peak of small sugars was very small until 4 hours of reaction. It was confirmed that the enzyme reaction was focused on the endo-type hydrolysis of starch and did not reach the production of small sugars including CD.

[Example 2: Production of Garaetteok with the addition of low molecular weight modified starch produced in Example 1]

In order to manufacture garaetteok with the low-molecular-weight modified starch manufactured in the above Example 1, the low-molecular-weight modified starch manufactured in Example 1 was added to rice flour so that it was 0.1 to 30 wt% of the 'sum of rice flour and modified starch', and 50 to 60% of moisture was sprinkled based on the weight of the entire mixture (sum of rice flour and modified starch) and mixed to knead. The uniformly mixed dough was injected using steam at 100°C or higher for 10 to 30 minutes to steam, and then the dough was put into an extruder and shaped into garaetteok, then transferred to an aging plate, and after aging for 24 hours or more using a cooler, it was immersed in 80% alcohol, and then packaged.

It is hereby disclosed that the present invention is a result of the project "Development of an extraction process for starch-containing natural products" (research period: July 1, 2020 to January 31, 2021) supported by the "LINC+ Project of the National Research Foundation of Korea."

Claims (5)

Step (a) of adding 10% (w/v) corn starch to a 50 mM, pH 6.0 buffer, dissolving and gelatinizing;
Step (b) of placing the starch gelatinized in the above step (a) in a constant temperature stirrer at 60°C and preheating for 5 minutes;
Step (c) of producing modified starch having a size of 5×10 3 Da without producing α-cyclodextrin by adding 0.645 U/mg substrate of cyclodextrin glucanotransferase after preheating in the above step (b) and inducing a reaction by performing an enzymatic reaction at ⁶⁰ °C for 0.5 to 4 hours;
After the above step (c), a step (d) of boiling the reaction solution for 20 minutes to stop the enzyme reaction; and
A method for producing modified starch for making rice cakes, characterized in that it comprises a step (e) of freeze-drying the liquid modified starch at -40°C after the above step (d).
delete delete A rice cake manufactured by adding modified starch for rice cake manufacturing having a size of 5×10 ³ Da manufactured by the method of claim 1 to rice flour, adding water, kneading, and steaming.
In paragraph 4,
The above rice cake,
A rice cake characterized in that it is manufactured by mixing 0.1 to 30 weight% of modified starch and 70 to 99.9 weight% of rice flour based on the weight sum of modified starch for rice cake manufacturing and rice flour.