KR20090066542A - Method for production of starch with a slow digestibility - Google Patents

Abstract

A method for preparing starch with improved digestibility is provided to slowly increase the blood-sugar level after a meal for obese or diabetic patients by mixing starch and sugar and adding amylosucrase to the mixture. Starch and sugar are suspended in water to prepare a suspension. Amylosucrase(E.C. 2.4.1.4) or amylosucrase-containing liquid is added to the suspension in order to induce an enzyme reaction. The suspension is boiled. The starch is selected among waxy corn starch, corn starch, waxy rice starch, normal rice starch, waxy potato starch and potato starch. The amylosucrase is derived from Neisseria polysaccharea. The amylosucrase-containing liquid represents a culture medium of transformed strains or homogenate. The enzyme reaction is performed at 25~35°C.

Description

Method for producing starch with increased digestibility {Method for production of starch with a slow digestibility}

The present invention relates to a method for preparing starch having improved plasticization, and more particularly, to a method for preparing starch having improved digestibility by mixing starch and sugar, and then reacting with addition of amylosucrase. will be.

Starch is a storage polysaccharide found in plants such as cereals and papers, and is a major source of carbohydrates in humans. Starch is divided into nutritionally rapidly digested starch (RDS), slowly digestible starch (SDS) and refractory starch (RS). Localized starch is starch that is completely digested in the small intestine but is slow to digest, and indigestible starch is starch which is not digested in the small intestine but fermented by microorganisms in the large intestine.

Much research has been done on the physiological benefits of digestive starch and indigestible starch, which has the health effects of slowly increasing postprandial blood sugar levels, while indigestible starch has been shown to affect colorectal cancer, hyperglycemia and hypercholesterolemia. Prevents and interferes with the accumulation of fat.

On the other hand, foods that rapidly increase blood sugar levels cause excessive insulin secretion and repetition, which causes insulin resistance due to pancreatic overload, causing insulin resistance, which is a fat starch that slowly increases blood sugar levels. Or diabetes can be effectively used in the diet.

In order to increase the localizable and indigestible content of starch, physical treatment, chemical treatment and enzymatic treatment are usually used alone or in combination. Among these, enzymatic treatment is environmentally friendly, safe and health-oriented for consumers. In addition, since only specific reactions can be induced, we can induce only the reactions we want, yields are high, there are few by-products of the reactions, and there is an advantage that the purification for use after treatment is easy.

Therefore, it would be required to develop a technology that can increase the degree of localization of starch by using an enzymatic reaction.

Accordingly, an object of the present invention is to develop and provide a technique capable of increasing the degree of localization of starch using an enzymatic reaction.

In order to achieve the above object, the present invention comprises the steps of preparing a suspension by suspending starch and sugar in water; (B) adding amylosucrase (EC 2.4.1.4) or a liquid containing the same to the suspension of step (a) to induce an enzymatic reaction; It provides a method of manufacturing.

Hereinafter, the problem solving means of the present invention will be described in more detail.

Generally, starch hydrolase is mainly used for the enzymatic treatment used for starch. However, in the present invention, a different approach from conventional enzymatic treatment is Neiserria. Amylosucrase (EC 2.4.1.4) isolated from polysaccharea ) is used to enhance the degree of localization of starch. Amylosucrase hydrolyzes sugar into fructose and glucose, which binds glucose to the non-reducing end of the receptor to synthesize α- (1 → 4) -glucan. Starches whose length of the chain has been lengthened by the method of the present invention exhibit localization.

The experimental group prepared by adding amylosucrase (AS) to sugar after adding sugar to starch increased the amount of aliphatic starch than the control, about 25% in the starch and about 8% in the normal starch. Increased respectively.

In addition, the yield of insoluble fraction after AS reaction was about 15% higher than that of normal starch, and the sugar used as substrate was mostly consumed after 15 hours in waxy starch and 40 hours in general starch.

In addition, as a result of analyzing the degree of polymerization (DP) of amylopectin branch chains, the long chain (> DP 20) increased and the short chain (≤DP 20) decreased by AS treatment, and the overall molecular weight also increased.

In addition, the X-ray diffractogram of the AS-treated starch showed Form B, but no peak was seen in the control group, and the relative crystallinity was also higher in the AS-treated starch than the control group.

In addition, the control group did not show any peak in the thermal change characteristic analysis, but the aging peak appeared in the AS-treated starch, the gel strength of the AS-treated starch was higher than the gel strength of the raw starch.

This result means that the starch can be increased by treating the starch with AS.

On the other hand, in the present invention, since the starch has a non-reducing terminal, any one may be used, but examples thereof include waxy corn starch, corn starch, glutinous rice starch, non-glutinous starch, waxy potato starch and potato starch. Either one can be used.

On the other hand, amylosucrase (EC 2.4.1.4) used in the present invention can separate the sugar into fructose and glucose, and then bind the glucose to α- (1 → 4)-at the non-reducing end of the starch. It means that there is, and if the reaction can be carried out as described above, it is not limited to those derived from a specific microorganism, for example Neiserria polysaccharea ( Neiserria polysaccharea ) can be used.

On the other hand, in the present invention can be used a liquid containing amylosucrase in addition to the purified amylosucrase, for example, the culture medium or strain lysate of the transformed strain to produce amylosucrase is have. In the case of purified enzymes, the cost is high. However, if the gene encoding amylosucrase is introduced into a strain and transformed, the culture solution or the lysate of the strain can be recovered and used to reduce costs. Because it is. In this case, the culture medium of the strain is used to discharge amylosucrase out of the strain, and the use of the strain crushed solution is to accumulate the inside of the amylosucrase without discharging it to the outside.

On the other hand, the method for producing a branched starch of the present invention preferably comprises a step (a), preferably further comprising the step of boiling the suspension (a-1); by boiling the accessibility of the amylosucrase substrate Because it is improved.

On the other hand, in the present invention, the enzymatic reaction of step (b) is preferably carried out at 25 ~ 35 ℃, because the reactivity of amylosucrase at this temperature condition is good.

On the other hand, the method for producing a plasticized starch of the present invention, after the enzymatic reaction of step (b), preferably further comprising the step (c) of obtaining an insoluble portion by centrifugation of the reaction solution; This is because the starch contains Mars.

In the present invention as described above, the starch can be prepared by mixing the sugar with starch, amylosucrase is added to the reaction to increase the localization, starch is improved in the post-prandial blood sugar level Because it has a health effect of increasing slowly, it can be effectively used in the diet of obese or diabetic patients.

Hereinafter, the content of the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited only to the following examples, and includes modifications of equivalent technical ideas.

Examples 1-6, Comparative example  1-6, Comparative example  11-16: Preparation of Starch

Examples 1 to 6 (indicated as 'AS-treated starch' in the following experiments), Waxy corn (Example 1), General corn (Example 2), Glutinous rice (Example 3), Non-glutinous rice (Example 4), Waxy potato (Example 5), Starch with improved localization was prepared using a general potato (Example 6).

Aqueous starch solution (2%, w / w) and 100 mM sugar were suspended in 100 mM sodium citrate buffer (pH 6.0). The suspension was boiled vigorously for 10 minutes to increase the accessibility of the enzyme to the substrate.

Thereafter, the mixture was cooled in a 30 ° C. constant temperature water bath, 2000 U of amylosucrase was added per 30 mL of the total solution, and reacted at 30 ° C. for 40 hours. After the reaction, the water-soluble and insoluble portions were centrifuged (10,000 × g, 10 minutes).

Thereafter, the insoluble portion was washed several times with distilled water in order to increase the purity of the plasticizable starch in the insoluble portion, and finally washed once more with ethanol.

Thereafter, the precipitate was lyophilized and ground into a mortar and pestle to be used in the following experiment.

Meanwhile, as a comparative example (indicated as 'cooked starch' or 'gelatinized starch' in the following experiment), the preparation of the lyophilized starch was performed only by boiling the suspension for 10 minutes, and Comparative Examples 1 to 6 (Comparative Example) 1: waxy corn, comparative example 2: general corn, comparative example 3: glutinous rice, comparative example 4: non-glutinous rice, comparative example 5: glutinous potato, comparative example 6: general potato)

In addition, as another comparative example (indicated as 'control' or 'control' in the following experiment), all the same process as the process for preparing the lyophilized starch, but the addition of enzymes only Comparative Examples 11-16 (Comparative Example 11: Waxy Corn, Comparative Example 12: General Corn, Comparative Example 13: Glutinous Rice, Comparative Example 14: Non-glutinous Rice, Comparative Example 15: Waxy Potato, Comparative Example 16: General Potato)

On the other hand, amylosucrase may be used as a purified enzyme purchased, in the present embodiment was used to isolate and express in a microorganism as follows.

First, Neiserria Amylosucrase gene isolated from polysaccharea ) was expressed in E. coli. Enzymes were purified from cell extracts using Ni-NTA affinity chromatography. Enzyme titers are described in van der Veen et al. (Van der veen, Bar A., Potocki-Veronese, G., Albenne, C., Joucla, G., Monsan, P., Remaud-Simeon, M., 2004. Combinatorial engineering to enhance amylosucrase performance: construction, selection, and screening of variant libraries for increase activity.FEBS Letters 560, 91-97). That is, 0.1 mL of 4% sugar, 0.1 mL of 1% glycogen, 0.05 mL of diluted enzyme, 0.25 mL of 100 mM sodium acetate buffer (pH 6.0) were mixed and reacted at 30 ° C. for 10 minutes to release the released fructose. Quantification was carried out using the (dinitrosalicylic acid) method. At this time, 1 unit (U) of amylosucrase was defined as the amount of enzyme required to consume 1 μmole of sugar per minute.

Experimental Example  1: Example and Comparative Example  Starch RDS , SDS , RS ) content

The prepared starch, control, and AS-treated starch prepared in Examples and Comparative Examples, 'rapidly digestible starch (RDS)', 'slowly digestible starch' , SDS) 'and' resistant starch (RS) 'were measured.

For the classification of 'rapidly digestible starch (RDS)', 'slowly digestible starch (SDS)' and 'resistant starch (RS)', The procedure was performed.

First, 0.75 mL of 100 mM sodium acetate buffer (pH 5.2) and 0.75 mL of digestive enzyme solution were added to 30 mg of the starch, and digested at 37 ° C. for 10, 20, 60, 120, and 240 minutes, respectively. Thereafter, the reaction was stopped by boiling for 10 minutes, and glucose in the supernatant was measured by the GOD-POD method. This experiment was divided into starch (RDS) rapidly digested within 10 minutes, digestible starch (SDS) digested between 10 and 240 minutes, and indigestible starch (RS) not digested after 240 minutes. At this time, the digestive enzyme solution was added 2 g of pancreatin (pancreatin) in 24 mL of distilled water, stirred for 10 minutes, centrifuged (1,500 × g, 10 minutes) and the supernatant (20 mL) was 0.4 mL of amyloglucosidase ( amyloglucosidase) and 3.6 mL distilled water in a beaker containing mixed and prepared was used.

The result of experiment by the above method is shown in Table 1.

RDS, SDS and RS Content by Starch Type and State Gelatinized starch Control AS processed starch Starch RDS SDS RS RDS SDS RS RDS SDS RS Waxy corn 74.0 ± 2.5 7.5 ± 0.4 18.5 ± 2.8 53.4 ± 1.3 5.2 ± 1.5 41.4 ± 0.3 24.8 ± 1.3 30.2 ± 1.0 45.0 ± 0.9 Normal corn 72.4 ± 0.7 7.1 ± 0.5 20.5 ± 0.4 49.6 ± 0.1 3.8 ± 0.6 46.7 ± 0.6 43.2 ± 1.0 12.7 ± 0.8 44.2 ± 1.5 Glutinous rice 73.9 ± 2.1 7.8 ± 1.7 18.4 ± 0.9 54.6 ± 0.7 4.9 ± 1.2 40.5 ± 0.6 21.2 ± 1.0 29.1 ± 1.2 49.8 ± 1.7 Normal rice 73.4 ± 1.6 6.5 ± 1.8 20.1 ± 2.9 52.7 ± 1.6 4.7 ± 1.2 42.6 ± 0.4 41.4 ± 1.5 12.4 ± 0.6 46.2 ± 1.3 Waxy potato 73.1 ± 2.0 4.1 ± 2.9 22.8 ± 1.3 50.0 ± 1.2 4.4 ± 1.4 45.6 ± 0.8 27.1 ± 1.6 24.2 ± 0.8 48.7 ± 1.7 Normal potato 70.6 ± 1.8 3.9 ± 1.2 25.4 ± 1.5 50.9 ± 1.1 3.7 ± 1.4 45.5 ± 0.9 37.2 ± 0.9 10.2 ± 2.0 52.6 ± 1.8

The RDS content of gelatinized starch showed a very high digestibility of 70% or more because the crystal structure of raw starch particles was destroyed during gelatinization. The control group showed RDS 50% and RS 40% because the digestibility of gelatinized starch decreased due to aging during the enzyme reaction time. RS content of AS-treated starch appeared similar to the control group, but RDS decreased and SDS increased.

In particular, the SDS content in the waxy starch (waxy starch) is about 25%, 8% in the normal starch (normal starch) higher than the control group in the increase in the SDS content, it can be seen that the difference between the starch and the normal starch.

Experimental Example  2: measuring digestibility over time

Digestion rate of the gelatinized starch, the control group, and the AS-treated starch from 10 to 240 minutes was investigated. The results were as shown in FIG. AS-treated starches showed slower digestion than gelatinized starches and controls. Gelatinized starch showed high digestibility of more than 70% in 10 minutes and the control group was digested more than 50%, and showed similar digestibility even after 240 minutes.

On the other hand, the starch of AS-treated starch digested about 20% by 10 minutes, gradually increased to about 40% by 120 minutes, and slightly over 50% by 240 minutes. Digestion of about 40% and gradual increase of about 50% until 240 minutes were observed, but the starch and normal starch showed different patterns.

Experimental Example  3: AS Analysis of insoluble and water soluble components of starch after treatment

In this experimental example, after the AS treatment, the components of the insoluble part and the water-soluble part of the starch were analyzed.

Analysis of the water soluble portion after AS treatment was performed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The column used was a 'Carbo-pack PA 1 anion-exchange column (4 × 250 mm, Dionex, Sunnyvale, CA, USA). 0.1 mL of the aqueous portion was filtered through a 0.45 μm membrane filter (Millipore) and then injected. The chromatography was allowed to flow 150 mM NaOH at 1 mL / min and the eluent was 600 mM sodium acetate (0-5 minutes, increased from 0 to 20%; 6-30 minutes, increased from 20 to 45%; 31-60 minutes, 45 Up to 55%; 61-80 minutes, 56 to 60%; 81-90 minutes, 61 to 65%; 91-95 minutes, 66 to 80%; 96-100 minutes, 81 to 100 Separated by%).

Table 2 below shows the yield of the insoluble portion and the component content of the water-soluble portion after the reaction, and the concentrations of fructose and sugar were analyzed by HPAEC-PAD.

Yield of insoluble portion and component content of water soluble portion Starch yield(%) Fructose (mM) Sugar (mM) AS / Raw Waxy corn 68.0 ± 1.3 96.8 ± 0.3 3.4 ± 0.1 1.8 ± 0.0 Common corn 52.7 ± 2.2 92.4 ± 0.1 6.1 ± 0.3 1.4 ± 0.1 Glutinous rice 69.1 ± 1.5 96.3 ± 0.2 2.5 ± 0.1 1.9 ± 0.0 Rice 57.1 ± 2.7 88.4 ± 0.6 7.7 ± 1.1 1.5 ± 0.1 Glutinous 66.7 ± 2.7 98.8 ± 0.5 2.3 ± 0.1 1.8 ± 0.1 General potato 50.7 ± 2.3 90.8 ± 0.4 7.1 ± 0.3 1.4 ± 0.1

After 40 hours of AS reaction, only fructose and sugar were present in the water-soluble fraction, indicating that only chain elongation of starch occurred. The initial sugar concentration was 100 mM and was consumed during the reaction, leaving approximately 2 to 3 mM of starch and 6 to 7 mM of normal starch. Since more sugar was used as a substrate for the chain elongation reaction, more chain elongation occurred in the starch, and as the sugar consumption increased, the concentration of fructose released by the hydrolysis of sugar increased. did. Therefore, the fructose concentration of the starch was higher than that of the general starch.

On the other hand, in Table 2, the yield was calculated by the following equation, which was about 15% higher than normal starch in the waxy starch.

In Table 2, the AS / Raw value is a ratio of the weight of starch before and after the reaction, which is a value that can be seen how many times after the reaction based on starch alone except for sugar. The starch increased about 2 times, and about 1.5 times for normal starch.

Experimental Example  4: sugar consumption analysis

The consumption of sugar and the production of fructose over time were measured by HPAEC-PAD and are shown in FIG. 3. In glutinous rice starch, sugar was almost consumed for 15 hours, but in nonglutinous rice starch, 93% of sugar was consumed for 40 hours. From this, AS reaction occurred more rapidly in glutinous rice starch. Similarly, fructose production was faster in glutinous rice.

Experimental Example  5: Amylosukrases  Investigation of chain length distribution by treatment

The chain length distribution of amylopectin affects the gelatinization temperature, enthalpy change and aging of starch (Jane, J., Chen, YY, Lee, LF, McPherson, AE, Wong, KS, Radosavljevic, M., and Kasemsuwan, T., 1999.Effects of Amylopectin Branch Chain Length and Amylose Content on the Gelatinization and Pasting Properties of Starch.Cereal Chemistry 76 (5), 629-637), Chain length distribution with HPAEC-PAD by cutting the branches of starch before and after AS treatment I looked at it.

In order to cut the branches of starch, an enzyme that hydrolyzes α-1,6 bond called isoamylase (Sigma) was used.

Starch (10 mg) was dissolved in 90% DMSO (0.5 mL), heated for 15 minutes, and distilled water (0.5 mL) was added. 0.49 mL of 0.2 M sodium acetate buffer (pH 3.5) was mixed with 0.5 mL of this solution, 0.01 mL of isoamylase was added thereto, and the mixture was reacted at 40 ° C for 16 hours. After boiling to stop the reaction, the mixture was centrifuged at 10,000 × g for 10 minutes and the supernatant was filtered through a 0.45 μm filter. The sample thus prepared was analyzed under the same conditions as described above. DP values from 1 to 7 were determined using a mixture of maltooligosaccharides (DP1-7, Sigma).

Experimental results were shown in Figure 3, where the chain length distribution was expressed as a percentage of the total peak area (Hanashiro, I., Abe, J., Hizukuri, S., 1996. A periodic distribution of the chain length of amylopectin as revealed by high-performance anion-exchange chromatography.Carbohydrate Research 283, 151-159).

Starch chain length increased after AS treatment. There were many short chains below DP20 before AS, but more long chains above DP20 after AS.

Table 3 divides the chain distribution by DP and summarizes the sum of the relative areas. The grouping by DP was Hanshiro (Hanashiro, I., Abe, J., Hizukuri, S., 1996. A periodic distribution of the chain length of amylopectin as revealed by high-performance anion-exchange chromatography.Carbohydrate Research 283, 151 -159).

Relative area and measurable longest DP depending on chain distribution Starch Relative area (%) Longest measurable DP DP6 ~ 12 DP13 ~ 24 DP25 ~ 36 ≥DP37 Glutinous rice Raw starch 38.0 50.6 8.9 2.5 52 AS processed starch 1.2 32.2 43.3 23.3 71 Rice Raw starch 33.7 52.9 10.0 3.5 54 AS processed starch 3.6 38.6 37.9 19.9 70 Glutinous Raw starch 27.1 55.9 11.5 5.5 59 AS processed starch 1.9 28.4 47.5 22.1 73 General potato Raw starch 24.6 55.5 13.4 6.5 55 AS processed starch 1.9 32.7 45.3 20.0 68

The short chain (DP6 ~ 12) was 24.6 ~ 38.0% and the long chain (≥DP37) was 2.5 ~ 6.5% in the raw starch, but the short chain (1.2 ~ 3.6% and long chain 19.9 ~ 23.3%) in the AS starch. . The largest portion of raw starch was DP13 ~ 24, while AS starch was DP25 ~ 36. The longest chain that could be measured was DP52 ~ 59 in raw starch, but DP68 ~ 73 in AS starch and increased DP15 after AS treatment.

Experimental Example  6: HPSEC - MALLS - RI  System AS Investigation of molecular weight change before and after treatment

The high molecular weight chromatographic multi-angle laser light scattering detector (HPSEC-MALLS-RI) was used to investigate the change of molecular weight according to AS treatment.

The sample (25 mg) was dissolved in 5 mL of DMSO, boiled for 15 minutes, and 25 mL of ethanol was added to precipitate starch. After centrifugation at 10,000 × g for 10 minutes, the precipitated starch is dissolved again in 5 mL 100 mM NaNO ₃ , boiled for 10 minutes, filtered in a 5.0 μm membrane filter (Millipore) and injected into the HPSEC-MALLS-RI system. It was. The concentration of the final filtered starch solution was measured by the phenol-sulfuric acid method.

The model name of the HPSEC-MALLS-RI system is' PU-2080 Plus (Jasco, Tokyo, Japan) ',' 200μL injector loop ',' degasser (NO-OX Vacuum Station, Alltech, Deerfield, IL, USA) ',' Differential refractive index detectors (Opti-Lab, Wyatt Technology, Santa Barbara, Calif., USA) and Shodex OH-Pak 804 and 806 columns (Showa Denko, Tokyo, Japan) were used. The mobile phase of the chromatography was NaNO ₃ (100 mM) with a flow rate of 0.4 mL / min and data was stored in the 'DAWN DSP / OptiLab' system and calculated using 'ASTRA software (Version 4.09.07, Wyatt Technology)'. .

The experimental results are shown in Table 4. The molecular weight of amylopectin can range from millions to hundreds of millions depending on the dissolution method or measurement technique (Park, JH, Kim, HJ, Kim, YH, Cha, HJ, Kim, YW, Kim, TJ, Kim YR, Park KH, 2007). The action mode of Thermus aquaticus YT-1 4-a-glucanotransferase and its chimeric enzymes introduced with starch-binding domain on amylose and amylopectin.Carbohydrate Polymers 67, 164-173), It was.

Starch M _w (× 10 ⁷ g / mol) Glutinous rice Raw starch 9.59 ± 1.44 AS processed starch 13.79 ± 2.46 Rice Raw starch 3.53 ± 0.51 AS processed starch 10.21 ± 1.95 Glutinous Raw starch 5.13 ± 0.72 AS processed starch 10.36 ± 1.76 General potato Raw starch 2.53 ± 0.25 AS processed starch 4.81 ± 0.68

The initial molecular weight of the starch was 2.53 ~ 9.5 × 10 ⁷ g / mol, and the AS-treated starch was 4.81 ~ 13.79 × 10 ⁷ g / mol, because the molecular weight was increased by the chain extension by the enzymatic reaction.

RI (refractive index) chromatogram of non-glucose starch can be divided into FI, which occupies mainly amylopectin, and FII, which mainly occupies amylose and intermediate molecules. It was found that (Fig. 4).

In addition, it can be seen from Table 5 that the molecular weight increase of the FI is very large, but the molecular weight increase of the FII is not large, and it can be seen that the chain extension reaction of AS occurred more than amylose in amylopectin.

M _w (× 10 ⁷ g / mol) Non-glutinous starch FI FII Raw starch 5.10 ± 0.73 1.40 ± 0.14 AS processed starch 13.75 ± 2.72 2.57 ± 0.31

Experimental Example  7: AS X-ray before and after treatment Diffractogram  Pattern analysis

X-ray before and after AS treatment using 'X-ray diffractometer (Model D5005, Bruker, Karlsruhe, Germany) (analysis parameters: 40 KV and 40 mA, CuK radiation λ = 0.15406, time constant = 4 sec, nickel filter)' The diffractogram pattern was analyzed. The sample was measured from 2 ° value 3 ° to 30 °. Relative crystallinity was calculated by the following equation.

5 shows the X-ray diffraction pattern of the control group before and after the AS treatment. The raw starch of glutinous and non-glutinous rice showed typical type A with peaks at 15, 17, 18 and 23 °, but changed to type B after AS treatment.

The raw starch of glutinous and ordinary potato showed typical type B with peaks at 5.5, 15, 17, 22, and 24 °, and type B, although peak intensity was weakened after AS treatment. On the other hand, no peaks were seen in all the controls, because they lost crystallinity during the heating process.

All starch treated AS showed type B regardless of the starch type, which showed the crystal structure of type B due to starch aging during chain elongation and enzymatic reaction. In the case of starch gel aged at room temperature, there was a study showing B-type crystal structure (Eerlingen, RC, Crombez, M., Delcour, JA, 1993. Enzyme-resistant starch.I. Quantitative and qualitative influence of incubation time and temperature of autoclaved starch on resistant starch formation.Cereal Chemistry 70 (3), 339-344; Kim et al., 1997.Acomparative study on retrogradation of rice starch gels by DSC, X-ray and alpha-amylase methods.Starch / St? rke 49 (Nr.2) S71-75), starch treated with AS in this experiment is a condition similar to that of the previous studies, considering the form of gel formed during the enzymatic reaction and the reaction temperature (30 ° C) similar to room temperature. It could be explained that the crystal structure of the type was shown.

On the other hand, the relative crystallinity is shown in Table 6, the control group showed a lower relative crystallinity than raw starch, but the AS-treated starch showed a high degree of crystallinity similar to raw starch. From this, it can be said that the stretched chain of AS treated starch reduces the amorphous region and increases the crystalline region. In addition, the formation of higher relative crystallinity can be attributed to the stronger and more dense structure of the AS-treated starch than the control (Cooke, D., Gidley, MJ, 1992. Loss of crystalline and molecular order during starch gelatinisation: origin of the enthalpic transition.Carbohydrate Research 227, 103-112).

Starch Raw starch (%) Control (%) AS (%)  Glutinous rice 48.1 10.7 47.5  Rice 40.7 18.6 39.1  Glutinous 49.1 11.4 44.4  General potato 46.0 19.6 40.0

Experimental Example  8: Differential Scanning Calorimeter  Used AS Changes in thermal properties before and after treatment

When the starch is heated in a differential scanning calorimeter, the crystallized amylopectin structure is released, showing peaks with enthalpy changes. The DSC (Diamond DSC, Perkin-Elmer, USA) was used to investigate the thermal characteristics before and after AS treatment. . Indium was used as a calibration standard, and 3 mg of sample and 15 mg of distilled water were placed in a Seiko high pressure stainless steel pan and placed at room temperature for one day. The sample pan was measured at a rate of 5 ° C./min from 30 ° C. to 130 ° C.

As a result, the thermal properties of raw starch, control group, and AS-treated starch are shown in Table 7.

Starch To (℃) Tp (℃) Tc (℃) Tc-To ΔH (J / g) Glutinous rice Raw starch 58.3 ± 0.1 64.9 ± 0.2  76.0 ± 0.4 17.6 ± 0.3 11.6 ± 0.2 AS processed starch 75.6 ± 0.5 88.7 ± 0.1 100.1 ± 0.4 24.4 ± 0.8  7.3 ± 0.5 Control Undetected Rice Raw starch 55.8 ± 0.7 62.6 ± 0.1  76.5 ± 0.1 20.7 ± 0.7  9.8 ± 0.2 AS processed starch 73.7 ± 0.8 88.8 ± 0.2 101.8 ± 1.0 28.1 ± 0.9  5.3 ± 0.3 Control Undetected Glutinous Raw starch 61.0 ± 0.2 65.7 ± 0.2  70.2 ± 0.2  9.1 ± 0.4 19.2 ± 1.5 AS processed starch 70.8 ± 0.4 88.8 ± 0.2 104.2 ± 0.5 33.4 ± 0.4  9.0 ± 0.5 Control Undetected General potato Raw starch 56.1 ± 0.4 59.9 ± 0.1  64.3 ± 0.3  8.2 ± 0.7 18.4 ± 0.7 AS processed starch 80.4 ± 0.6 89.3 ± 1.3 102.0 ± 0.1 21.6 ± 0.5  7.6 ± 0.5 Control Undetected

Raw starch showed peaks showing gelatinization characteristics. The control and AS-treated starches are samples that have already aged during the manufacturing process and then aged at 30 ° C for 40 hours. Therefore, the control and starch-treated starches should show peaks indicating aging characteristics. The degree is larger. Aging in the control group was judged to have no peak because the degree of aging was small enough not to be measured.

On the other hand, starch treated with AS showed an increased degree of aging due to increased chain length (Kalichevsky, MT, Orford, PD, and Ring, SG, 1990. The retrogradation and gelation of amylopectins from various botanical sources.Carbohydr.Res. 198, 49-55; Yuan, RC, Thompson, DB, and Boyer, CD, 1993. Fine structure of amylopectin in relation to gelatinization and retrogradation behavior of maize starches from three wx-containing genotypes in two inbred lines.Cereal Chem. 70 , 81-89.ichevsky et al., 1990; Yuan et al.), And other researchers reported changes in enthalpy of starch aged 2J / g and Tp 50 degrees under similar conditions (Kim). et al., 1997. Acomparative study on retrogradation of rice starch gels by DSC, X-ray and alpha-amylase methods.Starch / St®rke 49 (Nr. 2) S71-75; Kohyama, K., Matsukia, J. , Yasuib, T., Sasakia, T., 2004.A differential thermal analysis of the gelatinization and retrogradation of wheat starches with different amylopectin chain lengths.Carbohydrate Polymers 58, 71-77). In contrast, the AS-treated starch of this experiment showed a high enthalpy change of about 5-9 J / g and a Tp of about 88 degrees. This means that the crystal structure of AS-treated starch is more stable than other aged starches or controls. In general, amylose crystals are more stable than amylopectin (Shin, SI, Choi, HJ, Chung, KM, Hamaker, BR, Park, KH, Moon, TW, 2004. Slowly digestible starch from debranched waxy sorghum starch: preparation and properties Cereal Chemistry 81 (3), 404-408), the amylopectin chains with very long chains are similar to those of amylose. Could.

Experimental Example  9: AS Before and after treatment Rheological  Characteristic investigation

Rheological properties of starch gels were measured using 'oscillatory rheometer (Rheostress 1, Thermo HAAKE, Karlsruhe, Germany)' and 'corn-plate system (35mm diameter)'. First, starch suspension 2, 4, 6% was heated by high pressure at 121 ℃ for 15 minutes. The sample was placed on a rheometer and placed at 25 ° C. for one hour to form a gel and measured. Frequency range was 0.1 ~ 10Hz, it was carried out at 25 ℃.

6A and 6B are graphs showing changes in rheological properties before and after AS treatment. It was measured at 25 ° C. after gel formation with 2, 4, and 6% starch concentration, and showed a change in G ′ (storage rate) and G ”(loss modulus) when the frequency was changed from 0.1 to 10 Hz.

Modulus value increased with increasing starch concentration in all starches. In addition, the gel formation ability was improved by increasing all modulus values of starch after AS treatment. This is because elongated amylopectin chains exhibit similar properties as linear amylose with good gel-forming ability (Rolland-Sabate, A., Colonna, P., Potocki-Veronese, G., Monsan, P., Planchot, V.). , 2004.Elongation and insolubilisation of α-glucan by the action of Neisseria polysaccharea amylosucrase.Journal of Cereal Science 40, 17-30).

Raw starch had a solid form (G '> G ") in all starches at 6%, while solid starches in 2% of AS-treated starches.

Therefore, the AS-treated starch may represent the gel-forming ability of the raw starch even in a smaller amount than the raw starch, so the AS-treated starch was determined to be more efficient in terms of gel-forming ability.

1 shows in vitro digestibility of various starch types.

2 shows the consumption rate of sugar.

3 shows the chain length distribution of starch.

Figure 4 shows a refractive index (RI) chromatogram of non-glucose starch.

5 shows the X-ray diffractogram pattern of starch.

6a and 6b show the change in rheological properties of starch. 6a is for glutinous rice and non-glutinous rice starch, and 6b is for glutinous and regular potato starch.

Claims (7)

(A) suspending starch and sugar in water to prepare a suspension; (B) adding amylosucrase (EC 2.4.1.4) or a liquid containing the same to the suspension of step (a) to induce an enzymatic reaction; Manufacturing Method The method of claim 1, The starch is, Method of producing a starch enhanced starch, characterized in that any one selected from waxy corn starch, corn starch, glutinous rice starch, non-glutinous rice starch, glutinous potato starch and potato starch The method of claim 1, Amylosucrase, Neiserria polysaccharea ) method of producing starch with enhanced plasticity , characterized in that derived from The method of claim 1, The liquid containing amylosucrase, A method of preparing starch-enhanced starch, characterized in that the culture medium or strain lysate of the transformed strain to produce amylosucrase The method of claim 1, The method for producing a branched starch, After step (a), the step of boiling the suspension (a-1); a method of producing a starch enhanced starch, characterized in that it further comprises The method of claim 1, The enzyme reaction of step (b), Process for producing starch enhanced starch, characterized in that carried out at 25 ~ 35 ℃ The method of claim 1, The method for producing a branched starch, After the enzymatic reaction of step (b), centrifugation of the reaction solution to obtain an insoluble portion;

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