KR101693477B1 - Method for production of slowly digestible starch with alpha amylase - Google Patents

Abstract

The present invention relates to a method for producing a starch-digestible starch, which comprises treating an alpha amylase having an amino acid sequence of SEQ ID NO: 1 in starch, wherein the alpha amylase having an amino acid sequence of SEQ ID NO: It is possible to easily produce the starch-decomposable starch.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method of producing starch,

More particularly, the present invention relates to a method for producing a digestible starch using an alpha amylase having an amino acid sequence of SEQ ID NO: 1.

Starch is a storage polysaccharide contained in plants such as grains, papers, etc., and is a major source of human carbohydrates. Starch is divided into rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS). The hydrolyzable starch is a starch which is completely digested in the small intestine but has a slow digestion rate and the indigestible starch is a starch which is not digested in the small intestine but fermented by microorganisms in the large intestine.

Typical starches are converted to glucose in the body and are rapidly absorbed and then used or stored as energy. Such rapid digestion is accompanied by the problem of not feeling enough satiety and increasing the weight easily.

However, since the fatigueable starch has a slow rate of reaching the colon through the stomach and small intestine, the duration of satiety is long. In addition, it prevents blood sugar from rising sharply and is good for managing diabetes and prevents absorption of cholesterol, thereby preventing metabolic diseases such as hyperlipidemia.

In addition, foods that rapidly increase blood sugar levels cause excessive insulin secretion and repetition, resulting in 'insulin resistance' that does not play a role in insulin secretion due to overload of the pancreas. However, And diabetic patients.

On the other hand, as a method for producing the hydrogenated starch, a physical treatment, a chemical treatment and an enzymatic treatment are usually used alone or in combination. Enzymatic treatment is eco-friendly and safe and health-oriented for consumers. In addition, since only a specific reaction is performed, it is possible to induce only the desired reaction, and the yield is high, and the byproduct of the reaction is small.

Japanese Patent Registration No. JP 04945096 (registered on March 30, 2012) discloses a method for producing an indigestible dextrin, which comprises treating an aqueous solution of dextrin with glucoamylase and then treating glucose isomerase Is described. Korean Registered Patent No. 10-1426805 (Registered on Apr. 31, 2014) discloses a method for preparing a modified starch comprising: (a) preparing a suspension of modified starch modified by cross-linking; And (b) heat treating the suspension of the modified starch at a temperature of 130 to 180 ° C., wherein the heat treatment time is at least 20 seconds when the heat treatment temperature is 130 ° C. to 140 ° C. and the heat treatment temperature is 140 ° C. And the heat treatment time is at least 10 seconds when the temperature is from -30 to 180 DEG C. The present invention also relates to a process for producing an indigestible starch having improved processability.

It is an object of the present invention to develop and provide a method for producing a digestible starch by a simple and economical method.

In order to achieve the above object, the present invention provides a method for producing a digestible starch, which comprises hydrolyzing an alpha amylase having an amino acid sequence of SEQ ID NO: 1 into starch.

The hydrolyzed reaction product was obtained by treating the starch with the enzyme having the amino acid sequence of SEQ ID NO: 1 of the present invention, and the degree of the reaction of the alpha amylase was measured. As a result, the amino acid sequence of SEQ ID NO: It was confirmed that the enzyme-treated sample had a slower reaction rate than the control sample. In other words, it was confirmed that starch (RDS), which is easy to digest, was converted into the digestible starch (SDS) due to the enzyme treatment of the present invention.

In addition, since the enzyme having the amino acid sequence of SEQ ID NO: 1 of the present invention has a very fast reaction time and exhibits an optimum reaction rate at room temperature (around 20 DEG C), it is advantageous to economically produce the digestible starch in a short time have.

On the other hand, in the present invention, the alpha amylase is preferably Bifidobacterium ronggeom sub speaker system ronggeom JCM 1217 (Bifidobacterium longum subsp. longgum JCM 1217) (KCTC 3127).

In the present invention, the treatment of the alpha amylase is preferably performed at a temperature of 10 to 25 DEG C and a pH of 4.5 to 5.5.

According to the present invention, the starch can be hydrolyzed by treating with alpha amylase having the amino acid sequence of SEQ. ID.

In addition, the present invention is capable of producing the digestible starch in a short time at room temperature, and is very economical.

1 is a map of a recombinant vector into which an insert DNA of the present invention ( bllj- 0710) is inserted.
FIG. 2 is an SDS-PAGE electrophoresis image of purified amorphous alpha amylase (BiLA) enzyme.
Figure 3 shows TLC results of various amylases treated with the invention alpha amylase (BiLA) enzyme. (A) is a mixture of maltose (G2), maltotriose (G3), maltotetraose (G4), maltoheptaose (G7), alpha- cyclodextrin CD, beta-cyclodextrin, beta-CD, gamma-cyclodextrin, amylose, amylopectin, soluble starch, (pullulan)}, and (B) is a TLC result for p NPG5.
FIG. 4 is a graph showing the results of an experiment for confirming the optimal reaction temperature and pH of the present invention alpha amylase (BiLA) enzyme. (A) is the result of the experiment confirming the optimal reaction temperature of the BiLA enzyme, and (B) is the experiment result of confirming the optimum pH of the reaction of the BiLA enzyme.
FIG. 5 shows the results of confirming the reactivity ( k _cat / K _m ) of the present invention alpha amylase (BiLA) enzyme according to the substrate. (A) is soluble starch, (B) is amylopectin, (C) is amylose, (D) is corn starch, (E) is potato starch, .
Figure 6 shows the results of confirming the starch resolution of the alpha amylase (BiLA) enzyme of the present invention. 'control' is the BiLA enzyme-free group (control group), and 'rxn' is the BiLA enzyme-treated group.
Figure 7 shows the HPAEC results of starch solutions treated with the invention alpha amylase (BiLA) enzyme.
Figure 8 is the HPAEC results for the reaction product obtained by ethanol precipitation of the starch solution treated with the present invention alpha amylase (BiLA) enzyme.
FIG. 9 is a result of confirming the reactivity ( k _cat / K _m ) of the alpha amylase to the reaction product obtained by ethanol precipitation of the starch solution treated with the alpha amylase (BiLA) enzyme of the present invention. (A) is the result for the control group, (B) is the result for the present invention '20 minute sample treated with Alpha Amylase (BiLA) enzyme', and (C) .

Hereinafter, the structure of the present invention will be described in detail with reference to the following examples. However, the scope of the present invention is not limited to the following embodiments, and includes modifications of equivalent technical ideas.

[ Example  One: Bifidobacterium Long Sword Subspecies Long Sword JCM  1217 ( Bifidobacterium  longum subsp . longum JCM  1217) ( KCTC  3127) strain-derived alpha amylase gene Cloning  And tablets]

In this embodiment, Bifidobacterium ronggeom sub speaker system ronggeom JCM 1217 (Bifidobacterium longum subsp . longum Cloning alpha-amylase gene (bllj -0710) from JCM 1217) (KCTC 3127) strains, which was purified to.

(1) Polymerase chain reaction (PCR)

Bifidobacterium lematopsis subtilis JCM 1217 ( Bifidobacterium longum subsp. longum JCM 1217) were used to amplify the alpha amylase of the present invention.

5 μL of 10X EX Taq buffer, 4 μL of dNTP, 1 μL of a forward primer (see Table 1), 1 μL of a reverse primer (reverse primer, Table 1 below), DNA (KCTC 3127, Bifidobacterium longum subsp. longum JCM 1217) and 0.5 μL of EX Taq (total 50 μL) at 98 ° C. for 1 minute and then incubated at 98 ° C. for 10 seconds, at 55 ° C. for 30 seconds, at 72 ° C. for 1 minute and 30 seconds PCR was performed 30 times in total.

Forward primer
(forward primer) 5'-GTC GAC TTG GTT GTA AGC ATG GAT TCG-3 ' Reverse primer
(reverse primer) 5'-AAG CTT TCA GTA TTG ATA CGC TGT ACT GCC-3 '

(2) Purification of PCR products

After the above PCR reaction, the PCR mixture was added with 5 times as much volume of BNL buffer, and then subjected to voltexing. 1.5 times as much isopropanol as that of the BNL buffer was added, and the mixture was pipetted several times. gave. Then, it was transferred to a spin column and centrifuged at 11,000 rpm for 1 minute. Then, the separated lower layer was discarded, 700 μL of washing buffer was added, and the mixture was centrifuged at 11,000 rpm for 1 minute. Thereafter, the separated submerged solution was removed and centrifuged again at 11,000 rpm for 1 minute in the absence of any solution. After that, the upper part of the spin column was transferred to a microcentrifuge tube, and 30 μL of sterilized water was added. The sample was allowed to stand for 1 minute and then centrifuged at 11,000 rpm for 1 minute. Thereafter, the concentration was measured by nanodrop, and it was confirmed that the concentration of produced PCR product ( bllj- 0710) was 157.1 ng / μL.

(3) Restriction enzyme treatment

28 μL of DNA (bllj -0710) obtained above as a PCR product, 1.5 × K buffer, 7.5 μL, sterile water 10.5 μL, Sal I and 2 μL of Hind III was reacted at 37 ° C for 2 hours. As the vector, p6xHTKNd was used for restriction enzyme treatment in the same manner as described above. At this time, 1 μL of CIAP was added to the vector again and reacted at 37 ° C. for 1 hour.

(4) Gel extraction

5 μl of loading buffer was added to each of the insert DNA ( bllj- 0710) and vector (p6xHTKNd) treated with restriction enzymes and ligated to each other with 50% agarose gel using 1.2% agarose gel. V. Then, the band appeared by electrophoresis was cut and placed in a sterile microcentrifuge tube and weighed. Then, the BNL buffer, which is three times the measured weight, was vortexed and dissolved in a heating block at 55 ° C for 10 minutes. To this was added isopropanol, which is one time the weight of the gel, and pipetted several times. Then, each was transferred to a spin column and centrifuged at 11,000 rpm for 1 minute to remove the separated lower layer. Then, 700 μL of wash buffer was added, and centrifugation was performed at 11,000 rpm for 1 minute to remove the separated lower layer and centrifuged once more. Then, the upper portion of the spin column was transferred to a microcentrifuge tube, and 30 μL of sterilized water was added thereto. After allowing to stand for 1 minute, it was further centrifuged at 11,000 rpm for 1 minute. As a result of measurement of the concentration, it was confirmed that the inserted DNA was 11.47 ng / μL and the vector was 135.7 ng / μL.

(5) Ligation and transformation

Ligation (experimental), and self-ligation (self-ligation, control). In the ligation mixture, the concentration of insert DNA and vector should be at least three times higher than that of insert DNA (91.76 ng / μL), 1 μL of a 5-fold diluted vector (27 ng / μL), 9 μL of ligase .

On the other hand, the control self-ligation mixture was prepared by adding 1 μL of a 5-fold diluted vector, 8 μL of sterilized water and 9 μL of ligase instead of insert DNA.

The ligation mixture and the self-agitation mixture prepared above were reacted at 16 ° C for 30 minutes to prepare recombinant vectors, respectively (see FIG. 1). 1 is a map of a recombinant vector into which insert DNA ( bllj- 0710) prepared in the present invention is inserted.

On the other hand, Escherichia coli was transformed with the above recombinant vector using the CaCl ₂ method. 1 μL each of the ligation mixture and the self-ligation mixture was added to 200 μL of each competent cell (MC1061), and the mixture was reacted for 30 minutes on ice, 2 minutes on ice and 2 minutes on ice. Then, 800 μL of LB medium was added, and the mixture was incubated at 37 ° C. for 1 hour, followed by centrifugation at 5,000 × g for 1 minute. Then, 800 μL was discarded and the remaining 200 μL was plated on kanamycin medium and cultured at 37 ° C for 24 hours.

(6) Recovery of the present invention alpha amylase enzyme from transformed E. coli

Escherichia coli inserted with the lygated vector (experimental group) was inoculated into 1 L of LBK medium and cultured at 37 ° C for 20 hours at 200 rpm. Cells were recovered using a centrifuge. The recovered cells were resuspended in 50 mM NaOAc buffer solution at pH 6.0 and then subjected to cell disruption using ultrasound. The disruption obtained from the cell disruption was centrifuged at 11,000 rpm for 20 minutes, and the separated supernatant was taken and adsorbed on a Ni-NTA column to obtain the alpha amylase enzyme of the present invention. Thereafter, the separated enzyme was confirmed by electrophoresis (FIG. 2). 2 is an SDS-PAGE electrophoresis photograph of the separated enzyme of the present invention.

The thus isolated enzyme of the present invention was found to have the amino acid sequence of SEQ ID NO: 1, which will be referred to as 'BiLA enzyme' in the present invention.

[ Example  2: invention BiLA  Reaction test of enzyme by substrate]

In this Example, the reactivity of the BiLA enzyme according to the present invention was examined.

(maltose, G2, maltotriose (G3), maltotetraose (G4), maltoheptaose (G7), alpha- alpha Cyclodextrin, alpha-CD, beta-cyclodextrin, gamma-cyclodextrin, gamma-CD, amylose, amylopectin, , Soluble starch, pullulan) and 0.5 unit / mg of BiLA enzyme were added to the reaction mixture at 45 ° C for 12 hours, and thin layer chromatography (TLC) was performed. A).

In addition, 1% p NPG5 (p-nitrophenol- α-D-maltopentaoside) and 0.5 unit / mg of BiLA enzyme were added to a 50 mM NaOAC buffer solution of pH 6.0 at 45 ° C for 0.5, After each reaction for 24 hours, TLC was performed. The results are shown in Fig. 3 (B).

As a result of the experiment, it was confirmed that when maltoheptaose, amylopectin and soluble starch were used as a substrate, they reacted with BiLA enzyme (Fig. 3 (A)). FIG. 3 (A) is a graph showing the activity of the substrate of the present invention (FIG. 3) treated with an alpha amylase (BiLA) enzyme {maltose (G2), maltotriose (G3), maltotetraose (G4), maltoheptaose maltoheptaose, G7), alpha-cyclodextrin, alpha-CD, beta-cyclodextrin, beta-CD, gamma-cyclodextrin, gamma-CD, amylose ), Amylopectin, soluble starch, pullulan}.

In addition, when p NPG5 was used as a substrate, it was confirmed that p NPG5 was degraded by BiLA enzyme over time (Fig. 3 (B)). FIG. 3 (B) is a TLC result of the substrate ( p NPG5) treated with the present invention alpha amylase (BiLA) enzyme.

[ Example  3: invention BiLA  Optimal reaction temperature of enzyme pH  Confirmation experiment]

In this example, dinitrosalicylic acid (DNS) was used to determine the optimal reaction temperature and pH of the BiLA enzyme of the present invention.

(1) Experiments to determine optimum reaction temperature

Buffer (50 mM citric-NaOH, 50 mM NaOAC, 50 mM KH ₂ PO ₄ -NaOH) was added to the substrate {0.5% Soluble starch-starch, soluble (Showa, Japan)} and 0.5 unit / mg of BiLA enzyme were added, and the mixture was sampled every minute and then mixed with the sample to DNS ratio of 1: 3. After that, it was boiled to 10 ~ 60 ° C, and the absorbance was measured at 570 nm in an ELISA reader device.

As a result of the experiment, it was confirmed that the BiLA enzyme had the highest titer at 20 ° C (FIG. 4 (A)). Fig. 4 (A) shows the results of experiments in which the optimum reaction temperature of the alpha amylase (BiLA) enzyme of the present invention was confirmed.

(2) Optimal pH experiment

Buffer (50 mM citric-NaOH, 50 mM NaOAC, 50 mM KH ₂ PO ₄ -NaOH) was added to the substrate {0.5% Soluble Starch - starch, soluble (Showa, Japan)} and 0.5 unit / mg of BiLA enzyme were added to each sample every min and the ratio of sample to DNS was 1: 3. This was boiled for 5 minutes, then placed in an ELISA reader instrument and absorbance was measured at 570 nm.

As a result, it was confirmed that BiLA enzyme had the highest activity at pH 5 (FIG. 4B)). FIG. 4 (B) is a result of an experiment in which the optimum pH for the reaction of the present invention alpha amylase (BiLA) enzyme was confirmed.

[ Example  4: invention BiLA  Enzymatic Wrecker ( specific activity ) Measure]

In this example, the specific activity of the present invention was measured.

Cyclodextrin, beta-CD, gamma-cyclodextrin, and gamma-cyclodextrin were added to 50 mM NaOAc buffer, pH 5.0, Amylose, amylopectin, soluble starch, pullulan) and 0.5 unit / mg of BiLA enzyme were added to each well and reacted at 20 ° C for 3 minutes, 6 minutes, 9 Min, 12 min, and 15 min, and the ratio of sample to DNS was 1: 3. After boiling for 5 minutes, it was placed in an ELISA reader instrument and absorbance was measured at 570 nm.

Further, p NPG5 (p-nitrophenol-α-D-maltopentaoside) and BiLA enzyme were mixed in a ratio of 4: 5: 1 (buffer solution: pNPG5: enzyme) to a 50 mM NaOAC buffer solution at pH 5.0, And samples were taken at 3 minutes, 6 minutes, 9 minutes, 12 minutes and 15 minutes, and the absorbance was measured at 405 nm.

The results are shown in Table 2 below.

temperament Specific activity (U / mg) Soluble starch 18.62 Amylose 8.04 Amylopectin  2.02 p NPG5 0.0023 Pullulan N.D. Alpha-cyclodextrin (alpha-CD) N.D. Beta-cyclodextrin (beta-CD) N.D. Gamma-cyclodextrin (gamma-CD) N.D. N.D: not detected

As a result of the experiment, it was confirmed that BiLA enzyme showed the most excellent immunoglobulin at 18.62 U / mg in soluble starch. However, in the case of alpha-cyclodextrin, alpha-CD, beta-cyclodextrin, gamma-cyclodextrin, and pullulan, No potency was measured.

[ Example  5: invention BiLA  Reactivity of Enzymes ( k _cat / K _m ) Measure]

In this example, the reactivity ( k _cat / K _m ) of the present BiLA enzyme was measured.

The samples were incubated at 0, 2, 4, 6, 8, and 10 min with 1% substrate and 0.5 unit / mg BiLA enzyme in a 50 mM NaOAC buffer solution at pH 5.0. DNS ratio is 1: 3. This was boiled for 5 minutes and then placed in an ELISA reader instrument and absorbance was measured at 570 nm. At this time, the substrate was tested by concentration using soluble starch, amylopectin, amylose, corn starch and potato starch. The results are shown in Table 3 and FIG.

temperament _{k cat / K m (㎖ /} mg · sec) Soluble starch 1.87441 Amylopectin 0.373 Amylose 3.4191 Corn starch 0.4865 Potato starch 0.5774

As a result, k _cat / K _m in soluble starch was 1.87441 ml / mg · sec, amylopectin 0.373 ml / mg · sec, amylose 3.4191 ml / mg · sec, corn starch 0.4865 ml / mg · sec, Lt; RTI ID = 0.0 > mg / sec. ≪ / RTI >

It was confirmed that the reaction rate in amylose was the fastest, followed by the faster reaction rate in the soluble starch (FIG. 5). FIG. 5 shows the results of confirming the reactivity ( k _cat / K _m ) of the present invention alpha amylase (BiLA) enzyme according to the substrate. (A) is soluble starch, (B) is amylopectin, (C) is amylose, (D) is corn starch, (E) is potato starch, .

[ Example  6: invention BiLA  Determination of starch resolution of enzyme]

In this example, the starch resolution of the present BiLA enzyme was examined.

500 μL of the reaction mixture (corn starch added to final concentrations of 4, 8, and 16%) prepared by adding corn starch and 0.5 unit / mg of BiLA enzyme to a 50 mM NaOAC buffer solution at pH 5.0 was added at 20 ° C. for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, and 24 hours, respectively. As a control, BiLA enzyme-free treatment group was used.

As a result of the experiment, it was confirmed that the BiLA enzyme-treated solution had a lower turbidity (that is, clarified) as compared with the control over time. In particular, it was confirmed that the 4% starch reaction solution was the most decomposed and clearest as compared with the control group. In addition, the higher the starch concentration, the lower the degree of starch decomposition, and the visible difference was observed from the 2 hour reaction (Fig. 6). Figure 6 shows the results of confirming the starch resolution of the alpha amylase (BiLA) enzyme of the present invention. 'control' is the BiLA enzyme-free group (control group), and 'rxn' is the BiLA enzyme-treated group.

[ Example  7: invention BiLA  Enzyme treated starch Solution Geotoxicity  Confirm]

In this Example, the biodegradability of the starch solution treated with the BiLA enzyme of the present invention was confirmed.

(1) Manufacture of limit dextrin

To the 50 mM NaOAC buffer solution of pH 5.0, 1% starch substrate (soluble starch-starch, soluble (Showa, Japan)) and 0.5 unit / mg of BiLA enzyme were added and reacted at 20 ° C in 0, 20, After sampling, it was boiled and cooled for 5 minutes. Then, HPAEC (High Pressured Anion Exchange Chrpmatography) was performed.

As a result, maltodextrin having a degree of polymerization of 12 and 30 was found in the control group (untreated BiLA enzyme group), but it was confirmed that the biodegradable starch solution showed maltodextrin composition having a lower degree of polymerization than the control group (Fig. 7). Figure 7 shows the HPAEC results of starch solutions treated with the invention alpha amylase (BiLA) enzyme.

(2) Identification of reaction products through ethanol precipitation reaction

Two times volume of ethanol was added to the BiLA enzyme-treated starch solution (reaction product) and frozen for 5 hours. Thereafter, the mixture was centrifuged at 7,900 rpm and dried in an incubator at 37 ° C to obtain an enzyme-reacted starch (reaction product). 480 μL of 50 mM sodium acetate at pH 5.0 was added thereto, and 2 U of isoamylase was added thereto, followed by reaction at 40 ° C. for 4 hours. HPAEC was then performed.

Experimental results showed that maltodextrin was not found in the control (untreated BiLA enzyme group), but maltodextrin having a degree of polymerization of 3, 4, 13 was found in the BiLA enzyme treated starch solution (Fig. 8). Figure 8 shows the HPAEC results for the reaction product obtained by ethanol precipitation of the BiLA enzyme treated starch solution.

(3) Measurement of reactivity by alpha amylase ( k _cat / K _m )

Alpha amylase from porcine pancreas (commercially available alpha-amylase from porcine pancreas (commercially available enzyme-labeled amylase) was added to the reaction product obtained by the above ethanol precipitation (control group, starch solution reacted with BiLA enzyme for 20 minutes, starch solution reacted with BiLA enzyme for 12 hours) Sigma-Aldrich Co.) to measure the reaction rate.

Each substrate (Table 4 below) and alpha amylase were added to a 50 mM KHPO buffer solution at pH 7.0 and reacted at 20 ° C, which is the optimum temperature of alpha amylase. After the reaction, the samples were sampled at 0 minute, 2 minutes, 4 minutes, 6 minutes, 8 minutes, and 10 minutes, and DNS was added so that the samples and the DNS were in a ratio of 10: 1. After boiling for 5 minutes, the absorbance was measured at 570 nm in an ELISA reader instrument. The values of k _cat / K _m calculated from the measured values are shown in Table 4 and FIG.

temperament _{k cat / K m (㎖ /} mg · sec) Control group (untreated with BiLA enzyme) 0.66 BiLA enzyme-treated starch solution -20 minutes 0.44 BiLA enzyme-treated starch solution -12 hours 0.23

Experimental results, k _cat / K _m of the alpha-amylase of BiLA enzyme-treated starch to react for 12 hours BiLA enzyme treatment starches reacted for 0.23 ㎖ / mg · sec, 20 minutes, 0.44 ㎖ / mg · sec, the control group, 0.66 ㎖ / mg · sec (Fig. 9). FIG. 9 is a result of confirming the reactivity ( k _cat / K _m ) of the alpha amylase to the reaction product obtained by ethanol precipitation of the starch solution treated with the alpha amylase (BiLA) enzyme of the present invention. (A) is the result for the control group, (B) is for the BiLA enzyme treated starch solution (20 min), and (C) is the result for the BiLA enzyme treated starch solution (12 hr).

These results indicate that the samples treated with BiLA enzyme had lower resolution by alpha amylase than the control samples without BiLA enzyme, indicating that samples treated with BiLA enzyme had a slower digestibility, . In addition, among samples treated with BiLA enzymes, longer samples of BiLA enzyme were found to have a higher degree of lipidation.

<110> Industry Academic Cooperation Foundation, Hallym University <120> Method for production of slowly digestible starch with alpha          amylase <130> AP-2015-0053 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 431 <212> PRT <213> Bifidobacterium longum subsp. longum JCM 1217 (KCTC 3127) <400> 1 Val Val Val Ser Met Asp Ser Ser Arg Thr Met Pro Asp Trp Val Lys   1 5 10 15 Tyr Gly Val Phe Trp His Val Tyr Pro Leu Gly Phe Cys Gly Ala Asp              20 25 30 Ile Arg Pro Ala Gly Pro Arg Gln Phe His Gly Arg Gly Leu Asp Ala          35 40 45 Ile Ile Pro Trp Leu Glu Tyr Val Arg Asp Leu Gly Ala Ser Gly Leu      50 55 60 Leu Leu Gly Pro Val Phe Glu Ser Ala Thr His Gly Tyr Asp Thr Leu  65 70 75 80 Asp His Met His Ile Asp Val Arg Leu Gly Gly Asp Ala Ala Phe Asp                  85 90 95 Gln Leu Val Ala Ala Cys Arg Asp Met Gly Leu Gln Val Met Leu Asp             100 105 110 Gly Val Phe Asn His Val Ser Ser Asn His Pro Ala Val Gln Thr Ala         115 120 125 Leu Asp Glu Thr Ala Gly Arg Leu Asn Pro Ala Asp Asn Pro Trp His     130 135 140 Gly Leu Val Arg Ala His Val Gly Asp Asn Gly Glu Pro Ala Leu Asp 145 150 155 160 Val Phe Glu Gly His Gly Asp Leu Val Ala Leu Asp His Ser Ala Asp                 165 170 175 Glu Thr Val Asp Tyr Val Ala His Val Met Ser His Trp Leu Asp Arg             180 185 190 Gly Ala Gly Trp Arg Leu Asp Ala Ala Tyr Ala Val Pro Ser Pro         195 200 205 Phe Trp Ala Arg Val Leu Pro Gln Val Lys Ala Ala His Pro Tyr Ser     210 215 220 Trp Ile Phe Gly Glu Val Ile His Gly Asp Tyr Pro Arg Ile Ile Glu 225 230 235 240 Glu Ser Thr Met Asp Ser Ile Thr Gln Tyr Glu Leu Trp Lys Ser Ile                 245 250 255 Gln His Ala Leu Glu Thr Glu Asn Phe Phe Glu Leu Asp Trp Asn Leu             260 265 270 Lys Arg His Asn Ala Phe Leu Asp Ser Phe Val Pro Gln Thr Phe Ile         275 280 285 Gly Asn His Asp Val Thr Arg Ile Ala Ser Gln Ile Gly Pro Ala Lys     290 295 300 Ala Ala Leu Ala Leu Ala Val Leu Met Thr Val Gly Gly Val Ser Ser 305 310 315 320 Ile Tyr Tyr Gly Asp Glu Gln Gly Tyr Val Gly Val Lys Gln Glu Arg                 325 330 335 Phe Gly Gly Asp Asp Asp Val Arg Pro Lys Phe Pro Asp Ser Pro Ala             340 345 350 Glu Leu Ser Thr Leu Gly Glu Pro Thr Tyr Arg Leu His Gln Ala Leu         355 360 365 Ile Ala Leu Arg Arg Arg Asn Pro Trp Leu Leu Asp Ala Arg Thr Glu     370 375 380 Ala Val Lys Leu Glu Asn Lys His Phe Val Tyr Arg Ser Thr Ser Ala 385 390 395 400 Asp Ala Gln His Ser Leu Thr Val Asp Leu Asn Ile Glu Gln Ser Pro                 405 410 415 Thr Phe Thr Ile Arg Asn Ala Asp Gly Ser Thr Ala Tyr Gln Tyr             420 425 430

Claims (3)

Characterized in that the starch has an amino acid sequence of SEQ ID NO: 1 and is hydrolyzed by treating an alpha amylase derived from a strain of Bifidobacterium longum subsp. Longum JCM 1217 (KCTC 3127), Bacillus subtilis JCM 1217 (Method for producing starch of chemical conversion).
delete The method according to claim 1,
The treatment of the alpha amylase,
Wherein the hydrolysis is carried out at a temperature of 10 to 25 DEG C and a pH of 4.5 to 5.5.

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