KR20020066986A - Method for the production of thermostable and acid-stable oligosaccharide by using dextransucrase - Google Patents

Abstract

PURPOSE: Provided is a production method of thermostable and acid-stable oligosaccharide, which is used as a sweetening agent for beverage and foods, by using dextransucrase. CONSTITUTION: The production method of thermostable and acid-stable oligosaccharide is characterized by adding dextransucrase, which is derived from Leuconostoc mesenteroides strain, to a solution containing 0.5M-5M of sugar and additionally maltose then followed by reacting them and recovering produced oligosaccharide.

Description

Method for the production of thermostable and acid-stable oligosaccharide by using dextransucrase}

The present invention relates to a process for preparing oligosaccharides.

Recently, in order to compensate for problems such as caries, obesity, diabetes, and adult diseases caused by excessive intake of sugar and large amounts of existing sugar, a new type of alternative sugar, oligosaccharide of natural food material, has been developed through biotechnology.

In general, an oligosaccharide means a small sugar having 2 to 10 degree of polymerization (300 to 2,000 in molecular weight) in which a single sugar is dehydrated and condensed by a glycoside bond, regardless of the type of constituent sugar. Commercially produced oligosaccharides include fructooligosaccharides, isomaltooligosaccharides, maltooligosaccharides, galactooligosaccharides and the like, and research on soy oligosaccharides and xylo oligosaccharides is ongoing.

Fructooligosaccharides are sugars with 3 to 5 DPs made by combining one to three more fructose molecules with sugar using β-fructofuranosidase (FFase), a fructose transferase of sugar. The final product is produced by bleaching, filtration, desalting and concentration.

Isomaltooligosaccharide refers to a sugar to which the glucose molecule has α-1,6 bond. This is a saccharification process by liquefying the starch solution, which is a substrate, with α-amylase, and then acting β-amylase and transglucosidase. ) And the transition to produce at the same time.

Maltooligosaccharide refers to a sugar in which a glucose molecule has α-1,4 bonds and usually maltotriose or maltotetraose is a main component (50% or more of the total sugar content). The process is similar to isomaltooligosaccharides using starch solution as a substrate, except that α-amylase, β-amylase and pullulanase are used in the saccharification transition process.

Galactooligosaccharide refers to an oligosaccharide composed of galactose and glucose, and is prepared by adding galactose by reacting a sugar transferase (β-galactosidase) to lactose used as a substrate.

Commercially produced maltooligosaccharides are resistant to acids and heats like oligosaccharides composed of glucose, but they are less sweet.Fructooligosaccharides are sweeter but weaker to acids and heat. As necessary food additives, sweeteners, etc., there are restrictions on use.

Dextransucrase (EC 2.4.1.5) is a generic term for enzymes that synthesize glucans from sugar and is produced mainly from microorganisms of the genus Leuconostoc and Streptococcus . The reactor operation for the sugar of dextran sucrose is as follows.

n sucrose → (glucose) n-m-w + n-m fructose + m leucrose + w glucose

The main products of this enzymatic reaction are high molecular weight glucan and fructose of about 10 ⁷ to 10 ⁸ Da, and by-products of glucose and lucrose (5-O-α-D-glucopyranosyl-D-fructopyranose) are produced. .

Korean Patent Application No. 1998-24355 discloses a method for producing novel oligosaccharides using dextran sucrose obtained from a mutant strain of Leuconostoc mesenteroides using sugar as a substrate and maltose, genthiobiose, raffinose or lactose as receptors. Is disclosed. However, this method fixed the total carbohydrates at a low concentration of 100 mM and did not suggest how to produce oligosaccharides in high concentrations of sugar (500 mM-4M). In addition, this study proposes a method for producing oligosaccharides using only sugar without receptors. The oligosaccharides produced in this way have acid and heat resistance properties and have a sweet taste unlike the oligosaccharides prepared in Patent 24355.

It is an object of the present invention to provide oligosaccharides having strong acid and heat resistance.

Another object of the present invention is to provide a method for producing oligosaccharides having strong acid and heat resistance.

1 is a TLC result showing the pattern of the biosynthesized product when the dextran sucrose obtained from E. coli DH5α / pFMCM (KCTC 0859BP) and L. mesenteroides B-742 (ATCC 13146), respectively, in a high concentration sugar solution.

Lane 1, maltodextrin standard mixture;

Lane 2, maltodextrin standard mixture;

Lane 3, Panos

Lane 4, 4M sucrose + E. coli DH5α / pFMCM dextran sucrose;

Lane 5, 2M sucrose + E. coli DH5α / pFMCM dextran sucrose;

Lane 6, 2M sucrose + L. mesenteroides NRRL 742 dextran sucrose.

FIG. 2 is a TLC result of E. coli DH5α / pFMCM dextran sucrose showing the degree of use of sugar substrate and the size of biosynthetic dextran when maltose was added.

Lane 1, maltodextrin standard mixture;

Lane 2, isomaltodextrin standard mixture;

Lane 3, sucrose;

Lane 4, maltose;

Lane 5, 4M sucrose + dextran sucrose;

Lane 6, 3M sucrose + dextran sucrose;

Lane 7, 2M sucrose + dextran sucrose;

Lane 8, 1M sucrose + dextran sucrose;

Lane 9, 0.5 M sucrose + dextran sucrose;

Lane 10, 4M sucrose + 0.4M maltose + dextran sucrose;

Lane 11, 3M sucrose + 0.3M maltose + dextran sucrose;

Lane 12, 2M sucrose + 0.2M maltose + dextran sucrose;

Lane 13, 1 M sucrose + 0.1 M maltose + dextran sucrose;

Lane 14, 0.5 M sucrose + 0.05 M maltose + dextran sucrose;

Lane 15, 4M sucrose + 0.04M maltose + dextran sucrose;

Lane 16, 3M sucrose + 0.03M maltose + dextran sucrose;

Lane 17, 2M sucrose + 0.02M maltose + dextran sucrose;

Lane 18, 1 M sucrose + 0.01 M maltose + dextran sucrose;

Lane 19, 0.5 M sucrose + 0.005 M maltose + dextran sucrose;

Figure 3 is a TLC result of L. mesenteroides B-742 dextran sucrose, the degree of sugar substrate utilization and the size of biosynthetic dextran when maltose is added.

Lane 1, maltodextrin standard mixture;

Lane 2, isomaltodextrin standard mixture;

Lane 3, sucrose;

Lane 4, 2M sucrose + dextran sucrose

Lane 5, 2M sucrose + 0.2M maltose + dextran sucrose

Lane 6, 1M Sucrose + Dextran Sucrose

Lane 7, 1M sucrose + 0.1M maltose + dextran sucrose

Lane 8, 0.5M sucrose + dextran sucrose

Lane 9, 0.5M sucrose + 0.05M maltose + dextran sucrose

4 is a TLC result of L. mesenteroides B-1299 (ATCC 11449) dextran sucrose showing the degree of sugar substrate utilization and the size of biosynthetic dextran when maltose was added.

Lane 1, maltodextrin standard mixture;

Lane 2, isomaltodextrin standard mixture;

Lane 3, panose;

Lane 4, 0.5 M sucrose + 0.05 M maltose + dextran sucrose;

Lane 5, 0.5 M sucrose + 0.1 M maltose + dextran sucrose;

Lane 6, 0.5 M sucrose + 0.5 M maltose + dextran sucrose;

Lane 7, 0.5M sucrose + 1M maltose + dextran sucrose;

Lane 8, 0.5 M sucrose + 1.5 M maltose + dextran sucrose;

Lane 9, maltodextrin standard mixture;

Lane 10, isomaltodextrin standard mixture;

Lane 11, 3.09M sucrose + dextran sucrose;

Lane 12, 2.06M sucrose + dextran sucrose;

Lane 13, 1.55 M sucrose + dextran sucrose;

Lane 14, 1.03M sucrose + dextran sucrose;

Lane 15, 0.52M sucrose + dextran sucrose;

Lane 16, 0.1M Sucrose + Dextran Sucrose

5 is a TLC result of confirming acid resistance at various temperatures of an oligosaccharide prepared with E. coli DH5α / pFMCM dextran sucrose.

Lane 1, isomaltodextrin standard mixture;

Lane 2, oligosaccharide (4M sucrose + E. coli DH5α / pFMCM dextran sucrose);

Lanes 3-7, pH 2 in 40, 60, 80, 100, 120 ° C .;

Lanes 8-12, pH 3 in 40, 60, 80, 100, 120 ° C .;

Lanes 13-17, pH 4 in 40, 60, 80, 100, 120 ° C .;

Lanes 18-22, pH 5 in 40, 60, 80, 100, 120 ° C .;

Lane 23, maltodextrin standard mixture.

Figure 6 is a TLC result that can confirm the heat resistance of oligosaccharides prepared with E. coli DH5α / pFMCM dextran sucrose.

Lane 1, maltodextrin standard mixture;

Lane 2, isomaltodextrin standard mixture;

Lane 3, panose;

Lane 4, oligosaccharide (4M sucrose + E. coli DH5a / pFMCM dextran sucrose);

Lanes 5-6, 30 min at 120 ° C., 60 min;

Lanes 7-8, 30 min at 140 ° C., 60 min;

Lane 9-10, 30 minutes at 160 ° C., 60 minutes.

Figure 7 is a photograph showing the separation of high-branched oligosaccharides using two-dimensional thin-film chromatography method Lane 17: maltofulin series, lane 18: high-branched oligosaccharide, lane 19: high-branched oligosaccharide two-dimensional thin layer chromatography starting point, lane 20 : Isomaltoxtrin series, lane 21: high branched oligosaccharide, as developing solvent 2: 5: 1.5 (v / v / v) nitromethane / 1-propanol / water (first run) → 85: 20: 50: 50 (v / v / v / v) acetonitrile / ethylacetate / 1-propanol / water (second run) was used, 1-16: each constituent in the highly branched oligosaccharides was assigned a unique number.

FIG. 8 is a photograph showing analysis results of thin-film chromatography of decomposition products after various enzyme treatment of fractions separated from high-branched oligosaccharides. Lane 1: Maltodextrin series, lanes 2 and 19: high-branched oligosaccharide, lane 20: Isomaltodextrin series, (1) lanes 3-10: enzymatic reaction product of fraction 2 isolated from high-branched oligosaccharide, lanes 11-18: enzymatic reaction product of fraction 3 isolated from high-branched oligosaccharide, (2) lane 3- 10: Enzyme reaction product of fraction 8 isolated from high branched oligosaccharides, Lane 11-18: Enzyme reaction product of fraction 9 isolated from high branched oligosaccharides.

Each fraction was treated with enzymes in the following order: α-, β- and iso-amylase, α- and β-glucosidase, dextranase, α-amyloglucosidase and invertase.

9 is a photo showing the results of thin-film chromatography analysis of the decomposition product after the dextranase treatment to each fraction separated from the high-branched oligosaccharide, lane 1: isomaltoxtrin series, lane 2: maltodextrin series, lane 3: high-branched oligosaccharide, lane 4: dextranase degradation product of fractions separated from high-branched oligosaccharide, lane 5: decomtranation product after dextranase treatment with isomaltulose, lane 6: dextranase to leucrose Post-treatment degradation products, lane 7: high branched oligosaccharides, lane 8: isomaltooligosaccharides.

10 is a photograph showing the results of analysis of thin-film chromatography of the receptor reaction product using dextran sucrase of the separated high-branched oligosaccharide fractions, ①: maltotextrin series, ②: receptor product of isomaltulose (㉠ , ㉠ ', ㉠'') ^* , ③: Receptor product of leucose (㉡, ㉡') ^** , ④: Highly branched oligosaccharide ⑤: Isomaldextrin series, Lane 1-20: Fraction separated from high branched oligosaccharide Receptor reaction products of these dextran sucrases.

In the photograph, ㉠, ㉠ ', ㉠' ': the high-branched oligosaccharide fractions and the receptor reaction products of the fractions can be confirmed that the same as the receptor reaction product of isomaltulose, ㉡, ㉡': high-branched oligosaccharide fractions and fractions It can be confirmed that their receptor reaction product is consistent with the receptor reaction product of leucrose.

In order to achieve the above object, the present invention provides a method for producing an oligosaccharide having a strong acid resistance and heat resistance.

In another aspect, the present invention provides an oligosaccharide produced by the above production method.

The method of the present invention consists in recovering the oligosaccharide produced, a strain producing dextran sucrose derived from Leuconostoc mesenteroides species strain in a medium containing a high concentration of 0.5M-5M sugar.

Another method according to the invention consists in the addition of dextran sucrose derived from Leuconostoc mesenteroides species strain to a solution containing sugar at a high concentration of 0.5M-5M, followed by recovery of the oligosaccharides produced.

In the present invention, dextran sucrose refers to dextran sutrases derived from Leuconostoc mesenteroides as an enzyme synthesizing dextran industrially. For example, Leuconostoc mesenteroides NRRL B-742 (ATCC 13146), Leuconostoc mesenteroides NRRL B-1299 (ATCC 11449), Leuconostoc mesenteroides NRRL B-512F by Leuconostoc mesenteroides strains, these mutants or the inventors, such as (ATCC 10830) Dextran sucrose obtained from a transformant obtained by introducing a dextran watertrace gene isolated from a Leuconostoc mesenteroides strain or a mutant thereof, such as deposited E. coli DH5a / pFMCM (KCTC 0859BP).

By adding maltose in addition to sugar in the method of the present invention, it is possible to promote the production of oligosaccharides having heat resistance and acid resistance.

Example 1 Preparation of Enzyme Liquid

E. coli DH5a / pFMCM (KCTC 0859BP) was inoculated in LB liquid medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl) containing ampicillin (50 μg / ml) and incubated at 37 ° C. Only the cells were separated to prepare a crude enzyme solution, which was used as dextran sucrose enzyme solution.

L. mesenteroides NRRL B-742 was inoculated in LB medium containing 5 g of yeast extract, 5 g of peptone, 5 g of K ₂ HPO ₄ and 20 g of sucrose in 1 L of water and incubated at 37 ° C. Was prepared and used as the dextran sucrose enzyme solution.

L. mesenteroides NRRL B-1299 was inoculated in LB medium containing 5 g of yeast extract, 5 g of peptone, 5 g of K ₂ HPO ₄ and 20 g of sucrose in 1 L of water and incubated at 37 ° C. Was prepared and used as the dextran sucrose enzyme solution.

Example 2. Confirmation of Oligosaccharide Production Reaction and Composition of Reaction Products

In order to produce oligosaccharides, 4.5 M of sugar solution was prepared, and mixed with the enzyme obtained in Example 1, the final sugar concentration in the reactor of the enzyme reactor was 0.5M to 4.0M and reacted at 28 ° C.

The amount of enzyme used was from 0.1 U / ml to 10 U / ml and one unit of enzyme was expressed in μmol of fructose free from sugar per ml of enzyme per minute. Indicated. The reaction was performed until all the sugar in the reactor was consumed. The production of oligosaccharides and complete use of sugar were taken up in Merck K6F TLC plates with 1 μl of reaction solution and then run twice in MeNO / 1-propanol / water (2/5 / 2.5, v / v / v). The components of the separated carbohydrates were identified by using a coloring reagent containing 0.5% (w / v) α-naphthol and 5% (v / v) sulfuric acid in the TLC plate. Quantitative analysis of each carbohydrate was performed by NIH Image Program using a Macintosh (Power PC; 7100/80) computer. The composition of the produced product was also confirmed using HPLC.

After using E. coli DH5a / pFMCM dextran sucrose as an enzyme and reacting under the above synthetic conditions, the components and distribution of the synthesized product were confirmed by TLC and HPLC. The results are shown in FIGS. 1 and 2.

L. mesenteroides NRRL B-742 dextran sucrose was used as an enzyme, and 4M, 2M, and 1M sugars were used as a substrate, and then reacted under the above conditions. The composition and distribution of the synthetic product were confirmed by TLC. The results are shown in FIGS. 1 and 3.

After using L. mesenteroides NRRL B-1299 dextran sucrose as an enzyme and reacting with 1 unit of enzyme and the final sugar concentration from 3.09M to 0.1M under the above conditions, the composition and distribution of the synthetic product were confirmed by TLC. . Maltose was added at a concentration of 1/10 and 1/100 of the sugar concentration. The results are shown in FIG.

Example 3 Identification of Acid and Heat Resistance Characteristics of Oligosaccharides

To 1000 ml of 4M sucrose, 120 U (1 ml) of E. coli DH5a / pFMCM dextran sucrose obtained in Example 1 was added and reacted at 28 ° C. for a time to obtain an enzyme reaction solution.

50 ml of the enzyme reaction solution was adjusted to pH 2, 3, 4, and 5, respectively, and the mixture was divided into 5 parts, and left at 40, 60, 80, 100, and 120 ° C for 15 minutes, and then quenched. As a result of observing the pattern change of the oligosaccharide in the enzyme reaction solution using TLC, there was almost no change in the pattern of the oligosaccharide. The results are shown in FIG.

50 ml of the enzyme reaction solution was quenched after standing at 120, 140, and 160 ° C for 30 minutes and 1 hour, respectively. As a result of observing the pattern change of the oligosaccharide in the enzyme reaction solution using TLC, there was almost no change in the pattern of the oligosaccharide. The results are shown in FIG.

Enzyme reaction 50 ml were quenched after standing at 120, 140, and 160 ° C. for 30 minutes and 1 hour, respectively. As a result of observing the pattern change of the oligosaccharide in the enzyme reaction solution using TLC, there was almost no change in the pattern of the oligosaccharide. The results are shown in FIG.

Example 4: Isolation and Structure Analysis of Oligosaccharides

1) The oligosaccharides synthesized by dextran sucrase and high concentration of sugar were separated by sugar and structural analysis was performed by various methods to identify the components of oligosaccharides.

2) Experiment Method

a. Oligosaccharide Separation Using Thin Film Chromatography

After diluting the oligosaccharides using a silica gel plate for separation, 1 μl was added dropwise and developed using a developing solvent (acetonitrile / ethylacetate / 1-propanol / water = 85/20/50/50), and then the side surfaces were cut and separated. Carbohydrate components were identified using a coloring reagent containing 0.3% (w / v) N- (1-naphthyl) ethylenediamine and 5% (v / v) sulfuric acid. Compared to the non-colored silica gel plate after color development, the silica gel was collected by fractions and dissolved in the same developing solvent. After centrifugation, the supernatant was obtained, and after drying, it was dissolved in water and confirmed by separation using thin layer chromatography. It was used for.

b. Oligosaccharide Structure Verification

① Two-dimensional thin-film chromatography: For the structural analysis of oligosaccharides, two different development solvents were used to develop bidirectionally to determine the structure of pure sugars independently. Various developing solvents were developed in three different conditions (b → a, b → c, a → c) in different orders, and then separated from each other.

The oligosaccharides were first dropped and developed in the first developing solvent, then completely dried and then rotated 90 ° in a direction to drop standard materials at both ends and developed in a second developing solvent. The components of the separated carbohydrates were identified by using a coloring reagent containing 0.3% (w / v) N- (1-naphthyl) ethylenediamine and 5% (v / v) sulfuric acid in the TLC plate.

In the developed solvent, a) acetonitrile / ethyl acetate / 1-propanol / water = 85/20/50/50, b) nitromethane / 1-propanol / water = 2/5 / 1.5, c) acetonitrile / Ethyl acetate / 1-propanol / water = 85/20/50/70.

② mass spectrometry

The degree of polymerization of oligosaccharides was determined using a MALDI-TOF (Finnigan Lasermat 2000 mass spectrometers, CA, USA) mass spectrometer. Each fraction of the oligosaccharide and 1 μl of the oligosaccharide before separation were mixed with 1 μl 2,4-dihydrobenzoic acid (DHB) dissolved in acetonitrile as a support and then dried at 40 ° C. for analysis. In order to analyze the resulting peaks, isomaltoligosaccharide was also analyzed as a comparative substance.

(3) enzymes (α-, β-, iso-amylase, α-, β-glucosidase, dextranase, α-amyloglucosidase, invertase) obtained above 1 μl of the reaction solution was added to a silica gel plate in order to confirm the cut-off state after treating the oligosaccharide fractions in each reaction condition, and then nitromethane / 1-promanol / water (v / v / v / v, 2/5 / 1.5). The silica gel plate was identified by using a color developing reagent containing 0.3% (w / v) N- (1-naphthyl) ethylenediamine and 5% (v / v) sulfuric acid to identify the components of the carbohydrates and to decompose each enzyme. The structure was predicted as a result.

④ Acid hydrolysis: Each fraction was mixed with 1M hydrochloric acid, hydrolyzed at 100 ° C for 30 minutes, dried in vacuo, and dissolved in water. 1 μl of the reaction solution was added to a silica gel plate, followed by acetonitrile / water (v / v, 85 / 15) was developed twice, and then colored to confirm the composition of the monosaccharide.

⑤ Receptor reaction: The fractions obtained from each were mixed 1: 1 with dextran sucrase and 100 mM sugar and reacted for 12 hours at 28 ° C. After confirming the product by thin layer chromatography, the oligosaccharides were compared with oligosaccharides. It was confirmed that it was produced by each receptor reaction.

Experimental results are shown in FIGS. 7 to 10.

Figure 7 is a photograph showing the separation of high-branched oligosaccharides using two-dimensional thin-film chromatography method Lane 17: maltofulin series, lane 18: high-branched oligosaccharide, lane 19: high-branched oligosaccharide two-dimensional thin layer chromatography starting point, lane 20 : Isomaltoxtrin series, lane 21: high branched oligosaccharide, as developing solvent 2: 5: 1.5 (v / v / v) nitromethane / 1-propanol / water (first run) → 85: 20: 50: 50 (v / v / v / v) acetonitrile / ethylacetate / 1-propanol / water (second run) was used, 1-16: each constituent in the highly branched oligosaccharides was assigned a unique number.

Fractions 1, 4, 8, 12, 16, and polysaccharide were identified by comparison with the reference material (lanes 17 and 20).

FIG. 8 is a photograph showing analysis results of thin-film chromatography of decomposition products after various enzyme treatment of fractions separated from high-branched oligosaccharides. Lane 1: Maltodextrin series, lanes 2 and 19: high-branched oligosaccharide, lane 20: Isomaltodextrin series, (1) lanes 3-10: enzymatic reaction product of fraction 2 isolated from high-branched oligosaccharide, lanes 11-18: enzymatic reaction product of fraction 3 isolated from high-branched oligosaccharide, (2) lane 3- 10: Enzyme reaction product of fraction 8 isolated from high branched oligosaccharides, Lane 11-18: Enzyme reaction product of fraction 9 isolated from high branched oligosaccharides.

Each fraction was treated with enzymes in the following order: α-, β- and iso-amylase, α- and β-glucosidase, dextranase, α-amyloglucosidase and invertase.

Comparing the reaction products of the carbohydrate decomposing enzyme as described above, 2, 3, 5, 8, 9, 13, etc. confirmed the structure.

9 is a photo showing the results of thin-film chromatography analysis of the decomposition product after the dextranase treatment to each fraction separated from the high-branched oligosaccharide, lane 1: isomaltoxtrin series, lane 2: maltodextrin series, lane 3: high-branched oligosaccharide, lane 4: dextranase degradation product of fractions separated from high-branched oligosaccharide, lane 5: decomtranation product after dextranase treatment with isomaltulose, lane 6: dextranase to leucrose Post-treatment degradation products, lane 7: high branched oligosaccharides, lane 8: isomaltooligosaccharides.

The structures of 6, 7, 10, 11, 13, 14, and 15 were confirmed by comparing the structures of the compounds of dextranase and fructose.

Table 1 Decomposition products after treatment with various enzymes of products isolated from high-branched oligosaccharides

Processed enzymes Isolated Carbohydrate Components One 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 α-amylase - - - - - - - - - - - - - - - - β-amylase - - - - - - - - - - - - - - - - Iso-amylase - - - - - - - - - - - - - - - - α-glucosidase + + + + + + + + + + + + + + + + β-glucosidase - - - - - - - - - - - - - - - - Amyloglucosidase - - - + + + + + + + + + + + + + Invertase - - - - + - - - + - - - + - - - Stranase (composition of degradation product after dextranase treatment reaction) One + + + + - + + + + + + + + + + + 2 - - - - - + - - - - + - - - - - 3 - - - - - - + - - + - - - - - - 4 - - - - - - - + - + + + + + + + 5 - - - - - - - - - - - - + - - - 6 - - - - - - - - - - - - - + - - 7 - - - - - - - - - - - - - - + - 8 - - - - - - - - - - - - - - - +

"+" In the table means that the degradation product is present, "-" means that there is no decomposition product.

The above table can be confirmed by confirming the decomposition pattern of the enzymes in the contents of the table in the results of FIGS. 8 and 9 as a table.

10 is a photograph showing the results of analysis of thin-film chromatography of the receptor reaction product using dextran sucrase of the separated high-branched oligosaccharide fractions, ①: maltotextrin series, ②: receptor product of isomaltulose (㉠ , ㉠ ', ㉠'') ^* , ③: Receptor product of leucose (㉡, ㉡') ^** , ④: Highly branched oligosaccharide ⑤: Isomaldextrin series, Lane 1-20: Fraction separated from high branched oligosaccharide Half of the receptors of dextransukrases in the wild represent products.

In the photograph, ㉠, ㉠ ', ㉠' ': the high-branched oligosaccharide fractions and the receptor reaction products of the fractions can be confirmed that the same as the receptor reaction product of isomaltulose, ㉡, ㉡': high-branched oligosaccharide fractions and fractions It can be confirmed that their receptor reaction product is consistent with the receptor reaction product of leucrose.

Figure 10 was able to confirm the structure by comparing the receptor product synthesized by reacting small oligosaccharides with sugar using dextran sucrase.

From the above methods and results, the oligosaccharides synthesized in this patent are composed of more than 17 constituents. In comparison, the structure was defined as shown in Table 2.

[Table 2] Determination of structure and content of components of high-branched oligosaccharide

Isolated Fraction of Highly Branched Oligosaccharides Component Structure of components content(%) One D-fractose Frc 13.5 2 Isomaltulose (6-α-D-glucopyranosyl-D-fractopuranos) α-Glc (1 → 6) Frc 4.6 3 Leucross (5-α-D-glucopyranosyl-D-fractopyranose) α-Glc (1 → 5) Frc 12.4 4 Isomaltos α-Glc (1 → 6) Glc 7 5 3 ² -O-α- D-glucosyl isomaltose α-Glc (1 → 6) Glcα-Glc (1 → 3) 5.2 6 6-α-isomaltosyl-D-fractose α-Glc (1 → 6) α-Glc (1 → 6) Frc 3.7 7 5-α-isomaltosyl-D-fractopyranose α-Glc (1 → 6) α-Glc (1 → 5) Frc 4.3 8 Isomaltotriose α-Glc (1 → 6) α-Glc (1 → 6) Glc 6.3 9 3 ² -O-α- D-glucosyl-isomaltotriose α-Glc (1 → 6) α-Glc (1 → 6) Glcα-Glc (1 → 3) 2.9 10 6-α-isomaltotriosyl-D-fractose [α-Glc (1 → 6)] ₂ α-Glc (1 → 6) Frc 3.3 11 5-α-isomaltotriosyl-D-fractopyranose [α-Glc (1 → 6)] ₂ α-Glc (1 → 5) Frc 3.8 12 Isomaltotetraos [α-Glc (1 → 6)] ₃ Glc 6.2 13 3 ² -O-α- D-glucosyl-isomaltotetraose [α-Glc (1 → 6)] ₃ α-Glc (1 → 6) Glcα-Glc (1 → 3) 2.8 14 6-α-isomaltotetraosyl-D-fractose [α-Glc (1 → 6)] ₃ α-Glc (1 → 6) Frc 2.7 15 5-α-isomaltotetraosyl-D-fractopyranose [α-Glc (1 → 6)] ₃ α-Glc (1 → 5) Frc 2.7 16 Isomaltopentaos [α-Glc (1 → 6)] ₄ Glc 5.6 17 Oligosaccharides with a degree of polymerization of 5 or more 8.4 Polysaccharide Dextran [α-Glc (1 → 6)] _n Glc 4.6

Oligosaccharides can be produced from high concentrations of sugar by the method of the present invention. Since the oligosaccharide produced by the method of the present invention is resistant to acid and heat, it is suitable as a sweetener for foods such as beverages requiring acid resistance and heat resistance.

Claims (4)

A method for producing acid and heat resistant oligosaccharides, comprising culturing Leuconostoc mesenteroides strains under culture conditions containing 0.5M-5M sugar and recovering the produced oligosaccharides. A method for producing an acid resistant and heat resistant oligosaccharide comprising the reaction of dextran sucrose derived from Leuconostoc mesenteroides species strain to a solution containing 0.5M-5M sugar and recovering the produced oligosaccharide. The method according to claim 1 or 2, Said culture conditions or said solution further contains maltose. An oligosaccharide prepared by the method of claim 1.