KR100663065B1 - Production Method of Acid and Heat Resistant Oligosaccharides Using Levan Sucrase - Google Patents

Abstract

The present invention relates to a method for producing oligosaccharides resistant to acid and heat by using free enzymes with only high concentration of sugar, by mixing 0.5-4.0 M of sugar, preferably 2.7-4.0 M of sugar, from a strain of Leuconostoc mesenteroides . It is made to react by addition of the derived levansukraase and recover the oligosaccharides produced. It was confirmed that the recovered oligosaccharides inhibit the growth of plaque-forming bacteria and the activity of mutans sucrase. The oligosaccharides produced by the method of the present invention are suitable as functional sweeteners for foods such as beverages requiring acid and heat resistance. Do.

 Leuconosestock mesenteroides, acid-resistant oligosaccharides, heat-resistant oligosaccharides, levansukrases

Description

Method for the production of thermostable and acid-stable oligosaccharides using levansucrase

FIG. 1 shows thin-film chromatography showing the pattern of the biosynthesized product when 3M sugar solution was reacted with Levansukraase obtained from L. mesenteroides NRRL B-1355.

Mn, maltodextrin standard mixture;

IMn, isomaltodextrin standard mixture;

Lane 1, 1-Kestos

Lane 2, Nicetose

Lane 3, fructooligosaccharide mixture with branched bonds

Figure 2 is a TLC result showing the pattern of the biosynthetic product when reacted with 3M sugar solution L. mesenteroides B-1355 obtained from L. mesenteroides B-1355 in more detail by two-dimensional thin film chromatography.

Mn, maltodextrin standard mixture;

IMn, isomaltodextrin standard mixture;

Lane 5, high branch oligosaccharides

1-16: Individual components in high-branched oligosaccharides

Figure 3 is a result that can confirm the acid resistance at various temperatures of the fructooligosaccharide prepared with L. mesenteroides NRRL B-1355 Levansukraase.

-●-, L. mesenteroides fructooligosaccharide (pH 2) prepared with NRRL B-1355 Levansukraase

       -○-, commercially available fructooligosaccharide (pH 2)

-■-, fructooligosaccharide (pH 3) prepared with L. mesenteroides NRRL B-1355 Levansukraase

       -□-, commercially available fructooligosaccharide (pH 3)

-▲-, fructooligosaccharide (pH 4) prepared with L. mesenteroides NRRL B-1355 Levansukraase

       Commercially available fructooligosaccharides (pH 4)

Figure 4 is a result of confirming the thermal stability of the fructooligosaccharide prepared with L. mesenteroides NRRL B-1355 Levansukraase at various temperatures.

Black bar, left at each temperature for 30 minutes

White bar, left at each temperature for 60 minutes

Figure 5 is a result of confirming the stability after mixing the fructooligosaccharide prepared with L. mesenteroides NRRL B-1355 Levansukraase to commercially sold unsweetened carbonated beverage and allowed to stand at 55 ℃ 20 hours.

G, glucose

F, fructose

Lanes 1 and 2, before and after fixing of commercially available unsweetened soda

Lanes 3 and 4, before and after fixation of commercially available sugar-free soda

Lanes 5 and 6, before and after standing of the liquid mixture of the oligosaccharide of the present invention in a commercially available sugar-free carbonated beverage

Lanes 7 and 8, before and after politics of commercial fructo-oligosaccharides mixed with commercially available sugar-free soda

Figure 6 shows the oligosaccharide prepared with L. mesenteroides NRRL B-1355 Levansukraase S. mutans which is one of the bacteria forming plaque The results confirmed that it is effective in inhibiting growth. The left vertical axis represents the growth of S. mutans as absorbance values, and the right vertical axis measures the pH change of the growth medium as the bacteria grow. The horizontal axis represents the culture time of the bacteria.

-■-, L. mesenteroides NRRL B-1355 The degree of bacterial growth when S. mutans were cultured in a medium containing fructooligosaccharide prepared with Levansucrase (OD ₆₀₀ )

-□-, L. mesenteroides NRRL B-1355 The degree of bacterial growth when S. mutans were cultured in a medium without fructooligosaccharides prepared with levansucrase (OD ₆₀₀ )

PH of the medium when S. mutans were cultured in a medium containing fructooligosaccharide prepared with L. mesenteroides NRRL B-1355 Levansukraase

PH of the medium when S. mutans were cultured in a medium without fructooligosaccharide prepared with L. mesenteroides NRRL B-1355 Levansukraase

The present invention relates to a method for preparing oligosaccharides using a high concentration of sugar solution and the enzyme levan sucrase.

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.

Levansucrase (EC 2.4.1.10) is a generic term for enzymes that synthesize Levan from sugar and is produced mainly from microorganisms of the genus Leuconostoc and Pseudomonas . The reaction mechanism for the sugar of Levansukraase is as follows.

n sucrose → (fractose) n-m-w + n-m glucose

  + m ketoses, nythos + w fructose

The main products of this enzymatic reaction are fructans and glucose of high molecular weights of about 10 ⁷ ~ 10 ⁸ Da, and by-products of fructose, ketose and nitose 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, the method of the present invention discloses a method for producing fructooligosaccharides in which glucose is combined with fructose, which is not glucotooligosaccharide, using levansucrase rather than dextran sucrose. In addition, this study suggests a method for producing oligosaccharides using only sugar without any additional 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.

In Korean Patent Application No. 199-0017055, fructooligosaccharide was prepared by reacting Aspergillus snigger cells having glucose oxidase enzyme activity with a sugar solution by mixing with fructosyltransspirase enzyme. 5-10 units per gram of sugar and 12.5-62.5 mg of Aspergillus niger cells per gram of sugar were added and reacted with aeration and agitation. Fructooligosaccharide was synthesized at. However, in the method of the present invention, oligosaccharides were synthesized using 0.5-4M sugar, preferably 2.7-4.0M sugar, using only enzymes, not enzymes and bacteria, and the structure of fructooligosaccharide. In addition to the oligosaccharides of the new structure, including the oligosaccharides are different. The enzyme used was L. mesenteroides , which has not been published.

In addition, Korean Patent No. 1991-0024070 discloses immobilization of Oreobashidiflulanus MW-4218 (KFCC-10754) having fructosyltransspirase activity and β-galactosidase activity as alginate as a carrier. Using a sugar and lactose as a substrate at the same time to convert to fructooligosaccharide and galactooligosaccharide, respectively, a method for producing a complex oligosaccharide mixed with two oligosaccharides was proposed. The substrate solution was synthesized at pH 5-7, 40-60 ° C. containing 50-90% sugar and 10-60% lactose. However, the method of the present invention uses only one enzyme directly without using a cell carrier, using only 0.5-4M of one substrate sugar instead of two substrates, preferably 2.7-4.0M of sugar. The oligosaccharides were synthesized, and oligosaccharides having a structure different from that of Patent 1991-0024070 were synthesized. The origin of the enzyme used is also different.

In Korean Patent 1988-0013695, Aureobasidum pullulans (KFCC 10254) were cultured to immobilize cells in alginate to prepare fructosyltransferase-activated immobilized cells, and then to fixation cells and 30-95%, Preferably, 60-90% (W / V) sugar solution is reacted in a batchwise process at 10-30 hours intervals under a condition of pH 5-6 and a temperature of 45-55 ° C. in a continuous stirring reactor. Fructo oligosaccharides were prepared by fructosyltransferase activating cells. However, in the method of the present invention, using a free levansukraase, not an enzyme immobilized on a carrier, at a concentration of 0.5-4M, which is a high concentration sugar, preferably using only 2.7-4.0M sugar, pH 5-7, The reaction is carried out at 20-60 ° C. to synthesize fructooligosaccharide and oligosaccharide with a new structure. These oligosaccharides have acid and heat resistance.

Korean Patent No. 1993-0030374 discloses fructooligosaccharide by converting beta-fructosyltransferase derived from Oreobassidium, Aspergillus, Fusarium or Gleosporiium into a soy oligosaccharide mixture. A composite oligosaccharide of soy oligosaccharide and fructooligosaccharide was synthesized. In the method of the present invention, however, fructo oligosaccharides and oligosaccharides having a new structure are prepared using a single substrate of high concentration (0.5-4M; preferably 2.7-4.0M) using L. mesenteroides Levansukraase. An acid resistant and heat resistant oligosaccharide is synthesized.

In addition, Korean Patent No. 1991-0022176 discloses the addition of stevioside to a sugar solution, followed by the action of a cell or immobilized enzyme containing a plant or microorganism-derived fructosyltransferase to an aqueous solution substrate, followed by stirring at 30-70 ° C. After reacting, a complex sweetener containing fructooligosaccharide and fructosyl stevioside was synthesized. However, in the present invention, a free enzyme is used to synthesize acid and heat resistant oligosaccharides containing fructo oligosaccharides and oligosaccharides having a new structure using a single substrate of high concentration (0.5-4 M; preferably 2.7-4.0 M). . In addition, the levban sucrease used is L. mesenteroides , which is derived from different enzymes.

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

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

The present invention relates to oligosaccharides that are acid and heat resistant and contain lactose and glucose.

The present invention also relates to a method for producing oligosaccharides having strong acid and heat resistance.

In the method of the present invention, a solution containing sugar at a high concentration of 0.5-4M (preferably 2.7-4.0M) is added and reacted by adding Levansukraase, an enzyme derived from Leuconostoc mesenteroides species strain, to recover the oligosaccharides produced. It consists of doing In the present invention, levan sucrase refers to levan sucrase derived from Leuconostoc mesenteroides . For example, such as L. mesenteroides NRRL B-1355, L. mesenteroides NRRL B-742 (ATCC 13146), L. mesenteroides NRRL B-1299 (ATCC 11499), L. mesenteroides NRRL B-512F (ATCC 10830) L Lesban sukrases from mesenteroides strains, or mutant strains thereof, and Levan sukrases from recombinants of genes obtained from these bacteria. The recovered oligosaccharides were confirmed to have the effect of preventing tooth decay, and confirmed that they had heat resistance and acid resistance.

Example 1 Preparation of Enzyme Liquid

Leuconostoc mesenteroides B-1355 was added to LWG liquid medium [0.3% (w / v) yeast extract, peptone, 0.3% (w / v) K ₂ HPO ₄ , mineral solution (2% MgSO ₄ · 7H ₂ O, 0.1% sodium chloride, 0.1% FeSO ₄ · 7H ₂ O, 0.1% MnSO ₄ · H ₂ O, 0.13% CaCl 2 · 2H ₂ O, 2% (w / v) glucose) for 12 to 16 hours until OD ₆₀₀ reaches 3.0 After incubation, the cells were separated from the culture medium, and the supernatant was prepared as a coenzyme solution, which was then used as a levansukraase enzyme solution.

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

In order to produce oligosaccharides, a 4.5 M sugar solution was prepared, mixed with the enzyme obtained in Example 1 and reacted at 28 ° C. The final sugar concentration of the enzyme reactor was 0.5-4M, 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 glucose released from sugar per ml of enzyme per minute. Indicated. The reaction was carried out until the sugar in the reactor was exhausted. Production of oligosaccharides and full use of sugars were carried out in MeNO / 1-propanol / water (2/5 / 2.5, v / v / v) twice after taking 1 μl of the reaction solution and dropping onto a Merck K6F TLC plate. The components of the 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 using the NIH Image Program.

In addition, in order to identify the constituents of the oligosaccharide, using a different developing solvent to develop in both directions to determine the structure of the sugar by separating each sugar independently. Various developing solvents were developed under different conditions (a → b) in a different order, and then separated phases were confirmed.

Oligosaccharides were added dropwise to develop in the first developing solvent and after complete drying, the standard material was deposited at both ends and developed in a second developing solvent by rotating the orientation 90 °. 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 developing solvent used a) acetonitrile / ethyl acetate / 1-propanol / water = 85/20/50/50 (v / v / v / v), b) nitromethane / 1-propanol / water = 2 / 5 / 1.5 (v / v / v) was used.

The results are shown in FIGS. 1 and 2 and Table 1.

[Table 1] Comparison of Composition and Content Determination of Oligosaccharides of the Present Invention and Commercially Available Oligosaccharides (%)

Oligosaccharide Components Oligosaccharides of the Invention Commercially Available Fructooligosaccharides Glucose 30.26 (including fructose) 40.18 Sucrose 0 9.22 Palatinose 2.15 Leucrose 5.87 GF2 (kestose) 3.31 22.23 unknown product 3.19 GF3 (nystose) 5.47 15.05 unknown product 3.37  2.02 GF4 4.79 7.32 unknown product 1.75  1.01 unknown product 2.81 unknown product 2.91 GF5 2.46  2.97 unknown product 3.65 unknown product 2.78 GF6 4.85 unknown product 2.3 GF7 3.62 unknown product 1.20 GF8 2.78 GF9 0.85 GF10 1.20 Oligosaccharides Over GF10 4.88 Polysaccharide 3.55 total 100 100

Example 3 Identification of Acid and Heat Resistance Characteristics of Oligosaccharides

To the 1000 ml of 3M sucrose, 2400 U (1 ml) of levan sucralase obtained in Example 1 was added and reacted at 28 ° C. for 25 hours 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 oligosaccharide in the enzyme reaction solution using TLC, it was confirmed that it is stable to acidic condition compared to commercially available fructooligosaccharide. 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.

Example 4 Confirmation of Stability of Oligosaccharides in Unsweetened Carbonated Beverages

The oligosaccharides were mixed with a commercially available sugar-free carbonated beverage and allowed to stand at 55 ° C. for 20 hours to confirm the decomposition pattern using TLC. In comparison group, water was added instead of oligosaccharide, and sugar and commercially available fructooligosaccharide were added instead of oligosaccharide to compare the decomposition patterns of the components.

As a result, it was confirmed that after the reaction, commercially available fructooligosaccharides were decomposed into monosaccharides, but oligosaccharides of the present invention were not degraded. The results are shown in FIG.

Example 5 Confirmation of Influence on Growth and Acid Production of S. mutans of Oligosaccharide of the Present Invention

0.5% oligosaccharides were added to BHI liquid medium (0.5% glucose, 2% Brainheart infusion, 0.5% yeast extract, pH 7.0), which is a growth medium of S. mutans , and cultured at 37 ° C. The degree of acid production in the culture was measured. As a comparative group, water was added instead of the oligosaccharide of the present invention and compared.

As a result of confirming the change in the degree of growth and acid production according to the incubation time, it was confirmed that the addition of the oligosaccharide of the present invention inhibited the growth and acid production of S. mutans , which is plaque forming bacteria in the oral cavity. The results are shown in FIG.

The method of the present invention allows the production of fructooligosaccharides from high concentrations of sugar. Since fructooligosaccharide produced by the method of the present invention is resistant to acid and heat, it is suitable as a functional sweetener in foods such as beverages requiring acid resistance and heat resistance.

Claims (2)

A method for producing acid and heat resistant oligosaccharides comprising recovering oligosaccharides produced by adding and reacting levane sucrase derived from Leuconostoc mesenteroides NRRL B-1355 to a solution containing 0.5 to 4 M sugar. delete

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