JP2008173064A - Method for producing erlose - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing erlose by which the erlose is selectively obtained by using a microorganism catalyst without requiring complicated purification of enzyme in the production of the erlose. <P>SOLUTION: The method for producing the erlose includes using a raw material containing sucrose, and utilizing the microorganism catalyst obtained by culturing a microorganism belonging to Geobacillus stearothermophilus. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for highly selective production of erulose using a raw material containing sucrose and using a microbial catalyst derived from Geobacillus stearothermophilus.

  In recent years, with the diversification of eating habits and social life, consumers' interest in foods, food ingredients, etc. is increasing due to the improvement of health awareness. Among them, erulose is expected as a functional food material because it has anti-tactile properties, and also has attracted attention as a new type of sweetener because it has a taste similar to sucrose.

  Examples of experimental or industrial methods for producing erulose that have been attempted so far include a method for producing erulose using a purified enzyme, and a method for producing sucrose transfer sugar using purified bee-derived α-glucosidase. (Non-patent document 1) and a synthesis method by a transfer reaction using purified α-glucosidase derived from Kluyveromyces marxianus (non-patent document 2) have been reported. In addition to this, a method for producing sucrose-transferred sugars (including erulose), which uses cells obtained by culturing microorganisms belonging to the genus Bacillus as it is without using complicated steps such as enzyme purification. (Patent Document 1).

Japanese Unexamined Patent Publication No. 4-30796 Agric. Biol. Chem., 51 (7), 1859-1864, 1987 Waseda University Institute of Science and Engineering Report 117, 56-61, 1987

  The method for producing erulose using a purified enzyme is an effective method in terms of improving the erulose content in the produced trisaccharide. However, since an enzyme purification step for purifying a target enzyme from an organism containing an elulose-producing enzyme is required and the process becomes very complicated, the production method using this method is difficult to implement industrially. . Furthermore, in the method for producing erulose using the purified α-glucosidase derived from bees, if the enzyme reaction is not stopped at the beginning of the erulose synthesis reaction (after 3 hours), the decomposition of erulose will proceed remarkably and become an enzyme raw material. It is also difficult to acquire a large amount of bees, unlike microorganism-derived enzymes (Non-patent Document 1). In addition, there is a tendency similar to that of purified α-glucosidase derived from bees in the elulose synthesis reaction using purified α-glucosidase derived from Kluyveromyces marxianus. (Non-Patent Document 2).

  On the other hand, the method of using the cells obtained by culturing microorganisms as it is without any complicated steps such as enzyme purification can simplify the process compared to the method using purified enzymes. This is a very effective method. However, when conventionally known microorganisms are used, if microbial cells are used as they are without going through complicated enzyme purification steps, it is possible to obtain erulose as a main component due to the influence of contaminating enzymes other than erulose-producing enzyme. Not possible, and the production of trisaccharides other than erulose such as kestose in the produced trisaccharide will not only make it difficult to separate the contaminated trisaccharide from the final product, but also waste of raw materials due to contaminating enzymes. Will also occur.

  An object of the present invention is to provide a method for producing erulose, which can selectively obtain erulose using a microbial catalyst that does not require complicated enzyme purification in erulose production under such circumstances. It is.

  As a result of intensive investigations to solve these problems, the present inventors have used the microorganism itself as a catalyst without going through an enzyme purification step, and produced erulose with suppressed production of contaminating trisaccharides other than erulose. The method has been found and the present invention has been completed.

That is, this invention is a manufacturing method of the erulose shown to the following (1)-(8).
(1) A method for producing erulose, comprising using a microbial catalyst obtained by culturing microorganisms belonging to Geobacillus stearothermophilus using a raw material containing sucrose.
(2) The microorganism is Geobacillus stearothermophilus AKC-007 strain (Accession No. FERM P-21112), AKC-008 strain (Accession No. FERM P-21113) or AKC-009 strain (Accession No. FERM P-21114). The method for producing elulose according to 1).

(3) The method for producing erulose according to (1) or (2), wherein the erulose content in the produced trisaccharide is 50% or more.
(4) The method for producing erulose according to (1) or (2), wherein the erulose content in the produced trisaccharide is 75% or more.

(5) The method for producing erulose according to any one of (1) to (4), wherein the erulose concentration in the reaction solution is 2% by weight or more.
(6) The method for producing erulose according to any one of (1) to (4), wherein the erulose concentration in the reaction solution is 5% by weight or more.

(7) The method for producing erulose according to any one of (1) to (6), wherein the sucrose concentration in the raw material is 15% by weight or more.
(8) The method for producing erulose according to any one of (1) to (6), wherein the sucrose concentration in the raw material is 50% by weight or more.

  By using the present invention, it is possible to selectively produce erulose without going through complicated steps such as enzyme purification.

Hereinafter, the present invention will be specifically described.
When the present invention is used, the content of erulose in the produced trisaccharide can be improved to 50% or more, 75% or more under more preferable conditions, and 90% or more under more preferable conditions. When the content of erulose in the produced trisaccharide is low, not only is it difficult to purify erulose from the product, but also wasteful consumption of raw sucrose accompanying the production of contaminated trisaccharide becomes a serious problem. Furthermore, when the present invention is used, the concentration of erulose in the reaction solution can be 0.3% by weight or more, more preferably 2% by weight or more, and even more preferably 5% by weight or more. And can be easily separated from the reaction solution.

  The contaminated trisaccharide in the present invention is a trisaccharide oligosaccharide other than elulose, and examples thereof include maltotriose, isomaltotriose, melezitose, panose, isopanose, cellotriose, and kestose.

  As the microbial catalyst used for the production of erulose, microorganisms obtained by a usual culture method can be used, and it is not necessary to purify erulose synthase from the microorganisms. In some cases, a microorganism culture solution or a microorganism culture supernatant can be used. On the other hand, the microorganisms obtained by the culturing method can be used after washing with water or a buffer as required. For example, a culture solution of a cultured microorganism, a microorganism suspension obtained by centrifugation, washing with a buffer, an aqueous solution in which a microorganism or a processed microorganism product (for example, crushed microorganisms) is suspended or dissolved, or a microorganism Alternatively, a product obtained by immobilizing a processed microorganism product by a comprehensive method, a crosslinking method, or a carrier binding method can be used. Examples of the immobilization carrier used for immobilization include, but are not limited to, glass beads, silica gel, polyurethane, polyacrylamide, polyvinyl alcohol, carrageenan, alginic acid and the like.

As the microorganism used in the present invention, any microorganism belonging to Geobacillus stearothermophilus may be used, and any microorganism having an activity of synthesizing erulose from sucrose can be used. Preferably, Geobacillus stearothermophilus AKC-007 strain, AKC-008 strain, and AKC-009 strain are mentioned. In addition, the microorganism in the present invention may be a mutant obtained by using a microorganism belonging to Geobacillus stearothermophilus as a parent strain. Geobacillus stearothermophilus AKC-007, AKC-008, and AKC-009 were respectively registered on November 30, 2006 by the National Institute of Advanced Industrial Science and Technology Patent Biology Center (Higashi 1-chome Tsukuba, Ibaraki, Japan). It is deposited at address 1 center 6). The accession numbers are as follows.
AKC-007 strain (FERM P-21112)
AKC-008 strain (FERM P-21113)
AKC-009 strain (FERM P-21114)

  As a method for culturing microorganisms used in the present invention, ordinary aeration and agitation cultures or solid cultures are used, and generally used microorganism cultivation methods can be applied. As the medium, a synthetic medium or a natural medium containing a carbon source, a nitrogen source, an inorganic salt, a necessary nutrient source and the like necessary for the microorganism to grow well and to smoothly produce erulose synthase in the microorganism. Is mentioned. For example, as the carbon source, glucose, glycerol, sucrose, galactose, lactose, raffinose, cellobiose, erulose, organic acid, starch, olive oil, soybean oil and the like can be used. Examples of nitrogen sources include ammonium sulfate, ammonium nitrate, urea, amino acids, amines, ammonia, ammonium salts of various inorganic and organic acids, other nitrogen-containing compounds, peptone, tryptone, polypeptone, meat extract, yeast extract, cottonseed meal , Corn steep liquor, and soybean meal. Moreover, as inorganic salts, primary potassium phosphate, dibasic potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, manganese sulfate, copper sulfate, iron sulfate, calcium carbonate and the like are used. The culture temperature is preferably 25 to 80 ° C, more preferably 40 to 65 ° C, still more preferably 50 to 60 ° C. Moreover, PH of a culture medium can be selected in a wide range, for example, 3-9 are preferable.

  In the present invention, in order to improve the erulose concentration in the reaction solution immediately after the erulose synthesis reaction by the microbial catalyst, it is advantageous that the sucrose concentration of the raw sugar solution is higher. The raw sucrose concentration is preferably 15% by weight or more, more preferably 50% by weight or more, and still more preferably 70% by weight or more.

  Reaction temperature is 5-90 degreeC, More preferably, it is 30-80 degreeC, More preferably, it is 50-70 degreeC. When the reaction temperature is less than 5 ° C, the reaction rate is extremely low, and in the temperature range exceeding 90 ° C, the enzyme activity is rapidly deactivated and a large amount of microbial catalyst is required, which is not preferable. The reaction pH can be adjusted in a wide range, preferably pH 3 to 10, more preferably pH 4.5 to 8.5. A reaction pH of less than 3 or greater than 10 is not preferable because the deactivation of the catalyst is significantly accelerated. In the present invention, it is not seen that the decomposition of erulose proceeds significantly in the late stage of the reaction, but considering industrial use, the reaction time is preferably 20 minutes to 200 hours, more preferably 1 to 120 hours. However, the present invention is not limited to the above reaction conditions and reaction forms, and can be appropriately selected.

Elulose obtained in the present invention is measured by the following method.
[Measurement method of erulose content in the produced trisaccharide]
After completion of the erulose synthesis reaction, the reaction solution was diluted 25 times and held at 99 ° C. for 10 minutes to stop the reaction. After the reaction was stopped, microorganisms were removed by centrifugation, and the resulting reaction solution was quantified using high performance liquid chromatography (HPLC). For the measurement, a Hypercarb column manufactured by Thermo Electron was used, and RI was used as a detector. The erulose content in the produced trisaccharide was calculated from (the peak area of erulose) / (peak area of the produced trisaccharide) × 100 from the ratio of the peak areas detected in the HPLC analysis chart.

  As a method for purifying and separating the erulose produced by the method of the present invention, a commonly used purification method can be used. That is, for example, centrifugation, MF membrane or UF membrane treatment, filter press etc. removes the microbial catalyst, and buffering by chromatographic treatment such as cation exchange chromatography or anion exchange chromatography or desalting treatment such as dialysis. Removes salts, etc. brought in from liquids and culture media, and further uses cation exchange chromatography, anion exchange chromatography, high performance liquid chromatography, activated carbon chromatography, etc., and crystallization treatment using differences in solubility, etc. Elulose can be separated and purified according to other conventional methods. For chromatographic treatment, these methods may be used alone or in combination, and a moving bed method, a pseudo moving bed method, a multi-component separation simulated moving bed method, a multi-component separation circulation method, etc. may be used as appropriate. Can do. When these separation and purification methods are used, it is possible not only to separate erulose from other contaminating oligosaccharide components, but also from a plurality of oligosaccharides having various binding forms or different molecular weights. These purification methods of elulose and separation may be performed batchwise or continuously using a column or the like.

  Hereinafter, although an example explains concretely, the present invention is not limited at all by these examples.

Example 1
Geobacillus stearothermophilus AKC-007 strain (Accession No. FERM P-21112: Depositary; National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) cultured on TBAB (Tryptose Blood Agar Base) plate (Difco) at 55 ° C for 1 day To form colonies. The 1 platinum loop was inoculated into a medium-X (see Table 1 and Tables 2 to 4) 30 mL dispensed into a 150 mL Erlenmeyer flask and cultured at 55 ° C. and 150 rpm for 1 day.

  One day after the main culture, 10 mL of cultured cells were collected in a 15 mL tube. The 15 mL tube from which the culture solution was collected was centrifuged at 10,000 rpm, and the supernatant was removed. Next, 1 mL of 100 mM Na acetate buffer (pH 5) was added and resuspended, and then the suspension was transferred to a 2 mL eppen. After centrifuging the eppen again and removing the supernatant, add 300 μL of the raw sugar solution S80 (80% by weight of sucrose, 100 mM Na acetate (pH5)) and suspend the cells well with a vortex mixer to synthesize the sugar. The reaction was started. The sugar synthesis reaction was performed at a reaction temperature of 60 ° C. and a rotation speed of 1200 rpm. Two and 21 hours after the start of the sugar synthesis reaction, 20 μL of the reaction solution was recovered, mixed well with 480 μL of distilled water, and the enzyme was heat-inactivated at 99 ° C. for 10 minutes. Each diluted sugar solution was returned to room temperature and then subjected to HPLC analysis (Hypercarb column). Table 5 shows the erulose concentration in each reaction solution and the erulose content in the produced trisaccharide. In addition, the HPLC chart of the reaction for 21 hours is shown in FIG. Fractions corresponding to Peak-2 in FIG. 1 were collected and analyzed by 13C-NMR. As a result, it was confirmed that Peak-2 corresponds to erulose. The other peaks were contaminated oligosaccharides.

Example 2
Geobacillus stearothermophilus AKC-008 strain (Accession number FERM P-21113: Depositary; National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) is cultured on TBAB (Tryptose Blood Agar Base) plate (Difco) at 55 ° C for 1 day To form colonies. The one platinum loop was inoculated into a medium-X (see Table. 1) 30 mL dispensed into a 150 mL Erlenmeyer flask and cultured at 55 ° C. and 150 rpm for 1 day.

 One day after the main culture, 10 mL of cultured cells were collected in a 15 mL tube. The 15 mL tube from which the culture solution was collected was centrifuged at 10,000 rpm, and the supernatant was removed. Next, 1 mL of 100 mM Na acetate buffer (pH 5) was added and resuspended, and then the suspension was transferred to a 2 mL eppen. After centrifuging the eppen again and removing the supernatant, add 300 μL of the raw sugar solution S80 (80% by weight of sucrose, 100 mM Na acetate (pH5)) and suspend the cells well with a vortex mixer to synthesize the sugar. The reaction was started. The sugar synthesis reaction was performed at a reaction temperature of 60 ° C. and a rotation speed of 1200 rpm. Two and 21 hours after the start of the sugar synthesis reaction, 20 μL of the reaction solution was recovered, mixed well with 480 μL of distilled water, and the enzyme was heat-inactivated at 99 ° C. for 10 minutes. Each diluted sugar solution was returned to room temperature and then subjected to HPLC analysis (Hypercarb column). Table 5 shows the erulose concentration in each reaction solution and the erulose content in the produced trisaccharide.

Example 3
Geobacillus stearothermophilus AKC-009 strain (Accession No. FERM P-21114: Depositary Institution; National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) is cultured on TBAB (Tryptose Blood Agar Base) plate (Difco) at 55 ° C for 1 day To form colonies. One platinum loop was inoculated into a medium-X (see Table 1) 30 mL dispensed into a 150 mL Erlenmeyer flask and cultured at 55 ° C. and 150 rpm for 1 day.

 One day after the main culture, 10 mL of cultured cells were collected in a 15 mL tube. The 15 mL tube from which the culture solution was collected was centrifuged at 10,000 rpm, and the supernatant was removed. Next, 1 mL of 100 mM Na acetate buffer (pH 5) was added and resuspended, and then the suspension was transferred to a 2 mL eppen. After centrifuging the eppen again and removing the supernatant, add 300 μL of the raw sugar solution S80 (80% by weight of sucrose, 100 mM Na acetate (pH5)) and suspend the cells well with a vortex mixer to synthesize the sugar. The reaction was started. The sugar synthesis reaction was performed at a reaction temperature of 60 ° C. and a rotation speed of 1200 rpm. Two and 21 hours after the start of the sugar synthesis reaction, 20 μL of the reaction solution was recovered, mixed well with 480 μL of distilled water, and the enzyme was heat-inactivated at 99 ° C. for 10 minutes. Each diluted sugar solution was returned to room temperature and then subjected to HPLC analysis (Hypercarb column). Table 5 shows the erulose concentration in each reaction solution and the erulose content in the produced trisaccharide.

Example 4
In addition to the raw sugar solution S80, the raw sugar solution S62.5 (sucrose 62.5% by weight, 100 mM Na acetate buffer (pH5)), the raw sugar solution S40 (sucrose 40.0% by weight, 100 mM Na acetate buffer (pH 5) )), And a raw sugar solution S20 (sucrose 20.0% by weight, 100 mM Na acetate buffer (pH 5)) was used to carry out a sugar synthesis reaction in the same manner as in Example 1. In the sugar synthesis reaction performed using each raw sugar solution, 42 μl after the start of the sugar synthesis reaction, 20 μL of the reaction solution was collected, mixed well with 480 μL of distilled water, and the enzyme was heat-inactivated at 99 ° C for 10 minutes It was. The diluted sugar solution was returned to room temperature and then subjected to HPLC analysis (Hypercarb column). The results are shown in Table 6.

Example 5
In addition to the raw sugar solution S80, the raw sugar solution S62.5 (sucrose 62.5% by weight, 100 mM Na acetate buffer (pH5)), the raw sugar solution S40 (sucrose 40.0% by weight, 100 mM Na acetate buffer (pH 5) )), A raw sugar solution S20 (sucrose 20.0 wt%, 100 mM Na acetate (pH 5)) was used, and a sugar synthesis reaction was carried out in the same manner as in Example 2. In the sugar synthesis reaction performed using each raw sugar solution, 20 μL of the reaction solution was collected 114 hours after the start of the sugar synthesis reaction, mixed well with 480 μL of distilled water, and the enzyme was heat-inactivated at 99 ° C. for 10 minutes. It was. The diluted sugar solution was returned to room temperature and then subjected to HPLC analysis (Hypercarb column). The results are shown in Table 7.

FIG. 1 shows an HPLC chart of erulose synthesis solution by AKC-007 strain (reaction 21h).

Claims (8)

A method for producing erulose, comprising using a microbial catalyst obtained by culturing microorganisms belonging to Geobacillus stearothermophilus using a raw material containing sucrose. The microorganism is Geobacillus stearothermophilus AKC-007 strain (Accession number FERM P-21112), AKC-008 strain (Accession number FERM P-21113) or AKC-009 strain (Accession number FERM P-21114). The manufacturing method of the elulose of description. The method for producing erulose according to claim 1 or 2, wherein the erulose content in the produced trisaccharide is 50% or more. The method for producing erulose according to claim 1 or 2, wherein the erulose content in the produced trisaccharide is 75% or more. The method for producing erulose according to any one of claims 1 to 4, wherein the erulose concentration in the reaction solution is 2% by weight or more. The method for producing erulose according to any one of claims 1 to 4, wherein the erulose concentration in the reaction solution is 5% by weight or more. The method for producing erulose according to any one of claims 1 to 6, wherein the sucrose concentration in the raw material is 15% by weight or more. The method for producing erulose according to any one of claims 1 to 6, wherein the sucrose concentration in the raw material is 50% by weight or more.

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