JP2002125692A - METHOD FOR PRODUCING ETHYL-alpha-D-GLUCOSIDE - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an efficient method for producing high-purity ethyl-α-D- glucoside. SOLUTION: This method for producing ethyl-α-D-glucoside comprises making a Talaromyces dupontii-derived transglucosidase act on starch or a split product thereof in the presence of ethanol.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for efficiently producing ethyl-α-D-glucoside with high purity.

[0002]

2. Description of the Related Art Ethyl-α-D-glucoside is a taste component that contributes to the taste and richness of cooked foods, and has a refreshing sweetness like glucose and a unique bitterness. Is a component contained in trace amounts. As a method for producing ethyl-α-D-glucoside, Schizosaccharomyces pombe is allowed to act on an ethanol solution containing a saccharide (Japanese Patent Publication No. 5-56958), or α-glucosidase derived from Aspergillus niger is contained as a saccharide. Method to act on ethanol solution (Japanese Patent Publication No. 6-306)
No. 08) is known. However, the purity of the ethyl-α-D-glucoside obtained by these methods, in other words, the content relative to the sugar composition, is low, and a further separation operation is required to obtain high purity. For example, when maltose is used as a substrate, relatively high-purity ethyl-α-D-glucoside can be obtained, but even in this case, the content based on the sugar composition is 50% or less. In addition, in any method, when producing ethyl-α-D-glucoside, it is essential to prepare an ethanol solution containing a saccharide. In general, the solubility of carbohydrates in ethanol is low, and therefore, the drawback is that the higher the ethanol concentration, the lower the carbohydrate concentration available as a substrate. In view of such circumstances, the present applicant has proposed a highly pure and efficient method for producing ethyl-α-D-glucoside, which overcomes the disadvantages of the conventional method, in the presence of a microorganism having alcohol fermentation ability. Sugars,
An enzyme having α-1,6-glucosyltransferase activity on starch or its decomposed product, or a method for producing ethyl-α-D-glucoside characterized by reacting this enzyme with an amylase agent has been developed, and a patent application has already been filed. (Japanese Unexamined Patent Publication No. Hei 11
No. 243987).

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a more efficient method for producing ethyl-α-D-glucoside.

[0004]

Means for Solving the Problems The present inventors have proposed ethyl-α-D
-Talaromyces Dupont (Talaro) has been working diligently on the efficient and high-purity production of glucosides.
myces duponti), α-1,6-derived Aspergillus sp.
Ethyl-α is more efficiently used than when α-glucosidase, an enzyme having glucosyl transfer activity, is used.
It has been found that -D-glucoside can be produced, and the present invention has been completed. The transglucosidase is an enzyme disclosed in U.S. Patent Nos. 4,689,296 and 5,773,256 and the like, and the Talaromyces duponti FRI456 described therein.
6 obtained by growing it in an appropriate nutrient medium.

[0005] That is, the present invention relates to the use of Talalomyces in a sugar, starch or a decomposition product thereof in the presence of ethanol.
Ethyl-characterized by the action of transglucosidase derived from Talaromyces duponti.
The present invention provides a method for producing α-D-glucoside, and examples of the saccharide, starch, or a degradation product thereof include glucose, maltose, isomaltose, other oligosaccharides, and substances containing them. The present invention also includes the ethyl-α-D-glucoside-containing product obtained by the method of the present invention.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION As the saccharide, starch or its decomposed product used, glucose, maltose, isomaltose, panose and other oligosaccharides, or a mixture thereof, for example, a commercially available oligosaccharide mixture (trade name: isomalt 500 Showa Sangyo Co., Ltd., Trade name: Isomalt 9
00 Showa Sangyo Co., Ltd.), syrup and the like.
Further, saccharified rice flour commonly used in sake brewing is also preferably used. When the saccharide to be used contains an oligosaccharide having a relatively high degree of polymerization, such as an oligosaccharide mixture or a rice flour saccharified solution, for example, pentasaccharide or more, it is better to coexist α-amylase with the enzyme. Disaccharides that serve as substrates for
As the saccharide content increases, the yield of ethyl-α-D-glucoside increases. In some cases, liquefaction and saccharification enzymes such as starch and an amylase agent may be added to a reaction solution for carrying out the method of the present invention, and a saccharification solution may be directly prepared in the reaction solution.

In the method of the present invention, ethanol is added to a reaction solution at an appropriate concentration, for example, 10 to 60 v / w%, preferably,
An enzyme reaction is performed by adding 30 to 50 v / w%. Alternatively, alcohol fermentation may be performed in the reaction solution in the presence of a microorganism having alcohol fermentation ability to produce ethanol. The microorganism to be used is not particularly limited as long as it has alcohol fermentation ability, but from the viewpoint of high alcohol fermentation ability and alcohol resistance, yeasts for brewing, for example, sake yeast, wine yeast, beer yeast, etc. desirable.

The transglucosidase used in the present invention is, as described above, an enzyme disclosed in US Pat. Nos. 4,689,296 and 5,773,256 and the like. Talaromyces
duponti) It is obtained by growing FRI4566 or Talalomyces Dupont ATCC 20805 in an appropriate nutrient medium.

The conditions under which the enzyme acts are not particularly limited as long as the transglucosidase activity is exhibited, but the reaction temperature is usually 10 to 50 ° C., preferably 40 to 50 ° C. The pH of the reaction solution was pH 4.
0-7.0, preferably pH 4.5-5.5. Also,
When using a microorganism having an alcohol fermentation ability, for example, brewer's yeast, the culturing may be performed prior to the addition of transglucosidase, or may be performed simultaneously with the enzymatic reaction to improve the efficiency of producing ethyl-α-D-glucoside. Has no effect. Here, the following points must be taken into account when culturing yeast. That is, when a saccharified solution contains a nitrogen source and a trace component essential for yeast growth, such as a rice flour saccharified solution, the yeast may be directly inoculated into the reaction solution. A substrate that does not contain the components necessary for, for example,
When using high-purity maltose or the like, a nitrogen source, for example, yeast extract, polypeptone, or the like, or inorganic salts, for example, magnesium, potassium, or calcium phosphate, may be added to the reaction solution as necessary. Is desirable. The substrate concentration in the reaction solution is, for example, 5 to 30% by weight, preferably 10 to 20% by weight when maltose is used as the substrate.
It is. Even when using rice flour saccharified liquid, the sugar component content,
10-20% by weight is preferred. Further, the substrate addition is not limited to once. The production amount of ethyl-α-D-glucoside, the remaining amount of glucose or the remaining amount of isomaltose are determined by HP.
By performing analytical monitoring using LC,
For example, by adding at an interval of 2 to 3 days, it is possible to increase the production amount while maintaining high-purity ethyl-α-D-glucoside. According to the activity unit (U) using an α-glucosidase measurement kit manufactured by Kikkoman, 1-10 U was added per gram of substrate (maltose). As a result, 10 U / g maltose was sufficiently efficiently added to the enzyme. It was found that -α-D-glucoside could be produced.

The solution after the completion of the reaction has a concentration of 5 to 10% by weight and a purity of 90% depending on the concentration or the number of additions of the saccharide.
% Or more of ethyl-α-D-glucoside.
By removing yeast and the like from the solution by a commonly used operation such as centrifugation, high-purity ethyl-α-
A fermented liquor containing D-glucoside is obtained. This fermentation liquor
It can be used as it is as a flavoring agent for food. Also, distillation,
An ethyl-α-D-glucoside-containing liquid from which ethanol has been easily removed can be obtained by performing an evaporator, a concentration centrifugation, or the like. Furthermore, a syrup containing high purity ethyl-α-D-glucoside can be prepared by appropriately combining operations such as treatment with activated carbon, ion exchange chromatography, adsorption chromatography, and concentration, if necessary.
By adding this syrup to foods, beverages and alcoholic beverages, it can be used for improving the taste of sweetness, bitterness and flavor. For example, when a sake liquor test was performed on a sample in which this syrup was added to sake, opinions were obtained that pointed out a quick-acting sweetness, a slow-acting bitterness, and astringency, and were clearly different from the conventional sweetness of glucose. It has been found that sake with both sweet and bitter taste can be provided.

[0011]

EXAMPLES Next, the present invention will be described more specifically with reference to Reference Examples and Examples, but the present invention is not limited thereto. Reference Example 1 Preparation of enzyme preparation derived from Talalomyces duponti Maltose 2% by weight, wheat bran 1% by weight, malt extract (Difco) 0.5% by weight, bactopeptone (Difco) 0.5% by weight, yeast extra (D
ifco) 1% by weight, K ₂ HPO ₄ 0.1% by weight, MgSO
₄ 3 L of a medium containing 0.05% by weight and 0.01% by weight of CaCl ₂ was adjusted to pH 7.0 and sterilized. Then, preculture 1 of Talalomyces Dupont ATCC 20805
After inoculating 00 ml, the cells were cultured in a jar fermenter at a culture temperature of 37 ° C., an aeration rate of 1 vvm and a stirring speed of 350 rpm for 4 days. The culture is centrifuged to a pore size of 0.4
The cells were removed by filtration with a 5 μm membrane filter, and the supernatant was concentrated with an ultrafiltration membrane having a molecular weight cut off of 10,000, followed by lyophilization to prepare 2.4 g of a crude enzyme preparation.
When the α-glucosidase activity of this enzyme was measured by Kikkoman α-glucosidase measurement kit,
8.76 U / g powder.

Example 1 Comparison of productivity of ethyl-α-D-glucoside between enzyme preparation derived from Talalomyces dupont and enzyme preparation derived from Aspergillus niger 20 mM acetate buffer containing 10% by weight maltose concentration ( pH5.
0) ethanol at a concentration of 20, 30, 40 or 50 v
/ W%. To this, an enzyme preparation derived from Talalomyces Dupont ATCC 20805 or α-glucosidase “Amano” (derived from Aspergillus niger, manufactured by Amano Pharmaceutical Co., Ltd.) obtained in Reference Example 1 was added so as to have a concentration of 10 U / g maltose. Reacted. The sugar composition in the reaction solution was measured by HPLC. FIG. 1 shows ethyl-α-D-glycoside (hereinafter referred to as α-E) in the reaction solution.
G) (abbreviated as G) and the ratio of α-EG / glucose (hereinafter abbreviated as α-EG / Glc).

As is clear from FIG. 1, 20 to 50 v /
At each alcohol concentration of w%, the amount of α-EG produced by the enzyme preparation derived from Talalomyces duponti was larger than that of the enzyme preparation derived from Aspergillus niger, and α-EG / G
It can be seen that the ratio of lc is also high. In particular, alcohol concentration 5
At 0 v / w%, in the enzyme preparation derived from Talalomyces duponti, α-EG is 43.59 mg / ml, glucose is 49.21 mg / ml, and the ratio of α-EG / Glc is 0.89. In the enzyme preparation derived from Aspergillus niger, α-EG was 33.02 mg / ml, glucose was 77.12 mg / ml, and the ratio of α-EG / Glc was 0.43.

Usually, in the above reaction solution, the following reactions (1) to (3) are considered to occur in parallel by the action of α-glucosidase. (1) Hydrolysis reaction of maltose to two molecules of glucose (2) Glycosyl transfer reaction of maltose to produce glucose and isomaltoligosaccharide such as isomaltose and panose (3) One molecule of glucose and one molecule of α from maltose
Glycosyltransfer Reaction Generated by -EG If it is assumed that only the reaction (3) in which α-EG is generated occurs, one glucose molecule and one α-EG molecule are generated. That is, the ratio of α-EG / Glc in this reaction is 1.0 at the maximum. In this example, when the enzyme preparation derived from Aspergillus niger was used, the ratio of α-EG / Glc was about 0.4, whereas when the enzyme preparation derived from Talalomyces duponti was used, the ratio was 0.1%. 9, which is a value close to 1.0, which indicates that α-EG is generated in preference to other reactions. On the other hand, when the reaction was carried out using an enzyme agent derived from Aspergillus niger under the condition of an alcohol concentration of 80 v / w%, α-EG / Glc
Is increased to about 0.8, but the amount of α-EG generation is significantly reduced. Therefore, the enzyme preparation derived from Talaromyces duponti has a high production amount of α-EG and a small production amount of glucose as a by-product.
Optimum as an enzyme for G production.

[0015]

According to the present invention, it becomes possible to industrially produce ethyl-α-D-glucoside efficiently and with high purity.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the relationship between the amount of ethyl acetate in a reaction solution obtained when an enzyme preparation derived from T. duponti and an enzyme preparation derived from Aspergillus niger were used at various ethanol (Alc) concentrations. α-D-glycoside (EG) production amount and the ratio of EG / glucose (EG / G
lc).

Continued on the front page. (72) Inventor Toshiki Mineki 4-9, Imazu-Iizai-cho, Nishinomiya-shi, Hyogo Ozeki Research Institute Co., Ltd. (72) Inventor Kenji Ozeki 4-9, Imazu-Iizai-cho, Nishinomiya-shi, Hyogo Within the Research Institute, Inc. (72) Inventor Chieko Kumagai 4-9, Iizazu-cho, Imazu, Nishinomiya-shi, Hyogo Ozeki F-term within the Research Institute, Inc. (Reference) 4B015 MA03 4B064 AF41 CA21 CB30 CC03 CD09 CD19 DA10

Claims (3)

[Claims] 1. The method of claim 1 wherein a sugar, starch or a decomposition product thereof is added to Talaromyces Dupont in the presence of ethanol.
A process for producing ethyl-α-D-glucoside, which comprises reacting transglucosidase derived from myces duponti). 2. The method according to claim 1, wherein glucose, maltose, isomaltose, other oligosaccharides or a substance containing them is used as the saccharide, starch or its decomposed product.
The method for producing ethyl-α-D-glucoside according to the above. 3. An ethyl-α-D-glucoside-containing substance obtained by the method according to claim 1 or 2.