JPH0630608B2 - Method for producing ethyl-α-glucoside - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (1) Field of Industrial Application The present invention relates to an efficient method for producing ethyl-α-glucoside.

(2) Conventional technology and its problems Alcohol and mirin are used as drinks themselves, and are also used to improve taste in cooking and food processing. The present inventors have analyzed and studied which component of liquor or mirin has this effect. As a result, ethyl-α, which is one of trace components other than alcohol and glucose which are the main components, has been analyzed.
-It has been found that glucoside (hereinafter referred to as EG) is effective in improving the liveness (fish odor) of seafood and the smell of meat.

Conventionally, when maltose, maltotriose, starch syrup and the like obtained from molds, yeasts and the like in an alcohol solution are allowed to act on maltose, maltotriose, starch syrup and the like, a glucosyl group is transferred to alcohol to generate EG. It is known. For example, in the production of mirin, steamed sticky rice is mixed with rice malt and reacted in an alcohol solution of about 20%. At this time, it is considered that α-amylase in koji first decomposes starch to produce oligosaccharides mainly composed of maltose, and then produces EG by α-glucosidase. Similar effects occur during the brewing of sake to produce EG. However, EG generated under this condition
The concentration is low at around 1% at most.

(3) Means for solving the problems The inventors of the present invention diligently investigated the efficient production method of EG, and found that the enzymatic reaction of the α-glucosidase of fungi is considered to be such that the enzymatic reaction hardly proceeds in a high-concentration ethyl alcohol solution. In addition to unexpectedly excellent reactivity when acted on a specific substrate with, a new fact was discovered that the hydrolysis action hardly occurs, and only ethyl alcohol has a specific transfer action, thus increasing EG We were able to establish technology for efficient production. That is, α-glucosidase should react effectively even in a high concentration of alcohol, for example, an alcohol concentration of 40% or more. Also,
It was found that under these conditions, hydrolysis of the substrate, such as maltose, hardly occurred, and maltose produced mainly EG and glucose. Hereinafter, the present invention will be described in detail.

As a substrate in the present invention, in addition to maltose, industrially, a mixed sugar solution of oligosaccharides, for example, maltose, a mixture of maltotriose, starch syrup, and in some cases starch is used, and α-amylase is added to the reaction described above. You may make it parallel. Further, a sugar whose reducing end is hydrogen-reduced can also be used. In this case, since glucose is less produced, it can be adopted as a method for inexpensively producing EG containing no reducing sugar. In the present invention, the α-amylase decomposition product and the product obtained by reducing the reducing end with hydrogen are collectively referred to as a saccharide decomposition product.

The enzyme that can be used in this reaction is α-glucosidase, but among them, α-glucosidase produced by Asp.niger, a type of mold called α-1,6-glucosyltransferase, is preferable. The α-glucosidase derived from yeast is less preferable because it has lower alcohol resistance than that derived from mold. The enzyme can be used in both liquid culture and solid culture. The purified enzyme is of course good, but a crude enzyme can also be used. Rather, the crude enzyme is often found to be more stable in alcoholic solutions, probably due to the presence of impure proteins. However, if a large amount of glucoamylase is present in the enzyme preparation, another reaction occurs and the EG yield is reduced, which is not preferable.

Although the action conditions are not specified, for example, the action temperature was the highest at 45 ° C when the reaction was carried out for 24 hr in the presence of 50% alcohol, but even at 30 ° C, 80% of the amount of EG produced was recognized and can be used. . If the reaction is carried out at a lower temperature, the reaction rate will decrease, but the final amount of EG produced will not be affected. The working pH is in the range of 4-7 and the optimum pH is 6.0. The amount of enzyme added was 0.5 to 30 u (international unit) per gram (g) of substrate (maltose) for the experiment.
The addition of u / g may be sufficient for the purpose.

In the solution in which the reaction has been completed, alcohol, glucose and oligosaccharides are present in addition to EG, as can be seen from the analysis result of Sample A of Example 1 described later. As the food flavoring agent, it can be used as it is or after removing alcohol. A silica gel column is also effective in addition to the activated carbon column to remove substances other than EG.

(4) Action The reason why α-glucosidase derived from Asp. Niger reacts unexpectedly in high-concentration alcohol is not fully understood. However, since Asp.niger-derived α-glucosidase generally contains many sugar chains in its structure, it is in a state of being dissolved or gelated even in a high-concentration alcohol. Therefore, it is considered that the enzyme exhibits stable activity as compared with other enzymes. However, the phenomenon that they react well under such conditions and that the hydrolysis action is remarkably inhibited and only the EG formation reaction is promoted is the first one recognized in this study.

In the present invention, not only the hydrolysis action is inhibited, but also the production of isomaltose and panose, which are the main products of the rearrangement reaction in an aqueous solution, is not increased. Then, a new reaction of alcohol transfer occurred, and we discovered a new fact that this reaction takes precedence.

As an example, using 10% maltose as a substrate, commercially available α-glucosidase 3.9 u / g (international unit) was reacted in ethyl alcohol solutions of various concentrations at 40 ° C. and pH 5.8 for 24 hours, and then glucose was analyzed by HPLC, EG and other oligosaccharides were quantitatively analyzed. From the analysis results, it was judged that the following enzyme reactions shown in to occur in parallel. That is, hydrolysis of maltose into two glucoses Glycosylation from maltose to produce glucose and isomaltose and panose Glycosylation from maltose to produce glucose and EG Among these reactions, a small amount of isomaltose and panose formation Therefore, it can be ignored. It is considered that the ratio of EG / glucose (hereinafter referred to as EG / G) approaches 1.0 when the reaction is more inclined. If the hydrolysis action from the other side is prioritized, it is 0.
33 or less. Therefore, when the analysis result is displayed using this as an index, it becomes as shown in FIG. That is, as the alcohol concentration increases, the EG / G ratio increases, and when the alcohol concentration exceeds 40%, the ratio also exceeds 0.5.
When calculated backward from this value, it can be seen that the transfer action to alcohol occurs more than twice as quickly as the hydrolysis of maltose.

When the alcohol concentration is further increased, the EG / G ratio is also increased. The yield of EG decreases, but when 80% alcohol is used, E
The G / G ratio exceeds 0.8. This numerical value indicates that the hydrolysis-induced transfer action to alcohol occurs about 10 times. That is, in order to carry out the present invention, it is preferable that the alcohol concentration is 30% or more, in which the sugar transfer takes precedence over the hydrolysis of maltose. When maltotriose or maltotetraose is used as a substrate other than maltose,
α-Glucosidase releases glucose one by one from its non-reducing end and transfers it to alcohol. Therefore, it seems that the ratio of EG / G is naturally higher than that of maltose. The results are shown in Table 1, and as expected, the value of maltotetraose was about twice that of maltose.

Substrate concentration was rather problematic, as shown in FIG. 2, the lower the sugar concentration, the higher the EG yield and the EG / G ratio.

When the purified EG is treated with a commercially available mold (Asp. Niger) -derived α-glucosidase, it is decomposed into glucose and ethyl alcohol. However, this reaction rate is 1/100 or less compared to the maltose hydrolysis rate, which is extremely low. That is, maltose rapidly decomposes in an ethyl alcohol solution to produce E
G is produced, but the produced EG is hardly decomposed. It is for this reason that the EG once generated is accumulated in the reaction solution and stably exists. The EG obtained by this action is not decomposed by almond β-glucosidase, so α
Presumed to be binding. Furthermore, the structure could be confirmed by anomeric analysis by gas chromatography.

(5) Examples Example 1 Maltose 100 g was added to 60% ethyl alcohol 1000
1 g of a commercially available α-glucosidase preparation (400 u / g) derived from mold (Asp.niger) was added to the suspension and suspended.
The reaction was carried out at 0 ° C for 24 hours. The reaction solution was heated at 80 ° C. for 10 minutes to inactivate the enzyme, and then the supernatant obtained by removing proteins by centrifugation or filtration was used as sample A (850 m). Further, a part of this was taken and the alcohol was removed by distillation to obtain sample B (400 m). For both, EG, glucose, maltose, isomaltose, and other sugars were separated and quantified by HPLC. Separately, the total sugar value was measured by the phenol-sulfuric acid method, and the content% was calculated.

In both cases, the amounts of EG and glucose were almost the same, and the amounts of other sugars were small. Sample A contains alcohol and sample B can be used as an EG-containing sugar solution containing no alcohol as a taste improver for foods.

When a part of Sample B was taken and subjected to activated carbon column chromatography, purified EG was eluted in a 5-20% alcohol elution section. The yield by column chromatography was about 80%.

(6) Effects of the Invention According to the present invention, it has become possible to produce a large amount of ethyl-α-glucoside at a much lower cost than before.
Since ethyl-α-glucoside is a peculiar taste component as described above, its widespread use for cooking and seasoning is expected in the future.

[Brief description of drawings]

FIG. 1 ... Ethyl alcohol concentration-EG / G (or EG)
FIG. 10% maltose as a substrate and 3.9 u / g of mold-derived α-glucosidase were reacted in ethyl alcohol of various concentrations at 40 ° C. and pH 5.8 for 24 hours, and then subjected to HPLC.
It is a figure when EG and glucose are quantified by. The horizontal axis represents the ethyl alcohol concentration. The vertical axis represents the EG / G ratio in the product as a line graph with the numerical values shown on the left side, and the EG yield (%) from the substrate as a bar graph with the numerical values shown on the right side. FIG. 2 is a diagram showing the relationship between substrate concentration and EG / G (or EG). After using maltose of various concentrations as a substrate, mold-derived α-glucosidase 3.9 u / g was reacted in 50% concentration of ethyl alcohol at 50 ° C. and pH 5.8 for 24 hours, and then H
It is a figure when EG and glucose are quantified by PLC. The horizontal axis shows the substrate concentration. The vertical axis represents the EG / G ratio in the product as a line graph with the numerical values shown on the left side, and the EG yield (%) from the substrate as a bar graph with the numerical values shown on the right side.

Claims (2)

[Claims] 1. A saccharide or a decomposed product thereof is dissolved in a 30 to 80% ethyl alcohol solution, and α-derived from Asp.niger is dissolved in the solution.
-A method for producing an ethyl-α-glucoside, which comprises allowing an ethyl group to be introduced into a saccharide or a decomposition product thereof by causing a glucosidase (α-Glucosidase) to act. 2. Glucose as a saccharide or its decomposition product,
The method for producing ethyl-α-glucoside according to claim (1), characterized in that maltose, other oligosaccharides or substances containing them are used.