JP2001286294A - Method for producing l-arabinose, l-arabinose-containing enzyme treated product and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for easily and inexpensively producing L- arabinose and L-arabinose-containing enzyme treated product. SOLUTION: In the case that the enzyme showing the ability of isolating L-arabinose by acting on a natural product containing arabinan, arabinoxylan or arabinogalactan is acted on the natural product containing arabinan, arabinoxylan or arabinogalactan to produce L-arabinose, this method for producing L-arabinose features producing L-arabinose by directly acting the above enzyme without isolating and extracting arabinan, arabinoxylan or arabinogalactan from the above natural product.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing L-arabinose, an L-arabinose-containing enzyme-treated product, and a method for producing the same.

[0002]

2. Description of the Related Art L-arabinose is a non-caloric sweetener having a taste similar to that of sucrose and exhibiting poor absorption. In addition, since it inhibits an enzyme that hydrolyzes disaccharides such as sucrose, the effect of suppressing an increase in blood glucose level when sucrose is taken is also known. For this reason, attention has been focused on calorie-regulated foods for people who are worried about blood sugar levels, sweeteners, diabetic complication suppression, and the like. L-arabinose is a sugar that is also useful as a raw material for synthesizing pharmaceuticals. L-arabinose is present in hemicellulose of higher plants as arabinan, arabinoxylan, arabinogalactan and the like. In a free state, it is contained in fermented foods such as miso and liquor, instant coffee, etc., albeit in a trace amount.

Heretofore, L-arabinose has been produced by alkaline extraction of hemicellulose contained in corn fiber, gum arabic, beet pulp and the like, and acid-decomposing it. The acid decomposition method, which is a production method of these, has a high possibility that carcinogens that are unsuitable as food and pharmaceutical raw materials are generated, and requires a special reaction device. At present, this method cannot be said to be a sufficient production method, for example, since the post-processing is complicated. The operation of alkali-extracting various types of hemicellulose from natural raw materials requires a complicated operation and costs, and thus has been a major problem in inexpensively producing L-arabinose.

A method has been reported in which beet pulp is used as a raw material of L-arabinose, which is subjected to alkali heat treatment, precipitated with ethanol, and then extracted with arabinan, followed by acid decomposition or enzymatic decomposition (Japanese Patent Application Laid-open No. Hei 9-1997). −29
No. 9093, Journal of the Japanese Society of Agricultural Chemistry, Vol. 49, No. 6, 295-
305, 1975). However, this method has a problem that the operation of extracting arabinan from natural raw materials is complicated, and it has been considered that this method is not practical in terms of low L-arabinose yield and inexpensive industrial production. Was. Report by Spagnuolo et al. (Biotechnolo
gy and Bioengineering, Vol. 64, No. 6, 685-69
1, 1999), arabinose was obtained in high yield, but this method was carried out in addition to a deproteinization treatment in which beet pulp was allowed to act with a protease and filtration, followed by an autoclave treatment (at 121 ° C for 20 minutes). Since the processing is performed, the operation becomes complicated, and there is a problem in aiming for mass production. As a method for producing L-arabinose by acid decomposition, a method described in JP-A-11-313700 is known, but an apparatus for performing an acid treatment at 100 ° C. or more is required, or a neutralization operation is required. Or
The operation was complicated and the economy was low. The residue after obtaining arabinose was waste.

[0005]

As described above, while L-arabinose has high functionality, the conventional method for producing L-arabinose is expensive for practical use in applications such as food and pharmaceutical raw materials. It is a factor that hinders the development. An object of the present invention is to provide a method for inexpensively producing L-arabinose, an enzymatically treated L-arabinose-containing natural product containing arabinan, araginoxylan, and arabinogalactan, and a method for producing the same.

[0006]

Means for Solving the Problems The present inventors have made intensive studies to solve such problems, and have found that arabinan,
An enzyme having an activity of releasing L-arabinose by acting on a natural product containing arabinoxylan or arabinogalactan, without separating and extracting arabinan, arabinoxylan or arabinogalactan from arabinan, arabinoxylan or arabinogalactan-containing natural product. It has been found that L-arabinose can be produced at low cost by directly acting, and the present invention has been completed. That is, the first aspect of the present invention is to act on an enzyme having an activity of releasing L-arabinose by acting on a natural product containing arabinan, arabinoxylan or arabinogalactan, and acting on a natural product containing arabinan, arabinoxylan or arabinogalactan. Upon obtaining L-arabinose, without separating and extracting arabinan, arabinoxylan or arabinogalactan from the natural product,
The present invention is directed to a method for producing L-arabinose, wherein L-arabinose is obtained by directly acting the enzyme, and a second aspect of the present invention is a natural method containing arabinan, arabinoxylan or arabinogalactan. Thing is,
An L-arabinose-containing enzyme-treated product characterized by containing L-arabinose, and a third aspect of the present invention is to act on a natural product containing arabinan, arabinoxylan or arabinogalactan. An enzyme having an activity of releasing L-arabinose is directly acted on a natural product containing arabinan, arabinoxylan or arabinogalactan to release L-arabinose to obtain the L-arabinose-containing enzyme-treated product. The present invention provides a method for producing a treated product containing L-arabinose.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The contents of arabinan, arabinoxylan and arabinogalactan used in the present invention include apples, beets (sugar beets), soybeans, corns, rice, wheat and the like, as well as the remaining orange fiber and mandarin oranges. Examples include by-products such as juice lees, apple fiber, apple juice lees, beet fiber, beet pulp, peanut lees, rice bran, corn lees, soybean lees, corn fiber and groundnut oil grounds. The use of waste and by-products as raw material blades is a very preferable method not only from the viewpoint of low-cost production but also from the environmental protection aspect of effective use of industrial waste. The orange fiber or orange juice juice residue is a residue obtained by extracting juice from oranges or oranges, and contains about 3 to 10% of L-arabinose in the form of arabinan or the like. Apple fiber is a residue after extracting apple juice from apple and contains about 4 to 7% by weight of L-arabinose in the form of arabinan or the like. Beet pulp is a residue obtained by extracting beet sugar liquid from beets, and contains about 12 to 18% of L-arabinose in the form of arabinan or the like. Peanut meal is, for example, peanut kara, and contains about 5% L-arabinose in the form of arabinan or the like. The arabinans contained therein are characterized by having a linear structure in which L-arabinose is linked, and L-arabinose is generated relatively easily by the enzyme. In that regard, beet pulp, apple fiber, orange fiber, and the like, from which L-arabinose is easily released, can be said to be good raw materials. In addition, rice bran and corn fiber containing arabinoxylan, soybean meal containing arabinogalactan, and the like are also good raw materials. Any of these can be used as long as they occur in a normal exploitation operation, even if they are natural products having arabinan, arabinoxylan, or arabinogalactan of any origin and production method.

[0008] Examples of the enzyme having an activity of releasing L-arabinose by acting on an arabinan-containing substance used in the present invention include arabinan-degrading enzymes such as arabinase (arabanase) and arabinofuranosidase. The origin of arabinan-degrading enzymes is bacteria (Bacillus su
btilis, Streptomyces sp.), yeast (Rhodotorulas)
p.), filamentous fungi (Aspergillus niger, A.oryzae, A.pulv
erulentus, A. terreus, A. japonicus, A. flavus, Trich
oderma reesei, T. viride, Trichosporon penicillat
um, Rhizopus sp.), etc., but Aspergillus
Enzymes of origin are preferred. Particularly, an enzyme derived from Aspergillus niger is preferable.

[0009] These enzymes are produced in a culture supernatant or cells obtained by culturing the above-mentioned strains by a conventionally known method. In the present invention, any enzyme containing these enzymes is used. Fractions may be used. Further, if necessary, fractions containing these enzymes can be purified or partially purified by a conventional method and used. Alternatively, a commercially available enzyme may be used. In the present invention, it is particularly preferable to use arabinase and Sumiteam ARS manufactured by Shin Nippon Chemical Co., Ltd. as highly active commercially available enzymes.

Commercially available cellulase and hemicellulase enzyme agents such as xylanase, pectinase and galactanase may also show an activity to release L-arabinose when acted on arabinan, arabinoxylan and arabinogalactan-containing substances. Some of these enzymes have arabinan, arabinoxylan, and arabinogalactan-degrading activity in addition to the listed activities, and include arabinan, arabinoxylan, and arabinogalactan-degrading enzymes as impurities in addition to the main component enzyme. This is because they may be In the present invention, such an enzyme usually used as an enzyme other than arabinan, arabinoxylan, and arabinogalactan degrading enzyme acts on arabinan, arabinoxylan, and arabinogalactan-containing material to release L-arabinose. If it has activity, it can be used.

In the above case, the yield of L-arabinose can be increased by mixing two or more enzymes having these different activities.

In addition to the above, the addition of a maceration enzyme that destroys plant cells allows L
-It is desirable to add arabinose because it can increase the yield.

The amount of the enzyme having the activity of releasing L-arabinose by acting on arabinan, arabinoxylan, or arabinogalactan-containing material is not particularly limited, and may be used to degrade arabinan, arabinoxylan, and arabinogalactan in the raw material. Any amount is sufficient. For example, in the case of arabinase, 0.4 to 4000 per 100 g of raw material
A unit is desired, preferably 2 to 2000 units, and more preferably 4 to 1000 units. In this case, one unit is the amount of the enzyme that releases 1 μmol of arabinose per minute from linear arabinan.

In the method of the present invention, such an enzyme is allowed to act on arabinan, arabinoxylan, or arabinogalactan-containing material. In order to directly act, for example, a substance containing arabinan, arabinoxylan, or arabinogalactan is suspended in an aqueous medium, and an enzyme is added thereto, and the reaction is carried out with stirring or standing. The conditions for acting on the arabinan, arabinoxylan, and arabinogalactan-containing material are not particularly limited as long as they are conditions used in a normal enzyme reaction, and may be appropriately selected depending on the optimum working conditions of the enzyme used and other factors.

[0015] The reaction temperature is preferably a temperature at which the enzyme is not inactivated, and a temperature at which microorganisms are unlikely to grow in order to prevent spoilage. Specifically, 20 to 90
° C, preferably 40 to 80 ° C, more preferably 50 to 80 ° C.
75 ° C is good. Needless to say, it is desirable to carry out the reaction under the optimal conditions for the enzyme as the pH of the reaction solution.
The pH is preferably 2 to 9, preferably 2.5 to 8, and more preferably 3 to 6. The reaction time depends on the amounts of the arabinane, arabinoxylan, arabinogalactan-containing substance and the enzyme to be used, but it is usually preferable to set it to 3 to 48 hours in terms of work.

As the reaction proceeds, the arabinane, arabinoxylan and arabinogalactan components are hydrolyzed,
-Arabinose is produced and released. This can be used as it is, or further subjected to a heat treatment at 100 ° C. or higher, or further dried to obtain the second L-arabinose-containing enzyme-treated product of the present invention. Further, if the supernatant of the suspension after the reaction is collected, an L-arabinose sugar solution can be obtained. The L-arabinose thus obtained may be purified according to a standard method using various types of chromatography using an ion exchange resin, activated carbon, or the like. Also,
Crystalline L-arabinose may be obtained by adding hot ethanol to the obtained solution containing L-arabinose.

[0017]

Next, the present invention will be described in detail with reference to examples. Example 1 After suspending 200 g of apple fiber (manufactured by Nichiro Co., water content: 2.3%) in 2 L of water, Sumiteam AR was used.
S (arabinase manufactured by Shin Nippon Chemical Co., Ltd., titer: 40
(0 unit / mL), and reacted at 55 ° C. for 24 hours with stirring. After the completion of the reaction, the supernatant was filtered to obtain 1.9 L of a clear solution containing L-arabinose. The sugar in this solution was analyzed by high performance liquid column chromatography. The conditions for the analysis were as follows: TSKgel Amide-80 manufactured by Tosoh Corporation as an analytical column.
(4.6 mm ID x 25 cm), column temperature 80
Elution was performed with 80% acetonitrile at 80 ° C. and a flow rate of 0.8 mL / min. The sugar was detected by fluorescence detection of the benzamidine derivative, and the L-arabinose content was determined from the quantitative value of the standard product. As a result of analyzing the solution after the above reaction, 10 g of L-arabinose was accumulated in 1.9 L.

Next, this solution was treated with an anion exchange resin (Dowex SAR manufactured by The Dow Chemical Company,
OH ^- type, bed volume 100 ml), cation exchange resin (Dowex HCRW2 manufactured by The Dow Chemical Company, H ⁺ type bed volume 100 ml),
The solution was passed through activated carbon (diahop S80 manufactured by Mitsubishi Chemical Corporation, bed volume 100 ml) in this order, and a solution containing L-arabinose was recovered. The collected solution was concentrated by an evaporator until it became Brix 70, and 8.6 g of L
-A sugar solution containing arabinose was obtained.

Example 2 200 g of beet pulp (Cargill, 11% moisture) was suspended in 2 L of water, and then Sumiteam ARS (arabinase, manufactured by Shin Nippon Chemical Co., Ltd., titer: 400 units /
2 mL), and reacted under stirring at 55 ° C. for 24 hours. After the reaction was completed, the mixture was allowed to stand, and the supernatant was filtered to obtain 1.9 L of a clear solution containing L-arabinose. The sugar in this solution was analyzed in the same manner as in Example 1. As a result, 27 g of L-arabinose was accumulated in 1.9 L.

Next, 10 g of powdered activated carbon (manufactured by Takeda Pharmaceutical Co., Ltd., trade name: carbofin) was added to the solution, stirred for 20 minutes, and the solution containing L-arabinose was collected by filtration. Next, this solution was treated with an anion exchange resin (Diaion WA30, OH ^- type, manufactured by Mitsubishi Chemical Corporation, bed volume 100 ml), and a cation exchange resin (Diaion SK1B, H ⁺ type bed volume, manufactured by Mitsubishi Chemical Corporation 100 ml).
And the solution containing L-arabinose was recovered. After concentrating the collected solution with an evaporator until it becomes Brix 70, hot ethanol was added to a final concentration of 85.
%, A small amount of crystalline L-arabinose was added, and the mixture was allowed to stand at 4 ° C. The solution was filtered to obtain 25 g of crystalline L-arabinose. L obtained
-The melting point of arabinose was 157-160C.

Example 3 200 g of beet pulp (Cargill, moisture 11%) was added to 6
Two 100 ml of water and Sumiteam ARS (arabinase manufactured by Nippon Chemical Co., Ltd., strength 400 units / mL)
mL was added and reacted at 55 ° C. for 24 hours with stirring. After completion of the reaction, drying was performed at 70 ° C. for 24 hours using a box drier. 196 g of enzyme-treated beet pulp (9% water) was obtained. 1 g of this enzyme-treated beet pulp was suspended in 100 mL of water to extract a water-soluble fraction, and the free sugar in the enzyme-treated beet pulp was analyzed in the same manner as in Example 1. As a result, 14.5 g of L in 100 g of enzyme-treated beet pulp
-Arabinose had accumulated.

EXAMPLE 4 200 g of orange fiber (manufactured by Neutrinova Japan, moisture 2%) was mixed with 800 mL of water and Sumiteam AR.
S (arabinase manufactured by Shin Nippon Chemical Co., Ltd., strength 40
(0 unit / mL), and reacted under stirring at 55 ° C. for 24 hours. After completion of the reaction, drying was performed at 70 ° C. for 24 hours using a box drier. Enzyme-treated orange fiber 21
0 g (moisture 8%) was obtained. 1 g of the enzyme-treated orange fiber was suspended in 100 mL of water to extract a water-soluble fraction, and the free sugar in the enzyme-treated orange fiber was analyzed in the same manner as in Example 1. As a result, 9.3 g of L-arabinose was accumulated in 100 g of the enzyme-treated orange fiber.

Next, Japanese Patent Application Laid-Open No. 9-299093 is described.
For reference, SumiTeam ARS, which is desirable in the present invention,
A comparative example performed using the enzyme is described. Comparative Example 1 200 g of beet pulp (cargill, moisture 11%)
Add 600 ml of 2.5% calcium hydroxide solution and soak overnight
Soak, heat at 100 ° C for 12 hours, cool down to pH6
Adjusted and filtered. Ethanol is added to the obtained filtrate, and
A precipitate of labinan was obtained. Dissolve this in water again and add
Exchange resin (DIAION WA30, OH ^-Type, bed
Volume 100ml), cation exchange resin (Dai Eye)
ON SK1B, H⁺Bed volume 100ml)
After passing the solution through, it was dried to obtain 2.4 g of arabinan.

After suspending 2.4 g of arabinan in 50 ml of water, Sumiteam ARS (arabinase manufactured by Shin Nippon Chemical Co., Ltd., titer: 400 units / mL) was added to 0.04 g of the suspension.
25 mL was added and reacted at 55 ° C. for 24 hours with stirring.
After the reaction was completed, the mixture was allowed to stand, and the supernatant was filtered to obtain 47 ml of a clear solution containing L-arabinose. As a result of analyzing the sugar in this solution in the same manner as in Example 1, 47
1.3 g of L-arabinose were obtained in ml. In this method, 0.65% of L-arabinose with respect to the raw material is obtained. In contrast, in Example 2, 13.5% of L-arabinose was obtained with respect to the raw material, and the yield was high. According to the production method of the present invention, L-arabinose of 70% (% by mass) or more of L-arabinose contained in beet pulp could be released and recovered. The present invention has eliminated the decrease in L-arabinose recovery rate due to treatment such as alkali extraction.

Table 1 shows the arabinose yields of Examples 1 and 2 and Comparative Example 1. The arabinan content was determined by the following method. After adding 2 mL of 72% sulfuric acid to 20 mg of the raw material and stirring for 1 hour, this was diluted 20-fold,
Incubated for hours. The content of arabinose released by this treatment was measured by the method shown in Example 1, and this was taken as the arabinan content in the raw material. The amount of arabinose recovered refers to raw material 20
The amount (g) of purified arabinose obtained from 0 g is shown, and the yield indicates the ratio of the obtained purified arabinose to the arabinan present in the raw material.

[0026]

[Table 1]

[0027]

According to the production method of the present invention, arabinan, arabinoxylan, and arabinogalactan-containing natural products, which are inexpensive materials, are used.
An L-arabinose and an L-arabinose-containing enzyme-treated product can be easily and inexpensively manufactured without complicated operations such as previously removing arabinogalactan. In addition, the use of agricultural products, which have been discarded in the past, can be expanded, which greatly contributes to the increase in consumption, and can also reduce the environmental impact of disposal.

Continued on the front page (72) Inventor Yoshihiro Nishikawa 3-4-4 Nihonbashi Muromachi, Chuo-ku, Tokyo Unitika Ltd. Tokyo Head Office (72) Inventor Akemi Morimoto 3-4-4, Nihonbashi Muromachi, Chuo-ku, Tokyo Unitika Shares Tokyo head office F term (reference) 4B064 AF02 CA21 CB07 CC03 CD22 CD24 DA10

Claims (5)

[Claims] 1. An enzyme having an activity of releasing L-arabinose by acting on a natural product containing arabinan, arabinoxylan or arabinogalactan is caused to act on a natural product containing arabinan, arabinoxylan or arabinogalactan to produce L-arabinose. -When obtaining arabinose, L-arabinose is obtained by directly acting the enzyme without separating and extracting arabinan, arabinoxylan or arabinogalactan from the natural product, to obtain L-arabinose.
A method for producing arabinose. 2. The enzyme according to claim 1, wherein the enzyme having an activity of releasing L-arabinose by acting on a natural product containing arabinan, arabinoxylan or arabinogalactan is arabinan, arabinoxylan or arabinogalactan degrading enzyme. -A method for producing arabinose. 3. A natural product containing arabinan, arabinoxylan or arabinogalactan is orange fiber, tangerine juice lees, apple fiber, apple juice lees, beet fiber, beet pulp, peanut lees, rice bran, corn lees or soy lees. A method for producing L-arabinose according to claim 1. 4. An L-arabinose-containing enzyme-treated product, wherein the natural product containing arabinan, arabinoxylan or arabinogalactan contains L-arabinose. 5. An enzyme having an activity of releasing L-arabinose by acting on a natural product containing arabinan, arabinoxylan or arabinogalactan, directly acting on a natural product containing arabinan, arabinoxylan or arabinogalactan. A process for producing a treated product containing L-arabinose, characterized by obtaining L-arabinose-containing treated product according to claim 4 by releasing L-arabinose.