JP2003048901A - Long-chain xylooligosaccharide composition and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a xylooligosaccharide composition having high effects of intestinal function controlling actions due to arrival at the intestines without being decomposed and having not only characteristics of an oligosaccharide but also dietary fiberlike actions due to a long chain length. SOLUTION: (1) This xylooligosaccharide composition is a mixed composition of xylose polymers and consists essentially of a pentamer to an eicosamer of xylose. (2) The method for producing the long-chain xylooligosaccharide composition which is the mixed composition of the xylose polymers and consists essentially of the pentamer or a higher oligomer of xylose comprises enzymically or physicochemically treating a lignocellulose material as a raw material. The method is characterized by subjecting a complex comprising the xylooligosaccharide as a constituent component as an intermediate to a decomposition treatment.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a xylooligosaccharide used as a health food, a food additive, a drug / cosmetic additive, and the like. In particular, it relates to a xylooligosaccharide composition which is a polymer mixture of xylose and a method for producing the same.

[0002]

2. Description of the Related Art Due to the growing interest in public health,
Attempts to maintain good health through the foods eaten daily have received much attention. Especially polysaccharides such as dietary fiber and oligosaccharides,
These decomposition products are also fields that have been very researched in recent years because of their physiological activity exerted in digestive organs. Dietary fiber has historically been regarded as important as a food factor related to basic metabolism of the living body such as carbohydrate metabolism and lipid metabolism. Among functional foods, there are many that claim the action of this dietary fiber.

Dietary fiber has the function of improving fecality and adsorbing a carcinogen to dilute the carcinogen in the intestine, and has recently been regarded as an important human food factor. In addition, some conventional dietary fibers have the potential to improve blood hyperlipidemia and control human health by controlling glucose metabolism.
In particular, dietary fiber containing a large amount of acidic sugars such as sodium alginate, pectin, tamarind seed gum and guar gum is reported to reduce the amount of total cholesterol, phospholipids and triglyceride in blood and improve hyperlipidemia. (Rotenberg, S. And Jakobsen, P, E.: The effect of
dietary pectin on lipid composition of blood, ske
letal muscle and internal organs of rats. J. Nutr.,
108, 1384 (1978)) However, these dietary fibers are not sugars that suppress the growth of Escherichia coli and promote selective growth of Bifidobacteria. Rather, the mucosal cells of the small intestine are physically scraped off, often negatively affecting nutrient absorption.
(Cassidy, MM, Lightfood, FG, Grau, LE et al.:Effe
ct of chronic intake of dietary fibers on the ultr
asutractual topography of rat jejunum and colon:
a scanning electron microscopy study.Am.J.Cli.Nut
r., 112.6 (1982))

On the other hand, oligosaccharides have the function of maintaining a good condition of the stomach through the selective growth promoting effect of useful bacteria in the intestine, and are used in lactic acid bacteria drinks certified as foods for specified health use, such as chocolate. It is a useful sugar that is also used in confectioneries. In addition to human food applications, it also has applications as livestock feed. Further, it is also used as an emulsifier and a skin moisturizing component in the fields of medicine and sanitary products.

In general, most of oligosaccharides used for specified health foods have an intestinal regulating action, that is, the number of Escherichia coli which is an intestinal bad bacterium and the number of bacteria of the genus Clostridium which is an intestinal spoilage fermentative bacterium are reduced and the oligosaccharides are relatively reduced. It has the effect of increasing bifidobacteria, which are known as intestinal beneficial bacteria. Among various oligosaccharides, for example, xylooligosaccharide derived from wheat bran is famous. Regarding the action of xylooligosaccharides derived from wheat bran, it is said that while promoting the selective growth of Bifidobacterium, which is a favorable enterobacteria, the number of Escherichia coli, which is an intestinal bad bacterium, is relatively decreased. Escherichia coli and intestinal spoilage fermenters are known to produce carcinogens while growing in the intestine (Kanazawa et al .: Colonic bacterial flora-particularly on bile acid metabolism and colon carcinogenesis-. General Clinic, 26, 1042 ~ 1050 (197
7)) Therefore, it is important to reduce the number of Escherichia coli and intestinal spoilage-fermenting bacteria in the intestine when considering long-term health.

When an oligosaccharide is taken orally in anticipation of an intestinal regulating action, the decrease in the degree of polymerization of the oligosaccharide due to acid hydrolysis by gastric acid or an enzyme in the digestive juice is a serious problem. It is known that oligosaccharides are gradually reduced to a low molecular weight by hydrolysis with acids or enzymes and eventually decomposed into monosaccharides that can be assimilated even by Escherichia coli and putrefactive anaerobes belonging to the genus Clostridium. There is. However, the xylooligosaccharide composition containing a dimer or a trimer as a main component has relatively higher resistance to gastric acid than other oligosaccharides among oligosaccharides (Patent No. 2549638) and is not degraded so much. Is likely to be delivered to the intestines. When xylooligosaccharides are actually administered to humans, an intestinal regulating effect in vivo has been confirmed.

[0007] The xylooligosaccharides currently on the market are made from herbs such as wheat bran and corncob as raw materials, and the xylan main chain in these herbaceous plants has other sugars such as glucuronic acid as side chains. It has branches. From xylan having many side chains, oligosaccharides having only xylose as a constituent sugar can produce only those having a relatively low degree of polymerization. This is because the xylan chain, which is the main chain, is gradually reduced in molecular weight in the work process of removing the side chain. Currently there are 2 oligosaccharides that make up xylooligosaccharides on the market.
Most of them are mainly composed of a trimer or a trimer.

When xylan in a hemicellulose material derived from a natural product is enzymatically decomposed, a large difference appears in the decomposition products due to the difference in the xylanase which is the enzyme used. In general, xylanase derived from fungi such as mold and mushroom has relatively loose substrate specificity, and has a characteristic that the saccharification power is much higher than that of xylanase derived from bacteria. Therefore, when a xylo-oligosaccharide composition is produced using an enzyme solution derived from fungi as an enzyme, xylan, which is a substrate, is decomposed at once,
The abundance ratio of xylobiose in the xylooligosaccharide composition is 7
It makes a xylooligosaccharide composition such that it is 0% or more.

On the other hand, when a xylanase is produced using a xylanase produced by bacteria, the substrate specificity of the enzyme is greatly different from that of a fungal xylanase. It is known to produce xylo-oligosaccharides with a distribution from a certain xylobiose to a pentamer (Japanese Patent Laid-Open No. 1-25228).
No. 0).

[0010]

As described above, most of the oligosaccharides constituting xylo-oligosaccharides currently on the market are mainly composed of dimers and trimers.
It is recognized as a substance different from dietary fiber. The present invention has a long chain length of xylo-oligosaccharide, which has a high effect of controlling the intestine by reaching the intestine without being decomposed, and since the chain length is long, not only the characteristics of oligosaccharide but also the action of dietary fiber-like action. It is an object of the present invention to provide a xylo-oligosaccharide having the same.

[0011]

In order to solve the above problems, the present invention adopts the following configurations. That is, the present invention is the xylooligosaccharide composition of the following (1) to (2), and the present invention is a method for producing the xylooligosaccharide composition of the following (3) to (9).

(1) A xylooligosaccharide composition which is a mixed composition of xylose polymers, wherein the xylooligosaccharide composition comprises xylose pentamer to 20mer as a main component. (2) The xylooligosaccharide composition according to the above (1), wherein the average degree of polymerization of xylose is 7 to 20.

(3) A method for producing a long-chain xylooligosaccharide composition which is a mixed composition of xylose polymers and which contains xylose pentamer or more as a main component, which is prepared by using a lignocellulosic material as a raw material. In a method for producing a xylooligosaccharide composition by enzymatically or physicochemical treatment, a long-chain xylooligo characterized in that a complex having xylooligosaccharide as a constituent is used as an intermediate and the intermediate is decomposed. A method for producing a sugar composition.

(4) A method for producing a long-chain xylooligosaccharide composition characterized in that the lignocellulose is a chemical pulp in the method (3). (5) The method for producing a long-chain xylooligosaccharide composition according to the method (3) or (4), wherein the lignocellulose is treated with an enzyme or physicochemically is a hemicellulase. (6) The method for producing a long-chain xylooligosaccharide composition according to the method (5), wherein the hemicellulase is a xylanase. (7) In the method according to any one of (3) to (6) above,
A method for producing a long-chain xylooligosaccharide composition, wherein the complex containing the xylooligosaccharide as a constituent is a complex of xylooligosaccharide and lignin. (8) The method according to (7) above, wherein the complex having the xylooligosaccharide as a constituent component is a complex in which the xylooligosaccharide and the lignin are bound by a hydrolyzable chemical bond. A method for producing a long-chain xylooligosaccharide composition. (9) In the method according to any one of (3) to (8) above,
The method for producing a long-chain xylooligosaccharide composition, wherein the decomposition treatment is acid hydrolysis treatment.

A preferred embodiment of the present invention is as follows.
0) to (15). (10) The method for producing a long-chain xylooligosaccharide composition according to the above (8), wherein the complex is a complex in which a 2 to 20-mer of xylose and lignin are chemically bonded.

(11) In the enzyme treatment step of the above (5), xylan and / or hemicellulose is treated with hemicellulase, and the resulting sugar solution is subjected to acid hydrolysis treatment in the hydrolysis treatment step, followed by purification / separation step. In which the xylooligosaccharide component is separated and recovered from the treatment liquid obtained from the hydrolysis treatment step, the content of xylooligosaccharide having a degree of polymerization of 5 or less is 5% or less, and the average degree of polymerization is 7 or more, A method for producing a long-chain xylooligosaccharide composition, which is preferably 10 or more.

(13) The long chain xylooligosaccharide composition according to (12), wherein the hydrolysis treatment step is a step performed on a sugar solution obtained by concentrating the sugar solution obtained from the enzyme treatment step. Manufacturing method.

(14) The enzyme treatment step has a pH of 3 to 1.
The sugar solution adjusted to 0, preferably 5 to 9 is added to 10
The method for producing a long-chain xylooligosaccharide composition according to (5), which comprises a step of enzymatically treating at a temperature of from 90 ° C to 90 ° C, preferably from 30 ° C to 60 ° C. (15) In the hydrolysis treatment step, a sugar solution whose pH is adjusted to a value of about 3.5 or less, or a value of 105 ° C to
At a temperature of 150 ° C, preferably 110 ° C to 121 ° C, 1
The method for producing a long-chain xylooligosaccharide composition according to (9), which comprises a step of performing an acid hydrolysis treatment by heating for 5 minutes or more, preferably 30 minutes to 60 minutes.

(14) In the purification / separation step, the sugar solution obtained in the acid hydrolysis treatment step is purified by utilizing cation exchange resin column-anion exchange resin column-separated precipitation due to difference in solubility. The method for producing a xylo-oligosaccharide composition according to (11), which comprises a step.

The present inventors used xylose dimers when the enzyme used was a neutral thermophilic xylanase derived from the genus Bacillus and the raw material was a hardwood chemical pulp. To obtain a xylooligosaccharide composition having a distribution of xylooligosaccharides ranging from 20 to 20-mer, wherein the xylooligosaccharide composition contains a relatively large amount of pentamers to 20-mers having a large molecular weight, and further, this xylooligosaccharide It was found that the abundance ratio of xylobiose in the composition was about 10% or less of the total sugar amount in the xylooligosaccharide composition. Considering that when xylobiose is acid-hydrolyzed, intestinal harmful bacteria such as Escherichia coli are easily converted to xylose that can be assimilated, xylooligosaccharides generally have strong resistance to acid hydrolysis. A low abundance ratio is important for maintaining functionality.

The long-chain xylooligosaccharide composition produced by using lignocellulose derived from chemical pulp as a material is characterized by having a higher average degree of polymerization than xylooligosaccharide produced by using corncob or cottonseed shell as a raw material. When lignocellulose is used as a material, it is possible to some extent to prepare a new xylooligosaccharide at an arbitrary ratio of an average degree of polymerization of 2 to 5 by adjusting the enzyme used. However, as a new finding discovered this time, the most significant feature in producing a long-chain xylooligosaccharide is that "xylooligosaccharide-lignin complex" is used as an intermediate.

The xylooligosaccharide-lignin complex is a novel compound found in the treatment liquid of hardwood and softwood kraft pulps treated with an enzyme or a physicochemical treatment. This compound is a substance in which lignin is bound to a dimer to 20-mer xylooligosaccharide chain. Since lignin has an overwhelmingly large molecular weight with respect to xylooligosaccharide, it is sterically hindered and xylanase and xylosidase cannot decompose the xylooligosaccharide-lignin complex. Therefore, xylooligosaccharide-
The xylooligosaccharide in the lignin complex remains undegraded, and the relatively long chain xylooligosaccharide is present in the solution in a state of being bound to lignin. However, this xylooligosaccharide-lignin complex can be easily cleaved between the xylooligosaccharide and lignin by acid treatment with a dilute acid, and by utilizing this property, a xylooligosaccharide composition having a polymerization degree of 5 to 20 mer is formed. The thing can be easily manufactured.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be specifically described below. The long-chain xylooligosaccharide composition referred to in the present invention has a high content of xylooligosaccharide having a relatively high degree of polymerization in the oligosaccharide composition. Specifically, it means that the amount of xylooligosaccharide, which is a pentamer of xylose, is 80% by weight or more based on the total weight of sugar in the composition. When the average degree of polymerization of the long-chain xylooligosaccharide composition of the present invention is measured, the average degree of polymerization is 5 or more, preferably 7 or more, and more preferably 10 or more.

Among them, the xylooligosaccharide composition that is easy to produce and has excellent performance is mainly composed of a pentamer to a 20mer of xylose, preferably 7
Those whose main component is a monomer to a 20mer, more preferably,
The main component is a 10-mer to a 20-mer. Here, the expression "having a pentamer to a 20mer as a main component" means that the total weight of the pentamer to the 20mer is 50% by weight or more with respect to the total sugar weight. The quantity does not have to exist. Needless to say, it is possible to freely design and manufacture a xylooligosaccharide composition having a higher average degree of polymerization by changing the conditions of the enzyme used and the acid treatment.

The long-chain xylooligosaccharide composition of the present invention is a liquid containing a xylooligosaccharide having a large degree of polymerization by subjecting a lignocellulosic material to hemicellulase treatment and then acid-hydrolyzing the xylooligosaccharide-lignin complex with a dilute acid. Obtainable. When the pulp as lignocellulose is treated with hemicellulase, the pulp concentration is 1
It is carried out in the range of -30% by weight, preferably 2-15% by weight, but this is not the case when lignocellulose other than hardwood kraft pulp is used as a raw material for production. For example, regarding the xylan derived from lignocellulose, when the pulp concentration at the time of treatment is illustrated, wheat bran-derived xylan is 0.5 to 5%, preferably around 2%, and corncob-derived xylan is 1 to 10%, preferably 5%. Before and after, 1 to 10%, preferably around 5% for xylan derived from cottonseed shell, 0.2 to 5% for crushed corn pipe, preferably 1
%, For oat-derived xylan, it is 0.2 to 5%, preferably around 1%.

As a raw material lignocellulosic material from which the xylooligosaccharide composition of the present invention can be obtained, woods such as conifers and hardwoods are preferably used.
It may be a non-woody plant such as hemp, bagasse or rice,
It is not particularly limited. The pulp used in the present invention may be any of chemical pulp, mechanical pulp, deinking pulp, etc., but hardwood chemical pulp is preferable. As the cooking method for obtaining chemical pulp, kraft cooking, polysulfide cooking, soda cooking, known cooking methods such as alkaline sulphite cooking can be used, but considering pulp quality, energy efficiency, etc., kraft cooking method It is preferably used.

When a hardwood kraft pulp is used as the lignocellulosic material, it is desirable to first treat the pulp bleached in the alkaline oxygen bleaching step with hemicellulase, but the pulp after cooking or mechanical pulp is used as it is as a hemicellulase treatment raw material. May be.

It is well known that hemicellulose eluted from the pulp fibers in the cooking process is re-adsorbed on the surface of the pulp obtained by kraft cooking. 90% or more of the re-adsorbed hemicellulose has β1 of D-xylose.
→ It is a xylan composed of 4 bonds.
In hardwood kraft pulp, most of the side chains arabinose and glucuronic acid in hemicellulose are decomposed and removed. Further, 4-O-methylglucuronic acid in the side chain remains as a side chain, but is converted to hexenuronic acid under alkaline conditions. Since this hexenuronic acid is easily decomposed and removed by heating under acidic conditions, it does not pose a problem in the production of xylooligosaccharides. Unlike the xylan existing in the cell wall of normal plants, most of the side chains attached to xylan, which is the main chain of xylan, are extracted from the pulp during the pulp cooking process. Has been decomposed and removed. Therefore, readsorbed xylan has a much lower side chain retention rate than xylan in normal cell walls. However, since the xylo-oligosaccharide-lignin complex described below is formed, when the re-adsorbed xylan is decomposed and removed with xylanase, the xylo-oligosaccharide-lignin complex elutes into the pulp slurry, so that the reaction liquid becomes brown. A substance having an absorption of 280 nm exists in this brown reaction solution, which is considered to be the absorption derived from the aromatic ring of lignin.

The xylan content including the re-adsorbed portion accounts for about 20% of the absolute dry weight of hardwood kraft pulp. In the hemicellulase treatment, the enzyme acts on the hardwood kraft pulp and acts on all xylan including re-adsorbed components to lower the molecular weight thereof. For example, xylanase of Bacillus espi-S-2113 strain (see JP-A-8-224081)
In the case of using, the ratio of the constituent sugars of xylose and xylooligosaccharide produced in the treatment reaction solution is the largest in the range of 3 to 5 mer, and the oligosaccharide having a composition ratio of a small amount of monomers is produced.

The present inventors have already found that the xylooligosaccharide complex in which the xylooligosaccharide and the lignin-like substance are bound is present in the waste water obtained from the hemicellulase treatment step of hardwood kraft pulp. Furthermore, the xylooligosaccharide complex has found that a lignin-like substance is bound to an oligosaccharide having a relatively high degree of polymerization.
In this xylooligosaccharide complex, the xylooligosaccharide and the lignin-like substance can be easily separated and removed by treatment with a dilute acid, so that a xylooligosaccharide having a large degree of polymerization can be produced in large quantities at low cost.

At present, most of the enzymes used in large-scale enzyme treatment steps are hemicellulases, but any commercially available hemicellulases are used in the enzyme treatment step in the method for producing a xylooligosaccharide of the present invention. be able to. For example, trade name Cartazyme (made by Clariant), trade name Ecopulp (made by Rohm Enzyme),
Commercially available enzyme preparations such as product names Sumiteam (manufactured by Shin Nippon Chemical Industry Co., Ltd.) and pulpzyme (manufactured by Novo Nordix),
Uses xylanase produced by microorganisms such as Trichoderma spp., Thermomyces spp., Aureobasidium spp., Streptomyces spp., Aspergillus spp., Clostridium spp., Bacillus spp., Thermotoga spp., Thermoacus spp., Caldoceram spp. And Thermomomonospora spp. be able to.

The enzyme treatment temperature is in the range of 10 to 90 ° C., preferably 30 to 60 ° C., but a treatment temperature close to the optimum temperature of the enzyme is more preferable. In the case of a general enzyme, if the treatment temperature is lower than 10 ° C., the reaction becomes insufficient, and obtaining such a temperature itself requires a large cost, which is not suitable. On the other hand, if the temperature is higher than 90 ° C., the heat loss becomes large unless the treatment system is sealed, and in the case of a general enzyme, the enzyme itself is denatured and becomes inactive, which is not suitable.
The solution pH during the treatment is in the range of 3 to 10, preferably 5 to 9, but a pH close to the optimum pH of the enzyme is more preferable.
When a pulp obtained by bleaching hardwood kraft pulp with oxygen is treated with an enzyme to obtain a sugar solution, the optimum pH of the enzyme is closer to the alkaline side because the pH of the pulp tends to the alkaline side. The cost when adjusting is low and has an advantage. If it is necessary to adjust the pH, it goes without saying that the acid treatment may be carried out by adding an arbitrary acidic solution or alkaline solution for adjustment.

The sugar solution obtained by the enzyme treatment contains xylooligosaccharide (2 to 20 mer) and xylooligosaccharide complex. The sugar concentration in the enzyme-treated solution was 1 unit (1 unit) of xylanase produced by Bacillus sp. 2113 strain (incorporated administrative agency National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary Center strain FERM BP-5264) per pulp dry weight. Is an enzyme that releases 1 micromole of xylose per minute), and when added to a 10% strength pulp slurry and treated, about 3000 μg / m
1 (xylose conversion).

This sugar solution is concentrated using a membrane separation technique such as a charged NF membrane, other ultrafiltration membranes, reverse osmosis membranes, or concentration such as evaporation in consideration of the subsequent production process. It is also possible to perform the work of increasing the sugar concentration by the work. In fact, reducing the volume of sugar solution facilitates handling when processing a large amount of sugar solution in the subsequent purification step. In addition, the permeated liquid obtained by the operation of concentrating the membrane has a lower sugar concentration than the enzyme-treated liquid and is characterized by a small content of coloring organic matter such as lignin. Therefore, the permeate obtained in the membrane concentration step can be reused as industrial water in the pulp manufacturing step.

The sugar solution or the sugar solution after the concentration treatment step is subjected to acid hydrolysis treatment with a dilute acid to separate the xylooligosaccharide complex into a xylooligosaccharide and a lignin-like substance. As a method for adjusting the pH of the sugar solution, the pH of the sugar solution is adjusted to 3.5 by appropriately adding a mineral acid or an organic acid to the sugar solution.
Although it is generally adjusted to the vicinity, it is also possible to lower the pH by ion exchange by treating the sugar solution with a cation exchange resin such as Amberlite 200C (trade name, manufactured by Rohm and Haas Co.). Next, the sugar solution whose pH has been adjusted is heated to 105 ° C to 150 ° C, preferably 110 ° C.
The acid hydrolysis treatment is performed by heating in the range of ℃ to 121 ℃. The treatment time is 15 minutes or more, preferably 30 minutes to 60 minutes. When the heat treatment time is set to 90 minutes or longer, the decomposition of oligosaccharides into monosaccharides proceeds, which is not preferable. When the pH of the sugar solution is around 3.5, the xylooligosaccharide complex and the lignin-like substance, the xylooligosaccharide and hexenuronic acid, which is one of the side chains, can be separated and removed, but the xylooligosaccharide itself is hardly decomposed. Absent.

By this treatment, the lignin-like organic matter is decomposed and removed from the xylooligosaccharide complex and converted into xylooligosaccharide. The conversion efficiency from the xylooligosaccharide complex to the xylooligosaccharide under the treatment conditions of pH 3.5, 121 ° C., and 60 minutes is about 95%. At this time, a part of the monosaccharide is hydrolyzed and becomes a furfural-like substance, which is further condensed and precipitated. The lignin-like substance separated from the xylooligosaccharide complex similarly condenses under acidic conditions, becomes insoluble and precipitates. This insolubilized precipitate can be separated and removed not only by filtration with filter paper or diatomaceous earth but also by UF membrane, MF membrane, ceramic filter or the like.

The xylooligosaccharide composition obtained from the xylooligosaccharide complex as described above contains a novel xylooligosaccharide composition containing a high proportion of xylooligosaccharides having a relatively long chain length and a degree of polymerization of about 5-9. It is a thing. Also, 10
It also contains a small amount of monomers to 20-mers. The reason why xylooligosaccharides with a relatively high degree of polymerization are obtained is that xylooligosaccharide conjugates in the sugar solution obtained by enzyme treatment bind lignin-like substances to xylooligosaccharides with a chain length of about dimer to 20mer. Therefore, it is caused by avoiding unnecessary digestion by hemicellulase. When such a state is separated from the lignin-like substance by acid hydrolysis with a dilute acid, a xylooligosaccharide having a relatively long chain length is obtained.

The sugar solution obtained by acid hydrolysis contains, in addition to xylooligosaccharides, monosaccharides such as xylose and glucose, and organic substances such as lignin, furan compounds and furfural. In the step of separating and purifying only the xylooligosaccharide from the mixture of these organic substances, any conventional purification method such as ion exchange, molecular sieving, ethanol fractionation and membrane treatment may be used in combination. For example, in the purification method using a column in the order of cation exchange resin → anion exchange resin → activated carbon, the recovery rate of the purified xylooligosaccharide is about 70% when the acid-treated sugar solution as the starting material is 100%.

A method for producing a long-chain xylooligosaccharide composition using this purified xylooligosaccharide composition is carried out as follows. First, xylooligosaccharide is dissolved in water.
The concentration at this time is not particularly limited. However, preferably 0.5% to 50%, more preferably 5% to 10% xylooligosaccharide aqueous solution is desirable. The temperature for dissolution is not limited, but is preferably 10 ° C to 100 ° C, more preferably 40 ° C to 60 ° C. The standing time when the aqueous solution of xylooligosaccharide is allowed to stand and the long-chain xylooligosaccharide is separately collected as a precipitate is not particularly limited. It may be allowed to stand so as to obtain a sufficient precipitate, but preferably 12
More than time. The temperature during standing is not particularly limited, but is preferably 10 ° C to 40 ° C, more preferably 20 ° C.
The temperature is from 30 ° C to 30 ° C. For example, a long-chain xylooligosaccharide composition obtained by preparing an aqueous solution having a xylooligosaccharide concentration of 7% at a temperature of 60 ° C., then cooling to room temperature and then allowing it to stand for 24 hours has an average degree of polymerization of 12. It was 3. The long chain xylooligosaccharide composition recovered as a precipitate is concentrated,
It can be made into a powder, or can be dissolved again in water to give an aqueous solution. The recovery rate of the long-chain xylooligosaccharide composition that has been separately purified by utilizing the difference in solubility is about 3 when the starting material xylooligosaccharide composition is 100%.
%.

When the sugar solution containing the purified long-chain xylooligosaccharide was analyzed by ion chromatography (Dionex), it was found to be a sugar solution containing a dimer or at least a 20-mer or more xylooligosaccharide. .
When the weight of the organic component at this time was analyzed, the total amount of sugar in the absolute dry weight was 99% or more. In addition, ash content in the refined sugar solution was practically not detected by the measurement of ash content using a weighed crucible.

As described above, the novel long-chain xylooligosaccharide composition of the present invention has a large degree of polymerization obtained by separating and purifying a lignocellulosic material as a starting material from a reaction filtrate treated with hemicellulase. It is a xylooligosaccharide-containing composition. In addition, this long-chain xylooligosaccharide composition having a large degree of polymerization can be prepared from conventional xylose and xylose by using a physicochemical method such as enzyme or blasting.
The present invention also includes a method for producing xylose or xylobiose using this method, since it can be easily converted into xylooligosaccharides containing a monomer as a main component.

[0042]

The present invention will be specifically described below with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples. All percentages shown below are by weight unless otherwise specified, and the ratio of addition of pulp to pulp is the ratio of volume to absolutely dry weight of pulp. In addition, each measuring method is as follows.

(1) Quantification of total sugar amount: The total sugar amount was measured by the analytical curve D-
It was prepared by using xylose (Wako Pure Chemical Industries, Ltd.) and quantified by the phenol-sulfuric acid method (published by the "Quantitative method for reducing sugars" published by the Academic Press Center). (2) Quantification of reducing sugar amount: The reducing sugar amount was quantified by the Somogy-Nelson method ("Quantitative method for reducing sugar" published by the Academic Publishing Center) using a calibration curve prepared using D-xylose (Wako Pure Chemical Industries). did. (3) Determination of average degree of polymerization: Sample sugar solution was kept at 50 ° C. and centrifuged at 15,000 rpm for 15 minutes to remove insoluble matter, and the total sugar amount in the supernatant liquid was divided by the reducing sugar amount (both converted to xylose). The average degree of polymerization was determined.

(4) New xylooligosaccharide quantification method: The quantification method of oligosaccharide was performed by using an ion chromatograph (manufactured by Dionex), and the analytical column was also analyzed by using Carbo Pac PA-10 manufactured by Dionex. A 100 mM NaOH solution was used as the separation solvent, and sodium acetate was added to the above-mentioned separation solvent so that the concentration was 500 nM, and the solution ratio was as follows: separation solvent: elution solvent = 4:
A linear gradient of 6 was set and separated.

<Preparation of calibration curve> For preparation of the calibration curve, xylose (X), xylobiose (X2),
Xylotriose (X3), Xylotetraose (X
4) was used. In the above-mentioned analytical method, the peak area of these oligosaccharides per unit weight (this time, per 1 μg) becomes smaller in the order of xylobiose, xylotriose, and xylotetraose from xylose. Below, when a calibration curve was created based on the existing area per unit weight for the quantification of xylooligosaccharides in the absence of the standard product, the formula Y = 4 × 10 ⁷ × (X ^−0.6709 ) was obtained. . In this case, Y indicates the peak area of oligosaccharide per 1 μg, and X is the degree of polymerization of oligosaccharide.
(See Figure 1)

On the basis of this formula, xylopentaose (X5), xylohexaose (X6), in which no oligosaccharide standard is present, is hereinafter referred to as a 20-mer (X2) as xylooligosaccharide.
The area per 1 μg up to 0) was calculated and a calibration sheet was prepared and shown in Table 1.

[0047]

[Table 1]

In the following, the calibration sheet was used to calculate the concentration of xylooligosaccharide. 34 mg / m2 when xylo-oligosaccharide was determined as a neutral sugar by the phenol-sulfuric acid method
When the sample which was 1 was calculated with this calculation sheet, it was 3
It was 6.5 mg / ml. By using this calculation sheet, the concentration of xylooligosaccharide in which no preparation exists can also be calculated.
Also, the values obtained from this calculation sheet are very useful with relatively few errors.

(5) Definition of enzyme titer: The activity of xylanase used as an enzyme was measured by hippo xylan (manufactured by Sigma).
Was used. The enzyme titer is defined by measuring the reducing power of reducing sugars obtained by degrading xylan by xylanase, using the DNS method (“Quantitative method for reducing sugars” published by the Society Publishing Center), and The amount of enzyme that produces reducing power corresponding to xylose was set to 1 unit.

(6) Ion chromatograph analysis: An ion chromatograph (Dionex) was used for the analysis of xylooligosaccharides. Carbo Pac PA-10 (Dionex) was used for the analysis as a column suitable for sugar analysis.

Example 1 <Enzyme treatment step> A mixed hardwood chip consisting of 70% domestic hardwood chips and 30% eucalyptus wood was used as a raw material, and a kappa number of 20.1 and a pulp viscosity of 41c were obtained by kraft cooking.
A ps factory unbleached pulp was obtained. Next, oxygen delignification is performed to obtain a Kappa number of 9.6 and a pulp viscosity of 25.1c.
An oxygen delignified pulp of ps was obtained. 10 of this pulp
After filtering with a 0 mesh filter cloth and washing, the pulp concentration is 10
%, Adjusted to pH 8 by adding dilute sulfuric acid, and then Bacillus sp. S-2113 strain (Institute of Industrial Science and Technology, Patent Biological Depository Center, deposited strain FERM
Xylanase produced by BP-5264) was added so that the amount of xylanase was 1 unit / g of pulp, and the mixture was treated at 60 ° C. for 120 minutes. After the treatment, the residue was filtered through a 100-mesh filter cloth to separate pulp residue and the like, and a treatment liquid of 1050 liters (total sugar amount 3900 g) containing a total sugar concentration of 3700 mg / l was obtained. Then, NF membrane (Nitto Denko: NTR-745
0, membrane quality: sulfonated polyethersulfone system, salt inhibition rate 50%) and concentrated 40 times in volume ratio. This concentrate contains 2700 g of total sugar, and the total sugar recovery is 70%.
Met. As a result of analyzing the sugar in the concentrated solution by ion chromatography, a peak of the xylooligosaccharide complex was found after elution time of 24 minutes. This is shown in FIG.

<Acid hydrolysis treatment step> After adding sulfuric acid to 1,000 ml of the concentrated sugar solution obtained in the enzyme treatment step to adjust the pH to 3.5, the concentrated sugar solution was heated at 121 ° C. The reaction was carried out for 1 hour. The reaction product was used as a column for ion chromatography (Dionex: Carbo Pac PA-1).
As a result of analysis by ion chromatography using 0), it was found to contain a high concentration of xylooligosaccharide (dimer to 20mer).

<Purification / separation step of xylooligosaccharide> A sugar solution of xylooligosaccharide prepared in the acid hydrolysis treatment step (1
A column (inner diameter 36 m) filled with 10 ml of 17 mg / ml) and 1.2 g as the total sugar amount with a strongly acidic ion exchange resin (Amberlite 200C manufactured by Rohm and Haas).
m, length 150 mm). After collecting the xylooligosaccharides that passed through the column, the column was loaded on the same column filled with a weakly basic ion exchange resin (IRA67 manufactured by Rohm and Haas). The xylo-oligosaccharide obtained after passing through the column was decolorized by concentrating it and adding 80 mg of activated carbon (manufactured by Wako Pure Chemical Industries: product number 037-02115) to the xylo-oligosaccharide solution and stirring at 60 ° C. for 1 hour. After stirring, activated carbon was filtered through a 0.22 μm membrane filter to obtain a purified xylooligosaccharide solution. Absorption at wavelengths of 280 nm and 250 nm was not observed in the purified xylooligosaccharide solution, and the ultraviolet absorbing substance contained in the xylooligosaccharide solution after the acid treatment was removed. The residual rate of ash was also 0.1% or less based on the acid-treated xylooligosaccharide solution as the starting material. The recovery rate of xylooligosaccharide was 70.2%. The result of ion chromatography of this purified xylooligosaccharide is shown in FIG.

<Preparation of long-chain xylooligosaccharide> As a starting material, a neutral xylooligosaccharide having an average polymerization of 5.2 adjusted by the method described in JP 2000-333692 A was used as a starting material. 70 g of the starting material is dispersed in 1000 ml of ultrapure water and stirred well. The starting material does not completely dissolve in 1000 ml of pure water, and an apparently cloudy liquid is provided.
The cloudy liquid is completely dissolved when slowly heated to 60 ° C. When this completely dissolved aqueous solution is allowed to stand at room temperature for 24 hours, only insoluble long-chain oligosaccharides are precipitated. Therefore, when the supernatant is gently removed by a perista pump, only white long-chain xylooligosaccharides are recovered. The white precipitate had a sugar content of 497 mg. The white precipitate is completely dissolved when suspended in 50 ml of ultrapure water at 60 ° C and becomes a colorless and transparent liquid. By spray-drying this transparent liquid, a dry powdered long-chain xylooligosaccharide could be obtained. The weight of the long-chain xylooligosaccharide at this time was 455 mg. The analysis result of the long-chain xylooligosaccharide by ion chromatography is shown in FIG. The vertical axis represents the redox potential (nC), and the horizontal axis represents the elution time. It can be seen that the long-chain xylooligosaccharides are eluted around 17 minutes after the elution.

<Polymerization degree of long-chain xylooligosaccharide> The obtained long-chain xylooligosaccharide powder was dissolved in ultrapure water to prepare a 1% aqueous solution. The average degree of polymerization was determined by measuring the total sugar amount by the phenol-sulfuric acid method, and then measuring the reducing sugar amount in a 1% aqueous solution by the Somogene Nelson method. A calibration curve was prepared using D-xylose in all measurements. The average degree of polymerization was determined by dividing the total sugar amount per 1 ml by the reducing sugar amount per 1 ml. As a result, the degree of polymerization of the long-chain xylooligosaccharide produced by the above method was 12.3. This is a long-chain xylooligosaccharide having a higher degree of polymerization than the starting material having a degree of polymerization of 5.2.

Example 2 The long-chain xylooligosaccharide composition purified by the method of Example 1 (average degree of polymerization = 12.3) was used to test the intestinal function. SD rats, which had been on a diet containing 2% long-chain xylooligosaccharides for 4 weeks, showed selective growth of bifidobacteria in the intestine compared to the administration of a diet containing no long-chain xylooligosaccharides. The pH of the product was lowered, and the number of Bifidobacterium per 1 g of the cecal contents was actually increased. The result is shown in FIG.

On the other hand, the xylooligosaccharides to be administered were changed for each chain length, and the dietary fiber-like action of the xylooligosaccharides was examined for each chain length. At this time, SD rats containing 2% of oligosaccharides containing 70% or more xylobiose were administered for 4 weeks and 2% of long-chain xylooligosaccharides.
A comparison was made with a group of SD rats in which the added diet was administered for 4 weeks. When the blood characteristics of the rats in each group were compared, the total cholesterol, phospholipid, and triglyceride levels were significantly lower in the rats continuously administered with the long-chain xylooligosaccharide. The results regarding the amount of triglyceride are shown in FIG. In the figure, C indicates a rat in the oligosaccharide-free group, XB indicates a xylobiose administration group, XN indicates a xylooligosaccharide administration group having an average polymerization degree of 5.2, and XN10 indicates a long-chain xylooligosaccharide administration group.

From these results, it was found that long-chain xylooligosaccharides have an action of preventing hyperlipidemia in addition to the enhancement of selective growth of bifidobacteria, which is one of the physiological activities of ordinary xylooligosaccharides.

[0059]

INDUSTRIAL APPLICABILITY According to the present invention, a long-chain xylooligosaccharide composition having not only an intestinal regulating effect but also a hyperlipidemia-preventing effect is inexpensively supplied in a large amount. This novel xylooligosaccharide composition can be easily converted into xylobiose and xylose by treatment such as acid hydrolysis and enzymatic digestion. Also, as can be seen from the fact that xylooligosaccharides themselves have selective growth properties for bifidobacteria and are also used as materials for functional foods, they are highly safe for humans,
The xylooligosaccharide composition of the present invention is also a composition that can be sufficiently applied as a functional food material that is expected to have an intestinal regulating action, a cholesterol lowering action, and the like. Further, it is also useful as an additive to feed for livestock and cultivated fisheries. It should be further noted that the novel long-chain xylooligosaccharide having a high average degree of polymerization, when administered to SD male rats, has a surprising change in blood properties. In particular, the indices related to lipid metabolism such as total cholesterol, phospholipids, and triglycerides were reduced as compared with those when long-chain xylooligosaccharides were not administered. This is a characteristic of dietary fiber, and it is presumed that it exhibits a dietary fiber-like action because the chain length of xylooligosaccharides became longer. That is, it has the function of “having the functions of conventional low molecular weight xylooligosaccharides and dietary fiber”.

[Brief description of drawings]

FIG. 1 is a diagram showing a calibration curve for quantifying xylooligosaccharides.

FIG. 2 is a diagram showing a xylooligosaccharide complex in a concentrated solution.

FIG. 3 is a view showing xylooligosaccharide purified from a xylooligosaccharide-lignin complex.

FIG. 4 is a diagram showing a long-chain xylooligosaccharide newly prepared from a xylooligosaccharide composition by utilizing the difference in solubility.

FIG. 5 is a view showing the intestinal regulating action of long-chain xylooligosaccharides.

FIG. 6 is a diagram showing changes in blood properties among the effects of long-chain xylooligosaccharides for preventing hyperlipidemia.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61K 47/48 A61K 47/48 4C090 A61P 1/00 A61P 1/00 3/00 3/00 3/06 3 / 06 43/00 111 43/00 111 C07H 3/06 C07H 3/06 C12P 19/22 C12P 19/22 F term (reference) 4B064 AF04 AF11 CA21 CB01 CE11 DA10 4C057 AA02 BB04 4C076 BB01 CC16 CC21 CC40 EE58A EE59A 4C083 AD01 AD012 AD191 AD192 AD211 AD212 4C086 AA01 AA02 AA03 AA04 EA01 EA20 MA01 MA02 MA04 MA05 MA52 NA05 NA14 ZA69 ZC19 ZC33 4C090 AA01 AA04 BA04 BA51 BB10 BD41 CA43 DA09 DA23 DA27

Claims (9)

[Claims] 1. A xylooligosaccharide composition, which is a mixed composition of xylose polymers, wherein xylose pentamer to 2
A xylo-oligosaccharide composition mainly composed of a 0-mer. 2. The average degree of polymerization of the xylose polymer is 7 to 2.
The xylo-oligosaccharide composition according to claim 1, which is 0. 3. A mixed composition of xylose polymers,
And a method for producing a long-chain xylooligosaccharide composition mainly composed of a pentamer or more of xylose, which is a lignocellulosic material as a raw material and is enzymatically or physicochemically treated to obtain a xylooligosaccharide composition. A method for producing a long-chain xylooligosaccharide composition, which comprises using a complex having xylooligosaccharide as a constituent component as an intermediate and subjecting the intermediate to a decomposition treatment. 4. The method for producing a long-chain xylooligosaccharide composition according to claim 3, wherein the lignocellulose is a chemical pulp. 5. The method for producing a long-chain xylooligosaccharide composition according to claim 3, wherein the enzymatic treatment or physicochemical treatment of lignocellulose is a hemicellulase treatment. 6. The method for producing a long-chain xylooligosaccharide composition according to claim 5, wherein the hemicellulase is xylanase. 7. The long-chain xylooligosaccharide composition according to any one of claims 3 to 6, wherein the complex having the xylooligosaccharide as a constituent is a complex of xylooligosaccharide and lignin. Production method. 8. The length according to claim 7, wherein the complex having the xylooligosaccharide as a constituent component is a complex in which the xylooligosaccharide and the lignin are bonded by a chemical bond having hydrolyzability. A method for producing a chain xylooligosaccharide composition. 9. The method for producing a long chain xylooligosaccharide composition according to claim 3, wherein the decomposition treatment is acid hydrolysis treatment.