JP2006087390A - Method for separating acidic oligosaccharide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently, simply and selectively separating acidic oligosaccharides from a saccharide mixture solution containing acidic oligosaccharides and neutral components (neutral oligosaccharides and monosaccharides). <P>SOLUTION: In this method for selectively separating the acidic oligosaccharides from the saccharide mixture solution containing acidic oligosaccharides and the neutral components comprising neutral oligosaccharides and monosaccharides, the saccharide mixture solution is fed to a chromatographic separator in which a cationic ion-exchanging resin is used as a separating agent whereby the saccharide mixture solution is separated into an acidic oligosaccharide-rich fraction and a neutral components-rich fraction. In a preferred embodiment, the saccharide mixture solution is a hydrolzate of plant cell wall and the acidic oligosaccharides are saccharides prepared by allowing a weak acid to link to hardly digestive oligosaccharides. In addition, more than 90 wt.% of the oligosaccharides and monosaccharides in the separated neutral component-rich fraction preferably has a molecular weight of 680 or less. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for selectively separating acidic oligosaccharides from a mixed sugar solution containing acidic oligosaccharides and neutral components consisting of neutral oligosaccharides and monosaccharides, specifically, plant cell wall hydrolysates. The present invention relates to a method for selectively separating acidic oligosaccharides having useful properties such as intestinal bifidobacteria growth promoting action.

  Oligosaccharides are carbohydrates in which 2 to 10 monosaccharides are polymerized, and exist in various forms in nature. Some of the oligosaccharides that are indigestible, when taken orally, reach the large intestine without being substantially decomposed by the digestive fluid and are selectively assimilated by the intestinal bifidobacteria. Proliferates and improves the intestinal flora. In addition, the specific mechanism of indigestible oligosaccharides has not been clearly elucidated, but also has an effect of suppressing body fat accumulation.

  Conventionally, various oligosaccharides have been used as intestinal bifidobacterial growth factors and body fat accumulation inhibitory factors (Non-patent Documents 1 to 6, Patent Document 1), and these oligosaccharides are all sugars. It has some sweetness. However, some consumers retain a bad image of sweetness because of obesity and diabetes avoidance. Therefore, providing a bifido growth factor having a taste such as acidity in addition to sweetness has great market significance. Furthermore, when selling with emphasis on the effect of suppressing body fat accumulation, a taste different from sweet taste is generally easily accepted by consumers.

  In view of the current state of the prior art, the applicant has proposed an acidic oligosaccharide having a molecular structure in which a weak acid is bound to an indigestible oligosaccharide as an oligosaccharide having a sour taste instead of a sweet taste, in an application of the same date. Yes. It is said that this acidic oligosaccharide can be obtained, for example, by hydrolyzing plant cell walls such as corn seed coat with acid and / or enzyme.

  However, among oligosaccharides contained in plant cell wall hydrolysates, acidic oligosaccharides are about 30 to 40%, and the rest are neutral oligosaccharides to which weak acids are not bound. In addition, the hydrolyzate contains some monosaccharides as well as oligosaccharides. Therefore, in order to obtain acidic oligosaccharides, it is necessary to separate and remove neutral components (neutral oligosaccharides and monosaccharides) contained in the degradation product.

An adsorption method using an anion exchange resin has been conventionally used as a method for separating and removing neutral components in the degradation product of the plant cell wall. In this method, the decomposition sugar solution of the plant cell wall is passed through a column filled with an anion exchange resin. At that time, acidic components are adsorbed on the resin and remain in the column, while neutral components are not adsorbed on the resin. It is discharged as it is. The adsorbed acidic component can be eluted with a salt containing metal ions (Ca ²⁺ , Na ^+, etc.).

This method is suitable for obtaining high-purity acidic oligosaccharides, but since the acid bound to acidic oligosaccharides is a weak acid, it can be easily detached from the exchange resin and easily mixed with neutral oligosaccharides. Therefore, only a very small amount of acidic oligosaccharide is adsorbed on the anion exchange resin, resulting in a disadvantage that the capacity of the separation apparatus is increased and the efficiency is poor. Further, since a salt is used for detachment of the acidic oligosaccharide from the anion exchange resin, it is necessary to insert a desalting step after the separation step, and there is a drawback that the step becomes complicated.
Oku, Nutrition Journal, 44, 291 (1986) Ishikawa et al., Bifidos, 9, 5, (1995) Hara et al. , Bifdobact. Microflora. , 13, 51, (1994) Sakai et al., Starch Chemistry, 63, 167 (1990) Agheli et al. , J of Nutr. , 128, 1283, (1998) Hayashi et al., Medicine and Biology, 119, 15 (1989) Japanese Patent Laid-Open No. 10-290681

  The present invention was created in view of the current state of the prior art, and its purpose is to selectively select acidic oligosaccharides from a mixed sugar solution containing acidic oligosaccharides and neutral components (neutral oligosaccharides and monosaccharides). It is to provide an efficient and simple method of separation.

  In order to solve such problems, the present inventor has intensively studied a method for separating acidic oligosaccharides from a mixed sugar solution of acidic oligosaccharides and neutral components. It has been found that by providing a mixed sugar solution, a sugar solution mainly composed of acidic oligosaccharides can be obtained efficiently and simply, and the present invention has finally been completed.

  That is, according to the present invention, in the method for selectively separating acidic oligosaccharides from a mixed sugar solution containing acidic oligosaccharides and neutral components consisting of neutral oligosaccharides and monosaccharides, the cation exchange resin is separated as a separating agent. The method comprising the step of subjecting the mixed sugar solution to a chromatographic separation process used as a step, thereby separating the mixed sugar solution into a fraction rich in acidic oligosaccharides and a fraction rich in neutral components. Provided.

  According to another preferred embodiment of the present invention, the mixed sugar solution is a hydrolyzate of plant cell walls, and the acidic oligosaccharide is a sugar in which a weak acid is bound to an indigestible oligosaccharide.

  According to another preferred embodiment of the present invention, 90% by weight or more of the oligosaccharides or monosaccharides contained in the separated fraction rich in neutral components has a molecular weight (average molecular weight) of 680 or less.

  As a mixed sugar solution containing acidic oligosaccharides and neutral components used as raw materials in the separation method of the present invention, for example, a hydrolyzate of plant cell walls can be used. As the plant cell wall to be hydrolyzed, any material containing a large amount of acidic polysaccharide can be used. For example, corn seed coat (grain hull), which is a by-product during the production of corn starch, can be used. For the hydrolysis method, for example, acid hydrolysis and / or enzyme hydrolysis disclosed in JP-A-11-313700 may be used.

  In the separation method of the present invention, the composition of the acidic oligosaccharide in the mixed sugar liquid that is the raw material depends on the composition of the acidic polysaccharide in the plant cell wall used for the hydrolysis, but generally, it is an indigestible oligosaccharide. It is a sugar in which a weak acid (uronic acid such as glucuronic acid or ferulic acid) is bound. Due to the presence of this weak acid, the oligosaccharide can exhibit an acidity rather than the original sweetness of sugar. Further, the mode of binding of weak acids to oligosaccharides is generally considered to be a mode in which weak acid residues are present as oligosaccharide side chains.

  Oligosaccharides, which are basic skeletons of acidic oligosaccharides, are the main players in intestinal bifidobacteria growth promoting action and body fat accumulation inhibiting action. Although the average degree of polymerization of this oligosaccharide varies depending on the hydrolysis conditions of the plant cell wall, the average degree of polymerization is preferably 2 to 10 from the viewpoint of low viscosity and ease of handling of the resulting acidic oligosaccharide. . A more preferable range of the average degree of polymerization is 2 to 8, and a particularly preferable range is 2 to 7.

  The average degree of polymerization of the oligosaccharide can be controlled by appropriately adjusting the hydrolysis time and the pH of the reaction solution during the hydrolysis. In the present invention, the “indigestible oligosaccharide” means an oligosaccharide that is not substantially decomposed in the digestive fluid and therefore has a digestibility that is not absorbed in the small intestine and reaches the large intestine as it is. For example, xylooligosaccharide in which a plurality of xyloses are polymerized corresponds to this. Moreover, this oligosaccharide is not limited to a homo-oligosaccharide, and depending on the composition of the plant cell wall used for hydrolysis, it may be a hetero-oligosaccharide obtained by polymerizing two or more monosaccharides.

  The mixed sugar solution obtained by hydrolysis of the plant cell wall is decolored and desalted as desired before being subjected to chromatographic separation using a cation exchange resin, but the method is not limited at all. Moreover, when insoluble matters are contained in the impurities, it is desirable to remove them before chromatography.

  Decolorization and removal of contaminants can be carried out using activated carbon or chemically synthesized resin, but it is necessary to carry out decolorization and those that do not adsorb acidic oligosaccharides or under such conditions. Further, when the amount of coloring is high, the purification load can be reduced by preliminary decolorization with powdered activated carbon, activated clay, chitosan, chemically synthesized resin or the like. However, the particle size and method of use of activated carbon or chemically synthesized resin used for decolorization are not limited.

  The desalting treatment can be performed by sequentially passing the sugar solution through a cation exchange resin and an anion exchange resin, or by passing through a mix bed (mixed product of cation exchange resin and anion exchange resin). It is also possible to sequentially pass the sugar solution through a cation exchange resin, an anion exchange resin, and a mix bed. An electrodialysis membrane can also be used for the purpose of reducing the desalting load. Since the membrane used for the electrodialysis membrane has a pore size that allows salts to pass through and does not allow acidic oligosaccharides to pass through, the membrane is suitable for efficiently pre-desalting a sugar solution containing acidic oligosaccharides. The anion exchange resin used for desalting is not particularly limited, but it is necessary to use a weakly basic one that does not adsorb acidic oligosaccharides, or a weakly basic one that does not adsorb acidic oligosaccharides. The same applies to mixed beds.

  If the coloring component or impurities mask the resin used for chromatography and affect the separation, it is necessary to decolorize and eliminate the impurities in advance. Moreover, when the metal ion of the salt to contain exchanges with the metal ion of a cation exchange resin, and results in lowering | separating performance as a result, it is necessary to desalinate in advance. However, the desalting treatment is not limited to this when the degradation of the separation performance can be prevented by mixing counter ions in the eluent.

  Regardless of the presence or absence of decolorization and desalting treatment, the solid content concentration in the mixed sugar solution to be subjected to the chromatographic separation treatment is preferably as high as possible within a range that allows operation. Usually, it is desirable to use a sugar solution having a solid content concentration of 50% by weight or more, particularly 60 ± 5% by weight. In addition, the temperature of the sugar solution is preferably as high as possible in order to prevent the growth of microorganisms and reduce the viscosity. However, if the sugar solution is easily colored, be careful not to raise the temperature too much. There is a need. Usually, a sugar solution and water are supplied to the chromatography apparatus at 60 to 70 ° C.

The cation exchange resin used for the chromatographic separation treatment is preferably a Na ^{+ type} cation exchange resin, and particularly preferably a sulfonated styrene-divinylbenzene crosslinked copolymer in Na salt form. As the styrene-divinylbenzene crosslinked copolymer, a gel type is usually used. The lower the degree of crosslinking, the better the separation performance. In chromatography, the mixed sugar solution is separated into two fractions rich in acidic oligosaccharides and a fraction rich in neutral components, or two fractions rich in neutral components, and fractions rich in acidic oligosaccharides. The fraction is separated into three fractions, a fraction rich in neutral oligosaccharides and a fraction rich in monosaccharides. It is preferable that 90% by weight or more of the oligosaccharide or monosaccharide contained therein has a molecular weight of 680 or less in the separated fraction rich in neutral components.

  Chromatography can be performed according to the method described in JP 2000-201700 A. In order to fractionate the sugar liquid into a fraction rich in acidic oligosaccharides and a fraction rich in neutral components, the simulated moving bed method, that is, the separation agent is filled and the liquid circulates in the interior cyclically. Supply the sugar solution and water or eluent mixture as eluent to the packed bed, and simultaneously extract the fraction rich in acidic oligosaccharide and the fraction rich in neutral components from the bed, And it is preferable to carry out by the system which switches these supply ports and outlets to those downstream downstream. In this case, a standard method in which liquid is constantly circulated in the floor, and the supply of liquid to the floor and the extraction of liquid from the floor are continuously performed from the switching of the supply port and the outlet to the switching. Can also be performed. Also, a modification method (FIG. 2) in which only the liquid is circulated in the floor without supplying the liquid to the floor and extracting the liquid from the floor for a certain period of time between the switching of the supply port and the discharge port. And JP-A-2-49159).

Alternatively, according to the method described in JP-A-63-158105, the separating agent is filled and the front end and the rear end are connected by a liquid flow path so that the liquid circulates in the bed. It is also possible to carry out the following semi-continuous method using an apparatus equipped with a packed bed that can be moved in an automatic manner.
(I) A raw material supply step of supplying a sugar solution from the front end of the packed bed and extracting a fraction rich in neutral components from the rear end.
(Ii) A first desorption step of supplying water or a counter ion mixture from the front end of the packed bed and extracting a fraction rich in neutral components from the rear end.
(Iii) A second desorption step of supplying water or a counter ion mixture from the front end of the packed bed and extracting a fraction rich in acidic oligosaccharides from the rear end.
(Iv) The liquid in the bed is moved cyclically without supplying the liquid to the packed bed and withdrawing the liquid from the packed bed, and the zone where the acidic oligosaccharide and the neutral component are mixed is formed in the packed bed. Circulation process to move to the front end.

  In any case of chromatography, it is preferable to operate so that the fraction rich in acidic components contains 70% by weight or more, particularly 80% by weight or more of acidic oligosaccharides in the supplied sugar liquid. . Moreover, it is preferable to operate so that the solution rich in neutral components may contain 70% of the neutral components in the supplied sugar liquid. The supply ratio of water or counter ion mixture to sugar liquid is usually 5 times (volume ratio) or more, but the simulated moving bed method generally reduces the supply ratio of water or counter ion mixture. it can.

To fractionate a sugar solution into three fractions rich in acidic oligosaccharides, a fraction rich in neutral oligosaccharides, and a fraction rich in monosaccharides, the description of JP-A-63-158105 In accordance with the following, a packed bed in which the separating agent is filled and the front end and the rear end are connected by a liquid flow path so that the liquid can circulate in the bed is used. It is preferable to carry out by the semi-continuous system which repeats the cycle which consists of 1 process-4th process (refer also FIG. 3).
(I) A first step of supplying a sugar solution from the front end of the packed bed and extracting a fraction containing a large amount of neutral oligosaccharides from the rear end of the packed bed.
(Ii) Acid oligosaccharides, neutral oligosaccharides, and monosaccharides are mixed by circulating the liquid in the bed without supplying the liquid to the packed bed and withdrawing the liquid from the packed bed. A second step of moving the zone to the front end of the packed bed;
(Iii) A third step in which water or a counter ion mixture is supplied from the front end of the packed bed, and a fraction rich in monosaccharides and a fraction rich in acidic oligosaccharides are extracted in this order from the rear end of the packed bed.
(Iv) Acid oligosaccharides, neutral oligosaccharides, and monosaccharides are mixed by circulating the liquid in the bed without supplying the liquid to the packed bed and withdrawing the liquid from the packed bed. A fourth step of moving the zone to the front end of the packed bed.

  Several additional steps can be added to the above steps. For example, when it is desired to extract a specific component of a monosaccharide in a sugar solution with high purity, and when the purity required by the separation of three fractions is insufficient, before this, as a preliminary separation, Chromatography to separate monosaccharides may be inserted. For example, when the main composition of the sugar solution is acidic oligosaccharide, neutral oligosaccharide, D-xylose, L-arabinose, and L-arabinose is desired to have a purity of 80% or higher, For example, the liquid is separated into three fractions rich in acidic oligosaccharide and neutral oligosaccharide, fraction rich in D-xylose, and fraction rich in L-arabinose by the above-mentioned three-component separation, and then the acidic oligosaccharide A fraction rich in sugar and neutral oligosaccharide may be separated by the following chromatography. In that case, separation and purification of acidic oligosaccharides separates into two fractions rich in acidic oligosaccharides and a fraction rich in neutral components.

Examples Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

(1) Preparation of roughly purified mixed sugar solution Add oxalic acid to corn seed coat to adjust the pH to around 2 and pressurize at 130 ° C for 30 minutes to 2 hours to obtain a decomposed sugar solution It was. The obtained degraded sugar solution was treated with cell wall degrading enzyme and then treated with powdered activated carbon to perform decolorization and removal of impurities. The decolorized sugar solution was concentrated, and then the salt content was removed by 60% with an electrodialysis membrane. The preliminarily desalted sugar solution was desalted by passing it through an ion exchange resin by a conventional method. This sugar solution was subjected to a chromatographic separation treatment with a Ca-type cation exchange resin (UBK535 (Mitsubishi Chemical Corporation)) to substantially separate and remove the monosaccharide component to obtain a crude purified mixed sugar solution. When this crudely purified mixed sugar solution was analyzed by HPLC using a ligand exchange column in which Na form and Ca form were linked, it was a sugar chain (AO ₃₄ ) containing an acidic oligosaccharide component as a main component (34%). (Fig. 1, Table 1). Further, when the constituent sugars of this acidic oligosaccharide were examined, it was found to be a hetero sugar chain containing 10% glucuronic acid (Table 2).

(2) Separation of acidic oligosaccharides from crude mixed sugar solution by chromatographic separation using cation exchange resin as a separating agent Na ⁺ salt of sulfonated styrene-divinylbenzene crosslinked copolymer (gel type, average Modified simulated movement using an apparatus (Fig. 2) in which four packed beds packed with 338 mL each in a column with a particle diameter of 220 μm and an average capacity of 1.8 meq / mL packed in a column with an inner diameter of 27 mmφ are formed to form a circulation path. The crude purified mixed sugar solution (see Table 1) obtained in (1) was fractionated into a fraction rich in acidic oligosaccharides and a fraction rich in neutral components by the bed.
The operating conditions were as follows:
Sugar solution supply rate 54mL / hr
Water supply speed 675mL / hr
Extraction speed of fractions rich in neutral components 208mL / hr
Extraction rate of fraction rich in acidic oligosaccharides 170mL / hr
Flow rate of the circulation process 675 mL / hr
Supply-extraction process 12.4 minutes Circulation process 9.8 minutes Opening and closing of the valve (valve) until returning to the initial state by repeating four processes was as shown in the table of FIG. In the table, ◯ indicates the opened state of the valve, and × indicates the closed state.
As a result of fractionation, the composition shown in Table 3 could be obtained.

  From Table 3, it is clear that according to the present invention, acidic oligosaccharides can be efficiently separated from a mixed sugar solution of acidic oligosaccharides and neutral components by a simple process.

The gel filtration result of a corn seed husk acid decomposition product is shown. An example of the chromatography apparatus of a modified simulated moving bed system is shown. An example of a semi-continuous chromatography apparatus is shown. Shows opening and closing of valves on simulated moving floor.

Claims (4)

  In a method for selectively separating acidic oligosaccharides from a mixed sugar solution containing acidic oligosaccharides and neutral components consisting of neutral oligosaccharides and monosaccharides, the chromatographic separation process uses a cation exchange resin as a separating agent. A method comprising the step of providing the mixed sugar solution, thereby separating the mixed sugar solution into a fraction rich in acidic oligosaccharides and a fraction rich in neutral components.   2. The method according to claim 1, wherein the mixed sugar solution is a hydrolyzate of plant cell walls.   The method according to claim 1 or 2, wherein the acidic oligosaccharide is a sugar in which a weak acid is bound to an indigestible oligosaccharide. The method according to any one of claims 1 to 3, wherein 90% by weight or more of the oligosaccharide or monosaccharide contained in the separated fraction rich in neutral components has a molecular weight of 680 or less.

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