JPS62198694A - Purification of oligosaccharide - Google Patents

Abstract

PURPOSE:To stably fractionate an oligosaccharide, having good flavors and hardly suffering from occurrence of browing reaction, etc., from a liquid containing the oligosaccharide and protein, ash in addition to the sugar, by using strong acidic cation exchange resins with crosslinking degree within a specific range. CONSTITUTION:A liquid containing an oligosaccharide, proteins, amino-acid and ashes normally at 2-50% conc. (oligosaccharide concentration is preferably 1-15%) is treated with strongly acidic (preferably sulfonic acid type) cation exchange resins under 3-7pH condition normally at about 0.15-0.55 SV liquid velocity to fractionate the oligosaccharide from other components. As for the liquid containing the oligosaccharide, proteins, etc., a soybean whey, an UF- or RO-treated liquid thereof, a hydrous ethanol extraction liquid of de-fatted soybean, an exudate liquid of soybean, an aqueous medium extraction liquid of soybean such as 'YU' is bean curd production, a hydrolyzate of an enzyme part of manner, agar, guar gum, etc., etc., are utilized.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for stably fractionating and purifying oligosaccharides from a liquid containing proteins, amino acids, ash, and oligosaccharides.

(Prior Art) Known methods for purifying saccharides include methods using ion exchange resins and electrodialysis.

For example, in JP-A-60-63000, saccharides (monosaccharides, trisaccharides, oligosaccharides, etc.) are separated and
A method of purification is disclosed. Japanese Patent Publication No. 1986-1304
No. 00 discloses a method for separating and purifying saccharides using an ion exchange resin with a degree of crosslinking of 7% or more. Japanese Unexamined Patent Publication 1983
-43240 discloses a method for separating sugars by molecular exclusion chromatography.

In addition, as a method using the ion exclusion method, Japanese Patent Application Laid-Open No. 55-13
5600 uses a Na-type strongly acidic cation exchange resin with a degree of crosslinking of 4 to 6%, and a molasses solution with a sugar concentration of 25 to 35 Bx.
A method of separating sucrose and reducing sugar using an ion exclusion method is disclosed.

The present invention differs from conventional techniques in that it separates and purifies sugars and non-diabetic proteins (proteins, amino acids, peptides, etc.) and natural substances, rather than separating and purifying sugars.

On the other hand, methods for purifying oligosaccharides from liquids containing proteins, peptides, amino acids, ash, and oligosaccharides, such as soybean aqueous medium extracts such as soybean whey, are not well known.

Low sugar content (typically 55-65 on dry solids basis)
%), and relatively high in electrolytes such as protein, free amino acids, and ash (approximately 10-15% and 1%, respectively, in terms of dry solid content).
~2%, 20-35%), it is difficult to fractionate and purify sugars, and sugars are unstable (they easily cause browning reactions with proteins and amino acids and are easily decomposed), making them economical and practical. This is due to the difficulty in fractionating and purifying sugars.

(Problems to be Solved by the Invention) While investigating the fractionation and purification of heterooligosaccharides from soybean whey by electrodialysis, the present inventors found that: , free amino acids, etc. and heterooligosaccharides cause a browning reaction, and the resulting oligosaccharides take on a brown color. ■In addition, soybean whey usually has a solid content of about 2.5% and a sugar concentration of 1.3%. Because of its low level, it is necessary to concentrate it to the concentration required for electrodialysis treatment.
We found problems such as poor recovery of oligosaccharides even when membranes were used for the concentration. Also, when trying to separate heterooligosaccharides from soybean whey using ion exchange method, adsorption/desorption,
We have found that a large amount of acid or alkali is required for resin regeneration, neutralization, etc., which causes problems in wastewater treatment. Therefore, the present inventors aimed to stably fractionate and purify oligosaccharides (with very little browning or decomposition) that are unlikely to undergo Al browning reactions and therefore have good flavor and color.
b) The aim was to develop a method for fractionating and purifying oligosaccharides that would minimize the need for acids or alkalis for resin regeneration, neutralization, etc.

(Means for Solving the Problem) The present inventors have conducted extensive research to achieve the above-mentioned objective, and while proceeding with studies regarding soybean whey, found that the degree of crosslinking is 4 to 12%.
Pi (
We found that when soybean whey is fractionated under conditions 3 to 7, browning and decomposition of oligosaccharides due to reactions with amino acids and peptides can be suppressed and stable separation and purification can be achieved. Furthermore, we have completed the present invention by obtaining knowledge that can be applied not only to soybean whey but also to other heterooligosaccharide-containing liquids. That is, the present invention provides a method for purifying oligosaccharides, which is characterized by fractionating oligosaccharides from a liquid containing proteins, amino acids, ash, and oligosaccharides using a strongly acidic cation exchange resin with a degree of crosslinking of 4 to 12%. It is.

The liquid containing protein, amino acids, ash, and oligosaccharides used in the present invention is soybean whey obtained in the soybean protein manufacturing process, UFSRO treatment liquid of soybean whey, hydrous ethanol extract of soybean, milk fat soybean, soybean infusion liquid, and tofu manufacturing process. The so-called “Yu” of time
Contains soybean oligosaccharides, as well as mixtures of heterooligosaccharides, protein, ash, etc. For example, mannan, agar,
Enzymatic partial hydrolysates of heteropolysaccharides such as guar gum are ash-containing heterooligosaccharide liquids obtained by low concentration decomposition and neutralization.
It can be used in the present invention. For example, a solution obtained by neutralizing a 2% solution of konjac mannan with cellulase derived from Aspergillus niger (1.5% vs. substrate) at pH 5.0 and 50° C. for 15 hours can also be used. In addition, soybean whey usually has a sugar content of about 1.3%, crude protein of about 0.5%,
It is a hetero-oligosaccharide-containing liquid containing about 0.6% ash and usually about 48 parts of sucrose, about 12 parts of raffinose, and about 40 parts of stachyose, and oligosaccharides can be separated and purified by the present invention. .

As the strong acidic cation exchange resin used in the present invention, known strong acidic cation exchange resins can be used, but sulfonic acid type strong acid cation exchange resins are particularly suitable, such as Diaion MK-31S, Diamond Aeon 5K-102
(Mitsubishi Kasei side part), etc.

Further, the degree of crosslinking of the strongly acidic cation exchange resin is suitably 4 to 12%, preferably 4 to 10%. If the degree of crosslinking is less than 4%, the resin tends to swell and contract, making separation unstable. If the degree of crosslinking exceeds 12%, separation of oligosaccharides from other electrolytes becomes poor. Using such a strongly acidic cation exchange resin as a column or bed, oligosaccharides can be separated and purified using the Donnan effect in ion exclusion chromatography or the like.

Furthermore, in the present invention, in the case of oligosaccharides containing fructose such as a soybean oligosaccharide-containing solution, it is important to fractionate under conditions of pH 3 to 7, preferably 4 to 6. pH is 3
If the pH is less than 7, decomposition of oligosaccharides tends to occur, and if the pH exceeds 7, isomerization and decomposition of oligosaccharides tend to occur, making it difficult to stably fractionate oligosaccharides. For example, in the case of soybean whey, which contains proteins, free amino acids, natural substances, and other trace components and makes it difficult to stably fractionate soybean oligosaccharides, the degree of crosslinking is determined under conditions of pH 3 to 7. It can be stably purified by fractionation using a strongly acidic cation exchange resin of 4 to 12. In the case of oligosaccharides that do not contain fructose, isomerization and decomposition of oligosaccharides occur even at pH 3 to 8.5, so conditions of << pH 3 to 8.5 can be used.

In addition, the concentration of the oligosaccharide-containing solution used for fractionation is usually 2 to 50%.
(of which, the oligosaccharide concentration is usually 1-25%), the temperature is usually 10%
A temperature of 0°C or lower is suitable. If the oligosaccharide concentration is high, separation becomes poor, and if the oligosaccharide concentration is low, oligosaccharide decomposition and browning occur, which is undesirable. Therefore, the appropriate concentration of oligosaccharides is 1 to 25%, preferably 1 to 15%.

Further, it is preferable to adjust the concentration of the oligosaccharide-containing solution, the amount of the supplied solution, and the temperature depending on the degree of crosslinking. For example, when the degree of crosslinking is relatively low, about 4, the concentration of the soybean oligosaccharide-containing solution is as low as 2% (white sugar 1.2%), and the amount of supplied solution is 20% (V
/V%), and the temperature is preferably as low as about 15°C.On the other hand, when the degree of crosslinking is relatively high, about 8, the concentration of the oligosaccharide-containing liquid is as high as 10% (approximately 6% white sugar), and the temperature is as low as about 15°C. It is preferable that the liquid amount is as low as 8% per resin and the temperature as high as about 80°C. From the viewpoint of resin resistance and sugar stability, the temperature is 1.
It is preferable not to exceed 0°C. Incidentally, the flow rate is preferably slow, and the usual liquid flow rate is S, V, about o, 15~
Approximately 0.55 is appropriate.

In addition, water or hot water having the above-mentioned pH or an organic solvent such as ethanol can be used for passing the liquid. Incidentally, molecular weight fractionation is also possible to some extent by fractionating the effluent fraction.

The strongly acidic cation exchange resin used in the present invention can be continuously recycled and used for fractionation and purification of oligosaccharides, and when regenerating it, a dilute alkaline solution (for example, 5% NaOH solution) can be used.

Since the oligosaccharide-containing liquid fractionated as described above has a low sugar concentration (usually about 0.5 to 2%), it is better to concentrate or dry it. For concentration, known means such as heating concentration and membrane concentration can be used. Although it depends on the intended use, it is usually appropriate to concentrate it to 75% or more. Compared to electrodialysis and other conventional methods, the oligosaccharide-containing liquid fractionated by the method of the present invention is extremely unlikely to brown or decompose even under severe conditions such as sheet metal heating concentration, and has excellent flavor and color. It can be a sugar.

In addition, the oligosaccharide of the present invention has a low sweetness (and is white in color) and can be used in various foods and drinks. It has a wide range of uses, such as in various types of oligosaccharides.Also, depending on the type of oligosaccharide, it is effective as a bifidus factor.

In addition, the low tFj of the present invention can be further fractionated and purified into each component oligosaccharide using a known saccharide fractionation and purification method.

(Example) Embodiments of the present invention will be described below with reference to Examples.

Example 1 Soybean whey (concentration 2.5%, pH 5, white sugar content 1.3%, protein 0.6%, ash 0.6 %)250II1
) was adjusted to a temperature of 50°C and supplied to a column packed with a sulfonic acid type strongly acidic cation exchange resin (Diaion 5K-102, Mitsubishi Kasei side group) with a degree of crosslinking of 4% to a diameter of 4 cfflφ and a packed bed height of 138 cm. Using water at a temperature of 50°C, flow rate is 8f.
It was developed at fIl/min and fractionated into sugar and electrolyte (mainly consisting of protein and ash). The elution pattern at this time is shown in FIG. The vertical axis represents sugar concentration and electrical conductivity. However, the sugar concentration was determined by measuring the absorbance at 485 nm using the phenol-sulfuric acid method, and was expressed in terms of glucose. Further, electric conductivity was used as an index of electrolyte concentration, and m absorption/absorbance was used as an index. The absorbance was measured using a spectrophotometer (UV-260 manufactured by Shimabara@).

Each fraction shown in FIG. 1 was fractionated as shown in Table 1, and its sugar composition was identified and quantified using high performance liquid chromatography (manufactured by Waters). The results are shown in Table-1. In addition, the column uses CK CH for μBOND P,
The solvent used was acetonitrile:pure water=70×30.

(Left below) Table-1 Amount of liquid passed Type of sugar Ratio% 1200 to 1
400mj! Stachyose 70 Roughinose 5 Sucrose 25 1400-1600n+42 Stachyose 4
9 Raffinose 1 Sucrose 50 1600-1800 ml Sucrose 95 Fructose 5 A sugar solution with a high oligosaccharide content can be obtained depending on how the fractions are separated. Note that fructose is thought to be produced by decomposition of soybean oligosaccharides.

The fractionated sugar solution (1200-1800 mβ fraction in Figure 1) was heated and concentrated at 100°C (up to 75% concentration) to obtain soybean oligosaccharide syrup. The color was pale yellow and the flavor was good. Further, soybean oligosaccharide syrup was freeze-dried to obtain pale yellow crystalline soybean oligosaccharide. The dry solid content was 90% sugar, 3% crude protein, and 7% natural.

Example 2 In the same manner as in Example 1, soybean oligosaccharides were fractionated by changing the temperature at which soybean whey was applied to a sulfonic acid type strongly acidic cation exchange resin and the degree of crosslinking of the sulfonic acid type strongly acidic cation exchange resin. Of the elution patterns obtained, Figure 2 shows the crosslinking degree of 4.
%, the results at a temperature of 15°C are shown in Figure 3, with a degree of crosslinking of 8%,
The results at a temperature of 80°C are illustrated.

A temperature of 15 to 50°C is suitable for fractionation of soybean oligosaccharides when the degree of crosslinking is 4 to 6%, and a temperature of 50 to 80°C is suitable for a degree of crosslinking of 7 to 12%.
°C was suitable for fractionation of soybean oligosaccharides.

Example 3 In the same manner as in Example 2, soybean oligosaccharides were fractionated by changing the concentration of soybean whey applied to a sulfonic acid type strongly acidic cation exchange resin from 2 to 10%. However, soybean whey was concentrated at a low temperature to prevent browning and sugar decomposition.

Among the elution patterns obtained, Fig. 4 shows the results with a degree of crosslinking of 4% and a concentration of 2% and 4%, and Fig. 5 shows the results with a degree of crosslinking of 8% and a concentration of 2% and 4%.
The results at concentrations of 5% and 10% are illustrated.

At a cross-linking degree of 4-6%, a soybean whey concentration of 2-3% is suitable for the fractionation of soybean oligosaccharides, and at a cross-linking degree of 7-12%, a soybean whey concentration of 5-10% is suitable for the fractionation of soybean oligosaccharides. It was appropriate.

Example 4 In the same manner as in Example 1, soybean oligosaccharides were fractionated by changing the p8 applied to a sulfonic acid-type strongly acidic cation exchange resin of soybean whey to 3.5°7.

The results are shown in Figure 6.

When the soybean whey concentration was 2.5%, the temperature was 15°C, and the degree of crosslinking was 4%, p1)5 was most suitable for fractionation of soybean oligosaccharides.

Example 5 A 2% solution of commercially available konjac powder was adjusted to pH using a commercially available cellulase derived from Aspergillus (1.5% vs. konjac powder).
After hydrolyzing at 5, 0, and 50°C for 15 hours, the hydrolyzed solution was neutralized to pH 7 and heated to 50°C, and the same ion-excluding sulfonic acid type strong acid cation exchange resin column used in Example 1 (
The mixture was applied to an ion exchange resin (the degree of cross-linking was 8%) and fractionated into a sugar fraction and an electrolyte fraction.

(Effects) As detailed above, the present invention has made it possible to stably fractionate and purify oligosaccharides from a liquid containing oligosaccharides, proteins (including amino acids and peptides), ash, etc. It contributes to the development of industry.

[Brief Description of the Drawings] Fig. 1 is a drawing showing the column elution pattern of 2.5% soybean whey. 1□ shows the elution curve of sugar, 2... shows the elution curve of electrolyte, and 3-... shows the elution curve of protein. FIG. 2 is a diagram showing a column elution pattern of 2.5% soybean whey at a temperature of 15° C. and a degree of crosslinking of an ion exchange resin of 4%. 1□ shows the elution curve of sugar, 2... shows the elution curve of electrolyte, and 3-... shows the elution curve of protein. FIG. 3 is a drawing showing a column elution pattern of 2.5% soybean whey at a temperature of 80° C. and a degree of crosslinking of ion exchange resin of 8%. 1□ shows the elution curve of sugar, 2... shows the elution curve of electrolyte, and 3-... shows the elution curve of protein. FIG. 4 is a drawing showing column elution patterns of ion exchange resins with a crosslinking degree of 4% for soybean whey concentrations of 2% and 4%. 1□ shows the elution curves of sugars at 2% and 4% soybean whey concentrations, and 2... shows the elution curves of electrolytes at 2% soybean whey concentrations. 3... shows the elution curve of electrolyte with a soybean whey concentration of 4%. 4-- shows the elution curve of protein with a soybean whey concentration of 2%. 5-- shows the elution curve of protein with a soybean whey concentration of 4%. FIG. 5 is a drawing showing column elution patterns of soybean whey concentrations of 5% and 10% with ion exchange resins having a degree of crosslinking of 8%. 1□ shows the elution curve of sugar at a soybean whey concentration of 5%. 2□ shows the elution curve of sugar at a soybean whey concentration of 10%. 3... shows the elution curve of electrolyte with a soybean whey concentration of 5%. 4... shows the elution curve of electrolyte with a soybean whey concentration of 10%. 5-- shows the elution curve of protein with a soybean whey concentration of 5%. 6-- shows the elution curve of protein at a soybean whey concentration of 10%. FIG. 6 is a drawing showing the column elution pattern of soybean whey with a pH of 3.5.7. ■□ is the sugar elution curve of soy whey p) 13, 2□ is the sugar elution curve of soy whey p1) 5, 3□ is the soy whey pH
7 shows the sugar elution curve, 4... shows the electrolyte elution curve of soybean whey pH 3. 5... shows the electrolyte elution curve of soybean whey pH 5. 6 shows the electrolyte elution curve of soybean whey pH 7. 7-- shows the protein elution curve of soybean whey pH 3. 8-- shows the protein elution curve of soybean whey pH 5. 9-- shows the protein elution curve of soybean whey pH 7.

Claims (5)

[Claims] (1) A method for purifying oligosaccharides, which comprises fractionating oligosaccharides from a liquid containing proteins, amino acids, ash, and oligosaccharides using a strongly acidic cation exchange resin with a degree of crosslinking of 4 to 12%. (2) Claim No. 1 in which the oligosaccharide is a heterooligosaccharide (
Purification method described in section 1). (3) The purification method according to claim (1), wherein the liquid containing protein, amino acids, ash, and oligosaccharides is a soybean aqueous medium extract. (4) The purification method according to claim (1), in which the fractionation is carried out by ion exclusion chromatography. (5) The purification method according to claim (1), wherein the fractionation is carried out under conditions of pH 3 to 7.