JPS5995895A - Purification of fructo-oligosuccharide - Google Patents

Abstract

PURPOSE:To separate pure fructo-oligosaccharide useful as a raw material of a non-calorific food and an agent for the promotion of the proliferation of intestinal bifidus bacteria, in high yield, by treating a sugar composition with alpha- glucosidase, thereby selectively decomposing sucrose. CONSTITUTION:A sugar composition containing monosaccharides, sucrose, and fructo-oligosaccharide is treated with alpha-glucosidase. Only the sucrose is decomposed to monosaccharides and removed from the system by this treatment, and purified fructo-oligosaccharide can be obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for isolating and purifying a saccharide-oligosaccharide from a saccharide composition containing monosaccharides, sucrose, and fructooligosaccharides by allowing α-gluco/dase to act on the saccharide composition. ,
The present invention relates to a method for purifying fructooligosalts, which is characterized by decomposing and removing only sucrose into monosaccharides without decomposing fructooligosaccharides.

Fructooligosalts in the present invention include sucrose bound to one molecule of fructose/ζ trisaccharide (hereinafter referred to as GF2), and sucrose bound to two molecules of fructose (hereinafter referred to as GF2) and tetrasaccharide (hereinafter referred to as GF2). GF3), a pentasaccharide in which three fructose molecules are bonded to saccharide (hereinafter referred to as GF4), and mixtures thereof.

This fructooligosalt is very tactile, is not absorbed in the body, is non-active, and has excellent effects such as promoting the growth of bifidobacteria in the intestine and lowering lipids in the body. It is becoming clear that it has an effect.

These fructooligosaccharides can be produced, for example, by reacting sucrose with fructosyltransferase produced by plants or microorganisms, and the industrial reaction conditions are as follows:
It is preferable to carry out under the following conditions. That is, the concentration of 7-eu-close during the transfer reaction is set to 5 to 70, preferably 30 to 60. The pH of the reaction varies depending on the origin of the enzyme, but the pH of the reaction is between 4.0 and 7.0. Temperature 25~
65°C, preferably 50.-60°C. The amount of enzyme used is 5 to 200 units, preferably 20 to 80 units per nuclose 17. Here, the enzyme unit is 1.0 ml of 5% N-Eucrose solution, pH 5.
,0 ml of buffer! When 0.5m6 of the enzyme solution was added to the water and reacted at 40'C for 60 minutes, 2.5m6 of the reaction solution was added.
The amount of enzyme that produces 1 μmole of glucose in 60 minutes during /i is expressed as 1 unit.

After the rearrangement reaction is completed, the enzyme is deactivated by heating, decolorized with activated carbon, desalted with an ion exchange resin, and then concentrated to obtain the desired product. The transition composition can be analyzed using, for example, a Micro Bondapak CH column (manufactured by Waters Limited) using acetonitrile:water (80:20 (v/v)).
v) It can be carried out by a high performance liquid chromatography method using the solvent system of ()).

The composition of the sugar composition thus obtained was 28 parts glucose, 2 parts fructose, and 7 parts euucrose (
Sucrose) 11%, GF228, GF325%,
Although the CTF is 46%, the composition of each constituent sugar can take various values depending on the reaction conditions.

The oligosaccharide GF 2 is O-β-D-fructofuranosyl(2-1)-0-β-fructofuranoyl(
2→1)-α-D-curcopyranont.

0-β-D-fructofuranosyl-(2-6)-〇-β
-glucopyranone-ru(1→2)-β-D-fructofurano7do, 0-β-D-fructofuranone-ru(2→6)
)-〇-β-Fructofurano/Rue(2-1)-α-D
-glucopyranoside, etc., and as C7F3, O-
β-D-Fructofuranone Ru (2→[1-0-β-D
-Frucj-furanosyl]2→1)-α-D-glucopyranoside, 0-β-D-fructofuranosyl (2-6
)-0 [β-D-Fructofuranone Ru ('2-2))
-〇-α-1]-glucopyranosyl(]-2)-β-
There are D-fructofuranondo, etc., and GF4 is 0.
-β-D-fructofuranosyl-(2-C1-O-β-
D-fructofuranosyl-213-])-α-D-glucopyranoside and the like.

As a means for isolating and purifying fructooligosaccharides from the sugar composition obtained as described above, activated carbon chromatography and Imen exchange chromatography are generally used. Separation of sucrose from pentasaccharides or higher fructooligosaccharides, especially GF2, which is a trisaccharide, is poor, and if one attempts to isolate and purify fructooligosaccharides under such conditions, the yield will inevitably decrease ( (See Examples and Comparative Examples). However, if it is possible to decompose the sucrose and fructooligosaccharides in the sugar composition into monosaccharides (glucose, fructose) without decomposing them, the monosaccharides and pentasaccharides obtained as a result of such treatment can be By purifying the sugar solution containing only the above fructooligosaccharides using normal separation and purification means such as activated carbon chromatography,
Isolation and purification of oligosaccharides becomes easier, making it possible to improve yields and reduce costs.

The inventors conducted extensive research on this point and found that α-gluco/dase, a type of sugar ice-breaking enzyme, breaks down sucrose into monosaccharides, but does not have the effect of breaking down fructooligosaccharides. They discovered this and completed the present invention.

As such an enzyme, α-glucosidase present in the small intestines of rats and rabbits can be used, but from an industrial perspective, it is preferable to use a microbial enzyme, and as a result of a wide search, yeast -TΦ was used. Thesononoromyces 0 cerevisiai (Sa
ccharomyces cerevisiae)
We discovered that JAM4518 produces the desired enzyme. This enzyme was transferred to Zatucharomyces cerevizii I.
4518 strain at 28°C in a medium containing 15% maltose, 0.7% peptone, and 0.3% yeast extract.
It is produced within the bacterial cells by culturing for 4 to 48 hours, and its activity reaches 0.1 to 0.3 U/mg of the bacterial cells.

To obtain α-glucosidase activity, add 5 to 7 units per 17 of the reaction sugar composition (including monosaccharides, sucrose, and fructooligosaccharides) to adjust the concentration of the reaction solution to 5 to 50, preferably 5 to 30. pH 5-8, preferably 6-7, reaction temperature 30-50°C, preferably 35-40°C, 2-40°C.
By reacting for 4 hours, more than 80% of sucrose contained in the reaction sugar composition can be decomposed into monosaccharides consisting of glucose and fructose. In addition, the activity of α-glucosidase is determined by H, HALVOR3ON, etc.
iochem.

Biophys, Acta, 30 28 (] 95
8) Measured according to the method described in 1PH6, 8, 37
The amount of enzyme that produces 1 μm ole of paranitrophenol per minute from paranitrophenyl-α-D-glucopyranosite at °C is expressed as one unit.

The sugar solution thus obtained can be treated with conventional purification means to purify and isolate fructooligosaccharides in good yield. EXAMPLES The present invention will be explained in more detail by showing examples below.

Example: Zatucharomyces cerevizii IAM451s strain was mixed with 5% maltose, 0.7% peptone, and 0.3% fermented extract.
One platinum loop was inoculated into a medium containing the following, and cultured with shaking at 28°C for 24 hours. This is designated as the first seed mother liquor.

Next, 5 ml of the first seed mother liquor was added to the medium 5007I having the above composition.
(!) and cultured with shaking at 28°C for 24 hours. This is used as the second seed mother liquor. 500 ml of the second seed mother liquor was mixed with maltose 15%, peptone 0.7 part, and yeast U extract 03
Inoculate 15 t of culture medium containing the above-mentioned material, and incubate in a 30 t jar fermenter at 28°C and 350 rpm for 30 minutes.
Time aeration agitation culture was performed. After culturing, the bacterial cells are collected from the culture solution by centrifugation, and bacterial cells containing α-glucosidase 3 are collected.
I got 00g. The α-glucondase activity of bacterial cells is 0.2
Units/mg bacterial cells.

5.0 nog of a sugar composition consisting of 39.3% glucose, 10.4% sucrose, and 5.0.3 parts of fructooligosaccharide (including GF2, GF3, and GF4) was dissolved in water to make a 10 part solution. The PI-1 of the solution was set to 6.8. Next, 1,607 cells of the above-mentioned α-glucosidase-containing bacterial cells were added to this solution, and the mixture was reacted at 37°C for 3 hours with stirring.

After the reaction, the bacterial cells were removed by filtration, and the reaction solution was
% concentration. The sugar composition after the reaction was 49.6 units of monosaccharides/0.4 units of sucrose and 50.0 units of oligosaccharides.

Activated carbon for chromatography (manufactured by Takeda Pharmaceutical) 401q with a diameter of 55
15 t, :g of sugar solution packed in a cm column and subjected to the above α-glucosidase treatment.
Fill the pot with 2,200 tons of water at 5V=1, drain the pot with 1,500 tons of 5% ethanol solution. Next, elution was performed sequentially with 1,500 t of 20% ethanol solution.

Each eluate was divided into 001 fractions, and the sugar composition of each fraction was analyzed using high performance liquid chromatography. This fractionation diagram is shown in FIG.

Comparative Example Regarding a sugar solution that was not subjected to α-glucondase treatment, 15 kg of a 30% solution was charged and fractionated in the same manner as in the example. i.e. 2.2001 water, 1,5
Elution was carried out at 5V-1 with a 50% chetanol solution, 1,500t of a 10% ethanol solution, and 800t of a 20% ethanol solution. This fractionation diagram is shown in FIG.

As is clear from Fig. 2, which shows a solution that has not been treated with α-glucosidase, separation of sucrose and GF2 is poor in this method, and monosaccharides and fructooligosaccharides that do not contain sucrose are
It was obtained only in the 10% ethanol fraction and the 20% ethanol fraction. This fraction was concentrated and freeze-dried to obtain purified fructooligosaccharide 1,200F. The theoretical yield in Motoichi was 53 sections.

When the α-gluconodase treated solution was purified, as is clear from FIG. 1, fructooligosaccharides containing no monosaccharides and/or saccharides were obtained after the 5% heptanotanol fractionation. This fraction was xylemized and freeze-dried, resulting in 2.17 Of purified fructo-olicosaccharides. The theoretical yield in Motoichi was 96.

As described above, by performing the α-glucosidase treatment, the purification yield of fructo-olicosaccharide was improved by more than 80%.

[Brief explanation of the drawing]

FIG. 1 is a fraction diagram of a sugar composition purified by activated carbon chromatography using the method of the present invention, and FIG. 2 is a fraction diagram of a conventional method. 1 υ II) θ in Figure 20

Claims (1)

[Scope of Claims] 1. When isolating and purifying fructooligosaccharides from a sugar composition containing monosaccharides, sucrose and fructooligosalts, α-glucosidase is applied to the sugar composition to convert sucrose into monosaccharides. A method for purifying a fructooligosalt, which is characterized by decomposing it into 2. α-glucosidase is present in Satucharomyces se45
The purification method according to claim 1, wherein the enzyme is produced by No. 18. 3. A method for removing sucrose from a sugar solution containing monosaccharides, sucrose, and fructooligosaccharides without decomposing the fructooligosaccharides by allowing α-glucosidase to act on the sugar solution. 4 α-Glucosidaseca, Satucharomyces se451
8. The method according to claim 3, wherein the enzyme is produced by 8.