JPH04293494A - Production of fructose-containing oligosaccharide or glycoside - Google Patents

Abstract

PURPOSE:To efficiently produce a fructose-containing oligosaccharide or glycoside useful in the field of medicine and food at a low cost by treating sucrose with fructosyl group transferase and yeast deficient in invertase in the presence of an acceptor. CONSTITUTION:Sucrose is dissolved in water in the presence of an acceptor (e.g. lactose), reacted with beta-fructofuranosidase, a fructosyl group transferase produced by Arthrobacter sp. K-1 strain and simultaneously with yeast deficient in invertase at pH6.5-7.0 at 35 deg.C for 20 hours, the yeast deficient in invertase is removed by filtration with diatomaceous earth, the filtrate is treated with active carbon at 95 deg.C for 1 hour, purified with an ion exchange resin, concentrated and spray dried to give a fructose-containing oligosaccharide comprising powdery lactosylfructoside shown by the formula or a fructose-containing glycoside such as fructosyl stevioside.

Description

[Detailed description of the invention]

0001

TECHNICAL FIELD The present invention relates to a method for producing fructose-containing oligosaccharides or glycosides, and more particularly to a method for industrially producing fructose-containing oligosaccharides or glycosides efficiently and at low cost.

0002

[Prior Art and Problems to be Solved by the Invention] In recent years, with the increase in health consciousness, research has been conducted on the synthesis of oligosaccharides and useful glycosides with various physiological activities using glucosyl groups or fructosyl transferases. is being actively carried out. Coupling Sugar (registered trademark), fructooligosaccharide, palatinose, α-glucosyl stevioside, etc. are examples of products that have been put to practical use because they do not cause dental caries and have properties such as bifidobacteria growth factors.

[0003]Currently, Bacillus subtilis is used as a fructosyltransferase.
is) and levansucrase produced by Aspergillus niger.
), Penicillium
um oxalicum), Penicillium frequency
ans), Penicillium sp.
ium sp. ) β-Fructofuranosidase produced by molds such as K25, Arthrobacter sp.
Arthrobacter sp. ) β-fructofuranosidase produced by K-1 is known.

[0004] Xylosylfructoside and isomaltosylfructoside synthesized by these enzymes have anti-cariogenic properties, and fructooligosaccharides, galactosylfructoside and lactosylfructoside are also used as Bifidobacteria growth factors. It has the activity of Therefore, it is expected to have a wide range of applications in the pharmaceutical and food fields. The structure of lactosyl fructoside is shown in Figure 1.

[0005] However, in the oligosaccharide synthesis reaction using the above enzyme, the reaction reaches equilibrium due to product inhibition, so that only about 30% of the target substance, such as lactosyl fructoside, is obtained in the reaction solid content. do not have. Furthermore, this reaction solution containing about 30% of lactosyl fructoside has the disadvantage that it cannot be made into powder no matter what drying method is used.

[0006] There is also a method for producing fructose-containing oligosaccharides or glycosides using yeast with invertase activity, but in this method, the yeast decomposes the fructose-containing oligosaccharides or glycosides into receptors and fructose. This is not desirable because it causes Therefore, as a method for removing monosaccharides, it is possible to use an ultrafiltration membrane that removes only monosaccharides instead of yeast, but this method is still in the research stage, and if the receptor is a monosaccharide, The drawback is that it cannot be applied.

[0007]

[Means for Solving the Problems] Therefore, the present inventors have conducted extensive research in order to establish an efficient and practical method for producing fructose-containing oligosaccharides or glycosides, taking these circumstances into consideration. discovered that fructose-containing oligosaccharides or glycosides can be efficiently produced by allowing fructosyltransferase to act on sucrose in the presence of a receptor, and the resulting glucose is assimilated by invertase-deficient yeast. He completed his invention.

That is, the present invention provides a method for producing fructose-containing oligosaccharides or glycosides, which is characterized by allowing fructosyltransferase and invertase-deficient yeast to act on sucrose in the presence of a receptor.

In the present invention, fructosyltransferase acts on a fructose donor (sucrose) to hydrolyze it into glucose and fructose, and the fructose produced is converted into an acceptor (xylose, galactose, lactose, etc.).
Fructose-containing oligosaccharides or glycosides are produced by transferring glucose to and assimilating glucose by invertase-deficient yeast.

[0010] The fructose-containing oligosaccharides as used in the present invention include xylosylfructoside, galactosylfructoside, lactosylfructoside, sorbosylfructoside, fucosylfructoside, kestose, stachyose,
It means any one of erulose, raffinose, isomaltosylfructoside, xylobiosylfructoside, and arabinosylfructoside.

[0011] The term fructose-containing glycoside as used in the present invention means any one of fructosyl stevioside, fructosyl rubusoside and fructosyl rebaudioside.

The molecular entity that accepts fructose produced from the fructose donor by the enzymatic reaction of fructosyltransferase may be any one capable of producing the above fructose-containing oligosaccharide or glycoside. Specifically, for example, lactose, xylose, galactose, etc. are suitable.

[0013] Even when xylose or galactose is used as a receptor instead of lactose, the enzymatic reaction proceeds (Japanese Unexamined Patent Publication No. 3-27285).

[0014] The fructosyltransferase used in the present invention is Bacillus subtilis.
levansucrase produced by microorganisms such as Arthrobacter sp.
er sp. ), etc., and specifically, any β-fructofuranosidase produced by microorganisms such as Bacillus subtilis may be used.
is) levansucrase produced by Aspergillus niger,
Penicillium oxalicum
oxalicum), Penicillium frequentan
s), Penicilliu sp.
msp. ) β-Fructofuranosidase produced by molds such as K25, Arthrobacter sp.
hrobacter sp. ) K-1 (Unexamined Japanese Patent Publication No. 3-27
These include β-fructofuranosidase produced by No. 285).

[0015] Invertase-deficient yeast, which assimilates glucose produced by the enzymatic reaction of fructosyltransferase, does not assimilate xylose, and furthermore, does not assimilate galactose in the presence of glucose, which further promotes the enzymatic reaction. However, fructose-containing oligosaccharides or glycosides can be produced in large quantities. As the invertase-deficient yeast suitably used in the present invention, commercially available yeasts can be used. For example, LS-Y (manufactured by Oriental Yeast Industry Co., Ltd.) can be mentioned. The characteristics of LS-Y are shown below.

1) Monosaccharide assimilation ability Glucose assimilation ability → 720 mg glucose/h/g
wet yeast or higher 2) Degradability of sucrose Assimilation and fermentability of sucrose → None Invertase activity 0 units 3) Optimum assimilation temperature is optimal at 30 ± 2°C, and beyond this temperature range, assimilation activity decreases significantly. do.

In order to produce the desired fructose-containing oligosaccharide or glycoside, fructosyltransferase and invertase-deficient yeast are applied to a substrate solution in which sucrose (fructose donor) and fructose acceptor coexist. Just let it happen. When fructosyltransferase and invertase-deficient yeast are allowed to act, invertase-deficient yeast may be added to the reaction solution in which the enzyme has been deactivated after the fructosyltransferase reaction is completed, or the invertase-deficient yeast may be allowed to act on fructosyltransferase and invertase. The defective yeast may be added to the substrate solution at the same time and allowed to act.

Specifically, in the former case, the substrate concentration is preferably 40% (w/w), 7.5 to 10 units/g sucrose of fructosyltransferase is added, and 5%
After reacting at 0° C. to 60° C. and pH 6.5 to 7.0 for 9 to 24 hours, the enzyme is inactivated by heat. Next, 2% or more of invertase-deficient yeast is added to the preparation weight,
Let it work. At this time, the reaction temperature is 3
0°C is preferred. Further, the pH during the reaction may be within the range of 3 to 7. The reaction time varies depending on the amount of invertase-deficient yeast added. On the other hand, in the latter case, the substrate concentration is preferably 30 to 40% (w/w), 15 to 40 units of fructosyltransferase/g of sucrose is added, and invertase-deficient yeast is prepared at a rate of 2.
% or more and react. The temperature during the reaction is preferably 35°C if the optimum temperature of the enzyme is 35°C or higher, and the optimum temperature of the enzyme is suitable if it is below 35°C. In addition, the pH during the reaction should be the optimum pH of the enzyme if the optimum pH of the enzyme is 3 to 7.
is preferable, and for those outside this range, it is preferable to set the pH to 3 if it is on the acidic side, and to set it to pH 7 if it is on the alkaline side. The reaction time may be 6 to 30 hours.

The thus obtained reaction solution containing fructose-containing oligosaccharides or glycosides is subjected to diatomaceous earth filtration to remove invertase-deficient yeast, the enzyme is inactivated by heating, and activated carbon decolorization is carried out. The desired fructose-containing oligosaccharide or glycoside can be obtained by desalting and decolorizing using an ion exchange resin, and further purifying to a high degree of purity by chromatography such as activated carbon column chromatography.

Furthermore, in the present invention, by changing the ratio of sucrose and acceptor in the substrate solution, the sugar composition can be adjusted to be suitable for powdered products and liquid products. For example, if the product has a high sucrose content, it will be difficult to powder it, so the ratio of sucrose to acceptor can be changed to reduce the sucrose content in the substrate solution, whereas if the product has a high lactose (acceptor) content, it will be difficult to powder it. If it is in liquid form, lactose crystals will precipitate, so the ratio of sucrose to acceptor may be changed to suppress the lactose content in the substrate solution.

[0021]

[Example] Next, the present invention will be explained with reference to an example.

Example 1 The effect of adding invertase-deficient yeast LS-Y (manufactured by Oriental Yeast Co., Ltd.) on the production of lactosyl fructoside (hereinafter abbreviated as LS) was investigated. The test method is as shown below. In the method without adding LS-Y, 50.0 g of sucrose and 51.0 g of lactose were added.
Dissolve 6g to a final concentration of 40% (w/w),
Arthrobacter sp.
r sp. ) 7.5 units of β-fructofuranosidase produced by K-1 (hereinafter abbreviated as K-1 enzyme) per sucrose weight (g), pH 6.5, temperature 5
The reaction was carried out at 5°C for 20 hours. On the other hand, in the method adding LS-Y, sucrose 50.0g, lactose 51.6g
Dissolve K to a final concentration of 30% (w/w), and
-1 enzyme was added at 25 units per sucrose weight (g), and at the same time, LS-Y was added in an amount of 3% of the preparation weight to pH 6.
5 to 7.0, and the reaction was carried out at a temperature of 35°C for 20 hours. Here, one unit of enzyme activity was defined as the amount of enzyme that transferred 1 μmol of fructose per minute. Figure 2 shows the change over time in the amount of LS produced. Note that the amount of LS produced was measured using high performance liquid chromatography (HPLC). The same applies to the following examples. The HPLC conditions are shown below.

Column: TOSOH TSK-GEL
Amide-80 Mobile phase: 70% acetonitrile Flow rate: 1 ml/min Temperature: 35°C Detector: Differential refractometer

As is clear from FIG. 2, although the substrate concentration, the amount of enzyme added, and the reaction temperature are different, when LS-Y is used, the reaction equilibrium of the enzyme is clearly disrupted, and an increase in the yield of LS can be confirmed.

Example 2 15 kg of sucrose and 15 kg of lactose were added to a final concentration of 4.
0% (w/w) in tap water, and added 7.5 units of K-1 enzyme per sucrose weight (g).
The reaction was carried out at H6.5 and a temperature of 50° C. for 18 hours. Then,
The temperature was cooled to 30 °C, the concentration was diluted to 30% (w/w), and LS-Y was added to 5% of the weight of the preparation.
The reaction was carried out at 30°C for 18 hours. After the reaction, LS-Y was removed by diatomaceous earth filtration, treated with activated carbon at 95°C for 1 hour, and then treated with ion exchange resin (IRA-400, IR-120B).
It was purified with After concentrating this, spray dry it.22.
5 kg of powder product was obtained. Table 1 shows the sugar composition during the transfer reaction, during the yeast reaction, and in the powdered product.

Table 1──────
──────────────────────────
────── Sugar composition
Monosaccharide sucrose lactose
LS ──────────────────
────────────────── At the time of transfer reaction 17.0 23.0
26.0 34.0 During yeast reaction
──31.3
31.3 41.0 Powder products
2.0 26.1
32.3 39.6────────────
──────────────────────────
*The numbers in the table represent the content (%) in solid content.

Example 3 Sucrose and lactose were dissolved in 0.1M phosphate buffer (
K-1 enzyme was mixed and dissolved in equal amounts to a final concentration of 40% (w/w) (pH 6.5), and the K-1 enzyme was dissolved in sucrose weight (g).
At the same time, LS-Y was added in an amount of 2% of the charged weight, and the reaction was carried out at 30° C. for 72 hours. Figure 3 shows the change in sugar composition over time.

As is clear from FIG. 3, L
When SY acts, the reaction equilibrium of the enzyme is disrupted and the yield of LS is increased. Furthermore, the content of LS in the sugar was as high as 65%.

Example 4 45.0 g of sucrose and 56.8 g of lactose were dissolved in tap water to a final concentration of 30% (w/w).
1 enzyme was added in an amount of 15 units per sucrose weight (g), and at the same time, LS-Y was added in an amount of 3% of the weight of the preparation. The reaction was carried out at a temperature of 35°C for 28 hours. pH is 6.5~
It was adjusted using NaOH to fall within the range of 7.0. Figure 4 shows the change in sugar composition over time.

As is clear from FIG. 4, it was found that an amount of K-1 enzyme of 15 units per sucrose weight (g) was sufficient.

Example 5 1500 kg of sucrose (water content 0.03%) and 1850 kg of lactose (water content 3.1%) were mixed at a final concentration of 30% (w).
/w) in tap water, and added K-1 enzyme to 25 units per sucrose weight (g).
At the same time, 3% of the weight of LS-Y was added. temperature 35
℃, the pH was in the range of 6.5-7.0 with NaO
The mixture was adjusted using H and reacted. After 22 hours of reaction, activated carbon corresponding to 1% of the charged weight was added, and diatomaceous earth filtration was performed at room temperature to remove LS-Y. Next, activated carbon was added in an amount of 2% of the weight of the preparation and kept at 95°C for 30 minutes.
After cooling to 0°C, diatomaceous earth filtration was performed again. moreover,
The mixture was cooled to 35° C. and desalted and decolorized using an ion exchange resin. Concentrate this to a concentration of 55% (w/w) using an evaporator,
A powder product weighing 2360 kg was obtained by spray drying. The sugar composition of the obtained powder product is shown in Table 2.

Table 2----
──────────────────────────
──────── Sugar composition
Monosaccharide sucrose lactose
LS ────────────────
────────────────────
Powder product 3.5 7.
1 22.0 57.5────
──────────────────────────
────────*The numbers in the table represent the content (%) in solid content.

[0033]

According to the present invention, fructose-containing oligosaccharides or glycosides can be industrially produced efficiently at low cost. The fructose-containing oligosaccharides or glycosides obtained according to the present invention have anti-cariogenic properties and bifidobacterial growth activity, and are therefore useful in the fields of pharmaceuticals and foods.

[0034]

[Brief explanation of the drawing]

FIG. 1 shows the structure of lactosyl fructoside.

FIG. 2 shows changes over time in the amount of LS produced in Example 1.

FIG. 3 shows changes in sugar composition over time in Example 3.

FIG. 4 shows changes in sugar composition over time in Example 4.

Claims (4)

[Claims] 1. A method for producing fructose-containing oligosaccharides or glycosides, which comprises allowing fructosyltransferase and invertase-deficient yeast to act on sucrose in the presence of a receptor. [Claim 2] The fructose-containing oligosaccharide is xylosylfructoside, galactosylfructoside, lactosylfructoside, sorbosylfructoside, fucosylfructoside, kestose, stachyose, erulose,
2. The method for producing a fructose-containing oligosaccharide according to claim 1, which is any one of raffinose, isomaltosylfructoside, xylobiosylfructoside, and arabinosylfructoside. 3. The method for producing a fructose-containing glycoside according to claim 1, wherein the fructose-containing glycoside is any one of fructosyl stevioside, fructosyl rubusoside and fructosyl rebaudioside. Claim 4: The fructosyltransferase is Bacillus.
levansucrase produced by microorganisms such as Bacillus subtilis or Arthrobacter sp.
2. The method for producing a fructose-containing oligosaccharide or glycoside according to claim 1, wherein the fructose-containing oligosaccharide or glycoside is any one of β-fructofuranosidases produced by microorganisms such as ).