JPH02163091A - Production of fructose olilgosaccharide - Google Patents

Abstract

PURPOSE:To enable the simple production of the above high-concentration product in a shortened time with increased reaction rate by allowing a hydrolase to act on a sucrose-containing water-organic solvent system. CONSTITUTION:A hydrophobic organic solvent such as n-pentane is combined with 1 to 10vol.% of water to prepare a water-hydrophobic organic solvent system (A). Then, sucrose is added to the system A in an amount of 50 to 150wt.% of the water to prepare the substrate (B). Then, a hydrolase such as cellulase originating from Aspergillus niger (C) is added to the substrate (B) to effect the reaction at 10 to 65 deg.C, 3.5 to 7.0 pH under stirring to give reaction mixture (D). The product D is treated with deodorizer and purified by means of liquid chromatography to recover fructose oligosaccharide.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing fructose oligosaccharide, and more particularly to a method for producing fructose oligosaccharide used in sweet foods and health foods.

[Prior Art] Saccharides have traditionally been widely used as important sweeteners in the food industry, but recently new saccharides with physiological activity using enzymes derived from microorganisms have been developed one after another.

Among them, research on the synthesis of so-called oligosaccharides is particularly active, and various proposals have been made regarding, for example, methods for producing fructooligosaccharides.

For example, JP-A No. 60-41497 discloses that the genus Aureobacidium (^ureobacidium, sp.
Fructosyltransferase active bacterial cells are immobilized with β-1,3-1,6 glucan obtained by liquid culture of 1-glucan and aluminum sulfate or an aluminum compound, and sucrose (sucrose) is continuously injected into the immobilized bacterial cells. 1-Kestose and nystose
) is an inulin-type fructooligosaccharide (F
Discloses a method for producing fructo-oligosaccharides using immobilized bacterial cells, which converts them into ructo-of-fgo sugars. Furthermore, Japanese Patent Application Laid-open No. 60-27395 discloses that the genus Aureobasidium is cultured in a liquid culture to obtain cultured microbial cells or a culture solution containing the microbial cells, and the cultured microbial cells or the culture solution containing the microbial cells are Fructooligosaccharides that are added to a sucrose solution or subjected to a contact reaction to convert sucrose into inulin-type fructooligosaccharides containing 1-kesdose and nisdose as main components using fructosyltransferase, which is an intracellular transferase contained within the microbial cells. A manufacturing method is disclosed.

[Problems to be Solved by the Invention] However, the methods of JP-A-60-41479 and JP-A-60-27395 are both enzymatic reactions in water, and the reaction rate is slow and the reaction time is long. It takes
There was also the problem that the product could only be obtained at a low concentration.

The present invention has been made in view of the above points, and an object to be solved is to provide a method for producing fructose oligosaccharide that has a high reaction rate and can obtain a product in high concentration in a short reaction time. be.

[Means for Solving the Problems] According to the present invention, the production of fructose oligosaccharide is characterized in that sucrose is converted into fructose oligosaccharide by an enzymatic reaction using cellulase in a water-hydrophobic organic solvent system. A method is provided.

The present invention is characterized in that the reaction system is a water-hydrophobic solvent dispersion system and that a hydrolytic enzyme is used.

In other words, until now, only reactions in water have been studied;
Regarding enzymes, only transferases having sugar transfer activity have been studied.

In the present invention, any hydrophobic organic solvent can be used as long as it is not compatible with water. Specifically, n-pentane, cyclopentane, 2-methylbutane, methylcyclobutane, and n-hexane are used. , cyclohexane, 2-methylpentane, 2,2-dimethylbutane, 2,
3-dimethylbutane, methylcyclohexane, peptane, octane, 2.2.3-trimethylhebutane, 2,
2.4- Hydrocarbon solvents such as trimethylhebutane, nonane, decane, benzene, toluene, 0-xylene, m-xylene, p-xylene, ethylbenzene, cumene;
Petroleum ether, light benzine, ligroin, benzene for cleaning, etc. Solvents 1q as petroleum fractions Nidichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, 1,1,2.2-tetrachloroethane, trichloroethylene, Halogenated hydrocarbon solvents such as chlorobenzene and 0-dichlorobenzene; isoamyl alcohol, n-
Alcohol solvents having 5 or more carbon atoms such as octyl alcohol can be used.

Cellulase (cellulase) is a hydrolytic enzyme.
e) are mentioned as suitable ones, and examples of cellulase include Aspergillus niger (Aspergillus niger).
Cellulases derived from P. Iusniger and the like can be used.

The proportion of water in the water-hydrophobic organic solvent system is 1vo1%~
10vol% is preferable. If it is less than 1 vol%, the enzyme is likely to become inactive, and if it exceeds 10 vol%, the amount of product obtained will be reduced. In addition, sucrose is 5% relative to water.
It is preferable to use it in a proportion of 0 wt% to 150 wt%. If it is less than 50 wt%, the amount of product obtained is small, and 1
If it exceeds 50 wt%, the target oligosaccharide ratio will decrease.

The optimal temperature for enzyme reaction is a narrow range of around 37°C in an aqueous system, but in the present invention, the reaction proceeds in a wide temperature range of 70°C or less, preferably 10 to 65°C, more preferably 30 to 65°C.
It is 60'C. If the proportion of water used is small, the water will not freeze even below O'C, so the reaction can proceed even at -10C, for example. This is water bound lol
This is thought to be because it exists in the system as ater.

The convenient p t-+ for the enzyme reaction of the present invention is 3.5 to 7.
．． It is 0. A buffer solution is used to achieve this DH value range.

The reaction can be carried out, for example, by dispersing sucrose in a hydrophobic organic solvent with stirring, then dissolving cellulase in a buffer solution with a predetermined DH value, and adding this enzyme solution to the organic solvent. This is carried out by dispersing while stirring at a temperature.

After the reaction, when the stirring is stopped, the organic layer and the aqueous layer automatically phase separate, and the aqueous layer is concentrated to obtain a concentrated liquid containing the product.

Conventionally, this type of treatment had to be carried out in a closed system to prevent the intrusion of bacteria, but in the present invention, since a hydrophobic organic solvent is used, there is no need to sterilize the reaction system, and the reaction system is free from outside the system. It is blocked from bacteria and contamination by various bacteria is prevented.

In addition, in order to remove the organic solvent odor, the separated water droplets may be subjected to a deodorizing treatment using an adsorption treatment agent such as activated carbon or activated clay. In order to remove unreacted substances from the generated reaction mixture, for example, using liquid chromatography technology or the low solubility of the product in alcohols, alcohols are added to reduce the solubility of the product and cause it to precipitate. A method of separation is applied.

Note that if a surfactant is added to the reaction system, the water droplets will be dispersed very finely in the hydrophobic organic solvent and the reaction rate will be increased, so the use of the surfactant is not hindered.

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

Example 1 500/lit! 291 d of hexane and 9 g of sucrose were placed in an Erlenmeyer flask with a baffle plate and dispersed with stirring.

On the other hand, Aspergillus niger (A
acetate buffer solution (pl-1s, o, ionic strength IM
Cellulase 0.3 (1 (240 u)) was dissolved in >9 ml, and this enzyme aqueous solution was added to the Erlenmeyer flask mentioned above, and the enzymatic reaction was performed while stirring (600-1000 rpm). The composition (%) of high performance liquid chromatography (HPLC, column: 5hodex)
Quantitated using Sugar 5PIOIO). Enzyme activity was also measured using an analog substrate modified with ρ-nitrophenol. Figure 1 shows the change in sugar composition over time during the enzymatic reaction. This reaction takes place at a reaction temperature of 40°C.
I went there. After reaction time 2.3 hr, fructose oligosaccharide: 42.8%, sucrose 24.62%
%, glucose 27.7%, and fructose 4.8% (% is the ratio of the peak areas of l-1'PLC).

Looking at Figure 1, fructose oligosaccharide is 2.
It shows a peak after 3 hours and then gradually decreases,
On the other hand, it can be seen that fructose and glucose continue to increase, while sucrose continues to decrease. This shows that the component ratio of the product can be controlled by changing the reaction time.

Example 2 Dodecane 194d, cellulase 0.2g, buffer solution 67
! , 9 g of sucrose, and a reaction temperature of 61°C, but in the same manner as in Example 1.
Shown in the figure. The HPLC results at this time are shown in FIG.

After a reaction time of 2.25 hr, fructose oligosaccharide was found to be 37.9%, sucrose 26.5%, glucose 28.04%, and fructose 7.6% (% is the ratio of HPLC peak areas).

Furthermore, after a reaction time of 166 hours, the enzyme activity in the organic solvent was examined, and as a result of comparison with the enzyme activity (100%) in an aqueous system under the same conditions, it was found that 88.4% remained.

That is, it was found that the enzyme activity persisted for a long time in the water-hydrophobic organic solvent system, and the enzyme could be used repeatedly.

[Effects of the Invention] As is clear from the above explanation, the reaction rate in the method of the present invention is fast, the product can be obtained at high concentration in a short reaction time, and the reaction operation is simple. Furthermore, since an organic solvent is used, there is no need to sterilize the reaction system, the reaction system is isolated from bacteria, and contamination by various bacteria is prevented. Another advantage is that the component ratio of the product can be freely controlled by changing the reaction time.

[Brief explanation of the drawing]

Figure 1 is a graph showing changes over time of each component in the enzymatic reaction of Example 1, Figure 2 is a graph showing changes over time of each component in the enzyme reaction of Example 2, and Figure 3 is a graph showing the reaction time of Example 2. It is a graph showing HPLC results after 2.25 hours. Applicant Folkways Japan Co., Ltd. Agent
Patent Attorney Shinshi Arichika Department Agent Patent Attorney Kimi Tateno - Figure 2 Figure 1

Claims (1)

[Claims] 1. A method for producing fructose oligosaccharide, which comprises converting sucrose into fructose oligosaccharide by an enzymatic reaction using a hydrolase in a water-hydrophobic organic solvent system.