JP5680290B2 - Neokestose production method - Google Patents

Description

  The present invention relates to a method for efficiently producing neokestose, which is a trisaccharide of neofructooligosaccharide, using a fructan-containing composition such as an agricultural product or agricultural by-product as a raw material, and a fructan hydrolase used in the method.

  Some plants accumulate small to high molecular weight fructans as storage carbohydrates. Fructan is a general term for polysaccharides of trisaccharide or higher (inulin type) in which fructose is polymerized on one sucrose, and polysaccharides in which only fructose is polymerized, and are classified as water-soluble dietary fibers. Among them, asparagus roots, onion bulbs, and the like are rich in relatively low molecular weight inulin-type fructans such as 1-kestose and nystose and inulin neo-type fructans.

Inulin-type fructan is a fructose polymer having a structure in which the fructose chain is further extended by the Fru- (β2 → 1) -Fru bond, with the 2-position of another fructose bonded to the 1-position of the fructose residue of sucrose. (1-kestose type), the trisaccharide is 1-kestose, the tetrasaccharide is nystose, and the pentasaccharide is fructosyl nystose. These fructooligosaccharides are functional carbohydrates that have prebiotic effects (intestinal environment improvement, bowel movement improvement) and mineral absorption promotion effects, and are industrially produced using microbial enzymes (transferases). . The structure of the main inulin-type fructan is shown in FIG.
Levan type fructan is a fructose having a structure in which the second position of another fructose is bonded to the 6th position of the fructose residue of sucrose and the fructose chain is further extended by this Fru- (β2 → 6) -Fru bond. It is a polymer (6-kestose type).

  On the other hand, inulin neo-type fructan is composed of neokestose having a Fru- (β2 → 6) -Glc bond in which the fructose 2-position is bound to the 6-position of the glucose residue of sucrose as a basic skeleton. Fructose is a generic term for a fructose polymer (neokestose type) in which fructose is Fru- (β2 → 1) -Fru-bonded to a group and the fructose chain is extended. Neokestose, which is a trisaccharide of inulin neo-type fructans, is also called neofructooligosaccharide and is known to have functions such as prebiotic effects as well as fructooligosaccharide. The structure of the main inulin neo type fructan is shown in FIG.

  As a method for producing this neokestose, there is a method in which a microorganism-derived fructosyltransferase is allowed to act on sucrose and / or fructooligosaccharide (Patent Documents 1 and 2). In this case, neokestose is produced as a mixture with fructo-oligosaccharide, and also becomes a mixture with neo-fructo-oligosaccharide other than neokestose. In addition, there are a method using a plant-derived fructosyltransferase and a method of extracting an oligosaccharide from a plant (onion bulb, asparagus root, etc.) containing a neofructooligosaccharide. It becomes a mixture of neofructooligosaccharide and fructooligosaccharide.

  That is, in these methods, a separation operation is required to obtain each sugar alone. Examples of the fructooligosaccharide separation technique include various chromatographic separation methods using a cation exchange resin, and a 1-kestose and nystose separation technique (Patent Document 3) is disclosed. However, this method is a method for separating fructooligosaccharides having a large difference in molecular weight ratio. Trisaccharides and tetrasaccharides such as a mixture of 1-kestose and nystose can be separated, but 1-kestose is a trisaccharide. , 6-kestose and neokestose are difficult to separate, and in this case it is difficult to isolate neokestose.

  Thus, although microorganisms or plant-derived fructosyltransferases related to the production of neokestose have been found, high-purity neokestose has not been produced on an industrial scale due to the difficulty of its separation and purification technology and manufacturing costs. Is the current situation. Therefore, the functional research has not progressed sufficiently. In addition, industrially produced fructooligosaccharides that are actually marketed are mainly composed of 1-kestose or nystose, or are a mixture of fructooligosaccharides and neofructooligosaccharides.

JP 10-165192 A JP 2002-360238 A JP 2000-232878 A

  The present invention uses a fructan-containing composition (a composition containing a mixture of neofructooligosaccharide and fructooligosaccharide) as a raw material, and is capable of efficiently producing and isolating neokestose. An object is to provide an efficient manufacturing method.

  In order to achieve the above object, as a result of intensive studies, the inventors of the present invention analyzed a fructan-containing composition by decomposing the Fru- (β2 → 1) -Fru bond of fructan to produce Fru- (β2 → 6) -Glc of fructan. By treating with fructan hydrolase (for example, 1-FEH derived from burdock (fructan 1-exohydrolase) etc.), fructan other than neokestose is selectively decomposed and enzyme It has been found that only glucose, fructose, sucrose, and neokestose remain in the treatment liquid, and it has been found that high-purity neokestose can be efficiently produced simply by chromatographic separation (removal of glucose, fructose, and sucrose).

That is, the embodiment of the present invention is as follows.
(1) The fructan-containing composition is treated with a fructan hydrolase that decomposes Fru- (β2 → 1) -Fru bond of fructan and does not decompose Fru- (β2 → 6) -Glc bond of fructan and sucrose; A method for producing neokestose, which comprises chromatographic separation of the enzyme-treated solution.
(2) The method according to (1), wherein the fructan hydrolase is derived from burdock.
(3) The method according to (1) or (2), wherein the fructan hydrolase is 1-FEH (fructan 1-exohydrolase).
(4) The method according to (3), wherein the fructan hydrolase is 1-FEH (fructan 1-exohydrolase) having the amino acid sequence described in SEQ ID NO: 1 (FIG. 3).
(5) The fructan hydrolase comprises an amino acid sequence containing one or several amino acid substitutions, deletions, insertions or additions in the amino acid sequence shown in SEQ ID NO: 1 (FIG. 3), and 1-FEH (fructan) The method according to (3), which has an activity of 1-exohydrolase).
(6) The fructan hydrolase is a protein having an activity of 1-FEH (fructan 1-exohydrolase) encoded by a gene consisting of the base sequence described in SEQ ID NO: 2 (FIG. 4) ( The method according to 3).
(7) 1-FEH (fructan) encoded by a gene obtained by hybridization under stringent conditions in which fructan hydrolase uses DNA consisting of the base sequence shown in SEQ ID NO: 2 (FIG. 4) as a probe. The method according to (3), which is a protein having an activity of 1-exohydrolase).
(8) The method according to any one of (1) to (7), wherein the fructan-containing composition is an agricultural product and / or an agricultural by-product.
(9) The method according to (8), wherein the agricultural product and / or the agricultural by-product is derived from a lily family plant and / or an Asteraceae plant.
(10) According to (9), the agricultural product and / or agricultural by-product is derived from at least one selected from asparagus (for example, asparagus renewal waste strain), onion (for example, surplus production onion), and burdock. The method described.

(11) By treating the fructan-containing composition with the fructan-hydrolyzing enzyme according to any one of (1) to (7), fructan other than neokestose in the composition is selectively decomposed, A method to leave neokestose.
(12) Any one of (4) to (7), wherein fructan other than neokestose in the fructan-containing composition is selectively decomposed to leave neokestose as glucose, fructose, sucrose and fructan Fructan hydrolase 1-FEH (fructan 1-exohydrolase) according to one.

  According to the present invention, in the fructan-containing composition in which neofructooligosaccharide and fructooligosaccharide are mixed, inulin-type fructan containing 1-kestose which interferes with chromatographic separation of neokestose is converted into fructan hydrolyzed having a predetermined activity. It can be decomposed with a degrading enzyme, and inoline neo-type fructan of tetrasaccharide or higher can be decomposed with the enzyme to produce neokestose, and neokestose can be selectively left as fructan. Thereby, neokestose purification by chromatographic separation of the enzyme treatment liquid can be facilitated, and high-purity neokestose can be efficiently industrially produced (mass produced).

The structure of 1-kestose, nystose, fructosyl nystose of inulin type fructan was shown. Neoinstose, 1 ^F (1-β-D-fructofuranosyl) _m- 6 ^G (1-β-D-fructofuranosyl) _n sucrose (4b: m = 0, n = 2; 4c: m = 1 , n = 1; 5b: m = 0, n = 3; 5c: m = 2, n = 1; 5d: m = 1, n = 2). The amino acid sequence of novel 1-FEH obtained from burdock is shown (the upper part is represented by one letter symbol, the lower part is represented by three letter symbols). Glycosylation sites (3 sites) are underlined. The base sequence of the gene (aleh1, cDNA) encoding novel 1-FEH obtained from burdock is shown. The result of the high performance liquid anion exchange chromatography analysis of the hydrolysis reaction liquid (0, 2, 4, 8, 24 hr reaction liquid) of asparagus extracted sugar by recombinant 1-FEH is shown. (A) used 1U-ml of 1-FEH and (b) used 7U / ml of 1-FEH. The results of high-performance liquid anion exchange chromatography analysis of the onion-extracted sugar hydrolysis reaction solution (0, 2, 4, 8, 24 hr reaction solution) using recombinant 1-FEH are shown. (A) used 1U-ml of 1-FEH and (b) used 7U / ml of 1-FEH. The sugar composition change in the hydrolysis of asparagus extracted sugar and onion extracted sugar by recombinant 1-FEH is shown. The vertical axis shows the amount of each sugar, and the horizontal axis shows the reaction time. (A) uses 35 U / ml of 1-FEH for asparagus extracted sugar, (b) uses 7 U / ml of 1-FEH for asparagus extracted sugar, (c) uses 35 U / ml of 1-FEH for onion extracted sugar. 1 ml of 1-FEH was used for onion extract sugar. Various fructan concentrations contained in the extracted sugar of asparagus root were compared before and after treatment with recombinant 1-FEH (0 hr, left side of the bar graph) and after treatment (8 hr, right side of the bar graph). The result of chromatographic separation of neokestose using the preparative HPLC from the enzyme-treated solution obtained by treating asparagus root extracted sugar with recombinant 1-FEH is shown. (A) is an enzyme-treated solution stock solution, (b) is a 5-fold diluted enzyme-treated solution, (c) is a concentrated fraction of neokestose fraction obtained by chromatographic separation, G is glucose, F is fructose, S is sucrose, Nk represents neokestose.

  In the present invention, any fructan-containing composition that is a raw material for producing neokestose can be used as long as it contains inulin neo-type fructan, but asparagus root renewal waste strain, surplus production onion, other unused agricultural products, etc. Agricultural products represented by the above, agricultural by-products and mixtures thereof. In addition, the same lily family plants as asparagus and onions such as sea urchins, and agricultural products and agricultural by-products of asteraceae plants such as burdock can be used. Furthermore, a product obtained by treatment with fructosyltransferase using sucrose or the like as a substrate can be used as a raw material of the present invention if inulin neo type fructan is contained as a product. In the present invention, a mixed composition of inulin neo type fructan and inulin type fructan can be used as a raw material without exception.

  The fructan hydrolase used in the present invention decomposes Fru- (β2 → 1) -Fru bond (that is, decomposes the fructose polymerization portion of inulin-type fructan and inulin neo-type fructan having a tetrasaccharide or more), and Fru- Any fructan hydrolase having an activity that does not degrade (β2 → 6) -Glc bond and sucrose (that is, does not degrade neokestose) can be used. As the fructan hydrolase having such activity, for example, 1-FEH (fructan 1-exohydrolase) derived from burdock is exemplified, and 1-FEH having the amino acid sequence described in SEQ ID NO: 1 (FIG. 3) is used. It is preferable to do this.

  Fructan hydrolase is an enzyme that has been found in several kinds from edible plants such as chicory and asparagus, but it is one that breaks the Fru- (β2 → 1) -Fru bond of fructan, Fru- ( It is only confirmed that what decomposes | disassembles (beta) 2-> 6) -Fru coupling | bonding has been confirmed, and does not decompose | disassemble sucrose. That is, whether or not the Fru- (β2 → 6) -Glc bond, which is the neokestose basic skeleton of the enzyme, has been decomposed has not been known so far. Furthermore, the Fru- (β2 → 1) -Fru bond is decomposed and Fru is broken. A fructan hydrolase having an activity of not decomposing a-(β2 → 6) -Glc bond has not been known.

  As one of the fructan hydrolases that can be used in the present invention, the present inventors newly added 1-FEH consisting of the amino acid sequence described in SEQ ID NO: 1 (FIG. 3) derived from burdock and SEQ ID NO: encoding this 2 (FIG. 4), a gene comprising the base sequence described in SEQ ID NO: 1 was obtained, and the amino acid sequence comprising one or several amino acid substitutions, deletions, insertions or additions in the amino acid sequence described in SEQ ID NO: 1 -An enzyme having an activity of FEH, an enzyme having an activity of 1-FEH encoded by a gene obtained by hybridization under a stringent condition using a DNA comprising the nucleotide sequence of SEQ ID NO: 2 as a probe, and the like It can be used as well.

  An example of such industrial mass production of fructan hydrolase is recombinant protein production using microorganisms. Recombinant protein production can be performed using a conventional method. For example, a gene encoding the enzyme is inserted into a chromosomally integrated secretion expression vector, a methanol-assimilating yeast is transformed with this vector, and a transformant is obtained. It can be performed by culturing while inducing gene expression with methanol.

  In the treatment of the fructan-containing composition with fructan hydrolase, the enzyme reaction is performed at about 20 to 40 ° C. for 2 to 30 hours, preferably 8 to 24 hours after being added to the raw material. When the raw material is solid, pulverization is preferably performed, and an extract obtained by extracting saccharide from the solid raw material may be used as the reaction raw material. The amount of enzyme used is 5 to 50 U / ml, preferably 7 to 35 U / ml. By this enzyme treatment, the fructose polymerization part of inulin type fructan and inulin neo type fructan other than neokestose is decomposed.

  After the enzyme treatment, the enzyme deactivation treatment is performed by heating or the like. And when solid content remains in a process liquid, solid-liquid separation is performed by operation, such as centrifugation, and a liquid part (enzyme process liquid) is used for the next refinement | purification process.

In this enzyme treatment solution, sucrose and fructose, which are degradation products, and glucose, sucrose and fructose contained in the original raw material remain in addition to neokestose. Therefore, in order to obtain high-purity neokestose, the enzyme-treated solution is chromatographed to separate glucose, sucrose, fructose and neokestose. Examples of chromatographic separation methods include methods using high-performance liquid chromatography (HPLC) for preparation, but any chromatographic separation method can be used as long as it can separate and purify neokestose and glucose, sucrose, and fructose. It is.
Neokestose obtained by chromatographic separation can be used as it is as liquid high-purity neokestose, but it can also be made into a concentrate, pasted product, dried product (powder, granule) or the like by a conventional method.

  Thus, by using a fructan hydrolase typified by 1-FEH derived from burdock, a fructan mixed composition such as asparagus or onion extract sugar (inulin neo type, inulin type and / or levan type). By converting the composition contained in the mixture) into a sugar composition mainly composed of neokestose, sucrose, fructose, and glucose, and chromatographic separation thereof, large-scale preparation of high-purity neokestose can be facilitated. In addition, the present invention can be used as an effective utilization method of such unused resources because it can use the surplus produced onions and the roots of renewed waste strains of asparagus as production raw materials.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

(Acquisition of new fructan hydrolase gene derived from burdock and new fructan hydrolase encoded by this gene)
A novel 1-FEH gene (alleh1; full-length cDNA) derived from burdock was cloned from burdock total RNA using PCR method and RACE method (Rapid Amplification of cDNA end), and its nucleotide sequence was determined by the Sanger-deoxy method ( SEQ ID NO: 2, FIG. 4). The novel 1-FEH gene had an open reading frame consisting of 2,063 bp and 1,746 bp in base sequence. The amino acid sequence of the encoded protein was also determined from the base sequence (codon) of the open reading frame (SEQ ID NO: 1, FIG. 3). The amino acid sequence consisted of 581 residues and showed a high homology of 81% with 1-FEH derived from chicory.

  In order to obtain a large amount of protein encoded by the novel 1-FEH gene derived from burdock, the gene was inserted into a chromosome-integrated secretion expression vector pPLC2αβ (manufactured by Invitrogen). Methanol-assimilating yeast (Pichia pastoris) was transformed with this recombinant plasmid, and recombinant protein was secreted and produced in the culture solution using this transformant. Specifically, Pichia pastoris transformants were pre-cultured overnight at 29 ° C. in BMGY medium, then methanol-induced culture was performed in BMMY medium at 29 ° C. for 3 days, and methanol was added to 2% every day. The culture solution was centrifuged to collect the culture supernatant, and the recombinant protein contained in the culture supernatant was obtained.

  The enzyme activity was measured to determine the degradation activity for each substrate. The enzyme reaction was performed as follows. That is, 25 μl of McIlvaine buffer (pH 5.5), 50 μl of each 0.2M substrate solution (1-kestose, neokestose, sucrose) and 25 μl of enzyme solution were reacted at 30 ° C. for 30 minutes, and reacted by boiling for 3 minutes. Was stopped. For 1-FEH activity, 1 unit (U) was defined as the amount of enzyme that produces 1 μmol of fructose per minute using 1-kestose at a final concentration of 0.1 M as a substrate.

  As a result of the measurement, the 1-kestose decomposition activity was 70 U / ml. The relative activity of the hydrolysis activity of sucrose and neokestose was determined by setting the 1-kestose degradation activity as 100% and found to be 0.03 and 5.82%, respectively, and it was clear that there was little effect on sucrose and neokestose. It became.

(Hydrolysis of asparagus or onion extract sugar (fructan-containing composition) using recombinant 1-FEH)
Extracted sugar (fructan-containing composition) was prepared from asparagus roots and onion bulbs by hot ethanol extraction according to a conventional method. First, asparagus roots and onion bulbs were extracted with hot ethanol, the extract was lyophilized, and the lyophilized powder was dissolved in water to 320 mg / ml.

  The enzymatic reaction of the fructan-containing composition was performed as follows. 50 μl of fructan-containing solution extracted from asparagus and onion, 25 μl of McIlvaine buffer (pH 5.5), 25 μl of recombinant protein (35 U / ml (reaction solution (a)), 7 U / ml (reaction solution (b))) A total of 100 μl was reacted at 30 ° C. for 0, 2, 4, 8, and 24 hours, respectively, and the reaction was stopped by boiling for 3 minutes. After stopping the reaction, the sugar was quantified by high performance liquid anion exchange chromatography (HPAEC) analysis.

  High performance liquid anion exchange chromatography (HPAEC) analysis was performed using DX300 manufactured by DIONEX. Carbopac PA-1 (4 × 250 mm, registered trademark) was used as the column, and a pulsed amperometric detector was used as the detector for detection in the range of 0.3 μC. The column temperature was room temperature, 150 mM NaOH was used as an eluent, elution was performed with a sodium acetate concentration gradient (25 to 500 mM, 30 min), and the flow rate was 1 ml / min.

For the fructan-containing composition extracted from asparagus, fructans other than neokestose were efficiently hydrolyzed in both the results of the reaction liquids (a) and (b) (FIG. 5). In the reaction solution (a), the neokestose concentration at the start of the reaction increased to 54 mg / ml in the reaction for 8 hours, and in the reaction solution (b), the concentration increased to 51 mg / ml in the reaction for 24 hours ( FIG. 7). There was very little fructan other than neokestose in this reaction liquid. The fructose and sucrose concentrations that were hardly present at the start of the reaction were 24 hours of reaction, up to 104 mg / ml and 25 mg / ml in the reaction solution (a), respectively, and 83 mg / ml and 15 mg in the reaction solution (b), respectively. / Ml.
As a result, recombinant 1-FEH is allowed to act on the fructan-containing composition extracted from asparagus, and the fructan is decomposed to increase the concentration of neokestose by about 5 times. Mainly contains three types of sugars: fructose, sucrose, and neokestose. It was possible to convert it into a sugar composition suitable for separation as a component.

For the fructan-containing composition extracted from onion, fructans other than neokestose were efficiently hydrolyzed in both the reaction liquids (a) and (b) as in the case of asparagus (FIG. 6). In the reaction solution (a), the concentration of neokestose at the start of the reaction increased to 42 mg / ml in the reaction for 4 hours and in the reaction solution (b) to 44 mg / ml in the reaction for 8 hours ( FIG. 7). There was very little fructan other than neokestose in this reaction liquid. The fructose and sucrose concentrations at the start of the reaction were 20 mg / ml and 6 mg / ml, respectively, but in the reaction solution (a) up to 67 mg / ml and 22 mg / ml, respectively, the reaction solution (b) Increased to 51 mg / ml and 11 mg / ml.
As a result, recombinant 1-FEH was allowed to act on the fructan-containing composition extracted from onion, and the fructan was decomposed to increase the concentration of neokestose by about 1.5 times. Three types of sugars, fructose, sucrose, and neokestose, were obtained. It was possible to convert it to a sugar composition suitable for separation as the main component.

  In this example, the changes in sugar composition over time in hydrolysis of asparagus and onion sugar extract with recombinant 1-FEH are shown in a graph with the vertical axis representing the amount of each sugar and the horizontal axis representing the reaction time. (FIG. 7). Sugar was described with respect to glucose, fructose, sucrose, nystose, 1-kestose, neokestose, 4c, 4b. In any reaction, it was revealed that sugars other than neokestose, fructose, sucrose, and glucose decrease or disappear with time.

(Major preparation of neokestose)
Using a fructan-containing composition extracted from asparagus (asparagus root extract sugar) as a substrate, neokestose is considered to be the most effective condition for large-scale preparation of neokestose under the reaction conditions of 8 hours with a 35 U / ml enzyme solution. Was prepared in large quantities. Specifically, 20 ml of 320 mg / ml asparagus-fructan-containing solution, 10 ml of McIlvaine buffer (pH 5.5), and 10 ml of 35 U / ml enzyme solution were mixed, reacted at 30 ° C. for 8 hours, and boiled for 10 minutes. The reaction was stopped. The reaction product was quantified by HPAEC analysis as described in Example 2.

  As a result, the concentrations of neokestose, sucrose, and fructose in the reaction solution were 51 mg / ml, 24 mg / ml, and 155 mg / ml, respectively (FIG. 8). The concentration of other fructans (1-kestose, nystose, 4c, 4b) was 1 mg / ml or less, which was very small (FIG. 8).

  This reaction solution was subjected to preparative HPLC, and neokestose was isolated from the reaction solution using a preparative HPLC apparatus for the peak portion of neokestose. Specifically, HPLC system GULIVER (PU-1580, LG-1580-02, DG-1580-53, CO-1565RI-1530, registered trademark) manufactured by JASCO Corporation is used, and the column is TSKgel ODS-80Ts (20 × 250 mm, registered trademark) was used. The column temperature was 35 ° C. Pure water was used as an eluent and eluted at a flow rate of 3 ml / min, and a differential refractometer (RI-1530) was used for detection. Then, 2.0 g of neokestose could be isolated from 40 ml of the reaction solution by performing activated carbon column chromatography (FIG. 9).

  The present invention is summarized as follows.

  The present invention provides a method for efficiently isolating neokestose and producing high-purity neokestose using a fructan-containing composition (a composition containing a mixture of neofructooligosaccharide and fructooligosaccharide) as a material. Objective.

  Then, a fructan hydrolase having a predetermined activity is allowed to act on the fructan-containing composition to selectively decompose fructans other than neokestose, leaving only glucose, fructose, sucrose, and neokestose. High purity neokestose can be efficiently produced (industrial mass production) simply by chromatographic separation of the enzyme treatment solution.

Claims (9)

Treatment of fructan-containing composition with fructan hydrolase 1-FEH (fructan 1-exohydrolase) having the amino acid sequence shown in SEQ ID NO: 1, and chromatographic separation of the enzyme-treated solution, Method. The fructan-containing composition consists of an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, or additions in the amino acid sequence set forth in SEQ ID NO: 1, and exhibits 1-FEH (fructan 1-exohydrolase) activity. A method for producing neokestose, which comprises treating with a fructan hydrolase and chromatographic separation of the enzyme-treated solution. The fructan-containing composition is treated with a fructan hydrolase having 1-FEH (fructan 1-exohydrolase) activity encoded by the gene consisting of the base sequence shown in SEQ ID NO: 2, and the enzyme-treated solution is chromatographed. A method for producing neokestose characterized by the above. The activity of 1-FEH (fructan 1-exohydrolase) encoded by a gene obtained by hybridization under stringent conditions using as a probe a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 2 as a fructan-containing composition A method for producing neokestose, which comprises treating with a fructan hydrolase and chromatographic separation of the enzyme-treated solution.   The method according to any one of claims 1 to 4, wherein the fructan-containing composition is an agricultural product and / or an agricultural by-product.   The method according to claim 5, wherein the agricultural product and / or the agricultural by-product is derived from a lily family plant and / or a asteraceae plant family.   The method according to claim 6, wherein the agricultural product and / or the agricultural by-product is derived from at least one selected from asparagus, onion and burdock.   By treating a fructan-containing composition with the fructan hydrolase according to any one of claims 1 to 4, fructan other than neokestose in the composition is selectively decomposed to leave neokestose. Method.   The fructan hydrolase 1-FEH (1) according to any one of claims 1 to 4, wherein fructan other than neokestose in the fructan-containing composition is selectively degraded to leave neokestose. fructan 1-exohydrolase).

Images