Method for producing novel oligosaccharides and hexose trisaccharides

Abstract

We provide oligosaccharides with a novel structure. [Solution] The solution is β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose represented by formula (I). The β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose represented by formula (I) is useful as a novel oligosaccharide and can be used in food and beverage compositions. In addition, it can be used in pharmaceuticals and functional foods that exhibit effects such as improving the intestinal flora as a prebiotic, regulating bowel function, stimulating the immune system, and anti-caries effects.

Description

Technical Field

[0001] The present invention relates to a novel oligosaccharide in which a pyranose-type fructose residue is β-linked to the 6-position of the glucose at the non-reducing end side of gentiobiose and a method for producing hexose trisaccharides. More specifically, it relates to a novel oligosaccharide in which a pyranose-type fructose is β2-6-linked to the 6-position of the non-reducing end glucose residue of a disaccharide in which glucose and glucose are β1-6-linked, and a method for producing hexose trisaccharides.

Background Art

[0002] Recently, due to the increasing health consciousness of consumers, various functional foods, supplements, and pharmaceuticals have been developed. Among them, oligosaccharides are known as prebiotics that have the effect of increasing beneficial intestinal bacteria such as Bifidobacterium, and are widely used as one of the functional components. Such oligosaccharides have effects such as intestinal regulation (intestinal flora improvement effect), promotion of mineral absorption, anti-caries effect, and anti-allergic effect.

[0003] Oligosaccharides are also regarded as important nutritional components due to their digestibility and absorption in the body. Currently, many types of oligosaccharides are used as functional and nutritional components. However, due to the diversity of tastes and the increasing health consciousness, the acquisition of novel oligosaccharides and the development of their functions are demanded.

[0004] Regarding such novel oligosaccharides, conventionally, oligosaccharides in which a pyranose-type fructose and the reducing and non-reducing end glucoses of laminaribiose are β2-6-linked (Patent Document 1 and Non-Patent Document 1), oligosaccharides in which the fructose residue of sucrose and a pyranose-type fructose are β2-1-linked, oligosaccharides in which the glucose residue of sucrose and a pyranose-type fructose are β2-6-linked (Non-Patent Document 2), and further, those in which the fructose residue of sucrose and a pyranose-type fructose are β2-6-linked (Non-Patent Document 3) have been found. However, oligosaccharides (hexose trisaccharides) in which a pyranose-type fructose residue and the 6-position of the non-reducing end glucose of gentiobiose are β-linked are not known.

[0005] Furthermore, according to Example (4) of this specification, the NMR analysis described in Patent Document 1 and Non-Patent Document 1 does not allow for the analysis of the structure of oligosaccharides (hexose trisaccharides: sugar 1 in Example (2) of this specification) in which a pyranose-type fructose residue and the 6th position of the non-reducing terminal glucose of gentiobiose are β-linked, by subjecting the plant extract fermentation liquid to an activated carbon column.

[0006] Furthermore, β-D-fructopyranosyl(2→6)D-glucopyranose (oligosaccharide A), disclosed in Patent Document 1 and Non-Patent Document 1, has a completely different structure from oligosaccharides (hexose trisaccharides: sugar 1 in Example (2) of this specification) in which a pyranose-type fructose residue and the 6th position of the non-reducing terminal glucose of gentiobiose are β-linked.

[0007] Furthermore, according to Example (6) of this specification, the β-D-fructopyranosyl(2→6)-O-β-D-glucopyranosyl(1→3)D-glucopyranose (oligosaccharide B, the same applies hereinafter) and β-D-fructopyranosyl(2→6)-O-[β-D-glucopyranosyl(1→3)]D-glucopyranose (oligosaccharide C, the same applies hereinafter) disclosed in Patent Document 1 and Non-Patent Document 1 differ from oligosaccharides (hexose trisaccharides: sugar 1 in Example (2) of this specification) in which a pyranose-type fructose residue and the 6th position of the non-reducing terminal glucose of gentiobiose are β-linked, and differ in hydrophilicity and the enzymes required for decomposition. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Patent No. 3871222 [Non-patent literature]

[0009] [Non-Patent Document 1] Carbohydr.Res.,343,549-554(2008) [Non-Patent Document 2] Carbohydr.Res.,345,414-418(2010) [Non-Patent Document 3] J.Appl.Glycosci.,64,123-127(2017) [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] The present invention aims to provide an oligosaccharide having a novel structure. [Means for solving the problem]

[0011] As a result of diligent research, the inventors searched for and isolated novel oligosaccharides in plant extract fermentation liquid, and performed constituent sugar analysis, MALDI-TOF-MS analysis, and NMR analysis. They discovered that these oligosaccharides were novel oligosaccharides (hexose trisaccharides) that did not match any standard, and thus completed the following invention.

[0012] (1) The following formula (I): [ka] β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose, represented as β-D-fructopyranosyl-(2→6)-β-D-glucose.

[0013] (2) A food and beverage composition obtained by adding the β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose described in (1).

[0014] (3) A step of obtaining a plant extract by adding sucrose in an amount of 1 / 10 to 2 times the amount to the cut plant, The following formula (I): [ka] A process to produce β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose, represented by Subjecting the obtained plant extract fermentation broth to chromatography and eluting with water, and collecting the β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose A method for producing a hexose trisaccharide having the same

[0015] (4) The step of collecting the β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose is to subject the plant extract fermentation broth to activated carbon celite column chromatography, and sequentially perform stepwise gradients of distilled water, 5% ethanol, and 30% ethanol to elute saccharides. The eluted saccharides are subjected to hydrophilic interaction chromatography to detect the fraction containing the β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose, and the detected fraction is subjected to HPLC to collect the β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose. The production method according to (3).

Effects of the Invention

[0016] According to the present invention, the β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose represented by the formula (I) is useful as a novel oligosaccharide. In addition to being used as a food and beverage composition, it can also be used in pharmaceuticals, functional foods, etc. that exhibit effects such as improvement of intestinal flora as a prebiotic, intestinal regulation effect, immune activation effect, and anti-caries effect.

Brief Description of the Drawings

[0017] [Figure 1] It is a diagram showing a chart obtained by further subjecting the sugar 1 in Example (2) of this specification to an Amide-80 column. [Figure 2] It is a diagram showing a chart obtained by further subjecting the sugar 1 in Example (2) of this specification to HPLC using an ODS-100V column. [Figure 3]This figure shows a chart of the MALDI-TOF-MS results for sugar 1 in Example (2) of this specification. [Figure 4] This chart shows the results of NMR analysis of sugar 1 in Example (2) of this specification using H / D shift. [Figure 5] This figure shows the results of a digestion test of sugar 1, sucrose, and maltose in Example (2) of this specification using rat small intestinal enzymes. [Figure 6] This figure shows charts obtained by subjecting oligosaccharide B and oligosaccharide C, as well as sugar 1 of Example (2) of this specification, to an Amide-80 column. [Modes for carrying out the invention]

[0018] The following describes in detail the method for producing the novel oligosaccharide and hexose trisaccharide according to the present invention. The novel oligosaccharide according to the present invention is given by the following formula (I): [ka] This is β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose, represented as β-D-fructopyranosyl-(2→6)-β-D-glucose.

[0019] As described above, the novel oligosaccharide according to the present invention is a compound in which a pyranose-type fructose residue and the 6th position of the non-reducing end glucose of gentiobiose are β-linked. More specifically, it is a novel oligosaccharide in which pyranose-type fructose is β2-6 linked to the 6th position of the non-reducing end glucose residue of a disaccharide in which glucose and glucose are β1-6 linked.

[0020] Furthermore, the inventors have discovered that the novel oligosaccharides according to the present invention are produced or increased during the fermentation of plant extract fermentation liquid.

[0021] The novel oligosaccharides according to the present invention can be used in food and beverage compositions, as well as in pharmaceuticals and functional foods that exhibit effects such as improving the intestinal flora, regulating bowel function, stimulating the immune system, and anti-caries effects as prebiotics.

[0022] The novel oligosaccharides according to the present invention can be used in various forms: as a liquid containing sugar, concentrated as a syrup, or dried into a solid, powder, or granular form. Furthermore, they can be obtained by removing impurities such as other sugars through a purification process to obtain a high-sugar fraction, or by further purification to obtain a liquid, syrup, or crystallized product, or by molding it into various shapes such as powder, granules, spheres, short rods, plates, cubes, or tablets and solidifying it.

[0023] The solidified product can, if desired, be mixed with commonly used auxiliary ingredients such as bulking agents, excipients, binders, other sugars, sweeteners, flavoring agents, stabilizers, and emulsifiers.

[0024] Next, the method for producing hexose trisaccharides according to the present invention comprises the following steps (a), (b), and (c); (a) A step to obtain a plant extract solution by adding sucrose in an amount of 1 / 10 to 2 times the amount to the cut plant (plant extract preparation step), (b) A step to generate a novel oligosaccharide according to the present invention (the β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose) in a plant extract fermentation liquid obtained by naturally fermenting the obtained plant extract liquid (plant extract fermentation liquid preparation step), (c) A step to collect the novel oligosaccharide according to the present invention by subjecting the obtained plant extract fermentation liquid to chromatography and eluting with water (novel oligosaccharide collection step).

[0025] In the plant extract preparation step (a), "plant extract liquid" refers to an extract obtained by adding sucrose to cut plant material and utilizing the osmotic pressure of the added sucrose. The amount of sucrose added is preferably between 1 / 10 and 2 times the amount of the cut plant material, and particularly preferably equal to the amount of the cut plant material. Examples of such plants include fruits such as apples, bananas, strawberries, and oranges; vegetables such as carrots, radishes, ginger, burdock, cabbage, spinach, onions, tomatoes, cucumbers, eggplants, garlic, and perilla; mushrooms such as shiitake; seaweed such as kelp; and wild plants such as bamboo grass.

[0026] In the preparation step (b) of the plant extract fermentation liquid, "plant extract fermentation liquid" refers to a product obtained by fermenting the "plant extract liquid." Such fermentation can be carried out by natural fermentation, or, if desired, by maturation after natural fermentation. Natural fermentation is carried out mainly by yeast (e.g., microorganisms belonging to the genus Zygosaccharomyces, etc.) and lactic acid bacteria (e.g., microorganisms belonging to the genus Leuconostoc, etc.) by storing the "plant extract liquid" in the dark at 37°C.

[0027] High-performance liquid chromatography (HPLC) can be used as the chromatography method in the novel oligosaccharide extraction process (c). An example of a column that can be used in such HPLC is the ODS-100V column (manufactured by Tosoh Corporation).

[0028] The novel oligosaccharide extraction step (c) may be a step in which the plant extract fermentation liquid is subjected to activated carbon Celite column chromatography, and a stepwise gradient of distilled water, 5% ethanol, and 30% ethanol is sequentially performed to elute sugars including the novel oligosaccharide according to the present invention (the β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose), the eluted sugars are subjected to hydrophilic interaction chromatography to detect the fraction containing the novel oligosaccharide according to the present invention, and the detected fraction is subjected to HPLC to extract the novel oligosaccharide according to the present invention. An example of an activated carbon-Celite column that can be used in such activated carbon Celite column chromatography is one which is a commercially available activated carbon and Celite mixed and packed into a column. Furthermore, the mixing ratio of activated carbon and Celite is preferably about 1:4 to 4:1 by volume, and a mixture of activated carbon and Celite in equal amounts (1:1) is more preferable. Furthermore, an example of a column that can be used in such hydrophilic interaction chromatography is the Amide-80 (manufactured by Tosoh Corporation). Also, as mentioned above, an example of a column that can be used in such HPLC is the ODS-100V column (manufactured by Tosoh Corporation).

[0029] The following describes the method for producing novel oligosaccharides and hexose trisaccharides according to the present invention, based on examples. However, the technical scope of the present invention is not limited to the embodiments shown in these examples. [Examples]

[0030] (1) Preparation of plant extract fermentation liquid The raw materials for the fermented plant extract included fruits such as apples, bananas, strawberries, and oranges; vegetables such as carrots, radishes, ginger, burdock, cabbage, spinach, onions, tomatoes, cucumbers, eggplants, garlic, and perilla; mushrooms such as shiitake; seaweed such as kelp; and wild plants such as bamboo grass. These raw materials were cut into 2-3 cm wide pieces and placed in cedar barrels. Then, sucrose approximately equal in weight to the total weight of the raw materials was added and the mixture was extracted for one week. The resulting plant extract was naturally fermented in the dark at 37°C, and then aged for approximately six months under conditions of 37°C to obtain a viscous, brown fermented plant extract.

[0031] (2) Fractionation and purification of sugar 1 The plant extract ferment filtrate obtained in (1) was subjected to activated carbon Celite column chromatography (activated carbon:Celite = 1:1, 4.5 cm × 35 cm), and sugars were eluted by sequentially performing a stepwise gradient with distilled water, 5% ethanol, and 30% ethanol.

[0032] The sugars eluted by the aforementioned stepwise gradient of 30% ethanol were further subjected to Amide-80 (manufactured by Tosoh Corporation, column size: 7.8 mm × 30 cm, elution: 80% acetonitrile, column temperature: 80°C, flow rate: 2 mL / min, detection: differential refractometer) to elute oligosaccharides. Figure 1 shows the relationship between elution time and signal intensity. As indicated by the arrow in Figure 1, the oligosaccharide fraction was detected around 95 minutes of elution time in Figure 1. This eluted oligosaccharide was designated as "Sugar 1".

[0033] Next, the fraction at approximately 95 minutes of elution time in Figure 1 was collected and subjected to HPLC on an ODS-100V column (manufactured by Tosoh Corporation; column size: two 4.6 mm × 25 cm columns connected together; elution: distilled water; column temperature: room temperature; flow rate: 0.5 mL / min; detection: differential refractometer). The relationship between elution time and signal intensity is shown in Figure 2. As indicated by the arrows in Figure 2, the α-anomer and β-anomer fractions of sugar 1 were collected at approximately 33.7 minutes and 37 minutes of elution time in Figure 2, purified, and freeze-dried powder was obtained.

[0034] (3) Determination of the chemical structure The freeze-dried powder of sugar 1 isolated as a single component obtained in (2) was subjected to instrumental analysis, and its chemical structure was determined as follows. The freeze-dried powder of sugar 1 isolated as a single component obtained in (2) was subjected to acid hydrolysis with hydrochloric acid, and the constituent sugars were analyzed using HPAEC. Sugar 1 was released with a molar ratio of glucose to fructose of 2:1.

[0035] Next, the freeze-dried powder of sugar 1, isolated as a single component in (2), was subjected to mass spectrometry (MALDI-TOF-MS) in positive ion mode. The results are shown in Figure 3. As indicated by the arrows in Figure 3, sugar 1 is [M+Na] 527 + An ion peak was obtained. From this, it was confirmed that sugar 1 is a hexose trisaccharide.

[0036] Next, NMR analyses were performed for COSY, HSQC, HSQC-TOCSY, HMBC, 1D-TOCSY, and H / D shift. The results are shown in Table 1, which is a chemical shift table below.

[0037] [Table 1]

[0038] Here, Figure 4 shows the results of NMR analysis of sugar 1 using H / D shift (chart). As shown in Figure 4, it was revealed that the 1st and 3rd positions of the fructose residue (Fru) are free hydroxyl groups and are shifted. Furthermore, it has already been revealed by HMBC NMR analysis that the 6th position of the glucose residue (Glc) is bound to the 1st position of glucose' (GLC'). In addition, Fru6 does not shift because it forms a ring in the case of fructopyranose (Frup). Therefore, since GLC'6 does not shift, it was found that the 6th position of Glc is not a free hydroxyl group and is bound to the 2nd position of Frup.

[0039] Based on the above results, it was determined that sugar 1 is β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose, represented by formula (I) above.

[0040] (4) Structural analysis of sugar 1 by NMR analysis in Patent Document 1 and Non-Patent Document 1 We attempted to analyze the structure of sugar 1 using the NMR analyses described in Patent Document 1 and Non-Patent Document 1, namely, NMR analysis using COSY,HSQC,HSQC-TOCSY,HMBC,CT-HMBC,J-res HMBC (for oligosaccharide A), NMR analysis using COSY,HSQC,HSQC-TOCSY,CH2-HSQC-TOCSY,CT-HMBC (for oligosaccharide B), and NMR analysis using COSY,HSQC,HSQC-TOCSY,HMBC,CT-HMBC (for oligosaccharide C).

[0041] As a result, the correlation between the carbon at position 2 of Frup and the hydrogen at position 6 of Glc' overlaps with other signals, making it impossible to analyze the structure of sugar 1 using the NMR analysis described in Patent Document 1 and Non-Patent Document 1 (not shown).

[0042] In addition, in the NMR analysis of the above example (3), NMR analysis was performed using H / D shift, but this is 13 C-OD (heavy water displacement) or 13 When sugar 1 is dissolved in C-OH (light water), 13 This analytical method utilizes the shift of the carbon chemical shift. The chemical shift does not shift at the binding site because there is no -OH group. Comparing the signals after light water substitution of sugar 1, it was found that the chemical shift of the carbon at position Glc'6 hardly shifted, indicating that this is the binding site with the carbon at position Frup2. In other words, by using the NMR analysis method of example (3) above, it was possible to analyze the structure of sugar 1, which could not be analyzed by the NMR analysis methods described in Patent Document 1 and Non-Patent Document 1.

[0043] (5) Digestive Test To investigate the digestibility of glucose 1, digestion tests were conducted using artificial gastric juice, pancreatin, and rat small intestinal enzymes.

[0044] [5-1] Digestive test using artificial gastric juice A 50 mM HCl·KCl buffer (pH 2.0) was used as the artificial gastric fluid. 5 μL of the artificial gastric fluid was added to 10 μL of a 2.0% (w / v) aqueous solution of sugar 1, and the decomposition reaction was carried out at 37°C for 100 minutes. The decomposition reaction was then stopped by adding 6.2 μL of 10 mM NaOH to neutralize the mixture. The resulting reaction product was appropriately diluted with distilled water, and sugar 1 remaining in the diluted reaction product was detected by anion exchange liquid chromatography (HPAEC). The digestibility of sugar 1 was then calculated by setting the sugar 1 that had not undergone the decomposition reaction by the artificial gastric fluid to 100%.

[0045] [5-2] Digestive test with pancreatin To 20 μL of 50 mM Bis-Tris buffer (pH 6.6) containing 1 mM CaCl2 and 1.0% (w / v) sugar 1, 2 μL of a suspension of pancreatin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), prepared to have an amylase activity of 4 U / mL, was added. After the decomposition reaction was carried out at 37°C for 360 minutes, the reaction was stopped by heating at 100°C for 5 minutes. Similar to the test with artificial gastric juice, the obtained reaction product was appropriately diluted with distilled water, and the sugar 1 remaining in the diluted reaction product was detected by HPAEC. The digestibility of sugar 1 was then calculated by setting the sugar 1 that had not undergone the decomposition reaction by pancreatin to 100%.

[0046] [5-3] Digestive test using rat small intestinal enzymes 300 mg of rat small intestine acetone powder (manufactured by Sigma-Aldrich Japan LLC) was suspended in 2.7 mL of 10 mM sodium phosphate buffer (pH 7.0), homogenized in a glass homogenizer under ice conditions for 5 minutes, and then centrifuged at 9,000 xg for 15 minutes at 4°C. The resulting supernatant was used as the rat small intestine enzyme solution. 4 μL of the rat small intestine enzyme solution, prepared to have a maltase activity of 2.5 U / mL, was added to 20 μL of 10 mM sodium phosphate buffer (pH 6.8) containing 1.0% (w / v) sugar 1. The decomposition reaction was carried out at 37°C, and the progress of the decomposition was confirmed by collecting the reaction product over time. As a result, after 300 minutes, the reaction product was diluted with distilled water as appropriate, and the decomposition reaction was immediately stopped by heating at 100°C for 5 minutes. After detecting residual sugar 1 in the diluted reaction product using HPAEC, the digestibility of sugar 1 was determined by setting the sugar 1 that had not undergone degradation by the rat small intestinal enzyme to 100%. Similarly, digestion tests were performed by substituting sucrose and maltose for sugar 1, and the results were obtained.

[0047] [5-4]Result Table 2 shows the results of digestion tests of glucose 1 using artificial gastric juice, pancreatin, and rat small intestinal enzymes.

[0048] [Table 2]

[0049] As shown in Table 2, it was revealed that sugar 1 was hardly digested by artificial gastric juice and rat intestinal enzymes, and was only slightly digested by pancreatin.

[0050] On the other hand, Figure 5 shows the results of digestion tests of sugar 1, sucrose, and maltose using rat small intestinal enzymes. Figure 5 shows the residual rates of each substance over time as the digestion reaction time increases. As shown in Table 2 and Figure 5, it was revealed that sugar 1 is a poorly digestible oligosaccharide.

[0051] (6) HPLC analysis using an Amide-80 column The Amide-80 column is a separation method that utilizes the difference in hydrophilicity or hydrophobicity of substances. For oligosaccharides B and C, as well as sugar 1, disclosed in Patent Document 1 and Non-Patent Document 1, the elution time was confirmed by HPLC analysis using an Amide-80 column (manufactured by Tosoh Corporation, column size: 7.8 mm × 30 cm) (elution: 80% acetonitrile, column temperature: 80°C, flow rate: 2 mL / min, detection: differential refractometer). The results are shown in Figure 6.

[0052] As shown in Figure 6, the elution times of oligosaccharides B and C and sugar 1 differed significantly. Specifically, the elution time for oligosaccharide B was 51 minutes, for oligosaccharide C it was 65 minutes, and for sugar 1 it was 95 minutes. This clearly indicates that oligosaccharides B and C and sugar 1 have different hydrophilic properties. Furthermore, the bonds between glucose residues in oligosaccharides B and C are both β1-3 bonds, while the bonds between glucose residues in sugar 1 are β1-6 bonds. From this, it can be easily inferred that, considering digestibility by intestinal bacteria, the enzymes required for the breakdown of oligosaccharides B and C and sugar 1 are different.

Claims

1. Formula (I) below: 【Chemistry 1】 β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose, represented as β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose.

2. A food and beverage composition comprising the addition of β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose as described in claim 1.

3. A process to obtain a plant extract solution by adding sucrose in an amount of 1 / 10 to 2 times the amount to the cut plant, The following formula (I): 【Chemistry 1】 A step to produce β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose, represented by The process involves subjecting the obtained plant extract fermentation liquid to chromatography and eluting it with water to collect the β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose, and A method for producing hexose trisaccharides, comprising the characteristics of hexose trisaccharides.

4. The manufacturing method according to claim 3, wherein the step of obtaining the β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose is to subject the plant extract fermentation liquid to activated carbon Celite column chromatography, sequentially perform a stepwise gradient of distilled water, 5% ethanol, and 30% ethanol to elute sugars, subject the eluten sugars to hydrophilic interaction chromatography to detect a fraction containing the β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose, and subject the detected fraction to HPLC to obtain the β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose.

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