Method for producing novel oligosaccharides and hexose trisaccharides

Abstract

Provided is an oligosaccharide having a novel structure. SOLUTION: β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose is represented by formula (I). β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose represented by formula (I) is a useful novel oligosaccharide that can be used in food and beverage compositions, as well as in pharmaceuticals and functional foods that act as prebiotics to improve the intestinal flora, regulate the intestines, activate the immune system, and prevent dental caries.

Description

[Technical Field]

[0001] The present invention relates to a method for producing novel oligosaccharides and hexose trisaccharides in which a pyranose-type fructose residue is β-linked to the 6th position of the glucose residue at the non-reducing end of gentiobiose, and more specifically, to a method for producing novel oligosaccharides and hexose trisaccharides in which a pyranose-type fructose is β2-6-linked to the 6th position of the non-reducing end glucose residue of a disaccharide in which two glucoses are β1-6-linked. [Background technology]

[0002] Recently, with the increasing health consciousness of consumers, various functional foods, supplements, and pharmaceuticals have been developed, and among them, oligosaccharides are known as prebiotics that have the effect of increasing beneficial intestinal bacteria such as bifidobacteria, and are widely used as one of the functional ingredients. Such oligosaccharides have intestinal regulating effects (improving intestinal flora), promoting mineral absorption, anti-caries effects, and anti-allergic effects.

[0003] Oligosaccharides are considered important as nutritional components due to their digestibility and bioavailability. Currently, many types of oligosaccharides are used as functional and nutritional components. However, due to the diversification of tastes and increasing health consciousness, there is a demand for the acquisition of new oligosaccharides and the development of their functions.

[0004] Among such novel oligosaccharides, oligosaccharides have been discovered to date, including those in which a pyranose-type fructose is linked by a β2-6 bond to the reducing and non-reducing terminal glucose of laminaribiose (Patent Document 1 and Non-Patent Document 1), those in which a fructose residue of sucrose is linked by a β2-1 bond to a pyranose-type fructose, those in which a glucose residue of sucrose is linked by a β2-6 bond to a pyranose-type fructose (Non-Patent Document 2), and those in which a fructose residue of sucrose is linked by a β2-6 bond to a pyranose-type fructose (Non-Patent Document 3). However, no oligosaccharides (hexose trisaccharides) in which a pyranose-type fructose residue is linked by a β-bond to the 6th position of the non-reducing terminal glucose of gentiobiose are known.

[0005] According to Example (4) of the present specification, the NMR analysis described in Patent Document 1 and Non-Patent Document 1 cannot analyze the structure of an oligosaccharide (hexose trisaccharide: sugar 1 in Example (2) of the present specification) in which a pyranose-type fructose residue is β-linked to the 6-position of the non-reducing terminal glucose of gentiobiose by subjecting a plant extract fermentation liquid to an activated carbon column.

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

[0007] Furthermore, according to Example (6) of the present specification, 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), both of which are disclosed in Patent Document 1 and Non-Patent Document 1, differ in hydrophilicity and the enzyme required for decomposition from an oligosaccharide in which a pyranose-type fructose residue is β-linked to the 6th position of the non-reducing terminal glucose of gentiobiose (hexose trisaccharide: sugar 1 in Example (2) of the present specification). [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) Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide oligosaccharides having novel structures. [Means for solving the problem]

[0011] As a result of extensive research, the present inventors searched for and isolated novel oligosaccharides in plant extract fermentation broth, and conducted constituent sugar analysis, MALDI-TOF-MS analysis, and NMR analysis. As a result, they found that the oligosaccharides were novel oligosaccharides (hexose trisaccharides) that did not match any authentic samples, and thus completed the following invention.

[0012] (1) Formula (I): [ka] β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose,

[0013] (2) A food or drink composition comprising the β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose described in (1) above.

[0014] (3) adding sucrose in an amount of 1 / 10 or more and 2 times or less to the cut plant to obtain a plant extract; The obtained plant extract liquid is naturally fermented, and the plant extract fermentation liquid obtained contains the compound of the following formula (I): [ka] and producing β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose represented by the formula: subjecting the obtained plant extract fermentation liquid to chromatography and eluting with water to collect the β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose; The method for producing a hexose trisaccharide, comprising:

[0015] (4) The method according to (3), wherein the step of collecting β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose comprises subjecting the plant extract fermentation liquid to activated carbon Celite column chromatography, followed by a stepwise gradient of distilled water, 5% ethanol, and 30% ethanol to elute sugars, subjecting the eluted sugars to hydrophilic interaction chromatography to detect fractions containing β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose, and subjecting the detected fractions to HPLC to collect the β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose. [Effects of the Invention]

[0016] According to the present invention, β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose represented by the formula (I) is useful as a novel oligosaccharide, and can be used in food and beverage compositions. In addition, it can also be used as a prebiotic in pharmaceuticals and functional foods that improve the intestinal flora, regulate the intestines, activate the immune system, and prevent dental caries. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 shows a chart obtained by further subjecting sugar 1 in Example (2) of the present specification to an Amide-80 column. [Figure 2] FIG. 1 shows a chart obtained by further subjecting sugar 1 in Example (2) of the present specification to HPLC using an ODS-100V column. [Figure 3]FIG. 1 shows a chart of the results of MALDI-TOF-MS for sugar 1 in Example (2) of the present specification. [Figure 4] 1 shows a chart of the results of NMR analysis of sugar 1 in Example (2) of the present specification using H / D shifts. [Figure 5] FIG. 1 shows the results of a digestion test of sugar 1, sucrose, and maltose using rat small intestinal enzymes in Example (2) of the present specification. [Figure 6] FIG. 1 shows charts obtained by subjecting oligosaccharides B and C, and saccharide 1 in Example (2) of the present specification to an Amide-80 column. DETAILED DESCRIPTION OF THE INVENTION

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

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

[0020] The present inventors have also found that the novel oligosaccharides according to the present invention are produced or increased during the fermentation of a plant extract fermentation broth.

[0021] The novel oligosaccharides of the present invention can be used as food and beverage compositions, and can also be used as prebiotics in pharmaceuticals and functional foods that have effects such as improving the intestinal flora, regulating the intestines, stimulating the immune system, and preventing dental caries.

[0022] The novel oligosaccharides of the present invention can be used in the form of a liquid containing sugar, a concentrated syrup, or a dried solid, powder, or granule. Furthermore, they can be used in the form of a sugar-rich fraction obtained by removing contaminating sugars through a purification process, or in the form of a liquid, syrup, or crystallized form after further purification, or in the form of a powder, granule, sphere, short rod, plate, cube, tablet, or other solid form.

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

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

[0025] The "plant extract liquid" in the plant extract preparation step (a) refers to an extract liquid obtained by adding sucrose to cut plants 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 plants, and particularly preferably an amount equal to the amount of the cut plants. Examples of such plants include fruits such as apples, bananas, strawberries, and mandarin oranges; vegetables such as carrots, radishes, ginger, burdock, cabbage, spinach, onions, tomatoes, cucumbers, eggplants, garlic, and shiso; mushrooms such as shiitake mushrooms; seaweed such as kelp; and wild plants such as bamboo grass.

[0026] The "plant extract fermented liquid" in the plant extract fermented liquid preparation step (b) refers to a "plant extract liquid" that has been fermented. Such fermentation can be natural fermentation or, if desired, natural fermentation followed by aging. Natural fermentation is carried out by storing the "plant extract liquid" in a dark place at 37°C, primarily by yeast (e.g., microorganisms belonging to the genus Zygosaccharomyces) and lactic acid bacteria (e.g., microorganisms belonging to the genus Leuconostoc).

[0027] The chromatography used in the novel oligosaccharide isolation step (c) can be high performance liquid chromatography (HPLC), and 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 collection step (c) may involve subjecting the plant extract fermentation broth to activated carbon Celite column chromatography, sequentially using a stepwise gradient of distilled water, 5% ethanol, and 30% ethanol to elute saccharides containing the novel oligosaccharide of the present invention (the β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose), subjecting the eluted saccharides to hydrophilic interaction chromatography to detect fractions containing the novel oligosaccharide of the present invention, and subjecting the detected fractions to HPLC to collect the novel oligosaccharide of the present invention. Examples of activated carbon-Celite columns that can be used in such activated carbon-Celite column chromatography include those packed with a mixture of commercially available activated carbon and Celite. The volume ratio of the activated carbon to Celite is preferably about 1:4 to 4:1, and a mixture of equal amounts of activated carbon and Celite (1:1) is more preferred. Examples of columns that can be used in such hydrophilic interaction chromatography include Amide-80 (manufactured by Tosoh Corporation), and examples of columns that can be used in such HPLC include, as described above, ODS-100V column (manufactured by Tosoh Corporation).

[0029] The method for producing novel oligosaccharides and hexose trisaccharides according to the present invention will be described below with reference to examples, although the technical scope of the present invention is not limited to the embodiments shown in these examples. [Example]

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

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

[0032] The saccharides eluted with the 30% ethanol stepwise gradient were further subjected to Amide-80 (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 the oligosaccharides. The relationship between elution time and signal intensity is shown in Figure 1. As indicated by the arrow in Figure 1, an oligosaccharide fraction was detected around 95 minutes into the elution time in Figure 1. This eluted oligosaccharide was designated "Sugar 1."

[0033] Next, the fractions at approximately 95 minutes in elution time in Figure 1 were collected and subjected to HPLC on an ODS-100V column (Tosoh Corporation; column size: two connected 4.6 mm x 25 cm columns; 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, fractions of the α-anomer and β-anomer of sugar 1 were collected at approximately 33.7 minutes and 37 minutes in elution time in Figure 2, respectively, and purified to obtain a lyophilized powder.

[0034] (3) Determination of chemical structure The freeze-dried powder of sugar 1 isolated as a single component 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 in (2) was hydrolyzed with hydrochloric acid, and the constituent sugars were analyzed using HPAEC. Sugar 1 was found to have a glucose to fructose molar ratio of 2:1.

[0035] Next, the lyophilized 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 arrow in Figure 3, sugar 1 has a [M+Na] of 527. + This confirmed that sugar 1 was a hexose trisaccharide.

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

[0037] [Table 1]

[0038] Figure 4 shows the results (chart) of NMR analysis of sugar 1 using H / D shift. 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 NMR analysis using HMBC that the 6th position of the glucose residue (Glc) is bonded to the 1st position of glucose' (GLC'). Furthermore, in the case of fructopyranose (Frup), Fru6 does not shift because it forms a ring. Therefore, since GLC'6 does not shift, it is clear that the 6th position of Glc is not a free hydroxyl group and is bonded to the 2nd position of Frup.

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

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

[0041] As a result, the correlation between C at position 2 of Frup and H at position 6 of Glc' overlapped with other signals, and therefore the structure of sugar 1 could not be analyzed by the NMR analysis published in Patent Document 1 and Non-Patent Document 1 (not shown).

[0042] In the NMR analysis in Example (3) above, NMR analysis was carried out using H / D shifts. 13 C-OD (heavy water substitution) or 13 When sugar 1 is dissolved in C-OH (light water), 13 This analytical method utilizes the shift in the C chemical shift. This is because the absence of an -OH group at the bond site results in no shift in the chemical shift. Comparing the signals after water substitution of sugar 1, the chemical shift of the C at position Glc'6 hardly shifted, revealing that this is the bond site with the C at position Frup2. In other words, by using the NMR analysis method described in Example (3) above, it was possible to analyze the structure of sugar 1, which could not be analyzed using the NMR analysis methods described in Patent Document 1 and Non-Patent Document 1.

[0043] (5) Digestion test To examine the digestibility of Sugar 1, digestion tests of Sugar 1 using artificial gastric juice, pancreatin, and rat small intestinal enzymes were carried out.

[0044] [5-1] Digestion test using artificial gastric juice The artificial gastric fluid used was a 50 mM HCl·KCl buffer solution (pH 2.0). 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 digestion reaction was stopped by neutralizing with 6.2 μL of 10 mM NaOH at 37°C for 100 minutes. The resulting reaction mixture was diluted appropriately with distilled water, and the remaining sugar 1 in the diluted mixture was detected by anion exchange liquid chromatography (HPAEC). The digestibility of sugar 1 was calculated by setting the sugar 1 that had not been digested by the artificial gastric fluid as 100%.

[0045] [5-2] Digestion test using pancreatin To 20 μL of 50 mM Bis-Tris buffer (pH 6.6) containing 1 mM CaCl and 1.0% (w / v) sugar 1, 2 μL of a pancreatin (Fujifilm Wako Pure Chemical Industries, Ltd.) suspension prepared to an amylase activity of 4 U / mL was added. After a 360-minute degradation reaction at 37°C, the reaction was stopped by heating at 100°C for 5 minutes. As in the test using artificial gastric juice, the resulting reaction mixture was appropriately diluted with distilled water, and the remaining sugar 1 in the diluted reaction mixture was detected by HPAEC. The digestibility of sugar 1 was calculated by setting the sugar 1 that had not been decomposed by pancreatin as 100%.

[0046] [5-3] Digestion test using rat small intestinal enzymes 300 mg of rat small intestine acetone powder (Sigma-Aldrich Japan, LLC) was suspended in 2.7 mL of 10 mM sodium phosphate buffer (pH 7.0) and homogenized for 5 minutes on ice using a glass homogenizer. The resulting supernatant was then centrifuged at 9,000 x g 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, adjusted to 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 progress of the degradation reaction was monitored by sampling the reaction mixture over time at 37°C. After 300 minutes, the reaction mixture was diluted appropriately with distilled water and immediately heated to 100°C for 5 minutes to terminate the degradation reaction. After detecting the sugar 1 remaining in the diluted reaction mixture using HPAEC, the digestibility of sugar 1 was calculated by setting the sugar 1 that had not undergone decomposition by the rat small intestinal enzymes as 100%. In addition, similar digestion tests were performed using sucrose and maltose instead of sugar 1, and the results were obtained.

[0047] [5-4]Result The results of the digestion test of sugar 1 using artificial gastric juice, pancreatin, and rat small intestinal enzymes are shown in Table 2.

[0048] [Table 2]

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

[0050] The results of the digestion test of sugar 1, sucrose, and maltose using rat small intestinal enzymes are shown in Figure 5. Figure 5 shows the residual rate of each sugar over time as the decomposition reaction time increases. As shown in Table 2 and Figure 5, sugar 1 was found to be a difficult-to-digest oligosaccharide.

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

[0052] As shown in Figure 6, the elution times of oligosaccharides B, C, and saccharide 1 were significantly different. Specifically, the elution time of oligosaccharide B was 51 minutes, that of oligosaccharide C was 65 minutes, and that of saccharide 1 was 95 minutes. This revealed that oligosaccharides B, C, and saccharide 1 differ in hydrophilicity. Furthermore, the bonds between glucose residues in oligosaccharides B and C are both β1-3 bonds, whereas the bonds between glucose residues in saccharide 1 are β1-6 bonds. From this, it can be easily inferred that, when considering digestibility by intestinal bacteria, etc., the enzymes required for decomposition of oligosaccharides B, C, and saccharide 1 are different.

Claims

1. The following formula (I): 【Chemistry 1】 β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose represented by the formula:

2. A food or drink composition comprising the β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose according to claim 1.

3. a step of adding sucrose in an amount of 1 / 10 or more and 2 times or less to the cut plant to obtain a plant extract; The obtained plant extract liquid is naturally fermented, and the plant extract fermentation liquid obtained contains a compound represented by the following formula (I): 【Chemistry 1】 and producing β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose represented by the formula: the obtained plant extract fermentation liquid is subjected to activated carbon Celite column chromatography, and then a stepwise gradient of distilled water, 5% ethanol, and 30% ethanol is applied to elute sugars; the sugars eluted with the stepwise gradient of 30% ethanol are subjected to hydrophilic interaction chromatography to detect fractions containing the β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose; and the β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose is collected from the detected fractions. The method for producing a hexose trisaccharide, comprising:

4. The method of claim 3, further comprising the step of subjecting the detected fraction to HPLC to collect fractions of the α anomer and β anomer of β-D-fructopyranosyl-(2→6)-β-D-glucopyranosyl-(1→6)-D-glucose.

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