JP4674828B1 - α-D-fructofuranosyl- (2 → 6) -D-glucopyranose and method for producing the same - Google Patents

Abstract

【選択図】なしPROBLEM TO BE SOLVED: To provide a method for producing a disaccharide composed of fructose and glucose, wherein a fructose residue is α-bonded.
[Solution]
A sucrose is added to the cut plant so that it contains 1/10 to 2 times the weight of the raw material weight, and extracted using the osmotic pressure of sucrose to obtain a plant extract, By naturally fermenting the plant extract, α-D-fructofuranosyl (2 → 6) -D-glucopyranose represented by the formula (1) is produced in the fermented extract, and the fermentation broth is chromatographed. Α-D-fructofuranosyl (2 → 6) -D-glucopyranose is collected by applying to the chromatography and eluting with water . The disaccharide is indigestible.
[Chemical 1]

[Selection figure] None

Description

The present invention, alpha-fructoside relates to a manufacturing method of lifting one disaccharides, details a disaccharide consisting of fructose and glucose, that have a alpha linked fructose residues alpha-D-fructofuranosyl - ( 2 → 6) -D- Gurukopirano the scan process for the preparation of.

  Fructose and glucose having a 2-6 bond and a fructose residue having a β bond, that is, β-D-fructofuranosyl- (2 → 6) -D-glucopyranose is obtained from a hydrolyzate of neokestose. (Non-patent Document 1).

  In addition, the plant extract is naturally fermented, β-D-fructopyranosyl- (2 → 6) -D-glucopyranose is collected from the generated oligosaccharide, and the solution containing carbohydrates is collected. It has already been clarified by the present applicant that β-D-fructopyranosyl- (2 → 6) -D-glucopyranose is collected from a solution by acting β-fructofuranosidase (patent) Reference 1).

However, a disaccharide composed of fructose and glucose in which a fructose residue is α-linked is not known in nature .

Patent No. 3871222 Journal of Chromatography A, 920 (2001) 299-308.

The present invention, fructose residues linked alpha, and an object thereof is to provide a disaccharide ing from fructose and glucose.

The present inventors have searched for oligosaccharides of plant extracts fermented liquid is separated, TOF-MS analysis, gas chromatography methylated sugar - result of analysis and NMR analysis, was detected disaccharide. Disaccharide of this has been found to be produced during the fermentation of plant extract fermentation liquid. This disaccharide is an α-D-fructofuranosyl- (2 → 6) -D-glucopyranose (sugar 1) represented by the following formula (1) in which a fructose residue is bound to glucose by 2-6 by α-bonding. Also called).

In the method for producing a disaccharide represented by the formula (1) of the present invention, sucrose is added by adding sucrose so as to contain 1/10 to 2 times the amount of carbohydrates after cutting the plant. Extraction is performed using pressure to obtain a plant extract solution, and α-D-fructofuranosyl- (2 → 6) -D is obtained in the obtained plant extract fermentation solution by natural fermentation. -Glucopyranose is produced, α-D-fructofuranosyl- (2 → 6) -D-glucopyranose is collected by subjecting the fermentation broth to chromatography and elution with water .

  In the method for producing a disaccharide of the present invention, a method of collecting α-D-fructofuranosyl- (2 → 6) -D-glucopyranose from a plant extract fermentation broth is obtained by subjecting the plant extract fermentation broth to activated charcoal column chromatography. Added to the chromatography and eluted with water to obtain a fraction containing α-D-fructofuranosyl- (2 → 6) -D-glucopyranose, which was subjected to HPLC by α-D-fructofuranosyl -(2 → 6) -D-glucopyranose is separated.

  In the present specification, the “plant extract liquid” is used to mean an extract of a plant before fermentation. The term “plant extract fermented liquid” is used to mean a fermented plant extract.

The present invention provides a method for producing a disaccharide represented by the formula (1).

Addition of plant extract fermentation broth to activated charcoal celite column chromatography and addition of fraction eluted with water to Amide-80 (trade name, manufactured by Tosoh Corporation) It is a graph which shows. It is a graph which shows the relationship between elution time and signal intensity about what isolate | separated the fraction which eluted time 19.0-22.0 in FIG. 1 by performing HPLC using an ODS-80Ts column. It is a figure showing the chart of the mass spectrometry (MALDI-TOF-MS) in Positive ion mode about sugar 1. 1 is a chart showing a ¹ H-NMR analysis chart for sugar 1. FIG. It is a figure showing the chart of the < ¹³ > C-NMR analysis about the saccharide | sugar 1. FIG. It is a figure showing the chart of COSY about sugar 1. It is a figure showing the chart of E-HSQC about sugar 1. It is a figure showing the chart of HSQC-TOCSY about sugar 1. It is a figure showing the chart of HMBC about sugar 1. It is a figure showing the digestibility graph of saccharide | sugar 1. FIG.

(Manufacturing method of plant extract fermentation broth)
The plant extract fermentation broth is produced as follows. Plant materials include fruits such as apples, bananas, strawberries, mandarin oranges, orange peels, pears, pineapples, grapes, carrots, radishes, ginger, burdock, cabbage, spinach, onions, tomatoes, cucumbers, celery, peppers, black Mushrooms such as mappe palm, peas, eggplant, lotus root, pumpkin, lettuce, garlic, trefoil, udo, asparagus, perilla, turnip, chives, Chinese cabbage, salad vegetables, garlic, seri, leek, etc., mushrooms such as shiitake, enokitake And wild algae such as bears, clovers, cypresses, dandelions, psyllium, cedar leaves, parsley, weed buds, mugwort, todomatsu leaves, seaweeds such as kelp, seaweed, etc., two of these plant materials As described above, preferably, various types are used.

  The blending ratio of the above plant materials is, for example, 0.1 to 50% of the total weight of plant materials for apples, 0.1 to 50% for carrots, 0.05 to 40% for radishes, 0.05 to 40% for cabbage, It is desirable that the content is 0.01 to 30% for celery and cucumber, and 0.01 to 30% for banana, onion, burdock and spinach, but there is no particular limitation.

  Cut these plant materials into 1-5 cm width, preferably 1-4 cm width, most preferably 2-3 cm width, put all the raw materials in cedar barrels, mix this with sucrose, without pressing The plant extract solution is obtained by extracting from 3 days to 3 weeks using osmotic pressure. As for the mixing ratio of sucrose, it is desirable to include 1/10 amount or more and 2 times or less amount of saccharide as a whole. At the same time, a slight amount of sodium chloride may be added and mixed so that the concentration is about several ppm.

When the plant extract solution collected without pressing is stored at 37 ° C. in the dark, it is naturally fermented mainly by yeast (for example, microorganisms belonging to the genus Saccharomyces ) and lactic acid cocci (for example, microorganisms belonging to the genus Leuconostoc ). After fermentation, further ripening at 37 ° C. for about half a year or more yields a brown, viscous liquid plant extract fermentation broth.

( Disaccharide fractionation and purification)
The obtained plant extract fermentation solution through a column chromatography packed with activated carbon celite, alpha-D-fructofuranosyl represented in the previous Ri by the eluting with water following formula (1) - (2 → 6 ) -D-glucopyranose (sugar 1) can be obtained. The obtained eluate is further purified by separating the sugar 1 represented by the formula (1) using HPLC. Then it was freeze-dried to obtain the sugar 1 represented by powdery purified before following formula (1).

(Preparation of fermented plant extract)
As a raw material for the plant extract, a material having the following composition was cut into a width of 2 to 3 cm.

20% of apple plant material total weight
Carrot 16%
12% radish
Cabbage 10%
Celery 9%
9% cucumber
Banana 6%
Onion 6%
Burdock 6%
6% of spinach
An equivalent amount of sucrose is added to the total weight of these plant raw materials and extracted for one week. The resulting plant extract solution is naturally fermented and then aged at 37 ° C. for about half a year to obtain a brown viscous product. A viscous liquid plant extract fermentation broth was obtained.

(Fractionation and purification of unknown sugar components)
The fermented plant extract fermentation liquid obtained in the above step was added to activated carbon celite column chromatography (4.5 cm × 35 cm), and eluted sequentially with water, 5% ethanol, and 15% ethanol stepwise gradient.

  The fraction eluted with water was further treated with Amide-80 (trade name, 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) and eluted. The results are shown in FIG. 1 as a graph showing the relationship between elution time and signal intensity.

  Next, a fraction (the peak portion indicated by an arrow in FIG. 1) having an elution time of 19.0 to 22.0 minutes in FIG. Manufactured; column size: 4.6 mm × 25 cm, 4 elutions: distilled water, column temperature: room temperature, flow rate: 0.3 mL / min, detection: differential refractometer). The results are shown in FIG. 2 as a graph showing the relationship between elution time and signal intensity. The fraction (the peak portion indicated by the arrow in FIG. 2) having an elution time of 53.5 to 54.5 minutes in FIG. 2 was collected and purified to obtain a lyophilized powder.

(Chemical structure determination)
The lyophilized powder obtained in the above step was subjected to the following instrumental analysis, and its chemical structure was determined as follows.

As a result of mass spectrometry (MALDI-TOF-MS) of the lyophilized powder isolated as a single component obtained in the above step in the positive ion mode, the chart is shown in FIG. According to FIG. 3, the unknown component gave 365 [M + Na] ⁺ ion peaks. Therefore, it was confirmed that the unknown component was hexose disaccharide. The unknown hexose disaccharide is called sugar 1.

  After sugar 1 was acid hydrolyzed, HPAEC analysis was performed and the constituent sugars were examined. As a result, sugar 1 was released at a ratio of glucose to fructose of 1: 1. As a result of gas chromatographic analysis of a sample in which sugar 1 was methylated and decomposed in methanol by the method of Hakomori, methyl 1,3,4,6-tetra-O-methyl-D-fructofuranoside, methyl 2,3,4 Tri-O-methyl-D-glucoside was detected.

Next, sugar 1 was dissolved in heavy water and the following one-dimensional NMR analysis was performed. A chart of ¹ H-NMR analysis is shown in FIG. 4, and a chart of ¹³ C-NMR analysis is shown in FIG.

  Next, sugar 1 was dissolved in heavy water and the following two-dimensional NMR analysis was performed. FIG. 6 shows a COSY chart, FIG. 7 shows an E-HSQC chart, FIG. 8 shows an HSQC-TOCSY chart, and FIG. 9 shows an HMBC chart.

  From the above results, sugar 1 was determined to be α-D-fructofuranosyl- (2 → 6) -D-glucopyranose.

(Digestibility of sugar 1)
The degradability of the sugar 1 of the present invention by rat small intestine acetone powder was tested as follows.

  2.7 mL of 10 mM phosphate buffer (pH 7.0) was added to 300 mg of rat small intestine acetone powder, homogenized using a glass homogenizer, centrifuged at 10,000 × g for 15 minutes at 4 ° C., and the resulting supernatant was enzyme solution It was. The enzyme solution was diluted to 2.5 units per mL as maltase activity. 0.2 mL of the obtained diluted enzyme solution and 1 mL of a 1% aqueous solution of saccharide 1 obtained in Example 1 were mixed, reacted at 37 ° C. for a fixed time, and heated at 100 ° C. for 5 minutes to stop the reaction.

  FIG. 10 shows a graph representing the digestibility of sugar 1 (indicated by Δ), where the vertical axis represents the residual ratio of sugar (%) and the horizontal axis represents the reaction time. For comparison, the digestibility of maltose (indicated by ◆) and sucrose (indicated by ■) is also shown.

As a result of this test, the sugar 1 produced according to the present invention was hardly degraded. Therefore, it turns out that the saccharide | sugar 1 manufactured by this invention is an indigestible carbohydrate.

Since α-D-fructofuranosyl- (2 → 6) -D-glucopyranose produced by the present invention is a disaccharide oligosaccharide, it can be expected to be used as a food material or a pharmaceutical material.

Since the disaccharide produced according to the present invention is an indigestible carbohydrate, it is a food for improving the intestinal environment, improving hyperglycemic symptoms, and improving obesity and diabetic diseases caused by hyperglycemia. Expected to be used as a material and pharmaceutical material.

Claims (2)

A plant extract solution is obtained by adding sucrose so as to contain 1/10 to 2 times the amount of sugar in the cut plant, and extracting using the osmotic pressure of sucrose to obtain a plant extract solution. Is naturally fermented to produce α-D-fructofuranosyl- (2 → 6) -D-glucopyranose represented by the following formula (1) in the obtained plant extract fermentation broth, and the fermentation A method for producing a disaccharide, comprising collecting α-D-fructofuranosyl- (2 → 6) -D-glucopyranose by subjecting the solution to chromatography and elution with water .

  The method for collecting α-D-fructofuranosyl- (2 → 6) -D-glucopyranose in the method for producing a disaccharide according to claim 1, wherein a plant extract fermentation broth is added to activated carbon celite column chromatography. And fractions containing α-D-fructofuranosyl- (2 → 6) -D-glucopyranose were obtained by elution with water, and α-D-fructofuranosyl- (2 6) A method for producing a disaccharide, characterized by separating -D-glucopyranose.

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