JP2019094307A - β-D-GLUCOPYRANOSYL-(1→1)-D-FRUCTOSE AND USE THEREFOR - Google Patents

Abstract

To provide a novel disaccharide with glucose and fructose heled by glycosidic linkage.SOLUTION: The present invention provides β-D-glucopyranosyl-(1→1)-D-fructose. The β-D-glucopyranosyl-(1→1)-D-fructose is preferably crystaline one. The present invention also provides a food and drink composition containing β-D-glucopyranosyl-(1→1)-D-fructose, and a pharmaceutical composition. The present invention further provides a taste improver containing β-D-glucopyranosyl-(1→1)-D-fructose as an active ingredient, and an intestinal bacterial flora improver.SELECTED DRAWING: Figure 7

Description

  The present invention relates to a novel disaccharide, β-D-glucopyranosyl- (1 → 1) -D-fructose and uses thereof.

  A sugar in which two monosaccharide molecules form a glycosidic bond to form one molecule is called a disaccharide. The mainly known disaccharides include sucrose in which glucose and fructose are bound, lactulose in which galactose and fructose are bound, maltose in which two glucose molecules are bound, and trehalose.

  The above-mentioned sucrose is an α-1, 2 glycosidic bond of glucose and fructose, and as a disaccharide in which glucose and fructose are bonded in this manner, α-1, 6-glycosidic-linked palatinose (isomalt (Loose), α-1,3-glycosidic linked turanose, α-1,4-glycosidic linked maltulose are known.

  In addition to this, there are reports on disaccharides in which glucose and fructose are glycosidically linked (Patent Document 1 to Patent Document 4).

Patent 4583505 gazette Patent No. 4485602 Patent No. 4674828 Patent No. 4684361

  However, there has been no reported example of a disaccharide in which glucose and fructose are β-1, 1-glycosidic linked, that is, 1 position of glucose and 1 position of fructose are β linked.

  Therefore, an object of the present invention is to provide a novel disaccharide in which glucose and fructose are glycosidically linked.

  As a result of earnest studies to achieve the above object, the present inventors have found that among the products obtained by acting glucose and fructose as a substrate and causing β-glucosidase to act, β-D-glucopyranosyl, which is a novel disaccharide, We found (1 → 1) -D-fructose and completed the present invention.

  That is, the present invention provides β-D-glucopyranosyl- (1 → 1) -D-fructose represented by the following formula (1).

  In a preferred embodiment of the present invention, a crystalline β-D-glucopyranosyl- (1 → 1) -D-fructose represented by the above formula (1) is provided.

  The present invention also provides a composition for food and drink characterized by containing β-D-glucopyranosyl- (1 → 1) -D-fructose.

  The composition for food and drink of the present invention preferably contains 0.5% by mass or more of the β-D-glucopyranosyl- (1 → 1) -D-fructose.

  Furthermore, the present invention provides a pharmaceutical composition comprising β-D-glucopyranosyl- (1 → 1) -D-fructose.

  The pharmaceutical composition of the present invention preferably contains 0.5% by mass or more of the β-D-glucopyranosyl- (1 → 1) -D-fructose.

  Furthermore, the present invention provides a taste improving agent characterized by containing β-D-glucopyranosyl- (1 → 1) -D-fructose as an active ingredient.

  Furthermore, the present invention provides an intestinal flora improving agent characterized by containing β-D-glucopyranosyl- (1 → 1) -D-fructose as an active ingredient.

  According to the present invention, it is possible to provide β-D-glucopyranosyl- (1 → 1) -D-fructose which is a novel disaccharide in which position 1 of glucose and position 1 of fructose are β-linked.

  Since β-D-glucopyranosyl- (1 → 1) -D-fructose is a bitter-digestible indigestible carbohydrate, it is useful as a composition for food and drink, and a composition for medicine.

  In particular, since it exhibits a bitter taste, it is useful as an active ingredient of a taste improving agent, and since it is a nondigestible carbohydrate, it is useful as an active ingredient of an intestinal flora improving agent.

  In addition, the β-D-glucopyranosyl- (1 → 1) -D-fructose of the present invention has low solubility in water and can be easily crystallized, so a high purity product can be obtained relatively easily. .

It is a figure which shows the mass detector analysis chart of the crystal obtained by manufacture example 1. FIG. It is a figure which shows the chart of the < ¹ > H-NMR spectrum of the crystal obtained by the example 1 of manufacture. It is a figure which shows the chart of the < ¹³ > C-NMR spectrum of the crystal obtained by the example 1 of manufacture. It is a figure which shows the chart of the H-HCOSY spectrum of the crystal obtained by manufacture example 1. FIG. It is a figure which shows the chart of the E-HSQC spectrum of the crystal obtained by manufacture example 1. FIG. It is a figure which shows the chart of the HMBC spectrum of the crystal obtained by manufacture example 1. FIG. Structural formula (β-D-glucopyranosyl- (1 → 1) -β-D-fructo) of β-D-glucopyranosyl- (1 → 1) -D-fructose which is the main component of the sample obtained in Preparation Example 1 (A pyranose type). It is a figure which shows the residual ratio in artificial gastric juice of (beta) -D- glucopyranosyl- (1-> 1) -D-fructose and sucrose. It is a figure which shows the persistence by the digestive enzyme of (beta) -D- glucopyranosyl- (1-> 1) -D-fructose and sucrose. It is a figure which shows the assimilation test of (beta) -D- glucopyranosyl- (1-> 1) -D-fructose, sucrose, and fructooligosaccharide using B. catenulatum JCM 1194. FIG. It is a figure which shows the assimilation test of (beta) -D- glucopyranosyl- (1-> 1) -D-fructose, sucrose, and fructooligosaccharide using B. longum subsp. Longum JCM1217. It is a figure which shows the assimilability test of (beta) -D- glucopyranosyl- (1-> 1) -D-fructose, sucrose, and fructo-oligosaccharide using B. adolecentis JCM1275.

  In the β-D-glucopyranosyl- (1 → 1) -D-fructose (hereinafter also referred to as “Gβ1-1F”) represented by the following formula (1) of the present invention, the 1-position of glucose and the 1-position of fructose are It is a β-linked disaccharide.

  The method for producing Gβ1-1F of the present invention is not particularly limited, and various methods such as enzyme synthesis, chemical synthesis, decomposition, extraction and the like can be used, but D-glucose ( Hereinafter, it is preferable that the enzyme is caused to act on a carbohydrate raw material containing "glucose".) And D-fructose (hereinafter also referred to as "fructose"). The enzyme used in this case is preferably β-glucosidase.

  Glucose and fructose as raw materials are not particularly limited as long as they contain glucose and fructose, and may be an arbitrary mixture of glucose and fructose, for example, a sugar containing glucose such as sucrose and fructose as a constituent sugar It may be produced by degrading the quality, for example, it may be produced by isomerizing glucose or fructose to the other carbohydrate with a catalyst such as an isomerase. As such sugars, it is preferable to use isomerized sugars (glucose fructose sugar, fructose glucose sugar, high fructose liquid sugar) and invert sugar in consideration of availability and cost.

  The mass ratio of glucose to fructose to be used is preferably such that the mass ratio of glucose to fructose is 0.1: 10 to 10: 0.1 in terms of solid content, and more preferably 1:10 to 10: 1. And 1: 4 to 4: 1 are more preferable.

  There is no restriction | limiting in particular as (beta) -glucosidase to be used, The enzyme of various origin can be used. For example, β-glucosidase derived from almond, Aspergillus niger, Trichoderma reesei may be used, or enzymes separated and purified from cultured microorganisms may be used, and commercially available enzymes A formulation may be used. As the enzyme preparation, not only the β-glucosidase preparation but also a cellulase preparation in which β-glucosidase is mixed can be used.

  The conditions for causing β-glucosidase to act on glucose and fructose can be appropriately set in consideration of the optimum conditions of β-glucosidase used and the like. For example, an aqueous solution containing glucose and fructose and having a solid concentration of preferably 30 to 90% by mass, more preferably 50 to 80% by mass, is prepared, and the pH is preferably 4.0 to 8.0, more preferably Is preferably 5.0 to 7.0, and preferably 0.1 to 1000 mg, more preferably 1 to 250 mg, per 1 g of the solid content, preferably 30 to 80 ° C., more preferably 30 to 80 ° C. Can be acted by holding at 50 to 70 ° C., preferably for 1 to 100 hours, more preferably for 24 to 72 hours. The total content of glucose and fructose per solid content of the aqueous solution is preferably 30% by mass or more, more preferably 50% by mass or more, and still more preferably 70% by mass or more.

  The Gβ1-1F-containing composition obtained by causing β-glucosidase to act on glucose and fructose may remain in a liquid state, but can be powdered by a known method such as spray drying.

  In addition, the Gβ1-1F-containing composition can be subjected to a purification treatment such as filtration, decolorization, deodorization, desalting and the like by a conventional method, as necessary. In addition, extraction, centrifugation, crystallization, microbial assimilation, further fractionation by chromatography using activated carbon, porous carrier, hydrophobic resin, hydrophilic resin, ion exchange resin, adsorption resin, etc., dialysis, ultrafiltration, etc. The treatment of membrane fractionation or the like can be performed to increase the purity of G.beta..sub.1-1F.

  The Gβ1-1F of the present invention may be in a crystalline form (in a crystallized state). There is no particular limitation on the method of crystal formation, and methods such as temperature reduction, concentration, solvent addition (ethanol, methanol, acetone, etc.) and the like can be mentioned. In particular, Gβ1-1F can be easily crystallized by cooling and / or concentrating a Gβ1-1F-containing composition because the solubility in an aqueous solution is low. Thus, by crystallizing Gβ1-1F, it is possible to relatively easily obtain Gβ1-1F having a high purity.

  The Gβ1-1F of the present invention can also be used as a highly purified product, but can also be used as a sugar composition containing Gβ1-1F. In this case, the content of Gβ1-1F contained in the sugar composition is not particularly limited, but is preferably, for example, 0.5% by mass or more per solid content, and more preferably 1% by mass or more Preferably, it is more preferably 2% by mass or more, and most preferably 3% by mass or more. If the content of Gβ1-1F per solid content is less than 0.5% by mass, there is a disadvantage that the characteristics of Gβ1-1F can not be sufficiently imparted.

  Gβ 1-1 F is mainly present as β-D-glucopyranosyl- (1 → 1) -β-D-fructopyranose, but as described later, the structure changes via a chain-like structure, β-D- Glucopyranosyl- (1 → 1) -α-D-fructopyranose, β-D-glucopyranosyl- (1 → 1) -α-D-fructofuranose, and β-D-glucopyranosyl- (1 → 1) -β It is also present in trace amounts as D-fructofuranose.

  Gβ1-1F of the present invention, or a sugar composition containing the same can be used as a composition for food and drink. There is no restriction | limiting in particular as food-drinks used as object, All food-drinks which can be eaten can be mentioned. For example, the following food and drink are mentioned.

Non-alcoholic beverages (fruit juice-containing beverages, fruit juices, vegetable juices, carbonated beverages, isotonic beverages, amino acid beverages, sports beverages, coffee, coffee, coffee beverages, cocoa beverages, tea beverages, lactic acid bacteria beverages, milk beverages, nutritional drinks, non-alcoholic beverages Beverages such as beer, non-alcohol chuihi, non-alcoholic cocktail, near water, flavored water, etc., alcoholic beverages (beer, low-malt beer, liqueur, chuihai, sake, wine, fruit liquor, cocktail, distilled liquor etc.) ・ Ice cream Iced candy, sherbet, shaved ice, flappe, frozen yogurt, jelly, pudding, bavaroa, frozen sweet potato such as water gourd-Water syrup, pickled syrup, ice syrup, chocolate syrup, caramel syrup syrup, etc.-Flower pe Pastes such as peanut paste, fruit paste, butter cream, custard cream, etc.-Jams such as marmalade, fruit sauce, blueberry jam, strawberry jam, etc.-Bread such as bread, rolls, brioche, steamed bread, bean paste, cream bread, etc.・ Biscuits, crackers, cookies, waffles, muffins, sponge cakes, pies, etc. • Creams such as puffs, donuts, chocolates, chewing gum, caramel, nougat, candy such as confections • Rice crackers, hail, scalding, fertilizers, potatoes , Manju, Daifuku, Uiro, Japanese sweets such as jasper, castella, jasper, soy sauce, fish sauce, fish paste, miso, hiyashi, mayonnaise, dressing, three cups vinegar, tempura soup, noodle soup, worcester sauce, oyster sauce, Chap, grilled chicken sauce, grilled pork sauce, pickled sauce, sweet potato, spice, vinegar, sushi vinegar, calairue, Chinese ingredients, stew ingredients, soup ingredients, dumpling ingredients, combined seasonings, mirin, new mirin, Table salt, various condiments such as table sugar ・ Pasta sauce, meat sauce, tomato sauce, white sauce, demi-grass sauce, curry sauce, curry sauce, hayashi sauce, gravy sauce, hamburger sauce, salsa sauce, sauces such as steak sauce ・ Pickles pickles, pickle pickles , Pickled miso pickles, Fukujin pickles, pickled radish pickles, Nara pickles, Senzuke pickles, pickled plums etc.-Pickled mackerel raw materials, Chinese cabbage pickled mackerel raw materials, pickled vegetables such as kimchi raw material · Ham, bacon, sausage, hamburg, meatballs etc Meat products of meat ・ Fish meat ham, fish meat sausage, kamaboko, chikuwa, dried fish Fish products such as sea urchins, sea urchins, sea urchins, sea urchins, salted radish, dried vinegar kelp, seaweed radish, delicacies such as rice bran ・ Nori seaweed, wild vegetables, sea urchins, sea urchin, small fish, shellfish etc. , Boiled fish, potato salad, sugar beet food such as kelp roll ・ Dairy products, fish meat, meat meat, fruit, vegetables and other bottling products such as canned foods ・ Tempura, ton cutlet, fritters, fried food such as fried chicken, fried Tatsuta ・ Udon noodles Noodles such as buckwheat noodles, Chinese noodles, pasta, vermicelli, rice vermicelli, eggplant skin, chumeyem skin ・ Pudding mix, hot cake mix, instant juice, instant coffee, instant soup powder, instant food such as instant soup.

  Moreover, food and drink include food for specified health use, nutritive function food, food for elderly people, special purpose food, functional food, health supplement (supplement) and the like. The form of the food and drink in this case can be, for example, a powder form, a granular form, a tablet form, a capsule form, or a liquid form.

  The additive amount of the composition for food and drink according to the present invention to food and drink is preferably 0.002% by mass or more as Gβ1-1F, and may be 0.02% by mass or more. More preferably, it is more preferably 0.05% by mass or more, particularly preferably 0.1% by mass or more. If the addition amount as Gβ1-1F is less than 0.002% by mass, the characteristics of Gβ1-1F may not be sufficiently imparted.

  In addition, Gβ1-1F of the present invention or a sugar composition containing the same can be used as a pharmaceutical composition because a physiologically active effect described later is expected. That is, it can be used as a pharmaceutical raw material for producing the physiologically active effect described later. The medicine in this case is preferably a medicine for oral administration, but can also be applied to a medicine for external use. Specifically, as a dosage form of a pharmaceutical preparation for oral administration, a powder, a solid particle, a tablet, a capsule, a troche etc. as a solid preparation, and an internal solution, external solution, suspension as a liquid preparation , Emulsions, syrups and the like.

  The pharmaceutical composition of the present invention includes, in addition to Gβ1-1F, pharmaceutically acceptable common carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrants, solubilizers, You may contain suitably the mixing | blending component for various preparations, such as a solubilizing agent and an isotonic agent.

  The amount of the pharmaceutical composition of the present invention to be added to the pharmaceutical is preferably 0.02% by mass or more as Gβ1-1F, more preferably 0.1% by mass or more It is more preferable to be 0.2 mass% or more, and it is particularly preferable to be 1.0 mass% or more. If the addition amount as Gβ1-1F is less than 0.02% by mass, it may not be possible to sufficiently impart the characteristics of Gβ1-1F.

  In addition, Gβ1-1F can be used as a taste improving agent for food and drink or pharmaceuticals because Gβ1-1F has a bitter taste. The taste improver of the present invention is added to foods, foods and medicines to impart and enhance richness, impart and enhance texture, impart and enhance a sense of fruit juice, impart and enhance a taste of milk, and reduce ag It is expected to exert taste improving action such as taste improvement of high sweetness degree sweeteners. Further, the taste improver of the present invention can be used, for example, as a bitter taste imparting agent because it exhibits a bitter taste. In addition, as food-drinks applied, the various food-drinks which were mentioned above are mentioned. It is preferable to make it the addition amount to the food-drinks or the pharmaceutical as a taste improving agent be the addition amount as mentioned above as G (beta) 1-1F.

  In addition, as shown in Examples described later, Gβ1-1F has assimilability to Bifidobacterium more than or equal to fructooligosaccharide known as a prebiotics material, and thus Gβ1-1F of the present invention, Alternatively, the sugar composition containing it can be used as an intestinal flora improving agent, that is, a medicine for improving intestinal flora, food and drink. Here, the food and drink include food for specified health use, nutritive function food, food for elderly people, special purpose food, functional food, health supplement (supplement) and the like.

  In this case, the effective dosage of the agent for improving intestinal flora of the present invention is not particularly limited, but the dosage of Gβ1-1F is preferably 0.5 to 30 g per adult, and more preferably 2 to 10 g.

  Gβ 1-1 F is produced by the action of β-glucosidase on a raw material containing glucose and fructose which are nutritional components of humans and animals, so it has safety for humans and animals. There is.

  In addition, as shown in the examples described later, since Gβ 1-1 F is a nondigestible carbohydrate, it can not only be used for food and drink or medicine as a low-calorie material for diets, but also for fat accumulation suppression It can be used for food and drink and medicine for obesity improvement and / or prevention, metabolic syndrome treatment and / or prevention, etc.

  Furthermore, Gβ 1-1 F, like indigestible oligosaccharides, is an intestinal adjusting agent, a bowel movement improving agent, a mineral absorption promoter, an intestinal barrier function enhancer, a lipid metabolism improving agent, and the like because it promotes the growth of enterobacteria. It is expected to exert functions as an immunomodulator and an inhibitor of blood sugar elevation.

  The composition for food and drink, the pharmaceutical composition, the taste improving agent, and the agent for improving intestinal flora according to the present invention are other than G beta 1-1 F, as long as the effects of the present invention are not impaired. It may contain sugar. Other sugars may be monosaccharides or disaccharides or higher. In addition, it contains carbohydrates derived from raw materials (glucose sources used as raw materials, fructose sources, saccharides produced by the decomposition of glucose sources and fructose sources, saccharides produced by reaction of these saccharides, etc.) It may be.

  In the present invention, peak components detected at retention times of 37.8 minutes, 36.2 minutes and 34.5 minutes under the HPLC conditions shown in the following examples were judged as disaccharide components. In addition, the sugar composition in the present invention means the sugar composition (content of each saccharide (mass%)) per solid content.

<Production Example 1>
D-glucose (product name: anhydrous crystalline glucose, manufactured by Nippon Shokuhin Kako Co., Ltd.) (hereinafter referred to as "glucose"), D-fructose (manufactured by Nacalai Tesque, Inc.) (hereinafter referred to as "fructose"), And a mixture of equal weights of glucose and fructose were dissolved in ultrapure water so as to be 75 w / w% each. 1 M sodium acetate buffer (pH 5.0) was added to 10 mM. 14 mg / g-ds of beta-glucosidase (made by Toyobo Co., Ltd.) was added under 66 degreeC, and it was made to react, hold | maintaining for 72 hours. The sugar composition in the sample solution after each retention time was analyzed by HPLC.

  In HPLC analysis, two columns of column (300 mm × 8.0 mm ID) (product name: ULTRON PS-80 N. L, manufactured by Shinwa Kako Co., Ltd.) were connected, and the column temperature was set to 50 ° C. The solution was passed at 5 mL / min, and detection was performed using a differential refractive index detector (manufactured by Shimadzu Corporation).

  The results are shown in Table 1.

  When glucose was used as a carbohydrate source, the disaccharides at 34.5 minutes, the trisaccharides at 31.5 + 30.7 minutes, and the tetrasaccharide or higher peaks at 30 minutes earlier than 30 minutes increased. When fructose was used as a carbohydrate source, the sugar composition did not change substantially before and after the reaction. Similar to glucose, glucose and fructose, when using equal amounts of carbohydrates, respectively, are disaccharides at a retention time of 34.5 minutes, trisaccharides at 31.5 + 30.7 minutes, and four hours earlier than 30 minutes. The peaks above the sugar increased, and the peaks at the retention times 36.2 minutes and 37.8 minutes also increased.

  The sample solutions obtained using equal amounts of glucose and fructose as carbohydrate raw materials, respectively, were used as samples for the subsequent tests.

(1) Fractionation treatment Fractionation of the sample was performed by carbon-celite column chromatography. A slurry of activated carbon "purified white startle" (product name, manufactured by Osaka Gas Chemical Co., Ltd.) and "Celite 545" (product name, manufactured by Kanto Chemical Co., Ltd.) in equal weights and suspended in water is a glass column ( Model No .: XK-26 / 40 (manufactured by Pharmacia) (φ2.6 × 40 cm) was loaded, to which a 10 mL sample of Bx50 was loaded. The chromatography was performed at room temperature and the flow rate was 5 mL / min. First, ultra pure water was passed through to elute monosaccharides, and then 1.5 v / v% ethanol and 3.0 v / v% ethanol were passed through to elute unknown peaks and disaccharides. After confirming that disaccharide elution was complete, 50 v / v% ethanol was passed through to elute the trisaccharide or more sample.

  The eluate after passing through 1.5 v / v% ethanol was fractionated by a fixed amount, and concentrated to about B x 40 with a rotary evaporator (manufactured by Tokyo Rika Kikai Co., Ltd.).

  The sugar composition of the sample was analyzed by HPLC as described above, and the results are shown in Table 2.

  When the concentrated solution of the sample was stored overnight at 4 ° C., Fraction No. 3 containing a large amount of eluate having a retention time of 36.2 minutes. The formation of white crystals was confirmed in 1 to 3. As a result of analysis by HPLC, the white crystals were mainly composed of an unknown substance of disaccharide having a retention time of 36.2 minutes.

  The crystals of fraction No. 3 were redissolved in pure water, concentrated to about B x 40, and stored again overnight at 4 ° C. When crystals were formed again, the purity of the main component was improved. The purity was 99.3% by repeating the same operation again (Table 3).

(2) Identification of Crystals As a result of analyzing the obtained crystals with a mass detector (product name: 5610 mass detector, manufactured by Hitachi High-Tech Science), it has a molecular weight of 365 (subtracting 23 of sodium) 342, disaccharide It has been confirmed that there is (Figure 1).

Next, the crystals were analyzed using a 400 MHz nuclear magnetic resonance spectrometer (NMR) (product name: Ultrashield 400 PLUS, manufactured by BRUKER). The measurements were performed with ¹ H (FIG. 2) and ¹³ C (FIG. 3) as ^one dimension, H-HCOSY (FIG. 4), E-HSQC (FIG. 5) and HMBC (FIG. 6) as two dimensions (table 4). The analysis was carried out by dissolving the crystals in heavy water. A small amount of trimethylsilylpropanoic acid was added as an internal standard substance, and the peak derived from the substance was made 0 ppm.

  As a result of analysis and analysis, the main component of the sample was β-D-glucopyranosyl- (1 → 1) -β-D-fructopyranose (FIG. 7). In spite of the high purity sample, multiple minute peaks were confirmed in the NMR data. This is because β-D-glucopyranosyl- (1 → 1) -β-D-fructopyranose undergoes a ring-opening structure, β-D-glucopyranosyl- (1 → 1) -α-D-fructopyranose, β- It was considered that D-glucopyranosyl- (1 → 1) -α-D-fructofuranose, β-D-glucopyranosyl- (1 → 1) -β-D-fructofuranose is an isomer. Therefore, it was revealed that this sample is Gβ1-1F having β-D-glucopyranosyl- (1 → 1) -β-D-fructopyranose as a main structure.

(3) Confirmation of Taste Quality Gβ1-1F was dissolved in ultrapure water so as to be 10 w / w%, and when the taste quality was confirmed, a bitter taste was felt. That is, it turned out that G (beta) 1-1F is a carbohydrate which has a bitter taste.

(4) Artificial gastric fluid test The decomposition of Gβ 1-1 F by artificial gastric fluid was confirmed.

  The sample was dissolved in 16.7 mM HCl-KCl buffer (pH 2.0) to be 0.73 w / v% and kept at 37 ° C. The sample was sampled at 0, 30, 60 and 120 minutes, desalted by adding an ion exchange resin (product name: Amberlite MB4, Organo Corporation), filtered through a 0.45 μm filter, and analyzed by HPLC. The obtained peak area was divided by the peak area at 0 minutes of reaction, and the residual rate was calculated by multiplying by 100.

  For HPLC analysis, using a column (300 mm × 8.0 mm ID) (product name: ULTRON PS-80 N. L, manufactured by Shinwa Kako Co., Ltd.) and setting the column temperature to 50 ° C., the flow rate of ultra pure water is 0.9 mL The liquid was allowed to flow at / min, and detection was performed using a differential refractive index detector (manufactured by Shimadzu Corporation).

  From this result, it was found that Gβ1-1F was not degraded by artificial gastric juice as well as sucrose (FIG. 8).

(5) Digestibility test The digestibility by the digestive enzyme of G (beta) 1-1F was confirmed.

  After suspending 2 g of rat small intestine acetone powder (trade name, Sigma) in 20 mL of 45 mM sodium maleate buffer (pH 6.6), the supernatant was recovered by centrifugation (19000 g, 10 minutes). The activity of the resulting solution was measured and used as a digestive enzyme solution in the following in vitro digestibility test.

The activity of the digestive enzyme solution was measured as maltase activity. 40 μL of 0.1 M sodium acetate buffer (pH 6.0) was added to 15 μL of the digestive enzyme solution appropriately diluted with pure water, and kept at 37 ° C. The reaction was initiated by adding 45 μL of a 2 w / v% maltose solution to this. After 10 minutes, the reaction was stopped by adding 200 μL of 2 M Tris-HC1 buffer (pH 7.0), and adding 80 μL of glucose C-II Test Wako (product name, Wako Pure Chemical Industries, Ltd.) to 37 ° C. The color was maintained by holding for about 30 minutes. A ₄₉₂ was measured, and the amount of free glucose was calculated based on the glucose standard curve.

  A standard curve of glucose was prepared by adding 2 M Tris-HCl buffer solution (pH 7.0) and a coloring reagent to 100 μL of 0 to 0.01 w / v% glucose aqueous solution in the same manner as described above. The enzyme activity of 1 U was defined as the amount of enzyme that produces 2 μmol of glucose per minute under the above conditions.

  The sample is dissolved in 45 mM sodium maleate buffer (pH 6.6) to a final concentration of 0.45 w / v%, and the digestive enzyme solution prepared by the above method is added to give 86 U / g-ds, Hold at 37 ° C. The reaction was stopped by mixing 20 μL of the reaction solution with 200 μL of 2 M Tris-HC1 buffer, and sampling was appropriately performed. The glucose amount of the sampled solution was measured by the glucose oxidase method using a glucose C-II test Wako. The decomposition rate was defined as glucose mass × 2 / substrate mass × 100.

  As a result of the test, sucrose was rapidly degraded while Gβ1-1F was not degraded at all, which revealed that Gβ1-1F is an indigestible carbohydrate (FIG. 9).

(6) Assimilation test Test strains B. catenulatum JCM 1194, B. longum subsp. Longum JCM 1217, and B. adolecentis JCM 1275 (all are strains of Bifidobacterium (Bifidobacterium), JCM number is national Anaerobic jar and aneropeque / kenk (both product names, in the IL medium with glucose added at a final concentration of 15% as the sole carbon source, using R & D institute RIKEN BioResource Center, which is the deposit number of the Microbial Materials Development Office) The preculture was performed at 37 ° C. for 24 hours under anaerobic conditions using Mitsubishi Gas Chemical Co., Ltd.). Then, 1 v / v% of the preculture liquid is inoculated in 3.0 mL of ILS medium to which Gβ1-1F or fructo-oligosaccharide (Wako Pure Chemical Industries, Ltd.) is added to a final concentration of 1%, respectively, 37 ° C., anaerobic Culture was performed under conditions. As a negative control, sterilized ultrapure water was added instead of the carbon source. The assimilability is determined by bacterial growth, and the bacterial turbidity (OD 660) is measured using a simple OD monitor "mini photo 518R" (product name, manufactured by TAITEC) over time, and a growth curve is drawn. The

  In the assimilability test using B. catenulatum JCM 1194, the assimilability of Gβ1-1F was similar to fructooligosaccharide (FIG. 10). In the assimilability test using B. longum subsp. Longum JCM 1217, fructooligosaccharide showed assimilability, whereas Gβ1-1F showed little assimilation (FIG. 11). In the assimilability test using B. adolecentis JCM1275, the assimilability of Gβ1-1F showed higher assimilability than fructooligosaccharide (FIG. 12).

  Therefore, Gβ 1-1 F has assimilability to Bifidobacterium more than or equal to fructooligosaccharide known as a prebiotics material, suggesting the possibility that it can be used as prebiotics. In addition, some strains did not show assimilability, suggesting that specific bacterial species can be selectively grown.

<Production Example 2>
Adjust the pH of the cellulase preparation “Sumiteam ACL” (product name, Shin Nippon Chemical Industry Co., Ltd.) to 128 mg / g − Add ds, hold for 72 hours and let react.

  Thereafter, the same fractionation as described above was performed to obtain white crystals. As a result of analyzing the obtained crystals by HPLC, it was the same retention time as Gβ1-1F.

  Therefore, this method was also able to produce Gβ1-1F.

Claims (8)

Β-D-glucopyranosyl- (1 → 1) -D-fructose represented by the following formula (1).

  The crystalline β-D-glucopyranosyl- (1 → 1) -D-fructose according to claim 1. The composition for food-drinks characterized by containing (beta) -D- glucopyranosyl- (1-> 1) -D-fructose shown by following formula (1).

  The composition for food and drink according to claim 3, containing 0.5% by mass or more of the β-D-glucopyranosyl- (1 → 1) -D-fructose. A pharmaceutical composition comprising β-D-glucopyranosyl- (1 → 1) -D-fructose represented by the following formula (1).

  The pharmaceutical composition according to claim 5, which contains 0.5% by mass or more of the β-D-glucopyranosyl- (1 → 1) -D-fructose. The taste improving agent characterized by containing (beta) -D- glucopyranosyl- (1-> 1) -D-fructose shown by following formula (1) as an active ingredient.

The intestinal flora improvement agent characterized by containing (beta) -D-glucopyranosyl- (1-> 1) -D-fructose shown by following formula (1) as an active ingredient.

Images