JP2013059327A - METHOD OF PRODUCING OLIGOSACCHARIDE HAVING ALDONIC ACID RESIDUE AT REDUCING TERMINAL AND HAVING α1→6 GLUCOSIDE BOND OR β1→6 GLUCOSIDE BOND - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method which safely and easily oxidizes a hemiacetal hydroxyl group of oligosaccharides having an α1→6 glucoside bond or a β1→6 glucoside bond using highly safe microorganism cells to efficiently produce oligosaccharides having aldonic acid at the corresponding reducing terminal.SOLUTION: Cells of a microorganism belonging to acetic acid bacteria are brought into contact with oligosaccharides having a hemiacetal hydroxyl group and having an α1→6 glucoside bond or a β1→6 glucoside bond, whereby oligosaccharides having aldonic acid at the corresponding reducing terminal can be safely and efficiently produced.

Description

  The present invention relates to a method for producing an oligosaccharide having an aldonic acid residue at the reducing end and having an α1 → 6 glucoside bond or a β1 → 6 glucoside bond using a microbial cell belonging to an acetic acid bacterium.

  Conventionally, it is known that lactobionic acid, which is a disaccharide having an aldonic acid residue at the reducing end, has bifidobacteria selective growth activity (Patent Document 1) and has a mineral absorption promoting effect (Patent Document 2). Yes. On the other hand, isomaltobionic acid (α-D-glucopyranosyl- (1 → 6) -D-gluconic acid), isomaltoronic acid (α-D-glucopyranosyl- (1 → 6) -α-D-glucopyranosyl- (1 → 6) -D-gluconic acid), panotrionic acid α-D-glucopyranosyl- (1 → 6) -α-D-glucopyranosyl- (1 → 4) -D-gluconic acid), gentiobionic acid (β-D-glucopyranosyl) -(1 → 6) -D-gluconic acid) and other oligosaccharides having an aldonic acid residue at the reducing end and having a 1 → 6 glucoside bond are also known, such as food, medicine, cosmetics and feed Has been found to be useful as an additive used in

  As a specific method for obtaining an oligosaccharide having an aldonic acid residue at the reducing end and having an α1 → 6 or β1 → 6 glucoside bond, such as isomaltobionic acid, an oligosaccharide having a hemiacetal hydroxyl group is odorized. A method of applying electricity together with sodium bromide and a chemical oxidation method using bromine water (hydrobromic acid) are known. In addition, it has been reported that by using the cells of microorganisms belonging to the genus Pseudomonas, it can be obtained by oxidizing disaccharides such as lactose and maltose (Non-patent Document 1). It has also been reported that isomaltobionic acid can be produced from isomaltose by using the body (Non-patent Document 2).

  However, an oligosaccharide obtained by chemical synthesis or prepared by a microorganism belonging to the genus Pseudomonas, which is a pathogen, has an aldonic acid residue at the reducing end and has an α1 → 6 or β1 → 6 glucoside bond. It could not be used for food or feed applications.

  On the other hand, acetic acid bacteria, which are microorganisms belonging to the genus Acetobacter, Gluconobacter, and Gluconacetobacter, which are known as bacteria that oxidize carbohydrates, include vinegar, Since it is widely used for food production such as Nata de Coco, it is considered suitable for the production of oligosaccharides having an aldonic acid residue at the reducing end for food and feed applications. However, although these acetic acid bacteria can oxidize monosaccharides relatively efficiently, it is known that it is difficult to oxidize disaccharides such as lactose and maltose. Or, even if oxidized, only disaccharides having a β1 → 4 glucoside bond are known as substrates that can be oxidized. Furthermore, it has been reported that the reaction efficiency (reaction rate) is as low as about 10% or less of glucose (Non-Patent Documents 3 and 4).

  In addition, a method is known in which acetic acid bacteria are used to oxidize a hemiacetal hydroxyl group of a disaccharide such as lactose to produce a disaccharide having an aldonic acid residue at the reducing end, such as lactobionic acid ( For example, see Patent Documents 3 and 4). Since the disaccharide having an aldonic acid residue at the reducing end prepared by these methods is produced using acetic acid bacteria, it is suitable for use in food and feed applications.

  However, in the case of Patent Documents 3 and 4, oligosaccharides having an aldonic acid residue at the reducing end that can be produced include lactobionic acid, cellobionic acid, maltobionic acid, malto having α1 → 4 or β1 → 4 glucoside bond. Only triionic acid, maltotetraionic acid, maltopentanoic acid, and melionic acid are described. Furthermore, there are no reports on the production of oligosaccharides having an aldonic acid residue at the reducing end and an α1 → 6 or β1 → 6 glucoside bond using acetic acid bacteria. The oligosaccharide having an α1 → 4 or β1 → 4 glucoside bond and the oligosaccharide having an α1 → 6 or β1 → 6 glucoside bond are greatly different in steric structure, and accordingly, the reactivity by the enzyme is also greatly different. Therefore, at present, it cannot be inferred at all whether acetic acid bacteria can oxidize the hemiacetal hydroxyl group of an oligosaccharide having an α1 → 6 or β1 → 6 glucoside bond.

  Thus, development of a technique for efficiently producing an oligosaccharide having an aldonic acid residue at the reducing end and having an α1 → 6 or β1 → 6 glucoside bond is desired using a highly safe microorganism. However, the actual situation is that even the specific method has not been found from the prior art.

Japanese Patent No. 3559063 Japanese Patent No. 3501237 JP 2007-28917 A JP 2008-173065 A

Agric. Biol. Chem. 44 (7), 1505-1512 (1980) Journal of Bacteriol. Vol99, p.623 (1969) Agric. Biol. Chem. 45 (4), 851-861 (1981) J. Dairy Sci. 92, 25-34 (2009)

  Therefore, in view of these problems, an object of the present invention is to oxidize hemiacetal hydroxyl groups of oligosaccharides having an α1 → 6 or β1 → 6 glucoside bond safely and easily using a highly safe microorganism. Thus, it is to provide a method for efficiently producing an oligosaccharide having an aldonic acid residue at the reducing end.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have confirmed that safety is ensured by oligosaccharides having a hemiacetal hydroxyl group and having an α1 → 6 or β1 → 6 glucoside bond. It was found that an oligosaccharide having an aldonic acid residue at the reducing end can be produced safely and efficiently by contacting cells of a microorganism belonging to acetic acid bacteria.

  Furthermore, from the prior art, among the sugars listed in the examples of Patent Documents 3 and 4, lactose is one of the oligosaccharides that are most efficiently oxidized by the cells of microorganisms belonging to acetic acid bacteria. It is thought that there is. The present inventors compared the action of acetic acid bacteria on this lactose and the action of acetic acid bacteria on isomaltose and gentiobiose. As a result, it was found that the oxidation reaction efficiency of isomaltose and gentiobiose was 15 to 240 times or more that of lactose. And it is found that isomaltobionic acid and gentiobionic acid can be produced more efficiently by the cells of microorganisms belonging to acetic acid bacteria than any sugar described in Patent Documents 3 and 4, including lactobionic acid. It was.

The present invention has been completed by further studies based on these findings. That is, this invention provides the invention of the aspect hung up below.
(1) A microbial cell belonging to acetic acid bacteria is brought into contact with an oligosaccharide having a hemiacetal hydroxyl group and having an α1 → 6 or β1 → 6 glucoside bond, and the hemiacetal hydroxyl group of the oligosaccharide is oxidized. A method for producing an oligosaccharide having an aldonic acid residue at the reducing end.
(2) The method according to (1), wherein the microorganism belonging to the acetic acid bacterium is a microorganism belonging to the genus Acetobacter, Gluconobacter or Glucon acetobacter.
(3) The production method of (1) or (2), wherein the contact of the bacterial cells with the oligosaccharide is performed under a condition where the D-glucose concentration is 3% by mass or less.
(4) The production method according to any one of (1) to (3), wherein the oligosaccharide is isomaltose and / or isomaltotriose.
(5) The oligosaccharide is an isomaltoligosaccharide containing 2 or more types selected from the group consisting of isomaltose, isomaltotriose, isomalttetraose, and isomaltopentose. 3) Any manufacturing method.
(6) The production method of any one of (1) to (3), wherein the oligosaccharide is gentiobiose and / or gentiotriose.
(7) The method according to any one of (1) to (3), wherein the oligosaccharide is panose.
(8) Obtained by performing acetic acid fermentation by adding acetic acid bacteria to a fermentation raw material containing an oligosaccharide having a hemiacetal hydroxyl group and having an α1 → 6 or β1 → 6 glucoside bond and ethanol. A brewed vinegar containing an oligosaccharide having an aldonic acid residue at the reducing end.
(9) Acetic acid fermentation is performed by adding acetic acid bacteria to a fermentation raw material containing an oligosaccharide having a hemiacetal hydroxyl group and having an α1 → 6 or β1 → 6 glucoside bond and ethanol. And a method for producing a brewed vinegar containing an oligosaccharide having an aldonic acid residue at the reducing end.

  According to the present invention, the oligosaccharide having an α1 → 6 or β1 → 6 glucoside bond is used at the reducing end by using a microbial cell belonging to an acetic acid bacterium that has been used for food production and has been secured for a long time. An oligosaccharide having an aldonic acid residue can be produced safely and efficiently.

  Furthermore, when the D-glucose concentration is sufficiently reduced when contacting the cells of microorganisms belonging to acetic acid bacteria to an oligosaccharide having an α1 → 6 or β1 → 6 glucoside bond used as a raw material, It is possible to dramatically increase the production efficiency of oligosaccharides having an aldonic acid residue at the reducing end.

Isomaltose, isomaltotriose, panose, gentibiose and gentitriose are oxidized by the oxidation of the hemiacetal hydroxyl group, respectively, and isomaltobionic acid, isomaltorionic acid, panotriionic acid, gentiobionic acid and gentiotriionic acid, respectively. It is a figure which shows that is produced | generated. In Example 3, the reaction liquid using Acetobacter orientalis FERM P-21150 was subjected to TLC to confirm the presence or absence of isomaltobionic acid and lactobionic acid. In Example 7, it is the result of having confirmed the presence or absence of the production | generation of panotrionic acid, isomaltobionic acid, and lactobionic acid by using for TLC the reaction liquid using Luconobacter cerinus NBRC3267. In Example 5, it is the result of having analyzed the reaction liquid of Gluconobacter fratauri IFO3285 obtained in the preparation of the acetic acid microbial cell with a mass spectrometer. In Example 12, it is the result of measuring the amount of isomaltobionic acid produced | generated when Gluconobacter fratauri IFO3285 is made to contact with isomaltose in presence of each glucose density | concentration.

  The method for producing an oligosaccharide having an aldonic acid residue at the reducing end according to the present invention comprises a method for producing a microorganism belonging to acetic acid bacteria to an oligosaccharide having a hemiacetal hydroxyl group and having an α1 → 6 or β1 → 6 glucoside bond. It is characterized by contacting the body and oxidizing the hemiacetal hydroxyl group of the oligosaccharide. Hereinafter, the present invention will be described in detail. Hereinafter, in the present specification, “oligosaccharide having a hemiacetal hydroxyl group and having an α1 → 6 or β1 → 6 glucoside bond” is abbreviated as “raw oligosaccharide” and “residual end of aldonic acid residue”. The “oligosaccharide having a group and having an α1 → 6 glucoside bond or β1 → 6 glucoside bond” may be abbreviated as “target oligosaccharide”.

Raw material oligosaccharide and target oligosaccharide In the present invention, an oligosaccharide (raw material oligosaccharide) having a hemiacetal hydroxyl group and having an α1 → 6 or β1 → 6 glucoside bond is used as a raw material. Although it does not restrict | limit especially about the number of monosaccharides (monomer number) which comprises this raw material oligosaccharide, For example, 2-5, Preferably 2-3 are mentioned. Examples of the oligosaccharide having an α1 → 6 glucoside bond include isomaltose, isomaltotriose, isomalttetraose, isomaltopentose, isomaltoligosaccharide, panose and the like. Examples of the oligosaccharide having a β1 → 6 glucoside bond include gentiobiose and gentiotriose. Panose is a trisaccharide in which three molecules of D-glucose are bound by one α1 → 4 glucoside bond and one α1 → 6 glucoside bond. Among these raw material oligosaccharides, isomaltose and gentiobiose are preferable from the viewpoint of more efficiently producing the target oligosaccharide. These oligosaccharides may be used individually by 1 type, and may be used in combination of 2 or more type.

  Here, the isomalt-oligosaccharide includes two or more kinds selected from isomaltose, isomalttriose, isomalttetraose and isomaltopentose as components, and isomaltoligosaccharide having a longer chain length than those. May be included. The target oligosaccharide produced by using the isomaltoligosaccharide is an oligosaccharide having an aldonic acid residue at the reducing end corresponding to the isomaltoligosaccharide, and isomaltobionic acid, isomalttrionic acid, and isomalt It contains two or more selected from tetraonic acid and isomaltopentanic acid. The production method of isomalto-oligosaccharide used as a raw material includes α-glucosidase transfer reaction, dextrin / dextranase hydrolysis reaction, or dextran sucrase transfer reaction. Is not to be done.

  In the present invention, the hemiacetal hydroxyl group of the raw oligosaccharide used as the raw material is oxidized, and the carbon atom to which the hemiacetal hydroxy group is bonded is converted to a carboxyl group, and the target oligosaccharide corresponding to the raw oligosaccharide is obtained. Generate. Specifically, according to the present invention, as shown in FIG. 1, for example, it has an α1 → 6 or β1 → 6 glucoside bond represented by isomaltose, isomaltotriose, isomaltoligosaccharide, panose or gentiobiose. By oxidizing the hemiacetal hydroxyl group of the oligosaccharide, it is possible to produce isomaltobionic acid, isomaltrionic acid, isomaltoligoaldonic acid, panotrionic acid, or gentiobionic acid, respectively.

  According to the present invention, the target oligosaccharide is produced from the raw oligosaccharide under the action of acetic acid bacteria, and the aldonic acid residue of the produced oligosaccharide is converted into a salt form depending on the reaction conditions. Or a carboxyl group and a hydroxyl group may be dehydrated and condensed to form a lactone. That is, the target oligosaccharide produced by the present invention includes only oligosaccharides having an aldonic acid residue at the free reducing end, such as isomaltobionic acid, isomaltrionic acid, isomaltoligoaldonic acid, panotrionic acid, and gentiobionic acid. Or a salt of an oligosaccharide having an aldonic acid residue at the reducing end, such as isomaltobionate, isomalttrionate, isomaltoligoaldonate, panotrionate, gentiobionate, or isomaltobionolactone Lactones such as isomaltrionolactone, isomaltoligoaldonolactone, panotrionolactone, gentiobionolactone, and the like.

In the present invention, it is essential to use a microbial cell belonging to an acetic acid bacterium. In the prior art, it has not been known that the cells of microorganisms belonging to acetic acid bacteria oxidize oligosaccharides (raw oligosaccharides) having α1 → 6 or β1 → 6 glucoside bonds. Examples of the microorganisms belonging to the acetic acid bacteria include, for example, microorganisms belonging to the genus Acetobacter, Gluconobacter, Gluconacetobacter, Adiciphyllum, Acidmonam, and mutants and mutants of the microorganisms. Is mentioned.

  As the microorganisms belonging to the genus Acetobacter, specifically, Acetobacter pasteurianus, Acetobacter aceti, Acetobacter orientalis, Acetobacter indonesiensis Acetobacter cibinongensis and the like. Among these Acetobacter genera, Acetobacter pasterianus, Acetobacter aceti, and Acetobacter indonesiensis are preferable.

  Specific examples of microorganisms belonging to the genus Gluconobacter include Gluconobacter oxydance, Gluconobacter sphaericus, Gluconobacter frateurii, Gluconobacter frateurii, Gluconobacter frateurii, Selinus (Gluconobacter cerinus), Gluconobacter asaii, Gluconobacter thailandicus, Gluconobacter roseus, Gluconobacter roseus (Gluconobacter albidus) It is done. Among these genus Gluconobacter, preferably Gluconobacter oxydans, Gluconobacter sphaericus, Gluconobacter frateuri, Gluconobacter acai, Gluconobacter selinus, Gluconobacter albidas; More preferred are Gluconobacter oxydans, Gluconobacter sphaericus, and Gluconobacter acai.

  Specific examples of microorganisms belonging to the genus Gluconacetobacter include Gluconacetobacter xylinus, Gluconacetobacter hansenii, Gluconacetobacter liquefacience, and the like. . Of these genus Gluconacetobacter, Gluconacetobacter hanseni and Gluconacetobacter xylinus are preferable.

  Specific examples of microorganisms belonging to the genus Adisiphilum include Acidiphilium angustum, Acidiphilium organovorum, Acidiphilium cryptum, Acidiphilum rubrum, and Acidiphilium rubrum multivorum).

  Specific examples of microorganisms belonging to the genus Acidmonam include Acidomonas methanolicus.

  Among these microorganisms belonging to acetic acid bacteria, from the viewpoint of more efficiently producing the target oligosaccharide, preferably Acetobacter, Gluconobacter, Gluconacetobacter; more preferably Gluconobacter, Glucon Acetobacter genus; more preferably Gluconobacter oxydans, Gluconobacter sphaericus, Gluconobacter frateuri, Gluconobacter acai, Gluconobacter frateuri, Gluconobacter serinus, Gluconacetobacter Hanseni, Glucon Acetobacter xylinus; Gluconobacter oxydans, Gluconobacter sphaericus, Gluconobacter acai, Gluconobacter serinus, Gluconacetobacter Hanseni, Glucon Setobakuta-Kishirinusu and the like.

  These microorganisms belonging to acetic acid bacteria may be used singly or in combination of two or more.

  As a strain for obtaining the cells of microorganisms belonging to the above-mentioned acetic acid bacteria, there can be used foods using the natural world and acetic acid bacteria, and acetic acid bacteria isolated from production sites thereof. In addition, the University of Tokyo, Institute for Molecular Cell Biology, the National Institute for Product Evaluation Technology, the Independent Administrative Agency, RIKEN BioResource Center, etc. It is also possible to use strains conserved by traditional depository institutions.

  Among the microorganisms belonging to the genus Gluconobacter, the strains preserved by the official depository include, for example, Gluconobacter oxydance IFO 3292, Gluconobacter oxydance IFO 3293, Gluconobacter Bacter Oxydance IFO 3189, Gluconobacter Oxydance IFO 3244, Gluconobacter Oxydance IFO 3294, Gluconobacter Oxydance IFO 3462, Gluconobacter Oxydance IFO 3130, Gluconobacter Oxydance NBRC 3287 Gluconobacter sphaericus NBRC 12467 Gluconobacter frateurii IFO 3285 Gluconobacter NBRC 3171 Gluconobacter cerinus NBRC 3267 Serine IAM 1829, Gluconobacter Acai Gluconobacter asaii) IAM 14721, Gluconobacter, Thailand Randy dregs (Gluconobacter thailandicus) NBRC 3256, Gluconobacter roseus (Gluconobacter roseus) NBRC 3990, such as Gluconobacter-Arubidazu (Gluconobacter albidus) NBRC 3250, and the like. Among these strains, Gluconobacter oxydans IFO 3292, Gluconobacter oxydans IFO 3244, Gluconobacter sphaericus NBRC 12467, and Gluconobacter acai IAM 14721 are preferable.

  Among the strains of the genus Acetobacter, the strains conserved by the official depository include, for example, Acetobacter pasteurianus NBRC 3283, Acetobacter aceti NBRC 3281, Acetobacter orientalis (Acetobacter orientalis) ) KYG-22 (FERM P-21150), Acetobacter indonesiensis NBRC 16471, Acetobacter cibinongensis NBRC16605 and the like. Among these strains, Acetobacter pasterianus NBRC 3283, Acetobacter aceti NBRC 3281, and Acetobacter indonesiensis NBRC 16471 are preferable.

  Among the genus Gluconacetobacter, strains that are conserved by public depositories include, for example, Gluconacetobacter xylinus NBRC 3288, Gluconacetobacter xylinus NBRC 13693, Gluconaacetobacter Hanseni (Gluconacetobacter hansenii) NBRC 14816, Gluconacetobacter liquefacience IAM 1836, and the like. Among these strains, Gluconacetobacter hanseni NBRC 14816, Gluconacetobacter xylinus NBRC 3288, and Glucon acetobacter xylinus NBRC 13693 are preferable.

  When a hemiacetal hydroxyl group is oxidized by a microorganism belonging to an acetic acid bacterium, if the D-glucose of a certain concentration or more is contained in the environment where the microbial cells of the microorganism act, the target oligosaccharide It has been clarified by the present inventor that the production reaction may be inhibited and the production reaction may be delayed.

  In order to prevent this and efficiently produce the target oligosaccharide, it is preferable to remove or reduce D-glucose in the environment where the cells of microorganisms belonging to acetic acid bacteria act. Details will be described later. Further, in the case where D-glucose is mixed or there is a possibility that D-glucose may be mixed, when microorganisms belonging to acetic acid bacteria are brought into contact with the raw material oligosaccharide, D-glucose is present as the microorganism. It is desirable to select and use an acetic acid bacterium that can oxidize the reducing end of the raw oligosaccharide even under. Examples of such acetic acid bacteria include Gluconobacter genus, Gluconacetobacter genus; preferably Gluconobacter acai, Gluconobacter fratauri, Gluconacetobacter xylinus; More preferably, Gluconobacter acai It is done. Specific examples of such acetic acid bacteria include Gluconobacter acai IAM 14721, Gluconobacter frateuri IFO 3285, Gluconacetobacter xylinus NBRC 3288; preferably Gluconobacter acai IAM 14721.

Reaction conditions The method for producing an oligosaccharide having an aldonic acid residue at the reducing end of the present invention is carried out by bringing the raw material oligosaccharide into contact with the cells of a microorganism belonging to the acetic acid bacterium. When the oligosaccharide comes into contact with the cells of a microorganism belonging to the acetic acid bacterium, the hemiacetal hydroxyl group of the raw oligosaccharide is oxidized, and the oligosaccharide having an aldonic acid residue at the reducing end, which is the target product (target oligosaccharide) Produces.

  The method of bringing the microorganisms belonging to the acetic acid bacteria into contact with the raw oligosaccharide is not particularly limited, and examples thereof include a resting cell method and a fermentation production method. Hereinafter, the specific method will be described by dividing into the resting cell method and the fermentation production method.

<Pause cell method>
The resting cell method is a method in which acetic acid bacterial cells are brought into contact with the raw material oligosaccharide, which is a raw material, and the hemiacetal hydroxyl group of each raw material oligosaccharide is oxidized by an enzyme in the bacterial cell or on the cell membrane. This is a method for producing an oligosaccharide, in which cells of microorganisms belonging to acetic acid bacteria are cultured separately and oxidation of hemiacetal hydroxyl groups is performed separately.

  Examples of the microbial cells belonging to acetic acid bacteria used in the resting cell method include live microbial cells of acetic acid bacteria, or processed microbial cells or those immobilized on a carrier.

The culture of acetic acid bacteria is described below.
The culture medium is not particularly limited as long as it can be cultivated in the same manner as normal microorganisms, and those in which components such as a carbon source, a nitrogen source, vitamins, and minerals that can be assimilated by microorganisms are appropriately blended are used. Examples of carbon sources include monosaccharides such as glucose and fructose, oligosaccharides such as sucrose and maltose, polysaccharides, sugar alcohols, and glycerol. Common carbohydrates include corn starch and malt. Vegetable oils such as olive oil and corn oil are also included in the carbon source. Also, CSL (corn steep liquor) or soybean flakes may be used. In addition, carbon compounds such as alcohol, organic acid, and alkane may be used.

  As the nitrogen source, both inorganic and organic nitrogen can be used. As the inorganic nitrogen, ammonia salts, nitric acid bodies and the like are usually used. Organic nitrogen is usually given in the form of amino acids, proteins or urea. Other natural organic nitrogen complexes include CSL, soybeans, soybean flakes, peanut meal, cotton meal, distillers' solves, casein hydrolyzate, slaughterhouse waste, fish meal, yeast extract, etc. Can also be used.

  Regarding vitamins, vitamins necessary for the growth of microorganisms can be sufficiently supplied by using natural carbon sources and nitrogen sources, but calcium pantothenate, biotin, vitamin B1 and the like may be added as necessary.

  Examples of the mineral include magnesium, potassium, calcium, cobalt, copper, iron, manganese, molybdenum, and zinc.

  As other components, calcium carbonate may be added to the culture solution for pH control, or phosphate may be added to provide a buffering action. In order to control the pH of the culture system, ammonia, caustic soda, hydrochloric acid, sulfuric acid or the like may be added.

  Although culture conditions depend on the type of strain used, generally, a temperature of 20 to 35 ° C. and a pH of 4 to 8 are preferable. In order to adjust pH, hydrochloric acid, sodium hydroxide aqueous solution, calcium carbonate, or the like is used.

  Examples of the culture method in the resting cell method include stationary culture, shaking culture, and deep aeration and agitation culture. Of these, deep aeration and agitation culture is preferred from the viewpoint of culturing acetic acid bacteria in large quantities. Examples of the deep aeration stirring culture include a batch method, a sequential addition culture method, and a continuous culture method. When the culture is performed under such conditions, a sufficient amount of viable cells can be obtained in 15 to 168 hours after the culture.

  The viable cells of acetic acid bacteria obtained as described above can be used as they are after being collected and washed.

  Acetobacter cells can also be used as the treated cells. That is, cells treated with acetone, quaternary ammonium compounds, lauryl soda sulfate, Tween, or an enzyme that breaks down the specific binding of the cell walls of microorganisms; obtained by sublimating moisture under reduced pressure Freeze-dried microbial cells; crushed cells and cell membrane fractions physically crushed using a homogenizer and glass beads; and cell-free extracts that are supernatants of the crushed cells and enzymes extracted from these The crude enzyme solution etc. which were made can be used.

  In order to immobilize acetic acid bacterial cells or treated products on a carrier, a method of adsorbing to a substance such as a cellulose carrier, a ceramic carrier or a glass bead carrier; a gel-like substance having a lattice structure (for example, agar, calcium alginate, And carrageenan and a known method). By these methods, bacterial cells or treated bacterial cells of acetic acid bacteria having sugar oxidation activity can be repeatedly used.

  The raw oligosaccharide is dissolved in a reaction solvent, and the cells of microorganisms belonging to acetic acid bacteria obtained by culturing as described above are added, and if necessary, the reaction is performed while controlling the reaction temperature and the pH of the reaction solution, The hemiacetal hydroxyl group of the oligosaccharide is oxidized to produce the corresponding target oligosaccharide.

  The production of the target oligosaccharide by the resting cell method is performed in a solvent capable of oxidizing the hemiacetal hydroxyl group of the raw oligosaccharide. Examples of solvents that can be used in the resting cell method include aqueous solvents such as water and buffer solutions.

  In addition, the pH condition for producing the target oligosaccharide by the resting cell method is appropriately set according to the type of oligosaccharide or fungus used, for example, pH 3-9, preferably 4-6. Can be mentioned.

  What is necessary is just to set suitably about the reaction conditions at the time of producing | generating a target oligosaccharide by the resting cell method according to the said raw material oligosaccharide to be used, the kind of microbial cell, etc. As the reaction conditions, for example, the raw material oligosaccharide is preferably 0.5% by mass or more, more preferably 10 to 60% by mass, and still more preferably 20 to 50% by mass. In terms of OD660 nm, 0.1 or more is preferable, 1 to 500 is more preferable, and 10 to 100 is more preferable. Usually, 20 to 60 ° C., preferably 25 to 45 ° C., 3 to 120 hours, preferably 12 to 96 hours, more preferably 24 to 72 hours.

  As mentioned above, when oxidizing hemiacetal hydroxyl groups using bacterial cells of acetic acid bacteria, from the viewpoint of reaction efficiency, these reaction liquids do not contain D-glucose, or even if they contain it, they are suppressed to as little as possible. It is desirable. This is because when monosaccharides such as D-glucose are mixed in the reaction solution, these monosaccharides may inhibit the oxidation reaction and slow the reaction rate.

  In particular, isomaltoligosaccharides distributed as food often contain about 40% by mass of glucose. As described above, since the presence of glucose inhibits the oxidation reaction of isomaltooligosaccharide, it is preferable to select an isomaltooligosaccharide that does not contain glucose or has a low glucose content and use it as a substrate (raw oligosaccharide). .

  From the above viewpoint, contact of the bacterial body of the acetic acid bacterium with the raw oligosaccharide is performed in an environment where the D-glucose concentration is 3% by mass or less (that is, the D-glucose concentration in the reaction solution is 3% by mass or less). Preferably, it is carried out, and most preferably in an environment free from D-glucose.

  In order to remove or reduce D-glucose contained in the raw material, the raw material may be purified by subjecting the raw material to physical treatment such as chromatography, or microorganisms such as yeast, acetic acid bacteria, and lactic acid bacteria may be used. By contacting, only D-glucose may be assimilated and the concentration thereof may be reduced.

  Furthermore, in addition to the method for removing glucose, a raw material whose D-glucose content has been reduced in advance may be used. For example, if isomaltoligosaccharide is used as the raw material oligosaccharide, isomaltoligo prepared under conditions where glucose is difficult to be produced, such as hydrolysis of dextran with dextrin / dextranase or transfer reaction with dextran / sucrase. Sugar may be used.

  The step of removing and reducing D-glucose may be performed in advance before the oxidation reaction of the hemiacetal hydroxyl group, or may be performed simultaneously with the oxidation reaction.

  In addition, since the oxidation of the raw oligosaccharides by the bacterial cells of the acetic acid bacteria proceeds even in the presence of alcohol (for example, under the condition of ethanol concentration of about 7% by mass or less), The target oligosaccharide can be produced by adding the raw material oligosaccharide and the bacterial cell of the acetic acid bacterium, and by adding the raw material oligosaccharide and the bacterial cell of the acetic acid bacterium before or during the alcoholic fermentation. The target oligosaccharide can also be produced simultaneously with the progress of alcohol fermentation. Thus, the target oligosaccharide-containing fermented product can be produced by producing the target oligosaccharide in the alcohol fermented product. Examples of the alcohol fermented product that can contain the target oligosaccharide include beer, shochu, sake, wine, and whiskey.

<Fermentation production method>
The fermentation production method is described below.
The fermentation production method is a method for producing a target oligosaccharide by simultaneously oxidizing while culturing an acetic acid bacterium which is a microorganism used for an oxidation reaction. When fermentative production method is used, D-glucose is metabolized and decomposed by the action of acetic acid bacteria at the time of culturing. Therefore, reduction and removal of D-glucose and preparation of acetic acid bacteria that act as an oxidation catalyst, It is possible to carry out in the same culture tank. As a result, the manufacturing process can be simplified and made more efficient.

  In the fermentation production method, oligosaccharides having an aldonic acid residue at the reducing end are produced in a culture medium to which a nutrient source such as a carbon source or nitrogen source is added. About the kind etc. of the nutrient source added to this culture medium, it is the same as that used for culture | cultivation of the cell of an acetic acid bacterium in the said resting cell method. The culture medium used in the fermentation production method may be either liquid or solid. From the viewpoint of efficiently producing an oligosaccharide having an aldonic acid residue at the reducing end, a liquid is preferable. .

  The fermentation production method is carried out by adding the raw material oligosaccharide and adding an appropriate amount of the inoculum of the acetic acid bacterium or the preculture to the culture medium and culturing.

  In the fermentation production method, the concentration of the raw material oligosaccharide added to the culture medium is not particularly limited. However, considering productivity and the like, it is usually 0.5% by mass or more, preferably 10 to 60% by mass, and more preferably 20%. -50 mass% is mentioned.

  In the fermentation production method, the culture conditions are not particularly limited, but standing, shaking and deep aeration stirring are preferable from the viewpoint of aerobic reaction and increasing the yield of the target oligosaccharide. In the fermentation production method, the reaction time is not particularly limited, but it is preferable to select conditions under which the reaction is usually completed in 6 to 24 hours, preferably 6 to 12 hours. Moreover, as pH at the time of culture | cultivation, what is necessary is just to select suitably the pH suitable for the enzyme of the acetic acid bacterium used, but generally it is preferable to be the range of pH 4-8, and 5-7 are especially preferable. The culture temperature is not particularly limited as long as the enzyme of acetic acid bacteria is not inactivated, but is preferably 20 to 60 ° C, more preferably 25 to 45 ° C. When the reaction is performed under such conditions, a sufficient amount of product can be obtained in 6 to 24 hours.

  The acetic acid bacterium used in the present invention can produce acetic acid fermentation in the presence of ethanol, so that the target oligosaccharide can be produced in the presence of ethanol. By incorporating a method for producing an oligosaccharide having an aldonic acid residue (fermentation production method) into a brewed product, it becomes possible to produce a brewed vinegar containing the target oligosaccharide. That is, the target oligosaccharide-containing brewed vinegar can be produced by adding acetic acid bacteria to a fermentation raw material containing ethanol and the raw material oligosaccharide and performing acetic acid fermentation.

  Although content in particular of the ethanol in the said fermentation raw material is not restrict | limited, For example, 1-10 mass%, Preferably 3-7 mass% is mentioned.

  Moreover, the content of the raw material oligosaccharide in the fermentation raw material is not particularly limited, and for example, 0.5% by mass or more, preferably 10 to 60% by mass, and more preferably 20 to 50% by mass. Since brewed vinegar is usually produced through a saccharification process, an alcohol fermentation process, and an acetic acid fermentation process, when producing the objective oligosaccharide-containing brewed vinegar, before the saccharification process, during the saccharification process, before the alcohol fermentation process, alcohol What is necessary is just to add the said raw material oligosaccharide in a raw material at any timing in an acetic acid fermentation process before an acetic acid fermentation process during a fermentation process.

  Moreover, what is necessary is just to set similarly to the acetic acid fermentation currently employ | adopted by manufacture of normal brewing vinegar about the conditions of this acetic acid fermentation.

  Examples of the brewed vinegar that can contain the target oligosaccharide include various grain vinegars and fruit vinegars. Further, the concentration of the target oligosaccharide in the brewed vinegar can vary depending on the concentration of the raw material oligosaccharide in the fermentation raw material, the condition of acetic acid fermentation, etc., and thus cannot be uniformly defined. The mass% or more, Preferably it is 1-55 mass%, More preferably, 5-45 mass% is mentioned.

Separation and Recovery of Target Oligosaccharide The target oligosaccharide produced by bringing the acetic acid bacteria into contact with the raw material oligosaccharide is subjected to separation and purification treatment as necessary. Separation and recovery can be performed according to known methods. Specifically, after the production of the target oligosaccharide, if necessary, it is subjected to a step of removing bacterial cells of acetic acid bacteria by centrifugation, filtration, etc., and if necessary, a method for precipitating and collecting the target oligosaccharide, The target oligosaccharide is recovered by subjecting it to purification steps such as adsorption chromatography using activated carbon or porous organic resin particles, gel filtration, ion exchange resin chromatography and the like.

Uses of target oligosaccharides Oligosaccharides (target oligosaccharides) having an aldonic acid residue at the reducing end produced by the production method of the present invention are useful as pharmaceuticals, foods and drinks, and feeds. When the above-mentioned oligosaccharides, especially isomaltobionic acid, are used as pharmaceuticals, excipients, lubricants, diluents, pH regulators, preservatives, sweeteners, aromatics conventionally used in the field It can be administered orally or parenterally in the form of tablets, powders, wettable powders, emulsifiers using an agent, emulsifying dispersant and the like. Further, when isomaltobionic acid is used as a food or drink or feed, it can be used as it is, or added or mixed with other food or drink or feed.

  In addition, the target oligosaccharide-containing vinegar produced by incorporating the method for producing an oligosaccharide having an aldonic acid residue at the reducing end of the present invention (fermentation production method) into the brewing vinegar manufacturing process functions according to the target oligosaccharide. Since the property is enhanced, it is useful as a seasoning or food additive.

EXAMPLES Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto. In the examples,% represents mass%.
The analysis and identification method of the present invention will be described below.

High speed anion exchange chromatography with electrochemical detector (HPAEC-PAD)
In Examples, isomaltobionic acid, isomaltrionic acid, panotrionic acid, gentiobionic acid, and lactobionic acid were quantified using HPAEC-PAD under the following conditions. The amounts of isomaltobionic acid, isomaltrionic acid, panotrionic acid and gentiobionic acid produced are expressed in terms of the amount of lactobionic acid.

(HPAEC-PAD analysis conditions)
System: Dionex DX-500 (manufactured by Dionex)
Column: Carbopac PA-1 (Dionex)
Eluent: Linear concentration gradient method of 100 mM sodium hydroxide containing 100 mM sodium hydroxide and 1.0 M sodium acetate Conditions: Temperature: 35 ° C.
Flow rate: 1.0 mL / min Detector: Dionex model PADII electrochemical detector (Dionex)

  In the examples, the product was identified using thin layer chromatography, a mass spectrometer and a nuclear magnetic resonance apparatus under the following conditions.

[Mass spectrometer]
Mass spectrometer: API 2000 mass spectrometer (Applied Biosystems)
Ionization method: ESI (Electron-spray ionization) method Ion spray voltage: 5,000V
Detection: Negative ion mode

[Nuclear magnetic resonance apparatus (NMR)]
Nuclear magnetic resonance apparatus: AL-FT NMR apparatus (manufactured by JEOL Ltd.)
Solvent: D ₂ O

[Thin layer chromatography]
Plate: Silica gel 60 (Merck)
Developing solvent: ethyl acetate: acetic acid: water = 3: 1: 1 or pyridine: acetic acid: ethyl acetate: water = 5: 5: 1: 3
Color development: sulfuric acid: methanol = 1: 1

Preparation of acetic acid bacteria-1
In Examples 1, 2, 4-6, 8, and 10, acetic acid bacteria were prepared by the following method. In a test tube (18 mm × 200 mm), lactose 0.5% by mass, D-glucose 0.5% by mass, powdered yeast extract D-3 (Nippon Pharmaceutical Co., Ltd., Wako Code No. 390-00531) 0.5% by mass Polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd., Wako Code No. 394-00115) 0.5% by mass and 10 mL of medium A containing 0.1% by mass of magnesium sulfate / anhydride were dispensed and sterilized at 121 ° C. for 20 minutes. Inoculate a platinum loop of acetic acid bacteria (obtained from Fermentation Research Institute, National Institute of Technology and Evaluation of Molecular Cell Biology, University of Tokyo, etc.) into the test tube and shake at 27 ° C for 3 days. Culture was performed (240 reciprocations / min). Thereafter, the cells were collected by centrifuging the culture solution. The pH of the medium A was 6.0 to 7.0. The composition of medium A is shown in Table 1.

Preparation of acetic acid bacteria-2
In Example 3, Acetobacter orientalis cells were prepared by the method shown below. To a test tube (18 mm × 200 mm), 10 ml of medium C having the following composition was dispensed and sterilized at 121 ° C. for 20 minutes. One platinum loop of Acetobacter orientalis FERM P-21150 was inoculated into the test tube, and cultured with shaking at 27 ° C. and 240 reciprocations / min for 3 days (pre-culture). Next, 100 ml of medium C was placed in a 500 ml Sakaguchi flask, and 1.0 ml of the preculture solution obtained above was added thereto, and cultured with shaking at 27 ° C. and 120 reciprocations / min for 3 days. After cultivation, the cells were collected by centrifugation, washed twice with sterilized physiological saline, and the cells (wet cells) were collected.

Preparation of acetic acid bacteria-3
In Example 7, Gluconobacter cerinus cells were prepared by the method shown below. To a test tube (18 mm × 200 mm), 10 ml of medium C having the above composition was dispensed and sterilized at 121 ° C. for 20 minutes. One platinum loop of Gluconobacter cerinus NBRC3267 was inoculated into the test tube, and cultured with shaking at 27 ° C. and 240 reciprocations / min for 2 days (pre-culture). Next, 100 ml of the medium C was put into a 500 ml Sakaguchi flask, and 1.0 ml of the preculture solution obtained above was added thereto, and cultured with shaking at 27 ° C. and 120 reciprocations / min for 2 days. After cultivation, the cells were collected by centrifugation, washed twice with sterilized physiological saline, and the cells (wet cells) were collected.

Preparation of acetic acid bacteria 4
In Examples 9, 11, and 12, cells of each acetic acid bacterium were prepared by the following method. In a test tube (18 mm × 200 mm), 10 ml of the medium A having the above composition was dispensed and sterilized at 121 ° C. for 20 minutes. Inoculate a platinum loop of acetic acid bacteria (obtained from Fermentation Research Institute, National Institute of Technology and Evaluation of Molecular Cell Biology, University of Tokyo, etc.) into the test tube and shake at 27 ° C for 3 days. Culture was performed (240 reciprocations / min) (preculture). Next, separately, 10 mL of the medium A having the above composition was put into a test tube (18 mm × 200 mm), and 1.0 ml of the preculture solution obtained above was added thereto, and cultured with shaking at 27 ° C. and 240 reciprocations / min for 3 days. . After cultivation, the cells were collected by centrifugation, washed twice with sterilized physiological saline, and then collected.

Identification of isomaltobionic acid, isomaltrionic acid, panotrionic acid, and gentiobionic acid The cells were collected by centrifugation from 10 mL of the cultured solution of Acetobacter orientalis FERM P-21150. The bacterial cells were suspended in 4.5 ml of sterilized water containing 14 mg of calcium carbonate, and 0.5 ml of 10% isomaltose, 10% isomaltotriose, 10% panose, 10% gentiobiose was used as a substrate, respectively. In addition, the mixture was shaken at 27 ° C. for 3 days (240 reciprocations / minute) and then boiled for 5 minutes. This reaction solution was applied to a column packed with IRA-93ZU (Oregano) (equilibrium with 1N hydrochloric acid) which is a weak ion exchange resin, and the product was adsorbed on the column. The column was sufficiently washed with deionized water and then eluted with a linear concentration gradient method of 0-500 mM sodium chloride to purify isomaltionic acid, isomaltrionic acid, panotrionic acid and gentiobionic acid, respectively. By subjecting the purified oligoaldonic acid to TLC analysis and mass spectrometry, it was confirmed that they were isomaltobionic acid, isomaltrionic acid, panotrionic acid, and gentiobionic acid, respectively.

From the nuclear magnetic resonance analysis results ( ¹³ C- and ¹ H-NMR) of the obtained isomaltose oxidation product, carbon atoms were assigned. From this result, it was confirmed that the isomaltose oxide was isomaltobionic acid. The glucose residues (C-1 to C-6) and gluconic acid residues (C′-1 to C′-6) of isomaltobionic acid are converted into ¹³ C-NMR (D ₂ 0, δ in ppm) 179. 2 (C′-1), 98.7 (C-1), 74.7 (C′-2), 73.7 (C-3), 72.6 (C′-4), 72.4 ( C-5), 72.2 (C-2), 71.6 (C'-3), 70.2 (C-4), 70.0 (C'-5), 68.7 (C'- 6) and 61.1 (C-6).

Example 1
[Production of isomaltobionic acid and lactobionic acid using various acetic acid bacteria]
The respective cells obtained in the above-mentioned “Preparation of bacterial cells of acetic acid-1” (Acetobacter aceti NBRC3281, Acetobacter pasterianus NBRC3283, Acetobacter orientalis FERM P-21150, Gluconobacter oxydans IFO3292 , Gluconobacter oxydance IFO3293, Gluconobacter oxydance IFO3189, Gluconobacter oxydance IFO3244, Gluconobacter oxydance IFO3294, Gluconobacter oxydance IFO3462, Gluconobacter sphaericus NBRC12467, Glucono Bacter Roseus NBRC3990, Gluconobacter acai IAM14721, Gluconobacter frateuri NBRC3171, Gluconobacter frateuri IFO3285, Gluconobacter tylandicas NBRC3256, Gluconobacter albidas NBRC3250, Gluconobacter se 1/10 amount of Linus NBRC3267, Glucon Acetobacter Hanseni NBRC14816, Glucon Acetobacter xylinus NBRC3288, Glucon Acetobacter liquefaciens IAM1836) is 1.0 ml of 1% isomaltose (manufactured by Tokyo Chemical Industry Co., Ltd., “Code No. .I0231), or resuspended in 100 mM acetate buffer (pH 5.5) containing 1% lactose (manufactured by Wako Pure Chemical Industries, reagent special code No.128-00095), reacted at 27 ° C. for 240 reciprocations / minute The production amount in the reaction solution after shaking for isomaltobionic acid after 24 hours and after shaking for 72 hours for lactobionic acid was measured using HPAEC-PAD, respectively. The amount of maltobionic acid produced is shown in Table 3. From this result, by using bacterial cells of acetic acid bacteria, isomaltose is used as a raw material. It became clear that tobionic acid could be produced.

  Some of the above mentioned acetic acid bacteria (Acetobacter aceti NBRC3281, Acetobacter pasterianus NBRC3283, Gluconobacter oxydans IFO3292, Gluconobacter acai IAM14721, Gluconobacter frateuri IFO3285, Gluconobacter cerinus NBRC3267, Gluconacetobacter hanseni NBRC14816, Gluconacetobacter xylinus NBRC3288) were compared with the amount of isomaltobionic acid produced after 24 hours of reaction and the amount of lactobionic acid produced after 72 hours of reaction. The results are shown in Table 4. As is clear from Table 4, it was found that the amount of isomaltobionic acid produced by acetic acid bacteria was much higher than the amount of lactobionic acid produced under the same conditions.

  Furthermore, in some of the acetic acid bacteria described above, the amount of lactobionic acid produced per 24 hours was calculated from the amount of lactobionic acid produced after 72 hours of reaction, and compared with the amount of isomaltobionic acid produced after 24 hours of reaction. It was found that the acetic acid bacteria produced isomaltobionic acid with an efficiency of 15 to 240 times or more compared with lactobionic acid. The results are shown in Table 5. That is, the production efficiency of isomaltobionic acid per unit time using this acetic acid bacterium was significantly superior to the production efficiency of lactobionic acid under the same conditions.

  Furthermore, in the acetobacter genus that produces less isomaltobionic acid than the gluconobacter genus or the gluconoacetobacter genus, the medium A at the time of preparation of the cells is replaced with the medium B, and some of the above acetic acid bacteria are cultured did. Oxidation of 1% isomaltose by the same method as above, the production of isomaltobionic acid was 0.1 g / L for Acetobacter aceti NBRC3281 and 0.4 g / L for Acetobacter pasteria NBRC3283, respectively. Increased to 1.8 g / L and 4.4 g / L. That is, when the glucose in the medium was replaced with glycerin and the glucose content was reduced, the production efficiency was improved. The composition of the medium B is as shown in Table 1, and the pH was 6.0-7.0.

Example 2
[Production of isomaltobionic acid and lactobionic acid using various acetic acid bacteria]
The respective cells obtained in the above-mentioned “Preparation of bacterial cells of acetic acid-1” (Acetobacter orientalis FERM P-21150, Acetobacter indonesiensis NBRC16471, Acetobacter symbionogensis NBRC16605, Gluconobacter・ Oxydance IFO3130, Gluconobacter oxydans NBRC3287, Gluconobacter oxydance IFO 3292, Gluconobacter frauteuri IFO3171, Gluconobacter celine IAM1829, Gluconobacter albidas NBRC3250, Gluconobacter Roseus NBRC 3990 , Glucon Acetobacter xylinus NBRC13693, Glucon Acetobacter rewefaciens IAM1836) is resuspended in 200 μl of 100 mM acetate buffer (pH 5.5) containing 1% isomaltose or 1% lactose. Round trip / minute, 2 It was allowed to react at ℃. After 24 hours of reaction, the reaction solution was boiled to stop the reaction. The reaction solution was spotted on a TLC plate (silica gel 60, manufactured by Merck & Co., Inc.) and developed with a developing solvent of ethyl acetate: acetic acid: water = 3: 1: 1. After that, the TLC plate is dried, sprayed with a mixed solution of sulfuric acid and methanol (sulfuric acid: methanol = 1: 1), heated at 150 ° C. to cause color development, and visually checked for the presence of isomaltobionic acid and lactobionic acid. confirmed.

  The results obtained are shown in Table 6. Also from this result, it was confirmed that isomaltobionic acid can be produced from isomaltose by using acetic acid bacteria. Similarly to the case of Example 1, the amount of isomaltobionic acid produced under the same conditions It was confirmed that it was clearly higher than the amount of lactobionic acid produced in the plant.

Example 3
[Production of isomaltobionic acid and lactobionic acid using Acetobacter orientalis]
Acetobacter * obtained by the method using the above-mentioned medium C in “Preparation of bacterial cells of acetic acid bacteria-2” in 1 ml of 100 mM acetate buffer (pH 5.5) containing 1% isomaltose or 1% lactose 101.9 mg (wet weight) of cells of Orientalis FERM P-21150 was aseptically added, and the reaction was performed by shaking at 240 reciprocations / min at 27 ° C. for 24 hours. A part of the reaction solution was sampled 5, 24, 29 and 48 hours after the start of the reaction. The sampled reaction solution was subjected to boiling treatment for 5 minutes to stop the reaction. Thereafter, each reaction solution was spotted on a TLC plate (silica gel 60, manufactured by Merck & Co., Inc.) and developed with a developing solvent of ethyl acetate: acetic acid: water = 3: 1: 1. Thereafter, the TLC plate was dried, and a mixed solution of sulfuric acid and methanol (sulfuric acid: methanol = 1: 1) was sprayed, and the color was developed by heating at 150 ° C. to confirm the presence or absence of isomaltobionic acid and lactobionic acid. .

  The obtained results are shown in FIG. In FIG. 2, Iso is isomaltose, Isoa is isomaltobionic acid, Lac is lactose, and LacA is a spot presumed to be lactobionic acid. From this result, it was confirmed that Acetobacter orientalis has an action of oxidizing isomaltose to produce isomaltobionic acid. Further, similar to the results of Example 1, it was also confirmed that the amount of isomaltionic acid produced was clearly higher than the amount of lactobionic acid produced under the same conditions.

Example 4
[Production of isomaltrionic acid using various acetic acid bacteria]
Each 1/10 amount of some of the bacterial cells obtained in the above-mentioned “Preparation of bacterial cells of acetic acid bacteria-1” was replaced with 1.0 ml of 1% isomaltotriose (Seikagaku Corporation, Code No. .400473) in 100 mM acetate buffer (pH 5.5), and after shaking for 24 hours at 27 ° C. at 240 reciprocations / minute, isomaltrionic acid in the reaction solution was measured using HPAEC-PAD. . The results are shown in Table 7. From this result, it was confirmed that isomaltriotrionic acid can be produced using isomaltotriose as a raw material by using bacterial cells of acetic acid bacteria.

Example 5
[Oxidation of isomaltooligosaccharide mixture by contacting cells of various acetic acid bacteria]
Each 1/10 amount of some of the bacterial cells obtained in the above-mentioned “Preparation of bacterial cells of acetic acid bacteria-1” was added to 1.0 ml of 3% isomaltooligosaccharide (manufactured by Wako Pure Chemical Industries, Biochemistry). After resuspending in 100 mM acetate buffer (pH 5.5) containing reagent No. 090-03485 and shaking at 27 ° C. for 24 hours, the amounts of isomaltobionic acid and isomalttrionic acid in the reaction mixture were measured using HPAEC- The results are shown in Table 8. From this result, the bacterial cells of acetic acid bacteria oxidize isomaltose and isomaltotriose contained in isomaltoligosaccharides to produce isomaltobionic acid and isomalttrionic acid. It was confirmed that it can be converted to.

Example 6
[Production of panotrionic acid using various acetic acid bacteria]
Each 1/10 amount of some of the cells obtained in the above-mentioned “Preparation of bacterial cells of acetic acid bacteria-1” contains 1.0 ml of 1% panose (manufactured by Hayashibara Biochemical Laboratory, reagent). The suspension was resuspended in 100 mM acetate buffer (pH 5.5) and shaken at 27 ° C. The amount of panotrionic acid in the reaction solution 72 hours after the reaction was measured using HPAEC-PAD. Table 9 shows the amount of panotrionic acid produced. From this result, it was confirmed that the cells of acetic acid bacteria can oxidize panose and convert it into panotrionic acid.

Example 7
[Production of various sugar carboxylic acids using Gluconobacter cerinus]
Gluconobacter obtained in “Preparation of bacterial cells of acetic acid bacteria-3” in 0.5 ml of 100 mM acetate buffer (pH 5.5) containing 1% panose, 1% isomaltose, or 1% lactose 8.6 mg (wet weight) of cells of Serinus NBRC3267 was aseptically added, and the reaction was performed by shaking at 240 reciprocations / min at 27 ° C. for 24 hours. A part of the reaction solution was sampled at 8, 24, 48, 72 and 92 hours after the start of the reaction. The sampled reaction solution was subjected to boiling treatment for 5 minutes to stop the reaction. Thereafter, each reaction solution was spotted on a TLC plate (silica gel 60, manufactured by Merck & Co., Inc.) and developed with a developing solvent of ethyl acetate: acetic acid: water = 3: 1: 1. Then, the TLC plate is dried, sprayed with a mixed solution of sulfuric acid and methanol (sulfuric acid: methanol = 1: 1), heated at 150 ° C. to develop color, and whether or not panotrionic acid, isomaltobionic acid and lactobionic acid are generated. It was confirmed.

  The obtained results are shown in FIG. In FIG. 3, Pa is panose, PaA is panotrionic acid, Iso is isomaltose, IsoA is isomaltobionic acid, Lac is lactose, and LacA is a spot presumed to be lactobionic acid. From these results, it was confirmed that Gluconobacter cerinus has an action of oxidizing oligosaccharides such as panose and isomaltose to produce sugar carboxylic acids such as panotrionic acid and isomaltobionic acid. Moreover, from FIG. 3, it was also confirmed that the production amount of panotrionic acid and isomaltobionic acid is clearly higher than the production amount of lactobionic acid under the same conditions.

Example 8
[Production of gentiobionic acid using various acetic acid bacteria]
Each 1/10 amount of some of the bacterial cells obtained in the above-mentioned “Preparation of bacterial cells of acetic acid bacteria-1” was added to 1.0 ml of 3% gentio-oligosaccharide (Wako Pure Chemical Industries, Biochemical) The sample was resuspended in 100 mM acetate buffer (pH 5.5) containing Code No. 079-03791) and subjected to a shaking reaction at 27 ° C. The amount of gentiobionic acid in the reaction solution 24 hours after the reaction was measured using HPAEC-PAD. Table 10 shows the amount of gentiobionic acid produced. From this result, it was confirmed that the cells of acetic acid bacteria can oxidize gentiobiose and convert it into gentiobionic acid.

  Furthermore, FIG. 4 shows the result of analyzing the reaction solution of Gluconobacter frateuri IFO3285 obtained in the above-mentioned preparation of acetic acid bacteria with a mass spectrometer. As shown in FIG. 4, in addition to the signal derived from gentiobionic acid [m / z = 357], a signal derived from gentiotriionic acid [m / z = 519] was confirmed. Furthermore, the reaction solutions of the strains shown in Table 10 other than Gluconobacter frateuri IFO3285 were similarly analyzed with a mass spectrometer. As a result, a signal [m / z = 519] derived from gentiotrionic acid was confirmed in the reaction solutions of all strains except Acetobacter aceti NBRC3281. In addition, since Acetobacter aceti NBRC3281 also produces gentiobionic acid, it is thought that gentiotrionic acid can also be produced by extending the reaction time, increasing the concentration of cells used, and the like.

Example 9
[Reactivity specificity in the production of oligosaccharides having aldonic acid residues at the reducing end using various acetic acid bacteria]
Cells obtained in the above-mentioned "Preparation of bacterial cells of acetic acid bacteria-4" (Gluconobacter frateuri IFO3285, Gluconobacter oxydans IFO3292, Gluconobacter acai IAM14721, Acetobacter orientalis FERM P-21150 Gluconobacter tylandicus IFO3172) was suspended in 0.5 ml of physiological saline. The acetic acid bacteria suspension was diluted 1 to 10 times with physiological saline, 5 μl of the diluted solution, and 0.5 M carbohydrate solution (D-glucose, isomaltose, isomaltotriose, isomalttetraose, 20 μl of isomaltopentaose, gentiobiose, lactose) was added to the reaction solution A having the composition shown in Table 11 and reacted at 40 ° C. In addition, reaction time was unified between 2-10 minutes for every microbial cell. After the reaction, absorption at 530 nm is measured with a photometer UV-160A (manufactured by Shimadzu Corporation), and 2,6-dichlorophenolindophenol (ε ₅₃₀ = 7500 M ⁻¹ cm ⁻¹ ) reduced by oxidation of each carbohydrate. The amount of decrease was measured. After calculating the oxidative activity for each saccharide on each substrate of each acetic acid bacterium from the decrease in absorption, the oxidative activity for D-glucose was calculated from the value as 100, and the relative value of the oxidative activity for each saccharide was calculated.

  The results obtained are shown in Table 12. From this result, it was clarified that acetic acid bacteria have strong oxidizing activity against isomaltose and gentiobiose among oligosaccharides having α1 → 6 or β1 → 6 glucoside bonds. Also from these results, it was confirmed that acetic acid bacteria had a weak effect of oxidizing lactose.

Example 10 [Effect of glucose on isomaltobionic acid production using various acetic acid bacteria]
1/10 amount of each of the cells (Acetobacter aceti NBRC3281, Gluconobacter oxydans IFO3292, Gluconacetobacter xylinus NBRC3288) obtained in the above-mentioned "Preparation of acetic acid bacteria-1" It was resuspended in 100 mM acetate buffer (pH 5.5) containing 1.0 ml of 3% by mass isomaltoligosaccharide (manufactured by Wako Pure Chemical Industries, Biochemical reagent Code No.090-03485). Further, D-glucose was added so that the final concentration was 0% by mass, 3% by mass, 5% by mass, 10% by mass or 20% by mass, shaken at 27 ° C. for 24 hours, and then isomalt in the reaction solution. The amount of bioacid was quantified by HPAEC-PAD. The results are shown in Table 13. As apparent from Table 13, when the D-glucose concentration increased, the production of isomaltobionic acid was suppressed. In addition, when D-glucose was not contained, and when the content of D-glucose was 3% by mass, generation of isomaltobionic acid was confirmed in any strain. On the other hand, when the D-glucose concentration was 5% by mass, 10% by mass, and 20% by mass, the generation of isomaltobionic acid could not be confirmed in any strain.

Example 11
[Influence of glucose on isomaltobionic acid production using various acetic acid bacteria]
The cells obtained in the above “Preparation of bacterial cells of acetic acid bacteria-4” (Gluconobacter frateuri IFO3285, Gluconobacter oxydans IFO3292, Gluconobacter acai IAM14721, Gluconacetobacter xylinus NBRC3288) The suspension was suspended in 0.5 ml of physiological saline to prepare an acetic acid bacteria suspension (× 20). Then, the obtained acetic acid bacteria suspension (x20) was finally contained in a 10-fold diluted state, and 1% by mass of isomaltose or 1% by mass of isomaltotriose, 0% by mass, A 100 mM acetate buffer (pH 5.5) (reaction solution) containing 1% by mass, 3% by mass, or 5% by mass of D-glucose was prepared, and shaken at 27 ° C. for 6 hours at 240 reciprocations / min. After shaking, the amount of isomaltobionic acid or isomaltorionic acid in the reaction solution was quantified by HPAEC-PAD.

  The obtained results are shown in Table 14. From this result, in Gluconobacter frateuri, Gluconobacter oxydans, Gluconobacter acai, and Gluconacetobacter xylinus, the amount of isomaltobionic acid or isomaltriotrionic acid produced as the D-glucose concentration increased However, the formation of isomaltobionic acid or isomaltriotrionic acid was observed. In particular, in Gluconobacter acai, Gluconobacter frateuri and Gluconacetobacter xylinus, the production amount of isomaltobionic acid or isomalttrionic acid was maintained at a relatively high value even in the presence of D-glucose. .

Example 12
[Effect of glucose on isomaltobionic acid production using Gluconobacter frateuri]
The cell body of Gluconobacter frateuri IFO3285 obtained in the above-mentioned “Preparation of bacterial cells of acetic acid bacteria-4” was suspended in 0.5 ml of physiological saline to prepare an acetic acid bacteria suspension (× 20). . The resulting acetic acid bacteria suspension (x20) is then included in a final 10-fold diluted state, and 1 wt% isomaltose and 0 wt%, 1 wt%, 3 wt%, or A 500 mM acetate buffer (pH 5.5) (reaction solution) containing 5% by mass of D-glucose was prepared and shaken at 27 ° C. for 1 hour at 240 reciprocations / min. After shaking, the amount of isomaltobionic acid in the reaction solution was quantified by HPAEC-PAD. Moreover, it measured also about the pH of the reaction liquid after each reaction.

  The obtained results are shown in FIG. The pH of the reaction solution after each reaction was 5.5, which was unchanged from before the reaction. As shown in FIG. 5, as in Example 11, as the concentration of D-glucose increased, the amount of isomaltobionic acid produced decreased. That is, from the results of this test, it was found that pH is not affecting the production inhibition of isomaltobionic acid caused by the presence of D-glucose.

Example 13
[Isomaltobionic acid production by fermentation production method using various acetic acid bacteria]
Isomaltoligosaccharide (manufactured by Wako Pure Chemical Industries, Biochemical reagent Code No.090-03485) 2.0% by mass, lactose 0.5% by mass, D-glucose 0.5% by mass, powdered yeast extract D-3 (Nippon Pharmaceutical Co., Ltd., Wako Code No. 390-00531) 0.5% by mass, polypeptone (Nippon Pharmaceutical Co., Ltd., Wako Code No. 394-00115) 0.5% by mass, magnesium sulfate / anhydrous 0.1 Acetic acid bacteria (Acetobacter aceti NBRC3281, Acetobacter orientalis FERM P-21150, Gluconobacter oxydans IFO3292, Gluconobacter acai IAM14721, Gluconobacter frateuri IFO3285, Gluconacetobacter Hanseni NBRC14816) was inoculated with one platinum ear. After inoculation, the cells were cultured at 27 ° C. with shaking culture (240 reciprocations / minute). The isomaltobionic acid concentration in the fermentation liquid on the 3rd and 7th days of fermentation was measured by HPAEC-PAD. Table 15 shows the amount of isomaltobionic acid of each acetic acid bacterium.

Example 14
[Isomaltobionic acid production by acetic acid fermentation]
1% by mass of powdered yeast extract D-3 (Nippon Pharmaceutical Co., Ltd., Wako Code No. 390-00531), 1% by mass of polypeptone (Nippon Pharmaceutical Co., Ltd., Wako Code No. 394-00115), and 2% by mass of ethanol A platinum loop of acetic acid bacteria (Acetobacter aceti NBRC3281, Acetobacter pasterianus NBRC3283) cryopreserved in a glycerol stock was inoculated into 10 ml of a liquid medium, and allowed to stand at 27 ° C. for 3 days, followed by culture. Further, 1 ml of the obtained pre-culture solution was added to 0.2% by mass of powdered yeast extract D-3 (Nippon Pharmaceutical Co., Ltd., Wako Code No. 390-00531), polypeptone (Nippon Pharmaceutical Co., Ltd., Wako Code No. 394). -00115) Inoculated into 10 ml of a liquid medium containing 0.2% by mass, 0.2% by mass of D-glucose, 6% by mass of ethanol, and 1% by mass of acetic acid, and cultured with shaking at 27 ° C. for 31 hours (240 reciprocations / Min) and pre-cultured. Next, 50 μl of the obtained preculture solution was inoculated into a liquid medium containing 2% by mass of isomaltose, 0.2% by mass of D-glucose, 5% by mass of ethanol, and 1% by mass of acetic acid, and was treated at 27 ° C. for 12 days. Then, it was allowed to stand and subjected to acetic acid fermentation. The isomaltobionic acid concentration in the fermentation broth obtained by acetic acid fermentation for 12 days was measured by HPAEC-PAD.

  As a result, when Acetobacter aceti NBRC3281 was used, 0.0044% by mass of isomaltobionic acid was produced in the fermentation broth, and when Acetobacter pasterianus NBRC3283 was used, It was confirmed that 0.0033% by mass of isomaltobionic acid was produced. From this result, it was revealed that acetic acid bacteria can produce isomaltobionic acid even in the presence of alcohol. That is, during the fermentation process such as acetic acid fermentation, it is also possible to carry out the sugar aldonic acid production method of the present invention, which makes it possible to directly produce a fermented product containing isomaltobionic acid. .

Claims (9)

  It is characterized by contacting a microbial cell belonging to acetic acid bacteria with an oligosaccharide having a hemiacetal hydroxyl group and having an α1 → 6 or β1 → 6 glucoside bond, and oxidizing the hemiacetal hydroxyl group of the oligosaccharide. A method for producing an oligosaccharide having an aldonic acid residue at the reducing end.   The production method according to claim 1, wherein the microorganism belonging to the acetic acid bacterium is a microorganism belonging to the genus Acetobacter, Gluconobacter or Glucon acetobacter.   The production method according to claim 1 or 2, wherein the contact of the microbial cells with the oligosaccharide is performed under a condition where the D-glucose concentration is 3% by mass or less.   The production method according to claim 1, wherein the oligosaccharide is isomaltose and / or isomaltotriose.   The manufacturing method according to any one of claims 1 to 3, wherein the oligosaccharide is an isomaltoligosaccharide containing two or more selected from the group consisting of isomaltose, isomalttriose, and isomalttetraose. .   The manufacturing method in any one of Claims 1-3 whose said oligosaccharide is a gentiobiose and / or a gentiotriose.   The production method according to claim 1, wherein the oligosaccharide is panose.   It is obtained by performing acetic acid fermentation by adding acetic acid bacteria to a fermentation raw material containing an oligosaccharide having a hemiacetal hydroxyl group and having an α1 → 6 or β1 → 6 glucoside bond and ethanol. A brewed vinegar containing an oligosaccharide having an aldonic acid residue at the reducing end.   A reducing terminal, characterized in that acetic acid fermentation is performed by adding acetic acid bacteria to a fermentation raw material containing an oligosaccharide having a hemiacetal hydroxyl group and having an α1 → 6 or β1 → 6 glucoside bond and ethanol. A method for producing brewed vinegar containing an oligosaccharide having an aldonic acid residue.