WO2003046194A1 - NOVEL α-GALACTOSYL-CONTAINING NON-REDUCING DISACCHARIDES AND PROCESS FOR PRODUCING THE SAME - Google Patents

Abstract

Novel α-galactosyl-containing non-reducing disaccharides Galα1-1βGal and Galα1-1βGlc expected as useful food materials and medicinal materials which would not damage amino acids or proteins when added to foods or preserved. A process for producing the non-reducing disaccharide Galα1-1βGal which comprises treating galactose or a galactose-containing material with α-galactosidase, decomposing reducing sugars contained in the α-galactosyl-containing oligosaccharides thus obtained and separating the same; and a process for producing the non-reducing disaccharide Galα1-1βGlc which comprises starting with a material containing galactose and glucose, hydrolyzing Galα1-1βGal contained in the α-galactosyl-containing oligosaccharides obtained in the same manner as described above, decomposing the reducing sugars and separating the same.

Description

 ^ m -m

 TECHNICAL FIELD The present invention relates to a novel non-reducing disaccharide containing a 0; -galactosyl group and a method for producing the same.

The present invention relates to a non-reducing disaccharide, and methods for their preparation including the novel _alpha _ galactosyl groups, in particular, non-reducing disaccharide G a 1 α 1 - 1; . 3 G a 1 ( hereinafter , G a1 is galactose,) and G a1α1-1βG1c (hereinafter, G1c is glucose) and a method for producing them. Background art

 In recent years, trehalose (G1cα1-1-G1c), a type of non-reducing disaccharide, is non-reducing and has excellent heat and acid resistance. It is known that there is little adverse effect on other materials in food processing and preservation, such as the amino acid / protein is not damaged by a single reaction. In addition, it has excellent properties as a food material, such as a strong inhibitory effect on starch aging, protein denaturation, and lipid sleep. ''

 On the other hand, a carbohydrate having a non-reducing end with a galactosyl group is naturally known as the trisaccharide raffinose permeribose, which has strong bifidobacterial selective growth activity and anti-cariogenic activity, as well as immune cell activation. Various physiological functions such as action, anti-cancer effect, and improvement effect on atopic dermatitis have been reported. (Seipura Motoi-: Japanese Cancer Society-(General Assembly Article), 40, 13 2 (1 981), Taizo Nakura et al .: Food Industry, 2.2.28, 29, 1999).

Therefore, if a non-reducing disaccharide containing a monogalactosyl group can be synthesized, the above-mentioned properties of trehalose or the functions of raffinose and melibiose can be provided, and as a result, excellent food and pharmaceutical materials can be provided. Can be expected. However, non-reducing disaccharides containing an α-galactosyl group have conventionally been only Galal-laGlc described in JP-A-10-308488, and other sugars There is no known quality.

 Accordingly, the present invention is to provide a novel non-reducing disaccharide containing a galactosyl group, which is expected as a useful food material or pharmaceutical material, and to provide a simple production method thereof. Aim. Disclosure of the invention

 The present invention relates to a non-reducing disaccharide represented by the following formula (1).

上記非還元性二糖は、 ガラク トース又はガラク トースを含む物質に微 生物に由来するひ —ガラク トシダーゼを作用ざせ、 脱水縮合反応により α—ガラク トシル基を含むオリゴ糖を生成させ、 該オリ ゴ糖中の還元糖 の分解後、 分離操作することにより製造できる。 この製造方法において、 α—ガラク トシダーゼは、 脱水縮合反応の活性に優れるァスペルギルス '二ガー （A s p e r g i 1 1 u s n i g e r ) A P C— 9 3 1 9株 (寄託番号 ： F E RM B P— 7 6 8 0 ) 由来のものを用いることがで きる。 上記製造方法において生成するオリ ゴ糖中の還元糖の分解は、 ァ ルカリ分解により行うことができる。 また、 本発明は、 下記式 （2) で表される非還元性二糖に関する。

The non-reducing disaccharide is prepared by subjecting a galactose or a substance containing galactose to a hypergalactosidase derived from a microorganism to produce an oligosaccharide containing an α-galactosyl group by a dehydration condensation reaction. It can be produced by decomposing the reducing sugar in the sugar and then separating it. In this production method, α-galactosidase was derived from Aspergillus niger (A spergi 11 usniger) APC-9319 strain (deposit number: FE RM BP—7680) which has excellent dehydration condensation reaction activity. Can be used. The decomposition of the reducing sugar in the oligosaccharide produced in the above-mentioned production method can be performed by alkali decomposition. The present invention also relates to a non-reducing disaccharide represented by the following formula (2).

The non-reducing disaccharide is obtained by reacting a substance containing galactose and glucose with α-galactosidase derived from a microorganism to form an oligosaccharide containing a single galactosyl group by a dehydration condensation reaction. \ a1-1 βGa1 can be produced by hydrolyzing the reducing sugar in the oligosaccharide and then separating the oligosaccharide. In this production method, Hi-galactosidase was derived from Aspergillus niger APC-9319 strain (deposit number: FE RM BP-7680) which has excellent dehydration condensation reaction activity. Can be used. The hydrolysis of Galal to lj3Gal mixed in the above production method can be carried out with one galactosidase. Further, the decomposition of the reducing sugar in the oligosaccharide produced in the above-mentioned production method can be carried out by alcohol decomposition.

Examples of microorganisms that produce monogalactosidase include, but are not limited to, microorganisms such as the genus Aspergillus (A spergi 11 us), the genus Penicillium (Penici 11 ium), and the genus Trichoderma (Trichoderma). Α-galactosidase derived from microorganisms belonging to the genus Aspergillus (A spergi 11 us), including yeasts such as the genus Saccharomyces and bacteria belonging to the genus Bacillus. Is preferred. Among the above microorganisms, Aspergillus molds include Aspergillus

'ニ (A sp er g i 1 1 u s n i g e r

 Orize (A sp e r g i 1 1 u s o r y z a e), as z

 Punorebenorerentus (A spergi 1 1 uspulverulentus), as a genus of the genus Penicillium, Penicillium 'Citrinanum Penicillium 1 enium citrinum), Henicirium-Manore Chilean (Penicilli um mu lticolor), As the Trichoderma fungi, Trichoderma's viride (T richodermaviride) is preferable, and among them, Aspergillus ■ Niger (A spergillusniger) power is more preferable.

 Among the Aspernoregizoles' nigers (Aseergii11 uSniger), the Aspernoreginoles niger (Aspergir1 11 uSniger) APC-9319 strain is particularly preferred. This strain was obtained by the applicant based on the Bustust Treaty, the Patent Organism Depositary Center, National Institute of Advanced Industrial Science and Technology (IPOD, Japan). Deposit No. 1 No. 1 Central No. 6) and its deposit number is FE RM BP—7680 (transferred from domestic deposit to international deposit, original deposit date August 29, 2000, national deposit) Accession number F ERM P—1 800 3).

 The bacterial properties of this strain are described below.

Conidium head: spherical to radial, conidia (optical microscope): spherical to subspherical, smooth to slightly rough, conidia (electron microscopy): rough (with bumps), diameter (3.5 to 4) Leachate: little formation (transparent to brown), odor: almost none, sterigmata: two-stage, colony growth rate (malt extract agar medium, culture at 25 ° C):> 85 mm (7 days) Culture),> 85 mm '(cultured for 12 days), growth rate of colonies (deep cultivated agar medium, 25 ° C culture): 60-67 mm (cultured for 7 days), 65-73 mm (cultured for 7 days) 1 2 days culture), settlement 0212215 color (malt extract agar medium): black (front), colorless (back), settlement color (Vapeckeast agar): black to gray-black (front), talium-gray-yellow (back)

 This strain is classified into Aspergillus' Niker, Aspergillus nilu, niger, var. Niger, based on the conidial shape, the sterigmata, and the color of the colony.

_Α- galactosidase produced by this strain has extremely high dehydration condensation activity, and Candidaguilliermondii (C andidaguilliermondii), which is known to have the highest catalytic activity of dehydration condensation reaction among α-galactosidases in the past. ) An oligosaccharide containing an α-galatatosyl group can be produced at a high yield from α-galactosidase produced by the H-404 strain (deposit number: FERMP—11062) (WO 02 / 186 14), and correspondingly, the non-reducing disaccharide of the formula (1) or (2) can be produced in high yield.

In addition, as the genus Saccharomyces yeast, Saccharomyces cerevisiae Ne (S accharomycescervis ι ae), as is Nono Tenoresu bacteria belonging to the genus Bacillus' Megateri © beam (B acillusmegaterium) force Mashi ^¾ Nu Yu Rere.

As a method for producing α-galactosidase from each of the above microorganisms, solid culture or liquid culture is usually used. The solid culture medium may be wheat bran alone or wheat bran with various additives such as kinako, soybean flour, ammonium salt, nitrate, urea, glutamic acid, aspartic acid, polypeptone, corn steep liquor, meat. Organic and inorganic nitrogen compounds such as extracts, yeast extracts, and protein hydrolysates can be appropriately added and used. In addition, suitable inorganic salts can be added. In addition, as a culture medium for liquid culture, the microorganisms grow well and produce enzymes smoothly. A synthetic medium or a natural medium containing a carbon source, a nitrogen source, an inorganic salt, a necessary nutrient source, and the like necessary for production. For example, as the carbon source, starch or a carbohydrate such as a fraction thereof, roasted dextrin, processed starch, starch derivative, physically treated starch and a substance containing -starch or galactose can be used. Specific examples include soluble starch, corn starch, potato starch, sweet potato starch, dextrin, amylopectin, amylose, galataose, lactose, raffinose, and the like. Can be used in combination of two or more. Nitrogen sources include polypeptone, casein, meat extract, yeast extract, corn steep liquor or organic nitrogen source substances such as soybean or soybean meal extracts, ammonium sulfate, ammonium phosphate, etc. Amino acids such as inorganic nitrogen compounds and glutamic acid can be used, and these can be used alone or in combination of two or more. Examples of the inorganic salts include phosphates such as potassium phosphate and potassium phosphate, magnesium salts such as magnesium sulfate, calcium salts such as calcium chloride, and sodium salts such as sodium carbonate. Used alone or in combination of two or more.

 In the case of solid culture, the culture is carried out by stationary culture, and the culture is adjusted to pH 3 to 7, preferably 4 to 7, and the bacterial strain is inoculated into the culture. Incubate at 20-37 ° C for 1-10 days. After the cultivation, 1-galactosidase can be obtained from the culture extract as a crude enzyme precipitate by means such as ethanol precipitation.

In the case of liquid culture, the culture is performed under aerobic conditions such as shaking culture or aeration-agitation culture, and the medium is adjusted to a pH range of 4 to 10, preferably pH 5 to 8, and the temperature is adjusted. The cells are cultured at a temperature in the range of 10 to 40 ° C, preferably 25 to 37 ° C, for 24 to 96 hours. After the culture, the cells can be removed by centrifugation or other appropriate solid-liquid separation means to obtain a culture supernatant. Also the fungus The body can be treated physically or enzymatically to obtain a bacterial extract. Then, high-purity monogalactosidase can be obtained from these crude enzyme solutions by appropriately combining ammonium sulfate salting out, gel filtration, hydrophobic chromatography and the like.

 The enzymes used for the production of oligosaccharides containing a galactosyl group include, in addition to the enzyme preparations obtained as described above, extracts in the case of solid culture, and extracts in the case of liquid culture. Alternatively, a culture supernatant or an intracellular extract can be used as it is as an enzyme preparation. If necessary, an enzyme purified by a known method can also be used. It is also possible to use the cells as they are as an enzyme preparation. Alternatively, α-galactosidase mixed with a commercially available enzyme preparation, for example, a cellulase preparation ゃ protease preparation, can be used. In this case, the enzyme preparation can be used as it is, or a single galactosidase can be prepared from the enzyme preparation by various known methods. It can be used after purification.

 In addition, these enzymes or cells that produce the enzymes can be immobilized and used in a continuous or batch-wise manner in the reaction.

The raw material used for the reaction of α-galactosidase to produce the non-reducing disaccharide of the formula (1) is galactose or a substance containing galactose. Specific examples include galactose and hydrolysates of galactose and other compounds containing galactose such as lactose. These can be used alone or in combination. As galactose, there are not only commercial galatose, but also meribiose, mannino triose, raffinose, stachy, plante, benorenoscose, galactan, galactomannan, arabinogalactan, rhamnogalatatan, Α-galactosyl group or β-galactol such as galactolipid, ferulated galactose, galactovitol, galactosylglycerol, galactinol, lactose, ratatitol, lactulose, galactoligosaccharide, etc. Natural or synthetic oligosaccharides, glycosides or polysaccharides containing sil groups

(β-galactosidase, β-galactosidase, monogalactosidase, etc.) or galactose prepared from hydrolyzate with acid can be used.

 Examples of the hydrolyzate of a compound containing galactose include meribiose, mannino triose, raffinose, stachyose, planteose, verbascourse, galactan, galactomannan, arabinogalactan, rhamnogalactan, galactolipid, ferula oxidation. Α-galactose such as galactose, galactopinitol, galata tosyl glycerol, galactinol, lactose, ratatitol, lactulose, galata tori saccharide and the like; natural or synthetic oligos containing a 3-galact tosyl group; Those obtained by hydrolyzing sugars, glycosides or polysaccharides with enzymes (eg, 3-galactanase, β-galactosidase, α-galactosidase) or acids can be used as they are.

 Raw materials used for the reaction of α-galactosidase to produce the non-reducing disaccharide of the formula (2) include substances containing galactose and glucose. Specifically, a mixture of galactose and glucose is suitably used. Examples thereof include a mixture containing lactose and glucose, and a compound containing galactose and glucose at an appropriate ratio. Lactose hydrolyzate obtained by hydrolyzing inexpensive lactose with -galactosidase or an acid is preferably used as it is. The ratio of galactose to dulcose is not particularly limited.

Glucose that binds to galactose is not only commercially available glucose, but also starch, malto-oligosaccharide, isomaltoligo-sugar, nigerooligo-sugar, koji-oligosaccharide, cyclodextrin, trehalose, maltitol, cellulose, cellooligo-sugar , Or sophoro-oligo-sugar, laminario-oligo-sugar, gentio-oligosaccharide, etc. Hydrolysis of natural or synthetic oligosaccharides, glycosides or fertility, including enzymes, such as amylase, gnorecoamylase, cellulase, a-dalcosidase, β-g / recosidase, or acids Gnorecose prepared from the product can be used.

 α-Galactosidase is essentially a hydrolase, but if the concentration of galactose in the substrate is increased, it will also catalyze the reverse dehydration reaction of the hydrolysis reaction. Therefore, when a single galactosidase is allowed to act on a high concentration of galactose, it has the following structural formula: << — (G a)) n (n is usually an integer of 2 to 10 and G a1 is galactose) It produces an oligosaccharide containing an α-galactosyl group in a composition in which a large number of oligosaccharides such as disaccharide, trisaccharide, and tetrasaccharide are mixed.

G al _a l of non-reducing disaccharide of the formula of the present invention (1) -: L 0 G a Ui, can manufacture by performing decomposition and separation of reducing sugars oligosaccharide obtained above. The decomposition of the reducing sugar in the oligosaccharide can be carried out by alkali decomposition, for example, by adding an alkali such as sodium hydroxide or potassium hydroxide to the oligosaccharide. Galal — 1 Oligosaccharides other than i3Gal are degraded. The addition amount of alkali is preferably from 0.1 to 6. ON final concentration. The separation operation can be performed using a known separation means such as ion exchange chromatography, reverse phase chromatography, activated carbon column chromatography, and gel filtration column chromatography, but activated carbon mouth chromatography is preferred. .

 If glucose is present in the reaction system in addition to galactose, the structural formula of the combination of galactose and glucose (G a 1) n _G lc (where n is usually an integer from! To 9 and G lc is glucose) An oligosaccharide containing an α-galactosyl group is produced, and G a1 l 1 j3 G a1 is also present.

Galal-lj3Glc of the non-reducing disaccharide represented by the formula (2) of the present invention Hydrolyzes the mixed Galal-lj3Gal, and further decomposes and separates the reducing sugar in the α- (Gal) n-G1c oligosaccharide obtained above. It can be manufactured by performing. The hydrolysis of 110 Gal can be performed using an enzyme, and the enzyme is preferably -galactosidase.

 The amount of galactosidase to be added is appropriately determined in consideration of the conditions such as the origin and activity of the enzyme, which are appropriate for each enzyme. Decomposition of reducing sugars in oligosaccharides can be carried out by alkali decomposition, for example, by adding alkaline water such as sodium hydroxide or hydroxylic power to oligosaccharides. By this decomposition operation, oligosaccharides other than Galal-1 / 3G1c are decomposed. The added amount of the alkali is preferably 0.1 to 6. ON final concentration. The separation operation can be performed using a known separation means such as ion exchange chromatography, reverse phase chromatography, activated carbon column chromatography, and gel filtration column chromatography, but activated carbon mouth chromatography is preferred.

 In the production of the non-reducing disaccharides of the above (1) and (2), as the enzyme reaction proceeds, the oligosaccharide containing an α-galactosyl group synthesized by the dehydration condensation reaction is again subjected to monogalactosidase. The glycosyltransferase is also decomposed and the transglycosylation reaction occurs in parallel, so the transglycosylation also contributes to the synthesis of oligosaccharides containing a single galactosyl group.

 In addition, the galactose bond position and the number of bonds of the produced compound, or the ratio of these compounds, are affected by the composition of the raw material galactose and glucose, the origin of the enzyme used and the reaction conditions.

The reaction conditions for α-galactosidase vary depending on the enzyme used, but the reaction ρΗ is in the range of 3.0 to 10.0, preferably 4.0 to 9.0. The reaction temperature is desirably high in terms of solubility and reaction rate, and is usually in the range of 20 to 90 ° (preferably 40 to 80 ° C.). It usually varies from 1 to 150 hours, depending on the amount used. However, the present invention is not limited to the above conditions or only the reaction mode.

 Furthermore, in order to produce oligosaccharides containing a galactosyl group, the higher the concentration of the raw material galactose, the better.The galactose-glucose precipitates in the reaction system, and the galactose-glucose is supersaturated. It may be in a state, and is usually used at a concentration of 5 to 110% (w / v), and preferably at a concentration of 50 to 110% (w / V). BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a diagram showing a calibration curve when the molecular weight of α_galactosidase obtained by a production example in the best mode for carrying out the invention described below is measured by HPLC.

 FIG. 2 is a view showing the results of measuring, by SDS-PAGE, the molecular weight of α_galactosidase obtained in a production example in the best mode for carrying out the invention described below.

 FIG. 3 is a diagram showing a change over time of a dehydration condensation reaction using galactose as a raw material in the best mode for carrying out the invention described below. Hereinafter, the present invention will be described with reference to the drawings. BEST MODE FOR CARRYING OUT THE INVENTION

 The present invention will be described with reference to examples, but the present invention is not limited to the following examples.

Reference Example 1 (Paranitrophyl 0; a method for measuring α-galactosidase activity using galactoside as a substrate)

10 mM NO. Rani Torofue two Honoré α- galactosylceramide Toshido 0. 2m l and 4 0 mM buffer ([rho H are similar to that optimum PH for the enzyme) to 0. 2m l α _- Galata DOO 0.05 ml of the sidase solution was added, and the mixture was reacted at 40 ° C. for 10 minutes. After the reaction, the reaction was stopped by adding 0.5 ml of 0.2 M sodium carbonate, and the amount of released pentanol was measured by measuring the absorbance at 400 nm with a spectrophotometer. One unit (U) of the enzymatic activity was defined as the amount of the enzyme that liberated 1 micole of paraditrophenol per minute under these conditions. Reference Example 2 (Measuring activity of Hi-galactosidase using melibiose as substrate)

10 mM melibiose 0.2 ml and 40 mM buffer (pH is based on the optimal pH of enzyme) Add 0.25 ml of α-galactosidase solution to 0.05 ml At 40 ° C. for 10 minutes. Next, the reaction was stopped by heating at 100 ° C. for 10 minutes to stop the reaction, and the amount of generated glucose was measured using Roche Diagnostics Co., Ltd. F-kit (glucose Z fructose) or high performance liquid chromatography ( HP LC). One unit of enzyme activity ( ^UM ) was defined as the amount of enzyme that produced 1 micromole of glucose per minute under these conditions.

Production example (Production of Hi-ichi galactosidase)

90 ml of a liquid medium (pH 6.0) containing 5% wheat bran was placed in a 500 ml 1-volume flask, sterilized by autocrepe using a conventional method, and then subjected to Aspergillus. ■ Niger AP C—93 19 strains (deposit number: FE RM BP—7680) were inoculated and pre-cultured (seed culture) at 25 ° C. for 3 days. After adding 400 ml of water to 500 g of the bran and sterilizing, 1 Om1 of the preculture liquid was inoculated, stirred well, and then main-cultured at 25 ° C for 4 days. After the cultivation, the bran koji was crushed finely, 8 L of water was added thereto, and the mixture was extracted overnight at 4 ° C, and then filtered with a filter paper to obtain an extract filtrate. When the α-galactosidase activity of the obtained extract filtrate was measured, it was 3 units (U) per 1 ml of the extract filtrate. 1 L of 6 L of extraction filtrate is filtered with an ultrafiltration membrane (SIP made by Asahi Kasei Corporation) Then, ammonium sulfate was added to achieve 70% saturation, and salting out was performed. Subsequently, the precipitate was collected by centrifugation, dissolved in 500 ml of water, concentrated to 100 ml with an ultrafiltration membrane, and further added with 500 ml of water to 100 ml. After concentration, this operation was repeated three times to perform desalting. After desalting, freeze-drying was performed to obtain a freeze-dried powder (250 UZg).

 Ammonium sulfate was added to the extract filtrate obtained above so as to be 70% saturated and stirred, and then left at 4 ° C. for a while. The precipitate was collected by centrifugation, dissolved in 10 mM phosphate buffer (pH 6.0), concentrated with an ultrafiltration membrane (SIP manufactured by Asahi Kasei Corporation), and the buffer was reconstituted again. It was added and concentrated. This operation was repeated three times for desalting.

 Next, in order to perform ion-exchange chromatography, the sample was applied to a column of DAE-Toyopearl 65 M (manufactured by Tosoichi Co.) equilibrated with the same buffer. Subsequently, in order to perform hydrophobic chromatography, it was subjected to a petroleum yopal 650 M (manufactured by Tosoh I) in 50% saturated ammonium sulfate. The active fractions were collected and subjected to gel filtration chromatography. Topearl HW-55 was equilibrated with 50 mM acetate buffer (pH 5.5) containing 0.3 M sodium chloride. S (manufactured by East Souichi Co., Ltd.) These column chromatographs gave proteinaceous a-galactosidase.

The a-galactosidase obtained above has the following physicochemical properties. ① Action

 Catalyzes the reaction that hydrolyzes a-galactoside bonds to release D-galactose.

G a 1 α 1-OR + H ₂₀ → G a 1-O H + R-OH

(In the formula, G a1 a 1 OR represents a carbohydrate containing a single galactosyl group, G a 1〇H represents free galactose, and R—OH represents various sugars, alcohols and Compounds having a hydroxyl group such as fuynols are shown. )

 ② Substrate specificity

 It acts on meliviose, raffinose, stachyose, etc., which have a single galactosyl group at the non-reducing end, and on paranitrophenyl α-galactoside. Assuming that the decomposition rate of para-trophenyl hyalgalactoside as a substrate is 100, the relative rate of decomposition of meliviose is about 9.

③ Optimum ρ Η and ρ Η stability

 The optimal ρ で is 2.5 to 6.0. When left at 40 ° C for 1 hour, the pH is stable in the range of 3.5 to 8.0.

 ④Optimum temperature and temperature stability

 The optimum temperature at pH 4.5 (acetate buffer) is 60 ° C. It is stable up to 60 ° C when left at pH 4.5 (acetate buffer) for 15 minutes.

⑤Molecular weight and isoelectric point

 The molecular weight measured by a gel filtration method using a YMC-Pack Dio 1 — 200 column (manufactured by YMC) is 2,170,000, and the molecular weight measured by SDS-PAGE is 1 17 , 00 0 (FIGS. 1 and 2). The isoelectric point measured by isoelectric focusing is 4.2.

 This enzyme can be used for a-galactosita produced by Aspergillus niger (A spergi 11 usniger), which has been reported so far, and for 72,000 and 69,000 (all SDS -It is characterized by a higher molecular weight compared to (PAGE).

Example 1 (Production of G a l oi l— l j3 G a l of Formula (1))

Galactose (manufactured by Wako Pure Chemical Industries, Ltd.) 60 g and acetate buffer solution with a pH of 4.5 containing 2,100 U ^M of the galactosidase obtained in the production example 10 Om l (galactose concentration 6 0% (w / v) , enzyme concentration 3 5 U ^M - Gala click toast) was prepared and 3 0 hours reaction at 5 0 ° C. Figure 3 shows the time course of the reaction. The reaction solution was loaded on an activated carbon column, and oligosaccharide was eluted with water at a concentration gradient of galactose and ethyl alcohol of 0 to 30%. The oligosaccharide-eluted fraction was concentrated and dried to obtain 24 g of an oligosaccharide containing an α-galactosyl group. When this oligosaccharide was hydrolyzed with monogalactosidase or acid, only galactose was formed. It should be noted that, among α_galactosidases, Candida gilli enolemon tea (C andidagui 11 iermondii) H-404 strain, which is known to have the highest catalytic activity of the dehydration condensation reaction, is used. When oligosaccharides were produced under the same conditions as above, the yield was 14 g, a production example, and the yield of Hi-galactosidase obtained in this example was excellent.

Next, after dissolving 10 g of the oligosaccharide obtained above in 90 ml of deionized water, 10 ml of 2.0 N sodium hydroxide was added, and the mixture was treated at 100 ° C. for 30 minutes for reduction. Only sugar was degraded. After neutralization with hydrochloric acid, the treated solution was applied to an activated carbon column, and peak a was detected and fractionated. Structural analysis of the obtained peak a fraction by ¹ H—NMR and ¹³ C—NMR confirmed that peak a was Galal—lj3Gal of a non-reducing disaccharide. The attribution data is shown in Table 1. The numerical values are chemical shift values (δ) when TPS is used as an internal standard, and the numerical values in parentheses are binding constants.

C-l C-1 2 C-1 3 C-4 C-5 C-6 H-1 a-1 -Gal (1-1) 1-102.9 71.1 73.5 72.0 74.4 64.0 5.26

 (3.1Hz) β- -Gal 105.9 73.5 75.1 72.0 78.1 63.8 4.57

 (7.5 Hz) Example 2 (Production of Galal-l] 3Glc of formula (2))

Lactose (produced by Wako Pure Chemical Industries, Ltd.) 100 g is commercially available j3-galactosidase (Lactozym manufactured by Novozym Co., Ltd.) to obtain an equal mixture of galactose and glucose. P H4 containing the galactose and glucose in equal mixture 8 5 g and the α- galactosylceramide obtained in Production Example of Toshidaze ^{1, 5 0 0 U M.} 5 acetate buffer 1 00 m 1 (Brix 8 5 % (w / v), enzyme concentration 3 5 U ^M / g- galactose) were prepared and reacted for 8 7 h at 5 0 ° C. 27 g of oligosaccharide containing an α-galactosyl group was obtained from the reaction mixture by activated carbon column chromatography (Candida • = T lienolemon tea, C andidaguil ier iermondii). 16 g). The oligosaccharide containing the α-galactosyl group was hydrolyzed with an acid or α-galactosidase to produce only galactose and glucose.

Next, since the oligosaccharide obtained above contains a mixture of Galal-l ^ Gal, 20 g of this oligo was added to 100 ml of 20 mM phosphate buffer to facilitate purification. The solution was dissolved in a liquid (pH 6.5), and added with 45,000 LAU (unit of lactase from Novozym Co., Ltd.)], followed by addition of 3-galactosidase (Lactozym from Novozym Co., Ltd.). Further, 20 mM phosphate buffer (pH 6.5) was added to the solution so that the solution volume became S180 ml. This solution was treated at 40 ° C. for 24 hours to hydrolyze Gal-l] 3G a1 to D-galactose. Further, 20 ml of 2.0 N sodium hydroxide was added to the 13-galactosidase-treated solution, and the mixture was heated at 100 ° C for 30 minutes to decompose only the reducing sugar. After neutralization with hydrochloric acid, the treated solution was applied to an activated carbon column, and peak a was detected and fractionated. Structural analysis of the obtained peak a fraction by ¹ H-NMR and ¹³ C-NMR confirmed that peak a was a non-reducing disaccharide, Galal-ljS Glc. The attribution data is shown in Table 2. The values are chemical shift values (δ) when TPS is used as an internal standard, and the values in parentheses are binding constants. Table 2

71.1 73.4 .72.0 74.3 64.0 5.25 Cl C-2 C-1 3 C-1 4 C-5 C-6 Hl α--Gal (1-

71.1 73.4 .72.0 74.3 64.0 5.25

 (3.0Hz) β--G 1 c 105.4 75.9 77.2 73.2 78.2 64.0.4.63

 (7.5Hz) Industrial use o

 The novel non-reducing disaccharides represented by the formulas (1) and (2) of the present invention may cause amino acids and proteins to be damaged by the Maillard reaction caused by the reaction between the amino compound and the reducing sugar. Since it is not available, it is expected as a food material that has little adverse effect on other materials in food processing and storage, and as a pharmaceutical material.

 In addition, the novel method for producing a non-reducing disaccharide of the present invention is a method for producing a non-reducing disaccharide, which is derived from Aspergillus niger APC-9319 strain (FE RM BP-7680). By using galactosidase, each of the above non-reducing disaccharides can be produced at a high yield, and the enzyme is used as a source of organic fermentation and various enzyme agents for foods, and is excellent in safety.

Claims

The scope of the claims 1. Non-reducing disaccharide _c containing a galactosyl group represented by the following formula (1):

2. An α-galactosidase derived from a microorganism is allowed to act on galactose or a substance containing galactose, and an oligosaccharide containing an α-galactosyl group is generated by a dehydration condensation reaction, thereby decomposing the reducing sugar in the oligosaccharide. 2. The method for producing a non-reducing disaccharide containing an α-galactosyl group according to claim 1, which is followed by a separation operation.  3. The non-reducing disaccharide containing an α-galactosyl group according to claim 2, wherein the α-galata tosidase is derived from Aspernoreginoles niger (A spergi 11 sniger). Production method.  4. The claim 3 wherein the higarata tosidase is derived from Aspergillus niger (A spergi 11 usniger) AP C—93 19 strain (deposit number: FE RM BP-7680). The method for producing a non-reducing disaccharide containing an α-galactosyl group as described above.  5. A non-reducing composition comprising an α-galactosyl group according to any one of claims 2 to 4, wherein the reducing sugar in the oligosaccharide is decomposed using alkali. Production method of sex disaccharide.  6. The method for producing a non-reducing disaccharide containing a monogalactosyl group according to claim 5, wherein the alkali is sodium hydroxide. 7. A non-reducing disaccharide containing an α-galactosyl group represented by the following formula (2).

8. Microbial α-galactosidase is applied to a substance containing galactose and glucose to produce oligosaccharides containing α-galactosyl group by a dehydration condensation reaction. 8. The method for producing a non-reducing disaccharide containing an α-galactosyl group according to claim 7, wherein the hydrolysis is carried out and then the reducing sugar in the oligosaccharide is decomposed. Method.  9. The method for producing a non-reducing disaccharide containing an α-galactosyl group according to claim 8, wherein the <¾-galactosidase is derived from Aspergillus 2 niger (Aspergii11usniger).  10. The claim according to claim 9, wherein the α-galactosidase is derived from Aspergillus niger (A spergillusniger) APC-9319 strain (deposit number: FE RM BP-7680). A method for producing a non-reducing disaccharide containing an α-galactosyl group according to the above. 1 1. The method according to any one of claims 8 to 10, wherein hydrolysis of the mixed Galal-ljSGal is performed by β-galactosidase. A method for producing a non-reducing disaccharide containing a galactosyl group. 1 2. The α-galactosyl group according to any one of claims 8 to 11, wherein the reducing sugar in the oligosaccharide is decomposed with an alkali. A method for producing a non-reducing disaccharide comprising: 13. The method for producing a non-reducing disaccharide containing a monogalactosyl group according to claim 12, wherein the method is alkali alcohol or sodium hydroxide.