Method for producing indigestible oligosaccharides

Abstract

To provide methods for preparing functional (or indigestible) oligosaccharides derived from raw materials such as grains without artificial glucoside bond formation processes such as heat treatment and enzyme treatment, which becomes a psychological obstacle for consumers.SOLUTION: The problem is solved by a method for producing indigestible oligosaccharides comprising: preparing a gelatinized starch by gelatinizing a saccharide raw material; adding an enzyme to the gelatinized starch and hydrolyzing it to prepare a hydrolyzate; and centrifuging the hydrolyzate to obtain a supernatant.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for producing indigestible oligosaccharides by producing indigestible oligosaccharides from saccharide raw materials.

Background Art

[0002] Oligosaccharides are polymers of monosaccharides with a degree of polymerization of about 2 to 20, and there are various oligosaccharides due to differences in their constituent sugars and bonding modes.

[0003] In recent years, it has been revealed that oligosaccharides exhibit various biological activities and have attracted attention. In particular, indigestible oligosaccharides, which are characterized by low digestibility by various hydrolases, have attracted attention as a method for solving the problem of decreasing intake of dietary fiber, which has become a problem due to the Westernization of eating habits. In addition, oligosaccharides are said to be effective for maintaining health and preventing diseases, and are used not only for improving the intestinal microflora, which is called prebiotics, and as a low-cariogenic sweetener, but also for maintaining the quality of bread, and are widely used in various foods and beverages.

[0004] These indigestible oligosaccharides are produced by a roasting method or an enzymatic method using starch as a raw material. The roasting method is a method of forming complex branches by subjecting starch to high heat treatment under conditions containing a certain acid to cause hydrolysis and polymerization, and indigestible sugars are produced.

[0005] The oligosaccharides obtained by the roasting method have a pungent odor and are not preferable in terms of taste. However, there is a method for producing indigestible oligosaccharides by removing them by subjecting them to α-amylase treatment, glucoamylase treatment, transglucosidase treatment, hydrogenation treatment, desalting according to ion exchange, or chromatographic separation (Japanese Patent Publication No. 2-1,451,69, Japanese Patent Laid-Open No. 2-154,664).

[0006] On the other hand, methods for producing indigestible oligosaccharides by forming new glycosidic bonds through enzymatic action are also known. For example, there are methods for producing a mixture of gluco-oligosaccharides containing (α1→4) and (α1→6) glucosidic bonds using glucanotransferase (Japanese Patent Publication No. 2012-525840), and methods for synthesizing oligosaccharides containing (α1→2)(α1→3) bonds using heat-resistant glycosyltransferase (Japanese Patent Publication No. 2011-177118).

[0007] Other indigestible oligosaccharides include xylooligosaccharides obtained by xylan hydrolysis and agarooligosaccharides obtained by agar hydrolysis, but challenges include the limited availability of raw materials and the fact that the enzymes used for hydrolysis are specialized and expensive.

[0008] On the other hand, starches obtained from grains and potatoes contain few impurities, have almost no off-flavors, off-odors, or discoloration, and are inexpensive. However, these starches are easily digestible, and there is no method to produce indigestible oligosaccharide compositions using them as raw materials. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Patent No. 2-145169 [Patent Document 2] Japanese Patent Application Publication No. 2-154664 [Patent Document 3] Special Publication No. 2012-525840 [Patent Document 4] Japanese Patent Publication No. 2011-177118 [Overview of the project] [Problems that the invention aims to solve]

[0010] Oligosaccharides are used in a wide range of fields, but when it comes to ingested products, consumers tend to accept natural products more readily, as they have fewer psychological barriers. Therefore, a manufacturing method that utilizes glycosidic bonds contained in natural products and extracts oligosaccharides containing them is preferable to indigestible oligosaccharides produced by forming new glycosidic bonds.

[0011] Furthermore, some current manufacturing methods face challenges such as difficulty in obtaining raw materials, and the enzymes necessary for branching formation can be expensive or difficult to obtain.

[0012] Therefore, the present invention aims to provide a method for producing functional (or indigestible) oligosaccharides derived from raw materials such as grains themselves, without involving artificial glucosidic bond formation processes such as heat treatment or enzymatic treatment, which can be psychologically detrimental to consumers. [Means for solving the problem]

[0013] The inventors have been studying sake oligosaccharides because they have a distinctive structure (Journal of Bioscience and Bioengineering 124(2)171-177). From structural analysis of oligosaccharides with a degree of polymerization of 6 to 8, they found that some of these oligosaccharides have two α-1,6 linkage branches adjacent to the non-reducing end region of the α-1,4 linkage main chain. The three-dimensional structure of one of these, DP6-1, is shown in Figure 1. They discovered that DP6-1 has significantly lower digestibility by glucoamylase compared to isomaltoligosaccharides and maltooligosaccharides (Carbohydrate Polymers Volume 251, 1 January 2021, 116993).

[0014] Since such a branched structure pattern has not been found in starch, although the effects of glycosyl transfer and the like were investigated, it was found that adjacent branched structures contained in starch exist in rice starch, rather than being generated by glycosyl transfer. Since sake oligosaccharide DP6-1 having such a branch at the non-reducing end is indigestible, it has been considered that indigestible oligosaccharides can be produced by extracting the branched structure originally contained in starch without going through roasting or glycosyl transfer by enzymes. Furthermore, since it was found that the starch of a wide variety of grains contains the said branched structure, it was considered that there is also an advantage in that indigestible oligosaccharides can be prepared from various cereals containing starch, and the present invention was completed.

[0015] The present invention has been made based on such findings, and provides a method for producing indigestible oligosaccharides, which includes a step of preparing gelatinized starch by gelatinizing a saccharide raw material, a step of preparing a hydrolyzate by adding an enzyme to the gelatinized starch and hydrolyzing it, and a step of centrifuging the hydrolyzate to obtain a supernatant.

Advantages of the Invention

[0016] According to the present invention, safe indigestible oligosaccharides can be produced at low cost using a saccharide raw material with dietary habits.

Brief Description of the Drawings

[0017] [Figure 1] It is a stereostructure formula of indigestible oligosaccharides produced by the present invention. [Figure 2] It is a diagram showing the result of analyzing oligosaccharides extracted from an enzymatic hydrolyzate of rice by LC-TOF / MS. [Figure 3] It is a diagram showing the result of column purification and NMR analysis of the compound at the peak in FIG. 2. [Figure 4] It is a diagram showing the result of analyzing oligosaccharides extracted from enzymatic hydrolyzates of various saccharide raw materials by LC-TOF / MS.

Embodiments for Carrying Out the Invention

Examples

[0018] 1. Alpha - amylase digestion and glucoamylase digestion of rice 20 g of Yamada Nishiki (milled rice yield 70%) was thoroughly pulverized with a mixer and then suspended in 400 mL of 20 mM acetate buffer (pH 5.0). After treatment in a boiling water bath for 30 minutes, it was autoclaved for 30 minutes to achieve a gelatinized state. 1,000 U of porcine pancreatic alpha - amylase (manufactured by Sigma Aldrich) was added, and after digestion at 55°C for 24 hours, 40 U of glucoamylase (derived from Rhizopus, Toyobo Co., Ltd.) was added, and digestion was carried out at 40°C for 24 hours. After inactivating the enzyme by maintaining it in a boiling water bath for 10 minutes, it was centrifuged at 4°C for 10 minutes at 14,000 rpm and then passed through a membrane filter with a pore size of 0.45 μm to obtain a sample.

[0019] 2. Purification of rice starch - derived oligosaccharides 32 g of activated carbon for chromatography was added to the enzyme - digested sample and stirred well. After filtering with filter paper (No. 2, ADVANTEC) using a Buchner funnel, 2 L of Milli - Q water was added and filtered again. Next, 400 mL of 50% ethanol was added for elution, and the recovered solution was concentrated to 2 mL with an evaporator. Then, using a Shodex HILICpak VN - 50 10E column (10.0 mm×250 mm, particle size 5 μm, Showa Denko K.K.), isocratic elution with a mobile phase of 70% acetonitrile and a flow rate of 3.0 mL / min was carried out at 40°C. The sample injection volume was 50 μL, and fractionation was performed using a fraction collector (ADVANTEC CHF - 122SC).

[0020] 3. Analysis (1) LC - TOF / MS analysis For LC - TOF / MS analysis, a sample that was mixed with an equal amount of acetonitrile and then treated with a 0.45 - μm pore syringe filter was used.

[0021] An ACQUITY UPLC H-Class HPLC system (Waters Corporation, MA, USA) was used. A Shodex HILICpak VN-50 4D column (4.6 mm × 150 mm, particle size 5 μm, Showa Denko Corporation) was used, and isocratic elution of 70% acetonitrile was performed at a flow rate of 0.3 mL / min. The column temperature was 40°C. In the post-column elution, 75% acetonitrile containing 15 mM lithium chloride was mixed at a flow rate of 1 μL / min and introduced into the ESI. Desolvation temperatures were 280°C, desolvation gas flows were 800 L / h, and capillary voltage was 1.1 kV, with a detection mass range of m / z = 50–3,200.

[0022] A XevoG2-XS Q-TOF (Waters Corporation) was used for TOF / MS. Positive mode was selected for ionization using ESI, with desolvation temperatures of 600°C, desolvation gas flows of 1,200 L / h, and a capillary voltage of 3.0 kV. Waters MassLynx V4.1 was used for LC / MS operation and data analysis, with a scan time of 1 sec and a detection mass range of m / z = 50–700. The error range for m / z in EIC preparation was set to ±0.05 Da.

[0023] (2)NMR analysis The sample was dissolved in several mL of heavy water (99.8%) and then dried using an evaporator. This procedure was repeated to replace the hydrogen atoms in the hydroxyl groups with deuterium. This sample was dissolved in 650 μL of heavy water and injected into an NMR tube (4.22 mm × 180 mm, Shigemi Co., Ltd.). An NMR spectrum analyzer (JEOL ECZ 600R, JEOL Ltd.) was used. 1 The 1H-NMR spectrum was measured. Acetone (δ) was used as an internal standard. H :2.23 ppm, δ c A concentration of 30.5 ppm was used.

[0024] 4.Results Oligosaccharides purified from Yamada Nishiki rice were analyzed by LC-TOF / MS, and a clear peak with an elution time matching that of sake oligosaccharide DP6-1 was detected (Figure 2). Columnar purification of the compound at this peak and NMR analysis confirmed its match with sake oligosaccharide DP6-1. 1 A 1H-NMR spectrum was detected (Figure 3). This indicates that the compound is an oligosaccharide with the same structure as sake oligosaccharide DP6-1 (Figure 1). [Examples]

[0025] 1. Extraction of oligosaccharides containing adjacent branches from starches derived from various grains. As samples, commercially available reagents were used: corn-derived starch (Fujifilm Wako Pure Chemical Industries, Ltd.), wheat-derived starch (Fujifilm Wako Pure Chemical Industries, Ltd.), potato-derived starch (Fujifilm Wako Pure Chemical Industries, Ltd.), and rice-derived starch (Sigma-Aldrich). In addition, Thai rice (domestically produced in Thailand), pre-gelatinized rice (Iida Shoji Co., Ltd.), crushed Thai rice (domestically produced in Thailand) as indica rice, and joshinko (rice flour made from crushed non-glutinous rice) (Niigata Prefecture) and mochi rice flour (rice flour made from crushed non-glutinous rice) (Saga Prefecture) as japonica rice.

[0026] 0.5 g of the sample was suspended in 20 ml of 20 mM acetate buffer (pH 5.0), treated in boiling water for 30 minutes, then autoclaved at 120°C for 30 minutes, and immediately placed in a 40°C water bath to prepare gelatinized starch.

[0027] The gelatinized starch sample was enzymatically treated with 5U of porcine pancreatic α-amylase (Sigma Aldrich) and 40U of glucoamylase (derived from Rhizopus, Toyobo Co., Ltd.) at 40°C for 24 hours, and then the enzymatic reaction was stopped by treatment in boiling water for 10 minutes. Subsequently, the sample was centrifuged at 15,000 rpm at room temperature, and the supernatant was used as the sample.

[0028] 2.Analysis (1)LC-TOF / MS analysis For LC-TOF / MS analysis, the sample was mixed with an equal volume of acetonitrile and then processed through a syringe filter with a 0.45 μm pore size.

[0029] An ACQUITY UPLC H-Class HPLC system (Waters Corporation, MA, USA) was used. A Shodex HILICpak VN-50 4D column (4.6 mm × 150 mm, particle size 5 μm, Showa Denko Corporation) was used, and isocratic elution of 70% acetonitrile was performed at a flow rate of 0.3 mL / min. The column temperature was 40°C. In the post-column elution, 75% acetonitrile containing 15 mM lithium chloride was mixed at a flow rate of 1 μL / min and introduced into the ESI. Desolvation temperatures were 280°C, desolvation gas flows were 800 L / h, and capillary voltage was 1.1 kV, with a detection mass range of m / z = 50–3,200.

[0030] A XevoG2-XS Q-TOF (Waters Corporation) was used for TOF / MS. Positive mode was selected for ionization using ESI, with desolvation temperatures of 600°C, desolvation gas flows of 1,200 L / h, and a capillary voltage of 3.0 kV. Waters MassLynx V4.1 was used for LC / MS operation and data analysis, with a scan time of 1 sec and a detection mass range of m / z = 50–700. The error range for m / z in EIC preparation was set to ±0.05 Da.

[0031] To confirm the agreement of elution times with DP6-1, a sake oligosaccharide with adjacent branching, a sake sample containing DP6-1 was analyzed before and after the LC-TOF / MS analysis.

[0032] 3.Results A comparison of the mucochromatograms (Figure 4) revealed that in the corn-derived starch and Thai rice samples, peaks with different elution times from those of the sake oligosaccharide DP6-1 were detected. Therefore, DP6-1 with adjacent branches could not be extracted from these samples. However, in the other cereal samples, peaks with elution times consistent with those of DP6-1 were detected. Thus, it was shown that adjacent branches exist not only in rice but also in cereals such as wheat and potatoes, and that indigestible oligosaccharides containing these adjacent branches can be extracted from their enzymatic hydrolysates.

Claims

1. A method for producing a non-digestible oligosaccharide characterized by low digestibility by hydrolytic enzymes and having two α-1,6 linkage branches adjacent to the non-reducing end region of the α-1,4 linkage main chain, The process involves preparing gelatinized starch by gelatinizing a carbohydrate raw material, The process involves preparing a hydrolyzed product by adding α-amylase and glucoamylase as enzymes to the gelatinized starch and hydrolyzing it, The process of obtaining a supernatant liquid by centrifuging the hydrolyzed product, Having, A method for producing indigestible oligosaccharides.

2. The method for producing indigestible oligosaccharides according to claim 1, wherein the carbohydrate raw material is starch.

3. The method for producing indigestible oligosaccharides according to claim 1 or 2, wherein the carbohydrate raw material is at least one selected from the group consisting of cereals, potatoes, and processed products thereof.

4. A method for producing indigestible oligosaccharides according to any one of claims 1 to 3, wherein the enzyme is an enzyme derived from a filamentous fungus.

5. A method for producing indigestible oligosaccharides according to any one of claims 1 to 4, wherein the enzyme is derived from at least one selected from the group consisting of Aspergillus oryzae, Aspergillus sluthuensis, and Rhizopus oryzae.

6. A method for producing an indigestible oligosaccharide according to any one of claims 1 to 5, wherein the indigestible oligosaccharide has a structure represented by the following formula. 【Chemistry 1】

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