Methods for producing compositions containing sesaminol

Abstract

An object of the present invention is to provide a method for converting sesaminol glycoside to sesaminol and a method for producing a composition containing sesaminol from sesaminol glycoside. [Solution] Sesaminol glycoside or a composition containing sesaminol glycoside is enzymatically treated with α-amylase to cleave the β-1,2-glucosidic bond, β-1,6-glucosidic bond, and sesaminol-glucose β-glucosidic bond of the sesaminol glycoside.

Description

Technical Field

[0001] The present invention relates to a novel production method for producing sesaminol using a glycoside of sesaminol as a substrate.

Background Art

[0002] Sesame oil is more stable against oxidation than other vegetable oils. This is because several sesame-specific antioxidant components (lignans such as sesamol and sesaminol) are present in sesame oil. The most abundant lignans in sesame seeds are sesamin, sesamolin, pinoresinol, sesaminol, etc., and some of them exist as glycosides. Among these sesame lignans, sesaminol has particularly high antioxidant activity. Sesaminol exists as glycosides such as triglucoside [2,6-O-di(β-D-glucopyranosyl)-D-glucopyranosyl sesaminol], diglucoside, and monoglucoside in sesame seeds, and is also produced from sesamolin by an acid-catalyzed reaction in the purification process of sesame oil. Sesaminol exhibits some physiological activities favorable to human health in relation to its strong antioxidant activity. For example, sesaminol is a powerful inhibitor against the oxidation of low-density lipoprotein that causes circulatory system diseases, and it has also been reported to exhibit antitumor activity by inducing apoptosis in human lymphoid leukemia cells. These beneficial physiological activities of sesaminol are exerted exclusively in the form (aglycone) in which the sugar moiety is removed. Sesaminol aglycone is considered to contribute to some of the numerous beneficial physiological functions of sesame that have been revealed in recent years. By hydrolyzing sesaminol glycoside to produce a large amount of sesaminol, various developments such as health supplements become possible. Sesaminol is contained in a large amount in sesame oil cake (SOC) discharged during sesame oil production. Since SOC is often disposed of as waste, sesaminol can be produced using this SOC as an inexpensive raw material. In principle, a method of extracting lignan glycosides containing sesaminol glycoside from SOC with ethanol and producing their aglycone forms by β-glucosidase treatment is considered effective. However, sesaminol glycoside has a branched oligosaccharide structure containing a hardly decomposable β-1,2-bond in its structure, and the aromatic ring of sesaminol causes steric hindrance to the action of β-glucosidase. Therefore, it has been extremely difficult to decompose with conventional β-glucosidases. Thus, in order to realize the industrial production of sesaminol, it has been an important issue to find a method for efficiently removing the sugar moiety of the glycoside.

[0003] As a source of β-glucosidase for obtaining sesaminol from sesaminol glycoside, for example, a method using a microorganism of the genus Aspergillus or a fermentation method using a mixed culture of bacteria of the genus Bacillus and Enterococcus has been disclosed (Patent Documents 1 and 2). However, these methods still have low efficiency, such as requiring a long time to completely decompose sesaminol glycoside, and have not yet been put into practical use.

[0004] On the other hand, the applicant of the present application discovered the microorganism strain KB0549 in SOC and found that the β-glucosidase produced by the microorganism can efficiently produce sesaminol from sesaminol glycoside (Patent Document 3).

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Disclosure of the Invention

Problems to be Solved by the Invention

[0006] The present invention provides a method for more efficiently hydrolyzing sesaminol glycosides.

Means for Solving the Problems

[0007] The inventor of the present application searched for a method for more efficiently hydrolyzing sesaminol glycosides, and surprisingly newly found that sesaminol glycosides can be hydrolyzed with α-amylase instead of β-glucosidase, and completed the present invention. The present invention includes the following aspects. [1] A method for producing a composition containing sesaminol, comprising: enzymatically treating a sesaminol glycoside or a composition containing a sesaminol glycoside with α-amylase to cleave β-1,2-glucoside bonds, β-1,6-glucoside bonds, and β-glucoside bonds between sesaminol and glucose in the sesaminol glycoside. The production method (except when the α-amylase is contained in a living organism). [2] The production method according to [1], wherein the α-amylase is an α-amylase derived from Aspergillus niger Aspergillus niger ). [3] The production method according to any one of [1] to [2], wherein the composition containing the sesaminol glycoside is sesame seeds, sesame oil, sesame press cake, or an extract thereof. [4] The production method according to any one of [1] to [3], wherein the composition containing the sesaminol is a food, a cosmetic, a pharmaceutical, or a quasi-drug. [5] A method for enzymatically treating a sesaminol glycoside with α-amylase to cleave β-1,2-glucoside bonds, β-1,6-glucoside bonds, and / or β-glucoside bonds between sesaminol and glucose in the sesaminol glycoside. [6] The α-amylase is Aspergillus niger Aspergillus niger) The method described in [5], which is the native α-amylase.

Advantages of the Invention

[0008] According to the present invention, sesaminol can be efficiently produced from sesaminol glycoside.

Brief Description of the Drawings

[0009]

Figure 1

Modes for Carrying Out the Invention

[0010] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings, but the present invention is not necessarily limited thereto. The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of this specification, and those skilled in the art can easily reproduce the present invention from the description of this specification. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are shown for illustration or explanation, and the present invention is not limited thereto. It will be apparent to those skilled in the art that various modifications and variations can be made within the spirit and scope of the present invention disclosed in this specification based on the description of this specification.

[0011] As used herein, "comprising" may include "substantially comprising", "essentially comprising", "consisting essentially of", and "consisting of".

[0012] One embodiment of the present invention is a method for producing a composition containing sesaminol. Such a production method includes subjecting a sesaminol glycoside or a composition containing a sesaminol glycoside to enzymatic treatment with α-amylase to cleave the β-1,2-glucoside bond, β-1,6-glucoside bond, and / or β-glucoside bond between sesaminol and glucose of the sesaminol glycoside.

[0013] One embodiment of the present invention is a method for subjecting a sesaminol glycoside to enzymatic treatment with α-amylase to cleave the β-1,2-glucoside bond, β-1,6-glucoside bond, and / or β-glucoside bond between sesaminol and glucose of the sesaminol glycoside.

[0014] α-Amylase means an enzyme (enzyme number EC3.2.1.1) that hydrolyzes the α(alpha)-1→4 bond inside starch (sometimes called diastase, ptyalin, glycogenase, etc.). α-Amylase is usually present in living organisms (microorganisms), but in the present invention, the case where α-amylase is contained in a living organism (so-called fermentation method) is excluded. In one embodiment of the present invention, the α-amylase may be an α-amylase derived from Aspergillus niger ( Aspergillus niger ). It may be in an isolated state or a composition extracted by some method. In one embodiment of the present invention, such an α-amylase derived from Aspergillus niger is sold by Shin Nippon Chemical Industry Co., Ltd. under the name Sumiteam (registered trademark) AS-L.

[0015] A composition containing a sesaminol glycoside (including sesaminol triglucoside, sesaminol diglucoside, sesaminol monoglucoside) is not particularly limited as long as it contains a sesaminol glycoside, and may be sesame seeds, sesame oil, sesame press cake, or an extract thereof. The extract is not particularly limited, and may be extracted by a method usually performed using ethanol and / or water. The composition containing the produced sesaminol may be a product such as food, cosmetics, pharmaceuticals, or quasi-drugs. In one embodiment of the present invention, these products may be produced by including the manufacturing processes (such as column purification etc.) that are usually carried out after the enzymatic reaction.

[0016] Sesaminol triglucoside becomes sesaminol diglucoside (6-SDG or 2-SDG) when the β-1,2-glucoside bond or β-1,6-glucoside bond is cleaved. Alternatively, sesaminol triglucoside becomes sesaminol when the β-glucoside bond between sesaminol and glucose is cleaved. Sesaminol diglucoside (6-SDG or 2-SDG) becomes sesaminol monoglucoside when the β-1,6-glucoside bond or β-1,2-glucoside bond is cleaved. Alternatively, sesaminol diglucoside becomes sesaminol when the β-glucoside bond between sesaminol and glucose is cleaved. Sesaminol monoglucoside becomes sesaminol when the β-glucoside bond between sesaminol and glucose is cleaved. In one embodiment of the present invention, any or all of the above cleavage reactions may occur simultaneously or sequentially.

[0017] The enzymatic reaction time is not particularly limited, but preferably 1, 2, or 3 hours or more, and preferably 15, 20, 25, 30, or 45 hours or less. The initial substrate concentration is preferably 0.3, 0.5, 1, 1.5, or 2% by weight or more, and may be 0.5, 1.0, 2.0, 3.0, or 4.0% by weight or less. In one embodiment of the present invention, sesaminol can be recovered with a conversion efficiency from sesaminol glycoside to sesaminol (that is, the ratio of sesaminol to sesaminol glycoside and sesaminol after the enzymatic reaction) of 97%, 98%, or 99% or more.

[0018] Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited by these examples.

Examples

[0019] Example 1 1.1. Enzyme It was verified whether various sugar hydrolases used for the following food applications could hydrolyze sesaminol glycoside. The reaction temperatures were carried out under the conditions recommended in the catalog, respectively. <Enzymes related to α-glucan> α-Amylase: Sumizyme AS-L (Shin Nippon Chemical Industry Co., Ltd.): Reacted at 65°C for 3 hours β-Amylase: β-Amylase F "Amano" (Amano Enzyme Inc.): Reacted at 55°C for 3 hours Glucoamylase: Nagase Enzyme Agent N-40 (Nagase & Co., Ltd.): Reacted at 50°C for 3 hours <Enzymes related to 1,3-β-glucan> Cellulase: Sumizyme AC-G (Shin Nippon Chemical Industry Co., Ltd.): Reacted at 60°C for 3 hours β-Glucosidase: Sumizyme BGS-L (Shin Nippon Chemical Industry Co., Ltd.): Reacted at 50°C for 3 hours Inulinase: GODO-YNL (Godo Shusei Co., Ltd.): Reacted at 55°C for 3 hours Glucanase: Denazyme GEL-L1 / R (Nagase & Co., Ltd.): Reacted at 55°C for 3 hours <Enzymes related to hemicelluloses> Pectinase: Sumizyme AG2-L (Shin Nippon Chemical Industry Co., Ltd.): Reacted at 55°C for 3 hours Hemicellulase: Hemicellulase "Amano" 90 (Amano Enzyme Inc.): Reacted at 45°C for 3 hours Mannanase: Mannanase BGM "Amano" 10 (Amano Enzyme Inc.): Reacted at 55°C for 3 hours <Others> Invertase: Sumizyme INV-L (Shin Nippon Chemical Industry Co., Ltd.): Reacted at 50°C for 3 hours Purine nucleosidase: Sumizyme PNF-L (Shin Nippon Chemical Industry Co., Ltd.): Reacted at 55°C for 3 hours α-Galactosidase: Sumizyme AGS (Shin Nippon Chemical Industry Co., Ltd.): Reacted at 60°C for 3 hours β-Galactosidase: Denazyme GY2 (Nagase & Co., Ltd.): Reacted at 40°C for 3 hours Tannase: Sumitomo Team TAN (Shin Nippon Chemical Industry Co., Ltd.): Reaction at 55°C for 3 hours Lyase: Alginate lyase (Nagase Sangyo Co., Ltd.): Reaction at 50°C for 3 hours Protease: Sumitomo Team BNP-L (Shin Nippon Chemical Industry Co., Ltd.): Reaction at 55°C for 3 hours

[0020] 1.2. Test method To 40 μl of an aqueous solution containing 2 mg of STG purified from sesame press cake, 160 μl of an aqueous solution of the enzyme containing 20 mg or 20 μl of the enzyme was added, and the mixture was reacted at the optimal temperature of each enzyme for 3 hours. The enzyme reaction was stopped by heating at 98°C for 2 minutes. Then, 800 μl of 80% ethanol was added, and the mixture was subjected to high performance liquid chromatography (HPLC) under the following conditions to analyze sesaminol-related substances. HPLC: HITACHI Chromaster Column: Wakosil-II 5C18HG (4.6 * 250 mm, manufactured by Wako Pure Chemical Industries, Ltd.) Developing solvent: A; 10% acetonitrile + 0.1% trifluoroacetic acid, B; 80% acetonitrile + 0.1% trifluoroacetic acid, developed with a linear gradient of B from 10% to 100% (for 40 minutes). Flow rate: 0.80 ml / min Analysis wavelength: 280 nm The calibration curve was prepared using standard samples of sesaminol and sesaminol-related substances to identify the products and calculate their contents.

[0021] 1.3. Results The results are shown in Figure 1. As shown in Figure 1, there were several enzymes capable of dissociating glucose from STG, but only α-amylase could efficiently isolate sesaminol from STG.

[0022] Example 2 Large-scale preparation of sesaminol 180 ml of the above α - amylase was added to 620 ml of an aqueous solution containing 20 g of STG prepared from sesame press cake (initial concentration of STG substrate: 2.5%), and the mixture was reacted at 55 °C for 30 hours. Sesaminol was recovered by centrifugation etc. and used as the final product. When the final product was measured by HPLC in the same manner as above, the conversion rate of sesaminol was almost quantitative (100%), and the yield was 97% or more.

[0023] Comparative Example 1 Preparation of Sesaminol by Fermentation Method Sesaminol was prepared from STG prepared from sesame press cake by fermentation of the microorganism KB0549 strain found from SOC. The KB0549 strain was inoculated into 2 ml of an aqueous solution containing 0.03% STG, 1.0% tryptone, and 0.5% yeast extract and pre - cultured at 37 °C for 24 hours. 0.5 ml of the pre - culture solution was added to 100 ml of an aqueous solution containing 0.4%, 0.7%, or 1.0% STG and 1.0% tryptone and 0.5% yeast extract, respectively, and fermented at 37 °C for 72 hours. When the fermentation broth was measured by HPLC in the same manner as above, the conversion rates of sesaminol were 97%, 83%, and 60%, respectively.

Industrial Applicability

[0024] By using the present invention, it becomes possible to produce sesaminol from sesaminol glycoside on an industrial scale (especially for food applications).

Claims

1. A method for producing a composition containing sesaminol, comprising: enzymatically treating a composition containing a sesaminol glycoside or a sesaminol glycoside with α-amylase to cleave the β-1,2-glucoside bond, β-1,6-glucoside bond, and β-glucoside bond between sesaminol and glucose of the sesaminol glycoside; wherein the α-amylase is an α-amylase derived from Aspergillus niger; a production method (except when the α-amylase is contained in a living organism).

2. The production method according to claim 1, wherein the composition containing the sesaminol glycoside is sesame seeds, sesame oil, sesame press cake, or an extract thereof.

3. The production method according to claim 1, wherein the composition containing the sesaminol is a food, a cosmetic, a pharmaceutical, or a quasi-drug.

4. A method for enzymatically treating a sesaminol glycoside with α-amylase to cleave the β-1,2-glucoside bond, β-1,6-glucoside bond, and / or β-glucoside bond between sesaminol and glucose of the sesaminol glycoside, comprising: wherein the α-amylase is an α-amylase derived from Aspergillus niger; a method (except when the α-amylase is contained in a living organism).

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