Method for producing a fermented product containing ergothioneine and method for producing ergothioneine

Abstract

To provide methods for producing fermentation products containing ergothioneine and methods of making ergothioneine using microbial fermentation.SOLUTION: The method for producing fermentation products containing ergothioneine of the invention comprises culturing Aspergillus oryzae in a raw material mixture comprising one or more plant raw materials selected from the group consisting of leguminous plants and gramineous plants and one or more amino acid raw materials selected from the group consisting of amino acids and salt thereof and Aspergillus oryzae to obtain a fermentation product. The method of making ergothioneine of the invention comprises a step of obtaining ergothioneine from the fermentation product.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for producing a fermented product containing ergothioneine and a method for producing ergothioneine. Background Art

[0002] Ergothioneine is a sulfur-containing amino acid produced by microorganisms. Plants and animals cannot produce ergothioneine on their own, and accumulate ergothioneine ingested from microorganisms in their bodies.

[0003] Ergothioneine is known to have physiologically functions beneficial to the human body, such as antioxidant effect, skin-beautifying effect, and brain function improving effect.

[0004] Patent Document 1 describes a method for producing ergothioneine, which is characterized by culturing microorganisms such as actinomycetes, enteric bacteria, and yeast in which an ergothioneine biosynthetic gene is overexpressed, and obtaining ergothioneine produced extracellularly.

[0005] Patent Document 2 describes a method for producing ergothioneine by culturing a transformed Aspergillus microorganism that overexpresses a gene encoding an enzyme derived from an Aspergillus microorganism involved in the reaction of producing ergothioneine from histidine and cysteine. Prior Art Documents Patent Documents

[0006] Patent Document 1 International Publication WO2017 / 150304 Patent Document 2 International Publication WO2016 / 121285 Summary of the Invention Problem to be Solved by the Invention

[0007] There is currently no established method for mass-producing ergothioneine.

[0008] Therefore, this specification discloses a fermented product containing ergothioneine and a novel method for producing ergothioneine using microbial fermentation. [Means for solving the problem]

[0009] This specification discloses the following method as a novel method for producing ergothioneine-containing fermented products and ergothioneine using microbial fermentation.

[0010] (1) A process to obtain a fermented product containing ergothioneine by culturing koji mold in a raw material mixture containing one or more plant raw materials selected from the group consisting of leguminous plants and grasses, one or more amino acid raw materials selected from the group consisting of amino acids and salts thereof, and koji mold. A method for producing a fermented product containing ergothioneine. (2) The amino acid raw material is It contains one or more substances selected from the group consisting of cysteine, histidine, cystine, and γ-amino-n-butyric acid and their salts, or It contains one or more selected from ornithine and its salts, and one or more selected from methionine and its salts. The method described in (1). (3) The method according to (1) or (2), wherein the plant material contains one or more selected from the group consisting of soybeans, rice, and barley and processed products thereof. (4) The fermented product contains 25 ppm or more of ergothioneine, according to any one of (1) to (3). (5) A step of obtaining a fermented product containing ergothioneine by culturing koji mold in a raw material mixture containing one or more plant raw materials selected from the group consisting of leguminous plants and grasses, one or more amino acid raw materials selected from the group consisting of amino acids and salts thereof, and koji mold, and Steps to obtain ergothioneine from the fermented product. A method for producing ergothioneine, including the method described above. [Effects of the Invention]

[0011] A ferment containing ergothioneine and ergothioneine can be produced by the method disclosed herein. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 shows the results of Experiment 1. [Figure 2] Figure 2 shows the results of Experiment 2. [Modes for carrying out the invention]

[0013] <Method for producing fermented products containing ergothioneine> A method for producing a fermented product containing ergothioneine according to one embodiment of the present invention is: A process to obtain a fermented product containing ergothioneine by culturing koji mold in a raw material mixture containing one or more plant raw materials selected from the group consisting of leguminous plants and grasses, one or more amino acid raw materials selected from the group consisting of amino acids and salts thereof, and koji mold. It is characterized by including.

[0014] While the methods described in Patent Documents 1 and 2 produce ergothioneine using genetically modified microorganisms, the method according to this embodiment is particularly preferable because it does not require genetically modified microorganisms and can obtain an ergothioneine-containing fermented product using wild-type Aspergillus oryzae.

[0015] The plant raw material used in the method according to the present embodiment is at least one selected from the group consisting of leguminous plants and gramineous plants. As the plant raw material, a part of a plant body such as a seed of one or more plants selected from the group consisting of leguminous plants and gramineous plants, or a processed product obtained by processing a part of a plant body can be used. A seed is preferable as the part of the plant body. The part of the plant body such as a seed includes those in any form such as raw, dried product, and pulverized product.

[0016] As the leguminous plant, at least one selected from soybean and processed soybean products is preferable, and soybean seed or processed soybean seed product is particularly preferable. Examples of the processed soybean seed product include okara, soy milk, tofu, steamed soybean seed and the like, and okara is particularly preferable. The processed soybean seed product such as okara, soy milk, tofu, and steamed soybean seed may be either raw or dried, but a dried one is preferable.

[0017] As the gramineous plant, at least one selected from rice, barley and processed products thereof is preferable, and at least one selected from rice seed, barley seed and processed products thereof is particularly preferable. As the rice seed, for example, polished rice or brown rice in any form such as raw, dried product, and pulverized product can be used. As the barley seed, for example, barley seed in any form such as raw, dried product, and pulverized product can be used. Examples of the processed rice seed product and processed barley seed product include steamed rice seed and steamed barley seed.

[0018] The amino acid raw material used in the method according to the present embodiment is at least one selected from the group consisting of amino acids and salts thereof. As the salt of an amino acid, any salt of an amino acid with an organic acid, an inorganic acid, an organic base or an inorganic base can be used, and examples include hydrochloride, sulfate, sodium salt, potassium salt, magnesium salt, calcium salt, and ammonium salt of an amino acid. Further, for an amino acid having two or more amino groups or two or more carboxyl groups in one molecule, all of the two or more amino groups or carboxyl groups may form a salt, or only a part thereof may form a salt.

[0019] When the amino acid is an α-amino acid in which the α-carbon is a chiral carbon, it may be an L-isomer, a D-pair, or a mixture of both, but it is preferably an L-isomer.

[0020] The amino acid raw material used in the method according to this embodiment preferably contains one or more selected from the group consisting of ornithine, methionine, cysteine, histidine, cystine, γ-amino-n-butyric acid, and L-homocysteine ​​and their salts, and it is particularly preferable that it contains one or more selected from the group consisting of ornithine, methionine, cysteine, histidine, cystine, and γ-amino-n-butyric acid and their salts, or that it contains one or more selected from ornithine and its salts and one or more selected from methionine and its salts. Furthermore, it is particularly preferable that the amino acid raw material used in the method according to this embodiment contains one or more selected from methionine and its salts and one or more selected from amino acids other than methionine and their salts, one or more selected from cysteine ​​and its salts and one or more selected from amino acids other than cysteine ​​and their salts, one or more selected from cystine and its salts and one or more selected from amino acids other than cystine and their salts, or one or more selected from homocysteine ​​and its salts and one or more selected from amino acids other than homocysteine ​​and their salts. It is preferable to use these amino acid raw materials because a fermented product with a high ergothioneine concentration can be efficiently obtained. Ornithine, methionine, cysteine, histidine, cystine, and homocysteine ​​are preferably L-ornithine, L-methionine, L-cysteine, L-histidine, L-cystine, and L-homocysteine, respectively. When combining one or more substances selected from ornithine and its salts with one or more substances selected from methionine and its salts, for example, 1 to 100 parts by mass, preferably 1 to 20 parts by mass, of one or more substances selected from ornithine and its salts can be combined with 10 parts by mass of one or more substances selected from ornithine and its salts.

[0021] The koji mold used in the method according to this embodiment is not particularly limited, but for example, yellow koji such as Aspergillus oryzae and Aspergillus sojae, white koji such as Aspergillus luchensis and Aspergillus mut. kawachii, black koji such as Aspergillus var. awamori and Aspergillus niger, and red koji such as Monascus purpureus can be used, with yellow koji being particularly preferred. Here, Aspergillus oryzae is preferred as yellow koji, and Aspergillus var. awamori is preferred as black koji. As the koji mold, seed koji, in which koji mold is cultivated on a culture medium such as steamed rice, can be used.

[0022] The procedure for preparing the raw material mixture containing plant raw materials, amino acid raw materials, and Aspergillus oryzae used in the method according to this embodiment is not particularly limited. For example, the raw material mixture can be obtained by mixing the plant raw materials and amino acid raw materials first, and then mixing in Aspergillus oryzae. Alternatively, a raw material mixture for producing a second-stage fermented product can be obtained by culturing Aspergillus oryzae in the raw material mixture containing plant raw materials, amino acid raw materials, and Aspergillus oryzae, and then mixing the first-stage fermented product containing Aspergillus oryzae with additional plant raw materials (and additional amino acid raw materials as needed). Furthermore, the plant raw materials and / or amino acid raw materials may be added during the cultivation of Aspergillus oryzae as needed.

[0023] The concentration of amino acid raw materials in the raw material mixture is not particularly limited, but can be, for example, 0.01 to 10% by mass, preferably 0.20 to 5% by mass, and more preferably 0.50 to 3% by mass, relative to the total amount of the raw material mixture. When multiple types of amino acid raw materials are used, the total amount of the multiple types of amino acid raw materials relative to the raw material mixture can be within this range.

[0024] The concentration of plant material in the raw material mixture is not particularly limited, but the plant material (on a dry weight basis) can be, for example, 30 to 80% by mass, preferably 40 to 70% by mass, relative to the total amount of the raw material mixture.

[0025] The raw material mixture may further contain water and other components. The water content can be adjusted as appropriate and is not particularly limited, but can be, for example, 10 to 50% by mass, preferably 20 to 40% by mass, relative to the total amount of the raw material mixture.

[0026] The conditions for culturing Aspergillus oryzae in the raw material mixture are not particularly limited and can be adjusted as appropriate. For example, a temperature of 25 to 45°C, preferably 30 to 40°C, and a cultivation time of 40 to 120 hours, preferably 60 to 110 hours, more preferably 80 to 100 hours can be exemplified. Cultivation can be carried out by static culture.

[0027] The fermented product containing ergothioneine produced by the method according to this embodiment contains ergothioneine at a concentration of, for example, 25 ppm or more, preferably 30 ppm or more, more preferably 40 ppm or more, and even more preferably 50 ppm or more, relative to the total amount of the fermented product. According to the method according to this embodiment, it is possible to produce a fermented product containing such a high concentration of ergothioneine.

[0028] Examples of fermented products containing ergothioneine include the fermented product itself after cultivation, or concentrated or dried versions of the fermented product after cultivation.

[0029] <Manufacturing method for ergothioneine> Another embodiment of the present invention is, A step of obtaining a fermented product containing ergothioneine by culturing koji mold in a raw material mixture containing one or more plant raw materials selected from the group consisting of leguminous plants and grasses, one or more amino acid raw materials selected from the group consisting of amino acids and salts thereof, and koji mold, thereby fermenting the raw material mixture, and Steps to obtain ergothioneine from the fermented product. This invention relates to a method for producing ergothioneine, including the present invention.

[0030] The step of obtaining a fermented product containing ergothioneine can be carried out by the method for producing a fermented product containing ergothioneine according to the above-described embodiment of the present invention, so the explanation is omitted.

[0031] The process of obtaining ergothioneine from the fermented product is not limited to the process of obtaining ergothioneine as a purified product. Examples of the process of obtaining ergothioneine from the fermented product include extracting ergothioneine from the fermented product, or purifying or crudely purifying ergothioneine from the fermented product or its extract using concentration techniques such as chromatography, solvent fractionation, distillation, or crystal precipitation. [Examples]

[0032] Experiment 1: Production of ergothioneine-containing fermented product using okara (soy pulp) to which various amino acid raw materials have been added. Okara (soy pulp) was fermented with various amino acid raw materials and yellow koji (Aspergillus oryzae), and the concentration of ergothioneine in the fermented product was checked.

[0033] (Procedure for producing fermented products) The amino acid raw materials (ornithine and methionine used together, others used individually) in the amounts shown in the table below were dissolved in 30 ml of distilled water to create an aqueous solution. This solution was then added to 50 g of okara (dried okara, Yamami Co., Ltd.) and mixed uniformly. The resulting mixture was autoclaved at 121°C for 15 minutes.

[0034] The autoclaved mixture was transferred to a container (capacity 700-940 mL) and cooled to around 35°C. Then, 0.4 g of commercially available starter koji containing yellow koji (Aspergillus oryzae) (BF1 strain, for miso and amazake, Higuchi Matsunosuke Shoten Co., Ltd.) was inoculated and mixed uniformly to obtain the raw material mixture.

[0035] The container holding the raw material mixture was placed in a 35°C constant temperature incubator to cultivate yellow koji and ferment the raw material mixture.

[0036] After 43 or 67 hours from the start of cultivation, maintenance (opening the container lid and stirring the contents to loosen them) was performed, followed by a 2-hour drying period (cooling while allowing moisture to evaporate), resulting in fermented products with a cultivation time of 45 or 69 hours.

[0037] (Amino acid raw material)

[0038] [Table 1]

[0039] (Quantitative determination of ergothioneine) The ergothioneine (EGT) concentration in the fermented product was quantified using the following method.

[0040] (1) Preparation of the sample 0.5 g of the sample to be analyzed was placed in a 15 ml test tube, and 9.5 ml of 0.1% formic acid solution was added. The test tube was shaken for 30 mins, then centrifuged at 3000 rpm for 10 mins. 200 μl of the supernatant was placed on an ultrafiltration filter (Amicon Ultra-0.5 ml 3K, Merck Millipore) and centrifuged at 15000 rpm for 30 mins. 900 μl of 0.1% formic acid solution was added to 100 μl of the filtrate and mixed. The mixture was passed through a 0.2 μm filter and placed in a polypropylene vial to be used as the measurement sample.

[0041] (2) Preparation of samples for calibration curve A calibration curve sample was prepared by serially diluting ergothioneine (TRONTO RESEARCH CHEMICALS) standard with 0.1% formic acid solution.

[0042] (3)LCMS analysis conditions LC: Ultimate 3000 (Thermo Fisher Scientific) MS:Q Exactive Focus (Thermo Fisher Scientific) Analytical column: Discovery HS F5, 5μm, 250mm x 4.6mm, 5μm (SIGMA-ALDRICH) Column temperature: 40℃, injection volume: 2μl, flow rate: 0.3ml / min Mobile phase A: 0.1% formic acid solution (formic acid, Fujifilm Wako Pure Chemical Industries) Mobile phase B: Acetonitrile (LCMS grade, Fujifilm Wako Pure Chemical Industries) Mobile phase conditions:

[0043] [Table 2] MS conditions: Spray voltage 3.5kV, capillary temperature 250℃, MS scan range m / z 67-1005, ionization mode ESI positive, negative (switching)

[0044] (4) Analysis of LCMS data The precise mass of the ergothioneine proton adduct, 230.0957 [M+H], was extracted from the LC-MS total ion chromatogram, and its peak area was calculated.

[0045] [Table 3]

[0046] A calibration curve was created, and the ergothioneine concentration in the sample was calculated.

[0047] (result) Figure 1 shows the measurement results of ergothioneine concentration in the fermented product under each condition. The "control" in the figure represents the no-additive (no amino acid raw material added) sample.

[0048] These experimental results show that the concentration of ergothioneine increases significantly when cysteine, histidine, cystine, GABA (γ-amino-n-butyric acid), and a mixture of methionine and ornithine are added.

[0049] Experiment 2: Production of ergothioneine-containing fermented products using different plant raw materials Instead of okara (soy pulp) as in Experiment 1, polished rice or barley was used as the plant raw material to produce a fermented product containing ergothioneine.

[0050] For the milling process, we used raw non-glutinous rice (Hinohikari variety), which was soaked in water, drained, and then steamed.

[0051] For the barley used, we used raw barley (two-row barley variety).

[0052] An aqueous solution of 0.8g ornithine and 0.8g methionine dissolved in 30ml of distilled water was used, along with 50g of okara (soy pulp) or 50g of the above-mentioned polished rice or barley, and 0.4g of yellow koji starter. Fermentation was carried out using the same procedure as in Experiment 1, and the ergothioneine concentration in the fermented product was measured after 69 hours of incubation. The reagents used for ornithine and methionine were the same as those used in Experiment 1.

[0053] (result) Figure 2 shows the measurement results of ergothioneine concentration in the fermented product under each condition. Although not shown in the figure, the ergothioneine concentrations after 45 hours of fermentation using okara (soy pulp) and yellow koji made from barley, without added amino acid raw materials, were 13.80 ppm and 11.59 ppm, respectively.

[0054] Fermented products of polished rice or barley with added methionine and ornithine were shown to contain ergothioneine at a high concentration equivalent to that of fermented products of okara (soy pulp) with added methionine and ornithine.

Claims

1. A process to obtain a fermented product containing ergothioneine by culturing koji mold in a raw material mixture containing one or more plant raw materials selected from the group consisting of leguminous plants and grasses, one or more amino acid raw materials selected from the group consisting of amino acids and salts thereof, and koji mold. A method for producing a fermented product containing ergothioneine, including The aforementioned plant material is one or more selected from the group consisting of soybeans, rice, and barley, and processed products thereof. The aforementioned amino acid raw material is a raw material containing one or more selected from ornithine and its salts, and one or more selected from methionine and its salts. The aforementioned manufacturing method.

2. The method according to claim 1, wherein the fermented product contains 25 ppm or more of ergothioneine.

3. A step of obtaining a fermented product containing ergothioneine by culturing koji mold in a raw material mixture containing one or more plant raw materials selected from the group consisting of leguminous plants and grasses, one or more amino acid raw materials selected from the group consisting of amino acids and salts thereof, and koji mold, thereby fermenting the raw material mixture, and Steps to obtain ergothioneine from the fermented product. A method for producing ergothioneine, comprising: The aforementioned plant material is one or more selected from the group consisting of soybeans, rice, and barley, and processed products thereof. The aforementioned amino acid raw material is a raw material containing one or more selected from ornithine and its salts, and one or more selected from methionine and its salts. The aforementioned manufacturing method.

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