Fermented product of koji mold with high ergothioneine content and method for producing the same

Abstract

To provide a fermented product of koji mold containing ergothioneine at a high concentration and a method for producing the same.SOLUTION: The present invention provides a method for producing a fermented product of koji mold with high ERG content, by culturing using an ergothioneine high-yield koji mold strain. The resulting high-ERG-content koji mold fermented product contains 50 to 50,000 ppm of ERG and is utilized as a high-ergothioneine-content food material such as mycoprotein or rice koji, or as an intermediate material containing ergothioneine.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the production of a koji mold fermentation product containing ergothioneine at a high concentration. [Background technology]

[0002] Ergothioneine (hereinafter, also referred to as "ERG") is a sulfur-containing amino acid with a strong antioxidant effect, and has been found to exist in the living body of plants and animals. ERG has been reported to have not only a strong antioxidant effect, but also an elastase inhibitory effect and a tyrosinase inhibitory effect, and has been particularly attracting attention in the beauty and food fields, such as whitening and wrinkle prevention. It has also been found that ERG is involved in the biological oxidation defense system, and applications in the medical field have also been attempted. Recently, it has also been attracting attention in relation to longevity and mild dementia disorders, for example, because the ERG content decreases with age and ERG has an effect of mitigating telomere length reduction under oxidative stress. 5 mg / day of ERG is required to exert the effect of improving cognitive function in mild dementia disorders (Non-Patent Document 1).

[0003] Plants and animals cannot synthesize ERG, and it is believed that ERG in the body originates from ERG produced by microorganisms such as basidiomycetes. ERG is also found in some edible mushrooms of basidiomycetes, such as enokitake, oyster mushroom, shiitake, maitake, king oyster mushroom, and mushroom, and is known to be particularly abundant in Tamogitake. Foods other than mushrooms do not contain sufficient amounts of ERG to be effective. It is also known that Aspergillus oryzae produces it, but in order to ingest 5 mg of ERG per day, it is necessary to ingest more than 100 g of commonly sold koji per day.

[0004] Methods for producing ERG have been attempted, including extraction from basidiomycetes such as Pleurotus cornucopis, chemical synthesis, and fermentation using microorganisms, but extraction from basidiomycetes takes time to obtain raw materials and is not suitable for mass production. In order to obtain ERG in large quantities, fermentation using bacteria or yeast that can assimilate C1 compounds (Patent Document 1) and fermentation using microorganisms that overexpress ERG biosynthetic genes (Patent Documents 2 and 3) have been studied. Solid culture using Aspergillus oryzae that overexpresses ERG biosynthetic genes has also been attempted, with the production amount being 231 mg / kg (Non-Patent Document 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2016 / 104437 [Patent Document 2] International Publication No. 2017 / 150304 [Patent Document 3] International Publication No. 2019 / 240243 [Non-patent literature]

[0006] [Non-Patent Document 1] Pharmacology and Therapy (2020) Vol.48, Issue 4, p.685-697 [Non-Patent Document 2] BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY (2019) Vol.83, No.1, p.181‐184 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a koji mold fermentation product that has a high ergothioneine content and can be eaten as is to provide a food from which ergothioneine can be sufficiently ingested, or that can be used as a food ingredient or food raw material, and a method for producing the same. [Means for solving the problem]

[0008] As a result of extensive research into ERG-rich koji mold and foods made from this mold, the inventors developed or created a new ERG-rich koji mold strain and discovered that a fermented product obtained by culturing this new strain in a culture medium is an edible koji mold fermented product with a high ERG content, thus completing the present invention.

[0009] The present invention relates to a method for producing a koji mold fermentation product with high ergothioneine content as described in the following (1) to (3), a rice koji as described in (4), a mycoprotein or soy sauce koji as described in (5), and a koji mold fermentation product with high ergothioneine content as described in (6). (1) A method for producing a koji mold fermentation product with a high content of ergothioneine, comprising culturing an ergothioneine-rich koji mold strain in a medium. (2) The production method according to (2) above, which comprises a step of mixing a medium and a koji mold strain that highly produces ergothioneine, and culturing the mixture. (3) The method according to (1) or (2) above, wherein the medium is a medium composed of ingredients having a protein content of less than 5 to 10% (protein content according to the Food Nutritional Composition Table), or a medium composed of ingredients having a protein content of 10% or more. (4) A rice koji fermentation product with a high ergothioneine content produced by the production method described in (3) above, the rice koji being a koji fermentation product with a high ergothioneine content of 0.3 g ergothioneine or more per kg of dry koji fermentation product, obtained in a culture medium composed of a raw material with a protein content of less than 5 to 10%. (5) A mycoprotein or soy sauce koji that is a koji mold fermentation product with a high ergothioneine content produced by the production method described in (3) above, the mycoprotein or soy sauce koji being a koji mold fermentation product with a high ergothioneine content of 0.4 g ergothioneine or more per kg of dry koji mold fermentation product obtained in a culture medium composed of a raw material with a protein content of 10% or more. (6) An ergothioneine-rich koji mold fermentation product containing 50 to 50,000 ppm of ergothioneine. Effect of the Invention

[0010] The koji mold fermentation product of the present invention has a high ergothioneine content and can be used as a food, food ingredient, or food raw material from which ergothioneine can be sufficiently ingested by eating it as is. Furthermore, the high ergothioneine content results in a more delicious food with enhanced flavors such as umami, sweetness, and saltiness. Furthermore, the koji mold fermentation product of the present invention has a pleasant aroma because it has less of the odors specific to koji mold fermentation products, such as koji odor, bran odor, unproductive odor, and oxidized odor. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention relates to a method for producing an ERG-rich koji mold fermentation product using an ERG-rich koji mold strain as the koji mold, and to the obtained ERG-rich koji mold fermentation product. In the present invention, "%" means "% by weight" even if the unit is not specified, and 1 ppm is 0.0001% by weight.

[0012] As the koji mold that produces ERG and serves as the host of the ERG-rich koji mold strain of the present invention, any fungus belonging to the genus Aspergillus can be used. Examples include Aspergillus oryzae, Aspergillus sojae, Aspergillus niger, Aspergillus luchuensis, Aspergillus tamarii, etc. As the koji mold strain, for example, a strain available from a depository institution such as the Aspergillus oryzae RIB326 strain, a strain contained in a commercially available seed koji, or a strain obtained by isolating from a food and beverage production environment such as a sake or soy sauce brewery can be used. In addition, as the host, an ERG-high-producing mutant strain obtained by using a general method for mutagenesis of the wild-type strain or an Aspergillus oryzae strain into which a gene related to ERG biosynthesis has been introduced by genetic recombination can also be used, for example, an Aspergillus oryzae mutant strain into which the egtA gene has been introduced (Patent Document 3, Non-Patent Document 2).

[0013] In this specification, a koji mold strain that highly produces ERG refers to one that produces 5 to 10 times or more, preferably 20 times or more, of ERG when cultured in liquid at 20 to 30°C for 3 to 14 days (Puffmin SM medium (a solution prepared by adding 5 to 10% Puffmin SM (Kikkoman Corporation) to water and suspending it therein), pH unadjusted) compared to a non-mutated or non-transformed strain. Examples of this include an Aspergillus sojae strain in which the above-mentioned egtA gene is forcibly expressed, or an Aspergillus oryzae mutant strain having amino acid mutations or deletions in the W404 to Q515 region of the above-mentioned mutant AO090005000664 gene and its orthologous gene. The AO090005000664 gene was cloned from Aspergillus oryzae RIB40 strain, and has multiple conserved regions that are highly conserved with other species. In particular, the conserved region is the region corresponding to amino acid positions 411 to 470 of SEQ ID NO: 1, and by introducing a substitution mutation into the position corresponding to positions 428 and / or 459 in this conserved region, the expression of ERG in the mutant strain increases by 5 to 10 times or more. Similarly, the expression of ERG increases by 5 to 10 times or more when a deletion mutation is introduced in which 1 to 112 amino acids are deleted from the region corresponding to positions 404 to 515.

[0014] The ERG-rich koji mold fermentation product of the present invention is prepared by fermenting in a conventional solid or liquid medium for culturing koji mold. There are two forms of koji used industrially: solid koji obtained by directly growing koji mold on soybeans, wheat, rice, etc., and liquid koji obtained by growing koji mold in a liquid medium in which soybeans, wheat, rice, etc. are suspended. The koji mold fermentation product of the present invention includes solid koji and liquid koji. The fermentation conditions can be those normally used in this technical field.

[0015] The solid medium used to make solid koji includes a solid medium composed of raw materials with a protein content of less than 5 to 10% in the solid content (protein content in the Food Nutritional Composition Table), and a solid medium composed of raw materials with a protein content of 10% or more in the solid content (protein content in the Food Nutritional Composition Table). For example, rice (protein content 6%), which is a raw material with a protein content of less than 5 to 10%, and beans, fish, meat, algae, and processed products thereof, which are raw materials with a protein content of 10% or more, can be used as raw materials for such solid media. Examples of processed products containing 20% ​​or more of protein include defatted soybeans, fish meal, meat meal, and spirulina powder. The solid medium can be prepared by steaming or boiling the above-mentioned raw materials in boiling water and, if necessary, crushing them. Furthermore, examples of raw materials used in the liquid medium for producing liquid koji include wheat bran, rice, defatted soybeans, corn, soybean pulp, yeast extract, dextrin, and the like.

[0016] After fermentation is complete, the ERG-rich koji mold fermentation product is recovered by solid-liquid separation from the medium, concentrated using ion exchange resins or the like depending on the application, and can be used as is or dried, and seasoned as necessary, as an ERG-containing food material to serve as a raw material for other foods. Furthermore, by using a specific material for the solid or liquid medium and, if necessary, adding seasoning, the ERG-rich koji mold fermentation product obtained can be used as a food ingredient, for example, as a mycoprotein food, or as rice koji or soy sauce koji. In this way, the ERG-rich koji mold fermentation product of the present invention can be used as an intermediate raw material for foods or foods made from the koji mold fermentation product, such as fermented foods, fermented beverages, and fermented seasonings.

[0017] When a solid fermentation product with a high ERG content is produced using the high ERG-producing koji mold strain of the present invention, a solid fermentation product with a high ERG content can be produced that contains, for example, 0.3 g or 0.4 g ERG / 1 kg or more of solid fermentation product, preferably 0.5 g / 1 kg or more of solid fermentation product, more preferably 0.6 g / 1 kg or more of solid fermentation product, and even more preferably 0.8 g / 1 kg or more of solid fermentation product. Rice koji obtained in a culture medium composed of rice, a raw material with a protein content of less than 5 to 10%, becomes an ERG-rich koji mold fermentation product containing more than 0.3 g ERG / 1 kg of dried koji mold fermentation product. Similarly, soy sauce koji obtained in a culture medium with a protein content of 25% made from soybeans and wheat, and mycoprotein obtained in a culture medium composed of raw materials with a protein content of 10% or more, become ERG-rich koji mold fermentation products containing more than 0.4 g ERG / 1 kg of dried koji mold fermentation product.

[0018] When an ERG-rich koji mold liquid fermentation product is produced using the ERG-rich koji mold strain of the present invention, it is possible to produce an ERG-rich koji mold liquid fermentation product containing 50 to 50,000 ppm of ERG. For example, the koji mold fermentation product may contain 50 ppm or more, 100, 300, 500, or 1,000 ppm or more of ERG and 5%, 4, 3, 2, or 1% or less of ERG.

[0019] The concentration of ERG is measured by LCMS under the following conditions. (HPLC conditions) The HPLC analysis is carried out under the following conditions. Equipment: HPLC equipment: HPLC: Nexera series (Shimadzu Corporation) Column: COSMOSIL 2.5HILIC column 3.0 x 150 2.5 μm 3.0 mm I.D. x 15.0 cm (manufactured by Nacalai) Flow rate: 0.5mL / min Temperature: 40℃ Mobile phase: A) 0.1% (v / v) formic acid in water, B) 0.1 (v / v) formic acid in acetonitrile Isocratic: 0-10 minutes (B:80%) Injection volume: 5μL (Mass spectrometry) Equipment: LCMS-2020 (Shimadzu Corporation) (Mass spectrometry (LC-MS) analysis conditions) Ionization conditions: ESI+ MS conditions: SIM ERG: m / z230.1([M+H] + ) Retention time: Around 5 minutes EXAMPLES

[0020] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto. In the examples, "%" simply means "% by weight." For the sensory evaluation, samples at room temperature were used.

[0021] Example 1: Mycoprotein Manufacturing method Fermentation was carried out in a 30 L jar (Marubishi Bioengineering Co., Ltd.). Water was added to 1.5 kg of soybean pulp powder to make the volume 15 L, and 2.25 g of an antifoaming agent, Shin-Etsu Silicone (registered trademark) KM72F (Shin-Etsu Chemical Co., Ltd.), was added and sterilized at 123°C for 60 minutes. An enzyme solution filtered through a filter with a pore size of 0.22 μm was added so that the final concentration of arabinase was 0.6 U / ml and the final concentration of cellulase was 0.1 U / ml, and the mixture was reacted at 60°C for 2 hours with stirring. After the reaction, the mixture was cooled to 30°C. Preculture was carried out as follows. For each strain, 5 g of soybean pulp powder and 100 ml of water were added to one 500 ml baffled Erlenmeyer flask and autoclaved (121°C, 30 minutes). For the seed koji, an Aspergillus oryzae strain in which the G428S mutation had been introduced into the AO090005000664 gene (Example 1) and an Aspergillus oryzae strain in which the AO090005000664 gene is wild-type (Comparative Example 1) were used. 0.5-1 ml of glycerol stock of the seed bacteria was added, and the initial number of spores in the preculture solution was adjusted to 5 x 10 5 The preculture solution was inoculated to a concentration of 1 / ml. This preculture solution was cultured with shaking (30°C, 160 rpm) for 24 hours. The preculture solution was then inoculated into a 30 L jar and cultured at 30°C for 3 days. The aeration rate and stirring rate were started at 0.5 vvm and 250 rpm, and increased to 1 vvm and 330 rpm after 18 hours. After culture, the fermented product was heat sterilized (80°C, 30 minutes).

[0022] The heat-sterilized fermented product was dried for 1 minute in a drum dryer (manufactured by Katsuragi Kogyo Co., Ltd., product name "Drum Dryer D-00") set at an air gap of 0.3 mm and a surface temperature of 150°C. This is referred to as fermented soybean pulp. The ergothioneine content was 1.70 g / kg of the dried koji mold fermented product (Example 1) and 0.1 g / kg of the dried koji mold fermented product (Comparative Example 1). Using the fermented okara obtained by the above procedure, 16.7 g of the dried material was mixed with water in a ratio of 1:2 by weight, and then 3% soy sauce and 1% yeast extract were mixed and kneaded for 10 seconds with a kneading machine (Tiger Corporation, product name "Microcomputer Food Processor SKF-H101"). The kneaded sample was then molded into a size of 1.8 cm thick and 7.5 cm in inner diameter. 5 ml of oil was added to a frying pan and the molded sample was baked over low heat for 1 minute 20 seconds on each side, for a total of 2 minutes 40 seconds. The ergothioneine content was 1 g / kg dried koji mold fermented product (Example 1) and 0.05 g / kg dried koji mold fermented product (Comparative Example 1). A sensory evaluation was conducted by a panel of five experts on the taste of the mycoproteins of Comparative Example 1 and Example 1. All panelists evaluated that the mycoprotein of Example 1 had a stronger umami taste and was tastier. In addition, Example 1 had a weaker odor than Comparative Example 1, making it more suitable for processing.

[0023] [Example 2: Shio-koji] Rice koji production 1250g of polished rice was soaked overnight, transferred to a colander and drained for 2 hours. It was then wrapped in Tetoron (registered trademark) cloth (Toray Industries, Inc.) and steamed at 100°C for 40 minutes. The steamed rice was cooled to 45°C and inoculated with koji seed. Aspergillus oryzae strains in which the gene mutation G428S mutation was introduced into the AO090005000664 gene (Example 2) and Aspergillus oryzae strains in which the AO090005000664 gene is wild type (Comparative Example 2) were used as koji seed. It was wrapped in Pylen cloth and koji making was started under conditions of 35°C and 95% humidity. 19 hours after the start of koji making, the rice koji was loosened and wrapped in Pylen cloth again. When the product temperature reached 40°C, the Pylen cloth was spread to flatten the rice koji. After another 40 hours, the rice koji was loosened well and left to stand again. After 42 hours, the koji was discharged. The ergothioneine content was 0.35 g / kg of the dried koji mold fermentation product (Example 2) and 0.03 g / kg of the dried koji mold fermentation product (Comparative Example 2).

[0024] Making Shio-Koji 20 g of salt and 100 ml of hot water at 50° C. were added to 50 g of the obtained rice koji, and the mixture was reacted at 50° C. and 130 rpm for 14 hours. The ergothioneine content was 0.1 g / kg of the dried koji mold fermentation product (Example 2) and 0.01 g / kg of the dried koji mold fermentation product (Comparative Example 2). A sensory evaluation was carried out by a panel of four experts on the taste of the shio koji of Comparative Example 2 and Example 2, and all of the panelists evaluated that the shio koji of Example 2 had a stronger umami flavor and was more delicious.

[0025] [Example 3: Amazake] Amazake production 2.5 times the amount of 60°C hot water was added to 800g of rice koji obtained in Comparative Example 2 and Example 2, and saccharification reaction was carried out at 55°C and 200 rpm. After 16 hours, the Brix was adjusted to 18%, transferred to a heat-resistant container, and sterilized at 85°C for 30 minutes. The ERG content of the obtained amazake was 115 ppm (Example 3) and 7 ppm (Comparative Example 3). Example 3 had a stronger sweetness, a whiter color, a stronger chestnut aroma, and a better aroma due to less koji odor and rice bran odor compared to Comparative Example 3, and had a smoother mouthfeel due to fewer particles with large particle sizes.

[0026] [Example 4: Rice malt soaked in soy sauce] Manufacturing of soy sauce-pickled rice koji To 50 g of the rice koji obtained in Comparative Example 2 and Example 2, 4 g of salt and 75 ml of soy sauce were added and reacted for 14 hours at 50°C and 130 rpm. The ergothioneine content was 0.14 g / kg of the dried rice koji soaked in soy sauce (Example 4) and 0.01 g / kg of the dried rice koji soaked in soy sauce (Comparative Example 4). A sensory evaluation was conducted by a panel of four experts on the taste of the soy sauce-soaked rice koji of Comparative Example 4 and Example 4. All panelists evaluated that the soy sauce-soaked rice koji of Example 4 had a stronger umami flavor and was more delicious.

[0027] [Example 5: Sauce (hishiho)] Method of making sauce Equal amounts of steamed and denatured defatted soybeans and crushed roasted wheat were mixed, inoculated with seed koji, and koji was produced for 42 hours by a conventional method to obtain soy sauce koji. As the seed koji, an Aspergillus oryzae strain in which the G428S mutation was introduced into the AO090005000664 gene (Example 5) and an Aspergillus oryzae strain in which the AO090005000664 gene is wild type (Comparative Example 5) were used. 100 parts by mass of the obtained soy sauce koji were added to 130 parts by mass of salt solution (salt concentration 26% (w / v)), and fermented and aged at 25 to 30 ° C. for 150 days while appropriately stirring, following conventional moromi control. The ergothioneine content was 0.23 g / kg soy sauce (Example 5) and 0.04 g / kg soy sauce (Comparative Example 5). A sensory evaluation was carried out by a panel of four experts on the taste of the soy sauces of Comparative Example 5 and Example 5. All the panelists evaluated that the soy sauce of Example 5 had a stronger umami taste and was tastier. In addition, Example 5 had less stale odor and oxidized odor than Comparative Example 5.

[0028] [Example 6: Aspergillus fermented seasoning liquid] Method for producing koji-fermented seasoning liquid An Aspergillus oryzae strain with the G428S mutation introduced into the AO090005000664 gene was inoculated into a 2.15% dextrin, 4.5% yeast extract medium, and liquid cultured at 26°C for 7 days. The resulting culture solution was heated in a tank at 90°C for 30 minutes, and then filtered. The ERG in the resulting filtrate was adsorbed onto ion exchange resin UBK08 (manufactured by Mitsubishi Chemical Corporation) and eluted with ammonia water to obtain a koji mold fermented seasoning liquid. The ERG concentration in the resulting koji mold fermented seasoning liquid was 30,000 ppm. The koji-fermented seasoning liquid was found to have the effect of enhancing saltiness, sweetness, and umami.

[0029] [Test example: Shio-koji] A panel of four experts conducted a two-point discrimination test to evaluate the taste (umami, sweetness, bitterness) of a test product (ERG5) made by adding 5 ppm of ERG (Tetrahedron) to shio-koji (Plus Koji Nama Shio-koji, Marukome Co., Ltd.) and a control (ERG0) made by adding no ERG, compared to the control. The results of the sensory evaluation are shown in Table 1 below. Sensory evaluation ○: The taste is enhanced compared to the control. ◇: Tastes the same as the control.

[0030] [Table 1] The addition of 5 ppm ERG enhanced the umami and sweetness of shio-koji.

Claims

1. A method for producing an ergothioneine-rich koji mold fermented product, characterized by culturing an ergothioneine-high-producing koji mold strain in a medium as koji mold.

2. The production method according to Claim 1, comprising a step of mixing a medium and an ergothioneine-high-producing koji mold strain and culturing them for 1 to 3 days.

3. The production method according to Claim 1 or 2, wherein the medium is a medium composed of a raw material with a protein content of less than 5 to 10% or a medium composed of a raw material with a protein content of 10% or more.

4. An ergothioneine-rich koji mold fermented product produced by the production method according to Claim 3, which is obtained from a medium composed of a raw material with a protein content of less than 5 to 10%, and is an ergothioneine-rich koji mold fermented product containing 0.3 g or more of ergothioneine per 1 kg of dry koji mold fermented product, namely rice koji.

5. An ergothioneine-rich koji mold fermented product produced by the production method according to Claim 3, which is obtained from a medium composed of a raw material with a protein content of 10% or more, and is an ergothioneine-rich koji mold fermented product containing 0.4 g or more of ergothioneine per 1 kg of dry koji mold fermented product, namely mycoprotein or soy sauce koji.

6. An ergothioneine-rich koji mold fermented product containing 50 to 50,000 ppm of ergothioneine.

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