Method for producing γ-aminobutyric acid and its use

Abstract

This invention provides a highly versatile method for fermentation production of GABA with a relatively high conversion rate. [Solution] A microbial fermented product of vegetables, fruits, or grain materials containing 3300 mg / 100g or more of γ-aminobutyric acid, wherein the microorganisms are capable of functionally possessing glutamate decarboxylase. in A fermented product is provided. Preferably, the vegetable, fruit, or grain material is selected from the core, outer leaves, and peel. Also preferably, the vegetable, fruit, or grain material is off-grade.

Description

Technical Field

[0001] The present invention relates to a method for producing γ-aminobutyric acid and its utilization. The present invention is useful in the fields such as food production. Dairy

Background Art

[0002] The functional component γ-aminobutyric acid (GABA), which has been attracting attention in recent years, is known to be contained in specific fresh vegetables, and enriching GABA by processing vegetables has been studied (Non-Patent Documents 1 and 2).

[0003] Furthermore, the fermentation production of GABA is being considered. For example, Patent Document 1 describes a method for producing food containing γ-aminobutyric acid, characterized by inoculating a fermentation raw material containing glutamic acid or its salts with Lactobacillus hilgardii strain K-3 FERM P-18422, a lactic acid bacterium capable of producing γ-aminobutyric acid, and culturing it, and performing the fermentation treatment at an initial pH of 4.5 to 5.5. In this document, when 1 mL of pre-culture solution of strain K-3 was added to 1 L of GYP liquid medium containing 100 g of sodium glutamate and cultured at a temperature of 30°C, the γ-aminobutyric acid content in the culture solution was 48 g / L after 48 hours and 51 g / L after 72 hours, and the conversion rate to γ-aminobutyric acid was calculated to be 87% after 48 hours and 93% after 72 hours. Furthermore, Patent Document 2 describes a method for producing lactic acid fermented foods or seasonings with a high gamma-aminobutyric acid content, characterized by adding glutamic acid or a glutamic acid-containing substance and a strain of lactic acid bacteria capable of producing gamma-aminobutyric acid to a food or seasoning ingredient and carrying out lactic acid fermentation. This document also lists Lactobacillus brevis IFO3345 strain, Lactobacillus brevis IFO3960 strain, Lactobacillus brevis IFO12005 strain, Lactobacillus brevis IFO12520 strain, Lactobacillus brevis IFO13109 strain, and Lactobacillus brevis IFO13110 strain as lactic acid bacteria strains capable of producing gamma-aminobutyric acid. Furthermore, Patent Document 3 describes a method for producing GABA using fermentation by lactic acid bacteria capable of producing γ-aminobutyric acid, characterized in that glutamic acid is used as a substrate in a ratio of 25 parts by mass to 150 parts by mass per 100 parts by mass of sodium glutamate. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2003-070462 (Patent No. 3880820) [Patent Document 2] Japanese Patent Publication No. 2004-215529 (Japanese Patent No. 4344143) [Patent Document 3] Japanese Patent Publication No. 2023-117510 [Non-patent literature]

[0005] [Non-Patent Document 1] Ehime Prefecture Industrial Science Research Report No. 45, pp. 29-34 (2007) [Non-Patent Document 2] Changes in free amino acids in vegetables due to heat treatment with low-temperature steam: Journal of the Japanese Society for Food Chemistry Vol.41(1) 42-48(2008) [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Traditionally, when fermenting glutamic acid to produce GABA, the conversion rate to GABA remained relatively low unless a special strain of lactic acid bacteria was used. It is desirable to have a versatile method that can produce GABA through fermentation with a relatively high conversion rate. [Means for solving the problem]

[0007] To solve the above problems, the inventors attempted to add crushed vegetable liquid, such as cabbage, to a GABA fermentation production system. They found that this allowed for the production of GABA with a relatively high conversion rate. Furthermore, they discovered that this method of using vegetables as a GABA fermentation aid is effective with various fruits, vegetables, or grain materials, including parts that are normally discarded, such as cores, peels, and off-grade products, thus completing the present invention.

[0008] The present invention provides the following: [1] A microbial fermented product of vegetables, fruits, or grain materials containing 3300 mg / 100g or more of γ-aminobutyric acid, Microorganisms that possess glutamate decarboxylase in a functional manner in A certain fermented product. [2] The fermented product according to 1, wherein the vegetable, fruit, or grain material is selected from the core, outer leaves, and peel. [3] If the vegetable, fruit, or grain material is substandard, as described in 1 or 2. The Fermented product. [4] A vegetable, fruit, or grain material selected from a Brassicaceae or Solanaceae plant, as described in any one of items 1 to 3. The Fermented product. [5] A fermented product according to any one of items 1 to 4, wherein the microorganism is lactic acid bacteria. [6] A fermented product according to any one of items 1 to 5, wherein the microorganism is a lactic acid bacterium belonging to the genus Levilactobacillus. [7] A fermented product according to any one of items 1 to 6, wherein the microorganism is a lactic acid bacterium belonging to Levilactobacillus brevis. [8] Fermented product containing 3300 mg / 100g or more of γ-aminobutyric acid, obtained by a manufacturing method including the following steps: A fermentation process in which a microorganism functionally possessing glutamic acid decarboxylase is reacted with a culture medium containing 5-70% glutamic acid and vegetable, fruit, or grain material to obtain a lactic acid fermented product containing γ-aminobutyric acid. [9] A food composition comprising a fermented product as described in any one of items 1 to 8.

[10] The food composition according to 9, wherein the food composition includes fruits, vegetables or grains as raw materials, and the fermented product is a fermented product of materials derived from the fruits, vegetables or grains.

[11] A food composition according to 9 or 10, comprising 10 mg / 100 g of γ-aminobutyric acid.

[12] A method for producing gamma-aminobutyric acid, comprising the following steps: A step of converting glutamic acid in a culture medium to gamma-aminobutyric acid by acting on it with a microorganism functionally possessing glutamic acid decarboxylase, wherein the culture medium contains 5-70% vegetable, fruit, or grain material, thereby improving the conversion rate from glutamic acid to gamma-aminobutyric acid.

[13] In the step of applying lactic acid bacteria, the pH of the culture medium is set to 5 or less. adjustment The production method described in 12.

[14] Use of vegetable, fruit or grain material to improve the conversion rate from glutamate to gamma-aminobutyric acid in the fermentation production of gamma-aminobutyric acid by microorganisms functionally possessing glutamate decarboxylase.

[15] A fermentation aid composition containing vegetable, fruit, or grain material for improving the conversion rate to decarboxylates in the fermentation production of decarboxylates by microorganisms functionally possessing decarboxylase.

[16] The fermentation aid composition according to 15, wherein the decarboxylase is glutamate decarboxylase and the decarboxylated product is γ-aminobutyric acid.

[17] A method for producing a food composition, comprising the following steps: A process to obtain a fermented product containing γ-aminobutyric acid by reacting a microorganism functionally possessing glutamic acid decarboxylase with a culture medium containing 5-70% of a vegetable, fruit, or grain material selected from the core, outer leaves, peel, and off-grade products; A step of fortifying gamma-aminobutyric acid by mixing the obtained fermented product with a raw material containing the unfermented product of the vegetable or fruit. [Brief explanation of the drawing]

[0009] [Figure 1] This figure shows the GABA conversion rate when the concentration of the cabbage crushing liquid is changed in an embodiment of the present invention. [Modes for carrying out the invention]

[0010] The numerical range "X to Y" includes the values X and Y at both ends, unless otherwise specified. Food includes not only solid foods, but also liquid oral intake substances such as beverages and soups. It also includes not only those in the form taken as such (e.g., various cooked foods, supplements, drink agents), but also food additives, seasoning compositions, and beverage concentrates. Food further includes those for not only humans, but also non-human animals (pets, livestock, etc.). Food also includes, in addition to general foods (including so-called health foods), health functional foods (including foods with function claims, nutrient function foods, and foods for specified health uses). When expressing concentration or ratio (%, parts, etc.), it is based on mass, unless otherwise specified.

[0011] [Fermented product] As one embodiment, the present invention provides a microbial fermented product of a vegetable, fruit, or grain material containing γ-aminobutyric acid (GABA). GABA is a kind of amino acid relatively abundant in vegetables, fruits, grains, etc. In fermentative production, GABA is produced by by decarboxylation of glutamic acid by glutamic acid decarboxylase (GAD).

[0012] (Microorganism) The microorganism used in this embodiment is a microorganism that can function glutamic acid decarboxylase (GAD). A microorganism that can function glutamic acid decarboxylase (GAD) can be paraphrased as a microorganism that can express the GAD gene.

[0013] The type of microorganism is not particularly limited. In one aspect, as the microorganism, any one of lactic acid bacteria, Bifidobacterium, yeast, Chlorella, filamentous fungi, and Escherichia coli is used. Only one type of bacterium may be used, or multiple types may be used.

[0014] In one aspect, as the microorganism, any bacterium selected from lactic acid bacteria and Bifidobacterium (bacteria belonging to the genus Bifidobacterium) is used.

[0015] Many strains of lactic acid bacteria and bifidobacteria exhibit strain-specific gamma-aminobutyric acid (GABA) production capabilities via the glutamine decarboxylase (GAD) system, one of the bacterial amino acid-dependent acid resistance (AR) systems. Genomic studies have revealed that the GAD system, encoded by the gad operon for GABA production, exists in various species of lactic acid bacteria and bifidobacteria (Front. Microbiol., 14 February 2017 Sec. Food Microbiology Volume 8 https: / / doi.org / 10.3389 / fmicb.2017.00206).

[0016] Lactic acid bacteria that can be used include Acetilactobacillus genus, Agrilactobacillus genus, Amylolactobacillus genus, Apilactobacillus genus, Bombilactobacillus genus, Companilactobacillus genus, Dell aglioa, Fructilactobacillus, Furfurilactobacillus, Holzapfelia, Lacticaseibacillus, Lactiplantibacillus, Lactobacillus, Lapidilactobacillus Lactic acid bacteria belonging to the genera s, Latilactobacillus, Lentilactobacillus, Levilactobacillus, Ligilactobacillus, Limosilactobacillus, Liquorilactobacillus, Loigolactobacillus, Paralactobacillus, Paucilactobacillus, Schleiferilactobacillus, or Secundilactobacillus, preferably lactic acid bacteria belonging to the genus Levilactobacillus.

[0017] Lactobacillus(Lb.) brevis, Lb. plantarum、Lb. delbrueckii、Lb. helveticus、Lb. acidophilus、Lb. casei、Lb. paracasei、Lb. rhamnosus、Lb. reuteri、Lb. iners、Lb. gasseri、Lb. fermentum、Lb. salivarius、Lb. johnsonii、Lb. tiger、Lb. buchneri、Lb. sanfranciscensis、Lb. kunkeei、Lb. pentosus、Lb. jensenii、Lb. ruminis、Lb. crispatus、Lb. amylovorus、Lb. paralimentaryus、Lb. kefiranofaciens、Lb. oris、Lb. mucosae、Lb. florum、Lb. paraplantarum、Lb. curvatus、Lb. acidipiscis、Lb. fructorans、Lb. coryniformis、Leuconostoc(Leu.) mesenteroids、Leu. pseudomesenteroids、Leu. citreum、Leu. gelidum、Leu. lactis、Pediococcus(P.) acidilactici、P. pentosaceus、Lactococcus(Lc.) lactis、Lc. garvieae, Streptococcus thermophilus, Oenococcus oeni, Weissella cibaria, Lb. letter、Lb. plantarum、Lb. reuteri、Lb. fermentum、Lb. buchneri、Lb. oris、Lb. paraplantarum、Lc. lactis、Lc. garvieae、S. thermophilus and Lb. letter、Lb. reuteri、Lb. buchneri、Lb. oris、Lc. lactis、Lc. garvieaeである。

[0018] Examples of Bifidobacterium that can be used include Bifidobacterium (B.) longum, B. animalis, B. breve, B. bifidum, B. pseudolongum, B. dentium, B. adolescentis, B. thermacidophilum, B. thermophilum, B. pseudocatenulatum, B. kashiwanohense, B. asteroides, B. angulatum, B. catenulatum, B. scardovii, and B. gallicum.

[0019] In one embodiment, yeast is used as the microorganism. Yeast is known to be usable in the production of GABA-enriched food materials (Japanese Patent Publication No. 9-238650).

[0020] The yeast that can be used is, for example, any yeast belonging to a genera selected from the group consisting of Saccharomyces, Shizosaccharomyces, Pichia, Candida, Kluyveromyces, Williopsis, Debaryomyces, Galactomyces, Torulaspora, Rhodotorula, Yarrowia, and Zygosaccharomyces. The yeast is preferably baker's yeast used in bread making, Torula yeast used in the production of food and animal feed, or brewer's yeast used in beer making, due to their good growth rate, and more preferably a fungus belonging to Saccharomyces or Candida.

[0021] More specifically, the yeasts that can be used are Saccharomyces cerevisiae, Candida tropicalis, Candida lypolitica, Candida utilis, and Candida sake. Preferred examples are strains of Saccharomyces cerevisiae and Candida utilis.

[0022] In one embodiment, filamentous fungi are used as the microorganism. Filamentous fungi are a general term for fungi that are composed of tubular cells called hyphae. Brewing filamentous fungi are known to possess glutamate decarboxylase activity (J. Brew. Soc. Japan. 97(5):382-386 (2002)).

[0023] The filamentous fungi that can be used are those belonging to any genera selected from the group consisting of the genera Aspergillus and Monascus.

[0024] More specifically, the filamentous fungi that can be used are Aspergillus oryzae, Aspergillus sojae, Aspergillus kawachii, Aspergillus auamori, Monascus pmpureus, and Monascus pilosus.

[0025] In one embodiment, Escherichia coli is used as the microorganism. E. coli glutamine decarboxylase (GAD) is a hexameric enzyme, and it is known that there are two types: GAD-α and GAD-β. It is also known that it is readily induced in a culture medium containing glutamic acid (Journal of Molecular Catalysis B: Enzymatic. 10(1-3):67-79 (2000)).

[0026] In a particularly preferred embodiment, the microorganism used is a lactic acid bacterium. Preferably, the lactic acid bacterium belongs to the genus Levilactobacillus, and more preferably to Levilactobacillus brevis. Note that the genus Levilactobacillus is the name of a genus that has been reclassified according to the classification based on the reports of Zheng et al. (Int J Syst Evol Microbiol (2020) 70, 2782-2858) and Liu & Gu (Int J Syst Evol Microbiol (2020) 70, 6414-6417). Before the reclassification, it was classified in the genus Lactobacillus (https: / / www.nite.go.jp / nbrc / cultures / information / 20210201.html).

[0027] (Vegetables, fruits, or grains) In the embodiments, vegetables, fruits, or grain materials are used as fermentation raw materials. In relation to the present invention, when referring to vegetables, it includes not only vegetables as defined in the Japanese Food Standard Composition Table 2020 (8th Revised Edition), but also legumes, mushrooms, and algae as defined in the same table. In relation to the present invention, when referring to fruits, it includes not only fruits as defined in the same table 2, but also nuts and seeds as defined in the same table. In relation to the present invention, when referring to grains, it includes not only cereals as defined in the same standard composition table, but also potatoes and starches. Only one type of vegetable, fruit, or grain material may be used, or multiple types may be used.

[0028] The material may be the vegetable, fruit, or grain material itself, or it may be all or part of the plant individual of the vegetable, fruit, or grain material. Part of the plant individual may include roots, stems, leaves, flowers, fruits, seeds, cores, and peels, and may include both edible parts and non-edible parts that are usually discarded.

[0029] In one embodiment, vegetables, fruits, or grains substance Examples of materials include those from plants belonging to the Brassicaceae, Solanaceae, Amaryllidaceae, or Poaceae families, preferably Brassicaceae or Solanaceae.

[0030] Plants belonging to the Brassicaceae family include plants of the Brassica genus, such as rapeseed, mizuna, taisai, bok choy, komatsuna, turnip, Chinese cabbage, cabbage, broccoli, and wild mustard; plants of the Raphanus genus, such as radish; shepherd's purse, wasabi, etc.

[0031] Plants belonging to the Solanaceae family include plants of the Solanum genus, such as tomatoes, eggplants, potatoes, and pepino; plants of the Capsicum genus, such as chili peppers (bell peppers, paprika); and plants of the Lycium genus, such as goji berries.

[0032] Plants belonging to the Amaryllidaceae family include plants of the Allium genus in the Allioideae subfamily, such as onions, leeks, scallions, garlic, and shallots.

[0033] Plants belonging to the grass family include plants of the genus Poa, such as rice; plants of the genus Barley, such as barley; plants of the genus Wheat, such as wheat; and plants of the genus Millet, such as millet.

[0034] Other examples include plants belonging to the Asteraceae family, such as lettuce, Korean lettuce, and Chinese lettuce; plants belonging to the Lamiaceae family, such as basil; plants belonging to the Amaranthaceae family, such as spinach; and plants belonging to the Fabaceae family, such as soybeans, peas, green beans, broad beans, cowpeas, chickpeas, and mung beans.

[0035] In one embodiment, the vegetable, fruit, or grain material is selected from parts that are normally discarded or tend to be discarded, such as the core, outer leaves, and peel. Examples of such materials include the outer leaves and core of cabbage, the outer leaves of Chinese cabbage, onion peels, rice bran, and wheat bran.

[0036] Vegetables, fruits, or grains may be substandard. Generally, substandard products are those whose weight, volume, color, or shape differs from the standard for that product. Examples of substandard vegetables and fruits include tomatoes, cucumbers, radishes, cabbage, Chinese cabbage, mandarins, and lettuce.

[0037] Vegetables, fruits, or grain materials can be used in various forms, as long as they achieve the desired effect. They may be used as is, or as cut, shredded, or ground materials. Ground materials may be in paste or liquid form. In the case of paste or liquid form, the liquid portion (supernatant) and the other portion (sediment) may be separated and used separately. The desired effect can be achieved by both the supernatant and the precipitate.

[0038] (Glutamic acid) In relation to the present invention, when referring to glutamic acid, it includes food-acidifiable salts of glutamic acid and their solvates. From the viewpoint of solubility, glutamic acid is preferably in the form of sodium glutamate. Sodium glutamate may be in the form of monohydrate.

[0039] (GABA content in fermented products) The amount of GABA contained in the fermented product of this embodiment may vary, but for example, it may be 100 mg / 100g or more, 200 mg / 100g or more, 300 mg / 100g or more, 400 mg / 100g or more, 500 mg / 100g or more, 600 mg / 100g or more, 700 mg / 100g or more, 800 mg / 100g or more, 900 mg / 100g or more, 1000 mg / 100g or more, 1300 mg / 100g or more, 1600 mg / 100g or more, 2000 mg / 100g or more, 2500 mg / 100g or more, 3000 mg / 100g or more, 3500 mg / 100g or more, or 4000 mg / 100g or more.

[0040] Such fermented products are obtained by a manufacturing method that includes the following steps. A step of obtaining a lactic acid fermented product containing γ-aminobutyric acid by reacting a microorganism functionally possessing glutamic acid decarboxylase with a culture medium containing 5% or more, preferably 10% or more, more preferably 20% or more, for example 50% or more, of glutamic acid and vegetable, fruit, or grain material.

[0041] In the production of fermented products, vegetable, fruit, or grain materials can be crushed and made into a paste or liquid. Vegetable, fruit, or grain materials may contain glucose, free amino acids such as glutamic acid and GABA.

[0042] [Food composition] As one embodiment of the present invention, the present invention provides a food composition containing the above-mentioned fermented product. Food compositions can take various forms. Food compositions may also be refrigerated or frozen foods, which are cooked, cooled, and then refrigerated or frozen.

[0043] Examples of refrigerated and frozen foods include, for example, okonomiyaki, takoyaki, spring rolls, Chinese rice bowl (toppings), gyoza, shumai, xiaolongbao, steamed buns, happosai, twice-cooked pork, shredded pork with green peppers, stir-fried vegetables, kinpira, and chikuzenni, as well as fried products such as croquettes, minced meat cutlets, and shrimp cutlets, meat products such as hamburgers, noodles such as ankake udon, Nagasaki champon, yakisoba, spaghetti Napolitan, macaroni gratin, and lasagna, rice products such as cooked rice, takikomi gohan, fried rice, pilaf, and chirashi sushi, and wheat flour products such as pizza.

[0044] The amount of fermented product in a food composition varies depending on the type of food, but is typically 0.5% or more, preferably 0.75% or more, more preferably 0.9% or more, and can be 1% or more, 1.5% or more, 2% or more, 2.5% or more, 3% or more, 3.5% or more, 4% or more, or 4.5% or more, when converted to fermented product with a solid content of 6.21% relative to the food composition. The upper limit of the amount added is not limited as long as the sourness, saltiness, and aroma of the fermented product do not impair the flavor of the food in question. Depending on the food, for example, in the case of okonomiyaki, the flavor of the fermented product becomes noticeable when the amount exceeds 5%, so it is preferable to keep it at 4.5% or less, but it may also be 4% or less, 3% or less, 2% or less, or 1% or less. For mild-tasting foods that are prone to off-flavors and astringency, it is preferable to keep it at 3% or less, but it may also be 2.5% or less, 2% or less, 1.5% or less, or 1% or less. For foods with a strong flavor, the concentration is preferably 7% or less, but may also be 6% or less, 5% or less, 4% or less, or 3% or less.

[0045] In a preferred embodiment, the food composition contains vegetables, fruits, or grain materials as non-fermented ingredients, and the fermented products contained in the food composition are fermented products of said vegetables, fruits, or grain materials. An example of such a product is okonomiyaki, which contains cabbage as an ingredient and includes a fermented product of cabbage.

[0046] The GABA content in the food composition may vary, but for example, it may be 10 mg / 100g or more, 20 mg / 100g or more, 30 mg / 100g or more, 40 mg / 100g or more, 50 mg / 100g or more, 60 mg / 100g or more, 70 mg / 100g or more, 80 mg / 100g or more, 90 mg / 100g or more, 100 mg / 100g or more, 110 mg / 100g or more, 120 mg / 100g or more, 130 mg / 100g or more, 140 mg / 100g or more, or 150 mg / 100g or more.

[0047] [GABA manufacturing method] As one embodiment of this invention, the present invention provides a method for producing GABA.

[0048] The GABA production method of this embodiment includes the following steps. A step of converting glutamic acid in a culture medium to GABA by acting on it with a microorganism functionally possessing glutamic acid decarboxylase, wherein the culture medium contains 5-70% vegetables, fruits, or grain materials, thereby improving the conversion rate from glutamic acid to GABA.

[0049] In relation to the present invention, when referring to the GABA conversion rate, unless otherwise specified, it is calculated based on the amount of sodium glutamate monohydrate (molecular formula: C5H8NNaO4·H2O, molecular weight: 187.13) added to the culture medium and the amount of GABA recovered (molecular weight: 103.12), taking into account the molecular weights of both. For example, if 100g of sodium glutamate monohydrate is added to the culture medium and 55g of GABA is recovered, the GABA conversion rate is calculated to be 100%.

[0050] In the production method of this embodiment, the glucose concentration at the start of cultivation (sometimes called the initial stage) can be set as appropriate. According to the inventors' research, when glucose was not added to the culture medium at the start of cultivation, and only vegetables, fruits, or grain materials were used as the sugar source, the GABA conversion rate decreased, and the final GABA concentration was also lower. Therefore, it can be said that the conversion from glutamic acid to GABA can be promoted by supplementing with a sugar source, and that GABA can be produced even without adding a sugar source.

[0051] Therefore, the concentration of glucose added to the culture medium at the start of cultivation can be 0, 0.2-4%, 0.3-3%, 0.5-2%, or 0.7-1.5%.

[0052] In the production method of this embodiment, the concentration of glutamic acid at the start of culture can be adjusted as appropriate. According to the inventors' studies, there was almost no difference in the conversion rate when 5% of sodium glutamate was added at the start of culture compared to when 10% was added. Increasing the amount of glutamic acid added increased the amount of GABA produced, but tended to decrease the conversion rate.

[0053] From this perspective, when adding glutamic acid, the amount of sodium glutamate can be adjusted to concentrations such as 1-20%, 1-15%, 2-10%, 2-9%, 3-8%, 3-7%, and 4-6% of the culture medium.

[0054] In the production method of this embodiment, the pH of the culture medium after the start of cultivation can be adjusted as appropriate. According to the inventors' studies, it was found that the conversion rate increased when the pH was adjusted to 4 and when the pH was adjusted to 5, compared to when the pH was not adjusted after 24 hours of cultivation.

[0055] From this perspective, the pH of the culture medium after the start of cultivation can be set to 3-6, preferably 4-6, and can be set to 4.5-5.5.

[0056] [Utilization of vegetables, fruits, or grain materials, their waste materials, and substandard products] As one embodiment of this invention, the present invention provides a method for utilizing the above-mentioned vegetables, fruits, or grain materials, their waste materials, and off-spec products thereof.

[0057] The following are specific examples of how it can be used. (1) Use of vegetable, fruit, or grain material to improve the conversion rate from glutamic acid to gamma-aminobutyric acid in the fermentation production of gamma-aminobutyric acid by microorganisms functionally possessing glutamic acid decarboxylase. (2) A fermentation aid composition containing vegetables, fruits, or grain materials for improving the conversion rate to decarboxylates in the fermentation production of decarboxylates by microorganisms functionally possessing decarboxylase. Preferably, the fermentation aid composition wherein the decarboxylase is glutamate decarboxylase and the decarboxylate is γ-aminobutyric acid.

[0058] [Method for manufacturing GABA-enhanced food that reduces food waste] As one embodiment of this invention, the present invention provides a method for producing GABA-fortified food that reduces food waste.

[0059] The manufacturing method for such food products includes the following steps: A process to obtain a fermented product containing γ-aminobutyric acid by reacting a microorganism functionally possessing glutamic acid decarboxylase with a culture medium containing 5-70% of a vegetable, fruit, or grain material selected from the core, outer leaves, peel, and off-grade parts; A step of fortifying gamma-aminobutyric acid by mixing the resulting fermented product with a raw material containing the unfermented product of the vegetable, fruit, or grain.

[0060] Okonomiyaki is an example of a food product to which this manufacturing method is applied. [Examples]

[0061] [Experimental Method] 1) Method for measuring free amino acids Free amino acids were measured using HPLC with an amino acid analyzer. Glutamic acid (Glu) and gamma-aminobutyric acid (GABA) were also measured using this method.

[0062] (Pre-processing) 1. The sample was diluted to an appropriate concentration with 10% sulfosalicylic acid and shaken in a shaker at 200 vibrations / minute for 20 minutes. 2. Furthermore, the precipitate was removed by centrifugation at 3,000 rpm for 10 minutes. 3. The obtained supernatant was confirmed to have a pH of 1-2, and then centrifuged at 15,000 rpm for 5 minutes to remove the precipitate. 4. The obtained supernatant was passed through a 0.2 μm filter, injected into a vial, and subjected to the following measurements.

[0063] (Measurement conditions) Analyzer: HITACHI L-8900 Amino Acid Analyzer Column: #2622 ID 4.6x60mm Mobile phase: MCI BUFFER PH-1, MCI BUFFER PH-2, MCI BUFFER PH-3, MCI BUFFER PH-4 Reaction solution: Hitachi ninhydrin color development solution kit (ninhydrin solution and buffer solution) Flow rate: 0.4mL / min

[0064] 2) Method for measuring sugars Sugars were measured using the following method with HPLC.

[0065] (Pre-processing) 1. The sample was diluted with 3% perchloric acid to an appropriate concentration, and then sonicated at 40 kHz for 5 minutes to remove protein components. 2. The sample, which had been diluted to an appropriate volume with water, was adjusted to a pH of 5 or higher with a sodium hydroxide solution. 3. The precipitate was removed by centrifuging at 15,000 rpm for 5 minutes. 4. The obtained supernatant was passed through a 0.45 μm filter, injected into a vial, and subjected to the following measurements.

[0066] (Measurement conditions) Hitachi Chromaster series equipment Column: Shodex AsahiPak NH2P-4E 4.6×250mm Guard column Shodex AsahiPak NH2P-50G Column temperature 45℃ Reaction temperature: 150℃ Detection wavelength: UV365nm Flow rate Mobile phase 0.8mL / min Reaction solution 0.4 mL / min

[0067] The sugar was analyzed by eluting the sample using a concentration gradient of acetonitrile in the presence of phosphate, and then reacting it with a phenylhydrazine solution.

[0068] [Test Example 1: Production of Cabbage Crushing Liquid] Since the leaves of cabbage are used in the production of processed foods such as okonomiyaki, and the core is usually discarded, the following tests were conducted using the core as a raw material with the intention of reusing it.

[0069] The core portion was collected from cabbages, finely chopped, and then pulverized using a Mascoloider (Masuko Sangyo MKZA-10) to obtain a paste-like pulverized liquid. It was possible to pulverize it into a fine, yellowish-green, cream-colored paste-like fluid. The analytical values ​​are shown in the table below.

[0070] [Table 1]

[0071] We were able to prepare a cabbage pulverization liquid that can be used as a culture medium. The resulting product was a green, paste-like fluid that, while viscous, possessed sufficient fluidity.

[0072] [Test Example 2: Method for culturing microbial species] Lactic acid bacteria were cultured by inoculating the following culture media with 1 / 1000th of the culture medium volume, after each lactic acid bacterium had been suspended in a storage medium and frozen using a conventional method, and then allowing it to stand still at 30°C for 24 hours. For yeast extract, Highmax GL (Fuji Foods Industry Co., Ltd.) was used, and for glucose, Glu-Final (Sanei Sugar Refining Co., Ltd.) was used. Unless otherwise specified, Levilactobacillus brevis NBRC 12005 (available for purchase from the National Institute of Technology and Evaluation (NITE)) was used as the lactic acid bacterium.

[0073] [Table 2]

[0074] [Test Example 3: Lactic Acid Bacteria, Screening Results] Two bacterial species believed to possess glutamate decarboxylase (GAD) were cultured statically for 24 hours at 37°C in MRS liquid medium supplemented with 1% each of glutamine and sodium glutamate (MSG), and the free amino acid content was measured after the culture period.

[0075] [Table 3-1]

[0076] As described above, we confirmed that each of these can be converted from glutamic acid to gamma-aminobutyric acid (GABA).

[0077] [Test Example 4] (method) To the culture medium with the composition shown in the table below, 100g of seed culture solution was added to adjust the pH to 5.0, and then cultured at 30°C for 24 hours with stirring. After further adjustment of the pH to 4.0, cultured at 30°C for 48 hours. The GABA concentration and glutamic acid concentration were measured at this time. Highmax GL (Fuji Foods Industry Co., Ltd.) was used as the yeast extract, and Glu-Final (Sanei Sugar Refining Co., Ltd.) was used as the glucose. In addition, Gluace EO (Mitsubishi Corporation Life Sciences) was used as the monosodium glutamate.

[0078] The conversion rate from Glu to GABA was calculated by assuming that if the entire amount of added sodium glutamate monohydrate was converted, 55% of its weight would become GABA. The amount of amino acids was then calculated by converting the increased liquid volume during cultivation (due to pH adjustment, etc.) from the amino acid concentration, and the percentage of conversion from the originally added sodium glutamate was calculated.

[0079] [Table 3-2]

[0080] (result) During the exam, 17 hours, 24 hours, 41 Sampling was performed at 1 hour, 48 hours, 65 hours, and 72 hours, and GABA and glutamic acid concentrations were measured. The amounts of GABA and glutamic acid calculated from the culture medium volume are shown in the table below, and the conversion rate to GABA calculated at that time is shown in Table 3-3 and Figure 1. From around 24 hours, the GABA concentration increased in the group with more cabbage powder added, and at 65 hours, the cabbage addition concentrations of 20% and 50% showed almost the same value.

[0081] Furthermore, while the amount of remaining glutamate decreased as the reaction progressed, a difference began to appear around 24 hours, and a significant difference was observed at 41 hours. Subsequently, low values ​​were observed at 65 and 72 hours.

[0082] As the concentration of cabbage pulverized liquid was increased from no addition to 5% and then 20%, the GABA concentration increased, the residual glutamic acid concentration decreased, and the conversion rate increased. Incubating in a medium with 20% cabbage pulverized liquid for 72 hours yielded 59.2g of GABA and a high GABA conversion rate of 107.7%. Furthermore, in the case of a medium with the cabbage pulverized liquid increased to 50%, although the residual glutamic acid was low at 0.3g, the GABA amount was lower than in the 20% cabbage pulverized liquid medium (57.6g), resulting in a lower GABA conversion rate of 104.8%. still It showed high values.

[0083] [Table 3-3]

[0084] (Composition of the fermentation liquid) The composition of the fermentation liquid obtained after 72 hours of incubation in Example 2 above, to which 10% salt was added in order to improve its shelf life, is shown below. It had a brownish-yellow appearance, no particular off-flavors or odors, and possessed a good taste.

[0085] [Table 4]

[0086] [Test Example 5: Examination of Culture Conditions] Next, we investigated the culture conditions when using cabbage medium. 100g of lactic acid bacteria culture solution was added to the medium mixture shown in the table below and used for testing. Specifically, we examined the effects of adding glucose to the crushed cabbage liquid as a sugar source, the effects of pH adjustment, and the effects of the amount of glutamic acid added.

[0087] [Table 5-1]

[0088] After preparing the culture medium as described above, the cultures were incubated at 30°C with stirring. Examples 4 and 5 were incubated while adjusting the pH with 20% NaOH to ensure it did not fall below 5.0. On the other hand, in Examples 6 and 7, the pH was adjusted to ensure it did not fall below 5.0 from the start of incubation until 24 hours later, and then from 24 hours to 63 hours later, the pH was adjusted with 2N HCl to 4.0 for Example 6 and 5.0 for Example 7. Subsequently, the production status of GABA was confirmed by measuring amino acids, etc.

[0089] [Table 5-2]

[0090] The results are shown in the table above. The pH remained at a high value of around 6 in all cases. In Examples 4 and 5, with the exception of Examples 6 and 7, where the pH was adjusted to 4.0 and 5.0 respectively, little to no pH adjustment was performed, and the liquid volume was almost the same.

[0091] • The effect of using only crushed cabbage liquid as a sugar source versus adding glucose (comparison of Examples 4 and 5) The crushed cabbage liquid contains 2.6% sugar (Table 1), and we investigated the effect of adding approximately 0.9% glucose as an additional sugar source. When the bacteria were cultured using only crushed cabbage liquid as the sugar source without adding glucose, the conversion rate to GABA decreased, and the final GABA concentration was about 10% lower. This revealed that supplementing with crushed cabbage liquid and glucose as sugar sources promotes the growth of lactic acid bacteria, which in turn promotes the conversion of glutamic acid to GABA. It also showed that even when only crushed cabbage liquid is used as the sugar source without adding glucose, a certain level of conversion ability is maintained.

[0092] • Effects of pH adjustment (comparison of Example 5 with Examples 6 and 7) In Example 6, the pH was adjusted to 4 after 24 hours of culture, and in Example 7, the pH was adjusted to 5, compared to Example 5, where the pH was not adjusted to fall below 5.0 after 24 hours of culture. The effects of these adjustments were investigated. It was revealed that the conversion rate increased to 104.8% at pH 5.0 and 105.6% at pH 4.0. This indicates that pH is involved in the conversion rate of GABA, that adjusting the pH to the range of 4-5 is effective, and that adjusting the pH to 5 or below particularly improves the GABA conversion rate.

[0093] • Effect of glutamic acid addition (comparison of Example 2 and Example 6) In Example 2, the sodium glutamate concentration was 9.6%, whereas in this experiment (Example 6), it was 4.5%, half the concentration. In Example 2, the GABA conversion rate was 104.4% after 65 hours of culture, while in Example 6, it was 105.6% after 63 hours of culture. This indicates that despite the difference in the sodium glutamate concentration added at the start of culture, Example 2 (9.6%) and Example 6 (4.5%) yielded almost the same values. This suggests that, within the scope of this experiment, conversion to GABA occurs regardless of the concentration of added monosodium glutamate.

[0094] [Test Example 6: Analysis of the localization of effective components in the pulverized liquid] (method) The cabbage core was centrifuged (8,000 rpm x 20 minutes), and the supernatant and precipitate obtained were added to the GABA conversion medium in three states: "a mixture of supernatant and precipitate (original pulverized liquid)," "supernatant only," and "precipitate only," so that the amount of solid content equivalent to the original cabbage was the same.

[0095] To a culture medium with the composition shown in the table below, which had been pre-sterilized by heat sterilization at 120°C for 4 minutes, 100g of seed culture solution was added to adjust the pH to 5.0, and then the culture was incubated at 30°C for 24 hours with stirring. After further adjusting the pH to 4.0, the culture was incubated at 30°C for 41 hours. The GABA and glutamic acid concentrations were measured during this period.

[0096] [Table 6-1]

[0097] (result)

[0098] [Table 6-2]

[0099] Comparing Comparative Example 2 with each of the examples, the effect of improving the GABA conversion rate with the crushed cabbage liquid was observed in both the supernatant and the precipitate, but it was particularly strong in the supernatant than in the precipitate, suggesting that the soluble components possessing GABA conversion potential were particularly strongly localized.

[0100] [Test Example 7: Conversion Rate When Lactic Acid Bacteria are Changed] We investigated whether other lactic acid bacteria, besides those tested in this study, exhibited similar conversion rates. Since NBRC12520 was confirmed to have a glutamic acid to GABA conversion effect in Test Example 3, this bacterium was used in the test.

[0101] The test was performed using NBRC12520 in the same manner as in Example 2. <Bacterial strain used> Example 11 NBRC12520 In addition, as Comparative Example 3, NBRC12520 was cultured and analyzed without using the cabbage pulverization liquid.

[0102] [Table 7]

[0103] In NBRC12520, the conversion rate was low without the addition of the cabbage pulverized solution, reaching only 12.9% even after 72 hours of incubation. However, using the cabbage pulverized solution resulted in a high conversion rate. From the above, it can be seen that high conversion rates were observed not only with NBRC12005, which was used in the previous experiment, but also with NBRC12520.

[0104] This indicates that, in the case of bacterial strains possessing similar enzymes, the cabbage pulverized liquid is effective even if the strains are different. In particular, even in strains like NBRC12520, which possess the enzyme but have a low conversion rate, the conversion rate can be increased by adding bio-pulverized liquids such as cabbage pulverized liquid or other vegetables.

[0105] [Test Example 8: Conversion Rate When Vegetables Are Different] Next, we investigated whether similar effects were observed with the liquids obtained from crushing other vegetables.

[0106] (method) For crushing the vegetables, a blender (Panasonic MK-K48P) was used for all but the cabbage core and leaves, which were processed using a muscoloider. Cabbage leaves and Chinese cabbage were added to the initial culture medium at a rate of 50%, similar to the cabbage core. Cabbage leaves: Brix 5.0, mulberry color (pale yellow) Chinese cabbage: Brix 2.6, olive green (dark, dull yellowish-green) paste

[0107] The following vegetables were treated with Brix, with the same amount of Brix equivalent as cabbage core (50%) added. Cabbage core Brix: 2.2 Creamy paste • Nango Tomato (Minamiaizu Nango, Fukushima Prefecture) Brix: 5.8 Tomato's characteristic vermilion paste • Aokubi Daikon (Aomori Prefecture) Brix: 3.5 Flaxen color (yellowish light brown)

[0108] For all other cases, cultivation was carried out using the same method as in Example 3. Comparative Example 4 was also conducted in which no vegetable pulverized liquid was used.

[0109] [Table 8-1]

[0110] (Test results) In Comparative Example 4, the sample without vegetable pulverization liquid only achieved a conversion rate of 74.5% after 72 hours of incubation, whereas the cabbage core pulverization liquid examined so far achieved a conversion rate of 104.5%. Furthermore, pulverization liquids made from cabbage leaves, Chinese cabbage, tomatoes, and radishes all showed conversion rates of around 90-96%. This indicates that pulverization liquids made from sources other than cabbage core have a similar effect on improving the conversion rate as cabbage core, and that pulverization liquid made from cabbage core is particularly effective in improving the conversion rate.

[0111] [Table 8-2]

[0112] [Okonomiyaki] (method) Okonomiyaki containing GABA was manufactured using the following method.

[0113] The batter and fillings were mixed in a weight ratio of 40:60, and approximately 100g of the mixture was placed in a mold (12cm in diameter) and baked. (i) A mixture of solid ingredients such as wheat flour, sugar, seasonings, salt, spices, and thickeners. (ii) The raw materials of (i) were mixed with whole eggs, yam, water, and the cabbage fermentation liquid from the example. The cabbage fermentation liquid used was the fermentation liquid from Example 2 after 72 hours of culture, to which salt was added to make up 10%. (iii) Bake each side on a griddle at 200°C for 4 minutes. (iv) Removed from the metal plate and allowed to cool. (v) Frozen at -40°C. (vi) Stored at -18°C.

[0114] The stored samples were heated in a microwave oven at 600W for 7 minutes before being offered for tasting.

[0115] [Table 9]

[0116] [Table 10]

[0117] In Example A, the amount of GABA per sheet was set to 30 mg, and in Example B, it was set to 120 mg of GABA per sheet.

[0118] (result) In both cases, the okonomiyaki could be baked and frozen without any problems. Furthermore, upon tasting, the samples from Example A and Example B had a good taste compared to Comparative Example A, with no noticeable difference in flavor.

[0119] [Packaged Rice] We manufactured packaged rice. The rice was prepared according to the table below. It was sterilized by pressurized heat sterilization using conventional methods, aseptically packaged, stored at room temperature for one week, and then tasted after heating in a microwave oven. For the fermentation liquid, salt was added to the fermentation liquid from Example 2 after 72 hours of culture to a concentration of 13% (final GABA concentration of 3.0%).

[0120] [Table 11]

[0121] When fermentation liquid was added, there was no particular difference with 0.41g added (12.3mg GABA / piece) and 0.94g added (28.0mg GABA / piece), but with 1.67g added (50mg GABA / piece), the added salt had a noticeable effect. and I detected the expected saltiness, and with the addition of 3.4g (100mg of GABA per serving), the saltiness became more pronounced. Aside from the saltiness, there were no other noticeable issues.

Claims

1. A microbial fermented product of vegetables, fruits, or grain materials containing 3300 mg / 100g or more of γ-aminobutyric acid, A fermented product in which microorganisms functionally possess glutamate decarboxylase.

2. The fermented product according to claim 1, wherein the vegetable, fruit, or grain material is selected from the core, outer leaves, and peel.

3. The lactic acid fermented product according to claim 1, wherein the vegetable, fruit, or grain material is substandard.

4. The lactic acid fermented product according to claim 1, wherein the vegetable, fruit, or grain material is selected from plants of the Brassicaceae family or Solanaceae family.

5. The fermented product according to claim 1, wherein the microorganism is lactic acid bacteria.

6. The fermented product according to claim 1, wherein the microorganism is a lactic acid bacterium belonging to the genus Levilactobacillus.

7. The fermented product according to claim 1, wherein the microorganism is a lactic acid bacterium belonging to Levilactobacillus brevis.

8. Fermented product containing 3300 mg / 100g or more of γ-aminobutyric acid, obtained by a manufacturing method including the following steps: A fermentation process in which a microorganism functionally possessing glutamic acid decarboxylase is reacted with a culture medium containing 5-70% glutamic acid and vegetable, fruit, or grain material to obtain a lactic acid fermented product containing γ-aminobutyric acid.

9. A food composition comprising a fermented product according to any one of claims 1 to 8.

10. The food composition according to claim 9, wherein the food composition includes fruits, vegetables, or grains as raw materials, and the fermented product is a fermented product of materials derived from the fruits, vegetables, or grains.

11. The food composition according to claim 10, comprising 10 mg / 100 g of γ-aminobutyric acid.

12. A method for producing gamma-aminobutyric acid, including the following steps: A step of converting glutamic acid in a culture medium to gamma-aminobutyric acid by acting on it with a microorganism functionally possessing glutamic acid decarboxylase, wherein the culture medium contains 5-70% vegetable, fruit, or grain material, thereby improving the conversion rate from glutamic acid to gamma-aminobutyric acid.

13. The production method according to claim 12, wherein in the step of applying lactic acid bacteria, the pH of the culture medium is adjusted to 5 or less.

14. The use of vegetable, fruit, or grain material to improve the conversion rate from glutamic acid to gamma-aminobutyric acid in the fermentation production of gamma-aminobutyric acid by microorganisms functionally possessing glutamic acid decarboxylase.

15. A fermentation aid composition containing vegetable, fruit, or grain material for improving the conversion rate to decarboxylates in the fermentation production of decarboxylates by microorganisms functionally possessing decarboxylase.

16. The fermentation aid composition according to claim 15, wherein the decarboxylase is glutamate decarboxylase and the decarboxylated product is γ-aminobutyric acid.

17. A method for producing a food composition, comprising the following steps: A process to obtain a fermented product containing γ-aminobutyric acid by reacting a microorganism functionally possessing glutamic acid decarboxylase with a culture medium containing 5-70% of a vegetable, fruit, or grain material selected from the core, outer leaves, peel, and off-grade products; A step of fortifying γ-aminobutyric acid by mixing the obtained fermented product with a raw material containing the unfermented product of the vegetable or fruit.

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