JP7464940B2 - Onions enriched with γ-aminobutyric acid and cycloalliin and method for producing the same - Google Patents

Description

The present invention relates to lactic acid bacteria-fermented onions, which are obtained by lactic acid fermentation of heated onions under specific conditions and contain gamma-aminobutyric acid (hereinafter referred to as "GABA") and are enriched with functional components such as cycloalliin, and to a method for producing the same.

In recent years, interest in preventing lifestyle-related diseases and maintaining health has increased, and many related studies have been conducted, with increasing attention being paid to natural ingredients contained in or derived from fruits, vegetables, etc.

For example, onions are a vegetable eaten worldwide, and research into disease prevention and health maintenance is also progressing. Cycloalliin, which has antioxidant properties, is one of the main functional components contained in onions. When active oxygen is produced excessively in the body, it oxidizes lipids, proteins, DNA, etc. in cells, inducing cell death and causing aging and lifestyle-related diseases, and it is said that LDL-cholesterol oxidized by active oxygen is one of the factors that cause atherosclerosis. For this reason, there is a lot of interest in cycloalliin, which has antioxidant properties. Cycloalliin exists in the bulb part of onions as a cycloalliin precursor, and when heated to 90°C or higher, the precursor structure is cyclized to form cycloalliin (Patent Document 1). It has also been reported that lactic acid fermentation of onions increases their physiological activities, such as α-glucosidase inhibitory activity, lipase inhibitory activity, and radical scavenging activity (Patent Document 2).

Furthermore, a method has been reported in which fermentation of fruits such as mandarin oranges, peaches, grapes, strawberries, and pears, and vegetables such as carrots and tomatoes, or the juices thereof, is carried out to produce a fermented product containing GABA (Patent Document 3). GABA is a non-protein amino acid produced by decarboxylation of glutamic acid, and is known to be effective in alleviating hypertension symptoms. In addition, in recent years, it has been confirmed to be effective in suppressing stress and improving sleep. In anticipation of these various efficacies, GABA is used as an active ingredient in functional foods and supplements, and the demand for it is increasing. GABA can be produced by lactic acid fermentation using glutamic acid as a substrate, using lactic acid bacteria with high glutamic acid decarboxylase (hereinafter abbreviated as "GAD") activity (Non-Patent Documents 1 and 2). However, there is still a strong demand in this field for the development of new food materials that allow efficient intake of GABA.

JP 2005-289850 A JP 2005-097222 A Patent Document 1: WO2007/052806

Yokoyama, S. et al., J. Biosci. Bioeng, 93, 95-97 (2002). Nomura, M. et al., J. Dairy Sci., 81, 1846-1891 (1998).

Unlike the above-mentioned fruits such as mandarin oranges, peaches, grapes, strawberries, and pears, and vegetables such as carrots and tomatoes, it was not possible to produce GABA by lactic acid fermentation using onions alone as a raw material. Also, because onions naturally contain a lot of carbohydrates, there was concern that taking in an effective amount of functional ingredients such as cycloalliin would result in taking in too much carbohydrate.

The present invention aims to provide a method for producing GABA using onions as a raw material, as well as a method for reducing the carbohydrate content in onions and enriching them with functional components such as cycloalliin.

As a result of intensive studies to solve the above problems, the present inventors have found that lactic acid bacteria-fermented onions, obtained by lactic acid fermentation of heated onions in the presence of a nitrogen source and glutamic acid or a salt thereof using lactic acid bacteria having GAD activity, have a high content of GABA, a reduced content of soluble solids such as carbohydrates, and are enriched in functional components such as cycloalliin.

The present invention is based on these findings and includes the following aspects.
[1] Lactic acid bacteria-fermented onion containing 5 w/w % or more of γ-aminobutyric acid (GABA) based on the Brix solid content of the onion.
[2] The lactic acid bacteria-fermented onion according to [1], further containing 0.5 w/w % or more of cycloalliin based on the Brix solid content of the onion.
[3] Lactic acid bacteria-fermented onion according to [1] or [2], wherein the onion is an onion extract.
[4] A method for producing a lactic acid bacteria-fermented onion containing GABA, which comprises a step of culturing a lactic acid bacterium having glutamic acid decarboxylase activity in a medium containing heated onion, a nitrogen source, and glutamic acid or a salt thereof.
[5] The lactic acid bacteria is selected from Lactobacillus brevis, Lactococcus lactis, Lactobacillus hilgardii, Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus delbrueckii, Lactobacillus leichmannii, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, Enterococcus avium, Enterococcus casseliflavus, Streptococcus thermophilus, Streptococcus faecalis, Leuconostoc mesenteroides, and Bifidobacterium longum.
[6] The method according to [4] or [5], wherein the nitrogen source is one or more nitrogen sources selected from the group consisting of yeast extract, soy protein, corn hydrolysate, wheat hydrolysate, cotton extract, and whey protein.
[7] The method according to any one of [4] to [6], wherein the pH of the medium at the start of culture is greater than 4.
[8] The method according to any one of [4] to [7], wherein the medium further contains a divalent metal ion.
[9] The method according to [8], wherein the divalent metal ion is one or more ions selected from the group consisting of manganese ions and iron ions.
[10] The method according to any of [4] to [9], wherein the heated onion is a heated onion extract having a Brix solid content of 1 to 10 w/v %.
[11] The method according to any one of [4] to [10], wherein the lactic acid bacteria-fermented onion further contains cycloalliin.

The present invention provides a method for producing GABA using onions as a raw material, as well as a method for reducing the carbohydrate content in onions and enriching them with functional components such as cycloalliin.

The lactobacillus-fermented onions obtained by the present invention contain high concentrations of two functional ingredients, GABA and cycloalliin, and can be used as ingredients for medicines or functional foods, contributing to the prevention of lifestyle-related diseases and improving quality of life (QOL).

Fig. 1 is a graph showing the amount of GABA produced (conversion rate (%) from monosodium glutamate to GABA) in a lactic acid bacteria-fermented onion extract produced by adding a predetermined amount of yeast extract to a medium. The lactic acid bacteria used was Lactobacillus brevis NBRC3345 strain (left) or Lactobacillus brevis NBRC12005 strain (right). Fig. 2 is a graph showing the amount of GABA produced (conversion rate (%) from monosodium glutamate to GABA) in a lactic acid bacteria-fermented onion extract produced by adding manganese sulfate tetrahydrate to a medium. The lactic acid bacteria used was Lactobacillus brevis NBRC3345 strain (left) or Lactobacillus brevis NBRC12005 strain (right). Fig. 3 is a graph showing the amount of GABA produced (conversion rate (%) from monosodium glutamate to GABA) in lactic acid bacteria-fermented onion extracts produced in media having predetermined pH values. The lactic acid bacteria used were Lactobacillus brevis NBRC3345 strain (left) or Lactobacillus brevis NBRC12005 strain (right).

The present invention relates to a method for producing lactic acid bacteria-fermented onions that contain a high content of GABA and are enriched with functional components such as cycloalliin, and the method comprises the steps of culturing lactic acid bacteria having GAD activity in a medium containing heated onions, a nitrogen source, and glutamic acid or a salt thereof, and lactic acid fermenting the onions.

In the present invention, "onion" refers to an onion species (Allium cepa L.) that is generally used for food, and the type is not particularly limited. Existing common spring-sown onions (e.g., Kitamomiji 2000, Kitamomiji, Sapporo yellow, Polestar, Tsukihikari, Kitami yellow, Gekko 22, Okhotsk, etc.), autumn-sown onions (e.g., Osaka Maru, Sennan Koko, Senshu Chuko yellow, Satsuki, Momiji, etc.), and mutants thereof can be used. In the present invention, the edible bulb part (the part generally called "bulb" or "sphere") or a dried product thereof can be used as the onion, and it is preferable that the outer skin (thin brown skin) of the bulb part is not included. The inclusion of the outer skin may reduce the content of cycloalliin in the final product, lactic acid bacteria-fermented onion.

In the present invention, "onion" may be in the form of being cut to an appropriate size or shape, or in the form of being crushed, pulverized or ground, but is preferably in the form of an extract. By using onion in the form of an extract, efficient fermentation by lactic acid bacteria can be promoted.

In the present invention, "extract" refers to squeezed juice, extract, or a mixture thereof, or a concentrate or dried product of the squeezed juice, extract, or mixture thereof.

The "juice" can be prepared by crushing and squeezing onions, separating the liquid fraction from the solid fraction such as cell walls, and obtaining the liquid fraction. The liquid fraction can be separated from the solid fraction by conventional solid-liquid separation means such as centrifugation or filtration (e.g., diatomaceous earth filtration).

The "extract" can be prepared by extraction from onions using an extraction medium or steam distillation. As the extraction medium, an organic solvent, a solvent such as water, or a supercritical fluid such as supercritical carbon dioxide can be used. As the solvent, water, an alcohol such as ethanol, an organic solvent such as hexane or acetone, or a mixture of these can be used, preferably water, ethanol, or a mixture of these, or acetone, and particularly preferably water. When producing an extract by solvent extraction, onions are immersed in an appropriate amount of solvent (0.5 to 20 times the amount of onions by weight, for example 0.5 to 15 times or 1 to 10 times the amount), and appropriately stirred or allowed to stand to elute the solvent-soluble components into the solvent. There is no particular limitation on the extraction time, but it can be 5 to 360 minutes, for example 15 to 240 minutes or 30 to 120 minutes. The extraction temperature is not particularly limited, but can be in the range of 0°C to 125°C, for example, 25°C to 120°C, preferably 60°C to 120°C, more preferably 70°C to 120°C, even more preferably 80°C to 120°C, and particularly preferably 90°C to 120°C. After extraction, the solvent fraction containing the solvent-soluble components and the solid fraction such as cell walls are separated by the above-mentioned solid-liquid separation means, and the solvent fraction is obtained as an extract. If necessary, the obtained extract may be further purified or roughly purified by purification means such as chromatography (column chromatography, gas chromatography, high performance liquid chromatography (HPLC), supercritical fluid chromatography, etc.) and/or recrystallization. The onions used for extraction may be in the original form, cut to an appropriate size or shape, crushed, pulverized, or ground, or squeezed juice. In this specification, the "extract" may be referred to as "extract".

The "concentrate" can be obtained by concentrating the above-mentioned squeezed juice or extract or a mixture thereof. Here, concentration refers to reducing the liquid (water and/or extraction solvent) in the squeezed juice or extract or a mixture thereof to increase the content of soluble solids (sometimes referred to as "Brix solids content" in this specification). The "Brix solids content" can be measured using a commercially available sugar content meter or Brix meter, and is also called the Brix value. For example, vacuum evaporation concentration and membrane concentration can be used as a method of concentration. Vacuum evaporation concentration is generally called reduced pressure concentration. The degree of vacuum during concentration can be selected within a range that can increase the Brix solids content, and is not particularly limited. Membrane concentration can be performed using a membrane such as a reverse osmosis membrane (RO) or an ultrafiltration membrane (UF). The type of membrane used can be selected within a range that can increase the Brix solids content, and is not particularly limited.

The "dried product" can be obtained by drying the juice or extract or a mixture thereof or the concentrate using conventional drying methods (freeze drying, vacuum drying, heat drying, air drying, etc.).

In the present invention, heated onions are used. Heated onions contain cycloalliin, and therefore, by using heated onions, cycloalliin can be contained in the final product, lactic acid bacteria-fermented onions. The heat treatment of onions can be appropriately selected from the range of conditions that can produce cycloalliin, and can be performed, for example, at 70°C to 120°C, preferably 80°C to 120°C, more preferably 90°C to 120°C, for 15 minutes to 240 minutes, preferably 30 minutes to 120 minutes. The heat treatment may be dry heating or wet heating. The heat treatment can be performed at any stage before inoculation with lactic acid bacteria, and this treatment may be performed alone, or the heat treatment provided in the extraction step or sterilization step (including the sterilization step of the medium) may also serve as this treatment.

Heated onion can be included in the medium in any amount, but preferably in an amount such that the Brix solid content derived from the heated onion becomes 1 to 10 w/v %, preferably 1 to 5 w/v %, more preferably 2.5 w/v %. In order to achieve a desired content, any solvent such as water may be added together with the heated onion for dilution. If the amount of heated onion is more or less than the above amount, the growth of lactic acid bacteria and the production of GABA may become insufficient, and it may not be possible to obtain the desired final product, lactic acid bacteria-fermented onion.

In the present invention, the term "nitrogen source" refers to amino acids, proteins, etc., and any of those conventionally known as nitrogen sources for lactic acid bacteria culture media can be used. Examples of such nitrogen sources include (but are not limited to) yeast extract, soy protein, corn hydrolysate, wheat hydrolysate, cotton extract, whey protein, etc., and one or more nitrogen sources selected from these can be used in combination. The "nitrogen source" in the present invention is preferably yeast extract. By including yeast extract as a nitrogen source in the medium, the GABA content in the final product, lactic acid bacteria-fermented onion, can be particularly increased.

The nitrogen source can be included in the medium in any amount that allows for the production of GABA in lactic acid fermentation, for example, 0.1 to 5 w/v%, preferably 0.1 to 2 w/v%, and more preferably 0.2 to 1.5 w/v%.

In the present invention, "glutamic acid or a salt thereof" is included in the medium as a substrate in the GABA production reaction in lactic acid fermentation. Chemically, glutamic acid means L-glutamic acid, a type of amino acid, and its salts include sodium salts. The origin of glutamic acid or its salts is not limited, and it is possible to use glutamic acid produced by fermentation from molasses or starch, or glutamic acid produced by hydrolyzing food proteins with acids or enzymes, or food ingredients such as seasonings, processed seafood products, and tomatoes that contain free glutamic acid as a source of glutamic acid or its salts.

Glutamic acid or a salt thereof can be included in the medium in any amount that allows for the production of GABA in lactic acid fermentation, for example, 0.1 to 10 w/v%, e.g., 0.5 to 5 w/v%, or 1 to 3 w/v%.

In the present invention, the medium may further contain divalent metal ions. By including divalent metal ions in the medium, it is possible to enhance the growth and fermentation activity of lactic acid bacteria, and to increase the amount of GABA contained in the final product, lactic acid bacteria-fermented onion. Examples of divalent metal ions include (but are not limited to) Mn ²⁺ , Fe ²⁺ , Ca ²⁺ , Mg ²⁺ , Cu ²⁺ , Zn ²⁺ and the like, and one or more divalent metal ions selected from these may be used in combination. Preferred divalent metal ions are Mn ²⁺ and Fe ²⁺ , and particularly preferred is Mn ²⁺ . The divalent metal ions may be added to the medium in the form of inorganic salts or organic salts that can be used in medicines and foods and beverages. Examples of such salts include (but are not limited to) manganese sulfate, manganese chloride, ferric chloride, and hydrates thereof.

Divalent metal ions can be included in the medium in any amount that can enhance the growth and fermentation activity of lactic acid bacteria, for example, 0.0001 to 0.01 w/v%, 0.0005 to 0.01 w/v%, or 0.001 to 0.01 w/v%.

In the present invention, the pH value of the medium (meaning the pH value at the start of culture) can be any value at which lactic acid bacteria can grow and GABA can be produced in lactic acid fermentation. Preferably, the pH value of the medium is a value exceeding pH 4, more preferably pH 5 or higher (e.g., pH 5.1 or higher, pH 5.2 or higher, pH 5.3 or higher, pH 5.4 or higher, or pH 5.5 or higher). The upper limit of the pH value of the medium is not particularly limited, but can be pH 8 or lower (e.g., pH 7.5 or lower, pH 7.4 or lower, pH 7.3 or lower, pH 7.2 or lower, pH 7.1 or lower, or pH 7 or lower). In one aspect, the pH value of the medium in the present invention can be a value exceeding pH 4 and pH 8 or lower; pH 5 or higher and pH 8 or lower; pH 5.1 or higher and pH 8 or lower; pH 5.2 or higher and pH 8 or lower; pH 5.3 or higher and pH 8 or lower; pH 5.4 or higher and pH 8 or lower; or pH 5.5 or higher and pH 8 or lower. In another aspect, the pH value of the medium of the present invention can be greater than pH 4 and less than pH 7; greater than pH 5 and less than pH 7; greater than pH 5.1 and less than pH 7; greater than pH 5.2 and less than pH 7; greater than pH 5.3 and less than pH 7; greater than pH 5.4 and less than pH 7; or greater than pH 5.5 and less than pH 7.

Generally, when lactic acid fermentation is carried out, the pH value of the medium is set low (for example, about pH 4) to create an environment in which lactic acid bacteria can grow preferentially. On the other hand, in the present invention, the pH value of the medium is set to a value exceeding pH 4, thereby making it possible to significantly increase the GABA content in the final product, lactic acid bacteria-fermented onion.

The pH value of the medium can be adjusted by adding a pH adjuster that can be used in pharmaceuticals and food and beverages to the medium as appropriate. Examples of such pH adjusters include, but are not limited to, phosphoric acid, DL-malic acid, sodium lactate, sodium hydroxide, sodium carbonate, citric acid, and sodium citrate.

In addition to the above components, the medium may contain other components such as a carbon source, a nitrogen source different from those mentioned above, inorganic salts, animal and plant extracts, agar, etc., as necessary.

In the present invention, the medium can be prepared according to a conventional method. That is, the medium can be prepared by appropriately blending and mixing the above-mentioned heated onion, nitrogen source, glutamic acid or a salt thereof, and any solvent such as water as necessary, as well as divalent metal ions, pH adjusters, and other components as necessary, in the amounts described above, and sterilizing the mixture.

In the present invention, "lactic acid bacteria" refers to lactic acid bacteria having GAD activity. "GAD activity" (glutamic acid decarboxylase activity) refers to the activity of catalyzing the reaction of removing carbon dioxide from L-glutamic acid to produce GABA. Lactic acid bacteria having GAD activity that can be used in the present invention include, but are not limited to, lactic acid bacteria belonging to the genera Lactobacillus, Enterococcus, Streptococcus, Leuconostoc, and Bifidobacterium, such as Lactobacillus brevis, Lactococcus lactis, Lactobacillus hilgardii, Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus delbrueckii, and Lactobacillus subtilis. Examples of lactic acid bacteria include, but are not limited to, lactic acid bacteria belonging to Lactobacillus leichmannii, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, Enterococcus avium, Enterococcus casseliflavus, Streptococcus thermophilus, Streptococcus faecalis, Leuconostoc mesenteroides, Bifidobacterium longum, etc. In addition, as long as they have GAD activity, these mutant strains can also be used in the present invention.

In the present invention, "culturing" can be performed according to conventionally known conditions that allow lactic acid fermentation. That is, the above-mentioned lactic acid bacteria are added to the above-mentioned medium in an amount of 0.005 to 5 w/v%, preferably 0.01 to 2 w/v%, based on the wet cell weight, and the culture is performed under anaerobic conditions at 20°C to 50°C, preferably 25°C to 42°C. The temperature conditions can be adjusted by using a thermostatic bath, a mantle heater, a jacket, or the like. "Under anaerobic conditions" means a low-oxygen environment in which lactic acid bacteria can grow. For example, anaerobic conditions can be achieved by using an anaerobic chamber, an anaerobic box, or a sealed container or bag containing an oxygen scavenger, or by simply sealing the culture container. The culture can be performed by static culture, shaking culture, tank culture, etc., and the culture time is not particularly limited, but can be, for example, 3 hours to 14 days, for example, 6 hours to 10 days, or 12 hours to 5 days. The culture can be performed continuously or batchwise.

Cultivation can be stopped (lactic acid fermentation can be stopped) by conventional means such as treatment at high temperature (80°C or higher) for a short period of time (2 seconds to 5 minutes) or lowering the pH.

The lactobacillus-fermented onions thus produced may be further subjected to a refining step. The refining can be carried out by using a commonly used solid-liquid separation means (e.g., centrifugation, filtration, etc.) and by recovering the liquid portion (supernatant, filtrate).

The lactobacillus-fermented onions thus produced may be subjected to a sterilization treatment, if necessary. Sterilization can be performed by any commonly used means (e.g., heat sterilization, high-pressure sterilization, air current sterilization, etc.). In order to preserve the various components in the lactobacillus-fermented onions, it is preferable that the sterilization treatment be performed for as short a time as possible at as low a temperature as possible.

The lactobacillus-fermented onion thus produced can be in the form of a concentrate or a dry product, if necessary. The concentration can be carried out by reducing the water content in the lactobacillus-fermented onion. The above-mentioned vacuum evaporation concentration, membrane concentration, etc. can be used as the concentration method. The drying can be carried out by drying the lactobacillus-fermented onion or a concentrate thereof by a usual drying means (freeze drying, reduced pressure drying, heat drying, air drying, etc.). When concentrating or drying, the lactobacillus-fermented onion may be combined with other components such as excipients before the concentration or drying.

The lactic acid bacteria-fermented onion produced according to the present invention (hereinafter referred to as "lactic acid bacteria-fermented onion of the present invention") contains a high content of GABA, and contains 5 w/w%, 6 w/w%, 7 w/w%, 8 w/w%, 9 w/w%, 10 w/w%, 11 w/w%, 12 w/w%, 13 w/w%, 14 w/w%, 15 w/w% or more of GABA per Brix solid content.

The lactobacillus-fermented onions of the present invention also contain a high content of cycloalliin, with the amount being 0.5 w/w%, 0.6 w/w%, 0.7 w/w%, 0.8 w/w%, 0.9 w/w%, or 1 w/w% or more of cycloalliin per Brix solid content.

In the lactobacillus-fermented onion of the present invention, the carbohydrates contained in the onion are consumed during the lactic acid fermentation process, and the carbohydrate content is reduced, so that the content of functional components such as GABA and cycloalliin per Brix solid content can be increased.

The lactobacillus-fermented onion of the present invention can contain excipients, disintegrants, lubricants, binders, etc. that are acceptable for use as medicines or foods and beverages, and can be manufactured into a desired dosage form or shape.

Examples of excipients include glucose, starch, pregelatinized starch, dextrin, D-mannitol, D-sorbitol, lactose, corn starch, crystalline cellulose, low-substituted hydroxypropyl cellulose, sodium carboxymethylcellulose, gum arabic, white sugar, pullulan, light anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminometasilicate, etc.

Examples of disintegrants include starch, lactose, carboxymethylcellulose, calcium carboxymethylcellulose, croscarmellose sodium, sodium carboxymethylstarch, sucrose, light anhydrous silicic acid, and low-substituted hydroxypropylcellulose.

Examples of lubricants include sugar esters such as sucrose fatty acid esters and glycerin fatty acid esters, calcium stearate, magnesium stearate, stearic acid, stearyl alcohol, hydrogenated oils such as powdered vegetable oils and fats, waxes such as white beeswax, talc, silicic acid, silicon, etc.

Binders include, for example, sucrose, pregelatinized starch, gelatin, dextrin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, pullulan, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, etc.

If necessary, the lactic acid bacteria-fermented onion of the present invention can further contain other ingredients that are usually used in the production of medicines (including quasi-drugs) or food and drink, such as diluents, buffers, suspending agents, coating agents, preservatives, antiseptics, antioxidants, colorants, anti-aggregating agents, absorption promoters, solvents, solubilizers, isotonicity agents, stabilizers, flavorings, pH adjusters, fragrances, sweeteners, taste-imparting components and acidulants, which can be appropriately selected and blended according to the desired dosage form and shape.

The lactobacillus-fermented onion of the present invention can be provided in the form of a medicine (including quasi-drugs) or a food or drink.

The pharmaceutical preparation is not particularly limited in its form, but is preferably in a form suitable for oral administration. For example, solid compositions for oral administration (solid pharmaceutical preparations) may be in the form of tablets (including sugar-coated tablets), pills, capsules, fine granules, granules, troches, chewable tablets, drops, etc., while liquid compositions for oral administration (liquid pharmaceutical preparations) may be in the form of emulsions, solutions, suspensions, syrups, etc. These preparations can be formulated in the usual manner by appropriately mixing the lactobacillus-fermented onion of the present invention with the above-mentioned excipients and other ingredients according to the dosage form.

Medicines may be labeled as having efficacy and effects caused by GABA (e.g., suppression of blood pressure drops, mental stabilization, improvement of sleep, etc.) or efficacy and effects caused by cycloalliin (e.g., prevention of thrombotic diseases, etc.).

There are no particular limitations on the form of the food and drink, but examples include curry, mixed spices (curry powder, garam masala, etc.), furikake, seasoning, soup, Western-style simmered dishes (stew, chowder, hayashi, hash brown, bouillabaisse, etc.), sauce, dressing, snacks, noodles (ramen, udon, etc.), desserts, juice, tea, alcohol, tablets, tablets, chewable tablets, powders, powders, capsules, granules, drinks, and other health foods and drinks (supplements, dietary supplements, health supplements, nutritionally adjusted foods, etc.).

In addition to general foods and beverages, foods and beverages include health functional foods (foods for specified health uses (including conditional foods for specified health uses), nutritional functional foods, and foods with functional claims) and health foods, and these foods and beverages can be labeled as having efficacy and effects caused by GABA (e.g., suppression of blood pressure drop, mental stabilization, improvement of sleep, etc.) or efficacy and effects caused by cycloalliin (e.g., prevention of thrombotic diseases, etc.).
The present invention will now be described in more detail with reference to examples.

Example 1 Production of Lactic Acid Bacteria-Fermented Onion Extract Containing GABA and Cycloalliin Onion bulbs from which the outer skin had been removed were cut into suitable sizes, and 700 g of the cut onion bulbs were mixed with the same amount of water and heated at 80° C. for 10 minutes for extraction. The supernatant was then recovered to give an onion extract (hereinafter referred to as "onion extract") (1,300 g; Brix solid content 4.9 w/v %).

Then, the Brix solid content of the obtained onion extract was adjusted to 2.5 w/v %, and then 1 w/v % of powdered yeast extract (Powdered Yeast Extract D-3H (Nihon Seiyaku)) and 1 w/v % of monosodium glutamate monohydrate were added to the extract, sodium hydroxide solution was added to adjust the pH to 6.8, and the mixture was autoclaved at 120°C for 20 minutes.

The autoclaved onion extract was inoculated with pre-cultured Lactobacillus brevis NBRC12005 strain (obtained from the National Institute of Technology and Evaluation, Biotechnology Center (NBRC)) (hereinafter referred to as "NBRC12005 strain") at a ratio of 1 v/v%, and cultured at 30°C for 3 days to allow fermentation.

After the cultivation was completed, it was confirmed that the lactobacillus-fermented onion extract obtained contained 12 w/w% GABA and 0.74 w/w% cycloalliin, respectively, calculated as the amount per Brix solid content (Brix solid content after cultivation was 3.9 w/v%).

Example 2: Investigation of nitrogen source in lactic acid bacteria fermentation Onion bulbs from which the outer skin had been removed were cut into suitable sizes, and 1,400 g of water was added to 700 g of the cut onion bulbs. The mixture was heated at 115° C. for 30 minutes and extracted, and the supernatant was recovered to give an onion extract (2,000 g; Brix solid content 2.5 w/v %).

Next, 1 w/v % of soy protein hydrolysate (Soy peptone F (Fujifilm Wako Pure Chemical Industries, Ltd.)) and 1 w/v % of monosodium glutamate monohydrate were added to the onion extract obtained, and the pH was adjusted to 6.8 by adding sodium hydroxide solution, followed by autoclaving at 120°C for 20 minutes.

The autoclaved onion extract was inoculated with pre-cultured NBRC12005 strain at a ratio of 1 v/v %, and cultured at 30°C for 3 days to allow fermentation.

After the completion of the cultivation, it was confirmed that the obtained lactic acid bacteria-fermented onion extract contained 6 w/w% GABA and 0.5 w/w% cycloalliin, respectively, calculated as the amount per Brix solid content (Brix solid content after cultivation was 4.0 w/v%). From these results, it was confirmed that even when soybean protein hydrolysate was used as the nitrogen source instead of powdered yeast extract, a lactic acid bacteria-fermented onion extract containing GABA and cycloalliin at high rates could be obtained.

Example 3 Effect of Addition of Nitrogen Source on Lactic Acid Bacteria Fermentation Autoclaved onion extracts were prepared in the same manner as in Example 1 except for the amount of powdered yeast extract added. The powdered yeast extract was added in amounts of 0 w/v %, 0.25 w/v %, 0.5 w/v % or 1 w/v %.

Next, the NBRC12005 strain or Lactobacillus brevis NBRC3345 strain (obtained from NBRC) (hereinafter referred to as "NBRC3345 strain") that had been precultured in advance was inoculated at a ratio of 1 v/v%, and cultured at 30°C for 2 days to allow fermentation.

After the cultivation was completed, the conversion rate from monosodium glutamate monohydrate was calculated based on the GABA content in each lactobacillus-fermented onion extract.

The results are shown in Figure 1. In both the NBRC12005 and NBRC3345 strains, the addition of powdered yeast extract was confirmed to result in the production of GABA. On the other hand, because onions are poor in nitrogen sources that can be assimilated by lactic acid bacteria, it was confirmed that GABA is not produced when there is an insufficient amount of usable nitrogen source in the medium.

Example 4 Effect of Addition of Divalent Metal Ions on Lactic Acid Bacteria Fermentation An autoclaved onion extract was prepared in the same manner as in Example 1, except that manganese sulfate tetrahydrate was added as a source of divalent metal ions ( ^Mn ions). Manganese sulfate tetrahydrate was added to a concentration of 0.001 w/v %.

Next, the NBRC12005 strain or NBRC3345 strain, which had been precultured in advance, was inoculated at a ratio of 1 v/v%, and cultured at 30°C for 2 days to allow fermentation.

After the cultivation was completed, the conversion rate from monosodium glutamate monohydrate was calculated based on the GABA content in each lactobacillus-fermented onion extract.

The results are shown in Figure 2. It was confirmed that the addition of powdered yeast extract improved the amount of GABA produced in both the NBRC12005 strain and the NBRC3345 strain. It has been reported that the addition of Mn ²⁺ ions as a trace metal during lactic acid fermentation increases the growth and fermentation activity of lactic acid bacteria (Cheng X. et al., Appl Biochem Biotechnol. Nov; 174(5):1752-60(2014)). Since onion extract contains almost no Mn ²⁺ ions, it is suggested that the addition of Mn ²⁺ ions to onion extract improved the growth and fermentation activity of lactic acid bacteria, thereby improving the productivity of GABA.

Example 5 Increase in Proportion of Cycloalliin by Lactic Acid Bacteria Fermentation An autoclaved onion extract was prepared by the method described in Example 1 above, and the pre-cultured NBRC3345 strain was inoculated into this at a ratio of 1 v/v %, and cultured at 30° C. for 2 days to allow fermentation.

After the cultivation was finished, the cycloalliin content and the Brix solid content in the obtained lactic acid bacteria-fermented onion extract were measured, and the cycloalliin content per solid content was calculated.
The results are shown in Table 1 below.

In the onion extract after fermentation, an increase in the cycloalliin content was observed and a decrease in the Brix solid content was observed compared to the onion extract before fermentation, confirming an increase in the cycloalliin content per Brix solid content (1.3 times).

In the lactic acid fermentation of onion extract, bacteria assimilate and decompose the carbohydrates contained in onions, so that fermentation can be carried out without adding carbohydrates externally. Since the Brix solid content in onion extract decreases due to the decomposition of carbohydrates, an increase in the cycloalliin content per Brix solid content was confirmed. Until now, it was not clear how the content of functional components such as cycloalliin contained in onions changes through lactic acid fermentation. The results of this example showed that it is possible to increase the content of functional components such as cycloalliin and GABA by utilizing lactic acid fermentation.

Example 6: Investigation of pH in lactic acid bacteria fermentation Autoclaved onion extracts were prepared in the same manner as in Example 1 except that the adjusted pH values were different. The pH values were adjusted to pH 4, pH 5, pH 6 or pH 7 with a sodium hydroxide solution or hydrochloric acid.

Next, the NBRC12005 strain or NBRC3345 strain, which had been precultured in advance, was inoculated at a ratio of 1 v/v%, and cultured at 30°C for 2 days to allow fermentation.

After the cultivation was completed, the conversion rate from monosodium glutamate monohydrate was calculated based on the GABA content in each lactobacillus-fermented onion extract.

The results are shown in Figure 3. GABA production was confirmed at pH 4 to pH 7 for both NBRC12005 and NBRC3345 strains. In particular, higher GABA production was confirmed under relatively high pH conditions (pH 5 to pH 7) than under acidic conditions (pH 4).

Since lactic acid bacteria can grow under acidic conditions, lactic acid fermentation is generally carried out under acidic conditions where they can grow preferentially. On the other hand, in the production of GABA using lactic acid bacteria, it is considered preferable to culture the lactic acid bacteria under pH conditions where the lactic acid bacteria can grow and where the activity of GAD, a GABA-producing enzyme, becomes optimal. Therefore, it is considered that GABA is efficiently produced in onion extract when the pH at the start of fermentation is in the range of 5 to 7.

Claims (6)

A method for producing lactic acid bacteria-fermented onions containing GABA, comprising a step of culturing lactic acid bacteria having glutamic acid decarboxylase activity in a medium containing heated onions, a nitrogen source, manganese ions, and glutamic acid or a salt thereof, wherein the pH of the medium before autoclaving is 5 or higher. The lactic acid bacteria is Lactobacillus brevis, Lactococcus lactis, Lactobacillus hilgardii, Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus delbrueckii, Lactobacillus leichmannii, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fermentum, Enterococcus avium, Enterococcus casseliflavus, Streptococcus thermophilus, Streptococcus faecalis, Leuconostoc mesenteroides, Bifidobacterium longum. The method according to claim 1 , wherein the lactic acid bacteria is one or more selected from the group consisting of lactic acid bacteria. 3. The method according to claim 1 or 2 , wherein the nitrogen source is one or more nitrogen sources selected from the group consisting of yeast extract, soybean protein, corn hydrolysate, wheat hydrolysate, cotton extract, and whey protein. The method according to any one of claims 1 to 3 , wherein the pH of the medium before autoclaving is 6.8 or higher. The process according to any one of claims 1 to 4 , wherein the heated onion is a heated onion extract having a Brix solid content of 1 to 10 w/v %. The method according to any one of claims 1 to 5 , wherein the lactic acid bacteria-fermented onion further contains cycloalliin.