JP6991120B2 - Method for producing γ-aminobutyric acid - Google Patents

Description

NPMD NITE P-02790

The present invention relates to a novel lactic acid bacterium and a method for producing a food or drink containing γ-aminobutyric acid and γ-aminobutyric acid using the novel lactic acid bacterium.

γ-Aminobutyric acid (GABA) is a non-protein amino acid that is widely distributed in the biological world, and is known to function as an inhibitory neurotransmitter in higher animals (Non-Patent Document 1). In recent years, γ-aminobutyric acid has been known to have various physiological functions, and has been reported to have a blood pressure lowering effect, a brain function improving effect, a tranquilizing effect, and the like (Non-Patent Documents 2 to 4).

γ-Aminobutyric acid is contained in brown rice and some vegetables and fruits in foods, but only a trace amount is present in these, and foods containing an effective amount for fulfilling the above physiological functions There wasn't.
Therefore, various methods for increasing the content of γ-aminobutyric acid in foods have been studied, and many methods for converting glutamic acid in foods into γ-aminobutyric acid by fermentation of lactic acid bacteria have been taken (Patent Documents 1 to 7). ). As a fermentation raw material containing a large amount of glutamic acid, tomato fruits and processed products thereof (processed tomato products, etc.) are used.

Japanese Patent No. 4757569 Japanese Unexamined Patent Publication No. 2000-308457 Japanese Unexamined Patent Publication No. 2004-313032 Japanese Unexamined Patent Publication No. 2007-289008 Japanese Unexamined Patent Publication No. 2008-17703 Japanese Unexamined Patent Publication No. 2008-50269 Japanese Unexamined Patent Publication No. 2008-54555

Journal of Biological Engineering, 75, 239-244, 1997 Pharmacology and Treatment, 28, 529-533, 2000 Food and Development, 36, No. 6, 4-6, 2001 Journal of Japanese Society of Food Science and Engineering, 47, 596-603, 2000

Patent Document 1 shows that the conversion rate to γ-aminobutyric acid is not so high when a processed tomato product having a filtrate coloring degree of more than 0.20 at Brix 3% is used as a fermentation raw material in fermentation of lactic acid bacteria. There is. As described above, when the processed product of tomato fruit is used as a fermentation raw material, its heat history (filter coloring degree) affects the ability to produce γ-aminobutyric acid. Therefore, there are restrictions on the raw materials used, which is not preferable from the viewpoint of production cost when lactic acid bacteria fermentation of γ-aminobutyric acid is performed on an industrial scale.
Under such circumstances, it is an object of the present invention to provide a method for producing a food or drink containing γ-aminobutyric acid and γ-aminobutyric acid, which can use an inexpensive processed tomato product as a fermentation raw material without any limitation on the heat history. do.

As a result of diligent research by the present inventors, it has been found that the novel lactic acid bacterium Lactobacillus plantarum KB1253 strain can convert glutamic acid to γ-aminobutyric acid with high efficiency. Then, he came up with the idea that γ-aminobutyric acid and γ-aminobutyric acid-containing foods and drinks could be efficiently produced by fermenting a processed tomato product using the lactic acid bacterium, and completed the present invention.

That is, the first aspect of the present invention is a novel lactic acid bacterium Lactobacillus plantarum KB1253 strain.

Another aspect of the present invention is a composition containing a processed tomato product and a lactic acid bacterium Lactobacillus plantarum KB1253 strain. In this embodiment, the composition preferably contains γ-aminobutyric acid in an amount of 17.5 mM or more. Further, in this embodiment, the composition preferably contains 15.4 mM or less of glutamic acid. Further, the composition of this embodiment is preferably contained in foods and drinks.

Another aspect of the present invention is a method for producing γ-aminobutyric acid, which comprises a fermentation step of culturing the lactic acid bacterium Lactobacillus plantarum KB1253 strain in a glutamic acid-containing medium.
Another aspect of the present invention is a method for producing a γ-aminobutyric acid-containing food or drink, which comprises a fermentation step of fermenting a processed tomato product with a lactic acid bacterium Lactobacillus plantarum KB1253 strain. The Brix of the processed tomato product in this embodiment is preferably 5 to 20%.
The fermentation step in these production methods is preferably carried out at 30 to 40 ° C., and is preferably carried out for 12 hours or more. In these production methods, it is preferable to add glutamic acid and / or a salt thereof before or during the fermentation step.

INDUSTRIAL APPLICABILITY The present invention provides a novel lactic acid bacterium capable of converting glutamic acid to γ-aminobutyric acid with high efficiency. The lactic acid bacterium has a high heat history (filter coloring degree), for example, a filtrate coloring degree of Brix 3% is higher than 0.20, and even when a general-purpose processed tomato product is used as a fermentation raw material, the conversion is as high as 60% or more. Gamma-aminobutyric acid can be produced at a rate. Therefore, γ-aminobutyric acid and γ-aminobutyric acid-containing foods and drinks can be efficiently produced by using the processed tomato product as a fermentation raw material without being restricted by the heat history (filter coloring degree) and fermenting with the lactic acid bacteria. can do.
In such fermentation, glutamic acid is consumed and the concentration of glutamic acid in the processed tomato product after fermentation is low, so that it is suitable as a raw material or an additive for foods and drinks that requires a refreshing aftertaste with suppressed umami.

The graph which shows the time-dependent change of the γ-aminobutyric acid concentration in Example 1. FIG. The graph which shows the time-dependent change of the glutamic acid concentration in Example 1. FIG. The graph which shows the time-dependent change of the conversion rate from glutamic acid to γ-aminobutyric acid in Example 1. FIG. The graph which shows the time-dependent change of the γ-aminobutyric acid concentration in Example 2. The graph which shows the time-dependent change of the glutamic acid concentration in Example 2. The graph which shows the time-dependent change of the conversion rate from glutamic acid to γ-aminobutyric acid in Example 2. FIG. The graph which shows the time-dependent change of the γ-aminobutyric acid concentration in Example 3. The graph which shows the time-dependent change of the glutamic acid concentration in Example 3. The graph which shows the time-dependent change of the conversion rate from glutamic acid to γ-aminobutyric acid in Example 3. FIG. The graph which shows the time-dependent change of the conversion rate from glutamic acid to γ-aminobutyric acid in Example 4. FIG.

Next, an embodiment of the present invention will be described. However, the present invention is not limited to the following embodiments, and can be freely changed within the scope of the present invention.

The first aspect of the present invention is a novel lactic acid bacterium Lactobacillus plantarum KB1253 strain. Hereinafter, it will also be referred to as “lactic acid bacterium of the present invention”.
The lactic acid bacterium of the present invention can convert glutamic acid to γ-aminobutyric acid.

Lactobacillus plantarum KB1253 strain is a bacterium isolated from pickles. To investigate its genetic properties, the 16S rRNA gene sequence was identified by a conventional method. Furthermore, the nucleotide sequence of about 500 bp upstream of the 16S rRNA gene of Lactobacillus plantarum KB1253 strain was searched for homology by BLAST analysis in the database of the National Center for Biotechnology Information (NCBI).
As a result, the Lactobacillus plantarum KB1253 strain is 100% homologous to the Lactobacillus plantarum subspecies plantarum NBRC15891, which is the reference strain of Lactobacillus plantarum. It was confirmed that it is a lactic acid bacterium that has sex and belongs to Lactobacillus plantarum.

Lactobacillus plantarum KB1253 strain was released on October 3, 2018 by the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (postal code: 292-0818, address: 2-5-Kazusakamatari, Kisarazu City, Chiba Prefecture). A domestic deposit was made in Room 8122), and the receipt number NITE AP-02790 was given.

The lactic acid bacterium of the present invention is not limited to the strain itself deposited under the name of Lactobacillus plantarum KB1253 (also referred to as “deposited strain” for convenience), and is substantially equivalent to the deposited strain (“derivative strain”). Also referred to as "inducible strain"). The substantially equivalent bacterium is a bacterium of the same genus as the lactic acid bacterium of the present invention and has an activity of converting glutamic acid to γ-aminobutyric acid at a conversion rate as high as that of the deposited strain. Further, in substantially equivalent bacteria, the base sequence of the 16S rRNA gene has 99.5% or more, preferably 99.9% or more, more preferably 100% homology with the base sequence of the 16S rRNA gene of the above-mentioned deposit strain. And preferably have the same mycological properties as the deposited strain. Further, the lactic acid bacterium of the present invention is a mutant strain bred from a deposited bacterium or a bacterium substantially equivalent thereto by mutation treatment, gene recombination, selection of a natural mutant strain, etc., as long as the effect of the present invention is not impaired. May be. Examples of the breeding method include modification by a genetic engineering method and modification by mutation treatment. Mutation treatment includes X-ray irradiation, ultraviolet irradiation, and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethylmethanesulfonate (EMS), and methylmethanesulfonate (MMS). Treatment with a variegant such as, etc. can be mentioned. Examples of the naturally mutated strain from the deposited strain include strains naturally generated during the use of the deposited strain. Examples of such a strain include a mutant strain naturally generated by culturing a deposited strain (for example, subculture). The derivative strain may be constructed by one modification or by two or more modifications.

Another aspect of the present invention is a method for producing γ-aminobutyric acid, which comprises a fermentation step of culturing the lactic acid bacterium of the present invention in a glutamic acid-containing medium.
The lactic acid bacterium of the present invention can convert glutamic acid to γ-aminobutyric acid. Therefore, this aspect can be rephrased as a method for converting glutamic acid into γ-aminobutyric acid.

The glutamic acid-containing medium in this embodiment is not particularly limited as long as it contains glutamic acid, and a medium usually used for culturing lactic acid bacteria can be sterilized before use. That is, as the carbon source, for example, sugars such as glucose, galactose, lactose, arabinose, mannose, sucrose, starch, starch hydrolysate, and waste sugar honey can be used depending on the assimilation property. As the nitrogen source, for example, ammonium salts such as ammonia, ammonium sulfate, ammonium chloride and ammonium nitrate and nitrates can be used. Further, as the inorganic salts, for example, sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, ferrous sulfate and the like can be used. Further, organic components such as peptone, soybean flour, defatted soybean meal, meat extract and yeast extract may be used.
Further, a processed tomato product can also be used as the glutamic acid-containing medium, and in that case, it becomes an aspect of the method for producing a food or drink containing γ-aminobutyric acid, which will be described later.

The content of glutamic acid in the medium is not particularly limited, but the upper limit is preferably 400 mM or less, more preferably 160 mM or less, still more preferably 120 mM or less. The lower limit may be 1 mM or more, more preferably 10 mM or more.

The γ-aminobutyric acid produced according to this embodiment can be added to foods and drinks by any method.

Another aspect of the present invention is a method for producing a food or drink containing γ-aminobutyric acid, which comprises a fermentation step of fermenting a processed tomato product with the lactic acid bacterium of the present invention.

The processed tomato product in this embodiment refers to a processed tomato fruit, and the processing means thereof is not particularly limited. For example, tomato juice, ground, crushed, shredded, extracted, dried, heated, concentrated, centrifuged supernatant, clarified. Things etc. can be mentioned.
The tomato-treated product in this embodiment usually contains glutamic acid of 10.0 mM or more, more preferably 25.0 mM or more, still more preferably 30.0 mM or more. It should be noted that this is the concentration when used for fermentation, and when the tomato processed product is the above-mentioned dried product or concentrated product, it is the concentration when it is diluted with water or the like to be used for fermentation. In addition, it is the concentration of the portion excluding the insoluble solid content.

The Brix of the processed tomato product in this embodiment is preferably 5 to 20%, more preferably 10 to 15%. By using a substance in such a range, fermentation by the lactic acid bacterium of the present invention proceeds sufficiently, and the conversion rate from glutamic acid to γ-aminobutyric acid is further improved.

The insoluble solid content in the processed tomato product in this embodiment is preferably 5% by volume or less. When it is 5% by volume or less, fermentation by lactic acid bacteria proceeds sufficiently, and the conversion rate from glutamic acid to γ-aminobutyric acid is further improved. The insoluble solid content can be adjusted by filtration such as ordinary filtration, microfiltration, ultrafiltration, or centrifugation.
Here, the insoluble solid content is measured by the following method. 10 mL of the tomato processed product was placed in a centrifugal settling tube having a length of 105 mm, and the ratio of the volume of the precipitate to the total volume was measured when the tomato was centrifuged under the conditions of a turning radius of 14.5 cm, a rotation speed of 3000 rpm, and a time of 10 minutes. The value is an insoluble solid content.

The degree of coloration of the filtrate of the processed tomato product in this embodiment is not particularly limited.
Conventionally, when a processed tomato product having a high degree of filtrate coloring, for example, a filtrate coloring of Brix 3% higher than 0.20, is used as a fermentation raw material, fermentation by lactic acid bacteria proceeds, but the conversion rate from glutamic acid to γ-aminobutyric acid is high. It was supposed to be low. By using the lactic acid bacterium of the present invention, γ-aminobutyric acid can be produced with high efficiency even in a processed tomato product having a high degree of filtrate coloring. Therefore, any inexpensive processed tomato product can be used as a fermentation raw material without considering the heat history.

The degree of coloration of the filtrate of the processed tomato product in this embodiment may be high, but from the viewpoint of further improving the productivity of γ-aminobutyric acid, 0.02 to 1.22 is preferable in Brix 3%, and 0.02 to 0. 35 is more preferable.
Here, the degree of coloration of the filtrate is measured by the following method. First, the processed tomato product is adjusted to a sugar content of 3% using water or the like. Next, this is filtered using a filter paper (No. 5A manufactured by Advantech Co., Ltd.). A funnel-shaped glass filter (36060FNL3G4 manufactured by Asahi Techno Glass Co., Ltd.) containing a high-flow supercell (manufactured by Celite, sold by Wako Pure Chemical Industries, Ltd., Catalog No. 534-02315) with a thickness of about 5 mm is precoated with distilled water. do. The filtered tomato processed product is passed through this funnel-shaped glass filter, and further filtered with a membrane filter (DISMIC-25CS045AN manufactured by Advantech) having an opening diameter of 0.45 μm. The treated tomato product after filtration is measured for absorbance at 450 nm using a spectrophotometer (U-3310 manufactured by Hitachi, Ltd.), and the value is used as the filtrate coloring degree.

In the above-mentioned method for producing γ-aminobutyric acid and the method for producing γ-aminobutyric acid-containing food and drink, the lactic acid bacterium of the present invention is pre-cultured in an appropriate medium in advance, and the pre-culture is used as a glutamic acid-containing medium or a tomato-treated product. From the viewpoint of production efficiency and stability of γ-aminobutyric acid, it is preferable to add it to the main culture (fermentation step).

The preculture conditions are not particularly limited, but are preferably 8 to 48 hours at 30 to 40 ° C., for example. The number of lactic acid bacteria in the culture after preculture is preferably ¹⁰⁷ to ¹⁰⁹ cfu / mL.
A processed tomato product may be used as the medium in the preculture. Further, before the processed tomato product is subjected to pre-culture, it is usually sterilized in advance, and the conditions are not particularly limited, but it is preferable to treat the tomato product at 80 to 110 ° C. for 1 to 20 minutes.

The lactic acid bacterium preculture added in the main culture is preferably 0.1 to 20% by mass, particularly preferably 0.1 to 10% by mass, based on the glutamic acid-containing medium or the processed tomato product.
The temperature condition of the main culture (fermentation step) is preferably 30 to 40 ° C, more preferably 32 to 38 ° C. The pH is preferably 4.0 to 6.0. Further, the fermentation time is preferably 12 hours or more, more preferably 24 hours or more, and usually 96 hours or less is the fermentation end point.
The main culture (fermentation step) is preferably performed under anaerobic conditions, and for example, the culture can be performed while aerating an anaerobic gas such as carbon dioxide. Further, the cells may be cultured under slightly aerobic conditions such as liquid static culture.

It is preferable to add glutamic acid and / or a salt thereof before or during the main culture (fermentation step). This is to supplement the substrate and improve the amount of γ-aminobutyric acid converted.
The concentration of glutamic acid after the addition is not particularly limited, but is preferably 10 to 160 mM, more preferably 10 to 120 mM.
When glutamic acid is added during fermentation, the timing is not particularly limited, but is preferably 0 to 24 hours after the start of fermentation, and more preferably 0 to 12 hours after the start of fermentation.

The culture (fermented product) after the main culture has a high conversion rate of glutamic acid to γ-aminobutyric acid of at least 60% or more, and contains γ-aminobutyric acid in a high concentration.

The conversion rate from glutamic acid to γ-aminobutyric acid is calculated by the following mathematical formula (1). In formula (1), GABA refers to γ-aminobutyric acid. [GABA] ₀ represents the concentration of γ-aminobutyric acid (mM) at the start of fermentation. [GABA] _t represents the concentration (mM) of γ-aminobutyric acid at the time t after the start of fermentation. [Glu] ₀ represents the glutamic acid concentration (mM) at the start of fermentation.

The γ-aminobutyric acid-containing food or drink produced by the above-mentioned production method can be prepared in the form of any food or drink, such as tomato juice, tomato puree, or tomato sauce. Further, these may be added to fruits other than tomato, fruit juice, vegetable juice, soy milk, malt juice, milk, yogurt, seasonings, confectionery, supplements, and other foods and drinks.
Such a γ-aminobutyric acid-containing food or drink is an embodiment of a composition containing a processed tomato product and the lactic acid bacterium of the present invention, and such a composition is also an embodiment of the present invention.

In the composition containing the processed tomato product and the lactic acid bacterium of the present invention, the lactic acid bacterium may be a live bacterium or a dead bacterium, or may contain both a live bacterium and a dead bacterium. Further, a state in which lactic acid bacteria are crushed or a state in which a part of lactic acid bacteria is contained may be used.
The composition can be prepared in the form of any food or drink and can be added to any food or drink.

The composition is usually liquid and may contain γ-aminobutyric acid in an amount of 17.5 mM or more, preferably 21.2 mM or more, more preferably 27.6 mM or more. Further, it may be in the form of a semi-solid or solid such as a concentrated product or a dried product, and may contain γ-aminobutyric acid in an amount of 1.80 mg / g or more, 2.18 mg / g or more, more preferably 2.94 mg / g. Contains g or more.
The glutamic acid content in the composition is preferably 15.4 mM or less, more preferably 10.0 mM or less. Further, 2.27 mg / g or less is preferable, and 1.48 mg / g or less is more preferable. This results in a clean aftertaste when in the form of food and drink.

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

<Example 1>
American juice tomato paste (MSP.020CB, manufactured by Morningstar Japan) was diluted with water to Brix 10% (hereinafter referred to as "Brix 10% tomato processed product") to prepare a medium (fermentation raw material). The degree of coloration of the filtrate when the processed Brix 10% tomato product was adjusted to Brix 3% was 0.31.

10 mL of Brix 10% tomato processed product was placed in a φ18 mm test tube and sterilized by autoclave at 95 ° C. for 10 minutes. Lactic acid bacteria were inoculated into this and cultured at 30 ° C. for 18 hours (preculture). Lactic acid bacteria are described in L. Plantarum KB1253 strain, L. plantarum NBR
C3070 strain, L. Plantarum NBRC14712 strain, L. Plantarum NBRC14713 strain, L. Brevis NBRC3360 strain, L. Brevis NBRC12005 strain, L. Brevis NBRC3345 strain, or L. Limosilactobacillus JCM2762 strain was used.
Separately from the above, 200 mL of Brix 10% tomato processed product was placed in a 300 mL Erlenmeyer flask and sterilized by autoclaving at 95 ° C. for 10 minutes. To this, 2 mL of the preculture solution was added, and the cells were cultured at 36 ° C. for 72 hours (main culture).

1 mL of the medium was collected at 0, 12, 24, 48, and 72 hours after the start of the main culture. The collected medium is diluted 10-fold (volume) with 3% sulfosalicylic acid, filtered through a membrane filter (DISMIC-25CS045AN manufactured by Advantech), and γ- using an automatic amino acid analyzer (L-8800A manufactured by Hitachi, Ltd.). The amount of aminobutyric acid was measured. The amount of γ-aminobutyric acid was calculated from a calibration curve prepared using a γ-aminobutyric acid standard (manufactured by Across Organics). In addition, the amount of glutamic acid in the collected medium was measured using the above-mentioned automatic amino acid analyzer. Using these measured values, the conversion rate was calculated according to the mathematical formula (1).

The results are shown in FIGS. Even when the degree of coloration of the filtrate in Brix 3% of the processed tomato product as a fermentation raw material is as high as 0.31, L. The plantalum KB1253 strain was able to produce γ-aminobutyric acid with a high conversion rate. In addition, L. The plantarum KB1253 strain showed a high conversion rate as compared with other lactic acid bacteria strains.

<Example 2>
The lactic acid bacterium used is L. Fermentation was carried out in the same manner as in Example 1 except that only the plantarum KB1253 strain was used and the fermentation temperature was changed to 28 to 42 ° C.
The results are shown in FIGS. 4-6. A high conversion rate was observed under the condition that the fermentation temperature was 30 to 40 ° C.

<Example 3>
The lactic acid bacterium used is L. Fermentation was carried out in the same manner as in Example 1 except that only the plantarum KB1253 strain was used and the Brix of the medium (fermentation raw material) was changed to 1 to 25%.
The results are shown in FIGS. 7-9. A high conversion rate was observed under the condition that Brix was 5 to 20%.

<Example 4>
The lactic acid bacterium used is L. Fermentation was carried out in the same manner as in Example 1 except that only the plantarum KB1253 strain was used, the medium was changed to MRSBros (CM0359, manufactured by Oxoid), and the glutamic acid content was changed to 10 to 400 mM.
The results are shown in FIG. Conversion to γ-aminobutyric acid was observed in all glutamic acid contents, but a high conversion rate was observed especially under the condition of 10 to 160 mM.

INDUSTRIAL APPLICABILITY According to the present invention, a novel lactic acid bacterium capable of converting glutamic acid to γ-aminobutyric acid with high efficiency is provided. In addition, γ-aminobutyric acid and γ-aminobutyric acid-containing foods and drinks can be efficiently produced by fermenting with the above-mentioned lactic acid bacteria using a processed tomato product as a fermentation raw material without being restricted by the heat history (degree of coloration of the filtrate). A way to do it is provided.
The γ-aminobutyric acid-containing food and drink produced by the method of the present invention is suitable for food and drinks that require a refreshing aftertaste, health foods, and the like.

Claims (11)

Lactobacillus plantarum KB1253 strain. A composition containing a processed tomato product and a lactic acid bacterium Lactobacillus plantarum KB1253 strain. The composition according to claim 2, which contains 17.5 mM or more of γ-aminobutyric acid. The composition according to claim 2 or 3, which contains 15.4 mM or less of glutamic acid. A food or drink containing the composition according to any one of claims 2 to 4. A method for producing γ-aminobutyric acid, which comprises a fermentation step of culturing a lactic acid bacterium Lactobacillus plantarum KB1253 strain in a glutamic acid-containing medium. A method for producing a γ-aminobutyric acid-containing food or drink, which comprises a fermentation step of fermenting a processed tomato product with a lactic acid bacterium Lactobacillus plantarum KB1253 strain. The production method according to claim 7, wherein the Brix of the processed tomato product is 5 to 20%. The production method according to any one of claims 6 to 8, wherein the fermentation step is carried out at 30 to 40 ° C. The production method according to any one of claims 6 to 9, wherein the fermentation step is carried out for 12 hours or more. The production method according to any one of claims 6 to 10, wherein glutamic acid and / or a salt thereof is added before or during the fermentation step.

Images