JP2008054555A - NEW LACTOBACILLUS HAVING HIGH PRODUCTION CAPACITY OF gamma-AMINOBUTYRIC ACID, AND gamma-AMINO BUTYRIC ACID AND METHOD FOR PRODUCING LACTIC ACID FERMENTED FOOD BY USING THE SAME - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new lactobacillus having a high production capacity of γ-aminobutyric acid (GABA), a method for producing the GABA and method for producing a GABA-containing lactic acid fermented food by using the lactobacillus, and a glutamic acid decarboxylase (GAD) and nucleic acid encoding the same obtained from the lactobacillus. <P>SOLUTION: This new lactobacillus of Lactobacillus sp. L13 (NITE P-253) having a high production capacity of the GABA, the method for producing the GABA and method for producing the GABA-containing lactic acid fermented food by using the lactobacillus, and the GAD and nucleic acid encoding the same obtained from the lactobacillus are provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a novel lactic acid bacterium having high ability to produce γ-aminobutyric acid (hereinafter referred to as GABA), a method for producing GABA using the lactic acid bacterium, a method for producing a GABA-containing lactic acid fermented food, and glutamate decarboxylase obtained from the lactic acid bacterium (Hereinafter referred to as GAD) and a nucleic acid encoding the same.

  γ-aminobutyric acid (GABA) is a non-protein amino acid widely distributed in the living world, and is known to function as an inhibitory neurotransmitter in vivo (Non-patent Document 1). GABA has been known to have various physiological functions and has been reported to have effects such as blood pressure lowering action (Non-patent Document 2), arteriosclerosis prevention, liver function improvement, renal function improvement, mental stability and the like. (Non-Patent Document 3, Non-Patent Document 4).

  In order to utilize such a function of GABA, it was considered to first ingest a food material containing GABA. GABA is contained in foods such as brown rice, red rice bran, tea, some vegetables and fruits, etc., but these contain only a small amount of GABA (Non-patent Document 3) and express the original physiological functions. Was not a food containing an effective amount of GABA. Therefore, various methods for increasing the GABA content in foods have been studied. There are roughly two types of methods corresponding to the above-mentioned purpose proposed so far. One is a method of adding GABA or extracted GABA to food, and the other is to ferment the food using microorganisms such as lactic acid bacteria, or to produce GABA by causing an enzyme to act on glutamic acid in the food. Is the method.

  As a method for producing GABA added in the former method, in addition to a chemical synthesis method by amination of γ-halogenbutyric acid or hydrolysis of pyrrolidone, lactic acid bacteria producing GABA at a high concentration selected or bred in a natural material medium And fermenting using glutamate (Non-patent Document 3, Non-patent Document 5, Patent Document 1), and reacting glutamic acid or a raw material containing this with enzyme (glutamate decarboxylase (GAD)) to produce GABA at a high concentration A way to do it was proposed. In these methods, since GABA is added to existing foods later, there is a bad image of adding chemical additives, and there is a problem that the industrial production process of foods increases. Furthermore, when GABA is produced by fermentation, the flavor-causing substance as an impurity derived from the culture may be transferred to the food when added, and the original flavor of the food may be impaired, and microbial contamination may occur. Raised concerns. Therefore, unnaturalness in terms of production and quality in food production has occurred.

  On the other hand, in the latter method (Patent Document 2), when fermentation by lactic acid bacteria is used, fermentation by lactic acid bacteria does not always occur depending on food, and lactic acid bacteria do not always produce GABA even after fermentation. There was a problem. Moreover, the flavor of the said foodstuff may be impaired by fermentation by lactic acid bacteria depending on the case, and there was a restriction that lactic acid strains that satisfy the conditions for fermentation by lactic acid bacteria in the food product were very limited.

  So far, there has been a report on a strain isolated and bred from pickles such as kimchi as a lactic acid bacterium having a high ability to produce GABA (Patent Document 3). There is a possibility that such lactic acid bacteria can be used for the production of additive GABA as described above or for the production of GABA-containing foods, but in either case, a strain obtained from pickles such as kimchi that has a strong odor is used. In some cases, unpleasant flavors such as pickled odor and diacetyl odor may be added to the food.

  Since so-called Metnikov's theory of immortality regarding the relationship between yogurt, which is a typical food obtained by fermentation of lactic acid bacteria, and longevity, lactic acid bacteria and lactic acid fermented foods are thought to have a positive impact on human health, and epidemiological studies and Animal experiments including humans are being conducted. To date, effects such as stabilization of intestinal flora, reduction of blood cholesterol and suppression of carcinogenesis have been confirmed by ingestion of lactic acid bacteria. From the standpoint of food production, safety improvements such as suppression of food poisoning by lactic acid bacteria, salt habituation effect, and fermentation and flavor improvement by fermentation products are recognized.

  Pickles are foods that have been produced around the world from vegetables since ancient times. Traditionally, pickles are produced in Japan by fermentation, and it is considered that microorganisms such as lactic acid bacteria were involved in the fermentation process. That is, some conventional pickles are included in lactic acid fermented foods. Therefore, if such a pickle can be produced using fermentation by lactic acid bacteria that produce GABA, there is a possibility that a pickle as a functional fermented food containing GABA may be obtained.

  However, at present, it is said that the production of Japanese pickles by traditional fermentation methods is about 0.1% of the whole pickles, and most of the pickles are manufactured non-fermentatively. Advantages of the non-fermentative method include reduction of food hygiene problems, shortening of the production period, and uniform quality. Taking as an example the production of a thousand pickles that are a kind of Kyoto pickles, the simple main method of manufacturing is the overnight, where the current mainstream is the pickling period where salt is added to the raw vegetable turnip. On the other hand, the traditional law requires a period of one week. For these reasons, the non-fermentation method is considered more suitable for efficient industrial production of pickles.

  On the other hand, another reason why lactic acid fermentation is not used in the production of industrial pickles is that there is no starter suitable for pickles. That is, if a lactic acid bacterium that grows using plant components and does not adversely affect the flavor of food is obtained, it may be added to a raw vegetable as a starter to stably produce pickles. Moreover, if such lactic acid bacteria have the ability to produce GABA highly, fermented food containing GABA can be obtained efficiently and stably. Unlike cheese and yogurt, in the fermentation of pickles, generally starters are not used, but raw vegetables and microorganisms present in the production environment are used. For example, Patent Document 4 describes a method for producing pickles by adding a lactic acid bacterium isolated from kimchi as a starter, but it is unclear whether this can be used practically for pickle production. Moreover, it is not known whether the lactic acid bacteria described in this document have the ability to produce GABA.

JP 2000-210075 A Japanese Patent Laid-Open No. 3-236863 JP 2003-70462 A JP 2001-120173 A Yoshie Ueno et al., Kyoto M & T Center Information, 19999.6 Research Report Japan Food Science and Technology Journal, 49, 409-415 (2002) Osaka Bio-Environmental Science Research Report “GABA High-Concentration Fermented Extract” (2002) Yoshie Ueno et al., Kyoto M & T Center Information, 2001.6 Research Report Hayakawa Kiyoshi et al., Kyoto M & T Center Information, 1991.1 Research Report

  The present invention solves the problems of the prior art as described above. An object of the present invention is to provide a lactic acid bacterium having high GABA production ability, a method for producing GABA using the lactic acid bacterium, a method for producing a GABA-containing lactic acid fermented food, GAD obtained from the lactic acid bacterium, and a nucleic acid encoding the same. It is in.

  As a result of intensive studies, the present inventors have found that a novel lactic acid bacterium Lactobacillus sp. It was found that L13 (NITE P-253) has a high ability to produce GABA.

  As described above, most of the pickles are currently manufactured non-fermentatively, and the 1000 pickles are also manufactured by the vinegaring method not mainly based on lactic acid fermentation. There are few knowledge about the microorganisms which are concerned in fermentation, when a thousand pieces pickles are manufactured with a fermentation method like before, and there is no report about whether GABA is produced during a thousand pieces pickles by fermentation. Therefore, it was unexpected that a lactic acid bacterium having a high GABA production ability was obtained from the process of fermenting a thousand pieces. Thousands of pickles are not as strong as kimchi, so the lactic acid bacteria of the present invention obtained from the pickles are thought to have the advantage of not adding an unpleasant flavor to food. Moreover, since this lactic acid bacterium was obtained by pickling 1,000 pieces, it is considered that there is no problem in safety when used for food production.

  Furthermore, the present inventors have found that a lactic acid fermented food having excellent properties can be produced using the lactic acid bacterium, and a nucleic acid encoding GAD is cloned from the lactic acid bacterium to enable recombinant production of GABA. The present invention has been completed.

The present invention is:
[1] Lactobacillus Lactobacillus sp. Having the ability to produce γ-aminobutyric acid L13 (NITE P-253);
[2] Lactic acid bacteria Lactobacillus sp. A method for producing γ-aminobutyric acid, comprising culturing L13 (NITE P-253) and collecting γ-aminobutyric acid from the culture;
[3] The raw material of the fermented food is lactic acid bacteria Lactobacillus sp. A method for producing a lactic acid fermented food containing γ-aminobutyric acid, comprising a step of fermenting in the presence of L13 (NITE P-253);
[4] The method according to [3], wherein the lactic acid fermented food is pickled and the raw material of the fermented food is fogged;
[5] The raw material of the fermented food is lactic acid bacteria Lactobacillus sp. Γ-aminobutyric acid-containing lactic acid fermented food produced by a method comprising a step of fermenting in the presence of L13 (NITE P-253);
[6] The lactic acid fermented food according to [5], wherein the lactic acid fermented food is pickled and the raw material of the fermented food is fogged;
[7] A polypeptide comprising the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 7, and having glutamate decarboxylase activity;
[8] A nucleic acid encoding a polypeptide selected from the group consisting of the following and having glutamate decarboxylase activity:
(1) a nucleic acid comprising the base sequence of SEQ ID NO: 6;
(2) a nucleic acid comprising a base sequence in which one or several bases have been deleted, substituted or added in the base sequence of SEQ ID NO: 6;
(3) a nucleic acid that hybridizes with a nucleic acid comprising the base sequence of SEQ ID NO: 6 under stringent conditions;
[9] A method for producing a polypeptide having glutamate decarboxylase activity, comprising culturing cells containing the nucleic acid of [8], and collecting a polypeptide having glutamate decarboxylase activity from the culture;
[10] A method for producing γ-aminobutyric acid, comprising culturing cells containing the nucleic acid according to [8], and collecting γ-aminobutyric acid from the culture,
About.

  According to the present invention, there are provided a novel lactic acid bacterium having high GABA production ability, a method for producing GABA using the lactic acid bacterium, a method for producing a GABA-containing lactic acid fermented food, GAD obtained from the lactic acid bacterium, and a nucleic acid encoding the same.

γ-aminobutyric acid (GABA) is a non-protein amino acid widely distributed in the living world, and is biosynthesized from glutamic acid under the action of glutamic acid decarboxylase (GAD). It is known that GAD uses pyridoxal phosphate (PLP), which is a kind of vitamin B ₆ phosphate ester compound, as a coenzyme. GABA functions as an inhibitory neurotransmitter. GABA has physiological functions such as blood pressure lowering action, prevention of arteriosclerosis, improvement of liver function, improvement of kidney function, and mental stability. A preferred daily intake for adults to exert its efficacy is, for example, 10 to 500 mg, preferably 25 to 200 mg, and more preferably 50 to 100 mg.

  The present invention provides a lactic acid bacterium Lactobacillus sp. Having high ability to produce γ-aminobutyric acid. L13 (NITE P-253) (hereinafter referred to as L13 strain) is provided. The lactic acid bacteria of the present invention can be used for the production of GABA and GABA-containing lactic acid fermented foods. In this specification, a lactic acid bacterium refers to a bacterium that decomposes carbohydrates to produce lactic acid. There is no particular limitation on the genus to which the lactic acid bacteria belong, and examples include Lactobacillus genus.

  The lactic acid bacteria of the present invention were isolated as a strain having a high GABA production ability from a thousand pickles produced by a conventional fermentation method. Accordingly, lactic acid bacteria having a high GABA production ability can be obtained from lactic acid fermented foods such as pickled in the same manner as the L13 strain in accordance with the disclosure of the present specification. Such lactic acid bacteria are also included in the lactic acid bacteria of the present invention.

  The L13 strain is suggested to be different from Lactobacillus bacteria based on biochemical properties, but is a new species belonging to the Lactobacillus genus according to the genetic classification method based on the homology of the base sequence of 16S rDNA. It is concluded that The maximum identity between the 16S rDNA base sequence of the L13 strain and the known bacterial 16S rDNA base sequence was 98%. Therefore, bacteria showing the identity of the base sequence of 16S rDNA higher than 98% with the L13 strain are considered to belong to the same species as the L13 strain, and have a high possibility of having a high GABA production ability like the L13 strain. Such bacteria are also included in the lactic acid bacteria of the present invention.

  The present invention provides a method for producing GABA, comprising the steps of culturing L13 strain and collecting GABA from the culture. The culture conditions are not limited as long as the L13 strain grows well. As the medium, for example, a GYP medium can be used. The culture temperature is, for example, 10 to 35 ° C, preferably 15 to 30 ° C. The culture period is, for example, 1 to 10 days, preferably 2 to 5 days.

  Since GABA is biosynthesized from glutamic acid, GABA can be efficiently produced by culturing the L13 strain in the presence of glutamic acid or a salt thereof (for example, sodium glutamate). The L13 strain of the present invention has an excellent property that even if the glutamic acid concentration in the medium is increased, the conversion rate from glutamic acid to GABA does not decrease so much. Therefore, more GABA can be collected by adding more glutamic acid to the medium. The glutamic acid concentration in the medium is, for example, 1 to 20%, preferably 5 to 15%. The conversion rate from glutamic acid to GABA is, for example, 70 to 100%, preferably 80 to 100%, more preferably 85 to 100%.

  Moreover, GABA production ability can be further raised by maintaining the pH of a culture medium acidic. The medium pH is maintained at, for example, 3 to 7, preferably 4 to 6, more preferably about 5. Methods for maintaining medium pH during culture are known in the art.

  According to the method of the present invention, PLP known as a GAD coenzyme involved in GABA synthesis may be added to the medium, but the addition is not always necessary.

  Collection of GABA from the culture is performed according to a conventional method. For example, the culture supernatant obtained by removing the cells from the culture by centrifugation can be used as it is or after being concentrated, but it can be further purified by subjecting it to known purification means such as various chromatography. GABA can be obtained.

  GABA and glutamic acid can be measured by, for example, thin layer chromatography (TLC) using 1-butanol: acetic acid: water = 3: 2: 1 (v / v / v) as a developing solution, or a post column using orthophthalaldehyde. This can be done by detection in the law.

  The present invention provides a method for producing a GABA-containing lactic acid fermented food and a lactic acid fermented food produced by the method, including the step of fermenting the raw material of the fermented food in the presence of the L13 strain.

  In this specification, lactic acid fermentation refers to a phenomenon in which carbohydrates mainly generate lactic acid under the action of lactic acid bacteria. There are two types of lactic acid fermentation: homo-fermentation and hetero-fermentation. In homofermentation, two molecules of lactic acid are produced from one molecule of glucose. In heterofermentation, ethanol, carbon dioxide and the like are generated from glucose in addition to lactic acid. Homo-fermentation is preferred when efficient production of lactic acid is intended, but in the case of lactic acid fermentation of foods, it can also be expected that by-products generated in hetero-fermentation will have a good effect on the flavor of the food.

  In this specification, a lactic acid fermented food means a food in which lactic acid fermentation is included in the production process. Moreover, in this specification, the raw material of fermented food means the raw material provided to lactic acid fermentation for manufacture of lactic acid fermented food. That is, according to the method of the present invention, any fermented food product in which lactic acid bacteria are involved in the production process can be produced. Examples of such lactic acid fermented foods include processed meat products (ham, sausage, bacon, etc.), processed seafood products (boiled bonito, nare, etc.), dairy products (cheese, butter, yogurt, dairy drinks, etc.) , Processed vegetables and fruits (pickled products, liquor, etc.), processed cereal products (bread, liquor, etc.), processed bean products (miso, soy sauce, natto, okara processed products, etc.), processed seaweed (eg, kelp), etc. . Examples of pickles include a thousand pickles, shiba pickles, takuan, Chinese cabbage pickles, cucumber pickles, quick pickles, nuka miso pickles, sauerkraut and kimchi. Examples of the raw material for the fermented food include, in the case of vegetables, turnip, eggplant, cucumber, radish, Chinese cabbage, quick vegetable, cabbage, tomato and the like.

According to the method for producing a lactic acid fermented food of the present invention, the raw material of the fermented food is fermented in the presence of the L13 strain. This can be performed by adding the L13 strain as a starter to the raw material of the fermented food. That is, in one embodiment, the present invention provides the use of lactic acid bacteria as a starter in the production of lactic acid fermented foods. Fermentation is performed according to the conventional manufacturing method of each fermented food. The amount of lactic acid bacteria added, fermentation temperature, fermentation period, etc. will vary depending on the lactic acid fermented food to be produced, and those skilled in the art will appropriately set these conditions in consideration of the properties of the product (eg sensory evaluation, stability). Can be set. By increasing the addition amount of lactic acid bacteria and setting the fermentation temperature high, there is a possibility that the fermentation can be performed in a shorter period of time. However, it is preferable to ferment at a lower temperature in order to prevent contamination with bacteria. For example, a thousand pickles can be produced by inoculating under-soaked turnips with the L13 strain at a concentration of 1 × 10 ⁸ cells / ml at the stage of the main pickles and leaving them at 10 ° C. for 3 to 5 days.

  The lactic acid fermented food of the present invention contains GABA. Although it does not specifically limit, the content of GABA in the lactic acid fermented food is, for example, 0.05 to 1%, preferably 0.1 to 0.4%. In order to produce GABA, glutamic acid or a salt thereof (for example, sodium glutamate) may be added to the fermentation process, or added if the fermented food has a high glutamic acid or salt content (for example, eggplant, tomato, etc.). You don't have to.

  Lactic acid fermentation may be performed by a combination of a plurality of microorganisms. The lactic acid fermented food of the present invention may be carried out using the lactic acid bacteria L13 strain of the present invention alone or in combination with other microorganisms (for example, other lactic acid bacteria, yeast, filamentous fungi, etc.). .

  The present invention provides a method for producing GABA using a polypeptide having GAD activity, a nucleic acid encoding the polypeptide, and a cell containing the nucleic acid.

  For example, the polypeptide having GAD activity of the present invention consists of the amino acid sequence of SEQ ID NO: 7. Even a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 7 is included in the polypeptide of the present invention as long as it has GAD activity. The identity between the known amino acid sequence of GAD and the amino acid sequence of SEQ ID NO: 7 was 79.8% at the maximum. Therefore, a polypeptide having GAD activity consisting of an amino acid sequence of SEQ ID NO: 7 having an identity of, for example, 80% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95% or more, Included in the polypeptides of the present invention. Such polypeptides can be used for the conversion of glutamic acid to GABA.

  For example, a nucleic acid encoding a polypeptide having GAD activity of the present invention consists of the base sequence of SEQ ID NO: 6. Even a nucleic acid having a base sequence in which one or several bases are deleted, substituted or added in the base sequence of SEQ ID NO: 6 is included in the nucleic acid of the present invention as long as it encodes a polypeptide having GAD activity It is. Even a nucleic acid that hybridizes with a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 6 under stringent conditions is included in the nucleic acid of the present invention as long as it encodes a polypeptide having GAD activity. Examples of stringent hybridization conditions include 6 × SSC, 5 × Denhardt reagent, 0.5% (w / v) SDS, 1 μg / ml poly (A), 100 μg / ml salmon sperm DNA and labeled probe Incubation in solution at 68 ° C. is mentioned (J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 6.50-6.64 (2001) Cold Spring Harbor Laboratory Press).

  A polypeptide having GAD activity can be produced by a method comprising a step of culturing cells containing nucleic acids as described above and a step of collecting a polypeptide having GAD activity from the culture. In addition, GABA can be produced by a method including a step of culturing cells containing the nucleic acid as described above and a step of collecting GABA from the culture. A cell containing the nucleic acid of the present invention is obtained by introducing the nucleic acid of the present invention into a cell that can be used as a host together with a control sequence (promoter etc.) that naturally lies adjacent to an open reading frame encoding a polypeptide having GAD activity. be able to. Alternatively, the nucleic acid of the present invention may be operably linked to an appropriate control sequence (promoter) that functions in the host to be used, and then introduced into the host cell. As the host cell, any cell that can be used as a host for recombinant DNA technology is used. Examples of host cells include bacteria such as E. coli, Bacillus subtilis, and lactic acid bacteria. As a vector used for the introduction, any vector such as a plasmid vector or a bacteriophage vector can be used. Culturing is performed under conditions suitable for the host to be used. If expression is inducible, an appropriate inducer may be added during culture. In the case of producing GABA, GABA can be produced by culturing the above cells in the presence of glutamic acid or a salt thereof (for example, sodium glutamate), as in the case of L13 strain. The collection of GABA from the culture is performed in the same manner as in the case of the L13 strain.

  Whether the polypeptide encoded by the nucleic acid of the present invention has GAD activity depends on whether the nucleic acid is expressed in the host as described above and the resulting culture has an activity to convert glutamic acid to GABA. It can be confirmed by examining whether.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the scope of the present invention is not limited to these examples.

Separation of γ-aminobutyric acid-producing bacterium A γ-aminobutyric acid (hereinafter referred to as GABA) -producing bacterium was isolated from a pickled pickled solution produced by a traditional fermentation method and a ground product of turnip. Specifically, 5.0 kg of sodium chloride and 300 g of food decolorization enhancer Loving (Imai Kayei Shoten Co., Ltd.) are added to 70 kg of turnip slices and left to stand at 5 ° C. for 1 week for fermentation (underpickled). It was left overnight at 5 ° C. in a pickling solution consisting of seasoning liquid, sucrose, sodium glutamate, kelp, and vinegar (main pickling). GYP (1% glucose, 1% yeast extract, 0.5% polypeptone, 0.2% sodium acetate trihydrate containing 5% sodium glutamate using the resulting pickled liquor and turnip grind as samples. 20 ppm magnesium sulfate heptahydrate, 1 ppm manganese sulfate tetrahydrate, 1 ppm iron sulfate heptahydrate, 10 ppm sodium chloride, 50 ppm Tween 80, pH 6.8) applied to an agar medium at 30 ° C., 5 After a day of culture, a single colony was obtained. Next, each colony was inoculated into a GYP liquid medium and subjected to static culture at 30 ° C. for 2 days.

  The production of GABA was confirmed by thin layer chromatography (hereinafter, TLC) using the culture supernatant as a sample. As a thin layer, silica gel 60 F254 (manufactured by Merck) was used, and 1-butanol: acetic acid: water = 3: 2: 1 (v / v / v) was used for development. Further, ninhydrin was used for detection. An example of TLC results is shown in FIG. In the figure, lane 1 shows the results of TLC using GABA and a standard product of sodium glutamate (hereinafter, GluNa). In Lane 3, only GluNa spots are observed, whereas in Lane 2, GABA spots are observed. From this, it can be seen that the sample in Lane 2 contains GABA. Through the above operation, the L13 strain was obtained.

Examination of taxonomic position of isolate L13 First, the taxonomic position of L13 was examined. Analysis was requested from Techno Suruga. Table 1 shows the biochemical properties of the L13 strain.

  This strain was a Gram-positive bacilli, did not form spores, and did not show motility. Both the catalase reaction and the oxidase reaction were negative. These properties are consistent with the general properties of Lactobacillus bacteria, except that glucose is not oxidized or fermented.

  Next, it examined using the genetic classification method. Specifically, genomic DNA of the L13 strain was prepared, 16S rDNA was amplified by PCR using this as a template, and the DNA base sequence was determined. The sequences of primers 8-27 fwd and 1492 rev used in the PCR method are shown in SEQ ID NOs: 1 and 2, and the determined base sequence of 16S rDNA is shown in SEQ ID NO: 3.

  As a result of searching the DNA base sequence of 16S rDNA against DDBJ (DNA data bank of Japan) database, Lactobacillus hammesii (Valcheva, R., Korakli, M., Onno, B., Prevost, H., Ivanova, I. , Ehrmann, MA, Dousset, X., Ganzle, MG and Vogel, RF, Lactobacillus hammesii sp. Nov., Isolated from French sourdough Int. J. Syst. Evol. Microbiol. 55, 763-767 (2005)), Lactobacillus parabrevis (Vancanneyt, M., Naser, SM, Engelbeen, K., De Wachter, M., Van der Meulen, R., Cleenwerck, I., Hoste, B., De Vuyst, L. and Swings, J. Reclassification of Lactobacillus brevis strains LMG 11494 and LMG 11984 as Lactobacillus parabrevis sp. nov Int. J. Syst. Evol. Microbiol. 56, 1553-1557 (2006)). As a result of creating a molecular phylogenetic tree based on these sequence data, the L13 strain showed a different branch from the above Lactobacillus hammesii and Lactobacillus parabrevis. From these results, it was suggested that the L13 strain is a new species of bacteria belonging to the genus Lactobacillus.

  Based on the above biochemical and genetic properties, isolates were obtained from Lactobacillus sp. It was named L13. From August 2, 2006, this strain was registered with the patent microorganisms deposit center (NPMD), Kazusa Kamashizu 2-5-8, Kisarazu City, Chiba Prefecture 292-0818. Deposited under P-253.

GABA production of the L13 strain The GABA productivity of the L13 strain was compared with the Lactobacillus brevis IFO12005 strain. L. Brevis IFO12005 strain is known to produce GABA, and the present inventors use this lactic acid strain as a reference strain for GABA-producing lactic acid bacteria. Strain L13 or L. brevis IFO12005 strain was inoculated into GYP medium containing various concentrations of GluNa, cultured at 30 ° C., and GABA content was measured over time.

  Glutamic acid (Glu) and GABA were quantified as follows. Detection was performed by a post-column method using orthophthalaldehyde. The system used was high performance liquid chromatography (HPLC, Prominence, Shimadzu Corp.) using a strongly acidic ion exchange resin column Shim-pack Amino-Na. Detection was performed using a fluorescence detector (Ex 348 nm, Em 450 nm), and an amino acid standard solution (H type, Wako Pure Chemical Industries) was used as a standard solution.

  The results are shown in FIG. L. Brevis IFO12005 produces up to about 1% GABA regardless of the initial sodium glutamate concentration in the medium (FIG. 2A), whereas L13 produces GABA depending on the initial GluNa concentration in the medium, When the initial GluNa concentration was 15%, a maximum of 5.7% GABA could be produced.

  The GABA conversion rates of the two strains are summarized as shown in Table 2. L. Brevis IFO 12005 strain showed a significant decrease in GABA conversion rate when the initial GluNa concentration increased, but the GABA conversion rate of the L13 strain was not greatly affected by the initial GluNa concentration, and a conversion rate of about 85% was obtained. .

  Lactobacillus paracasei has been reported as a lactic acid bacterium with high GABA productivity (Noriko Komatsuzaki, Jun Shimaa, Shinichi Kawamoto, Hiroh Momose, Toshinori Kimura, Production of g-aminobutylic acid (GABA) by Lactobacillus paracasei isolated from traditional fermented foods, Food Microbiology 22, 497504 (2005)). This bacterium has been reported to have a GABA producing ability of up to 3.1% (initial glutamic acid concentration: 7.4%, conversion rate: 60.4%). Comparing the GABA productivity of this strain with that of the L13 strain, It was revealed that about twice as much GABA as paracasei can be produced. As described above, the L13 strain exhibits a very high GABA productivity as compared with known GABA high-producing lactic acid bacteria.

  One of the physiological significance of microorganisms producing GABA is thought to be protection against medium acidification. Focusing on this point, the effect on maintaining GABA productivity by the L13 strain was investigated by maintaining the culture medium pH during culture to be acidic (pH 5). The result is shown in FIG. In FIG. 3, A is GABA when the L13 strain was cultured without pH adjustment in the same manner as described above except that the initial glutamic acid concentration was 12% (the actually used sodium glutamate monohydrate concentration was converted to the glutamic acid concentration). B shows the results of measuring the content of GluNa over time, and B shows the results of culturing while maintaining the medium pH at 5. As shown in FIG. 3, it succeeded in producing about 6.7% GABA from glutamic acid having an initial concentration of 12%. The conversion rate was about 85%.

  At this time, GluNa in the medium completely disappeared after 5 days of culture. Moreover, it can be said that it is one of the merit on GABA production that it was not necessary to add pyridoxal phosphate (PLP), which is a coenzyme of glutamate decarboxylase (GAD), to the medium during GABA production.

Manufacture of thousand pieces by fermentation using L13 strain Using the L13 strain as a starter, we made a thousand pieces of pickle by fermentation and tried to produce GABA in the product at the same time. The examination was carried out on a small scale of 6L. That is, after immersing the turnip in a 0.2% acetic acid solution and washing the same, sodium chloride and a food decolorization enhancing agent Loving were added in the same manner as in Example 1 and submerged at 4 ° C. overnight. This pickle was prepared so that the initial bacterial cell concentration (pickling solution) was 1 × 10 ⁸ cells / ml, and was allowed to stand at 10 ° C. for 6 days (performed in duplicate). As a control, a series not inoculated with L13 was also prepared. After the start of this pickling, sampling was conducted every day until 6 days, and glutamic acid and GABA were quantified in addition to the sensory test.

  Samples were prepared as follows. 40 ml of purified water is added to 10 g of 1000 pieces, and homogenized (ACE homogenizer (Nippon Seiki), 10,000 rpm, 5 minutes), and the centrifuged supernatant after boiling for 5 minutes is diluted with buffer (trisodium citrate buffer, pH 2.2). ). GABA was quantified by the method described in Example 3 using the prepared sample.

  The results are shown in FIG. As shown in FIG. 4, about 0.1% of GABA was produced after 3 days. Furthermore, after about 5 to 6 days, a maximum of about 0.4% GABA was produced. After 4 days, growth of various bacteria was observed.

  The sensory test was conducted by four panelists, and three levels of evaluation (◯, Δ, ×) and comments were obtained regarding odor and taste. The results are shown in Table 3.

  As shown in Table 3, in the sensory test, there was a fresh scent like fruit until the product after 3 days, and the taste was also a sweet taste but a refreshing taste with no aftertaste. It was found to have properties.

  In addition, GABA is said to be able to enjoy its effect when ingested 50 mg per day for an adult. One thousand pieces soaked is about 30 g. For example, if it is a thousand pieces soaked after 3 days under the above conditions, it contains about 0.1% GABA.

5-1. Cloning of Glutamate Decarboxylase (GAD) Gene A primer for PCR was prepared with reference to a previously reported GAD gene, and a GAD gene (gadB gene) was obtained by PCR according to a conventional method. The sequences of primers L13 GAD fwd and L13 GAD rev used in the PCR method are shown in SEQ ID NOs: 4 and 5, and the determined base sequence of the gadB gene is shown in SEQ ID NO: 6. Lactobacillus sp. The base sequence of the gadB gene of the L13 strain had an open reading frame of 1410 bp. The amino acid sequence deduced from this open reading frame is shown in SEQ ID NO: 7.

5-2. Comparison of L13 strain-derived GAD and previously reported GAD Using the amino acid sequence (L13gadb) inferred from the gadB gene derived from the L13 strain, a homology search was performed on the DDBJ database using the FASTA program or the like. The results are shown in Table 4.

References etc.
* Unpublished data (our data); accession number: AB258458
1) Nomura M., Nakajima I., Fujita Y., Kobayashi M., Kimoto H., Suzuki I., Aso H .; Lactococcus lactis contains only one glutamate decarboxylase gene. Microbiology, 145: 1375-1380 (1999).
2) Bolotin A., Wincker P., Mauger S., Jaillon O., Malarme K., Weissenbach J., Ehrlich SD, Sorokin A., The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. Lactis IL1403, Genome Res. 11: 731-753 (2001).
3) Sanders JW, Leenhouts K., Burghoorn J., Brands JR, Venema G., Kok J .; A chloride-inducible acid resistance mechanism in Lactococcus lactis and its regulation. Mol. Microbiol. 27: 299-310 (1998) .
4) Nomura, M., Kobayashi, M., Ohmomo, S., and Okamoto, T., Inactivation of the Glutamate Decarboxylase Gene in Lactococcus lactis subsp. Cremoris, Appl. Envir. Microbiol. 66: 2235-2237 (2000) .
5) Park KB, Oh SH., Cloning, sequencing and expression of a novel glutamate decarboxylase gene from a newly isolated lactic acid bacterium, Lactobacillus brevis OPK-3. Bioresour Technol. Feb 22, (2006) [Epub ahead of print] 6 ) Kleerebezem M, Boekhorst J, van Kranenburg R, Molenaar D, Kuipers OP, Leer R, Tarchini R, Peters SA, Sandbrink HM, Fiers MW, Stiekema W, Lankhorst RM, Bron PA, Hoffer SM, Groot MN, Kerkhoven R, de Vries M, Ursing B, de Vos WM, Siezen RJ., Complete genome sequence of Lactobacillus plantarum WCFS1., Proc. Natl. Acad. Sci. US A. 100 (4): 1990-1995 (2003).
7) Blattner FR, Plunkett G 3rd, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, Gregor J, Davis NW, Kirkpatrick HA, Goeden MA, Rose DJ, Mau B, Shao Y., The complete genome sequence of Escherichia coli K-12, Science, 277 (5331): 1453-1474 (1997).

  As shown in Table 4, the deduced amino acid sequence of L13gadB was particularly highly homologous to that of UenogadB (Lactobacillus brevis IFO12005) and showed 79.8% identity. As described above, the present inventors use this lactic acid strain as a reference strain for GABA-producing lactic acid bacteria. Among GADs derived from other microorganisms, Lc. lactis lac (Lactococcus lactis ssp. lactis IL1403), Lc. It showed 52.7% and 52.2% identity with the amino acid sequence of lactis cre (Lactococcus lactis subsp. cremoris), respectively.

  Furthermore, for these sequences, multiple alignment was created using analysis software called BioEdit 7.0.1 and Clustal X 1.83. The results are shown in FIGS. Even in this case, L13gadB and UenogadB formed a group distinctly different from other GAD proteins.

The differences between these GADs and the amino acid sequences of other GADs are as follows:
(1) The portion corresponding to residues 19-21 (-EKT-), 120-124 residues (-KFGD-), 161-163 residues (-DLH-) of L13gadB is deleted except for the above two (FIGS. 5-1, 5-2);
(2) The valine 178 residue of L13gadB is cysteine except for the above two (FIG. 5-2);
(3) The above two cysteine residues are 5 while the others are 6 (except that E. coli GAD (Ecoli gadB, Ecoli gadA) has 10 cysteines)) FIGS. 5-1 to 5-4). A disulfide bond is formed between the two cysteines and plays an important role in the formation of the protein conformation. The difference in the number of cysteines suggests the possibility of different conformations between these groups.

  On the other hand, the common feature of alignment is aspartic acid residue (D289, FIG. 5-1) considered essential for activity and important for substrate binding, and lysine residue (K289) to which pyridoxal phosphate, a coenzyme, binds. FIG. 5-3) is highly preserved.

  From the above results, Lactobacillus sp. It was suggested that the gadB gene of the L13 strain encodes active glutamate decarboxylase (GAD) in the same manner as known GAD. It was also confirmed by the multiple alignment analysis of L13 GAD that there were many differences from the previously reported GAD.

  According to the present invention, there are provided a novel lactic acid bacterium having high GABA production ability, a method for producing GABA using the lactic acid bacterium, a method for producing a GABA-containing lactic acid fermented food, GAD obtained from the lactic acid bacterium, and a nucleic acid encoding the same.

It is a figure which shows an example of the result of thin layer chromatography. Lactobacillus sp. It is a figure which shows the GABA production amount of L13 strain | stump | stock and Lactobacillus brevis IFO12005 strain | stump | stock. It is a figure which shows the effect with respect to GABA productivity of maintaining culture medium pH acidic during culture | cultivation. It is a figure which shows the GABA production amount during a 1000-sheet pickling fermentation. It is a figure which shows the multiple alignment of various GAD amino acid sequences. It is a figure which shows the multiple alignment of various GAD amino acid sequences. It is a figure which shows the multiple alignment of various GAD amino acid sequences. It is a figure which shows the multiple alignment of various GAD amino acid sequences.

SEQ ID NO: 1: A primer 8-27 fwd for amplification of 16S rDNA
SEQ ID NO: 2: A primer 1492 rev for amplification of 16S rDNA
SEQ ID NO: 4: A primer L13 GAD fwd for amplification of L13 gadB gene
SEQ ID NO: 5: A primer L13 GAD rev for amplification of L13 gadB gene

Claims (10)

  Lactobacillus Lactobacillus sp. having the ability to produce γ-aminobutyric acid L13 (NITE P-253).   Lactic acid bacteria Lactobacillus sp. A method for producing γ-aminobutyric acid, comprising a step of culturing L13 (NITE P-253) and a step of collecting γ-aminobutyric acid from the culture.   The raw material of the fermented food is lactic acid bacteria Lactobacillus sp. A method for producing a lactic acid fermented food containing γ-aminobutyric acid, comprising a step of fermenting in the presence of L13 (NITE P-253).   The production method according to claim 3, wherein the lactic acid fermented food is pickled in a thousand pieces, and the raw material of the fermented food is fog.   The raw material of the fermented food is lactic acid bacteria Lactobacillus sp. A γ-aminobutyric acid-containing lactic acid fermented food produced by a method comprising a step of fermenting in the presence of L13 (NITE P-253).   The lactic acid fermented food according to claim 5, wherein the lactic acid fermented food is pickled in a thousand pieces, and the raw material of the fermented food is fog.   A polypeptide comprising the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 7, and having glutamate decarboxylase activity. A nucleic acid encoding a polypeptide selected from the group consisting of and having glutamate decarboxylase activity:
(1) a nucleic acid comprising the base sequence of SEQ ID NO: 6;
(2) a nucleic acid comprising a base sequence in which one or several bases have been deleted, substituted or added in the base sequence of SEQ ID NO: 6;
(3) A nucleic acid that hybridizes with a nucleic acid comprising the base sequence of SEQ ID NO: 6 under stringent conditions.   A method for producing a polypeptide having glutamate decarboxylase activity, comprising culturing cells containing the nucleic acid according to claim 8 and collecting a polypeptide having glutamate decarboxylase activity from the culture. A method for producing γ-aminobutyric acid, comprising a step of culturing cells containing the nucleic acid according to claim 8 and a step of collecting γ-aminobutyric acid from the culture.

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