WO2006064791A1

WO2006064791A1 - Methods of producing ϝ-aminobutyric acid and extract containing ϝ-aminobutyric acid

Info

Publication number: WO2006064791A1
Application number: PCT/JP2005/022851
Authority: WO
Inventors: Masato Kawaguchi; Koujun Sato; Sachiko Tsuchiya; Hisae Matsunami; Rieko Hukayama; Isao Horiuchi
Original assignee: Japan Applied Microbiology Research Institute Ltd.
Priority date: 2004-12-14
Filing date: 2005-12-13
Publication date: 2006-06-22
Also published as: JPWO2006064791A1

Abstract

[PROBLEMS] To provide novel methods of producing Ϝ-aminobutyric acid and an extract containing Ϝ-aminobutyric acid by using a lactic acid bacterium. [MEANS FOR SOLVING PROBLEMS] A method of producing Ϝ-aminobutyric acid which comprises using Lactobacillus reuteri and producing Ϝ-aminobutyric acid from glutamic acid or its salt. A method of producing an extract containing Ϝ-aminobutyric acid which comprises co-culturing lactic acid bacteria at least containing Lactobacillus reuteri together with a yeast in a medium containing glutamic acid or its salt and then obtaining an extract containing Ϝ-aminobutyric acid by subjecting the co-culture medium to solid-liquid separation.

Description

Specification

Method for producing γ-aminobutyric acid and γ-aminobutyric acid-containing extract

Technical field

The present invention relates to a method for producing a novel γ-aminobutyric acid and a γ-aminobutyric acid-containing extract using lactic acid bacteria.

Background art

[0002] It is well known that there are lactic acid bacteria having the ability to produce γ-aminobutyric acid (GABA) having various pharmacological actions including antihypertensive effect from gnoretamic acid or a salt thereof. Patent Document 1 discloses such lactic acid bacteria as Lactobacillus casei (Lactobacillus casei), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Lactobacillus helveticus, Lactobacillus helveticus (Lactobacillus helveticus), obacillus brevis) and the like.

[0003] However, not all lactic acid bacteria having the ability to produce γ_aminobutyric acid are known, but it is significant to search for a lactic acid bacterium having a better ability to produce γ_aminobutyric acid. It is.

[0004] Also, in recent years, extracts obtained by solid-liquid separation from a cultivated lactic acid bacterium and yeast strain in a medium made from soybeans have attracted attention as health foods that have the effect of regulating the intestinal environment. (For example, see Patent Document 2).

[0005] However, there is a demand for an extract having a much better effect than the previously known culturing extract of lactic acid bacteria and yeast.

Patent Document 1: JP 2001-120179 A

Patent Document 2: Japanese Patent Application Laid-Open No. 11-106276

Disclosure of the invention

Problems to be solved by the invention

[0006] Accordingly, an _{object of the} present invention is to _provide a novel method for producing a _bis -aminobutyric acid and γ-aminobutyric acid-containing _extrude using lactic acid bacteria.

Means for solving the problem [0007] As a result of intensive studies in view of the above points, the present inventors have found that Lactobacillus reuteri (Lact obacillus reuteri), which is known as a lactic acid bacterium that produces Reuterin, which is a kind of antibacterial protein. It has been found that it has a much better ability to produce γ-aminobutyric acid than lactic acid bacteria having the ability to produce γ-aminobutyric acid.

[0008] _Ί of the present invention was made based on the above findings - manufacturing method of Amino butyric acid as claimed in claim 1 Symbol placement, using Lactobacillus reuteri (Lactobacillus reuteri), glutamate or _I from a salt thereof -A method for producing aminobutyric acid.

In addition, the method for producing a _Ί -aminobutyric acid-containing extract of the present invention comprises a lactic acid bacterium containing at least Lactobacillus reuteri (Lactobacillus reuteri) in a medium containing gnoretamic acid or a salt thereof, as defined in claim 2. This is a method for producing a γ-aminobutyric acid-containing extract obtained by solid-liquid separation from a yeast coculture.

The method according to claim 3 is the method according to claim 2, wherein a medium containing soybean as a raw material is used as the medium containing glutamic acid or a salt thereof.

In addition, the γ-aminobutyric acid-containing extract of the present invention comprises, as described in claim 4, lactic acid bacteria and yeast strains containing at least Lactobacillus reuteri in a medium containing glutamic acid or a salt thereof. It is obtained by solid-liquid separation from the cultivated culture.

The health food of the present invention comprises, as described in claim 5, the γ-aminobutyric acid-containing extract according to claim 4 as an active ingredient.

The γ-aminobutyric acid-containing composition of the present invention, as described in claim 6, coexists with lactic acid bacteria and yeasts containing at least Lactobacillus reuteri in a medium containing glutamic acid or a salt thereof. It is obtained by culturing.

The invention's effect

[0009] According to the present invention, novel _it using a lactic acid bacterium - may provide Amino acid-containing E manufacturing method kiss - Amino acid and _I.

Brief Description of Drawings

FIG. 1 is a graph showing the amount of γ-aminobutyric acid in 100 ml of the culture supernatant of each of the 13 lactic acid bacteria strains in Example 1.

FIG. 2 shows the effect of adjusting the intestinal environment of the extract containing γ-aminobutyric acid in Example 2 It is fu.

BEST MODE FOR CARRYING OUT THE INVENTION

[0011] (1) Method for producing γ_aminobutyric acid

Specific strains of Lactobacillus reuteri that can be used in the present invention include, but are not limited to, a virulent strain including, for example, the strain of RIKEN Deposit Number JCM2762. .

[0012] In the present invention, y-aminobutyric acid is used to cultivate Lactobacillus reuteri (Lactobacillus reuteri) using a liquid medium for lactic acid bacteria to which, for example, gnoretamic acid or a salt thereof (sodium salt or the like) as a substrate is added. By doing so, it can be obtained from the culture solution. The amount of glutamic acid or its salt added to the liquid medium for lactic acid bacteria is, for example, 0.2% (w / v) to 6.0% (w / v), preferably 0.5% (w / v) to 5.0% (w / v). v). The liquid medium for lactic acid bacteria is a general medium used for liquid culture of lactic acid bacteria such as GYP medium.For example, sugars such as glucose, fructose, and maltose, yeast extract, polypeptone, sodium acetate, and various minerals can be used. Including. The culture conditions are, for example, a temperature of 25 ° C to 38 ° C, a culture time of 24 hours to 96 hours, and in particular, an optimum temperature of 37 ° C to 38 ° C and a stationary phase arrival time of 48 hours are desirable. . Acquisition of γ-aminobutyric acid from the culture solution can be performed by a purification means such as liquid chromatography, membrane filtration, ultrafiltration, or nanofiltration.

In the present invention, γ-aminobutyric acid is a lactobacillus reuteri (Lactobacillus reuteri) in various food materials (for example, soybeans described later) containing glutamic acid as a substrate or a salt thereof as a constituent component. It may be produced by inoculating and culturing.

[0014] The γ_aminobutyric acid produced by the present invention can be used as a raw material for pharmaceuticals and health foods by a conventional method.

[0015] (2) Method for producing γ-aminobutyric acid-containing extract

As a specific strain of Lactobacillus reuteri contained in at least the lactic acid bacterium used in the present invention, the above-mentioned strain of RIKEN Deposit No. JCM2762 can be cited. It is not done. Lactic acid bacteria In addition to Lactobacillus reuteri, for example, Ratatococcus' La

, Lactobacillus acidophilus, Enterococcus faecalis, etc. Examples of yeasts that can be used in the present invention include Saccharomyces cerevisiae (Saccharomyces cerevisiae). , Ir Carroses' Unis Hoffs (Saccharomyces unisporus), Saccharomyces' Ma ¹ ~ Ayue (Saccharomvces martiniae), Tonorefus, Hu '

These may be used alone or in combination.

[0017] The medium containing gnoretamic acid or a salt thereof (such as sodium salt) may be any medium as long as it can cultivate both lactic acid bacteria and yeast. Examples thereof include a culture medium containing soybean containing glutamic acid as a raw material. Specific examples of the medium containing soybean as a raw material include those prepared by adding glucose or yeast extract to a powder obtained by crushing soybean.

[0018] Co-cultivation of lactic acid bacteria and yeast at least containing Lactobacillus reuteri in a medium containing glutamic acid or a salt thereof can be performed, for example, as follows. First, a lactic acid bacterium containing at least Lactobacillus reuteri is inoculated into a medium containing glutamic acid or a salt thereof and cultured. For example, inoculation may be performed at 0.5% to 2% (v / v) per bacterial species, for example, at 25 ° C to 45 ° C for 12 hours to 72 hours. As a result, fermentation of the medium components by lactic acid bacteria proceeds, and γ-aminobutyric acid is produced from glutamic acid by the action of Lactobacillus reuteri. In addition, the production of organic acids such as lactic acid and acetic acid lowers the ρΗ of the medium, making it suitable for culturing yeast. Therefore, the yeast is then inoculated into the medium (inoculation amount is 0.5% to 2% for example), and further cultured at 25 ° C to 45 ° C for 12 hours to 72 hours, for example. As a result, γ-aminobutyric acid is further produced from the glutamic acid produced by fermentation with yeast by the action of Lactobacillus reuteri. It should be noted that lactic acid bacteria and yeasts containing at least Lactobacillus reuteri are pre-cultured using, for example, GYP medium and YM medium (for example, 10 ⁶ (ΛαΑη1˜ 1θ ίΙιΑη1)) and co-culture In order to make the treatment more effective, multiple sets of lactic acid bacteria and yeast co-cultivation cultures (at least one co-cultivation culture is a lactic acid bacteria and yeast co-culture of at least Lactobacillus reuteri) May be mixed and further cultured in co-cultivation (mixing ratio is ex: '1: 0.5-1.5).

[0019] The _Ί -aminobutyric acid-containing extract of the present invention can be _obtained by, for example, solid-liquid separating a co-culture of lactic acid bacteria and yeast containing at least Lactobacill us reuteri by centrifugation or filtration. It can be obtained as the supernatant. The _ァ -aminobutyric acid-containing extract of the present invention can be used as a drink by adjusting the taste by adding it as it is or diluted with water, and adding acetic acid or citrate as necessary. In addition to the effect of preparing the intestinal environment obtained by co-culturing lactic acid bacteria and yeast by formulating them into tablets or granules, or by kneading the powdered product into bread or the like. It is possible to eat and drink as a health food having the effect of improving hypertension due to the action of y-aminobutyric acid. In addition, the co-culture of lactic acid bacteria and yeast containing at least Lactobacillus reuteri, which is positioned as a raw material for producing the γ-aminobutyric acid-containing extract of the present invention, itself contains γ_aminobutyric acid. Eating as a composition

Example

[0020] Hereinafter, the method for producing γ-aminobutyric acid and γ-aminobutyric acid-containing extract of the present invention will be described in more detail with reference to Examples, but the present invention should not be construed as being limited to the following description.

Example 1: Method for producing y-aminobutyric acid

(A) Culture of lactic acid bacteria

1. Test strain

13 strains of lactic acid bacteria listed in Table 1 owned by Okenken Co., Ltd. were used.

[0022] [Table 1] Lb.brevis (RIFY5111T)

Lb.casei (IF015883)

Lb. fermentum (RIFY5127)

Lb.helveticus W:

Lb.plantarum (RIFY5 HOT: Type Strain)

Lb.reuteri (JCM2762)

Lc. Lactis (NBRC12007)

Stc. Lactis (unknown)

Stc. Thermophilus (NBRC13957)

Leuc.mesenteroides (RIFY5030T)

Ped.pentosaceus (JCM5890)

Ec.faecal is (IAM10065)

Sib. Inulinus (unknown)

*) In Katsuko, it means the storage organization control number from which the strain originates.

[0023] 2. Liquid medium for lactic acid bacteria for producing γ-aminobutyric acid

Each component was mixed and dissolved in deionized water, and autoclaved at 121 ° C for 15 minutes, and a medium with sodium glutamate added to the GYP medium with the composition shown in Table 2 and Table 3 was used.

[0024] [Table 2]

[0025]

[0026] 3. Preparation of preculture

Collect 1 platinum loop colony from a lactic acid bacteria plate that has been streaked in advance, and aseptically inoculate 30 ml to 100 ml of the above-mentioned liquid medium, and then leave it for 1 to 2 days in a 37 ° C incubator. did.

[0027] 4. Preparation of the main culture

Inoculate 1% (ν / ν) to 3% (ν / ν) of the pre-cultured liquid medium into the liquid medium in which the main culture is performed. Absorbance OD was 0.01 to 0.02), and after inoculation, the cells were statically cultured in a 37 ° C incubator for 4 days. [0028] 5. Sorting of culture supernatant

The main culture medium was centrifuged at 6000 rpm for 5 minutes, and separated into a centrifugal supernatant and bacterial cell precipitate.

[0029] (B) Amino acid analysis of culture supernatant

Free amino acid analysis in the culture supernatant obtained as described above was performed according to a post-column system using high performance liquid chromatography (HPLC). The analysis specimen used was a culture supernatant diluted 50-fold with 0.2 M citrate buffer (pH 2.2). If the culture supernatant is rich in protein, add 80% (v / v) ethanol and gently agitate to denature and aggregate the proteins, and then centrifuge them to remove them as precipitates. The centrifuged supernatant was diluted 50-fold with 0.2M citrate buffer (pH 2.2) to prepare an analytical sample. Analytes were filtered through a 0.2 zm filter and 20 μl was injected into the HPLC. The analysis conditions were as shown in Table 4.

[0030] [Table 4]

[0031] (C) Results

The amount of γ-aminobutyric acid in 100 ml of the culture supernatant of each of the 13 lactic acid bacteria is shown in Fig. 1 (blank in the figure means the amount of γ-aminobutyric acid in 100 ml of liquid medium not inoculated with lactic acid bacteria). As can be seen in Fig. 1, the ability of Lactobacillus reuteri to produce γ-aminobutyric acid is markedly superior to that of other lactic acid bacteria. It was.

[0032] Example 2: Method for producing γ-aminobutyric acid-containing extract

(Ii) Co-culture of lactic acid bacteria and yeast

1. Test strain Two combinations of the following two types of lactic acid bacteria and one type of yeast were used.

(Combination 1)

• Lactic acid bacteria: Lactobacillus reuteri (JCM2762)

• Lactic acid bacteria: Lattococus lactis (NBRC12007)

• Yeast: Saccharomyces martiniae (IFO0752)

(Combination 2)

'Lactobacillus acidophilus (JCM1132) • Lactic acid bacteria: Enterococcus faecalis (NBRC14714)

• Yeast: Saccharomyces cerevisiae (IFO0725)

(¾ The number at the end in Katsuko means the preservation agency control number from which the strain originates.

[0033] 2. Medium used

For pre-culture of lactic acid bacteria, GYP medium prepared by mixing and dissolving the components shown in Table 5 in deionized water was used. YM medium prepared by mixing and dissolving the components shown in Table 6 in deionized water was used for preculture of yeast. For co-cultivation of lactic acid bacteria and yeast, a soybean medium prepared by mixing and dissolving the components shown in Table 7 in deionized water was used. All media were autoclaved at 121 ° C for 20 minutes before use.

[0034] [Table 5] Glucose! 1.0¾ (w / v)

Yeast extract i 0.5¾ (w / v)

Polypepton! 0.5¾ (w / v)

Anhydrous sodium acetate • 0.5¾ (/ v)

Inorganic salt solution 0.25¾ (w / v)

*) See Table 3

[0035]: Table 6]

Glucose i 1.0¾ (v / v)

Yeast extract 0.3¾ (v / v)

Malt extract 0.3% (w / v)

Polybepenne i 0.5¾ (w / v)

[0036] ： Translation

Soybean powder: 10.0¾ (w / v)

Glucose 1.0% (w / v)

Yeast extract 1.0 (w / v)

[0037] 3. Preparation of preculture After cryopreserving and inoculating the GYP medium with lees and lactic acid bacteria, they were statically cultured in a 30 ° C incubator for 24 hours to prepare a preculture solution of about 10 ⁸ cfli / ml. Similarly, each of the yeasts that had been stored frozen was inoculated into YM medium, and left to stand for 24 hours in an incubator at 30 ° C. to prepare a preculture solution of about 10 ⁸ cfu / ml.

[0038] 4. Production of symbiotic culture

First, about 1% (ν / ν) of each lactic acid bacteria pre-culture solution for the combination 1 lactic acid bacteria and yeast, inoculated in a 30 ° C incubator for 24 hours, The yeast preculture was inoculated with about 1% (ν / ν) of soybean medium, and further cultured for 48 hours in a 30 ° C incubator for co-cultivation. Combination 2 lactic acid bacteria and yeast were cocultured in the same manner. Next, combine the lactic acid bacteria and yeast co-culture of combination 1 with the lactic acid bacteria and yeast co-culture of combination 2 at a ratio of 1: 1, and let stand in an incubator at 30 ° C for 24 hours. By culturing, the desired co-cultivation culture was obtained by co-culturing

[0039] 5. Preparation of γ-aminobutyric acid-containing extract

The co-cultured culture obtained as described above was autoclaved at 121 ° C for 20 minutes, and then centrifuged at 500 ° C. for 10 minutes to obtain a γ-aminobutyric acid-containing extract as a centrifugal supernatant. .

[0040] (Β) Amino acid analysis of γ-aminobutyric acid-containing extract

Analysis of free amino acids in the γ-aminobutyric acid-containing extract obtained as described above was carried out in the same manner as described in Example 1.

[0041] The amount of amino acids contained in the γ_aminobutyric acid-containing extract is shown in Table 8 together with the amount of amino acids contained in the soybean medium and the amount of amino acids contained in the supernatant of the combined lactic acid bacteria and yeast cultivated yeast. As is apparent from Table 8, it was found that the extract obtained by coculturing lactic acid bacteria and yeast containing Lactobacillus reuteri contained a large amount of γ-aminobutyric acid.

[0042] [Table 8] Soybean medium Aminobutyric acid-containing extract Combination 2 supernatant Aspartic acid 38.7 19.8 4.2 Threonine 24.8 11.9 0.4 Serine 29.9 10.3 0.2

Glutamic acid 71.3 11.0 5.5 Glycine 20.2 10.7 1.3 Alanine 50.7 23.9 3.5 Parin 39.6 18.7 0.5

Cystine 1.6 0.3 Not detected Methionine 10.0 3.9 0.2 Isoleucine 29.7 13.7 0.1 Leucine 46.4 20.8 0.3 Tyrosine 10.8 5.0 0.4 Phenylalanine 28.1 13.0 0.5 Histidine 8.9 5.5 0.8 Lysine 27.6 16.0 1.5

Tribute fan 8.5 4.0 Zhu out Arginine 49.8 0.9 0.9 Proline 16.5 8.6 1.3

7 "-aminobutyric acid 12.4 36.0 2.0

Unit: mg / 100ml

[0043] (C) Effect of y-aminobutyric acid-containing extract to improve intestinal environment

The y-aminobutyric acid-containing extract was forcibly orally administered to mice for 14 days with a gastric sonde, and the aerobic intestinal bacteria in the stool were analyzed. Group and enterococci group) were compared. The experimental method is as follows. Before the administration and on the 14th day after administration, fresh stool of mice was collected and weighed. After appropriately diluting the fecal sample with a diluent, each of the samples was added to the McConkey agar medium, which is a selective differentiation medium for coliforms, the LBS agar medium, which is a selective medium for lactic acid bacteria, and the EF agar medium, which is a selective medium for enterococci. / i Painted one by one. Colonies were counted after culturing at 37 ° C for 24-48 hours, and the number of bacteria per stool weight was calculated. As a result, there was no significant change in the lactic acid bacteria group that was an indicator of good bacteria, but there was an increasing tendency for the enterococci group and a decreasing tendency for the coliform group that was an indicator of bad bacteria. It was confirmed that the aminoaminobutyric acid-containing extract has an effect of adjusting the intestinal environment (see Fig. 2).

[0044] (D) Processing example of y-aminobutyric acid-containing extract (granule)

16 g of lyophilized powder of ァ -aminobutyric acid-containing extract, 29 g of starch, 55 g of lactose and 100 g in total were mixed uniformly, and granulated according to a conventional method.

Industrial applicability

[0045] The present invention relates to novel γ-aminobutyric acid and γ-aminobutyric acid-containing extract using lactic acid bacteria. The present invention has industrial applicability in that a manufacturing method can be provided.

Claims

The scope of the claims

[1] A method for producing γ-aminobutyric acid from glutamic acid or a salt thereof using Lactobacillus reuteri.

[2] _Koji obtained by solid-liquid separation from a co-culture of lactic acid bacteria and yeast containing at least Lactobacillus reuteri in a medium containing glutamic acid or a salt thereof

-Method for producing an aminoaminobutyric acid-containing extract.

[3] The method according to claim 2, wherein a medium containing soybean as a raw material is used as the medium containing gnoretamic acid or a salt thereof.

[4] γ-Aminobutyric acid-containing extract obtained by solid-liquid separation from a co-culture of lactic acid bacteria and yeast containing at least Lactobacillus reuteri (Lactobacillus reuteri) in a medium containing glutamic acid or a salt thereof .

[5] A health food comprising the γ-aminobutyric acid-containing extract according to claim 4 as an active ingredient.

[6] A γ-aminobutyric acid-containing composition obtained by co-culturing a lactic acid bacterium and yeast containing at least Lactobacilli reuteri (Lactobacilli! S reuteri) in a medium containing glutamic acid or a salt thereof.