JP4332247B2 - A lactic acid bacterium having high ability to produce γ-aminobutyric acid, a fermented food containing a high amount of γ-aminobutyric acid using the lactic acid bacterium, and a method for producing the same. - Google Patents

Description

【００２２】
その結果、２種類の混合培養においても１００ｍｇ／１００ｇ以上のＧＡＢＡ含有発酵乳を得た（図２）。また、この結果より、組み合わせる菌種が同じブルガリカス種であっても、菌株によりＧＡＢＡ生産能に関する相性に差があることが分かった。
【００２３】
この時、混合培養に用いたブルガリカス種各菌株単菌によるＧＹＰ培地（１％ＭＳＧ含有）におけるＧＡＢＡ生産能は図３に示すようにかなり低いものであった。
【００２４】
しかし、図２の結果程度のＧＡＢＡへの変換率では、発酵液中に高い濃度のＬ−グルタミン酸が残存し、“うま味”の強い発酵液となり、乳酸菌発酵特有のサワー感に欠ける食品となる。
【００２５】
そこで、発酵促進のために脱脂粉乳培地に植物由来成分（例えば、豆、人参、ピーマン、カボチャ、セロリ、ほうれん草、キャベツ、トマトなどを原料とする食品）を添加し、ＧＡＢＡ生産性の向上を図った。この時の植物由来成分の例として豆乳を使用したときの処方を表２に示す。なお、スターターはブレビスＴＹ４１４とブルガリカスタイプストレインの混合培養系を例とする。
【００２６】
【表２】

【００２７】
なお、表２において、「ブレビスＴＹ４１４スターター」とは、２．０％ブドウ糖、１．０％豆由来ペプチド、０．５％酵母エキスの配合の水溶液を１２１℃、１５分にて滅菌したものにブレビスＴＹ４１４を添加し、３０℃、２４時間培養したものをいう。
また、表２において、「ブルガリカスタイプストレインスターター」とは、８．３％脱脂粉乳溶液（１２１℃、１５分滅菌）にブルガリカスタイプストレインを添加し、３７℃、２４時間培養したものをいう。
【００２８】
表２を処方例とし、各混合培養におけるＧＡＢＡ生産量を図４に示す。その結果、新たにブレビスＴＹ４１４とブルガリカスＴＹ３９３（サンプルＡ３ｂ）、ブレビスＴＹ４１４とブルガリカスタイプストレイン（サンプルＡ４ｂ）、ブレビスＩＦＯ１２００５とブルガリカスＴＹ３９３（サンプルＣ３ｂ）の混合培養において、高濃度のＧＡＢＡが得られた。
更に、ＧＡＢＡ生産性をＧＹＰ培地（１％ＭＳＧ含有）発酵時レベルに上げるために、脱脂粉乳の代わりにホエーパウダーを使用することによりＧＡＢＡの生産能が上がった。また、ホエーパウダーの他に濃縮ホエーについても同様の効果が得られる。なお、その他に乳製品として、クリームパウダー、バターミルパウダー、濃縮乳、脱脂濃縮乳、無糖練乳、無糖脱脂練乳、加糖練乳、加糖脱脂練乳、全粉乳加糖粉乳、調整粉乳についての使用も考えられる。
【００２９】
上記で検討した発酵物のなかでもブレビスＴＹ４１４とブルガリカスタイプストレインの混合培養（サンプルＡ４ｃ）にて得られたＧＡＢＡ含有発酵物については、ＭＳＧ若しくはＬ−グルタミン酸をほぼ１００％の効率でＧＡＢＡに変換していた。また、ブレビスＴＹ４１４とブルガリカスＴＹ３９３の混合培養（サンプルＡ３ｃ）、ブレビスＩＦＯ１２００５とブルガリカスＴＹ３９３の混合培養（サンプルＣ３ｃ）においてもＧＡＢＡ生産量が増し、ＧＡＢＡ含有量もかなり高いものであった（図５）。
さらに、サンプルＡ４ｃにおける経時のＧＡＢＡ生産量の変化をみたところ、５０時間過ぎたぐらいからＧＡＢＡの生産量が急激に上がり、７０時間前後で定常期をむかえることが分かった（図６）。
【００３０】
また、この時の培地の配合と発酵条件は、乳製品としてホエーパウダー、植物由来成分として豆乳、Ｌ−グルタミン酸供給源としてＭＳＧを使用した時を例とし表３に示す。
【００３１】
【表３】

【００３２】
なお、表３において、「炭酸ナトリウム」は、ｐＨ調整用であり、「ブレビスＴＹ４１４スターター」とは、２．０％ブドウ糖、１．０％豆由来ペプチド、０．５％酵母エキスの配合の水溶液を１２１℃、１５分にて滅菌したものにブレビスＴＹ４１４を添加し、３０℃、２４時間培養したものであり、「ブルガリカスタイプストレインスターター」とは、８．３％脱脂粉乳溶液（１２１℃、１５分滅菌）にブルガリカスタイプタイプストレインを添加し、３７℃、２４時間培養したものをいう。
【００３３】
表３のように発酵条件は検討の結果、発酵時の２種の乳酸菌株バランスの推移がＧＡＢＡ生産に最も好ましい、温度２８〜３２℃、初発ｐＨ６．０〜６．５とした。
【００３４】
こうして得られた発酵物をエキス化や濃縮、若しくはパウダー化することで更に高濃度のＧＡＢＡを含有する発酵物が得られる（図７）。また、それぞれの製法例は表４に示すとおりである。
【００３５】
【表４】

【００３６】
このようなＧＡＢＡ高含有発酵物は、ＧＡＢＡを安全かつ、高濃度で摂取できるもので、更には、風味的にも嗜好性が高いものであるため、日常的に手軽に食することができる。
【００３７】
また、このことより、例えば、当該発酵物を含む食品を日常的に摂取することにより、積極的な健康保持効果が期待でき、産業上の利用価値が極めて高いものである。
【００３８】
【発明の効果】
以上詳述したとおり、この発明によれば、短期間に効率よく、高含有のＧＡＢＡを生産することができ、しかも、これを食品として安全に用いることができる。
【図面の簡単な説明】
【図１】各乳酸菌株によるＧＹＰ培地（１％ＭＳＧ含有）発酵後のＧＡＢＡ含有量を示す棒グラフである（培養条件：温度３０℃、ｐＨ無調整、培養７２時間）。
【図２】各乳酸菌株の混合培養における１０％脱脂粉乳培地（１％ＭＳＧ含有）発酵後のＧＡＢＡ含有量を示す棒グラフである（培養条件：温度３０℃、ｐＨ無調整、培養７２時間）。
【図３】ＧＡＢＡ生産菌との混合培養に使用した乳酸菌株によるＧＹＰ培地（１％ＭＳＧ含有）発酵後のＧＡＢＡ含有量を示す棒グラフである（培養条件：温度３０℃、ｐＨ無調整、培養７２時間）。
【図４】各乳酸菌株の混合培養における表２を処方例とする発酵物のＧＡＢＡ含有量をを示す棒グラフである。
【図５】各乳酸菌株の混合培養における表３を処方例とする発酵物のＧＡＢＡ含有量をを示す棒グラフである。
【図６】表３を処方例とするブレビスＴＹ４１４とブルガリカスタイプストレインの混合培養におけるＧＡＢＡ生産量とＬ−グルタミン酸量の経時変化を示す折線図である。
【図７】表３を処方例とする発酵物を各加工法（表４に加工例を示す）により加工したときのＧＡＢＡ含有量を示す棒グラフである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a lactic acid bacterium having a high production ability of γ-aminobutyric acid (GABA), which has been attracting attention as a compound having an inhibitory effect on blood pressure, a highly health-oriented food containing GABA using the lactic acid bacterium, and the food. Relates to the manufacturing method.
[0002]
[Prior art]
GABA is an amino acid that is widely distributed in nature, and is usually contained in foods, tea, vegetables, etc., and is produced in vivo by decarboxylation from L-glutamic acid, and in tissues. Exists. Unlike many other amino acids, GABA is a non-protein constituent amino acid, but has a physiologically important function. That is, GABA is not only ingested as a food by the body, but also promotes the excretion of sodium ions into the urine due to excessive intake of salt, and has a blood pressure lowering effect (Hubert C. Stanton, Archint. Pharmacodyn., 143, p195-204, 1963) are known to contribute to important regulatory actions.
[0003]
As food containing GABA, GABA-enhanced tea leaves were developed as Gabaron tea (Journal of Japanese Society for Agricultural Chemistry, 61, NO.11, p1449-1451, 1987, Shokai 63-103285), and rats were used. Experiments have confirmed that Gabalon tea has an effect of lowering blood pressure and promoting the discharge of sodium ions into urine. In addition, it has recently been clarified that the main active ingredient of red yeast rice which has been used for medicinal meals and the like since its antihypertensive effect has been remarkable for a long time is GABA.
[0004]
[Problems to be solved by the invention]
However, the food containing GABA has a limited number of types such as a small number of vegetables, and even the food containing GABA has a small content, and although GABA is an effective ingredient, There are few opportunities to be ingested as food.
[0005]
Therefore, as a method for artificially increasing the intake of GABA, GABA may be produced by microorganisms such as Escherichia coli and added to food. However, GABA produced by E. coli has a safety problem when used in food. As described above, when GABA produced by microorganisms is used in foods, the type of bacteria producing GABA becomes a problem, and bacteria capable of producing them safely and in large quantities are required.
[0006]
Here, for example, as disclosed in Japanese Patent No. 2704493, Lactobacillus plantum or brewing moromi containing the same is added, and fish soy sauce moromi, L-glutamic acid-containing savory sauce, its compressed solution, and fish soy sauce are fermented. You can get food. However, since the fermentation takes time, the productivity is poor, and since the raw material, L-glutamic acid, remains considerably, the amount of addition is not sufficient for use in the case where “umami” addition such as soft drinks is not preferable. Limited or cannot be added.
Therefore, bacteria and production methods that produce GABA from sodium L-glutamate (MSG) or L-glutamic acid in a short time and at a high rate are desired.
[0007]
[Means for Solving the Problems]
In the present invention, from the viewpoint of safety as a food, the production capacity of GABA by microorganisms conventionally used for food production such as fermented milk is used.
Moreover, in order not to destroy the flavor peculiar to lactic acid bacteria fermentation, almost 100% conversion from MSG, L-glutamic acid, or L-glutamic acid-containing food (for example, food made from beans, tomatoes, seaweed, etc.) to GABA is performed. The conditions (selection of lactic acid strain, selection of combination of lactic acid strain, blending of fermentation medium, setting of initial pH, setting of fermentation temperature and fermentation time) were searched.
[0008]
As a result, a new lactic acid strain with high GABA productivity was discovered, and further, selection of a combination of lactic acid strains (mixed culture of lactic acid bacteria that improved GABA productivity), composition of fermentation medium, setting of initial pH, fermentation The inventors have found that GABA can be efficiently obtained by setting the temperature and fermentation time, and have reached the present invention.
[0009]
That is, the novel lactic acid bacterium Lactobacillus brevis TY414 (FERM P-16910) having high GABA production ability of the present invention has the following mycological properties.
Bacterial morphology when cultured in MRS liquid medium for the first time at pH 5.7, 30 ° C. for 16 hours (1) Bacterial morphology Spider (2) Gram staining Positive (3) Motility None (4) Spore None (5) Catalase None (6) facultative anaerobic (7) growth temperature range 20-40 ° C
(8) Growth pH range 3.8 to 9.4
(9) Lactic acid fermentation Heterotype (10) Optical rotation of lactic acid DL
(11) Fermentative strong fermentation of sugar → L-arabinose, ribose, D-xylose, galactose, glucose, fructose, α-methyl-D-glucoside, maltose, melibiose weak fermentation → mannitol, N acetylglucosamine, seriobiose, gluconate, 2-ketogluconate, 5-ketogluconate
Furthermore, the present invention is also characterized by a mixed culture of Lactobacillus brevis TY414 and Lactobacillus delbrueckii subsp. Bulgaricus IFO13953 (Bulgaricus type strain) for the purpose of improving GABA productivity. .
[0012]
Furthermore, the present invention is a mixture of a dairy product (or a plant component added thereto) and a food containing sodium L-glutamate (MSG) or L-glutamic acid or L-glutamic acid, The method is also characterized by a short-term production method of a fermented product with a high GABA content by the fermentation action of any of the above lactic acid bacteria.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the production of GABA by the lactic acid bacteria of the present invention will be described in more detail.
[0014]
An appropriate amount of a test sample is sampled, diluted and suspended in physiological saline, and then mixed and cultured on bromcresol, purple, and plate count agar medium (BCP medium). Pure separation of lactic acid bacteria is performed by repeating the operation of streaking and culturing on an agar medium.
[0015]
There are reports that 20 lactic acid bacterial strains and GABA producing ability were recognized from the laboratory-preserved strains of the applicant obtained in this way (Journal of Biotechnology, 75, NO.4, p239-244, 1997). Lactobacillus 4 strains {Lactobacillus brevis (Lactobacillus brevis) IFO3345, Lactobacillus brevis IFO3960, Lactobacillus brevis IFO12005, Lactobacillus brevis IFO12520} were each cultured in GYP medium (containing 1% MSG), and comparison of GABA production ability Went.
[0016]
As a result, remarkable GABA production ability was recognized among 5 strains of brevis TY414, brevis IFO3345, brevis IFO3960, brevis IFO12005 and brevis IFO12520. FIG. 1 shows the GABA production ability of these five strains.
[0017]
In addition, these quantifications are carried out by membrane filtration of each fermentation broth, followed by rough purification with Sepppack, thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and F kit L-glutamic acid {L-glutamic acid quantification. Kit (Boehringer Mannheim)}. The conditions for TLC and HPLC are as follows.
(1) TLC conditions:
Plate; Funacell SF (cellulose plate)
Developing solvent; isopropanol: water: acetic acid = 50: 45: 5
Rf value; GABA = 0.91, MSG = 0.80
(2) HPLC condition column: DAISO SP-120-5-ODS-BP (4.6 mmφ × 150 mmL)
Detector d; Waters M484 (UV)
Wavelength: 210nm
Elution solvent; water: phosphoric acid = 99.9: 0.1
Flow rate: 1 ml / min
Temperature: 40 ° C
[0018]
As a result of the screening shown in FIG. 1, the strain was narrowed down to 3 lactic acid bacteria (brevis TY414, brevis IFO3960, brevis IFO12005) that efficiently produce GABA by fermenting GYP medium (containing 1% MSG). Hereinafter, the fermentation conditions are examined for these three strains.
[0019]
Fermentation was attempted using 10% nonfat dry milk (containing 1% MSG) as a starting material in order to adjust the fermented products for these three lactic acid strains, but fermentation was weak and therefore almost no conversion to GABA was observed.
[0020]
Therefore, an attempt was made to improve fermentation and GABA production capacity by mixing and culturing other types of lactic acid bacteria. FIG. 2 shows the amount of Lactobacillus delbrueckii subsp. Bulgaricus TY028, Lactobacillus delbrueckii subsp. Indicates. Each fermented product name is shown in Table 1.
[0021]
[Table 1]

[0022]
As a result, 100 mg / 100 g or more of GABA-containing fermented milk was obtained in the two types of mixed culture (FIG. 2). Moreover, even if the combined bacterial species was the same Bulgaricus species, it was found from these results that there was a difference in the affinity for GABA production ability depending on the strain.
[0023]
At this time, the GABA production ability in the GYP medium (containing 1% MSG) by each single strain of Bulgaricus spp. Used in the mixed culture was considerably low as shown in FIG.
[0024]
However, with the conversion rate to GABA of the level shown in FIG. 2, a high concentration of L-glutamic acid remains in the fermentation liquid, resulting in a fermented liquid with a strong “umami taste” and a food lacking the sour feeling peculiar to lactic acid bacteria fermentation.
[0025]
Therefore, plant-derived ingredients (for example, foods made from beans, carrots, peppers, pumpkins, celery, spinach, cabbage, tomatoes, etc.) are added to the skim milk medium to promote fermentation to improve GABA productivity. It was. Table 2 shows the formulation when soymilk is used as an example of the plant-derived component at this time. An example of the starter is a mixed culture system of brevis TY414 and Bulgaricus type strain.
[0026]
[Table 2]

[0027]
In Table 2, “Brevis TY414 Starter” refers to an aqueous solution containing 2.0% glucose, 1.0% bean-derived peptide, and 0.5% yeast extract sterilized at 121 ° C. for 15 minutes. This refers to a culture in which brevis TY414 was added and cultured at 30 ° C. for 24 hours.
In Table 2, “Bulgaricus type strain starter” refers to a product obtained by adding Bvlgaricus type strain to 8.3% skim milk powder solution (121 ° C., sterilized for 15 minutes) and culturing at 37 ° C. for 24 hours. .
[0028]
Table 2 is a formulation example, and the GABA production amount in each mixed culture is shown in FIG. As a result, a high concentration of GABA is newly obtained in a mixed culture of Brevis TY414 and Bulgaricus TY393 (sample A3b), Brevis TY414 and Bulgaricus type strain (sample A4b), Brevis IFO12005 and Bulgaricus TY393 (sample C3b). It was.
Furthermore, in order to raise GABA productivity to the level at the time of fermentation of GYP culture medium (containing 1% MSG), GABA productivity was increased by using whey powder instead of skim milk powder. In addition to whey powder, the same effect can be obtained with concentrated whey. As other dairy products, use of cream powder, butter mill powder, concentrated milk, defatted concentrated milk, sugar-free condensed milk, sugar-free defatted condensed milk, sweetened condensed milk, sweetened defatted condensed milk, whole powdered milk powdered powdered milk, adjusted powdered milk is also considered. It is done.
[0029]
Among the fermented products examined above, MSG or L-glutamic acid is converted to GABA with an efficiency of almost 100% for fermented products containing GABA obtained by mixed culture of brevis TY414 and Bulgaricus type strain (sample A4c). Was. Also, in the mixed culture of brevis TY414 and Bulgaricus TY393 (sample A3c) and in the mixed culture of brevis IFO12005 and Bulgaricus TY393 (sample C3c), the GABA production increased and the GABA content was also considerably high (FIG. 5). ).
Furthermore, when the change in GABA production over time in sample A4c was observed, it was found that the production of GABA suddenly increased after about 50 hours, and the steady phase was reached around 70 hours (FIG. 6).
[0030]
In addition, the composition and fermentation conditions of the medium at this time are shown in Table 3 as an example when whey powder is used as a dairy product, soy milk is used as a plant-derived component, and MSG is used as an L-glutamic acid supply source.
[0031]
[Table 3]

[0032]
In Table 3, “sodium carbonate” is for pH adjustment, and “brevis TY414 starter” is an aqueous solution containing 2.0% glucose, 1.0% bean-derived peptide, and 0.5% yeast extract. Was sterilized at 121 ° C. for 15 minutes and brevis TY414 was added and cultured at 30 ° C. for 24 hours. “Bulgaricus type strain starter” is an 8% skim milk solution (121 ° C., 15 minutes sterilized) and added with Bulgaricus type strain and cultured at 37 ° C. for 24 hours.
[0033]
As shown in Table 3, as a result of the examination of the fermentation conditions, the transition of the balance of the two lactic acid bacterial strains during fermentation was most preferable for GABA production, with a temperature of 28 to 32 ° C. and an initial pH of 6.0 to 6.5.
[0034]
A fermented product containing a higher concentration of GABA can be obtained by extracting, concentrating, or powdering the fermented product thus obtained (FIG. 7). Each example of the production method is as shown in Table 4.
[0035]
[Table 4]

[0036]
Such a fermented product with a high GABA content is safe and can be ingested at a high concentration. Furthermore, since it is highly palatable in flavor, it can be easily eaten on a daily basis.
[0037]
In addition, for example, by taking a food containing the fermented product on a daily basis, a positive health maintenance effect can be expected, and the industrial utility value is extremely high.
[0038]
【The invention's effect】
As described in detail above, according to the present invention, high-content GABA can be produced efficiently in a short period of time, and this can be safely used as food.
[Brief description of the drawings]
FIG. 1 is a bar graph showing GABA content after fermentation of GYP medium (containing 1% MSG) by each lactic acid strain (culture conditions: temperature 30 ° C., pH unadjusted, culture 72 hours).
FIG. 2 is a bar graph showing GABA content after fermentation of 10% nonfat dry milk medium (containing 1% MSG) in mixed culture of each lactic acid strain (culture conditions: temperature 30 ° C., pH unadjusted, culture 72 hours).
FIG. 3 is a bar graph showing GABA content after fermentation using a GYP medium (containing 1% MSG) by a lactic acid strain used for mixed culture with GABA-producing bacteria (culture conditions: temperature 30 ° C., pH unadjusted, culture 72). time).
FIG. 4 is a bar graph showing the GABA content of a fermented product having Table 2 as a formulation example in mixed culture of each lactic acid bacterial strain.
FIG. 5 is a bar graph showing the GABA content of a fermented product having Table 3 as a formulation example in mixed culture of each lactic acid bacterial strain.
FIG. 6 is a polygonal diagram showing temporal changes in GABA production amount and L-glutamic acid amount in mixed culture of brevis TY414 and bulgaricus type strain with Table 3 as a formulation example.
FIG. 7 is a bar graph showing the GABA content when fermented products having Table 3 as formulation examples are processed by various processing methods (processing examples are shown in Table 4).

Claims (3)

A novel lactic acid bacterium Lactobacillus brevis TY414 (FERM P-16910) having the ability to produce γ-aminobutyric acid having the following mycological properties.
Bacterial morphology when cultured in MRS liquid medium for the first time at pH 5.7, 30 ° C. for 16 hours (1) Bacterial morphology Spider (2) Gram staining Positive (3) Motility None (4) Spore None (5) Catalase None (6) facultative anaerobic (7) growth temperature range 20-40 ° C
(8) Growth pH range 3.8 to 9.4
(9) Lactic acid fermentation Heterotype (10) Optical rotation of lactic acid DL
(11) Fermentation of sugar Strong fermentation → L-arabinose, ribose, D-xylose, galactose, glucose, fructose, α-methyl-D-glucoside, maltose, melibiose weak fermentation → mannitol, N acetylglucosamine, seriobiose, gluconate, 2-ketogluconate, 5-ketogluconate   A mixed culture method for the purpose of improving the productivity of γ-aminobutyric acid between the novel lactic acid bacterium Lactobacillus brevis TY414 according to claim 1 and Lactobacillus delbrukki Bulgaricus IFO13953. The fermentation medium is a mixture of a dairy product or a product obtained by adding a plant component thereto and a food containing sodium L-glutamate, L-glutamic acid, or L-glutamic acid,
The short-term manufacturing method of the fermented material with high content of (gamma) -aminobutyric acid by the fermenting effect of the lactic acid bacteria of Claim 1, or the fermenting effect of the mixed culture method of Claim 2.

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