JP2003070462A - LACTOBACILLUS WITH ABILITY TO PRODUCE 7%. gamma-AMINOBUTYRIC ACID, AND METHOD FOR PRODUCING FOOD USING THE LACTOBACILLUS - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing γ-aminobutyric acid-rich food. SOLUTION: The food, i.e., a fermented product containing γ-aminobutyric acid is obtained by inoculating a fermentation raw material containing glutamic acid or a salt thereof with a Lactobacillus hilgardii K-3 strain (FERM P-18422) isolated from kimchi (a type of Korean pickles) and then culturing the thus isolated K-3 strain.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to decarboxylation of glutamic acid by the action of glutamate decarboxylase (glutamate decarboxylase) to produce γ-aminobutyric acid (GAB
A) a lactic acid bacterium capable of producing γ-aminobutyric acid,
It also relates to a method for producing a food containing γ-aminobutyric acid using the lactic acid bacterium.

[0002]

2. Description of the Related Art γ-Aminobutyric acid is also called GABA,
It is a type of non-protein amino acid widely distributed in nature, and is commonly contained in foods such as tea, vegetables, and cereals. In the living body, glutamic acid is decarboxylated and produced, and in the human body, especially in the substantia nigra in the brain,
It is known to be present in high concentrations in the basal ganglia, hypothalamus, etc.

As a physiological action of γ-aminobutyric acid, it is well known that blood vessels are expanded to lower blood pressure. In addition, γ-aminobutyric acid activates blood flow in the brain,
It functions to increase the oxygen supply to brain cells and promote metabolic functions, and plays an important role in the central nervous system as a major inhibitory neurotransmitter. And
As a drug, it has been applied to the treatment of headache, tinnitus, etc. due to stenosis or blockage of cerebral blood vessels. It has also been reported that administration of γ-aminobutyric acid to the human body contributes to enhancement of learning ability and promotion of long-term memory.

Therefore, if γ-aminobutyric acid is ingested as a food, the above-mentioned physiological action is expected. Therefore, a food having an increased γ-aminobutyric acid content can be obtained by subjecting the food to an appropriate treatment. Some have been proposed. For example, known are food materials obtained by dipping embryos such as rice in water (Japanese Patent No. 2810993) and gabaron tea obtained by anaerobically treating tea leaves (Japanese Utility Model Publication No. 63-103285).

On the other hand, as a method for producing γ-aminobutyric acid-containing foods using food-use microorganisms, Lactobacillus (L
Those using lactic acid bacteria of the genus actobacillus (Japanese Patent No. 2704493, JP 2000-308A).
No. 457, JP 2000-210075, etc.)
There are many reports such as those using yeast and koji molds (JP-A-10-165191, JP-A-11-103825, etc.) and those using yeast or chlorella (JP-A-9-238650).

[0006]

However, the food having an increased γ-aminobutyric acid content by the above-mentioned conventional method has a higher content than that before the treatment, but 100 g of the food The content of γ-aminobutyric acid per unit is less than 1000 mg, that is, the content rate in food is 1%
Less than With this content ratio, for example, γ-
It is still insufficient to add such foods containing aminobutyric acid to foods and drinks such as soups, prepared foods, pickles and confectionery and to use them widely as raw materials for processed foods.

The present invention has been made in view of the above circumstances, and provides a lactic acid bacterium having a γ-aminobutyric acid-producing ability, which can obtain a food containing a high proportion of γ-aminobutyric acid. It is also an object of the present invention to provide a method for producing a food which can produce a food containing a high proportion of γ-aminobutyric acid using the lactic acid bacterium.

[0008]

Means for Solving the Problems The present inventor has conducted a search for lactic acid bacteria having a high γ-aminobutyric acid-producing ability in fermented and brewed foods, and as a result, Lactobacillus hirugardei (Lactobacillus) isolated from kimchi was found.
A strain belonging to us hirgardii) has a high ability to produce γ-aminobutyric acid, and if the strain is used and is cultivated in a culture solution containing 10 wt% sodium glutamate, for example, about 5 wt% It was found that a culture broth containing a high concentration of γ-aminobutyric acid was obtained, and that a fermentation broth containing 15% by weight or more of γ-aminobutyric acid was obtained simply by filtering and concentrating the culture. The present invention has been completed.

That is, the invention according to claim 1 is a lactic acid bacterium capable of producing γ-aminobutyric acid, which is a strain isolated from kimchi and whose growth is promoted by adding glutamic acid or a salt thereof to the medium. It is characterized by being a strain belonging to Lactobacillus hirugardi that produces γ-aminobutyric acid by the action of glutamate decarboxylase.

The invention according to claim 2 is the lactic acid bacterium according to claim 1, wherein the strain is Lactobacillus hilgardi K-3 strain FERM P-18422 (hereinafter simply referred to as "K-3 strain"). Characterize.

The invention according to claim 3 is a method for producing a food using a lactic acid bacterium capable of producing γ-aminobutyric acid, wherein the fermentation raw material containing glutamic acid or a salt thereof is added to the lactic acid bacterium according to claim 1 or 2. Is inoculated and cultured to obtain a fermented product containing γ-aminobutyric acid.

The invention according to claim 4 is characterized in that, in the manufacturing method according to claim 3, the fermentation treatment is carried out at an initial pH of 4.5 to 5.5. In this way, the lactic acid bacteria
Its growth is most promoted and its ability to produce γ-aminobutyric acid is improved.

The invention according to claim 5 is the method according to claim 3 or 4, wherein the fermented product is a fermented liquid, and the fermented liquid is filtered, concentrated and dried to obtain γ-.
It is characterized in that a powder containing aminobutyric acid is obtained.

According to a sixth aspect of the present invention, in the production method according to the third or fourth aspect, the fermented product is a fermented liquor, and the fermented liquor is concentrated and then added to food or beverage to obtain γ- A feature is to obtain a food or drink containing aminobutyric acid.

The invention according to claim 7 is characterized in that, in the manufacturing method according to claim 5, the powder is added to food or beverage to obtain a food or drink containing γ-aminobutyric acid.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION Preferred embodiments of the present invention will be described below.

The lactic acid bacterium according to the present invention is a strain belonging to Lactobacillus hilgardi isolated from Kimchi, and its growth is promoted by the addition of glutamic acid or its salts to the medium. -It has the ability to produce aminobutyric acid at a high rate. That is, this kimchi-derived lactic acid bacterium has 1
When cultured in a liquid medium containing 00 g of sodium glutamate at a temperature of 30 ° C. for 48 hours, 45 g / L or more of γ-aminobutyric acid can be produced. The lactic acid bacterium is preferably the K-3 strain.

The lactic acid bacterium is isolated from kimchi by the following method. That is, kimchi was added with 10 g / kg of sodium glutamate and fermented and matured as a separation source, and lactic acid bacteria were separated using a BCP-added plate count agar medium (manufactured by Nissui Co., Ltd.) for separating lactic acid bacteria. Was cultured in a GYP liquid medium containing 10 g / L of sodium glutamate, and a strain having a large production amount of γ-aminobutyric acid was selected.

Table 1 shows the mycological properties of the K-3 strain isolated as described above.

[Table 1]

Using the lactic acid bacterium derived from kimchi described above, a fermentation raw material containing glutamic acid or a salt thereof is inoculated with the lactic acid bacterium and cultured to produce a fermented food containing γ-aminobutyric acid. As the lactic acid bacterium, a lactic acid bacterium isolated and cultured from Kimchi and subcultured is used, but the same lactic acid bacterium contained in Kimchi may be used as it is or may be newly isolated and used. Also, any of freeze-dried cells, cryopreserved strains and liquid culture medium may be used, but the one pre-cultured in a fermentation liquid or liquid medium containing 1% or more glutamic acid from the day before use is used. It is preferable.

Glutamic acid used in this method for producing fermented foods chemically refers to L-glutamic acid, which is one of the amino acids, and is a food additive such as glutamic acid, sodium glutamate, and other glutamate which has a use as a seasoning. Further, any of glutamic acid obtained by hydrolyzing food protein with acid or enzyme may be used. Further, foods containing free glutamic acid such as seasonings, processed marine products and tomatoes may be used as they are. However, in order to obtain a food containing a larger amount of γ-aminobutyric acid, it is necessary to use a fermentation raw material containing a larger amount of glutamic acid.

The fermentation medium contains foods containing glucose such as glucose, fructose and maltose, and vitamins and minerals such as yeast extract and meat extract in addition to the above-mentioned glutamic acid for the growth of lactic acid bacteria. It is preferable. Further, food additives such as emulsifiers, stabilizers and pH adjusters may be used.

The container used for fermentation treatment can be washed and sterilized by heating and can be used for food production, regardless of size or material, but a structure in which various bacteria are not easily mixed. Those having the above are preferable.

The conditions of fermentation treatment are as follows. That is, the fermentation temperature is preferably 20 ° C to 30 ° C, the fermentation time is, for example, 48 hours to 75 hours, and the initial pH is
Is preferably 4.0 to 7.0. However, when fermentation is performed using a fermentation medium containing a high concentration of 5% by weight or more of sodium glutamate, the initial p
Most preferred is H4.5-5.5. For example, K-3 in a GYP liquid medium containing 4% by weight of sodium glutamate.
When the strain was cultured, the amount of γ-aminobutyric acid produced after 72 hours was about 2% by weight at both the initial pH of 6.3 and the initial pH of 5.0, whereas 10% by weight of sodium glutamate was used. K-3 in GYP liquid medium containing
When the strain was cultured, the amount of γ-aminobutyric acid produced after 72 hours was about 3% by weight at the initial pH of 6.3, while it reached about 5% by weight at the initial pH of 5.0. Also,
The pH of the fermented liquor rises with the growth of the K-3 strain, and reaches a neutral range of pH 7 to 8 when 24 hours to 48 hours have passed since the start of the fermentation treatment. Therefore, after about 24 hours have passed from the start of the fermentation process, the pH of the fermented liquid is adjusted to about 5 again to continue the fermentation process.
By doing so, the growth of lactic acid bacteria can be further promoted, and the rate and efficiency of γ-aminobutyric acid production can be increased.

The fermented broth containing γ-aminobutyric acid obtained by culturing lactic acid bacteria may be used as it is by adding it to various beverages and foods, or may be subjected to treatment steps such as filtration, concentration and drying. Therefore, the content of γ-aminobutyric acid may be further increased before use. In this way, without going through purification steps such as chromatography,
By the method that can be carried out relatively inexpensively with little waste and by-products, liquid or powdery high γ-aminobutyric acid-rich foods that have a high concentration of γ-aminobutyric acid and have wide applications as raw materials such as processed foods. Obtainable. In addition, various kinds of beverages and foods containing a large amount of γ-aminobutyric acid, for example, foods and drinks having a γ-aminobutyric acid concentration of 15% by weight or more can be obtained by passing through treatment steps such as filtration, concentration, and drying.

The above-mentioned filtration step can be carried out by using ordinary filtration equipment for food processing using filter paper, filter cloth and the like, and even if a filter aid such as diatomaceous earth, cellulose or activated carbon is used. Good. The concentration step may be performed using equipment such as a vacuum concentrator, a vacuum concentrator, a distillation pot, a freeze concentrator, or the like, and the water in the fermentation liquor may be simply evaporated in a pan that has been heated. The drying step is preferably performed by an efficient and hygienic method such as spray drying or freeze drying. Further, in order to obtain a food having a higher content of γ-aminobutyric acid, a method such as liquid chromatography may be used in addition to the above steps.
Further, by concentrating or drying the above-mentioned fermentation liquid, a liquid or powdery food containing γ-aminobutyric acid is obtained, and by mixing or secondary processing those foods, γ-aminobutyric acid was contained. Beverages and food are available.

In addition to the above-mentioned components, foods high in γ-aminobutyric acid usually include excipients, sweeteners, thickeners, proteins, peptides, lipids, polysaccharides, sugars, salts, etc.
It may contain ingredients used in foods.

Γ-in the obtained fermented liquid or fermented food
The content of aminobutyric acid may be measured using an analytical instrument such as high performance liquid chromatography or an amino acid measuring instrument, or an analytical reagent using an enzyme.

[0029]

EXAMPLES More specific examples to which the present invention is applied will be described below.

[Restoration from preserved strain] When using the K-3 strain which has been stored for a long time in a frozen or freeze-dried state,
After the K-3 strain was added to sterile water to form a suspension, the suspension was inoculated using a platinum loop into a GYP agar medium having the composition shown in Table 2, and the temperature was 30 ° C. for 24 hours. Incubate. When milky white colonies are confirmed on the agar medium, the K-3 strain can be used as it is for the subsequent culture. K-3
The strain can be stored on an agar medium at a temperature of 4 ° C to 10 ° C for 4 weeks.

[0031]

[Table 2]

[Preculture] The K-3 strain on the GYP agar medium was subjected to 5 to 100 mL of a GYP liquid medium using a platinum loop (the standard agar was removed from the GYP agar medium having the component composition shown in Table 1). Inoculate for 24 hours at a temperature of 30 ° C,
Let stand and culture. When the turbidity (absorbance at a wavelength of 660 nm) of the culture solution reaches 1.0 or more, the culture solution can be used for the subsequent fermentation.

[Method for measuring γ-aminobutyric acid]
100 with 2N sodium citrate buffer (pH 2.2)
After diluting 1- to 10,000-fold, the solid matter was removed by centrifugation or filtration to obtain a measurement sample. The respective contents of γ-aminobutyric acid and glutamic acid were measured by a high performance liquid chromatograph under the following conditions according to a conventional method for measuring the amino acid content. Equipment used: High-performance liquid chromatograph LC-9A manufactured by Shimadzu Corporation Analytical column: Strongly acidic cation exchange resin column Shin
-Pack Isc-07 Na type moving layer buffer: amino acid moving layer kit manufactured by Shimadzu Corporation Na type moving layer flow rate: 0.3 ml / min Reaction layer 1: 0.04% sodium hypochlorite solution (pH
10 ml of boric acid-carbonic acid buffer solution to 0.2 ml of hypochlorous acid) Reaction layer 2: amino acid analysis kit O manufactured by Shimadzu Corporation
Flow rate of PA reagent reaction layer: 0.2 ml / min Detection: Fluorescence detection Ex348 nm, Em450 nm

Example 1 GYP liquid medium containing 100 g of sodium glutamate (Table 3 shows the composition of ingredients)
1 mL of the preculture liquid of the K-3 strain was added to 1 L, and the mixture was cultured at a temperature of 30 ° C. The content of γ-aminobutyric acid in the culture solution is
It was 48 g / L after 48 hours and 51 g / L after 72 hours. The content of glutamic acid is 8 after 48 hours.
g / L, 2 g / L after 72 hours. In addition, 100
Since the amount of γ-aminobutyric acid when 100 g of sodium glutamate (monohydrate) is 100% converted to γ-aminobutyric acid is about 55 g, the conversion rate to γ-aminobutyric acid is relatively 48%. It can be expressed as 87% after hours and 93% after 72 hours.

[0035]

[Table 3]

Comparative Example Lactobacillus derived from wine
Hilgardee JCM1155 strain was used and cultured under the same conditions as in Example 1. As a result, the content of γ-aminobutyric acid in the obtained culture solution was 0.05 g / L or less after both 48 hours and 72 hours.

[Example 2] 50 L of fermentation raw material 1 (the composition of which is shown in Table 4) sterilized by heating at a temperature of 90 ° C for 10 minutes
50 mL of the preculture liquid of the K-3 strain was inoculated into and was cultured at a temperature of 30 ° C. for 3 days. The content of γ-aminobutyric acid in the fermentation liquid after the fermentation was 32 g / L. 9 of this fermented liquid
Heat sterilize at a temperature of 0 ° C. for 10 minutes, add activated carbon to decolorize it, add a filter aid containing diatomaceous earth, and filter,
After concentrating with a vacuum concentrator, it was pulverized with a spray dryer. The content of γ-aminobutyric acid in the obtained powdered food was 16% by weight.

[0038]

[Table 4]

[Example 3] Fermented seasoning liquid raw material (component composition is shown in Table 5) 5 sterilized by heating at a temperature of 90 ° C for 10 minutes
50 mL of the preculture liquid of the K-3 strain was inoculated into 0 L and cultured at a temperature of 30 ° C. for 3 days. The content of γ-aminobutyric acid in the fermented liquid after the fermentation was 26 g / L. This fermented liquid was heat-sterilized at a temperature of 90 ° C. for 10 minutes and then concentrated using a vacuum concentrator. The content of γ-aminobutyric acid in the obtained fermented seasoning liquid was 16% by weight.

[0040]

[Table 5]

Further, 100 mL of the obtained fermented seasoning liquid was added to 10 kg of kimchi raw material containing salted Chinese cabbage and aged in a refrigerator for 1 week. The content of γ-aminobutyric acid in the obtained kimchi was 152 mg per 100 g. This amount corresponds to 95% of the added γ-aminobutyric acid.

[Example 4] Fermentation raw material 2 heat-sterilized at a temperature of 90 ° C for 10 minutes (the composition of ingredients is shown in Table 6) 1,0
00 L was inoculated with 1 L of the preculture liquid of the K-3 strain and cultured at a temperature of 30 ° C. for 3 days. The content of γ-aminobutyric acid in the fermented liquid after the fermentation was 26 g / L. This fermented liquid is sterilized by heating at 90 ° C for 10 minutes, decolorized by adding activated carbon to it, filtered by adding a filter aid containing diatomaceous earth, concentrated by using a vacuum concentrator, and then sprayed with dextrin. It was pulverized using a dryer. The content of γ-aminobutyric acid in the obtained powdered food was 30% by weight. It concentrated using the vacuum concentrator. The content of γ-aminobutyric acid in the obtained fermented seasoning liquid was 16% by weight.

[0043]

[Table 6]

[Example 5] 100 of fermentation raw material 3 (the composition of which is shown in Table 7) sterilized by heating at 90 ° C for 10 minutes
L was inoculated with 100 mL of the preculture liquid of the K-3 strain, cultivated at a temperature of 30 ° C. for 24 hours, and then the pH was readjusted to 5.0,
The culture was continued for another 2 days. Γ in fermented liquid after fermentation
-The content of aminobutyric acid was 54 g / L. This fermented liquid was sterilized by heating at a temperature of 90 ° C. for 10 minutes, filtered by adding a filter aid containing diatomaceous earth, concentrated using a vacuum concentrator, and then pulverized using a freeze dryer. The content of γ-aminobutyric acid in the obtained powdered food was 70% by weight.

[0045]

[Table 7]

[0046]

When the lactic acid bacterium capable of producing γ-aminobutyric acid of the invention according to claim 1 is used, a fermentation raw material containing glutamic acid or a salt thereof at a high concentration is subjected to fermentation treatment,
A food containing a high proportion of γ-aminobutyric acid can be obtained.

With the lactic acid bacterium of the invention according to claim 2, the above-mentioned effect according to the invention of claim 1 can be reliably obtained.

According to the manufacturing method of the invention of claim 3,
Foods containing a high proportion of γ-aminobutyric acid can be produced.

In the production method of the invention according to claim 4, since the growth of lactic acid bacteria is most promoted and the ability to produce γ-aminobutyric acid is improved, the food containing γ-aminobutyric acid in a high proportion is more efficient. Is obtained.

In the manufacturing method of the invention according to claim 5, γ-
A powdery food product containing a high proportion of aminobutyric acid is obtained.

In the production method according to the sixth aspect of the present invention, the concentrated liquid of the fermentation broth is added to obtain a food or drink containing a high proportion of γ-aminobutyric acid.

In the manufacturing method of the invention according to claim 7, γ-
A powder containing a high proportion of aminobutyric acid was added to give γ
-Foods and drinks containing a high proportion of aminobutyric acid are obtained.

Continued front page    (72) Inventor Yoshie Ueno             21-27 cents, Uzumasa Yasui Road Road, Ukyo-ku, Kyoto             Paulia Uzumasa II 102 (72) Inventor Shinji Higashiguchi             24-5 Gonishimachi, Kichijoin Ishiharado, Minami-ku, Kyoto-shi               Pharma Foods Co., Ltd. (72) Inventor Kim Takehisa             24-5 Gonishimachi, Kichijoin Ishiharado, Minami-ku, Kyoto-shi               Pharma Foods Co., Ltd. F term (reference) 4B018 LB01 LB03 LB08 MD19 MD86                       ME04 ME14 MF13                 4B065 AA30X BA21 BB02 BB03                       BB12 BB15 BB29 CA17 CA41

Claims (7)

[Claims] 1. A strain isolated from kimchi, the growth of which is promoted by the addition of glutamic acid or a salt thereof to the medium, and Lactobacillus hirgardii that produces γ-aminobutyric acid by the action of glutamate decarboxylase. A lactic acid bacterium having the ability to produce γ-aminobutyric acid. 2. The lactic acid bacterium capable of producing γ-aminobutyric acid according to claim 1, wherein the strain is Lactobacillus hilgardi K-3 strain FERM P-18422. 3. A food product, characterized in that a fermentation raw material containing glutamic acid or a salt thereof is inoculated with the lactic acid bacterium according to claim 1 or 2 and cultured to obtain a fermented product containing γ-aminobutyric acid. Manufacturing method. 4. The method for producing a food according to claim 3, wherein the fermentation treatment is performed at an initial pH of 4.5 to 5.5. 5. The method for producing a food product according to claim 3, wherein the fermented product is a fermented liquid, and the fermented liquid is filtered, concentrated and dried to obtain a powder containing γ-aminobutyric acid. . 6. The fermented product is a fermented liquor, and the fermented liquor is concentrated and then added to food or beverage to obtain a food or drink containing γ-aminobutyric acid.
A method for producing the described food. 7. Adding the powder to a food or beverage,
The method for producing a food according to claim 5, wherein a food or drink containing γ-aminobutyric acid is obtained.