JP2023171280A - METHOD FOR PRODUCING COMPOSITION CONTAINING HIGH CONCENTRATION OF γ-AMINOBUTYRIC ACID - Google Patents

Abstract

To develop a technique that can efficiently produce γ-aminobutyric acid at a low cost and utilize this technique to provide food and drink containing a high concentration of γ-aminobutyric acid inexpensively.SOLUTION: The present invention provides a method for producing a composition, preferably food and drink, containing a high concentration of γ-aminobutyric acid. This method involves coculturing Lactococcus lactis JCM20101 and Lactobacillus helveticus at 27-37°C. The Lactobacillus helveticus may be Lactobacillus helveticus JCM1004.SELECTED DRAWING: Figure 2

Description

There is an application for exception to loss of novelty.

The present invention relates to a method for producing a composition with a high content of γ-aminobutyric acid, and more specifically, by co-cultivating a specific strain belonging to Lactococcus lactis and Lactobacillus helveticus having the ability to produce protease. The present invention relates to a method for producing a composition containing a high content of γ-aminobutyric acid.

In recent years, γ-aminobutyric acid (GABA) has attracted attention as a functional material. GABA is known to play an important role as an inhibitory neurotransmitter in the mammalian body, and is also an active ingredient that has physiological activities such as hypotensive, diuretic, and tranquilizing effects. Therefore, it is considered desirable to incorporate GABA into the daily diet. GABA can be produced by converting glutamic acid into GABA by the action of glutamate decarboxylase (optimum pH 4 to 5).

As a manufacturing technology for foods containing high GABA content, for example, Patent Document 1 discloses the production of γ-aminobutyric acid-containing foods and drinks, which is characterized by culturing a mixture of microorganisms capable of producing protease and microorganisms capable of producing glutamic acid decarboxylase. A law is proposed. Specifically, the GABA production concentration in Lactococcus lactis YIT2027 alone culture (3 days) was 15 mg/100 mL, but in the mixed culture of Lactococcus lactis YIT2027 and Lactobacillus casei YIT9029 (3 days) The GABA production concentration increased more than twice to 38 mg/100 mL.

Japanese Patent Application Publication No. 2000-14356

However, since GABA is very expensive as a product, it is difficult to incorporate foods containing a large amount of GABA into daily life. Even with the technique of Patent Document 1, the amount of GABA produced is only about twice that of the case of monoculture, and the production cost of GABA remains high.

Therefore, an object of the present invention is to develop a technology that can efficiently produce GABA at low cost, and to provide compositions, particularly food and drink products, containing GABA at high concentrations at low cost.

Therefore, the present inventors conducted extensive studies to solve the above problems, and as a result, by co-cultivating Lactococcus lactis JCM20101, which has the ability to produce GABA, and Lactobacillus helveticus, which has the ability to produce protease, It was discovered that the amount of GABA produced was significantly increased, and the present invention was completed.

That is, the present invention provides a method for producing a composition with a high content of γ-aminobutyric acid, which is characterized by co-cultivating Lactococcus lactis JCM20101 and Lactobacillus helveticus at 27 to 37°C.

Here, the Lactobacillus helveticus may be Lactobacillus helveticus JCM1004.

Further, the co-cultivation can be performed in a medium containing milk as a main component.

Further, the amount of Lactococcus lactis JCM20101 inoculated into the medium may be 0.2 to 10% (w/w), and the amount of Lactobacillus helveticus inoculated into the medium may be 0.1 to 10%. (w/w).

Further, the medium may contain 0.25 to 10% (w/w) of glutamate.

Furthermore, the composition with high γ-aminobutyric acid content can be used as a food or drink with high γ-aminobutyric acid content.

According to the present invention, GABA can be produced efficiently and inexpensively by co-cultivating Lactococcus lactis JCM20101 and Lactobacillus helveticus having protease-producing ability, and therefore, a composition containing GABA at a high concentration, In particular, food and beverages can be provided.

It is a graph comparing the amount of GABA produced by co-cultivation using different Lactococcus lactis (Test Example 2). The vertical axis shows the GABA production amount (mg/100ml) after 24 hours of culture, and the horizontal axis shows the test plot. It is a graph showing the influence of the inoculation amount of Lactococcus lactis JCM20101 on the GABA production amount in co-culture (the inoculation amount of Helveticus bacteria is fixed at 1%) (Test Example 3). The vertical axis shows the GABA production amount (mg/100ml) after 24 hours of culture, and the horizontal axis shows the test plot (inoculated amount of JCM20101). Moreover, the white bar graph shows the case of co-culture at 33°C, and the black bar graph shows the case of co-culture at 37°C. It is a graph showing the influence of culture temperature on GABA production amount in co-culture (Test Example 6). The vertical axis shows the GABA production amount (mg/100ml) after 24 hours of culture, and the horizontal axis shows the culture temperature (°C). It is a graph showing the influence of the glutamate concentration in the medium on the GABA production amount in co-culture (Test Example 7). The vertical axis shows the GABA production amount (mg/100ml) after 24 hours of culture, and the horizontal axis shows the GluNa concentration. It is a graph showing the influence of culture time on GABA production amount in co-culture (Test Example 8). The vertical axis shows the GABA production amount (mg/100ml), and the horizontal axis shows the culture time (h).

The present invention will be explained in detail below.

The method for producing a composition with high γ-aminobutyric acid content according to the present embodiment is characterized by co-cultivating Lactococcus lactis JCM20101 and Lactobacillus helveticus at 27 to 37°C.

Lactococcus lactis JCM20101 has the ability to produce glutamic acid decarboxylase and can convert glutamic acid in the medium to GABA, but the amount of GABA produced when cultured alone is small. JCM20101 is a facultative anaerobe like the common Lactococcus lactis, and is a diplococcal streptococcus. Furthermore, JCM20101 has a low protease production ability, and the optimum growth temperature is around 30°C.

Lactobacillus helveticus is a lactic acid bacterium that has a low ability to produce glutamic acid decarboxylase and has an optimal growth temperature around 37°C. Examples of Lactobacillus helveticus used in this embodiment include Lactobacillus helveticus JCM1004, JCM1006, JCM1120, CP790, CP611, and CP615. Particularly preferred is Lactobacillus helveticus JCM1004. However, the present invention is not limited to these strains, and any strain belonging to Lactobacillus helveticus can be used. Lactobacillus helveticus can be used in combination of two or more strains.

The medium used for the co-culture is not particularly limited as long as it is a liquid medium for lactic acid bacteria, and for example, a medium containing milk as a main component or a synthetic medium can be used. Examples of "medium containing milk as a main component" include liquid media containing milk components as a main component, such as animal milk such as cow's milk, whey, and skim milk. Examples of the "synthetic medium" include GLYP medium and MRS medium. Preferably, from the viewpoint of production cost, raw milk or animal milk subjected to various sterilization treatments such as UHT, LL, and HTST, such as UHT milk, can be used as the medium.

It is desirable that the medium contains glutamate as a raw material for GABA. As the glutamate, sodium glutamate (GluNa) or the like can be used. The glutamate concentration in the medium can be generally 0.25 to 10% (w/w), preferably 0.25 to 1% (w/w), and 0.5 to 1% (w/w). w/w) is more preferable. If the glutamate concentration exceeds 10%, a characteristic flavor will be felt, which is not preferable. Furthermore, if the glutamate concentration is less than 0.25%, the amount of GABA produced decreases, which is not preferable.

The two types of lactic acid bacteria may be inoculated into the medium at the same time, or Lactobacillus helveticus may be inoculated first, and then Lactococcus lactis JCM20101 may be inoculated. Inoculation may also be performed in the reverse order. Inoculation of the lactic acid bacteria can be performed by pre-cultivating each lactic acid bacteria in an appropriate medium and adding an appropriate amount of the culture solution to the medium. At this time, the medium used for pre-culture may have a different composition from the medium used for co-culture, and is not particularly limited as long as it is a medium that can activate each lactic acid bacterium. The medium used for pre-culturing Lactococcus lactis JCM20101 preferably contains glucose as a nutrient source, but is not limited thereto. Furthermore, it is desirable that the culture solution serving as the inoculum contains at least 10 ⁸ cells/ml of any of the above-mentioned lactic acid bacteria.

The amount of Lactococcus lactis JCM20101 inoculated into the medium in co-culture can be preferably 0.2 to 10% (w/w), more preferably 0. It can be .5 to 6% (w/w), more preferably 1 to 5% (w/w).

Further, the amount of Lactobacillus helveticus inoculated into the medium can be usually 0.1 to 10% (w/w), and from the viewpoint of flavor, preferably 0.1 to 4% (w/w). ), more preferably 0.3 to 3% (w/w), even more preferably 0.5 to 2% (w/w), especially 1 to 2% (w/w). can do.

Co-culture using the two types of lactic acid bacteria needs to be carried out at a temperature of 20 to 45° C. in order to achieve a higher GABA production amount than when cultured alone. In addition, in order to adjust the growth balance of these lactic acid bacteria so that GABA production by JCM20101 is maximized and the growth of Lactobacillus helveticus is moderately suppressed, the temperature conditions should be 27 to 37°C. is preferable, more preferably 30 to 35°C, further preferably 32 to 34°C.

In addition, when Lactobacillus helveticus grows actively, the pH of the medium rapidly decreases, and the GABA conversion efficiency by the action of glutamic acid decarboxylase decreases. It is desirable to perform co-cultivation under the above temperature conditions.

Co-culture in this embodiment can be carried out by inoculating the two types of lactic acid bacteria into the medium and culturing them under the temperature conditions. Here, the culture method is not particularly limited, and for example, a stationary culture method, a shaking culture method, a stirring culture method, etc. can be adopted. The culture time may be changed depending on the GABA concentration in the medium, but it can usually be 16 hours or more, preferably 16 hours or more and 72 hours or less, and more preferably 16 hours or more and 48 hours or less. preferable. If manufacturing cost and time permit, the culture time is preferably 40 hours or more and 48 hours or less, but it can also be 16 hours or more and 30 hours or less, 16 hours or more and 24 hours or less, or 20 hours or more and 24 hours or less. .

By co-cultivating in this way, a GABA-rich composition can be obtained as a culture. This GABA-rich composition has a GABA concentration after 24-hour culture, preferably 100 mg/100 ml or more, more preferably 200 mg/100 ml or more, even more preferably 300 mg/100 ml or more, particularly preferably 400 mg/100 ml or more, Particularly preferably 500 mg/100 ml or more.

The GABA-rich composition can be used in foods and drinks such as fermented milk, milk drinks, lactic acid bacteria drinks, and soft drinks. The GABA-rich composition contains not only GABA but also peptides and free amino acids which are generally excised from proteins in the medium by the action of protease possessed by Lactobacillus helveticus. For example, casein-degrading peptides and various free amino acids are known to have various physiological activities, and therefore various benefits can be expected by ingesting the GABA-rich composition as a food or drink.

According to this embodiment, by co-cultivating Lactococcus lactis JCM20101 and Lactobacillus helveticus at 27 to 37°C in a milk-based medium, a significantly high GABA content can be achieved in a short period of time. Food and drink products can be manufactured at low cost.

The present invention will be specifically explained below by giving Examples and Comparative Examples. In addition, in the following, "%" means weight % (w/w) unless otherwise specified.

(Test Example 1) Selection of GABA-producing Lactic Acid Bacteria Lactococcus lactis strains shown in Table 1 that have the potential to produce GABA were obtained from the RIKEN BioResource Center (JCM), and their GABA-producing abilities were compared.

-Culture conditions The above eight strains were cultured to produce GABA. That is, GLYP medium, which is a general-purpose medium for L. lactis (per 1 L, glucose 5.0 g, lactose 5.0 g, yeast extract 10.0 g, polypeptone 5.0 g, sodium acetate trihydrate 2.0 g, magnesium sulfate heptahydrate) 0.2 g of manganese sulfate tetrahydrate, 0.01 g of iron sulfate heptahydrate, 0.01 g of sodium chloride; pH 6.8) were inoculated with 10% of the above bacterial strain. It was cultured alone for 24 hours at an appropriate temperature.

・Screening by thin layer chromatography The culture of each bacterial strain obtained above was centrifuged (9000 rcf x 15 minutes) to collect the supernatant, filtered through a 0.45 μm membrane to use as a sample, and subjected to thin layer chromatography. GABA was detected by photography. The composition of the developing solution was n-butanol: glacial acetic acid: pure water = 3:2:1, and it was heated at 100° C. for 10 minutes before use. As a result, GABA was detected in three strains: JCM20101, JCM20399, and JCM20312.

- Quantitative analysis by HPLC Subsequently, detailed GABA concentrations in the cultures of the three strains mentioned above were measured by HPLC method. That is, the culture solution was centrifuged to collect the supernatant, and ethanol was mixed with it to create a 75% ethanol fraction. This fraction was subjected to derivatization treatment by adding phenylisothiocyanate and triethylamine, and the lower layer was filtered through a 0.45 μm membrane and used as an analytical sample for HPLC analysis. HPLC conditions are as follows.

(HPLC conditions)
Column: Wakosil-PTC φ4.0mm x 200mm
Wavelength: 254nm
Elution solvent: Amino acid eluent A (acetonitrile:water = 6:94)
Amino acid eluent B (acetonitrile:water = 60:40)
Flow rate: 1ml/min
Column temperature: 40℃

-Results Table 2 shows the measurement results by HPLC. Although it was a close difference, JCM20101 was found to have the highest GABA production ability.

(Test Example 2) Co-culture test with Lactobacillus helveticus For the three strains of Lactococcus lactis that were found to produce GABA in Test Example 1, a co-culture test with Lactobacillus helveticus was conducted, and the amount of GABA produced was compared the impact on As Lactobacillus helveticus having protease-producing ability, JCM1004 obtained from RIKEN BioResource Center (JCM) was used.

・Method In pre-culture, each strain of Lactococcus lactis (JCM20101, JCM20399, JCM20312) was inoculated at 2% and cultured alone at 33°C for 24 hours to obtain a bacterial solution. Regarding Lactobacillus helveticus, 1% was inoculated into a 10% skim milk medium, and cultured alone at 33° C. for 24 hours to obtain a bacterial solution. For co-culture, 1% Lactococcus lactis and 1% Lactobacillus helveticus were added to 10% skim milk medium supplemented with 2% GluNa, and co-cultured at 33°C for 24 hours. went. As a control, monoculture of Lactococcus lactis alone was carried out under the same conditions except that the amount of Lactococcus lactis inoculated was 2%. For each culture obtained above, the GABA concentration was measured by HPLC method. HPLC analysis was performed in the same manner as in Test Example 1.

-Results The results are shown in Table 3 and Figure 1. In FIG. 1, the vertical axis shows the GABA production amount (mg/100ml) after 24-hour culture, and the horizontal axis shows the test plot. When co-cultivated with Lactobacillus helveticus, all strains of Lactococcus lactis exhibited a GABA-enhancing effect approximately 5 times greater in 24 hours than when cultured alone. In particular, JCM20101 showed a remarkable GABA-enhancing effect of 90 times or more upon co-culture (Table 3, Fig. 1). In the following test examples, JCM20101 was used as Lactococcus lactis having GABA-producing ability.

(Test Example 3) Co-culture test of Lactococcus lactis JCM20101 and Lactobacillus helveticus Using the combination of Lactococcus lactis JCM20101 and Lactobacillus helveticus JCM1004, which had the best results in Test Example 2, two types of microorganisms were used in the culture medium. Changes in the amount of GABA produced were investigated by changing the amount of addition.

・Method For pre-culture, Lactococcus lactis JCM20101 was inoculated into 10% skim milk medium supplemented with 1% glucose, and Lactobacillus helveticus JCM1004 was inoculated into 10% skim milk medium, and cultured alone at 33°C for 24 hours. , and a bacterial solution. The lactic acid bacteria concentrations of the obtained bacterial solutions were all 1×10 ⁹ to 1×10 ¹⁰ cells/ml. Next, the above bacterial solution was inoculated in the amount shown in Table 4 below into a 10% skim milk medium supplemented with 2% GluNa, and co-cultured at 33°C or 37°C for 24 hours. Regarding the obtained culture, the GABA concentration was measured by the HPLC method in the same manner as in Test Example 1. As a control, a culture in which 1% of JCM20101 was inoculated and cultured alone at 33°C or 37°C was used.

-Results and discussion The results are shown in Table 4 and Figure 2. In FIG. 2, the vertical axis shows the GABA production amount (mg/100ml) after 24 hours of culture, and the horizontal axis shows the test plot (inoculation amount of JCM20101) (the inoculation amount of Helveticus bacteria was fixed at 1%). Moreover, the white bar graph shows the case of co-culture at 33°C, and the black bar graph shows the case of co-culture at 37°C.

As a result of co-culturing JCM20101 and Lactobacillus helveticus in various amounts added to the medium, the amount of GABA produced increased significantly. In particular, when the inoculation amount of Lactobacillus helveticus JCM1004 is fixed at 1%, the amount of GABA produced is highest when the inoculation amount of Lactococcus lactis JCM20101 is 1% and the culture temperature is 33°C, and JCM20101 monoculture It increased 90 times compared to the time (control) (Table 4, Figure 2). Furthermore, when the inoculum amount of JCM20101 was fixed at 10%, the amount of GABA produced was highest when the inoculum amount of JCM1004 was 10% (Table 4). In addition, when the ratio of the inoculum amount of JCM20101 and JCM1004 is 1:1, when the culture temperature is 33°C, the GABA production amount is higher when the inoculum amount to the JCM1004 medium is 1% than when it is 10%. However, when the culture temperature was 37°C, the amount of GABA produced was higher when the inoculum amount of JCM1004 was 10% (Table 4).

When the inoculum amount of JCM1004 was fixed at 1%, the amount of GABA produced was higher when the culture temperature was 33°C than at 37°C. However, when the inoculum amount of JCM1004 was 5% or more, GABA production was greater at a culture temperature of 37°C than at 33°C. However, since Helveticus bacteria has a higher lactic acid production ability than Lactis bacteria and has a stronger ability to lower pH, if the amount of JCM1004 inoculated is 5% or more, the pH will drop immediately and JCM20101 will not be able to sufficiently produce GABA. It was considered. In fact, when the amount of JCM1004 inoculated was 5% or more, the pH of the culture after 24 hours of cultivation was 3.5 or less, and the sour taste was too strong to be suitable for use as a food.

From these results, in this embodiment, when JCM20101 and Lactobacillus helveticus are co-cultivated, the amount of GABA produced is significantly greater than when culturing them alone, regardless of the amount of the two types of lactic acid bacteria added to the medium (amount of inoculation). was shown to increase.

The amount of JCM20101 added to the medium is preferably 0.2 to 10%, more preferably 0.5 to 6%, even more preferably 1 to 5%, and the highest GABA-enhancing effect is particularly achieved when the amount is 1 to 4%. was shown to be obtained.

Further, the amount of Lactobacillus helveticus added to the medium can be 0.1 to 10%, preferably 0.1 to 4%, more preferably 0.3 to 3%, even more preferably 0.5%. It was shown that the highest GABA-enhancing effect can be obtained especially when the concentration is 1 to 2%.

(Test Example 4) GABA production test in various protease-treated media The Helveticus bacteria used for co-culture in the above test examples are generally known to have high protease activity. Therefore, the GABA-enhancing effect when a protease-treated medium was used instead of Helveticus was investigated.

- Materials Protease HF "Amano" and Protease M "Amano" manufactured by Amano Enzyme Co., Ltd. were used as protease preparations. Lactococcus lactis JCM20101 was used as a GABA-producing bacterium.

・Method For pre-culture, Lactococcus lactis JCM20101 was inoculated into 10% skim milk medium supplemented with 1% glucose, and Lactobacillus helveticus JCM1004 was inoculated into 10% skim milk medium, and cultured alone at 33°C for 24 hours. This was used as a bacterial solution. Next, 0.1% of protease HF or protease M, which is the recommended addition amount, was added to the 10% skim milk medium supplemented with 2% GluNa, and protease treatment was performed at 50° C. for 24 hours. Thereafter, heat treatment was performed at 90° C. for 1 minute to stop the enzyme reaction, and a protease-treated medium was prepared. This protease-treated medium was inoculated with 2% of the JCM20101 bacterial solution prepared above, and cultured at 33°C for 24 hours.

Regarding the obtained culture, the GABA concentration was measured by the HPLC method in the same manner as in Test Example 1. As a control, 2% JCM20101 bacterial suspension and 1% Lactobacillus helveticus JCM1004 bacterial suspension were added to 10% skimmed milk medium supplemented with 2% GluNa without protease treatment, and a culture was co-cultured at 33°C for 24 hours. was used.

-Results and discussion The results are shown in Table 5. In the medium supplemented with Protease M, the amount of GABA produced increased to some extent, although not as much as in the case of co-culture (Table 5). However, in cultures using protease-treated media, whey and casein were completely separated. I also felt a bitter taste, probably due to too much decomposition of the milk protein. Thus, when a protease-treated medium was used, it was not suitable for use as a food product.

On the other hand, in the case of co-cultivation, no separation of the culture was observed, the texture was like that of fermented milk with a loose curd, and there was no problem in terms of flavor. Therefore, when considering the use as food (particularly fermented milk), it was found that the method of co-cultivation with Helveticus bacteria is more suitable than the protease preparation.

(Test Example 5) Co-culture test of Lactococcus lactis JCM20101 with other than Lactobacillus helveticus So far, it has been shown that co-culture of JCM20101 and Helveticus bacteria brings about a GABA-enhancing effect. Therefore, it was investigated whether the effect of increasing GABA production could be obtained by co-cultivating JCM20101 with lactic acid bacteria other than Helveticus.

- Materials As lactic acid bacteria, A) Lactococcus lactis JCM20101, B) Lactobacillus helveticus JCM1004, C) Lactobacillus gasseri JCM1025, and D) Lactobacillus casei 431 were used. All JCM strains were obtained from RIKEN BioResource Center (JCM). Lactobacillus casei 431 was obtained from Nozawa Gumi Co., Ltd.

- Method In pre-culturing, JCM20101 was cultured at 33°C for 24 hours in a 10% skim milk medium supplemented with 1% glucose to obtain a bacterial solution. JCM1025 was cultured in a 10% skim milk medium at 37°C for 24 hours to obtain a bacterial solution. Lactobacillus helveticus JCM1004 and Lactobacillus casei 431 were cultured alone in a 10% skim milk medium at 30°C for 24 hours to obtain bacterial solutions. Next, the bacterial suspension prepared above was inoculated into a 10% skim milk medium supplemented with 1% GluNa in the combinations and amounts shown in Table 6 below, and co-cultured at 33°C or 37°C for 24 hours. Regarding the obtained culture, the GABA concentration was measured by the HPLC method in the same manner as in Test Example 1.

-Results and discussion The results are shown in Table 6. In co-cultivation of JCM20101 and Helveticus bacteria, the GABA enhancement effect was remarkable when the culture temperature was 33°C. On the other hand, in co-culture with pairs other than Helveticus bacteria, no increase in GABA production was observed at any temperature range (Table 6).

The optimal growth temperatures for Lactococcus lactis and Helveticus are different, with the former being around 30°C and the latter around 37°C. The combination of lactic acid bacteria of this embodiment is advantageous in that growth control such as increasing the activity of only one of the bacteria can be easily performed by adjusting the culture temperature.

On the other hand, co-culture with Lactobacillus casei, which is generally said to have high protease activity, did not show any GABA enhancing effect (Table 6). This may be because various factors, such as insufficient culture time and progress of acid production, are different from those of Helveticus bacteria, and as a result, the GABA converting enzyme of JCM20101 does not work, and GABA production does not proceed. .

In addition, Lactobacillus gasseri does not have the ability to produce proteases and cannot release glutamic acid, the raw material for GABA production, from proteins contained in the culture medium, so even if co-cultured with JCM20101, the amount of GABA produced will increase. It is thought that it was not connected.

From the above results, it was shown that Lactobacillus helveticus is suitable as a pair of JCM20101 in the co-culture of this embodiment.

(Test Example 6) Effect of changes in culture temperature in co-culture on GABA production amount The culture temperature in co-culture of Lactococcus lactis JCM20101 and Lactobacillus helveticus was varied to examine the effect on GABA production amount.

- Materials Lactococcus lactis JCM20101 and Lactobacillus helveticus JCM1004 were used as lactic acid bacteria.

・Method: For pre-culture, JCM20101 was inoculated into 10% skim milk medium supplemented with 1% glucose, and JCM1004 was inoculated into 10% skim milk medium, and the lactic acid bacteria were activated by culturing them alone at 33°C for 24 hours. The resulting material was used as a bacterial solution. Next, 1% each of JCM20101 and JCM1004 bacterial solutions were inoculated into a 10% skim milk medium supplemented with 2% GluNa, and co-cultured for 24 hours under the temperature conditions shown in Table 7 and FIG. 3. Regarding the obtained culture, the GABA concentration was measured by the HPLC method in the same manner as in Test Example 1.

-Results and discussion The results are shown in Table 7 and Figure 3. In FIG. 3, the vertical axis shows the GABA production amount (mg/100ml) after 24 hours of culture, and the horizontal axis shows the culture temperature (°C). As a result, an increase in the amount of GABA produced by co-cultivation was observed at culture temperatures from 30°C to 40°C (Table 7, Figure 3). At culture temperatures below 25°C and above 40°C, the amount of GABA produced up to 24 hours of culture was relatively low, but this is because Helveticus bacteria cannot grow at low temperatures, and at high temperatures. It seemed that the effect was that Lactis bacteria could no longer grow (or it took a long time).

The flavor, appearance, etc. of the culture were as follows. At temperatures below 25°C, it took time for the bacteria to grow, and no acid coagulation was observed within 24 hours, but the umami flavor was felt due to the presence of GluNa. At 30-45°C, fermentation progressed in 24 hours, resulting in a loose curd-like texture like fermented milk. The flavor caused by GluNa was also felt. Although data is not shown, at culture temperatures above 45°C, fermentation was sufficient within 24 hours and acid coagulation was observed, but the characteristic flavor of Helveticus bacteria (acrid or bitter flavor) was felt. Ta.

As shown in Table 6 of Test Example 5, it was thought that at around 33°C, the growth balance of the two types of microorganisms (balance of supply and demand of nutrients) was particularly good, and the amount of GABA produced was also increased. In addition, in this temperature range, the flavor, appearance, etc. of the culture were at a level that did not pose any problem as a food or drink (fermented milk). Therefore, the temperature conditions during co-cultivation in this embodiment can be 27 to 40°C, preferably 27 to 37°C, more preferably 30 to 37°C, even more preferably 30 to 35°C, and even more preferably 32 to 34°C. ℃ was shown to be particularly preferred.

(Test Example 7) Examination of optimal glutamate concentration in culture medium In order to examine optimal medium conditions, a co-cultivation test was conducted in media with different glutamate concentrations. Lactococcus lactis JCM20101 and Lactobacillus helveticus JCM1004 were used as lactic acid bacteria.

・Materials A 10% skimmed milk medium containing GluNa was prepared by adding 0% (control), 0.25%, 1%, 2%, 3%, and 5% of monosodium glutamate (GluNa) to 10% skimmed milk. .

・Method For pre-culture, JCM20101 was inoculated into 10% skim milk medium supplemented with 1% glucose, and JCM1004 was inoculated into 10% skim milk medium, and cultured alone at 33°C for 24 hours to obtain a bacterial solution. . Next, 1% each of JCM20101 and JCM1004 bacterial solutions were inoculated into the prepared 10% skim milk medium supplemented with GluNa at each concentration (0 to 5%), and co-cultured at 33°C for 24 hours. Regarding the obtained culture, the GABA concentration was measured by the HPLC method in the same manner as in Test Example 1.

-Results The results are shown in Table 8 and Figure 4. In FIG. 4, the vertical axis shows the GABA production amount (mg/100ml) after 24 hours of culture, and the horizontal axis shows the GluNa concentration. As a result, at all GluNa concentrations from 0% to 5%, the GABA production enhancing effect by co-culture was observed. The amount of GABA produced did not increase depending on the GluNa concentration, and the amount of GABA produced reached the maximum when the GluNa concentration was 3%.

The decrease in GABA production when GluNa exceeded 3% was thought to be due to growth inhibition due to the increase in salt concentration. That is, the concentration of glutamic acid, which is a raw material for GABA, increased, but the concentration of sodium also increased, so it was thought that the salt killed the lactic acid bacteria or stopped their growth before they produced GABA.

Furthermore, even with 0% GluNa, GABA production progressed, albeit in a small amount, because the bacterial solution added at the start of co-cultivation contained a small amount of GluNa, which may have been used as a raw material for GABA production by JCM20101.

Furthermore, if the amount of GluNa added is too large, the glutamine odor and salty taste will become strong, making the product unsuitable for use as a food product. Therefore, it was considered that the GluNa concentration in the medium is preferably 0.25 to 2%, and more preferably 0.25 to 1%.

(Test Example 8) Effect of change in culture time in co-culture on GABA production amount The effect on GABA production amount was investigated by changing the culture time in co-culture of Lactococcus lactis JCM20101 and Lactobacillus helveticus.

- Materials Lactococcus lactis JCM20101 and Lactobacillus helveticus JCM1004 were used as lactic acid bacteria.

- Method In pre-culture, JCM20101 was cultured alone in a 10% skim milk medium supplemented with 1% glucose, and JCM1004 was cultured alone in a 10% skim milk medium at 33°C for 24 hours to obtain a bacterial solution. Next, 1% each of JCM20101 and JCM1004 bacterial solutions were inoculated into a 10% skim milk medium supplemented with 2% GluNa, and co-cultivation was started at 33°C. GABA concentrations were measured by HPLC in the same manner as in Test Example 1 for cultures collected at the start of culture (0 hours) and 8, 12, 16, 24, 48, and 72 hours after the start of culture.

-Results and discussion The results are shown in Table 9 and Figure 5. In FIG. 5, the vertical axis shows the GABA production amount (mg/100ml), and the horizontal axis shows the culture time (h). As a result, the amount of GABA produced finally started to increase after about 16 hours of culture, and a rapid increase in the amount of GABA produced was observed between 16 and 24 hours of culture, but the rate of increase rapidly decreased after 48 hours of culture ( Table 9, Figure 5).

Therefore, the culture time for co-culture can be 16 hours or more, preferably 16 to 24 hours from a cost-effective perspective, but if manufacturing costs and time permit, culture for 48 to 72 hours. By doing so, a very high amount of GABA can be produced.

Although the embodiments and examples of the present invention have been described above in detail with reference to the drawings, the specific configuration is not limited to these, and design changes that do not depart from the gist of the present invention may be made. include.

For example, in the above example, JCM20101 was used as Lactococcus lactis having GABA-producing ability, but derivative strains of JCM20101 may also be used if a significant GABA-enhancing effect can be obtained by co-culturing them with Lactobacillus helveticus. can be used as the GABA-producing bacterium in the present invention.

Further, for example, in the above embodiment, skim milk was used as the medium, but the medium is not limited to this, and milk with various sterilization histories such as LL milk, HTST milk, and UHT milk, and unpasteurized raw milk may also be used. can be used as a co-culture medium in the present invention. Furthermore, a medium containing milk as a main component such as animal milk or whey other than cow's milk, or a synthetic medium such as GLYP medium can also be used as a medium for co-cultivation in the present invention.

Claims (11)

A method for producing a composition with a high content of γ-aminobutyric acid, which comprises co-cultivating Lactococcus lactis JCM20101 and Lactobacillus helveticus at 27 to 37°C. The method for producing a composition with a high content of γ-aminobutyric acid according to claim 1, wherein the Lactobacillus helveticus is Lactobacillus helveticus JCM1004. The method for producing a composition with a high content of γ-aminobutyric acid according to claim 1 or 2, wherein the co-cultivation is performed in a medium containing milk as a main component. The amount of inoculation of Lactococcus lactis JCM20101 to the medium is 0.2 to 10% (w/w), and the amount of inoculation of Lactobacillus helveticus to the medium is 0.1 to 10% (w/w). The method for producing a composition with a high content of γ-aminobutyric acid according to claim 3. The method for producing a composition with a high content of γ-aminobutyric acid according to claim 3, wherein the medium contains 0.25 to 10% (w/w) of glutamate. The method for producing a composition with a high content of γ-aminobutyric acid according to claim 4, wherein the medium contains 0.25 to 10% (w/w) of glutamate. The method for producing a composition with a high content of γ-aminobutyric acid according to claim 1 or 2, wherein the composition with a high content of γ-aminobutyric acid is a food or drink product with a high content of γ-aminobutyric acid. 4. The method for producing a composition with a high content of γ-aminobutyric acid according to claim 3, wherein the composition with a high content of γ-aminobutyric acid is a food or drink product with a high content of γ-aminobutyric acid. 5. The method for producing a composition with a high content of γ-aminobutyric acid according to claim 4, wherein the composition with a high content of γ-aminobutyric acid is a food or drink product with a high content of γ-aminobutyric acid. The method for producing a composition with a high content of γ-aminobutyric acid according to claim 5, wherein the composition with a high content of γ-aminobutyric acid is a food or drink product with a high content of γ-aminobutyric acid. The method for producing a composition with a high content of γ-aminobutyric acid according to claim 6, wherein the composition with a high content of γ-aminobutyric acid is a food or drink product with a high content of γ-aminobutyric acid.