KR20160112227A - Method for production of GABA with lactic acid bacteria through L-glutamic acid feeding - Google Patents

Abstract

The present invention relates to a method for producing gamma-aminobutyric acid (GABA), by culturing lactobacillus in a culture medium containing L-glutamic acid and then injecting solid glutamic acid in a batch-feeding type, in accordance with pH after a certain period of time. According to the present invention, it is possible to produce high-concentration and high-purity GABA efficiently.

Description

[0001] The present invention relates to a method for producing L-glutamic acid from a lactic acid bacterium by feeding L-

The present invention relates to a method for producing gamma aminobutyric acid (hereinafter referred to as " GABA ") at a high concentration from lactic acid bacterium by injecting glutamic acid into a feed system. More specifically, a method of efficiently producing a high concentration and high purity of GABA by culturing lactic acid bacterium in a medium containing L-glutamic acid, adding a glutamic acid in a solid state to a fermentation system in conjunction with pH after a lapse of a predetermined time .

      'GABA' is an abbreviation of 'γ-Aminobutyric acid (GABA)'. It is a nonprotein constituent amino acid. -COOH bonded to α-carbon of L-glutamic acid is glutamic acid decarboxylase decarboxylase (GAD).

GABA is contained in the human nervous system and blood, and most of them are present in the bone marrow of the brain, increasing the neurotransmitter called acetylcholine, and physiological functions such as promoting brain function. In addition, long-term use of blood pressure lowering effect, diuretic effect, antidepressant, antioxidant, weight loss, and alcohol poisoning treatment have been known. Gabba acts as a neurotransmitter in non-human higher animals, invertebrates and insects, is widely distributed in the plant kingdom, and is found in most tissues of plants.

On the other hand, a method for industrially producing Gabas includes a synthesis method, an enzymatic method, and a fermentation method.

The 'synthesis method' is advantageous in that the reaction is simple and the cost is lower than other methods. However, since the solvent used is not suitable for the use of the food, the use of the solvent is limited only for the use of the food.

The 'enzyme method' is a method of converting L-glutamic acid (hereinafter referred to as GA) into decarboxylated GABA by catalytic action of glutamic acid decarboxylase (GAD). The enzyme may be a purified enzyme, or a GAD-containing crude enzyme solution obtained by culturing a lactic acid bacterium and then disrupting the cell membrane may be used. At this time, the production cost of the enzyme can be remarkably reduced by mass-expressing GAD derived from foreign microorganisms in E. coli and recovering the crude enzyme solution. However, when an enzyme derived from a recombinant strain is used for a food, it is not preferable because it is troublesome to verify the safety and obtain permission from the related organization.

On the other hand, 'fermentation method' is to produce gaba through fermentation of microorganisms, and fermentation method using lactic acid bacteria is representative. Since lactic acid bacteria are recognized as GRAS (Generally Recognized As Safe), there is a great advantage in that GABA produced by lactic acid fermentation has no restriction in use for food use.

The method using lactic acid bacteria fermentation also uses glutamic acid decarboxylase (GAD) produced through lactic acid bacteria culture, and L-glutamic acid (GA) is externally added to the substrate to obtain GABA. Most of the lactic acid bacteria have GAD enzymes. Among them, especially, there are many bacteria with high GAD activity in Lactobacillus (Korean Patent No. 10-0631857). The GAD produced by Lactobacillus is present in the cells. The GA permeates the cell membrane, enters the cell, is converted to GABA by GAD, and the converted GABA is exchanged with GA 1: 1. It goes through.

      On the other hand, conventionally, as a method for obtaining high concentration and high purity of GABA, L-glutamic acid is converted to gamma aminobutyric acid by an enzyme reaction, and a reaction solution containing gammaaminobutylic acid at a high concentration is purified to obtain high purity gammaaminobutyric acid (Korean Patent No. 10-0857215). However, this method requires disruption of the cell membrane or the use of purified enzyme. When the reaction time is prolonged, the activity of glutamic acid decarboxylase (GAD) sharply drops, so that the enzyme reaction must be completed in a short period of time.

On the other hand, the production of GABA by lactic acid fermentation does not require cell membrane disruption and has a great advantage that it can be used for food. In order to economically produce high concentration of GABA, L-glutamic acid (GA) Sodium glutamate (MSG) must be added in large amounts during the initial or during the incubation. For fed-batch fermentation, the substrate is usually dissolved in water and fed to the fermenter in aqueous solution. However, the solubility of L-glutamic acid (GA) is low at about 3 g / l at room temperature, so that it is impossible to carry out fed-batch culture in which feeding solution of a high concentration is required. Therefore, there is a fundamental problem in which productivity and economical efficiency are low.

(MSG), which is high in solubility, was added several times during fermentation as an alternative to L-glutamic acid (GA), but the amount of GABA produced in 60 hours was only 11.1% (Haixing Li et al. , Microbial Cell Factories - MICROB CELL FACT, vol. 9, no. 1, pp. 85-7, 2010).

The reason for using MSG instead of GA in the above paper is that MSG has a high solubility in water of 740 g / l, which is advantageous in that it can be put into a sterilized liquid state. Generally, when carbon dioxide is generated during the conversion of GA to GABA, the pH of the culture solution rises to 6.0 or higher and the conversion reaction to GABA is stopped or decelerated. To maintain the conversion reaction, the pH of the culture solution should be lowered to about 5.0 do. However, when MSG is added, the pH is rather increased, and additional acid should be added. However, when an acid is added to adjust the pH of the reaction solution, a large amount of salt is generated in proportion to the content of the produced GABA, so that a high concentration or high purity of GABA can not be obtained.

In addition, this method requires a purification process such as an ion exchange resin process and recrystallization in order to remove the generated salt, and involves a problem such as yield reduction and generation of waste water. It is. That is, in the conventional method using lactic acid fermentation, the content of Gabba in the solid content could not exceed 50%, and the purity of the product did not exceed 20%.

Korean Patent No. 10-0857215 (registered on September 1, 2008), in which the present applicant has registered, converts L-glutamic acid into gamma aminobutyric acid by an enzyme reaction and purifies the reaction solution containing gamma aminobutyric acid in a high concentration A process for producing high purity gamma aminobutyric acid is described.

Production of gamma-aminobutyric acid by Lactobacillus brevis NCL912 using fed-batch culture method ", Microbial Cell Factories - MICROB CELL FACT, vol 9, no. 1, pp. 85-7, Lactobacillus brevis NCL912 using fed-batch fermentation) "is described.

The present invention relates to a method for efficiently producing a high concentration and high purity of GABA which is suitable for use in foods by using lactic acid bacteria fermentation and which is in a 'solid state' in which the lactic acid bacteria live or are in a resting period and relatively long- L-glutamic acid (GA) of the present invention is added in an equilibrium manner, thereby providing a method for producing high-purity and high-purity Gabas.

The present invention relates to a method for producing γ-aminobutyric acid (GABA) by inoculating a culture medium with a lactic acid bacterium inoculated with the culture medium, wherein the culture is carried out by supplying solid medium L-glutamic acid continuously or intermittently to the medium And maintaining the pH of the culture medium at 3.5 to 6.0. The present invention also provides a method for producing gamma aminobutyric acid. FIG. 1 shows a process for the production of GABA of the present invention. In the case of a conventional GABA product using lactic acid fermentation, the GABA content is only 20%. However, when the fed-batch culture using solid state L-glutamic acid according to the present invention is used, A product of 80 to 99% in content can be efficiently produced.

When the pH of the medium is maintained at 3.5 to 6.0 as in the present invention, the lactic acid bacteria absorb L-glutamic acid into the cells, and the absorbed L-glutamic acid is converted to glutamic acid decarboxylase (GAD) After the conversion, the cells are discharged to the outside of the cell. The pH range of 3.5 ~ 6.0 is acidic, and the growth of other contaminants is not smooth at this range of pH. Therefore, even if 'L-glutamic acid' having a very low solubility is supplied to a medium in a solid state (preferably powder) rather than a liquid state, contamination of the culture liquid does not occur.

The ability to add L-glutamic acid (GA) to the medium in a solid state, rather than a liquid phase, is a great advantage in terms of productivity, because it allows fed-batch culture. Fed-batch culture is a culture method that continuously or intermittently feeds a substrate during culture to maximize the production of reactants. In order to maximize the production of reactants, the substrate must be continuously fed into the medium. In the case of general oil-price cultivation, substrates are sterilized by dissolving the substrate in a solution (water) at a high concentration due to contamination problems, and then supplied to the medium in a sterilized state.

However, in the case of 'L-glutamic acid', since the solubility thereof is remarkably low, there is a problem that it can not be fed in a liquid phase. However, in the case of the present invention, since the pH of the culture medium is maintained in the acidic state of 3.5 to 6.0 during the culture, the load on the strain is less, and L-glutamic acid can be supplied in a solid state. Through the experiments of the present invention, it was confirmed that GABA can be produced at a high concentration using L-glutamic acid in a solid state as a substrate in a state where contamination of the strain is minimized.

Further, in the present invention, since the pH of the culture medium is automatically lowered by adding L-glutamic acid, the lactic acid bacteria can continue to convert L-glutamic acid into GABA without addition of an additional acid. That is, in order for the lactic acid bacteria to convert L-glutamic acid to GABA, the pH should be maintained in an acidic state of 3.5 to 6.0. When L-glutamic acid is added as in the present invention, the pH naturally drops. Therefore, You do not need to.

In the present invention, lactic acid bacteria are used, and since lactic acid bacteria are strains confirmed to be food-compatible, any of them may be used. However, it is preferable to use lactobacillus of the genus Lactobacillus expressing the highly active decarboxylase (GAD). In this case, the Lactobacillus bacteria (Lactobacillus) in lactic acid bacteria is one example, Lactobacillus brevis (Lactobacillus brevis), Lactobacillus hilgardii ), Lactobacillus planta ( Lactobacillus plantarum ).

The medium used in the present invention may be any medium that can grow lactic acid bacteria. However, in order to induce the production of glutamic acid decarboxylase (GAD) in lactic acid bacterium cells, it is preferable that glutamic acid (GA) or glutamate salt is already added to the medium before the start of cultivation. The glutamate salt added to the medium may be K or ammonium salt in addition to the sodium salt. The amount of GA added or GA equivalent of GA salt is preferably 1.0 to 6.0% (w / v), more preferably 3.0 to 4.0% (w / v). When the GA concentration is lower than 1.0% (w / v), the GAD expression level is relatively low, and when the GA concentration is higher than 6.0% (w / v), the growth of the microorganism is inhibited.

In the method for producing gamma aminobutyric acid of the present invention, when the lactic acid bacteria are cultured and the cell concentration is more than 4 as OD ₆₀₀ (optical density at ₆₀₀ nm) in the solid state, L- It is good to supply. That is, when the bacterial concentration of the lactic acid bacteria is measured by absorbance, it is preferable that the absorbance value of OD ₆₀₀ measured at a wavelength of 600 nm is 0.2 or more by diluting the collected culture medium 20 times. Cell density OD ₆₀₀ If L-glutamic acid is supplied in a state where the value is lower than 4, it is undesirable because the yield is lowered.

On the other hand, according to the present invention, when the bacterial concentration of the lactic acid bacteria reaches a concentration of OD ₆₀₀ value of 4 or more, it is resistant to contamination from the outside, and even when GA is opened for 3 to 5 days, No problems were found. It is presumed that this is due to bacteriocin produced by lactic acid bacteria, resistance to contamination of lactic acid bacteria, low pH, high concentration of L-glutamic acid (GA) and GABA.

On the other hand, the supply of L-glutamic acid is more preferably an OD ₆₀₀ It is preferable that the value is 6 to 24, more preferably, the OD ₆₀₀ value is 6 to 16. When L-glutamic acid is supplied in an excessively high OD ₆₀₀ value, the specific activity of glutamic acid decarboxylase (GAD) activity is low, and fermentation period is long, and there is no profit.

The OD ₆₀₀ value of 1.0 corresponds to the wet cell mass of 20 to 30 g / l although it differs depending on the kind of lactic acid bacteria and the growth state.

In the method for producing gamma aminobutyric acid of the present invention, it is preferable that the culture is preferably maintained at pH 3.5 to 6.0 during the culture by adding L-glutamic acid in a solid state. Considering that the growth of lactic acid bacterium cells and the optimal pH of GAD are in the vicinity of 4.5 ~ 5, the pH range during cultivation is preferably 3.5 ~ 6.0.

In the present invention, it is preferable to add L-glutamic acid (GA) when the lactic acid bacteria reaches a predetermined cell density (OD ₆₀₀ value of 4 or more). When the manhole or nozzle of the fermenter is opened and a solid state GA is added, (Preferably from 4.6 to 5.0). When the applied GA is converted to GABA, carbon dioxide is generated and discharged. The pH gradually increases. If the pH exceeds 6.0 (more preferably, the pH exceeds 5.0), it is preferable to lower the pH by adding GA again. At least the pH should be lowered to 6.0 or less to induce the conversion of L-glutamic acid to the GABARA.

The total amount of L-glutamic acid (GA) to be added is preferably 150 to 600 g / l based on the amount of the initial culture, preferably 300 to 500 g / l. If the amount of GA is less than 150 g / l, the economical efficiency is lowered. If the amount of GA is more than 600 g / l, the activity of glutamic acid decarboxylase (GAD) is remarkably decreased and the time for conversion becomes too long.

When the total dosage of GA is about 150 g / l or more, the lactic acid bacteria stops growing and enters the resting period. When the total dosage of GA is over 400 g / l, the lactic acid bacteria start to die, and when it exceeds 600 g / l, the lactic acid bacteria die almost. According to the present invention, the time point at which the lactic acid bacteria die and the time point at which the conversion rate of GA to GABA decrease sharply coincide with each other.

Although the dosage period of the GA varies depending on the kind of lactic acid bacteria and the growth state, it is preferable to add the GA over a period of 18 to 96 hours, preferably 36 to 72 hours. As time goes by, the gas is accumulated, and as the gas is accumulated, the conversion speed of the gas to the gas is gradually lowered.

The method of inputting the GA may be manually performed by the operator several times, or may be automated by constructing a system comprising a hopper, a rotary valve, a feeder, and a control device as shown in Fig. Fig. 3 shows a schematic diagram of a device for automatically supplying glutamic acid powder in solid state in conjunction with pH.

The method of producing gamma aminobutyric acid of the present invention may further include a step of sterilizing the culture medium and spray drying after the supply of L-glutamic acid is completed. In this case, the method may further include a step of removing the lactic acid bacterium cells by filtration after sterilization and before spray drying. If a high purity of GABA is not required and a small amount of L-glutamic acid (GA) may remain in the product, it may be sterilized and spray dried immediately to obtain a powdered product or a crystalline product. At this time, it is possible to obtain a powder or crystalline powder having a grain content of 80% or more and a residual GA of 10% or less.

The method of producing gamma aminobutyric acid of the present invention may further include adding an acid after the supply of L-glutamic acid is completed. Thus, by lowering the concentration of the unreacted GA in the culture medium, the purity of GABA can be increased. The acid can be, for example, an inorganic acid or an organic acid. The method may further include the step of adding an acid to convert the unreacted glutamic acid into GABA, sterilizing the culture liquid, and spray drying. Further, it may further include a step of removing lactic acid bacterium cells through sterilization and filtration before spray drying.

The acid is preferably added at the late stage of fermentation in which the conversion rate of GABARA by the glutamic acid decarboxylase (GAD) is slow, and the conversion rate of L-glutamic acid (GA) to GABARA is approximately 4.5 g / l / Hour " or less. In this case, GA injection is stopped, and instead, acid is added to lower the pH to 4.5 to 5.3, thereby promoting the conversion of remaining GA to GABA in the culture medium. Acids can be both inorganic and organic acids. Specific examples of the inorganic acid include hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid and mixtures thereof. Specific examples of the organic acid include L-aspartic acid, alpha-ketoglutaric acid, lactic acid, citric acid, tartaric acid, fumaric acid, Acetic acid, gluconic acid, 2-ketogluconic acid, 5-ketogluconic acid, kojic acid or a mixture thereof.

When the pH is raised to 5.3 to 5.8 as the conversion to Gabarone, it is advisable to lower the pH by adding acid again. In this manner, the acid is added until the pH is no longer increased. The residual GA concentration was 0.2 ~ 1.2% (w / v) when the pH was no longer increased, and the concentration of GABA was 100 ~ 400g / l though it varied depending on the amount of GA added. At this concentration, the crystallization operation of 1 to 2 times can easily obtain a purity of 98% or more. As an example of the purification process, the method disclosed in the patent 10-0857215 may be used. In other words, a high-purity Gabar product having a total content of 98% or more can be obtained through sterilization, removal of cells, decoloration, concentration and crystallization.

According to the present invention, the lactic acid bacteria fermentation in which L-glutamic acid (GA) in a solid state is added in a cost-effective manner makes it possible to efficiently and economically produce Gabas with high concentration or high purity while being suitable for use in foods.

Fig. 1 shows the manufacturing process of the present invention.
Fig. 2 shows the lactic acid bacteria culturing pattern and the gas generation pattern over time in Example 2 of the present invention. Fig.
Fig. 3 shows a schematic diagram of a device for automatically supplying glutamic acid powder in solid state in conjunction with pH.
Fig. 4 shows the lactic acid bacteria cultivation pattern over time and the fed-on feeding pattern and the Gabba generation pattern of the solid glutamic acid powder in Example 3 of the present invention.
Fig. 5 shows the inflow-feeding pattern and the gaba generation pattern of the lactic acid bacteria cultivation pattern and the sodium glutamate (MSG) with the lapse of time in Example 4 of the present invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples or experimental examples. However, the scope of the present invention is not limited to the following examples or experimental examples, but includes modifications of equivalent technical ideas.

[ Example  One: Lactobacillus Brevis  ( Lactobacillus brevis ) OPK3  ( KCCM  80047)

Lactobacillus brevis ) OPK3 (KCCM 80047) is a lactic acid bacterium isolated from kimchi and is a patent strain excellent in the production of GABA (Korean Patent No. 10-0631857).

500 ml of MRS medium was prepared in a 1 L Erlenmeyer flask, sterilized at 120 ° C for 15 minutes and cooled. Then, Lactobacillus brevis brevis OPK3 (KCCM 80047) were inoculated and cultured at 35 ° C for 15 hours.

On the other hand, in a 30 L fermenter to be used for the experiment, 15 L of MRS medium containing GA according to the initial GA concentration (Table 1) was weighed, adjusted to the pH (Table 1) and sterilized. Subsequently, the medium was cooled, inoculated with seed bacterium, and cultured for 18 hours in an anaerobic state at 32 DEG C and a rotation speed of 100 rpm. The incubation temperature was 35 ° C. The culture results after 18 hours of inoculation with the culture pH and initial GA concentration were as shown in Table 1.

Lactobacillus brevis Comparison of pH and 'initial GA in culture medium' between cultured OPK3 (KCCM 80047) and residual GA concentration
Culture pH (GA concentration = 3.5% (w / v)) Initial GA concentration (pH = 5.0) 4.0 5.0 6.0 2% (w / v) 3.5% (w / v) 5% (w / v) The wet cell concentration (g / l) 16.8 14.3 18.4 14.9 14.3 12.9 Residual GA concentration (g / l) 1.5% 0.5% 2. 0% 0.2% 0.5% 1.3%

[ Example  2: Lactobacillus Flora Room  ( Lactobacillus plantarum )]

500 ml of MRS medium was prepared in a 1 L Erlenmeyer flask, sterilized at 120 ° C for 15 minutes, cooled, and then transferred to Lactobacillus plantarum ) and inoculated at 35 ° C for 15 hours.

Separately, 15 L of MRS medium containing GA 1% (w / v) was prepared and sterilized in a 30 L fermenter. After the medium was cooled, the seeds were inoculated and cultured for 18 hours in an anaerobic state at 35 ° C and an initial pH of 6.0 at a revolution of 100 rpm.

As a result of culturing, a wet cell concentration of 12 g / l and a Gabba concentration of 1800 mg / l were obtained (Fig. 2). Fig. 2 shows the lactic acid bacterium culture pattern and the gaba generation pattern with the lapse of time in Example 2 of the present invention. Fig.

[ Example  3: L-Glutamic acid ( GA )of Oil price formula  High concentration by input Gabba Example  One]

500 ml of the seed culture medium was prepared in the same manner as in Example 1, and Lactobacillus brevis OPK3 (KCCM 80047) was inoculated thereon and cultured at 35 ° C for 15 hours. 15 L of a MRS medium containing a GA concentration of 3.5% (w / v) in a 30 L fermenter was prepared, sterilized and cooled in the same manner as in Example 1, and inoculated with the pre-cultivated seeds at 35 ° C, The culture was started in an anaerobic state at 100 rpm.

After 18 hours of incubation, solid L-glutamic acid (GA) was added again to stop the addition at a pH of about 5.0. When the pH rose to 5.5, GA was added again. This process was repeated, and the fermentation was terminated after 64 hours from the start of the culture.

The pH, the amount of cell mass, the amount of produced GABA and the like with the passage of the fermentation time are shown in Fig. Fig. 4 shows the lactic acid bacteria cultivation pattern over time and the fed-on feeding pattern and the Gabba generation pattern of the solid glutamic acid powder in Example 3 of the present invention. The final fermentation liquid volume was 17.6 liters. The total input GA amount was 5100 g and the generated GABA was 3478 g.

[ Example  4: Of sodium glutamate (MSG) Oil price formula  Due to input Gabba  produce]

15 liters of an MRS medium containing 3.5% (w / v) of GA was prepared in a 30 L fermentor in the same manner as in Example 3, and 500 ml of lactic acid bacteria (Lactobacillus brevis OPK3 (KCCM 80047) Respectively. MSG (Monosodium glutamate) in solid state was added 18 hours after the inoculation. As the pH rose during the incubation, the pH was automatically adjusted to 5.0 using 2N sulfuric acid. After 64 hours from the start of the culture, the fermentation was terminated.

The final amount of fermentation broth was about 19.1L. The total input MSG amount was 5,595 g (4,400 g converted to GA amount) and the generated GABA was 2253 g (Fig. 5). Fig. 5 shows the inflow-feeding pattern and the gaba generation pattern of the lactic acid bacteria cultivation pattern and the sodium glutamate (MSG) with the lapse of time in Example 4 of the present invention.

[ Example  5: L-Glutamic acid ( GA )of Oil price formula  High concentration by input Gabba Example  2]

After 500 ml of the seed culture medium was prepared in the same manner as in Example 1, Lactobacillus heligandi (Lactobacillus hilgardii), And the cells were cultured for 15 hours at 35 ° C. Thereafter, 15 L of an MRS medium containing a GA concentration of 3.5% (w / v) in a 30 L fermenter was prepared, sterilized and cooled in the same manner as in Example 1, and then pre-cultured seeds were inoculated thereinto to start culturing. After 18 hours from the start of the culture, the GA was started to be injected in the same manner as in Example 3.

As a result of the fermentation, the final amount of the fermentation broth was about 17.2 liters. The total input GA amount was 4,450 g and the produced GABA was 2,828 g.

[ Example  6: L-Glutamic acid ( GA )of Oil price formula  High concentration by input Gabba Example  3]

In the same manner as in Example 1, 4000 ml of MRS seed culture medium was prepared by 1 L each in four 2 L flasks, sterilized and cooled, inoculated with Lactobacillus brevis OPK3 (KCCM 80047), and cultured at 35 캜 for 15 hours in a stationary culture Respectively.

250 L of an MRS medium containing a GA concentration of 2.5% (w / v) was prepared, sterilized and cooled in the same manner as in Example 3, and 4 L of pre-cultivated seeds were inoculated in a 500 L fermenter, At a rotation speed of 75 rpm in an anaerobic state.

An OD ₆₀₀ value of 7 was obtained within 15 hours after the start of the culture. From this time, the solid state GA was added and when the pH reached 4.9, the addition was stopped. When the pH rose to 5.6, GA was again added. This process was repeated. Thereafter, the fermentation was terminated when 101 hours had elapsed after the start of the culture. The final amount of fermentation broth was 320L. The total input GA amount was 147kg, and the produced Gaba was 99.2kg.

[ Example  7: High concentration Gabba  From the contained culture medium Gabba  Preparation of Powder]

After completion of the culture, 1 L of the culture solution of Example 3 was taken, and 6 N caustic soda was added to adjust the pH to 6.8. Thereafter, the mixture was heat-treated at a temperature of 80 DEG C for 15 minutes, cooled to 55 DEG C, added with 10 g of diatomaceous earth and 5 g of activated carbon, and stirred for 3 hours. Thereafter, the filter paper was filtered under vacuum using a small size filter. After filtration, a pale brown transparent filtrate was obtained. The filtrate was 32 Bx.

This was spray dried in a small spray dryer to obtain 240 g of brown powder. At this time, the hot air temperature was 180 占 폚 and the exhaust temperature was 95 占 폚. The moisture content of the obtained brown powder was 3.2%, the content of the grains in the solid was 85%, and the GA was 6.3%.

[ Example  8: High concentration Gabba  From the contained culture medium, Gabba  Produce]

The pH of the fermentation broth was adjusted to 4.9 by adding 2N hydrochloric acid to the fermentation broth of Example 3. The pH of the fermentation broth was adjusted to 4.9 after 5 hours and then the pH was adjusted to 4.9 by addition of 2N hydrochloric acid. Respectively. At this time, the residual GA concentration was 0.85%. Thereafter, 1 L of the culture was taken, ammonia was added, and the pH was adjusted to 6.8. Thereafter, the mixture was heat-treated at a temperature of 80 캜 for 15 minutes, cooled to 55 캜, added with 10 g of diatomaceous earth and 5 g of activated carbon, and stirred for 3 hours. Thereafter, the filter paper was filtered under vacuum using a small size filter. Thereafter, the filtrate was diluted 3-fold, and the measured permeability (T%) of the filtrate was 95%.

The filtrate was concentrated to 55 Brix under a vacuum of 700 mmHg using a glass rotary evaporator and transferred to a glass vessel with a lid and jacket. The sample was evaporated at a vacuum degree of 680 mmHg while slowly stirring in a glass vessel. At the time of crystallization, evaporation was stopped, the vacuum was released, cooled and 95% ethanol was added. The final concentration of ethanol was 80%.

The solution was cooled continuously until the temperature of the crystal liquid reached 10 占 폚, and then the solution was desalted by a centrifugal separator and washed twice with a wash solution whose ethanol content was adjusted to 80% (v / v). The wet cake obtained above was dried in hot air dryer at 80 占 폚 for 1 hour with hot air.

The apparent specific gravity of the dried crystals was 0.70, the yield of dried crystals was 71%, and the content of Gabba and ammonium ions in the dried crystals were 99.1% and 185 ppm, respectively.

Claims (12)

In producing gamma-aminobutyric acid (GABA) by inoculating lactic acid bacteria in culture medium and culturing,
The above-
L-glutamic acid in a solid state is continuously or intermittently supplied to the medium during culturing, and the pH of the medium is maintained at 3.5 to 6.0.
The method according to claim 1,
The lactic acid bacteria,
Lactobacillus bacteria (Lactobacillus) in lactic acid bacteria in the method of the gamma amino-butyric acid according to claim production.
3. The method of claim 2,
The Lactobacillus bacteria (Lactobacillus) in lactic acid bacteria,
Lactobacillus brevis , Lactobacillus hilgardii , Lactobacillus < RTI ID = 0.0 & gt; plantarum , and plantarum .
The method according to claim 1,
The above-
Glutamic acid or glutamic acid salt at a concentration of 1 to 6% (w / v) in terms of L-glutamic acid prior to initiation of culture.
The method according to claim 1,
The solid-state L-glutamic acid may be,
When the lactic acid bacterium is a culture cell density exceeds 4 to OD ₆₀₀ value, the method of production of gamma-amino-butyric acid, comprising a step of supplying the culture medium.
The method according to claim 1,
The above-
Wherein the pH of the culture medium is kept at 3.5 to 6.0 during the culture by adding L-glutamic acid in a solid state.
The method according to claim 1,
The method of producing gamma-aminobutyric acid comprises:
After the supply of L-glutamic acid is terminated,
A method for producing gamma aminobutyric acid, which comprises sterilizing the culture medium and spray-drying it.
8. The method of claim 7,
The method of producing gamma-aminobutyric acid comprises:
And removing lactic acid bacterium cells by filtration after sterilization and spray drying.
The method according to claim 1,
The method of producing gamma-aminobutyric acid comprises:
Wherein the acid is further added after the supply of the L-glutamic acid is completed.
10. The method of claim 9,
Preferably,
Wherein the aminobutyric acid is an inorganic acid or an organic acid.
10. The method of claim 9,
The method of producing gamma-aminobutyric acid comprises:
Acid was added to convert unreacted glutamic acid to GABA,
A method for producing gamma aminobutyric acid, which comprises sterilizing the culture medium and spray-drying it.
12. The method of claim 11,
The method of producing gamma-aminobutyric acid comprises:
And removing lactic acid bacterium cells by filtration after sterilization and spray drying.

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