JP2007049910A - Method for producing gamma-aminobutyric acid and production apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To increase the conversion ratio from glutamic acid or glutamic acid salt to γ-aminobutyric acid without adding a particular coenzyme. <P>SOLUTION: γ-Aminobutyric acid is produced batchwise by adding glutamic acid or a glutamic acid salt to a solution incorporated with a material containing glutamic acid decarboxylase to effect decarboxylation reaction with the glutamic acid decarboxylase. The addition of glutamic acid or glutamic acid salt in the batch reaction process is carried out dividedly to keep the substrate concentration of glutamic acid or glutamic acid salt below a warning level. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a production method and production apparatus for γ-aminobutyric acid.

  γ-Aminobutyric acid is an amino acid having functions such as an antihypertensive action, an arteriosclerosis inhibiting action, and a tranquilizing action, and has attracted attention as a health food ingredient and a medical ingredient. As a method for producing γ-aminobutyric acid, a method in which a material containing glutamic acid decarboxylase is added to a glutamic acid or glutamic acid solution is known. According to this method, the carboxyl group of glutamic acid or glutamate is decarboxylated by the catalytic action of glutamic acid decarboxylase to produce γ-aminobutyric acid. Known materials containing glutamic acid decarboxylase include rice germ, rice bran containing germ, pumpkin, carrot and other plants, and lactic acid bacteria and koji molds.

  Patent Document 1 discloses a method for producing γ-aminobutyric acid using rice germ or rice bran containing germ as a glutamate decarboxylase source. Rice bran and rice bran containing embryos are discharged in large quantities as a by-product during rice milling and have the advantage that they can be obtained at low cost. This Patent Document 1 also discloses that pyridoxal phosphate, which is a coenzyme, is added to the glutamate decarboxylase source to increase the amount of γ-aminobutyric acid produced.

Patent Document 2 also discloses a method for producing γ-aminobutyric acid using rice bran as a glutamic acid decarboxylase source. At that time, a yeast containing a small amount of pyridoxalphosphate which is a coenzyme is added, and γ-aminobutyric acid is added. It is described that the production amount of is increased.
JP 2000-201651 A Japanese Patent Laid-Open No. 2003-245093

  In the method described in Patent Document 1, the conversion rate to γ-aminobutyric acid may be remarkably reduced depending on the concentration of glutamic acid or glutamate as a substrate. This drawback can be remedied by adding a coenzyme pyridoxal phosphate. However, pyridoxal phosphate is not currently recognized as a food additive, and γ-aminobutyric acid for foods using pyridoxal phosphate cannot be produced. In the future, even when pyridoxal phosphate is recognized as a food additive, a method of consuming expensive pyridoxal phosphate is not a good idea. Since the method described in Patent Document 2 uses yeast as a coenzyme source, there is no problem as a food additive. However, in this method, since butyric acid is generated as a by-product, a post-treatment step for separating butyric acid from the generated γ-aminobutyric acid is required, and there is a disadvantage that the manufacturing cost increases.

  The object of the present invention is to produce γ-aminobutyric acid that eliminates the disadvantages of the prior art and can increase the conversion rate of glutamic acid or glutamate to γ-aminobutyric acid without adding pyridoxal phosphate or yeast. It is to provide a method and a manufacturing apparatus.

  In order to achieve the above-described object, the method for producing γ-aminobutyric acid according to the present invention includes adding glutamic acid or glutamate to a solution to which a material containing glutamic acid decarboxylase has been added, and removing the glutamic acid decarboxylase using the glutamic acid decarboxylase. In the method for producing γ-aminobutyric acid in which γ-aminobutyric acid is produced batchwise by a carbonic acid reaction, the glutamic acid or glutamate is dividedly added to the solution in the course of the batchwise reaction.

  The method for producing γ-aminobutyric acid according to the present invention is characterized in that the material containing glutamic acid decarboxylase is rice germ or rice bran containing germ. In the method for producing γ-aminobutyric acid, it is desirable to adjust the amount of glutamic acid or glutamate added so that the concentration of glutamic acid or glutamate in the solution is always 30 g / L or less.

  The apparatus for producing γ-aminobutyric acid according to the present invention generates γ-aminobutyric acid by adding glutamic acid or glutamate to a solution to which a material containing glutamic acid decarboxylase is added, and reacting in a batch manner for a predetermined time. Reaction temperature vessel, temperature adjusting means for keeping the temperature of the solution in the reaction vessel in a range of 30 to 50 ° C., and keeping the pH of the solution in the reaction vessel in a range of 4.5 to 6.5 PH adjusting means, substrate introducing means for dividing and adding glutamic acid or glutamate in the process of batch reaction in the reaction vessel, membrane separation means for filtering the solution after completion of the batch reaction in the reaction vessel, It is characterized by comprising.

  In the present invention, the reason why the substrate glutamic acid or glutamate (hereinafter sometimes simply referred to as substrate) is added in the course of batch reaction is based on the following basic experimental results. In the basic experiment, in order to grasp the productivity of γ-aminobutyric acid, the initial concentration of the substrate was changed in the range of 10 to 60 g / L, and the conversion rate after 48 hours of reaction (the substrate glutamate or glutamate was γ- The ratio of aminobutyric acid converted) was examined. In addition, rice germ was used as a glutamic acid decarboxylase source, and the amount added was constant at 220 g / L, which is the maximum amount of rice germ that can be well stirred in water in any experiment.

  FIG. 2 is a graph showing the results of basic experiments, in which the horizontal axis represents the initial concentration of the substrate and the vertical axis represents the conversion rate. As apparent from FIG. 2, the conversion rate greatly depends on the initial concentration of the substrate, and the conversion rate is almost 100% when the initial concentration of the substrate is 30 g / L or less. However, it was found that when the initial concentration of the substrate was 40 g / L, the conversion rate decreased to 90% or less, and thereafter the conversion rate decreased as the initial concentration of the substrate was increased. Thus, the reason why the conversion rate decreases when the initial concentration of the substrate is high has not been fully elucidated. If the substrate concentration is high, it is presumed that some kind of disorder occurs with respect to glutamate decarboxylase and the activity of glutamate decarboxylase decreases.

  The present invention is based on the results of the above basic experiment, and the substrate glutamate or glutamate is used so that the initial concentration of the substrate and the substrate concentration in the reaction system in the subsequent batch reaction become a predetermined value, preferably 30 g / L or less. In the batch reaction process.

  According to the production method and production apparatus for γ-aminobutyric acid according to the present invention, glutamic acid or glutamate which is a substrate is divided in a batch reaction and charged into a reaction system. For this reason, the substrate concentration in the reaction system can be suppressed to a warning value (eg, 30 g / L) or less throughout the batch reaction, and batch conversion with a high conversion rate avoiding substrate failure caused by the high substrate concentration. Can be realized.

  Rice germ and rice bran are inexpensive materials that are discharged in large quantities from a rice mill. Therefore, γ-aminobutyric acid can be produced at low cost by using rice germ or rice bran containing germ as a glutamate decarboxylase source. In addition, rice germ and rice bran itself contain glutamic acid, and there is an advantage that it can be used as a part of a substrate for producing γ-aminobutyric acid.

  FIG. 1 is a system diagram showing an embodiment of a method and apparatus for producing γ-aminobutyric acid according to the present invention. The reaction vessel 10 is provided with a chemical introduction path 12, and glutamic acid or glutamate can be introduced into the reaction container 10 from the pipe line 14 via the injection pump 16. Further, water (warm water) 18 and a material 20 containing glutamic acid decarboxylase (for example, rice germ or rice bran containing embryo) can be introduced from the introduction path 12.

  A jacket 22 is attached to the outer periphery of the reaction vessel 10, and the temperature of the solution stuck in the reaction vessel 10 is maintained at a set value by supplying a heat medium such as hot water from the conduit 24 into the jacket 22. Can do. The heat medium supplied into the jacket 22 is discharged from the pipe line 26. A stirrer 28 is attached to the reaction vessel 10, and water (warm water), rice bran, a substrate and the like charged into the reaction vessel 10 from the charging path 12 are mixed and stirred by the stirrer 28. Further, a pH meter 30 for measuring the pH of the internal solution is attached to the reaction vessel 10, and a pH adjusting agent is supplied from the pH adjusting means 32 so that the pH of the solution measured by the pH meter 30 becomes a set value. Added. A discharge pipe 36 having an open / close valve 34 is connected to the bottom of the reaction vessel 10, and a membrane having a microfiltration membrane (or ultrafiltration membrane) 40 via a pump 38 on the downstream side of the discharge pipe 36. Separation means 42 is arranged.

  The manufacturing apparatus having the above configuration is operated by a batch system. First, water (warm water) 18 is charged into the reaction vessel 10 and a temperature of water (warm water) stuck to the reaction vessel 10 through a heat medium through the jacket 22 is within a range of 30 to 50 ° C., preferably 35 to 40. Hold at ° C. Next, for example, rice bran is charged into the reaction vessel 10 as the material 20 containing glutamate decarboxylase from the charging path 12. Rice bran is an inexpensive material that is discharged in large quantities from rice mills, so it is an effective source of glutamate decarboxylase. Since glutamate decarboxylase in rice bran is mainly derived from rice germ, it is also effective to use rice germ as a material containing glutamate decarboxylase. Rice germ can be easily separated from rice bran by sieving the rice bran. From the conduit 14, glutamic acid or glutamate as a substrate is charged into the reaction vessel 10 as a solution or in the form of crystal powder.

Then, by driving the stirrer 28, the charged water (warm water), rice bran, and the substrate are mixed, and the temperature of the mixed solution is maintained in the above temperature range. As a result, glutamate decarboxylase contained in the substrate and rice bran comes into contact with each other, and glutamate or glutamate as a substrate is converted to γ-aminobutyric acid by a decarboxylation reaction by glutamate decarboxylase. By continuing this decarboxylation reaction for a predetermined time, most of the initially charged substrate is converted to γ-aminobutyric acid. In order to increase the production rate of γ-aminobutyric acid, pH control of the internal solution is important. Therefore, the pH adjusting means 32 controls the amount of the pH adjusting agent so that the pH of the internal solution measured by the pH meter 30 is in the range of 4.5 to 6.5, preferably 5 to 6.
The substrate, glutamic acid or glutamate, is divided into the reaction vessel 10 even during the batch reaction.

  FIG. 3 is an explanatory diagram showing a first model of the substrate charging method, in which the horizontal axis indicates the elapsed time, and the vertical axis indicates the substrate concentration or the total amount charged. This first model shows a case where, for example, when the cumulative amount of substrate is 60 g / L, this is equally divided into three. That is, initially, 20 g / L of substrate is charged into the reaction vessel 10. The reaction does not occur until about 8 hours have elapsed from the initial charging, but thereafter, as shown in FIG. 2, the decarboxylation reaction is actively performed and the conversion to γ-aminobutyric acid proceeds. For this reason, the substrate concentration decreases with time as shown by the broken line.

  Therefore, after 18 hours, as shown by the solid line, the second substrate is introduced, and the cumulative amount of substrate introduced is 40 g / L. Since most of the substrate initially charged is converted to γ-aminobutyric acid after 18 hours, the substrate concentration in the reaction vessel 10 is also a warning value indicated by a two-dot chain line even after the second substrate is charged. (For example, 30 g / L) is not exceeded. For this reason, the decarboxylation reaction proceeds actively even after the second substrate injection without suffering from substrate damage caused by the high substrate concentration. In the same way, the substrate is introduced for the third time after 28 hours, the cumulative amount of substrate is set to the target of 60 g / L, and thereafter, the batch reaction is completed 48 hours later without introducing the substrate.

  FIG. 4 is an explanatory view showing a second model of the substrate charging method. In this second model, when the cumulative amount of substrate input is 60 g / L as described above, 20 g / L of one third is initially input, and the remaining two thirds are applied 6 times after 12 hours. Divided into 4 and every 4 hours. According to this method, fluctuations in the substrate concentration in the reaction vessel 10 are reduced, and stable operation is possible.

  FIG. 5 is an explanatory view showing a third model of the substrate charging method. In this third model, when the cumulative amount of substrate is similarly 60 g / L, 20 g / L of one third is initially introduced, and the remaining two thirds are elapsed time 12 hours to 34 hours. Introduce continuously at a constant flow rate. According to this method, the fluctuation of the substrate concentration in the reaction vessel 10 is further reduced, and stable operation is possible.

  In any of the charging methods shown in FIGS. 3 to 5, the substrate concentration in the reaction vessel 10 does not exceed a warning value (for example, 30 g / L) indicated by a two-dot chain line in the entire batch reaction. A batch reaction with a high conversion rate that avoids substrate damage caused by a high concentration can be realized. Note that when performing these charging methods, the driving of the infusion pump 16 provided in the pipe line 14 may be controlled by a controller having a timer and a substrate flow rate adjusting function.

  As shown in FIGS. 3 to 5, the batch reaction is completed in, for example, 48 hours in total, but a reaction time of about 8 hours is required in order for the last substrate to be fully converted to γ-aminobutyric acid. And Therefore, it is desirable to complete the loading of the substrate to be loaded last at least 8 hours before the end of the reaction.

  When the batch reaction for one batch is completed, the on-off valve 34 arranged at the lower part in the reaction vessel 10 is opened, and the solution in the reaction vessel 10 is sent to the membrane separation means 42 by the pump 38. In the membrane separation means 42, a solution containing γ-aminobutyric acid that has permeated through the microfiltration membrane (or ultrafiltration membrane) 40 is taken out from the conduit 44 as a product. Solids such as rice bran and rice germ are separated from the solution by a microfiltration membrane (or ultrafiltration membrane) 40 and discharged out of the apparatus as used material 46.

  As described above, according to the production method and production apparatus for γ-aminobutyric acid of this embodiment, glutamic acid or glutamate as a substrate is divided and introduced into the reaction vessel 10 during the batch reaction. For this reason, since the substrate concentration in the reaction vessel 10 does not exceed a warning value (for example, 30 g / L) in the entire batch reaction, a batch with a high conversion rate that avoids substrate failure caused by a high substrate concentration. A reaction can be realized. Further, when rice germ containing rice germ or germ is used as a glutamate decarboxylase source, γ-aminobutyric acid can be produced at low cost. Rice germ and rice bran itself contain glutamic acid, and there is an advantage that it can be used as a part of a substrate for producing γ-aminobutyric acid.

  For this reason, according to the manufacturing method and manufacturing apparatus of γ-aminobutyric acid of this embodiment, it is possible to manufacture γ-aminobutyric acid with a high conversion rate from glutamic acid or glutamate without adding pyridoxalphosphoric acid or yeast. Can be realized. However, the present invention does not exclude the addition of pyridoxal phosphate or yeast as a coenzyme component, and these coenzyme components may be added as necessary in order to further increase productivity. In the present embodiment, the case where rice germ or rice bran containing germ is used as the glutamate decarboxylase source has been described. However, the present invention is not limited to this, and can also be applied to the case where other plants or microorganisms are used as a material containing glutamate decarboxylase.

Experimental Example In the first experiment, 1.5 kg (50 g / L) of sodium glutamate (crystal powder) was added to 30 L of tap water at 40 ° C. and dissolved completely, and then 6.6 kg (220 g / L) of rice germ. ) Was added and allowed to react with stirring. The solution 48 hours after the start of the reaction was filtered with a filter having a pore size of 0.5 μm, and then the concentration of γ-aminobutyric acid in the solution was analyzed by high performance liquid chromatography. From the analyzed concentration of γ-aminobutyric acid, the conversion rate to γ-aminobutyric acid with respect to sodium glutamate added was calculated. As a result, the conversion rate was 74%.

  In the second experiment, 450 g (15 g / L) of sodium glutamate was added to 30 L of tap water at 40 ° C. and completely dissolved, and then 6.6 kg (220 g / L) of rice germ was added and the reaction was conducted while stirring. Started. After 13 hours from the start of the reaction, 450 g (15 g / L) of sodium glutamate was added. Further, 21 hours after the start of the reaction, 600 g (20 g / L) of sodium glutamate was added, and the total amount of sodium glutamate added was 1.5 kg (50 g / L) as in the first experiment. The solution for 48 hours after the start of the reaction was treated and analyzed in the same manner as in the first experiment, and the conversion rate was calculated. As a result, the conversion rate was 86%.

  In the third experiment, after adding 480 g (16 g / L) of sodium glutamate to 30 L of tap water at 40 ° C. and completely dissolving it, 6.6 kg (220 g / L) of rice germ was added and the reaction was carried out with stirring. Started. From 8 hours to 24 hours after the start of the reaction, 60 g (2 g / L) of sodium glutamate was added every hour, and the total amount of sodium glutamate added was 1.5 kg (50 g / L) as in the first experiment. did. The solution for 48 hours after the start of the reaction was treated and analyzed in the same manner as in the first experiment, and the conversion rate was calculated. As a result, the conversion rate was 99%.

  As is clear from the first to third experiments, the second and third experiments according to the present invention show a higher conversion rate than the first experiment according to the prior art. Further, it was found that the conversion rate was further improved by increasing the number of divisions as in the third experiment and adding the substrate frequently.

BRIEF DESCRIPTION OF THE DRAWINGS It is a systematic diagram which shows embodiment of the manufacturing method and manufacturing apparatus of (gamma) -aminobutyric acid which concern on this invention. It is a graph which shows a basic experiment result. It is explanatory drawing which showed the 1st model of the substrate injection | throwing-in method. It is explanatory drawing which showed the 2nd model of the substrate injection | throwing-in method. It is explanatory drawing which showed the 3rd model of the substrate injection | throwing-in method.

Explanation of symbols

10 ......... Reaction vessel, 12 ......... Input path, 14 ......... Substrate (for substrate), 16 ......... Infusion pump, 18 ......... Water (warm water), 20 ...... Glutamate decarboxylase 22 ......... jacket, 28 ......... stirrer, 30 ......... pH meter, 32 ......... pH adjusting means, 34 ......... open / close valve, 36 ......... discharge pipe, 38 ......... pump , 40... Microfiltration membrane (or ultrafiltration membrane), 42.

Claims (4)

  In a method for producing γ-aminobutyric acid, in which glutamic acid or glutamate is added to a solution to which a material containing glutamic acid decarboxylase is added, and γ-aminobutyric acid is produced batchwise by a decarboxylation reaction using the glutamic acid decarboxylase, A process for producing γ-aminobutyric acid, wherein the glutamic acid or glutamate is dividedly added to the solution during the batch reaction.   The method for producing γ-aminobutyric acid according to claim 1, wherein the material containing glutamic acid decarboxylase is rice germ or rice bran containing germ.   The γ-aminobutyric acid according to claim 1 or 2, wherein an input amount of the glutamic acid or glutamate is adjusted so that a concentration of glutamic acid or glutamate in the solution is always 30 g / L or less. Manufacturing method.   A reaction vessel for producing γ-aminobutyric acid by adding glutamic acid or glutamate to a solution to which a material containing glutamic acid decarboxylase has been added and reacting batchwise for a predetermined time, and the temperature of the solution in the reaction vessel Temperature adjusting means for maintaining the pH in the range of 30 to 50 ° C., pH adjusting means for maintaining the pH of the solution in the reaction container in the range of 4.5 to 6.5, and batch reaction in the reaction container Γ-aminobutyric acid, characterized in that it comprises a substrate loading means for dividing and feeding the glutamic acid or glutamate in the course of the step, and a membrane separation means for filtering the solution after completion of the batch reaction in the reaction vessel Manufacturing equipment.

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