KR101298111B1 - Preparation Method of GABA(γ-Amino butyric acid)- Enriched Rice Bran Extract - Google Patents

Abstract

In the present invention, L-glutamic acid dissolved in rice bran extract is used as a substrate, and glutamic acid decarboxylase and pyridoxal phosphate as biocatalysts. It relates to a method for preparing an extract.

Description

Preparation Method of GABA (γ-Amino butyric acid) -Enriched Rice Bran Extract}

The present invention relates to a method for producing Gaba-enhanced rice bran extract, more specifically, by dissolving glutamic acid in a solution obtained by extracting the rice bran by-products of rice bran and rice eyes as a solvent, glutamic acid decarboxylase (L-Glutamic acid decarboxylase) and a coenzyme pyridoxal phosphate to convert to gaba to a rice bran extract containing a high concentration of gaba.

Rice bran contains a lot of vitamins, minerals and many bioactive substances. By the way, 500,000 tons of rice bran (rice bran) generated in Korea is a fact that most of the added value is used as a feed or organic compost. Rice bran contains fats and fats, tocopherols and various unsaturated fatty acids in addition to vitamins and minerals; Lipophilic substances such as tocotrienols and gamma-oryzanol; It contains water-soluble substances such as pichin acid, gaba, ceramide, lecithin and inositol.

Γ-Aminobutylic acid (GABA) is a non-protein constituent amino acid. GABA is contained in the human nervous system and blood. Most of it is present in the bone marrow of the brain, increasing the neurotransmitter called acetylcholine. Promotes physiological activities such as. In addition, GABA is known to have anti-stress, antihypertensive, concentration enhancement, diet effect, and is especially contained in germinated brown rice. GABA also acts as a neurotransmitter in higher animals, invertebrates and insects other than humans and is widely distributed in the plant kingdom and found in most tissues of plants (Navayan & Nair, 1990; Tompson et al., 1952). Fowden, 1981; Hunt, 1981). Meanwhile, various foods and health functional foods using anti-stress, antihypertensive and other functionalities of Gabba have been released, and its application range has been expanded to feed additives and phytonutrients, and recently, nylon-4, a biodegradable fiber. It is also attracting attention as a key raw material.

The method of extracting rice bran is well established and does not require special skills. In other words, the washed rice bran is extracted for 4 hours to 12 hours at a concentration of 5% to 10% by weight at a temperature of 50 to 80 grams at a rate of 50 to 100 grams per liter of water before hand-making, and the solid content by compression filtration and the like. By removing the rice bran extract is obtained. Solid content of the rice bran extract obtained at this time is 1 ~ 4 Bx. This rice bran extract is one of the basic elements of the substrate solution used in the method for producing Gaba-enhanced rice bran extract of the present invention.

Methods of industrially producing GABA include synthetic methods, extraction methods, enzyme methods, fermentation methods, and the like. In the case of the synthesis method, the solvent used in many cases is not allowed in the food hygiene method, and the extraction method has a disadvantage in that the manufacturing cost of Gaba is high and the amount of waste is generated. Enzyme and fermentation, on the other hand, are economical and can produce Gabba for food. In the enzymatic method, as shown in FIG. 1, -COOH bound to α-carbon of L-glutamic acid is decarbonized by catalysis of glutamic acid dicarboxylase (hereinafter referred to as decarboxylase) to biosynthesize Gaba. As a conventional technique utilizing rice bran extract, it is disclosed to prepare a fermented rice bran extract containing Gabba by adding a carbon source, a nitrogen source, and monosodium glutamate (MSG) to the rice bran extract, inoculating the lactic acid bacteria and incubating for a predetermined time ( Republic of Korea Patent Publication No. 10-2006-0042583). However, this method does not grow if the fermentation time is longer than 48 hours and the concentration of glutamic acid to be added to the medium is too high. Therefore, the production amount of Gabba in the fermentation broth is relatively low within 5.7% (550mM), and the fermentation equipment is operated every time. There is a disadvantage in that the manufacturing cost is high. In addition, the content of Gabba contained in rice bran is relatively low, such as 100ppm, so that the production of rice bran extract containing Gabba at a high concentration has been recognized as an industrially difficult task.

The inventors of the present invention intensively studied a variety of physiologically active substances derived from rice bran and a technique capable of economically manufacturing rice bran extract containing a high concentration of Gabba, as a result of overcoming the disadvantages of the prior art rice bran strengthened rice bran We have completed the method of producing extracts efficiently.

The present invention is to solve the above problems, extract useful bioactive substances from rice bran, glutamic acid in the extracted solution and glutamic acid decarboxylase (L-Glutamic acid Decarboxylase) and pyridoxal phosphate enzyme and coenzyme respectively It is an object of the present invention to provide a method for producing rice bran extract fortified by using.

In addition, the present invention is the desired concentration from a low concentration to a high concentration in a relatively short time, using a decarboxylase and pyridoxal phosphate using a decarboxylase and pyridoxate phosphate as a main raw material containing rice bran and glutamic acid, which contains part of the rice snow generated during the process of rice planting An object of the present invention is to provide a method for producing a variety of rice bran extract containing Gabba.

In addition, an object of the present invention is to provide a method for producing Gaba-enhanced rice bran extract that can be made in various forms such as powder, liquid, granules according to the processing method.

In order to achieve the above object, the present invention is a rice bran extract and L- glutamic acid as a substrate, decarboxylase and pyridoxal phosphate (Pyridoxal phosphate) by adding enzyme and coenzyme respectively to convert the glutamic acid to GABA (GABA) It is characterized by the production method of rice bran extract.

In addition, the present invention comprises the steps of preparing a substrate solution containing rice bran extract and L- glutamic acid; Adding decarboxylase and pyridoxalphosphate to the substrate solution to initiate an enzyme reaction; And after the start of the enzymatic reaction, characterized in that it comprises the step of additionally adding L- glutamic acid in the substrate solution.

In addition, the present invention is characterized in that the decarboxylase is selected from the group consisting of lactic acid bacteria, E. coli, Corynebacterium, and Bacillus.

In addition, the present invention is characterized in that the activity of the decarboxylase is more than 100 Unit / g-cell.

In addition, the present invention is characterized in that the input amount of the pyridoxal phosphate is 1 to 10 micromoles per 1000 units of decarboxylase.

In addition, the present invention is characterized in that the total amount of L- glutamic acid added is 50g ~ 700g per liter of the total substrate solution.

In addition, the present invention is characterized in that after the end of the enzyme reaction, the concentration of residual L- glutamic acid in the substrate solution is less than 1% by weight.

In addition, the present invention is characterized in that the form of bran enhanced rice bran extract is liquid, powder or granules.

Schematic diagram of the manufacturing process of the GABA enhanced rice bran extract of the present invention is as shown in FIG. As shown in FIG. 2, the present invention includes a step of injecting glutamic acid into the rice bran extract, or glutamic acid-dissolved rice bran extract, in which decarboxylase and pyridoxal phosphate are separately prepared. It is done. According to the present invention, by adding the decarboxylase and coenzyme pyridoxal phosphate prepared separately to the rice bran extract, and glutamic acid corresponding to the desired Gab content, by adding the Gaba content in the reaction solution more than 20% by weight, the Gab content relative to the solid content The rice bran extract of more than 90% by weight can be efficiently produced through a simple enzymatic reaction without undergoing a complicated fermentation process. In addition, according to the present invention, the enzyme reaction time depends on the amount of enzymatically added and the amount of glutamic acid, and more than 98% by weight of glutamic acid added within 3 hours to 24 hours may be converted to Gava.

Hereinafter will be described in detail the manufacturing method of the GABA enhanced rice bran extract of the present invention.

1. Rice Bran Extract

Rice bran extract used in the present invention is not particularly limited in its manufacturing method, but when extracted with alcohol to inhibit the enzyme reaction and gives a bad smell to the final product. On the other hand, when the extraction using water, the temperature and extraction time of the hot water used for extraction is preferably 50 to 90 ℃, 2 to 12 hours, respectively, preferably 60 to 80 ℃, 4 to 8 hours, respectively. After the extraction is completed, the rice bran extract is removed from the insoluble solids by filtration. Solid content dissolved in rice bran extract is 1 ~ 4Bx. The solid content dissolved in the rice bran extract can be adjusted through the concentration process before the enzyme reaction.

On the other hand, the present inventors investigated the effect of the rice bran extract on the activity of the decarboxylase in order to build a rice bran extract production system enhanced by Gaba, the effect on the activity of the decarboxylase of the rice bran extract when water as a control It was less than ± 5% compared to the little effect.

2. Substrate solution

As a substrate solution to be used in the enzyme reaction of the present invention, a rice bran extract or a rice bran extract in which L-glutamic acid is dissolved may be used. Preferably, the rice bran extract in which L-glutamic acid is dissolved.

In the present invention, rice bran extract in which L-glutamic acid is dissolved may be prepared as follows. In the rice bran extract or the rice bran extract concentrated to a predetermined concentration, the concentration of L-glutamic acid is about 1% to 10% by weight, and the pH and temperature are adjusted to the reaction conditions of the decarboxylase while stirring. For example, when the decarboxylase is derived from lactic acid bacteria and Escherichia coli, the optimum pH and the optimum temperature are shown as 5.3 and 37 ° C., respectively, and it is preferable to adjust the pH and temperature of the substrate solution to this range. On the other hand, in the case of L- glutamic acid is a pKa value of 2.2 is not soluble in water, but is dissolved completely by adjusting the pH to 5.0 with caustic soda. Accordingly, the substrate solution of the present invention is preferably a rice bran extract in which 1 to 10% by weight of L-glutamic acid is dissolved and the pH is adjusted to 5.0.

3. Decarboxylase

The higher the deactivation enzyme used in the present invention, the better the inactivation, and can be used irrespective of its origin, and among them, it is preferable that it is derived from lactic acid bacteria, E. coli, Corynebacterium genus or Bacillus genus.

The deactivation of the decarboxylase of the present invention is preferably 500 U / g-cell or more, preferably 1000 IU / g-cell or more. If the inactivity is low, a large amount of enzyme is required and the reaction time is long, and the advantages pursued by the present invention disappear.

In addition, decarboxylase obtained through genetic engineering can be used depending on its inactivity.

On the other hand, the pH range in which the decarboxylase of the present invention acts is 3.5 to 6, preferably the pH range is 4.5 to 5.5. When the pH is below 3.5 or above 6, the enzyme reaction hardly occurs. For example, when the decarboxylase is derived from lactic acid bacteria and Escherichia coli, the optimal pH is 5.3.

4. Pyridoxal phosphate

In the present invention, pyridoxal phosphate is essential as a coenzyme of decarboxylase. The concentration used per 1000 Units of enzyme activity is preferably 1 to 10 micromoles, more preferably 4 to 6 micromoles. If the amount of coenzyme is too small, the rate of enzymatic reaction decreases significantly, and if it is too high, the color of the product becomes dark.

5. Manufacturing Method of Rice Bran Extract with Enhanced Gaba

In the present invention, the method for producing Gaba-enhanced rice bran extract is characterized in that glutamic acid is added at a low cost after decarboxylase and pyridoxal phosphate are added to rice bran extract or glutamic acid-dissolved rice bran extract. Preferably, after the decarboxylase and pyridoxal phosphate are added to the substrate solution containing the rice bran extract and L-glutamic acid to initiate the enzymatic reaction, L-glutamic acid may be added more advantageously with increasing pH.

Specifically, the temperature of the rice bran extract (that is, the substrate solution) in which 1 to 10% by weight of L-glutamic acid is dissolved and the pH is adjusted to 5.0 is adjusted to 37 ° C., followed by enzymatic reaction by adding decarboxylase and pyridoxalphosphate. This is disclosed. As the enzyme reaction proceeds, the pH of the substrate solution increases as Gaba is generated, and thus the reaction slows down and eventually stops. To continue the reaction, an acid should be added. When the pH is about 5.4, L-glutamic acid (crystal powder) is added to adjust the pH to about 5.0 again. This operation is repeated until the desired Gaba concentration is reached, and the volume of the substrate solution increases with the addition of L-glutamic acid. At this time, the total amount of L- glutamic acid added is preferably 50g ~ 700g per liter of the total substrate solution. If the total amount of L-glutamic acid to be added is within the above range, it is possible to obtain a high concentration of Gaba while minimizing the generation of salt. After the addition of L-glutamic acid as a pH adjusting agent is completed, the reaction is carried out by adding an acid as another pH adjusting agent, which may be used without limitation as long as the acid has a pH adjusting function regardless of inorganic acid or organic acid. have. Here, specific examples of the inorganic acid may include hydrochloric acid, sulfuric acid, acetic acid, phosphoric acid, or a mixture thereof, and specific examples of the organic acid may include L-aspartic acid, alpha ketoglutaric acid, lactic acid, citric acid, tartaric acid, fumaric acid, and malic acid. , Itaconic acid, acetic acid, gluconic acid, 2-ketogluconic acid, 5-ketogluconic acid, kojic acid, or a mixture thereof.

In the present invention, the concentration of residual L-glutamic acid in the substrate solution after the end of the enzyme reaction is less than 1% by weight. On the other hand, the present invention can convert 600Kg glutamic acid to Gabba based on 1000 liters of the substrate solution, wherein the produced Gabba is about 413Kg and the conversion rate is 98.3%.

In the present invention, the GABA-enriched rice bran extract obtained through the enzymatic reaction is subjected to microfiltration using diatomaceous earth to remove insoluble solids, including enzymes, and then to various forms such as liquid, powder and granules through purification and processing. Gaba can be processed into fortified rice bran extract. Gaba-enhanced rice bran extract of the present invention can be used in food, feed, cosmetics, phytonutrients and the like.

The present invention converts glutamic acid to Gabba by adding decarboxylase and pyridoxal phosphate to glutamic acid-dissolved rice bran extract, and acts as a simple enzyme without complex fermentation process. The reaction can be produced efficiently.

In addition, according to the present invention, rice bran extract fortified with Gaba in a very short time within 3 hours to 24 hours can be produced efficiently and economically.

Figure 1 shows the production reaction by the enzyme method of Gaba.
Figure 2 is a view showing the manufacturing process of the GABA enhanced rice bran extract powder of the present invention.
Figure 3 is a graph showing the effect of the concentration of PLP on the activity of the decarboxylase of the present invention.

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

Manufacturing example  1: Preparation of Rice Bran Extract

7 kg of rice bran within 24 hours of incubation, 3.5Kg each in two extracts, washed with 10 liters of water, immersed in 100 liters of water, and the temperature was raised to 70 ℃ and pumped for 8 hours. Circulated. The extraction cloth was taken out, cooled to 55 ° C., and then diatomaceous earth 1.5Kg and activated carbon 0.25Kg were added and stirred for 2 hours. Filtration was carried out using a press filter. About 90 liters of light brown and obfuscated liquid were obtained. The solid concentration of the filtrate was 1.8 brix. The filtrate thus obtained was used as rice bran extract.

Manufacturing example  2 : Of substrate solution  Produce

50 kg of the rice bran extract prepared in Preparation Example 1 was added 2.5 Kg of glutamic acid while adding caustic soda (98%, granular) while stirring to adjust the pH and temperature to 5.0 and 37 ° C., respectively, as a substrate solution.

Manufacturing example  3: Decarboxylase  Produce

The lactic acid bacteria and Escherichia coli were cultured in a conventional manner using a 70 liter fermenter to generate decarboxylase, and the cells were separated after the incubation. The isolated cells lysed the cell wall / cell membrane by repeating the freeze thaw cycle three times. The results of measuring the activity of the obtained decarboxylase are shown in Table 1 below. One unit of activity was defined as the amount of 1 micromole of Gabba produced per minute.

Host Lactobacillus Genetic Recombinant Escherichia coli Origin of Decarboxylase Lactobacillus brevis Escherichia coli Inactive 510 Unit / g-cell 1490 Unit / g-cell

Manufacturing example  4 : Pyridoxalphosphate ( PLP ) stock Solution  Produce

1 g of PLP was dissolved in distilled water to a total volume of 100 mL. 1 mL of stock solution corresponds to 40.5 micromoles.

Example 1 Preparation of Gaba-Reinforced Rice Bran Extract Using Decarboxylase Derived from Lactic Acid Bacteria (Gaba Content 20 wt%)

To 2 liters of substrate solution, 1.0 mL of PLP stock solution and 20 g of decarboxylase derived from lactic acid bacteria were added (about 10,000 units). When the decarbonation reaction proceeded and the pH of the substrate solution reached 5.4, glutamic acid was added until the pH reached 5.0. The operation of injecting glutamic acid in conjunction with the pH change was continued, and a total of 290 g of glutamic acid was added, and the pH was adjusted to 5.0 with 20% hydrochloric acid. When the pH reached 5.4, the pH was adjusted to 5.0 with 20% hydrochloric acid. The pH was adjusted twice with hydrochloric acid and the pH no longer changed. The volume of the reaction solution was increased to 2.3 liters, and the concentrations of Gabba and glutamic acid were measured to be 9.1 wt% and 0.2 wt%, respectively. The reaction solution was heat-treated at 65 ° C. for 10 minutes to inactivate the enzyme, and then 20 g of diatomaceous earth and 5 g of activated carbon were added to the reaction solution and stirred at 55 ° C. for 2 hours. The filter was filtered under reduced pressure under a vacuum of 600 mmHg using a silkworm. The pH of the filtrate was adjusted from 5.3 to 7.0 using caustic soda. The filtrate was concentrated under reduced pressure under a vacuum degree of 680 mmHg using a rotary evaporator. 0.9 l of a light brown clear concentrate was obtained. The resulting concentrate was a light brown clear liquid rice bran extract containing 3.5% by weight of water-soluble solids derived from rice bran, 20% by weight of GABA, 0.5% by weight of salt, and 0.25% by weight of glutamic acid.

Example  2: derived from E. coli Decarboxylase  Used, Gabba  Preparation of Fortified Rice Bran Extract ( Gab content  57% by weight)

To 2 liters of the substrate solution, 4.0 mL of PLP stock solution and 30 g (about 40000 Unit) of decarboxylase derived from E. coli were added. When the decarbonation reaction proceeded and the pH of the substrate solution reached 5.4, glutamic acid was added until the pH reached 5.0. The operation of injecting glutamic acid in conjunction with the pH change was continued, and a total of 1100 g of glutamic acid was added, and the pH was adjusted to 5.0 with 20% hydrochloric acid. When the pH reached 5.4, the pH was adjusted to 5.0 with 20% hydrochloric acid. The pH was adjusted three times with hydrochloric acid and the pH no longer changed. The volume of the reaction solution was increased to 2.7 liters and the concentrations of Gaba and glutamic acid were measured to be 28.2 wt% and 0.1 wt%, respectively. The reaction solution was heat-treated at 65 ° C. for 10 minutes to inactivate the enzyme, and then 30 g of diatomaceous earth and 7.5 g of activated carbon were added to the reaction solution and stirred at 55 ° C. for 2 hours. The filter was filtered under reduced pressure under a vacuum of 400 mmHg using a silkworm. The filtrate was concentrated under reduced pressure using a rotary evaporator under a vacuum degree of 680 mmHg. 1.2 liters of a light brown clear concentrate were obtained. The resulting concentrate was a light brown clear liquid rice bran extract containing 3.5% by weight of water-soluble solids derived from rice bran, 57.0% by weight of Gaba, 1.0% by weight of salt, and 0.15% by weight of glutamic acid.

Example  3: Pyridoxalphosphate  Effect of Concentration

This example was carried out to determine the effect of the concentration of pyridoxalphosphate in the production of Gaba-enriched rice bran extract.

To 1 liter of substrate solution, 0, 0.1, 0.2, 0.4, 0.8, 1.6 mL of PLP stock solution and 8 g of decarboxylase derived from lactic acid bacteria were added (about 4000 Unit). 1 mL of PLP solution corresponds to 40.5 micromoles. After 10 minutes, a sample was taken, the reaction was stopped with 5N caustic soda, and the resulting Gabba was measured by HPLC. The decarboxylase activity calculated from the amount of Gava produced is shown in FIG. 3. As shown in FIG. 3, it was found that the dosage of pyridoxal phosphate was preferably 1 to 10 micromoles per 1000 units of decarboxylase.

Example  4 : Gabba  Preparation of Powdered Rice Bran Extract Powder Gab content  90 wt%)

800 mL of the liquid rice bran extract obtained in Example 2 was diluted to 40 brix and then spray dried using hot air. As a result of drying the inlet hot air temperature at 180 ° C and the outlet temperature at 105 ° C, 380 g of a powder having good brown flowability was obtained. The obtained powder was a rice bran extract powder fortified with GABA, and the GABA content was 92.4% by weight.

Example  5: Gabba  Preparation of Fortified Rice Bran Extract Granules Gab content  60% by weight)

Laba rotary while spraying 90 mL of an aqueous solution adjusted to 60% alcohol by mixing 300 g of the GABA-enhanced rice bran extract powder obtained in Example 4 with 150 g of dextrin (MAX1000) and adding water to alcohol containing 95% alcohol Rice bran extract granules were prepared using a granulator. 400 g of the obtained granules were dried at 60 ° C. for 12 hours using a drying oven, and the average particle size of the granules was 0.2 mm and the moisture content was 0.5%. As a result of analyzing the Gaba content, it was 61.3 wt%.

Claims (8)

delete Preparing a substrate solution by dissolving L-glutamic acid in rice bran extract;
Adding decarboxylase and pyridoxalphosphate to the substrate solution as an enzyme and a coenzyme, respectively, to initiate an enzyme reaction; And
After the start of the enzymatic reaction, further comprising the step of additionally adding L- glutamic acid in the substrate solution,
The total amount of the added L- glutamic acid is 50g ~ 700g per liter of the total substrate solution characterized by the Gaba-enhanced rice bran extract manufacturing method.
The method of claim 2,
The decarboxylase is derived from lactic acid bacteria, E. coli derived, Corynebacterium derived from the genus Bacillus genus derived from the group consisting of Gaba-enhanced rice bran extract.
The method of claim 2,
The decarboxylase activity is Gaba-enhanced rice bran extract, characterized in that more than 100 Unit / g-cell.
The method of claim 2,
The amount of the pyridoxal phosphate dose is 1 to 10 micromoles per 1000 units of decarboxylase, Gaba-enhanced rice bran extract manufacturing method.
delete The method of claim 2,
A method for producing Gaba-enriched rice bran extract, characterized in that the concentration of residual L- glutamic acid in the substrate solution after the end of the enzyme reaction is less than 1% by weight. The method of claim 2,
Form of the GABA-enhanced rice bran extract is a method of producing a GABA-enhanced rice bran extract, characterized in that the liquid.

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