KR100687599B1 - Method for Preparing ???? Using By-Products from Rice Polishing - Google Patents

Abstract

The present invention relates to a method for producing GABA using rice bran by-products rice bran and / or rice snow and lactic acid bacteria, more specifically, (a) rice bran, rice snow, a mixture of rice bran and rice snow, rice bran extract, rice snow extract, and Preparing a medium containing a natural component selected from the group consisting of a mixture of rice bran and rice bran; (b) adding MSG (monosodium glutamate) to the medium and inoculating the medium with lactic acid bacteria; And (c) culturing the lactic acid bacteria, converting MSG to GABA by glutamate decarboxylase possessed by the lactic acid bacteria to produce GABA, thereby producing a GABA (γ-Aminobutyric acid) by lactic acid bacteria. to provide.

GABA, rice bran, rice snow, lactic acid bacteria, culture, fermentation

Description

Method for Preparing Baa Using By-Products from Rice Polishing}             

1 is a schematic view showing one embodiment of a process for preparing GABA according to the solid culture method of the method of the present invention.

Figure 2 is a schematic diagram showing an embodiment of the process for producing GABA according to the liquid culture method of the method of the present invention.

The present invention relates to a method for producing GABA, and more particularly to a method for producing GABA using rice bran by-products rice bran and / or rice snow and lactic acid bacteria.

GABA (γ-Aminobutyric acid) is a nonprotein amino acid that is well known as the major inhibitory neurotransmitter of the central nervous system in animals. GABA is known to be involved in many physiological mechanisms to stimulate the blood flow of the brain in animals and to increase the oxygen supply to promote the metabolism of brain cells, and also to regulate the production of prolactin and secretion of growth hormone. It is known to be effective in lowering blood pressure and pain relief, and thus has a high pharmacological interest.

As GABA is known to play an important role in the prevention of high blood pressure and dementia, interest in not only as a medicine but also as a functional food material has recently been increasing. According to such interest, researches to increase the content of GABA naturally in various materials have been actively conducted. The green leaves of green tea are left in N ₂ gas for 6-8 hours or rice germ and brown rice are immersed in water at a constant temperature. The method produces green tea and GABA-containing rice germ and germinated brown rice with increased GABA content.

However, the GABA content in the product manufactured by this method does not exceed 0.5% (w / w) in terms of the weight of the product, and the form of the product is both insoluble, indicating a limitation in the development of the product using GABA. . In order to solve this problem, a product was recently developed in Japan, in which MSG (monosodium glutamate) added to a substrate using lactic acid bacteria was converted to GABA using decarboxylase, and only the GABA accumulated in the culture solution was recovered and concentrated. . The GABA produced using lactic acid bacteria is capable of producing a high concentration of the product according to the post-fermentation process, and has the advantage of producing a completely dissolved product. The researchers have successfully screened lactobacillus strains capable of producing GABA from kimchi (see Patent Application No. 2003-5828).

The present inventors have diligently researched to establish an efficient GABA production system using lactic acid bacteria, and when the lactic acid bacteria were cultured using rice bran and / or rice snow, which are by-products produced during the milling of rice, GABA-high content products can be obtained. By confirming that, this invention was completed.

Accordingly, it is an object of the present invention to provide a method for preparing GABA (γ-Aminobutyric acid) using rice bran and / or rice bran and lactic acid bacteria.

Another object of the present invention is to provide a method for preparing GABA-containing powder.

Another object of the present invention is to provide a method for preparing a GABA-containing solution.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for preparing GABA (γ-Aminobutyric acid) by lactic acid bacteria, comprising the following steps: (a) rice bran, rice bran, mixture of rice bran and rice bran, rice bran extract, Preparing a medium containing a natural ingredient selected from the group consisting of rice eye extract, and rice bran and a mixed extract of rice bran; (b) adding MSG (monosodium glutamate) to the medium and inoculating the medium with lactic acid bacteria; And (c) culturing the lactic acid bacteria to convert MSG to GABA by glutamate decarboxylase possessed by the lactic acid bacteria to produce GABA.

The present inventors conducted various media composition experiments to build an efficient GABA production system using lactic acid bacteria, and as a result, when culturing lactic acid bacteria using rice bran and / or rice snow, which are by-products produced during the milling of rice. In addition to obtaining a GABA-rich culture solution, it was confirmed that a culture solution containing a large amount of excellent nutrients derived from rice bran (or rice bran) was obtained. Rice bran, which is included as an essential ingredient in the medium used in the present invention, may be partially used to produce oil, but most of them are used for feed, and in the case of rice snow, its use is not used except for reproductive addition.

In the method of the present invention, glutamate decarboxylase is involved in converting a monosodium glutamate (MSG) substrate into GABA, and this enzyme is expressed in lactic acid bacteria. The lactic acid bacterium used in the present invention is not limited as long as it expresses glutamate decarboxylase, and is preferably a strain of the genus Lactobacillus, most preferably Lactobacillus sakei and Lactobacillus brevis . to be.

The medium used in the present invention is a medium containing a natural component selected from the group consisting of rice bran, rice snow, rice bran and rice snow mixture, rice bran extract, rice snow extract, and rice bran and rice snow mixed extract.

In the present invention, the quality of the rice milling by-products play an important role in determining the yield of the final GABA product, so it is advantageous to receive raw materials continuously from the milling factories of the same Nonghyup to maintain the same quality of rice bran and rice ginseng. In the case of using rice bran and rice glen from, there is a slight difference in the yield of GABA but it is not a big problem for fermentation itself.

Rice bran and rice bran are preferably washed before use as a component of the medium, to remove pesticides remaining on the surface. Most residual pesticides are mostly removed during the removal of rice hulls, and almost no residual pesticides are detected in rice bran and rice bran in areas that allow limited use of water-soluble pesticides. Do. The washing process is preferably to use cold water of less than 30 ℃, it is advantageous to treat in as short time as possible within 30 minutes to prevent the leakage of water-soluble nutrients. If the temperature of the wash water is more than 30 ℃ and the time required for the washing is more than 30 minutes, nutrients of rice bran and rice snow may be lost.

On the other hand, the method of the present invention can be largely divided into solid culture and liquid culture.

First, in the case of solid culture, a carbon source, a nitrogen source, a trace element and / or a surfactant are added to a natural component, i.e., rice bran and / or rice bran medium components obtained from rice milling, if necessary, followed by MSG and water. The addition of water facilitates the mixing of the various components added, preferably with a final moisture content of 10-40% after the addition of water. When the moisture is low, the added ingredients are difficult to mix evenly, and when the moisture is too high, a high cost is required for drying the final product.

On the other hand, in the case of solid culture, rice bran and / or rice bran is used as it is, but in liquid culture, rice bran and / or rice bran extract is used.

Obtaining the extract can be carried out using a variety of extraction solvents known in the art. Available extraction solvents include (a) water, (b) anhydrous or hydrous lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) a mixed solvent of the lower alcohols with water, (d) Acetone, (e) ethyl acetate, (f) chloroform, (g) butyl acetate and (h) 1,3-butylene glycol, but water is most preferred, given the safety of the final product and the culture of lactic acid bacteria. Even in the case of using the extract suitable for the present invention can be obtained sufficiently.

When the extract of rice bran and / or rice bran is prepared using water, the amount of water used is 2-20 times, preferably 5-15 times. If the amount of water is too small, there is a disadvantage that the extraction efficiency is low, on the contrary, if the amount of water is too high, the concentration of the active ingredient after extraction is low, when used as a fermentation source may cause a low GABA conversion rate. The extraction temperature of nutrients is suitable for 40 ℃ -70 ℃. If the temperature is low, the extraction time of water-soluble nutrients is long, and if the temperature is over 70 ℃, starch of rice bran or rice snow is gelatinized, which makes it difficult to separate useful ingredients. . The extraction time is suitably 3-24 hours, preferably 6-18 hours with stirring at a speed of 30-100 rpm. If the extraction time is insufficient, the concentration of nutrients is insufficient, if the extraction time is too long can be a burden on the process. Extracted rice mill by-products are obtained as a nutrient component of the medium for GABA production after the supernatant is obtained by centrifugation. At this time, the removal of solids may proceed after incubation, and if the solubility of the final product is not a problem, it is possible to dry and commercialize.

According to a preferred embodiment of the present invention, the medium of the present invention further comprises a carbon source, a nitrogen source, a trace element, a surfactant or a mixture thereof. More preferably a carbon source and a nitrogen source, most preferably a carbon source, a nitrogen source and a trace element (or surfactant).

Available as a carbon source in the medium of the present invention is selected from the group consisting of glucose, sucrose, maltose, fructose, lactose, xylose, galactose, arabinose or a combination thereof, preferably sucrose, fructose Tose, glucose, galactose, arabinose and lactose, more preferably sucrose, fructose and glucose, most preferably sucrose. The amount of carbon source used is preferably 1-20% by weight of the natural component, more preferably 2-10% by weight, most preferably 3-6% by weight.

Usable as a nitrogen source in the medium of the present invention is selected from the group consisting of yeast extract, soytone, peptone, bee extract, tryptone, cachetone and combinations thereof, preferably yeast extract, Peptone, tryptone and soy, more preferably yeast extract and soyton, and most preferably yeast extract. The amount of the nitrogen source used is preferably 1-20% by weight of the natural component, more preferably 1-10% by weight, most preferably 2-4% by weight.

Usable as trace elements in the medium of the present invention are selected from the group consisting of magnesium sulfate, sodium acetate, manganese sulfate, ferric sulfate, calcium chloride and combinations thereof. The amount of the trace element used is preferably 0.001-1% by weight of the natural component, more preferably 0.01-1% by weight, and most preferably 0.03-0.3% by weight.

One of the characteristics of the medium of the present invention is that a surfactant can be used as a trace component. In the case of using such a surfactant, the need for the trace element in the medium can be eliminated. Preferably, the surfactant used in the medium of the present invention is a polyalcohol fatty acid (eg monostearicin glycine), polyethyleneglycol fatty acid (eg stearic acid polyoxyl), polyoxyethylene alcohol (eg Raulmacrogol), Nonionic surfactants selected from the group consisting of sorbitan fatty acids (eg sorbitan oleate), polyoxyethylene sorbitan fatty acids (eg polysorbate) and combinations thereof, more preferably polyoxyethylene sorbitan fatty acids And most preferably polysorbate. When the medium is added by adding a nonionic surfactant, the lactic acid bacteria may be cultured normally even when the trace element is not added. The amount of the nonionic surfactant used as the trace component is preferably 0.01-1% by weight of the natural ingredient, more preferably 0.01-1% by weight, most preferably 0.05-0.3% by weight.

The amount of MSG added in step (b) of the process of the invention is preferably 1-30% by weight of the natural component, more preferably 1-20% by weight, most preferably 1-15% by weight. If the amount of MSG is too large, MSG which is not converted to GABA may remain in the final product and affect the flavor of the product. If the amount of MSG is too small, there is a problem that the GABA content of the final product is lowered.

Rice milling by-products mixed with all ingredients (medium and MSG) are then preferably sterilized. At this time, it is preferable to proceed for 15-30 minutes at a temperature of 60-121 ℃ to minimize nutrient destruction, if the temperature is too low or short time, the sterilization effect is reduced, if the temperature is too high or the sterilization time is long The destruction of nutrients worsens.

Lactobacillus is inoculated into the rice by-product-containing medium after the sterilization is completed. The inoculation amount is preferably 10 ⁵ -10 ⁸ cfu / g or 10 ⁵ -10 ⁸ cfu / ml after the inoculation. In the number of bacteria, the incubation time for the production of GABA is longer, and inoculation of more bacteria is burdened on the production of spawn. The inoculated rice island product-containing medium is mixed evenly and proceeds to fermentation at 20-35 ° C., but fermentation does not easily occur at a temperature below that, and heat-weak lactic acid bacteria die at higher temperatures to produce GABA. The time for incubating the lactic acid bacteria is preferably 48-90 hours, more preferably 48-80 hours, and most preferably 48-72 hours.

According to another aspect of the present invention, the present invention comprises the steps of (i) removing the insoluble component from the GABA-containing lactic acid culture medium obtained according to the method of the present invention described above; And (ii) mixing an excipient in the culture solution from which the insoluble component is removed; And (iii) provides a method for producing a GABA-containing powder comprising the step of drying the product of step ii).

According to another aspect of the present invention, the present invention comprises the steps of (i) removing insoluble components from the GABA-containing lactic acid culture medium obtained according to the method of the present invention; And (ii) concentrating the culture solution from which the insoluble component is removed.

The GABA-containing culture medium prepared according to the above-described GABA preparation method of the present invention undergoes a filtration process to remove insoluble components. Subsequently, after the addition of the excipient according to the GABA content of the final product and the drying process, the product is dried by-product (powder). Maltodextrin, skim milk powder, soluble starch, lactose, casein, etc. are mainly used as excipients, and can be dried by freeze drying, spray drying, reduced pressure drying and hot air drying.

If the final product is to be obtained in solution, the culture solution from which the insoluble components are removed is concentrated. Concentration can be carried out through a variety of concentration methods known in the art, it can be easily carried out using a vacuum evaporator (vacuum evaporator).

The fermented rice bran extract by fermentation extract (rice bran fermentation extract and rice bran fermentation extract) obtained by the method of the present invention has a significantly higher GABA content as compared to the conventionally developed GABA-containing products, as well as solubility and It can be used both as insoluble materials. It is to be understood that the GABA-containing products developed by the present invention have many advantages when compared to the conventionally developed GABA-containing products having low GABA content and being limited in their use because they are mostly insoluble in the solid state. Can be.

In addition, by using rice milling by-products that have many nutrients and cannot find their use, they can make the most of the nutrients contained in rice bran and rice bran, and they are expensive high-quality health foods recently recognized for their functionality. The present invention has the meaning of producing using lactic acid bacteria. In addition, by using domestic agricultural products can not only replace GABA imported into the country, but also means that it produces functional materials that can be exported.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Preparation of GABA-Containing Rice Bran Using Solid Culture

After washing 1 kg of rice bran purchased from Nonghyup direct milling plant in 3 L of water, 50 g of sucrose, 20 g of yeast extract and 1 g of polysorbate polysorbate were added. MSG (monosodium glutamate) was added followed by water to sterilize for 25 minutes at 70 ° C. with a total water content of 30%. Lactobacillus sakei B2-16 ( Lactobacillus ) and Lactobacillus brevis B3 incubated in MRS broth (deMan Rogosa Sharpe broth) for 18 hours after cooling the temperature of sterilized rice bran medium to 30 ° C -18, Bioben Co., Ltd. was inoculated to have an initial bacterial concentration of 10 ⁸ cfu / ml, and then mixed evenly in a sterile bag and incubated for 24 hours, 48 hours and 72 hours in a 30 ℃ incubator. The cultured rice bran dough was dried in an oven at 40 ° C. and the GABA content in the rice bran was measured. The content of GABA was measured according to Example 8 using a method of Ibolya et al. (Ibolya Molnar-Perl and Aniko Vasanits, Journal of chromatography A , 835: 73-91 (1999)) modified using reversed phase HPLC.

As shown in Table 1, the GABA content of the final product increased in proportion to the amount of MSG added, but no increase in GABA content was observed from 20% or more. This means that the added lactic acid bacteria have a limit on the amount of decarboxylase produced as they grow using rice bran, and when 30% is added, the yield decreases. It is judged to be a phenomenon. GABA production according to the incubation time for fermentation did not show a significant difference after 48 hours, but less than half in the case of 24 hours, the production of GABA began after the growth of lactic acid bacteria.

GABA production was not significantly different according to the type of inoculated strains. However, when low concentrations of MSG were added, Lactobacillus sakei showed higher GABA production, and Lactobacillus brevis had higher conversion as MSG was added. Could. This is believed to be due to the production capacity of decarboxylase resulting from the differences in the species of lactic acid bacteria.

Example 2: Preparation of GABA High Content Powder Using Rice Bran Extract

1 kg of rice bran purchased from Nonghyup direct mill (RPC) was washed in 3 L of cold water, and then immersed in 10 L of water and extracted at 60 ° C. for 12 hours while stirring at 30 rpm. The extracted rice bran extract was centrifuged to remove solids, and then 3% sucrose, 2% yeast extract, and 0.1% polysorbate were added, and MSG was added at different concentrations and sterilized at 121 ° C. for 15 minutes. 50 ml of Lactobacillus sakei culture incubated for 12 hours in MRS medium in sterilized rice bran extract medium was adjusted to an initial bacterial concentration of 10 ⁷ cfu / ㎖ and then incubated at 30 ℃ for 72 hours. After fermentation of the cultured fermentation broth at 80 ° C for 20 minutes, the cells and insoluble solids were removed using a membrane separator, and maltodextrin was added to adjust the final solids content to 20%, followed by spray drying, to remove the various kinds of GABA contents. Was prepared. The experimental results are shown in Table 2.

As shown in Table 2, when the incubation time was less than 48 hours, it was found that the production of GABA was not sufficient, and there was no increase in the content of GABA even after 72 hours. It was found to be suitable for the production of. In addition, the GABA content of the final product increases according to the amount of MSG added, but the increase rate of GABA production was decreased at the MSG addition amount of 15% or more, and more GABA content was added when 20% or more MSG was added. There was no increase and the GABA content decreased due to the inhibition of fermentation according to MSG concentration. Therefore, the maximum amount of MSG added to minimize the residual amount of MSG and increase the production of GABA was 15%.

Inoculation Incubation time (hr) MSG addition amount (%, w / w) GABA content (%, w / w) Lactobacillus sakei 24 One 0.13 10 1.22 20 1.18 30 0.81 48 One 0.32 10 2.44 20 2.65 30 1.37 72 One 0.35 10 2.58 20 2.61 30 1.52 Lactobacillus brevis 24 One 0.11 10 1.19 20 1.28 30 0.84 48 One 0.29 10 2.25 20 2.44 30 1.31 72 One 0.25 10 2.11 20 2.50 30 1.42

Incubation time (hr) MSG addition amount (%, w / w) GABA content (%, w / w) 12 One 0.1 5 0.3 10 1.1 15 0.9 20 0.8 24 One 0.5 5 3.3 10 10.1 15 9.9 20 8.8 48 One 1.7 5 9.5 10 18.1 15 17.9 20 12.8 72 One 1.8 5 9.6 10 18.2 15 18.8 20 13.5 96 One 1.7 5 9.6 10 18.2 15 18.3 20 12.5

Example 3: Preparation of GABA High Content Powder Using Rice Eye Extract

10 kg of rice snow purchased from Nonghyup direct mill was washed in 50 L of cold water, and then immersed in 80 L of water and extracted at 55 ° C. for 18 hours while stirring at 30 rpm. After centrifugation of the extracted rice bran extract to remove solids, transfer 1 L each to an Erlenmeyer flask and select sucrose, soytone, and polysorbate as carbon, nitrogen, and trace elements, respectively, 3%, 2%, and 0.1%. After the separation was added to the concentration and when not added, 7% MSG was added and sterilized for 15 minutes at 121 ℃. Inoculated with 1000 ㎖ of Lactobacillus Sakei culture incubated for 16 hours in MRS medium in the sterilized rice eye extract medium to adjust the initial bacterial concentration to 10 ⁸ cfu / ㎖ and incubated at 30 ℃ for 72 hours. After fermentation of the cultured fermentation broth at 80 ° C for 20 minutes, the cells and insoluble solids were removed using a membrane separator, and maltodextrin was added to adjust the final solids content to 20%, followed by spray drying. Was prepared. The experimental results are shown in Table 3.

Carbon source Nitrogen source Trace elements GABA content (%, w / w) O O O 15.3 O O X 12.5 O X O 7.8 O X X 5.3 X O O 3.9 X O X 2.4 X X O 1.9 X X X 1.1

As shown in Table 3, when the carbon source, the nitrogen source and the trace element were added to the rice eye extract, it was possible to prepare the rice eye extract having a high GABA content, but when one of the three was omitted, the GABA content was lowered. In particular, when the carbon source was not supplemented, the GABA production decreased drastically, and even when the nitrogen source was omitted, the amount decreased by nearly 50%. Therefore, it can be seen that the production of GABA using rice bran and rice bran requires supplementation of an appropriate amount of carbon source, nitrogen source and trace components.

Example 4: Changes in GABA Content with Addition of Rice Bran and Rice Bran Nutrients

500 g of rice bran and rice bran, respectively, purchased from Nonghyup direct factory, were washed in 2 L of cold water, and then put in 5 L of water and extracted at 60 ° C. for 12 hours. Centrifuge the extracted rice bran and rice bran extract to remove solids, transfer 1 L each to Erlenmeyer flask, add 3% sucrose, 1% yeast extract, and 0.1% sodium acetate, and add MSG of 10% at 121 ℃. Sterilization for 15 minutes. In another Erlenmeyer flask, add 1 liter of distilled water without using rice bran and rice snow extract, add 3% sucrose, 1% yeast extract, and 0.1% sodium acetate, add 10% MSG, and sterilize at 121 ℃ for 15 minutes. It was. Each sterilized flask was inoculated with Lactobacillus sakei culture incubated for 12 hours in MRS medium to adjust the initial bacterial concentration to 10 ⁸ cfu / ㎖ and then incubated at 30 ℃ for 70 hours. Table 4 shows the results of measuring the GABA content of the culture broth prepared by using distilled water instead of rice bran and rice bran fermentation broth and rice by-product extract.

Badge composition Final cell count (cfu / ml) PH after fermentation GABA concentration (mM) Rice bran extract, sucrose, yeast extract, sodium acetate, MSG 2.7 × 10 ⁹ 6.55 544 Rice Eye Extract, Sucrose, Yeast Extract, Sodium Acetate, MSG 2.5 × 10 ⁹ 6.48 529 Distilled water, sucrose, yeast extract, sodium acetate, MSG 6.4 × 10 ⁸ 4.87 10

As shown in Table 4, unlike the case of using rice by-products of rice bran and rice snow extract, the culture of lactic acid bacteria was added to the distilled water by adding other media components. Not converted. This result indicates that the rice by-product added to the medium contains essential ingredients in the production of enzymes for converting MSG to GABA during fermentation.

Example 5: Change of GABA Content with Extraction Temperature of Rice Bran and Rice Bran Nutrients

Rice snow and 1 kg of rice bran purchased from Nonghyup direct factory were washed in 4 L of cold water, and then extracted into 12 L of water for 12 hours. Centrifuged rice bran and rice bran extracts under each condition to remove solids, transfer 1 L to Erlenmeyer flasks, add 2% sucrose, 1% yeast extract, and 0.05% magnesium sulfate, and then add 10% MSG. Sterilization for 15 minutes at 121 ℃. Inoculated with Lactobacillus brevis culture medium incubated in MRS medium for 12 hours in rice bran and rice bran extract medium was adjusted to an initial bacterial concentration of 10 ⁸ cfu / ㎖ and then incubated at 30 ℃ for 72 hours. The GABA content of the rice bran and rice bran fermentation broths after the incubation was measured. The measurement results are shown in Table 5.

In Table 5, the yield of GABA was similar for both rice and rice bran, but when the solid content was high after extraction, the production of GABA was high and the final pH of the fermentation broth increased when the production of GABA was high. This means that the extraction of many nutrients from rice bran or rice bran is a favorable condition for the production of GABA, and is consistent with the findings that the pH increases because GABA consumes H ⁺ ions in the medium.

When the temperature is low, the extraction efficiency decreases, and at 90 ° C, the extraction solid content is not only low but also the production of GABA is low. This is because the starch contained in rice bran or rice flakes is gelatinized. This is because it is lowered. Therefore, the extraction temperature of rice bran and rice bran for producing GABA is judged to be suitable for 40-70 ℃. In addition, the number of microorganisms in the culture medium was not significantly related to the production of GABA, but showed a tendency to increase when the extracted solid content was high.

Raw material Extraction temperature (℃) Extract solid content (%) Final cell count (cfu / ml) PH after fermentation GABA concentration (mM) Rice eyes 10 1.13 2.0 × 10 ⁹ 5.18 218 40 2.25 1.5 × 10 ⁹ 6.32 444 70 3.12 2.3 × 10 ⁹ 6.66 500 90 2.28 2.2 × 10 ⁹ 5.77 383 Rice bran 10 1.33 2.4 × 10 ⁹ 4.98 250 40 2.65 2.5 × 10 ⁹ 6.19 480 70 4.00 2.8 × 10 ⁹ 6.75 550 90 1.23 2.5 × 10 ⁹ 5.55 400

Example 6: Changes in GABA Content with Extraction Concentrations of Rice Bran and Rice Bran

5 kg of rice bran and rice bran were each washed in 20 L of cold water, and extracted at 60 ° C. for 12 hours while varying the amount of water. The rice bran and rice bran extracts extracted under each condition were centrifuged to remove solids, followed by 4% sucrose, 2% yeast extract, and 0.1% polysorbate, followed by 10% MSG and sterilization at 121 ° C. for 15 minutes. Inoculated with lactobacillus brevis culture incubated in MRS medium for 18 hours in rice bran and rice bran extract medium was adjusted to an initial bacterial concentration of 10 ⁷ cfu / ㎖ and then incubated at 30 ℃ for 72 hours. After culturing the fermented broth at 80 ° C. for 20 minutes and removing the cells and insoluble solids using a membrane separator, the GABA content was measured, and the results are shown in Table 6.

In the case of GABA production using rice bran and rice bran extract, if the solid content of the extract is high depending on the extraction temperature, sufficient amount of nutrient is supplied to produce GABA, but GABA production is higher when extracting with less water. The content was lowered. This is believed to be due to the fermentation inhibition phenomenon due to the viscosity of the fermentation broth and excessive nutrients. However, when the fermentation was completed, the number of lactic acid bacteria was high, but this also did not show a result proportional to the production of GABA.

Therefore, when producing GABA using rice bran and rice bran, the concentration of the most preferred extract was found to use 5 times-15 times of water.

Raw material Hydrogen amount (multiple) PH after fermentation Final cell count (cfu / ml) GABA (mM) Rice eyes One 5.18 3.2 × 10 ⁹ 218 5 6.22 2.5 × 10 ⁹ 444 10 6.66 2.3 × 10 ⁹ 489 15 6.17 1.8 × 10 ⁹ 483 20 4.33 3.4 × 10 ⁸ 256 Rice bran One 5.03 3.2 × 10 ⁹ 218 5 6.33 2.5 × 10 ⁹ 444 10 6.56 2.3 × 10 ⁹ 489 15 6.47 1.8 × 10 ⁹ 483 20 4.11 4.9 × 10 ⁸ 256

Example 7 Preparation of GABA-Containing Materials Using Precursor Rice Bran and Rice Bran Mixture Extracts

Rice and rice bran were mixed 5 kg each, washed, 50 L of water was added, and extracted at 60 ° C. for 15 hours. The extracted rice bran and rice bran mixed extract were centrifuged to remove solids, followed by 2% sucrose, 2% yeast extract, and 0.05% polysorbate, followed by sterilization at 121 ° C. for 15 minutes by the addition of 10% MSG. Inoculated with lactobacillus brevis culture medium incubated in MRS medium for 18 hours in rice bran and rice bran mixed extract medium was adjusted to an initial bacterial concentration of 10 ⁷ cfu / ㎖ and then incubated at 30 ℃ for 72 hours. After culturing the fermented broth at 80 ° C for 20 minutes, the cells and insoluble solids were removed using a membrane separator, and maltodextrin was added to adjust the final solids content to 20%. Rice bran and rice bran mixed fermentation extracts were prepared.

Example 8 Preparation of GABA-High Containing Solution of Rice Bran Raw Materials

In order to make GABA high content of rice bran extract for mixing in the beverage, 3 kg of rice bran was put in 33 L water and extracted at 65 ° C. for 18 hours. After extraction, the solids were removed by centrifugation, and 3% of sucrose, 1.5% of yeast extract, and 0.1% of polysorbate were added, followed by 9% of MSG, followed by sterilization for 15 minutes at 121 ° C. The sterilized rice bran extract medium was inoculated with Lactobacillus sakei culture incubated for 12 hours in MRS medium to adjust the initial bacterial concentration to 10 ⁸ cfu / ㎖ and then incubated at 30 ℃ for 72 hours. The cultured fermentation broth was sterilized at 80 ° C for 20 minutes, and then cells and insoluble solids were removed using a membrane separator, and then concentrated to 10 times at a temperature of 15 torr and 60 ° C using a vacuum evaporator. A solution of at least% was prepared.

Example 9 Quantitative Analysis of GABA Using Reversed Phase HPLC

Quantitative analysis of GABA in this example was determined using reverse phase HPLC (HPLC Waters) as follows: Analytical conditions using reverse phase HPLC were established with reference to what Ibolya et al reported. First, the sample was centrifuged at 8000 rpm for 10 minutes, and then the supernatant was filtered through a membrane filter and diluted in tertiary distilled water to an appropriate concentration. The thus prepared samples o - au taldi aldehyde (o -phthaldialdehyde: OPA) free with - after derivatization process of the column reaction was carried out reversed-phase HPLC. OPA solution (pH 9.3) was prepared by mixing 5.0 mL of methanolic OPA, 20 mL borate buffer (pH 9.9) and 50 μl 2-mercaptoethanol. Methanolic OPA was prepared by dissolving 2.56 g of OPA in 50 ml of methanol.Borate buffer was mixed with 0.2 M boric acid and 0.2 M sodium hydroxide 50:50 (v / v), followed by 0.2 M potassium chloride. Used. The OPA solution was used after 2 hours and stable until about one week after preparation. 380 μl of the prepared OPA solution and 120 μl of the sample were mixed well and allowed to react at room temperature for about 8 minutes. If left at room temperature for too long, reverse phase HPLC may have different peak shape or area value due to instability of the derivative. Therefore, reverse phase HPLC was performed without standing at room temperature for 1-2 hours or longer. Injected.

XTerra column (Waters, RP18 5 m, 4.6 mm 150 mm) was used as HPLC column, 0.05 M sodium acetate (pH 7.2) as solvent A and 0.1 M sodium acetate, acetonitrile (HPLC grade) as mobile phase. And methanol (HPLC grade) mixed at 46:44:10 (v / v) (pH 7.2) as solvent B, respectively. The concentration gradient of the mobile phase was 100% of solvent A. After 30 minutes, solvent B became 100%. After 40 minutes, solvent B was 100%. After 45 minutes, solvent A was 100 again. % And solvent A was adjusted to 100% until 60 minutes later. The flow rate of the mobile phase was fixed at 1 ml / min and U. V. 358 nm. GABA was detected with a detector. Under these conditions, the retention times of GABA and glutamate were 21.01 and 9.89 minutes, respectively, and the limit of detection was 0.1 mM. In addition, when the concentration of GABA exceeds 10 mM, the detection limit of the detector is exceeded, so it was diluted and measured accordingly.

As described in detail above, the present invention provides a method for preparing GABA (γ-Aminobutyric acid) using rice bran and / or rice bran and lactic acid bacteria. The present invention also provides a process for preparing GABA-containing powders and solutions. Rice bran by-product fermentation extracts (rice bran fermentation extracts and rice bran fermentation extracts) obtained by the method of the present invention have a significantly higher GABA content than conventionally developed GABA-containing products as well as solubility and It can be used both as insoluble materials. In addition, according to the present invention, while using a nutritious by-products that have a lot of nutrients, but can not increase the value added because they can not find its use, while maximizing the nutritional ingredients contained in rice bran and rice snow, high-quality health food recently recognized its functionality Expensive GABA can be produced in large quantities.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (12)

(Iii) preparing a medium comprising any one selected from the group consisting of rice bran, rice bran, a mixture of rice bran and rice bran, rice bran extract, rice bran extract, and a mixture of rice bran and rice bran; (Ii) adding MSG (monosodium glutamate) to the medium prepared in step (iii) and inoculating the medium with lactic acid bacteria Lactobacillus sakei; And (Iii) Lactobacillus sakei consisting of culturing the lactic acid bacteria inoculated in the step (ii) to convert the MSG to GABA by the glutamate decarboxylase possessed by the lactic acid bacteria to produce GABA Method for preparing GABA (γ-Aminobutyric acid) by). delete delete The method of claim 1, wherein the medium further comprises a carbon source, a nitrogen source, a trace element, a surfactant, or a mixture thereof. The method of claim 4, wherein the carbon source is selected from the group consisting of glucose, sucrose, maltose, fructose, lactose, xylose, galactose, arabinose or combinations thereof. 5. The method of claim 4, wherein the nitrogen source is any one selected from the group consisting of yeast extract, soytone, peptone, beep extract, tryptone, cachetone and combinations thereof. 5. The method of claim 4, wherein the trace element is any one selected from the group consisting of magnesium sulfate, sodium acetate, manganese sulfate, ferric sulfate, calcium chloride and combinations thereof. The method of claim 4, wherein the surfactant is any one selected from the group consisting of polyalcohol fatty acid, polyethylene glycol fatty acid, polyoxyethylene alcohol, sorbitan fatty acid, polyoxyethylene sorbitan fatty acid, and combinations thereof. . The method of claim 1, wherein the amount of MSG added is 1-15% by weight of the natural ingredient medium. The method of claim 1, wherein the culturing of step (c) is carried out for 48-72 hours. (Iii) removing the insoluble components from the GABA-containing lactic acid culture medium obtained according to any one of claims 1 and 4 to 10; And (Ii) mixing the excipients into the culture medium from which the insoluble components prepared in step (iii) have been removed; And (Iii) a method for producing a GABA-containing powder, comprising drying a culture solution containing an excipient prepared from step (ii). (Iii) removing the insoluble components from the GABA-containing lactic acid culture medium obtained according to any one of claims 1 and 4 to 10; And (Ii) concentrating the culture solution from which the insoluble component prepared in step (iii) has been removed.

Images