KR101477696B1 - Novel Lactobacillus sp. for brown rice fermentation and brown rice fermentation method using thereof - Google Patents

Abstract

The present invention relates to a method for producing fermented brown rice by identifying a new strain of Lactobacillus genus for brown rice fermentation and fermenting brown rice using the same. Using the novel strain of Lactobacillus genus for the brown rice fermentation of the present invention and the fermented brown rice manufacturing method using the same, it is possible to survive the lactic acid bacteria and the yeast for a considerable period of time in the brown rice fermentation, Particularly, the effect of significantly increasing the production of gamma-aminobutyric acid (GABA). GABA has been reported to be capable of controlling various physiological mechanisms such as sedation effect, antistress effect and blood pressure, normalizing function of renal function, suppression of increase of blood cholesterol and triglyceride, obesity, and visual acuity improvement. It would be useful for the development of various medicines, health supplements and functional beverages. In addition, when the strain of the present invention is used as a strain of the genus Lactobacillus, it is possible to produce a GABA high-content fermented brown rice only by adding glutamic acid at a low concentration, so that it can be used for simple and economical functional fermented rice mass production.

Description

(Novel Lactobacillus sp. ≪ RTI ID = 0.0 > l. ≪ / RTI > for brown rice fermentation and brown rice fermentation method using thereof}

The present invention relates to a method for producing fermented brown rice by identifying a new strain of Lactobacillus genus for brown rice fermentation and fermenting brown rice using the same.

In general, the process of shredding rice husks and rice bran layers to make them look good and eat rice is called rice bran. The brown rice is the rice bran layer only, which is composed of three parts: seed bran, perilla, and endosperm. Vitamins, minerals and other nutrients are high in content of fiber is rich in the prevention of adult diseases is a health food material has been attracting attention. In addition, brown rice regulates unbalanced nutrients in the body, strengthens the bones, nourishes in the process of fermentation in the colon, inhibits the development of colorectal cancer, lowers the cholesterol in the blood, .

However, brown rice has a weak absorption of digestion in the body, fermented brown rice fermented brown rice is widely used as a health food. On the other hand, if brown rice is fermented, functional materials that have not been previously found in brown rice may be produced or the amount of production thereof may increase. Its representative material is gamma-aminobutyric acid (GABA).

GABA is an abbreviation of gamma-aminobutyric acid. It has been known for a long time that it exists widely in the plant kingdom, but it was first known in the 1950's that it exists in the brain in animals. It has been confirmed that it is biosynthesized in glutamic acid, and has an action to suppress nerve excitement, and has a sedative effect, antistress effect, blood pressure, and renal function normalizing action. These GABAs play an important role in the central nervous system as inhibitory neurotransmitters and have been developed as drugs for improving brain metabolism, such as improving cerebral blood flow.

In animals, it is the main inhibitory neurotransmitter of the central nervous system, accounting for about 30% of total neurotransmitters, and is known to exist at a high concentration of about 200-1000 times that of other neurotransmitters. GABA is a non - amino acid that is produced by the activity of glutamate decarboxylase (GAD) enzyme using glutamate, a precursor. GABA is not only an important neurotransmitter in the central nervous system, but also involved in the regulation of various physiological mechanisms such as inhibition of blood cholesterol and triglycerides, obesity, and visual acuity, and is involved in the regulation of growth hormone. It is the substance that is received. Especially, when fermenting brown rice, it is known that functional components such as GABA are increased by various enzymes.

Therefore, it is expected that the development of new strains capable of effectively fermenting brown rice under these conditions by establishing the conditions of brown rice fermentation will be highly applicable to pharmaceuticals, health supplements and functional beverages, and development of such new strains is required.

1. Korean Patent Publication No. 10-1991-0011168 2. Korean Patent Publication No. 10-1984-0000640 3. Korean Patent Publication No. 10-2011-0107011

An object of the present invention is to identify and provide novel strains capable of effectively fermenting brown rice under these conditions by setting conditions for fermentation of brown rice.

Another object of the present invention is to provide an optimum method for producing fermented brown rice using the new strain.

It is still another object of the present invention to provide a fermented brown rice mass production method in which the content of GABA, which is a functional substance, is increased by using the new strain.

In order to achieve the above object, the present invention provides a food-effective Lactobacillus ( Lactobacillus sp . ) KFCC11545P strain.

In one embodiment of the present invention, the food may be brown rice.

In one embodiment of the present invention, the strain may ferment brown rice to increase the amount of GABA (gamma-aminobutyric acid) produced.

The present invention also relates to a pharmaceutical composition comprising (a) Lactobacillus sp . ) Culturing a strain of KFCC11545P; And (b) culturing the cultured Lactobacillus genus sp . ) A step of inoculating a strain of KFCC11545P into brown rice and fermenting the fermented brown rice.

In one embodiment of the present invention, the Lactobacillus sp . ) The KFCC11545P strain is cultured on an MRS liquid medium, which liquid medium may comprise MSG at a concentration of 0.1-5%, based on the total weight.

In one embodiment of the present invention, the Lactobacillus sp . ) The KFCC11545P strain is cultured on an MRS liquid medium, wherein the liquid medium comprises glutamic acid at a concentration of 0.1 to 5% based on the total weight.

In one embodiment of the present invention, the fermentation step is compared to the brown rice 100g genus Lactobacillus (Lactobacillus sp . ) KFCC11545P strain 1 x 10 < ⁹ > to 1 x 10 < ¹¹ >

In one embodiment of the present invention, the fermentation step is carried out using the Lactobacillus sp . ) KFCC11545P strain and yeast can be inoculated together.

In one embodiment of the present invention, the yeast is Saccharomyces cerevisiae S. cerevisiae strain KACC30008.

In one embodiment of the present invention, the Lactobacillus sp . ) Strain KFCC11545P and Saccharomyces cerevisiae The S. cerevisiae strain KACC30008 can be inoculated at a ratio of 1: 1 (v / v).

The present invention also relates to a method for producing glutamic acid, comprising the steps of (i) adding a solution containing glutamic acid to a solution of Lactobacillus sp . ) A step of preparing a suspension by adding and dispersing KFCC11545P strain; (Ii) inoculating the strain by spraying the prepared suspension into brown rice; And (iii) fermenting the inoculated brown rice. The present invention also provides a method for mass production of fermented brown rice containing GABA.

In one embodiment of the present invention, the suspension sprayed on the brown rice in the step (ii) may contain glutamic acid at a concentration of 0.1 to 0.7% (w / w) based on the weight of the brown rice.

In one embodiment of the present invention, the strains inoculated in (ⅱ) step is Lactobacillus genus per microscopic 1g (Lactobacillus sp . ) It may be inoculated with the strain KFCC11545P 1 × 10 ⁹ to 1 × 10 ^11.

In one embodiment of the present invention, in the step (iii), the fermentation may be performed at a temperature of 28 ° C to 32 ° C for 20 hours to 30 hours.

In one embodiment of the present invention, the GABA may be contained in an amount of 1.0 to 1.8 mg per 1 g of brown rice.

Using the novel strain of Lactobacillus genus for the brown rice fermentation of the present invention and the fermented brown rice manufacturing method using the same, it is possible to survive the lactic acid bacteria and the yeast for a considerable period of time in the brown rice fermentation, Particularly, the effect of significantly increasing the production of gamma-aminobutyric acid (GABA). GABA has been reported to be capable of controlling various physiological mechanisms such as sedation effect, antistress effect and blood pressure, normalizing function of renal function, inhibition of increase of blood cholesterol and triglyceride, obesity, and visual acuity improvement. It would be useful for the development of various medicines, health supplements and functional beverages.

In addition, when the strain of the present invention is used as a strain of the genus Lactobacillus, it is possible to produce a GABA high-content fermented brown rice only by adding glutamic acid at a low concentration, so that it can be used for simple and economical functional fermented rice mass production.

Figure 1 shows the results of GABA production of various Kimchi isolates using TLC (1: 100 μM MSG, 2: 100 μM GABA, MRS containing 3: 5% MSG, and MRS containing 4: 5% MSG lb. brevis KACC14481, 5: GU240, 6-14: Kimchi-isolated lactic acid bacteria samples (GU414, GU498, GU1029, GU1062, GU1063, GU1064, ED141, ED143, PM85).
Fig. 2 shows the result of observation of GU240 strain under a microscope (left: no staining, right: gram staining).
Figure 3 shows the result of confirming colony form (left) and exopolysaccharide production (right) of GU240 strain.
Figure 4 shows the results of confirming the growth of GU240 according to various temperatures.
FIG. 5 shows the results of confirming the growth of GU240 according to various initial pH conditions.
FIG. 6 shows the result of confirming the degree of growth of various concentrations of salt of GU240.
7 shows the results of confirming the production of GABA in fermented brown rice using GU240 (A, immediately after inoculation of the strain; B, 48 hours fermented brown rice sample; 1, glutamic acid; 29.43 g); 2, MSG (33.82 g) GABA (20.62 ㎍); 4, the control-strains not inoculated (ddH ₂ O); 5, the control (10 mM glutamic acid); 6 , the control (1% MSG); 7, the control (3% MSG); 8, experimental Lb zymae GU240 one strain inoculated _{(ddH 2 O);. 9} , experimental (10 mM glutamic acid); 10 , experimental group (1% MSG); 11, experimental group (3% MSG); 12, Lb. zymae GU240 culture supernatant).

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are for illustrative purposes only and that the scope of the present invention is not limited by these embodiments.

< Example  1>

Establishment of brown rice fermentation condition using lactic acid bacteria of various genus

In order to find lactic acid bacteria suitable for brown rice fermentation, the present inventors have used Lactobacillus Genus, Leuconostoc And Pediococcus And lactic acid bacteria were used in the experiment. A total of 13 lactic acid bacteria were selected and each strain was used as an inoculum strain for the brown rice fermentation experiment. 7 species of Lactobacillus genus, 5 species of Leuconostoc genus and 1 genus of Pediococcus genus were used respectively. Species names and standard strain numbers are shown in Table 1 below.

Lactic acid bacteria used for brown rice fermentation Strain name Lactobacillus casei ATCC4646 Lactobacillus brevis 2.14 Lactobacillus amylovorus B4540 Lactobacillus fermentum ATCC14931 Lactobacillus paraplantarum C7 Lactobacillus plantarum ATCC8014 Lactobacillus plantarum ATCC10241 Leuconostoc mesenteroides ATCC10830 Leuconostoc mesenteroides ATCC9135 Leuconostoc citreum ATCC49370 Leuconostoc gelidum ATCC49366 Leuconostoc mesenteroides SY1 Pediococcus pentosaceus NRRL D-14009

<1-1> Condition setting of brown rice fermentation process

Microscopic 13 kinds of lactic acid bacteria used in the fermentation process are inoculated into 50 ml MRS broth, respectively, incubated with each 2 × centrifuged culture broth equal to 10 ¹⁰ bacteria count obtained cell clusters and then (4 ℃, 12,000 rpm, 15 minutes) Respectively. The cell clusters were completely dissolved in 30 ml of distilled water and inoculated in brown rice. Brown rice was produced in the year 2000 in Gyangyang, Gyeongnam Province. 100 g of brown rice were each poured into 500 ml bottles, and then the above-mentioned lactic acid bacteria (2 × 10 ¹⁰ ) dissolved in water was added thereto and mixed well using a sterilized spoon. Thereafter, the mixture was fermented at 30 DEG C for 12 hours.

<1-2> Drying and storage conditions of fermented brown rice

The brown rice samples which had been fermented through Example <1-1> were spread on a 15-cm diameter Petri dish and dried at 45 ° C for 36 hours. After drying, each sample was divided into two 50 ml conical tubes and stored at 4 ° C (refrigerated) and at room temperature, respectively. To determine the viable cell counts during 30 days of storage, 10 g of samples were collected on days 3, 9, 18 and 30 of storage and used for the measurement of viable counts by the stepwise dilution method.

<1-3> Fermented brown rice Viable cell count  Research

Fermented brown rice samples were collected six times in total, and samples were collected at the time of completion of fermentation, at the time of completion of drying, at the end of drying, at 4 ° C. and room temperature, and at 3, 9, 18 and 30 days, Changes were observed. To 10 g of the sample, 90 ml of the peptone was added and homogenized for 2 minutes using a stomacher. The homogenized samples were subjected to step dilution and cultured on MRS plate medium supplemented with cycloheximide (50 μg / ml, for the selection of lactic acid bacteria) for 24 hours at 30 ° C. The number of colonies was counted, Respectively.

It was to determine the results shown in Table 2. As a result, that yieoteuna initial inoculation bacteria are lactic acid bacteria of 2 × ¹⁰ 10 per 100 g rice, 12 hours after fermentation, Lb. plantarum It was confirmed that the number of viable cells increased about 10 times in all strains except ATCC8014. After drying, 10 ⁷ -10 ¹⁰ viable cell counts per 100 g of brown rice were observed depending on the strains. Lb. plantarum At the end of fermentation, ATCC8014 did not increase the viable cell count, but after drying, it was 10 ¹⁰ per 100 g of brown rice. Leu . gelidum At the end of fermentation, ATCC49366 and P. pentosaceus NRRL D-14009 increased the number of viable bacteria by about 10 times, and after drying, decreased to 10 ¹⁰ viable cells per 100 g of brown rice. On the other hand, the decrease in other strains was slightly larger.

On the third day of storage, the number of viable cells per 100 g of brown rice was 10 ⁷ -10 ¹⁰ , indicating that the viable count was maintained or decreased by about 10 times as compared with the sample immediately after drying. The longer the storage time of the number of viable cells was gradually reduced but the beam slightly different in the decline by each strain, the number of live cells according to the storage temperature is stored three days did not significantly differ, Lb. casei ATCC 4646, Lb. brevis Some Lactobacillus, such as 2.14 The results showed that the viable cell count at room temperature was lower than that stored at 4 ℃. On the other hand, in case of storage at room temperature, fungal contamination was observed as the storage period became longer. In the case of storage samples at 30 days, the number of viable cells in some strains is 10 ⁷ -10 ⁶ per 100 g of brown rice, but Leu . citreum ATCC 49370, Leu . gelidum ATCC 49366 and P. pentosaceus NRRL D-14009 showed higher viable counts than other strains at 10 ⁸ -10 ⁹ . Lb. casei ATCC4646, Lb. brevis And 2.14 Lb. plantarum Three strains of ATCC8014 were found in other Lactobacillus And the number of viable cells was relatively high at 10 ⁸ compared to the genus.

Based on the above results, the present inventors determined that the above-mentioned lactic acid bacteria strains were suitable for use as a seed of brown rice fermentation.

Changes in viable cell counts during fermentation, drying and storage of fermented brown rice using various lactic acid bacteria (per 100 g of brown rice samples) No Strain inoculation
Strain Primary sample Secondary
Sample The third sample,
Day 3 of storage Fourth sample
Day 9 of storage 5th sample
Save 18 days 6th sample
Save 30 days 4 ℃ storage Room temperature storage 4 ℃ storage Room temperature storage 4 ℃ storage Room temperature storage 4 ℃ storage Room temperature storage # 108 Lb. casei ATCC4646 2.01 × ¹⁰ 2.5 × 10 ¹¹ 7.1 x 10 ⁹ 2.6 x 10 ⁹ 1.3 x 10 ⁹ 8.4 × 10 ⁸ 4.0 × 10 ⁷ 3.8 × 10 ⁸ 6.8 × 10 ⁶ 1.4 x 10 ⁸ Fungal contamination # 110 Lb. brevis 2.14 2.0 × 10 ¹⁰ 1.8 × 10 ¹¹ 7.6 × 10 ⁹ 5.2 × 10 ⁹ 1.1 × 10 ⁹ 1.8 × 10 ⁹ 1.2 × 10 ⁷ 1.0 × 10 ⁹ 4.7 × 10 ⁶ 6.4 × 10 ⁸ Fungal contamination # 118 Lb. amylovorus B4540 2.0 × 10 ¹⁰ 1.4 × 10 ¹¹ 1.3 x 10 ⁸ 1.1 x 10 ⁸ 9.0 × 10 ⁷ 1.0 × 10 ⁸ 4.7 × 10 ⁷ 8.0 × 10 ⁷ 3.4 × 10 ⁷ 5.9 x 10 ⁷ 1.5 x 10 ⁷ # 209 Lb. fermentum ATCC14931 2.0 × 10 ¹⁰ 2.5 × 10 ¹¹ 1.1 x 10 ⁸ 3.2 × 10 ⁷ 6.8 × 10 ⁷ 7.5 × 10 ⁶ 1.0 x 10 ⁷ 7.1 × 10 ⁶ Fungal contamination 1.7 × 10 ⁷ Fungal contamination # 284 Lb. paraplantarum C7 2.0 × 10 ¹⁰ 1.9 × 10 ¹¹ 1.7 x 10 ⁸ 2.4 × 10 ⁷ 5.4 × 10 ⁷ 1.6 × 10 ⁷ 2.2 x 10 ⁷ 1.3 × 10 ⁶ 1.6 × 10 ⁶ 1.0 × 10 ⁶ Fungal contamination # 317 Lb. plantarum ATCC8014 2.0 × 10 ¹⁰ 1.8 × 10 ¹⁰ 1.5 × 10 ¹⁰ 6.2 × 10 ⁹ 6.7 × 10 ⁹ 3.1 × 10 ⁶ 2.6 x 10 ⁶ 2.9 × 10 ⁶ 2.0 × 10 ⁶ 1.4 × 10 ⁶ 1.1 × 10 ⁶ # 318 Lb. plantarum ATCC10241 2.0 × 10 ¹⁰ 1.2 × 10 ¹¹ 4.8 x 10 ⁸ 2.2 x 10 ⁸ 4.3 × 10 ⁸ 2.5 x 10 ⁷ 2.3 × 10 ⁷ 2.3 × 10 ⁶ 2.1 × 10 ⁶ 1.2 × 10 ⁶ Fungal contamination # 235 Leu . mesenteroides ATCC10830 2.0 × 10 ¹⁰ 1.9 × 10 ¹¹ 1.5 x 10 ⁷ 6.9 × 10 ⁶ 5.5 × 10 ⁶ 5.0 × 10 ⁶ 4.7 × 10 ⁶ 4.7 × 10 ⁶ 4.1 × 10 ⁶ 1.5 × 10 ⁶ Fungal contamination # 240 Leu . mesenteroides ATCC9135 2.0 × 10 ¹⁰ 1.2 × 10 ¹¹ 2.1 x 10 ⁸ 1.8 x 10 ⁷ 6.4 × 10 ⁷ 9.1 × 10 ⁶ 4.6 × 10 ⁷ 2.7 × 10 ⁶ 1.3 x 10 ⁷ 2.9 × 10 ⁶ Fungal contamination # 313 Leu . citerum ATCC49370 2.0 × 10 ¹⁰ 2.9 × 10 ¹¹ 1.7 x 10 ⁹ 1.1 × 10 ⁹ 2.2 x 10 ⁹ 1.4 x 10 ⁹ 1.8 × 10 ⁹ 1.1 × 10 ⁹ 7.4 × 10 ⁸ 1.7 x 10 ⁹ 7.9 × 10 ⁸ # 314 Leu . gelidum ATCC49366 2.0 × 10 ¹⁰ 2.7 × 10 ¹¹ 1.0 × 10 ¹⁰ 1.2 × 10 ¹⁰ 1.0 × 10 ¹⁰ 9.1 × 10 ⁸ 9.5 × 10 ⁸ 5.6 x 10 ⁸ 5.3 × 10 ⁸ 4.9 × 10 ⁸ 5.2 × 10 ⁷ # 285 Leu . mesenteroides SY1 2.0 × 10 ¹⁰ 1.5 × 10 ¹¹ 4.5 × 10 ⁹ 7.2 × 10 ¹⁰ 2.1 × 10 ⁹ 1.7 x 10 ⁸ 2.9 × 10 ⁸ 1.5 × 10 ⁸ 2.0 x 10 ⁸ 9.4 × 10 ⁷ Fungal contamination

< Example  2>

Establishment of brown rice fermentation condition using lactic acid bacteria and yeast

After selecting the four strains having relatively high viable counts as a result of the experiment for setting the first fermentation condition using the above-mentioned various kinds of lactic acid bacteria, the inventors of the present invention mixed the selected strains with the yeast Fermentation, and in this case, the fermentation using a single strain.

In addition, the present inventors have confirmed that this can help solve the problem of odor caused by prolonged fermentation. On the other hand, when the fermentation time and the water content to be added were variously set, the number of viable bacteria and pH change were checked.

First, the inventors of the present invention found that four kinds of lactic acid bacteria ( P. pentosaceus NRRL D-14009, Leu . Citreum ATCC 49370, Leu . gelidum ATCC49366 and Lb. plantarum ATCC 8014) and Saccharomyces yeast cerevisiae KACC30008 strain was mixed with 1: 1 (v / v) and used for fermentation. The total number of lactic acid bacteria and yeast were 5.0 × 10 ^{10, and} 1.0 × 10 ¹¹ bacteria were inoculated into 100 g of brown rice.

On the other hand, the present inventors experimented with 15, 30 and 50 ml of distilled water added to 100 g of brown rice. In the same manner as in Example 1, the cell clusters corresponding to the number of inoculated cells were dissolved in sterilized distilled water of 15, 30, and 50 ml, respectively, and then added to 100 g of brown rice and mixed with a sterilized spoon. Fermentation was carried out at 30 ℃ for 10 days to investigate the difference in the number of lactic acid bacteria in fermentation time. Samples were collected at intervals of 2 days to measure the viable cell count and pH, and the sampling method was the same as that described in Example 1.

As can be seen in the following Tables 3 and 4, the number of lactic acid bacteria was about twice as high as that of inoculated lactic acid bacteria on the second day of fermentation, and about 10 ¹⁰ viable cell counts were maintained until the 10th day of fermentation I could confirm. On the other hand, yeast maintained about 10 ⁸ viable cell counts from the second day of fermentation, but decreased slightly and decreased to 10 ⁷ on the 10th day of fermentation. It was judged that the growth of yeast was decreased by lactic acid bacteria. Lactic acid bacteria were also detected in the control group not inoculated with the strain, suggesting that the lactic acid bacteria present in the brown rice itself proliferated.

On the other hand, when the pH was changed, the pH was slightly lowered from 7.2 to 7.0 in the immediately after inoculation of each strain, but the pH gradually decreased as the fermentation progressed. It remained at pH 4.2 to 4.9.

Comparing the number of viable cells according to the addition of distilled water, 15% distilled water added samples showed low viable count, but 30% and 50% did not show any significant difference. The pH decreased as the amount of water added increased, which is thought to be due to the increase in the number of lactic acid bacteria. On the 10th day after fermentation, the number of viable cells decreased more than that on the 2nd day. Based on the results, fermentation of brown rice with lactic acid bacterium for 2 days or more is not preferable in the number of lactic acid bacteria.

Changes in viable cell count during fermentation period by mixed fermentation of yeast and lactic acid bacteria (per 100g of brown rice sample) Strain Water addition amount
(ml) Inoculation number 2nd day of fermentation On the fourth day of fermentation 6th day of fermentation Eighth day of fermentation On the 10th day of fermentation Lactobacillus leaven Lactobacillus leaven Lactobacillus leaven Lactobacillus leaven Lactobacillus leaven Control
(Lactic acid bacteria and yeast
Not inoculated) 15 - 3.0 x 10 ⁴ - 1.2 × 10 ⁸ - 2.4 × 10 ¹⁰ - 2.1 × 10 ¹⁰ - 1.8 × 10 ⁹ - 30 - 4.2 × 10 ⁵ - 4.2 × 10 ⁸ - 8.4 × 10 ¹⁰ - 3.1 × 10 ¹⁰ - 2.1 × 10 ⁹ - 50 - 5.0 × 10 ⁵ - 4.7 × 10 ⁸ - 3.4 × 10 ¹⁰ - 1.2 × 10 ¹⁰ - 1.6 × 10 ⁹ - P. pentosaceus NRRL D-14009
+ S. cerevisiae KACC30008 15 1.0 × 10 ¹¹ 2.0 × 10 ¹¹ 3.1 x 10 ⁸ 2.4 × 10 ¹⁰ 7.7 x 10 ⁸ 9.9 x 10 ⁹ 2.4 × 10 ⁹ 1.0 × 10 ¹⁰ 3.5 × 10 ⁷ 2.6 × 10 ¹⁰ 2.5 × 10 ⁶ 30 1.0 × 10 ¹¹ 2.9 × 10 ¹¹ 2.9 × 10 ⁸ 7.6 × 10 ¹⁰ 5.8 x 10 ⁸ 7.0 × 10 ¹⁰ 1.9 × 10 ⁹ 1.9 × 10 ¹⁰ 5.9 x 10 ⁷ 3.1 × 10 ¹⁰ 2.9 × 10 ⁶ 50 1.0 × 10 ¹¹ 3.2 × 10 ¹¹ 2.4 × 10 ⁸ 7.8 × 10 ¹⁰ 4.2 × 10 ⁸ 6.5 × 10 ¹⁰ 1.3 x 10 ⁹ 1.5 × 10 ¹⁰ 6.3 × 10 ⁷ 2.9 × 10 ¹⁰ 2.8 × 10 ⁶
Leu . citerum ATCC49370
+ S. cerevisiae KACC30008 15 1.0 × 10 ¹¹ 1.5 × 10 ¹¹ 2.2 x 108 3.1 × 10 ¹⁰ 7.1 x 10 ⁸ 2.9 × 10 ¹⁰ 9.1 × 10 ⁸ 2.2 × 10 ¹⁰ 4.5 × 10 ⁶ 1.9 × 10 ¹⁰ 1.2 × 10 ⁶ 30 1.0 × 10 ¹¹ 3.0 × 10 ¹¹ 2.1 x 10 ⁸ 5.9 × 10 ¹⁰ 5.8 x 10 ⁸ 5.7 × 10 ¹⁰ 8.5 × 10 ⁸ 1.9 × 10 ¹⁰ 3.5 × 10 ⁶ 2.2 × 10 ¹⁰ 1.1 × 10 ⁶ 50 1.0 × 10 ¹¹ 2.8 × 10 ¹¹ 1.6 x 10 ⁸ 6.3 × 10 ¹⁰ 4.6 × 10 ⁸ 5.9 × 10 ¹⁰ 1.1 × 10 ⁹ 2.4 × 10 ¹⁰ 4.1 × 10 ⁶ 2.1 × 10 ¹⁰ 1.3 × 10 ⁶
Leu . gelidum ATCC49366
+ S. cerevisiae KACC30008 15 1.0 × 10 ¹¹ 3.7 × 10 ¹⁰ 2.4 × 10 ⁸ 6.7 × 10 ¹⁰ 3.6 × 10 ⁸ 8.9 × 10 ¹⁰ 4.5 × 10 ⁸ 1.3 × 10 ¹⁰ 3.8 × 10 ⁷ 1.6 × 10 ¹⁰ 1.5 x 10 ⁷ 30 1.0 × 10 ¹¹ 2.0 × 10 ¹¹ 2.0 x 10 ⁸ 6.3 × 10 ¹⁰ 3.2 x 10 ⁸ 4.8 × 10 ¹⁰ 3.9 × 10 ⁸ 1.9 × 10 ¹⁰ 2.2 x 10 ⁷ 2.1 × 10 ¹⁰ 1.2 × 10 ⁷ 50 1.0 × 10 ¹¹ 2.5 × 10 ¹¹ 2.5 x 10 ⁸ 7.1 × 10 ¹⁰ 2.9 × 10 ⁸ 5.9 × 10 ¹⁰ 3.1 x 10 ⁸ 2.3 × 10 ¹⁰ 4.3 × 10 ⁷ 1.9 × 10 ¹⁰ 2.1 × 10 ⁷ Lb. plantarum ATCC8014
+ S. cerevisiae KACC30008 15 1.0 × 10 ¹¹ 2.4 × 10 ¹⁰ 4.4 × 10 ⁸ 3.2 × 10 ¹⁰ 3.8 × 10 ⁸ 4.8 × 10 ¹⁰ 3.5 × 10 ⁸ 1.6 × 10 ¹⁰ 1.0 × 10 ⁸ 1.7 × 10 ¹⁰ 4.7 × 10 ⁷ 30 1.0 × 10 ¹¹ 2.4 × 10 ¹¹ 4.2 × 10 ⁸ 4.6 × 10 ¹⁰ 7.2 x 10 ⁸ 4.7 × 10 ¹⁰ 1.1 × 10 ⁹ 1.4 × 10 ¹⁰ 4.6 × 10 ⁸ 1.3 × 10 ¹⁰ 5.4 × 10 ⁷ 50 1.0 × 10 ¹¹ 1.1 × 10 ¹¹ 3.6 × 10 ⁸ 1.8 × 10 ¹⁰ 6.6 x 10 ⁸ 1.7 × 10 ¹⁰ 1.6 × 10 ⁹ 9.6 × 10 ⁹ 4.8 x 10 ⁸ 1.1 × 10 ¹⁰ 5.1 x 10 ⁷

PH change during fermentation period by mixed fermentation of yeast and lactic acid bacteria (per 100 g of brown rice sample) Strain moisture
Amount added (ml) Immediately after inoculation Fermentation
Day 2 Fermentation
Day 4 Fermentation
Day 6 Fermentation
Day 8 Fermentation
Day 10 Control
(Lactic acid bacteria and yeast
Not inoculated) 15 7.21 5.78 5.21 4.92 4.49 4.18 30 7.20 5.71 5.19 4.46 4.29 4.22 50 7.21 5.47 5.18 4.44 4.42 4.12 P. pentosaceus NRRL D-14009
+ S. cerevisiae KACC30008 15 6.96 4.78 4.35 4.33 4.37 4.46 30 6.95 4.71 4.30 4.31 4.37 4.78 50 6.95 4.61 4.28 4.24 4.43 4.93 Leu . citerum ATCC49370
+ S. cerevisiae KACC30008 15 6.96 4.93 4.64 4.61 4.58 4.58 30 6.95 4.53 4.34 4.23 4.25 4.27 50 6.96 4.54 4.23 4.22 4.78 4.79 Leu . gelidum ATCC49366
+ S. cerevisiae KACC30008 15 7.02 5.55 4.97 4.65 4.54 4.48 30 6.98 4.74 4.23 4.16 4.29 4.31 50 6.97 4.54 4.20 4.17 4.28 4.34 Lb. plantarum ATCC8014
+ S. cerevisiae KACC30008 15 7.07 6.09 5.78 5.54 5.38 5.08 30 7.04 5.35 4.35 4.23 4.23 4.26 50 7.04 4.66 4.28 4.15 4.17 4.20

< Example  3>

GABA  produce Lactic acid bacteria  Selection

GABA is a substance that has functions such as inhibition of blood pressure rise and improvement of blood flow and functions such as inhibition of blood cholesterol and triglyceride increase, obesity and visual acuity, and is contained in a large amount in brown rice. On the other hand, if the brown rice is fermented, the production amount of GABA can be further increased. Therefore, the inventors of the present invention have found that by inoculating a lactic acid bacterium having GABA production ability using the brown rice fermentation conditions set forth in Examples 1 and 2, And tried to increase the functionality.

<3-1> Isolation of lactic acid bacteria in kimchi

For the isolation of GABA - producing lactic acid bacteria to be used for brown rice fermentation, lactic acid bacteria were isolated from kimchi samples. Kimchi purchased various kinds of kimchi from local large - scale mart and traditional market and used it as a sample. Kimchi samples were classified into the conditions of purchase, kimchi, and chilled storage period, so that lactic acid bacteria could be separated to obtain various lactic acid bacteria strains. In each sample, 20 g of kimchi and kimchi broth were removed, 80 ml of peptone was added, homogenized for 2 minutes, and stepwise dilution was performed using the number of peptones. Thereafter, the cells were cultured on MRS plate medium and cultured at 30 ° C for 24 hours, and then various kinds were selected according to the colony type.

As a result, a total of 2800 lactic acid bacteria were isolated. Each strain was stored at -70 ° C and -20 ° C with 30% glycerol. For use in the experiment, MRS liquid medium was first inoculated and cultured.

<3-2> TLC Through GABA  produce Lactic acid bacteria  Selection

In order to select GABA - producing strains among 2,800 lactic acid bacteria isolated from kimchi, first selection experiment was conducted using TLC method. First, 560 strains were inoculated into MRS medium containing 5% MSG and cultured at 30 ° C for 48 hours. The following culture supernatant was subjected to TLC to confirm the generation of GABA. For the mixed samples in which the GABA spot was confirmed, 5 strains were separately cultured again to confirm which strain produced GABA.

The concrete method of the TLC experiment is as follows. 1 ml of MRS liquid medium and incubated overnight at 37 ° C. The cells were cultured in MRS liquid medium supplemented with 5% monosodium glutamate (MSG, Cat no G1626, Sigma-aldrich, USA) Respectively. The culture was centrifuged (4 ° C, 12,000 rpm, 5 minutes) to obtain culture supernatant and used in the experiment. 1 μl of the culture supernatant was dispensed on a plate of TLC Silica gel 60 F254 (25 Glass plate, 20 × 20 cm, Merck Millipore, Cat No. 105715). 1 μl of 100 mM MSG and 1 μl of 100 mM GABA were mixed with the standard material and the results were compared. TLC plates were developed using butanol: acetic acid: water = 4: 1: 1 as a developing solvent. After development, the TLC plate was sufficiently dried and then developed using 0.2% ninhydrin to compare the GABA spot with the substrate MSG spot.

As a result, the present inventors could confirm by TLC that many lactic acid bacteria produce GABA. As a positive control, GU240, which had better GABA production ability than Lactobacillus brevis KACC 14481, was finally selected. As shown in the TLC result shown in Fig. 1, GU240 (lane 5) is different from other strains (lane 6-14) with Lb. it was found that more GABA was produced than brevis KACC 14481 (lane 4).

<3-3> GABASE Using GABA  Confirm creation

Quantitative analysis of GABA produced by GABase enzyme was performed on GU240 and GU498 strains which showed excellent GABA production by TLC analysis. Lb as a positive control is produced GABA identified. brevis KACC14481 were distributed at Rural Development Administration. As a negative control group that does not produce GABA, various kinds of lactic acid bacteria ( Leuconostoc mesenteroides ATCC10830, Lactobacillus plantarum C7 and Lactococcus lactis B2) were selected randomly and the results were compared.

Quantitative analysis of GABA was measured by GABase method. Each strain was inoculated and cultured in MRS liquid medium supplemented with 5% MSG and cultured at 30 ° C for 48 hours. The culture was centrifuged (4 ° C, 1,500 × g, 15 min) and the culture supernatant was used for the experiment. The sample was stored at -70 ° C until used. 400 μl of methanol was added to 100 μl of the culture supernatant of each strain, mixed thoroughly, and then extracted and reacted at 25 ° C for 30 minutes using a water bath. After that, it was dried under low-temperature vacuum for 2 hours by using a speed vacuum concentrator, 1 ml of 70 mM LaCl ₃ was added, and the mixture was shaken for 15 minutes. Then, centrifuge (4 ° C, 12,000 rpm, 5 minutes), and 800 μl of the supernatant is transferred into a new E-tube. Then, 160 μl of a 1 M KOH solution was added to the supernatant, shaken for 5 minutes, and centrifuged (4 ° C., 12,000 rpm, 5 minutes) to use the supernatant. 200 μl of 50 mM potassium pyrophosphate (pH 8.6), 150 μl of 4 mM NADP ⁺ (Sigma), 50 μl of 1 unit GABase [ Pseudomonas fluorescens (Sigma)] and 50 μl of 20 mM α-ketoglutarate were added to 550 μl of the pre- 1 ml of the total reaction solution was added thereto, and the mixture was reacted at 37 ° C for 60 minutes, and the absorbance at 340 nm was measured. Quantification of GABA using GABase was performed using the GABA standard curve (Abs = 0.6034 X - 0.0002, R ² = 0.9975).

As a result, the results as shown in Table 5 were obtained. The GABA content was expressed in mg per 1 ml of the culture supernatant. The strain GU240 produced 4.12 mg of GABA per ml of culture supernatant and showed about 68-84 times higher GABA production when compared to the 0.05-0.06 ㎎ values of the common lactic acid bacteria. GABA producing ability is already well known Lb. brevis And 2.65 times higher level of GABA than 1.55 mg of KACC14481. GU498 is Lb to generate 1.82 ㎎ GABA. brevis It was slightly higher than KACC14481. Therefore, the present inventors once again confirmed that GU240 is a very excellent GABA-producing strain through quantitative analysis.

Lb. Quantitative analysis of GABA in culture supernatant of zymae GU240 strain Strain GABA production
(mg / ml) GABA conversion yield (%) Control group Leuconostoc mesenteroides ATCC10830 0.05 - Lactobacillus plantarum C7 0.06 - Lactococcus lactis B2 0.06 - Lactobacillus brevis KACC14481 1.55 5.08 Experimental Section Lactobacillus zymae GU240 4.12 13.51 LAB GU498 1.82 5.97

< Example  4>

GABA  Investigation of characteristics of excellent strains

The present inventors isolated the GU240 strain having excellent GABA-producing ability from the kimchi through the above Example 3 and tried to identify GU240 lactic acid bacteria.

<4-1> GU240 The identification of

First, to observe the morphology of GU240 lactic acid bacteria, no staining and Gram staining were performed and observed with a microscope. OD ₆₀₀ The culture medium with a value of 1.5 was cut into thin slices using a platinum slice, fixed with heat, and observed with a microscope (no staining). Gram staining was performed in the same manner as staining without staining, followed by staining according to Gram staining method And observed with a microscope. As a result, the results as shown in FIG. 2 were confirmed. As a result of observing each sample at 400 times, Gram stain showed purple color and Gram-positive staphylococci were observed.

In order to confirm colony shape and exopolysaccharide formation of GU240, GU240 and control groups were spotted together on MRS plate and 5% sucrose-containing MRS plate. The morphology was examined by streaking in each medium. Whether or not exopolysaccharide was formed was confirmed by spotting each strain on a 5% sucrose MRS plate. Weissella cibaria 33 was used as a control to confirm the production of exopolysaccharide. As a result, the results as shown in Fig. 3 were confirmed. The GU 240 was found to form a circular round white bloom on the MRS medium, and compared with the control group W. cibaria 33 on a medium containing sucrose, Respectively.

We also tried to analyze the 16S rRNA gene sequence for the identification of GU240. The chromosomal DNA was recovered by recovering the cells from 1.5 ml of the culture. Chromosomal DNA was used as a template and PCR was performed using the universal primers 27F (5-AGAGTTTGATCMTGGCTCAG-3) and 1492R (5-GGTTACCTTGTTACGACTT-3) for 16S rRNA gene amplification and Ex Taq enzyme of Takara Co. under the following conditions (Table 6). The resulting 1.5 kb PCR product was separated from the gel after electrophoresis, gel extracted using the Favorgen kit, and then subjected to nucleotide sequence analysis by Cosmojin Tech.

PCR conditions for amplification of 16s rRNA gene of GU240 No Temp. Time Component volume One 94 05: 00 Template 1.0 μl 2 94 00: 30 primer (27 F) -10 pM 1.0 μl 3 60 00: 30 primer (1492R) -10 pM 1.0 μl 4 72 02: 00 10 x dNTP 5.0 μl 5 Go to step 2, repeat 29 times 10 × ex taq buffer 5.0 μl 6 72 10:00 D.W. 36.5 μl 7 4 10:00 ex-taq (2.5U) 0.5 μl End Total volume 50 μl

As a result, there were obtained the results shown in Table 7, GU240 is Lb. zymae, Lb. brevis, Lb. acidifarinae 99% homology to the three species, indicating that these three species are very similar. For the identification of GU240, further rec A gene sequencing and API kit were used. In addition, for the distinction of three species, growth was examined in MRS liquid media supplemented with D-arabitol as a carbon source. Lb. with zymae Lb. acidifarinae are available on the D-Arabitol as a carbon source but, Lb. It is known that brevis can not be used (Vancanneyt et al., International Journal of Systematic and Evolutionary Microbiology (2005), 55, 615-620). GU240 the present inventors case Growth the D-arabitol as a substrate are GU240 is Lb. brevis Lb instead. It was determined to zymae, end-Lb. zymae .

Identification of GU240 strain using 16S rRNA gene sequence Description Max ident Lb. zymae strain: LMG22198 = R-18615 16s ribosomal RNA,
종열 99% Lb. brevis 16s ribosomal RNA,
종열 99%
Lb. acidifarinae strain: LMG22200 = R-19065 16s ribosomal RNA,
종열 99%

Finally, we investigated the fermentation of 49 carbohydrate substrates of GU240 using API 50 CHL kit (BioMerieux, France). This is because the pH indicator is contained in the medium, and when the fermented carbohydrate forms an acid during the incubation period, the pH is lowered and the positive and negative are read by the color change. After incubation in API 50 CHL media using GU240 medium, 50 CHL strip tube containing each carbon source was added and mineral oil was added to make anaerobic conditions suitable for fermentation. Culturing was carried out at 37 ° C for 24 hours to 48 hours And the results were confirmed.

As a result, the results as shown in Table 8 were obtained, which was identified as Lactobacillus brevis with a homology of 99.8%. But in this case because it has two different kind of data is not yet accumulated in the API database, Lb. zymae And Lb. acidifarinae is Lb. It is known to have one difference from brevi s. I.e., Lb. zymae and Lb. acidifarinae are using D-arabitol as a carbon source, but Lb. Brevis is known to be unable to use it. GU240 strain grows with D-arabitol as a substrate and grows at a salt concentration of 10% . Therefore, Lb. acidifarinae is to determine on the basis of that it can not grow in the salt concentration of 7% or more GU240 is Lb. zymae . Lb. zymae The results of investigating the carbon source utilization of various compounds of GU240 are shown in Table 9.

Identification of GU240 strain using API 50CHL kit Strain % ID T Index The opposite biochemical characteristics (Test against) Lactobacillus brevis 1 99.8 0.29 GAL 99% SAC 81% MLZ 14% GEN 99% TUR 14% 5KG 14% Lactobacillus plantarum One 0.1 0.0 DXYL 2% GAL 92% SOR 78% LAC 99% SAC 88% GEN 98% 5KG 0%

Lb. Carbohydrate availability of zymae GU240 carbohydrate Availability of GU240 One Maltose + 2 Glucose + 3 Rhamnose - 4 Lactose - 5 Trehalose + 6 Mannose + 7 Sucrose - 8 Sorbitol - 9 Inulin - 10 D-Xylose - 11 D-Arabinose - 12 Cellobiose + 13 Raffinose - 14 Esculin + 15 Manitol + 16 D-arabitol +

On the other hand, using API ZYM kit, Lb zymae As a result of confirming the presence or absence of the enzyme titer of GU240, it was confirmed to have a titer of leucine arylamidase, β-glucosidase and N-acetyl-β-glucosaminidase, and the results are shown in Table 10.

Lb. Enzyme activity of zymae GU240 enzyme temperament GU240's
Enzyme activity One Leucine arylamidase L-leucyl-2-naphthylamide + 2 β-glucosidase 6-Br-2-naphthyl- [beta] -D-glucopyranoside + 3 N-acetyl-β-glucosaminidase 1-naphthyl-N-acetyl- [beta] -D-glucosaminide + 4 a-galactosidase 6-Br-2-naphthyl- [alpha] -D-galactopyranoside - 5 β-glucosidase Naphthol-AS-BI- [beta] -D-glucuronide -

In conclusion, the present inventors isolated a strain having excellent GABA production ability from kimchi. Through the morphology, genetics and biochemical characteristics of the strain, the strain was identified as Lactobacillus zymae. In the present invention, the strain was designated as' Lactobacillus zymae GU240 (hereinafter simply referred to as 'GU240'). In addition, this strain was deposited with KFCC (Korea Microbiological Research Center) on November 23, 2012, and received the deposit number KFCC11545P.

<4-2> GU240 ( Lb . zymae ) Growth characteristics

The inventors have seen examine the growth characteristics of the newly identified GU240 (Lb. Zymae). To investigate the growth characteristics of GU240 according to temperature, 100 ml of MRS broth was inoculated with 1 ml (1%, v / v) of GU240 culture medium and incubated at 4, 10, 15, 20, 37, Lt; / RTI &gt; The culture was recovered at 3, 6, 12, and 24 hour intervals and the absorbance was measured at 600 nm using a spectrophotometer. As a result, the graph shown in FIG. 4 was obtained, that is, at 37 and 42 ° C, there was no difference in the growth curve of GU240, and after about 10 hours, the stopper was reached. Growth was delayed up to 10 hours at 20 ℃, and then growth started after 24 hours. Proliferation was slower at 10 ℃ and 15 ℃ than at other temperatures. After 72 hours, however, OD ₆₀₀ reached 1.6 as at other temperatures. On the other hand, at 4 ℃, growth was delayed until 24 hours and gradually increased, reaching 0.8 at 120 hours.

Meanwhile, the present inventors prepared 100 ml of MRS broth in order to investigate the growth characteristics according to the pH of GU240, adjusted the pH from 3.0 to 10.0, and sterilized by autoclaving. The sterilized medium was sufficiently cooled at room temperature, 1 ml of GU240 medium (1%, v / v) was inoculated into each medium, and the culture was recovered at intervals of 3, 6, 12, Were measured. As a result, the graph shown in FIG. 5 was obtained. In the pH 5.0-9.0 MRS medium except for the initial pH 3.0 and 10.0, GU240 reached the stationary phase after about 12 hours from the start of the culture. The initial pH 4.0-6.0 of the medium at 12 hours OD ₆₀₀ This value was reached more than 1.6 pH 7.0, 8.0 and 9.0 in the medium is rather low of 1.5, 1.4 and 1.2 of OD ₆₀₀ Respectively. Did not substantially grow in the medium of initial pH 3.0, and the initial pH 10.0 is reached 12 hours to 0.6 gradually decrease in OD ₆₀₀ 0.4 Respectively.

In order to examine the growth depending on the salt concentration (NaCl), the present inventors prepared a culture medium by adding NaCl to 100 ml of MRS broth to a predetermined salt concentration (2, 4, 6, 8, 10, 12, 15% Respectively. Each medium was inoculated with 1 ml of GU240 medium (1%, v / v), and the culture was recovered at 3, 6, 12, and 24 hours intervals and absorbance was measured at 600 nm. As a result, there was obtained a graph as shown in FIG. 6, GU240 does not significantly differ to rapidly proliferate salt concentration up to 2-8% OD ₆₀₀ Value reached 1.6 or more, but almost no proliferation was observed at 12% and 15%. Growth was slow at 10% salt concentration, reaching a stopping factor of 1.4 at 48 hours and then 1.2 at 240 hours. The point at which growth is slower in the salt concentration of 10% is reported Lb. zymae Match the characteristics.

< Example  5>

GABA Productive  Excellent GU240 Brown rice fermentation experiment using

The present inventors conducted a brown rice fermentation experiment with a strain of GU240 having excellent GABA production ability as a seed. GU240 strains were inoculated at 1.0 × 10 ¹¹ , and brown rice samples inoculated with no strains were used as a control. 200 g of brown rice was placed in sterilized 500 ml bottle, and 50 ml of distilled water containing 1.0 x 10 &lt; ¹¹ &gt; bacteria was added and mixed with sterilized spoon. In addition to the control group containing only sterilized distilled water, a total of eight samples including a brown rice sample containing glutamic acid (10 mM) and MSG (1% and 3%) as substrates were tested. Brown rice was fermented at 30 ℃ for 48 hours. After fermentation, it was spread evenly on Petri dish of 15 ㎝ in diameter and dried at 40 ℃ for 24 hours. The dried samples were collected in 50 ml conical tube and stored at 4 ℃ and room temperature for 12 days. The viable cell count, pH, TA and GABA contents were measured at 3 days intervals.

<5-1> GU240  According to the duration of brown rice fermentation process using strain Viable cell count  change

First, the viable cell counts were confirmed as described in Examples 1-3, and the results are shown in Table 11 below. The initial number of inoculated bacteria was 1.0 × 10 ¹⁰ and increased to 1.5-2 times in most of the samples until 48 hours of fermentation. However, in the samples containing 3% MSG, it increased slightly until 24 hours of fermentation and then decreased at 48 hours.

After drying at 40 ° C, the number of viable cells was reduced by 10 times compared to the fermentation sample for 48 hours. After 6 days of storage, the number of viable cells was not significantly different from the number of viable cells at the time of completion of drying. Thereafter, the number of viable cells decreased slightly after 12 days of storage but was found to be very small. The GU240 inoculum was reduced to about 100 times as much as the number of inoculated bacteria, but it was confirmed that a considerable amount of bacteria still remained at about 10 ⁹ .

Lb. Changes of viable cell counts during the fermentation period of brown rice using zymae GU240 strain Strain Inoculation number 24h fermentation 48h fermentation Dry Day 3 of storage Day 6 of storage Day 9 of storage Day 12 of storage 4 ℃ storage Room temperature storage 4 ℃ storage Room temperature storage 4 ℃ storage Room temperature storage 4 ℃ storage Room temperature storage ¹ control ddH2O - 4.2 × 10 ⁵ 3.8 × 10 ⁵ 1.8 × 10 ⁵ 1.6 × 10 ⁵ 1.3 x 10 ⁵ 6.4 × 10 ⁴ 2.4 × 10 ⁴ 4.4 × 10 ⁴ 2.2 x 10 ⁴ 2.1 x 10 ⁴ 6.3 × 10 ³ 10 mM glutamic acid - 3.7 × 10 ⁵ 3.6 × 10 ⁵ 1.4 x 10 ⁵ 1.5 × 10 ⁵ 1.5 × 10 ⁵ 5.2 × 10 ⁴ 3.1 x 10 ⁴ 3.9 × 10 ⁴ 1.9 × 10 ⁴ 1.4 x 10 ⁴ 4.9 × 10 ³ 1% MSG - 4.3 × 10 ⁵ 4.1 × 10 ⁵ 1.2 × 10 ⁵ 1.1 × 10 ⁵ 1.3 x 10 ⁵ 6.1 × 10 ⁴ 3.5 × 10 ⁴ 3.4 × 10 ⁴ 2.7 × 10 ⁴ 1.4 x 10 ⁴ 5.7 x 10 ³ 3% MSG - 2.7 × 10 ⁵ 2.2 x 10 ⁵ 1.1 × 10 ⁵ 8.9 × 10 ⁴ 8.2 × 10 ⁴ 2.9 × 10 ⁴ 1.4 x 10 ⁴ 3.6 × 10 ⁴ 1.5 x 10 ⁴ 2.7 × 10 ⁴ 2.8 × 10 ^{3 2} Experimental Section ddH2O 1.0 × 10 ¹¹ 2.4 × 10 ¹¹ 2.9 × 10 ¹¹ 2.1 × 10 ¹⁰ 2.3 × 10 ¹⁰ 2.1 × 10 ¹⁰ 7.4 × 10 ⁹ 7.1 x 10 ⁹ 5.3 × 10 ⁹ 4.7 × 10 ⁹ 3.9 × 10 ⁹ 2.7 × 10 ⁹ 10 mM glutamic acid 1.0 × 10 ¹¹ 1.3 × 10 ¹¹ 1.2 × 10 ¹¹ 3.4 × 10 ¹⁰ 3.1 × 10 ¹⁰ 3.3 × 10 ¹⁰ 6.9 × 10 ⁹ 3.7 × 10 ⁹ 4.8 × 10 ⁹ 2.8 × 10 ⁹ 4.0 × 10 ⁹ 1.9 × 10 ⁹ 1% MSG 1.0 × 10 ¹¹ 2.5 × 10 ¹¹ 2.3 × 10 ¹¹ 1.9 × 10 ¹⁰ 1.7 × 10 ¹⁰ 1.6 × 10 ¹⁰ 6.4 × 10 ⁹ 2.9 × 10 ⁹ 5.1 x 10 ⁹ 3.2 × 10 ⁹ 3.8 × 10 ⁹ 3.1 × 10 ⁹ 3% MSG 1.0 × 10 ¹¹ 1.2 × 10 ¹¹ 8.0 × 10 ¹⁰ 6.4 × 10 ⁹ 5.9 × 10 ⁹ 5.7 x 10 ⁹ 6.1 × 10 ⁸ 1.6 x 10 ⁸ 3.9 × 10 ⁸ 6.1 × 10 ⁷ 1.5 × 10 ⁸ 4.2 × 10 ⁷

¹ Lb. Zymae GU240 strain was not inoculated

² Lb. zymae GU240 strain was inoculated at a rate of 1 × 10 ¹¹ per 100 g of brown rice

* Number of viable cells is expressed as viable count per 100g of brown rice

<5-2> GU240  According to the duration of brown rice fermentation process using strain pH  And pH measurement

The present inventors measured the pH in the step of fermenting brown rice with the strain GU240 inoculated into brown rice. The pH of the homogenized sample liquid obtained for the measurement of viable cell count was measured using a pH meter, and the acidity was measured as follows. Fifty microliters of fermented brown rice samples were added to 90 ml of distilled water and homogenized. 50 ml of the homogenized sample was placed in a beaker, and then 500 μl of 1% phenolphthalein was added. The mixture was titrated with 0.1 N NaOH for 30 seconds I took the time to the end. The acidity was calculated using the NaOH ㎖ required to reach the end point. The calculation is as follows.

Acidity = consumed NaOH (ml) x 0.9 / weight of fermented brown rice (g)

As shown in Table 12, the initial pH and TA values immediately after the inoculation of the strain were 6.8-6.9 and 0.14-0.16, respectively. As the fermentation progressed, the pH and the TA were increased to 5.2-5.4 and TA 0.34 -0.35 and for 48 hours fermentation samples were pH 4.7-4.9 and TA 0.39-0.42. After fermentation and drying, the pH rose to 6.2-6.5 and the TA value dropped to 0.15-0.18. Thereafter, pH and TA values were maintained during the 12 - day storage period.

Lb. Changes in pH and Acidity of Zymae GU240 Strains during Brown Rice Fermentation Strain Immediately after inoculation
(pH / TA) 24h
Fermentation
(pH / TA) 48h
Fermentation
(pH / TA) Dry
(pH / TA) On the third day of storage (pH / TA) On the 6th day of storage (pH / TA) On the 9th day of storage (pH / TA) On the 12th day of storage (pH / TA) 4 ℃ storage Room temperature storage 4 ℃ storage Room temperature storage 4 ℃ storage Room temperature storage 4 ℃ storage Room temperature storage ¹ control ddH2O 6.86
0.15 5.66
0.28 5.68
0.31 6.30
0.17 6.41
0.16 6.44
0.15 6.47
0.16 6.49
0.15 6.49
0.14 6.51
0.14 6.55
0.13 6.49
0.14 10 mM glutamic acid 6.80
0.14 5.72
0.26 5.54
0.29 6.43
0.17 6.63
0.14 6.58
0.15 6.69
0.15 6.81
0.13 6.58
0.13 6.63
0.14 6.61
0.14 6.65
0.14 1% MSG 6.91
0.14 5.61
0.26 5.45
0.30 6.48
0.15 6.56
0.16 6.87
0.14 6.62
0.15 6.74
0.14 6.56
0.14 6.62
0.13 6.58
0.14 6.73
0.14 3% MSG 6.78
0.15 5.78
0.27 5.61
0.28 6.69
0.16 6.49
0.17 6.62
0.15 6.58
0.16 6.57
0.14 6.51
0.15 6.62
0.15 6.54
0.13 6.65
0.15 ² Experimental Section ddH2O 6.80
0.14 5.21
0.35 4.71
0.41 6.21
0.18 6.31
0.21 6.23
0.21 6.27
0.19 6.34
0.22 6.37
0.19 6.46
0.22 6.47
0.21 6.38
0.22 10 mM glutamic acid 6.85
0.15 5.24
0.34 4.92
0.39 6.29
0.16 6.45
0.18 6.41
0.18 6.44
0.18 6.42
0.20 6.32
0.19 6.41
0.22 6.55
0.20 6.43
0.20 1% MSG 6.88
0.16 5.36
0.35 4.67
0.42 6.39
0.16 6.55
0.17 6.53
0.17 6.47
0.18 6.41
0.19 6.34
0.19 6.39
0.20 6.58
0.22 6.32
0.22 3% MSG 6.85
0.16 5.44
0.34 4.98
0.39 6.46
0.15 6.41
0.16 6.43
0.18 6.38
0.19 6.29
0.19 6.34
0.20 6.44
0.21 6.52
0.21 6.41
0.21

¹ Lb. Zymae GU240 strain was not inoculated

² Lb. zymae GU240 strain was inoculated at a rate of 1 × 10 ¹¹ per 100 g of brown rice

<5-3> GU240  In a Brown Rice Fermentation Process Using Strain GABA  Confirm creation

GABA production was determined by two methods: a qualitative method using TLC method and a quantitative method using GABase.

TLC results Lb 1% and 3% is added MSG brown rice while not made at all the GABA produced in the control group, as shown in Figure 7. In the samples fermented with zymae GU240, spot was observed at the same position as GABA standard material. The approximate amount of GABA was estimated by comparing to the amount of GABA standard spot (20.62 μg). In particular, the intensity and location of spots 6, 7, 10 and 11 in Figure 7 indicate that MSG is converted to GABA I could confirm.

Table 13 shows the results of quantitative analysis by GABase enzyme. Lb. Among the brown rice samples fermented by inoculation with zymae GU240, GABA production was observed in MSG added samples. In 48 hours of fermentation, 2.39 ㎎ / g of brown rice and 2.56 ㎎ of GABA were added . The conversion rates of MSG to GABA were 39.1% and 13.99%, respectively. Only water is added to the rice, without strain or immunization Lb no MSG added. zymae Compared with the value of 0.18 ㎎ in the case of adding only GU240, it can be estimated that GABA was produced from MSG during fermentation. GABA was maintained at 2.15 mg and 2.07 mg, respectively, after 12 days of storage or storage at room temperature. In MSG 1% supplemented medium, GABA production similar to that of 3% medium was shown. The rice was added man 10 mM glutamic acid without strain is inoculated after 48 hours fermentation GABA content was 0.18 mg This was Lb with 10 mM glutamic acid. and zymae , respectively. This suggests that a lower amount of GABA than 1% MSG is produced from 10 mM glutamic acid. After storage for 12 days in cold storage or at room temperature, the contents of goverberry were 0.57 mg and 0.53 mg, respectively.

MSG Lb and the glutamic acid in a MRS medium supplemented with different concentrations. GABA was quantified for the culture medium in which zymae GU240 was cultured. The results are shown in Table 14. As can be seen from the results, GABA production was higher than in the brown rice fermentation experiment. This is unlike the brown Lb. zymae is considered to be a better growth in MRS medium. In 1% MSG medium, 5.55 mg of GABA was produced and the conversion rate was 90.2%. The lower the concentration of MSG, the higher the conversion rate. That is, the conversion rate is 100% at less than 0.5% and 4.42 mg of GABA at 0.75%, and the conversion rate is 96.7%. When MSG concentration was higher than 1%, GABA production was decreased. In the glutamic acid supplemented medium, the amount of GABA production increased with increasing glutamic acid concentration but the conversion rate decreased. When the content of glutamic acid was 0.75%, 5.51 mg of GABA was produced and the conversion was 104%. This concentration was considered to be a suitable concentration for GABA production.

These results suggest that it is appropriate to add glutamic acid to the brown rice for the purpose of GABA production rather than MSG.

Lb. Changes of GABA level during the fermentation period of brown rice using zymae GU240 strain sample Immediately after inoculation 24h
Fermentation 48h
Fermentation Dry Day 3 of storage Day 6 of storage Day 9 of storage Day 12 of storage 4 ℃ storage Room temperature
Save 4 ℃ storage Room temperature
Save 4 ℃ storage Room temperature
Save 4 ℃ storage Room temperature
Save ¹ control ddH2O ³ 0.19 0.18 0.18 0.19 0.14 0.14 0.15 0.14 0.14 0.13 0.15 0.14 10 mM glutamic acid 0.16 0.23 0.26 0.29 0.22 0.25 0.22 0.21 0.22 0.21 0.22 0.21 1% MSG 0.26 0.37 0.36 0.33 0.26 0.25 0.27 0.24 0.24 0.23 0.25 0.24 3% MSG 0.28 0.36 0.36 0.26 0.21 0.20 0.21 0.20 0.21 0.19 0.19 0.20 ² Experimental Section ddH2O 0.13 0.18 0.18 0.29 0.24 0.22 0.22 0.21 0.22 0.21 0.23 0.22 10 mM glutamic acid 0.32 0.83 0.89 0.59 0.64 0.63 0.63 0.59 0.61 0.57 0.57 0.53 1% MSG 0.16
⁴ 2.62% 1.90
32.06% 2.39
39.19% 2.24
36.73% 2.21
36.24% 2.17
35.59% 2.21
36.24% 2.13
35.01% 2.18
35.80% 2.14
35.17% 2.15
35.34% 2.07
34.02% 3% MSG 0.22
1.21% 2.01
10.98% 2.56
13.99% 2.41
13.17% 2.25
12.30% 2.26
12.35% 2.17
11.86% 2.19
11.97% 2.15
11.75% 2.15
11.75% 2.12
11.59% 1.99
10.88%

¹ Lb. zymae GU240 strain Not inoculated

² Lb. zymae GU240 strain was inoculated at a concentration of 1 × 10 ⁹ per g of brown rice

³ mg GABA / brown rice sample (fresh weight)

⁴ GABA conversion yield (%)

Lb in a MRS medium, unlike the MSG and glutamic acid concentrations. GABA production of zymae GU240 MSG, glutamic acid concentration (%) GABA production
(mg / ml) GABA conversion yield (%) MSG 0.1 0.68 111.52 0.25 1.61 105.61 0.5 3.46 113.39 0.75 4.42 96.67 1.0 5.55 90.20 2.0 5.05 41.41 3.0 5.02 27.44 4.0 4.98 20.41 5.0 3.97 13.02 Glutamic acid 0.1 0.81 115.58 0.25 1.98 113.01 0.5 3.98 113.58 0.75 5.51 104.83 1.0 5.94 84.75 2.0 7.79 55.58 3.0 9.12 43.37 4.0 11.41 40.70 5.0 13.03 37.18

< Example  6>

GU240  Brown rice fermentation process using strain and low concentration glutamic acid

Ganoderma lucidum fermentation was carried out under the condition of low concentration of glutamic acid as a seed strain of GU240 strain having excellent GABA production ability. GU240 strains were inoculated in a total volume of 3.0 × ^{10 11} , and two control strains were used, in which the strain was not inoculated and the strains were inoculated without addition of glutamic acid. 300 g of brown rice was placed in a sterilized 500 ml bottle, 75 ml of distilled water containing 3.0 x ^{10 11} of GU240 was added, and the mixture was thoroughly mixed with sterilized spoon. Glutamic acid as a precursor was added to distilled water at different concentrations (0.1%, 0.3%, 0.5% and 0.7%). Experiments were conducted on 6 brown rice samples in which GU240 strain was inoculated and glutamic acid was added at different concentrations. Brown rice was fermented at 30 ℃ for 24 hours. After fermentation, it was spread evenly on a Petri dish of 15 ㎝ in diameter and dried at 40 ℃ for 24 hours. The dried samples were collected in 50 ml conical tube and stored at 4 ° C for 5 weeks, and were collected at intervals of 1-2 weeks to measure viable cell count and GABA content.

<6-1> GU240  Microbial fermentation process with low concentration of glutamic acid Viable cell count  change

First, the viable cell count was confirmed as described in Example 1-3, and the results are shown in Table 15 below. The total number of bacteria in the initial inoculation was 3.0 × 10 ¹¹ , which was 1.0 × 10 ⁹ in terms of 1 g of brown rice. At the completion of the fermentation for 24 hours, the samples increased by about 4-5 times to 3.9-4.7 × 10 ⁹ . However, as the storage period became longer, the viable cell counts decreased. The amount of GU240 inoculum decreased to 6.9-7.3 × 10 ⁷ after 1 week of storage, and the decrease after that was somewhat small. After 5 weeks of storage, the viable cell count of each sample was 1.2-2.2 × 10 ⁶ . After 5 weeks, the number of inoculated samples decreased about 500-1,000 times compared to the initial number of inoculated bacteria, but when compared with 3.1 × 10 ² , which was 5 weeks after the 5 hours of GU240 non-inoculated samples, This is worthy of reference to the manufacture of health food using fermented brown rice.

Lb. Changes in viable cell counts during fermentation using zymae GU240 and low concentration glutamic acid sample Number of viable cells (per CFU / g of brown rice) Immediately after inoculation 24 hours after fermentation Save one day Storage 1 week Storage 3 weeks Save 5 weeks A ¹ control 1 - 2.7 x 10 ³ 1.9 × 10 ³ 3.8 × 10 ² 3.2 x 10 ² 3.1 x 10 ² B ² Controls 2 1.1 × 10 ⁹ 4.2 × 10 ⁹ 1.2 × 10 ⁹ 7.3 × 10 ⁷ 4.3 × 10 ⁷ 1.7 × 10 ⁶ C 0.1% glutamic acid 1.0 × 10 ⁹ 3.9 × 10 ⁹ 1.5 x 10 ⁹ 7.1 x 10 ⁷ 3.9 × 10 ⁷ 1.4 × 10 ⁶ D 0.3% glutamic acid 1.3 x 10 ⁹ 4.7 × 10 ⁹ 1.7 x 10 ⁹ 6.9 × 10 ⁷ 4.5 × 10 ⁷ 2.2 x 10 ⁶ E 0.5% glutamic acid 1.2 × 10 ⁹ 4.5 × 10 ⁹ 1.3 x 10 ⁹ 7.3 × 10 ⁷ 2.8 x 10 ⁷ 1.2 × 10 ⁶ F 0.7% glutamic acid 1.1 × 10 ⁹ 4.3 × 10 ⁹ 1.2 × 10 ⁹ 7.0 × 10 ⁷ 3.3 × 10 ⁷ 1.5 × 10 ⁶

¹ Lb. zymae GU240 strain Inoculated, Glutamic acid-free

² Lb. zymae GU240 strain was inoculated at 1 x 10 &lt; ⁹ &gt; per gram of brown rice, and glutamic acid-free

<6-2> GU240  Microbial fermentation process with low concentration of glutamic acid GABA  Confirm creation

GABA production was determined by quantitative method using GABase as described above.

Table 16 shows the results of quantitative analysis by GABase enzyme. Lb. Of the brown rice samples fermented with zymae GU240, GABA production was confirmed in glutamic acid added samples. GABA production of 1.70 ㎎ and 2.14 ㎎ was observed at 0.52 ㎎ / g of brown rice and at 1.22 ㎎, 0.5% and 0.7% of 0.3% added samples, respectively, in the addition of 0.1% glutamic acid at 24 hours of fermentation. GABA conversion rates of glutamic acid were 75.59, 58.10, 48.62 and 43.68%, respectively. Addition of water only to the rice, without strains or vaccinated Lb no glutamic acid was added. zymae GABA contents were 0.14 ㎎ and 0.17 ㎎, respectively. These results suggest that GABA was produced from glutamic acid by GU240 strain. After storage for 5 weeks, the values of 0.44 mg, 1.01 mg, 1.25 mg and 1.66 mg, respectively, were maintained at 73.5% -84.6% level, indicating a considerable storage stability. The higher the concentration of glutamic acid, the higher GABA production, but the lower the conversion rate.

Lb. Changes in GABA content during the fermentation of brown rice using zymae GU240 and low concentration glutamic acid sample GABA production (mg / g of brown rice) / conversion rate (%) Immediately after inoculation 24 hours after fermentation Save one day Storage 1 week Storage 3 weeks Save 5 weeks A ¹ control 1 0.09 0.14 0.17 0.19 0.21 0.19 B ² Controls 2 0.13 0.17 0.13 1.13 0.06 0.05 C 0.1% glutamic acid
(6.79 mM) 0.13 0.52
(75.59%) 0.50
(71.54%) 0.47
(68.14%) 0.46
(66.18%) 0.44
(64.07%) D 0.3% glutamic acid
(20.39 mM) 0.13 1.22
(58.10%) 1.03
(49.41%) 1.04
(49.50%) 1.07
(51.34%) 1.01
(48.23%) E 0.5% glutamic acid
(33.98 mM) 0.13 1.70
(48.62%) 1.22
(34.83%) 1.20
(34.45%) 1.24
(35.60%) 1.21
(34.68%) F 0.7% glutamic acid
(47.57 mM) 0.13 2.14
(43.68%) 1.74
(35.54%) 1.78
(36.31%) 1.71
(34.96%) 1.66
(33.84%)

¹ Lb. zymae GU240 strain Inoculated, Glutamic acid-free

² Lb. zymae GU240 strain was inoculated at 1 x 10 &lt; ⁹ &gt; per gram of brown rice, and glutamic acid-free

< Example  7>

GU240  Application of fermented brown rice mass production process using strain

The germination and fermentation experiments of GU240 strain, which has excellent GABA production ability, were conducted under the condition of mass production of the strain as a seed. 0.7 kg of glutamic acid was dissolved in 25 L of distilled water, and then the strain GU240 was suspended in 25 L by 1.0 × 10 ¹⁴ . This was inoculated by spraying 100 kg of brown rice uniformly. That is, the inoculum of glutamic acid was adjusted to 0.7% (w / w) and the GU240 number per gram of brown rice was 1.0 × 10 ⁹ . The inoculated brown rice was fermented at 30 ℃ for 24 hours in the fermentation chamber, and then dried at 40 ℃ for 24 hours. The viable cell count and GABA production confirmation experiment on these fermented brown rice samples were carried out. The fermented brown rice samples obtained by mass production were collected at room temperature for 12 weeks and were collected at intervals of 1-4 weeks to measure viable cell count and GABA content.

The number of live cells was confirmed as described in Examples 1-3, and the generation of GABA was confirmed using the quantitative method using the GABase mentioned above.

Table 17 shows the results of measurement of viable cell count and quantitation of GABA. The strain GU240 was reduced from 1 × 10 ⁹ per gram of brown rice, which was the initial dose, to about 1.64 × 10 ^{8 in the} first week of storage, and decreased to about 6.62 × 10 ⁷ in three weeks of storage. Thereafter, it decreased slowly to about 4.24 × 10 ^{5 at} 12 weeks of storage, but there were still many. Importantly, it was confirmed that GABA was produced from glutamic acid by fermentation through GU240 strain even in mass production. In the first week of storage, the conversion was 1.34 mg, which was 36.14%. As the storage period was longer, the GABA concentration decreased to 1.29 mg (26.36%) after 12 weeks of storage. In particular, the results from mass production using 100 kg brown rice were almost the same when compared with the result of GABA production in the fermentation using the small amount (300 g) of the above-mentioned Table 15. That is, when the fermentation was carried out with 0.7% glutamic acid in a small amount of production, there was no difference between 1.78 mg of GABA content and 1.77 mg of 1 week after fermentation. In small volume production, 1.66 mg after 5 weeks and 1.51 mg after 6 weeks in mass production were also similar.

It was shown that the high content of GABA fermented brown rice could be mass produced by the addition of the developed GU240 strain and a relatively small amount of glutamidic acid (0.7% or less), indicating that it is economically feasible to mass produce GABA high content fermented brown rice That significance is great.

Lb. Number of viable cells and GABA during the mass fermentation of brown rice using zymae GU240 sample Storage 1 week Storage 2 weeks Storage 3 weeks Save 4 weeks Storage 6 weeks Save 8 weeks Save 12 weeks Live bacteria
(CFU per gram of brown rice) 1.64 x 10 ⁸ 1.60 x 10 ⁸ 6.62 × 10 ⁷ 4.72 × 10 ⁷ 6.74 x 10 ⁶ 3.94 × 10 ⁶ 4.24 × 10 ⁵ GABA production
(mg / gram of brown rice) 1.77 1.73 1.65 1.64 1.51 1.39 1.29 Conversion Rate
(%) 36.14 35.32 33.73 33.57 30.87 28.53 26.36

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Korea Microorganism Conservation Center (Domestic) KFCC11545P 20121123

Claims (15)

Lactobacillus zymae GU240 (KFCC11545P) strain with food-pharmacological activity. The method according to claim 1,
Lactobacillus zymae GU240 (KFCC11545P) strain characterized in that the food is brown rice. 3. The method of claim 2,
The strain Lactobacillus zymae GU240 (KFCC11545P) strain is characterized by fermenting brown rice to increase the amount of GABA (gamma-aminobutyric acid) produced. (a) culturing a strain of Lactobacillus zymae GU240 (KFCC11545P); And
(b) fermenting the cultured Lactobacillus zymae GU240 (KFCC11545P) strain in a brown rice and fermenting the fermented brown rice. 5. The method of claim 4,
Wherein the Lactobacillus zymae GU240 (KFCC11545P) strain is cultured on an MRS liquid culture medium, wherein the liquid culture medium comprises MSG at a concentration of 0.1-5% based on the total weight. 5. The method of claim 4,
Wherein the Lactobacillus zymae GU240 (KFCC11545P) strain is cultured on an MRS liquid culture medium, wherein the liquid culture medium comprises glutamic acid at a concentration of 0.1 to 5% based on the total weight. 5. The method of claim 4,
Wherein the fermentation step comprises inoculating 1 × 10 ⁹ to 1 × 10 ¹¹ CFU (colony forming unit) of the Lactobacillus zymae GU240 (KFCC11545P) strain against 100 g of brown rice. 5. The method of claim 4,
Wherein the fermentation step comprises inoculating a strain of Lactobacillus zymae GU240 (KFCC11545P) and yeast together. 9. The method of claim 8,
Wherein the yeast is a Saccharomyces Mrs. Celebi Asia (Saccharomyces cerevisiae ) KACC30008 strain. 10. The method of claim 9,
Wherein the Lactobacillus zymae GU240 (KFCC11545P) strain and the Saccharomyces cerevisiae KACC30008 strain are inoculated at a ratio of 1: 1 (v / v). (I) preparing a suspension by adding a Lactobacillus zymae GU240 (KFCC11545P) strain to a solution containing glutamic acid and dispersing it;
(Ii) inoculating the strain by spraying the prepared suspension into brown rice; And
(Iii) fermenting the inoculated brown rice. 12. The method of claim 11,
Wherein the suspension sprayed on the brown rice in step (ii) comprises glutamic acid at a concentration of 0.1 to 0.7% (w / w) based on the weight of the brown rice. 12. The method of claim 11,
Wherein the strain inoculation in step (ii) is inoculated with 1 x 10 &lt; ⁹ &gt; to 1 x 10 &lt; ¹¹ &gt; CFU (colony forming unit) of Lactobacillus zymae GU240 (KFCC11545P) strain per gram of brown rice. Fermented brown rice mass production method. 12. The method of claim 11,
Wherein the fermentation in step (iii) is performed at a temperature of 28 ° C to 32 ° C for 20 hours to 30 hours. 15. The method according to any one of claims 11 to 14,
Wherein said GABA is contained in an amount of 1.0 to 1.8 mg per 1 g of brown rice.

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