KR101407231B1 - Method for producing fermented herb extract with high GABA content using Lactobacillus helveticus RMK85 - Google Patents

Abstract

The present invention relates to a method for producing a GABA-enhanced fermentation plant extract using Lactobacillus helveticus RMK85, comprising the steps of: preparing a starter by culturing Lactobacillus helveticus in a medium; Inoculating the Lactobacillus helveticus starter with a crude drug extract containing 0.1 to 10% by weight glucose and 0.1 to 5% by weight yeast extract; And a step of fermenting the inoculated herbal medicine extract, wherein the gamma-aminobutyric acid (GABA) is promoted. Specifically, the medium is further supplemented with 0.1 to 1% by weight of monosodium glutamate (MSG). Also provided is a fermented herbal medicine extract having enhanced GABA produced by the above method.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for producing a GABA-enhanced fermented plant extract using Lactobacillus helveticus RMK85,

The present invention relates to Lactobacillus < RTI ID = 0.0 > The present invention relates to a method for producing a fermented herbal medicine extract in which gamma-aminobutyric acid (GABA) is promoted using helveticus RMK85.

Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter, which is known to inhibit blood pressure buildup, improve visual acuity, and calm anxiety and nervousness. In animals, it is distributed in the brain, heart, lung, kidney, etc. In the case of plants, it is mainly found in germinated brown rice and green tea. Hypnotists, examinees, and alcohol-consuming sufferers suffering from stress are reported to have low levels of GABA in the brain and blood, and a severe shortage of GABA in the body is known to cause seizures, convulsions, and epilepsy.

As the function of GABA is known, interest as functional food materials as well as pharmaceuticals is increasing. Currently, GABA is slightly contained in brown rice, green tea, malt, and cabbage, but its content is low and it is difficult to expect physiological activity by the amount of natural food. In order to increase the GABA content of these plants, GABA content in the plant was increased by anaerobic treatment of barley germinated in the preparation of tea leaves and barley malt, and glutamic acid addition during brown germination (Chang et al., 1992; Park et al., 2001; Yokoyama et al., 2002). However, since there is a limit to the production of GABA using plant-based materials, many studies using microorganisms for mass production of GABA have been conducted. Among them, lactic acid bacteria are one of the most beneficial microorganisms that can be used by human beings, and they have been used widely in fermented dairy products, such as soy sauce, kimchi, salted fish, sausages and medicines. However, studies on the isolation and improvement of strains producing GABA have been made in various aspects, but studies on foods or functional beverages having increased contents of GABA have not been studied yet.

On the other hand, Korean Patent No. 0404921 discloses a preparation method of tea ginseng which has ameliorating effect for forgetfulness, but it is not mentioned about fermentation using lactic acid bacteria or GABA as a total ginseng mixed with oriental herb medicine such as Japanese papaya and licorice. Accordingly, the inventors of the present invention have completed the present invention by preparing fermented herbal medicine extracts in which the content of GABA was enhanced through lactic acid fermentation of extracts of Seokchangpo, native and ginseng, which are used as main medicines of Sogwangju.

It is an object of the present invention to provide a method for producing a fermented herbal medicine extract in which gamma-aminobutyric acid (GABA) is promoted using lactobacillus Lactobacillus helveticus starter.

Another object of the present invention is to provide a GABA-enhanced fermented herbal medicine extract prepared by the above method.

In order to accomplish the above object, the present invention provides a method for culturing Lactobacillus helveticus, comprising culturing Lactobacillus helveticus in a culture medium to prepare a starter; Inoculating the Lactobacillus helveticus starter with a crude drug extract containing 0.1 to 10% by weight glucose and 0.1 to 5% by weight yeast extract; And a step of fermenting the inoculated herbal medicine extract, wherein the gamma-aminobutyric acid (GABA) is promoted. Specifically, the medium is further supplemented with 0.1 to 1% by weight of monosodium glutamate (MSG).

In the present invention, the term "starter" refers to a culture medium of microorganisms used for producing a fermented product. Therefore, the types of starter microorganisms determine the characteristics of the product and have an important influence on the quality of the product. Bacteria, fungi, yeast, etc., which are used as starters in microorganisms, can be used alone or in combination.

The medium is preferably a Lactobacillus MRS medium, but is not particularly limited as long as it is a medium suitable for culturing the strain.

If the amount of MSG added is less than 0.1 wt%, the precursor necessary for GABA production may be insufficient. If the amount of MSG is more than 1.0 wt%, MSG not converted to GABA may remain.

In the present invention, the herbal extract of the base paper, and baekbokryeong seokchangpo 1: 1: wherein the hot-water extract at a weight ratio of 1, wherein said Lactobacillus helveticus is Lactobacillus helveticus coarse coarse (Lactobacillus helveticus RMK85 (KACC91721P).

In the present invention, "Sungmyunggang" is a herb medicine composition composed of four natural herbal medicines, such as white ginseng, lavender, liquorice, and licorice root, which is called " If you forget to forget and eat for a long time, you will be able to memorize a thousand words a day. "(Source: Naver Encyclopedia). In the present invention, 300 g of raw paper, 300 g of Baekbokryeong, and 300 g of Seokchangpo were added to a hot water machine together with 13,000 mL of water and heated at 120 캜 for a total of 3 hours to prepare a herbal extract or a common mint.

The inoculating step is characterized in that 1 to 10% (v / v) of the Lactobacillus helveticus starter is inoculated, and the step of fermenting comprises fermenting at 25 to 42 ° C for 12 to 72 hours .

When the inoculation amount of the Lactobacillus helveticus starter is less than 1% (v / v), gamma-aminobutyric acid can not be produced in a high content due to low concentration of the strain, and if it exceeds 10% (v / v) The fermentation may occur.

In addition, the present invention provides a fermented herbal medicine extract having enhanced GABA produced by the above method.

The inventors of the present invention prepared fermented herbal medicine extracts by enhancing the content of gamma-aminobutyric acid (GABA) through lactic acid fermentation of herbal medicine extracts (Seokchangpo, native and extracts) and found that a high content of GABA It is possible to increase the possibility of developing functional beverages and foods containing them.

FIG. 1 shows the results of TLC in which MSG was added (0.5%) and GABA production was confirmed by strain type when not added.
FIG. 2 shows TLC results showing the ability of GABA production according to the ratio of MRS medium and herbal medicine extract.
FIG. 3 shows the results of TLC showing the ability of GABA production according to the concentration of yeast extract.
FIG. 4 shows TLC results confirming GABA production ability according to starter inoculation conditions.
FIG. 5 shows a fermentation process diagram for preparing a fermented herbal medicine extract having enhanced GABA content.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

< Example  1> Depending on the strain type GABA Generation  Confirm

1. Culture method of strain

Five strains isolated from crude oil, Enterococcus faecalis K48, Lactobacillus helveticus RMK85, Enterococcus faecium K36, Lactobacillus acidophilus RMK 567, Streptococcus macedonicus K49 The cells were cultured so that a single colony appeared on Lactobacilli MRS agar medium (Difco co., Ltd.), then cultured in MRS medium broth supplemented with 0.5% monosodium glutamate (MSG) And cultured at 30 ° C for 24 hours.

The composition of the MRS medium used for strain isolation of the present invention  ingredient Component Ratio (grams / liter) Proteose Peptone # 3 10.0 Beef extract 10.0 Yeast extract 5.0 Dextrose 20.0 Twin 80 1.0 Ammonium citrate 2.0 Sodium acetate 5.0 Magnesium sulfate 0.1 Manganese sulfate 0.05 Dipotassium phosphate 2.0

2. TLC measurement method

The MSG contained in the sample and the produced GABA content were confirmed by TLC (Silica gel 60 F254, Merck). 1 mL of the culture was concentrated 4 times using a speed vaccum and 2 μL was added to the TLC plate. In the analysis, the developing solvent was acetic acid: n-butyl alcohol: distilled water (2: 6: 2, v / v / v) And a 0.2% ninhydrin solution was sprayed with the coloring reagent. The spot was identified by color development at 95 ° C for 5 minutes. As a result, Lactobacillus helveticus RMK85 showed the highest ability to produce GABA, and that GABA production was increased by addition of MSG (FIG. 1).

< Example  2> MRS  Depending on the concentration of the medium GABA Generation  Confirm

To confirm the GABA production ability according to the MRS medium concentration, 300 g of raw cotton, 300 g of Baekbok-ryeong and 300 g of Seokchangpo were poured into a hot water pot with 13,000 mL of water and heated for 3 hours at 120 ° C. for 3 hours to prepare MRS medium % (v / v). As a strain, Lactobacillus helveticus RMK85 strain, which has excellent GABA production ability, was used. This strain was added to 5% (v / v) starter cultured at 30 ° C for 24 hours in MRS medium, Lt; / RTI &gt;

The pH and total acidity of each sample were measured by centrifuging each sample in 10 mL of the supernatant using a pH meter (Digital pH meter 420A +, Thermo Orion Beverly, MA, USA) The acidity was calculated by taking 10 mL of the supernatant and converting the amount used for titration with 0.1 N-NaOH to the lactic acid content (%) until the pH reached 8.3.

MRS medium concentration pH Total acidity (%) 0% 4.02 0.2 20% 3.85 0.58 40% 3.85 0.9 60% 3.95 1.35 80% 4.02 1.68 100% 4.04 1.93

As a result, it was confirmed that the higher the concentration of MRS medium, the better the production of GABA (Fig. 2). However, the MRS medium was judged as not suitable for food, and the influence of carbon source and nitrogen source Only the nutrient source was added to the herbal medicine extract. The decrease in pH and the increase in total acidity are due to Lactobacillus &lt; RTI ID = 0.0 &gt; The means and the helveticus) strains RMK85 growth is determined to increase GABA production when the growth of the strain hwalbalhal (Table 2). That is, when the strain is active, the activity of the glutamate decarboxylase (Glutamate decarboxylase) of the strain becomes active, thereby decomposing the substrate, monosodium glutamate (MSG), resulting in increased GABA production.

< Example  3> Yeast extract  Depending on concentration GABA Generation  Confirm

When 2% of yeast extract was added to 2% of glucose and lactic acid fermentation was performed at 1 ~ 2.5% concentration, GABA production ability was measured by TLC. When 2% of glucose and 2% of yeast extract were added, (Fig. 3). As a result of measurement of pH and total acidity, it was confirmed that there was no problem in growth of the strain (Table 3).

Therefore, 2% of glucose and 2.5% of yeast extract were used as the basic medium for herb extract.

Yeast extract concentration pH Total acidity (%) One% 3.37 1.35 1.5% 3.33 1.78 2% 3.42 1.89 2.5% 3.46 1.99

< Example  4> starter starter ) Depending on the vaccination condition GABA Production capacity  Confirm

GABA production ability was determined by adding starter inoculation method to 2% of glucose and 2.5% of yeast extract. MRS medium supplemented with Lactobacillus &lt; RTI ID = 0.0 &gt; helveticus RMK85 strain was inoculated with 5% starter and 0.5% MSG was added to MRS medium to obtain Lactobacillus helveticus ) Starter cultured in RMK85 strain was inoculated into a 5% inoculum and a starter Lactobacillus ( Lactobacillus) helveticus RMK85 cells were collected and enriched and tested under inoculation conditions (Table 4). As a result, it was confirmed that the herbal medicine extract using starter with 0.5% MSG had the highest GABA content (FIG. 4).

Sample pH Total acidity (%)
(Total acidity) Absorbance (660 nm) Herbal extract +
Glucose 2% +
YE 2.5% Starter 5% 3.50 1.50 2.717 MSG 0.5%
Starter 5% 3.58 1.40 2.713 Mycelium 3.47 1.40 2.884

The GABA content in the fermented herbal extract fermented by each of the above conditions was analyzed by HPLC. As a result, it was confirmed that GABA was increased about 3 times or more after fermentation compared to the fermented herbal medicine extract (Table 5).

Condition GABA content (ug / mL) Herbal extract 31.40 Herbal extract + glucose 2%
+ YE 2% + Starter 5% 45.44 Herbal extract + glucose 2%
+ YE 2.5% +
Starter 5% (MSG 0.5%) 105.86

Agricultural Biotechnology Research Institute KACC91721 20120523

Claims (7)

Culturing Lactobacillus helveticus RMK85 (KACC91721P) in a medium to prepare a starter;
A step of inoculating the herbal medicine extract comprising the extract of Lactobacillus helveticus starter with 0.1 to 10% by weight glucose and 0.1 to 5% by weight of yeast extract; And
A method for producing a fermented herbal medicine extract, wherein gamma-aminobutyric acid (GABA) is enhanced, comprising fermenting the herbal herb extract. [2] The method of claim 1, wherein the culture medium further comprises 0.1 to 1% by weight of mono sodium glutamate (MSG). The method according to claim 1 or 2, wherein the herbal medicine extract is hot water-extracted at a weight ratio of 1: 1: 1 of raw paper, Baekbokryeong and Seokchangpo. delete The method according to claim 1 or 2, wherein the inoculating step comprises inoculating 1 to 10% (v / v) of the Lactobacillus helveticus starter. The process according to any one of claims 1 to 5, To 72 hours after the fermentation. delete

Images