KR20140051543A - Method for producing fermented herb extract with high gaba content using lactobacillus plantarum k154 - Google Patents

Abstract

The present invention relates to a method for manufacturing a fermented herb extract with a high gamma-aminobutyric acid (GABA) content using lactobacillus plantarum K154, and provides the method for manufacturing a fermented herb extract with a high GABA content using lactobacillus plantarum K154 including: a step of preparing a starter by cultivating lactobacillus plantarum K154 in a medium; a step of inoculating the lactobacillus plantarum starter into a plant extract including 0.1-10 wt% of glucose and 0.1-5 wt% of a yeast extract; and a step of fermenting the inoculated herb extract. More specifically, 0.1-5 wt% of mono sodium glutamate (MSG) is additionally added to the medium. Moreover, provided is the fermented herb extract with an enhanced GABA content manufactured by the method.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for producing a GABA-enhanced fermented plant extract using Lactobacillus plantarum K154,

The present invention relates to the use of Lactobacillus plantarum The present invention relates to a method for producing a fermented plant extract in which gamma-aminobutyric acid (GABA) is promoted using K154.

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 present inventors have completed the present invention by preparing a fermented plant extract having enhanced GABA content through lactic acid fermentation of extracts of Seokchangpo, native and ginseng extracts, which are used as main medicaments of Gomyeongmang.

It is an object of the present invention to provide a method for producing a fermented plant extract in which gamma-aminobutyric acid (GABA) is promoted using Lactobacillus plantarum K154 starter, which is a lactic acid bacterium.

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

In order to accomplish the above object, the present invention provides a method for producing Lactobacillus plantarum plantarum ) to prepare a starter; Inoculating the Lactobacillus plantarum starter with a plant 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 plant extract, wherein the gamma-aminobutyric acid (GABA) is promoted. Specifically, the medium is supplemented with 0.1 to 5% 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 required for GABA production may be insufficient. If the amount of MSG is more than 5.0 wt%, MSG not converted to GABA may remain.

In the present invention, the plant extract is characterized by extracting raw paper, Baekbokryeong and Seokchangpo at a weight ratio of 1: 1: 1, wherein the Lactobacillus plantarum is Lactobacillus plantarum plantarum ) K154 (KACC91727P).

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 ground paper, 300 g of Baekbokryeong, and 300 g of Seokchangpo were added to a water pan with 13,000 mL of water and heated at 120 캜 for a total of 3 hours to prepare a plant extract or a common mint.

Also, the inoculating step is characterized by inoculating 1 to 10% (v / v) of the lactobacillus plantarum starter, wherein the step of fermenting is characterized by fermentation at 25 to 42 캜 for 1 to 7 days .

When the inoculum amount of the lactobacillus plantarum starter is less than 1% (v / v), it is impossible to produce gamma-aminobutyric acid in a high content due to the low concentration of the strain. When the amount exceeds 10% (v / v) There may be a problem.

The present invention also provides a fermented plant extract having enhanced GABA produced by the above method.

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

Figure 1 shows that when MSG is added (0.5%) and not added, Lactobacillus plantarum plantarum ) K66 and Lactobacillus plantarum K154 strain.
FIG. 2 shows TLC results confirming GABA production ability according to fermentation time.
FIG. 3 is a TLC result showing the change in GABA productivity according to the heat treatment conditions.
FIG. 4 shows a fermentation process diagram for preparing a fermented plant 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> GABA  Screening of the producing strains ( screening )Through GABA  Production Optimization Strain Selection

1. Culture method of strain

Lactobacillus isolated from kimchi ( Lactobacillus plantarum) K66 and Lactobacillus tarum Plan (Lactobacillus plantarum ) strain K154 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 plantarum Lactobacillus tarum Plan (Lactobacillus more plantarum) K66 plantarum strain K154 exhibited a good GABA production ability, and MSG production significantly increased GABA production (FIG. 1).

< Example  2> Depending on fermentation time GABA Generation  Confirm

To determine the optimal conditions for GABA production according to the fermentation time, 300 g of ground rice, 300 g of Baekbokwolgyeong and 300 g of Seokchangpo were added to a hot water tank with 13,000 mL of water and heated for 3 hours at 120 ° C for a total of 3 hours, 2.5% of yeast extract and 0.5% of glucose were added and sterilized. Then, 10% (v / v) of Lactobacillus plantarum K154 starter cultured at 30 ° C for 24 hours was added to the MRS medium with 2% MSG and fermented for 7 days.

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. As a result, the pH increased and the acidity decreased as the fermentation proceeded, indicating acidification (Table 2). On the other hand, GABA content was significantly increased on day 3 than on day 1, and the size of spot after day 3 was not significantly different, indicating that the fermentation conditions for 3 days were optimal (FIG. 2).

Fermentation time ( day ) pH Total acidity ( Total asitity ) (%) One 3.77 1.13 3 3.85 1.08 5 3.82 1.04 7 3.86 1.03

< Example  3> HPLC Using GABA  dose

The high performance liquid chromatograph used for the analysis was a Hewlett Packard 1100 Series. The column was equipped with a Waters Nova-Pac C18 4 μm (3.9 x 300 mm) and mounted on a column oven maintained at 46 ° C to allow for reproducible separation under constant temperature conditions. Two buffers were used for mobile phase. Buffer A used 140 mM NaHAc, 0.1% TEA, 6% CH ₃ CN, pH 6.1, and buffer B was 60% CH ₃ CN. Gradient conditions for separation were as shown in Table 3 and analyzed at a flow rate of 1.0 mL for 30 minutes. Detection was performed at a wavelength of 254 nm using a UV-VIS detector (HP 1100 Series).

Solvent gradient condition time % B Flow rate (ml / min) 0 0 One 12 8 One 13 12 One 15.2 20 One 22.5 46 One 22.72 100 1.5 23.2 100 1.5 25 100 1.5 25.7 100 1.5 26 0 1.5 29 0 1.5 30 0 One

The GABA contents of fermented mung mungang were analyzed by HPLC to evaluate the difference in GABA content between 1 and 3 days after fermentation. After fermentation, it was confirmed that GABA was increased to about 60-fold or more (Table 4 and Table 5).

Free amino acid content after fermentation ug / mL AA Gun mang tang However,
+ YE 2.5% +
Starter 10% (MSG 2%)
1day However,
+ YE 2.5% +
Starter 10% (MSG 2%)
3day Cys 0.00 14.48 41.23 ASP 352.58 332.42 335.98 GLU 949.29 732.26 25.56 ASN 292.13 404.97 383.58 SER 210.14 228.36 149.40 GLN 11.66 0.00 11.13 GLY 163.68 164.29 156.03 HIS 68.78 69.82 74.55 ARG 310.53 319.47 324.91 THR 222.54 215.16 214.83 ALA 594.60 565.02 540.91 GABA 31.40 1356.57 1942.84 PRO 153.66 130.67 123.48 TYR 159.21 159.56 154.46 VAL 440.02 428.19 417.06 MET 130.46 116.81 112.69 Cys2 0.00 0.00 0.00 ILE 353.45 343.70 327.57 LEU 610.91 584.43 556.56 PHE 313.09 330.56 311.75 TRP 160.88 254.45 234.27 LYS 203.82 237.99 222.57 TOTAL 5746.88 6989.18 6661.34

Condition GABA content (ug / mL) Gun mang tang 31.40 Gum myeongdang + glucose 0.5% + YE 2.5% +
Starter 10% (MSG 2%)
1day 1356.57 Gum myeongdang + glucose 0.5% + YE 2.5% +
Starter 10% (MSG 2%)
3day 1942.84

< Example  4> Fermentation Marsh DPPH (2,2- 피덴 -One- picryl - hydrazyl ) Radical  Scatters

The antioxidant capacity of the extract was measured by DPPH radical scavenging activity modified by Moreno et al. The free radical scavenging activity of the sample was determined by measuring the reducing power against DPPH, which is a stable radical. Each sample was dissolved in 99% methanol, and 800 μL of the diluted diluted solution and 0.15 mM DPPH in 20% 200 μL of the solution was left for 30 minutes, and the absorbance was measured at 517 nm. The free radical scavenging activity of each sample extract was expressed as RC50, the concentration of the sample required to reduce the absorbance of the control without addition of the sample to ½ (Table 6). BHA was used for activity comparison. The DPPH radical scavenging ability of Sungmyung-tang was not high, but it was confirmed that it increased as the fermentation progressed.

Sample RC ₅₀ value (%)
DPPH scavenging ^{1 )} Gun mang tang 311.25 ± 6.24 ²⁾ Fermented mushroom  One day 240.52 + - 4.36 Fermented mushroom  3 day 208.46 ± 21.52

¹⁾ Concentration required for 50% reduction of DPPH at 30 min after starting the reaction.

²⁾ Each value is mean ± SD (n ≥ 3).

< Example  5> Depending on the heat treatment conditions GABA  Confirmation of content change

When commercializing fermented mushrooms, it is necessary to heat-treat them to prevent pollution of the germs and proliferation of viable bacteria. Therefore, heat treatment was performed at 85 ° C for 15 minutes, 30 minutes, and 45 minutes to find the most efficient conditions. Then, viable cell counts were measured and it was observed whether the GABA content was changed by TLC method. As a result of counting viable cell counts, no colony appeared in all the plates, and TAB showed no difference in GABA content. It was confirmed that heating for 15 minutes at 85 ° C was the optimum heat treatment condition (FIG. 3).

Agricultural Biotechnology Research Institute KACC91727 20120706

Claims (7)

Culturing Lactobacillus plantarum in a medium to prepare a starter;
Inoculating the Lactobacillus plantarum starter with a plant extract containing 0.1 to 10% by weight glucose and 0.1 to 5% by weight yeast extract; And
A method for producing a fermented plant extract having enhanced gamma-aminobutyric acid (GABA) comprising fermenting the inoculated plant extract. The method of claim 1, wherein the culture medium further comprises 0.1-5 wt% monosodium glutamate (MSG). 3. The method according to claim 1 or 2, wherein the plant extract is hot water-extracted at a weight ratio of 1: 1: 1 of raw paper, Baekbokryeong and Seokchangpo. 3. The method according to claim 1 or 2, wherein said Lactobacillus plantarum is selected from the group consisting of Lactobacillus plantarum ) K154 (KACC91727P). The method according to claim 1 or 2, wherein the inoculating step comprises inoculating 1 to 10% (v / v) of the Lactobacillus plantarum starter. 3. The method of claim 1 or 2, wherein the fermenting step is performed at 25 to &lt; RTI ID = 0.0 &gt; 42 C & To 7 days after the fermentation. A GABA-enhanced fermented plant extract prepared by the method of any one of claims 1 to 6.

Images