KR20140117188A - Novel strains Lactobacillus plantarum K154 with high gamma-aminobutyric acid activity - Google Patents

Abstract

The present invention relates to a lactobacillus plantarum K154 (KACC91727P) strain having excellent gamma-aminobutyric acid (GABA) producibility. As bile resistance, pH resistance and GABA producibility are more excellent than the previously found lactic acid, if the strain is cultivated and then used, functional food such as an extract of fermented plants containing high GABA can be manufactured.

Description

Novel strains Lactobacillus plantarum K154 with high gamma-aminobutyric acid activity < RTI ID = 0.0 >

The present invention relates to a novel Lactobacillus plantarum capable of producing gamma-aminobutyric acid plantarum ) K154 strain.

Gamma-aminobutyric acid (GABA) is a nonprotein amino acid that is distributed in nature such as microorganisms, plants and animals. GABA is a metabolite of plants and microorganisms produced by the decarboxylation of glutamic acid. It functions as an inhibitory neurotransmitter in the brain, so as to calm anxiety and nervousness caused by stress. Traditional foods made from microbial fermentation contain GABA. GABA is a safe, environmentally friendly ingredient and has potential for development as a new health functional food.

The synthesis of GABA is caused by glutamate decarboxylase, the optimal fermentation conditions being based mainly on the biochemical properties of the enzyme. The microorganisms that mainly produce GABA are lactic acid bacteria, which are bacteria that act as probiotics in the intestines, preventing them from growing food-causing pathogens. Factors predominantly affecting the production of GABA through microbial fermentation are temperature, pH, fermentation time and media composition. Therefore, studies on the mass production of GABA by optimizing these factors during the cultivation of various microorganisms are being actively carried out. 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 with amounts of natural food.

A study on the lactic acid bacteria that produce GABA Lactococcus to (Nomura et al. International Journal of Systematic Bacteriology. 49, 163, 1999) lactis has been shown to be capable of producing GABA, but it has not been used in fermented plant extracts as a high GABA-producing lactic acid bacterium.

On the other hand, methods for producing GABA-producing strains and gamma-aminobutyric acid using them are disclosed in Korean Patent Application Nos. 10-2010-0068711, Korean Patent No. 0549094, Korean Patent No. 0631857 and Korean Patent No. 0755508 However, the present inventors have found that Lactobacillus plantarum plantarum ) K154 strain. Accordingly, the present inventors have isolated Lactobacillus plantarum K154 from Kimchi and confirmed that the strain has excellent GABA production ability, and completed the present invention.

It is an object of the present invention to provide a novel Lactobacillus plantarum plantarum ) K154 (KACC91727P).

In order to achieve the above object, the present invention is gamma-aminobutyric acid (gamma-aminobutyric acid; GABA) capable of producing excellent Lactobacillus plan tarum (Lactobacillus plantarum ) K154 (KACC91727P).

The Lactobacillus plantarum of the present invention plantarum ) K154 (KACC91727P) was isolated from kimchi and screened and identified according to the method of the following examples. The identification of the microorganisms was based on his morphological, physiological and biochemical characteristics.

The present inventors collected and used different kimchi from different regions and were inoculated in a modified MRS medium (Table 1) in which NaN ₃ was added by diluting with a dilution of peptone, in a planar state by 0.1 ml, and cultured at 37 ° C for 48 hours , And each isolate was purely isolated from the transformed MRS medium and then transformed into yellow as a potential lactic acid bacterium. The selected strains were plated on the electrotransformed MRS medium and cultured aerobically to isolate pure strains. As a result of the identification of the pure isolates, the strain was Gram - positive bacillus and showed good growth regardless of presence or absence of oxygen, and negative for catalase and motility. Also, it was grown at 15 ° C and 45 ° C, and it was confirmed that it did not produce ammonia from gas and alginic acid from glucose and belonged to Lactobacillus .

On the other hand, 49 kinds of sugar fermentation tests were carried out to confirm the species names. As a result, 25 kinds of acids such as glucose were produced, and the results were obtained by inputting into the ATB identification system and using 16S rRNA gene sequence (Gene Sequence) Were found to be Lactobacillus plantarum .

Therefore, the inventors of the present invention found that a strain having excellent GABA-producing ability, isolated from kimchi, was transformed into Lactobacillus plantarum plantarum ) K154.

In addition, the strain is characterized by having bile resistance and acid resistance. The bile resistance was measured by measuring the lactic acid bacterium growth on the MRS medium. As a result, it was 0.5 log , But it was found to be resistant to bile. In addition, the results of the test for pH resistance showed that there was almost no influence even at pH 2, which is stronger than 6.4, which is the pH of the control.

The present inventors have found that Lactobacillus plants ( Lactobacillus The plant strain K154 (KACC91727P) is superior to the conventionally found lactic acid bacteria in biliary properties, pH tolerance and GABA production ability. Therefore, when the strain is cultured and used, a functional food such as a fermented plant extract containing a high content of GABA Can be manufactured.

Brief Description of the Drawings Fig. 1 is a schematic view of a Lactobacillus plantarum ) K154 was inoculated on the MRS medium.
FIG. 2 is a graph showing the activity of Lactobacillus plantarum ) K154 was inoculated on the MRS medium.
FIG. 3 shows the effect of Lactobacillus plantarum plantarum ) K154.

Hereinafter, the present invention will be described in more detail with reference to the following examples. It is to be understood, however, that these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

< Example  1> Isolation of microorganisms

Kimchi samples were collected from different localities and different households, and were inoculated with 0.3 ml of topical lotion in modified MRS medium (Table 1) supplemented with bromocresol purple and sodium azide And then cultured at 37 DEG C for 48 hours. Then, each of the herringbone was purely separated from the electro-transformed MRS medium, and the yellowish-transformed broth was selected as a potential lactic acid bacterium. The selected strains were plated on the electrotransformed MRS medium three times and cultured aerobically.

The composition of the modified MRS medium used for the microbial separation in the present invention  ingredient Component Ratio (grams / liter)  Proteose Peptone # 3  10.0 Beef extract  10.0 Yeast extract 5.0  Lactose 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 Sodium azide 0.25 Bromocresol purple 0.04

< Example  2> GABA  Generated vegetable Lactic acid bacteria  Selection

The MSG was added to the MRS medium at the level of 7 strains of 900 ug / ml or more, and the pH and GABA production were measured after culturing at 37 ° C for 18 hours. The results are shown in Table 2 below.

PH and GABA production by MSG addition level Strains 1% MSG added MSG 2% added MSG added 3% GABA (ug / ml) pH GABA (ug / ml) pH GABA (ug / ml) pH K4 703.91 + - 81.28 4.09 1061.36 ± 28.28 4.16 1501.69 + - 46.93 4.37 K66 1141.91 + - 71.86 4.04 1224.65 ± 48.99 4.23 1830.18 + - 46.93 4.43 K154 1548.61 + 81.28 4.04 1704.28 ± 28.66 4.19 2017.89 ± 0.00 4.42 K174 328.49 + - 66.36 4.42 821.23 + - 99.54 4.3 1689.40 ± 66.36 4.6 K177 782.13 ± 27.09 4.13 1042.42 + 49.64 4.45 2096.11 ± 270.94 4.47 K200 398.88 +/- 33.18 4.04 821.23 + - 33.18 4.21 1814.54 ± 27.09 4.42 K256 625.70 + - 71.68 4.06 821.23 + - 99.54 4.25 1384.37 ± 99.54 4.48

As shown in Table 2, the strain K154 isolated from Lactobacillus was selected as a strain having a high GABA production ability because the amount of GABA produced was the most stable and increased compared to other strains.

< Example  3> Identification of microorganisms

The taxonomic identification of microorganisms was carried out according to the method of Hammes et al. [The prokaryotes, 1563-1578, 2nd Edition, Springer-Verlag Co. (1992)]. These strains were Gram positive, Bacillus, well grown regardless of presence or absence of oxygen, and negative for catalase and motility. Also, it grew at 15 ℃ and 45 ℃ and did not produce ammonia from glucose and gas and arginine and belonged to Lactobacillus genus. When 49 kinds of sugar fermentation tests were carried out using API 50CHL kit (API bioMerieux, France) to determine the species, acid was generated from 25 species such as glucose, and it was inputted into the ATB identification system, The result obtained using the Gene Sequence was confirmed to be Lactobacillus plantarum. A strain having excellent ability to produce GABA isolated from kimchi was obtained from Lactobacillus plantarum plantarum K154, deposited with the Korean Agency for Research on Bioscience and Biotechnology (KACC) on July 6, 2012 under accession number KACC 91727P.

The physiological and biochemical characteristics of the novel microorganism Lactobacillus plantarum K154 of the present invention Gram reaction
Cell morphology
Spore formation
motility
Aerobic growth
Anaerobic growth
Catalase reaction
Grow at 15 ℃
Grow at 45 ° C
Gas production from glucose
Ammonia production from arginine +
Bacillus
-
-
+
+
-
+
+
-
- Party ability Glycerol
Erythritol
D-arabinose
L-arabinose
Ribos
Glucose
Fructose
Mannos
Sorbos
Rams North
Dissytol
Inositol
Mannitol
Sorbitol
a-Methyl-D-mannose
a-Methyl-D-glucoside
N-acetylglucosamine
Amigalline
Arbutin
Esculin
Salincin
Cellobiose
Maltose
Lactose
Melibiose -
-
-
+
+
+
+
+
-
+
-
-
+
+
+
-
+
+
+
+
+
+
+
+
+ D-Cirrus
L-Cirrus
Adonitol
beta -methyl-D-thiazole
Galactose
Sucrose
Trehalose
Inulin
Melitos
Raffinos
Starch
Glycogen
Silica
Gentiobios
D-Turanos
D-lysos
D-tagatose
D-Fucose
L-Fucose
D-arabitol
L-arabitol
Gluconate
2-keto-gluconate
5-keto-gluconate
-
-
-
-
+
+
+
-
+
-
-
-
-
+
+
-
-
-
-
-
-
+
-
-

In order to confirm whether GABA is suitable as an enhanced fermented plant extract starter, Lactobacillus plantarum plantarum ) K154 was used for the test while cultivating in the mungol.

< Example  4> Microbial growth test

The growth of lactic acid bacteria was tested by measuring viable cell count and pH. The viable cell counts were obtained by inoculating 1 ml of lactic acid bacteria in 150 ml of MRS liquid medium and 1 ml of 0.1% peptone solution at 34, 37, and 40 ° C for 3 hours. agar plate, incubated at 35 ° C for 48 hours, and the pH change was measured by temperature and time. The results are shown in FIG. 1 and FIG. As can be seen in FIGS. 1 and 2, the optimum temperature was 37 ° C, and it took 13 hours to reach pH 4.3-4.4, which is the optimum pH condition of the fermented product.

< Example  5> Antibiotic resistance test

The antimicrobial resistance test was carried out by using a tryptic soy broth (Difco, USA), and the growth inhibition was examined by a 2-fold dilution method to determine the lowest inhibitory concentration (MIC) .

Antibiotic susceptibility of Lactobacillus plantarum K154 Antibiotic The lowest inhibitory concentration (占 퐂 / ml) Aminoglycoside system Amikacin 160 Gentamicin 80 Kanamycin 200 Neomycin 200 Streptomycin * 200 beta -lactam system Penicillin-G * 160 Methacillin 320 Oxacillin 60 Ampicillin 160 Gram-positive spectrum Bashtrasin * 30 Rifampicin 480 Novobaiosin 7.5 Lincomycin 50 Gram-voice spectrum Polymycin B * 2,400 Broad spectrum Chloramphenicol 40 Vancomycin * 3,200

*: units / ml

As can be seen in Table 4, Lactobacillus plantarum K154 is more resistant to vancomycin and polymyxin B than to other antibiotics, while susceptible to novobiocin and bacitracin.

< Example  6> Enzyme activity test

The enzyme activity was determined by diluting the strain cultured in MRS liquid medium at 37 ° C for 18 hours with physiological saline to prepare a sample of 10 ⁵ - 10 ⁶ cfu / ml and then using API ZYM kit (API bioMerieux, France) Incubated at 37 ° C for 5 hours, and then subjected to enzyme reaction. The enzyme activity is shown in Table 5 as the value of 0-5 compared with the standard color trademark.

Comparison of Enzyme Activities of Lactobacillus plantarum K154 enzyme Enzyme activity Alkaline phosphatase
Esterase (C4)
Esterase lipase (C8)
Lipase (C14)
Leucine arylamidase
Valine arylamidase
Cysteine aryl amidase
Trypsin
Chymotrypsin
Acid phosphatase
Naphthol-AS-BI-phosphohydrolase
? -galactosidase
? -galactosidase
β-glucuronidase
alpha -glucosidase
? -glucosidase
N-acetyl- beta -glucosamine amine
? -mannosidase
? -fucosidase 0
0
0
0
5
4
One
0
0
One
3
0
5
0
4
5
3
0
0

*: Standard color is specified in steps from 0 to 5, 0 is negative and 5 is maximum intensity. 1 shows 5 nano moles, 2 has 10 nano moles, 3 has 20 nano moles, 4 has 30 nano moles, and 5 has an enzyme activity of 40 nano moles or more.

As shown in Table 5, Lactobacillus plantarum K154 showed high enzyme activity in leucine arylamidase,? -Galactosidase and? -Glucosidase. In the case of β-glucuronidase, which is a carcinogenic enzyme that converts benzopyrene into a carcinogenic substance, it has no enzyme activity and its safety can be confirmed.

< Example  7> Bile tolerance  exam

The results of the bile resistance test according to the method of Gilliland and Walker (Gilliland & Walker, J. Dairy Sci., 73, 905, 1990) are shown in FIG. As can be seen in FIG. 3, after the incubation for 7 hours, there was a slight inhibition at the difference of 0.5 log between the addition and the absence of bile, but it was shown to be resistant to bile.

< Example  8> pH  Immunity test

The pH tolerance test was carried out according to the method of Clark et al. (Clark et al., Cultured Dairy Products J., 28 (4), 11, 1993).

Comparison of Lactobacillus plantarum K154 resistance after 3 hours in hydrochloric acid solution [Unit: CFU per ml (Log number)] Culture time (hr) pH 2 pH 3 pH 4 pH 6.4 0 8.16 8.22 8.20 8.29 One 8.21 8.22 8.21 8.30 2 8.22 8.26 8.21 8.32 3 8.20 8.25 8.22 8.30

As can be seen from the above table 6, it showed acid resistance due to almost no influence even at pH 2, which is a strong acid, compared with the control pH of 6.4.

< Example  9> Antimicrobial activity test

The antimicrobial activity test was carried out according to Gilliland & Speck, J. Food Prot., 40 (12), 820, 1977).

Inhibition of food poisoning by Lactobacillus plantarum K154 in MRS medium (*) Food poisoning bacteria Growth Food poisoning bacteria ^a Lactobacillus plantarum K154 ^a % Inhibition
CFU / ml CFU / ml Escherichia coli
( Escherichia coli ) 5.8 x 10 ⁴ 4.7 × 10 ⁴ 19.0 Salmonella typhimurium
( Salmonella typhimurium ) 3.7 × 10 ⁵ 3.0 × 10 ⁵ 18.9 Stefiro Cocus Oreus
( Staphyloccous aureus ) 2.2 x 10 ⁶ 1.9 × 10 ⁶ 13.6

* Initial number of Lactobacillus plantarum K154: 2.4 × 10 ⁶ CFU / ml

^a Measurement after incubation at 37 ° C for 6 hours

As can be seen in Table 7, when Lactobacillus plantarum K154 was transformed into Escherichia coli coli ) and 19% inhibition of Salmonella typhimurium ( Salmonella typhimurium ), showing a similar inhibitory effect. Staphylococcus aureus (13.6%) was slightly less inhibitory than other food poisoning bacteria.

< Example  10> Gun mang tang  Following fermentation GABA  Content change

Glucose 0.5% and chongmyeongtang chongmyeongtang, yeast extract (Yeast Extract) 2.5%, the starter (Starter) 10%, MSG by 2% skim milk and MSG content Lactobacillus plan tarum (Lactobacillus plantarum) was inoculated to K154 10% and incubation at 37 ℃ 24 hours and 72 hours As a result of measuring a GABA content are given in Table 8.

Condition GABA content (ug / mL) Gun mang tang 31.40 Gum myeongdang + glucose 0.5% + YE 2.5% +
Starter 10% (MSG 2%), culture for 24 hours 1356.57 Gum myeongdang + glucose 0.5% + YE 2.5% +
Starter 10% (MSG 2%) 72 hours culture 1942.84

Lactobacillus plantarum ( Lactobacillus plantarum ) K154 and fermentation time increased with increasing GABA content.

Agricultural Biotechnology Research Institute KACC91727 20120706

Claims (2)

Lactobacillus plantarum ( Lactobacillus plantarum), which has excellent ability to produce gamma-aminobutyric acid (GABA) plantarum) K154 (KACC91727P) strain. The method according to claim 1, wherein the strain has bile resistance and acid resistance. Lactobacillus plantarum plantarum) K154 (KACC91727P) strain.

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