KR101485970B1 - Novel Lactobacillus sp. AML 2-1 strain, a method of producing polyamines and Gamma-aminobutyric acid using Lactobacillus sp. AML 2-1 and use of polyamines and Gamma-aminobutyric acid - Google Patents

Abstract

The present invention relates to a method for producing Lactobacillus sp. Lactobacillus sp. AML 2-1 ( Lctobacillus sp. sp. AML 2-1) with a substrate ornithine to produce a polyamine and a functional neurotransmitter, gamma aminobutyric acid.

Description

Novel Lactobacillus sp. AML 2-1 strain, a method for producing polyamines and gamma aminobutyric acid using the same, and use of polyamines and gamma-aminobutyric acid {Novel Lactobacillus sp. AML 2-1 strain, a method of producing polyamines and Gamma-aminobutyric acid using Lactobacillus sp. AML 2-1 and use of polyamines and Gamma-aminobutyric acid}

The present invention relates to novel Lactobacillus sp. AML 2-1 strain ( Lactobacillus sp. (AML 2-1 strain: Accession No .: KCTC 12048BP), a method for preparing polyamines and gamma-aminobutyric acid using the same, and use of polyamines and gammaaminobutyric acid. More specifically, the present invention relates to a method for producing Generally Recognized As Safe, a novel microbial strain, Lactobacillus sp. AML 2-1, is isolated and identified, and a method for producing polyamines such as gamma aminobutyric acid and biogenic amines, Gamma-aminobutyric acid. ≪ / RTI >

Polyamines are a kind of biogenic amines, nitrogen compounds mainly formed by the decarboxylation of amino acids, the amination of aldehydes and ketones, and the transamination of amino acids, and are generally necessary to maintain high intestinal function and immune system metabolism.

Biogenic amines are low molecular weight and are a common component of these cells because they are synthesized in microbial, plant and animal metabolic processes (Estefan et al ., Int . J. Food Microbiol. 2011; Brink et al ., Int . J. Food Microbiol . 1990). Bigenenic amines in food and beverages are produced by the enzymatic action of raw materials and the decanoic action of amino acids of microorganisms. The microorganisms (lactic acid bacteria) producing biogenic amines include Lactobacillus brevis , Lactobacillus buchnerii , Lactobacillus curvatus , Lactobacillus carnis , Lactobacillus divergens and Lactobacillus hilgardii are known and are found in meat, meat products and fermented foods (Maijala et al ., Meat Sci . , 1993; Sara et al., Int . J. Food Microbiol . 1999). Various kinds of amino acids are produced, but putrycin, spermidine and spermine belonging to polyamines are indispensable components of all living cells in particular (Romain et al ., Pediatric Res . 1992; Bard et al ., J. Nutr . Biochem . 1993).

Fermented foods have been reported to enhance the antioxidant system and strengthen the immune system to enhance the body's defense, and have potential for various functional materials (Magwamba et al ., J. Food Port . 2010). Fermented foods are excellent fermenting microorganisms and fermented foods that contain substances that are extremely nutritious as well as physiologically functional. Therefore, it is possible to switch to industrial groups with high added value by using microbial resources having specific functions existing in traditional fermented foods.

A method for producing gamma aminobutyric acid using a strain of Lactobacillus sp. Isolated from a fermented food or the like is disclosed in Korean Patent Laid-Open No. 10-2004-0069437 (Publication Date: 2004.08.06, Registration No.: 1005490940000, (KFCC-11321), which has the ability to separate gamma-aminobutyric acid (GABA) from kimchi,

Korean Patent Laid-Open No. 10-2007-0052831 (Publication date: May 23, 2007, No. 1007538640000, Registered on August 24, 2007) discloses a lactobacillus A strain of OPM-2 (Lactobacillus buchnery OPM-2, accession number KFCC 11353P) is described,

Korean Patent Application No. 10-2006-0041691 (filed on May 5, 2006, No. 1007555080000, issue date: Aug. 29, 2007) discloses a strain Lactobacillus crispus sp. Having excellent milk clotting ability and GABA producing ability separated from crude oil (Lactobacillus crispatus) RMK567 (KCCM 10745P).

1. Korean Patent Laid-Open No. 10-2004-0069437 (Publication Date: 2004.08.06, Registration No .: 1005490940000, Registered Date: 2006.01.26) 2. Korean Patent Publication No. 10-2007-0052831 (Publication Date: May 23, 2007, Registration No.: 1007538640000, Registered Date: Aug. 24, 2007) 3. Korean Patent Application No. 10-2006-0041691 (Filing Date: 2006.05.09, Registration No .: 1007555080000, Registered Date: Aug. 29, 2007)

The present invention seeks to isolate and identify GRAS-class microorganisms producing polyamines (Putrescine, Spermidine, Spermine) and gamma-aminobutyric acid from the fermented seafood, which is a traditional fermented food in Korea, and using polyamines and gammaaminobutyric acid .

In the present invention, a microorganism producing polyamine as a biogenic amine and producing gamma aminobutyric acid was separated from fermented fermented salted fish and production of polyamines was confirmed by adding 0.5% (w / v) of substrate (ornithine) The resulting microorganism was named Lactobacillus sp. AML 2-1 ( Lactobacillus sp. AML 2-1).

That is, a main object of the present invention is to provide a novel strain producing polyamines and gamma-aminobutyric acid.

It is still another object of the present invention to provide a method for producing a polyamine or gamma aminobutyric acid by culturing the novel strain in a culture medium supplemented with ornithine 0.5% (w / v) in MRS medium, Butyric acid. ≪ / RTI >

Unlike conventional polyamine-producing bacteria that produce a substance, the substrate is formed and then the other strain is added to produce a polyamine. The Lactobacillus sp. AML 2-1 strain isolated from Korean traditional fermented food fermented foodstuff, AML 2-1, Which is a strain that produces polyamines through the steps of putricin, spumidine and spermine, and also produces gamma aminobutyric acid (GABA) among the major metabolites. Therefore, the polyamine and gamma aminobutyric acid can be more efficiently produced using the Lactobacillus sp. AML2-1 strain of the present invention.

Fig. 1 is a graph showing the activity of the Lactobacillus sp. AML 2-1 ( Lactobacillus sp. AML 2-1).
2 is a photograph of Lactobacillus sp. AML 2-1 ( Lactobacillus sp. AML 2-1) taken by scanning electron microscope.
FIG. 3A shows the Lactobacillus sp. AML 2-1 ( Lactobacillus sp. 3B is a HPLC graph showing the production of polyamine by AML 2-1), and FIG. 3B is a HPLC graph showing the production of polyamine by MRS medium + substrate (Ornithine 0.5% (w / v) AML 2-1 ( Lactobacillus sp. AML 2-1). ≪ tb >< TABLE >
Fig. 4 is a graph showing the Lactobacillus sp. AML 2-1 ( Lactobacillus sp. AML 2-1). ≪ / RTI >
FIG. 5A shows the Lactobacillus sp. AML 2-1 ( Lactobacillus sp. AML 2-1) in terms of DPPH, and Fig. 5B is a graph showing the antioxidative activity of the polyamine-producing culture broth of Lactobacillus sp. AML 2-1 ( Lactobacillus sp. (Flavonoid content: ppm), and Fig. 5C is a graph showing the antioxidative activity of the polyamine-producing culture broth of Lactobacillus sp. AML 2-1 ( Lactobacillus sp. AML 2-1) (polyphenol content unit: ppm), and FIG. 5D is a graph showing the antioxidative activity of the polyamine- AML 2-1 ( Lactobacillus sp. AML 2-1) in terms of SOD-like activity.
FIG. 6A is a photograph of the Lactobacillus sp. AML 2-1 ( Lactobacillus sp. The polyamine produced the culture medium of AML 2-1) the footage Pseudomonas (Pseudomonas putida ) KCTC 2198, and Fig. 6B and Fig. 6C are photographs showing that they do not have antimicrobial activity in Lactobacillus sp. AML 2-1 ( Lactobacillus sp. AML 2-1) has antimicrobial activity on Bacillus subtillus KCTC 1914 and E. coli KCTC 1041. The polyamine-

In order to achieve the above object, the present invention provides Lactobacillus sp. AML 2-1, which produces polyamines and gamma-aminobutyric acid.

The present invention provides the basis for the antioxidative and antimicrobial activities of polyamines and gamma-aminobutyric acid containing the microbial culture solution of the present invention as an active ingredient.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example  One : Polyamine and  Isolation and selection of gamma aminobutyric acid-producing bacteria

In order to isolate GRAS-grade microorganisms producing polyamines and gamma-aminobutyric acid, Korean traditional fermented foods such as fermented seafood were collected and used as a source of microorganism, suspended and diluted in sterilized physiological saline, and proteose peptone 10.0 , 10.0 g of beef extract, 5.0 g of yeast extract, 20.0 g of glucose, 1.0 g of Polysorbate 80, 2.0 g of ammonium citrate, 5.0 g of sodium acetate, 0.1 g of magnesium sulfate, 0.05 g of manganese sulfate, 2.0 g of diphotassium phosphate and 18 g / l of agar was inoculated on an MRS agar medium , ≪ / RTI > 37 for 2 days to isolate the microorganisms.

To isolate strains producing polyamine and gamma aminobutyric acid from pure strains, 1 g of a bacterial sample was added to 10 ml of sterilized distilled water using conventional salted fish as a sample, and 100 μl of the sample was added to a MRS solid medium And cultured in a 37 ° C incubator for 2 days. The colonies grown in the incubator for 2 days were selected based on the colonies whose color changed to yellow. The selected strains were inoculated on a MRS liquid medium supplemented with 5% ornithine (W / V) substrate, cultured for 3 days at 37 ° C for 3 days, pretreated with Dansylation (0.5 ml of 0.5% HClO _{4 in} 1 ml of sample, After centrifugation, the supernatant was diluted to 1: 2: 1 with Dansyl chloride and Na ₂ CO ₃ saturated solution, and the reaction was induced at 60 for 1 hour. The proline solution (250 mg / ml) was added to the culture supernatant Chloroform: Developed with a 5: 1 dilution of triethylamine (5: 1), followed by UV at 254 nm to detect putricin, spumidine and spermine. Gamma aminobutyric acid (GABA) The solution was loaded on TLC and developed with 5: 2: 2 solution of n-buthanol, acetic acid and deionized water. The ninhydrin solution was sprayed and dried. A strain that simultaneously produces gamma aminobutyric acid (GABA) Species were selected.

Then, 16S rRNA (base analysis) sequencing was performed to identify the bacterium producing polyamines and gamma aminobutyric acid. The morphological characteristics of the strains were confirmed by scanning electron microscope (SEM ) (Figs. 1 and 2). The isolated, identified microorganism was transformed into Lactobacillus sp. AML 2-1, and deposited with the Korea Research Institute of Bioscience and Human Technology (KCTC 12048BP) on April 4, 2011.

Example  2: Using the above antagonistic bacteria Polyamine  ( Putricin , Spudmin , Spermine ) Culturing conditions for production

MRS media conditions were supplemented with ornithine 0.5% (w / v) substrate to produce Lactobacillus sp. AML 2-1 ( Lactobacillus sp. AML 2-1) was cultivated at 30 ° C. for 3 days. As shown in FIG. 3, the polyamine was produced in the culture medium, and the production of gamma aminobutyric acid was examined. As a result, gamma aminobutyric acid was produced in the culture solution as shown in FIG.

Example  3: Lactobacillus sp. AML  2-1 ( Lactobacillus sp . AML  2-1) Measurement of antioxidative activity of culture medium

Total polyphenol content, DPPH radical scavenging activity, total flavonoid content, and SOD - like activity of isolates were measured.

Total polyphenol content was measured by Folin-Denis method, which is generally known to be involved in anticancer activity. That is, 1 ml of a 2% Na ₂ CO ₃ solution was added to 50 μl of the supernatant by centrifugation, and 50 μl of Folin &Ciocalteu's phenol reagents were mixed. After reacting at room temperature for 30 minutes, the absorbance was measured at 760 nm . Tannic acid (Sigma co., USA) was used as a reference material and the total polyphenol content was calculated from the calibration curve prepared by the same method as the sample.

     DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity was measured by partially modifying Blois et al. In other words, 800 μl of DPPH solution was added to 200 μl of the supernatant by centrifugation, and the mixture was reacted at room temperature for 10 minutes, and the absorbance was measured at 525 nm. At this time, DPPH radical scavenging activity was shown by comparing the difference of absorbance between the sample and the non - additive.

Total flavonoid content was measured by the modified Davis method. 4 ml of 90% diethylene glycol was added to 400 μl of sample, and 40 μl of 1N NaOH was added. After reacting at 37 ° C for 1 hour, absorbance was measured at 420 nm. Rutin was used as a reference material.

Superoxide dismutase (SOD) - like activity was measured by measuring the amount of pyrogallol that catalyzed the conversion of hydrogen peroxide to SOD - like activity. To 10 μl of the sample were added 130 μl of Tris-HCl buffer (50 mM Tris [hydroxymethyl] amino-methane + 10 mM EDTA) and 10 μl of 7.2 mM pyrogallol at pH 8.5 and reacted at 25 ° C for 10 minutes. 10 μl of 1N HCl And the reaction was stopped. The amount of pyrogallol oxidized in the reaction solution was measured at 420 nm. The SOD - like activity was expressed as a percentage (%) of the difference between the absorbance of the culture solution and the undiluted Katsui.

SOD-like activity = [1- (absorbance of sample-added absorbance / untreated absorbance)] × 100

As a result, it was investigated that the DPPH radical scavenging activity showed a high DPPH radical scavenging activity of 90% or more regardless of the addition of the substrate (ornithine) in Lactobacillus sp. AML 2-1 (Fig. 5A).

The total flavonoid content was 53-60 ppm when the substrate was not added, 76-86 ppm when the substrate was added, and increased when the substrate was added. The content of flavonoid was found to be higher than that of the polyamine (Fig. 5B).

The total polyphenol content of Lactobacillus sp. AML 2-1 was 243-278 ppm when the substrate was not added and 276-292 ppm when the substrate was added. (Fig. 5B).

As a result of SOD-like activity, when the substrate was not added in Lactobacillus sp. AML 2-1 at early stage, it was decreased by 50% after 43 days at 3 ppm and increased from 38 ppm at 44 ppm by adding substrate . In comparison with Vitamin C 100 ppm, SOD activity was found to be about 1/2, and SOD activity was observed when compared with vitamin C concentration. The possibility of using SOD activity as a functional food additive or functional cosmetic additive (Fig. 5D).

Example  4 : Lactobacillus  genus AML  2-1 ( Lactobacillus sp . AML  2-1) Measurement of antimicrobial activity of culture medium

Lactobacillus sp. The antimicrobial activity of AML 2-1 was measured by a commonly known disk diffusion method. After culturing the Lactobacillus sp. AML 2-1 strain (ornithine 0.5% (w / w) substrate), the culture was centrifuged and 100 μl of the culture supernatant was loaded on each test disk. After drying, And the degree of antimicrobial activity was investigated (see Table 1 below).

As a result, Lactobacillus sp. AML 2-1 showed no antimicrobial activity in Pseudomonas putida KCTC 2198 (Fig. 6A), Bacillus subtilis subtillus) and KCTC 1914 in Escherichia coli (E. coli) KCTC 1041 exhibited the antibacterial activity (Fig. 6b, Fig. 6c).

Strain name AML 2-1 AML 2-1 + substrate MRS badge P. putida KTCT2198 - - - B. subtilis KCTC1914 1.68mm 0.35mm - E. coli KCTC1041 0.86 mm - -

Korea Biotechnology Research Institute KCTC12048BP 20111104

<110> andong national university industry-Academic cooperation foundation <120> Novel Lactobacillus sp. AML 2-1 strain, a method of producing          polyamines and Gamma-aminobutyric acid using Lactobacillus sp.          AML 2-1 and use of polyamines and Gamma-aminobutyric acid <130> 20111111 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1396 <212> DNA <213> Lactobacillus AML2-1 <400> 1 cgagcttccg ttgaatgacg tgcttgcact gatttcaaca atgaagcgag tggcgaactg 60 gtgagtaaca cgtggggaat ctgcccagaa gcaggggata acacttggaa acaggtgcta 120 ataccgtata acaacaaaat ccgcatggat tttgtttgaa aggtggcttc ggctatcact 180 tctggatgat cccgcggcgt attagttagt tggtgaggta aaggcccacc aagacgatga 240 tacgtagccg acctgagagg gtaatcggcc acattgggac tgagacacgg cccaaactnc 300 tacgggaggc agcagtaggg aatcttccac aatggacgaa agtctgatgg agcaatgccg 360 cgtgagtgaa gaagggtttc ggctcgtaaa actctgttgt taaagaagaa cacctttgag 420 agtaactgtt caagggttga cggtatttaa ccagaaagcc acggctaact acgtgccagc 480 agccgcggta atacgtaggt ggcaagcgtt gtccggattt attgggcgta aagcgagcgc 540 aggcggtttt ttaagtctga tgtgaaagcc ttcggcttaa ccggagaagt gcatcggaaa 600 ctgggagact tgagtgcaga agaggacagt ggaactccat gtgtagcggt ggaatgcgta 660 gatatatgga agaacaccag tggcgaaggc ggctgtctag tctgtaactg acgctgaggc 720 tcgaaagcat gggtagcgaa caggattaga taccctggta gtccatgccg taaacgatga 780 gtgctaagtg ttggagggtt tccgcccttc agtgctgcag ctaacgcatt aagcactccg 840 cctggggagt acgaccgcaa ggttgaaact caaaggaatt gacgggggcc cgcacaagcg 900 gtggagcatg tggtttaatt cgaagctacg cgaagaacct taccaggtct tgacatcttc 960 tgccaatctt agagataaga cgttcccttc ggggacagaa tgacaggtgg tgcatggttg 1020 tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttattat 1080 cagttgccag cattcagttg ggcactctgg tgagactgcc ggtgacaaac cggaggaagg 1140 tggggatgac gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg 1200 acggtacaac gagttgcgaa gtcgtgaggc taagctaatc tcttaaagcc gttctcagtt 1260 cggattgtag gctgcaactc gcctacatga agttggaatc gctagtaatc gcggatcagc 1320 atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc atgagagttt 1380 gtaacaccca aagccg 1396

Claims (6)

A Lactobacillus sp. AML 2-1 strain (Accession No .: KCTC 12048BP) having a nucleotide sequence of SEQ ID NO: 1 and having polyamine-producing ability.
Lactobacillus sp. AML 2-1 strain (Accession No .: KCTC 12048BP) having the nucleotide sequence of SEQ ID NO: 1 and having gamma aminobutyric acid production ability. Culturing the Lactobacillus sp. AML 2-1 strain (accession number: KCTC 12048BP) of claim 1 in a culture medium supplemented with ornithine 0.5% (w / v) substrate in MRS medium; And collecting the polyamine produced in the culture broth.
Culturing the Lactobacillus sp. AML2-1 strain (accession number: KCTC 12048BP) of claim 2 in a culture medium supplemented with ornithine 0.5% (w / v) substrate in MRS medium; And collecting the gamma-aminobutyric acid produced in the culture solution.
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