KR20230060470A - Novel Lactobacillus plantarum D2-3 strain and use thereof - Google Patents

Abstract

The present invention relates to a novel Lactobacillus plantarum D2-3 strain and its use, wherein the tannin hydrolyzate obtained by treating the strain with a tannin compound forms SOD1 (superoxide dismutase 1) G93A mutant protein filaments By confirming that it effectively inhibits, it can be usefully used as a composition for inhibiting filament formation of the SOD1 G93A mutant protein.

Description

Novel Lactobacillus plantarum D2-3 strain and use thereof {Novel Lactobacillus plantarum D2-3 strain and use thereof}

The present invention relates to a novel Lactobacillus plantarum D2-3 strain and its use.

Tannins are water-soluble phenolic products that can precipitate naturally occurring proteins from aqueous solutions. Tannins can be classified into hydrolysable tannins that are hydrolyzed when heated with dilute acids and condensed tannins that polymerize with hydrolyzed tannins to form water-insoluble phlobaphene. Tannins inhibit the growth of many microorganisms and have resistance to microbial attack. In general, molds, yeasts, and some aerobic bacteria are most suitable for tannin degradation, but tannin degradation by some anaerobic bacteria has also been reported.

Tannins are also known as anti-nutrients that hinder absorption of nutrients and minerals, but many beneficial effects on health have also been reported. Representatively, it has excellent antioxidant activity, suppresses inflammation, improves blood vessel, heart and cancer diseases, inhibits the growth of fungi, yeast, bacteria and viruses, and shows improvement effects on urinary tract infections, skin diseases, diabetes, etc. . Therefore, adequate intake of tannins can be beneficial to human health.

1. Republic of Korea Patent Publication No. 10-2019-0005157 (2019.01.15. Publication)

An object of the present invention is to provide a Lactobacillus plantarum D2-3 strain deposited under accession number KCTC 15134BP.

Another object of the present invention is tannin hydrolysis comprising the step of obtaining a tannin hydrolyzate by treating a Lactobacillus plantarum D2-3 strain extract or a culture thereof deposited with accession number KCTC 15134BP in a tannin compound. is to provide a way

Another object of the present invention is to provide a composition for inhibiting filament formation of SOD1 (superoxide dismutase 1) G93A mutant protein, containing tannin hydrolyzate obtained through the tannin hydrolysis method as an active ingredient.

Another object of the present invention is SOD1, comprising at least one tannin compound selected from the group consisting of epigallocatechin gallte (EGCG), gallic acid, tannic acid and ellagic acid as an active ingredient ( superoxide dismutase 1) To provide a composition for suppressing filament formation of G93A mutant protein.

Another object of the present invention is to add one or more tannin compounds selected from the group consisting of epigallocatechin gallte (EGCG), gallic acid, tannic acid and ellagic acid to Lactobacillus plantarum ( Lactobacillus plantarum ). ) to provide a composition for inhibiting SOD1 (superoxide dismutase 1) G93A mutant protein filament formation, comprising a culture solution co-cultured with the addition of the strain as an active ingredient.

In order to achieve the above object, the present invention provides a Lactobacillus plantarum D2-3 strain deposited with accession number KCTC 15134BP.

In addition, the present invention is a tannin hydrolysis method comprising the step of obtaining a tannin hydrolyzate by treating a Lactobacillus plantarum D2-3 strain extract or a culture thereof deposited with accession number KCTC 15134BP in a tannin compound. provides

In addition, the present invention provides a composition for inhibiting filament formation of SOD1 (superoxide dismutase 1) G93A mutant protein, comprising tannin hydrolyzate obtained through the tannin hydrolysis method as an active ingredient.

In addition, the present invention is a superoxide dismutase (SOD1) containing at least one tannin compound selected from the group consisting of epigallocatechin gallte (EGCG), gallic acid, tannic acid and ellagic acid as an active ingredient. 1) Provided is a composition for inhibiting G93A mutant protein filament formation.

In addition, the present invention relates to one or more tannin compounds selected from the group consisting of epigallocatechin gallte (EGCG), gallic acid, tannic acid and ellagic acid, Lactobacillus plantarum strain It provides a composition for inhibiting SOD1 (superoxide dismutase 1) G93A mutant protein filament formation, containing the co-cultured culture medium as an active ingredient.

According to the present invention, by confirming that the tannin hydrolyzate obtained by treating the novel Lactobacillus plantarum D2-3 strain with a tannin compound effectively inhibits SOD1 (superoxide dismutase 1) G93A mutant protein filament formation , SOD1 G93A mutant protein can be usefully utilized as a composition for inhibiting filament formation.

1 is a 16S rRNA sequencing result of Lactobacillus plantarum D2-3 strain.
Figure 2 is a result of comparing and analyzing tannaise expression levels of nine Lactobacillus plantarum strains, including the Lactobacillus plantarum D2-3 strain.
Figure 3 is a result of analyzing the PCR product of the tannaise enzyme of the Lactobacillus plantarum D2-3 strain by electrophoresis.
FIG. 4 shows the result of cloning the Lactobacillus plantarum D2-3 tannaise gene into the pET15b vector by electrophoresis.
5 is a result of analyzing the nucleotide sequence of the tannaise gene cloning of the Lactobacillus plantarum D2-3 strain.
6 is a result of mass expression of tannase enzyme of Lactobacillus plantarum D2-3 strain and analysis by SDS-PAGE (sodium dodecyl-sulfate polyacrylamide gel electrophoresis).
7 is a result of analyzing the purified tannaise enzyme by SDS-PAGE (sodium dodecyl-sulfate polyacrylamide gel electrophoresis).
8 is a result of analyzing purified tannaise enzyme by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).
9 is a result of analyzing the tannic acid degradation ability of the tannase enzyme of the purified Lactobacillus plantarum D2-3 strain through a clear zone.
10 is a result of HPLC analysis of EGCG decomposition products by the tannaise enzyme of the purified Lactobacillus plantarum D2-3 strain.
11 is a result of analyzing the filament formation inhibitory activity of SOD1 (superoxide dismutase 1) wild-type protein by a tannin-based natural product by Thioflavin T assay (ThT).
12 is a result of analyzing the filament formation inhibitory activity of the SOD1 G93A mutant protein by a tannin-based natural product by Thioflavin T assay.
13 is a result of analyzing the filament formation inhibitory activity of SOD1 protein by a tannin-based natural product with a negative-staining electron microscope (electron microscopy; EM).
14 is a result of analyzing the filament formation inhibitory activity of the SOD1 G93A mutant protein by tannin-based natural products in SK-N-SH neurons.
15 is a result of analyzing the filament formation inhibitory activity of the SOD1 G93A mutant protein by the culture product of Lactobacillus strains and tannin-based natural products by Thioflavin T assay.

Hereinafter, the present invention will be described in more detail.

The present invention provides a Lactobacillus plantarum D2-3 strain deposited under accession number KCTC 15134BP.

The strain may exhibit tannase activity.

In addition, the present invention is a tannin hydrolysis method comprising the step of obtaining a tannin hydrolyzate by treating a Lactobacillus plantarum D2-3 strain extract or a culture thereof deposited with accession number KCTC 15134BP in a tannin compound. provides

The tannin compound may be at least one selected from the group consisting of epigallocatechin gallte (EGCG), gallic acid, tannic acid, and ellagic acid, but is not limited thereto.

In addition, the present invention provides a composition for inhibiting filament formation of SOD1 (superoxide dismutase 1) G93A mutant protein, comprising tannin hydrolyzate obtained through the tannin hydrolysis method as an active ingredient.

The tannin hydrolyzate may be one or more selected from the group consisting of epigallocatechin gallte (EGCG), gallic acid, tannic acid, and ellagic acid, but is not limited thereto.

In addition, the present invention is a superoxide dismutase (SOD1) containing at least one tannin compound selected from the group consisting of epigallocatechin gallte (EGCG), gallic acid, tannic acid and ellagic acid as an active ingredient. 1) Provided is a composition for inhibiting G93A mutant protein filament formation.

In addition, the present invention relates to one or more tannin compounds selected from the group consisting of epigallocatechin gallte (EGCG), gallic acid, tannic acid and ellagic acid, Lactobacillus plantarum strain It provides a composition for inhibiting SOD1 (superoxide dismutase 1) G93A mutant protein filament formation, containing the co-cultured culture medium as an active ingredient.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

[Example 1] Strain Isolation and Identification

Isolated culture of Lactobacillus plantarum strains was performed using LBS (Lactobacillus Selection) selective medium from samples obtained by securing traditional fermented foods. Through PCR and sequencing of the 16S rRNA of the strain isolated through the selective medium, as shown in Figure 1, the isolated strain is Lactobacillus plantarum ( Lactobacillus plantarum D2-3; hereinafter referred to as D2-3) strain It was confirmed that The 16S rRNA sequence of the D2-3 strain is shown in Table 1 below.

16S rRNA of Lactobacillus plantarum D2-3 strain CAAATCTGGTCACCTTATGCGGCTGGTTCCTAAAAGGTTACCCCACCGACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGCTTTAAGAGATTAGCTTaCTCTCGCGAGTTCGCAAcTCGTTGTaCCAT CCATtGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGtTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAATCTCTTAGATTTGCATAGTA TGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCATTCATCGTTTACGGTATGGACTACCAGGGTATCTAATCCTGTTTGCTACCCATACTTTCGAGCCTCAGCGTCA GTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCTTTCACATCAGACTTAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTT AGCCGTGGCTTTCTGGTTAAATACCGTCAATACCTGAACAGTTACTCTCAGATATGTTCTTCTTTAACAACAGAGTTTTACGAGCCGAAACCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGATTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCTACGTATCATTGCCATGGTGAG CCGTTACCCCACCATCTAGCTAATACGCCGCGGGACCATCCAAAAGTGATAGCCGAAGCCATCTTTCAAGCTCGGACCATGCGGTCCAAGTTGTTATGCGGTATTAGCATCTGTTTCCAGGTGTTATCCCCCGCTTCTGGGCAGGTTTCCCACGTGTTACTCACCAGTTCGCCACTCACTCAAATGTAAATCATGATGCAAGCACCAATCAATACCAGAGTTCGTTCGACTTGCCATGTAA TTAGGG

[ Example 2] D2-3 strain tannaise Gene expression measurement

In order to confirm the expression level of the tannaise enzyme of the isolated D2-3 strain, 8 strains of Lactobacillus plantarum derived from 8 different traditional fermented foods and the tannaise of the standard strain distributed from ATCC (American Type Culture Collection) Enzyme expression levels were compared and analyzed. The tannaise enzyme expression level was compared with the relative mRNA expression level for each strain using qRT-PCR, which can be quantified in real time. The primer set used for qRT-PCR was prepared by referring to a previous study (Natalia et al., (2014) Applied and Environmental Microbiology, 80(10), 2991-2997). Primer sequences of the prepared primer set are shown in Table 2 below. As a result, as shown in FIG. 2 , it was confirmed that the tannaise expression level of the D2-3 strain was the highest among a total of 9 strains including the D2-3 strain.

Target F/R Sequence (5’-3’) ldh F AAC CGC GAC AAT GTT TTG ATT R TTG TGA ACG GCA GTT TCA GTG T tannase F TGC GCT ACC GTG GGA TAT TC R AAT CCA GGA AAA TAA ATC GCC TAA

[Example 3] Tannaise gene amplification of strain D2-3

The tannaise enzyme nucleotide sequence of strain D2-3 was obtained through the NCBI GenBank database (http://www.ncbi.nlm.nih.gov/genbank/). Based on the obtained nucleotide sequence, a primer set for tannaise gene amplification was prepared by attaching specific restriction enzymes (XhoI and BamHI) to both ends of the gene. The tannaise enzyme nucleotide sequence of the D2-3 strain is shown in Table 3 below. Thereafter, as shown in FIG. 3, PCR was used to obtain tannaise enzyme amplification products of the D2-3 strain and confirmed by electrophoresis. PCR conditions are shown in Table 4 below.

Target F/R Sequence (5’-3’) tannase F AAA CTC GAG ATG AGT AAC CGA TTG ATT TTT GAT GCT GAC TGG CT R AAA GGA TCC TCA TTG GCA CAA GCC ATC AAT CCA GGA AAA TAA AT

Step Temperature(℃) Time Cycle Initial denaturation 95 3min One Denaturation 98 20sec 30 Annealing 55 15sec 30 Extension 72 3sec 30 final extension 72 90sec One

[Example 4] Cloning of tannaise gene into pET15b vector

The tannaise gene amplified by PCR was double digested with two restriction enzymes (XhoI and BamHI) to obtain insert DNA. After cloning the obtained insert DNA into the XhoI and BamHI parts in the pET15b vector using the Quick Ligation Kit (BioLabs, USA), transformation was performed in E. coli [ E. coli BL21(DE3)], and ampicillin ), the plasmid was isolated and purified by selecting colonies on a solid medium containing. In order to confirm the plasmid, after double digestion with XhoI and BamHI, as a result of confirming by electrophoresis, as shown in FIG. 4, it was confirmed that the transformation was successful.

[Example 5] Confirmation of cloned tannaise gene sequence

As shown in FIG. 5, the transformants confirmed by electrophoresis after cloning was completed were confirmed by using the universal primer of the pET15b vector to confirm the sequence of the final cloning product. The sequence of the universal primer is shown in Table 5 below.

F/R Sequence (5’-3’) Forward pBRrevBam GGTGATGTCGGCGATATAGG Reverse T7 terminator GCTAGTTATTGCTCAGCGG

[Example 6] Mass expression of tannaise

For mass expression of the cloned tannaise gene, E. coli [ E. coli BL21(DE3)] into which the tannaise gene of the D2-3 strain was introduced was inoculated into 5ml LB (containing 50 μg/ml ampicillin) medium and incubated at 37°C for 12 days. A seed culture was prepared by culturing for a period of time. Sub-culture was performed by inoculating 1% of the spawn in 100 ml of fresh LB medium (containing 50 μg/ml ampicillin). After incubation at 37℃ until the OD ₆₀₀ of the sub-culture is 0.6~0.8, IPTG was added to a final concentration of 1 mM, followed by mass expression at 30℃ for 24 hours. As shown in FIG. 6, it was confirmed through SDS-PAGE that the tannase enzyme was expressed in large quantities.

[Example 7] Isolation and purification of expressed tannase enzyme

Since the His-Tag is encoded in the pET15b vector used for the large-scale expression of the tanase enzyme, His-Tag can be used for purification when exogenous genes are expressed. Therefore, in order to obtain a large amount of tannase enzyme, first, the supernatant was removed by centrifuging the mass-expressed culture medium at 4° C. and 10,000 × g for 30 minutes. After removing the supernatant, the remaining bacterial pellet was resuspended in 5ml of lysis buffer [50mM monosodium phosphate (NaH ₂ PO ₄ ), 300mM sodium chloride (NaCl) and 10mM imidazole], and then at 200W. The strain was disrupted by sonication for a total of 10 minutes with a 10sec/10sec pulse. In order to obtain the supernatant of the disrupted strain, only the supernatant was separated by centrifugation for 30 minutes at 4 ° C and 10,000 × g conditions. In order to purify only the tannaise enzyme, a wash buffer and an elution buffer were prepared by changing the imidazole concentration to 50 mM and 500 mM, respectively, in the composition of the lysis buffer. After flowing lysis buffer corresponding to 5 times the capacity to the Ni-NTA column, the separated supernatant sample was slowly flowed. 10 times the column volume of wash buffer was slowly flowed to remove other proteins not bound to the column. Elution buffer 5 times the column capacity was slowly flowed to purify the His-Tag-attached tannase enzyme, and as shown in FIG. 7, the purified product was confirmed by SDS-PAGE. In order to confirm that the tannaise enzyme was purified without impurities, as a result of MALDI-TOF MS analysis, as shown in FIG. 8, it was confirmed that a single peak was detected, thereby confirming that the enzyme was well purified without impurities.

[Example 8] Analysis of tannic acid degradation ability of purified tannase enzyme

In order to confirm the tannic acid decomposition ability of the purified tannase enzyme, it was analyzed whether the tannic acid was hydrolyzed on a solid medium. A 2% tannic acid solution dissolved in purified water was evenly dispensed on the BHI solid medium containing 0.5% yeast extract, and then reacted for 20 minutes to make an opaque medium. As a result of confirming tannic acid decomposition through a clear zone by dotting the purified D2-3 tannaise enzyme thereon, it was confirmed that tannic acid decomposition activity was exhibited by the purified tannaise enzyme, as shown in FIG. 9 . In addition, as a result of HPLC analysis of EGCG (Epigallocatechin gallte) decomposition by purified tannaise enzyme, as shown in FIG. 10, it was confirmed that EGCG was decomposed into gallic acid.

[ Example 9] on tannins by superoxide dismutase One( SOD1 ) Analysis of inhibition of filament formation by wild-type protein

In order to confirm the filament formation inhibitory activity of superoxide dismutase 1 (hereinafter referred to as SOD1) wild-type protein and SOD1 G93A mutant protein of tannin-based natural products, Thioflavin T assay (ThT) and negative-staining electron microscope (EM) were confirmed. As a result, as shown in FIGS. 11, 12 and 13, it was confirmed that the tannin-based natural product showed the filament formation inhibitory activity of the SOD1 G93A mutant protein. In addition, as shown in FIG. 14, it was confirmed that the tannin-based natural product exhibited the filament formation inhibitory activity of the SOD1 G93A mutant protein in SK-N-SH nerve cells.

[ Example 10] Lactobacillus strains and Tannin system by culture products of natural products SOD1 G93A mutant protein filament formation inhibition assay

Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) and Lactobacillus plantarum ( Lactobacillus plantarum ) In order to confirm the filament formation inhibitory activity of the SOD1 G93A mutant protein by co-culture products of tannin-based natural products, Thioflavin T assay (ThT) was performed did As a result, as shown in FIG. 15, the culture solution obtained by adding and culturing the Lactobacillus plantarum strain to EGCG, tannic acid and gallic acid was superior to the tannin-based natural product not co-cultured with the strain. It was confirmed that the SOD1 G93A mutant protein showed excellent filament formation inhibitory activity.

Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (8)

Lactobacillus plantarum deposited under accession number KCTC 15134BP ( Lactobacillus plantarum ) D2-3 strain. The method according to claim 1, wherein the strain is characterized in that exhibiting the tannin acid degrading enzyme (tannase) activity. Tannin hydrolysis method comprising the step of obtaining a tannin hydrolyzate by treating a Lactobacillus plantarum D2-3 strain extract or a culture thereof deposited with accession number KCTC 15134BP in a tannin compound. The hydrolysis method according to claim 3, wherein the tannin compound is at least one selected from the group consisting of epigallocatechin gallte (EGCG), gallic acid, tannic acid, and ellagic acid. A composition for inhibiting filament formation of SOD1 (superoxide dismutase 1) G93A mutant protein, comprising a tannin hydrolyzate obtained through the tannin hydrolysis method according to claim 3 as an active ingredient. The composition according to claim 5, wherein the tannin hydrolyzate is at least one selected from the group consisting of epigallocatechin gallte (EGCG), gallic acid, tannic acid, and ellagic acid. Superoxide dismutase 1 (SOD1) G93A mutant protein containing at least one tannin compound selected from the group consisting of epigallocatechin gallte (EGCG), gallic acid, tannic acid, and ellagic acid as an active ingredient A composition for inhibiting filament formation. EGCG (epigallocatechin gallte), gallic acid (gallic acid), tannin acid (tannic acid) and ellagic acid (ellagic acid) was added to one or more tannin compounds selected from the group consisting of Lactobacillus plantarum ( Lactobacillus plantarum ) strain and co-cultivated A composition for inhibiting SOD1 (superoxide dismutase 1) G93A mutant protein filament formation, comprising a culture medium as an active ingredient.

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