KR20150057636A - Composition comprising cyclic dipeptides and DL-3-phenyllactic acid for antibacterial, antifungal and antiviral agents - Google Patents

Abstract

The present invention provides an antibacterial, antifungal and antiviral composition comprising cyclic dipeptides and DL-3-phenyllactic acid, which has a antibacterial, antifungal and antiviral activity. The composition is an active compound separated from culture supernatant liquid of lactobacillus plantarum LBP-K10, and exhibits higher antibacterial, antifungal and antiviral effects than a conventional composition using one type of cyclic dipeptide.

Description

Antibacterial, antifungal and antiviral compositions comprising cyclic dipeptides and DL-3-phenyllactic acid (Composition containing cyclic dipeptides and DL-3-phenyllactic acid for antibacterial, antifungal and antiviral agents)

The present invention relates to antibacterial, antifungal and antiviral compositions comprising a cyclic dipeptide and DL-3-phenyllactic acid.

Lactic acid bacteria are Gram-positive bacteria of any anaerobic and non-spore formation and are resistant to gram-positive and gram-negative bacteria and fungi caused by many types of fermented food originating from raw materials including animals and plants. Suitable as probiotics and active substances in It was proposed to be generally used as a vivo antagonist [1]. The importance of antimicrobial properties of secondary metabolites secreted from lactic acid bacteria can be attributed to the presence of several types of bacteriocins and bacteriocin-like peptide molecules and small molecules including lactic acid, volatile acids, hydrogen peroxide, carbon dioxide, diacetyl, (2). Among the various kinds of lactic acid bacteria, the functional roles of antibiotics and metabolites of Lactobacillus strains have been reported [3].

The present inventors have previously isolated cis -cyclo (L-leucine-L-proline) and cis -cyclo (L-phenylalanine-L-proline) against influenza A virus and found that the plant pathogen Ganoderma boninense ) And fungi Candida albicans ( Candida cyclo (L- -L-proline-valine) and the cis - - albicans) system to inhibit [21] a cycloalkyl (L- phenylalanine -L- proline) was isolated. In previous reports, cis -cyclo (L-leucine-L-proline) has been associated with multiple drug-resistant Staphylococcus aureus 11471, Streptococcus pneumoniae 14596, and Salmonella typhimurium 12219 [22]. In addition, the antimicrobial activity of typhimurium 12219 has been reported.

In this study, inventors have found a mixture consisting of 7 cyclic dipeptide as a kind of small material from the Lactobacillus plan culture supernatant of tareom (Lb. Plantarum) LBP-K10 derived from a traditionally fermented kimchi in Korea , Antimicrobial, antifungal and antiviral activity, and completed the present invention.

In Korean Patent Application No. 10-2011-0064091 (filed on June 29, 2011), the present inventors' prior patents have disclosed that a cis-cyclo (L-phenylalanine-L-proline) And has an antifungal activity specific to fungi.

Nishanth Kumar S et al. Have reported that cyclo- (. Pro-Leu), cyclo- (.-Pro-.- Met) and cyclo- (Pharm Biol. 2013 Sep. 19). ≪ RTI ID = 0.0 >

It is an object of the present invention to provide antibacterial, antifungal and antiviral compositions having antimicrobial, antifungal and antiviral activities including cyclic peptides and DL-3-phenyllactic acid.

In order to achieve the above object, a first aspect of the present invention provides an antimicrobial composition having an antimicrobial activity comprising a cyclic dipeptide and DL-3-phenyllactic acid.

More specifically, the cyclic dipeptide are cis-bicyclo (L- tyrosine -L- proline), cis-bicyclo (L- serine -L- proline), cis-bicyclo (L- leucine -L- proline), cis-bicyclo (L- valine -L- proline), cis-bicyclo (L- leucine -L- hydroxyproline) cis-bicyclo (L- phenylalanine -L- alanine), cis-bicyclo (L- methionine-proline ) And cis -cyclo (L-phenylalanine-L-proline).

More specifically, the bacteria are gram-positive or gram-negative bacteria and, more specifically, the gram-than-positive or gram-negative bacteria is Bacillus subtilis , Staphylococcus aureus , Streptococcus pneumoniae , Eschericia coli , Salmonella typhimurium or Shigella dysenterii .

A second aspect of the present invention provides a composition for an antifungal agent having an antifungal activity comprising a cyclic dipeptide and DL-3-phenyllactic acid.

More specifically, the cyclic dipeptide are cis-bicyclo (L- tyrosine -L- proline), cis-bicyclo (L- serine -L- proline), cis-bicyclo (L- leucine -L- proline), cis-bicyclo (L- valine -L- proline), cis-bicyclo (L- leucine -L- hydroxyproline) cis-bicyclo (L- phenylalanine -L- alanine), cis-bicyclo (L- methionine-proline ) And cis -cyclo (L-phenylalanine-L-proline).

More specifically, the fungus is Ganoderma boninens, a pathogenic strain of plants, or Candida albicans, a human pathogenic strain.

A third aspect of the present invention provides an antiviral composition having an antiviral activity comprising a cyclic dipeptide and DL-3-phenyllactic acid.

More specifically, the cyclic dipeptide are cis-bicyclo (L- tyrosine -L- proline), cis-bicyclo (L- serine -L- proline), cis-bicyclo (L- leucine -L- proline), cis-bicyclo (L- valine -L- proline), cis-bicyclo (L- leucine -L- hydroxyproline) cis-bicyclo (L- phenylalanine -L- alanine), cis-bicyclo (L- methionine-proline ) And cis -cyclo (L-phenylalanine-L-proline).

More specifically, the virus is influenza A (H3N2) virus.

The antimicrobial, antifungal and antiviral compositions having antimicrobial, antifungal and antiviral activity according to the present invention are substances isolated from the culture supernatant of Lactobacillus plantarum LBP-K10, which is remarkably effective compared to conventional antibiotics Respectively.

The complex active compounds isolated from the culture supernatant of Lactobacillus plantarum LBP-K10 according to the present invention showed higher antibacterial, antifungal and antiviral activity than those using the conventional cyclic peptides.

Figure 1 shows a structural analysis of fractions from Lactobacillus plantarum LBP-K10. (a) - (h) correspond to F1-F17 by electrical ionization and chemical ionization using gas chromatography-mass spectrometry. The molecular formula of the active compounds are: cis F1, F6 and F11, respectively -cycloalkyl (L- tyrosine -L- _{proline), C 14 H 16 N 2} O 3 [21]; Of F2 and F9 cis-bicyclo (L- serine -L- _{proline), C 8 H 12 N 2} O 3 [21]; Cis -cyclo (L-leucine-L-proline), C ₁₁ H ₁₈ N ₂ O ₂ [20-22] of F3, F4, F12 and F13; F5 and F7 cis-bicyclo (L- valine -L- _{proline), C 10 H 16 N 2} O 2 as [21]; F8 of cis-bicyclo (L- leucine -L- _{hydroxyproline), C 11 H 18 N 2} O 3; F10 of the cis-bicyclo (L- phenylalanine -L- _{alanine), C 12 H 14 N 2} O 2; F14 of the cis-bicyclo (L- _{methionine-proline), C 10 H 16 N 2} O 2 S 1; F15 DL-3- phenyl lactic acid of, C ₉ H ₁₀ O ₃ ; System of F16 and F17 - cycloalkyl (L- phenylalanine -L- _{proline), C 14 H 16 N 2} O 2 [20-22].
Figure 2 summarizes the indicated active compounds in F1 to F17. Structural units separated by chemical bonds were separated by dashes.
Figure 3 shows the structural analysis of active compounds against multiple drug-resistant bacteria, pathogens and influenza A virus using X-ray crystallography. (a) a crystal of F7 corresponding to an antifungal compound, (c) a crystal of F13 corresponding to an antibacterial and antiviral compound, (e) a crystal of F17 corresponding to an antibacterial, antifungal and antiviral compound, Is inactive F8, and (i) is inactive F14. The X-ray crystal structure of these fractions was specified in appropriate proportions of methanol and methylene chloride. (b), (d), (f), (h) and (j) show the inferred structures of F7, F8, F13, F14 and F17 by X-ray crystallography. Carbon (grayish white), hydrogen (white), oxygen (gray) and nitrogen (dark gray).
Figure 4 shows the overall profile of the purified material from Lactobacillus plantarum LBP-K10. All fractions were low-molecular substances including cyclic dipeptide and DL-3-phenyllactic acid.
Figure 5 shows the HPLC profile from Lactobacillus plantarum LBP-K10 and other isolated lactic acid bacteria. Lactic acid bacteria is Lactobacillus sakei LBP-S01, Lactobacillus plantarum / pentos LBP-S02, Lactococcus lactis LBP-S03, Lactococcus lactis LBP-S06, Staphylococcus sciuri LBP-S07, Leuconostoc mesenteroides LBP-K06, Lactobacillus plantarum LBP-K10, Weissella cibaria LBP-K15 and Weissella confusa LBP-K16. Chromatographic analysis of Lactobacillus plantarum LBP-K10 was performed at 210 nm (b) and 260 nm (c) every 8 hours.
Figure 6 compares the culture supernatant using Amberlite IRA-67 and the eluate of the culture supernatant of Lactobacillus plantarum LBP-K10.
Figure 7 shows a similar profile of the cyclic dipeptide in the eluate of the complete culture supernatant of the LBP-K10 and the culture supernatant from which the organic acid or the sugar was removed after using Amberlite IRA-67 or Purolite A420S.
Figure 8 shows the anti-cancer activity for the powder from the organic acid or sugar free culture supernatant. (a) and (b) illustrate the activity against Ganoderma boninense using various concentrations of the organic acid-removed culture supernatant obtained from Amberlite IRA-67 chromatography for 3.5 or 7 days. (c) is the transfer of the myoides from the existing a and b to the new PDA version. This was done for 14 days. in In the in vitro assay, the range of 3.45-6.9% of the powder from the organic supernatant without organic acid was found to have the killing effect of Ganoderma boninens. (d) shows the effect of curing and growth inhibition on the powder from the culture supernatant without organic acid. On the fourth day, 5.0% of the powder from the organic supernatant without the organic acid was added to Ganoderma boninenes. This experiment was observed for 20 days. After 4 days of growth, the 5.0% of the powder from the culture supernatant without organic acid was treated to observe the curing and growth inhibitory effect of Ganoderma boninens.
Figure 9 shows the antiviral activity of the culture supernatant without organic acid. (a), (b), and (c) - (f) show the concentration of the culture supernatant without organic acid at 0.5% (7.176 mg / ml) and 1.0% / ml), 1.5% (21.528 mg / ml) and 2.0% (28.704 mg / ml), and incubated with 0.7% DMEM agarose inoculated with virus infection medium and influenza A virus (2.8 x 10 ⁷ PFU / ml) . MDCK (Madin-Darby canine kidney) epithelial cells were infected with influenza A virus (H3N @) and cultured for 60 hours. All experiments were performed three times independently.

Figure 10 shows the structural analysis of 17 fractions from Lactobacillus plantarum LBP-K10. (a) - (q) correspond to F1-F17 by electrical ionization and chemical ionization using gas chromatography-mass spectrometry. The EI and CI values of each fraction are shown. Among these fractions, the ESI-CID mass spectra of the antibacterial, antifungal and antiviral substances are as follows. (a) F1, cis -cyclo (L-Tyr-L-Pro) Cis-cyclo (L-Ser-L-Pro ), (c) F3, cis-cyclo (L-Leu-L-Pro ), (d) F4, cis-cyclo (L-Leu-L-Pro ), ( e) F5, cis-cyclo (L-Val-L-Pro ), (f) F6, cis-cyclo (L-Tyr-L-Pro ), (g) F7, cis-cyclo (L-Val-L- Pro), (h) F8, cis-cyclo (L-Leu-L-Hyp ), (i) F9, cis-cyclo (L-Ser-L-Pro ), (j) F10, cis-bicyclo (L- Phe-L-Ala), ( k) F11, cis-cyclo (L-Tyr-L-Pro ), (l) F12, cis-cyclo (L-Leu-L-Pro ), (m) F13, cis- cyclo (L-Leu-L-Pro ), (n) F14, cis-cyclo (L-Met-L-Pro ), (o) F15, DL-3-phenyllactic acid, (p) F16, cis-bicyclo ( L-Phe-L-Pro) and (q) F17, cis- cycloo (L-Phe-L-Pro).

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention. It will be obvious.

Experimental Method

1. Strain

Lactobacillus plantarum LBP-K10, an antibacterial and antiviral strain, was isolated from fermented Chinese cabbage, mustard leaves and stalks, and stonecrop, grown on MRS (de Man, Rogosa and Sharpes) agar plates, 16S rRNA sequencing using a primer of genuine bacteria for polymerase chain reaction (PCR) was performed as proposed in [20].

For antifungal assays, the Ganoderma boninens isolate (GMR3) and Candida albicans were raised and maintained as previously described [21].

Drug multi-resistant Gram-positive and Gram-negative bacterial strains were prepared to observe antimicrobial activity as previously proposed [22].

Plaque assays for antiviral activity were carried out using Madin Darby canine kidney (MDCK) and influenza A (H3N2) virus as host cells [20].

2. Fractions of antimicrobial and antiviral compounds

The three-day culture supernatant of Lactobacillus plantarum LBP K-10 was concentrated by lyophilization as previously described, extracted with methylene chloride, and isolated using a high performance liquid chromatography system [20].

3. Mass analysis

To investigate the electronic ionization and chemical ionization of the fractions, a gas chromatography-mass spectrometer was used as described previously [20].

4. X- Lay Crystallography

Crystals of the fractions were measured at 95 K with a synchrotron radiation (λ = 0.66999 Å) at the ADSC Quantum-210 detector in a silicon (111) double crystal monochromator (DCM) at the Pohang Accelerator Laboratory in Korea And the diffraction data were determined [20]. F-7, cis -cyclo (L-valine-L-proline) was identified as CCDC 937497 [21], F8, cis -cyclo -L- hydroxyproline) is CCDC 937498, F13, cis-bicyclo (L- leucine -L- proline) is CCDC 937533 [20], F14, DL-3- phenyl lactic acid CCDC 951328, and F17, cis-bicyclo (L-phenylalanine-L-proline) is CCDC 937534 [20].

5. Cultures without organic acids and sugars Supernatant  Ready

Amberlite IRA-67 [23, 24] and Purolite A420S [25-27] were used as a weak anion exchanger by the recovery of organic acids to remove the interference of organic acids in the culture supernatant from Lactobacillus plantarum LBP-K10 [ 23, 24]. Before performing the weak anion exchange chromatography, Amberlite IRA-67 resin (resin) has been washed according to the purpose of the experiment OH ^- to be converted to form ^[23,24] or Cl. OH ^- , the free base form of Amberlite IRA-67 resin recovered a large amount of lactic acid by washing the resin with some strain. 3.0 kg of Amberlite IRA-67 packed column (10 cm x 100 cm) was washed sequentially with 2.0 N sodium hydroxide, distilled water, 1.5 N hydrochloric acid, distilled water to pH 7.0, 1.5 N sodium hydroxide and finally distilled water Or less). After Amberlite IRA-67 was prepared as described above, the culture supernatant was poured into Amberlite IRA-67 at a flow rate of 15 ml / min, resulting in unattached fractions of the culture supernatant.

In order to remove the sugar of the first eluents from the culture supernatant after performing Amberlite IRA-67 chromatography, Purolite A420S resin was used as described above. After performing a weak anion exchange chromatography, the Purolite A420S resin should be prepared as follows before use. Purolite A420S should be washed, regenerated and converted to Cl ^- form. A 3.0 kg Purolite A420S packed column (10 cm x 100 cm) was in turn regenerated with 15% sodium chloride containing 0.5% sodium hydroxide and distilled water. The first eluent from Amberlite IRA-67 was applied to Purolite A420S at a flow rate of 15 ml / min. After performing Amberlite IRA-67, the unattached fractions were freeze-dried in a freeze dryer. The resulting powder was extracted with methylene chloride. The methylene chloride extract was freeze-dried and dissolved in an appropriate amount of sterilized water for future antimicrobial or antiviral assays. All samples were stored at 4 ° C.

6. Antibacterial, antifungal and antiviral Assay

All experiments were performed with culture supernatants without fractions or organic acids.

The antimicrobial activity was observed every 24 hours after seed inoculation using a minimum inhibitory concentration (MIC), and antifungal assays against Ganoderma boninenes and Candida albicans were performed as previously described Was performed [21,22].

MDCK cells for plaque assay are then reared in complete medium in DMEM (Dulbecco's Modified Eagle Medium), as set out in conventional cell culture plate (90 x 15 mm) and a half of ⁵ x 10 5 cells from the surface even in the filled single layer 90 of -95% and transferred to a 6-well culture dish [20].

Example  1. Identification of separation substances from kimchi

Based on the inventor's discovery that the culture supernatant of the isolated Lactobacillus plantarum LBP-K10 exhibits strong antibacterial, antifungal and antiviral activity from the antagonistic test results [20-22], in the present invention, .

Example  2. Culture Supernatant  Fraction and identification

Fifteen different fractions designated as F1-F17, isolated from the cultured supernatant of Lactobacillus plantarum LBP-K10 cultured for three days, designated as F1-F17, containing multiple drug-resistant bacteria, pathogens and active compounds against influenza A virus, As a freeze-dried powder, according to the chromatographic separation pattern as given in [20]. The predicted segmentation pattern of electrical ionization corresponding to the m / z of the [M + 1] ⁺ value and the molecular ion mass of all fractions of F1-F17 is 261, 185, 211, 211, 195, 261, 197, 227 , 185, 219, 261, 211, 211, 228, 169, 245, and 245 (Table 1).

From the results of the mass resolution through high resolution by electron impact, we find that all fractions are diketopiperazines with seven proline-containing groups common to 154 as m / z of electronic ionization-mass spectrometry (EI-MS) . In addition, one kind of non-proline-containing cyclic dipeptide and non-cyclic dipeptidyl materials have been identified (Figures 1 and 2).

Example  3. Structural determination of antimicrobial, antifungal and antiviral substances

To determine the three-dimensional structure of the active substance from the culture supernatant against bacteria, fungi and viruses, the crystal data, data collection, refinement and statistics of these active fractions statistics were confirmed in the studies of the present inventors (FIG. 3 and Tables 2 to 6) [20-22]. In addition, all fractions and active compounds from Lactobacillus plantarum LBP-K10 were summarized (Figure 4 and Table 7).

Furthermore, in order to compare antimicrobial and antiviral compounds, we have identified all the cyclic dipeptides and small substances isolated from lactic acid bacteria by fractionated HPLC chromatogram (FIG. 5). The total compounds from nine different lactic acid bacteria showed equally divided patterns (Figure 5). This result suggests that lactic acid bacteria produce and secrete similar metabolites that function as antimicrobial, antifungal, and antiviral agents (FIG. 5).

Example  4. Culture In the supernatant  Removal of organic acids and sugars

The present inventors confirmed that an organic acid including lactic acid, butyric acid, acetic acid, formic acid and propionic acid has a recovery rate of 96.9% through Amberlite IRA-67 anion exchanger (Table 8). Antifungal, antifungal and antiviral substances were investigated in the culture supernatant containing no organic acid. Lactic acid and acetic acid were removed to 96.88% and 99.6%, respectively. In addition, other removed organic acids and DL-3-phenyllactic acid are shown in Table 8. From the culture supernatant of Lactobacillus plantarum LBP-L10, the total amount of the cyclic dipeptide complex in powder state (Table 9) and the amount of cyclic dipeptide (Table 10) were shown using the methylene chloride extraction method. After performing Amberlite IRA-67 and Purolite A420S resin by HPLC analysis, the present inventors confirmed the cyclic dipeptide and DL-3-phenyllactic acid (Fig. S1). All fractions after elution with Amberlite IRA-67 and Purolite A420S resin were obtained (Fig. S2). From these results, it was assumed that the organic acid was effectively removed by Amberlite IRA-67 and Purolite A420S resin, and the fraction not adhered to the resin contained antibacterial, antifungal and antiviral substances containing other impurities.

Example  5. Culture without organic acids or sugars Supernatant  Antibacterial, antifungal and antiviral activity

We investigated the antimicrobial activity of Gram-positive and Gram-negative bacteria (Table 11) and multi-drug-resistant strains (Table 12) using culture supernatants without organic acids. The proliferation of Ganoderma boninens and Candida albicans was significantly inhibited by treating the culture supernatant without organic acids or sugars at the concentrations shown in Table 13 and S3. In addition, the antiviral effect of this complex mixture was seen in influenza A virus (Tables 14-14 and S4).

Depending on the significant antibacterial, antifungal and antiviral effects of the organic acid or sugar-free culture supernatant, the culture supernatant was assumed to be a complex of small substances containing cyclic dipeptides.

Finally, the present inventors have found that complexes of other concentrations, so-called organic acid and sugar-removed mixtures, contain seven proline-containing cyclic dipeptides, one non-proline-containing cyclic dipeptide and DL- Phenyllactic acid, which would be a notable antibiotic compared to conventional antibiotics and vaccines.

Table 1 shows the mass spectrometry using EI and CI by GC-MS of the fractions.

Fractions m / z of [M + 1] ⁺ m / z (%) of EI-MS Predicted molecules F1
(6.5-7.0) ^a 261.0 (28.9), 61.2 (12.2), 70.2 (80.1), 76.2 (16.2), 85.2 (100.0), 86.2 (31.9), 91.2 (84.2), 98.2 (16.1), 99.2 (7.8), 111.3 128.3 (45.5), 141.3 (8.8), 148.3 (21.3), 154.3 (45.9), 155.3 (10.5), 170.4 (35.3), 184.4 (11.3), 196.4 (12.5), 244.5 Cis-bicyclo (L- tyrosine -L- _{proline), C 14 H 16 N 2} O 3 F2
(7.0-7.5) ^a 185.0 55.1 (18.7), 69.2 (30.2), 70.2 (44.4), 74.2 (2.6), 83.2 (52.7), 98.2 (20.4), 111.2 (75.9), 126.3 (8.7), 154.3 (100.0), 155.3 184.4 (1.3) Cis-bicyclo (L- serine -L- _{proline), C 8 H 12 N 2} O 3 F3
(7.5-7.8) ^a 211.0 (17.6), 69.3 (68.6), 83.3 (41.7), 86.3 (10.6), 98.3 (17.0), 111.3 (57.3), 114.3 (3.7), 126.4 (9.1), 140.4 154.4 (100), 155.4 (10.9), 168.5 (9.8), 210.6 (17.6) Cis -cyclo (L-leucine-L-proline), C ₁₁ H ₁₈ N ₂ O ₂ F4
(7.8-8.1) ^a 211.0 (14.2), 58.2 (4.6), 68.2 (14.8), 69.2 (37.6), 70.3 (100), 71.3 (20.6), 83.3 (7.7), 86.3 (10.1), 97.3 (58.6), 98.3 111.3 (9.4), 112.3 (8.4), 114.3 (21.3), 125.4 (34.1), 126.3 (33.4), 140.4 (10.2), 154.4 (31.0), 168.5 (63.9), 194.5 Cis -cyclo (L-leucine-L-proline), C ₁₁ H ₁₈ N ₂ O ₂ F5
(8.1-9.0) ^a 195.0 54.9 (14.2), 56.0 (13.5), 68.0 (14.1), 69.0 (25.2), 70.0 (100.0), 71.0 (10.3), 83.9 (17.6), 84.9 (13.6), 85.9 (10.0), 95.9 96.9 (21.7), 97.9 (8.2), 106.9 (28.7), 111.9 (23.9), 113.9 (39.3), 124.9 (21.3), 125.9 (9.9), 139.9 (7.6), 140.9 (4.2), 153.9 154.9 (2.0), 155.9 (4.6), 167.9 (45.3), 168.9 (8.5), 169.9 (10.2), 193.9 (41.0), 194.9 (5.9) Cis-bicyclo (L- valine -L- _{proline), C 10 H 16 N 2} O 2 F6
(9.5-10.5) ^a 261.0 144.1 (1.2), 125.1 (4.1), 114.1 (0.2), 152.1 (2.3), 260.2 (12.4), 244.2 (1.2), 224.2 108.1 (4.6), 107.1 (38.7), 91.1 (2.1), 77.1 (3.3), 71.1 (1.4), 70.1 Cis-bicyclo (L- tyrosine -L- _{proline), C 14 H 16 N 2} O 3 F7
(12.2-13.2) ^a 197.0 156.1 (1.4), 155.1 (16.3), 154.0 (100.0), 153.1 (14.3), 152.1 (2.1), 16.1 (1.3), 139.1 (1.1), 138.1 (6.2), 137.1 (2.1), 125.1 (44.8), 114.1 (3.2), 113.1 (1.1), 112.1 (2.0), 100.1 (0.3), 99.1 (3.2), 98.1 (8.0), 97.1 (3.3), 96.1 (2.4), 84.1 (2.8), 83.1 (2.3), 82.1 (1.8), 76.1 73.1 (5.2), 72.1 (48.0), 71.1 (7.1), 70.1 (86.7), 69.1 (15.2), 68.1 (8.4) Cis-bicyclo (L- valine -L- _{proline), C 10 H 16 N 2} O 2 F8
(14.0-15.0) ^a 227.0 22.0 (5.1), 213.0 (1.26), 181.0 (7.60), 166.9 (3.2), 154.0 (11.8), 91.0 (8.1), 77.0 (62.0), 61.0 Cis -cyclo (L-leucine-L-hydroxyproline), C ₁₁ H ₁₈ N ₂ O ₃ F9
(15.0-16.5) ^a 185.0 (5.0), 99.0 (8.0), 98.0 (4.7), 128.0 (100), 127.0 (3.3), 113.0 (3.7), 91.0 (6.2), 86.0 (15.0), 85.0 (6.4), 84.0 (5.7), 83.0 (3.4), 71.1 (7.0), 70.1 (10.4), 69.0 (7.8), 60.0 57.0 (13.2), 56.0 (6.0), 55.0 (7.4) Cis-bicyclo (L- serine -L- _{proline), C 8 H 12 N 2} O 3 F10
(17.0-18.5) ^a 219.0 128.9 (12.6), 182.0 (3.0), 168.9 (5.4), 154.0 (77.1), 141.0 (8.1), 128.0 (41.2), 125.9 (24.5), 125.0 (13.9), 122.0 (17.2), 76.0 (100.0), 71.0 (11.2), 70.0 (47.3), 100.0 (17.2), 99.0 (13.5), 98.0 (12.0), 97.0 69.0 (25.3), 58.0 (11.3), 57.0 (42.6) Cis -cyclo (L-phenylalanine-L-alanine), C ₁₂ H ₁₄ N ₂ O ₂ F11
(19.0-20.0) ^a 261.0 260.1 (12.9), 244.1 (21.6), 169.1 (9.3), 168.1 (8.7), 167.1 (6.8), 155.1 (11.1), 154.1 (100.0), 153.1 (20.7), 152.1 125.1 (30.0), 120.1 (10.0), 114.0 (10.8), 111.1 (25.8), 107.1 (19.7), 98.1 (10.8), 97.1 (11.1), 92.1 (7.8), 91.1 (38.7), 86.1 74.1 (7.3), 72.1 (6.7), 71.1 (11.7), 77.0 (8.1), 74.0 (7.8) 60.0 (7.2), 57.1 (18.1), 56.0 (13.4), 55.0 (20.0), 70.1 (61.9), 69.1 (22.9), 68.0 Cis-bicyclo (L- tyrosine -L- _{proline), C 14 H 16 N 2} O 3 F12
(20.5-22.0) ^a 211.0 156.0, 156.0, 155.0, 154.0, 153.0, 5.3.0, 125.0, 11.9, 16.0, 98.0 (2.8), 86.0 (18.2), 70.0 (48.5), 55.0 (3.3) Cis -cyclo (L-leucine-L-proline), C ₁₁ H ₁₈ N ₂ O ₂ F13
(22.5-23.5) ^a 211.0 105.0 (19.0), 103.1 (13.2), 91.0 (100.0), 87.1 (66.0), 128.0 57.1 (13.5), 76.0 (40.2), 75.1 (22.5), 71.1 (7.7), 70.1 (20.9), 69.1 (76.2), 65.1 (12.1), 57.1 Cis -cyclo (L-leucine-L-proline), C ₁₁ H ₁₈ N ₂ O ₂ F14
(24.0-24.5) ^a 228.0 105.0 (19.0), 103.1 (13.2), 91.0 (100.0), 87.1 (66.0), 128.0 57.1 (13.5), 76.0 (40.2), 75.1 (22.5), 71.1 (7.7), 70.1 (20.9), 69.1 (76.2), 65.1 (12.1), 57.1 Cis-bicyclo (L- methionine -L- _{proline), C 10 H 16 N 2} O 2 S 1 F15
(24.5-25.0) ^a 167 168.1 (0.3), 167.1 (1.9), 166.1 (13.6), 154.1 (5.4), 149.1 (7.5), 148.1 (58.6), 147.1 (10.4), 130.1 (2.6), 129.1 121.1 (15.9), 120.1 (4.2), 104.1 (2.9), 103.1 (22.9), 102.1 (1.7), 93.1 (2.3), 92.1 (27.2), 91.1 (100), 79.1 (3.6), 77.1 70.1 (4.2), 65.1 (21.9), 63.1 (4.9), 51.1 (9.3) DL-3-phenyllactic acid,
C ₉ H ₁₀ O ₃ F16
(26.0-27.0) ^a 245.0 (100.0), 154.0 (79.8), 153.0 (55.5), 125.0 (90.5), 91.0 (41.7), 70.1 (64.9) Cis-bicyclo (L- phenylalanine -L- _{proline), C 14 H 16 N 2} O 2 F17
(30.0-31.0) ^a 245.0 244.1 (97.5), 153.0 (82.0), 125.1 (100.0), 91.0 (52.3), 70.1 (46.3) Cis-bicyclo (L- phenylalanine -L- _{proline), C 14 H 16 N 2} O 2

^a Retention time (min)

Table 2 shows the crystal data, data collection, refinement, and phasing statistic of F7.

Cis -Cyclo (L-valine-L-proline) (antifungal activity) Crystal Data Chemical formula C ₂₀ H ₃₂ N ₄ O ₄ M _r 392.50 Crystal system, space group Orthormbic, P 2 ₁ 2 ₁ 2 ₁ Temperature (K) 100 (2) a, b, c (A) 5.6240 (11), 10.257 (2), 34.240 (7) a , b , g (DEG) 90, 90, 90 V (Å ³ ) 1975.1 Z 4 m (mm ^-1 ) 0.045 F (000) 848 Crystal size (mm) 0.15 x 0.05 x 0.05 Data Collection Diffractometer ADSC Quantum 210 2D SMC, PAL-6B θ _max (°) 2.88 to 28.00 No. of measured, independent and observed [ I > 2σ ( I )] reflections 21052, 5927 R _int 0.0604 Refinement ^{R [F 2> 2σ (F} 2)], wR (F 2), (all data) S 0.0324, 0.0822, 0.0338, 0.0836 No. of reflections 5927 No. of parameters 284 No. of restrains 0 H-atom treatment H atoms treated by a mixture of independent and constrained refinement Δρ _max , Δρ _min (e Å ^-3 ) 0.369, -0.279

Table 3 shows F13 crystal data, data collection, refinement, and phasing statistic.

Cis -Cyclo (L-leucine-L-proline) (antimicrobial and antiviral activity) Crystal data Chemical formula C ₁₁ H ₁₈ N ₂ O ₂ M _r 210.277 Crystal system, space group Orthorhombic, P 2 ₁ 2 ₁ 2 ₁ Temperature (K) 100 a, b, c (A) 6.2820, 9.2440, 19.6250 a , b , g (DEG) 89.998, 90.003, 90.022 V (Å ³ ) 1139.64 Z 4 m (mm ^-1 ) 0.08 F (000) 456 Crystal size (mm) 0.1 x 0.3 x 0.5 Data collection Diffractometer ADSC Quantum 210 CCD, PAL-6B θ _max (°) No. of measured, independent and observed [ I > 2σ ( I )] reflections 4345, 1399, 1362 R _int 0.0543 Refinement ^{R [F 2> 2σ (F} 2)], wR (F 2), (all data) S 0.1120, 0.2878, 2.8194 No. of reflections 1399 No. of parameters 136 No. of restrains 0 H-atom treatment H atoms treated by a mixture of independent and constrained refinement Δρ _max , Δρ _min (e Å ^-3 ) 1.18, -0.40

Table 4 shows the crystal data, data collection, refinement, and phasing statistic of F17.

Cis - cyclo (L-phenylalanine-L-proline) (antibacterial, antifungal and antiviral activity) Crystal data Chemical formula C ₁₄ H ₁₆ N ₂ O ₂ M _r 244.12 Crystal system, space group Monoclinic, P 2 ₁ Temperature (K) 100 a, b, c (A) 5.592, 10.008, 10.681 a , b , g (DEG) 90, 87.634, 90 V (Å ³ ) 597.25 Z 2 m (mm ^-1 ) 0.089 F (000) 260 Crystal size (mm) 0.1 x 0.3 x 0.5 Data collection Diffractometer ADSC Quantum 210 CCD, PAL-6B θ _max (°) 27 No. of measured, independent and observed [ I > 2σ ( I )] reflections 2220, 1314, 1254 R _int 0.069 Refinement ^{R [F 2> 2σ (F} 2)], wR (F 2), (all data) S 0.1019, 0.2667, 2.519 No. of reflections 1312 No. of parameters 164 No. of restrains One H-atom treatment H atoms treated by a mixture of independent and constrained refinement Δρ _max , Δρ _min (e Å ^-3 ) 1.09, -0.44

Table 5 shows the crystal data, data collection, refinement, and phasing statistic of F8.

Cis -Cyclo (L-leucine-L- Hydroxyproline ) (No activity) Crystal data Chemical formula C ₁₁ H ₁₈ N ₂ O ₃ M _r 226.27 Crystal system, space group Orthorhmbic, P 2 ₁ 2 ₁ 2 ₁ Temperature (K) 100 (2) a, b, c (A) 6.2780 (13), 9.4440 (19), 19.674 (4) a , b , g (DEG) 90, 90, 90 V (Å ³ ) 1166.5 (4) Z 4 m (mm ^-1 ) 0.046 F (000) 488 Crystal size (mm) 0.30 x 0.30 x 0.03 Data collection Diffractometer ADSC Quantum 210 2D SMC, PAL-6B θ _max (°) 3.16 to 27.991 No. of measured, independent and observed [ I > 2σ ( I )] reflections 12546, 3475 R _int 0.0491 Refinement ^{R [F 2> 2σ (F} 2)], wR (F 2), (all data) S 0.0287, 0.0762, 0.0295, 0.0768 No. of reflections 3475 No. of parameters 155 No. of restrains 0 H-atom treatment H atoms treated by a mixture of independent and constrained refinement Δρ _max , Δρ _min (e Å ^-3 ) 0.358, -0.220

Table 6 shows F15 crystal data, data collection, refinement and phasing statistic.

DL -3- Phenyllactic acid  (No activity) Crystal data Chemical formula C ₉ H ₁₀ N ₂ O ₃ M _r 166.17 Crystal system, space group Orthorhombic, P 2 ₁ 2 ₁ 2 ₁ Temperature (K) 100 a, b, c (A) 5.844, 8.316, 16.963 a , b , g (DEG) 90, 90, 90 θ _max (°) 824.4 (3) Z 4 m (mm ^-1 ) 0.077 F (000) 352 Crystal size (mm) 0.11 x 0.11 x 0.05 Data collection Diffractometer ADSC Quantum 210 CCD, PAL-6B θ _max (°) 3.777 to 27.991 No. of measured, independent and observed [ I > 2σ ( I )] reflections 10365, 2835 R _int 0.0336 Refinement ^{R [F 2> 2σ (F} 2)], wR (F 2), (all data) S 0.0290, 0.0771, 0.0300, 0.0778 No. of reflections 2835 No. of parameters 111 No. of restrains 0 H-atom treatment H atoms treated by a mixture of independent and constrained refinement Δρ _max , Δρ _min (e Å ^-3 ) 0.431, -0.211

Table 7 shows antibacterial, antifungal and antiviral activities against Lactobacillus plantarum LBP-K10.

Fraction m / z of [M + 1] ⁺ The Molecule Antimicrobial activity F1 261.0 C ₁₄ H ₁₆ N ₂ O ₃ Cis -cyclo (L-tyrosine-L-proline) antifungal F2 185.0 C ₈ H ₁₂ N ₂ O ₃ Cis -cyclo (L-serine-L-proline) antifungal F3 211.0 C ₁₁ H ₁₈ N ₂ O ₂ Cis -cyclo (L-leucine-L-proline) antibacterial, antifungal, and antiviral F4 211.0 C ₁₁ H ₁₈ N ₂ O ₂ Cis -cyclo (L-leucine-L-proline) antibacterial, antifungal, and antiviral F5 195.0 C ₁₀ H ₁₆ N ₂ O ₂ Cis -cyclo (L-valine-L-proline) antifungal F6 261.0 C ₁₄ H ₁₆ N ₂ O ₃ Cis -cyclo (L-tyrosine-L-proline) antifungal F7 197.0 C ₁₀ H ₁₆ N ₂ O ₂ Cis -cyclo (L-valine-L-proline) antifungal F8 227.0 C ₁₁ H ₁₈ N ₂ O ₃ Cis -cyclo (L-leucine-L-hydroxyproline) - F9 185.0 C ₈ H ₁₂ N ₂ O ₃ Cis -cyclo (L-serine-L-proline) antifungal F10 219.0 C ₁₂ H ₁₄ N ₂ O ₂ Cis -cyclo (L-phenylalanine-L-alanine) - F11 261.0 C ₁₄ H ₁₆ N ₂ O ₃ Cis -cyclo (L-tyrosine-L-proline) antifungal F12 211.0 C ₁₁ H ₁₈ N ₂ O ₂ Cis -cyclo (L-leucine-L-proline) antibacterial, antifungal, and antiviral F13 211.0 C ₁₁ H ₁₈ N ₂ O ₂ Cis -cyclo (L-leucine-L-proline) antibacterial, antifungal, and antiviral F14 228.0 C ₁₀ H ₁₆ N ₂ O ₂ S ₁ Cis -cyclo (L-methionine-proline) - F15 167.0 C ₉ H ₁₀ O ₃ DL-3-phenyllactic acid antifungal F16 245.0 C ₁₄ H ₁₆ N ₂ O ₂ Cis -cyclo (L-phenylalanine-L-proline) antibacterial, antifungal, and antiviral F17 245.0 C ₁₄ H ₁₆ N ₂ O ₂ Cis -cyclo (L-phenylalanine-L-proline) antibacterial, antifungal, and antiviral

Table 8 shows the recovery of organic acids using Amberlite IRA-67, a weak anion exchanger.

matter Quantity (gl- ¹ ) Adsorbed rate (%) Not adsorbed rate (%) Organic acid Lactic acid 6.78 (+/- 0.57) 96.88 3.12 Acetic acid 1.19 (+/- 0.11) 99.6 0.4 Formic acid 0.23 (0.08) 100 0 Propionic acid 0.65 (+/- 0.24) 100 0

Table 9 shows the total amount of purified material in Lactobacillus plantarum LBP-K10.

Perform Total quantity  ( mg / L) Culture without organic acid Supernatant / wo methylene chloride extraction, a 28,335.2 / w methylene chloride extraction, b 517.6 yield,% (b / a x 100) 1.8 Culture without sugar Supernatant / wo methylene chloride extraction, c 32,538. / w methylene chloride extraction, d 351.9 yield,% (d ÷ c x 100) 1.1 Organic acids and sugars  Do not have  culture Supernatant / wo methylene chloride extraction, e 18,964.5 / w methylene chloride extraction, f 411.5 yield,% (f ÷ e x 100) 2.2

Table 10 shows the total amount of cyclic dipeptide and DL-3-phenyllactic acid in Lactobacillus plantarum LBP-K10.

compound culture Supernatant Culture without organic acid Supernatant Sugar-free culture supernatant Organic acid and sugar free culture Supernatant Total quantity in Lactobacillus plantarum LBP-K10 mg / L μM mg / L μM mg / L μM mg / L μM Cis -cyclo (L-leucine-L-proline) 12.6 59.7 9.8 46.5 10.5 49.8 5.7 27.2 Cis -cyclo (L-phenylalanine-L-proline) 8.8 36.1 9.0 36.7 3.7 15.2 6.7 27.3 Cis -cyclo (L-tyrosine-L-proline) 27.7 106.4 23.6 90.7 27.6 106.1 15.0 57.8 Cis -cyclo (L-serine-L-proline) 4.4 23.9 4.0 21.7 2.7 14.7 2.1 11.3 Cis -cyclo (L-valine-L-proline) 10.1 51.7 15.3 78.1 17.4 88.6 12.7 64.8 Cis -cyclo (L-leucine-L-hydroxyproline) 3.0 13.4 6.6 29.3 6.3 27.7 0.9 3.9 Cis -cyclo (L-phenylalanine-L-alanine) 1.0 4.5 2.7 12.2 10.3 47.2 0.6 2.7 Cis -cyclo (L-methionine-L-proline) 2.1 9.2 0.4 1.8 1.6 6.9 0.3 1.5 DL-3-phenyllactic acid 19.8 119.2 3.8 23.2 14.9 89.7 3.2 19.2

Table 11 shows the antimicrobial activity of an organic acid or sugar-free culture supernatant from Lactobacillus plantarum LBP-K10 against Gram-positive and Gram-negative indicator strains.

Indicator strains Culture without organic acid Supernatant Culture without sugar Supernatant Organic acid and sugar free culture Supernatant Active concentration of mg complex Bacillus subtilis 0.133 0.146 0.125 Staphylococcus aureus 0.133 0.146 0.125 Streptococcus pneumoniae 0.133 0.146 0.125 Eschericia . coli 0.133 0.146 0.125 Salmonella typhimurium 0.133 0.146 0.125 Shigella dysentery 0.133 0.146 0.125

Table 12 compares the antimicrobial activity of organic acid or sugar-free culture supernatants from Lactobacillus plantarum LBP-K10 against multimeric drug-resistant gram-positive and gram-negative bacteria.

Multi - drug resistant strains Culture without organic acid Supernatant Culture without sugar Supernatant Organic acid and sugar free culture Supernatant Active concentration of mg complex Staphylococcus aureus 11471 (KNIH) ^a 0.133 0.146 0.125 Streptococcus pneumoniae 14596 (KNIH) ^a 0.133 0.146 0.125 Salmonella Typhimurium 12219 (KNIH) ^b 0.133 0.146 0.125

^a Multidrug-resistant gram-positive bacteria is supported by Korea National Institute of Health.

^b Multidrug-resistant gram-negative bacteria are supported by Korea National Institute of Health.

Table 13 compares the antifungal activity of the organic acid or sugar-free culture supernatant from Lactobacillus plantarum LBP-K10 against Ganoderma johninens and Candida albicans.

Fungal strains Culture without organic acid Supernatant Culture without sugar Supernatant Organic acid and sugar free culture Supernatant Active concentration of mg complex (%) Plant pathogenic strain G. boninense 103.52 (3.45) 71.70 (3.59) 92.25 (3.28) Human pathogenic strain C. albicans 51.76 (1.73) 47.84 (2.39) 61.55 (2.18)

Table 14 shows the antiviral activity of an organic acid or sugar-free culture supernatant from Lactobacillus plantarum LBP-K10 against influenza A (H3N2) virus.

Sample Sample ratio (%) Plaque number (± SD) Inhibition ratio (%) Quantity (mg / ml) Untreated cells 0 76.6 (+ - 5.7) 0 0 Cells treated with culture supernatant without organic acids 0.5 71 (+/- 2.6) 7.3 7.176 1.0 46.3 (+ - 4.6) 39.6 14.352 1.5 34.6 (占 6.1) 54.9 21.528 2.0 25 (± 2.6) 67.4 28.704

Table 15 shows the antiviral activity of an organic acid or sugar-free culture supernatant from Lactobacillus plantarum LBP-K10 against influenza A (H3N2) virus.

Sample Sample ratio (%) Plaque number (± SD) Inhibition ratio (%) Quantity (mg / ml) Untreated cells 0 79.1 (+/- 3.4) 0 0 Cells treated with culture supernatant without sugars 0.5 73.4 (+/- 6.5) 7.2 7.176 1.0 43.1 (+/- 5.5) 45.5 14.352 1.5 32.7 (+/- 4.2) 58.7 21.528 2.0 29.2 (+/- 2.9) 63.1 28.704

Table 16 shows the antiviral activity of an organic acid or sugar-free culture supernatant from Lactobacillus plantarum LBP-K10 against influenza A (H3N2) virus.

Sample Sample ratio (%) Plaque number (± SD) Inhibition ratio (%) Quantity (mg / ml) Untreated cells 0 82.5 (+ - 4.9) 0 0 Cells treated with culture supernatant without organic acids and sugars 0.5 76.2 (+/- 2.6) 7.6 7.176 1.0 50.4 (+ - 4.1) 38.9 14.352 1.5 40.5 (+ - 4.7) 50.9 21.528 2.0 24.9 (+ - 4.6) 69.8 28.704

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Claims (11)

An antimicrobial activity comprising a cyclic dipeptide and DL-3-phenyllactic acid. According to claim 1, wherein said cyclic dipeptide is cis-bicyclo (L- tyrosine -L- proline), cis-bicyclo (L- serine -L- proline), cis-bicyclo (L- leucine -L- proline) cis-bicyclo (L- valine -L- proline), cis-bicyclo (L- leucine -L- hydroxyproline) cis-bicyclo (L- phenylalanine -L- alanine), cis-bicyclo (L- methionine- Proline) and cis -cyclo (L-phenylalanine-L-proline). The antimicrobial composition according to claim 1, wherein the bacterium is any one of Gram-positive and Gram-negative bacteria. The method of claim 3, wherein the gram-positive and gram-negative bacteria is Bacillus subtilis , Staphylococcus aureus , Streptococcus pneumoniae , Eschericia coli , Salmonella typhimurium , and Shigella dysentery . < RTI ID = 0.0 > 11. < / RTI > A cyclic dipeptide, and a DL-3-phenyllactic acid. The method of claim 5, wherein the dipeptide is a cyclic cis-bicyclo (L- tyrosine -L- proline), cis-bicyclo (L- serine -L- proline), cis-bicyclo (L- leucine -L- proline) cis-bicyclo (L- valine -L- proline), cis-bicyclo (L- leucine -L- hydroxyproline) cis-bicyclo (L- phenylalanine -L- alanine), cis-bicyclo (L- methionine- Proline) and cis -cyclo (L-phenylalanine-L-proline). 6. The antifungal composition according to claim 5, wherein the fungus is Ganoderma boninens, a pathogenic strain of a plant. The composition for an antifungal agent according to claim 5, wherein the fungus is Candida albicans, which is a pathogenic bacterium of a human. An antiviral activity comprising a cyclic dipeptide and DL-3-phenyllactic acid. The method of claim 9, wherein said cyclic dipeptide is cis-bicyclo (L- tyrosine -L- proline), cis-bicyclo (L- serine -L- proline), cis-bicyclo (L- leucine -L- proline) cis-bicyclo (L- valine -L- proline), cis-bicyclo (L- leucine -L- hydroxyproline) cis-bicyclo (L- phenylalanine -L- alanine), cis-bicyclo (L- methionine- Proline) and cis -cyclo (L-phenylalanine-L-proline). The antiviral composition according to claim 9, wherein the virus is an influenza A (H3N2) virus.

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