WO2013002439A1 - Antifungal composition comprising cis-cyclo(l-phe-l-pro) having genus ganoderma fungus-specific antifungal activity - Google Patents

Abstract

The present invention provides an antifungal composition comprising cis-cyclo(L-Phe-L-Pro) as an active ingredient and having Genus Ganoderma fungus-specific antifungal activity, an insecticide composition, and a method for producing an antifungal agent for Genus Ganoderma fungi. The present invention exhibits superior antifungal activity to the Genus Ganoderma fungi (especially, to Ganoderma boninense fungi) and thus can be applied to various industrial fields requiring removal and prevention of Genus Ganoderma fungi, and is economically advantageous in terms of usage due to the antifungal activity even with a small amount. In addition, the present invention provides basic material as a biological agent regarding cis-cyclo(L-Phe-L-Pro) to the insecticide industry which uses biological agents.

Description

[Correction 16.09.2011] according to Rule 26. Antifungal composition comprising cis-cyclo (L-phenylalanine-L-proline) having specific antifungal activity against fungi in genus ganoderma

The present invention relates to novel uses of cis- cyclo (L-phenylalanine-L-proline). More specifically, the present invention includes cis- cyclo (L-phenylalanine-L-proline) ( cis- cyclo (L-Phe-L-Pro)) as an active ingredient, and the fungus of genus Ganoderma It relates to an antifungal composition characterized by having a specific antifungal activity.

In general, lactic acid fermentation efficacy depends primarily on organic acid accumulation and substrate oxidation. ^[1,2,3] Metabolites such as acetaldehyde, diacetyl compounds, hydrogen peroxide and carbon dioxide are important for inhibiting the growth of bacteria, pathogens and rot. ^[1,2,3] It has also been reported that non-protein substrates inhibit Gram-negative bacteria as well as Gram-positive and fungal bacteria. ^[4]

The first low molecular weight antibiotic, Reutericyclin, was purified from Lactobacillus reuteri LTH2584 isolated from fermented bread. Ryutercycline is somewhat sensitive to microorganisms such as Gram-positive bacteria, Gram-negative bacteria, yeasts and fungi. ^[5] Lactic acid bacteria (LAB) have been used for a long time in various foods for humans and animals, and they are useful for fermenting, storing and storing food like yeast. ^[1,2,3]

LABs generally secrete extracellular antagonist compounds to combat many microorganisms and alter the extracellular environment by endogenous metabolite byproducts. ^[6] Among the compounds excreted by LAB, various kinds of antimicrobial metabolites can be classified into bacteriocins having low molecular weight of less than 1000 and molecular weights of less than 1000. ^[7] Many studies of antimicrobial activity by LAB have focused on bacteriocins. The bacteriocin produced by LAB is one of a heterogeneous group of antimicrobial peptides and proteins that varies over a broad spectrum in terms of activity, mode of action, molecular weight, genetic origin and biochemical properties, ^[8,9] This class of peptides and proteins has been classified into three groups based on the sequence of mature peptides and prepeptides. ^{[10,11,12,13]}

Pediosin is a type of bacteriocin known to be produced by Pediococcus pentosaceus . ^[14] Most of these compounds contain cationic molecules and small sized substrates. ^[15], and these compounds may generally kill several gram positive pathogenic strains, such as Bacillus subtilis (Bacillus subtilis), Listeria species (Listeria species) and Staphylococcus aureus (Staphylococcus aureus). ^{[16,17,18] In} addition, some bacteriocins can kill food pathogenic strains such as Escherichia coli and Salmonella spp. ^[19] Proline-containing 2,5-diketopiperazine, a cyclic dipeptide, has been intensively investigated. ^{[6,20,22,23,24,25,26,27]} Many of these cyclic or ^{cyclodipeptides} have been considered to act as substrates in terms of many biological events and phenomena. ^{[22,23,24,25,26] In} particular, the potential therapeutic and prophylactic efficacy of such cyclic dipeptides has been shown to have a variety of antibiotic activities against Bacteria, fungi, viruses and cancer. ^{[6,23,26,27,28]} These antimicrobial cyclic dipeptides have also been identified in yeast, lichen and fungal cultures. ^{[20,29] Separation} of cyclo (1-leusil-1-propyl) and cyclo (1-phenylalanyl-1-prolyl) from Streptomyces species has already been reported. ^{[20,24,25,29]} Antimicrobially active cyclic peptides exist everywhere in nature and influence the wide variety of biological targets for humans. ^[22,23,25]

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The inventors have sought to find novel substances having good antifungal activity against fungi, particularly fungi of the genus Ganoderma . A cycloalkyl (L- phenylalanine -L- proline) represents the (cis -cyclo (L-Phe- L-Pro)) having excellent antifungal activity against fungi of the genus Kano Derma (Genus Ganoderma) - As a result, the dipeptide of cis By clarifying, this invention was completed.

Therefore, an object of the present invention comprises cis- cyclo (L-phenylalanine-L-proline) ( cis- cyclo (L-Phe-L-Pro)) as an active ingredient, and to the fungus of genus Ganoderma It is an object of the present invention to provide an antifungal composition characterized by having specific antifungal activity.

Another object of the present invention comprises cis- cyclo (L-phenylalanine-L-proline) ( cis- cyclo (L-Phe-L-Pro)) as an active ingredient, and to the fungus of genus Ganoderma It is an object of the present invention to provide a pesticide composition characterized by having specific antifungal activity.

Still another object of the present invention is to provide a method for producing an antifungal agent against fungi of genus Ganoderma .

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to an aspect of the present invention, the present invention comprises cis- cyclo (L-phenylalanine-L-proline) ( cis- cyclo (L-Phe-L-Pro)) as an active ingredient, Genus Ganoderma It provides an antifungal composition, characterized in that it has a specific antifungal activity against the fungi.

The inventors have sought to find novel substances having good antifungal activity against fungi, particularly fungi of the genus Ganoderma . A cycloalkyl (L- phenylalanine -L- proline) represents the (cis -cyclo (L-Phe- L-Pro)) having excellent antifungal activity against fungi of the genus Kano Derma (Genus Ganoderma) - As a result, the dipeptide of cis By clarifying, this invention was completed.

Cis contained as an effective ingredient in the present invention -cycloalkyl (L- phenylalanine -L- proline) are cis in nature - including a cycloalkyl (L- phenylalanine -L- proline), cis-bicyclo (L- -L-phenylalanine Chemical formula for -proline) is shown in FIG. 22 in the context of the present invention.

The present invention is characterized by having excellent antifungal activity against fungi of genus Ganoderma.

According to a preferred embodiment of the present invention, the present invention comprises at least one genus Ganoderma selected from the group consisting of Ganoderma boninense , Ganoderma applanatum and Ganoderma zonatum . It has antifungal activity against fungi, and more preferably has antifungal activity against Ganoderma boninense .

System included as an active ingredient of the present invention -cycloalkyl (L- phenylalanine -L- proline) is a system separated from the lactic acid - include cycloalkyl (L- phenylalanine -L- proline).

Preferably, cis- cyclo (L-phenylalanine-L-proline) included as an active ingredient in the present invention is composed of the genus Pediococcus , Lactobacillus, and Leuconostoc . It is isolated from one or more lactic acid bacteria selected from the group.

When the cis- cyclo (L-phenylalanine-L-proline) included as an active ingredient in the present invention is isolated from the pediccoccus lactic acid bacterium, the bacterium pediococcus is Pediococcus pentosaceus , Pediococcus acidilactici , Pediococcus cellicola , Pediococcus claussenii , Pediococcus damnosus , Pediococcus ethanoliduus Pediococcus ethanolidurans), Phedi OKO to kusu Ino Pina tooth (Pediococcus inopinatus), Phedi OKO kusu wave bulruseu (Pediococcus parvulus) and Phedi OKO kusu steel Lacey (Pediococcus stilesii) comprises a group of one or more Phedi OKO kusu in lactic acid bacteria selected from the consisting of. Preferably, when the cis- cyclo (L-phenylalanine-L-proline) included as an active ingredient in the present invention is isolated from the pepiococcus lactic acid bacterium, the Pediococcus bacterium is Pediococcus pentosaceus bacterium. to be.

When the cis- cyclo (L-phenylalanine-L-proline) included as an active ingredient in the present invention is isolated from Lactobacillus of Lactobacillus , Lactobacillus of Lactobacillus plantarum ( Lactobacillus plantarum ), Lactobacillus kimchi ( Lactobacillus kimchii ), Lactobacillus rhamnosus , Lactobacillus casei , Lactobacillus acidophilus , Lactobacillus acidophilus , Lactobacillus brevis , Lactobacillus buccinerus Lactobacillus Lactobacillus fermentum , Lactobacillus helveticus ( Lactobacillus helveticus ), Lactobacillus reuteri and Lactobacillus genus Lactic acid bacteria selected from the group consisting of Lactobacillus sakei . . Preferably, when the cis- cyclo (L-phenylalanine-L-proline) included as an active ingredient in the present invention is isolated from Lactobacillus spp., Lactobacillus spp. Is Lactobacillus plantarum.

Included as an active ingredient in the present inventionSheathWhen cyclo (L-phenylalanine-L-proline) is separated from the lactic acid bacteria of the leuconosstock, the lactic acid bacteria of the leuconosstock areLeuconostoc mesenteroides), Ryukono Stock Kimchi (Leuconostoc kimchii), Leukonostock lactis (Leuconostoc lactis), Due to leukonostock (Leuconostoc inhae), Leukonostock Gelidum (Leuconostoc gelidum), Leukonostock Paramesenteroides (Leuconostoc paramesenteroides), Leukonostock Citrium (Leuconostoc citreum), Leukono stock pseudometheroidesLeuconostoc pseudomesenteroides) And leukonostock Holzappelli (Leuconostoc holzapfeliiLactic acid bacteria of the genus Lyukonostok selected from the group consisting of Preferably, included as an active ingredient in the present inventionSheathWhen cyclo (L-phenylalanine-L-proline) is separated from lactic acid bacteria of the genus Leukonostock, the genus of the genus Leukonostock is leukonostock mesenteroides.

As used herein, the term "antifungal activity" refers to the growth inhibition, growth inhibition and killing action of the fungus, and the term "fungal" is used interchangeably with the "fungus" in the present specification.

According to another aspect of the present invention, the present invention comprises cis- cyclo (L-phenylalanine-L-proline) ( cis- cyclo (L-Phe-L-Pro)) as an active ingredient, Genus Ganoderma It provides a pesticide composition characterized in that it has a specific antifungal activity against the fungi.

The composition of the present invention also provides an agrochemical composition comprising an agrochemically effective amount of cis- cyclo (L-phenylalanine-L-proline) of the present invention described above.

More preferably, the pesticide composition of the present invention comprises an agrochemically effective amount of cis- cyclo (L-phenylalanine-L-proline); And (b) at least one component or additive selected from the group consisting of agrochemically acceptable solvents, carriers, emulsifiers and dispersants. The term of the terms "agricultural effective amount" is the above-mentioned cis-against fungi of the cyclo (L- phenylalanine -L- proline) (cis -cyclo (L-Phe -L-Pro)) in Kano Derma (Genus Ganoderma) of By an amount sufficient to achieve antifungal efficacy or activity.

When the composition of the present invention is prepared as a pesticide composition, the pesticide composition of the present invention includes an agriculturally acceptable solvent. Such solvents include water, aromatic solvents (eg Solvesso products, xylene), paraffins (eg mineral oil fractions), alcohols (eg methanol, butanol, pentanol, benzyl alcohol), ketones (eg For example cyclohexanone, gamma-butyrolactone), pyrrolidone (NMP, NOP), acetates (glycol diacetate), glycols, fatty acid dimethylamides, fatty acids and fatty acid esters (in principle, solvent mixtures may be used) Etc. are included.

In addition, when the composition of the present invention is made of a pesticide composition, the pesticide composition of the present invention includes an agriculturally acceptable carrier. For example, such carriers include, for example, ground natural minerals (eg kaolin, clay, talc, chalk) and ground synthetic minerals (eg highly dispersed silica, silicates).

In addition, when the composition of the present invention is prepared as a pesticide composition, the pesticide composition of the present invention includes an agriculturally acceptable emulsifier. For example, the emulsifiers include nonionic and anionic emulsifiers (eg, polyoxyethylene fatty alcohol ethers, alkylsulfonates and arylsulfonates).

In addition, when the composition of the present invention is prepared as a pesticide composition, the pesticide composition of the present invention includes an agriculturally acceptable dispersant. For example, the propellant includes lignin-sulfite waste liquor and methylcellulose.

The present invention provides a system including the above-mentioned anti-fungal composition, so pesticide composition comprising cyclo (L- phenylalanine -L- proline) (cis -cyclo (L-Phe -L-Pro)) as an active ingredient, in common with the two The matters are omitted in order to avoid excessive description of this specification.

According to another aspect of the present invention, the present invention comprises the steps of (a) culturing the lactic acid bacteria to prepare a lactic acid bacteria culture medium; And (b) concentrating the cis- cyclo (L-phenylalanine-L-proline) in the culture medium at an effective concentration for antifungal, wherein the method for producing an antifungal agent against fungi of Genus Ganoderma . to provide.

Lactic acid bacteria in step (a) in the present invention Phedi OKO kusu in (Pediococcus), Lactobacillus (Lactobacillus) and is in flow Pocono stock in lactic acid bacteria selected from the group consisting of (Leuconostoc) or more.

Preferably, when using the Pediococcus spp. As a lactic acid bacterium in step (a) of the present invention, the Pediococcus spp. Is Pediococcus pentosaceus , Pediococcus asidyllactic acid ( Pediococcus acidilactici , Pediococcus cellicola , Pediococcus claussenii , Pediococcus damnosus , Pediococcus ethanolidurans Pediococcus ethanolidurans the blood or tooth (Pediococcus inopinatus), Phedi OKO kusu wave bulruseu (Pediococcus parvulus) and Phedi OKO kusu steel Lacey (Pediococcus stilesii) comprises a group of one or more lactic acid bacteria selected from the genus Phedi OKO kusu made. More preferably, the genus Pediococcus is Pediococcus pentosaceus.

Preferably, when the Lactobacillus genus bacteria are used as lactic acid bacteria in step (a) of the present invention, the Lactobacillus genus is Lactobacillus plantarum , Lactobacillus kimchii , Lactobacillus kimchii , Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), Lactobacillus casei , Lactobacillus acidophilus , Lactobacillus brevis , Lactobacillus Buchyne , Lactobacillus buchneri , Lactobacillus pertusum L. , Lactobacillus helveticus , Lactobacillus reuteri and Lactobacillus sakei , and one or more Lactobacillus genus lactic acid bacteria selected from the group consisting of. More preferably, the genus Lactobacillus is Lactobacillus plantarum bacteria.

Preferably, in the case of using the present invention in the step (a) of the lactic acid bacteria as lactic acid bacteria, the genus Leukono stock bacteria are leukonostock mesenteroides (Leuconostoc mesenteroides), Ryukono Stock Kimchi (Leuconostoc kimchii), Leukonostock lactis (Leuconostoc lactis), Due to leukonostock (Leuconostoc inhae), Leukonostock Gelidum (Leuconostoc gelidum), Leukonostock Paramesenteroides (Leuconostoc paramesenteroides), Leukonostock Citrium (Leuconostoc citreum), Leukono stock pseudometheroidesLeuconostoc pseudomesenteroides) And leukonostock Holzappelli (Leuconostoc holzapfeliiLactic acid bacteria of the genus Lyukonostok selected from the group consisting of More preferably, the genus Leukonostock is leukonostock mesenteroides.

In the present invention, the step of preparing the lactic acid bacteria culture medium in step (a) can be carried out using a medium composition and additives available in the art.

Step of concentration in step (b) from the culture broth in the present invention are cis-bicyclo (L- phenylalanine -L- proline) (cis -cyclo (L-Phe -L-Pro)) and the separation step of purification by concentration or Concentrating the culture comprising cis- cyclo (L-phenylalanine-L-proline).

Separation and purification of cis- cyclo (L-phenylalanine-L-proline) in step (b) of the present invention is carried out through various known methods.

For example, fractions obtained by passing the water through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. It is carried out through the purification method.

The expression "concentrated to an effective concentration" in the present invention means that the lactic acid bacteria culture medium containing cis- cyclo (L-phenylalanine-L-proline) or cis- cyclo (L-phenylalanine-L-proline) is genus Ganoderma . It means to concentrate to the concentration showing the antifungal activity against the fungi of.

For example, the cis-bicyclo (L- phenylalanine -L- proline) or the sheath contained in the culture-cell density of nenseu (Ganoderma boninense) effective Kano Thomas I of cyclo (L- phenylalanine -L- proline) is 50.24 mm ² ( 16π) at 6.82 mM (20.47 μmol / 3 ml medium).

The present invention provides a system including the above-mentioned antifungal composition because it is a method of producing cyclo (L- phenylalanine -L- proline) (cis -cyclo (L-Phe -L-Pro)), the common information and the two are present Omitted to avoid undue description of the specification.

The features and advantages of the present invention are summarized as follows:

(i) The present invention includes cis- cyclo (L-phenylalanine-L-proline) ( cis- cyclo (L-Phe-L-Pro)) as an active ingredient and against fungi of Genus Ganoderma . It provides an antifungal composition, agrochemical composition and antifungal production method for fungi of genus Ganoderma, characterized by having specific antifungal activity.

(ii) The present invention exhibits excellent antifungal activity against the fungi of Genus Ganoderma , and thus the need for the removal and prevention of fungi (particularly, Ganoderma boninense fungi) in Ganoderma genus . It can be applied to various industrial fields, and in particular, since it shows antifungal activity even in a small amount, it has economic advantages in terms of use.

(iii) The present invention also provides basic data as biological agents for cis- cyclo (L-phenylalanine-L-proline) in the pesticide industry using biological agents.

1 is a result of comparing the antimicrobial activity in the plant extract. The disk containing the culture solution was prepared as follows. Disc 1 is Wishella Sibaria (W. cibaria) LBP-B06 medium containing the culture medium, disk 2 is Lactobacillus sakei (L. sakei) The disk containing the LBP-S02 culture medium, disk 3 is Lactobacillus kimchi (L. kimchii) LBP-B02 medium containing the culture medium, disc 4 is Lactobacillus plantarum (Lb. plantarum) A disk containing LBP-K10 culture medium, and the disk 5 is Lactobacillus citreum LBP-K11 (L. citreumLBP-K11) disk containing the culture medium, and disk 6 is Leukonostock Mesenteroides LBP-K06 (L. mesenteroidesLBP-K06) is a disk containing the culture. The left panel is Bacillus subtilis (Bacillus subtilis) And the right panel shows Escherichia coli (Escherichia coli) It is the result using the indicator strain. All experiments were performed three times independently.

2 is Lactobacillus plantarum The normal growth of LBP-K10 is a graph showing the absorbance and pH change at 600 nm. After 28 hours, pH (closed circle) and OD₆₀₀(open circle) was maintained for 2 weeks and the pH was kept at pH 3.8 (left panel). In addition, Lactobacillus plantarum Changes in the antimicrobial activity during normal growth of LBP-K10 were observed by a disk diffusion assay (right panel). I was.

3 is a result of the growth inhibition ability of pathogenic bacteria. Using the culture medium, Gram-positive bacteria ( B. subtilis, L. monocytogens and S. aureus ), Gram-negative bacteria ( S. typhimurium, S. sonnei and E. coli ) and pathogenic Candida albicans ( C. albicans) ) Was observed in the agar plate.

4 shows the Lactobacillus plantarum Results of thermal stability of nonproteinaceous antimicrobial agents derived from the culture of LBP-K10. Gram-positive bacteria (B. subtilis) And Gram-negative bacteria (E. coliThe antimicrobial activity of the nonproteinaceous material was similar to that of the heat-treated medium under various conditions.

Figure 5 shows the characteristics of the culture medium having the effect of a non-protein antimicrobial when treated with proteolytic enzymes. Various anti-proteinases were treated to observe antimicrobial activity. Proteinase K and chymotrypsin were treated in the culture medium, wherein the culture medium was treated with YM-1 cut-off (YM1C) and YM-1 supernatant (YM1S), and as a control, as described in the following Examples based on molecular weight 1,000. The experiment was divided.

6 is CH₂Cl₂Lactobacillus Planta Room This is the result of extracting the LBP-K10 culture solution. a is CH₂Cl₂Antibacterial activity of the supernatant after extraction with (methylene chloride), b is CH₂Cl₂Shows the antimicrobial activity of the culture supernatant, c is CH₂Cl₂It shows the antibacterial activity of the lower layer solution extracted with (methylene chloride).

FIG. 7 shows the results of semi-prep HPLC analysis of Lactobacillus plantarum LBP-K10 using ZORBAX C18 octadedosil silica hydrophobic resin. Each fraction was collected by liquid-liquid extraction. These analyzes were recorded at ultraviolet wavelengths 210, 260 and 280 nm, respectively. All fractionated materials could be divided into about 10 each. The ten fractionated materials thus collected were collected and concentrated as described in the Examples below.

8 and 9 show the antifungal activity of the substances isolated from Leukonostock Mesenteroides LBP-K06 and Lactobacillus plantarum LBP-K10. It shows the antifungal activity of the substances isolated from. Ganoderma BoniniensGanoderma boninenseMycelium) was inoculated on a PDA (potato dextrose agar) plate, and growth was observed at 28 ° C for 3 days (initial inoculated ganoderma). Mycelium size is in the shape of a circle with a diameter of 8.0 mm per well).

10 and 11 show the inhibitory activity against the opportunistic / pathogenic fungi of substances isolated from Leukonostock Mesenteroides LBP-K06 and Lactobacillus plantarum LBP-K10. Candida albicas (Candida albicans) Cells were seeded on SD agar plates, which were minimal nutrient medium, and growth was observed for 3 days at 28 ° C. (Initially inoculated Candida cells were 1 × 10 per well).⁴dog).

12 is Pediococcus pentosaceus.,Lactobacillus plantarum LBP-K10 And chromatograms of the substances fractionated in Leukonostock Mesenteroides LBP-K06. All strains are CH₂Cl₂ Fractionation was performed using extraction.

FIG. 13 is a result of chromatograms of substances fractionated from various lactic acid bacteria identified separately. Semi-prep HPLC chromatograms of the various lactic acid bacteria strains identified using the same extraction method were observed. The strains used in this experiment were Pediococcus pentosaceus MCPP, Leukonostock mesenteroides LBP-K06, Lactobacillus plantarum LBP-K10, Weilscilla sibaria (Weissella cibaria) LBP-K15, WelCella Confusa (Weissella confusaLBP-K16, Lactobacillus sakei (Lactobacillus sakei) LBP-S01, Lactobacillus plantarum / pentos (Lactobacillus plantarum / pentosLBP-S02, lactococcus lactis (Lactococcus lactisLBP-S03, lactococcus lactis (Lactococcus lactis) LBP-S06 and Staphylococcus shiuri (Staphylococcus sciuri) LBP-S07 was used.

FIG. 14 shows the results of analyzing N10 of Lactobacillus plantarum LBP-K10 using electron ionization (EI).

FIG. 15 is a result of analyzing N10 of Lactobacillus plantarum LBP-K10 using chemical ionization (CI).

FIG. 16 shows the results of analyzing N10 of Lactobacillus plantarum LBP-K10 using ¹ H-nuclear magnetic resonance method (500 MHz, in 100% DMSO).

FIG. 17 shows the results of 2D HSQC analysis using ¹ H / ¹³ C nuclear magnetic resonance (600 MHz, in 100% MeOD) at N10 of Lactobacillus plantarum LBP-K10.

18 and 19 show ¹ H / ¹ H nuclear magnetic resonance (600 MHz, 100% DMSO (FIG. 18) and 10% D ₂ O containing DMSO in N10 of Lactobacillus plantarum LBP-K10 (FIG. 19)) is the result of 2D COSY analysis.

20 and 21 show expected structural formulas of N10 in Lactobacillus plantarum LBP-K10 using X-ray diffraction.

FIG. 22 finally shows the structure of P10 isolated from Lactobacillus plantarum LBP-K10. Separate antifungal material is C ₁₄ H ₁₆ O ₂ system with the molecular formula N ₂ - was confirmed by cycloalkyl (L- phenylalanine -L- proline) (cis -cyclo (L-Phe -L-Pro).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, and the scope of the present invention according to the gist of the present invention is not limited by these examples.

Example

Materials and methods

Isolation of experimental strains and lactic acid bacteria

Various lactic acid bacteria were separated into fresh kimchi, dolmul kimchi and Chinese cabbage kimchi. Each of the prepared materials was ground with a blender, and freshly divided kimchi and dolmen kimchi with and without 5% NaCl and stored at 4 ° C, 22 ° C and 30 ° C, respectively. All plant experimental groups were sampled for 30 days at intervals of one day, and the concentration of each experimental group was appropriately diluted in sterile distilled water and plated on MRS (de Mann Rogosa Sharpe) solid medium, which is a lactic acid bacteria separation medium, and then, at 30 ° C. Incubate for about 3 days. After incubation, the well-separated cells were selected and subjected to 16s rDNA sequencing, and cultured in liquid MRS medium for comparison with genomic information.

Identification of Isolated Lactobacillus

In this experiment, lactic acid bacteria were identified by 16S rDNA sequencing to identify lactic acid bacteria isolated from fermented plants. Primers for PCR (Polymerase Chain Reaction) include primers targeting general sedative bacteria, 27F (5'-AGA GTT TGA TCM TGG CTC AG-3 '(SEQ ID NO: 1) and 1492R (5'-GGY TAC CTT) GTT ACG ACT T-3 '(SEQ ID NO: 2)) The PCR reaction was subjected to pre-denaturation for 5 minutes at 94 ° C., followed by denaturation at 94 ° C. for 1 minute. ), Annealing at 55 ° C. for 1 minute, and extension at 1 minute at 72 ° C. for 30 times, followed by extra-extension at 72 ° C. for 7 minutes, and stored at 4 ° C. 16S rDNA amplified by 0.7% agarose gel electrophoresis after polymerase chain reaction, and DNA sequencing was analyzed.The homology of 16S rDNA sequencing of selected strains was determined by NCBI's nucleotide BLAST program. Comparison was made using http://www.ncbi.nlm.nih.gov .

Medium and culture conditions

In this experiment, Lactobacillus plantarum LBP-K10 ( Lactobacillus plantarum LBP-K10) identified from Chinese cabbage kimchi was used as the main strain. The isolated Lactobacillus plantarum LBP-K10 lactic acid bacteria were passaged in MRS broth (Difco, Laboratories, Detroit, MI).

   As a lactic acid bacterium used to find antibacterial substances, Pediococcus pentosaceus (Pediococcus pentosaceus),Lactobacillus plantar roomLactobacillus plantarum) And leukonostock mesenteroides (Leuconostoc mesenteroides), And as an indicator strain for measuring antimicrobial activity, Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Salmonella typhimurium (Salmonella typhimurium), Shigella Sonei (Shigella sonnei), Staphylococcus Areus (Staphylococcus areus) And Listeria innocuaListeria innocua) And the like. The seed strains (seed inoculums) inoculated with an appropriate amount of lactic acid bacteria in MRS broth were used, and the seed strains were inoculated again to 0.1-0.2% in MRS liquid medium and then cultured at 30 ° C. for 3 days. For the antimicrobial activity test, Listeria genus (Listeria Most pathogenic strains were cultured in Luria-Bertani (LB) broth, except Listia spp. It was.

Antimicrobial Activity Assessment

In order to determine the antimicrobial activity, broth microdilution test, disk diffusion assay and antagonism test were performed.

Each experimental method is as follows.

The medium dilution method was prepared by concentrating the culture solution, and then examined the antibacterial activity of the lactic acid bacteria culture medium through serial dilution.

Antagonism is an experiment to determine the antimicrobial activity of lactic acid bacteria. After lactic acid bacteria used in the experiment for 24 hours to measure the absorbance at 600 ㎚ was adjusted to 1 and 1 ㎖ dropping in MRS medium was incubated at 30 ℃ for 24 hours. The indicator strain incubated for 3-5 hours in advance was mixed with 1% soft agar and overlayed, and then incubated at an appropriate temperature for the indicator strain for 24 hours. The presence or absence of antimicrobial activity was confirmed by confirming the growth inhibitory ring produced after the culture.

In the disk diffusion method, the culture medium and the antimicrobial material were dipped on a 6 mm paper disk (Toyo Roshi kaisha, ltd), and the indicator strain was prepared by incubating for 3-5 hours. Inoculant was inoculated with 1% of indicator strain in liquid agar in a liquid state, and then the disk was placed and incubated at the incubation temperature of the indicator strain for 24 hours. The antimicrobial activity was confirmed through the diameter (mm) of the growth resistant ring produced after the culture.

Changes of pH and Antimicrobial Activity According to Growth

Lactobacillus plantarum grown overnight LBP-K10 was inoculated at 1.0% in modified MRS broth and cultured at 30 °. After inoculation, absorbance was measured at 600 nm every 4 hours until entering the stationary phase. After centrifugation, the supernatant was obtained to measure pH and antibacterial activity. Antimicrobial activity over time was obtained by a disk diffusion method once every 24 hours for a week after inoculation.

Enzyme treatment

In order to compare the effects of proteinaceous and nonproteinaceous agents on the pathogenic bacteria, the molecular weight of 1000 and below is separated by using the acetate membrane YM-1 (Amicon, Inc., USA). Proteinase K (proteinase K, Sigma-Aldrich, Inc., USA), chymotrypsin, Boehringer Mannheim GmbH., Germany, and trypsin, Sigma-Aldrich, Inc., USA) was treated at a final concentration of 1 mg / ml to observe sensitivity. Except for proteinase K, at room temperature (25 ° C), proteins treated with proteinase K were incubated at 37 ° C for one hour and then heat treated at 95 ° C for 5 minutes to remove enzyme activity. Antimicrobial activity was measured by the disk diffusion method.

Heat treatment

Lactobacillus plantarum to investigate the effect of heat on antimicrobial substances The day 3 culture of LBP-K10 was heat treated by heating or sterilizing at 100 ° C. for 60 minutes and 120 minutes and at 121 ° C. for 15 minutes, respectively. In addition, after heating to room temperature, the culture medium that was not heat treated as a control was used as a control, the antimicrobial activity was measured by the disk diffusion method.

Preparation for the isolation of antifungal substances

Lactobacillus plantarum Modified MRS (mMRS) was used to separate antifungal material from LBP-K10. mMRS is 10 g of peptone per liter, (NH₄)₂HC₆H₅O₇ 2 g, NaC₂H₃O₂ 5 g, MnSO₄ 0.1 g, MnSO₄(H₂O) 0.05 g, K₂HPO₄ 2 g, 5 g of yeast extract and 20 g of glucose were made and glucose was isolated and sterilized. Lactobacillus plantarum LBP-K10 was incubated in mMRS for 3 days at 30 ° C and centrifuged at 8,000 rpm for 30 minutes. Experiment).

Then, the culture solution was freeze-dried and concentrated about 10 times, and then liquid-liquid extraction was performed with 5 times the concentration of CH ₂ Cl ₂ (methylene chloride). The mixture of the culture solution and CH ₂ Cl ₂ was stored at room temperature for one day, and then allowed to stand until the aqueous solution layer and the organic solvent layer were separated using a separatory funnel. When the layers were separated well, the stopper was opened, the CH ₂ Cl ₂ layer was taken out, and the organic solvent was removed at 55 ° C. using an evaporator. Aqueous solutions were also received separately and used for experiments comparing antifungal activity. The organic solvent was removed and the remaining extract (MCK10) was dissolved in an appropriate amount of distilled water and filtered through a 0.22 μm filter. This eluate was separated again by eluting with 100% methyl alcohol through an SPE cartridge using C18 SPE (Solid phase extraction; Waters Sep-pak C18 plus cartridge, Millipore Corp., Marlborou, Mass.). The SPE cartridge was activated with 100% methyl alcohol and washed with 100% distilled water before passing the liquid material to elute. The solution to be eluted was then passed through SPE and washed again with 100% distilled water. Finally, the eluted solution was collected by passing SPE colum through 100% methyl alcohol, and the organic solvent was concentrated by evaporating an organic solvent through an evaporator and then dissolved in an appropriate amount in 100% sterilized water.

Isolation of Antifungal Substances by High Performance Liquid Chromatography (HPLC) Analysis

The filtered MCK10 culture was filtered through a 0.22 μm acetate cellulose membrane for analysis of antifungal substances, and then analyzed and separated through a high-performance liquid chromatography (semi-prep HPLC system, Agilent 1200 series, USA) analysis equipment based on the filtrate. And an ODS C-18 column (octadedosyl-C18 hydrophobic semi-preparative column; 9.4 x 250 mm, Agilent, USA) was analyzed using reverse phase high pressure liquid chromatography. The temperature of the column was kept constant at 40 ° C. and the mobile phase was operated for 35 minutes under conditions of 67% tertiary distilled water, 30% methanol and 3% acetonitrile. Mobile phases were detected at each 210 nm, 260 nm and 280 nm wavelength at a flow rate of 1.5 ml / min. Each peak detected in the analysis was prepared by fractions, and the organic solvent was removed using an evaporator, and then freeze-dried to obtain a sample.

Determination of Antifungal Activity

Each fraction material separated through a high performance liquid chromatography system was collected and concentrated using a lyophilizer and antifungal activity was measured using a 6 well cell culture plate (SPL Lifescience, Korea). Antifungal activity was measured by applying two strains, Candida albicans and Ganoderma boninense , to the 6-well plate. First, 2.5 ml of SD minimal nutrient medium (2% D-glucose, 0.5% (NH ₄ ) ₂ SO ₄ , 0.17% yeast nitrogen base without amino acids and (NH ₄ ) ₂ SO ₄ ) per well: YNB; Becton Dickinson, USA) was mixed with 1.8% microagar (Difco, USA) to identify the antifungal activity against Candida albicans, and 4% PDA (Potato-Dextrose Agar, Kisanbiotech Co., Ltd.) for Ganoderma boninines. The antifungal activity was measured by using the same method as Korea, and the higher activity among the materials separated by peaks was obtained in a single peak after more detailed separation according to the aforementioned method, followed by GC-MS, elemental analysis and NMR structure analysis. Was used for the structural analysis.

Analysis by electron ionization (EI) and chemical ionization (CI)

Agilent 6890 series GC equipped with 7679 series automatic liquid sampler to observe mass numbers of purely separated materials from Lactobacillus plantarum by electron impact ionization and chemical ionization Agilent Technologies, Waldron, Germany) was used for this experiment. Mass spectrometry was also performed using a high-resolution mass spectrometer (Jeol JMS-700, Tokyo, Japan). Both gas chromatography and high magnification mass spectrometers used JMS-700 M station software (JEOL JMS-700, Tokyo, Japan).

^One H, ¹³ C, HSQC and 2D-COSY Nuclear Magnetic Resonance Analysis

A 1.0 mg sample of pure material isolated from Lactobacillus plantarum was dissolved in 0.6 ml dimethyl sulfoxide (DMSO) containing D ₂ O and D ₂ O, and then the proton and carbon structures, the bonds, and the sequence of binding were analyzed by NMR spectroscopy. It was confirmed using. The wavelength used for nuclear magnetic resonance was measured using a Bruker AVANCE-500 spectrometer equipped with a 300 K CryoProbe16 using XWIN-NMR 3.5 software. ^[20,38,39] All two-dimensional nuclear magnetic resonance methods were subjected to Bruker's standard pulse sequence. The results by nuclear magnetic resonance analysis were analyzed using the XWIN-NMR 3.5 software package (Karlsruhe, Germany). Nuclear magnetic resonance is a Z gradient, using a 5 mm broadband inverse detector (BBI) for proton nuclear magnetic resonance at a frequency of 500.13 MHz, and carbon nuclear magnetic resonance at a frequency of 125.77 MHz for the AVANCE 500 spectrometer (Bruker). -Biospin). All nuclear magnetic resonance wavelengths were recorded at 293 K in heavy water, including heavy water and DMSO. ^[40]

Structure analysis through component element analysis

An elemental analyzer (CE Instruments EA1110, EA1112) was used to analyze the constituent elements of the purely isolated material in Lactobacillus plantarum. Elemental analyzers were performed for carbon, hydrogen, oxygen and nitrogen analysis. It was also used for the explosive evaluation through oxygen balance (OB). Oxygen balance was calculated through element ratios such as carbon, hydrogen, oxygen and nitrogen.

Crystal structure analysis using X-ray diffraction

Crystal structure analysis using X-ray diffraction was performed to investigate the three-dimensional structure of the purely separated material in Lactobacillus plantarum. 20.0 mg of the separated material sample was dissolved in 35% ethyl alcohol and 65% CH ₂ Cl _2, and then left in a dehumidifying dryer to observe the crystallization process. The resulting sample crystals were obtained at a wavelength of 1.6 통해 through a Bruker Proteum 300 CCD at Beamline 6B in Pohang Accelerator Laboratory.

Experiment result

Identification of Lactic Acid Bacteria in Plants

Many kinds of lactic acid bacteria were isolated from various Korean botanical materials, gat kimchi, sedum kimchi and general kimchi. In this experiment, a total of 400 lactic acid bacteria were isolated from these plant materials, and 200 strains with antifungal activity were selected through an antagonism method. Selected strains were subjected to PCR to identify via 16S rDNA sequencing. As a result of sequencing, strains of the genus Leukonostok, Lactobacillus, Lactococcus and Weissella were isolated and identified as a whole (Table 1). Among all the materials, the genus Ryuconostock and Lactobacillus were separated, and Lactobacillus Lactobacillus was also isolated from. By time The genus Ryuconosstock was mainly isolated between 1-2 days of fermentation, and as time passed (2-3 days), Lactobacillus genus was isolated as a major lactic acid bacteria strain.

Table 1

Strain	Number of strains in plant material
	Freshly kimchi	Sedum kimchi	Kimchi
Leuconostoc spp.	93	10	28
Lactobacillus spp.	14	8	17
Lactococcus spp.	-	One	-
Way in Gela (Weisella spp.)	2	14	18

Comparison of Antimicrobial Activity of Lactobacillus Isolated and Identified

Lactic acid bacteria have antimicrobial activity by producing a variety of secondary metabolites in growth. In order to identify the most active strains among the identified lactic acid bacteria and to carry out further experiments, the antimicrobial activity of each strain was compared through the antagonism method and the broth microdilution method (Table 2). .

In addition, as a result of the antimicrobial activity experiment using the disk diffusion assay (wasteella sibaria LBP-B06 (W. cibariaLBP-B06), Lactobacillus sakei LBP-S01 (L. sakeiLBP-S01), Lactobacillus Kimchi LBP-B02 (L. kimchiiLBP-B02), Lactobacillus plantarum LBP-K10 (L. plantarumLBP-K10), Lactobacillus citreum LBP-K11 (L. citreumLBP-K11) and Leukonostock Mesenteroides LBP-K06 (L. mesenteroidesIt was observed that the antibacterial activity of LBP-K06) was very high (Table 2). As the test strain, an indicator strain used in the antibacterial activity test of lactic acid bacteria, Escherichia coli (Gram negative bacteria)Escherichia coli) And Gram-positive bacteria Bacillus subtilis (Bacillu subtilis) Was used. In addition, as shown in Figure 1, Lactobacillus plantarum among the lactic acid bacteria identified through the above experiments It was confirmed that the best activity of LBP-K10. Therefore, in this experiment, Pediococcus pentosaceus (Pediococcus pentosaceus), Leukonostock Mesenteroides LBP-K06 and Lactobacillus plantarum Future experiments were conducted using LBP-K10 as the experimental strain.

TABLE 2

Plant material	Strain	Antagonism test a	MIC b, c	Cell sorting group identified by nucleotide sequence
Freshly kimchi	LBP-B01	++	+++	Lb. sakei
	LBP-B02	++	++	L. kimchii
	LBP-B03	++	++	L. mesenteroides
	LBP-B04	++	++	L. mesenteroides
	LBP-B05	+++	++	L. paramesenteroides
	LBP-B06	+++	++	W. cibaria
Sedum kimchi	LBP-S01	++	+++	Lb. sakei
	LBP-S02	+++	+++	Lb. plantarum
	LBP-S03	++	++	Lc. lactis
	LBP-S04	++	++	L. citreum
	LBP-S05	++	++	L. citreum
	LBP-S06	+	++	L. lactis
	LBP-S08	++	++	W. hellenica
Kimchi	LBP-K01	++	+++	Lb. plantarum
	LBP-K03	++	++	L. citreum
	LBP-K04	++	-	L. citreum
	LBP-K05	++	++	L. holzapfelii
	LBP-K06	+++	++	L. mesenteroides
	LBP-K07	++	++	L. pseudomesenteroides
	LBP-K08	++	+	L. mesenteroides
	LBP-K09	++	++	Lb. brevis
	LBP-K10	+++	+++	Lb. plantarum
	LBP-K11	++	++	L. citreum
	LBP-K12	+++	++	L. mesenteroides
	LBP-K13	++	++	L. mesenteroides
	LBP-K14	++	++	L. pseudomesenteroides
	LBP-K15	+++	++	W. cibaria
	LBP-K16	++	++	W. confusa

a means diameter indicating an antimicrobial activity range (indicator strain: Bacillus subtilis). + Means diameter <15 mm, ++ means diameter <22 mm, and +++ means diameter ≧ 22 mm.

b is the minimum inhibitory concentration (MIC).

c means the magnification of the minimum inhibitory concentration. + Means 1 fold, ++ means 0.5 fold, +++ means 0.25 fold (indicator strain: Bacillus subtilis).

Isolation and Characterization of Lactobacillus Planta Room LBP-K10

The 16S rDNA sequence of Lactobacillus plantarum LBP-K10 was compared with the base sequence of NCBI using the BLAST program, and found to be 100% identical to that of Lactobacillus plantarum IMAU10173 (data not shown). In order to examine the pH change and growth curve of Lactobacillus plantarum LBP-K10 over time, the OD ₆₀₀ value was observed simultaneously for 28 hours, which showed a significant change in pH and the growth of lactic acid bacteria (left panel of FIG. 2). . Growth and pH change were inversely proportional to each other. In the case of growth curve, it was entered into the log phase from 8 hours after inoculation, and after 28 hours, it was confirmed to be in stagnant phase. After entering the plateau, it was confirmed that the pH value remained almost constant, and the growth was also constant (left panel of FIG. 2).

In order to determine the effect on the antimicrobial activity over time, the experiment was carried out by taking the culture solution for 3 days each day. As a result, activity began to appear from day 1, and continued to increase on day 2, and the highest antimicrobial activity was observed on day 3, and this activity was maintained for about 2 weeks (right panel in FIG. 2). In the case of pH, it was decreased continuously during normal growth, and the growth appeared to be around pH 4.1 before and after the stagnation period and then remained constant.

Lactobacillus plantarum In order to determine whether LBP-K10 has an antimicrobial activity against pathogenic bacteria, the antimicrobial activity was confirmed through a disk diffusion method as described above for various strains (FIG. 3). Lactobacillus plantarum performed here Culture of LBP-K10 was performed by performing a total of three independent disk diffusion method to determine the growth inhibition ring (mm) activity for each strain. Lactobacillus plantarum The culture medium of LBP-K10 was Bacillus subtilis (Bacillus subtilis), Staphylococcus aureus (Staphylococcus aureus) And Listeria innocuaListeria innocuaSalmonella typhimurium (as well as Gram-positive bacteria)Salmonella typhimuruium), Shigella Sonei (Shigella sonnei), Streptococcus pneumoniae (Streptococcus pneumonia) And Escherichia coli (Escherichia coliGram-negative bacteria such as), and Candida albicans (Candida                 albicansActivity was also observed in fungi such as) (FIG. 3).

In addition, to confirm that the antimicrobial material is stable to heat, heat was applied under various conditions, and then the antimicrobial activity test on the culture solution (FIG. 4). The heat-treated culture did not change the antimicrobial activity. The activity of the culture medium was observed using the Gram-positive bacillus Bacillus subtilis and the Gram-negative bacterium Salmonella typhimurium, and the experimental groups were similar to the control group, regardless of the conditions in which the heat was applied. It was confirmed to show an aspect. In addition, the antimicrobial activity was maintained even after heating to 121 ?? in a sterilizer for 15 minutes. As a result, it was confirmed that the material having antimicrobial activity was stable without being denatured by heat (FIG. 4).

In order to determine whether the antimicrobial substance in the culture medium is affected by protease (proteinase K, chymotrypsin and trypsin), the experiment was performed after the enzyme was treated in the culture solution (FIG. 5). The cultures were incubated for 72 hours for the experiment, and before the enzyme treatment, the cultures were cut off with acetate membrane YM-1 (Amicon, USA) to separate the proteinaceous and nonproteinaceous portions, It divided into the above. As a control group, a sample (YM1S) having a molecular weight of 1000 or more and a sample (YM1C) having a molecular weight of 1000 or less were prepared as a culture solution (CS) and an experimental group, and the final concentrations of the enzymes were treated to 1 mg / ml. . ^[55] When the protease treatment, it was confirmed that the surface strain of Bacillus subtilis SJ result antimicrobial activity of CS and YM1S was observed in Escherichia coli as a growth stop ring reduction (Fig. 5). In particular, the activity of proteinase K was almost unchanged in the control group CS, but the activity was decreased by 10.5% when treated with trypsin and 6.5% by chymotrypsin. Escherichia coli showed an antimicrobial activity of proteinase K and trypsin about 7.0% compared to the control group when compared to Bacillus subtilis (FIG. 5). Dividing by molecular weight 1000 based on the acetate membrane YM-1, it can be seen that the decrease in the antimicrobial activity in CS and YM1S is mostly due to the degradation of proteinaceous substances.

The overall antimicrobial activity was reduced, but the degree was insignificant, and heat and protease experiments showed that most of the activity was maintained, suggesting that the antimicrobial activity was a nonproteinaceous material (Fig. 4 and 5).

Isolation and Characterization of Antifungal Substances

Pure separation of the antimicrobial material of Lactobacillus plantarum was carried out with the supernatant cultured for 3 days based on various experimental results. The separation process was performed by liquid-liquid extraction using CH ₂ Cl ₂ to separate the antimicrobial material from the culture medium, and then fractionated using semi-prep HPLC. The collected fractions were collected by HPLC chromatography. It was confirmed (FIGS. 6 and 7). In this experiment, in order to carry out these experiments, a lower layer extracted broth using a CH ₂ Cl ₂ to remove the unnecessary portion such as the D- lactic acid impurities or acid in a pure separation of the antimicrobial substance, the culture supernatant was extracted with CH ₂ Cl ₂ The antimicrobial activity of the culture medium not extracted with and CH ₂ Cl ₂ was compared by liquid-liquid extraction and applied to subsequent experiments (FIG. 6).

Through this experiment, all the antimicrobial material of the culture solution was performed after the experiment with the lower layer solution after extraction of CH ₂ Cl ₂ (Fig. 6). That is, with the concentrated culture solution is not the above the described method the culture of CH ₂ Cl ₂ extracted supernatant and the lower layer liquid, and the CH ₂ Cl ₂ extract confirm the antibacterial activity test results, CH ₂ Cl ₂ extraction supernatant with CH ₂ Cl _It was observed that the antimicrobial activity of the concentrated culture broth _{2 without} extraction was similar, and the lower layer had much higher antimicrobial activity than the other two experimental groups (FIG. 6). Therefore, the separation of the antimicrobial material was found to be more efficient to experiment with the lower layer of CH ₂ Cl ₂ extraction (Fig. 6).

In addition, the experimental group prepared through such a pretreatment process was concentrated to an appropriate amount of the fractions fractionated in the semi-prep HPLC chromatogram and carried out an antimicrobial activity experiment, and identified 10 fractions in the chromatogram of Lactobacillus plantarum. And, the peaks coming out during each time period were collected by fractionation to check the antimicrobial activity of each fraction (Fig. 7 and Table 3).

TABLE 3

Indicator strain	MIC a	Fraction	Cell class identified by nucleotide sequence
Bacillus subtilis	+++	M8	Lc. mesenteroides LBP-K06
	+++	N8	Lb. plantarum LBP-K10
	+++	P8	P. pentosaceus MCPP
Escherichia collie	++	M8	Lc. mesenteroides
Staphylococcus aureus	++	M8	Lc. mesenteroides
Streptococcus pneumoniae	++	M8	Lc. mesenteroides
Shigella descenteri	++	M8	Lc.mesenteroides

a means minimum inhibitory concentration (MIC). + Means 1x, ++ means 0.5x, and +++ means 0.25x.

Results for antifungal activity are shown in FIGS. 8 to 11.

Find out the effect of fractionated pure isolates on the fungi of all strains of the common chromatogram in all fractions of Pediococcus pentosaceus, Lactobacillus plantarum, and leukonostock mesenteroides. To see, first, the effect of Ganoderma boninense on the antifungal effect was observed (Figs. 8 to 9). Ganoderma boninens pre-incubated for 10 days was prepared in the form of an 8 mm diameter circle, and 2.5 ml of PDA (potato dextrose agar) was inoculated into a 6-well plate, followed by incubation at 28 ° C. for 3 days (FIG. 8). To Figure 9). As a result, the activities of M3, M4, M6a, M7b and M10 were significantly higher, and among them, the antifungal activity of the M10 fraction was the highest (FIGS. 8 to 9). In addition, in order to examine the antifungal activity of Candida albicans in the minimal nutrient medium, the overnight culture of the seed cultures of the Candida albicans on the SD agar plate of the minimum nutrient medium was 1 × 10 ⁴ . After inoculation and incubation at 28 ° C. for 3 days, antifungal activity was observed (FIGS. 10 and 11). As a result, only the activity of the M10 fraction was observed, unlike the results of ganoderma boninines, and the activity was determined to be very high (Figs. 10 and 11).

The pH of each fractionated material was close to the neutral pH except for the acidic material identified by fractionation in the front of the chromatogram (Table 4). These results were found in Pediococcus pentosaceus, Lactobacillus plantarum and leukonostock. All showed similar trends in Mesenteroides (FIGS. 8-11, and Table 4).

Table 4

Fraction number
division	P1	P2	P3	P4	P5	P6a	P6b	P7a	P7b	P8	P9	P10
g / l	-	5.88	-	-	-	14.5	2.0	5.0	7.3	2.2	4.2	10.7
pH	-	3.8	7.6	7.9	9.6	9.2	8.9	8.8	8.6	8.5	8.2	8.1
-: No activity.

In addition, the chromatograms were analyzed by HPLC with the various lactic acid bacteria identified in this experiment based on the results of the minimum medium concentration method. Pediococcus pentosaceus, Lactobacillus plantarum and leukonostock mesenteroides The distribution of fractional substances was found to be similar (Fig. 5a). Chromatograms in other lactic acid bacteria strains also showed similar fractional trends as Pediococcus pentosaceus, Lactobacillus plantarum and leukonostock mesenteroides (FIG. 12). These results show that lactic acid bacteria grow and produce antifungal substances and the resulting metabolites are similar to each other (FIGS. 12 and 13).

Structural Analysis of Antifungal Substances

P10, N10 and M10 of Pediococcus pentosaceus, Lactobacillus plantarum and Leukonostock mesenteroides isolated by HPLC were prepared in powder form with a freeze dryer to analyze the structure of the antifungal substance. Molecular weight was determined by direct chromatography (DIP, Direct Insertion Probe) as described in Experimental Materials and Methods (Agilent 6890 series GC, Agilent Technologies, Waldron, Germany; high-resolution mass spectrometer, JEOL JMS-700, Tokyo , Japan) (GC-MS) and analyzed (Figs. 14 to 15).

The analysis was performed by electron ionization (EI) and chemical ionization (CI), and the correlation with reference values was determined. The reference values of the molecular weights of P10 and N10 were investigated at a molecular weight of 244. As a result of analysis by electron ionization (EI) and chemical ionization (CI), Pediococcus pentosaceus and Lactobacillus plantar All rooms were found to be 244 (FIGS. 14-15).

P10, N10, and M10 using 500 MHz magnetic resonance to identify more accurate molecular structures and to compare analyzes by previous experiments, electron ionization (EI) and chemical ionization (CI) The structure of was observed (FIGS. 16-21). P10 treated with 100% DMSO and P10 fractions mixed with 10% heavy water (D ₂ O) with DMSO were analyzed by ¹ H nuclear magnetic resonance method and ¹³ C nuclear magnetic resonance method (FIGS. 16 to 21).

Also¹³C /^OneH 2D HSQC (500 MHz, 100% DMSO and 10% D₂The spectra of O-containing DMSO) nuclear magnetic resonance spectroscopy were obtained from the isolates of isolated Pediococcus pentosaceus, Lactobacillus plantarum and leukonostock mesenteroides. The results for carbon-hydrogen of M10, N10 and P10^OneH nuclear magnetic resonance method¹³It was shown to be consistent with the results of previous experiments analyzed by C nuclear magnetic resonance (FIGS. 16 and 17). P10 treated with 100% DMSO purely isolated from Lactobacillus plantarum with 100% DMSO showed a typical nitrogen-hydrogen peak at 8.0 ppm (Figure 16), but 10% D₂In case of M10 containing O, the 8.0 ppm peak disappears from the nitrogen-hydrogen peak to heavy water.^OneH /¹³C NMR (600 MHz, 100% MeOD) was confirmed via HSQC spectra (FIG. 17).

Chemical shifts of P10 through ¹³ C nuclear magnetic resonance revealed 14 carbons in DMSO and DMSO samples containing 10% heavy water (FIG. 17). ^As a result of ¹³ C nuclear magnetic resonance method, the P10 fractions were consistent with the reference values for the analysis results by electron impact ionization and chemical ionization (FIG. 17).

In addition, after analyzing the samples dissolved in DMSO containing 100% DMSO and 10% D ₂ O in ¹ H / ¹ H 2D COSY nuclear magnetic resonance, the P10 and N10 structures could be expected (Figs. 18 and 19). . Based on the experimental results, elemental components were analyzed by the component analyzer, and the element ratios shown in Table 5 were obtained.

Table 5

ingredient	ratio (%)	Detected mass	Calculated number	A predicted number	Calculated mass
carbon	66.9970	163.4727	13.6227	14	168
nitrogen	11.2623	27.4800	1.9628	2	28
Hydrogen	6.3883	15.5875	15.5874	16	16
Oxygen	13.1100	31.9884	1.9992	2	32
sulfur	0.0000	0.0000	0.0000	0.0000	0.0000
total	97.7576	238.5286	-	-	244

Also, the crystal structure analysis results using X-ray diffraction and various structure analysis results were cis-cyclo (L-phenylalanine-L-proline) ( cis- cyclo (L-Phe-L) having a structure of C ₁₄ H ₁₆ O ₂ N ₂ . -Pro)) (Figs. 20 and 21). The final chemical structures of P10, N10 and M10 of Pediococcus pentosaceus, Lactobacillus plantarum and Leukonostock mesenteroides isolated by HPLC to analyze the structure of the antifungal substance are shown in FIG. 22.

ESI-CID mass spectrum m / z 245 [MH] ⁺ base peak, 153 [MH-C ₇ H ₈ ] ⁺ , 125 [MH-C ₇ H ₈ -CO] ⁺

¹ H NMR (DMSO-d ₆ ) δ 1.41-1.45 (1H at C-5 , m), 1.69 -1.74 (2H at C-4 , m), 1.98-2.03 (1H at C-5 , m), 3.01 -3.08 (2H at C-10, m), 3.24 - 3.42 (2H at C-3, m), 4.05 - 4.08 (1H at C-6, m), 4.33 - 4.35 (1H at C-9, m) , 7.17-7.27 (5H at phenyl group , m) 7.98 (1H at N-8 , s).

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

references

[1] Schillinger, U., and W. H. Holzapfel. 1996. Guidelines for manuscripts on bacteriocins of lactic acid bacteria. Int J Food Microbiol. 33: iii-v.

[2] Bottazzi, V. 1988. An introduction to rod-shaped lactic-acid bacteria. Biochimie. 70: 303-315.

[3] Atrih, A., N. Rekhif., AJ Moir., A. Lebrihi, and G. Lefebvre. 2001.Mode of action, purification and amino acid sequence of plantaricin C19, an anti-Listeria bacteriocin produced by Lactobacillus plantarum C19. Int J Food Microbiol. 68: 93-104.

[4] Ocana, VS, AA Pesce De Ruiz Holgado., And ME Nader-Maczas. Characterization of a bacteriocin-like substance produced by a vaginal Lactobacillus salivarius strain. Appl Environ Microbiol. 65: 5631-5635.

[5] Ganzle, M. G., A. Holtzel., J. Walter., G. Jung, and W. P. Hammes, 2000. Characterization of reutericyclin produced by Lactobacillus reuteri LTH2584. Appl Environ Microbiol. 66: 4325-4333.

[6] Lindgren, S. E., and W. J. Dobrogosz. 1990. Antagonistic activities of lactic-acid bacteria in food and feed fermentations. FEMS Microbiol. Rev. 87: 149-163.

[7] Niku-Paavola, ML, A. Laitila, T. Mattila-Sandholm, and A. Haikara. New types of antimicrobial compounds produced by Lactobacillus plantarum . J Appl Microbiol. 86: 29-35.

[8] Abee, T., L., Krockel., And C, Hill. 1995. Bacteriocins: modes of action and potentials in food preservation and control of food poisoning. Int J Food Microbiol. 28: 169-185.

[9] De Vuyst, L., and F. Leroy. 2007. Bacteriocins from lactic acid bacteria: production, purification, and food applications. J Mol Microbiol Biotechnol. 13: 194-199.

[10] Nes, I. F., and H. Holo. 2000. Class II antimicrobial peptides from lactic acid bacteria. Biopolymers. 55: 50-61.

[11] Nes, I. F., D. B. Diep., L. S. Havarstein., M. B. Brurberg, V. Eijsink, and H. Holo. 1996. Biosynthesis of bacteriocins in lactic acid bacteria. Antonie Van Leeuwenhoek. 70: 113-128.

[12] Nes, I. F., and J. R. Tagg. 1996. Novel lantibiotics and their pre-peptides. Antonie Van Leeuwenhoek. 69: 89-97.

[13] Parente, E., and A. Ricciardi. 1999. Production, recovery and purification of bacteriocins from lactic acid bacteria. Appl Microbiol Biotechnol. 52: 628-638.

[14] Tominaga, T., and Y. Hatakeyama. 2007. Development of innovative pediocin PA-1 by DNA shuffling among class IIa bacteriocins. Appl Environ Microbiol. 73: 5292-5299.

[15] Klaenhammer, T. R. 1993. Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiol Rev. 12: 39-85.

[16] Joerger, MC, and TR Klaenhammer. 1990. Cloning, expression, and nucleotide sequence of the Lactobacillus helveticus 481 gene encoding the bacteriocin helveticin. J Bacteriol. 172: 6339-6347.

[17] Wu, CW, LJ Yin, and ST Jiang. 2004. Purification and characterization of bacteriocin from Pediococcus pentosaceus ACCEL . J Agric Food Chem. 52: 1146-1151.

[18] Nardi, R. M., M. M. Santoro., J. S. Oliveira., A. M. Pimenta., V. P. Ferraz., L. C. Benchetrit, and J. R. Nicoli. 2005. Purification and molecular characterization of antibacterial compounds produced by Lactobacillus murinus strain L1. J Appl Microbiol. 99: 649-656.

[19] Stevens, KA, BW Sheldon., NA Klapes, and TR Klaenhammer. 1991. Nisin treatment for inactivation of Salmonella species and other gram-negative bacteria. Appl Environ Microbiol. 57: 3613-3615.

[20] Graz, M., A, Hunt., H. Jamie., G. Grant, and P. Milne. 1999. Antimicrobial activity of selected cyclic dipeptides. Pharmazie. 54: 772-775.

[21] Strom, K., S., S. Jorgen., B, Anders, and S. John. 2002. Lactobacillus plantarium Mi-LAB 393 produce the antifungal cyclic dipeptides cyclo (L-Phe-L-Pro) and cyclo (L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid. Appl Environ Microbiol. 68: 4322-4327.

[22] O'Neill, J. C., and H. E. Blackwell. 2007. Solid-phase and microwave-assisted syntheses of 2,5-diketopiperazines: small molecules with great potential. Comb Chem High Throughput Screen. 10: 857-876.

[23] Prasad, C. 1995. Bioactive cyclic dipeptides. Peptides. 16: 151-164.

[24] Rhee, K. H., K. H. Choi., C. J. Kim., And C. H. Kim. 2001. Identification of Streptomyces sp. AMLK-335 producing antibiotic substance inhibitory to VRE (vancomycin-resistant enterococci). J Micribiol Biotechnol. 11: 469-474.

[25] Rhee KH. 2002. Isolation and characterization of Streptomyces sp. KH-614 producing anti-VRE (vancomycin-resistant enterococci) antibiotics. J Gen Appl Microbiol. 48: 321-327.

[26] Stark, T., and T. Hofmann. 2005. Structures, sensory activity, and dose / response functions of 2,5-diketopiperazines in roasted cocoa nibs (Theobroma cacao). J Agric Food Chem. 53: 7222-7231.

[27] Strm, K., J. Sj. Gren., A. Broberg, and J. Schn.rer. 2002. Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo (L-Phe-L-Pro) and cyclo (L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid. Appl Environ Microbiol. 68: 4322-4327.

[28] Trabocchi, A., D. Scarpi., And A. Guarna. 2008. Structural diversity of bicyclic amino acids. Amino Acids. 34: 1-24.

[29] Ottenheijm, HC J, JDM Herscheid., GPC Kerkhoff, and TF Spende. 1976. Preparation of episulfide DKPS from ZN ²⁺ catalysed cyclization procedure. J Org Chem. 41: 3433-3438.

[30] Woo, P. C., S. K. Lau., J. L. Teng., H. Tse, and K. Y. Yuen. 2008 and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories. Clin Microbiol Infect. 14: 908-934.

[31] Holzapfel, W. H., P. Haberer., R. Geisen., J. Bj.rkroth, and U. Schillinger. 2001. Taxonomy and important features of probiotic microorganisms in food and nutrition. Am J Clin Nutr. 73: 365S-373S.

[32] Huys, G., K. D'Haene, and J. Swings. Influence of the culture medium on antibiotic susceptibility testing of food-associated lactic acid bacteria with the agar overlay disc diffusion method. Lett Appl Microbiol. 34: 402-406.

[33] Mathur, S., and R. Singh. 2005. Antibiotic resistance in food lactic acid bacteria-a review. Int J Food Microbiol. 105: 281-295.

[34] Messens, W., and V. L. De. 2002.Inhibitory substances produced by Lactobacilli isolated from sourdoughs-a review. Int J Food Microbiol. 72: 31-43.

[35] T ㆆ lgyessy, P., and J. Hrivnak. 2006. Analysis of volatiles in water using headspace solid-phase microcolumn extraction. J Chromatogr A. 1127: 295-297.

[36] Ahi, S., I. M. Reddy., A. Beotra., R. Lal., S. Jain, and T. Kaur. 2008. Sample purification procedure for confirmation of 3'-hydroxy stanozolol by gas chromatography / high resolution mass spectrometry. Indian J Pharmacol. 40: 164-169.

[37] Reynolds, W. F., and R. G. Enr. 2002. Choosing the best pulse sequences, acquisition parameters, postacquisition processing strategies, and probes for natural product structure elucidation by NMR spectroscopy. Review. J Nat Prod. 65: 221-244.

[38] Lu, TL, CS Chen., FL Yang., JM Fung., MY Chen., SS Tsay., J. Li., W. Zou, and SH Wu. 2004.Structure of a major glycolipid from Thermus oshimai NTU-063. Carbohydr Res. 339: 2593-2598.

[39] Baranovskii, AV, RP Litvinovskaya., MA Aver'kova., NB Khripach, and VA Khripach. 2007. NMR spectra of androstane analogs of brassinosteroids. Appl Spectros. 74: 642-650.

[40] Patel, S., N. Kasoju., U. Bora, and A. Goyal. 2010. Structural analysis and biomedical applications of dextran produced by a new isolate Pediococcus pentosaceus screened from biodiversity hot spot Assam. Bioresour Technol. 101: 6852-6855.

[41] Lonvaud-Funel, A., and Joyeux, A. 1993. Antagonism between lactic acid bacteria of wines: inhibition of Leuconostoc oenos by Lactobacillus plantarum and Pediococcus pentosaceus . Food Microbiology. 10: 411-419.

[42] Olasupo, N. A. 1996. Bacteriocins of Lactobacillus plantarum strains from fermented foods. Folia Microbiol (Praha). 41: 130-136.

[43] Molin, G. 2001. Probiotics in foods not containing milk or milk constituents, with special reference to Lactobacillus plantarum 299v. Am J Clin Nutr. 73: 380 S-385 S.

[44] Jamuna, M., and K. Jeevaratnam. 2004. Isolation and partial characterization of bacteriocins from Pediococcus species. Appl Microbiol Biotechnol. 65: 433-439.

[45] Gourama, H., and LB Bullerman. 1995. Inhibition of growth and aflatoxin production of Aspergillus flavus by Lactobacillus species. Journal of Food Protection. 58: 1249-1256.

[46] Kim, J., J. Chun., And HU Han. 2000. Leuconostoc kimchii sp. nov., a new species from kimchi. Int J Syst Evol Microbiol. 50: 1915-1919.

[47] Skytt, E., A. Haikara, and T. Mattila-Sandholm. 1993. Production and characterization of antibacterial compounds produced by Pediococcus damnosus and Pediococcus pentosaceus.J Appl Bacteriol. 74: 134-142.

[48] Aguilar, C., C. Vanegas, and B. Klotz. 2010. Antagonistic effect of Lactobacillus strains against Escherichia coli and Listeria monocytogenes in milk. J Dairy Res. 3: 1-8.

[49] Ganzle, M. G. 2004. Reutericyclin: biological activity, mode of action, and potential applications. Appl Microbiol Biotechnol. 64: 326-332.

[50] Bernbom, N., TR Licht., P. Saadbye., FK Vogensen, and B. N ㆈ rrung. 2006. Lactobacillus plantarum inhibits growth of Listeria monocytogenes in an in vitro continuous flow gut model, but promotes invasion of L. monocytogenes in the gut of gnotobiotic rats. Int J Food Microbiol. 108: 10-14.

[51] Loessner, M., S. Guenther., S. Steffan, and S. Scherer S. A pediocin-producing Lactobacillus plantarum strain inhibits Listeria monocytogenes in a multispecies cheese surface microbial ripening consortium. Appl Environ Microbiol. 69: 1854-1857.

[52] Van Reenen, CA, ML Chikindas., WH Van Zyl, and LM Dicks. 2003. Characterization and heterologous expression of a class IIa bacteriocin, plantaricin 423 from Lactobacillus plantarum 423, in Saccharomyces cerevisiae . Int J Food Microbiol. 81: 29-40.

[53] van Reenen, C. A, LM Dicks, and ML Chikindas. Isolation, purification and partial characterization of plantaricin 423, a bacteriocin produced by Lactobacillus plantarum . J Appl Microbiol. 84: 1131-1137.

[54] Ennahar, S., O. Assobhel, and C. Hasselmann. Inhibition of Listeria monocytogenes in a smear-surface soft cheese by Lactobacillus plantarum WHE 92, a pediocin AcH producer. J Food Prot. 61: 186-191.

[55] Pangsomboon, K., S. Bansal., GP Martin., P. Suntinanalert, S. Kaewnopparat, and T. Srichana. 2009. Further characterization of a bacteriocin produced by Lactobacillus paracasei HL32. J Appl Microbiol. 106: 1928-1940.

Claims (14)

1. Cis-bicyclo with (L- phenylalanine -L- proline) (cis-cyclo (L- Phe-L-Pro)) the specific antifungal activity against fungi including, as an active ingredient, and Kano Derma in (Genus Ganoderma) Antifungal composition, characterized in that.
2. According to claim 1, wherein the genus of genus Ganoderma is one or more genus selected from the group consisting of Ganoderma boninense, Ganoderma applanatum and Ganoderma zonatum Antifungal composition, characterized in that the fungus.
3. The antifungal composition according to claim 2, wherein the fungus of the genus Ganoderma is ganoderma boninense.
4. It contains cis-cyclo (L-Phe-L-Pro) as an active ingredient and has specific antifungal activity against the fungus of Genus Ganoderma. Agrochemical composition, characterized in that.
5. 5. The pesticide composition of claim 4, wherein the pesticide composition further comprises one or more components or additives selected from the group consisting of solvents, carriers, emulsifiers and dispersants.
6. (A) culturing the lactic acid bacteria to prepare a lactic acid bacteria culture medium; And

   (b) a method for producing an antifungal agent against fungi of genus Ganoderma, comprising the step of concentrating the cis-cyclo (L-phenylalanine-L-proline) in the culture at an effective concentration for antifungal.
7. The method of claim 6, wherein the lactic acid bacterium is selected from the group consisting of Pediococcus, Lactobacillus, Leukonostoc, Weissella, and Lactococcus. Antifungal production method, characterized in that one or more lactic acid bacteria.
8. The method of claim 7, wherein the genus Pediococcus is Pediococcus pentosaceus (Pediococcus pentosaceus), Pediococcus acidilactici (Pediococcus acidilactici), Pediococcus cellicola, Pediococcus Pediococcus claussenii, Pediococcus damnosus, Pediococcus ethanolidurans, Pediococcus inopinatus, Pediococcus parcous parcous pucous parcous Pediococcus stillesi (Pediococcus stilesii) Antifungal production method characterized in that the at least one Pediococcus genus selected from the group consisting of.
9. According to claim 7, wherein the genus Lactobacillus Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus kimchii, Lactobacillus rhamnosus (Lactobacillus rhamnosus), Lactobacillus casei (Lactobacillus casei), Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus fermentum, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus helveticus, Lactobacillus reuteri) and Lactobacillus sakei (Lactobacillus sakei) Antifungal production method, characterized in that at least one Lactobacillus genus selected from the group consisting of.
10. According to claim 7, wherein the genus Leukonostock is Leukonostoc mesenteroides (Leuconostoc mesenteroides), Leukonostoc kimchii (Leuconostoc kimchii), Leuconostoc lactis (Leuconostoc lactis), ), Leuconostoc gelidum, Leuconostoc paramesenteroides, Leuconostoc citreum, Leuconostoc pseudomesenteroides and Leuconostoc pseudomesenteroides A method for producing an antifungal agent, characterized in that it is one or more of the genus Leukonostock selected from the group consisting of Leuconostoc holzapfelii.
11. According to claim 7, wherein the genus Bacillus (Weissella cibaria), Weissella confusa (Weissella confusa) and Weissella hellenica (Weissella hellenica) is one or more Bacillus genus selected from the group consisting of. Antifungal production method characterized in that.
12. According to claim 7, wherein the genus Lactococcus is at least one selected from the group consisting of Lactococcus lactis (Lactococcus lactis), Lactococcus plantarum and Lactococcus piscium (Lactococcus piscium) Antifungal production method, characterized in that the genus Lactococcus.
13. According to claim 6, wherein the genus of the genus Ganoderma is one or more genus selected from the group consisting of Ganoderma boninense, Ganoderma applanatum and Ganoderma zonatum Antifungal production method, characterized in that the fungus.
14. The method of claim 13, wherein the genus fungus is Ganoderma boninense (Ganoderma boninense).

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