WO2013002439A1 - Composition antifongique comprenant du cis-cyclo(l-phe-l-pro), qui possède une activité antifongique spécifique contre les champignons du genre ganoderma - Google Patents

Composition antifongique comprenant du cis-cyclo(l-phe-l-pro), qui possède une activité antifongique spécifique contre les champignons du genre ganoderma Download PDF

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WO2013002439A1
WO2013002439A1 PCT/KR2011/004770 KR2011004770W WO2013002439A1 WO 2013002439 A1 WO2013002439 A1 WO 2013002439A1 KR 2011004770 W KR2011004770 W KR 2011004770W WO 2013002439 A1 WO2013002439 A1 WO 2013002439A1
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lactobacillus
genus
antifungal
pediococcus
ganoderma
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PCT/KR2011/004770
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English (en)
Korean (ko)
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강사욱
곽민규
유예
권준오
오은선
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서울대학교 산학협력단
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Priority to MYPI2013702565A priority Critical patent/MY174593A/en
Priority to PCT/KR2011/004770 priority patent/WO2013002439A1/fr
Publication of WO2013002439A1 publication Critical patent/WO2013002439A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/401—Proline; Derivatives thereof, e.g. captopril
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics

Definitions

  • 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.
  • lactic acid fermentation efficacy depends primarily on organic acid accumulation and substrate oxidation.
  • Metabolites such as acetaldehyde, diacetyl compounds, hydrogen peroxide and carbon dioxide are important for inhibiting the growth of bacteria, pathogens and rot.
  • non-protein substrates inhibit Gram-negative bacteria as well as Gram-positive and fungal bacteria.
  • 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.
  • 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.
  • LABs generally secrete extracellular antagonist compounds to combat many microorganisms and alter the extracellular environment by endogenous metabolite byproducts.
  • 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.
  • 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.
  • 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) -
  • the dipeptide of cis By clarifying, this invention was completed.
  • 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 .
  • 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.
  • 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) -
  • the dipeptide of cis By clarifying, this invention was completed.
  • Cis contained as an effective ingredient in the present invention 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.
  • 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).
  • 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.
  • 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
  • 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.
  • 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 .
  • 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.
  • the lactic acid bacteria of the leuconosstock are 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 pseudometheroides Leuconostoc pseudomesenteroides ) And leukonostock Holzappelli ( Leuconostoc holzapfelii Lactic acid bacteria of the genus
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the pesticide composition of the present invention includes an agriculturally acceptable solvent.
  • 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.
  • the pesticide composition of the present invention 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.
  • such carriers include, for example, ground natural minerals (eg kaolin, clay, talc, chalk) and ground synthetic minerals (eg highly dispersed silica, silicates).
  • the pesticide composition of the present invention 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.
  • the emulsifiers include nonionic and anionic emulsifiers (eg, polyoxyethylene fatty alcohol ethers, alkylsulfonates and arylsulfonates).
  • the pesticide composition of the present invention 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.
  • 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
  • cyclo L- phenylalanine -L- proline
  • cis -cyclo L-Phe -L-Pro
  • 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.
  • 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 kus
  • 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
  • one or more Lactobacillus genus lactic acid bacteria selected from the group consisting of. More preferably, the genus Lactobacillus is Lactobacillus plantarum 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 pseudometheroides Leuconostoc pseudomesenteroides ) And leukonostock Holzappelli ( Leuconostoc holzapfelii Lactic acid bacteria of the genus Lyukonostok selected from the group
  • 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).
  • step (b) of the present invention Separation and purification of cis- cyclo (L-phenylalanine-L-proline) in step (b) of the present invention is carried out through various known methods.
  • 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.
  • concentration of an effective concentration 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.
  • 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 ( 16 ⁇ ) at 6.82 mM (20.47 ⁇ mol / 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 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.
  • 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.
  • the present invention also provides basic data as biological agents for cis- cyclo (L-phenylalanine-L-proline) in the pesticide industry using biological agents.
  • Disc 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 )
  • disk 3 is Lactobacillus kimchi ( L. kimchii ) LBP-B02 medium containing the culture medium
  • disc 4 is Lactobacillus plantarum ( Lb. plantarum )
  • the disk 5 is Lactobacillus citreum LBP-K11 ( L.
  • LBP-K11 citreum LBP-K11
  • disk 6 is Leukonostock Mesenteroides LBP-K06 ( L. mesenteroides LBP-K06) is a disk containing the culture.
  • the left panel is Bacillus subtilis ( Bacillus subtilis )
  • the right panel shows Escherichia coli ( Escherichia coli ) It is the result using the indicator strain. All experiments were performed three times independently.
  • 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 OD 600 (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.
  • 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.
  • a is CH 2 Cl 2 Antibacterial activity of the supernatant after extraction with (methylene chloride)
  • b is CH 2 Cl 2 Shows the antimicrobial activity of the culture supernatant
  • c is CH 2 Cl 2 It 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.
  • 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). 4 dog).
  • 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 confusa LBP-K16, Lactobacillus sakei ( Lactobacillus sakei ) LBP-S01, Lactobacillus plantarum / pentos ( Lactobacillus plantarum / pentos LBP-S02, lactococcus lactis ( Lactococcus lactis LBP-S03, lactococcus lactis ( Lactococcus lac
  • 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.
  • 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).
  • 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.
  • MRS de Mann Rogosa Sharpe
  • lactic acid bacteria were identified by 16S rDNA sequencing to identify lactic acid bacteria isolated from fermented plants.
  • Primers for PCR 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.
  • 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 .
  • Lactobacillus plantarum LBP-K10 Lactobacillus plantarum LBP-K10 identified from Chinese cabbage kimchi was used as the main strain.
  • Pediococcus pentosaceus As a lactic acid bacterium used to find antibacterial substances, Pediococcus pentosaceus ( Pediococcus pentosaceus ) , Lactobacillus plantar room Lactobacillus 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 innocua Listeria innocua ) And the like.
  • Escherichia coli Escherichia coli
  • Bacillus subtilis Bacillus subtilis
  • Salmonella typhimurium Salmonella
  • seed inoculums 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.
  • Listeria genus Listeria Most pathogenic strains were cultured in Luria-Bertani (LB) broth, except Listia spp. It was.
  • 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 nm was adjusted to 1 and 1 ml dropping in MRS medium was incubated at 30 °C 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.
  • 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.
  • 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.
  • 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.
  • Lactobacillus plantarum Modified MRS was used to separate antifungal material from LBP-K10.
  • mMRS is 10 g of peptone per liter, (NH 4 ) 2 HC 6 H 5 O 7 2 g, NaC 2 H 3 O 2 5 g, MnSO 4 0.1 g, MnSO 4 (H 2 O) 0.05 g, K 2 HPO 4 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).
  • 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).
  • 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 ⁇ m 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.
  • 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.
  • the filtered MCK10 culture was filtered through a 0.22 ⁇ m 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.
  • 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.
  • 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.
  • 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.
  • 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 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 ⁇ through a Bruker Proteum 300 CCD at Beamline 6B in Pohang Accelerator Laboratory.
  • lactic acid bacteria were isolated from various Korean botanical materials, gat kimchi, sedum kimchi and general kimchi.
  • 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.
  • strains of the genus Leukonostok, Lactobacillus, Lactococcus and Weissella were isolated and identified as a whole (Table 1).
  • the genus Ryuconostock and Lactobacillus were separated, and Lactobacillus Lactobacillus was also isolated from.
  • 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.
  • Lactic acid bacteria have antimicrobial activity by producing a variety of secondary metabolites in growth.
  • the antimicrobial activity of each strain was compared through the antagonism method and the broth microdilution method (Table 2). .
  • 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.
  • Escherichia coli Gram negative bacteria
  • Bacillus subtilis Bacillu subtilis
  • 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.
  • 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).
  • 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).
  • 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).
  • 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 innocua Listeria innocua Salmonella typhimurium (as well as Gram-positive bacteria) Salmonella typhimuruium ), Shigella Sonei ( Shigella sonnei ), Streptococcus pneumoniae ( Streptococcus pneumonia ) And Escherichia coli ( Escherichia coli Gram-negative bacteria such as), and Candida albicans ( Candida albicans Activity was also observed in fungi such as) (FIG. 3).
  • 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.
  • protease proteinase K, chymotrypsin and trypsin
  • 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. .
  • CS culture solution
  • YM1C sample having a molecular weight of 1000 or less
  • 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).
  • 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
  • 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).
  • EI electron ionization
  • CI chemical ionization
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne une composition antifongique comprenant du cis-cyclo(L-Phe-L-Pro) en tant que principe actif et possédant une activité antifongique spécifique contre les champignons du genre Ganoderma. L'invention porte en outre sur une composition insecticide et sur un procédé de production d'un agent antifongique pour les champignons du genre Ganoderma. La composition de la présente invention fait ressortir une activité antifongique supérieure contre les champignons du genre Ganoderma (en particulier les champignons Ganoderma boninense) ; elle peut ainsi être appliquée dans différents domaines industriels qui nécessitent l'élimination et la prévention de champignons du genre Ganoderma, et se révèle économiquement avantageuse en termes d'utilisation grâce à son activité antifongique, même en petite quantité. En outre, l'invention porte sur une substance de base en tant qu'agent biologique concernant le cis-cyclo(L-Phe-L-Pro), appliquée à l'industrie insecticide qui utilise des agents biologiques.
PCT/KR2011/004770 2011-06-29 2011-06-29 Composition antifongique comprenant du cis-cyclo(l-phe-l-pro), qui possède une activité antifongique spécifique contre les champignons du genre ganoderma WO2013002439A1 (fr)

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MYPI2013702565A MY174593A (en) 2011-06-29 2011-06-29 Antifungal composition comprising cis-cyclo(l-phe-l-pro) having genus ganoderma fungus-specific antifungal activity
PCT/KR2011/004770 WO2013002439A1 (fr) 2011-06-29 2011-06-29 Composition antifongique comprenant du cis-cyclo(l-phe-l-pro), qui possède une activité antifongique spécifique contre les champignons du genre ganoderma

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CN110325197A (zh) * 2016-08-30 2019-10-11 (株)库恩生物 用于防止脱发或促进毛发生长的包括展示脂肪分解作用的菌株的组合物
CN110776899A (zh) * 2019-11-26 2020-02-11 西南石油大学 一种高温高盐油藏原位乳化增黏体系及其应用
EP3508568A4 (fr) * 2016-08-30 2020-04-22 Coenbio Co., Ltd. Souche de leuconostoc holzapfelii ayant pour effets de prévenir la perte des cheveux, de favoriser la pousse des cheveux ou d'améliorer la fonction sexuelle, et composition la comprenant
WO2023152569A1 (fr) * 2022-02-14 2023-08-17 Croda International Plc Adjuvants agrochimiques

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EP1386543A1 (fr) * 2001-12-20 2004-02-04 Medipharm AB Nouvelle souche bacterielle et utilisation

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EP1386543A1 (fr) * 2001-12-20 2004-02-04 Medipharm AB Nouvelle souche bacterielle et utilisation

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MAGNUSSON, J. ET AL.: "Broad and complex antifungal activity among environmental isolates of lactic acid bacteria", FEMS MICROBIOLOGY LETTERS, vol. 219, 2003, pages 129 - 135, XP002993413, DOI: doi:10.1016/S0378-1097(02)01207-7 *
SCHNURER, J. ET AL.: "Antifungal lactic acid bacteria as biopreservatives", TRENDS IN FOOD SCIENCE & TECHNOLOGY, vol. 16, 2005, pages 70 - 78, XP025324765, DOI: doi:10.1016/j.tifs.2004.02.014 *
STROM, K. ET AL.: "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", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 68, 2002, pages 4322 - 4327 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110325197A (zh) * 2016-08-30 2019-10-11 (株)库恩生物 用于防止脱发或促进毛发生长的包括展示脂肪分解作用的菌株的组合物
EP3508568A4 (fr) * 2016-08-30 2020-04-22 Coenbio Co., Ltd. Souche de leuconostoc holzapfelii ayant pour effets de prévenir la perte des cheveux, de favoriser la pousse des cheveux ou d'améliorer la fonction sexuelle, et composition la comprenant
EP3508208A4 (fr) * 2016-08-30 2020-07-29 Coenbio Co., Ltd. Composition permettant de prévenir la chute des cheveux ou de favoriser la pousse des cheveux, comprenant des souches présentant un effet de lipolyse
EP3816304A3 (fr) * 2016-08-30 2021-07-21 Coenbio Co., Ltd. Composition permettant de prévenir la chute des cheveux ou de favoriser la pousse des cheveux, comprenant des souches présentant un effet de lipolyse
CN110776899A (zh) * 2019-11-26 2020-02-11 西南石油大学 一种高温高盐油藏原位乳化增黏体系及其应用
WO2023152569A1 (fr) * 2022-02-14 2023-08-17 Croda International Plc Adjuvants agrochimiques

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