KR102095200B1 - Novel fungus Penicillium dokdoense and use thereof - Google Patents

Abstract

The present invention is a novel fungus, Penicillium, Penicillium dokdoense ) and various compositions utilizing the same, which can inhibit nitric oxide production, and thus can be used for improvement, prevention and treatment of inflammation or inflammatory diseases.

Description

New strain Penicillium dokdoense and use thereof

The present invention relates to a new Penicillium strain, Penicillium dokdoense , and to various anti-inflammatory compositions in particular with various techniques utilizing the same.

When a tissue or organ is damaged due to a wound or infection, the damaged area is healed by inflammation, which is one of the biological defense mechanisms. There are various types of inflammation, such as chronic inflammation or acute inflammation, and the occurrence factors are very diverse. In some cases, inflammation continues to be a serious disease.

When inflammation occurs, cytokines, chemokines, interferons, colony-stimulating factors, lysozymes, proteases, growth factors, eicosanoids, nitric oxides in macrophages with innate immune activity oxide, NO) and various inflammation control factors. Among the inflammation control factors produced in macrophages, nitric oxide is an important factor for signaling related to cytotoxicity or cell proliferation inhibition by bacterial, viral, and cancer cells, which are factors causing inflammation, or excessive nitric oxide due to excessive activity of macrophages Rather, there is a risk of causing various diseases.

Compositions for improving or treating inflammation by regulating the production of inflammatory regulators have been studied in various ways, such as natural compounds extracted from plants, artificial synthetic compounds, or components produced by microorganisms as active ingredients. For example, Korean Patent Publication No. 10-2016-0060884 discloses an anti-inflammatory composition using an extract of a specific tree, and Korean Patent Publication No. 10-2015-0146331 discloses an anti-inflammatory composition using a microorganism.

Republic of Korea Patent Publication No. 2016-0060884 Republic of Korea Patent Publication No. 2015-0146331

The present invention is a novel fungus, Penicillium, Penicillium dokdoense ), and provides pharmaceutical composition, health functional food, cosmetic composition, feed, antifungal agent, and antibacterial agent to prevent or treat anti-inflammatory and inflammatory diseases using the same.

The present invention is Penicillium dokdoense ) (accession number: KACC 93309p).

The present invention is Penicillium dokdoense ) (Accession number: KACC 93309p), Penicillium dokdoense ) (Accession No .: KACC 93309p) and Penicillium dokdoense ) (Accession No .: KACC 93309p) provides a pharmaceutical composition for the prevention or treatment of inflammatory and inflammatory diseases comprising at least one selected from the group consisting of culture filtrate.

The present invention is Penicillium dokdoense ) (Accession number: KACC 93309p), Penicillium dokdoense ) (Accession No .: KACC 93309p) and Penicillium dokdoense ) (Accession number: KACC 93309p) provides an anti-inflammatory health functional food composition comprising at least one selected from the group consisting of culture filtrate.

The present invention is Penicillium dokdoense ) (Accession number: KACC 93309p), Penicillium dokdoense ) (Accession No .: KACC 93309p) and Penicillium dokdoense ) (Accession number: KACC 93309p) provides an anti-inflammatory cosmetic composition comprising at least one selected from the group consisting of culture filtrate.

The present invention is Penicillium dokdoense ) (Accession number: KACC 93309p), Penicillium dokdoense ) (Accession No .: KACC 93309p) and Penicillium dokdoense ) (Accession number: KACC 93309p) provides an antifungal or antibacterial pharmaceutical composition comprising at least one selected from the group consisting of culture filtrate.

Penicillium , a novel fungus of the present invention dokdoense ) (Accession No .: KACC 93309p), its culture fluid or its culture filtrate can control nitric oxide production to effectively treat and prevent inflammation, as well as show proteolytic activity, antifungal activity and antibacterial It is expected to be used for the treatment and prevention of diseases related to this activity.

Figure 1 is a new fungus Penicillium Dokdoens ( Penicillium) dokdoense ) Phylogenetic tree of CNUFC-DDS11-1 and CNUFC-DDS11-2 strains, constructed using a complex data set for BenA and CaM by Citrina section-based the maximum likelihood analysis, Penicillium Corylophilum was used as an outgroup. The number of nodes (nodes) represents the bootstrap value (over 50%) of 1000 replications, the new taxonomic group is indicated in blue, and the strain used as a standard strain (ex-type) is shown in bold.
2 is a new fungus, Penicillium, Penicillium dokdoense ) shows the morphological structure of the CNUFC-DDS11-1 (holotype) strain (ad: obverse view, eh: reverse view). [a and e are colonies in yeast extract sucrose agar (YES) medium, b and f are colonies in malt extract agar (MEA) medium, c and g are colonies in Czapek yeast autolysate agar (CYA) medium, d and h shows colonies in creatine sucrose agar (CREA) medium, i ~ l shows verticillate conidiophore and phialide conidia, and n shows conidia. The scale bar is 20 µm for i, 10 µm for j to l, 5 µm for m, and 3 µm for n.
3 is Penicillium dokdoense ) shows cell proliferation of macrophages RAW264.7 according to CNUFC-DDS11-1 culture filtrate concentrations (50 μl / ml, 100 μl / ml).
4 is Penicillium dokdoense ) CNUFC-DDS11-1 shows the inhibitory effect of nitric oxide (NO) production by the culture filtrate.
5 is Penicillium dokdoense ) (CNUFC-DDS11-1) shows the effect of reducing free radicals by the culture filtrate (MFI: mean fluorescence intensity).
6 is Penicillium dokdoense ) CNUFC-DDS11-1 shows the effect of reducing the expression of TNF-α and IL-1β by the culture filtrate.
7 is Penicillium dokdoense ) It shows the enzyme activity, antifungal activity and antibacterial activity of CNUFC-DDS11-1.
A) Clear zone around colonies formed by culturing Penicillium Dokdoensu CNUFC-DDS11-1 in skim milk agar, B) Alternaria Alternaria alternata ) antifungal activity of penicillium dokone CNUFC-DDS11-1 against EML-BLDF1-4, and C) penicillium dokone CNUFC-DDS11-1 against Staphylococcus aureus ATCC Antibacterial activity.

Hereinafter, the present invention will be described in detail.

Unless otherwise defined, terms used in this specification should be interpreted as contents generally understood by a person having ordinary skill in the relevant field. The drawings and examples in the present specification are for those skilled in the art to easily understand and implement the present invention. In the drawings and examples, contents that may obscure the subject matter of the invention may be omitted, and the present invention is limited to the drawings and examples. It does not work.

The present invention is a novel fungus, Penicillium, Penicillium dokdoense ) (accession number: KACC 93309p) and various uses thereof. In particular, Penicillium of the present invention dokdoense ) is very effective for use in antifungal or antibacterial, including prevention, improvement and treatment of anti-inflammatory and inflammatory diseases.

Penicillium , a novel fungus of the present invention dokdoense ) is separated and identified from soil collected from the East Sea (eastern region) of Dokdo located in the East Sea of Korea, and Penicillium dokdoense ) Named CNUFC-DDS11-1 and CNUFC-DDS11-2, deposited on July 23, 2018 with the Depository No. KACC 93309p to the Microorganism Bank of Korea (KACC).

Penicillium , a novel fungus of the present invention dokdoense ) has a high correlation with P. terrigenum and P. copticola , but it shows slow growth in CYA medium and rapid growth in MEA medium, which is distinct from these. In addition, from the morphological point of view, Penicillium Dokdoen's is the number of P. terrigenum and P. copticola of Metulae and Phialide per base . More, P. terrigenum and P. copticola produced divaricate conidiophores that do not appear, and conidia have distinct features from spherical to hemispherical or elliptic.

Penicillium of the present invention dokdoense ) has a variety of biologically active effects, can be produced in large quantities of a specific substance can be utilized as a variety of compositions, such as pharmaceutical compositions, food, cosmetic, feed, antibiotics. In particular, the penicillium doxens and substances produced therefrom have a function to suppress or control inflammation, and thus can be used for prevention, treatment, and improvement of various anti-inflammatory compositions and inflammatory diseases.

According to an embodiment of the present invention, Penicillium dokdoense (Accession No .: KACC 93309p) is not only a cell itself, but also a culture medium of Penicillium dokdoense (Accession No .: KACC 93309p), Penny The culture filtrate obtained by filtering the culture solution of Penicillium dokdoense (Accession No: KACC 93309p) may also have various biological activity effects.

In addition, penicillium doxens and its culture broth or culture filtrate has enzyme activity capable of decomposing sugars and proteins. For example, cellulose, hemicellulose, starch and protein can be decomposed through such enzyme activity. However, the scope of enzyme activity is not limited thereto.

The enzyme having proteolytic activity among the enzymes contained in the penicillium doxens and its culture medium, or the culture filtrate, is used in the food industry for cheese ripening, protein hydrolyzate production, and bread production. It is considered an important enzyme activity in the industry. Studies on various types of enzymes of Penicillium spp. Have been conducted to date, but few studies have been reported on proteolytic enzymes.

Penicillium , a novel fungus of the present invention dokdoense ) has antifungal or antibacterial activity, and can be used as various compositions such as pharmaceutical compositions, food, cosmetics, feed, and antibiotics.

According to an embodiment of the present invention, Penicillium dokdoense CNUFC-DDS11-1 was newly confirmed to exhibit decomposition activity against proteins, and is a new penicillium species belonging to the section Citrina. Penicillium dokdoense ) CNUFC-DDS11-1 suggested the potential for enzyme production.

Penicillium according to an embodiment of the present invention dokdoense ) CNUFC-DDS11-1 is an Alternaria that produces four mycotoxins, ALT, AOH, ATX-1, and AME. alternata ) EML-BLDF1-4 was confirmed to have antifungal activity.

Penicillium according to an embodiment of the present invention dokdoense ) It was confirmed that the organic solvent extract of CNUFC-DDS11-1 has antibacterial activity against the pathogen Staphylococcus aureus ATCC.

In addition to the culture medium and the filtrate of the culture medium, a lyophilized powder of the culture medium, a lyophilized powder of the culture filtrate, or a solution in which the lyophilized powder is dissolved may be used in various fields.

In the present invention, the culture medium may include a substance produced by the cell, the cell itself, and the medium by culturing the cell in the medium.

In the present invention, the culture filtrate may include a supernatant obtained by centrifuging the culture solution, and a liquid obtained by filtering the supernatant.

Various compositions, methods of manufacture of food of the present invention penny room Solarium islets Enschede (Penicillium dokdoense) (Accession No: KACC 93309p) the culturing in the culture medium and Penny room Solarium islets Enschede (Penicillium dokdoense ) (Accession number: KACC 93309p) may be obtained by culturing to obtain a culture medium and may be formulated.

The method for preparing a composition of the present invention may further include the step of formulating the supernatant by centrifuging the culture medium to obtain a supernatant of the culture medium in which the layer separation has occurred.

The method for preparing the composition of the present invention may further include the step of formulating a culture filtrate obtained by filtering the supernatant of the culture medium in which the layer separation has occurred by centrifuging the culture medium.

Penicillium in the present invention ( Penicillium The medium for cultivating dokdoense (accession number: KACC 93309p) is in MEA (Blakeslee's malt extract agar) medium, YES (yeast extract sucrose agar) medium, CREA (Czapek yeast autolysate agar) medium, and PDA (potato dextrose agar) medium. The medium may be selected, but is not limited thereto. According to an embodiment of the present invention is preferably MEA medium, growth may be slow in CYA and YES medium.

The composition according to the present invention may be particularly effective in inhibiting and regulating the production of inflammatory cytokines, reactive oxygen species (ROS) and nitric oxide (NO). Specifically, according to the present invention, penicillium dokone, its culture medium or its filtrate can effectively suppress TNF-α and IL-1β, cytokines whose expression is increased by inflammation, resulting in excessive inflammation. And it can effectively treat and prevent various diseases that can progress from it. In addition, according to the present invention, penicillium doxens, a culture solution thereof, or a filtrate thereof can effectively inhibit nitric oxide and reactive oxygen species and control their production, and thus may be particularly effective in inhibiting inflammatory reactions. Prevention and treatment of various inflammatory diseases may also be possible.

In the present invention, the inflammatory disease is not particularly limited as long as it is an inflammatory disease associated with inflammatory cytokines such as nitric oxide. Preferably atopy, asthma, gastric ulcer, gastritis, hepatitis, crohn's disease, cystitis, nephritis, rheumatism, arthritis, pneumonia, inflammatory allergies, conjunctivitis, rhinitis, otitis media, sore throat, gout, tendinitis, myositis, acute It may be any one selected from the group consisting of inflammatory diseases and chronic inflammatory diseases, but is not limited thereto.

When preparing a dietary supplement or a food additive in the composition according to the present invention, its form or type is not particularly limited and may be prepared by mixing with other ingredients capable of increasing anti-inflammatory activity. There is no particular limitation on the type of food, and for example, it may include foods commonly produced and sold, such as drinks, vitamins, various beverages, processed meats, processed sausages, processed noodles, processed fats and oils, candy and gum.

Preferably, it is good to prepare it as a drink agent or other beverage form composition, and it may contain various flavoring agents such as sweeteners as additional ingredients to help intake. Various flavors such as monosaccharides, fructose, and glucose may include carbohydrates and sugar alcohols such as sorbitol and xylitol. Examples of sweeteners include not only natural sweeteners, but also synthetic sweeteners such as saccharin.

In the case of preparing the pharmaceutical composition of the composition according to the present invention, its form or type is not particularly limited and may be prepared in various dosage forms depending on the method of administration or intake. In addition, when preparing pharmaceutical compositions, excipients, carriers or diluents commonly used in the field may be further included. As an example of the formulation, various formulations such as an external preparation or an injection may be possible in addition to oral forms such as tablets, syrups, capsules, and powders. Examples of carriers or excipients include various compounds including sugar alcohols such as sorbitol, xylitol or maltiol, monosaccharides, polysaccharides, gelatin, water, alcohol, starch, edible rubbers, mineral oils, and mixtures thereof. The type of wetting agents, surfactants, excipients, fillers or binders generally used in formulation is also not particularly limited.

Hereinafter, the present invention will be described in more detail through examples, and the present invention is not limited by the following examples.

Identification of fungi

Example 1-1. Sampling and separation for identification of new fungi

A soil sample of 10 to 15 cm in depth was collected from Dongdo on Dokdo Island located in the East Sea of Korea. The collected sample was transferred to a sterilized 50 ml conical tube and stored at 4 ° C. Fungi were separated by a serial dilution plating method. Specifically, 1 g of sample soil was mixed with 9 ml of sterile distilled water, shaken for 15 minutes at room temperature, and then diluted stepwise from 10 ^-1 to 10 ^-4 . 0.1 ml of each dilution was transferred to potato glucose agar medium (PDA medium) (39 g potato dextrose agar [Becton, Dickinson, and Co., Sparks, MD, USA]) and incubated at 25 ° C. for 3 to 7 days. To isolate pure cultures, individual colonies of various types were transferred to PDA plates. The purely isolated strain was stored in 20% glycerol at -80 ° C at the Chonnam National University Fungal Collection (CNUFC) in Gwangju, Korea, and used as CNUFC-DDS11-1 and CNUFC-DDS11-2.

Example 1-2. DNA extraction, PCR and sequencing

Genomic DNA was directly extracted from the isolated mycelia of mycelia using Solgent Genomic DNA prep kit (Solgent Co. Ltd., Daejeon, Korea). The internal transcribed spacer (rDNA ITS) using primer pairs ITS1 / ITS4 (White et al, 1990), Bt2a / Bt2b (Glass & Donaldson 1995) and Cmd5 / Cmd6 (Samson et al, 2014) S1-5.8S-ITS2 The region, BenA partial gene encoding beta tubulin and the CaM partial gene encoding calmodulin were amplified and used (Table 1).

Each PCR amplification mixture (total volume, 20 μl) contained 2 μl of strain DNA template (10 ng), 1.5 μl of each primer (5 pM / μl); 1 μl of Accupower®PCR premix (Bioneer Corp., Daejeon, Korea) and 14 μl of sterile water containing Taq DNA polymerase, dNTP, buffer and tracking dye. The PCR product was purified with Accuprep® PCR purification kit (Bioneer) according to the manufacturer's instructions. DNA sequencing was performed using an ABI 3700 automated DNA sequencer (Applied Biosystems Inc., Foster City, CA, USA).

[Table 1] PCR  Used for amplification and sequencing primer

Example  1-3. Phylogenetic analysis

Clustal_X v.1.83 (Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment) for sequencing results and sequence data obtained from the GenBank database (see [Table 2]) aided by quality analysis tools.Nucleic Acids Research: 4876-82) and Bioedit v. 5.0.9.1 software (Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98 / NT.Nucleic.Acids Symp Ser 1999; 41: 95-8). Phylogenetic analysis was performed using MEGA 6 software (Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution 30: 2725-2729).

Phylogenetic counts, including CNUFC-DDS11-1, CNUFC-DDS11-2, and Citrina section related species, were determined using a complex data set for BenA and CaM by the maximum likelihood analysis. Constituent, Penicillium corylophilum The sequence was selected out group.

Sequence matching rates were obtained by Basic Local Alignment Search Tool (BLASTn) (NCBI) search for each strain. The reliability of the internal branch was evaluated using a p-distance alternative model using 1000 bootstrap replicates.

Taxon name Collection no.
(Strain no.) GenBank accession No. BenA CaM P. anatolicum NRRL5820 (T) P. anatolicum CBS47966 JN606849 JN606571 P. anatolicum CBS47866 JN606848 JN606570 P. argentinense CBS130371 (T) JN606815 JN606549 P. argentinense DTO18B1 JN606809 JN606552 P. argentinense DTO 132D5 JN606806 JN606542 P. copticola CBS127355 (T) JN606817 JN606553 P. copticola CBS127356 JN606805 JN606532 P. dokdoense CNUFC - DDS11 -One MH243037 MH243031 P. dokdoense CNUFC - DDS11 -2 MH243038 MH243032 P. euglaucum CBS323.71 (T) JN606856 JN606550 P. euglaucum DTO16G1 JN606808 JN606550 P. gallaicum CBS167.81 (T) JN606837 JN606548 P. gallaicum CBS41869 JN606845 JN606567 P. gallaicum CBS 16481 JN606836 JN606547 P. paxilli CBS360.48 (T) JN606844 JN606566 P. paxilli CBS547.77 JN606857 JN606574 P. paxilli CBS162.96 JN606804 JN606545 P. paxilli CBS 117191 JN606831 JN606535 P. roseopurpureum CBS266.29 (T) JN606838 JN606556 P. roseopurpureum CBS281.39 JN606839 JN606557 P. roseopurpureum CBS 127027 JN606824 P. roseopurpureum CBS 127028 JN606825 P. sanguifluum CBS 14883 JN606835 P. sanguifluum CBS 127032 (T) JN606819 JN617681 P. sanguifluum CBS 118020 JN606832 JN606536 P. sanguifluum CV 1865 JX141021 JX157340 P. shearii CBS290.48 (T) JN606840 JN606560 P. shearii CBS578.70 JN606852 JN606575 P. shearii CBS 51373 JN606851 JN606573 P. shearii CBS 118059 JN606834 JN606538 P. terrigenum CBS127354 (T) JN606810 JN606583 P. terrigenum JM1453 KP235308 KP235298 P. terrigenum JM439 KP235309 KP235297 P. cf. terrigenum DTO 19H8 JN606818 JN606554 P. corylophilum CBS 330.79 GU944519 GU944607

Example  1-4. Morphological analysis

The strains isolated for detailed morphology studies were Blakeslee's malt extract agar (MEA; Blakeslee, 1915), yeast extract sucrose agar (YES; Frisvad, 1981), Czapek yeast autolysate agar (CYA; Pitt, 1979), and Cultured in creatine sucrose agar (CREA; Frisvad 1981). Plates (3 replicates for each medium) were inoculated at three-point. After incubation for 7 days, growth was confirmed using Rayner's color charts (the color charts of Rayner (1970)) at 5 ° C, 10 ° C, 15 ° C, 20 ° C, 25 ° C, 30 ° C, 35 ° C, and 37 ° C in the dark. The sample was mounted on a lactophenol solution (Junsei Chemical Co. Ltd., Tokyo, Japan) and observed with an Olympus BX51 microscope equipped with a DIC optical device (Olympus, Tokyo, Japan).

The micro-homogenous structure was observed with a scanning electron microscope (SEM) (Hitachi S4700; Hitachi, Tokyo, Japan). The isolated strain was fixed in 2.5% paraformaldehyde-glutaraldehyde in 0.05M phosphate buffer (pH 7.2) for 2 hours, and then washed with cacodylate buffer (Junsei Chemical Co. Ltd.) Did. Cell membranes were preserved by immobilizing the sample in 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA, USA) diluted with chacodil hydrochloric acid for 1 hour. The sample was washed again with a carcodil hydrochloric acid buffer, dehydrated with ethanol (graded ethanol) (Emsure, Darmstadt, Germany) and isoamyl acetate (Junsei Chemical Co. Ltd.) and dried in a fume hood Ordered. Finally, the samples were sputter coated with gold and observed using a Hitachi S4700 field emission scanning electron microscope (FE-SEM) at the Korea Basic Science Institute.

Example  1-5. Phylogenetic analysis result

Analysis of the related strain phylogenetic relationship of the new strain was conducted through analysis of phylogenetic relationship in the ITS, BenA, and CaM genes between the new strain and related species. According to the results of s rDNA sequence comparison with the related species extracted from GeneBank, Penicillium dokdoense ) Deposited in the NCBI database with CNUFC-DDS11-1 and CNUFC-DDS11-2, and analyzed using this (see [Table 2]). Analysis was performed using ITS (SEQ ID NO: 1), BenA (SEQ ID NO: 2) and CaM (SEQ ID NO: 3) genes.

When ITS rDNA sequences of CNUFC-DDS11-1 and CNUFC-DDS11-2 strains were compared with related species extracted from GenBank, sequencing results by BLASTn search showed that CNUFC-DDS11-1 and CNUFC-DDS11-2 were Penicillium sp . E3 (GenBank accession number FJ471589) and 98.4% (548/557 bp) showed the closest association.

BenA sequences of P. terrigenum MUT 5749 and P. terrigenum CBS 127345 showed sequence homology of 96.9% (444/458 bp) and 96.8% (429/443 bp) with BenA of isolates, respectively. The CaM sequences of P. terrigenum DTO 9D4 and JM439 showed sequence homology with 97.1% (505/520 bp) and 96.4% (396/411 bp) of CaM of isolates, respectively.

Phylogenetic trees were deduced from the data set for the relevant species (FIG. 1) belonging to the Citrina section extracted from BenA and CaM and GenBank of CNUFC-DDS11-1 and CNUFC-DDS11-2. Phylogenetic analysis put the isolated fungi in the Citrina section, and the isolated fungi formed separate clades to separate the isolated fungi from the Penicillium. Showed that it was paper.

New strains by slower growth in CYA medium was separated from P. terrigenum and P. copticola, P. terrigenum showed a rapid growth in YES and CYA.

The number of fialides per basal etiology (Metulae) and basal erythema was much higher than that observed in P. terrigenum and P. copticola . In addition, the new strain produced a double divaricate conidia that is not in P. terrigenum and P. copticola . The conidia form of the new strain was distinguished from the elliptical form P. terrigenum from spherical to hemispherical or elliptical.

Comparison of the characteristics of the new strain and P. terrigenum and P. copticola could be summarized as in [Table 3] below.

New strains and P. terrigenum  And P. copticola of  Characteristics comparison Character New strain P. copticola  ^a P. terrigenum  ^b Conidiophores Biverticillate, monoverticillate, or divaricate Biverticillate, and monoverticillate Biverticillate Phialides Ampuliform, 3 - 9 per metula, 6.7 - 11.5 × 2.0 - 3.5 μm
Ampulliform to cylindrical, 7.5-9 × 2.0-3.0 μm Ampulliform to cylindrical, 7.5-9 × 2.5-3.5 μm Metulae 2 - 5 per stipe, 10.5 - 17.5 × 2.5 - 4.2 μm 2-4 per stipe, 12-16 × 2.0-3.5 μm 3-7 per stipe, (10-) 12-16 × 2.0-3.5 μm Conidia Rough-walled, globose to subglobose, ellipsoidal, 2.0 - 3.5 μm × 2.0 - 3.0 μm. Broadly ellipsoidal, smooth, 2.5-3.0 × 2.0-2.5 μm Broadly ellipsoidal, smooth, 2.0-3.0 × 2.0-2.5 μm Sclerotia Absent Absent Absent Reverse color on CYA Yellowish white with yellow brown in center. Pale beige or creme Pale or creme Colonies grown on CYA at 25 ^o C 23.5-26 mm 31-37 mm 28-36 mm Colonies grown on CYA at 37 ^o C No growth No growth No growth

Penicillium, a new fungus dokdoense ) Named CNUFC-DDS11-1 and CNUFC-DDS11-2, of which Penicillium dokdoense ) CNUFC-DDS11-1 was deposited with the Agribusiness Administration Microbial Bank (KACC).

2-1. new Penicillium Dokdo Nance ( Penicillium dokdoense ) CNUFC - DDS11 Confirm anti-inflammatory effect of -1

Confirmation of the anti-inflammatory effect of the new penicillium doxens was confirmed by macrophages stimulated and activated by the endotoxin substance LPS (lipopolysaccharide). The anti-inflammatory effect was confirmed by measuring nitric oxide (NO), free radicals, TNF-α and IL-1β.

Dulbecco's modified Eagle's medium (DMEM), antimyotic-antibiotics, fetal bovine serum (FBS), and lipopolysaccharide (LPS), MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2 -(4-sulfophenyl) -2H-tetrazolium) was purchased from Invitrogen-Gibco (New York, USA) and the rest of the chemicals were purchased from Merck, Sigma or Junsei.

Macrophage RAW 264.7 cells were seeded in 96 well-plates at 2 × 10 ⁴ cells / well and incubated at 37 ° C. in humid atmosphere at 5% CO for 24 hours. After 24 hours, 50 μL of the medium and 50 μL of the diagnostic reagents containing MTS and PMS [mix ratio 20: 1 (MTS: PMS)] were mixed. 100 μl of the supernatant was transferred three times to a 96-well plate and the absorbance of the formed formazan was measured at 490 nm with a microplate reader.

Nitric oxide (NO) was measured by analyzing the level of nitrite using the Griess reagent system. Specifically, it is as follows. RAW 264.7 cells were seeded in 12 well-plates at a density of 2 × 10 ⁵ cells / well and stimulated with LPS (500 ng / ml) for 24 hours, followed by 30 μl of cell culture supernatant with the same volume of Griess reagent in a 96-well plate. Was incubated at room temperature for 10 minutes. A nitrite standard (0.1M sodium nitrite) was prepared according to the Griess reagent system, and the absorbance was measured at 540 nm using a microplate reader. Two experiments were conducted in three replicates.

Reactive oxygen species (ROS) were measured using DCFDA (2 ′ 7′diacetate). Specifically, RAW 264.7 cells were seeded in 12 well-plates at a density of 2 × 10 ⁵ cells / well and stimulated with LPS (500 ng / ml) for 24 hours, the medium was removed, and the cells were treated with 10 μM DCFDA for 30 minutes in the dark. Cultured at 37 ° C. Cells were then washed, re-suspensioned in FACs buffer and analyzed with a FACs Calibur flow cytometer.

TNF-α and IL-1β were measured by isolating total RNA using TRISOL reagent, and synthesizing cDNA using the isolated RNA, followed by RT-PCR (real time polymerase chain reaction). Primers used for RT-PCR are shown in [Table 4] below.

[Table 4]

All measurements are expressed as the mean of 3 repeat measurements ± standard error. Statistical analysis was performed by ANOVA (analysis of variance) by Tukey's test.

Among the results of confirming the anti-inflammatory effect of penicillium dokone using macrophages, in terms of cytotoxicity, penicillium dokone showed no cytotoxicity against macrophage RAW 264.7 at 50 μg / mL (based on culture supernatant) and 100 μg / mL Showed a slight cytotoxicity against macrophage RAW 264.7 (Fig. 3).

When treating the culture filtrate of Penicillium Dokdoen's with LPS-stimulated macrophages RAW 264.7, the level of nitrite was 7.5 μM, which confirmed the activity of inhibiting NO production (FIG. 4).

When the culture filtrate of Penicillium Dokdoen's was treated with LPS-stimulated macrophage RAW 264.7, it was confirmed that the production of ROS was significantly reduced (FIG. 5).

It was confirmed that the mRNA expression of TNF-α and IL-1β significantly increased when LPS alone was treated when the culture filtrate of Penicillium Dokdoen's was treated with LPS-stimulated macrophage RAW 264.7 (FIG. 6).

2-2. new Penicillium Dokdo Nance ( Penicillium dokdoense ) CNUFC - DDS11 Extraction of active compound from -1

Penicillium dokdoensu CNUFC-DDS11 of the present invention was cultured in a potato dextrose broth (PDB) at 25 ° C for 10 days. After incubation, the culture medium was replaced with Whatman filter paper No. Filtered by 2. The culture filtrate was extracted three times at room temperature with 150 mL of the volume of ethyl acetate (EtOAc). The ethyl acetate layer was concentrated at 40 ° C. in a rotary evaporator to remove the ethyl acetate solvent. Evaporation of the organic extract gave 0.04 g.

2-3. new Penicillium Dokdo Nance ( Penicillium dokdoense ) CNUFC - DDS11 -1 proteolytic activity ( proteolytic  activity)

The agar plate containing 2% agar (agar) was inoculated with the Penicillium Dokdoens CNUFC-DDS11-1 strain of the present invention, and supplemented with 2% skim milk. Plates were incubated at 25 ° C for 4 days (3-5 days).

The screening results for proteolytic activity of the new Penicillium dokdoence are shown in Figure 7A. From this, the penicillium doxens of the present invention produced proteolytic enzymes after incubation for 24 hours, which was confirmed from a clear zone surrounding the colony.

2-4. new Penicillium Dokdo Nance ( Penicillium dokdoense ) CNUFC - DDS11 -1 antifungal activity ( antifungal  activity)

The antifungal activity of Penicillium Dokdoen's CNUFC-DDS11-1 of the present invention was measured by a dual culture method in PDA (potato dextrose agar) medium.

Alternaria , a fungal pathogen alternata ) The mycelial plug of EML-BLDF1-4 was located in the center of the PDA plate, and the Penicillium Dokdoen's CNUFC-DDS11-1 culture of the present invention was plated 2.5 cm from the pathogenic fungus.

All plates were cultured for 6 days (5-7 days) in a dark room at 25 ° C, and each experiment was repeated three times.

Subsequently, the width of the inhibition zone between the pathogen and the Penicillium Dokdoensu CNUFC-DDS11-1 strain was measured in millimeters and analyzed as antifungal activity.

The screening results for the antifungal activity of the new Penicillium dokdoensu are shown in Figure 7B.

From this, it was confirmed that the penicillium dokone of the present invention has an antifungal activity that inhibits mycelial growth of the fungal pathogen Alternaria alternata EML-BLDF1-4.

2-5. new Penicillium Dokdo Nance ( Penicillium dokdoense ) CNUFC - DDS11 -1 antimicrobial activity

The antibacterial activity of Penicillium Dokdoen's CNUFC-DDS11-1 of the present invention was measured by the paper disc method for Staphylococcus aureus ATCC, a pathogen.

A fresh culture solution of the pathogen was inoculated on the surface of a LB (luria-bertani) agar medium, and 0.10 mg / disc (0.05 to 0.25 mg / disc), which is a test concentration of the compound dissolved in acetone, was sterilized on a paper disk having a diameter of 6 mm. Was loaded.

The inoculated plate was cultured at 25 ° C. for 24 hours, the experiment was repeated 3 times, and the growth inhibition zone around the paper disc was measured and recorded.

The screening results for the antibacterial activity of the new Penicillium dokdoensu is shown in Figure 7B.

From this, ethyl acetate extracts of Staphylococcus aureus Gram-positive bacteria that cause human disease from Penny room Solarium islets Enschede of the present invention (Staphylocuccus aureus ) It was confirmed that there is an antibacterial activity that inhibits growth against ATCC.

Microbiological Bank of the National Academy of Agricultural Science KACC93309 20180723

<110> University Industry Liaison Office Of Chonnam National University <120> Novel fungus Penicillium dokdoense and use thereof <130> P18030120190 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 576 <212> DNA <213> Artificial Sequence <220> <223> MG906868 Penicillium dokdoense CNUFC-DDS11-1 sp. nov. [ITS          region] <400> 1 aggatctata ccgcagtaga gagacctctg ggtccagcct cccacccgtg ttttccgaac 60 cctgttgctt cggcgggccc gcctcacggc cgccgggggg cttctgcccc cgggcccgcg 120 cccgccgaag acacctgtga acgctgtctg aagttgcagt ctgagaaact agctaaatta 180 gttaaaactt tcaacaacgg atctcttggt tccggcatcg atgaagaacg cagcgaaatg 240 cgataactaa tgtgaattgc agaattcagt gaatcatcga gtctttgaac gcacattgcg 300 ccctctggta ttccggaggg catgcctgtc cgagcgtcat tgctgccctc aagcacggct 360 tgtgtgttgg gcccccgccc ccccccgcac cgggggggcg ggcccgaaag gcagcggcgg 420 caccgcgtcc ggtcctcgag cgtatggggc ttcgtcaccc gctcttgtag gcccggccgg 480 cgccagccga ccccccctca atctattttt tcaggttgac ctcggatcag gtagggatac 540 ccgctgaact taagcatatc ataagccggc aggaaa 576 <210> 2 <211> 452 <212> DNA <213> Artificial Sequence <220> <223> MH243037 Penicillium dokdoense CNUFC-DDS11-1 sp. nov. (BenA gene          [encoding beta tubulin]) <400> 2 ggtgcgttgc tgcatcttga tcatcattgt tggatttacg gggcaatata ctgattaatt 60 tcacaggcaa accattgctg gcgagcacgg ccttgatggc gatggacagt gagttcttct 120 cgacaaagtt ttgatttcca agaatggcgg tctgatattt ttgggcagct acaacggtac 180 ttccgacctc cagctggagc gcatgaacgt ctacttcaac cacgtaagtc aacccaacaa 240 tatcaattgc acttgtgaca tcgactctaa ttgatttgct ctcttttttg tcaataggct 300 tccggtgaca agtatgttcc ccgcgctgtt ctggtcgatc tggagcccgg taccatggac 360 gctgtccgtg ccggtccctt cggcaagctt ttccgtcccg acaacttcgt cttcggccag 420 tctggtgctg gtaacaactg ggccaagggt ca 452 <210> 3 <211> 517 <212> DNA <213> Artificial Sequence <220> <223> MH243031 Penicillium dokdoense CNUFC-DDS11-1 sp. nov. (CaM gene          [encoding calmodulin]) <400> 3 agcgtaggtg atcattatag gctgacgggt tatttgtgat cgacaggaca aggatggcga 60 tggtgagtgc agtcgtgtct aaacaaatcg ggtctctttc acgggcggca tttctattgc 120 tgcgattggg gctccgtcct tccctcttta aacgcagcta atggatatgt gatcaatagg 180 acaaattacc accaaggagc tcggcaccgt catgcgctcc ctcggccaga acccctccga 240 gtccgagctg caggatatga tcaacgaggt tgacgccgat aacaatggca ccattgattt 300 ccctggtacg atacccgcaa cctcagatcc tcttcctttc ttcccccatc ctccatgctg 360 cctcccacga taggatcaat attgacatgc gcctcacaga gttcttgacc atgatggccc 420 gtaagatgaa ggataccgac tctgaggagg agatccgtga ggcattcaag gttttcgacc 480 gcgacaacaa cggcttcatt tccgccgctg agctgcg 517 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> GAPDH Forward <400> 4 cccttattga cctcaactac atggt 25 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GAPDH Reverse <400> 5 gaggggccat ccacagtctt ctg 23 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha Forward <400> 6 catcttctca aaattcgagt gacaa 25 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> TNF-alpha Reverse <400> 7 tgggagtaga caaggtacaa ccc 23 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> IL-1beta Forward <400> 8 caaccaacaa gtgatattct ccatg 25 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL-1beta Reverse <400> 9 gatccacact ctccagctgc a 21

Claims (6)

Penicillium dokdoense CNUFC-DDS11-1 (Accession number: KACC 93309p). According to claim 1,
The penicillium dokdoensu CNUFC-DDS11-1 (accession number: KACC 93309p) is a penicillium dokdoensu CNUFC-DDS11-1 (accession number: KACC 93309p) characterized by inhibiting the production of nitrogen oxide (NO). Penicillium dokdoense CNUFC-DDS11-1 ( Penicillium dokdoense CNUFC-DDS11-1) (Accession No .: KACC 93309p), Penicillium dokdoense CNUFC-DDS11-1 ( Penicillium dokdoense CNUFC-DDS11-1) (Accession No .: KACC 93309p) and Penicillium dokdoense CNUFC-DDS11-1 ( Penicillium dokdoense CNUFC-DDS11-1) (Accession No .: KACC 93309p). Pharmaceutical composition for the prevention or treatment of inflammatory diseases. Penicillium dokdoense CNUFC-DDS11-1 ( Penicillium dokdoense CNUFC-DDS11-1) (Accession No .: KACC 93309p), Penicillium dokdoense CNUFC-DDS11-1 ( Penicillium dokdoense CNUFC-DDS11-1) (Accession No .: KACC 93309p) and Penicillium dokdoense CNUFC-DDS11-1 ( Penicillium dokdoense CNUFC-DDS11-1) (Accession No .: KACC 93309p). Anti-inflammatory health functional food composition. Penicillium dokdoense CNUFC-DDS11-1 ( Penicillium dokdoense CNUFC-DDS11-1) (Accession No .: KACC 93309p), Penicillium dokdoense CNUFC-DDS11-1 ( Penicillium dokdoense CNUFC-DDS11-1) (Accession No .: KACC 93309p) and Penicillium dokdoense CNUFC-DDS11-1 ( Penicillium dokdoense CNUFC-DDS11-1) (Accession No .: KACC 93309p). Cosmetic composition for anti-inflammatory. Penicillium dokdoense CNUFC-DDS11-1 ( Penicillium dokdoense CNUFC-DDS11-1) (Accession No .: KACC 93309p), Penicillium dokdoense CNUFC-DDS11-1 ( Penicillium dokdoense CNUFC-DDS11-1) (Accession No .: KACC 93309p) and Penicillium dokdoense CNUFC-DDS11-1 ( Penicillium dokdoense CNUFC-DDS11-1) (Accession No .: KACC 93309p). , Anti-fungal pharmaceutical composition against Alternaria alternata or Staphylococcus aureus .

Images