A penicillium metabolite and use in the preparation of an antibacterial agent

Abstract

The application discloses a penicillium metabolic product and application thereof in preparation of bacteriostatic agents, and the penicillium Lp.PY-01 metabolic product of the application has a MIC of 0.3906 mg / mL for inhibiting staphylococcus aureus and a MIC of 0.7813 mg / mL for inhibiting candida albicans. As a natural bacteriostatic agent, the penicillium metabolic product is expected to provide a more sustainable and environment-friendly solution for the agricultural and medical fields.

Description

(I) Technical Field

[0001] This invention relates to an antibacterial agent with inhibitory ability against Staphylococcus aureus (CMCC(B)26003) and Candida albicans (CMCC(F)98001), specifically to a Penicillium Lp.PY-01 metabolite and its application in the preparation of the antibacterial agent. (II) Background Technology

[0002] *Ilex pubescens* Hook. et Arn, also known as hairy holly or June frost, is a plant belonging to the genus *Ilex* in the family Aquifoliaceae. It flowers from April to May and fruits from August to November. It is distributed in Anhui and Zhejiang provinces of China. Studies have found that the main chemical components of *Ilex pubescens* include triterpenoid saponins, iridoid glycosides, lignins, phenylpropanoids, phenolic acids, flavonoids, and chlorogenic acids. *Ilex pubescens* is a commonly used traditional Chinese medicine in southern China, possessing antioxidant, anti-inflammatory, antiviral, antitussive, expectorant, heat-clearing, detoxifying, swelling-reducing, and analgesic effects. Furthermore, extracts of *Ilex pubescens* have been reported to have anti-fatigue and antibacterial properties.

[0003] Plant endophytes are a specific class of microorganisms that parasitize parts of the intercellular spaces, organs, or tissues of healthy plants throughout their entire life cycle or at certain stages of their life cycle, without causing any abnormal symptoms or diseases in the host plant. Endophytes with diverse biological activities can be isolated from plants, some of which can produce products similar to plant secondary metabolites through secondary metabolic pathways. The diversity of secondary metabolites produced by different plant endophytes provides opportunities for the discovery of novel antimicrobial compounds. This is of great significance for combating emerging pathogens and drug-resistant strains.

[0004] Compared to synthetic antimicrobial agents, secondary metabolites produced by plant endophytes are generally more environmentally friendly. Their environmental impact during production and decomposition is relatively small, thus offering potential advantages in sustainable agriculture and ecosystem management. (III) Summary of the Invention

[0005] The purpose of this invention is to provide a Penicillium metabolite and its application in the preparation of antibacterial agents. The metabolite produced by fermentation of Penicillium Lp.PY-01 has the ability to inhibit Staphylococcus aureus (CMCC(B)26003) and Candida albicans (CMCC(F)98001). As a natural antibacterial agent, it is expected to provide a more sustainable and environmentally friendly solution for the agricultural and medical fields.

[0006] The technical solution adopted in this invention is:

[0007] This invention provides a metabolite of Penicillium macrosclerotiorum Lp.PY-01, which is prepared by the following method: (1) The fermentation broth of Penicillium macrosclerotiorum Lp.PY-01 is ultrasonically crushed and then filtered. The filtrate and the residue are extracted with ethyl acetate, and the ethyl acetate phases are combined and concentrated to constant weight to obtain the crude metabolite. Penicillium macrosclerotiorum Lp.PY-01 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO:M 20232576, deposited on December 15, 2023, deposited at: Wuhan University, Wuhan, China, 430072, China; (2) The crude metabolite from step (1) was dissolved in ethyl acetate and subjected to silica gel column chromatography. The elution was performed using a petroleum ether-ethyl acetate gradient with volume ratios of 100:0, 100:1, 100:2, 100:5, 100:10, 100:20, 50:50, and 0:100. The elution rate was 5 mL / min and the elution volume was 2 to 3 column volumes. (2) The eluent with a volume ratio of 100:2 petroleum ether-ethyl acetate was concentrated to dryness and recorded as component E.1; (3) Component E.1 from step (2) was dissolved in ethyl acetate and subjected to silica gel column chromatography again. The mobile phase was eluted with petroleum ether-ethyl acetate at a volume ratio of 10:1-0:1. The eluent with a volume ratio of 8:1 to 4:1 was concentrated to dryness and recorded as component E.1-2; (4) Component E.1-2 from step (3) was recrystallized with ethyl acetate. The crystals were collected to obtain the metabolites of Penicillium Lp.PY-01.

[0008] Further, the fermentation broth in step (1) is prepared as follows: Penicillium Lp.PY-01 is inoculated into the fermentation medium and cultured at 28℃ and 180r / min for 5 days to obtain the fermentation broth; the fermentation medium consists of: 10g / L peptone, 40g / L sucrose, distilled water as solvent, and pH 5.6.

[0009] Further, the crude metabolic product in step (1) is prepared as follows: the fermentation broth is subjected to ultrasonic cell disruption treatment for 300 cycles at 405W for 3 seconds and 4 seconds intervals, and then filtered. The filtrate is extracted three times with 1 volume of ethyl acetate. The filter residue is soaked in ethyl acetate for 12 hours and then filtered. The ethyl acetate phases are combined and concentrated to constant weight to obtain the crude metabolic product.

[0010] Further, step (2) involves dissolving the crude metabolic product from step (1) in ethyl acetate, adding silica gel, mixing thoroughly, and then vacuum drying to obtain the silica gel for adsorbing the sample; the silica gel for adsorbing the sample is then loaded onto a silica gel chromatographic column; the mass ratio of the silica gel to the crude fermentation product is 1:1.

[0011] Further, step (3) involves dissolving component E.1 from step (2) in ethyl acetate, adding silica gel, mixing thoroughly, and then vacuum drying to obtain silica gel for adsorbing component E.1; the silica gel for adsorbing component E.1 is then loaded onto a silica gel chromatographic column; the mass ratio of component E.1 to silica gel is 1:1.

[0012] The present invention also provides the application of the metabolite of Penicillium Lp.PY-01 in the preparation of antibacterial agents, wherein the antibacterial agents include Staphylococcus aureus antibacterial agents or Candida albicans antibacterial agents.

[0013] The Staphylococcus aureus includes Staphylococcus aureus CMCC(B)26003, and the Candida albicans includes Candida albicans CMCC(F)98001.

[0014] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0015] This invention provides a metabolite extracted from the fermentation of Penicillium strain Lp.PY-01, and the application of the metabolite as an antibacterial agent. The metabolite exhibits an inhibitory MIC of 0.3906 mg / mL against Staphylococcus aureus (CMCC(B)26003) and 0.7813 mg / mL against Candida albicans (CMCC(F)98001). This invention prepares the antibacterial agent through microbial fermentation of Penicillium strain Lp.PY-01. It is a natural component with high safety and holds promise for development into a natural antibacterial product. (iv) Description of the attached drawings

[0016] Figure 1 Phylogenetic tree of Penicillium Lp.PY-01.

[0017] Figure 2 Photographs showing the antibacterial activity of Penicillium Lp.PY-01 metabolites against Staphylococcus aureus (A) and Candida albicans (B).

[0018] Figure 3 The structural formula of Palmitic acid, a major component of the metabolites of Penicillium Lp.PY-01.

[0019] Figure 4 Metabolites of Penicillium Lp.PY-01 1 H-NMR spectrum.

[0020] Figure 5 Metabolites of Penicillium Lp.PY-01 13 C-NMR spectrum.

[0021] Figure 6 MS spectrum of metabolites of Penicillium Lp.PY-01. (V) Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0023] The hairy holly (Ilex pubescens Hook.et Arn) described in this invention, also known as black holly or June frost, is a plant of the genus Ilex in the family Aquifoliaceae. It flowers from April to May and fruits from August to November, and is distributed in Anhui, Zhejiang and other places in China.

[0024] The ultrapure water described in this invention refers to water with a resistivity of 18 MΩ*cm (25℃). Apart from water molecules, the water contains almost no impurities, bacteria, viruses, dioxins, or other organic matter, as well as minerals and trace elements required by the human body.

[0025] Example 1: Obtaining and identifying Penicillium Lp.PY-01

[0026] 1. Plant Sample Collection: Fresh, healthy branches of *Ilex pubescens* were collected from Hangzhou, Zhejiang Province. Leaves were washed with tap water for 10 minutes, then rinsed once with a 75% ethanol aqueous solution. After soaking in a 2% sodium hypochlorite aqueous solution for 10 minutes, the leaves were repeatedly washed with sterile water, then soaked in a 75% ethanol aqueous solution for 15 minutes, and rinsed three times with sterile water. The rinsing solution was collected, and the rinsed leaves were dried on sterile absorbent paper to obtain surface-sterilized leaves. In a laminar flow hood, sterile PDA agar plates were placed as blank control 1 to check the cleanliness of the laminar flow hood; the collected rinsing solution was inoculated onto sterile PDA agar plates as blank control 2 for rinsing solution testing; and the surface-sterilized leaves were rolled once in sterile PDA agar plates and then removed as blank control 3 for screening sterile tissue blocks using plant tissue imprinting.

[0027] 2. Screening and purification of endophytic fungi: In a sterile laminar flow hood, the sterilized leaves of *Ilex pubescens* from step 1 were cut into thin slices as sterile tissue and inoculated into PDA medium. The slices were then incubated upside down at 30°C. When endophytic hyphae appeared growing outwards along the tissue cut, the samples were compared with blank controls 1, 2, and 3. Using the tip hyphae selection method, colonies of different morphologies were streaked and inoculated into PDA medium. After single colonies grew, they were streaked again into sterile PDA medium. This process was repeated multiple times until the colony morphology was consistent and only one type of fungus was growing, indicating that purification was complete. Twelve fungal strains were obtained. Strain 1 colonies were white and cottony with a dark brown underside; strain 2 colonies were milky white, round, smooth, and slightly convex. Strain 11 has neat edges, relatively hard colonies, and its metabolites have no obvious color; strain 3 is white and fluffy with a red back; strain 4 is white and fluffy with long hyphae that later turn pale yellow; strain 5 is grayish-black and fluffy with a black base; strain 6 is white and rough solid with pale yellow spores on the surface and a light brown back; strain 7 is white and fluffy with a yellowish-brown back; strain 8 is grayish-brown with a smooth, uneven surface and no bumps, and its metabolites have no obvious color; strain 9 is pure black, slightly raised, with a dry, rough surface, irregular shape, and relatively hard texture; strain 10 is milky white, with small, slightly raised colonies, a smooth and moist surface, and irregular edges; strain 11 is pure white and cottony; strain 12 is dark green and powdery, producing a distinctly yellow substance in the culture medium. Strain 12 is designated as strain Lp.PY-01.

[0028] PDA medium composition: 200g potato, 20g glucose, 20g agar, 1000mL distilled water, pH 7.0.

[0029] 3. Total DNA Extraction: Lp.PY-01 strain was inoculated into PDA medium and incubated upside down at 30℃ for 5 days. Genomic DNA was extracted using the Ezup column-based fungal genomic DNA extraction kit (purchased from Sangon Biotech (Shanghai) Co., Ltd., product number: B518259-0050, and related operating instructions): ① Take 50-100 mg of fresh fungi or 20 mg of dried fruiting bodies or hyphae, grind them thoroughly into powder in liquid nitrogen, and place them in a 1.5 mL centrifuge tube. Add 200 μL of Buffer Digestion, 2 μL of β-mercaptoethanol, and 20 μL of Proteinase K solution sequentially, vortex to mix, and incubate at 65℃ for 1 h until complete cell lysis. ② Add 100 μL of Buffer PF, invert to mix thoroughly, and incubate at -20℃ for 5 min. ③ Centrifuge at 10000 rpm for 5 min at room temperature, and transfer the supernatant to a new 1.5 mL centrifuge tube. ④ Add 200 μL of Buffer PF. BD, mix thoroughly by inverting. ⑤ Add 200 μL of anhydrous ethanol and mix thoroughly by inverting. ⑥ Place the adsorption column into the collection tube, and use a pipette to add all the solution and translucent fibrous suspension into the adsorption column. Let it stand for 2 min, then centrifuge at 10000 rpm at room temperature for 1 min, and discard the waste liquid in the collection tube. ⑦ Place the adsorption column back into the collection tube, add 500 μL of PW Solution, centrifuge at 10000 rpm for 30 s, and discard the waste liquid in the collection tube. ⑧ Place the adsorption column back into the collection tube, add 500 μL of Wash Solution, centrifuge at 10000 rpm for 30 s, and discard the waste liquid in the collection tube. ⑨ Place the adsorption column back into the collection tube, centrifuge at 12000 rpm at room temperature for 2 min, and remove the residual Wash Solution. ⑩ Remove the adsorption column, place it in a new 1.5 ml centrifuge tube, and add 50 μL of TE. Let the buffer stand for 3 minutes, then centrifuge at 12,000 rpm for 2 minutes at room temperature to collect the DNA solution. The extracted DNA can be used immediately for the next experiment or stored at -20°C.

[0030] 4. ITS sequence amplification of strain Lp.PY-01: The internal transcription region sequence was amplified using the universal fungal amplification primers ITS1 and ITS4.

[0031] ITS1:5'-TCCGTAGGTGAACCTGCGG-3'

[0032] ITS4:5'-TCCTCCGCTTATTGATATGC-3'

[0033] Table 1 PCR reaction system

[0034]

[0035] PCR amplification program: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles, 72℃ extension for 10 min, and storage at 4℃.

[0036] 5. Confirmation of PCR product: Mix 2.5 μL of PCR product with 0.5 μL of Loading Buffer and spot the mixture onto a 1.2% agarose gel. Electrophoresis is performed at 120V for 20 minutes. Observe the bands in the gel imaging system. If a band of about 600-750 bp appears, the amplification is preliminarily judged to be successful.

[0037] 6. Sequencing of PCR products: The PCR products were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The ITS sequence of strain Lp.PY-01 is shown in SEQ ID NO.1.

[0038] 7. Data Analysis: The sequence of strain Lp. PY-01 was compared with sequences in GenBank using BLAST. BLAST search showed that the ITS sequence of strain Lp. PY-01 had a 99.28% similarity to the sequence of Penicillium macrosclerotiorum (GenBank accession number NR156585.1). A phylogenetic tree was constructed as shown below. Figure 1 As shown, by Figure 1 The support rate is 99%. Based on the comparison of genetic affinity, strain Lp.PY-01 was identified as belonging to the genus Penicillium and named Penicillium macrosclerotiorum Lp.PY-01. It is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20232576, deposit date December 15, 2023, and address: Wuhan University, Wuhan, China, 430072, China.

[0039] Example 2: Isolation of metabolites from Penicillium Lp.PY-01

[0040] 1. Strain revival and activation: Penicillium Lp.PY-01, which was stored at 4℃, was inoculated into PDA medium and cultured in a 28℃ incubator for 5 days;

[0041] 2. Preparation of metabolites of Penicillium Lp.PY-01: In a clean bench, using a sterile punch, 5mm diameter mycelial cakes of the Lp.PY-01 strain from step 1 were made along the colony edge and inoculated into a 1500mL Erlenmeyer flask containing 300mL of fermentation medium. The flasks were incubated at 28℃ and 180r / min for 5 days, with uninoculated fermentation medium serving as a blank control. The fermentation broth was then subjected to ultrasonic cell disruption using a JY92-IIDN ultrasonic cell disruptor at 405W, with 300 cycles of 3s operation followed by 4s intervals. The resulting broth was then filtered to obtain the filtrate and residue. The filtrate was extracted three times with 1 volume of ethyl acetate until no significant color change was observed visually, yielding the ethyl acetate phase. The residue was soaked in ethyl acetate at room temperature for 12 hours and then filtered to obtain the ethyl acetate phase. The ethyl acetate phases were combined and concentrated and dried to constant weight using a rotary evaporator to obtain 60 g of crude metabolite of Penicillium Lp.PY-01 (abbreviated as PY-A), which was stored at -20℃.

[0042] The fermentation medium consisted of 10 g / L peptone, 40 g / L glucose, and distilled water as the solvent. The culture medium was sterilized using a high-pressure steam autoclave at 121°C for 20 min.

[0043] 3. Separation and purification of crude metabolic products: (1) Weigh 60g PY-A, dissolve it in 10-15mL ethyl acetate, add 60g silica gel (200-300 mesh), mix well, and dry in a vacuum desiccator to obtain silica gel for adsorbing samples. The silica gel containing the adsorbed sample was loaded into a silica gel column (8cm*60cm) with a column volume of 3 / 4. The column was eluted sequentially with a gradient of petroleum ether-ethyl acetate (100:0, 100:1, 100:2, 100:5, 100:10, 100:20, 50:50, 0:100, v / v) at a rate of 5mL / min and a volume of 2 column volumes. The eluents were collected to obtain eluents E.1 to E.8, a total of 8 components. The eluent with a volume ratio of 100:2 petroleum ether-ethyl acetate was concentrated to dryness and recorded as component E.1, 2.4g; (2) 2.4g of component E.1 was dissolved in ethyl acetate and 2.5g of silica gel was added and mixed. After homogenization, vacuum drying was performed to obtain silica gel containing adsorbed component E.1. Silica gel containing adsorbed component E.1 was loaded onto a silica gel column (4cm*30cm, 3 / 4 column capacity). Elution was performed using petroleum ether-ethyl acetate with volume ratios of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1, respectively. The elution rate was 5mL / min, and the elution volume was 2 column volumes. The collected eluents with volume ratios of 8:1 to 4:1 were concentrated to dryness and recorded as component E.1-2, 1.36g. (3) Component E.1-2 was recrystallized with ethyl acetate to obtain transparent crystals, yielding 62mg of Penicillium Lp.PY-01 metabolites. The sample was dissolved in deuterated chloroform and analyzed by nuclear magnetic resonance spectroscopy and mass spectrometry. The spectral data are available in [reference needed]. Figure 4-6 The main component is Palmitic acid, and its structure is shown below. Figure 3 As shown.

[0044] Example 3: Determination of the inhibitory effect of Penicillium Lp.PY-01 metabolites on Staphylococcus aureus (CMCC(B)26003) and Candida albicans (CMCC(F)98001).

[0045] 1. Sample solution:

[0046] In a clean bench, 25 mg of the Penicillium Lp.PY-01 metabolite prepared according to the method in Example 2 was weighed and dissolved in 1 mL of DMSO. After filtration through a microporous membrane (0.22 μm), the solution was used as a stock solution. The 25 mg / mL stock solution was diluted with DMSO using the two-fold dilution method to obtain sample solutions of different concentrations (25 mg / mL, 12.5 mg / mL, 6.25 mg / mL, 3.125 mg / mL, 1.5625 mg / mL, and 0.7813 mg / mL).

[0047] 2. Staphylococcus aureus

[0048] Place five sterile Oxford cups in each blank sterile culture dish using forceps. Then pour in a layer of melted sterile LB medium and allow it to cool and solidify. Next, add a layer containing 10... 6 Melted LB solid medium containing CFU / mL Staphylococcus aureus (CMCC(B)26003) was allowed to solidify again. The Oxford cup was then removed using sterile forceps to obtain the sample wells. 120 μL of different concentrations of sample solution (25 mg / mL, 12.5 mg / mL, 6.25 mg / mL, 3.125 mg / mL, 1.5625 mg / mL, 0.7813 mg / mL) were added to wells 1-6 using a pipette. 120 μL of DMSO was added to well '-' as a blank control; 120 μL of 1.2 mg / L kanamycin was added to well '+' as a positive control. The culture dishes were incubated at 37°C for 12-24 hours, and the appearance of a clear zone around the sample wells was observed. The results are shown in [Table data would be inserted here]. Figure 2 A; MIC = 0.3906 mg / mL.

[0049] 3. Candida albicans

[0050] In step 2, *Staphylococcus aureus* was replaced with *Candida albicans* CMCC(F)98001. Sample solution concentrations were 6.25 mg / mL, 3.125 mg / mL, 1.5625 mg / mL, 0.7813 mg / mL, 0.3906 mg / mL, and 0.1953 mg / mL. Nystatin was used as the positive control. The culture dishes with the samples were incubated at 25°C for 12–24 hours, with all other procedures performed as before. Results are shown in […]. Figure 2 In the case of B, MIC = 0.7813 mg / mL.

Claims

1. A metabolite of Penicillium Lp. PY-01, characterized in that, The metabolites were prepared as follows: (1) The fermentation broth of Penicillium Lp. PY-01 was ultrasonically crushed and then filtered. The filtrate and the residue were extracted with ethyl acetate, and the ethyl acetate phases were combined and concentrated to constant weight to obtain the crude metabolites; Penicillium ( Penicillium macrosclerotiorum Lp. PY-01, deposited at the China Center for Type Culture Collection, accession number: CCTCC NO:M 20232576; (2) The crude metabolite from step (1) was dissolved in ethyl acetate and subjected to silica gel column chromatography. The elution was carried out with a gradient of petroleum ether-ethyl acetate at volume ratios of 100:0, 100:1, 100:2, 100:5, 100:10, 100:20, 50:50, and 0:100, with an elution rate of 5 mL / min and an elution volume of 2 to 3 column volumes. The eluent with a volume ratio of 100:2 petroleum ether-ethyl acetate was concentrated to dryness and recorded as component E.

1. (3) Component E.1 from step (2) was dissolved in ethyl acetate and subjected to silica gel column chromatography again. The elution was carried out with a volume ratio of petroleum ether-ethyl acetate at 10:1 to 0:

1. The eluent with a volume ratio of 8:1 to 4:1 was concentrated to dryness and recorded as component E.1-2. (4) Component E.1-2 was recrystallized with ethyl acetate and the crystals were collected to obtain the metabolite of Penicillium Lp. PY-01.

2. The Penicillium Lp.PY-01 metabolite as described in claim 1, characterized in that, The fermentation broth in step (1) is prepared as follows: Penicillium Lp. PY-01 is inoculated into the fermentation medium and cultured at 28 ℃ and 180 r / min for 5 days to obtain the fermentation broth; the fermentation medium consists of: 10 g / L peptone, 40 g / L sucrose, distilled water as solvent, and pH 5.

6.

3. The Penicillium Lp.PY-01 metabolite as described in claim 1, characterized in that, The crude metabolic product in step (1) is prepared as follows: the fermentation broth is subjected to ultrasonic cell disruption treatment for 300 cycles at 405 W for 3 working seconds and 4 intermittent seconds. The broth is then filtered. The filtrate is extracted three times with 1 volume of ethyl acetate. The filter residue is soaked in ethyl acetate for 12 h and then filtered. The ethyl acetate phases are combined and concentrated to constant weight to obtain the crude metabolic product.

4. The Penicillium Lp.PY-01 metabolite as described in claim 1, characterized in that, Step (2) involves dissolving the crude metabolic product from step (1) in ethyl acetate, adding silica gel, mixing thoroughly, and then vacuum drying to obtain the silica gel for adsorbing the sample. The silica gel for adsorbing the sample is then loaded onto a silica gel chromatographic column. The mass ratio of the silica gel to the crude fermentation product is 1:

1.

5. The Penicillium Lp.PY-01 metabolite as described in claim 1, characterized in that, Step (3) involves dissolving component E.1 from step (2) in ethyl acetate, adding silica gel, mixing thoroughly, and then vacuum drying to obtain silica gel containing component E.

1. The silica gel containing component E.1 is then loaded onto a silica gel chromatographic column.

6. The use of the Penicillium Lp. PY-01 metabolite as described in claim 1 in the preparation of an antibacterial agent.

7. The application as described in claim 6, characterized in that, The antibacterial agent is a Staphylococcus aureus antibacterial agent or a Candida albicans antibacterial agent.

8. The application as described in claim 7, characterized in that, The Staphylococcus aureus includes Staphylococcus aureus ( Staphylococcus aureus )CMCC(B)26003.

9. The application as described in claim 7, characterized in that, The Candida albicans includes Candida albicans ( Candida albicans )CMCC(F)98001.

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