Polypeptide compound produced by fermentation of penicillium, preparation method therefor, and use thereof
Patent Information
- Application Number
- PCT/CN2025/094597
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-03-25
- Filing Date
- 2025-05-13
- Publication Date
- 2026-10-01
Smart Images
Figure CN2025094597_01102026_PF_FP_ABST
Abstract
Description
A polypeptide compound produced by Penicillium fermentation, its preparation method and application Technical Field
[0001] This invention relates to the field of microbial development and application technology, specifically to a polypeptide compound produced by Penicillium fermentation, its preparation method, and its application. Background Technology
[0002] The overuse and misuse of antibiotics both domestically and internationally have led to the rapid emergence of drug-resistant bacteria and genes, significantly reducing the therapeutic potential of antibiotics against various pathogens in humans and animals, and seriously threatening the effectiveness of antibiotic treatment and human health. Deep infections caused by drug-resistant bacteria have become a new challenge for anti-infective therapy in the new century. As early as 2011, the World Health Organization proposed a "global strategy to control bacterial resistance," elevating the issue of bacterial resistance to a global crisis. The situation of bacterial resistance in clinical practice in my country is extremely serious, with high detection rates of various common multidrug-resistant and pan-drug-resistant bacteria. Major drug-resistant bacteria include methicillin-resistant Staphylococcus aureus (MRSA) and macrolide-resistant Streptococcus (MRSP). Furthermore, the number of carbapenem-resistant Klebsiella pneumoniae has been rising rapidly in recent years, all of which pose significant challenges to clinical treatment. The rapid spread of multidrug-resistant bacteria makes the prevention, control, and treatment of bacterial infections even more challenging, but the development of new structural and mechanism-based antimicrobial drugs in my country lags significantly behind the current situation of multidrug resistance.
[0003] Therefore, it is urgent to expand new antibiotic resources and find new antibacterial drugs to cope with the increasingly serious situation of multidrug resistance. Summary of the Invention
[0004] The purpose of this invention is to provide a method for preparing polypeptide compounds produced by Penicillium fermentation, so as to solve the problems mentioned in the background art.
[0005] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:
[0006] A method for preparing polypeptide compounds produced by Penicillium fermentation, comprising the following steps:
[0007] Penicillium was inoculated into a liquid fermentation medium and subjected to static fermentation to obtain a fermentation supernatant. The liquid fermentation medium comprised the following components by mass concentration: mannitol 1%-3%, maltose 1%-3%, glucose 0.5%-1.5%, monosodium glutamate 0.5%-1.5%, KH2PO4 0.01%-0.1%, MgSO4·7H2O 0.01%-0.05%, yeast extract 0.1%-0.5%, corn steep liquor 0.05%-0.15%, with the remainder being distilled water.
[0008] The fermentation supernatant was extracted and then subjected to vacuum distillation to obtain a crude extract.
[0009] The crude extract was separated by silica gel normal-phase column chromatography, and the fraction was collected.
[0010] The fraction was purified by high performance liquid chromatography to obtain polypeptide compounds.
[0011] Preferably, the Penicillium is Penicillium pulvillorum JW13-16, deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 41147.
[0012] Preferably, before inoculating Penicillium into the liquid fermentation medium, Penicillium is first subjected to a large-scale culture; wherein, the large-scale culture uses PDA solid medium, the temperature of the large-scale culture is 28°C, the environment of the large-scale culture is a microbial biochemical incubator, and the large-scale culture time is 3-5 days.
[0013] Preferably, the extraction process uses ethyl acetate and the extraction temperature is 22-27℃.
[0014] Preferably, the pressure of vacuum distillation is -0.1 MPa, and the water bath temperature of vacuum distillation is 20-30℃.
[0015] Preferably, in silica gel normal phase column separation, the mobile phase is a mixed solvent of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of (1-3):(7-9).
[0016] Preferably, in the high performance liquid chromatography purification, the mobile phase is a 60% methanol aqueous solution and the retention time is 21.6 min.
[0017] Another object of the present invention is to provide a polypeptide compound prepared by the above-described preparation method.
[0018] Preferably, the structural formula of the polypeptide compound is Formula I:
[0019] .
[0020] Another object of the present invention is to provide the use of the above-mentioned polypeptide compound in the preparation of a medicament for treating drug-resistant bacteria.
[0021] This invention employs a liquid fermentation medium containing mannitol, maltose, glucose, monosodium glutamate, KH2PO4, MgSO4·7H2O, yeast extract, and corn steep liquor to ferment and train Penicillium, followed by purification to obtain polypeptide compounds. Bioactivity screening revealed that these polypeptide compounds exhibit great application potential in anti-drug resistant bacteria activity. Attached Figure Description
[0022] Figure 1 shows the HPLC fingerprint of the crude extract obtained in the embodiment of the invention.
[0023] Figure 2 is an MS chromatogram of the polypeptide compounds obtained in the embodiments of the invention.
[0024] Figure 3 shows the polypeptide compounds obtained in the embodiments of the invention. 1 H-NMR spectrum.
[0025] Figure 4 shows the polypeptide compounds obtained in the embodiments of the invention. 13 C-NMR spectrum. Detailed Implementation
[0026] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0027] Example 1: This example provides a method for preparing polypeptide compounds produced by Penicillium fermentation, which includes the following steps:
[0028] S1. Penicillium JW13-16, with preservation number CGMCC No.41147, was first cultured on PDA solid medium at 28℃ in a microbial biochemical incubator for 4 days. Then, it was inoculated into liquid fermentation medium and cultured statically at 28℃ for 30 days to obtain the fermentation supernatant. The PDA solid medium was prepared as follows: 200g of peeled and diced potatoes were boiled in distilled water for 20 minutes, filtered through gauze to obtain the extract, and 20g of glucose and 18g of agar were added to the extract. The volume was then adjusted to 1L with water and sterilized by steam at 121℃ under high pressure for 30 minutes. The liquid fermentation medium comprises the following components by mass concentration: mannitol 2%, maltose 2%, glucose 1%, monosodium glutamate 1%, KH2PO4 0.05%, MgSO4·7H2O 0.03%, yeast extract 0.3%, corn steep liquor 0.1%, and the balance being 1L of distilled water, pH=6.5; the liquid fermentation medium is preferably contained in a 500mL Erlenmeyer flask.
[0029] S2. The fermentation supernatant was extracted with ethyl acetate for 12 hours at a temperature of 25°C. This extraction process facilitates the dissolution of polypeptide compounds in the fermentation supernatant in ethyl acetate. Then, vacuum distillation was performed at a pressure of -0.1 MPa and a water bath temperature of 25°C to obtain a crude extract. The crude extract was analyzed using a fingerprint chromatogram, and the gradient elution method is as follows:
[0030] 0-5 minutes: 10% methanol aqueous solution (volume concentration);
[0031] 5-45 minutes: 10%-100% methanol aqueous solution (by volume);
[0032] 45-55 minutes: pure methanol solution;
[0033] The fingerprint chromatogram analysis results are shown in Table 1. According to the fingerprint chromatogram analysis, the compound represented by the chromatographic peak at 31.19 minutes is a polypeptide compound.
[0034] S3. The crude extract is separated by vacuum silica gel normal phase column and the fraction is collected; wherein the stationary phase is silica gel; the mobile phase is a mixed solvent of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 2:8.
[0035] S4. The above fraction is purified by high performance liquid chromatography to obtain polypeptide compounds; wherein, in the high performance liquid chromatography purification, the mobile phase is 60% methanol aqueous solution and the retention time is 21.6 min.
[0036] The obtained polypeptide compounds were subjected to 1H / 1C NMR and mass spectrometry analyses. The mass spectrometry (MS) spectra are shown in Figure 2, the 1H NMR spectra are shown in Figure 3, and the 13C NMR spectra are shown in Figure 4. The NMR data analysis identified the compounds as polypeptides, with the structural formula I:
[0037] .
[0038] The peptide compounds obtained above were tested for their antimicrobial activity against drug-resistant bacteria. Ciprofloxacin was used as a positive control. The results of the antimicrobial activity test showed that the minimum inhibitory concentration (MIC) of the peptide compounds against MRSA was 1.25 μg / mL.
[0039] Example 2: This example provides a method for preparing polypeptide compounds produced by Penicillium fermentation, which includes the following steps:
[0040] S1. Penicillium JW13-16, with preservation number CGMCC No.41147, was first cultured on PDA solid medium at 28℃ in a microbial biochemical incubator for 3 days. Then, it was inoculated into liquid fermentation medium and cultured statically at 28℃ for 30 days to obtain the fermentation supernatant. The PDA solid medium was prepared as follows: 200g of peeled and diced potatoes were boiled in distilled water for 20 minutes, filtered through gauze to obtain the extract, and 20g of glucose and 18g of agar were added to the extract. The volume was then adjusted to 1L with water and sterilized by steam at 121℃ under high pressure for 30 minutes. The liquid fermentation medium comprises the following components by mass concentration: mannitol 1%, maltose 1%, glucose 0.5%, monosodium glutamate 0.5%, KH2PO4 0.01%, MgSO4·7H2O 0.01%, yeast extract 0.1%, corn steep liquor 0.05%, and the balance being 1L of distilled water; the liquid fermentation medium is preferably contained in a 500mL Erlenmeyer flask.
[0041] S2. The above fermentation supernatant was extracted with ethyl acetate for 12 hours at an extraction temperature of 22°C. This process facilitates the dissolution of polypeptide compounds in the fermentation supernatant in ethyl acetate. Then, vacuum distillation was performed at a pressure of -0.1 MPa and a water bath temperature of 20°C to obtain the crude extract.
[0042] S3. The crude extract is separated by vacuum silica gel normal phase column and the fraction is collected; wherein the stationary phase is silica gel; the mobile phase is a mixed solvent of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 1:9.
[0043] S4. The above fraction is purified by high performance liquid chromatography to obtain polypeptide compounds; wherein, in the high performance liquid chromatography purification, the mobile phase is 60% methanol aqueous solution and the retention time is 21.6 min.
[0044] Example 3: This example provides a method for preparing polypeptide compounds produced by Penicillium fermentation, which includes the following steps:
[0045] S1. Penicillium JW13-16, with preservation number CGMCC No.41147, was first cultured on PDA solid medium at 28℃ in a microbial biochemical incubator for 5 days. Then, it was inoculated into liquid fermentation medium and cultured statically at 28℃ for 30 days to obtain the fermentation supernatant. The PDA solid medium was prepared as follows: 200g of peeled and diced potatoes were boiled in distilled water for 20 minutes, filtered through gauze to obtain the extract, and 20g of glucose and 18g of agar were added to the extract. The volume was then adjusted to 1L with water and sterilized by steam at 121℃ under high pressure for 30 minutes. The liquid fermentation medium comprises the following components by mass concentration: mannitol 3%, maltose 3%, glucose 1.5%, monosodium glutamate 1.5%, KH2PO4 0.1%, MgSO4·7H2O 0.05%, yeast extract 0.5%, corn steep liquor 0.15%, and the balance being 1L of distilled water; the liquid fermentation medium is preferably contained in a 500mL Erlenmeyer flask.
[0046] S2. The above fermentation supernatant was extracted with ethyl acetate for 12 hours at an extraction temperature of 27°C, which facilitated the dissolution of polypeptide compounds in the fermentation supernatant in ethyl acetate. Then, vacuum distillation was performed at a pressure of -0.1 MPa and a water bath temperature of 30°C to obtain the crude extract.
[0047] S3. The crude extract is separated by vacuum silica gel normal phase column chromatography, and the fraction is collected; wherein the stationary phase is silica gel; the mobile phase is a mixed solvent of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 3:7.
[0048] S4. The above fraction is purified by high performance liquid chromatography to obtain polypeptide compounds; wherein, in the high performance liquid chromatography purification, the mobile phase is 60% methanol aqueous solution and the retention time is 21.6 min.
[0049] Example 4: This example provides a method for preparing polypeptide compounds produced by Penicillium fermentation, which includes the following steps:
[0050] S1. Penicillium JW13-16, with preservation number CGMCC No.41147, was first cultured on PDA solid medium at 28℃ in a microbial biochemical incubator for 4 days. Then, it was inoculated into liquid fermentation medium and cultured statically at 28℃ for 30 days to obtain the fermentation supernatant. The PDA solid medium was prepared as follows: 200g of peeled and diced potatoes were boiled in distilled water for 20 minutes, filtered through gauze to obtain the extract, and 20g of glucose and 18g of agar were added to the extract. The volume was then adjusted to 1L with water and sterilized by steam at 121℃ under high pressure for 30 minutes. The liquid fermentation medium comprises the following components by mass concentration: mannitol 1.5%, maltose 1.5%, glucose 0.8%, monosodium glutamate 0.8%, KH2PO4 0.04%, MgSO4·7H2O 0.02%, yeast extract 0.2%, corn steep liquor 0.08%, and the balance being 1L of distilled water; the liquid fermentation medium is preferably contained in a 500mL Erlenmeyer flask.
[0051] S2. The above fermentation supernatant was extracted with ethyl acetate for 12 hours at an extraction temperature of 24°C. This process facilitates the dissolution of polypeptide compounds in the fermentation supernatant in ethyl acetate. Then, vacuum distillation was performed at a pressure of -0.1 MPa and a water bath temperature of 24°C to obtain the crude extract.
[0052] S3. The crude extract is separated by vacuum silica gel normal phase column and the fraction is collected; wherein the stationary phase is silica gel; the mobile phase is a mixed solvent of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 2:8.
[0053] S4. The above fraction is purified by high performance liquid chromatography to obtain polypeptide compounds; wherein, in the high performance liquid chromatography purification, the mobile phase is 60% methanol aqueous solution and the retention time is 21.6 min.
[0054] Example 5: This example provides a method for preparing polypeptide compounds produced by Penicillium fermentation, which includes the following steps:
[0055] S1. Penicillium JW13-16, with preservation number CGMCC No.41147, was first cultured on PDA solid medium at 28℃ in a microbial biochemical incubator for 4 days. Then, it was inoculated into liquid fermentation medium and cultured statically at 28℃ for 30 days to obtain the fermentation supernatant. The PDA solid medium was prepared as follows: 200g of peeled and diced potatoes were boiled in distilled water for 20 minutes, filtered through gauze to obtain the extract, and 20g of glucose and 18g of agar were added to the extract. The volume was then adjusted to 1L with water and sterilized by steam at 121℃ under high pressure for 30 minutes. The liquid fermentation medium comprises the following components by mass concentration: mannitol 2.5%, maltose 2.5%, glucose 1.2%, monosodium glutamate 1.2%, KH2PO4 0.08%, MgSO4·7H2O 0.04%, yeast extract 0.4%, corn steep liquor 0.12%, and the balance being 1L of distilled water; the liquid fermentation medium is preferably contained in a 500mL Erlenmeyer flask.
[0056] S2. The above fermentation supernatant was extracted with ethyl acetate for 12 hours at an extraction temperature of 26°C. This process facilitates the dissolution of polypeptide compounds in the fermentation supernatant in ethyl acetate. Then, vacuum distillation was performed at a pressure of -0.1 MPa and a water bath temperature of 26°C to obtain the crude extract.
[0057] S3. The crude extract is separated by vacuum silica gel normal phase column and the fraction is collected; wherein the stationary phase is silica gel; the mobile phase is a mixed solvent of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of 2:8.
[0058] S4. The above fraction is purified by high performance liquid chromatography to obtain polypeptide compounds; wherein, in the high performance liquid chromatography purification, the mobile phase is 60% methanol aqueous solution and the retention time is 21.6 min.
[0059] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.
Claims
1. A method for preparing polypeptide compounds produced by Penicillium fermentation, characterized in that, Includes the following steps: Penicillium was inoculated into a liquid fermentation medium and subjected to static fermentation to obtain a fermentation supernatant. The liquid fermentation medium comprised the following components by mass concentration: mannitol 1%-3%, maltose 1%-3%, glucose 0.5%-1.5%, monosodium glutamate 0.5%-1.5%, KH2PO4 0.01%-0.1%, MgSO4·7H2O 0.01%-0.05%, yeast extract 0.1%-0.5%, corn steep liquor 0.05%-0.15%, with the remainder being distilled water. The fermentation supernatant was extracted and then subjected to vacuum distillation to obtain a crude extract. The crude extract was separated by silica gel normal-phase column chromatography, and the fraction was collected. The fraction was purified by high performance liquid chromatography to obtain polypeptide compounds.
2. The method for preparing polypeptide compounds produced by Penicillium fermentation according to claim 1, characterized in that, The Penicillium species in question is Penicillium JW13-16, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41147.
3. The method for preparing polypeptide compounds produced by Penicillium fermentation according to claim 1, characterized in that, Before inoculating Penicillium into the liquid fermentation medium, Penicillium is first cultured on a scale-up basis. The scale-up culture uses PDA solid medium, the temperature is 28℃, the environment is a microbial biochemical incubator, and the culture time is 3-5 days.
4. The method for preparing polypeptide compounds produced by Penicillium fermentation according to claim 1, characterized in that, Extraction was performed using ethyl acetate at a temperature of 22-27℃.
5. The method for preparing polypeptide compounds produced by Penicillium fermentation according to claim 1, characterized in that, The pressure for vacuum distillation is -0.1 MPa, and the water bath temperature for vacuum distillation is 20-30℃.
6. The method for preparing polypeptide compounds produced by Penicillium fermentation according to claim 1, characterized in that, In silica gel normal phase column separation, the mobile phase is a mixed solvent of petroleum ether and ethyl acetate, with a volume ratio of (1-3):(7-9).
7. The method for preparing polypeptide compounds produced by Penicillium fermentation according to claim 1, characterized in that, In the high performance liquid chromatography purification, the mobile phase was 60% methanol aqueous solution, and the retention time was 21.6 min.
8. A polypeptide compound prepared by any one of claims 1-7.
9. The polypeptide compound according to claim 8, characterized in that, The structural formula of the polypeptide compound is Formula I: .
10. The use of a polypeptide compound as described in claim 8 or 9 in the preparation of a medicament for treating drug-resistant bacteria.