Antimicrobial peptide sesquimycin and its application

Abstract

This invention discloses an antimicrobial peptide, sesquimycin, and its applications. Specifically, it involves eight sesquimycin-like polypeptide compounds. Compounds 1-8 exhibit varying degrees of antimicrobial activity, with compound 2 possessing broad-spectrum antimicrobial activity. Its minimum inhibitory concentration (MIC) against *Staphylococcus aureus* ATCC 6538 and *Bacillus cereus* CMCC(B)63301 reaches 20 μg / mL, and its MIC against *Enterococcus faecalis* 160119481, *Salmonella typhi* CMCC(B)50071, and *Salmonella typhimurium* CMCC(B)50115 is 40 μg / mL. Given that *Staphylococcus*, *Bacillus*, *Enterococcus*, and *Salmonella* are common foodborne pathogens posing a serious threat to food safety, compounds 1-8 can be used to prepare novel antimicrobial peptides for application in the field of food safety preservation.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an antimicrobial peptide, sesquimycin, and its application in antibacterial and / or antiseptic purposes. Background Technology

[0002] The global food system faces a persistent threat from bacterial pathogens, particularly Gram-negative Enterobacteriaceae. According to the World Health Organization, more than 600 million people worldwide fall ill annually from contaminated food, resulting in 420,000 deaths. These pathogens are of particular concern due to their tendency to form biofilms, their strong ability to survive in the environment, and their increasing resistance to conventional interventions. The emergence and spread of multidrug-resistant strains have significantly reduced the antibacterial efficacy of many existing food-grade preservatives, a problem exacerbated by the overuse of antibiotics in agriculture. Currently used chemical preservatives (such as nitrites and organic acids) suffer from narrow antibacterial spectra, a tendency to induce resistance, and potential health risks. Meanwhile, the use of antibiotics in agriculture is strictly regulated due to their role in exacerbating clinical resistance. At the same time, with increasing consumer awareness of health and safety, the market is increasingly favoring food additives with natural ingredients, transparent processing, and clean labeling. This trend is driving the food industry to seek new antibacterial agents that combine safety, broad-spectrum antibacterial activity, and low resistance to induce resistance, in order to address the dual challenges of food safety and public health.

[0003] Compared to traditional antibiotics that act on a single target, antimicrobial peptides, as key effector molecules of innate immunity, exert broad-spectrum bactericidal activity primarily through rapid disruption of bacterial cell membrane integrity or action on intracellular targets. This multi-mechanism mode of action significantly reduces the risk of bacterial resistance. Furthermore, antimicrobial peptides typically exhibit high selectivity for prokaryotic cells, good safety profiles for human cells, and are biodegradable and easily modified to enhance stability, making them highly suitable for use as food preservatives. For example, the antimicrobial peptide nisin has been successfully used to inhibit Listeria in dairy products, demonstrating its practical application value in the food industry. However, existing antimicrobial peptides still face limitations in terms of activity, stability, and cost in complex food matrices, necessitating the discovery of novel antimicrobial peptides with novel structures, stronger activity, and better applicability. Summary of the Invention

[0004] The purpose of this invention is to provide an antimicrobial peptide, sesquimycin, and its application in antibacterial and / or antiseptic applications.

[0005] An antimicrobial peptide sesquimycin, wherein the antimicrobial peptide sesquimycin is selected from one or more of the following compounds 1-8:

[0006]

[0007]

[0008]

[0009]

[0010]

[0011] .

[0012] The antimicrobial peptide sesquimycin has a pharmaceutically acceptable salt.

[0013] A food preservative comprising the antimicrobial peptide sesquiformin and / or a pharmaceutically acceptable salt thereof.

[0014] The use of the sesquiformin or a pharmaceutically acceptable salt of the sesquiformin in antibacterial and / or antiseptic applications.

[0015] The bacteria are Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Salmonella typhi, or Salmonella typhimurium.

[0016] The beneficial effects of this invention: The sesquimycin discovered in this invention has been confirmed through antibacterial experiments to have significant growth inhibitory effects on Gram-positive bacteria of the spp. *Staphylococcus*, *Enterococcus*, and *Bacillus*, as well as Gram-negative bacteria of the spp. *Salmonella*. Compound 2 exhibits the strongest activity, with a minimum inhibitory concentration (MIC) of 20 μg / mL against *Staphylococcus aureus* ATCC 6538 and *Bacillus cereus* CMCC(B) 63301, and an MIC of 40 μg / mL against *Enterococcus faecalis* 160119481, *Salmonella typhi* CMCC(B) 50071, and *Salmonella typhimurium* CMCC(B) 50115. This indicates that sesquimycin possesses broad-spectrum and significant antibacterial activity, and has broad application prospects in the field of food safety preservation. Attached Figure Description

[0017] Figure 1 The image shows the HRESIMS spectrum of compound 2.

[0018] Figure 2 The image shows the 1H-NMR spectrum of compound 2.

[0019] Figure 3 The image shows the 13C-NMR spectrum of compound 2.

[0020] Figure 4 The image shows the 1H-1H TOCSY spectrum of compound 2.

[0021] Figure 5 This is the HSQC spectrum of compound 2.

[0022] Figure 6 The image shows the HMBC spectrum of compound 2.

[0023] Figure 7 The image shows the ROESY spectrum of compound 2. Detailed Implementation

[0024] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0025] The strains used in the following examples Sesquicillium sp. QL0466 was derived from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences, and is preserved in the inventor's laboratory. Analytical grade methanol and ethanol used in the experiment were produced by Sinopharm Chemical Reagent Co., Ltd. Chromatographic grade methanol, acetonitrile, and formic acid were produced by Fisher Chemical. D101 type macroporous adsorption resin was produced by Shanghai Maclean Biochemical Technology Co., Ltd. CHROMATOREX C18 MB 100-40 / 75 reversed-phase silica gel packing was produced by Fuji Silicon Chemical Co., Ltd., Japan. ZORBOX Eclipse Plus C18 reversed-phase analytical ultra-high performance liquid chromatography column (2.1 mm × 50 mm, 1.8 µm) and ZORBOX RX-C18 reversed-phase semi-preparative high performance liquid chromatography column (9.4 mm × 250 mm, 5 µm) were purchased from Agilent Technologies, USA. The BUCHI R-300 rotary evaporator, BUCHI I-300 touchscreen central control unit, BUCHI V-300 PTFE membrane vacuum pump, and BUCHI B-300 electric thermostatic water bath were all manufactured by BUCHI GmbH, Switzerland. The cryogenic coolant circulation pump was manufactured by Shanghai Zhixin Experimental Instrument Technology Co., Ltd. The analytical high-performance liquid chromatograph (Agilent 1290 Infinity II), the semi-preparative high-performance liquid chromatograph (Agilent 1260 Infinity II), the HRESI-TOF mass spectrometer (Agilent 6530), and the 600 MHz nuclear magnetic resonance spectrometer (Agilent DD2) were manufactured by Agilent Technologies, Inc., USA. The AutoFlex MALDI-TOF mass spectrometer was manufactured by Bruker GmbH, Germany. The deuterated reagents used in the nuclear magnetic resonance spectroscopy were manufactured by Cambridge I.L. (CIL), Inc., USA.

[0026] Example 1: Preparation and structural analysis of compounds 1-8

[0027] 1.1 Isolation and purification of compounds 1-8

[0028] On PDA medium Sesquicillium sp. QL0466 was activated and incubated statically at 28°C for 5 days. Fungal spores were collected using sterile water and diluted to 1×10⁻⁶. 6 / mL concentration. The spore suspension was inoculated into 12 L of PDA medium and cultured statically at 28℃ for 14 days. The fermentation medium was collected, and the spores, along with the bacterial cells, were cut into small pieces and placed in a 10 L extraction vessel. An equal volume of 90% ethanol was added for ultrasonic extraction (0.5 h each time, repeated 3 times). After filtration, the supernatant was collected and concentrated to approximately 1 L. The extract was then loaded onto a macroporous adsorption resin using a wet method, followed by gradient elution with water, 50% methanol-water, and methanol, for 3 column volumes (2.5 L per column) for each gradient. The methanol eluent was further separated by medium-pressure reversed-phase ODS column chromatography, using a methanol-water gradient elution (v / v, 60:40 → 100:0), yielding 5 fractions (Fr.A–Fr.E). Analysis by MALDI-TOF-MS and analytical high-performance liquid chromatography determined that the sesquicols were mainly enriched in fraction Fr.D. The fraction Fr.D was separated by semi-preparative high-performance liquid chromatography (HPLC) on a COSMOSIL Cholester column (5 μm, 10 mm × 250 mm) using methanol / water (v / v 86:14) as the mobile phase, yielding six subfractions: FrD1 to FrD6. Fraction FrD1 was isocratically eluted using methanol-water (containing 0.1% formic acid, v / v 82:18) as the mobile phase at a flow rate of 2.5 mL / min and a detection wavelength of 210 nm, ultimately yielding compounds 1 (6.7 mg), 2 (6.4 mg), 3 (2.1 mg), and 4 (11.5 mg). Fraction FrD3 was eluted isocratically with methanol-water (containing 0.1% formic acid, v / v 55:45) as the mobile phase at a flow rate of 2.5 mL / min and a detection wavelength of 210 nm, ultimately yielding compounds 5 (3.6 mg), 6 (7.3 mg), and 7 (1.6 mg). Fraction FrD5 was eluted isocratically with methanol-water (containing 0.1% formic acid, v / v 88:12) as the mobile phase at a flow rate of 2.5 mL / min and a detection wavelength of 210 nm, ultimately yielding compound 8 (4.9 mg).

[0029] 1.2 Structural analysis of compounds 1-8

[0030] The structures of compounds 1-8 were identified by analyzing high-resolution mass spectrometry, one-dimensional and two-dimensional nuclear magnetic resonance spectra as follows:

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038] The structural identification data of compounds 1-8 are as follows:

[0039] Compound 1: White powder, [a]25 D −2.67 (c 0.60, CH3OH); UV (CH3OH) l max (log e 204 (4.46) nm, the proton and carbon NMR spectra are shown in Tables 1 and 2, HRESIMS m / z 1151.7403 [M + H] + (calcd for C 55 H 99 N 12 O 14 , m / z 1151.7409).

[0040] Compound 2: White powder, [a]25 D +2.20 (c 1.18, CH3OH); UV (MeOH) l max (log e 204 (4.54) nm, the proton and carbon NMR data are shown in Tables 1 and 2, HRESIMS m / z 1165.7560 [M + H] + (calcd for C 56 H 101 N 12 O 14 , m / z 1165.7566).

[0041] Compound 3: White powder, [a]25 D +0.83 (c 0.24, CH3OH); UV (MeOH)l max (log e 204 (4.54) nm, the proton and carbon NMR data are shown in Tables 1 and 2, HRESIMS m / z 1165.7563 [M + H] + (calcd for C 56 H 101 N 12 O 14 , m / z 1165.7566).

[0042] Compound 4: White powder, [a]25 D −4.35 (c 0.69, CH3OH); UV (MeOH) l max (log e 204 (4.43) nm, the proton and carbon NMR data are shown in Tables 1 and 2, HRESIMS m / z 1165.7567 [M + H] + (calcd for C 56 H 101 N 12 O 14 , m / z 1165.7566).

[0043] Compound 5: White powder, [a]25 D +3.24 (c 0.37, CH3OH); UV (MeOH) l max (log e 203 (4.36) nm, the proton and carbon NMR spectra are shown in Tables 3 and 4, HRESIMS m / z 1179.7727 [M + H] + (calcd for C 57 H 103 N 12 O 14 , m / z 1179.7722).

[0044] Compound 6: White powder, [a]25 D +1.82 (c 0.66, CH3OH); UV (MeOH) l max (log e 204 (4.52) nm, the proton and carbon NMR data are shown in Tables 3 and 4, HRESIMS m / z 1179.7727 [M + H]+ (calcd for C 57 H 103 N 12 O 14 , m / z 1179.7722).

[0045] Compound 7: White powder, [a]25 D +2.36 (c 0.55, CH3OH); UV (MeOH) l max (log e 204 (4.45) nm, the proton and carbon NMR data are shown in Tables 3 and 4, HRESIMS m / z 1193.7874 [M + H] + (calcd for C 58 H 105 N 12 O 14 , m / z 1193.7879).

[0046] Compound 8: White powder, [a]25 D +1.76 (c 0.17, CH3OH); UV (MeOH) l max (log e 203 (4.32) nm, the proton and carbon NMR spectra are shown in Tables 3 and 4, HRESIMS m / z 1193.7876 [M + H] + (calcd for C 58 H 105 N 12 O 14 , m / z 1193.7879).

[0047] Table 1. 1H NMR spectra of compounds 1-4 (test solvent: deuterated dimethyl sulfoxide, recorded at 600 MHz)

[0048]

[0049] a Multiple peaks due to signal overlap

[0050] Table 2. Carbon NMR data of compounds 1-4 (test solvent: deuterated dimethyl sulfoxide, recorded at 150 MHz)

[0051]

[0052] Table 3. 1H NMR spectra of compounds 5–8 (test solvent: deuterated dimethyl sulfoxide, recorded at 600 MHz)

[0053]

[0054] a Multiple peaks due to signal overlap

[0055] Table 4. Carbon NMR spectra of compounds 5-8 (test solvent: deuterated dimethyl sulfoxide, recorded at 150 MHz)

[0056]

[0057] Example 2: Antibacterial activity test of compounds 1-8

[0058] 2.1 Microbial strains

[0059] Gram-positive bacteria: Staphylococcus aureus ATCC 6538, Enterococcus faecalis 160119481 and Bacillus cereus CMCC(B) 63301.

[0060] Gram-negative bacteria: Salmonella enteritidis CMCC(B) 50041, Salmonella typhi CMCC(B) 50071 and Salmonella typhimurium CMCC(B) 50115.

[0061] 2.2 Antibacterial activity test

[0062] Antimicrobial assays were performed in 96-well plates using a micro-dilution method. Compounds 1-8 were serially diluted twofold in LB medium and then inoculated with the bacterial suspension to be tested. After incubation, the lowest compound concentration that completely inhibited visible bacterial growth was recorded as the minimum inhibitory concentration (MIC). All experiments were independently repeated three times.

[0063] The results showed that most compounds exhibited significant growth inhibitory effects against Gram-positive bacteria (including Staphylococcus aureus, Enterococcus faecalis, and Bacillus cereus), and also showed significant activity against some Gram-negative bacteria (such as Salmonella enteritidis, Salmonella typhi, and Salmonella typhimurium). Among all the compounds with antibacterial activity, compound 2 showed the broadest antibacterial spectrum, with a minimum inhibitory concentration (MIC) of 20 μg / mL against Staphylococcus aureus ATCC 6538 and Bacillus cereus CMCC(B) 63301, and an MIC of 40 μg / mL against Enterococcus faecalis 160119481, Salmonella typhimurium CMCC(B) 50071, and Salmonella typhimurium CMCC(B) 50115.

[0064] Table 5. Antibacterial activity (MIC, mg / mL) of compounds 1-8

[0065]

[0066] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. An antimicrobial peptide, bishemiamycin, characterized in that, The antibacterial peptide bamicin is selected from one or several of the following compounds 1-8: 、 、 、 、 、 、 、 。 2. A pharmaceutically acceptable salt of the antibacterial peptide bamicin according to claim 1.

3. A food preservative, characterized by, A pharmaceutical composition comprising the antibacterial peptide bamicin according to claim 1 and / or a pharmaceutically acceptable salt of the antibacterial peptide bamicin according to claim 2.

4. Use of the bamicin according to claim 1 or a pharmaceutically acceptable salt of the bamicin according to claim 2 for antibacterial and / or antiseptic purposes.

5. Use according to claim 4, characterized in that, The bacteria is Staphylococcus aureus, Bacillus cereus, Enterococcus faecium, Salmonella typhi or Salmonella typhimurium.

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