A fructus mume polypeptide with anti-drug resistant bacteria activity and application thereof

Abstract

The present application belongs to the technical field of peptide preparation, and particularly relates to a Fructus mume polypeptide with anti-drug resistant bacteria activity and application thereof. The amino acid sequence of the Fructus mume polypeptide provided by the present application is SEQ ID NO. 1. The Fructus mume polypeptide provided by the present application has excellent anti-drug resistant bacteria activity, and by virtue of the property, the Fructus mume polypeptide can be added as an active ingredient into a water environment treatment agent, a disinfectant or an antibacterial composition, and used for controlling drug resistant microorganisms in a water environment such as natural water, reclaimed water, industrial circulating cooling water and landscape water.

Description

Technical Field

[0001] This invention belongs to the field of functional polypeptide preparation technology, specifically relating to a plum polypeptide with anti-drug-resistant bacterial activity and its application. Background Technology

[0002] Ume (dried plum) is the dried, nearly mature fruit of the plum tree (Prunus mume), a plant in the Rosaceae family. Ancient Chinese medical texts, such as the *Shennong Bencao Jing*, record its effects as astringent to the lungs, intestines, and as an anti-parasitic agent, and as a saliva-generating agent. Modern pharmacological studies have shown that ume is rich in organic acids, flavonoids, and protein peptides, exhibiting significant potential in antibacterial, antioxidant, and anti-inflammatory properties. However, current research and development of the active ingredients in ume remains significantly limited, particularly in the systematic study of its derived peptides against drug-resistant microorganisms, which restricts the development and application of its high-value-added products.

[0003] Currently, the control of drug-resistant microorganisms in aquatic environments faces three major bottlenecks: 1) Long-term presence of sub-inhibitory concentrations of antibiotic residues in water bodies (originating from medical wastewater, pharmaceutical wastewater, aquaculture wastewater, etc.), leading to widespread drug resistance in environmental microorganisms such as *Pseudomonas aeruginosa* and *Escherichia coli*; 2) Decreased effectiveness of traditional disinfection technologies (such as chlorine disinfection and ultraviolet light) against some drug-resistant bacteria, and chemical disinfectants easily generate toxic byproducts (such as trihalomethanes); 3) Safety and environmental residue issues with mainstream chemical disinfectants on the market, and long-term use further exacerbates the spread of drug resistance. In particular, carbapenem-resistant *Pseudomonas aeruginosa* (CRPA) is listed as a Level 1 (critical priority) pathogen on the World Health Organization's "Priority Pathogens" list, and is widely detected in aquatic environments (such as reclaimed water, rivers, and cooling water), posing a serious threat to public health and safety. Therefore, there is an urgent need to use modern biotechnology to directionally prepare *Prunus mume* polypeptides with clear anti-drug-resistant bacterial activity to overcome technical barriers and achieve high-value utilization of *Prunus mume* resources. Summary of the Invention

[0004] The purpose of this invention is to provide a polypeptide prepared from dried plum that has anti-drug resistant bacteria activity, especially a polypeptide with highly efficient inhibitory activity against carbapenem-resistant Pseudomonas aeruginosa (CRPA), thereby improving the high-value utilization of dried plum resources.

[0005] This invention first provides a ume polypeptide, the amino acid sequence of which is shown in SEQ ID NO: 1 of the sequence listing;

[0006] In another aspect, the present invention also provides the application of the aforementioned ume polypeptide in the reprocessing of antibacterial products.

[0007] Furthermore, the antibacterial product is a product used for the prevention and control of bacteria in water bodies;

[0008] The water body mentioned refers to natural water bodies, reclaimed water, industrial circulating cooling water, or landscape water.

[0009] The bacteria in question are carbapenem-resistant Pseudomonas aeruginosa.

[0010] The present invention also provides an antibacterial composition for use in aquatic environments, comprising the aforementioned ume polypeptide.

[0011] The ume polypeptide provided by this invention has excellent anti-drug-resistant bacteria activity, especially strong inhibitory activity against carbapenem-resistant Pseudomonas aeruginosa (CRPA). Utilizing this property, the ume polypeptide can be added as an active ingredient to water treatment agents, disinfectants, or antibacterial compositions to control CRPA in various water environments, ensuring aquatic biosafety and public health safety. Attached Figure Description

[0012] Figure 1 Molecular docking diagram of ume peptide and outer membrane protein OprD. Detailed Implementation

[0013] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0014] Example 1: Preparation of Prunus mume polypeptide with anti-drug-resistant bacteria activity

[0015] Take dried plum pulp powder, add deionized water at a material-to-liquid ratio of 1:40 (g / mL), mix well, add 2.5% neutral protease, adjust to pH 7.0, and enzymatically hydrolyze in a 55℃ water bath with shaking for 3 hours. Inactivate the enzymes by heating the hydrolysate at 95℃ for 10 minutes, centrifuge at 12000 r / min for 15 minutes, collect the supernatant, and freeze-dry to obtain crude plum polypeptide extract.

[0016] Weigh 10.00 g of crude extract and dilute to 100 mL with ultrapure water to prepare a 100 mg / mL stock solution. Separation was performed using a reversed-phase high-performance liquid chromatography (RP-HPLC) system with a Dubhe C18 preparative column (250 mm × 20 mm, 10 μm), a flow rate of 8 mL / min, and a detection wavelength of 220 nm. Gradient elution with methanol-water was performed, and the 8%, 25%, 45%, and 75% methanol eluates were collected and designated as F1, F2, F3, and F4, respectively. Each fraction was concentrated and lyophilized for later use.

[0017] The antimicrobial activity of various components of the *Prunus mume* polypeptide was screened using the micro-broth dilution method. *Carbapenem-resistant Pseudomonas aeruginosa* (CRPA) was used as the indicator strain (isolated from water samples from an urban water treatment plant), and the minimum inhibitory concentration (MIC) of each component was determined. The results showed that component F2 exhibited a significant antimicrobial activity against drug-resistant bacteria, with an MIC value of 32 μg / mL, significantly superior to the control group and other experimental groups. This result indicates that component F2 is rich in *Prunus mume* polypeptide with antimicrobial activity. Specific experimental data are shown in Table 1.

[0018] Table 1: Antimicrobial activity (MIC values) of different components of ume peptides against drug-resistant bacteria

[0019] MIC (μg / mL) >256 128 256 32 64 128

[0020] The structure of the Prunus mume polypeptide F2 fraction was determined using an LCMS-8050 triple quadrupole LC-MS system. Chromatographic separation was performed using a Halo C18 reversed-phase column (100 mm × 2.1 mm, 2.7 μm), maintained at 35 °C, with a mobile phase flow rate of 0.25 mL / min. Mobile phase A was 0.05% formic acid aqueous solution, and mobile phase B was 0.05% formic acid acetonitrile solution. The gradient elution program was as follows: 0–3 min to maintain 8% B, 3–8 min to linearly increase from 8% B to 15% B, 8–15 min to increase from 15% B to 35% B, and 15–20 min to increase from 35% B to 60% B. Mass spectrometry was performed using an electrospray ionization source in positive ion scanning mode. The ion spray voltage was 3.5 kV, the nebulizer gas flow rate was 8.0 L / min, the drying gas flow rate was 10.0 L / min, the collision gas flow rate was 9.0 L / min, the interface temperature was 280°C, the desolvation tube temperature was 250°C, and the heating module temperature was 350°C. The mass scan range was 100–1800 m / z. The obtained mass spectrometry data were sequence aligned and identified using proteomics software, and a total of 38 valid peptides were identified.

[0021] The bacterial cell membrane is a key target for antimicrobial peptides. Antimicrobial peptides interact electrostatically with the negatively charged lipopolysaccharides on the bacterial cell membrane surface through their positively charged amino acid residues, inserting themselves into the membrane to form transmembrane channels. This disrupts membrane integrity, leading to the leakage of intracellular contents such as potassium ions and ATP, ultimately causing bacterial death. This single membrane disruption mechanism makes it difficult for bacteria to develop drug resistance through simple gene mutations.

[0022] To verify the interaction mechanism between the active ingredient and the bacterial cell membrane, this invention employs a molecular docking strategy combining AutoDockFR and Rosetta FlexPepDock. This method first examines the flexible conformational changes of the target protein using AutoDockFR, then refines the binding conformation and accurately assesses the binding free energy using Rosetta FlexPepDock. Finally, based on a comprehensive score, the binding mode and interaction strength between the ume peptide and the target are predicted, providing a molecular-level mechanistic basis for its anti-drug-resistant bacterial efficacy. The prediction results are shown in Table 2. Six peptides have high comprehensive scores, suggesting anti-drug-resistant bacterial activity. Among them, the peptide shown in SEQ ID NO.1 has the highest comprehensive score, suggesting the best anti-drug-resistant bacterial activity.

[0023] Table 2: Comprehensive Score Table for Plum Peptide Molecular Docking

[0024] 1 GPIGQPGPTGPSGER -9.1 -12.8 2 WVKLPGGTHHIER -8.0 -9.5 3 IEELETEAEHERQA -7.4 -8.2 4 AVRNTLIEGRGEFS -6.1 -7.6 5 VAIEDKVAKGL -5.3 -5.5 6 TNDIALIKLPSPVSL -5.1 -5.4

[0025] The above molecular docking results confirm at the molecular level that the ume peptide exerts its anti-CRPA activity by disrupting the bacterial cell membrane (targeting the OprD outer membrane protein).

[0026] Example 2: Activity assay of ume peptide (SEQ ID NO.1) against carbapenem-resistant Pseudomonas aeruginosa (CRPA).

[0027] 1) Synthesis of the target polypeptide sequence

[0028] The target polypeptide sequence (SEQ ID NO.1: GPIGQPGPTGPSGER) was successfully prepared using solid-phase synthesis technology.

[0029] 2) Experimental strains and materials

[0030] Experimental strain: Carbapenem-resistant Pseudomonas aeruginosa.

[0031] Experimental sample: Ume polypeptide shown in SEQ ID NO.1, prepared by solid-phase synthesis.

[0032] Culture media: Mueller-Hinton broth (MHB) and Mueller-Hinton agar (MHA).

[0033] (3) Determination of minimum inhibitory concentration (MIC)

[0034] Following the Clinical Laboratory Standards Institute (CLSI) microbroth dilution method, the synthetic peptides were dissolved in sterile water and serially diluted to different concentrations (0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256 μg / mL). The diluted peptides were then added to 96-well plates, with each well containing a logarithmic growth phase CRPA bacterial suspension (final concentration approximately 5 × 10⁻⁶). 5 (CFU / mL), incubate at 35°C for 18–24 hours, and the lowest concentration at which no bacterial growth is observed to the naked eye is the MIC value.

[0035] (4) Determination of minimum bactericidal concentration (MBC)

[0036] Take 100 μL of culture medium from the wells where no visible growth is observed in the MIC assay, spread it on MHA solid medium, and incubate at 35°C for 24 hours. The lowest concentration that kills 99.9% of the original inoculum colonies is taken as the MBC value.

[0037] (5) Dose-response relationship experiment

[0038] A control group (without peptide) and different dose groups (16, 32, 64, 128 μg / mL) were set up to determine the inhibition rate of CRPA.

[0039] Inhibition rate (%) = (1 − (OD) 600 control group −OD 600 Blank group) / (OD) 600 Experimental group −OD 600 (Blank group) × 100%

[0040] Antimicrobial activity evaluation using the microbroth dilution method showed that the peptide had a significant inhibitory effect on carbapenem-resistant Pseudomonas aeruginosa (CRPA), exhibiting a clear dose-response relationship. As shown in Table 3, the MIC value of this Prunus mume peptide against CRPA was 32 μg / mL, and the MBC value was 64 μg / mL. When the peptide concentration reached 64 μg / mL, the inhibition rate against CRPA exceeded 99.9%. These results fully demonstrate that the synthesized peptide has significant antimicrobial bioactivity against carbapenem-resistant Pseudomonas aeruginosa.

[0041] Table 3: MIC and MBC of ume peptide (SEQ ID NO.1) against CRPA

[0042] CRPA 32 64

[0043] Table 4: Inhibition rate of different doses of ume peptide on CRPA

[0044] control group 0 0 low-dose group 16 68.5 medium dose group 32 95.2 High-dose group 64 99.9 Ultra-high dose group 128 99.9

[0045] Example 3: The bactericidal effect of ume peptides on CRPA in different aquatic matrices.

[0046] Three types of water samples were collected: ① river water; ② reclaimed water; ③ cooling circulating water. Carbapenem-resistant Pseudomonas aeruginosa (CRPA) was added exogenously to each water sample to a final concentration of approximately 10. 5 The concentration was increased to CFU / mL, and then ume peptide (SEQ ID NO. 1) was added to a final concentration of 50 mg / L. The mixture was incubated at 25°C for 60 minutes, and the number of surviving CRPA bacteria was determined. The results showed that ume peptide exhibited stable bactericidal effects in different aquatic substrates, with a bactericidal rate of over 99.9%. Specific data are shown in Table 5.

[0047] Table 5: Bactericidal effect of ume peptides on CRPA in different aquatic matrices

[0048] River water <![CDATA[1.2 × 10 5 ]]> <10 >4.1 Reclaimed water <![CDATA[1.5 × 10 5 ]]> 1.5 × 10² 3.0 Cooling circulating water <![CDATA[1.0 × 10 5 ]]> <10 >4.0

[0049] In summary, the *Prunus mume* polypeptide provided by this invention exhibits good water solubility and can be prepared on a large scale using solid-phase synthesis technology. This polypeptide demonstrates strong inhibitory activity against carbapenem-resistant *Pseudomonas aeruginosa* (CRPA), a critical priority pathogen listed by the World Health Organization in aquatic environments, with a MIC value reaching 32 μg / mL. Molecular docking studies further confirmed that this polypeptide exerts its bactericidal effect through a membrane disruption mechanism targeting the outer membrane protein OprD. This polypeptide can be widely applied to the control of CRPA in aquatic environments such as natural water bodies, reclaimed water, industrial circulating cooling water, and landscape water, ensuring aquatic biosafety and public health safety, and possesses significant environmental application value and promising industrialization prospects.

Claims

1. A type of ume polypeptide, characterized in that, The amino acid sequence of the polypeptide is SEQ ID NO:

1.

2. The application of the ume polypeptide described in claim 1 in the reprocessing of antibacterial products.

3. The application as described in claim 2, characterized in that, The antibacterial product is used for the prevention and control of bacteria in water.

4. The application as described in claim 3, characterized in that, The water body mentioned can be a natural water body, reclaimed water, industrial circulating cooling water, or landscape water.

5. The application as described in claim 3, characterized in that, The bacteria in question are carbapenem-resistant Pseudomonas aeruginosa.

6. An antibacterial composition for use in aquatic environments, characterized in that, The composition contains the ume polypeptide of claim 1.

7. A method for purifying and sterilizing water, characterized in that, The method involves using the antibacterial composition of claim 6 for purification treatment.

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