Zinc chelating peptide as well as preparation method and application thereof

Abstract

The invention relates to a zinc chelating peptide. The amino acid sequence of the zinc chelating peptide is HAES. The polypeptide part of the zinc chelating peptide has high zinc chelating activity, a nitrogen atom in a histidine imidazole group at an N terminal is coordinated with Zn < 2 + > through lone pair electrons, and meanwhile, a carboxyl oxygen atom of serine at a C terminal can be used as an electron donor and is further coordinated with Zn < 2 + >, so that the zinc chelating peptide is obtained. The obtained zinc chelating peptide enables the polypeptide HAES which does not have antibacterial activity originally to show excellent broad-spectrum antibacterial activity and biological membrane dispersion effect. In addition, HAES is chelated with Zn < 2 + > through a coordinate bond, so that the stability of polypeptide is remarkably improved, and the defect that a peptidyl antibacterial agent is easily hydrolyzed by protease is overcome. The zinc chelating peptide provided by the invention provides a potential antibiotic substitute for controlling food-borne pathogenic bacteria, and also provides a potential application scheme for prolonging the shelf life of food.

Description

Technical Field

[0001] The present invention relates to the technical field of food microbiology, and particularly to a zinc-chelated peptide, a preparation method thereof, and an application thereof. Background Art

[0002] With the increasingly serious problem of drug resistance caused by the abuse of antibiotics, traditional antibiotics face bottlenecks in dealing with drug-resistant bacteria. Therefore, there is an urgent need to develop new antibacterial agents. Zinc has been widely used since ancient Egypt because of its antibacterial properties, and zinc is crucial for cell function and metabolism. Zn2+ plays an important role in cell membrane and DNA structures and participates in key cellular processes such as electron transfer and catalysis. However, excessive Zn2+ may have a fatal impact on cells. Compared with antibiotics, Zn2+ can be distinguished by the differences in bacterial and mammalian metal transport systems, which makes zinc-based antibacterial materials ideal long-term antibacterial agents, and their toxic side effects depend on the dose of Zn2+.

[0003] Although there have been studies on inorganic zinc antibacterial agents, inorganic zinc (such as nano-sized zinc oxide) is prone to precipitation and oxidation, may produce toxic side effects, and cannot provide long-lasting antibacterial activity. In recent years, antibacterial peptides have received extensive attention because they are not easily induced to produce drug-resistant strains, but they are easily affected by proteases and serum in vivo, resulting in reduced bioavailability. Zinc-chelated peptide is an innovative antibacterial agent that combines inorganic zinc and antibacterial peptides, which can improve the stability of polypeptides and effectively control the release of Zn2+, overcoming the precipitation problem of inorganic zinc. However, the research on zinc-chelated peptides is still in its initial stage and further exploration is urgently needed.

[0004] Food industry by-products such as fish scales, fish skins, and bones are rich in proteins, but they are often discarded due to their low economic value. Therefore, the research and development of fish-scale-derived zinc-chelated peptides not only provide innovative solutions for the food and pharmaceutical industries, but also contribute to the efficient utilization of resources and environmental protection. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a zinc-chelated peptide, a preparation method thereof, and an application thereof.

[0006] The technical solution adopted by the present invention is as follows:

[0007] On the one hand, the present invention provides a zinc-chelated peptide, and the amino acid sequence of the zinc-chelated peptide is HAES.

[0008] A zinc-chelating peptide proposed by the present invention has a high zinc-chelating activity in its polypeptide part. The nitrogen atom in the histidine imidazole group at the N-terminus coordinates with Zn2+ through a lone pair of electrons. At the same time, the carboxyl oxygen atom of serine at the C-terminus can act as an electron donor to further coordinate with Zn2+. The resulting zinc-chelating peptide enables HAES, which originally has no antibacterial activity, to exhibit excellent broad-spectrum antibacterial activity and biofilm dispersion effect. In addition, HAES chelates with Zn2+ through a coordination bond, significantly improving the stability of the polypeptide and avoiding the deficiency that peptide-based antibacterial agents are easily hydrolyzed by proteases. The zinc-chelating peptide of the present invention provides potential antibiotic alternatives for controlling foodborne pathogens and also provides potential application solutions for extending the shelf life of food.

[0009] On the other hand, the present invention provides a method for preparing the zinc-chelating peptide as described in the above technical solution, including the following steps: mixing an aqueous polypeptide solution with an inorganic zinc solution, adding an organic solvent for chelation reaction, washing the obtained reaction product, centrifuging to collect the precipitate, and freeze-drying to obtain the zinc-chelating peptide HAES@Zn.

[0010] In the method for preparing the zinc-chelating peptide provided by the present invention, the polypeptide can use fish scales as a natural protein source, providing a sustainable source for the green ligand of the zinc-chelating peptide, promoting the reuse of food industry by-products, and having simple chelation process operation, low cost and good safety.

[0011] Preferably, the concentration of the aqueous polypeptide solution is 0.1 - 0.15 g / mL, the concentration of the inorganic zinc solution is 2 mol / L, and the mass ratio of the polypeptide to the inorganic zinc is 0.13 - 0.15:1.

[0012] Preferably, the temperature of the chelation reaction is 30 - 40 °C, and the chelation reaction time is 12 - 16 h.

[0013] On yet another aspect, the present invention provides an application of the zinc-chelating peptide as described in the above technical solution or the zinc-chelating peptide prepared by the preparation method as described in any one of the above technical solutions in the preparation of an antibacterial agent.

[0014] Preferably, the objects to which the antibacterial agent acts include Gram-negative bacteria and Gram-positive bacteria.

[0015] The zinc-chelating peptide of the present invention has broad-spectrum antibacterial activity and can effectively inhibit a variety of Gram-negative bacteria (such as Vibrio alginolyticus 2512, TJ-2, Vibrio parahaemolyticus 2503, etc.) and Gram-positive bacteria (such as methicillin-resistant Staphylococcus aureus, Listeria monocytogenes 19115, etc.). Among them, it has good inhibitory activity against both planktonic cells and biofilm strains of Vibrio alginolyticus 2512, and has good potential in the preparation of antibacterial agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 For the structural characterization of the polypeptide and zinc-chelated peptide in Example 1, where A is the secondary mass spectrum of the polypeptide HAES, and B is the Fourier transform infrared spectrum of the polypeptide HAES and the zinc-chelated peptide HAES@Zn;

[0017] Figure 2 For the minimum inhibitory concentration (MIC) graph of the zinc-chelated peptide HAES@Zn against Vibrio alginolyticus 2512 in Example 1;

[0018] Figure 3 For the effect of the zinc-chelated peptide HAES@Zn on the anti-biofilm activity against Vibrio alginolyticus 2512 in Example 1;

[0019] Figure 4 For the antibacterial spectrum of the zinc-chelated peptide HAES@Zn in Example 1;

[0020] Figure 5 For the effect of the zinc-chelated peptide HAES@Zn on the hemolytic activity of mouse red blood cells in Example 1;

[0021] Figure 6 For the cytotoxicity effect of the zinc-chelated peptide HAES@Zn on mouse macrophages RAW 264.7 in Example 1;

[0022] Figure 7 For the stability of the zinc-chelated peptide HAES@Zn in Example 1. Detailed implementation mode

[0023] To better understand the above technical solution, the exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a clearer and more thorough understanding of the present invention and to be able to fully convey the scope of the present invention to those skilled in the art.

[0024] On the one hand, the present invention provides a zinc-chelated peptide, and the amino acid sequence of the zinc-chelated peptide is HAES.

[0025] On the other hand, the present invention provides a preparation method of a zinc-chelated peptide as described in the above technical solution, including the following steps: mixing the polypeptide aqueous solution and the inorganic zinc solution, adding an organic solvent for chelation reaction, washing the obtained reaction product, centrifuging to collect the precipitate, and freeze-drying to obtain the zinc-chelated peptide HAES@Zn.

[0026] In the embodiment of the present invention, the polypeptide can be purified from the hydrolyzate of Lutjanus erythropterus scale protein to obtain the peptide sequence and synthesized by solid-phase synthesis technology, which is not particularly limited herein. See the secondary mass spectrum of HAES Figure 1A. Additionally, in the embodiments of the present invention, anhydrous ethanol is used as the organic solvent, which can accelerate the action of Zn2+-capturing polypeptide; inorganic zinc is ZnSO4·7H2O.

[0027] In a preferred embodiment of the present invention, the concentration of the polypeptide aqueous solution is 0.1 - 0.15 g / mL, the concentration of the inorganic zinc solution is 2 mol / L, and the mass ratio of the polypeptide to the inorganic zinc is 0.13 - 0.15:1.

[0028] In a preferred embodiment of the present invention, the temperature of the chelation reaction is 30 - 40 °C, and the chelation reaction time is 12 - 16 h.

[0029] On the other hand, the present invention provides an application of a zinc chelate peptide as described in the above technical solution or a zinc chelate peptide prepared by the preparation method of any one of the above technical solutions in the preparation of an antibacterial agent.

[0030] In a preferred embodiment of the present invention, the objects to which the antibacterial agent acts include Gram-negative bacteria and Gram-positive bacteria.

[0031] Example 1

[0032] A preparation method of a zinc chelate peptide includes the following steps:

[0033] Mix a polypeptide aqueous solution (1.21 g of polypeptide HAES dissolved in 10 mL of ultrapure water) with an inorganic zinc solution (15 mL, 2 mol / L ZnSO4·7H2O), add 2.5 times the volume of anhydrous ethanol of the mixed solution for chelation reaction, react at 35 °C for 16 h, then wash the obtained reaction product with 9 times the volume of anhydrous ethanol, centrifuge to collect the precipitate, and freeze-dry to obtain the zinc chelate peptide HAES@Zn.

[0034] Perform Fourier transform infrared spectroscopy on the above-mentioned polypeptide HAES and the zinc chelate peptide HAES@Zn, and the results are recorded in Figure 1 B.

[0035] The results are as Figure 1 shown in B. After HAES coordinates with Zn2+ to form a chelate, the position of its characteristic absorption peak shifts, and the absorption intensity also changes. The amide I band generated by the stretching vibration of C=O, after coordinating with Zn2+, its absorption peak shifts from 1660 cm -1 to 1630 cm -1 . After coordinating with Zn2+, the absorption peaks of HAES at 1540 cm -1 and 1440 cm -1 significantly weaken or even disappear, indicating that -COO- participates in the covalent binding of Zn2+. Similarly, the absorption peak of HAES related to the stretching vibration of the -C-O bond (1190 cm -1and 1140 cm -1 ) shifted towards 1100 cm -1 , possibly due to the formation of -C-O-Zn complex. The in-plane vibration of the O═CN bond caused the absorption peak at 626 cm -1 to shift towards 600 cm -1 , indicating that the coordination of Zn2+ may increase the electron cloud density of the oxygen adjacent to C═O in HAES. This series of spectral changes indicates that amino nitrogen, carboxyl oxygen, and carbonyl oxygen are all involved in the coordination of Zn2+.

[0036] The performance of the above zinc-chelated peptide HAES@Zn was measured, and the results were recorded in Figures 2 - 7 .

[0037] (1) Antibacterial activity of HAES@Zn against Vibrio alginolyticus 2512

[0038] Test method: The MIC was determined by the microbroth dilution method. The specific operation is as follows: First, add 50 μL of LB broth to a 96-well plate, and then add 50 μL of HAES@Zn to the first column of the 96-well plate and mix well. Gradient dilution was carried out along the second to tenth columns using the two-fold dilution method, and then 50 μL of the strain 2512 suspension (1×10 5 CFU / mL) was added. The eleventh column was used as a blank control (50 μL of medium), and the twelfth column was used as a negative control (50 μL of medium + 50 μL of sterile water). Incubate at 30 °C in the dark for 24 h, and the lowest concentration corresponding to no bacterial growth was the MIC of HAES@Zn.

[0039] The results are as Figure 2 shown. The MIC of HAES@Zn against Vibrio alginolyticus 2512 was 73.86 μg / mL.

[0040] (2) Anti-biofilm activity of HAES@Zn against Vibrio alginolyticus 2512

[0041] Test method: Determined by crystal violet staining. Add cover slips to a 6-well plate, take 200 μL of activated Vibrio alginolyticus 2512 and culture it in LB broth. After incubating at 30 °C for 12 h, add HAES@Zn and incubate at 30 °C for 24 h. Gently wash each well 3 times with phosphate buffer (PBS, pH = 7.2). Air-dry the adherent cells at 60 °C for 30 min, then stain with 200 μL of 0.1% (w / v) crystal violet at room temperature for 5 min, and gently wash each well again with PBS (pH = 7.4). After staining, dissolve the stain in glacial acetic acid for 10 min. Subsequently, measure the absorbance at 600 nm using a microplate reader and observe with an optical microscope.

[0042] Figure 3The results showed that HAES@Zn not only had an inhibitory effect on the planktonic Vibrio alginolyticus 2512, but also could inhibit the biofilm formation of strain 2512, indicating its potential in pathogen inhibition.

[0043] (3) Antibacterial spectrum of HAES@Zn against pathogenic bacteria

[0044] Test method: Vibrio alginolyticus 2512, Vibrio alginolyticus TJ-2, Vibrio parahaemolyticus 2503, Vibrio harveyi 2510, Escherichia coli K88, Pseudomonas aeruginosa PAO1, Listeria monocytogenes 19115, Bacillus cereus 63302, Enterococcus faecalis 29122, methicillin-resistant Staphylococcus aureus 43300 (MRSA) were used as indicator bacteria respectively. The above-mentioned indicator bacteria (1×10 7 CFU / mL) were inoculated on LB agar medium by mixed bacteria pour plate method. Then, holes were punched with a 6 mm puncher, and 50 μL of HAES@Zn (50 mg / mL) was added into the holes. The plates were placed in a biochemical incubator for 24 h, and the diameter of the inhibition zone (mm) was evaluated by the cross method.

[0045] From Figure 4 it can be seen that HAES@Zn had antibacterial activity against all the above ten foodborne pathogenic bacteria, showing broad-spectrum antibacterial activity. Among them, the inhibitory activity was as follows: Vibrio harveyi 2510 was the strongest, with an inhibition zone diameter of 28.13±0.53 mm; Vibrio alginolyticus 2512 was the second (28.03±0.31 mm); Escherichia coli K88 (25.30±0.51 mm) and MRSA (25.13±0.62 mm) followed closely; and the activity against Pseudomonas aeruginosa PAO1 was the worst (20.17±0.41 mm).

[0046] (4) Hemolytic activity of HAES@Zn on mouse red blood cells

[0047] Determination method: The hemolytic activity of HAES@Zn was studied by the red blood cell lysis method. First, fresh mouse blood was prepared into a 4% red blood cell suspension. 500 μL of the red blood cell suspension was added to a 1.5 mL centrifuge tube, and then 500 μL of PBS (negative control), 0.1% Triton X-100 (positive control) and different concentration gradients of HAES@Zn were added to each centrifuge tube respectively. Then, the mixture was incubated in a 37 °C constant temperature incubator for 4 h. Subsequently, the mixture was centrifuged, and the supernatant was transferred to a 96-well plate, and the absorbance was measured at OD540.

[0048] From Figure 5 it can be known that HAES@Zn did not cause hemolysis of mouse red blood cells, indicating its good biocompatibility.

[0049] (5) Cytotoxicity of HAES@Zn against mouse macrophages RAW264.7

[0050] Determination method: The cytotoxicity of HAES@Zn against RAW264.7 cells was determined by the thiazolyl blue colorimetric method. The specific steps are as follows: After resuscitating the cryopreserved cells, inoculate them into the medium (containing 10% fetal bovine serum and 1% double antibody) and subculture (37 °C, 5% CO2); and adjust its concentration to 2 - 4×10 5 cells / mL with the medium; Take 50 μL of the cell suspension and 50 μL of different concentrations of antibacterial agents and incubate them in a 96-well plate for 24 h (37 °C, 5% CO2), then add 25 μL of MTT (5 mg / mL) to the 96-well plate and continue to incubate for 4 h; After the incubation, discard the supernatant, dissolve the crystals at the bottom of the well with 100 μL of dimethyl sulfoxide, and measure the absorbance at 570 nm.

[0051] Figure 6 The results showed that at the MIC concentration, HAES@Zn had good biocompatibility with RAW264.7 cells, and the cell survival rate > 80%.

[0052] (6) Stability analysis of HAES@Zn

[0053] Determination method: Use 1 mg / mL pepsin solution (pH = 2.0) and 1 mg / mL trypsin solution (pH = 7.0) to simulate gastric fluid digestion and intestinal fluid digestion respectively. First, prepare the HAES@Zn solution with ultrapure water and adjust the pH of the solution to 2.0. Then, add the pepsin solution to it so that the mass ratio of pepsin to HAES@Zn in the system is 1:50, and place it at 37 °C and 200 rpm for 2 h, and adjust the pH of the solution to 7.0, and add the trypsin solution. At this time, the mass ratio of trypsin to HAES@Zn is 1:25, and continue to react at 37 °C and 200 rpm for 2 h. After the reaction, place it in a boiling water bath at 100 °C to inactivate the enzyme. Then centrifuge and take the supernatant, and refer to the agar diffusion method for antibacterial activity determination.

[0054] As Figure 7 shown, after in vitro simulated gastrointestinal digestion, the inhibitory activity of HAES@Zn against strain 2512 did not change significantly, indicating that HAES@Zn has excellent stability.

[0055] In summary, the zinc chelated peptide HAES@Zn of the present invention has excellent biocompatibility, stability, broad-spectrum antibacterial activity and biofilm dispersion effect.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A zinc-chelating peptide, characterized in that, The amino acid sequence of the zinc chelated peptide is HAES.

2. A method for preparing the zinc-chelated peptide as described in claim 1, characterized in that, It includes the following steps: Mix the polypeptide aqueous solution with the inorganic zinc solution, add an organic solvent for chelation reaction, wash the obtained reaction product, centrifuge to collect the precipitate, and freeze-dry to obtain the zinc chelated peptide HAES@Zn.

3. The preparation method of the zinc-chelated peptide according to claim 2, characterized in that, The concentration of the polypeptide aqueous solution is 0.1 - 0.15 g / mL, the concentration of the inorganic zinc solution is 2 mol / L, and the mass ratio of the polypeptide to the inorganic zinc is 0.13 - 0.15:

1.

4. The preparation method of the zinc chelated peptide according to claim 2, wherein, The temperature of the chelation reaction is 30 - 40 °C, and the chelation reaction time is 12 - 16 h.

5. Use of the zinc chelated peptide as claimed in claim 1 or the zinc chelated peptide prepared by the preparation method as claimed in any one of claims 2 - 4 in the preparation of an antibacterial agent.

6. The application according to claim 5, wherein The objects on which the antibacterial agent acts include Gram-negative bacteria and Gram-positive bacteria.

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