Zinc chelating peptide with antibacterial activity and preparation method and application thereof

Abstract

The invention relates to a zinc chelating peptide with antibacterial activity. The amino acid sequence of the zinc chelating peptide is HLG. According to the zinc chelating peptide with the antibacterial activity, the polypeptide part has high zinc affinity, nitrogen atoms in a histidine imidazole group at the N end are coordinated with Zn < 2 + > through lone pair electrons, meanwhile, carboxyl oxygen atoms of glycine at the C end can serve as electron donors and are further coordinated with Zn < 2 + >, and the zinc chelating peptide with the antibacterial activity is obtained. The obtained zinc chelating peptide enables HLG which does not have antibacterial activity originally to show excellent broad-spectrum antibacterial activity. HLG is chelated with Zn < 2 + > through a coordinate bond, so that a synergistic interaction effect can be achieved, on one hand, the stability of polypeptide is remarkably improved, and the defect that a peptidyl antibacterial agent is easily hydrolyzed by protease is overcome; on the other hand, the release of Zn < 2 + > is effectively controlled, the precipitation problem of inorganic zinc is solved, and the antibacterial durability is improved. In addition, the zinc chelating peptide also has excellent biocompatibility and biofilm eradication effect, and has important application value in the fields of food safety and sustainable agriculture.

Description

Technical Field

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

[0002] Food safety is the biggest livelihood issue. Among them, microbial contamination is the main factor affecting food safety. However, with the increasingly serious problem of drug resistance caused by the abuse of antibiotics, traditional antibiotics face bottlenecks in dealing with drug-resistant bacteria.

[0003] In the research process of antibiotic substitutes, it is found that peptide-metal ion chelates have antibacterial activity. Their inhibition mechanisms against bacteria mainly include destroying the cytoplasmic membrane structure resulting in the leakage of intracellular contents and bacterial apoptosis, and targeting intracellular nucleic acids, proteins and other biological macromolecules through the cytoplasmic membrane to affect cell metabolism, etc., showing great application potential in food preservation. The characteristics of different peptide-metal ion chelates are different, and there are also significant differences in functional effects. At present, the research on peptide-metal ion chelates with antibacterial activity is still not deep enough.

[0004] A large amount of fish scale by-products are generated during the fish processing process. Due to the low market value of these by-products, there are often problems such as direct discard and low processing utilization rate. Therefore, it has very important practical significance and application prospects to research and develop fish scale-derived peptide-metal ion chelates with high antibacterial efficacy. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a zinc-chelated peptide with antibacterial activity, 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 with antibacterial activity, and the amino acid sequence of the zinc-chelated peptide is HLG.

[0008] A zinc-chelated peptide with antibacterial activity proposed by the present invention has a high zinc affinity 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 glycine at the C-terminus can act as an electron donor to further coordinate with Zn2+. The resulting zinc-chelated peptide enables HLG, which originally has no antibacterial activity, to exhibit excellent broad-spectrum antibacterial activity. HLG chelates with Zn2+ through a coordination bond to play a synergistic effect. On the one hand, it significantly improves the stability of the polypeptide and avoids the deficiency that peptide-based antibacterial agents are easily hydrolyzed by proteases. On the other hand, it effectively controls the release of Zn2+, overcomes the precipitation problem of inorganic zinc, and improves the antibacterial persistence. In addition, the zinc-chelated peptide of the present invention also has excellent biocompatibility and biofilm eradication effect, and has important application value in the fields of food safety and sustainable agriculture.

[0009] On the other hand, the present invention provides a preparation method of a zinc-chelated peptide with antibacterial activity as described in the above technical solution, including the following steps: mixing a polypeptide solution and an inorganic zinc solution, adding absolute ethanol, and carrying out a chelation reaction to obtain the zinc-chelated peptide HLG@Zn with antibacterial activity.

[0010] The preparation method adopted by the present invention has a simple process and can use fish scales as a green ligand source for zinc-chelated peptides, which has reference significance for the intensive processing and high-value utilization of fish scales.

[0011] Preferably, the concentration of the 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 conditions of the chelation reaction are as follows: the temperature is 30 - 40 °C, and the time is 12 - 16 h.

[0013] Preferably, it further includes post-treatment of the product of the chelation reaction, and the post-treatment includes: washing the product with absolute ethanol, centrifuging to collect the precipitate, and freeze-drying.

[0014] On yet another aspect, the present invention provides an application of a zinc-chelated peptide with antibacterial activity as described in the above technical solution or a zinc-chelated peptide with antibacterial activity prepared by the preparation method as described in any one of the above technical solutions in the preparation of a medicament for anti-Gram-negative bacteria and / or Gram-positive bacteria.

[0015] The zinc-chelated peptide of the present invention has broad-spectrum antibacterial activity and can 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, Enterococcus faecalis 29212, etc.). 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 polypeptide HLG, and B is the Fourier transform infrared spectrum of polypeptide HLG and zinc-chelated peptide HLG@Zn;

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

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

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

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

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

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

[0023] The following combines the attached drawings to detail the specific embodiments of the present invention, but does not make any limitations on the claims of the present invention.

[0024] On the one hand, an embodiment of the present invention provides a zinc-chelated peptide with antibacterial activity, and the amino acid sequence of the zinc-chelated peptide is HLG.

[0025] On the other hand, an embodiment of the present invention provides a preparation method of a zinc-chelated peptide with antibacterial activity as described in the above technical solution, including the following steps: mixing the polypeptide solution and the inorganic zinc solution, adding absolute ethanol, and performing a chelation reaction to obtain the zinc-chelated peptide HLG@Zn with antibacterial activity.

[0026] In the embodiment of the present invention, the polypeptide can be purified from the hydrolyzate of Lutjanus erythropterus fish scale protein to obtain the peptide sequence and synthesized by solid-phase synthesis technology, which is not particularly limited herein. Refer to Figure 1 A for the secondary mass spectrum of HLG. In addition, adding absolute ethanol during the chelation reaction in the present invention can play a role in accelerating the capture of Zn2+ by the polypeptide; the inorganic zinc uses ZnSO4·7H2O.

[0027] In a preferred embodiment of the present invention, the concentration of the 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.

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

[0029] In a preferred embodiment of the present invention, it further includes post-treatment of the product of the chelation reaction. The post-treatment includes: washing the product with absolute ethanol, centrifuging to collect the precipitate, and freeze-drying.

[0030] Another aspect of the embodiments of the present invention provides an application of a zinc-chelated peptide with antibacterial activity as described in the above technical solution or a zinc-chelated peptide with antibacterial activity prepared by the preparation method of any one of the above technical solutions in the preparation of a medicament for resisting Gram-negative bacteria and / or Gram-positive bacteria.

[0031] Example 1

[0032] A preparation method of a zinc-chelated peptide with antibacterial activity includes the following steps:

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

[0034] Perform Fourier transform infrared spectroscopy on the above-mentioned polypeptide HLG and the zinc-chelated peptide HLG@Zn, and analyze its chelation mechanism.

[0035] As Figure 1 shown in B, after HLG coordinates with Zn2+ to form a chelate, the position of its characteristic absorption peak shifts, and the absorption intensity also changes. The absorption peak in the range of 3500 cm -1 ~2900 cm -1 reflects the stretching vibrations of N-H and O-H. This phenomenon is due to the nitrogen atom providing a lone pair of electrons to Zn2+, resulting in the absorption peak shifting from 2960 cm -1 to 3360 cm -1 . The amide I band (1670 cm -1 ) generated by the C=O stretching vibration shifts its absorption peak to 1650 cm -1 after coordinating with Zn2+. After coordinating with Zn2+, the absorption peak of HLG at 1560 cm -1and 1440 cm -1 The absorption peaks at -1 disappeared, indicating that -COO- participated in the covalent binding of Zn2+. Similarly, the absorption peak related to the stretching vibration of the -C-O bond in HLG (1190 cm -1 ) shifted to 1100 cm -1 , probably due to the formation of -C-O-Zn complex. The absorption peak at 627 cm -1 caused by the in-plane vibration of the O═CN bond shifted to 601 cm -1 , indicating that the coordination of Zn2+ may increase the electron cloud density of the O adjacent to C═O in HLG. This series of spectral changes indicates that amino nitrogen, carboxyl oxygen, and carbonyl oxygen all participated in the coordination of Zn2+.

[0036] The above zinc chelated peptide HLG@Zn was subjected to performance measurement, and the results were recorded in Figures 2 - 7 .

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

[0038] Measurement method: The MIC was determined by the microbroth dilution method. The specific operation was as follows: First, 50 μL of LB broth was added to the 96-well plate, and then 50 μL of HLG@Zn was added to the first column of the 96-well plate and mixed evenly. Gradient dilution was performed 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). It was cultured at 30 °C for 24 h, and the lowest concentration corresponding to no bacterial growth was the MIC of HLG@Zn.

[0039] The results are as Figure 2 shown. The MIC of HLG@Zn against Vibrio alginolyticus 2512 was 75.01 μg / mL.

[0040] (2) Antibiofilm activity of HLG@Zn against Vibrio alginolyticus 2512

[0041] Determination method: The determination was carried out by crystal violet staining. A cover glass was added to a 6-well plate. 200 μL of activated Vibrio alginolyticus 2512 was taken and cultured in LB broth. After incubation at 30 °C for 12 h, HLG@Zn was added, and then incubated at 30 °C for 24 h. Each well was gently washed 3 times with phosphate buffer (PBS, pH = 7.2). The adherent cells were air-dried at 60 °C for 30 min, and then stained with 200 μL of 0.1% (w / v) crystal violet at room temperature for 5 min, and each well was gently washed again with PBS (pH = 7.4). After staining, the stain was dissolved in glacial acetic acid for 10 min. Subsequently, the absorbance at 600 nm was measured using a microplate reader, and observed with an optical microscope.

[0042] Figure 3 The results showed that HLG@Zn not only had an inhibitory effect on planktonic Vibrio alginolyticus 2512, but also inhibited the biofilm formation of strain 2512.

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

[0044] Determination 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 (MRSA) 43300 were used as indicator bacteria respectively. The above-mentioned indicator bacteria (1×10 7 CFU / mL) were mixed and poured onto a plate and inoculated on LB agar medium. Subsequently, a hole was punched with a 6 mm puncher, and 50 μL of HAES@Zn (50 mg / mL) was added to the hole, and then placed in a biochemical incubator for 24 h. The diameter (mm) of the inhibition zone was evaluated by the cross method.

[0045] Figure 4 The results showed that HLG@Zn had antibacterial activities against foodborne pathogenic bacteria such as Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, methicillin-resistant Staphylococcus aureus, etc. The activity levels were as follows: Vibrio alginolyticus 2512 was the strongest, at 28.60 ± 0.43 mm, followed by 2510 (26.70 ± 0.29 mm), MRSA (26.40 ± 0.51 mm) and Vibrio alginolyticus TJ-2 (24.43 ± 0.49 mm), and the activity against Pseudomonas aeruginosa PAO1 was the worst, at 20.43 ± 0.58 mm.

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

[0047] Determination method: The hemolytic activity of HLG@Zn was studied by the erythrocyte lysis method. First, fresh mouse blood was prepared into a 4% erythrocyte suspension. 500 μL of the erythrocyte 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 HLG@Zn were added to each centrifuge tube. Then, the mixture was incubated in a constant temperature incubator at 37 °C 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] Figure 5 The results showed that HLG@Zn did not cause hemolysis of mouse erythrocytes.

[0049] (5) Cytotoxicity of HLG@Zn against mouse macrophage RAW264.7

[0050] Determination method: The toxicity of HLG@Zn against RAW264.7 cells was determined by the thiazolyl blue colorimetric method. The specific steps were as follows: The cryopreserved cells were resuscitated and inoculated in a medium (containing 10% fetal bovine serum and 1% double antibody) for subculture (37 °C, 5% CO2); and its concentration was adjusted to 2 - 4×10 5 cells / mL with the medium; 50 μL of the cell suspension and 50 μL of different concentration antibacterial agents were taken and incubated in a 96-well plate for 24 h (37 °C, 5% CO2), and then 25 μL of MTT (5 mg / mL) was added to the 96-well plate and incubated for another 4 h; after the incubation, the supernatant was discarded, and the crystals at the bottom of the well were dissolved with 100 μL of dimethyl sulfoxide, and the absorbance was measured at 570 nm.

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

[0052] (6) Stability analysis of HLG@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 HLG@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 HLG@Zn in the system is 1:50, and place it at 37 °C and 200 rpm for reaction for 2 h. Then adjust the pH of the solution to 7.0 and add the trypsin solution. At this time, the mass ratio of trypsin to HLG@Zn is 1:25, and continue to react at 37 °C and 200 rpm for 2 h. After the reaction is completed, place it in a boiling water bath at 100 °C to inactivate the enzyme. Subsequently, centrifuge to take the supernatant, and refer to the agar diffusion method for antibacterial activity determination.

[0054] Figure 7 The results show that after in vitro simulated gastrointestinal digestion, the antibacterial activity of HLG@Zn against strain 2512 has not changed significantly, and it has excellent stability.

[0055] In summary, the zinc-chelated peptide HLG@Zn with antibacterial activity of the present invention has excellent biocompatibility, stability, broad-spectrum antibacterial activity and biofilm eradication effect, provides potential antibiotic alternatives for controlling foodborne pathogens, and also provides potential application solutions for extending the shelf life of aquatic products.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; 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 make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A zinc-chelating peptide with antibacterial activity, characterized in that, The amino acid sequence of the zinc chelated peptide is HLG.

2. A method for preparing a zinc-chelating peptide with antibacterial activity as described in claim 1, characterized in that, It includes the following steps: mixing the polypeptide solution and the inorganic zinc solution, adding absolute ethanol, and carrying out a chelation reaction to obtain the zinc chelated peptide HLG@Zn with antibacterial activity.

3. The preparation method of the zinc-chelated peptide with antibacterial activity according to claim 2, wherein, The concentration of the 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.

4. The preparation method of the zinc-chelated peptide with antibacterial activity according to claim 2, characterized in that, The conditions of the chelation reaction are as follows: the temperature is 30 - 40 °C, and the time is 12 - 16 h.

5. The preparation method of the zinc-chelated peptide with antibacterial activity according to claim 2, characterized in that, It also includes post-treatment of the product of the chelation reaction, and the post-treatment includes: washing the product with absolute ethanol, centrifuging to collect the precipitate, and freeze-drying.

6. Use of a zinc chelated peptide with antibacterial activity as described in claim 1 or a zinc chelated peptide with antibacterial activity prepared by the preparation method as described in any one of claims 2 - 5 in the preparation of a medicament for anti-Gram-negative bacteria and / or Gram-positive bacteria.

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