Fish scale polypeptide chelated zinc as well as preparation method and application thereof

Abstract

The invention relates to fish scale polypeptide chelated zinc. The amino acid sequence of the fish scale polypeptide chelated zinc is HMGPR. According to the fish scale polypeptide chelated zinc disclosed by the invention, a polypeptide ligand HMGPR does not have antibacterial activity, but a nitrogen atom in a histidine imidazole group at an N end of the polypeptide ligand HMGPR is coordinated with Zn < 2 + > through lone pair electrons, and meanwhile, a carboxyl oxygen atom of arginine at a C end is used as an electron donor to form coordination with Zn < 2 + >, so that the fish scale polypeptide chelated zinc HMGPR (at) Zn is endowed with broad-spectrum antibacterial activity. Besides, HMGPR is chelated with Zn < 2 + > through a coordinate bond, so that the stability of the polypeptide is remarkably improved, and the defect that a peptidyl antibacterial agent is easily hydrolyzed by protease is overcome. The fish scale polypeptide chelated zinc can serve as a novel antibacterial preservative to be applied to the food industry, microbial infection is effectively prevented, and meanwhile the additional value and the comprehensive social value of the aquatic product industry are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of food microbiology, and particularly relates to a scale polypeptide chelated zinc and its preparation method and application. Background Art

[0002] In recent years, the abuse of antibiotics in food has caused a series of food safety problems. Drug-resistant bacteria not only threaten human health but also hinder the sustainable development of the industry. Therefore, it has become a consensus to develop healthier and more efficient antibiotic alternatives.

[0003] Polypeptide metal chelates have high development value and application prospects due to their activities such as antioxidant, antibacterial, lipid-lowering, and hypoglycemic effects, and have become a research hotspot at home and abroad. Zinc is called the "life element" and is an essential trace element for all biological systems. When a polypeptide chelates with zinc ions, it can effectively enhance biological activity, significantly improve the stability of the polypeptide, and effectively control the release of Zn2+, overcoming the precipitation problem of inorganic zinc. However, so far, the research on peptide zinc chelates is still in its initial stage and further exploration is urgently needed.

[0004] By-products such as fish scales and fish skins generated during fish processing contain rich proteins. Except for a small part used in the processing of fish bait and feed, the vast majority have not been reasonably developed and utilized. Therefore, using fish scales as raw materials to research and develop a peptide zinc chelate with antibacterial activity not only has biological potency but also can contribute to the efficient utilization of resources and environmental protection. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a scale polypeptide chelated zinc and its preparation method and application.

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

[0007] On the one hand, the present invention provides a scale polypeptide chelated zinc, and the amino acid sequence of the scale polypeptide chelated zinc is HMGPR.

[0008] For the scale polypeptide chelated zinc proposed by the present invention, its polypeptide ligand HMGPR itself does not have antibacterial activity, but 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 arginine at the C-terminus acts as an electron donor to form a coordination with Zn2+, thereby endowing the scale polypeptide chelated zinc HMGPR@Zn with broad-spectrum antibacterial activity. In addition, HMGPR 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 scale polypeptide chelated zinc of the present invention can be used as a new antibacterial preservative in the food industry, effectively preventing and controlling microbial infections while increasing the added value and comprehensive social value of the aquatic product industry.

[0009] On the other hand, the present invention provides a method for preparing fish scale polypeptide chelated zinc as described in the above technical solution, comprising the following steps: mixing a fish scale polypeptide solution and a zinc source compound solution, adding anhydrous ethanol, and performing a chelation reaction to obtain the fish scale polypeptide chelated zinc HMGPR@Zn.

[0010] The method for preparing fish scale polypeptide chelated zinc provided by the present invention uses fish scales as a natural protein source for polypeptides, which can solve the comprehensive utilization of waste fish scales in aquatic product processing. Moreover, the chelation process is simple in operation and good in safety, and is suitable for industrial production.

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

[0012] Preferably, the temperature of the chelation reaction 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 anhydrous ethanol, centrifuging to collect the precipitate, and freeze-drying.

[0014] On yet another aspect, the present invention provides an application of the fish scale polypeptide chelated zinc as described in the above technical solution or the fish scale polypeptide chelated zinc prepared by the preparation method described in any one of the above technical solutions in the preparation of antibacterial products.

[0015] Preferably, the antibacterial product is used for preventing, improving or treating diseases related to Gram-negative bacteria and / or Gram-positive bacteria infections.

[0016] The fish scale polypeptide chelated zinc of the present invention has broad-spectrum antibacterial activity and can effectively inhibit a variety of Gram-negative bacteria (such as Vibrio alginolyticus TJ-2, Vibrio parahaemolyticus 2503, Vibrio harveyi 2510, etc.) and Gram-positive bacteria (such as methicillin-resistant Staphylococcus aureus, Listeria monocytogenes 19115, etc.), and has broad application prospects in the preparation of antibacterial products. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 For the structural characterization of fish scale polypeptide and fish scale polypeptide chelated zinc in Example 1, wherein, A is the secondary mass spectrum of fish scale polypeptide HMGPR, and B is the Fourier transform infrared spectrum of fish scale polypeptide HMGPR and fish scale polypeptide chelated zinc HMGPR@Zn;

[0018] Figure 2 For the minimum inhibitory concentration (MIC) graph of fish scale polypeptide chelated zinc HMGPR@Zn against Vibrio alginolyticus 2512 in Example 1;

[0019] Figure 3 To study the effect of HMGPR@Zn, a zinc chelate of fish scale polypeptide in Example 1, on the anti-biofilm activity against Vibrio alginolyticus 2512;

[0020] Figure 4 To study the antibacterial spectrum of HMGPR@Zn, a zinc chelate of fish scale polypeptide in Example 1;

[0021] Figure 5 To study the effect of HMGPR@Zn, a zinc chelate of fish scale polypeptide in Example 1, on the hemolytic activity of mouse red blood cells;

[0022] Figure 6 To study the cytotoxicity of HMGPR@Zn, a zinc chelate of fish scale polypeptide in Example 1, on mouse macrophage RAW 264.7;

[0023] Figure 7 To study the stability of HMGPR@Zn, a zinc chelate of fish scale polypeptide in Example 1. Detailed implementation manners

[0024] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings and embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0025] One aspect of the embodiments of the present invention provides a zinc chelate of fish scale polypeptide, and the amino acid sequence of the zinc chelate of fish scale polypeptide is HMGPR.

[0026] Another aspect of the embodiments of the present invention provides a preparation method of a zinc chelate of fish scale polypeptide as described in the above technical solution, including the following steps: mixing a fish scale polypeptide solution and a zinc source compound solution, adding absolute ethanol, and performing a chelation reaction to obtain HMGPR@Zn, a zinc chelate of fish scale polypeptide.

[0027] In the embodiments of the present invention, the fish scale polypeptide can be purified from the hydrolysate of Lutjanus erythropterus fish scale protein to obtain a peptide sequence and synthesized by solid-phase synthesis technology, which is not particularly limited herein. See the secondary mass spectrum of HMGPR in Figure 1 A. In addition, absolute ethanol is introduced in the chelation reaction of the present invention, which can accelerate the capture of Zn2+ by the polypeptide; the zinc source compound is specifically ZnSO4·7H2O.

[0028] In a preferred embodiment of the present invention, the concentration of the fish scale polypeptide solution is 0.1 - 0.15 g / mL, the concentration of the zinc source compound solution is 2 mol / L, and the mass ratio of the fish scale polypeptide to the zinc source compound is 0.13 - 0.15:1.

[0029] In a preferred embodiment of the present invention, the temperature of the chelation reaction is 30 - 40 °C and the time is 12 - 16 h.

[0030] 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.

[0031] Another aspect of the embodiments of the present invention provides an application of the fish scale polypeptide chelated zinc as described in the above technical solution or the fish scale polypeptide chelated zinc prepared by the preparation method of any one of the above technical solutions in the preparation of antibacterial products.

[0032] In a preferred embodiment of the present invention, the antibacterial product is used for preventing, improving or treating diseases related to Gram-negative bacteria and / or Gram-positive bacteria infections.

[0033] Example 1

[0034] A preparation method of fish scale polypeptide chelated zinc includes the following steps:

[0035] Mix the fish scale polypeptide solution (1.21 g of polypeptide HMGPR dissolved in 10 mL of ultrapure water) with the zinc source compound solution (15 mL of 2 mol / L ZnSO4·7H2O), add 2.5 times the volume of absolute ethanol of the mixed solution, carry out the chelation reaction, react at 35 °C for 16 h, wash the product with 9 times the volume of absolute ethanol, centrifuge to collect the precipitate, and freeze-dry to obtain fish scale polypeptide chelated zinc HMGPR@Zn.

[0036] Perform Fourier transform infrared spectroscopy on the above-mentioned fish scale polypeptide HMGPR and fish scale polypeptide chelated zinc HMGPR@Zn, and analyze its chelation mechanism.

[0037] From Figure 1 B, it can be seen that after HMGPR with high zinc affinity activity 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 ~3000 cm -1 reflects the stretching vibration 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 3290 cm -1 to 3360 cm -1 . After the amide I band generated by the C=O stretching vibration coordinates with Zn2+, its absorption peak shifts from 1670 cm-1 Shifted to 1630 cm -1 . After coordination with Zn2+, the absorption peaks of HMGPR at 1550 cm -1 and 1440 cm -1 disappeared, indicating that -COO- participated in the covalent binding of Zn2+. Similarly, the absorption peaks (1200 cm -1 and 1140 cm -1 ) related to the stretching vibration of the -C-O bond in HMGPR shifted to 1080 cm -1 , probably due to the formation of a -C-O-Zn complex. The absorption peak at 627 cm -1 caused by the in-plane vibration of the O═CN bond shifted to 598 cm -1 , indicating that Zn2+ coordination may increase the electron cloud density of the O adjacent to C═O in HMGPR. This series of spectral changes indicates that amino nitrogen, carboxyl oxygen, and carbonyl oxygen all participated in the coordination of Zn2+.

[0038] The performance of the above-mentioned scale polypeptide chelated zinc HMGPR@Zn was measured, and the results were recorded in Figures 2 - 7 .

[0039] (1) Antibacterial activity of HMGPR@Zn against Vibrio alginolyticus 2512

[0040] Determination 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 HMGPR@Zn to the first column of the 96-well plate and mix well. Gradient dilution was performed along columns 2 to 10 using the two-fold dilution method, and then 50 μL of the strain 2512 suspension (1×10 5 CFU / mL) was added. Column 11 was used as a blank control (50 μL of medium), and column 12 was used as a negative control (50 μL of medium + 50 μL of sterile water). Incubate at 30 °C for 24 h, and the lowest concentration corresponding to no bacterial growth was the MIC of HMGPR@Zn.

[0041] As can be seen from Figure 2 , the MIC of HMGPR@Zn against Vibrio alginolyticus 2512 was 78.59 μg / mL.

[0042] (2) Anti-biofilm activity of HMGPR@Zn against Vibrio alginolyticus 2512

[0043] Determination method: Measured by crystal violet staining. Add cover slips to 6-well plates, take 200 μL of activated Vibrio alginolyticus 2512 and culture it in LB broth. After incubating at 30 °C for 12 h, add HMGPR@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.

[0044] It can be seen from Figure 3 that HMGPR@Zn not only has an inhibitory effect on planktonic Vibrio alginolyticus 2512 but also can inhibit the biofilm formation of strain 2512.

[0045] (3) Antibacterial spectrum of HMGPR@Zn against pathogenic bacteria

[0046] Determination method: Use 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, Staphylococcus aureus 6538, methicillin-resistant Staphylococcus aureus (MRSA) 43300 as indicator bacteria. Mix and pour plate inoculate the above indicator bacteria (1×10 7 CFU / mL) into LB agar medium, then punch holes with a 6 mm puncher and add 50 μL of HMGPR@Zn (50 mg / mL) into the holes, and place it in a biochemical incubator for 24 h. Evaluate the diameter (mm) of the inhibition zone using the cross method.

[0047] It can be seen from Figure 4 that HMGPR@Zn has antibacterial activity against foodborne pathogenic bacteria such as Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, and methicillin-resistant Staphylococcus aureus. The activity levels are as follows: Vibrio harveyi 2510 is the strongest, with a diameter of 31.21 ± 0.47 mm, followed by Escherichia coli K88 (27.13 ± 0.71 mm), MRSA (25.73 ± 0.79 mm) and Listeria monocytogenes 19115 (25.40 ± 0.29 mm), and the activity against Staphylococcus aureus 6538 is the worst, with a diameter of 15.37 ± 0.32 mm.

[0048] (4) Hemolytic activity of HMGPR@Zn on mouse red blood cells

[0049] Determination method: The hemolytic activity of HMGPR@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 HMGPR@Zn were added to each centrifuge tube. Then, it 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.

[0050] It can be seen from Figure 5 that HMGPR@Zn does not induce hemolysis of mouse erythrocytes.

[0051] (5) Cytotoxicity of HMGPR@Zn to mouse macrophage RAW264.7

[0052] Determination method: The cytotoxicity of HMGPR@Zn to RAW264.7 cells was determined by the thiazolyl blue colorimetric method. The specific steps are as follows: After the cryopreserved cells were resuscitated, they were inoculated into a medium (containing 10% fetal bovine serum and 1% double antibody) and subcultured (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 concentrations of 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 wells were dissolved with 100 μL of dimethyl sulfoxide, and the absorbance was measured at 570 nm.

[0053] It can be seen from Figure 6 that at the MIC concentration, HMGPR@Zn has good biocompatibility with RAW 264.7 cells, and the cell survival rate > 80%.

[0054] (6) Stability analysis of HMGPR@Zn

[0055] 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 HMGPR@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 HMGPR@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 HMGPR@Zn is 1:25, and continue the reaction 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.

[0056] It can be seen from Figure 7 that after in vitro simulated gastrointestinal digestion, the antibacterial activity of HMGPR@Zn against strain 2512 did not change significantly.

[0057] In summary, the fish scale polypeptide chelated zinc HMGPR@Zn of the present invention has excellent biocompatibility and 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.

[0058] 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 kind of zinc chelated with fish scale polypeptide, characterized in that, The amino acid sequence of the fish scale polypeptide chelated zinc is HMGPR.

2. A preparation method of the zinc-polypeptide chelate from fish scales as described in claim 1, characterized in that, It includes the following steps: Mix the fish scale polypeptide solution and the zinc source compound solution, add absolute ethanol, and carry out a chelation reaction to obtain the fish scale polypeptide chelated zinc HMGPR@Zn.

3. The preparation method of the zinc polypeptide chelate of fish scales according to claim 2, characterized in that, The concentration of the fish scale polypeptide solution is 0.1 - 0.15 g / mL, the concentration of the zinc source compound solution is 2 mol / L, and the mass ratio of the fish scale polypeptide to the zinc source compound is 0.13 - 0.15:

1.

4. The preparation method of the zinc-polypeptide chelate of fish scales according to claim 2, characterized in that, The temperature of the chelation reaction is 30 - 40 °C, and the time is 12 - 16 h.

5. The preparation method of the zinc-polypeptide chelate of fish scale according to claim 2, wherein 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 the fish scale polypeptide chelated zinc as described in claim 1 or the fish scale polypeptide chelated zinc prepared by the preparation method as described in any one of claims 2 - 5 in the preparation of antibacterial products.

7. The application according to claim 6, characterized in that, The antibacterial product is used for preventing, improving or treating diseases related to Gram-negative bacteria and / or Gram-positive bacteria infections.

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