Fish skin source antibacterial peptide, composition and application thereof
By combining fish skin enzymatic hydrolysis technology with artificial intelligence prediction models, fish skin-derived antimicrobial peptide compositions were screened and synthesized, solving the problems of low efficiency and poor effectiveness in existing fish skin-derived antimicrobial peptide research, and achieving efficient and safe antimicrobial effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANJING TECH UNIV
- Filing Date
- 2026-01-27
- Publication Date
- 2026-04-21
AI Technical Summary
Current research on antimicrobial peptides derived from fish skin relies on traditional technical routes, resulting in long experimental cycles, high costs, low screening efficiency and hit rate. Furthermore, single antimicrobial peptides have problems such as limited antimicrobial spectrum, high antibacterial concentration, or insufficient stability in application.
By combining fish skin enzymatic hydrolysis technology with artificial intelligence prediction models, antimicrobial peptides with excellent antibacterial activity were screened out, and antimicrobial peptide compositions were synthesized through the Fmoc solid-phase peptide synthesis method to optimize their synergistic effect and improve the antibacterial effect.
Efficient screening and combination optimization were achieved to obtain antimicrobial peptide compositions with good antimicrobial activity, which significantly improved the antimicrobial effect and application potential of antimicrobial peptides, and showed good biocompatibility and safety to mammalian cells.
Smart Images
Figure CN121895409A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a fish skin-derived antimicrobial peptide, its composition, and its application. Background Technology
[0003] Antimicrobial peptides (AMPs) are a class of short peptide molecules naturally produced by organisms, typically composed of 10–50 amino acid residues. They possess advantages such as broad-spectrum antibacterial activity, rapid onset of action, and low likelihood of inducing drug resistance. They exert their effects primarily by disrupting cell membranes or interfering with various key physiological processes, exhibiting diverse targets and a low probability of drug resistance development, making them important candidates for next-generation antimicrobial agents.
[0004] Fish skin, scales, and bones, byproducts of aquatic product processing, are rich in collagen and are important raw materials for the preparation of natural bioactive peptides. Enzymatic hydrolysis of fish skin protein can yield a large number of short peptide fragments, some of which have been proven to have antibacterial activity. Furthermore, fish skin fragments offer significant advantages in terms of availability, safety, and sustainability, aligning with the development trend of green and high-value utilization.
[0005] However, current research on antimicrobial peptides derived from fish skin largely relies on the traditional technical route of "enzymatic hydrolysis-separation-identification-activity verification," which requires repeated separation and individual testing of complex peptide mixtures. This approach is time-consuming, costly, and results in low screening efficiency and hit rate when faced with a large number of potential peptide sequences. Furthermore, single antimicrobial peptides often suffer from limited antimicrobial spectrum, high inhibitory concentrations, or insufficient stability in practical applications, hindering their further development. Summary of the Invention
[0006] Objective of the Invention: Addressing the problems existing in the prior art, the objective of this invention is to provide a fish skin-derived antimicrobial peptide, its composition, and its applications. This invention combines fish skin proteolytic technology with an artificial intelligence prediction model to achieve efficient screening and synergistic combination optimization of fish skin-derived peptides, obtaining an antimicrobial peptide composition with good antimicrobial activity and application potential. This provides an efficient and scalable technical solution for the development of novel antimicrobial agents.
[0007] Technical solution: To achieve the above-mentioned objectives, the present invention adopts the following technical solution.
[0008] In a first aspect, the present invention provides a fish skin-derived antimicrobial peptide, wherein the fish skin-derived antimicrobial peptide is selected from FSP-1, FSP-2, FSP-3 or FSP-4, and the amino acid sequences of FSP-1, FSP-2, FSP-3 and FSP-4 are shown in sequence as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
[0009] Specifically, the fish skin-derived antimicrobial peptide is derived from the pepsin hydrolysate of fish skin protein.
[0010] Secondly, the present invention provides a method for synthesizing the fish skin-derived antimicrobial peptide, which mainly uses the Fmoc solid-phase polypeptide synthesis method.
[0011] Thirdly, the present invention provides a fish skin-derived antimicrobial peptide composition, wherein the fish skin-derived antimicrobial peptide composition is selected from at least two of antimicrobial peptides FSP-1, FSP-2, FSP-3 and FSP-4, and the amino acid sequences of FSP-1, FSP-2, FSP-3 and FSP-4 are as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 in sequence.
[0012] As a specific implementation, the fish skin-derived antimicrobial peptide composition is selected from two of the antimicrobial peptides FSP-1, FSP-2, FSP-3 and FSP-4, and the mass ratio of the two antimicrobial peptides is 1:10 to 10:1, preferably 1:5 to 5:1.
[0013] As a preferred embodiment, the fish skin-derived antimicrobial peptide composition is selected from a combination of antimicrobial peptides FSP-1 and FSP-2.
[0014] As a specific implementation scheme, the effective viable bacteria concentration in the fish skin-derived antimicrobial peptide composition is ≥4.8×10⁻⁶. 9 CFU / g.
[0015] As a specific implementation, the fish skin-derived antimicrobial peptide composition is in the form of a formulation, which is selected from solid dosage forms or solution dosage forms.
[0016] Fourthly, the present invention provides the application of the fish skin-derived antimicrobial peptide and the fish skin-derived antimicrobial peptide composition in the preparation of products that inhibit pathogenic bacteria.
[0017] As a specific implementation plan, the pathogens include Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus cereus, Salmonella, or Cronobacter sakazakii.
[0018] Beneficial Effects: Compared with existing technologies, this invention combines fish skin enzymatic hydrolysis technology with artificial intelligence prediction models to screen for novel antimicrobial peptides with excellent antimicrobial activity. Furthermore, this invention provides a composition containing the aforementioned antimicrobial peptides, optimizing the combination of these antimicrobial peptides to achieve a significant synergistic effect, thereby significantly improving the antimicrobial efficacy. The resulting antimicrobial peptides and compositions have good application potential. Attached Figure Description
[0019] Figure 1This diagram illustrates the antibacterial effect of hydrolysates obtained from fish skin protein via pepsin hydrolysis on various indicator bacteria.
[0020] Figure 2 This is a schematic diagram of the antimicrobial peptide prediction model based on a protein language model.
[0021] Figure 3 This is a schematic diagram illustrating the antibacterial effects of the synthesized peptide against various indicator bacteria.
[0022] Figure 4 A schematic diagram of scanning electron microscopy observation of the effects of the screened antimicrobial peptides on bacterial cell morphology.
[0023] Figure 5 This is a schematic diagram of the docking model between antimicrobial peptides and key bacterial target proteins.
[0024] Figure 6 This is a schematic diagram of the stability analysis of the antimicrobial peptide-target protein complex using molecular dynamics simulation. Detailed Implementation
[0025] The present invention will be further described in detail below with reference to specific embodiments. Those skilled in the art should understand that any equivalent modifications or substitutions made to the following embodiments without departing from the inventive concept should fall within the protection scope of the present invention.
[0026] Example 1 Screening of antimicrobial peptides 1. Preparation and pretreatment of fish skin protein raw materials Fresh fish skin is selected as the raw material. After removing the attached fish scales and impurities, it is repeatedly rinsed with running tap water.
[0027] Soak the cleaned fish skin in 60 ℃ hot water for 10 minutes to remove surface lipids, then rinse twice with deionized water.
[0028] The processed fish skin was freeze-dried in a freeze dryer for 24 hours. After freeze-drying, it was pulverized using a pulverizing device until it passed through a 60-mesh sieve to obtain fish skin protein powder, which was then sealed and stored at −20 ℃ for later use.
[0029] 2. Enzymatic hydrolysis of fish skin protein Weigh 10.0 g of the prepared fish skin protein powder, add 100 mL of deionized water, and prepare a protein solution with a mass fraction of 10% (w / v).
[0030] Adjust the pH of the solution to 2.0 using 1 mol / L HCl.
[0031] Pepsin was added under magnetic stirring conditions, with an enzyme-to-substrate mass ratio of 2% (w / w).
[0032] The reaction system was placed in a constant temperature water bath at 37 ℃ and enzymatically hydrolyzed for 6 h under stirring at 300 rpm.
[0033] After the enzymatic hydrolysis is complete, the reaction solution is heated in a 90 ℃ water bath for 15 min to terminate the enzyme reaction.
[0034] After the reaction solution was cooled to room temperature, it was centrifuged at 4 ℃ and 8000 × g for 10 min, and the supernatant was collected.
[0035] The resulting supernatant was freeze-dried to obtain the fish skin protein pepsin hydrolysate, denoted as CPH-Pep.
[0036] 3. Preliminary screening of antibacterial activity of fish skin protein hydrolysates Weigh the obtained CPH-Pep, dissolve it in LB medium, and prepare a series of solutions with mass concentrations of 32, 64, 128, 256, 512, and 1024 μg / mL.
[0037] Select Escherichia coli ( Escherichia coli ATCC 25922), Klebsiella pneumoniae ( Klebsiella pneumoniae ATCC 13883), Staphylococcus aureus ( Staphylococcus aureus ATCC 29213), Bacillus cereus ( Bacillus cereus ATCC 14579), Salmonella ( Salmonella enterica ATCC 14028) and Enterobacter sakazakii ( Cronobacter sakazakii ATCC 29544) is used as an indicator bacterium.
[0038] The strain was inoculated into LB liquid medium and cultured at 37 ℃ and 200 rpm until the logarithmic growth phase (OD600≈0.5), then diluted to a final concentration of 1×10⁻⁶. 6 CFU / mL.
[0039] In a 96-well plate, 100 μL of bacterial culture and 100 μL of CPH-Pep solution at different concentrations were added to each well. The plates were incubated at 37°C for 24 h. The antibacterial effect was evaluated by measuring changes in OD600. The results are as follows: Figure 1As shown, the hydrolysate (CPH-Pep) obtained from the enzymatic hydrolysis of fish skin protein by pepsin exhibited certain antibacterial activity against various indicator bacteria, and the antibacterial effect increased with increasing concentration. The inhibitory effect on Staphylococcus aureus, Bacillus cereus, and Escherichia coli was particularly significant, and at higher concentrations, it could significantly reduce the growth level of the bacterial culture. These results indicate that the pepsin hydrolysate of fish skin protein is rich in peptides with potential antibacterial activity, providing a basis for subsequent screening of antimicrobial peptides.
[0040] 4. Artificial intelligence-based screening of antimicrobial peptides The obtained peptide amino acid sequences are input into the antimicrobial peptide prediction model.
[0041] The prediction model extracts peptide sequence features based on the protein language model ESM2 and combines it with a binary classification neural network based on the Transformer architecture to predict whether a peptide has antibacterial potential. The structure of the prediction model is as follows: Figure 2 As shown.
[0042] Candidate antimicrobial peptide sequences were obtained by using a prediction probability ≥ 0.90 as the screening threshold.
[0043] name Sequence (N–C) Length (aa) Molecular weight (Da) Theoretical pI FSP-1 KYCFKIKGAL 10 1170.47 9.63 FSP-2 VSKVPASF 8 833.97 8.72 FSP-3 GINKISALHL 10 1065.27 8.76 FSP-4 SAAAAAKF 8 735.83 8.47 5. Prediction and combination screening of antimicrobial peptide synergistic effects The selected candidate antimicrobial peptides are paired and input into the antimicrobial peptide synergistic effect prediction model.
[0044] The model, based on a deep learning algorithm, evaluates the synergistic antibacterial potential of different combinations of antimicrobial peptides when used together, and outputs a synergistic score.
[0045] Based on an antimicrobial peptide synergy prediction model, peptide combination analysis was performed on candidate antimicrobial peptides, and the model output synergy scores for different combinations under combined use conditions. The results showed that FSP-1‖FSP-2, FSP-2‖FSP-3, and FSP-1‖FSP-2‖FSP-3 ranked in the top three in synergy scores, predicting that they have high synergistic antibacterial potential when used in combination.
[0046] To verify the reliability of the model's predictions, the antimicrobial peptide combinations with high synergistic scores were synthesized and in vitro antibacterial experiments were conducted. The antibacterial effects of different antimicrobial peptides and their combinations against various indicator bacteria are shown below. Figure 3 As shown in the figure. The results indicate that, compared with single antimicrobial peptides, the combination of antimicrobial peptides with higher synergistic scores exhibited stronger antibacterial activity under the same concentration conditions, and the growth of bacteria was more significantly inhibited, thus preliminarily verifying the accuracy of the model prediction results.
[0047] Example 2: Synthesis of antimicrobial peptides and preparation of the composition The antimicrobial peptides screened in Example 1 were synthesized using the Fmoc solid-phase peptide synthesis method.
[0048] After synthesis, the peptide was cleaved with trifluoroacetic acid to remove the side chain protecting groups, and then precipitated with cold diethyl ether to obtain the crude peptide.
[0049] The crude peptide was purified by high performance liquid chromatography with a purity of ≥90%, and the molecular weight was confirmed to be correct by mass spectrometry.
[0050] Two antimicrobial peptides were mixed in a 1:1 mass ratio, dissolved in sterile water, and freeze-dried to obtain a fish skin-derived antimicrobial peptide composition.
[0051] Example 3: Biosafety Evaluation of Antimicrobial Peptides in Mammalian Cells To further evaluate the biosafety of the screened fish skin-derived antimicrobial peptides in application, this embodiment conducts in vitro cytotoxicity experiments on representative antimicrobial peptides to verify their effects on mammalian cells.
[0052] Human umbilical vein endothelial cells (HUVECs) were selected as the model cells. Cells were cultured in DMEM medium containing 10% fetal bovine serum at 37 °C and 5% CO2 until the logarithmic growth phase. Cells were then seeded into 96-well cell culture plates, with approximately 5 × 10⁶ cells per well. 3 One cell was cultured for 24 hours to allow it to adhere to the culture vessel.
[0053] The original culture medium was then discarded, and culture media containing different concentrations of antimicrobial peptides, including FSP-1, FSP-2, FSP-3, and FSP-4, were added. The concentrations of the antimicrobial peptides were set to 1, 2, 4, and 8 times their minimum inhibitory concentration (MIC); the control group received only culture media without antimicrobial peptides. Each treatment group had three parallel wells.
[0054] After culturing cells for another 24 h, cell viability was assessed using a CCK-8 assay kit. 10 μL of CCK-8 solution was added to each well, and the cells were incubated in the dark for 2 h. The absorbance (OD) was then measured at 450 nm. 450 Cell viability is calculated using the following formula: Cell viability (%) = (Experimental group OD) 450 / Control group OD 450 ) × 100%.
[0055] CCK-8 assay results showed that within the tested concentration range, the survival rate of HUVEC cells in each antimicrobial peptide treatment group remained above 85%. When the antimicrobial peptide concentration was 1 to 4 times its minimum inhibitory concentration (MIC), the cell survival rate was not significantly different from that of the control group; even at a concentration of 8 times the MIC, the cell survival rate was still above 80%, and no obvious cytotoxic effects were observed. These results indicate that the fish skin-derived antimicrobial peptides screened in this invention exhibit good biocompatibility and safety in mammalian cells while exerting antimicrobial activity, providing a basis for further application development.
[0056] Example 4: In vitro antimicrobial evaluation of the antimicrobial peptide composition The antimicrobial peptide composition obtained in Example 2 was dissolved in a culture medium to prepare different concentration gradients.
[0057] The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the antimicrobial peptide composition against *Escherichia coli*, *Staphylococcus aureus*, *Bacillus subtilis*, *Klebsiella pneumoniae*, *Cronobacter sakazakii*, and *Salmonella* were determined using the microbroth dilution method. The specific method is as follows: Each indicator bacterium was inoculated into LB broth and cultured at 37 °C with shaking until the logarithmic growth phase. The bacterial suspension concentration was then adjusted so that the final concentration after adding the culture to a 96-well plate was approximately 5 × 10⁻⁶. 5 CFU / mL. The antimicrobial peptide composition was prepared as an initial concentration solution using sterile culture medium and serially diluted twofold in 96-well plates. An equal volume of bacterial suspension was added to each well. Blank control group, growth control group, and antimicrobial peptide treatment group were set up. The culture conditions were 37 ℃ for 24 h.
[0058] After cultivation, the turbidity of the bacterial solution was observed and combined with OD. 600 The results were used to determine the bacterial growth status. The lowest concentration of antimicrobial peptide that completely inhibited visible bacterial growth was defined as the minimum inhibitory concentration (MIC). Samples from the MIC and higher concentration groups were spread onto LB solid medium without antimicrobial peptides and incubated at 37 °C for 24 h. Colony formation was then observed. The lowest concentration of antimicrobial peptide that reduced the colony count by ≥99.9% compared to the control group was defined as the minimum bactericidal concentration (MBC).
[0059] The MIC and MBC results of the synergistic combination of antimicrobial peptides are shown in the table below. The results showed that compared with the dipeptide combination, the tripeptide combination FSP-1‖FSP-2‖FSP-3 exhibited lower MIC values against various indicator bacteria, especially against *Escherichia coli*, *Bacillus cereus*, and *Salmonella*, where the MIC decreased to 16 μg / mL, demonstrating stronger antibacterial activity. In the dipeptide combination, the overall MIC and MBC of FSP-1‖FSP-2 against various strains were lower than those of FSP-2‖FSP-3. For example, in *Bacillus cereus* and *Cronobacter sakazakii*, the MIC of FSP-1‖FSP-2 was significantly lower than that of FSP-2‖FSP-3. These results indicate that there is a certain degree of synergistic effect among different fish skin-derived antimicrobial peptides, and reasonable combination can significantly improve their antibacterial and bactericidal effects.
[0060] .
[0061] Example 5: Effect of antimicrobial peptide composition on bacterial morphology Take bacteria in the logarithmic growth phase and incubate them with the antimicrobial peptide composition for 2 hours.
[0062] After incubation, the cells were fixed with 2.5% glutaraldehyde for 12 h, washed with PBS, and dehydrated with a gradient of 30%–100% ethanol.
[0063] After critical point drying and gold sputtering, the morphological changes of bacteria were observed using scanning electron microscopy. The results of the scanning electron microscopy observation are as follows: Figure 4 As shown, compared with the control group whose bacteria had smooth surfaces and intact structures, the bacterial cells treated with the antimicrobial peptide composition exhibited obvious shrinkage, collapse, and rupture on their surfaces, with some cell contents leaking out and cell morphology severely damaged. This indicates that the antimicrobial peptide composition can exert its antibacterial effect by disrupting the bacterial cell structure.
[0064] Example 6: Molecular simulation analysis of the mechanism of action of antimicrobial peptides The bacterial cell wall synthesis-related protein MurA and the cell division protein FtsZ were selected as target proteins.
[0065] Molecular docking software was used to perform docking analysis between the antimicrobial peptide and the target protein to obtain the initial complex structure. The molecular docking results are as follows: Figure 5 As shown, the antimicrobial peptides can stably bind to the functional regions of MurA and FtsZ proteins, forming various hydrogen bonds and hydrophobic interactions with the target proteins, suggesting that the antimicrobial peptides may exert their antibacterial effect by interfering with bacterial cell wall synthesis and cell division processes.
[0066] A 100 ns molecular dynamics simulation was performed on the complex to analyze its stability and interaction characteristics. The results of the molecular dynamics simulation are as follows: Figure 6 As shown, during the 100 ns simulation, the RMSD value of the antimicrobial peptide-target protein complex remained stable overall, with no obvious structural dissociation, indicating that the antimicrobial peptide and target protein formed a relatively stable binding conformation, further supporting its potential mechanism of action.
Claims
1. A fish skin-derived antimicrobial peptide, characterized in that, The fish skin-derived antimicrobial peptide is selected from FSP-1, FSP-2, FSP-3 or FSP-4, and the amino acid sequences of FSP-1, FSP-2, FSP-3 and FSP-4 are shown in sequence as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:
4.
2. The fish skin-derived antimicrobial peptide according to claim 1, characterized in that, The fish skin-derived antimicrobial peptides are derived from pepsin hydrolysates of fish skin protein.
3. The method for synthesizing the fish skin-derived antimicrobial peptide according to claim 1, characterized in that, The main method used is the Fmoc solid-phase peptide synthesis method.
4. A fish skin-derived antimicrobial peptide composition, characterized in that, The fish skin-derived antimicrobial peptide composition is selected from at least two of the antimicrobial peptides FSP-1, FSP-2, FSP-3 and FSP-4, and the amino acid sequences of FSP-1, FSP-2, FSP-3 and FSP-4 are shown in sequence as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:
4.
5. The fish skin-derived antimicrobial peptide composition according to claim 4, characterized in that, The fish skin-derived antimicrobial peptide composition is selected from two of the antimicrobial peptides FSP-1, FSP-2, FSP-3 and FSP-4, and the mass ratio of the two antimicrobial peptides is 1:10 to 10:1, preferably 1:5 to 5:
1.
6. The fish skin-derived antimicrobial peptide composition according to claim 4, characterized in that, The fish skin-derived antimicrobial peptide composition is selected from a combination of antimicrobial peptides FSP-1 and FSP-2.
7. The fish skin-derived antimicrobial peptide composition according to claim 4, characterized in that, The effective viable bacteria concentration in the fish skin-derived antimicrobial peptide composition is ≥4.8×10⁻⁶. 9 CFU / g.
8. The fish skin-derived antimicrobial peptide composition according to claim 4, characterized in that, The fish skin-derived antimicrobial peptide composition is in the form of a formulation, which is selected from solid dosage forms or solution dosage forms.
9. The use of the fish skin-derived antimicrobial peptide according to claim 1 or 2, or the fish skin-derived antimicrobial peptide composition according to any one of claims 4-7, in the preparation of products that inhibit pathogenic bacteria.
10. The application according to claim 9, characterized in that, The pathogens include Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus cereus, Salmonella, or Cronobacter sakazakii.