A novel antibacterial peptide T1, a preparation method thereof and application of the antibacterial peptide T1 in preserving salmon

Abstract

The application provides a novel antibacterial peptide T1, a preparation method thereof and application of the antibacterial peptide T1 in salmon preservation, and belongs to the technical field of food preservation. The antibacterial peptide T1 is composed of 11 amino acids, and the amino acid sequence is GLKPGLHGFHV. The antibacterial peptide T1 can be obtained from an active fraction F3 of a third elution peak of a microbial fermentation product through separation and purification. The antibacterial peptide T1 contains basic amino acid residues and has a positive electric property under physiological conditions. The novel antibacterial peptide T1 plays an antibacterial role through multiple mechanisms such as destroying cell membrane structure, interfering with bacterial metabolic processes and inhibiting virulence-related functions, so as to reduce the risk of drug resistance of microorganisms. The antibacterial peptide has antibacterial activity on Listeria monocytogenes, and the minimum inhibitory concentration is 3.5 mg / mL. When the antibacterial peptide is applied to the cold storage and preservation process of salmon, the proliferation of pathogenic bacteria and spoilage bacteria can be inhibited, the microbial load can be reduced, and the quality deterioration can be delayed, so that the cold storage shelf life of the salmon is significantly prolonged, and the safety of the product is improved.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and food preservation technology, specifically to a novel antimicrobial peptide T1, its preparation method, and its application in the preservation of salmon. Background Technology

[0002] With the increasing demand for aquatic products, salmon has become one of the important refrigerated ready-to-eat aquatic products due to its high nutritional value and excellent taste. However, salmon is rich in protein, unsaturated fatty acids, and water, making it highly susceptible to microbial contamination and quality deterioration under refrigeration conditions. This leads to spoilage, shortened shelf life, and seriously affects food safety and economic value.

[0003] In salmon and other chilled aquatic products, pathogenic and spoilage bacteria such as Listeria monocytogenes exhibit strong resistance to low temperatures and biofilm formation capabilities, enabling them to continuously grow and reproduce under refrigeration conditions. This makes them a significant factor contributing to aquatic product spoilage and food safety risks. Existing research indicates that Listeria monocytogenes not only causes food spoilage but may also lead to serious foodborne illnesses, posing a potential threat to public health. Therefore, developing safe, efficient, and suitable antimicrobial preservation technologies for chilled aquatic products is of great importance.

[0004] Currently, salmon preservation mainly relies on chemical preservatives, natural extracts, or physical preservation techniques. For example, commonly used chemical preservatives include organic acids and their salts, nitrites, etc., but their long-term use may raise safety concerns and easily trigger consumer concerns about food additives. While natural plant extracts possess certain antibacterial activity, they generally suffer from insufficient stability, significant flavor interference, limited antibacterial spectrum, or high cost.

[0005] Furthermore, while physical preservation technologies such as refrigeration, modified atmosphere packaging, and irradiation can slow down microbial growth, their inhibitory effects on cold-resistant microorganisms and biofilms are limited, and they cannot fundamentally solve the problem of microbial contamination. Antimicrobial peptides, as a class of widely sourced and structurally diverse natural active substances, have gradually attracted attention in the food preservation field due to their diverse mechanisms of action, low likelihood of inducing drug resistance, and good biocompatibility. However, the application of existing antimicrobial peptides in food systems still faces many challenges, such as insufficient antimicrobial activity, limited stability, poor adaptability to complex food matrices, high production costs, and unstable actual preservation effects. Therefore, there is an urgent need to develop a novel antimicrobial peptide with high antimicrobial activity, good safety, and suitability for aquatic product preservation to meet practical application needs.

[0006] Against this backdrop, how to screen or construct a novel antimicrobial peptide and effectively apply it to the preservation system of refrigerated aquatic products such as salmon in order to effectively inhibit pathogenic and spoilage bacteria, extend shelf life, and improve food safety remains a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] To overcome the problems existing in related technologies, the purpose of this invention is to provide a novel antimicrobial peptide T1, its preparation method, and its application in the preservation of salmon. The novel antimicrobial peptide T1 exhibits strong antimicrobial activity and significantly inhibits foodborne pathogens such as Listeria monocytogenes. The antimicrobial peptide T1 has diverse mechanisms of action, is less likely to induce drug resistance, and provides stable preservation effects, significantly extending the shelf life of salmon.

[0008] A novel antimicrobial peptide T1 and its application in the preservation of fresh salmon, wherein the novel antimicrobial peptide T1 has the amino acid sequence shown in SEQ ID NO:1.

[0009] In a preferred embodiment of the present invention, the novel antimicrobial peptide T1 is composed of 11 amino acid residues, has a net charge of +1, a hydrophobicity parameter H=0.582, and a hydrophobic moment μH=0.266; the novel antimicrobial peptide T1 has hydrophobic and dispersive amphiphilic characteristics.

[0010] This invention also provides a method for preparing a novel antimicrobial peptide T1, comprising: Using lactic acid bacteria as the fermentation strain, fermentation was carried out in a culture system containing protein substrate, and the antimicrobial peptide T1 was obtained by separation and purification. Alternatively, antimicrobial peptide T1 can be prepared using solid-phase peptide synthesis technology.

[0011] In a preferred embodiment of the present invention, the fermentation of lactic acid bacteria in a culture system containing a protein substrate, followed by separation and purification, yields the antimicrobial peptide T1, comprising: Mix defatted soybean meal with water at a mass-volume ratio of 1:12, boil for 15 minutes, and then filter. The activated lactic acid bacteria were inoculated into soybean meal substrate, and 2% glucose by weight was added. The mixture was fermented at 37°C for 5 days to obtain the fermentation broth. The fermentation broth was centrifuged, evaporated and concentrated, and then freeze-dried to obtain crude peptide samples. The crude peptide sample was prepared into a crude peptide solution, and the crude peptide solution was filtered using a 5 kDa ultrafiltration membrane to collect the fraction with a value less than 5 kDa. The fraction was purified by preparative reversed-phase high-performance liquid chromatography to obtain antimicrobial peptide T1.

[0012] In a preferred embodiment of the present invention, a first elution peak, a second elution peak, and a third elution peak are selected during the purification process. The third elution peak appears at a time longer than the first and second elution peaks, and the appearance time of the third elution peak is greater than 14 min and less than 15 min. The diameter of the inhibition zone formed by the component of the third elution peak is larger than the diameter of the inhibition zone formed by the component of the first elution peak, and the diameter of the inhibition zone formed by the component of the third elution peak is larger than the diameter of the inhibition zone formed by the component of the second elution peak.

[0013] In a preferred embodiment of the present invention, the novel antimicrobial peptide T1 is used to prepare a food preservative to inhibit the growth of microorganisms in salmon.

[0014] In a preferred embodiment of the present invention, the microorganism includes any one or more of Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, and Pseudomonas.

[0015] In a preferred embodiment of the present invention, the minimum inhibitory concentration of the novel antimicrobial peptide T1 is 3.5 mg / mL.

[0016] In a preferred embodiment of the present invention, the effects of the novel antimicrobial peptide T1 on the cell membrane include altering the surface charge properties of the cell membrane, enhancing the membrane permeability of the cell membrane, and regulating the energy metabolism of the cell membrane.

[0017] In a preferred embodiment of the present invention, the novel antimicrobial peptide T1 is used to reduce the total bacterial count and TVB-N content of salmon during the salmon preservation process.

[0018] The beneficial effects of this invention are as follows: The novel antimicrobial peptide T1 provided by this invention is a polypeptide in the third elution peak (F3 fraction) obtained during the separation of small molecule peptides from ultrafiltration of microbial fermentation products. The amino acid sequence of T1 is GLKPGLHGFHV, and its minimum inhibitory concentration (MIC) against Listeria monocytogenes is 3.5 mg / mL. The novel antimicrobial peptide T1 contains numerous protonable residues, making it more readily positively charged under physiological conditions. This facilitates initial electrostatic adsorption to the negatively charged Listeria cell surface, thereby enhancing its antimicrobial effect. Due to its low MIC, the novel antimicrobial peptide T1 provided by this invention exhibits significant inhibitory effects against foodborne pathogens such as Listeria monocytogenes, and is particularly suitable for cold-resistant microorganisms that maintain strong survival capabilities under refrigeration conditions. It can effectively reduce the number of pathogenic and spoilage bacteria in salmon. The novel antimicrobial peptide T1 exerts its antimicrobial effect through multiple mechanisms, including disrupting cell membrane structure, interfering with bacterial metabolic processes, and inhibiting virulence-related functions, thereby reducing the risk of microbial resistance. Applying antimicrobial peptide T1 to the preservation process of salmon can effectively reduce the total bacterial count and TVB-N content, delay quality deterioration, and thus significantly extend the refrigerated shelf life of salmon and improve product safety. Attached Figure Description

[0019] Figure 1 This is a diagram showing the overall analysis results of the F3 component polypeptide of the present invention, wherein... Figure 1 A represents the inhibition zones of different components. Figure 1 B represents protein information for component F3. Figure 1 C represents the molecular weight distribution of component F3; Figure 1 D represents the overall distribution of molecular weight and isoelectric point of component F3; Figure 2 These are the mass spectra of three candidate polypeptides of the present invention; wherein, Figure 2 a represents the mass spectrum of the T4 polypeptide. Figure 2 b is the mass spectrum of the T1 polypeptide. Figure 2 c represents the mass spectrum of the T3 polypeptide; Figure 3 This is a schematic diagram of the α-helix wheel of the antimicrobial peptide T1 of the present invention; Figure 4 This is a schematic diagram of the elution peaks of the F3 component during the elution separation process of the present invention. Detailed Implementation

[0020] Preferred embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While preferred embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0021] Example 1: Amino acid sequence and physicochemical properties of novel antimicrobial peptide T1 like Figure 1 As shown, this embodiment provides a novel antimicrobial peptide T1, which has an amino acid sequence as shown in SEQ ID NO:1, namely GLKPGLHGFHV.

[0022] The novel antimicrobial peptide T1 consists of 11 amino acid residues with a net charge of +1, a hydrophobicity parameter H=0.582, and a hydrophobic moment μH=0.266; the novel antimicrobial peptide T1 has hydrophobic and dispersive amphiphilic characteristics.

[0023] Figure 3 This is a schematic diagram of the α-helix of the novel antimicrobial peptide T1 (GLKPGLHGFHV). Lysine (K) is shown in dark blue, histidine (H) in light blue, the hydrophobic amino acids leucine (L), valine (V), and phenylalanine (F) in yellow, proline (P) in green, and glycine (G) in gray. The N-terminus and C-terminus are indicated by red letters.

[0024] The chemical structural formula of the novel antimicrobial peptide T1 prepared in this invention is as follows:

[0025] The novel antimicrobial peptide T1 is a short-chain antimicrobial peptide. Helical wheel analysis revealed the absence of a continuous, distinct hydrophobic surface, indicating moderate hydrophobicity and dispersible amphiphilicity. In vitro antibacterial experiments showed that the novel antimicrobial peptide T1 significantly inhibited Listeria monocytogenes, with a minimum inhibitory concentration (MIC) of 3.5 mg / mL. Based on sequence composition and physicochemical parameters, it is speculated that the positively charged residues of the novel antimicrobial peptide T1 may promote its interaction with negatively charged components on the bacterial surface, while hydrophobic / aromatic residues may participate in membrane interface binding, thus relating to its antimicrobial activity.

[0026] Example 2: Preparation method of novel antimicrobial peptide T1 Preparation of novel antimicrobial peptide T1 by lactic acid bacteria fermentation: using Lactobacillus paracasei Lactobacillus paracasei FX-6 was used as the fermentation strain to prepare crude peptide samples. This strain was derived from a traditional fermented dairy product system and is deposited at the China Center for Type Culture Collection (CCTCC; accession number: M2014043). Before the experiment, FX-6 was inoculated into MRS medium and statically cultured at 37°C for 8 h to obtain activated bacterial solution for subsequent fermentation experiments.

[0027] The crude peptides were prepared according to the previous method, by mixing defatted soybean meal and water at a ratio of 1:12 (w / v), boiling for 15 min, and then filtering. In this invention, w / v represents the mass-volume percentage. The activated peptides... Lactobacillus paracasei FX-6 (1 × 10 8 CFU was inoculated into a soybean meal substrate, and 2% (w / v) glucose was added. Fermentation was carried out at 37°C for 5 days. The fermentation broth was centrifuged at 4000 rpm for 20 min, and the supernatant was collected, concentrated by rotary evaporation, and then lyophilized. The resulting powder was filtered through a 0.22 μm filter membrane as the crude peptide sample.

[0028] The crude peptide solution was fractionated using a 5 kDa ultrafiltration membrane (Pellicon XL, Millipore, USA), and the <5 kDa fraction was collected. This fraction was precipitated with 70% ethanol (4 °C, overnight), and the supernatant was lyophilized and purified by preparative reversed-phase high-performance liquid chromatography (RP-HPLC). Separation was performed on a C18 column using mobile phase A of 2% acetonitrile aqueous solution containing 0.1% formic acid and mobile phase B of 80% acetonitrile solution containing 0.1% formic acid. Linear gradient elution was performed using 4–8% mobile phase B (80 min), and the elution peak was monitored at 214 nm.

[0029] Antimicrobial peptides in the samples were identified using high-resolution liquid chromatography-tandem mass spectrometry (LC-MS / MS). The lyophilized samples were dissolved in the mass spectrometry loading buffer and analyzed using a Vanquish Neo liquid chromatography system coupled with a timsTOF HT mass spectrometer.

[0030] Liquid chromatography was performed using a C18 column (15 cm × 100 μm, 1.7 μm) with a gradient elution program of: 0–1 min, 8–17% mobile phase B; 1–5.5 min, 17–55% mobile phase B; 5.5–7 min, 55–99% mobile phase B; 7–8 min, 99% mobile phase B. The corresponding flow rates were 1, 0.4, 0.7, and 1 μL / min. Mass spectrometry data were acquired in DDA-PASEF mode with an ion source voltage of 1600 V. The primary and secondary scan ranges were both 180–1800 m / z, and the cycle time was 0.39 s.

[0031] Peptide sequences obtained from MS / MS spectra were compared with those of soybean proteins retrieved from the UniProt database. Lactobacillus paracaseiThe relevant amino acid sequences were compared and verified. Subsequently, candidate peptides were further screened using various online antimicrobial peptide prediction tools, including CAMPR3 (http: / / www.camp3.bicnirrh.res.in / ), AntiBP2 (http: / / crdd.osdd.net / raghava / antibp2 / ), the antimicrobial peptide database APD (http: / / aps.unmc.edu / AP / ), the antimicrobial activity and peptide structure database DBAASP (https: / / dbaasp.org / tools?page=general-prediction), and MultiPep (https: / / agbg.shinyapps.io / MultiPep / ), to evaluate their potential antimicrobial properties.

[0032] To further screen candidate antimicrobial peptides with potential antimicrobial activity based on peptide prediction screening, and to evaluate their potential interactions with key functional proteins of Listeria monocytogenes at the structural level, molecular docking analysis was conducted. Three-dimensional structures of candidate antimicrobial peptides were constructed using Chem3D Ultra 14.0, and energy minimization was performed. Subsequently, AutoDockTools 1.5.7 was used to dehydrate, hydrogenate, and set rotatable bonds in the peptide structures, and the results were saved as pdbqt files.

[0033] Selected Listeria monocytogenes ( Listeria monocytogenes The penicillin-binding protein PBP4 (PDB ID: 3ZG8) and the surface-invasive protein InlA (PDB ID: 1O6T) were used as molecular docking target proteins. The structures of these proteins were obtained from the RCSB Protein Data Bank database. Before docking, water molecules and co-crystallization ligands were removed from the protein structures, and hydrogenation was performed using AutoDockTools 1.5.7, exporting the results as a receptor pdbqt file.

[0034] Molecular docking was performed using AutoDock Vina software. Each candidate antimicrobial peptide was simulated for docking with PBP4 and InlA, with each peptide-protein system undergoing 20 independent docking calculations. The binding free energy obtained from docking was used as an evaluation metric to compare the binding affinity between different peptides and target proteins.

[0035] Solid-phase peptide synthesis of antimicrobial peptide T1: Three identified and screened antimicrobial peptides (AMPs) were prepared by solid-phase peptide synthesis at Medef Biotechnology Co., Ltd. (Suzhou, Jiangsu Province, China). The synthesized peptides were lyophilized and stored at -20°C. The purity and molecular weight of the synthesized peptides were analyzed using HPLC-MS, and the results showed that the purity was higher than 95%. Furthermore, mass spectrometry analysis further confirmed the molecular weight and structure of the peptides.

[0036] Example 3 Analysis of Elution Peaks This embodiment uses *Listeria monocytogenes* as the target bacterium to further analyze the core active peptide components that exert inhibitory effects in the fermentation products. Following the aforementioned crude peptide preparation and separation process, the <5 kDa small molecule peptide components obtained by ultrafiltration were separated by preparative reversed-phase high-performance liquid chromatography (RP-HPLC). Although the components smaller than 5 kDa are still relatively complex in composition, RP-HPLC can effectively separate and enrich them based on the differences in hydrophobicity and polarity of peptide molecules. Three main elution peaks were obtained during the chromatographic separation process, named F1, F2, and F3, respectively. F1 is the first elution peak, F2 is the second elution peak, and F3 is the third elution peak.

[0037] like Figure 4 As shown, the first elution peak appears after 9 min but less than 10 min. The second elution peak appears after 12 min but less than 13 min. The third elution peak appears after 14 min but less than 15 min.

[0038] Subsequently, the antibacterial activities of different elution peaks were preliminarily screened to compare their inhibitory differences against various bacteria. The results showed significant differences in the antibacterial spectrum and inhibitory intensity among the different peak components. Among them, the F3 component exhibited relatively strong antibacterial activity, with minimum inhibitory concentrations (MICs) of 16 mg / mL, 16 mg / mL, and 8 mg / mL against *Escherichia coli*, *Staphylococcus aureus*, and *Listeria monocytogenes*, respectively.

[0039] like Figure 1 As shown in Figure A, the inhibitory effect on Listeria monocytogenes was further evaluated using the disc diffusion method. The results showed that the diameter of the inhibition zone formed by the F3 component was significantly larger than that of other elution peaks, indicating that it had a more significant inhibitory effect on Listeria monocytogenes.

[0040] Based on the combined results of MIC assays and inhibition zone experiments, fraction F3 exhibited the most significant activity against Listeria monocytogenes, suggesting that it may contain enriched antimicrobial peptides that play a key role in this fermentation system. Therefore, fraction F3 was selected as the representative active fraction for subsequent systematic evaluation of its antimicrobial activity and investigation of its mechanism of action.

[0041] Example 4: Whole-body analysis of F3 component peptides based on peptidomics Given that purified fraction F3 exhibited the most significant antibacterial activity against Listeria monocytogenes, LC-MS / MS peptidomics was further employed to systematically analyze this fraction and comprehensively characterize its peptide composition. Raw MS / MS data were processed using PEAKS Studio software, and peptide identification was performed by searching the UniProt database. Figure 1 As shown in B, a total of 3459 reliably matched MS / MS spectra were obtained, and 220 non-redundant peptides were finally identified and merged into 402 proteomes, indicating that the F3 component has high peptide composition complexity.

[0042] From the perspective of the protein level corresponding to peptide annotation, its molecular weight distribution is as follows: Figure 1 As shown in Figure C, most related proteins are concentrated in the 10-60 kDa range, reflecting that the low molecular weight peptides detected in this fermentation system are mainly associated with medium molecular weight proteins. Further analysis of the physicochemical characteristics of the peptides themselves... Figure 1 D shows the overall distribution of molecular weight and isoelectric point of the identified peptides. The results show that these peptides are generally low in molecular weight, mainly concentrated in the range of 0.7-3.8 kDa, and the isoelectric point range is 4-12, indicating that the F3 component contains both acidic and basic peptides.

[0043] The identified peptides are mainly composed of 6-31 amino acid residues, with a corresponding molecular weight range of 743-3766 Da. Overall, the F3 component constitutes a diverse set of peptides with low molecular weight and wide distribution of charge characteristics, providing a reliable data basis for the prediction, screening and functional verification of antimicrobial peptides.

[0044] Example 5: Predictive Screening of Antimicrobial Peptides Based on Bioinformatics Bioinformatics prediction tools have been widely used for the preliminary assessment of the potential antibacterial activity of peptide sequences. In this invention, PeptideRanker is first used to screen all peptide sequences obtained from peptidomics identification, retaining only peptides with a prediction score greater than 0.5 as candidate sequences with potential biological activity. This threshold is considered to effectively enrich short peptide sequences with functional potential while reducing the probability of irrelevant peptides entering subsequent analyses.

[0045] Subsequently, the candidate peptides obtained from the initial screening were submitted to multiple antimicrobial peptide prediction platforms for further analysis, including APD3, CAMPR3, AntiBP2, DBAASP, and MULTIPEP. These platforms evaluate the antimicrobial potential of peptides based on different algorithm models and training datasets (such as support vector machines, random forests, or deep learning models). Therefore, the judgment results for the same peptide may differ across different prediction tools. To improve the reliability and consistency of the screening results, this study integrated and analyzed the results obtained from the five prediction tools and removed repetitive sequences. Only when the same peptide was consistently judged to have antimicrobial peptide characteristics on at least three prediction platforms was it included in the further screening scope. Based on this, the candidate sequences were further optimized and screened by combining the prediction scores and the physicochemical characteristics of the peptides. Existing research has shown that typical antimicrobial peptides usually have physicochemical characteristics such as an overall positive charge and high hydrophobicity. These properties help them bind to the negatively charged bacterial cell membrane through electrostatic interactions and further insert into the lipid bilayer to exert their antibacterial effect. Based on the above theory, the physicochemical properties analysis of the screened candidate peptides revealed that all 17 candidate peptides obtained were positively charged and had a hydrophobicity ratio of over 30%, which is consistent with the structural characteristics of classic antimicrobial peptides.

[0046] Penicillin-binding protein PBP4 and surface protein InlA are involved in cell wall-related processes and surface-related functions, respectively, in Listeria monocytogenes, and are representative key functional proteins. Based on this, this study selected PBP4 (PDB ID: 3ZG8) and InlA (PDB ID: 1O6T) as molecular docking targets to further screen 17 candidate antimicrobial peptides obtained from the aforementioned bioinformatics prediction screening. Molecular docking analysis was performed on all candidate peptides with PBP4 and InlA, and their potential antimicrobial activity was evaluated based on their binding free energy. The results showed that all 17 candidate peptides could form stable bindings with both target proteins, and the binding free energies were all negative, indicating that these peptides have the structural potential to interact with key functional proteins of Listeria monocytogenes.

[0047] Among all candidate peptides, T1, T3, and T4 exhibited relatively low binding free energies on both target proteins, indicating strong binding affinity. Specifically, T1 (GLKPGLHGFHV) showed a binding free energy of -10.95 kcal / mol on InlA and stable binding on PBP4 (-7.41 kcal / mol), ranking first among the 17 candidate peptides. Meanwhile, T3 and T4 also showed superior docking scores on PBP4 and InlA, and their positive charge to hydrophobicity ratios conformed to the typical physicochemical characteristics of antimicrobial peptides.

[0048] Example 6: Verification of the antimicrobial activity of synthesized antimicrobial peptides against Listeria monocytogenes Three candidate peptides, T1, T3, and T4, were synthesized using a solid-phase synthesis method, such as... Figure 2 As shown in (b), the amino acid sequence of polypeptide T1 is GLKPGLHGFHV. Figure 2 As shown in (c), the amino acid sequence of polypeptide T3 is LVVSKNKPLVVQF. Figure 2 As shown in (a), the amino acid sequence of polypeptide T4 is GGSVLSGFSKHFL.

[0049] Peptide T3 showed no significant antibacterial activity within the tested concentration range, while peptide T1 exhibited stronger antibacterial activity, with a minimum inhibitory concentration (MIC) of 3.5 mg / mL. Peptide T4 also showed inhibitory activity against Listeria monocytogenes, but its antibacterial ability was relatively weak, with an MIC of 7 mg / mL.

[0050] Further comparison of the sequence characteristics of the three peptides reveals that the differences in antibacterial activity may be closely related to the amino acid composition and physicochemical properties of the peptides. Peptide T1 contains more protonable residues (such as Lys and His), making it more likely to carry a positive charge under physiological conditions. This facilitates initial electrostatic adsorption to the negatively charged Listeria cell surface, thereby enhancing its antibacterial effect. In contrast, peptide T4 has a higher proportion of polar and flexible residues (such as Gly and Ser), which may reduce its conformational stability and effective interaction with the cell membrane, resulting in weakened antibacterial activity. Although peptide T3 contains a certain number of hydrophobic residues, its overall charge distribution and amphiphilicity may be insufficient to support effective cell membrane binding or disruption, thus failing to exhibit significant antibacterial activity.

[0051] In summary, the results of this study indicate that even short peptides of similar length can exhibit significantly different antibacterial activities against Listeria monocytogenes due to subtle differences in sequence composition. Among them, peptide T1 showed the best antibacterial performance and was therefore selected for subsequent mechanistic studies.

[0052] Example 7: Application of novel antimicrobial peptide T1 in the preservation of smoked salmon (1) Changes in appearance and color: Sensory qualities (such as color and texture) are important indicators of changes in the quality of smoked salmon. Under refrigeration at 4℃, with the extension of storage time, the control group samples gradually showed phenomena such as darkening of color, weakening of luster, and increase of surface mucus, showing a clear trend of quality deterioration. In contrast, the appearance changes of the T1 treatment group samples were relatively slow, and the overall color remained relatively stable. The results show that the treatment with the novel antimicrobial peptide T1 can delay the changes in the appearance quality of smoked salmon during refrigeration, thereby improving its sensory status to a certain extent.

[0053] (2) Microbial Count: Listeria monocytogenes is considered a representative risk microorganism in smoked salmon. It can grow under low-temperature conditions, making its growth monitoring crucial. With prolonged storage, the number of microorganisms in the control group samples increased rapidly. By day 6 of storage, the total colony count (TVC) was close to 6.84–7.12 log CFU / g, and continued to rise during subsequent storage, indicating gradual and significant spoilage. In contrast, the microbial proliferation in the T1-treated group samples slowed significantly. The TVC growth rates in the 1 / 2×MIC and 1×MIC groups were lower than those in the control group, and the overall levels remained relatively low. By day 10 of storage, the TVC in the control group rose to 7.21–7.59 log CFU / g, while the 1 / 2×MIC and 1×MIC groups were 5.45–5.74 and 4.33–5.36 log CFU / g, respectively, demonstrating the significant inhibitory effect of T1 on microbial growth.

[0054] Based on the aforementioned in vitro experimental results, it can be inferred that the antibacterial effect of T1 in the smoked salmon system may be related to its disruption of bacterial cell membrane structure and inhibition of biofilm formation. The novel antimicrobial peptide T1 reduced the total bacterial count and TVB-N content in salmon during preservation. TVB-N represents volatile basic nitrogen and is commonly used as an important indicator for evaluating protein degradation and spoilage during fish storage. In contrast, TVB-N accumulation in the T1-treated group was significantly slowed. By day 10 of storage, the TVB-N content in the 1 / 2×MIC group and the 1×MIC group was approximately 5.31–5.97 and 4.88–5.22 mg / 100 g, respectively, significantly lower than the control group, indicating that T1 treatment can delay protein degradation in smoked salmon during refrigerated storage. Combined with the microbial counting results, it can be inferred that the inhibitory effect of T1 on TVB-N accumulation may be related to its ability to slow microbial growth and reduce the rate of protein degradation, thereby reducing the formation of volatile nitrogenous substances such as ammonia and amines.

[0055] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, apparatus, article, or method that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, apparatus, article, or method. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, apparatus, article, or method that includes that element.

[0056] The above description is only a preferred embodiment of this application and does not limit the patent scope of this application. Any equivalent structural or procedural changes made based on the content of this application's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of this application.

Claims

1. A novel antimicrobial peptide T1, characterized in that, The novel antimicrobial peptide T1 has the amino acid sequence shown in SEQ ID NO:

1.

2. The novel antimicrobial peptide T1 according to claim 1, characterized in that, The novel antimicrobial peptide T1 consists of 11 amino acid residues with a net charge of +1, a hydrophobicity parameter H=0.582, and a hydrophobic moment μH=0.266; the novel antimicrobial peptide T1 has hydrophobic and dispersive amphiphilic characteristics.

3. The method for preparing the novel antimicrobial peptide T1 according to claim 1, characterized in that, include: Using lactic acid bacteria as the fermentation strain, fermentation was carried out in a culture system containing protein substrate, and the antimicrobial peptide T1 was obtained by separation and purification. Alternatively, antimicrobial peptide T1 can be prepared using solid-phase peptide synthesis technology.

4. The method according to claim 3, characterized in that, The method involves fermenting lactic acid bacteria in a culture system containing a protein substrate, followed by separation and purification to obtain the antimicrobial peptide T1, which includes: Mix defatted soybean meal with water at a mass-volume ratio of 1:12, boil for 15 minutes, and then filter. The activated lactic acid bacteria were inoculated into soybean meal substrate, and 2% glucose by weight was added. The mixture was fermented at 37°C for 5 days to obtain the fermentation broth. The fermentation broth was centrifuged, evaporated and concentrated, and then freeze-dried to obtain crude peptide samples. The crude peptide sample was prepared into a crude peptide solution, and the crude peptide solution was filtered using a 5 kDa ultrafiltration membrane to collect the fraction with a value less than 5 kDa. The fraction was purified by preparative reversed-phase high-performance liquid chromatography to obtain antimicrobial peptide T1.

5. The method according to claim 4, characterized in that, During the purification process, a first elution peak, a second elution peak, and a third elution peak were selected. The third elution peak appeared at a time longer than the first and second elution peaks, with the appearance time of the third elution peak being greater than 14 min and less than 15 min. The diameter of the inhibition zone formed by the component of the third elution peak was larger than that formed by the component of the first elution peak, and the diameter of the inhibition zone formed by the component of the third elution peak was larger than that formed by the component of the second elution peak.

6. The application of the novel antimicrobial peptide T1 according to claim 1 in the preservation of salmon, characterized in that, A food preservative was prepared using the novel antimicrobial peptide T1 to inhibit the growth of microorganisms in salmon.

7. The application according to claim 6, characterized in that, The microorganisms include any one or more of Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, and Pseudomonas.

8. The application according to claim 6, characterized in that, The minimum inhibitory concentration of the novel antimicrobial peptide T1 is 3.5 mg / mL.

9. The application according to claim 6, characterized in that, The effects of the novel antimicrobial peptide T1 on the cell membrane include altering the surface charge properties of the cell membrane, enhancing cell membrane permeability, and regulating cell membrane energy metabolism.

10. The application according to claim 6, characterized in that, The novel antimicrobial peptide T1 is used to reduce the total bacterial count and TVB-N content of salmon during the preservation process.

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