Antibacterial peptide and application thereof

Abstract

The application discloses an antibacterial peptide and application thereof, and the antibacterial peptide is selected from any one of polypeptides shown in the following: SEQ ID NO:1: MRCFKRLIKVFLIARKR; SEQ ID NO:2: MWIAANKTLFSSRKMMAIICVVVLIFPGIAAA; and SEQ ID NO:3: MSRSKATCLYFFRCAINALT. The antibacterial peptide, a preparation method and application thereof are obtained through screening and optimization design on a genome sequence of lactobacillus plantarum, a series of novel antibacterial peptides are obtained, compared with other antibacterial peptides, the antibacterial peptide LP2140 has better antibacterial effect, can effectively inhibit the growth of shewanella putrefaciens, can be used for water product preservation, can delay spoilage and deterioration and prolong a shelf life, provides a new thought for water product preservation technology, and provides theoretical support and practical reference for food safety and quality guarantee.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to an antimicrobial peptide and its applications. Background Technology

[0002] *Lactobacillus plantarum* is the most widely distributed species in the *Lactobacillus* genus and is currently widely used in the food and feed industries. *Lactobacillus plantarum* has strong acid resistance and is a generally recognized as safe (GRSA) microorganism, considered the most important and widely used species among lactic acid bacteria. Plantaricin, synthesized during the metabolism of *Lactobacillus plantarum*, is a natural antimicrobial peptide that exhibits inhibitory activity against both Gram-negative and Gram-positive bacteria. It is safe, unlikely to induce drug resistance, and leaves no residue in food, making it a promising candidate for applications in the food and feed industries. The development of novel plantaricin and its applications has become a research hotspot both domestically and internationally.

[0003] However, the difficulty in screening plant lactobacilliin, low yield, and cumbersome isolation and purification steps have hindered its industrial production and application. In recent years, the development of microbial genomics has enabled the discovery of novel plant lactobacilliin at the gene level of plant lactic acid bacteria. The establishment of large peptide databases has brought new methods for the development of novel plant lactobacilliin. Artificial intelligence algorithms can autonomously learn sequence characteristics and screen potential antimicrobial peptides from a large-scale genome by identifying characteristic sequences from the genome, even identifying the correlation characteristics of short sequences with low homology. Artificial intelligence algorithms mainly include machine learning and deep learning, primarily predicting biological activity through amino acid sequences, utilizing several prediction algorithms such as artificial neural networks (ANN), quantitative matrix (QM), random forest, and support vector machine (SVM). Lee et al. developed a quantitative structure-activity relationship (QSAR) model using machine learning to effectively screen and optimize the sequences of antimicrobial peptides, achieving the function of predicting molecular biological activity. Boone et al. combined genetic algorithms with rough set theory to design antimicrobial peptides. This method provides a clear boundary for the physicochemical properties of active antimicrobial peptide sequences that are not present in inactive sequences, and found that antimicrobial peptides synthesized using this method are easier to synthesize than those in the APD3 database. Wang et al. designed short sequences of antimicrobial peptides with potential anti-E. coli activity by constructing an LSTM generative model (Long Short-Term Memory, a type of RNN model) and a bidirectional LSTM classification model. The validation accuracy of the classification model was 81.6%–88.9%, and the accuracy of classifying novel antimicrobial peptides as antibacterial drugs was as high as 70.6%–91.7%, indicating that LSTM is an effective tool for finding novel antimicrobial peptides. Sharma et al., based on deep learning features, used the SVM algorithm to construct the AniAMPpred model to identify possible antimicrobial proteins in animal genomes. Meanwhile, the connection between artificial intelligence and synthetic biology is becoming increasingly close. Researchers are using artificial intelligence algorithms to design and optimize the coding sequences of antimicrobial peptides, further improving their bactericidal activity. By introducing artificial intelligence algorithms, rapid prediction and optimized screening of the antibacterial activity of *Lactobacillus plantarum* peptides can be achieved, greatly improving screening efficiency and accuracy. However, there are currently few reports on research into AI-assisted discovery of antimicrobial peptides in *Lactobacillus plantarum*.

[0004] Shewanella putrefaciens is a Gram-negative bacillus with strong putrefactive properties, making it one of the dominant spoilage bacteria in aquatic products. It can still grow even under low-temperature storage conditions, posing a significant threat to aquatic products. Shewanella putrefaciens metabolizes muscle protein in aquatic products, decarboxylating amino acids to produce biogenic amines such as putrescine and cadaverine, and generating odorous substances such as H2S. The biofilm formed by Shewanella putrefaciens can cause a sticky surface on fish. Simultaneously, Shewanella putrefaciens is a rare opportunistic human pathogen, primarily associated with intra-abdominal, skin, and soft tissue infections, and can also cause sepsis, potentially leading to death. Currently, Shewanella putrefaciens infections usually require antibiotic treatment; however, antibiotic resistance in Shewanella is increasing, exhibiting resistance to various drugs including β-lactams, quinolones, aminoglycosides, and macrolides. Therefore, there is an urgent need to find a natural antibacterial agent to address this challenge.

[0005] Marine fish are highly nutritious and considered one of the most important foods in the human diet. However, once caught and taken ashore, they are prone to surviving due to sudden changes in environmental factors such as air pressure, temperature, and dissolved oxygen. After a fish dies, cellular metabolism ceases, and the goblet cells in the epidermis stop secreting mucus containing antibacterial substances. Furthermore, fish meat, with its short muscle fibers and high water content, is easily spoiled by microbial growth during storage, resulting in significant losses. Spoilage of fish meat is typically caused by three factors: autolytic enzyme decomposition, fat oxidation, and microbial growth; among these, microbial growth and metabolism are the main causes of spoilage. Studies have shown that Shewanella, with Shewanella as a typical example, is a major specific spoilage bacterium in marine fish, playing a dominant role in the spoilage process. Currently, the country places great emphasis on food safety. Due to the food safety risks associated with chemically synthesized preservatives, the development of novel, green, and safe lactic acid bacteria-derived bacteriocins for the preservation of marine fish is of great significance.

[0006] Therefore, to address the aforementioned technical issues, it is necessary to provide a novel antimicrobial peptide that can achieve both preservation and food safety. Summary of the Invention

[0007] The purpose of this invention is to provide an antimicrobial peptide, its preparation method, and its application. Through screening and optimization of the genome sequence of Lactobacillus plantarum, a novel antimicrobial peptide was obtained, which can effectively inhibit the growth of Shewanella putrefactive bacteria, thereby preserving fish without causing food safety issues.

[0008] To achieve the above objectives, a specific embodiment of the present invention provides the following technical solution:

[0009] On one hand, the present invention provides an antimicrobial peptide or a derivative thereof, wherein the antimicrobial peptide is selected from any one of the following polypeptides: SEQ ID NO:1: MRCFKRLIKVFLIARKR; SEQ ID NO:2: MWIAANKTLFSSRKMMAIICVVVLIFPGIAAA; SEQ ID NO:3: MSRSKATCLYFFRCAINALT.

[0010] In one or more embodiments of the present invention, the antimicrobial peptide is the polypeptide shown in SEQ ID NO:1: MRCFKRLIKVFLIARKR.

[0011] In one or more embodiments of the present invention, the antimicrobial peptide or its derivative is designed based on the genome optimization of Lactobacillus plantarum MMB-07, the accession number of which is CGMCC NO.20032.

[0012] On the other hand, the present invention also provides the application of the above-mentioned antimicrobial peptide or its derivatives in preservation, preferably in the preservation of aquatic products.

[0013] In another aspect, the present invention also provides the use of the above-mentioned antimicrobial peptide or its derivative in the preparation of antimicrobial drugs.

[0014] In one or more embodiments of the present invention, the antibacterial drug is used for preservation and freshness protection, preferably for the preservation and freshness protection of aquatic products.

[0015] In another aspect, the present invention also provides the application of the above-mentioned antimicrobial peptide or its derivatives in inhibiting microorganisms.

[0016] In one or more embodiments of the present invention, the microorganism is Shewanella putrefactive.

[0017] On the other hand, an antimicrobial drug includes the aforementioned antimicrobial peptide or its derivatives, and a pharmaceutically acceptable carrier.

[0018] In one or more embodiments of the present invention, the antimicrobial agent preserves food by inhibiting Shewanella putrefactive bacteria.

[0019] Compared with existing technologies, the antimicrobial peptides, their preparation methods, and applications of this invention, through the screening and optimization design of the genome sequence of *Lactobacillus plantarum*, combined with artificial intelligence analysis, have yielded a series of novel antimicrobial peptides. Experiments have shown that the antimicrobial peptide LP2140 exhibits better antimicrobial effects compared to other antimicrobial peptides. LP2140 can effectively inhibit the growth of *Shewanella putrefactiveis*, thereby maintaining the quality of aquatic products such as fish during storage, delaying spoilage, and extending shelf life. Furthermore, since it originates from *Lactobacillus plantarum* MMB-07, there are no issues related to drug resistance or safety, providing new ideas for aquatic product preservation technology and offering theoretical support and practical reference for food safety and quality assurance. In addition, by combining artificial intelligence analysis of the genome sequence and optimizing the design to obtain antimicrobial peptides that are easier to synthesize, this invention provides insights for the development of new antimicrobial peptides. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a structural characterization diagram of Embodiment 2 of the present invention;

[0022] Figure 2 The circular dichroism chromatogram of Example 2 of the present invention;

[0023] Figure 3 This is a graph showing the effect of LP2150 on the growth of Shewanella putrefactive bacteria in Example 3 of the present invention.

[0024] Figure 4 This is an electron micrograph showing the effect of LP2150 on the morphology of Shewanella putrefactive in Example 3 of the present invention.

[0025] Figure 5 This is a sensory evaluation diagram of the preservation effect of LP2150 on large yellow croaker surimi in Example 4 of the present invention;

[0026] Figure 6 This is a graph showing the effect of LP2150 on the total bacterial count in large yellow croaker surimi in Example 4 of the present invention;

[0027] Figure 7 This is a graph showing the effect of LP2150 on the TVB-N value of large yellow croaker surimi in Example 4 of the present invention;

[0028] Figure 8This is a graph showing the effect of LP2150 on the TBA value of large yellow croaker surimi in Example 4 of the present invention;

[0029] Figure 9 This is a graph showing the effect of LP2150 on the pH value of large yellow croaker surimi in Example 4 of the present invention;

[0030] Figure 10 This is a graph showing the effect of LP2150 on the water-holding capacity of large yellow croaker surimi in Example 4 of the present invention;

[0031] Figure 11 Figure A shows the effect of LP2150 on large yellow croaker surimi in Example 4 of the present invention. Figure B shows the effect on brightness, Figure C shows the effect on red-green hue, and Figure D shows the effect on whiteness. Detailed Implementation

[0032] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0033] Unless otherwise specified, all materials and reagents used in this article are available from legitimate commercial sources.

[0034] Unless otherwise specified, "ORF2150" in this article refers to "LP2150".

[0035] Example 1. Screening and synthesis of antimicrobial peptide sequences

[0036] Using the genomic data of *Lactobacillus plantarum* MMB-07 (details of which are described in the authorized patent CN111979141B, accession number CGMCC NO.20032), the small open reading frames (sorfs) in the *Lactobacillus plantarum* genome sequence were first specifically identified using the MiPepid logistic regression model with 4-mer characteristics. The selected sorrfs were translated into 45,385 peptide sequences. The SignalP server was used to predict the signal peptide, which was then removed from the sequence, filtering out 302 secretory peptides. Subsequently, the TMHMM server was used to predict the ability of these peptides to be secreted into the extracellular space, thereby filtering out 44 transmembrane peptides. The deep neural network model DNN, which combines convolutional and recurrent layers, will be used to predict and identify potential antimicrobial peptides. Finally, the antimicrobial peptides ORF2150 (SEQ ID NO:1, MRCFKRLIKVFLIARKR), ORF25500 (SEQ ID NO:2, MWIAANKTLFSSRKMMAIICVVVLIFPGIAAA), and ORF39896 (SEQ ID NO:3, MSRSKATCLYFFRCAINALT) will be selected and chemically synthesized.

[0037] Example 2. Structural characterization and secondary structure content determination of antimicrobial peptide LP2150

[0038] 1. Peptide structure characterization: The theoretical molecular weight, hydrophobicity, and electrostatic charge of the antimicrobial peptide were calculated online using the website https: / / aps.unmc.edu / prediction; helical wheel projection was performed online using https: / / heliquest.ipmc.cnrs.fr / cgi-bin / ComputParams.py; and the peptide structure diagram was obtained online using https: / / www.allpeptide.com / jiegoutu.html. The results are as follows: Figure 1 And as shown in Table 1 below.

[0039] Table 1 Key Physicochemical Parameters of LP2150

[0040]

[0041] The chemical structural formula of LP2150 is:

[0042]

[0043] Experimental results, through CCS calculation and comparative analysis, revealed that LP2150 exhibits a random structure with the strongest positive charge (+7), the lowest hydrophilicity (Grand average of 0.171), and a relatively high lipid index (114.71). These structural characteristics and physicochemical properties indicate that LP2150 is a potential optimal antimicrobial peptide. The theoretical molecular weight values ​​of the peptides matched the measured values, and the purity of all compounds was above 95%, indicating successful synthesis.

[0044] 2. Determination of circular dichroism (CD)

[0045] Sample LP2150 was dissolved in 0.01 mol / L phosphate buffer (pH 7.0) to prepare a 0.2 mg / mL protein solution. Circular dichroism chromatograms were then analyzed using a JASCO-815 circular dichroism chromatogram. The wavelength range was 195–250 nm, the scan rate was 100 nm / min, the sample cell path length was 0.1 nm, and the phosphate buffer was used as a blank control. Circular dichroism chromatograms were expressed as mean ellipticity [θ], in degrees per centimeter (deg·cm). 2 / dmol, CD chromatogram data were analyzed using CDPRO software, and the relative content of secondary structures was evaluated using the bestsel platform. The results are as follows: Figure 2 As shown.

[0046] from Figure 2 It can be seen that the CD spectrum of LP2150 exhibits a continuous negative peak in the 190–250 nm band, with the peak maximum (mdeg minimum) mainly located around 199 nm. This band primarily represents the characteristic region of the Random structure, indicating that the secondary structure of LP2150 is predominantly Random. The fitting results for LP2150 also show that its relative contents of α-helix, β-sheet, β-turn, and Random are 5.59%, 26.57%, 19.18%, and 48.65%, respectively.

[0047] The secondary structures of ORF25500 and ORF39896 were characterized using the same method, and the content of the secondary structures was determined. The specific structures are not shown.

[0048] Example 3. Effect of antimicrobial peptides on the growth of Shewanella putrefactive bacteria.

[0049] 1. Effects of antimicrobial peptides on the growth of Shewanella putrefactive bacteria

[0050] The concentration of Shewanella putrefaciens CN32 bacterial suspension was adjusted to OD600 of 0.1. LP2150 was added to final concentrations of 0, 1 / 4 MIC, 1 / 2× MIC, 1× MIC, and 2× MIC. The suspensions were then incubated at 28°C with shaking. The absorbance of the bacterial suspension was measured at 600 nm every 2 hours. The results showed that the minimum inhibitory concentration (MIC) of LP2150 against Shewanella putrefaciens was 1 μg / mL, the MIC of ORF25500 against Shewanella putrefaciens was 16 μg / mL, and the MIC of ORF39896 against Shewanella putrefaciens was 4 μg / mL.

[0051] Among them, the results of the effect of LP2150 on the growth of Shewanella putrefactive bacteria are as follows: Figure 3 As shown, from Figure 3 It can be seen that in the control group (without LP2150), *Shewanella putrefactive* entered the logarithmic growth phase after 2 hours and began to enter the stationary phase after 20 hours. LP2150 at concentrations of 1 MIC and 2 MIC completely inhibited bacterial growth, while LP2150 at concentrations of 1 / 4 MIC and 1 / 2 MIC delayed both the logarithmic and stationary phases. When the bacteria finally reached the stationary phase, their concentration was significantly lower than that of the control group, indicating that LP2150 has a significant inhibitory effect on the growth of *Shewanella putrefactive*.

[0052] 2. Effects of LP2150 on the morphology of Shewanella putrefactive bacteria

[0053] LP2150 was added to the bacterial suspension resuspended in PBS to a final concentration of 2 MIC (Minimum Inhibitory Concentration, MIC). An equal volume of PBS buffer was added to the control group. The mixture was incubated at 28°C for 2 hours, and then the remaining LP2150 was removed with PBS. The bacterial suspension was fixed overnight at 4°C in 2.5% glutaraldehyde electron microscopy fixative.

[0054] Scanning Electron Microscope (SEM): After washing three times with PBS, the sample was dehydrated using a gradient of 30%, 50%, 70%, 80%, 90%, 100%, and 100% ethanol, and then replaced three times with tert-butanol. After drying, the sample was plated with gold using an ion sputtering instrument, and the morphology of the cells was observed using a scanning electron microscope.

[0055] Transmission Electron Microscopy (TEM): After washing three times with PBS, fixation with 1% osmium tetroxide was performed, followed by gradient dehydration with ethanol, each gradient lasting 20 min. Cells were then embedded in epoxy resin, ultrasectioned, and double-stained with uranium acetate and lead citrate. Cell morphology was observed using TEM. Results are shown below. Figure 4 As shown.

[0056] from Figure 4 It can be seen that the untreated *Shewanella putrefactive* cells are plump and typically rod-shaped, with intact cell morphology and dense internal structure. Figure 4 (A in the text). After treatment with 2×MIC LP2150 for 2 hours, many bacterial cells showed obvious deformation, and the cell surface exhibited significant shrinkage. Figure 4 (B in the original text) indicates that LP2150 can act on the cell wall structure of Shewanella putrefactive bacteria. Transmission electron microscopy (TEM) was used to further observe the ultrastructure and intracellular changes of Shewanella putrefactive bacteria. Untreated bacteria had intact cell walls tightly attached to the cell membrane, and their internal tissues were dense (…). Figure 4 (C in the text). After treatment with 2×MIC LP2150 for 2 hours, the bacterial cytoplasm became loose, plasmolysis occurred, intracellular components leaked out, and the cells lost their basic structure. Figure 4 (D in the middle).

[0057] The above results indicate that LP2150 significantly disrupts the integrity and intracellular structure of Shewanella putrefactive bacteria cells, leading to bacterial death. This is consistent with the results of the experiments on cell membrane and cell wall integrity mentioned above.

[0058] Example 4. Preservation effect of LP2150 on large yellow croaker surimi

[0059] All experiments were conducted in triplicate, and SPSS software was used for analysis. One-way ANOVA and Duncan's test were used to determine statistically significant differences (P < 0.05).

[0060] 1. Preparation of fish paste

[0061] Fresh large yellow croaker (purchased from Hualong Supermarket, Wanda Plaza, Haizhou District, Lianyungang) was cleaned and the meat was placed in a blender to make fish paste. Different amounts of LP2150 (0%, 0.02%, and 0.04%) were then added and mixed thoroughly. These were labeled as Group A and Group B, and stored at 4℃. The sample without LP2150 treatment served as the control, designated as Group CK. Samples were taken on days 0, 2, 4, 6, and 8 to determine the total bacterial count, volatile basic nitrogen, TBARS value, pH value, water-holding capacity, and sensory evaluation.

[0062] 2. Sensory evaluation

[0063] During storage, fish undergo changes in appearance and odor due to the action of microorganisms and enzymes. Therefore, sensory evaluation can be used to determine the degree of spoilage of large yellow croaker. Referring to the sensory evaluation standards for fish surimi in GB / T 36187-2018 "Frozen Fish Surimi", an evaluation table (Table 1) was developed based on the odor, texture, elasticity, and color of the large yellow croaker surimi. [1] An evaluation panel of 10 graduate students trained in sensory evaluation scored the students according to an evaluation form, and the result was the average of the overall scores.

[0064] Table 1 Sensory Evaluation Table of Yellow Croaker Paste

[0065]

[0066]

[0067] The results are as follows Figure 5 As shown, from Figure 5 It can be seen that the sensory scores of the surimi decreased with the extension of storage time. Compared with the control group, the addition of LP2150 resulted in better sensory evaluation and superior surimi quality. This is because LP2150 inhibits the growth and reproduction of microorganisms to a certain extent, delaying the spoilage of the superior surimi, indicating that LP2150 can effectively maintain the sensory quality of the surimi.

[0068] 3. Total bacterial count

[0069] The total bacterial count was determined according to GB 4789.2-2022, "National Food Safety Standard - Microbiological Examination of Food: Determination of Total Colony Count". 5g of large yellow croaker surimi was weighed and added to 45mL of sterile physiological saline, then homogenized. The mixture was then serially diluted 10-fold with sterile physiological saline. 100μL of each of three suitable dilutions was plated onto plate count agar (PCA) and incubated at 30℃ for 72h. The total viable count (TVC) was then calculated, and the result was expressed as lg(CFU / g). [2] .

[0070] The results are as follows Figure 6 As shown, from Figure 6It can be seen that the total bacterial count of the surimi in both the control group and the LP2150-added group gradually increased with the extension of storage time, and the total bacterial counts of the 0.02% LP2150 and 0.04% LP2150 groups were lower than those in the control group. According to GB10136-2015 "Animal-Derived Aquatic Products," the total bacterial count of surimi products should not exceed 51 g CFU / g. The total bacterial count of the control group after storage for more than 6 days was 6.26 g CFU / g, exceeding the national standard. The experimental groups were 4.71 and 4.22 g CFU / g, respectively, both within the national standard. After storage for more than 8 days, the total bacterial counts of the 0.02% LP2150 and 0.04% LP2150 groups were 5.71 and 5.15 g CFU / g, respectively, exceeding the national standard. This result is similar to that of mandarin fish balls treated with Xinyang Maojian polyphenols. [7] Therefore, LP2150 can effectively inhibit bacterial growth in large yellow croaker surimi and extend the shelf life of the surimi.

[0071] 4. Determination of TVB-N value

[0072] The determination was performed according to GB 5009.228-2016, "National Food Safety Standard: Determination of Volatile Basic Nitrogen in Food". A semi-micro nitrogen determination method was used. 5g of large yellow croaker surimi was weighed into an Erlenmeyer flask, 50mL of distilled water was added, and the mixture was stirred and shaken at 25℃ for 30min before filtration. 10mL of the filtrate was mixed with 10mL of MgO (10g / L) suspension and distilled. The distillate was absorbed using 10mL of boric acid absorption solution (20g / L) containing a mixed indicator solution of methyl red (2g / L) and methylene blue (1g / L). Titration was performed with 10mmol / L standard hydrochloric acid, and a reagent blank was prepared simultaneously. The TVB-N content in the sample was calculated according to formula (1). [3] .

[0073] X=[(V1-V2)×0.01×14] / [m×(V / V0)]×100 Formula (1)

[0074] in,

[0075] V1 represents the volume of hydrochloric acid standard titration solution consumed by the test solution, in milliliters (mL);

[0076] V2 represents the volume of hydrochloric acid standard titration solution consumed by the reagent blank, in milliliters (mL);

[0077] m represents the sample mass in grams (g) or the sample volume in mL.

[0078] V represents the volume of filtrate accurately drawn, in milliliters (mL);

[0079] V0 represents the total volume of the sample solution, in milliliters (mL);

[0080] The results are as follows Figure 7 As shown, from Figure 7 It can be seen that the TVB-N values ​​of all three groups of samples showed a gradual upward trend during storage, with the LP2150-added groups consistently showing lower values ​​than the control group. The rate of increase was slow and relatively small in the early stages of storage; however, with prolonged storage, the rate of increase in TVB-N values ​​in the surimi products significantly increased. This was mainly due to the rapid and increased number of microorganisms in the later stages of storage, leading to protein degradation and a sharp rise in TVB-N values. In the control group, the TVB-N content in the surimi was 23.12 mg / 100g on day 4 of storage, and reached 31.24 mg / 100g on day 6, exceeding the national standard. The surimi with 0.02% LP2150 added had a TVB-N content of 26.51 mg / 100g on day 6 and 31.19 mg / 100g on day 8, also exceeding the national standard. The fish paste with added 0.04% LP2150 had a TVB-N content of 28.66 mg / 100g on day 8, which was close to the upper limit.

[0081] The above results are consistent with the total colony count results, indicating that LP2150 can inhibit bacterial growth and inhibit the decomposition of proteins by microorganisms.

[0082] 5. Determination of Thiobarbituric Acid

[0083] Refer to Lu et al. [4] The method was modified slightly to determine the thiobarbituric acid value of large yellow croaker surimi. Take 5g of surimi into a sterile homogenizing bag, add 25mL of 7.5% trichloroacetic acid solution to homogenize, centrifuge at 8000r / min for 10min, take 2mL of supernatant and mix with 2mL of 0.01mol / L TBA solution, boil in water for 40min, take it out and cool naturally, measure the absorbance value at 532nm and 600nm respectively, substitute into formula (2) to calculate, and the result is expressed as the mass fraction of malondialdehyde (MDA), unit mg / kg.

[0084] TBA (mg / kg) = [(A 532 -A 600 )×0.05×72.6] / [155×m]×1000 Formula (2)

[0085] Where m is the sample mass (g),

[0086] A 532 and A 600 These are the absorbances at 532nm and 600nm, respectively.

[0087] The results are as follows Figure 8 As shown, from Figure 8It can be seen that the TBA values ​​of all fish paste samples increased with the increase of storage time, and the oxidation degree of fish paste with added LP2150 was lower than that of the blank group. The results indicate that LP2150 has strong antioxidant properties and can effectively inhibit the oxidation of lipids in fish paste, thus playing a role in preservation.

[0088] 6. pH Measurement

[0089] The pH value of food was determined according to GB5009.237-2016 "Determination of pH value of food". Take 5g of fish paste into a sterile homogenizing bag, add 40mL of distilled water, homogenize, centrifuge at 8000r / min for 10min, take the supernatant and measure the pH value with a pH meter. Perform the experiment 3 times and take the average value.

[0090] The results are as follows Figure 9 As shown, from Figure 9 It can be seen that the pH value of the large yellow croaker surimi generally showed a trend of first decreasing and then increasing throughout the storage process. This may be because in the early stage of storage, the glycogen in the surimi was broken down into pyruvate by glycolysis, and then converted into lactic acid, leading to a decrease in pH value. In the middle and late stages of storage, the pH value increased due to the conversion into alkaline ammonia compounds produced by protein decomposition under bacterial action. The pH change trend of the surimi with added LP2150 was lower than that of the control group, indicating that LP2150 can inhibit the growth of bacteria in the surimi and delay the change in pH of the fish meat.

[0091] 7. Water-holding capacity measurement

[0092] See Majumdar et al. [5] The water-holding capacity of large yellow croaker surimi was determined by the method described in the article. Approximately M1 of the surimi was wrapped in double-layered filter paper and placed in a centrifuge tube. It was centrifuged at 8000 r / min for 15 min and then removed from the filter paper. The mass after centrifugation was M2. The water-holding capacity was calculated according to formula (3).

[0093] Water holding capacity (%) = M2 / M1 × 100% Formula (3)

[0094] The results are as follows Figure 10 As shown, from Figure 10 It can be seen that the water-holding capacity of all samples decreased with prolonged storage time. Compared with the control group, the water-holding capacity of the surimi with added LP2150 was higher than that of the control group, and the higher the concentration, the better the effect. The results indicate that LP2150 can inhibit the growth of bacteria in surimi, delay the ability of bacteria to destroy the structure of surimi muscle cells and degrade proteins, and help maintain the water-holding capacity of surimi.

[0095] 8. Whiteness Measurement

[0096] Whiteness measurement references Mi Hongbo, etc. [6]The method was modified slightly. The same position of the fish paste was taken and its L* (brightness), a* (red / green), and b* (yellow / blue) were measured using a colorimeter. The results were repeated 3 times in parallel, and the whiteness was calculated according to formula (4).

[0097]

[0098] The results are as follows Figure 11 As shown, from Figure 11 It can be seen that with prolonged storage time, the L*, a*, and W values ​​of both the control group and the LP2150-added large yellow croaker surimi decreased, while the b* value increased. The trend of surimi whiteness change is similar to the color change trend of large yellow croaker reported in the literature. The change in L* during storage may be related to factors such as protein dissolution, fat and protein oxidation; the decrease in a* value may be due to the presence of Fe in the fish meat. 2+ Oxidized to Fe 3+ The addition of LP2150 resulted in a yellowish-green color; the change in b* value was mainly attributed to the browning reaction caused by oxidation. Compared with the control group, the whiteness (W) value of the surimi with added LP2150 was significantly higher than that of the control group. The results indicate that LP2150 inhibits the oxidation of fats and the activity of microbial enzymes in large yellow croaker surimi during storage, and can effectively maintain the color of large yellow croaker surimi and slow down the spoilage of large yellow croaker.

[0099] In summary, this invention evaluated the effect of adding different concentrations of the antimicrobial peptide LP2150 during the production of large yellow croaker surimi on the quality of the surimi during storage. Under storage conditions of 4℃, the indicators (total bacterial count, TVB-N value, TBA value, pH, water-holding capacity, whiteness, and sensory score) of each group of surimi samples were measured and compared to analyze the effect of LP2150 on stabilizing the quality of the surimi during cold storage. The results showed that, compared with the control group, surimi with added LP2150 could inhibit the growth of microorganisms, effectively inhibit the increase in the total bacterial count, slow down protein decomposition and fat oxidation, delay the rise in pH, TBA, and TVB-N values, improve the water-holding capacity of the surimi, and maintain the color of the large yellow croaker surimi. Adding LP2150 extended the shelf life of the large yellow croaker surimi at 4℃ from 6 days to 8 days. Therefore, the addition of LP2150 can improve the quality of large yellow croaker surimi during storage, delay spoilage and extend shelf life, provide new ideas for aquatic product preservation technology, and provide theoretical support and practical reference for food safety and quality assurance.

[0100] The antimicrobial peptide LP2150 of the present invention has the following significance:

[0101] 1. Extended shelf life: By adding LP2150, the shelf life of fish paste can be effectively extended (from 6 days to 8 days), which is of great significance for the sale and transportation of aquatic products, especially in the circulation of fresh seafood, and can reduce economic losses caused by short shelf life.

[0102] 2. Improve product quality: Antimicrobial peptide LP2150 can effectively inhibit the growth of microorganisms, improve the water retention, color and sensory quality of surimi, making the final product more competitive in the market, attracting more consumers, and thus increasing sales.

[0103] 3. Enhance the competitiveness of fisheries: By extending the shelf life of aquatic products and improving their quality, fishery producers can not only better position their products in the market and increase added value, but also enhance their market competitiveness and explore new market opportunities.

[0104] 4. Improving Aquatic Product Preservation Technology: Experiments have shown that the antimicrobial peptide LP2150 can significantly inhibit the growth of microorganisms in large yellow croaker surimi and delay spoilage, thus providing new ideas and effective methods for the storage and preservation of aquatic products. This has important practical significance for improving the safety and edible value of aquatic products.

[0105] 5. Promote research in the field of antimicrobial peptides: The results of this experiment provide a foundation for subsequent research. Related studies can explore the application of antimicrobial peptides in other aquatic products or foods, thereby promoting the development of food science and technology.

[0106] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0107] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0108] References

[0109] [1] National Health and Family Planning Commission of the People's Republic of China. National Food Safety Standard for Frozen Fish Paste: GB / T 36187-2018 [S]. Beijing: China Standards Press, 2018.

[0110] [2] National Health and Family Planning Commission of the People's Republic of China. National Food Safety Standard: Determination of Total Colony Count in Food Microbiology: GB 4789.2-2016 [S]. Beijing: China Standards Press, 2016.

[0111] [3] National Health and Family Planning Commission of the People's Republic of China. National Food Safety Standard: Determination of Volatile Basic Nitrogen in Food: GB 5009.228-2016 [S]. Beijing: China Standards Press, 2017.

[0112] [4]Lu T,Haiyin L,Jinfeng Z,et al.Preparation and coating applicationofγ-polyglutamic acid hydrogel to improve storage life andqualityofshiitakemushrooms[J].FoodControl,2021,130.

[0113] [5]KR M, Apurba S, Bahni D, et al. Effect ofgarlic extract on physical, oxidative and microbial changes during refrigerated storage ofrestructuredproduct from Thai pangas(pangasianodon hypophthalmus)surimi.[J].Journaloffoodscience and technology, 2015, 52(12):7994-8003.

[0114] [6] Mi Hongbo, Wang Cong, Zhao Bo, et al. Effects of soybean oil, flaxseed oil and perilla seed oil on the quality of grass carp surimi [J]. Food Industry Technology, 2017, 38(18):60-64,73.

[0115] [7] Bai Xiaozhou, Wang Tianlin. Effect of polyphenol-protamine compound preservative on the quality of chilled mandarin fish balls from Xinyang Maojian tea[J]. China Food Additives, 2024, 35(03):181-187.DOI:10.19804 / j.issn1006-2513.2024.3.022.

Claims

1. An antimicrobial peptide, wherein the antimicrobial peptide is the polypeptide represented by SEQ ID NO:1: MRCFKRLIKVFLIARKR.

2. The antimicrobial peptide according to claim 1, characterized in that, The antimicrobial peptide was designed based on the genome optimization of Lactiplantibacillus plantarum MMB-07, whose accession number is CGMCC NO.20032.

3. The application of the antimicrobial peptide according to any one of claims 1 to 2 in the preservation of surimi products.

4. The use of the antimicrobial peptide according to any one of claims 1 to 2 in the preparation of a drug against Shewanella putrefaction.

5. The application according to claim 4, characterized in that, The drug is used for the preservation and freshness of surimi products.

6. The use of the antimicrobial peptide according to any one of claims 1 to 2 in inhibiting microorganisms in surimi products, wherein the microorganism is Shewanella putrefactive.

7. An antimicrobial agent comprising the antimicrobial peptide of any one of claims 1 to 2, and a pharmaceutically acceptable carrier.

8. The antibacterial drug according to claim 7, characterized in that, The antibacterial drug preserves food by inhibiting Shewanella putrefactive bacteria.

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