Antibacterial peptide and application thereof

Abstract

The invention discloses an antibacterial peptide and application thereof. The antibacterial peptide is selected from any one of the following polypeptides: SEQ ID NO: 1: MRCFKRLIKVFLIARKR, SEQ ID NO: 2: MRCFKRLIKVFLIARKR, and SEQ ID NO: 3: MRCFKRLIKVFLIARKR. The molecular marker is shown as SEQ ID NO: 2: MWIAANKTLFSSRKMMAIICVVLIFPGIAAA, and the molecular marker is shown as SEQ ID NO: 2. And the SEQ ID NO: 3: MSRSKATCLYFFRCAINALT is shown in the description. According to the antibacterial peptide as well as the preparation method and the application thereof, a series of novel antibacterial peptides are obtained through screening and optimization design of a genome sequence of lactobacillus plantarum, and compared with other antibacterial peptides and the antibacterial peptide LP2140, the antibacterial peptide has a better antibacterial effect, can effectively inhibit growth of shewanella putrefaciens, can prevent aquatic products from being preserved and fresh, and has a good application prospect. The invention provides a new thought for the preservation technology of aquatic products, and provides theoretical support and practical reference for food safety and quality assurance.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and specifically relates to an antimicrobial peptide and application thereof. Background Art

[0002] Lactobacillus plantarum is the most widely distributed species of Lactobacillus and has been widely used in the food and feed industry. Lactobacillus plantarum has strong acid resistance and is a generally recognized as safe (GRSA) microorganism. It is considered to be the most important and widely used species of lactic acid bacteria. Plantaricin is synthesized during the metabolism of Lactobacillus plantarum. It is a natural antimicrobial peptide that exhibits antibacterial activity against both Gram-negative and Gram-positive bacteria. It is safe, not easy to develop drug resistance, and has no residue in food. It has broad application prospects in the fields of food and feed. The development of new plantaricin and its application has become a hot topic of research at home and abroad.

[0003] However, the difficulty in screening plant lactobacillus, low yield, and cumbersome separation and purification steps restrict its industrial production and application. In recent years, with the development of microbial genomics, it has become possible to mine new plant lactobacillus from the gene level of plant lactic acid bacteria. The establishment of a large number of peptide databases has brought new methods for the development of new plant lactobacillus. Artificial intelligence algorithms can autonomously learn sequence features and screen out potential antimicrobial peptides from large-scale genomes by identifying characteristic sequences from the genome, and even identifying the correlation features of short sequences with low homology. Artificial intelligence algorithms mainly include machine learning and deep learning, which mainly predict biological activity through amino acid sequences, using artificial neural networks (ANN), quantitative matrices (QM), random forests (Random Forest) and support vector machines (SVM) among several prediction algorithms. Lee et al. developed a quantitative structure-activity relationship (QSAR) model through machine learning to effectively screen and optimize the sequence of antimicrobial peptides, so as to predict the biological activity of molecules. Boone et al. combined genetic algorithms with rough set theory to design antimicrobial peptides. The use of this method provides a clear boundary for the physical and chemical properties that the active sequences of antimicrobial peptides have and the inactive sequences do not have. It was found that the antimicrobial peptides synthesized by this method are easier to synthesize than the antimicrobial peptides in the APD3 database. Wang et al. designed short sequences of antimicrobial peptides with potential anti-E. coli activity by constructing an LSTM generation model (Long Short-Term Memory, a RNN model) and a bidirectional LSTM classification model. The verification accuracy of the classification model was 81.6% to 88.9%, and the accuracy of classifying new antimicrobial peptides as antibacterial drugs was as high as 70.6% to 91.7%, indicating that LSTM is an effective tool for finding new antimicrobial peptides. Sharma et al., based on the characteristics of deep learning, used the SVM algorithm to construct the AniAMPpred model to identify antimicrobial proteins that may exist in animal genomes. At the same time, the connection between artificial intelligence and synthetic biology is becoming increasingly close. Researchers use artificial intelligence algorithms to design and optimize the coding sequences of antimicrobial peptides to further improve the bactericidal activity of antimicrobial peptides. By introducing artificial intelligence algorithms, rapid prediction and optimized screening of the antibacterial activity of plant lactobacillus can be achieved, greatly improving the screening efficiency and accuracy. However, there are few reports on the research of artificial intelligence-assisted mining of antimicrobial peptides from plant lactobacillus.

[0004] Shewanella putrefaciens is a Gram-negative bacillus with a strong ability to cause corruption. It is one of the dominant spoilage bacteria in aquatic products. It can still grow even under low-temperature storage conditions, posing a great threat to aquatic products. Shewanella putrefaciens can metabolize muscle protein in aquatic products, decarboxylate amino acids to produce biogenic amines such as putrescine and cadaverine, and produce odorous substances such as H2S. The biofilm formed by Shewanella putrefaciens can cause the surface of the fish to become sticky. At the same time, Shewanella putrefaciens is a rare opportunistic human pathogen, mainly associated with intraperitoneal, skin and soft tissue infections, and can also cause septicemia, which may even be fatal. At present, Shewanella putrefaciens infections usually require antibiotic treatment, but the antibiotic resistance of Shewanella is increasing, and it is resistant to a variety of drugs such as β-lactams, quinolones, aminoglycosides, and macrolides. Therefore, there is an urgent need to find a natural antibacterial substance to meet this challenge.

[0005] Marine fish have high nutritional value and are considered the most important food in the human diet. However, after being caught and brought ashore, they are not easy to survive due to sudden changes in environmental factors such as air pressure, temperature, and dissolved oxygen. After the fish dies, cell metabolism stops, and the epidermal goblet cells stop secreting mucus containing antibacterial substances. In addition, fish meat is prone to spoilage due to microbial growth during storage due to short muscle fibers and rich in water, causing huge losses. The spoilage of fish meat is usually caused by three factors: autolytic enzyme decomposition, fat oxidation, and microbial growth; among them, microbial growth and metabolism are the main causes of fish meat spoilage. Studies have shown that Shewanella, represented by Shewanella putrefaciens, is the main specific spoilage bacteria in marine fish and plays a leading role in the spoilage process of fish meat. At present, the country attaches great importance to food safety. Because chemical synthetic preservatives have food safety risks, it is of great significance to explore new, green, and safe lactic acid bacteria-derived bacteriocins for the preservation of marine fish.

[0006] Therefore, in view of the above technical problems, it is necessary to provide a new type of antimicrobial peptide so as to achieve the goal of preservation and freshness while meeting food safety. Summary of the invention

[0007] The purpose of the present invention is to provide an antimicrobial peptide and a preparation method and application thereof. By screening and optimizing the genome sequence of Lactobacillus plantarum, a new type of antimicrobial peptide is obtained, which can play a role in preserving fish by effectively inhibiting the growth of Shewanella putrefaciens, and there is no food safety problem.

[0008] In order to achieve the above object, a technical solution provided by a specific embodiment of the present invention is as follows:

[0009] In one aspect, the present invention provides an antimicrobial peptide or a derivative thereof, wherein the antimicrobial peptide is selected from any one of the polypeptides shown below: SEQ ID NO: 1: MRCFKRLIKVFLIARKR; SEQ ID NO: 2: MWIANKTLFSSRKMMAIICVVVLIFPGIAAA; SEQ ID NO: 3: MSRSKATCLYFFRCAINALT.

[0010] In one or more embodiments of the present invention, the antimicrobial peptide is a polypeptide represented by 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, and the accession number of MMB-07 is: CGMCC NO.20032.

[0012] On the other hand, the present invention also provides a use of the antimicrobial peptide or its derivatives in preservation and freshness preservation, preferably in the preservation and freshness preservation of aquatic products.

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

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

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

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

[0017] In yet another aspect, an antibacterial drug comprises the above antibacterial peptide or its derivative, and a pharmaceutically acceptable carrier.

[0018] In one or more embodiments of the present invention, the antimicrobial drug preserves freshness by inhibiting Shewanella putrefaciens.

[0019] Compared with the prior art, the antimicrobial peptides and their preparation methods and applications of the present invention are screened and optimized by the genome sequence of Lactobacillus plantarum, and combined with artificial intelligence analysis to obtain a series of novel antimicrobial peptides, and the antimicrobial peptide LP2140 is found through experiments to have better antimicrobial effects than other antimicrobial peptides. LP2140 can effectively inhibit the growth of Shewanella putrefaciens, thereby improving the quality of aquatic products such as fish during storage, delaying spoilage and extending the shelf life; and because it comes from Lactobacillus plantarum MMB-07, there are no drug resistance and safety issues, providing new ideas for the preservation technology of aquatic products, and providing theoretical support and practical reference for food safety and quality assurance; in addition, due to the combination of artificial intelligence analysis of the genome sequence, the optimization design obtains an antimicrobial peptide that is easier to synthesize, providing ideas for the development of new antimicrobial peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 It is a structural representation diagram of Example 2 of the present invention;

[0022] Figure 2 is the circular dichroism spectrum of Example 2 of the present invention;

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

[0024] Figure 4 This is an electron microscope image showing the effect of LP2150 on the morphology of Shewanella putrefaciens 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 yellow croaker surimi in Example 4 of the present invention;

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

[0029] Fig. 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] Fig.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] Fig.11 Figure 4 is a graph showing the effect of LP2150 on large yellow croaker surimi in Example 4 of the present invention, wherein Figure A is a graph showing the effect on brightness, Figure B is a graph showing the effect on red-green hues, Figure C is a graph showing the effect on yellow-blue hues, and Figure D is a graph showing the effect on whiteness. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0033] Unless otherwise specified, the materials and reagents in this article can be purchased from regular channels.

[0034] Unless otherwise specified, "ORF2150" herein refers to "LP2150".

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

[0036] Using the genome data of Lactobacillus plantarum MMB-07 (its detailed information has been recorded in the authorized patent CN111979141B, and the deposit number of this strain is CGMCC NO.20032), the logistic regression model MiPepid with 4-mer characteristics is first used to specifically identify the sorf (small open reading frame, small open reading frame) in the Lactobacillus plantarum genome sequence. The selected sorf will be translated into 45,385 peptide sequences, and the SignalP server will be used to predict the signal peptide, which is then removed from the sequence to filter out 302 secreted peptides. Subsequently, the TMHMM server will be used to predict the ability of peptide secretion to the extracellular space, thereby filtering out 44 transmembrane peptides. A deep neural network model DNN combined with 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) were 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 through the website https: / / aps.unmc.edu / prediction and the helical wheel projection was performed online at https: / / heliquest.ipmc.cnrs.fr / cgi-bin / ComputParams.py; the peptide structure diagram was obtained online at https: / / www.allpeptide.com / jiegoutu.html. The results are as follows Figure 1 and as shown in the following Table 1.

[0039] Table 1 Key physical and chemical parameters of LP2150

[0040]

[0041] The chemical structure of LP2150 is:

[0042]

[0043] The experimental results show that through CCS calculation and comparative analysis, LP2150 exists in a Random structure, with the strongest positive charge of +7, the lowest hydrophilicity of Grand average value of 0.171, and a large fat index of 114.71. These structural characteristics and physicochemical properties indicate that LP2150 is a potential optimal antimicrobial peptide. The theoretical molecular weight of the peptide matches the measured value, and the purity of all compounds is above 95%, indicating that the synthesis was successful.

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

[0045] The sample LP2150 was dissolved in 0.01 mol / L phosphate buffer at pH 7.0 to prepare a 0.2 mg / mL protein solution, and the circular dichroism spectrum was measured on a JASCO-815 circular dichroism spectrometer. The wavelength range was 195-250 nm, the scanning rate was 100 nm / min, the sample cell optical path was 0.1 nm, and the phosphate buffer was used as a blank control. The circular dichroism spectrum is expressed as the average ellipticity [θ], the unit is deg·cm 2 / dmol, CD chromatogram data were analyzed by CDPRO software, and the relative content of secondary structure was evaluated using the bestsel platform. The results are shown in Figure 2 shown.

[0046] from Figure 2 It can be seen that the CD spectrum of LP2150 has a continuous negative peak in the 190-250nm band, and the peak maximum (mdeg minimum) is mainly located near 199nm. The above band is mainly the characteristic area of ​​Random structure, indicating that the secondary structure of LP2150 is mainly Random. The fitting results of LP2150 also show that the 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 and their secondary structure contents were determined according to the same method, but the specific structures are not shown.

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

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

[0050] The concentration of Shewanella putrefaciens CN32 bacterial suspension was adjusted to OD600 of 0.1, and LP2150 was added at final concentrations of 0, 1 / 4MIC, 1 / 2×MIC, 1×MIC, and 2×MIC. The suspension was placed in an incubator and shaken at 28°C. The absorbance of the bacterial solution at a wavelength of 600nm was measured every 2 hours. The results showed that the minimum inhibitory concentration of LP2150 against Shewanella putrefaciens CN32 was 1μg / mL, the minimum inhibitory concentration of ORF25500 against Shewanella putrefaciens was 16μg / mL, and the minimum inhibitory concentration of ORF39896 against Shewanella putrefaciens was 4μg / mL.

[0051] Among them, the results of the effect of LP2150 on the growth of Shewanella putrefaciens were as follows: Figure 3 As shown, from Figure 3 It can be seen that in the control group (no LP2150 added), Shewanella putrefaciens entered the logarithmic growth phase after 2 hours of growth and entered the stable phase after 20 hours. LP2150 at concentrations of 1MIC and 2MIC can completely inhibit bacterial growth, and LP2150 at concentrations of 1 / 4MIC and 1 / 2MIC delays the logarithmic growth phase and stable phase of the bacteria. When the bacteria finally reach the stable phase, the concentration is significantly lower than that of the control group, indicating that LP2150 has a significant inhibitory effect on the growth of Shewanella putrefaciens.

[0052] 2. Effect of LP2150 on the morphology of Shewanella putrefaciens

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

[0054] Scanning Electron Microscope (SEM): After washing with PBS for three times, dehydrate with 30%, 50%, 70%, 80%, 90%, 100%, and 100% ethanol in a gradient manner, replace with tert-butanol for three times, and after the sample is dried, gold-plated with an ion sputtering instrument, and the morphology of the cells is observed by scanning electron microscopy.

[0055] Transmission Electron Microscope (TEM): After washing with PBS three times, fix with 1% osmium tetroxide, and then dehydrate with ethanol gradient, each gradient treatment for 20 minutes. Then, embed after epoxy resin infiltration, ultramicrosection, double staining with uranyl acetate and lead citrate, and observe cell morphology under transmission electron microscopy. The results are as follows Figure 4 shown.

[0056] from Figure 4 It can be seen that the untreated Shewanella putrefaciens bacteria are full and have a typical long rod shape, complete cell morphology, and dense internal tissue ( Figure 4 After being treated with 2×MIC LP2150 for 2 h, many bacteria were obviously deformed and the cell surface showed obvious wrinkles ( Figure 4 B), indicating that LP2150 can act on the wall membrane structure of Shewanella putrefaciens. Transmission electron microscopy (TEM) was used to further observe the ultrastructure and intracellular changes of Shewanella putrefaciens. Bacteria not treated with LP2150 had intact cell walls tightly attached to the cell membrane and dense internal tissues ( Figure 4 C). After being treated with 2×MIC LP2150 for 2 hours, the cytoplasm of the bacteria became loose, plasmolysis occurred, intracellular components leaked out, and the cells lost their basic structure ( Figure 4 D).

[0057] The above results show that LP2150 has a significant destructive effect on the integrity and intracellular organization of Shewanella putrefaciens cells, leading to bacterial death. This is consistent with the above-mentioned results of the cell membrane and cell wall integrity experiments.

[0058] Example 4. Effect of LP2150 on the preservation of large yellow croaker surimi

[0059] The following experiments were performed three times and analyzed using SPSS software. One-way ANOVA and Duncan test showed statistically significant differences (P<0.05).

[0060] 1. Preparation of Surimi

[0061] Fresh yellow croaker (purchased from Baozhen Hualong Supermarket, Wanda Plaza, Haizhou District, Lianyungang) was slaughtered and the clean meat was taken out and placed in a blender to make fish paste. Then different amounts of LP2150 (0%, 0.02%, 0.04%) were added and mixed evenly. The samples were marked as group A and group B respectively and stored at 4°C. The samples without LP2150 were used as the control and recorded as group CK. The total number of colonies, volatile basic nitrogen, TBARS value, pH value, water holding capacity, sensory evaluation and other indicators of the samples were measured on days 0, 2, 4, 6 and 8.

[0062] 2. Sensory evaluation

[0063] During storage, the appearance and smell of fish will change due to the action of microorganisms and enzymes, so sensory evaluation can be used to judge the degree of spoilage of large yellow croaker. With reference to the sensory evaluation standards of fish surimi in GB / T 36187-2018 "Frozen Fish Surimi", an evaluation table was developed based on the smell, tissue state, elasticity, and color of large yellow croaker surimi (Table 1). [1] The evaluation team consisted of 10 postgraduate students who had received sensory evaluation training. They scored according to the evaluation form, and the result was the average of the comprehensive scores.

[0064] Table 1 Sensory evaluation of yellow croaker surimi

[0065]

[0066]

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

[0068] 3. Total colony count

[0069] Refer to GB 4789.2-2022 "National Food Safety Standard for Food Microbiology Inspection and Determination of Total Colony Count" to determine the total colony count. Weigh 5g of large yellow croaker surimi, add 45mL of sterile saline, and homogenize. Dilute with sterile saline in a 10-fold gradient, select three appropriate dilution gradient samples, and spread 100μL of each on plate count agar (PCA). After culturing at 30℃ for 72h, count the total viable count (TVC), and the result is expressed in lg (CFU / g). [2] .

[0070] The results are as follows Figure 6 As shown, from Figure 6It can be seen that the total colony counts of the surimi in the control group and the LP2150-added group gradually increased with the extension of storage time, and the total colony counts of the 0.02% LP2150 and 0.04% LP2150 groups were lower than those of the control group. According to the provisions of GB10136-2015 "Animal Aquatic Products", the total colony count of surimi products cannot exceed 5lg CFU / g. The total colony count of the control group after storage for more than 6 days was 6.26lg CFU / g, which has exceeded the national standard. The total colony counts of the experimental groups were 4.71 and 4.22lg CFU / g, respectively, which did not exceed the national standard. After storage for more than 8 days, the total colony counts of the 0.02% LP2150 and 0.04% LP2150 groups were 5.71 and 5.15lg CFU / g, respectively, which exceeded the national standard. This result is similar to that of the mandarin fish balls treated with Xinyang Maojian polyphenols. [7] It can be seen that LP2150 can effectively inhibit the growth of bacteria in yellow croaker surimi and extend the shelf life of surimi.

[0071] 4. Determination of TVB-N value

[0072] Refer to GB 5009.228-2016 "National Food Safety Standard Determination of Volatile Basic Nitrogen in Foods" for determination. Using the semi-micro nitrogen determination method, weigh 5g of large yellow croaker minced fish into a conical flask, add 50mL of distilled water, stir well, and filter after constant temperature oscillation at 25°C for 30min. Take 10mL of the filtrate and mix it with 10mL of MgO (10g / L) suspension for distillation. Use 10mL of boric acid absorption solution (20g / L) containing a mixed indicator solution of methyl red (2g / L) and methylene blue (1g / L) to absorb the evaporated material, and titrate with 10mmol / L standard hydrochloric acid, and make a reagent blank at the same time. Calculate the content of TVB-N in the sample 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 the 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 the filtrate accurately aspirated, 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 the three groups of samples showed a gradual upward trend during storage, and the LP2150-added group was less than the control group. The rising speed was slow in the early stage of storage and the difference was not large; with the extension of storage time, the growth rate of TVB-N value in surimi products increased significantly, mainly because the microbial reproduction speed was fast and the number increased in the later stage of storage, and the protein was destroyed, which caused the TVB-N value of surimi to rise sharply. On the 4th day of storage in the control group, the TVB-N content in surimi was 23.12 mg / 100g, and on the 6th day, the TVB-N content reached 31.24 mg / 100g, which exceeded the national standard. The TVB-N content of surimi with 0.02% LP2150 was 26.51 mg / 100g on the sixth day, and the TVB-N content was 31.19 mg / 100g on the eighth day, which exceeded the national standard. The TVB-N content of surimi with 0.04% LP2150 added on the eighth day was 28.66 mg / 100 g, which was close to the upper limit.

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

[0082] 5. Determination of Thiobarbituric Acid

[0083] Reference Lu et al. [4] The thiobarbituric acid value of yellow croaker surimi was determined by using the method with slight modifications. 5 g of surimi was put into a sterile homogenizing bag, and 25 mL of 7.5% trichloroacetic acid solution was added to homogenize, and the mixture was centrifuged at 8000 r / min for 10 min. 2 mL of the supernatant was mixed with 2 mL of 0.01 mol / L TBA solution, and the mixture was placed in a boiling water bath for 40 min. After the mixture was taken out and cooled naturally, the absorbance values ​​were measured at 532 nm and 600 nm, respectively, and the results were calculated by substituting them into formula (2). The results were expressed as the mass fraction of malondialdehyde (MDA) in 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 are the absorbance at 532nm and 600nm respectively.

[0087] The results are as follows Figure 8 As shown, from Figure 8It can be seen that with the increase of storage time, the TBA value of each group of surimi samples showed an upward trend, and the oxidation degree of surimi added with LP2150 was lower than that of the blank group. The results show that LP2150 has strong antioxidant properties and can effectively inhibit the oxidation of lipids in surimi, playing a role in preserving freshness.

[0088] 6. pH determination

[0089] Refer to GB5009.237-2016 "Determination of pH value of food" for determination. 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 3 experiments and take the average value.

[0090] The results are as follows Fig. 9 As shown, from Fig. 9 It can be seen that the pH value of yellow croaker surimi showed a general trend of decreasing first and then increasing during the entire storage process. This may be because in the early stage of storage, the glycogen in the yellow croaker surimi was decomposed into pyruvic acid by glycolysis and then converted into lactic acid, resulting in 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 the action of bacteria. The pH change trend of surimi with LP2150 was lower than that of the control group, indicating that LP2150 can inhibit the growth of bacteria in surimi and delay the change of fish pH.

[0091] 7. Water holding capacity determination

[0092] Reference Majumdar et al. [5] The water holding capacity of yellow croaker surimi was determined by the method in . The surimi was taken as M1, wrapped with double-layer filter paper and placed in a centrifuge tube. Centrifuged at 8000r / min for 15min, then taken out 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 Fig.10 As shown, from Fig.10 It can be seen that as the storage time increases, the water holding capacity of each group of samples shows a downward trend. Compared with the control group, the water holding capacity of the surimi with LP2150 added is higher than that of the control group, and the higher the concentration added, the better the effect. The results show that LP2150 can inhibit the growth of bacteria in surimi, delay the ability of bacteria to destroy the muscle cell structure and protein degradation of surimi, and help maintain the water holding capacity of surimi.

[0095] 8. Determination of whiteness

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

[0097]

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

[0099] In summary, the present invention adds different concentrations of antimicrobial peptide LP2150 during the production process of large yellow croaker surimi to evaluate the effect of LP2150 on the quality of surimi during storage. Under 4°C storage conditions, the indexes (total colony count, TVB-N value, TBA value, pH, water holding capacity, whiteness and sensory score) in each group of surimi samples were measured and compared to analyze the effect of LP2150 on stabilizing the quality of surimi during refrigeration. The results show that compared with the control group, the surimi with LP2150 can inhibit the growth of microorganisms, effectively inhibit the increase of the total colony count in the surimi, slow down the decomposition of protein and the oxidation of fat, delay the increase of pH value, TBA value and TVB-N value, improve the water holding capacity of surimi and the ability to maintain the color of large yellow croaker surimi, and the addition of LP2150 can extend the shelf life of large yellow croaker surimi at 4°C from 6d to 8d. Therefore, the addition of LP2150 can improve the quality of large yellow croaker surimi during storage, delay spoilage and prolong the shelf life, provide new ideas for the preservation technology of aquatic products, 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. Extend the shelf life: By adding LP2150, the shelf life of surimi can be effectively extended (from 6 days to 8 days), which is of great significance for the sales and transportation of aquatic products, especially in the circulation of fresh seafood, which can reduce the economic losses caused by the short shelf life.

[0102] 2. Improve product quality: Antimicrobial peptide LP2150 can effectively inhibit the growth of microorganisms, improve the water holding capacity, color and sensory quality of fish paste, 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 storage period and improving the quality of aquatic products, fishery producers can not only better position their products in the market and increase added value, but also enhance their market competitiveness and open up new market opportunities.

[0104] 4. Improve the preservation technology of aquatic products: Through experiments, it was found that the antimicrobial peptide LP2150 can significantly inhibit the growth of microorganisms in yellow croaker surimi and delay spoilage, thus providing new ideas and effective means 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 basis for subsequent research. Related research 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 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 the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0107] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes 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 Frozen Surimi: 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 for food microbiological examination-Determination of total colony count: 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 Foods: 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, linseed oil and perilla seed oil on the quality of grass carp surimi [J]. Food Industry Science and Technology, 2017, 38(18): 60-64, 73.

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

Claims

1. 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: MWIANKTLFSSRKMMAIICVVVLIFPGIAAA; SEQ ID NO: 3: MSRSKATCLYFFRCAINALT.

2. The antimicrobial peptide or its derivative according to claim 1, characterized in that: The antimicrobial peptide is a polypeptide shown in SEQ ID NO: 1: MRCFKRLIKVFLIARKR.

3. The antimicrobial peptide or derivative thereof according to claim 1 or 2, characterized in that: The antimicrobial peptide or its derivative is obtained by optimizing the genome design of Lactobacillus plantarum MMB-07, and the accession number of MMB-07 is CGMCC NO.20032.

4. Use of the antimicrobial peptide or its derivative according to any one of claims 1 to 3 for preservation and freshness preservation, preferably for the preservation and freshness preservation of aquatic products.

5. Use of the antimicrobial peptide or its derivative according to any one of claims 1 to 3 in the preparation of antimicrobial drugs.

6. The use according to claim 5, characterized in that: The antibacterial drug is used for preservation and freshness preservation, and is preferably used for the preservation and freshness preservation of aquatic products.

7. Use of the antimicrobial peptide or derivative thereof according to any one of claims 1 to 3 for inhibiting microorganisms.

8. The use according to claim 7, characterized in that: The microorganism is Shewanella putrefaciens.

9. An antibacterial drug comprising the antibacterial peptide or its derivative according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.

10. The antibacterial drug according to claim 9, characterized in that: The antimicrobial drug can preserve freshness by inhibiting the spoilage of Shewanella.

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