Scylla antibacterial peptide Scyhepcin135-151 and application thereof in preservation and fresh keeping

Abstract

The invention discloses a blue crab antibacterial peptide Scyhepcin135-151 and application of the blue crab antibacterial peptide Scyhepcin135-151 in preservation and fresh keeping. The amino acid sequence of the blue crab antibacterial peptide Scyhepcin135-151 is as shown in SEQ ID NO.01. The preservative has broad-spectrum antibacterial activity, is high in safety, has a fruit preservative effect, is excellent in thermal stability, is natural and environment-friendly, and has great application potential.

Description

Technical Field

[0001] The present invention belongs to the field of marine molecular biology and specifically relates to a blue crab antimicrobial peptide Scyhepcin. 135-151 And its application in anti-corrosion and preservation. Background Art

[0002] Food safety is a major global concern, and the threat posed by foodborne fungal pathogens and their toxic metabolites is particularly prominent. According to the Food and Agriculture Organization of the United Nations (FAO), approximately 25% of agricultural products are lost worldwide each year due to fungal contamination, with fruit rotting due to post-harvest pathogenic fungi being particularly common. Penicillium expansum is one of the main fungi that cause fruit rot and is widely distributed in fruits such as apples, grapes, and citrus. The fungus invades through wounds on the surface of the fruit, causing not only rotting the fruit and reducing its commercial value, but also producing toxins such as patulin. Patulin is a heat-resistant neurotoxin that cannot be destroyed by conventional cooking. High-dose intake can cause symptoms such as nausea and vomiting. Long-term low-dose exposure may lead to immunosuppression, teratogenicity, and even carcinogenicity, posing a serious threat to human health.

[0003] Currently, the primary method for controlling fruit rot and fungal toxin contamination is the use of chemical preservatives, such as sodium benzoate and potassium sorbate. However, these chemical preservatives have significant limitations. First, long-term use may lead to fungal resistance, reducing their effectiveness. Second, chemical residues may pose potential hazards to human health, especially in sensitive populations. Furthermore, with consumers' increasing demands for food safety and health, the safety of chemical preservatives has been questioned. Therefore, the development of novel, natural food preservatives has become a research hotspot.

[0004] Antimicrobial peptides (AMPs) are a class of naturally occurring antimicrobial substances produced by organisms. Due to their broad-spectrum antimicrobial activity, low toxicity, and resistance to drug resistance, they are considered an ideal alternative to chemical preservatives. AMPs are widely found in plants, animals, and microorganisms, and possess diverse antibacterial, antifungal, and antiviral properties. Marine organisms, with their unique habitats and rich biodiversity, are an important source for discovering new antimicrobial peptides. Scylla paramamosain, an economically important crab, may contain a variety of peptides with antimicrobial activity. Research on Scylla paramamosain antimicrobial peptides could provide new technical approaches for fruit preservation and promote the development and application of natural preservatives.

[0005] While some antimicrobial peptides have been used in prior art for antimicrobial research, their application in foodborne fungi (such as Penicillium expansum) and preservation remains insufficient. Existing natural preservatives still need to improve their antimicrobial spectrum, stability, and effectiveness. Therefore, there is a need for a highly effective, safe, and suitable natural antimicrobial peptide suitable for fruit preservation to overcome the shortcomings of traditional chemical preservatives. Summary of the Invention

[0006] The present invention aims to overcome the defects of the prior art and provide a kind of blue crab antimicrobial peptide Scyhepcin 135-151 .

[0007] Another object of the present invention is to provide the above-mentioned blue crab antimicrobial peptide Scyhepcin 135-151 Application in fruit preservation.

[0008] The technical solutions of the present invention are as follows:

[0009] Scyhepcin, a blue crab antimicrobial peptide 135-151 , and its amino acid sequence is shown in SEQ ID NO.01.

[0010] The above-mentioned blue crab antimicrobial peptide Scyhepcin 135-151 Use in preparing fruit preservative composition.

[0011] In a preferred embodiment of the present invention, the fruit is apple.

[0012] A fruit preservative composition characterized in that its active ingredient includes the above-mentioned blue crab antibacterial peptide Scyhepcin 135-151 .

[0013] In a preferred embodiment of the present invention, the fruit is apple.

[0014] The above-mentioned blue crab antimicrobial peptide Scyhepcin 135-151 Use of the blue crab antibacterial peptide Scyhepcin in preparing an antibacterial composition 135-151 It has inhibitory and killing effects on Enterococcus faecium, Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas stutzeri, Acinetobacter baumannii and Shigella flexneri.

[0015] An antibacterial composition, the active ingredient of which includes the above-mentioned blue crab antibacterial peptide Scyhepcin 135-151 , the blue crab antimicrobial peptide Scyhepcin 135-151 It has inhibitory and killing effects on Enterococcus faecium, Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas stutzeri, Acinetobacter baumannii and Shigella flexneri.

[0016] The above-mentioned blue crab antimicrobial peptide Scyhepcin135-151 Use of the blue crab antibacterial peptide Scyhepcin in preparing an antifungal composition 135-151 It has inhibitory and killing effects on Pichia pastoris, Candida albicans, Cryptococcus neoformans, Aspergillus niger, Aspergillus ochra, Aspergillus fumigatus, Aspergillus flavus, Penicillium expansum, Fusarium oxysporum and Fusarium graminearum.

[0017] An antifungal composition, the active ingredient of which includes the above-mentioned blue crab antimicrobial peptide Scyhepcin 135-151 , the blue crab antimicrobial peptide Scyhepcin 135-151 It has inhibitory and killing effects on Pichia pastoris, Candida albicans, Cryptococcus neoformans, Aspergillus niger, Aspergillus ochra, Aspergillus fumigatus, Aspergillus flavus, Penicillium expansum, Fusarium oxysporum and Fusarium graminearum.

[0018] The beneficial effects of the present invention are:

[0019] 1. The present invention consists of 17 amino acids, the molecular formula is C 92 H 150 N 34 O 21 S1, with a molecular weight of 2.10 kilodaltons, contains four positively charged amino acid residues. Based on the charge of the amino acid residues, the isoelectric point of this antimicrobial peptide is predicted to be 11.82, with a hydrophobicity of 39.0%. It is a cationic peptide with good water solubility and broad application prospects.

[0020] 2. The present invention has significant inhibitory and killing effects on a variety of food-borne fungi (such as Pichia pastoris, Candida albicans, Aspergillus niger, Penicillium expansum, etc.), providing an efficient means for fruit preservation.

[0021] 3. The present invention has no cytotoxicity to human embryonic kidney cells (HEK-293T), normal human liver cells (L02) and mouse mononuclear macrophages (RAW264.7), proving its safety in application.

[0022] 4. The present invention can effectively inhibit the hyphae growth and microbial count of fungi such as Penicillium expansum in the lesion area of ​​apple fruit, delay the rot process, extend the shelf life of the fruit, and reduce economic losses.

[0023] 5. The present invention still maintains good antibacterial activity after high-temperature treatment and is suitable for a variety of processing scenarios.

[0024] 6. As a natural antimicrobial peptide derived from mud crab, the present invention avoids the problem of chemical preservative residues and meets the development needs of green food.

[0025] 7. The excellent performance of the present invention in fruit preservation gives it a broad market application prospect and can provide a safe and efficient solution for the food preservation industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The antimicrobial peptide Scyhepcin in Example 3 of the present invention 135-151 Sporicidal kinetics curve of Penicillium expansum. Figure 1 In the figure, the horizontal axis is time (h) and the vertical axis is spore survival rate (%).

[0027] Figure 2 The antimicrobial peptide Scyhepcin in Example 4 of the present invention 135-151 Thermostability diagram of antimicrobial activity against Penicillium expansum. Figure 2 In the figure, the horizontal axis is time (h) and the vertical axis is OD 600 value.

[0028] Figure 3 The antimicrobial peptide Scyhepcin in Example 5 of the present invention 135-151 Transmission electron microscopy observation of Penicillium expansum.

[0029] Figure 4 The cck8 method for detecting the antimicrobial peptide Scyhepcin in Example 6 of the present invention 135-151 Cytotoxicity test diagram. The horizontal axis is the antibacterial peptide Scyhepcin 135-151 The vertical axis is the concentration (μM), and the vertical axis is the cell proliferation rate (%).

[0030] Figure 5 It shows that the detection of Scyhepcin antimicrobial peptide in Example 7 of the present invention 135-151 Effects on the diameter of apple lesions and the number of microorganisms in the lesions. In Figure A, the horizontal axis is the treatment time (days), and the vertical axis is the lesion diameter (mm); in Figure B, the horizontal axis is the antimicrobial peptide concentration (μg / mL), and the vertical axis is the microbial colony count (CFU / g). DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further illustrated and described below through specific implementation methods in conjunction with the accompanying drawings.

[0032] Example 1

[0033] The antimicrobial peptide Scyhepcin in this example 135-151 The amino acid sequence is WRRRRGLSEGRLGWLVC (SEQ ID NO.01).

[0034] The existing solid phase chemical synthesis method can be used to obtain the blue crab antimicrobial peptide Scyhepcin with a purity of more than 95%. 135-151 The antimicrobial peptide Scyhepcin in this example 135-151We commissioned GenScript Biotech to synthesize the peptide using solid phase synthesis and provide information on its molecular weight and HPLC. 135-151 The physical and chemical parameters are shown in Table 1:

[0035] Table 1 Scyhepcin, an antimicrobial peptide from blue crab 135-151 Physical and chemical parameters

[0036]

[0037] Example 2 Scyhepcin 135-151 Determination of minimum inhibitory concentration and minimum bactericidal concentration

[0038] This example is to compare the blue crab antimicrobial peptide Scyhepcin obtained in Example 1 135-151 The minimum inhibitory concentration and minimum bactericidal concentration were determined, and the strains involved included Enterococcus faecium, Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas stutzeri, Acinetobacter baumannii, Shigella flexneri, Pichia pastoris, Candida albicans, Cryptococcus neoformans, Aspergillus niger, Aspergillus ochraceus, Aspergillus fumigatus, Aspergillus flavus, Penicillium expansum, Fusarium oxysporum and Fusarium graminearum. All of the above strains were purchased from the Culture Collection Center of Institute of Microbiology, Chinese Academy of Sciences.

[0039] The specific method is as follows:

[0040] (1) The preserved Enterococcus faecium, Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas stutzeri, Acinetobacter baumannii and Shigella flexneri were spread on nutrient broth plates and cultured inverted; Pichia pastoris, Candida albicans and Cryptococcus neoformans were spread on YPD plates and cultured inverted; Aspergillus niger, Aspergillus ochraceus, Aspergillus fumigatus, Aspergillus flavus, Penicillium expansum, Fusarium oxysporum and Fusarium graminearum were spread on PDA plates and cultured inverted.

[0041] (2) Pick colonies from each plate and inoculate them into the corresponding liquid culture medium, then place them in a constant temperature shaker for overnight culture.

[0042] (3) Collect the cells by centrifugation and resuspend them in sterile 10 mM sodium phosphate buffer (pH = 7.4). Then dilute the bacteria with MH liquid medium and the yeast fungi with 1640 liquid medium to make the final concentration of the cells 5 × 10 5 CFU / mL; dilute the fungus with 20% PDW liquid medium to make the final concentration of the fungus 2×10 4 CFU / mL.

[0043] (4) Scyhepcin 135-151The powder was dissolved in sterilized MilliQ water, filtered through a 0.22 μM filter membrane, and then diluted serially to protein concentrations of 3 μM, 6 μM, 12 μM, 24 μM, 48 μM, and 96 μM, and placed on ice for later use.

[0044] (5) On a 96-well cell culture plate, set up a blank control group, a negative control group, and an experimental group for each test bacterium, with three replicates for each group:

[0045] a Blank control group: 50 μL of the protein sample to be tested (Scyhepcin 135-151 ) and 50 μL culture medium;

[0046] b Negative control group: 50 μL sterile MilliQ water and 50 μL bacterial suspension;

[0047] c Test group: 50 μL of protein sample to be tested (Scyhepcin 135-151 ) and 50 μL bacterial suspension.

[0048] (6) Place the 96-well cell culture plate in the corresponding incubator at a suitable temperature for bacteria and fungi, culture for 1-2 days, and observe the MIC results of the experimental group to be tested; after the experimental group to be tested is pipetted and mixed, an appropriate amount of bacterial liquid is dropped onto the corresponding solid culture medium plate, and cultured inverted at a suitable temperature for 1-2 days, and the MBC results are observed.

[0049] Scyhepcin 135-151 The antibacterial activity results are shown in Table 2, which show that the compound has broad-spectrum antibacterial activity.

[0050] Table 2 Antimicrobial peptide Scyhepcin 135-151 Antibacterial activity

[0051]

[0052] Note: MIC: Minimum inhibitory concentration (μM), expressed as ab. a: The highest protein concentration at which bacterial growth is visible; b: The lowest protein concentration at which no bacterial growth is visible. MBC: Minimum bactericidal concentration (μM), representing the lowest protein concentration capable of killing 99.9% of bacteria.

[0053] Example 3 Scyhepcin 135-151 Sporicidal kinetic curve

[0054] In this example, Penicillium expansum was selected as the test bacteria, and the antimicrobial peptide Scyhepcin obtained in Example 1 was tested. 135-151 The sporicidal kinetics were determined.

[0055] The specific method is similar to the antibacterial activity determination described in Example 2.135-151 After co-incubation with fungal spores for a certain period of time, take an appropriate amount of the co-incubated bacterial suspension, dilute it, and spread it on a PDA plate. Incubate it at 28°C for 1-2 days and count the colonies. 135-151 The kinetic curve of the sporicidal activity against Penicillium expansum is as follows: Figure 1 Scyhepcin 135-151 At a final concentration of 24 μM, 99% of Penicillium expansum spores were killed within 12 h.

[0056] Example 4 Scyhepcin 135-151 Antimicrobial activity Thermal stability

[0057] In this example, Penicillium expansum was selected as the test bacteria, and the antimicrobial peptide Scyhepcin obtained in Example 1 was tested. 135-151 The antibacterial activity and thermal stability were determined.

[0058] The specific method is similar to the antibacterial activity determination described in Example 2. 135-151 The final concentration was 1 times MSC (MSC: minimum sporicidal concentration (μM)), and the antimicrobial peptide Scyhepcin 135-151 Heat in boiling water at 100°C for 10 min, 20 min, 30 min and 60 min, and place on ice for later use. 135-151 Incubate with the test bacteria for 48 hours and continuously monitor OD using a microplate reader 600 The result is as follows Figure 2 As shown, Scyhepcin 135-1510 After continuous heat treatment at 100℃ for 60min, it still retained good antibacterial activity.

[0059] Example 5 Transmission electron microscopy observation of Scyhepcin 135-151 Changes in fungal morphology and structure after treatment

[0060] In this example, Penicillium expansum was selected as the test bacteria, and the antimicrobial peptide Scyhepcin obtained in Example 1 was observed using a transmission electron microscope. 135-151 The morphological structure of the fungus after treatment. The preparation of the transmission electron microscopy sample was carried out in the following steps:

[0061] (1) Dilute the polypeptide solution to 4×MSC with sterile water (final polypeptide concentration is 2×MSC) and place on ice until ready for use; mix equal volumes of spore suspension and polypeptide in an EP tube and incubate at 28°C for 4 h.

[0062] (2) Fixation: After incubation, centrifuge and remove the supernatant with a pipette. Add glutaraldehyde fixative to each sample, pipette and mix thoroughly, then place in a refrigerator at 4°C overnight for 12 hours.

[0063] (3) Take out the spore suspension fixed with glutaraldehyde overnight and centrifuge it. Aspirate the supernatant, mix the precipitate by pipetting, and transfer all the spore liquid to the mold. After absorbing all the supernatant liquid, quickly fill the mold tube with agar to seal the bacterial sample. After the sample is completely solidified, cut the sample into small pieces with a blade, add glutaraldehyde, and fix it in a refrigerator at 4℃ overnight.

[0064] (4) Sample preparation: Wash three times with PBS, soaking for 15 minutes each time. After washing, proceed to the subsequent sample preparation steps and observe under a transmission electron microscope.

[0065] The results are as follows Figure 4 As shown in the figure, the fungus in the control group has a complete internal structure and a smooth surface. 135-151 The treatment caused significant changes in the cell morphology of fungal spores, including cell membrane damage, cytoplasmic cavitation and massive leakage of bacterial contents.

[0066] Example 6 Scyhepcin 135-151 Cytotoxicity assay

[0067] In this example, human embryonic kidney cells (HEK-293T), normal human liver cells (L02) and mouse mononuclear macrophages (RAW264.7) were selected to test the antimicrobial peptide Scyhepcin obtained in Example 1. 135-151 The cytotoxicity of the cells was determined as follows:

[0068] (1) Collect human renal epithelial cells, normal human liver cells and mouse mononuclear macrophages in good growth state, count them with a cell counter and adjust the cell concentration to 1×10 5 The cell suspension was mixed and 100 μL of cell suspension was added to each well of a 96-well cell culture plate. The plate was placed in an incubator with appropriate cell conditions and cultured until more than 80% of the cells adhered to the wall.

[0069] (2) Carefully aspirate the culture medium and add different concentrations of Scyhepcin 135-151 The cells were cultured in an incubator suitable for cell conditions for 24 h.

[0070] (3) After 24 hours, 10 μL of CCK-8 solution was added to each well and incubated for 1 hour. 450 Measure absorbance at nm and evaluate Scyhepcin 135-151 cytotoxicity.

[0071] The results are as follows Figure 4 As shown, 96 μM Scyhepcin 135-151 It has no cytotoxicity to human renal epithelial cells, normal human hepatocytes and mouse monocytes and macrophages.

[0072] Example 7 Scyhepcin 135-151 Evaluation of apple fruit antiseptic

[0073] This example detects the antimicrobial peptide Scyhepcin obtained in Example 1. 135-151 The antiseptic effect on apples. The specific methods are as follows:

[0074] The peptide solution was diluted with sterile water to a concentration of 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, and 800 μg / mL and placed on ice until ready for use. Several apple fruits of similar size and maturity were randomly selected for each group. Three wounds were made with a punch at equal distances near the equator of each fruit, with a depth of approximately 3-4 mm. 25 μL of peptide was injected and air-dried for 2 h. A control group and a blank group were also set up. The concentration of the Penicillium expansum spore suspension was adjusted to 1×10 5 cells / mL. 25 μL of spore suspension was injected into each wound and incubated at 20°C and 30-40% relative humidity for 7 days. During the incubation period, the surface lesions of the apple fruit were observed daily, and the diameter of the lesions was measured and recorded using a vernier caliper. After the 7-day incubation period, samples were collected from each treatment group and the colony counts were counted.

[0075] The results are as follows Figure 5 As shown, Scyhepcin 135-151 The inhibitory effect on apple lesion rings caused by Penicillium expansum shows a dose effect and has an antiseptic and fresh-keeping effect.

[0076] The above description is merely a preferred embodiment of the present invention and therefore cannot be used to limit the scope of the present invention. In other words, equivalent changes and modifications made within the scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A blue crab antimicrobial peptide Scyhepcin 135-151 , characterized in that: Its amino acid sequence is shown in SEQ ID NO.

01.

2. The blue crab antimicrobial peptide Scyhepcin according to claim 1 135-151 Use in preparing fruit preservative composition.

3. The use according to claim 2, characterized in that: The fruit is apple.

4. A fruit antiseptic composition, characterized in that: The active ingredient includes the blue crab antimicrobial peptide Scyhepcin according to claim 1 135-151 .

5. A fruit preservative composition according to claim 4, characterized in that: The fruit is apple.

6. The blue crab antimicrobial peptide Scyhepcin according to claim 1 135-151 Use in the preparation of an antibacterial composition, characterized in that: Scyhepcin 135-151 It has inhibitory and killing effects on Enterococcus faecium, Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas stutzeri, Acinetobacter baumannii and Shigella flexneri.

7. An antibacterial composition, characterized in that: The active ingredient includes the blue crab antimicrobial peptide Scyhepcin according to claim 1 135-151 , the blue crab antimicrobial peptide Scyhepcin 135-151 It has inhibitory and killing effects on Enterococcus faecium, Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas stutzeri, Acinetobacter baumannii and Shigella flexneri.

8. The blue crab antimicrobial peptide Scyhepcin according to claim 1 135-151 Use in the preparation of an antifungal composition, characterized in that: Scyhepcin 135-151 It has inhibitory and killing effects on Pichia pastoris, Candida albicans, Cryptococcus neoformans, Aspergillus niger, Aspergillus ochra, Aspergillus fumigatus, Aspergillus flavus, Penicillium expansum, Fusarium oxysporum and Fusarium graminearum.

9. An antifungal composition, characterized in that: The active ingredient includes the blue crab antimicrobial peptide Scyhepcin according to claim 1 135-151 , the blue crab antimicrobial peptide Scyhepcin 135-151 It has inhibitory and killing effects on Pichia pastoris, Candida albicans, Cryptococcus neoformans, Aspergillus niger, Aspergillus ochra, Aspergillus fumigatus, Aspergillus flavus, Penicillium expansum, Fusarium oxysporum and Fusarium graminearum.

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