Bacillus vesiculosus antimicrobial peptides and their application in food preservation

Abstract

This invention relates to Bacillus vesiculosus antimicrobial peptides and their application in food preservation, belonging to the field of biotechnology. The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO: 1. The antimicrobial peptide provided by this invention is a novel antimicrobial peptide in structure, enriching the antimicrobial peptide database; this antimicrobial peptide can effectively inhibit the normal growth of Fusarium oxysporum, has a broad antimicrobial spectrum, and has potential development and application prospects; this antimicrobial peptide can effectively extend the refrigerated shelf life of dragon fruit, and has good application prospects in fruit preservation.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to Bacillus belyssus antimicrobial peptides and their application in food preservation. Background Technology

[0002] Fungal diseases refer to infections of fruits and vegetables by pathogenic fungi before or after harvest, leading to spoilage and deterioration. Common fungal diseases, such as stem rot, dragon fruit canker, and gray mold, cause serious economic losses to crops. With the improvement of people's living standards, lifestyles and consumption patterns have changed significantly, and people are more inclined to adopt green, efficient, environmentally friendly, and biosafe methods to reduce losses of fruits and vegetables before and after harvest.

[0003] Compared to traditional physical control methods, the application of Bacillus in plant disease control is more in line with current needs. Bacillus is a type of aerobic or facultative anaerobic Gram-positive bacillus capable of spore production, possessing diverse physiological characteristics, and widely distributed. Bacillus has low nutritional requirements, grows rapidly, and can produce spores to resist harsh environments, providing a foundation for its industrial production and application. Bacillus species are non-pathogenic, do not contain exogenous toxins or endotoxins, and can secrete various antimicrobial peptides, reducing the drug resistance of plant pathogens and inducing plant disease resistance. This has resulted in a long history of safe application in the food industry.

[0004] Bacillus, as a typical representative of Gram-positive bacteria, exhibits a complex metabolic network not only in its basic energy metabolism but also prominently in the secretion of various antimicrobial peptides of significant biotechnological importance, making it a core group in microbial resource development. These antimicrobial peptides can promote plant growth and provide biological control against biotic and abiotic stresses. Approximately 4%-10% of the coding region in the Bacillus genome (about 15-30 gene clusters) is dedicated to the synthesis of antimicrobial peptides, encompassing functional components such as nonribosomal peptides and polyketides. Although these components do not directly participate in bacterial growth and reproduction, they occupy an important position in microbial competition through functions such as antibacterial activity, signal transduction, and environmental adaptation.

[0005] Although a large number of Bacillus antimicrobial peptides have been discovered, the database of Bacillus antimicrobial peptides remains insufficient. Mangrove environments are characterized by extreme stresses such as high salinity, periodic immersion, anoxic conditions, complex organic matter, and intense interspecific competition. Bacillus species surviving under these harsh conditions may evolve highly efficient and novel secondary metabolites, especially antimicrobial peptides with unique structures and diverse mechanisms of action, in order to compete for ecological niches and resources. Therefore, this invention screens Bacillus belesii, a species producing antimicrobial peptides, from mangroves and provides information on Bacillus belesii antimicrobial peptides and their applications in food preservation. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide Bacillus vesiculosus antimicrobial peptides and their application in food preservation. The aim is to provide an antimicrobial peptide and a food preservative with food preservation functions.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0008] In the first aspect, Bacillus belyssus antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0009] The antimicrobial peptide GAB712 from Bacillus belye was extracted with ethyl acetate, and the active components were enriched using an AKTA pure protein purifier. High-performance liquid chromatography (HPLC) was used to separate and purify the antimicrobial peptide GAB712 from the active components. LC-MS / MS analysis and comparative analysis with the UniProtKB reference proteomes and the Antimicrobial Peptide Database confirmed that the amino acid sequence of the antimicrobial peptide GAB712 is as shown in SEQ ID NO:1, thus identifying it as a novel antimicrobial peptide.

[0010] The beneficial effects of this invention are as follows: the antimicrobial peptide provided by this invention is a novel antimicrobial peptide in structure, enriching the antimicrobial peptide database; this antimicrobial peptide can effectively inhibit the normal growth of Fusarium oxysporum, has a broad antimicrobial spectrum, and has potential development and application prospects; this antimicrobial peptide can effectively extend the refrigerated shelf life of cooked food, and has good application prospects in food preservation.

[0011] Based on the above technical solution, the present invention can be further improved as follows.

[0012] Furthermore, the antimicrobial peptide was prepared by fermentation of Bacillus velezensis K3, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 23, 2025, with accession number GDMCC No: 66202 and taxonomic name Bacillus velezensis.

[0013] The applicant screened a novel strain with strong antagonistic activity against Fusarium oxysporum from mangrove soil. Through 16S rDNA sequencing and phylogenetic tree construction analysis, combined with colony and cell morphology characteristics, strain K3 was identified as Bacillus velezensis. This strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on April 23, 2025, with accession number GDMCC No: 66202, classification name: Bacillus velezensis, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0014] Secondly, the gene for the antimicrobial peptide of Bacillus vesicularis, wherein the gene for the antimicrobial peptide of Bacillus vesicularis encodes the antimicrobial peptide of Bacillus vesicularis.

[0015] Thirdly, a recombinant plasmid, wherein the recombinant plasmid includes the gene for the Bacillus belyssus antimicrobial peptide. The plasmid may be pET28a, but is not limited to this plasmid, and may also be other expression vectors.

[0016] Fourthly, recombinant microorganisms, including the recombinant plasmids.

[0017] The recombinant microorganism is *Escherichia coli*. It can also be another host.

[0018] Fifthly, the application of the Bacillus vesicularis antimicrobial peptide, or the gene of the Bacillus vesicularis antimicrobial peptide, or the recombinant plasmid, or the recombinant microorganism in food preservation.

[0019] Furthermore, the food includes any one of fruits, vegetables, and aquatic products.

[0020] Sixthly, a food preservative comprising the aforementioned Bacillus vesiculosus antimicrobial peptide.

[0021] Furthermore, the food preservative also includes a food science-acceptable carrier and / or one or at least two functional additives.

[0022] Furthermore, the food preservative achieves food preservation by killing Fusarium oxysporum. Attached Figure Description

[0023] Figure 1 The figure shows the effect of protease on the antifungal activity of Bacillus belysin K3; different letters in the figure indicate significant differences (P<0.05).

[0024] Figure 2 The graph shows the results of the minimum inhibitory concentration (MIC) determination of the antimicrobial peptide GAB712 of Bacillus belyssus; the left graph shows the inhibition of GAB712 against Fusarium oxysporum at concentrations of 64 mg / mL, 32 mg / mL, 16 mg / mL, and 8 mg / mL, and the right graph shows the inhibition of Fusarium oxysporum at concentrations of 64 mg / mL, 32 mg / mL, 16 mg / mL, and 8 mg / mL. Figure 4 Inhibition of antimicrobial peptide GAB712 against Fusarium oxysporum at concentrations of mg / mL, 2 mg / mL, 1 mg / mL, and 0 mg / mL;

[0025] Figure 3 The high-performance liquid chromatography chromatogram of the antimicrobial peptide GAB712 from Bacillus belyssus;

[0026] Figure 4The mass spectrum of the antimicrobial peptide GAB712 from Bacillus belyssus;

[0027] Figure 5 The mass spectrum of the antimicrobial peptide GAB712 from Bacillus belyssus;

[0028] Figure 6 The figure shows the effect of Bacillus belysin's antimicrobial peptide GAB712 on the rot rate and lesion area of ​​dragon fruit; (a) is a comparison of dragon fruit on day 0 and day 11; (b) is the rot rate statistics; (c) is the lesion area statistics.

[0029] Figure 7 Figure showing the effect of Bacillus vesiculosus antimicrobial peptide GAB712 on the weight loss rate of dragon fruit;

[0030] Figure 8 Figure showing the effect of Bacillus vesiculosus antimicrobial peptide GAB712 on TSS in dragon fruit;

[0031] Figure 9 Figure showing the effect of Bacillus vesiculosus antimicrobial peptide GAB712 on the TA of dragon fruit;

[0032] Figure 10 Figure showing the effect of Bacillus vesiculosus antimicrobial peptide GAB712 on MDA content in dragon fruit;

[0033] Figure 11 Figure showing the effect of Bacillus vesiculosus antimicrobial peptide GAB712 on SOD activity in dragon fruit;

[0034] Figure 12 Figure showing the effect of Bacillus vesiculosus antimicrobial peptide GAB712 on the POD activity of dragon fruit;

[0035] Figure 13 The effect of Bacillus vesiculosus antimicrobial peptide GAB712 on the CAT activity of dragon fruit is shown in the figure. Detailed Implementation

[0036] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0037] Description of the source of materials and reagents:

[0038] Fusarium oxysporum GDMCC NO.3.696 was purchased from Guangdong Provincial Microbial Culture Collection Center.

[0039] Nutrient agar, LB broth, and potato dextrose agar were purchased from Beijing Luqiao Technology Co., Ltd.; pepsin, trypsin, and papain were purchased from Aladdin Biochemical Technology Co., Ltd.; glutaraldehyde and ethyl acetate were analytical grade and purchased from Xilong Scientific Co., Ltd.; guaiacol was analytical grade and purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; malondialdehyde content detection kit, superoxide dismutase (SOD) activity detection kit, and peroxidase (POD) activity detection kit were purchased from Beijing Box Biotechnology Co., Ltd.

[0040] Example 1: Obtaining and identifying Bacillus K3.

[0041] 1.1 Obtaining Bacillus K3:

[0042] Using *Fusarium oxysporum* as an indicator bacterium, a strain of *Bacillus* K3 with strong antifungal activity was screened from mangrove mud soil samples from Zhanjiang and Guangxi. *Bacillus* K3 was activated by glycerol tubes for two generations and inoculated into 1000 mL of LB broth at a 3% inoculum. Fermentation was carried out at 36℃ for 24 h. After fermentation, the mixture was centrifuged at 8000 r / min and 4℃ for 20 min, discarding the bacterial cells and retaining the fermentation broth. The pH of the fermentation supernatant was first adjusted to the optimal pH for protease. Then, papain, trypsin, and pepsin were added to treat the fermentation supernatant until the final enzyme concentration reached 10 mg / mL. After incubation at 50℃ for 4 h, the pH of the original fermentation supernatant was adjusted back. The antifungal activity was determined using the Oxford cup method. The fermentation supernatant without protease was used as a blank control to determine whether the antifungal substance of *Bacillus* K3 was a polypeptide or protein.

[0043] The results are as follows Figure 1 As shown, the single-layer Oxford cup experiment revealed that the antibacterial activity of the fermentation supernatant against Fusarium oxysporum was reduced after treatment with papain, trypsin and pepsin for 4 h, respectively. Therefore, it can be determined that the main antibacterial substance of Bacillus K3 is polypeptide or protein.

[0044] 1.2 Molecular identification of Bacillus bereaves K3:

[0045] Single colonies of Bacillus K3 were picked with sterile toothpicks for colony PCR amplification. Universal bacterial primers 27F and 1492R were used as PCR primers. The total reaction volume was 30 µL: 15 µL 2×MightyAmp Buffer, 0.75 µL MightyAmp DNA Polymerase, 0.75 µL each of forward and reverse primers, and 12.75 µL ddH2O. The PCR reaction program was: 94℃ for 4 min; 94℃ for 30 s, 55℃ for 20 s, 72℃ for 1.5 min, 30 cycles; 72℃ for 10 min, and stored at 4℃. The PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained sequences were compared with the EZBioCloud and NCBI databases, and a phylogenetic tree was constructed using MEGA 5.0.

[0046] The results showed that after 16S rDNA sequencing, strain K3 yielded a full-length 1428 bp 16S rRNA gene sequence. BLAST alignment analysis using the NCBI GenBank database showed that this sequence had the highest similarity (99.71%) to the type strain of Bacillus velezensis, CR-502(T)AY603658. Using MEGA 11.0 software to construct a neighbor-joining method, gene sequences from several different strains with similarity exceeding 97% were selected for analysis. K3 clustered with Bacillus velezensis, indicating the closest phylogenetic relationship between the two. Based on the colony morphology and cell structure, strain K3 could be identified as Bacillus velezensis. Strain K3 was deposited at the Guangdong Provincial Microbial Culture Collection Center on April 23, 2025, and classified as Bacillus velezensis K3, with accession number GDMCC No: 66202. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Bacillus velezensis K3 is referred to as Bacillus velezensis K3.

[0047] Example 2: Preparation, purification and identification of antimicrobial peptide GAB712.

[0048] 2.1 Preparation of antimicrobial peptide GAB712:

[0049] Bacillus belye K3 was activated by glycerol tubes for two generations and inoculated into 1000 mL of LB broth at a 3% inoculum. Fermentation was carried out at 36°C for 24 h. After fermentation, the cells were centrifuged at 8000 r / min and 4°C for 20 min, and the fermentation broth was discarded. The fermentation broth was mixed with ethyl acetate at a 1:1 ratio and extracted at 28°C and 150 r / min for 12 h. After standing and separation, the upper organic phase was collected, and the lower phase was extracted twice more with ethyl acetate at a 1:1 ratio. The organic phases were then mixed and vacuum evaporated and concentrated in a rotary evaporator at 40°C. The residual solid was collected as the antimicrobial peptide GAB712.

[0050] To determine the minimum inhibitory concentration (MIC) of the antimicrobial peptide GAB712 on the mycelial growth of *Fusarium oxysporum*, this experiment employed the Oxford cup method to determine the inhibition zone. *Fusarium oxysporum* spore suspension was added to unsolidified sterilized PDA medium, and the final concentration was adjusted to 1 × 10⁻⁶. 6 After thorough mixing with cfu / mL, the solution was poured into sterile Petri dishes. Subsequently, the antimicrobial peptide GAB712 was dissolved in sterile water to prepare solutions with final concentrations of 0 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, 8 mg / mL, 16 mg / mL, 32 mg / mL, and 64 mg / mL. 100 μL of each solution was injected into Oxford cups. The plates were placed at 4°C to allow the antimicrobial peptide solution to pre-diffuse in the culture medium for 4 h. Afterward, the plates were transferred to a 30°C incubator and incubated for 48 h, with sterile water as a control group. By observing the formation of inhibition zones, the minimum inhibitory concentration of the antimicrobial peptide GAB712 on the mycelial growth of *Fusarium oxysporum* was determined; this was the lowest concentration of the component used to produce inhibition zones.

[0051] pass Figure 2 The minimum inhibitory concentration (MIC) of the antimicrobial peptide GAB712 against Fusarium oxysporum was 1 mg / mL, with an inhibition zone diameter of 8.55 mm. The higher the concentration, the larger the inhibition zone diameter. This indicates that the antimicrobial effect of GAB712 is concentration-dependent, and a concentration of 64 mg / mL of GAB712 resulted in an inhibition zone of 25.31 mm, effectively inhibiting the growth of Fusarium oxysporum.

[0052] 2.2 Purification of antimicrobial peptide GAB712:

[0053] First, the AKTA purifier piping system and circulation pump were cleaned with Buffer A (0.1% formic acid) and Buffer B (acetonitrile, chromatographic grade), respectively. Then, the Ultimate® XB-C18 high-performance liquid chromatography preparative column (10µm, 300Å, 10×250 mm) was connected to the AKTA purifier body at a low flow rate. The column was then equilibrated with Buffer A for 30 min, with the flow rate controlled at 1 mL / min. Then, the crude extract obtained above was filtered through a 0.22μm aqueous microporous membrane, and 2 mL of the filtrate was loaded onto the sample. Elution program: (1) 0-10 min, 2%B; (2) 10-30 min, 2%-10%B; (3) 30-33 min, 10%-60%B; (4) 33-40 min, 60%B; (5) 40-43 min, 60%-2%B; (6) 43-48 min, 2%B. The eluent flow rate was controlled at 0.8 mL / min, the detection wavelength was 214 nm, and 2 mL was collected per minute. After collecting the eluent, the eluents located at the same absorption peak were combined, and the samples were freeze-dried into powder using a vacuum freeze dryer and stored at 4 °C. The antibacterial activity was measured using Fusarium oxysporum as an indicator bacterium.

[0054] The extract was purified using high-performance liquid chromatography (LC-20AD) with an Inertsil ODS-3 column (4.6 mm × 250 mm, 5 μm). Mobile phase A was ultrapure water, and mobile phase B was acetonitrile. The sample was filtered through a 0.22 μm microporous membrane and then eluted isocratically. The mobile phase ratio was A:B = 40:60, the flow rate was 0.4 mL / min, the injection volume was 10 μL, and the absorption wavelength was 214 nm. The mobile phase corresponding to the retention times of each absorption peak was collected. After removing acetonitrile by nitrogen blowing at 35 °C, the sample was freeze-dried, and the antibacterial activity was measured using *Fusarium oxysporum* as an indicator bacterium.

[0055] The crude extract, obtained by ethyl acetate extraction and rotary evaporation concentration, was dissolved in methanol. 1 mL of the sample was then subjected to chromatographic purification. Fractions were collected according to their elution times, and after vacuum freeze-drying, the sample solution was reconstituted with ultrapure water. Antibacterial activity assays against *Fusarium oxysporum* revealed that only the fractions corresponding to collection tubes 7-12 showed antibacterial effects. This active fraction was selected for secondary purification, and high-performance liquid chromatography (HPLC) was used to optimize separation parameters, providing a high-purity sample for subsequent structural identification of the active fraction. The HPLC purification results are shown below. Figure 3As shown, the target component exhibits two characteristic absorption peaks, with few impurities and a stable baseline. The separation effect between each peak is good. Each fraction was concentrated by vacuum nitrogen blowing and freeze-dried. The antibacterial experiment of Fusarium oxysporum confirmed that only the main peak had an antibacterial effect. The separated substance corresponding to the main peak was named antimicrobial peptide GAB712.

[0056] 2.3 Structural analysis of antimicrobial peptide GAB712:

[0057] Accurately transfer 100 μL of sample into an EP tube, add 1 μL of 1M dithiothreitol (DTT) solution to a final DTT concentration of 10 mmol / L, and reduce in a water bath at 56 °C for 1 h. Then add 2 μL of 1M iodoacetamide (IAM) solution to a final IAM concentration of 20 mmol / L, and alkylate at room temperature in the dark for 40 min. To quench excess IAM, add another 1 μL of 1M DTT solution to a final DTT concentration of 10 mmol / L. After the reaction is complete, desalt and purify using a C18 stage-tip column, and remove the organic solvent by vacuum drying at 45 °C to finally obtain the desalted target product.

[0058] The processed sample peptide fragments were analyzed using liquid chromatography-mass spectrometry.

[0059] (1) Pre-column: PEPMAP NEO C18 300μm×5 mm analytical column: 150μm id×170 mm, packing: Reprosil-Pur 120 C18-AQ 1.9μm;

[0060] (2) Mobile phase A: 0.1% formic acid;

[0061] (3) Mobile phase B: 0.1% formic acid, 80% ACN;

[0062] (4) Flow rate: 600 mL / min;

[0063] (5) Analysis time: 66 min;

[0064] (6) Specific chromatographic conditions were as follows: Primary mass spectrometry parameters: Resolution: 120000; AGCtarget: Standard; Maximum IT: 20 ms; Scanrange: 300 to 1800 m / z; Secondary mass spectrometry parameters: Resolution: 15000; AGCtarget: Standard; Maximum IT: 22 ms; Cycle time: 2 s; Collision Energy: 30. Raw data were obtained after mass spectrometry acquisition. The raw mass spectrometry files were analyzed by searching the UniProtKB database for all "Bacillus" data using MaxQuant software. Further multi-dimensional sequence analysis was performed using the Antimicrobial Peptide Database.

[0065] The raw mass spectrometry data were processed using the Thermo Scientific Xcalibur 4.3 platform to obtain a first-order mass spectrum. Figure 4 ), secondary mass spectrum ( Figure 5 Based on the fragmentation pattern, the structure of GAB712 was deduced, and the amino acid sequence of GAB712 was inferred as ISVYPSQPLVS (SEQ ID NO: 1) based on the ionic strength of the fragment ions. It was found that the antimicrobial peptide GAB712 showed no homology with other reported antimicrobial peptides. Comparative analysis in the antimicrobial peptide APD database showed a maximum similarity of only 46.15% with known antimicrobial peptides, thus confirming it as a novel antimicrobial peptide.

[0066] Example 3: Application of Bacillus vesiculosus antimicrobial peptide GAB712 in food preservation.

[0067] 3.1 Experimental Grouping:

[0068] Fresh, commercially available dragon fruits of similar ripeness were purchased and processed on the same day. They were first manually sorted to remove those with pests, diseases, or mechanical damage, ensuring consistency in variety, ripeness, and physical condition. The dragon fruits in each treatment group were rinsed thoroughly with running tap water, air-dried, disinfected with alcohol, washed three times with sterile water, and then air-dried again. Under sterile conditions, three standard wounds (3×3 mm) were prepared on one side of the dragon fruit using sterile toothpicks, and the oozing fluid was aspirated with a pipette. Three treatment groups were set up. Treatment group 1 (T1): After surface disinfection, 5 μL of Fusarium oxysporum spore suspension was injected into the wells of the dragon fruit, and the fruit was placed in a 25℃ incubator in the dark for 24 h to allow for full absorption of the Fusarium oxysporum spore suspension. Subsequently, 5 μL of the antimicrobial peptide GAB712 (3×MIC) filtered through a 0.22 μm membrane was injected into each well. Treatment group 2 (T2): After surface sterilization, 5 μL of the antimicrobial peptide GAB712 (3×MIC) filtered through a 0.22 μm membrane was injected into the wells. The fruits were then incubated at 25℃ in the dark for 24 h, followed by the injection of 5 μL of Fusarium oxysporum spore suspension into the wells. Control group (CK): After surface sterilization, 5 μL of sterile water was injected into the wells. The fruits were then incubated at 25℃ in the dark for 24 h, followed by the injection of 5 μL of Fusarium oxysporum spore suspension into the wells. The incubation time after inoculation was recorded at 0, 3, 5, 7, 9, and 11 days. Six dragon fruits were selected for each treatment and incubated at 25℃ in the dark.

[0069] 3.2 Determination of decay rate, weight loss rate, and lesion area:

[0070] If dragon fruit shows obvious signs of rot, its rot rate and the extent of rot in the affected areas are determined. During storage, the affected area and weight are recorded at 0, 3, 5, 7, 9, and 11 days after treatment. The affected area is calculated by measuring the diameter of the lesions at 9 points, using formula (1):

[0071] Lesion area (S) = 3.14 × (lesion diameter / 2) 2 (1);

[0072] If the diameter of the lesion exceeds 10 mm, the dragon fruit is considered rotten, and the number of rotten dragon fruits in each group is recorded for statistical analysis.

[0073] The formulas for calculating the decay rate and weight loss rate are equations (2) and (3):

[0074] Rot rate (%) = (total number of rotten fruits / total number of fruits) × 100% (2);

[0075] Weight loss rate (%) = (initial fruit weight - weight at each time point) / initial fruit weight × 100% (3).

[0076] according to Figure 6 Experimental data (a) to (c) show that the antimicrobial peptide GAB712 treatment significantly inhibited the development of postharvest diseases in dragon fruit. Specifically, the control group showed rot symptoms on day 5 after inoculation with Fusarium oxysporum, reaching 100% rot rate by day 11, with an average lesion area of ​​614.71 mm². In contrast, the T1 and T2 treatment groups showed significantly lower lesion severity at the same time point. The final rot rate of the T1 group was 85%, with a lesion area of ​​417.56 mm², while the T2 group further reduced to a rot rate of 75% and a lesion area of ​​397.88 mm². This suggests that when preventive treatment is performed before pathogen infection, an appropriate concentration of the antimicrobial peptide GAB712 can effectively intervene in the pathogenic process of Fusarium oxysporum.

[0077] like Figure 7 As shown, the weight loss rate of all treatment groups increased with the storage process, confirming the continuous water loss of dragon fruit. However, there were significant differences in weight loss rates among the different treatment groups, with the control group showing a higher weight loss rate than the treatment groups, the most significant difference occurring on day 11. Specifically, the weight loss rate of the dragon fruit control group reached 4.58%, while the T1 and T2 treatment groups were controlled at 2.50% and 3.04%, respectively. This result indicates that treatment with the antimicrobial peptide GAB712 can effectively control fruit water evaporation, maintaining the fruit's plumpness and freshness.

[0078] 3.3 Determination of soluble solids and titratable acids:

[0079] Soluble solids (TSS) content was measured using a digital handheld refractometer, expressed as a percentage (%). Each treatment had three replicates. A slight modification was made to the pH potential method according to the national standard GB / T12456-2008. 2.5 g of fruit pulp was homogenized with 4 mL of deionized water, and the volume was adjusted to 50 mL using a volumetric flask. After filtration through filter paper, the filtrate was titrated with 0.1% sodium hydroxide to determine the titratable acid (TA) concentration, with the final titration pH being 8.2. Results are expressed as a percentage (citric acid equivalent).

[0080] The effects of antimicrobial peptide GAB712 on the soluble solids (TSS) and titratable acid (TA) content of dragon fruit during storage, such as... Figure 8 and Figure 9 As shown: Figure 8 The soluble solids content of dragon fruit also showed a gradual decreasing trend during storage. After 7-11 days of storage, the soluble solids content of treated dragon fruit decreased... Figure 9Similarly, the levels of soluble solids and titratable acid in dragon fruit were well maintained. While the effect of antimicrobial peptide GAB712 on maintaining titratable acid was limited in the early stages of storage, it effectively delayed the loss of titratable acid as storage time increased. These results indicate that antimicrobial peptide GAB712 can effectively inhibit the decrease in soluble solids and titratable acid content during dragon fruit storage, thus helping to maintain the quality and flavor of the fruit.

[0081] 3.4 Determination of malondialdehyde (MDA) content:

[0082] The working solution was prepared according to a tissue mass (g) to extract volume (ml) ratio of 1:10. The samples were homogenized in an ice bath, centrifuged at 8000 g for 10 min at 4℃, and the supernatant was collected and placed on ice for testing. The changes in malondialdehyde (MDA) content in the fruit were determined using a malondialdehyde (MDA) content detection kit.

[0083] Figure 10 The changes in MDA content in dragon fruit after treatment with the antimicrobial peptide GAB712 are shown. Figure 10 As can be seen, the MDA content in the control group (CK) continuously increased throughout the storage period, reaching its highest value on day 12; the MDA content in the T1 treatment group first increased and then decreased, showing a fluctuating trend; while the T2 treatment group showed a more stable change, with its MDA content consistently lower than that of the CK and T1 groups. This indicates that preventative treatment is more effective in controlling MDA accumulation. The MDA content in the experimental groups treated with the antimicrobial peptide GAB712 was lower than that in the control group, indicating that the antimicrobial peptide GAB712 can effectively slow down lipid peroxidation in fruit tissue and reduce cell membrane damage while controlling fungal invasion, thereby helping to maintain fruit quality and extend shelf life.

[0084] 3.5 Determination of superoxide dismutase (SOD):

[0085] The working solution was prepared according to a tissue mass (g) to extraction liquid volume (ml) ratio of 1:10. After sample treatment, the solution was homogenized in an ice bath. Subsequently, the sample was centrifuged at 8000 g for 10 min at 4°C, and the supernatant was collected and placed on ice for subsequent detection. Superoxide dismutase (SOD) was determined using a kit method. All kits were purchased from Beijing Box Biotechnology Co., Ltd., and enzyme activity was determined according to the kit instructions.

[0086] The results are as follows Figure 11 As shown, the SOD activity of all control and treatment groups initially increased and then decreased during storage, reaching a peak on day 7. The overall SOD activity of dragon fruit treated with the antimicrobial peptide GAB712 was generally higher than that of the control group, demonstrating better antioxidant capacity, mitigating oxidative stress, and helping to extend the shelf life of the fruit.

[0087] 3.6 Determination of catalase activity (POD):

[0088] The POD enzyme activity in the sample was determined using the guaiacol colorimetric method. 80 µL of crude enzyme extract was gently mixed with 280 µL of 0.3% guaiacol solution. After heating in a 30 °C water bath for 10 min, 0.1 M 30 µL of H₂O₂ solution was added to each well of the microplate before measurement. Then, 270 µL of the sample to be tested was added, and the absorbance was quickly measured at 470 nm. Measurements were taken every 30 seconds for a total of 10 measurements. The plate was zeroed using distilled water.

[0089] Studies on the effects of different treatments on postharvest fruit POD activity showed that ( Figure 12 The POD activity of dragon fruit showed a dynamic change during storage, initially increasing and then decreasing. In the dragon fruit POD activity experiment, the T2 treatment group showed the most significant preservation effect, with its POD activity reaching a peak of 107.5 U·g on day 9. -1 The activity of the T2 treatment group was increased by 75.19% and 5.47% compared to the CK group and T1 treatment group, respectively, and then entered the activity decay stage, indicating that the T2 treatment group could effectively activate the antioxidant capacity of dragon fruit.

[0090] 3.7 Determination of catalase (CAT):

[0091] The working solution was prepared according to a tissue mass (g) to extraction liquid volume (ml) ratio of 1:10. After sample processing, the solution was homogenized in an ice bath. Subsequently, the sample was centrifuged at 8000 g for 10 min at 4°C, and the supernatant was collected and placed on ice for subsequent detection. Catalase (CAT) was determined using a kit method. All kits were purchased from Beijing Box Biotechnology Co., Ltd., and enzyme activity was determined according to the kit instructions.

[0092] Experiments show that ( Figure 13 Treatment with the antimicrobial peptide GAB712 significantly enhanced the CAT activity of dragon fruit during storage. The CAT activity in the T2 treatment group reached its peak earlier and remained at a high level thereafter; while the activity in the T1 treatment group fluctuated more significantly and was generally lower than that in the control group. Treatment with the antimicrobial peptide GAB712 can improve the fruit's ability to scavenge reactive oxygen species, effectively delaying quality deterioration during storage.

[0093] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Bacillus belysinian antimicrobial peptide, characterized in that, The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO:

1.

2. The gene for Bacillus belyssus antimicrobial peptides, characterized in that, The gene encoding the Bacillus beryl antimicrobial peptide as described in claim 1.

3. A recombinant plasmid, characterized in that, The recombinant plasmid includes the gene for the Bacillus vesiculosus antimicrobial peptide as described in claim 2.

4. Recombinant microorganisms, characterized in that, The recombinant microorganism includes the recombinant plasmid as described in claim 3.

5. The application of the Bacillus vesiculosus antimicrobial peptide of claim 1, or the gene of the Bacillus vesiculosus antimicrobial peptide of claim 2, or the recombinant plasmid of claim 3, or the recombinant microorganism of claim 4 in food preservation.

6. The application according to claim 5, characterized in that, The food in question is fruit.

7. A food preservative, characterized in that, The food preservative includes the Bacillus vesiculosus antimicrobial peptide as described in claim 1.

8. The food preservative according to claim 7, characterized in that, The food preservative also includes a food science-acceptable carrier and / or one or at least two functional additives.

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