Broad-spectrum bacteriostatic bacteriocin Y-30 and application thereof

Abstract

The application belongs to the technical field of food microorganisms, and particularly relates to a broad-spectrum bacteriocin Y-30 and application thereof, wherein the amino acid sequence of the bacteriocin Y-30 is shown as SEQ ID NO. 1. The bacteriocin Y-30 has excellent thermal stability and pH tolerance. In addition, the combination of the bacteriocin Y-30 and 1.0% chitosan can effectively reduce the number of Listeria monocytogenes and the total number of viable bacteria, and maintain the quality index of fruits. These results show that the bacteriocin Y-30 can effectively inhibit the growth of foodborne pathogens, and can be used as a promising natural preservative to replace chemical preservatives in the agricultural and food industries.

Description

Technical Field

[0001] This invention belongs to the field of food microbiology technology, specifically relating to a broad-spectrum antibacterial bacteriocin Y-30 and its applications. Background Technology

[0002] Foodborne pathogens are the leading cause of significant deterioration in food safety, with common pathogens including pathogenic Escherichia coli (E. coli). Escherichiacoli Staphylococcus aureus ( Staphylococcus aureus Listeria monocytogenes ( ) Listeria monocytogenes ) and Salmonella ( Salmonella These pathogens invade the human body through the food we eat every day, and can cause mild symptoms such as diarrhea, vomiting, and skin infections, or serious illnesses that can endanger life, such as meningitis, sepsis, and miscarriage during pregnancy.

[0003] Nisin is a 34-amino acid polypeptide produced by *Streptococcus lactis*. As an antibacterial agent, it is widely used in the food, cosmetic, and pharmaceutical industries. However, its activity is significantly reduced or even completely lost under alkaline conditions, and it only exhibits strong inhibitory effects against Gram-positive bacteria; it has no effect on *Listeria monocytogenes* and no effect on Gram-negative bacteria. Despite its significant advantages, nisin's inherent limitations necessitate the development of a novel bacteriocin with broad-spectrum antibacterial activity. Summary of the Invention

[0004] The purpose of this invention is to provide a broad-spectrum antibacterial bacteriocin Y-30, which solves the problems existing in the prior art.

[0005] The technical solution adopted in this invention is: This invention provides a broad-spectrum antibacterial bacteriocin Y-30, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] A second aspect of the present invention provides a nucleic acid molecule that encodes the bacteriocin Y-30.

[0007] A third aspect of the present invention provides a preservative comprising the bacteriocin Y-30 or the nucleic acid molecule described above; the preservative further comprises chitosan.

[0008] Preferably, the preservative is prepared by: Chitosan was dissolved in 1% glacial acetic acid to prepare a chitosan solution with a mass fraction of 0.5% to 2.0%. The preservative is obtained by dissolving bacteriocin Y-30 in chitosan solution to achieve a final concentration of bacteriocin Y-30 of 64 μg / mL to 256 μg / mL.

[0009] Preferably, the preservative also includes food-acceptable excipients.

[0010] Preferably, the acceptable excipients in the food include at least one of emulsifiers, pH adjusters, solubilizers, and stabilizers.

[0011] Preferably, the emulsifier includes at least one of polysorbate 80 and polyethylene glycol octylphenyl ether.

[0012] Preferably, the pH adjuster includes at least one of acetic acid and sodium hydroxide.

[0013] Preferably, the co-solvent includes at least one of propylene glycol, hexanediol, and pentanediol.

[0014] Preferably, the stabilizer includes at least one of sodium EDTA and ascorbic acid.

[0015] A fourth aspect of the present invention provides the application of the bacteriocin Y-30, the nucleic acid molecule, and / or the preservative, wherein the application refers to any one of the following: 1) Preparation of formulations for inhibiting Gram-positive bacteria; 2) Preparation of formulations for inhibiting Gram-negative bacteria; 3) Prepare formulations for extending the shelf life of fruits.

[0016] Preferably, the Gram-positive bacteria include at least one of Listeria monocytogenes, Micrococcus luteus, and Bacillus.

[0017] Preferably, the Gram-negative bacteria include at least one of Escherichia coli, Pseudomonas aeruginosa, and Salmonella enteritidis.

[0018] Preferably, the fruit includes at least one of strawberry, apple and pear.

[0019] The bacteriocin Y-30 described in this invention was isolated from Enterobacter spp. strain Y-30, and its proposed classification is named as follows: Leuconostoc mesenteroides Y-30 was deposited at the China Center for Type Culture Collection (CCTCC) on April 24, 2026, with accession number CCTCC NO:M 2026809, at Wuhan University, Wuhan, China.

[0020] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a broad-spectrum antibacterial bacteriocin Y-30, the amino acid sequence of which is shown in SEQ ID NO.1. This invention isolates and identifies a novel bacteriocin Y-30 (molecular weight 573 Da), which exhibits excellent thermostability and pH tolerance, demonstrating broad-spectrum antibacterial activity against a variety of Gram-negative and Gram-positive bacteria, such as Listeria monocytogenes, Micrococcus luteus, Bacillus cereus, Escherichia coli, and Pseudomonas aeruginosa. By disrupting the cell membrane, it induces leakage of intracellular nucleic acids and proteins, altering cell morphology and inhibiting bacterial growth. Furthermore, bacteriocin Y-30 exhibits a targeted interaction with the major groove structure of Listeria monocytogenes. The genomic DNA of Listeria monocytogenes ATCC 19114 is specifically enriched in the AG enrichment region. Its hemolysis rate is much lower than that of nisin, ensuring its safe application in the food industry. Furthermore, bacteriocin Y-30 combined with 1.0% chitosan can reduce the population of Listeria monocytogenes. Reducing Listeria monocytogenes in strawberries can optimize storage quality indicators (such as spoilage rate and weight loss rate) and extend the shelf life of strawberries. These results indicate that bacteriocin Y-30 + 1.0% chitosan, as a composite natural preservative, has promising applications as an alternative to chemical preservatives in agriculture and the food industry. Attached Figure Description

[0021] Figure 1 The colony morphology, Gram staining results, and phylogenetic tree of strain Y-30 are presented. (a) Colony morphology; (b) Gram staining results; (c) Phylogenetic tree.

[0022] Figure 2 The antimicrobial kinetic curve of strain Y-30 is shown.

[0023] Figure 3 The results of RP-HPLC, LC-MS and LC-MS / MS analysis of bacteriocin Y-30 are shown. (a) RP-HPLC analysis; (b) LC-MS analysis; (c) LC-MS / MS analysis.

[0024] Figure 4 The effect of bacteriocin Y-30 on the growth curve and time-bactericidal curve of Listeria monocytogenes; (a) Growth curve; (b) Time-sterilization curve.

[0025] Figure 5 This describes the leakage of substances from Listeria monocytogenes after the action of bacteriocin Y-30. (a) Nucleic acid leakage; (b) Protein leakage.

[0026] Figure 6 The effects of bacteriocin Y-30 on the morphology and ultrastructure of Listeria monocytogenes ATCC 19114 cells; (a) Cell morphology of the control group; (b) Cell morphology after treatment with bacteriocin Y-30; (c) Cell ultrastructure of the control group; (d) Cell ultrastructure after treatment with bacteriocin Y-30.

[0027] Figure 7 To illustrate the interaction between bacteriocin Y-30 and Listeria monocytogenes genomic DNA; (a) Gel retardation experiment, where CK: Listeria monocytogenes genomic DNA; lane 1: bacteriocin Y-30 treatment group with 2×MIC concentration; lane 2: bacteriocin Y-30 treatment group with 4×MIC concentration; (b) Molecular model of the interaction between bacteriocin Y-30 and DNA; (c) A magnified view of the binding site in (b).

[0028] Figure 8 The effects of different treatment groups on the number of Listeria monocytogenes ATCC 19114 bacteria and TVC in strawberries; (a) Listeria monocytogenes ATCC 19114 bacterial count; (b) strawberry TVC.

[0029] Figure 9 The effects of combined use of bacteriocin Y-30 and chitosan on different indicators of strawberries during storage; (a) Strawberry rot index; (b) weight loss rate; (c) L* value; (d) firmness; (e) total sugar content (TSS); (f) total acidity (TA). Detailed Implementation

[0030] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0031] The inventive concept of this invention is as follows: This invention isolates and identifies a *Enterobacter* strain Y-30 with broad-spectrum bacteriocin production capacity from traditional Chinese fermented fish, a food made by hand. The novel bacteriocin Y-30 was subsequently purified. Bacteriocin Y-30 exhibits superior properties compared to nisin, overcoming the application limitations of nisin and demonstrating great potential as a natural preservative.

[0032] To evaluate the preservation effect of bacteriocin Y-30 as a food preservative, this invention uses bacteriocin Y-30 alone and in combination with chitosan as a food preservative to preserve strawberries, in order to explore its efficacy, thereby providing a basis for the development of novel natural compound preservatives, and ultimately replacing chemical preservatives in agriculture and the food industry.

[0033] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0034] Example 1: A broad-spectrum antibacterial bacteriocin Y-30 and its application, as detailed below: 1. Materials and Methods.

[0035] 1.1 Strains and culture media.

[0036] Leuconostoc mesenteroides Y-30 ( Leuconostoc mesenteroides The strain was collected from fermented sour and spicy fish made by farmers in Bengbu City, Anhui Province, China. The strain was inoculated into De Man Rogosa Sharpe (MRS) broth medium and then incubated at 37°C for 48 hours in a laboratory incubator. The indicator bacterium was Listeria monocytogenes. L. monocytogenes ATCC 19114) and Micrococcus luteus ( M. luteus CMCC 28001), preserved in our laboratory. Incubated at 37°C with continuous shaking at 180 rpm. The fungal strain was cultured on potato dextrose agar (PDA). To prepare the corresponding solid medium, 1.5% (w / v) agar was added to MRS or nutrient broth (NB).

[0037] 1.2 Isolation and purification of bacteriocin-producing bacteria.

[0038] 1.2.1 Isolation and purification of bacteriocin-producing bacteria in acid-fermented fish.

[0039] This invention employs the plate dilution method used by Sidhu et al.: 1 g of sample is placed in 9 mL of sterile physiological saline and thoroughly mixed by shaking. The resulting turbid liquid is then serially diluted, with each dilution increment being 10. -4 10 -5 10 -6 and 10 -7The diluted solution was spread onto solid MRS medium containing 0.3% CaCO3 and incubated at 37°C for 48 h. After incubation, single colonies with calcium-soluble zones were screened for streak plating purification, and the purification process was repeated at least three times. The purified single colonies were then incubated in MRS broth at 37°C for 48 h. Strains with good antibacterial activity were screened using the agar plate diffusion method, with Listeria monocytogenes as the indicator bacterium. Antibacterial activity was assessed by measuring the diameter of the inhibition zone. In addition, the pH of the cell-free supernatant (CFS) was adjusted to 7.0 using 6 mol / L sodium hydroxide solution to eliminate acid interference. Finally, the antibacterial activity of Listeria monocytogenes (CFS) was evaluated. L. monocytogenes The single colony that showed the best inhibitory effect was named strain Y-30.

[0040] 1.2.2 Identification of strain Y-30.

[0041] Genomic DNA extraction experiments for molecular identification were performed according to the method described by Islam et al. The 16S rRNA gene of strain Y-30 was amplified by polymerase chain reaction (PCR) using primers (27F, 1492R), with the strain's genomic DNA as a template. Subsequently, the PCR amplification products were purified using a gel extraction kit before sequencing. The obtained sequences were analyzed using BLAST (Basic Local Alignment Search) in NCBI (http: / / www.ncbi.nlm.nih.gov / blast), and a phylogenetic tree was constructed using neighbor-joining in MEGA 12.0 software.

[0042] 1.2.3 Determination of growth curve and antibacterial activity curve of strain Y-30.

[0043] Based on the method described by Pan et al., with appropriate adjustments, activated bacterial cultures (1%, v / v) were inoculated into three Erlenmeyer flasks, each with 800 mL of MRS broth, and incubated at 37°C. 5 mL samples were collected every 4 h, and the absorbance of the bacterial cultures was measured at 600 nm at each time point. The bacterial cultures from each culture cycle were centrifuged at 8000 rpm for 5 min. The pH of the supernatant was measured using a pH meter. Micrococcus luteus (Micrococcus luteus) was detected using the agar plate diffusion method. M. luteus The Y-30 strain was used as an indicator strain to test its antimicrobial activity. Three replicates were performed for each sample.

[0044] 1.3 Study on purification of bacteriocins produced by strain Y-30.

[0045] The purification and identification of bacteriocin Y-30 were performed according to the method of Zhao et al., with some modifications made based on actual conditions: Y-30 strain was placed in 2 L of MRS broth and cultured at 37°C for 48 h to prepare CFS. Subsequently, the mixture was stirred continuously at 4°C for 12 h, and ammonium sulfate was slowly added to the CFS to promote precipitation. The precipitate was collected by centrifugation and resuspended in 20 mM disodium hydrogen phosphate-citric acid (DHPCA) buffer (pH 4.0). The agar diffusion method was used to identify the bacteria *Micrococcus luteus* (…). M. luteus The antibacterial activity of the target strain was assessed. Further purification of the active sample was performed using gel filtration chromatography on a Sephadex G-50 column (80 × 2.0 cm) with DHPCA buffer for isocratic elution. All eluted fractions were collected, and their antibacterial activity was determined by agar diffusion. After analysis of the maximum absorption wavelength of the most active fraction, this fraction was further purified by high-performance liquid chromatography (HPLC, LC-20, Shimadzu) to separate the bioactive compounds. Elution was performed using a linear gradient of acetonitrile (containing 0.1% TFA) and ultrapure water at a flow rate of 0.6 mL / min over 40 min, reducing the water content from 95% to 5%. The antibacterial activity was then determined by agar diffusion.

[0046] 1.4. Determination of peptide chain sequence.

[0047] To identify the target active substance, matrix-assisted laser desorption / ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS) was selected as the analytical tool. Based on the peptide fragmentation nomenclature method established by Manneberg et al., the amino acid composition of the purified target active substance was analyzed using an automated amino acid analyzer (U3000, Thermo Fisher Scientific, USA), and its amino acid sequence was determined using an automated protein sequencer (PPSQ-31A, Shimadzu, Japan).

[0048] 1.5 Stability of bacteriocin Y-30.

[0049] Following the method described by Zhao et al., with appropriate modifications, the effects of temperature, pH, and enzyme treatment on its antibacterial activity were investigated. Bacteriocin Y-30 was heat-treated at 40℃, 60℃, and 80℃ for 30 min each time; or treated at 121℃ under 15 psi pressure for 20 min. For long-term thermal stability analysis, the samples were stored at 4℃ for 120 days.

[0050] In addition, the bacteriocin aliquots were adjusted to a pH range of 2.0 to 10.0 with hydrochloric acid (1 mol / L) or sodium hydroxide (1 mol / L), and then incubated in a water bath at 37°C for 1 h.

[0051] Enzyme sensitivity was assessed by co-incubation with the corresponding enzymes (papain 600 U / mg, Sigma; trypsin 1.5 KU / mg, Sigma; pepsin 2.5 KU / mg, Sigma). The final concentration of each enzyme was adjusted to 40 U / mL, followed by incubation at 37°C for 30 min. Enzyme inactivation was achieved by immersing the reaction mixture in a 100°C water bath for 5 min. The control group consisted of bacteriocin samples at pH 4.0 that were neither heated nor enzyme-treated. The antibacterial activity of each treated sample was then assessed using the agar diffusion method. All experiments were performed in triplicate.

[0052] 1.6 Antibacterial spectrum and minimum inhibitory concentration (MIC) of bacteriocin Y-30.

[0053] The minimum inhibitory concentration (MIC) of bacteriocin Y-30 was determined strictly in accordance with the guidelines issued by the Clinical and Laboratory Standards Institute (CLSI). Listeria monocytogenes ATCC 19114 strain in logarithmic growth phase was collected and washed with DHPCA buffer. Cells were resuspended in fresh NB broth and diluted 100-fold using NB broth. Simultaneously, serial dilutions of bacteriocin Y-30 were prepared in DHPCA buffer. Subsequently, 100 μL of each bacteriocin Y-30 dilution was mixed with an equal volume of the diluted bacterial suspension and placed in each well of a sterile 96-well microplate. Nisin and DHPCA buffer served as positive and negative controls, respectively. After incubation at 37°C for 24 h, bacterial growth was assessed by measuring optical density (OD). 600 A Varioskan Flash spectrophotometer was used. MIC (Minimum Inhibitory Concentration) is defined as the lowest concentration that can effectively inhibit the growth of the indicator strain. Meanwhile, Listeria monocytogenes ATCC19114 was replaced by other strains as the indicator strain, and the minimum inhibitory concentrations (MICs) of bacteriocins Y-30 and Nisin were determined using the same method.

[0054] 1.7 Safety assessment of bacteriocin Y-30.

[0055] The safety of bacteriocin Y-30 was evaluated using qualitative and quantitative assays. In this invention, agar plates supplemented with 7% (v / v) sheep blood were prepared. Subsequently, 200 μL of bacteriocin samples of different concentrations were added to each well and incubated at 37°C for 48 h. Phosphate-buffered saline (PBS) and 1% Triton X-100 were used as negative and positive controls, respectively. The presence of a clear hemolytic zone (β-hemolysis) indicated hemolytic activity; conversely, the absence of a hemolytic zone (γ-hemolysis) or the appearance of a green halo (α-hemolysis) around the colony indicated that the bacteriocin lacked hemolytic activity.

[0056] The hemolytic activity of the isolated bacteriocinolone peptides was quantitatively analyzed using 96-well plates. 2% blood cell suspension was prepared with PBS and serially diluted using the same procedure as for determining the minimum inhibitory concentration (MIC). The negative control group used PBS buffer, and the positive control group used 1% Triton X-100. After incubating the 96-well plates at 37°C for 3 hours, the optical density (OD) was measured. 540 The sample size was quantitatively analyzed using a microplate reader.

[0057] 1.8 Mechanism of action of bacteriocin Y-30 against Listeria monocytogenes.

[0058] 1.8.1 Determination of growth curve and time-kill curve.

[0059] (1) The growth curve determination experiment was based on the method of Meng et al., with minor modifications: Listeria monocytogenes was inoculated with the following: L. monocytogenes Fresh LB broth was seeded into 96-well plates at 200 μL per well and then incubated at 37°C for 2 h.

[0060] 200 μL of bacteriocin Y-30 was added to the test wells at final concentrations of 1 / 2×MIC and 1×MIC, serving as the treatment group; simultaneously, an equal volume of DHPCA buffer was added to the control wells, serving as the control group. Each group contained three replicates. After incubating the 96-well plate in a 37°C automated biochemical incubator for 12 h, OD... 600 Quantitative analysis was performed using a microplate reader (Varioskan LUX, ThermoFisher, USA).

[0061] (2) The time-lethal curve determination experiment was conducted according to the method of Du et al. With slight modifications: after three rounds of washing, the density of 200 μL was 10. 8 CFU / mL Listeria monocytogenes ( L. monocytogenes The cells were resuspended in DHPCA buffer.

[0062] 200 μL of bacteriocin Y-30 was added to the system at final concentrations of 1×MIC and 2×MIC, respectively, and the mixture was incubated at 37°C for continuous culture. Nisin (1×MIC) and DHPCA buffer were designated as positive and negative controls, respectively, as alternatives to bacteriocin Y-30. Samples were collected at fixed time points using plate counting to determine the number of viable colonies. All experiments were repeated three times.

[0063] 1.8.2 Protein and nucleic acid leakage.

[0064] Listeria monocytogenes cells were washed three times, resuspended in DHPCA buffer, and then bacteriocin Y-30 was added to final concentrations of 1×MIC and 2×MIC, respectively. The resulting mixtures were incubated in a 37°C water bath for predetermined time points (0h, 3h, 6h, 9h, and 12h), and then centrifuged at 5000×g for 10 min to separate CFS. Subsequently, the nucleic acid and protein content in the cell-free supernatant leaked from Listeria monocytogenes cells was determined using a UV-Vis spectrophotometer.

[0065] 1.8.3 Scanning electron microscope (SEM) and transmission electron microscope (TEM).

[0066] Bacteriocin Y-30 at a final concentration of 1 × minimum inhibitory concentration (MIC) was applied to OD. 600 Listeria monocytogenes was 0.4. L. monocytogenes The cells were incubated in a mixture at 37°C for 1 h. After washing, Listeria monocytogenes (…) L. monocytogenes Cells were fixed with 2.5% glutaraldehyde at 4°C for 12 h. The samples were dehydrated using a series of pre-cooled ethanol solutions for 15 min, followed by tert-butanol for further processing. After drying and gold-palladium coating, cell morphology was imaged and analyzed using a Regulus 8100 scanning electron microscope system (Hitachi, Japan). A negative control group was established using DHPCA buffer instead of bacteriocin Y-30.

[0067] Listeria monocytogenes ( L. monocytogenes The cell preparation steps for transmission electron microscopy (TEM) observation are as follows: Initially, Listeria monocytogenes (… L. monocytogenesCells were perfused with acetone and then embedded in epoxy resin for 24 h. The polymerization reaction was then carried out in a metal bath maintained at 60°C for 48 h, after which the samples were sectioned using an ultramicrotome. The sections were then stained with lead citrate and uranyl acetate, and observed using a transmission electron microscope (HT 7900, Hitachi, Japan). This invention uses the same negative control design, but replaces bacteriocin Y-30 with DHPCA buffer.

[0068] 1.8.4 Effect of bacteriocin Y-30 on the DNA of Listeria monocytogenes strains.

[0069] Genomic DNA was extracted from Listeria monocytogenes in its exponential growth phase, and quantitative and qualitative analyses were performed using an ultra-micro spectrophotometer and 1.2% agarose gel electrophoresis. DNA samples (146.3 ng / μL) were mixed with bacteriocin Y-30 at concentrations of 128 μg / mL and 256 μg / mL, and incubated in a metal bath for 1 h. Subsequently, electrophoretic mobility shift analysis was used to detect and analyze the treated samples.

[0070] Molecular docking analysis was performed using AutoDock software to simulate the binding process, with Lamarck's genetic algorithm used for the random search method. The B-DNA sequence (CGCGAATTCGCG)2, obtained from the protein database (PDB ID: 1BNA), was selected as the DNA model. All molecular docking experiments were strictly performed according to the method proposed by Ricci and Netz.

[0071] SEQ ID NO.2: CGCGAATTCGCG.

[0072] 1.9. Preservation of strawberries.

[0073] The bacteriocin Y-30 used in this invention can be obtained by either HPLC purification or chemical synthesis, and verification has shown that there is no difference in efficacy between the two methods. Therefore, this invention does not restrict the source of bacteriocin Y-30.

[0074] The chitosan used in this invention was purchased from Merck, CAS#: 9012-76-4, catalog number 448869-50G.

[0075] 1.9.1 Sample preparation.

[0076] In the preparation of chitosan solutions, chitosan (95% degree of deacetylation) is dissolved in 1% (volume ratio) glacial acetic acid to prepare chitosan solutions with concentrations of 0.5% (w / v) and 1.0% (w / v).

[0077] Bacteriocin Y-30 was added to the chitosan solution to achieve a final concentration of 1×MIC (64 μg / mL). This invention included six experimental groups using different preservative regimens, as detailed below:

[0078] (1) Sterile water, designated as the control group; (2) Bacteriocin Y-30: 1×MIC, denoted as A1; (3) 0.5% chitosan: 0.5% C, denoted as A2; (4) 1.0% chitosan: 1.0% C, denoted as A3; (5) 1×MIC bacteriocin Y-30 combined with 0.5% chitosan: 1×MIC Y-30 + 0.5%C, denoted as A4; (6) 1×MIC bacteriocin Y-30 combined with 1.0% chitosan: 1×MIC Y-30 + 1.0% C, denoted as A5.

[0079] Strawberries were purchased from a fruit supermarket in Huaibei. Fruits of uniform size were selected and divided into 12 groups of 20 strawberries each. Six groups underwent 30 minutes of full ultraviolet irradiation to inactivate surface microorganisms; subsequently, 100 μL of Listeria monocytogenes in its exponential growth phase was added. L. monocytogenes ) suspension (to 10 5 A solution of CFU / mL was evenly applied to the surface of the strawberries, followed by 30 minutes of natural air drying. These strawberries were then immersed in the six preservatives mentioned above and kept at 4°C for 10 minutes. After 30 minutes of cooling with circulating cold air, all samples were sealed in sterile low-density polyethylene bags and stored at 4°C. Relevant indicators were measured on days 0, 3, 6, 9, 12, and 15 of storage. The remaining six groups of strawberries underwent no pretreatment and were similarly immersed in the six preservatives, with subsequent experimental procedures following the same steps. All experiments were performed in triplicate.

[0080] 1.9.2 Determination of Listeria monocytogenes count and total viable count (TVC).

[0081] The microbial counting method was based on the method used by de Jesus Salas-Mendez et al., with slight modifications: For Listeria monocytogenes detection, 10 g of strawberry sample was weighed, mixed with 90 mL of sterile physiological saline, and homogenized using a laboratory homogenizer to prepare a uniform bacterial suspension. The suspension was then spread onto PALCAM agar plates and incubated at 37°C for 24 hours, after which Listeria monocytogenes colonies were counted.

[0082] 1.9.3 Sensory evaluation.

[0083] Sensory evaluation was conducted by a trained panel of 10 judges, focusing on quality attributes such as strawberry color, flavor, and appearance. The sensory evaluation used a 10-point sensory rating scale as shown in Table 1.

[0084] Table 1 Sensory Evaluation Scoring Criteria 1.9.4 Decay Index.

[0085] The decay index is determined by classifying the severity of decay into four levels (0-3) based on the area of ​​decay. The decay index is directly proportional to the degree of fruit decay. The calculation formula is as follows:

[0086] Decay Index = .

[0087] M i and N i These represent the degree of rot and the number of strawberries at that degree; M is the highest degree of rot; N is the total number of strawberries in each group.

[0088] Based on the size of the rotten area, the degree of fruit rot is divided into four levels: Grade 0: The rotten area accounts for 0% of the fruit area, which means fresh strawberries without rot or damage; Grade 1: Rotten area accounts for 1% to 25% of the total strawberry area; Level 2: Rotten area accounts for 26% to 50% of the total strawberry area; Level 3: The rotten area accounts for more than 50% of the total strawberry area.

[0089] 1.9.5 Weight loss rate.

[0090] Six representative strawberry samples were randomly selected from each group, and their fresh weight was recorded at predetermined sampling time points. All measurements were repeated three times. The calculation formula is as follows:

[0091] Weightlessness rate = .

[0092] Where w1 is the initial mass (g) of the strawberries, and w2 is the mass (g) of the strawberries after storage.

[0093] 1.9.6. Color and hardness.

[0094] Based on the method established by Adhikari et al., the color of strawberry peel was measured using a colorimeter, and the L* value in the CIELAB color space was recorded. L* represents lightness. Each measurement was independently repeated three times.

[0095] Based on the method of Salas-Mendez et al., and after calibration, the hardness of strawberry flesh was measured using a digital hardness tester, with a 1 cm sample taken. 2 The peel samples exposed the pulp tissue, and measurements were repeated three times at different locations on each fruit.

[0096] 1.9.7 Total soluble solids (TSS) and titratable acid (TA).

[0097] Total soluble solids (TSS) in strawberry samples were determined according to the method described by Tumbarski et al. Strawberries were ground into a pulp, and the juice was separated by filtration through sterile gauze. A portable refractometer was zeroed and calibrated with deionized water. Two drops of strawberry juice were added to the prism surface, and the TSS value was immediately read. Each sample was measured in triplicate, and the results are expressed as the average.

[0098] The titratable acid content was determined using the method of Salas-Mendez et al.: 10 g of strawberry pulp sample was accurately weighed and transferred to a beaker, then diluted with 50 mL of deionized water. After homogenization, the mixture was brought to a final volume of 100 mL in a volumetric flask and allowed to stand for 30 min. 25 mL of the supernatant was transferred to an Erlenmeyer flask, and 3 drops of 1% phenolphthalein ethanol solution were added as an indicator. Titration was performed using 0.1 mol / L standardized sodium hydroxide solution until a pale pink endpoint was reached (lasting 30 s). The calculation formula is as follows:

[0099] Titratable acid content (%) = .

[0100] Where V represents the total volume of the strawberry extract (mL); C represents the concentration of the sodium hydroxide standard solution (mol / L); V NaOH V is the volume of sodium hydroxide used in the titration (mL); f is the conversion factor (1 mmol of sodium hydroxide is equivalent to 0.070 g of malic acid); s m represents the volume (mL) of strawberry extract used in the titration; m is the mass (g) of the strawberry sample.

[0101] 1.10 Statistical Analysis.

[0102] All experiments had three replicates, and data were statistically analyzed using SPSS Statistics 20.0 (IBM). Significant differences were assessed using analysis of variance (ANOVA). P (≤0.05), results are expressed as mean ± standard deviation.

[0103] 2. Results.

[0104] 2.1 Isolation and identification of strain Y-30.

[0105] like Figure 1 As shown in (a), single colonies were isolated from a handmade dilute solution of fermented fish with sour and spicy flavors using multiple rounds of plating and streak plating. The obtained colonies were milky white, moist, glossy, and round, surrounded by a calcification zone. The presence of this calcification zone indicates that the strain has acid-producing ability. Gram staining of the strain revealed blue-purple oval cells under microscopic examination. Figure 1 (b)). Phylogenetic tree analysis based on strain Y-30 ( Figure 1 (c)), strain Y-30 and Leuconostoc mesenteroides qz-547 (GenBank accession number: HF562942.1) showed high homology and belonged to the same evolutionary branch. Based on this, strain Y-30 was identified as *Leuconostoc mesenteroides*. Leuconostoc mesenteroides Its 16S rRNA gene sequence has been submitted to the GenBank database, accession number PX963688.

[0106] 2.2 Growth curve and antibacterial activity of Leuconostoc mesenteroides Y-30.

[0107] The growth curve of *Leuconostoc mesenteroides* Y-30 exhibits an S-shaped pattern. After an initial lag phase, the strain enters the logarithmic growth phase at 4 hours, followed by the exponential growth phase, gradually reaching the stationary phase by 12 hours, at which point the OD... 600 The pH value was in the range of 2.3–2.5. During the fermentation of strain Y-30, the pH value of CFS decreased sharply within 4–12 hours, then the rate of decrease slowed down, eventually stabilizing at around 4.1. According to the inhibition zone results, the antibacterial activity of CFS of strain Y-30 increased rapidly within 4–12 hours, then slowly increased and stabilized within the range of 20.9 mm–21.3 mm. The above results are shown in […]. Figure 2 .

[0108] 2.3 Purification and identification of bacteriocin Y-30.

[0109] The crude antibacterial extract was concentrated by ammonium sulfate precipitation and then purified by Sephadex G-50 gel filtration chromatography. Antibacterial activity tracking of each collected fraction revealed that fraction 39 exhibited significant antibacterial activity and a characteristic absorption peak at 220 nm. The fraction with the highest activity was further purified by HPLC, showing a single activity peak at a retention time of 18.653 min. Figure 3 (a)). This single-peak component was collected and analyzed by mass spectrometry; its quasi-molecular ion peak [M+H] was observed. + Its m / z is 574.2861, thus determining its molecular weight to be 573 Da ( ). Figure 3 (b)). TOF-MS / MS secondary mass spectrometry analysis ( Figure 3(c) shows that the parent ion fragmentation at m / z 574.2861 produced a series of characteristic fragments, including b-type fragment ions (b2–b5) with m / z 457.1355, 360.1544, 273.0617, and 186.0902, and y-type fragment ions (y2, y5) with m / z 459.0177 and 215.1391. Based on the peptide fragmentation nomenclature proposed by Roepstorff and Fohlman, combined with amino acid composition analysis (containing Asp, Ala, Ser, Pro, and Val), the amino acid sequence of this component was confirmed as N-aspartic acid-alanine-serine-serine-proline-valine-OH. Finally, this antimicrobial peptide was named bacteriocin Y-30, and its sequence is shown in SEQ ID NO.1.

[0110] SEQ ID NO.1: DASSPV.

[0111] 2.4 Stability of bacteriocin Y-30.

[0112] Bacteriocin Y-30 exhibits excellent thermal stability. After treatment at 40°C, 60°C, and 80°C for 30 min, its relative antibacterial activity remained above 90%; even after treatment under autoclaving at 121°C for 20 min, its residual activity was still as high as 85.11%. In addition, this bacteriocin also has excellent low-temperature storage performance; after storage at 4°C for 120 days, its residual antibacterial activity still reached 92.58%.

[0113] The antibacterial activity of bacteriocin Y-30 exhibits a clear pH dependence. This bacteriocin demonstrates high stability within the pH range of 2.0–5.0, maintaining a relative antibacterial activity above 90%. However, its activity begins to decrease significantly when the pH rises above 7.0, and is completely lost at pH 9.0 and 10.0. These results indicate that bacteriocin Y-30 possesses excellent stability under acidic and neutral conditions, but its activity is highly sensitive to alkaline environments.

[0114] Furthermore, bacteriocin Y-30 exhibited significant resistance to both trypsin and papain. Its antibacterial activity was retained after treatment with these two enzymes, with the residual activity after trypsin treatment reaching as high as 90.23%, indicating that this bacteriocin has strong resistance to protease hydrolysis.

[0115] 2.5. Inhibitory effect of bacteriocin Y-30 on Listeria monocytogenes ATCC 19114 and its safety assessment.

[0116] The minimum inhibitory concentration (MIC) of bacteriocin Y-30 against Listeria monocytogenes ATCC 19114 was 64 μg / mL. Qualitative hemolysis tests showed that bacteriocin Y-30 did not induce hemolysis (no clear zone around colonies) within the concentration range of 1×MIC to 16×MIC (i.e., 64 μg / mL to 1024 μg / mL). Notably, when the concentration was increased to 32×MIC (2048 μg / mL), an inhibitory halo appeared around the colonies, but this phenomenon was judged to be non-hemolytic. In conclusion, bacteriocin Y-30 demonstrated good safety across the entire tested concentration range.

[0117] The quantitative hemolysis test results further support the above conclusions. As the concentration of bacteriocin Y-30 increased from 1×MIC to 32×MIC, its hemolysis rate gradually increased from 0.5% to 1.79% (specifically: 1×MIC: 0.5%; 2×MIC: 0.71%; 4×MIC: 0.89%; 8×MIC: 1.09%; 16×MIC: 1.34%; 32×MIC: 1.79%). As a control, the hemolysis rate of bacteriocin Nisin A, which is generally considered to be generally safe (GRAS), was less than 2%. It is noteworthy that even at the highest concentration used in this experiment (32×MIC), the hemolysis rate of bacteriocin Y-30 (1.79%) was still lower than that of Nisin A, indicating that the concentration required to induce hemolysis is much higher than the concentration at which it exerts its antibacterial activity. According to the criteria for hemolysis testing (0-5% hemolysis rate is considered non-toxic), the above results fully demonstrate that bacteriocin Y-30 has good safety and the potential to be developed into a novel biological food preservative.

[0118] 2.6 Antibacterial spectrum of bacteriocin Y-30.

[0119] This invention uses the minimum inhibitory concentration (MIC) method to evaluate the antibacterial activity of bacteriocin Y-30. The results (as shown in Table 2) indicate that bacteriocin Y-30 exhibits potent antibacterial activity against both Gram-positive and Gram-negative bacteria, but shows no inhibitory effect against fungi. In contrast, Nisin, used as a control, has a relatively narrow antibacterial spectrum, with its activity limited to Gram-positive bacteria.

[0120] Table 2 Antibacterial spectrum of bacteriocin Y-30 Note: In Table 2, - indicates no inhibitory activity. Each well has a diameter of 6 mm, and 50 μL of bacteriocin Y-30 was added to each well.

[0121] 2.7 Mechanism of action of bacteriocin Y-30 against Listeria monocytogenes.

[0122] 2.7.1 Inhibitory effect of bacteriocin Y-30 on Listeria monocytogenes.

[0123] The minimum inhibitory concentrations (MICs) of bacteriocin Y-30 and Nisin A against Listeria monocytogenes were 64 μg / mL and 16 μg / mL, respectively, indicating that their antibacterial activities against this bacterium are on the same order of magnitude.

[0124] Results of bactericidal kinetic experiments ( Figure 4 This further confirmed the above observations. Compared with the 1 / 2×MIC treatment group, the 1×MIC treatment group showed complete inhibition of bacterial growth, as evidenced by the optical density (OD)... 600 No significant increase was observed during the 12-hour incubation period. Specifically, after treatment with 1×MIC of Nisin or bacteriocin Y-30, the colony count rapidly decreased to 1 log within 120 minutes. 10 Below CFU. Notably, when the concentration of bacteriocin Y-30 was increased to 2×MIC, all Listeria monocytogenes were completely inactivated within 90 min.

[0125] The above results indicate that bacteriocin Y-30 has rapid and long-lasting antibacterial activity against Listeria monocytogenes, and this activity is time- and dose-dependent.

[0126] 2.7.2 Effect of bacteriocin Y-30 on the permeability of Listeria monocytogenes membrane.

[0127] The effect of bacteriocin Y-30 on the cell membrane permeability of Listeria monocytogenes, such as Figure 5 As shown in the figure, compared with the control group, the concentrations of nucleic acids and proteins in the extracellular fluid of bacteria showed an overall increasing trend after treatment with 1×MIC bacteriocin Y-30. When the treatment concentration was increased to 2×MIC, similar but more significant changes were observed. These results indicate that bacteriocin Y-30 can severely disrupt the cell membrane integrity of Listeria monocytogenes, leading to leakage of intracellular proteins and DNA, and this effect shows a clear dose-dependent relationship.

[0128] 2.7.3 Effects of bacteriocin Y-30 on the cell structure of Listeria monocytogenes.

[0129] Scanning electron microscopy (SEM) observation results Figure 6This study revealed the destructive mechanism of bacteriocin Y-30 on Listeria monocytogenes. Compared with the smooth and intact control group, bacteria treated with 1×MIC bacteriocin Y-30 exhibited typical damage characteristics: surface shrinkage and intercellular adhesion; at the ultrastructural level, the cytoplasm became irregular, the cell wall underwent degenerative changes, the membrane boundary disappeared, and the overall structure was severely damaged. These results confirm that bacteriocin Y-30 inhibits the target bacteria by inducing changes in cell morphology and ultrastructural damage.

[0130] 2.7.4 Interaction between bacteriocin Y-30 and Listeria monocytogenes cell DNA.

[0131] The interaction between bacteriocin Y-30 and Listeria monocytogenes ATCC 19114 DNA was verified using gel retardation experiments and molecular docking simulations. Results are shown below. Figure 7 .

[0132] Gel retardation assay: As the concentration of bacteriocin Y-30 increased, the fluorescence intensity in the sample wells gradually increased, while the brightness of the DNA bands continuously decreased until they disappeared completely. When the concentration reached 256 μg / mL, all DNA was trapped in the sample wells, and no DNA band migration was observed, confirming that bacteriocin Y-30 can directly bind to the target DNA.

[0133] Molecular docking simulation: Analysis based on 100 successful docking results showed that bacteriocin Y-30 preferentially binds to the major groove of DNA rich in AG bases, with specific binding sites involving G4, A5, A6, G16, A17 and T19 bases.

[0134] The above results not only confirm that bacteriocin Y-30 can bind to the genomic DNA of Listeria ATCC 19114 in vitro, but also suggest that bacteriocin Y-30 may enter the cell and interact with intracellular substances.

[0135] 2.8. Preservation of strawberries.

[0136] 2.8.1 Microbiological analysis.

[0137] The effect of bacteriocin Y-30 combined with chitosan on the inhibitory effect of microorganisms in strawberries is as follows: Figure 8 As shown.

[0138] Listeria monocytogenes count: Throughout the storage period, the colony counts in all treatment groups (A1–A5) were significantly lower than those in the control group. Except for groups A4 and A5, the colony counts in the other treatment groups increased with prolonged storage time. Notably, the colony counts in groups A4 and A5 dropped to 0 CFU / g on day 6. Using 5 log CFU / g as the standard threshold, the control group, groups A1, A2, and A3 exceeded this limit on days 9, 12, 9, and 15, respectively.

[0139] Total bacterial count: TVC increased with prolonged storage time in all groups, and the control group consistently had higher TVC levels than all treatment groups throughout the storage period. On day 9, the TVC levels of the control group, A1, A2, and A3 groups all exceeded the microbial safety limits. In contrast, the TVC levels of groups A4 and A5 were significantly lower than the standard threshold throughout the storage period (P<0.05). On day 15 of storage, the TVC levels of groups A4 and A5 were 4.84 log CFU / g and 4.28 log CFU / g, respectively, representing decreases of 3.56 log CFU / g and 4.12 log CFU / g compared to the control group.

[0140] The above results indicate that the synergistic formulation of bacteriocin Y-30 with 0.5% or 1.0% chitosan can effectively inhibit the growth and reproduction of microorganisms and block the spread of foodborne pathogens (especially Listeria monocytogenes).

[0141] 2.8.2 Sensory analysis.

[0142] The sensory evaluation results of the strawberry samples (including color, aroma, and morphological characteristics) are shown in Table 3. On day 0, all samples exhibited a uniform bright red color, characteristic aroma, no water stains, and fresh calyxes without signs of wilting. During storage, the sensory scores of the control group, A1 group, A2 group, and A3 group all decreased significantly. P < 0.05 The control group showed the most significant decrease in shelf life. In contrast, the sensory scores of groups A4 (bacteriocin Y-30 + 0.5% chitosan) and A5 (bacteriocin Y-30 + 1.0% chitosan) remained at 6 or higher throughout the 15-day storage period. These results indicate that the combination of bacteriocin Y-30 with 0.5% or 1.0% chitosan can effectively extend the shelf life of strawberries.

[0143] Table 3 Sensory evaluation of strawberry samples during storage at 4℃ Note: Significant differences in values ​​within the same column ( P < 0.05 Those marked with different lowercase letters are significantly different within the same row. P < 0.05(These are marked with different capital letters.)

[0144] 2.8.3 Analysis of decay and weight loss rate.

[0145] Using strawberry rot rate as an indicator to monitor preservation performance during storage provides a direct reflection of fruit quality. For example... Figure 9 As shown in (a), the decay rate of the control group and all treatment groups gradually increased with prolonged storage time. Notably, the decay rate of the control group was consistently higher than that of the treatment groups throughout the storage period. On day 15, the decay rates of group A4 and group A5 were 24.37% and 19.84%, respectively, significantly lower than those of the other treatment groups.

[0146] The trend of weight loss rate is as follows Figure 9 As shown in (b), the weight loss rate of all strawberry samples increased with prolonged storage time. However, all treatment groups exhibited a slower rate of water loss, with less weight loss than the control group. On day 15, the weight loss rates of groups A4 and A5 were 21.83% and 19.26%, respectively, which were 19.92% and 22.49% lower than the control group.

[0147] The above results indicate that this compound formulation effectively extends the shelf life of strawberries by slowing down moisture loss and inhibiting decay.

[0148] 2.8.4 Color and hardness analysis.

[0149] Color is one of the key indicators for evaluating strawberry quality. For example... Figure 9 As shown in (c), the L* values ​​of all samples decreased with prolonged storage time, and the color difference between the control group and each treatment group gradually increased. Among them, the color difference between groups A4 and A5 and the control group was the most significant.

[0150] Regarding firmness, the firmness of the fruit in all groups gradually decreased throughout the storage period. Figure 9 (d). However, the samples treated with the bacteriocin-chitosan combination showed a slower softening rate. By day 15, the fruit firmness of groups A4 and A5 was 46.74% and 49.48% higher than that of the control group, respectively. This indicates that the combination treatment can effectively inhibit fruit softening.

[0151] 2.8.5 Analysis of soluble solids and acidity.

[0152] The trends of changes in soluble solids and acidity are as follows: Figure 9 As shown. Regarding soluble solids ( Figure 9(e) The content of soluble solids in all strawberry samples showed an increasing trend, with the control group showing a significantly faster growth rate than the treatment groups. On day 15, the soluble solids content of group A4 and group A5 was 11.53% and 11.27%, respectively, both lower than that of the control group and groups A1, A2, and A3.

[0153] Regarding titratable acidity ( Figure 9 (f) The content of all samples showed a gradual decreasing trend throughout the storage period, with group A5 exhibiting the slowest rate of decrease. On day 15, the titratable acid content of group A5 was 0.46%, which was 0.17 percentage points higher than that of the control group, demonstrating the best flavor retention effect.

[0154] The technical features described in the above embodiments can be combined arbitrarily. For the sake of simplicity, this specification does not list all possible combinations. However, any combination of these technical features that does not substantially contradict each other should be considered within the scope of this specification.

[0155] The above-described embodiments are merely illustrative of several specific implementations of the present invention, and while the descriptions are quite detailed, they should not be construed as limiting the scope of protection of this invention. It should be noted that those skilled in the art can make various modifications, improvements, or equivalent substitutions without departing from the concept of the present invention, and these all fall within the scope of protection of this invention.

Claims

1. A broad-spectrum antibacterial bacteriocin Y-30, characterized in that, The amino acid sequence of the bacteriocin Y-30 is shown in SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the bacteriocin Y-30 as described in claim 1.

3. A preservative, characterized in that, The preservative comprises bacteriocin Y-30 as described in claim 1 or the nucleic acid molecule as described in claim 2; The preservatives also include chitosan.

4. The preservative as described in claim 3, characterized in that, The preparation method of the preservative is as follows: Chitosan was dissolved in 1% glacial acetic acid to prepare a chitosan solution with a mass fraction of 0.5% to 2.0%. The preservative is obtained by dissolving bacteriocin Y-30 in chitosan solution to achieve a final concentration of bacteriocin Y-30 of 64 μg / mL to 256 μg / mL.

5. The preservative as described in claim 4, characterized in that, The preservatives also include food-grade additives.

6. The application of bacteriocin Y-30 according to claim 1, the nucleic acid molecule according to claim 2, and / or the preservative according to claim 3, characterized in that, The application refers to any one of the following: 1) Preparation of formulations for inhibiting Gram-positive bacteria; 2) Preparation of formulations for inhibiting Gram-negative bacteria; 3) Prepare formulations for extending the shelf life of fruits.

7. The application as described in claim 6, characterized in that, The Gram-positive bacteria include at least one of Listeria monocytogenes, Micrococcus luteus, and Bacillus.

8. The application as described in claim 6, characterized in that, The Gram-negative bacteria include at least one of Escherichia coli, Pseudomonas aeruginosa, and Salmonella enteritidis.

9. The application as described in claim 6, characterized in that, The fruit includes at least one of strawberries, apples, and pears.

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