A plant lactobacillus 9, a compound preservative containing plant lactobacillus 9 and its application

Abstract

This invention relates to the field of food antibacterial and preservative technology, specifically to a plant lactobacillus 9, a compound preservative containing plant lactobacillus 9, and their applications. The plant lactobacillus 9 is produced from *Lactobacillus plantarum* with the preservation number CCTCC NO: M 2026808. Lactobacillus plantarum The amino acid sequence of this plant lactobacillus 9 is N-Thr-Asp-Gly-Ala-Ala-Phe-OH. This plant lactobacillus 9 exhibits strong antibacterial activity against Listeria monocytogenes, Bacillus subtilis, Micrococcus luteus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas fluorescens, Salmonella typhimurium, and Pseudomonas aeruginosa, and demonstrates outstanding stability. It is a safe and reliable biological preservative. Its effectiveness is significantly enhanced when combined with chitosan, making it a potential alternative to chemical preservatives for controlling foodborne pathogens in the agricultural and food industries and extending the shelf life of meat and meat products.

Description

Technical Field

[0001] This invention relates to the field of food antibacterial and preservative technology, specifically to a plant lactobacillus 9, a composite preservative containing plant lactobacillus 9, and their applications. Background Technology

[0002] Listeria monocytogenes is an important foodborne pathogen that can survive under various environmental conditions, including extreme pH, low temperature, high salinity, and dryness. This bacterium spreads to humans through contaminated fruits, seafood, meat, dairy products, and other food items, causing serious and even fatal infections. It poses a particularly serious threat to the elderly, children, pregnant women, and those with weakened immune systems. Therefore, controlling the spread of foodborne pathogens, especially Listeria monocytogenes, is urgently needed.

[0003] Currently, the food industry widely uses chemical preservatives to control microbial contamination, but these pose potential health risks and may also reduce the nutritional value and sensory quality of food. Therefore, the need to develop natural biological preservatives is becoming increasingly urgent.

[0004] Bacteriocins are bioactive antimicrobial peptides or proteins synthesized by bacterial ribosomes and are generally considered safe substances. Among them, bacteriocins produced by *Lactobacillus plantarum* are characterized by biodegradability, lack of drug resistance, and low hemolytic activity. In recent years, numerous studies have confirmed that bacteriocins can inhibit the growth of pathogenic bacteria in meat products, dairy products, and fruits and vegetables. However, currently only nisin has been approved for commercial use by the U.S. Food and Drug Administration. This bacteriocin has a narrow antibacterial spectrum, effective only against Gram-positive bacteria, and lacks antibacterial activity under alkaline conditions, thus limiting its application. Therefore, there is an urgent need to develop novel bacteriocins with broad-spectrum antibacterial activity, wide pH adaptability, and high thermal stability, while simultaneously elucidating their mechanisms of action against pathogenic bacteria, laying the foundation for the approval and industrial application of novel bacteriocins. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a plant lactobacillus 9, a composite preservative containing plant lactobacillus 9, and their applications.

[0006] The present invention specifically adopts the following technical solution: In a first aspect, the present invention provides a plant lactobacillus 9 with the amino acid sequence N-Thr-Asp-Gly-Ala-Ala-Phe-OH.

[0007] Most bacteriocins have a molecular weight greater than 2 kDa. Compared to conventional bacteriocins, small molecule bacteriocins have become a research hotspot due to their unique biological characteristics and mechanisms of action. Representative small molecule bacteriocins include Lactobacillus plantarum GZ1-27 (975 Da), Lactobacillus plantarum W3-2 (618.26 Da), and bacteriocin JS17 (652.37 Da). Significantly different from large molecule bacteriocins, small molecule bacteriocins typically cannot form classic pore structures on bacterial cell membranes. Their potential antibacterial pathways mainly involve: specifically binding to cell wall or cell membrane precursors, inhibiting cell wall and cell membrane synthesis, and disrupting bacterial structural integrity; they can also penetrate the bacterial cell to enter the intracellular space, interacting with functional enzymes and genomic DNA, thereby interfering with normal bacterial physiological metabolic processes. Currently, the exact molecular mechanisms of action of these small molecule bacteriocins still require further investigation and elucidation.

[0008] This invention isolates and screens *Lactobacillus plantarum* strain 9 from fermentation mash, and then prepares a novel class IId bacteriocin, named *Lactobacillus plantarum*in 9, with a molecular weight of only 581 Da. This bacteriocin possesses broad-spectrum antibacterial activity, wide pH adaptability, and excellent thermal stability, effectively inhibiting various Gram-positive bacteria (*Listeria monocytogenes*, *Bacillus subtilis*, *Micrococcus luteus*, and methicillin-resistant *Staphylococcus aureus*) and Gram-negative bacteria (*Escherichia coli*, *Pseudomonas fluorescens*, *Salmonella typhimurium*, and *Pseudomonas aeruginosa*).

[0009] Secondly, the present invention provides a *Lactobacillus plantarum* that produces the aforementioned *Lactobacillus plantarum* 9. Lactobacillus plantarum The *Lactobacillus plantarum* is deposited at the China Center for Type Culture Collection (CCTCC), with accession number M2026808 and accession date April 24, 2026.

[0010] Thirdly, the present invention provides a gene encoding the plant lactobacillus 9.

[0011] Fourthly, the present invention provides the use of the plant lactobacillus 9, the plant lactobacillus, or the gene in the preparation of antibacterial and / or preservative products.

[0012] Furthermore, the antibacterial effect includes the inhibition of Gram-positive bacteria and / or Gram-negative bacteria.

[0013] Furthermore, the Gram-positive bacteria include Listeria monocytogenes (… Listeria monocytogenes Bacillus subtilis ( Bacillus subtilis Micrococcus luteus ( Micrococcus luteus), methicillin-resistant Staphylococcus aureus (MRSA) Staphylococcus aureus MRSA).

[0014] Furthermore, the Gram-negative bacteria include Escherichia coli (Escherichia coli). Escherichia coli ), Fluorescent Pseudomonas ( Pseudomonas fluorescens Salmonella typhimurium ( Salmonella typhimurium ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ).

[0015] Fifthly, the present invention provides a composite preservative, which is composed of the aforementioned plant lactobacillus 9, chitosan and water; wherein the concentration of plant lactobacillus 9 is 32 μg / mL and the mass fraction of chitosan is 0.5%~1.0%.

[0016] Sixthly, the present invention provides the application of the composite preservative in inhibiting pathogens in food, delaying food spoilage, and extending the shelf life of food.

[0017] Furthermore, the food products include meat products.

[0018] Furthermore, the application method involves soaking meat products in the composite preservative.

[0019] The beneficial effects of this invention are: The plant-derived lactobacillus 9 of this invention exhibits strong antibacterial activity against Listeria monocytogenes, Bacillus subtilis, Micrococcus luteus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas fluorescens, Salmonella typhimurium, and Pseudomonas aeruginosa, with outstanding stability: 77.98% activity is retained after treatment at 121℃ for 20 min, >88% activity is retained after storage at 4℃ for 120 days, and ≥70.24% activity is retained at pH 2-9. Compared to nisin, which is inactivated under neutral / alkaline conditions, it has superior thermal stability and pH adaptability. This bacteriocin can be degraded by pepsin, proteinase K, and papain, and is non-hemolytic (within 32 × MIC), making it safe and reliable as a biological preservative.

[0020] The antibacterial activity of plant lactobacillus 9 of the present invention is concentration- and time-dependent. It exerts its effect by adsorbing onto the target cell membrane, forming pores, causing cytoplasmic leakage and morphological changes. At the same time, it selectively binds to the genomic DNA of sensitive bacteria and targets the AT enrichment groove, forming a dual antibacterial mechanism of membrane disruption + DNA binding, which is the unique mode of action of this bacteriocin.

[0021] Chicken breast preservation trials confirmed that *Lactobacillus plantarum* 9, used alone, can inhibit microbial growth during storage, and its effect is significantly enhanced when combined with chitosan. Treatment with 1 × MIC *Lactobacillus plantarum* 9 + 1.0% chitosan significantly reduced the total bacterial count in chicken breast throughout the entire process, completely inactivating *Listeria monocytogenes* within 3 days, effectively controlling foodborne pathogens. This compound system also significantly improved quality indicators of chicken breast such as pH, TVB-N, brightness, and storage loss rate. Sensory evaluation showed that the shelf life was extended to 15 days, with a preservation effect superior to nisin.

[0022] In summary, the composite preservative of this invention is a highly promising composite natural preservative that can replace chemical preservatives, control foodborne pathogens in the agricultural and food industry, and extend the shelf life of meat and meat products. Attached Figure Description

[0023] Figure 1 Characteristic analysis of strain 9: (a) colony morphology, (b) Gram staining results, (c) phylogenetic tree.

[0024] Figure 2 To investigate the effect of enzyme treatment on the antibacterial activity of *Lactobacillus plantarum* 9 supernatant; (a) proteinase K, (b) pepsin, (c) papain; *Listeria monocytogenes* was used as the indicator bacterium. 1: 45 μL *Lactobacillus plantarum* 9 supernatant (pH 6.0) + 5 μL proteinase K solution (1 mg / mL, final volume ratio 9:1); 2: 45 μL *Lactobacillus plantarum* 9 supernatant (pH 6.0) + 5 μL pepsin solution (1 mg / mL, final volume ratio 9:1); 3: 45 μL *Lactobacillus plantarum* 9 supernatant (pH 6.0) + 5 μL papain solution (1 mg / mL, final volume ratio 9:1). Control group (CK): 45 μL *Lactobacillus plantarum* 9 supernatant (pH 6.0) + 5 μL sterile water.

[0025] Figure 3 The growth curve and antibacterial activity curve of *Lactobacillus plantarum* 9 were obtained, with *Listeria monocytogenes* ATCC 19114 as the indicator bacterium.

[0026] Figure 4 Purification and characterization of plant lactobacillus 9. (a) RP-HPLC chromatogram, (b) LC-MS chromatogram, (c) LC-MS / MS chromatogram, (d) molecular weight determination by Tricine-SDS-PAGE.

[0027] Figure 5Qualitative analysis of the hemolytic activity of plant lactobacillus 9; the experimental treatment groups included: negative control group (phosphate-buffered saline, PBS), positive control group (1% Triton X-100), and plant lactobacillus 9 at 1, 2, 4, 8, 16, and 32 times the minimum inhibitory concentration (MIC).

[0028] Figure 6 The effect of plant lactobacillus 9 on the growth curve (a) and bactericidal curve (b) of Listeria monocytogenes ATCC 19114.

[0029] Figure 7 The effects of plant lactobacillus 9 on nucleic acid and protein leakage and cell membrane integrity in Listeria monocytogenes ATCC 19114 cells; (a) nucleic acid leakage; (b) protein leakage; (c) DHPCA buffer treatment (control group); (d) 1×MIC plant lactobacillus 9 treatment; (e) 2×MIC plant lactobacillus 9 treatment.

[0030] Figure 8 The effects of plant lactobacillusin 9 on the morphology and ultrastructure of Listeria monocytogenes ATCC 19114 were investigated. Note: Cells treated as follows are shown in the images for each group: (a, d) DHPCA buffer (control group), (b, e) 1×MIC plant lactobacillusin 9, (c, f) 2×MIC plant lactobacillusin 9. Cell morphological changes (ac) and cell ultrastructural changes (df) were observed respectively.

[0031] Figure 9 (a) Agarose gel electrophoresis showing DNA treated with DHPCA buffer (CK, control), 2×MIC lactobacillus 9 (lane 1), and 4×MIC lactobacillus 9 (lane 2); (b, c) Molecular model of the interaction between lactobacillus 9 and DNA.

[0032] Figure 10The effects of Lactobacillus plantarum 9 alone and in combination with chitosan on the number of Listeria monocytogenes ATCC 19114 colonies and total viable count (TVC) in chicken breast during storage; (a) Listeria monocytogenes ATCC 19114 colony count; (b) TVC. Treatment groups: control group (sterile water control group), T1 group (1×MIC Lactobacillus plantarum 9 alone), T2 group (0.5% chitosan alone), T3 group (1.0% chitosan alone), T4 group (1×MIC Lactobacillus plantarum 9 + 0.5% chitosan combined), T5 group (1×MIC Lactobacillus plantarum 9 + 1.0% chitosan combined), and T6 group (1×MIC Nisin alone).

[0033] Figure 11 The effects of using Lactobacillus plantarum 9 alone and in combination with chitosan on pH, total volatile basic nitrogen (TVB-N) content, exhaust loss rate, and mild meat quality of chicken breast during storage were investigated. (a) pH; (b) TVB-N content; (c) exhaust loss rate; (d) mild meat quality. Treatment groups: control group (sterile water control), T1 group (1×MIC Lactobacillus plantarum 9 alone), T2 group (0.5% chitosan alone), T3 group (1.0% chitosan alone), T4 group (1×MIC Lactobacillus plantarum 9 + 0.5% chitosan), T5 group (1×MIC Lactobacillus plantarum 9 + 1.0% chitosan) and T6 group (1×MIC Nisin alone). Detailed Implementation

[0034] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0035] Example 1: Obtaining the bacterial strain and isolating, identifying, and validating the antibacterial activity of bacteriocins. 1. Method 1.1 Strains and Culture Conditions Lactobacillus plantarum 9 was isolated from fermented mash samples from the Kouzijiao Old Town brewing workshop in Huaibei City, Anhui Province, and cultured statically in MRS broth at 37℃ for 48 h. Listeria monocytogenes ATCC 199114, preserved in the laboratory, was cultured with shaking at 37℃ and 180 rpm in LB broth. Solid culture media were prepared by adding 1.8% (w / v) agar to the corresponding broths.

[0036] 1.2 Isolation and purification of bacteriocinogens 1.2.1 Screening of bacteriocin-producing bacteria The mash sample was serially diluted tenfold with sterile 0.9% (w / v) sodium chloride solution. 100 μL of the appropriate dilution (10...) was then taken. -1 ~10 -7 The bacterial suspension was spread onto MRS agar plates containing 0.3% calcium carbonate and incubated at 37°C for 48 h. Single colonies were picked and inoculated into MRS broth for 48 h, and cell-free supernatant was collected. 50 μL of the neutralized cell-free supernatant was added to the wells of LB agar plates inoculated with Listeria monocytogenes. After incubation, inhibition zones were observed, and their diameters were measured to assess antibacterial activity. The initial positive strains were rescreened using a double-layer agar method. Indicator strains included Micrococcus luteus CMCC 28001, Listeria monocytogenes ATCC 19114, Pseudomonas fluorescens ATCC 49642, and Salmonella enteritidis CICC 21527.

[0037] 1.2.2 Organic Acid and Hydrogen Peroxide Elimination Test The cell-free supernatant was adjusted to pH 6.0 with 1 mol / L sodium hydroxide to neutralize the organic acids, with the untreated supernatant serving as a control. For the supernatant that still exhibited antibacterial activity after neutralization, a catalase solution (9:1 volume ratio) with a final concentration of 1 mg / mL was added. The control group received an equal volume of enzyme-free diluent. The antibacterial titer against Listeria monocytogenes ATCC 19114 was determined using the agar well diffusion method. Each experiment was repeated three times.

[0038] 1.2.3 Protease Sensitivity Assay Cell-free supernatants that still exhibited antibacterial activity after the above exclusion tests were treated with pepsin, proteinase K, and papain, respectively, incubated at 37°C for 1 h, and then heated at 100°C for 5 min to inactivate the enzymes. Untreated supernatants served as controls. Residual antibacterial activity against Listeria monocytogenes ATCC 19114 was determined using the agar well diffusion method, with each test repeated three times.

[0039] 1.3 Identification of strain 9 Strain 9, exhibiting significant antibacterial activity, was classified and identified using morphological, biochemical, and molecular biological methods. Gram staining combined with microscopic observation of morphological characteristics was performed. The 16S rDNA gene was amplified by PCR using universal primers 27F and 1492R, purified, and sequenced. The DNA was then compared with the GenBank database using BLASTN, and a neighbor-joining phylogenetic tree was constructed using MEGA 12 software to determine the strain's evolutionary position.

[0040] 1.4 Growth kinetics, acid production and bacteriocin synthesis characteristics of Lactobacillus plantarum 9 Activated *Lactobacillus plantarum* 9 was inoculated into 1 L of fresh MRS broth at a 1% (v / v) inoculation rate and incubated statically at 37°C for 48 h. 5 mL samples were taken every 4 h to measure the absorbance (OD) at 600 nm. 600 The antibacterial activity against Listeria monocytogenes ATCC 19114 was determined by centrifugation and the cell-free supernatant was collected. The diameter of the inhibition zone was measured at each time point, and the experiment was repeated three times.

[0041] 1.5 Purification of bacteriocins *Lactobacillus plantarum* 9 was inoculated into 2 L of MRS broth and incubated at 37 °C for 48 h. The culture was then centrifuged at 10,000 rpm for 15 min to obtain a cell-free supernatant. The supernatant was extracted with an equal volume of ethyl acetate, and the organic phase was collected. The supernatant was concentrated by rotary evaporation at 35 °C, and purified by reversed-phase high-performance liquid chromatography (RP-HPLC) after scanning the entire wavelength range. A C18 column was used, with a linear gradient elution of acetonitrile (containing 0.1% trifluoroacetic acid) from 5% to 95% for 43 min at a flow rate of 0.6 mL / min. Each elution peak was collected, and after solvent removal, the antibacterial activity was detected by agar diffusion method to determine the purified active component.

[0042] 1.6 Identification of bacteriocins The molecular weight of the purified bacteriocin was estimated using Tricine-SDS-PAGE. The target active component was identified using matrix-assisted laser desorption / ionization time-of-flight tandem mass spectrometry, the amino acid composition was determined using an automated amino acid analyzer, and the primary structure (amino acid sequence) of the active substance was resolved using an automated protein sequencer.

[0043] 1.7 Antibacterial activity of plant lactobacillus 9 The MIC of plant lactobacillus 9 for indicator strains was determined using the broth microdilution method. Logarithmic growth phase indicator bacteria were collected, washed and resuspended in DHPCA buffer, and then diluted 100-fold in LB broth to prepare working bacterial suspensions. Plant lactobacillus 9 was serially diluted two-fold with DHPCA buffer. 100 μL of each dilution and 100 μL of the bacterial suspension were added to sterile 96-well plates. Nisin was used as a positive control, and DHPCA buffer as a negative control. The plates were incubated at 37°C for 24 h, and the OD was measured using a multi-mode microplate reader. 600 To assess bacterial growth, all experiments were repeated three times.

[0044] 1.8 Stability of plant lactobacillus 9 The stability of plant lactobacillusin 9 under different environmental conditions was systematically evaluated. Thermal stability: treatment at 40, 60, 80, and 100℃ for 30 min, and at 121℃ for 20 min; long-term thermal stability: storage at 4℃ for 120 days. pH stability: pH was adjusted to 2.0–9.0 with 1 mol / L sodium hydroxide or hydrochloric acid, incubated at 37℃ for 4 h, and then adjusted back to pH 4.0. Enzyme sensitivity: residual antibacterial activity was measured after mixing with proteinase K, pepsin, trypsin, and papain (final concentration 1 mg / mL), incubating at 37℃ for 4 h, with an untreated sample as a control. All tests were repeated three times.

[0045] 1.9 Hemolytic test of plant lactobacillus 9 Hemolysis tests were performed according to the method of Margalho et al. Blood agar plates were prepared by adding 7% (v / v) defibrinated sheep blood to the basal culture medium. 100 μL of plant lactobacillus 9 solution of different concentrations was added to the wells of the plates and incubated at 37°C for 24 h. Phosphate buffer was used as a negative control and 1% Triton X-100 was used as a positive control. The presence of hemolytic clear zones around the wells was observed.

[0046] 2. Results 2.1 Screening and Identification of Strain 9 Six strains with significant antibacterial activity were obtained through secondary screening, among which strain 9 exhibited the strongest antibacterial activity (as shown in Table 1) and was selected as the strain for subsequent experiments. The colonies formed by this strain on MRS agar plates were milky white, raised, with a smooth, rounded surface and regular edges, exhibiting the typical colony morphology of lactic acid bacteria (see Table 1). Figure 1 a). Microscopic examination after Gram staining revealed that the strain was a Gram-positive bacillus (see...). Figure 1 b). 16S rRNA gene sequencing showed that the sequence of this strain had 99% similarity to the known strain *Lactobacillus plantarum* MKNK10 (GenBank accession number PV405243.1) (see [link to article]). Figure 1 c). Based on morphological and physiological characteristics and phylogenetic analysis, it was identified as *Lactobacillus plantarum*. The 16S rRNA gene sequence of this strain has been submitted and obtained GenBank accession number PZ151899.

[0047] Table 1. Antibacterial activity of the 6 strains obtained from the secondary screening 2.2 Identification of antimicrobial metabolites of Lactobacillus plantarum 9 Organic acid and hydrogen peroxide exclusion tests showed that cell-free supernatant treated with neutralized organic acids and catalase still exhibited a significant inhibition zone against Listeria monocytogenes, with no significant difference compared to the control group, indicating that organic acids and hydrogen peroxide are not the primary antibacterial substances. After digestion with proteinase K, trypsin, and papain, the antibacterial activity was completely lost, while the control group retained its activity. Figure 2 The antibacterial component was confirmed to be a protein, and it was named plantaricin 9.

[0048] 2.3 Growth, acid production and bacteriocin synthesis characteristics of Lactobacillus plantarum 9 like Figure 3 As shown, strain 9 rapidly entered the logarithmic phase after inoculation into MRS liquid medium: 4–8 h was the pre-logarithmic phase, 8–16 h was the mid-logarithmic phase, and 16–20 h was the post-logarithmic phase; it entered the stationary phase after 20 h, with OD... 600 The maximum value reached 8.356. During fermentation, the pH value continuously decreased with the growth of the strain, from the initial value of 6.1 to 3.72 after 24 h, indicating that strain 9 continuously produced acid during metabolism. The synthesis of antimicrobial substances began after 8 h of culture, and the diameter of the inhibition zone reached a maximum of 21.48 mm at 40 h.

[0049] 2.4 Determination of molecular weight and amino acid sequence of plant lactobacillus 9 Reversed-phase high-performance liquid chromatography (RP-HPLC) yielded a single active peak after elution at 20.127 min, confirming the complete purification of plant lactobacillus 9. Figure 4 (a); Electrophoresis showed that its band was much lower than the 3.3 kDa label, indicating a small molecule antimicrobial peptide (a). Figure 4 The mass-to-charge ratio was 582.2195, corresponding to a molecular weight of 581 Da, as determined by tandem mass spectrometry (LC-MS / MS). Figure 4 b); Tandem mass spectrometry analysis of fragment ions ( Figure 4 (c) Combined with Edman degradation N-terminal sequencing, the amino acid sequence was determined to be N-Thr-Asp-Gly-Ala-Ala-Phe-OH, which was consistent with the amino acid composition analysis results. Comparison with the NCBI database showed that this sequence had no significant homology with known bacteriocins, indicating it is a novel class IId bacteriocin.

[0050] 2.5 Antibacterial spectrum and minimum inhibitory concentration (MIC) of plant lactobacillusin 9 As shown in Table 2, plant lactobacillus 9 has an inhibitory effect on both Gram-positive and Gram-negative bacteria; nisin has an inhibitory effect on Gram-positive bacteria, but no inhibitory effect on Gram-negative bacteria.

[0051] Table 2. Minimum inhibitory concentrations (MICs) of plant lactobacillus 9 against different indicator strains. Note: ATCC: American Type Culture Collection; CMCC: China Center of Medicine Culture Collection; CICC: China Center of Industrial Culture Collection; GIMCC: Guangdong Institute of Microbiology Culture Center; AS: Institute of Microbiology, Chinese Academy of Science; -: No inhibition.

[0052] 2.6 Stability and safety of plant lactobacillus 9 As shown in Table 3, plant lactobacillus 9 exhibits excellent thermostability: 82.97% activity was retained after treatment at 100℃ for 30 min, and 77.98% activity was retained after treatment at 121℃ for 20 min; 88.09% activity was retained after storage at 4℃ for 120 days. It also exhibits wide pH adaptability: ≥70.24% activity was retained at pH 2.0–9.0, and ≥93.87% activity was retained at pH < 6.0. Regarding enzyme sensitivity: it is stable to trypsin treatment (retaining 82.73% activity) and is completely inactivated by pepsin, proteinase K, and papain.

[0053] Table 3. Stability of Lactobacillus plantarum 9 a: The average of three parallel measurements.

[0054] b: Residual activity compared to the untreated control group.

[0055] c: CK control group: The sample storage conditions were 25 ℃, pH 4.0, and the concentration of plant lactobacillus 9 was 0.1 mg / mL.

[0056] d: Single hole diameter 6 mm.

[0057] Listeria monocytogenes ATCC19114 was used as the indicator strain in this experiment; in the agar diffusion test, 60 μL of purified plant lactobacillus 9 was added to each well.

[0058] Hemolytic test showed ( Figure 5 At concentrations of 1×~32× MIC, plant lactobacillus 9 showed no hemolytic zone, while the positive control showed a clear hemolytic zone, confirming that the bacteriocin is non-hemolytic and has good safety.

[0059] Example 2: The antibacterial mechanism of plant lactobacillusin 9 against Listeria monocytogenes 1. Method 1.1 Growth curve and time-sterilization curve The effect of plant lactobacillusin 9 on the growth kinetics of Listeria monocytogenes was investigated. Logarithmic-phase Listeria monocytogenes was inoculated into LB broth diluted to an appropriate concentration. 100 μL of the bacterial suspension was added to 96-well plates and incubated at 37°C for 2 h. Then, plant lactobacillusin 9 was added at final concentrations of 0.5×, 1×, and 2× MIC. The control group received an equal volume of DHPCA buffer. The plates were then incubated statically at 37°C for 12 h, and the OD values ​​were recorded. 600 Values ​​were measured three times.

[0060] The bactericidal kinetics of plant lactobacillusin 9 against Listeria monocytogenes were evaluated. Plant lactobacillusin 9 at 1× and 2× MIC concentrations was added to logarithmic-phase bacterial cultures. Samples of 1 mL were taken at 0, 15, 30, 60, 90, 120, 150, 180, 210, and 240 min, serially diluted tenfold, and spread onto LB agar plates. The plates were incubated at 37°C for 24 h, and bacterial counts were performed. A negative control was added with an equal volume of DHPCA buffer, and a positive control was treated with 1× MIC nisin (CAS#: 1414-45-5). All assays were repeated three times.

[0061] 1.2 Detection of cell membrane permeability and integrity The study determined the leakage of proteins and nucleic acids caused by cell membrane damage. Listeria monocytogenes in mid-logarithmic growth phase were collected, washed, and resuspended in DHPCA buffer. The samples were then treated with 1× and 2× MIC Lactobacillus plantarum 9 and incubated at 37°C for 0, 3, 6, 9, and 12 h. The control group received only DHPCA buffer. Samples were taken at each time point, centrifuged at 5000×g for 15 min at 4°C, and the supernatant was collected and filtered through a 0.22 μm sterile filter. The absorbance was measured using a UV spectrophotometer.

[0062] The damage to the cell membrane of Listeria monocytogenes caused by SYTO9 and propidium iodide staining combined with laser confocal microscopy was observed. Bacterial cells were treated with 1× and 2× MIC SYTO9 and incubated at 37℃ for 20 min. The cells were then collected by centrifugation and resuspended in DHPCA buffer. An equal volume of SYTO9 and propidium iodide was mixed, and 3 μL of the mixed dye was added to 1 mL of the bacterial suspension. The mixture was incubated at 37℃ in the dark for 30 min, and observed using laser confocal microscopy.

[0063] 1.3 Morphological observation using scanning electron microscopy and transmission electron microscopy Morphological changes in Listeria monocytogenes treated with plant lactobacillusin 9 were observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Logarithmic-phase cells were adjusted to OD. 600 =0.4, treated with 1× and 2× MIC plant lactobacillus 9 in a 37℃ water bath for 1 h, washed with phosphate buffer, centrifuged at 5000×g for 10 min at 4℃ to collect the cells, and fixed with 2.5% glutaraldehyde at 4℃ for 12~16 h.

[0064] Scanning electron microscopy (SEM) samples were dehydrated with gradient ethanol, treated with tert-butanol, critical point dried, and sputter-coated with gold. The changes in bacterial surface morphology were observed, with DHPCA buffer treatment serving as a control. Transmission electron microscopy (TEM) samples were dehydrated with gradient ethanol, infiltrated with acetone, embedded in epoxy resin, polymerized at 60℃ for 48 h, ultrathinly sectioned, and stained with uranyl acetate and lead citrate. The ultrastructural changes in the bacterial cells were observed, with the control group undergoing the same treatment.

[0065] 1.4 Interactions with genomic DNA To investigate the interaction between plant lactobacillusin 9 and Listeria monocytogenes genomic DNA. Genomic DNA was extracted from logarithmic-phase Listeria monocytogenes ATCC 19114, and its integrity was assessed by 1.2% agarose gel electrophoresis. The concentration was determined to be 192.5 ng / μL. DNA samples were mixed with 2× and 4× MIC plant lactobacillusin 9 and incubated at 37℃ for 1 h. DNA binding and structural modification were analyzed by gel migration assay.

[0066] 1.5 Molecular docking simulation Based on the results of gel migration assays, molecular docking simulations were performed using AutoDock 4.2 software, with Lamarck's genetic algorithm as the search strategy, to explore the binding mode and mechanism of action of plant lactobacillusin 9 with DNA. The B-DNA dodecamer sequence (PDB ID: 1BNA) from the protein database was used as the receptor model.

[0067] 2. Results 2.1 Growth inhibition and bactericidal activity 0.5×MIC plant lactobacillus 9 can inhibit the early growth of Listeria monocytogenes; 1×MIC completely inhibits growth, with OD within 12 h. 600 No significant increase; OD after 12 h of treatment with 2× MIC 600 Reduced to 0 ( Figure 6 (a).

[0068] The bactericidal activity was concentration- and time-dependent: 2×MIC treatment for 30 min resulted in a decrease of 3.9 lg CFU / mL in bacterial count, with no viable bacteria after 150 min; 1×MIC treatment for 90 min resulted in a decrease of 3.26 lg CFU / mL in bacterial count, with no viable bacteria after 210 min. Figure 6 (b).

[0069] 2.2 Disruption of cell membrane integrity and increased permeability like Figure 7 As shown in Figures ab, treatment with 1×MIC (32 μg / mL) of plant lactobacillus 9 significantly increased the concentrations of nucleic acid and protein in the bacterial supernatant compared to the control group, reaching a peak at 9 h; the effect was even more pronounced with 2×MIC (64 μg / mL), confirming that it disrupted cell membrane integrity, leading to leakage of intracellular substances. Laser confocal microscopy revealed that as the concentration of plant lactobacillus 9 increased, the bacterial cells changed from green to red, indicating a dose-dependent damage to the cell membrane, ultimately leading to cell death. Figure 7 ce).

[0070] 2.3 Scanning electron microscopy and transmission electron microscopy analysis The control group had smooth and flat cell membrane surfaces. Figure 8 a); 1× MIC treatment resulted in cell shrinkage and morphological changes ( Figure 8 b); 2× MIC treatment exacerbated bacterial cell membrane shrinkage, deformation, and damage ( Figure 8 c). Transmission electron microscopy showed that the control group had dense and uniform cytoplasm, and intact cell walls and cell membranes. Figure 8 d); 1× MIC treatment resulted in cell wall degradation, blurred membrane boundaries, and irregular intracellular structures ( Figure 8 (e); 2× MIC treatment resulted in cell wall rupture, cell membrane disintegration, cytoplasmic leakage, and heterogeneous contents. Figure 8 (f), confirming that bacteriocins enter the cell and interact with intracellular components.

[0071] 2.4 Interaction with Listeria monocytogenes DNA like Figure 9As shown in Figure a, with the increase of plant lactobacillus 9 concentration, the fluorescence intensity in the well increased, and the DNA band gradually weakened and disappeared; after treatment with 4× MIC (128 μg / mL), the DNA was completely retained in the well and no band was observed, confirming that it can effectively bind bacterial DNA. Figure 9 Consistent results were obtained in bc. Molecular docking simulations of 100 repetitions showed that plant lactobacillus 9 binds to the purine-rich region of the major groove of DNA, acting on the G16, A17, T7, A6, and A5 base pairs. Its binding ability to bacterial DNA was verified in vitro.

[0072] Example 3: The Preservative Effect of Plant Lactobacillus 9 Combined with Chitosan on Chicken Breast 1. Method 1.1 Sample Preparation and Experimental Design Fresh, skinless, boneless chicken breast was purchased from the supermarket and delivered to the laboratory within 30 minutes in an ice box. Fat and connective tissue were aseptically removed, and the meat was cut into uniform slices of 3×3 cm and 10±0.5 g each. Chitosan with a deacetylation degree of 95% was dissolved in 1% (v / v) glacial acetic acid solution and mixed with plant lactobacillus 9 to a final concentration of 1× MIC to prepare 7 treatment solutions: (1) sterile water (control group); (2) 1× MIC plant lactobacillus 9 alone (T1); (3) 0.5% chitosan alone (T2); (4) 1.0% chitosan alone (T3); (5) 1× MIC plant lactobacillus 9 + 0.5% chitosan (T4); (6) 1× MIC plant lactobacillus 9 + 1.0% chitosan (T5); (7) 1× MIC nisin (T6).

[0073] Chicken breast was divided into 14 groups. Seven groups were irradiated with ultraviolet light for 30 min, then immersed in a Listeria monocytogenes ATCC 19114 bacterial suspension for 10 min, drained, and then immersed in seven different preservative solutions at 4℃ for 10 min. The remaining seven groups were not irradiated with ultraviolet light or inoculated with bacteria, but were only immersed in the same preservative solution. All samples were placed in sterile homogenization bags and stored at 4℃. Quality and microbiological indicators were measured at 0, 3, 6, 9, 12, and 15 days.

[0074] 1.2 Determination of microbial quantity, pH value and TVB-N content Total bacterial count and Listeria monocytogenes count in chicken breast were determined using the plate count method: 10 g of sample was homogenized with 90 mL of sterile physiological saline, serially diluted tenfold, and 100 μL of the appropriate dilution was plated onto counting agar (total bacterial count) and PALCAM agar (Listeria monocytogenes), respectively, and incubated at 37℃ for 24 h for counting. pH value was directly measured from the homogenized sample; TVB-N content was determined by steam distillation. After filtration, the sample suspension was distilled using an automatic Kjeldahl nitrogen analyzer and titrated with 0.01 mol / L hydrochloric acid for quantification.

[0075] 1.3 Storage Loss Rate Determine the storage loss rate of chicken breast. Record the initial mass of the sample m1. On each sampling day, gently wipe the surface moisture of the sample with absorbent paper and weigh it m2. Calculate the storage loss rate according to the formula: Storage loss rate (%) = [(m1-m2) / m1]×100.

[0076] 1.4 Brightness Measurement The brightness (L value) of chicken breast was measured using a colorimeter. ， An L value of 0 represents black, and 100 represents white.

[0077] 1.5 Sensory Evaluation Ten professionally trained evaluators were recruited to conduct sensory evaluations of chicken breast based on color, texture, and aroma. A 10-point preference scale was used, with 10 points being the best (excellent), 0 points being the worst (very poor), and 5-10 points being the acceptable range.

[0078] 1.6 Data Analysis All experiments were repeated three times. Data are expressed as mean ± standard deviation. One-way ANOVA was performed using SPSS 20.0 software. Duncan's multiple range test was used to determine differences between groups. P < 0.05 was considered statistically significant.

[0079] 2. Results 2.1 Microbial Analysis like Figure 10 As shown in Figure a, during the storage period, except for groups T4 and T5, the number of Listeria monocytogenes increased in all groups, and the bacterial count in the treatment groups was consistently lower than that in the control group. No live bacteria were detected in group T4 on day 6 and in group T5 on day 3; the bacterial count in the control group exceeded the poultry standard limit (6 lg CFU / g) on ​​day 9. Figure 10As shown in b, the total bacterial count increased in all groups, with the highest in the control group. Groups T4 and T5 remained significantly below the standard limit throughout the process, with the total bacterial count on day 15 being 4.26 and 6.17 lg CFU / g lower than the control group, respectively. Groups T1, T2, T3, and T6 exceeded the standard limit on days 9, 15, 12, 15, and 12, respectively, confirming that the combination of plant lactobacillus 9 and 1.0% chitosan can effectively inhibit the proliferation of microorganisms and foodborne pathogens, delay the spoilage of chicken breast, and extend its shelf life.

[0080] 2.2 pH value analysis like Figure 11 As shown in Figure a, the pH of all groups increased during the storage period, with the control group showing a significantly higher pH than the treatment group. The control group, T1, T2, and T6 groups exceeded the pH standard limit of 6.6 on days 12, 15, 15, and 15, respectively; groups T4 and T5 remained within the acceptable range throughout the entire process. The pH increase in chicken breast meat was due to alkali production from protein degradation. The treatment group inhibited microbial proliferation, slowed protein decomposition, and delayed the pH increase.

[0081] 2.3 TVB-N Content Analysis like Figure 11 As shown in b, the initial TVB-N content of the samples ranged from 4.54 to 6.23 mg / 100 g, indicating excellent quality. During the storage period, the content in each group gradually increased, with the control group showing significantly higher levels than the treatment group. On day 15, the control group, T1, T2, and T6 groups all exceeded the standard limit of 20 mg / 100 g; while the T4 and T5 groups maintained low levels throughout the process, confirming that the compound treatment inhibited the growth of putrefactive bacteria and delayed protein decomposition.

[0082] 2.4 Storage Loss Rate like Figure 11 As shown in Figure c, the loss rate of each group gradually increased during the storage period, with the control group having the highest loss rate. On day 15, the loss rates of the control group, T1, T2, T3, T4, T5, and T6 groups were 11.53%, 9.38%, 10.45%, 8.26%, 5.98%, 5.01%, and 10.17%, respectively. The T5 group had the lowest loss rate and the best preservation effect.

[0083] 2.5 Brightness Measurement like Figure 11 As shown in d, the control group had high initial brightness, which decreased significantly during storage and the meat color gradually darkened; the treatment groups, especially T4 and T5, maintained high brightness throughout the process, confirming that the compound treatment can maintain the color stability of chicken breast and improve sensory quality.

[0084] 2.6 Sensory Analysis Sensory evaluation of chicken breast samples was conducted based on odor, color, and texture. The results are shown in Table 4. On day 0, the sensory scores of all groups were ≥9.77, which was completely acceptable. The scores gradually decreased during the storage period, with the control group showing the fastest decline. The control group, T1, T2, T3, and T6 groups dropped below the acceptable score of 5 on days 9, 12, and 12, respectively. The scores of T4 and T5 groups were >5 throughout the entire storage period, confirming that the combination of plant lactobacillus 9 with 0.5% and 1.0% chitosan can effectively extend the shelf life of chicken breast.

[0085] Table 4 Sensory evaluation of chicken breast samples during storage at 4°C Note: Control group: sterile water; T1: 1×MIC lactobacillus plantarum 9; T2: 0.5% calcium chloride; T3: 1.0% calcium chloride; T4: 1×MIC lactobacillus plantarum 9 + 0.5% calcium chloride; T5: 1×MIC lactobacillus plantarum 9 + 1.0% calcium chloride; T6: 1×nisin MIC value. Within the same column: different lowercase letters, P < 0.05; Within the same row: different uppercase letters, P < 0.05.

[0086] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0087] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

Claims

1. A plant lactobacillus 9, characterized in that, The amino acid sequence is N-Thr-Asp-Gly-Ala-Ala-Phe-OH.

2. A type of *Lactobacillus plantarum* producing the plant lactobacillus 9 of claim 1 (… Lactobacillus plantarum ), characterized in that, The *Lactobacillus plantarum* was deposited at the China Center for Type Culture Collection (CCTCC) with accession number M 2026808 and accession date April 24, 2026.

3. A gene encoding plant lactobacillus 9 as described in claim 1.

4. The use of the plant lactobacillus 9 of claim 1, the plant lactobacillus of claim 2, or the gene of claim 3 in the preparation of antibacterial and / or preservative products.

5. The application according to claim 4, characterized in that, The antibacterial effect includes the inhibition of Gram-positive bacteria and / or Gram-negative bacteria.

6. The application according to claim 5, characterized in that, The Gram-positive bacteria include Listeria monocytogenes (…). Listeria monocytogenes Bacillus subtilis ( Bacillus subtilis Micrococcus luteus ( Micrococcus luteus ), methicillin-resistant Staphylococcus aureus (MRSA) Staphylococcus aureus MRSA).

7. The application according to claim 5, characterized in that, The Gram-negative bacteria include Escherichia coli (Escherichia coli) Escherichia coli ), Fluorescent Pseudomonas ( Pseudomonas fluorescens Salmonella typhimurium ( Salmonella typhimurium ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ).

8. A composite preservative, characterized in that, It is composed of plant lactobacillus 9 as described in claim 1, chitosan and water; wherein the concentration of plant lactobacillus 9 is 32 μg / mL and the mass fraction of chitosan is 0.5%~1.0%.

9. The application of the composite preservative according to claim 8 in inhibiting pathogens in food, delaying food spoilage, and extending the shelf life of food.

10. The application according to claim 9, characterized in that, The food product includes meat products; the application method is to soak the meat products in the compound preservative.

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