Lactobacillus paracasei subsp. paracasei with antibacterial and fermentation improvement properties, its bacteriocin and applications

Abstract

The application belongs to the technical field of microbial fermentation, and provides Lactobacillus paracasei subsp. paracasei with bacteriostatic and fermentation improvement characteristics, as well as a bacteriocin and application thereof. The classification name of the Lactobacillus paracasei subsp. paracasei is Lactobacillus paracasei subsp. paracasei, which has been preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on June 25, 2025, the address of the preservation center is Building 59, 5th Floor, 100, Martyrs' Road, Guangzhou, and the preservation number is GDMCC No: 66588. The YCL29 bacteriocin produced by the application exhibits a significant inhibitory effect on Vibrio parahaemolyticus, and the starter prepared by the strain can be used to improve the flavor quality and metabolic regulation of fermented fish tea, thereby providing a new solution for developing a local starter with the functions of inhibiting raw material source pathogenic bacteria and regulating fermentation quality.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to Lactobacillus paracasei subsp. paracasei, which has antibacterial and fermentation-improving properties, as well as its bacteriocins and applications. Background Technology

[0002] Fish and other aquatic products are highly susceptible to contamination by pathogenic microorganisms such as Vibrio parahaemolyticus during harvesting, processing, and natural fermentation. As a typical Gram-negative halophilic bacterium, Vibrio parahaemolyticus is not only a major cause of diseases in aquaculture but also a key pathogen in causing foodborne acute gastroenteritis in humans. In traditional fish tea and other natural fermentation systems, if the pathogens carried by the raw materials are not effectively suppressed in the early stages of fermentation, it will not only pose serious food safety risks but also interfere with the natural succession of the microbial community, leading to technical problems such as uncontrollable fermentation processes, large fluctuations in product quality, and prominent undesirable flavors (such as fishy or ammonia-like odors).

[0003] Although antibiotics have been used for antibacterial purposes in recent years, the spread of drug resistance caused by antibiotic abuse and the formation of biofilms of pathogens have severely limited the effectiveness of traditional antibacterial methods in complex fermentation systems. Summary of the Invention

[0004] This invention aims to provide *Lactobacillus paracasei* subsp. paracasei with antibacterial and fermentation-improving properties, along with its bacteriocin and applications, to effectively address the problems existing in the prior art. The YCL29 bacteriocin developed in this invention exhibits significant inhibitory effects against *Vibrio parahaemolyticus*, providing a new solution for developing indigenous starter cultures that combine the functions of inhibiting pathogens from raw materials with regulating fermentation quality.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention provides a subsp. paracasei of Lactobacillus paracasei with antibacterial and fermentation-improving properties, classified as Lactobacillus paracasei subsp. paracasei, which was deposited on June 25, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCCNo: 66588.

[0007] This invention provides a bacteriocin derived from the above-mentioned Lactobacillus paracasei subsp. paracasei, the amino acid sequence of which is shown in SEQ ID NO.26; the molecular weight is 1308.6705 Da.

[0008] Preferably, the bacteriocin is prepared by the following method: fermentation of *Lactobacillus paracasei* subsp. *paracasei* to obtain a cell-free supernatant; extraction with organic reagents to obtain a crude bacteriocin extract; and finally separation and purification of the crude bacteriocin extract by ultrafiltration, gel chromatography, and reversed-phase high-performance liquid chromatography.

[0009] More preferably, the method specifically includes:

[0010] 1) Fermentation: The Lactobacillus paracasei subsp. paracasei was fermented in MRS liquid medium at 37 ℃ for 24 h to obtain fermentation broth. The fermentation broth was filtered through a 0.22 μm filter membrane to obtain cell-free supernatant.

[0011] 2) Organic extraction: After thoroughly mixing ethyl acetate and cell-free supernatant in a certain proportion, the mixture was allowed to stand and separate into layers. The organic phase and aqueous phase were then removed separately. The organic phase was then removed by rotary evaporation at 45 °C to obtain the crude extract of bacteriocin.

[0012] 3) Ultrafiltration: The crude extract of bacteriocins was purified by fractional ultrafiltration using ultrafiltration centrifuge tubes with molecular weights of 30 kDa, 10 kDa and 3 kDa.

[0013] 4) Gel chromatography: Based on the ultrafiltration results, dextran gel G-100 gel chromatography was used to further purify the ultrafiltration product obtained in step 3).

[0014] 5) Reversed-phase high-performance liquid chromatography (RP-HPLC) separation and purification: The purified components collected in step 4) were filtered through a microporous membrane and then detected by reversed-phase high-performance liquid chromatography (RP-HPLC) with a UV detector; wavelength: 280 nm; injection volume: 20 μL; flow rate: 0.5 mL / min; mobile phases were phase B: acetonitrile solution containing 0.1% trifluoroacetic acid and phase C: aqueous solution containing 0.1% trifluoroacetic acid.

[0015] The present invention also provides an antibacterial agent / fermenting agent comprising the above-mentioned Lactobacillus paracasei subsp. paracasei.

[0016] This invention also provides the application of the above-mentioned antibacterial agent / fermenting agent in improving the flavor quality and metabolic regulation of fermented fish tea.

[0017] Furthermore, the fermenting agent can effectively inhibit the growth of Vibrio spp. during the fermentation process of fish tea, optimize the microbial community structure, improve the balance of organic acids and amino acids in the product, significantly enhance pleasant flavors including fruit and nutty aromas, and reduce unpleasant flavors including sourness, ammonia, and fishiness.

[0018] The beneficial effects of this invention are as follows:

[0019] This invention isolated a strain of *Lactobacillus paracasei* subsp. YCL29 from Hainan fish tea, and subsequently purified a novel bacteriocin with a molecular weight of 1308.6705 Da and an amino acid sequence of AVSVGGVATNFRM (SEQ ID NO. 26). This bacteriocin exhibits good UV resistance and heat resistance (121 ℃, 30 min). Treatment with pepsin, trypsin, α-amylase, lipase, and papain showed no significant difference in antibacterial activity against *Vibrio parahaemolyticus*, indicating its high applicability and adaptability. Furthermore, this bacteriocin can achieve highly efficient inhibition of the source pathogen by disrupting cell membrane integrity and downregulating the expression of key virulence genes in *Vibrio parahaemolyticus*. The minimum inhibitory concentration (MIC) against *Vibrio parahaemolyticus* is 5.54 μg / mL, which is more effective than some previously reported antimicrobial peptide bacteriocins.

[0020] It is worth noting that this bacteriocin-producing strain, as a native fermentation agent, can effectively inhibit the growth of Vibrio spp. during fish tea fermentation, optimize the microbial community structure, increase the content of organic acids and amino acids in the product, significantly improve pleasant flavors such as fruit and nutty aromas, and reduce unpleasant sensory characteristics such as sourness, ammonia, and fishy smells, thereby increasing public acceptance of fish tea. This invention provides a new solution for developing native fermentation agents that combine the functions of inhibiting pathogens from raw materials and regulating fermentation quality, which is of great significance for ensuring the safety and quality stability of traditional fermented aquatic products. Attached Figure Description

[0021] Figure 1 Colony morphology (A) and Gram staining (B) of Lactobacillus paracasei subsp. YCL29 were characterized.

[0022] Figure 2 For the antibiotic susceptibility assessment of Lactobacillus paracasei subsp. paracasei YCL29, Figure A shows the susceptibility to ciprofloxacin, levofloxacin, vancomycin, and erythromycin, while Figure B shows the susceptibility to gentamicin, kanamycin, amikacin, ofloxacin, norfloxacin, and streptomycin.

[0023] Figure 3 Safety assessment of Lactobacillus paracasei subsp. YCL29.

[0024] Figure 4 Phylogenetic tree of Lactobacillus paracasei subsp. YCL29.

[0025] Figure 5 This is a complete genome map of bacteriocin YCL29.

[0026] Figure 6 This is a secondary mass spectrum of the peptide of bacteriocin YCL29.

[0027] Figure 7 This is a diagram illustrating the secondary structure of bacteriocin YCL29.

[0028] Figure 8 The effects of different factors on the antibacterial stability of bacteriocin YCL29 against Vibrio parahaemolyticus were investigated. A: The effect of ambient temperature on the antibacterial activity of bacteriocin; B: The effect of UV irradiation duration on the antibacterial activity of bacteriocin; C: The effect of protease on the antibacterial activity of bacteriocin.

[0029] Figure 9 This is a graph showing the minimum inhibitory concentration (MIC) of bacteriocin YCL29 against Vibrio parahaemolyticus.

[0030] Figure 10 The effect of bacteriocin YCL29 on the cell micromorphology of Vibrio parahaemolyticus.

[0031] Figure 11 The effect of bacteriocin YCL29 on the expression of virulence genes in Vibrio parahaemolyticus.

[0032] Figure 12 The changes in physicochemical and microbiological characteristics of *Lactobacillus paracasei* subsp. *paracasei* YCL29 starter culture during fish tea fermentation; A: pH value; B: Change in total acid content (g / L); C: Change in lactic acid bacteria count (log 10 CFU / g); D: Change in total bacterial count (log) 10 CFU / g).

[0033] Figure 13 Changes in nutritional and quality components and color of fermented fish tea made with Lactobacillus paracasei subsp. paracasei YCL29; A: Changes in organic acid content (g / L); B: Changes in amino acid content (g / L); C: Product brightness ( D: Product popularity ( E: Product yellowness ( ).

[0034] Figure 14 A dynamic evolution diagram of the dominant bacterial community in fish tea fermented with Lactobacillus paracasei subsp. paracasei YCL29 starter culture.

[0035] Figure 15 A comparative diagram of volatile flavor compounds in fish tea fermented with Lactobacillus paracasei subsp. paracasei YCL29 starter culture.

[0036] Figure 16 A graph showing the differences in aldehyde and alcohol content in fish fermented with Lactobacillus paracasei subsp. paracasei YCL29 starter culture.

[0037] Figure 17Sensory quality changes of fish tea fermented with Lactobacillus paracasei subsp. paracasei YCL29 starter culture; A: Electronic tongue radar image of fermented fish tea after 30 days; B: Electronic tongue evaluation heatmap of fermented fish tea after 30 days; C: Electronic nose radar image of fermented fish tea after 30 days; D: Electronic nose evaluation heatmap of fermented fish tea after 30 days. Detailed Implementation

[0038] The present invention will now be described in detail with reference to specific embodiments. The following specific embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any way.

[0039] Example

[0040] This embodiment mainly includes the following aspects:

[0041] I. Isolation and Purification of Lactic Acid Bacteria

[0042] Single colonies producing calcium-dissolving zones were picked using the dilution plate method, isolated and purified, and numbered YCL29. They were then stored at -80 °C.

[0043] II. Morphological identification and safety testing of lactic acid bacteria

[0044] Strain YCL29 was streaked onto MRS solid medium and incubated at 37 °C for 48 h. Colony morphology was observed and recorded. Single colonies were picked, Gram-stained, and observed under an oil immersion microscope. Using Staphylococcus aureus ATCC 6538 as a positive control and sterile PBS as a negative control, strain YCL29 was inoculated onto Columbia blood agar plates using the diffusion method, and hemolysis was observed. Antibiotic susceptibility of strain YCL29 was determined using the disk ring diffusion method.

[0045] 16S rRNA Molecular Biological Identification: Genomic DNA of strain YCL29 was extracted using a rapid DNA extraction kit (catalog number DP302-02, Beijing Tiangen Biotech). Using the extracted DNA as a template, PCR amplification was performed using universal bacterial primers: upstream primer 27F (5′-AGAGTTTGATCCTGGCTCAG-3′, SEQ ID NO.23) and downstream primer 1492R (5′-TACGGYTACCTTGTTACGACTT-3′, SEQ ID NO.24). The total amplification volume was 25 μL, containing 1.0 μL of DNA template, 1.0 μL each of 10 μmol / L upstream and downstream primers, 12.5 μL of Taq PCR premix, and 9.5 μL of ddH2O. The amplification program was set as follows: 94 ℃ pre-denaturation for 2 min, 94 ℃ denaturation for 1 min, 60 ℃ annealing for 1 min, 72 ℃ extension for 90 s, for 30 cycles, and a final extension at 72 ℃ for 10 s. Incubate at 4 ℃ for min; take the bands and send them to Shanghai General Biotechnology Co., Ltd. for sequencing. Perform BLAST homology comparison on the sequenced sequences in the NCBI GenBank database to draw a phylogenetic tree.

[0046] III. Isolation and purification of YCL29 bacteriocin

[0047] The bacteriocin of Lactobacillus paracasei subsp. paracasei YCL29 (hereinafter referred to as YCL29 bacteriocin) was isolated and purified by organic solvent extraction, ultrafiltration, gel chromatography, and reversed-phase high-performance liquid chromatography. The specific steps are as follows:

[0048] 3.1 Organic Extraction Method

[0049] Lactobacillus paracasei subsp. paracasei YCL29 was fermented in MRS liquid medium at 37 ℃ for 24 h to obtain fermentation broth. The fermentation broth was filtered through a 0.22 μm filter membrane to obtain cell-free supernatant. Ethyl acetate was thoroughly mixed with the cell-free supernatant at a ratio of 7:5, and the mixture was allowed to stand for separation. The organic phase and aqueous phase were taken out separately. The organic phase was rotary evaporated at 45 ℃ to remove ethyl acetate, thus obtaining the crude extract of bacteriocins.

[0050] 3.2 Purification by centrifugation and ultrafiltration

[0051] The crude bacteriocin extract was purified by fractional ultrafiltration using ultrafiltration centrifuge tubes with molecular weights of 30 kDa, 10 kDa, and 3 kDa.

[0052] 3.3 Purification by gel chromatography

[0053] Based on the ultrafiltration results, dextran gel G-100 gel chromatography was selected for further purification of the active component obtained in the previous step. 10.0 g of dextran gel G-100 powder was soaked overnight to allow for full swelling, and then packed into a column with phosphate buffer for 2 h to equilibrate. The sample loading volume was 4.0 mL, and the elution peak was collected at a flow rate of 1 mL / min.

[0054] 3.4 Reversed-phase high-performance liquid chromatography separation and purification

[0055] The collected purified fraction was filtered through a microporous membrane and then analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) with a UV detector; wavelength: 280 nm; injection volume: 20 μL; flow rate: 0.5 mL / min; mobile phase: phase B: acetonitrile (containing 0.1% trifluoroacetic acid) and phase C: water (containing 0.1% trifluoroacetic acid). Absorption peaks were collected in fractions according to elution time for identification of antibacterial activity and molecular weight.

[0056] IV. Determination of molecular weight of YCL29 bacteriocin

[0057] The precise molecular weight of the purified bacteriocin was determined using liquid chromatography-mass spectrometry; its secondary structure was analyzed by circular dichroism spectroscopy.

[0058] HPLC-MS / MS analysis: After reduction, alkylation, desalting, and vacuum drying, the bacteriocin extract was analyzed and identified for polypeptide molecular weight and amino acid sequence using HPLC-MS / MS. The column (150 μm id × 170 mm, packing: Reprosil-Pur 120 C18-AQ 1.9 μm) was equilibrated with 4% buffer B using buffer A (0.1% trifluoroacetic acid, 100% water) and buffer B (0.1% formic acid, 80% acetonitrile). Samples were loaded via an autosampler, and bacteriocin was eluted at a flow rate of 600 mL / min.

[0059] Circular dichroism detection: The secondary structure of the peptides was determined using a circular dichroism spectrometer. Peptide solutions were prepared using PBS to simulate bacterial membrane and water conditions. Analysis was performed at ambient temperature using a wavelength range of 190–260 nm, a scan rate of 100 nm / min, and a bandwidth of 1 nm.

[0060] V. Stability Study of YCL29 Bacteriocin

[0061] Different proteases (lipase, α-amylase, trypsin, pepsin, catalase, and papain) were selected and mixed at a final concentration of 1000 U / mL, then incubated at 37 °C for 3 h. YCL29 bacteriocin was placed under a 30W UV lamp, and samples were taken at 15 min, 30 min, 60 min, and 120 min. YCL29 bacteriocin was then treated at ambient temperatures of 40 °C, 60 °C, 80 °C, 100 °C, and 121 °C for 30 min, and then placed at room temperature. Untreated YCL29 bacteriocin served as the control group. The antibacterial activity of YCL29 bacteriocin was determined using the double-layer agar plate method.

[0062] VI. Determination of Minimum Inhibitory Concentration (MIC)

[0063] The minimum inhibitory concentration (MIC) of Vibrio parahaemolyticus was determined using the 96-well plate broth two-fold dilution method. LB broth was used as the diluent, and the plates were incubated at 37 °C for 12 h. The lowest bacteriocin concentration with turbidity similar to that of the control group was observed visually and taken as the MIC of YCL29 bacteriocin against Vibrio parahaemolyticus.

[0064] VII. Effects of YCL29 bacteriocin on the morphology of Vibrio parahaemolyticus

[0065] The Vibrio parahaemolyticus suspension was co-incubated with YCL29 bacteriocin (final concentrations of 0×MIC, 1 / 2×MIC, and 1×MIC) for 4 h. The bacterial pellet was collected, washed with PBS, and then resuspended in 2.5% glutaraldehyde and fixed at 4 °C. After discarding the supernatant, the sample was washed and dehydrated sequentially with 20%, 50%, 70%, 90%, 95%, and 100% ethanol in a gradient. Finally, the sample was dried at the cutoff point. The treated sample was fixed on a scanning electron microscope stage, sputter-coated with gold, and the morphological changes of the bacteria were observed.

[0066] VIII. Effects of YCL29 bacteriocin on the expression of virulence genes in Vibrio parahaemolyticus

[0067] The primer sequences are shown in Table 1.

[0068] Table 1 Primer sequence listing

[0069] The effect of YCL29 bacteriocin on the transcriptional levels of virulence-related genes in Vibrio parahaemolyticus was determined using RT-qPCR. Vibrio parahaemolyticus bacterial suspension was prepared, and bacteriocin was added to final concentrations of 0×MIC, 1 / 2×MIC, and 1×MIC. The suspension was then incubated at 37 °C for 4 h to allow for sufficient interaction between the bacteria and the bacteriocin. After centrifugation, the bacterial cells were washed with PBS, and the precipitate was collected. Total RNA was extracted using a bacterial total RNA extraction kit (Catalog No. R6950, Omega, USA). High-purity RNA samples were stored at -80 °C for later use. Reverse transcription was performed using a fourth-generation one-strand cDNA synthesis kit (HiScript IV 1st Strand cDNA Synthesis Kit (+gDNA wiper), Catalog No. R412, Novizan, Nanjing). Using recA as an internal reference gene, quantitative analysis of the target gene was performed using a real-time PCR instrument (Bio-Rad Laboratories, USA). The amplification reaction program was: 95℃ pre-denaturation for 3 min, followed by 40 cycles (95℃ for 10 s, 60℃ for 30 s). After amplification, the cycle number (Ct) value of each gene in different groups was analyzed, and a 2-1 time-varying PCR method was used. -ΔΔCt The relative expression levels of the target virulence genes were analyzed.

[0070] IX. Application of YCL29 bacteriocin

[0071] 9.1 Production process of fish tea fermentation

[0072] The experiment included two fermentation groups: a natural fermentation group (control group) and a YCL29 inoculum group (YCL29 group). Three-year-old, 5kg grass carp were slaughtered, gutted, washed, and cut into pieces. The pieces were marinated with 4% (w / v) salt and 3% (w / v) ginger for 2 hours. Wuchang rice was rinsed, steamed until cooked but not mushy, and cooled to room temperature. The marinated fish pieces and rice were layered in a 500mL glass jar and fermented at a constant temperature of 30℃ for 30 days to obtain the fish tea product.

[0073] Preparation of YCL29 fermentation spawn: YCL29 strain, preserved at -80 ℃, was continuously activated for two generations in MRS broth. The activated bacterial solution was inoculated into 50 mL of MRS broth and cultured at 37 ℃ and 160 rpm with shaking for 24 h. The bacterial cells were collected by centrifugation at 4 ℃ and 8000 rpm for 10 min. The cells were washed three times with sterile physiological saline solution and resuspended. The concentration of the bacterial suspension was adjusted to 10. 7 CFU / mL. A 5% (v / v) inoculum was uniformly added to the YCL29 group, while the control group was replaced with an equal volume of sterile physiological saline. Aseptic samples were collected at fermentation days 0 (initial), 3, 6, 12, 20, and 30 (endpoint), with three biological replicates per group. Samples were used for subsequent analysis.

[0074] 9.2 Determination of Fish Tea Indicators

[0075] 9.2.1 Determination of physicochemical properties

[0076] Color difference measurement: During the fermentation process, the fish pieces in the fish tea were cut into 1.5 cm × 1.5 cm × 1.5 cm samples to be tested. The color difference meter was used to measure the color change of the fish meat. Each sample group was tested in triplicate.

[0077] pH and total acid determination: Weigh 1 g of fermentation sample, add 4 mL of deionized water, vortex mix, centrifuge and collect the supernatant, and measure the pH value using a pH meter; the total acid was determined in accordance with the Chinese national standard GB12456-2021 "Determination of total acid in food".

[0078] Microbial colony counting: Lactic acid bacteria and total colony count were performed according to the plate dilution plating method. Weigh 1 g of fermentation sample, add 4 mL of sterile physiological saline, vortex, and perform 10-fold serial dilutions before plate colony counting.

[0079] Determination of free amino acids: Add 2 mL of sample to a centrifuge tube containing 15 mL of 0.05 g / mL trichloroacetic acid, vortex, sonicate, allow to stand at room temperature, centrifuge, and collect the supernatant. Adjust the pH to 2.0 with NaOH solution, bring to volume with ultrapure water, filter through an aqueous filter membrane, and determine by high performance liquid chromatography (HPLC). Use sodium citrate buffer (0.1 mol / L, pH 3.2-4.9) as the mobile phase.

[0080] Organic acid determination: Weigh 5 g of sample, add 20 mL of ultrapure water for homogenization, ultrasonically extract for 10 min, centrifuge, take the supernatant and filter it through a filter membrane, and quantitatively determine lactic acid, tartaric acid, malic acid, succinic acid and citric acid by high performance liquid chromatography.

[0081] 9.2.2 Microbiome and Flavor Metric Determination

[0082] Microbiome: 5 g of fish tea sample was collected during fermentation, added to 20 mL of sterile physiological saline, vortexed to mix, centrifuged, and the supernatant was discarded, leaving the precipitate as the enriched microbial sample. Total metagenomic DNA of the fermentation microbial community was extracted using the EZNA® Soil DNA Kit according to the manufacturer's instructions. The hypervariable region of the bacterial 16S rRNA gene V3-V4 was amplified using primers 338F and 806R. The purified PCR products were sequenced end-to-end on an Illumina NovaSeq 6000 sequencing platform according to the manufacturer's instructions.

[0083] Flavor group determination: Volatile components were extracted from the fish tea samples using headspace solid-phase microextraction (HS-SPME) and detected by gas chromatography-mass spectrometry (GC-MS). The measured mass spectra were compared with the NIST 17 standard mass spectral library, and the retention index (RI) was calculated using the C8-C39 n-alkanes series. These results were then compared with reported values ​​in the literature to complete the qualitative identification of the volatile components. Quantitative analysis was performed using 4-methyl-2-pentanol as an internal standard.

[0084] 9.2.3 Sensory evaluation

[0085] Electronic tongue: Weigh 5 g of fish tea sample, add distilled water, mix well, centrifuge, take the supernatant, dilute to volume, filter through a filter membrane, and then load onto the instrument. Use an alternating detection mode for sample and washing solution; sampling time is 120 s, and a stable signal between 100-120 s is used for analysis.

[0086] Electronic nose: This experiment used the Heracles II ultra-fast gas chromatography electronic nose system from Alpha MOS, France, equipped with two parallel metal capillary columns of different polarities (MXT-5 and MXT-1701) and a dual flame ionization detector (FID). The column heating rate reached 10 °C / s, and the average analysis time for each sample was about 5 min.

[0087] X. Statistics and Analysis

[0088] All data are expressed as mean ± standard deviation (X ± SD) of three independent replicates. All statistical analyses were performed using SPSS 22.0 (IBM SPSS Statistics, Armonk, NY, USA). Plotting was performed using Origin 2021 (Origin Lab, Northampton, MA, USA). One-way ANOVA was used to evaluate the statistical significance of differences among groups. Different letters indicate significant differences among parameters (p < 0.05), and the same letter indicates no significant differences among parameters (p > 0.05).

[0089] XI. Results and Analysis

[0090] 11.1 Identification and Safety Testing of Bacteriocin-Producing Lactic Acid Bacteria

[0091] Lactic acid bacteria strain identification: morphological identification and safety test results are as follows Figure 1 As shown in Figure 1A, the cells of strain YCL29 are rod-shaped, the colonies are milky white, opaque, with smooth edges, and a wrinkled and glossy surface. Figure 1Figure B shows a purple Gram stain, indicating a Gram-positive bacterium with the basic phenotype of lactic acid bacteria. As shown in Figures A and B of 2, strain YCL29 exhibits some sensitivity to levofloxacin, norfloxacin, ofloxacin, and erythromycin, but strong resistance to the other six antibiotics. Furthermore, in Figure 3, the control strain *Staphylococcus aureus* ATCC 6538 showed a clear hemolytic area, while strain YCL29 did not exhibit hemolysis. These results indicate that strain YCL29 does not produce hemolytic toxins, possesses strong broad-spectrum resistance, and is a highly safe, low-sensitivity Gram-positive bacterium suitable for bacteriocin extraction and subsequent research.

[0092] The 16S rRNA gene sequence of strain YCL29 is shown in SEQ ID No. 25.

[0093] Mean Nucleic Acid Similarity (ANI) is one of the most powerful measures for determining the closeness of bacterial genome phylogenetic relationships. fastANI, based on the average of comparisons of all orthologous protein sequences in a genome quality check, reflects the evolutionary distance between genomes. When ANI > 95%, it indicates that two genomes belong to the same species. Table 2 shows that the strain *Lactobacillus paracasei* subsp. *paracasei* (Note: "Lacticaseibacillus" is the latest name after the renaming of "Lactobacillus") has an ANI score of 98.2056. 16S rRNA molecular biological identification and genome ANI analysis confirm that this strain is *Lactobacillus paracasei* subsp. *paracasei*, and its taxonomic name is *Lactobacillus paracasei* subsp. *paracasei*. The phylogenetic tree is shown below. Figure 4 As shown. This strain was deposited on June 25, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC NO: 66588.

[0094] Table 2. Statistical table of average nucleic acid similarity analysis results

[0095] 11.2 Molecular weight and identification results of YCL29 bacteriocin

[0096] First, the complete genome map of *Lactobacillus paracasei* subsp. *paracasei* YCL29 (Figure 5) was analyzed, and this map was used as a database for comparison and analysis of the raw mass spectrometry acquisition maps. The probability of antimicrobial peptides was predicted using random forest in CAMPR4 software (http: / / www.camp.bicnirrh.res.in / ). Target peptides were screened based on spectral confidence and peak intensity. Subsequently, when searching for bacteriocins of *Lactobacillus paracasei* subsp. *paracasei* YCL29 in the NCBI, UniProt, and PAPD3 databases, no homology was found with other reported bacteriocins. Simultaneously, its secondary structure was analyzed using circular dichroism spectroscopy. Finally, the molecular weight of bacteriocin YCL29 was determined to be 1308.6705 Da, and the amino acid sequence was AVSVGGVATNFRM (Figure 6); its secondary structure contained only α-helices (100%), with no other conformations. Figure 7 This characteristic indicates that YCL29 bacteriocins are highly stable small molecule peptides with the advantages of high efficiency and easy synthesis.

[0097] 11.3 Stability of YCL29 bacteriocin

[0098] Figure 8 illustrates the effects of common enzymes, UV irradiation, and ambient temperature in the food system on the inhibitory activity of YCL29 bacteriocin against Vibrio parahaemolyticus. In the thermal stability test (Figure 8, A), YCL29 bacteriocin exhibited good stability at 40 °C, 60 °C, and 100 °C. At 40 °C and 60 °C, the activity of YCL29 bacteriocin was not significantly different from the control group, and was even stronger. After treatment at 80 °C and 121 °C for 30 min, it still maintained good antibacterial activity. In the UV irradiation stability test (Figure 8, B), the antibacterial activity of YCL29 bacteriocin was found to be not significantly different from the control group; in fact, the antibacterial effect of YCL29 bacteriocin was even better after UV irradiation for 15 min and 120 min. Treatment with pepsin, trypsin, α-amylase, lipase, and papain showed that the antibacterial activity of YCL29 bacteriocin remained at a high level but decreased slightly (Figure 8, C), indicating that YCL29 bacteriocin can exert effective antibacterial effects against different food processing techniques. Many food processing processes involve high-temperature sterilization and ultraviolet irradiation; this bacteriocin exhibits good enzyme stability, ultraviolet irradiation stability, and thermal stability, making it a promising candidate for food additives and biological preservatives.

[0099] 11.4 Minimum inhibitory concentration of YCL29 bacteriocin

[0100] The minimum inhibitory concentration (MIC) is an effective indicator for describing antibacterial efficacy. The MIC of YCL29 bacteriocin against Vibrio parahaemolyticus is 5.54 μg / mL, which is close to or lower than the previously reported MIC values ​​of bacteriocins produced by lactic acid bacteria, indicating that this bacteriocin is a more effective bacteriocin (Figure 9).

[0101] 11.5 Effects of YCL29 bacteriocin on the morphology of Vibrio parahaemolyticus

[0102] The morphological changes of Vibrio parahaemolyticus before and after treatment were observed using scanning electron microscopy (Figure 10). The results showed that untreated Vibrio parahaemolyticus existed with intact and clear rod-shaped structures, without significant damage. When the bacteriocin concentration reached 1 / 2×MIC, collapse of some cell surfaces was observed, with wrinkling and deformation of cell morphology, and pores appearing on the surface of a small number of cells. As the bacteriocin concentration was further increased to 1×MIC, the cell membrane ruptured significantly, the wrinkling and collapse of cells intensified, the surface pores became more obvious, and cell debris floated around. These results indicate that bacteriocin significantly damages the cell membrane of Vibrio parahaemolyticus through a pore-bursting mechanism, leading to membrane dysfunction and inducing the release of cytoplasmic components, and this effect is clearly dose-dependent.

[0103] 11.6 Effects of YCL29 bacteriocin on the expression of virulence genes in Vibrio parahaemolyticus

[0104] like Figure 11 As shown, RT-qPCR analysis revealed that, compared with the control group, the expression of *Vibrio parahaemolyticus*-related virulence genes was significantly downregulated after bacteriocin treatment. At a concentration of 1 / 2×MIC, the expression levels of *Puva* (a key iron metabolism gene for *Vibrio parahaemolyticus* adaptation to the environment and host colonization), VP_RS10835 (a gene involved in metabolic regulation), flaE and flgA (flag assembly-related genes), and trH and tdH (hemolysin regulation genes) were downregulated to 0.65-fold, 0.62-fold, 0.88-fold, 0.91-fold, 0.94-fold, and 0.16-fold, respectively, compared with the control group. When the bacteriocin concentration increased to the MIC, the expression of the above genes was further inhibited, with expression levels decreasing to 0.34-fold, 0.21-fold, 0.54-fold, 0.57-fold, 0.62-fold, and 0.11-fold, respectively, compared with the control group. Furthermore, the expression of three key genes in Vibrio parahaemolyticus—toxR, luxS, and flgL—was regulated by bacteriocin concentration. These three genes did not show significant downregulation at a concentration of 1 / 2×MIC, but their expression levels were significantly downregulated compared to the control group when the concentration was increased to the MIC, decreasing by 0.87-fold, 0.67-fold, and 0.91-fold, respectively. These gene expression changes indicate that bacteriocin YCL29 can effectively inhibit the virulence of Vibrio parahaemolyticus.

[0105] 11.7 Application of YCL29 bacteriocin

[0106] 11.7.1 Physicochemical property analysis

[0107] Changes in the physicochemical properties of fish tea during fermentation (including pH value, total acid content, lactic acid bacteria count, and total bacterial count) such as Figure 12 As shown, both the YCL29 group and the control group showed a gradual decrease followed by an increase in pH value. Figure 12 (Figure A) The total acid content increased and then decreased simultaneously. Figure 12 The trend is shown in Figure B of Figure 12. Throughout the fermentation cycle, the YCL29 group consistently maintained a lower pH and a higher total acid content. Figure C in Figure 12 shows the changes in the viable count of lactic acid bacteria in the two groups during fermentation. At the beginning of fermentation, the lactic acid bacteria count in the YCL29 group was significantly higher than that in the control group (p < 0.05), reaching a peak on day 3, and then slightly decreasing from day 6 to day 30 before stabilizing. *Lactobacillus paracasei* subsp. *paracasei* YCL29 plays a role in regulating the microbial community, stabilizing the number of lactic acid bacteria, and maintaining product stability. Figure D in Figure 12 shows the changes in the total bacterial count of the two groups during the 0-30 days of fish tea fermentation. The total microbial counts of the control group and the YCL29 group showed a significant difference. At the beginning of fermentation (day 0), the total microbial count in the YCL29 group was significantly higher than that in the control group, indicating that the inoculated strain effectively increased the initial microbial load of the system, providing sufficient seed bacteria for fermentation initiation. On day 3 of fermentation, the total number of microorganisms in both groups reached its peak, with the YCL29 group showing a significantly higher number than the control group. This indicates that inoculation with the YCL29 starter culture can rapidly proliferate and dominate the fermentation process, accelerating microbial succession in the system. This result demonstrates that inoculation fermentation not only increases the number of microorganisms in the mid-fermentation stage but also maintains higher and more stable microbial activity throughout the later stages. Furthermore, this helps to continuously produce metabolic products such as organic acids, inhibiting the growth of unwanted microorganisms and ensuring the stability of the fermentation system and the consistency of product quality.

[0108] 11.7.2 Nutritional quality and color difference analysis

[0109] Figure 13 shows the nutritional quality and color difference analysis of fermented fish tea. The composition of organic acids and amino acids during fermentation is an important indicator of the quality characteristics of fish tea products. Organic acids produced by lactic acid bacteria fermentation can inhibit the growth of harmful microorganisms and improve the flavor quality of fermented foods. Within 30 days of fermentation, five organic acids were detected in both groups of samples: tartaric acid, malic acid, lactic acid, succinic acid, and citric acid (Figure A in Figure 13). Figure 13 As shown in Figure A, inoculating the fermentation group can enrich the organic acid composition of fish tea and increase its content.

[0110] As shown in Figure 13B, there were significant differences in the amino acid composition and content between the control group and the YCL29 group during fermentation. The total amino acid content in both groups increased with prolonged fermentation time, with the YCL29 group showing a higher total amino acid content than the control group in the later stages of fermentation (20-30 days). Cysteine, phenylalanine, and alanine were the main amino acids in both groups, with the cysteine ​​and alanine contents in the YCL29 group significantly higher than those in the control group (p < 0.05). Cysteine ​​is a sulfur-containing amino acid closely related to food flavor formation. These results indicate that inoculation fermentation can effectively promote the accumulation of these sulfur-containing and aromatic flavor precursor amino acids. Simultaneously, the free alanine content showed an upward trend with prolonged fermentation time, reflecting a more vigorous trend in microbial protein degradation and amino acid synthesis metabolism in the fermentation system, which is beneficial for human absorption. (Brightness) ), redness ( ) and yellowness ( The color parameters of fermented fish tea are an important sensory indicator affecting consumers' acceptance of its quality. Significant differences in color parameters were observed between the control group and the YCL29 group during fermentation. At the beginning of fermentation, the brightness of the control group (…) was… ) and yellowness ( The redness was slightly higher than that of the YCL29 group, while the redness ( The two groups were similar. On day 3 of fermentation, the YCL29 group... and The values ​​of the YCL29 group were significantly higher than those of the control group (p < 0.05) and remained at a relatively high level throughout the fermentation cycle; meanwhile, the values ​​of the YCL29 group were significantly higher than those of the control group (p < 0.05) and remained at a relatively high level throughout the fermentation cycle. The value turned negative after 6 days, significantly lower than the control group (p < 0.05). These results indicate that inoculation with *Lactobacillus paracasei* subsp. *paracasei* YCL29 starter culture significantly enhances the brightness and yellowness of fish tea while inhibiting the reddening trend. This makes the product brighter and more golden in color, effectively improving the sensory color quality of the fermented product (Figures C, D, and E in Figure 13).

[0111] 11.7.3 Microbiome and Flavor Profile Analysis

[0112] The bacterial community structure during fish tea fermentation was analyzed at the phylum and genus levels (Figure 14). The results showed that the dominant phyla during fermentation were Bacteroidetes, Firmicutes, and Proteobacteria. At the genus level, 43 bacterial genera had an average relative abundance greater than 1%. In the control group, the dominant genera were *Pediococcus* and *Morganella*, with *Pediococcus* peaking on day 30 and *Morganella* peaking on day 3 and then gradually decreasing. *Vibrio* had a higher proportion in the early stages of fermentation and gradually decreased with the fermentation process. In the YCL29 group, the dominant genera were *Lacticaseibacillus* and *Lactiplantibacillus*, with *Lacticaseibacillus* having the highest proportion. Compared with the control group, the abundance of *Vibrio* was significantly reduced in the early stages of fermentation in the YCL29 group, indicating that inoculation with YCL29 can effectively inhibit the growth of *Vibrio* and reduce the risk associated with *Vibrio parahaemolyticus*. This effect is related to its bacteriocin-producing antibacterial mechanism. The above results indicate that inoculation with Lactobacillus paracasei subsp. paracasei YCL29 starter culture can effectively regulate the bacterial community structure and succession trajectory during fish tea fermentation, which is of great significance for improving the uniformity, safety and quality stability of fermented products.

[0113] Volatile compounds are a key indicator of the flavor of fish tea. This study analyzed them using headspace-solid phase microextraction-gas chromatography-mass spectrometry (HSP-GC-MS), identifying 107 compounds, including 19 alcohols, 30 acids, 11 esters, 7 ketones, 5 phenols, and 35 other substances. During fermentation, the total volatile component content of the YCL29 group was significantly higher than that of the control group (Figure 15). Furthermore, the aldehyde and alcohol content of the YCL29 group was higher than that of the control group (Figure 16), indicating that the higher aldehyde and alcohol content in the YCL29 group contributed floral and fruity aromas to the fish tea.

[0114] 11.7.4 Sensory Analysis

[0115] The taste characteristics of fish tea prepared in the YCL29 group and the control group on day 30 of fermentation were analyzed using electronic tongue technology. Radar chart analysis ( Figure 17 Figure A shows that the umami and sweetness of group YCL29 were higher than those of the control group, while the sourness, bitterness, and saltiness were more pronounced in the control group. Hierarchical clustering heatmap ( Figure 17 Figure B in the middle section further validated these differences. These results indicate that inoculation with Lactobacillus paracasei subsp. paracasei YCL29 can effectively regulate the flavor spectrum of fermented fish tea, with the microorganisms breaking down proteins into small umami peptides and enhancing the sweetness of the fish tea while reducing the intensity of the traditional pickled flavor.

[0116] The volatile flavor characteristics of fish tea prepared from the YCL29 group and the control group on day 30 of fermentation were analyzed using electronic nose technology. Radar chart analysis ( Figure 17 The middle figure (Figure C) shows that the YCL29 group had higher levels of positive flavor compounds such as 2-hexanol, 2-methylpentanal, hexanal, and valeric acid, while the control group had a more significant off-flavor compound, 1-butanamine. Hierarchical clustering heatmap ( Figure 17 The differences were further verified by the figure (Figure D), and the intragroup repeatability was good. These results indicate that inoculation with Lactobacillus paracasei subsp. paracasei YCL29 can effectively regulate the volatile flavor profile of fermented fish tea, enhance pleasant flavors such as fresh fruit and nutty aromas, and suppress unpleasant flavors such as ammonia and fishy odors.

[0117] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A *Lactobacillus paracasei* subsp. *paracasei* with antibacterial and fermentation-improving properties, characterized in that, The specimen, classified as Lactobacillus paracasei subsp. paracasei, was deposited on June 25, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCCNo: 66588.

2. A bacteriocin, characterized in that, Derived from Lactobacillus paracasei subsp. paracasei according to claim 1, the bacteriocin has the amino acid sequence shown in SEQ ID NO. 26 and a molecular weight of 1308.6705 Da.

3. The bacteriocin according to claim 2, characterized in that, The bacteriocins are prepared by the following method: cell-free supernatant is obtained by fermentation with *Lactobacillus paracasei* subsp. *paracasei*; crude bacteriocin is then obtained by extraction with organic reagents; and finally, the crude bacteriocin is separated and purified by ultrafiltration, gel chromatography, and reversed-phase high-performance liquid chromatography.

4. The bacteriocin according to claim 3, characterized in that, The method specifically includes: 1) Fermentation: The Lactobacillus paracasei subsp. paracasei was fermented in MRS liquid medium at 37 ℃ for 24 h to obtain fermentation broth. The fermentation broth was filtered through a 0.22 μm filter membrane to obtain cell-free supernatant. 2) Organic extraction: After thoroughly mixing ethyl acetate and cell-free supernatant in a certain proportion, the mixture was allowed to stand and separate into layers. The organic phase and aqueous phase were then removed separately. The organic phase was then removed by rotary evaporation at 45 °C to obtain the crude extract of bacteriocin. 3) Ultrafiltration: The crude extract of bacteriocins was purified by fractional ultrafiltration using ultrafiltration centrifuge tubes with molecular weights of 30 kDa, 10 kDa and 3 kDa. 4) Gel chromatography: Based on the ultrafiltration results, dextran gel G-100 gel chromatography was used to further purify the ultrafiltration product obtained in step 3). 5) Reversed-phase high-performance liquid chromatography (RP-HPLC) separation and purification: The purified components collected in step 4) were filtered through a microporous membrane and then detected by reversed-phase high-performance liquid chromatography (RP-HPLC) with a UV detector; wavelength: 280 nm; injection volume: 20 μL; flow rate: 0.5 mL / min; mobile phases were phase B: acetonitrile solution containing 0.1% trifluoroacetic acid and phase C: aqueous solution containing 0.1% trifluoroacetic acid.

5. A fermenting agent, characterized in that, It includes Lactobacillus paracasei subsp. paracasei as described in claim 1.

6. The application of the fermenting agent according to claim 5 in improving the flavor quality and metabolic regulation of fermented fish tea.

7. The application according to claim 6, characterized in that, The fermenting agent can effectively inhibit the growth of Vibrio spp. and optimize the microbial community structure during the fermentation process of fish tea.

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