Lactococcus lactis with antagonistic activity and intestinal colonization capability and application thereof

Abstract

The invention provides lactococcus lactis with antagonistic activity and intestinal colonization ability and application thereof, and belongs to the technical field of biological prevention and treatment. The lactococcus lactis GCC10 screened by the invention is good in biological safety and strong in antagonistic bacteriostatic activity, can be colonized in intestinal tracts, shows excellent probiotic characteristics, provides an excellent germplasm resource for prevention and control of fish bacterial diseases, and promotes research and development of new prevention and control products for reducing resistance and replacing resistance for aquatic animals.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology, and specifically relates to a strain of Lactococcus lactis with antagonistic activity and intestinal colonization ability and its application. Background Technology

[0002] Grass carp, with its rapid growth, strong adaptability, high nutritional value, and delicious meat, has become one of the most widely farmed freshwater fish in my country and even worldwide. However, due to increased stocking density, deterioration of the aquatic environment, and unbalanced feed nutrition, bacterial diseases are becoming increasingly prevalent in grass carp farming. Diseases such as hemorrhagic septicemia caused by Aeromonas hydrophila, enteritis caused by Edwardsiella tarda, bacterial gill rot caused by Flavobacterium columnare, and vibriosis caused by Vibrio mimicry threaten the healthy farming of grass carp in my country and cause significant economic losses. Traditional disease control measures mainly rely on chemical drugs such as antibiotics and disinfectants. Long-term overuse of chemical drugs easily leads to drug residue problems and the development of bacterial resistance, which not only affects the quality and safety of aquatic products but also disrupts the ecological balance of aquatic bodies. Therefore, finding safe and effective antibiotic-reduced and antibiotic-alternative products has become a research hotspot in the current aquaculture industry.

[0003] In recent years, probiotics have received widespread attention in the aquaculture industry as a green and safe biological control method. However, many probiotics currently on the market are derived from terrestrial animals, and their adaptability and safety to water and fish are often inferior to those of fish-derived probiotics. Therefore, developing and applying fish-derived probiotics with high biosafety and good biocontrol effects is of significant practical importance for the healthy farming of grass carp. Summary of the Invention

[0004] This invention screened a strain of Lactococcus lactis with antagonistic activity and intestinal colonization ability from the intestines of healthy grass carp. Through strain identification, biological characteristic analysis, biosafety assessment and potential biocontrol analysis, it was shown that the isolated strain has good biosafety and probiotic characteristics. This invention not only enriches the germplasm resource bank of aquatic animal-derived probiotics, but also provides a new product for the ecological control of bacterial diseases in grass carp.

[0005] The present invention discloses a strain of Lactococcus lactis with antagonistic activity and intestinal colonization ability, which is classified as Lactococcus lactis GCC10 and was deposited at Guangdong Provincial Microbial Culture Collection Center on June 24, 2025, with accession number GDMCC No: 66583.

[0006] This invention collects intestinal and surface mucus from healthy grass carp. After homogenizing the intestinal tissue and mucus, lactic acid bacteria are isolated and purified using MRS medium. First, the antagonistic and hemolytic activities of the isolated strains are measured to screen for the candidate strain GCC10, which exhibits good antagonistic activity and does not cause hemolysis. The morphological characteristics and taxonomic position of the strain are analyzed, and its extracellular enzyme activity, drug sensitivity, bile tolerance, intestinal colonization ability, biocontrol effects, and biosafety are determined. The biosafety and potential probiotic functions of strain GCC10 are evaluated through whole-genome sequencing.

[0007] The results showed that the isolated strain GCC10 was Gram-positive, with cells arranged singly or in pairs, and was identified as *Lactococcus lactis*. This bacterium lacked hemolytic activity but possessed extracellular protease activity, bile tolerance, and intestinal colonization ability. Pathogenicity tests showed good safety after intraperitoneal injection and gavage infection of grass carp. Biocontrol efficacy analysis showed that strain GCC10 had good protective effects against *Aeromonas hydrophila*, *Aeromonas vesiculosus*, and *Aeromonas temperate* infections in grass carp. Antimicrobial susceptibility testing showed that strain GCC10 was sensitive to vancomycin, tetracycline, erythromycin, chloramphenicol, enrofloxacin, florfenicol, doxycycline, sulfamethoxazole, and sulfamethoxazole + trimethoprim. Whole-genome sequencing analysis revealed that strain GCC10 does not contain virulence genes such as cylA, gelE, esp, agg, gelE, hyl, and ace. However, it contains genes related to stress response, bile salt tolerance, acid tolerance, immune regulation, proteolysis, and adhesion factors, as well as gene clusters related to lactococcal production. Therefore, this invention screened a *Lactococcus lactis* strain GCC10 with good biocompatibility, strong antagonistic and antibacterial activity, and the ability to colonize the intestines. This strain exhibits excellent probiotic characteristics, providing a superior germplasm resource for the control of bacterial diseases in fish and promoting the development of new antibiotic-free and antibiotic-alternative control products for aquatic animals. Based on the antibacterial spectrum of strain GCC10, it showed the best antibacterial effect against *Aeromonas tempera*, and also exhibited good antibacterial activity against *Brachystomia melanogaster*, *Aeromonas hydrophila*, and *Aeromonas vesiculosus*. Attached Figure Description

[0008] Figure 1 This is a colony morphology diagram of the Lactococcus lactis GCC10 strain of the present invention;

[0009] Figure 2 Gram staining morphology of the Lactococcus lactis GCC10 strain of the present invention (×1000x);

[0010] Figure 3 The hemolytic activity diagram of the Lactococcus lactis GCC10 strain of the present invention is shown.

[0011] Figure 4The phylogenetic tree of the Lactococcus lactis GCC10 strain of the present invention is constructed based on 16S rRNA;

[0012] Figure 5 The image shows the results of the protease activity test of the Lactococcus lactis GCC10 strain of the present invention.

[0013] Figure 6 The inhibition zone of the Lactococcus lactis GCC10 strain of the present invention against common aquatic pathogens;

[0014] Figure 7 The figure shows the bile tolerance results of the *Lactococcus lactis* GCC10 strain of the present invention; in the figure: ***, p<0.01; **, p<0.01; *, p<0.05; the initial bacterial concentration was 10. 9 CFU / mL;

[0015] Figure 8 The image shows the colony PCR identification results of recombinant strain GCC10-pNZ8148. In the image, M represents DL2000 Marker; 1, 2, 3, 4, 5 represent the PCR amplification fragments of pNZ8148 plasmid.

[0016] Figure 9 The results of the intestinal colonization assay for the recombinant strain GCC10-pNZ8148 are shown in the figure. ** indicates p < 0.01; * indicates p < 0.05. The concentration of the bacterial culture administered via gavage was 10... 9 CFU / g, gavage dose was 0.2 mL / tail; the control group did not receive gavage.

[0017] Figure 10 KEGG annotation and classification statistics of Lactococcus lactis GCC10 strain of the present invention;

[0018] Figure 11 This is a schematic diagram of a gene cluster related to the synthesis of an antagonistic substance in the genome of the Lactococcus lactis GCC10 strain of the present invention. Detailed Implementation

[0019] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments.

[0020] 1. Materials and Methods

[0021] 1.1 Materials and Equipment

[0022] The healthy grass carp were purchased from a seedling farm in Huadu District, Guangzhou City, and temporarily raised in pond cages for 2 weeks before being used after being checked for health. Indicator bacteria include *Photobacterium damselae* Bt2402, *Aeromonas hydrophila* Ci2426, *Aeromonas sobria* Ci2403, *Aeromonas veronii* (ACVA07), *Edwardsiella ictaluri* HS1601, *Edwardsiella piscicida* Ci2319, *Edwardsiella anguillarum* Ci2325, *Flavobacterium columnare* FC001, *Flavobacterium psychrophilum* Ci2402, and standard strains *Staphylococcus aureus* ATCC25923 and *Escherichia coli*. Both coli ATCC25922 and coli are deposited in this laboratory.

[0023] Brain heart and brain broth (BHI) was purchased from BD Biosciences, Inc. (USA). MRS broth and hydrolyzed casein peptone (MH) broth were purchased from Guangdong Huankai Microbial Co., Ltd. Skim milk powder and agar powder were purchased from Saiguo Biotechnology (Guangzhou) Co., Ltd. Columbia blood agar was purchased from Guangzhou Dijing Microbial Technology Co., Ltd. Antimicrobial susceptibility testing discs were purchased from Hangzhou Microbial Reagent Co., Ltd. PCR reagents were purchased from Takara Bio Engineering (Dalian) Co., Ltd. Bacterial genomic DNA extraction kits were purchased from Tiangen Biotech (Beijing) Co., Ltd. Primer synthesis and sequencing services were provided by Guangzhou Aiji Biotechnology Co., Ltd. Bacterial whole genome sequencing was commissioned to Beijing Biomarker Biotechnology Co., Ltd.

[0024] 1.2 Test Methods

[0025] 1.2.1 Isolation and purification of strains

[0026] Healthy 2-year-old grass carp (body weight 1.23±0.31 kg / fish) were harvested. Under aseptic conditions, the intestines were removed, and the contents were chopped and placed in 50 mL centrifuge tubes. 10 mL of sterile 0.65% physiological saline was added, and the mixture was vortexed to mix. 200 μL of the stock solution, 10-fold and 100-fold dilutions were plated on MRS agar plates, with each concentration repeated three times. The plates were incubated at 28℃ for 24–48 h.

[0027] Mucus was collected from the body surface of grass carp and placed in a 50 mL centrifuge tube. 10 mL of sterile 0.65% physiological saline was added, and the mixture was vortexed to mix. 200 μL of the stock solution, 10-fold dilutions, and 100-fold dilutions were cultured on MRS agar plates for bacterial isolation. Single colonies grown on MRS solid medium were picked and further purified. The purified strains were added to 80% glycerol and stored at -80°C.

[0028] Using Aeromonas hydrophila Ci2426 as an indicator bacterium, antagonistic strains were screened using the spot inoculation method. Target strains with large antagonistic inhibition zones and no hemolysis on blood agar plates were selected as candidate strains. Among them, strain GCC10 was selected as a candidate probiotic for strain identification and functional evaluation due to its large inhibition zone and non-hemolysis.

[0029] 1.2.2 Morphological identification of strains

[0030] The purified bacterial strain was inoculated onto BHI solid medium and cultured at 28°C for 24 hours. The morphology, size, concavity, and transparency of the colonies were observed. Single colonies were picked and inoculated onto BHI liquid medium and cultured at 28°C and 200 rpm in a shaker until the logarithmic growth phase. The bacterial solution was then prepared into smears, stained with Gram stain, and the bacterial morphology was observed under a microscope.

[0031] 1.2.3 Hemolytic activity

[0032] Hemolytic activity is a key indicator for evaluating the pathogenicity of potential probiotics. Hemolytic bacteria can disrupt the host's erythrocyte membrane structure by secreting hemolysin, causing tissue damage or systemic infection. The International Committee for Microbiological Standards in Food (ICMSF) recommends screening non-hemolytic (γ-hemolytic) strains as candidate probiotics using a blood agar test. After activation, the selected strains are inoculated onto Columbia blood agar plates and incubated at 28°C for 24 hours, during which their hemolytic activity is observed. Complete hemolysis is defined as β-hemolysis (a clear zone appears around the colony); incomplete hemolysis is defined as α-hemolysis (only hemoglobin deposition occurs around the colony without a clear zone); and no hemolysis is defined as γ-hemolysis.

[0033] 1.2.4 Molecular biological identification of isolated strains

[0034] Genomic DNA was extracted from bacterial strain GCC10 using a bacterial genomic DNA extraction kit. The 16S rRNA gene of strain GCC10 was amplified using universal primers 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ (SEQ ID NO.1) and 1492R: 5′-TTCAGCATTGTTCCATTGG-3′ (SEQ ID NO.2). The PCR reaction mixture (50 μL) consisted of: 25 μL 2×Taq Master Mix, 22 μL ddH2O, 1 μL upstream primer 27F, 1 μL downstream primer 1492R, and 1 μL DNA template. The PCR program was: 95℃ pre-denaturation for 4 min, 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 1.5 min, 35 cycles, followed by a final extension at 72℃ for 10 min. PCR products were detected by 1.0% agarose gel electrophoresis, and positive products were sequenced. Sequencing results were compared for homology in the GenBank database using BLASTN, and a phylogenetic tree was constructed using MEGA11.0 software and the neighbor-joining method.

[0035] 1.2.5 Detection of extracellular enzyme activity

[0036] Selective culture media for protease, amylase, and lipase were prepared. The protease medium was prepared as follows: first, prepare BHI medium with 3% agar powder and sterilize at 121℃ for 25 min; then add skim milk powder to a final concentration of 1.5% (sterilize at 115℃ for 15 min), pH = 7.0–7.2, and then pour into plates. The amylase medium was prepared as follows: add 1.5% soluble starch and 1.5% agar to BHI medium, pH = 7.0–7.2, sterilize at 121℃ for 25 min, and then pour into plates. The lipase medium formula was as follows: 10g peptone, 5g yeast extract, 5g sodium chloride, 0.1g calcium chloride, 10mL Tween-80, 1000mL distilled water, 15g agar powder, and autoclave at 121℃ for 20 min. The Oxford cup method was used to detect whether the isolated strains possessed extracellular protease, amylase, and lipase activities. Add 200 μL of bacterial culture (MCF = 0.5) to protease, lipase, and amylase media respectively, and incubate at 28°C for 48 h. Measure the diameter of the hydrolysis zone. Repeat the experiment three times.

[0037] 1.2.6 Antibacterial spectrum test

[0038] The antagonistic effect of strain GCC10 against common aquatic pathogens was determined using the Oxford cup method. Indicator bacteria (excluding *Flavobacterium psychrophilum* and *Flavobacterium columnare*) were inoculated into BHI liquid medium and cultured at 28°C and 180 rpm for 12 h. The activated indicator bacteria were collected, washed twice with sterile physiological saline, and the bacterial culture was adjusted to 1.5 × 10⁻⁶. 8CFU / mL. Spread 100 μL of indicator bacterial suspension evenly onto BHI solid medium perforated with Oxford cups. Add 150 μL of GCC10 bacterial suspension to each well (28℃, incubate for 24 h), and incubate upright at 28℃ for 24 h. Measure and record the diameter of the inhibition zone (mm). The inhibition spectrum assays for *Flavobacterium psychrophilum* and *Flavobacterium columnare* were performed using the same method, but both strains were cultured on Shieh medium at 15℃ and 28℃, respectively. Sterile physiological saline was used as the control group. Each treatment was repeated three times.

[0039] 1.2.7 Drug susceptibility testing

[0040] The minimum inhibitory concentration (MIC) of strain GCC10 against antimicrobial agents (vancomycin, tetracycline, erythromycin, kanamycin, chloramphenicol, enrofloxacin, neomycin, florfenicol, doxycycline, sulfamethoxazole, and trimethoprim + sulfamethoxazole) was determined using a two-fold dilution method. First, 50 μL of MRS liquid medium was added to each well of a 96-well plate. Second, 50 μL of antibiotic was added to the first column and serially diluted twofold, with drug concentrations of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 mg / L in each column, respectively. Finally, 50 μL of the diluted bacterial suspension was inoculated into each well to achieve a final bacterial concentration of 5 × 10⁻⁶ mg / L. 5 CFU / mL. Three replicates were set up for each drug. The positive control group received bacterial culture without antibiotics, and the negative control group received antibiotics only.

[0041] 1.2.8 Biosafety Testing

[0042] The experiment included an intraperitoneal injection group, a gavage group, and a negative control group, with three replicates per group. Grass carp (average weight 18.30±1.45g) were used as the experimental fish. Fifteen fish were randomly selected for each replicate. The bacterial suspension concentration was set at 1.0×10⁻⁶. 9 CFU / mL, administered via gavage and intraperitoneal injection, respectively. In the experimental group, each fish received 200 μL of GCC10 bacterial solution via intraperitoneal injection and gavage, while in the control group, each fish received 200 μL of physiological saline via intraperitoneal injection or gavage. The fish were observed for 7 consecutive days, with infection symptoms and mortality recorded daily.

[0043] 1.2.9 Bile tolerance

[0044] Healthy grass carp (5.3–6.7 kg / fish) were harvested, and bile was extracted using a sterile syringe (bile samples from 3 healthy grass carp were pooled). The experiment included a bile group and a 0.65% saline group. GCC10 bacterial strain cultured to the logarithmic growth phase was resuspended in sterile saline, and the bacterial concentration was adjusted to 10. 9CFU / mL, at a ratio of 1%, 20 μL of bacterial solution was added to 2 mL of bile and 0.65% sterile saline, respectively, to achieve a final bacterial concentration of 10. 7 CFU / mL. Incubate at 28℃ with shaking at 180 rpm. Repeat each group three times, and take 100 μL of bacterial culture at 2, 4, 6, 8, 12 and 24 h, dilute 10 times and plate for counting.

[0045] 1.2.10 Intestinal colonization capacity

[0046] Preparation of recombinant strain: The recombinant strain *Lactococcus lactis* GCC10 (denoted as GCC10-pNZ8148) carrying the chloramphenicol resistance plasmid pNZ8148 was obtained by electroporation. Specifically, plasmid pNZ8148 was electroporated into GCC10 competent cells, and the cells were selected for chloramphenicol resistance. Several colonies were randomly selected on chloramphenicol-resistant plates and amplified using pNZ8148-specific primers 8148F: 5′-CTATCTTGAGAAAGTATTGGTAA-3′ (SEQ ID NO.3) and 8148R: 5′-AATTGCTTTATCAACTGCTGC-3′ (SEQ ID NO.4). PCR products were detected by 1.0% agarose gel electrophoresis. Finally, a strain successfully transformed pNZ8148 into GCC10 was selected, namely GCC10-pNZ8148. The strain was passaged in antibiotic-free BHI liquid medium as follows: GCC10-pNZ8148 positive single clones were picked and cultured in antibiotic-free MRS liquid medium at 28℃ and 180 rpm for 12 h. The resulting bacterial culture was the first generation. Subsequently, every 12 h, 200 μL of the bacterial culture was inoculated into 20 mL of antibiotic-free MRS liquid medium. 100 μL of each generation was aspirated for plasmid stability testing.

[0047] Forty healthy grass carp (body weight: 23.5±3.3g) were housed in a 200L glass aquarium. The experimental group (N=20 fish) each grass carp was gavaged with 0.2mL of GCC10-pNZ8148 bacterial solution (1.0×10⁻⁶). 9The control group (N=20 fish) received 0.2 mL of 0.65% sterile saline via gavage. Before the experiment, the mid- and hindgut of three grass carp were randomly selected to detect lactic acid bacteria content. The intestinal contents were separated from the intestinal wall, and the intestinal wall was rinsed with sterile saline. The intestinal contents and intestinal wall were homogenized separately, and the undiluted solution and a 10-fold dilution were plated on chloramphenicol-resistant MRS plates for colony counting. The fish were fed twice daily for 7 days. At 12h, 24h, 48h, 36h, 120h, and 168h after the start of the experiment, three grass carp were randomly selected, and intestinal contents and intestines (without contents) were collected from each fish. The intestinal wall was rinsed with sterile saline. The intestinal contents and intestines were homogenized separately, and the undiluted solution and a 10-fold dilution were plated on chloramphenicol-resistant MRS plates (chloramphenicol 25 μg / mL) and incubated at 28℃ for 24h for colony counting. Meanwhile, single colonies were randomly selected from the plates and verified by PCR to determine whether they were strain GCC10-pNZ8148.

[0048] 1.3 Genome Sequencing and Analysis

[0049] The GCC10 strain was inoculated into BHI medium and cultured overnight to the logarithmic growth phase. The bacterial cells were collected, and genomic DNA was extracted for whole-genome sequencing analysis. Whole-genome sequencing was performed using the Nanopore sequencing platform by Beijing Biomarker Biotechnology Co., Ltd.

[0050] 1.3.1 Genome Assembly and Sequence Analysis

[0051] Genome assembly: The strain GCC10 was assembled using Hifiasm software, and Pilon v1.22 software was used to further correct errors using second-generation data to obtain a more accurate genome.

[0052] Gene prediction: coding genes were predicted using Prodigal v2.6.3 software; non-coding gene rRNA was predicted using Infernal v1.1.3 software based on the Rfam database.

[0053] 1.3.2 Genome Functional Annotation

[0054] Gene function annotation results were obtained by performing BLAST alignment analysis between the predicted gene sequences and functional databases such as Nr, KEGG, Pfam, Swiss-Prot, TrEMBL, GO, and eggNOG. Furthermore, the predicted gene protein sequences were also analyzed using BLAST alignment analysis with functional databases such as the Transporter Protein Database (TCDB), Virulence Factor Database (VFDB), and Antibiotic Resistance Gene Database (CRDB) to obtain corresponding annotation results.

[0055] 1.3.3 Analysis of gene clusters related to antagonistic substances

[0056] The antiSMASH v5.0.0 online analysis was used to predict antagonistic gene clusters related to the synthesis of antimicrobial substances in the genome of strain GCC10. Based on the preliminary online prediction results, the corresponding antimicrobial substance synthesis-related genes were downloaded and compared one by one to determine whether antagonistic genes in the GCC10 genome had been deleted or mutated. In addition, the proportion of antagonistic gene clusters to the full length of the genome was analyzed based on the predicted antagonistic gene sequences.

[0057] 1.3.4 Annotation of genes related to probiotic function

[0058] The database is used to screen for genes known to have beneficial functions. Information from all general databases is then compiled. If a gene or function is annotated in any of these databases, it is considered to have the corresponding function.

[0059] 2. Results and Analysis

[0060] 2.1 Morphological observation of the isolated strains

[0061] Numerous bacterial strains were isolated from the intestines and mucus of grass carp using MRS plates, and the isolated strains were numbered. Furthermore, Aeromonas hydrophila was used as an indicator bacterium to determine the antibacterial activity of the isolated strains. Finally, the strain with the strongest antibacterial activity was screened and designated GCC10.

[0062] The isolated strain formed milky-white, round colonies with raised, neat edges on BHI agar medium. Figure 1 Gram-positive, and under microscopic examination, they appear as round or oval cocci, arranged singly or in pairs. Figure 2 ).

[0063] 2.2 Hemolytic activity

[0064] After culturing on blood agar plates for 24 hours, strain GCC10 did not develop a β-hemolytic zone, while the control strain Bacillus pumilus YB12 showed a clear hemolytic zone, indicating that strain GCC10 has no hemolytic activity. Figure 3 ).

[0065] 2.3 Molecular biological identification

[0066] The results of the comparative analysis of the 16S rRNA gene sequence of the isolated strain showed that ( Figure 4 The isolated strain GCC10 showed 100% similarity to *Lactococcus lactis* (GenBank sequence number CP065984). Based on morphological observation, colony characteristics, and 16S rRNA molecular identification, the isolated strain GCC10 was preliminarily identified as *Lactococcus lactis*.

[0067] 2.4 Extracellular enzyme activity

[0068] Extracellular enzyme activity assays of strain GCC10 showed that the bacterium exhibited a clear zone on the skim milk powder culture medium, with a diameter of 22.55 mm. Figure 5 The presence of extracellular protease activity indicates that the strain possesses extracellular protease activity. However, the absence of lipolysis and starch hydrolysis clear zones indicates that the strain lacks extracellular lipase and amylase activity.

[0069] 2.5 Antibacterial spectrum

[0070] According to the antibacterial spectrum of strain GCC10 (Table 1), it showed the best antibacterial effect against Aeromonas hydrophila, with an average inhibition zone diameter of 24.36 mm. It also exhibited good antibacterial activity against *Bacillus psychrophilus*, *Aeromonas hydrophila*, and *Aeromonas vesiculosus*, with an average inhibition zone diameter of 18.53 mm. However, this strain showed no antibacterial effect against *Edwardsiella piscinoides* and *Edwardsiella eelii*. The antibacterial test results of strain GCC10 against Ci2402 (*Flavobacterium psychrophilum*), ACVA07 (*Aeromonas vesiculosus*), ATCC25922 (*Escherichia coli*), ATCC25923 (*Staphylococcus aureus*), FC001 (*Flavobacterium columnare*), HS1601 (*Edwardsiella piscinoides*), Bt2402 (*Bacillus psychrophilus*), and Ci2426 (*Aeromonas hydrophila*) are as follows: Figure 6 As shown.

[0071] Table 1. Antibacterial effects of GCC10 strain against common aquatic pathogens.

[0072]

[0073]

[0074] 2.6 Drug susceptibility testing

[0075] According to the standards of the "Guidelines for Identification and Safety Evaluation of Microorganisms and Fermentation Products for Direct Feeding," strain GCC10 is sensitive to four antibiotics: vancomycin, tetracycline, erythromycin, and chloramphenicol, but resistant to aminoglycosides such as kanamycin and streptomycin. This invention also tested the minimum inhibitory concentration (MIC) of commonly used aquaculture antibiotics. The results showed that GCC10 was sensitive to six antibiotics: enrofloxacin, florfenicol, doxycycline, sulfamethoxazole, and trimethoprim-sulfamethoxazole, but resistant to kanamycin and neomycin (Table 2).

[0076] Table 2. MICs of GCC10 strain against different antimicrobial agents

[0077]

[0078] 2.7 Biosafety

[0079] The experimental results showed that during the experiment, the injection concentration was 10. 9 At CFU / mL, no deaths or other abnormalities were observed in grass carp in both the gavage and intraperitoneal injection groups, indicating that the GCC10 strain has good biosafety for grass carp.

[0080] 2.8 Bile tolerance

[0081] like Figure 7 As shown, strain GCC10 is tolerant of bile and can survive in bile. The colony count increased from 10 at 2 hours. 7 CFU / mL decreased to 10 5 CFU / mL, strain GCC10 began to proliferate in bile starting from hour 6, reaching a colony count of 10 at hour 24. 6 CFU / mL.

[0082] 2.9 Intestinal colonization capacity

[0083] After plasmid pNZ8148 was electroporated into GCC10 strain, colony PCR was performed for amplification, and the results were detected by agarose gel electrophoresis. Figure 8 As shown, by Figure 8 It was found that the GCC10-pNZ8148 strain amplified a specific band of approximately 310 bp, the same size as the predicted band, indicating that the recombinant strain GCC10-pNZ8148 was successfully constructed. The plasmid pNZ8148 showed a plasmid loss rate of less than 20% after six passages in antibiotic-free liquid medium, indicating that the plasmid can be relatively stably passaged in the GCC10 strain, and this recombinant strain can be used for intestinal colonization experiments.

[0084] The count of GCC10-pNZ8148 strain in grass carp intestinal homogenate and its contents is shown in the figure. Figure 9 Before the experiment, no GCC10-pNZ8148 strain was detected in the intestinal contents and intestinal wall homogenate of the group that did not receive gavage. Twelve hours after gavage administration of GCC10, the number of Lactococcus lactis in the intestinal contents and intestinal tissue remained above 10. 6 CFU / g, the number of strains decreased continuously over time, dropping to 10 after 168 hours. 2 CFU / g, and GCC10-PNZ8148 was no longer detected in intestinal tissue and contents after 168 hours. Observation revealed that the number of strains in the intestinal wall homogenate was significantly lower than that in the intestinal contents after 48 hours.

[0085] 2.10 Biocontrol Effects

[0086] To evaluate the protective effect of GCC10 against Aeromonas hydrophila Ci2426 infection, healthy grass carp (body weight: 18.4±5.2g) were housed in three glass tanks containing 100L of water, 10 fish per tank. The experiment was divided into three groups: a blank control group, where each grass carp was intraperitoneally injected with 0.2mL of sterile 0.65% saline; and a positive control group, where each fish was intraperitoneally injected with 0.2mL of Ci2426 (1×10⁻⁶ g / mL). 7 CFU / mL) bacterial suspension; each grass carp in the probiotic group was injected intraperitoneally with 0.2 mL of a mixed bacterial suspension, which consisted of GCC10 (1×10⁻⁶ CFU / mL). 8 CFU / mL) and Ci2426 (1×10) 7 Composition (CFU / mL). During the experiment, fish were fed twice daily, with a total feed amount of approximately 3% of their body weight. Disease and mortality were observed and recorded daily for two consecutive weeks.

[0087] The experimental results showed that the cumulative mortality rate of grass carp in the probiotic group was 30%, the cumulative mortality rate of grass carp in the positive control group was 90%, and the cumulative mortality rate of grass carp in the blank control group was 0. The relative protection rate of probiotic GCC10 against grass carp infected with Aeromonas hydrophila was 62.5%, indicating that intraperitoneal injection of GCC10 strain can reduce the lethality of pathogen Ci2426 in grass carp.

[0088] Using a similar experimental scheme as described above, the protective effects of mixed infection of GCC10 with Aeromonas versicolor ACVA07 and Aeromonas sobria Ci2403 on grass carp were evaluated. The results showed that the relative protection rates of GCC10 strain against Aeromonas versicolor and Aeromonas sobria infection in grass carp were 75.0% and 71.4%, respectively.

[0089] 2.11 Genome Sequencing

[0090] 2.11.1 Sequence Assembly and Annotation

[0091] Sequencing of strain GCC10 yielded 45,612 clean reads, with an N50 length of 10,454 bp, a genome size of 2,359,028 bp, and a G+C content of 34.98%. The GCC10 genome contains 2,233 coding genes.

[0092] COG database annotation of protein-coding genes in the *Lactococcus lactis* GCC10 genome revealed 1874 functionally annotated genes, with high abundance in genes related to amino acid transport and metabolism (171), carbohydrate transport and metabolism (168), transcription (154), translation, ribosome structure and bioformation (154), replication, recombination and repair (106), and inorganic ion transport and metabolism (106). Nr database annotation showed that 97.34% of the genes in *Lactococcus lactis* GCC10 were mapped to *Lactococcus lactis*, and 0.95% to the *Lactococcus* genus. KEGG database annotation revealed 1312 genes annotated in *Lactococcus lactis* GCC10. Figure 10 It can be seen that the annotated genes can be divided into three main categories: environmental information processing, metabolic-related processes, and genetic information processing, which can be further subdivided into 19 KEGG secondary functions. Among them, the most genes are related to global metabolic pathways, while the fewest are related to environmental information processing. There are 108 genes related to amino acid metabolism and 64 genes related to carbohydrate metabolism pathways.

[0093] The predicted genes were annotated using the CARD database to identify two antibiotic resistance genes: aadA4 (GE000618), lmrD (GE000305), and lmrC (GE000304), which are associated with resistance to aminoglycosides and lincosamides. Further analysis revealed that the identified resistance genes are located on the genome, and no mobile genetic elements or transposase genes were found upstream or downstream of the resistance genes aad4, lmrD, and lmrC.

[0094] Common virulence-related genes such as cylA, gelE, esp, agg, gelE, hyl, and ace were compared and analyzed (similarity ≥80%). The results showed that the genome sequence of strain GCC10 did not contain the above-mentioned virulence genes.

[0095] 2.11.2 Analysis of Antagonistic Substance-Related Gene Clusters

[0096] Online analysis using antiSMASH v5.0.0 software revealed that the GCC10 genome contains four gene clusters involved in the production of secondary metabolites, such as... Figure 11As shown, the lactococcin gene cluster is located at positions 1,706,159-1,716,488 nt in the genome. The brown-marked portion represents the core biosynthetic gene (GE001629), whose product belongs to the lactococcin family. It lies between GE001628 and GE001630, where GE001628 and GE001630 are different genes, respectively. Blastp comparison of the protein sequence of this core biosynthetic gene further confirmed a high degree of similarity to lactococcin family bacteriocins, suggesting that this bacteriocin may be related to the antibacterial activity of *Lactococcus lactis* GCC10.

[0097] 2.11.3 Annotation of genes related to probiotic function

[0098] Previous research has shown that most *Lactococcus lactis* strains that can become potential probiotics possess genes related to probiotic functions, such as tolerance to different temperatures, aggregation ability, adhesion ability, antibacterial ability, and antioxidant capacity. During the screening of relevant genes, it was found that some genes were not annotated in all databases. To prevent omissions, this invention aggregated information from all common databases; if a gene or function was annotated in any of these databases, it was considered to have the corresponding function. Among 2233 coding genes, 69 genes related to probiotic functions were annotated. Specific information is shown in Table 3.

[0099] Table 3. Annotation of genes related to probiotic function in strain GCC10

[0100]

[0101]

[0102] 3. Discussion

[0103] Lactococcus lactis has attracted attention due to its diverse phenotypic differences and various antagonistic mechanisms against pathogenic microorganisms, such as the production of antimicrobial secondary metabolites, competition for adhesion sites, and nutrients. Lactococcus lactis can synthesize a variety of metabolites, such as organic acids, hydrogen peroxide, lysozyme, and bacteriocins, which play important roles in disease inhibition. To further analyze the potential probiotic or antagonistic properties of the strain, this study evaluated the safety, probiotic properties, and potential biocontrol functions of Lactococcus lactis GCC10 using phenotypic and functional gene prediction methods.

[0104] The absence of hemolytic activity in isolates is a prerequisite for screening potential probiotics. Studies have shown that strains with β-hemolytic activity (such as some bacteria in the genera *Bacillus* and *Streptococcus*) may cause hemolytic anemia or secondary pathogen invasion risks in farmed animals. Therefore, in vitro tests were first conducted to determine the hemolytic activity of *Lactococcus lactis* GCC10, and the results showed that this strain did not have hemolytic activity. Artificial infection experiments also showed good safety. Furthermore, genomic prediction revealed that strain GCC10 does not possess common virulence genes (cylA, gelE, esp, agg, gelE, hyl, ace), suggesting that this strain has high biocompatibility.

[0105] Besides hemolytic activity and virulence genes, antibiotic susceptibility testing is also crucial for screening potential probiotics. Antibiotic resistance (AMR) is a major threat to global public health; therefore, the safety assessment of probiotics needs to pay particular attention to whether they carry resistance genes. If these strains carry or spread resistance genes during application, it may exacerbate the spread of drug-resistant pathogens. Studies have found that some lactic acid bacteria (such as Lactobacillus and Bifidobacterium) contain mobile resistance genes that can be transferred via transposons or plasmids, thus posing a potential health risk. In vitro drug susceptibility testing showed that strain GCC10 is resistant to aminoglycoside antibiotics. Genome annotation results revealed two classes of antibiotic resistance genes: aadA4 (GE000618), lmrD (GE000305), and lmrC (GE000304), which are associated with resistance to aminoglycosides and lincosamides. The mechanism of action of the aadA4 gene is to inactivate antibiotics, thereby achieving resistance to aminoglycoside antibiotics. The lmrC and lmrD genes reduce the intracellular concentration of antibiotics by effluxing antibiotic molecules, ultimately leading to antibiotic resistance in the strain. This invention found that GCC10 does not carry plasmids, the identified resistance genes are located on the genome, and no mobile genetic elements or transposase genes were found upstream or downstream of the resistance genes aad4, lmrC, and lmrD, suggesting a very low risk of horizontal transmission of these resistance genes. Furthermore, to improve the safety of this strain for industrial application, gene editing technology can be used to delete or mutate the aforementioned resistance genes, further enhancing the strain's biosafety.

[0106] Strain GCC10 also exhibited protease activity. The ability of probiotics to produce proteases typically indicates their capacity to hydrolyze protein molecules in feed, thereby enhancing intestinal function and improving nutrient absorption efficiency. Currently, plant proteins in aquatic feeds often contain anti-nutritional factors (such as phytic acid and tannins), which inhibit protein absorption. Probiotic-secreted proteases can break down these anti-nutritional factors, reducing their impact on protein digestion and further improving the nutritional value of the feed. The GCC10 genome also contains the proteolysis-related gene prtC (GE001976), which encodes a cell wall-binding serine protease that plays an important role in probiotics. This enzyme can cleave complex proteins (such as casein) into small peptides and amino acids, providing nutrients for bacteria and promoting strain growth. Furthermore, these peptide products may also exert biological activities, such as antibacterial activity or regulation of intestinal immune function, thereby enhancing the colonization and adaptability of *Lactococcus lactis* in the intestine, indicating that strain GCC10 has the potential to become a feed additive in fermented plant protein sources.

[0107] This invention predicts a gene cluster (GE001623-GE001632) related to lactococcin synthesis. Through annotation analysis using the TCDB database, the secretion mechanism of lactococcin in *Lactococcus lactis* was inferred. Gene annotation results show that the GCC10 genome contains multiple transporter genes related to lactococcin secretion, including the lactococcin A (GE001629) encoding gene and the ATP-binding cassette (ABC) superfamily-related transporter gene (GE001632). Lactococcin A belongs to the pore-forming toxin family and can form pores on the target cell membrane, thereby causing cell membrane rupture and target cell death. This characteristic suggests that lactococcin may have the potential for transmembrane action, directly penetrating or attaching to the cell membrane through pores. Secondly, gene annotation results also revealed the presence of a transporter (GE001632) associated with the ABC transporter superfamily in *Lactococcus lactis*, which is related to an active transport mechanism driven by PP bond hydrolysis. ATP-binding cassette (ABC) transporters are a widely distributed active transport system that uses ATP hydrolysis to provide energy for transporting various substrates, including proteins and small molecules, from the intracellular to the extracellular space. Therefore, this transporter may play a crucial role in the active transport and secretion of lactococcin A. In summary, the pore-forming characteristics of lactococcin A and the ABC transporter-mediated secretion mechanism may synergistically constitute its complete transmembrane transport and secretion pathway. Antagonism assays also showed that strain GCC10 exhibits antagonistic effects against *Aeromonas tempera*, *Bacillus melanogaster*, *Aeromonas hydrophila*, and *Aeromonas vesiculosus*. This indicates that strain GCC10 possesses the ability to antagonize common aquatic pathogens, and its antagonistic substances have significant research value in the prevention and control of bacterial diseases in aquatic animals. This study found that strain GCC10 has no antagonistic activity against Edwardsiella spp. Both Aeromonas and Edwardsiella are Gram-negative bacteria. Typically, the outer membrane of Gram-negative bacteria (composed of lipopolysaccharide and protein) acts as a barrier against bacteriocins from Gram-positive bacteria, thus most lactococcins are insensitive to them. It is speculated that the differences between Aeromonas and Edwardsiella may stem from differences in membrane structure or receptors; the relevant mechanisms require further in-depth analysis. Studies have shown that subtle sequence differences in the man-PTS components (IIC / IID subunits) between different bacterial species may determine the targeting specificity of bacteriocins. If the man-PTS structure or membrane lipid composition of Edwardsiella differs from that of Aeromonas, lactococcin A is difficult to bind and form pores.

[0108] Bile tolerance is a crucial indicator for screening probiotics, directly impacting their colonization and function in the host gut. Bile salt tolerance is a key characteristic for successful probiotic colonization in the gut. Based on the bile tolerance test results in this study, strain GCC10 exhibited good bile salt tolerance. Under bile conditions, strain GCC10 was initially inhibited; however, it subsequently adapted to the environment and achieved restorative proliferation after 6 hours, reaching 10^6 cells / day at 24 hours. 6 CFU / mL. This result indicates that strain GCC10 not only survives under bile stress but also exhibits strong environmental adaptability. Possible mechanisms include the strain's own bile salt hydrolase (BSH) activity, membrane structure stabilization mechanisms, and regulation of internal pH or extracellular secretion release. Bile salt hydrolase can bind bile acids, thereby reducing their destructive effect on the cell membrane, helping the strain maintain cell integrity and achieve proliferation. Intestinal colonization experiments also support its probiotic potential: after gavage into grass carp intestines, strain GCC10 survived for at least 168 hours. Experimental results showed that 12 hours after gavage, the colony count of strain GCC10 in the intestinal contents and intestinal wall remained at approximately 10. 6 The CFU / g level indicates that this strain possesses effective short-term colonization ability, enabling it to temporarily colonize and exert its effects within the host. The detection of probiotic-related genes also predicted a gene related to bile salt tolerance (cfa). These results collectively suggest that strain GCC10 exhibits good physiological tolerance and short-term intestinal colonization ability, indicating high probiotic application value. This also lays a solid foundation for subsequent research.

[0109] Studies have shown that most *Lactococcus lactis* strains that can be considered potential probiotics possess genes related to probiotic functions, such as tolerance to different temperatures and environments, aggregation ability, adhesion ability, antibacterial ability, and antioxidant capacity. This study also annotated many related genes. Most of these genes are related to immune regulation, suggesting that GCC10, as a feed additive or microbial preparation, may improve the immunity of aquatic animals, thereby reducing bacterial disease outbreaks. Furthermore, multi-database annotation of genes within the gene cluster revealed many genes related to the adaptability and survival ability of lactic acid bacteria under stress (ctsR, msrA, clpB, clpP) and genes related to the excretion of extracellular toxins or antimicrobial substances. This also indicates that *Lactococcus lactis* GCC10 can adapt to and survive in adverse environments.

[0110] 4. Conclusion

[0111] This study isolated a strain of *Lactococcus lactis* GCC10 with antagonistic antibacterial activity from the intestines of healthy grass carp. This bacterium lacks hemolytic activity but exhibits extracellular protease activity, broad-spectrum antibacterial activity, bile tolerance, and intestinal colonization ability. Biosafety tests showed that strain GCC10 was not pathogenic to grass carp at high infectious doses, and it was sensitive to common antimicrobial agents such as tetracyclines, amyl alcohols, macrolides, quinolones, and sulfonamides, indicating good biosafety. Furthermore, the bacterium showed good tolerance to grass carp bile and could colonize the grass carp intestine for a relatively long time. Co-infection of grass carp with the pathogen by strain GCC10 also showed excellent protective effects. Whole-genome sequencing analysis revealed the presence of genes related to stress response, bile salt tolerance, acid tolerance, immune regulation, proteolysis, and adhesion factors, as well as predicted gene clusters related to lactococcalin synthesis, indicating strong probiotic potential. In summary, this study has screened out a strain of Lactococcus lactis with good probiotic function and good biosafety, providing a new germplasm resource for the development of microbial preparations for aquaculture.

Claims

1. A strain of Lactococcus lactis with antagonistic activity and intestinal colonization ability, designated GCC10, and classified as Lactococcus lactis, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 24, 2025, with accession number GDMCC No: 66583.

2. The *Lactococcus lactis* as described in claim 1, characterized in that, The lactococcus lactis strain has antibacterial effects against Aeromonas hydrophila, Aeromonas merantii, Aeromonas hydrophila, Aeromonas vernix, Edwardsiella tarda, Staphylococcus aureus, Escherichia coli, Flavobacterium columnare, and Flavobacterium psychrophilum.

3. The use of Lactococcus lactis as described in claim 1 in the preparation of biocontrol agents for preventing and controlling harmful bacteria in aquatic bodies or bacterial diseases in aquatic animals.

4. The application as described in claim 3, characterized in that, The aquatic animal in question is a fish.

5. The application as described in claim 3, characterized in that, The bacterial diseases in aquatic animals are caused by one or more of the following: Aeromonas hydrophila, Aeromonas velutipes, Aeromonas versicolor, Edwardsiella tarda, Flavobacterium columnare, and Flavobacterium psychrophilum.

6. The application of Lactococcus lactis as described in claim 1 as an additive in the preparation of aquatic feed.

7. The application as described in claim 6, characterized in that, The feed is a fermented plant protein source feed.

8. A feed, characterized in that, Add the lactococcus lactis as described in claim 1.

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