Fish-derived plant lactobacillus and application thereof

Abstract

The invention relates to the technical field of microorganisms, and provides a plant lactobacillus strain which is separated from an intestinal tract sample of wild fish in a Danjiangkou reservoir. Experiments prove that the strain has relatively high acid resistance and bile salt tolerance, and has certain bacteriostatic ability to vibrio parahaemolyticus, vibrio alginolyticus, aeromonas veronii, aeromonas cainea and aeromonas hydrophila. The invention also provides an aquatic feed composition comprising such a strain. The aquatic feed composition containing the DY-JY3 strain can promote growth of largemouth black bass, improve immunity and digestive enzyme activity and adjust intestinal flora, so that the aquatic feed composition has remarkable potential in the aspect of being a probiotic candidate strain in aquaculture application, and a new microbial intervention strategy is provided for anti-infection treatment of fishes. The strain and the composition provided by the invention have wide application prospects in the field of aquatic products.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Lactobacillus flocculation, a fish-derived plant, and its applications. Background Technology

[0002] Probiotics are a class of live microorganisms that are beneficial to the health of the host. Since their definition in the 1970s, they have been widely used in the fields of human and animal health. Early probiotic research mainly focused on species such as Lactobacillus and Bifidobacterium, and they were widely used in regulating gastrointestinal function and enhancing immunity. However, traditional probiotics, such as terrestrial-derived lactic acid bacteria and yeast, have certain limitations in the application of fish farming. They may cause ecological imbalance or intestinal damage due to incompatibility with the native gut microbiota of fish. Screening for probiotics suitable for the fish gut has become a key research direction in aquaculture. In recent years, with the development of large-scale water conservancy projects, the structure of the aquatic microbial community in the water-receiving areas has changed, providing new resources for the screening of probiotics for the fish gut. By tapping into these potential high-quality probiotics, the development of highly efficient and adaptable probiotic preparations for aquaculture is expected to become an effective way to improve the gut health of fish, enhance immunity, and improve aquaculture efficiency.

[0003] my country is a major aquaculture country, accounting for over 60% of the world's total aquaculture production. However, the aquaculture process faces challenges such as high feed costs and frequent disease outbreaks. While existing formulated feeds have improved growth efficiency and feed conversion ratios to some extent, they remain expensive. Under intensive aquaculture models, high mortality rates caused by various pathogens result in significant losses to the industry, and the overuse of antibiotics has led to increased drug resistance and drug residues, necessitating the urgent need for green alternatives.

[0004] Lactic acid bacteria, a type of probiotic, has demonstrated various benefits in fish farming. These include improving the intestinal microecology, enhancing nutrient absorption, increasing specific growth rates and reducing feed conversion ratios, stimulating intestinal villus development, inhibiting pathogen adhesion, and activating both non-specific and specific immunity to improve disease resistance. However, traditional probiotics may not fully adapt to the intestinal environment. Screening native probiotics from the intestines of largemouth bass or local waters to develop highly adaptable formulations plays a crucial role in addressing feed utilization and disease control issues. Against this backdrop, this invention screens safe and highly effective native lactic acid bacteria strains from the intestines of wild fish in the Danjiangkou Reservoir. Utilizing their antibacterial properties, these strains optimize fish feed formulations and replace antibiotics, providing the industry with a green and sustainable solution for healthy aquaculture. Summary of the Invention

[0005] Based on the above issues, the applicant isolated and identified a strain of *Lactobacillus plantarum* from the intestines of wild fish in Danjiangkou Reservoir. This strain exhibits excellent growth performance, high acid resistance and bile salt tolerance, and certain antibacterial abilities against *Vibrio alginolyticus*, *Vibrio parahaemolyticus*, *Aeromonas vesiculosus*, *Aeromonas vulgaris*, and *Aeromonas hydrophila*. This strain can promote fish growth, enhance immunity, increase intestinal digestive enzyme activity, and regulate intestinal flora, thus demonstrating significant potential as a candidate probiotic strain for aquaculture applications and providing a new microbial intervention strategy for the anti-infection treatment of largemouth bass.

[0006] On one hand, the present invention provides a strain of Lactiplantibacillus plantarum, LP-DJY3, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251939, deposit date September 1, 2025. The address of the depository is Wuhan University, Wuhan, China, postcode: 430072, telephone: 027-68754052.

[0007] In this invention, Lactiplantibacillus plantarum LP-DJY3 can also be abbreviated as LP-DJY3, DJY3 or DJ-JY3, all of which refer to Lactiplantibacillus plantarum LP-DJY3.

[0008] In addition, *Lactobacillus plantarum* can also be called *Lactobacillus plantarum*.

[0009] On the other hand, the present invention also provides a method for culturing the above-mentioned *Lactobacillus plantarum*, the method comprising the step of culturing the strain using a culture medium.

[0010] In one embodiment, the culture medium is MRS medium; the culture temperature is 25℃-35℃, preferably 28℃; the culture time is 4h-48h; and the culture pH is 4-10, for example, 5, 6, 7, 8, or 9.

[0011] The *Lactobacillus plantarum* of this invention, when grown to the logarithmic phase, has an OD... 600 It can reach 10 or more, and its growth capacity far exceeds that of previously reported Lactobacillus plantarum.

[0012] On the other hand, the present invention also provides the use of the above-mentioned strains in inhibiting pathogens or in the preparation of reagents for inhibiting pathogens.

[0013] In one embodiment, the pathogen is selected from one or more of Vibrio parahaemolyticus, Vibrio alginolyticus, Aeromonas verrucosa, Aeromonas hydrophila, and Aeromonas vulgaris.

[0014] On the other hand, the present invention also provides the application of the above-mentioned strains in promoting fish growth or in the preparation of feed for promoting fish growth.

[0015] In one embodiment, the fish is selected from largemouth bass or butterfly carp.

[0016] On the other hand, the present invention also provides the use of the above-mentioned strains in the preparation of reagents for improving fish immunity, improving fish digestibility, or improving fish antioxidant capacity.

[0017] On the other hand, the present invention also provides the use of the above-mentioned strains in the preparation of reagents for treating and / or preventing fish pathogen infections.

[0018] In one embodiment, the pathogen is selected from Aeromonas versicolor.

[0019] In one embodiment, the fish is selected from largemouth bass.

[0020] Beneficial Effects: The *Lactobacillus plantarum* DJ-JY3 strain provided by this invention has multiple beneficial effects on largemouth bass farming. The isolated DJ-JY3 strain exhibits excellent in vitro probiotic properties, and its strong tolerance to low pH and high bile salts is a key prerequisite for its crossing of the digestive tract barrier and successful colonization. (The text then abruptly shifts to a seemingly unrelated topic about a specific strain, "5×10," which appears to be a fragment from a different document.) 8 After being added to the feed at a CFU / g dose and fed continuously for 42 days, the experimental group of largemouth bass showed statistically significant improvements in several core indicators: DJ-JY3 may have improved growth performance by increasing digestive enzyme activity, optimizing nutrient digestion and absorption, and promoting host growth; it increased nutrient absorption by increasing the height of intestinal villi, thereby expanding the absorption surface area; DJ-JY3 enhanced non-specific immunity by increasing LZM and AKP activity, and effectively alleviated oxidative stress by enhancing antioxidant capacity; and it could also effectively enhance the host's ability to resist infection; intestinal flora analysis showed that DJ-JY3 significantly increased the abundance of Lactobacillus plantarum in the experimental group, and it became the most dominant bacterial group and successfully colonized. It may have constructed a healthier intestinal microenvironment by regulating the microecological balance, inhibiting pathogens, or promoting the growth of symbiotic bacteria. In summary, Lactobacillus plantarum DJ-JY3 demonstrates significant application value due to its excellent in vitro tolerance and antibacterial ability, as well as its comprehensive promoting effects on the growth, digestion, intestinal structure, immunity, antioxidant capacity, disease resistance, and intestinal microbiota regulation of largemouth bass. Future research can focus on optimizing its large-scale production process and evaluating its application under different aquaculture conditions. Attached Figure Description

[0021] Figure 1 Isolation and identification of Lactobacillus plantarum DJ-JY3 (A: colony morphology; B: Gram staining results; C: phylogenetic tree constructed based on 16S rRNA gene sequence).

[0022] Figure 2 Growth curve and acid production capacity of Lactobacillus plantarum DJ-JY3.

[0023] Figure 3 Lactobacillus plantarum does not produce hemolysis (A: Lactobacillus plantarum DJ-JY3; B: Aeromonas hydrophila).

[0024] Figure 4 Lactobacillus plantarum DJ-JY3 exhibits in vitro antibacterial activity against (A: Vibrio parahaemolyticus, B: Vibrio alginolyticus, C: Aeromonas verrucosa, D: Aeromonas hydrophila, E: Aeromonas vulgaris).

[0025] Figure 5 Acid and bile salt tolerance of Lactobacillus plantarum DJ-JY3 (A: acid tolerance survival rate; B: choline tolerance survival rate).

[0026] Figure 6 Lactobacillus plantarum DJ-JY3 promotes the growth of largemouth bass.

[0027] Figure 7 Lactobacillus plantarum DJ-JY3 promotes the growth of butterfly carp.

[0028] Figure 8 Feeding largemouth bass with Lactobacillus plantarum DJ-JY3 enhances the non-specific immunity of their serum, liver, and intestines.

[0029] Figure 9 Feeding with Lactobacillus plantarum DJ-JY3 increases the activity of digestive enzymes (A: pepsin; B: lipase; C: amylase) in the liver and intestines of largemouth bass.

[0030] Figure 10 Feeding with Lactobacillus plantarum DJ-JY3 can improve the antioxidant capacity of serum, liver, and intestines in largemouth bass.

[0031] Figure 11 Feeding with Lactobacillus plantarum DJ-JY3 helps protect the intestinal morphology of largemouth bass.

[0032] Figure 12 The effects of DJ-JY3 on gut microbial diversity in largemouth bass (A: α-diversity analysis; B: β-diversity analysis).

[0033] Figure 13 Effects of DJ-JY3 on the intestinal microbial composition of largemouth bass (phylum level).

[0034] Figure 14 Effects of DJ-JY3 on the intestinal microbial composition of largemouth bass (genus level).

[0035] Figure 15 LEfSe analysis of gut microbiota.

[0036] Figure 16 The mortality rate of largemouth bass infected with Aeromonas versicolor. Detailed Implementation

[0037] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0038] Example 1: Isolation, screening and identification of Lactobacillus plantarum DJ-JY3

[0039] 1. Strains Isolation and Screening

[0040] Intestinal contents of wild fish from Danjiangkou Reservoir were ground and serially diluted 10-100 times. 50 μL of each diluted sample was plated on MRS solid medium and purified by multiple streak plating. The culture was incubated at 28℃ for 24 h. Single colonies of different morphologies of lactic acid bacteria were selected and purified twice by streak plating. Colony morphology was observed under a microscope. Results are shown below. Figure 1 As shown in Figure A. Figure 1 A represents the colony characteristics of DJ-JY3, which are uniform in size, round, smooth, slightly convex, and milky white colonies.

[0041] 2. Morphological identification

[0042] Gram staining was used to stain *Lactobacillus plantarum* DJ-JY3, and its morphology was observed under a microscope. The specific steps are as follows: *Lactobacillus plantarum* DJ-JY3 was inoculated into MRS solid medium and incubated at 28°C for 24 hours. A small amount of DJ-JY3 was picked up and evenly spread on a glass slide to form a bacterial film with a diameter of about 1 cm. The slide was allowed to dry naturally, and then fixed by quickly passing it through an alcohol lamp flame 3-4 times. Add ammonium oxalate crystal violet stain to the area coated with bacterial film, cover the film, and stain for 1-2 minutes. Rinse slowly with distilled water, add iodine solution, cover the film, and allow to act for 1-2 minutes. Rinse with distilled water again, then add 95% ethanol for destaining for 20-30 seconds, until the ethanol flowing out is no longer purple. Immediately rinse with distilled water, add safranin stain, and stain for 2-3 minutes. Rinse with distilled water again, then blot the slide dry with absorbent paper. Place the slide on the microscope stage, first locate the target under low magnification, then observe under oil immersion. Gram staining microscopy results show Gram-positive bacteria such as... Figure 1 B, in the form of short rods, exists singly or in pairs.

[0043] 3. Molecular identification

[0044] Molecular identification was performed using 16S rDNA. Universal primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; and 1492R: 5'-GGTTACCTTGTTACGACTT-3' were used to amplify and sequence the isolated strains.

[0045] The reaction system (20 μL) consisted of: 10 μL high-fidelity Taq DNA polymerase, 0.5 μL upstream primer 27F, 0.5 μL downstream primer 1492R, 8 μL DEPC water, and 1 μL DJ-JY3 bacterial culture. The reaction program was as follows: pre-denaturation at 94℃ for 3 minutes, denaturation at 94℃ for 30 seconds, annealing at 56.5℃ for 30 seconds, extension at 72℃ for 1 minute, and total extension at 72℃ for 10 minutes. The reaction products were subjected to electrophoresis, and PCR products matching the band size were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were analyzed using BLAST in the GenBank database.

[0046] PCR product sequencing results: The PCR amplification products of the strain were identified by 1% agarose gel electrophoresis, with band sizes of approximately 1500 bp. BLAST sequence comparison analysis of the sequencing results in the GenBank database showed that strain DJ-JY3 clustered with *Lactiplantibacillus plantarum* MT604806.1 within the same clade, exhibiting 100% homology. Figure 1 C. Based on its morphological, physiological and biochemical characteristics and molecular identification results, this strain was identified as *Lactobacillus plantarum*.

[0047] The strain was deposited at the China Center for Type Culture Collection (CCTCC) and named Lactiplantibacillus plantarum LP-DJY3, with accession number CCTCC NO: M 20251939, deposit date September 1, 2025. The address of the depository is Wuhan University, Wuhan, China, 430072, China, and the telephone number is 027-68754052.

[0048] In this invention, *Lactiplantibacillus plantarum* LP-DJY3 can be used, and it can also be abbreviated as LP-DJY3, DJY3, or DJ-JY3, all of which refer to *Lactiplantibacillus plantarum* LP-DJY3. Additionally, *Lactiplantibacillus plantarum* can also be called *Lactobacillus plantarum*.

[0049] Example 2: Biological activity of DJ-JY3

[0050] 1. Growth curve and acid production capacity of Lactobacillus plantarum DJ-JY3

[0051] The strain *Lactobacillus plantarum* DJ-JY3 was inoculated into MRS liquid medium and cultured at 28℃ for 24 hours to prepare the seed culture. A suitable amount of MRS liquid medium was added to a sterile Erlenmeyer flask, and the seed culture was inoculated at a rate of 1% and cultured at 28℃ for 24 hours. Every 2 hours or 1 hour, 1 mL of the bacterial culture was taken, with uninoculated MRS liquid medium as a blank control, and the OD value was measured at 600 nm. This was repeated three times, and the average value was taken. A growth curve of *Lactobacillus plantarum* DJ-JY3 was plotted with culture time as the x-axis and OD600 as the y-axis. After each OD value measurement, the pH of the bacterial culture was measured using a pH meter, and the data was recorded. This was repeated three times, and the average value was taken. A curve of acid production capacity was plotted with culture time as the x-axis and pH as the y-axis. By detecting the absorbance and pH value of the bacterial culture at 600 nm at different times, the growth curve and acid production curve of *Lactobacillus plantarum* DJ-JY3 were plotted. The results showed that... Figure 2 As shown, *Lactobacillus plantarum* DJ-JY3 exhibits a growth stasis phase from 0 to 4 hours, but acid production has already begun. From 4 to 16 hours, the absorbance increases significantly, indicating rapid growth. The absorbance reaches over 10, marking the start of the logarithmic growth phase. This phase also coincides with a rapid pH decrease. After 16 hours, it enters a stationary growth phase, where the pH value stabilizes at around 3.7, down from an initial 5.9. Compared to other reported strains, *Lactobacillus plantarum* DJ-JY3 demonstrates superior growth performance, achieving a cell concentration of 10⁻⁶ cells after entering the logarithmic growth phase. 10 CFU / mL or higher.

[0052] 2. Lactobacillus plantarum does not produce hemolysis.

[0053] The safety of *Lactobacillus plantarum* DJ-JY3 was determined using a hemolysis test. The isolated *Lactobacillus plantarum* DJ-JY3 was cultured overnight in MRS liquid medium to prepare a fresh bacterial suspension. The activated fresh *Lactobacillus plantarum* suspension and *Aeromonas hydrophila* were streaked onto blood agar plates, and the plates were labeled with names and numbers. The plates were then incubated overnight at 28°C for 24 hours, and the presence of hemolysis was observed. Results are as follows: Figure 3 As shown, the left figure ( Figure 3 A) After DJ-JY3 was cultured on a blood agar plate, the colonies were milky white with no hemolysis observed around them, as shown in the right figure. Figure 3 B) Colonies of Aeromonas hydrophila have a distinct hemolytic zone on blood agar plates, and the area around the colony is transparent.

[0054] 3. Lactobacillus plantarum DJ-JY3 exhibits in vitro antibacterial activity.

[0055] To explore the in vitro antibacterial effect of *Lactobacillus plantarum* DJ-JY3, DJ-JY3 was inoculated into 5 mL of MRS liquid medium and incubated statically at 28°C for 18-24 h. The culture medium was then removed, and the culture was centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was filtered through a 0.22 μm filter to remove residual bacteria and impurities, yielding a cell-free clear solution of DJ-JY3. Under aseptic conditions, five indicator bacteria—*Vibrio alginolyticus*, *Vibrio parahaemolyticus*, *Aeromonas vulgaris*, *Aeromonas vesiculosus*, and *Aeromonas hydrophila*—were inoculated into LB liquid medium and incubated overnight at 28°C. The medium was then diluted with physiological saline to a final concentration of 10 μL. 7 CFU / mL. Spread 100 μL evenly onto LB solid medium; punch wells using a 10-100 μL sterile pipette tip (6 mm diameter), then use the tip to remove the medium from the agar wells; add cell-free, clear DJ-JY3 solution to the wells, repeating for three wells as one group. Include one well as a physiological saline control; incubate at 28℃ for 24 h, and measure the diameter of the inhibition zone. Results are as follows. Figure 4 As shown, DJ-JY3 exhibited varying degrees of antibacterial effects against the indicator strains *Vibrio parahaemolyticus* (inhibition zone diameter: 21.33 mm), *Vibrio alginolyticus* (inhibition zone diameter: 16.37 mm), *Aeromonas hydrophila* (inhibition zone diameter: 15.67 mm), *Aeromonas vesiculosus* (inhibition zone diameter: 18.32 mm), and *Aeromonas vulgaris* (inhibition zone diameter: 15.63 mm). The antibacterial effect against *Vibrio parahaemolyticus* and *Aeromonas vesiculosus* was particularly strong.

[0056] 4. Acid and bile salt tolerance test of Lactobacillus plantarum DJ-JY3

[0057] The growth status of Lactobacillus plantarum DJ-JY3 under different pH values ​​and different concentrations of bile salts was investigated to determine its acid and bile salt tolerance and evaluate its probiotic properties. Prepare an appropriate amount of MRS medium, add different concentrations of choline chloride to the medium to prepare MRS medium containing 0.1%, 0.3%, 0.5%, and 0.7% bile salts. Dispense each concentration into three test tubes containing 5 mL of medium and autoclave for later use. Inoculate *Lactobacillus plantarum* DJ-JY3 into MRS liquid medium and incubate at 28°C for 24 hours. Collect the cells by centrifugation at 5000 rpm for 2 minutes and wash twice with sterile PBS. Inoculate the bacterial suspension at a 2% inoculation rate into MRS liquid medium with different pH values, different bile salt concentrations, and natural medium, and incubate at 28°C for 3 hours. Perform 10-fold serial dilutions of the cultured bacterial suspension, and spread 100 μL onto MRS solid medium. Incubate at 28°C for 24 hours. Perform viable colony testing on plates with colony counts between 30 and 300. Repeat each sample three times. Viable colony counts are expressed as CFU / mL. Results are as follows: Figure 5 As shown, strain DJ-JY3 can survive in environments with different pH levels and bile salt concentrations. The survival rate of DJ-JY3 increases with increasing pH. The left figure shows the acid tolerance test. The lowest survival rate (6.8%) was observed at pH 2, while the survival rates reached 16.8% and 46.1% at pH 3 and 4, respectively. At pH 5 and 6, the survival rates reached 84.1% and 97.4%, respectively. The right figure shows the choline tolerance test. DJ-JY3 showed survival rates of 95.4% and 89.1% in 0.1% and 0.3% choline salt concentrations, respectively. In 0.5% and 0.7% choline salt concentrations, the survival rates reached high levels of 57.9% and 57.8%, respectively, indicating that DJ-JY3 has good bile salt tolerance.

[0058] Example 3: The beneficial effects of DJ-JY3 on largemouth bass

[0059] 1. Animal safety study of Lactobacillus plantarum DJ-JY3

[0060] Animal safety experiments were conducted to provide scientific evidence for the rational use of *Lactobacillus plantarum* DJ-JY3 in animal feed and to determine its safety. The activated strain was washed with sterile PBS and diluted to a concentration of 2 × 10⁻⁶. 9 CFU / ml and 2×10 8 CFU / ml; Largemouth bass were purchased from a nearby fish fry farm. Healthy, undamaged largemouth bass fry weighing 6.7±0.2g were selected for a safety experiment. After being temporarily raised for one week, the largemouth bass fry were divided into three groups of 10 fish per group. Each group was injected intraperitoneally with 50μL of *Lactobacillus plantarum* DJ-JY3 bacteria at a concentration of 2×10⁻⁶. 8CFU / mL, 2×10 9 CFU / mL, while the control group was injected with an equal volume of sterile PBS; the health status and survival rate of largemouth bass injected with Lactobacillus plantarum DJ-JY3 were observed for 2 weeks, and their tissues were dissected and observed for lesions. After two weeks of continuous observation, the survival rate of largemouth bass injected with the DJ-JY3 strain was 100%, their growth was normal, and no histopathological changes were found in their internal organs after dissection.

[0061] 2. Effects of Lactobacillus plantarum DJ-JY3 on the growth performance of largemouth bass

[0062] The effect of *Lactobacillus plantarum* DJ-JY3 on the growth performance of largemouth bass was investigated by feeding them a diet containing DJ-JY3. The activated bacterial solution of DJ-JY3 was resuspended in PBS by centrifugation to prepare a bacterial solution with a concentration of 5 × 10⁻⁶. 8 The experimental feed (CFU / g) was dried in an air-conditioned room and then stored in sealed bags at 4℃. The experimental feed was prepared every two days, with the control feed serving as the basal diet. Largemouth bass fry were temporarily housed in the aquaculture room for one week before the experiment to ensure their health. 180 fry of uniform size were selected and randomly divided into two groups: an experimental group and a control group. Each group had three tanks, with 30 fish per tank. Fish were fed twice daily (8:00 AM and 5:00 PM) at 3% of their body weight for six weeks, with weekly weight measurements recorded. The rearing conditions were: water temperature 25±1℃, pH 7.5±0.1, nitrite <0.05mg / L, ammonia nitrogen <0.5mg / L, dissolved oxygen not less than 6.0mg / L, and water changed every two days. The number and total weight of largemouth bass in each tank were recorded before and weekly after the start of the feeding experiment. Figure 6 As shown, the weight gain of largemouth bass fed continuously for six weeks was significantly different (P<0.01).

[0063] 3. Effects of Lactobacillus plantarum DJ-JY3 on the growth performance of butterfly carp

[0064] On July 8, 2025, two 5m×5m×1m net cages were selected at the Jinzhuang Reservoir fish farm in Nanyang City. Each cage contained 235 butterfly carp, each initially weighing 1.75kg. The control group was fed normal feed, while the experimental group was fed feed sprayed with Lactobacillus plantarum DJ-JY3. The fish were manually fed twice daily (8:00 AM and 5:00 PM). Body weight was measured and recorded at 2 and 4 weeks. After 2 weeks, the control group weighed 4.06kg, and the experimental group weighed 4.5kg. After 4 weeks, the control group weighed 6.74kg, and the experimental group weighed 7.79kg. The difference in body weight gain was statistically significant (P<0.01). Figure 7 ).

[0065] 4. Detection of serum, liver, and intestinal immune markers in largemouth bass.

[0066] The effects of feeding largemouth bass with a diet containing *Lactobacillus plantarum* DJ-JY3 on their immune capacity were determined by detecting the activities of lysozyme (LZM) and alkaline phosphatase (AKP) in serum, liver, and intestines. A lysozyme (LZM) assay kit (Nanjing Jiancheng Bioengineering) was used to prepare the bacterial suspension and standards. The sample was diluted two-fold, and 200 μL was added to the corresponding bacterial suspension and mixed thoroughly. The mixture was incubated at 37°C for 15 min, then immediately placed in an ice-water bath below 0°C for 3 min. 200 μL of each sample was added to a 96-well plate, and the absorbance (A) was measured at 530 nm using a microplate reader. The lysozyme content (μg / mL) was calculated using the formula: Lysozyme content (μg / mL) = (Tassay - Tblank) / (Tstandard - Tblank) × Cstandard × N. The reaction solution was prepared using an alkaline phosphatase (AKP) test kit (Nanjing Jiancheng Bioengineering). The sample was diluted two-fold, and 50 μL was added to the corresponding reaction solution. The mixture was incubated at 37°C for 15 min, then 1.5 ml of colorimetric reagent was added, and the mixture was immediately mixed and allowed to stand at room temperature for 10 min. 200 μL of the reaction solution was then pipetted into each well of a 96-well plate, and the absorbance (A) was read at 520 nm using a microplate reader. The AKP activity was calculated using the formula: AKP activity (Kinsh units / 100 mL) = (Aassay / Astandard) × Cstandard × Vstandard × 100 mL / Vsample × N. The results are as follows: Figure 8 After feeding largemouth bass with Lactobacillus plantarum DJ-JY3 for 6 weeks, the activity of lysozyme (LZM) and alkaline phosphatase (AKP) in the serum, liver and intestine of the experimental group of largemouth bass was significantly higher than that in the control group.

[0067] 5. Effects of Lactobacillus plantarum DJ-JY3 on the activity of digestive enzymes in the liver of largemouth bass

[0068] The effects of feeding largemouth bass with a diet containing *Lactobacillus plantarum* DJ-JY3 on digestive enzymes were determined by detecting the activities of amylase, pepsin, and lipase in the liver and intestines. Amylase test kits (Nanjing Jiancheng Bioengineering), lipase assay kits (Nanjing Jiancheng Bioengineering), and pepsin kits (Nanjing Jiancheng Bioengineering) were used according to the instructions to detect the activities of amylase, pepsin, and lipase in the liver of largemouth bass. The results are as follows: Figure 9 As shown, after feeding largemouth bass with Lactobacillus plantarum DJ-JY3 for 6 weeks, the activities of digestive enzymes (pepsin, lipase and amylase) in the liver and intestine of largemouth bass were significantly higher than those in the control group.

[0069] 6. Determination of antioxidant capacity of Lactobacillus plantarum DJ-JY3 in serum, liver, and intestines of largemouth bass.

[0070] The effects of feeding largemouth bass with a diet containing *Lactobacillus plantarum* DJ-JY3 on their antioxidant capacity were determined by detecting the activities of CAT, GSH, MDA, and SOD in the serum, liver, and intestines of the fish. The activities of CAT, GSH, MDA, and SOD in the serum of largemouth bass were measured according to the manufacturer's instructions using a cellular malondialdehyde (MDA) assay kit (Nanjing Jiancheng Biotechnology Co., Ltd.), a total superoxide dismutase (SOD) assay kit (Nanjing Jiancheng Biotechnology Co., Ltd.), a reduced glutathione (GSH) assay kit (Nanjing Jiancheng Biotechnology Co., Ltd.), and a catalase (CAT) assay kit (Nanjing Jiancheng Biotechnology Co., Ltd.). The results are as follows: Figure 10 After feeding largemouth bass with Lactobacillus plantarum DJ-JY3 for 6 weeks, the activities of GSH, SOD, and CAT in the serum and intestines of the experimental group were significantly higher than those in the control group. The activities of SOD and CAT in the liver were also significantly higher than those in the control group. The activities of MDA in the serum, intestines, and liver were all significantly lower than those in the control group. This indicates that adding Lactobacillus plantarum DJ-JY3 to the feed of largemouth bass can improve the antioxidant capacity of largemouth bass.

[0071] 7. Effects of Lactobacillus plantarum DJ-JY3 on the histopathological morphology of largemouth bass intestines

[0072] The histopathological changes in the intestine of largemouth bass were observed by hematoxylin-eosin (H&E) staining, which directly revealed the changes in intestinal structure and morphology, and provided a basis for evaluating the probiotic effect of feeding with Lactobacillus plantarum DJ-JY3. Largemouth bass intestinal tissue was fixed in 4% paraformaldehyde at 4°C for 12 h. Then, it was dehydrated and embedded in paraffin using the following methods: (formalin 37°C, 10 min; formalin 37°C, 20 min; 80% ethanol 37°C, 30 min; 90% ethanol 37°C, 30 min; 95% ethanol 37°C, 1.5 h; 95% ethanol 37°C, 1.5 h; anhydrous ethanol 37°C, 1 h; anhydrous ethanol 37°C, 1 h; xylene 37°C, 20 min; xylene 37°C, 20 min; paraffin 65°C, 20 min; paraffin 65°C, 30 min; paraffin 65°C, 30 min; paraffin 65°C, 30 min). Subsequently, 2 μm sections were prepared using a microtome and baked at 65°C for 30 min. The sections were then processed according to the following method: (xylene 5 min, ... The staining process involved the following steps: xylene 3 min, 100% ethanol 30 s, 100% ethanol 30 s, 95% ethanol 30 s, 90% ethanol 30 s, rinsing with tap water for 10-30 s, hematoxylin staining for 10-15 min, rinsing with running water for 20 s, differentiating sections with 1% hydrochloric acid ethanol for 10 s, rinsing with running water for 15 min, staining with 1% eosin for 3 min, followed by a series of staining steps: 90% ethanol 30 s, 95% ethanol 30 s, 95% ethanol 30 s, 100% ethanol 30 s, 100% ethanol 30 s, 100% ethanol 30 s, carbolic acid xylene 30 s, xylene 30 s, xylene 30 s, xylene 30 s. The sections were mounted with neutral resin, and the stained intestinal tissue sections were observed under a microscope. The results were evaluated using SPSS software. Figure 11 As shown, after feeding largemouth bass with *Lactobacillus plantarum* DJ-JY3 in their feed for six weeks, the intestinal tissue structure of the fish showed a significant increase in villus height compared to the control group. While villus width increased and muscle layer thickness decreased, the difference was not statistically significant. *Lactobacillus plantarum* DJ-JY3 can improve the growth performance of largemouth bass, possibly by altering intestinal structure to promote nutrient digestion and absorption.

[0073] 8. Effects of feeding Lactobacillus plantarum DJ-JY3 on the diversity and composition of the gut microbiota of largemouth bass

[0074] The effect of feeding *Lactobacillus plantarum* DJ-JY3 on the gut microbiota of largemouth bass was determined by analyzing the microbial diversity of the intestinal contents. Largemouth bass fed for six weeks were placed on ice, dissected, and the intestines were removed. The intestinal contents were gently scraped out and immediately placed in cryovials, collected, and transported to the laboratory on dry ice for temporary storage at -80°C. Total genomic DNA was extracted from the microbial community using the soil DNA kit (Omega Bio-tek, Norcross, GA, US). Samples were sent to Shanghai Meiji Biomedical Technology Co., Ltd. for PCR amplification, sequencing library construction, and high-throughput sequencing data analysis. Results are as follows: Figure 12 As shown, DJ-JY3 induced a decrease in gut microbiota abundance and diversity, but there was no significant difference between the two groups. PCoA analysis revealed a clear separation between the microbiota of the experimental and control groups. Figure 13 As shown, the abundance of Firmicutes in the DJ-JY3 group was significantly higher than that in the control group. Figure 14 As shown, at the genus level, the abundance of *Lactobacillus plantarum* in group DJ-JY3 was significantly higher than that in the control group. Figure 15 The results show that feeding DJ-JY3 enabled Lactobacillus plantarum to colonize the intestines well and become the most dominant bacteria, thereby altering the intestinal microbial structure. In contrast, the abundance of harmful bacteria Mammaliicoccus and Bacteroides was relatively increased in the control group.

[0075] 9. Artificial infection experiment

[0076] The inhibitory effect of feeding *Lactobacillus plantarum* DJ-JY3 on pathogen infection in animals was investigated to provide data support for its production and application. Activated *Aeromonas vesiculosus* was centrifuged at 5000g for 10 min, washed three times with sterile PBS, and the concentration was adjusted to 2×10⁻⁶. 8 CFU / mL; Largemouth bass were infected via intraperitoneal injection. Twelve largemouth bass were selected for each group, and each fish was injected with 50 μL. Symptoms and mortality were observed and recorded daily for one week, and the mortality rate was calculated. Results are as follows: Figure 16 As shown, the survival rate of largemouth bass fed with DJ-JY3 was significantly higher than that of the blank control group. This indicates that *Lactobacillus plantarum* DJ-JY3 has a certain inhibitory effect on *Aeromonas vesiculosus* infection in vivo.

[0077] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A strain of *Lactiplantibacillus plantarum* LP-DJY3, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251939, deposited on September 1, 2025. The address of the depository is Wuhan University, Wuhan, China, 430072, China. Telephone: 027-68754052.

2. A method for culturing *Lactobacillus plantarum* as claimed in claim 1, the method comprising the step of culturing the strain using a culture medium.

3. The method according to claim 2, characterized in that, The culture medium is MRS medium, the culture temperature is 25℃-35℃, the culture time is 4h-48h, and the culture pH is 4-10.

4. The use of *Lactobacillus plantarum* as described in claim 1 in inhibiting pathogens or in the preparation of reagents for inhibiting pathogens.

5. The application according to claim 4, characterized in that, The pathogens are selected from one or more of Vibrio parahaemolyticus, Vibrio alginolyticus, Aeromonas verrucosa, Aeromonas hydrophila, and Aeromonas vulgaris.

6. The use of *Lactobacillus plantarum* as described in claim 1 in promoting fish growth or in the preparation of feed for promoting fish growth.

7. The application according to claim 6, characterized in that, The fish species were selected from largemouth bass or butterfly carp.

8. The use of the *Lactobacillus plantarum* as described in claim 1 in the preparation of reagents for improving fish immunity, fish digestibility, or fish antioxidant capacity.

9. The use of *Lactobacillus plantarum* as described in claim 1 in the preparation of reagents for treating and / or preventing pathogenic infections in fish.

10. The application according to claim 9, characterized in that, The pathogen was selected from Aeromonas versicolor.

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