Lactobacillus plantarum bacteriostatic agent and application thereof

Abstract

The invention discloses a lactobacillus plantarum bacteriostatic agent and application thereof, and relates to the technical field of microbial engineering. According to the lactobacillus plantarum LP2301 provided by the invention, fermentation liquor of the lactobacillus plantarum LP2301 can be used for effectively inhibiting aquatic pathogenic bacteria streptococcus agalactiae, limiting the growth of the streptococcus agalactiae, reducing the use of antibiotics, ensuring food safety and guaranteeing the health of animals and human beings by secreting metabolites such as bacteriostatic proteins and organic acids. Secondly, the invention provides an application of the lactobacillus plantarum bacteriostatic agent, and the lactobacillus plantarum LP2301 is compounded into the bacteriostatic agent; through multi-target cooperation of the cortex phellodendri extract and the rosmarinic acid, the antibacterial efficiency of single probiotics is remarkably optimized; active ingredients of cortex phellodendri directionally destroy a biological membrane barrier of streptococcus agalactiae and disintegrate an adhesion defense system of pathogenic bacteria; rosmarinic acid blocks a bacterial energy metabolism pathway synchronously, so that pathogens lose proliferative power; and exclusive bacteriocin secreted by the LP2301 accurately breaks down a cell membrane structure, so that fatal killing is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial engineering, in particular to a Lactobacillus plantarum antibacterial agent and application thereof. Background Art

[0002] In the field of traditional aquaculture disease prevention and control, systemic infections caused by Streptococcus agalactiae have become a major threat to the healthy development of the industry. This pathogen can form stubborn biofilms in aquatic animals, evade the effects of conventional drugs, and secrete virulence factors that damage gill tissue and hepatopancreatic function, leading to high mortality rates in juveniles, stunted growth in adults, and decreased reproductive capacity. Existing technologies often rely on single prevention and control methods: chemical antibiotics are prone to induce drug resistance and their residues harm the ecological environment; plant extracts, while relatively safe, have defects such as a narrow antibacterial spectrum and a short duration of action; probiotic preparations, while able to regulate the intestinal microecology, have difficulty penetrating the pathogen's biofilm barrier and are ineffective in controlling outbreaks of infection. Especially when Streptococcus agalactiae forms a dominant population in aquaculture water, its strong adhesion characteristics accelerate the spread of pathogens, causing the spread of epidemics across ponds or even regions. Traditional solutions often fail to address one issue while focusing on the other. Summary of the Invention

[0003] The purpose of the present invention is to provide a Lactobacillus plantarum antibacterial agent and application thereof, so as to solve the problems existing in the prior art.

[0004] In order to solve the above technical problems, the present invention provides the following technical solution: a Lactobacillus plantarum antibacterial agent, comprising the following preparation steps:

[0005] (1) Phellodendron amurense was ground into 40 mesh, 70% ethanol was added at a solid-liquid ratio of 1:15, and reflux extraction was performed twice at 85°C, each time for 1-2 hours; the extracts were combined, concentrated under reduced pressure until there was no alcohol smell, and freeze-dried to obtain the Phellodendron amurense extract;

[0006] (2) Rosemary leaves were extracted with supercritical CO2, and the extract was purified by macroporous resin AB-8, followed by ethanol gradient elution. The 60% ethanol elution fraction was collected, rotary evaporated, and freeze-dried to obtain high-purity rosmarinic acid;

[0007] (3) Add the Phellodendron chinense extract and rosmarinic acid to 120 mL of PBS buffer solution with a pH of 7.8, shake at 200 rpm in a 45°C water bath for 20 min, and then blend. After mixing, continue shaking in a 45°C water bath for 30 min to prepare a blend solution; slowly add the bacterial suspension to the blend solution and operate in an ice bath at 0-4°C; then add the trehalose solution that has been pre-cooled to 0-4°C, and emulsify it with a homogenizer at 8000 rpm for 5 min; vacuum freeze-dry, first pre-freeze at -40°C for 4 h, and then sublimate at 25°C for 24 h to prepare a composite antibacterial agent.

[0008] Furthermore, in step (2), the extraction pressure is 35 MPa and the temperature is 45°C.

[0009] Furthermore, the preparation method of the bacterial suspension in step (3) is as follows: the bacterial suspension is centrifuged at 6000 rpm for 10 min at 0-4°C to collect bacterial mud; the bacterial mud is then resuspended in sterile physiological saline with a concentration of 0.85% NaCl precooled to 0-4°C to adjust the concentration to 1×10 11 CFU / mL was used to prepare the bacterial suspension.

[0010] Furthermore, the strain in the bacterial suspension is Lactobacillus plantarum LP2301 strain, classified and named Lactobacillus plantarum. The strain was deposited in the General Microbiology Center of China Culture Collection Administration on September 4, 2024, with the address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO.31854.

[0011] Furthermore, the bacterial liquid is a bacterial liquid prepared by expanding the Lactobacillus plantarum LP2301 bacteria in MRS broth.

[0012] Furthermore, the concentration of the trehalose solution in step (3) is 5%.

[0013] Furthermore, in the step (3), the mass ratio of the Phellodendron amurense extract, rosmarinic acid and the Lactobacillus plantarum LP2301 suspension is 5-15:6-8:40-60.

[0014] Furthermore, any one of the aforementioned Lactobacillus plantarum antibacterial agents is used in the preparation of an aquatic feed antibacterial agent.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention provides an application of a Lactobacillus plantarum antibacterial agent, in which Lactobacillus plantarum LP2301 is compounded to prepare an antibacterial powder as an aquatic feed additive. Through the multi-target synergy of Phellodendron amurense extract and rosmarinic acid, the antibacterial efficacy of a single probiotic is significantly optimized. The active ingredients of Phellodendron amurense targetably destroy the biofilm barrier of Streptococcus agalactiae, disintegrating the pathogen's adhesion defense system. Rosmarinic acid simultaneously blocks the bacterial energy metabolism pathway, causing the pathogen to lose its proliferation power. The exclusive bacteriocin secreted by LP2301 accurately penetrates the cell membrane structure, achieving lethal killing. The three synergistically not only completely disintegrate the resistance mechanism of the pathogen, but also greatly improve the depth and speed of antibacterial efficiency, providing fundamental immune protection for aquatic animals. DETAILED DESCRIPTION

[0017] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0018] In order to more clearly illustrate the method provided by the present invention, the following examples are provided in detail, and the test methods for each index are as follows:

[0019] Antibacterial activity of the antibacterial agent: After sterilizing the LB agar medium at high temperature and high pressure, pour it into a culture dish, with 15 mL in each culture dish (lower layer). After solidification, a sterile Oxford cup (inner diameter 6 mm, outer diameter 8 mm, height 10 mm) is placed vertically on the surface of the culture medium and gently pressed so that it contacts the culture medium without a gap. 5-10 mL of the culture medium is poured into the culture dish (upper layer); after it is completely solidified, the Oxford cup is taken out; a sterile cotton swab is dipped in an appropriate amount of Streptococcus agalactiae liquid and evenly coated on the LB solid plate medium in step 3; the antibacterial agents prepared in the examples and comparative examples are placed on the solid culture medium, and the antibacterial rate against Streptococcus agalactiae is observed.

[0020] Example 1

[0021] (1) Phellodendron amurense was ground into 40 mesh, 70% ethanol was added at a material-liquid ratio of 1:15, and reflux extraction was performed twice at 85°C, each time for 1 hour. The extracts were combined, concentrated under reduced pressure until there was no alcohol smell, and freeze-dried at -40°C for 5 hours to obtain the Phellodendron amurense extract.

[0022] (2) Rosemary leaves were extracted with supercritical CO2 at a pressure of 35 MPa and a temperature of 45°C. The extract was purified by macroporous resin AB-8 and then eluted with an ethanol gradient. The 60% ethanol elution fraction was collected, rotary evaporated, and freeze-dried at -40°C for 3 h to obtain high-purity rosmarinic acid.

[0023] (3) The experiment was conducted in a dairy farm in Daqing City, Heilongjiang Province, with a dairy cow inventory of 3,000 heads and an average annual milk production of 10,900 kg for lactating cows. The lactating cows were kept in semi-open barns and milked three times a day. Lactobacillus plantarum was isolated from the milk of 74 healthy cows. The selected 74 cows had not received antibiotic treatment and had an average parity of 3. Before collecting milk samples, the milking area was disinfected. The collected milk samples were placed in an ice bath and Lactobacillus plantarum was isolated within 3 hours. In a sterile environment, 10 μL of milk sample was evenly spread on MRS solid culture medium, allowed to stand for 10 minutes, and then inverted and cultured in 37°C culture medium for 12 hours. The different morphological characteristics of the colonies were used to identify the isolates. For preliminary screening, carefully pick a single colony with an inoculating loop and culture it in a new MRS solid medium in a 37°C incubator for 12 hours using the three-line inoculation method. Culture the purified single colony in MRS broth for 12 hours until the bacterial solution becomes turbid. If a single colony cannot be picked, use an inoculating loop to pick the mixed colony and continue to culture it in a new MRS medium using the three-line inoculation method until a single colony can be picked and expanded to obtain Lactobacillus plantarum LP2301 bacteria. Centrifuge the obtained bacterial solution at 6000 rpm for 10 minutes at 0°C to collect the bacterial sludge. Then resuspend the bacterial sludge with sterile saline with a concentration of 0.85% NaCl precooled to 0°C and adjust the concentration to 1×10 11 CFU / mL to prepare the Lactobacillus plantarum LP2301 bacterial suspension;

[0024] (4) Add 120 mL of PBS buffer solution with a pH of 7.8 to the Phellodendron amurense extract and rosmarinic acid, respectively, shake at 200 rpm in a 45°C water bath for 20 min, then blend, and continue shaking in a 45°C water bath for 30 min after mixing to obtain a blend solution; slowly add the LP2301 bacterial suspension to the blend solution, and operate in an ice bath at 0°C; then add 8% trehalose by mass of the solution that has been pre-cooled to 0°C, and emulsify at 8000 rpm in a homogenizer for 5 min; vacuum freeze-dry, first pre-freeze at -40°C for 4 h, and then sublimate at 25°C for 24 h to obtain a composite antibacterial agent; wherein the mass ratio of the Phellodendron amurense extract, rosmarinic acid and Lactobacillus plantarum LP2301 bacterial suspension is 5:6:40.

[0025] Example 2

[0026] (1) Phellodendron amurense was ground into 40 mesh, 70% ethanol was added at a material-liquid ratio of 1:15, and reflux extraction was performed twice at 85°C, each time for 1.5 h. The extracts were combined, concentrated under reduced pressure until there was no alcohol smell, and freeze-dried at -40°C for 5 h to obtain the Phellodendron amurense extract.

[0027] (2) Rosemary leaves were extracted with supercritical CO2 at a pressure of 35 MPa and a temperature of 45°C. The extract was purified by macroporous resin AB-8 and then eluted with an ethanol gradient. The 60% ethanol elution fraction was collected, rotary evaporated, and freeze-dried at -40°C for 3 h to obtain high-purity rosmarinic acid.

[0028] (3) The experiment was conducted in a dairy farm in Daqing City, Heilongjiang Province, with a dairy cow inventory of 3,000 heads and an average annual milk production of 10,900 kg for lactating cows. The lactating cows were kept in semi-open barns and milked three times a day. Lactobacillus plantarum was isolated from the milk of 74 healthy cows. The selected 74 cows had not received antibiotic treatment and had an average parity of 3. Before collecting milk samples, the milking area was disinfected. The collected milk samples were placed in an ice bath and Lactobacillus plantarum was isolated within 3 hours. In a sterile environment, 10 μL of milk sample was evenly spread on MRS solid culture medium, allowed to stand for 10 minutes, and then inverted and cultured in 37°C culture medium for 15 hours. The different morphological characteristics of the colonies were used to identify the isolates. For preliminary screening, carefully pick a single colony with an inoculating loop and culture it in a new MRS solid medium in a 37°C incubator for 15 hours using the three-line inoculation method. Culture the purified single colony in MRS broth for 15 hours until the bacterial solution becomes turbid. If a single colony cannot be picked, use an inoculating loop to pick a mixed colony and continue to culture it in a new MRS medium using the three-line inoculation method until a single colony can be picked and expanded to obtain Lactobacillus plantarum LP2301 bacteria. Centrifuge the obtained bacterial solution at 6000 rpm for 10 minutes at 2°C to collect the bacterial sludge. Then resuspend the bacterial sludge with sterile saline with a concentration of 0.85% NaCl precooled to 2°C and adjust the concentration to 1×10 11 CFU / mL to prepare the Lactobacillus plantarum LP2301 bacterial suspension;

[0029] (4) Add the Phellodendron amurense extract and rosmarinic acid to 120 mL of PBS buffer solution with a pH of 7.8, shake at 200 rpm in a water bath at 45°C for 20 min, and then blend. After mixing, continue shaking in a water bath at 45°C for 30 min to obtain a mixed solution; slowly add the LP2301 bacterial suspension to the mixed solution, and operate in an ice bath at 2°C; then add 8% trehalose by mass of the solution that has been pre-cooled to 2°C, and emulsify at 8000 rpm in a homogenizer for 5 min; vacuum freeze-dry, first pre-freeze at -40°C for 4 h, and then sublimate at 25°C for 24 h to obtain a composite antibacterial agent; wherein the mass ratio of Phellodendron amurense extract, rosmarinic acid and Lactobacillus plantarum LP2301 bacterial suspension is 10:7:50.

[0030] Example 3

[0031] (1) Phellodendron amurense was ground into 40 mesh, 70% ethanol was added at a material-liquid ratio of 1:15, and reflux extraction was performed twice at 85°C, each time for 2 hours. The extracts were combined, concentrated under reduced pressure until there was no alcohol smell, and freeze-dried at -40°C for 5 hours to obtain the Phellodendron amurense extract.

[0032] (2) Rosemary leaves were extracted with supercritical CO2 at a pressure of 35 MPa and a temperature of 45°C. The extract was purified by macroporous resin AB-8 and then eluted with an ethanol gradient. The 60% ethanol elution fraction was collected, rotary evaporated, and freeze-dried at -40°C for 3 h to obtain high-purity rosmarinic acid.

[0033] (3) The experiment was conducted in a dairy farm in Daqing City, Heilongjiang Province, with a dairy cow inventory of 3,000 heads and an average annual milk production of 10,900 kg for lactating cows. The lactating cows were kept in semi-open barns and milked three times a day. Lactobacillus plantarum was isolated from the milk of 74 healthy cows. The selected 74 cows had not received antibiotic treatment and had an average parity of 3. Before collecting milk samples, the milking area was disinfected. The collected milk samples were placed in an ice bath and Lactobacillus plantarum was isolated within 3 hours. In a sterile environment, 10 μL of milk sample was evenly spread on MRS solid culture medium, allowed to stand for 10 minutes, and then inverted and cultured in 37°C culture medium for 18 hours. The different morphological characteristics of the colonies were used to identify the isolates. For preliminary screening, carefully pick a single colony with an inoculating loop and culture it in a new MRS solid medium in a 37°C incubator for 18 hours using the three-line inoculation method. Culture the purified single colony in MRS broth for 18 hours until the bacterial solution becomes turbid. If a single colony cannot be picked, use an inoculating loop to pick a mixed colony and continue to culture it in a new MRS medium using the three-line inoculation method until a single colony can be picked and expanded to obtain Lactobacillus plantarum LP2301 bacteria. Centrifuge the obtained bacterial solution at 6000 rpm for 10 minutes at 4°C to collect the bacterial slurry. Then resuspend the bacterial slurry in sterile saline with a concentration of 0.85% NaCl precooled to 4°C and adjust the concentration to 1×10 11 CFU / mL to prepare the Lactobacillus plantarum LP2301 bacterial suspension;

[0034] (4) Add the Phellodendron amurense extract and rosmarinic acid to 120 mL of PBS buffer solution with a pH of 7.8, shake at 200 rpm in a 45°C water bath for 20 min, and then blend. After mixing, continue shaking in a 45°C water bath for 30 min to prepare a blended solution; slowly add the LP2301 bacterial suspension to the blended solution, and operate in an ice bath at 4°C; then add 8% trehalose by mass of the solution that has been pre-cooled to 4°C, and emulsify at 8000 rpm in a homogenizer for 5 min; vacuum freeze-dry, first pre-freeze at -40°C for 4 h, and then sublimate at 25°C for 24 h to obtain a composite antibacterial agent; wherein the mass ratio of Phellodendron amurense extract, rosmarinic acid and Lactobacillus plantarum LP2301 bacterial suspension is 15:8:60.

[0035] Comparative Example 1

[0036] The difference between Comparative Example 1 and Example 3 is that there is no step (1), and step (4) is changed to: rosmarinic acid is added to 120 mL of PBS buffer solution with a pH of 7.8, and the mixture is shaken in a water bath at 45° C. at a speed of 200 rpm for 20 min to obtain a rosmarinic acid solution; LP2301 bacterial suspension is slowly added to the rosmarinic acid solution, and the mixture is operated in an ice bath at 2° C.; then trehalose with a mass% of 8% of the solution precooled to 4° C. is added, and the mixture is emulsified at 8000 rpm by a homogenizer for 5 min; vacuum freeze drying is performed, first pre-freezing at -40° C. for 4 h, and then subliming at 25° C. for 24 h to obtain a composite antibacterial agent; wherein the mass ratio of rosmarinic acid to the Lactobacillus plantarum LP2301 bacterial suspension is 8:60; and the remaining steps are the same as those in Example 3.

[0037] Comparative Example 2

[0038] The difference between Comparative Example 2 and Example 3 is that there is no step (2), and step (4) is changed to: add the Phellodendron chinense extract to 120 mL of PBS buffer solution with a pH of 7.8, and shake it in a water bath at 45°C at a speed of 200 rpm for 20 minutes to obtain a Phellodendron chinense extract solution; slowly add the LP2301 bacterial suspension to the Phellodendron chinense extract solution, and operate in an ice bath at 2°C; then add 8% trehalose by mass of the solution pre-cooled to 4°C, and emulsify it at 8000 rpm in a homogenizer for 5 minutes; vacuum freeze-dry, first pre-freeze at -40°C for 4 hours, and then sublimate at 25°C for 24 hours to obtain a composite antibacterial agent; wherein, the mass ratio of the Phellodendron chinense extract to the Lactobacillus plantarum LP2301 bacterial suspension is 15:60; the remaining steps are the same as in Example 3.

[0039] Comparative Example 3

[0040] The difference between Comparative Example 3 and Example 3 is that there is no step (3), and step (4) is changed to: add 120 mL of PBS buffer solution with pH = 7.8 to the Phellodendron chinense extract and rosmarinic acid, respectively, shake in a water bath at 45°C at a speed of 200 rpm for 20 min, and then blend. After mixing, continue to shake in a water bath at 45°C for 30 min to obtain a blended solution, and then add 8% trehalose by mass of the solution pre-cooled to 4°C, and emulsify at 8000 rpm in a homogenizer for 5 min; vacuum freeze-dry, first pre-freeze at -40°C for 4 h, and then sublimate at 25°C for 24 h to obtain a composite antibacterial agent; wherein, the mass ratio of Phellodendron chinense extract and rosmarinic acid is 15:8; the remaining steps are the same as Example 3.

[0041] Effect Examples

[0042] Table 1 below shows the performance analysis results of the antibacterial agents of Examples 1 to 3 of the present invention and Comparative Examples 1 to 3.

[0043] Table 1

[0044] Antibacterial rate (%) Diameter of inhibition zone (mm) Biofilm removal rate (%) Example 1 82.5 22.7 83.6 Example 2 86.7 25.1 87.2 Example 3 94.3 28.9 93.5 Comparative Example 1 60.2 17.3 68.4 Comparative Example 2 52.9 14.6 60.8 Comparative Example 3 35.7 10.4 47.2

[0045] From the comparison of the experimental data on the antibacterial properties of the examples and the comparative examples, it can be found that the present invention provides a Lactobacillus plantarum LP2301, whose fermentation broth can effectively inhibit the aquatic pathogen Streptococcus agalactiae by secreting antibacterial proteins, organic acids and other metabolites, limit the growth of Streptococcus agalactiae, reduce the use of antibiotics, ensure food safety, and protect the health of animals and humans. The present invention provides an application of a Lactobacillus plantarum antibacterial agent, and compound Lactobacillus plantarum LP2301 to prepare an antibacterial agent as an aquatic feed additive; through the multi-target synergy of Phellodendron chinense extract and rosmarinic acid, the antibacterial efficacy of a single probiotic is significantly optimized; the active ingredients of Phellodendron chinense directionally destroy the biofilm barrier of Streptococcus agalactiae and disintegrate the adhesion defense system of the pathogen; rosmarinic acid simultaneously blocks the bacterial energy metabolism pathway, causing the pathogen to lose its proliferation power; and the exclusive bacteriocin secreted by LP2301 accurately penetrates the cell membrane structure to achieve fatal killing. The synergy of the three not only completely disintegrates the resistance mechanism of the pathogen, but also greatly improves the depth and speed of the antibacterial efficiency, providing root immune protection for aquatic animals.

[0046] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be encompassed therein. Any reference in a claim should not be construed as limiting the claim to which it relates.

Claims

1. A plant lactobacillus antibacterial agent, characterized in that The method comprises the following preparation steps: (1) Phellodendron amurense was ground into 40 mesh, 70% ethanol was added at a solid-liquid ratio of 1:15, and reflux extraction was performed twice at 85°C, each time for 1-2 hours; the extracts were combined, concentrated under reduced pressure until there was no alcohol smell, and freeze-dried to obtain the Phellodendron amurense extract; (2) Rosemary leaves were extracted with supercritical CO2, and the extract was purified by macroporous resin AB-8, followed by ethanol gradient elution. The 60% ethanol elution fraction was collected, rotary evaporated, and freeze-dried to obtain high-purity rosmarinic acid; (3) Add the Phellodendron chinense extract and rosmarinic acid to 120 mL of PBS buffer solution with a pH of 7.8, shake at 200 rpm in a 45°C water bath for 20 min, and then blend. After mixing, continue shaking in a 45°C water bath for 30 min to prepare a blend solution; slowly add the bacterial suspension to the blend solution and operate in an ice bath at 0-4°C; then add the trehalose solution that has been pre-cooled to 0-4°C, and emulsify it with a homogenizer at 8000 rpm for 5 min; vacuum freeze-dry, first pre-freeze at -40°C for 4 h, and then sublimate at 25°C for 24 h to prepare a composite antibacterial agent.

2. A plant lactobacillus antibacterial agent according to claim 1, characterized in that, In step (2), the extraction pressure is 35 MPa and the temperature is 45°C.

3. A plant lactobacillus antibacterial agent according to claim 1, characterized in that, The preparation method of the bacterial suspension in step (3) is as follows: the bacterial suspension is centrifuged at 6000 rpm for 10 min at 0-4°C to collect bacterial mud; the bacterial mud is then resuspended in sterile physiological saline with a concentration of 0.85% NaCl pre-cooled to 0-4°C to adjust the concentration to 1×10 11 CFU / mL was used to prepare the bacterial suspension.

4. A plant lactobacillus antibacterial agent according to claim 3, characterized in that, The strain in the bacterial suspension is the Lactobacillus plantarum LP2301 strain, which was deposited in the General Microbiology Center of the China Culture Collection Administration on September 4, 2024, with the address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCCNO.31854.

5. A plant lactobacillus antibacterial agent according to claim 3, characterized in that, The bacterial liquid is prepared by expanding and culturing Lactobacillus plantarum LP2301 cells in MRS broth.

6. A plant lactobacillus antibacterial agent according to claim 1, characterized in that, The concentration of the trehalose solution in step (3) is 5%.

7. A plant lactobacillus antibacterial agent according to claim 1, characterized in that, In the step (3), the mass ratio of the Phellodendron amurense extract, rosmarinic acid and the Lactobacillus plantarum LP2301 bacterial suspension is 5-15:6-8:40-60.

8. Use of the Lactobacillus plantarum antibacterial agent according to any one of claims 1 to 7 in the preparation of an aquatic feed antibacterial agent.