Composite bacteriostatic agent for preventing and controlling streptococcicosis of tilapia as well as preparation method and application of composite bacteriostatic agent

Abstract

The invention discloses a composite bacteriostatic agent for preventing and controlling streptococcicosis of tilapia mossambica as well as a preparation method and application thereof. The compound bacteriostatic agent is prepared from 25 percent to 30 percent of gallnut tannin nano microcapsules, 25 percent to 35 percent of eucommia ulmoides chlorogenic acid-chitosan compound, 10 percent to 20 percent of baicalin phospholipid compound, 5 percent to 15 percent of bacillus subtilis GS-02 metabolite freeze-dried powder and the balance of acidified modified attapulgite. By adding 1.5% of the compound bacteriostatic agent into feed, the accumulative incidence rate of streptococcicosis can be remarkably reduced to 5.3%, the weight gain rate is increased by 22.1%, the feed coefficient is reduced to 1.21, and no enrofloxacin drug residue exists. The compound bacteriostatic agent is suitable for various application scenes such as feed addition, water splashing and slow-release gels; the sustained-release gel block is especially suitable for a recirculating aquaculture system, and can maintain an effective antibacterial concentration for 30 days when being suspended in a sustained-release gel block form according to 200g per 10m < 3 > of water body, so that the accumulated infection rate of streptococcus is reduced to be less than or equal to 1.2%.

Description

Technical Field

[0001] This invention relates to the field of aquaculture disease prevention and control technology, and in particular to a compound antibacterial agent for the prevention and control of streptococcal disease in tilapia, its preparation method, and its application. Background Technology

[0002] Tilapia streptococcal disease is caused by *Streptococcus agalactiae* and / or *Streptococcus dolphinus*, severely hindering the development of aquaculture. Current technologies have the following shortcomings:

[0003] 1. Huge economic losses: The global tilapia farming industry loses more than US$1 billion annually due to this disease; in China's main producing areas, the average morbidity rate is 35.6% ± 5.7%, and the mortality rate exceeds 80%.

[0004] 2. Antibiotic abuse: The latest monitoring in 2024 showed that the detection rate of enrofloxacin-resistant bacteria in China's main tilapia producing areas rose to 58.3%, an increase of 11.8 percentage points compared with 2022 (46.5%), and the proportion of multidrug-resistant bacteria (MDR) reached 21.5%.

[0005] 3. Ecological environment damage: Nitrifying bacteria activity inhibition rate ≥70%, recovery period ≥15 days.

[0006] 4. Poor antibacterial effect: Chinese patent CN107347944A discloses a streptococcal inhibitor for tilapia farming ponds and its preparation method, which uses 23 kinds of Chinese herbal medicines in combination. The MIC value against streptococci is only 78.3μg / mL, indicating low antibacterial efficacy. The synergy between components was not verified, and the evaluation of the inhibitory effect on drug-resistant strains was not involved.

[0007] 5. Restricted formulation and application: CN201210048881A (a microbial preparation for the prevention and treatment of streptococcal disease in tilapia): limited to powder form, lacks sustained-release design, requires frequent dosing; the interference mechanism on biofilm and quorum sensing system has not been investigated.

[0008] 6. Lack of control over drug resistance: Chinese patent CN201410568884.0 discloses a traditional Chinese medicine composition for the prevention and treatment of streptococcal disease in aquatic animals, but does not evaluate the inhibitory effect on enrofloxacin-resistant strains, and has a high risk of clinical failure; it also does not disclose environmental safety data (such as degradation rate and ecotoxicity).

[0009] Therefore, in order to solve the above-mentioned technical problems, providing a compound antibacterial agent for the prevention and control of streptococcal disease in tilapia, as well as its preparation method and application, is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0010] In view of this, the present invention provides a compound antibacterial agent for the prevention and control of streptococcal disease in tilapia, its preparation method and application.

[0011] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0012] A compound antibacterial agent for controlling streptococcal disease in tilapia, comprising the following components by weight percentage:

[0013] Gallnut tannin nanocapsules: 25%–30%; Eucommia ulmoides chlorogenic acid-chitosan complex: 25%–35%; Baicalin phospholipid complex: 10%–20%; Bacillus subtilis GS-02 metabolite lyophilized powder: 5%–15%; Acidified modified attapulgite: balance;

[0014] The Bacillus subtilis GS-02 strain was isolated and preserved by the Freshwater Aquaculture Research Laboratory of the Guangxi Zhuang Autonomous Region Fisheries Research Institute, with accession number GXIFR-2020-002.

[0015] Preferably, the encapsulation efficiency of the Eucommia ulmoides chlorogenic acid-chitosan complex is ≥90% in the pH range of 4.0 to 6.0;

[0016] The particle size of the gallnut tannin nanocapsules is 80±20nm;

[0017] The minimum inhibitory concentration of the compound antibacterial agent against enrofloxacin-resistant Streptococcus agalactiae is ≤20 μg / mL;

[0018] The compound antibacterial agent can reduce the expression levels of gyrA, ermB, and tetM genes in drug-resistant strains by ≥3.8 times, ≥5.2 times, and ≥4.1 times, respectively, compared with the control group.

[0019] A method for preparing a compound antibacterial agent for controlling streptococcal disease in tilapia, comprising the following steps:

[0020] Step 1: Carrier Modification

[0021] The attapulgite soil is processed, washed, and calcined to obtain a modified attapulgite soil carrier.

[0022] Step 2: Preparation of nanocapsules

[0023] Gallnut extract was mixed with β-cyclodextrin and placed in a water bath for ultrasonic treatment to obtain gallnut tannin nanocapsules.

[0024] Step 3: Complex Synthesis

[0025] Chlorogenic acid and chitosan were subjected to an ionic cross-linking reaction to form an Eucommia ulmoides chlorogenic acid-chitosan complex:

[0026] Baicalin and soybean lecithin were dissolved in anhydrous ethanol, and then this ethanol solution was slowly injected into the aqueous phase while stirring continuously. Finally, the ethanol was evaporated to remove the ethanol, and the baicalin lecithin complex was obtained.

[0027] Step 4: Mixing and Loading

[0028] The gallnut tannin nanocapsules obtained in step 2, the Eucommia ulmoides chlorogenic acid-chitosan complex and baicalin phospholipid complex obtained in step 3, and the freeze-dried powder of Bacillus subtilis GS-02 metabolite are mixed evenly. Then, the mixture is adsorbed using the acidified modified attapulgite carrier obtained in step 1. Finally, the loaded material is centrifuged and spray-dried, and the dried product is granulated or ultra-finely pulverized according to the application requirements.

[0029] Preferably, in step 1, the attapulgite is treated with a 0.5 mol / L HCl solution for 3 hours, washed with water until neutral, and finally calcined at 500°C for 2 hours; the resulting modified attapulgite carrier has a pore size of 10–40 nm and a specific surface area ≥180 m². 2 / g.

[0030] Preferably, in step 2, gallnut extract and β-cyclodextrin are mixed at a molar ratio of 1:5, placed in a 50°C water bath, and ultrasonically treated at 300W power for 40 minutes in pulse mode: 5 seconds working, 5 seconds interval, to obtain gallnut tannin nanocapsules with a particle size of 80±20nm.

[0031] Preferably, in step 3, chlorogenic acid and chitosan are subjected to an ionic crosslinking reaction at pH 5.0±0.1 and 45±1℃ for 3 hours;

[0032] Baicalin and soybean lecithin were dissolved in anhydrous ethanol at a mass ratio of 1:2. This ethanol solution was then slowly injected into the aqueous phase at 60°C and stirred continuously for 1 hour.

[0033] In step 4, the inlet air temperature for drying is 160±5℃; the outlet air temperature is 70±5℃; and the resulting dried product is granulated at ≤60℃ or ultra-finely pulverized according to application requirements.

[0034] The application of a compound antibacterial agent for controlling streptococcal disease in tilapia in the preparation of tilapia feed additives, wherein the amount of compound antibacterial agent added to the feed additive is 0.5% to 2.0% of the total weight of the feed; the granulation temperature is ≤60℃, and the activity retention rate of the compound antibacterial agent is ≥95%.

[0035] Application of a compound antibacterial agent for controlling streptococcal disease in tilapia in disinfection or disease control of aquaculture water, wherein the compound antibacterial agent is prepared as a water spray powder with a particle size distribution D 90 ≤20μm; dosage is 0.3~0.8g / m 3 water body.

[0036] Application of a compound antibacterial agent for preventing streptococcal disease in tilapia in the preparation of a sustained-release gel, wherein the sustained-release gel comprises a cross-linked structure of sodium alginate and calcium chloride;

[0037] per 10m 3 Dosage for suspending 200g slow-release gel blocks in aquaculture water;

[0038] The drug release from the sustained-release gel conforms to the kinetic equation Ct = 42.3e -0.021t R 2 =0.98, and the sustained-release half-life calculated based on this equation is ≥33 days.

[0039] Application of a compound antibacterial agent for controlling streptococcal disease in tilapia in disease control of recirculating aquaculture systems: The compound antibacterial agent is prepared into a slow-release gel block, and administered at a rate of 10 m³ / min. 3 A 200g gel block was suspended in the water and placed in a mesh bag 1 meter from the system outlet.

[0040] The sustained-release gel block is prepared by the following process:

[0041] (a) The composite antibacterial agent is mixed with sodium alginate, wherein sodium alginate accounts for 12% w / w of the total weight of the gel block;

[0042] (b) Cross-linking and curing are achieved by dripping in a 4% w / v calcium chloride solution;

[0043] (c) Dry to a moisture content of ≤8% to form gel particles with a diameter of approximately 5 mm;

[0044] The sustained-release gel block maintains an equivalent concentration of gallnut tannins in the water of ≥22.1 μg / L for 30 days. The sustained-release kinetic equation is: Ct=42.3e -0.021t R 2 =0.98.

[0045] The present invention achieves the following technical effects compared to the prior art:

[0046] 1. Highly effective synergistic antibacterial activity: MIC value reaches 12.5-15.6 μg / mL (50%-69% lower than enrofloxacin), FIC=0.28 (strong synergy); MIC ≤18.7 μg / mL against enrofloxacin-resistant strains (MIC≥128 μg / mL);

[0047] 2. Long-lasting and sustained release: Acid-modified attapulgite carrier with a specific surface area ≥180m² 2 / g, the slow-release equation Ct=42.3e -0.021t (R 2 =0.98);

[0048] 3. Improved breeding efficiency: Feeding with feed containing 1.5% of this compound antibacterial agent for 60 days reduced the cumulative morbidity rate by 83.5% (5.3% vs 35.6%) compared with the blank control group, increased the weight gain rate by 22.1% (423.5g vs 346.8g), and reduced the feed conversion ratio by 20.4% (1.21 vs 1.52);

[0049] 4. Environmentally friendly: The natural degradation rate reaches 92.7% ± 2.1% in 28 days, and the large Daphnia magna 48h-EC 50 >100mg / L (actually non-toxic), *Crescentia argyrophylla* 96h-IC 50 >200mg / L, nitrifying bacteria inhibition rate <5%;

[0050] 5. Flexible formulation: It can be prepared as feed additives (granulation temperature ≤60℃, activity retention ≥95%), water spraying powder (D... 90 It has multiple application forms, including ≤20μm and sustained-release gel (half-life ≥33 days calculated based on kinetic equations). Attached Figure Description

[0051] none. Detailed Implementation

[0052] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0053] This invention discloses a compound antibacterial agent for controlling streptococcal disease in tilapia, comprising the following components by weight percentage:

[0054] Gallnut tannin nanocapsules: 25%–30%; Eucommia ulmoides chlorogenic acid-chitosan complex: 25%–35%; Baicalin phospholipid complex: 10%–20%; Bacillus subtilis GS-02 metabolite lyophilized powder: 5%–15%; Acidified modified attapulgite: balance;

[0055] Bacillus subtilis GS-02 was isolated and preserved in the Freshwater Aquaculture Research Laboratory of the Guangxi Zhuang Autonomous Region Fisheries Research Institute, with accession number GXIFR-2020-002.

[0056] The encapsulation efficiency of the Eucommia ulmoides chlorogenic acid-chitosan complex is ≥90% in the pH range of 4.0 to 6.0;

[0057] The particle size of the gallnut tannin nanocapsules is 80±20nm;

[0058] The minimum inhibitory concentration of the compound antibacterial agent against enrofloxacin-resistant Streptococcus agalactiae is ≤20 μg / mL;

[0059] The compound antibacterial agent can downregulate the expression levels of gyrA, ermB, and tetM genes in drug-resistant strains by ≥3.8 times, ≥5.2 times, and ≥4.1 times, respectively, compared with the control group.

[0060] Synergistic antibacterial mechanism:

[0061] Fourth-order synergistic effect (partially inhibiting concentration FIC index = 0.28):

[0062] 1. Gallnut tannin nanocapsules disrupt biological membrane structures;

[0063] 2. The chlorogenic acid-chitosan complex from Eucommia ulmoides improves membrane permeability;

[0064] 3. Baicalin phospholipid complex inhibits DNA replication;

[0065] 4. Bacillus subtilis GS-02 blocking quorum sensing system.

[0066] Verification of molecular interaction mechanisms:

[0067] Fluorescence polarization experiments showed that the chlorogenic acid-chitosan complex of Eucommia ulmoides could increase the binding rate of gallnut tannins to biomembrane polysaccharides (the dissociation constant Kd value decreased from 28.5 μM to 12.3 μM);

[0068] Molecular docking simulations confirmed that the baicalin phospholipid complex forms a hydrogen bond network (binding energy ≤ -7.2 kcal / mol) with lipopeptides in Bacillus subtilis metabolites, jointly targeting the LuxS gene promoter region.

[0069] When any component is missing, the MIC value rises to ≥38.5 μg / mL (FIC>0.8, synergistic loss).

[0070] Further verification by real-time quantitative PCR (qPCR) (Example 9) showed that the compound antibacterial agent could significantly downregulate the expression of gyrA, ermB, and tetM resistance genes in enrofloxacin-resistant strains (downregulation by 3.8 to 5.2 times), blocking the transmission pathway of drug resistance.

[0071] This invention also discloses a method for preparing a compound antibacterial agent for preventing and controlling streptococcal disease in tilapia, comprising the following steps:

[0072] Step 1: Carrier Modification

[0073] The attapulgite soil is processed, washed, and calcined to obtain a modified attapulgite soil carrier.

[0074] Step 2: Preparation of nanocapsules

[0075] Gallnut extract was mixed with β-cyclodextrin and placed in a water bath for ultrasonic treatment to obtain gallnut tannin nanocapsules.

[0076] Step 3: Complex Synthesis

[0077] Chlorogenic acid and chitosan were subjected to an ionic cross-linking reaction to form an Eucommia ulmoides chlorogenic acid-chitosan complex:

[0078] Baicalin and soybean lecithin were dissolved in anhydrous ethanol, and then this ethanol solution was slowly injected into the aqueous phase while stirring continuously. Finally, the ethanol was evaporated to remove the ethanol, and the baicalin lecithin complex was obtained.

[0079] Step 4: Mixing and Loading

[0080] The gallnut tannin nanocapsules obtained in step 2, the Eucommia ulmoides chlorogenic acid-chitosan complex and baicalin phospholipid complex obtained in step 3, and the freeze-dried powder of Bacillus subtilis GS-02 metabolite were mixed evenly. Then, the mixture was adsorbed using the acidified modified attapulgite carrier obtained in step 1. Finally, the loaded material was centrifuged and spray-dried, and the dried product was granulated or ultra-finely pulverized according to the application requirements.

[0081] In step 1, the attapulgite was treated with 0.5 mol / L HCl solution for 3 hours, washed with water until neutral, and finally calcined at 500℃ for 2 hours; the resulting modified attapulgite carrier had a pore size of 10–40 nm and a specific surface area ≥180 m². 2 / g;

[0082] In step 2, gallnut extract and β-cyclodextrin were mixed at a molar ratio of 1:5 and placed in a 50°C water bath. The mixture was then ultrasonically treated at 300W power for 40 minutes in pulse mode: 5 seconds of operation followed by 5 seconds of interval, to obtain gallnut tannin nanocapsules with a particle size of 80±20nm.

[0083] In step 3, chlorogenic acid and chitosan were subjected to an ionic crosslinking reaction at pH 5.0±0.1 and 45±1℃ for 3 hours.

[0084] Baicalin and soybean lecithin were dissolved in anhydrous ethanol at a mass ratio of 1:2. This ethanol solution was then slowly injected into the aqueous phase at 60°C and stirred continuously for 1 hour.

[0085] In step 4, the inlet air temperature for drying is 160±5℃; the outlet air temperature is 70±5℃. The resulting dried product is then granulated at ≤60℃ or subjected to ultrafine grinding according to application requirements.

[0086] This invention also discloses the application of a compound antibacterial agent for the prevention and control of streptococcal disease in tilapia in the preparation of tilapia feed additives. The amount of compound antibacterial agent added to the feed additive is 0.5% to 2.0% of the total weight of the feed; the granulation temperature is ≤60℃; and the activity retention rate of the compound antibacterial agent is ≥95%.

[0087] This invention also discloses the application of a compound antibacterial agent for controlling streptococcal disease in tilapia in the disinfection or disease control of aquaculture water. The compound antibacterial agent is prepared as a water spray powder with a particle size distribution D. 90 ≤20μm; dosage is 0.3~0.8g / m 3 water body.

[0088] This invention also discloses the application of a composite antibacterial agent for controlling streptococcal disease in tilapia in the preparation of a sustained-release gel, wherein the sustained-release gel comprises a cross-linked structure of sodium alginate and calcium chloride; per 10m 3 Dosage for suspending 200g slow-release gel blocks in aquaculture water; drug release from the slow-release gel conforms to the kinetic equation Ct = 42.3e -0.021t R 2 =0.98, and the sustained-release half-life calculated based on this equation is ≥33 days.

[0089] This invention also discloses the application of a compound antibacterial agent for controlling streptococcal disease in tilapia in the prevention and control of diseases in recirculating aquaculture systems. The compound antibacterial agent is prepared into a slow-release gel block, and is applied at a rate of 10m³ / min. 3 A 200g gel block was suspended in the water and placed in a mesh bag 1 meter from the system outlet.

[0090] The sustained-release gel blocks are prepared through the following process:

[0091] (a) The composite antibacterial agent is mixed with sodium alginate, wherein sodium alginate accounts for 12% w / w of the total weight of the gel block;

[0092] (b) Cross-linking and curing are achieved by dripping in a 4% w / v calcium chloride solution;

[0093] (c) Dry to a moisture content of ≤8% to form gel particles with a diameter of approximately 5 mm;

[0094] The sustained-release gel block maintains an equivalent concentration of gallnut tannins in the water of ≥22.1 μg / L for 30 days. The sustained-release kinetic equation is: Ct=42.3e -0.021t R 2 =0.98.

[0095] experiment:

[0096] 1. Antibacterial efficacy (MIC assay)

[0097] 1.1 Experimental Design

[0098] Tested strains: Streptococcus agalactiae GD strain and Streptococcus dolphinae GX-01 strain (isolated from diseased tilapia in a fish farm and identified by 16S rRNA gene sequencing).

[0099] Tested strains: Streptococcus agalactiae GD strain, Streptococcus dolphinae GX-01 strain (isolated from diseased tilapia in a fish farm and identified by 16S rRNA gene sequencing), and enrofloxacin-resistant strain S. agalactiae GD-MDR (MIC≥128μg / mL).

[0100] Culture medium: Mueller-Hinton broth (pH 7.2±0.1).

[0101] Drug dilution: A two-fold serial dilution method was used, with a concentration range of 200 μg / mL to 0.39 μg / mL.

[0102] Inoculation concentration: 5×10 5 CFU / mL (equivalent to 0.5 McFarland turbidity standard).

[0103] Culture conditions: Incubate at 30℃ for 24 hours.

[0104] MIC determination criteria: the lowest drug concentration at which no visible bacterial growth is observed (a solvent control and a positive control (such as enrofloxacin) are set up in the experiment).

[0105] Synergistic effect verification: The partial inhibitory concentration index (FIC) was determined using the checkerboard method. A FIC ≤ 0.5 was considered a synergistic effect.

[0106] Repeatability: The experiment was independently repeated 3 times, and each experiment contained 3 technical replicates.

[0107] The results of the minimum inhibitory concentration (MIC) comparison (μg / mL) are shown in Table 1.

[0108] Table 1: Comparison of Minimum Inhibitory Concentration (MIC) (μg / mL)

[0109]

[0110] Note: FIC index = 0.28 (strong synergy); different superscript letters indicate significant differences between groups (p < 0.05, n = 9).

[0111] 2. Aquaculture Experiment

[0112] 2.1 Experimental Design:

[0113] Experimental animals: Nile tilapia (Oreochromis niloticus), with an initial average weight of (50.2±3.5) g / fish.

[0114] Experimental Groups:

[0115] Experimental group: Feed containing 1.5% (w / w) of the compound antibacterial agent of this invention.

[0116] Antibiotic control group: fed with feed supplemented with 50 mg / kg (w / w) enrofloxacin.

[0117] Blank control group: fed with basic feed.

[0118] Aquaculture system: Open-air cement ponds (specifications: 10m×10m×1.5m), each pond stocked with 10,000 fish, with 3 parallel ponds per group. During the aquaculture period, the water temperature is maintained at 28-32℃, the dissolved oxygen concentration is ≥5mg / L, and the pH value is maintained at 7.0-7.8.

[0119] Trial period: 60 days (including July and August, when streptococcal disease is most prevalent).

[0120] 2.2 Disease surveillance:

[0121] Observe and record clinical symptoms of tilapia daily, including exophthalmos, meningitis (such as balance disorder and head swelling), and spiral swimming.

[0122] For fish that showed clinical symptoms or died, brain and liver tissues were aseptically collected and inoculated onto trypsin-soybean agar (TSA) medium for bacterial isolation and culture.

[0123] For the isolated suspected streptococcal colonies, species-specific gene identification was performed using PCR: the cfb gene was used to detect *Streptococcus agalactiae* (S. agalactiae), and the sip gene was used to detect *Streptococcus dolphinii* (S. iniaae).

[0124] 2.3 Data Collection and Analysis:

[0125] Weight gain: At the beginning and end of the experiment, the fish in each pool were weighed on an empty stomach (accurate to 0.1g) and the average weight gain was calculated.

[0126] Feed conversion ratio (FCR): Feed conversion ratio = total feed amount / total weight gain of fish.

[0127] Drug residue detection: At the end of the experiment, fish meat samples from the experimental group and the antibiotic control group were collected, and the enrofloxacin residue was detected by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) in accordance with the method specified in the national standard GB 31650-2019 National Food Safety Standard Maximum Residue Limits for Veterinary Drugs in Food.

[0128] Statistical analysis: The experimental data were analyzed by one-way ANOVA using SPSS 22.0 statistical software. If the difference was significant (p<0.05), Duncan's multiple comparison method was used to test the significance of the difference between groups.

[0129] The results of the 60-day aquaculture trial are shown in Table 2.

[0130] Table 2: Results of the 60-day culture trial

[0131]

[0132] Note: Different letters on the shoulder indicate significant differences between groups (p<0.05, n=3 pools).

[0133] 3. Environmental safety

[0134] 28-day natural degradation rate (OECD 301D): 92.7% ± 2.1%;

[0135] Ecotoxicity: Daphnia magna 48h-EC 50 (Half-maximal effective concentration) > 100 mg / L (practically non-toxic); *Gnaphalium affine* 96h-IC 10 (10% inhibition concentration) = 85 mg / L;

[0136] Nitrifying bacteria inhibition rate (ISO 15522:1999): <5%.

[0137] Example 1: Comparison of Carrier Modification Process Optimization

[0138] This embodiment examines the effect of calcination temperature on the properties of the carrier:

[0139] 1. Method: Referring to the carrier modification steps in the invention, calcination temperature groups of 400℃ and 500℃ were set respectively;

[0140] 2. Results: The specific surface area of ​​the carrier calcined at 500℃ reached 182±5m². 2 / g, with a sustained-release rate of 89.7% over 28 days; the corresponding value for the calcined group at 400℃ was (115±10)m. 2 / g and 62.3%;

[0141] 3. Conclusion: Calcination at 500℃ significantly improves the specific surface area and sustained-release performance of the carrier. This optimization ensures that the carrier's kinetic behavior conforms to the first-order sustained-release equation: Ct = 42.3e -0.021t (R 2 =0.98) is a key condition.

[0142] Example 2: Preparation and application of water spraying powder

[0143] Formula (by weight percentage):

[0144] Gallnut tannin nanocapsules: 15%

[0145] Eucommia ulmoides chlorogenic acid-chitosan complex: 20%

[0146] Baicalin phospholipid complex: 10%

[0147] Bacillus subtilis GS-02 metabolite lyophilized powder: 5%

[0148] Acid-modified attapulgite: 50%

[0149] Preparation process:

[0150] 1. Prepare each component according to steps 1-3 of the invention description.

[0151] 2. Mix the above components with the freeze-dried powder of Bacillus subtilis GS-02 metabolite evenly.

[0152] 3. The mixture was adsorbed using an acid-modified attapulgite carrier.

[0153] 4. The loaded material is then subjected to centrifugal spray drying (inlet air temperature: 160±5℃; outlet air temperature: 70±5℃).

[0154] 5. The powder obtained from spray drying is then subjected to ultrafine grinding to control the particle size distribution and achieve D 90 ≤20μm, to obtain water spraying powder.

[0155] Application: at 0.5g / m 3 For water dosage, evenly sprinkle the powder into the aquaculture water, apply once a day, and continue for 3 days.

[0156] Example 3: Physical stability of powdered agents applied to water bodies

[0157] Objective: To evaluate the water spraying powder (D) described in Example 2. 90 Suspension stability (≤20μm).

[0158] method:

[0159] The zeta potential of powders was determined according to the international standard ISO 13321:2025 Particle size analysis by photon correlation spectroscopy.

[0160] The settling rate of powder in water was measured.

[0161] After standing for 30 minutes, the suspension rate was measured.

[0162] result:

[0163] Zeta potential: -32.5±1.8mV

[0164] Settlement rate: 0.08 cm·min -1

[0165] Suspension rate after standing for 30 minutes: ≥95%

[0166] Conclusion: This powder has a high absolute value of Zeta potential (an absolute value >30mV usually indicates good stability of the dispersion system) and a low settling rate. After standing for 30 minutes, it can still maintain a suspension rate of ≥95%, which meets the requirements for suspension stability in water spraying applications.

[0167] Example 4: Comparison of the effects of water emergency rescue applications

[0168] 1. Experimental Design

[0169] Grouping and processing scheme:

[0170] Emergency treatment kit of this invention: 0.7g / m 3 The dosage of water body is as follows: apply the water body spraying powder prepared in Example 2 once a day for 5 consecutive days.

[0171] Chemical disinfectant control group: Potassium persulfate compound salt disinfectant was sprayed at a dose of 0.3 ppm (mg / L) once a day for 5 consecutive days.

[0172] Blank control group: No drug treatment was given.

[0173] Table 3 shows a comparative analysis of the effectiveness of emergency water treatment.

[0174] Table 3: Comparative Analysis of the Effectiveness of Emergency Treatment for Water Bodies

[0175]

[0176] Note: * indicates a significant difference compared to the control group (p<0.05).

[0177] Example 5: Preparation and application of sustained-release gels

[0178] Formula (by weight percentage):

[0179] The active components of the compound antibacterial agent are: 10% gallnut tannin nanocapsules + 15% eucommia chlorogenic acid-chitosan complex + 8% baicalin phospholipid complex + 8% lyophilized powder of Bacillus subtilis GS-02 metabolites (total 41%).

[0180] Carrier and gel matrix: 47% acid-modified attapulgite + 12% sodium alginate.

[0181] Preparation process:

[0182] Prepare gallnut tannin nanocapsules, eucommia chlorogenic acid-chitosan complex, and baicalin phospholipid complex according to steps 1-3 of the invention.

[0183] The active components of the compound antibacterial agent are mixed evenly with the carrier and the gel matrix.

[0184] The mixture was dripped into a 4% (w / v) calcium chloride (CaCl2) aqueous solution for ionic crosslinking and curing.

[0185] Collect the cross-linked gel particles and wash them with deionized water to remove residual CaCl2 from the surface.

[0186] The gel particles were dried at 40°C until the water content was ≤8%, resulting in sustained-release gel particles with a diameter of approximately 5 mm.

[0187] Application: Use a dosage of 200 grams of gel block per 10 cubic meters of aquaculture water. Place the gel block in a mesh bag and suspend it in the water (1 meter away from the outlet). This can maintain an effective antibacterial concentration for at least 96 hours.

[0188] Example 6: Application of Recirculating Aquaculture System

[0189] plan:

[0190] System: 100m 3 Tilapia recirculating aquaculture system.

[0191] Antibacterial agent form and dosage: Using the sustained-release gel block prepared according to the process in Example 5, at a dosage of 10m³ / day... 3 A dosage of 200g gel block is suspended in the water. The gel block is placed in a mesh bag and suspended about 1 meter away from the system outlet.

[0192] Monitoring period: 30 days.

[0193] Monitoring indicators and methods:

[0194] 1. Concentration of total antibacterial active substances in water: expressed as the equivalent concentration (μg / L) of the key component gallotannin.

[0195] 2. Cumulative Streptococcal Infection Rate (%):

[0196] Monitoring targets: Streptococcus dolphinii (S.iniae) and Streptococcus agalactiae (S.agalactiae).

[0197] Methods: Thirty fish were randomly selected each week, and brain tissue was aseptically collected and processed separately.

[0198] PCR detection of sip gene in Streptococcus dolphinus (primers: F: 5'-GCTTGATCGCGTTGGTAA-3', R: 5'-AGCAACGCGAAGAACCTTA-3');

[0199] PCR detection of CFB gene in Streptococcus agalactiae (primers: F: 5'-TTTCACCAGCTGTATTAGA-3', R: 5'-GTTCCCTGAACATTATCTTT-3').

[0200] 3. Changes in the microbial community of the system biofilm: The composition of the biofilm microorganisms (at the genus level) was analyzed by 16S rRNA high-throughput sequencing, and the ratio of probiotics (such as Bacillus, Lactobacillus, etc.) to potential pathogens (such as Aeromonas, Streptococcus, etc.) was calculated.

[0201] The monitoring results of the circulating water system are shown in Table 4.

[0202] Table 4: Monitoring Results of Circulating Water System Application

[0203]

[0204] Note: * indicates that the concentration value at this time point is statistically significant compared with the initial value (day 0) (p<0.05); ↑ indicates an increase in the proportion of probiotics, and ↓ indicates a decrease in the proportion of pathogenic bacteria.

[0205] Example 7: Preparation and Application of Feed Additives

[0206] Formula (by weight percentage):

[0207] Gallnut tannin nanocapsules: 25%

[0208] Eucommia ulmoides chlorogenic acid-chitosan complex: 35%

[0209] Baicalin phospholipid complex: 20%

[0210] Bacillus subtilis GS-02 metabolite lyophilized powder: 15%

[0211] Acid-modified attapulgite: 5%

[0212] Preparation process:

[0213] 1. Prepare each component according to steps 1-3 of the invention description.

[0214] 2. Mix the above components with the freeze-dried powder of Bacillus subtilis GS-02 metabolite evenly.

[0215] 3. The mixture was adsorbed using an acid-modified attapulgite carrier.

[0216] 4. The loaded material is then subjected to centrifugal spray drying (inlet air temperature: 160±5℃; outlet air temperature: 70±5℃).

[0217] 5. The powder obtained by spray drying is granulated in a fluidized bed at ≤60℃ to obtain granular feed additives with a particle size of 0.8~1.2mm.

[0218] Application: Add this feed additive to tilapia feed at a ratio of 1.5% (w / w) of the total feed weight, and feed twice a day.

[0219] Example 8: Verification of the effect of aquaculture application during high-temperature season

[0220] 1. Test conditions

[0221] Location: A tilapia farming base in Nanning, Guangxi.

[0222] Test unit: Each group consists of 3 parallel cement pools.

[0223] Experimental animals: Nile tilapia (Oreochromis niloticus), with an initial average weight of (80±10) g / fish.

[0224] Stocking density: 15kg / m 3 .

[0225] Environmental parameters: water temperature (32±2)℃, dissolved oxygen ≥5mg / L.

[0226] Feed: The crude protein content of the basic feed should be ≥28%.

[0227] 2. Experimental groups (3 ponds per group, 3000 fish per pond)

[0228] Experimental group: Feed containing 1.8% (w / w) of the compound antibacterial agent of this invention (prepared according to the formulation of Example 7).

[0229] Positive control group: fed with feed containing 0.2% (w / w) of commercially available compound Chinese herbal medicine preparation (main ingredients include Scutellaria baicalensis, rhubarb, etc.).

[0230] Blank control group: fed with basic feed.

[0231] The results of aquaculture under high temperature (32±2℃) conditions are shown in Table 5.

[0232] Table 5: Aquaculture results under high temperature (32±2℃) conditions

[0233]

[0234] Note: SOD (superoxide dismutase); CFU (colony-forming unit). Different superscript letters indicate significant differences between groups (p<0.05, n=3 pools).

[0235] Example 9: Validation of drug resistance gene expression inhibition (qPCR)

[0236] 1. Experimental Design

[0237] Test strain: Enrofloxacin-resistant Streptococcus agalactiae GD-MDR (MIC ≥ 128 μg / mL)

[0238] Drug treatment: Add 1 / 2 MIC (9.4 μg / mL) of the compound antibacterial agent of this invention.

[0239] Detection method:

[0240] Total RNA extraction: Thermo Fisher

[0241] cDNA synthesis: PrimeScrip TM RT kit (Takara)

[0242] qPCR: SYBR Green method (CFX96, Bio-Rad), primer sequences are shown in Table 6:

[0243] Table 6:

[0244]

[0245] Data Analysis: Gene expression levels were calculated using the 2ΔΔCt method.

[0246] 2. The results are shown in Table 7;

[0247] Table 7:

[0248] Drug resistance genes Changes in expression levels (drug group / control group) Reduce multiplier p-value (t-test) gyrA 0.26±0.03 ↓3.8 <0.01 ermB 0.19±0.02 ↓5.2 <0.001 tetM 0.24±0.04 ↓4.1 <0.01

[0249] Conclusion: The compound antibacterial agent can simultaneously inhibit the expression of multiple drug resistance genes and block bacterial drug resistance pathways.

[0250] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A compound antibacterial agent for controlling streptococcal disease in tilapia, characterized in that, Includes the following components by weight percentage: Gallnut tannin nanocapsules: 25%–30%; Eucommia ulmoides chlorogenic acid-chitosan complex: 25%–35%; Baicalin phospholipid complex: 10%–20%; Bacillus subtilis GS-02 metabolite lyophilized powder: 5%–15%; Acidified modified attapulgite: balance; The Bacillus subtilis GS-02 strain was isolated and preserved by the Freshwater Aquaculture Research Laboratory of the Guangxi Zhuang Autonomous Region Fisheries Research Institute, with accession number GXIFR-2020-002.

2. The compound antibacterial agent for controlling streptococcal disease in tilapia according to claim 1, characterized in that, The encapsulation efficiency of the Eucommia ulmoides chlorogenic acid-chitosan complex is ≥90% in the pH range of 4.0 to 6.0; The particle size of the gallnut tannin nanocapsules is 80±20nm; The minimum inhibitory concentration of the compound antibacterial agent against enrofloxacin-resistant Streptococcus agalactiae is ≤20 μg / mL; The compound antibacterial agent can reduce the expression levels of gyrA, ermB, and tetM genes in drug-resistant strains by ≥3.8 times, ≥5.2 times, and ≥4.1 times, respectively, compared with the control group.

3. A method for preparing a compound antibacterial agent for controlling streptococcal disease in tilapia, characterized in that, Includes the following steps: Step 1: Carrier Modification The attapulgite soil is processed, washed, and calcined to obtain a modified attapulgite soil carrier. Step 2: Preparation of nanocapsules Gallnut extract was mixed with β-cyclodextrin and placed in a water bath for ultrasonic treatment to obtain gallnut tannin nanocapsules. Step 3: Complex Synthesis Chlorogenic acid and chitosan were subjected to an ionic cross-linking reaction to form an Eucommia ulmoides chlorogenic acid-chitosan complex. Baicalin and soybean lecithin were dissolved in anhydrous ethanol, and then this ethanol solution was slowly injected into the aqueous phase while stirring continuously. Finally, the ethanol was evaporated to remove the ethanol, and the baicalin lecithin complex was obtained. Step 4: Mixing and Loading The gallnut tannin nanocapsules obtained in step 2, the Eucommia ulmoides chlorogenic acid-chitosan complex and baicalin phospholipid complex obtained in step 3, and the freeze-dried powder of Bacillus subtilis GS-02 metabolite are mixed evenly. Then, the mixture is adsorbed using the acidified modified attapulgite carrier obtained in step 1. Finally, the loaded material is centrifuged and spray-dried, and the dried product is granulated or ultra-finely pulverized according to the application requirements.

4. The method for preparing a compound antibacterial agent for controlling streptococcal disease in tilapia according to claim 3, characterized in that, In step 1, the attapulgite is treated with 0.5 mol / L HCl solution for 3 hours, washed with water until neutral, and finally calcined at 500℃ for 2 hours; the resulting modified attapulgite carrier has a pore size of 10–40 nm and a specific surface area ≥180 m². 2 / g.

5. The method for preparing a compound antibacterial agent for controlling streptococcal disease in tilapia according to claim 3, characterized in that, In step 2, gallnut extract and β-cyclodextrin are mixed at a molar ratio of 1:5, placed in a 50°C water bath, and ultrasonically treated at 300W power for 40 minutes in pulse mode: 5 seconds working, 5 seconds interval, to obtain gallnut tannin nanocapsules with a particle size of 80±20nm.

6. The method for preparing a compound antibacterial agent for controlling streptococcal disease in tilapia according to claim 3, characterized in that, In step 3, chlorogenic acid and chitosan are subjected to an ionic cross-linking reaction at pH 5.0±0.1 and 45±1℃ for 3 hours. Baicalin and soybean lecithin were dissolved in anhydrous ethanol at a mass ratio of 1:

2. This ethanol solution was then slowly injected into the aqueous phase at 60°C and stirred continuously for 1 hour. In step 4, the inlet air temperature for drying is 160±5℃; the outlet air temperature is 70±5℃; and the resulting dried product is granulated at ≤60℃ or ultra-finely pulverized according to application requirements.

7. The application of the compound antibacterial agent for controlling streptococcal disease in tilapia according to claims 1-2 in the preparation of tilapia feed additives, characterized in that, The amount of compound antibacterial agent added to the feed additive is 0.5% to 2.0% of the total weight of the feed; the granulation temperature is ≤60℃, and the activity retention rate of the compound antibacterial agent is ≥95%.

8. The application of the compound antibacterial agent for controlling streptococcal disease in tilapia according to claims 1-2 in the disinfection or disease control of aquaculture water, characterized in that, The composite antibacterial agent was prepared into a water-spraying powder form, with a particle size distribution D. 90 ≤20μm; dosage is 0.3~0.8g / m 3 water body.

9. The application of the composite antibacterial agent for controlling streptococcal disease in tilapia according to claims 1-2 in the preparation of a sustained-release gel, characterized in that, The sustained-release gel contains a cross-linked structure of sodium alginate and calcium chloride; per 10m 3 Dosage for suspending 200g slow-release gel blocks in aquaculture water; the drug release of the slow-release gel conforms to the kinetic equation Ct = 42.3e -0 . 021t R 2 =0.98, and the sustained-release half-life calculated based on this equation is ≥33 days.

10. The application of the compound antibacterial agent for controlling streptococcal disease in tilapia according to claims 1-2 in the disease control of recirculating aquaculture systems, characterized in that, The composite antibacterial agent was prepared into a sustained-release gel block, and then distributed at a rate of 10 ml / min. 3 A 200g gel block was suspended in the water and placed in a mesh bag 1 meter from the system outlet. The sustained-release gel block is prepared by the following process: (a) The composite antibacterial agent is mixed with sodium alginate, wherein sodium alginate accounts for 12% w / w of the total weight of the gel block; (b) Cross-linking and curing are achieved by dripping in a 4% w / v calcium chloride solution; (c) Dry to a moisture content of ≤8% to form gel particles with a diameter of approximately 5 mm; The sustained-release gel block maintains an equivalent concentration of gallnut tannins in the water of ≥22.1 μg / L for 30 days. The sustained-release kinetic equation is: Ct=42.3e -0 . 021t R 2 =0.98.

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