An additive composition for alleviating tissue damage of fish caused by fluorine exposure and a method of preparing the same

Abstract

The application provides an additive composition for relieving fish tissue damage caused by fluorine exposure and a preparation method thereof. The additive composition is compounded from active ingredient nanoparticles, rare earth shell glucosamine chelate salt and a complex microbial preparation. The active ingredient nanoparticles are prepared by self-assembly of selenomethionine, astaxanthin, chicoric acid and lecithin. The complex microbial preparation is prepared by uniformly mixing Bacillus subtilis, Rhodopseudomonas palustris and Spirulina platensis. In the additive composition, the rare earth shell glucosamine chelate salt releases rare earth ions to complex fluorine ions to generate inert fluorides, the complex microbial preparation secretes exopolysaccharides to adsorb and discharge fluorine ions, and the active ingredient nanoparticles quench active oxygen and protect brain tissue and intestinal barrier. The additive composition can significantly relieve fish tissue damage caused by fluorine exposure, enhance the anti-fluorine stress ability, improve the growth performance and survival rate, and is suitable for preparing fish feed additives.

Description

Technical Field

[0001] This invention belongs to the field of feed additive preparation technology, specifically relating to an additive composition for alleviating tissue damage in fish caused by fluoride exposure and its preparation method. Background Technology

[0002] Fluorine, a widely distributed trace element in nature, is chemically reactive and possesses strong oxidizing power. It typically exists in the environment in the form of inorganic or organic fluorine compounds, exhibiting both nutritional and toxic properties. Adequate fluorine intake is crucial for the health of organisms, effectively preventing tooth decay and maintaining normal bone development. However, under the combined influence of natural volcanic eruptions, geological activities, and human activities such as mining, smelting, and fertilizer application, fluorine continuously enters aquatic environments in the form of inorganic or organic compounds. This has resulted in fluorine concentrations in aquatic environments far exceeding the natural levels of 0.01-0.3 mg / L in unpolluted waters. In some high-fluoride groundwater, the fluorine content can even reach 79.2 mg / L, becoming a key risk factor threatening aquatic ecosystems.

[0003] Water bodies serve as the fundamental environment for aquatic life, and the growth and development of fish are directly affected by the aquatic environment, with fluoride pollution having a particularly significant impact. Existing research indicates that fluoride and its derivatives produce various toxic effects on fish. Long-term exposure to high fluoride levels leads to fluoride accumulation in fish organs and tissues, causing harmful effects. For example, fluoride exposure can cause abnormalities in gill lamellae structure, such as rod-like formation, mucinous metaplasia, and hyperplasia, and interfere with ion transport function in the gill tissue, thereby impairing respiration and osmotic pressure regulation. Fluoride toxicity can damage the intestinal barrier, affecting the balance of intestinal flora and normal metabolic activity, increasing pathogenic bacteria and decreasing beneficial bacteria, thus disrupting intestinal permeability and local immune stability. Due to its neurotoxicity, fluoride can penetrate the blood-brain barrier and accumulate in fish brain tissue. The brain tissue, with its high metabolic activity and high lipid content, is highly susceptible to oxidative damage, leading to oxidative stress, apoptosis, and neurological dysfunction. Furthermore, fluoride can severely interfere with and damage the reproductive performance and endocrine system of fish, significantly limiting their reproduction, development, and normal growth.

[0004] Existing technologies for mitigating fluoride exposure-induced tissue damage in fish include reducing fluoride concentration in water through physical and chemical means and enhancing fish resistance through nutritional intervention using functional feed additives. However, these interventions focus on the migration, accumulation, and toxic damage of fluoride within the fish. Furthermore, research on mitigating fluoride hazards in fish often employs single-component methods, resulting in limited effectiveness. For example, while nano-selenium can reduce intracellular fluoride content by decreasing cell membrane permeability, it suffers from a narrow safe dosage window and potential toxicity risks. Inorganic mineral materials, although capable of adsorbing fluoride ions in fish tissues to form low-solubility complexes and reduce fluoride bioavailability, exhibit low adsorption capacity, poor selectivity, and susceptibility to interference from coexisting ions. Therefore, there is an urgent need to research a composite additive composition for antagonizing fluoride exposure-induced tissue damage in fish, which would provide an intervention strategy for healthy fish farming in high-fluoride water environments. Summary of the Invention

[0005] Technical Problem to be Solved: To address the aforementioned technical problems, the present invention aims to provide an additive composition and its preparation method for mitigating tissue damage in fish caused by fluoride exposure. This additive composition is formulated from active ingredient nanoparticles, rare earth chitosan chelate salts, and a composite microbial preparation. The active ingredient nanoparticles are prepared by self-assembly of selenomethionine, astaxanthin, chicoric acid, and lecithin. The composite microbial preparation is prepared by uniformly mixing Bacillus subtilis, Rhodopseudomonas palustris, and Spirulina platensis. This invention solves the problems of limited adsorption capacity, poor selectivity for fluoride ion adsorption, inability to efficiently remove fluoride ions from fish, and easy loss of feed nutrients and decreased feed utilization associated with traditional inorganic mineral adsorbents used to alleviate fluoride toxicity in fish.

[0006] Technical solution: An additive composition for alleviating tissue damage in fish caused by fluoride exposure, comprising the following components by weight: 5-10 parts of active ingredient nanoparticles, 10-20 parts of rare earth chitosan chelate salt, and 20-35 parts of compound microbial preparation. A method for preparing an additive composition for mitigating tissue damage in fish caused by fluoride exposure includes the following steps: S1. The active ingredient nanoparticles are self-assembled from selenomethionine, astaxanthin, chicoric acid and lecithin by an antisolvent method; the mass ratio of selenomethionine, astaxanthin, chicoric acid and lecithin is (1-3):(2-5):(1-3):(8-15). The specific steps are as follows: ① Dissolve selenomethionine, astaxanthin, and chicoric acid in anhydrous ethanol to prepare a mixed solution with a concentration of 5-15 mg / mL, and dissolve lecithin in 4% ethanol aqueous solution to prepare a lecithin solution with a concentration of 10-30 mg / mL; ② Mix the mixed solution and the lecithin solution at a volume ratio of 1:(1-3), then emulsify by high-speed shearing, ultrasonically treat, evaporate to remove alcohol, and electrostatic spray dry to obtain the final product; S2. Mix Bacillus subtilis bacterial solution, Rhodopseudomonas palustris bacterial solution and Spirulina platensis in a ratio of (2-5) mL: (1-4) mL: (1-3) g to obtain a compound microbial preparation. S3. The additive composition is obtained by mixing and drying the composite microbial preparation, active ingredient nanoparticles and rare earth chitosan chelate salt.

[0007] Furthermore, the conditions for high-speed shear emulsification are a rotation speed of 8000-12000 rpm and an emulsification time of 20-40 min; the conditions for ultrasonic treatment are a power of 200-400 W and a time of 10-20 min; and the conditions for electrostatic spray drying are an electrostatic voltage of 5-20 kV, an inlet air temperature of 60-80℃, an outlet air temperature of 40-50℃, and a feed rate of 5-15 mL / min.

[0008] Furthermore, the concentration of Bacillus subtilis in the S2 culture is (2-5.5)×10⁻⁶. 8 The concentration of *Rhodopseudomonas palustris* bacterial culture was (0.5-3) × 10⁻³ CFU / mL. 8 The concentration of CFU / mL and Spirulina platensis used is (1-5)×10⁻⁶. 7 cells / mL.

[0009] The above-described additive composition for mitigating fluoride exposure-induced tissue damage in fish is used in the preparation of fish feed, wherein the additive composition is added to the fish feed at an amount of 0.5-3.0%. Beneficial effects

[0010] 1. The additive composition prepared in this invention consists of active ingredient nanoparticles, rare earth chitosan chelate salts, and a composite microbial preparation. When applied to fish feed, this additive composition can alleviate tissue damage in fish caused by fluoride exposure and enhance the overall stress resistance and physiological function of the fish. The rare earth chitosan chelate salts in the additive composition can be degraded by digestive enzymes in the fish's stomach, releasing lanthanum and cerium rare earth ions. These ions capture fluoride ions in the gastrointestinal tract through ion complexation and coordination precipitation, generating inert rare earth fluorides. Furthermore, the composite microbial preparation consists of *Rhodopseudomonas palustris*, *Bacillus subtilis*, and *Spirulina platensis*. The extracellular polysaccharide polymers secreted by *Bacillus subtilis* and *Spirulina platensis* not only serve as a carrier for the inert rare earth fluorides but also adhere to fluoride ions in the fish's gastrointestinal tract through their active groups, reducing the risk of fluoride deposition and toxic effects in the fish's body. This allows the fluoride ions to be directly excreted with the fish's feces, solving the problem of limited adsorption and poor selectivity of traditional inorganic mineral materials, which leads to nutrient loss and reduced utilization in feed.

[0011] 2. The lecithin outer layer of the active ingredient nanoparticles in the additive composition of this invention can bind to the intestinal mucosa of fish and release selenomethionine, astaxanthin, and chicoric acid. On the one hand, it can quench reactive oxygen species produced in fish under fluoride exposure, and on the other hand, it can exert its effects in fish brain tissue by crossing the blood-brain barrier, reducing the oxidative stress level of fish brain tissue. At the same time, it can alleviate the inhibitory effect of fluoride toxicity on endogenous antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase in fish brain tissue, regulate the activity of endogenous antioxidant enzymes in fish brain tissue to enhance the oxidative stress resistance of fish, thereby achieving the purpose of improving the damage to fish brain tissue caused by fluoride exposure. On the other hand, compound microbial agents can also colonize the fish's intestines. Bacillus subtilis produces quorum sensing signal molecules that can promote the proliferation of beneficial bacteria and the secretion of short-chain fatty acids in the intestinal tissue. Secondary metabolites such as 5-ALA and coenzyme factors produced by Rhodopseudomonas palustris, as well as phycocyanin and spirulina polysaccharides from Spirulina platensis, can further enhance the barrier function of the fish's intestinal tissue, alleviate the adverse effects of fluoride toxicity on the intestinal tissue, improve the intestinal permeability of fish, and protect the integrity of the blood-brain barrier, thereby achieving the effect of healthy growth of fish.

[0012] 3. The additive composition of the present invention can alleviate the adverse effects of long-term fluoride exposure on the intestines and brain tissue of fish, reduce the accumulation level of fluoride in the fish body, regulate the normal metabolism of the fish body, and significantly improve the fish's resistance to fluoride stress, survival rate and growth performance. It is beneficial to provide technical reference and theoretical support for the physiological protection of fish under fluoride stress, and also provides an effective nutritional intervention strategy for the health protection of fish farmed in fluoride-polluted waters. Attached Figure Description

[0013] Figure 1 The weight gain rate of carp in the CK group, F group, Examples 1-7 and Comparative Examples 1-5 during the feeding period; Figure 2 The growth rate of carp in the CK group, F group, Examples 1-7 and Comparative Examples 1-5 during the feeding period; Figure 3 The specific growth rates of carp in the CK group, F group, Examples 1-7 and Comparative Examples 1-5 during the feeding period; Figure 4 The total swimming distance of carp in the CK group, F group, Examples 1-7 and Comparative Examples 1-5 during the feeding period; Figure 5 The D-lactic acid content in the serum of carp in the CK group, F group, Examples 1-7 and Comparative Examples 1-5 during the feeding period; Figure 6 The activity of diamine oxidase in the serum of carp in the CK group, F group, Examples 1-7 and Comparative Examples 1-5 during the feeding period; Figure 7 The content of intestinal mucin MUC2 in carp during feeding was measured in the CK group, F group, Examples 1-7 and Comparative Examples 1-5. Figure 8 The short-chain fatty acid content of carp in the CK group, F group, Examples 1-7 and Comparative Examples 1-5 during the feeding period; Figure 9 The changes in antioxidant-related indicators in carp brain tissue were measured after 30 days of feeding in the CK group, F group, Examples 1-7 and Comparative Examples 1-5 groups, where A represents MDA, B represents T-SOD, C represents CAT, D represents GSH-Px and E represents GSH. Figure 10 The changes in antioxidant-related indicators in the brain tissue of carp in the CK group, F group, Examples 1-7 and Comparative Examples 1-5 after 60 days of feeding were shown. A represents MDA, B represents T-SOD, C represents CAT, D represents GSH-Px and E represents GSH. Figure 11 The changes in antioxidant-related indicators in the brain tissue of carp in the CK group, F group, Examples 1-7 and Comparative Examples 1-5 after 90 days of feeding were shown. A represents MDA, B represents T-SOD, C represents CAT, D represents GSH-Px and E represents GSH. Detailed Implementation

[0014] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments: The bacterial strains used in this invention are: Bacillus subtilis ATCC6633 purchased from Wuhan Gray Algae Biotechnology Co., Ltd., Rhodopseudomonas palustris ATCC17001 purchased from Hangzhou Baosai Biotechnology Co., Ltd., and Spirulina platensis purchased from Shanghai Guangyu Biotechnology Co., Ltd. Rare earth chitosan chelate was purchased from Jiangxi Chunke Technology Co., Ltd. Example 1

[0015] A method for preparing an additive composition for mitigating tissue damage in fish caused by fluoride exposure includes the following steps: S1. Weigh 1.00g selenomethionine, 2.00g astaxanthin, and 1.00g chicoric acid and dissolve them in anhydrous ethanol to prepare a mixed active ingredient ethanol solution with a concentration of 5mg / mL; separately, dissolve 8.00g lecithin in a 4% (v / v) ethanol aqueous solution to prepare a lecithin solution with a concentration of 10mg / mL; mix the two solutions at a volume ratio of 1:1 and emulsify them at high speed at 8000rpm for 20min, then ultrasonically treat them at 200W power for 10min, remove the ethanol by rotary evaporation, and then electrostatically spray dry them under the conditions of electrostatic voltage 5kV, inlet air temperature 60℃, outlet air temperature 40℃, and feed rate 5mL / min to obtain active ingredient nanoparticles; S2. Take 20 mL of a 2.0 × 10⁻⁶ solution. 8 CFU / mL Bacillus subtilis bacterial suspension, 10 mL concentration 0.5×10 8 CFU / mL Rhodopseudomonas palustris bacterial suspension, 10 mL concentration 1.0×10 7 A compound microbial preparation was prepared by stirring and mixing Spirulina platensis cells / mL. S3. Weigh 5g of active ingredient nanoparticles and 10g of rare earth chitosan chelate salt, then mix them with 30mL of compound microbial preparation and dry to obtain the additive composition. Example 2

[0016] A method for preparing an additive composition for mitigating tissue damage in fish caused by fluoride exposure includes the following steps: S1. Weigh 1.50g selenomethionine, 3.00g astaxanthin, and 1.50g chicoric acid and dissolve them in anhydrous ethanol to prepare a mixed active ingredient ethanol solution with a concentration of 10mg / mL; separately, dissolve 10.00g lecithin in a 4% (v / v) ethanol aqueous solution to prepare a lecithin solution with a concentration of 20mg / mL; mix the two solutions at a volume ratio of 1:3 and emulsify them at 9000rpm for 25min, then sonicate them at 300W for 15min, remove the ethanol by rotary evaporation, and then electrostatically spray dry them under the conditions of 10kV electrostatic voltage, 65℃ inlet air temperature, 40℃ outlet air temperature, and 8mL / min feed rate to obtain active ingredient nanoparticles; S2. Take 30 mL of a 3.0 × 10⁻⁶ solution. 8 CFU / mL Bacillus subtilis bacterial suspension, 20 mL concentration 1.0×10 8 CFU / mL Rhodopseudomonas palustris bacterial suspension, 15 mL concentration 2.0×10 7 A compound microbial preparation was prepared by stirring and mixing Spirulina platensis cells / mL. S3. Weigh 10g of active ingredient nanoparticles and 12g of rare earth chitosan chelate salt, then mix them with 25mL of compound microbial preparation and dry to obtain the additive composition. Example 3

[0017] A method for preparing an additive composition for mitigating tissue damage in fish caused by fluoride exposure includes the following steps: S1. Weigh 2.00g selenomethionine, 4.00g astaxanthin, and 2.00g chicoric acid and dissolve them in anhydrous ethanol to prepare a mixed active ingredient ethanol solution with a concentration of 15mg / mL; separately, dissolve 12.00g lecithin in a 4% (v / v) ethanol aqueous solution to prepare a lecithin solution with a concentration of 20mg / mL; mix the two solutions at a volume ratio of 1:2 and emulsify them at 10000rpm for 15min, then ultrasonically treat them with 200W power for 15min, remove the ethanol by rotary evaporation, and then electrostatically spray dry them under the conditions of electrostatic voltage 15kV, inlet air temperature 70℃, outlet air temperature 45℃, and feed rate 5mL / min to obtain active ingredient nanoparticles; S2. Take 20 mL of a 4.5 × 10⁻⁶ solution. 8 CFU / mL Bacillus subtilis bacterial suspension, 15 mL concentration 2.0×10 8 CFU / mL Rhodopseudomonas palustris bacterial suspension, 10 mL concentration 2.0×10 7 A compound microbial preparation was prepared by stirring and mixing Spirulina platensis cells / mL. S3. Weigh 5g of active ingredient nanoparticles and 15g of rare earth chitosan chelate salt, then mix them with 30mL of compound microbial preparation and dry to obtain the additive composition. Example 4

[0018] A method for preparing an additive composition for mitigating tissue damage in fish caused by fluoride exposure includes the following steps: S1. Weigh 2.00g selenomethionine, 3.50g astaxanthin, and 2.00g chicoric acid and dissolve them in anhydrous ethanol to prepare a mixed active ingredient ethanol solution with a concentration of 5mg / mL; separately, dissolve 12.00g lecithin in a 4% (v / v) ethanol aqueous solution to prepare a lecithin solution with a concentration of 15mg / mL; mix the two solutions at a volume ratio of 1:1 and emulsify them at high speed at 10000rpm for 25min, then sonicate them at 350W power for 15min, remove the ethanol by rotary evaporation, and then electrostatically spray dry them under the conditions of electrostatic voltage 15kV, inlet air temperature 70℃, outlet air temperature 40℃, and feed rate 7mL / min to obtain active ingredient nanoparticles; S2. Take 35 mL of a 4.0 × 10⁻⁶ solution. 8 CFU / mL Bacillus subtilis bacterial suspension, 25 mL concentration 1.5×10 8 CFU / mL Rhodopseudomonas palustris bacterial suspension, 20 mL concentration 1.5×10 7 A compound microbial preparation was prepared by stirring and mixing Spirulina platensis cells / mL. S3. Weigh 8g of active ingredient nanoparticles and 15g of rare earth chitosan chelate salt, then mix them with 25mL of compound microbial preparation and dry to obtain the additive composition. Example 5

[0019] A method for preparing an additive composition for mitigating tissue damage in fish caused by fluoride exposure includes the following steps: S1. Weigh 3.00g selenomethionine, 4.00g astaxanthin, and 3.00g chicoric acid and dissolve them in anhydrous ethanol to prepare a mixed active ingredient ethanol solution with a concentration of 15mg / mL; separately, dissolve 15.00g lecithin in a 4% (v / v) ethanol aqueous solution to prepare a lecithin solution with a concentration of 30mg / mL; mix the two solutions at a volume ratio of 1:2 and emulsify them at high speed at 12000rpm for 30min, then ultrasonically treat them with 350W power for 20min, remove the ethanol by rotary evaporation, and then electrostatically spray dry them under the conditions of electrostatic voltage 20kV, inlet air temperature 75℃, outlet air temperature 50℃, and feed rate 15mL / min to obtain active ingredient nanoparticles; S2. Take 25 mL of a 5.5 × 10⁻⁶ solution. 8 CFU / mL Bacillus subtilis bacterial suspension, 20mL concentration 3×10 8 CFU / mL Rhodopseudomonas palustris bacterial suspension, 15 mL concentration 4.5×10 7 A compound microbial preparation was prepared by stirring and mixing Spirulina platensis cells / mL. S3. Weigh 10g of active ingredient nanoparticles and 20g of rare earth chitosan chelate salt, then mix them with 35mL of compound microbial preparation and dry to obtain the additive composition. Example 6

[0020] A method for preparing an additive composition for mitigating tissue damage in fish caused by fluoride exposure includes the following steps: S1. Weigh 2.50g of selenomethionine, 4.50g of astaxanthin, and 2.50g of chicoric acid and dissolve them in anhydrous ethanol to prepare a mixed active ingredient ethanol solution with a concentration of 10mg / mL; separately, dissolve 13.00g of lecithin in a 4% (v / v) ethanol aqueous solution to prepare a lecithin solution with a concentration of 20mg / mL; mix the two solutions at a volume ratio of 1:1 and emulsify them at high speed of 10000rpm for 25min, then sonicate them at 350W power for 20min, remove the ethanol by rotary evaporation, and then electrostatic spray dry them under the conditions of electrostatic voltage of 15kV, inlet air temperature of 65℃, outlet air temperature of 45℃, and feed rate of 10mL / min to obtain active ingredient nanoparticles; S2. Take 45 mL of a solution with a concentration of 4.0 × 10⁻⁶. 8 CFU / mL Bacillus subtilis bacterial suspension, 35mL concentration 2×10 8 CFU / mL Rhodopseudomonas palustris bacterial suspension, 25 mL concentration 3.0×10 7 A compound microbial preparation was prepared by stirring and mixing Spirulina platensis cells / mL. S3. Weigh 8g of active ingredient nanoparticles and 15g of rare earth chitosan chelate salt, then mix them with 30mL of compound microbial preparation and dry to obtain the additive composition. Example 7

[0021] A method for preparing an additive composition for mitigating tissue damage in fish caused by fluoride exposure includes the following steps: S1. Weigh 2.8g of selenomethionine, 4.5g of astaxanthin, and 2.80g of chicoric acid and dissolve them in anhydrous ethanol to prepare a mixed active ingredient ethanol solution with a concentration of 15mg / mL; separately, dissolve 10g of lecithin in a 4% (v / v) ethanol aqueous solution to prepare a lecithin solution with a concentration of 20mg / mL; mix the two solutions at a volume ratio of 1:3 and emulsify them at 11000rpm for 40min, then ultrasonically treat them with 250W power for 20min, remove the ethanol by rotary evaporation, and then electrostatically spray dry them under the conditions of electrostatic voltage 20kV, inlet air temperature 75℃, outlet air temperature 50℃, and feed rate 10mL / min to obtain active ingredient nanoparticles. S2. Take 40 mL of a solution with a concentration of 4.5 × 10⁻⁶. 8 CFU / mL Bacillus subtilis bacterial suspension, 25 mL concentration 2.5×10 8 CFU / mL Rhodopseudomonas palustris bacterial suspension, 28 mL concentration 4.0×10 7 A compound microbial preparation was prepared by stirring and mixing Spirulina platensis cells / mL. S3. Weigh 5g of active ingredient nanoparticles and 19g of rare earth chitosan chelate salt, then mix them with 35mL of compound microbial preparation and dry to obtain the additive composition. Comparative Example 1

[0022] The difference between this comparative example and Example 6 is that rare earth chitosan chelate salt is not added; the remaining operations are the same as in Example 6. Comparative Example 2

[0023] The difference between this comparative example and Example 6 is that astaxanthin and chicoric acid are not added; the remaining operations are the same as in Example 6. Comparative Example 3

[0024] The difference between this comparative example and Example 6 is that lecithin is not added; the remaining operations are the same as in Example 6. Comparative Example 4

[0025] The difference between this comparative example and Example 6 is that no compound microbial preparation is added; the remaining operations are the same as in Example 6. Comparative Example 5

[0026] The difference between this comparative example and Example 6 is that Spirulina platensis is not added to the compound microbial preparation; the remaining operations are the same as in Example 6. Performance testing

[0027] Validation test of additive composition to alleviate tissue damage in fluoride-exposed fish Seven hundred juvenile carp (female:male = 1:1) were acclimatized for 7 days in a culture system with a temperature of 24±1℃, pH 7.0-7.4, light-dark ratio of 14:10, and dissolved oxygen of 5.0-7.0 mg / L. Then, they were exposed to fluoride using a semi-static water contact method. The acclimatized juvenile carp were randomly divided into 13 groups: the control group (CK) was fed aerated tap water and a basic diet; the F group was exposed to water containing 80 mg / L sodium fluoride and fed a basic diet; groups 1-7 (Examples) and 1-5 (Comparative Examples) were fed a basic diet supplemented with 2.0% sodium fluoride and simultaneously exposed to water containing 80 mg / L sodium fluoride for 90 days. During the experiment, the daily feed intake was 5% of the fish's body weight, and uneaten feed and excrement were promptly removed. The water was changed every three days.

[0028] Samples were collected on days 30, 60, and 90 after treatment to measure fish growth indicators, including weight gain rate, growth rate, and specific growth rate. Before each sampling, three male and three female carp were randomly selected from each group for a free-swimming test, and the total swimming distance was measured. Samples were collected and analyzed immediately after the test. At the end of the experiment (90 days), intestinal and brain tissues were collected from six carp in each group, flash-frozen in liquid nitrogen, and stored at -80℃. Intestinal permeability, intestinal mucin MUC2 protein content, and short-chain fatty acids were measured in carp intestinal tissue; malondialdehyde (MDA) content and the activities of SOD, CAT, GSH-Px, and GSH were measured in carp brain tissue.

[0029] Depend on Figure 1 , Figure 2 , Figure 3 and Figure 4 It can be seen that during the 90-day feeding period, all indicators of the CK group were at their optimal levels, indicating that carp can maintain normal growth, development, and movement under fluoride-free conditions. However, the growth indicators and movement of carp in the F group were significantly deteriorated, fully demonstrating the severe inhibitory effect of 80 mg / L sodium fluoride exposure on the growth performance and behavior of carp. The physiological indicators of carp in Examples 1-6 were significantly better than those in Comparative Examples 1-5, indicating that the active ingredients and compound microbial preparations contained in the additive composition prepared in this invention can alleviate the inhibitory effects of fluoride exposure on the growth and movement of carp.

[0030] Depend on Figure 5 , Figure 6 , Figure 7 , Figure 8It was found that during the 90-day feeding period, the intestinal tissue indicators of the CK group remained at normal physiological levels, indicating that the intestinal barrier function of carp was intact, the mucosal structure was normal, and the intestinal microbial metabolism was active under fluoride-free conditions. Compared with the CK group, the intestinal tissue indicators of carp in the F group deviated from normal levels and continued to deteriorate with the extension of exposure time, indicating the severe destructive effect of sodium fluoride exposure on the intestinal barrier function of carp. In particular, the 6th example group was able to effectively maintain the stable recovery of intestinal barrier function, demonstrating a significant and lasting technical effect in alleviating fluoride exposure-induced intestinal tissue damage in fish, and providing an effective nutritional intervention strategy for protecting the intestinal health of fish farmed in fluoride-polluted waters.

[0031] Depend on Figure 9 , Figure 10 , Figure 11 It was found that in group F carp brain tissue, MDA was significantly increased, and the levels of endogenous antioxidant enzymes (T-SOD, CAT, GSH-Px) and GSH were significantly decreased, and brain tissue damage accumulated with prolonged exposure time. Examples 1-6 could alleviate fluoride-induced oxidative damage in carp brain tissue to varying degrees, especially example 6, which showed the best alleviating effect. However, comparative example 1 lacked rare earth chitosan chelating salts. Due to the lack of rare earth chitosan chelating salts, fluoride ions could not be effectively complexed and excreted from the body, resulting in the inability of the active ingredient nanoparticles and compound microbial preparations to effectively block the accumulation of fluoride. The effects were as follows: the active ingredient nanoparticles in Comparative Examples 2 and 3 lacked astaxanthin, chicoric acid, or lecithin loading. This is because the active ingredients without lecithin shell protection have reduced efficiency in penetrating the blood-brain barrier and targeting brain tissue, or limited ability to quench free radicals generated by fluoride accumulation in brain tissue, resulting in poor alleviation of antioxidant-related indicators in carp brain tissue. Comparative Examples 4 and 5 did not contain compound microbial preparations or lacked components, which directly affected the metabolism and barrier stability of carp intestinal tissue, thus adversely affecting antioxidant-related enzymes in carp brain tissue.

[0032] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. An additive composition for mitigating tissue damage in fish caused by fluoride exposure, characterized in that, By weight, it includes the following components: 5-10 parts active ingredient nanoparticles, 10-20 parts rare earth chitosan chelate salt, and 20-35 parts compound microbial preparation; The active ingredient nanoparticles are made of selenomethionine, astaxanthin, chicoric acid and lecithin; The compound microbial preparation is made from Bacillus subtilis, Rhodopseudomonas palustris, and Spirulina platensis.

2. The additive composition for mitigating fluoride exposure-induced tissue damage in fish according to claim 1, characterized in that, The mass ratio of selenomethionine, astaxanthin, chicoric acid and lecithin is (1-3):(2-5):(1-3):(8-15).

3. The method for preparing an additive composition for mitigating fluoride exposure-induced tissue damage in fish according to claim 1, characterized in that, Includes the following steps: S1. The active ingredient nanoparticles are self-assembled from selenomethionine, astaxanthin, chicoric acid and lecithin via an antisolvent method. S2. Mix Bacillus subtilis bacterial solution, Rhodopseudomonas palustris bacterial solution and Spirulina platensis to obtain a compound microbial preparation; S3. The additive composition is obtained by mixing and drying the compound microbial preparation, active ingredient nanoparticles and rare earth chitosan chelate salt.

4. The method for preparing an additive composition for mitigating fluoride exposure-induced tissue damage in fish according to claim 3, characterized in that: The specific operation of S1 is as follows: ① Dissolve selenomethionine, astaxanthin, and chicoric acid in anhydrous ethanol to prepare a mixed solution with a concentration of 5-15 mg / mL, and dissolve lecithin in 4% ethanol aqueous solution to prepare a lecithin solution with a concentration of 10-30 mg / mL; ② Mix the mixed solution and the lecithin solution, then emulsify by high-speed shearing, sonicate, evaporate to remove alcohol, and electrostatic spray dry to obtain the final product.

5. The method for preparing an additive composition for alleviating fluoride exposure-induced tissue damage in fish according to claim 4, characterized in that: The conditions for high-speed shear emulsification are: rotation speed 8000-12000 rpm, emulsification time 20-40 min; the conditions for ultrasonic treatment are: power 200-400W, time 10-20 min; the conditions for electrostatic spray drying are: electrostatic voltage 5-20kV, inlet air temperature 60-80℃, outlet air temperature 40-50℃, and feed rate 5-15mL / min.

6. The method for preparing an additive composition for mitigating fluoride exposure-induced tissue damage in fish according to claim 3, characterized in that: The ratio of Bacillus subtilis culture, Rhodopseudomonas palustris culture and Spirulina platensis in S2 is (2-5) mL: (1-4) mL: (1-3) g.

7. The method for preparing an additive composition for mitigating fluoride exposure-induced tissue damage in fish according to claim 3, characterized in that: The concentration of Bacillus subtilis in the S2 culture is (2-5.5)×10⁻⁶. 8 The concentration of *Rhodopseudomonas palustris* bacterial culture was (0.5-3) × 10⁻³ CFU / mL. 8 The concentration of CFU / mL and Spirulina platensis used is (1-5)×10⁻⁶. 7 cells / mL.

8. The application of the additive composition according to claim 1 for mitigating fluoride exposure-induced tissue damage in fish in the preparation of fish feed.

9. The application according to claim 7, characterized in that, The additive composition is added to fish feed at a rate of 0.5-3.0%.

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