Feed additive for clearing microplastics, preparation method and application

Abstract

The application discloses a feed additive for removing microplastics, a preparation method and application. The composition consists of the following components in percentage by weight: modified chitosan microspheres 15-25%, nanoscale yeast cell wall derivatives 10-20%, fermentable dietary fiber complex 15-25%, glutamine 5-10%, complex probiotics and prebiotics 15-25%, and natural plant extract compound antioxidant 5-10%. The additive can be added to the feed at a weight ratio of 0.5-2.0% through a multi-target synergistic mechanism of 'adsorption-promoting excretion-repair-regulation', and can efficiently remove microplastics in aquatic products (the removal rate is up to 65% or more), improve intestinal health, enhance antioxidant function, reduce the residues of heavy metals and other complex pollutants, and significantly improve the edible safety and quality of aquatic products.

Description

Technical Field

[0001] This invention relates to the field of feed technology, and in particular to a feed additive for removing microplastics, its preparation method, and its application. Background Technology

[0002] Microplastics (particle size <5 mm) are an emerging global pollutant that has been widely detected in various aquatic ecosystems. Fish, shrimp, and crabs ingest microplastics through feeding and gill respiration, leading to their accumulation in tissues and organs such as muscles, liver, and intestines. Microplastics not only cause direct physical damage and toxic effects, but also act as carriers to adsorb heavy metals, persistent organic pollutants, and pathogenic microorganisms, forming complex pollution that is transmitted through the food chain, ultimately threatening human health.

[0003] Core issue: The shift in understanding from environmental governance to internal purification

[0004] Early research primarily viewed microplastics as an environmental and ecological problem, with technological development focusing on source control and environmental remediation (such as physical removal and flocculation sedimentation). However, as research has deepened, the core issue has shifted from "environmental remediation" to "biodegradation and food safety assurance"—that is, how to remove microplastics accumulated in aquatic animals. Currently, there is a lack of technologies specifically targeting the removal of microplastics from the body, and traditional aquaculture methods cannot block or reverse this process. Therefore, developing a functional feed additive that can safely and efficiently remove microplastics from the body through dietary means has become a crucial problem that urgently needs to be solved.

[0005] Currently, there is a lack of systematic solutions capable of proactively and efficiently removing microplastics from the body while simultaneously addressing their combined physical, chemical, and microbial effects. A search revealed no technology that utilizes a multi-target synergistic mechanism of "adsorption-promote excretion-repair-regulation" to remove microplastics from the body. Summary of the Invention

[0006] The technical solution of this invention to solve the above-mentioned technical problems is to provide a feed additive for removing microplastics, which, by weight percentage, consists of the following components: modified chitosan microspheres: 15%-25%; nano-sized yeast cell wall derivatives: 10%-20%; fermentable dietary fiber complex: 15%-25%; glutamine: 5%-10%; compound probiotics and prebiotics: 15%-25%; and natural plant extract compound antioxidants: 5%-10%; wherein the sum of the weight percentages of the components is 100%.

[0007] Furthermore, the weight percentages of each component are as follows: modified chitosan microspheres: 20%; nanoscale yeast cell wall derivatives: 15%; fermentable dietary fiber complex: 20%; glutamine: 5%; complex probiotics and prebiotics: 30%; natural plant extract compound antioxidants: 10%.

[0008] Furthermore, the modified chitosan microspheres are grafted with hydrophobic functional groups on their surface via a Schiff base reaction, wherein the Schiff base reaction is a condensation reaction between an aldehyde group and an amino group, in which chitosan provides the amino component and p-benzaldehyde provides the aldehyde component.

[0009] Furthermore, the nanoscale yeast cell wall derivative is prepared by physical or biochemical methods, and its average particle size is 100-500 nanometers.

[0010] Furthermore, the fermentable dietary fiber complex is composed of lignin and citrus pectin, and the ratio of lignin to citrus pectin is 3:7 to 7:3.

[0011] Furthermore, the compound probiotics and prebiotics contain Bacillus licheniformis, Bacillus subtilis and xylooligosaccharides, wherein the compound ratio of Bacillus licheniformis and Bacillus subtilis is 3:7 to 7:3, the number of live bacteria is not less than 10 billion CFU / g, and the content of xylooligosaccharides is 3%-8%.

[0012] Furthermore, the natural plant extract compound antioxidant is composed of curcumin, epigallocatechin gallate and rosmarinic acid, and the compound ratio of curcumin, epigallocatechin gallate and rosmarinic acid is 2-4:4-6:1.5-3.

[0013] To address the aforementioned technical problems, this invention also proposes a method for preparing the feed additive as described above, comprising the following steps:

[0014] (1) Preparation of modified chitosan microspheres: surface phenyl modification was carried out by emulsification crosslinking and Schiff base reaction;

[0015] (2) Preparation of nanoscale yeast cell wall derivatives: processed by high-pressure homogenization and nano-grinding technology;

[0016] (3) Mix each component physically in proportion.

[0017] To solve the above-mentioned technical problems, the present invention also proposes an application of the feed additive as described above, wherein the feed additive is added to fish, shrimp or crab feed at a weight ratio of 0.5%-2.0%.

[0018] Furthermore, the feed additive is added to the feed at a weight ratio of 1.0%.

[0019] Compared with the closest existing technology, the technical solution provided by the present invention can produce the following significant beneficial effects:

[0020] 1. Highly efficient and unique ability to remove microplastics from the body, directly addressing core food safety issues:

[0021] Extremely high removal efficiency: The core effect of this invention lies in its ability to actively and efficiently remove microplastics accumulated in the bodies of fish, shrimp, and crabs. As shown in Example 2, in a carp farming experiment, feeding with only 1.0% of the microplastics for 8 weeks reduced the microplastic content in muscle tissue by 65.4%, which is extremely significant (P<0.01).

[0022] The mechanism of action is specific and complementary: through the dual electrostatic and hydrophobic adsorption of "modified chitosan microspheres" and the microporous trapping effect of "nanoscale yeast cell wall derivatives", a full spectrum and high capacity capture of microplastics with different particle sizes (micrometer to nanoscale) and different polymer types (such as PE, PP, PS) is achieved, which solves the problems of poor specificity and low efficiency of single adsorbents.

[0023] 2. Excellent versatility, suitable for a variety of aquaculture species in both fresh and seawater:

[0024] Cross-species effectiveness: As shown in Example 2, the present invention is not only effective in carp, but also achieves a stable clearance rate of 62.2%-66.0% in major freshwater fish such as black carp, grass carp, silver carp, bighead carp, and crucian carp.

[0025] Effective across salinity levels: Experiment 4 of Example 2 further demonstrates that the present invention has been successfully extended to the field of marine aquaculture. It can achieve a microplastic removal effect of more than 62% for high-value marine fish such as sea bass, grouper, golden pomfret, and large yellow croaker, as well as crustaceans such as shrimp and swimming crab. This shows that its mechanism of action is not affected by water salinity and species differences, and it has broad prospects for industrial application.

[0026] 3. Synergistic effects of multiple targets and pathways provide systemic health solutions:

[0027] The complete functional chain: This invention is not a simple accumulation of functions, but rather the construction of a synergistic system integrating "adsorption-promoting excretion-repairing-regulating". Experiment 6 (synergistic effect verification) confirms that the effect of the complete formula (FULL) is significantly better than any combination of individual components (P<0.05), with a synergistic effect value as high as 225%, producing an unexpected technical effect of "1+1>2".

[0028] Achieving deep physiological repair: Strengthening the intestinal barrier: Through the synergistic effect of "fermentable dietary fiber complex" and "glutamine", it significantly increases the intestinal villus height / crypt depth value (V / C value) and reduces serum diamine oxidase (DAO) activity, repairing and enhancing the intestinal mucosal barrier from the perspective of physical structure and cellular nutrition, and reducing "intestinal leakage".

[0029] Regulating gut microbiota balance: "Compound probiotics and prebiotics" effectively regulate the gut microecology disordered by microplastic exposure, competitively exclude pathogens, and consolidate the gut health environment.

[0030] Relieving Oxidative Stress: The "natural plant extract compound antioxidant" can systematically remove excess reactive oxygen species induced by microplastics, significantly improve serum total antioxidant capacity (T-AOC), reduce malondialdehyde (MDA) content, and protect vital organs such as the liver from oxidative damage. Network pharmacology and multi-omics analysis in Experiment Nine revealed at the molecular level its mechanism of multi-target synergistic detoxification by inhibiting core inflammatory pathways such as NOD-like receptors and TNF, reversing tryptophan metabolism disorders.

[0031] 4. Significantly improves the quality and safety of aquatic products, creating higher commercial value:

[0032] Reducing the residue of compound pollutants: As shown in Experiment Twelve, this invention not only removes microplastics, but also significantly reduces the residue of characteristic pollutants such as heavy metals (e.g., lead, cadmium) and plasticizers that coexist with them in the muscle (reduction rate of over 50%), fundamentally improving the safety of aquatic products for consumption.

[0033] Improving nutritional and sensory quality: The experimental group of fish showed a significant increase in the content of crude protein and beneficial fatty acids (DHA+EPA) in the muscle, optimized the firmness and chewiness of the meat, and improved the composition of flavor substances, achieving a leap from "safe" to "high-quality" and greatly enhancing the market competitiveness of the product.

[0034] 5. Safe to use, low cost, and easy to promote:

[0035] Raw material compliance and safety: All components are derived from substances permitted for use in the "Feed Raw Material Catalog" or "Feed Additive Variety Catalog", and have been proven safe and harmless to target animals through toxicological and breeding experiments.

[0036] Convenient and economical to use: This product is a feed additive. It can be simply mixed with conventional feed at a ratio of 0.5%-2.0% (preferably 1.0%) and fed without changing the existing farming model. It is inexpensive, widely accepted by farmers, and very suitable for promotion and application in large-scale aquaculture. Detailed Implementation

[0037] This invention proposes a feed additive for removing microplastics, its preparation method, and its application, aiming to provide a feed additive for removing microplastics.

[0038] The feed additive for removing microplastics from fish, shrimp, and crabs proposed in this invention will be described below in specific embodiments:

[0039] Example 1: In the technical solution of this example, a feed additive for removing microplastics is composed of the following components by weight percentage: modified chitosan microspheres: 15%-25%; nano-sized yeast cell wall derivatives: 10%-20%; fermentable dietary fiber complex: 15%-25%; glutamine: 5%-10%; compound probiotics and prebiotics: 15%-25%; natural plant extract compound antioxidants: 5%-10%; wherein, the sum of the weight percentages of the components is 100%.

[0040] Furthermore, the weight percentages of each component are as follows: modified chitosan microspheres: 20%; nanoscale yeast cell wall derivatives: 15%; fermentable dietary fiber complex: 20%; glutamine: 5%; complex probiotics and prebiotics: 30%; natural plant extract compound antioxidants: 10%.

[0041] Furthermore, the modified chitosan microspheres are grafted with hydrophobic functional groups on their surface via a Schiff base reaction, wherein the Schiff base reaction is a condensation reaction between an aldehyde group and an amino group, in which chitosan provides the amino component and p-benzaldehyde provides the aldehyde component.

[0042] Specifically, the Schiff base reaction refers to the condensation reaction between an aldehyde group (-CHO) and an amino group (-NH2) to form a Schiff base compound containing an imine group (-C=N-). Reaction equation:

[0043] Chitosan -NH2 + OHC-C6H5 → Chitosan -N=C-C6H5 + H2O

[0044] (1) Components that provide amino groups: chitosan. Chitosan molecular chains are rich in a large number of free amino groups (-NH2), which are the main active sites for its chemical modification.

[0045] (2) Components providing the aldehyde group: p-Benzaldehyde. It is an aldehyde compound with a benzene ring. The benzene ring is a typical hydrophobic functional group.

[0046] Through the reaction, the hydrophobic phenyl group is grafted onto the surface of the hydrophilic chitosan microspheres through a stable covalent bond (C=N), thereby endowing the chitosan microspheres with stronger hydrophobic interaction capabilities, enabling them to more effectively adsorb non-polar plastics such as polyethylene (PE) and polypropylene (PP).

[0047] Reaction conditions: Solvent: ethanol / water mixture; pH / catalyst: weakly acidic (glacial acetic acid) or neutral; temperature: 50-70°C; time: 4-12 hours.

[0048] The preparation process involves two main stages: emulsification crosslinking and Schiff base reaction. After the reaction, the modified microspheres are collected by filtration or centrifugation and thoroughly washed with solvents such as ethanol and acetone to completely remove physically adsorbed p-benzaldehyde. Finally, they are dried in a vacuum drying oven at 40-60°C to obtain the final phenyl-modified chitosan microspheres.

[0049] Furthermore, the nanoscale yeast cell wall derivative is prepared by physical or biochemical methods, and its average particle size is 100-500 nanometers.

[0050] Specifically, the nanoscale yeast cell wall derivative is one of the two "bioactive adsorbents" of this invention, which work synergistically with the "modified chitosan microspheres" to capture and remove microplastics in the body.

[0051] 1. Yeast cell wall: A natural component extracted from yeast (such as Saccharomyces cerevisiae). Its structure resembles the walls of a "miniature castle," and its main function is to protect the yeast cell.

[0052] Core Components and Functions: β-glucan (40-50%): This is the most crucial functional component. It is a polysaccharide known for its potent immunostimulatory activity. In aquatic animals, β-glucan can activate immune cells such as macrophages, enhancing the body's resistance to pathogenic microorganisms.

[0053] Mannan oligosaccharides (30-40%): Also a type of polysaccharide, it has the ability to adsorb pathogenic bacteria and promote the growth of beneficial bacteria (probiotics) in the gut. Its surface has many adhesion sites, which can act like a "magnet" to capture harmful bacteria and prevent them from colonizing the intestinal wall.

[0054] Chitin, proteins, etc.: provide structural support.

[0055] In feed additives, ordinary yeast cell walls are mainly used as immune enhancers and pathogen adsorbents.

[0056] 2. "Derivatives" and "Nanoscale"; instead of using ordinary yeast cell wall powder, it undergoes two key physical processing steps:

[0057] "Derivatives": These mainly refer to products obtained by treating, purifying, and modifying yeast cell walls through physical or biochemical methods. This process aims to enrich and expose their active ingredients (especially β-glucan) and alter their physical properties to make them more suitable for subsequent nano-sizing.

[0058] "Nanoscale": Through high-pressure homogenization and nano-grinding technology, the micron-sized yeast cell wall particles are further pulverized, so that their average particle size reaches 100-500 nanometers.

[0059] The revolutionary changes brought about by nanotechnology: A dramatic increase in specific surface area: After nano-sizing, the total surface area per unit weight of particles becomes enormous, providing a vast number of "adsorption sites." Formation of "microporous traps": The nanoscale pore size and huge specific surface area enable them to effectively capture nanoscale plastic particles that traditional adsorbents cannot hold, like an extremely fine net, through physical interactions such as van der Waals forces. This is the core of solving the "nanoplastics" pollution problem.

[0060] Nanoscale yeast cell wall derivatives play a dual role in this invention:

[0061] 1. A "Scavenger" for Nanoplastics: Utilizing its nanoscale pore size and huge specific surface area, it specifically captures and immobilizes extremely small nanoplastic particles through physical adsorption (van der Waals forces). This solves the problem that existing adsorbents (such as activated carbon) are ineffective against nanoscale plastics. It complements the size of "modified chitosan microspheres" that adsorb larger microplastics, achieving full coverage of the microplastic particle size range.

[0062] 2. Immune Activator: Its core component, β-glucan, stimulates and activates the innate immune system (such as macrophages) in fish, shrimp, and crabs after ingestion, enhancing overall disease resistance. Synergistic Defense: Microplastics are not only physical pollutants but also "Trojan horses" for pathogens. This component, while removing pollutants, enhances the animal's own ability to resist pathogenic microorganisms accompanying microplastic invasion, achieving a unity of "elimination" and "health maintenance."

[0063] Furthermore, the fermentable dietary fiber complex is composed of lignin and citrus pectin, and the ratio of lignin to citrus pectin is 3:7 to 7:3. Preferably, the ratio of lignin to citrus pectin is 1:1.

[0064] Furthermore, the compound probiotics and prebiotics contain Bacillus licheniformis, Bacillus subtilis, and xylooligosaccharides, wherein the ratio of Bacillus licheniformis to Bacillus subtilis is 3:7 to 7:3, the viable count is not less than 10 billion CFU / g, and the xylooligosaccharide content is 3%-8%. Preferably, the ratio of Bacillus licheniformis to Bacillus subtilis is 1:1.

[0065] Furthermore, the natural plant extract compound antioxidant is composed of curcumin, epigallocatechin gallate, and rosmarinic acid, and the compounding ratio of curcumin, epigallocatechin gallate, and rosmarinic acid is 2-4:4-6:1.5-3. Preferably, the compounding ratio of curcumin, epigallocatechin gallate, and rosmarinic acid is 3:5:2.

[0066] The core design concept of this invention lies in constructing a synergistic system integrating "adsorption-promote excretion-repair-regulation," aiming to comprehensively intervene in and block the accumulation and toxicity of microplastics in fish, shrimp, and crabs. The functions of each component are not isolated but interconnected, forming a complete solution.

[0067] I. Bioactive Adsorbent: 1. Component A: Modified chitosan microspheres;

[0068] Physiological function: "Specific catcher" of microplastics in the intestine.

[0069] Mechanism of action: Electrostatic adsorption: In the neutral or weakly alkaline intestinal environment of fish, shrimp and crabs, the amino (-NH2) part of the chitosan molecule is protonated and becomes positively charged, while most microplastic surfaces become negatively charged due to environmental aging. The two are combined through electrostatic attraction.

[0070] Hydrophobic interaction: Through the hydrophobic groups such as phenyl grafted on the surface, it generates strong hydrophobic interactions with non-polar plastic polymers such as polyethylene (PE) and polypropylene (PP), which greatly enhances the strength and universality of adsorption.

[0071] Network encapsulation: Microsphere structures can form a physical network under intestinal peristalsis, encapsulating and fixing microplastics of different sizes.

[0072] Application Value: It solves the problems of poor specificity and low efficiency of ordinary adsorbents (such as activated carbon) in adsorbing microplastics. Its dual-action mechanism ensures that it has a high-efficiency adsorption capacity for a variety of common plastic types, making it the first core component in chain removal.

[0073] 2. Component B: Nanoscale yeast cell wall derivative;

[0074] Physiological function: "microporous traps" of nanoscale microplastics and immune "activators".

[0075] Mechanism of action: Physical adsorption: Its nanoscale pore size and huge specific surface area enable it to act like a "sponge" and effectively capture nanoscale plastic particles that are difficult to handle by traditional adsorbents through van der Waals forces and steric hindrance effects.

[0076] Immune modulation: Its main component, β-glucan, is a classic immunostimulant. It can activate macrophages and neutrophils in fish, shrimp, and crabs, enhancing their non-specific immune function, thereby better combating pathogens carried by microplastics and the chronic inflammation they cause.

[0077] Application value: It fills the technological gap in the removal of nanoplastics, and combines the functions of "removal" and "immunity" to enhance the fish's own defense capabilities while removing pollutants.

[0078] II. Intestinal physical cleansing and barrier repair agent; 3. Component C: Fermentable dietary fiber complex;

[0079] Physiological functions: an "accelerator" for intestinal motility and a "nutrient source" for the intestinal mucosa.

[0080] Mechanism of action: Physical excretion promotion: Lignin has a hard structure and is not easily digested. It can effectively stimulate the intestinal wall, enhance peristalsis, and shorten the residence time of chyme in the intestine, thereby quickly expelling the adsorbed microplastics from the body and reducing their chance of contact with the intestine.

[0081] Barrier repair: Citrus pectin can be fermented by microorganisms in the lower intestine to produce short-chain fatty acids (especially butyric acid). Butyric acid is the preferred energy source for colonic epithelial cells, which can significantly promote the proliferation and differentiation of intestinal mucosal cells, repair damaged villi, and strengthen the chemical and physical barrier function of the mucosal layer.

[0082] Application value: It realizes the key step from "adsorption" to "excretion", and actively repairs the intestines worn by microplastics, reducing the risk of microplastics and attached pollutants penetrating the intestinal barrier and entering the body.

[0083] 4. Component D: Glutamine; Physiological function: "Special nutrition" for intestinal epithelial cells.

[0084] Mechanism of action: It serves as an important energy source for rapidly proliferating cells (such as intestinal epithelial cells and lymphocytes), directly participating in and promoting the synthesis and repair of the intestinal mucosa. It can enhance the expression of tight junction proteins between intestinal cells, structurally and functionally "strengthening" the intestinal barrier and reducing the occurrence of leaky gut syndrome.

[0085] Application value: In synergy with component C, it provides direct and crucial nutritional support for the repair of the intestinal barrier, especially under the continuous physical stimulation caused by microplastics, ensuring the integrity and health of the intestine.

[0086] III. Microbial Regulation and Detoxification Agents; 5. Component E: Complex Probiotics and Prebiotics;

[0087] Physiological function: "Regulator" of gut microbiota and "site competitor" of microplastics.

[0088] Mechanism of action: Competitive exclusion: Supplemented probiotics (such as Bacillus) can colonize and multiply in the intestine, competing with foreign harmful bacteria and microplastics for attachment sites on the intestinal wall, reducing the chance of microplastics acting as "Trojan horses" to carry pathogens into the intestine.

[0089] Microbial balance: Probiotics inhibit the growth of harmful bacteria through their metabolites (such as bacteriocins). Prebiotics (xylooligosaccharides) selectively promote the proliferation of beneficial bacteria, both those already present in the body and those added from external sources, working together to restore the gut microbiota structure, which has been disrupted by microplastic exposure, to a healthy balance.

[0090] Aids digestion: Probiotics can secrete a variety of digestive enzymes, improving overall feed utilization and gut health.

[0091] Application value: It addresses the "secondary disaster" caused by microplastics—gut flora imbalance, strengthens fish health from a microecological level, and breaks the vicious cycle between pollutants and diseases.

[0092] 6. Component F: A blend of natural plant extracts and antioxidants; Physiological function: The body's "fire brigade" against oxidative stress. Mechanism of action: Free radical scavenging: Curcumin, EGCG, and rosmarinic acid are all potent natural antioxidants that can directly neutralize excess reactive oxygen species (ROS) induced by microplastics in the body, blocking the lipid peroxidation chain reaction. Anti-inflammatory effect: These components also have significant anti-inflammatory activity, inhibiting the overactivation of inflammatory signaling pathways such as NF-κB, and reducing inflammatory damage to tissues such as the liver and intestines. Synergistic effect: The combination of the three can act on different stages and targets of oxidative stress, producing a synergistic antioxidant effect of "1+1+1>3".

[0093] Application value: It provides an efficient detoxification solution for the core toxic mechanism of microplastics—oxidative stress, protecting the function of vital organs in fish, shrimp, and crabs, and improving their survival and health quality under pollution stress.

[0094] The application value of this invention lies in the organic integration of the functions of the above six components, forming a complete in vivo microplastic reduction and control chain: inlet adsorption (A+B) → intermediate excretion promotion (C) and barrier repair (C+D) → internal environment regulation (E) and detoxification (F). In practical applications, this product can be added to conventional feed at a ratio of 0.5%-2.0% to achieve the following without changing the existing aquaculture model: (1) Improve the safety of aquatic products: significantly reduce microplastic residues in edible tissues, directly addressing consumers' concerns about food safety. (2) Promote healthy animal farming: reduce disease occurrence and improve feed utilization and survival rate by improving intestinal health and antioxidant function. (3) Address the reality of environmental pollution: provide an effective risk management tool for open or semi-open aquaculture environments where microplastic pollution is difficult to completely avoid. Therefore, this invention is not only a technological innovation, but also a comprehensive solution for the sustainable development of aquaculture and food safety assurance, with extremely high market transformation potential and industry promotion value.

[0095] Example 2: A feed additive for removing microplastics, comprising the following raw materials by weight percentage: modified chitosan microspheres (component A): 20 kg; nanoscale yeast cell wall derivatives (component B): 15 kg; fermentable dietary fiber complex (lignin: citrus pectin = 1:1, component C): 20 kg; glutamine (component D): 5 kg; compound probiotics and prebiotics (Bacillus licheniformis: Bacillus subtilis = 1:1, viable count ≥ 10 billion CFU / g, and containing 5% xylooligosaccharides, component E): 30 kg; natural plant extract compound antioxidants (curcumin: EGCG: rosmarinic acid = 3:5:2, component F): 10 kg;

[0096] Experiment 1, Preparation Method: All the above components were placed into a 500L three-dimensional motion mixer and mixed at 20-30 rpm for 50 minutes to ensure thorough and uniform mixing. After discharging, the mixture was packaged in aluminum foil bags (5kg per bag), sealed, and stored in a cool, dry place to obtain the functional feed additive of this invention.

[0097] Experiment 2: Application and Effect Verification in Carp Farming:

[0098] 1. Experimental Design: 300 healthy carp fry with an initial weight of (50.5±2.1) grams were randomly divided into two groups, with three replicates in each group. The control group was fed a basal diet, while the experimental group was fed an experimental diet containing 1.0% of the additive of this invention.

[0099] 2. Basic feed: In order to accurately evaluate the effect of the functional feed additives described in this invention, a nutritionally complete basic feed that does not contain any known substances that may affect microplastic adsorption or metabolism is required.

[0100] (1) Basic feed formulation: The basic feed consists of the following raw materials by weight percentage (%):

[0101] Table 2-1: Basic Feed Formulation

[0102]

[0103] Notes: ① Critical Control Points: To ensure the experiment was not interfered with, all plant protein sources (soybean meal, rapeseed meal) and fiber sources (wheat bran) underwent microplastic testing before use, confirming extremely low background values. The vitamin premix was specifically designed to be free of any artificial antioxidants to avoid affecting the evaluation of the antioxidant function of the additives in this invention.

[0104] ②Preparation process: All raw materials are crushed and passed through an 80-mesh sieve, and mixed in a stepwise manner to ensure uniformity. After adding fish oil, soybean oil and an appropriate amount of water, the mixture is extruded into 2.0mm diameter pellets using a twin-screw extruder. The pellets are dried at 55℃ until the moisture content is below 10%, cooled, sealed in packaging, and stored in a -20℃ refrigerator for later use.

[0105] (2) Vitamin premix formulation: Add 10 grams of vitamin premix (i.e., 1.0% addition) per kilogram of basal feed to provide the following vitamin content. This formulation is based on the NRC (2011) nutritional requirements for fish and with reference to the standards for major farmed freshwater fish in my country, and common synthetic antioxidants (such as ethoxyquinoline BHT / BHA) are deliberately omitted to avoid interfering with the evaluation of the antioxidant function of the additives of this invention.

[0106] Table 2-2: Vitamin Premix Formulation (Content per kg of premix)

[0107]

[0108] (3) Mineral premix formulation: Add 10 grams of mineral premix (i.e., 1.0% addition) per kilogram of basic feed to provide the trace elements necessary for fish growth.

[0109] Table 2-3: Mineral Premix Formulation (Content per Kilogram of Premix)

[0110]

[0111] Notes: ① Nutritional balance: The above vitamin and mineral premix formula, combined with the main ingredients in the basal feed such as fishmeal and soybean meal, ensures that the experimental fish receive comprehensive and balanced nutrition throughout the experiment, meeting their needs for normal growth and physiological metabolism. This eliminates the possibility that malnutrition could lead to a decline in physiological function, thereby indirectly affecting microplastic metabolism.

[0112] ② Experimental rigor: The detailed description of the premix reflects the meticulousness and standardization of the experimental design, enabling any fellow researchers to accurately replicate the experiment based on this formula, thus enhancing the credibility and verifiability of the research results of this invention.

[0113] ③ Purity of Functional Evaluation: Special emphasis is placed on the absence of exogenous antioxidants in the vitamin premix, which is crucial. This ensures that when evaluating the antioxidant effects of the additives of this invention (especially component F), the observed increases in indicators such as serum T-AOC are entirely attributable to the effects of the additives of this invention, rather than the contribution of the basal feed.

[0114] (4) Basic feed nutrient level (theoretical calculation value);

[0115] Table 2-4: Nutritional Levels of Basal Feed (Dry Matter Basis)

[0116]

[0117] Application Notes: (1) The control group (CTRL) was fed the above-mentioned basic feed. The feeds of each experimental group were based on this basic formula, with the microcrystalline cellulose replaced by the functional feed additives described above in equal amounts. For example, when preparing feed containing 1.0% additive, the microcrystalline cellulose content in the formula was adjusted from 8.0% to 7.0%, and 1.0% of the additives of this invention were added. This method ensured that all experimental feeds maintained consistency in major nutrient levels and energy, so that any observed physiological differences could be attributed to the effect of the additives rather than changes in basic nutrients.

[0118] (2) The basic feed formula provided in this embodiment is scientific and reasonable, meets the nutritional needs of carp, and provides a reliable and clean "background" for verifying the effectiveness of the present invention through strict raw material control, which fully ensures the accuracy and persuasiveness of the experimental results.

[0119] 3. Culture Environment: The culture was conducted in an indoor recirculating aquaculture system, with each aquarium having a capacity of 300L. To simulate a polluted environment, polyethylene (PE) microplastics (particle size 10-100μm) at a concentration of 1000 particles / L were continuously added to the system. The culture experiment lasted for 8 weeks.

[0120] 4. Sample Collection and Testing: After the experiment, 6 fish were randomly selected from each box. The dorsal muscle tissue was taken and processed using the internationally recognized alkaline digestion method (10M KOH, 60°C, 24h). Subsequently, polymer identification was performed by microscopic counting combined with Fourier transform infrared spectroscopy (μFT-IR) to accurately determine the quantity and type of microplastics in the muscle.

[0121] 5. Results: (1) Microplastic content: The average microplastic content in the muscle of carp in the experimental group was (1.8 ± 0.5) particles / g of tissue, while that in the control group was (5.2 ± 1.1) particles / g of tissue. The experimental group was 65.4% lower than that in the control group, and the difference was highly significant (P < 0.01) (see Table 2-5).

[0122] Table 2-5: Results of Microplastic Content Analysis in Carp Muscle Tissue

[0123]

[0124] Note: Data are expressed as mean ± standard deviation; different lowercase letters in the superscript of data in the same row indicate extremely significant differences (P < 0.01).

[0125] Table 2-6: Results of intestinal tissue morphology and serum antioxidant index determination in carp

[0126]

[0127] Notes: ①Intestinal morphology data were obtained through tissue sections and image analysis software, with ≥30 intact villi measured in each group.

[0128] ② Serum antioxidant indicators were measured using the corresponding reagent kits from Nanjing Jiancheng Biotechnology Research Institute.

[0129] ③ Data are expressed as mean ± standard deviation; different lowercase letters in the superscript of data from the same row indicate significant differences (P < 0.05).

[0130] (2) Intestinal health indicators: The results showed (see Tables 2-6, 2-7, and 2-8) that the intestinal villi structure of the carp in the experimental group was intact and tightly arranged, and the villi height / crypt depth value was significantly higher than that of the control group (P < 0.05).

[0131] Table 2-7: Results of intestinal morphology measurements in carp

[0132]

[0133] *Note: Compared with the control group, the villus height and villus height / crypt depth values ​​were significantly increased in the experimental group (P < 0.05).

[0134] Table 2-8: Intestinal Histological Scoring Results

[0135]

[0136] *Note: Scoring criteria: 1 = severe abnormality, 2 = moderate abnormality, 3 = mild abnormality, 4 = normal; * indicates P < 0.05 compared with the control group.

[0137] (3) Antioxidant capacity: The malondialdehyde content in the serum and liver of carp in the experimental group was significantly reduced, while the activities of total superoxide dismutase and glutathione peroxidase were significantly increased (P < 0.05), indicating that the overall antioxidant capacity was effectively improved (see Table 2-6).

[0138] The conclusion of this experiment is that the functional feed additive of the present invention can safely and efficiently reduce the microplastic accumulation level in the muscle of farmed carp, and simultaneously improve their intestinal health and antioxidant function, with significant comprehensive effects.

[0139] Experiment 3: Application verification in other major freshwater fish (black carp, grass carp, silver carp, bighead carp, and crucian carp):

[0140] 1. Experimental objective: To verify whether the functional feed additive described in this invention has the same effect on significantly reducing the microplastic content in the bodies of other major freshwater farmed fish (black carp, grass carp, silver carp, bighead carp, and crucian carp) besides carp, and to examine its wide applicability and universality.

[0141] 2. Experimental design: Fish species: Select healthy, uniformly sized fry of grass carp, silver carp, bighead carp, and crucian carp.

[0142] Aquaculture environment: Aquaculture was conducted in separate recirculating aquaculture systems to simulate suitable water temperature, dissolved oxygen, and pH conditions for the growth of each fish species. A mixed microplastic (containing PE, PP, and PS, with a particle size of 10-150 μm) at a concentration of 800 particles / L was continuously added to the water in all systems.

[0143] Experimental grouping and feed: Each type of fish was randomly divided into two groups: the control group was fed its special basic feed (same as the basic feed in Experiment 2); the experimental group was fed the experimental feed with 1.0% of the additive of this invention added.

[0144] Breeding cycle: 60 days.

[0145] Testing indicators: After the culture period, dorsal muscle tissue was collected from each fish species, and the microplastic content was determined using alkaline digestion-micro Fourier transform infrared spectroscopy (μFT-IR). Simultaneously, serum samples were collected to determine total antioxidant capacity (T-AOC).

[0146] 3. Experimental Results:

[0147] Table 3-1: Effects of the present invention on microplastic content and antioxidant capacity in different freshwater fish species

[0148]

[0149] Note: Data are expressed as mean ± standard deviation; different letters on the subscript of data from the same species indicate significant differences (P < 0.05).

[0150] 4. Results and Conclusions: (1) Verification of Universality: As shown in Table 3-1, in five major freshwater fish species with different diets and ecological niches, the microplastic content in their muscle tissue was significantly reduced after feeding with feed containing 1.0% of the additive of this invention (P < 0.05), with a reduction rate between 62.2% and 66.0%, which is highly consistent with the effect in carp (65.4%). It is particularly noteworthy that silver carp and bighead carp, as filter feeders, filter a large amount of water through their gill rakers, and their initial microplastic accumulation is significantly higher than that of other fish species (the content in the control group is as high as 6.90 and 6.35). However, the additive of this invention can also reduce the microplastic content in their bodies by about 65%, which strongly proves that the additive of this invention also has an excellent removal effect on filter feeders with high exposure risk.

[0151] (2) Universality of physiological health benefits: The total serum antioxidant capacity (T-AOC) of all five fish experimental groups was significantly higher than that of their respective control groups. This indicates that the additive of the present invention can not only effectively remove microplastics, but also universally improve the systemic antioxidant level of different fish species and alleviate microplastic-induced oxidative stress. This beneficial effect has universality across fish species.

[0152] (3) Correlation analysis of mechanism of action and fish species adaptability: The main targets of this invention are the intestinal environment (adsorption, excretion promotion, repair, and microbial regulation) and systemic antioxidant defense in fish. Although these fish species have different diets, their basic intestinal structure and physiological functions (such as peristalsis, absorption, mucosal barrier, and microbial colonization) and liver detoxification and metabolic mechanisms are conserved and similar. Therefore, based on the multi-target mechanism of action of intestinal and systemic health, this invention can overcome dietary differences and exert a stable and efficient effect in different fish species.

[0153] Conclusion of this experiment: This experiment fully demonstrates that the provided functional feed additive can be safely and effectively applied to major freshwater aquaculture fish such as grass carp, silver carp, bighead carp, and crucian carp, and can stably achieve the dual goals of reducing microplastic accumulation in the body and enhancing the body's antioxidant capacity. This indicates that the invention does not possess species-specificity and has broad applicability, providing a solid scientific basis for its large-scale promotion and application in aquaculture, and greatly expanding the scope of protection and market value of the invention. A removal rate of 62.2%-66.0% was achieved in freshwater fish such as grass carp, silver carp, bighead carp, and crucian carp, proving the universality of the invention.

[0154] Experiment 4: Extended application verification in marine aquaculture:

[0155] 1. The purpose of this experiment is to verify the effectiveness and universality of the functional feed additive described in this invention in typical marine aquaculture fish, including pond culture (sea bass, grouper, turbot) and cage culture (golden pomfret, large yellow croaker, cobia), and to examine its ability to cope with microplastic pollution under different salinity and aquaculture environments.

[0156] 2. Experimental Design: (1) Experimental Fish Species and Farming Model:

[0157] Pond rearing group: Sea bass, grouper, and turbot were selected and reared in standardized outdoor seawater aquaculture ponds.

[0158] Net cage rearing group: Golden pomfret (ovoid pomfret), large yellow croaker, and cobia were selected and reared in nearshore wind and wave resistant net cages.

[0159] Experimental environment: All aquaculture environments were natural seawater, which inevitably contained background microplastics.

[0160] Experimental grouping and feed: Each type of fish was randomly divided into two groups: the control group was fed with basic marine fish feed; the experimental group was fed with experimental feed containing 1.2% of the additive of this invention (considering the possible metabolic differences of marine fish, the amount of additive was slightly increased).

[0161] ① General marine fish basic feed formula: By weight percentage (%), the basic feed consists of the following ingredients:

[0162] Table 4-1: Common Basic Feed Formulations for Marine Fish

[0163]

[0164] ② Vitamin and mineral premix formulation: Vitamin premix: Add 15 grams of vitamin premix per kilogram of basal feed (i.e., 1.5% addition).

[0165] Table 4-2: Formula for Vitamin Premix for Marine Fish (Content per Kilogram of Premix)

[0166]

[0167] Mineral premix: Add 20 grams of mineral premix per kilogram of basal feed (i.e., 2.0% addition).

[0168] Table 4-3: Formulation of mineral premix for marine fish (content per kilogram of premix)

[0169]

[0170] ③ Basic feed nutrient level (theoretical calculation value);

[0171] Table 4-4: Nutritional Levels of Basal Feed (Dry Matter Basis)

[0172]

[0173] ④ Application Notes: In this experiment, the control group (CTRL) was fed the aforementioned basic feed. The feeds for each experimental group were based on this basic formula, with the functional feed additive described above replacing the microcrystalline cellulose in an equal amount. For example, when preparing a feed containing 1.2% additive, the microcrystalline cellulose content in the formula was adjusted from 2.49% to 1.29%, and 1.2% of the additive of this invention was added. This method ensured that all experimental feeds maintained consistent levels of major nutrients and energy, thus demonstrating that the observed physiological differences were entirely attributable to the effect of the additive. This formula is designed specifically for the physiological characteristics of marine fish, with high protein and fat content, and fortified with key vitamins (such as VC and VE) and minerals, providing a reliable and nutritionally balanced benchmark for verifying the effectiveness of this invention in marine fish.

[0174] (4) Breeding cycle: 70 days.

[0175] (5) Detection indicators: After the breeding is completed, samples are taken to detect the microplastic content in the back muscles and the serum total antioxidant capacity (T-AOC) is measured as a health indicator.

[0176] 3. Experimental Results:

[0177] Table 4-5: Effects of the present invention on microplastic content and antioxidant capacity in different marine fish species

[0178]

[0179] Notes: (1) Data are expressed as mean ± standard deviation; different letters on the subscript of data of the same species indicate significant differences (P < 0.05). (2) Turbot is a bottom-dwelling fish, and its liver is more likely to accumulate lipid-soluble pollutants. Therefore, liver indicators were measured, and the results also showed a highly significant decrease.

[0180] 4. Results and Conclusions: (1) Verification of effectiveness across salinity and aquaculture modes: This experiment is the first to apply the additive of this invention to marine fish. As shown in Tables 4-5, regardless of whether it is in a more controllable pond aquaculture environment or a more open net cage aquaculture environment, the additive of this invention shows a stable and efficient microplastic removal effect on all six economically valuable marine fish species, with a reduction rate between 62% and 64%, which is highly consistent with the effect in freshwater fish. This strongly proves that the mechanism of action of this invention is not affected by the salinity of the water. Its core function—adsorbing microplastics in the intestine, promoting their excretion with feces, and protecting intestinal health—also works perfectly in the digestive system of marine fish.

[0181] (2) Adaptability to special habits and high-value fish species: For bottom-dwelling fish such as turbot with a high liver metabolic load, this invention not only significantly reduces microplastics in muscles, but also has a more prominent effect on liver clearance (66.9%). This indicates that this invention can effectively reduce the pollutant burden on major metabolic organs and has important health significance. For valuable cage-cultured fish species such as large yellow croaker and cobia, which have extremely high requirements for feed quality and health status, this invention effectively removes microplastics while significantly improving their antioxidant levels. This has significant application value for reducing aquaculture risks, improving survival rates and product quality.

[0182] (3) Conclusion on the universality and market expansion of the technology: The target points of this invention (intestinal physicochemical environment, intestinal flora, and systemic antioxidant system) are common and conserved physiological systems in fish. Therefore, its effects can be seamlessly extended from freshwater fish to marine fish, and from ponds to cages. This embodiment greatly broadens the scope of protection and application scenarios of this invention, making it a universal solution covering major aquaculture species in both freshwater and marine environments. This is not only a major technological breakthrough, but also signifies huge market potential and commercial value, providing a practical and feasible technical tool for addressing the global problem of microplastic pollution in seafood.

[0183] Experiment 5: Validation of cross-species (shrimp, swimming crab) aquaculture application;

[0184] 1. Experimental objective: To verify the effect of the functional feed additive described in this invention on the removal of microplastics in typical marine crustaceans (shrimp and swimming crab) and to evaluate its application potential.

[0185] 2. Experimental Design: (1) Experimental Animals and Culture Environment: Litopenaeus vannamei and Portunus trituberculatus larvae were selected and cultured in an indoor recirculating aquaculture system. The water temperature, salinity, and pH were adjusted to their suitable growth range. Mixed microplastics (containing PE and PP with a particle size of 10-150 μm) at a concentration of 800 particles / L were continuously added to the culture water to simulate a microplastic pollution environment.

[0186] (2) Experimental grouping and feed: Each animal was randomly divided into two groups: the control group was fed its own basic feed; the experimental group was fed the experimental feed with 1.2% of the additive of this invention added. The breeding period was 56 days, which is a common period for studies on the accumulation and excretion of microplastics in crustaceans.

[0187] (3) Basic feed formulation: ① Formulation design principles, nutritional balance: meeting the core nutritional needs of shrimp and swimming crab larvae to adult stages, including protein, fat, carbohydrates, cholesterol, and phospholipids. Raw material standardization: all raw materials must pass microplastic background testing to ensure extremely low background values ​​that do not interfere with experimental results. No interfering components: no artificial antioxidants (such as BHT and BHA) are added to the vitamin premix. Process suitability: it has good binding and water stability to adapt to the feeding habits of shrimp and crabs. ② Basic feed formulation (weight percentage, %).

[0188] Table 5-1: Composition of General Basic Feed Formulation

[0189]

[0190] ③ Vitamin and Mineral Premix Formulation: Vitamin Premix (content per kg of premix); Vitamin A: 2,800,000 IU (Vitamin A acetate); Vitamin D3: 600,000 IU (Cholecalciferol); Vitamin E: 25,000 mg (DL-α-tocopherol acetate, 50%); Vitamin K3: 1,500 mg (MSB); Vitamin C: 30,000 mg (L-ascorbic acid-2-phosphate, 35%); Vitamin B1: 3,500 mg (Thiamine hydrochloride); Vitamin B2: 6,000 mg (Riboflavin); Vitamin B6: 4,500 mg (Pyridoxine hydrochloride); Vitamin B2 ... 12 : 25 mg (cyanocobalamin); Calcium pantothenate: 12,000 mg (D-calcium pantothenate); Niacin: 30,000 mg (nicotinamide); Folic acid: 1,200 mg; Biotin: 60,000 mg (D-biotin, 2%); Inositol: 60,000 mg; Carrier: wheat bran, up to 1,500 g; ④ Mineral premix (content per kilogram of premix); Magnesium: 20,000 mg (magnesium sulfate); Iron: 4,000 mg (ferrous sulfate); Zinc: 8,000 mg (zinc sulfate); Manganese: 3,000 mg (manganese sulfate); Copper: 800 mg (copper sulfate); Cobalt: 100 mg (cobalt chloride); Selenium: 50 mg (sodium selenite); Iodine: 400 mg (calcium iodate); Carrier: zeolite powder, up to 2,000 g.

[0191] ⑤ Feed Preparation Process: Crushing and Sieving: Crush all raw materials and pass them through a 100-mesh sieve. Mixing: Use a step-by-step expansion method for thorough mixing to ensure uniformity. Feeding Sequence: First, add trace components (vitamins, minerals, etc.) and carriers (microcrystalline cellulose, bran) for premixing, then add protein raw materials, and finally add oils. Pelletizing and Curing: Add an appropriate amount of water (about 30%) and extrude pellets with a diameter of 1.5mm (shrimp) or 2.0mm (swimming crab) using a twin-screw extruder. Drying and Packaging: Dry at 45-50℃ until the moisture content is below 10%, cool, seal in packaging, and store in a -20℃ refrigerator.

[0192] ⑥ Theoretical nutritional level (dry matter basis): Crude protein: 45.0%; Crude fat: 8.5%; Crude ash: 11.0%; Total phosphorus: 1.3%; n-3 HUFA: >1.5% (especially EPA and DHA).

[0193] ⑦ Application Instructions: In the aquaculture experiment, the control group (CTRL) was fed the above-mentioned basic feed. The feed for each experimental group was based on this basic formula, with the functional feed additive described above replacing the microcrystalline cellulose in an equal amount. For example, when preparing feed containing 1.2% additive, the microcrystalline cellulose content in the formula was adjusted from 5.49% to 4.29%, and 1.2% of the additive of this invention was added.

[0194] (4) Sample collection and testing: After the aquaculture was completed, samples were taken (whole muscle of shrimp; gills, muscle and hepatopancreas of swimming crab). Microplastics were extracted, counted and polymer types were identified by alkaline digestion-density flotation combined with micro Fourier transform infrared spectroscopy (μFT-IR). At the same time, the survival rate, molting cycle and frequency of the experimental animals were recorded, and fecal samples were collected for microplastic content analysis.

[0195] 3. Experimental Results:

[0196] Table 5-2: Effects of the additive of the present invention on microplastic content and growth indicators in shrimp and crabs

[0197]

[0198] * indicates a significant difference compared to the control group (P < 0.05).

[0199] Table 5-3: Detection results of microplastic content in shrimp and crab feces:

[0200]

[0201] * indicates a significant difference compared to the control group (P < 0.05).

[0202] 4. Results Analysis: (1) Highly efficient removal effect: The additive of the present invention can significantly reduce the content of microplastics in the muscle and hepatopancreas of shrimp and crab, with a removal rate of over 64%. This indicates that the "adsorption-promoting excretion" mechanism of the present invention is also effective in crustaceans. (2) Effect on key metabolic organs: The hepatopancreas of crab is the main organ for digestion and detoxification, and the initial accumulation of microplastics is much higher than that in muscle. The microplastic content in the experimental group was significantly reduced, indicating that the present invention can effectively reduce the pollutant load on metabolic organs, which is crucial for maintaining animal health. (3) Promote excretion pathway: Combined with the detection of shrimp and crab feces, it was found that the microplastic content in the feces of the experimental group was significantly higher than that in the control group. This confirms that the present invention can effectively promote the excretion of microplastics through the digestive tract with feces. In addition, the observed increase in the frequency of molting of shrimp suggests that the present invention may indirectly promote the excretion of microplastics accumulated in the epidermis and gills of crustaceans through the important physiological process of molting by improving the body's health. (4) Animal health and safety: During the experiment, no abnormal deaths occurred in any group of animals, the survival rate of the experimental group was good, and the growth indicators were normal. This indicates that the additive of the present invention is safe for marine-farmed shrimp and crabs at the recommended dosage.

[0203] 5. Conclusion of this experiment: The functional feed additive of this invention can safely and efficiently remove microplastics from the bodies of marine-cultured shrimp and swimming crabs, with a removal effect comparable to that observed in fish. This further expands the application scope of this invention, provides an effective technical solution for addressing the microplastic pollution problem in marine-cultured crustaceans, and enhances the universality and market application potential of the patent.

[0204] Experiment 6: Verification of the synergistic effect of each component:

[0205] 1. Experimental objective: To verify the synergistic effect of the components in the additive of this invention, and to clarify the significant advantages of the complete formula compared with a single component or a combination of components in reducing the microplastic content in fish and improving physiological indicators.

[0206] 2. Experimental Design: Experimental Animals and Breeding Environment: Similar to Experiment 2, healthy carp fry were selected and cultured in aquariums continuously supplemented with PE microplastics (1000 particles / L) for 8 weeks. Experimental Feed: 1.0% of different types of additives were added to the basal feed to form the following 6 experimental groups: Control Group (CTRL): Basal feed.

[0207] Single adsorbent group (SA): basal feed + 1.0% (component A + component B).

[0208] Adsorption + Excretion-Promoting Group (AP): Basal feed + 1.0% (Component A + Component B + Component C).

[0209] Adsorption + Repair Group (AR): Basic feed + 1.0% (Component A + Component B + Component D).

[0210] Partial Functional Group (PF): Basic feed + 1.0% (Component A + Component B + Component C + Component D). It includes the "adsorption-promote excretion-repair" chain, but lacks the "regulation-detoxification" chain.

[0211] The complete formula of this invention consists of: basal feed + 1.0% (components A + B + C + D + E + F).

[0212] 3. Detection indicators and methods: (1) Microplastic content: Same as Experiment 2, determine the number of microplastic particles in the back muscles.

[0213] (2) Intestinal barrier function: The activity of diamine oxidase (DAO) in serum was measured. DAO is a marker enzyme in intestinal mucosal cells. When the intestinal barrier is damaged, a large amount of DAO is released into the blood. Therefore, serum DAO activity is positively correlated with the degree of intestinal damage.

[0214] (3) Systemic antioxidant capacity: The total antioxidant capacity (T-AOC) of serum was measured.

[0215] (4) Synergistic effect value calculation: The synergistic effect value is calculated to quantify the effect of "1+1>2". The following general formula is used for calculation: Synergistic effect value = [(actual effect difference - theoretical superposition effect) / theoretical superposition effect] ×100%; where: actual effect difference = actual value of the control group - actual value of the FULL group; theoretical superposition effect = the expected effect difference estimated by the linear superposition model based on the effect of each independent module in the control group.

[0216] 4. Experimental Results:

[0217] Table 6-1: Effects of different additive formulations on physiological indicators of carp (synergistic effect verification)

[0218]

[0219] Note: Data are expressed as mean ± standard deviation; different letters in the same column indicate significant differences (P < 0.05).

[0220] Table 6-2: Additive composition and microplastic content in carp muscle

[0221]

[0222] Table 6-3: Summary of Synergistic Effect Values ​​of Each Component in the Invention

[0223]

[0224] 5. Results and Conclusions: (1) Regarding the microplastic removal effect: The SA group (adsorption only) showed a certain effect, proving that adsorption is the foundation. The AP group (adsorption + excretion promotion) and the AR group (adsorption + repair) were significantly better than the SA group (P < 0.05), proving that "excretion promotion" and "repair" can effectively improve the removal efficiency. The PF group (adsorption + excretion promotion + repair) showed a further significant improvement, indicating that the extension of the functional chain is crucial. The FULL group (complete formula) was significantly better than all other groups (P < 0.05), and the microplastic content was reduced to the lowest level. This proves that without the "regulation and detoxification" function of components E and F, even if the preceding steps are perfected, the best removal effect cannot be achieved, highlighting the necessity of the complete formula.

[0225] (2) Regarding intestinal barrier protection: Serum DAO activity showed a gradual and significant decreasing trend from the CTRL to the FULL group. The FULL group had the lowest DAO activity, which was significantly lower than that of the PF group (P < 0.05). This indicates that the addition of probiotics (E) and antioxidants (F) played a key role in alleviating intestinal inflammation and protecting mucosal integrity, which was not present in the PF group, thus achieving a deeper level of intestinal protection.

[0226] (3) Regarding overall antioxidant capacity: The T-AOC index also showed a gradual increasing trend from the CTRL to the FULL group. Only the FULL group reached the highest T-AOC level, which was significantly higher than that of the PF group (P < 0.05). This directly proves the irreplaceable role of component F (a compound antioxidant of natural plant extracts) in enhancing the body's overall resilience, as well as the best effect brought about by its synergistic effect with other components.

[0227] (4) Synergistic effect value calculation: ① Core comparison: FULL group vs PF group (to prove the synergistic contribution of E+F modules); Measured effect difference: PF group content - FULL group content = 2.45 - 1.80 = 0.65 particles / gram; Theoretical superposition effect estimation: PF group already includes A+B+C+D, and its effect is 2.45. Based on PF group, if the functions of E and F are simply superimposed, their theoretical contribution should be approximately equal to the effect increment they exhibit in other part combinations. Based on the data of AP group (3.05) and AR group (2.95), the independent contribution estimates of E and F are small, and their theoretical superposition effect is conservatively estimated to be about 0.20 particles / gram. Synergistic effect value calculation: = [(0.65-0.20) / 0.20]×100%=(0.45 / 0.20)×100%=225%.

[0228] Conclusion: Adding modules E and F to the PF group resulted in a significantly better actual cleansing effect than expected, with a synergistic effect value as high as 225%, indicating that E and F had a strong synergistic effect with the front-end system.

[0229] ② Secondary comparison: PF group vs. SA group (proving the synergistic contribution of C+D modules): Measured effect difference: SA group content - PF group content = 3.85 - 2.45 = 1.40 particles / gram; Theoretical superposition effect estimation: Based on the data of SA group (3.85), AP group (3.05), and AR group (2.95), the theoretical independent contribution of C and D is approximately 0.95 particles / gram. Synergistic effect value calculation: = [(1.40 - 0.95) / 0.95] × 100% = (0.45 / 0.95) × 100% = 147%.

[0230] Conclusion: Adding modules C and D to the SA group also produced a significant synergistic effect, with an effect size of 147%.

[0231] 6. Synergistic Effect Conclusion: ① This experiment fully demonstrates that the six components of this invention constitute an organic whole, none of which can be omitted. Components A and B perform the removal task. Components C and D ensure the removal and repair of the removed substances. Components E and F maintain internal environmental stability and resist secondary damage from toxins. The efficacy of any single component or partial combination is significantly lower than that of the complete formula. There is a clear positive synergistic effect among the components, which together constitute the core technical advantage of this invention in efficiently and comprehensively solving the problem of microplastic pollution in fish. This embodiment provides strong experimental data support for the inventiveness and progress of this invention.

[0232] ② This experiment confirms a strong positive synergistic effect among the functional modules in the additive of this invention. The absence of any functional module leads to a significant decrease in the overall effect. The effect of the full combination is not a simple summation of the functions of each component, but rather produces an emergent effect of "1+1+1+1+1+1 >> 6". The synergistic effect value of up to 225% strongly demonstrates that the specific combination of this invention produces unexpected technical effects, which cannot be easily obtained by those skilled in the art through conventional reasoning or simple reference to existing technology, fully reflecting the outstanding substantive features and significant progress of this invention.

[0233] Experiment 7: Dosage-Effect Experiment of Each Functional Module of the Additive

[0234] 1. Experimental objective: To accurately investigate the contribution of the three functional modules (bioactive adsorbent, intestinal physical cleansing and barrier repair agent, and microbial regulation and detoxification agent) in the additive of this invention to the microplastic removal effect under different dosage ratios, determine the optimal dosage ratio range of each module, and optimize the overall combination.

[0235] 2. Experimental Design: (1) Experimental Animals and Breeding Environment: Same as Experiment 2, carp fry were selected and raised in water polluted by microplastics for 8 weeks. (2) Experimental Design Principle: The "single-factor variable method" was adopted. Under the premise of keeping the total dose of the whole formula at 1.5%, the dose of a certain functional module was systematically adjusted, and the doses of the other two modules were adjusted proportionally to observe the effect of the dose change of the module on the overall effect. (3) Six components of the additive: A: Modified chitosan microspheres Function: As a core bioadsorbent, it specifically adsorbs microplastics through the electrostatic and hydrophobic effects imparted by surface modification. B: Nanoscale yeast cell wall derivatives Function: As an auxiliary adsorbent and immune enhancer, it captures nanoscale plastics using nanoscale pores. Its β-glucan component can activate the body's immunity. C: Fermentable dietary fiber complex Function: As a physical scavenger, it stimulates intestinal peristalsis and accelerates the excretion of adsorbed microplastics from the body. D: Glutamine Function: As a barrier repair agent, it is a key nutrient for intestinal mucosal cells and directly promotes the repair of damaged intestinal epithelium. E: Compound probiotics and prebiotics function: As a microbial regulator, they competitively inhibit harmful bacteria and regulate the intestinal microecological balance by colonizing beneficial bacteria and providing them with nutrients. F: Natural plant extracts combined with antioxidants function: As a detoxifier, they eliminate excess reactive oxygen species induced by microplastics through their potent antioxidant and anti-inflammatory activities, alleviating systemic oxidative stress. These six components together constitute the synergistic four-in-one technology system of this invention: "adsorption-promote elimination-repair-regulation". (4) Experimental Groups: The following 7 experimental groups were set up: FULL-1.5: Optimal All-Round Formula Group (A+B: 0.5%, C+D: 0.5%, E+F: 0.5%); AB-High: High Adsorbent Group (A+B: 0.7%, C+D: 0.4%, E+F: 0.4%); AB-Low: Low Adsorbent Group (A+B: 0.3%, C+D: 0.6%, E+F: 0.6%); CD-High: High Ovulation-Promoting and Repairing Agent Group (A+B: 0.4%, C+D: 0.7%, E+F: 0.4%); CD-Low: Low Ovulation-Promoting and Repairing Agent Group (A+B: 0.6%, C+D: 0.3%, E+F: 0.6%); EF-High: High Regulation and Detoxification Agent Group (A+B: 0.4%, C+D: 0.4%, E+F: 0.4%). 0.7%); EF-Low: Low-regulation antidote group (A+B: 0.6%, C+D: 0.6%, E+F: 0.3%).

[0236] 3. Detection indicators: (1) Core indicator: Microplastic content in back muscles. (2) Functional indicators: Adsorption efficiency: The number of free microplastics in intestinal contents that have not been expelled. Emptying rate: The rate at which intestinal chyme empties. Antioxidant level: Malondialdehyde (MDA) content in the liver.

[0237] 4. Experimental Results:

[0238] Table 7-1: Effect of different dosage ratios of each functional module on the microplastic removal effect

[0239]

[0240] Notes: (1) Different letters in the same column indicate significant differences (P < 0.05). (2) The removal rate is calculated based on the CTRL group (5.20 particles / g). (3) The number of free microplastics is a relative value; the lower the value, the higher the adsorption efficiency.

[0241] 5. Results and Mechanism Analysis: (1) Dosage Effect of Bioactive Adsorbents (A+B): Low-dose defects: The effect of the AB-Low group decreased significantly, and the number of free microplastics was the highest, proving that insufficient adsorption dose is the key bottleneck limiting the removal efficiency. High-dose redundancy: The effect of the AB-High group was not significantly better than FULL-1.5, indicating that the adsorbent dose of 0.5% is close to saturation, and further increasing it has limited contribution to improving the final removal effect. The optimal dose range is 0.5%-0.7%.

[0242] (2) Dose-effect of intestinal physical clearance and barrier repair agent (C+D): Low dose drawback: The intestinal emptying rate of the CD-Low group was significantly the lowest, resulting in the inability of adsorbed microplastics to be expelled in time, prolonging the retention time in the intestine and affecting the overall clearance efficiency. High dose effect: The CD-High group had the highest emptying rate, but the final clearance effect was no different from that of the preferred group, indicating that a dose of 0.5% was sufficient to ensure effective emptying. The optimal dose range is 0.5%-0.7%.

[0243] (3) Dosage-effect relationship of microbial regulation and detoxification agents (E+F): Low-dose hazards: The EF-Low group showed poor efficacy and extremely high MDA content in the liver, indicating that the body was under intense oxidative stress at low doses. This internal damage severely restricted the efficiency of preceding functional modules. High-dose benefits: The EF-High group showed excellent performance in scavenging effect and antioxidant indicators, indicating a certain potential for dose increase. The optimal dose range is 0.5%-0.7%, and a moderate increase can be considered.

[0244] 6. Experimental Conclusion: This experiment confirms, from the perspective of each functional module, that:

[0245] (1) The three functional modules of “adsorption-promoting excretion-repair-regulation” in this invention are closely related. Insufficient dosage of any module will become a “shortcoming” in the overall effect.

[0246] (2) The functional modules in the whole formula are compounded in a ratio of 0.5% : 0.5% : 0.5% (total dose 1.5%), which achieves the best synergistic balance and the highest return on investment.

[0247] (3) The weight percentage range of “30%-50% bioactive adsorbent, 25%-40% intestinal physical cleansing and barrier repair agent, and 20%-30% microbial regulator and detoxifier” (based on a total addition of 1.0%-2.0%) is scientific and necessary. Exceeding or falling below this range will disrupt the synergistic balance of the system and lead to a decrease in effectiveness.

[0248] Experiment 8: Dosage-Effect Experiment of Additives

[0249] 1. Experimental objective: To investigate the effects of the functional feed additive of the present invention at different dosages on the removal of microplastics in farmed fish and on their physiological health, and to determine its optimal effective dosage range.

[0250] 2. Experimental Design: Experimental Animals and Breeding Environment: Three hundred healthy carp fry with an initial weight of (50.5±5.1) grams were selected and cultured in an indoor recirculating aquaculture system. Polyethylene (PE) microplastics (particle size 10-100 μm) at a concentration of 1000 particles / L were continuously added to the culture water. The breeding cycle was 8 weeks. Experimental Feed: Different proportions of the additive of this invention were added to the standard basal feed (formula as above) to form the following 5 experimental groups: Control Group (CTRL): Basal feed + 0% additive. Low-dose group (LOW): Basal feed + 0.5% additive. Medium-dose group (MID): Basal feed + 1.0% additive. High-dose group (HIGH): Basal feed + 1.5% additive. Ultra-high-dose group (SUPER): Basal feed + 2.0% additive.

[0251] 3. Detection Indicators and Methods: Core Performance Indicators: After the rearing period, dorsal muscle tissue was collected, and the microplastic content was determined using alkaline digestion-micro Fourier transform infrared spectroscopy (μFT-IR). Physiological Health Indicators: Gut Health: Midgut tissue was prepared into sections, and the villus height / crypt depth ratio (V / C value) was measured. Antioxidant Capacity: Serum total antioxidant capacity (T-AOC) was measured. Growth Performance: Weight Gain Rate (WGR) and Feed Conversion Ratio (FCR) were calculated.

[0252] 4. Experimental Results:

[0253] Table 8-1: Effects of different additive dosages on microplastic content and growth performance in carp

[0254]

[0255] Note: Different letters in the superscript of data in the same column indicate significant differences (P < 0.05). Clearance rate is calculated based on the CTRL group.

[0256] Table 8-2: Effects of different additive dosages on physiological health indicators of carp

[0257]

[0258] Note: Different letters in the superscript of data in the same column indicate significant differences (P < 0.05).

[0259] 5. Results Analysis: (1) Dose-response relationship: The microplastic removal effect increased significantly with the increase of additive dosage. From 0.5% to 1.0%, the removal rate jumped from 43.3% to 65.4%, indicating that 1.0% is a key effective dosage point. When the dosage exceeds 1.0% (1.5% and 2.0% groups), although the removal rate still increased slightly, there was no statistically significant difference compared with the 1.0% group (P > 0.05), indicating that the effect entered a plateau period. (2) Determination of the optimal dosage range: An additive dosage of 1.0% can achieve excellent microplastic removal effect (>65%) and significantly improve intestinal health, antioxidant capacity and growth performance. 1.0%-1.5% is the optimal dosage window, within which the effect reaches its peak and remains stable. Considering cost-effectiveness, 1.0% is the recommended optimal economic dosage. (3) Safety confirmation: Even at an ultra-high dose of 2.0%, the growth, feed efficiency and various physiological indicators of the test fish did not show any abnormalities or inhibition, indicating that the additive of the present invention has good safety within the experimental dose range.

[0260] 6. Conclusions of this experiment: (1) There is a clear dose-response relationship between the dosage of the additive of this invention and the scavenging effect. (2) 0.5%-2.0% is the effective dosage range, of which 1.0% is the key effective dosage. (3) It is recommended to use an addition amount of 0.5%-2.0% in production practice, of which 1.0% is the optimal choice, which can ensure the best scavenging effect while obtaining good economic returns.

[0261] Experiment 9: Systematic Analysis of Synergistic Mechanisms Based on Network Pharmacology and Multi-omics:

[0262] 1. Experimental objective: To elucidate the mechanism of action of the additives of this invention in synergistically clearing microplastics and alleviating their toxicity through multiple targets and pathways by integrating network pharmacology, transcriptomics and metabolomics at the molecular level using a "predictive-validation" systems biology strategy.

[0263] 2. Experimental Design and Methods: (1) Sample Source: Carp liver and intestinal tissues from the control group (CTRL), microplastic exposure group (MP), and the additive intervention group of this invention (MP+FULL) in Experiment 2 were collected. (2) Network Pharmacology Prediction: Screening of Active Ingredients: Curcumin, epigallocatechin gallate (EGCG), rosmarinic acid, chitosan oligosaccharide, and β-1,3 / 1,6-glucan were selected as representative active molecules in this invention. Target Prediction: The active ingredient targets were predicted using the SwissTargetPrediction and PharmMapper databases. Network Construction: The intersection with the microplastic toxicity targets was taken to obtain common targets. A "component-target-pathway" network was constructed using Cytoscape, and GO and KEGG enrichment analysis was performed. (3) Multi-omics Validation: Transcriptomics: mRNA sequencing was performed on liver tissue to analyze differentially expressed genes (DEGs). Non-targeted Metabolomics: UPLC-MS analysis was performed on liver tissue to identify differentially expressed metabolites (DAMs). Integration analysis: Correlation analysis of transcriptomic and metabolomic data to construct a molecular-metabolite regulatory network.

[0264] 3. Experimental Results: 3.1 Network Pharmacology Prediction of Core Pathways: KEGG enrichment analysis showed that this invention was significantly enriched in the NOD-like receptor signaling pathway (P=3.2E-08), TNF signaling pathway (P=4.5E-07), and PI3K-Akt signaling pathway (P=6.1E-06), predicting that it would function by regulating inflammatory responses, apoptosis, and tight junctions. Core targets included AKT1, TNF, IL6, MAPK1, and CASP3. 3.2 Key Findings Validated by Multi-omics: Transcriptomics: Compared with the MP group, the expression of key inflammatory genes in the NOD-like receptor pathway (such as Nlrp3 and Il1b) was significantly downregulated in the MP+FULL group; while the expression of intestinal tight junction protein genes (such as Occludin and ZO-1) was significantly upregulated. This is highly consistent with network pharmacology predictions. Metabolomics: The MP+FULL group significantly reversed the tryptophan metabolism disorder induced by microplastics, with a highly significant decrease in the kynurenine / tryptophan ratio (P<0.01), a key indicator for measuring inflammation and oxidative stress in the body. Metabolomic analysis revealed that differentially expressed genes and metabolites were significantly associated with arachidonic acid and glycerophospholipid metabolism pathways. In the MP+FULL group, levels of pro-inflammatory metabolites (such as leukotriene B4) decreased, while levels of membrane repair-related phospholipid metabolites increased.

[0265] Table 9-1: Enrichment analysis of core targets and pathways predicted by network pharmacology (Top 10)

[0266]

[0267] Table 9-2: Statistics and Analysis of Differentially Expressed Genes by Transcriptomics

[0268]

[0269] Note: DEGs are defined as genes with |log2FC|>1 and FDR<0.05.

[0270] Table 9-3: Changes in the expression of key inflammation and barrier-related genes (qPCR validation)

[0271]

[0272] Note: Different letters in the superscript of the same data indicate significant differences (P < 0.05).

[0273] Table 9-4: Key Differential Metabolite Changes in Metabolomics

[0274]

[0275] Table 9-5: Key Association Pairs in Gene-Metabolite Integration Analysis

[0276]

[0277] 4. Experimental conclusions and mechanism explanation: (1) Systemic inhibition of inflammatory pathways: Through network pharmacology prediction and transcriptomics verification, this invention specifically inhibits two core inflammatory signaling pathways, NOD-like receptor and TNF, and significantly reduces the expression of NLRP3, a key component of the inflammasome, and downstream pro-inflammatory factors IL-1β and TNF-α. (2) Reprogramming of metabolic homeostasis: Metabolomics data show that this invention significantly reverses the tryptophan metabolism disorder caused by microplastics, reducing the kynurenine / tryptophan ratio from 3.85 to 0.42. This key indicator change proves that this invention effectively alleviates systemic inflammation and oxidative stress. (3) Active repair of the intestinal barrier: Integrated analysis of transcriptomics and metabolomics reveals a positive regulatory loop of "microbiota-metabolites-genes": This invention promotes the production of butyrate by intestinal microbiota → butyrate upregulates the expression of tight junction protein (OCLN, ZO-1) → enhances intestinal barrier function → reduces the penetration of microplastics and toxins. These data, from a systems biology perspective, fully reveal the molecular mechanism by which this invention achieves comprehensive control of microplastic toxicity through the synergistic action of multiple targets and pathways, providing in-depth scientific evidence for its outstanding technical effects.

[0278] Summary of Mechanism of Action: This invention, through its group of active ingredients, works synergistically on three core aspects: inflammation, oxidative stress, and intestinal barrier: (1) Systemic Inhibition of Inflammation: By downregulating NOD-like receptors and TNF signaling pathways, it inhibits the activation of inflammasomes and the release of downstream pro-inflammatory factors. (2) Reversal of Metabolic Disorders: By regulating tryptophan metabolism, it reduces the activity of the kynurenine pathway, alleviating systemic inflammation and oxidative stress. (3) Strengthening of Physical Barriers: By upregulating the expression of tight junction proteins, it repairs the intestinal mucosal structure damaged by microplastics, reducing the penetration of microplastics and attached contaminants.

[0279] Experiment 10: Comparison of the effects of the present invention and existing technical solutions:

[0280] 1. Experimental objective: By comparing the complete composition of this invention with existing feed additive solutions with similar functions, this invention aims to quantitatively evaluate its significant advantages in reducing microplastic content and improving overall physiological function in fish, thereby demonstrating the technological advancement of this invention.

[0281] 2. Comparison Scheme Selection: Based on patent and literature searches, the following two representative schemes were selected as comparisons: Comparison Scheme 1 (C1): A probiotic preparation (mainly containing Bacillus and Lactobacillus) for improving water quality based on patent CN 114081099A. This scheme represents the "environmental remediation" approach, aiming to indirectly influence pollutants by improving water quality. Comparison Scheme 2 (C2): A polysaccharide probiotic compound feed additive (mainly containing compound probiotics) based on patent CN 110881571A. This scheme represents the "intestinal health" approach and is the prior art closest to the functional positioning of this invention.

[0282] 3. Experimental Design: (1) Experimental Animals and Breeding Environment: Same as Experiment 2. Carp fry were selected and raised in water containing PE microplastics (1000 particles / L) for 8 weeks. (2) Experimental Groups and Feeds: Control Group (CTRL): Basic feed. Control Scheme 1 (C1): Basic feed + 1.0% probiotic preparation of CN 114081099A. Control Scheme 2 (C2): Basic feed + 1.0% polysaccharide probiotic compound feed additive of CN 110881571A. Full Formula of the Invention (FULL): Basic feed + 1.0% additive of the present invention. (3) Detection Indicators: Core effect indicator: Microplastic content in back muscle. Intestinal health indicators: Vulvar height / crypt depth (V / C value); serum diamine oxidase (DAO) activity (DAO activity is positively correlated with the degree of intestinal barrier damage). Antioxidant indicator: Serum total antioxidant capacity (T-AOC).

[0283] 4. Experimental Results:

[0284] Table 10-1: Comparison of the effects of the present invention and existing technical solutions

[0285]

[0286] Note: Different letters in the superscript of the same data indicate significant differences (P < 0.05).

[0287] 5. Comparative Analysis and Conclusions: 5.1 Comparative Analysis of Core Effects (Microplastic Removal): Control Scheme 1 (C1): No significant reduction effect on microplastic content in fish muscle (P > 0.05). This indicates that improving external water quality solely through microorganisms is insufficient to effectively remove microplastics already in the fish's body, confirming the limitations of its "indirect effect." Control Scheme 2 (C2): Showed a certain reduction effect (approximately 21%), possibly due to its improvement of intestinal peristalsis and barrier function, which helps reduce the absorption and retention of microplastics. This demonstrates the partial effectiveness of the "intestinal health" approach, but the effect is limited. The Full-Scale Solution of this Invention (FULL): Showed a highly significant reduction effect (65.4%), with a removal efficiency more than three times that of the C2 group. This fully demonstrates that the multi-mechanism synergistic whole-chain intervention strategy of this invention ("adsorption-promote excretion-repair-regulation") has a fundamental advantage over simple "environmental remediation" or "intestinal health" schemes in addressing the specific problem of microplastic accumulation in the body. 5.2 Comparative Analysis of Comprehensive Physiological Benefits: Regarding gut health, this invention significantly outperformed group C2 in both increasing the V / C ratio and reducing serum DAO activity, indicating a more comprehensive and thorough effect in repairing the intestinal barrier. In terms of antioxidant capacity, this invention also demonstrated a significant advantage, proving its stronger ability to alleviate oxidative stress induced by microplastics. 5.3 Summary of Differences in Technical Solutions:

[0288] Table 10-2: Comparison of Technical Solutions and Mechanisms

[0289]

[0290] 6. Experimental conclusions: (1) Specificity of target and effect: Existing technical solutions each have their own focus, but none are specifically designed for "in vivo microplastics", so the effect is limited. This invention is the first systematic functional feed additive solution built to solve this problem. (2) Advanced and synergistic technical mechanism: The control scheme has a relatively simple mechanism, while this invention integrates multiple mechanisms through ingenious component design, forming a complete technical system with synergistic effect, which is the fundamental reason for its excellent effect. (3) Significant technical progress: Whether in core indicators (microplastic clearance rate) or comprehensive physiological benefits, this invention has achieved unexpected technical progress far exceeding that of the closest existing technology (control scheme 2). Such progress cannot be easily obtained by those skilled in the art through conventional optimization based on existing technology.

[0291] Experiment 11: Compliance and Safety Analysis of Core Components of Each Part

[0292] 1. Purpose of the analysis: To systematically evaluate the compliance of each core component used in the functional feed additive of this invention with the relevant national laws, regulations and feed safety standards, and to demonstrate its biosafety to farmed fish and end consumers by combining literature and experimental data.

[0293] 2. Analysis Basis: Regulatory Standards: Ministry of Agriculture and Rural Affairs of the People's Republic of China's "Catalogue of Feed Raw Materials," "Catalogue of Feed Additives," GB 13078 "Feed Hygiene Standard," and related revision announcements. Safety Data Sources: Publicly published toxicological studies, food safety assessment reports, and safety data of physiological indicators involved in the embodiments of this invention.

[0294] 3. Analysis and Results: 3.1 Compliance and safety assessment of each component;

[0295] Table 11-1: Compliance and Safety Analysis of Core Components for Each Part

[0296]

[0297] 3.2 Overall safety assessment of the composition:

[0298] (1) Regulatory compliance: All components of this invention are derived from substances permitted to be used in the "Feed Raw Material Catalog" or "Feed Additive Variety Catalog". Their scope of use and target animal categories for aquatic animals comply with national regulations, and the overall formula is fully compliant.

[0299] (2) Toxicological safety (based on literature): Acute toxicity: All components are low-toxicity or practically non-toxic substances. The median lethal dose of this composition is expected to be much greater than 5000 mg / kg body weight, which is classified as non-toxic. Genotoxicity: None of the selected components have been reported to have genotoxicity. Subchronic toxicity: In Experiment II (8 weeks of culture) and Experiment X (dose-effect), the liver function indicators (such as AST, ALT), kidney function indicators (blood urea nitrogen), and blood physiological indicators of the experimental group fish were all within the normal physiological range, with no abnormal deviations from the control group, indicating that long-term intake has no organ toxicity.

[0300] (3) Safety in target animals (based on this experiment): In the examples, even in the high-dose groups (e.g., 1.4%-1.5% addition), no growth inhibition, decreased feed intake, or abnormal mortality was observed in the experimental fish. On the contrary, all experimental groups showed improved physiological conditions, such as stronger antioxidant capacity and healthier intestinal structure. This, in turn, proves that the additive of the present invention is highly safe and beneficial to target animals (freshwater and marine fish) within the effective dosage range.

[0301] (4) Residue and food safety: The mechanism of action of this invention is physical clearance and physiological regulation. Its core components (such as chitosan microspheres, dietary fiber, and probiotics) are not absorbed by the gastrointestinal tract, so they will not leave residues in edible tissues such as muscle. The absorbable trace components (such as glutamine and plant antioxidant metabolites) are themselves normal participants in the metabolism of organisms or natural food components, and there are no safety concerns.

[0302] The microplastic content in the final product (fish meat) is significantly reduced, which greatly improves its food safety and commercial value.

[0303] 4. Conclusion of this experiment: All components of the functional feed additive of this invention comply with national feed regulations, and there is no use of prohibited substances or use beyond the permitted scope. Combined with publicly available toxicological data and the results of the invention's own aquaculture experiments, the composition is safe and harmless to target animals (various freshwater and marine fish) at the recommended dosage, and can improve the safety of the final aquatic products by reducing contaminant residues. Therefore, this invention possesses a high degree of compliance and biosafety, laying a solid foundation for its industrial production and market application.

[0304] Experiment 12: Evaluation of the effect on improving the quality and food safety of farmed fish products:

[0305] 1. Experimental objective: To systematically evaluate the comprehensive impact of feeding the additive of this invention on the nutritional quality, sensory characteristics and food safety of farmed fish (represented by carp and sea bass), and to quantify its actual benefits in improving the commercial value and food safety level of aquatic products.

[0306] 2. Experimental Design: (1) Experimental Fish and Groups: Fish bodies from Experiment 2 (carp) and Experiment 4 (sea bass) were tested after the aquaculture experiment. (2) Test Samples: Control Group (CTRL): Fish bodies fed with basic feed. Experimental Group (FULL): Fish bodies fed with feed containing 1.0% (carp) or 1.2% (sea bass) of the additive of this invention. (3) Test Indicators and Methods: ① Nutritional Quality, Basic Nutritional Components: The crude protein, crude fat, moisture and ash content of the back muscle were determined using the national standard method. Amino Acid Composition: The content of 17 amino acids was determined using an amino acid analyzer, and the essential amino acid index (EAAI) was calculated. Fatty Acid Composition: The fatty acid profile in the muscle was determined using gas chromatography, with a focus on the content of EPA (C20:5n-3) and DHA (C22:6n-3). ② Sensory Quality, Texture Characteristics (TPA): The hardness, elasticity, chewiness and resilience of the muscle were determined using a texture analyzer. Flavor compounds: Volatile flavor compounds were analyzed using an electronic nose and headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). ③ Food safety and heavy metal residues: The contents of lead (Pb), cadmium (Cd), mercury (Hg), and arsenic (As) in muscle were detected using inductively coupled plasma mass spectrometry (ICP-MS). Plasticizer residues: The residual amounts of microplastic-characteristic adsorbed pollutants such as phthalates (PAEs) were detected.

[0307] 3. Experimental Results:

[0308] Table 12-1: Effects of the present invention on the nutritional quality of carp and sea bass muscle

[0309]

[0310] Table 12-2: Effects of the present invention on the safety indicators of fish muscle for consumption

[0311]

[0312] Note: Σ plasticizer refers to the total reference limit for several common plasticizers such as DBP and DEHP. Different superscript letters indicate significant differences within groups (P < 0.05).

[0313] 4. Results Analysis and Conclusions: 4.1 Significantly Improved Nutritional Quality: Protein and Amino Acids: The crude protein content and essential amino acid index (EAAI) of the fish muscle in the experimental group were significantly higher than those in the control group. This indicates that the present invention promotes protein deposition and enhances the nutritional value of fish meat by improving intestinal health and overall metabolic status. Fat and Fatty Acids: The crude fat content in the experimental group was moderately reduced, but the content of beneficial fatty acids (DHA+EPA) increased significantly. This may be because the additives in the present invention regulate fat metabolism, reduce unnecessary body fat accumulation, and promote the retention and synthesis of high-quality unsaturated fatty acids, making the fish meat "higher quality and healthier".

[0314] 4.2 Fundamental Improvement in Food Safety: Reduced Heavy Metal Residues: The levels of heavy metal residues such as lead and cadmium in the muscle of the experimental group were significantly lower than those in the control group. This strongly demonstrates that the additive of this invention, by adsorbing and accelerating the excretion of pollutants from the environment, not only removes microplastics themselves but also significantly reduces the accumulation of heavy metals coexisting with microplastics or entering the body through other pathways in the muscle. Extremely Significant Reduction in Plasticizer Residues: This is the most direct evidence. Plasticizers are typical adhering pollutants of microplastics. The plasticizer content in the muscle of the experimental group was reduced by more than 50%, directly proving that this invention can reduce the transmission of microplastics and their "cargo" toxins into the food chain from the source.

[0315] 4.3 Sensory quality was optimized: Texture analysis showed a moderate increase in the firmness and chewiness of the fish meat in the experimental group, which may be related to the increased protein content and optimized fat content, resulting in a better taste. Flavor analysis revealed a decrease in the content of some volatile substances related to "earthy" taste in the fish meat of the experimental group, while the content of substances related to "fresh and clean" taste increased. This may be the result of the combined effect of improved gut microbiota and reduced oxidative stress.

[0316] The overall conclusion of this experiment is that the application of the additive of this invention achieves a leap from "ensuring survival" (removing pollutants and maintaining health) to "improving quality" (optimizing nutrition and enhancing flavor). It not only solves the safety hazard of microplastic pollution but also proactively enhances the nutritional value and sensory experience of aquatic products, producing safer, higher-quality, and more market-popular aquatic products. This "quality improvement and efficiency enhancement" effect provides an extremely strong market driving force and consumer value proposition for the commercial promotion of this invention.

[0317] Example 3: A method for preparing a feed additive, comprising the following steps:

[0318] (1) Preparation of modified chitosan microspheres: surface phenyl modification was carried out by emulsification crosslinking and Schiff base reaction;

[0319] (2) Preparation of nanoscale yeast cell wall derivatives: processed by high-pressure homogenization and nano-grinding technology;

[0320] (3) Mix each component physically in proportion.

[0321] Example 4: An application of the feed additive, wherein the feed additive is added to fish, shrimp or crab feed at a weight ratio of 0.5%-2.0%.

[0322] Furthermore, the feed additive is added to the feed at a weight ratio of 1.0%.

Claims

1. A feed additive for clearing microplastics, characterized in that, consists of, by weight percentage: modified chitosan microspheres: 20%; nanoscale yeast cell wall derivative: 15%; fermentable dietary fiber complex: 20%; glutamine: 5%; complex probiotics and prebiotics: 30%; natural plant extract compound antioxidant: 10%; the modified chitosan microspheres are surface grafted with hydrophobic functional groups through Schiff base reaction, which is a condensation reaction between aldehyde groups and amino groups, wherein chitosan provides the amino component and p-methyl benzaldehyde provides the aldehyde component.

2. The feed additive according to claim 1, characterized in that, the nanoscale yeast cell wall derivative is prepared by physical or biochemical methods and has an average particle size of 100-500 nanometers.

3. The feed additive according to claim 1, characterized in that, the fermentable dietary fiber complex is compounded from lignin and citrus pectin, and the compounding ratio of lignin to citrus pectin is 3:7 to 7:

3.

4. The feed additive according to claim 1, characterized in that, the complex probiotics and prebiotics contain Bacillus licheniformis, Bacillus subtilis, and xylo-oligosaccharide, wherein the compounding ratio of Bacillus licheniformis to Bacillus subtilis is 3:7 to 7:3, the viable bacterial count is not less than 100 billion CFU / g, and the content of xylo-oligosaccharide is 3%-8%.

5. The feed additive according to claim 1, characterized in that, the natural plant extract compound antioxidant is compounded from curcumin, epigallocatechin gallate, and rosemary acid, and the compounding ratio of curcumin to epigallocatechin gallate to rosemary acid is 2-4:4-6:1.5-3.

6. A method of preparing the feed additive according to any one of claims 1 to 5, characterized in that, comprising the following steps: (1) preparing modified chitosan microspheres: surface phenyl modification through emulsion crosslinking and Schiff base reaction; (2) preparing nanoscale yeast cell wall derivative: treatment by high-pressure homogenization and nanomilling technology; (3) physically mixing the components uniformly in proportion.

7. Use of the feed additive according to any one of claims 1 to 5, characterized in that, The feed additive is added to fish, shrimp, or crab feed at a weight ratio of 0.5%-2.0%.

8. Use according to claim 7, characterized in that, The feed additive is added to the feed at a weight ratio of 1.0%. The feed additive is added to the feed at a weight ratio of 1.0%.

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