Pelteobagrus fulvidraco composite functional feed additive as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of feed additives, and discloses a compound functional feed additive for pelteobagrus fulvidraco as well as a preparation method and application of the compound functional feed additive. The invention creatively provides a multi-target synergistic compound functional feed additive for pelteobagrus fulvidraco by scientifically compounding the antibacterial peptide, the eucommia ulmoides extract, the eucalyptus essential oil and the sodium butyrate and supplementing a carrier. The composite functional feed additive is not simply superposed in functions of all the components, but systematically improves the health level and disease resistance of the pelteobagrus fulvidraco from multiple aspects of pathogen inhibition, immunoregulation, oxidative stress resistance, intestinal barrier repair and the like through a synergistic effect, and can replace or reduce the use of antibiotics.

Description

Technical Field

[0001] This invention belongs to the field of feed additive technology, specifically relating to a compound functional feed additive for yellow catfish, its preparation method, and its application. Background Technology

[0002] Yellow catfish ( Pelteobagrus fulvidraco Yellow catfish, also known as yellow bream or yellow bullhead, is a small freshwater economic fish belonging to the genus *Pelteobagrus* in the family Bagridae. It is widely distributed in major river systems in China, including the Yangtze, Yellow, and Pearl Rivers. Its flesh is tender, delicious, and boneless, and rich in amino acids and unsaturated fatty acids, making it highly nutritious and an important specialty aquaculture species. With expanding market demand, the yellow catfish farming industry has developed rapidly, with a total national output reaching 565,400 tons in 2020. Intensive, high-density farming has become the mainstream model.

[0003] Under high-density aquaculture conditions, the primary problem facing yellow catfish is frequent disease outbreaks. Due to their lack of scales and relatively weak skin barrier function, they are highly susceptible to bacterial diseases in high-density, stressful environments. These diseases, such as septicemia and enteritis caused by streptococci and Edwardsiella, often lead to large-scale mortality and severe economic losses. Furthermore, common non-specific health problems during aquaculture, such as discolored or rotten bodies, abnormal body color, liver lesions (e.g., yellow or green liver), and indigestion, also seriously affect the appearance, quality, and survival rate of marketable fish. These problems are not only related to direct pathogen infection, but more fundamentally stem from oxidative stress, decreased immune function, and liver and intestinal damage caused by high-density aquaculture environments. These three factors are interconnected, creating a vicious cycle.

[0004] Traditionally, aquaculture farmers have relied on adding antibiotics to feed to prevent and treat bacterial diseases. However, the long-term and excessive use of antibiotics has led to increased drug resistance in pathogens, reduced effectiveness in prevention and treatment, drug residues that threaten the safety of aquatic products and consumer health, and their metabolites that pollute aquatic environments and disrupt ecological balance. Therefore, finding safe and effective antibiotic alternatives has become an important direction in aquatic feed research and development.

[0005] Currently, research on feed additives for the healthy aquaculture of yellow catfish has made some progress. These mainly include microecological preparations, antimicrobial peptides, traditional Chinese medicine and its extracts, plant essential oils, and functional oligosaccharides. However, existing technologies still have the following shortcomings: 1) Single function, unable to address comprehensive challenges: Most additives focus on antibacterial, antioxidant, or growth-promoting effects, making it difficult to address the comprehensive health problems faced by yellow catfish in high-density aquaculture from multiple targets and pathways, such as antibacterial, antioxidant, liver protection, intestinal health, and immune regulation. Diseases and health problems in yellow catfish are often the result of multiple intertwined factors, and single-function additives have limited effectiveness. 2) Unclear synergistic effects and lack of scientific formulation: Existing technologies are mostly simple combinations of different functional substances, failing to deeply explore the scientific formulation and synergistic mechanisms between different components (such as antimicrobial peptides, plant essential oils, plant extracts, and intestinal regulators). Simple physical mixing may not achieve a "1+1>2" effect, and may even reduce efficacy due to antagonistic effects. 3) Insufficient comprehensive protection of the "liver-gut axis": The liver and intestines are the core organs for nutritional metabolism, immune defense, and detoxification in fish, and are closely linked and mutually influential through the "liver-gut axis." Existing solutions often focus only on one aspect (such as protecting only the liver or only promoting gut health), failing to form a systematic protection plan for liver and gut health, which is the core of solving key problems such as "discoloration," liver disease, and digestive disorders in yellow catfish. 4) Insufficient systematic regulation of oxidative stress: Oxidative stress is a core factor leading to decreased immunity and tissue damage. Existing technologies mostly rely on exogenous antioxidants (such as vitamins C and E), and the design for systematic intervention at multiple levels, such as scavenging free radicals, enhancing the activity of endogenous antioxidant enzymes (such as SOD and CAT), and inhibiting lipid peroxidation, is not in-depth enough, making it difficult to fundamentally enhance the body's antioxidant defense system.

[0006] Therefore, the market urgently needs a feed additive that can act on multiple targets and in a systemic manner, possessing multiple compound functions such as direct antibacterial, highly effective antioxidant, immune enhancement, liver protection, and intestinal barrier repair, to fundamentally improve the disease resistance and health level of yellow catfish, and ensure aquaculture efficiency and food safety. Summary of the Invention

[0007] The present invention aims to at least solve one of the technical problems existing in the prior art. The present invention provides a compound functional feed additive that can systematically enhance the immunity of yellow catfish, improve liver and intestinal health, and effectively prevent and control bacterial diseases, so as to replace or reduce the use of antibiotics.

[0008] The first objective of this invention is to provide a compound functional feed additive.

[0009] The second objective of this invention is to provide a method for preparing the compound functional feed additive of the first aspect of this invention.

[0010] The third aspect of this invention aims to provide the application of the compound functional feed additive of the first aspect of this invention in the preparation of feed.

[0011] The fourth aspect of the present invention is to provide a feed.

[0012] The fifth aspect of this invention aims to provide the application of the compound functional feed additive of the first aspect of this invention or the feed of the fourth aspect of this invention in aquaculture.

[0013] The sixth aspect of this invention aims to provide the application of the compound functional feed additive of the first aspect of this invention or the feed of the fourth aspect of this invention in the preparation of products that improve the antibacterial ability and liver and intestinal protection functions of aquatic animals.

[0014] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a feed additive comprising an antimicrobial peptide, Eucommia ulmoides extract, eucalyptus oil, sodium butyrate, and a carrier.

[0015] In some embodiments of the present invention, the antimicrobial peptide includes cephalosporin antimicrobial peptide.

[0016] In some embodiments of the present invention, the amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO:1.

[0017] In some embodiments of the present invention, the carrier includes at least one of rice husk powder, wheat bran, peanut shell powder, corn cob powder, zeolite powder, and diatomaceous earth; preferably rice husk powder.

[0018] In some embodiments of the present invention, the Eucommia ulmoides extract is an aqueous extract of Eucommia ulmoides leaves.

[0019] In some embodiments of the present invention, the Eucommia ulmoides extract is prepared by a method comprising the following steps: mixing Eucommia ulmoides leaves with water and decocting to obtain the Eucommia ulmoides extract.

[0020] In some embodiments of the present invention, the mass-to-volume ratio of the Eucommia ulmoides leaves to water is 1:(4-10), such as any one of the values ​​of 1:4, 1:5, 1:6, 1:7 or 1:8, or any range formed by both.

[0021] In some embodiments of the present invention, the simmering time is 1-4 hours, such as any value of 1, 2, 3 or 4 hours or a range formed by any two of them.

[0022] In some embodiments of the present invention, the Eucommia ulmoides extract is prepared by a method comprising the following steps: mixing Eucommia ulmoides leaves with water and decocting; separating the solid and liquid, collecting the filtrate, and mixing the residue with water and decocting; separating the solid and liquid, collecting the filtrate, and mixing the residue with water and decocting; combining the collected filtrates, concentrating and drying to obtain the Eucommia ulmoides extract.

[0023] In some embodiments of the present invention, the feed additive comprises 5-40 parts by weight of antimicrobial peptide; further comprising 10-35 parts, such as any value or a range formed by any combination of 10, 15, 20, 25, 30 or 35 parts.

[0024] In some embodiments of the present invention, the feed additive comprises, by weight, 5-40 parts of Eucommia ulmoides extract; further, 10-35 parts, such as any value or a range formed by any two of 10, 15, 20, 25, 30 or 35 parts.

[0025] In some embodiments of the present invention, the feed additive comprises 5-25 parts by weight of eucalyptus oil; further comprising 5-20 parts, such as any value or a range formed by any two of 5, 7, 9, 10, 12, 14, 15, 17, 19 or 20 parts.

[0026] In some embodiments of the present invention, the feed additive comprises, by weight, 5-25 parts of sodium butyrate; further, 5-20 parts, such as any value or a range formed by any two of 5, 7, 9, 10, 12, 14, 15, 17, 19 or 20 parts.

[0027] In some embodiments of the present invention, the feed additive comprises 5-80 parts by weight of carrier; further comprising 10-70 parts, such as any value or a range formed by any two of 10, 20, 30, 40, 50, 60 or 70 parts.

[0028] The compound functional feed additive provided by this invention uses antimicrobial peptides, eucalyptus oil, Eucommia ulmoides extract, sodium butyrate, and rice husk powder as its main functional components, with rice husk powder added to act as a carrier and provide functional assistance. The core function of eucalyptus oil lies in its broad-spectrum antibacterial activity and positive impact on the host gut. Its main active ingredient, 1,8-cineole, exerts its antibacterial effect through multiple mechanisms, including disrupting the integrity of pathogen cell membranes, inhibiting quorum sensing signaling systems, and interfering with biofilm formation. Animal experimental studies provide direct evidence for this: in vitro studies have shown that eucalyptus oil exhibits a strong inhibitory effect on drug-resistant Escherichia coli strains, with a minimum inhibitory concentration as low as 3.13-35 µg / mL, and effectively inhibits biofilm formation. Furthermore, mouse model studies reveal that eucalyptus oil intervention can significantly increase gut microbiota diversity and promote the proliferation of short-chain fatty acid-producing and neurotransmitter precursor-producing bacteria, suggesting that it can influence the host's physiological state by regulating the gut-brain axis.

[0029] Antimicrobial peptides, as natural immune effector molecules, have had their functional mechanisms studied in considerable depth. Through their cationic amphiphilic structure, they bind to the negatively charged bacterial cell membrane, forming transmembrane pores that lead to leakage of cell contents and cell death. This physical killing mechanism makes it difficult for bacteria to develop drug resistance. Furthermore, antimicrobial peptides can also act as immunomodulators, chemotactically attracting macrophages and neutrophils, and regulating the expression of inflammatory factors and apoptosis pathways.

[0030] Eucommia ulmoides extract is rich in chlorogenic acid, flavonoids, and other polyphenolic compounds. Theoretically, it can exert antioxidant effects by directly scavenging free radicals and activating cellular defense pathways, particularly by activating the Keap1-Nrf2 / ARE signaling pathway to upregulate the expression of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT). Simultaneously, it can inhibit excessive inflammatory responses by suppressing pathways such as NF-κB. Regarding animal application evidence, a patent shows that combining Eucommia ulmoides leaf extract with xylanase as a feed additive can effectively improve feed utilization and production performance in piglets. Although this evidence does not directly measure its antioxidant indicators, it provides indirect support for its functional applications in animal nutrition.

[0031] Sodium butyrate, the sodium salt of butyrate, is a preferred energy source for fish intestinal epithelial cells. It plays a key role through multiple mechanisms, including improving intestinal physical structure, enhancing digestive function, regulating gut microbiota balance, strengthening the immune barrier, and improving stress resistance. Specifically, it significantly promotes intestinal villus development, increasing the absorptive surface area; stimulates the secretion of digestive enzymes such as proteases and amylases, improving feed utilization; selectively inhibits harmful bacteria and promotes the proliferation of beneficial bacteria, optimizing gut microbiota balance; strengthens intestinal barrier function by enhancing tight junctions and increasing the activity of antioxidant enzymes (such as SOD and CAT); and regulates immune responses to reduce inflammation.

[0032] The compound functional feed additive provided by this invention is a feed additive that can act on multiple targets and in a systemic manner, and has multiple compound functions such as direct antibacterial, high-efficiency antioxidant, immune enhancement, liver protection and intestinal barrier repair. It can fundamentally improve the disease resistance and health level of yellow catfish, and ensure aquaculture efficiency and food safety.

[0033] A second aspect of the present invention provides a method for preparing the composite functional feed additive of the first aspect of the present invention, comprising the following steps: mixing antimicrobial peptides, Eucommia ulmoides extract, eucalyptus oil, sodium butyrate and a carrier to obtain the feed additive.

[0034] A third aspect of the present invention provides the application of the compound functional feed additive of the first aspect of the present invention in the preparation of feed.

[0035] In some embodiments of the present invention, the feed includes aquaculture feed.

[0036] In some embodiments of the present invention, the aquatic animal is the yellow catfish.

[0037] A fourth aspect of the present invention provides a feed comprising the compound functional feed additive of the first aspect of the present invention and a base feed.

[0038] In some embodiments of the present invention, the feed additive is present in the feed at a content of 0.5wt%-1.5wt%, such as any value or a range formed by any two of 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt%, 1.1wt%, 1.2wt%, 1.3wt%, 1.4wt%, or 1.5wt%.

[0039] In some embodiments of the present invention, the basic feed includes fish meal, corn gluten meal, chicken meal, soybean meal, wheat starch, soybean oil, fish oil, calcium dihydrogen phosphate, vitamin premix, and mineral premix.

[0040] In some embodiments of the present invention, the vitamin premix comprises the following components: vitamin A, vitamin B1, vitamin B2, vitamin B6, and vitamin B6. 12 It contains at least one of the following: vitamin C, vitamin D3, vitamin E, vitamin K, folic acid, biotin, inositol, nicotinamide, and choline.

[0041] In some embodiments of the present invention, the vitamin premix comprises the following components per 1 kg of feed: vitamin B1 15-25 mg / kg, vitamin B2 15-20 mg / kg, vitamin B6 25-35 mg / kg, vitamin B... 120.05-0.15mg / kg, Vitamin C 1500-2500mg / kg, Vitamin D3 4-8mg / kg, Vitamin E 80-120mg / kg, Vitamin K 5-15mg / kg, Folic acid 10-25mg / kg, Biotin 0.5-2mg / kg, Inositol 150-250mg / kg, Nicotinamide 150-250mg / kg, and Choline 800-1200mg / kg.

[0042] In some embodiments of the present invention, the mineral premix comprises at least one of the following components: MnSO4, MgSO4, FeSO4, CoSO4, Na2SeO3, CuSO4, KCl, NaCl, ZnSO4, H2CaIO4, and CoCl2.

[0043] In some embodiments of the present invention, the mineral premix comprises the following components per 1 kg of feed: MnSO4 40-70 mg / kg, MgSO4 700-900 mg / kg, FeSO4 200-250 mg / kg, CoSO4 0.5-1.5 mg / kg, Na2SeO3 0.5-1.5 mg / kg, CuSO4 10-20 mg / kg, KCl 2000-3000 mg / kg, NaCl 400-500 mg / kg, ZnSO4 150-200 mg / kg, H2CaIO4 500-600 mg / kg, and CoCl2 8-15 mg / kg.

[0044] In some embodiments of the present invention, the basic feed comprises the following components by weight percentage: 20%-40% fish meal, 10%-20% corn gluten meal, 10%-20% chicken meal, 10%-20% soybean meal, 10%-20% wheat starch, 2%-10% soybean oil, 1%-5% fish oil, 1%-5% calcium dihydrogen phosphate, 1%-5% vitamin premix, and 1%-5% mineral premix.

[0045] In some embodiments of the present invention, the basic feed comprises the following components by weight percentage of aquatic feed: 20%-30% fish meal, 12%-18% corn gluten meal, 15%-20% chicken meal, 10%-15% soybean meal, 15%-20% wheat starch, 2%-6% soybean oil, 2%-4% fish oil, 1%-3% calcium dihydrogen phosphate, 1%-3% vitamin premix, and 1%-2% mineral premix.

[0046] In some embodiments of the present invention, the crude protein content of the aquatic feed is 40wt%-45wt%, and the crude fat content is 9wt%-11wt%.

[0047] In some preferred embodiments of the present invention, the crude protein content of the aquatic feed is 41.5wt%-42.5wt%, and the crude fat content is 9.5wt%-10wt%.

[0048] In some embodiments of the present invention, the feed has the function of improving the antibacterial ability and liver and intestinal protection of aquatic animals.

[0049] In some embodiments of the present invention, the feed includes aquatic feed.

[0050] In some embodiments of the present invention, the feed includes yellow catfish farming feed.

[0051] In some embodiments of the present invention, the feed preparation includes the following steps: mixing compound functional feed additives with base feed, extruding and granulating the mixture, and drying it to obtain feed.

[0052] A fifth aspect of the present invention provides the application of the compound functional feed additive described in the first aspect of the present invention, or the aquatic feed described in the third aspect, in aquaculture.

[0053] In some embodiments of the present invention, the aquatic animal is a fish, preferably a yellow catfish.

[0054] A sixth aspect of the present invention provides the application of the compound functional feed additive of the first aspect of the present invention or the feed of the fourth aspect of the present invention in the preparation of products that improve the antibacterial ability and liver and intestinal protection functions of aquatic animals.

[0055] In some embodiments of the present invention, the aquatic animal is a fish, preferably a yellow catfish.

[0056] The beneficial effects of this invention are: This invention creatively provides a multi-target, synergistic compound functional feed additive for yellow catfish by scientifically compounding antimicrobial peptides, Eucommia ulmoides extract, eucalyptus oil, and sodium butyrate, and supplementing it with a carrier. This compound functional feed additive is not a simple sum of the functions of its components, but rather, through synergistic effects, systematically improves the health and disease resistance of yellow catfish from multiple levels, including pathogen inhibition, immune regulation, anti-oxidative stress, and intestinal barrier repair, and can replace or reduce the use of antibiotics.

[0057] Experiments show that adding the compound functional feed additive of this invention to the basal feed of yellow catfish at 0.5wt%-1.5wt% significantly enhances serum lysozyme and complement C3 activity, thus strengthening innate immunity. Regarding antioxidant stress, it significantly upregulates the expression of hepatic antioxidant genes (sod1, cat, gpx1) and downregulates the expression of endoplasmic reticulum stress gene (grp78), thereby strengthening antioxidant defense at its source. From the perspective of intestinal health, it significantly reduces serum D-lactate levels and regulates the balance of pro-inflammatory / anti-inflammatory factors in the intestine, maintaining the intestinal barrier and immune homeostasis. Finally, in challenge experiments, it significantly improves the survival rate of yellow catfish against *Streptococcus dolphinii*. The compound functional feed additive of this invention, when used in aquaculture, can significantly improve the antibacterial ability of yellow catfish and has hepatoprotective and intestinal-protective effects. Attached Figure Description

[0058] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a comparative graph showing the effects of compound functional feed additives on the activity of serum lysozyme in yellow catfish.

[0059] Figure 2 A comparative graph showing the effects of compound functional feed additives on serum complement C3 activity in yellow catfish.

[0060] Figure 3 A comparative graph showing the effects of compound functional feed additives on serum complement C4 activity in yellow catfish.

[0061] Figure 4 This is a comparative graph showing the effects of compound functional feed additives on serum D-lactic acid content in yellow catfish.

[0062] Figure 5 A comparative graph showing the effects of compound functional feed additives on the activity of superoxide dismutase in the liver of yellow catfish.

[0063] Figure 6 A comparative graph showing the effects of compound functional feed additives on the activity of catalase in the liver of yellow catfish.

[0064] Figure 7 A comparative graph showing the effects of compound functional feed additives on malondialdehyde content in the liver of yellow catfish.

[0065] Figure 8 This is a comparative diagram showing the effects of compound functional feed additives on the expression of antioxidant-related genes in the liver of yellow catfish.

[0066] Figure 9 This is a comparative diagram showing the effects of compound functional feed additives on the expression of intestinal immune-related genes in yellow catfish.

[0067] Figure 10A comparative study on the effects of compound functional feed additives on the disease resistance of yellow catfish. Detailed Implementation

[0068] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0069] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0070] The antimicrobial peptide used in this embodiment of the invention is cephalosporin antimicrobial peptide, whose amino acid sequence is: KWKIFKKIEKVGRNIRNGIIKAGPAVAVLGEAKAL (SEQ ID NO:1).

[0071] The Eucommia ulmoides leaf extract used in this embodiment of the invention is obtained by extraction steps including the following: (1) Wash the Eucommia ulmoides leaves, dry them and grind them to obtain Eucommia ulmoides leaf powder; (2) Boil 1000g of the above Eucommia ulmoides leaf powder in 8L, 6L and 4L of water for 1 hour in sequence; then filter and collect the filtrate. (3) The recovered filtrate was further boiled to concentrate the filtrate to 200 mL; then the concentrate was freeze-dried for 48 h under the following conditions: cold trap temperature -30 °C and internal pressure of the drying zone below 20 Pa. The Eucommia ulmoides extract was obtained after drying.

[0072] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0073] Example 1 A compound functional feed additive, by weight, comprises 10 parts antimicrobial peptide, 10 parts Eucommia ulmoides extract, 5 parts eucalyptus oil (Shandong Longchang Animal Health Products Co., Ltd., item number: XT-300L, the same below), 5 parts sodium butyrate and 70 parts rice husk powder.

[0074] The preparation method of the above-mentioned compound functional feed additive includes mixing antimicrobial peptides, Eucommia ulmoides extract, eucalyptus oil, sodium butyrate and rice husk powder evenly to obtain the compound functional feed additive.

[0075] Example 2 A compound functional feed additive, by weight, comprises 20 parts antimicrobial peptide, 20 parts Eucommia ulmoides extract, 10 parts eucalyptus oil, 10 parts sodium butyrate and 40 parts rice husk powder.

[0076] The preparation method of the above-mentioned compound functional feed additive includes mixing antimicrobial peptides, Eucommia ulmoides extract, eucalyptus oil, sodium butyrate and rice husk powder evenly to obtain the compound functional feed additive.

[0077] Example 3 A compound functional feed additive, by weight, comprises 30 parts antimicrobial peptide, 30 parts Eucommia ulmoides extract, 15 parts eucalyptus oil, 15 parts sodium butyrate and 10 parts rice husk powder.

[0078] The preparation method of the above-mentioned compound functional feed additive includes mixing antimicrobial peptides, Eucommia ulmoides extract, eucalyptus oil, sodium butyrate and rice husk powder evenly to obtain the compound functional feed additive.

[0079] Comparative Example 1 A feed additive comprising, by weight, 20 parts antimicrobial peptides and 80 parts rice husk powder.

[0080] The preparation method of the above-mentioned compound functional feed additive includes mixing antimicrobial peptides and rice husk powder evenly to obtain the compound functional feed additive.

[0081] Comparative Example 2 A feed additive comprising, by weight, 20 parts Eucommia ulmoides extract and 80 parts rice husk powder.

[0082] The preparation method of the above-mentioned compound functional feed additive includes mixing Eucommia ulmoides extract and rice husk powder evenly to obtain the compound functional feed additive.

[0083] Comparative Example 3 A feed additive comprising, by weight, 10 parts eucalyptus oil and 90 parts rice husk powder.

[0084] The preparation method of the above-mentioned compound functional feed additive includes mixing eucalyptus oil and rice husk powder evenly to obtain the compound functional feed additive.

[0085] Comparative Example 4 A feed additive comprising, by weight, 10 parts sodium butyrate and 90 parts rice husk powder.

[0086] The preparation method of the above-mentioned compound functional feed additive includes mixing sodium butyrate and rice husk powder evenly to obtain the compound functional feed additive.

[0087] Effect Example To verify the effectiveness of the additives of this invention, the feed additives prepared in Examples 1-3 and Comparative Examples 1-4 were mixed with basal feeds to prepare eight experimental aquatic feeds, numbered M1, M2, M3, CE, EU, EO, and SB, respectively. Simultaneously, an equal amount of rice husk powder was used to replace the functional feed additives and mixed with the basal feeds as a blank control group, numbered Control. The crude protein content of all feeds ranged from 41.5 wt% to 42.5 wt%, and the crude fat content ranged from 9.5 wt% to 10 wt%. Specific formulations are shown in Table 1.

[0088] Table 1 Aquatic Feed Formulation 1-8

[0089] Table 1 shows that the vitamin premix contains the following components: Vitamin B1 20 mg / kg (i.e., 20 mg of Vitamin B1 per kg of feed, the same below), Vitamin B2 20 mg / kg, Vitamin B6 30 mg / kg, Vitamin B2, Vitamin B6, Vitamin B2 ... 12 0.1 mg / kg, Vitamin C 2000 mg / kg, Vitamin D 35 mg / kg, Vitamin E 100 mg / kg, Vitamin K 10 mg / kg, Folic acid 15 mg / kg, Biotin 1 mg / kg, Inositol 200 mg / kg, Nicotinamide 200 mg / kg, Choline 1000 mg / kg.

[0090] Table 1 shows that the mineral premix contains the following components: MnSO4 60 mg / kg (i.e., 60 mg MnSO4 per kg of feed, the same below), MgSO4 800 mg / kg, FeSO4 220 mg / kg, CoSO4 0.91 mg / kg, Na2SeO3 1 mg / kg, CuSO4 15 mg / kg, KCl 2500 mg / kg, NaCl 500 mg / kg, ZnSO4 180 mg / kg, H2CaIO4 600 mg / kg, and CoCl 2 10 mg / kg.

[0091] The above-mentioned aquatic feeds 1-8 were used in the farming of yellow catfish to investigate the comprehensive effects of the feed additives contained in Examples 1-3 and Comparative Examples 1-4 on improving the immunity, antioxidant capacity, intestinal health, and disease resistance of yellow catfish, as detailed below: The experiment was conducted in experimental net cages at the Zhongshan Triangle R&D and Experiment Base of Guangdong Haida Group. Four hundred and eighty thousand healthy, uniformly sized yellow catfish juveniles with an average weight of (20.3 ± 0.1) g were selected. The juveniles were temporarily raised for two weeks to acclimatize to the environment before the experiment. They were then starved for 24 hours before the formal experiment. The fish were randomly assigned to 24 2m × 2m experimental net cages, with 200 fish per cage. These 24 cages were randomly divided into eight treatment groups, with three replicate cages per group. The eight treatment groups were fed the eight experimental feeds (M1, M2, M3, CE, EU, EO, SB, and Control), respectively. Feeding was conducted twice daily at 05:00 and 19:00, with a full-feeding period of eight weeks. During the rearing period, water temperature, dissolved oxygen, pH, and other water quality indicators were controlled within the suitable growth range for yellow catfish.

[0092] After the culture experiment, all experimental fish were starved for 24 hours. Three uniformly sized fish were randomly selected from each net cage for tail vein blood collection. The blood was allowed to stand overnight at 4°C, then centrifuged at 3000 rpm for 10 minutes to prepare serum for subsequent immunochemical assays. After blood collection, the fish were quickly dissected, and liver and intestinal tissue samples were collected, flash-frozen in liquid nitrogen, and stored at -80°C for subsequent biochemical and gene expression analysis. Additionally, 20 fish were randomly selected from each treatment group for a challenge experiment. Twenty uniformly sized experimental fish from each treatment group were used for the *Streptococcus dolphinus* challenge experiment (including the following steps: using a 1 mL syringe to inject 0.1 mL of *Streptococcus dolphinus* bacterial solution (concentration 1×10⁻⁶)). 5 (CFU / mL) was injected intraperitoneally into the yellow catfish, and then the fish were transferred to a 300L aquarium. They were observed for 7 consecutive days, and mortality was recorded daily. During the challenge period, the water was not changed and the fish were not fed.

[0093] The determination indicators and methods are as follows: (1) Serum immune indicators: Using commercial kits, strictly follow the instructions to determine the activity of lysozyme, complement C3 and complement C4 in serum. (2) Serum intestinal barrier function indicators: Using commercial kits, determine the D-lactic acid content in serum to assess intestinal permeability. (3) Liver antioxidant and oxidative damage indicators: Take liver tissue homogenate and use commercial kits to determine the activity of superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA). (4) Liver and intestinal gene expression analysis: Total RNA was extracted from liver and intestinal tissues using the TRIzol method and cDNA was synthesized by reverse transcription. Table 2 shows the brand and catalog number of the purchased kits. Using the primers listed in Table 3, the expression level of related genes was detected by real-time fluorescence quantitative PCR. β-actin was used as the internal reference gene, and 2^ (-ΔΔCt)The relative expression levels of genes were calculated using this method. Table 3 shows the primer sequences used for real-time quantitative PCR detection.

[0094] Table 2. Brands and catalog numbers of reagent kits used.

[0095] Table 3 Primer sequences for real-time quantitative PCR detection of oxidative stress and immune-related gene expression levels.

[0096] Experimental data are expressed as mean ± standard error (n=3). One-way ANOVA was performed using SPSS 20.0 software. If the differences were statistically significant (…),… p If the value is less than 0.05, then Tukey's test is used for multiple comparisons.

[0097] Figure 1 This is a comparative graph showing the effects of various compound functional feed additives on the serum lysozyme activity of yellow catfish. The same letter in the graph represents no significant difference. p >0.05). Among the groups fed aquatic diets M1, M2, M3, CE, EU, EO, SB, and Control, the serum lysozyme activities of yellow catfish were 201.6 U / mL, 268.4 U / mL, 228.3 U / mL, 155.7 U / mL, 186.5 U / mL, 162.4 U / mL, 168.9 U / mL, and 132.1 U / mL, respectively. The M2 group had the highest activity, while the Control group had the lowest. All additive groups showed higher activity than the Control group, with significant increases in the M2, M3, and M1 groups, while the increases in the CE, EU, EO, and SB groups were smaller. Figure 1 ).

[0098] Figure 2 This is a comparative graph showing the effects of various compound functional feed additives on serum complement C3 activity in yellow catfish. In the groups fed aquatic diets M1, M2, M3, CE, EU, EO, SB, and Control, the complement C3 levels were 2.16 mg / mL, 2.34 mg / mL, 2.52 mg / mL, 1.58 mg / mL, 1.79 mg / mL, 1.65 mg / mL, 1.61 mg / mL, and 1.38 mg / mL, respectively. The M3 group had the highest levels, while the Control group had the lowest. The M1, M2, and M3 groups were significantly higher than the Control group and other additive groups, while the CE, EU, EO, and SB groups were slightly higher than the Control group, but the differences were small. Figure 2 ).

[0099] Figure 3This is a comparative graph showing the effects of various compound functional feed additives on complement C4 activity in the serum of yellow catfish. In the groups fed aquatic diets M1, M2, M3, CE, EU, EO, SB, and Control, the complement C4 activities were 1.18 mg / mL, 1.12 mg / mL, 1.2 mg / mL, 1.08 mg / mL, 1.15 mg / mL, 1.28 mg / mL, 1.16 mg / mL, and 1.14 mg / mL, respectively. The EO group had the highest activity, and the CE group had the lowest. The values ​​in each group were similar to those in the Control group, with no significant trend. Figure 3 ).

[0100] Figure 2 and Figure 3 The results show that after adding the compound functional feed additives provided by this invention (Examples 1-3) to the basic feed, the regulatory effects of different compound functional feed additives on the innate immune system of fish were systematically evaluated by detecting three key immune indicators in the serum of yellow catfish: lysozyme, complement C3, and C4. Lysozyme, as the first line of defense of innate immunity, can hydrolyze peptidoglycan in bacterial cell walls and directly exert antibacterial effects. Data show that the activity of group M2 was significantly higher than that of all other groups, indicating that the compound functional feed additive formula may effectively stimulate the activity and secretory function of immune cells such as macrophages, thereby most powerfully enhancing the body's direct lysis ability against pathogenic microorganisms. The complement system is an important humoral immune component connecting innate and adaptive immunity. Among them, C3 is the core hub and amplifier of the three complement activation pathways. Its increased content usually means that the complement system is fully activated and can more efficiently perform functions such as opsonization, chemotaxis, and bactericidal membrane attack complexes. This indicates that the compound functional feed additives of this invention (Examples 1-3) can effectively activate the complement system and enhance the body's humoral immune response. The results showed no statistically significant difference in serum complement C4 levels among the groups. p >0.05), indicating that the compound functional feed additive of the present invention has no significant effect on the early activation components of the classical complement pathway.

[0101] Figure 4This is a comparative graph showing the effects of various compound functional feed additives on serum D-lactic acid content in yellow catfish. In the groups fed aquatic diets M1, M2, M3, CE, EU, EO, SB, and Control, the D-lactic acid contents were 5.42 μmol / g, 4.15 μmol / g, 5.89 μmol / g, 6.23 μmol / g, 5.67 μmol / g, 6.08 μmol / g, 6.18 μmol / g, and 7.84 μmol / g, respectively. Serum D-lactic acid content is a sensitive indicator for assessing the integrity of the intestinal mucosal barrier; the lower the content, the lower the intestinal permeability and the better the barrier function. The Control group had the highest D-lactic acid content. All additive groups had lower D-lactic acid content than the Control group, with the M2 group having the lowest content, significantly lower than all other experimental groups. p <0.05%. Although the D-lactic acid content in the comparative CE, EO, and SB groups was lower than that in the Control group, it was significantly higher than that in the M2 group ( p <0.05)( Figure 4 The results demonstrate that the compound functional feed additive of Example 2 of this invention is most effective in maintaining the integrity of the intestinal barrier of yellow catfish and reducing the risk of intestinal leakage.

[0102] Figure 5 This study compared the effects of various compound functional feed additives on superoxide dismutase (SOD) activity in the liver of yellow catfish. In the groups fed aquatic diets M1, M2, M3, CE, EU, EO, SB, and Control, the liver SOD activities were 125.4 U / mg, 168.2 U / mg, 142.7 U / mg, 105.3 U / mg, 128.6 U / mg, 110.5 U / mg, 108.9 U / mg, and 92.6 U / mg, respectively. The M2 group had the highest SOD activity, significantly higher than the other groups; the Control group had the lowest. All additive groups showed higher SOD activity than the Control group, with the M2, M3, and M1 groups showing the most significant increases, while the CE, EO, and SB groups showed relatively lower increases. Figure 5 ).

[0103] Figure 6 This study compares the effects of various compound functional feed additives on catalase activity in the liver of yellow catfish. Among the groups fed aquatic diets M1, M2, M3, CE, EU, EO, SB, and Control, the liver catalase activities were 35.6 U / mg, 42.3 U / mg, 31.8 U / mg, 29.4 U / mg, 33.2 U / mg, 30.7 U / mg, 28.9 U / mg, and 24.1 U / mg, respectively. Group M2 showed the highest catalase activity, while the Control group showed the lowest. Except for slightly lower levels in the CE, EO, and SB groups, all other additive groups showed an increase compared to the Control group, especially the M2 group. Figure 6 ).

[0104] Figure 7 This study compared the effects of various compound functional feed additives on MDA content in the liver of yellow catfish. In the groups fed aquatic diets M1, M2, M3, CE, EU, EO, SB, and Control, the liver MDA content was 3.28 nmol / mg, 2.51 nmol / mg, 3.65 nmol / mg, 3.92 nmol / mg, 3.41 nmol / mg, 3.78 nmol / mg, 3.87 nmol / mg, and 4.36 nmol / mg, respectively. As a product of lipid peroxidation, lower MDA values ​​indicate less oxidative damage. The M2 group had the lowest MDA content, while the Control group had the highest. Most additive groups had lower MDA levels than the Control group, but the CE, EO, and SB groups had slightly higher levels or were close to the Control group, suggesting that the M2 group may have been the most effective in reducing oxidative stress. Figure 7 ).

[0105] based on Figure 5 , Figure 6 and Figure 7 The data show that the compound functional feed additive of Example 2 (Group M2) has outstanding efficacy in enhancing the antioxidant defense of yellow catfish liver and protecting hepatocytes from oxidative damage. This indicates that the compound functional feed additive of Example 2 of the present invention can most effectively reduce oxidative stress damage to yellow catfish liver.

[0106] Figure 8 This is a comparative diagram showing the effects of various compound functional feed additives on the expression of genes related to antioxidant stress in the liver of yellow catfish. Figure 8It was found that the expression of key antioxidant and endoplasmic reticulum stress-related genes in the liver differed significantly among the different feed groups. Specifically, the relative expression levels of superoxide dismutase (SOD1) were: M1 group 1.65, M2 group 2.08, M3 group 1.52, CE group 1.41, EU group 1.58, EO group 1.47, SB group 1.44, and Control group 1.22. The expression levels of catalase (CAT) were: 1.72, 2.16, 1.58, 1.47, 1.65, 1.53, 1.50, and 1.28, respectively. The expression levels of glutathione peroxidase (GPX1) were: 1.58, 1.94, 1.46, 1.36, 1.52, 1.41, 1.38, and 1.18, respectively. The expression levels of the endoplasmic reticulum stress marker gene (grp78) showed the opposite trend, at 1.24, 0.92, 1.38, 1.52, 1.32, 1.45, 1.49, and 1.86, respectively. Based on the above data, compared with the Control group, feeding with the compound functional feed additive of this invention (M1, M2, and M3 groups) significantly upregulated the expression levels of key antioxidant genes sod1, cat, and gpx1 in the liver of yellow catfish. p <0.05). Among them, the M2 group showed the largest upregulation, with the relative expression levels of sod1, cat, and gpx1 reaching 2.08 times, 2.16 times, and 1.94 times that of the control group, respectively. Simultaneously, the expression of the endoplasmic reticulum stress marker gene grp78 was significantly downregulated to the lowest level in the M2 group. This molecular-level evidence corroborates the aforementioned results on enzyme activity and MDA content, confirming at the gene transcription level that the compound functional feed additive of this invention (especially Example 2) can systematically activate the body's endogenous antioxidant defense network and effectively alleviate organelle stress, thereby protecting hepatocytes.

[0107] Figure 9 This is a comparative graph showing the effects of various compound functional feed additives on the expression of intestinal immune-related genes in yellow catfish. The darker the color, the higher the gene expression level. Figure 9It was found that the expression of key intestinal immune and inflammation-related genes differed significantly among the groups fed different diets. The relative expression levels of p65-NFκB, a core transcription factor for inflammatory responses, were: M1 group 0.67, M2 group 0.28, M3 group 0.74, CE group 0.82, EU group 0.71, EO group 0.98, SB group 1.10, and Control group 1.00. The expression levels of the key pro-inflammatory cytokine TNF-α were: 0.66, 0.48, 0.74, 0.81, 0.90, 0.85, 0.95, and 1.00, respectively. The expression levels of the pro-inflammatory mediator IL-1β were: 0.67, 0.48, 0.55, 0.81, 0.30, 0.78, 0.85, and 1.00, respectively. The expression levels of the anti-inflammatory factor IL-10 showed the opposite trend, at 1.3, 2.3, 2.6, 1.3, 1.6, 1.23, 1.3, and 1.00, respectively. The results indicate that the compound functional feed additive of this invention has a significant regulatory effect on the intestinal immune balance of yellow catfish. Compared with the Control group, feeding with the M2 diet most effectively downregulated the gene expression of the pro-inflammatory core transcription factor p65-NFκB and its downstream effectors TNF-α and IL-1β, while most effectively upregulating the expression of the anti-inflammatory factor IL-10. This bidirectional regulatory pattern of "inhibiting pro-inflammatory and enhancing anti-inflammatory" indicates that the compound functional feed additive of this invention (especially Example 2) can effectively maintain intestinal immune homeostasis and reduce inflammatory damage. The regulatory effects of the comparative CE, EO, and SB groups in this regard were relatively limited.

[0108] Figure 10 A comparative chart showing the effects of various compound functional feed additives on the disease resistance of yellow catfish, such as... Figure 10 As shown, after challenge with Streptococcus dolphins, the cumulative survival rates of groups M1, M2, and M3 fed with the compound functional feed additive of this invention after 7 days (65%, 80%, and 70%, respectively) were significantly higher than those of the Control group (30%). p <0.05%. Among them, the survival rate of group M2 was the highest (80%), significantly better than all other experimental groups. While the survival rates of the comparative CE, EU, EO, and SB groups (55%, 55%, 60%, and 55%, respectively) were slightly higher than those of the control group, they were significantly lower than those of group M2. p <0.05). The results of this challenge test directly prove that the compound functional feed additive provided by the present invention can significantly improve the resistance of yellow catfish to important bacterial pathogens (Dolphin Streptococcus), and the formulation in Example 2 (M2) has the best effect.

[0109] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. A feed additive comprising antimicrobial peptides, Eucommia ulmoides extract, eucalyptus oil, sodium butyrate, and a carrier.

2. The feed additive according to claim 1, characterized in that, The antimicrobial peptide includes cephalosporin antimicrobial peptide; and / or, the carrier includes at least one of rice husk powder, wheat bran, peanut shell powder, corn cob powder, zeolite powder, and diatomaceous earth.

3. The feed additive according to claim 2, characterized in that, The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO:

1.

4. The feed additive according to any one of claims 1-3, characterized in that, The Eucommia ulmoides extract is an aqueous extract of Eucommia ulmoides leaves; Preferably, the Eucommia ulmoides extract is prepared by the following method: Eucommia ulmoides leaves are mixed with water and decocted to obtain the Eucommia ulmoides extract.

5. The feed additive according to any one of claims 1-3, characterized in that, By weight, the feed additive includes 5-40 parts of antimicrobial peptides; and / or By weight, the feed additive comprises 5-40 parts of Eucommia ulmoides extract; and / or By weight, the feed additive comprises 5-25 parts eucalyptus oil; and / or By weight, the feed additive comprises 5-25 parts sodium butyrate; and / or The feed additive comprises 5-80 parts carrier by weight.

6. A method for preparing the feed additive according to any one of claims 1-5, comprising the following steps: mixing antimicrobial peptides, Eucommia ulmoides extract, eucalyptus oil, sodium butyrate and a carrier to obtain the feed additive.

7. The use of the feed additive according to any one of claims 1-5 in the preparation of feed.

8. A feed comprising the feed additive and basal feed as described in any one of claims 1-5; Preferably, the feed additive is present in the feed at a content of 0.5 wt%-1.5 wt%; Preferably, the basic feed includes fish meal, corn gluten meal, chicken meal, soybean meal, wheat starch, soybean oil, fish oil, calcium dihydrogen phosphate, vitamin premix, and mineral premix.

9. The use of the feed additive according to any one of claims 1-5 or the feed according to claim 8 in aquaculture.

10. The use of the feed additive according to any one of claims 1-5 or the feed according to claim 8 in the preparation of products that improve the antibacterial ability and liver and intestinal protection functions of aquatic animals.

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