Preparation method of antibacterial macrobrachium rosenbergii feed additive and application thereof

Abstract

This invention belongs to the field of feed additive technology. It discloses a method for preparing an antibacterial feed additive for giant freshwater prawns and its application. The invention provides a feed additive containing a probiotic complex, a mineral-essential oil complex, an immune-enhancing complex, and compound trace elements. This feed additive achieves highly efficient inhibition of pathogens such as parahemolytic bacteria through a four-pronged mechanism of action: probiotics inhibit bacterial growth, plant-derived components synergistically kill bacteria, minerals adsorb toxins, and trace elements enhance immunity and the intestinal barrier. This significantly reduces the incidence and mortality of bacterial diseases in giant freshwater prawns. Furthermore, this invention solves the technical problem of the difficulty in coexisting probiotics and plant essential oils in existing technologies, exhibiting significant synergistic effects, good stability, and green safety, making it suitable for the entire process of giant freshwater prawn farming.

Description

Technical Field

[0001] This invention belongs to the field of feed additive technology, specifically relating to a method for preparing an antibacterial feed additive for giant freshwater prawns and its application. Background Technology

[0002] Giant freshwater prawns are one of the most important economically important freshwater aquaculture species in the world, renowned for their large size, rapid growth, wide diet, and high nutritional value, leading to large-scale aquaculture industries in tropical and subtropical regions globally. However, with increasingly intensive and high-density farming practices, bacterial diseases caused by pathogens such as Aeromonas and other bacteria have become a significant bottleneck restricting the healthy and sustainable development of the industry.

[0003] In intensive aquaculture environments, the accumulation of uneaten feed and excrement leads to water quality deterioration, creating conditions for the proliferation of opportunistic pathogens. These pathogens invade shrimp through water and feeding pathways, severely disrupting the physiological homeostasis of the giant freshwater prawn. On one hand, bacterial infection induces the production of large amounts of reactive oxygen species (ROS), leading to oxidative stress. This is primarily manifested in the inhibition of the activity of key antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) in the hepatopancreas, a core metabolic and immune organ of the shrimp. Simultaneously, the consumption of non-enzymatic antioxidants (such as glutathione) is exacerbated, reducing the body's ability to scavenge free radicals. The direct consequence is lipid peroxidation of the hepatopancreas cell membrane, damaging cell structure and function, thereby affecting nutrient metabolism, energy supply, and detoxification functions.

[0004] On the other hand, pathogens and their toxins can strongly activate the shrimp's innate immune system. Giant freshwater prawns lack adaptive immunity; their defense relies entirely on an innate immune network composed of hemolymphocytes, the prophenoloxidase system, antimicrobial peptides, and lectins. Bacterial infection triggers an overreaction in this network, leading to abnormal and continuous activation of the prophenoloxidase (proPO) system in the hemolymph, producing large amounts of cytotoxic quinones that damage the prawn's own tissues. Simultaneously, the expression of key genes related to immune defense in the hepatopancreas and hemocells (such as certain antimicrobial peptide and lysozyme genes) may exhibit a disordered state of initial upregulation followed by decline. This uncontrolled immune hyperactivity followed by immunosuppression results in excessive immune depletion and functional disorder in the shrimp, ultimately leading to a loss of the ability to clear pathogens. This manifests as decreased feed intake, weakened vitality, lesions on the body surface and gills, and, under high-density conditions, large-scale mortality.

[0005] To control bacterial diseases, traditional aquaculture has long relied on the direct use of antibiotics and chemical disinfectants in feed or water. The drawbacks of this practice are becoming increasingly apparent: first, it leads to the rapid development of drug resistance in pathogens, rendering treatments ineffective; second, it indiscriminately kills beneficial microorganisms in the water, disrupting the ecological balance of aquaculture; and third, it leaves drug residues in shrimp, threatening food safety and public health. Therefore, developing green functional additives that can replace antibiotics and control diseases by enhancing the shrimp's own immunity and antioxidant capacity has become an urgent technological need for the industry.

[0006] Currently, research on green control of bacterial diseases largely focuses on alternatives such as probiotics, prebiotics, herbal extracts, and antimicrobial peptides. However, these solutions face significant challenges in practical application: single probiotics have low colonization rates in the intestines of giant freshwater prawns, slow onset of action, and their effectiveness is greatly affected by environmental factors such as water quality and temperature, resulting in poor stability; many plant extracts have complex compositions, narrow effective dosage ranges, and are easily dissolved in aquaculture water; furthermore, when probiotics are directly compounded with plant-derived antimicrobial components, the plant components often have a bactericidal effect on the probiotics, leading to product inactivation. Meanwhile, bioactive ingredients such as antimicrobial peptides suffer from high production costs and are easily degraded and inactivated during feed processing and in the intestines. Therefore, overcoming these limitations and developing efficient, stable, cost-controllable functional feed additives that can precisely regulate the immune-antioxidant system of giant freshwater prawns is a key technical challenge in breaking through the bottleneck of bacterial disease control. Therefore, developing a special antibacterial feed additive for giant freshwater prawns that can synergistically leverage the advantages of probiotics and plant-derived components while simultaneously addressing component compatibility issues is of significant practical importance. Summary of the Invention

[0007] This invention aims to solve at least one of the technical problems existing in the prior art. It provides an antibacterial synergistic feed additive for giant freshwater prawns, which achieves highly efficient inhibition of pathogenic bacteria and comprehensive improvement of the health level of giant freshwater prawns through a four-fold mechanism of action: "probiotics occupying space for bacterial inhibition - plant-derived components synergistically killing bacteria - modified minerals adsorbing toxins - trace elements enhancing immunity and intestinal barrier".

[0008] The first aspect of the present invention is to provide a feed additive.

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

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

[0011] The fourth aspect of this invention is to provide aquaculture feed.

[0012] 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 a probiotic complex, a mineral-essential oil complex, an immune-enhancing complex, and a complex of trace elements. The mineral-essential oil complex is prepared from gallnut extract, carvacrol and attapulgite. The immune-enhancing complex comprises chitosan oligosaccharide and yeast β-glucan.

[0013] In some embodiments of the present invention, the probiotic complex is a microencapsulated compound probiotic.

[0014] In some embodiments of the present invention, the compound probiotics include Bacillus subtilis and Clostridium butyricum.

[0015] Bacillus subtilis can secrete antimicrobial peptides to inhibit bacterial growth, while Clostridium butyricum can produce butyric acid to repair the intestinal mucosa.

[0016] In some embodiments of the present invention, the ratio of viable Bacillus subtilis to Clostridium butyricum is (1-4):1, such as any ratio of 1:1, 2:1, 3:1 or 4:1 or any range formed by both.

[0017] In some embodiments of the present invention, the microencapsulated compound probiotics also include a wall material.

[0018] In some embodiments of the present invention, the wall material comprises starch and sodium alginate.

[0019] In some embodiments of the present invention, the starch comprises porous starch.

[0020] In some embodiments of the present invention, the microencapsulated compound probiotics are prepared by a method comprising: encapsulating the compound probiotics with a wall material to obtain microencapsulated compound probiotics.

[0021] In some embodiments of the present invention, the preparation method of the microencapsulated compound probiotics includes the following steps: (1) Bacillus subtilis and Clostridium butyricum were inoculated into LB liquid medium and RCM liquid medium respectively. After expansion culture, the bacterial sludge was collected by centrifugation. 1-3 times the mass of skim milk powder aqueous solution was added to the bacterial sludge to prepare Bacillus subtilis bacterial solution and Clostridium butyricum bacterial solution respectively. (2) Mix Bacillus subtilis bacterial solution with Clostridium butyricum bacterial solution, add porous starch to obtain mixed bacterial solution; (3) Using microcrystalline cellulose blank pellets as the mold core particles, put them into a fluidized bed coating machine, spray the mixed bacterial solution onto the surface of the pellet core, and obtain bacterial core particles; (4) The bacteria-containing core particles are fed into a fluidized bed coating machine for double-layer microencapsulation: the first layer of coating uses a porous starch aqueous solution as the coating liquid to form a porous starch inner layer; the second layer of coating uses a sodium alginate aqueous solution as the coating liquid to form a sodium alginate outer layer. (5) After coating, the product is dried with hot air, cooled and discharged to obtain the probiotic complex.

[0022] In some embodiments of the present invention, the amount of porous starch added in step (2) is 0.05-0.2 times the mass of the mixed bacterial solution, such as any ratio or range formed by any two of 0.05, 0.07, 0.09, 0.1, 0.15 and 0.2 times.

[0023] In some embodiments of the present invention, the porous starch aqueous solution in step (4) has a mass fraction of 7%-9% and is used in an amount of 45%-55% of the mass of the bacterial core particles.

[0024] In some embodiments of the present invention, the sodium alginate aqueous solution in step (4) has a mass fraction of 1%-3% and is used in an amount of 45%-55% of the mass of the bacterial core particles.

[0025] In some embodiments of the present invention, the hot air drying conditions in step (5) are hot air drying at 40-50°C for 5-15 minutes; further, hot air drying at 42-46°C for 8-12 minutes.

[0026] In some embodiments of the present invention, the viable count of Bacillus subtilis in the probiotic complex is (2-4) × 10⁻⁶. 9 CFU / g.

[0027] In some embodiments of the present invention, the viable count of Clostridium butyricum in the probiotic complex is (1-2) × 10⁻⁶. 9 CFU / g.

[0028] In some embodiments of the present invention, the weight ratio of the gallnut extract, carvacrol and attapulgite is (2-6):1:(7-25); further, it is (2-5):1:(10-20); and even further, it is (3-5):1:(10-15).

[0029] Gallnut extract is rich in tannins, which have a strong inhibitory effect on bacteria. Carvacrol can destroy bacterial cell membranes, and attapulgite can adsorb bacterial toxins and protect the liver and pancreas. At the same time, it can also serve as a carrier to load plant-derived active ingredients.

[0030] In some embodiments of the present invention, the attapulgite is modified attapulgite, that is, attapulgite that has undergone high-temperature activation treatment.

[0031] In some embodiments of the present invention, the modified attapulgite has a specific surface area ≥ 150 m². 2 / g, cation exchange capacity ≥30 mmol / 100g; further modified attapulgite has a specific surface area of ​​180 m². 2 / g, cation exchange capacity 35 mmol / 100g.

[0032] In some embodiments of the present invention, the gallnut extract is an alcoholic extract of gallnut.

[0033] In some embodiments of the present invention, the gallnut extract contains at least 50 wt% tannins; further, it contains at least 55 wt% tannins.

[0034] In some embodiments of the present invention, the gallnut extract is prepared by the following method: gallnut galls are crushed, ethanol solution is added according to the material-liquid ratio, reflux extraction is performed, concentration is achieved, and drying is carried out to obtain the gallnut extract.

[0035] In some preferred embodiments of the present invention, the feed-liquid ratio is 1:(8-12), such as any one of 1:8, 1:9, 1:10, 1:11 or 1:12 or a range formed by any two of them.

[0036] In some preferred embodiments of the present invention, the ethanol solution is a 50%-70% ethanol solution.

[0037] In some preferred embodiments of the present invention, the reflux extraction conditions are reflux extraction at 60℃~80℃ for 1-2 hours, repeated 2-3 times.

[0038] In some preferred embodiments of the present invention, the concentration is made to a relative density of 1.10-1.20 (measured at 60°C).

[0039] In some preferred embodiments of the present invention, the drying includes spray drying with an inlet temperature of 160-180°C and an outlet temperature of 70-80°C.

[0040] In some embodiments of the present invention, the carvacrol is obtained from natural extraction or prepared by synthesis, and has a purity of ≥95%.

[0041] In some embodiments of the present invention, the mineral-essential oil complex is prepared by a method comprising: embedding gallnut extract and carvacrol into the interlayer of attapulgite using solid-phase shearing technology, thereby obtaining the mineral-essential oil complex.

[0042] Solid-phase shearing technology embeds plant essential oils between mineral layers, with an essential oil retention rate of ≥85% (stored at 40℃ for 30 days), high-temperature granulation resistance, and long shelf life.

[0043] In some embodiments of the present invention, the solid-phase shearing technology includes using a disc-shaped mechanical chemical reactor, with process parameters of: disc gap 0.1-0.5 mm, grinding temperature ≤50℃, and 5-30 cycles of grinding.

[0044] In some preferred embodiments of the present invention, the process parameters are: grinding disc gap 0.2-0.4 mm, grinding temperature ≤45℃, and 10-20 cycles of grinding; further, grinding disc gap 0.2-0.3 mm, grinding temperature ≤45℃, and 10-15 cycles of grinding.

[0045] In some embodiments of the present invention, the weight ratio of chitosan oligosaccharide to yeast β-glucan is (1-5):1, such as any ratio of 1:1, 2:1, 3:1, 4:1 or 5:1 or any range of both.

[0046] In some embodiments of the present invention, the chitosan oligosaccharide has a degree of polymerization of 2-20, a molecular weight of ≤3000 Da, and a water solubility of ≥95%; further, the chitosan oligosaccharide has a degree of polymerization of 5-10, a molecular weight of ≤2000 Da, and a water solubility of ≥95%.

[0047] In some embodiments of the present invention, the yeast β-glucan contains at least 70 wt% β-glucan.

[0048] Chitosan oligosaccharides can activate the phenol oxidase system of giant freshwater shrimp and enhance innate immunity, while β-glucan can enhance the expression of immune-related genes.

[0049] In some embodiments of the present invention, the composite trace elements include copper and zinc.

[0050] In some embodiments of the present invention, the copper and zinc are respectively organic chelated copper (copper methionine, containing 15%-20% copper element) and organic chelated zinc (zinc methionine, containing 15%-20% zinc element). Copper methionine, as a cofactor of phenol oxidase and superoxide dismutase, significantly enhances the activity of immune enzymes; zinc methionine maintains intestinal integrity and promotes tight junctions of the intestinal epithelium.

[0051] In some embodiments of the present invention, the weight ratio of copper to zinc is 1:(1-6), such as any one of 1:1, 1:2, 1:3, 1:4, 1:5 or 1:6 or any range of both.

[0052] In some embodiments of the present invention, the feed additive comprises, by weight, 10-30 parts of probiotic complex, 20-50 parts of mineral-essential oil complex, 5-20 parts of immune-enhancing complex, and 3-15 parts of complex trace elements.

[0053] In some embodiments of the present invention, the feed additive comprises 20-25 parts by weight of a probiotic complex, such as any one of 20, 21, 22, 23, 24 or 25 parts or a range formed by any two of these values.

[0054] In some embodiments of the present invention, the feed additive comprises 30-40 parts by weight of a mineral-essential oil complex, such as any value or a range formed by any two of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 parts.

[0055] In some embodiments of the present invention, the feed additive comprises, by weight, 8-15 parts of an immune-enhancing complex, such as any value or range formed by any combination of 8, 9, 10, 11, 12, 13, 14 or 15 parts.

[0056] In some embodiments of the present invention, the feed additive comprises 5-10 parts by weight of a complex trace element, such as any value or range formed by any combination of 5, 6, 7, 8, 9 or 10 parts.

[0057] A second aspect of the present invention provides a method for preparing the feed additive of the first aspect of the present invention, comprising the following steps: mixing a probiotic complex, a mineral-essential oil complex, an immune-enhancing complex, and a complex of trace elements at a temperature below 30°C to obtain the feed additive.

[0058] A third aspect of the present invention provides the application of the feed additive of the first aspect of the present invention in the preparation of animal feed.

[0059] In some embodiments of the present invention, the aquaculture includes aquaculture.

[0060] In some embodiments of the present invention, the aquatic products include shrimp, such as prawns and giant freshwater prawns.

[0061] In some embodiments of the present invention, the amount of the feed additive added is 0.1wt%-5wt% of the animal feed, such as any value or a range formed by any two of 0.1wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt% or 5wt%.

[0062] Adding the feed additive provided by this invention to aquaculture feed can inhibit bacterial diseases in giant freshwater prawns and improve their innate immunity.

[0063] A fourth aspect of the present invention provides a livestock feed, comprising the feed additive of the first aspect of the present invention.

[0064] In some embodiments of the present invention, the aquaculture feed also includes a basic feed.

[0065] In some embodiments of the present invention, the amount of the feed additive added is 0.1wt%-5wt% of the animal feed, such as any value or a range formed by any two of 0.1wt%, 0.5wt%, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt% or 5wt%.

[0066] In some embodiments of the present invention, the aquaculture includes aquaculture.

[0067] In some embodiments of the present invention, the aquatic products include shrimp, such as prawns and giant freshwater prawns.

[0068] The beneficial effects of this invention are: This invention provides a feed additive containing a probiotic complex, a mineral-essential oil complex, an immune-enhancing complex, and a compound trace element. This feed additive achieves highly efficient inhibition of pathogens such as parahemolytic bacteria through a four-pronged mechanism of action: probiotics inhibit bacterial growth, plant-derived components synergistically kill bacteria, minerals adsorb toxins, and trace elements enhance immunity and the intestinal barrier. This significantly reduces the incidence and mortality of bacterial diseases in giant freshwater prawns. Furthermore, this invention solves the technical challenge of coexistence between probiotics and plant essential oils in existing technologies, exhibiting significant synergistic effects, good stability, and green safety, making it suitable for the entire process of giant freshwater prawn farming.

[0069] Specifically, it has the following beneficial effects: Significant synergistic effect: Through the triple action of Bacillus subtilis secreting antimicrobial peptides, gallnut tannin coagulating bacterial proteins, and carvacrol disrupting cell membranes, it has a strong inhibitory effect on bacteria (such as parahemolytic bacteria).

[0070] Dual-target protection of the liver, pancreas, and intestines: Modified attapulgite adsorbs bacterial toxins to protect the liver and pancreas; Clostridium butyricum produces butyric acid to repair the intestinal mucosa; zinc methionine promotes tight junctions of the intestinal epithelium; and copper methionine enhances the activity of immune enzymes, thus constructing a complete protective barrier.

[0071] Synergistic effect of trace elements: Copper methionine and zinc methionine are combined. Copper acts as a coenzyme for phenol oxidase (PO) and superoxide dismutase (SOD), while zinc acts as a coenzyme for another subtype of SOD and maintains the intestinal barrier. The two work together to enhance antioxidant and immune defense capabilities.

[0072] Solving the problem of bacteria-drug antagonism: By using probiotic double-layer microencapsulation and mineral-loaded plant essential oil isolation technology, the stable coexistence of live bacteria and antibacterial ingredients in the same product is achieved.

[0073] Outstanding immune-enhancing effect: Chitosan oligosaccharide and β-glucan synergistically activate the phenol oxidase system of Macrobrachium rosenbergii, significantly increasing the activity of phenol oxidase (PO) and superoxide dismutase (SOD), thereby enhancing the innate immunity of Macrobrachium rosenbergii.

[0074] Green and safe: Contains no antibiotics and no drug residues; uses organic trace elements with low addition levels, reducing environmental pollution and meeting the requirements of green farming. Detailed Implementation

[0075] 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.

[0076] 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.

[0077] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0078] Example 1 A feed additive for giant freshwater prawns, by weight, comprises 20 parts of probiotic complex, 40 parts of plant-derived disease-resistant and carrier complex, 10 parts of immune-enhancing complex, and 6 parts of trace element synergistic complex. The probiotic complex is a microencapsulated compound probiotic, prepared by the following methods: (1) Bacillus subtilis ( Bacillus subtilis CPCC 160016) and Clostridium butyricum ( Clostridium buttermilkCPCC 160032 was provided by the China Pharmaceutical Microbial Culture Collection Center. Bacillus subtilis and Clostridium butyricum were inoculated into LB liquid medium and RCM liquid medium, respectively, and cultured three times. The bacterial solutions were centrifuged at 10,000 r / min for 10-15 min, the supernatant was removed, and bacterial sludge was collected. Two times the weight of the bacterial sludge (15% skim milk powder aqueous solution) was added to obtain bacterial solutions of both strains. Bacillus subtilis and Clostridium butyricum bacterial solutions were mixed at a volume ratio of 2:1 to obtain a mixed bacterial solution. Porous starch (LD1067, Shaanxi Langde Biotechnology Co., Ltd.) of 1 / 10 of its mass was added to the mixed bacterial solution and mixed evenly. Microcrystalline cellulose blank pellet cores (TONCELLUS® TP102, particle size 106-212μm) were used as the matrix (the amount of matrix added was equal to the mass of the mixed bacterial solution). The mixed bacterial solution and matrix were fed into a Freund FC-LAB 3 multi-functional fluidized bed coating machine (standard configuration with bottom spray Wurster system). The inlet air temperature was set to 50-60℃ and the atomization pressure to 1.5 bar to granulate and obtain bacterial core particles. (2) The above-mentioned bacteria-containing core particles were fed into a Freund FC-LAB 3 multi-functional fluidized bed coating machine (standard configuration with bottom spray Wurster system) for double-layer microencapsulation coating: First coating: Prepare a porous starch aqueous solution with a mass fraction of 8% as the coating solution. Set the inlet air temperature to 50℃, the atomization pressure to 1.8 bar, and the inlet air volume to 35 m³ / h. Atomize the coating solution through the bottom spray nozzle and spray it onto the fluidized particles. The amount of coating solution used is 50% of the mass of the bacterial core particles, forming a porous starch inner layer. Second coating layer: After the first coating layer is completed, the coating solution is switched to a 2% sodium alginate aqueous solution, maintaining an inlet air temperature of 50℃ and an atomization pressure of 1.8 bar. The amount of coating solution used is 50% of the mass of the bacterial core particles, forming the sodium alginate outer layer. After coating, the product is dried with hot air at 45℃ for 10 minutes, cooled, and discharged to obtain the probiotic complex. After the above double-layer microencapsulation coating process, the obtained probiotic complex was tested using the plate count method, and the viable count of Bacillus subtilis in the finished product was 3.5 × 10⁻⁶. 9 (CFU / g) and Clostridium butyricum viable count 1.6 × 10⁻⁶ 9 CFU / g; The plant-derived disease-resistant and carrier complex is a mineral-essential oil complex, prepared by the following method: Dried gallnut galls are pulverized and passed through a 40-mesh sieve to obtain coarse gallnut powder. A 70% ethanol solution is added at a material-to-liquid ratio of 1:12 (g / mL), and the mixture is refluxed three times at 75℃ for one hour each time. The extracts are combined, filtered, concentrated under reduced pressure, and then spray-dried (inlet temperature 160℃, outlet temperature 80℃) to obtain gallnut extract. The measured tannin content in this gallnut extract is 55 wt%. Eight parts by weight of gallnut extract and two parts by weight of carvacrol (98% purity) are mixed thoroughly. Modified attapulgite (attapulgite activated at high temperature, with a specific surface area of ​​180 m²) is added. 2 30 parts of a mineral-essential oil complex (i.e., the weight ratio of gallnut extract, carvacrol, and modified attapulgite was 4:1:15) were processed in a disc-shaped mechanochemical reactor (refer to patent number: ZL 95 2 42817.2) with a disc gap of 0.3 mm, a grinding temperature of ≤45℃, and cyclic grinding for 15 cycles, each for 3 minutes, to obtain the mineral-essential oil complex. The immune-enhancing complex was prepared by the following method: 2 parts by weight of chitosan oligosaccharide (degree of polymerization 5-10, molecular weight ≤2000 Da, water solubility ≥95%, purchased from Guangzhou Anrui Food Ingredients Co., Ltd.) and 1 part by weight of yeast β-glucan (purchased from Shanxi Hongchuang Biotechnology Co., Ltd., SXHC-0709-164, β-glucan purity 70%, the same below) (i.e., the weight ratio of chitosan oligosaccharide to β-glucan is 2:1), mixed evenly to obtain the immune-enhancing complex; The trace element synergistic complex was prepared by the following method: 1.5 parts by weight of copper methionine (copper content 17wt%) and 4.5 parts by weight of zinc methionine (zinc content 17wt%) (i.e., the weight ratio of copper methionine to zinc methionine is 1:3) were mixed evenly to obtain the trace element synergistic complex.

[0079] The preparation method of the above-mentioned giant freshwater prawn feed additive includes the following steps: in a 25℃ low-temperature mixer (Jiangyin Xiangda Machinery SHJ-2000), a probiotic complex, a plant-derived disease-resistant and carrier complex, an immune-enhancing complex, and a trace element synergistic complex are added in sequence and mixed for 15 minutes to obtain the giant freshwater prawn feed additive.

[0080] Example 2 A feed additive for giant freshwater prawns, by weight, comprises 15 parts of a probiotic complex, 30 parts of a plant-derived disease-resistant and carrier complex, 15 parts of an immune-enhancing complex, and 8 parts of a trace element synergistic complex. The preparation method of the probiotic complex is the same as in Example 1; The preparation method of the plant-derived disease resistance and carrier complex differs from that in Example 1 only in that the weight ratio of gallnut extract, carvacrol and modified attapulgite is 5:1:10. The preparation method of the immune-enhancing complex differs from that in Example 1 only in that the weight ratio of chitosan oligosaccharide and β-glucan is 1:1. The preparation method of the trace element synergistic complex differs from that in Example 1 only in that the weight ratio of copper methionine and zinc methionine is 1:2. The preparation method of the above-mentioned giant freshwater prawn feed additive is the same as in Example 1.

[0081] Example 3 A feed additive for giant freshwater prawns, comprising, by weight, 25 parts of a probiotic complex, 35 parts of a plant-derived disease-resistant and carrier complex, 8 parts of an immune-enhancing complex, and 5 parts of a trace element synergistic complex. The preparation method of the probiotic complex is the same as in Example 1; The preparation method of the plant-derived disease resistance and carrier complex differs from that in Example 1 only in that the weight ratio of gallnut extract, carvacrol and modified attapulgite is 3:1:15. The preparation method of the immune-enhancing complex differs from that in Example 1 only in that the weight ratio of chitosan oligosaccharide to β-glucan is 3:1. The preparation method of the trace element synergistic complex differs from that in Example 1 only in that the weight ratio of copper methionine and zinc methionine is 1:4. The preparation method of the above-mentioned giant freshwater prawn feed additive is the same as in Example 1.

[0082] Example 4 A feed additive for giant freshwater prawns differs from Example 1 only in that: in the preparation method of the plant-derived disease-resistant and carrier complex, gallnut extract and carvacrol are not subjected to solid-phase shearing treatment (i.e., not ground with modified attapulgite in a disc-shaped mechanical chemical reactor), but are simply physically mixed.

[0083] Comparative Example 1 A feed additive for giant freshwater prawns, compared with Example 1, differs only in that it does not contain a probiotic complex, and the missing amount is supplemented by modified attapulgite, that is, 20 parts of modified attapulgite are used to replace 20 parts of the probiotic complex.

[0084] The preparation method of the above-mentioned giant freshwater prawn feed additive is the same as in Example 1.

[0085] Comparative Example 2 A feed additive for giant freshwater prawns, compared with Example 1, differs only in that it does not contain a plant-derived disease resistance and carrier complex. The missing amount is supplemented by modified attapulgite, that is, 40 parts of modified attapulgite are used to replace 40 parts of the plant-derived disease resistance and carrier complex.

[0086] The preparation method of the above-mentioned giant freshwater prawn feed additive is the same as in Example 1.

[0087] Comparative Example 3 A feed additive for giant freshwater prawns, compared with Example 1, differs only in that it does not contain an immune-enhancing complex, and the missing amount is supplemented by modified attapulgite, that is, 10 parts of modified attapulgite are used to replace 10 parts of the immune-enhancing complex.

[0088] The preparation method of the above-mentioned giant freshwater prawn feed additive is the same as in Example 1.

[0089] Comparative Example 4 A feed additive for giant freshwater prawns, compared with Example 1, differs only in that it does not contain the trace element synergistic complex, and the missing amount is supplemented by modified attapulgite, that is, 6 parts of modified attapulgite are used to replace 6 parts of the trace element synergistic complex.

[0090] The preparation method of the above-mentioned giant freshwater prawn feed additive is the same as in Example 1.

[0091] Example 1: In vitro antibacterial experiment The effects of feed additives for giant freshwater prawns in Examples 1-4 and Comparative Examples 1-4 on parahemolytic bacteria (Gastrolytic bacteria) were determined using the micro-broth dilution method. Vibrio parahaemolyticus The minimum inhibitory concentration (MIC) of Vibrio parahaemolyticus was determined using the following experimental procedure: A standard strain of Vibrio parahaemolyticus (ATCC 17802) was provided by the Guangdong Provincial Microbial Culture Collection Center. 1 g of the feed additive sample for Macrobrachium rosenbergii was weighed, added to 50 mL of sterile physiological saline, shaken to mix, and extracted at 4℃ for 12 h. The sample was then centrifuged at 1000 r / min for 10 min, and the supernatant was collected to obtain the original sample solution (equivalent to an additive concentration of 20 mg / mL). The original sample solution was diluted to a series of concentrations (10, 5, 2.5, 1.25, 0.625, 0.3125 mg / mL) using MH broth medium via a two-fold dilution method. Vibrio parahaemolyticus was cultured to the logarithmic growth phase, and the bacterial concentration was adjusted to 1×10⁻⁶. 6 CFU / mL. In a 96-well plate, add 100 μL of sample solution of varying concentrations and 100 μL of bacterial culture to each well. Set up a positive control (containing bacteria but without additives) and a negative control (sterile and additive-free). Incubate at 37℃ for 24 h. The lowest sample concentration at which no bacterial growth is observed visually is the MIC. Each experiment is repeated three times, and the average value is taken.

[0092] The results showed that the MIC (microinhibitory factor) of the feed additive for *Macrobrachium rosenbergii* in Example 1 was 1.35 mg / mL, in Example 2 it was 1.25 mg / mL, in Example 3 it was 1.67 mg / mL, and in Example 4 it was 1.67 mg / mL. The MIC for Comparative Example 1 was 3.33 mg / mL, in Comparative Example 2 it was 6.67 mg / mL, in Comparative Example 3 it was 2.92 mg / mL, and in Comparative Example 4 it was 1.35 mg / mL. These results indicate that the addition of probiotic complexes, plant-derived disease-resistant and carrier complexes, and immune-enhancing complexes can improve the antibacterial effect of feed additives against *Macrobrachium rosenbergii*.

[0093] Example 2: Stability Experiment The feed additives for giant freshwater prawns from Examples 1 and 4 were stored in a 40°C constant temperature incubator for 30 days. The carvacrol content in the feed was determined according to TSXSL11—2022 "Determination of Carvacrol and Thymol Content in Feed by High Performance Liquid Chromatography". The carvacrol retention rate was calculated. The experiment was repeated three times, and the result was the average of the three experiments. Carvacrol retention rate (%) = Ct / C0 × 100%, where: Ct—carvacrol content in the sample after t days of storage (mg / kg); C0—initial carvacrol content in the sample (mg / kg).

[0094] Table 1. Retention rate of carvacrol in feed

[0095] Note: Data in the table is "mean ± standard deviation" (n=3). Compared to Example 4, each Example 1... P <0.05. Carvacrol retention rate (%) = Ct / C0 × 100%, where: Ct — Carvacrol content in the sample after storage for t days (mg / kg).

[0096] The results showed that after 30 days of storage, the carvacrol retention rate in the feed additive for giant freshwater prawns in Example 1 was 87.3%, while the carvacrol retention rate in the feed additive for giant freshwater prawns in Example 4 was only 52.6% (Table 1), indicating that solid-phase shearing treatment significantly improved the stability of plant essential oils (i.e., carvacrol).

[0097] Example 3: Protection against viral challenge in giant freshwater prawns 1200 healthy giant freshwater prawns (initial weight 5±0.5g) were randomly divided into 10 groups, with 3 replicates per group and 40 prawns per replicate. They were cultured in 1m×1m×1m outdoor pond cages. The control group was fed a basal diet. The example groups were fed a basal diet supplemented with 1wt% of the feed additives from Examples 1-4 (referred to as Example 1-Example 4, respectively). The comparative groups were fed a basal diet supplemented with 1wt% of the feed additives from Comparative Examples 1-4. The antibiotic group was fed a basal diet supplemented with 0.02wt% enrofloxacin. The feed formulations are shown in Table 2. The crude protein content of each group's feed was 41.5wt%, and the crude fat content was 7.5wt%.

[0098] Feeding lasted for 30 days, twice daily at 8:00 AM and 5:00 PM, with a daily feeding rate of 5% of body weight. The morning feeding accounted for 30% of the total daily feed, and the afternoon feeding for 70%. Samples were taken weekly on Sundays to determine the feeding amount for the following week. At the end of the feeding experiment, the average body weight was 16 grams, and the survival rate was 90%. Twenty healthy giant freshwater prawns were taken from each net cage, and each prawn was intraperitoneally injected with 0.1 mL of parahemolytic bacteria (1.0 × 10⁻⁶). 8 The animals were challenged with CFU / mL and then placed in a 30cm×40cm×40cm aquarium. During this period, they were fed the corresponding group feed once a day at 17:00, with the feed amount being 3% of their body weight. Continuous oxygenation was maintained, and 50% of the water was changed every day. The mortality rate was recorded within 7 days.

[0099] Table 2. Experimental substrate formulation for giant freshwater prawns

[0100] The vitamin premix contains the following components: Vitamin A 900,000 IU / kg, Vitamin D 200,000 IU / kg, Vitamin E 4,500 mg / kg, Vitamin K3 220 mg / kg, Vitamin B1 320 mg / kg, Vitamin B2 1,090 mg / kg, Vitamin B5 2,000 mg / kg, Vitamin B6 500 mg / kg, Vitamin B12 1.6 mg / kg, Vitamin C 5,000 mg / kg, Calcium pantothenate 1,000 mg / kg, and Folic acid 165 mg / kg. The mineral premix contains the following components: CuSO4·5H2O 2 g / kg, FeSO4·7H2O 25 g / kg, ZnSO4·7H2O 22 g / kg, and MnSO4·4H2O. 7g / kg, Na2SeO30.04g / kg, KI0.026g / kg, CoCl2·6H2O 0.1g / kg.

[0101] The results are shown in Table 3. These results indicate that the mortality rate of giant freshwater prawns in Examples 1-3 was significantly lower than that in the control groups ( P The concentration of the antimicrobial compound in the feed additive was <0.05%, and there was no significant difference compared with the antibiotic group, with a relative protection rate of 80.4%-82.4%. However, in Example 4, the plant-derived antimicrobial component and the carrier complex were not subjected to solid-phase shearing treatment during the preparation process, resulting in poor stability of the plant-derived antimicrobial component in the complex, which reduced the protective effect of the feed additive on giant freshwater prawns.

[0102] Table 3 Mortality rate of giant freshwater prawns after viral challenge

[0103] Note: Data in the table are "mean ± standard deviation" (n=3). Compared with the control group, each example group... P <0.05; Compared with the comparative example groups, each example group has <0.05. P <0.05. Mortality rate = number of deaths / total number × 100%, relative protection rate = (1 - mortality rate of treatment group / mortality rate of control group) × 100%.

[0104] Example 4: Immunoenzyme Activity Assay In Example 3, after culture of *Macrobrachium rosenbergii*, six prawns were randomly selected from each group and challenged with the virus according to the method described in Example 3. Six hours after challenge (without mortality), hepatopancreas was collected, and the activities of phenol oxidase (PO) and superoxide dismutase (SOD) were measured. Phenol oxidase (PO) activity was measured using a polyphenol oxidase (PPO) test kit (catalog number: A136-1-1) produced by Nanjing Jiancheng Bioengineering Institute. Superoxide dismutase (SOD) activity was measured using a total superoxide dismutase (T-SOD) test kit (hydroxylamine method, catalog number: A001-1) produced by Nanjing Jiancheng Bioengineering Institute.

[0105] The results showed that the activities of PO and SOD in each example group were significantly higher than those in the control group and each comparative example group. P <0.05). Among them, the immune enhancement effect of the Example 1 group (preferred ratio) was the most prominent, with PO and SOD activities being 3.2 times and 2.5 times that of the control group, respectively (Table 4).

[0106] Table 4. Hepatopancreatic Immune Enzyme Activities in Giant Freshwater Prawns

[0107] Note: Data in the table are "mean ± standard deviation" (n=6). Compared with the control group, each example group... P <0.05; Compared with the comparative example groups, each example group has <0.05. P <0.05.

[0108] The embodiments of the present invention have been described in detail, but the present invention is not limited to the above embodiments. Various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A feed additive comprising a probiotic complex, a mineral-essential oil complex, an immune-enhancing complex, and a complex of trace elements; wherein The mineral-essential oil complex is prepared from gallnut extract, carvacrol and attapulgite; The immune-enhancing complex comprises chitosan oligosaccharide and yeast β-glucan.

2. The feed additive according to claim 1, characterized in that, The probiotic complex is a microencapsulated compound probiotic; Preferably, the compound probiotics include Bacillus subtilis and Clostridium butyricum; Preferably, the ratio of viable Bacillus subtilis to Clostridium butyricum is (1-4):1; Preferably, the microencapsulated compound probiotics further include a wall material; Preferably, the microencapsulated compound probiotics are prepared by the following method: encapsulating the compound probiotics with a wall material to obtain microencapsulated compound probiotics.

3. The feed additive according to claim 1, characterized in that, The weight ratio of gallnut extract, carvacrol and attapulgite is (2-6):1:(7-25); Preferably, the gallnut extract is an alcoholic extract of gallnut; Preferably, the mineral-essential oil complex is prepared by the following method: embedding gallnut extract and carvacrol into the interlayer of attapulgite using solid-phase shearing technology to obtain the mineral-essential oil complex; Preferably, the solid-phase shearing technology includes a disc-shaped mechanical chemical reactor with the following process parameters: disc gap 0.1-0.5 mm, grinding temperature ≤50℃, and 5-30 cycles of grinding.

4. The feed additive according to any one of claims 1 to 3, characterized in that, The weight ratio of chitosan oligosaccharide to yeast β-glucan is (1-5):1; Preferably, the degree of polymerization of the chitosan oligosaccharide is 2-20, and the molecular weight is ≤3000 Da.

5. The feed additive according to claim 4, characterized in that, The composite trace elements include copper and zinc; Preferably, the copper and zinc are organically chelated copper and organically chelated zinc, respectively; Preferably, the weight ratio of copper to zinc is 1:(1-6).

6. The feed additive according to claim 4, characterized in that, By weight, the feed additive includes 10-30 parts of probiotic complex, 20-50 parts of mineral-essential oil complex, 5-20 parts of immune-enhancing complex, and 3-15 parts of complex trace elements.

7. A method for preparing the feed additive according to any one of claims 1-6, comprising the following steps: mixing a probiotic complex, a mineral-essential oil complex, an immune-enhancing complex, and a complex of trace elements at a temperature below 30°C to obtain the feed additive.

8. The use of the feed additive according to any one of claims 1-6 in the preparation of livestock feed; Preferably, the aquaculture includes aquaculture; Preferably, the amount of the feed additive added is 0.1wt%-5wt% of the livestock feed.

9. A livestock feed, comprising the feed additives according to any one of claims 1-6.

10. The cultured feed of claim 9, wherein, The aquaculture feed also includes basic feed; Preferably, the amount of the feed additive added is 0.1wt%-5wt% of the livestock feed.

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