An immune enhancer for larvae of pterygoplichthys pardalis and a preparation method and application thereof

Abstract

This invention relates to the field of aquaculture technology, and in particular to an immune enhancer for juvenile large yellow croaker (Culter alburnus), its preparation method, and its application. The immune enhancer provided by this invention, through the compounding of specific components in a specific ratio, achieves the synergistic effect of polypeptides, immunopolysaccharides, antioxidants, and nutritional factors, while also considering rapid absorption, immune regulation, and high-temperature environmental stability. It is suitable for the immune enhancement needs of large yellow croaker juveniles under high-temperature seawater conditions of 29-31 °C, and can enhance the non-specific and specific immunity of juveniles under high-temperature seawater conditions, improving disease resistance and survival rate.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, and in particular to an immune enhancer for fry of large yellow croaker from the Naozhou group, its preparation method, and its application. Background Technology

[0002] The large yellow croaker (Croatia naanensis) is a high-quality economic fish species unique to the South China Sea. It is characterized by its delicious flesh, tolerance to high temperatures (optimal growth temperature 29-31 ℃), and high commercial value, making it an important aquaculture species for deep-sea farming and marine ranching. However, during artificial breeding, especially in the fry rearing stage, the fish's immune system is not yet fully developed, making it susceptible to Vibrio (…). Vibrio harveyi , V. alginolyticus Streptococcus ( Streptococcus iniae Pathogens such as viruses and other pathogens can cause a decline in survival rates, severely restricting industrial development.

[0003] Current methods for enhancing the immunity of fry mainly rely on the addition of immunopolysaccharides, vitamins, and minerals, but these methods suffer from slow onset of action, limited efficacy, and difficulty adapting to high-temperature seawater environments. In recent years, bioactive peptides have shown advantages in aquatic animal immune regulation due to their small molecular weight, rapid absorption, ability to directly regulate the expression of immune-related genes, and multiple functions including antibacterial, antioxidant, and growth-promoting effects. However, there is currently no bioactive peptide-based compound immune enhancement program specifically designed for the high-temperature environment and immune developmental stage of large yellow croaker fry. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides an immune enhancer for juvenile large yellow croaker, its preparation method, and its application. This invention achieves the synergistic effect of polypeptides, immunopolysaccharides, antioxidants, and nutritional factors through a scientifically formulated ratio, balancing rapid absorption, immune regulation, and high-temperature stability, thus meeting the immune enhancement needs of large yellow croaker juveniles under high-temperature seawater conditions of 29-31 ℃.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides an immune enhancer for fry of large yellow croaker (Croatia acutissima), comprising the following components by weight percentage: 3.0%-6.0% marine fish skin collagen polypeptide, 1.0%-3.0% astaxanthin polypeptide, 1.0%-2.0% soybean antimicrobial peptide, 0.5%-1.0% β-glucan, 0.5%-1.5% seaweed polysaccharide, 0.5%-1.0% vitamin C, and the balance being a carrier; wherein the marine fish skin collagen polypeptide is a polypeptide powder with a molecular weight of 500-1000 Da obtained by enzymatic hydrolysis of deep-sea fish skin with papain and alkaline protease; the astaxanthin polypeptide is a polypeptide powder with a molecular weight of <800 Da obtained by enzymatic hydrolysis of waste from Antarctic krill and / or shrimp with a complex protease; and the soybean antimicrobial peptide is a polypeptide powder with a molecular weight of 500-1500 Da obtained by enzymatic hydrolysis of soybean protein with trypsin.

[0006] Preferably, the carrier is corn starch and / or low-fat fish meal.

[0007] Preferably, the moisture content of the carrier is ≤10% and the fineness is ≥60 mesh.

[0008] Preferably, the vitamin C is vitamin C coated powder.

[0009] This invention provides a method for preparing the seedling immune enhancer described in the above technical solution, comprising the following steps: Fresh deep-sea fish skin, papain, and alkaline protease were mixed and enzymatically hydrolyzed at pH 7.5-8.2 and 45-55 ℃ for 3-6 h. After hydrolysis, the mixture was separated by ultrafiltration with a molecular weight cutoff of 800-1200 Da and dried to obtain marine fish skin collagen polypeptides with a molecular weight of 500-1000 Da and a moisture content of ≤6%. The mass ratio of papain to alkaline protease was 1:0.5-2, preferably 1:1. The papain activity was ≥8.0×10⁻⁶. 5 U / g; the alkaline protease activity is ≥3.0×10⁻⁶. 5 U / g; the total mass of the papain and alkaline protease is 1.0-4.0 wt.%, preferably 2.0 wt.%, of the dry weight of fresh deep-sea fish skin; Using Antarctic krill and / or shrimp waste as raw materials, a complex protease is added and enzymatically hydrolyzed for 4-7 h at pH 7.2-7.8 and 45-52 ℃. The resulting product is then separated and dried using an ultrafiltration membrane with a molecular weight cutoff of 600-1000 Da to obtain astaxanthin polypeptides with a molecular weight <800 Da. The amount of the complex protease added is 1.5-4.0 wt.% of the dry weight of the raw materials, preferably 2.5 wt.%, and the total activity of the complex protease is ≥5.0 × 10⁻⁶. 5 U / g; Soybean protein and trypsin are mixed and enzymatically hydrolyzed at pH 7.5-8.5 and 45-55 ℃ for 4-8 h, followed by nanofiltration separation to obtain soybean antimicrobial peptides with a molecular weight of 500-1500 Da; the amount of trypsin added is 1.0-3.5 wt.% of the dry weight of soybean protein, preferably 2.0 wt.%, and the activity of the trypsin is ≥2,500 BAEE U / mg; The marine fish skin collagen peptides, astaxanthin peptides, and soybean antimicrobial peptides are mixed with β-glucan, seaweed polysaccharide, and vitamin C in a certain proportion to obtain the seedling immune enhancer.

[0010] Preferably, the drying method includes spray drying; the conditions for spray drying include: inlet air temperature 125±5 ℃, outlet air temperature ≤60 ℃, and spray pressure 0.6-0.8 MPa.

[0011] Preferably, the ambient temperature for mixing is not higher than 30 ℃, the relative humidity is ≤50%, the mixing time is 10-15 min, and the mixing uniformity is ≤5%.

[0012] This invention provides the application of the seedling immune enhancer described in the above technical solution in one or more of the following: 1) Improve the immunity of Naozhou yellow croaker; 2) Improve the disease resistance of Naozhou yellow croaker; 3) Improve the antioxidant capacity of Naozhou yellow croaker; 4) Improve the survival rate of Naozhou yellow croaker.

[0013] This invention provides a method for cultivating seedlings of the large yellow croaker (Cyprinus naanensis), comprising the following steps: In the process of raising juvenile large yellow croaker fry, the fry immune enhancer is mixed with the feed and fed once a day; the fry immune enhancer is 1.0%-1.2% of the dry weight of the feed; the fry immune enhancer is the fry immune enhancer described in the above technical solution.

[0014] Preferably, the seedling immune enhancer and feed are moistened with fresh water and / or seawater, stirred evenly, and fed within 30 minutes.

[0015] Beneficial effects: The seedling immune enhancer provided by this invention achieves the synergistic effect of polypeptides, immunopolysaccharides, antioxidants and nutritional factors through the compounding of specific components in a specific ratio. It takes into account rapid absorption, immune regulation and high temperature environment stability, and is suitable for the immune enhancement needs of large yellow croaker seedlings in seawater at high temperature conditions of 29-31 ℃. It can enhance the non-specific and specific immunity of seedlings under high temperature seawater conditions, and improve disease resistance and survival rate. Detailed Implementation

[0016] This invention provides an immune enhancer for fry of large yellow croaker (Croatia acutissima), comprising the following components by weight percentage: 3.0%-6.0% marine fish skin collagen polypeptide, 1.0%-3.0% astaxanthin polypeptide, 1.0%-2.0% soybean antimicrobial peptide, 0.5%-1.0% β-glucan, 0.5%-1.5% seaweed polysaccharide, 0.5%-1.0% vitamin C, and the balance being a carrier; wherein the marine fish skin collagen polypeptide is a polypeptide powder with a molecular weight of 500-1000 Da obtained by enzymatic hydrolysis of deep-sea fish skin with papain and alkaline protease; the astaxanthin polypeptide is a polypeptide powder with a molecular weight of <800 Da obtained by enzymatic hydrolysis of waste from Antarctic krill and / or shrimp with a complex protease; and the soybean antimicrobial peptide is a polypeptide powder with a molecular weight of 500-1500 Da obtained by enzymatic hydrolysis of soybean protein with trypsin.

[0017] In one embodiment, the carrier is corn starch and / or low-fat fish meal.

[0018] In one embodiment, the moisture content of the carrier is ≤10%, and the fineness is ≥60 mesh.

[0019] In one embodiment, the vitamin C is vitamin C coated powder.

[0020] In one embodiment, the deep-sea fish skin is deep-sea cod skin. This invention selects deep-sea cod skin as the raw material for preparing marine fish skin collagen polypeptides, which allows the molecular weight to be more stably concentrated in the 500-1000 Da range after enzymatic hydrolysis. This results in advantages such as high enzymatic hydrolysis yield (purity ≥85%), fewer impurities, and higher content of hydrophobic amino acids, which is more beneficial for immune regulation.

[0021] The peptides in the seedling immune enhancer provided by this invention can promote the construction of non-specific immune barriers. Among them, marine fish skin collagen peptides and astaxanthin peptides can be rapidly absorbed and stimulate intestinal goblet cells to secrete mucin, enhance the intestinal mucus layer, and reduce pathogen attachment and invasion. Soybean antimicrobial peptides and β-glucan work synergistically to significantly increase lysozyme (LZM) activity and complement C3 and C4 levels, accelerating pathogen clearance. Active peptides can activate NF-κB and MAPK signaling pathways, upregulate IL-1β and TNF-α in the early stage to initiate inflammatory responses, and increase IL-10 in the later stage to inhibit excessive inflammation and prevent tissue damage. Peptides can act as immune adjuvants to promote B cell differentiation and IgM synthesis, significantly enhancing specific immune memory. Astaxanthin peptides can increase GSH-Px and SOD activity, reduce MDA levels, and alleviate oxidative stress under high temperature and high density aquaculture.

[0022] The seedling immune enhancer provided by this invention achieves the synergistic effect of polypeptides, immunopolysaccharides, antioxidants and nutritional factors through the compounding of specific components in a specific ratio. It takes into account rapid absorption, immune regulation and high temperature environment stability, and is suitable for the immune enhancement needs of large yellow croaker seedlings in seawater at high temperature conditions of 29-31 ℃. It can enhance the non-specific and specific immunity of seedlings under high temperature seawater conditions, and improve disease resistance and survival rate.

[0023] Based on the above advantages, the present invention provides a method for preparing the seedling immune enhancer described in the above technical solution, comprising the following steps: Fresh deep-sea fish skin, papain, and alkaline protease were mixed and enzymatically hydrolyzed at pH 7.5-8.2 and 45-55 ℃ for 3-6 h. After hydrolysis, the mixture was separated by ultrafiltration with a molecular weight cutoff of 800-1200 Da and dried to obtain marine fish skin collagen polypeptides with a molecular weight of 500-1000 Da and a moisture content of ≤6%. The mass ratio of papain to alkaline protease was 1:0.5-2, preferably 1:1. The papain activity was ≥8.0×10⁻⁶. 5 U / g; the alkaline protease activity is ≥3.0×10⁻⁶. 5 U / g; the total mass of the papain and alkaline protease is 1.0-4.0 wt.%, preferably 2.0 wt.%, of the dry weight of fresh deep-sea fish skin; Using Antarctic krill and / or shrimp waste as raw materials, a complex protease is added and enzymatically hydrolyzed for 4-7 h at pH 7.2-7.8 and 45-52 ℃. The resulting product is then separated and dried using an ultrafiltration membrane with a molecular weight cutoff of 600-1000 Da to obtain astaxanthin polypeptides with a molecular weight <800 Da. The amount of the complex protease added is 1.5-4.0 wt.% of the dry weight of the raw materials, preferably 2.5 wt.%, and the total activity of the complex protease is ≥5.0 × 10⁻⁶. 5 U / g; Soybean protein and trypsin are mixed and enzymatically hydrolyzed at pH 7.5-8.5 and 45-55 ℃ for 4-8 h, followed by nanofiltration separation to obtain soybean antimicrobial peptides with a molecular weight of 500-1500 Da; the amount of trypsin added is 1.0-3.5 wt.% of the dry weight of soybean protein, preferably 2.0 wt.%, and the activity of the trypsin is ≥2,500 BAEE U / mg; The marine fish skin collagen peptides, astaxanthin peptides, and soybean antimicrobial peptides are mixed with β-glucan, seaweed polysaccharide, and vitamin C in a certain proportion to obtain the seedling immune enhancer.

[0024] In one embodiment, fresh deep-sea fish skin, papain, and alkaline protease were mixed and enzymatically hydrolyzed for 4 h at pH 7.8±0.1 and 50±1 ℃. After hydrolysis, the mixture was separated by an ultrafiltration membrane with a molecular weight cutoff of 1000 Da and dried to obtain marine fish skin collagen polypeptides with a molecular weight of 500-1000 Da and a moisture content of ≤6%. The mass ratio of papain to alkaline protease was 1:1; the enzyme activity of the papain was ≥8.0×10⁻⁶. 5 U / g; the alkaline protease activity is ≥3.0×10⁻⁶. 5 U / g; the weight of the papain and alkaline protease is 2.0 wt.% of the dry weight of fresh deep-sea fish skin.

[0025] In one implementation method, Antarctic krill and / or shrimp waste are used as raw materials. After enzymatic hydrolysis with a complex protease for 5 hours at pH 7.5±0.1 and 48±1℃, the astaxanthin polypeptide with a molecular weight cutoff of 800 Da is separated by ultrafiltration and dried to obtain astaxanthin polypeptides with a molecular weight <800 Da. The amount of the complex protease added is 2.5 wt.% of the dry weight of the raw materials, and the total activity of the complex protease is ≥5.0 × 10⁻⁶. 5 U / g; As one implementation method, soybean protein and trypsin are mixed and enzymatically hydrolyzed at pH 8.0 and 50 ℃ for 6 h, followed by nanofiltration separation to obtain soybean antimicrobial peptides with a molecular weight of 500-1500 Da; the trypsin is 2.0 wt.% of the dry weight of soybean protein, and the activity of the trypsin is ≥2,500 BAEE U / mg.

[0026] As one implementation method, the drying method includes spray drying; the conditions for spray drying include: inlet air temperature 125±5 ℃, outlet air temperature ≤60 ℃, and spray pressure 0.6-0.8 MPa.

[0027] As one implementation method, the ambient temperature for mixing is not higher than 30 ℃, the relative humidity is ≤50%, the mixing time is 10-15 min, and the mixing uniformity is ≤5%.

[0028] Based on the above advantages, the present invention provides the application of the seedling immune enhancer described in the above technical solution in one or more of the following: 1) Improve the immunity of Naozhou yellow croaker; 2) Improve the disease resistance of Naozhou yellow croaker; 3) Improve the antioxidant capacity of Naozhou yellow croaker; 4) Improve the survival rate of Naozhou yellow croaker.

[0029] Based on the above advantages, the present invention provides a method for raising seedlings of large yellow croaker from the Naozhou group, comprising the following steps: In the process of raising juvenile large yellow croaker fry, the fry immune enhancer is mixed with the feed and fed once a day; the fry immune enhancer is 1.0%-1.2% of the dry weight of the feed; the fry immune enhancer is the fry immune enhancer described in the above technical solution.

[0030] As one implementation method, the seedling immune enhancer and feed are moistened with fresh water and / or seawater, stirred evenly, and fed within 30 minutes to avoid the inactivation of active peptides and vitamin C due to hydrolysis or oxidation.

[0031] As one implementation method, the addition ratio of seedling immune enhancer can be adjusted according to the seedling growth and immune indicators. If the immune indicators are lower than 90% of the initial value, the addition ratio can be increased by 0.1%-0.2% in the next cycle, and vice versa. This ensures that non-specific and specific immunity are synergistically enhanced during the rapid development stage of the seedling immune system, thereby significantly improving the survival rate and reducing the incidence of diseases.

[0032] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of an immune enhancer for seedlings of large yellow croaker (Cyprinus naanensis), its preparation method, and its application, is provided by the present invention, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0033] Preparation Example The sources of the reagents in this embodiment of the invention are as follows: The papain enzyme activity for preparing marine fish skin collagen peptides is ≥8.0×10⁻⁶. 5 U / g, purchased from Nanning Pangbo Biotechnology Co., Ltd.; The alkaline protease activity for preparing marine fish skin collagen peptides is ≥3.0×10⁻⁶. 5 U / g, purchased from Nanning Pangbo Biotechnology Co., Ltd. The total activity of the complex protease used to prepare astaxanthin peptides is ≥5.0×10⁻⁶. 5 U / g, purchased from Nanning Pangbo Biotechnology Co., Ltd.; The trypsin activity for preparing soybean antimicrobial peptides was ≥2,500 BAEE U / mg, purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number T8170; the enzyme activity of the trypsin used in the preparation of soybean antimicrobial peptides is expressed as BAEE-U, that is, the amount of enzyme that causes an increase of 0.001 per minute in absorbance at 253 nm due to hydrolysis of N-benzoyl-L-arginine ethyl ester (BAEE) under the conditions of pH 8.0 and 25 ℃ is defined as 1 U.

[0034] β-glucan was obtained from yeast cell walls, with a purity of ≥80% and a solubility of ≥95%, and was purchased from Xi'an Tianguangyuan Biotechnology Co., Ltd. Seaweed polysaccharide is derived from brown algae and is obtained by hot water extraction, ethanol precipitation and vacuum drying. The sulfate ester content is ≥18%. It was purchased from Shaanxi Xintianyu Biotechnology Co., Ltd. The vitamin C coated powder was prepared by spray condensation method, with a coating rate of ≥95%, an effective ingredient content of ≥35%, a particle size of ≤150 μm, and a damp heat resistance of ≥85%. It was purchased from Qingdao Master Biotechnology Co., Ltd. Low-fat fish meal (carrier), moisture content ≤10%, fineness ≥60 mesh, purchased from Rongcheng Lanrun Biotechnology Co., Ltd.

[0035] Example 1 An immune enhancer comprises the following components in weight percentage: 5% marine fish skin collagen peptides, 2% astaxanthin peptides, 1.5% soybean antimicrobial peptides, 0.8% β-glucan, 1.0% seaweed polysaccharide, 0.8% vitamin C coated powder, and low-fat fish meal to make up to 100%.

[0036] The preparation method of the immune enhancer is as follows: Fresh deep-sea cod skin was selected as raw material. Papain and alkaline protease were used at a mass ratio of 1:1 and enzymatically hydrolyzed for 4 h at pH 7.8±0.1 and 50±1 ℃. The enzyme dosage was 2.0 wt.% of the dry weight of the substrate. After enzymatic hydrolysis, the product was separated by an ultrafiltration membrane with a molecular weight cutoff of 1000 Da and spray-dried (inlet air temperature 125±5 ℃, outlet air temperature ≤60 ℃, spray pressure 0.6-0.8 MPa) to obtain a powder with a molecular weight of 500-1000 Da, a purity of ≥85%, and a moisture content of ≤6%, which is marine fish skin collagen polypeptide. Using shrimp waste as raw material, 2.5wt.% of a complex protease was added and enzymatically hydrolyzed for 5 h under the conditions of pH 7.5±0.1 and 48±1 ℃. The mixture was then separated by an ultrafiltration membrane with a molecular weight cutoff of 800 Da and spray-dried to obtain a powder with a molecular weight of <800 Da, an astaxanthin content of ≥2.5%, and a purity of ≥80%, which is astaxanthin polypeptide. Using defatted and peeled soybean protein as raw material, the soybean antimicrobial peptide was obtained by enzymatic hydrolysis with trypsin at pH 8.0 and 50 ℃ for 6 h, with an enzyme dosage of 2.0 wt.% of the substrate dry weight. Nanofiltration separation yielded a polypeptide powder with a molecular weight of 500-1500 Da, which is the soybean antimicrobial peptide. The soybean antimicrobial peptide showed antibacterial activity against bacteria in a standard in vitro test. Vibrio harveyi The antibacterial rate can reach ≥85%, exhibiting significant antibacterial activity; The polypeptide powder, β-glucan, seaweed polysaccharide, and vitamin C coating powder were weighed according to the formula ratio and mixed for 10-15 minutes in a clean room with a temperature not exceeding 30 ℃ and a relative humidity ≤50%, using a horizontal ribbon mixer, with a mixing uniformity (coefficient of variation) ≤5%. After mixing, the product was packaged into an aluminum-plastic composite high-barrier packaging bag, vacuumed under a vacuum degree ≥0.08MPa, and sealed with 99.9% nitrogen gas. It was then stored in a dry environment at 4-8 ℃, with a shelf life of up to 6 months and an active ingredient attenuation rate ≤10%. This preparation process ensures the structural integrity and functional stability of each active ingredient and avoids the loss of immune activity caused by high temperature and humidity.

[0037] Example 2 Healthy, uninjured juvenile Naozhou croaker (30 days old, average total length 4.1±0.3 cm, weight 0.74±0.05 g) were selected from a marine hatchery along the coast of Naozhou Island, Zhanjiang, Guangdong Province. After disinfection by immersion in a 0.5% sea salt solution for 1 minute, the juveniles were released into the pond. The juveniles were randomly divided into an experimental group and a control group, with three replicates per group and 1000 juveniles per replicate, at a stocking density of 1000 juveniles / m². 3 The seedling tray has a volume of 20m³. 3 The pool bottom is equipped with a microporous aeration device and is continuously aerated to maintain dissolved oxygen ≥6.0 mg / L.

[0038] Water quality conditions: The water temperature is 29.5±0.3 ℃ throughout the process, the salinity is 28±0.5‰, the pH is 7.9-8.2, the total ammonia nitrogen is ≤0.2mg / L, the nitrite nitrogen is ≤0.05 mg / L, and the daily water exchange volume is 30% of the total tank volume, which is replaced with clean seawater that has been filtered through 50 μm sand and disinfected by ultraviolet light.

[0039] Feed and Immunostimulant Administration: The experimental group was fed the immunostimulant prepared in Example 1. Before feeding, the immunostimulant was weighed at 1.0% of the dry weight of the feed, moistened with fresh water at 4% of the dry weight of the feed, and mixed at low speed for 3 minutes to ensure the preparation was evenly adhered to the feed surface. After standing for 5 minutes, it was fed immediately, once a day at 8:30 am. The addition ratio was 1.0% for the first 15 days, and increased to 1.1% for the next 15 days, for a continuous period of 30 days. The feed amount was 6-8% of the daily body weight, and all feed was consumed within 30 minutes after feeding. The control group was fed only an equal amount of compound feed without the immunostimulant.

[0040] Monitoring and sampling: 20 fish per replicate were randomly selected every 7 days, and their body weight and total length were measured. Blood was collected from the tail vein, and serum was separated by centrifugation at 3500 rpm for 10 min. Lysozyme (LZM) activity, immunoglobulin M (IgM) level, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, and malondialdehyde (MDA) content were measured using commercial kits.

[0041] Pathogen challenge test: After feeding, 60 seedlings were randomly selected from each group and injected intramuscularly. Vibrio harveyi (1×10) 7 0.1 mL / tail (CFU / mL) was used as a control group, and 20 tails were used as a blank control (injected with an equal volume of sterile PBS as baseline healthy controls to exclude stress-induced death during injection). Under the same conditions, the tails were observed for 7 days and the cumulative mortality rate was recorded. Vibrio harveyi The pathogens isolated and identified were deposited at Guangdong Ocean University and published in the literature [Xu Luxi, Gu Haoming, Xu Xinlan, et al. Isolation and identification of Vibrio harveyi from grouper[J]. Anhui Agricultural Sciences, 2025, 53(2): 71-76].

[0042] The results are shown in Tables 1-5.

[0043] Table 1. Results of weight and total length measurements of fish in different groups.

[0044] Table 2. Detection results of lysozyme and immunoglobulins in different groups of fish.

[0045] Table 3. Detection results of SOD, GSH-Px and MDA in different groups of fish.

[0046] Table 4. Survival rate and feeding results of different groups of fish

[0047] Table 5 Results of pathogen challenge tests in different groups of fish

[0048] The results showed that the initial body weight and total length of the experimental and control groups were consistent throughout the experimental period, indicating that the groups were comparable. With the extension of the rearing period, the experimental group consistently outperformed the control group in both body weight and total length. During the 30-day experimental period, the lysozyme activity in the experimental group was significantly higher than that in the control group by 31.6%. P <0.05%, IgM level increased by 19.7% ( PThe activity of SOD and GSH-Px increased by 22.5% and 18.7% respectively, while the MDA content decreased by 17.3%, and the survival rate increased by 8.6 percentage points compared with the control group. In the pathogen challenge test, the mortality rate of the experimental group was 21.7%, which was significantly lower than that of the control group (45.3% P<0.05), and the mortality rate was reduced by about 52%.

[0049] Example 3 The experiment was conducted in a marine industrialized recirculating aquaculture system (RAS) seedling rearing workshop in Guangdong Province. Healthy juvenile large yellow croaker (30 days old, average total length 4.0 ± 0.2 cm, weight 0.72 ± 0.04 g) were selected and disinfected by immersion in a 0.5% sea salt solution for 1 min before being introduced into the RAS system. The juveniles were randomly divided into experimental and control groups, with three replicates per group and 2000 juveniles per replicate, at a stocking density of 2000 juveniles / m². 3 The seedling pond has a volume of 20 m³. 3 .

[0050] Circulating water system conditions: The system adopts a three-layer mechanical filtration (50 μm sand filter + activated carbon + biological ceramic particles), equipped with a protein separator and ultraviolet sterilization device, water temperature 29.6±0.3 ℃, salinity 28±0.4‰, pH 7.9-8.1, dissolved oxygen 6.5-7.0 mg / L, total ammonia nitrogen ≤0.15 mg / L, nitrite nitrogen ≤0.04 mg / L, and 10% fresh water is added daily.

[0051] Feed and immune enhancer administration: The immune enhancer formula was the same as in Example 1. For the first 15 days, it was mixed with feed at 1.0% of the dry weight, increasing to 1.1% for the next 15 days, once daily (8:30 AM). When mixing with feed, use dechlorinated seawater at 29-31°C (4% of the dry weight of the feed) to moisten the mixture, stirring at low speed for 3 minutes to ensure even adhesion. Let it stand for 5 minutes before feeding. The daily feed intake was 6%-7% of the daily body weight, to be consumed within 30 minutes. The control group was fed only the same amount of basal feed.

[0052] Monitoring and sampling: Same as in Example 2.

[0053] Pathogen challenge test: Same as Example 2.

[0054] Results: The survival rate of the experimental group was 9.3 percentage points higher than that of the control group. P <0.05), lysozyme activity increased by 29.8%, IgM level increased by 18.4%, SOD and GSH-Px activities increased by 21.2% and 17.6%, respectively, and MDA content decreased by 15.8%. In the challenge trial, the mortality rate in the experimental group was 19.8%, significantly lower than the 42.5% in the control group. P <0.05).

[0055] In summary, the immune enhancer of this invention demonstrates significant advantages in the cultivation of large yellow croaker fry. In Examples 2 (coastal net cage conditions) and 3 (factory-scale recirculating aquaculture system conditions), the lysozyme activity of the experimental group fry increased by 31.4% and 29.8% respectively compared to the control group, the immunoglobulin M level increased by 19.7% and 18.4%, the antioxidant enzyme SOD activity increased by 22.6% and 21.2%, the GSH-Px activity increased by 18.9% and 17.6%, and the malondialdehyde content decreased by 17.3% and 15.8%. Regarding survival rate, Examples 2 and 3 showed increases of 8.6 and 9.3 percentage points respectively compared to the control group.

[0056] In the pathogen challenge trial, the cumulative mortality rates in the experimental groups were 21.7% (Example 2) and 19.8% (Example 3), significantly lower than the 45.3% in the control group. P <0.05%, a decrease of approximately 52%-56%. Statistical analysis showed that the above-mentioned differences in immunity, physiology, and survival rate were all statistically significant. P <0.05), and showed a stable upward trend under different aquaculture modes, proving that the immune enhancer provided by the present invention can not only maintain its activity under high temperature seawater conditions, but also effectively enhance the non-specific immunity, specific immunity and antioxidant capacity of seedlings, thereby significantly reducing the incidence of diseases and increasing the survival rate.

[0057] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An immune enhancer for fry of large yellow croaker from the Naozhou group, characterized in that, The product comprises the following components by weight percentage: 3.0%-6.0% marine fish skin collagen peptides, 1.0%-3.0% astaxanthin peptides, 1.0%-2.0% soybean antimicrobial peptides, 0.5%-1.0% β-glucan, 0.5%-1.5% seaweed polysaccharide, 0.5%-1.0% vitamin C, and the balance being a carrier; wherein the marine fish skin collagen peptides are polypeptide powders with a molecular weight of 500-1000 Da obtained by enzymatic hydrolysis of deep-sea fish skin with papain and alkaline protease; wherein the astaxanthin peptides are polypeptide powders with a molecular weight of <800 Da obtained by enzymatic hydrolysis of waste from Antarctic krill and / or shrimp with a complex protease; and wherein the soybean antimicrobial peptides are polypeptide powders with a molecular weight of 500-1500 Da obtained by enzymatic hydrolysis of soybean protein with trypsin.

2. The seedling immune enhancer according to claim 1, characterized in that, The carrier is corn starch and / or low-fat fish meal.

3. The seedling immune enhancer according to claim 2, characterized in that, The moisture content of the carrier is ≤10%, and the fineness is ≥60 mesh.

4. The seedling immune enhancer according to claim 1, characterized in that, The vitamin C mentioned is vitamin C coated powder.

5. The method for preparing the seedling immune enhancer according to any one of claims 1-4, characterized in that, Includes the following steps: Fresh deep-sea fish skin, papain, and alkaline protease were mixed and enzymatically hydrolyzed at pH 7.5-8.2 and 45-55 ℃ for 3-6 h. After hydrolysis, the mixture was separated by ultrafiltration with a molecular weight cutoff of 800-1200 Da and dried to obtain marine fish skin collagen polypeptides with a molecular weight of 500-1000 Da and a moisture content of ≤6%. The mass ratio of papain to alkaline protease was 1:0.5-2, and the papain activity was ≥8.0×10⁻⁶. 5 U / g; the alkaline protease activity is ≥3.0×10⁻⁶. 5 U / g; the total mass of the papain and alkaline protease is 1.0-4.0 wt.% of the dry weight of fresh deep-sea fish skin. Using Antarctic krill and / or shrimp waste as raw materials, a complex protease was added and enzymatically hydrolyzed for 4-7 h at pH 7.2-7.8 and 45-52 ℃. The resulting product was then separated and dried using an ultrafiltration membrane with a molecular weight cutoff of 600-1000 Da to obtain astaxanthin polypeptides with a molecular weight <800 Da. The amount of the complex protease added was 1.5-4.0 wt.% of the dry weight of the raw materials, and the total activity of the complex protease was ≥5.0 × 10⁻⁶. 5 U / g; Soybean antimicrobial peptides with a molecular weight of 500-1500 Da were obtained by enzymatic hydrolysis of a mixture of soybean protein and trypsin at pH 7.5-8.5 and 45-55 ℃ for 4-8 h, followed by nanofiltration separation. The amount of trypsin added was 1.0-3.5 wt.% of the dry weight of soybean protein, and the activity of the trypsin was ≥2,500 BAEE U / mg. The marine fish skin collagen peptides, astaxanthin peptides, and soybean antimicrobial peptides are mixed with β-glucan, seaweed polysaccharide, and vitamin C in a certain proportion to obtain the seedling immune enhancer.

6. The preparation method according to claim 5, characterized in that, The drying method includes spray drying; the conditions for spray drying include: inlet air temperature 125±5 ℃, outlet air temperature ≤60 ℃, and spray pressure 0.6-0.8 MPa.

7. The preparation method according to claim 5, characterized in that, The ambient temperature for mixing is no higher than 30 ℃, the relative humidity is ≤50%, the mixing time is 10-15 min, and the mixing uniformity is ≤5%.

8. The use of the seedling immune enhancer according to any one of claims 1-4 or the seedling immune enhancer prepared by the preparation method according to any one of claims 5-7 in one or more of the following: 1) Improve the immunity of Naozhou yellow croaker; 2) Improve the disease resistance of Naozhou yellow croaker; 3) Improve the antioxidant capacity of Naozhou yellow croaker; 4) Improve the survival rate of Naozhou yellow croaker.

9. A method for cultivating fry of the large yellow croaker from the Naozhou group, characterized in that, Includes the following steps: In the process of raising juvenile large yellow croaker, the juvenile immune enhancer is mixed with the feed and fed once a day; the juvenile immune enhancer is 1.0%-1.2% of the dry weight of the feed; the juvenile immune enhancer is the juvenile immune enhancer according to any one of claims 1-4 or the juvenile immune enhancer prepared by the preparation method according to any one of claims 5-7.

10. The seedling cultivation method according to claim 9, characterized in that, Use fresh water and / or seawater to moisten the seedling immune enhancer and feed, mix well, and feed within 30 minutes.

Images