Immune-enhanced fermented functional feed for prawns and preparation method thereof

Abstract

The invention provides a prawn immunity-enhanced fermented functional feed and a preparation method thereof. Belongs to the field of animal feed. The feed comprises a basal feed and a feed additive, the feed additive comprises the following components: lactobacillus plantarum fermentation liquor, passion flower flavone and an immature bitter orange extract. Three functional components including the lactobacillus plantarum fermentation liquor, the passion flower flavone and the immature bitter orange extract are fused, a synergistic interaction system is constructed through a definite proportion, and the immunity of prawns is remarkably improved; the sources and the content of effective components of the immature bitter orange extract and the lactobacillus plantarum fermentation liquor are strictly limited, and the controllable product quality is ensured; the preparation process of the feed is simple, the key growth stage of shrimp culture is accurately positioned, and a comprehensive solution integrating probiotic regulation and control, plant active ingredient immune activation and process feasibility is provided for healthy culture of prawns.

Description

Technical Field

[0001] The invention belongs to the field of animal feed, and in particular relates to a shrimp immunity-enhancing fermented functional feed and a preparation method thereof. Background Art

[0002] Shrimp is an important economic species in my country's aquaculture sector. The selection of shrimp farming species in my country is based on ecological adaptability, growth performance and economic value. Currently, the species farmed on a large scale mainly include the following three categories: (1) Penaeus vannamei Originating from the Pacific coast of Central America, it is the world's largest shrimp species, and also the species with the largest farming area and the highest yield in my country. Its core characteristics are: Strong ecological adaptability: It can survive in water bodies with a salinity of 0.5-40, and can be farmed in both seawater and freshwater through desalination technology, adapting to my country's diverse farming environments from coastal to inland areas; Excellent growth performance: Under suitable conditions, the breeding cycle is about 3-4 months to reach commercial size (body length 12-15cm), and the average daily weight gain can reach 0.1-0.2g; Economic traits are stable: the meat is firm, the meat yield can reach more than 60%, and it has strong disease resistance, making it suitable for high-density intensive farming.

[0003] (2) Giant tiger prawn (Penaeus monodon) Also known as grass shrimp, it is a tropical and subtropical species that is widely farmed along the coast of South my country and Southeast Asia. Its notable characteristics include: Individuals are large: the maximum body length can reach over 30cm, and a single tail can weigh up to 300g. The commercial specifications are generally 20-50g / tail; Diverse and wide-ranging diet: They have a high demand for feed protein (40%-45% in the juvenile stage) and can consume animal bait (fish, shellfish) and plant bait (soybean meal, wheat bran). Environmental tolerance: Suitable water temperature is 25-30℃, salinity is 10-25, sensitive to low temperature (growth stagnates when water temperature is below 18℃).

[0004] (3) Chinese white shrimp (Fenneropenaeuschinensis) Also known as the Oriental shrimp, the Oriental shrimp is a premium native species of my country, primarily found in the Yellow and Bohai Seas. A typical marine shrimp, it thrives in salinity levels of 25-30°C and is challenging to domesticate in freshwater. Its cultivation cycle is approximately 5-6 months, with a commercial weight of 15-20g per shrimp. Its tender flesh is highly valued in the market. However, due to disease and environmental changes, the cultivation area has significantly decreased in recent years compared to that of Penaeus vannamei and Penaeus monodon, with the main focus now being on stocking and ecological aquaculture.

[0005] Among common aquatic products, shrimp farming has relatively high environmental requirements. Its environmental sensitivity, parameter control precision, and stability requirements are significantly higher than those of most farmed fish, shellfish, and other species. This difference is mainly due to the physiological characteristics, ecological habits, and farming model characteristics of shrimp: (1) Sensitivity to water quality parameters: Shrimp have a narrower tolerance range As crustaceans, shrimp's physiological metabolism (such as molting, growth, and respiratory cycle) is more sensitive to fluctuations in water quality parameters, and the suitable range of core indicators is significantly narrower than that of most fish: In terms of water temperature, the suitable range for shrimp is 18-35℃, and the optimum temperature is 25-30℃. They have poor tolerance to low temperatures and will stop feeding when the water temperature is below 18℃. In high temperature environments, they are prone to death due to lack of oxygen. The suitable water temperature for common fish such as grass carp is 10-32℃, and the optimum is 20-28℃, and they have a wider range of tolerance to temperature fluctuations.

[0006] In terms of dissolved oxygen, shrimp require dissolved oxygen in water ≥5mg / L, and bottom water ≥3mg / L. The suffocation point is higher, and shrimp are prone to death in a low oxygen environment for a long time. Most fish, such as grass carp, can survive normally when the dissolved oxygen is ≥3 mg / L, and can even tolerate a low oxygen environment of 1.5 mg / L in the short term.

[0007] In terms of pH value, the suitable pH value of water for shrimp is 7.5-8.6. They are sensitive to acidic water and are prone to stress reactions when the pH value is lower than 7. The suitable pH range for fish is 6.5-8.5, and they are more adaptable to acid-base fluctuations.

[0008] Regarding the limits of harmful substances such as ammonia nitrogen and nitrite, shrimp are required to have ammonia nitrogen ≤ 0.5mg / L and nitrite ≤ 0.1mg / L, with a lower tolerance threshold. The upper limit of fish's tolerance to ammonia nitrogen can reach 1.0 mg / L, and nitrite ≤ 0.5 mg / L can meet basic survival needs.

[0009] For marine aquaculture species, such as whiteleg shrimp, although they can adapt to water bodies with a salinity of 0.5 through desalination technology, they are extremely sensitive to salinity fluctuations in the fry stage and require a daily salinity change of ≤3, otherwise it may be fatal; most marine fish have a wider salinity tolerance range and are more adaptable to short-term fluctuations.

[0010] (2) Resistance to stress and stress response: Shrimp have weaker self-regulation ability Prawns lack the buoyancy-regulating organs of fish, such as the swim bladder, and their circulatory system is open (mainly sinusoids). They have extremely poor self-buffering ability against sudden changes in the environment, and their stress response is more intense and the consequences are more serious.

[0011] In terms of physical stress, turbid water (transparency <20cm) will cause shrimp to have visual feeding obstruction, and mud and sand will easily adhere to the gills and cause infection; while omnivorous fish such as carp and crucian carp can perceive the environment through their lateral lines and can still feed normally in turbid water.

[0012] During chemical stress, when the concentration of hydrogen sulfide in the aquaculture water exceeds 0.01 mg / L, the gill tissue of the shrimp will be rapidly damaged, leading to death from lack of oxygen; while low-oxygen-tolerant fish such as tilapia can assist breathing through their skin and can tolerate higher concentrations of harmful substances in the short term.

[0013] In terms of biological stress, shrimp are extremely sensitive to farming density. In high-density environments, they are prone to killing each other due to competition for territory (especially during the molting period), so the density needs to be strictly controlled (up to 200,000 fish / mu in high-level ponds); while grass carp, silver carp, etc. can tolerate higher densities (conventional ponds can reach 10,000-20,000 fish / mu), and the risk of group conflict is lower.

[0014] (3) Farming density and environmental regulation intensity: Shrimp rely on more precise management Shrimp farming is mainly based on intensive and high-density operations. The biological load per unit water body is much higher than that of traditional fish farming, and the requirements for environmental control technology are more stringent.

[0015] In terms of oxygen demand, the oxygen consumption rate of shrimp is significantly higher than that of fish of the same weight (the oxygen consumption rate of shrimp weighing 1g is about 0.15mg / h, while that of grass carp is about 0.08mg / h). High-level ponds need to be equipped with 1.5-2kW / mu of oxygenation equipment, and need to operate uninterruptedly for 24 hours; while the oxygenation power of conventional fish farming is only 0.5-1kW / mu, and intermittent oxygenation at night can meet the demand.

[0016] In terms of bottom soil management, shrimp are benthic organisms, and leftover bait and feces tend to accumulate at the bottom of the pond. They need to be treated with bottom soil improvers (such as calcium peroxide and zeolite powder) every week, otherwise pathogenic bacteria such as Vibrio will breed. However, the farming of mid- and upper-layer fish has lower requirements for bottom soil, and silt removal once a month is sufficient to maintain water stability.

[0017] In terms of water change frequency, the self-purification ability of shrimp farming water is weak, and 10%-20% of the water needs to be changed every week, and even 5% every day during high temperature periods; while ecological fish farming can achieve low water changes (monthly water changes <5%) through the "fish-grass-snail" symbiotic system, relying on natural ecological cycles to purify water quality.

[0018] (4) Correlation between diseases and the environment: Shrimp diseases are more likely to break out due to environmental deterioration. The immune mechanism of shrimp is relatively simple (lack of specific antibodies), and environmental deterioration will directly lead to the collapse of disease resistance, and the risk of disease outbreak is much higher than that of fish.

[0019] Among environmentally induced diseases, deterioration of water quality (such as excessive nitrite) can trigger "stealth death disease" (hepatopancreatic necrosis) in shrimp, with a mortality rate of over 80%; while hepatobiliary syndrome in fish is mostly caused by long-term nutritional imbalance, and sudden changes in the environment are only a secondary cause.

[0020] In terms of pathogen transmission conditions, the main pathogenic bacteria of shrimp (such as Vibrio) reproduce extremely quickly in eutrophic water bodies (one generation every 20 minutes at a water temperature of 25°C), and improper environmental regulation can easily lead to epidemics of diseases; while most viral diseases of fish (such as grass carp hemorrhagic disease) rely more on host contact for transmission and have a lower direct dependence on water quality.

[0021] In terms of differences in drug resistance, shrimp have thin shells and exposed gills, and even slightly higher concentrations of disinfectants will cause damage; while fish have mucus protection on their body surfaces and can tolerate higher concentrations of environmental disinfectants, making disease prevention and control measures more flexible.

[0022] Due to their fragile physiological structure, special metabolic characteristics and the need for intensive farming models, shrimp have significantly higher requirements for water quality stability, parameter accuracy and regulation intensity than conventional fish, shellfish and other aquatic products; therefore, how to reduce shrimp's sensitivity to the environment through various means is an important issue in shrimp farming. Summary of the Invention

[0023] The purpose of the present invention is to provide a shrimp immune-enhancing fermented functional feed and a preparation method thereof, which forms a triple immune-enhancing system through the synergistic effect of Lactobacillus plantarum fermentation and plant extracts.

[0024] A fermented functional feed for enhancing immunity of shrimp, comprising a basic feed and a feed additive; the feed additive comprises the following components in an amount calculated as a percentage by mass of the basic feed: Lactobacillus plantarum fermentation liquid 2-3% Passionflower flavonoids 0.4-0.8% Citrus aurantium extract 0.6-1.2%.

[0025] Preferably, the Lactobacillus plantarum fermentation broth is obtained by fermentation with MRS culture medium, and the total acidity of the Lactobacillus plantarum fermentation broth is ≥1.5%.

[0026] The Lactobacillus plantarum fermentation liquid is obtained by fermenting artichoke leaves with Lactobacillus plantarum, and comprises the following steps: S1: Select fresh artichoke leaves, soak them in sterilizing solution and then wash them with water until they are neutral; S2: Blanch the washed leaves in hot water, cool them down, and then add at least 3 times the weight of water to make pulp to obtain pulp; S3: Add the following components to the slurry and adjust the initial pH to 6.0-6.5 to obtain a culture medium; At least 1% of the leaf mass as a carbon source; A nitrogen source of at least 0.3-0.5% of the leaf mass; S4: inoculating activated Lactobacillus plantarum into the culture medium and performing anaerobic fermentation at a temperature of 34-37°C; S5: After the fermentation is completed, sterilize and terminate the fermentation, separate the solid and liquid, and take the liquid part. Test the total acidity of the Lactobacillus plantarum fermentation liquid. If the total acidity is ≥1.5%, it is the Lactobacillus plantarum fermentation liquid.

[0027] Preferably, the Citrus aurantium extract is obtained by water extraction of dried young fruits of Citrus aurantium L. and its cultivated varieties.

[0028] Preferably, the content of Citrus aurantium flavonoids in the Citrus aurantium extract is ≥50%.

[0029] The present invention also provides a method for preparing the aforementioned immune-enhancing fermented functional feed for shrimp, which comprises uniformly mixing the feed additive with the basic feed; the basic feed refers to a standardized compound feed designed to meet the basic nutritional needs of shrimp at different growth stages during shrimp farming. It is the main feed form consumed by shrimp on a daily basis and is also the core material basis for achieving nutritional supply, growth regulation, and ensuring farming efficiency.

[0030] The third object of the present invention is to provide a use of the aforementioned immune-enhancing fermented functional feed for shrimp, wherein the immune-enhancing fermented functional feed for shrimp is a feed for shrimp farming in the post-larval stage.

[0031] In the present invention: The core formula achieves synergistic enhancement through the scientific combination of three immune-active ingredients. Among them, Lactobacillus plantarum fermentation broth provides active probiotics and organic acids, which directly enhance the intestinal barrier function of shrimp and inhibit pathogenic bacteria; passionflower flavonoids and Citrus aurantium extract can significantly promote the growth of shrimp through their synergistic effect; and increase the survival rate of shrimp under ammonia nitrogen stress and reduce oxidative damage to the hepatopancreas; the three form a full-pathway protection mechanism of "microecological regulation-antiviral-immune activation".

[0032] In order to ensure the beneficial effects of each additive, the total acidity of Lactobacillus plantarum fermentation broth is clearly limited to ≥1.5%. Total acidity is a key indicator of the fermentation effect of lactic acid bacteria. ≥1.5% can ensure the content of organic acids (such as lactic acid) in the fermentation broth to ensure its regulatory and antibacterial effects on intestinal flora.

[0033] The present invention also found that when the fermentation substrate of the Lactobacillus plantarum fermentation broth is limited to artichoke leaves, its high content of inulin (15-20%) can be fully utilized as a prebiotic to promote the proliferation of Lactobacillus plantarum. At the same time, the chlorogenic acid in the leaves is fermented and degraded into highly active derivatives; this can further enhance the intestinal barrier function of shrimp and inhibit the effect of pathogenic bacteria.

[0034] To ensure the safety and efficiency of the artichoke leaf fermentation process using Lactobacillus plantarum, each step of the present invention serves a clear purpose: S1: Soaking in a sterilizing solution to remove pathogens such as Salmonella from the leaf surface; S2: Blanching to simultaneously inactivate browning enzymes and soften the fibers; S3: Adding carbon and nitrogen sources and adjusting the pH to 6.0-6.5 to optimize the bacterial growth environment; S4: Anaerobic fermentation to eliminate contamination by foreign bacteria and ensure efficient homolactic acid production; S5: A total acidity of ≥1.5% is the endpoint, achieving both functional and antiseptic properties without the need for additional additives. By establishing key control points such as "sterilization to terminate fermentation," "solid-liquid separation to extract liquid," and "total acidity ≥1.5%," a closed loop is formed from fermentation termination to finished product testing, ensuring both the safety (sterilization to remove foreign bacteria) and efficacy (acidity meets the standard) of the final fermentation broth.

[0035] The Citrus aurantium extract used in this invention is specifically derived from the dried young fruits of Citrus aurantium and its cultivated varieties (Rutaceae). This raw material is identified through plant taxonomy, avoiding compositional differences that can arise from using closely related species (such as Citrus aurantium), thereby ensuring the stability of the extract's active ingredients (such as flavonoids). The high synephrine content of Citrus aurantium (with an immunostimulatory potency five times that of sweet orange) synergizes with Passiflora flavonoids to form an antioxidant network, significantly upregulating the expression of antimicrobial peptide genes in shrimp. Furthermore, the quantitative standard avoids the risk of immune failure associated with low flavonoid content (less than 30%).

[0036] In terms of application, this invention specifically applies to the "mid-stage and adult stages" of shrimp, which aligns with the physiological laws of shrimp: the mid-stage is a critical period for immune system development, while the adult stage faces the disease pressures of high-density farming (such as Vibrio infection and environmental stress). Supplementing with immune-enhancing feed at this stage can specifically improve disease resistance and survival rate, avoiding a mismatch between function and demand. Excluding the juvenile stage (where intestinal function is fragile) prevents high-dose additives from irritating the juvenile shrimp's intestines. This demonstrates respect for the physiological characteristics of different growth stages and enhances the scientific and safe nature of feed application.

[0037] The present invention integrates three functional ingredients, namely, Lactobacillus plantarum fermentation broth, passionflower flavonoids and Citrus aurantium extract, to construct a synergistic enhancement system through a clear ratio, which can significantly enhance the immunity of shrimp. The sources and effective ingredient contents of Citrus aurantium extract and Lactobacillus plantarum fermentation broth are strictly limited to ensure controllable product quality. The feed preparation process is simple and accurately locates the key growth stages of shrimp farming, providing a comprehensive solution for healthy shrimp farming that combines probiotic regulation, immune activation of plant active ingredients and process feasibility. DETAILED DESCRIPTION

[0038] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0039] In the description of the present invention, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature specified as "first" or "second" may explicitly or implicitly include one or more of the specified features. In the description of the present invention, "plurality" means two or more, unless otherwise specifically defined.

[0040] Unless otherwise specified, the experimental methods used in the specific embodiments are all conventional methods, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0041] Unless otherwise specified, %, and percentages are all mass percentages.

[0042] Some of the raw materials used in the present invention come from the following sources: Passionflower flavonoids are the whole herb extract of Passiflora caerulea L., a plant of the genus Passiflora in the family Passifloraceae, and its flavonoid content is ≥10%.

[0043] Citrus aurantium extract is obtained by water extraction of dried young fruits of Citrus aurantium L. and its cultivated varieties. The flavonoid content of Citrus aurantium is detected by HPLC and divided into Citrus aurantium extracts with different contents of 30%, 50%, and 90%.

[0044] The plant lactobacillus is Lactobacillus plantarum P-8.

[0045] The basic feed contains the following components by mass: 20 parts of fish meal, 25 parts of soybean meal, 15 parts of peanut meal, 22 parts of wheat flour, 3 parts of gluten, 3 parts of squid paste, 2.6 parts of fish oil, 1.5 parts of phospholipid oil, 1.5 parts of multivitamins, and 1.5 parts of multiminerals.

[0046] Each kilogram of multivitamin includes: Vitamin A 4000000IU, Vitamin D3 2000000IU, Vitamin E 30g, Vitamin K3 10g, Vitamin B15g, Vitamin B2 15g, Vitamin B6 8g, Vitamin B12 0.02g, Niacin 40g, Calcium Pantothenate 25g, Folic Acid 2.5g, Inositol 150g and Biotin 0.08g; Each kilogram of complex minerals includes: 12g magnesium sulfate monohydrate, 90g sodium chloride, 3g metallic copper, 1g ferrous sulfate monohydrate, 0.06g calcium iodate, 0.16g cobalt methionine, 10g zinc sulfate monohydrate and 0.0036g sodium selenite. Example 1

[0047] The preparation of the culture medium Lactobacillus plantarum fermentation liquid comprises the following steps: S1: Activate the Lactobacillus plantarum strain, inoculate it into MRS liquid medium, and culture it at 37°C for 18 hours until OD600 ≥ 1.5 to obtain seed solution; S2: Inoculate the seed solution into the optimized MRS medium at a volume ratio of 5%, and perform anaerobically fermentation in a nitrogen-filled fermentation tank at a temperature of 37±0.5℃; In addition to the basic formula, the optimized MRS medium also has the following additional supplements: additional 10 g / L glucose; 1g / L calcium carbonate; S3: During the fermentation process, the pH value was monitored in real time. When the pH was ≤ 4.0, alkali solution (10% NaOH) was automatically added to maintain the pH at 4.5-5.0. The addition of alkali solution was stopped after 24 hours of fermentation. The fermentation was stopped when the total acidity was ≥ 1.5%. The temperature was immediately raised to 85°C and maintained for 15 minutes to inactivate the bacteria. The Lactobacillus plantarum fermentation liquid was obtained. Example 2

[0048] The preparation of artichoke plant lactobacillus fermentation liquid comprises the following steps: S1: Select fresh artichoke leaves and soak them in 0.1% (w / v) hydrogen peroxide solution for 10 minutes to sterilize. Then rinse with plenty of water until the pH is neutral and there is no residual odor of disinfectant.

[0049] S2: Blanch the washed leaves in 90°C hot water for 5 minutes, immediately cool to room temperature with cold or running water, and then add sterile water at least 3 times the weight of the leaves to pulp; obtain pulp.

[0050] S3: The following components are added to the slurry (all calculated based on the initial mass of fresh artichoke leaves): Carbon source: glucose, added at 1% of leaf mass Nitrogen source: yeast extract powder, added at 0.3% of leaf mass Adjust the initial pH to 6.0 (using 1M NaOH or 1M HCl solution) to obtain a culture medium; S4: Inoculate the culture medium with a 5% (v / v) inoculum of activated Lactobacillus plantarum suspension and perform anaerobic fermentation at a temperature of 34-37°C. S5: After 24 hours of fermentation, the total acidity of the Lactobacillus plantarum fermentation liquid was sampled and tested every 6 hours. When the total acidity was ≥1.5%, the fermentation was completed. The fermentation liquid was heated to 70-75°C and kept for 15 minutes for pasteurization to terminate the fermentation. Then centrifugation is performed to separate the solid and liquid, and the supernatant is taken to obtain artichoke Lactobacillus plantarum fermentation liquid. Example 3

[0051] Whiteleg shrimp farming experiment (weight gain) Whiteleg shrimp (Penaeus vannamei) were cultured using an ecological recirculation system. Each pond contained 200 shrimp, with three replicates per feed group. Post-larval shrimp with an average length of 3.0 ± 0.20 cm and an average weight of 0.65 ± 0.04 g were stocked uniformly at a density of 50 shrimp / m³.

[0052] Feed the shrimp daily at 7:00 AM, 12:00 PM, and 6:00 PM. The daily feed intake is approximately 4% of the shrimp's weight, with minimal or no excess. Remove any leftover bait and excrement promptly. The culture cycle is 60 days.

[0053] After feeding the shrimp with 10 groups of feed for 60 days, the growth performance of the shrimp in different treatment groups, such as final weight, weight gain rate, specific growth rate, and feed utilization rate, were measured. The test results are shown in Table 1.

[0054] The feeds for different groups were as follows: Group 1: pure basal feed.

[0055] Group 2: 100 portions of basic feed; 2 portions of Lactobacillus plantarum fermentation broth from Example 1.

[0056] Group 3: 100 portions of basic feed; 2 portions of Lactobacillus plantarum fermentation broth from Example 2.

[0057] Group 4: 100 portions of basic feed; 2 portions of Lactobacillus plantarum fermentation broth from Example 2, and 0.4 portions of passion fruit flavonoids.

[0058] Group 5: 100 portions of basic feed; 2 portions of Lactobacillus plantarum fermentation broth from Example 2, 0.4 portions of passionflower flavonoids, and 0.6 portions of Citrus aurantium immaturum extract (flavonoids 30%).

[0059] Group 6: 100 portions of basic feed; 2 portions of Lactobacillus plantarum fermentation broth from Example 2, 0.4 portions of passionflower flavonoids, and 0.6 portions of Citrus aurantium immaturum extract (flavonoids 50%).

[0060] Group 7: 100 portions of basic feed; 2 portions of Lactobacillus plantarum fermentation broth from Example 2, 0.4 portions of passionflower flavonoids, and 0.6 portions of Citrus aurantium immaturum extract (flavonoids 90%).

[0061] Group 8: 100 portions of basic feed; 3 portions of Lactobacillus plantarum fermentation broth from Example 2, 0.8 portions of passionflower flavonoids, and 1.2 portions of Citrus aurantium immaturum extract (flavonoids 30%).

[0062] Group 9: 100 portions of basic feed; 3 portions of Lactobacillus plantarum fermentation broth from Example 2.

[0063] Group 10: 100 parts of basic feed; 1.2 parts of passionflower flavonoids, 1.8 parts of Citrus aurantium extract (flavonoids 30%).

[0064] Table 1

[0065] In Table 1: Weight gain rate (%) = (final weight - initial weight) / initial weight × 100%; Feed utilization (%) = (final weight - initial weight) / feed intake × 100%; Survival rate (%) = final number of shrimp tails / initial number of shrimp tails × 100%.

[0066] Specific growth rate (%) = (ln final weight - ln initial weight) / number of experimental days × 100%; The initial weight was 0.65±0.04g.

[0067] Example 4 Ammonia nitrogen stress experiment on white shrimp After the breeding experiment, 200 shrimps in the grow-out stage were collected from each group as subjects for the ammonia nitrogen stress experiment.

[0068] Sodium nitrite (NaNO2) was used as the source of nitrite nitrogen, and the nitrite nitrogen concentration was adjusted to 15 mg / L by adding an appropriate amount of NaNO2 (analytical grade) to seawater.

[0069] During the experiment, feed was adjusted based on feeding patterns, and dead individuals were promptly removed and cleaned. Nitrite nitrogen concentrations were adjusted every 12 hours to maintain the desired concentration. The experimental period was 48 hours.

[0070] After the experiment, the shrimp were immersed in 8 CFU / ml Vibrio harveyi (LD50 is 107 CFU / ml) and the mortality rate within 120 hours was analyzed.

[0071] After the stress experiment, 30 shrimps were randomly selected from each group, and blood samples were drawn from the heart using a sterile syringe. The collected blood samples were mixed and centrifuged (4°C, 3500 r / min, 30 min) to fully remove impurities. The supernatant was frozen in liquid nitrogen for later use.

[0072] The detection indicators include glutathione peroxidase (GSH-Px), catalase (CAT), nitric oxide synthase (NOS), pyruvate kinase (PK), and succinate dehydrogenase (SDH).

[0073] At the end of the experiment, hepatopancreas tissue was harvested from an ice tray and placed in an Eppendorf centrifuge tube (30 shrimp were randomly selected and mixed to form one sample). After mincing, a certain amount of tissue was weighed and placed in a homogenization tube. Nine volumes of physiological saline were added and the homogenization was centrifuged in a refrigerated centrifuge at 3000 rpm for 15 minutes at 4°C. The supernatant was collected for analysis. Detection parameters included adenosine deaminase (ADA), aspartate aminotransferase (GOT), and alanine aminotransferase (GPT) activities; the results are shown in Table 2.

[0074] Table 2

[0075] The experimental data in Table 2 show that glutathione peroxidase (GSH-Px) and catalase (CAT) in serum are the core antioxidant enzymes for the body to eliminate reactive oxygen species (ROS), and their activities directly reflect the ability of shrimp to cope with oxidative stress.

[0076] The control group (Group 1) was fed only a basal diet without antioxidant or immune-enhancing ingredients. Under the dual pressures of ammonia nitrogen stress (nitrite nitrogen 15 mg / L) and Vibrio infection, the body produced large amounts of ROS (such as superoxide anions and hydrogen peroxide), but antioxidant enzyme synthesis was insufficient, resulting in significantly lower GSH-Px (348.56 μmol / L) and CAT (26.84 U / mL) activities than those in the other groups. Oxidative damage accumulated, ultimately leading to a 120-hour mortality rate of 68.5%.

[0077] Compound additive group (Groups 5 and 8): Adding Lactobacillus plantarum fermentation broth, passionflower flavonoids and Citrus aurantium extract at the same time, the antioxidant enzyme activity increased significantly. The reason is: Passiflora flavonoids and Citrus aurantium flavonoids (natural polyphenols) can directly activate the expression of antioxidant enzyme genes, promote the synthesis of GSH-Px and CAT, and enhance the efficiency of ROS scavenging; The Lactobacillus plantarum fermentation broth (Example 2, artichoke leaf fermentation) contains metabolites such as organic acids and short-chain fatty acids, which can regulate the balance of intestinal flora, reduce endotoxin production, and indirectly reduce oxidative stress; Group 9 contained only Lactobacillus plantarum fermentation broth and lacked direct antioxidant components of flavonoids. Its GSH-Px and CAT activities were lower than those of groups 5 and 8. Group 10 contained only flavonoids and lacked the fermentation broth's regulation of intestinal flora. Its antioxidant enzyme activity was still weaker than that of the combined group, indicating that a single component cannot completely replace the synergistic effect of the combined group.

[0078] Nitric oxide (NO) produced by nitric oxide synthase (NOS) has a broad-spectrum antibacterial effect and is an important effector molecule of shrimp innate immunity; its activity directly reflects the body's ability to eliminate pathogens (Vibrio harveyi).

[0079] The NOS activity in groups 5 and 8 was significantly increased: flavonoids can stimulate the expression of NOS genes in immune cells (such as blood lymphocytes) and promote NO synthesis; Lactobacillus plantarum fermentation broth enhances the activity of immune cells by regulating intestinal mucosal immunity. The two synergistically enhance antibacterial ability, thus significantly reducing the 120-hour mortality rate.

[0080] The NOS activity in the control group and the single additive group was low: lack of immune activation by flavonoids and immune regulation by fermentation broth, insufficient NO production, weak inhibitory ability against Vibrio, leading to increased mortality.

[0081] Pyruvate kinase (PK) is involved in glycolysis, and succinate dehydrogenase (SDH) is involved in the tricarboxylic acid cycle (aerobic respiration energy supply). The activities of both reflect the energy metabolism efficiency of shrimp under stress.

[0082] Groups 5 and 8 showed higher PK and SDH activity: Ammonia stress can inhibit respiratory chain function, leading to insufficient energy supply. Lactobacillus plantarum fermentation broth contains easily digestible small-molecule nutrients (such as amino acids and oligosaccharides) that improve nutrient absorption. Flavonoids reduce oxidative damage to mitochondria and maintain the activity of energy metabolism enzymes, ensuring efficient energy supply under stress and supporting immune responses and physiological activities.

[0083] The control group and the single additive group had weak energy metabolism: insufficient nutrient absorption and mitochondrial damage, limited energy supply and inability to effectively respond to stress and infection, leading to a decline in physiological functions.

[0084] The hepatopancreas is the core organ of shrimp metabolism and detoxification. Adenosine deaminase (ADA) is involved in purine metabolism (reflecting cellular immune activity), while aspartate aminotransferase (GOT) and alanine aminotransferase (GPT) are markers of liver damage (increased enzyme activity indicates liver cell damage).

[0085] The hepatopancreatic indices of groups 5 and 8 were better: low ADA activity, low GOT and GPT activity. The reasons are: The organic acids in Lactobacillus plantarum fermentation broth can lower intestinal pH, reduce ammonia absorption, and reduce the detoxification burden of the hepatopancreas; The antioxidant effects of flavonoids protect hepatopancreatic cells from oxidative damage, maintain cell membrane integrity, and reduce transaminase release; The compound addition maintains the normal function of the hepatopancreas through the dual effects of "reducing toxicity and protecting cells".

[0086] The hepatopancreas was severely damaged in the control group: the activities of ADA, GOT, and GPT were the highest in Group 1, indicating that ammonia nitrogen accumulation and oxidative damage led to hepatopancreatic dysfunction, further exacerbating the decline in the body's resistance to stress.

[0087] Group 8, which used a higher dosage of additives than Group 5, showed improved performance in various indicators (antioxidant enzyme, NOS, and energy metabolism enzyme activity) and a lower mortality rate, indicating that within the experimental dosage range, appropriately increasing the proportion of the combined additives can enhance the effect. This further validates the synergistic effect of the three ingredients: the fermentation broth improves the intestinal environment and nutrient utilization, while the flavonoids enhance antioxidant and immune activation. The combination of the two forms a positive cycle far superior to the effects of each individual ingredient.

[0088] The above detailed description is a specific description of one feasible embodiment of the present invention. This embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not depart from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A shrimp immune-enhancing fermented functional feed, characterized in that: It includes basic feed and feed additives; the amount of the feed additives added is calculated as a percentage of the mass of the basic feed and includes the following components: Lactobacillus plantarum fermentation liquid 2-3% Passionflower flavonoids 0.4-0.8% Citrus aurantium extract 0.6-1.2%.

2. The immune-enhancing fermented functional feed for shrimp according to claim 1, characterized in that: The Lactobacillus plantarum fermentation liquid is obtained by fermentation with an MRS culture medium, and the total acidity of the Lactobacillus plantarum fermentation liquid is greater than or equal to 1.5%.

3. The immune-enhancing fermented functional feed for shrimp according to claim 2, characterized in that: The Lactobacillus plantarum fermentation liquid is obtained by fermenting artichoke leaves with Lactobacillus plantarum.

4. The immune-enhancing fermented functional feed for shrimp according to claim 3, characterized in that: The preparation of the plant lactobacillus fermented liquid comprises the following steps: S1: Select fresh artichoke leaves, soak them in sterilizing solution and then wash them with water until they are neutral; S2: Blanch the washed leaves in hot water, cool them down, and then add at least 3 times the weight of water to make pulp to obtain pulp; S3: Add the following components to the slurry and adjust the initial pH to 6.0-6.5 to obtain a culture medium; At least 1% of the leaf mass as a carbon source; A nitrogen source of at least 0.3-0.5% of the leaf mass; S4: inoculating activated Lactobacillus plantarum into the culture medium and performing anaerobic fermentation at a temperature of 34-37°C; S5: After the fermentation is completed, sterilize and terminate the fermentation, separate the solid and liquid, and take the liquid part. Test the total acidity of the Lactobacillus plantarum fermentation liquid. If the total acidity is ≥1.5%, it is the Lactobacillus plantarum fermentation liquid.

5. The immune-enhancing fermented functional feed for shrimp according to claim 1, characterized in that: The Citrus aurantium extract is obtained by water extraction of dried young fruits of Citrus aurantium L. and its cultivated varieties of the Rutaceae family.

6. The immune-enhancing fermented functional feed for shrimp according to claim 5, characterized in that: The content of citrus aurantium flavonoids in the citrus aurantium extract is ≥50%.

7. The method for preparing the immune-enhancing fermented functional feed for shrimp according to any one of claims 1 to 6, characterized in that: Mix the feed additive with the basic feed evenly.

8. The use of the shrimp immune-enhancing fermented functional feed according to any one of claims 1 to 6, characterized in that: The immune-enhancing fermented functional feed for shrimp is feed for shrimp farming after the post-larval stage.

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