Feed additive for improving antibacterial ability of haliotis diversicolor as well as preparation method and application of feed additive

Abstract

The invention provides a feed additive capable of improving the antibacterial ability of haliotis diversicolor and a preparation method and application thereof.The preparation method of the feed additive comprises the steps that an ulva lactuca raw material is properly treated, polysaccharide components in the ulva lactuca raw material are extracted, and the obtained ulva lactuca polysaccharide is scientifically proportioned and mixed with a proper feed carrier and other nutritional ingredients; after the feed additive is fed, the ulva polysaccharide can play a role in the abalone body, enhance the non-specific and specific immune response of the abalone and improve the removal capacity of vibrio alginolyticus, so that diseases caused by pathogenic bacteria are effectively prevented and controlled, and the morbidity and mortality in the abalone culture process are reduced. By using the feed additive disclosed by the invention, the health condition and the growth performance of abalones can be improved, the use amount of antibiotics is reduced, the culture risk and the environmental pollution are reduced, the quality and the safety of abalone products can also be improved, and the feed additive has important practical significance and popularization value for promoting the sustainable development of the abalone culture industry.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of feed additives, in particular to a feed additive for improving the antibacterial ability of abalone and a preparation method and application thereof. BACKGROUND

[0002] Abalone, especially green abalone (Haliotis diversicolor Reeve) Haliotis discus hannai ♀ x H. fulgens ♂ , is a kind of marine mollusk with high economic and nutritional value. Its aquaculture industry occupies an important position in the world, especially in Asian countries, and has become an important pillar industry for the economic development of coastal areas and the income increase of fishermen. Green abalone is one of the main breeding varieties due to its fast growth rate and delicious meat. However, with the popularization of intensive and high-density breeding mode, although the yield is improved, it also brings a series of severe challenges. Among them, the disease problem is one of the main bottlenecks restricting the sustainable and healthy development of green abalone breeding industry. The invasion of various pathogens leads to high morbidity and mortality of green abalone, causing huge economic losses and risks to breeders.

[0003] Among the various pathogenic bacteria that threaten the health of green abalone, Vibrio alginolyticus Vibrio alginolyticus is a conditional pathogen widely existing in seawater environment, which has significant pathogenicity to a variety of aquatic animals including green abalone. This bacterium can cause a variety of diseases in green abalone, including but not limited to digestive system lesions, soft tissue ulceration, shell margin erosion, etc. These symptoms often manifest as reduced feeding, slow growth, and in severe cases, can lead to mass mortality of green abalone in a short period of time, even destroying the entire breeding batch. The strong reproductive capacity and wide distribution of Vibrio alginolyticus in the environment make the green abalone breeding industry face the threat of its infection for a long time. Especially in high-density breeding environment, the stress level of green abalone increases, and the immune function decreases, making it more susceptible to Vibrio alginolyticus infection, leading to the outbreak and prevalence of diseases, which poses a serious threat to the breeding of green abalone.

[0004] For a long time, the main means to deal with Vibrio disease of green abalone is to use antibiotics and other antibacterial drugs for prevention and treatment. When the disease breaks out, breeders often add antibiotics or sprinkle drugs in the feed to control the spread of the disease. However, over-reliance on antibiotics has brought many drawbacks. First, bacteria, including Vibrio alginolyticus that causes disease in green abalone, are increasingly resistant to commonly used antibiotics, leading to a gradual decrease in the efficacy of the drugs, and even failure, making disease control more and more difficult. Second, the problem of antibiotic residues in green abalone has attracted much attention. These residues may enter the human body through the food chain, posing potential risks to consumers' health, and also causing strict regulation of drug residues in aquatic products by domestic and foreign markets, limiting the export and sale of green abalone products. Third, the large-scale use of antibiotics into the water body will pollute the breeding environment, destroy the balance of water micro-ecosystem, and possibly affect the health of other non-target organisms.

[0005] Given the limitations and potential hazards of traditional antibiotic-based disease control methods, developing safe, environmentally friendly, and efficient new disease control strategies has become a research hotspot and important development direction in the current abalone farming industry. Among these, enhancing the abalone's own immunity or disease resistance to resist pathogen invasion is a more sustainable and healthy approach. Finding natural substances with immunomodulatory or antibacterial activities and applying them as feed additives in abalone farming is considered an important way to achieve this goal.

[0006] Therefore, the purpose of this invention is to solve the problems of existing green abalone ( Haliotis discus hannai ♀ x H. fulgens ♂ During the cultivation process, Vibrio alginolyticus ( Vibrio alginolyticus The diseases caused by antibiotics are becoming increasingly serious, and traditional prevention and control methods, such as the use of antibiotics, have prominent drawbacks such as drug resistance, drug residues and environmental pollution. Therefore, we provide a natural, safe, efficient and environmentally friendly solution. Summary of the Invention

[0007] To address the aforementioned problems, this invention aims to provide a feed additive that can significantly improve the antibacterial ability of green abalone, especially its effective resistance to Vibrio alginolyticus invasion, as well as its simple and feasible preparation method and application.

[0008] To achieve the above-mentioned technical objectives, the technical solution adopted by this invention is as follows: In a first aspect, the present invention provides a simple and feasible method for preparing Ulva polysaccharide, comprising the following steps: S1: Dissolve Ulva lactuca powder in distilled water at a ratio of 1:40; S2: Extraction was performed at 60℃ with ultrasonic assistance at 45 kHz and 200 W power for 1 h, followed by filtration. This process was repeated twice. S3: Centrifuge to collect the supernatant, add ethanol, and precipitate overnight; S4: Centrifuge to collect the precipitate and freeze-dry it. Dissolve the freeze-dried product in water and heat at 60°C until completely dissolved. S5: After dissolving, add 400×V / 50000 g of neutral protease (stored at room temperature), where V represents the solution volume; enzymatically hydrolyze in a 55℃ water bath for 2 h, inactivate the enzyme in a 100℃ water bath for 10 min, then centrifuge to discard the precipitate and take the supernatant. S6: Dialyze for 48 h (8k-14k Da), concentrate and freeze-dry to obtain crude sugar, then prepare a 1% polysaccharide solution, add 2.7 mL of concentrated sulfuric acid per liter of solution, and acid hydrolyze in a 100℃ water bath for 1.5 h; after cooling, centrifuge to remove impurities, then add 1.0 mol / L NaOH to adjust to neutral; S7: Concentrate and precipitate with alcohol for more than 8 hours, take the supernatant, and freeze-dry to obtain small molecule Ulva polysaccharide.

[0009] Further, the volume ratio of the supernatant to the ethanol in step S3 is 1:4, and the concentration of the ethanol is 95%.

[0010] Further, the mass ratio of the freeze-drying to the water in step S4 is 1:100; and the solution obtained in step S6 is mixed with ethanol in a volume ratio of 1:1 in step S7, and alcohol precipitation is performed for more than 8 hours.

[0011] In a second aspect, the present application provides a green abalone antibacterial feed additive containing ulvich polysaccharide, which comprises any one of the ulvich polysaccharide and the feed component, and the proportion of the ulvich polysaccharide in the feed additive is 40%. The feed component comprises fish meal, soybean protein concentrate, soybean meal, flour, high-gluten flour, kelp powder, shell powder, calcium dihydrogen phosphate, choline chloride, fish oil, soybean oil, multi-vitamin premix, and multi-mineral premix.

[0012] Further, the multi-vitamin premix comprises vitamin A, 250000 IU; riboflavin, 750 mg; vitamin B6, 400 mg; vitamin B12, 1 mg; vitamin B1, 250 mg; vitamin K3, 250 mg; folic acid, 125 mg; biotin, 10 mg; a-tocopherol, 2.5 g; myo-inositol, 8000 mg; calcium pantothenate, 1250 mg; nicotinic acid, 2000 mg; choline chloride, 8000 mg; vitamin D3, 45000 IU; vitamin C, 7000 mg. The multi-mineral premix comprises ZnSO4·7H2O, 0.04 g; CaCO3, 37.9 g; KCl, 5.3 g; KI, 0.04 g; NaCl, 2.6 g; CuSO4·5H2O, 0.02 g; CoSO4·7H2O, 0.02 g; FeSO4·7H2O, 0.9 g; MnSO4·H2O, 0.03 g; MgSO4·7H2O, 3.5 g; Ca(HPO4)2·2H2O, 9.8 g.

[0013] Further, the ulvich polysaccharide is a crude polysaccharide or a partially purified polysaccharide, which is obtained by pretreating fresh or dried ulvich raw materials such as washing, drying, and crushing, and then extracting and obtaining the ulvich polysaccharide product from the ulvich by using appropriate extraction and purification processes. The extraction process can use one or a combination of hot water extraction, enzymatic hydrolysis, and acid hydrolysis, and subsequent steps such as alcohol precipitation, dialysis, concentration, and drying to improve the purity and activity of the polysaccharide.

[0014] Preferably, the ulvich polysaccharide is prepared by the above-mentioned method for preparing ulvich polysaccharide.

[0015] In a third aspect, the present application also provides application of the Ulva polysaccharide feed additive in the preparation of a feed for improving the antibacterial ability of Haliotis discus hannai.

[0016] Further, the antibacterial ability includes the ability to resist Vibrio alginolyticus.

[0017] In a fourth aspect, the present application provides a feed for improving the antibacterial ability of Haliotis discus hannai, and the raw material composition of the feed is shown in Table 1. Table 1: Experimental feed formula

[0018] The multi-vitamin premix comprises vitamin A, 250000IU; riboflavin, 750mg; vitamin B6, 400mg; vitamin B12, 1mg; vitamin B1, 250mg; vitamin K3, 250mg; folic acid, 125mg; biotin, 10mg; a-tocopherol, 2.5g; myo-inositol, 8000mg; calcium pantothenate, 1250mg; nicotinic acid, 2000mg; choline chloride, 8000mg; vitamin D3, 45000IU; vitamin C, 7000mg; The multi-mineral premix comprises ZnSO4·7H2O, 0.04 g; CaCO3, 37.9 g; KCl, 5.3 g; KI, 0.04 g; NaCl, 2.6 g; CuSO4·5H2O, 0.02 g; CoSO4·7H2O, 0.02 g; FeSO4·7H2O, 0.9 g; MnSO4·H2O, 0.03 g; MgSO4·7H2O, 3.5 g; Ca(HPO4)2·2H2O, 9.8 g; After the components in the feed are uniformly mixed, further post-processing such as granulation, drying, and coating can be performed as needed to obtain a tablet-shaped feed that is convenient to store and feed.

[0019] Compared with the prior art, the present application has the following remarkable beneficial effects: 1) effectively improving the antibacterial ability of Haliotis discus hannai against Vibrio alginolyticus. The core of the present application is to apply Ulva polysaccharide to improve the antibacterial performance of Haliotis discus hannai. After being ingested and absorbed by the abalone as a feed additive, the Ulva polysaccharide can systematically regulate the physiological and immune state of the abalone. Through research, it is found that feeding the Ulva polysaccharide feed additive of the present application can significantly improve the activity of key digestive enzymes and immune-related enzymes in the hepatopancreas of Haliotis discus hannai. Further tests against Vibrio alginolyticus have shown that feeding the Ulva polysaccharide feed additive of the present application can significantly reduce the morbidity and mortality of Haliotis discus hannai after infection with Vibrio alginolyticus, and improve its survival rate, indicating that it has a clear and efficient prevention and control effect on Vibrio alginolyticus.

[0020] 2) Improve the digestive system health and physiological state of green abalone. Before the attack, the tissue section observation of the liver and intestine of green abalone shows that feeding the additive of the application helps to maintain the normal tissue structure of the liver and intestine and reduce pathological damage. The oil red O staining results show that adding ulvan polysaccharide can regulate the lipid metabolism of the liver and improve the health of the liver. These improvements in physiology and tissue structure provide a better physiological basis for the abalone to resist pathogenic bacteria invasion.

[0021] 3) The preparation process is advanced, which ensures efficient use of active ingredients and stable product performance. The application adopts a multi-step combined extraction and purification process of ultrasonic-assisted extraction, enzymatic hydrolysis, acid hydrolysis, and dialysis, aiming to improve the extraction rate (target > 60%) and purity of specific active polysaccharide components in ulva, and obtain small molecule ulva polysaccharide which is easy for abalone to absorb and utilize and has stronger biological activity. The 40% purity ulva polysaccharide obtained is scientifically proportioned and uniformly mixed with the basic feed raw materials (see Table 1), and is made into sheet feed which is easy for abalone to eat. The whole process optimization from raw material treatment, polysaccharide fine extraction to scientific preparation of feed additive maximizes the biological activity of ulva polysaccharide and its effective delivery in abalone, thereby systematically enhancing the antibacterial ability and overall health level of green abalone.

[0022] 4) The application provides a natural, safe and environmentally friendly disease prevention and control means. Ulva polysaccharide is derived from natural seaweed, has good biocompatibility and safety, has no toxic side effects, is not easy to produce drug residues in abalone, and meets the current consumer pursuit of food safety and health. Compared with chemically synthesized antibiotics, the additive of the application is friendly to the cultivation environment and will not cause water pollution and ecological damage, which meets the concept of green and sustainable cultivation.

[0023] 5) The application helps to reduce the risk and economic loss of cultivation. By effectively controlling vibrio alginolyticus disease, improving the disease resistance and survival rate of abalone, and reducing the death and growth stagnation caused by disease, the additive of the application can significantly reduce the risk and economic loss in the cultivation process, improve the yield and benefit of cultivation.

[0024] 6) The preparation process of the application is mature and feasible, and is easy to realize industrial production. The extraction of ulva polysaccharide and the preparation method of the feed additive are relatively simple and controllable in cost, which is conducive to the scale application and promotion of the product.

[0025] In summary, the ulva polysaccharide-containing feed additive and its preparation method of the application enhance the antibacterial ability and overall health level of green abalone through multiple pathways, providing a new strategy for green, efficient and sustainable prevention and control of vibrio alginolyticus disease, which provides strong support for improving the health level and economic benefit of green abalone cultivation. The application makes full use of the biological activity of marine natural products, which meets the trend of developing the aquaculture industry towards ecology and health. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The effects of adding Ulva polysaccharide on liver and intestinal endoplasmic reticulum stress and inflammation in Abalone rotundus, and on lipid accumulation in the liver and pancreas of Abalone rotundus.

[0028] in, Figure 1 A represents H&E staining of the liver; Figure 1 B represents intestinal H&E staining; Figure 1 C is an Oil Red O stained section of liver and pancreas tissue; Figure 1 D represents visualization data of hepatopancreatic vacuolation; Figure 1 E represents the quantitative analysis of the relative intensity of Oil Red O staining in hepatopancreatic sections. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be particularly noted that the following embodiments are for illustrative purposes only and do not limit the scope of the invention. Similarly, the following embodiments are only some, not all, embodiments of the present invention, and all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] This invention relates to a feed additive containing Ulva prolifera polysaccharide to enhance the immunity of abalone and its preparation method, including the preparation of Ulva prolifera polysaccharide and the mixing of Ulva prolifera polysaccharide with other feed components. The preparation method of Ulva prolifera polysaccharide can employ various methods known in the art, such as hot water extraction, enzymatic hydrolysis, and acid hydrolysis, followed by purification steps to obtain Ulva prolifera polysaccharide products of different purities. The obtained Ulva prolifera polysaccharide can be mixed with a feed carrier to prepare a premixed additive, or directly added to abalone basal feed in a certain proportion.

[0031] Specifically, the preparation method of Ulva polysaccharide in the following examples is as follows: S1: Dissolve Ulva lactuca powder in distilled water at a ratio of 1:40; S2: Extraction was performed at 60℃ with ultrasonic assistance at 45 kHz and 200 W power for 1 h, followed by filtration. This process was repeated twice. S3: Centrifuge and collect the supernatant, then add 95% ethanol at a ratio of 1:4 and precipitate overnight. S4: Centrifuge to collect the precipitate and freeze-dry it. Dissolve the freeze-dried product in water at a ratio of 1:100 and heat at 60°C until completely dissolved. S5: After dissolving, add 400×V / 50000 g of neutral protease (stored at room temperature) (V represents the solution volume); enzymatically hydrolyze in a 55℃ water bath for 2 h, inactivate the enzyme in a 100℃ water bath for 10 min, centrifuge to discard the precipitate and take the supernatant. S6: Dialyze for 48 h (8k-14k Da), concentrate and freeze-dry to obtain crude sugar, then prepare a 1% polysaccharide solution, add 2.7 mL of concentrated sulfuric acid per liter of solution, and acid hydrolyze in a 100℃ water bath for 1.5 h; after cooling, centrifuge to remove impurities, then add 1.0 mol / L NaOH to adjust to neutral; S7: Mix the solution obtained in step S6 with ethanol at a volume ratio of 1:1, concentrate and precipitate for more than 8 hours, take the supernatant, and freeze-dry to obtain small molecule Ulva polysaccharide.

[0032] Effect of feed containing Ulva polysaccharide on the antibacterial ability of Abalone rotundifolia This embodiment aims to evaluate, through a 60-day sea feeding trial, the effects of different levels of the Ulva polysaccharide-containing feed of this invention on the growth performance and health status of green abalone, as well as its subsequent effects on Vibrio alginolyticus (Villus alginolyticus). Vibrio alginolyticus The impact on resistance.

[0033] 1. Experimental animals and breeding environment Select green abalone that are healthy and relatively uniform in size, purchased from specific aquaculture farms. Haliotis discus hannai ♀ x H. fulgens ♂ Abalone were used as experimental animals. Prior to the experiment, the abalone underwent a period of acclimatization rearing on marine aquaculture rafts. The initial average weight of the abalone used in the formal experiment was 34.48 ± 0.5 g.

[0034] The experiment was conducted in a marine aquaculture area using standard abalone cages fixed to a raft. The culture environment was natural seawater conditions. Environmental parameters (such as water temperature, pH, and total ammonia nitrogen) were monitored and recorded during the experiment and controlled within the suitable range for the growth of green abalone. Specifically, the water temperature was controlled at 18-22℃, pH at 7-8, and total ammonia nitrogen concentration below 0.4 mg / L. The dissolved oxygen level in the culture area generally met the requirements of the abalone.

[0035] 2. Preparation of experimental feed A basic abalone diet was prepared as a control group diet (0% addition of Ulva prostrata polysaccharide). The basic diet formula is shown in the table below.

[0036] Experimental feed formulation

[0037] Multivitamin premix contains vitamin A, 250000 IU; riboflavin, 750 mg; vitamin B6, 400 mg; vitamin B12, 1 mg; vitamin B1, 250 mg; vitamin K3, 250 mg; folic acid, 125 mg; biotin, 10 mg; a-tocopherol, 2.5 g; myo-inositol, 8000 mg; calcium pantothenate, 1250 mg; niacin, 2000 mg; choline chloride, 8000 mg; vitamin D3, 45000 IU; vitamin C, 7000 mg; Multimineral premix contains ZnSO4·7H2O, 0.04 g; CaCO3, 37.9 g; KCl, 5.3 g; KI, 0.04 g; NaCl, 2.6 g; CuSO4·5H2O, 0.02 g; CoSO4·7H2O, 0.02 g; FeSO4·7H2O, 0.9 g; MnSO4·H2O, 0.03 g; MgSO4·7H2O, 3.5 g; Ca(HPO4)2·2H2O, 9.8 g.

[0038] According to the technical scheme of the present application, test feed containing different addition levels of Ulva polysaccharide is prepared. In this example, four test groups are set. The pre-prepared Ulva polysaccharide is accurately weighed according to the proportion of the total weight of the basic feed, and then added to the basic feed raw materials. The Ulva polysaccharide addition levels of the test groups (calculated based on the dry weight of the feed) are 0.25%, 0.5%, 0.75% and 1.0% respectively. After the Ulva polysaccharide is fully mixed with other feed raw materials, the feed is processed into sheet-shaped feed by a feed processing equipment, dried to a specified moisture content, sealed and packaged, and stored in a cool and dry place.

[0039] The test grouping design is as follows: Control group (Control Group): fed with basic feed with 0% addition of Ulva polysaccharide.

[0040] Test group 1 (EP-0.25%): fed with feed with 0.25% addition of Ulva polysaccharide.

[0041] Test group 2 (EP-0.5%): fed with feed with 0.50% addition of Ulva polysaccharide.

[0042] Test group 3 (EP-0.75%): fed with feed with 0.75% addition of Ulva polysaccharide.

[0043] Test group 4 (EP-1.0%): fed with feed with 1.00% addition of Ulva polysaccharide.

[0044] Three parallel repeats are set for each treatment group, and 14 abalone are raised in each repeat. The total number of abalone used in the test is 5 groups x 14 abalones / group x 3 = 210 abalones.

[0045] 3. Feeding management The feeding trial lasted for 60 days. During the trial, abalones in each group were managed in separate rearing units. The feeding frequency was once every four days, and the feeding time was fixed at 15:30-16:30 pm. The amount of feed fed each time was adjusted according to the feeding behavior of abalones, aiming to meet their feeding needs and minimize leftover feed. The rearing units were regularly inspected to clean up leftover feed and excrement, ensuring a clean rearing environment. The activity status, feeding behavior, and any abnormal behavior of abalones were observed and recorded. The amount of feed fed in each group was recorded.

[0046] 4. Sample collection and initial measurement at the end of the feeding trial After the 60-day feeding trial, 12 green abalones were randomly and accurately sampled from each parallel repetition in each group (including the control group and the four experimental groups) for the measurement of various indicators at the end of the trial.

[0047] The sampled abalones were subjected to the following weighing and tissue collection: Individual total weight: The total weight of each abalone with shell was weighed.

[0048] Whole meat weight: The abalone soft body was completely peeled off from the shell, and the wet weight of the soft body (i.e., whole meat weight) was weighed.

[0049] By comparing with the initial body weight, the growth performance indicators such as weight gain rate and specific growth rate were calculated.

[0050] Liver weight: The liver tissue was carefully peeled off, and its wet weight was weighed. The liver-somatic index (the proportion of liver weight to whole meat weight) was calculated.

[0051] Tissue collection: Blood samples were collected (e.g., through foot vein or heart blood collection). Liver and intestinal tissues were collected. Part of the liver and intestinal tissues were fixed with 4% paraformaldehyde solution for subsequent histological sectioning and staining (including H&E staining and oil red O staining of the liver). The foot muscle samples collected were used for subsequent meat quality indicator determination. The remaining liver and intestinal tissues and blood samples were quickly aliquoted and then frozen in liquid nitrogen before being transferred to a -80°C refrigerator for storage, for subsequent determination of liver enzyme activity, qPCR, and blood biochemical indicators.

[0052] At the same time, detailed analysis was conducted on the collected samples, including: Histopathological observation and oil red O staining: H&E staining was performed on the fixed and embedded liver and intestinal tissue sections to observe the cell morphological structure and whether there were inflammation or pathological damage. Oil red O staining was performed on the liver frozen sections to evaluate the degree of liver lipid deposition.

[0053] Liver pancreas, serum enzyme activity determination: The activity of various enzymes in liver pancreas homogenate or serum was determined, including digestive enzymes (a-amylase, endo-β-1, 4-glucanase cellulase, trypsin), antioxidant enzymes (CAT, GSH-Px, T-AOC, MDA), and immune-related enzymes (ACP, LZM, POO, LPS).

[0054] Routine nutritional ingredients: The crude protein content (Kjeldahl nitrogen determination method) and crude fat content (Soxhlet extraction method) of the muscle sample were determined according to national standard or industry standard methods.

[0055] Amino acid content: The muscle sample was subjected to acid hydrolysis to release the amino acids in the protein, and then an automatic amino acid analyzer was used to determine the content of various amino acids (including essential and non-essential amino acids), and the total amino acid content was calculated.

[0056] 5. Vibrio alginolyticus challenge test and post-challenge observation After the end of the feeding test period, the abalones used for the challenge test were carefully transferred from the sea farming unit to independent water buckets, with each container corresponding to one parallel replicate of each treatment group, and ensuring that the water quality conditions were maintained within the suitable range for H. discus (water temperature 18-22℃, salinity 28-32‰, pH 7.8-8.3, dissolved oxygen >6 mg / L, ammonia nitrogen <0.3 mg / L).

[0057] Vibrio alginolyticus strain (e.g., pathogenic strain isolated from sick H. discus) was cultured in suitable 2216E medium at 28℃ to the logarithmic growth phase. The bacterial cells were collected by centrifugation, washed with sterile seawater or physiological saline and resuspended, and the concentration of the bacterial solution was adjusted to the preset challenge dose by measuring the optical density value (OD 600 ) and combining the previously prepared standard curve.

[0058] Each abalone was artificially infected and challenged by intraperitoneal injection. The injection dose was set at 0.07 mL of Vibrio alginolyticus bacterial solution per abalone, with a concentration of 1.05 x 10 8 cfu·mL -1 The injection operation was designed to minimize stress and damage to the abalones.

[0059] After the challenge treatment, the abalones were returned to their respective test containers for further feeding. The health status, activity, and symptoms of each group of abalones were closely observed, such as decreased vitality, soft body atrophy, and body surface ulceration.

[0060] Sample collection at 24 hours post-challenge: At 24 hours post-challenge, 12 samples were randomly collected from each replicate in each group. These samples were used for early post-challenge response analysis, including but not limited to: blood sample collection for serum immune parameter determination; hepatopancreas and intestinal tissue collection for immune-related enzyme activity detection and RNA extraction for immune-related gene qPCR analysis.

[0061] Survival observation post-challenge: The remaining abalones were continued to be raised under the same conditions for a 7-day survival experiment. All test containers were checked at regular intervals every day, and the number of deaths in each group was recorded. The dead abalones were removed in time to avoid polluting the water quality. The observation was recorded until the end of the experiment on the 7th day after the challenge.

[0062] 6. Post-challenge parameter determination and analysis The samples collected at different time points after the challenge and the survival data at the end of the challenge period were used to determine and analyze the following parameters: Cumulative mortality rate: Based on the number of deaths recorded each day during the challenge period (7 days), the cumulative mortality rate of each group of abalones (the percentage of the number of deaths to the initial number of challenges) was calculated. This is a key indicator for evaluating the effect of feed additives against Vibrio alginolyticus.

[0063] Enzyme activity parameter determination: biochemical and immune parameters (LZM, ACP, AKP, PO) in plasma, biochemical and immune parameters (T-SOD, CAT, GSH-Px, T-AOC) in hepatopancreas, oxidative damage parameters (MDA), and digestive enzyme parameters (AMY, CEL, LPS, TRY) were determined.

[0064] 7. Data processing and statistical analysis All collected growth performance data, enzyme activity data, gene expression data, pathological data, etc. were sorted out. Suitable statistical software was used for statistical analysis of the data in each group, such as one-way ANOVA, to determine whether there were significant differences between different treatment groups. Different letters in the same row of the table indicate significant differences (P < 0.05), and the same letter indicates no significant difference (P > 0.05). The statistical results were clearly displayed in the form of charts.

[0065] Table 2 shows the effect of Ulva polysaccharide supplementation on the survival rate of H. discus before and after the challenge. Before the challenge, the number of each group was 42, and the survival rate of the control group was the lowest, while the highest was 97.67% with a 0.75% addition. After the challenge, the number of each group was 24, and the survival rate of the control group was the lowest, while the survival rate of the 0.50% and 0.75% addition groups was 100%. The preliminary analysis of the survival rate after the challenge being higher than that before the challenge in some groups may be due to the difference in stress resistance of the same batch of abalones, and the number of abalones after the challenge was much less than that before the challenge.

[0066] Table 2 Survival rate of abalone

[0067] Table 3 shows the effect of adding ulva polysaccharide on the growth performance of green abalone. At the end of the feeding test, the final body weight (FBW), weight gain rate (WGR), specific growth rate (SGR), and feed efficiency (FE) of the 0.50% and 0.75% addition groups were significantly different from those of the control group, and the hepatosomatic index (HSI) of the 0.75% addition group was also significantly different from that of the control group.

[0068] Table 3 Physiological indicators of abalone

[0069] Table 4 shows the effect of adding ulva polysaccharide on the serum and hepatopancreas biochemical indicators of green abalone. In terms of immune function-related enzyme activity, the lysozyme (LZM) activity was best at 0.25% addition, which was 18.32% higher than that of the control group; the acid phosphatase (ACP) activity was best at 0.75% addition, which was 51.68% higher than that of the control group; the alkaline phosphatase (AKP) activity was best at 1.00% addition, which was 12.60% higher than that of the control group, but there was no statistically significant difference; and the phenol oxidase (PO) activity was best at 0.50% addition, which was 65.14% higher than that of the control group. In terms of antioxidant system function, the superoxide dismutase (SOD) activity was best at 0.25% addition, which was 16.35% higher than that of the control group; the catalase (CAT) activity was best at 0.25% addition, which was 98.24% higher than that of the control group; the glutathione peroxidase (GSH-Px) activity was best at 0.50% addition, which was 88.92% higher than that of the control group; the total antioxidant capacity (T-AOC) was best at 0.75% addition, which was 173.61% higher than that of the control group (0.50% addition also showed excellent performance, with an increase of 154.17%, both of which were statistically optimal); and the malondialdehyde (MDA) content (the lower the better) was best at 0.25% addition, which was 40.52% lower than that of the control group. In terms of digestive enzyme activity, the amylase (AMY) activity was best at 0.25% addition, which was 27.59% higher than that of the control group; the cellulase (CEL) activity was best in the control group, and none of the addition groups showed an improvement; the lipase (LPS) activity was best at 0.50% addition, which was 50.74% higher than that of the control group; and the trypsin (TRY) activity was best in the control group, and none of the addition groups showed a statistically significant improvement.

[0070] Table 4 Enzyme activity indicators of abalone

[0071] Table 5 is the effect of supplementing ulva polysaccharide on the serum and hepatopancreas biochemical indicators of green abalone after challenge with vibrio alginolyticus. It can be seen that, in the immune-related enzyme activity, the activity of alkaline phosphatase (AKP) is best at 0.50% addition, which is increased by 243.26% compared with the control group; in the immune function-related enzyme activity, the activity of lysozyme (LZM) is best at 0.50% addition, which is increased by 46.35% compared with the control group; in the antioxidant system function, the content of malondialdehyde (MDA) (the lower the better) is best at 0.75% addition, which is reduced by 41.90% compared with the control group (the addition of 1.00% also performs very close, with a decrease of 41.07%, both of which are the best groups in statistics).

[0072] Table 5 Enzyme activity index of abalone after challenge

[0073] Figure 1 The effect of adding ulva polysaccharide on the endoplasmic reticulum stress and inflammation in the liver and intestine of green abalone and the effect on the lipid accumulation in the hepatopancreas of green abalone. Figure 1 A is the H&E staining of liver, Figure 1 B is the H&E staining of intestine. By observing the H&E staining of liver, it can be clearly seen that, compared with the control group, the overall basophilic cells in the ulva group are reduced, that is, the inflammatory response is reduced, the number of refractive globules is reduced, that is, the metabolic abnormalities / cell damage is reduced, and the vacuolization is significantly reduced, that is, the cell damage is reduced. By observing the H&E staining of intestine, it can be clearly seen that, compared with the control group, the overall vacuolization in the ulva group is significantly reduced, that is, the cell damage is reduced. For details, see Figure 1 D is the visualization data of vacuolization, the increase of intestinal villus height and width increases the contact area between the intestine and the real object to promote nutrient absorption, the increase of intestinal muscle layer thickness provides better support and protection for the intestine, helps to maintain the integrity of the intestine, and promotes digestion and nutrient absorption. Figure 1 C is the oil red O staining section of hepatopancreas tissue, Figure 1 E is the quantitative analysis of oil red O staining relative intensity in hepatopancreas section. The study shows that, by observing the oil red O staining section of hepatopancreas Figure 1 A, a large number of dense red lipid droplets can be seen in the hepatopancreas cells of the control group (Control group), indicating that the lipid accumulation is more serious; after treatment of ulva polysaccharide S2 group and S3 group, the number and area of red lipid droplets in cells are significantly reduced, and the color is obviously lighter. Combined with the quantitative analysis of oil red O staining relative intensity Figure 1 E, it is found that the addition of ulva polysaccharide has a significant effect on the lipid content of the hepatopancreas of green abalone.

[0074] Table 6 is the influence of supplementing adding ulva polysaccharide on the body composition of green abalone, based on the previous study, the optimal group S2 is determined to ensure the realization of precise nutrition regulation. It can be seen that compared with the control group, the moisture of the experimental group abalone is increased by 2.83%, the ash is reduced by 9.35%, the crude fat is increased by 46.25%, the crude protein is increased by 28.20%, the free fatty acid is reduced by 21.86%, and the amino acid is increased by 1.58%. Combined with the above data, the significant increase of crude protein and crude fat can effectively improve the nutritional value, the decrease of free fatty acid further improves the flavor and quality of abalone, and the slight increase or decrease of the remaining indicators makes the abalone meat better in taste and retains the original flavor.

[0075] Table 6 abalone body composition index

[0076] In summary, the present application extracts polysaccharide components from the raw material of ulva by proper treatment, and scientifically matches and mixes the obtained ulva polysaccharide with suitable feed carriers and other nutritional components, to ensure the uniform distribution and stability of active ingredients in the feed, avoid the degradation or unevenness of effective components leading to different effects, and form a feed additive product that is easy for abalone to eat and absorb. Considering the feeding habit of abalone during preparation, the mixture is finally made into a sheet-shaped feed that is easy for abalone to eat and absorb. After feeding the feed additive, ulva polysaccharide can play a role in the body of abalone, enhance its non-specific and specific immune response, and improve its ability to eliminate vibrio alginolyticus, thereby effectively preventing and controlling diseases caused by the pathogenic bacteria, reducing the morbidity and mortality during abalone breeding. The use of the feed additive of the present application not only helps to improve the health status and growth performance of abalone, reduces the amount of antibiotics used, reduces the risk of breeding and environmental pollution, but also improves the quality and safety of abalone products, and has important practical significance and popularization value for promoting the sustainable development of abalone breeding industry.

[0077] The above only describes some embodiments of the present application, and does not limit the protection scope of the present application, and any equivalent device or equivalent process transformation based on the content of the specification and drawings, or direct or indirect application in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A method for preparing Ulva polysaccharide, characterized in that, Includes the following steps: S1: Dissolve Ulva lactuca powder in distilled water at a ratio of 1:40; S2: Extraction was performed at 60℃ with ultrasonic assistance at 45 kHz and 200 W power for 1 h, followed by filtration. This process was repeated twice. S3: Centrifuge to collect the supernatant, add ethanol, and precipitate overnight; S4: Centrifuge to collect the precipitate and freeze-dry it. Dissolve the freeze-dried product in water and heat at 60°C until completely dissolved. S5: After dissolving, add 400×V / 50000 g of neutral protease, where V represents the solution volume; hydrolyze in a 55℃ water bath for 2 h, inactivate the enzyme in a 100℃ water bath for 10 min, centrifuge to discard the precipitate and take the supernatant. S6: Dialyze 8k-14kDa for 48 h, concentrate and freeze-dry to obtain crude sugar, then prepare a 1% polysaccharide solution, add 2.7 mL of concentrated sulfuric acid per liter of solution, and acid hydrolyze in a 100℃ water bath for 1.5 h; after cooling, centrifuge to remove impurities, then add 1.0 mol / L NaOH to adjust to neutral; S7: Concentrate and precipitate with alcohol for more than 8 hours, take the supernatant, and freeze-dry to obtain small molecule Ulva polysaccharide.

2. The method for preparing Ulva polysaccharide according to claim 1, characterized in that, In step S3, the volume ratio of the supernatant to ethanol is 1:4, and the concentration of the ethanol is 95%.

3. The method for preparing Ulva polysaccharide according to claim 1, characterized in that, In step S4, the mass ratio of freeze-dried liquid to water is 1:100; in step S7, the solution obtained in step S6 is mixed with ethanol at a volume ratio of 1:1 and precipitated for more than 8 hours.

4. A feed additive containing Ulva polysaccharide to enhance the antibacterial ability of green abalone, characterized in that, The feed additive includes any one of Ulva polysaccharide and feed components, wherein the proportion of Ulva polysaccharide in the feed additive is 40%; The feed components include fish meal, soybean protein concentrate, soybean meal, flour, high-gluten flour, kelp powder, shell powder, calcium dihydrogen phosphate, choline chloride, fish oil, soybean oil, multivitamin premix, and multimineral premix.

5. The feed additive containing Ulva polysaccharide for improving the antibacterial ability of green abalone according to claim 4, characterized in that, The multivitamin premix contains: Vitamin A, 250,000 IU; Riboflavin, 750 mg; Vitamin B6, 400 mg; Vitamin B12, 1 mg; Vitamin B1, 250 mg; Vitamin K3, 250 mg; Folic acid, 125 mg; Biotin, 10 mg; α-Tocopherol, 2.5 g; Inositol, 8,000 mg; Calcium pantothenate, 1,250 mg; Niacin, 2,000 mg; Choline chloride, 8,000 mg; Vitamin D3, 45,000 IU; and Vitamin C, 7,000 mg. The multi-mineral premix contains ZnSO4·7H2O, 0.04 g; CaCO3, 37.9 g; KCl, 5.3 g; KI, 0.04 g; NaCl, 2.6 g; CuSO4·5H2O, 0.02 g; CoSO4·7H2O, 0.02 g; FeSO4·7H2O, 0.9 g; MnSO4·H2O, 0.03 g; MgSO4·7H2O, 3.5 g; and Ca(HPO4)2·2H2O, 9.8 g.

6. The feed additive containing Ulva polysaccharide for improving the antibacterial ability of green abalone according to claim 4, characterized in that, The polysaccharide from *Ulva prostrata* is either crude polysaccharide or partially purified polysaccharide.

7. The feed additive containing Ulva polysaccharide for improving the antibacterial ability of green abalone according to claim 6, characterized in that, The Ulva polysaccharide is prepared by the preparation method according to any one of claims 1 to 3.

8. The application of the Ulva polysaccharide feed additive as described in any one of claims 4 to 7 in the preparation of feed that enhances the antibacterial ability of abalone.

9. The application according to claim 8, characterized in that, The antibacterial ability includes the ability to resist Vibrio alginolyticus.

10. A feed for enhancing the antibacterial ability of green abalone, characterized in that, The raw material composition of the feed is shown in the table below: The multivitamin premix contains vitamin A, 250,000 IU; riboflavin, 750 mg; vitamin B6, 400 mg; and vitamin B12, 1 mg. Vitamin B1, 250 mg; Vitamin K3, 250 mg; Folic acid, 125 mg; Biotin, 10 mg; α-Tocopherol, 2.5 g; Inositol, 8000 mg; Calcium pantothenate, 1250 mg; Niacin, 2000 mg; Choline chloride, 8000 mg; Vitamin D3, 45000 IU; Vitamin C, 7000 mg; The multi-mineral premix contains: ZnSO4·7H2O, 0.04 g; CaCO3, 37.9 g; KCl, 5.3 g; KI, 0.04 g; NaCl, 2.6 g; CuSO4·5H2O, 0.02 g; CoSO4·7H2O, 0.02 g; FeSO4·7H2O, 0.9 g; MnSO4·H2O, 0.03 g; MgSO4·7H2O, 3.5 g; and Ca(HPO4)2·2H2O, 9.8 g. After the components of the feed are mixed evenly, they are processed into flake feed through a post-processing step.

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