Turbot complex immune enhancer, its preparation method and application

Abstract

The application provides a turbot compound immune enhancer, a preparation method and application thereof. The application discloses application of a composition containing a series of components in preparation of a turbot immune enhancer, and the immune enhancer can effectively improve expression of a proinflammatory related gene of the turbot, reduce a number of bacteria in a liver or an intestinal tract, enhance a bactericidal capacity of the turbot, enhance an antioxidant stress capacity of the turbot, relieve liver damage of the turbot after being infected by bacteria, and relieve tissue damage of the turbot after being infected by bacteria. The immune enhancer component is simple, environment-friendly, low in cost and ideal in effect, and provides a new scheme for improving a breeding level of marine fish and improving a survival rate.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture feed additive technology, specifically relating to a turbot compound immune enhancer, its preparation method and application. Background Technology

[0002] With the increasing depletion of natural fishery resources, the aquaculture industry, as a traditional sector, has experienced rapid and effective development in modern times, highlighting its important role in society, the economy, and people's lives. However, due to the limited availability of water and soil resources, increasing production by expanding aquaculture area is unsustainable. Therefore, large-scale, intensive, and high-density farming models have gradually become the mainstream of China's fish farming industry. However, with the steady development of aquaculture, various diseases have become increasingly prominent, seriously affecting aquaculture yields and growth. Disease problems have become a significant factor restricting the healthy development of marine aquaculture.

[0003] Chemotherapy, represented by antibiotics, has played a positive role in controlling and preventing various diseases. However, the negative impacts of these disease control measures, such as environmental pollution, the emergence of drug-resistant pathogens, and drug residues in aquatic products, are becoming increasingly serious.

[0004] To curb the increasing prevalence of diseases in farmed fish caused by various environmental factors and rapid increases in stocking density, and to promote the sustainable development of marine aquaculture, the Food and Agriculture Organization of the United Nations (FAO), drawing on the successful experiences of fisheries development in developed countries, advocates the "System Management Approach" (SMA) approach to prevent various diseases. Enhancing the immunity of fish is also a way to respond to this advocacy.

[0005] Turbot (Scophthalmus maximus) and other fish species are among the pillar industries of marine fish farming. With the continuous expansion of farming scale and the increasing intensification of farming practices, disease problems during the farming process are also increasing year by year, especially bacterial diseases such as Edwardsiella tarda and Vibrio anguillarum. At the same time, the inappropriate addition of antibiotics in recent years has led to a continuous increase in bacterial resistance, seriously jeopardizing the green and healthy development of the industry.

[0006] To enhance the immunity of aquatic animals and prevent the occurrence of diseases, the use of immune enhancers has become an important prevention and control strategy in the aquaculture industry. Summary of the Invention

[0007] The purpose of this invention is to provide a turbot-based immune enhancer, its preparation method, and its application.

[0008] In a first aspect of the invention, the use of a composition in the preparation of a turbot immune enhancer is provided, wherein the composition comprises the following components: β-glucan, mannan oligosaccharide, astragalus polysaccharide, lecithin, and fish oil, preferably further comprising phytase; and, by weight percentage, the composition comprises:

[0009]

[0010] Preferably, the composition further includes:

[0011] Phytase: 1-5%.

[0012] In one or more embodiments, the immune enhancement is an enhancement of the ability to resist infection by Edwardsiella piracetam and / or Aeromonas salmonii.

[0013] In one or more embodiments, the immune enhancer increases the expression of pro-inflammatory genes in turbot, preferably including (but not limited to): il1b, il6, and tnf.

[0014] In one or more embodiments, the number of bacteria in turbot tissue is reduced, preferably, the number of bacteria in the liver or intestines is reduced.

[0015] In one or more embodiments, the lysozyme activity of turbot tissue is increased, thereby enhancing the bactericidal ability of turbot; preferably, the lysozyme activity of the liver or intestine is increased.

[0016] In one or more embodiments, the catalase activity of turbot tissues is increased, thereby enhancing the turbot's antioxidant stress resistance; preferably, the catalase activity in the liver or intestines is increased.

[0017] In one or more embodiments, liver damage in turbot following bacterial infection is alleviated.

[0018] In one or more embodiments, the tissue damage in turbot following bacterial infection is alleviated, preferably including intestinal tissue damage.

[0019] In one or more embodiments, the composition comprises, by weight percentage:

[0020]

[0021] Preferably, the composition further includes:

[0022] Phytase: 1.5–4%.

[0023] In one or more embodiments, the turbot is a fish susceptible to Edwardsiella piscicida or Aeromonas salmonicidas.

[0024] In another aspect of the invention, a composition is provided, said composition being a turbot immune enhancer, comprising, by weight percentage:

[0025]

[0026] Preferably, the composition further includes:

[0027] Phytase: 1-5%.

[0028] In one or more embodiments, the composition comprises, by weight percentage:

[0029]

[0030] Preferably, the composition further includes:

[0031] Phytase: 1.5–4%;

[0032] Preferably, the components of the composition are mixed in water.

[0033] In another aspect of the present invention, a method for preparing a composition is provided, said composition being a turbot immune enhancer, the method comprising: mixing β-glucan, mannan oligosaccharide, astragalus polysaccharide and lecithin, preferably further comprising phytase, adding fish oil to the mixture, then adding purified water, homogenizing and emulsifying to obtain the composition; wherein, the content of each component by weight percentage is:

[0034]

[0035] In one or more embodiments, a homogenizer is used for homogenization and emulsification.

[0036] In one or more embodiments, the homogenizer is stirred at 7600 r / min for 5-10 min to fully homogenize and emulsify the mixture.

[0037] In another aspect of the invention, the use of the aforementioned composition in the preparation of fish feed is provided; preferably, the fish feed is turbot feed.

[0038] In another aspect of the invention, a fish feed is provided, the feed comprising: (a) a fish base feed; and

[0039] (b) Any of the compositions described above.

[0040] In one or more embodiments, (b) and (a) are mixed at a volume mass ratio of 3 to 10% (V / m); preferably, (b) and (a) are mixed at a volume mass ratio of 4 to 8% (V / m).

[0041] In one or more embodiments, (b) is mixed with (a) at a volume-to-mass ratio of 4.5, 5, 5.5, 6, 6.5, 7 or 7.5% (V / m).

[0042] In one or more embodiments, the phrase “(b) and (a) are mixed in a volume-to-mass ratio of 3 to 10% (V / m)” is equivalent to: (b) and (a) in a volume-to-mass ratio of 3 to 10:100 (wherein if the volume is in mL, then the mass is in g).

[0043] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0044] Figure 1 Gene expression of pro-inflammatory genes in the tissues of turbot fed different diets.

[0045] Figure 2 Bacterial colonization in the liver (left) and hindgut (right) of turbot fed different diet groups.

[0046] Figure 3 Lysozyme activity in the liver (left) and hindgut (right) of turbot fed different diet groups.

[0047] Figure 4 The activity of catalase in the spleen of turbot fed with different feed groups.

[0048] Figure 5 MDA content in the liver of turbot fed with different diets.

[0049] Figure 6 Pathological sections of the hind intestine of turbot fed with different feed groups. Detailed Implementation

[0050] Through in-depth research, the inventors have revealed the application of a composition consisting of a series of components in the preparation of an immune enhancer for turbot. This immune enhancer can effectively increase the expression of pro-inflammatory genes in turbot, reduce the number of bacteria in the liver or intestines, enhance the bactericidal ability of turbot, enhance the antioxidant stress capacity of turbot, alleviate liver damage in turbot after bacterial infection, and alleviate tissue damage in turbot after bacterial infection. The immune enhancer of this invention has simple and readily available components, is environmentally friendly, low in cost, and has ideal effects, providing a new solution for improving the aquaculture level and survival rate of marine fish.

[0051] the term

[0052] As used herein, “containing,” “having,” or “including” includes “comprising,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0053] As used herein, “about,” “approximately,” “roughly,” or “basically” generally refers to a specific value or range, such as within 20%, preferably within 10%, or even more preferably within 5%. The values ​​used herein are approximate and mean that unless explicitly stated otherwise, the terms “about,” “approximately,” “roughly,” or “basically” can be inferred to apply.

[0054] As used herein, "primary fermentation" and "secondary fermentation" refer to seed tank fermentation. "Tertiary fermentation" refers to fermentation for production purposes. Unless otherwise stated, "fermentation" and "cultivation" in the claims and specification of this invention refer to "tertiary fermentation."

[0055] Immune enhancers

[0056] As mentioned earlier, marine pathogens such as Edwardsiella piscicida or Aeromonas salmonicida can infect fish, causing infectious diseases in them.

[0057] In existing technologies, aquatic immune enhancers are relatively limited in variety, mainly consisting of immunopolysaccharides; and most products are in powder form, resulting in a single dosage form; some products have complex and diverse ingredients, leading to high costs and less than ideal effects. The inventors of this invention are dedicated to improving aquatic immune enhancers, considering both fish growth performance and immune response capabilities. They utilize β-glucan, mannooligosaccharides, astragalus polysaccharides, lecithin, and fish oil, preferably with the addition of phytase for further formulation. An emulsification and homogenization method is used to prepare a liquid immune enhancer, allowing for better integration with feed and improved feeding effects. Results show that β-glucan, mannooligosaccharides, astragalus polysaccharides, lecithin, and fish oil have good immune-enhancing effects, while the addition of phytase further improves the efficacy of the immune enhancer, giving turbot stronger antioxidant stress resistance and anti-infection capabilities.

[0058] Based on the above-mentioned new discovery of the inventors, the present invention provides a novel composition for enhancing the immunity of marine fish (especially turbot), comprising: β-glucan, mannan oligosaccharide, astragalus polysaccharide, lecithin and fish oil, preferably further comprising phytase.

[0059] In a preferred embodiment of the present invention, the amounts of the components comprising the turbot immune enhancer by weight percentage are shown in Table 1.

[0060] Table 1

[0061]

[0062]

[0063] As a preferred embodiment of the present invention, the immune enhancer comprises: 2% β-glucan, 2% mannan oligosaccharide, 2% astragalus polysaccharide, 3% lecithin and 10% fish oil, and preferably also includes 2% phytase.

[0064] Phytase can catalyze the hydrolysis of phytic acid in plant-based raw materials. However, there is currently no information on its application in enhancing the infection resistance of fish.

[0065] In the immune enhancer of the present invention, the various components are combined with each other in appropriate amounts to achieve a synergistic effect and thus a significant immune enhancement effect.

[0066] The dosage forms of the immune enhancers can be diverse, including but not limited to: emulsions, aqueous solutions, emulsifiable concentrates, wettable powders, lyophilized agents, sprayable solutions, oily or aqueous dispersions, suspensions, powders, granules, or microcapsules. It should be understood that any dosage form that allows the composition of the present invention to be delivered to the object to be treated while retaining all or part of its activity is desirable. Preferred dosage forms are those that are easily mixed with feed, such as emulsions (emulsions) formed by homogenization and capable of good mixing with feed.

[0067] Concentrated compositions contain higher levels of active ingredients, such as 20–90 wt%, while diluted compositions and those actually used contain lower levels of active ingredients, typically 0.00005–0.5 wt%. In addition, other suitable chemical agents, synergists, trace elements, stabilizers, binders, wetting agents, dispersants, emulsifiers, penetrants, solvents, fillers, and other commonly used components may be included. The compositions of this invention may also contain other active ingredients, such as digestive aids.

[0068] The immune enhancer of the present invention has a simple formulation of each component, readily available raw materials, a simple preparation method, and low cost. However, it can be effectively and efficiently utilized by marine fish, especially turbot, to give the fish good immunity. It is very suitable as a feed additive for marine fish, especially turbot.

[0069] application

[0070] The present invention also provides an application of the above-mentioned immune enhancer in the preparation of fish feed (such as fish fry feed). Preferably, the liquid immune enhancer can be added to the basic feed for turbot fry at a volume-to-mass ratio of 3-10% (V / m).

[0071] The present invention also provides a fish feed comprising: a basic fish feed; and the immune enhancer described in the present invention.

[0072] As a preferred embodiment of the present invention, the above-mentioned components are homogenized and emulsified to prepare an immune enhancer. The liquid immune enhancer is added to the basic feed of turbot seedlings at a ratio of 5% (V / m). The immune response of turbot seedlings can be enhanced by short-term feeding for 4 weeks, and the effective protection period can exceed 7 weeks.

[0073] This invention also provides a kit / packaging for enhancing the immunity of marine fish, particularly turbot, including the aforementioned immune enhancer. The kit typically includes packaging / container. The kit usually also includes instructions for use, which may describe methods for using the immune enhancer, or, for example, methods for preparing the immune enhancer of this invention, to facilitate formulation and application by those skilled in the art.

[0074] This invention also provides a kit for enhancing the immunity of marine fish, particularly turbot, comprising the aforementioned fish feed. The kit typically includes packaging / containers. The kit usually also includes instructions for use, which may describe methods for using the immune enhancer, or, for example, methods for preparing the immune enhancer of this invention, to facilitate formulation and application by those skilled in the art.

[0075] Compared with the prior art, the main beneficial effects of the present invention are:

[0076] The immune enhancer of this invention is a combination formulation, which has a significant effect on enhancing immunity. The β-glucan, mannan oligosaccharide, astragalus polysaccharide, lecithin and fish oil used in this invention work synergistically and significantly improve the immune response of juvenile turbot and alleviate tissue damage compared to using polysaccharides or oligosaccharides alone.

[0077] The immune enhancer of the present invention is characterized by simple preparation, convenient use, safety and non-toxicity, no drug resistance, and no pollution. It also has the effects of antibacterial and antimicrobial properties, and can improve the immunity of aquatic animals.

[0078] The immune enhancer of the present invention uses inexpensive and readily available raw materials that are non-toxic and pollution-free, will not induce drug resistance in fish, will not pollute water bodies, and has a non-specific protective effect.

[0079] The preparation method and application of the immune enhancer of the present invention are simple and convenient to operate, and are suitable for mass production and application.

[0080] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0081] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0082] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0083] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0084] Materials and methods

[0085] Test reagents

[0086] TRIzol, isopropanol, All-in-One First-Strand cDNA Synthesis SuperMix, MonAmpSYBR Green qPCR Mix (Low ROX), catalase assay kit, Micrococcus lysate, lipid oxidation (MDA) assay kit, hematoxylin and eosin (HE) staining kit, anhydrous ethanol, xylene.

[0087] Test equipment

[0088] Tissue homogenizer, QuantStudio3, multi-functional microplate reader, electronic scale, paraffin microtome, grinder, grinding rod.

[0089] Experimental feed

[0090] Control group: basal feed (purchased from Shandong Zhenghe Feed Co., Ltd.)

[0091] Experimental Group A: An immune enhancer was added to the basal feed (Example 1), with the volume-to-mass ratio of the immune enhancer to the basal feed being 5% (V / m).

[0092] Experimental Group B: An immune enhancer was added to the basal feed (Example 2), with the volume-to-mass ratio of the immune enhancer to the basal feed being 5% (V / m).

[0093] Experimental turbot

[0094] Healthy, undamaged, uniform in size, and weighing 3-5g per fish.

[0095] Experimental feeding

[0096] Three to five grams of turbot were selected from the breeding base and fed with a basic feed and an experimental feed containing active substances (the compound immune enhancer of Example 1 or Example 2) for four weeks.

[0097] After feeding, 100 fish from each experimental group were selected and transported to the laboratory for temporary rearing for 3 weeks before bacterial challenge and immune response index testing.

[0098] Challenge experiment

[0099] A challenge experiment was conducted on fish that had been fed a compound immune enhancer for 3 weeks using Edwardsiella piscicida and Aeromonas salmonicida. Edwardsiella piscicida bacterial culture was inoculated at 1% in TSB medium containing 50 μg / ml polymyxin and cultured at 28°C and 220 rpm for 12 h. Primary seed culture was inoculated at 1% in TSB medium containing 50 μg / ml polymyxin and cultured overnight at 28°C and 220 rpm. Aeromonas salmonicidas was inoculated at 1% in TSB medium and cultured at 22°C and 220 rpm for 24 h. Primary seed culture was inoculated at 1% in TSB medium and cultured overnight at 22°C and 220 rpm.

[0100] Bacterial concentration was determined using NanoDrop. When the OD reached 3, bacteria were collected by centrifugation at 5000 rpm for 5 min. Forty turbot were randomly selected from each group for immersion challenge, with an immersion volume of 60 L and a bacterial concentration of 2*10⁻⁶. 8The concentration of CFU / ml was increased, and the soaking time was 1 hour. After soaking, the fish was washed with seawater and placed in a fish tank for further observation.

[0101] Sample collection and testing

[0102] Sampling was performed on day 3 post-infection to detect the expression of pro-inflammatory cytokine-related genes in the tissues. The sampling procedure was as follows: Nine fish were randomly selected from each group and divided into three parts. The hindgut, gills, liver, spleen, and head kidney were placed in extraction tubes containing lysis beads. 1 ml of TRIzol was added, and the tissues were ground in a tissue homogenizer to extract RNA, which was then reverse transcribed into cDNA. The expression of pro-inflammatory genes such as il1b, il6, and tnf was detected by qPCR.

[0103] Sampling was performed on day 7 post-challenge to detect bacterial colonization in the tissues. The sampling procedure was as follows: Nine fish were randomly selected from each group and divided into three portions. The liver and hindgut were placed in a grinding tube, and 400 μL of 1% Triton X-100 solution was added for immersion. The mixture was then ground using a grinder. The grinding solution was serially diluted with PBS, and 5 μL was used for plate counting.

[0104] Sampling was performed on day 7 post-infection to detect lysozyme activity in the tissues. The sampling procedure was as follows: Nine fish were randomly selected from each group and divided into three portions. The liver and hindgut were placed in grinding tubes, lysis buffer was added, and the mixture was ground using a grinder. After centrifugation, the supernatant was collected to detect lysozyme activity.

[0105] Sampling was performed on day 7 post-infection to detect antioxidant stress kinase activity in tissues. The specific sampling steps were as follows: Nine fish were randomly selected from each group and divided into three portions. The spleen was removed and placed in a 1.5 ml EP tube. Lysis buffer was added, and the mixture was ground with a grinder. After centrifugation, the supernatant was collected to detect catalase activity.

[0106] Sampling was performed on the 7th day after the challenge to detect the malondialdehyde (MDA) content in the tissue. The specific sampling steps were as follows: Nine fish were randomly selected from each group and divided into three parts. The liver was placed in a 1.5ml EP tube, lysis buffer was added, and the liver was ground with a grinder. After centrifugation, the supernatant was collected to detect the MDA content in the liver.

[0107] Sampling was performed on day 21 after the challenge to detect intestinal pathology. The specific sampling steps were as follows: Two fish were taken from each group, and the hindgut of the fish was dissected and immersed in tissue fixation solution for subsequent paraffin section preparation, HE staining, and tissue morphology observation.

[0108] Example 1: Preparation of compound immune enhancers 1 and 1'

[0109] A compound immune enhancer 1 and 1' for fish was prepared, comprising the components shown in Table 2 by weight percentage.

[0110] Table 2

[0111] Components Enhancer 1 Enhancer 1' β-glucan 2％ 1.8％ Mann oligosaccharides 2％ 2.3％ Astragalus polysaccharides 2％ 1.7％ Phytase 2％ 2.5％ Lecithin 3％ 2.6％ fish oil 10％ 12％ margin purified water purified water

[0112] The preparation of the above-mentioned compound immune enhancer for fish includes the following steps: β-glucan, mannan oligosaccharide, astragalus polysaccharide, phytase and lecithin are weighed according to the required weight and added to fish oil, and purified water is added. Then, a homogenizer is used to stir at 7600 r / min for 5-10 minutes to make it fully mixed.

[0113] The prepared liquid immune enhancer was added to the basic feed of fish fry (such as turbot) at a volume-to-mass ratio of 5% (V / m).

[0114] Example 2: Preparation of compound immune enhancers 2 and 2'

[0115] A compound immune enhancer 2 and 2' for fish was prepared, comprising the components shown in Table 3 by weight percentage.

[0116] Table 3

[0117] Components Enhancer 2 Enhancer 2' β-glucan 2％ 2.2％ Mann oligosaccharides 2％ 1.8％ Astragalus polysaccharides 2％ 2.5％ Lecithin 3％ 3.5％ fish oil 10％ 8.5％ margin purified water purified water

[0118] The preparation method of the above-mentioned compound immune enhancer for fish includes the following steps: weighing β-glucan, mannan oligosaccharide, astragalus polysaccharide and lecithin according to the required weight, adding them to fish oil, and adding purified water, and stirring with a homogenizer at 7600 r / min for 5-10 min to make them fully mixed.

[0119] The prepared liquid immune enhancer was added to the basic feed of turbot seedlings at a volume-to-mass ratio of 5% (V / m).

[0120] Example 3: Effects of immune enhancers on gene expression of pro-inflammatory genes in fish

[0121] After feeding turbot with a basal diet and different immune enhancers (for 4 weeks), feeding was stopped for 3 weeks before challenge with the virus. Three days after infection, the gene expression of pro-inflammatory genes such as il1b, il6, and tnf in various tissues of turbot, including liver, spleen, head kidney, hind intestine, and gills, was measured.

[0122] The results are as follows Figure 1 As shown. By Figure 1 It can be seen that compared with the control group fed with ordinary feed, the expression of genes such as il1b, il6, and tnf in experimental groups A and B was significantly increased. Furthermore, the expression of genes such as il1b, il6, and tnf in the spleen, head kidney, and gills in experimental group A, which was supplemented with phytase, was significantly higher than that in experimental group B, which contained only polysaccharides and oligosaccharides.

[0123] Therefore, the compound immune enhancer of the present invention can significantly increase the expression of pro-inflammatory genes in turbot, and the addition of phytase further enhances this effect.

[0124] Example 4: Analysis of the effect of immune enhancers on the bactericidal ability of fish

[0125] 1. Bacterial gradation analysis of fish liver and intestines

[0126] After feeding a basal diet and different immune enhancers (for 4 weeks), followed by a 3-week feeding halt, turbot were challenged with *Edwardsiella piscicida* and *Aeromonas salmonicida*, respectively, for 7 days. Liver samples were then measured. Figure 2 (Left image) and hindgut ( Figure 2 The bacterial colonization is shown in the right figure.

[0127] Depend on Figure 2 It can be seen that, compared with the control group fed with ordinary feed, experimental groups A and B showed significantly lower liver bacterial counts 7 days after infection with Aeromonas salmonicidas and significantly lower intestinal bacterial counts 7 days after infection with Edwardsiella piscicida, with significant differences from the control group.

[0128] Furthermore, the number of bacteria in each tissue of experimental group A was significantly lower than that of experimental group B.

[0129] Meanwhile, bacterial colonization analysis was performed on experimental group A' (containing compound immune enhancer 1', with a volume-to-mass ratio of 5% to the basal feed) and experimental group B' (containing compound immune enhancer 2', with a volume-to-mass ratio of 5% to the basal feed). Similarly, the number of intestinal bacteria was significantly reduced 7 days after Edwardsiella piscicida infection, and the number of bacteria in each tissue of experimental group A' was significantly lower than that of experimental group B'.

[0130] 2. Analysis of lysozyme activity in fish liver and intestines

[0131] Lysozyme activity in the liver (left) and hindgut (right) of turbot 7 days after infection with Edwardsiella piscicida was determined after feeding a basal diet and different immune enhancers (4 weeks of feeding) followed by a 3-week feeding cessation.

[0132] Depend on Figure 3 It can be seen that after feeding with feed containing immune enhancers for 4 weeks and Edwardsiella piscicida challenged for 7 days, the liver and hindgut lysozyme activities of experimental groups A and B were significantly increased, and experimental group A was significantly higher than that of experimental group B.

[0133] Therefore, the fish compound immune enhancer of the present invention can significantly enhance the bactericidal ability of turbot.

[0134] Example 5: Analysis of the effect of immune enhancers on the antioxidant stress capacity of fish

[0135] Figure 4 The study investigated the catalase activity in the spleen of turbot 7 days after infection with Edwardsiella piscicida, following a 4-week feeding period with a basal diet and various immune enhancers, followed by a 3-week feeding cessation.

[0136] Depend on Figure 4 It can be seen that after feeding with feed containing immune enhancers for 4 weeks, followed by a 3-week feeding stoppage and then challenge with Edwardsiella piscicida, the activity of catalase in the liver and hindgut of experimental groups A and B was significantly increased 7 days after challenge, and the activity of catalase in experimental group A was higher than that in experimental group B.

[0137] Therefore, the fish compound immune enhancer of the present invention can significantly enhance the antioxidant stress resistance of turbot.

[0138] Example 6: Analysis of the effect of immune enhancers on liver damage in fish

[0139] After feeding a basal diet and different immune enhancers (for 4 weeks), followed by a 3-week feeding halt, turbot were challenged with the virus. Seven days after infection with Edwardsiella piscicida, the malondialdehyde (MDA) content in the liver of turbot was measured.

[0140] Depend on Figure 5 It can be seen that after feeding with feed containing immune enhancers for 4 weeks and Edwardsiella piscicida challenged for 7 days, the MDA content in the liver of the control group was significantly higher than that of the experimental groups A and B, and the MDA content in the experimental group B was higher than that in the experimental group A. The MDA content was positively correlated with the degree of liver damage.

[0141] Therefore, the fish compound immune enhancer of the present invention can significantly alleviate liver damage in fish after bacterial infection.

[0142] Example 7: Analysis of the effect of immune enhancers on intestinal tissue damage in fish

[0143] After feeding a basal diet and different immune enhancers (for 4 weeks), feeding was stopped for 3 weeks before challenge with the virus. The hindgut of turbot was obtained 21 days after infection with Edwardsiella piscicida, and pathological sections were prepared and observed.

[0144] Depend on Figure 6It can be seen that: after feeding with feed containing immune enhancers for 4 weeks, Edwardsiella piscicida challenged and infected for 21 days, the control group had severe villus loss, while the experimental groups A and B had intact intestinal structural barriers after infection, with very significant relief effects, and experimental group A was the best.

[0145] Therefore, the fish immune enhancer of the present invention can significantly alleviate tissue damage in turbot after challenge.

[0146] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. The application of the composition in the preparation of turbot immune enhancer, comprising the following components: β-glucan, mannan oligosaccharide, astragalus polysaccharide, lecithin, fish oil and phytase; The composition comprises, by weight percentage: The aforementioned immune enhancement includes: an enhanced ability to resist infection by Edwardsiella piracetam and Aeromonas salmonidae; a reduction in the number of bacteria in the liver or intestinal tissue of turbot after infection by Edwardsiella piracetam and Aeromonas salmonidae; an alleviation of liver damage in turbot after infection by Edwardsiella piracetam and Aeromonas salmonidae; and an alleviation of intestinal tissue damage in turbot after infection by Edwardsiella piracetam and Aeromonas salmonidae. The immune enhancement is achieved by increasing the expression of pro-inflammatory genes in turbot after infection with Edwardsiella tarda and Aeromonas salmonidae, including il1b, il6, and tnf; increasing lysozyme activity in turbot tissues; enhancing the bactericidal ability of turbot; and increasing lysozyme activity in the liver or intestines.

2. The application as described in claim 1, characterized in that, The immune enhancement mentioned refers to increasing the activity of catalase in the liver or intestinal tissue of turbot, thereby enhancing the turbot's ability to resist oxidative stress.

3. The application as described in claim 1, characterized in that, The composition comprises, by weight percentage:

4. The application as described in claim 1, characterized in that, The turbot mentioned is infected with Edwardsiella tarda (a bacterium that kills fish). Edwardsiellapiscicida Susceptible fish, kills Aeromonas salmonii ( Aeromonas salmonicidas Susceptible fish.

5. The use of a composition in the preparation of fish feed; The fish feed is turbot feed, characterized in that... The composition is a turbot immune enhancer, comprising, by weight percentage: The aforementioned immune enhancement includes: an enhanced ability to resist infection by Edwardsiella piracetam and Aeromonas salmonidae; a reduction in the number of bacteria in the liver or intestinal tissue of turbot after infection by Edwardsiella piracetam and Aeromonas salmonidae; an alleviation of liver damage in turbot after infection by Edwardsiella piracetam and Aeromonas salmonidae; and an alleviation of intestinal tissue damage in turbot after infection by Edwardsiella piracetam and Aeromonas salmonidae. The immune enhancement is achieved by increasing the expression of pro-inflammatory genes in turbot after infection with Edwardsiella faecium and Aeromonas salmonidae, including il1b, il6, and tnf. It can increase the lysozyme activity in turbot tissues, enhance the bactericidal ability of turbot, and increase the lysozyme activity in the liver or intestines.

6. The application as described in claim 5, characterized in that, The composition comprises, by weight percentage: The components of the composition are mixed in water.

7. A method for preparing a composition, said composition being a turbot immune enhancer, said method comprising: β-glucan, mannan oligosaccharide, astragalus polysaccharide, lecithin, and phytase were mixed, fish oil was added to the mixture, and then purified water was added. The mixture was homogenized and emulsified to obtain a composition. The content of each component, by weight percentage, is as follows: The aforementioned immune enhancement includes: an enhanced ability to resist infection by Edwardsiella piracetam and Aeromonas salmonidae; a reduction in the number of bacteria in the liver or intestinal tissue of turbot after infection by Edwardsiella piracetam and Aeromonas salmonidae; an alleviation of liver damage in turbot after infection by Edwardsiella piracetam and Aeromonas salmonidae; and an alleviation of intestinal tissue damage in turbot after infection by Edwardsiella piracetam and Aeromonas salmonidae. The immune enhancement is achieved by increasing the expression of pro-inflammatory genes in turbot after infection with Edwardsiella tarda and Aeromonas salmonidae, including il1b, il6, and tnf; increasing lysozyme activity in turbot tissues; enhancing the bactericidal ability of turbot; and increasing lysozyme activity in the liver or intestines.

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