Embedding type fry living body transportation anti-stress agent and preparation method and application thereof

Abstract

The application belongs to the technical field of fish live transportation, and particularly relates to a embedding type fry live transportation anti-stress agent and a preparation method and application thereof. The preparation method comprises the following steps: S1, mixing gelatinized starch and an acrylic acid solution, then adding a eugenol solution and / or a vitamin C solution, and uniformly mixing to obtain a solution A; S2, under an inert atmosphere, adding a saturated aqueous solution of beta-cyclodextrin to the solution A, and uniformly mixing to obtain a solution B; S3, irradiating the solution B, and after the irradiation is completed, standing overnight, then performing suction filtration, washing, and drying to obtain a complex particle of beta-cyclodextrin embedding eugenol and / or vitamin C. The beta-cyclodextrin is reacted with starch and acrylic acid to generate a beta-cyclodextrin polymer embedding the efficacy component eugenol or vitamin C, which can improve the stability and solubility of the efficacy component, delay the release speed of the efficacy component, make the efficacy component play a long-acting role, and then be conducive to reducing the stress response of fry transportation and improving the survival rate.

Description

Technical Field

[0001] This invention relates to the field of live fish transport technology, and in particular to an encapsulated anti-stress agent for live fish fry transport, its preparation method, and its application. Background Technology

[0002] Live animal transport has become a common method of transporting aquatic products. However, it inevitably causes stress to aquatic animals, such as jumping, collisions, sudden increases in oxygen consumption, increased mucus secretion, and abnormal physiological and biochemical indicators. During live fish transport, numerous stressors trigger stress responses, including ammonia nitrogen stress, crowding stress, vibration stress, and temperature stress. These factors can affect the body functions and meat quality of fish to varying degrees, leading to skin damage, endocrine disorders, decreased non-specific immunity, and weakened disease resistance, even causing death. Fish fry are small and have poor tolerance to environmental stress, making them more sensitive to environmental pressures. Transport stress directly impacts their survival rate and growth after transport. Currently, various measures are in place to alleviate fish stress and improve survival rates during fry transport, such as reducing activity and metabolism through low-temperature transport or adding anti-stress agents.

[0003] Currently, stress relief agents used during fish transportation generally involve adding antioxidants or anesthetics, such as vitamin C (VC) and clove oil (EU). However, these methods are often ineffective due to the limited availability and specificity of the substances. For example, clove oil (EU) is a broad-spectrum antibacterial agent with antibacterial, anti-inflammatory, and antioxidant properties. It is commonly used as a food preservative and a fish anesthetic. Adding clove oil can reduce the metabolic capacity and antioxidant capacity of the liver and gills, thus maintaining good water quality during transportation. However, clove oil is highly volatile and poorly soluble in water, resulting in a short duration of action and difficulty in maintaining a stable effect over a long period. Furthermore, excessively high concentrations can increase lipid peroxidation levels in the gills. Especially for fish fry, the use of clove oil is itself a form of environmental stress. If clove oil is applied directly to fish fry, the stress can be too intense, and even low concentrations can lead to the death of the fry. For example, vitamin C (VC), as a strong reducing agent, can mediate redox reactions in the body, eliminate excess free radicals, and reduce damage to the body caused by toxic substances and environmental stimuli. It plays a role in detoxification, enhancing immunity, and resisting stress, thereby improving the survival rate of fish. However, VC is unstable and easily oxidized. To maintain its antioxidant effect, it needs to be continuously replenished during transportation, resulting in significant waste and economic costs.

[0004] In response to the above-mentioned problems, there is an urgent need to provide a method that can provide long-term effectiveness, low cost, and effectively alleviate fish transport stress, reduce oxidative damage, and improve survival rates. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention provides an embedded anti-stress agent for live fish fry transportation and its preparation method. The prepared anti-stress agent can effectively improve the antioxidant capacity of fish, enhance non-specific immunity, reduce tissue damage, and also adsorb ammonia nitrogen in water, thereby reducing the stress response during fish fry transportation, extending transportation time, and improving survival rate. It has good application prospects in live transportation practice.

[0006] To achieve the above objectives, the present invention is specifically implemented through the following technical solutions:

[0007] The first aspect of this invention provides a method for preparing an embedded anti-stress agent for live transport of fish fry, comprising the following steps:

[0008] S1. Mix gelatinized starch with acrylic acid solution, then add eugenol solution and / or vitamin C solution, and mix well to obtain solution A;

[0009] S2. Under an inert atmosphere, add a saturated aqueous solution of β-cyclodextrin to solution A and mix well to obtain solution B;

[0010] S3. Irradiate the solution B, allow it to stand overnight after irradiation, then filter, wash, and dry to obtain inclusion complex particles of β-cyclodextrin encapsulating eugenol and / or vitamin C; wherein, in the inclusion complex particles, the mass ratio of eugenol, vitamin C, and β-cyclodextrin is 1:0.5-8:50-800, and the mass ratio of starch, acrylic acid, and β-cyclodextrin is 1:0.6-10:1.26-18.144.

[0011] Furthermore, the mass ratio of eugenol, vitamin C, and β-cyclodextrin is 1:2-8:20-800, and even more specifically, the mass ratio of eugenol, vitamin C, and β-cyclodextrin is 1:5:681.

[0012] Further, the mass ratio of the starch, the acrylic acid, and the β-cyclodextrin is 1:2.4:4.536-18.144. Even further, the mass ratio of the starch, the acrylic acid, and the β-cyclodextrin is 1:2.4:13.62.

[0013] Furthermore, the inclusion complex particles contain eugenol and vitamin C.

[0014] Further, in step S1, the method for preparing the gelatinized starch includes: dissolving starch in deionized water, heating under stirring conditions, and then keeping it warm under reflux to fully gelatinize the starch; wherein the stirring rate is 100-300 rpm and the temperature is raised to 85±3℃.

[0015] Further, in step S1, the acrylic acid solution is an aqueous solution of acrylic acid, the eugenol solution is an ethanolic solution of eugenol, and the vitamin C solution is an aqueous solution of vitamin C.

[0016] Further, in step S2, the method for preparing the β-cyclodextrin saturated aqueous solution includes: adding β-cyclodextrin to preheated distilled water and stirring to prepare a β-cyclodextrin saturated aqueous solution; wherein the temperature of the preheated distilled water is 30-50℃.

[0017] Furthermore, in step S3, 0-9 kGy is irradiated using a cobalt source.

[0018] The second aspect of the present invention provides an embedded fish fry live transport anti-stress agent prepared by the method described above.

[0019] The third aspect of the present invention provides the application of the encapsulated anti-stress agent for live transport of fish fry as described above in the live transport of fish fry.

[0020] Furthermore, the fish fry include yellow catfish fry.

[0021] The advantages and positive effects of this invention are as follows:

[0022] 1. This invention involves reacting β-cyclodextrin with starch and acrylic acid to generate a β-cyclodextrin polymer that encapsulates the active ingredient eugenol or vitamin C. This significantly enhances the encapsulation effect of β-cyclodextrin on eugenol and vitamin C, improves the stability and solubility of the active ingredients, slows down the release rate of the active ingredients, and prolongs their duration of action, thus facilitating a long-lasting effect.

[0023] 2. This invention combines β-cyclodextrin with eugenol or vitamin C, which can significantly enhance the effects of the active ingredients in anesthetizing fish fry, slowing down their metabolic activity, clearing free radicals in the fry, and alleviating oxidative damage to the fish. This reduces the fish's response to external stimuli, delays the rise in serum cortisol and blood glucose levels and the increase in antioxidant enzyme activity, reduces damage to gills and liver tissue induced by live transport, and enhances the fish's non-specific immune system. Moreover, the encapsulated structure is also conducive to adsorbing ammonia nitrogen in the water, which can reduce ammonia nitrogen pollution and water quality deterioration during transport. This enhances the stress resistance of fish fry during transport, greatly extends the transport time, improves the survival rate during transport, and largely ensures muscle quality. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying 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.

[0025] Figure 1 This is a time-varying release curve of the active ingredients of the inclusion complex particles containing β-cyclodextrin encapsulated with eugenol and / or vitamin C in an embodiment of the present invention.

[0026] Figure 2 The effects of adding different additives on water quality and pH during transportation in embodiments of the present invention;

[0027] Figure 3 The effects of adding different additives on ammonia nitrogen in water during transportation in embodiments of the present invention;

[0028] Figure 4 The effect of adding different additives on serum glucose (GLU) concentration in embodiments of the present invention;

[0029] Figure 5 The effects of adding different additives on serum cortisol (COR) concentration in embodiments of the present invention;

[0030] Figure 6 The effects of adding different additives on serum immunoglobulin G (IgG) concentration in embodiments of the present invention;

[0031] Figure 7 The effects of adding different additives on the activity of liver superoxide dismutase (SOD) in embodiments of the present invention;

[0032] Figure 8 The effects of adding different additives on the activity of hepatic catalase (CAT) in embodiments of the present invention;

[0033] Figure 9 The effects of adding different additives on the concentration of malondialdehyde (MDA) in the liver in embodiments of the present invention;

[0034] Figure 10 The effects of adding different additives on the activity of liver lysozyme (LZM) in embodiments of the present invention;

[0035] Figure 11The images show liver tissue sections of juvenile yellow catfish transported for 24 hours according to an embodiment of the present invention. Figures (a)-(g) represent the control group, EU-5mg / β-CD group, EU-10mg / β-CD group, VC-20mg / β-CD group, VC-50mg / β-CD group, EU-10mg+VC-50mg / β-CD group, and β-CD group, respectively. The arrows indicate blood (bc), hepatocyte edema (hs), portal vein branches containing erythrocytes (hpv), focal necrosis of liver tissue (fn), and vacuolation (va).

[0036] Figure 12 This is a section of gill tissue from juvenile yellow catfish transported for 24 hours, as per an embodiment of the present invention. Figures (a)-(g) represent the control group, EU-5mg / β-CD group, EU-10mg / β-CD group, VC-20mg / β-CD group, VC-50mg / β-CD group, EU-10mg+VC-50mg / β-CD group, and β-CD group, respectively. The arrows indicate columnar cells (pc), epithelial cells (ec), aneurysms (an), hyperplasia (hp), lamellar curl (cl), and edema (ed).

[0037] Figure 13 This invention illustrates the effect of different additives on the expression level of the heat shock protein HSP70 gene in various embodiments.

[0038] Figure 14 This invention illustrates the effect of different additives on TNF-α gene expression levels in various embodiments.

[0039] Figure 15 The effects of different additives on IL-1β gene expression levels in embodiments of the present invention. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0041] Based on the information contained herein, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0042] To better understand the invention and not to limit its scope, all figures indicating amounts, percentages, and other numerical values ​​used in this invention should, in all cases, be understood to be modified by the word "approximately." Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods. Furthermore, the terms "comprising," "including," "containing," "having," and similar words are non-limiting in meaning, allowing for the addition of other steps and components that do not affect the result.

[0043] It should be emphasized that the use of “and / or” in this document should be interpreted as referring to a specific disclosure of each of the two specified features or components, with or without the other. For example, “A and / or B” would be considered to include the following cases: (i) A, (ii) B, and (iii) A and B.

[0044] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0045] This invention provides a method for preparing an encapsulated anti-stress agent for live transport of fish fry, comprising the following steps:

[0046] S1. Mix gelatinized starch with acrylic acid solution, then add eugenol solution and / or vitamin C solution, and mix well to obtain solution A;

[0047] S2. Under an inert atmosphere, add a saturated aqueous solution of β-cyclodextrin to solution A and mix well to obtain solution B;

[0048] S3. Irradiate the solution B, let it stand overnight after irradiation, then filter, wash and dry to obtain inclusion complex particles of β-cyclodextrin encapsulating eugenol and / or vitamin C.

[0049] To reduce the instantaneous concentration of eugenol (EU) or maintain the stability of vitamin C (VC) so that both can act stably and continuously on fish fry, this invention employs a β-cyclodextrin (β-CD) encapsulation method for sustained release. β-cyclodextrin is a natural, non-toxic cyclic oligosaccharide with a unique hydrophobic inner cavity and a hydrophilic outer surface, possessing a relatively hydrophobic center and a relatively hydrophilic surface. Embedding the active ingredient in the hydrophobic center of β-CD can stabilize the active ingredient, reduce oxidation, passivate photosensitivity and thermosensitivity, reduce toxicity and volatility, and improve solubility and bioavailability. Furthermore, it can slow down the release rate of the active ingredient, prolonging its duration of action and thus enabling a long-lasting effect. This invention first mixes gelatinized starch with extended molecular chains with an acrylic acid solution, then adds EU and / or VC solutions, and finally adds a saturated aqueous solution of β-CD. EU and / or VC are initially encapsulated within the hydrophobic cavities of the β-CD. Subsequently, under irradiation conditions, acrylic acid polymerizes with starch and β-cyclodextrin to form a β-cyclodextrin polymer. After filtration, washing, and drying, the β-cyclodextrin-encapsulated eugenol and / or vitamin C inclusion complex particles become solids, with EU and / or VC embedded within the β-cyclodextrin polymer. On one hand, the formation of the β-cyclodextrin polymer's polymer network chain improves the encapsulation strength; on the other hand, the hydrogen bonds formed between gelatinized starch and β-cyclodextrin due to the simultaneous presence of CO- and NH2- ions also contribute to improved polymer structural stability. This significantly enhances the encapsulation effect of β-CD on EU and VC, making them more stable in water, extending their release time, and substantially prolonging the effectiveness of EU and VC. By examining the effects of transportation on the physiological, biochemical, and immune indicators of juvenile yellow catfish, this invention combines β-CD with VC or EU to enhance the effects of the active ingredients in anesthetizing the fry, slowing their metabolic activity, clearing free radicals, and alleviating oxidative damage. This results in reduced or absent responses to external stimuli, delays the rise in serum cortisol and blood glucose levels and antioxidant enzyme activity, mitigates damage to gills and liver tissue induced by live transport, and enhances the non-specific immune system of the fish. Furthermore, the encapsulated structure facilitates the adsorption of ammonia nitrogen in the water, reducing ammonia nitrogen pollution and water quality deterioration during transport. This enhances the stress resistance of the fry during transport, significantly extends transport time, improves survival rate, and largely preserves muscle quality.

[0050] Under normal circumstances, the effect of a single substance is limited. Adding vitamins without eugenol results in fish fry not being anesthetized, having poor sedation effects, and exhibiting high stress levels, which reduces survival rates. Similarly, adding eugenol without vitamins leads to poor immune function and increased liver congestion in transported fish fry, also resulting in lower survival rates. To better maintain the relative stability of the fish's physiological and biochemical indicators and alleviate the adverse effects of transport stress on fish fry, this invention preferably includes both eugenol and vitamin C in the inclusion compound particles. Preliminary experiments revealed that when the concentration of clove oil is too high, the fish fry will be deeply anesthetized and die directly; when the concentration is too low, it is ineffective. Excessive vitamin addition is wasteful, while insufficient amounts are ineffective in providing immune protection. Therefore, appropriate amounts of eugenol and vitamin C can help reduce stress in fish. On the one hand, encapsulating effective amounts of eugenol and vitamin C into the β-cyclodextrin polymer to create a sustained-release material can prevent the oxidation and volatilization of eugenol and vitamin C, improve their stability and solubility, and exert antioxidant and anesthetic / sedative effects. On the other hand, vitamin C can also promote the solubility of eugenol, and the two can act as antioxidants to each other, enhancing their respective stability. Thus, the combination of eugenol and vitamin C can simultaneously increase the concentration and stability of the active ingredients in the encapsulation particles. Consequently, only a small amount of chemical components are needed to produce effects in a relatively short time, enhancing their antioxidant, anesthetic, and sedative effects, greatly reducing transport stress damage to fish fry, and significantly improving survival rates. Optionally, in the inclusion complex particles, the mass ratio of eugenol, vitamin C, and β-cyclodextrin is 1:0.5-8:50-800. For example, the amount of eugenol is 11.34 mg-45.36 mg, the amount of vitamin C is 22.68 mg-90.72 mg, and the amount of β-cyclodextrin is 2.268 g-9.072 g. Preferably, the mass ratio of eugenol, vitamin C, and β-cyclodextrin is 1:2-8:20-800; more preferably, it is 1:5:681.

[0051] The ratio of starch, acrylic acid, and β-cyclodextrin affects the formation of the β-cyclodextrin polymer. Excessive acrylic acid and starch content can lead to acrylic acid self-polymerization, producing numerous small acrylic acid aggregates that cannot be grafted onto cyclodextrin, resulting in excessive impurities and poor encapsulation. Therefore, the content of acrylic acid and starch should be appropriate. Optionally, the mass ratio of starch, acrylic acid, and β-cyclodextrin in the inclusion complex particles is 1:0.6-10:1.26-18.144. For example, the amount of starch is 0.5-1.8 g, the amount of acrylic acid is 1.2-5.0 g, and the amount of β-cyclodextrin is 2.268-9.072 g. Preferably, the mass ratio of starch, acrylic acid, and β-cyclodextrin is 1:2.4:4.536-18.144; more preferably, it is 1:2.4:13.62.

[0052] In step S1, the method for preparing the gelatinized starch includes: dissolving starch in deionized water, heating under stirring conditions, then keeping warm and refluxing to fully gelatinize the starch, and cooling to room temperature for later use after gelatinization.

[0053] The stirring rate is 100-300 rpm, and the temperature is raised to 85±3℃.

[0054] In some preferred embodiments, the starch is tapioca starch.

[0055] Optionally, in step S1, the acrylic acid solution is an aqueous solution of acrylic acid, the eugenol solution is an eugenol ethanol solution, and the vitamin C solution is an aqueous solution of vitamin C. That is, the solvent for the acrylic acid solution and the vitamin C solution is water, and the solvent for the eugenol solution is ethanol.

[0056] In step S2, the method for preparing the β-cyclodextrin saturated aqueous solution includes: adding β-cyclodextrin to preheated distilled water and stirring to prepare a β-cyclodextrin (β-CD) saturated aqueous solution.

[0057] The temperature of the preheated distilled water is 30-50℃, preferably 40℃.

[0058] It is understood that the polymerization reaction of acrylic acid can be induced by generating active free radicals through irradiation. Therefore, the present invention does not impose special limitations on the irradiation source and irradiation dose. In a specific embodiment, in step S3, 0-9 kGy is irradiated using a cobalt source.

[0059] In step S3, filtration, washing, and drying are all conventional operations in the art. For example, the product is washed twice with distilled water, dried under vacuum at 45°C, ground, and sieved to obtain inclusion complex particles of β-cyclodextrin encapsulated with eugenol and / or vitamin C.

[0060] The fish fry mentioned above include yellow catfish fry.

[0061] Another embodiment of the present invention provides an embedded fish fry live transport anti-stress agent prepared by the method described above.

[0062] The method for preparing the embedded fish fry live transport anti-stress agent described above has the same advantages over the prior art as the method for preparing the embedded fish fry live transport anti-stress agent described above, and will not be repeated here.

[0063] Another embodiment of the present invention provides the application of the encapsulated anti-stress agent for live fish fry transport as described above in the live transport of fish fry.

[0064] The advantages of the embedded anti-stress agent for live fish fry transport are the same as those of the preparation method of the embedded anti-stress agent for live fish fry transport as described above compared with the prior art, and will not be repeated here.

[0065] In some preferred embodiments, the amount of the embedded anti-stress agent for live fry transport is 0.1-5 g / L, preferably 0.5-3 g / L, and more preferably 1 g / L, where the aforementioned amount refers to the mass of the anti-stress agent per liter of water. At this dosage, the increase in serum cortisol, lysozyme, and blood glucose levels, as well as the increase in the activity of oxidases and stress proteins in fry serum, can be effectively delayed, mitigating damage to gills and liver tissue induced by live transport, enhancing the non-specific immune system of fish, and improving transport survival rates.

[0066] The present invention will be further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments, unless otherwise specified, are generally performed according to the conditions recommended by the manufacturer.

[0067] Example

[0068] 1. Preparation of embedded anti-stress agents for live transport of fish fry

[0069] S1. Place 0.5g of cassava starch in a three-necked flask, add deionized water to dissolve it, and heat to 85±3℃ under stirring at 200rpm to keep it warm and reflux to fully gelatinize the cassava starch. After gelatinization, cool to room temperature, take 1.2g of acrylic acid and dissolve it in 10mL of distilled water, and slowly add it dropwise to the aforementioned three-necked flask containing gelatinized cassava starch. Heat the three-necked flask in a water bath, and introduce nitrogen gas into one end to ensure an inert atmosphere. Under a certain speed and temperature, add an anhydrous ethanol solution containing 10mg of eugenol (EU) dropwise over 5min. Then slowly add an aqueous solution of vitamin C (VC) containing 50mg of vitamin C (VC) and mix well to obtain solution A.

[0070] S2. Add 50 mL of distilled water preheated to 40 °C to a 100 mL beaker, add 6.81 g of β-cyclodextrin (β-CD), stir well to prepare a β-CD saturated aqueous solution, slowly add solution A to the beaker containing the β-CD saturated aqueous solution, stir at 200-800 r / min for 1-4 h, blow with nitrogen for 5 min, and mix well to obtain solution B;

[0071] S3. Using cobalt source irradiation solution B, the irradiation dose was 3 kGy. After irradiation, the solution was left to stand overnight at 4°C. Then, it was filtered, washed twice with distilled water, dried under vacuum at 45°C, ground, and sieved to obtain uniform β-cyclodextrin-encapsulated eugenol and / or vitamin C inclusion complex particles, named EU-10mg+VC-50mg / β-CD.

[0072] EU+VC / β-CD inclusion complex particles, EU / β-CD inclusion complex particles, and VC / β-CD inclusion complex particles were prepared using the above method. It should be noted that the difference between the EU / β-CD inclusion complex particles and the VC / β-CD inclusion complex particles and the EU+VC / β-CD inclusion complex particles mentioned above is that the corresponding substance is not added in step S1.

[0073] Five inclusion complexes were finally prepared: (1) β-cyclodextrin encapsulating eugenol, with eugenol content of 5 mg, EU-5mg / β-CD; (2) β-cyclodextrin encapsulating eugenol, with eugenol content of 10 mg, EU-10mg / β-CD; (3) β-cyclodextrin encapsulating vitamin C, with vitamin C content of 20 mg, VC-20mg / β-CD; (4) β-cyclodextrin encapsulating vitamin C, with vitamin C content of 50 mg, VC-50mg / β-CD; (5) β-cyclodextrin encapsulating 10 mg eugenol and 50 mg vitamin C, a complex inclusion complex EU-10mg+VC-50mg / β-CD.

[0074] 2. Inclusion complex sustained-release performance test

[0075] Accurately measure 50 mL each of three buffer solutions (prepared from KH₂PO₄ and K₂HPO₄) with pH values ​​of 5, 7, and 9 into 100 mL stoppered Erlenmeyer flasks. Place tea bags (6 cm × 8 cm) containing the inclusion complex into the Erlenmeyer flasks until submerged. Gently shake the flasks to ensure homogeneity of the eugenol in the medium. Sustained release was performed at 25 °C. After 0.5, 1, 2, 3, 6, 12, 24, 30, 48, and 72 h, 5 mL of liquid was collected, and its absorbance was measured using a UV-Vis spectrophotometer. The EU content of the sustained release was obtained according to the standard curve. After each sample was taken, 5 mL of buffer solution was added to the Erlenmeyer flask. All experiments were performed in triplicate, and the average value was taken as the final value.

[0076] Sustained-release assays were performed using three inclusion complexes: EU-5mg / β-CD, EU-10mg / β-CD, and EU-10mg + VC-50mg / β-CD. The sustained-release results are as follows: Figure 1As shown in the figure, the inclusion complexes all exhibit slow-release properties in aqueous solutions. EU is poorly soluble in water and has a certain degree of volatility. The inclusion complexes increase the water solubility of EU and prolong its effective time. In the initial release phase (≤3h), the release amount of EU is relatively large, with EU-5mg / β-CD and EU-10mg / β-CD releasing even faster, reaching 52% and 51% respectively within 2h. In the middle release phase, the release rate of EU slows down. EU-5mg / β-CD and EU-10mg / β-CD both reach their peak release point after 24h, releasing 73% and 77% respectively, while EU-10mg+VC-50mg / β-CD only reaches release equilibrium after 30h. As the release time continues, some of the released EU volatilizes, and the concentration begins to decrease slowly. The aforementioned data demonstrate that β-CD polymer encapsulation can effectively prolong the duration of eugenol's effect. The lower the encapsulation amount, the faster the eugenol volatilizes in the later stages. The addition of vitamin C (EU-10mg+VC-50mg / β-CD) further prolongs the release time of eugenol and improves the release efficiency, because VC can promote the dissolution of eugenol and maintain its stability.

[0077] 3. Simulated transport test of fish fry

[0078] The juvenile yellow catfish used in this experiment were purchased from Jingzhou City, Hubei Province, China, and were raised for 15 days in the Fish Research Laboratory of the Institute of Agricultural and Sideline Products Processing and Nuclear Agriculture Technology, Hubei Academy of Agricultural Sciences.

[0079] Fry acclimatization: The water temperature was maintained at 20±2℃, and 1 / 3 of the water was changed daily with fresh water. The fry were fed twice daily with commercial feed (expanded yellow catfish feed, purchased from Tongwei Co., Ltd.) (containing 35% crude protein), at a rate of 3% of their total weight. They were reared in a 12h / 12h light / dark cycle. Dissolved oxygen and ammonia levels were monitored daily and controlled to concentrations of 7.5±0.35 mg / L and 0.23±0.04 mg / L, respectively. All experiments described in this study were conducted under the authorization of the Experimental Animal Resources Committee of the Hubei Academy of Agricultural Sciences.

[0080] Fish fry transportation: After the fish fry acclimatized to the environment, 2000 fish of similar size (weight 15.74±1.04g, body length 10.26±0.87cm) were selected and fasted for 24 hours before transportation. The fish fry were added to the water at a fish-to-water ratio of 1:2, followed by the addition of 1g of additive / L of water. The additives were EU-5mg / β-CD, EU-10mg / β-CD, VC-20mg / β-CD, VC-50mg / β-CD, EU-10mg+VC-50mg / β-CD, and β-CD, respectively. A control group was set up without any additives. Each experimental group had three replicates. The fish were placed in plastic boxes (22cm×14cm×15cm) containing 1L of water with an oxygen concentration greater than 6±1.0mg / L, and continuously aerated. Samples were collected at 4h, 8h, and 24h of transportation.

[0081] Sample Collection: Temperature, pH, and dissolved oxygen were measured directly after transport using a water quality analyzer (HQ40d; Hach, Shanghai, China). Nine fish (three replicates per group) were collected from each of the seven groups, and water samples were collected simultaneously. 1 mL of blood was collected from the tail vein of the experimental fish using a non-heparinized syringe, centrifuged at 3000 rpm for 10 min at 4°C, and the plasma was collected and stored at -20°C for subsequent testing. The liver and gills were removed and stored at -80°C for further analysis. The liver and third gill were removed, soaked in 4% paraformaldehyde, and stored at 4°C until use. The remaining fish were placed in a recirculating aquaculture system after sampling. Fish survival was checked after 36 h.

[0082] 3.1 Survival rate of juvenile yellow catfish and water quality during transportation

[0083] Table 1 records the effects of various additives on the survival rate of juvenile yellow catfish during transportation. Transportation for 4 hours had no significant effect on fry mortality. After 24 hours, significant differences in fry survival rates emerged. The VC-10mg / β-CD group had the lowest survival rate at only 78%. The EU-10mg + VC-50mg / β-CD group showed the advantage, having the highest cumulative survival rate, followed by the EU-10mg / β-CD group (93%).

[0084] Table 1. Effects of six different additives on the survival rate of juvenile yellow catfish at different time points during transportation.

[0085]

[0086] 3.2 Water quality during transportation

[0087] Figure 2The effects of various additives on water pH during transport are shown. pH values ​​for all groups ranged from 6.08 to 6.94, fluctuating with increasing transport time. There were no significant differences in the average pH values ​​between the groups, and they converged after 24 hours.

[0088] During fish transportation, water quality may change rapidly, especially when fish density is high. If ammonia nitrogen content increases, the oxygen-carrying capacity of the blood decreases, which can easily lead to fish hypoxia and death. Figure 3 The effects of various additives on ammonia nitrogen levels in water during transport were shown. The total ammonia nitrogen levels in each additive group were lower than those in the control group. The total ammonia nitrogen levels increased rapidly during transport. The EU-10mg+VC-50mg / β-CD group had the lowest ammonia nitrogen levels after 24 hours of transport, and no significant deterioration in water quality was observed.

[0089] 3.3 Serum stress markers

[0090] The following indicators were analyzed using commercial kits according to the manufacturer's instructions. Specifically, the fish serum cortisol kit was purchased from Purity Biotechnology Co., Ltd. (Wuhan, China), catalog number Y-106379; the serum glucose kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), catalog number GLU A154-1-1, using the glucose oxidase-microplate method; and the serum immunoglobulin G (IgG) kit was purchased from ZOKEYO (Wuhan, China), catalog number Y-106379.

[0091] Glucose is an essential energy source for various life activities in fish. Blood glucose levels regulate cortisol release and glucose homeostasis through glycogen metabolism and gluconeogenesis. Fish use blood glucose as their primary energy source, a process regulated by the nervous and endocrine systems. Blood glucose levels increase significantly when subjected to transport stress. Figure 4 The effects of various additives on serum glucose (GLU) concentration were shown. Serum GLU levels in each group showed a gradual upward trend over time, followed by a decrease. The β-CD group and the control group showed a continuous increase over time, reaching their highest level at 24 hours of transport, significantly higher than other experimental groups (p<0.05). The EU-10mg + VC-50mg / β-CD group maintained a low blood glucose level throughout transport, indicating that eugenol and VC can effectively inhibit the increase in serum blood glucose during the transport of juvenile yellow catfish and reduce the degree of stress response.

[0092] Plasma cortisol is one of the most commonly used stress indicators in fish, and the severity of fish stress can be represented by the degree of increase in COR. Figure 5The effects of various additives on serum cortisol (COR) concentration were shown. Throughout the 24-hour live transport process, serum cortisol (COR) levels in all groups showed an increasing trend, indicating that the fry responded to stress and secreted large amounts of COR. The EU-10mg + VC-50mg / β-CD group maintained a consistently low COR level throughout the live transport process, and the serum COR level in yellow catfish fry was the lowest among the experimental groups, which to some extent suggests a less intense stress response in the fry.

[0093] It is rich in IgG and widely distributed. It can easily penetrate capillary tissue and plays a vital role in the defense mechanism of fish mucosal immunity. Figure 6 The effects of various additives on serum immunoglobulin G (IgG) concentrations were shown. Throughout the 24-hour live transport process, serum IgG levels steadily increased over a period of time, with the IgG level in the EU-5mg / β-CD group being significantly higher than other groups (p<0.05). All additive groups showed a continuous increase in IgG levels over time, while the IgG level in the EU-10mg+VC-50mg / β-CD group did not change significantly and remained at a low level. This indicates that the combined use of eugenol and vitamin C can keep yellow catfish fry in a sedated state during live transport, and that IgG is produced and remains stable as a resistance mechanism when yellow catfish fry are exposed to environmental stress.

[0094] 3.4 Liver stress indicators

[0095] Superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and lysozyme (LZM) were analyzed using commercial kits according to the manufacturer's instructions. Specifically, the liver superoxide dismutase kit (SOD A001-3) was purchased from Nanjing Jiancheng Biotechnology Institute (Nanjing, China) and analyzed using the WST-1 method; the catalase kit (CAT A007-1-1) was purchased from Nanjing Jiancheng Biotechnology Institute (Nanjing, China) and analyzed using the ammonium molybdate method; the malondialdehyde kit (MDA A003-1) was purchased from Nanjing Jiancheng Biotechnology Institute (Nanjing, China) and analyzed using the TBA method; and the lysozyme kit (LZM A050-1-1) was purchased from Nanjing Jiancheng Biotechnology Institute (Nanjing, China) and analyzed using the turbidimetric method.

[0096] Under normal conditions, the production and scavenging of free radicals in fish are in equilibrium, thanks to the combined action of SOD and CAT enzymes. However, when environmental factors change, the production of reactive oxygen species (ROS) far exceeds consumption as transport time increases, leading to a series of stress responses in the fish and causing varying degrees of damage. When the amount of free radicals produced exceeds the amount that can be scavenged, SOD and CAT enzymes also increase significantly to scavenge them. Increased SOD and CAT enzyme levels indicate increased stress. Adding EU-10mg + VC-50mg / β-CD significantly reduced fish stress, resulting in significantly lower stress levels in the fish 24 hours after transport compared to the control group. Consequently, SOD and CAT levels decreased. SOD reacted excess ROS to generate hydrogen peroxide (H2O2), and then CAT catalyzed H2O2 to produce harmless H2O and O2. EU-10mg + VC-50mg / β-CD not only scavenges free radicals but also resists lipid peroxidation.

[0097] Figure 7 The effects of various additives on superoxide dismutase (SOD) activity were shown. The figure shows that the VC-50mg / β-CD group exhibited the highest SOD activity in fish fry after 4 hours of transport, reaching 256.28 U / mg prot, significantly higher than the control group (P<0.05) and also higher than the activities of other experimental groups, decreasing with prolonged transport time. After 24 hours of transport, the β-CD group showed the highest SOD activity compared to other experimental groups, at 234.86 U / mg prot, indicating that oxidative stress intensified with prolonged survival transport time. The SOD activities of the remaining experimental groups were all lower than the control group, and the EU-10mg + VC-50mg / β-CD group consistently showed the lowest activity throughout the entire transport process, suggesting that the combined use of eugenol and vitamins can more quickly eliminate oxidative stress.

[0098] Figure 8 The effects of various additives on catalase (CAT) activity were shown. The CAT content of each additive group showed a trend of first increasing and then decreasing with increasing transport time. After 4 hours of transport, the EU-10mg + VC-50mg / β-CD group had the lowest CAT content, which was significantly lower than that of the other groups (P<0.05).

[0099] Figure 9The effects of various additives on malondialdehyde (MDA) concentration were shown. MDA can indicate the content of lipid peroxides to some extent, and the trends of MDA and CAT were similar across the groups. Excessive stress leads to the production of a large number of free radicals in the body, which SOD and CAT cannot clear in time. Peroxides accumulate, forming products of lipid peroxidation, resulting in a significant increase in MDA. Transport stress causes considerable damage to the fish. As the duration of additive application increases, free radicals are slowly cleared, the MDA content in the body begins to decrease, lipid peroxides begin to decrease, and the damage to the body diminishes. Previous studies have found that eugenol can protect cell membrane lipids from oxidation and inhibit the formation of hydroxyl free radicals. Therefore, it is speculated that eugenol exerts a protective function during the anesthesia of yellow catfish fry, stimulating the oxidative defense capacity of tissue cells, thereby rapidly resisting oxidative damage caused by transport stress.

[0100] Figure 10 The effects of various additives on lysozyme (LZM) activity were shown. Fish lysozyme acts on the peptidoglycan layer of bacterial cell walls, leading to bacterial lysis. It exhibits opsonin activity, limited antiviral and antitumor activities, and can activate the complement system and phagocytes. Fish lysozyme activity is relatively strong and increases with other stresses, participating in a wide range of defense mechanisms and playing a crucial role in fish immune defense. Higher LZM levels indicate stronger stress, thus triggering stronger defenses and causing greater damage to the fish. In the control group, LZM levels increased over time during transport, reaching a maximum after 24 hours. The LZM content in the EU-10mg+VC-50mg / β-CD group remained at a low level 8 hours after transportation, and increased slightly after 24 hours, but was still much lower than that in the control group. This indicates that the compound additive had a good protective effect on the fish in the first 8 hours of transportation, increasing the fish's immunity and stress resistance. The protective effect slowed down after 24 hours, but the overall level remained at a low level throughout the process, significantly lower than all other groups (P<0.05).

[0101] 3.5 Observation of tissue damage sections

[0102] The liver and gill tissue damage was observed through HE tissue sections. The specific procedure is as follows:

[0103] 1) Tissue collection: Fresh tissue is fixed in fixative for at least 24 hours. The tissue is removed from the fixative and trimmed with a scalpel in a fume hood. The trimmed tissue and corresponding labels are then placed in a dehydration box.

[0104] 2) Dehydration and wax impregnation: Place the dehydration box into the dehydrator for gradient dehydration. 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol I for 30 minutes, anhydrous ethanol II for 30 minutes, benzene for 5-10 minutes, xylene I for 5-10 minutes, xylene II for 5-10 minutes, molten paraffin I at 65℃ for 1 hour, molten paraffin II at 65℃ for 1 hour, molten paraffin III at 65℃ for 1 hour.

[0105] 3) Embedding: The paraffin-impregnated tissue is embedded in an embedding machine. First, the molten paraffin is placed into the embedding frame. Before the paraffin solidifies, the tissue is removed from the dehydration box and placed into the embedding frame according to the embedding surface requirements, and the corresponding label is attached. The tissue is cooled on a -20°C freezing stage. After the paraffin solidifies, the paraffin block is removed from the embedding frame and trimmed.

[0106] 4) Sectioning: Place the trimmed wax block on a -20℃ freezing stage to cool, then place the cooled wax block on a paraffin microtome to section it to a thickness of 4μm. Float the sections on 40℃ warm water in a slide to flatten the tissue, then lift the tissue onto a glass slide and bake it in a 60℃ oven. After the wax has melted from the water, remove the slide and store it at room temperature for later use.

[0107] Histological sections of the liver can better assess the extent of liver damage. Necrosis is closely related to the body's oxidative stress; lipid peroxidation leads to membrane damage, and these oxidized forms may result in hepatocyte necrosis. Figure 11Images of liver tissue sections from juvenile yellow catfish transported for 24 hours are shown. Figures (a)-(g) represent the control group, EU-5mg / β-CD group, EU-10mg / β-CD group, VC-20mg / β-CD group, VC-50mg / β-CD group, EU-10mg+VC-50mg / β-CD group, and β-CD group, respectively. Arrows indicate blood (bc), hepatocyte edema (hs), portal vein branches containing erythrocytes (hpv), focal necrosis of liver tissue (fn), and vacuolation (va). The images show that in the control group, the size of the liver interstitial tissue was reduced, the boundaries of the liver lobules were indistinct, and the hepatocytes were round or slightly oval, tightly packed, filled with blood cells and multiple congestion points. The EU-10mg / β-CD group had more congestion points, lighter cell staining, disordered cell arrangement, and even observed hepatocyte vacuolation. In the VC-20mg / β-CD group, hepatocytes were clearly and orderly arranged, but multiple areas of congestion were observed in the liver tissue. The VC-50mg / β-CD group and the β-CD group showed the worst performance, with blurred tissues and even focal necrosis of the liver tissue. The results indicate that adding VC or β-CD alone has a poor protective effect on liver tissue, resulting in significant liver necrosis. Adding clove oil alone also causes stem cell vacuolation. Only the EU-10mg + VC-50mg / β-CD group showed clear and orderly arranged hepatocytes, and only a few congestion points appeared after transportation. Therefore, the EU-10mg + VC-50mg / β-CD group had a better relieving effect on liver tissue after transportation.

[0108] Gills are the main respiratory organs for gas exchange in fish, and they also play a role in maintaining osmotic pressure balance, removing ammonia and nitrogen excretions, and regulating the body's pH value. The integrity of gill tissue morphology is crucial for the normal life activities of fish. Figure 12Images of gill tissue sections from juvenile yellow catfish transported for 24 hours are shown. Figures (a)-(g) represent the control group, EU-5mg / β-CD group, EU-10mg / β-CD group, VC-20mg / β-CD group, VC-50mg / β-CD group, EU-10mg+VC-50mg / β-CD group, and β-CD group, respectively. Arrows indicate columnar cells (pc), epithelial cells (ec), aneurysms (an), hyperplasia (hp), lamellar curling (cl), and edema (ed). In the control group, gill filaments underwent lamellar curling and cell proliferation after transport. All experimental groups showed gill filament abnormalities after transport, indicating varying degrees of damage to the gill tissue structure of juvenile yellow catfish, including mucus cell swelling and hyperplasia, gill lamellae bending and breakage, and erythrocyte damage. The VC-20mg / β-CD group showed the most gill filament hyperplasia and the greatest degree of gill filament bending. In the EU-10mg / β-CD group, gill filament congestion and edema were observed. The VC-50mg / β-CD group showed the least gill filament curling, but some edematous tissue and aneurysms were observed. This indicates that adding VC alone cannot prevent gill filament proliferation, and adding EU alone will also cause gill filament congestion. Only the EU-10mg+VC-50mg / β-CD group, which simultaneously embeds EU and VC, showed the least cell proliferation in gill tissue sections, with clearly visible epithelial cells and column cells, and less gill filament curling, indicating that the EU-10mg+VC-50mg / β-CD group was most effective for yellow catfish fry. Overall, the EU-5mg / β-CD group and the EU-10mg+VC-50mg / β-CD group showed relatively mild gill tissue edema.

[0109] 3.6. Relative transcriptional levels of genes related to the TLR and NLR signaling pathways.

[0110] RNA was extracted from liver samples using an RNA extraction kit (Servicebio, Wuhan, China, catalog number G3013), and its concentration, purity, and absorbance were determined using Nanodrop 2000 software. RNA was diluted to a final concentration of 200 ng / µL. Complementary DNA (cDNA) was synthesized using a cDNA synthesis kit (Servicebio, Wuhan, China, catalog number G3330) according to the manufacturer's protocol. Relative transcriptional levels of genes associated with the TLR and NLR signaling pathways were determined by quantitative real-time polymerase chain reaction (qRT-PCR) using the following procedure: 95°C for 30 sec per cycle, 95°C for 15 sec, 60°C for 30 sec, for 40 cycles. β-actin was used as a reference gene. Relative transcript levels were determined using the 2-ΔΔCT method. Primers used are listed in Table 2.

[0111] Table 2. Sequences of oligonucleotide primers used for real-time quantitative PCR in this embodiment.

[0112]

[0113] Note: HSP, heat shock protein; TNF-α, tumor necrosis factor α; IL-1β, interleukin-1β; bp, base pair.

[0114] Heat shock proteins (HSPs) are indicator proteins of stress. HSPs are highly conserved, and their expression is significantly upregulated under stress. They are frequently used to reflect the degree of stress and can serve as molecular biomarkers to assess the dangerous state of tissues and cells. This study uses the relative expression levels of heat shock proteins to reflect the stress response of juvenile yellow catfish before and after transportation. Under normal conditions, HSP levels are low and stable, but their expression is upregulated after exposure to stress. Hsp70, on the other hand, is not expressed or expressed in small amounts under normal conditions, but its expression increases rapidly after cellular stress. Figure 13 The effects of various additives on the expression level of the heat shock protein HSP70 were shown. After transportation, the HSP70 gene level was upregulated in the control group of yellow catfish fry, while it was downregulated in the additive groups, especially the EU-10mg+VC-50mg / β-CD group, which showed the largest decrease in HSP70 gene expression, significantly lower than the control group (P<0.05). This indicates that the addition of inclusion complex particles reduced the stress caused by transportation to yellow catfish fry.

[0115] IL-1β and TNF-α are immune signaling molecules and are commonly used non-specific immune marker genes in fish. Their expression levels are upregulated under stress. The expression levels of TNF-α and IL-1β genes indicate the ability of liver cells to resist inflammatory responses. Figure 14-15The effects of various additives on the expression levels of TNF-α and IL-1β genes were shown. Tumor necrosis factor (TNF) is one of the most potent bioactive factors that can directly cause tumor cell death, but it has no significant toxicity to normal cells. TNF not only has a cytotoxic effect on tumor cells, but also participates in various pathological physiological processes such as antiviral infection and inflammation. Overall, the additives reduced the expression level of the TNF-α gene, thus reducing the inflammatory response in fish during transportation. In contrast, the groups with 10 mg clove oil and 20 mg vitamin C alone showed upregulation of TNF-α gene expression after 8 hours of transportation, indicating that clove oil and vitamin C alone cannot completely resist inflammatory factors and cause an inflammatory response in fish. The EU-10 mg + VC-50 mg / β-CD group showed downregulation after both 8 and 24 hours of transportation, and the expression level of the TNF-α gene decreased with increasing transportation time, indicating that the compound additives reduced inflammation and promoted a decrease in the expression levels of TNF-α and IL-1β genes.

[0116] In summary, this invention alleviates the stress caused by transporting juvenile yellow catfish by adding different types and concentrations of β-CD inclusion complexes to the transport water. By examining the effects of transport on the physiological, biochemical, and immune indicators of juvenile yellow catfish, it was found that after 4 hours of transport, the liver oxidative stress index was affected, but not as severely as after 24 hours. β-CD inclusion complexes containing vitamin C or eugenol can effectively alleviate the stress response, mitigating damage and apoptosis induced by transport to the gill and liver tissues, delaying the rise in serum cortisol and blood glucose levels, inhibiting the increase in antioxidant enzyme activity, enhancing the antioxidant capacity of the juveniles, preventing water quality deterioration, and eliminating stress responses more quickly, thereby improving the stress resistance of the juveniles and increasing their survival rate during transport. The EU-10mg + VC-50mg / β-CD group showed lower levels of oxidative stress markers (SOD and CAT) compared to other additive groups. Liver and gill tissue sections showed the least tissue damage in the EU-10mg + VC-50mg / β-CD group. Serum COR and GLU stress markers were also lowest in the EU-10mg + VC-50mg / β-CD group, indicating the best efficacy and lower post-transport mortality. Furthermore, the downregulation of HSP70, TNF-α, and IL-1β genes reduced inflammatory responses and lowered infection rates, reflecting the efficacy of the compound additive in anti-inflammatory and immune-boosting effects.

[0117] It should be noted that all values ​​in this embodiment are expressed as Mean ± SE and analyzed using SPSS V.19 (IBM, Armonk, NY, USA). Data distribution was assessed using the Shapiro-Wilk test, and the uniformity of variance was assessed using the Levene test. T-tests were used to compare the changes in fish in the six additive groups at each time period. Efficacy regression analysis (density × time) was used to assess the transport effect (Vidal et al., 2008). Stress and immune parameters were analyzed using two-way ANOVA (treatment × sampling time), and Duncan's test was used to test for significant differences between treatments, with statistical significance defined as P < 0.05.

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an encapsulated anti-stress agent for live transport of yellow catfish fry, characterized in that, Includes the following steps: S1. Mix gelatinized starch with acrylic acid solution, then add eugenol solution and vitamin C solution, and mix well to obtain solution A; S2. Under an inert atmosphere, add a saturated aqueous solution of β-cyclodextrin to solution A and mix well to obtain solution B; S3. Irradiate the solution B, let it stand overnight after irradiation, then filter, wash and dry to obtain inclusion complex particles of β-cyclodextrin encapsulated with eugenol and vitamin C; In the inclusion complex particles, the mass ratio of eugenol, vitamin C, and β-cyclodextrin is 1:5:681; the mass ratio of starch, acrylic acid, and β-cyclodextrin is 1:2.4:13.

62.

2. The method for preparing the encapsulated anti-stress agent for live transport of yellow catfish fry according to claim 1, characterized in that, In step S1, the method for preparing the gelatinized starch includes: dissolving starch in deionized water, heating under stirring conditions, and then keeping it warm under reflux to fully gelatinize the starch; wherein the stirring rate is 100-300 rpm and the temperature is raised to 85±3℃. In step S2, the method for preparing the β-cyclodextrin saturated aqueous solution includes: adding β-cyclodextrin to preheated distilled water and stirring to prepare a β-cyclodextrin saturated aqueous solution; wherein the temperature of the preheated distilled water is 30-50℃; In step S3, 3 kGy is irradiated using a cobalt source.

3. An encapsulated anti-stress agent for live transport of yellow catfish fry, characterized in that, It is prepared by the method for preparing the embedded fish fry live transport anti-stress agent according to any one of claims 1-2.

Images