A polysaccharide copper, a polysaccharide copper gel and a preparation method and application thereof

Abstract

The present application belongs to the field of biological medicine, and relates to aquaculture, in particular to a copper compound, in particular to a polysaccharide copper, polysaccharide copper gel and a preparation method and application thereof. The preparation method of the polysaccharide copper or polysaccharide copper gel comprises the following steps: dissolving polysaccharide, starch and trisodium citrate in water, stirring uniformly to obtain a mixed solution; slowly adding a hydrochloric acid solution of CuCl2 into the mixed solution, adjusting pH, and then performing water bath heating reaction; centrifuging, dialyzing and freeze-drying the obtained product to obtain polysaccharide copper; and concentrating the dialysate to a concentration of 1.01-1.35 g / mL to obtain polysaccharide copper gel. The copper gel prepared by the present application has good antibacterial activity on gram-positive bacteria and gram-negative bacteria, can slowly release copper, has low acute toxicity to mice, and has no obvious toxicity to zebra fish larvae.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, relates to aquaculture, and specifically relates to a copper complex, particularly a polysaccharide copper, a polysaccharide copper gel, its preparation method and application. Background Technology

[0002] Copper sulfate is widely used as an insecticide in aquaculture in my country. It not only kills fish parasites but also has a strong killing effect on harmful organisms such as moss and cyanobacteria. However, gills are important respiratory organs for fish and come into direct contact with the aquaculture water. Excessive concentrations of copper ions in the water can first enter the fish's body through the gills, causing toxicity. Studies have shown that copper ions can cause congestion and dilation of capillaries inside the gills, inflammation and edema of epidermal cells, and in severe cases, even cell necrosis. High concentrations of the inorganic heavy metal copper ions can cause chronic poisoning in aquatic animals, even leading to death. Furthermore, the concentration of free copper ions in the water affects the survival rate of fish embryos, the malformation rate of newly hatched fry, and the mortality rate. However, if the concentration of copper ions in the water is too low, it will not have an effective insecticidal or bactericidal effect. Especially in ornamental fish farming, due to the small size of the aquarium, it is difficult to accurately control the concentration of copper ions in the water. Publication number CN106135324A discloses a disinfectant for aquaculture water, which uses a combination of chlorine dioxide, dibromohydantoin, quicklime, garlic, Sichuan pepper, copper sulfate, and ferrous sulfate as a disinfectant, employing the direct addition of copper sulfate. Publication number CN105923720A discloses a method for killing bacteria, viruses, and microorganisms in aquaculture water using copper and silver ions, where copper and silver ions are also added directly. Furthermore, in aquaculture, antibiotics are often used in large quantities to reduce the harm of bacteria, fungi, and other microorganisms to fish. For example, the aquatic feed in publication number CN110510757A directly adds various chemical substances and antibiotics. Antibiotics have wide diffusion in water, easily leading to a continuous increase in antibiotic residues in the surrounding environment and the emergence of drug-resistant bacteria. Metal ion antibacterial agents do not cause microorganisms to develop drug resistance. In addition, there are currently no safe and effective copper supplements for copper deficiency, a rare human disease. Therefore, there is an urgent need for a complex that can slowly and steadily release copper, exert a long-lasting antibacterial effect while having low toxicity, in order to meet the growing market demand. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention proposes a polysaccharide copper, a polysaccharide copper gel, its preparation method, and its applications. The technical solution of this invention is achieved as follows: a method for preparing polysaccharide copper or a polysaccharide copper gel, comprising the following steps: (1) Dissolve the polysaccharide, starch and trisodium citrate in water and stir until a mixed solution is obtained; (2) The hydrochloric acid solution of CuCl2 is slowly added dropwise to the mixed solution in step (1), the pH is adjusted and the reaction is heated in a water bath. The resulting product is centrifuged and dialyzed. The dialysate is then freeze-dried to obtain a polysaccharide copper complex. The dialysate is concentrated to a concentration of 1.01-1.35 g / mL and allowed to stand to obtain a polysaccharide copper gel.

[0004] In step (1) above, the mass ratio of polysaccharide, starch and trisodium citrate is (0-1):(0.1-5):1, where the polysaccharide is Chinese yam polysaccharide.

[0005] The above mixed solution is a clear and transparent solution that is fully dissolved.

[0006] In step (2) above, the molar ratio of CuCl2 to trisodium citrate in step (1) is (0.1-10):1.

[0007] The pH value is 5.5-10.5; the water bath heating temperature is 30-100℃ and the time is 1-48 h.

[0008] The polysaccharide copper gel prepared by the above method can slowly release polysaccharide copper in aqueous solution. The released polysaccharide copper is a macromolecular complex and is spherical.

[0009] The above-mentioned polysaccharide copper gel is used in aquaculture.

[0010] The above-mentioned polysaccharide copper is used in the preparation of copper supplements or antibacterial agents for copper deficiency diseases in animals or humans.

[0011] The present invention has the following beneficial effects: 1. This invention is the first to prepare a copper polysaccharide gel from Chinese yam. In aqueous solution, the copper polysaccharide gel can slowly release copper complexes over 48 hours, avoiding the toxicity to aquatic organisms caused by a sudden increase in copper ion concentration in the water. The released copper polysaccharide is a macromolecular complex that is spherical after freeze-drying. The released copper polysaccharide has low toxicity and good antibacterial properties, and can be used for antibacterial purposes in fish farming.

[0012] 2. The polysaccharide copper prepared by this invention has been shown to have low toxicity through zebrafish toxicity experiments. It is expected to be used as a copper supplement or antibacterial agent for copper deficiency diseases in animals or humans, filling the gap in the lack of low-toxicity copper preparations for the rare disease "copper deficiency".

[0013] 3. In this application, the infrared spectra of copper polysaccharide from Chinese yam and the corresponding Chinese yam polysaccharide were compared, and the C=O stretching vibration peak at 1743.8 cm⁻¹ in the polysaccharide was clearly visible. -1 The disappearance of the peak indicates that a coordination reaction has occurred; the stretching vibration peak of the glycosidic bond in the polysaccharide decreased from 1627.9 cm⁻¹. -1 It shifted to a lower wavenumber of 1612.5 cm. -1This indicates that the glycosidic bond has reacted. Furthermore, the saturated CH stretching vibration peak in the polysaccharide is at 2947.2 cm⁻¹. -1 The disappearance of the peak indicates a change in the adjacent hydroxyl group, masking the peak. The in-plane bending vibration of the hydroxyl group βOH is at 1396.46 cm⁻¹. -1 The peak position was significantly shifted to lower wavenumbers compared to the corresponding peak position of the polysaccharide. This indicates that the polysaccharide-copper complex prepared in this application holds promise for use in the preparation of blue pigments or dyes due to its low toxicity. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 The effect of pH on copper gel of Chinese yam polysaccharide.

[0016] Figure 2 This is a dialysis image of copper polysaccharide from Chinese yam.

[0017] Figure 3 This is a graph showing the amount of precipitate in the copper reaction of Chinese yam polysaccharide.

[0018] Figure 4 The effect of concentration on the copper gel of Chinese yam polysaccharide.

[0019] Figure 5 The effect of temperature on copper gel of Chinese yam polysaccharide.

[0020] Figure 6 The effect of reaction time on the copper gel of Chinese yam polysaccharide.

[0021] Figure 7 The image shows the copper infrared spectrum of Chinese yam polysaccharide.

[0022] Figure 8 The image shows the copper ultraviolet spectrum of Chinese yam polysaccharide.

[0023] Figure 9 This is a scanning electron microscope image of copper polysaccharide from Chinese yam.

[0024] Figure 10 The thermogravimetric curve of copper in Chinese yam polysaccharide.

[0025] Figure 11 XRD pattern of copper polysaccharide from Chinese yam.

[0026] Figure 12 The amount of food consumed by mice in each group.

[0027] Figure 13 This image shows the antibacterial effect of copper-containing polysaccharides from Chinese yam.

[0028] Figure 14 Photos of zebrafish in the normal group and the drug-treated group. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1 The steps of the synthesis process of copper gel polysaccharide from Dioscorea opposita in this embodiment are as follows: Take a certain amount of Chinese yam polysaccharide and water-soluble starch (mass ratio 1:1), add a certain amount of water, heat and stir thoroughly to dissolve the Chinese yam polysaccharide and water-soluble starch completely to form a clear solution. After cooling to room temperature, add the same number of moles of copper chloride and sodium citrate to make the concentration of both 0.15 mol / L. Adjust the pH value to 6.5, 7.5, 8.5 and 9.5 respectively, heat in a water bath at 70℃ for 1.5 hours. After the reaction is complete, centrifuge at 4000 r / min for 5 min, take the supernatant for dialyzing, concentrate the dialysate under reduced pressure, and place the concentrate at room temperature to obtain blue Chinese yam polysaccharide copper gel.

[0031] Using a single-factor investigation method, the analysis of five factors revealed that pH had the greatest impact on the experimental results. The coordination ability of the ligand to the metal is related to its stability constant, which is strongly affected by pH. The lower the pH, the greater the acid effect and the smaller the stability constant. The acid effect decreases as the solution pH increases, but when the solution pH is too high, metal ions tend to hydrolyze with hydroxide ions in the solution. Simultaneously, a strongly alkaline environment causes the hydrogen bonds between polysaccharide and starch molecules to break, all of which affect the stability of the complex. Therefore, selecting a suitable pH is crucial for the formation of the complex. When the pH is low, dialysis after the reaction yields a large amount of white precipitate, indicating that the complex formed at this time is unstable. When pH > 10, a large amount of white, viscous substance appears during pH adjustment, easily adhering to the pH meter, and dialysis after the reaction yields a small amount of light blue precipitate. When the pH is too high, a blue precipitate forms during the reaction. pH is crucial for the formation of copper gel from Dioscorea opposita polysaccharide. Figure 1 It is evident that the gels formed at pH 6.5 and 7.5 have greater fluidity, while the gels formed at pH 8.5 and 9.5 have better solidification properties and can be stabilized when inverted.

[0032] Different reactant ratios significantly affect the product. For example, a certain amount of Chinese yam polysaccharide and water-soluble starch (mass ratio 1:1) are taken, and a certain amount of water is added. The mixture is heated and stirred thoroughly until the Chinese yam polysaccharide and water-soluble starch are completely dissolved, forming a clear solution. After cooling to room temperature, copper chloride and sodium citrate are added to make the copper chloride concentration 0.12 mol / L and the sodium citrate concentrations 0.08 mol / L and 0.3 mol / L, respectively. The pH is adjusted to 8.5, and the mixture is heated in a water bath at 70℃ for 1.5 hours. After the reaction is complete, the mixture is centrifuged at 4000 r / min for 5 min, and the supernatant is dialyzed. The dialysate is concentrated under reduced pressure, and the concentrate is left at room temperature to obtain the final product. When the copper ion ratio is low, stratification easily occurs during dialysis, indicating that the copper stability of the prepared Chinese yam polysaccharide is relatively poor. Figure 2 As shown. However, when the proportion of copper ions is too high, insoluble copper hydroxide precipitate is easily formed during the reaction, such as... Figure 3 As shown. A stable complex can only be formed when the proportions of the reactants are appropriate.

[0033] The concentration of reactants has a significant impact on the yield. For example, a certain amount of Chinese yam polysaccharide and water-soluble starch (mass ratio 1:1) are taken, and a certain amount of water is added. The mixture is heated and stirred thoroughly until the Chinese yam polysaccharide and water-soluble starch are completely dissolved, forming a clear solution. After cooling to room temperature, equimolar amounts of copper chloride and sodium citrate are added to achieve concentrations of 0.05 mol / L, 0.10 mol / L, 0.15 mol / L, and 0.20 mol / L, respectively. The pH is adjusted to approximately 8.5, and the mixture is heated in a water bath at 70℃ for 1.5 hours. After the reaction is complete, the mixture is centrifuged at 4000 r / min for 5 min. The supernatant is dialyzed, and the dialysate is concentrated under reduced pressure. The concentrate is then left at room temperature to obtain the final product. When the concentration of the copper chloride solution is low, the yield decreases sharply due to the low reactant concentration. This may be because the low reactant concentration reduces the degree of reaction, resulting in significant losses during dialysis. Furthermore, the low concentration is not conducive to gel formation. Figure 4 As shown, when the concentration of the copper chloride solution is too high, copper is easily hydrolyzed during the reaction, resulting in the formation of a large amount of insoluble substances and a decrease in yield.

[0034] Temperature also affects the reactants. For example, a certain amount of Chinese yam polysaccharide and water-soluble starch (mass ratio 1:1) are taken, and a certain amount of water is added. The mixture is heated and stirred thoroughly to completely dissolve the Chinese yam polysaccharide and water-soluble starch, forming a clear solution. After cooling to room temperature, copper chloride and sodium citrate are added to make their concentrations both 0.15 mol / L. The pH is adjusted to approximately 8.5. The mixture is then heated in a water bath at 30℃, 50℃, 70℃, and 90℃ for 1.5 hours respectively. After the reaction is complete, the mixture is centrifuged at 4000 r / min for 5 min. The supernatant is dialyzed, and the dialysate is concentrated under reduced pressure. The concentrate is then left at room temperature to obtain the final product. As the temperature increases, the complexation reaction proceeds more completely, and the reaction yield reaches its maximum. However, when the temperature continues to rise, insoluble substances are easily formed during the reaction, reducing the yield. Furthermore, excessively low temperatures are not conducive to gel formation. Figure 5 As shown.

[0035] A certain amount of Chinese yam polysaccharide and water-soluble starch (mass ratio 1:1) were added to a certain amount of water. The mixture was heated and stirred thoroughly until the Chinese yam polysaccharide and water-soluble starch were completely dissolved, forming a clear solution. After cooling to room temperature, copper chloride was added to a concentration of 0.12 mol / L, and sodium citrate to a concentration of 0.15 mol / L. The pH was adjusted to approximately 8.5. The mixture was heated in a water bath at 70℃ for 0.5, 1.0, 2.0, and 3.0 hours, respectively. After the reaction was complete, the mixture was centrifuged at 4000 r / min for 5 min. The supernatant was dialyzed, and the dialysate was concentrated under reduced pressure. The concentrate was left at room temperature to obtain the final product. With prolonged reaction time, the yield of the coordination reaction increased, and those with more complete reactions formed gels, such as... Figure 6 As shown. However, when the reaction time is too long, the reaction yield decreases rapidly with the extension of time. This is because the increase in reaction time makes it easier for polysaccharide and starch molecules to break down in alkaline solution, and the combination of copper ions and hydroxide ions in the solution increases, making it easier to form insoluble substances, resulting in a decrease in yield.

[0036] Example 2 The preparation method of copper polysaccharide from Chinese yam includes the following steps: Add water to Chinese yam polysaccharide and water-soluble starch (at a mass ratio of 1:1), heat and stir thoroughly to completely dissolve the Chinese yam polysaccharide and water-soluble starch, forming a clear solution. After cooling to room temperature, add copper chloride and sodium citrate to make the concentration of both 0.15 mol / L, adjust the pH to about 8.5, and react in a water bath at 70℃ for 1.5 hours. After the reaction is complete, centrifuge at 4000 r / min for 5 min, take the supernatant and dialyze it. The dialysate obtained is directly freeze-dried to obtain Chinese yam polysaccharide copper.

[0037] The infrared spectrum of the obtained copper polysaccharide from Chinese yam is shown below. Figure 7As shown, the infrared spectra of copper polysaccharide from Chinese yam differ significantly from those of the corresponding Chinese yam polysaccharide. The C=O stretching vibration peak in the polysaccharide is at 1743.8 cm⁻¹. -1 The disappearance of the peak indicates that a coordination reaction has occurred. The stretching vibration peak of the glycosidic bond in the polysaccharide decreased from 1627.9 cm⁻¹. -1 It shifted to a lower wavenumber of 1612.5 cm. -1 This indicates that the glycosidic bond has reacted. Furthermore, the saturated CH stretching vibration peak in the polysaccharide is at 2947.2 cm⁻¹. -1 The disappearance of the peak indicates a change in the adjacent hydroxyl group, masking the peak. The in-plane bending vibration of the hydroxyl group βOH is at 1396.46 cm⁻¹. -1 Compared to the corresponding peak position of polysaccharides, there was a significant shift to lower wavenumbers. The considerable difference in the infrared spectra of polysaccharides and polysaccharide copper provides, from one perspective, evidence of the formation of a new substance.

[0038] The copper chloride solution and the reacted yam polysaccharide copper solution were measured using a UV-Vis spectrophotometer in the range of 500–900 nm. The results are shown in the figure. Figure 8 The maximum absorption wavelength of copper chloride is around 800 nm. The product obtained from the coordination reaction has maximum ultraviolet absorption at 728 nm, while copper chloride has little absorption at 728 nm. This proves that copper participated in the coordination reaction and a new substance was formed.

[0039] The dialysate of copper polysaccharide from Chinese yam was directly freeze-dried, and its microstructure was observed, such as... Figure 9 As shown, the spheres are formed by the accumulation of small particles, with a diameter of about 2 micrometers, and their microstructures are significantly different from those of the corresponding Chinese yam polysaccharides and starches.

[0040] The thermogravimetric analysis curve of copper in Chinese yam polysaccharide was compared with that of the corresponding mixture of raw materials starch and Chinese yam polysaccharide. Figure 10 It can be seen that the introduction of copper ions disrupts the thermal stability of the polysaccharide structure. Before 200℃, the sample loses water. Starting at 200℃, the mass decreases rapidly, stabilizing around 270℃, indicating poor thermal stability of the polysaccharide-copper complex, which decomposes rapidly starting at 200℃. This may be because copper ions and iron participate in coordination in different ways. In the case of *Dioscorea opposita* polysaccharide-iron complex, the polysaccharide forms a central polyhydroxy iron core, with polysaccharides encapsulating it layer by layer. Therefore, the thermogravimetric curve of *Dioscorea opposita* polysaccharide-iron complex shows a gradual decrease in mass with increasing temperature, without a clear water loss stage or a weight loss stage of combustible organic matter. However, *Dioscorea opposita* polysaccharide-copper complex may not form a polyhydroxy metal core structure; instead, copper ions directly participate in the coordination reaction, causing thermal instability of the sugar chain molecular structure. This also suggests the formation of a new complex.

[0041] The XRD curve of copper from Chinese yam polysaccharide was compared with that of the mixture of raw Chinese yam polysaccharide and starch. Figure 11 It can be seen that the two have different peak positions and different peak shapes, but neither of them has sharp peaks, indicating an amorphous structure, which also indirectly demonstrates that a new complex is formed.

[0042] Results of copper ion release: The copper standard solution (spectral pure) was diluted to corresponding concentrations respectively to prepare standard curve data for copper ion determination as shown in Table 1, and the standard curve equation y = 83413x + 6606.9, R = 0.9999 was obtained.

[0043] Table 1 Standard curve of copper ions It can be seen from the data in Table 2 that the copper yam polysaccharide gel can slowly release the copper complex in aqueous solution within 48 h, avoiding the toxicity to aquatic organisms caused by a sudden increase in copper ion concentration in the water body.

[0044] Table 2 Release profile of copper ions Examples of implementation effects 1. Acute toxicity test SPF Kunming mice, half male and half female, weighing 20~22 g, were provided by the Henan Animal Experiment Center (Animal Certificate No.: SCXK (Yu) 2015-0004). Before the test, the mice were adaptively reared for 1 week, with free access to drinking water and food, and their health status was observed. The intragastric administration dose was formulated according to the WHO acute toxicity classification standard for exogenous chemicals (The User's Manual for the IPCS Health and Safety Guides, 1996). 40 SPF mice were randomly divided into 4 groups according to the intragastric administration dose of copper yam polysaccharide, 10 mice in each group, half male and half female. The doses were increased in the order of 200, 240, 288, 345, 414 mg / kg body weight (BW), with a dose progression factor of 0.20, and the control group was given the same amount of normal saline by the same method. The mice were fasted but allowed free access to water for 12 h before intragastric administration. The food intake, clinical symptoms and mortality were recorded, and the median lethal dose (LD 50 ) was calculated. The observation lasted for 14 days after administration. The results showed that no abnormal changes were observed in the mice of the control group. All the mice in the low-dose group (Group 1) showed toxic reactions, including mental hyperactivity, increased activity, frequent climbing in the cage, and restlessness. The dosed mice in Groups 2 to 4 showed symptoms such as reduced activity, limb tremors, and dyspnea, and some of them died subsequently. In the high-dose group (Group 5), all the mice curled up and trembled continuously after administration and eventually died. The SPSS software was used to calculate the median lethal dose (LD 50The LD50 concentration was approximately 275.9 g / kg, with a 95% confidence interval of (248.0, 306.9), indicating that although copper polysaccharide from Dioscorea opposita has some toxicity, it is less than that of inorganic copper chloride (oral LD50 in rats). 50 (mg / kg): 140), the toxicity has been significantly reduced.

[0045] Table 3 Acute copper toxicity test of Dioscorea opposita polysaccharide (n=10) The difference in body weight between day 14 post-administration and day 1 post-administration was calculated in mice. Compared with the blank control group, there was no significant difference in body weight gain among the different dosage groups (P>0.05). Compared with the blank control group, groups 3-5 showed a greater decrease in food intake after administration; almost no food was consumed at 24 hours, and food intake was approximately 75% of that of normal mice at 48 hours. After 72 hours, food intake was roughly equivalent across all groups. The results are as follows... Figure 12 As shown.

[0046] Fourteen days after administration, blood was collected from the abdominal aorta, and the organs were dissected. The heart, liver, spleen, and kidneys were weighed, recorded, and the organ index of each organ was calculated: Organ index (%) = organ weight / body weight × 100%. Compared with the control group, the heart, liver, spleen, and kidney indices of surviving mice in each dose group were not significantly different (P>0.05). The results are shown in Table 4.

[0047] Table 4 Comparison of organ indices in mice of different groups (x±s) 2. Results of antibacterial test Staphylococcus aureus is a typical representative of Gram-positive bacteria, and Escherichia coli is a typical representative of Gram-negative bacteria. Therefore, the antibacterial effect of copper polysaccharide from Dioscorea opposita against these two bacteria was tested. Samples were placed in the wells of agar plates for a screening test, and the antibacterial effect was judged based on the size of the inhibition zone. Copper polysaccharide from Dioscorea opposita showed good antibacterial activity against both S. aureus and E. coli. Figure 13 As shown, the inhibition zone gradually increases with increasing sample concentration. The minimum inhibitory concentration (MIC) method yielded a value of 12 mg / mL for both *S. aureus* and *E. coli*, indicating that the copper polysaccharide from *Dioscorea opposita* has antibacterial effects against both Gram-positive and Gram-negative bacteria.

[0048] 3. Results of zebrafish toxicity test Male and female zebrafish were raised separately under standard conditions of 14 hours of light / 10 hours of darkness and 28°C, and fed regularly with pelleted food and brine shrimp. For egg collection, healthy, sexually mature zebrafish were placed in a mating tank at a 1:1 or 1:2 ratio of males to females, and fertilized eggs were obtained the following day between 9 and 10 am. After disinfection and cleaning, the fertilized eggs were transferred to zebrafish embryo culture water and cultured under controlled light at 28°C. (Zebrafish farming and rearing system (Beijing Aisheng Technology Co., Ltd.))

[0049] Embryos at 24 hpf (hours post-fertilization) were demembranes using 1 mg / mL streptomycin. Normal zebrafish embryos were selected under a stereomicroscope and carefully transferred to 24-well plates, 10 embryos per well. A blank control group (embryo culture water containing 0.5% DMSO) and a 10 mg / L test compound group were established. After drug administration, the embryos were placed in a light incubator (28℃) for continued controlled-light culture. Twenty-four hours after drug administration, the mortality rate of juvenile fish in each experimental group was observed and recorded (the presence of heartbeat was used as the criterion for determining juvenile survival). The spontaneous activity of the juvenile fish was observed under a microscope. The juvenile fish were anesthetized with 1 mmol / L tricaine solution, and side-view photographs were taken with a fixed focus. Morphological developmental indicators of the zebrafish juveniles were recorded, such as body shape, brain, facial structure, notochord, somites, tail, fins, heart, and pigmentation. The effects of the compound on the developmental toxicity of zebrafish juveniles were evaluated.

[0050] The mortality rate in the blank control group was 1.67±2.89%, while that in the drug-treated group was 2.03±2.89%. Due to individual differences and the high early mortality rate of juvenile fish, a mortality rate of <5% in the blank control group was considered sufficient for this batch of juvenile fish to be suitable for the experiment. No obvious abnormalities were observed in the spontaneous activity or development of any part of the juvenile fish in either group. Figure 14 As shown, there was no significant difference between the two groups, and this polysaccharide copper gel can be used for sterilization in ornamental fish farming.

[0051] 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, improvements, etc., 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 a polysaccharide copper gel, characterized in that, The steps are as follows: (1) Dissolve polysaccharide, starch and trisodium citrate in water, heat to dissolve, and stir until homogeneous to obtain a mixed solution; (2) The hydrochloric acid solution of CuCl2 is slowly added dropwise to the mixed solution in step (1), the pH is adjusted and the reaction is heated in a water bath. The resulting product is centrifuged and dialyzed. The dialysate is concentrated to a certain concentration to obtain polysaccharide copper gel. In step (1), the mass ratio of polysaccharide, starch and trisodium citrate is 1:(0.1-5):1, wherein the polysaccharide is Chinese yam polysaccharide; The mixed solution is a fully dissolved, clear, and transparent solution; In step (2), the molar ratio of CuCl2 to trisodium citrate in step (1) is (0.1-10):1; The pH value is 8.5-10.5; the water bath heating reaction temperature is 70-100℃ and the time is 1-48 h; The concentration after concentration is 1.01-1.35 g / mL.

2. The polysaccharide copper gel prepared by the method of claim 1, characterized in that: The polysaccharide copper in the polysaccharide copper gel is a macromolecular complex that can be retained by dialysis bags and becomes spherical after lyophilization.

3. The application of the polysaccharide copper gel according to claim 2 in aquaculture.

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