A zonula occludens toxin (zot) mutant that is more stable and has improved immunogenicity

Abstract

The application discloses a viscosity removing agent for sticky fertilized eggs based on magnesium-calcium ion chelation, which is an aqueous solution of a magnesium-calcium ion chelator. The sticky fertilized eggs are only needed to be soaked in the viscosity removing agent for 8-12 minutes with slight stirring, so as to be chelated with magnesium-calcium metal ions to form stable complexes, thereby destroying the combination of magnesium-calcium metal ions and surface proteins or polysaccharides of the fertilized eggs, effectively weakening the adhesion of the surface of the fertilized eggs, and avoiding the influence of the ventilation of the fertilized eggs due to the adhesion of the fertilized eggs. Meanwhile, the viscosity removing agent can reduce the attachment of impurities in the incubation process, and avoid the phenomenon of microbial breeding and disease transmission caused by the attachment of impurities. In addition, the viscosity removing agent can effectively reduce the mechanical damage of the fertilized eggs caused by mud, talcum powder or strong stirring, effectively protect the integrity of the structure of the fertilized eggs, and overcome the adverse effects of the suspension of particles in water on the transparency of the water body and the dissolved oxygen level.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture, and particularly relates to a de-adhesive agent for adhesive fertilized eggs based on magnesium-calcium ion chelation. Background Technology

[0002] In aquaculture, the rate of artificial breeding directly affects the yield. However, due to the strong adhesiveness of the egg surface of some aquatic animals, the resulting fertilized eggs are adhesive fertilized eggs, such as those of the rockfish (Hexagrammos oysterii). Hexagrammos otakii Figure 1 Adhesive fertilized eggs, such as those with adhesive fertilization, directly affect the artificial breeding rate of aquatic animals. One reason is that adhesive fertilized eggs easily cause the egg masses to clump together, affecting the aeration of the fertilized eggs; another reason is that adhesive fertilized eggs are prone to adhering to impurities during the hatching process, leading to the growth of microorganisms and the spread of diseases, thus reducing the hatching rate and survival rate of the fertilized eggs. Therefore, researchers have been working on de-adhesive treatment for adhesive fertilized eggs.

[0003] Existing de-adhesion methods mostly employ mud, talcum powder, or mechanical stirring. Mud and talcum powder primarily work by physically adsorbing and covering the fertilized eggs to break the adhesion between them. However, during operation, the mud or talcum powder particles can cause mechanical damage to the fertilized eggs due to improper control of stirring intensity and time. Furthermore, mud and talcum powder particles suspended in water reduce water transparency and affect dissolved oxygen levels, thus creating environmental stress on the fertilized eggs. Mechanical stirring involves strong agitation, which can easily damage the surface of the fertilized eggs, affecting their quality and hatching rate.

[0004] Magnesium-calcium ion chelating agents are substances that can chelate with magnesium and calcium metal ions to form stable complexes, such as sodium tripolyphosphate (STPP), which are commonly used as food additives and industrial raw materials. To date, there are no reports of using magnesium-calcium ion chelating agents as de-adhesive agents for sticky fertilized eggs. Summary of the Invention

[0005] This invention addresses the aforementioned technical problems in the prior art by providing a chelating agent for adhesive fertilized eggs based on magnesium-calcium ion chelation.

[0006] The technical solution of the present invention is: a chelating agent for adhesive fertilized eggs based on magnesium-calcium ion chelation, characterized in that it is an aqueous solution of magnesium-calcium ion chelating agent.

[0007] The preferred magnesium-calcium ion chelating agent is sodium tripolyphosphate.

[0008] The preferred mass concentration of the magnesium-calcium ion chelating agent is 0.5-3%.

[0009] The preferred mass concentration of the magnesium-calcium ion chelating agent is 1-2%.

[0010] The preferred aqueous solution of the magnesium-calcium ion chelating agent has a temperature of 14-16℃ and a pH value of 7.5-8.2.

[0011] This invention is the first to confirm the presence of Mg 2+ and Ca 2+ The key factor leading to the strong adhesion of the egg surface is magnesium-calcium ion chelating agent; therefore, an aqueous solution of magnesium-calcium ion chelating agent is used as a de-adhesive for adhesive fertilized eggs. Simply immerse the fertilized eggs in the de-adhesive agent for 8-12 minutes with gentle stirring. This allows the fertilized eggs to chelate with magnesium-calcium metal ions to form a stable complex, thereby disrupting the binding of magnesium-calcium metal ions to proteins or polysaccharides on the surface of the fertilized eggs. This effectively weakens the adhesion of the fertilized egg surface, preventing the fertilized eggs from clumping together and affecting aeration. Simultaneously, it reduces the adhesion of impurities during incubation, preventing microbial growth and disease transmission caused by impurities. Furthermore, this invention effectively reduces mechanical damage to the fertilized eggs caused by mud, talcum powder, or vigorous stirring, effectively protecting the structural integrity of the fertilized eggs. It also overcomes the adverse effects of particles suspended in water on water transparency and dissolved oxygen levels. In summary, this invention effectively improves the de-adhesion efficiency of adhesive fertilized eggs, increases the hatching rate and survival rate of adhesive fertilized eggs, and thus improves the artificial breeding rate and yield of aquatic animals, making it suitable for large-scale production applications. Attached Figure Description

[0012] Figure 2 This invention confirms that Mg 2+ and Ca 2+ This is a micrograph of the fish eggs that make the eggs of the Ōtaki Hexagrammos viscous.

[0013] Figure 1 This is a schematic diagram illustrating the effect of the present invention on the de-adhesion of adhesive fertilized eggs of the rockfish *Hexagrammos ogakii*. Detailed Implementation Example 1

[0014] The present invention discloses a chelating agent for adhesive fertilized eggs based on magnesium-calcium ion chelation, which is an aqueous solution of sodium tripolyphosphate with a mass concentration of 1%, an aqueous solution temperature of 14-16℃, and a pH value of 7.5-8.2.

[0015] Example 2:

[0016] The present invention provides a magnesium-calcium ion chelation-based adhesive fertilized egg de-adhesion agent, which is an aqueous solution of sodium tripolyphosphate with a mass concentration of 2%, an aqueous solution temperature of 14-16℃, and a pH value of 7.5-8.2.

[0017] Example 3:

[0018] The present invention provides a chelating agent for adhesive fertilized eggs based on magnesium-calcium ion chelation, which is an aqueous solution of sodium tripolyphosphate with a mass concentration of 3%, an aqueous solution temperature of 14-16℃, and a pH value of 7.5-8.2.

[0019] experiment:

[0020] 1. Experiment on metal ion concentration gradient in Hexagrammos ogakii eggs.

[0021] Eggs of *Hexagrammos ogami* were divided into four treatment groups (groups 1-4), and each group was immersed for equal time in different concentrations of NaCl, KCl, CaCl2, and MgCl2. Group 1 was immersed in NaCl solutions with concentrations of 10 mmol / L, 20 mmol / L, 30 mmol / L, and 50 mmol / L; Group 2 was immersed in KCl solutions with concentrations of 10 mmol / L, 20 mmol / L, 30 mmol / L, and 50 mmol / L; Group 3 was immersed in CaCl2 solutions with concentrations of 5 mmol / L, 10 mmol / L, 15 mmol / L, and 30 mmol / L; and Group 4 was immersed in MgCl2 solutions with concentrations of 5 mmol / L, 10 mmol / L, 15 mmol / L, and 30 mmol / L. Microscopic observation was performed after immersion treatment, and the results are as follows: Figure 1 As shown. Figure 1 Images (A)-(D) show the microscopic observation results of fish eggs in Group 1 after treatment with different concentrations of NaCl solution. The results indicate that the viscosity of the Hexagrammos otakii eggs did not increase, suggesting that the NaCl concentration gradient... + Unactivated fish eggs further develop stickiness; Figure 1 (E)-(H) are microscopic observations of fish eggs from the second group after treatment with KCl solutions of different concentrations. The results show that the viscosity of the Hexagrammos otakii eggs did not increase, indicating that the KCl concentration gradient... + Unactivated fish eggs further develop stickiness; Figure 1 (I)-(L) are the microscopic observation results of fish eggs in the third group after treatment with CaCl2 solutions of different concentrations. The results show that the viscosity of the Hexagrammos ogakii eggs is enhanced, indicating that the fish eggs are susceptible to the effects of CaCl2. 2+ It is very sensitive to stimuli, and Ca at various concentration gradients + It can activate the fish eggs to further produce adhesiveness; Figure 2 (M)-(P) are the microscopic observation results of fish eggs in group 4 after treatment with MgCl2 solutions of different concentrations. The results show that the viscosity of the Hexagrammos ogakii eggs is enhanced, indicating that the fish eggs are more resistant to MgCl2. 2+ It is very sensitive to stimuli, and Mg at various concentration gradients 2+ It can activate the fish eggs to further produce adhesiveness;

[0022] The experimental results prove that Mg 2+ and Ca 2+ are the key metal ions for making the eggs of Hexagrammos otakii sticky.

[0023] 2. Experiment of de-sticking treatment of the fertilized eggs of Hexagrammos otakii by the de-sticking agent of Example 1 of the present application

[0024] The de-sticking agent prepared in Example 1 was taken, and the fertilized eggs of Hexagrammos otakii after treatment with CaCl2 and MgCl2 solutions in the above-mentioned Experiment 1 were collected, respectively, and then were soaked in the de-sticking agent at a concentration of 200-300 g / L for 8 minutes with slight stirring to promote the separation of the sticky substances on the surface of the eggs; the temperature in the soaking process was controlled at 14-16℃, and the pH value was kept at 7.5-8.2, and the physicochemical conditions were kept consistent with the incubation process to ensure the viability and quality of the fertilized eggs; after the soaking treatment, the fertilized eggs were gently rinsed with physiological saline or deionized water for 3 times to remove the residual sodium tripolyphosphate solution.

[0025] The fertilized eggs of Hexagrammos otakii after the de-sticking treatment were observed under a microscope, and the de-sticking effect was as shown in Figure 2 . ​ The results show that the surface of the fertilized eggs of Hexagrammos otakii after the soaking treatment by the de-sticking agent of Example 1 of the present application is not sticky, and the integrity rate of the fertilized eggs reaches more than 98%, which can effectively improve the de-sticking efficiency of the fertilized eggs and the subsequent hatching rate and survival rate of the fertilized eggs.

Claims

1. Use of an aqueous solution of a magnesium-calcium ion chelator for the preparation of a viscous zygote debulking agent, characterized in that: The viscous fertilized egg is a fertilized egg of Hexagrammos otakii.

2. Use of a solution of a magnesium-calcium ion chelator in water according to claim 1 for the preparation of a zonadhesiveness de-zonadhesiveness agent, characterized in that The magnesium-calcium ion chelator is sodium tripolyphosphate.

3. Use of a solution of a magnesium-calcium ion chelator in water according to claim 1 for the preparation of a zonadhesiveness de-zonadhesiveness agent, characterized in that The magnesium-calcium ion chelator has a mass concentration of 0.5-3%.

4. Use of a solution of a magnesium-calcium ion chelator in water according to claim 3 for the preparation of a zonadhesiveness de-zonadhesiveness agent, characterized in that The magnesium-calcium ion chelator has a mass concentration of 1-2%.

5. Use of a solution of a magnesium-calcium ion chelator in water according to claim 4 for the preparation of a zonadhesiveness de-zonadhesiveness agent, characterized in that The aqueous solution of the magnesium-calcium ion chelator has a temperature of 14-16℃ and a pH value of 7.5-8.2.

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