Application of vitamin B2 in preparation of product for inhibiting or killing microsporidia
By using vitamin B2 at different concentrations to treat or feed microsporidia spores in vitro, the problem of the difficulty in safely and effectively inhibiting or killing microsporidia in existing technologies has been solved, achieving significant inactivation effects and low-cost operation.
Patent Information
- Application Number
- CN202511753389.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-26
- Publication Date
- 2026-02-24
AI Technical Summary
Existing technologies are insufficient to safely and effectively suppress or kill microsporidia, especially those microsporidia that are widely present in farmed animals such as silkworms, bees, fish, and shrimp, which affect healthy aquaculture.
Vitamin B2 (VB2) is used as a product to inhibit or kill microsporidia. It is added externally or fed to the animal to utilize its inactivation effect on microsporidia spores at different concentrations, especially showing significant effects on shrimp hepatocellular carcinoma, Helen's encephalitis microsporidia, silkworm microsporidia, and rabbit encephalitis microsporidia.
Vitamin B2 significantly improved the in vitro inactivation rate of microsporidian spores at different concentrations, maintaining safety for farmed animals. It is also simple to operate, low in cost, and does not require complex chemical reagents or expensive instruments.
Smart Images
Figure CN121550232A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of aquaculture technology, and in particular relates to the application of vitamin B2 in the preparation of products that inhibit or kill microsporidia. Background Technology
[0002] Microsporidia are a class of single-celled eukaryotic microorganisms with unique morphology and life cycle. Microsporidia infect a wide range of hosts, including farmed animals such as silkworms, bees, fish, and shrimp, threatening healthy aquaculture. How to safely and effectively suppress microsporidia has become an urgent technical problem to be solved. Summary of the Invention
[0003] The purpose of this invention is to provide the application of vitamin B2 in the preparation of products that inhibit or kill microsporidia, which can safely and effectively inhibit microsporidia spores.
[0004] This invention provides the application of vitamin B2 in the preparation of products that inhibit or kill microsporidia.
[0005] The present invention also provides the use of vitamin B2 in the preparation of drugs that inhibit microsporidia infection.
[0006] Preferably, the microsporidia include one or more of the following: shrimp hepatocellular carcinoma, Helen's encephalitis microsporidia, silkworm microsporidia, and rabbit encephalitis microsporidia.
[0007] Preferably, the vitamin B2 is applied to the digestive tract and / or in vitro.
[0008] The present invention also provides a method for inhibiting or killing microsporidian spores in vitro, comprising the following steps: Vitamin B2 is added to environments or samples containing microsporidia and / or microsporidia spores to inhibit or kill them.
[0009] Preferably, the environment includes the digestive tract and water.
[0010] This invention also provides a method for inhibiting or killing microsporidian spores in the digestive tract, comprising the following steps: Feeding farmed animals infected with microsporidia with foods containing vitamin B2 can inhibit or kill the microsporidia.
[0011] Preferably, the vitamin B2 is used in the form of a vitamin B2 solution; the working concentration of the vitamin B2 is ≥0.2 μg / μL.
[0012] Preferably, the working concentration of vitamin B2 is 0.2, 0.25, 0.4, 0.5, 1 or 1.5 μg / μL.
[0013] Preferably, the inhibition or killing time is 1 to 48 hours.
[0014] This invention provides the application of vitamin B2 (VB2) in the preparation of products that inhibit or kill microsporidia. VB2 can inactivate mature microsporidia spores, thereby inhibiting microsporidia infection. From a safety perspective, appropriate amounts of VB2 are harmless to humans and animals and can be used as a nutritional supplement for animals. Furthermore, as an essential water-soluble vitamin, VB2's metabolic pathway is well-understood; it participates in redox reactions through flavin enzyme systems (such as FAD / FMN) and is naturally excreted from the body without any risk of residue. In addition, VB2 inhibits microsporidia (such as...) Ecytonucleospora hepatopenaei , Encephalitozoon hellem , Nosema bombycis and Encephalitozoon cuniculi It maintains broad-spectrum activity while being non-toxic to farmed animals. VB2 has been verified to significantly improve the in vitro inactivation rate of microsporidian spores. Attached Figure Description
[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.
[0016] Figure 1 The mortality rate of shrimp hepatocystis spores after 12 hours of in vitro incubation with the drug; Figure 2 The mortality rate of Helen's encephalitis microsporidia after 24 hours of in vitro drug incubation; Figure 3 The mortality rate of silkworm microsporidia after 24 hours of in vitro incubation with the drug. Detailed Implementation
[0017] This invention provides the application of vitamin B2 in the preparation of products that inhibit or kill microsporidia.
[0018] The chemical structural formula of vitamin B2 is shown in Formula I: Formula I.
[0019] The present invention also provides the use of vitamin B2 in the preparation of drugs that inhibit microsporidia infection.
[0020] In one embodiment, the microsporidia include one or more of the following: shrimp hepatocellular carcinoma, Helen's encephalitis microsporidia, silkworm microsporidia, and rabbit encephalitis microsporidia.
[0021] In one implementation, the spores include mature spores.
[0022] In one implementation, the vitamin B2 is applied to the digestive tract and / or in vitro.
[0023] The present invention also provides a method for inhibiting or killing microsporidian spores in vitro, comprising the following steps: Vitamin B2 is added to environments or samples containing microsporidia to inhibit or kill the microsporidia.
[0024] In one embodiment, the environment includes the digestive tract and water. In another embodiment, the concentration of microsporidia spores in the environment or sample is 10. 6 per μL.
[0025] This invention also provides a method for inhibiting or killing microsporidian spores in the digestive tract, comprising the following steps: Feeding farmed animals infected with microsporidia with foods containing vitamin B2 can inhibit or kill them.
[0026] In one embodiment, the vitamin B2 is used in the form of a vitamin B2 solution; the working concentration of the vitamin B2 is ≥0.2 μg / μL.
[0027] As one implementation, the working concentration of vitamin B2 is 0.2, 0.25, 0.4, 0.5, 1 or 1.5 μg / μL.
[0028] As another implementation, when the microsporidia is Enterocytozoa hepatis (EHP), the working concentration of vitamin B2 is 0.2, 0.4 or 1 μg / μL, and more specifically 1 μg / μL. This concentration can significantly improve the in vitro inactivation rate of EHP spores, and can both ensure the effectiveness of vitamin B2 and avoid excessive consumption of vitamin B2.
[0029] As another implementation, when the microsporidia is *Microsporidia helenii* (… Encephalitozoon hell When the working concentration of vitamin B2 is 0.25 or 1.5 μg / μL, and more specifically 1.5 μg / μL, this concentration can significantly improve… E. hellem The in vitro inactivation rate of spores can ensure the effectiveness of vitamin B2 while avoiding excessive consumption of vitamin B2.
[0030] As another implementation, when the microsporidia is *Bombyx mori* microsporidia (… Nosema bombycis When the working concentration of vitamin B2 is 0.25, 0.5, 1 or 1.5 μg / μL, and further 1.5 μg / μL, this concentration can significantly improve the in vitro inactivation rate of silkworm microsporidia spores, and can both ensure the effectiveness of vitamin B2 and avoid excessive consumption of vitamin B2.
[0031] As one implementation method, the inhibition or killing time is 1 to 24 hours, and more specifically 12 hours. This time ensures the effectiveness of vitamin B2 while avoiding excessive consumption of vitamin B2.
[0032] This invention uses vitamin B2 to inhibit or kill microsporidian spores. The process is simple, the equipment requirements are low, and there is no need to use complex chemical reagents or expensive instruments, thus resulting in low operating costs.
[0033] To further illustrate the present invention, the application of vitamin B2 provided by the present invention in the preparation of products that inhibit or kill microsporidia is described in detail below with reference to the accompanying drawings and embodiments, but these descriptions should not be construed as limiting the scope of protection of the present invention.
[0034] Example 1 Verification of vitamin B2 (VB2) in vitro against shrimp hepatocellular carcinoma (VLCC) Ecytonucleospora hepatopenia ,Original Enterocytozoon hepatopenaei The inhibitory effect of EHP spores was investigated to clarify its inactivation potential and concentration dependence.
[0035] The main materials and reagents are listed in Table 1.
[0036] Table 1. Main materials and reagents used in Example 1
[0037] I. Methods 1. Spore treatment preparation Extraction of Enterocytozoa hepatis from shrimp: Hepatopancreatic tissue from EHP-positive shrimp was ground and filtered through a 100 μm sieve. Impurities were removed by gradient centrifugation to obtain a concentration of 10. 6 A spore suspension of 1 spore per μL was aliquoted into multiple centrifuge tubes for later use.
[0038] 2. VB2 Drug Processing Settings: Three experimental groups were set up, with VB2 solution added to a final concentration of 0.2 μg / μL, 0.4 μg / μL, and 1 μg / μL respectively, while keeping the total amount of spores consistent; a control group (water group) was also set up as a control.
[0039] 3. Incubation conditions: All samples were incubated at room temperature for 12 hours.
[0040] 4. Staining detection: After incubation, equal volumes of three fluorescent staining solutions (SYTOX Green, PI, FB28) were added. After incubation in the dark for 5-10 minutes, fluorescence microscopy was used for observation and image acquisition, and the proportion of dead spores was recorded.
[0041] 1) Staining agents: fluorescent whitening agent FB28 (1 μM), SYTOX Green (5 μM), propidium iodide (1.5 μM).
[0042] 2) Procedure: Mix the spore suspension with the staining agent, incubate in the dark for 5-10 minutes, and observe the activity under a fluorescence microscope. Take pictures and record the results using ultraviolet, blue, and white light channels.
[0043] 3) Statistical formula: Spore mortality rate = (Number of dead spores / Total number of spores) × 100% 5. Data Statistics: ImageJ was used for image analysis. Ten fields of view were randomly selected for counting, with three replicates per group. The percentage of dead spores was calculated, and one-way ANOVA was performed.
[0044] II. Results 1. Mortality rate of spores at 0 h The mortality rate of crudely extracted EHP spores was 10.8%. If the mortality rate is below 20%, subsequent drug incubation experiments will be conducted.
[0045] 2. Mortality rate of spores over 12 hours After 12 h of in vitro treatment, the mortality rate of EHP spores in the water control group was 11.8%. The mortality rates after 12 h of incubation with 0.2 μg / μL, 0.4 μg / μL, and 1 μg / μL vitamin B2 were 27.4%, 37%, and 52.13%, respectively. This indicates that vitamin B2 has a certain inactivation effect on EHP spores in vitro. (See [link to relevant documentation]). Figure 1 .
[0046] III. Conclusion The results showed that vitamin B2 had a significant in vitro inactivation effect on EHP spores, exhibiting a dose-dependent effect. At higher concentrations, vitamin B2 significantly reduced the activity of EHP spores, and the inactivation effect increased with increasing concentration. However, vitamin B2 is slightly soluble in water, with a solubility of 100–300 mg / L at 27.5°C. The higher concentrations of vitamin B2 in the EHP drug test were in suspension form.
[0047] Example 2 Verify the effect of vitamin B2 (VB2) on Helen's encephalitis microsporidia (Vitamin B2) under in vitro conditions. Encephalitozoon hell , E. hellem The inhibitory effect on spore activity was investigated to clarify its inactivation potential and concentration dependence.
[0048] See Table 2 for the main materials and reagents.
[0049] Table 2. Main materials and reagents used in Example 2
[0050] I. Methods 1. Spore extraction and purification Extraction of Helen's encephalitis microsporidia: The culture medium of rabbit kidney cell line infected with Helen's encephalitis microsporidia was purified by centrifugation to obtain the required concentration of 10. 6 Spores were collected at a concentration of 1 spore per μL. The spores were resuspended in PBS and aliquoted into multiple centrifuge tubes for later use.
[0051] 2. VB2 Drug Processing Settings: Three experimental groups were set up, with VB2 solution added to a final concentration of 0.25 μg / μL and 1.5 μg / μL respectively, while keeping the total amount of spores consistent; an untreated control group (water group) was also set up as a control.
[0052] 3. Incubation conditions: All samples were incubated in a 37°C incubator for 24 hours without shaking, allowing the reaction to proceed statically.
[0053] 4. Staining detection: After incubation, equal volumes of three fluorescent staining solutions (SYTOX Green, PI, FB28) were added. After incubation in the dark for 30 minutes, fluorescence microscopy was used for observation and image acquisition, and the proportion of dead spores was recorded.
[0054] 5. Data Statistics: ImageJ was used for image analysis. Ten fields of view were randomly selected for counting, with three replicates per group. The percentage of dead spores was calculated, and one-way ANOVA was performed.
[0055] II. Results After 24 h of vitamin B2 treatment, the mortality rate in the control group was 0.4%, while the mortality rates of *Microsporidium helenii* after 24 h of incubation with 0.25 μg / μL and 1.5 μg / μL vitamin B2 were 54.7% and 88.1%, respectively. (See also...) Figure 2 .
[0056] III. Conclusion The results show that VB2 has... E. hellem The spores exhibited a significant in vitro inactivation effect, with a concentration-dependent increasing trend, indicating that VB2 can significantly reduce the activity of microsporidian spores at higher concentrations.
[0057] Example 3 Verify the effect of vitamin B2 (VB2) on silkworm microsporidia (Bombyx mori) under in vitro conditions. Nosema bombycis The inhibitory effect on spore activity was investigated to clarify its inactivation potential and concentration dependence.
[0058] See Table 3 for the main materials and reagents.
[0059] Table 3. Main materials and reagents used in Example 3
[0060] I. Methods 1. Spore extraction and purification Silkworm Microsporidia Extraction: The midgut of infected silkworms was ground, filtered, and purified by differential centrifugation to obtain the desired concentration of 10. 6 Spores were collected at a concentration of 1 spore per μL. The spores were resuspended in PBS and aliquoted into multiple centrifuge tubes for later use.
[0061] 2. VB2 Drug Processing Settings: Three experimental groups were set up, with VB2 solution added to final concentrations of 0.25 μg / μL, 0.5 μg / μL, 1 μg / μL, and 1.5 μg / μL respectively, while maintaining the same total spore count; an untreated control group (water group) was also set up as a control.
[0062] 3. Incubation conditions: All samples were incubated at room temperature for 24 hours without shaking, allowing the reaction to proceed statically.
[0063] 4. Staining detection: After incubation, equal volumes of three fluorescent staining solutions (SYTOX Green, PI, FB28) were added. After incubation in the dark for 30 minutes, fluorescence microscopy was used for observation and image acquisition, and the proportion of dead spores was recorded.
[0064] 5. Data Statistics: ImageJ was used for image analysis. Ten fields of view were randomly selected for counting, with three replicates per group. The percentage of dead spores was calculated, and one-way ANOVA was performed.
[0065] II. Results After 24 h of vitamin B2 treatment, the mortality rate in the control group was 0.2%. The mortality rates of silkworm microsporidia after 24 h of incubation with 0.25 μg / μL, 0.5 μg / μL, 1 μg / μL, and 1.5 μg / μL vitamin B2 were 16.8%, 22.9%, 35.5%, and 40.3%, respectively. (See also...) Figure 3 .
[0066] III. Conclusion The results showed that VB2 had a significant in vitro inactivation effect on silkworm microsporidia, and the effect increased in a concentration-dependent manner. At higher concentrations, it could significantly reduce the activity of microsporidia spores.
[0067] Example 4 To verify the inhibitory effect of vitamin B2 (VB2) on the spore activity of Microsporidium helenii encephalitis and Microsporidium rabbitii encephalitis at the cellular level, and to clarify its inactivation potential and concentration dependence.
[0068] I. Methods 1. Spore extraction and purification Microsporidia extraction: The culture medium of rabbit kidney cell lines (RK13 cells) infected with Helen's encephalitis microsporidia and rabbit encephalitis microsporidia was purified by centrifugation to obtain the required concentration of 10. 6 Spores were collected at a concentration of 1 spore per μL. The spores were resuspended in PBS and aliquoted into multiple centrifuge tubes for later use.
[0069] 2. Spore incubation and cell poisoning Two types of encephalitis microsporidia were incubated with a vitamin B2 solution within a safe range for 24 h, and the vitamin B2 solution was removed by washing with PBS. RK13 cells in 24-well plates were counted, and spores were added to the wells at a ratio of 5:1 to infect the cells.
[0070] 3. Sample collection Cell samples were collected at 3 and 5 days of infection, and total DNA was extracted from the precipitate using the OMEGA Tissue DNA Extraction Kit. Simultaneously, the 3-day and 5-day cell samples were fixed with 4% paraformaldehyde, incubated at room temperature for 15 min, washed three times with PBST, stained with a fluorescent whitening agent diluted 1000 times (1 mg / mL), and photographed under an inverted fluorescence microscope.
[0071] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.
Claims
1. Application of vitamin B2 in the preparation of products that inhibit or kill microsporidia.
2. Application of vitamin B2 in the preparation of drugs that inhibit microsporidia infection.
3. The application according to claim 1 or 2, characterized in that, The microsporidia include one or more of the following: shrimp hepatocellular carcinoma, Helen's encephalitis microsporidia, silkworm microsporidia, and rabbit encephalitis microsporidia.
4. The application according to claim 1 or 2, characterized in that, The vitamin B2 is used in the digestive tract and / or in vitro.
5. A method for inhibiting or killing microsporidian spores in vitro, characterized in that, Includes the following steps: Vitamin B2 is added to environments or samples containing microsporidia and / or microsporidia spores to inhibit or kill them.
6. The method according to claim 5, characterized in that, The environment includes bodies of water.
7. A method for inhibiting or killing microsporidian spores in the digestive tract, characterized in that, Includes the following steps: Feeding farmed animals infected with microsporidia with food containing vitamin B2 can inhibit or kill the microsporidia in the digestive tract of the farmed animals.
8. The method according to claim 5 or 7, characterized in that, The vitamin B2 is used in the form of a vitamin B2 solution; the working concentration of the vitamin B2 is ≥0.2 μg / μL.
9. The method according to claim 8, characterized in that, The working concentration of vitamin B2 is 0.2, 0.25, 0.4, 0.5, 1 or 1.5 μg / μL.
10. The method according to claim 5 or 7, characterized in that, The inhibition or killing time is 1 to 48 hours.