Application of bacillus amyloliquefaciens E35 in preparation of grouper iridovirus infection resistant product
By applying Bacillus amyloliquefaciens E35 and its fermented crude extract, the problem of prevention and control of iridovirus in grouper has been solved, achieving efficient, safe, and environmentally friendly virus control and growth promotion effects, and is suitable for the prevention and control of iridovirus in grouper.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGXI ACAD OF MARINE SCI (GUANGXI MANGROVE RES CENT)
- Filing Date
- 2026-01-15
- Publication Date
- 2026-05-12
AI Technical Summary
Grouper iridovirus is a viral disease that spreads rapidly and has a high mortality rate. Existing prevention and control measures, such as vaccines and antibiotics, have limited effectiveness and pose problems of drug residues and ecological pollution. There is an urgent need for green and safe prevention and control strategies.
Using Bacillus amyloliquefaciens E35 and its fermented crude extract, the host's immunity is regulated and the antiviral ability of the fish is enhanced by inhibiting the adsorption, invasion and replication of grouper iridovirus. The drug is administered orally or mixed with feed to optimize the intestinal microecology, inhibit harmful bacteria and promote digestion and absorption.
It effectively inhibits the invasion and proliferation of grouper iris virus, improves fish survival rate, enhances immunity, reduces drug residues and environmental pollution, promotes healthy fish growth, is easy to operate, and is safe with no side effects.
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Figure CN122005618A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of antiviral microorganisms in aquatic organisms, and in particular to the application of Bacillus amyloliquefaciens E35 in the preparation of products resistant to grouper iridovirus infection. Background Technology
[0002] Grouper, prized for its tender flesh and rich nutritional content, is a high-quality marine aquaculture fish in my country, possessing both high nutritional value and market demand. According to the "2025 China Fisheries Statistical Yearbook," annual grouper production has been increasing year by year. However, intensive aquaculture has led to frequent outbreaks of grouper diseases, among which viral diseases are highly contagious and have high mortality rates. The Singapore grouper iridovirus (SGIV) is particularly harmful, causing symptoms such as splenomegaly and systemic hemorrhage. Currently, there are no effective prevention and control measures for grouper iridovirus disease, causing economic losses to the grouper aquaculture industry and hindering its sustainable development.
[0003] Current prevention and control methods mainly include vaccines and antibiotic therapies. However, SGIV infection can cause immune system dysfunction and immunosuppression, leading to secondary or mixed infections with bacteria such as Vibrio harveyi and Brilliantobacterium medullaris. This results in poor cross-protection between heterologous strains, limiting the protective efficacy of existing vaccines. Furthermore, traditional antibiotic therapies are not only ineffective against iridovirus diseases but also easily lead to or enhance bacterial resistance. Excessive antibiotic use in aquatic environments causes drug residues, resulting in water pollution and ecosystem damage. The use of antibiotics also leads to their accumulation in aquatic products and transmission to humans through the food chain, posing a threat to human health. Therefore, there is an urgent need to find alternative prevention and control strategies that can effectively control pathogens such as SGIV while retaining antibiotic-like growth-promoting properties. Summary of the Invention
[0004] In view of the serious harm of grouper iridovirus in grouper aquaculture and the current status of prevention and control, this application provides the application of Bacillus amyloliquefaciens E35 in the preparation of products resistant to grouper iridovirus infection.
[0005] In the first aspect, this application provides the application of Bacillus amyloliquefaciens E35 in the preparation of products against grouper iridovirus infection, using the following technical solution: Application of Bacillus amyloliquefaciens E35 in the preparation of products against grouper iridovirus infection. The Bacillus amyloliquefaciens E35 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 20, 2024, with the accession number GDMCC65657 and the Latin name Bacillus amyloliquefaciens.
[0006] Bacillus amyloliquefaciens is a microorganism that can metabolize a variety of antibacterial substances and is beneficial to the host. It is recognized as a probiotic in aquaculture, but under traditional research paradigms, researchers typically do not directly apply it to the prevention and control of grouper iridovirus. This is mainly because probiotic research and applications have long focused on the biocontrol of bacterial diseases and their beneficial functions, limiting current research to areas already explored and applied. Based on its mechanism of action, it has not been applied to the prevention and control of grouper iridovirus disease.
[0007] Grouper infected with grouper iridovirus (SGIV) suffers from hematopoietic tissue necrosis, and SGIV spreads horizontally within fish populations, negatively impacting the health of aquaculture. This application, through related experimental research, found that Bacillus amyloliquefaciens E35 can effectively prevent and control SGIV. Specifically, Bacillus amyloliquefaciens E35 can inhibit the adsorption, invasion, and replication of SGIV within cells, and can enhance the host cell's antiviral capacity by regulating host immunity. Furthermore, Bacillus amyloliquefaciens E35 has a strong ability to secrete hydrolytic enzymes such as amylase, protease, and cellulase, potentially promoting digestion and absorption in farmed animals, helping to maintain a healthy intestinal barrier in fish, and indirectly improving the overall health of fish through competitive exclusion of other harmful microorganisms. This enhances the fish's defense against grouper iridovirus, delays further SGIV infection, and improves fish survival rates. The above-mentioned effects of Bacillus amyloliquefaciens E35 can inhibit the invasion and proliferation of viruses in marine aquaculture fish such as grouper iris virus, thereby effectively preventing and controlling viral diseases in marine aquaculture fish such as grouper iris virus.
[0008] Meanwhile, Bacillus amyloliquefaciens E35 and its metabolites are non-toxic, residue-free, green, and safe, with negligible negative impacts on fish. Furthermore, Bacillus amyloliquefaciens E35 has good palatability and can be administered orally or mixed with feed, eliminating the need to catch fish and greatly reducing stress and physical damage to them. After oral administration, Bacillus amyloliquefaciens E35 can directly enter the intestines, facilitating rapid colonization and optimizing the intestinal microecological balance, inhibiting harmful bacteria, promoting nutrient digestion and absorption, and thus strengthening the intestinal barrier function.
[0009] Preferably, the product includes at least one of live Bacillus amyloliquefaciens E35 or crude fermented Bacillus amyloliquefaciens E35 extract.
[0010] Preferably, the preparation method of the crude extract of Bacillus amyloliquefaciens E35 fermentation includes the following steps: Bacillus amyloliquefaciens E35 is inoculated into ordinary beef extract peptone liquid culture medium and cultured in a shaker at 30°C for 36-48 hours. After centrifugation to remove the bacterial cells, the fermentation supernatant is obtained. The fermentation supernatant is treated with 2 mol / L de1 hydrochloric acid to adjust the pH to 2-3, and then allowed to stand at 4°C for 8-12 hours. The precipitate is then collected by centrifugation to obtain the crude extract of Bacillus amyloliquefaciens E35 fermentation.
[0011] The Bacillus amyloliquefaciens E35 and its crude fermentation extract in this application have no toxic side effects on marine aquaculture fish such as grouper and golden pomfret. Furthermore, the crude fermentation extract of Bacillus amyloliquefaciens E35 can directly inhibit the infection of Singapore grouper iridovirus SGIV, with an inhibition rate of up to 84%.
[0012] Preferably, the grouper iridovirus is Singapore grouper iridovirus SGIV.
[0013] Grouper iridovirus, also known as "marine foot-and-mouth disease," can cause high mortality rates in grouper, resulting in significant economic losses for the grouper aquaculture industry. Currently, there are no commercially available effective antiviral drugs for grouper iridovirus disease. This application presents an anti-grouper iridovirus product prepared using Bacillus amyloliquefaciens E35 or its crude fermentation extract. Related experiments have shown that this product has significant preventive and therapeutic effects against Singapore grouper iridovirus (SGIV), and can significantly delay the disease progression in infected grouper, increasing the survival rate of infected grouper.
[0014] Preferably, the product can inhibit the envelope protein in Singapore grouper iridovirus. VP19 or / and major capsid proteins MCP Gene expression.
[0015] Preferably, the product can improve the survival rate of fish.
[0016] Preferably, the product includes any one of microbial preparations and veterinary drugs.
[0017] Secondly, this application provides a microbial preparation for resisting grouper iridovirus infection, employing the following technical solution: A microbial preparation for resisting grouper iridovirus infection, characterized in that it comprises live Bacillus amyloliquefaciens E35 or crude fermented extract of Bacillus amyloliquefaciens E35; the Bacillus amyloliquefaciens E35 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 20, 2024, with accession number GDMCC 65657.
[0018] Thirdly, this application provides a fish medicine for treating grouper iridovirus infection, using the following technical solution: A fish medicine for treating grouper iridovirus infection, characterized in that it comprises live Bacillus amyloliquefaciens E35 or crude fermented extract of Bacillus amyloliquefaciens E35; the Bacillus amyloliquefaciens E35 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 20, 2024, with accession number GDMCC 65657.
[0019] In summary, this application has the following beneficial effects: (1) Highly effective antiviral: Bacillus amyloliquefaciens E35 and its fermented crude extract can inhibit the adsorption, invasion and replication of viruses in marine aquaculture fish such as grouper iris virus, and effectively prevent and treat viral diseases in marine aquaculture fish such as iris virus; Bacillus amyloliquefaciens E35 and its fermented crude extract have no toxic side effects on marine aquaculture fish such as grouper and golden pomfret, and the fermented crude extract of Bacillus amyloliquefaciens E35 can directly inhibit grouper iris virus, with an inhibition rate of up to 84%.
[0020] (2) Growth promotion potential: Bacillus amyloliquefaciens E35 has a strong ability to secrete hydrolytic enzymes such as amylase, protease and cellulase, and has the potential to promote the digestion and absorption of farmed animals, which is beneficial to maintaining the intestinal health of fish, promoting the healthy growth of farmed animals, and enhancing the fish's resistance to iridovirus.
[0021] (3) Easy to use: It can be administered orally or mixed with feed, which is simple to operate, has no stress on farmed animals, and has high economic benefits.
[0022] (4) Safe and environmentally friendly: The strain and its metabolites have no toxic side effects, are antiviral, have no residues, and are green and safe. Attached Figure Description
[0023] Figure 1 The effects of different concentrations of Bacillus amyloliquefaciens E35 fermentation crude extract on GS of grouper spleen cells.
[0024] Figure 2 To investigate the in vitro antiviral effect of crude extract of Bacillus amyloliquefaciens E35 fermentation against grouper iridovirus SGIV infection: (a) Observation of cytopathic effect; (b) Expression level of MCP and VP19 genes of grouper iridovirus; (c) Crystal violet staining results.
[0025] Figure 3 The protective effect of feeding live Bacillus amyloliquefaciens E35 on SGIV-infected grouper: (a) Cumulative mortality rate graph; (b) Survival curve graph. Detailed Implementation
[0026] To better understand and implement this application, the technical solutions of this application will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only some of the embodiments of this application, and not all of them.
[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application.
[0028] Unless otherwise stated, all numerical values for the amounts of expressed components, reaction conditions, etc., used in the specification and claims are to be understood as being modified by the term "about". Therefore, unless otherwise indicated, the numerical parameters set forth herein are approximate values that can be varied to obtain the desired performance.
[0029] The word “and / or” as used in this article refers to one or all of the elements mentioned.
[0030] The terms "include" and "contain" as used in this article cover both cases where only the mentioned elements exist and cases where other unmentioned elements exist in addition to the mentioned elements.
[0031] All percentages in this application are weight percentages unless otherwise stated.
[0032] Unless otherwise stated, the terms “a,” “an,” “an,” and “the” as used in this specification are intended to include “at least one” or “one or more.” For example, “a component” refers to one or more components, and therefore more than one component may be considered and may be employed or used in the implementation of the described embodiments.
[0033] The technical features of the technical solution provided in this application will be further clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.
[0034] Example 1: Preparation of crude extract from Bacillus amyloliquefaciens E35 fermentation Bacillus amyloliquefaciens E35 was inoculated into ordinary beef extract peptone liquid medium and cultured in a shaker at 30°C for 48 hours. After the culture was completed, the bacterial cells were removed by centrifugation, and the fermentation supernatant was collected. 2 mol / L hydrochloric acid was added to the fermentation supernatant for acidification, and the pH of the fermentation supernatant was adjusted to 2. Obvious flocculent precipitate was observed to form. The supernatant was then allowed to stand at 4°C for 10 hours, and then centrifuged to collect the precipitate, obtaining the crude fermentation extract of Bacillus amyloliquefaciens E35. After freeze-drying, a powdered solid crude fermentation extract was obtained and stored at -80°C for later use.
[0035] Example 2: Preparation of crude fermentation extract of Bacillus amyloliquefaciens E35 Weigh the powdered solid fermentation crude extract obtained in Example 1 above, reconstitute it with sterile water, and adjust the volume to a concentration of 10 mg / mL to obtain the fermentation crude extract mother liquor; adjust the pH to 7.0 ± 0.2 with 2 mol / L NaOH aqueous solution, then filter it through a 0.22 μm filter membrane for sterilization, dispense it into sterile centrifuge tubes, and store it at -20℃ for later use in Examples 3-5.
[0036] Example 3: Cytotoxicity assay of crude fermentation extract of Bacillus amyloliquefaciens E35 The cells used in this embodiment are grouper spleen cells (GS).
[0037] (1) Seed GS cells in 96-well plates and cultured at 28°C until they grow into a monolayer. (2) Take the mother liquor of the fermented crude extract obtained in Example 2 and perform serial dilution with serum-free culture medium. Select 2.5 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL and 0 mg / mL (control group: CON) as the concentration of the diluent to obtain fermented crude extract diluents of different concentrations; (3) Four replicates were set up for each concentration, and 100 μL of different dilutions were added to each well and incubated in an incubator at 28°C for 48 h; (4) Observe the lesions of each group of cells under a light microscope and take pictures for preservation; (5) Cell viability was detected using the CCK-8 assay: The culture medium in each well was discarded, and the cells were washed once with sterile PBS. 100 μL of CCK-8 solution was added to each well, and the cells were incubated at 28°C for 4 hours in the dark. The OD value of each well was measured using a microplate reader at a wavelength of 450 nm. Cell viability was calculated according to the following formula. The results are shown in [Figure number missing]. Figure 1 ; Cell viability is calculated using the following formula: Cell viability / % = OD value of fermentation crude extract dilution group / OD value of cell control group × 100%.
[0038] Based on the above experimental results, the highest safe concentration was determined to be the diluted crude fermentation extract when there was no significant difference in cell viability compared to the control group and no obvious lesioning effect on the cells, which was 0.5 mg / mL.
[0039] Example 4: Evaluation of the in vitro efficacy of crude extract of Bacillus amyloliquefaciens E35 fermentation against grouper iridovirus SGIV. The cells used in this embodiment were GS cells. A 10 mg / mL mother liquor of the crude fermentation extract was diluted with serum-free medium to 0.5, 0.25, and 0.125 mg / mL to obtain different concentrations of diluted crude fermentation extract, and the inhibitory effect on SGIV at the corresponding concentrations was detected.
[0040] 1.1 qPCR detection of gene expression related to SGIV replication (1) Seed GS cells in 12-well plates and cultured at 28°C until they grow into a monolayer. (2) Take 800 μL of serum-free culture medium and 0.5, 0.25 and 0.125 mg / mL of crude fermentation extract dilution, add 2-5 μL of SGIV virus solution to each tube respectively, i.e. SGIV and E35+SGIV groups, and set up a blank control group CON. Incubate at 4℃ for 2h. (3) Discard the culture medium in the monolayer cell culture plate, wash once with sterile PBS, and add each tube after incubation at 4°C to each well of the cell plate and continue to culture at 28°C. (4) Observe the cytopathic effects of each group under a light microscope from 12 hours to 48 hours and take photos for preservation; (5) Discard the culture medium from each well, collect cells with trizol, extract total RNA from the samples and perform reverse transcription, amplify the viral envelope protein (VP) and major capsid protein (MCP) genes of SGIV using specific primers, using β-actin as an internal control, and analyze the expression of MCP and VP19 genes. The experiment was performed in triplicate; primer sequence information is detailed in Table 1. Results are shown in [Table 1]. Figure 2 a and Figure 2 b.
[0041] Table 1 qPCR primer sequences
[0042] 1.2 Evaluation of the anti-SGIV activity of Bacillus amyloliquefaciens E35 fermentation crude extract using crystal violet staining method (1) Seed GS cells in 12-well plates and cultured at 28°C until they grow into a monolayer. (2) Take 400 μL of serum-free culture medium and 0.5, 0.25 and 0.125 mg / mL of crude fermentation extract dilution, add 1-2.5 μL of SGIV virus solution to each tube respectively, namely SGIV and E35+SGIV group, and set up blank control group CON, and incubate at 4℃ for 2h. (3) Discard the culture medium in the monolayer cell culture plate, wash once with sterile PBS, and add each tube after incubation at 4°C to the cell plate and continue incubation at 28°C. (4) Observe the cytopathic effects of each group under a light microscope starting from 12 hours until 48 hours. (5) Discard the culture medium in each well and add 400 μL of 4% paraformaldehyde to each well for cell fixation for 1 h; (6) Remove paraformaldehyde by adding 300 μL of crystal violet solution to each well for staining. Take photos and observe after 6 hours. See below for results. Figure 2 c.
[0043] Results from in vitro cell experiments Figure 2 The results of the cell-level infection experiment showed that, at a safe concentration, the cytopathic effect induced by SGIV infection in the treated group (diluted fermentation crude extract) was significantly weaker than that in the untreated group (CON). The Bacillus amyloliquefaciens E35 fermentation crude extract significantly inhibited SGIV replication. After co-incubation for 48 hours, significant cell disruption was observed in the untreated group, while the cytopathic effect (CPE) in the treated group's GS cells was significantly reduced. Figure 2 a, 2c), and the expression of genes related to SGIV infection, namely viral protein (VP) and major capsid protein (MCP), was significantly reduced, indicating that the crude extract of Bacillus amyloliquefaciens E35 fermentation has a direct inhibitory effect on grouper iridovirus, with an inhibition rate of 84%. Figure 2 (b) indicates that the crude fermentation extract of Bacillus amyloliquefaciens E35 can inhibit SGIV-infected cells in a concentration-dependent manner.
[0044] Example 5: Evaluation of the in vivo efficacy of Bacillus amyloliquefaciens E35 against grouper iridovirus SGIV. (1) Amplify Bacillus amyloliquefaciens E35 in ordinary beef extract peptone liquid medium, and spray it evenly on the surface of the feed to make the bacterial count in the feed 2×10 8 -5×10 8 cfu / g, which is the Bacillus amyloliquefaciens E35 feeding group; at the same time, control group 1 and control group 2 were set up, and the same volume of PBS solution was sprayed evenly. The feed amount of each group was 3% of the body weight of the experimental fish, and the fish were fed twice a day. The feed was prepared fresh for each use, and the feeding cycle was 40 days. (2) After feeding, feeding was stopped for 1 day. Control group 1 (CON group) and Bacillus amyloliquefaciens E35 feeding group (E35 group) were injected with 300 μL of SGIV virus solution via intraperitoneal injection. Control group 2 was injected with an equal amount of PBS solution (PBS group). Each group had 2 replicates, with 10 fish in each group. (3) After SGIV infection, fish characteristics and mortality were observed and recorded daily until 7 days later, and the mortality rate of each group was calculated.
[0045] The results of the live feeding experiment are shown below Figure 3 The results showed that the mortality rate in control group 2 (PBS group) was 0%, the mean cumulative mortality rate in control group 1 (CON group) was 55%, and the mean cumulative mortality rate in the experimental group (E35 group) was 38.1%. Figure 3 a); Furthermore, feeding with live Bacillus amyloliquefaciens E35 can reduce the mortality rate of SGIV infection and delay the mortality process caused by SGIV infection, effectively maintaining the survival of fish over a long period of time. Figure 3 (b) This demonstrates that the Bacillus amyloliquefaciens E35 of this application can effectively inhibit SGIV infection and can be applied to the prevention and treatment of grouper iridovirus infection.
[0046] In summary, the live Bacillus amyloliquefaciens E35 and its crude fermentation extract provided in this application exhibit good anti-SGIV activity both in vitro and in vivo in water. This allows for the development of green products for the prevention and control of grouper iridovirus disease, such as microbial preparations or veterinary drugs. Applying these products to the prevention and control of grouper iridovirus is expected to alleviate the harm of iridovirus to grouper aquaculture, reduce secondary pollution and residues in the aquaculture environment caused by chemical drug use, and improve the quality and safety of grouper and other aquaculture products.
[0047] The above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application, but such modifications or substitutions are all within the scope of protection of this application.
Claims
1. The application of Bacillus amyloliquefaciens E35 in the preparation of products against grouper iridovirus infection, characterized by: The Bacillus amyloliquefaciens E35 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 20, 2024, with accession number GDMCC 65657.
2. The application of Bacillus amyloliquefaciens E35 as described in claim 1 in the preparation of products resistant to grouper iridovirus infection, characterized in that: The product includes at least one of live Bacillus amyloliquefaciens E35 or crude fermented Bacillus amyloliquefaciens E35 extract.
3. The application of Bacillus amyloliquefaciens E35 as described in claim 1 in the preparation of products resistant to grouper iridovirus infection, characterized in that: The grouper iridovirus mentioned is the Singapore grouper iridovirus.
4. The application of Bacillus amyloliquefaciens E35 as described in claim 1 in the preparation of products resistant to grouper iridovirus infection, characterized in that: The product can inhibit the envelope protein of Singapore grouper iridovirus. VP19 or / and major capsid proteins MCP Gene expression.
5. The application of Bacillus amyloliquefaciens E35 as described in claim 1 in the preparation of products resistant to grouper iridovirus infection, characterized in that: The product can improve the survival rate of fish.
6. The use of Bacillus amyloliquefaciens E35 as described in any one of claims 1-5 in the preparation of products against grouper iridovirus infection, characterized in that: The products include any one of microbial preparations and veterinary drugs.
7. A microbial preparation for resisting grouper iridovirus infection, characterized in that: Includes at least one of live Bacillus amyloliquefaciens E35 or the crude fermentation extract of Bacillus amyloliquefaciens E35; The Bacillus amyloliquefaciens E35 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 20, 2024, with accession number GDMCC 65657.
8. A fish medicine for treating grouper iridovirus infection, characterized in that: Includes at least one of live Bacillus amyloliquefaciens E35 or the crude fermentation extract of Bacillus amyloliquefaciens E35; The Bacillus amyloliquefaciens E35 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 20, 2024, with accession number GDMCC 65657.