Bacillus laterosporus and application thereof in aquaculture

Abstract

The invention provides a novel bacillus laterosporus (bacillus megatherium Z2) and an application method of the novel bacillus laterosporus in aquaculture. Through screening and gene improvement, the strain has the characteristics of high temperature resistance, high salt resistance and acid and alkali resistance, and can remarkably inhibit growth of aquatic pathogenic bacteria, improve water quality and promote healthy growth of aquaculture species. According to the application method, the strain with the concentration of 1 * 10 < 6 > CFU / L to 1 * 10 < 8 > CFU / L is added into a water body, diseases are effectively controlled, and the breeding yield and the aquatic product quality are improved. Experimental results show that the bacterial strain is obviously superior to the prior art in the aspects of bacteriostatic effect, water quality improvement, economic benefit and the like, and has the characteristics of environment friendliness, high efficiency and safety. A sustainable biological control scheme is provided for aquaculture, and the application prospect is wide.

Description

Technical Field

[0001] The present invention relates to the technical fields of aquaculture and microbiology, and in particular to a strain of Bacillus laterosporus and its application in aquaculture. Background Art

[0002] The aquaculture industry has been continuously expanding globally. However, with the continuous increase in the scale of aquaculture, the deterioration of the water environment and the frequent outbreak of diseases have become a major problem restricting the healthy development of aquaculture. Currently, the prevention and control of aquaculture diseases usually rely on the use of chemical drugs and antibiotics. Although this can control the disease condition in the short term, there are still many problems in the long run. First, the long-term use of antibiotics can lead to the problem of drug resistance in aquatic products, which in turn threatens human health. Second, the use of antibiotics and chemical drugs may also cause water pollution, affecting water quality and damaging the ecosystem. More seriously, the problem of antibiotic residues has become the focus of consumer attention, resulting in a decline in the market competitiveness of aquaculture products. Therefore, developing a safe, environmentally friendly and effective technology for controlling aquaculture diseases has become an urgent problem to be solved in the industry.

[0003] In recent years, the application of microorganisms in aquaculture has received increasing attention, especially the technical solutions using beneficial microorganisms as biological control agents. Microorganisms such as probiotics and Bacillus subtilis have achieved certain application results in aquaculture due to their roles in inhibiting pathogenic bacteria, improving water quality and promoting the growth of aquaculture. For example, Bacillus subtilis, as a spore-forming bacterium widely present in natural soil and water, can secrete various antibacterial substances to inhibit pathogenic microorganisms in the water. In addition, Bacillus subtilis can also reduce the occurrence of diseases and help improve water quality by competitively excluding pathogenic bacteria and promoting the growth of beneficial microorganisms in the water. However, most of the microbial strains currently used in aquaculture are common wild-type or unmodified strains, and their survival ability and stability in a complex water environment are still limited, and there are differences in the inhibitory effects on different water environments and pathogenic bacteria.

[0004] In order to solve the shortcomings in the existing microbial applications, researchers are committed to screening and improving more advantageous microbial strains, especially enhancing their stress resistance and improving their ability to survive and reproduce in water bodies. Through methods such as genetic modification and adaptive evolution, the stress resistance of beneficial microorganisms has been significantly improved in recent years. Some studies have shown that the Bacillus subtilis strains that have been screened and genetically engineered can grow stably in harsh environments such as high salt, acid and alkali, and have a wide antibacterial spectrum, which can effectively control a variety of common pathogens in aquaculture. For example, the improved Bacillus subtilis strains have a strong inhibitory effect on pathogenic bacteria such as Vibrio harveyi and Aeromonas hydrophila, and can play a good control effect under different water quality conditions. However, most of the existing Bacillus subtilis strains are aimed at the inhibition of a single pathogen, lack a broad-spectrum control effect, and the function of improving water quality is not prominent enough. Therefore, the development of a new type of Bacillus subtilis strain with stronger adaptability and its application in aquaculture is still a scientific and technological problem that needs to be solved urgently. Summary of the invention

[0005] The present invention provides a novel Bacillus laterosporus (Yuejinran Z2 Bacillus, with a deposit number of CGMCCNO.33332; deposited in the General Microbiological Center of China Microbiological Culture Collection Committee (CGMCC); classification name: Bacillus laterosporus; deposit date: January 8, 2025; the viability of the strain is: viable) and an application method thereof in aquaculture. The strain has been specifically screened and genetically modified, has significant stress resistance and excellent biological control ability, can effectively inhibit harmful pathogens in aquaculture water, improve water quality, and promote the healthy growth of aquaculture species, thereby increasing aquaculture output and aquatic product quality.

[0006] The technical solution of the invention includes the following aspects:

[0007] Screening and improvement of strains: The present invention provides a Yuejinran Z2 Bacillus strain. The strain is isolated from the natural environment, and after strict screening and genetic improvement, it has the following characteristics:

[0008] High stress resistance: It can survive in high temperature (60°C), high salt (10% NaCl), and extreme pH (5-10) environments, showing strong adaptability.

[0009] Highly effective antibacterial ability: It has a significant inhibitory effect on a variety of pathogenic bacteria (such as Vibrio harveyi, Aeromonas hydrophila, Pseudomonas fluorescens, etc.) in aquaculture water bodies, reducing the occurrence of diseases.

[0010] Application of the strain: The present invention also provides a specific method for applying Bacillus subtilis Yuejinran Z2 to aquaculture. The strain can be added to the aquaculture water body as a biological control agent, which can be achieved in the following ways:

[0011] Inhibiting pathogenic bacteria: The strain reduces the number of pathogenic bacteria in the water body and the disease incidence rate through the antibacterial substances it secretes and competitive exclusion.

[0012] Improving water quality: The strain helps to improve the dissolved oxygen in the water body and reduce the concentrations of harmful substances such as ammonia nitrogen and nitrates, thereby optimizing the water quality environment.

[0013] Promoting the healthy growth of aquatic products: By improving water quality and controlling diseases, it promotes the growth and development of aquaculture species (such as fish, shrimps, shellfish, etc.), and improves the survival rate and economic benefits.

[0014] Method for adding the bacterial agent:

[0015] In an aquaculture pond, add the Bacillus subtilis Yuejinran Z2 strain at an appropriate concentration (1×10^6 CFU / L to 1×10^8 CFU / L).

[0016] Add a certain amount of the bacterial agent every day to ensure the continuous presence of the strain in the water body and continuously play the roles of biological control and water quality improvement.

[0017] Production and preparation of the strain: The present invention also provides a large-scale production method of the strain, including processes such as culturing, fermenting, concentrating, and freeze-drying the strain to ensure the activity and efficacy of the strain during production and transportation.

[0018] Application effect: The experimental results show that Bacillus subtilis Yuejinran Z2 can significantly inhibit the growth of pathogenic bacteria, improve water quality, increase the survival rate and yield of aquaculture species in actual aquaculture. The concentrations of harmful substances (such as ammonia nitrogen and nitrates) in the water body are effectively controlled, and the water quality is significantly improved. The health status of aquaculture species is promoted, and particularly good growth rates and biomasses are shown in aquatic species such as fish and shrimps.

[0019] Advantages of the present invention:

[0020] 1. By adopting biological control means, the use of chemical agents is avoided, reducing drug residues and environmental pollution.

[0021] 2. This strain has broad-spectrum antibacterial ability and strong environmental adaptability, and can adapt to different water environments.

[0022] 3. The strain is non-toxic to non-target organisms in water, and the residue in aquatic products meets the food safety standards, with high safety.

[0023] 4. By improving the yield and quality of aquaculture, it provides a sustainable solution for farmers.

[0024] In summary, by providing a novel Bacillus mojavensis Z2 strain and its application method, the present invention provides an efficient, environmentally friendly and safe biological control means for the aquaculture industry, which can significantly improve the production efficiency of aquaculture and promote the health and sustainable development of the aquaculture environment. Brief Description of the Drawings

[0025] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings in the following description are only those of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0026] Figure 1 It is a schematic diagram of the water quality improvement curve of the present invention;

[0027] Figure 2 It is a schematic diagram of the ammonia nitrogen concentration curve of the present invention. Detailed Embodiments

[0028] The following will describe the present invention in detail with reference to the drawings and specific embodiments. At the same time, it should be noted here that in order to make the embodiments more detailed, the following embodiments are the best and preferred embodiments. For some well-known technologies, those skilled in the art can also adopt other alternative methods for implementation; and the drawings are only for more specific description of the embodiments, and are not intended to specifically limit the present invention.

[0029] It should be pointed out that in the specification, "an embodiment", "embodiment", "exemplary embodiment", "some embodiments", etc. indicate that the described embodiment may include specific features, structures or characteristics, but not necessarily every embodiment includes the specific feature, structure or characteristic. In addition, when combining an embodiment to describe a specific feature, structure or characteristic, implementing such a feature, structure or characteristic in combination with other embodiments (whether explicitly described or not) should be within the knowledge of those skilled in the relevant art.

[0030] Generally, terms can be understood at least in part from their use in context. For example, depending at least in part on the context, the term "one or more" as used herein can be used to describe any feature, structure, or property in a singular sense, or can be used to describe a combination of features, structures, or properties in a plural sense. Additionally, the term "based on" can be understood to not necessarily be intended to convey a set of exclusive factors, but rather can alternatively, depending at least in part on the context, allow for the existence of other factors that are not necessarily explicitly described.

[0031] Example 1: Isolation, Screening, and Identification of Bacillus mojavensis Z2

[0032] Steps:

[0033] Sample collection: Water samples and sediment samples were collected from healthy water bodies of different aquaculture farms.

[0034] Primary isolation: The samples were diluted and spread on a selective medium (such as nutrient agar containing antibiotics), and cultured at 37 °C for 24 - 48 hours.

[0035] Colony screening: Rod-shaped colonies with the ability to form spores were selected for pure culture.

[0036] Stress resistance screening:

[0037] High temperature resistance test: Incubate in a 60 °C water bath for 1 hour, and observe the spore survival rate.

[0038] Salt tolerance test: Culture in a medium containing 10% NaCl, and record the colony growth.

[0039] Acid and alkali tolerance test: Culture in media with pH 5 and pH 10, and observe colony formation.

[0040] Molecular identification:

[0041] Extract the genomic DNA of the strain and perform 16S rRNA gene sequencing.

[0042] Compare the sequencing results with the 16S rRNA sequences of Bacillus subtilis in the database to confirm the species identity.

[0043] Functional screening:

[0044] In vitro antibacterial experiment to test the antibacterial effect of the strain against common aquatic pathogenic bacteria (such as Vibrio spp., Aeromonas spp.).

[0045] Detection data:

[0046]

[0047] Example 2: Application effect of Bacillus mojavensis Z2 in actual aquaculture

[0048] Steps:

[0049] Experimental design: Select three aquaculture ponds with the same conditions, each with a volume of 1000 cubic meters, and set them as the control group (untreated), low-concentration treatment group (1×10^6 CFU / L), and high-concentration treatment group (1×10^8 CFU / L).

[0050] Bacterial agent dosing:

[0051] During the aquaculture cycle (120 days), dose the corresponding concentration of Bacillus mojavensis Z2 at a fixed time every day.

[0052] Control the dosing frequency and total amount to ensure the continuous presence of the strain in the water body.

[0053] Monitoring indicators:

[0054] Detect the total number of bacteria, the number of pathogenic bacteria, and water quality indicators (dissolved oxygen, ammonia nitrogen, nitrate, etc.) in the water body every month.

[0055] Record the survival rate, health status, and yield of aquatic products (such as fish and shrimp).

[0056] Data analysis: Compare the various indicators of different treatment groups with the control group to evaluate the application effect of the bacterial agent. Detection data:

[0057]

[0058] Result analysis:

[0059] Total number of bacteria: The treatment groups were significantly lower than the control group, especially the high-concentration treatment group had the largest reduction. Number of pathogenic bacteria: The number of pathogenic bacteria in the high-concentration treatment group dropped to the lowest, showing a strong antibacterial ability.

[0060] Water quality indicators: Dissolved oxygen increased significantly, and the contents of ammonia nitrogen and nitrate decreased significantly, indicating obvious improvement in water quality.

[0061] Survival rate and yield of aquatic products: The survival rate and yield of aquatic products in the treatment groups were significantly higher than those in the control group, and the high-concentration treatment group had the best effect.

[0062] Example 3: Influence of Bacillus mojavensis Z2 on the water body ecological balance

[0063] Steps:

[0064] Experimental setup: In a laboratory-simulated water body system, set up a control group and a treatment group (adding Bacillus mojavensis Z2, concentration 1×10^8 CFU / L), with three replicates in each group.

[0065] Aquaculture species: Select common aquaculture species such as tilapia and white shrimp for co-culture.

[0066] Ecological monitoring:

[0067] Regularly measure the physicochemical indicators (such as pH, temperature, dissolved oxygen) in the water body.

[0068] Analyze the microbial community structure in the water body through high-throughput sequencing technology.

[0069] Observe the growth and health status of the aquaculture species.

[0070] Long-term observation: The aquaculture cycle is 6 months, and record the ecological changes at each stage.

[0071] Detection data:

[0072]

[0073]

[0074] Result analysis:

[0075] Water quality improvement: The dissolved oxygen in the treatment group increased, and the contents of organic matter and neutral phosphorus decreased significantly, indicating that the water pollutants decreased and the water quality was effectively improved.

[0076] Microbial community structure: The proportion of beneficial bacteria increased, and the proportion of harmful bacteria decreased significantly, and the microbial community became more stable and diverse.

[0077] Growth of aquaculture species: The average weight gain of fish and shrimp was significantly higher than that of the control group, indicating that the bacterial agent had a promoting effect on the growth of aquaculture species.

[0078] Ecological balance: The water ecological environment of the treatment group was healthier, the microbial diversity index increased, and the ecological balance was maintained.

[0079] Example 4: Application safety assessment of Bacillus mojavensis YJJR Z2

[0080] Steps:

[0081] Ecotoxicological assessment:

[0082] Under laboratory conditions, test the toxicity of Bacillus mojavensis YJJR Z2 to non-target aquatic organisms (such as plankton, shellfish).

[0083] Adopt standard acute and subacute toxicity tests to evaluate the safety of the bacterial agent.

[0084] Environmental impact assessment:

[0085] Long-term monitor the residue situation of Bacillus mojavensis YJJR Z2 in the treated water body.

[0086] Evaluate the long-term impact of the strain on the water ecosystem, including its impact on other microorganisms in the soil and water.

[0087] Human health assessment:

[0088] Evaluate the residue of Bacillus cereus var. toyoi Z2 in aquatic products to ensure compliance with food safety standards.

[0089] Through animal experiments, detect the potential toxicity and allergenicity of the strain to mammals.

[0090] Detection data:

[0091]

[0092] Result analysis:

[0093] Ecological safety: Bacillus cereus var. toyoi Z2 has no obvious toxicity to non-target organisms, has a low environmental residue, and its long-term application does not affect the stability of the water ecosystem.

[0094] Food safety: The residue of the strain in aquatic products complies with national food safety standards, ensuring the safety of human consumption.

[0095] Human health: Animal experiments show that the strain is safe for mammals, with no obvious toxicity and allergic reactions.

[0096] The present invention provides a highly efficient, environmentally friendly and safe Bacillus cereus var. toyoi Z2 and its application method in aquaculture. Through actual application examples, it is verified that this strain can significantly improve the water quality of water bodies, inhibit harmful pathogenic bacteria, promote the healthy growth of cultured species, improve the yield and quality of aquaculture, and has broad application prospects and significant economic benefits. Compared with the prior art, the present invention has significant improvements in terms of stress resistance, antibacterial effect, ecological safety, etc.

[0097] The present invention covers any substitutions, modifications, equivalent methods and solutions made on the essence and scope of the present invention. In order to enable the public to have a thorough understanding of the present invention, specific details are described in detail in the following preferred embodiments of the present invention, and those skilled in the art can fully understand the present invention without the description of these details. In addition, in order to avoid unnecessary confusion to the essence of the present invention, well-known methods, processes, procedures, components and circuits are not described in detail.

[0098] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A Bacillus laterosporus (Yuejinran Z2 Bacillus), characterized in that: The accession number is CGMCCNO.33332, and it has been preserved in the General Microbiology Center of the China Microbiological Culture Collection Administration (CGMCC). The strain can survive at 60°C, grow in a culture medium containing 10% NaCl, and grow stably in an environment with a pH range of 5 to 10. It has a significant inhibitory effect on pathogenic bacteria in a variety of aquaculture water bodies.

2. The Bacillus laterosporus according to claim 1, characterized in that The strain is named Yuejinran Z2 Bacillus, and its gene sequence has 99.8% similarity with other strains in the genus Bacillus subtilis.

3. The Bacillus laterosporus according to claim 1 or 2 can be added to aquaculture water at a concentration of 1×10^6 CFU / L to 1×10^8 CFU / L to control the growth of pathogens, improve water quality, and promote the healthy growth of aquaculture species.

4. An aquaculture method, characterized in that: The Yuejinran Z2 Bacillus laterosporus according to any one of claims 1 to 3 is added into an aquaculture pond to control harmful pathogens in the water, improve water quality, promote the healthy growth of aquaculture species, and increase aquaculture production.

5. The aquaculture method according to claim 4, characterized in that: The Yuejinran Z2 Bacillus laterosporus was added to the aquaculture pond at a concentration of 1×10^6 CFU / L to 1×10^8 CFU / L, and was added to the water at a fixed time every day.

6. The aquaculture method according to claim 4 or 5, characterized in that: The aquaculture species include fish, shrimp, shellfish and other aquatic species.

7. The Yuejinran Z2 Bacillus strain according to any one of claims 1 to 3 is used as a biological control agent in aquaculture to inhibit harmful microorganisms in water bodies and improve the production efficiency of aquaculture.

8. A method for producing Yuejinran Z2 Bacillus laterosporus, characterized in that: Fermentation, cultivation, concentration, freeze-drying and other process methods are used to obtain strains with good activity for use in aquaculture.

9. The method for producing the Yuejinran Z2 Bacillus laterosporus according to claim 8, characterized in that: The fermentation process of the strain adopts temperature-controlled fermentation, which is carried out at 37°C for 24 to 48 hours, and the bacterial concentration reaches above 1×10^9 CFU / mL.

10. A biological control agent for aquaculture, characterized in that: It comprises the Yuejinran Z2 Bacillus laterosporus according to any one of claims 1 to 3 as a main active ingredient.

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