Bacillus megaterium strain for increasing stress tolerance of aquatic animals and application thereof

Abstract

The application discloses a bacillus megaterium strain for increasing adversity tolerance of aquatic animals and application thereof. The bacillus megaterium strain is named bacillus megaterium SCAU-Ohb1, the strain has been preserved in the Guangdong Microbial Culture Collection Center, the preservation number is GDMCC NO.62119, and the preservation date is December 13, 2021. The bacillus megaterium SCAU-Ohb1 is isolated from fish intestinal tracts and is safe to fish bodies. After the fermentation liquor of the bacillus megaterium SCAU-Ohb1 is mixed with a basic daily ration and is used for feeding freshwater fish, the activity of important antioxidant enzymes in the fish bodies can be significantly improved, the salt tolerance of gill tissues of the freshwater fish under acute salt stress can be increased, and the survival rate of the fish bodies can be improved. The application provides a new method for improving adversity tolerance of aquatic animals, has important significance for the aquaculture industry, and is expected to improve production efficiency and reduce losses.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbiology, more particularly to a bacillus megaterium strain for increasing the stress tolerance of aquatic animals and its application. BACKGROUND

[0002] In aquaculture, aquatic animals face various environmental stresses such as temperature changes, oxygen concentration changes, pH fluctuations, salt stress, water pollution, etc. By improving the stress tolerance of aquatic animals, their adaptability to environmental stress can be enhanced, thereby improving their production performance and economic benefits, and reducing losses during cultivation.

[0003] Salinity is an important ecological factor affecting the living environment of aquatic organisms such as fish, and is also an important abiotic factor affecting their various physiological activities. Changes in salinity force fish to adjust their physiological activities to maintain the dynamic balance of body fluid osmotic pressure, and can directly affect fish respiration, metabolism, digestion, growth and even survival. Improving the salt tolerance of fish can treat parasitic diseases or other salt-sensitive fish diseases, and can provide a theoretical basis for salt acclimation of fish such as sea or brackish water tilapia farming, thereby fully tapping the potential of the aquaculture industry.

[0004] Current research on the salinity adaptation mechanism of fish mainly focuses on the fish itself, and there is little known about the interaction between fish intestinal microorganisms and the host or the mechanism of responding to environmental and pathogenic stress. The research on the extent of improvement of fish growth and stress tolerance by fish microorganisms is very limited. Bacillus can secrete extracellular enzymes such as amylase, cellulase, protease, etc., promote the degradation of nutrients in feed that are difficult for the animal body to digest and absorb, such as cellulose, xylan, pectin, etc., facilitate the absorption of the animal body, and improve feed utilization. Feeds supplemented with Bacillus can be more effectively digested and absorbed by aquatic animals, which helps the digestion process, feed utilization, and assimilation of dietary components, improves the health status and growth performance of aquatic animals, and can also regulate the production of effective molecules with bactericidal activity in the cellular and humoral components of the immune system of aquatic animals, causing the up-regulation of the host's non-specific or specific immune system, directly inhibiting or reducing the incidence of pathogenic diseases, and improving the immune capacity of aquatic animals. Bacillus can also be used as a water quality improver to improve the water quality of the cultivation environment, degrade or transform feed residues and metabolic waste in the cultivation pond, reduce or eliminate harmful substances such as NH3-H, H2S, NO2-N in water, inhibit the growth of pathogenic bacteria, and achieve the purpose of improving water quality.

[0005] In recent years, Bacillus such as Bacillus licheniformis, Bacillus subtilis and Bacillus coagulans have been increasingly studied in the field of aquaculture, but there has been no progress in the research on improving the salt tolerance of aquatic animal hosts by Bacillus. Therefore, there is an urgent need in the art to develop strains or microbial preparations that can significantly improve the salt tolerance of aquatic animals under stress.

[0006] Bacillus megaterium is a gram-positive bacterium with relatively large shape, strict anaerobic, easy to produce spores with special structure, and has the characteristics of acid, alkali, high temperature (100℃), extrusion resistance, etc., so it has strong stability in feed processing and granulation environment. It is reported that the expression of proVWX operon of Bacillus megaterium is significantly increased under drought conditions, and the biosynthesis of intracellular compatible solutes such as betaine, tetrahydropyrimidine, spermidine and putrescine is significantly increased, indicating that Bacillus megaterium has certain salt tolerance. Some Bacillus megaterium has high bacteriostatic effect on many pathogenic bacteria and can effectively improve the salt and alkali stress tolerance of plants, but not all Bacillus megaterium can be used as feed additives. Some Bacillus megaterium that can effectively improve the salt and alkali stress tolerance of plants have toxicity or pathogenicity, and some Bacillus megaterium cannot colonize in animals, and cannot play the function of benefiting animals.

[0007] There is no related research on the effect of Bacillus megaterium on improving the stress tolerance of aquatic animals. SUMMARY

[0008] The purpose of the present application is to overcome the shortcomings of the prior art and provide a Bacillus megaterium strain that increases the stress tolerance of aquatic animals and its application.

[0009] The first purpose of the present application is to provide a Bacillus megaterium strain SCAU-Ohb1 that increases the stress tolerance of aquatic animals.

[0010] The second purpose of the present application is to provide a product for improving the stress tolerance of aquatic animals.

[0011] The third purpose of the present application is to provide a method for improving the stress tolerance of aquatic animals.

[0012] In order to achieve the above purpose, the present application is realized by the following technical scheme:

[0013] A Bacillus megaterium strain SCAU-Ohb1 that increases the stress tolerance of aquatic animals, which is classified and named as Bacillus megaterium The Bacillus megaterium strain SCAU-Ohb1 was deposited at the Guangdong Microbial Culture Collection Center on December 13, 2021, and the deposit number is GDMCC NO.62119.

[0014] Preferably, the aquatic animal is a freshwater aquaculture animal.

[0015] More preferably, the aquatic animal is a freshwater fish.

[0016] More preferably, the aquatic animal is tilapia.

[0017] Preferably, the increase in aquatic animal stress tolerance is to increase the aquatic animal's tolerance to salt stress.

[0018] The application of Bacillus megaterium SCAU-Ohb1 in the preparation of products that enhance the stress tolerance of aquatic animals should also be within the scope of protection of this invention.

[0019] Preferably, the improvement of aquatic animal stress tolerance refers to improving the tolerance of freshwater fish to salt stress.

[0020] More preferably, the freshwater fish is tilapia.

[0021] Preferably, the product is a feed additive or aquatic feed.

[0022] A product for improving the stress tolerance of aquatic animals, the product comprising the Bacillus megaterium SCAU-Ohb1.

[0023] Preferably, the improvement of aquatic animal stress tolerance refers to improving the tolerance of freshwater fish to salt stress.

[0024] More preferably, the freshwater fish is tilapia.

[0025] The application of the product in improving the stress tolerance of aquatic animals should also be within the scope of protection of this invention.

[0026] Preferably, the improvement of aquatic animal stress tolerance refers to improving the tolerance of freshwater fish to salt stress.

[0027] More preferably, the freshwater fish is tilapia.

[0028] Preferably, when the product is applied, the concentration of Bacillus megaterium SCAU-Ohb1 is at least 1 × 10⁻⁶. 7 CFU / ml.

[0029] A method for improving the stress tolerance of aquatic animals, the method comprising the Bacillus megaterium SCAU-Ohb1, or the product thereof.

[0030] Preferably, the concentration of Bacillus megaterium SCAU-Ohb1 during application is at least 1 × 10⁻⁶. 7 CFU / ml.

[0031] Preferably, the aquatic animal is a freshwater fish.

[0032] More preferably, the freshwater fish is tilapia.

[0033] Preferably, the application is performed by mixing the feed into the feed as a feed additive.

[0034] Preferably, the application can be a single application, repeated application, or continuous application.

[0035] Preferably, the application can be performed at any stage of the freshwater animal's life cycle.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] This invention discloses a strain of Bacillus megaterium that enhances the stress tolerance of aquatic animals. The Bacillus megaterium, isolated from the intestines of tilapia, is named Bacillus megaterium SCAU-Ohb1. This strain is safe and harmless to fish and possesses multiple key genes for compatibility with solutes, enabling it to produce various organic osmotic agents, such as betaine and proline. These organic osmotic agents can participate in regulating the osmotic system of fish, stabilizing the macromolecular structure and function of fish under salt stress, thereby maintaining osmotic pressure regulation and ion balance in the fish.

[0038] Experimental results showed that the fermentation broth of Bacillus megaterium SCAU-Ohb1 significantly increased important antioxidant enzymes such as SOD and CAT, which have the function of scavenging reactive oxygen species, in fish under acute salt stress. By scavenging reactive oxygen species, it protected cell integrity and improved the salt tolerance of tilapia gill tissue and the survival rate of the fish under acute salt stress. Therefore, feeding freshwater fish with fermentation broth of Bacillus megaterium SCAU-Ohb1 mixed with their basal diet can improve their physiological adaptability and enhance their survival ability under adverse conditions, which is of great significance to aquaculture. Attached Figure Description

[0039] Figure 1 This is a cluster analysis diagram used for strain identification.

[0040] Figure 2This is a genomic map of Bacillus megaterium SCAU-Ohb1. The outermost ring indicates the genome size, with each scale representing 5 kb. The second and third rings represent genes on the positive and negative strands of the genome, respectively, with different colors representing different COG functional classifications. The fourth ring shows repetitive sequences. The fifth ring shows tRNA and rRNA, with blue for tRNA and purple for rRNA. The sixth ring shows GC content, with light yellow areas indicating regions with GC content higher than the genome average, and higher peak values ​​indicating greater differences from the average GC content. Blue areas indicate regions with GC content lower than the genome average. The innermost ring shows GC-skew, with dark gray representing regions where G content is greater than C content and red representing regions where C content is greater than G content.

[0041] Figure 3 Mortality analysis of tilapia under high salt stress after adding different Bacillus strains.

[0042] Figure 4 Tissue sections of gill filaments in tilapia before and after acute salt stress (high-salt environment) with Bacillus megaterium feeding were obtained. Figure A shows the gill filament tissue section of the control group without Bacillus megaterium before acute salt stress; Figure B shows the gill filament tissue section of the control group without Bacillus megaterium after acute salt stress; Figure C shows the gill filament tissue section of the group fed with Bacillus megaterium before acute salt stress; Figure D shows the gill filament tissue section of the group fed with Bacillus megaterium after acute salt stress; Figure E shows the gill filament tissue section of the group fed with Bacillus spheroidae before acute salt stress; and Figure F shows the gill filament tissue section of the group fed with Bacillus spheroidae after acute salt stress. Red arrows indicate chloride cells, and blue arrows indicate gill filament bending and deformation.

[0043] Figure 5 Feeding tilapia with Bacillus megaterium affected liver antioxidant-related indicators before and after acute salt stress (freshwater). Figure A shows the change in hepatic malondialdehyde activity; Figure B shows the change in hepatic superoxide dismutase activity.

[0044] Figure 6 To analyze the mortality rate of tilapia under high salt stress 3 days after the addition of Bacillus megaterium. Detailed Implementation

[0045] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0046] As used herein, the terms “invention strain”, “invention Bacillus megaterium SCAU-Ohb1” and “Bacillus megaterium” refer to the strain with accession number GDMCC NO.62119 and its subcultured or derived strains.

[0047] The term “salt stress tolerance” as used in this article refers to improved survival rate under salt stress, or improved tolerance of aquatic animals to salt stress, or improved adaptability and / or survival ability of aquatic animals in the face of salinity changes and / or abnormal salinity environments.

[0048] Example 1 Bacillus megaterium ( B. megaterium Isolation, purification and identification of SCAU-Ohb1

[0049] I. Experimental Methods

[0050] Take tilapia and scrape the intestinal contents into a sterile 10 mL centrifuge tube using tweezers. Add an appropriate amount of LB liquid medium and mix well. Place the centrifuge tube in a water bath at 75℃~80℃ for 15~20 min. After serial dilution, select a suitable gradient mixture and spread it on LB solid plates. Incubate in a 37℃ biochemical incubator for 24~48 h, and select the bacterial strains from the resulting plate.

[0051] The selected bacterial strains were identified using 16S rDNA gene sequencing. The specific isolation steps were as follows: the strains were inoculated into 5 mL of LB medium and cultured at 37°C and 200 rpm for 24 hours. After centrifugation at 10,000 rpm for 1 min, the bacterial cells were collected in 1.5 mL centrifuge tubes. Genomic DNA was extracted from the bacterial samples, and the target region of the 16S rDNA was amplified by PCR using the universal 16S rDNA primers 27F (SEQ ID NO: 1) and 1525R (SEQ ID NO: 2). The PCR amplification system is shown in Table 1.

[0052] Table 1 PCR amplification system

[0053]

[0054] After sequencing, the amplified products were compared with the nucleotide sequence homology of the relevant sequences already registered in GenBank.

[0055] II. Experimental Results

[0056] Homology comparison analysis of the selected strains revealed that the 16S rDNA sequence of a strain named SCAU-Ohb1 was identical to that of the standard strain of Bacillus megaterium. Bacillus megaterium The 16S rDNA sequence similarity of ATCC 14581 and NR117473 reached 99.93%, as shown in the cluster analysis diagram.Figure 1 As shown. Therefore, the strain named SCAU-Ohb1 was identified as Bacillus megaterium (Bacillus megaterium). B. megaterium The strain is Bacillus megaterium SCAU-Ohb1. This Bacillus megaterium SCAU-Ohb1 strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 13, 2021, with accession number GDMCC NO.62119.

[0057] Example 2 Bacillus megaterium ( B. megaterium SCAU-Ohb1 genome sequencing, assembly annotation, and statistics of major compatibility solute-related genes

[0058] I. Experimental Methods

[0059] In this embodiment, the terms "invention strain", "invention Bacillus megaterium SCAU-Ohb1" and "Bacillus megaterium", etc., refer to the strain with accession number GDMCC NO.62119 and its passaged strains or derivative strains.

[0060] Bacillus megaterium B. megaterium The SCAU-Ohb1 whole genome was sequenced, assembled, and annotated by Beijing Biomarker Biotechnology Co., Ltd. The sequencing experimental workflow was performed according to the standard protocol provided by Oxford Nanopore Technologies (ONT), including sample quality control, library construction, library quality control, and library sequencing. Information analysis mainly included the following steps: raw data quality control, genome assembly, genome component analysis and functional annotation, and genome mapping analysis.

[0061] II. Experimental Results

[0062] Bacillus megaterium B. megaterium The SCAU-Ohb1 genome assembly Scaffold / Contig length was 5053924 bp, and the number of predicted coding genes was 5440. The results are shown in Table 2.

[0063] Table 2. Statistical analysis of genome assembly and coding gene prediction results of Bacillus megater SCAU-Ohb1

[0064]

[0065] Using the genomic information obtained from assembly and prediction, such as tRNA, rRNA, repetitive sequences, GC content, and gene function information, the software Circos v0.66 is used to draw a visualized genome map. Figure 2 This allows for a clearer exploration of the positional relationships between genomic components. The genome contains 14 16S rRNA sequences, all of which can be compared with 100% similarity.B. megaterium .

[0066] Compatible solutes mainly refer to small organic molecules that are polar, easily soluble, uncharged within the physiological pH range, and capable of accumulating in high concentrations within cells without hindering important cellular activities and metabolism. Compatible solutes have a protective effect on microorganisms under hyperosmolar conditions and can also serve as nutrients and energy sources within cells. As shown in Table 3, a statistical analysis of genes related to the production of compatible solutes in the whole genome of this bacterium revealed that the genome of *Bacillus megaterium* SCAU-Ohb1 contains one betaine dehydrogenase gene, three pyrroline-5-carboxylic acid reductase genes, and three proline dehydrogenase genes. Another *Bacillus* strain, also isolated from the intestine of tilapia and identified as *Bacillus spheroidans* (strain number SCAU-090), with its 16S rDNA sequence registered in GenBank as MK281536, lacks the betaine dehydrogenase gene but possesses one pyrroline-5-carboxylic acid reductase gene and three proline dehydrogenase genes. The whole-genome analysis predicts that Bacillus megaterium SCAU-Ohb1 has the ability to produce or accumulate the organic penetrants betaine and proline.

[0067] Table 3. Statistics of key genes for compatibility solutes in the whole genome of Bacillus megaterium SCAU-Ohb1

[0068]

[0069] Example 3 Safety test of Bacillus megaterium

[0070] I. Experimental Methods

[0071] In this embodiment, the terms "invention strain", "invention Bacillus megaterium SCAU-Ohb1" and "Bacillus megaterium", etc., refer to the strain with accession number GDMCC NO.62119 and its passaged strains or derivative strains.

[0072] The *Bacillus spheroidae* described in this embodiment was isolated from the intestine of tilapia. The strain number is SCAU-090, and its 16S rDNA sequence is registered in Genbank as MK281536.

[0073] Preparation of fermentation broths of Bacillus megaterium SCAU-Ohb1 and Bacillus spheroidae: Bacillus megaterium SCAU-Ohb1 and Bacillus spheroidae were inoculated into LB liquid medium (pH=7.3~7.6) and cultured in a shaker at 160 rpm and 30℃ for 24 h. Bacterial counts were performed, and the bacterial concentration was prepared to 1×10⁻⁶ using sterile LB medium. 8 CFU / mL solution.

[0074] Feed preparation: This study used pretreated commercial fish feed (Tongwei Co., Ltd., China) as the basal diet. The ingredients of the commercial fish feed were 38% crude protein, 3% crude fiber, 4% crude fat, 15% crude ash, 10% water, and 1% phosphorus.

[0075] Experimental grouping: 600 juvenile tilapia (about 3g) were divided into three groups: the experimental group with Bacillus megaterium SCAU-Ohb1, the control experimental group with Bacillus spheroides lysine-containing bacteria, and the control group without bacteria.

[0076] The experimental group supplemented with Bacillus megaterium SCAU-Ohb1 was prepared by spraying LB broth containing SCAU-Ohb1 into the basal diet of tilapia, resulting in a bacterial count of 1×10⁻⁶. 7 CFU / mg feed;

[0077] The control group, which included *Bacillus spheroides*, was prepared by spraying LB broth containing *Bacillus spheroides* into the basal diet of tilapia, resulting in a bacterial count of 1×10⁻⁶. 7 CFU / mg feed;

[0078] The control group without bacterial culture was sprayed with sterile LB broth into the basal diet of tilapia.

[0079] Unused feed should be packaged in sealed containers and labeled, and then stored at -20°C.

[0080] Three groups of tilapia fry were fed equal amounts of feed containing Bacillus megaterium SCAU-Ohb1, Bacillus spheroides lysine, and no bacteria, respectively. They were fed twice daily, morning and evening, and weighed after 21 days to investigate the effect of Bacillus megaterium SCAU-Ohb1 on the weight gain of tilapia.

[0081] II. Experimental Results

[0082] As shown in Table 4, the results of tilapia weight change showed that after feeding tilapia fry with Bacillus megaterium and Bacillus spheroidae for 21 days, there was no significant difference in weight gain between the two groups of tilapia and the control group without bacteria (P>0.05). This indicates that Bacillus megaterium did not significantly hinder weight gain during tilapia growth, proving the safety of Bacillus megaterium SCAU-Ohb1 for tilapia.

[0083] Table 4. Effects of Bacillus megaterium SCAU-Ohb1 and Bacillus spheroidans on body weight gain in tilapia.

[0084]

[0085] Example 4: Effect of Bacillus megaterium SCAU-Ohb1 on the tolerance of tilapia to high salt stress

[0086] I. Experimental Methods

[0087] In this embodiment, the terms "invention strain", "invention Bacillus megaterium SCAU-Ohb1" and "Bacillus megaterium", etc., refer to the strain with accession number GDMCC NO.62119 and its passaged strains or derivative strains.

[0088] The *Bacillus spheroidae* described in this embodiment was isolated from the intestine of tilapia. The strain number is SCAU-090, and its 16S rDNA sequence is registered in Genbank as MK281536.

[0089] The preparation of Bacillus megaterium SCAU-Ohb1 fermentation broth and Bacillus spheroidans fermentation broth, as well as feed preparation, are the same as in Example 3.

[0090] 1) Experimental Grouping

[0091] Experimental grouping: Tilapia fry (about 3g) were divided into three groups: the experimental group with Bacillus megaterium SCAU-Ohb1, the control experimental group with Bacillus spheroides lysine-containing bacteria, and the control group without bacteria.

[0092] The experimental group supplemented with Bacillus megaterium SCAU-Ohb1 was prepared by spraying LB broth containing SCAU-Ohb1 into the basal diet of tilapia, resulting in a bacterial count of 1×10⁻⁶. 7 CFU / mg feed;

[0093] The control group, which included *Bacillus spheroides*, was prepared by spraying LB broth containing *Bacillus spheroides* into the basal diet of tilapia, resulting in a bacterial count of 1×10⁻⁶. 7 CFU / mg feed;

[0094] The control group without bacterial culture was sprayed with sterile LB broth into the basal diet of tilapia.

[0095] Unused feed should be packaged in sealed containers and labeled, and then stored at -20°C.

[0096] Three groups of tilapia fry were fed equal amounts of the diet supplemented with Bacillus megaterium SCAU-Ohb1, Bacillus spheroides lysine-containing feed, and no feed, respectively. They were fed twice daily, morning and evening. After 21 days, they were weighed, and tilapia of uniform size were selected for subsequent acute salinity stress experiments.

[0097] 2) Acute salt stress

[0098] Tilapia were divided into freshwater and 2.5% saline experimental groups, with the addition of Bacillus megaterium SCAU-Ohb1, the addition of Bacillus spheroides lysine, and the control group without bacteria. Each experimental group was stocked with 60 tilapia of similar weight. The mortality rate of tilapia after acute salt stress was counted, and the tolerance of tilapia to salt stress in different experimental groups was analyzed.

[0099] During the acute salt stress experiment, no feed was given, and the number of fish deaths during the experiment was recorded. The main purpose was to measure the cumulative mortality rate of tilapia after acute salt stress, which was used as an indicator of salt stress tolerance.

[0100]

[0101] 3) Sample collection, histochemical staining, and enzyme activity assay

[0102] Sampling began at a semi-lethal level of high salt stress. The fish surface was disinfected with 70% ethanol, and sampling was performed under aseptic conditions. Liver tissue from five tilapia under the same experimental conditions was collected from each sample tube for antioxidant enzyme activity detection, and gill tissue samples from five tilapia under the same experimental conditions were placed in 4% formalin solution for tissue section analysis.

[0103] All animals are handled in accordance with EU regulations concerning the protection of laboratory animals.

[0104] 4) Chemical staining of gill tissue

[0105] The gill tissue samples were removed from 4% formalin solution, dehydrated with alcohol of different concentrations, cleared in xylene solution, embedded in paraffin, cut into 5 µm thin sections, stained with hematoxylin and eosin (HE), and finally the images were acquired and photographed.

[0106] 5) Measurement of liver antioxidant enzyme activity index

[0107] Following the instructions for enzyme activity measurement of the kit from Nanjing Jiancheng Biotechnology Co., Ltd., enzyme activity was measured in tilapia tissues from the experimental group supplemented with *Bacillus megaterium* SCAU-Ohb1, the control group supplemented with *Bacillus spheroides*, and the control group without bacteria. The measurement procedure is briefly as follows: the tissues were homogenized in pre-cooled physiological saline, centrifuged at 4°C, and the supernatant was collected. The activities of superoxide dismutase (SOD) and malondialdehyde (MDA) in the tilapia liver tissue were measured. The enzyme activity data are expressed as U / mL (SOD) and nmol / mL (MDA), respectively.

[0108] II. Experimental Results

[0109] (1) Analysis of the tolerance of tilapia to salt stress after adding Bacillus megaterium for 21 days

[0110] Experimental results are as follows Figure 3 As shown, after 2 hours of salt stress, the mortality rate in the control group without bacteria was 17%, the mortality rate in the experimental group with *Bacillus spheroides* was 12%, and the mortality rate in the experimental group with *Bacillus megaterium* SCAU-Ohb1 was 5%.

[0111] At 3 h, the mortality rate in the control group without bacteria was 95%, the mortality rate in the experimental group with Bacillus spheroides lysine added was 62%, and the mortality rate in the experimental group with Bacillus megaterium SCAU-Ohb1 added was 36%.

[0112] At 3.5 h, all members of the control group had died. The mortality rate of the experimental group with Bacillus spheroides lysine added was 83%, while the mortality rate of the experimental group with Bacillus megaterium SCAU-Ohb1 added was 62%.

[0113] The results show that, compared with no bacterial supplementation, the addition of Bacillus spheroides lysine and Bacillus megaterium to the basal diet improved the salt stress tolerance of tilapia. Furthermore, the experimental group supplemented with Bacillus megaterium SCAU-Ohb1 showed even higher salt stress tolerance. This indicates that the ability of Bacillus megaterium SCAU-Ohb1 to produce or accumulate organic osmotic agents helps fish maintain normal cellular water and ion balance, thereby enhancing their tolerance to salt stress.

[0114] (2) Morphological analysis of gill tissue sections of tilapia after acute salt stress

[0115] Experimental results are as follows Figure 4 As shown, the morphological structure of gill tissue sections indicates that the gill filaments of fish are relatively intact, symmetrical, and naturally extended under freshwater conditions. Figure 4 A, Figure 4 C and Figure 4 E), while the control group without bacteria after acute salt stress ( Figure 4 B) and the control group with added spherical lysine-containing Bacillus (B) Figure 4 The gill filaments of fish in group F showed more deformation and curvature, and the number of chloride cells was relatively increased. However, the experimental group with added Bacillus megaterium SCAU-Ohb1 ( Figure 4D) Although the gill filaments were not as uniform and orderly as in freshwater conditions, they were basically without curvature, and no significant increase in chloride cells was observed. This indicates that after salt stress, compared to the control group without bacterial supplementation and the experimental group with Bacillus spheroides lysine supplementation, the experimental group with Bacillus megaterium SCAU-Ohb1 showed less damage to the gill tissue, and the gill filaments were arranged more neatly and with greater integrity. This suggests that the active substances in the Bacillus megaterium SCAU-Ohb1 fermentation broth have beneficial effects on the fish, producing or accumulating organic osmotic agents, regulating the osmotic balance inside and outside cells, thereby helping the fish maintain normal cellular water and ion balance, reducing the degree of gill tissue damage and disordered arrangement, and improving overall tolerance to high salt stress. In other words, adding Bacillus megaterium SCAU-Ohb1 to a normal diet can promote the adaptation of fish to high salinity environments and improve their tolerance to salt stress; the high salt stress tolerance of tilapia was enhanced.

[0116] (3) Analysis of the antioxidant capacity of tilapia liver after acute salt stress

[0117] The results are as follows Figure 5 As shown, malondialdehyde (MDA) activity is an indicator of the degree of oxidative damage in organisms. In the control group without bacterial strains, MDA activity increased significantly before and after acute salinity stress. In contrast, the experimental group with *Bacillus megaterium* SCAU-Ohb1 and the control group with *Bacillus spheroides* showed significantly decreased MDA activity before and after acute salinity stress compared to the control group without bacterial strains. This indicates that *Bacillus megaterium* SCAU-Ohb1 can reduce lipid peroxidation products in tilapia cell membranes after salt stress, thus alleviating oxidative damage to the cell membrane.

[0118] Superoxide dismutase (SOD) is an important intracellular antioxidant enzyme, and its activity reflects the strength of the cell's protective system. After acute salt stress treatment, compared to the untreated group, the SOD activity in the experimental group supplemented with *Bacillus megaterium* SCAU-Ohb1 and the control group supplemented with *Bacillus spheroides* showed extremely significant and significantly increased activity, respectively. Furthermore, between the freshwater group and the acute salt stress treatment group, compared to the untreated group, the SOD activity in both the experimental group supplemented with *Bacillus megaterium* SCAU-Ohb1 and the control group supplemented with *Bacillus spheroides* showed significant increases. This indicates that *Bacillus megaterium* SCAU-Ohb1 can significantly enhance the antioxidant capacity of tilapia and reduce the degree of oxidative stress caused by salt stress.

[0119] Therefore, it can be seen that adding Bacillus megaterium SCAU-Ohb1 to the basal diet can improve the antioxidant capacity of tilapia and reduce the degree of oxidative stress caused by salt stress.

[0120] Example 4: Protective effect of short-term feeding of Bacillus megaterium on tilapia under high salt stress.

[0121] I. Experimental Methods

[0122] In this embodiment, the terms "invention strain", "invention Bacillus megaterium SCAU-Ohb1" and "Bacillus megaterium", etc., refer to the strain with accession number GDMCC NO.62119 and its passaged strains or derivative strains.

[0123] The *Bacillus spheroidae* described in this embodiment was isolated from the intestine of tilapia. The strain number is SCAU-090, and its 16S rDNA sequence is registered in Genbank as MK281536.

[0124] The preparation of Bacillus megaterium SCAU-Ohb1 fermentation broth and Bacillus spheroidans fermentation broth, as well as feed preparation, are the same as in Example 3.

[0125] 1) Experimental Grouping

[0126] Six hundred juvenile tilapia (approximately 30g) were divided into three groups: an experimental group supplemented with Bacillus megaterium SCAU-Ohb1, a control group supplemented with Bacillus spheroides lysine, and a control group without bacteria.

[0127] The experimental group supplemented with Bacillus megaterium SCAU-Ohb1 was prepared by spraying LB broth containing SCAU-Ohb1 into the basal diet of tilapia, resulting in a bacterial count of 1×10⁻⁶. 7 CFU / mg feed;

[0128] The control group, which included *Bacillus spheroides*, was prepared by spraying LB broth containing *Bacillus spheroides* into the basal diet of tilapia, resulting in a bacterial count of 1×10⁻⁶. 7 CFU / mg feed;

[0129] The control group without bacterial culture was sprayed with sterile LB broth into the basal diet of tilapia.

[0130] Unused feed should be packaged in sealed containers and labeled, and then stored at -20°C.

[0131] Three groups of tilapia fry were fed equal amounts of the diet supplemented with Bacillus megaterium SCAU-Ohb1, Bacillus spheroides lysine-containing feed, and no feed, respectively. They were fed twice daily, morning and evening. After three days, tilapia of uniform size were weighed and selected for subsequent acute salinity stress experiments.

[0132] 2) Acute salt stress

[0133] Tilapia fed diets supplemented with *Bacillus megaterium* SCAU-Ohb1, a control group supplemented with *Bacillus spheroides* lysine, and a control group without bacterial supplementation were divided into freshwater and 2.5% saline experimental groups. Each group contained 120 tilapia with no significant difference in body weight. Mortality rates after acute salt stress were recorded to analyze differences in salt tolerance. No feed was given during the acute salt stress experiment, and the number of fish deaths was recorded. The cumulative mortality rate after acute salt stress was the primary indicator of tilapia tolerance.

[0134]

[0135] II. Experimental Results

[0136] Experimental results are as follows Figure 6 As shown, at 5 hours, the mortality rate of the control group without bacteria was 24%, the mortality rate of the experimental group with Bacillus spheroides lysine added was 7%, and the mortality rate of the experimental group with Bacillus megaterium SCAU-Ohb1 added was 2%; at 5.5 hours, the mortality rate of the control group without bacteria was 42%, the mortality rate of the experimental group with Bacillus spheroides lysine added was 24%, and the mortality rate of the experimental group with Bacillus megaterium SCAU-Ohb1 added was 12%, indicating that short-term feeding with Bacillus megaterium can reduce the mortality rate of tilapia fry.

[0137] The onset time of mortality under acute salt stress differed between 3 and 21 days of feeding with Bacillus megaterium SCAU-Ohb1. This difference was related not only to the feeding period but also to the size of the tilapia. Generally, the longer the feeding period and the larger the fish, the stronger their tolerance to high salt stress. Overall, feeding with Bacillus megaterium SCAU-Ohb1 significantly improved the fish's tolerance to high salt stress and enhanced their antioxidant capacity.

[0138] In summary, the whole genome of *Bacillus megaterium* SCAU-Ohb1 contains key genes for compatible solute metabolism, such as betaine dehydrogenase and pyrroline-5-carboxylic acid reductase, which may produce compatible solutes and thus participate in the regulation of osmotic pressure and ion balance in fish. Experimental results show that feeding tilapia with *Bacillus megaterium* SCAU-Ohb1 significantly increases the activity of important antioxidant enzymes such as SOD (superoxide dismutase), which has the function of scavenging reactive oxygen species, in fish under acute salt stress. It also improves the salt tolerance of tilapia gill tissue and the survival rate of the fish under acute salt stress. In other words, adding *Bacillus megaterium* SCAU-Ohb1 to the basal diet of fish can improve the tolerance of tilapia to high salt stress and enhance the antioxidant capacity of the fish.

[0139] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of Bacillus megaterium SCAU-Ohb1 that enhances the stress tolerance of aquatic animals, characterized in that, The Bacillus megaterium SCAU-Ohb1 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on December 13, 2021, with accession number GDMCC NO: 62119. The genome of the Bacillus megaterium SCAU-Ohb1 contains one betaine dehydrogenase gene, three pyrroline-5-carboxylic acid reductase genes, and three proline dehydrogenase genes.

2. The application of Bacillus megaterium SCAU-Ohb1 as described in claim 1 in the preparation of products that enhance the stress tolerance of aquatic animals, characterized in that, The improvement of aquatic animal stress tolerance refers to improving the tolerance of freshwater fish to salt stress.

3. The application according to claim 2, characterized in that, The product is a feed additive or aquatic feed.

4. A product for improving the stress tolerance of aquatic animals, characterized in that, The product contains Bacillus megaterium SCAU-Ohb1 as described in claim 1; The improvement of aquatic animal stress tolerance refers to improving the tolerance of freshwater fish to salt stress.

5. The application of the product according to claim 4 in improving the stress tolerance of aquatic animals, characterized in that, The improvement of aquatic animal stress tolerance refers to improving the tolerance of freshwater fish to salt stress.

6. A method for improving the stress tolerance of aquatic animals, characterized in that, The method includes applying Bacillus megaterium SCAU-Ohb1 as described in claim 1, or the product as described in claim 4; The improvement of aquatic animal stress tolerance refers to improving the tolerance of freshwater fish to salt stress.

7. The method according to claim 6, characterized in that, When applying, the concentration of Bacillus megaterium SCAU-Ohb1 should be at least 1 × 10⁻⁶. 7 CFU / ml.

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