Bacillus megaterium A-27 as well as microecological preparation and preparation method thereof

Abstract

The invention relates to bacillus megaterium A-27, a microecological preparation thereof and a preparation method of the microecological preparation, the bacillus megaterium is preserved in the China Center for Type Culture Collection, the strain preservation number is CCTCC NO: M 20241848, the classification name is bacillus megaterium A-27, and the Latin name is bacillus megaterium A-27. The microecological preparation is prepared by fermenting bacillus megatherium A-27. The invention also discloses a preparation method of the microecological preparation. A microecological preparation which is prepared from the bacterial strain through a corresponding method and is suitable for aquaculture acts on freshwater fishes in a feed additive mode, has broad-spectrum fish pathogenic bacterium inhibition activity, and particularly has a remarkable effect of resisting aeromonas hydrophila infection on the freshwater fishes; meanwhile, the strain can be stably colonized in freshwater fish bodies, the immunocompetence of the freshwater fishes is effectively improved, and a certain effect of promoting the growth of the fishes is achieved.

Description

Technical Field

[0001] The present invention relates to the field of microecological preparations, and in particular to a strain of Bacillus megaterium A-27, a microecological preparation thereof and a preparation method thereof. Background Art

[0002] The rapid growth of the aquaculture industry has greatly promoted economic prosperity and the diversification of food culture, but it has also brought severe tests to environmental sustainability. Ensuring the sustainable development of the aquaculture industry, especially under the premise of maintaining the health of aquatic ecology and the self-recovery capacity of water bodies, has become a common focus of attention within and outside the industry. Achieving a balance aims to promote the sustainable and healthy development of the aquaculture industry, ensure that humans can obtain safe and nutritious aquatic products, and effectively avoid irreversible damage to the aquatic ecosystem. Chemical drugs such as antimicrobial agents, anthelmintics and antibiotics are currently widely used in the aquaculture industry. Although they have guaranteed the health of aquatic animals to a certain extent, they have also brought problems that cannot be ignored. These drug residues may accumulate in downstream areas through water circulation, affecting water quality safety; at the same time, their enrichment in aquatic animals poses a potential risk to human health and exacerbates the emergence of drug-resistant pathogens.

[0003] The transformation of fish farming mode from the past extensive farming method with complex trophic levels and a relatively natural environment to the current farming mode with simplified trophic levels and refined management has achieved a significant expansion of production scale, but it is also accompanied by a significant problem: the natural resilience of the farming environment has weakened, and the natural immunity of fish has been suppressed, thereby increasing their sensitivity to bacterial diseases. In response to this challenge, with the in-depth study of bacterial pathogens of fish, a variety of response strategies have emerged to reduce the impact of diseases, including but not limited to chemotherapeutic agents, natural Chinese herbal medicines, preventive vaccines, and microecological preparations using antagonistic bacteria and other diversified prevention and control methods. Gao et al. isolated a Bacillus megaterium S379, which can effectively degrade nitrite in aquaculture wastewater and has positive hydrolase activity for starch, casein, cellulose and fat (GAO J, GAO D, LIU H, et al. Biopotentiality of high efficient aerobicdenitrifier Bacillus megaterium S379 for intensive aquaculture water qualitymanagement[J]. Journal of environmental management, 2018, 222: 104-11.). Sathishkumar et al. studied the antagonistic effect of Lactobacillus plantarum and Bacillus megaterium on Aeromonas hydrophila. The experimental results showed that the combination of the two bacteria can effectively improve the survival rate of catfish (SATHISHKUMAR R, THIRUMALAIKUMAR E, RAJESWARI MV, et al. Extraction, statistical optimization, and immunomodulatory activity of exopolysaccharide from seaweed-associated Bacillus megateriumDSKPDF CMST3[J]. Biomass Conversion and Biorefinery, 2022: 1-14.). Bacillus megaterium has potential application value in aquaculture. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a strain of Bacillus megaterium A-27, which has a good antagonistic effect on a variety of common pathogens of fish and can enable fish to resist infection by a variety of fish bacterial diseases.

[0005] The technical problem to be further solved by the present invention is to provide a microecological preparation of Bacillus megaterium A-27 and a preparation method thereof, wherein the microecological preparation can enable fish to resist infection by a variety of fish bacterial diseases and is suitable for fish disease prevention and treatment in aquaculture.

[0006] The technical solution adopted by the present invention to solve its technical problems is as follows: a strain of Bacillus megaterium A-27, which was deposited in the China Center for Type Culture Collection on August 26, 2024, with a strain collection number of CCTCC NO: M 20241848; it is classified and named: Bacillus megaterium A-27, and its Latin name is Bacillus megaterium A-27.

[0007] The 16S rDNA sequence of Bacillus megaterium A-27 of the present invention is shown in the sequence listing SEQ ID No. 1.

[0008] The Bacillus megaterium A-27 of the present invention was screened from a soil sample of West Lake Park, Longjing Road, West Lake District, Hangzhou City, Zhejiang Province.

[0009] The present invention further solves the technical problem by adopting the following technical solution: a microecological preparation of Bacillus megaterium A-27, suitable for aquaculture, is fermented by Bacillus megaterium A-27 with a strain collection number of CCTCC NO: M 20241848. The antibacterial active substances in the fermentation liquid of the strain have good medium and high temperature, acid and alkali, ultraviolet irradiation and protease stability, and are relatively stable in actual production applications; it has a good inhibitory effect on freshwater fish pathogens, can promote fish growth, and improve the immunity of fish.

[0010] Furthermore, the microecological preparation is a liquid preparation or a solid preparation.

[0011] Furthermore, the aquaculture animals are freshwater fish.

[0012] Furthermore, the freshwater fish is crucian carp, grass carp or carp.

[0013] The present invention further solves the technical problem by adopting the following technical solution: a method for preparing a microecological preparation suitable for aquaculture, comprising the following steps:

[0014] (1) Inoculation and activation: transferring the Bacillus megaterium A-27 stored slant strain to an activated slant culture medium for activation to obtain an activated seed solution; the activated slant culture medium is LB liquid culture medium;

[0015] The activation culture conditions are as follows: temperature 28-30°C, liquid volume 10-30% of the container volume, and shaking speed 120-140 rpm;

[0016] (2) First expansion culture: The seed liquid activated in step (1) is inoculated into a fermentation tank at an inoculation rate of 1 to 2% for first expansion culture;

[0017] The fermentation medium formula for the first expansion culture is: tryptic soy bean broth (TSB) 42-45 g / L, glucose 8-10 g / L, yeast powder 8-9 g / L, magnesium sulfate heptahydrate 2.0-2.2 g / L; sterilized at 115°C for more than 30 min;

[0018] The culture conditions are as follows: the entire culture process is monitored online, the dissolved oxygen concentration is 35-55%, the temperature is 28-30°C, the culture is more than 24 hours (preferably 24-26 hours), and the defoaming agent is added online in real time;

[0019] (3) Second expansion culture: inoculating the seed liquid of the first expansion culture in step (2) into a fermentation tank filled with fermentation medium at an inoculum amount of 10-15% for fermentation culture, wherein the fermentation medium formula is the same as the fermentation medium formula of the first expansion culture in step (2); the culture conditions are: dissolved oxygen concentration of 35-55%, temperature of 28-30° C., culture for more than 24 hours (preferably 24-26 hours), and adding defoaming agent online in real time;

[0020] Furthermore, in step (3), the fermentation tank is preferably filled with a fermentation medium equivalent to 65%-70% of the volume of the fermentation tank, and more preferably a 500L-1000L fermentation tank.

[0021] (4) Concentration and collection: After the culture is completed, the fermentation product is collected in a tank and concentrated to obtain a microecological liquid bacterial agent, or spray-dried to obtain a microecological solid preparation.

[0022] The identification and research methods of Bacillus megaterium A-27 of the present invention are as follows.

[0023] (A) Observation of cell morphological characteristics of Bacillus megaterium A-27;

[0024] (ii) Identification using the 16S rDNA gene;

[0025] (III) Physical and chemical properties testing of antibacterial active substances of Bacillus megaterium A-27;

[0026] (iv) Detection of colonization of Bacillus megaterium A-27 in grass carp;

[0027] (V) Effects of Bacillus megaterium A-27 as a feed additive on the growth and disease resistance of fish, especially grass carp.

[0028] The invention has the following beneficial effects: (1) The 16S rDNA gene sequence is determined to be highly homologous to Bacillus megaterium ATCC 14581, and is named Bacillus megaterium A-27 (strain collection number: CCTCC NO: M20241848; classification name: Bacillus megaterium A-27, Latin name: Bacillus megaterium A-27), and has the following beneficial effects on Aeromonas hydrophila (Ah), Aeromonas veronii (Aeromonas veronii), Aeromonas sobria (Aeromonas megaterium A-27), and has the following beneficial effects on Aeromonas hydrophila (Ah), Aeromonas veronii (Aeromonas veronii), Aeromonas megaterium (Aeromonas megaterium A-27), and has the following beneficial effects on Aeromonas hydrophila (Ah), Aeromonas veronii (Aeromonas megaterium A-27), Aeromonas megaterium A-27, and Aeromonas megaterium A-27. sobria) and other freshwater fish pathogens; (2) Bacillus megaterium A-27 can survive and colonize in fish and is harmless to fish, which lays a foundation for the strain to play a stable role in disease prevention and growth promotion in fish farming; (3) Adding Bacillus megaterium A-27 to feed fish as a feed additive can promote the growth rate and feed efficiency of fish, improve the immunity of fish, and enhance the resistance of fish to pathogens; (4) The method of preparing Bacillus megaterium A-27 into a microecological preparation is relatively simple and has a low production cost. At the same time, it can effectively reduce the risks of drug residues and pathogenic bacteria resistance caused by the abuse of antibiotics, and has good application prospects.

[0029] SEQ ID №1:

[0030]

[0031] Description of Microbiological Deposit

[0032] The Bacillus megaterium A-27 of the present invention was deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China) on August 26, 2024, with the strain accession number of CCTCC NO: M 20241848; the classification name is: Bacillus megaterium A-27; the Latin name: Bacillus megaterium A-27. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a diagram showing the antibacterial effect of Bacillus megaterium A-27 of the present invention on fish pathogens;

[0034] Figure 2 These are observation diagrams of the morphological characteristics of Bacillus megaterium A-27 of the present invention; a: observation diagram of the plate colony morphology of strain A-27; b: observation diagram of strain A-27 under a phase contrast microscope; c: observation diagram of strain A-27 under Gram staining; d, e: observation diagrams of strain A-27 under a scanning electron microscope;

[0035] Figure 3 The phylogenetic tree diagram is constructed from the 16SrDNA sequence of Bacillus megaterium A-27 of the present invention;

[0036] Figure 4 It is a RAST strain identification point map of the whole genome of Bacillus megaterium A-27 of the present invention;

[0037] Figure 5 It is a RAST species identification circle diagram of the whole genome of Bacillus megaterium A-27 of the present invention;

[0038] Figure 6 This is a diagram of the physical and chemical properties of the antibacterial active substance of Bacillus megaterium A-27 of the present invention (*, P < 0.05; **, P < 0.01; ***, P < 0.001);

[0039] Figure 7 This is a diagram showing the effect of the fermentation supernatant of Bacillus megaterium A-27 of the present invention on liver cells of grass carp (LB: culture medium treatment; Ah: treatment with the fermentation supernatant of Aeromonas hydrophila AhX040 strain; A-27: treatment with the fermentation supernatant of Bacillus megaterium A-27 strain);

[0040] Figure 8 This is a graph showing the effect of Bacillus megaterium A-27 of the present invention on the survival rate of L8824 grass carp liver cells;

[0041] Fig. 9It is an observation (400×) diagram of tissue infection slices of grass carp after in vitro injection of Bacillus megaterium A-27 of the present invention and a comparison diagram of the control group;

[0042] Fig.10 This is a diagram of the colonization and protection of the Bacillus megaterium A-27 labeled strain in the grass carp observed by the small animal living imaging system of the present invention;

[0043] Fig.11 This is a graph showing the results of analyzing the effect of Bacillus megaterium A-27 of the present invention on the expression of growth-related genes in grass carp muscle (* indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001);

[0044] Fig.12 This is a graph showing the results of analyzing the effect of Bacillus megaterium A-27 of the present invention on the enzyme activity of nonspecific immune factors in grass carp serum (* indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001);

[0045] Fig.13 This is a graph showing the results of the analysis of the effect of Bacillus megaterium A-27 of the present invention on the expression of antioxidant-related genes in grass carp tissues (* indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001);

[0046] Fig.14 This is a graph showing the results of the analysis of the effect of Bacillus megaterium A-27 of the present invention on the expression of immune-related genes in grass carp tissues (* indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001);

[0047] Fig.15 This is a survival curve of grass carp fed with Bacillus megaterium A-27 of the present invention and challenged with Ah X040;

[0048] Fig.16 This is a diagram showing the distribution of Ah X040mCherry in grass carp observed by the small animal imaging system and the inhibition of Ah X040 by the Bacillus megaterium A-27 probiotic preparation. DETAILED DESCRIPTION

[0049] The present invention will be further described below in conjunction with the embodiments and drawings.

[0050] Unless otherwise specified, the chemical reagents used in the embodiments of the present invention were obtained through conventional commercial channels.

[0051] Unless otherwise specified in the present invention, the percentages are all by mass.

[0052] (I) Screening of Bacillus megaterium A-27 and determination of antibacterial spectrum

[0053] Dissolve about 10g of soil sample in 90ml of sterile water and shake thoroughly, then dilute to 10 by 10-fold gradient dilution method. -1 , 10 -2 , 10 -3 , 10 -4 100 μL of each gradient sample was pipetted onto LB solid culture plates and evenly spread with a sterile applicator. The plates were inverted and cultured in a constant temperature incubator at 30°C overnight. The dominant single colony was picked and streaked and purified again. The fish pathogen Aeromonas veronii AvX005, Aeromonas hydrophila, Aeromonas sobria, Plesiomonas shigelloide, Aeromonas salmonicida, Aeromonas allosaccharophila, Aeromonas caviae, Citrobacter freundii, and Edwardsiella tarda were used as indicator bacteria, and the antibacterial activity of the fermentation supernatant against the above pathogens was detected by the punch method (for results, see Figure 1 ) and found a strain with good antibacterial effect on 9 fish pathogens, named Bacillus megaterium A-27.

[0054] (II) Cell morphological characteristics of Bacillus megaterium A-27

[0055] When strain A-27 was streaked on LB solid medium, it was observed that the colony of A-27 was medium-sized, smooth and moist, transparent, convex in the middle and irregular at the edge; under an optical microscope, a typical rod-shaped structure was observed, which was purple after Gram staining, indicating that it was a Gram-positive bacterium; under a scanning electron microscope, its ultrastructure was found to be rod-shaped as a whole, with regular elliptical shapes at both ends. The above observations are consistent with the morphological characteristics of typical Bacillus (see Figure 2 ).

[0056] (III) Identification of 16S rDNA gene of Bacillus megaterium A-27

[0057] The strain A-27 was inoculated into liquid CSM medium and cultured at 30°C for 24 h. The bacteria were collected by centrifugation at 12,000 rpm for 2.5 min. The genomic DNA of the strain A-27 was extracted using the Ezup column bacterial genomic DNA extraction kit (Shanghai Sangon Biotechnology Co., Ltd.) and the 16SrDNA gene was amplified using primers.

[0058] 16SrDNA F: 5′-AGAGTTTGATCCTGGCTCAG-3′;

[0059] 16SrDNA R: 5′-ACGGCTACCTTGTTACGACTT-3′.

[0060] The 16SrDNA gene sequence was amplified using the above primers, and the expected length of the sequence was about 1500 bp.

[0061] PCR reaction system (20 μL): sterile double distilled water, 14 μL; 10× Buffer, 2 μL; dNTP, 1.6 μL; Bf-R (10 μM), 0.6 μL; Bf-F (10 μM), 0.6 μL; genomic template, 1 μL; PrimerSTAR DNA Polymerase (Takara), 0.2 μL;

[0062] PCR reaction program: pre-denaturation at 95°C for 5 min; 30 cycles: 95°C, 45 sec; 55°C, 45 sec; 72°C, 1.5 min; 72°C, 10 min.

[0063] After the 16SrDNA gene PCR product was detected by 1.0% agarose gel electrophoresis, the successfully amplified product was sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing.

[0064] The 16SrDNA gene sequence of strain A-27 was sequenced and showed to be 1488 bp in length. The amplified sequencing results were analyzed by BLAST comparison on NCBI, and the phylogenetic tree of the strain was constructed by the neighbor-joining method in MEGA X software (see Figure 3 ).

[0065] (IV) RAST identification of Bacillus megaterium A-27 whole genome

[0066] After the whole genome of strain A-27 was sequenced, the whole genome sequence of strain A-27 and its closest relative strain was compared online using the RAST database, and dot plots and circle plots were drawn (see Figure 4 , Figure 5 ), the similarity between strain A-27 and them was only 82.91%. Combining the morphological characteristics and molecular biological identification of strain A-27, the strain was further identified as a new species of Bacillus megaterium and named Bacillus megaterium A-27.

[0067] (V) Analysis of the physicochemical properties of antibacterial active substances of Bacillus megaterium A-27

[0068] (1) Thermal stability

[0069] The strain A-27 was inoculated into liquid CSM medium and cultured at 30°C and 120rpm for 24h. The fermentation supernatant was treated in a constant temperature water bath at 20°C, 30°C, 50°C, 70°C, 90°C, and 100°C for 1h. The antibacterial activity of the treated fermentation broth against Aeromonas hydrophila was detected by the punch method. The untreated supernatant was used as a control. Each treatment was repeated three times. (See Figure 6 ).

[0070] (2) Acid and alkali tolerance

[0071] The strain A-27 was inoculated into CSM fermentation medium at 30°C and 120rpm for 24h, and the pH of the fermentation supernatant was adjusted to 1, 3, 5, 7, 9, and 11 for 1h, and then the pH was adjusted back to the original pH. The antibacterial activity of the treated fermentation broth against Aeromonas hydrophila was detected by the punch method, and the untreated supernatant was used as a control. Each treatment was repeated three times.

[0072] (3) UV light tolerance

[0073] The strain A-27 was inoculated into CSM fermentation medium at 30°C and 120rpm for 24h. The fermentation supernatant was irradiated under ultraviolet light for 30min, 60min, 90min, 120min, 150min, and 180min, respectively. The antibacterial activity of the treated fermentation broth against Aeromonas hydrophila was detected by punching method. The untreated supernatant was used as control. Each treatment was repeated three times (see Figure 6 ).

[0074] (4) Protease resistance

[0075] The strain A-27 was inoculated into CSM fermentation medium at 30℃ and 120rpm for 24h. 40μL protease (20mg / mL) was added to 400μL fermentation supernatant. The reaction was terminated at 37℃ after reacting at 52℃ for 1h. The antibacterial activity of the treated fermentation broth against Aeromonas hydrophila was detected by punching method. The untreated supernatant was used as control. Each treatment was repeated three times. (See Figure 6 ).

[0076] It can be seen that the antibacterial active substances in the fermentation broth of this strain have good medium-high temperature, acid-base, ultraviolet irradiation and protease stability.

[0077] (VI) Preparation of Bacillus megaterium A-27 microecological preparation

[0078] (1) Inoculation and activation: transferring the Bacillus megaterium A-27 stored slant strain to an activated slant culture medium for activation to obtain an activated seed solution; the activated slant culture medium is LB liquid culture medium, and the activation culture conditions are: temperature 30° C., liquid volume 30% of the container volume, and shaking speed 140 rpm;

[0079] (2) First expansion culture: The seed liquid activated in step (1) is inoculated into a fermentation tank at an inoculum amount of 2% for the first expansion culture; the fermentation medium formula is: 45 g / L tryptic soy broth (TSB), 10 g / L glucose, 9 g / L yeast powder, and 2.2 g / L magnesium sulfate heptahydrate; the culture conditions are: the entire culture process is monitored online, the dissolved oxygen concentration is 55%, the temperature is 30°C, the culture is for 24 hours, and the defoaming agent is added online in real time;

[0080] (3) Second expansion culture: the seed solution expanded in step (2) is inoculated into a 500 L fermentation tank filled with 70% fermentation medium at an inoculum rate of 15%, and the fermentation medium formula is the same as the fermentation medium formula described in step (2); the culture conditions are: dissolved oxygen concentration of 55%, temperature of 30° C., culture for 24 hours, and real-time online addition of defoaming agent;

[0081] (4) Concentration and collection: After tanking, the fermentation products are collected and concentrated to obtain a microecological liquid bacterial agent, or spray-dried to obtain a microecological solid preparation.

[0082] (VII) Application of Bacillus megaterium A-27 in fish farming

[0083] Example 1: Safety test of Bacillus megaterium A-27 on grass carp

[0084] (1) In vitro cytotoxicity test

[0085] In order to investigate whether strain A-27 has toxic effects on grass carp liver cells, the fermentation supernatant of strain A-27 was co-incubated with grass carp liver cells L8824. Figure 7 As shown in Figure 2, the supernatant of strain A-27 fermentation was co-incubated with L8824 cells for 12 hours, and the cells grew well, with complete shape and clear outline, and still maintained a full and high-density growth state after 24 hours. The MTT test results are shown in Figure 2. Figure 8 As shown, the survival rate of 10-60MOI (MOI in Chinese means infection multiplicity, which refers to the ratio of strain A-27 to the number of cells, and 10-60MOI refers to the gradient of infection multiplicity) strain A-27 after co-incubation with grass carp liver cells was not significantly different from that of the control group, and the cell survival rate remained above 95%. The above results show that the cell-free supernatant of strain A-27 is not toxic to grass carp liver cells, and does not affect the normal growth and reproduction of cells, and the cell safety is relatively high.

[0086] (2) Grass carp safety and tissue infection section observation

[0087] The grass carp in the experiment were divided into three groups, with 15 grass carp in each group. Group A was injected with normal saline; Group B was injected with 1×10 8cfu / mL Ah X040 (Aeromonas hydrophila); Group C: injected with 1×10 8 cfu / mL strain A-27; after the injection, the survival and movement of grass carp were observed every day, and basic feed was fed at 2% of body weight every day for 7 days. The results after one week are shown in Table 1. Compared with the grass carp injected with normal saline in the experimental group (Group C) injected with bacterial suspension A-27 and the control group (Group A), both showed normal living conditions and no death occurred. The grass carp in the control group (Group B) injected with Aeromonas hydrophila had swollen abdomen and congested fins, and the mortality rate reached 100%. Subsequently, the tissues of grass carp in Group A (Control), Group B (Ah), and Group C (A-27) were sliced ​​and observed. The results are as follows Fig. 9 As shown in the results, it was found that the tissue cells of grass carp infected with A-27 and the control group were full, with intact structures and no obvious organic lesions, while the liver cells of grass carp infected with Aeromonas hydrophila showed large-area vacuolar degeneration, separation from surrounding tissues, and cell apoptosis and shedding; large gaps were seen in the endothelial cells of the splenic sinus, and the basement membrane was incomplete; the cells of the intestinal serosa were stained darker, the cells were condensed, and the structure was destroyed. This is consistent with the results of the in vitro injection test, which further shows that strain A-27 is safe for grass carp and provides theoretical support for the production of microecological preparations in aquaculture.

[0088] Table 1 Safety test of strain A-27 on grass carp

[0089]

[0090] Note: Group A: injected with normal saline; Group B: injected with 1×10 8 cfu / mL Ah X040 (Aeromonas hydrophila); Group C: injected with 1×10 8 cfu / mL strain A-27.

[0091] Example 2: Colonization and protective effect of Bacillus megaterium A-27 in grass carp

[0092] The experimental fish were divided into two groups and fed with feed at 2% of the grass carp body weight. The control group was fed with ordinary feed; the experimental group was fed with 1×10 8 cfu / g A-27 GFP The bacteria-mixed feed was used, with 30 fish in each group and repeated. The fluorescence distribution in the grass carp of the experimental group was observed by the small animal in vivo imaging system. The experiment lasted for two weeks (see Fig.10 ).

[0093] Fig.10 The present invention is a small animal in vivo imaging system to observe the colonization and protection of Bacillus megaterium A-27 labeled strain in grass carp; the results show that feeding strain A-27 EGFPOn the 3rd day, the fluorescence signal can be observed in the small animal imaging system, and the fluorescence signal increases with time. The fluorescence signal first appears in the gills of grass carp and then deepens and extends to the back half of the abdomen (see Fig.10 This result shows that strain A-27 can stably colonize in grass carp, laying the foundation for the subsequent study of the effects of strain A-27 on the growth and immune defense ability of grass carp.

[0094] Example 3: Effects of Bacillus megaterium A-27 on growth and disease resistance of grass carp

[0095] Grass carp fry with the same body weight and length were selected and divided into an experimental group and a control group, with 50 fish in each group and repeated. Before the experiment, the grass carp were acclimated by feeding normal feed for one week, and then the control group was fed with ordinary feed, and the experimental group was fed with a concentration of 1×10 8 cfu / g of A-27 bacteria mixed feed every 2 days, and the indoor temperature was controlled at 25±2℃. After the experiment lasted for 30 days, 6 grass carp were randomly selected from each group to cut off their tails and take blood. At the same time, muscle, liver, spleen, and intestinal tissues were collected and stored in an ultra-low temperature refrigerator at -80℃ for later use.

[0096] (1) Effects of Bacillus megaterium A-27 on the growth performance of grass carp

[0097] The A-27 strain was added to the feed and fed to grass carp for 30 days, and the changes in body weight were analyzed. The initial body weight of grass carp was measured at the beginning of the experiment. 0 (g), the final weight of grass carp was measured after feeding for 30 days. t (g) The weight gain rate (WGR), specific growth rate (SGR) and survival rate (SR) of grass carp were determined according to the following formulas.

[0098] WGR(%)=100×[(W t -W 0 ) / W 0 ]; SGR (%) = 100 × (lnW t -lnW 0 ) / T; SR (%) = number of fish at the end of the test / number of fish at the beginning of the test × 100%. T represents the production test cycle.

[0099] The results are shown in Table 2. The WGR and SGR of grass carp in the control group were 10.5% and 0.33%, respectively, and the WGR and SGR of grass carp in the fed group were 16.14% and 0.50%, respectively, which were significantly higher than those in the control group (P < 0.001). It can be inferred that the body weight and feed utilization of grass carp increased significantly after the A-27 strain was added to the feed.

[0100] Fluorescence quantitative PCR was used to analyze the expression levels of growth-related genes in grass carp muscle. The expression levels of insulin-like growth factor-1 (IGF-1) and insulin-like growth factor-2 (IGF-2) in the grass carp muscle of the experimental group were significantly higher than those in the control group (P < 0.001), which were 1.77 times and 2.45 times of the control group, respectively. The expression levels of myostatin-1 (MSTN-1) and myostatin-2 (MSTN-2) in the grass carp of the experimental group were significantly lower than those in the control group (P < 0.001), which were downregulated to 0.26 times and 0.45 times of the control group, respectively. The experimental results show that strain A-27 can increase the expression level of growth-promoting factors in grass carp and reduce the expression level of growth-inhibiting factors, which provides a molecular explanation for the promotion of grass carp growth and development by strain A-27 (see Fig.11 ).

[0101] Table 2 Growth performance parameters of grass carp

[0102]

[0103] Note: * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001

[0104] It can be seen that the Bacillus megaterium A-27 of the present invention has a promoting effect on the growth of grass carp.

[0105] (2) Effects of Bacillus megaterium A-27 on nonspecific immune parameters in grass carp serum

[0106] After the A-27 strain was continuously fed to grass carp for 30 days, the serum of the experimental fish was collected and the ACP and AKP enzyme activities were measured. Compared with the grass carp in the control group, the enzyme activity of the grass carp in the feeding group was significantly increased. The concentration of acid phosphatase (ACP) in the serum was 304.32U / L, and its activity was 8.13 times that of the control group. The acid phosphatase activity (ACP) in the feeding group was also significantly higher than that in the control group (36.3U / L; P<0.001), and its activity was 3.23 times that of the control group. The experimental results show that strain A-27 can increase the enzyme activity level and thus enhance the relevant nonspecific immune response ability of grass carp (see Fig.12 ).

[0107] (3) Effect of strain A-27 on the expression level of antioxidant factors in grass carp

[0108] After the A-27 strain was added to the feed and fed to grass carp for 30 days, the expression levels of antioxidant-related genes Nrf2 and Keap1 in the liver, spleen, intestine and other tissues of the two groups of experimental fish were measured. Fig.14 This is the analysis result of the effect of Bacillus megaterium A-27 of the present invention on antioxidant-related genes in grass carp tissues (* indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001); compared with the control group, the expression levels of Keap1 in the liver, spleen, and intestine were significantly upregulated, which were 5.51 times, 5.15 times, and 15.49 times that of the control group, respectively (P < 0.001), and the expression level of Nrf2 in the spleen was upregulated, which was 1.77 times that of the control group (P < 0.05). In general, compared with the control group, the relevant antioxidant factors of various tissues of grass carp in the experimental group were upregulated to varying degrees, indicating that strain A-27 can activate the expression of antioxidant-related genes in grass carp and improve the antioxidant capacity of grass carp (see Fig.13 , Fig.13 1 is an analysis result of the effect of Bacillus megaterium A-27 of the present invention on the expression of antioxidant-related genes in grass carp tissues). Fig.13 A, B, and C represent the expression levels of antioxidant-related genes Nrf2 and Keap1 in the liver, spleen, and intestinal tissues of grass carp, respectively.

[0109] (4) Effect of strain A-27 on the expression level of grass carp immune factors

[0110] After the A-27 strain was added to the feed and fed to grass carp for 30 days, the expression levels of immune-related genes IL-8, IgM, C3, TNF-α, MyD-88, and TLR-4 in the liver, spleen, intestine and other tissues of the two groups of experimental fish were measured. Fig.14 This is a graph showing the results of the analysis of the effects of Bacillus megaterium A-27 on immune-related tissues in grass carp (* indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001); in the liver, the expression levels of IL-8, IgM, C3, and TNF-α in the experimental group were significantly higher than those in the control group, which were 3.91 times, 5.74 times, 7.18 times, and 9.00 times of those in the control group, respectively, while the expression levels of MyD-88 and TLR-4 were significantly downregulated (P < 0.001) ( Fig.14 A); The expression levels of IL-8, C3, and TLR-4 in the spleen of the experimental group were significantly higher than those of the control group, which were 3.29 times, 3.70 times, and 6.74 times higher than those of the control group, respectively. There were no significant differences in the expression levels of TNF-α and MyD-88 between the experimental group and the control group, and the expression level of IgM was significantly downregulated ( Fig.14B); the expression levels of IgM, C3, TLR-4, TNF-α, and MyD-88 in the experimental group were significantly higher than those in the control group, which were 5.45 times, 6.14 times, 1.89 times, 1.83 times, and 9.99 times higher than those in the control group, respectively. The expression level of IL-8 was significantly downregulated ( Fig.14 In summary, the immune factors of various tissues of grass carp in the experimental group were increased to varying degrees compared with those in the control group through feeding with strain A-27, indicating that strain A-27 can regulate the expression level of immune-related genes in grass carp organs and tissues and improve the immunity of grass carp (see Fig.14 ).

[0111] Example 4: Protective test of Bacillus megaterium A-27 on grass carp

[0112] Grass carp fry with the same body weight and length were selected and divided into an experimental group and a control group, with 50 fish in each group and repeated. Before the experiment, the grass carp were acclimated by feeding normal feed for one week, and then the control group was fed with ordinary feed, and the experimental group was fed with a concentration of 1×10 8 cfu / g of A-27 bacteria mixed feed was fed every 2 days, and the indoor temperature was controlled at 25±2℃. After feeding grass carp for 30 days, the two groups of grass carp were injected with 200μL of 1×10 8 cfu / mL of Ah X040, and the survival of grass carp in the control group and the experimental group was observed and recorded every day for one week. The survival curve of grass carp is shown in Fig.15 As shown in the results, the mortality rate of grass carp in the control group was as high as 60.97% on the first day of the experiment, and all died after 5 days; the mortality rate of grass carp in the feeding group was only 34.72% after 1 day of infection, which was significantly lower than that of the control group. The survival rate of grass carp was 13.89% on the seventh day and remained until the end of the experiment. This shows that strain A-27 can alleviate the toxic effects of Aeromonas hydrophila on grass carp to a certain extent, thereby reducing the mortality rate of grass carp after infection with Ah X040.

[0113] Example 5: Probiotic preparation prepared by Bacillus megaterium A-27 promotes the growth of grass carp

[0114] The test fish were divided into two groups and fed with feed at 2% of the grass carp's body weight. The control group was fed with ordinary feed; the experimental group was fed with a microecological preparation prepared by Bacillus megaterium A-27 mixed with feed, and the amount of microecological preparation prepared by Bacillus megaterium A-27 added was equivalent to 2% of the feed weight. There were 30 fish in each group, and repeated experiments were set up. The experiment lasted for two weeks. The experimental group grass carp was fed with Bacillus megaterium A-27 microecological solid preparation, and the results are shown in Table 3. The growth rate and specific growth rate of the grass carp in the experimental group were significantly higher than those in the control group. It can be inferred that the microecological preparation prepared by Bacillus megaterium A-27 promotes the growth of grass carp.

[0115] Table 3A-27 Growth performance parameters of grass carp after treatment with probiotics

[0116]

[0117] Example 6 Antagonistic effect of the probiotic preparation prepared by Bacillus megaterium A-27 on Aeromonas hydrophila in grass carp

[0118] Grass carp with uniform body size were randomly divided into 2 groups, with 30 fish in each group. They were acclimated for 7 days and starved for 24 hours. The grass carp in the control group and the experimental group were intraperitoneally injected with 100 μL of 1×10 6 CFU / mL of AvX040 mCherry In the following 7 days, the infection status of the two groups of grass carp was regularly observed using a small animal live imaging system. After the 7th day, the grass carp in the experimental group were fed with A-27 microecological solid preparations, and their mortality and fluorescent colonization were observed. The observation period was 7 days.

[0119] The results showed that on the fifth day after infection, red fluorescence signals began to appear in the gills of the two groups of grass carp, and on the seventh day, a large area of ​​red fluorescence appeared in the abdomen of the grass carp in the control group, indicating that the Ah X040 pathogen first invaded the gills and then spread to the abdomen. In contrast, the red fluorescence signal of the grass carp in the experimental group fed with the A-27 microecological solid preparation was weaker, and no large area of ​​red fluorescence appeared in the abdomen. The A-27 microecological preparation effectively antagonized the invasion of the Ah X040 pathogen in the grass carp, indicating that the strain A-27 can alleviate the infection of Aeromonas hydrophila in the grass carp (refer to Fig.16 ). Fig.16 This is a diagram showing the distribution of Ah X040mCherry in grass carp observed by the small animal imaging system and the inhibition of Ah X040 by the Bacillus megaterium A-27 probiotic preparation.

Claims

1. A strain of Bacillus megaterium A-27, characterized in that: It was deposited in the China Center for Type Culture Collection on August 26, 2024, and the strain collection number is CCTCC NO: M 20241848; the classification name is: Bacillus megaterium A-27, and the Latin name is Bacillus megaterium A-27.

2. The Bacillus megaterium A-27 according to claim 1, characterized in that The 16SrDNA sequence of Bacillus megaterium A-27 is shown in the sequence table SEQ ID No.

1.

3. A microecological preparation, characterized in that: The invention is suitable for aquaculture and is prepared by fermenting the Bacillus megaterium A-27 with the strain preservation number of CCTCC NO: M 20241848 as claimed in claim 1 or 2.

4. The microecological preparation according to claim 3, characterized in that: The microecological preparation is a liquid preparation or a solid preparation.

5. The microecological preparation according to claim 3 or 4, characterized in that: The aquaculture animals are freshwater fish.

6. The microecological preparation according to claim 5, characterized in that: The freshwater fish is crucian carp, grass carp or carp.

7. A method for preparing the probiotic preparation according to claim 3 or 4, characterized in that: The following steps are involved: (1) Inoculation and activation: The Bacillus megaterium A-27 stored slant strain was transferred to the activated slant culture medium for activation to obtain an activated seed solution; (2) First expansion culture: The seed liquid activated in step (1) is inoculated into a fermentation tank at an inoculation rate of 1 to 2% for first expansion culture; (3) Second expansion culture: inoculating the seed solution of the first expansion culture in step (2) into a fermentation tank containing fermentation medium at an inoculum rate of 10-15%; (4) Concentration and collection: After tanking, the fermentation products are collected and concentrated to obtain a microecological liquid bacterial agent, or spray-dried to obtain a microecological solid preparation.

8. The method for preparing a probiotic preparation according to claim 7, characterized in that: In step (1), the activated slant culture medium is LB liquid culture medium, and the activation culture conditions are: temperature 28-30° C., liquid volume 10-30% of the container volume, and shaking speed 120-140 rpm.

9. The method for preparing a probiotic preparation according to claim 7 or 8, characterized in that: In step (2), the fermentation medium formula is: 42-45 g / L tryptic soy bean liquid medium, 8-10 g / L glucose, 8-9 g / L yeast powder, 2.0-2.2 g / L magnesium sulfate heptahydrate, and sterilized at 115°C for more than 30 min; And / or the culture conditions are: the entire culture process is monitored online, the dissolved oxygen concentration is 35-55%, the temperature is 28-30°C, the culture is more than 24 hours, and the defoaming agent is added online in real time.

10. The method for preparing a probiotic preparation according to claim 7 or 8, characterized in that: In step (3), the fermentation medium formula is the same as the fermentation medium formula described in step (2); And / or the culture conditions are: dissolved oxygen concentration of 35-55%, temperature of 28-30°C, culture for more than 24 hours, and real-time online addition of defoaming agent; And / or in step (3), the fermentation tank is filled with fermentation medium equivalent to 65%-70% of the volume of the fermentation tank.

Images