Salt-tolerant bacillus for freshwater fish culture and application thereof

Abstract

The invention provides a salt-tolerant bacillus for freshwater fish culture. The preservation number of the salt-tolerant bacillus is CGMCC (China General Microbiological Culture Collection Center) No.32389. The salt-tolerant bacillus CAG2307-32 strain provided by the invention has no pathogenicity to crucian carps, can be widely applied to culture production of freshwater fishes such as crucian carps and the like, can effectively regulate and control water quality, promote fish growth and enhance antioxidant and immune levels of fish bodies, and has great application potential.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotic screening and application, and specifically relates to a halotolerant bacillus for freshwater fish farming and application thereof. Background Art

[0002] The breeding model of freshwater aquaculture species such as crucian carp (Carassius auratus) presents a high-yield trend, but it is also accompanied by high risks and high investment. Problems such as water quality deterioration, high incidence of diseases, slow growth and high costs caused by excessive stocking density and improper feeding have brought great pressure to farmers.

[0003] Bacillus sp. is a probiotic widely used in aquaculture. However, there are still many problems in its application, and the actual efficacy of commercial Bacillus products is not ideal. First, because of its unknown origin, it cannot be well colonized in the host body. Secondly, many strains have poor heat resistance, which leads to insufficient activity when added to feed. The safety issues of many probiotics still need to be investigated. In addition, the functions of probiotics are mostly concentrated in one aspect, which cannot solve many problems in the breeding process.

[0004] In the breeding process of freshwater fish such as crucian carp, relatively little attention is paid to the above issues. Therefore, in order to screen and obtain probiotics suitable for the breeding of freshwater fish such as crucian carp, it is necessary to screen out indigenous bacteria that are efficient, versatile and easy to colonize from the intestines of healthy hosts, evaluate their heat resistance and safety, evaluate their application effects, and explore their application methods. This is of great significance for promoting the healthy breeding of freshwater fish such as crucian carp and increasing breeding income. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of bacillus halophilus for freshwater fish farming and its application, wherein the provided bacillus halophilus has a highly efficient nitrogen reduction effect, can secrete digestive enzymes, and has good heat resistance and safety. The application methods of water spraying or feed addition can improve the feed utilization rate of crucian carp, enhance the antioxidant and immune levels of crucian carp, thereby effectively regulating water quality and promoting the healthy growth of crucian carp.

[0006] The Bacillus halotolerans CAG2307-32 strain provided by the present invention was deposited at the General Microbiology Center of the China Microbiological Culture Collection Committee of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on October 28, 2024, with a deposit number of CGMCC No. 32389.

[0007] The 16S rDNA sequence of the CAG2307-32 strain is SEQ ID NO: 1;

[0008] The present invention also provides a use of the CAG2307-32 strain, which is the use in preparing a bacterial preparation for freshwater fish farming;

[0009] The present invention also provides a bacterial preparation for freshwater fish farming, wherein the bacterial preparation contains live bacteria of the CAG2307-32 strain.

[0010] The present invention also provides another use of the CAG2307-32 strain, which is use as a feed additive.

[0011] The present invention also provides a feed for freshwater fish breeding, wherein the live bacteria of the CAG2307-32 strain are added.

[0012] Another aspect of the present invention provides a method for aquaculture of freshwater fish, which comprises spraying the fermentation bacterial liquid of the CAG2307-32 strain into the aquaculture water.

[0013] The salt-tolerant Bacillus CAG2307-32 strain provided by the present invention is not pathogenic to crucian carp, can be widely used in the breeding and production of freshwater fish such as crucian carp, can effectively regulate water quality, promote fish growth, and enhance the antioxidant and immune levels of fish, and has great application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Enzyme production effect diagram of strain CAG2307-32;

[0015] Figure 2 :Colony morphology and molecular classification of strain CAG2307-32;

[0016] Figure 3 :CAG2307-32 strain safety identification hemolysis test diagram and survival rate after infection diagram;

[0017] Figure 4 : The nitrogen reduction effect of spraying CAG2307-32 strain on water bodies;

[0018] Figure 5 :The effect of spraying CAG2307-32 strain on intestinal digestive enzymes of crucian carp;

[0019] Figure 6 :The effect of spraying CAG2307-32 strain on the liver antioxidant and immune enzymes of crucian carp;

[0020] Figure 7:The effect of adding CAG2307-32 strain to feed on digestive enzymes in the intestine of crucian carp;

[0021] Figure 8 :The effect of adding CAG2307-32 strain to feed on the antioxidant and immune enzymes in crucian carp liver. DETAILED DESCRIPTION

[0022] The present invention is described in detail below in conjunction with examples and drawings. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention.

[0023] Example 1: Screening and identification of probiotic Bacillus

[0024] 1) Strain isolation and screening

[0025] Strain isolation: In July 2023, healthy crucian carp were collected from the crucian carp breeding pond of Jiangsu Zhengyuan Chuanghui Agricultural Science and Technology Development Co., Ltd. in Yancheng City. The crucian carp intestines were dissected and obtained under sterile operation, ground and diluted to an appropriate concentration in sterile physiological saline, and 100 μL of homogenate dilution was drawn with a sterile pipette tip and smeared on the surface of freshwater fish agar (FWA) medium (beef extract 2.5g, glucose 1g, NaCl 5g, MgSO4 0.05g, peptone 5g, yeast extract 2.5g, K2HSO4 0.2g, agar 15g, H2O1000mL, pH 7.2-7.4), and cultured at 30°C for 24h. Use a sterile inoculation loop to pick single colonies with different morphology, size, color, etc. on the surface of FWA medium, and use the three-step streak method to inoculate on the surface of new FWA medium, continue to culture, and purify multiple times until a pure strain is obtained.

[0026] Screening of nitrogen-reducing bacteria: The above 33 strains were cultured in FW medium (30°C) for 24 h, and then the bacterial cultures were adjusted to OD 600 =1.0, and inoculated into NH4 at a 1% inoculation rate + -N (glucose 1.05 g, NH4Cl 0.08 g, MgSO4·7H20 0.2 g, KH2PO4 0.05 g, Na2HPO4 0.5 g, NaCl 5 g, H2O 1000 mL, pH 6.0-7.0), NO2 - -N (glucose 0.25 g, NaNO2 0.025 g, MgSO4·7H20 0.2 g, KH2PO4 0.05 g, Na2HPO4 0.5 g, NaCl 5 g, H2O 1000 mL, pH 6.0-7.0) and NO3 --N (glucose 0.5g, KNO3 0.072g, MgSO4·7H20 0.2g, KH2PO4 0.05g, Na2HPO4 0.5g, NaCl 5g, H2O 1000mL, pH 6.0-7.0) medium, the control group was not inoculated with any strains, and cultured on a constant temperature shaker (30℃, 180r / min) for 24h. The indophenol blue spectrophotometry was used to detect the NH4 + The degradation ability of NO2 was tested by N-(1-naphthyl)-ethylenediamine spectrophotometry. - The degradation ability of NO3 - -N degradation ability.

[0027] Screening of enzyme-producing bacteria: The above 33 strains were inoculated with a sterile inoculation loop on amylase (10 g peptone, 5 g yeast extract, 5 g NaCl, 10 g soluble starch, 20 g agar, 1000 mL H2O), protease (1 g glucose, 3 g beef extract, 5 g NaCl, 2 g CaCl2, 1 g L-tyrosine, 5 g casein, 20 g agar, 1000 mL H2O) and lipase (10 g peptone, 0.1 g CaCl2, 5 g NaCl, 10 mL Tween-80, 16 g agar, 1000 mL H2O, pH 7.2-7.4) culture media, and cultured at 30°C for 24 h, and the presence or absence of degradation zones was observed and recorded.

[0028] Results: Thirty-three pure strains were isolated from the intestines of healthy crucian carp and numbered CAG2307-1 to CAG2307-33.

[0029] Among them, 23 strains were screened to be resistant to NH4 + -N, NO2 - -N and NO3 - -N strains, among which CAG2307-2, CAG2307-4, CAG2307-11 and CAG2307-32 had better effects, and CAG2307-32 had the best effect. + -N, NO2 - -N and NO3 - -N degradation rates were 95.36%, 99.75% and 80.32% respectively (see Table 1).

[0030] From the above 33 strains, 5 strains (CAG2307-3, CAG2307-7, CAG2307-11, CAG2307-13 and CAG2307-32) were screened and obtained to have amylase, protease and lipase activities. Combined with the nitrogen reduction results, strain CAG2307-32 has the best potential probiotic effect, which can not only reduce nitrogen efficiently, but also produce obvious hydrolysis circles on amylase, protease and lipase culture media (see Figure 1 ).

[0031] Table 1: Data table of the degradation effect of strains on different nitrogen sources

[0032]

[0033]

[0034] 2) Temperature tolerance of strains

[0035] The strains CAG2307-11 and CAG2307-32, which have both high-efficiency nitrogen reduction activity and enzyme production activity, were selected for high temperature tolerance experiments. The two activated strains were inoculated into FW medium at a 1% inoculation rate, and each strain was inoculated into 3 test tubes. They were cultured at 30°C for 24 hours, and the number of live bacteria was detected by plate count method. Then, they were placed in a water bath at 80°C, and samples were taken at 30 and 60 minutes, and the number of live bacteria was detected by plate count method, and the survival rate of the strains was calculated.

[0036] The results showed that strains CAG2307-11 and CAG2307-32 both had a certain tolerance to high temperatures of 80°C, but the high temperature tolerance of CAG2307-32 was significantly higher than that of CAG2307-11, whether at 30 minutes or 60 minutes.

[0037] Table 2: Survival rate of strains at different times under 80°C (%)

[0038]

[0039] Note: * indicates significant difference (P<0.05)

[0040] 3) Strain identification

[0041] The strain CAG2307-32, which has both efficient nitrogen reduction activity and enzyme production activity and is resistant to high temperature, was selected for morphological observation and 16S rDNA sequencing identification. The CAG2307-32 strain was inoculated onto the surface of the FWA medium with a sterile inoculation loop and the strain morphology was observed after culturing at 30°C for 24 h. The genomic DNA of the CAG2307-32 strain was extracted using the Ezup column bacterial genomic DNA extraction kit (Shanghai, China), and the 16S rDNA gene of the CAG2307-32 strain was amplified. The nucleotide sequences of the primers used were: 27F (5'-AGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTT ACGACTT-3'). The 16S rDNA sequence of the CAG2307-32 strain was compared on the ribosomal database (http: / / rdp.cme.msu.edu / index.jsp), and the target phylogenetic tree was constructed using the software MEGA11.

[0042] After culturing CAG2307-32 on FWA medium (30°C) for 24 h, the colonies were irregular in shape, dirty white, opaque, with rough surfaces and edges (see Figure 2 ).

[0043] The 16S rDNA gene of strain CAG2307-32 was sequenced to obtain a 1499 bp amplification product with the following sequence:

[0044] GTTTGATTCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAG

[0045] CGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTA

[0046] ACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGA

[0047] ACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGC

[0048] ATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGG

[0049] GTGATCGGCCACACTGGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGG

[0050] GAATCTTCCGCAATGGAACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTT

[0051] CGGATCGTAAAGCTCTGTTGTTTAGGGGAAACAAGTACCGTTCGAATAGGGCGGTACCTTG

[0052] ACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGG

[0053] TGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGGA

[0054] TGTGAAAGCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAGACTTGAGTGCAG

[0055] AAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAAACC

[0056] AGTGGCGAAGGCACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGC

[0057] GAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGG

[0058] TTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCA

[0059] AGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAAT

[0060] TCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGA

[0061] CGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAG

[0062] ATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGG

[0063] GCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATC

[0064] ATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAA

[0065] CCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGA

[0066] CTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCG

[0067] GGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGT

[0068] AACCTTTTAGGAGCCAGCCGCCGAAGGTGGGACAGATGATTGGGGTGAAGTCGTAA

[0069] (SEQ ID NO: 1).

[0070] Through phylogenetic analysis, strain CAG2307-32 was identified as a Bacillus strain, with the highest similarity to Bacillus halodurans ( Figure 2 ).

[0071] 4) Biosafety evaluation of strain CAG2307-32

[0072] Hemolytic assay: CAG2307-32 strain was inoculated into FW medium (30°C, 180 rpm) and cultured for 24 h. The OD 600 =1.0, take 10 μL of bacterial solution and add it onto a sterile filter paper and place it on the surface of blood agar medium. The blood agar plate was purchased from Barrett Biotechnology (Zhengzhou) Co., Ltd., and cultured at 30°C for 24 hours to observe whether there is a hemolytic zone.

[0073] Antibiotic sensitivity test: CAG2307-32 strain was inoculated into FW medium (30°C, 180 r / min) and cultured for 24 h. The OD 600 =1.0, take 100 μL and spread it evenly on FWA medium, stick the drug sensitivity paper, which was purchased from Hangzhou Microbiological Reagent Co., Ltd., and culture at 30℃ for 24h, measure the diameter of the inhibition zone and judge the drug sensitivity according to the CLSI 2011 standard.

[0074] Injection infection experiment: Healthy crucian carp larvae were selected as the materials for the injection infection experiment, with a body length of (8.74±0.85) cm and a body weight of (9.19±2.98) g. Crucian carp were temporarily kept in an indoor recirculating aquaculture system for 7 days, and then stopped feeding for 24 hours, and then intraperitoneal injection infection was performed. Each fish was injected with 100 μL of CAG2307-32 strain (1×10 8 CFU / mL), pathogen ZKW3 strain (1×10 8 CFU / mL) bacterial suspension and saline. The pathogen ZKW3 strain was screened from crucian carp breeding water and has been confirmed to be a crucian carp pathogen, serving as the positive control group; saline was the negative control group, and 30 fish were injected into each group. During the experiment, the fish were fed normally, dissolved oxygen was ≥5mg / L, the average water temperature was 25.4℃, and the observation lasted for 7 days, and the mortality of the tested fish was recorded.

[0075] The results showed that the strain CAG2307-32 was γ-hemolytic on the blood plate, i.e., non-hemolytic ( Figure 3 ); The results of antibiotic sensitivity experiments showed that the CAG2307-32 strain was resistant to co-trimoxazole, moderately sensitive to cefazolin, and sensitive to 20 antibiotics including oxacillin, carbenicillin, cefoperazone, and amikacin (see Table 3); after the injection of CAG2307-32 strain and normal saline, no disease or death occurred in crucian carp.

[0076] Table 3: Antibiotic sensitivity results of CAG2307-32 strain

[0077]

[0078]

[0079] Note: R stands for resistance, I stands for intermediate sensitivity, and S stands for sensitivity.

[0080] Based on the above safety evaluation results, it is proved that the CAG2307-32 strain is safe for crucian carp and can be used in crucian carp farming and production.

[0081] Example 2: Effect of spraying CAG2307-32 strain in crucian carp farming

[0082] 1) Crucian carp farming management

[0083] Young silver crucian carp with healthy body surface and uniform size were selected as experimental materials, with an average body length of (10.82±0.65) cm and an average weight of (18.45±0.43) g. The probiotic CAG2307-32 strain screened in the early stage was used as the test strain. In addition, it was compared with the other two strains of bacteria ZKF17 and ZKW7 screened by our team in the early stage. Both strains were identified as Bacillus. The silver crucian carp was divided into 4 groups (control group, CAG2307-32 group, ZKF17 group and ZKW7 group), with 3 replicates in each group and 10 fish in each replicate. The experiment was carried out in a white polyethylene aquaculture tank with an actual aquaculture water body of 30L for 26 days. During the experiment, compound feed was fed twice a day (9:00 and 17:00), and the daily feeding amount was 3% of the fish body weight. The bacterial solution was sprayed once every 3 days to each group (except the control group) to reach a final concentration of 1×10 6 During the culture period, the water temperature was 21.1-23.3℃, ​​and the water was changed every 7 days, with 30% of the water changed each time.

[0084] 2) Water quality monitoring during crucian carp farming

[0085] During the breeding period, water samples were taken at regular intervals to determine the NH4 + -N, NO2 - -N and NO3 - -N concentration.

[0086] The results showed that the three strains had a strong effect on NO2 - -N and NO3 - -N have a certain degradation effect. Among them, CAG2307-32 has the most obvious effect. - -N and NO3 - -N concentration was always maintained at the lowest level. CAG2307-32 strain had no effect on NO2 - -N and NO3 - -N maximum degradation rates were 54.81% and 43.88% ( Figure 4). The above results show that spraying three strains of probiotics on water can reduce the nitrogen content in water to a certain extent. In comparison, strain CAG2307-32 has the best cleaning effect on water quality.

[0087] 3) Determination of crucian carp growth performance

[0088] After the breeding experiment, the fish were fasted for 24 hours, and the weight and length of each group of experimental fish were measured. The relative weight gain rate, specific growth rate, fatness and survival rate were calculated. The calculation formula is as follows:

[0089] Relative weight gain rate (%) = [(final body weight) - (initial body weight) / (initial body weight)] × 100%;

[0090] Specific growth rate (% / d) = [(ln(final body weight) - ln(initial body weight) / (number of experimental days)] × 100%;

[0091] Fullness(g / cm 3 ) = 100 × (final body weight) / (final body length) 3 ;

[0092] Survival rate (%) = (final number of fish) / (initial number of fish)×100%.

[0093] The results showed that compared with the control group, the final body weight of crucian carp in the CAG2307-32 group, ZKF17 group and ZKW7 group was significantly increased, among which the final body weight of crucian carp in the CAG2307-32 group was the highest (see Table 4); the final body length of crucian carp in the CAG2307-32 group and ZKF17 group was significantly higher than that in the control group, and the CAG2307-32 strain had the most obvious effect; compared with the control group, the CAG2307-32 strain could significantly increase the relative weight gain rate and specific growth rate of crucian carp, while the other two strains had no significant difference compared with the control group. The above results show that among all experimental groups, the CAG2307-32 strain has the most significant growth-promoting effect on crucian carp.

[0094] Table 4: Effects of spraying CAG2307-32 strain on the growth of crucian carp

[0095]

[0096]

[0097] Note: Different superscripts indicate significant differences in the data in the same row (P<0.05).

[0098] 4) Determination of digestive enzyme activity in the intestine of crucian carp

[0099] After the breeding experiment, 9 experimental fish were randomly selected from each group, dissected on ice, and the midgut tissue was collected and stored at -80°C. According to the operating requirements of the lipase, amylase and trypsin test kit instructions, the midgut digestive enzyme activity of each group of experimental fish was determined.

[0100] The results showed that spraying CAG2307-32 and ZKF17 strains in water could significantly increase the intestinal amylase activity of crucian carp, but among the three probiotics, only spraying CAG2307-32 strain could significantly increase the trypsin activity of crucian carp ( Figure 5 ). The above shows that spraying CAG2307-32 strain into water can effectively promote the digestion and absorption of feed starch and protein by crucian carp.

[0101] 5) Determination of antioxidant and immune enzyme activities in crucian carp liver

[0102] After the breeding experiment, 9 experimental fish were randomly selected from each group, dissected on ice, and the liver tissue was collected and stored at -80°C. According to the operating requirements of the superoxide dismutase, reduced glutathione and alkaline phosphatase test kit instructions, the antioxidant and immune enzyme activities of the liver of each group of experimental fish were determined.

[0103] The results showed that spraying CAG2307-32 strain in water could significantly increase the superoxide dismutase activity in crucian carp liver, while the other two strains had no significant effect on superoxide dismutase activity; although ZKW7 strain could also increase the reduced glutathione content and acid phosphatase activity in crucian carp liver, the effect was significantly inferior to that of CAG2307-32 strain ( Figure 6 ). Among them, superoxide dismutase has the functions of removing reactive oxygen, anti-oxidation and relieving inflammation; reduced glutathione can remove excess oxygen free radicals in the body and play an important role in the body's anti-oxidative stress; acid phosphatase is a non-specific phosphohydrolase and an important component of blood cell lysosomal enzymes, which plays an important role in the body's defense against external microbial invasion. The above results show that spraying CAG2307-32 strains in water can effectively improve the antioxidant and immune levels of crucian carp.

[0104] Example 3: Effect of adding CAG2307-32 strain to feed in crucian carp farming

[0105] 1) Production of feed rich in CAG2307-32 strain

[0106] Tongwei fish extruded feed 180 (Wuxi Tongwei Biotechnology Co., Ltd.) was used as the experimental feed. After testing, the crude protein content of the feed was (30.77±0.17)%, the crude fat content was (9.34±0.93)%, the crude ash content was (22.39±2.59)%, and the moisture content was (9.52±0.26)%.

[0107] The CAG2307-32 strain was cultured in FW medium (30°C, 180 r / min) for 24 h, centrifuged (4000 r / min) to remove the supernatant, and then the bacterial solution concentration was adjusted with sterile saline. The bacterial solution was evenly added to the feed by spraying to a final concentration of 1×10 8 CFU / mL, the control group feed was supplemented with an equal amount of sterile saline. The feed was dried at 40℃ and stored at -20℃, with a batch of feed made every 7 days.

[0108] 2) Crucian carp farming management

[0109] Healthy and uniform-sized albiculture juvenile silver crucian carp were selected as experimental materials and randomly divided into 2 groups (CAG2307-32 group and control group), with 4 replicates in each group and 10 fish in each replicate. The experiment was conducted in a circulating water system with an actual culture water volume of 35L for 35 days. During the experiment, the control group was fed with a basic compound feed (with physiological saline added), and the CAG2307-32 group was fed with a feed with CAG2307-32 strain added. The initial daily feeding amount was 3% of the fish body weight, which was adjusted according to the feeding situation. The feeding amount of the two groups remained the same. During the culture period, the water temperature was (24.4±0.06)℃, and the water was changed every 3 days, with 30% of the water changed each time.

[0110] 3) Determination of crucian carp growth performance

[0111] After the breeding experiment, the fish were fasted for 24 hours, the body weight and length of each group of experimental fish were measured, and the relative weight gain rate, specific growth rate and feed coefficient were calculated.

[0112] The results showed that adding CAG2307-32 strain to the feed could significantly increase the final body weight and relative weight gain rate of crucian carp, and significantly reduce the feed coefficient of crucian carp (see Table 5), indicating that CAG2307-32 strain can effectively improve the digestion and absorption capacity of crucian carp for feed and promote its growth.

[0113] Table 5: Effect of adding CAG2307-32 strain to feed on the growth of crucian carp

[0114]

[0115] Note: * indicates significant difference (P<0.05).

[0116] 4) Determination of digestive enzyme activity in the intestine of crucian carp

[0117] After the breeding experiment, 9 experimental fish were randomly selected from each group, dissected on ice, and the midgut tissue was collected and stored at -80°C. According to the operating requirements of the lipase, amylase and trypsin test kit instructions, the midgut digestive enzyme activity of each group of experimental fish was determined.

[0118] The results showed that adding CAG2307-32 strain to feed could significantly increase the activities of lipase and amylase in the intestine of crucian carp ( Figure 7 ), indicating that adding CAG2307-32 strain to feed can effectively improve the digestion and absorption capacity of crucian carp.

[0119] 5) Determination of antioxidant and immune enzyme activities in crucian carp liver

[0120] After the breeding experiment, 9 experimental fish were randomly selected from each group, dissected on ice, and the liver tissue was collected and stored at -80°C. According to the operating requirements of the superoxide dismutase, reduced glutathione, catalase, malondialdehyde, alkaline phosphatase and acid phosphatase test kit instructions, the antioxidant and immune enzyme activities of the liver of each group of experimental fish were determined.

[0121] The results showed that adding CAG2307-32 strain to feed could significantly increase the activities of superoxide dismutase, reduced glutathione, catalase and acid phosphatase in the liver of crucian carp, and significantly reduce the concentration of malondialdehyde in the liver of crucian carp ( Figure 8 ). Superoxide dismutase, reduced glutathione and catalase are all important antioxidant enzymes. The higher the activity, the stronger the body's antioxidant capacity. Malondialdehyde is the product of peroxidation in the body and an important marker of cell membrane damage. Its reduced content indicates that the body's antioxidant capacity is enhanced. Alkaline phosphatase is similar to acid phosphatase and plays an important role in the body's immune defense.

[0122] The above results show that the CAG2307-32 strain screened by the present invention can effectively improve the antioxidant and immune levels of crucian carp.

Claims

1. A halotolerant Bacillus, characterized in that The deposit number of the halotolerant Bacillus is CGMCC No.32389.

2. The halotolerant Bacillus according to claim 1, wherein The 16S rDNA sequence of the halotolerant Bacillus is SEQ ID NO:

1.

3. Use of the halotolerant Bacillus according to claim 1 in preparing a bacterial preparation for freshwater fish farming.

4. A bacterial preparation for freshwater fish farming, characterized in that: The bacterial preparation contains live bacteria of the halotolerant Bacillus according to claim 1.

5. Use of the halotolerant Bacillus according to claim 1 as a feed additive.

6. A feed for freshwater fish farming, characterized in that: The live bacteria of the halotolerant Bacillus according to claim 1 are added to the feed.

7. A method for breeding freshwater fish, characterized in that: The method is to spray the fermentation bacterial liquid of the halotolerant Bacillus according to claim 1 into the aquaculture water.

8. The method according to claim 7, characterized in that The method is to cultivate freshwater crucian carp.

Images