Bacillus halodurans for freshwater fish farming and application thereof

Abstract

The application provides a salt-tolerant bacillus for freshwater fish culture, and the preservation number is CGMCC No.32389.The salt-tolerant bacillus CAG2307-32 strain provided by the application has no pathogenicity to crucian carp, can be applied in a large amount in the freshwater fish culture production such as crucian carp, can effectively regulate water quality, promote fish growth, and enhance the oxidation resistance and immune level of fish, and has great application potential.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of probiotic screening and application, and particularly relates to a salt-tolerant Bacillus sp. for freshwater fish farming and application thereof. BACKGROUND

[0002] The farming mode of freshwater farming species such as Carassius auratus presents a high yield situation, but is accompanied by high risk and high input characteristics. Overly high farming density, water quality deterioration caused by improper feeding, high incidence of diseases, slow growth, and high cost, etc. have brought great pressure to farmers.

[0003] Bacillus sp. is a probiotic widely used in aquaculture, but there are still many problems in its application. The actual efficacy of commercial Bacillus sp. products is not ideal. First, the unknown source of Bacillus sp. cannot be well colonized in the host body. Second, many strains have poor heat resistance, resulting in insufficient activity when added to feed. The safety of many probiotics still needs to be investigated. In addition, the functions of probiotics are mainly concentrated in one aspect, and they cannot well solve many problems in the farming process.

[0004] In the farming process of freshwater fish such as Carassius auratus, less attention is paid to the above problems. Therefore, in order to screen probiotics suitable for application in the farming of Carassius auratus and other freshwater fish, it is necessary to screen indigenous bacteria with high efficiency, diverse functions and easy colonization from the intestinal tract of healthy hosts, evaluate their heat resistance and safety, evaluate their application effect, and explore their application mode. This has important significance for promoting the healthy farming of Carassius auratus and other freshwater fish and improving the farming income. SUMMARY

[0005] The purpose of the present application is to provide a salt-tolerant Bacillus halotolerans for freshwater fish farming and its application. The provided salt-tolerant Bacillus halotolerans has high nitrogen reduction efficiency and can secrete digestive enzymes, has good heat resistance and safety. The application mode of water spraying or feed addition can improve the feed utilization rate of Carassius auratus, enhance the antioxidant and immune levels of Carassius auratus, thereby effectively regulating the water quality and promoting the healthy growth of Carassius auratus.

[0006] The salt-tolerant Bacillus halotolerans CAG2307-32 strain provided by the present application was preserved in the China General Microbiological Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, on October 28, 2024, and the preservation number is CGMCC No. 32389.

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

[0008] The application also provides a use of the CAG2307-32 strain in the preparation of a bacterial preparation for freshwater fish culture.

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

[0010] The application also provides another use of the CAG2307-32 strain as a feed additive.

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

[0012] The application also provides a method for freshwater fish culture, which is to sprinkle fermentation bacteria liquid of the CAG2307-32 strain in the culture water.

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

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

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

[0016] Figure 3 : Hemolysis experiment diagram and survival rate diagram after infection of safety identification of CAG2307-32 strain;

[0017] Figure 4 : Nitrogen reduction effect diagram of water body sprinkled with CAG2307-32 strain;

[0018] Figure 5 : Effect diagram of CAG2307-32 strain sprinkled in water body on intestinal digestive enzymes of crucian carp;

[0019] Figure 6 : Effect diagram of CAG2307-32 strain sprinkled in water body on antioxidant and immune enzymes of crucian carp liver;

[0020] Figure 7: The effect diagram of feed added with CAG2307-32 strain on the intestinal digestive enzyme of crucian carp;

[0021] Figure 8 : The effect diagram of feed added with CAG2307-32 strain on the antioxidant and immune enzyme of liver of crucian carp. DETAILED DESCRIPTION

[0022] The present application will be described in greater detail by way of example and with reference to the accompanying drawings wherein:

[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 Zhengyuanchuanghui Agricultural Science and Technology Development Co., Ltd., and the intestinal tract of the crucian carp was obtained under aseptic operation. The intestinal tract was ground and diluted to an appropriate concentration in sterile saline, and 100 μL of the homogenate diluent was smeared on the surface of freshwater fish agar (FWA) medium (beef extract 2.5 g, glucose 1 g, NaCl 5 g, MgSO4 0.05 g, peptone 5 g, yeast extract 2.5 g, K2HSO4 0.2 g, agar 15 g, H2O 1000 mL, pH 7.2-7.4). The culture was incubated at 30°C for 24 hours. Single colonies with different morphology, size, and color were picked from the surface of the FWA medium using a sterile inoculation loop, and a three-step streaking method was used to inoculate the surface of a new FWA medium. The culture was continued to be incubated, and the strain was purified multiple times until a pure strain was obtained.

[0026] Nitrogen-reducing bacteria screening: After the above 33 strains were cultured in FWA medium (30°C) for 24 hours, each bacterial solution was adjusted to OD 600 = 1.0 with normal saline, and inoculated into NH4 + -N (glucose 1.05 g, NH4Cl 0.08 g, MgSO4·7H2O 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, MgSO4·7H2O 0.2 g, KH2PO4 0.05 g, Na2HPO4 0.5 g, NaCl 5 g, H2O 1000 mL, pH 6.0-7.0), and NO3 -NH4 + -N degradation ability, the NO2 - -N degradation ability of each strain was detected by chromotropic acid method. - -N degradation ability.

[0027] Enzyme-producing bacteria screening: The above 33 strains were inoculated with sterile inoculation ring on amylase (peptone 10 g, yeast extract 5 g, NaCl 5 g, soluble starch 10 g, agar 20 g, H2O 1000 mL), protease (glucose 1 g, beef extract 3 g, NaCl 5 g, CaCl2 2 g, L-tyrosine 1 g, casein 5 g, agar 20 g, H2O 1000 mL) and lipase (peptone 10 g, CaCl2 0.1 g, NaCl 5 g, Tween-80 10 mL, agar 16 g, H2O 1000 mL, pH 7.2-7.4) medium, and observed and recorded whether there was a degradation ring after 24 h incubation at 30℃.

[0028] Results: 33 pure strains were isolated from the intestinal tract of healthy crucian carp, numbered CAG2307-1 to CAG2307-33.

[0029] Among them, 23 strains of NH4 + -N, NO2 - -N and NO3 - -N all have degradation effect, among which CAG2307-2, CAG2307-4, CAG2307-11 and CAG2307-32 strains have better effect, and CAG2307-32 strain has the best effect, NH4 + -N, NO2 - -N and NO3 - -N degradation rate 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 to have amylase, protease and lipase activities. Combined with the nitrogen reduction results, the potential probiotic effect of strain CAG2307-32 was the best, which could efficiently reduce nitrogen and produce obvious hydrolysis rings on amylase, protease and lipase media (see Figure 1 ).

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

[0032]

[0033]

[0034] 2) Temperature tolerance of the strain

[0035] Strains CAG2307-11 and CAG2307-32 with high nitrogen reduction activity and enzyme production activity were selected for high temperature tolerance experiment. The activated 2 strains were inoculated into FW medium at 1% inoculation amount, 3 test tubes for each strain, cultured at 30°C for 24h, then detected the viable bacteria number by plate counting method, and then placed in 80°C water bath, sampled at 30 and 60min, detected the viable bacteria number by plate counting method, and calculated the survival rate of the strain.

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

[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 with high nitrogen removal activity and enzyme production activity and high temperature resistance was selected for morphological observation and 16S rDNA sequencing identification. The strain CAG2307-32 was inoculated on the surface of FWA medium by sterile inoculation ring, and the strain morphology was observed after 24 h culture at 30°C. The genomic DNA of the strain CAG2307-32 was extracted by Ezup column bacterial genomic DNA extraction kit (Shanghai, China), and the 16S rDNA gene of the strain CAG2307-32 was amplified. The nucleotide sequence of the primers used was: 27F (5'-AGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTCGACTT-3'). The 16S rDNA sequence of the strain CAG2307-32 was compared on the ribosome database (http: / / rdp.cme.msu.edu / index.jsp), and the target phylogenetic tree was constructed by using the software MEGA11.

[0042] After the strain CAG2307-32 was cultured on FWA medium (30°C) for 24 h, the colony shape was irregular, dirty white, opaque, and the surface and edge were rough (see Figure 2 ).

[0043] The 16S rDNA gene sequencing of the strain CAG2307-32 obtained 1499 bp amplification product, and the sequence was as follows:

[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] By phylogenetic analysis, the CAG2307-32 strain was determined to be a Bacillus strain, with the highest similarity to Bacillus halodurans Figure 2 ).

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

[0072] Hemolytic experiment: CAG2307-32 strain was inoculated in FW medium (30℃, 180r / min) for 24h, adjusted to OD 600 =1.0, 10μL bacterial solution was added on sterile filter paper and placed on the surface of blood agar medium, which was purchased from Beryt Biology Technology (Zhengzhou) Co., Ltd., and cultured at 30℃ for 24h, and whether hemolytic ring was produced was observed.

[0073] Antibiotic sensitivity experiment: CAG2307-32 strain was inoculated in FW medium (30℃, 180r / min) for 24h, adjusted to OD 600 =1.0, 100μL was uniformly coated on FWA medium, and antibiotic sensitivity paper was pasted, which was purchased from Hangzhou Microbial Reagent Co., Ltd., and cultured at 30℃ for 24h, then the diameter of inhibition zone was measured and the antibiotic sensitivity was judged according to CLSI 2011 standard.

[0074] Injection infection experiment: healthy crucian carp juveniles with body length of (8.74±0.85) cm and body weight of (9.19±2.98) g were selected as injection infection materials. After being temporarily cultured in indoor circulating water culture system for 7d, the fish were starved for 24h, and then injected intraperitoneally. Each fish was injected with 100μL CAG2307-32 strain (1×10 8 CFU / mL), pathogenic bacteria ZKW3 strain (1×10 8 CFU / mL) and normal saline. The pathogenic bacteria ZKW3 strain was screened from crucian carp culture water and had been confirmed as a pathogenic bacteria of crucian carp, which was used as a positive control group; the normal saline was a negative control group, and 30 fish were injected in each group. During the experiment, the fish were normally fed, the dissolved oxygen was ≥5mg / L, the average water temperature was 25.4℃, and the fish were observed for 7d, and the death of the test fish was recorded.

[0075] The results showed that CAG2307-32 strain showed γ-hemolysis on blood plate, i.e. no hemolysis( Figure 3 ); the antibiotic sensitivity experiment results showed that CAG2307-32 strain was resistant to compound sulfamethoxazole, moderately sensitive to cefazolin, and sensitive to 20 kinds of antibiotics such as oxacillin, carbenicillin, cefoperazone and amikacin (see Table 3); after injection of CAG2307-32 strain and normal saline, no disease and death occurred in crucian carp.

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

[0077]

[0078]

[0079] Note: R is resistant, I is moderately sensitive, S is sensitive.

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

[0081] Example 2: Application effect of CAG2307-32 strain sprayed in water in crucian carp breeding

[0082] 1) Management of crucian carp breeding

[0083] Healthy and uniform-sized juvenile A. argenteus were selected as experimental materials, with an average body length of (10.82 ± 0.65) cm and an average body weight of (18.45 ± 0.43) g. The probiotic CAG2307-32 strain selected in the previous stage was used as the test strain, and the other two strains ZKF17 and ZKW7 selected in the previous stage were used for comparison. Both strains were identified as Bacillus. The A. argenteus were 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 conducted in white polyethylene breeding tanks, with an actual water volume of 30 L and a duration of 26 d. During the experiment, the fish were fed twice a day (9:00 and 17:00) with a daily feeding amount of 3% of their body weight. The bacterial solution was sprayed once every 3 days in each group (except the control group) to achieve a final concentration of 1 × 10 6 CFU / mL. During the breeding period, the water temperature was 21.1-23.3℃, and the water was changed every 7 days, with a 30% replacement rate each time.

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

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

[0086] The results showed that all three strains had certain degradation effects on NO2 - -N and NO3 - -N. Among them, CAG2307-32 had the most obvious effect, and the concentrations of NO2 - -N and NO3 - -N in the CAG2307-32 group remained at the lowest level. The maximum degradation rates of CAG2307-32 strain on NO2 - -N and NO3 - -N were 54.81% and 43.88%, respectively. Figure 4). The above results show that the water body splashing three probiotics can reduce the nitrogen content of water body to a certain extent, and compared with the other two strains, CAG2307-32 strain has the best cleaning effect on water quality.

[0087] 3) Growth performance determination of crucian carp

[0088] After the end of the cultivation experiment, the fish body weight and body length of each group were measured after fasting for 24 h, and the relative weight gain rate, specific growth rate, condition factor 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)] x 100%;

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

[0091] Condition factor (g / cm 3 ) = 100 x (final body weight) / (final body length) 3 ;

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

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

[0094] Table 4: Results of CAG2307-32 strain splashed in water on the growth of crucian carp

[0095]

[0096]

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

[0098] 4) Determination of intestinal digestive enzyme activity of crucian carp

[0099] After the end of the experiment, randomly selected 9 fish from each group, dissection on ice, collect the intestine tissue and store at -80℃. According to the operation requirements of the kit instructions of lipase, amylase and trypsin, the activity of digestive enzymes in the intestine of each group of fish was determined.

[0100] The results showed that the water spraying CAG2307-32 strain and ZKF17 strain could significantly improve the amylase activity in the intestine of crucian carp, but only the CAG2307-32 strain could significantly improve the trypsin activity in the intestine of crucian carp. Figure 5 The above results showed that the water spraying CAG2307-32 strain could effectively promote the digestion and absorption of feed starch and protein in crucian carp.

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

[0102] After the end of the experiment, randomly selected 9 fish from each group, dissection on ice, collect the liver tissue and store at -80℃. According to the operation requirements of the kit instructions of superoxide dismutase, reduced glutathione and alkaline phosphatase, the antioxidant and immune enzyme activity in the liver of each group of fish was determined.

[0103] The results showed that the water spraying CAG2307-32 strain could significantly improve the superoxide dismutase activity in the liver of crucian carp, while the other two strains had no significant effect on the superoxide dismutase activity. Although ZKW7 strain could also improve the content of reduced glutathione and the activity of acid phosphatase in the liver of crucian carp, the effect was not as good as that of CAG2307-32 strain. Figure 6 The superoxide dismutase has the functions of scavenging active oxygen, antioxidant and relieving inflammation. The reduced glutathione can scavenge excess oxygen free radicals in the body and plays an important role in antioxidant stress. The acid phosphatase is a non-specific phosphoric acid hydrolytic enzyme and an important component of blood cell lysosome enzyme, which plays an important role in defending against the invasion of external microorganisms. The above results showed that the water spraying CAG2307-32 strain could effectively improve the antioxidant and immune level of crucian carp.

[0104] Example 3: Application effect of feed added with CAG2307-32 strain in crucian carp culture

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

[0106] The feed 180 for fish (Tongwei Biotechnology Co., Ltd. of Wuxi) was used as the test feed. The detection results showed that the crude protein was (30.77±0.17)%, the crude fat was (9.34±0.93)%, the crude ash was (22.39±2.59)%, and the moisture was (9.52±0.26)%.

[0107] CAG2307-32 strain was cultured in FW medium (30℃, 180r / min) for 24h, and then centrifuged (4000r / min) to remove the supernatant. The bacterial solution was adjusted to a concentration of 1x10 8 CFU / mL, and the control group was added with the same amount of sterile saline. The feed was dried at 40℃ and stored at -20℃. A batch of feed was prepared every 7 days.

[0108] 2) Carassius auratus breeding management

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

[0110] 3) Carassius auratus growth performance determination

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

[0112] The results showed that the addition of CAG2307-32 strain in feed could significantly improve the final body weight and relative weight gain rate of Carassius auratus, and significantly reduce the feed coefficient (see Table 5), indicating that CAG2307-32 strain could effectively improve the digestion and absorption capacity of Carassius auratus and promote its growth.

[0113] Table 5: Results of the effect of feed added with CAG2307-32 strain on the growth of Carassius auratus

[0114]

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

[0116] 4) Carassius auratus intestinal digestive enzyme activity determination

[0117] After the breeding experiment, 9 experimental fish were randomly selected from each group and dissected on ice. The intestinal tissue was collected and stored at -80℃. The intestinal digestive enzyme activity of the experimental fish in each group was determined according to the operation requirements of the lipase, amylase, and trypsin test kits.

[0118] The results show that the addition of CAG2307-32 strain in feed can significantly improve the lipase and amylase activity of crucian carp intestine Figure 7 , indicating that the addition of CAG2307-32 strain in feed can effectively improve the digestion and absorption capacity of crucian carp to feed.

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

[0120] After the end of the cultivation experiment, 9 experimental fish were randomly selected from each group on ice, and the liver tissue was collected and stored at -80℃. According to the operation requirements of the superoxide dismutase, reduced glutathione, catalase, malondialdehyde, alkaline phosphatase and acid phosphatase test kits, the antioxidant and immune enzyme activity of the liver of the experimental fish in each group was determined.

[0121] The results show that the addition of CAG2307-32 strain in feed can significantly improve the superoxide dismutase, reduced glutathione, catalase and acid phosphatase activity 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 important antioxidant enzymes, and the higher the activity, the stronger the antioxidant capacity of the body; malondialdehyde is the product of peroxidation in the body, and is an important marker of cell membrane damage in the body, and its content decreases, indicating that the antioxidant capacity of the body is enhanced; alkaline phosphatase and acid phosphatase are similar and play an important role in the body's immune defense.

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

Claims

1. A salt-tolerant Bacillus, characterized in that, The salt-resistant Bacillus ( Bacillus halotolerans The accession number of the object is CGMCC No. 32389.

2. The use of the salt-tolerant Bacillus of claim 1 in the preparation of bacterial preparations for freshwater fish farming.

3. A bacterial preparation for freshwater fish farming, characterized in that, The bacterial preparation contains live bacteria of the salt-tolerant Bacillus as described in claim 1.

4. The application of the salt-tolerant Bacillus as described in claim 1 as a feed additive.

5. A feed for freshwater fish farming, characterized in that, The feed contains live bacteria of the salt-tolerant Bacillus as described in claim 1.

6. A method for freshwater fish farming, characterized in that, The method involves spraying the fermentation liquid of the salt-tolerant Bacillus as described in claim 1 into the aquaculture water.

7. The method as described in claim 6, characterized in that, The method described is for raising freshwater crucian carp.

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