Bacillus pumilus and application thereof in aquaculture

Abstract

The invention relates to the technical field of functional microorganism screening and application, and particularly provides novel bacillus pumilus and application thereof in aquaculture. The preservation number of the bacillus pumilus is CCTCC (China Center For Type Culture Collection) NO: M2024808, and the bacillus pumilus can be used for remarkably inhibiting various pathogenic bacteria, reducing the content of ammoniacal nitrogen and nitrite nitrogen in an aquaculture water body and improving the water quality, and also has very strong degradation capability on microcystin. The strain can effectively improve the growth performance of aquatic animals, enhance the immunity and oxidation resistance of the aquatic animals, and can be widely applied to the field of aquatic feed production.

Description

Technical Field

[0001] The invention relates to the technical field of functional microorganism screening and application, and in particular to a strain of Bacillus pumilus and application thereof in aquaculture. Background Art

[0002] In recent years, the aquaculture industry has developed rapidly, and it is estimated that by 2027, the output of aquaculture will exceed 100 million tons. However, the high-density and intensive farming of aquaculture can easily lead to deterioration of water quality, frequent aquatic diseases, and cause large economic losses. Probiotics are defined as living microorganisms that are beneficial to the health of the host when given in appropriate quantities. They colonize in the gastrointestinal tract and inhibit pathogens, thereby improving intestinal flora and enhancing animal immunity. Probiotics can also increase the activity of digestive enzymes such as lipase, amylase and protease in aquatic animals, promote digestion and absorption, and thus improve the growth performance of aquatic animals. The application of probiotics in aquaculture can achieve green and sustainable development of aquaculture, which has important economic and social value.

[0003] Commonly used probiotics in aquaculture include lactic acid bacteria, Bacillus, yeast, and light and bacteria. Lactic acid bacteria are resistant to acid and bile salts, which enables them to survive in the intestinal system. At the same time, the bacteriocins produced by lactic acid bacteria have an inhibitory effect on common fish pathogens. Studies have found that both Lactococcus lactis and Lactobacillus plantarum can exert their antibacterial effects and inhibit the adhesion of Aeromonas hydrophila and Aeromonas salmonicida to the intestinal mucus of rainbow trout. The lactic acid bacteria that are more widely used in aquaculture are mainly Lactobacillus acidophilus, Lactococcus lactis, Lactobacillus rhamnosus, and Lactobacillus plantarum.

[0004] Bacillus has the functions of producing antibiotics, bacteriocins, and competing with pathogenic microorganisms for adhesion sites, inhibiting the growth of pathogens, and promoting the growth of beneficial bacteria in the intestines, thereby enhancing animal immunity and bacteriocins. As a probiotic, Bacillus produces antioxidant enzymes such as SOD and glutathione, which effectively eliminate free radicals and improve the antioxidant capacity of animals. Bacillus can also produce a variety of enzymes, which can effectively metabolize a variety of lipids, proteins and carbohydrates, increase the activity of animal digestive enzymes, promote animal growth and development, and increase daily mass gain. At present, the most widely used Bacillus in aquaculture are Bacillus subtilis, Bacillus licheniformis and Bacillus cereus.

[0005] The proteins and vitamins contained in yeast can provide nutrients for aquatic animals. Studies have found that yeast can remain active at a pH of 2.5 and has good acid resistance. Yeast has been shown to have no hemolytic activity and is a safe probiotic that can be used in aquaculture. Yeast also has the effect of inhibiting the growth of pathogens. Agarbati et al. reported that yeast strains have antibacterial activity against Candida albicans, Escherichia coli, Staphylococcus aureus and Salmonella enterica. At present, the yeast added in aquaculture is mainly brewer's yeast and marine red yeast.

[0006] Photosynthetic bacteria are rich in nutrients such as protein, vitamins and carotenoids, and are also palatable and easily absorbed. They are often added to the feed of aquatic animals such as sea cucumbers, shellfish, shrimps and crabs. In addition, photosynthetic bacteria have a strong decomposition ability and can decompose toxic substances such as ammonia nitrogen, nitrite nitrogen and hydrogen sulfide in water, promote the circulation of organic matter, and are widely used in water purification.

[0007] In actual production, factors such as the source, type, dosage and duration of probiotics will affect the effectiveness of probiotics. The continuous search for the most suitable probiotic strains for the growth of aquatic animals is still a current research hotspot. Summary of the invention

[0008] The purpose of the present invention is to provide a novel strain of Bacillus pumilus and its application in aquaculture. The Bacillus pumilus is selected from the healthy and vigorous intestine of grouper, can significantly inhibit pathogenic bacteria, improve the water quality of aquaculture water, promote the growth of aquatic animals, improve immune function, and enhance the immunity and antioxidant capacity of aquatic animals, and can be widely used in the field of aquatic feed.

[0009] On the one hand, the present invention relates to a strain of Bacillus pumilus CZX17 (Bacillus pumilus CZX17), which was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on April 29, 2024, and its deposit number is CCTCCNO: M2024808.

[0010] The invention also relates to the application of Bacillus pumilus CZX17 in the production of aquatic feed.

[0011] The present invention also relates to the application of Bacillus pumilus CZX17 in the production of antibacterial agents.

[0012] The invention also relates to the application of Bacillus pumilus CZX17 in water purification.

[0013] The present invention also relates to an aquatic feed, comprising the Bacillus pumilus CZX17 bacterial powder.

[0014] The amount of live bacteria in the bacterial powder is not less than 1010 CFU / g.

[0015] The addition amount of Bacillus pumilus CZX17 in the aquatic feed is 20-30 kg / ton.

[0016] The Bacillus pumilus CZX17 screened by the present invention can produce high levels of amylase and protease, and the activities of amylase and protease in its fermentation supernatant are as high as 11.23U / mL and 257U / mL, respectively; it can effectively inhibit pathogenic bacteria such as Vibrio harveyi, Vibrio parahaemolyticus, Streptococcus agalactiae, Pseudomonas, Oligotrophomonas, Citrobacter freundii and Aeromonas hydrophila, among which the antibacterial effect on Vibrio harveyi, Vibrio parahaemolyticus and Citrobacter freundii is the strongest, and the diameters of the inhibition zones are 2.68cm, 2.23cm and 2.41cm, respectively, achieving unexpected technical effects.

[0017] The Bacillus pumilus CZX17 can rapidly reduce the pH of water. After 24 hours of treatment, the contents of ammonia nitrogen and nitrite nitrogen in the water decreased by 97.5% and 93.3% respectively, which is very effective and is conducive to reducing water pollution and improving the quality of aquaculture water. The strain also has a strong ability to degrade microcystin. After 72 hours of treatment, the degradation rate of microcystin reached 48.92%, achieving unexpected technical results.

[0018] The Bacillus pumilus CZX17 can be added to aquatic feed, which can significantly improve feed utilization, improve the growth performance of aquatic animals, and enhance their antioxidant capacity and immunity. Compared with the control group, the growth rate and specific growth rate of the grouper fed with Bacillus pumilus CZX1 powder increased by 15.77% and 14.75% respectively, the feed coefficient decreased by 8.2%, the concentrations of blood glucose and triglycerides in plasma decreased by 14.22% and 11.52% respectively, the alkaline phosphatase activity increased by 15.93%, the mass concentration of complement C3 increased by 36.84%, and the total antioxidant capacity of serum increased by 14.08% (P<0.05), of which the superoxide dismutase activity and peroxidase activity increased by 1.3%. The activities of hydrogenase, peroxidase and glutathione reductase increased by 20.16%, 21.23%, 22.59% and 88.24%, respectively, and the molar concentration of malondialdehyde decreased by 20.18%. The total antioxidant capacity of the serum of Penaeus vannamei in the experimental group fed with Bacillus pumilus CZX17 powder increased by 160.36%, among which the superoxide dismutase activity, peroxidase activity and lysozyme activity increased by 41.60%, 48.66%, and 347.66%, respectively, and the effect was very significant.

[0019] The Bacillus pumilus CZX17 can be widely used in aquaculture as a feed additive, water quality improver, etc., can significantly improve the growth performance and immunity of aquatic animals, help reduce the occurrence of diseases, increase economic benefits, and has broad prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the colony morphology of Bacillus pumilus CZX17;

[0021] Figure 2 This is the growth curve of Bacillus pumilus CZX17. DETAILED DESCRIPTION

[0022] The equipment and reagents used in the embodiments of the present invention can be selected from any commercially available one. For the specific methods or materials used in the embodiments, those skilled in the art can make conventional replacement selections based on the technical ideas of the present invention and according to existing technologies, and are not limited to the specific records of the embodiments of the present invention.

[0023] The culture medium selected in the embodiment has the following specific formula:

[0024] 2216E culture medium: 5g sodium alginate, 1g glucose, 1g sodium tripolyphosphate, 0.5g magnesium sulfate heptahydrate, 0.1g calcium chloride tetrahydrate, 0.1g ammonium sulfate, 0.01g iron sulfate heptahydrate, 0.01g manganese sulfate heptahydrate, 0.01g zinc sulfate heptahydrate, 0.005g copper sulfate heptahydrate, 0.5g potassium dihydrogen sulfate, 5g peptone, 1000ml distilled water, pH 7.2-7.4, prepare solid culture medium by adding 15-20g agar, and sterilize at 121℃ for 20min.

[0025] Nutrient broth medium (1L): 10g peptone, 3g beef extract, 5g sodium chloride. Add the above ingredients to distilled water, make up to 1000mL, heat to dissolve, adjust pH to 7.3±0.2; sterilize at 121℃ for 20min after aliquoting. (Note: solid medium is based on this plus 1.5% agar powder).

[0026] TSB medium: 17g tryptone, 3g soybean papain hydrolysate, 5g sodium chloride, 2.5g glucose monohydrate, 2.5g potassium dihydrogen phosphate, 1000ml distilled water, pH 7.1-7.5. Prepare solid culture medium by adding 13-20g agar and sterilize at 121℃ for 20min.

[0027] Skim milk 2216E solid culture medium: Add 1% skim milk to 2216E culture medium, prepare solid culture medium by adding 1.2%-1.5% agar powder, and sterilize at 121℃ for 20 minutes.

[0028] Potato starch 2216E solid medium: Add 0.3% potato starch to 2216E medium, prepare solid medium by adding 1.2%-1.5% agar powder, and sterilize at 121°C for 20 minutes.

[0029] TW-80 2216E solid culture medium: Add 0.8% TW-80 to 2216E culture medium, prepare solid culture medium by adding 1.2%-1.5% agar powder, and sterilize at 121℃ for 20 minutes.

[0030] The present invention is further described below in conjunction with specific embodiments.

[0031] Example 1 Isolation, screening and identification of strains

[0032] 1. Sample

[0033] Healthy and vigorous grouper fish were collected from a farm in Hainan Province, and their intestines were removed under aseptic conditions for subsequent separation.

[0034] 2. Screening method

[0035] Take the intestinal sample of the grouper and put it into a grinder, put it into physiological saline, homogenize it, centrifuge it at low speed, so that the bacteria in the sample are fully dispersed in the upper layer of physiological saline, dilute it 10 times in a gradient, inoculate it on 2216E and TSB solid culture media, and culture it at 28℃ for 24-48h. Select uniform and clear single colonies and purify the bacteria by streaking. The single colonies are named CZX01, CZX02, CZX03,..., CZX50 in turn.

[0036] A single colony was picked up with an inoculation needle and spotted on skim milk 2216E solid medium, potato starch 2216E solid medium and TW-80 2216E solid medium, and cultured at 35°C for 3 days.

[0037] After 3 days of culture, after simple pretreatment, the presence and diameter of transparent dissolution ring and white precipitation ring of colony on skim milk 2216E solid medium, potato starch 2216E solid medium and TW-80 2216E solid medium were observed. Finally, the applicant screened out 38 enzyme-producing bacteria, including 32 protease-producing strains, 26 lipase-producing strains, and 29 amylase-producing strains. In addition, 16 strains of bacteria (CZX02, CZX04, CZX05, CZX10, CZX13, CZX16, CZX17, CZX20, CZX23, CZX25, CZX29, CZX32, CZX35, CZX38, CZX45, CZX49) showed three extracellular enzyme activities. The subsequent screening will be carried out based on its antibacterial ability.

[0038] Using Vibrio harveyi and Vibrio parahaemolyticus as indicator bacteria, the 16 strains showing extracellular enzyme activity selected above were screened for beneficial bacteria using the plate antagonism method. The bacterial solution to be screened was inoculated on a plate coated with indicator bacteria, cultured at a constant temperature of 28°C, and observed within 48 hours whether an antibacterial transparent zone or a covered zone appeared around the inoculation area. Finally, the applicant screened a total of 8 pathogenic antagonistic bacteria that had varying degrees of inhibitory effects on the above two pathogens. The specific results are shown in Table 1.

[0039] Table 1 Antibacterial performance of potential probiotic strains

[0040]

[0041] Note: Inhibition zone diameter (φ) > 2cm is extremely sensitive, 1.5cm < φ ≤ 2cm is highly sensitive, 1cm < φ ≤ 1.5cm is moderately sensitive, 0.5cm < φ ≤ 1cm is low sensitive, and φ ≤ 0.5cm is insensitive.

[0042] It can be seen from the data in Table 1 that, among the 8 pathogenic antagonistic bacteria screened by the present invention, strain CZX17 has the strongest comprehensive antibacterial ability against Vibrio harveyi and Vibrio parahaemolyticus.

[0043] 3. Strain identification

[0044] (1) Colony morphology characteristics

[0045] The above strain CZX17 was re-streaked and cultured. The colonies were off-white, with smooth and neat edges, clear edges, no elongation, and no root-like structure. The surface was sticky and the cells were flat. It was Gram-positive and could be preliminarily determined to be Bacillus. The colony morphology was shown in Figure 1 .

[0046] (2) Physiological and biochemical identification

[0047] According to the instructions for bacterial biochemical identification, strain CZX17 was inoculated into the reaction tube and cultured at 37°C for 24 hours. As shown in Table 2, based on the biochemical identification results and the bacterial biochemical identification code book, the strain was determined to be Bacillus.

[0048] Table 2 Biochemical identification results of strain CZX17

[0049] project result project result glucose + Malonate - lactose - Citrate - Galactose + Nitrate reduction - maltose + 6.5% high salt broth Growth Mannitol + <![CDATA[H2S]]> - Rhamnose - Phenylalanine - sucrose + Urea - Sorbitol + Sportiness +

[0050] Note: "+" means positive, corresponding color reaction occurs; "-" means negative, no color reaction occurs.

[0051] (3) The genomic DNA of the strain CZX17 was extracted, and the 16S rRNA sequence was amplified by PCR technology. The 16S rRNA sequence was similar to that of several published strains of Bacillus pumilus by sequencing BLAST comparison analysis. The strain CZX17 was identified as Bacillus pumilus, which was consistent with the biochemical identification results.

[0052] 4. Strain preservation

[0053] The above strain CZX17 was named Bacillus pumilus CZX17 (Bacillus pumilus CZX17), and was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on April 29, 2024, with the deposit number being CCTCC NO: M2024808.

[0054] Example 2 Growth characteristics and enzyme production capacity analysis of Bacillus pumilus CZX17

[0055] 1. Analysis of growth characteristics of Bacillus pumilus CZX17

[0056] The strain stored at -80°C was activated and resuspended, and inoculated into nutrient broth medium (pH 7.3±0.2) at an inoculum size of 2%, and cultured at 30°C with shaking at 180 r / min. Samples were taken at 0, 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 21, 24, 27, 30, and 33 h, and the absorbance value at OD600 nm was measured. There were 3 parallels in each group, and the growth curve of the strain was drawn. The growth curve of Bacillus brevis CZX17 is shown in Figure 2 .

[0057] 2. Analysis of enzyme production characteristics of Bacillus pumilus CZX17

[0058] The bacterial liquid of Bacillus pumilus CZX17 was inoculated into 200 mL of nutrient broth medium at a 1% inoculation amount, cultured at 37°C and 180 r / min for 24 h, centrifuged at 4°C and 5000 r / min for 10 min, and the supernatant was collected. The enzymatic activities of amylase and protease in the supernatant were determined by the following methods, respectively. The results are shown in Table 3.

[0059] (1) The DNS colorimetric method was used to determine the amylase activity.

[0060] Definition of amylase activity unit: Under the conditions of 40°C and pH 6.0, the amount of enzyme that releases 1 μmol of reducing sugar from 1% (mass fraction) soluble starch per minute is defined as 1 enzyme activity unit (U).

[0061] (2) The protease activity was determined by the Folin-phenol method.

[0062] Definition of protease activity unit: Under the conditions of 40°C and pH 7.5, the amount of enzyme that hydrolyzes casein to produce 1 μg of tyrosine per minute is defined as 1 enzyme activity unit (U).

[0063] Table 3 Enzyme production capacity of Bacillus pumilus CZX17

[0064] Strain name Amylase activity / (U / mL) Protease activity / (U / mL) Bacillus pumilus CZX17 11.23±0.35 257±4.33

[0065] As can be seen from Table 3, the Bacillus pumilus CZX17 screened by the present invention can produce high levels of amylase and protease, and the amylase and protease activities in its fermentation supernatant are as high as 11.23 U / mL and 257 U / mL, respectively, achieving unexpected technical effects.

[0066] Example 3 Antibacterial effect of Bacillus pumilus CZX17 on common pathogenic bacteria

[0067] The fermentation supernatant of Bacillus pumilus CZX17 was filtered through a 0.22 μm microporous filter membrane to obtain a filtrate; the antibacterial ability of the filtrate against 5 bacteria (Streptococcus agalactiae, Pseudomonas, Stenotrophomonas, Citrobacter freundii and Aeromonas hydrophila) was determined by the Oxford cup method. The amount of filtrate added to the Oxford cup was 100 μL. After filling, the culture medium was cultured at 37°C with the front side facing up for 16-18 hours, and the results were observed and the size of the inhibition zone was measured. The results are shown in Table 4.

[0068] Table 4 Antibacterial effect of Bacillus pumilus CZX17 on 5 kinds of bacteria

[0069] Bacterial species Bacillus pumilus inhibition zone diameter (cm) Streptococcus agalactiae 1.61±0.19 Pseudomonas 1.53±0.12 Stenotrophomonas 1.52±0.21 Citrobacter freundii 2.41±0.23 Aeromonas hydrophila 1.52±0.15

[0070] From the results in Table 4, it can be seen that Bacillus pumilus CZX17 has a strong antibacterial effect on five bacteria, including Streptococcus agalactiae, Pseudomonas, Stenotrophomonas, Citrobacter freundii and Aeromonas hydrophila. Among them, the antibacterial effect on Citrobacter freundii is the strongest, with the diameter of the inhibition zone reaching 2.41 cm, achieving unexpected technical results.

[0071] Example 4 Degradation effect of Bacillus pumilus CZX17 on microcystin

[0072] The activated Bacillus pumilus CZX17 bacterial liquid was inoculated into 2216E medium containing 1.39 mg / L microcystin at a volume ratio of 3%, and cultured at 35°C and 220 r / min for 72 hours. 5 mL of the fermentation liquid was taken every 24 hours, centrifuged, and the supernatant was filtered through a 0.22 μm filter membrane to detect the content of microcystin. The results are shown in Table 5.

[0073] Table 5 Degradation effect of Bacillus pumilus CZX17 on microcystins

[0074] Cultivation time Microcystin content (mg / L) 24h 1.10 48h 0.71 72h 0.52

[0075] From the results in Table 5, it can be seen that the Bacillus pumilus CZX17 provided by the present invention can effectively degrade microcystin, and the degradation rate of microcystin reaches 62.59% at 72 hours, achieving an unexpected technical effect.

[0076] Example 5 Degradation effect of Bacillus pumilus CZX17 on ammonia nitrogen and nitrite nitrogen in aquaculture water

[0077] The Bacillus pumilus CZX17 screened by the present invention is activated and expanded for culture, and then liquid fermentation is performed, and bacterial sludge is collected by centrifugation, a starch carrier is added, and the bacterial powder with a live bacterial count exceeding 10 billion / g is prepared by drying.

[0078] The experimental water was taken from a grouper culture pond in Jiaozhou City, Qingdao. The ammonia nitrogen content in the water was 1.05±0.07 mg / L, and the nitrite nitrogen content was 0.12±0.03 mg / L. The experimental container was a plastic barrel with a volume of 10 L of water.

[0079] Experimental groups:

[0080] (1) Control group: no Bacillus pumilus CZX17 bacterial solution was sprayed;

[0081] (2) Experimental group: Spray Bacillus pumilus CZX17 at a rate of 0.5 g / L.

[0082] 24 hours after spraying the bacterial powder, the water quality was analyzed, and the analysis indicators included pH, ammonia nitrogen and nitrite nitrogen content. The results are shown in Table 6.

[0083] Table 6 Degradation effect of Bacillus pumilus CZX17 on ammonia nitrogen and nitrite nitrogen

[0084] project Control group Experimental Group pH 8.1±0.3 7.9±0.3 Ammonia nitrogen (mg / L) 1.05±0.07 0.026±0.002 Nitrite nitrogen (mg / L) 0.12±0.03 0.008±0.001

[0085] As shown in Table 6, compared with the control group, the pH of the water in the experimental group sprayed with Bacillus pumilus CZX17 powder decreased, and the contents of ammonia nitrogen and nitrite nitrogen decreased by 97.5% and 93.3%, respectively, with a very significant effect. This shows that the Bacillus pumilus CZX17 screened by the present invention can effectively improve water quality and reduce water pollution.

[0086] Example 6 Application of Bacillus pumilus CZX17 in the cultivation of grouper

[0087] 1. Experimental location

[0088] A recirculating aquaculture workshop in Jiaozhou, Qingdao.

[0089] 2. Experimental Grouping

[0090] (1) Control group: fed with basic formula feed;

[0091] (2) Experimental group: 3% Bacillus pumilus CZX17 powder (10 10 CFU / g) of basic formula feed.

[0092] 3. Experimental conditions

[0093] 180 groupers were selected, and three breeding barrels were used as parallel controls in the control group and the experimental group, with 30 groupers in each breeding barrel. Before the formal experiment, they were temporarily raised for 15 days to adapt to the experimental environment. The water temperature of the breeding water was controlled at 25.0±1.0℃, the salinity was 24-26, the ammonia nitrogen concentration was not higher than 0.1mg / L, the nitrite concentration was not higher than 0.05mg / L, the pH was 8.0-8.3, and the dissolved oxygen concentration was 7.0-8.5mg / L. Feed was fed once at 8:30 and 16:30 every day, and the feeding amount was until a little feed was seen, and the residual bait and feces were siphoned off. The experimental feeding cycle was 75d.

[0094] On the 75th day of the experiment, the weight and length of the fish in each group were weighed, and the growth rate, specific growth rate, feed coefficient and survival rate were calculated. The experimental grouper was deprived of food for 24 hours and anesthetized with 40 mg / L eugenol. When sampling, 1 mL of medical syringe was rinsed with 1% sodium heparin, and venous blood was drawn from the fish. The blood was centrifuged at 3000 rpm for 10 min at 4 °C, and the supernatant was aspirated for the determination of blood indicators. The grouper was dissected, and about 1.0 g of liver tissue was cut and stored at -20 °C for the determination of liver enzyme activity, total antioxidant capacity, and malondialdehyde concentration.

[0095] 4. Experimental results

[0096] (1) Growth performance analysis

[0097] Table 7 Growth performance parameters of grouper after feeding for 75 days

[0098]

[0099]

[0100] Note: Data in the same column with different letters indicate significant differences (P<0.05), while data with the same letters or no letters indicate no significant differences (P>0.05).

[0101] As shown in Table 7, compared with the control group, the growth rate and specific growth rate of the grouper fed with Bacillus pumilus CZX1 powder increased by 15.77% and 14.75% respectively, and the feed coefficient decreased by 8.2%. This shows that the Bacillus pumilus CZX17 provided by the present invention can effectively promote the growth of farmed animals and improve feed utilization, with a very significant effect.

[0102] (2) Plasma biochemical and immune indicators

[0103] ROCHE p800 fully automatic biochemical analyzer (Germany) was used to detect plasma biochemical indices, including total protein, blood glucose, triglyceride concentration and alkaline phosphatase activity. The mass concentrations of plasma complement C3 and C4 were determined according to the instructions of the corresponding kit (purchased from Nanjing Jiancheng Bioengineering Institute). The experimental results are shown in Table 8.

[0104] Table 8 Effects of Bacillus pumilus CZX17 on plasma biochemical and immune parameters of Platycodon grandis Note: Data in the same column with different letters indicate significant differences (P<0.05), while data with the same letters or no letters indicate no significant differences (P>0.05).

[0105] As can be seen from Table 8, compared with the control group, the concentrations of blood glucose and triglyceride in the plasma of the experimental group of grouper fed with Bacillus pumilus CZX17 powder decreased by 14.22% and 11.52%, respectively, and the alkaline phosphatase activity increased by 15.93%, with significant effects. The results of immune indexes showed that the mass concentration of complement C3 in the plasma of the grouper in the experimental group was significantly higher than that in the control group, which increased by 36.84%, indicating that the Bacillus pumilus CZX17 provided by the present invention can significantly improve the immunity of grouper.

[0106] (3) Liver antioxidant index

[0107] The liver superoxide dismutase activity, catalase activity, peroxidase activity, glutathione reductase activity, glutathione peroxidase activity, total antioxidant capacity and malondialdehyde concentration were determined according to the instructions of the antioxidant enzyme detection kit (purchased from Nanjing Jiancheng Bioengineering Institute). The results are shown in Table 9.

[0108] Table 9 Effects of Bacillus pumilus CZX17 on antioxidant capacity of grouper liver Note: Data in the same column with different letters indicate significant differences (P<0.05), while data with the same letters or no letters indicate no significant differences (P>0.05).

[0109] As shown in Table 9, compared with the control group, the total antioxidant capacity of the group of grouper fed with Bacillus pumilus CZX17 powder increased by 14.08% (P<0.05), the superoxide dismutase activity, catalase activity, peroxidase activity and glutathione reductase activity in serum increased by 20.16%, 21.23%, 22.59% and 88.24% respectively; the molar concentration of malondialdehyde decreased by 20.18% (P<0.05), and the effect was very significant. It is thus shown that the Bacillus pumilus CZX17 provided by the present invention can significantly improve the antioxidant capacity and active oxygen scavenging ability of grouper, thereby helping to reduce the accumulation of lipid peroxides and reduce tissue loss.

[0110] Example 7 Application of Bacillus pumilus CZX17 in the cultivation of white shrimp

[0111] 1. Experimental location

[0112] A recirculating aquaculture workshop in Jiaozhou, Qingdao.

[0113] 2. Experimental Grouping

[0114] (1) Control group: fed with basic formula feed;

[0115] (2) Experimental group: 2% Bacillus pumilus CZX17 powder (10 10 CFU / g) of basic formula feed.

[0116] 3. Experimental conditions

[0117] Three aquariums were set up in each group as parallel, and 60 white shrimps were cultured in each aquarium. They were fed four times a day (8:00, 12:00, 16:00, and 20:00 every day), and 3-4% of the shrimp body weight was fed every day. The feed was adjusted at any time according to the feeding amount. During the experiment, the water temperature of the culture water was controlled at 26.0±1.0℃, the salinity was 21-23, the ammonia nitrogen concentration was not higher than 0.1mg / L, the nitrite concentration was not higher than 0.05mg / L, the pH was 8.0-8.3, and the dissolved oxygen concentration was ≥8.2mg / L. After each feeding, the residual material and feces were sucked out by siphoning. The culture cycle was 60d.

[0118] After the breeding cycle, the white shrimp was fasted for 24 hours, and then the white shrimp was dissected, blood was taken from the shrimp heart, and serum was centrifuged to measure serum superoxide dismutase, peroxisome, lysosome activity, total antioxidant capacity, etc. The results are shown in Table 10.

[0119] Table 10 Effects of Bacillus pumilus CZX17 on immunity of Penaeus vannamei As shown in Table 10, compared with the control group, the total antioxidant capacity of the serum of Penaeus vannamei in the experimental group fed with Bacillus pumilus CZX17 powder increased by 160.36% (p < 0.05), among which the superoxide dismutase activity, peroxidase activity, and lysozyme activity increased by 41.60%, 48.66%, and 347.66% (p < 0.05), respectively, and the effect was very significant. It can be seen that adding Bacillus pumilus CZX17 provided by the present invention to the feed can effectively enhance the antioxidant capacity of Penaeus vannamei and enhance its immunity.

[0120] In summary, the high-yield amylase and protease of Bacillus pumilus CZX17 screened by the present invention can quickly reduce the content of ammonia nitrogen and nitrite nitrogen in aquaculture water, effectively improve the water quality; can significantly inhibit the growth of various pathogenic bacteria such as Vibrio harveyi, Vibrio parahaemolyticus, Streptococcus agalactiae, Pseudomonas, Oligotrophomonas, Citrobacter freundii and Aeromonas hydrophila; and also has a strong degradation ability for microcystin. Bacillus pumilus CZX17 can be widely used in aquaculture, can significantly improve feed utilization, improve the growth performance of aquatic animals, enhance their antioxidant capacity and immunity, help reduce the occurrence of diseases, improve economic benefits, and has broad application prospects.

Claims

1. A Bacillus pumilus, characterized in that The preservation number of the Bacillus pumilus is CCTCC NO: M2024808.

2. Use of Bacillus pumilus as claimed in claim 1 in aquatic feed production.

3. Use of Bacillus pumilus as claimed in claim 1 in the production of antibacterial agents.

4. Application of Bacillus pumilus in water purification as claimed in claim 1.

5. An aquatic feed, characterized in that: The aquatic feed contains the Bacillus pumilus according to claim 1.

6. The aquatic feed according to claim 5, characterized in that: The amount of live bacteria of Bacillus pumilus in the aquatic feed is at least 10 10 CFU / g.

7. The aquatic feed according to claim 5 or 6, characterized in that: The addition amount of Bacillus pumilus in the aquatic feed is 20-30 kg / ton.

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