A strain of Bacillus licheniformis XA07 and its application

Abstract

The invention discloses a strain of Bacillus licheniformis XA07 and its application in the cultivation of white shrimp, belonging to the technical field of microorganisms and aquaculture. The deposit number of the strain is: CCTCC NO: M2024810. The Bacillus licheniformis XA07 of the invention can effectively inhibit the reproduction of pathogenic bacteria in aquatic animals by colonizing in the intestines of the aquatic animals, improve the microecology of the gastrointestinal flora, enhance immunity and disease resistance, and improve the survival rate and growth rate; at the same time, it can also effectively reduce the content of ammonia nitrogen and nitrite nitrogen in the aquaculture water body, purify the water quality, and has broad application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms and aquaculture, and more particularly to a strain of Bacillus licheniformis and application thereof in the cultivation of Penaeus vannamei. Background Art

[0002] Litopenaeus vannamei belongs to the Penaeidae family, and its scientific name is Penaeus vannamei. It is one of the three species with the highest production of farmed shrimp in the world. The white shrimp is large, thin-shelled and fat, with a meat yield of more than 65%. The meat is tender, delicious, and rich in protein. It is very popular in domestic and foreign markets and has high economic value. It grows fast and can reach the specifications for listing in 60 days. It has low nutritional requirements, strong disease resistance, and a wide range of salt adaptability (0-45%). It can be cultivated in pure fresh water, brackish water, and seawater. It can grow from natural sea areas to freshwater ponds without geographical limitations. It also has the advantages of high temperature resistance and strong disease resistance. It is a high-quality variety of marine shrimp and a very promising farmed shrimp. Based on the above characteristics, the artificial cultivation of white shrimp is very extensive and is the pillar industry of marine aquaculture in my country.

[0003] However, due to the deterioration of the breeding environment in recent years, the scale of high-density and intensive development of aquaculture has been growing, and shrimps have problems such as intestinal diseases, weak immunity, and slow growth. As a result, large-scale epidemic outbreaks and mass deaths often occur in the breeding process of white shrimp, causing huge economic losses. The traditional disease prevention and control methods with antibiotics and chemical drugs as the main means can no longer meet the needs. The reason is that on the one hand, the large-scale use of antibiotics and chemical drugs has led to drug resistance in pathogens, reduced prevention and control effects, and significantly increased prevention and control costs; on the other hand, the presence of drug residues directly affects the health of consumers and public safety. Therefore, it is of great significance to strengthen the research on disease prevention technology of white shrimp.

[0004] Probiotics have broad application prospects in the field of feed preparation due to their ability to colonize in the digestive tract of animals, regulate the host immune system and intestinal flora microecological balance, inhibit pathogens, and thus improve the host's immunity and disease resistance; and can produce a variety of metabolites and enzymes, decompose complex feed ingredients, and promote the digestion and absorption of nutrients such as protein, carbohydrates and fats. Prior art CN104164392B discloses a strain of Bacillus licheniformis, which can effectively reduce the content of ammonia nitrogen in water and reduce the feed-to-meat ratio of shrimp, but its probiotic effect is relatively limited, it cannot stably colonize in the animal digestive tract, fails to inhibit pathogens, and has no significant effect on enhancing the immunity and disease resistance of aquatic animals.

[0005] Therefore, it is of great significance to find new probiotics that can not only promote the growth of aquatic animals, but also inhibit pathogens, improve animal immunity and survival rate, and purify aquaculture water. Summary of the invention

[0006] In view of the above problems, the present invention provides a Bacillus licheniformis XA07 and applications thereof.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A strain of Bacillus licheniformis XA07, classified and named Bacillus licheniformis, was deposited in the China Center for Type Culture Collection on April 29, 2024, with the deposit number CCTCC NO: M2024810, and the deposit address is Wuhan University, Wuhan, China.

[0009] Another object of the present invention is to provide application of Bacillus licheniformis XA07 in aquaculture.

[0010] Another object of the present invention is to provide the use of Bacillus licheniformis XA07 in promoting and / or improving the growth of Penaeus vannamei.

[0011] Another object of the present invention is to provide the use of Bacillus licheniformis XA07 in the preparation of feed or additives for promoting or improving the growth condition of Penaeus vannamei.

[0012] Another object of the present invention is to provide a culture of Bacillus licheniformis XA07 or a processed product thereof.

[0013] Another object of the present invention is to provide a feed additive, which comprises the above-mentioned Bacillus licheniformis XA07 and / or a culture of Bacillus licheniformis XA07 or a processed product thereof.

[0014] Another object of the present invention is to provide the use of Bacillus licheniformis XA07 in the preparation of aquaculture water purification preparations.

[0015] Another object of the present invention is to provide a water purification preparation, which contains the above-mentioned Bacillus licheniformis XA07 and / or a culture of Bacillus licheniformis XA07 or a processed product thereof.

[0016] It can be seen from the above technical scheme that, compared with the prior art, the present invention discloses a new probiotic, Bacillus licheniformis XA07, which can effectively colonize in the intestine of white shrimp, effectively inhibit the reproduction of its pathogenic bacteria, improve the gastrointestinal flora microecology, enhance immunity and disease resistance, and improve its survival rate and growth rate; at the same time, it can also effectively reduce the content of ammonia nitrogen and nitrite nitrogen in aquaculture water, purify water quality, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 :Colony morphology characteristics of Bacillus licheniformis XA07;

[0018] Figure 2 :Construction of phylogenetic tree of Bacillus licheniformis strains;

[0019] Figure 3 : Antioxidant activity test results of Bacillus licheniformis XA07;

[0020] Figure 4 : The bile salt tolerance test result of Bacillus licheniformis XA07;

[0021] Figure 5 : pH tolerance test result of Bacillus licheniformis XA07;

[0022] Figure 6 : The results of the high temperature resistance test of Bacillus licheniformis XA07;

[0023] in, Figure 3-6 Different lowercase letters represent p < 0.05. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] The reagents involved in the embodiments of the present invention are all purchased from commercial channels, and the methods not mentioned are conventional experimental methods and will not be described in detail here.

[0026] The following reagents are illustrative:

[0027] 2216 culture medium: 5 g peptone, 1 g yeast extract powder, 0.01 g ferric phosphate, 1 L seawater (salinity 35), pH 7.6 ± 0.2, sterilization at 121 ° C for 20 min.

[0028] TSB medium: 17 g trypticase, 5 g sodium chloride, 3 g soybean papain hydrolysate, 2.5 g potassium dihydrogen phosphate, 2.5 g glucose, 1 L distilled water, pH 7.3±0.2, sterilize at 21°C for 20 min.

[0029] LB liquid medium: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, 1 L distilled water, pH 7.0 ± 0.2, sterilized at 21 °C for 20 min.

[0030] Amylase liquid enzyme production medium: 20g starch, 20g peptone, 5.0g Na2HPO4, 1g MgSO4, 0.1g NaCl, 1L distilled water, pH 7.2±0.2, sterilization at 115℃ for 20min.

[0031] Cellulase liquid enzyme production medium: 10g CMC-Na, 5g peptone, 10g yeast powder, 10g NaCl, 1L distilled water, pH 5.6±0.2, sterilization at 115℃ for 20min.

[0032] Protease liquid enzyme production culture medium: 40g glucose, 20g peptone, 1.4gNa2HPO4, 0.6g CaCl2, 0.4gMgSO4, 1L distilled water, pH 7.2±0.2, sterilization at 115℃ for 20min.

[0033] Solid culture medium is prepared by adding 2% agar to the corresponding liquid culture medium.

[0034] Xylo-oligosaccharide (CAS No. 87-99-0, purchased from Shaanxi Denis Biotechnology Co., Ltd.);

[0035] VC sodium salt (CAS No. 134-03-2, purchased from Shandong Chenda Chemical Co., Ltd.);

[0036] Soluble starch (CAS No. 9005-84-9, purchased from Langfang Kangpu Huiwei Technology Co., Ltd.);

[0037] Skim milk powder was purchased from Shanghai Dongzhen Trading Co., Ltd.

[0038] Example 1 Isolation, screening and identification of strains

[0039] 1. Isolation and screening of strains

[0040] The shrimps were collected from the white shrimp breeding shed in Hetao Street, Chengyang. The intestines of healthy and vigorous shrimps were removed by aseptic operation, put into a grinder, added with physiological saline, homogenized, centrifuged at 3000r / min for 5min, so that the bacteria in the intestinal sample were fully dispersed in the upper physiological saline, and diluted with sterile water for 10 3 The strains were inoculated on 2216E, TSB and LB solid culture media respectively and cultured at 28℃ for 36h-48h. Uniform and clear single colonies were selected and streaked to purify until there were no foreign bacteria. A total of 24 single colonies were selected and named XA01, XA02, XA03, XA24 in turn, and stored in glycerol at -80℃ for future use.

[0041] With Vibrio parahaemolyticus and Vibrio harveyi as indicator bacteria, the bacterial liquid of the above 24 single colonies was spotted on the 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 spotted area. Finally, a total of 8 pathogenic antagonistic bacteria were screened, which had varying degrees of inhibitory effects on the above two pathogens. The specific results are shown in Table 1.

[0042] Table 1 Antibacterial performance of potential probiotic strains

[0043]

[0044] As shown in Table 1, among the 8 pathogenic antagonistic bacteria screened by the present invention, strain XA07 has the strongest comprehensive antibacterial ability against Vibrio parahaemolyticus and Vibrio harveyi, and the diameters of the inhibition zones against Vibrio parahaemolyticus and Vibrio harveyi are 2.19 cm and 1.94 cm, respectively.

[0045] 2. Identification of strains

[0046] (1) Identification of colony morphology

[0047] Use an inoculation loop to sample strain XA07 stored in glycerol at -80℃, inoculate it into LB liquid culture medium, and culture it in a shaking incubator at 37℃ and 180r / min for 24h, then activate it 2-3 times to obtain activated strains. Inoculate the activated seed bacterial liquid into LB liquid culture medium at a 1% inoculation rate, and culture it in a shaking incubator at 37℃ and 180r / min for 18h until the logarithmic phase, to obtain XA07 bacterial suspension. Dilute the strain XA07 bacterial suspension with sterile water for 10 5 times, take 100 μL of the dilution and evenly spread it on LB solid medium, and culture it in a 28°C incubator for 36 hours. Figure 1 ,Depend on Figure 1 It can be seen that the colonies are milky white, 1 to 2 mm in diameter, flat in shape, with neat edges and a smooth and moist surface.

[0048] (2) Molecular Biological Identification

[0049] Pick a single colony of strain XA07 into a 1.5 mL centrifuge tube, add 50 μL of sterile water, vortex mix, place in -196°C liquid nitrogen and 100°C water bath for repeated freeze-thaw 3 times (10 min / time), centrifuge at 12000 rpm at 4°C for 10 min, and take the supernatant, which is the DNA extract of strain XA07. The DNA extracts extracted above were subjected to PCR amplification using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1) and 1492R (5'-TACGGTTACCTTGTTACGACTT-3', SEQ ID NO.2) as primers. The reaction system for PCR amplification was as follows: 4 μL of DNA template, 0.7 μL of each upstream and downstream primer at a concentration of 10 μM, 17.5 μL of PCR SuperMix, and ddH2O supplemented to 35 μL; the PCR amplification program was as follows: pre-denaturation at 94°C for 3 min; 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 5 min; and storage at 4°C.

[0050] The amplified product was purified by 2% agarose gel electrophoresis using a gel extraction kit (Beckman CoulterGenomics, Danvers, MA, USA). After sequencing, the 16S rRNA sequence of strain XA07 is shown in SEQ ID NO. 3. By BLAST comparison analysis, the 16S rRNA sequence similarity between strain XA07 and several published strains of Bacillus licheniformis was as high as 97%. The phylogenetic tree was constructed using the Neighborjionig (NJ) method, and the results are shown in Figure 2 As shown, it was determined to be Bacillus licheniformis.

[0051] Example 2 Determination of tolerance of strains

[0052] 1. Antibiotic sensitivity test

[0053] Take 200 μL of the XA07 bacterial suspension in Example 1 on the surface of the LB solid plate, spread the bacterial solution evenly on the plate with a spreading rod, and use sterile tweezers to stick erythromycin, ampicillin, tetracycline, polymyxin B, penicillin, cephalothin, ceftriaxone and oxacillin on the solid plate. After culturing at 37°C for 72 hours, the diameter of the inhibition zone was measured. The judgment standard was: the diameter of the inhibition zone ≥2.0 cm was antibiotic sensitive. The results are shown in Table 2.

[0054] Table 2 Results of XA07 sensitivity test to antibiotics

[0055] antibiotic Diameter of inhibition zone (cm) Erythromycin 2.74±0.17 Ampicillin 3.82±0.11 tetracycline 3.60±0.06 Polymyxin B 2.63±0.10 penicillin 3.62±0.20 Ceftriaxone 2.23±0.09 Ceftriaxone 3.47±0.34 Oxacillin 2.15±0.04

[0056] As shown in Table 2, strain XA07 is sensitive to the above eight commonly used antibiotics, which indicates that the strain is safe and has the potential to be used in the preparation of biological products or feed additives.

[0057] 2. Antioxidant Activity

[0058] Take 10 mL of the XA07 bacterial suspension in Example 1, centrifuge at 8000 r / min for 10 min, and filter the supernatant through 0.22 μm, which is recorded as the supernatant group. Wash the filtered residue with sterile water and resuspend it to obtain the XA07 bacterial solution for later use. Divide the resuspended XA07 bacterial solution into two parts, adjust the bacterial cell concentration of one part to 10 9 The other part was disrupted by ultrasonic ice bath for 15 min, then centrifuged at 8500 r / min for 2 min at 4 °C, and the supernatant was collected and recorded as the cell-free extract group.

[0059] Take 0.5 ml of each of the three groups of solutions as the test solution, add them into 1 mL of 0.2 mmol / L freshly prepared DPPH-anhydrous ethanol solution, mix well, react at room temperature in the dark for 30 min, centrifuge at 6000 r / min for 10 min, take the supernatant and measure the absorbance value As at a wavelength of 517 nm. In the blank group, replace the DPPH solution with an equal volume of anhydrous ethanol to measure the absorbance value, which is recorded as Ab; in the control group, replace the test solution with an equal volume of distilled water, measure the absorbance value, which is recorded as Ac. According to the formula: DPPH free radical scavenging rate (%) = [1-(As-Ab) / Ac] × 100, calculate the DPPH free radical scavenging rates of the solutions in the three groups above, and the results are shown in the table. Figure 3 .

[0060] like Figure 3 As shown, the DPPH free radical scavenging rate was supernatant group > bacterial cell group > cell-free extract group, among which the DPPH free radical scavenging rate of the supernatant group was 84.0%, which was much higher than that of the other two groups, indicating that the XA07 strain had good in vitro antioxidant properties.

[0061] 3. Bile salt resistance, pH resistance, and high temperature resistance tests

[0062] Whether probiotics can colonize and survive in animals is mainly related to their acid and bile resistance. As a feed additive, whether it can withstand high temperatures in industrial production is also crucial. Therefore, the XA07 bacterial suspension (preparation method is the same as in Example 1) was subjected to bile resistance, pH resistance and high temperature resistance tests, and the specific process is as follows:

[0063] Bile salt tolerance test: XA07 bacterial suspension was inoculated with 2% (volume fraction) in LB liquid medium with bile salt concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 g / L. After constant temperature culture at 37°C for 24 hours, the absorbance of the bacterial solution at a wavelength of 600 nm was measured using a spectrophotometer. Three parallels were set for each bile salt concentration group. The specific results are as follows: Figure 4 shown.

[0064] pH tolerance test: XA07 bacterial suspension was inoculated into LB liquid culture medium with pH values ​​of 1, 3, 5, 7, 9, and 11 at a volume fraction of 5%. After constant temperature culture at 37°C for 24 hours, the absorbance of the bacterial solution at a wavelength of 600 nm was measured using a spectrophotometer. Three parallel experiments were set for each pH group. The specific results are shown in the figure. Figure 5 shown.

[0065] High temperature resistance test: Take 6 groups of LB liquid culture medium, each group of liquid culture medium is inoculated with 5% (volume fraction) of XA07 bacterial suspension, and then water bath for 15 minutes at 37, 50, 60, 70, 80, and 90℃. After the water bath, culture at 37℃ for 24 hours, and measure the absorbance of the bacterial solution at a wavelength of 600nm with a spectrophotometer. Each group is set up with 3 parallels. The specific results are as follows: Figure 6 shown.

[0066] Depend on Figure 4 It can be seen that with the increase of bile salt concentration, the proliferation ability of strain XA07 weakened. When the bile salt concentration was 0.2-0.4 g / L, the OD of XA07 bacterial solution was 600 =1.19~1.16, which was not significantly different from the blank control group (bile salt concentration 0g / L); when the bile salt concentration was 0.6g / L, 0.8g / L, and 1.0g / L, the OD of XA07 bacterial solution 600 They were 0.90, 0.61 and 0.42 respectively, which were 23.72%, 48.31% and 64.41% lower than those of the blank control group; however, after constant temperature culture at 37°C for 24 hours, strain XA07 had the ability to proliferate in LB liquid culture medium with a bile salt concentration of 0-1.0 g / L.

[0067] Depend on Figure 5 It can be seen that after culturing at 37°C for 24 hours, the proliferation rate of strain XA07 decreased with increasing or decreasing pH compared with the neutral pH 7 condition. 600 They were 1.24, 1.62, and 1.30, respectively, which were significantly higher than the culture environments with pH values ​​of 1, 3, and 11, and still had strong proliferation ability in an environment with a pH value of 5. However, strain XA07 still had proliferation ability in the range of pH﹤7.

[0068] Depend on Figure 6 It can be seen that the strain XA07 can continue to proliferate after being treated at different temperatures, but its growth rate is significantly inhibited as the treatment temperature increases. Compared with the 37℃ treatment group, the OD of the XA07 strain treated with high temperature (70-90℃) after culturing for 24h was 600 =0.72~0.60, which were significantly reduced; while the final concentration of bacterial solution in the 37, 50, and 60℃ treatment groups was OD 600 They are 1.18, 1.16 and 1.04 respectively, and are growing well.

[0069] Therefore, Bacillus licheniformis XA07 has the potential to resist adverse environmental factors such as low pH and bile salts in the digestive tract, which is conducive to colonization in the animal digestive tract.

[0070] Example 3 Determination of enzyme production capacity of strains

[0071] XA07 strain (OD 600 =1.0) was inoculated into LB liquid culture medium at a 2% inoculum volume fraction, and cultured at 37°C and 160 r / min for 12 h to obtain seed solution, which was stored at 4°C for later use.

[0072] 1. Amylase production capacity test

[0073] (1) Plotting of glucose standard curve: Prepare 1 mg / mL glucose standard solution and add each solution to test tubes 0 to 5 according to Table 3. Mix well and boil in a boiling water bath for 10 min to develop color. Cool to room temperature and dilute to 10 mL. Measure the absorbance of the solution in each test tube at 680 nm using an ultraviolet spectrophotometer. Draw a standard curve with glucose concentration as the horizontal axis and absorbance as the vertical axis.

[0074] Table 3 Amount of solution added to each test tube during the drawing of the glucose standard curve

[0075] Test tube number Glucose standard solution / mL Distilled water / mL DNS reagent / mL 0 0 2 2 1 0.2 1.8 2 2 0.4 1.6 2 3 0.6 1.4 2 4 0.8 1.2 2 5 1.0 1.0 2 6 1.2 0.8 2

[0076] (2) Preparation of crude enzyme solution: Count using a hemocytometer and adjust the seed solution concentration to 1×10 7 cells / mL, inoculated into amylase liquid enzyme production medium at a rate of 1%, cultured at 37°C, 160r / min with shaking for 20h, centrifuged at 8000r / min for 10min, and the supernatant was taken as the crude enzyme solution.

[0077] (3) Determination of amylase activity: 0.9 mL of 1% soluble starch solution was used as substrate. After preheating for 10 min, 0.1 mL of the above crude enzyme solution was added. The mixture was mixed and reacted for 10 min. 1.5 mL of DNS solution was added. The mixture was placed in a boiling water bath for 15 min. The mixture was cooled in running water and the absorbance at a wavelength of 540 nm was measured. The reducing sugar content was calculated based on the glucose standard curve, and the amylase activity was calculated to be 84.437 U / mL.

[0078] 2. Cellulase production capacity test

[0079] (1) The drawing of glucose standard curve is the same as the step (1) of amylase production capacity detection.

[0080] (2) Preparation of crude enzyme solution: Count using a hemocytometer and adjust the seed solution concentration to 1×10 7 cells / mL, inoculated into the cellulase liquid enzyme production medium at a rate of 1%, cultured at 37°C, 160r / min with shaking for 20h, centrifuged at 8000r / min for 10min, and the supernatant was taken as the crude enzyme solution.

[0081] (3) Determination of cellulase activity: Take another test tube, add 0.5 mL of crude enzyme solution and 1.5 mL of citric acid buffer, add 1.5 mL of DNS reagent to test tube No. 0 shown in Table 3 as a control, place the test tube in a 50°C water bath for 10 min, add 50 mg of filter paper, keep warm for 1 h, take it out, and immediately add 1.5 mL of DNS reagent to test tubes No. 1-6 in Table 3, place in a boiling water bath for 5 min, cool and make up to 20 ml, and measure the absorbance at 540 nm.

[0082] Cellulase activity calculation formula: FPA = M*V*5.56 / (A*B*T)

[0083] M is the glucose content (mg); V is the fixed volume (mL); T is the reaction time (h); A is the amount of enzyme added (mL); B is the substrate mass (mg); 5.56 is the amount of 1 mg of glucose substrate (μmoL).

[0084] Calculation showed that the cellulase activity of strain XA07 reached 73.306U / mL.

[0085] 3. Protease production capacity detection

[0086] (1) Preparation of tyrosine standard curve: According to Table 4, add 1 mL of 0-50 μg / mL tyrosine standard solution into test tubes 0-5, respectively, and then add 5 mL of 0.4 mol / L Na2CO3 solution and 1 mL of Folin phenol reagent. Mix well and place in a water bath at 37°C for 10 min. After cooling to room temperature, measure the absorbance at 680 nm. Draw a standard curve with tyrosine concentration as the horizontal axis and absorbance as the vertical axis.

[0087] Table 4 Amount of solution added to each test tube during the drawing of the tyrosine standard curve

[0088] Test tube number Tyrosine standard solution (mL) <![CDATA[0.4 mol / L Na2CO3 solution (mL)]]> Folin phenol reagent (mL) 0 1(0μg / mL) 5 1 1 1(10μg / mL) 5 1 2 1(20μg / mL) 5 1 3 1(30μg / mL) 5 1 4 1(40μg / mL) 5 1 5 1(50μg / mL) 5 1

[0089] (2) Preparation of crude enzyme solution: Count using a hemocytometer and adjust the seed solution concentration to 1×10 7 cells / mL, inoculated into the protease liquid enzyme production medium at a rate of 1%, cultured at 37°C, 160r / min with shaking for 20h, centrifuged at 8000r / min for 10min, and the supernatant was taken as the crude enzyme solution.

[0090] (3) Protease activity assay: add 1 mL of crude enzyme solution preheated at 40°C for 5 min and 1 mL of 1% casein solution preheated at 40°C for 5 min to tubes 1 to 5 respectively, mix well and incubate at 40°C for 10 min, add 2 mL of TCA reagent (0.4 mol / L); add 2 mL of TCA reagent (0.4 mol / L) to tube 0, and the other steps are the same as those of tubes 1 to 5. Centrifuge all tubes at 4500 r / min for 5 min, take 1 mL of supernatant, add 5 mL of Na2CO3 solution (0.4 mol / L) and 1 mL of Folin-phenol solution, keep at 40°C for 20 min, and measure the absorbance at 680 nm.

[0091] Protease activity calculation formula: X = OD680*K*V*N / T

[0092] K is the absorption constant of the tyrosine standard curve; V is the reaction volume (mL); T is the reaction time (min); N is the dilution multiple.

[0093] The calculation showed that the protease activity of strain XA07 reached 95.362U / mL.

[0094] In summary, Bacillus licheniformis XA07 has the ability to produce amylase, cellulase and protease, and the activity of each enzyme is relatively high, which is 84.437U / mL, 73.306U / mL and 95.362U / mL, respectively, and can be used for large-scale fermentation production of bio-enzymes.

[0095] Example 4 Determination of the effect of Bacillus licheniformis XA07 on the growth and immune characteristics of white shrimp

[0096] 30 mL of the XA07 bacterial suspension prepared in Example 1 was placed in a 50 mL centrifuge tube and centrifuged at 4°C and 8000 r / min for 15 min. The supernatant was discarded, the bacterial sludge was collected, and 8% of oligoxylose, 4% of sodium VC, 14% of soluble starch and 8% of skim milk powder were added in sequence and mixed evenly. After pre-freezing at -80°C for 4 h, vacuum freeze-dried for 24 h at a cold trap temperature of -70°C and a vacuum degree of 1 Pa, Bacillus licheniformis XA07 bacterial powder (live bacteria amount 10 10 CFU / g).

[0097] A control group and a probiotic group were set up, and three parallels were set up in each group, with 50 white shrimp seedlings of uniform size in each parallel. The shrimp Minghai feed (purchased from Maoming Wanhe Livestock and Aquatic Feed Co., Ltd.) was used as the basic feed. The control group was fed with the basic feed, and the probiotic group was fed with the above-mentioned Bacillus licheniformis XA07 powder at a mass ratio of 3‰ in the basic feed. The feeding experiment was carried out in an indoor recirculating aquaculture system, with a breeding cycle of 20 days. The feeding amount was fed according to 4-6% of the average body weight of white shrimp, and it was fed once in the morning and evening every day. During the experiment, the bottom was sucked and discharged every morning, and aeration was continued to ensure that the dissolved oxygen content in the water was ≥6mg / L, the salinity was between 8-9, the pH was 7.8-8.1, and the water temperature was maintained at 24-26℃. After the breeding experiment, the number and weight of white shrimp seedlings in the control group and the probiotic group were measured, and the survival rate, weight gain rate, specific growth rate, etc. were calculated; and tail vein blood was taken to measure serum acid phosphatase, alkaline phosphatase, and superoxide dismutase. The results are shown in Table 5.

[0098] Survival rate = N t / N0*100%

[0099] Weight gain rate = (W t -W0) / W0*100%

[0100] Specific growth rate = (lnW t -lnW0) / t*100%

[0101] N t : Final digit; N0: Initial digit; W t : Final body weight; W0: Initial body weight; t: Number of days of culture

[0102] Table 5 Statistics of the effects of Bacillus licheniformis XA07 on the growth and immune characteristics of white shrimp

[0103]

[0104]

[0105] Different lowercase letters represent p < 0.05

[0106] As shown in Table 5, compared with the control group, in the probiotic group added with Bacillus licheniformis XA07, the weight gain rate, specific growth rate, survival rate, acid phosphatase activity, alkaline phosphatase activity and superoxide dismutase activity of white shrimp increased by 42.22%, 34.23%, 23.49%, 23.99%, 26.09% and 24.15%, respectively. It can be seen that the Bacillus licheniformis XA07 provided by the present invention can significantly promote the growth of white shrimp, and effectively improve the immunity of white shrimp, which is beneficial to resist bacteria and reduce the morbidity. Therefore, Bacillus licheniformis XA07 can be widely used in the cultivation of white shrimp as a probiotic.

[0107] Example 5 Determination of the Purification Capacity of Bacillus licheniformis XA07 on Aquaculture Water

[0108] A control group and a probiotic group were set up, and three parallels were set up for each group, and each parallel was 500 mL of aquaculture water from a shrimp farm in Weihai City. 0.3 g of Bacillus licheniformis XA07 powder (preparation method is the same as Example 4) was added to the probiotic group, and no addition was made to the control group. The experimental water temperature was 24-26°C, and aeration was continued during the period. After 24 hours, the ammonia nitrogen content and nitrite nitrogen content in the water body were respectively measured, and the specific results are shown in Table 6.

[0109] Table 6 Statistics of the results of the determination of aquaculture water purification by Bacillus licheniformis XA07

[0110] Group Ammonia nitrogen content (mg / mL) Nitrite nitrogen content (mg / mL) Control group <![CDATA[7.46±0.43 a ]]> <![CDATA[0.37±0.05 a ]]> Probiotics group <![CDATA[1.02±0.16 b ]]> <![CDATA[0.03±0.01 b ]]>

[0111] Different lowercase letters represent p < 0.05

[0112] As shown in Table 6, after adding Bacillus licheniformis XA07 powder to the aquaculture water for 24 hours, the ammonia nitrogen content in the water was reduced by 86.33% compared with the control, and the nitrite nitrogen content was reduced by 91.89% compared with the control.

[0113] In summary, the Bacillus licheniformis XA07 provided by the present invention can effectively inhibit the reproduction of pathogenic bacteria in aquatic animals, improve the gastrointestinal flora microecology of aquatic animals, enhance immunity and disease resistance, improve the utilization rate of feed by aquatic animals, and improve their survival rate and growth rate. At the same time, it can also effectively solve the problem of excessive ammonia nitrogen and nitrite nitrogen content in aquaculture water, and has broad application prospects.

[0114] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0115] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Bacillus licheniformis XA07, characterized in that: It was classified and named Bacillus licheniformis, and was deposited in the China Center for Type Culture Collection on April 29, 2024. The deposit number is CCTCC NO: M2024810, and the deposit address is Wuhan University, Wuhan, China.

2. An application of the Bacillus licheniformis XA07 according to claim 1 in the cultivation of whiteleg shrimp.

3. Use of the Bacillus licheniformis XA07 according to claim 1 in promoting and / or improving the growth of Penaeus vannamei.

4. Use of the Bacillus licheniformis XA07 according to claim 1 in preparing feed or additives for promoting or improving the growth of Penaeus vannamei. A culture comprising the Bacillus licheniformis XA07 according to claim 1 or a processed product thereof.

6. A feed, characterized in that: Comprising the Bacillus licheniformis XA07 according to claim 1 and / or the culture or a processed product thereof according to claim 5.

7. A feed additive, characterized in that: The feed additive comprises the Bacillus licheniformis XA07 according to claim 1 and / or the culture or a processed product thereof according to claim 5.

8. Use of the Bacillus licheniformis XA07 according to claim 1 in preparing an aquaculture water purification preparation.

9. A water purification preparation, characterized in that: Comprising the Bacillus licheniformis XA07 according to claim 1 and / or the culture or a processed product thereof according to claim 5.

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