Pelteobagrus fulvidraco intestinal catfish source cetella and application thereof in improving ammonia tolerance of fish
By developing the intestinal catfish, the problem of increased ammonia and tissue ammonia in yellow catfish was solved in the high ammonia environment, the ammonia tolerance and survival rate of fish were improved, and the fish was achieved. Microecological balance of aquaculture systems.
Patent Information
- Application Number
- CN202411346546.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-26
- Publication Date
- 2025-02-07
AI Technical Summary
In high ammonia environment, yellow catfish are prone to increased blood ammonia and tissue ammonia, resulting in a decrease in survival rate and immune dysfunction. The prior art is difficult to effectively improve the tolerance of fish to high ammonia.
A strain of Cetobacillus YC01, a yellow catfish intestinal catfish, can reduce the blood ammonia and tissue ammonia levels in fish under high ammonia environments, regulate the intestinal flora, and improve the ammonia tolerance of fish.
By using the Cetobacillus YC01 strain, the survival rate of yellow catfish in high ammonia environment was significantly improved, and the blood ammonia content was reduced, the microecological balance of the fish farming system was maintained, and it has important application value.
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Figure CN119391561A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a catfish-derived cetacean from the intestine of yellow catfish and an application thereof in improving the ammonia tolerance of fish. Background Art
[0002] Ammonia nitrogen is one of the important pollutants in the aquatic animal breeding water environment. It exists in the form of ionized ammonia and non-ionized ammonia. Non-ionized ammonia is the main toxic form, which can cause farmed animals to have reduced food intake, slow growth, and poor immunity. Long-term exposure to a high ammonia nitrogen environment can cause an imbalance in the antioxidant system of aquatic animals, trigger immune and metabolic dysfunction, and ultimately cause liver and intestinal tissue damage. Long-term exposure to an ammonia-containing environment will increase the sensitivity of fish to pathogenic bacteria. The U.S. Environmental Protection Agency (EPA) warns that exposure to an environment with 17 mg / L of total ammonia nitrogen for one hour can cause acute ammonia poisoning, while continuous exposure to 1.9 mg / L of total ammonia nitrogen for one month may lead to chronic ammonia poisoning, which will have adverse effects on aquatic organisms. Data from an ammonia accumulation ecosystem experiment under natural conditions showed that under the pond farming model, the annual average total ammonia nitrogen content gradient was 0.3 to 20.6 mg / L, which has exceeded the tolerance threshold of fish. The accumulation of ammonia in aquaculture ponds and closed-loop aquaculture systems may pose a potential risk to the aquaculture industry because it may lead to the outbreak of aquatic animal diseases and a sharp drop in production. The reduction of ammonia nitrogen by adding probiotics such as Bacillus to water bodies has achieved initial results in the aquaculture industry, so it is particularly important to develop a new probiotic to improve fish tolerance to ammonia nitrogen. Summary of the invention
[0003] The technical problem to be solved by the present invention is to provide a catfish-derived cetacean from the intestine of yellow catfish, which has the function of reducing blood ammonia and tissue ammonia of yellow catfish in a high-ammonia environment and improving the survival rate of yellow catfish in a high-ammonia environment, and its application in improving the ammonia tolerance of fish.
[0004] The technical solution adopted by the present invention to solve the above-mentioned technical problems is: a yellow catfish intestinal catfish-derived Cetobacterium somerae, which is a YC01 strain, classified and named Cetobacterium somerae, and was preserved in the Guangdong Provincial Microbial Culture Collection Center on April 25, 2024, with a preservation number of GDMCC No.64562.
[0005] The invention also provides application of catfish-derived cetacean from the intestine of yellow catfish in improving the ammonia tolerance of fish.
[0006] The invention also provides the use of catfish-derived cetacean from the intestine of yellow catfish in the preparation of a probiotic additive for yellow catfish breeding.
[0007] Compared with the prior art, the invention has the following advantages: the invention discloses a yellow catfish intestinal catfish-derived Cetobacterium YC01 strain and an application thereof for improving fish ammonia tolerance; the strain is isolated from the yellow catfish intestinal tract, cultured at 37°C for 96 hours, and a single colony appears on a solid culture medium, which is transparent, round, slightly irregular, convex, and has a diameter of 1.0-2.0 mm; the strain also has the function of reducing blood ammonia and tissue ammonia of the yellow catfish under a high-ammonia environment, can regulate normal intestinal flora of the body, maintain the microecological balance of the yellow catfish breeding system, and improve the survival rate of the yellow catfish under high-ammonia conditions, thereby playing an important role in improving the yellow catfish's ability to cope with high-ammonia environments; the strain is used as a probiotic additive for intensive breeding of yellow catfish, which can improve the survival rate of the yellow catfish under high-ammonia conditions; therefore, the Cetobacterium SomeraeYC01 is used in shrimp farming, where it can quickly become the dominant flora and promote the stability of the host's intestinal flora. It has significant economic value, especially in the development of new green feed additives for yellow catfish and research on antibiotic substitutes.
[0008] The above-mentioned yellow catfish intestinal catfish-derived Cetobacterium somerae is the YC01 strain, classified and named Cetobacterium somerae. It was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 25, 2024, with the collection number GDMCC No. 64562, and the collection address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Figure 1 The colony morphology of Bacillus cereus YC01, including (A) the colony characteristics of Bacillus cereus YC01Z on the culture medium, (B) the morphological characteristics of Bacillus cereus YC01Z under the microscope;
[0010] Figure 2 For the functional evolution analysis of Cetiella YC01 plasmid 6 and Cetiella NK01;
[0011] Figure 3 For the functional evolution analysis of Cetiella YC01 plasmid 7 and Cetiella NK01;
[0012] Figure 4 To investigate the effects of adding Bacillus subtilis YC01 on the survival rate and blood ammonia of Pelteobagrus fulvidraco under acute high-level ammonia stress. DETAILED DESCRIPTION
[0013] The present invention is further described in detail below with reference to the accompanying drawings.
[0014] Example 1
[0015] 1. Isolation and purification of the strain of the present invention
[0016] The intestinal contents of yellow catfish were placed in a fastidious anaerobic liquid culture medium at 37°C for 96 h, and the obtained culture medium was diluted to 10 -3 , 10 -4 , 10 -5 , take 150 μl of each dilution, apply it to the fastidious anaerobic solid culture medium, and place it in a constant temperature incubator at 37°C for 96 hours. Select colonies with single and regular colony morphology for repeated purification and culture until a pure strain is obtained. Select a single colony with a pure strain for 16S rRNA gene sequence analysis, morphology and Gram staining observation.
[0017] The above-mentioned fastidious anaerobic liquid culture medium formula, ingredients (g / L): tryptic peptone 12.0, peptone 5.0, yeast extract powder 3.0, beef extract powder 3.0, soluble starch 1.0, sodium chloride 5.0, pH 7.3±0.2, 25℃. Test principle: tryptic peptone, peptone, beef extract powder, yeast extract powder and soluble starch provide carbon and nitrogen sources, vitamins and growth factors, and sodium chloride maintains a balanced osmotic pressure. Usage: Weigh 29.0g of culture medium, heat and dissolve in 1000mL of distilled water, autoclave at 121℃ for 15 minutes, and set aside. Note: The culture medium contains a small amount of starch. If it is not heated and boiled to dissolve before sterilization, a small amount of white precipitate may appear after cooling after sterilization.
[0018] 2. Identification of bacterial species
[0019] 2.1 Morphological observation
[0020] The selected strains were streaked onto fastidious anaerobic solid culture medium, placed in a 37°C constant temperature incubator for anaerobic culture for 96 hours, and their colony morphology was observed.
[0021] Depend on Figure 1 A shows that the colony morphology of Bacillus cereus YC01 cultured in caustic anaerobic medium is: single round colony, transparent, slightly irregular, convex, with a diameter of 1.0-2.0mm. Figure 1 B shows that after Gram staining, the rod-shaped bacteria are Bacillus subtilis YC01, and no other bacteria appear in the field of view.
[0022] 2.2 Whole genome sequencing analysis
[0023] A single colony of Bacillus cereus YC01 was inoculated into a harsh anaerobic liquid culture medium, cultured anaerobically at 37°C for 96h, and centrifuged at 4°C and 6000r / min for 5min to collect the bacteria. High-quality DNA was extracted using a genome extraction kit and a DNA library was constructed. The DNA was sequenced using the PromethION sequencer based on the third-generation sequencing technology OxfordNanopore Technology for single-molecule sequencing, and the whole genome of the strain was sequenced with the MGISEQ-2000 system in the PE150 read length mode for double-end sequencing. The results of whole genome sequencing showed that 10 genes in the bacteria were involved in the utilization of ammonia in yellow catfish. YC01 has the function of converting toxic ammonia into non-toxic arginine and glutamine, and at the same time has the function of further decomposing arginine and glutamine. In addition, it also has the function of producing argininosuccinic acid. The bacteria has a full set (16) of glycolysis genes and has the ability to produce acetyl-CoA and pyruvate. In addition, the bacteria also has a full set (19) of genes that use acetyl coenzyme A to produce acetic acid, propionic acid and butyric acid, which has the function of increasing short-chain fatty acids in the intestines of fish.
[0024] 2.3 Identification of the strains of the present invention
[0025] The chromosome of Cetiella YC01 (NCBI, GenBank number: GCA_036324545.1) and 7 plasmids were subjected to evolutionary analysis with the reference genome of Cetiella (NK01, NCBI, GenBank number: GCA_024506395.1) with the greatest similarity on NCBI. The results showed that the 6th and 7th plasmids of Cetiella YC01 were most closely related to NK01 because they both came from freshwater fish ( Figure 2 and Figure 3 In addition, the average nucleotide identity (OrthoANIu) between the sixth and seventh plasmids (p6 and p7) and NK01 was 93.11, which was lower than 95%, because the ANI value between the same species is generally above 95% (Tables 1 and 2). Therefore, sequence analysis showed that there were differences in the sequences of Cetiella YC01 and NK01, and YC01 was a new species of the genus Cetiella.
[0026] Table 1 Average nucleotide similarity between plasmid 6 of Cetiella YC01 and Cetiella NK01
[0027] project value OrthoANIuvalue(%) 87.27 NK01tig00007 Length 64260 Cetop6 length 47940 Average alignment length 2213 NK01 coverage (%) 3.44 Ceto plasmid 7 coverage (%) 4.62
[0028] Table 2 Average nucleotide similarity between plasmid 7 of Cetiella YC01 and Cetiella NK01
[0029] project value OrthoANIuvalue(%) 93.11 NK01tig00007 Length 64,260 Cetop7 length 44,880 Average alignment length 10,461 NK01 coverage (%) 16.28 Ceto plasmid 7 coverage (%) 23.31 .
[0030] Example 2
[0031] This experiment was conducted at the pilot plant of the College of Oceanography of Ningbo University. A total of 12 70L yellow catfish culture experimental tanks were set up, of which 3 tanks were used as the control group and 3 were used as the control group for the control group. 7 CFU / mL, 10mL) was added to the group, 125mg / L total ammonia nitrogen was added to the three barrels as the ammonia stress group, and the last three barrels were simultaneously added with Cetiella YC01 (1×10 7 CFU / mL, 10mL) and 125mg / L total ammonia nitrogen as the Cephalosporin + ammonia stress group. Pelteobagrus fulvidraco fry were uniformly released, with 30 fry in each experimental bucket. Activation and addition of bacterial agent: The strain of preserved bacteria (lyophilized powder) was dissolved in sterile water, then spread on Columbia blood agar medium, incubated at 37℃ for 96 hours without oxygen for activation. After the bacterial lawn grew, pure bacteria were picked with an inoculation loop and inoculated in 10mL sterile water until the concentration was 1×10 7 CFU / mL(OD 600 Value = 1.0) was added to the aquaculture water. No food was allowed during the experiment. The experimental period was 7 days. After the experiment, the survival rate of the yellow catfish was counted and the blood ammonia content was measured. The study found that the addition of Bacillus cereus YC01 under high ammonia stress can significantly increase the survival rate of yellow catfish in a high ammonia environment (from 50% to 75%). Figure 4 A). And reduce the increase of blood ammonia caused by ammonia stress (from 1200μmol / L to 600μmol / L, Figure 4 B).
[0032] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.
Claims
1. A yellow catfish intestinal catfish-derived cetacean, characterized in that: The strain is the YC01 strain, classified and named Cetobacterium somerae, and was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 25, 2024, with the collection number GDMCC No: 64562.
2. A use of the catfish-derived Ceti bacillus from the intestine of yellow catfish as claimed in claim 1 in improving the ammonia tolerance of fish.
3. A use of the catfish-derived cetacean from the intestines of yellow catfish as claimed in claim 1 in the preparation of a probiotic additive for yellow catfish farming.