Vibrio marinus adhesivus LvS229 in the intestine of penaeid shrimp, probiotic preparation, feed and application

Abstract

The present invention belongs to the field of microorganisms, and particularly relates to a Vibrio adhesivibacter LvS229 from shrimp intestinal mud, a probiotic preparation, a feed and their applications. The present invention isolated and identified a strain of Tenacibaculum lutimaris LvS229 strain from the intestine of Litopenaeus vannamei that survived after being stressed by the semi-lethal concentration of nitrite for 72 hours. The strain was deposited at the China Center for Type Culture Collection on December 20, 2024, with the deposit number CCTCC NO: M20242874, and it can improve the tolerance of shrimp to high concentrations of nitrite and reduce the mortality rate of shrimp. The present invention also provides a probiotic preparation and a shrimp farming feed containing Tenacibaculum lutimaris the LvS229 strain.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and particularly relates to a Bacillus adhaerens LvS229 in shrimp intestine sea mud, a probiotic preparation, a feed and applications thereof. Background Art

[0002] Litopenaeus vannamei Penaeus vannamei , also known as whiteleg shrimp or Pacific white shrimp, belongs to the class Malacostraca, order Decapoda, family Penaeidae, and genus Litopenaeus. It has the advantages of fast growth rate, strong disease resistance, wide temperature and salinity tolerance ranges, and high protein content, and has become an important economic species for shrimp farming in China.

[0003] In recent years, with the rapid development of intensive shrimp farming at high density, the problem of excessive nitrite concentration in the farming process has occurred commonly. The main sources of nitrite include the input of excessive nitrogen sources (such as high-nitrogen feeds) and the imbalance of nitrification and denitrification processes in the farming system. In addition, excessive farming density, accumulation of excreta, and improper water quality management will further exacerbate the accumulation of nitrite in the water body.

[0004] High concentrations of nitrite have significant negative impacts on the organizational structure, growth and development, and immune defense of shrimp. Research shows that nitrite can accumulate in the intestine and hepatopancreas of shrimp, resulting in damage to their organizational structure. For example, the intestinal epithelial cells are arranged loosely, broken or even shed; the hepatopancreas shows phenomena such as damage to the connective tissue of hepatic tubules, infiltration of blood cells, and narrowing of the lumen. As a strong oxidant, nitrite can also oxidize hemocyanin to generate deoxyhemocyanin, resulting in the loss of oxygen-carrying function and tissue hypoxia, and ultimately leading to slow growth and decreased survival rate of shrimp. In addition to its toxic effect on shrimp itself, nitrite also affects the intestinal flora of shrimp. It is found that exposure to an environment with high concentrations of nitrite will reduce the richness and diversity of the intestinal flora of Litopenaeus vannamei, resulting in the disruption of the flora balance. At the same time, Photobacterium and Vibrio the relative abundances of opportunistic pathogens such as

[0005] Probiotic preparations have been widely used in aquaculture to improve the farming environment, enhance the immunity of aquatic animals, and promote the growth and development of animals by synthesizing beneficial metabolites. For example, in the farming of Litopenaeus vannamei, adding Lactobacillus , Enterococcus and other probiotics can enhance the activity of hemolymph cells of shrimp, improve the immune response of shrimp, and reduce the susceptibility to diseases. Currently, probiotics publicly used in aquaculture, such as Stutzerimonas sp. A12-2 (CN117866824A), Arthrobacter sp. DYS3-4 (CN104862260B), and Halomonassp. CYQ1-6-1 (CN104911130B), etc., are isolated from the environment and mainly reduce the stress pressure of nitrite on Litopenaeus vannamei by utilizing nitrite in the water body. However, most of the probiotics currently applied are heterologous strains. Although these probiotics can play beneficial roles in a short time, they may be recognized as "foreign invaders" by the host immune system, triggering immune responses or inflammatory reactions. Developing safer and more stable probiotics to improve the nitrite tolerance of shrimp during farming is a technical problem urgently to be solved in this field. There is no report yet on isolating probiotics from the in-situ intestine of Litopenaeus vannamei that can improve the nitrite tolerance of shrimp. Summary of the Invention

[0006] To solve the above technical problems, in the present invention, a strain of Marinobacter adhaerens was isolated and identified from the intestine of Litopenaeus vannamei that survived after being stressed by the 72-hour median lethal concentration of nitrite. Tenacibaculum lutimaris Strain LvS229, and it was verified that it can significantly improve the survival rate of shrimp under high-concentration nitrite conditions.

[0007] The present invention provides a Marinobacter adhaerens from the intestine of shrimp ( Tenacibaculum lutimaris ), which is named Tenacibaculum lutimaris LvS229, and was deposited at the China Center for Type Culture Collection on December 20, 2024, with the deposit number CCTCC NO: M20242874.

[0008] The present invention also provides the application of the Marinobacter adhaerens from the intestine of shrimp ( Tenacibaculum lutimaris ), which is used to improve the tolerance of shrimp to the nitrite in the aquaculture water body, or to improve the survival rate of shrimp in the aquaculture water body containing nitrite. The nitrite in the present invention refers to the concentration of nitrite in the aquaculture water body that is the 72-hour median lethal concentration of Litopenaeus vannamei.

[0009] Further, the shrimp is Litopenaeus vannamei.

[0010] Further, the concentration of the Marinobacter adhaerens from the intestine of shrimp ( Tenacibaculum lutimaris ) in the aquaculture water body ≥ 10 6 CFU / mL.

[0011] The present invention also provides a probiotic preparation, which contains the Marinobacter adhaerens from the intestine of shrimp ( Tenacibaculum lutimaris ).

[0012] Further, the probiotic preparation is used for shrimp farming.

[0013] The present invention also provides a feed for shrimp farming, which contains the Marinobacter adhaerens from the intestine of shrimp ( Tenacibaculum lutimaris ).

[0014] Compared with the prior art, the beneficial effects of the present invention at least include:

[0015] 1. The Tenacibaculum lutimaris LvS229 provided by the present invention is Tenacibaculum a new strain of the genus, which can improve the tolerance of shrimp to nitrite and reduce the mortality rate of shrimp.

[0016] 2. Tenacibaculum lutimaris LvS229 is the first probiotic isolated in situ from the intestine of Litopenaeus vannamei. Using it in shrimp farming can reduce the triggering of immune responses or inflammatory reactions and is safer and more stable. Description of the Drawings

[0017] Figure 1 Relative abundances of the genus after nitrite stress Tenacibaculum in the intestinal bacterial communities of surviving and dead shrimp.

[0018] Figure 2 is Tenacibaculum lutimaris a schematic diagram of the key metabolic pathways of LvS229.

[0019] Figure 3 is Tenacibaculum lutimaris the situation of LvS229 metabolizing nitrite.

[0020] Figure 4 is Tenacibaculum lutimaris the hemolytic property of LvS229; Figure 4 in which a is the hemolytic ring formed by a strain with β-hemolytic property on a blood agar medium containing defibrinated sheep blood, Figure 4 b in Tenacibaculum lutimaris is the colony formed by LvS229 on a blood agar medium containing defibrinated sheep blood.

[0021] Figure 5 is the effect on the survival rate of shrimp by adding Tenacibaculum lutimaris LvS229 to the shrimp farming water body for 7 consecutive days.

[0022] Figure 6 is Tenacibaculum lutimaris the sensitivity of LvS229 to four types of antibiotics. The diameter of the inhibition zone reflects the high or low sensitivity of the strain to antibiotics. Among them, AMP is ampicillin, C is chloramphenicol, CN is gentamicin, and TE is tetracycline.

[0023] Figure 7 is the effect on the survival rate of Litopenaeus vannamei under nitrite after adding Tenacibaculum lutimaris LvS229 to the farming water body. Detailed Embodiments

[0024] To make the objectives, technical solutions, and beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only illustrative descriptions of the specific embodiments of the present invention, intended to explain the present invention, and do not constitute a limitation to the present invention.

[0025] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values, and these ranges or values should be understood to include those close to these ranges.

[0026] Example 1: Tenacibaculum Enrichment in the intestinal flora of Litopenaeus vannamei that survived after nitrite stress

[0027] Litopenaeus vannamei (5.49 ± 1.20 cm) was acutely stressed with the 72-hour half-lethal concentration of nitrite. After 72 hours of stress, the intestinal tissues of dead and surviving shrimp were taken respectively, quickly frozen in liquid nitrogen and stored in an ultra-low temperature freezer at -80 °C. The total genomic DNA of the intestinal microbiota of the two groups of shrimp was extracted using a fecal genomic DNA extraction kit (Tiangen, China), and the DNA quality and concentration were evaluated by 1% agarose gel electrophoresis and Nanodrop spectrophotometer. The V3-V4 region of the bacterial 16S rRNA gene was amplified using the 341F / 806R primers. The PCR amplification system included: 15 μL of Phusion® High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA), 0.2 μM F / R primers, and 10 ng of DNA template, and was quantified to 50 µL volume with distilled water. The PCR amplification program was: pre-denaturation at 98 °C for 60 s; denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, extension at 72 °C for 30 s, for 30 cycles; final extension at 72 °C for 5 min. The purified PCR products were used to construct a 16S rRNA gene amplicon sequencing library and sequenced on the Illumina NovaSeq platform using the PE250 strategy to generate paired-end reads. The sequencing depth for each sample was 50,000 reads. The paired reads were merged according to the overlapping fragments using the FLASH software, and low-quality reads with a base quality lower than 20 and a proportion exceeding 30% were filtered out by fastp. The high-quality reads after quality control were processed for dereplication and denoising using QIIME2 to generate amplicon sequence variants (ASVs) with a sequence similarity of 100%. The abundance distribution of ASVs in different samples was statistically analyzed, and they were hierarchically classified and annotated based on the SILVA database. The results of the genus-level analysis showed that TenacibaculumGenus bacteria were significantly enriched in the intestinal bacterial community of surviving shrimp after nitrite stress (as Figure 1 shown).

[0028] Example 2: Isolation from the intestine of surviving Litopenaeus vannamei Tenacibaculum Genus strains

[0029] Dissect the surviving Litopenaeus vannamei under sterile conditions, and place its intestinal tissue in 1×PBS buffer. Use a sterile grinding rod to thoroughly grind the intestinal tissue to release the microorganisms therein. Dilute the grinding solution into a series of concentration gradients (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 ). Take 100 μL of the diluted solution from each gradient and spread it on Marine 2216E agar plates, and culture at 28 °C for 96 hours. After the colonies grow out, use a sterile inoculation loop to pick different-colored and -shaped single colonies, transfer them to a new Marine 2216E agar plate for isolation and purification, and obtain purified strains through continuous passage (7-8 generations). Pick a single colony as the template for PCR amplification, and use the universal primers 27F and 1492R for the amplification of the bacterial 16S rRNA gene. The PCR amplification system contains: 15 μL of Q5 2× Master Mix (NEB), 1 μL of F / R primers, 8 μL of sterile water, and a single colony. The PCR amplification program is: pre-denaturation at 98 °C for 30 s; denaturation at 98 °C for 10 s, annealing at 60 °C for 30 s, extension at 72 °C for 45 s, 30 cycles; final extension at 72 °C for 2 min. The amplified product is sent to Sangon Biotech Co., Ltd. for Sanger sequencing. Retrieve and align the 16S rRNA gene sequences obtained by sequencing in the RDP classifier and NCBI databases to identify the species of the isolated strains. A bacterium numbered LvS229 and belonging to Tenacibaculum Genus was selected for subsequent research.

[0030] Example 3: Tenacibaculum Whole-genome sequencing and species annotation of sp. LvS229

[0031] Inoculate the LvS229 strain into 2216E liquid medium and culture it at 28 °C and 180 rpm until OD 600Reached 0.5. The genomic DNA of bacteria was extracted using the SDS method, and its concentration and quality were evaluated using Qubit 4.0 (Thermo Scientific) and 1% agarose gel electrophoresis. The qualified DNA was used to construct a sequencing library and sequenced on the Illumina NovaSeq6000 platform (Novogene, China), and finally 2 Gb of sequencing data was obtained. The raw data was filtered using the fastp software to remove low-quality reads with a base quality lower than 20 and a proportion exceeding 30% to obtain high-quality data. The genome was assembled using the Megahit software, setting the k-mer parameters to 61, 71, 81, 91, 101, 111, and 121, and filtering out Contigs with a length less than 1 kb to construct Tenacibaculum a draft genome of sp. LvS229. The integrity and contamination of the genome were evaluated by CheckM, and the number of tRNAs and rRNAs in the genome was predicted using Prokka. Tenacibaculum The genome size of sp. LvS229 is 2.56 Mb, containing 41 Contigs. The genome integrity and contamination are 98.71% and 0.84% respectively, containing 45 tRNAs and 4 rRNA genes, and the GC content is 32.52%. Based on the whole-genome classification of the GTDB-TK database, the isolated strain was determined to be Tenacibaculum lutimaris , so this strain was named Tenacibaculum lutimaris LvS229.

[0032] Example 4: Tenacibaculum lutimaris Functional Genomic Analysis and Experimental Verification of LvS229

[0033] Based on the codons of prokaryotes, the Prodigal software was used to predict the complete open reading frames (ORFs) in the Tenacibaculum lutimaris genome of LvS229. A total of 2536 complete ORFs were predicted, and the nucleic acid sequences encoding proteins and the corresponding protein sequences were obtained. The protein sequences were aligned with the KEGG database using Diamond BLASTp to complete the functional annotation of the protein sequences encoded by the genome. The annotated genes were mapped to metabolic pathways to analyze the integrity of the metabolic pathways and the metabolic potential. The results of the genome analysis showed that Tenacibaculum lutimaris LvS229 has nirK and norB genes that can reduce toxic nitrite (NO2 - -) to non-toxic nitrous oxide (N2O) and excrete it outside the cell (as shown in Figure 2 ). To verify its nitrite metabolism ability, Tenacibaculum lutimarisLvS229 was inoculated into a liquid medium with 20 mg / L nitrite as the sole nitrogen source, and the nitrite concentration in the medium was measured every 24 hours. The results showed that this strain had strong nitrite metabolism ability and could metabolize more than 90% of the nitrite in the medium within 72 hours (as Figure 3 shown).

[0034] Example 5: Tenacibaculum lutimaris Other probiotic characteristics of LvS229

[0035] Non-hemolytic, non-biotoxic and high antibiotic sensitivity are important criteria for evaluating the safety of probiotics. To verify Tenacibaculum lutimaris the probiotic potential of LvS229, its safety was systematically evaluated. First, LvS229 was inoculated into 2216E liquid medium and cultured in a constant temperature shaker at 28 °C and 180 rpm for 24 hours to obtain a bacterial solution in the logarithmic growth phase. 5 μL of the bacterial solution was dropped onto the surface of a blood agar medium containing defibrinated sheep blood and cultured at 28 °C for 48 h to observe whether a hemolytic ring was formed. At the same time, a strain with β-hemolysis was selected as the positive control. Second, the LvS229 bacterial solution was centrifuged at 5000 rpm for 10 minutes, then resuspended with sterile seawater and added to the culture water of Litopenaeus vannamei to make the concentration of LvS229 in the water reach 10 6 CFU / mL. One-third of the seawater was replaced daily and fresh bacterial solution was supplemented for 7 consecutive days, and the death of the shrimp was observed at the same time. Finally, the LvS229 bacterial solution was evenly spread on a Marine 2216E agar plate, and antibiotic sensitivity tests were carried out using drug sensitivity test papers containing four different types of antibiotics, namely ampicillin (10 μg), chloramphenicol (30 μg), tetracycline (30 μg), and gentamicin (10 μg). The sensitivity of the LvS229 strain to the four types of antibiotics was evaluated according to the diameter of the inhibition zone. The results showed that no hemolytic ring was formed on the defibrinated sheep blood agar medium by LvS229, indicating that it did not have hemolytic property (as Figure 4 shown). In the shrimp culture experiment, no shrimp death was observed for 7 consecutive days, and all individuals grew normally and had good health conditions (as Figure 5 shown). The results of the antibiotic sensitivity test showed that LvS229 was highly sensitive to ampicillin, chloramphenicol and tetracycline, while it had strong resistance to gentamicin (as Figure 6 shown). It can be seen that Tenacibaculum lutimaris LvS229 of the present invention has probiotic properties and can be used to prepare probiotic preparations.

[0036] Example 6: Effect of adding Tenacibaculum lutimaris LvS229 on the survival rate of shrimp under nitrite exposure

[0037] Inoculate LvS229 into 2216E liquid medium and culture it in a constant temperature shaker at 28 °C and 180 rpm for 24 hours to obtain a bacterial solution in the logarithmic growth phase. Subsequently, centrifuge the bacterial solution at 5000 rpm for 10 min, discard the supernatant, and resuspend the bacterial pellet with sterile seawater. Randomly divide the prawns (9.71 ± 0.61 cm) into a control group and an LvS229 group, with three replicates in each group. Add the resuspended bacterial solution to the culture water of the prawns in the LvS229 group to make its concentration reach 10 6 CFU / mL. For the prawns in the control group, add an equal amount of sterile seawater. Replace one-third of the culture water every day, and supplement the LvS229 group with fresh bacterial solution to maintain the concentration of the LvS229 strain in the water. The control group is supplemented with an equal amount of sterile seawater. During the experiment, control the culture conditions as salinity 30‰, pH 7.5 - 8.0, temperature 28 ± 2 °C, and dissolved oxygen > 5 mg / L. Feed sterile bait three times a day, in the morning, middle, and evening, and the feeding amount is 5% of the prawn body weight. After 7 days of the experiment, transfer the prawns in the LvS229 group and the control group to fresh sterile seawater respectively, and add nitrite to the water of both groups to make its concentration reach the 72-hour median lethal concentration of the prawns. Observe and record the death conditions of the prawns in the LvS229 group and the control group every 12 hours. The results show that adding Tenacibaculum lutimaris LvS229 to the prawn culture water can significantly improve the survival rate of prawns under nitrite (as Figure 7 shown). The Tenacibaculum lutimaris bacterial cells of LvS229 of the present invention can also be mixed with ordinary prawn bait to prepare a prawn feed containing probiotics, which is added to the prawn culture water to improve the survival of prawns.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and do not constitute a limitation to the content of the present invention. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A Vibrio mytili strain LvS229 that adheres to shrimp intestine and sea mud, Tenacibaculum lutimaris characterized in that It was deposited at the China Center for Type Culture Collection on December 20, 2024, with the deposit number CCTCC NO: M20242874.

2. The application of Vibrio maritimus LvS229 adherent to the intestine of penaeid shrimp in penaeid shrimp farming according to claim 1, characterized in that, It is used to improve the tolerance of shrimp to nitrite in aquaculture water, or to improve the survival rate of shrimp in aquaculture water containing nitrite.

3. The application according to claim 2, wherein The shrimp is Litopenaeus vannamei.

4. The application according to claim 2, wherein The concentration of Vibrio maritimus LvS229 in the shrimp intestine in the aquaculture water body ≥ 10 6 CFU / mL.

5. A probiotic preparation, characterized in that, It contains Tenacibaculum lutimaris LvS229 from the intestine of the shrimp described in claim 1.

6. The probiotic preparation according to claim 5, wherein, The probiotic preparation is used for shrimp aquaculture.

7. A feed for prawn farming, characterized in that, It contains Tenacibaculum lutimaris LvS229 from the intestine of the shrimp described in claim 1.

8. The feed for prawn farming according to claim 7, characterized in that, The shrimp is Litopenaeus vannamei.

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