Prawn intestinal tract adhesive mud bacillus LvS229, probiotic preparation, feed and application

Abstract

The invention belongs to the field of microorganisms, and particularly relates to prawn intestinal tract adhesive marine mud bacillus LvS229, a probiotic preparation, a feed and application of the prawn intestinal tract adhesive marine mud bacillus LvS229. According to the invention, a Tenciaculum lutimaris LvS229 strain is separated and identified from intestinal tracts of litopenaeus vannamei surviving after stress of nitrite at half lethal concentration for 72 hours, the Tenciaculum lutimaris LvS229 strain is preserved in China Center for Type Culture Collection on December 20, 2024, the preservation number is CCTCC NO: M20242874, the tolerance of the litopenaeus vannamei to high-concentration nitrite can be improved, and the death rate of the litopenaeus vannamei can be reduced. The invention also provides a probiotic preparation containing the Tencibaculum lutimaris LvS229 strain, and a prawn culture feed containing the Tencibaculum lutimaris LvS229 strain.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and specifically relates to a shrimp intestinal marine mud sticking bacillus LvS229, a probiotic preparation, a feed and an application thereof. Background Art

[0002] Litopenaeus vannamei Litopenaeus vannamei ), also known as whiteleg shrimp or white-limbed shrimp, belongs to the class Crustacea, order Decapoda, family Penaeidae, and genus Penaeus. It has the advantages of fast growth, strong disease resistance, wide range of temperature and salt adaptability, and high protein content. It has become an important economic variety for shrimp farming in my country.

[0003] In recent years, with the rapid development of high-density intensive shrimp farming, the problem of excessive nitrite concentration in the farming process has become common. The main sources of nitrite include excessive nitrogen input (such as high-nitrogen feed) and the imbalance of nitrification and denitrification in the farming system. In addition, excessive farming density, accumulation of excrement and improper water quality management will further aggravate the accumulation of nitrite in the water.

[0004] High concentrations of nitrite have significant negative effects on the tissue structure, growth and development, and immune defense of shrimp. Studies have shown that nitrite can accumulate in the intestines and hepatopancreas of shrimp, causing damage to its tissue structure. For example, intestinal epithelial cells are loosely arranged, broken, or even fall off; the hepatopancreas shows damage to the connective tissue of the hepatic ducts, blood cell infiltration, and narrowing of the lumen. As a strong oxidant, nitrite can also oxidize hemocyanin to produce deoxyhemocyanin, resulting in loss of oxygen-carrying function and inducing tissue hypoxia, ultimately leading to slow growth and decreased survival rate of shrimp. In addition to being toxic to the shrimp itself, nitrite also affects the intestinal flora of shrimp. Studies have found that exposure to high concentrations of nitrite reduces the richness and diversity of the intestinal flora of Penaeus vannamei, leading to the destruction of the balance of the flora. At the same time, Photobacterium and Vibrio The increase in the relative abundance of opportunistic pathogens further increases the risk of disease infection.

[0005] Probiotic preparations have been widely used in aquaculture to improve the breeding environment, enhance the immunity of aquatic animals, and promote animal growth and development by synthesizing beneficial metabolites. For example, in the cultivation of Litopenaeus vannamei, adding Lactobacillus , Enterococcus Probiotics such as can enhance the activity of shrimp blood lymphocytes, improve the immune response of shrimp, and reduce disease susceptibility. Currently, probiotics publicly used in aquaculture, such as Stutzerimonas sp. A12-2 (CN117866824A), Arthrobacter sp. DYS3-4 (CN104862260B), and Halomonassp. CYQ1-6-1 (CN104911130B) and others are isolated from the environment, and mainly use nitrite in the water to reduce the nitrite stress on the vannamei shrimp. However, most of the probiotics currently used are heterologous strains. Although these probiotics can play a beneficial role in a short period of time, they may be recognized as "foreign invaders" by the host immune system, triggering immune responses or inflammatory responses. The development of safer and more stable probiotics to improve the nitrite tolerance of shrimp during aquaculture is a technical problem that needs to be solved in this field. At present, there is no report on the in situ isolation of probiotics from the intestine of vannamei shrimp that can improve the nitrite tolerance of shrimp. Summary of the invention

[0006] In order to solve the above technical problems, the present invention isolates and identifies a strain of Bacillus sphaeroides from the intestines of Penaeus vannamei that survived nitrite stress at a half-lethal concentration for 72 hours. Tenacibaculum lutimaris The LvS229 strain was verified to be able to significantly improve the survival rate of shrimp under high concentration nitrite conditions.

[0007] The present invention provides a shrimp intestinal marine mud sticking bacillus ( Tenacibaculum lutimaris ), named Tenacibaculum lutimaris LvS229 was deposited in 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 shrimp intestinal marine mud sticking bacteria ( Tenacibaculum lutimaris ) is used in shrimp farming to improve the tolerance of shrimp to nitrite in aquaculture water, or to improve the survival rate of shrimp in nitrite aquaculture water. The nitrite in the present invention refers to the nitrite concentration in the aquaculture water that is the half lethal concentration of Penaeus vannamei in 72 hours.

[0009] Furthermore, the prawn is Penaeus vannamei.

[0010] Furthermore, the shrimp intestinal mud sticking bacteria ( Tenacibaculum lutimaris ) concentration in aquaculture water ≥10 6 CFU / mL.

[0011] The present invention also provides a probiotic preparation, comprising the shrimp intestinal mud sticking bacillus ( Tenacibaculum lutimaris ).

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

[0013] The present invention also provides a feed for shrimp farming, comprising the shrimp intestinal tract Adhesibacterium marine mud ( Tenacibaculum lutimaris ).

[0014] Compared with the prior art, the beneficial effects of the present invention include at least: 1. The present invention provides Tenacibaculum lutimaris LvS229 is Tenacibaculum The new strain of the genus can improve the tolerance of shrimp to nitrite and reduce the mortality of shrimp.

[0015] 2. Tenacibaculum lutimaris LvS229 is the first probiotic isolated in situ from the intestine of Penaeus vannamei. It can be used in shrimp farming to reduce the triggering of immune or inflammatory responses and is safer and more stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 After nitrite stress Tenacibaculum Relative abundance of genera in the intestinal bacterial communities of living and dead shrimp.

[0017] Figure 2 for Tenacibaculum lutimaris Schematic diagram of the key metabolic pathways of LvS229.

[0018] Figure 3 for Tenacibaculum lutimaris The metabolism of nitrite by LvS229.

[0019] Figure 4 for Tenacibaculum lutimaris Hemolytic activity of LvS229; Figure 4 Figure a is a hemolytic ring formed by a β-hemolytic strain on blood agar medium containing defibrinated sheep blood. Figure 4 b is Tenacibaculum lutimaris Colonies of LvS229 formed on blood agar containing defibrinated sheep blood.

[0020] Figure 5 Add for 7 consecutive days Tenacibaculum lutimaris The effect of LvS229 on the survival rate of shrimp in shrimp farming water.

[0021] Figure 6 for Tenacibaculum lutimaris The sensitivity of LvS229 to four types of antibiotics. The diameter of the inhibition zone reflects the sensitivity of the strain to the antibiotics. AMP is ampicillin, C is chloramphenicol, CN is gentamicin, and TE is tetracycline.

[0022] Figure 7 Add to aquaculture water Tenacibaculum lutimaris Effect of LvS229 on the survival rate of Penaeus vannamei under nitrite. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and beneficial effects of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary descriptions of the specific embodiments of the present invention, and are intended to be used to explain the present invention, but do not constitute a limitation of the present invention.

[0024] The endpoints of ranges and any values ​​disclosed herein are not limited to the exact range or value, and these ranges or values ​​should be understood to include approximations to these ranges.

[0025] Embodiment 1: Tenacibaculum The genera are enriched in the intestinal flora of Penaeus vannamei surviving nitrite stress Litopenaeus vannamei (5.49 ± 1.20 cm) was acutely stressed with nitrite at a 72-hour half-lethal concentration. After 72 hours of stress, the intestinal tissues of dead and surviving shrimp were collected, quickly frozen in liquid nitrogen, and stored in a -80 °C ultra-low temperature freezer. 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 341F / 806R primers. The PCR amplification system included: 15 μL Phusion® High-Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA), 0.2 μM F / R primers, and 10 ng DNA template, which was quantified to 50 μL volume with distilled water. The PCR amplification program was as follows: 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, 30 cycles; final extension at 72 °C for 5 min. The purified PCR products were used to construct a sequencing library of 16S rRNA gene amplified fragments 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. FLASH software was used to merge paired reads based on overlapping fragments, and low-quality reads with a base quality of less than 20 and a ratio of more than 30% were filtered out using fastp. The high-quality reads after quality control were de-chimerized and denoised using QIIME2 to generate amplicon sequence variations (ASVs) with a sequence similarity of 100%. The abundance distribution of ASVs in different samples was statistically analyzed, and hierarchical classification annotations were performed based on the SILVA database. The results of genus-level analysis showed that Tenacibaculum Bacteria of the genus Pseudomonas were significantly enriched in the intestinal bacterial community of shrimp that survived nitrite stress (e.g.Figure 1 as shown).

[0026] Example 2: Isolation from the intestine of live Penaeus vannamei Tenacibaculum strains Surviving Penaeus vannamei were dissected under sterile conditions, and their intestinal tissues were taken and placed in 1× PBS buffer. A sterile grinding rod was used to fully grind the intestinal tissue to release the microorganisms in it. The grinding solution was diluted into a series of concentration gradients (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 ), 100 μL of each gradient dilution was spread on the Ocean 2216E agar plate and cultured at 28 ℃ for 96 hours. After the colonies grew, single colonies of different colors and morphologies were picked with a sterile inoculation loop and transferred to a new Ocean 2216E agar plate for separation and purification, and purified strains were obtained by continuous passage (7-8 generations). Single colonies were picked as templates for PCR amplification and amplified using universal primers 27F and 1492R of bacterial 16S rRNA gene. The PCR amplification system included: 15 μL Q5 2× Master Mix (NEB), 1 μL F / R primers, 8 μL sterile water and single colonies. The PCR amplification program was: 98 ℃ pre-denaturation for 30s; 98 ℃ denaturation for 10s, 60 ℃ annealing for 30s, 72 ℃ extension for 45s, 30 cycles; 72 ℃ final extension for 2 min. The amplified products were sent to Sangon Biotech Co., Ltd. for Sanger sequencing. The 16S rRNA gene sequences obtained by sequencing were searched and compared in the RDP classifier and NCBI databases, and the isolated strains were identified. Tenacibaculum Subsequent studies on bacteria of the genus were conducted.

[0027] Embodiment 3: Tenacibaculum Whole genome sequencing and species annotation of sp. LvS229 The LvS229 strain was inoculated into 2216E liquid medium and cultured at 28 °C and 180 rpm until OD 600Reached 0.5. The bacterial genomic DNA was extracted using the SDS method, and its concentration and quality were evaluated using Qubit 4.0 (Thermo Scientific) and 1% agarose gel electrophoresis. Qualified DNA was used to construct a sequencing library and sequenced on the Illumina NovaSeq6000 platform (Novo, China), ultimately obtaining 2 Gb of sequencing data. The raw data was filtered out using fastp software to remove low-quality reads with a base quality of less than 20 and a ratio of more than 30% to obtain high-quality data. The genome was assembled using Megahit software, with k-mer parameters set to 61, 71, 81, 91, 101, 111, and 121, and Contigs with a length of less than 1 kb were filtered out to construct Tenacibaculum The draft genome of sp. LvS229 was evaluated by CheckM for completeness and contamination, and Prokka was used to predict the number of tRNA and rRNA in the genome. Tenacibaculum The genome size of sp. LvS229 is 2.56 Mb, containing 41 contigs, with a genome integrity and contamination of 98.71% and 0.84%, respectively. It contains 45 tRNA and 4 rRNA genes, and a GC content of 32.52%. The whole genome classification based on the GTDB-TK database determined that the isolated strain was Tenacibaculum lutimaris Therefore, this strain was named Tenacibaculum lutimaris LvS229.

[0028] Embodiment 4: Tenacibaculum lutimaris Functional genomic analysis and experimental verification of LvS229 Based on the codons of prokaryotes, Prodigal software was used to Tenacibaculum lutimaris The complete open reading frames (ORFs) in the genome of LvS229 were predicted, and 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 compared with the KEGG database through Diamond BLASTp to complete the functional annotation of the protein sequences encoded by the genome. The annotated genes were mapped to the metabolic pathways to analyze the integrity and metabolic potential of the metabolic pathways. The results of the genome analysis are shown in Tenacibaculum lutimaris LvS229 has nirK and norB Genes that convert toxic nitrite (NO2 - ) is reduced to non-toxic nitrous oxide (N2O) and excreted from the cell (e.g. 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 concentration of nitrite in the medium was measured every 24 hours. The results showed that the strain had a strong nitrite metabolism ability and could metabolize more than 90% of the nitrite in the medium within 72 hours (e.g. Figure 3 as shown).

[0029] Embodiment 5: Tenacibaculum lutimaris Other probiotic properties of LvS229 Non-hemolytic, non-biological toxicity and high antibiotic sensitivity are important criteria for evaluating the safety of probiotics. Tenacibaculum lutimaris The probiotic potential of LvS229 and its safety were systematically evaluated. First, LvS229 was inoculated into 2216E liquid culture 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 added to the surface of the blood agar medium containing defibrinated sheep blood and cultured at 28 °C for 48 hours to observe whether a hemolytic ring was formed. At the same time, a strain with β-hemolytic activity was selected as a positive control. Secondly, the LvS229 bacterial solution was centrifuged at 5000 rpm for 10 minutes, then resuspended in sterile seawater and added to the Vannamei shrimp farming water to make the concentration of LvS229 in the water reach 10 6 CFU / mL. One-third of the seawater was replaced and fresh bacterial solution was added every day for 7 consecutive days, and the death of shrimp was observed at the same time. Finally, the LvS229 bacterial solution was evenly spread on the Marine 2216E agar plate, and an antibiotic sensitivity experiment was carried out using drug-sensitive paper containing four different types of antibiotics, namely ampicillin (10ug), chloramphenicol (30ug), tetracycline (30ug), and gentamicin (10ug). The sensitivity of the LvS229 strain to the four types of antibiotics was evaluated based on the diameter of the inhibition zone. The results showed that LvS229 did not form a hemolytic ring on the defibrinated sheep blood agar medium, indicating that it is not hemolytic (such as Figure 4 In the shrimp farming experiment, no shrimp deaths were observed for 7 consecutive days, and all individuals grew normally and were in good health (as shown in Figure 2). Figure 5 The results of antibiotic sensitivity test showed that LvS229 was highly sensitive to ampicillin, chloramphenicol and tetracycline, but highly resistant to gentamicin (as shown in Figure 6 As shown). It can be seen that the present invention Tenacibaculum lutimaris LvS229 has probiotic properties and can be used to prepare probiotic preparations.

[0030] Example 6: Addition Tenacibaculum lutimaris Effects of LvS229 on the survival rate of shrimp exposed to nitrite 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. Subsequently, the bacterial solution was centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the bacterial precipitate was resuspended in sterile seawater. The shrimp (9.71 ± 0.61 cm) were randomly divided into a control group and a LvS229 group, with three replicates in each group. The resuspended bacterial solution was added to the culture water of the LvS229 group shrimp to a concentration of 10 6 CFU / mL. The control group of shrimps were given an equal amount of sterile seawater. One-third of the culture water was replaced every day, and fresh bacterial liquid was added to the LvS229 group to maintain the concentration of LvS229 strain in the water. The control group was supplemented with an equal amount of sterile seawater. During the experiment, the culture conditions were controlled to be 30‰ salinity, pH 7.5-8.0, temperature 28 ± 2℃, and dissolved oxygen>5mg / L. Sterile bait was fed three times a day in the morning, noon and evening, and the feed amount was 5% of the shrimp body weight. After 7 days of the experiment, the shrimps in the LvS229 group and the control group were transferred to fresh sterile seawater, and nitrite was added to the water of the two groups to a concentration that reached the 72-hour half-lethal concentration of shrimp. The mortality of shrimps in the LvS229 group and the control group was observed and recorded every 12 hours. The results showed that adding nitrite to the shrimp culture water Tenacibaculum lutimaris LvS229 can significantly improve the survival rate of shrimp under nitrite (such as Figure 7 The present invention Tenacibaculum lutimaris The LvS229 bacteria can also be mixed with ordinary shrimp bait to prepare shrimp feed containing probiotics, which can be added to the shrimp breeding water to improve the survival of the shrimp.

[0031] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and do not constitute a limitation on the content of the present invention. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A shrimp intestinal sea mud sticking Bacillus LvS229, characterized in that: It was deposited in the China Center for Type Culture Collection on December 20, 2024, with the deposit number being CCTCC NO: M20242874.

2. The use of the shrimp intestinal mud sticking Bacillus LvS229 in shrimp farming as claimed in claim 1, characterized in that: Used to improve the tolerance of shrimp to nitrite in aquaculture water, or to improve the survival rate of shrimp in nitrite in aquaculture water.

3. The use according to claim 2, characterized in that The prawn is Penaeus vannamei.

4. The use according to claim 2, characterized in that The concentration of the shrimp intestinal mud sticky bacteria LvS229 in the aquaculture water is ≥10 6 CFU / mL.

5. A probiotic preparation, characterized in that: It comprises the shrimp intestinal marine mud sticking Bacillus LvS229 described in claim 1.

6. The probiotic preparation according to claim 5, characterized in that The probiotic preparation is used for shrimp farming.

7. A feed for shrimp farming, characterized in that: It comprises the shrimp intestinal marine mud sticking Bacillus LvS229 described in claim 1.

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

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