Marine organism envelope bacterium capable of producing TDA, probiotic preparation, feed and application

Abstract

The invention belongs to the field of microorganisms, and particularly relates to a marine organism envelope bacterium for producing TDA, a probiotic preparation, a feed and application. The invention provides a marine organism envelop bacterium capable of producing TDA, namely Epibacterium mobile 2-1-1, which is a new strain of Epibacterium, can be used for producing TDA, and has an obvious inhibition effect on Vibrio owensii ems001 (Vibrio owensii ems001), and the marine organism envelop bacterium is a marine organism envelop bacterium capable of producing TDA, namely Epibacterium mobile 2-1-1, which is a new strain of Epibacterium and can be used for producing TDA. The compound has the prospect of serving as a natural antibacterial agent, and pathogenic bacteria vibrio in aquaculture are effectively controlled. The invention also provides an application of the Epibacterium mobile 2-1-1 in the antibacterial and TDA production of the aquaculture. The invention also provides the application of the Epibacterium mobile 2-1-1 in the antibacterial and TDA production of the aquaculture. The invention also discloses a probiotic preparation containing the Epibacterium mobile 2-1-1, and a feed for aquaculture. The probiotic preparation comprises the Epibacterium mobile 2-1-1.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to marine biofilm bacteria producing TDA, a probiotic preparation, a feed and applications. Background Art

[0002] Acute hepatopancreatic necrosis disease (AHPND), commonly known as "stealing death disease" or "early death syndrome", is one of the most serious diseases in shrimp farming worldwide. AHPND is mainly caused by pathogenic Vibrio, which carries a plasmid (pVA1) containing genes encoding toxins PirA and PirB proteins. This toxic protein complex will cause tissue necrosis and functional failure of hepatopancreatic and intestinal cells, leading to a high mortality rate of Penaeus vannamei, causing serious economic losses to the shrimp farming industry. Among them, Vibrio erwinii ( Vibrio owensii ) is a pathogenic strain that causes AHPND in Penaeus vannamei.

[0003] In the field of aquaculture research, probiotics have been widely used to inhibit the growth of opportunistic pathogens and reduce the prevalence of viruses due to their environmental friendliness and high efficiency. Bacillus )、Lactobacillus( Lactobacillus ), Enterococcus spp. Enterococcus ), Pseudomonas ( Pseudomonas ) and other probiotics have been used in aquaculture. These probiotics can inhibit the growth of pathogenic Vibrio and even cause its death by secreting antibacterial substances such as antibiotics, peptides, and bacteriocins. TDA, the full name of which is tropodithietic acid, is a small broad-spectrum antibiotic produced by marine bacteria. Epibacterium There are reports of TDA production by bacteria of the genus.

[0004] Existing aquaculture probiotics mainly improve the immune response of Penaeus vannamei to reduce the host's susceptibility to pathogens. There are no probiotics that can directly inhibit the growth of Vibrio erwinii and be applied to aquaculture. Summary of the invention

[0005] In order to solve the above technical problems, the present invention has discovered a strain from marine biofilms that can inhibit the growth of Vibrio erwinii by secreting the antibiotic TDA. Epibacterium strains of the genus, providing a new path for the development of probiotics against pathogenic strains in aquaculture.

[0006] The present invention provides a TDA-producing marine biofilm bacterium named Microbacterium motile surface ( Epibacterium mobile) 2-1-1, deposited in China Center for Type Culture Collection on December 18, 2024, with the deposit number CCTCC NO: M 20242840.

[0007] The present invention also provides the TDA-producing marine biofilm bacteria for use in the antibacterial effect on aquaculture pathogenic bacteria.

[0008] Furthermore, the aquaculture is Penaeus vannamei farming, and the antibacterial is anti-Vibrio erwinia ( Vibrio owensii ).

[0009] The present invention also provides the use of the TDA-producing marine biofilm bacteria for producing TDA.

[0010] The present invention also provides a probiotic preparation comprising the TDA-producing marine biofilm bacteria.

[0011] Furthermore, the probiotic preparation is used for antibacterial purposes in aquaculture.

[0012] Furthermore, the aquaculture is Penaeus vannamei farming, and the antibacterial is anti-Vibrio erwinia ( Vibrio owensii ).

[0013] The present invention also provides a feed for aquaculture, comprising the TDA-producing marine biofilm bacteria.

[0014] Furthermore, the feed is Penaeus vannamei feed.

[0015] Compared with the prior art, the beneficial effects of the present invention include at least: 1. The motile surface microbacterium provided by the present invention Epibacterium mobile 2-1-1 is Epibacterium A novel strain of the genus that produces TDA and has been shown to be effective against AHPND Vibrio Vibrio owensii ems001 It has a significant inhibitory effect.

[0016] 2. Microbacterium spp. on the surface of sports Epibacterium mobile 2-1-1 has the potential to be used as a natural antimicrobial agent to effectively control pathogenic Vibrio in aquaculture, reduce the use of antibiotics, and help reduce antibiotic resistance in pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Bacillus motile Epibacterium mobile 2-1-1 and others Epibacterium Relatedness of strains; Figure 2 Bacillus motile Epibacterium mobile2-1-1 Distribution of TDA synthesis genes in the genome 1; Figure 3 Bacillus motile Epibacterium mobile 2-1-1 Distribution of TDA synthesis genes in the genome 2; Figure 4 Bacillus motile Epibacterium mobile 2-1-1 HPLC-MS / MS test results of synthetic TDA, Figure 4 A in the figure shows the change of [TDA + H]+ abundance over time in HPLC. Figure 4 In the figure, B is the ion detected at the peak; Figure 5 is the detected TDA ion fragment; Figure 6 is the fragmentation pattern of TDA in HPLC-MS analysis; Figure 7 Bacillus motile Epibacterium mobile 2-1-1 Pair Vibrio owensii The antibacterial effect of ems001. DETAILED DESCRIPTION

[0018] 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.

[0019] 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.

[0020] Example 1: Separation from marine biofilms Epibacterium Marine biofilms were collected from the surface of plastic debris in the subtidal zone of Maidao, Qingdao, Shandong, China (36°05' N, 120°43' E) using sterile cotton swabs. The collected samples were suspended in sterile seawater and immediately transferred to the laboratory. After vortexing for 15 min, the bacterial cells were resuspended in sterile marine broth 2216E medium and diluted into a series of gradients (10 0 , 10 -1 , 10 -2 , 10 -3 , 10 -4 and 10 -5). 100 μL of the dilution was spread on the Marine 2216E agar plate and incubated at 28 °C until colonies were visible. The grown single colonies were subcultured on the Marine 2216E agar plate for more than 8 generations, and then the single colonies were inoculated into the 2216E liquid culture medium. The bacterial liquid grown for 24 hours was collected by centrifuge (12000 rpm, 5min). After centrifugation, the supernatant was discarded, and the obtained bacterial precipitate was used to extract genomic DNA. Next, the universal primers of the 16S rRNA gene, 27F and 1492R, were used to PCR amplify the 16S rRNA gene fragment of the DNA. The PCR products were Sanger sequenced at Sangon Biotechnology Co., Ltd. (Shanghai, China) to obtain the full-length 16S rRNA gene, and then the 16SrRNA gene was Blastn compared and analyzed on the NCBI website to determine the classification of each strain, and the strain numbered 2-1-1 was selected. Epibacterium Conduct follow-up experiments.

[0021] Embodiment 2: Epibacterium mobile 2-1-1 Whole-Genome Sequencing The above obtained Epibacterium Tianjin Novogene Bioinformatics Technology Co., Ltd. performed genomic DNA extraction and next-generation sequencing. Next-generation sequencing was performed on the Illumina NovaSeq 6000 platform, and 2 Gb of offline data was finally obtained. The offline data was removed using NGS QC Toolkit (v2.0) with a low-quality base (Phred Quality Score < 20) content greater than 30% of the reads. The offline data after quality control was assembled using Megahit (v1.2.9) with clean reads of k-mers of 61, 71, 81, 91, 101, 111, and 121, and contigs with bases less than 2 kb were removed after assembly to obtain Epibacterium mobile The completeness and contamination of the genome were assessed using CheckM (v1.1.2), and the number of tRNA and rRNA in the genome was predicted using Prokka (v1.13). According to the Genome Taxonomy database, GTDB-TK (v1.3.0) was used to Epibacterium mobileThe genome of 2-1-1 was annotated for species. Based on the codons of prokaryotes, Prodigal (v2.6.3) was used to predict the complete ORF of all genomes, output the nucleic acid sequences involved in protein coding in the genome and the encoded protein sequences, and BLASTp was used to compare the output protein sequences with the sequences in the KEGG protein database, and the results with the highest alignment for each sequence were returned, thereby completing the annotation of the protein sequences encoded by the genome. The genome of 2-1-1 contains 55 contigs, with a size of 4.46 Mb, a GC content of 59.10%, a completeness and contamination of 99.47% and 0.18%, respectively, and the numbers of tRNA, rRNA genes and ORFs are 51, 3 and 4210, respectively. The whole genome classification based on the GTDB-TK database determined that the isolated strain was a microbacterium motile. Epibacterium mobile , which was numbered 2-1-1 in this study, so this strain was named Microbacterium motilum Epibacterium mobile 2-1-1.

[0022] Example 3: Phylogenetic evolutionary relationships Download 6 reported Epibacterium The complete genome of the genus was used as a reference to Epibacterium mobile 2-1-1 Construction of phylogenetic tree (such as Figure 1 ). The 16S rRNA sequences of the above genomes were extracted using Barrnap (v0.9), and these sequences were aligned with ClustalW and MEGA (v7.0) was used to construct a phylogenetic tree based on the maximum likelihood method. Vibrio owensii The 16S rRNA sequence of ems001 is an outgroup. The phylogenetic tree construction results show that all Epibacterium Clustered into the same branch. 2-1-1 and Epibacterium sp. MM17-32 is in the same monophyletic clade with a Bootstrap value of 94%. Epibacterium mobile This is consistent with the taxonomic results in Example 2, and 2-1-1 is determined to be Epibacterium A new strain of the genus.

[0023] Embodiment 4: Epibacterium mobile Analysis of TDA synthesis-related genes in 2-1-1 and HPLC-MS / MS detection of antibacterial compound TDA According to the KEGG results of 2-1-1 in Example 2, Figures 2 - 3As shown in the figure, the unit of gene fragment size is bp. Two slashes indicate that the gene is located in different contigs. Genes without color markings represent genes that are not involved in the synthesis of TDA. 16 important genes related to TDA synthesis were retrieved, including katG , amiE , paaK , paaA , paaB , paaC , paaD , paaE , paaG , paaZ , tdaA , tdaB , tdaC , tdaD , tdaE and tdaF , located in 7 Contigs. paaG and amiE The copy number of the remaining genes is 1.

[0024] High performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used to detect whether 2-1-1 could synthesize TDA. After the strain was statically cultured at 28°C for 4 days, it was transferred to a 500 mL conical flask, an equal volume of acidified ethyl acetate (0.1% formic acid) was added, and ultrasonic vibration was performed for 20 min. It was extracted three times with acidified ethyl acetate, and the organic phase was retained and concentrated under reduced pressure. The concentrated organic phase was dissolved in 2 mL of methanol, treated with ultrasonic vibration, and centrifuged at 8000 rpm for 5 min. The supernatant was filtered through a 0.22 μm organic nylon membrane, and the final filtrate was collected. The compounds were separated on a C18 column (100 mm × 2.1 mm; particle size: 1.8 μm) with acetonitrile (solvent A) and 0.1% formic acid solution (solvent B) as the mobile phase, and the column temperature was maintained at 35°C. Gradient elution was applied with a flow rate of 0.3 mL / min and a total time of 32 min. The proportion of solvent A was 10% at the beginning, increased to 50% at 10 min, increased to 90% at 15 min and maintained until 25 min, and then decreased back to 10% between 25.5 and 32 min. The injection volume was 1 μL, and the electrospray mass spectrometry data were recorded in the positive ion (ESI+) mode with a mass range of m / z 100 to 1700. The capillary voltage and cone voltage were set to 4 kV and 100 V, respectively. The sheath gas heater was 350 °C, the sheath gas flow was 11 L / min, the gas flow was 5 L / min, and the gas temperature was 350 °C. Collision induced dissociation (CID) was performed using collision energies of 10, 20, 40, and 60 eV, with argon as the collision gas. The raw data were imported into Agilent Masshunter Qualitative Analysis software, and the identification workflow of unknown compounds was established using the software wizard settings and method templates. In the full scan mass spectrum, the protonated molecules [M + H] of the compounds were detected in the positive ion mode. + The identities of these unique compounds were further confirmed by accurate mass determination of the precursor ions and their corresponding mass spectral fragment ions. An ion with m / z 212.9671 was detected at 7.5 min, which corresponds to the protonated molecule of TDA [TDA + H] + (like Figure 4 as shown). Figure 4 A is [TDA + H] in HPLC + Changes in abundance over time. Figure 4B is the ion detected at the peak. Figure 5 is the detected TDA ion fragment. [TDA + H] + The abundance is 2.92 × 10 6 , and 6 TDA ion fragments were detected (such as Figure 6 ). The precursor ion of 212.9671 m / z generates product ions including m / z 194.9561, 166.9608, 150.9660, 138.9657, and 110.9886. This fragmentation pattern indicates that the protonated molecule undergoes a dehydration reaction [M + H - H2O] + , generating a stable carbocation with m / z 194.9561. The subsequent loss of 28 Da from the carbocation represents the loss of a neutral CO molecule, forming the Tropodithietane cation with m / z 166.9608 [M + H - H2O - CO] + Further loss of CO (28 Da) generates the six-membered benzo-dithietane cation at m / z 138.9657. Subsequent reduction and loss of a hydrogen atom form the 2-Mercapto-2, 4-cyclopentadien-1-yl methyl cation at m / z 110.9886. The product ion at m / z 150.9960 is the CID fragmentation ion of TDA.

[0025] Embodiment 5: Epibacterium mobile 2-1-1 Pair Vibrio owensii Antibacterial experiment of ems001 Will Epibacterium mobile 2-1-1 Cultivate the culture medium in 2216E liquid medium at 28°C for 4 days. Sterilize the 2216 solid agar medium, cool it to 45-50°C, and then add the culture medium grown in 2216E liquid medium for 24 hours at a volume ratio of 1:100. Vibrio owensii ems001 bacterial solution. Vibrio owensii A sterile hole was made in the center of the 2216 solid agar culture plate of ems001 using a sterile hole puncher. 20 μL of 2-1-1 culture solution was added to the hole as the treatment group, and the same volume of 2216 marine culture medium was added to the hole as the control group. After culturing for 24 h, the formation of the inhibition zone was observed. The antibacterial experiment showed that 2-1-1 could inhibit Vibrio owensii ems001, and a clear inhibition zone with a diameter of about 2 cm was observed (e.g. Figure 7 The results show that the metabolites of strain 2-1-1 can inhibit pathogenic bacteria Vibrio owensii growth.

[0026] In summary, the HPLC-MS / MS test results showed that the strain Epibacterium mobile 2-1-1 can synthesize the antibacterial compound TDA and inhibit pathogenic strains Vibrio owensii The discovery shows that Epibacterium mobile The prospect of 2-1-1 as a potential natural antimicrobial agent can effectively inhibit pathogenic Vibrio in aquaculture without relying on chemical drugs, reduce the use of antibiotics, and help reduce the drug resistance of pathogens.

[0027] 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 TDA-producing marine biofilm bacterium, characterized in that: Microbacterium motilum Epibacterium mobile 2-1-1, deposited in China Center for Type Culture Collection on December 18, 2024, with the deposit number CCTCC NO: M 20242840.

2. Antibacterial application of the TDA-producing marine biofilm bacteria as claimed in claim 1 in aquaculture.

3. The antibacterial use according to claim 2, characterized in that: The aquaculture is Penaeus vannamei farming, and the antibacterial is anti-Vibrio erwinii Vibrio owensii .

4. Use of the TDA-producing marine biofilm bacteria according to claim 1 for producing TDA.

5. A probiotic preparation, characterized in that: The method comprises the TDA-producing marine biofilm bacteria according to claim 1.

6. The probiotic preparation according to claim 5, characterized in that The probiotic preparation is used for the antibacterial effect on aquaculture pathogenic bacteria.

7. The probiotic preparation according to claim 6, characterized in that The aquaculture is Penaeus vannamei farming, and the antibacterial is anti-Vibrio erwinii Vibrio owensii .

8. A feed for aquaculture, characterized in that: The method comprises the TDA-producing marine biofilm bacteria according to claim 1.

9. A feed for aquaculture according to claim 8, characterized in that: The feed is Penaeus vannamei feed.

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