Vibrio-resistant probiotic bacteria for coral health farming and application

Abstract

The present application belongs to the technical field of probiotic agents, and discloses a kind of anti-vibrio pathogenic probiotic for coral healthy cultivation and application, the anti-vibrio pathogenic probiotic for coral healthy cultivation is alternation bacterium strain ZD22-1, preserved in Guangdong Microbial Preservation Center GDMCC on May 8, 2021, address is Guangzhou, Guangdong Province, First Field Road, 100, Building 59, 5th Floor, Guangdong Institute of Microbiology, and the preservation strain number is GDMCC No:61647.The coral probiotic Alteromonas sp.ZD22-1 screened in the present application has antagonistic effect on vibrio pathogen of coral and has antioxidant potential.The present application develops marine microecological preparation-anti-vibrio pathogenic probiotic, which can effectively treat coral diseases and albinism caused by vibrio pathogen, improve coral immunity and disease resistance, and has broad application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of probiotic agents, and particularly relates to a Vibrio pathogenic probiotic for healthy cultivation of corals. BACKGROUND

[0002] Currently, ornamental corals, sea anemones and other ornamental fish organisms are sensitive to fluctuations in seawater environment and pathogenic Vibrio, which can easily lead to coral pathogenesis and cause a large number of corals to bleach and ornamental fish organisms to die. In view of the bleaching and disease problems of marine cnidarians, DMSP metabolites and active microorganisms thereof exhibit strong antibacterial and stress resistance potential, and many reports have appeared in recent years and have attracted much attention. It is a major development trend of marine microecological preparations in the future, and the key points are briefly summarized as follows: Since most marine ornamental cnidarians do not have the same movement ability as other higher animals, in order to survive, they have obtained a special mechanism for resisting environmental changes, bleaching and disease and other adversity in long-term evolution, i.e. DMSP metabolic active substances, including DMS, AA (acrylic acid), DMSO, TDA and other unknown compounds, and active microorganisms thereof play an important role in resisting bleaching, antibacterial properties, acquired immunity and abiotic stress. There is no microecological preparation based on DMSP metabolic active substances in the marine aquarium market. Methyl sulfide is an important carbon source and sulfur source for microorganisms, which can control and regulate microbial population and biomass through carbon source and sulfur source, and also has antibacterial properties and activates chemical defense. Acrylic acid is mainly adsorbed on the mucus layer, and research has confirmed that the mucus layer is a clear sterile zone. In addition, DMS and TDA produced by degradation of DMSP have antibacterial and antiviral activity in water. 50% to 80% of DMSP / DMS in the ocean is converted through bacterial metabolic processes. DMSP active microorganisms not only have outstanding contributions to enhancing the antioxidant protection and antibacterial properties of coral host cells, but also participate in the biochemical cycles of sulfur and nitrogen elements in marine organisms, as well as the synthesis of essential amino acids, coenzymes, vitamins and glutathione (GST) and the like. The isolated methyl sulfide active microorganisms are relatively few, and may only account for a small part of marine microorganisms, and have considerable utilization potential in the future.

[0003] Therefore, based on DMSP metabolic active microorganisms, strains with antibacterial activity against Vibrio are screened, and a microecological preparation is prepared, which will help to maintain the health of marine ornamental corals and improve the vitality of organisms by additionally supplementing the corresponding active substances and active bacterial agents. There is no microecological preparation based on DMSP metabolic active substances in the marine aquarium market.

[0004] Through the above analysis, the problems and defects of the prior art are that there is no microecological preparation based on DMSP metabolic active substances in the marine aquarium market. SUMMARY

[0005] In view of the problems existing in the prior art, the present application provides a Vibrio pathogenic probiotic for coral healthy aquaculture and application.

[0006] The present application is achieved in that the Vibrio pathogenic probiotic for coral healthy aquaculture is Alteromonas strain ZD22-1, which is a probiotic isolated from the tissue of Chinese clavularia, and was preserved in Guangdong Microbial Preservation Center GDMCC on May 8, 2021, at address No. 59, Building 5, Institute of Microbiology of Guangdong, 100, Martyrs' Road, Guangzhou, Guangdong, with preservation number GDMCC No: 61647.

[0007] Further, the Vibrio pathogenic probiotic for coral healthy aquaculture is a marine DMSP active bacterial group obtained by culturing in a medium with dimethyl sulfoxide propionate as the only carbon source.

[0008] Further, the Vibrio pathogenic probiotic for coral healthy aquaculture has a suitable culture temperature of 25-28℃ and a salinity of 30-35‰.

[0009] Further, the Vibrio pathogenic probiotic for coral healthy aquaculture is identified as Alteromonas sp. through PCR amplification and sequencing of 16S rDNA gene primers 27F / 1492R and microbial morphology.

[0010] Further, under the condition of circulating seawater culture, the half-life period of Alteromonas sp. ZD22-1 bacterial cells is 13 days.

[0011] Further, the Vibrio pathogenic probiotic for coral healthy aquaculture has an antagonistic effect on Vibrio pathogens of corals. It can be applied in the aquaculture of seawater corals, fish, shrimp, and shellfish, and has a treatment and prevention effect on diseases caused by pathogenic Vibrio.

[0012] Further, the Vibrio pathogenic probiotic for coral healthy aquaculture has high catalase activity and certain antioxidant potential. It can be applied in the aquaculture of seawater corals, fish, shrimp, and shellfish, and has a significant effect on treating acute stress reactions caused by the environment, such as coral bleaching and abnormal metabolism of other marine organisms caused by high temperature, pH change, salinity change, and light change.

[0013] Furthermore, the genome of the probiotic strain for coral health cultivation that is resistant to Vibrio pathogens contains multiple functional genes related to sulfur or nitrogen cycles, including DmdA, dddD, dddL, nifH, nirK, and dddP. PCR amplification results are used to verify the functions of the strain.

[0014] Another object of the present invention is to provide an anti-Vibrio pathogen probiotic agent using the aforementioned anti-Vibrio pathogen probiotic for healthy coral aquaculture, wherein the agent consists of Alteromonas sp. ZD22-1 cells at a final concentration of not less than 1×10⁻⁶. 12 Zooxanthindella type D cells / mL, final concentration not less than 1×10⁻⁶ 8 Composition of DMSP / DMS / AA, vitamins, amino acids, trace elements, CaCl2, and artificial seawater per cell / mL.

[0015] Another object of the present invention is to provide a method of using the aforementioned anti-Vibrio pathogen probiotic agent, wherein the method of using the anti-Vibrio pathogen probiotic agent includes:

[0016] Add 10 mL of bacterial solution per 100 L of water to ensure a total active microbial community count ≥ 5 × 10⁻⁶. 10 / L; Add the bacterial agent every 10-15 days to keep the number of bacteria in the water stable; store at low temperature of 4-8℃; shelf life of 6 months; shake well before use to ensure even distribution of bacteria.

[0017] Based on the above technical solutions and the technical problems solved, please analyze the advantages and positive effects of the technical solution to be protected by this invention from the following aspects:

[0018] First, addressing the technical problems existing in the prior art and the difficulty in solving them, this paper closely analyzes, in conjunction with the technical solution to be protected by this invention and the results and data obtained during the research and development process, how the technical solution of this invention solves the technical problems, and the inventive technical effects brought about by solving these problems. The specific description is as follows:

[0019] The present application utilizes microbiology and molecular biology technology to isolate and identify marine DMSP metabolic active bacteria, and according to the anti-Vibrio and metabolic traits, a coral probiotic strain with anti-Vibrio is screened, and belongs to the field of probiotic agents; the screened coral probiotic strain, Alteromonas sp. ZD22-1, has a strain preservation number of GDMCC61647, and the bacterial antagonism experiment proves that Alteromonas sp. ZD22-1 has antagonistic effect on Vibrio pathogen of corals and has certain antioxidant potential. Under the condition of circulating seawater culture, the half-life period of Alteromonas sp. ZD22-1 is 13 days. The marine micro-ecological preparation, anti-Vibrio pathogen probiotic, is developed, and the preparation formula components include Alteromonas sp. ZD22-1, D-type chlorella, DMSP / DMS / AA, vitamins, amino acids, trace elements, CaCl2, artificial seawater and the like. The present application can effectively treat coral diseases and whitening conditions caused by Vibrio pathogens, improve the immunity and disease resistance of corals, and is expected to be widely used in ecological breeding and healthy breeding of ornamental corals.

[0020] Secondly, the technical solution is regarded as a whole or from the perspective of the product, the technical solution to be protected by the present application has technical effects and advantages, which are described as follows:

[0021] The screened coral probiotic Alteromonas sp. ZD22-1 of the present application has antagonistic effect on Vibrio pathogen of corals and has certain antioxidant potential through bacterial antagonism experiment. Meanwhile, the present application develops the marine micro-ecological preparation, anti-Vibrio pathogen probiotic, which can effectively treat coral diseases and whitening conditions caused by Vibrio pathogens, improve the immunity and disease resistance of corals, and has broad application prospect.

[0022] The anti-Vibrio pathogen probiotic and the preparation thereof for coral healthy breeding provided by the present application can effectively treat coral diseases and whitening conditions caused by Vibrio pathogens, improve the immunity and disease resistance of corals, and can also be used in ecological healthy breeding of other marine organisms, including fish, shellfish, snails and shrimp, and the like, and has the function of treating Vibrio diseases.

[0023] Thirdly, the creativity of the claims of the present application is also embodied in the following aspects:

[0024] (1) The expected income and commercial value of the technical solution of the present application after transformation are as follows:

[0025] The application can be converted into a commercialized probiotic agent for coral culture or aquaculture, and has high commercial value.

[0026] (2) The technical scheme of the application fills the technical blank in the industry at home and abroad:

[0027] There is no specific product for the treatment of Vibrio disease of corals on the domestic market at present. The anti-Vibrio probiotic agent converted by the technical scheme of the application for treating coral diseases caused by pathogenic Vibrio will have independent intellectual property rights and can fill the blank of disease control technology in coral culture in China.

[0028] (3) The technical scheme of the application solves the technical problems that people have long been eager to solve but have failed to obtain success:

[0029] Due to the weak automatic regulation ability of the micro-seawater circulation culture ecosystem, the cultured organisms are easily infected by pathogenic Vibrio, resulting in various diseases. Disease has been a big problem that is difficult to overcome in the feeding of ornamental corals and other cnidarians, and the general "sea people" are deeply troubled by it, often paying heavy economic and energy costs. The anti-Vibrio pathogen probiotic for coral health culture of the application solves the technical problems that people have long been eager to solve but have failed to obtain success, such as high cost and difficulty in treating Vibrio disease in coral culture.

[0030] (4) The technical scheme of the application overcomes technical prejudice:

[0031] The application develops the corresponding marine micro-ecological preparation from the deep biological problems of "albinism" and "disease" of marine ornamental organisms (coral, sea anemone and other cnidarians), and overcomes the technical prejudice of difficulty in treating Vibrio disease in the culture of marine ornamental organisms. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical scheme of the embodiments of the application, the drawings required to be used in the embodiments of the application will be briefly introduced as follows. Obviously, the drawings described below are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0033] Figure 1 is a schematic diagram of the antagonistic experiment results of Alteromonas sp. strain ZD22-1 and pathogenic Vibrio provided by the embodiments of the application.

[0034] Figure 2 is a vitality cycle determination diagram of Alteromonas sp. ZD22-1 provided by the embodiments of the application. DETAILED DESCRIPTION

[0035] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and not to limit the present application.

[0036] In view of the problems in the prior art, the present application provides a Vibrio pathogenic probiotic for coral health cultivation and application, which will be described in detail below with reference to the accompanying drawings.

[0037] I. Explanation of embodiments. In order to enable those skilled in the art to fully understand how the present application is specifically implemented, this part is an explanation of the embodiments of the technical solutions claimed.

[0038] Embodiment 1

[0039] The present application utilizes microbiology and molecular biology techniques to isolate and identify marine DMSP metabolically active bacteria, and according to the anti-Vibrio and metabolic properties, a coral probiotic strain with anti-Vibrio is screened, which belongs to the field of probiotic agents. The screened coral probiotic, Alteromonas sp. ZD22-1, strain accession number: GDMCC61647, has been confirmed by bacterial antagonism experiments that Alteromonas sp. ZD22-1 has antagonistic effect on Vibrio pathogen of coral and has certain antioxidant potential. Under the condition of circulating seawater culture, the half-life period of Alteromonas sp. ZD22-1 is 13 days. A marine micro-ecological preparation, anti-Vibrio pathogenic probiotic, is developed, and the preparation formula includes Alteromonas sp. ZD22-1, D-type zooxanthellae, DMSP / DMS / AA, vitamins, amino acids, trace elements, CaCl2, artificial seawater, etc. The present application can effectively treat coral diseases and albinism caused by Vibrio pathogen, improve the immunity and disease resistance of coral, and is expected to be widely used in ecological cultivation and health cultivation of ornamental coral.

[0040] The probiotic Alteromonas sp. ZD22-1 isolated from the tissue of Chinese encrusting corals is preserved in Guangdong Microbial Culture Collection Center (GDMCC) on May 8, 2021, and the address is Guangdong Institute of Microbiology, No. 59 Building, 5th Floor, Guangzhou Institute of Microbiology, Guangzhou, Guangdong Province, China, and the preservation strain number is GDMCC No: 61647.

[0041] The Alteromonas sp. ZD22-1 provided by the embodiment of the present application is a kind of marine DMSP active bacteria group obtained by separation and culture through a culture medium with dimethyl sulfonyl propionate (DMSP) as the only carbon source; the suitable culture temperature is 25-28 DEG C, and the salinity is 30-35 ‰.

[0042] The PCR amplification and sequencing of 16S rDNA gene primers 27F / 1492R, combined with microbial morphological identification, are Alteromonas sp.

[0043] The antagonistic experiment of the bacteria proves that the strain has antagonistic effect on the vibrio pathogen of corals.

[0044] The detection of catalase content shows that the strain has high catalase activity and certain antioxidant potential.

[0045] The PCR identification of functional genes shows that the strain genome contains multiple functional genes related to sulfur cycle or nitrogen cycle, including DmdA, dddD, dddL, nifH, nirK and dddP genes, and the PCR amplification results thereof are used to test the functions of the strain.

[0046] Under the condition of circulating seawater culture, the half-life period of the Alteromonas sp. ZD22-1 strain is 13 days.

[0047] The marine microecological preparation, i.e., the anti-vibrio pathogen probiotic, is developed based on the strain, and the preparation formula components include the Alteromonas sp. ZD22-1 strain, and the final concentration is not less than 1×10 12 cells / mL, the chlorella D type, the final concentration is not less than 1×10 8 cells / mL, DMSP / DMS / AA, vitamins, amino acids, trace elements, CaCl2, artificial seawater and the like.

[0048] The anti-vibrio pathogen probiotic and the preparation thereof for coral health cultivation provided by the embodiment of the present application can effectively treat the coral diseases and whitening conditions caused by the vibrio pathogen, improve the immune function and disease resistance of the coral, and can also be used in ecological health cultivation of other marine organisms, including fish, shellfish, snails and shrimp in the field of aquatic cultivation, and has the function of treating vibrio diseases.

[0049] The preparation method of the preparation is as follows: (1) the use method is that 10 mL of the bacterial liquid is added into 100 L of water body, so that the total number of active microbial flora is greater than or equal to 5×10 10(1) Storage: The product should be stored at 4-8°C; (2) Shelf life: 6 months; (3) Use: Add the product to the water body once every 10-15 days to keep the number of bacteria stable; (4) Shake well before use to ensure uniform distribution of the bacteria.

[0050] Example 2: Screening and identification of DMSP-active bacteria

[0051] 1. Preparation of the base medium

[0052] Table 1. Medium formula

[0053]

[0054] The above base medium was placed in a sterilization pot for sterilization treatment, and a 90 mm culture dish was used for pouring plate preparation.

[0055] 2. Coral samples

[0056] Tissue was extracted from the coral (Pocillopora damicornis), and the tissue was ground into a coral tissue slurry sample. 5 mL of the sample was taken in a conical flask containing the culture solution for enrichment culture and was labeled. 5 mL of seawater sample was taken from the coral culture pool in a conical flask containing the culture solution for enrichment culture and was labeled.

[0057] 3. Isolation and culture of DMSP-active bacteria

[0058] The enrichment culture solution was diluted and plated from the skeleton, tissue, and seawater, respectively, and the dilution factor was selected to be 10-3-10-5. Two solid culture media were plated for each concentration, and the numbers were recorded (all operations must be performed in a clean bench). After sealing with a sealing film, the plates were inverted and further cultured in a 28°C incubator for about 2 days. The formation and growth of colonies on the plates were observed, and different colony characteristics were classified and selected. Then, the colonies were inoculated using an inoculation needle to obtain first-generation purified bacteria, and the numbers of each bacterium were recorded. The bacteria were further cultured in a 28°C incubator for about 2 days. Single colonies were selected from the first-generation bacteria for inoculation to obtain second-generation purified bacteria, and the numbers were recorded. The bacteria were further cultured in a 28°C incubator for about 2 days. Single colonies were selected from the second-generation bacteria for inoculation to obtain third-generation purified bacteria, and the numbers were recorded.

[0059] 4. Bacterial strain preservation

[0060] Prepare 2216E liquid medium and sterilize. Take 5 mL 2216E liquid medium in a sterile 10 mL centrifuge tube, take the third generation of purified bacteria into the centrifuge tube, and record the number. After sealing with sealing film, perform enrichment culture at 28°C in a shaker for about 2 days. Add 300 μL of glycerol to a sterile 1 mL centrifuge tube, then add 500 μL of the bacterial solution after enrichment culture, and record the number. After sealing with sealing film, store at -80°C. The remaining bacterial solution is used for subsequent experiments.

[0061] The DMSP active bacteria obtained by the above method are subjected to 16s gene species identification, and the 16S rDNA fragment of the bacteria is subjected to PCR amplification and sequencing using specific primers 27F / 1492R. Based on the sequencing results and morphological analysis, the strain is finally identified as Alteromonas sp. ZD22-1, and the strain is preserved in the Guangdong Microbial Culture Collection Center.

[0062] The 16s sequence (see SEQ ID NO: 9) is as follows:

[0063]

[0064] Example 3: PCR identification of functional genes of Alteromonas sp. ZD22-1

[0065] The DNA of Alteromonas sp. ZD22-1 is subjected to PCR amplification using the primers in Table 2, and the PCR reaction program is 94°C pre-denaturation for 1 min; 94°C denaturation for 30 s, 54°C annealing for 45 s, 72°C extension for 1 min 30 s, 35 cycles; 72°C extension for 10 min; 4°C storage. The obtained PCR product is subjected to agarose gel electrophoresis for 30 min. Then the PCR results are observed and photographed in a gel imaging system. It is identified and analyzed that the genome of the strain contains multiple functional genes related to sulfur cycle or nitrogen cycle, including dddD (see SEQ ID NO: 1), dddL (see SEQ ID NO: 2), dddP (see SEQ ID NO: 3), 16S (see SEQ ID NO: 4), nifH (see SEQ ID NO: 5), nirK (see SEQ ID NO: 6), dmdA (see SEQ ID NO: 7), DmdA (see SEQ ID NO: 8), etc. The PCR amplification results are used to verify the functions of the strain.

[0066] Table 2: Primers of functional genes

[0067]

[0068] Example 4: Antagonistic effect test of Alteromonas sp. ZD22-1 against Vibrio spp.

[0069] Prepare 2216E solid medium (1.5% agar) and sterilize. Take 300 μL of the enriched culture of V. harveyi and evenly spread on the solid medium. Select the dilution concentration of 10 -3 Then evenly divide the 2216E solid medium into four equal parts, place a sterile filter paper piece with a diameter of about 0.6 cm in each area, and inoculate the purified bacteria on the filter paper piece with 20 μL of bacterial solution per piece. After sealing with a sealing film (without inversion), incubate in a 28°C incubator for 24 h. It is identified that Alteromonas sp. ZD22-1 has strong antagonistic potential against V. harveyi (see Figure 1 ).

[0070] Example 5: Determination of the activity cycle of Alteromonas sp. ZD22-1

[0071] Centrifuge the bacterial solution obtained by culturing Alteromonas sp. ZD22-1 to the stationary phase at 6000 rpm to collect the bacterial cells, dilute the bacterial cells with artificial seawater (35‰) to a target concentration of OD600 = 1.0, and the concentration is about 1 x 10 9 cfu / mL, and put the target concentration of bacterial cells into a coral tank containing sterilized seawater. Detect the residual concentration of the microorganisms put into the coral tank by using an ATP fluorescence detector, and the detection frequency is 2-3 times per 3 days. The ATP fluorescence detector is based on the firefly luminescence principle, and uses the "luciferase-luciferin system" to rapidly detect adenosine triphosphate (ATP). Since all living cells contain a constant amount of ATP, the ATP content can clearly indicate the concentration or biomass of microorganisms in the sample. For specific operation methods, refer to the instrument use instructions. Set the same amount of sterile water as a blank control. The experimental results show that the average ATP fluorescence intensity (RLU) of Alteromonas sp. ZD22-1 bacterial cells after being put in is 180, the average RLU on the 13th day is 90, indicating that the half-life of the activity of Alteromonas sp. ZD22-1 is 13 days, and the average RLU on the 22nd day is close to the blank control, indicating that the biological residual of Alteromonas sp. ZD22-1 tends to 0. In order to ensure that Alteromonas sp. ZD22-1 has strong biological activity and disease resistance in the developed seawater, based on the experimental results, set the frequency of putting in to be 10-15 days per cycle (see Figure 2 ).

[0072] II. Application Examples. To demonstrate the inventiveness and technical value of the technical solution of this invention, this section provides application examples of the technical solution of the claims on specific products or related technologies.

[0073] Application Example 1:

[0074] The main ingredients of the anti-Vibrio pathogen probiotic formula for coral aquaculture are: anti-Vibrio pathogen probiotic Alteromonas sp. ZD22-1, Zooxanthellae type D, DMSP / DMS / AA, vitamins, amino acids, trace elements, CaCl2, artificial seawater, etc. It is available in two sizes: 1 liter / bottle and 500 ml / bottle.

[0075] Specific preparation steps:

[0076] 1) Collect Alteromonas sp. ZD22-1 cells by centrifugation at 5000-7000 rpm.

[0077] 2) Sterilize seawater with a salinity of 30-35 under high temperature and high pressure conditions;

[0078] 3) Add Alteromonas sp. ZD22-1 cells to the sterilized seawater, with a final concentration of not less than 1×10⁻⁶. 12 Cells / mL, Zooxanthus type D, final concentration not less than 1×10 8 Cells / mL, DMSP / DMSO / AA final concentration 100-1000μm, appropriate amount of multivitamins and amino acids, appropriate amount of vitamins, appropriate amount of trace elements.

[0079] In addition, regarding the precautions for using marine microecological preparations—anti-Vibrio pathogen probiotics:

[0080] 1) Usage: Add 10 mL of bacterial solution per 100 L of water to ensure a total active microbial count ≥ 5 × 10⁻⁶. 10 / L;

[0081] 2) Usage cycle: Add the bacterial agent every 10 to 15 days to keep the number of bacteria in the water stable;

[0082] 3) Storage conditions: It should be stored at a low temperature of 4-8℃;

[0083] 4) Validity period: 6 months;

[0084] 5) Shake well before use to ensure even distribution of bacteria.

[0085] Application Example 2.

[0086] Applying Alteromonas sp. ZD22-1 to aquaculture water or adding it to feed can eliminate pathogenic Vibrio, improve water quality, reduce reactive oxygen species, enhance the disease resistance of fish and shrimp, and increase yield. The following points should be noted when using Alteromonas sp. ZD22-1: (1) Use in moderation. When using in fish ponds, mix 1-5 grams of Alteromonas with crushed dry fertilizer mud per cubic meter of water and spread it evenly in the fish pond. Afterwards, every 10-20 days, use 1-2 grams of Alteromonas per cubic meter of water and sprinkle it throughout the pond after diluting the water. When used for disease prevention and control, it can be used continuously. Use 2-10 grams per cubic meter of water in the fish pond and sprinkle it throughout the pond after diluting the water. (2) When adding it to feed for fish and shrimp, mix it in at a ratio of 0.5-1%.

[0087] Application Example 3

[0088] In aquaculture, a compound bacterial agent is prepared using the alternating monocellular strain Alteromonas sp. ZD22-1, combined with commercially available strains of photosynthetic bacteria, Bacillus subtilis, lactic acid bacteria, and yeast. First, these strains are separately inoculated onto solid culture media for activation. Single colonies of the activated strains are then inoculated onto their respective liquid culture media and cultured at 37℃ for 48 hours. The cultured strains are then mixed in a 1:1:1:1:1 ratio and inoculated into a fermenter, fermented at 37℃ for 24 hours to prepare a bacterial suspension with a pH of 5.0–7.0 and a viable count of 1 × 10⁻⁶. 8 ~1×10 10 CFU / mL indicates the complete preparation of the compound bacterial agent. Store in a cool, dry place.

[0089] The obtained compound probiotic agent was proliferated in seawater. The preparation method of the agent in seawater (taking 100L as an example): (1) Move the 100L plastic white bucket to a place in direct sunlight outdoors (the temperature in summer is greater than 25 ℃, and it can be propagated indoors in winter. It needs to be heated to keep the propagation temperature between 10℃ and 30℃. Add about 50L of artificial seawater (well water or tap water can be used. Tap water needs to be left to stand for a period of time to prevent the residual chlorine in the tap water from damaging the growth of bacteria. Then prepare it with sea salt with a salinity of 25-35‰); (2) Add the weighed culture medium, 2 kg of brown sugar and 5L of compound probiotic agent product to the 100L white bucket, stir it evenly with a clean stick, and then fill the plastic white bucket with seawater until it is full. Seal and ferment for 3 days. If the temperature is lower than 20 ℃ or the weather is cloudy and rainy, the bacterial fermentation does not achieve the expected effect. The propagation time can be extended by one or two days.

[0090] III. Evidence of the effects of the embodiments. The embodiments of the present application have achieved some positive effects in the development or use process, and indeed have great advantages compared with the prior art. The following content is described in combination with the data, graphs, etc. of the test process.

[0091] Anti-vibrio pathogenic probiotic Alteromonas sp. ZD22-1 inoculation test and physiological index detection of stressed corals by temperature and pathogens:

[0092] a. Active bacteria inoculation test and coral temperature stress experiment

[0093] Two temperature settings (25-26℃ and 29-30℃) and three experimental groups of Pocillopora damicornis treated with Alteromonas sp. ZD22-1 inoculation were set up for comparison: 1) non-inoculated probiotic and vibrio control group (CT); 2) inoculated probiotic experimental group (PT); 3) inoculated probiotic + vibrio experimental group (C+PT). The specific experimental steps are as follows: first, the corals in the above 1-3 groups were acclimated at normal temperature 25℃ for 10 days, and the samples were detected and analyzed. Then the temperature was raised to 29℃ for feeding, and the samples were detected and analyzed on the 15th and 20th days. The experimental results show that after 20 days of feeding at 29℃, the white rate of corals in the CT group is significantly higher than that in the C+PT group and the PT group. This shows that Alteromonas sp. ZD22-1 has the effect of treating vibrio disease and high temperature stress.

[0094] b. Detection of physiological and health indicators of experimental corals

[0095] The samples in the above experimental stages were detected, the DNA of coral tissue and marine samples was extracted, and the changes in microbial community structure of coral tissue and seawater in each experimental treatment were analyzed by 16S tag sequencing; the ROS level of each treated coral was detected by DCFH-DA reactive oxygen ROS fluorescent probe under laser confocal microscope. The experimental results show that the relative abundance of vibrio in the CT group is significantly higher than that in the C+PT group and the PT group. The ROS level of the CT group is significantly higher than that of the C+PT group and the PT group, indicating that Alteromonas sp. ZD22-1 has the effect of resisting vibrio pathogens and ROS.

[0096] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any modification, equivalent replacement and improvement made by any person skilled in the art within the technical scope disclosed by the present application, as long as it is within the spirit and principles of the present application, should be covered within the protection scope of the present application.

[0097] Sequence Listing

[0098] <110> Lin Zhenyue, Zhang Junli, Wang Wei, Chen Jianming

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[0143] Gly Thr lie Cys Ala Arg lie Thr lie Thr Gly Gly Gly Ala Tyr Gly 1 5 10 15

[0145] Thr Thr Cys lie Ala Thr lie Cys Lys lie Thr Cys lie Ala Thr lie 20 25 30

[0147] Ala lie Arg Thr Thr Asp Gly Gly 35 40

[0149] <210> 8

[0150] <211> 37

[0151] <212> DNA

[0152] <213> Artificial Sequence

[0153] <400> 8

[0154] tattggtata gctatgatta aataaaaggt aaatcgc 37

[0155] <210> 9

[0156] <211> 700

[0157] <212> DNA

[0158] <213> Artificial Sequence

[0159] <400> 9

[0160] gactaactac ttcttttgca tcccactccc atggtgtgac gggcggtgtg tacaaggccc 60

[0161] gggaacgtat tcaccgcagt attctgacct gcgattacta gcgattccga cttcatggag 120

[0162] TCGAGTTGCA GACTCCAATC CGGACTACGA CATTCTTTAA GGGGTCCGCT CCACATC ACT 180

[0163] GTCTCGCTTC CCTCTGTAAA TGCCATTGTA GCACGTGTGT AGCCCTACAC GTAAGGGCCA 240

[0164] TGATGACTTG ACGTCGTCCC ACACCTTCCT CCGGTTTGTC ACCGGCAGTC TCCTTAGAGT G 300

[0165] CCCAACTTAA GGCTGGCAAC TAAGGACAAG GGTTCGCTCG TTGCGGGACT TAACCCAAC 360

[0166] ATCTCACGAC ACGAGCTGAC GACAGCCATG CAGCACCTGT GTCTGAGTTC CCGAAGGCAC 420

[0167] CAAACTATCT CTAGAAACTT CTCAGCATGT CAAGTGTAGG TAAGGTTCTT CGCgttgcat 480

[0168] Cgaattaaac cacatgctcc accgcttgtg cgggcccccg tcaattcatt tgagttttaa 540

[0169] CCTTGCGGCC GTACTCCCCA GGCGgtctac ttagcgcgtt AGCTTCGCTA CGCACGCCTT 600

[0170] AAAGACACAC ACAGCTAGTA GACAGCgttt acggtgtgga ctaccagggt atctaatcct 660

[0171] GTTcgctacc cacactttcg cacatgagcg tcagtctttg 700

[0172] The above describes the specific embodiments of the present application in combination with the accompanying drawings, but is not a limitation on the scope of protection of the present application, and those skilled in the art should understand that various modifications or changes made by those skilled in the art on the basis of the technical solutions of the present application without creative labor are still within the scope of protection of the present application. SEQUENCE LISTING <110> Lin Zhenyue, Zhang Junli, Wang Wei, Chen Jianming <120> A kind of anti-vibrio pathogenic probiotic for coral healthy culture and application <160> 9 <170> SIPOSequenceListing 1.0 <210> 1 <211> 38 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 1 accaacgtca ttgcaggact gtgcgtgttc ttccggtg 38 <210> 2 <211> 40 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 2 ctgggaatac ggctacgaga gttcaagatc agcgatccgg 40 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 3 gcagctctgg aacgcccata gcatcaggca gccgtatttc 40 <210> 4 <211> 39 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 4 agagtttgat cctggctcag ggttaccttg ttacgactt 39 <210> 5 <211> 40 <212> PRT <213> Artificial Sequence (Artificial Sequence) <400> 5 tgcgatccga aagccgactc atggccatca tttcaccgga 40 <210> 6 <211> 37 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 6 atcatggtcc tgccgcgttg gtgttagact agctccg 37 <210> 7 <211> 40 <212> PRT <213> Artificial Sequence (Artificial Sequence) <400> 7 Gly Thr Ile Cys Ala Arg Ile Thr Ile Thr Gly Gly Gly Ala Tyr Gly 1 5 10 15 Thr Thr Cys Ile Ala Thr Ile Cys Lys Ile Thr Cys Ile Ala Thr Ile 20 25 30 Ala Ile Arg Thr Thr Asp Gly Gly 35 40 <210> 8 <211> 37 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 8 tattggtata gctatgatta aataaaaggt aaatcgc 37 <210> 9 <211> 700 <212> DNA <213> Artificial Sequence <400> 9 gactaactac ttcttttgca tcccactccc atggtgtgac gggcggtgtg tacaaggccc 60 gggaacgtat tcaccgcagt attctgacct gcgattacta gcgattccga cttcatggag 120 tcgagttgca gactccaatc cggactacga cattctttaa ggggtccgct ccacatcact 180 gtctcgcttc cctctgtaaa tgccattgta gcacgtgtgt agccctacac gtaagggcca 240 tgatgacttg acgtcgtccc caccttcctc cggtttgtca ccggcagtct ccttagagtg 300 cccaacttaa ggctggcaac taaggacaag ggttgcgctc gttgcgggac ttaacccaac 360 atctcacgac acgagctgac gacagccatg cagcacctgt gtctgagttc ccgaaggcac 420 caaactatct ctagaaactt ctcagcatgt caagtgtagg taaggttctt cgcgttgcat 480 cgaattaaac cacatgctcc accgcttgtg cgggcccccg tcaattcatt tgagttttaa 540 ccttgcggcc gtactcccca ggcggtctac ttagcgcgtt agcttcgcta cgcacgcctt 600 aaagacacac acagctagta gacagcgttt acggtgtgga ctaccagggt atctaatcct 660 gttcgctacc cacactttcg cacatgagcg tcagtctttg 700

Claims

1. A probiotic against vibrio pathogenic for the health of coral farming, characterized in that, The anti-vibrio pathogenic probiotic bacteria for coral health culture is Alteromonas sp. ZD22-1, and the preservation number is GDMCC No: 61647. The anti-vibrio pathogenic probiotic bacteria for coral health culture is Alteromonas sp. ZD22-1, and the preservation number is GDMCC No: 61647.

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