A strain of Oceanobacillus timonensis and its application in aquaculture

Abstract

The present invention belongs to the field of microorganisms, and specifically relates to a strain of Oceania Timonii Bacillus YZS-M02 and its application in aquaculture. The Oceania Timonii Bacillus YZS-M02 is classified and named Oceania Timonii Bacillus ( Oceanobacillus timonensis ) YZS-M02, which was deposited with the General Microbiology Center of the China Microorganism Culture Collection Committee on September 3, 2020, with the deposit number CGMCC No. 20593. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The aquaculture microbial preparation prepared using the Oceanobacillus timonii YZS-M02 has the ability to purify aquaculture water and increase the survival rate and yield of aquatic animals, which is beneficial to aquaculture and profitability.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and relates to a strain of Oceania timonii and application of the strain in aquaculture. Background Art

[0002] In recent years, the aquaculture industry has experienced rapid growth and increasing intensification. Harmful algae and pathogens have proliferated in many aquaculture ponds, severely damaging the aquaculture ecosystem and leading to frequent aquaculture diseases. As a new type of green, environmentally friendly product, probiotics play an irreplaceable role in regulating the animal microbiome, preventing disease, improving feed conversion efficiency, promoting animal health, and protecting the ecological environment. Currently, probiotics have been accepted as green feed additives and are widely used in aquaculture. Among the most widely used probiotics on the market are probiotics based on yeast, lactic acid bacteria, and Bacillus. These probiotics antagonize intestinal pathogens, maintain and regulate intestinal microbial balance, synthesize vitamins and produce various digestive enzymes in the host, provide nutrition, and prevent the invasion of pathogens. Probiotics can also promote animal growth, improve feed conversion efficiency, and enhance animal immunity. Therefore, the discovery and utilization of functional microorganisms is a crucial component and foundation for the effectiveness of effective probiotics. Summary of the Invention

[0003] The purpose of the present invention is to provide a strain of Oceania timonii and application thereof in aquaculture.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] The application of Oceanobacillus timonensis YZS-M02 in the preparation of aquaculture microbial preparations. The Oceanobacillus timonensis YZS-M02 is classified and named Oceanobacillus timonensis YZS-M02. It was deposited in the General Microbiology Center of the China Culture Collection Administration on September 3, 2020, with the deposit number CGMCC No. 20593. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the aquaculture microbial preparation has the effect of purifying aquaculture water and improving the survival rate and yield of aquatic animals.

[0006] An aquaculture microbial preparation containing Bacillus thimonens YZS-M02, wherein the aquaculture microbial preparation is a liquid preparation, and the content of live bacteria of Bacillus thimonens YZS-M02 in the aquaculture microbial preparation is in the range of (2 to 5)×10 8 cfu / mL.

[0007] The above-mentioned aquaculture microbial preparation containing Bacillus timonioides YZS-M02 is used in purifying aquaculture water and improving the survival rate and yield of aquatic animals.

[0008] The significant advantages of the present invention are:

[0009] Timon Oceanic Bacillus YZS-M02 can effectively improve the transparency of aquaculture water and has a significant water purification effect; it can also increase the survival rate and yield of aquaculture products. DETAILED DESCRIPTION

[0010] In order to make the contents of the present invention easier to understand, the technical solutions of the present invention are further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0011] In the following examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from biological or chemical reagent companies.

[0012] The formula of the culture medium required in the following examples is as follows:

[0013] NA medium: peptone 10 g, beef extract 3 g, NaCl 5 g, agar 15 g, water 1000 mL; pH 7.0-7.2.

[0014] NB medium: peptone 10 g, beef extract 3 g, NaCl 5 g, water 1000 mL; pH 7.0-7.2.

[0015] Example 1: Isolation, screening and identification of Bacillus timonensis

[0016] (1) Separation and screening process:

[0017] Using the gradient dilution method, 319 isolates were isolated from Fujian mangroves. The isolated endophytic bacteria were purified using the three-zone striping method. Purification was confirmed by microscopic examination. Purified bacteria were numbered, and individual colonies were picked and transferred to NA medium slants for future use. The isolated strains were then assayed for protease, lipase, amylase, and cellulase activity, as well as for ammonia nitrogen, nitrite, sulfide, and COD degradation. Ultimately, an endophytic bacterium with strong activity was identified and labeled YZS-M02.

[0018] (2) Colony characteristics and colony morphology:

[0019] After YZS-M02 was cultured on a NA medium plate for 24 hours, colonies were formed that were light yellow, dull, round, non-flowing, Gram-positive, with spores and no capsule; the colony diameter was 3-5 mm.

[0020] (3) Physiological and biochemical characteristics:

[0021] YZS-M02 catalase reaction was negative, VP assay was negative, MR assay was positive, glucose acid production test was negative, glucose gas production test was negative, citrate test was negative, nitrate reduction was positive, starch hydrolysis was negative, indole test was negative, malonic acid assay was negative, and H2S production test was negative.

[0022] (4) 16S rDNA sequence analysis

[0023] The nucleotide sequence of the 16S rDNA gene of strain YZS-M02 is listed in SEQ ID NO. 1. Comparison of the 16S rDNA sequence with sequences in the GenBank database revealed that YZS-M02 and Oceanobacillus timonensis share a common clade, with a 16S rDNA sequence similarity of 99.72% to Oceanobacillus timonensis (MT192497.1). Based on colony morphology, physiological and biochemical characteristics, and 16S rDNA sequence analysis, strain YZS-M02 was preliminarily identified as Oceanobacillus timonensis.

[0024] Example 2: Application of Oceanobacillus timonensis YZS-M02 in Purification of Water for Litopenaeus vannamei Culture After activation of Oceanobacillus timonensis YZS-M02 on a slant of NA culture medium, a loopful of the bacterium was inoculated into NB culture medium and cultured at 28°C and 180 rpm for 72 h. The culture solution was diluted with sterile water to prepare a solution containing 1×10 8 cfu / mL of bacterial solution for use.

[0025] The whiteleg shrimp in the factory-style indoor winter shed culture pond was selected. The whiteleg shrimp in the culture pond were about one month old. 8 A 200 mL solution of Oceania Bacillus timonens YZS-M02 was used. The control tank was treated with no microbial agents or other similar products. The test tank was treated with 20 mL of the solution daily for two consecutive days. The 20 mL solution was evenly distributed throughout the test tank, and an aerator was operated for two hours after application. Water color was regularly monitored for changes, and the water exchange rate was reduced based on water quality. No other biological, chemical, or disinfectant products that could affect the results were added during the test.

[0026] The results showed that the water transparency of the test pond treated with the bacterial solution of Oceanobacillus timonens YZS-M02 gradually improved over time. The water exchange volume of the test pond decreased by 27% on the second day after the application of the bacterial solution, and the total water exchange volume in the following three days decreased by 35%, which was significantly different from that of the control pond. The water color of the test pond treated with the bacterial solution of Oceanobacillus timonens YZS-M02 was light in color, which was significantly different from the light brown water color of the control pond.

[0027] Table 1 Purification effect of Timon oceanic Bacillus YZS-M02 on white shrimp aquaculture water

[0028]

[0029] Example 3: Effect of Bacillus timonioides YZS-M02 on the survival rate and yield of whiteleg shrimp culture

[0030] After Oceanobacillus timonensis YZS-M02 was activated on the slant of NA medium, a loop was picked and inoculated into NB medium. The culture was shaken at 28°C and 180 rpm for 72 h. The culture solution was diluted with sterile water to prepare a solution containing 1×10 8 cfu / mL of bacterial solution for use.

[0031] The whiteleg shrimp culture pond (high-level pond) was selected and two treatments were set up: experimental pond: 1×10 8 cfu / mL of Bacillus thimensis YZS-M02 bacterial solution; control ponds: no microbial agents or other similar products were applied. The average stocking weight of the test and control ponds was 4.6 grams per fish, with a culture area of ​​0.5 mu (approximately 1.5 acres) per pond and a culture duration of 90 days. The Bacillus thimensis YZS-M02 bacterial solution was mixed evenly with feed in a proportional manner before feeding. In the early stages of the culture phase (stocking to 60 days of culture), the Bacillus thimensis YZS-M02 bacterial solution was mixed with feed at a ratio of 5% (volume to mass ratio); in the late stages of culture phase (60 days of culture to harvest), the Bacillus thimensis YZS-M02 bacterial solution was mixed with feed at a ratio of 8% (volume to mass ratio). Water clarity, ammonia nitrogen, and nitrite nitrogen were regularly measured. No other biological, chemical, or disinfectant products, which could affect the results, were added during the experiment.

[0032] The results showed that the water transparency of the test ponds treated with the bacterial solution of Oceanobacillus timonens YZS-M02 was significantly improved, and the concentrations of ammonia nitrogen and nitrite nitrogen in the water were significantly reduced; the survival rate of whiteleg shrimp after application of the bacterial solution of Oceanobacillus timonens YZS-M02 was 92%, an increase of 16.46% compared with the control shrimp ponds; the qualified rate of normal shrimp bodies in the test ponds after application of the bacterial solution of Oceanobacillus timonens YZS-M02 was 69%, an increase of 23.21% compared with the control ponds; and the yield per mu of shrimp ponds increased by 19%.

[0033] Table 2 Effects of Bacillus timonioides YZS-M02 on water purification for Penaeus vannamei aquaculture

[0034]

[0035] Table 3 Effects of Bacillus timonioides YZS-M02 on the survival rate and yield of whiteleg shrimp

[0036]

[0037] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. The application of Bacillus thuringiensis YZS-M02 in the preparation of aquaculture microbial preparations is characterized by: The Timon Oceanic Bacillus YZS-M02 is classified and named Timon Oceanic Bacillus ( Oceanobacillus timonensis )YZS-M02 was deposited in the General Microbiology Center of the China Culture Collection Administration on September 3, 2020, with the deposit number CGMCC No. 20593, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the aquaculture microbial preparation has the effect of improving the transparency of water for whiteleg shrimp farming, and improving the survival rate and yield of whiteleg shrimp farming.

2. An aquaculture microbial preparation comprising Bacillus timonioides YZS-M02, characterized in that: The Timon Oceanic Bacillus YZS-M02 is classified and named Timon Oceanic Bacillus ( Oceanobacillus timonensis )YZS-M02 was deposited in the General Microbiology Center of China Culture Collection Administration on September 3, 2020, with the deposit number CGMCC No. 20593, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

3. The aquaculture microbial preparation according to claim 2, characterized in that: The aquaculture microorganism preparation is a liquid preparation.

4. The aquaculture microbial preparation according to claim 3, characterized in that: The content of viable bacteria of Bacillus timoniosum YZS-M02 in the aquaculture microbial preparation is in the range of 2×10 8 ~5×10 8 cfu / mL.

5. The use of the aquaculture microbial preparation according to claim 2 in purifying aquaculture water and improving the survival rate and yield of aquatic animals, characterized in that: The method of purifying aquaculture water is to improve the transparency of water for Penaeus vannamei aquaculture, and the method of improving the survival rate and yield of aquatic animals is to improve the survival rate and yield of Penaeus vannamei aquaculture.

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