Pseudoalteromonas angularis for treating aquaculture water of penaeus vannamei boone

Abstract

The invention provides a strain of pseudoalteromonas angustifolia for treating a culture water body of penaeus vannamei boone. The preservation number of the pseudoalteromonas angustifolia is CCTCC (China Center for Type Culture Collection) NO: M20241364. The screened pseudoalteromonas angustifolia can effectively treat ammonia nitrogen in a penaeus vannamei aquaculture water body in a high-temperature season, and due to the fact that the pseudoalteromonas angustifolia is separated from intestinal tracts of penaeus vannamei, the penaeus vannamei cannot be damaged under the condition of high-concentration use of the pseudoalteromonas angustifolia; therefore, the feed can be used for a long time in a penaeus vannamei breeding environment.

Description

Technical Field

[0001] The invention belongs to the technical field of biological product preparation, and mainly relates to a strain of Pseudoalteromonas elsdenii used for treating water for breeding of white shrimp. Background Art

[0002] With the rapid economic development, people's living standards have been greatly improved, and the consumption of aquatic products as a source of high-quality protein has increased year by year. Intensive high-density aquaculture has solved the contradiction between the small breeding area and the large demand for aquatic products to a certain extent. However, the environmental pollution caused by economic development has restricted the development of aquaculture. Due to the deterioration of the breeding water environment caused by long-term high-density breeding, it is often accompanied by the breeding of harmful microorganisms and serious aquaculture diseases, which brings great harm to the aquaculture industry.

[0003] Water treatment is very important for the cultivation of Penaeus vannamei, especially in the high temperature period of summer, its impact is more significant. As an important economic shrimp, Penaeus vannamei is very sensitive to the water quality of the breeding environment. High-quality water environment can provide good growth conditions, affecting the shrimp's feeding, metabolism, digestion and immune function.

[0004] During the high temperature period in summer, due to the large temperature difference between day and night, the vigorous metabolism of aquatic animals, the bottom water is prone to hypoxia, and the water quality changes rapidly, all of these pose a challenge to the growth of white shrimp. Therefore, it is crucial to regulate the water quality in a timely manner to ensure a stable breeding environment. This includes adding new water in an appropriate amount, strengthening the detection of major indicators such as dissolved oxygen, pH, ammonia nitrogen, and nitrite in the pond water environment, and taking countermeasures such as water changes and oxygenation. Through these measures, the growth of pathogenic microorganisms can be effectively inhibited, the circulation of organic matter in the breeding water can be promoted, and an ideal living environment can be created for farmed animals.

[0005] In addition, during the high temperature period in summer, organic matter such as excrement, leftover bait and dead organisms of white shrimp continue to accumulate, polluting the bottom and affecting the water quality. Therefore, water quality management centered on improving the bottom is also one of the key measures for the success of shrimp farming. Using microbial preparations to raise shrimp and establish an ecological and healthy breeding model can maintain the ecological balance of the water body, improve the balance of bacteria and algae in the shrimp pond, thereby maintaining good water color and transparency, and providing a stable water quality environment for the growth of shrimp. Summary of the invention

[0006] The present invention aims to provide a strain of Pseudoalteromonas elsdenii for treating water for Penaeus vannamei aquaculture, which can effectively treat ammonia nitrogen in water for Penaeus vannamei aquaculture in high temperature seasons, and because the strain is separated from the intestinal tract of Penaeus vannamei, it will not cause harm to the Penaeus vannamei even when used in high concentrations, and thus can be used for a long time in a Penaeus vannamei aquaculture environment.

[0007] The Pseudoalteromonas espejiana S157 strain provided by the present invention was deposited in the China Type Culture Collection of Wuhan University, Wuhan, China on June 24, 2024, with the deposit number CCTCCNO: M20241364.

[0008] The present invention also provides a use of the Pseudoalteromonas espejiana S157 strain, which is the use of degrading ammonia nitrogen in aquaculture water.

[0009] In another aspect, the present invention also provides the use of the screened Pseudoalteromonas elsdenii in preparing products for breeding of Penaeus vannamei.

[0010] The product is a live bacteria preparation.

[0011] The Pseudoalteromonas elsdenii S157 strain screened by the present invention can effectively degrade the nitrogen content in the aquaculture water, thereby effectively maintaining the quality of the aquaculture water, thereby promoting the survival and growth of Penaeus vannamei. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 :The culture morphology of Pseudoalteromonas elsdenii;

[0013] Figure 2 :Hemolytic detection chart of strain S157 (A is buffer control, B, C, D are 10 6 , 10 7 , 10 8 CFU / mL).

[0014] Figure 3 : The ability of strain S157 to degrade ammonia nitrogen at different temperatures. DETAILED DESCRIPTION

[0015] In order to obtain a probiotic strain derived from Penaeus vannamei, a strain capable of effectively treating ammonia nitrogen in Penaeus vannamei culture water in high temperature seasons was screened out from the intestine of healthy Penaeus vannamei in the present invention.

[0016] The present invention is described in detail below in conjunction with embodiments and drawings.

[0017] Example 1: Isolation and identification of strains

[0018] 1. Isolation of strains

[0019] Take healthy white shrimp fry, wash the shrimp body with tap water, and then disinfect its surface with 75% alcohol. Use a sterile 5mL syringe needle to pick the complete intestine of the shrimp, remove the digested material, rinse it with phosphate buffered saline (PBS, pH 7.4) 3 times, take 1g of intestinal tissue, and add 1mL PBS. Under sterile conditions, use an electric homogenizer to evenly disperse the sample. Use the dilution coating method to spread the intestinal homogenate on the 2216E plate, select the plate with clear and dispersed colonies, pick a single colony for purification, and then store the strain in glycerol for use after pure culture.

[0020] 2. Study on the enzymatic properties of the strain

[0021] The amylase, cellulase and protease production capacity of the strains was determined by the spot method. The test strains were spotted on starch medium, cellulase production medium and seawater casein agar on the culture medium plates, cultured at 15°C, and Lu's iodine solution and Congo red solution were added after 4 days and cultured for 7 days.

[0022] A total of 208 bacterial strains were isolated from the shrimp fry, including 115 strains producing amylase, 91 strains producing cellulase, 64 strains producing protease, and 7 strains producing three enzymes (Table 1).

[0023] Table 1: Statistics of strains producing three enzymes

[0024]

[0025]

[0026] 3. Screening of strains with ammonia nitrogen reduction effect

[0027] The strain obtained by preliminary screening was inoculated into a culture medium containing ammonia nitrogen, and cultured at 30°C and 120 rpm for 24 hours. The obtained culture solution was used as seed solution. Then, this sub-liquid was inoculated into the ammonia nitrogen culture medium again at a ratio of 3%, and continued to be cultured at 30°C and 120 rpm. Every 5 hours, part of the fermentation liquid was taken out and separated by centrifugation at 12,000 rpm for 3 minutes, and then the ammonia nitrogen content in the supernatant was determined, and the ammonia nitrogen removal rate was calculated accordingly. Finally, the strain with the strongest ammonia nitrogen degradation ability was saved.

[0028] Ammonia nitrogen removal rate / % = (1-C1 / C0) × 100

[0029] Where: C0, initial ammonia nitrogen concentration; C1, residual ammonia nitrogen concentration.

[0030] The residual ammonia nitrogen of the strains obtained in the initial screening was measured every 5 hours (Table 2). Five strains showed the ability to degrade ammonia nitrogen at 15 hours, of which only one strain (S157) showed the ability to degrade ammonia nitrogen at 5 hours, and the degradation effect was the best after 25 hours, so this strain (S157) was selected to continue the experiment.

[0031] Table 2: Effect of strains on degradation of ammonia nitrogen

[0032]

[0033] Example 2: Molecular biological identification and physicochemical properties of strain S157

[0034] 1. Identification of isolated strains

[0035] The screened S157 strain was inoculated into 2216E liquid medium for overnight culture, and bacterial genomic DNA and 16SrRNA gene were extracted. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., which were bacterial universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). 25μL reaction system: 2×Mix Ex Taq 12.5μL, 27F (10μmol / L), 1492R (10μmol / L) each 1μL, DNA template 1μL, double distilled water 9.5μL. The reaction conditions were 96℃5min; 96℃30s, 55℃45s, 72℃90s, a total of 33 cycles; 72℃10min. PCR products were detected by agarose gel electrophoresis, and Sangon Biotech (Shanghai) Co., Ltd. was entrusted to sequence and splice.

[0036] The obtained DNA sequences were compared for sequence homology in the public database provided by EzBio Cloud (http: / / www.ezbiocloud.net / eztaxon), and the sequences with the highest similarity were selected from the results to preliminarily determine the species. It was determined that the sequence similarity between the S157 strain and Pseudoalteromonas elsdenii was the highest.

[0037] The S157 strain was named Pseudoalteromonas espejiana and was deposited in the China Type Culture Collection, Wuhan University, Wuhan, China on June 24, 2024, with the deposit number CCTCC NO:M20241364.

[0038] 2. Physical and chemical properties of Pseudoalteromonas elsdenii

[0039] The isolated and purified bacteria were streaked onto 2216E plate culture medium and cultured at 28°C for 18 h before observing the characteristics of the colonies and performing Gram staining. Physiological and biochemical identification was performed using the methods in the Manual of Common Bacterial Systematic Identification. Figure 1 The morphological diagram of the strain is round and raised, with a smooth and opaque surface and clear edge boundaries. The Gram staining test was negative, and the bacterial morphology was rod-shaped. As shown in Table 3, the methyl red reaction, gelatin liquefaction, catalase, citrate utilization, and starch hydrolysis of Pseudoalteromonas elsdenii were all positive, while catalase, glucose oxidation and fermentation, nitrate reduction, indole test, and VP test were all negative.

[0040] Table 3 Physiological and biochemical test results of Pseudoalteromonas elsdenii S157 strain

[0041]

[0042]

[0043] 3. Hemolytic test

[0044] The isolate S157 was inoculated on a 2216E plate, and a single colony was picked and cultured in a shaker at different concentrations (10 6 , 10 7 , 10 8 CFU / mL) of bacterial suspension were inoculated on 5% sheep blood agar plates for hemolysis experiment, with buffer as negative control. The plates were cultured at 15°C for 48h, and the production of hemolysin was determined by the formation of transparent circles around the colonies. Figure 2 The results showed that no hemolytic ring was produced in the bacterial solution of each concentration, indicating that strain S157 was not hemolytic.

[0045] 4. Safety test

[0046] Four hundred healthy 15-day-old Penaeus vannamei fry were randomly divided into four groups and placed in plastic buckets containing 50 L of sand-filtered seawater. In the experimental group, three buckets of water were added with S157 bacterial suspension to a bacterial concentration of 1 × 10 6 , 1×10 7 , 1×10 8 CFU / mL; no potential probiotics were added to the water of the control group. The shrimp fry were observed for 49 days and the incidence and mortality of shrimp fry were recorded.

[0047] According to the results of enzyme production, antagonism and hemolysis tests, the potential probiotic S157 strain was subjected to an immersion test. The results showed (Table 4) that the survival rate of shrimp seedlings in the control group was not lower than that in the test group, indicating that the strain had good survival at different concentrations (1.0×10 6 , 1.0×10 7, 1.0×10 8 CFU / mL) had no harm to the shrimp fry in the experimental group 1; the survival rate of the shrimp fry in the experimental group 1 was significantly improved compared with that in the control group (P < 0.05), confirming that the potential probiotic S157 can improve the survival of white shrimp.

[0048] Table 4: Statistical table of pathogenicity of potential probiotic S157 to white shrimp

[0049]

[0050] 5. Degradation performance of S157 strain on ammonia nitrogen in water

[0051] In order to test the ability of S157 strain to degrade ammonia nitrogen in the water of Penaeus vannamei culture, two groups were set up, one at normal temperature (25℃) and the other at high temperature (35℃), with 4 water tanks in each group, containing 10 4 , 10 5 , 10 6 CFU / mL of S157 strain and 10 6 CFU / mL of Enterobacter cloacae K7 (which has been reported to have a good degradation effect on ammonia nitrogen, with a preservation number of CCTCC NO: M2024937) and 3L of white shrimp farming water, with 3 replicates in each group. After 25 hours, samples were taken to determine the ammonia nitrogen mass concentration, and the ammonia nitrogen removal rate was calculated.

[0052] Figure 3 The results showed that after 25 hours, all strains in the normal temperature (25℃) group showed good ability to degrade ammonia nitrogen, with degradation rates above 90%. At high temperature (35℃), Pseudomonas elsdenii S157 still maintained good ammonia nitrogen degradation performance, while the ammonia nitrogen degradation performance of Enterobacter cloacae K7 was significantly lower than that of S157 (P<0.05).

[0053] Example 3: Application of strain S157 in the cultivation of Penaeus vannamei

[0054] Healthy white shrimp fry were selected from the white shrimp breeding base, and the larvae were randomly distributed into plastic buckets containing 50L sand-filtered seawater after selection. A control group without the addition of potential probiotics and three concentration groups (1×10 4 CFU / mL, 1×10 6 CFU / mL and 1×10 8 CFU / mL), with 3 parallels in each group. The shrimps were tested on the 10th day after the addition of potential probiotics, the 25th day of the juvenile stage, and the 55th day of the adult stage. Twenty individuals were taken from each parallel to measure the body length and calculate the specific growth rate.

[0055] Table 5: Effects of different concentrations of S157 strain on the growth rate of white shrimp

[0056]

[0057] As shown in Table 5, on the 10th day of growth of Penaeus vannamei, there was no significant difference in the growth rate of each concentration group of S157 strain. On the 25th and 55th days, the growth rate of Penaeus vannamei in experimental group 2 was significantly higher than that in other groups (P < 0.05), while the growth rates of experimental groups 1 and 3 were not significantly different from those in the control group (P > 0.05).

[0058] The above results show that the strain S157 screened by the present invention is identified as Pseudoalteromonas elsdenii, which can efficiently degrade the ammonia nitrogen content in the water, thereby reducing the stress response of cultured white shrimp, thereby improving the growth rate, and can be developed and applied as a microecological preparation for white shrimp.

Claims

1. A Pseudoalteromonas elsdenii, characterized in that The deposit number of the Pseudoalteromonas elsdenii is CCTCCNO:M 20241364.

2. Use of the Pseudoalteromonas elsdenii described in claim 1 in degrading ammonia nitrogen in aquaculture water.

3. The use according to claim 2, characterized in that The aquaculture water body is the aquaculture water body for Penaeus vannamei.

4. Use of the Pseudoalteromonas elsdenii according to claim 1 in preparing products for breeding of Penaeus vannamei.

5. The use according to claim 4, characterized in that The product is a bacterial preparation.

6. A bacterial preparation, characterized in that The bacterial preparation contains live bacteria of Pseudoalteromonas elsdenii as claimed in claim 1.

7. A method for degrading ammonia nitrogen in water for breeding Penaeus vannamei, characterized in that: The method is to use the Pseudoalteromonas elsdenii described in claim 1 to degrade ammonia nitrogen in water.

8. The method according to claim 7, characterized in that The method is to use the bacterial preparation described in claim 6 to degrade water.

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