Rhodopseudomonas sphaeroides as well as microecological preparation and application thereof

Abstract

The invention discloses a Rhodopseudomonas sphaeroides capable of producing coenzyme Q10 at high yield. Belongs to the technical field of microorganism application, and particularly relates to rhodopseudomonas sphaeroides and a microecological preparation and application thereof. According to the invention, a strain of Rhodopseudomonas sphaeroides for producing coenzyme Q10 is taken as an original strain, and is subjected to ultraviolet mutagenesis treatment to obtain a strain of which the yield of coenzyme Q10 is remarkably improved. After the culture medium is optimized, the content of the coenzyme Q10 in the fermentation liquor reaches 505mg / L. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.27269. When fermentation liquor of the strain is mixed with feed to feed penaeus vannamei, the capability of resisting ammonia nitrogen, nitrite, oxygen deficit and low-salt environment stress can be enhanced, and the survival rate of cultivation and transportation is increased.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial application, and particularly relates to a strain of Rhodopseudomonas sphaeroides and a microecological preparation and application thereof. Background Art

[0002] At present, the high-density farming and excessive feeding of white shrimp in Vannamei have led to insufficient dissolved oxygen in the water and deterioration of water quality, such as excessive ammonia nitrogen and nitrite. Ammonia nitrogen refers to the free ammonia (NH3) and ammonium ions (NH4 + ) in the form of nitrogen. Free ammonia is toxic to aquatic animals by affecting their osmotic pressure balance and biochemical reactions in the body, and its toxicity increases with the increase of pH and water temperature. In severe cases, it can cause aquatic animals to become excited, lose balance, convulse, and even die. Nitrite is an important intermediate product of the nitrogen cycle in the ecosystem and a common pollutant in aquaculture. High concentrations of nitrite in the water environment pose a serious threat to the health of aquatic animals and can accumulate in the blood, leading to disorders in various physiological processes of aquatic animals such as respiration, cardiovascular, and endocrine, and even causing death from lack of oxygen and suffocation.

[0003] Photosynthetic bacteria are rich in nutrients. They are not only excellent single-cell proteins, but also contain a variety of vitamins, carotenoids and coenzyme Q 10 Coenzyme Q 10 It is a natural antioxidant and free radical scavenger in the body. It has the effect of anti-aging and improving the body's immunity. Therefore, breeding high-yield coenzyme Q 10 The photosynthetic bacteria and their use in shrimp farming are of great significance for enhancing shrimp's resistance to external stress and improving its survival rate. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a new strain of Rhodopseudomonas sphaeroides.

[0005] The spherical Rhodopseudomonas CGMCC No. 27269 provided by the present invention is obtained by mutation breeding of a spherical Rhodopseudomonas from a seawater pond, and the strain has high coenzyme Q production 10 After the culture medium was optimized, the fermentation broth coenzyme Q 10 The content reaches 505mg / L.

[0006] The Rhodopseudomonas sphaeroides provided by the present invention was deposited on May 5, 2023 in the General Microbiology Center of the China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a deposit number of CGMCC No. 27269.

[0007] Furthermore, the 16S rRNA sequence of the Rhodopseudomonas sphaeroides is shown in the sequence listing SEQ ID No. 1.

[0008] The present invention also provides a microecological preparation containing the Rhodopseudomonas spherica. Specifically, the microecological preparation contains the fermentation liquid or bacterial powder of the Rhodopseudomonas spherica.

[0009] The present invention also provides a fermentation medium for the Rhodopseudomonas spherical, specifically:

[0010] Sodium acetate 1-3g / L, ammonium chloride 1-5g / L, yeast powder 0.5-2.5g / L, tyrosine 75mg / L, potassium dihydrogen phosphate 0.5g / L, calcium chloride 0.1g / L, magnesium chloride 0.5g / L, sodium chloride 1g / L;

[0011] The seed liquid of the spherical red pseudomonas is inoculated into a transparent container filled with fermentation medium at an inoculation rate of 5-10%, and cultured at a temperature of 28-35° C. under light for 48-72 hours to obtain the fermentation liquid of the spherical red pseudomonas.

[0012] Furthermore, the Rhodopseudomonas sphaeroides powder is vacuum freeze-dried powder or spray-dried powder.

[0013] The present invention also provides the use of the Rhodopseudomonas spherica or a microecological preparation containing the Rhodopseudomonas spherica in enhancing the resistance of shrimps to adverse environments (such as ammonia nitrogen stress, nitrite stress, hypoxia stress, and low salt stress).

[0014] Specifically, the spherical Rhodopseudomonas or its microecological preparation is mixed with feed to feed the shrimp.

[0015] The present invention also provides a method for preparing coenzyme Q by using the Rhodopseudomonas spherica or a microecological preparation containing the Rhodopseudomonas spherica. 10 Application in.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention provides a new strain of Rhodopseudomonas sphericalensis. After the fermentation medium is optimized, the coenzyme Q 10 The content reaches 505mg / L. After experimental verification, this strain can enhance shrimps' resistance to adverse environments such as ammonia nitrogen stress, nitrite stress, hypoxia, and low salt stress, and improve the survival rate of breeding and transportation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 For CoQ 10 Detection standard curve graph;

[0019] Figure 2Optimize the test results of carbon source types for single factor experiments;

[0020] Figure 3 The results of the optimization test for the optimal carbon source concentration in the single factor experiment;

[0021] Figure 4 Optimize the test results of nitrogen source types for single factor experiments;

[0022] Figure 5 The results of the optimization test for the optimal nitrogen source concentration in the single factor experiment;

[0023] Figure 6 Optimize test results for single factor experiments with multiple culture species;

[0024] Figure 7 Optimize test results for the best composite culture concentration for single factor experiments;

[0025] Figure 8 Optimize test results for additional additives in single factor experiments;

[0026] Fig. 9 Optimize test results for additional additive concentrations in single factor experiments;

[0027] Fig.10 For CoQ 10 Content changes with fermentation time. DETAILED DESCRIPTION

[0028] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or manufacturer recommendations. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased from the market.

[0029] Example 1: Production of Coenzyme Q 10 Photosynthetic bacteria screening

[0030] 1. Isolation and screening of photosynthetic bacteria

[0031] (1) Enrichment medium ( / L): NaHCO3 2.0 g, yeast powder 1 g, K2HPO4 0.2 g, NH4Cl 1 g, NaAc 0.3 g, MgSO4·7H2O 0.2 g, NaCl 0.2 g.

[0032] (2) Basic fermentation medium ATYP ( / L): KH2PO4 0.5 g, NH4Cl 1 g, CaCl2 0.1 g, MgCl2.6H2O 0.5 g, NaCl 1 g, NaAc 1 g, yeast powder 0.5 g, peptone 0.3 g.

[0033] Take the sediment sample collected from the shrimp pond, add it to the anaerobic tube containing 10mL of enrichment medium, cover it with 1-2cm of paraffin oil, and culture it under light at 30℃ until the liquid turns red. Dilute the enrichment liquid that turns red 10 times, take 10 -3 ~10 -5 100 μL of the diluted liquid was evenly spread on the enrichment medium agar plate, and cultured in an anaerobic incubator at 30°C for 5 to 7 days until red colonies grew, and purified 2 to 3 times. The purified photosynthetic bacterial strain was inoculated into ATYP liquid medium at 30°C for 72 hours of static light culture, and stored in glycerol at -80°C (Note: pH 7.0, NaHCO3 was filtered and sterilized). Select photosynthetic bacterial strains with fast growth rate and uniform and stable fermentation liquid for coenzyme Q 10 Detection.

[0034] 2. Coenzyme Q 10 Detection Methods

[0035] (1) Coenzyme Q 10 Extraction: Take a single colony and inoculate it into an anaerobic tube containing basic ATYP medium, and culture it at 30℃ for 72h. Inoculate it into 500mL ATYP medium sterilized with 10% inoculum, and culture it at 30℃ for 72h. Collect 400mL of photosynthetic bacteria fermentation liquid by centrifugation at 5000rpm for 10min. Discard the supernatant, add 40mL acetone to the bacteria, break the cell wall by ultrasonic for 10min, oscillate at room temperature for 30min, and centrifuge at 8000rpm for 10min. Stand and separate to obtain the supernatant. Dry it on a rotary evaporator at 60℃, dissolve it in 10mL of anhydrous ethanol, and store it away from light for testing.

[0036] (2) Coenzyme Q 10 Content detection: 5mgQ 10 The standard product (Sigma) was dissolved in a 25mL n-hexane volumetric flask, shaken to the scale, and the final concentration was 200μg / mL. 1.25mL, 2mL, 2.5mL, and 5mL of the stock solution were measured and placed in a 10mL volumetric flask, and diluted to the scale with n-hexane to obtain standard solutions with concentrations of 25μg / mL, 40μg / mL, 50μg / mL, and 100μg / mL, respectively. Chromatographic column Diamonsil C18 (200mm*4.6mm, id5μm), mobile phase: methanol: anhydrous ethanol (30:70), liquid phase detection wavelength 275nm, column temperature 25℃, injection 20μL, flow rate 1mL / min. Sample injection detection made a curve of the relationship between the absorption peak area (y) and concentration (x) at a wavelength of 275nm: y=11693x+37319 (see Figure 1 The sample to be tested was diluted 10 times with anhydrous ethanol and the injection volume was 20 μL. 10The highest content was 74.7 mg / L, as shown in Table 1. The test results of each strain are shown in the following table.

[0037] Table 1 CoQ of fermentation broth of different photosynthetic bacterial strains 10 Test results

[0038]

[0039] Example 2: Production of Coenzyme Q 10 Mutation breeding and identification of photosynthetic bacteria Rh09

[0040] The starting strain was Rhodopseudomonas sphaeroides Rh09, which was subjected to UV-nitrosoguanidine combined mutagenesis. The specific process was as follows:

[0041] Growth curve measurement: Activate Rhodopseudomonas sphericalensis Rh09 twice, inoculate into 500mL ATYP liquid culture medium, culture at 30℃ with light, and measure OD value every two hours. Draw growth curve with time as the abscissa and OD value as the ordinate. At the same time, measure bacterial concentration with spiral automatic inoculator.

[0042] Preparation of bacterial suspension: After activating the starting strain twice, inoculate it into 500 mL ATYP liquid medium and culture it at 30°C for 72 hours according to the growth curve. Centrifuge at 5000 rpm for 10 minutes to collect the cells of the photosynthetic bacterial fermentation liquid. Wash it with sterile saline for 3 times to make 10 7 CFU / mL~10 8 CFU / mL bacterial suspension for later use.

[0043] Ultraviolet mutagenesis lethality: 3-5 mL of bacterial suspension was placed in a sterile culture dish with a diameter of 6 cm, placed under a 20W ultraviolet lamp, and irradiated at a distance of 28 cm. Under stirring, irradiation was performed for 10s, 20s, 40s, 60s, 120s, and 240s. The mutagenesis experimental group was appropriately diluted according to the mutagenesis time, and the fermentation liquid of the wild-type strain was diluted to 10 -6 , 10 -7 , 10 -8 As a control, three parallels were made for each dilution, spread on solid culture medium and cultured at 30°C for 3 to 5 days, and the mortality rate was calculated. The results are shown in Table 2.

[0044] Table 2 Ultraviolet induced mortality

[0045]

[0046] Ultraviolet mutagenesis: Select the mutagenesis time with a lethality rate of 70% to 85%, and perform ultraviolet mutagenesis on the starting strain. Take 3 to 5 mL of bacterial suspension and place it in a sterile culture dish with a diameter of 6 cm. Place it under a 20W ultraviolet lamp with an irradiation distance of 28 cm. Under stirring, the irradiation time is 20s and 40s for a mutagenesis time with a lethality rate of 70% to 85%. Inoculate the mutated bacterial solution into fresh liquid culture medium and culture it at 30°C for 72h. After the bacterial solution becomes turbid, spread it for separation, select 100 to 300 strains with a significant change in colony morphology from the starting strain, and compare the survival rate of the mutation. Store the 20 to 70 strains with a higher survival rate at 4°C, and then perform coenzyme Q 10 Content detection (according to the method in Example 1), among which the strain numbered Rh09-40 performed best, coenzyme Q 10 The content reached 224.1 mg / L.

[0047] Nitrosoguanidine mutagenesis lethality: The superior mutant strain Rh09-40 selected by UV mutagenesis was activated to produce 10 7 CFU / mL~10 8 CFU / mL bacterial suspension, nitrosoguanidine solution was prepared at a ratio of 10mg NTG: 1mL acetone, and the bacterial suspension was treated at a concentration of 0.3g / L, 30℃, 100r / min, and the treatment time was 30min, 40min, 50min, and 60min respectively. The mutagenesis experimental group was appropriately diluted according to the mutagenesis time, and the fermentation broth of the wild-type strain was diluted to 10 -6 , 10 -7 , 10 -8 As a control, three replicates were made for each dilution, plated on solid culture medium, and cultured at 30°C for 3 to 5 days. The lethality was calculated and the results are shown in Table 3.

[0048] Table 3 Nitrosoguanidine mutagenesis lethality

[0049]

[0050] Nitrosoguanidine mutagenesis: Select a mutagenesis time with a lethality rate of 70% to 85%, and subject the starting strain to nitrosoguanidine mutagenesis. Prepare a nitrosoguanidine solution at a ratio of 10 mg NTG: 1 mL acetone, and treat the bacterial suspension at a concentration of 0.3 g / L, 30°C, and 100 r / min for 40 minutes. Inoculate the mutated bacterial solution into a fresh liquid culture medium and culture it at 30°C for 72 hours. After the bacterial solution turns bright red, spread it for separation, and select 100 to 200 strains with a significant change in colony morphology from the starting strain, and compare the survival rate of the mutation. Store the 20 to 70 strains with a higher survival rate at 4°C, and then perform coenzyme Q 10 Content detection (according to the method in Example 1), among which the strain numbered Rh09-40-6 had the best result, coenzyme Q 10The content reached 380.97 mg / L and was named RhM09.

[0051] The 16S rDNA fragment of strain RhM09 was amplified using the bacterial 16S universal primers 5'-gagagtttgatcctggctcag-3' and 5'-cggctaccttgttacgactt--3'. The template DNA was extracted according to the instructions of the bacterial DNA extraction kit. The reaction system was 20 μL: 2 μL of 10×PCR buffer, 0.5 μL of each primer (20 μmol / L), 2 μL of dNTP (2.5 mmol / L), 0.2 μL of Taq enzyme (5 U / μL), 1 μL of template DNA, and water was added to 20 μL. Reaction conditions: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles, and 72℃ extension for 5 min.

[0052] After 16S rDNA sequencing of strain RhM09, the obtained gene sequence was compared in the NCBI database (sequence table SEQ ID No. 1), and the strain was identified as Rhodopseudomonas spherical. The obtained Rhodopseudomonas spherical was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on May 5, 2023, with the deposit address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No. 27269.

[0053] Example 3 High-yield Coenzyme Q by Rhodopseudomonas sphaeroides CGMCC No.27269 10 Medium optimization

[0054] To obtain high concentration of CoQ 10 The photosynthetic bacteria fermentation broth was based on ATYP medium, and the carbon source, nitrogen source, composite culture and additional additives of CGMCC No.27269 medium were further optimized by single factor method.

[0055] Carbon source types: NaAc, glucose, sucrose, sodium bicarbonate, and the optimal carbon source concentration gradient was set as: 0.5 g / L, 1.5 g / L, 2.5 g / L, 3.5 g / L, and 4.5 g / L.

[0056] Types of nitrogen sources: ammonium sulfate, ammonium chloride, urea. The optimal nitrogen source concentration setting gradient is: 1.0g / L, 1.5g / L, 2.0g / L, 2.5g / L, 3.0g / L.

[0057] Composite culture: yeast powder, beef powder, peptone. The optimal composite culture concentration setting gradient: 0.5g / L, 1.5g / L, 2.5g / L, 3.5g / L.

[0058] Additional additives phenylalanine, tyrosine, methionine and concentration settings: 25mg / L, 75mg / L, 125mg / L, 175mg / L, 225mg / L.

[0059] The bacterial culture and coenzyme Q were carried out according to the method in Example 1. 10 Detection.

[0060] The results confirmed that fermentation of RhM09 produced Q 10 The best carbon source is sodium acetate (see Figure 2 ), the optimal concentration is 2.5 g / L (see Figure 3 ); the best nitrogen source is ammonium chloride (see Figure 4 ), the optimal concentration is 1.5g / L (see Figure 5 ); the best composite culture is yeast powder (see Figure 6 ), the optimal concentration is 1.5 g / L (see Figure 7 ); the best additional accelerator is tyrosine (see Figure 8 ), the optimal concentration is 75 mg / L (see Fig. 9 ), based on the above, the optimal fermentation time was determined to be 56h (see Fig.10 ). Ammonium chloride, sodium acetate and yeast powder were selected for three-factor three-level response surface analysis, and the coenzyme Q10 content was determined at 56h of RhM09 fermentation. The response surface analysis factors and levels are shown in Table 4, and the response surface experimental design results are shown in Table 5.

[0061] Table 4 Response surface analysis factors and levels

[0062]

[0063] Table 5 Response surface experimental design results

[0064]

[0065] According to the model prediction, when NH4Cl 1.16g / L, NaAc 2.87g / L, yeast powder 1.22g / L, CoQ 10 The maximum content is 510.259 mg / L.

[0066] Verification experiment: The culture medium was prepared according to the theoretical composition: NH4Cl 1.16g / L, NaAc 2.87g / L, yeast powder 1.22g / L, tyrosine 75mg / L, KH2PO4 0.5g / L, CaCl2 0.1g / L, MgCl2·6H2O 0.5g / L, NaCl 1g / L. After testing, CoQ 10 The content detection value was 505 mg / L, which was 32.56% higher than before optimization.

[0067] Example 4 Preparation of Rhodopseudomonas sphaeroides CGMCC No. 27269 bacterial powder (vacuum freeze drying)

[0068] The seed liquid of the spherical red pseudomonas is inoculated into a transparent container filled with a fermentation medium according to an inoculation amount of 5-10%, and the temperature is 28-35° C. and the light is cultured for 56 hours to obtain a fermentation liquid of the spherical red pseudomonas, wherein the composition and content of the fermentation medium are: NH4Cl 1.16g / L, NaAc 2.87g / L, yeast powder 1.22g / L, tyrosine 75mg / L, KH2PO4 0.5g / L, CaCl2 0.1g / L, MgCl2·6H2O 0.5g / L, and NaCl 1g / L.

[0069] The fermentation liquid prepared as above was centrifuged in a high-speed refrigerated centrifuge at 5000rpm, 4°C, for 10min. After centrifugation, the supernatant was discarded to obtain bacterial sludge. 5.0% sucrose, 1.0% sodium glutamate, and 1.0% ascorbic acid aqueous solution were added to the bacterial sludge and mixed evenly, and 50% skimmed milk powder was added. The ratio of bacterial sludge to water was 1:2, first placed in a -80°C ultra-low temperature refrigerator for freezing, then placed in a vacuum freeze dryer for drying for 24 to 30 hours, taken out, crushed, and passed through a 60-mesh sieve. The number of viable bacteria was 1×10 10 CFU / g bacterial powder, CoQ 10 The content is 6.8~7.5mg / g.

[0070] Example 5 Preparation of Rhodopseudomonas sphaeroides CGMCC No.27269 bacterial powder (spray drying)

[0071] 15% porous starch and 10% cyclodextrin were added to the fermentation liquid prepared in Example 4, mixed, and spray-dried. The inlet air temperature was 125°C, the exhaust air temperature was 50°C, the atomizer speed was 15000-18000 rpm, and the CoQ 10 The content is 9.2~10.6mg / g.

[0072] Test Example 1: Application of Rhodopseudomonas sphaeroides CGMCC No.27269 in enhancing shrimp resistance to ammonia nitrogen, nitrite, hypoxia and low salt stress

[0073] RhM09 was fermented in the optimal medium according to Example 3, with a seed liquid inoculation amount of 10%, and irradiation at 30°C for 5 days to obtain a fermentation liquid. Before the experiment, the water temperature, pH, ammonia nitrogen content, nitrite nitrogen content, dissolved oxygen content and salinity of natural seawater, and the weight of shrimp were measured, as shown in Table 6.

[0074] Table 6 Natural seawater parameters

[0075]

[0076] (1) Ammonia nitrogen stress test:

[0077] Take 20.0g of ammonium chloride and dissolve it in 1L of distilled water to obtain a 20g / L ammonium chloride solution. Take four white experimental barrels, put 10L of seawater (aquaculture water can be used) in each, add 500mL of 20g / L ammonium chloride solution to each, and the ammonium chloride content in the water body is 1g / L. After mixing, keep sufficient dissolved oxygen.

[0078] 15 shrimps with the same growth conditions were placed in the experimental group and the control group, with two parallel groups in each group. 50mL RhM09 fermentation liquid was added to the experimental group. No addition was made to the control group. The shrimp status was observed every 60 minutes and the mortality rate was recorded. As shown in Table 7, the mortality rate of shrimps in the control group gradually increased with time, reaching 100% at 5 hours; while shrimps in the experimental group began to die at 3 hours. Although the mortality rate increased with time, it was far lower than the 100% mortality rate of the control group. Therefore, photosynthetic bacteria RhM09 can alleviate the stress of ammonia nitrogen on shrimps.

[0079] Table 7 Results of shrimp ammonia nitrogen stress test

[0080]

[0081] (2) Nitrite stress test:

[0082] Take 20.0g of sodium nitrite and dissolve it in 1L of distilled water to obtain a 20g / L sodium nitrite solution. Take four white experimental barrels, put 10L of seawater (aquaculture water can be used) in each, add 300mL of 20g / L sodium nitrite solution to each, and the sodium nitrite content in the water body is 0.6g / L. After mixing, keep sufficient dissolved oxygen.

[0083] Take 15 shrimps with the same growth conditions and put them in the experimental group and the control group, with two parallels in each group. The experimental group was added with 50mL RhM09 fermentation liquid. The control group was not added. The shrimp status was observed every 60 minutes and the mortality rate was recorded. As shown in Table 8, shrimps began to die in the control group at 1 hour, and the mortality rate gradually increased with time, reaching 26.67% at 8 hours of the experiment. However, shrimps began to die in the experimental group at 6 hours. At 8 hours, the mortality rate of shrimps in the experimental group (13.33%) was still lower than that in the control group (26.67%), indicating that photosynthetic bacteria RhM09 can alleviate the stress of nitrite on shrimps.

[0084] Table 8 Results of nitrite stress test on shrimp

[0085]

[0086]

[0087] (3) Hypoxia stress

[0088] Take four white experimental buckets, put 10L seawater (breeding water can be used) in each, take 15 shrimps with the same growth status and put them in the experimental group and the control group, two in each group, and the water body is not oxygenated. 50mL RhM09 fermentation liquid is added to the experimental group, and no addition is made to the control group. The dissolved oxygen content of the water body during the experiment is recorded, and the state of the shrimp is observed every 60 minutes and the mortality rate is recorded. As shown in Table 9, with the extension of time, the mortality rate of shrimps in the control group has been increasing, reaching 100% at 6h. Although the mortality rate of shrimps in the experimental group has also increased, it has been lower than that of the control group. The mortality rate of shrimps in the experimental group at 6h of the experiment is only 46.67%. Therefore, photosynthetic bacteria RhM09 can enhance the tolerance of shrimps to hypoxic conditions.

[0089] Table 9 Results of shrimp hypoxia stress test

[0090]

[0091] (4) Low salt stress

[0092] Take four white experimental buckets, put 10L of tap water in each, and aerate 24 hours in advance to discharge chlorine from the water. Dilute natural seawater to a salinity of 10 with tap water that has been aerated for 24 hours in advance, and measure the salinity of the experimental water with a refractometer. Take 15 shrimps with the same growth conditions and put them in the experimental group and the control group, with two parallel groups in each group. 50mL of RhM09 fermentation liquid is added to the experimental group, and no addition is made to the control group. Observe the state of the shrimp every 60 minutes and record the cumulative mortality rate. As shown in Table 10, shrimp deaths began to appear in the control group at 4 hours, and the mortality rate of shrimp reached 33.33% at 7 hours; shrimp deaths began to appear in the experimental group at 6 hours, and the mortality rate was only 6.67% at 8 hours. Therefore, photosynthetic bacteria RhM09 can enhance the tolerance of shrimp to low salt conditions.

[0093] Table 10 Results of low salt stress test on shrimp

[0094]

[0095] Test Example 2: Improving the survival rate of commercial shrimp during transportation

[0096] Three days before the shrimp were caught, the RhM09 fermentation liquid was continuously mixed with the shrimp in pond 1, and the fermentation liquid was added at a ratio of 1‰; the feed fed to the shrimp in pond 2 was not mixed with the fermentation liquid of Rhodopseudomonas spherical RhM09. Feeding was stopped on the day of fishing. The shrimps in the two ponds were caught separately into buckets and aerated in time to make the water in the bucket tumble. The fine-mesh mesh was fixed at the mouth of the bucket to prevent the shrimp from shaking out. The shrimps were transported from the Tianjin breeding base to the Chongqing Aquatic Products Wholesale Market, and the survival rates of the shrimps were tested and compared. The reduced survival rate of shrimps caused by high-density and long-distance transportation is a prominent problem affecting the benefits of shrimp farming. As shown in Table 11, feeding shrimps with the microecological preparation mixed with feed can increase the survival rate of shrimps by 12% during transportation.

[0097] Table 11 Results of shrimp transportation survival test

[0098]

[0099] Sequence Listing

[0100] SEQ ID No.1

[0101]

Claims

1. A strain of Rhodopseudomonas sphaeroides, whose deposit number is CGMCC No.27269.

2. The Rhodopseudomonas sphaeroides strain according to claim 1, characterized in that Its 16S rRNA sequence is shown in Table SEQID No.

1.

3. A microecological preparation containing the Rhodopseudomonas sphaeroides strain according to claim 1 or 2.

4. The microecological preparation according to claim 3, characterized in that: The microecological preparation contains the fermentation liquid or bacterial powder of the Rhodopseudomonas spherical.

5. The microecological preparation according to claim 4, characterized in that: The preparation method of the fermentation liquid of the spherical red pseudomonas is as follows: the seed liquid of the spherical red pseudomonas is inoculated into a transparent container filled with a fermentation medium according to an inoculation amount of 5-10%, and the temperature is 28-35°C and the light is cultured for 48-72 hours to obtain the fermentation liquid of the spherical red pseudomonas, wherein the composition and content of the fermentation medium are: sodium acetate 1-3g / L, NH4Cl 1-5g / L, yeast powder 0.5-2.5g / L, tyrosine 75mg / L, KH2PO4 0.5 g / L, CaCl2 0.1g / L, MgCl2 . 6H2O 0.5g / L, NaCl 1g / L.

6. The microecological preparation according to claim 4, characterized in that: The Rhodopseudomonas sphaeroides powder is vacuum freeze-dried powder or spray-dried powder.

7. Use of the Rhodopseudomonas sphaeroides according to claim 1 or 2 in the preparation of a feed additive for enhancing shrimp resistance to ammonia nitrogen, nitrite, low salt or hypoxia stress environments.

8. Use of the probiotic preparation according to any one of claims 3 to 6 in the preparation of a feed additive for enhancing shrimp resistance to ammonia nitrogen, nitrite, low salt or hypoxia stress environments.

9. The Rhodopseudomonas sphericalensis according to claim 1 or 2 is used to prepare coenzyme Q 10 Application in.

10. The probiotic preparation according to any one of claims 3 to 6 in the preparation of coenzyme Q 10 Application in.

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