Rhodopseudomonas spheroides, microecological preparation thereof and application thereof

Abstract

The application discloses a spherical red pseudomonas with high coenzyme Q 10 production capacity, and belongs to the technical field of microorganism application. The application particularly relates to a spherical red pseudomonas, a microecological preparation thereof and application. The application takes a spherical red pseudomonas with coenzyme Q 10 production capacity as a starting strain, and obtains a strain with significantly improved coenzyme Q 10 production capacity through ultraviolet mutagenesis treatment. After a culture medium is optimized, the content of coenzyme Q 10 in a fermentation liquor reaches 505 mg / L. The strain has been preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 27269. The fermentation liquor of the strain is mixed with feed to feed South American white prawns, so that the South American white prawns can enhance the resistance to ammonia nitrogen, nitrite, hypoxia and low-salt environmental stress, and improve the survival rate during breeding and transportation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial application, and particularly relates to a Rhodopseudomonas sphaeroides, a microecological preparation thereof and application. BACKGROUND

[0002] At present, high-density culture and over-feeding of Penaeus vannamei result in insufficient dissolved oxygen and water quality deterioration such as excessive ammonia nitrogen and nitrite in water. Ammonia nitrogen refers to nitrogen in the form of free ammonia (NH3) and ammonium ion (NH4 + ) in water. Free ammonia has toxic effects on aquatic animals by affecting osmotic pressure balance, biochemical reactions in the body, and the toxicity increases with the increase of pH and water temperature, which can cause excitement, loss of balance, convulsions and even death of aquatic animals. Nitrite is an important intermediate product in the nitrogen cycle of the ecosystem, and is also a common pollutant in aquaculture. High concentration of nitrite in water environment seriously threatens the health of aquatic animals, and can accumulate in large amounts in the blood, causing physiological processes such as respiration, cardiovascular and endocrine disorders, and even causing anoxic asphyxia death.

[0003] Photosynthetic bacteria have rich nutritional components, and are not only excellent single-cell proteins, but also contain various vitamins, carotenoids and coenzyme Q 10 . Coenzyme Q 10 is a natural antioxidant and in vivo free radical scavenger. It has the effects of anti-aging and improving the body's immunity. Therefore, breeding photosynthetic bacteria with high coenzyme Q 10 yield and using them in shrimp culture have important significance for enhancing the resistance of shrimps to external stress and improving the survival rate of culture. SUMMARY

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

[0005] The Rhodopseudomonas sphaeroides CGMCC No.27269 provided by the present application is obtained by mutagenesis breeding from a Rhodopseudomonas sphaeroides strain from a seawater pond. The strain has the ability to produce high coenzyme Q 10 , and the coenzyme Q 10 content of the fermentation broth reaches 505mg / L after optimization of the culture medium.

[0006] The Rhodopseudomonas sphaeroides provided by the present application was preserved in the China General Microbiological Culture Collection Center on May 5, 2023, and the address is No.3, Beichen West Road, Yard 1, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, and the preservation number is CGMCC No.27269.

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

[0008] The present application also provides a microecological preparation containing the Rhodopseudomonas sphaeroides.

[0009] The present application also provides a fermentation medium of the Rhodopseudomonas sphaeroides, in particular:

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

[0011] The seed liquid of the Rhodopseudomonas sphaeroides is inoculated into a transparent container containing the fermentation medium at an inoculation amount of 5-10%, and cultured at a temperature of 28-35°C under light for 48-72 h, to obtain the fermentation liquid of the Rhodopseudomonas sphaeroides.

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

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

[0014] In particular, the Rhodopseudomonas sphaeroides or the microecological preparation thereof is used to feed shrimps by mixing with feed.

[0015] The present application also provides an application of the Rhodopseudomonas sphaeroides or the microecological preparation containing the Rhodopseudomonas sphaeroides in preparing coenzyme Q 10 .

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

[0017] The present application provides a new Rhodopseudomonas sphaeroides, and after optimization of the fermentation medium, the content of coenzyme Q 10 in the fermentation liquid of the strain reaches 505 mg / L. Through experiments, it is verified that the strain can enhance the resistance of shrimps 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 DRAWINGS

[0018] Figure 1 CoQ 10 standard curve graph;

[0019] Figure 2Optimization results of carbon source type for single factor experiment

[0020] Figure 3 Optimization results of optimal carbon source concentration for single factor experiment

[0021] Figure 4 Optimization results of nitrogen source type for single factor experiment

[0022] Figure 5 Optimization results of optimal nitrogen source concentration for single factor experiment

[0023] Figure 6 Optimization results of composite culture type for single factor experiment

[0024] Figure 7 Optimization results of optimal composite culture concentration for single factor experiment

[0025] Figure 8 Optimization results of additional additive type for single factor experiment

[0026] Figure 9 Optimization results of additional additive concentration for single factor experiment

[0027] Figure 10 Optimization results of CoQ 10 content with fermentation time. DETAILED DESCRIPTION

[0028] The embodiments of the present application will be described in detail below with examples, but those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. The specific conditions not noted in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments not noted for the manufacturer are all the conventional products available in the market.

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

[0030] 1. Photosynthetic bacteria isolation and screening

[0031] (1) Enrichment medium (per L): NaHCO32.0 g, yeast powder 1 g, K2HPO40.2 g, NH4Cl 1 g, NaAc 0.3 g, MgSO4·7H2O 0.2 g, NaCl 0.2 g.

[0032] (2) Basic fermentation medium ATYP (per L): KH2PO40.5 g, NH4Cl 1 g, CaCl20.1 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 samples collected from the shrimp ponds, add to the anaerobic tube containing 10 mL enrichment medium, cover with 1-2 cm paraffin oil, 30°C light culture until the liquid turns red. 10-fold gradient dilution of the red enrichment liquid, take 10 -3 ~10 -5 Dilution liquid 100 μL evenly coated in enrichment medium agar plates, 30°C anaerobic incubator light culture 5-7d until red colonies grow, purified 2-3 times. Pick the purified photosynthetic bacterial strains inoculated in ATYP liquid medium 30°C static light culture 72h, -80°C glycerol preservation (note: pH 7.0, NaHCO3 filter sterilization). Select the fast growth, stable fermentation broth uniform photosynthetic bacterial strains for coenzyme Q 10 detection.

[0034] 2、Coenzyme Q 10 detection method

[0035] (1) Coenzyme Q 10 extraction: take a single colony inoculated in anaerobic tube containing ATYP base medium, 30°C light culture 72h. Inoculated in sterile 500 mL ATYP medium with 10% inoculation, 30°C light culture 72h. 5000 rpm centrifugation 10 min to collect 400 mL photosynthetic bacterial fermentation broth. Discard the supernatant, add 40 mL acetone to the bacterial body, ultrasonic wave broken wall 10 min, room temperature oscillation 30 min, 8000 rpm centrifugation 10 min. Separate the supernatant by standing.

[0036] (2) Coenzyme Q 10 content detection: 5 mg Q 10 10 standard (Sigma) was dissolved in 25 mL n-hexane volumetric flask, shake well to the mark, the final concentration was 200 μg / mL. 1.25 mL, 2 mL, 2.5 mL, 5 mL of stock solution was placed in 10 mL volumetric flask, n-hexane was diluted to the mark to get the concentration of 25 μg / mL, 40 μg / mL, 50 μg / mL, 100 μg / mL standard solution. Chromatographic column Diamonsil C18 (200 mm*4.6 mm, i.d. 5 μm), mobile phase: methanol: anhydrous ethanol (30:70), liquid phase detection wavelength 275 nm, column temperature 25°C, injection 20 μL, flow rate 1 mL / min. Injection detection of the relationship between the absorption peak area (y) and concentration (x) at wavelength 275 nm: y = 11693x + 37319 (see Figure 1 ). The sample was diluted 10 times with anhydrous ethanol, and the injection amount was 20 μL. After detection, the coenzyme Q 10The content was the highest, up to 74.7 mg / L, see Table 1. The test results of each strain are as follows in the table.

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

[0038]

[0039] Example 2: Coenzyme Q production 10 Mutagenic selection and identification of photosynthetic bacteria Rh09

[0040] The starting strain was Rhodopseudomonas spheroides Rh09, which was subjected to ultraviolet-nitroso guanidine combined mutagenesis, and the specific process was as follows:

[0041] Measurement of growth curve: Rhodopseudomonas spheroides Rh09 was activated twice and inoculated into 500 mL ATYP liquid medium, and the OD value was measured every two hours during 30°C static light culture. The horizontal coordinate is time and the vertical coordinate is OD value, and the growth curve is drawn. At the same time, the bacterial concentration was measured by spiral automatic inoculator.

[0042] Preparation of bacterial suspension: the starting strain was activated twice and inoculated into 500 mL ATYP liquid medium, and according to the growth curve, 30°C light culture was carried out for 72 h. The bacterial body of photosynthetic bacterial fermentation broth was collected by centrifugation at 5000 rpm for 10 min. It was washed with sterile normal saline for 3 times to prepare 10 7 CFU / mL~10 8 CFU / mL bacterial suspension for standby.

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

[0044] Table 2 Ultraviolet mutagenesis lethality

[0045]

[0046] UV mutagenesis breeding: choose mutagenesis time with lethal rate of 70%~85%, mutagenize the starting strain, take 3~5 mL bacterial suspension in a sterile petri dish with a diameter of 6 cm, place it under a 20 W UV lamp with an irradiation distance of 28 cm, and irradiate it for 20 s and 40 s under stirring conditions, i.e. the mutagenesis time with a lethal rate of 70%~85%. Then inoculate the mutagenized bacterial solution into fresh liquid medium and culture it at 30°C for 72 h. After the bacterial solution turns turbid, perform coating separation, select 100~300 strains with a change in colony morphology greater than that of the starting strain, and compare the survival rates of the mutants. Store 20~70 strains with a higher survival rate at 4°C, and then perform coenzyme Q 10 content detection (according to the method in Example 1), wherein the strain numbered Rh09-40 performs best, and the coenzyme Q 10 content reaches 224.1 mg / L.

[0047] Nitrosoguanidine mutagenesis lethal rate: activate the excellent mutant strain Rh09-40 mutagenized by UV mutagenesis, and prepare 10 7 CFU / mL~10 8 CFU / mL bacterial suspension. Prepare a nitrosoguanidine solution at a ratio of 10 mg NTG: 1 mL acetone, treat the bacterial suspension at a concentration of 0.3 g / L, 30°C, and 100 r / min, and the treatment time is 30 min, 40 min, 50 min, and 60 min. Dilute the experimental group according to the mutagenesis time as appropriate, and dilute the wild-type strain fermentation broth to 10 -6 , 10 -7 , 10 -8 as a control. Perform three parallel tests at each dilution, coat them on a solid medium, and culture them at 30°C for 3~5 days. Calculate the lethal rate, and the results are shown in Table 3.

[0048] Table 3 Nitrosoguanidine mutagenesis lethal rate

[0049]

[0050] Nitrosoguanidine mutagenesis breeding: choose mutagenesis time with a lethal rate of 70%~85%, mutagenize the starting strain by nitrosoguanidine mutagenesis, prepare a nitrosoguanidine solution at a ratio of 10 mg NTG: 1 mL acetone, treat the bacterial suspension at a concentration of 0.3 g / L, 30°C, and 100 r / min, and the treatment time is 40 min. Then inoculate the mutagenized bacterial solution into fresh liquid medium and culture it at 30°C for 72 h. After the bacterial solution turns bright red, perform coating separation, select 100~200 strains with a change in colony morphology greater than that of the starting strain, and compare the survival rates of the mutants. Store 20~70 strains with a higher survival rate at 4°C, and then perform coenzyme Q 10 content detection (according to the method in Example 1), wherein the strain numbered Rh09-40-6 performs best, and the coenzyme Q 10The content reached 380.97 mg / L, and was named RhM09.

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

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

[0053] Example 3 High production of coenzyme Q by Rhodopseudomonas spheroides CGMCC No. 27269 10 Medium optimization

[0054] In order to obtain a photosynthetic bacterial fermentation broth containing high concentration of CoQ 10 Based on the ATYP medium, the single factor method was used to further optimize the carbon source, nitrogen source, complex culture, and additional additives of the CGMCC No. 27269 culture medium.

[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] Nitrogen source types: ammonium sulfate, ammonium chloride, and urea, and the optimal nitrogen source concentration gradient was set as 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L, and 3.0 g / L.

[0057] Complex culture: yeast powder, beef powder, and peptone, and the optimal complex culture concentration gradient was set as 0.5 g / L, 1.5 g / L, 2.5 g / L, and 3.5 g / L.

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

[0059] Bacterial culture and coenzyme Q were performed according to the method described in Example 1. 10 Testing.

[0060] The results confirmed that fermentation of RhM09 produced Q. 10 The optimal carbon source is sodium acetate (see Figure 2 The optimal concentration is 2.5 g / L (see...). Figure 3 The optimal nitrogen source is ammonium chloride (see...). Figure 4 The optimal concentration is 1.5 g / L (see...). Figure 5 The optimal compound culture is yeast extract (see...); Figure 6 The optimal concentration is 1.5 g / L (see...). Figure 7 The optimal additional promoter is tyrosine (see...); Figure 8 The optimal concentration is 75 mg / L (see...). Figure 9 Based on the above, the optimal fermentation time was determined to be 56 hours (see...). Figure 10 A three-factor, three-level response surface methodology was used to analyze the coenzyme Q10 content of RhM09 after 56 hours of fermentation. The factors and levels of the response surface methodology are shown in Table 4, and the results of the response surface experimental design are shown in Table 5.

[0061] Table 4 Factors and Levels in Response Surface Analysis

[0062]

[0063] Table 5 Results of Response Surface Experimental Design

[0064]

[0065] According to model predictions, when NH4Cl is 1.16 g / L, NaAc is 2.87 g / L, and yeast extract is 1.22 g / L, CoQ 10 The maximum concentration is 510.259 mg / L.

[0066] Verification experiment: The culture medium was prepared according to the theoretical composition: NH4Cl 1.16 g / L, NaAc 2.87 g / L, yeast extract 1.22 g / L, tyrosine 75 mg / L, KH2PO4 0.5 g / L, CaCl2 0.1 g / L, MgCl2·6H2O 0.5 g / L, NaCl 1 g / L. The CoQ20 was tested... 10 The content was detected at 505 mg / L, which is 32.56% higher than before optimization.

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

[0068] The seed liquid of the Rhodopseudomonas spheroides was inoculated into a transparent container containing a fermentation medium at an inoculation amount of 5-10%, the temperature was 28-35°C, and the light culture was performed for 56 h to obtain a fermentation liquid of the Rhodopseudomonas spheroides, wherein the fermentation medium was composed of NH4Cl 1.16 g / L, NaAc 2.87 g / L, yeast powder 1.22 g / L, tyrosine 75 mg / L, KH2PO40.5 g / L, CaCl20.1 g / L, MgCl2·6H2O 0.5 g / L, and NaCl 1 g / L.

[0069] The fermentation liquid prepared above was centrifuged by a high-speed refrigerated centrifuge at 5000 rpm and 4°C for 10 min. After centrifugation, the supernatant was discarded to obtain a bacterial slurry. 5.0% sucrose, 1.0% sodium glutamate, and 1.0% ascorbic acid aqueous solution were added to the bacterial slurry, and 50% skimmed milk powder was added. The bacterial slurry and water were added at a ratio of 1:2, and then were frozen in a -80°C ultra-low temperature refrigerator and dried in a vacuum freeze dryer for 24-30 h. The powder was obtained by crushing and passing through a 60-mesh sieve. The obtained powder had a viable cell count of 1×10 10 CFU / g, and a CoQ 10 content of 6.8-7.5 mg / g.

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

[0071] The fermentation liquid prepared in Example 4 was mixed with 15% porous starch and 10% cyclodextrin, and then was subjected to spray drying at an inlet temperature of 125°C and an exhaust temperature of 50°C, and the atomizer was rotated at a speed of 15000-18000 rpm. The obtained powder had a CoQ 10 content of 9.2-10.6 mg / g.

[0072] Test Example 1: Application of Rhodopseudomonas spheroides CGMCC No. 27269 in enhancing the resistance of shrimps to ammonia nitrogen, nitrite, anoxia, and low-salt stress

[0073] RhM09 was fermented according to the optimal medium of Example 3, and the seed liquid was inoculated at an inoculation amount of 10%, and the light culture was performed at 30°C for 5 days to obtain a fermentation liquid. The water temperature, pH, ammonia nitrogen content, nitrite nitrogen content, dissolved oxygen content, and salinity of the natural seawater were determined before the experiment, and the body weight of the shrimps was determined, as shown in Table 6.

[0074] Table 6 Parameters of natural seawater

[0075]

[0076] (1) Ammonia nitrogen stress test:

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

[0078] Take 15 prawns with consistent growth conditions in the experimental group and the control group, each group with two parallel, the test group adds 50 mL of RhM09 fermentation broth. The control group does not add. Observe the state of prawns and record the mortality every 60 min. As shown in Table 7, the mortality of prawns in the control group gradually increases with time, and reaches 100% at 5h; while the test group starts to appear prawn death at 3h, and although the mortality is also increasing with time, it is far lower than the 100% mortality of the control group. Therefore, photosynthetic bacteria RhM09 can reduce the stress of ammonia nitrogen on prawns.

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

[0080]

[0081] (2) Nitrite stress test:

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

[0083] Take 15 prawns with consistent growth conditions in the experimental group and the control group, each group with two parallel, the test group adds 50 mL of RhM09 fermentation broth. The control group does not add. Observe the state of prawns and record the mortality every 60 min. As shown in Table 8, the control group starts to appear prawn death at 1h, and the mortality gradually increases with time, and reaches 26.67% at 8h of test. While the test group starts to appear prawn death at 6h, and the mortality of prawns in the test group (13.33%) is still lower than that of the control group (26.67%) at 8h, indicating that photosynthetic bacteria RhM09 can reduce the stress of nitrite on prawns.

[0084] Table 8 Results of prawn nitrite stress test

[0085]

[0086]

[0087] (3) Hypoxia stress

[0088] Take four white experimental barrels, each 10 L of seawater (can be used for aquaculture water), take the growth of 15 prawns each placed in the experimental group and the control group, each group of two parallel, water without oxygen. The test group added 50 mL RhM09 fermentation broth, the control group without adding. Record the experimental water dissolved oxygen content, every 60 min observation and record the mortality rate of prawns. As shown in Table 9, the mortality rate of prawns in the control group increased with time, to 6h reached 100%. While the mortality rate of prawns in the test group also increased, but has been lower than the control group, test 6h test group prawn mortality rate was only 46.67%. Therefore, photosynthetic bacteria RhM09 can enhance the tolerance of prawns to hypoxia.

[0089] Table 9 Prawn hypoxia stress test results

[0090]

[0091] (4) low salt stress

[0092] Take four white experimental barrels, each 10 L of tap water, 24 h in advance for aeration, discharge water chlorine. The tap water diluted with natural seawater to a salinity of 10, measured by refractive salinity meter. Take the growth of 15 prawns each placed in the experimental group and the control group, each group of two parallel, test group added 50 mL RhM09 fermentation broth, the control group without adding. Every 60 min observation and record the cumulative mortality rate of prawns. As shown in Table 10, the control group 4h began to appear prawn death, 7h prawn mortality rate reached 33.33%; test group 6h began to appear prawn death, to 8h mortality rate was only 6.67%. Therefore, photosynthetic bacteria RhM09 can enhance the tolerance of prawns to low salt conditions.

[0093] Table 10 Prawn low salt stress test results

[0094]

[0095] Test Example 2: improve the survival rate of commodity shrimp transportation

[0096] Pond 1 in the three days before the capture of prawns, with RhM09 fermentation broth continuously mixed with prawns, fermentation broth added proportion of 1 ‰; Pond 2 prawns fed feed without RhM09 fermentation broth. The day of capture stopped feeding. Two ponds of prawns were caught separately into the barrel and timely oxygenation to make the water in the barrel to roll, barrel mouth fixed fine mesh screen, to prevent the prawns shake out. From Tianjin aquaculture base to Chongqing aquatic wholesale market, test and compare the survival rate of prawns. High density, long distance transportation of prawns leads to a decrease in survival rate is a prominent problem affecting the yield of prawn culture. As shown in Table 11, the use of the microbial preparation mixed with prawns can improve the survival rate of prawns by 12% during transportation.

[0097] Table 11. Results of shrimp transport survival test

[0098]

[0099] Sequence Listing

[0100] SEQ ID No. 1

[0101]

Claims

1. A Rhodopseudomonas sphaeroides strain, with the accession number of CGMCC No. 27269.

2. The spheroid Rhodopseudomonas strain of claim 1, characterized in that, The 16S rRNA sequence of which is shown in Table SEQ ID No.

1.

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

4. The microecological preparation according to claim 3, characterized in that, The microecological preparation contains a fermentation broth or a bacterial powder of the Rhodopseudomonas sphaeroides strain.

5. The microecological preparation according to claim 4, characterized in that, The preparation method of the fermentation liquor of the Rhodopseudomonas sphaeroides comprises the following steps: inoculating the seed liquid of the Rhodopseudomonas sphaeroides into a transparent container filled with a fermentation medium at an inoculation amount of 5-10%, and culturing under light irradiation at a temperature of 28-35 ℃ for 48-72 h, so that the fermentation liquor of the Rhodopseudomonas sphaeroides is obtained, wherein the fermentation medium comprises the following components and in the following amounts: sodium acetate 1-3 g / L, NH4Cl 1-5 g / L, yeast powder 0.5-2.5 g / L, tyrosine 75 mg / L, KH2PO4 0.5 g / L, CaCl2 0.1 g / L, MgCl2 . 6H2O 0.5 g / L, and NaCl 1 g / L.

6. The microecological preparation according to claim 4, characterized in that, The bacterial powder of the Rhodopseudomonas sphaeroides strain is a vacuum freeze-dried bacterial powder or a spray-dried bacterial powder.

7. Use of the Rhodopseudomonas sphaeroides strain of claim 1 or 2 in the preparation of a feed additive for enhancing the resistance of shrimps to ammonia nitrogen, nitrite, low-salt or anoxic stress environment.

8. Use of the microecological preparation of any one of claims 3 to 6 in the preparation of a feed additive for enhancing the resistance of shrimps to ammonia nitrogen, nitrite, low-salt or anoxic stress environment.

9. Use of the spherical Pseudomonas rhodozyma of claim 1 or 2 for the production of coenzyme Q 10 .

10. Use of the microecological preparation according to any one of claims 3 to 6 for the preparation of coenzyme Q 10 .

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