Streptomyces pseudogriseogriseus XH-1 as well as microecological preparation and preparation method of streptomyces pseudogriseogriseus XH-1

Abstract

The invention relates to streptomyces pseudogriseogriseus XH-1 as well as a microecological preparation and a preparation method thereof, the streptomyces pseudogriseogriseus XH-1 is preserved in the China Center for Type Culture Collection on August 26, 2024, and the strain preservation number is CCTCC NO: M 20241850; the invention discloses streptomyces pseudogriseolus XH-1, which is classified and named as streptomyces pseudogriseolus XH-1, and the Latin name of the streptomyces pseudogriseolus XH-1 is Streptomyces pseudogriseolus XH-1. The microecological preparation is prepared by fermenting streptomyces pseudogriseus XH-1. The invention further discloses a preparation method of the streptomyces pseudogriseogriseus XH-1 microecological preparation. By feeding fish with fermentation liquor with proper concentration of the strain, the immune performance, especially the specific immune performance, of the fish can be remarkably improved, and a microecological preparation prepared from the strain has an antibacterial effect on common fish pathogenic bacteria such as aeromonas veronii, plesiomonas shigelloides and aeromonas amylophila at the same time; when the streptomyces pseudogriseogriseus XH-1 is added into feed to feed fishes, the streptomyces pseudogriseogriseus XH-1 is found to be capable of remarkably improving the expression of specific immune factors and improving the disease resistance of the fishes.

Description

Technical Field

[0001] The present invention relates to the field of microecological preparations, and in particular to a strain of Streptomyces pseudogreyii XH-1, a microecological preparation thereof and a preparation method thereof. Background Art

[0002] As an important source of human nutrition, the increase in demand for aquatic products has promoted the expansion of aquaculture. High-density aquaculture is the most mainstream aquaculture model. While providing huge economic benefits to aquaculture operators, it has also led to frequent fish diseases, among which bacterial diseases of fish are particularly prominent. Common pathogens of fish bacterial diseases include Edwardsiella, Vibrio, Aeromonas, Pseudomonas, etc., which have the characteristics of wide prevalence, high mortality, and long duration, causing serious losses to aquaculture. For the problem of bacterial diseases of fish, common solutions include phage therapy, vaccine therapy, and Chinese herbal medicine therapy, etc. However, the above methods have problems such as strong specificity, high development difficulty, and insignificant effect. Therefore, a more common solution is to put antibiotic chemical drugs into the aquaculture water. Although antibiotic drugs have the advantages of fast effect and broad antibacterial spectrum, they also have obvious disadvantages. Putting a large amount of antibiotics into the aquaculture water will not only pollute the water environment, but also produce strains resistant to antibiotics in pathogenic bacteria, which will reduce or even eliminate the therapeutic effect of antibiotics. Summary of the invention

[0003] The technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art and provide a strain of Streptomyces pseudo-light gray XH-1, its microecological preparation and its preparation method. The Streptomyces pseudo-light gray XH-1 and the microecological preparation prepared by the Streptomyces pseudo-light gray have antibacterial activity against a variety of fish pathogens and can effectively improve the immunity level of fish.

[0004] The technical solution adopted by the present invention to solve its technical problems is as follows: a strain of Streptomyces pseudogriseolus XH-1 was deposited in the China Center for Type Culture Collection on August 26, 2024, with a strain collection number of CCTCC NO: M 20241850; it was classified and named: Streptomyces pseudogriseolus XH-1, with a Latin name of Streptomyces pseudogriseolus XH-1.

[0005] The 16S rRNA sequence of Streptomyces pseudogreinii XH-1 of the present invention is shown in the sequence table SEQ ID No. 1.

[0006] The pseudo-light gray Streptomyces XH-1 of the present invention was screened from 3 soil samples from Baopo Road, Jiyang District, Sanya City, Hainan Province. The antibacterial active substances in the fermentation broth of the strain have stable physical and chemical properties, can withstand high temperatures of 70°C, are insensitive to ultraviolet radiation and proteases, have antibacterial effects on a variety of fish pathogens including Aeromonas vernix, Shigella-like Pleistomonas, and Aeromonas anomala saccharophila, and can effectively improve the immunity, especially the specific immunity, of fish, especially crucian carp, and have high application value in healthy fish breeding.

[0007] The Streptomyces pseudogreyii XH-1 of the present invention can be fermented to prepare microecological preparations for use in actual production.

[0008] Furthermore, Streptomyces pseudogreyii XH-1 and its microecological preparations can be applied in aquaculture.

[0009] Furthermore, the microecological preparation is a liquid bacterial preparation or a solid bacterial preparation.

[0010] Furthermore, the aquaculture animals are freshwater fish.

[0011] Furthermore, the freshwater fish is crucian carp.

[0012] The microecological preparation of Streptomyces pseudolicheniformis XH-1 of the invention is prepared by fermenting Streptomyces pseudolicheniformis XH-1 with a strain collection number of CCTCC NO: M 20241850.

[0013] The preparation method of the microecological preparation of Streptomyces pseudogreyii XH-1 of the present invention comprises the following steps:

[0014] (1) Inoculation and activation: The preserved slant strain of Streptomyces pseudogreyii XH-1 was transferred to CSM liquid medium for activation, and the Streptomyces XH-1 activated in CSM liquid medium was placed in Gao's solid medium No. 1 for streak culture, and a single clone was selected for fermentation culture in AM6 liquid medium to obtain an activated seed liquid;

[0015] Furthermore, in step (1), the formula of CSM liquid culture medium is: 40-50 g / L tryptone soy broth, 8-12 g / L glucose, 8-10 g / L yeast powder, 2-2.4 g / L magnesium sulfate heptahydrate, dissolved in pure water to 1 L, sterilization conditions: above 115°C, more than 30 min.

[0016] Furthermore, in step (1), the formula of CSM liquid culture medium is: 45 g / L tryptone soy broth, 10 g / L glucose, 9 g / L yeast powder, 2.2 g / L magnesium sulfate heptahydrate, dissolved in pure water to 1 L, sterilization conditions: 115°C, 30 min.

[0017] Furthermore, in step (1), the formula of Gao's solid culture medium No. 1 is: soluble starch 18-22 g / L, potassium nitrate 0.8-1.2 g / L, sodium chloride 0.4-0.6 g / L, dipotassium hydrogen phosphate trihydrate 0.4-0.6 g / L, magnesium sulfate heptahydrate 0.4-0.6 g / L, ferrous sulfate heptahydrate 0.005-0.015 g / L, agar 1.6-2.0 g / 100 mL, dissolved in pure water to 1 L, sterilization conditions: above 115°C, more than 30 min.

[0018] Furthermore, in step (1), the formula of Gao's solid culture medium No. 1 is: soluble starch 20g / L, potassium nitrate 1g / L, sodium chloride 0.5g / L, dipotassium hydrogen phosphate trihydrate 0.5g / L, magnesium sulfate heptahydrate 0.5g / L, ferrous sulfate heptahydrate 0.01g / L, agar 1.8g / 100mL, dissolved in pure water to 1L, sterilization conditions: 115°C, 30min.

[0019] Furthermore, in step (1), the formula of AM6 liquid culture medium is: soluble starch 18-22 g / L, glucose 8-12 g / L, bacteriological peptone 4-6 g / L, yeast extract 4-6 g / L, calcium carbonate 4-6 g / L, dissolved in pure water to 1 L, pH 7.2±0.5, sterilization conditions: above 115°C, more than 30 min.

[0020] Further, in step (1), the formula of AM6 liquid culture medium is: soluble starch 20g / L, glucose 10g / L, bacteriological peptone 5g / L, yeast extract 5g / L, calcium carbonate 5g / L, dissolved in pure water to 1L, pH 7.2±0.5, sterilization conditions: 115°C, 30min.

[0021] Furthermore, in step (1), the activation culture conditions of the CSM liquid culture medium are: temperature 28-30° C., liquid volume 10-15% of the container volume, shaking speed 180-200 rpm, and activation culture time 48-50 h.

[0022] Furthermore, in step (1), the streak culture conditions of Gao's solid medium No. 1 are as follows: temperature 28-30° C., and streak culture time is 70-72 h.

[0023] Furthermore, in step (1), the AM6 liquid culture medium fermentation culture conditions are: temperature 28-30°C, shaker speed 180-200 rpm, and fermentation culture time 96-120 h.

[0024] (2) First expansion culture: The seed liquid activated in step (1) is inoculated into a fermentation tank at an inoculation rate of 1 to 2% for first expansion culture to obtain a seed liquid for first expansion culture;

[0025] Furthermore, in step (2), the culture medium in the fermentation tank is the AM6 liquid culture medium described in step (1).

[0026] Furthermore, in step (2), the fermentation culture conditions of the AM6 liquid culture medium for the first expanded culture are: temperature 28-30°C, dissolved oxygen concentration 35-55%, fermentation tank agitator speed 180-200rpm, fermentation culture time 168-192h, the entire culture process is monitored online, and the defoaming agent is added online in real time.

[0027] (3) inoculating the seed liquid obtained from the first expanded culture in step (2) into a fermentation tank containing a fermentation medium at an inoculum amount of 10 to 15% for a second expanded culture to obtain a fermentation product obtained from the second expanded culture;

[0028] Furthermore, in step (3), the fermentation medium is the AM6 liquid medium described in step (1).

[0029] Furthermore, in step (3), the fermentation culture conditions of the AM6 liquid culture medium for the second expanded culture are: temperature 28-30°C, dissolved oxygen concentration 35-55%, fermentation tank agitator speed 180-200rpm, fermentation medium volume 65%-70% of the maximum volume of the fermentation tank, fermentation culture time 168-192h, the entire culture process is monitored online, and defoaming agent is added online in real time.

[0030] (4) Concentration and collection: After the fermentation is completed, the fermentation product of the second expanded culture is concentrated by rotary evaporation to obtain a microecological liquid bacterial agent, or concentrated by rotary evaporation and then freeze-dried (preferably using a freeze dryer) to obtain a microecological solid bacterial agent.

[0031] The identification and research methods of Streptomyces pseudogreinii XH-1 of the present invention are as follows.

[0032] (A) Observation of cell morphological characteristics of strain Streptomyces pseudogreinii XH-1;

[0033] (ii) Identification using the 16S rRNA gene;

[0034] (III) Physical and chemical properties of antibacterial active substances of Streptomyces pseudogreyii XH-1;

[0035] (iv) Effect of Streptomyces pseudolicheniformis XH-1 as a feed additive on the disease resistance of fish;

[0036] (V) Analysis of the antibacterial activity of the microecological liquid preparation and crude extract of fermentation broth of Streptomyces pseudogreinii XH-1 against 12 common fish pathogens;

[0037] (VI) Whole genome analysis of Streptomyces pseudogreinii XH-1.

[0038] Beneficial effects of the present invention:

[0039] The strain was identified as Streptomyces pseudogriseolus (strain collection number: CCTCC NO: M20241850; classified name: Streptomyces pseudogriseolus XH-1, Latin name: Streptomyces pseudogriseolusXH-1) through 16S rRNA gene sequence determination. Streptomyces pseudogriseolus XH-1 has good antibacterial effects on many common fish pathogens such as Aeromonas vernix, Shigella-like Pseudomonas, and Aeromonas anomala.

[0040] Streptomyces pseudolicheniformis XH-1 does not cause harm to fish, especially crucian carp, and can effectively improve the immune level of fish, especially crucian carp, especially the specific immune performance. When Streptomyces XH-1 is added to the feed for feeding fish, it is found that it can significantly increase the expression of specific immune factors and improve the disease resistance of fish.

[0041] The method for preparing a microecological preparation from Streptomyces pseudogreyni XH-1 is simple and convenient, and can replace chemical drugs such as antibiotics as a treatment for common fish diseases.

[0042] The 16S rRNA sequence of Streptomyces pseudogreinii XH-1 of the present invention is shown in the sequence table SEQ ID No. 1.

[0043] SEQ ID №1:

[0044] GGAGGATGGGGGGGGGCCTTACCATGTCAAGTCGGAACGATGAACCACTT 50CGGTGGGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCT 100GCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATACTGATCA150TCTTGGGCATCCTTGGTGATCGAAAGCTCCGGCGGTGCAGGATGAGCCCG 200CGGCCTATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGACGACGGG 250TAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCA300GACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCC350TGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTC 400TTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGG 450CTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCGAGCGTTGTCC 500GGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCGCGTCGGTTGTG 550AAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAG 600TTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATA650TCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGC 700TGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCC 750ACGCCGTAAACGGTGGGCACTAGGTGTGGGCGACATTCCACGTCGTCCGT 800GCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGC 850TAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGG900CTTAATTCGACGCAACGCGAAGAACCTTACCAAAGGCTTGACATACACCGG 950AAAACCCTGGAGACAGGGTCCCCCTTGTGGTCGGTGTACAGGTGTGCAT 1000GGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAG 1050CGCAACCCTTGTCCCGTGTTGCCAGCAGGCCCTTGTGGTGCTGGGGACTC 1100ACGGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGT 1150

[0045] CATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTAC 1200AATGAGCTGCGATACCGCGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCA1250GTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTA1300ATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACA1350CCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAAC 1400CCCTTGTGGGAGGGAGCTGTCGAAAAGGCGGA1432 Microorganism preservation instructions

[0046] The Streptomyces pseudogriseolus XH-1 of the present invention is deposited in China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China), with a strain accession number of CCTCC NO: M 20241850; the classification name is: Streptomyces pseudogriseolus XH-1; the Latin name is: Streptomyces pseudogriseolus XH-1. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a diagram showing the antibacterial effect of the fermentation supernatant of Streptomyces pseudogreyensis XH-1 of the present invention on 6 fish pathogens;

[0048] Figure 2 The morphological observation diagram of Streptomyces pseudogreyii XH-1 of the present invention; a: morphological diagram of XH-1 on Gao's I; b: morphological observation and Gram staining diagram of XH-1 under upright microscope; C: morphological observation diagram of XH-1 under cold field scanning electron microscope;

[0049] Figure 3 It is a phylogenetic tree diagram constructed from the 16S rRNA sequence of Streptomyces pseudogreinii XH-1 of the present invention;

[0050] Figure 4 1 is a graph showing the physical and chemical properties of the antibacterial active substance of Streptomyces pseudogreinii XH-1 of the present invention; 2 is a graph showing the effects of temperature, ultraviolet irradiation and protease on the antibacterial activity of the supernatant of the fermentation liquid of the strain XH-1;

[0051] Figure 5 and Figure 6 This is a diagram showing the effect of the microecological preparation prepared by Streptomyces pseudogreyii XH-1 of the present invention on the activity of immune-related enzymes in the serum of crucian carp;

[0052] Figure 7-10 This is a diagram showing the results of an analysis of the effect of the microecological preparation prepared by Streptomyces pseudogreyii XH-1 of the present invention on the expression of some immune-related genes in crucian carp serum;

[0053] Fig.11 This is a diagram showing the effect of Streptomyces pseudolicheniformis XH-1 of the present invention on the tissue structure of crucian carp;

[0054] Fig.12 This is a graph showing the effect of Streptomyces pseudogreyii XH-1 of the present invention on the survival rate of crucian carp;

[0055] Fig.13 This is a graph showing the antibacterial effects of Streptomyces pseudogreyii XH-1 microecological liquid preparation and fermentation broth crude extract on 12 common fish pathogens;

[0056] Fig.14 It is the whole genome composition map of Streptomyces pseudogreyii XH-1 of the present invention;

[0057] Fig.15 It is a statistics of the annotation results of the protein encoded by Streptomyces pseudogreysii XH-1 of the present invention in various databases;

[0058] Fig.16 It is the prediction result of the antiSMASH secondary metabolite gene cluster of Streptomyces pseudolight gray XH-1 of the present invention. DETAILED DESCRIPTION

[0059] The present invention will be further described below in conjunction with the embodiments and drawings.

[0060] Unless otherwise specified, the chemical reagents used in the embodiments of the present invention were obtained through conventional commercial channels.

[0061] The Streptomyces pseudogriseolus XH-1 of the present invention is deposited in China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China), with a strain accession number of CCTCC NO: M 20241850; the classification name is: Streptomyces pseudogriseolus XH-1; the Latin name is: Streptomyces pseudogriseolus XH-1.

[0062] (I) Screening of Streptomyces pseudogreyii XH-1 and determination of its antibacterial spectrum

[0063] Soil samples taken from Baopo Road, Jiyang District, Sanya City, Hainan Province were dissolved and mixed with sterile water to obtain a soil sample stock solution, and the soil sample stock solution was gradient diluted with sterile water to obtain 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Take 100 μL of the soil sample dilution and evenly spread it on Gao's No. 1 solid culture medium, invert it and culture it in a 30℃ incubator for 3 days. Pick out the single clone with similar morphology to actinomycetes and transfer it to a new Gao's No. 1 solid culture medium for purification and culture for 3 days. The common pathogenic bacteria of fish are Edwardsiella tarda, Citrobacterfreundii, Plesiomonas shigelloide, Erwinia spp., Aeromonas salmonicida, Shewanella xiamenensis, Aeromonas hydrophila, Aeromonas caviae, Aeromonas veronii AvX005, Aeromonas sobria, Pseudomonas plecoglossicida, Aeromonas saccharophila, Aeromonas spp. allosaccharophila) was used as the indicator bacteria, and the antibacterial effect of the purified supernatant of each actinomycete fermentation broth on the above 12 common pathogenic bacteria of fish was detected by well method. A strain with good antibacterial effect on 6 common pathogenic bacteria of fish was found and named XH-1 (see Figure 1 ).

[0064] (II) Morphological characteristics of strain XH-1

[0065] Strain XH-1 was streaked on Gao's solid medium No. 1 and cultured for 3 days. Its single colony was white as a whole with silver-gray edges. The colonies were of different sizes, opaque and dry on the surface. Under the oil immersion lens of an upright microscope, it was found that the mycelium of strain XH-1 was well-branched. After being activated with CSM liquid activation medium, strain XH-1 was purple after Gram staining, which determined that strain XH-1 was a Gram-positive bacterium. Strain XH-1 was observed under a cold-field scanning electron microscope, and it was found that the mycelium of this strain was well-developed, the spore-producing ability was strong, the spore chain was spiral, and thorn-like structures that were not found on the mycelium could be observed on the spores (see Figure 2 ).

[0066] (III) 16S rRNA gene identification of strain XH-1

[0067] The strain XH-1 was inoculated into CSM liquid culture medium for activation culture at 30°C and 180rpm for 2 days. After the culture, 1 mL was taken into a 1.5 mL EP tube for centrifugation, and the bacterial pellet was retained. The genomic DNA of the strain XH-1 was extracted using the Ezup column bacterial genomic DNA extraction kit, and the 16S rRNA gene of the strain XH-1 was amplified.

[0068] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'

[0069] 1492R:5'-CGGTTACCTTGTTACGACTT-3'

[0070] The 16S rRNA gene sequence was amplified using the above primers, and the expected length of the sequence was about 1500 bp.

[0071] PCR reaction system (20 μL): sterile double distilled water, 14 μL; 10× Buffer, 2 μL; dNTP, 1.6 μL; Bf-F (10 μM), 0.6 μL; Bf-R (10 μM), 0.6 μL; genomic template, 1 μL; PrimerSTAR DNA Polymerase (Takara), 0.2 μL;

[0072] PCR reaction program: pre-denaturation at 95°C for 5 min; 30 cycles: 95°C, 45 sec; 55°C, 45 sec; 72°C, 1.5 min; 72°C, 10 min.

[0073] After the 16S rRNA gene PCR products were detected by 1.0% agarose gel electrophoresis, the successfully amplified products were sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing.

[0074] The 16S rRNA gene sequence of strain XH-1 was sequenced and showed to be 1432 bp in length. After BLAST analysis, the phylogenetic tree of the strain was constructed using the neighbor-joining method in MEGA X software ( Figure 3 ).

[0075] (IV) Analysis of the physicochemical properties of antibacterial active substances of Streptomyces pseudogreyii XH-1

[0076] The pseudo-light gray Streptomyces XH-1 was activated and cultured using CSM liquid culture medium, and then transferred to AM6 liquid culture medium at a ratio of 1% for fermentation. After culturing for 168 to 192 hours, the fermentation broth was centrifuged at 8000 rpm for 10 minutes, and the supernatant of the fermentation broth was retained. The supernatant of the fermentation broth of Streptomyces XH-1 was analyzed for thermal stability, ultraviolet radiation tolerance, and common protease tolerance.

[0077] Thermal stability analysis: Take 4 1.5mL EP tubes, add 500μL of the supernatant of the fermentation broth of Streptomyces pseudogreyensis XH-1 to each tube, and incubate them in a water bath at 30℃, 50℃, 70℃, and 90℃ for 1h. After the incubation, wait for the supernatant of the fermentation broth to return to room temperature. Use Aeromonas vernix as the indicator bacteria and use the well trap method to detect the antibacterial activity of the supernatant of the fermentation broth. Set three groups of replicates at each temperature ( Figure 4 ).

[0078] Ultraviolet irradiation tolerance analysis: Take 6 1.5mL EP tubes, add 500μL of the supernatant of the fermentation broth of Streptomyces pseudogreyensis XH-1 to each tube, and place them under ultraviolet light for irradiation. The irradiation time is controlled at 10min, 20min, 30min, 40min, 50min, and 60min respectively. After the irradiation, Aeromonas vernix is ​​used as the indicator bacteria and the well trap method is used to detect the antibacterial activity of the fermentation broth supernatant. Three groups of replicates are set for each ultraviolet irradiation time ( Figure 4 ).

[0079] Common protease tolerance analysis: Take 3 1.5mL EP tubes, add 500μL of the fermentation supernatant of Streptomyces pseudogreyensis XH-1 to each tube, add 40μL of 20mg / mL proteinase K, 40μL of 20mg / m trypsin, and 40μL of 20mg / m pepsin, respectively, and incubate for 1h (proteinase K incubation temperature: 50℃, trypsin and pepsin incubation temperature: 37℃), wait for the fermentation supernatant to return to room temperature after the incubation, use Aeromonas veronii as the indicator bacteria and use the well trap method to detect the antibacterial activity of the fermentation supernatant, set three groups of replicates for each enzyme ( Figure 4 ).

[0080] From the above experiments, it can be seen that the fermentation broth of strain XH-1 can still have strong antibacterial activity at a high temperature of 70°C, and ultraviolet irradiation and common proteases have no effect on its antibacterial activity. The antibacterial substances of strain XH-1 have relatively stable properties.

[0081] (V) Preparation of microecological preparations of Streptomyces pseudogreyii XH-1

[0082] The microecological preparation of Streptomyces pseudogriseus XH-1 in this embodiment is prepared by fermenting Streptomyces pseudogriseus XH-1 with a strain collection number of CCTCC NO: M20241850.

[0083] The preparation method of the microecological preparation of Streptomyces pseudogreyii XH-1 of this embodiment comprises the following steps:

[0084] (1) Inoculation and activation: The preserved slant strain of Streptomyces pseudogreyii XH-1 was transferred to CSM liquid medium for activation, and the Streptomyces XH-1 activated in CSM liquid medium was placed in Gao's solid medium No. 1 for streak culture, and a single clone was selected for fermentation culture in AM6 liquid medium to obtain an activated seed liquid;

[0085] The formula of CSM liquid culture medium is: TSB 45g / L, glucose 10g / L, yeast powder 9g / L, magnesium sulfate heptahydrate 2.2g / L, dissolved in pure water to 1L, sterilization conditions: 115℃, 30min;

[0086] Gao's solid culture medium formula is: soluble starch 20g / L, potassium nitrate 1g / L, sodium chloride 0.5g / L, dipotassium hydrogen phosphate trihydrate 0.5g / L, magnesium sulfate heptahydrate 0.5g / L, ferrous sulfate heptahydrate 0.01g / L, agar 1.8g / 100mL, dissolved in pure water to 1L, sterilization conditions: 115℃, 30min;

[0087] The formula of AM6 liquid culture medium is: 20g / L soluble starch, 10g / L glucose, 5g / L bacteriological peptone, 5g / L yeast extract, 5g / L calcium carbonate, dissolved in pure water to 1L, pH7.2±0.5, sterilization conditions: 115℃, 30min; the activation culture conditions of the CSM liquid culture medium are: temperature 30℃, liquid filling volume is 10% of the container volume, shaking speed 180-200rpm, and activation culture time is 48h; the streaking culture conditions of Gao's No. 1 solid culture medium are: temperature 30℃, streaking culture time is 72h; the fermentation culture conditions of AM6 liquid culture medium are: temperature 30℃, shaking speed 180-200rpm, and fermentation culture time is 96h.

[0088] (2) First expansion culture: The seed liquid activated in step (1) is inoculated into a fermentation tank at a 1% inoculation rate for first expansion culture to obtain a seed liquid for first expansion culture;

[0089] The culture medium is the AM6 liquid culture medium described in step (1); the fermentation culture conditions of the AM6 liquid culture medium for the first expansion culture are: temperature 30°C, dissolved oxygen concentration 35-55%, fermentation tank agitator speed 180-200rpm, fermentation culture time 168h, the entire culture process is monitored online, and the defoaming agent is added online in real time.

[0090] (3) inoculating the seed liquid obtained from the first expanded culture in step (2) into a fermentation tank containing a fermentation medium at an inoculum rate of 10% for a second expanded culture to obtain a fermentation product obtained from the second expanded culture;

[0091] The culture medium is the AM6 liquid culture medium described in step (1); the fermentation culture conditions of the AM6 liquid culture medium for the second expanded culture are: temperature 30°C, dissolved oxygen concentration 35-55%, fermentation tank agitator speed 180-200rpm, fermentation culture medium volume 70% of the maximum volume of the fermentation tank, fermentation culture time 180h, the entire culture process is monitored online, and defoaming agent is added online in real time.

[0092] (4) Concentration and collection: After the fermentation is completed, the fermentation product of the second expanded culture is concentrated by rotary evaporation to obtain a microecological liquid bacterial agent, or after rotary evaporation concentration, it is freeze-dried using a freeze dryer to obtain a microecological solid bacterial agent.

[0093] (VI) Application of Streptomyces pseudogreyii XH-1 in fish farming

[0094] Example: Effects of Streptomyces pseudogreyii XH-1 on the immune performance of crucian carp

[0095] In this experiment, the domesticated crucian carp with uniform weight distribution were divided into two groups, including a control group and an experimental group. Each group had 10 crucian carp with uniform weight distribution. Three parallel replicates were set for the control group and the experimental group. The crucian carp were fed with feed at 1% of their initial body weight, once in the morning and once in the evening. The control group was fed with ordinary feed, and the experimental group was fed with Streptomyces pseudolividus XH-1 bacteria on the basis of ordinary feed, with an addition amount of 1×10 7 CFU / g, feeding period is 30 days.

[0096] (1) Effects of Streptomyces pseudogreyii XH-1 on immune-related enzyme activities in crucian carp

[0097] After feeding, the activities of immune-related enzymes in the serum of crucian carp in the control group and the experimental group were measured (see Figure 5 and Figure 6). The results showed that the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), glutathione reductase (GSH) and catalase (CAT) in the experimental group fed with Streptomyces pseudogreyensis XH-1 were increased compared with the control group, among which the activities of acid phosphatase, alkaline phosphatase and catalase increased significantly, indicating that Streptomyces XH-1 can effectively improve the immune ability of crucian carp.

[0098] (2) Effects of Streptomyces pseudogreyii XH-1 on the expression of immune-related genes in crucian carp

[0099] After continuous feeding of crucian carp for 30 days, the liver, kidney, spleen and intestine of the control group and the experimental group were taken to measure the gene expression levels of Ig M, Ig T, IL-10 and C3 in the liver, kidney, spleen and intestine of the control group and the experimental group, respectively (see Figure 7-10 ). The results showed that compared with the control group, the expression of Ig M in the liver, kidney, spleen and intestine of the experimental group crucian carp was significantly increased, and the expression of Ig T in the liver, spleen and intestine of the experimental group crucian carp was significantly increased, while the expression level in the kidney remained similar to that of the control group; for the expression level of IL-10, it was found that its expression in the liver and spleen of the experimental group was significantly upregulated, and there was no significant difference in the kidney and intestine; C3, as a factor involved in both specific and nonspecific immune responses, was expressed at higher levels in the liver, kidney, spleen and intestine of the experimental group crucian carp than in the control group. The above results also show that Streptomyces XH-1 can significantly improve the immunity of crucian carp, especially the specific immunity.

[0100] (3) Effects of Streptomyces pseudogreyii XH-1 on the tissue structure of crucian carp

[0101] After feeding crucian carp for 30 consecutive days, the liver, kidney, spleen, intestine and gills of the control group and experimental group were taken for tissue section observation. The results showed that the experimental group fed with feed containing Streptomyces XH-1 had no obvious difference in cell morphology from the experimental group of crucian carp and showed no symptoms, indicating that Streptomyces XH-1 would not damage the tissue structure of crucian carp. By observing the intestinal structure of crucian carp in the experimental and control groups through sectioning, it was found that the thickness of the intestinal mucosal muscle layer of crucian carp in the experimental group was greater than that in the control group, and the columnar cell morphology was fuller than that in the control group. The intestinal mucosa and columnar cells are a physical barrier for fish to deal with pathogens, indicating that Pseudomonas aeruginosa XH-1 can promote the barrier function of the intestine, and also shows that Pseudomonas aeruginosa XH-1 can improve the immune ability of crucian carp (see Fig.11 ).

[0102] (4) Experiment on the protective effect of Streptomyces pseudogreyii XH-1 on crucian carp

[0103] After continuous feeding of crucian carp for 30 days, the control group and experimental group were subjected to a challenge experiment, in which the group injected with 100 μL PBS and fed with ordinary feed was designated as the negative control group, the group injected with 100 μL Aeromonas veronii and fed with ordinary feed was designated as the positive control group, and the group injected with 100 μL Aeromonas veronii and fed with feed mixed with strain XH-1 was designated as the experimental group. The challenge method was intraperitoneal injection of 100 μL Aeromonas veronii. The control group and experimental group after the challenge were observed continuously for 7 days, and the death of crucian carp in each group was recorded. Finally, the survival rate of crucian carp in each group was calculated to determine the protection rate of Streptomyces pseudo-light gray XH-1 on crucian carp. The results showed that the survival rate of the experimental group fed with feed containing Streptomyces pseudo-light gray XH-1 was higher than that of the control group, proving that Streptomyces pseudo-light gray XH-1 can improve the resistance of crucian carp to Aeromonas veronii (see Fig.12 ).

[0104] (5) Test on the inhibitory effect of Streptomyces pseudogreyii XH-1 microecological preparation and crude extract of fermentation broth supernatant on 12 common fish pathogens

[0105] CSM liquid culture medium was used to activate Streptomyces pseudo-light gray XH-1 for 2 days. After the activation, it was transferred to AM6 liquid culture medium for shake flask fermentation for 6 days. After the fermentation, half of the fermentation liquid was taken for rotary evaporation and concentration. After the evaporation and concentration, the pseudo-light gray Streptomyces XH-1 microecological liquid preparation was obtained. At the same time, the supernatant of the other half of the fermentation liquid after centrifugation was extracted, that is, an equal volume of ethyl acetate was added for overnight extraction. After the extraction, the organic phase was taken for rotary evaporation and concentration. After the rotary evaporation, 10 ml of methanol was added to the rotary evaporation flask for dissolution to obtain a crude extract of the fermentation liquid. 100 μL of each test sample was taken to test the antibacterial effects of the microecological liquid preparation and crude extract of the fermentation broth of Streptomyces pseudo-light gray on 12 common fish pathogens. In the antibacterial test of the microecological liquid preparation of Streptomyces pseudo-light gray, 100 μL of sterile water was set as the control, and in the antibacterial test of the crude extract of the fermentation broth of Streptomyces pseudo-light gray, 100 μL of methanol was set as the control (to distinguish it from the previous control, this control group was named crude extract solvent methanol). The results showed that the microecological liquid preparation of Streptomyces pseudo-light gray XH-1 had an antibacterial effect of 100 μL of sterile water, and the crude extract of the fermentation broth of Streptomyces pseudo-light gray was an antibacterial effect of 100 μL of methanol. 1 The microecological liquid preparation has antibacterial activity against 8 of the 12 common fish pathogens, including Aeromonas hydrophila, Aeromonas anomala, Aeromonas salmonicida, Aeromonas vernix, Pseudomonas ayutans, Aeromonas sobria, Ossomonas shigellae, and Shewanella xiamenensis. The antibacterial test is better than that of the supernatant of the fermentation broth of Streptomyces XH-1. The crude extract of the fermentation broth has antibacterial activity against all 12 common fish pathogens, indicating that Streptomyces XH-1 can be used to prepare microecological liquid preparations to treat or prevent common fish diseases (reference Fig.13 ).

[0106] (VII) Whole genome analysis of Streptomyces pseudogreyii XH-1

[0107] The whole genome of Streptomyces pseudo-light gray XH-1 was sequenced on the PromethION platform. After quality control and assembly of the offline data, the whole genome size of Streptomyces pseudo-light gray XH-1 was 7,448,841bp, with a GC ratio of 72.59%, including a genome size of 7,360,314bp and a plasmid size of 88,527bp. The gene structure of the whole genome was annotated, and a total of 6,622 complete CDSs were obtained. AntiSMASH was used to predict and analyze the gene clusters of Streptomyces pseudo-light gray XH-1, and it was found that there were 18 secondary metabolite synthesis gene clusters in the whole genome of Streptomyces pseudo-light gray XH-1, including gene clusters related to the synthesis of potential antibacterial active substances such as naphthymycin, flavonoids, and sefomycin, indicating that Streptomyces pseudo-light gray XH-1 has strong potential for drug development (see Fig.14 , 15 , 16).

Claims

1. A strain of Streptomyces pseudogreyii XH-1, characterized in that: It was deposited in the China Center for Type Culture Collection on August 26, 2024, with the strain accession number of CCTCC NO: M 20241850; the classification name is: Streptomyces pseudogriseolus XH-1, Latin name Streptomyces pseudogriseolus XH-1.

2. The Streptomyces pseudogreyii XH-1 according to claim 1, characterized in that Its 16S rRNA sequence is shown in the sequence table SEQ ID No.

1.

3. The microecological preparation of Streptomyces pseudogreyii XH-1 as claimed in claim 1 or 2, characterized in that: The product is prepared by fermentation of Streptomyces pseudogreyni XH-1 with a strain collection number of CCTCC NO: M 20241850.

4. The microecological preparation of Streptomyces pseudogreyii XH-1 according to claim 3, characterized in that: Probiotic preparations are liquid or solid bacterial preparations.

5. The method for preparing the probiotic preparation of Streptomyces pseudogreyii XH-1 as claimed in claim 3 or 4, characterized in that: The following steps are involved: (1) Inoculation and activation: The preserved slant strain of Streptomyces pseudolicheniformis XH-1 was transferred to CSM liquid medium for activation, and the Streptomyces pseudolicheniformis XH-1 activated in CSM liquid medium was placed in Gao's solid medium No. 1 for streak culture, and a single clone was selected for fermentation culture in AM6 liquid medium to obtain an activated seed liquid; (2) First expansion culture: The seed liquid activated in step (1) is inoculated into a fermentation tank at an inoculation rate of 1 to 2% for first expansion culture to obtain a seed liquid for first expansion culture; (3) inoculating the seed liquid obtained from the first expanded culture in step (2) into a fermentation tank containing a fermentation medium at an inoculum amount of 10 to 15% for a second expanded culture to obtain a fermentation product obtained from the second expanded culture; (4) Concentration and collection: After the fermentation is completed, the fermentation product of the second expanded culture is concentrated by rotary evaporation to obtain a microecological liquid bacterial agent, or concentrated by rotary evaporation and then freeze-dried to obtain a microecological solid bacterial agent.

6. The method for preparing the probiotic preparation of Streptomyces pseudogreyii XH-1 according to claim 5, characterized in that: In step (1), the formula of CSM liquid culture medium is: 40-50 g / L tryptone soy broth, 8-12 g / L glucose, 8-10 g / L yeast powder, 2-2.4 g / L magnesium sulfate heptahydrate, dissolved in pure water to 1 L, sterilization conditions: above 115°C, more than 30 min; and / or, in step (1), the formula of Gao's No. 1 solid culture medium is: 18-22 g / L soluble starch, 0.8-1 .2g / L, sodium chloride 0.4-0.6g / L, potassium hydrogen phosphate trihydrate 0.4-0.6g / L, magnesium sulfate heptahydrate 0.4-0.6g / L, ferrous sulfate heptahydrate 0.005-0.015g / L, agar 1.6-2.0g / 100mL, dissolved in pure water to 1L, sterilization conditions: above 115°C, more than 30min; and / or, in step (1), the formula of AM6 liquid culture medium is: Soluble starch 18-22 g / L, glucose 8-12 g / L, bacteriological peptone 4-6 g / L, yeast extract 4-6 g / L, calcium carbonate 4-6 g / L, dissolved in pure water to 1 L, pH 7.2±0.5, sterilization conditions: above 115°C, more than 30 min; and / or, in step (1), the activation culture conditions of the CSM liquid culture medium are: temperature 28-30°C, liquid volume is 10-15% of the container volume, shaking speed is 180-200 rpm, and activation culture time is 48-50 h; and / or, in step (1), the streaking culture conditions of the Gao's No. 1 solid culture medium are: temperature 28-30°C, streaking culture time is 70-72 h; and / or, in step (1), the fermentation culture conditions of the AM6 liquid culture medium are: temperature 28-30°C, shaking speed is 180-200 rpm, and fermentation culture time is 96-120 h.

7. The method for preparing the probiotic preparation of Streptomyces pseudogreyii XH-1 according to claim 5 or 6, characterized in that: In step (2), the culture medium in the fermenter is the AM6 liquid culture medium described in step (1); and / or, in step (2), the fermentation culture conditions of the AM6 liquid culture medium for the first expanded culture are: temperature 28-30°C, dissolved oxygen concentration 35-55%, fermenter agitator speed 180-200rpm, fermentation culture time 168-192h, the entire culture process is monitored online, and the defoaming agent is added online in real time.

8. The method for preparing the probiotic preparation of Streptomyces pseudogreyii XH-1 according to claim 5 or 6, characterized in that: In step (3), the fermentation medium is the AM6 liquid medium described in step (1); and / or, in step (3), the fermentation culture conditions of the AM6 liquid medium for the second expanded culture are: temperature 28-30°C, dissolved oxygen concentration 35-55%, fermentation tank agitator speed 180-200rpm, fermentation medium volume 65%-70% of the maximum volume of the fermentation tank, fermentation culture time 168-192h, the entire culture process is monitored online, and defoaming agent is added online in real time.

9. Use of Streptomyces pseudogreinii XH-1 as claimed in claim 1 or 2 in aquaculture.

10. Use of the microecological preparation of Streptomyces pseudogreysii XH-1 as claimed in claim 3 or 4 in aquaculture.

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