A strain of Streptomyces pseudogriseus XH-1, its microecological preparation and its preparation method

Abstract

A strain of Streptomyces pseudo-light gray XH-1, its microecological preparation and its preparation method. The Streptomyces pseudo-light gray XH-1 of the present invention was deposited in the China Center for Type Culture Collection on August 26, 2024, with the strain deposit number CCTCC NO: M 20241850; the classification name is: Streptomyces pseudo-light gray XH-1, the Latin name Streptomyces pseudogriseolus XH‑1. Its microecological preparation is made by fermenting Streptomyces pseudogriseus XH‑1. The present invention also includes a method for preparing a microecological preparation of Streptomyces pseudogriseus XH‑1. Feeding fish with a fermentation liquid of an appropriate concentration of the strain can significantly improve the immune performance of the fish, especially the specific immune performance. The microecological preparation prepared by the strain also has an antibacterial effect on common fish pathogens such as Aeromonas vernix, Shigella-like Pseudomonas, and Aeromonas anomala. When Streptomyces pseudogriseus XH‑1 is added to the feed to feed fish, it is found that it can significantly increase the expression of specific immune factors and improve the disease resistance of fish.

Description

Technical Field

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

[0002] As a vital source of human nutrition, the increasing demand for aquatic products has driven the expansion of aquaculture. High-density aquaculture is the most common farming model, providing significant economic benefits to aquaculture operators but also leading to frequent fish diseases, particularly bacterial fish diseases. Common pathogens of fish bacterial diseases include Edwardsiella, Vibrio, Aeromonas, and Pseudomonas. These diseases are characterized by widespread prevalence, high mortality rates, and long duration, causing significant losses to aquaculture. Common solutions to bacterial fish diseases include phage therapy, vaccines, and traditional Chinese medicine. However, these approaches suffer from high specificity, high development difficulty, and limited effectiveness. Therefore, a more common solution is to inject antibiotics into aquaculture water. While antibiotics offer advantages such as rapid effectiveness and broad antimicrobial spectrum, they also have significant drawbacks. Large doses of antibiotics in aquaculture water not only pollute the water environment but also contribute to the emergence of antibiotic-resistant strains among pathogenic bacteria, reducing or even eliminating the effectiveness of antibiotic treatment. 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 pseudogriseus XH-1, a microecological preparation thereof and a preparation method thereof. The Streptomyces pseudogriseus XH-1 and the microecological preparation prepared from the Streptomyces pseudogriseus have antibacterial activity against a variety of fish pathogens and can effectively improve the immune level of fish.

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

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

[0006] The strain, Streptomyces pseudoglaucoma XH-1, was screened from three soil samples collected from Baopo Road, Jiyang District, Sanya City, Hainan Province. The antibacterial active substances in the fermentation broth of this strain exhibit stable physical and chemical properties, are heat-resistant to 70°C, are insensitive to UV radiation and proteases, and exhibit antibacterial effects against various fish pathogens, including Aeromonas vernix, Pleistomonas shigelloides, and Aeromonas anomala. It also effectively enhances the immunity, especially the specific immunity, of fish, particularly crucian carp, and has high application value in healthy fish farming.

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

[0008] Furthermore, Streptomyces pseudogriseus 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 pseudogriseus XH-1 is prepared by fermenting Streptomyces pseudogriseus XH-1 with a strain collection number of CCTCC NO: M 20241850.

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

[0014] (1) Inoculation and activation: The preserved strain of Streptomyces pseudogreyensis XH-1 was transferred to CSM liquid medium for activation. The Streptomyces XH-1 activated in CSM liquid medium was placed in Gao's solid medium No. 1 for streak culture and then inverted for culture. A single clone was selected and fermented in AM6 liquid medium to obtain the 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 20 g / L, potassium nitrate 1 g / L, sodium chloride 0.5 g / L, dipotassium hydrogen phosphate trihydrate 0.5 g / L, magnesium sulfate heptahydrate 0.5 g / L, ferrous sulfate heptahydrate 0.01 g / L, agar 1.8 g / 100 mL, dissolved in pure water to 1 L, sterilization conditions: 115 ° C, 30 min.

[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, above 30 min.

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

[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, shaker speed 180-200 rpm, and activation culture time 48-50 h.

[0022] Furthermore, in step (1), the streak culture conditions of Gao's No. 1 solid culture medium are: 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 1-2% for the first expansion culture to obtain the seed liquid for the 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 180-200 rpm, fermentation culture time 168-192 h, the entire culture process is monitored online, and 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 rate of 10-15% 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 as follows: temperature 28-30°C, dissolved oxygen concentration 35-55%, fermentor agitator speed 180-200 rpm, fermentation culture medium volume 65%-70% of the maximum volume of the fermentor, fermentation culture time 168-192 h, 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 after rotary evaporation and concentration, it is freeze-dried (preferably using a freeze dryer) to obtain a microecological solid bacterial agent.

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

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

[0033] (2) Identification using the 16S rRNA gene;

[0034] (3) Physical and chemical properties of antibacterial active substances of Streptomyces pseudogriseus XH-1;

[0035] (4) Effects of Streptomyces pseudogriseus XH-1 as a feed additive on fish disease resistance;

[0036] (5) Analysis of the antibacterial activity of Streptomyces pseudogriseus XH-1 microecological liquid preparation and fermentation broth crude extract against 12 common fish pathogens;

[0037] (6) Whole genome analysis of Streptomyces pseudogriseus XH-1.

[0038] Beneficial effects of the present invention:

[0039] The 16S rRNA gene sequence was used to identify the strain as Streptomyces pseudogriseus (strain collection number: CCTCC NO: M20241850; classification name: Streptomyces pseudogriseus XH-1, Latin name: Streptomyces pseudogriseolus XH-1), Streptomyces pseudogriseus XH-1 has a good antibacterial effect on many common fish pathogens such as Aeromonas wiseri, Shigella-like bacteria, and Aeromonas anomala.

[0040] Streptomyces pseudogriseus XH-1 does not pose a hazard to fish, especially crucian carp, and can effectively improve their immune systems, particularly their specific immune function. Adding Streptomyces XH-1 to fish feed has been shown to significantly increase the expression of specific immune factors, improving their disease resistance.

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

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

[0043] SEQ ID No. 1:

[0044] GGAGGATGGGGGGGGGCCTTACCATGTCAAGTCGGAACGATGAACCACTT 50

[0045] CGGTGGGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCT 100

[0046] GCACTCTGGGACAAGCCCTGGAAAACGGGGTCTAATACCGGATACTGATCA 150

[0047] TCTTGGGCATCCTTGGTGATCGAAAGCTCCGGCGGTGCAGGATGAGCCCG 200

[0048] CGGCCTATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGACGACGGG 250

[0049] TAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCA 300

[0050] GACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCC 350

[0051] TGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTC 400

[0052] TTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGG 450

[0053] CTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCGAGCGTTGTCC 500

[0054] GGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCGCGTCGGTTGTG 550

[0055] AAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAG 600

[0056] TTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATA 650

[0057] TCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGC 700

[0058] TGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCC 750

[0059] ACGCCGTAAACGGTGGGCACTAGGTGTGGGCGACATTCCACGTCGTCCGT 800

[0060] GCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGC 850

[0061] TAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGG 900

[0062] CTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGG 950

[0063] AAAACCCTGGAGACAGGGTCCCCCTTGTGGTCGGTGTACAGGTGGTGCAT 1000

[0064] GGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAG 1050

[0065] CGCAACCCTTGTCCCGTGTTGCCAGCAGGCCCTTGTGGTGCTGGGGACTC 1100

[0066] ACGGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGT 1150

[0067] CATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTAC 1200

[0068] AATGAGCTGCGATACCGCGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCA 1250

[0069] GTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTA 1300

[0070] ATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACA 1350

[0071] CCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCGGTGGCCCAAC 1400

[0072] CCCTTGTGGGAGGGAGCTGTCGAAAAGGCGGA 1432

[0073] Description of Microbial Deposit

[0074] The Streptomyces pseudogriseus XH-1 of the present invention is deposited in the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China) with a strain accession number of CCTCC NO: M 20241850; its classification name is Streptomyces pseudogriseus XH-1; its Latin name is: Streptomyces pseudogriseolus XH-1. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 This is a graph showing the antibacterial effect of the fermentation supernatant of Streptomyces pseudogriseus XH-1 of the present invention on six fish pathogens;

[0076] Figure 2 These are morphological observations of Streptomyces pseudogriseus XH-1 of the present invention; a: morphological observation of XH-1 on Gao's I microscope; b: morphological observation and Gram staining of XH-1 under an upright microscope; C: morphological observation of XH-1 under a cold-field scanning electron microscope;

[0077] Figure 3 This is a phylogenetic tree diagram constructed from the 16S rRNA sequence of Streptomyces pseudogriseus XH-1 of the present invention;

[0078] Figure 4 1. ...

[0079] Figure 5 and Figure 6 This is a graph showing the results of an analysis of the effects of the probiotic preparation prepared by Streptomyces pseudogriseus XH-1 of the present invention on the activities of immune-related enzymes in the serum of crucian carp;

[0080] Figure 7-10 This is a graph showing the results of an analysis of the effect of the probiotic preparation prepared by Streptomyces pseudogriseus XH-1 of the present invention on the expression of some immune-related genes in crucian carp serum;

[0081] Figure 11 This is a diagram showing the effect of Streptomyces pseudogriseus XH-1 of the present invention on the tissue structure of crucian carp;

[0082] Figure 12 This is a graph showing the effect of Streptomyces pseudogriseus XH-1 of the present invention on the survival rate of crucian carp;

[0083] Figure 13 This is a graph showing the antibacterial effect of Streptomyces pseudogriseus XH-1 microecological liquid preparation and fermentation broth crude extract on 12 common fish pathogens;

[0084] Figure 14 This is the complete genome composition map of Streptomyces pseudogriseus XH-1 of the present invention;

[0085] Figure 15 It is a statistical analysis of the annotation results of the protein encoded by Streptomyces pseudogriseus XH-1 of the present invention in various databases;

[0086] Figure 16 This is the prediction result of the antiSMASH secondary metabolite gene cluster of Streptomyces pseudogriseus XH-1 of the present invention. DETAILED DESCRIPTION

[0087] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0088] Unless otherwise specified, the chemical reagents used in the embodiments of the present invention were obtained through conventional commercial channels.

[0089] The Streptomyces pseudogriseus XH-1 of the present invention is deposited in the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China) with a strain accession number of CCTCC NO: M 20241850; its classification name is Streptomyces pseudogriseus XH-1; its Latin name is: Streptomyces pseudogriseolus XH-1.

[0090] (1) Screening of Streptomyces pseudogreyensis XH-1 and determination of its antibacterial spectrum

[0091] Soil samples taken from Baopo Road, Jiyang District, Sanya City, Hainan Province were dissolved and mixed with sterile water to obtain a soil sample solution, which was then gradiently 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 solid medium No. 1, invert it and culture it in a 30℃ incubator for 3 days. Pick out the single colony with similar morphology to actinomycetes and culture it on a new Gao's solid medium No. 1 for 3 days. Edwardsiella tarda ), Citrobacter freundii ( Citrobacter freundii ), Plesiomonas shigelloides ( Plesiomonas shigelloide ), Erwinia ( Erwinia spp. ), Aeromonas salmonicida ( Aeromonas salmonicida ), Shewanella xiamenensis ( Shewanella xiamenensis ), Aeromonas hydrophila ( Aeromonas hydrophila ), Aeromonas caviae ( Aeromonas caviae), Aeromonas wilkeri ( Aeromonas veronii ) AvX005, Aeromonas sobria ( Aeromonas sobria )、Pseudomonas ayuensis( Pseudomonas plecoglossicida ), Aeromonas anomala saccharophila ( Aeromonas 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-well method. A strain with good antibacterial effect on 6 common pathogenic bacteria of fish was found and named XH-1 (see Figure 1 ).

[0092] (II) Morphological characteristics of strain XH-1

[0093] Strain XH-1 was streaked onto Gao's solid medium No. 1 and cultured for 3 days. The single colonies were white in color with silver-gray edges, of varying sizes, opaque, and dry on the surface. Observation under an upright oil immersion microscope revealed that the mycelium of strain XH-1 was well-branched. After activation with CSM liquid activation medium, strain XH-1 was purple after Gram staining, indicating that strain XH-1 was a Gram-positive bacterium. Observation of strain XH-1 under a cold-field scanning electron microscope revealed that the mycelium of this strain was well-developed, with a strong spore-producing ability. The spore chains were spiral-shaped, and thorn-like structures that were not present on the mycelium were observed on the spores (see Figure 2 ).

[0094] (III) 16S rRNA gene identification of strain XH-1

[0095] Strain XH-1 was inoculated into CSM liquid medium for activation culture at 30°C and 180 rpm for 2 days. After the culture was completed, 1 mL was taken and placed in a 1.5 mL EP tube for centrifugation. The bacterial pellet was retained and the genomic DNA of strain XH-1 was extracted using the Ezup column-based bacterial genomic DNA extraction kit. The 16S rRNA gene of strain XH-1 was amplified.

[0096] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'

[0097] 1492R:5'-CGGTTACCTTGTTACGACTT-3'

[0098] The 16S rRNA gene sequence was amplified using the above primers, and the expected length of the sequence was about 1500 bp.

[0099] 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;

[0100] PCR reaction program: pre-denaturation at 95°C for 5 min; 30 cycles of: 95°C, 45 sec; 55°C, 45 sec; 72°C, 1.5 min; 72°C, 10 min.

[0101] The 16S rRNA gene PCR products were detected by 1.0% agarose gel electrophoresis, and the successfully amplified products were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing.

[0102] The 16S rRNA gene sequence of strain XH-1 was sequenced to be 1432 bp in length. BLAST analysis was performed and the phylogenetic tree of the strain was constructed using the neighbor-joining method in MEGA X software ( Figure 3 ).

[0103] (IV) Analysis of the physicochemical properties of antibacterial active substances from Streptomyces pseudogriseus XH-1

[0104] Streptomyces pseudogriseus XH-1 was activated in CSM liquid medium and then transferred to AM6 liquid medium at a ratio of 1% for fermentation. After culturing for 168 to 192 h, the fermentation broth was centrifuged at 8000 rpm for 10 min, and the supernatant of the fermentation broth was retained. The supernatant of the Streptomyces XH-1 fermentation broth was analyzed for thermal stability, ultraviolet radiation tolerance, and common protease tolerance.

[0105] Thermal stability analysis: Take four 1.5 mL EP tubes and add 500 μL of the supernatant of the fermentation broth of Streptomyces pseudogriseus XH-1 to each tube. Incubate in a water bath at 30°C, 50°C, 70°C, and 90°C for 1 h. After the incubation, wait for the supernatant of the fermentation broth to return to room temperature. Use Aeromonas vernix as an indicator bacteria and use the well-trap method to detect the antibacterial activity of the fermentation broth supernatant. Set three groups of replicates at each temperature ( Figure 4 ).

[0106] Ultraviolet irradiation tolerance analysis: Take 6 1.5 mL EP tubes, add 500 μL of the supernatant of the fermentation broth of Streptomyces pseudogreyensis XH-1 to each tube, and place them under ultraviolet irradiation for 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min, respectively. After the irradiation, the antibacterial activity of the fermentation broth supernatant was detected by the well trap method using Aeromonas vernix as the indicator bacteria. Three groups of replicates were set for each ultraviolet irradiation time ( Figure 4 ).

[0107] Common protease tolerance analysis: Take three 1.5 mL EP tubes, add 500 μL of the supernatant of the fermentation broth of Streptomyces pseudogriseus XH-1 to each tube, add 40 μL of 20 mg / mL proteinase K, 40 μL of 20 mg / mL trypsin, and 40 μL of 20 mg / mL pepsin, respectively, and incubate for 1 hour (proteinase K incubation temperature: 50 ℃, trypsin and pepsin incubation temperature: 37 ℃). After the incubation, wait for the fermentation broth supernatant to return to room temperature, and use Aeromonas vernix as an indicator bacteria and the well-trap method to detect the antibacterial activity of the fermentation broth supernatant. Set three replicates for each enzyme ( Figure 4 ).

[0108] 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.

[0109] (V) Preparation of microecological preparations of Streptomyces pseudogreyens XH-1

[0110] 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.

[0111] The preparation method of the microecological preparation of Streptomyces pseudogriseus XH-1 of this embodiment comprises the following steps:

[0112] (1) Inoculation and activation: The preserved strain of Streptomyces pseudogreyensis XH-1 was transferred to CSM liquid medium for activation. The Streptomyces XH-1 activated in CSM liquid medium was placed in Gao's solid medium No. 1 for streak culture and then inverted for culture. A single clone was selected and fermented in AM6 liquid medium to obtain the activated seed liquid.

[0113] The formula of CSM liquid medium is: TSB 45 g / L, glucose 10 g / L, yeast powder 9 g / L, magnesium sulfate heptahydrate 2.2 g / L, dissolved in pure water to 1 L, sterilized at 115 °C for 30 min;

[0114] Gao's solid medium No. 1 is formulated as follows: soluble starch 20 g / L, potassium nitrate 1 g / L, sodium chloride 0.5 g / L, dipotassium hydrogen phosphate trihydrate 0.5 g / L, magnesium sulfate heptahydrate 0.5 g / L, ferrous sulfate heptahydrate 0.01 g / L, agar 1.8 g / 100 mL, dissolved in pure water to 1 L, sterilized at 115°C for 30 min.

[0115] The formula of AM6 liquid culture medium is as follows: 20 g / L soluble starch, 10 g / L glucose, 5 g / L bacteriological peptone, 5 g / L yeast extract, 5 g / L calcium carbonate, dissolved in pure water to 1 L, pH 7.2±0.5, sterilization conditions: 115°C, 30 min; the activation culture conditions of the CSM liquid culture medium are: temperature 30°C, liquid volume 10% of the container volume, shaker speed 180-200 rpm, and activation culture time 48 h; the streak culture conditions of Gao's No. 1 solid culture medium are: temperature 30°C, streak culture time 72 h; the fermentation culture conditions of AM6 liquid culture medium are: temperature 30°C, shaker speed 180-200 rpm, and fermentation culture time 96 h.

[0116] (2) First expansion culture: The seed liquid activated in step (1) is inoculated into a fermentation tank at a 1% inoculation rate for the first expansion culture to obtain the seed liquid for the first expansion culture;

[0117] 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 180-200 rpm, fermentation culture time 168 h, the entire culture process is monitored online, and defoaming agent is added online in real time.

[0118] (3) inoculating the seed liquid obtained from the first expanded culture in step (2) into a fermentation tank containing fermentation medium at a 10% inoculation rate for a second expanded culture to obtain a fermentation product obtained from the second expanded culture;

[0119] 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 180-200 rpm, fermentation medium volume 70% of the maximum volume of the fermentation tank, fermentation culture time 180 h, the entire culture process is monitored online, and defoaming agent is added online in real time.

[0120] (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.

[0121] (6) Application of Streptomyces pseudogreyensis XH-1 in fish farming

[0122] Example: Effects of Streptomyces pseudogriseus XH-1 on the immune function of crucian carp

[0123] In this experiment, the domesticated crucian carp with uniform weight distribution were divided into two groups, a control group and an experimental group. Each group consisted of 10 crucian carp with uniform weight distribution. Three replicates were set for both 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, while the experimental group was fed with Streptomyces pseudoglaucoma XH-1 bacteria in an amount of 1×10 7 CFU / g, feeding period is 30 days.

[0124] (1) Effects of Streptomyces pseudogriseus XH-1 on the activities of immune-related enzymes in crucian carp

[0125] After the feeding, the activities of immune-related enzymes in the serum of crucian carp in the control group and 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 pseudogriseus XH-1 bacteria were all increased compared with the control group. Among them, the activities of acid phosphatase, alkaline phosphatase, and catalase increased more significantly, indicating that Streptomyces XH-1 can effectively improve the immune ability of crucian carp.

[0126] (2) Effects of Streptomyces pseudogriseus XH-1 on the expression of immune-related genes in crucian carp

[0127] After feeding crucian carp for 30 days, the liver, kidney, spleen and intestine of the control and experimental groups were collected to measure the gene expression levels of IgM, IgT, IL-10 and C3 in the liver, kidney, spleen and intestine of the control and experimental groups, respectively (see Figure 7-10The results showed that compared with the control group, the expression of IgM in the liver, kidney, spleen, and intestine of the experimental group crucian carp was significantly increased. The expression of IgT in the liver, spleen, and intestine of the experimental group crucian carp was also significantly increased, while the expression level in the kidney remained similar to that of the control group. As 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, while there was no significant difference in the kidney and intestine. C3, 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. These results also indicate that Streptomyces XH-1 can significantly enhance the immune capacity of crucian carp, especially its specific immune capacity.

[0128] (3) Effects of Streptomyces pseudogreyii XH-1 on the tissue structure of crucian carp

[0129] After 30 days of continuous feeding of crucian carp, tissue sections of the liver, kidney, spleen, intestine, and gills of the control and experimental groups were taken for observation. The results showed that the experimental group fed with the feed containing Streptomyces XH-1 had no significant difference in cell morphology from the experimental group of crucian carp in the five tissues mentioned above, and no symptoms were shown, indicating that Streptomyces XH-1 did not damage the tissue structure of crucian carp. By observing the intestinal structure of the experimental and control groups of crucian carp through sectioning, it was found that the thickness of the intestinal mucosal muscular layer of the experimental group of crucian carp was greater than that of the control group, and the columnar cell morphology was fuller than that of 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 indicate that Pseudomonas aeruginosa XH-1 can improve the immune ability of crucian carp (see Figure 11 ).

[0130] (4) Experiment on the protective effect of Streptomyces pseudogreyii XH-1 on crucian carp

[0131] After feeding crucian carp for 30 consecutive days, the control group and experimental group were challenged with the virus. 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 vernis and fed with ordinary feed was designated as the positive control group, and the group injected with 100 μL Aeromonas vernis 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 vernis. The control group and experimental group were observed continuously for 7 days after the challenge, 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 vernis (see Figure 12 ).

[0132] (5) Test on the inhibitory effect of the microecological preparation of Streptomyces pseudogriseus XH-1 and the crude extract of the fermentation liquid supernatant on 12 common fish pathogens. The Streptomyces pseudogriseus XH-1 was activated and cultured using CSM liquid culture medium for 2 days. After the activation, it was transferred to AM6 liquid culture medium for shake flask fermentation for a second time. The culture time was 6 days. After the fermentation, half of the fermentation liquid was taken for rotary evaporation and concentration. After the evaporation and concentration were completed, the microecological liquid preparation of Streptomyces pseudogriseus XH-1 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 was completed, the organic phase was taken for rotary evaporation and concentration. After the rotary evaporation was completed, 10 ml of methanol was added to the rotary evaporation flask for dissolution to obtain the crude extract of the fermentation liquid. 100 μL of each test sample was taken to test the antibacterial effect of the microecological liquid preparation and fermentation broth crude extract of Pseudomonas aeruginosa XH-1 on 12 common fish pathogens. In the antibacterial test of the microecological liquid preparation of Pseudomonas aeruginosa XH-1, the control was set as 100 μL of sterile water, and in the antibacterial test of the crude extract of fermentation broth of Pseudomonas aeruginosa XH-1, the control was set as 100 μL of methanol (to distinguish it from the previous control, this group of control groups was named crude extract solvent methanol). The results showed that Pseudomonas aeruginosa XH-1 had an antibacterial effect on 12 common fish pathogens. 1. The microecological liquid preparation has antibacterial activity against 8 of the 12 common fish pathogens, including Aeromonas hydrophila, Aeromonas anomala, Aeromonas salmonicida, Aeromonas versii, Pseudomonas ayutans, Aeromonas sobria, Pseudomonas shigelloides, 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 Figure 13 ).

[0133] (VII) Whole genome analysis of Streptomyces pseudogriseus XH-1

[0134] The whole genome of Streptomyces pseudogriseus XH-1 was sequenced on the PromethION platform. After quality control and assembly of the offline data, the total genome size of Streptomyces pseudogriseus XH-1 was 7,448,841 bp, with a GC ratio of 72.59%. The genome size was 7,360,314 bp and the plasmid size was 88,527 bp. 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 gene clusters of Streptomyces pseudogriseus XH-1, and 18 secondary metabolite synthesis gene clusters were found in the whole genome of Streptomyces pseudogriseus XH-1, including gene clusters related to the synthesis of potential antimicrobial active substances such as naphthymycin, flavomycin, and sefamicin. This indicates that Streptomyces pseudogriseus XH-1 has strong potential for drug development (see ). Figure 14 、15 、16）。

Claims

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

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

1.

3. The probiotic preparation of Streptomyces pseudogreyens XH-1 according to claim 1 or 2, characterized in that The product is prepared by fermentation of Streptomyces pseudogriseus XH-1 with a strain collection number of CCTCC NO: M 20241850.

4. The microecological preparation of Streptomyces pseudogreysii 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 pseudogreyens 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 pseudogriseus XH-1 was transferred to CSM liquid medium for activation. The Streptomyces pseudogriseus XH-1 activated in CSM liquid medium was placed in Gao's solid medium No. 1 for streak culture, and a single clone was picked and fermented in AM6 liquid medium to obtain the 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 1-2% for the first expansion culture to obtain the seed liquid for the 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 rate of 10-15% 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 proecological preparation of Streptomyces pseudogreyens XH-1 according to claim 5, characterized in that: In step (1), the formula of CSM liquid culture medium is: 40-50 g / L of tryptone soy broth, 8-12 g / L of glucose, 8-10 g / L of yeast powder, 2-2.4 g / L of magnesium sulfate heptahydrate, dissolved in pure water and fixed 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 of soluble starch, 0.8-1.2 g / L of potassium nitrate, 0.4-0.6 g / L of sodium chloride, 0.4-0.6 g / L of potassium hydrogen phosphate trihydrate, 0.4-0.6 g / L of magnesium sulfate heptahydrate, 0.005-0.015 g / L of ferrous sulfate heptahydrate, 1.6-2.0 g / 100 mL of agar, dissolved in pure water and fixed to 1 L, sterilization conditions: above 115°C, 30 min or more; 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 ℃, above 30 min; and / or, in step (1), the activation culture conditions of the CSM liquid culture medium are: temperature 28-30 ℃, liquid volume is 10-15% of the container volume, shaking speed 180-200 rpm, activation culture time is 48-50 h; and / or, in step (1), the streaking culture conditions of Gao's No. 1 solid culture medium are: temperature 28-30 ℃, 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 ℃, shaking speed 180-200 rpm, and fermentation time 96-120 h.

7. The method for preparing the proecological preparation of Streptomyces pseudogreyens XH-1 according to claim 5 or 6, characterized in that, In step (2), the culture medium in the fermentation tank 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%, fermentation tank agitator speed 180-200 rpm, fermentation culture time 168-192 h, the entire culture process is monitored online, and defoaming agent is added online in real time.

8. The method for preparing the proecological preparation of Streptomyces pseudogreyens 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%, fermentor agitator speed 180-200 rpm, fermentation medium volume 65%-70% of the maximum volume of the fermentor, fermentation culture time 168-192 h, the entire culture process is monitored online, and defoaming agent is added online in real time.

9. Use of the Streptomyces pseudogriseus XH-1 according to claim 1 or 2 in aquaculture.

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

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