Streptomyces giganteus L1, microecological preparation thereof and preparation method of microecological preparation

Abstract

The invention relates to a streptomyces giganteus L1, a microecological preparation thereof and a preparation method of the microecological preparation, the classification name of the streptomyces giganteus L1 is streptomyces giganteus L1, the streptomyces giganteus L1 is preserved in China Center for Type Culture Collection on December 12, 2024, and the preservation number of the strain is CCTCC NO: M 20242802. The microecological preparation prepared by fermenting the streptomyces giganteus L1 is suitable for aquaculture, can effectively inhibit freshwater fish pathogenic bacteria such as aeromonas veronii, aeromonas hydrophila, aeromonas salmonicida and aeromonas cavisiae, and has broad-spectrum activity of inhibiting various fish pathogenic bacteria. The feed additive is added into feed to feed crucian, can be stably colonized in crucian bodies and is safe to fishes, the growth rate of the crucian and the feed efficiency are improved, and the immunity of the crucian bodies and the resistance of the crucian bodies to fish pathogenic bacteria are enhanced.

Description

Technical Field

[0001] The present invention relates to a microorganism Streptomyces and a microecological preparation thereof, and in particular to a strain of Streptomyces ohiya L1, a microecological preparation thereof and a preparation method thereof. Background Art

[0002] The rapid development of aquaculture has provided the world with a rich diet and created huge economic value. However, with the expansion of aquaculture scale and the increase in intensiveness, a series of problems have also emerged, especially the frequent occurrence of diseases and the deterioration of the water environment. Although traditional disease prevention and control measures have improved aquaculture efficiency to a certain extent, they have also brought many negative effects. The irrational use of chemicals not only destroys the ecological balance of aquaculture waters, but also promotes the development of drug resistance in pathogens, reduces the immunity of aquaculture organisms, and increases disease mortality. What is more serious is that these drug residues may be passed to humans through the food chain, posing a potential threat to human health.

[0003] Given the limitations of traditional disease prevention and control, there is an urgent need to explore more environmentally friendly and sustainable disease prevention and control methods. Among them, probiotics have attracted much attention due to their unique biological characteristics and ecological functions. In the field of aquaculture, probiotics can not only effectively inhibit the growth of harmful pathogens, but also promote the growth and development of farmed organisms and improve their immunity, thereby achieving effective prevention and control of diseases.

[0004] Actinomycetes, as a special class of probiotics, are able to decompose complex macromolecules, produce substances with antimicrobial activity, and form spores to cope with changing environmental conditions. These characteristics make actinomycetes have great application potential in aquaculture, especially in the prevention and treatment of diseases.

[0005] However, despite the broad application prospects of actinomycetes in aquaculture, research on their isolation, screening, identification and application effect evaluation is still relatively lagging behind. The number of strains available is also quite limited.

[0006] CN 114854631 A disclosed a sponge-derived biocontrol Streptomyces ITBBZKa5 on August 5, 2022. Although it can also be used for the prevention and control of aquatic animal diseases, it is mainly used for the prevention and control of plant diseases such as banana wilt pathogen, banana wilt pathogen No. 4 physiological subspecies, banana anthracnose pathogen, papaya anthracnose pathogen, papaya brown stem rot pathogen, papaya ulcer pathogen, mango anthracnose pathogen, pepper anthracnose pathogen, pepper corynepora leaf spot pathogen, cowpea gray spot pathogen, cowpea stem base rot pathogen, date palm microspore pseudodisk multihirsutum, coconut fruit rot pathogen, rice blast pathogen, wheat fusarium wilt pathogen, cotton verticillium wilt pathogen, rubber anthracnose pathogen, cucumber root rot pathogen, pitaya fruit rot pathogen, litchi downy mildew pathogen and ivy anthracnose pathogen. Although it has a certain antibacterial effect on pathogenic bacteria of aquaculture animal tilapia, Streptococcus agalactiae, Streptococcus iniae, and Vibrio harveyi, it is far lower than the control kanamycin, and the antibacterial effect is not satisfactory.

[0007] CN 116590167 A disclosed a Streptomyces and its application in preventing and controlling aquatic animal diseases on August 15, 2023. The Streptomyces rochei strain 2-85 provided therein is co-cultured with halodurous Cladosporium and a fermentation product is obtained by adjusting the culture medium conditions. The fermentation product is separated and purified to obtain an active compound having an antagonistic effect against parasitic Saprolegnia. The antibacterial spectrum is narrow and the antibacterial effect is also poor. The scope of application is limited. Summary of the invention

[0008] 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 ohiya L1. The microecological preparation prepared by the Streptomyces has antagonistic effects on a variety of common pathogens of fish, has a broad antibacterial spectrum, strong protective power, and can resist infection of a variety of bacterial diseases of aquatic animals, especially fish.

[0009] The technical problem to be further solved by the present invention is to provide a Streptomyces microecological preparation suitable for preventing and treating fish diseases in aquaculture.

[0010] A further technical problem to be solved by the present invention is to provide a method for preparing the microecological preparation of Streptomyces ovale.

[0011] The technical solution adopted by the present invention to solve its technical problem is: a strain of Streptomyces omiyaensis L1, which is classified and named as Streptomyces omiyaensis. L1, and was deposited in the China Center for Type Culture Collection on December 12, 2024, with a strain collection number of CCTCC NO: M 20242802.

[0012] The 16S rRNA sequence of Streptomyces ohimiya L1 of the present invention is shown in the sequence table SEQ ID No. 1.

[0013] The Streptomyces Ōmiya L1 of the present invention was screened from soil samples in Sichuan Province.

[0014] The technical solution adopted by the present invention to further solve the technical problem is as follows: a Streptomyces microecological preparation suitable for aquaculture is fermented by Streptomyces Ōmiya L1 with a strain collection number of CCTCC NO: M 20242802. The antibacterial active substances in the fermentation liquid of the strain have good medium and high temperature, ultraviolet irradiation and protease stability, and are relatively stable in actual production applications; it has a good inhibitory effect on freshwater fish pathogens, can promote fish growth, and improve fish immunity.

[0015] Furthermore, the microecological preparation is a liquid preparation or a solid preparation.

[0016] Furthermore, the aquaculture animals are freshwater fish.

[0017] Furthermore, the freshwater fish is crucian carp, grass carp, bighead carp, silver carp or crucian carp.

[0018] The present invention further solves the technical problem by adopting a technical solution: a method for preparing a microecological preparation suitable for aquaculture, comprising the following steps:

[0019] (1) Inoculation and activation: The stored slant strain of Streptomyces ohmiya L1 was transferred to a shake flask seed activation medium for activation to obtain an activated seed liquid; the activated slant medium was a CSM liquid medium having a formula of: 15 g / L tryptone, 5 g / L soy peptone, 5 g / L sodium chloride, 10 g / L glucose, 9 g / L yeast powder, and 2.2 g / L MgSO4·7H2O.

[0020] (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;

[0021] The fermentation medium formula is: soluble starch 15-25 g / L, glucose 5-15 g / L, CaCO3 2-8 g / L, yeast extract 2-8 g / L, bacteriological peptone 2-6 g / L, pH value 7.2-7.4;

[0022] The preferred formula of the fermentation medium is: soluble starch 10g / L, glucose 10g / L, CaCO3 5g / L, yeast extract 5g / L, bacteriological peptone 5g / L; pH value is 7.2-7.4;

[0023] The culture conditions are as follows: the entire culture process is monitored online, the dissolved oxygen concentration is 40-50%, the culture temperature is 28-30°C, the culture time is 72-96h, and the defoaming agent is added online in real time;

[0024] (3) Second expansion culture: inoculate the seed solution of the first expansion culture in step (2) into a fermentation tank filled with fermentation medium at an inoculum rate of 10-15% for fermentation culture, and the fermentation medium formula is the same as the fermentation medium formula in step (2);

[0025] The culture conditions are as follows: dissolved oxygen concentration of 40-50%, culture temperature of 28-30°C, culture time of 96-168h, and real-time online addition of defoaming agent.

[0026] Furthermore, in step (3), the fermentation tank is preferably filled with a fermentation medium equivalent to 65%-70% of the volume of the fermentation tank, and more preferably a 500L-1000L fermentation tank.

[0027] (4) Concentration and collection: After the culture is completed, the fermentation product is collected in a tank and concentrated to obtain a microecological liquid bacterial agent, or spray-dried to obtain a microecological solid preparation.

[0028] The identification and research methods of Streptomyces ohiya L1 of the present invention are as follows.

[0029] (A) Observation of cell morphological characteristics of strain Streptomyces Ōmiya L1;

[0030] (ii) Identification using the 16S rRNA gene;

[0031] (III) Detection of the physical and chemical properties of antibacterial active substances of Streptomyces ohmiya L1;

[0032] (IV) Detection of colonization of Streptomyces Ōmiya L1 in crucian carp;

[0033] (V) Effects of Omiya Streptomyces L1 as feed additive on the growth and disease resistance of crucian carp.

[0034] The invention has the following beneficial effects: (1) the 16S rRNA gene sequence is determined to identify the strain as Streptomyces omiyaensis. L1, with a strain collection number of CCTCC NO: M 20242802; the strain is effective against Aeromonas veronii, Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas caviae, and Aeromonas naevi. caviae) and other freshwater fish pathogens; (2) Omiya Streptomyces L1 can survive and colonize in fish bodies without causing any harm to fish, laying a foundation for the strain to play a stable role in preventing disease and promoting growth in fish farming; (3) Adding Omiya Streptomyces L1 to feed fish as a feed additive can promote the growth rate and feed efficiency of fish, improve the immunity of fish, and enhance the resistance of fish to pathogens; (4) The method of preparing Omiya Streptomyces L1 into a microecological preparation is relatively simple and has a low production cost. It can also effectively reduce the risks of drug residues and pathogenic bacteria resistance caused by the abuse of antibiotics, and has good application prospects.

[0035] SEQ ID №1:

[0036]

[0037]

[0038] Description of Microbiological Deposit

[0039] The Streptomyces omiyaensis. L1 of the present invention was deposited in the China Center for Type Culture Collection (CCTCC for short, address: Wuhan University, Wuhan, China) on December 12, 2024, and the strain deposit number is CCTCCNO: M 20242802. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a diagram showing the antibacterial effect of Streptomyces Ōmiya L1 on fish pathogens;

[0041] Figure 2 Observation diagram of morphological characteristics of Streptomyces Ōmiya L1 of the present invention; a: observation diagram of plate colony morphology of strain L1; b: observation diagram of strain L1 under phase contrast microscope; c: observation diagram of Gram staining of strain L1; d, e: observation diagram of strain L1 under scanning electron microscope;

[0042] Figure 3 The phylogenetic tree diagram is constructed from the 16S rRNA sequence of Streptomyces Ōmiya L1 of the present invention;

[0043] Figure 4 This is a diagram of the observation of liver, kidney, spleen and intestine tissue sections of crucian carp after feeding with Omiya Streptomyces L1 of the present invention;

[0044] Figure 5 This is a diagram of the physical and chemical properties of the antibacterial active substance of Streptomyces ohmiya L1 of the present invention (*, P < 0.05; **, P < 0.01; ***, P < 0.001);

[0045] Figure 6 This is a diagram of the colonization and protection of the L1 labeled strain of Streptomyces Ōmiya in crucian carp observed by the small animal living imaging system of the present invention;

[0046] Figure 7 This is a graph showing the effect of the microecological preparation prepared by Streptomyces L1 of the present invention on the activity of immune-related enzymes in the serum of crucian carp (*, P < 0.05; **, P < 0.01; ***, P < 0.001);

[0047] Figure 8 This is a diagram showing the antagonistic effect of the microecological preparation prepared by Streptomyces L1 on Aeromonas hydrophila in crucian carp observed by the small animal living imaging system of the present invention.

[0048] Fig. 9 This is a diagram of the assembly result of the Streptomyces Ōmiya L1 genome of the present invention.

[0049] Fig.10 It is a statistical diagram of the prediction results of the biosynthetic gene cluster of the secondary metabolites of Streptomyces ohmiya L1 of the present invention. DETAILED DESCRIPTION

[0050] The present invention will be further described below in conjunction with the embodiments and drawings.

[0051] Unless otherwise specified, the chemical reagents used in the embodiments of the present invention were obtained through conventional commercial channels.

[0052] (I) Screening of Streptomyces Ōmiya L1 and determination of antibacterial spectrum

[0053] 120 soil samples from Hunan, Sichuan, Hainan and other regions were thoroughly mixed in sterile water, ultrasonically treated for 30 min, and diluted to 10% of the original mixture according to the gradient dilution method. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5, spread on a Goll's plate, culture at 30℃ for 5 days, observe the growth status of the strain, pick out obvious actinomycete-like single clones, streak continuously on a new Goll's plate, purify the picked strains, and use fish pathogens Aeromonas veronii, Aeromonas salmonicida, Aeromonas hydrophila, Plesiomonas shigelloide, Aeromonas sobria, Aeromonas allosaccharophila, and Aeromonas caviae as indicator bacteria to detect the antibacterial activity of the fermentation supernatant against the above pathogens using the plate well method (for results, see Figure 1 ) and found a broad-spectrum antibacterial strain with good antibacterial effect on the 7 fish pathogens, named Streptomyces omasum L1.

[0054] (II) Cell morphological characteristics of Streptomyces Ōmiya L1

[0055] The strain L1 was streaked on Gao's solid medium No. 1. On the Gao's No. 1 plate, it was observed that the colonies of strain L-1 were small and irregularly round, with wrinkled and convex surfaces, dry and opaque, irregular edges, and white spores. Under a phase contrast microscope, it was observed that the mycelium of strain L-1 was relatively developed and had many branches. Under a cold-field scanning electron microscope, it was observed that the mycelium of strain L-1 was developed and the spores were clearly separated. Based on the above morphological characteristics, strain L1 can be preliminarily identified as an actinomycete (see Figure 2 ).

[0056] (III) Identification of the 16S rRNA gene of Streptomyces Ōmiya L1

[0057] The strain L1 was inoculated into liquid Gould's medium and cultured at 30°C and 120 rpm for 3 days. The bacteria were collected and the genomic DNA of the strain L1 was extracted using the Ezup column bacterial genomic DNA extraction kit (Shanghai Sangon Biotechnology Co., Ltd.) and the 16S rRNA gene amplification primers were used.

[0058] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0059] 1492R: 5'-ACGGCTACCTTGTTACGACTT-3'.

[0060] The 16S rRNA gene sequence was amplified with the above primers, and the expected length of the sequence was about 1500 bp. The specific parameters of PCR are shown in Tables 1 and 2.

[0061] Table 1 PCR reaction system

[0062]

[0063] Table 2 PCR amplification cycles

[0064]

[0065] 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.

[0066] The 16S rRNA gene sequence of strain L1 was sequenced and showed to be 1419 bp in length. The amplified sequencing results were compared and analyzed by BLAST on NCBI. The phylogenetic tree of the strain was constructed by the neighbor-joining method using the software MEGAX (see Figure 3 ).

[0067] (IV) Effects of Streptomyces Ōmiya L1 on the tissue structure of crucian carp

[0068] The conventional feed group (30 days) and the group supplemented with 1×10 8 CFU / mlL1 feed group (30 days), added bacteria 1×10 9 CFU / mlL1 feed group (30 days), strain 1×10 8 CFU / mlL1 feeding (30 days) followed by poison challenge (7 days) and the liver, kidney, spleen and intestine immune tissues of the poison challenge (7 days) group.

[0069] The results showed that adding 1×10 8 CFU / mlL1 feed group (30 days), added bacteria 1×10 9 The tissue status of the group fed with 1×10 CFU / mlL1 (30 days) was consistent with that of the group fed with conventional feed (30 days). The liver, kidney, spleen, and intestinal tissue cells were tightly arranged without pathological symptoms. The intestinal cells of the challenge group injected with Aeromonas hydrophila were continuously destroyed, the intestinal villi atrophied and the mucosa fell off, the spleen tissue area structure was discrete and necrotic, the kidney tissue lost its original organ cavity structure, and the liver cells were arranged in disorder. 8 The lesions of the experimental group were significantly reduced after feeding (30 days) with CFU / mlL1 and then challenged with the virus (7 days). This shows that strain L1 is relatively safe for treating infected crucian carp and is effective in alleviating the lesions of crucian carp viscera infected with Aeromonas hydrophila (see Figure 4 ).

[0070] (V) Analysis of the physicochemical properties of antibacterial active substances of Streptomyces ohmiya L1

[0071] (1) Thermal stability

[0072] The strain L1 was inoculated into Am6 medium, and cultured at 28°C and 120 rpm for 3 days. The culture was centrifuged at 10,000 rpm for 10 minutes. The supernatant was divided into 4 groups. 1 ml was taken from each group and placed in a water bath at 40°C, 60°C, 80°C, and 100°C for 1 hour, respectively, and then returned to room temperature. A control group (fermentation supernatant was not treated) was set up, and an antibacterial experiment was carried out using Aeromonas vernix as an indicator bacteria. The culture was placed upright at 30°C for 24 hours, and the antibacterial changes of each group were observed (see Figure 6 ).

[0073] (2) UV resistance

[0074] The strain L1 was inoculated into liquid Am6 medium and cultured at 30°C and 120 rpm for 3 days. The fermentation liquid was irradiated under ultraviolet light for 60 minutes. Aeromonas vernix was used as a pathogen indicator bacteria and cultured upright at 30°C for 24 hours. The antibacterial changes of each group were observed (see Figure 6 ).

[0075] (3) Protease resistance

[0076] The strain L1 was inoculated into liquid Am6 medium and cultured at 30°C and 120 rpm for 3 days. Proteinase K, trypsin and pepsin were used for treatment at 37°C for 1 h, respectively. Aeromonas vernix was used as a pathogen indicator bacteria and cultured upright at 30°C for 24 h. The antibacterial changes of each group were observed (see Figure 6 ).

[0077] It can be seen that the antibacterial active substances in the fermentation broth of this strain have good medium-high temperature, ultraviolet irradiation and protease stability.

[0078] (VI) Preparation of microecological preparations of Streptomyces ohmiya L1

[0079] (1) Inoculation and activation: The stored slant strain of Streptomyces ohmiya L1 was transferred to a shake flask seed activation medium for activation to obtain an activated seed liquid; the activated slant medium was a CSM liquid medium having a formula of: 15 g / L tryptone, 5 g / L soy peptone, 5 g / L sodium chloride, 10 g / L glucose, 9 g / L yeast powder, and 2.2 g / L MgSO4·7H2O.

[0080] The activation culture conditions are as follows: temperature 29°C, liquid volume 20% of the container volume, and shaking speed 160 rpm;

[0081] (2) First expansion culture: The seed liquid activated in step (1) is inoculated into a fermentation tank at an inoculum rate of 1.5% for the first expansion culture;

[0082] The fermentation medium formula is: soluble starch 20g / L, glucose 10g / L, CaCO3 5g / L, yeast extract 5g / L, bacteriological peptone 4g / L, pH 7.3;

[0083] The preferred formula of the fermentation medium is: soluble starch 10g / L, glucose 10g / L, CaCO3 5g / L, yeast extract 5g / L, bacteriological peptone 5g / L, pH value is 7.3;

[0084] The culture conditions are as follows: the entire culture process is monitored online, the dissolved oxygen concentration is 45%, the culture temperature is 29°C, the culture time is 85h, and the defoaming agent is added online in real time;

[0085] (3) Second expansion culture: The seed liquid of the first expansion culture in step (2) is inoculated into a fermentation tank containing a fermentation medium at an inoculum amount of 12% for fermentation culture. The fermentation medium formula is the same as the fermentation medium formula in step (2); the culture conditions are: dissolved oxygen concentration of 45%, temperature of 29° C., culture for 132 hours, and real-time online addition of defoaming agent;

[0086] The fermentation tank is filled with fermentation medium equivalent to 68% of the volume of the fermentation tank; the volume of the fermentation tank is 800L.

[0087] (4) Concentration and collection: After the culture is completed, the fermentation product is collected and concentrated to obtain a microecological liquid bacterial agent; the liquid bacterial agent is spray-dried to obtain a microecological solid preparation.

[0088] (VII) Application of Streptomyces Ōmiya L1 in fish farming

[0089] Application Example 1: Colonization and protective ability of Streptomyces Ōmiya L1 in crucian carp

[0090] The crucian carps of uniform body size were randomly divided into 2 groups, 30 in each group, and repeated. The two groups of crucian carps were acclimated for 7 days, and the experiment was carried out when the state of each group of crucian carps was stable. The control group was fed with ordinary feed, and the experimental group was fed with a concentration of 1×10 8 CFU / ml of strain L1 EGFP The feeding amount was 2% of the crucian carp's body weight. After 15 days of continuous feeding, three crucian carps from the experimental group were randomly selected every 1 day to observe the strain L1 using a small animal imaging system. EGFP Colonization in crucian carp.

[0091] The results showed that feeding strain L1 EGFP On the first day, the fluorescence signal could be observed in the small animal imaging system, and the fluorescence signal continued to increase over time. The fluorescence signal first appeared in the gills and the front half of the abdomen of the crucian carp, and then continued to deepen and extend to the back half of the abdomen (see Figure 6). After 15 days of continuous observation, the crucian carp grew well. This result shows that strain L1 can stably colonize in the body of crucian carp and has no toxic effect on crucian carp.

[0092] Example 2: Effects of Streptomyces Ōmiya L1 on growth and disease resistance of crucian carp

[0093] The crucian carps with uniform body size were randomly divided into 6 frames, including 3 frames for the experimental group and 3 frames for the control group, with 30 crucian carps in each frame. The experiment was carried out after the crucian carps were domesticated for 7 days and their state was stable. The control group was fed with ordinary feed, and the experimental groups were fed with a concentration of 1×10 8 CFU / mL and 1×10 9 The strain L1 with a CFU / mL was fed at a dose of 2% of the crucian carp's body weight for 30 consecutive days, with feed given twice a day at 8:00 and 20:00, and the indoor temperature was controlled at 25±2℃.

[0094] (1) Effects of Streptomyces Ōmiya L1 on the growth performance of crucian carp

[0095] The weight of crucian carp before grouping was taken as the initial weight (W0), and the weight of crucian carp fed for 30 days was taken as the final weight (W t ), feeding time (t), feed intake (g), initial number of crucian carp in the experiment (n0), and final number of crucian carp in the experiment (n1) were used to calculate the weight growth rate (WGR), specific growth rate (SGR), feed efficiency (FE), and survival rate (SR): WGR = ((W t -W0) / W0)×100%, SGR=((lnW t -lnW0) / t)×100%, FE=((W t -W0) / g)×100%, SR=(n1 / n0)×100% (see Table 3).

[0096] Table 3 Growth performance parameters of crucian carp

[0097]

[0098] Note: Ⅰ: control group; II: 1x10 8 CFU / mL; Ⅲ: 1x10 9 CFU / mL

[0099] It can be seen that the Streptomyces L1 of the present invention has a significant promoting effect on the growth of crucian carp.

[0100] (2) Effects of Streptomyces Ōmiya L1 on nonspecific immune parameters in crucian carp serum

[0101] Strain L1 was added to the feed and fed to crucian carp for 30 days, 1x10 8 CFU / mL feeding group and 1x10 9 The levels of lysozyme, catalase, total superoxide dismutase, glutathionease, and alkaline phosphatase in the serum of the CFU / mL feeding group were significantly higher than those of the control group (P < 0.001). 9 CFU / mL feeding group was 1x10 8 CFU / mL feeding group compared to 1x10 9 The levels of lysozyme, catalase, alkaline phosphatase and acid phosphatase in serum of the CFU / mL feeding group were higher than 1x10 8 CFU / mL feeding group, in which the acid phosphatase content was significantly higher than 1x10 8 CFU / mL feeding group. Phosphatase plays a key role in the regulatory mechanism of the organism. Alkaline phosphatase can increase the concentration of inorganic phosphate through catalytic reactions. This substance is essential for the synthesis of ATP. As the level of inorganic phosphate increases, this helps to increase the metabolic rate of crucian carp, thereby enhancing the function of its immune system. This improvement in immunity is an important factor for crucian carp to ensure its healthy growth. In summary, feeding strain L1 can significantly increase the activity of non-specific immune indicators such as lysozyme, catalase, total superoxide dismutase, glutathionease, and alkaline phosphatase in crucian carp, thereby improving the immune level of the fish.

[0102] (3) Experiment on the protective effect of Streptomyces Ōmiya L1 on crucian carp

[0103] In order to study the protective effect of strain L1 on crucian carp infected with Aeromonas veroni, the experimental group was injected with 100 μL of Aeromonas veroni intraperitoneally into crucian carp fed with strain L1 mixed feed for 30 days. The control group was fed with basic feed and injected with the same amount of Aeromonas veroni. The living conditions of crucian carp in the experimental and control groups were observed within 7 days and the deaths were recorded. The study found that the survival rate of crucian carp in the control group fed with basic feed was 20% 7 days after infection, while the survival rate of crucian carp fed with 1×10 L1 strain was 20% at 7 days after infection. 8 The survival rate of crucian carp in the experimental group of the CFU / g group was 50%, which was significantly higher than that of the control group (see Table 4).

[0104] Table 4 Experiment on the protective effect of Streptomyces daimiya L-1 on crucian carp

[0105]

[0106] (VIII) Antagonistic effect of microecological preparations prepared by Streptomyces Ōmiya L1 against Aeromonas hydrophila in crucian carp

[0107] Carassius auratus with uniform body size were randomly divided into 2 groups, 30 in each group. Carassius auratus were acclimated for 7 days and starved for 24 hours. The carassius auratus in the control group and the experimental group were intraperitoneally injected with 100 μL of 1×10 8 CFU / mL of Av mCherry The strain Av was observed every 1 day using a small animal imaging system. mCherry The colonization and distribution of the carp were observed for 7 consecutive days. After the 7th day, the carp in the experimental group were fed with a concentration of 1×10 8 The distribution and brightness changes of red fluorescence in crucian carp were observed using a small animal imaging system every 1 day for 7 consecutive days.

[0108] The results showed that the fluorescence intensity of Aeromonas hydrophila in crucian carp increased over time. On the first day, the fluorescence signal first appeared in the gills, and on the third day, it began to spread and the signal continued to increase. 8 CFU / mL of strain L1, the small animal imaging system found that the fluorescence signal of crucian carp fed with strain L1 was significantly weakened compared with the control group, indicating that strain L1 can alleviate the infection of Aeromonas veronis in crucian carp (refer to Figure 8 ).

[0109] (IX) Assembly and characteristics of the Streptomyces Ōmiya L1 genome

[0110] After optimizing the genome assembly of the quality-controlled data, the final genome was obtained. The genome consists of 2 contigs with a size of 8563093 bp. The assembly results are statistically (refer to Fig. 9 ). The strain L1 was analyzed online using the online platform antiSMASH to predict its genome secondary metabolite synthesis gene clusters. This analysis was conducted from the nuclear genome tig00001 and plasmid tig00002 of strain L1. The analysis results are shown in the table. A total of 34 secondary metabolite synthesis gene clusters were obtained, of which 33 synthesis gene clusters were predicted in the nuclear genome tig00001 (reference Fig.10 ), of which there are 9 gene clusters associated with the synthesis of non-ribosomal polypeptide (NRPS) synthetase, 6 gene clusters related to polyketide synthase (TPKS), 4 gene clusters related to terpenes synthesis, and 4 gene clusters related to lanthipeptides synthesis. It reveals that the new strain L1 of Streptomyces Ōmiya has diverse biosynthetic potential, indicating that strain L1 is a Streptomyces with rich secondary metabolites.

Claims

1. A strain of Streptomyces ohimiya L1, characterized in that: The classification name of this Streptomyces is: Streptomyces omiyaensis. L1, which was deposited in the China Center for Type Culture Collection on December 12, 2024, and the strain collection number is CCTCC NO: M 20242802.

2. A microecological preparation suitable for aquaculture, characterized in that: The product is prepared by fermenting the Omiya Streptomyces L1 strain with the strain collection number of CCTCC NO: M 20242802 as described in claim 1.

3. The microecological preparation suitable for aquaculture according to claim 2, characterized in that: The microecological preparation is a liquid preparation or a solid preparation.

4. The microecological preparation suitable for aquaculture according to claim 2 or 3, characterized in that: The aquaculture animals are freshwater fish.

5. The microecological preparation suitable for aquaculture according to claim 4, characterized in that: The freshwater fish is crucian carp, grass carp, bighead carp or silver carp.

6. A method for preparing a microecological preparation suitable for aquaculture as claimed in claim 2 or 3, characterized in that: The following steps are involved: (1) Inoculation and activation: The stored slant strain of Streptomyces Ōmiya L1 was transferred to the seed activation medium of the shake flask for activation to obtain the activated seed liquid; (2) First expansion culture: The seed solution activated in step (1) is inoculated into a fermentation tank for first expansion culture to obtain expansion culture solution I; (3) Second expansion culture: inoculate the expansion culture solution I in step (2) into a fermentation tank containing fermentation medium at an inoculum amount of 10-15%, and culture again to obtain expansion culture solution II; (4) Concentration and collection: After the tank is placed, the expanded culture medium II is collected and concentrated to obtain a microecological liquid preparation; the solid preparation is obtained by filtering the liquid preparation with a ceramic membrane, collecting the filtrate, and then spray drying the filtrate.

7. The method for preparing a microecological preparation suitable for aquaculture according to claim 6, characterized in that: In step (1), the shake flask seed activation medium is CSM liquid medium, and the formula is: 15g / L tryptone, 5g / L soy peptone, 5g / L sodium chloride, 10g / L glucose, 9g / L yeast powder, 2.2g / L MgSO4.7H2O; the activation process conditions are: temperature 28-30°C, liquid volume 10-30% of the container volume, and shaker speed 150-180rpm.

8. The method for preparing a microecological preparation suitable for aquaculture according to claim 6 or 7, characterized in that In step (2), the inoculation amount of the seed liquid inoculated into the fermentation tank is 1-2% of the volume of the culture medium; the solvent of the fermentation medium used for the first expansion culture is water, and the formula is: soluble starch 15-25g / L, glucose 5-15g / L, CaCO3 2-8g / L, yeast extract 2-8g / L, bacteriological peptone 2-6g / L; The culture conditions are as follows: the entire culture process is monitored online, the dissolved oxygen concentration is 40-50%, the culture temperature is 28-30°C, the culture time is 72-96h, and the defoaming agent is added online in real time.

9. The method for preparing a microecological preparation suitable for aquaculture according to claim 6 or 7, characterized in that: In step (3), the fermentation medium formula is the same as the fermentation medium formula described in step (2); The culture conditions are as follows: dissolved oxygen concentration of 40-50%, culture temperature of 28-30°C, culture time of 96-168h, and real-time online addition of defoaming agent.

10. The method for preparing a microecological preparation suitable for aquaculture according to claim 6 or 7, characterized in that: In step (3), the fermentation tank is filled with fermentation medium equivalent to 65-70% of the volume of the fermentation tank.

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