Streptomyces Q15, microecological preparation thereof and preparation method of microecological preparation

Abstract

The invention relates to a streptomyces Q15, a microecological preparation thereof and a preparation method of the microecological preparation, the streptomyces Q15 is preserved in China Center for Type Culture Collection on October 17, 2024, the strain preservation number is CCTCC NO: M 20242237, the classification name is streptomyces Q15, and the Latin name is Streptomyces sp.Q15. The invention also discloses a preparation method of the microecological preparation, the streptomyces Q15 is preserved in China Center for Type Culture Collection on October 17, 2024, and the strain preservation number is CCTCC NO: M 20242237. The microecological preparation prepared by fermenting streptomyces Q15 is suitable for aquaculture, and can effectively inhibit various freshwater fish pathogenic bacteria such as aeromonas hydrophila, aeromonas veronii, edwardsiella tarda and the like; the broad-spectrum fish pathogenic bacterium inhibition activity is realized; the strain can be stably colonized in freshwater fish bodies, is harmless to fishes, and can enhance the immunity of the freshwater fish, so that the resistance of the freshwater fish such as crucian and the like to fish pathogenic bacteria is remarkably improved.

Description

Technical Field

[0001] This invention relates to a strain of Streptomyces, its microecological preparation and preparation method thereof, specifically to a strain of Streptomyces Q15 used for aquaculture, its microecological preparation and preparation method thereof. Background Technology

[0002] With the rapid development of high-density aquaculture, it has not only brought significant economic returns and more diverse dietary choices to society, but has also sparked widespread concern about the industry's sustainable development, especially environmental sustainability. We aim for an aquaculture model that can continuously provide healthy, safe, and beneficial aquaculture for humans, while protecting aquatic ecosystems and ensuring that the self-purification capacity of water bodies is not compromised. High-density farming and inadequate disease management measures have led to outbreaks of serious diseases, especially bacterial diseases, which pose a significant challenge to the sustainable development of aquaculture. Currently, the treatment of bacterial diseases in aquaculture generally relies on fast-acting synthetic antibiotics and chemical drugs. However, this reliance brings a series of problems: long-term use of these drugs not only damages the farming environment but also promotes drug resistance in many pathogens, reduces the immunity of farmed animals, increases their disease mortality rate, and may transmit drug residues to humans through aquatic products, increasing the risk of infectious diseases and threatening human health. Given the increasingly serious problem of drug resistance caused by antibiotic overuse, there is an urgent need to develop new drugs to effectively combat bacterial diseases in fish to ensure the healthy development of aquaculture and food safety.

[0003] Microbial preparations developed using high-tech methods such as microbial technology and biotechnology are currently considered to be non-toxic and green products that are well-suited for improving aquaculture water quality, replacing antibiotics in disease prevention and control, promoting the growth of aquatic animals, and improving nutrient utilization.

[0004] Streptomyces are a class of microorganisms with broad application potential. In aquaculture, Streptomyces have been extensively studied and applied in the development of microecological preparations. Studies have shown that the antibacterial substances and bioactive substances they produce can effectively control pathogens in water bodies and improve the health of aquatic animals. Related technical literature (including patent literature) reports an increasing number of applications since the beginning of this century, especially in recent years. Examples include: a strain of *Streptomyces algaecide* N1-32 reported in CN 110331103 A, its microecological preparation and preparation method; a strain of *Streptomyces termitans* N-15 reported in CN 114426935 A, its microecological preparation and preparation method; a strain of *Streptomyces rubrum* Z1-26 reported in CN 113621533 A, its microecological preparation and preparation method; a strain of *Streptomyces Virginiais* W18 reported in CN114561315 A, its microecological preparation and preparation method; a strain of *Streptomyces saarmycin* reported in CN 115975846 A, its microecological preparation and preparation method; and a strain of *Streptomyces* reported in CN 116590167 A and its application in the prevention and control of diseases in aquatic animals, etc.

[0005] CN 110468086 A discloses a strain of Streptomyces griseus HL-50 and a compound microbial agent prepared with Eucalyptus polybractea essential oil. It claims that "this compound microbial agent can not only effectively repel various parasites infecting freshwater fish, such as Gyrodactylus, Trichodina, Chilodonella, Sporozoa, Ichthyophthirius multifiliis, and ciliates, but can also be taken internally to regulate the balance and health of the microbial community in fish; while repelling parasites, this compound microbial agent can also effectively inhibit the reproduction of harmful bacteria in freshwater fish farming water, reduce bacterial infections, and thus improve fish growth. Furthermore, this microbial agent is safe to use, does not produce any toxic side effects, is simple to prepare, environmentally friendly, has a wide range of applications, and has a very good parasite-repelling effect, which can save aquaculture costs." However, the study only conducted deworming trials on freshwater fish. While claiming to "effectively inhibit the reproduction of harmful bacteria in freshwater fish farming water, reduce bacterial infections, and thus improve fish growth," no related tests on inhibiting bacterial infections were performed. Therefore, its effectiveness in inhibiting the reproduction of harmful bacteria in freshwater fish farming water is highly questionable. Furthermore, the disclosed compound microbial agent requires mixing with Eucalyptus polybrachium oil, which originates from distant Australia, making the raw material and preparation costs of Eucalyptus polybrachium oil obviously higher. Since the compound microbial agent is mixed with Eucalyptus polybrachium oil, which also has deworming properties, its deworming effect should be the result of the synergistic effect of Streptomyces grayi HL-50 and Eucalyptus polybrachium oil, rather than the effect of Streptomyces grayi HL-50 alone. Logically, it can be inferred that the deworming effect of Streptomyces grayi HL-50 alone would be less than ideal, let alone effective in preventing and treating bacterial diseases in fish.

[0006] In addition, existing microbial and microecological preparations have some other problems in application, such as poor activity stability of strains, limited types of strains, narrow selection range, and uncertainty in environmental adaptability.

[0007] Therefore, more in-depth and comprehensive research is needed on microbial strains and microecological preparations to increase the number of new microbial strains and microecological preparations, so as to provide more biological means for the prevention and control of diseases in aquatic fish. 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 novel Streptomyces strain. The microecological preparation prepared from this Streptomyces strain has a good antagonistic effect on a variety of common pathogens in fish and can effectively resist the infection of a variety of bacterial diseases in fish.

[0009] A further technical problem to be solved by the present invention is to provide a Streptomyces microecological preparation suitable for the prevention and treatment of fish diseases in aquaculture and its preparation method.

[0010] The technical solution adopted by the present invention to solve its technical problem is as follows: A strain of Streptomyces Q15 was deposited at the China Center for Type Culture Collection on October 17, 2024, with accession number CCTCC NO:M20242237.

[0012] The 16S rRNA sequence of Streptomyces Q15 of the present invention is shown in SEQ ID NO. 1 of the sequence listing.

[0013] The Streptomyces Q15 strain of this invention was screened from 87 soil samples from Yunnan Province, Hainan Province, and other regions.

[0014] The technical solution adopted by this invention to further solve its technical problem is as follows: A Streptomyces microecological preparation suitable for aquaculture, which is fermented from Streptomyces Q15 with preservation number CCTCC NO:M 20242237. The antibacterial active substances in the fermentation broth of this strain have good stability under medium and high temperature, ultraviolet irradiation and protease, and are relatively stable in actual production applications; it has a good inhibitory effect on pathogenic bacteria of freshwater fish, 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 mentioned include, but are not limited to, crucian carp, grass carp, or common carp.

[0018] The technical solution adopted by this invention to further solve its technical problem is as follows: A method for preparing a microecological preparation suitable for aquaculture, comprising the following steps:

[0019] (1) Inoculation and activation: The Streptomyces Q15 preservation slant culture was transferred to the shake flask seed activation medium for activation to obtain the activated seed liquid;

[0020] Furthermore, the activated slant culture medium is CSM liquid culture medium with the following formula: 15 g / L tryptone, 5 g / L soybean peptone, 5 g / L sodium chloride, 10 g / L glucose, 9 g / L yeast extract, and 2.2 g / L MgSO4·7H2O.

[0021] (2) First expansion culture: The activated seed liquid from step (1) is inoculated into the fermenter at an inoculation rate of 1-2% for the first expansion culture;

[0022] Furthermore, the fermentation medium formula is as follows: 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 7.0-7.4;

[0023] Furthermore, the preferred formulation of the fermentation medium is: 10 g / L soluble starch, 10 g / L glucose, 5 g / L CaCO3, 5 g / L yeast extract, 5 g / L bacteriological peptone, and pH 7.0–7.4;

[0024] Furthermore, the cultivation conditions are as follows: the entire cultivation process is monitored online, dissolved oxygen concentration is 35-55%, temperature is 28-30℃, cultivation time is 72-96 hours, and defoamer is added online in real time;

[0025] (3) Second expansion culture: The seed liquid from the first expansion culture in step (2) is inoculated into a fermenter containing fermentation medium at an inoculation rate of 10-15%. The fermentation medium formula is the same as the fermentation medium formula in step (2).

[0026] Furthermore, the cultivation conditions are: dissolved oxygen concentration 35-55%, temperature 28-30℃, cultivation time 96-168h, and real-time online replenishment of defoamer;

[0027] Furthermore, in step (3), the fermenter preferably contains fermentation medium equivalent to 65%-70% of its volume. More preferably, a 500L-1000L fermenter is used.

[0028] (4) Concentration and collection: After the culture is completed, the fermentation product is collected and concentrated to obtain a microecological liquid agent, or a microecological solid preparation is obtained by spray drying.

[0029] The identification and research methods of Streptomyces Q15 of this invention are as follows.

[0030] (I) Observation of the cell morphology characteristics of strain Streptomyces Q15;

[0031] (ii) Identification using the 16S rRNA gene;

[0032] (III) Detection of the physicochemical properties of the antibacterial active substance of Streptomyces Q15;

[0033] (iv) Colonization detection of Streptomyces Q15 in crucian carp;

[0034] (V) Effects of Streptomyces Q15 as a feed additive on the growth and disease resistance of crucian carp.

[0035] The beneficial effects of this invention are as follows: (1) It was identified as Streptomyces sp. Q15 by 16S rRNA gene sequence determination (strain preservation number: CCTCCNO: M 20242237; classification name: Streptomyces sp. Q15, Latin name: Streptomyces sp. Q15), which has a good antibacterial effect on a variety of freshwater fish pathogens such as Aeromonas hydrophila, Aeromonas vernix, and Edwardsiella tarda; (2) Streptomyces Q15 can survive and colonize in fish without harming them, laying the foundation for this strain to play a stable role in disease prevention and growth promotion in fish farming; (3) Adding Streptomyces Q15 as a feed additive to feed fish 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 Streptomyces Q15 into a microecological preparation is relatively simple and has a low production cost. At the same time, it can effectively reduce the risks of drug residues and drug resistance of pathogens caused by antibiotic abuse, and has good application prospects.

[0036] SEQ ID No. 1:

[0037]

[0038]

[0039] Description of Microbial Preservation

[0040] The Streptomyces Q15 of this invention was deposited on October 17, 2024, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China), with accession number CCTCC NO:M 20242237; classified as Streptomyces Q15; Latin scientific name: Streptomyces sp.Q15. Attached Figure Description

[0041] Figure 1This is a diagram illustrating the antibacterial effect of Streptomyces Q15 of the present invention against fish pathogens;

[0042] Figure 2 These are microscopic views of the morphological characteristics of Streptomyces Q15 of the present invention; a: Observation of the morphology of strain Q15 on a plate; b: Observation of strain Q15 under phase contrast microscopy; c: Observation of strain Q15 under Gram staining; d, e: Observation of strain Q15 under scanning electron microscopy.

[0043] Figure 3 This is a phylogenetic tree diagram constructed from the 16S rRNA sequence of Streptomyces Q15 of this invention;

[0044] Figure 4 These are comparative microscopic images showing the effects of Streptomyces Q15 of this invention on the tissue structure of crucian carp.

[0045] Figure 5 The physicochemical properties analysis diagram of the antibacterial active substance of Streptomyces Q15 in this invention is shown in the figure (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

[0046] Figure 6 This invention provides a small animal in vivo imaging system for observing the colonization and protective force of Streptomyces Q15-labeled strains in crucian carp.

[0047] Figure 7 The figure shows the results of the analysis of the effect of the microecological preparation of Streptomyces Q15 of this invention on the activity of immune-related enzymes in crucian carp serum (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

[0048] Figure 8 This is a diagram showing the antagonistic effect of a microecological preparation made from Streptomyces Q15 on Aeromonas hydrophila in crucian carp, observed using the small animal in vivo imaging system of this invention.

[0049] Figure 9 This is the result of the assembly of the Streptomyces Q15 genome in this invention.

[0050] Figure 10 This invention is a prediction of the gene cluster for the biosynthesis of secondary metabolites of Streptomyces Q15. Detailed Implementation

[0051] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0052] Unless otherwise specified, the chemical reagents used in the embodiments of this invention are obtained through conventional commercial channels.

[0053] (I) Screening and Antibacterial Spectrum Determination Tests for Streptomyces Q15

[0054] Eighty-seven soil samples from Yunnan Province, Hainan Province, and other regions were thoroughly mixed in sterile water and diluted to 10% of the original mixture using a serial dilution method. -1 10 -2 10 -3 10 -4 10 -5 The bacteria were spread on Gao's agar plates and incubated at 30°C for 5 days. The growth status of the strains was observed, and single colonies of actinomycetes were picked and inoculated onto fresh culture medium for purification. Using *Aeromonas veronii* AvX005, *Aeromonas hydrophila*, *Aeromonas caviae*, *Aeromonas salmonicida*, *Aeromonas allosaccharophila*, *Edwardsiella tarda*, *Aeromonas sobria*, and *Plesiomonas shigelloide* as indicator bacteria, the antibacterial activity of the fermentation supernatant against these pathogens was detected using the plate well trap method (results are shown in [link to results]). Figure 1 A strain with good antibacterial effect against eight fish pathogens was discovered and named Streptomyces Q15.

[0055] (II) Cell morphological characteristics of Streptomyces Q15

[0056] Streptomyces Q15 was streaked onto Gao's No. 1 solid medium. The colonies of strain Q15 were observed to be opaque and white, with neat edges and a granular, scattered distribution. Spore growth was abundant, and the colonies were tightly bound to the medium, making them difficult to pick up. Phase-contrast microscopy revealed well-developed, branched mycelium. Gram staining showed a purple color, confirming strain Q15 as Gram-positive. Cold field scanning electron microscopy revealed numerous branched aerial hyphae with well-developed, spirally coiled structures. Based on these morphological characteristics, strain Q15 was preliminarily identified as an actinomycete (see [link to relevant documentation]). Figure 2 ).

[0057] (III) Identification of the 16S rRNA gene of Streptomyces Q15

[0058] Streptomyces Q15 was inoculated into liquid Gao's medium and cultured at 30°C and 120 rpm for 4 days. The bacterial cells were collected, and genomic DNA was extracted from strain Q15 using the Ezup column-based bacterial genomic DNA extraction kit (Shanghai Sangon Biotech Co., Ltd.). 16S rRNA gene amplification primers were used.

[0059] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0060] 1492R: 5'-ACGGCTACCTTGTTACGACTT-3'.

[0061] The 16S rRNA gene sequence was amplified using the primers described above, with an expected sequence length of approximately 1500 bp. Specific PCR parameters are shown in Tables 1 and 2.

[0062] Table 1 PCR reaction system

[0063]

[0064] Table 2 PCR amplification cycles

[0065]

[0066] After the PCR products of the 16S rRNA gene were detected by 1.0% agarose gel electrophoresis, the successfully amplified products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0067] The 16S rRNA gene sequence of strain Q15 was sequenced to be 1423 bp in length. The amplified sequencing results were analyzed by BLAST alignment on NCBI, and a phylogenetic tree of this strain was constructed using the neighbor-joining method in MEGAX software (see [link to MEGAX]). Figure 3 ).

[0068] (IV) Effects of Streptomyces Q15 on the tissue structure of crucian carp

[0069] For the group fed with conventional feed (30 days), 1×10 of bacterial strain was added. 9 Feed group with CFU / mlQ15 (30 days), bacterial culture 1×10 9 Liver, kidney, spleen, intestines, and immune tissues of patients fed with CFU / mlQ15 for 30 days followed by challenge for 7 days and challenged for 7 days.

[0070] The results showed that the tissue condition of the feed group (30 days) supplemented with 1×10⁹ CFU / ml Q15 of bacterial strain was consistent with that of the conventional feed group (30 days). Liver, kidney, spleen, and intestinal cells were tightly packed, with no pathological symptoms. In the challenge group, injection of Aeromonas hydrophila caused discontinuous disruption of intestinal cells, atrophy of intestinal villi, and mucosal sloughing. The spleen showed structural dispersion and necrosis, the kidneys lost their original tubular structure, and hepatocytes were disordered. The bacterial strain supplemented with 1×10⁹ CFU / ml Q15... 9 The experimental group fed with CFU / ml Q15 for 30 days followed by challenge with the virus for 7 days showed a significant reduction in lesions. This indicates that strain Q15 is relatively safe for treating infected crucian carp and effectively alleviates visceral lesions in crucian carp infected with Aeromonas hydrophila.

[0071] (V) Physicochemical Properties Analysis of the Antibacterial Activity of Streptomyces Q15

[0072] (1) Thermal stability

[0073] Strain Q15 was inoculated into Am6 medium and cultured at 28°C with shaking at 120 rpm for 5 days. After centrifugation at 13000 rpm for 10 minutes, the supernatant was divided into four groups. One ml of each group was placed in water baths at 30°C, 50°C, 70°C, and 90°C for 1 hour, respectively, and then allowed to return to room temperature. A control group (fermentation supernatant untreated) was included. Antimicrobial inhibition experiments were conducted using *Aeromonas hydrophila* as an indicator bacterium. The cultures were incubated upright at 30°C for 24 hours, and the antimicrobial changes in each group were observed (results are shown in [link to results]). Figure 6 ).

[0074] (2) UV tolerance

[0075] Strain Q15 was inoculated into liquid Am6 medium and cultured at 30℃ and 120 rpm for 5 days. The fermentation broth was then irradiated under a UV lamp for 10 to 60 minutes. Aeromonas hydrophila was used as the pathogen indicator bacterium, and the mixture was incubated upright at 30℃ for 24 hours. Antibacterial changes in each group were observed. The antibacterial activity of the fermentation supernatant after different UV irradiation times was detected using the plate trap method (results are shown in [link to results]). Figure 6 ).

[0076] (3) Protease tolerance

[0077] Strain Q15 was inoculated into liquid Am6 medium and cultured at 30℃ and 120 rpm for 5 days. It was then treated with proteinase K, trypsin, and pepsin at 37℃ for 1 hour. Aeromonas hydrophila was used as the pathogen indicator bacterium, and the culture was incubated upright at 30℃ for 24 hours. Antimicrobial activity was observed in each group. The antimicrobial activity of the fermentation supernatant after treatment with different proteases was detected using the plate trap method (results are shown in [link to results]). Figure 6 ).

[0078] Depend on Figure 6 It is evident that the antibacterial active substances in the fermentation broth of Streptomyces Q15 of this invention exhibit good stability under medium and high temperatures, ultraviolet irradiation, and protease.

[0079] (vi) Preparation of Streptomyces Q15 probiotic preparation

[0080] (1) Inoculation and activation: The Streptomyces Q15 preservation slant culture was transferred to the shake flask seed activation medium for activation to obtain the activated seed liquid; the activation slant medium was CSM liquid medium with the following formula: 15 g / L tryptone, 5 g / L soybean peptone, 5 g / L sodium chloride, 10 g / L glucose, 9 g / L yeast powder, and 2.2 g / L MgSO4·7H2O.

[0081] The activation culture conditions are: temperature 29℃, liquid volume 20% of container volume, and shaker speed 160 rpm.

[0082] (2) First expansion culture: The activated seed liquid from step (1) is inoculated into the fermenter at an inoculation rate of 1-2% for the first expansion culture;

[0083] The fermentation medium formula is as follows: 10 g / L soluble starch, 10 g / L glucose, 5 g / L CaCO3, 5 g / L yeast extract, 5 g / L bacteriological peptone, pH 7.2;

[0084] The cultivation conditions were as follows: the entire cultivation process was monitored online, dissolved oxygen concentration was 45%, temperature was 29℃, cultivation time was 85h, and defoamer was added online in real time;

[0085] (3) Second expansion culture: The seed liquid from the first expansion culture in step (2) is inoculated into a fermenter containing fermentation medium at an inoculation rate of 10-15%. The fermentation medium formula is the same as the fermentation medium formula in step (2). The culture conditions are: dissolved oxygen concentration 45%, temperature 28℃, culture for 135h, and defoamer is added online in real time.

[0086] In step (3), the fermenter is filled with fermentation medium equivalent to 65% of the fermenter's volume. The fermenter has a volume of 1000L.

[0087] Concentration and Collection: After cultivation, the product is placed in a tank, collected, concentrated, and a microecological liquid inoculant is obtained (the liquid inoculant can be spray-dried to obtain a microecological solid preparation).

[0088] (VII) Application Experiment of Streptomyces Q15 in Fish Farming

[0089] Application Experiment Example 1: Colonization and Protective Power of Streptomyces Q15 in Crucian Carp

[0090] Uniformly sized crucian carp were randomly divided into two groups of 10 fish each, with replicates. Both groups of crucian carp were acclimatized for 7 days until their condition stabilized before the experiment. The control group was fed a standard diet, while the experimental groups were fed a diet with a concentration of 1×10⁻⁶. 9 CFU / ml strain Q15 EGFP The feeding amount was 2% of the crucian carp's body weight. After 15 days of continuous feeding, three crucian carp from the experimental group were randomly selected every day to observe the strain Q15 using a small animal imaging system. EGFP Colonization within the crucian carp.

[0091] The results showed that feeding strain Q15 EGFPOn day 1, a fluorescent signal could be observed in the small animal imaging system, and this signal continuously increased over time. The fluorescent signal first appeared in the gills and the anterior half of the abdomen of the crucian carp, and then continued to deepen and extend towards the posterior half of the abdomen (see...). Figure 6 After 15 days of continuous observation, the crucian carp showed good growth. This result indicates that strain Q15 can stably colonize crucian carp without causing toxicity, laying the foundation for further research on the effects of strain Q15 on the growth and immune defense capabilities of crucian carp.

[0092] Application Experiment Example 2: Effects of Streptomyces Q15 on the growth and disease resistance of crucian carp

[0093] Uniformly sized crucian carp were randomly divided into 6 frames, with 3 frames for the experimental group and 3 frames for the control group. Each frame contained 10 crucian carp. After acclimatization for 7 days until the crucian carp were stable, the experiment was conducted. The control group was fed ordinary feed, while the experimental group was fed a feed with a concentration of 1×10⁻⁶. 9 CFU / mL and 1×10 10 The strain Q15 with a CFU / mL content was fed to the crucian carp at a rate of 2% of their body weight for 30 consecutive days, twice a day at 8:00 and 20:00, with the indoor temperature controlled at 25±2℃.

[0094] (1) Effects of Streptomyces Q15 on the growth performance of crucian carp

[0095] The initial weight (W0) of the crucian carp before grouping was taken as the initial weight, and the final weight (W0) of the crucian carp after 30 days of feeding was taken as the final weight. t The following formulas 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 9 CFU / mL; III: 1x10 10CFU / mL

[0099] It is evident that the novel Streptomyces Q15 of this invention promotes the growth of crucian carp.

[0100] (2) Effects of Streptomyces Q15 on non-specific immune markers in crucian carp serum

[0101] Streptomyces Q15 strain was added to the feed and fed to crucian carp for 30 days at a dose of 1 x 10. 9 The serum alkaline phosphatase (AKP) and acid phosphatase (ACP) activities in the CFU / mL feeding group were significantly higher than those in the control group (P < 0.001). 9 CFU / mL feeding group compared to x10 10 Compared to the CFU / mL feeding group, the high concentration of alkaline phosphatase (AKP) showed higher enzyme activity than the low concentration group, while the high concentration group of acid phosphatase (ACP) showed lower activity but still higher than the control group. Phosphatases play a crucial role in the regulatory mechanisms of organisms. Alkaline phosphatase can increase the concentration of inorganic phosphate through catalytic reactions, a substance essential for ATP synthesis. The increase in inorganic phosphate levels helps to enhance the metabolic rate of crucian carp, thereby strengthening its immune system function. This enhanced immunity is an important factor in ensuring the healthy growth of crucian carp. In short, AKP phosphatase promotes metabolism and immunity in crucian carp by increasing the content of inorganic phosphate in their bodies, thus ensuring their robust growth. 1x10 9 The activities of glutathione (GSH), catalase (CAT), total superoxide dismutase (T-SOD), and lysozyme (LZM) in the serum of the CFU / mL feeding group were significantly higher than those in the control group (P < 0.01). However, in the high-concentration feeding group, the enzyme activities were all decreased to varying degrees. The increased T-SOD activity helps the body scavenge superoxide anion free radicals and protects cells from damage (see [link to relevant documentation]). Figure 7 This indicates that feeding with strain Q15 can enhance the activity of non-specific immune indicators such as SOD and AKP in crucian carp, thereby improving the fish's immune level and enabling grass carp to better adapt to the external environment, playing a significant role in the fish's immune defense.

[0102] (3) Protective test of Streptomyces Q15 on crucian carp

[0103] To investigate the protective efficacy of strain Q15 against *Aeromonas hydrophila* infection in crucian carp, the experimental group received intraperitoneal injections of 200 μL of *Aeromonas hydrophila* after being fed a basal diet for 30 days. Control group 1 received an equal volume of PBS, while control group 2 received an equal volume of *Aeromonas hydrophila* after being fed a basal diet. The survival rate and mortality of the crucian carp were observed for 7 days after infection. The study found that the survival rate of the control group fed a basal diet was 20% 7 days after infection, while the survival rate of the Q15 strain was significantly higher at a concentration of 1 × 10⁻⁶ μL. 9 CFU / g and 1×10 10 The survival rates of crucian carp in the CFU / g experimental groups were 40% and 62%, respectively, which were significantly higher than those in the control group (see Table 4).

[0104] Table 4. Protective effect of strain Q15 on crucian carp

[0105]

[0106] (VIII) Antagonistic effect of the microecological preparation made from Streptomyces Q15 on Aeromonas hydrophila in crucian carp

[0107] Uniformly sized crucian carp were randomly divided into two groups of 15 fish each. The fish were acclimatized for 7 days and then starved for 24 hours. Both the control and experimental groups were intraperitoneally injected with 100 μL of a 1×10⁻⁶ concentration. 8 Ahm CFU / mL Ch erry, using a small animal imaging system, observed strain Ah every 1 day mCherry The colonization and distribution of the fish within the crucian carp were observed continuously for 7 days. After the 7th day, the experimental group of crucian carp were fed a feed concentration of 1×10⁻⁶. 9 Q15 at CFU / mL was used to observe the distribution and brightness changes of red fluorescence in crucian carp every 1 day using a small animal imaging system for 7 consecutive days.

[0108] The results showed that the fluorescence intensity of *Aeromonas hydrophila* in crucian carp increased progressively over time. The fluorescence signal first appeared in the gills on day 1, and began to spread and intensify on day 3. When the experimental group of crucian carp was fed 1×10⁻⁶ bacteria on day 7... 9 CFU / mL of strain Q15 was observed using a small animal imaging system to significantly reduce the fluorescence signal in crucian carp fed strain Q15 compared to the control group, indicating that strain Q15 can alleviate Aeromonas hydrophila infection in crucian carp (refer to...). Figure 8 ).

[0109] (ix) Genome assembly and characteristics of Streptomyces Q15

[0110] After optimizing the quality-controlled data through genome assembly, the final genome was obtained. The genome consists of 4 contigs, with a size of 9,335,044 bp. For assembly result statistics, please refer to [link to relevant documentation]. Figure 9 The antiSMASH platform was used to analyze strain Q-15 online to predict its genomic secondary metabolite synthesis gene clusters. This analysis examined both the nuclear genome tig00001 and plasmid tig00002 of strain Q-15. The results are shown in the table. A total of 34 secondary metabolite synthesis gene clusters were obtained, of which 33 were predicted in the nuclear genome tig00001 (see table). Figure 10 Among them, five gene clusters are associated with the synthesis of nonribosomal polypeptide (NRPS) synthases, four gene clusters are associated with polyketide synthases (TPKS), four gene clusters are associated with terpene synthesis, and four gene clusters are associated with lanthanum peptide synthesis. This reveals the diverse biosynthetic potential of Streptomyces Q-15, indicating that strain Q-15 is a novel Streptomyces strain with abundant secondary metabolites.

Claims

1. A strain of Streptomyces Q15, characterized in that: Classification name: Streptomyces Q15 ( Streptomyces sp.Q15), was deposited in the China Center for Type Culture Collection on October 17, 2024, with the deposit number CCTCC NO: M 20242237.

2. A microecological preparation suitable for aquaculture, characterized in that: It is a microecological preparation prepared by fermenting the Streptomyces Q15 strain with the deposit number CCTCC NO: M 20242237 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 or 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 Q15 was transferred to the seed activation medium in a shake flask for activation to obtain an activated seed solution; (2) the first expansion culture: the step (1) inoculated into the activated seed solution into the fermenter for the first expansion culture, the expansion culture medium I; (3) Second expansion culture: The expanded culture medium I obtained in step (2) was inoculated into a fermenter containing a fermentation medium at an inoculum amount of 10 to 15%, and cultured again to obtain expanded culture medium II; (4) Concentration and collection: After tanking, 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 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, the formula is: tryptone 15g / L, soy peptone 5g / L, sodium chloride 5g / L, glucose 10g / L, yeast powder 9g / L, MgSO4.7H2O2.2g / L; the activation culture 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 15-25 g / L, glucose 5-15 g / L, CaCO3 2-8 g / L, yeast extract 2-8 g / L, bacteriological peptone 2-6 g / L; The culture conditions are as follows: the entire culture process is monitored online, the dissolved oxygen concentration is 35-55%, the temperature is 28-30°C, the culture 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: dissolved oxygen concentration of 35-55%, temperature of 28-30°C, culture for 96-168 hours, 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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