A strain of Streptomyces D-6, its microecological preparation and preparation method thereof

Abstract

A strain of Streptomyces D-6, its microecological preparation and its preparation method, the Streptomyces D-6 was deposited in the China Center for Type Culture Collection on June 13, 2024, with a deposit number of CCTCC NO: M 20241201; the classification name is: Streptomyces D-6, Latin name Streptomyces sp.D‑6. Its microecological preparation is fermented by the Streptomyces D‑6. The present invention also includes a method for preparing the Streptomyces D‑6 microecological preparation. The present invention also includes the application of the Streptomyces D‑6 and its microecological preparation in aquaculture. The Streptomyces D‑6 and its microecological preparation of the present invention have good antibacterial effects on common fish pathogens such as Shigella-like Pseudomonas, Aeromonas vernix, Aeromonas abnormal saccharophilus, and Shewanella xiamenensis; feeding fish, especially crucian carp, with Streptomyces D‑6 as a feed additive can improve the growth performance and immunity of freshwater fish, especially crucian carp, and can also improve its intestinal flora.

Description

Technical Field

[0001] The invention relates to a strain of Streptomyces D-6, and in particular to a strain of Streptomyces D-6, a microecological preparation thereof and a preparation method thereof. Background Art

[0002] Aquaculture is developing rapidly around the world and has become one of the most sustainable projects for providing food for mankind. However, aquaculture is now facing unbearable fish health problems. High-density fish breeding and deteriorating water quality have created an ideal environment for pathogen reproduction and infection, causing a significant increase in fish morbidity. The high mortality and morbidity rates of fish have caused huge economic losses to the aquaculture industry, seriously hindering the efficient, economical and sustainable development of aquaculture.

[0003] With the deepening of research on fish disease prevention and control, more and more innovative means have emerged, and the popular means mainly involve antibiotics, vaccines and Chinese herbal medicines. However, although antibiotics have shown great advantages in defending against pathogens, the dysbiosis of the intestinal microbiome caused by antibiotics during treatment creates conditions for existing pathogenic microorganisms in the intestine, which is extremely unfavorable for the sustainable development of aquaculture. There are still some limitations in the development and use of fish vaccines in aquaculture. For example, during the use of attenuated live vaccines, it is necessary to ensure that they do not return to their original virulence. At present, the mechanism of action of most Chinese herbal medicines is still unclear, and their growth stage and growth conditions have a great influence on the active ingredients, making it difficult to fully develop the application of Chinese herbal medicines in aquaculture.

[0004] Probiotics are one of the most promising disease control products in the aquaculture industry. Compared with other preparations such as antibiotics, they can have beneficial effects on the host and improve environmental parameters. Their use in aquaculture can enhance fish growth and immune function to fight fish diseases and increase fish survival rates. They are non-toxic, harmless, green and environmentally friendly.

[0005] However, there are few reports on the application of Streptomyces as probiotics in aquaculture. Summary of the invention

[0006] 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 D-6, its microecological preparation and a preparation method thereof; the Streptomyces and its microecological preparation have good antagonistic effects on a variety of common fish pathogens and can be used to resist infection by a variety of pathogenic bacteria.

[0007] The technical solution adopted by the present invention to solve its technical problems is as follows: a strain of Streptomyces D-6, deposited in the China Center for Type Culture Collection (abbreviated as "CCTCC") on June 13, 2024, with a preservation number of CCTCC NO: M 20241201; classified and named: Streptomyces D-6, Latin name Streptomyces sp.D-6.

[0008] The Streptomyces D-6 of the present invention was screened from the soil of Jiemu Creek in Yuanling County, Hunan Province. The 16S rRNA gene sequence of the Streptomyces D-6 of the present invention is shown in Table SEQ ID No.1.

[0009] The Streptomyces D-6 of the present invention is used as an antagonistic bacterium to fish pathogenic bacteria.

[0010] The Streptomyces D-6 of the present invention can be prepared into a microecological preparation for use.

[0011] The microecological preparation of Streptomyces D-6 of the invention is prepared by fermenting Streptomyces D-6 with a strain collection number of CCTCC NO: M 20241201.

[0012] Preferably, the microecological preparation is a liquid bacterial preparation or a solid bacterial preparation.

[0013] Preferably, the microecological preparation is used in aquaculture.

[0014] Preferably, the aquaculture animal is a freshwater fish; more preferably, it is crucian carp, grass carp or carp.

[0015] The preparation method of the microecological preparation of Streptomyces D-6 of the present invention comprises the following steps:

[0016] (1) Inoculation and activation: Inoculate Streptomyces D-6 on a slant Gao's medium No. 1, pick a single clone and culture it in AM6 medium for fermentation, and collect the fermentation seed liquid;

[0017] (2) First expansion culture: inoculating the fermentation seed liquid obtained in step (1) into a fermentation tank containing a fermentation medium for first expansion culture to obtain seed liquid I for expansion culture;

[0018] (3) Second expansion culture: inoculating the expanded culture seed solution I obtained in step (2) into a fermentation tank filled with fermentation medium, and performing a second expansion culture to obtain expanded culture solution II;

[0019] (4) Concentration and collection: The expanded culture fluid II obtained in step (3) is collected and concentrated to obtain a liquid bacterial agent; the solid bacterial agent is obtained by spray drying the liquid bacterial agent or the expanded culture fluid II.

[0020] Preferably, in step (2), the inoculation amount of the fermentation seed liquid inoculated into the fermenter is 1.0-1.5% of the volume of the fermentation medium.

[0021] Preferably, in step (2), the solvent of the fermentation medium for the expanded culture is water, and the formula is: 4-6 g / L bacteriological peptone, 4-6 g / L yeast extract, 4-6 g / L calcium carbonate, 8-12 g / L glucose, and 15-25 g / L soluble starch.

[0022] Preferably, in step (2), the dissolved oxygen content of the first expansion culture is 40-50%.

[0023] Preferably, in step (2), the temperature of the first expansion culture is 28-30°C.

[0024] Preferably, in step (2), the first expansion culture time is 48 to 72 hours.

[0025] Preferably, in step (2), a fermentation defoamer is used during the culture process to avoid foaming.

[0026] Preferably, in step (3), the inoculation amount of the expanded culture seed solution I is 10-15% of the volume of the fermentation medium.

[0027] Preferably, in step (3), the solvent of the fermentation medium for the expanded culture is water, and the formula is: 4-6 g / L bacteriological peptone, 4-6 g / L yeast extract, 4-6 g / L calcium carbonate, 8-12 g / L glucose, and 15-25 g / L soluble starch.

[0028] Preferably, in step (3), the dissolved oxygen content of the second expanded culture is 40-50%.

[0029] Preferably, in step (3), the temperature of the second expansion culture is 28-30°C.

[0030] Preferably, in step (3), the time of the second expansion culture is 108 to 144 hours.

[0031] Preferably, in step (3), a fermentation defoamer is used during the culture process to avoid foaming.

[0032] Beneficial effects of the present invention:

[0033] (1) The Streptomyces D-6 of the present invention has a good antibacterial effect on various fish pathogens such as Shigella-like Pseudomonas, Aeromonas veronii, Aeromonas anomala, and Shewanella xiamenensis.

[0034] (2) The fermentation broth of Streptomyces D-6 is stable in nature, and intraperitoneal injection of this strain can inhibit the growth of Aeromonas veronii in fish.

[0035] (3) Streptomyces D-6 can be used as a feed additive to feed fish. Feeding can improve the growth performance of fish and enhance the resistance of fish to pathogens.

[0036] (4) The method of preparing Streptomyces D-6 into a microecological preparation is simple and has a low production cost. It can also effectively reduce the hazards such as drug residues caused by the abuse of antibiotics and has good application prospects.

[0037] The 16S rRNA gene sequence of Streptomyces D-6 of the present invention is shown in Table SEQ ID No.1.

[0038] SEQ ID No.1:

[0039]

[0040]

[0041] Description of Microbiological Deposit

[0042] The Streptomyces D-6 of the present invention was deposited in the China Center for Type Culture Collection (CCTCC for short, address: Wuhan University, Wuhan, China) on June 13, 2024, and the strain collection number is CCTCC NO: M 20241201; the classification name is: Streptomyces D-6; Latin name: Streptomyces sp.D-6. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a diagram showing the antibacterial effect of Streptomyces D-6 of the present invention on fish pathogens.

[0044] Figure 2 These are observation pictures of the morphological characteristics of Streptomyces D-6 of the present invention, a: observation picture on a Goll plate; b: observation picture under an upright microscope (1000×); c: Gram staining picture; d: observation picture under a scanning electron microscope (10000×); e: observation picture under a scanning electron microscope (20000×).

[0045] Figure 3 The phylogenetic tree diagram is constructed using the 16S rRNA sequence of Streptomyces D-6 of the present invention.

[0046] Figure 4 It is an analysis diagram of the antibacterial active substance of Streptomyces D-6 of the present invention and the influence of temperature.

[0047] Figure 5 It is an analysis diagram of the antibacterial active substance of Streptomyces D-6 of the present invention and the influence of ultraviolet irradiation.

[0048] Figure 6 It is an analysis diagram of the effects of the antibacterial active substances of Streptomyces D-6 and proteases of the present invention.

[0049] Figure 7 This is a colonization analysis diagram of crucian carp observed by a small animal living imaging system using Streptomyces D-6 of the present invention.

[0050] Figure 8 This is a distribution result diagram of the effect of Streptomyces D-6 of the present invention on the activities of immune-related enzymes in the serum of crucian carp, a: alkaline phosphatase activity and acid phosphatase activity; b: catalase activity and superoxide dismutase activity; c: reduced glutathione content at different feeding concentrations.

[0051] Fig. 9 This is a diagram showing the results of analyzing the effect of Streptomyces D-6 of the present invention on the expression of immune-related genes in crucian carp, a: liver; b: kidney; c: spleen; d: intestine.

[0052] Fig.10 This is an analysis diagram of the protective effect of Streptomyces D-6 of the present invention on crucian carp.

[0053] Fig.11 This is a diagram of the antagonism analysis of the microecological preparation prepared by Streptomyces D-6 against Aeromonas veronii visualized by the small animal imaging system of the present invention. DETAILED DESCRIPTION

[0054] The present invention will be further described below in conjunction with the embodiments and drawings.

[0055] Unless otherwise specified, the reagents used in the examples of the present invention were obtained through conventional commercial channels.

[0056] The Streptomyces D-6 of the present invention was deposited in the China Center for Type Culture Collection on June 13, 2024, with the strain accession number of CCTCC NO: M 20241201; the classification name is: Streptomyces D-6; Latin name: Streptomyces sp.D-6.

[0057] Example

[0058] The screening and identification of Streptomyces D-6 of the present invention are as follows.

[0059] (I) Screening of Streptomyces D-6 and determination of antibacterial spectrum

[0060] Soil samples from Jiemuxi, Yuanling County, Hunan Province were dissolved in an appropriate amount of sterile water, and after gradient dilution, they were spread on solid Goller plates and incubated in a constant temperature incubator at 30°C for 5 days. Single colonies with typical actinomycete morphology were picked and inoculated on new plates for streak culture, and repeated isolation and purification were performed. The fish pathogens Aeromonas veronii, Citrobacter freundii, Edwardsiella tarda, Aeromonas allosaccharophila, Erwinia spp., Plesiomonas shigelloide, Shewanella xiamenensis, and Aeromonas hydrophila were used as indicator bacteria, and the fermentation supernatant was tested for its antibacterial activity against the above fish pathogens by the punch method (see Figure 1 ), and found that the Streptomyces D-6 screened by the inventors had a good antibacterial effect on the above 8 fish pathogens, and the diameters of the inhibition zones were all over 16 mm. Among them, the antibacterial effect on Shigella-like Pleistomonas was the strongest, and the average inhibition zone diameter could reach 25.5 mm.

[0061] (II) Cell morphological characteristics of Streptomyces D-6

[0062] Streptomyces D-6 colonies on Gao's No. 1 culture plate are snow-white, irregular in shape, uniform in size, dense and wrinkled in texture, dry and opaque, and the back of the colonies is light pink. Under an upright microscope and a scanning electron microscope, the hyphae are dense, multi-branched and thin in diameter, and the spores are wrinkled inward (see Figure 2 ). The above results all indicate that Streptomyces D-6 conforms to the morphological characteristics of typical Streptomyces.

[0063] (III) Identification of the 16S rRNA gene of Streptomyces D-6

[0064] Streptomyces D-6 was inoculated into AM6 liquid culture medium, and the cells were collected after culturing for 48 h. The genomic DNA of strain L-82 was extracted using the Ezup column bacterial genomic DNA extraction kit (Shanghai Shenggong Biotechnology Co., Ltd.), and the 16S rRNA gene was amplified using primers.

[0065] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'

[0066] 1492R:5'-CGGTTACCTTGTTACGACTT-3'

[0067] The 16SrRNA gene sequence was amplified using the above primers, and the expected length of the sequence was about 1500 bp.

[0068] PCR reaction system (20 μL): sterile double distilled water, 14 μL; 10× Buffer, 2 μL; dNTP, 1.6 μL; Bf-R (10 μM / L), 0.6 μL; Bf-F (10 μM / L), 0.6 μL; genomic template, 1 μL; Primer STAR DNA polymerase (Takara), 0.2 μL;

[0069] PCR reaction program: pre-denaturation at 95°C for 5 min; 30 cycles: 95°C, 45 sec; 55°C, 45 sce; 72°C, 1.5 min; 72°C, 10 min.

[0070] 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.

[0071] The 16S rRNA gene sequence of Streptomyces D-6 was sequenced and showed a length of 14291 bp. The sequencing results were uploaded to the EzBiocloud database for homology comparison with known 16S rRNA sequences, and BLAST online comparison was performed at the same time. The strains with higher homology were summarized and downloaded, and the MEGA software was used to analyze the Neighbor-Joining method to construct a phylogenetic tree (bootstrap value was 1000) (see Figure 3 ). The 16S rRNA gene sequence of Streptomyces D-6 of the present invention is shown in Table SEQ ID No.1.

[0072] (IV) Strain genome ANI and dDDH comparison

[0073] Bacterial ANI and dDDH comparison values ​​are key data for bacterial identification. Therefore, on the basis of obtaining the 16S rRNA phylogenetic tree and strain genome information, the bacterial species with the closest relationship to the test strain was determined through the 16S rRNA phylogenetic tree, and the whole genome data of the closest related bacterial species were downloaded from the GenBank or EzBioCloud database. The data were submitted to the ANI computing platform to calculate the average nucleotide identity (ANI) and the GGDC website (https: / / ggdc.dsmz.de / ) for digital DNA-DNA hybridization (dDDH) analysis. The results showed that the dDDH value and ANI value of strain D-6 with S. xanthophaeus strain NRRL B-5414 were 28.90% and 85.90%, respectively, and the dDDH value and ANI value with S. lavendulae subsp. Lavendulae strain NRRL B-2774 were 29.20% and 86.11%, respectively, both lower than d The threshold values ​​of DDH and ANI for distinguishing new strains were 70% and 95% (Table 1), which further indicated that strain D-6 was a new species of Streptomyces, and was finally named Streptomyces D-6; Latin name: Streptomyces sp.D-6.

[0074] Table 1 Comparison of molecular biological characteristics of strain D-6 and the most similar strains

[0075]

[0076] (V) Analysis of the physicochemical properties of antibacterial active substances of Streptomyces D-6

[0077] (1) Thermal stability

[0078] The strain D-6 was inoculated into AM6 liquid culture medium and cultured in a constant temperature shaker at 30°C for 72 to 96 hours, 12,000 rpm, and 10 minutes. The obtained supernatant was treated at different temperature conditions (20°C, 40°C, 60°C, 80°C, and 100°C) for 1 hour, and an antibacterial experiment was carried out using Aeromonas vermifuge as the indicator bacteria. The untreated fermentation broth supernatant was used as the control group, and the antibacterial experiment was carried out using the punching method. The size of the inhibition circle was measured after 12 hours, and repeated experiments were set. The results showed that the antibacterial activity did not change below 60°C, the antibacterial activity decreased at 80°C, and the antibacterial activity was completely lost at 100°C, indicating that the antibacterial effect of strain D-6 was relatively stable within 20 to 80°C. It is inferred that the strain can tolerate medium and high temperatures for a short period of time (test results refer to Figure 4 ).

[0079] (2) UV resistance

[0080] The strain D-6 was inoculated into AM6 liquid culture medium and cultured in a constant temperature shaker at 30°C for 72 to 96 hours, 12,000 rpm for 10 minutes. The obtained fermentation supernatant was exposed to ultraviolet light for 15 minutes, 30 minutes, 45 minutes and 60 minutes respectively. Aeromonas vernix was used as the indicator bacteria and the untreated fermentation supernatant was used as the control group. The perforation method was used for inhibition. The size of the inhibition zone was measured after 12 hours and repeated experiments were set. The results showed that compared with the untreated fermentation broth, the antibacterial activity of the experimental group after ultraviolet treatment did not decrease, indicating that the active substances produced during the fermentation process of strain D-6 have good tolerance to ultraviolet light irradiation (test results refer to Figure 5 ).

[0081] (3) Protease resistance

[0082] The strain D-6 was inoculated into AM6 liquid culture medium and cultured in a constant temperature shaker at 30°C for 72 to 96 hours, 12,000 rpm for 10 minutes. The fermentation supernatant was incubated with proteinase K at 55°C for 30 minutes; the fermentation supernatant was incubated with pepsin and trypsin at 37°C for 30 minutes respectively; and then treated at 85°C for 5 minutes to terminate the enzyme reaction. Aeromonas vernix was used as the indicator bacteria and the untreated fermentation supernatant as the control group. The punching method was used for inhibition. The size of the inhibition zone was measured after 12 hours, and replicates were set. The results showed that the antibacterial effect of the treated fermentation supernatant was not affected compared with the antibacterial effect of the untreated fermentation supernatant (test results refer to Figure 6 ).

[0083] (VI) Preparation of Streptomyces D-6 microecological preparation

[0084] The microecological preparation of Streptomyces D-6 of the present invention is prepared by fermenting Streptomyces D-6 with a strain collection number of CCTCC NO: M 20241201, and specifically comprises the following steps:

[0085] (1) Inoculation and activation: Inoculate the new strain of Streptomyces D-6 on a slant Gao's medium No. 1, pick a single clone and culture it in AM6 medium for fermentation, and collect the fermentation seed liquid;

[0086] (2) First expansion culture: The fermentation seed liquid obtained in step (1) is inoculated into a fermentation tank with a fermentation medium for the first expansion culture. The inoculation amount of the fermentation seed liquid inoculated into the fermentation tank is 1.0% of the volume of the fermentation medium. The solvent of the fermentation medium for expansion culture is water, and the medium formula is: bacteriological peptone 5g / L, yeast extract 5g / L, calcium carbonate 5g / L, glucose 10g / L, soluble starch 20g / L. The dissolved oxygen content of the expansion culture is 45%. The temperature of the first expansion culture is 28-30°C, and the culture time is 48-72h. The defoaming agent is added online in real time; the seed liquid I for expansion culture is obtained;

[0087] (3) Second expansion culture: The seed liquid of the first expansion culture obtained in step (2) is inoculated into a fermentation tank filled with fermentation medium, and the second expansion culture is carried out to obtain expansion culture liquid II; the seed liquid inoculation amount of the second expansion culture is 10% of the volume of the culture medium, the solvent of the fermentation medium of the expansion culture is water, and the formula of the fermentation medium is: bacteriological peptone 4-6g / L, yeast extract 4-6g / L, calcium carbonate 4-6g / L, glucose 8-12g / L, soluble starch 15-25g / L. The dissolved oxygen content of the expansion culture is 40-50%. The temperature of the second expansion culture is 28-30°C, and the culture time is 108-144h. The defoaming agent is added online in real time.

[0088] (4) Concentration and collection: After the tank is placed, the expanded culture medium II obtained in step (3) is collected and concentrated to obtain a microecological liquid bacterial agent, or spray-dried to obtain a microecological solid bacterial agent.

[0089] (VII) Application of Streptomyces D-6 in fish farming

[0090] Example 1: Colonization analysis of Streptomyces D-6 in crucian carp

[0091] Crucian carp with the same physiological state were randomly divided into 2 groups, 10 in each group, and acclimated for 7 days to adapt to the environment. EGFP (1×10 8 CFU / mL) of feed, and the control group was fed with ordinary feed twice a day, and the amount of feed each time was 2% of the crucian carp body weight. After the experiment started, the small animal living imaging system was used to observe on the 0th day, the 1st day, the 3rd day and the 5th day.

[0092] The results are as follows Figure 7As shown, fluorescent signals began to appear in the gills of crucian carp on the first day, indicating that strain D-6 successfully entered the body of crucian carp, and subsequent observations of crucian carp on the 3rd and 5th days showed that the green fluorescent signal gradually moved to the abdomen, and it was speculated that with longer feeding observations, the green fluorescent signal in the crucian carp would be further strengthened, indicating that the survival ability of strain D-6 would not be affected by the internal environment of crucian carp during the positioning process, and it could stably colonize in the body of crucian carp, which further strengthened the application value of strain D-6 as a probiotic for fish.

[0093] Example 2: Effects of Streptomyces D-6 on Growth and Disease Resistance of Crucian Carp

[0094] Crucian carps of similar size were randomly divided into two experimental groups and one control group, with 30 fish in each group, and were housed in fish boxes with a water volume of 60 L. The experimental crucian carps were acclimated for 7 days to adapt to the experimental environment. Water was aerated for 48 hours and the water temperature was controlled at 25°C. The feeding method was 1×10 8 CFU / mL and 1×10 9 CFU / mL of Streptomyces D-6 were mixed with feed and fed to crucian carp, while the control group was fed with ordinary feed twice a day, with the amount of feed each time being 2% of the crucian carp's body weight for 30 days.

[0095] (1) Effects of Streptomyces D-6 on the growth performance of crucian carp

[0096] Record the initial body weight (W) before experimental feeding i ), and the final weight of crucian carp was recorded after the experiment (W f ). WGR (body weight growth rate), SGR (body weight growth rate) and SR (survival rate) were calculated using the following formulas.

[0097] WGR=((Wf-Wi) / Wi)×100%

[0098] SGR=((lnWf-lnWi) / T)×100%

[0099] SR = (number of fish at the end of the experiment / number of fish at the beginning of the experiment) × 100%

[0100] The above Wi, Wf, and T represent the average body weight before feeding, the average body weight after feeding, and the feeding time, respectively (see Table 2 for the test results).

[0101] Table 2 Growth performance parameters of crucian carp

[0102]

[0103] In the table, group I: normal feeding; group II: 1×10 8 CFU / g D-6 bacteria, mixed with feed at a ratio of 2%; Group III: 1×109 CFU / g D-6 bacteria, mixed with feed at a ratio of 2%. The feed amount for each group was 2% of the crucian carp's body weight, and the crucian carp was fed for 30 days.

[0104] The results in Table 2 show that the weight growth rate of crucian carp fed with normal feed was 18.22%, and the specific growth rate was 0.56%. The weight growth rates of the two experimental groups fed with D-6 bacteria were 19.86% and 22.14%, and the specific growth rates were 0.60% and 0.67%, respectively. There were no abnormalities in the living conditions of the three groups of crucian carp during the experiment, indicating that the strain D-6 can effectively enhance the growth performance of crucian carp to a certain extent.

[0105] (2) Effects of Streptomyces D-6 on nonspecific immune parameters in crucian carp serum

[0106] Streptomyces D-6 was used as a feed additive to feed crucian carp for 30 days, and then the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), catalase (CAT), total superoxide dismutase (T-SOD) and glutathione (GSH) in the serum of crucian carp were determined, and the changes of nonspecific immune indexes in serum of crucian carp after different treatments were analyzed (the test results refer to Figure 8 ). The activities of AKP, ACP and CAT in the experimental group fed with Streptomyces D-6 were increased, and the enzyme activity of the high-concentration feeding group was lower than that of the low-concentration feeding group; the glutathione reductase content of the feeding group was also increased; the T-SOD activity of the D-6 feeding group was higher than that of the control group but not significantly higher. The above results show that feeding feed containing Streptomyces D-6 can increase the activity of nonspecific immune indicators of crucian carp and improve the immune level of fish.

[0107] (3) Effects of Streptomyces D-6 on the expression of immune-related genes in crucian carp tissues

[0108] Streptomyces D-6 was used as a feed additive to feed crucian carp for 30 days, and the expression levels of immune-related genes in the liver, kidney, spleen and intestine of crucian carp were analyzed (the test results refer to Fig. 9), in the liver, the expression levels of IgM, C3, and LSZ genes were all upregulated, among which the LSZ gene was upregulated more significantly, while the pro-inflammatory factor IL-8 that induces inflammation in the low-concentration D-6 feeding group had no significant change; in the kidney, the expression levels of IgM and C3 genes were significantly upregulated, while the LSZ and IL-8 genes had no significant changes; in the spleen, the expression levels of IgM and C3 genes in the low-concentration D-6 feeding group were significantly upregulated compared with the control group, while the expression level of IgM gene in the high-concentration D-6 feeding group was increased, the expression level of C3 gene was significantly decreased, the expression level of LSZ gene had no significant change, and the expression level of IL-8 gene in the feeding group was decreased compared with the control group; in the intestine, the expression levels of IgM, C3, and LSZ genes were significantly increased compared with the control group, while the expression level of IL-8 gene was significantly decreased. After feeding with strain D-6, related immune factors showed different degrees of up-regulation or down-regulation, indicating that strain D-6 can affect the state of the immune organs of crucian carp and may also cause local inflammatory reactions, but it can also recruit immune cells to enrich here and protect the body.

[0109] (4) Experiment on the protective effect of Streptomyces D-6 on crucian carp

[0110] Streptomyces D-6 was used as a feed additive to feed crucian carp, and Aeromonas veronii was used to challenge the bacteria and the mortality of crucian carp in each group was continuously observed.

[0111] Group I: normal feeding; Group II: 1×10 8 CFU / g D-6 bacteria, mixed with feed at a ratio of 2%; Group III: 1×10 9 CFU / g D-6 bacteria, mixed with feed at a ratio of 2%. The amount of feed for each group was 2% of the crucian carp's body weight. Fig.10 After 30 days of feeding, 1×10 8 CFU / 100ul Aeromonas veronii was used to challenge and the mortality of crucian carp in each group was continuously observed.

[0112] Control: Before the experiment, some crucian carp in each group were injected with PBS to ensure that the injection method and other environmental factors would not affect the experiment. The results showed that the survival rate of the PBS-injected group remained at 100%, indicating that the injection method and other factors had no effect on the experiment. Therefore, the PBS injection here is to exclude the interference of other factors such as the injection method on the experiment.

[0113] The results showed that the survival rate of the PBS injection group remained at 100%, indicating that the injection process and other factors such as the environment would not affect the survival of crucian carp; the poison-challenged group began to die from the first day, and the number of subsequent deaths gradually increased until it stabilized on the seventh day, with a final survival rate of 20%. The survival rate of the low-concentration experimental group fed with strain D-6 was 45% on the seventh day after the poison-challenged group, and the survival rate of the high-concentration experimental group reached 50% (test results refer to Fig.10 ), indicating that strain D-6 can improve the survival rate of crucian carp after infection and effectively protect crucian carp.

[0114] Example 3: Antagonistic effect of the probiotic preparation prepared by Streptomyces D-6 on Aeromonas veronis in crucian carp

[0115] In order to explore the antagonistic effect of the microecological preparation prepared by Streptomyces D-6 on Aeromonas veroni in crucian carp, crucian carp with the same physiological state were randomly divided into two groups, 10 in each group, and acclimated for 7 days to adapt to the environment. EGFP (1×10 8 CFU / g) of fermented bacteria, mixed with feed at a ratio of 2%; the control group was fed with ordinary feed twice a day, and the amount of each feed was 2% of the crucian carp's body weight. After the experiment began, the small animal live imaging system was used to observe the crucian carp on the 0th, 1st, 3rd and 5th day. The crucian carp was then challenged with the red fluorescent strain. (1×10 8 CFU / mL) were injected intraperitoneally into crucian carp, with each tail injected with 100 μL, and the cells were observed using a small animal living imaging system on the 0th, 1st, 3rd, and 5th day after injection.

[0116] The results are as follows Fig.11 As shown, the probiotic D-6 was fed EGFP Red fluorescence signals appeared in the bodies of crucian carp in the experimental group and the control group fed with ordinary feed, indicating that the infection was successful. The red fluorescence in the control group gradually strengthened, while that in the experimental group showed a weakening trend, indicating that the microecological preparation D-6 can effectively resist the proliferation of Aeromonas veroni in crucian carp.

Claims

1. A strain of Streptomyces D-6, characterized in that: It was deposited in China Center for Type Culture Collection on June 13, 2024, with the deposit number CCTCC NO: M 20241201; the classification name is: Streptomyces D-6, Latin name Streptomyces sp.D-6.

2. The microecological preparation of Streptomyces D-6 according to claim 1, characterized in that The product is prepared by fermentation of Streptomyces D-6 with a strain collection number of CCTCC NO: M 20241201.

3. The microecological preparation of Streptomyces D-6 according to claim 2, characterized in that The microecological preparation is a liquid bacterial preparation or a solid bacterial preparation.

4. The method for preparing the probiotic preparation of Streptomyces D-6 according to claim 2 or 3, characterized in that: The following steps are involved: (1) Inoculation and activation: Inoculate Streptomyces D-6 on a slant Gao's medium No. 1, pick a single clone and place it in AM6 medium for fermentation, and collect the fermentation seed liquid; (2) First expansion culture: inoculating the fermentation seed liquid obtained in step (1) into a fermentation tank containing a fermentation medium for first expansion culture to obtain seed liquid I for expansion culture; (3) Second expansion culture: inoculating the expanded culture seed solution I obtained in step (2) into a fermentation tank filled with fermentation medium, and performing a second expansion culture to obtain expanded culture solution II; (4) Concentration and collection: The expanded culture fluid II obtained in step (3) is collected and concentrated to obtain a liquid bacterial agent; the solid bacterial agent is obtained by spray-drying the liquid bacterial agent or the expanded culture fluid II.

5. The method for preparing the probiotic preparation of Streptomyces D-6 according to claim 4, characterized in that: In step (2), the inoculation amount of the fermentation seed liquid inoculated into the fermentation tank is 1.0-1.5% of the volume of the fermentation medium; and / or, in step (2), the solvent of the fermentation medium for the expanded culture is water, and the formula is: bacteriological peptone 4-6 g / L, yeast extract 4-6 g / L, calcium carbonate 4-6 g / L, glucose 8-12 g / L, soluble starch 15-25 g / L.

6. The method for preparing the probiotic preparation of Streptomyces D-6 according to claim 4 or 5, characterized in that: In step (2), the dissolved oxygen content of the first expanded culture is 40-50%; and / or, in step (2), the temperature of the first expanded culture is 28-30° C.; and / or, in step (2), the time of the first expanded culture is 48-72 h; and / or, in step (2), a fermentation defoamer is used during the culture process to avoid foaming.

7. The method for preparing the probiotic preparation of Streptomyces D-6 according to claim 4 or 5, characterized in that: In step (3), the inoculation amount of the expanded culture seed solution I is 10-15% of the volume of the fermentation medium; and / or, in step (3), the solvent of the expanded culture fermentation medium is water, and the formula is: bacteriological peptone 4-6 g / L, yeast extract 4-6 g / L, calcium carbonate 4-6 g / L, glucose 8-12 g / L, soluble starch 15-25 g / L.

8. The method for preparing the probiotic preparation of Streptomyces D-6 according to claim 4 or 5, characterized in that: In step (3), the dissolved oxygen content of the second expanded culture is 40-50%; and / or, in step (3), the temperature of the second expanded culture is 28-30° C.; and / or, in step (3), the time of the second expanded culture is 108-144 h; and / or, in step (3), a fermentation defoamer is used during the culture process to avoid foaming.

9. Use of Streptomyces D-6 as claimed in claim 1 in crucian carp breeding.

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