CN114426935B

CN114426935B - Streptomyces termitis N-15 and microecological preparation as well as preparation method and application thereof

Info

Publication number: CN114426935B
Application number: CN202111324319.6A
Authority: CN
Inventors: 夏立秋; 彭雅楠; 易敢峰; 丁学知
Original assignee: Hunan Normal University
Current assignee: Qiansheng Shenzhen Kechuang Group Co ltd
Priority date: 2021-11-10
Filing date: 2021-11-10
Publication date: 2023-07-04
Anticipated expiration: 2041-11-10
Also published as: CN114426935A

Abstract

Streptomyces termitis N-15 and a microecological preparation, and a preparation method and application thereof, wherein the Streptomyces termitis N-15 (Streptomyces termitum N-15) is preserved in China Center for Type Culture Collection (CCTCC) with a strain preservation number of M2019218. The Streptomyces termitis N-15 is obtained by screening from bottom mud of a fish pond in Zhang Jiang region of Hunan province. The microecological preparation prepared by fermenting the streptomyces termitis N-15 is suitable for aquaculture, can inhibit the growth of various fish pathogenic bacteria such as aeromonas hydrophila, aeromonas verrucosa, edwardsiella tarda and the like, and can be used as a feed additive for feeding freshwater fishes such as grass carp, crucian carp and the like, so that the growth rate and the bait efficiency of the grass carp can be improved, the immunity of the freshwater fishes can be enhanced, and the resistance of the freshwater fishes such as the grass carp and the like to the fish pathogenic bacteria can be obviously improved.

Description

Streptomyces termitis N-15 and microecological preparation as well as preparation method and application thereof

Technical Field

The invention relates to a microbial streptomyces termite, in particular to a streptomyces termite N-15 strain, a microecological preparation prepared by fermenting the same, and a preparation method and application thereof.

Background

In recent decades, aquaculture yields continue to increase rapidly, and at the same time, due to the increase in the stocking density of intensive fish ponds and the lack of disease control measures, major diseases have been outbreaks, in which bacterial diseases are the major diseases, greatly impeding the healthy development of the aquaculture industry. So far, for bacterial diseases, quick and effective synthetic antibiotics and various chemicals are often selected for treatment. However, due to the unreasonable use of such drugs, not only the breeding environment is destroyed, but many pathogenic bacteria generate drug resistance, so that the immunity of the aquaculture animals is reduced or the aquaculture animals die, and even the drugs remained in the aquatic products can be transferred to the human body, thereby generating infectious diseases and constituting a risk for human health. In order to overcome the drug-resistant pathogenic bacteria of the continuous abuse of antibiotics in aquaculture, the development of new drugs for resisting fish bacterial diseases is urgent.

The probiotics can resist pathogens, promote growth and stimulate immune response of a host to infection, and are effective substitutes for preventing and controlling various diseases in aquaculture. Actinomycetes are an important resource library of various active molecules with novel structures, have the advantages of degrading macromolecules such as starch and protein, secreting antibacterial active substances and producing heat-resistant spores, and are expected to be applied to disease control in aquaculture industry. Thus, actinomycetes have great potential to become aquaculture probiotics.

Streptomyces lyzolyticus (Streptomyces amritsarensis N1-32) obtained from soil in the Yankee lake wetland in Hunan province as a feed additive can increase the expression level of immune-related genes in immune tissues of grass carp, thereby protecting the grass carp from infection by pathogenic bacteria (see "Li Y, hu S, gong L, pan L, li D, cao L, khan T A, yang Y, peng Y, ding X, yi G, liu S, xia L.Isolating a new Streptomyces amritsarensis N1-32 against Fish pathogens and determining its effects on disease resistance of grass carp[J ]. Fish Shellfish Immunol,2020,98 (632-640"). Sunish et al evaluates actinomycetes as immunostimulants for feeding litopenaeus vannamei, and shows that 6 immune factors (alpha-2 macroglobulin, para-astaxanthin 3, transglutaminase, alanine, capsid protein and peroxidase) are significantly up-regulated after challenge (see "Sunish, K.S., M.Biji, P.Rosamma, N.S.Sudheer, K.Sreedharan, A.Mohandas, I.B.Singh, et al, marine actinomycetes Nocardiopsis alba MCCB 110has immunomodulatory property in the tiger shrimp Penaeus monodon[J ]. Fish & shellfish immunology,2020, 125-132").

However, there has been no report on the application of Streptomyces termitis to disease control in aquaculture so far.

Disclosure of Invention

The invention aims to solve the technical problems of overcoming the defects in the prior art and providing the Streptomyces termitis N-15 which has good antagonism to various fish pathogenic bacteria.

The invention further aims to solve the technical problem of providing a microecological preparation which is suitable for aquaculture and can prevent and treat fish diseases.

The invention further aims to solve the technical problem of providing a preparation method of the microecological preparation, which is easy and convenient to operate.

The technical scheme adopted by the invention for solving the technical problems is that a strain of Streptomyces termitis N-15 (Streptomyces termitum N-15) is preserved in China Center for Type Culture Collection (CCTCC) with the strain preservation number of M2021726; the classification is named: streptomyces termitis N-15.

The 16S rRNA sequence of the Streptomyces termitid N-15 is shown in a sequence table SEQ ID No. 1.

The Streptomyces termitis N-15 is obtained by screening from bottom mud of a fishpond in the Zhangjia area of Hunan province.

The technical scheme adopted by the invention for further solving the technical problems is that the microecological preparation is prepared by fermenting Streptomyces termitis N-15 with the strain preservation number of CCTCC NO: M2021726.

Further, the antibacterial active substances in the strain fermentation broth are as follows: cha Ermei element (calcomycin A) with a relative molecular weight of 701.4.

Further, the microecological preparation is a liquid preparation or a solid preparation.

Research shows that the microecological preparation of the invention can inhibit the growth of various fish pathogenic bacteria such as aeromonas hydrophila, aeromonas veronii, edwardsiella tarda and the like, and the physicochemical properties of antibacterial active substances in the strain fermentation liquor are insensitive to high temperature, strong acid, strong alkali and protease.

The technical scheme adopted by the invention for further solving the technical problems is that the preparation method of the microecological preparation comprises the following steps:

(1) Inoculating Streptomyces termitis N-15 to a culture medium, and selecting mature spores to culture in an Am6 shake flask seed culture medium to obtain seed liquid;

(2) Inoculating the seed liquid obtained in the step (1) into a fermentation tank filled with a fermentation medium for expansion culture to obtain an expansion-cultured seed liquid;

(3) Inoculating the seed liquid obtained in the step (2) for the expanded culture into a fermentation tank filled with a fermentation medium for culture, and supplementing an antifoaming agent on line in real time during the culture to obtain a fermentation product;

(4) Concentrating the fermentation product obtained in the step (3) to obtain a microecological liquid preparation; or filtering the microecological liquid preparation with ceramic membrane, collecting filtrate, and spray drying the filtrate to obtain microecological powder preparation.

Further, in the step (2), the seed liquid is inoculated into the fermentation tank in an inoculation amount of 1-1.5% of the volume of the fermentation medium; the formulation of the fermentation medium for the expansion culture is glucose 0.5-2.0 g, caCO ₃ 0.2 to 0.5g, 1 to 3g of soluble starch, 0.1 to 0.5g of yeast extract and 0.2 to 0.6g of bacteriological peptone.

In the step (3), the inoculation amount of the seed liquid for the expansion culture is 8-10% of the volume of the fermentation medium; the formula of the fermentation medium is glucose 0.5-2.0 g, caCO ₃ 0.2 to 0.5g, 1 to 3g of soluble starch, 0.1 to 0.5g of yeast extract and 0.2 to 0.6g of bacteriological peptone.

Further, in the step (3), the dissolved oxygen amount of the culture is 35-55%; the temperature of the culture is 30-40 ℃; the culture time is 48-60 h.

In the step (3), the fermentation medium is filled in the fermentation tank in an amount of 70-75% of the total volume of the fermentation tank, and the volume of the fermentation tank is 500-600L.

The invention researches the characteristic of Streptomyces termitis N-15 as fish disease control, and the identification and research method is as follows:

the bacterial morphological characteristics of the Streptomyces termitis N-15 are observed;

(II) identifying by using a 16S rRNA gene;

thirdly, evaluating the safety of the Streptomyces termitis N-15 on grass carp;

fourthly, feeding the grass carp with Streptomyces termitis N-15 as a feed additive;

and fifthly, separating and structurally identifying active substances of the streptomyces termitis N-15.

The microecological preparation prepared by using the streptomyces termitis N-15 is suitable for aquaculture, particularly freshwater fish culture, and has no toxic or harmful effects on grass carp liver cells and grass carp bodies; the microecological preparation of the streptomyces termitis N-15 is used as a feed additive to feed freshwater fishes such as grass carp and crucian carp, so that the growth rate and the bait efficiency of the grass carp can be improved, and meanwhile, the immunity of the freshwater fishes can be enhanced, so that the resistance of the freshwater fishes such as the grass carp to fish pathogenic bacteria can be obviously improved. The fish protective agent has the broad-spectrum effect of inhibiting fish pathogenic bacteria, the protective rate reaches 50%, and after the fish protective agent acts on the fish body, the fish protective agent can effectively resist the infection of aeromonas hydrophila on freshwater fishes such as grass carp, crucian carp and the like, and has the best protective effect on the grass carp.

Compared with the prior art, the invention has the following beneficial effects:

(1) The separation and identification of the identified active substances by the 16S rRNA gene sequence determination has good antibacterial effects on aeromonas hydrophila, aeromonas veronii, edwardsiella tarda and the like;

(2) The microecological preparation prepared by fermenting the Streptomyces termitis N-15 strain has great potential in aquatic products, after the Streptomyces termitis N-15 strain is used as a feed additive for feeding grass carp, the activities of serum acid phosphatase and alkaline phosphatase of the grass carp are measured, and simultaneously, the anti-nuclear factor related factors 2-Kelch-like epichlorohydrin related protein 1 (Keap 1) of main immune organs such as liver, kidney, spleen and head kidney and the like are analyzed, immune related genes (Toll-like receptor 4 genes, immunoglobulin M genes, marrow-like differentiation factors 88, complement C3 and lysozyme genes LSZ) are also analyzed, so that the activities of alkaline phosphatase and acid phosphatase in serum of grass carp fed with the Streptomyces termitis N-15 strain microecological preparation are obviously increased, the expression level of the immunity related genes of the grass carp is also up-regulated in different immune organs, and the Streptomyces termitis N-15 strain microecological preparation can enhance the resistance of pathogenic bacteria, the grass carp to aeromonas, the survival rate of the grass carp can be obviously improved (50-60%), and the freshwater fish can effectively resist infection of the grass carp, such as the freshwater fish and the like;

(3) The separation and identification of the active substances can be used as a probiotic preparation with great development potential in aquaculture, and the separation and identification of the antibacterial active substances in the streptomyces albus N-15 fermentation broth are as follows: chalcomycin A (Cha Ermei element) with molecular weight of 701.4 and chemical formula C ₃₅ H ₅₆ O ₁₄ The method comprises the steps of carrying out a first treatment on the surface of the The invention also detects the antibacterial activity of the Chalcomycin A on 5 common pathogenic bacteria of fish for the first time, which shows that the Chalcomycin A has good antibacterial activity and potential aquatic product application value.

Description of the preservation of microorganisms:

streptomyces termitis N-15 of the invention is preserved in China center for type culture collection (CCTCC for short, address: university of Wuhan, china) in 2021, and has a strain preservation number of CCTCC NO: M2021726, and is classified and named: streptomyces termitis N-15, latin brand name Streptomyces termitum N-15.

Drawings

FIG. 1 is a view showing morphological characteristics of Streptomyces termitis N-15 according to the present invention.

FIG. 2 is a scanning electron microscope view of Streptomyces termitid N-15 according to the present invention.

FIG. 3 is a phylogenetic tree constructed based on the 16S rDNA sequence of strain N-15 according to the present invention.

FIG. 4 is a graph showing the growth of Streptomyces termitid N-15 according to the present invention.

FIG. 5 is a graph showing the isolation and identification of the peaks of Streptomyces termitid N-15 activity according to the present invention.

FIG. 6 is a HPLC separation of Streptomyces termitilis N-15 metabolite of the present invention.

FIG. 7 is a first-order mass spectrum of the inhibitory activity peak Z3 of Streptomyces termitis N-15 according to the present invention.

FIG. 8 is a carbon spectrum of the Streptomyces termitilis N-15 bacteriostatic active substance according to the invention.

FIG. 9 is a hydrogen spectrum of the Streptomyces termitilis N-15 bacteriostatic active substance according to the invention.

FIG. 10 is a structural formula of a Streptomyces termitilis N-15 bacteriostatic active substance according to the present invention.

FIG. 11 is a diagram of the biosynthetic gene cluster of Streptomyces termitis N-15 bacteriostatic active substances according to the invention.

FIG. 12 is a bar graph showing the changes in activity of the enzyme associated with the humoral immunity of Streptomyces termitis N-15 to grass carp according to the present invention.

FIG. 13 is a bar graph showing the changes in expression of genes related to the immunity of Streptomyces termitis N-15 to grass carp liver according to the present invention.

FIG. 14 is a bar graph showing the changes in expression of kidney immune-related genes of Streptomyces termitis N-15 of the present invention against grass carp.

FIG. 15 is a bar graph showing the changes in expression of spleen immune-related genes of Streptomyces termitis of the present invention against grass carp.

FIG. 16 is a bar graph showing the changes in expression of genes related to the immunity of Streptomyces termitis N-15 to grass carp head kidney according to the present invention.

FIG. 17 is a plot of the survival rate of grass carp after the Streptomyces termitis N-15 attacks Aeromonas veronii according to the present invention.

Detailed Description

The invention will be further described with reference to the drawings and the specific examples.

The chemical reagents used in the examples of the present invention, unless otherwise specified, were all obtained by conventional commercial means.

Collecting, separating, purifying and screening process of Streptomyces termitid N-15

Collecting soil samples of different provinces in China, weighing 5g of sediment, adding into 45mL of sterile water, shaking uniformly by a vortex oscillator, diluting by a 10-time gradient dilution method, and selecting 10 ^-4 、10 ^-5 、10 ^-6 Three dilutions of 0.1mL of the dilution were each coated with potassium dichromate (30X 10 concentration ^-6 g/mL) was cultured on a plate of Gao's I at 30℃for 5 days, different colonies were selected by the difference in shape and color of the colonies on the plate, streaked on the plate for purification culture, the procedure was repeated 3 times, and the purified strain was subjected to AM6 liquid culture and then stored in an ultra-low temperature refrigerator at-80 ℃.

(II) cell morphology characterization of Streptomyces termitid N-15

140 strains are obtained through separation and purification, and one excellent strain (table 1) with good antagonism to various fish pathogenic bacteria is selected and named as N-15. Actinomycetes N-15 are streaked on a Gao I solid culture medium, after culturing for 4 days at 30 ℃, large colonies and compact texture are observed, spores are produced in a rich way, middle bulges are formed, aerial hyphae are milky white, the hyphae in the basal are yellowish and accompanied with yellow soluble pigment production (see figure 1), the strain N-15 is observed under a scanning electron microscope to have broken spore filaments, and spores are cylindrical and have wrinkled surfaces (see figure 2).

TABLE 1 Excellent strains having good antagonism against various fish pathogenic bacteria

Note that: "-" means no antagonistic effect; "+" indicates antagonism

(III) homologous sequence analysis of N-15 Strain 16S rRNA Gene

Spores of the strain N-15 were inoculated in a liquid medium of Gao I (30 ℃ C., 125 rpm) for shake culture for 4d, then fresh bacterial liquid was collected by centrifugation in an EP tube, and genome of the strain N-15 was extracted using a bacterial genomic DNA extraction kit purchased from Shanghai Biotechnology company, and the 16S rRNA gene amplification primer sequence was:

27F：5′-AGAGTTTGATCCTGGCTCAG-3′；

1492R：5′-ACGGCTACCTTGTTACGACTT-3′，

the gene sequences corresponding to the strains are amplified by the primers, and the expected lengths of the sequences are about 1443bp respectively.

PCR reaction System (20. Mu.L): sterile double distilled water, 12 μl;5 Xbuffer, 4. Mu.L; dNTP, 1.6. Mu.L; bf-R (10. Mu.M), 0.6. Mu.L; bf-F (10. Mu.M), 0.6. Mu.L; genome template, 1 μl; primerSTAR DNA Polymerase (Takara), 0.2. Mu.L;

PCR reaction procedure: pre-denaturation at 95 ℃ for 5min;30 cycles: 95 ℃ for 45sec;55 ℃,45sec;72 ℃ for 1.5min;72℃for 10min.

The 16S rRNA gene PCR product was detected and recovered by 1.0% agarose gel electrophoresis. And then connecting the purified PCR product with a pMD-18T vector, transforming into escherichia coli DH5 alpha competent cells, screening for ampicillin resistance, picking positive transformants to extract plasmids, and sending to Shanghai Biotechnology Co.

The 16S rDNA sequence of strain N-15 was sequenced to a length of 1529bp, and the amplified sequencing results were analyzed by BLAST alignment at NCBI, using the Kimura-2-Parameter model of MEGA6.0, and using adjacency (NJ) to construct a phylogenetic tree (see FIG. 3). The strain is estimated to belong to Streptomyces termitum subspecies according to BLAST results and phase contrast microscope observation, and is named Streptomyces termitum N-15 and Streptomyces termitis N-15.

LB medium (/ L): 10g of sodium chloride, 10g of peptone, 5g of yeast powder, 20g of agar (liquid medium is not added), and the pH is 6.8-7.2;

the specific implementation process comprises the following steps: selecting N-15 spores with good growth state on a Gaoshi flat plate, growing in an AM6 liquid culture medium until the growth is in a logarithmic phase to serve as seed liquid, transferring the seed liquid of the strain N-15 to a multi-bottle containing 20mL of the AM6 fermentation culture medium for shake culture at an inoculum size of 1%, taking out 3 bottles every 12 hours, centrifuging at 9000rpm for 10min to collect thalli, washing the thalli with PBS for 2 times, drying the thalli with an oven to obtain moisture, weighing the dry weight of actinomycetes N-15, recording an average value, repeating the operation for 3 times, and drawing a biomass curve of the actinomycetes N-15.

The growth curve of the Streptomyces termitis strain N-15 is shown in figure 4, and the result shows that when fermentation starts, each nutrient-rich strain in the bottle can grow and reproduce rapidly, the growth logarithmic phase is reached when the strain is cultured for 84 hours, the strain is in a stable growth state in 84 hours-110 hours, and the decay phase appears after 120 hours of fermentation.

(IV) separation and purification of antibacterial active substances of Streptomyces termitis N-15 strain

Strain N-15 was inoculated into AM6 fermentation medium for 4d fermentation, followed by a total fermentation of 30L. And repeatedly centrifuging the fermented streptomyces N-15 culture solution for 3 times, collecting a fermentation supernatant, adding an equal volume of ethyl acetate for extraction, repeatedly oscillating in a separating funnel to enable the extraction to be full, collecting an organic phase after 6 hours, extracting an aqueous phase once again, collecting all the organic phases together, rotationally evaporating an organic solvent at 37 ℃, and adding 20mL of methanol to dissolve the organic phase as a crude extract of active substances of the strain N-15.

Dissolving the crude extract with DMSO, filtering with 0.22 μm organic phase filter membrane, loading 200 μl into RESOURCETM RPC chromatographic column (3 mL,15 μm, 6.4X 100mm,GE Healthcare, U.S.) and rapidly purifying with biomolecule purification system

Avant 25 was subjected to primary isolation. The mobile phase A is acetonitrile with 20% volume fraction, the mobile phase B is acetonitrile with 80% volume fraction, 12 column volumes are subjected to linear gradient elution (0% -100% of the mobile phase B) at a flow rate of 2mL/min, and the monitoring wavelength is 190-640 nm. The elution procedure is shown in Table 2, the collection method is to collect the peaks by peak, freeze-dry the collected peaks, and detect the antibacterial activity of the peaks on the Aeromonas veronii to obtain the crude active product.

TABLE 2

Primary Avant 25 separation method

The collected freeze-dried sample was redissolved in methanol, filtered, and subjected to high performance liquid chromatography (Agilent 1290) using a TC-C18 column (15 μm, 4.6X105 mm, agilent) at a monitoring wavelength of 250nm. Mobile phase: phase a was 5% acetonitrile by volume, phase B was 95% acetonitrile by volume, flow rate was 1mL/min, and separated mobile phase a: b=5% 95% acetonitrile/ultrapure water; the flow rate was 1mL/min. Elution procedure as shown in table 3, the activity peaks were collected (see fig. 5) and lyophilized in a freeze concentration centrifuge.

Activating fish pathogenic bacteria such as aeromonas hydrophila, aeromonas veronii, aeromonas salmonicida, aeromonas miltiorrhizae and Shigella, taking 100 mu L of bacterial liquid on a solid LB culture medium plate, uniformly coating the solid LB culture medium plate by using a coater, airing the solid LB culture medium plate in an ultra-clean bench, and placing a filter paper sheet on the surface of the plate. The activity peak (10. Mu.L) was added dropwise to a filter paper sheet, and incubated overnight at 30℃to observe the bacteriostatic effect (see FIG. 6). The active peaks were mass identified (see fig. 7) and their structure was further determined in combination with nuclear magnetic resonance techniques (see fig. 8-10). Finally, the active substance biosynthesis gene cluster was mined by whole genome sequencing (see FIG. 11).

TABLE 3 high performance liquid chromatography separation and purification method

Preparation of Streptomyces termitilis N-15 fermentation inoculant (microecological preparation)

(1) Seed of Streptomyces termitis N-15 inclined plane is cultured in a first shaking flask of Gaoshan seed culture medium (soluble starch 20g/L; naCl 0.5g/L; KNO) ₃ 1g/L；K ₂ HPO ₄ ·3H ₂ O 0.5g/L；MgSO ₄ ·7H ₂ O 0.5g/L；FeSO ₄ ·7H ₂ O0.01 g/L) for 72 hours under the culture condition of 28 ℃ and 180rpm to obtain seed liquid;

(2) Inoculating the seed solution obtained in the step (1) into a 20L fermentation tank according to an inoculum size of 1 percent for expansion culture, wherein the formula of a fermentation medium is (1L): glucose 1g, caCO ₃ 0.5g, 2g of soluble starch, 0.5g of yeast extract and 0.5g of bacteriological peptone; the liquid loading amount of the fermentation culture medium is 70%, the whole culture process is monitored on line, the dissolved oxygen concentration is controlled to be 45%, the temperature is controlled to be 28 ℃, the fermentation culture is carried out for 84 hours, the defoaming agent is supplemented on line in real time, and the OD600 is 4.0, so that the seed liquid for the expanded culture is obtained;

(3) Inoculating the seed liquid obtained in the step (2) in an expanded culture manner into a 100L fermentation tank with the liquid loading amount of 70% according to the inoculation amount of 10%, wherein the fermentation medium formula is the same as that of the fermentation medium formula obtained in the step (2), and culturing for 84 hours at the temperature of 28 ℃ with the dissolved oxygen amount of 45%, and adding an antifoaming agent on line in real time to obtain a fermentation product;

The obtained fermentation microecological preparation of the Streptomyces termitid N-15 strain is directly sprayed on a culture water body or mixed with feed and the like for application.

Application of Streptomyces termitid N-15 microecological preparation in aquatic products

Example 1: will 10 ¹⁰ After 100g of feed is mixed into 100g of microecological powder preparation of cfu streptomyces termite N-15 for feeding grass carp, the powder preparation resists fish pathogenic aeromonas veronii disease

120 healthy grass carp (7-12 g) to be tested are randomly divided into 6 groups and are fed into a cylindrical laboratory cultivation box (with the diameter of 1m and the water volume of 300L), and feed which is 2% of the weight of the grass carp is fed every day for 2 weeks, so that the health of the grass carp is ensured, and the grass carp is tested after stopping feeding for 24 hours. During the test, the illumination period is 12L/12D, the water temperature is 25+/-2 ℃, the pH of the water body is 7.2+/-0.5, and the feed is Tongwei 156 granule puffed material which is purchased from Tongwei Co., ltd.

And (3) test design: control group (CK): feeding sterilized fish feed, additive group (N-15): feeding with 1×10 concentration ⁸ cfu/g live bacteria feed of Streptomyces termitid N-15 strain; three replicates were set for each group, after 28d feeding, the fish were starved for 24h, 5 grass carp were randomly taken from each group, weighed, anesthetized with MS-222, blood was taken from the tail, and the alkaline phosphatase and acid phosphatase activities in the serum were measured using the kit (alkaline phosphatase activity measuring kit, acid phosphatase activity measuring kit was purchased from nanjing built biosystems, inc) (see fig. 12), and a significant increase in alkaline phosphatase activity in the grass carp serum was found. Rapidly dissecting in ice tray under sterile environment, taking liver, kidney, spleen and head kidney tissues, respectively extracting total RNA of each tissue, designing primers (see table 5), detecting immune related gene expression changes of each tissue by qRT-PCR (see fig. 13, 14, 15 and 16), and significantly up-regulating gene expression of IgM, C3, TLR-4, keap1, myD-88 and LSZ by adding groups into grass carp spleen and tail kidney. The expression level of IgM genes in immune organs except the liver is obviously up-regulated. Injection into fish body of 1×10 ⁸ cfu/mL of aeromonas veronii, 0.2mL per tail, and observing and counting the death number of the fish (see figure 17), wherein the protection rate of the grass carp reaches 50% -60%.

TABLE 4 Effect of feed on growth Properties of grass carp after Streptomyces termitis N-15 micro-ecological preparation was added

TABLE 5 qRT-PCR primer sequences

Example 2: prevention and treatment of fish aeromonas veronii by using streptomyces termitis microecological preparation in feed addition mode

And (3) test design: control group: feeding sterilized fish feed, additive group: feeding with 1×10 concentration ⁸ cfu/g live bacteria feed of Streptomyces termitid N-15 strain; three replicates were set for each group, and after 28d feeding, the grass carp was injected at a concentration of 1X 10 after 28d feeding ⁸ cfu/mL of Aeromonas veronii was injected at a dose of 0.2 mL/tail. The fish death number was counted by continuous observation for 2 weeks (see fig. 17), and the protection rate for grass carp reached 50% -60%.

Sequence listing

<110> Hunan university of teachers and students

<120> Streptomyces termitis N-15 and microecological preparation, and preparation method and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1443

<212> DNA

<213> Streptomyces termitilis N-15 (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 1

acgctggcgg cgtgcttaac acatgcaagt cgaacgatga agcccttcgg ggtggattgt 60

ggcgaacggg tgagtaacag ctggttgaaa gctccggcgg tgaaggatga gcccgcgcct 120

atcagcttgt tggtggggta atggcctacc aaggcgacgc tggttgaaag ctccggggtg 180

aaggatgagc ccgcggccta tcagcttgtt ggtggggtaa tggcctacca aggcgagacg 240

ggtaagccgg cctgagaggg cgaccggcca cactgggact gagacacggc ccagatccta 300

cgggaggcag cagtggggaa tattgcacaa tgggcgaaag cctgatgcag cgacccgcgt 360

gagggatgac ggccttcggg ttgtaaacct ctttcagcag ggaagaagcg agatgacggt 420

acctgcagaa gaagcgcggc taactacgtg ccagcagccg cggtaatacg tgggcgcaag 480

cgttgttccg gaattattgg gcgtaaagag ctcgtaggcg gcttgtcacg tgggtgtaaa 540

gcccggggct taaccccggg tctgcatccg atacgggcag gctagagtgg gtaggggaga 600

tcggaattcc tggtgtagcg gtgaaatgcg agatatcagg aggaacacgg tggcgaaggc 660

ggatctctgg gccattactg acgctgagga gcgaaagcgt ggggaggaac aggattagat 720

accctggtag tccacgccgt aaacgttggg aactaggtgt tggcgcattc cacgtcgtcg 780

gtgccgcagc taacgcatta agttccccgc ctggggagta cggcgcaagg ctaaaactca 840

aaggaattga cgggggcccg cacaagcagc ggagcatgtg gctaattcga cgcaacgcga 900

agaaccttac caaggcttga catataccgg aaagcattag agtagtgccc cccttgtggt 960

cggtatacag gtggtgcatg gctgtcgtca gctcgtgtcg tagatgttgg gttaagtccc 1020

gcaacgagcg caacccttgt cctgtgttgc cagcatgccc tcggggtgat ggggactcac 1080

aggagaccgc cggggtcaac tcggaggaag gtggggacgc gtcaagtcat catgcccctt 1140

atgtcttggg ctgcacacgt gctacaatgg ccggtacaag agctgcgatg ccgcgaggcg 1200

gagcgaatct caaaaagccg gtctcagttc ggattgggtc tgcaactcga ccccatgaag 1260

tcggagttgc tagtaatcgc agatcagcat tgctgcgtga atacgttccc gggccttgta 1320

cacaccgccc gtcacgtcac gaaagtcggt aacaccgaag ccggtggccc aaccccttgt 1380

gggagggagc tgtcgaaggt gggaccagcg attgggacga agtcgtaaca aggtagccgt 1440

acc 1443

Claims

1. Streptomyces termitis N-15%Streptomyces termitumN-15) is preserved in China Center for Type Culture Collection (CCTCC) with the strain preservation number of M2021726.

2. A microecological preparation prepared by fermenting Streptomyces termitis N-15 with a strain preservation number of CCTCC NO: M2021726 as claimed in claim 1.

3. The probiotic formulation according to claim 2, characterized in that it is a liquid formulation or a solid formulation.

4. A method of preparing a microecological formulation as claimed in claim 2 or 3, comprising the steps of:

(1) Inoculating Streptomyces termitis N-15 to a culture medium, and selecting mature spores to shake flask seed culture medium for culturing to obtain seed liquid;

(3) Inoculating the seed liquid obtained in the step (2) for expanding culture into a fermentation tank filled with a fermentation medium for culturing to obtain a fermentation product;

5. The method of claim 4, wherein in the step (2), the seed solution is inoculated into the fermentation tank in an amount of 1.0 to 1.5% by volume of the fermentation medium; the formulation of the fermentation medium for the enlarged culture is glucose 0.5-2.0 g, caCO ₃ 0.2-0.5 g, 1-3 g of soluble starch, 0.1-0.5 g of yeast extract and 0.2-0.6 g of bacteriological peptone.

6. The method according to claim 4 or 5, wherein in the step (3), the seed liquid for the expansion culture is inoculated in an amount of 8 to 10% of the volume of the fermentation medium; the formula of the fermentation medium is glucose 0.5-2.0 g, caCO ₃ 0.2-0.5 g, 1-3 g of soluble starch, 0.1-0.5 g of yeast extract and 0.2-0.6 g of bacteriological peptone.

7. The method for producing a microecological preparation according to claim 4 or 5, wherein in the step (3), the amount of dissolved oxygen in the culture is 35 to 55%; the temperature of the culture is 30-40 ℃; the culture time is 48-60 h.

8. The method for producing a microecological preparation according to claim 6, wherein in the step (3), the amount of dissolved oxygen in the culture is 35 to 55%; the temperature of the culture is 30-40 ℃; the culture time is 48-60 h.

9. Use of a probiotic according to claim 2 or 3 in aquaculture; the aquatic animals are freshwater fish.