CN113621533A - Streptomyces rubiginosus Z1-26, microecological preparation and preparation method thereof - Google Patents
Streptomyces rubiginosus Z1-26, microecological preparation and preparation method thereof Download PDFInfo
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Abstract
Streptomyces brickkaiensis Z1-26, microecological preparation and preparation method thereof, the Streptomyces brickkaiensis Z1-26 (Streptomyces lateritiusZ1-26) is preserved in China center for type culture Collection, and the preservation number of the strains is CCTCC NO: M2021705. The microbial ecological agent prepared by fermenting the streptomyces bricorubidus Z1-26 is suitable for aquaculture and is used as a feed additiveThe protective rate of the compound preparation is up to 60 percent when the compound preparation is applied to freshwater fishes such as crucian, grass carp, carp and the like, wherein the compound preparation has a remarkable effect of resisting infection of aeromonas hydrophila on the freshwater fishes, and can effectively improve the growth performance and the immunity of the freshwater fishes.
Description
Technical Field
The invention relates to a microorganism streptomyces bricorubidus, in particular to a streptomyces bricorubidus strain Z1-26, a microbial ecological agent and a preparation method thereof.
Background
In recent decades, the fish farming industry in China has been developed rapidly, and the scale and the level of intensification of fish farming have been rapidly expanded and improved. However, as the number and density of cultured fishes rapidly increase, the residual baits and excretions cannot be treated in time, and the water body with deteriorated water quality cannot be replaced in time, so that various diseases of the cultured fishes frequently occur. The bacteria, which are the main pathogens for cultivating fishes, can cause about 57.6 percent of biogenic diseases, and cause huge economic loss to the breeding industry.
At present, the prevention and treatment of bacterial diseases in fish culture mainly use antibiotics. However, in practical applications, the irrational use of antibiotics not only increases the resistance of pathogenic microorganisms, but also seriously harms human health and ecological environment. The probiotics is one of the products replacing antibiotics, can enhance the immunity of the cultured fishes so as to reduce the incidence rate of diseases in the culture process, does not generate toxic or side effect in the use process, and can effectively improve the living environment of the cultured fishes.
At present, probiotics commonly used in the aquaculture process mainly comprise photosynthetic bacteria, bacillus, lactic acid bacteria, saccharomycetes, nitrobacteria and the like, and the reports of applying actinomycetes as probiotics to aquaculture are few.
The actinomycetes have the ultra-strong potential of synthesizing abundant secondary metabolites and unique activity of inhibiting pathogenic bacteria, so the actinomycetes have important application value in aquaculture as probiotics. In recent years, actinomycetes are gradually applied to disease control in aquaculture, and certain effect is achieved. In 2008, Iwatsuki et al have shown that actinomycete strains K01-0509 are capable of producing inhibitors of The type III secretion system against infection by gram-negative bacteria such as Yersinia spp, Salmonella spp, Shigella spp, Pseudomonas spp (Shigella spp) and Pseudomonas spp (see "Iwatsuki, M, Uda R, Yoshijima H, Ui H, Shiomi K, Matsumoto A, Takahashi Y, Abe A, Tomoda H, Omura S. Guadinominines, type III secretion system in vivo, produced by microorganism sp.01-0509 [ J ] of The genus Anotipe, 229, 2008: (222. 13). Li et al added Streptomyces amrithrensis N1-32(Streptomyces amrithrensis) to the feed to feed grass carp, found that the strain can regulate the expression of immune factors of grass carp, improve the immunity of grass carp, and enhance the resistance of grass carp to Aeromonas veronii (Aeromonas veronii) (see "Li, Y.P.,. Hu SB, Gong L, Pan LF, Li DJ, Cao LN, Khan TA, Yang YH, Peng YN, Ding XZ, Yi GF, Liu SJ, Xia LQ. Isolating a new Streptomyces amsarhrensis N1-32 against bacterium on stress resistance of grass carp [ J ]. Fish & Shellfish & shotfish, 98: 632"). The researches show that the actinomycetes have better inhibiting and killing effects on aquatic pathogenic microorganisms and have the potential of becoming a novel microorganism fishing medicament.
However, reports of the streptomyces bricorubidus applied to aquaculture are rare, and particularly, the streptomyces bricorubidus applied to fish disease control as probiotics is not reported.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects in the prior art and provide the streptomyces bricorubidus which has good antagonistic action on common pathogenic bacteria of various fishes and can resist the infection of bacterial diseases of various fishes.
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 simple and convenient to operate.
The technical scheme for solving the technical problem is that a Streptomyces erythreus strain Z1-26(Streptomyces lateriticus Z1-26) is preserved in China center for type culture collection with the strain preservation number of CCTCC NO: M2021705.
The 16S rRNA sequence of the streptomyces erythreus Z1-26 is shown in a sequence table SEQ ID No. 1.
The streptomyces bricorubidus Z1-26 of the invention is screened from soil in the area of Hebei province.
The invention further solves the technical problem by adopting the technical scheme that the microbial ecological agent is prepared by fermenting the streptomyces erythrorhizogenes Z1-26 with the strain preservation number of CCTCC NO: M2021705.
Further, the microecological preparation is a liquid preparation or a solid preparation.
Further, the microecologics can be used in aquaculture.
Further, the aquaculture animals are freshwater fish.
Further, the freshwater fish is crucian, grass carp or carp.
The antibacterial active substance in the strain fermentation liquor has good medium-high temperature, acid-base, ultraviolet irradiation and protease stability, and is stable in practical production and application; has good effect of inhibiting pathogenic bacteria of freshwater fishes, can promote the growth of the fishes, and improves the immunity of the fishes.
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 and activating: transferring the Streptomyces rubiginosus Z1-26 preservation slant culture strain to an activated slant culture medium for activation to obtain a seed solution;
(2) first amplification culture: inoculating the seed solution obtained in the step (1) into a fermentation tank filled with a fermentation medium for first amplification culture to obtain a culture solution for the first amplification culture;
(3) and (3) second amplification culture: inoculating the seed liquid obtained in the step (2) in the first amplification culture into a fermentation tank filled with a fermentation culture medium, and culturing again to obtain an amplification culture liquid;
(4) Concentrating and collecting: collecting the enlarged culture solution obtained in the step (3), and concentrating to obtain a micro-ecological liquid microbial inoculum; or spray drying the micro-ecological liquid microbial inoculum to obtain the micro-ecological solid preparation.
Further, in the step (1), the activated slant culture medium is a TSB liquid culture medium; the activation culture conditions are as follows: the temperature is 28-30 ℃, the liquid loading amount is 10-30% of the volume of the container, and the rotating speed of the shaking table is 180-200 rpm.
Further, in the 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 formula of the fermentation medium for the first amplification culture is 5-12 g/L of glucose, 3-5 g/L of bacteriological peptone, 15-25 g/L of soluble starch, 3-6 g/L of yeast extract and CaCO 3 2～5g/L。
Further, in the step (2), the dissolved oxygen of the first amplification culture is 35-55%; the temperature of the first amplification culture is 28-30 ℃; and the time of the first amplification culture is 72-96 h, and the defoaming agent is supplemented on line in real time.
Further, in the step (3), the inoculation amount of the seed liquid for the first amplification culture is 10-15% of the volume of the culture medium; the fermentation medium comprises 5-12 g/L of glucose, 3-5 g/L of bacteriological peptone, 15-25 g/L of soluble starch, 3-6 g/L of yeast extract and CaCO 3 2～5g/L。
Further, in the step (3), the cultured dissolved oxygen is 35-55%; the culture temperature is 28-30 ℃; the culture time is 96-132 h, and the defoaming agent is supplemented on line in real time.
Further, in the step (3), the amount of the fermentation medium filled in the fermentation tank is 70-75% of the volume of the fermentation tank, and the volume of the fermentation tank is 500-600L.
The identification and research method of the streptomyces bricorubidus Z1-26 of the invention is as follows:
firstly, observing the cytomorphological characteristics of the strain Z1-26;
(II) identifying by using 16S rRNA gene;
(III) detecting the physicochemical property of the streptomyces brichami Z1-26 bacteriostatic active substance;
(IV) colonizing and detecting the streptomyces bricorubidus Z1-26 in the crucian body;
and (V) observing the influence of the streptomyces bricorubidus Z1-26 as a feed additive on the growth and disease resistance of crucian.
Compared with the prior art, the invention has the following beneficial effects:
(1) the strain is identified as a new strain of the Streptomyces bricanthus through 16S rRNA gene sequence determination (the strain preservation number is CCTCC NO: M2021705; the classification is named as Streptomyces bricanthus Z1-26; the Latin scientific name is Streptomyces lateriticus Z1-26; and the bacterial strain has good antibacterial effect on various freshwater fish pathogenic bacteria such as aeromonas hydrophila, aeromonas veronii, edwardsiella tarda and the like;
(2) The streptomyces erythropolis Z1-26 can survive and colonize in fish bodies, does not damage fishes, and lays a foundation for the strain to play a stable disease prevention and growth promotion role in fish culture;
(3) the streptomyces bricorubidus Z1-26 is added into feed as a feed additive to feed fishes, so that the growth rate of fish bodies can be promoted, the feed efficiency is improved, the immunity of the fish bodies is enhanced, and the resistance of the fish bodies to pathogenic bacteria is enhanced;
(4) the method for preparing the microbial ecological agent from the streptomyces erythropolis Z1-26 through fermentation is simple and convenient, the production cost is low, meanwhile, the risks of drug residue, pathogenic bacteria resistance and the like caused by antibiotic abuse can be effectively reduced, and the application prospect is good.
Description of the preservation of the microorganism
The Streptomyces cubilofaciens Z1-26(Streptomyces lateriticus Z1-26) is preserved in China center for type culture Collection (CCTCC for short, the address: Wuhan, Wuhan university, China) at 6-9 months in 2021, and the preservation number of the strain is CCTCC NO: M2021705.
Drawings
FIG. 1 is a diagram of the bacteriostatic effect of Streptomyces cubiloensis Z1-26 on fish pathogenic bacteria.
FIG. 2 is an observation picture of the morphological characteristics of the Streptomyces cubilofaciens Z1-26 of the invention.
FIG. 3 is a phylogenetic tree diagram constructed from the 16S rDNA sequence of Streptomyces rubiginus Z1-26 of the present invention.
FIG. 4 is an analysis diagram of the effect of the bacteriostatic active substance Z1-26 of Streptomyces cubiloensis of the invention on temperature.
FIG. 5 is an analysis diagram of the effect of the bacteriostatic active substance Z1-26 of Streptomyces cubiloensis of the invention on ultraviolet irradiation.
FIG. 6 is an analysis chart of the effect of the bacteriostatic active substance of Streptomyces brichamensis Z1-26 and protease.
FIG. 7 is an analysis diagram of the effect of the bacteriostatic active substance Z1-26 of Streptomyces cubiloensis of the invention on pH value.
FIG. 8 is a diagram of a small animal living body imaging system for observing the colonization and protection force of the Streptomyces rubiginis Z1-26 marker strain in the crucian body.
FIG. 9 is a graph showing the results of analysis of the effect of the microecological preparation prepared from Streptomyces rubiginis Z1-26 of the present invention on the expression of the gene MSTN associated with the inhibition of muscle growth in crucian muscles.
FIG. 10 is a diagram showing the results of analysis of the effect of the microecological preparation prepared from Streptomyces rubiginis Z1-26 of the present invention on the IGF expression of the relevant gene for promoting muscle growth of crucian.
FIG. 11 is a diagram showing the results of analysis of the effect of the microecological preparation prepared from Streptomyces rubiginus Z1-26 of the present invention on the activity of the serum acid phosphatase of crucian.
FIG. 12 is a diagram showing the results of analysis of the effect of the microbial ecological agent prepared by Streptomyces rubiginis Z1-26 on the activity of alkaline phosphatase in the serum of crucian.
FIG. 13 is a diagram of the results of analysis of the effect of the microecological preparation prepared from Streptomyces rubiginus Z1-26 on the enzymatic activity of the serum superoxide dismutase of crucian.
FIG. 14 is a diagram showing the analysis result of the influence of the microecological preparation prepared from Streptomyces rubiginus Z1-26 of the present invention on the activity of the serum lysozyme enzyme of crucian carp.
FIG. 15 is a diagram showing the results of analysis of the effect of the microecological preparation prepared from Streptomyces rubiginis Z1-26 of the present invention on the expression of immune-related genes in the liver of crucian carp.
FIG. 16 is a diagram showing the analysis result of the influence of the microecological preparation prepared from Streptomyces rubiginus Z1-26 of the present invention on the expression of immune related genes in the kidney of crucian.
FIG. 17 is a graph showing the results of analysis of the effect of the microbial ecological agent prepared by Streptomyces rubiginis Z1-26 of the present invention on the expression of immune-related genes in the spleen of Carassius auratus.
Detailed Description
The invention is further illustrated by the following examples and figures.
The chemical reagents used in the examples of the present invention, unless otherwise specified, are commercially available in a conventional manner.
Screening of streptomyces bricorubidus Z1-26 and determination test of bacteriostasis spectrum
Mixing soil samples from Hebei province in sterile water, gradient diluting, spreading on Gao's plate, culturing at 30 deg.C for 5d, observing growth state of strain, selecting actinomycete sample, inoculating to new culture medium, purifying selected strain, and detecting whether the strain has good bacteriostatic effect on pathogenic bacteria of Oxyhydrophila (Aeromonas hydrophila), Aeromonas sobria (Aeromonas sobria), Aeromonas caviae (Aeromonas caviae), Aeromonas veroniae (Aeromonas veronii), Aeromonas anophaga (Aeromonas aromatica), Edwardsiella tarda), Erwinia sp, Citrobacter freundii (Citrobacter freundiii), Salmonella frewanensis (Shewanella Xiamenensis), Plesiomonas sp. and Plesioshis sp. with the strain of Bacillus strain as indication strain, wherein the strain has good bacteriostatic effect on pathogenic bacteria of Bacillus strain of Botrytis found by fermentation method, and the strain has good bacteriostatic effect on bovine spongiella strain of Bacillus sp. origin, and the strain is found by use of Bacillus subtilis 1, is named as Z1-26.
Cytomorphological characteristics of Streptomyces brichamensis Z1-26
Bacterial colonies of the strain Z1-26 on the Gao's first plate are in concentric rings, are dry, opaque and powdery, are tightly combined with a culture medium and are not easy to pick up. The bacterial strain is gram-positive bacteria. Z1-26 was observed under a scanning electron microscope to have developed mycelium, thicker mycelium, and more and shorter branches (see FIG. 2). As can be seen, strain Z1-26 exhibited morphological characteristics consistent with those typical of actinomycete colonies and thalli.
(III) identification of 16S rRNA gene of Streptomyces cubiloensis Z1-26
The strain Z1-26 was inoculated into liquid high-grade medium, cultured at 30 ℃ and 120rpm for 4 days, the cells were collected, the genomic DNA of the strain Z1-26 was extracted using an Ezup column type bacterial genomic DNA extraction kit (Shanghai Bioengineering Co., Ltd.), and 16S rRNA gene amplification primers were used.
27F：5′-AGAGTTTGATCCTGGCTCAG-3′；
1492R：5′-ACGGCTACCTTGTTACGACTT-3′。
The 16S rRNA gene sequence was amplified with the above primers and the expected length of the sequence was about 1500 bp.
PCR reaction (20. mu.L): sterile double distilled water, 14 μ L; 10 × Buffer, 2 μ 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 μ L; PrimerSTAR DNA Polymerase (Takara), 0.2. mu.L;
PCR reaction procedure: pre-denaturation at 95 ℃ for 5 min; 30 cycles: 95 ℃ for 45 sec; 55 ℃ for 45 sec; 72 ℃ for 1.5 min; 72 ℃ for 10 min.
After the 16S rRNA gene PCR product is detected by 1.0% agarose gel electrophoresis, the successfully amplified product is sent to Shanghai biological engineering Co., Ltd for sequencing.
The 16S rRNA gene sequence of the strain Z1-26 is shown to be 1428bp in length by sequencing, and the amplification sequencing result is analyzed by BLAST comparison on NCBI, and a phylogenetic tree of the strain is constructed by a adjacency method in software MEGA 7.0.26 (see figure 3). Closest to Streptomyces lateriticus strain LMG 19372.
Analysis of physicochemical properties of (IV) Streptomyces brichamii Z1-26 bacteriostatic active substance
(1) Thermal stability
The Streptomyces cubilophilus Z1-26 strain is inoculated into a liquid AM6 culture medium, the culture is carried out for 5d at 30 ℃ and 120rpm, fermentation liquor is respectively taken and treated for 1h at the temperature of 30 ℃, 50 ℃, 70 ℃ and 90 ℃, aeromonas hydrophila is taken as a pathogen indicator, and the antimicrobial activity of fermentation supernatant after different temperature treatment is detected by an oxford cup method (see figure 4).
(2) Acid-base resistance
The Streptomyces cubilophilus Z1-26 strain is inoculated into a liquid AM6 culture medium, the culture is carried out for 5d at 30 ℃ and 120rpm, fermentation liquor is respectively taken and treated for 1h under the conditions that the pH value is 1, 3, 5, 7, 9, 11 and 13, the original pH value is recovered, aeromonas hydrophila is taken as a pathogen indicator, and the antimicrobial activity of fermentation supernatant after different pH values are detected by an oxford cup method (see figure 7).
(3) Resistance to ultraviolet light
Inoculating the Streptomyces cubilophilus Z1-26 strain into a liquid AM6 culture medium, culturing at 30 ℃ and 120rpm for 5d, taking the fermentation liquid to irradiate under an ultraviolet lamp for 10min, 20min, 30min, 40min, 50min and 60min respectively, taking aeromonas hydrophila as a pathogenic indicator, and detecting the bacteriostatic activity of the fermentation supernatant after different ultraviolet irradiation time treatments by using an Oxford cup method (see figure 5).
(4) Protease resistance
The streptomyces bricorubidus Z1-26 strain is inoculated into a liquid AM6 culture medium, cultured for 5d at 30 ℃ and 120rpm, treated for 1h at 37 ℃ by using protease K and trypsin respectively, aeromonas hydrophila is used as a pathogen indicator, and the antimicrobial activity of fermentation supernatant subjected to different protease treatments is detected by an oxford cup method (see figure 6).
(V) preparation of Streptomyces cubilofaciens Z1-26 microecological preparation
(1) Inoculating and activating: transferring the Streptomyces rubiginosus Z1-26 preservation slant strain to an activated slant culture medium for activation to obtain activated seed liquid; the activated slant culture medium used is TSB liquid culture medium, and the conditions of activated culture are as follows: the temperature is 30 ℃, the liquid loading amount is 15 percent of the volume of the container, and the rotating speed of the shaking table is 200 rpm;
(2) first amplification culture: inoculating the activated seed solution obtained in the step (1) into a fermentation tank filled with a fermentation culture medium according to the inoculation amount of 1% for carrying out first amplification culture to obtain a seed solution for the first amplification culture; the formula of the fermentation medium is as follows: 10g/L glucose, 5g/L bacteriological peptone, 20g/L soluble starch, 5g/L yeast extract, CaCO 3 5g/L; the culture conditions were: the whole culture process is monitored on line, the dissolved oxygen concentration is 45%, the temperature is 30 ℃, the culture is carried out for 84h, and the defoaming agent is supplemented on line in real time;
(3) and (3) second amplification culture: inoculating the seed solution obtained in the step (2) in the first amplification culture into a 500L fermentation tank filled with 70% of fermentation medium according to the inoculation amount of 10% to obtain an amplification culture solution; the formula of the fermentation medium is the same as that of the fermentation medium in the step (2); the culture conditions were: the dissolved oxygen concentration is 45%, the temperature is 30 ℃, the culture is carried out for 120h, and the defoaming agent is supplemented online in real time;
(4) concentrating and collecting: collecting the enlarged culture solution obtained in the step (3), and concentrating to obtain a micro-ecological liquid microbial inoculum; or spray drying the micro-ecological liquid microbial inoculum to obtain the micro-ecological solid preparation.
Application of streptomyces bricanthus Z1-26 in fish culture
a. Colonization and protection of Streptomyces rubrorubidus Z1-26 in crucian
Selecting healthy crucian carps with consistent sizes, randomly dividing the crucian carps into 1 control group and 1 experimental group, wherein 10 carps in each group are arranged repeatedly. Feeding 7 days before experiment, acclimatizing to make crucian adapt to experiment environment, aerating 48 hr water at 25 deg.C, feeding basic feed for crucian in control group, and feeding crucian in experiment group with 1 × 10 concentration 9cfu/g green fluorescent gene marker strain Z1-26EGFPThe feed of (3) is fed for 2 times a day with the feed dosage of 2% of the body weight, and the small animal living body imaging observation is carried out every 2d (see figure 8), and the experiment lasts for 14 d. Performing challenge test two weeks later, wherein the test adopts intraperitoneal injection method, and the control group and the experimental group are injected with 200 μ L of 1 × 106AhX040 of cfu/mLmCherryThe bacterial suspension was subjected to in vivo imaging observation of the small animals every 1d (see FIG. 8), and was continuously examined for 7 d.
The experimental result shows that the feed is fed with Z1-26EGFPAfter the strain is 1d, a green fluorescence signal is detected on gills of the crucian, green fluorescence is detected on the abdomen of the crucian along with the prolonging of feeding time, and Z1-26EGFPThe bacterial strain is gradually increased in the crucian body, and the green fluorescence signal is gradually enhanced. AhX040mCherryDetecting red fluorescence at 1d after toxin attack, gradually increasing red fluorescence signal in crucian fed with common feed along with time, feeding with additive Z1-26EGFPThe red fluorescence signal in the crucian body of the feed of the strain is not obviously strengthened. The results show that the strain Z1-26 of the inventionEGFPCan survive and colonize in crucian body, and can inhibit strain AhX040 in crucian bodymCherryAgainst infection by Aeromonas hydrophila.
b. Influence of Streptomyces rubrorubidus Z1-26 on growth and disease resistance of crucian
Selecting healthy crucian carps with consistent sizes, randomly dividing the crucian carps into 1 control group and 2 experimental groups, wherein each group has 27 tails, and setting the groups to be repeated. Feeding 7 days before experiment, acclimatizing to make crucian adapt to experiment environment, aerating 48 hr water at 25 deg.C, feeding common feed for crucian in control group, and feeding crucian in 2 experiment groups with 1 × 10 concentration8cfu/g and 1X 109cfu/g of Z1-26 strain feed, the feeding amount of the feed is about 2% of the body weight, 2 times per day, and the experiment lasts for 30 days.
(1) Influence of Streptomyces rubrorubidus Z1-26 on growth performance of crucian
The experiment uses crucian carp to be weighed at experiment 1d and 30d respectively. Weight Gain Rate (WGR), Specific Growth Rate (SGR), Feed Efficiency (FE) and Survival Rate (SR) were calculated using the following formulas: WGR (%) ═ 100 × (Wt-W0)/W0; SGR (%) ═ 100 × (lnWt-lnW 0)/T; FE (%) ═ 100 × (Wt-W0)/(feed intake); SR (%) ═ 100 × (end of run crucian tail)/(start of run crucian tail) (see table 1). Then, the expression level of the muscle growth related factor is analyzed by fluorescence quantitative PCR, the expression level of the muscle growth inhibition related gene MSTN in the muscle is obviously reduced (see figure 9), and the expression level of the muscle growth promotion related gene IGF is obviously increased (see figure 10)
TABLE 1 crucian growth Performance parameters
Note: different superscripts on the same row indicate significant differences (P <0.05)
(2) Influence of Streptomyces rubrorubidus Z1-26 on non-specific immunity index of crucian serum
The Z1-26 strain was added to the feed to feed crucian for 30d, and the serum of crucian was analyzed for alkaline phosphatase (AKP), acid phosphatase (ACP), Lysozyme (LZM), and superoxide dismutase (SOD) activities (see fig. 11-fig. 14). Researches show that after the crucian carp is fed by the feed containing the Z1-26 strain, the enzyme activities of AKP, SOD and LZM in the crucian carp serum are obviously improved, and the ACP activity is higher than that of a control group, but the ACP activity is not obviously different.
(3) Influence of Streptomyces rubiginosus Z1-26 on expression of immune-related genes in crucian tissues
The Z1-26 strain is added into feed to feed crucian for 30d, and the expression level of immune related genes in the liver, kidney and spleen of crucian is analyzed (see figure 15-figure 17). Researches show that after the feed containing the Z1-26 strain is fed, the IgM expression level is obviously improved, the C3 expression level is obviously improved in the liver and the spleen, the LZM expression level is obviously improved in the kidney, the expression level of an inflammatory factor IL-1 beta is obviously reduced, and the immunity is improved.
(4) Protection test of streptomyces adoxorubi Z1-26 on crucian
After the Z1-26 strain is added into feed to feed crucian carp for 30d, and after using aeromonas hydrophila to counteract poison, the living state and death of each group of crucian carp in 7d are continuously observed and recorded (see Table 2). The research shows that the survival rate of the control group crucian fed with the basic feed is 10 percent at 7 days after the toxin attack, and the addition amount of the Z1-26 strain is 1 multiplied by 10 8cfu/g and 1X 109The survival rate of the crucian in the cfu/g group is obviously higher than that of the control group, and is respectively 50% and 60%.
TABLE 2 protective experiment of crucian by Z1-26 strain
Sequence listing
<110> university of Master in Hunan
<120> Streptomyces rubiginosus, microecological preparation thereof and preparation method thereof
<130> 2021.07.08
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1428
<212> DNA
<213> Streptomyces brickluyangensis Z1-26(2 Ambystoma laterale x Ambystoma jeffersonanum)
<400> 2
gggcaggcgc gtgcttacac atgcagtcga acgatgaagc ccttcggggt ggattagtgg 60
cgaacgggtg agtaacacgt gggcaatctg cccttcactc tgggacaagc cctggaaacg 120
gggtctaata ccggataaca ccggcttccg catggaagct ggttgaaagc tccggcggtg 180
aaggatgagc ccgcggccta tcagcttgtt ggtggggtaa tggcctacca aggcgacgac 240
gggtagccgg cctgagaggg cgaccggcca cactgggact gagacacggc ccagactcct 300
acgggaggca gcagtgggga atattgcaca atgggcgaaa gcctgatgca gcgacgccgc 360
gtgagggatg acggccttcg ggttgtaaac ctctttcagc agggaagaag cgaaagtgac 420
ggtacctgca gaagaagcgc cggctaacta cgtgccagca gccgcggtaa tacgtagggc 480
gcaagcgttg tccggaatta ttgggcgtaa agagctcgta ggcggcttgt cacgtcgggt 540
gtgaaagccc ggggcttaac cccgggtctg catccgatac gggcaggcta gagtgtggta 600
ggggagatcg gaattcctgg tgtagcggtg aaatgcgcag atatcaggag gaacaccggt 660
ggcgaaggcg gatctctggg ccattactga cgctgaggag cgaaagcgtg gggagcgaac 720
aggattagat accctggtag tccacgccgt aaacgttggg aactaggtgt tggcgacatt 780
ccacgtcgtc ggtgccgcag ctaacgcatt aagttccccg cctggggagt acggccgcaa 840
ggctaaaact caaaggaatt gacgggggcc cgcacaagca gcggagcatg tggcttaatt 900
cgacgcaacg cgaagaacct taccaaggct tgacatatac cggaaagcat tagagatagt 960
gccccccttg tggtcggtat acaggtggtg catggctgtc gtcagctcgt gtcgtgagat 1020
gttgggttaa gtcccgcaac gagcgcaacc cttgtcctgt gttgccagca tgcccttcgg 1080
ggtgatgggg actcacagga gaccgccggg gtcaactcgg aggaaggtgg ggacgacgtc 1140
aagtcatcat gccccttatg tcttgggctg cacacgtgct acaatggccg gtacaaagag 1200
ctgcgatgcc gtgaggcgga gcgaatctca aaaagccggt ctcagttcgg attggggtct 1260
gcaactcgac cccatgaagt cggagttgct agtaatcgca gatcagcatt gctgcggtga 1320
atacgttccc gggccttgta cacaccgccc gtcacgtcac gaaagtcggt aacacccgaa 1380
gccggtggcc caaccccttg tgggagggag ctgtcgaagt gacgagtt 1428
Claims (10)
1. Streptomyces cubilofaciens Z1-26(Streptomyces lateritiusZ1-26) and is preserved in China center for type culture Collection, and the preservation number of the strains is CCTCC NO: M2021705.
2. A microbial ecological agent prepared by fermenting Streptomyces cubilose Z1-26 with the strain preservation number of CCTCC NO: M2021705 as claimed in claim 1.
3. The microecological formulation according to claim 2, wherein the microecological formulation is a liquid formulation or a solid formulation.
4. The microecological formulation according to claim 2 or 3, wherein the microecological formulation is for use in aquaculture.
5. The microecological formulation according to claim 4, wherein the aquaculture animal is a freshwater fish.
6. A method for preparing a microecological formulation according to any one of claims 2 to 5, comprising the steps of:
(1) Inoculating and activating: transferring the Streptomyces rubiginosus Z1-26 preservation slant culture strain to an activated slant culture medium for activation to obtain a seed solution;
(2) first amplification culture: inoculating the seed solution obtained in the step (1) into a fermentation tank filled with a fermentation medium for first amplification culture to obtain a culture solution for the first amplification culture;
(3) and (3) second amplification culture: inoculating the seed liquid obtained in the step (2) in the first amplification culture into a fermentation tank filled with a fermentation culture medium, and culturing again to obtain an amplification culture liquid;
(4) concentrating and collecting: collecting the enlarged culture solution obtained in the step (3), and concentrating to obtain a micro-ecological liquid microbial inoculum; or spray drying the micro-ecological liquid microbial inoculum to obtain the micro-ecological solid preparation.
7. The method for producing a microecological agent according to claim 6, wherein in the step (1), the activated slant medium is TSB broth; the activation culture conditions are as follows: the temperature is 28-30 ℃, the liquid loading amount is 10-30% of the volume of the container, and the rotating speed of the shaking table is 180-200 rpm.
8. The method for preparing a microecological preparation according to claim 6 or 7, wherein in the step (2), the seed solution is inoculated into the fermenter in an amount of 1-2% by volume of the culture medium; the formula of the fermentation medium for the first amplification culture is 5-12 g/L of glucose, 3-5 g/L of bacteriological peptone, 15-25 g/L of soluble starch, 3-6 g/L of yeast extract and CaCO 3 2~5 g/L。
9. The method for preparing a microecological agent according to any one of claims 6 to 8, wherein in step (2), the dissolved oxygen of the first scale-up culture is 35 to 55%; the temperature of the first amplification culture is 28-30 ℃; the time of the first amplification culture is 72-96 h.
10. The method for preparing a microecological preparation according to any one of claims 6 to 9, wherein in step (3), the seed solution inoculation amount of the first expansion culture is 10 to 15% of the volume of the culture medium; the fermentation medium comprises 5-12 g/L of glucose, 3-5 g/L of bacteriological peptone, 15-25 g/L of soluble starch, 3-6 g/L of yeast extract and CaCO32-5 g/L; the dissolved oxygen amount of the culture is 35-55%; the culture temperature is 28-30 ℃; the culture time is 96-132 h.
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| CN114292785A (en) * | 2021-12-29 | 2022-04-08 | 暨南大学 | Marine streptomyces and application thereof in preventing and controlling aeromonas hydrophila from infecting aquatic animals |
| CN114908013A (en) * | 2022-05-19 | 2022-08-16 | 中国水产科学研究院黑龙江水产研究所 | Shewanella manshurica for producing DDP-IV inhibitor and application thereof |
| CN115197972A (en) * | 2022-09-13 | 2022-10-18 | 中国农业科学院农产品加工研究所 | Anti-aging streptomyces bricorubidus extract and preparation method thereof |
| CN115975846A (en) * | 2022-08-02 | 2023-04-18 | 岳阳渔美康生物科技有限公司 | Streptomyces saadriamycin, microecological preparation thereof and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114292785A (en) * | 2021-12-29 | 2022-04-08 | 暨南大学 | Marine streptomyces and application thereof in preventing and controlling aeromonas hydrophila from infecting aquatic animals |
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| CN114908013A (en) * | 2022-05-19 | 2022-08-16 | 中国水产科学研究院黑龙江水产研究所 | Shewanella manshurica for producing DDP-IV inhibitor and application thereof |
| CN115975846A (en) * | 2022-08-02 | 2023-04-18 | 岳阳渔美康生物科技有限公司 | Streptomyces saadriamycin, microecological preparation thereof and preparation method thereof |
| CN115975846B (en) * | 2022-08-02 | 2023-10-03 | 岳阳渔美康生物科技有限公司 | Streptomyces saramycin, microecological preparation and preparation method thereof |
| CN115197972A (en) * | 2022-09-13 | 2022-10-18 | 中国农业科学院农产品加工研究所 | Anti-aging streptomyces bricorubidus extract and preparation method thereof |
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