CN113621533B - Streptomyces erythropolis Z1-26, microecological preparation and preparation method thereof - Google Patents
Streptomyces erythropolis Z1-26, microecological preparation and preparation method thereof Download PDFInfo
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- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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Abstract
Streptomyces erythropolis Z1-26, microecological preparation and preparation method thereof, wherein the Streptomyces erythropolis Z1-26%Streptomyces lateritiusZ1-26) is preserved in China Center for Type Culture Collection (CCTCC) with the strain preservation number of M2021705. The microecological preparation prepared by fermenting the Streptomyces brick red mold Z1-26 is suitable for aquaculture, acts on freshwater fishes such as crucian, grass carp, carp and the like in a feed additive mode, has a protection rate of 60%, has a particularly remarkable effect of resisting aeromonas hydrophila infection on freshwater fishes, and can effectively improve the growth performance and the immunity of freshwater fishes.
Description
Technical Field
The invention relates to a microorganism streptomyces erythropolis, in particular to a streptomyces erythropolis Z1-26, a microecological preparation and a preparation method thereof.
Background
In recent decades, the development of the fish farming industry in China has been rapid, and the scale and the intensive level of fish farming have been rapidly expanded and improved. However, with the rapid expansion of the number and density of farmed fish, residual baits and excreta cannot be treated in time, and water with deteriorated quality cannot be replaced in time, so that various diseases of farmed fish frequently occur. Bacteria, which are the main pathogens for fish farming, can cause approximately 57.6% of biogenic diseases, causing a great economic loss to the farming industry.
At present, the prevention and treatment of bacterial diseases in fish culture mainly uses antibiotics. However, in practical applications, the unreasonable use of antibiotics not only increases the resistance of pathogenic microorganisms, but also severely jeopardizes human health and ecological environment. The probiotics are used as one of products for replacing antibiotics, so that the immunity of the cultured fishes can be enhanced, the occurrence rate of diseases in the culturing process is reduced, toxic and side effects cannot be generated in the using process, and the living environment of the cultured fishes can be effectively improved.
At present, the common probiotics species in the aquaculture process mainly comprise photosynthetic bacteria, bacillus, lactobacillus, saccharomycetes, nitrifying bacteria and the like, and few reports on the application of actinomycetes as probiotics in aquaculture are provided.
Actinomycetes have the potential of superstrong synthesis of abundant secondary metabolites and unique activity of inhibiting pathogenic bacteria, so that the actinomycetes have important application value as probiotics in aquaculture. In recent years, actinomycetes are gradually applied to prevention and control of aquaculture diseases, and certain effects are achieved. Studies by iwotsuki et al have shown that actinomycete strain K01-0509 is capable of producing a type iii secretion system inhibitor to combat infection with gram-negative bacteria of the genus yersinia (yersinaspp.), salmonella (Salmonella spp.), shigella (Shigella spp.), pseudomonas (Pseudomonas spp.) (see "iwotsuki, M, uchida R, yoshimma H, ui H, shiomim K, matsumoto a, takahashi Y, abe a, tomoda H, omura s.guandinamines, type III secretion system inhibitors, produced by Streptomyces.k 01-0509 j., [ The Journal of Antibiotics,2008,61 (4): 222-229"), which have a type iii secretion system virulence system. Li et al added Streptomyces alginolyticus N1-32 (Streptomyces amritsarensis) to feed for grass carp, found that the strain was able to modulate expression of grass carp immune factor, enhance grass carp immunity, enhance grass carp resistance to Aeromonas verrucosa (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 amritsarensis N-32 against Fish pathogens and determining its effects on disease resistance of grass carp[J ]. Fish & Shellfish Immunology,2020, 98:632-640"). The research shows that the actinomycetes have better inhibiting and killing effects on aquatic pathogenic microorganisms and have the potential of becoming a novel microbial fishing drug.
However, reports of Streptomyces brick red applied to aquaculture are rare, and particularly, the application of Streptomyces brick red as probiotics to fish disease control has not been reported yet.
Disclosure of Invention
The invention aims to solve the technical problems of overcoming the defects in the prior art and providing the Streptomyces brick red mold which has good antagonism on various common pathogenic bacteria of fishes and can resist infection of various bacterial diseases of 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 provide a preparation method of the microecological preparation, which is easy to operate.
The invention solves the technical problems by adopting the technical proposal that a strain of Streptomyces erythropolis Z1-26 (Streptomyces lateritius Z1-26) is preserved in China center for type culture collection, and the strain preservation number is CCTCC NO: M2021705.
The 16S rRNA sequence of the Streptomyces griseus Z1-26 is shown in a sequence table SEQ ID No. 1.
Streptomyces brick red mold Z1-26 of the present invention is used for screening soil from the Hebei province area.
The technical scheme adopted by the invention for further solving the technical problems is that the microbial ecological agent is prepared by fermenting Streptomyces erythropolis 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 animal is a freshwater fish.
Further, the freshwater fish is crucian, grass carp or carp.
The antibacterial active substances in the strain fermentation liquor have better medium-high temperature, acid-base, ultraviolet irradiation and protease stability, and are more stable in actual production and application; has good inhibiting effect on pathogenic bacteria of freshwater fish, and can promote fish growth and improve the immunity of fish.
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) Inoculation and activation: transferring the Streptomyces brick red Z1-26 deposited slant strain to an activating slant culture medium for activation to obtain seed liquid;
(2) First expansion culture: inoculating the seed liquid obtained in the step (1) into a fermentation tank filled with a fermentation medium for primary expansion culture to obtain a culture liquid for primary expansion culture;
(3) Second expansion culture: inoculating the seed liquid obtained in the step (2) for the first time to a fermentation tank filled with a fermentation medium, and culturing again to obtain an enlarged culture liquid;
(4) Concentrating and collecting: collecting the enlarged culture solution obtained in the step (3), and concentrating to obtain a microecological liquid microbial inoculum; or spray drying the microecological liquid microbial inoculum to obtain the microecological 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 seed solution is inoculated into a fermentation tankThe inoculation amount of the strain is 1-2% of the volume of the culture medium; the formulation of the fermentation medium for the first expansion culture is 5-12 g/L glucose, 3-5 g/L bacteriological peptone, 15-25 g/L soluble starch, 3-6 g/L yeast extract and CaCO 3 2～5g/L。
Further, in the step (2), the dissolved oxygen amount of the first expansion culture is 35-55%; the temperature of the first expansion culture is 28-30 ℃; the time of the first expansion culture is 72-96 h, and the defoaming agent is supplemented on line in real time.
Further, in the step (3), the seed liquid inoculation amount of the first expansion culture is 10-15% of the volume of the culture medium; the formula of the fermentation medium is 5-12 g/L glucose, 3-5 g/L bacteriological peptone, 15-25 g/L soluble starch, 3-6 g/L yeast extract and CaCO 3 2～5g/L。
Further, in the step (3), the dissolved oxygen amount of the culture is 35-55%; the temperature of the culture is 28-30 ℃; the culture time is 96-132 h, and the defoamer is supplemented on line in real time.
Further, in the step (3), the amount of the fermentation medium in the fermenter is 70 to 75% of the volume of the fermenter, and the volume of the fermenter is 500 to 600L.
The identification and research method of the Streptomyces brick red Z1-26 is as follows:
observing the morphological characteristics of cells of the strain Z1-26;
(II) identifying by using a 16S rRNA gene;
(III) physical and chemical property detection of the streptomyces erythropolis Z1-26 antibacterial active substances;
(IV) detecting the colonization of the streptomyces erythropolis Z1-26 in the crucian body;
and fifthly, observing the influence of the streptomyces rubus Z1-26 serving as a feed additive on the growth and disease resistance of the crucian.
Compared with the prior art, the invention has the following beneficial effects:
(1) The strain is identified as a new strain of Streptomyces brick red (strain preservation number: CCTCC NO: M2021705; classification name: streptomyces brick red Z1-26, latin' S name: streptomyces lateritius Z-26), and has good antibacterial effect on pathogenic bacteria of various freshwater fishes such as aeromonas hydrophila, aeromonas vernii, edwardsiella tarda, and the like;
(2) The Streptomyces brick red mold Z1-26 can survive and colonize in fish bodies, has no harm to fish, and lays a foundation for the strain to exert stable disease prevention and growth promotion effects in fish culture;
(3) The Streptomyces brick red Z1-26 provided by the invention is used as a feed additive to be added into feed for feeding fishes, so that the growth rate of fish bodies can be promoted, the feed efficiency can be improved, the immunity of the fish bodies can be enhanced, and the resistance of the fish bodies to pathogenic bacteria can be enhanced;
(4) The method for preparing the microecological preparation from the streptomyces erythropolis Z1-26 through fermentation is simple and convenient, has low production cost, can effectively reduce the risks of drug residues, pathogenic bacteria drug resistance and the like caused by abuse of antibiotics, and has good application prospect.
Description of the preservation of microorganisms
Streptomyces erythropolis Z1-26 (Streptomyces lateritius Z1-26) of the invention are preserved in China center for type culture collection (CCTCC for short, address: university of Wuhan, china) at 2021, and strain preservation number is CCTCC NO: M2021705.
Drawings
FIG. 1 is a graph showing the bacteriostatic effect of Streptomyces brick red Z1-26 of the present invention on fish pathogenic bacteria.
FIG. 2 is a view showing morphological characteristics of Streptomyces erythropolis Z1-26 according to the present invention.
FIG. 3 is a phylogenetic tree diagram of the 16S rDNA sequence construction of Streptomyces lividans Z1-26 according to the invention.
FIG. 4 is a graph showing the analysis of the influence of the bacteriostatic active substance of Streptomyces erythropolis Z1-26 according to the present invention on temperature.
FIG. 5 is a graph showing the analysis of the influence of the bacteriostatic active substance of Streptomyces erythropolis Z1-26 according to the present invention and ultraviolet irradiation.
FIG. 6 is a graph showing the analysis of the effect of the bacteriostatic active substance of Streptomyces erythropolis Z1-26 according to the present invention on protease.
FIG. 7 is a graph showing the analysis of the influence of the bacteriostatic active substances and pH value of Streptomyces erythropolis Z1-26 according to the present invention.
FIG. 8 is a diagram of a small animal in vivo imaging system observing the colonization and protection of Streptomyces erythropolis Z1-26 marker strains of the present invention in vivo in crucian.
FIG. 9 is a graph showing the analysis result of the effect of the microecological preparation prepared by Streptomyces brick red mold Z1-26 on the expression of related gene MSTN inhibiting the growth of the muscle in the muscle of the crucian.
FIG. 10 is a graph showing the analysis result of the effect of the microecological preparation prepared by Streptomyces brick red mold Z1-26 on the IGF expression of the related genes for promoting muscle growth of crucian.
FIG. 11 is a graph showing the result of analysis of the effect of the microecological preparation prepared by Streptomyces brick red mold Z1-26 on the activity of the crucian serum acid phosphatase.
FIG. 12 is a graph showing the results of analysis of the effect of the microecological preparation prepared by Streptomyces brick red mold Z1-26 on the activity of the serum alkaline phosphatase of crucian.
FIG. 13 is a graph showing the results of analysis of the effect of the microecological preparation prepared from Streptomyces brick red Z1-26 on the enzyme activity of superoxide dismutase in the serum of Carassius auratus.
FIG. 14 is a graph showing the results of analysis of the effect of the microecological preparation prepared by Streptomyces brick red Z1-26 on the activity of the serum lysozyme of crucian.
FIG. 15 is a graph showing the result of analysis of the effect of the microecological preparation prepared from Streptomyces brick red Z1-26 on the expression of immune-related genes in the liver of crucian.
FIG. 16 is a graph showing the results of analysis of the effect of the microecological preparation prepared from Streptomyces brick red Z1-26 on the expression of immune-related genes in the kidney of crucian carp.
FIG. 17 is a graph showing the results of analysis of the effect of the microecological preparation prepared from Streptomyces brick red Z1-26 on the expression of immune-related genes in the spleen of crucian.
Detailed Description
The invention is further described below with reference to examples and figures.
The chemical reagents used in the examples of the present invention, unless otherwise specified, were all obtained by conventional commercial means.
Screening of Streptomyces erythropolis Z1-26 and determination test of antibacterial spectrum
Soil samples from the Hebei province area are fully and evenly mixed in sterile water, the mixture is coated on a Gao's plate after gradient dilution, the culture is carried out for 5 days at 30 ℃, the growth state of the strain is observed, actinomycete samples are picked up and inoculated on a new culture medium for purifying the picked strain, and bacterial activities of pathogenic bacteria of fish, such as Aeromonas hydrophila (Aeromonas hydrophila), aeromonas miltiorrhizae (Aeromonas sobria), aeromonas guinea (Aeromonas caviae), aeromonas veronii (Aeromonas veronii), abnormal Aeromonas hydrophila (Aeromonas allosaccharophila), edwardsiella tarda (Edwardsiella tarda), erwinia spp.), citrobacter freundii (Citrobacter freundii), shewanella mandshurica (Shewanella xiamenensis) and Shigella (Plesiomonas shigelloide) are detected by using an oxford cup method, and a strain with good bacterial inhibition effect on 10 pathogenic bacteria of fish is found and named as Z1-26.
Cell morphology characterization of Streptomyces erythropolis Z1-26
Bacterial colony of the strain Z1-26 on a Gaoshan first plate is concentric and annular, is dry, opaque and powdery, has tight combination with a culture medium and is not easy to pick up. The strain is gram positive. Under a scanning electron microscope, the mycelium of the Z1-26 strain was observed to develop, and the mycelium was thicker, more branched and shorter (see FIG. 2). As can be seen, the morphological characteristics exhibited by strains Z1-26 are consistent with the typical characteristics of actinomycete colonies and thalli.
16S rRNA Gene identification of Streptomyces lividans Z1-26
The strain Z1-26 was inoculated into a liquid high-temperature medium, cultured at 30℃and 120rpm for 4d, the cells were collected, genomic DNA of the strain Z1-26 was extracted using Ezup column type bacterial genomic DNA extraction kit (Shanghai Biotechnology Co., ltd.), and the 16S rRNA gene amplification primers were used.
27F：5′-AGAGTTTGATCCTGGCTCAG-3′；
1492R：5′-ACGGCTACCTTGTTACGACTT-3′。
The 16S rRNA gene sequence was amplified using the above primers, and the expected length of the sequence was about 1500bp.
PCR reaction System (20. Mu.L): sterile double distilled water, 14 μl;10 Xbuffer, 2. 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.
After the PCR product of the 16S rRNA gene is detected by 1.0% agarose gel electrophoresis, the amplified product is sent to Shanghai Bioengineering Co., ltd for sequencing.
The 16S rRNA gene sequence of strain Z1-26 was sequenced to a length of 1428bp, and the amplified sequencing results were analyzed by BLAST alignment at NCBI, and a phylogenetic tree of the strain was constructed by the adjacency method in software MEGA 7.0.26 (see FIG. 3). Closest to Streptomyces lateritius strain LMG 19372.
Physicochemical property analysis of Streptomyces erythropolis Z1-26 antibacterial active substance
(1) Thermal stability
The Streptomyces brick red Z1-26 strain is inoculated into a liquid AM6 culture medium, and cultured for 5 days at 30 ℃ and 120rpm, fermentation liquid is respectively taken and treated for 1 hour at the temperature of 30 ℃, 50 ℃, 70 ℃ and 90 ℃, aeromonas hydrophila is taken as a pathogenic indicator, and the oxford cup method is used for detecting the antibacterial activity of fermentation supernatant after different temperature treatments (see figure 4).
(2) Acid and alkali tolerance
Streptomyces brick red Z1-26 strain is inoculated into a liquid AM6 culture medium, and cultured for 5 days at 30 ℃ and 120rpm, fermentation liquor is respectively treated for 1h under the conditions of pH of 1, 3, 5, 7, 9, 11 and 13, the original pH is recovered, aeromonas hydrophila is used as a pathogenic indicator, and the antibacterial activity of fermentation supernatant after different pH treatments is detected by using an oxford cup method (see figure 7).
(3) Ultraviolet light resistance
The Streptomyces brick red Z1-26 strain is inoculated into a liquid AM6 culture medium, cultured for 5 days at 30 ℃ and at 120rpm, fermentation liquid is taken and irradiated under an ultraviolet lamp for 10min, 20min, 30min, 40min, 50min and 60min respectively, aeromonas hydrophila is taken as a pathogenic indicator, and the antibacterial activity of fermentation supernatant after being treated by different ultraviolet irradiation times is detected by using an oxford cup method (see figure 5).
(4) Protease tolerance
Streptomyces brick red Z1-26 strain is inoculated into a liquid AM6 culture medium, cultured for 5 days at 30 ℃ and 120rpm, treated with proteinase K and trypsin at 37 ℃ for 1 hour respectively, and the antibacterial activity of fermentation supernatant after different proteinase treatments is detected by using an oxford cup method by taking aeromonas hydrophila as a pathogenic indicator (see figure 6).
Preparation of Streptomyces erythropolis Z1-26 microecological preparation
(1) Inoculation and activation: transferring the Streptomyces brick red Z1-26 deposited slant strain to an activating slant culture medium for activation to obtain activated seed liquid; the used activated slant culture medium is TSB liquid culture medium, and the conditions of the activated culture are as follows: the temperature is 30 ℃, the liquid loading amount is 15% of the volume of the container, and the rotating speed of the shaking table is 200rpm;
(2) First expansion culture: inoculating the activated seed liquid obtained in the step (1) into a fermentation tank filled with a fermentation medium according to an inoculum size of 1% for primary expansion culture to obtain seed liquid for primary expansion 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 and CaCO 3 5g/L; the culture conditions are as follows: the whole culture process is monitored on line, the dissolved oxygen concentration is 45%, the temperature is 30 ℃, the culture is carried out for 84 hours, and the defoamer is added on line in real time;
(3) Second expansion culture: inoculating the seed solution obtained in the step (2) for the first time into a 500L fermentation tank filled with 70% of fermentation medium according to 10% of inoculation amount to obtain an enlarged culture solution; the fermentation medium formula is the same as the fermentation medium formula in the step (2); the culture conditions are as follows: the dissolved oxygen concentration is 45%, the temperature is 30 ℃, the culture is carried out for 120 hours, and the defoamer is added on line in real time;
(4) Concentrating and collecting: collecting the enlarged culture solution obtained in the step (3), and concentrating to obtain the microecological liquid microbial inoculum; or spray drying the microecological liquid microbial inoculum to obtain the microecological solid preparation.
Application of Streptomyces erythropolis Z1-26 in fish culture
a. Colonization and protective force of Streptomyces brick red Z1-26 in crucian body
Healthy crucian carps with the same size are selected and randomly divided into 1 control group and 1 experiment group, 10 crucian carps are arranged in each group, and repetition is arranged. Feeding 7d before experiment, domesticating to adapt the crucian to experimental environment, wherein aeration water is used for 48h in the experiment, the water temperature is controlled at about 25 ℃, the crucian of the control group is fed with basic feed, and the crucian of the experimental group is fed with basic feed with concentration of 1×10 9 cfu/g green fluorescent gene marker strain Z1-26 EGFP The feed amount was 2% of the body weight, and the animal was fed 2 times daily, and the in vivo imaging observation was performed every 2d (see fig. 8), and the experiment was continued for 14d. Two weeks later, an toxicity attack test was performed by intraperitoneal injection, and 200. Mu.L of 1X 10 concentration was injected into both the control group and the experimental group 6 AhX040 of cfu/mL mCherry The bacterial liquid was subjected to in vivo imaging observation of small animals at 1d intervals (see FIG. 8), and the detection was continued for 7d.
Experimental results show that Z1-26 is fed EGFP After the strain 1d, a green fluorescence signal is detected at the gill part of the crucian, and along with the extension of feeding time, the abdomen of the crucian starts to detect green fluorescence, Z1-26 EGFP The strain is gradually increased in the crucian body, and the green fluorescent signal is gradually enhanced. AhX040 mCherry Red fluorescence is detected in 1d after toxicity attack, and along with the extension of time, the red fluorescence signal in the crucian body fed with common feed is gradually enhanced, and Z1-26 is added in the feeding process EGFP The red fluorescent signal in the crucian body of the feed of the strain is not obviously enhanced. The results show that the strain Z1-26 of the invention EGFP Can survive and colonise in the crucian, and can inhibit the strain AhX040 in the crucian mCherry Against infection by aeromonas hydrophila.
b. Influence of Streptomyces brick red Z1-26 on crucian growth and disease resistance
Healthy crucian carps with the same size are selected and randomly divided into 1 control group and 2 experimental groups, 27 tails are arranged in each group, and repetition is arranged. Feeding 7d before experiment, domesticating to adapt crucian to experimental environment, and making in experimentThe water with aeration for 48h and water temperature controlled at about 25 ℃ is used for feeding common feed to crucian of control group in experiment, and the crucian of 2 test groups are respectively fed with feed with concentration of 1×10 8 cfu/g and 1X 10 9 The feed of the Z1-26 strain with cfu/g, the feeding amount of the feed is about 2% of the body weight, the experiment lasts for 30d 2 times a day.
(1) Influence of Streptomyces brick red Z1-26 on crucian growth performance
The experimental crucian was weighed at experiments 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× (test end crucian mantissa)/(test start crucian mantissa) (see table 1). Subsequent fluorescent quantitative PCR analysis of the expression level of muscle growth-related factor, the expression level of the related gene MSTN inhibiting muscle growth in muscle was significantly reduced (see FIG. 9), and the expression level of the related gene IGF promoting muscle growth was significantly increased (see FIG. 10)
Table 1 crucian growth performance parameters
Note that: the different superscripts of the same row represent significant differences (P < 0.05)
(2) Influence of Streptomyces brick red Z1-26 on serum nonspecific immunity index of crucian
The strain Z1-26 was added to feed for feeding crucian 30d, and the activity of alkaline phosphatase (AKP), acid phosphatase (ACP), lysozyme (LZM) and superoxide dismutase (SOD) in the crucian serum was analyzed (see FIGS. 11-14). The research shows that after the feed containing the Z1-26 strain is fed, the AKP, SOD, LZM enzyme activity in the crucian serum is obviously improved, and the ACP activity is higher than that of a control group, but no obvious difference exists.
(3) Influence of Streptomyces brick red Z1-26 on immune related gene expression in crucian tissue
The Z1-26 strain is added into feed for feeding crucian 30d, and the expression level of immune related genes in the liver, kidney and spleen of the crucian is analyzed (see fig. 15-17). The research shows that after the feed containing the Z1-26 strain is fed, igM expression level is obviously improved, C3 expression level is obviously improved in liver and spleen, LZM expression level is obviously improved in kidney, and expression level of inflammatory factor IL-1 beta is obviously reduced, thereby improving immunity.
(4) Protective test of Streptomyces brick red Z1-26 on crucian
After adding the strain Z1-26 into the feed to feed crucian for 30d, after using aeromonas hydrophila to attack toxin, the living state and death condition of each group of crucian in 7d are continuously observed and recorded (see table 2). The study shows that the survival rate of the crucian carp of the control group fed with the basic feed is 10% at 7d after the toxicity is removed, and the addition amount of the Z1-26 strain is 1 multiplied by 10 8 cfu/g and 1X 10 9 The survival rate of crucian in the experimental group of cfu/g group is significantly higher than that of the control group, which are respectively 50% and 60%.
TABLE 2 protective experiments of Z1-26 strains on Carassius auratus
Sequence listing
<110> Hunan university of teachers and students
<120> Streptomyces brick red, its microecological preparation and preparation method thereof
<130> 2021.07.08
<160> 1
<170> SIPOSequenceListing 1.0
<210> 2
<211> 1428
<212> DNA
<213> Streptomyces erythropolis Z1-26 (2 Ambystoma laterale x Ambystoma jeffersonianum)
<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 (11)
1. Streptomyces erythropolis Z1-26%Streptomyces lateritiusZ1-26) is preserved in China Center for Type Culture Collection (CCTCC) with the strain preservation number of M2021705.
2. A microecological preparation prepared by fermenting Streptomyces erythropolis Z1-26 with a strain of CCTCC NO: M2021705 as set forth 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 probiotic according to claim 2 or 3, characterized in that it is used in aquaculture.
5. The probiotic according to claim 4, characterized in that the aquaculture animal is a crucian.
6. A method for preparing a microecological formulation according to any one of claims 2 to 5, comprising the steps of:
(1) Inoculation and activation: transferring the Streptomyces brick red Z1-26 deposited slant strain to an activating slant culture medium for activation to obtain seed liquid;
(2) First expansion culture: inoculating the seed liquid obtained in the step (1) into a fermentation tank filled with a fermentation medium for primary expansion culture to obtain a culture liquid for primary expansion culture;
(3) Second expansion culture: inoculating the seed liquid obtained in the step (2) for the first time to a fermentation tank filled with a fermentation medium, and culturing again to obtain an enlarged culture liquid;
(4) Concentrating and collecting: collecting the enlarged culture solution obtained in the step (3), and concentrating to obtain a microecological liquid microbial inoculum; or spray drying the microecological liquid microbial inoculum to obtain the microecological solid preparation.
7. The method of claim 6, wherein in step (1), the activated slant medium is TSB liquid 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.
8. The method of claim 6, wherein in the step (2), the seed liquid is inoculated into the fermenter in an amount of 1 to 2% by volume of the medium; the formula of the fermentation medium for the first expansion culture is 5-12 g/L glucose, 3-5 g/L bacteriological peptone, 15-25 g/L soluble starch, 3-6 g/L yeast extract and CaCO 3 2~5 g/L。
9. The method for preparing a microecological preparation according to any one of claims 6 to 8, wherein in step (2), the dissolved oxygen amount of the first expansion culture is 35 to 55%; the temperature of the first expansion culture is 28-30 ℃; the time of the first expansion culture is 72-96 h.
10. The method of preparing a microbial ecological agent according to any one of claims 6 to 8, wherein in step (3), the seed liquid inoculum size of the first expansion culture is 10 to 15% of the volume of the culture medium; the formula of the fermentation medium comprises 5-12 g/L glucose, 3-5 g/L bacteriological peptone, 15-25 g/L soluble starch, 3-6 g/L yeast extract and CaCO 3 2-5 g/L; the oxygen dissolving amount of the culture is 35-55%; the temperature of the culture is 28-30 ℃; the culture time is 96-132 h.
11. The method for preparing a microecological preparation according to claim 9, wherein the steps of(3) The seed liquid inoculation amount of the first expansion culture is 10-15% of the volume of the culture medium; the formula of the fermentation medium comprises 5-12 g/L glucose, 3-5 g/L bacteriological peptone, 15-25 g/L soluble starch, 3-6 g/L yeast extract and CaCO 3 2-5 g/L; the oxygen dissolving amount of the culture is 35-55%; the temperature of the culture is 28-30 ℃; the culture time is 96-132 h.
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