CN115975846A - Streptomyces saadriamycin, microecological preparation thereof and preparation method thereof - Google Patents
Streptomyces saadriamycin, microecological preparation thereof and preparation method thereof Download PDFInfo
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Abstract
Sha AmeiStreptomyces saxadoxus, which is preserved in China center for type culture Collection at 23.6.6.2022 with the preservation number of CCTCC M2022952 and is classified and named as Sha A streptomycin S1-6, and the Latin literature name is Sha A streptomycin S1-6, and a microecological preparation and a preparation method thereofStreptomyces zaomyceticusAnd S1-6. The invention also comprises application of the streptomyces sakadiae, a microecological preparation prepared from the streptomyces sakadiae and a preparation method of the microecological preparation. The bacterial strain has good bacteriostatic effect on a plurality of freshwater fish pathogenic bacteria such as aeromonas hydrophila, aeromonas veronii, edwardsiella tarda and the like; can survive and colonize in the fish body, and has no harm to the fish; the feed additive can be added into feed to feed fish, and has effects of enhancing fish immunity and enhancing resistance of fish to pathogenic bacteria.
Description
Technical Field
The invention relates to streptomyces sapadriamycin, and particularly relates to streptomyces sapadriamycin, a microecological preparation of the streptomyces sapadriamycin and a preparation method of the microecological preparation.
Background
In recent years, the development trend of the aquaculture industry in China is rising, the scale of the aquaculture industry is continuously enlarged, and the income is continuously increased, but due to unscientific and reasonable behaviors of abusing fishery drugs, feeds and the like, various diseases occur in the aquaculture, particularly fish culture, the infection is rapid and cannot be recovered, so that the income of the aquaculture industry is sharply reduced.
For the prevention and treatment of fish bacterial diseases, at present, antibiotic therapy is mainly used, antibiotics can not only treat diseases, but also promote the growth of fishes, but once the dosage of the antibiotics is excessive, a lot of side effects can be generated, especially, the blind increase of the dosage of the antibiotics in the breeding process of grass carps can cause pathogenic bacteria in grass carps to enhance the drug resistance to the antibiotics, and even thoroughly immunize the grass carps against the antibiotics. The occurrence of drug-resistant pathogenic bacteria not only increases the breeding cost, but also increases the difficulty of preventing and treating aquatic diseases, and once the drug-resistant pathogenic bacteria flow into the market, the health and safety of human beings can be threatened. The antibiotic drugs are likely to remain in aquatic products during the cultivation process, and the antibiotic drugs can be accumulated in the bodies of consumers after the consumers eat the aquatic products for a long time, so that the health of the human bodies is harmed.
The microecological preparation is the most promising and sustainable preparation for preventing and treating the bacterial diseases of the grass carps at present, and compared with antibiotics and other preparations, the microecological preparation has the characteristics of no toxic or side effect, no drug residue, low manufacturing cost, good prevention and treatment effect, environmental friendliness, no environmental pollution and the like.
However, the existing microecologics which can achieve better effects in aquaculture are few in types, narrow in application range and not ideal in prevention and treatment effects, and further intensive research is needed.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects in the prior art and provide a streptomyces sapadriamycin, a microecological preparation thereof and a preparation method thereof; the Sha A streptomycin and the microecological preparation thereof have good antagonistic effect on pathogenic bacteria of various fishes and can resist bacterial disease infection of various fishes.
The technical scheme adopted by the invention for solving the technical problems is as follows: a streptomyces saradriamycin is classified and named as Sha A streptomyces saramygdalinus S1-6, and the Latin literature name isStreptomyces zaomyceticusS1-6, deposited in China center for type culture Collection (CCTCC for short) at 23.6.2022 with the collection number of CCTCC M2022952.
The streptomyces saxadoxorubicin S1-6 is screened from soil in Qinhuangjima city, hebei. The 16S rRNA sequence of the streptomyces saxadoxorubicin S1-6 is shown in a sequence table SEQ ID No. 1.
The invention relates to application of streptomyces saadriamycin serving as antagonistic bacteria of fish pathogenic bacteria.
The streptomyces saadriamycin of the invention can be prepared into a microecological preparation for use.
Preferably, the probiotic is a liquid or solid formulation.
Preferably, the probiotic is used in aquaculture.
Preferably, the aquaculture animal is a freshwater fish; more preferably crucian, grass carp or carp.
The preparation method of the microecological preparation comprises the following steps:
(1) Inoculating and activating: transferring Sha A streptomycin S1-6 slant strain to shake flask seed activation culture medium for activation to obtain seed liquid;
(2) First amplification culture: inoculating the seed solution obtained in the step (1) into a fermentation tank filled with a fermentation medium for amplification culture to obtain an amplification culture solution I;
(3) And (3) second amplification culture: inoculating the expanded culture solution I obtained in the step (2) into a fermentation tank filled with a fermentation culture medium, and culturing again to obtain an expanded culture solution II;
(4) Concentrating and collecting: collecting the enlarged culture solution II obtained in the step (3), and concentrating to obtain a liquid preparation;
the solid preparation is obtained by filtering the liquid preparation with ceramic membrane, collecting filtrate, and spray drying the filtrate.
Preferably, in step (1), the shake flask seed activation medium is Am6 liquid medium.
Preferably, in step (1), the activation conditions are: the temperature is 28-30 ℃, and the rotating speed of the shaking table is 100-150 rpm/min.
Preferably, in the step (2), the inoculation amount of the seed solution into the fermenter is 1 to 2% of the volume of the culture medium.
Preferably, in step (2), the solvent of the fermentation medium used in the first amplification culture is water, and the formula is as follows: 1-3 g/L of soluble starch, 0.5-2 g/L of glucose and CaCO 3 0.2 to 0.5 g/L, 0.1 to 0.5 g/L of yeast extract and 0.2 to 0.6 g/L of bacteriological peptone.
Preferably, in the step (2), the dissolved oxygen amount in the first scale-up culture is 40 to 50%.
Preferably, in step (2), the temperature of the first amplification culture is 28-30 ℃.
Preferably, in the step (2), the time for the first amplification culture is 48 to 72 h.
Preferably, in step (2), a fermentation antifoaming agent is used during the cultivation to avoid foaming.
Preferably, in step (3), the inoculation amount of the expanding culture solution I is 10 to 15% of the volume of the culture medium.
Preferably, in step (3), the solvent of the fermentation medium is water, and the formulation 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。
Preferably, in the step (3), the dissolved oxygen amount of the culture is 40 to 50%.
Preferably, in step (3), the temperature of the culture is 28 to 30 ℃.
Preferably, in the step (3), the culturing time is 72 to 96 h.
Preferably, in step (3), a fermentation antifoaming agent is used during the cultivation to avoid foaming.
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 obvious effect of inhibiting pathogenic bacteria of freshwater fishes, can promote the growth of the fishes and improve the immunity of the fishes to the pathogenic bacteria. Through the microecological preparation, the Sha A streptomycin S1-6 can be concisely and conveniently added into the environment needing to be used, such as the microecological preparation is mixed into feed or added into a water body environment.
Compared with the prior art, the invention has the following beneficial effects:
(1) The bacterial strain has obvious bacteriostatic effect on a plurality of freshwater fish pathogenic bacteria such as aeromonas hydrophila, aeromonas veronii, edwardsiella tarda and the like, wherein the bacteriostatic effect on abnormal aeromonas saccharivorans is best, and the diameter of a bacteriostatic zone reaches 23.70 mm;
(2) The streptomyces saxadoxorubicin S1-6 can survive and colonize in a fish body, and does not harm the fish;
(3) The streptomyces saxadoxorubicin S1-6 is added into feed as a feed additive to feed fishes, so that the immunity of the fish bodies can be enhanced, and the resistance of the fish bodies to pathogenic bacteria can be enhanced; after being fed to grass carp as feed additive, the immune related gene in grass carpTNF-αIs significantly up-regulated in the spleen;C3is significantly up-regulated in the expression level in the kidney and spleen;IL-8the expression level of (b) is significantly up-regulated in kidney, spleen and intestinal tract;LSZthe expression level of the polypeptide is obviously increased in the liver, the kidney and the spleen;Keap1the expression level of (a) is significantly up-regulated in the liver and kidney;
(4) The microecological preparation prepared from Sha A streptomycin S1-6 or streptomycin saxaxomycete S1-6 is mixed into feed for feeding fish, and the growth of the fish can be remarkably promoted.
(5) The method for preparing the micro-ecological preparation from Sha A streptomycin S1-6 by fermentation is simple and convenient, the production cost is low, and meanwhile, the risks of drug residues, pathogenic bacteria drug resistance and the like caused by antibiotic abuse can be effectively reduced.
Description of the microbial deposits
The Streptomyces saxadoxorubicin of the invention S1-6 (S)Streptomyces zaomyceticus S1-6) stored in the chinese culture collection center (CCTCC, address: wuhan, wuhan university, china), the preservation number of the strain is CCTCC M2022952.
Drawings
FIG. 1 is a diagram of the bacteriostatic effect of streptomyces saxadoxorubicin S1-6 on fish pathogenic bacteria.
FIG. 2 is an observation picture of the morphological characteristics of Streptomyces saxadoxus S1-6 of the present invention.
FIG. 3 is a phylogenetic tree diagram constructed from the 16S rDNA sequence of Streptomyces saxadoxus S1-6 of the present invention.
FIG. 4 is an analysis diagram of the effect of the bacteriostatic active substance S1-6 of Streptomyces saxadoxus of the present invention on temperature.
FIG. 5 is an analysis diagram of the influence of bacteriostatic active substances of streptomyces saxadoxorubicin S1-6 and the pH value.
FIG. 6 is an analysis chart of the influence of the bacteriostatic active substance S1-6 of streptomyces saxadoxorubicin and ultraviolet irradiation.
FIG. 7 is an analysis diagram of the effect of the bacteriostatic active substances of Streptomyces saxadoxus S1-6 of the present invention on proteases.
FIG. 8 is a graph of a small animal in vivo imaging system observing the colonization profile of the Streptomyces saxadoxus S3 marker strain of the present invention in grass carp.
FIG. 9 is a diagram of a small animal in vivo imaging system observing the protection of grass carp by Streptomyces saxadoxorubicin S1-6 of the present invention.
FIG. 10 is a diagram showing the results of analysis of the effect of the microecological preparation prepared from Streptomyces saxadoxus S1-6 of the present invention on the activity of serum acid phosphatase in grass carp.
FIG. 11 is a diagram showing the results of analysis of the effect of the microecological preparation prepared from Streptomyces saxadoxus S1-6 of the present invention on the activity of grass carp serum alkaline phosphatase.
FIG. 12 is a diagram of the results of the analysis of the effect of the microecological preparation prepared from Streptomyces saxadoxorubicin S1-6 on the enzymatic activity of grass carp serum superoxide dismutase.
FIG. 13 is a diagram showing the results of analysis of the effect of the microecologics prepared from Streptomyces saxadoxus S1-6 of the present invention on the lysozyme activity of grass carp serum lysozyme.
FIG. 14 is a diagram showing the results of analysis of the effect of the microecologics prepared from Streptomyces saxadoxorubicin S1-6 on the enzymatic activity of serum catalase in grass carp.
FIG. 15 is a diagram showing the results of analysis of the effect of the microecological preparation prepared by Streptomyces saxadoxorubicin S1-6 on the expression of immune-related genes in the liver of grass carp.
FIG. 16 is a diagram showing the analysis result of the influence of the microecological preparation prepared from Streptomyces saxadoxorubicin S1-6 on the expression of immune-related genes in the kidney of grass carp.
FIG. 17 is a graph showing the results of analysis of the effect of the microecologics prepared from Streptomyces saxadoxorubicin S1-6 on the expression of immune-related genes in the spleen of grass carp.
FIG. 18 is a diagram showing the results of analysis of the effect of the microecologics prepared from Streptomyces saxadoxus S1-6 of the present invention on the expression of immune-related genes in the intestinal tract of grass carp.
Detailed Description
The invention is further illustrated by the following examples and figures.
The reagents used in the examples of the present invention, unless otherwise specified, were obtained from conventional commercial sources.
Screening of Sha A mycin streptomyces S1-6 and determination test of bacteriostasis spectrum
Mixing soil sample from Qinhuang island city, hebei in sterile water, gradient diluting, spreading on Gao's plate, culturing at 30 deg.C for 5 days, observing growth state of strain, selecting actinomycetes sample, inoculating to new culture medium, purifying, and culturing with fish pathogenic bacteria Aeromonas hydrophila (A)Aeromonas hydrophila) Aeromonas sobria: (Aeromonas sobria) Aeromonas caviae (A. Mey.), (B. Mary.), (Aeromonas caviae) Aeromonas veronii (A), (B) and (C)Aeromonas veronii) Aeromonas anomala: (A) A. ParadoxusAeromonas allosaccharophila) Slow and slow Edwardsiella (A), (B) and (C)Edwardsiella tarda) Erwins and Wen bacteria (A), (B)Erwinia spp.) Shewanella xiamenensis: (Xiamensis) ((Xiamensis))Shewanella xiamenensis) Plesiomonas shigelloides (A), (B), (C)Plesiomonas shigelloide) As an indicator bacterium, the bacteriostatic activity of the fermentation supernatant on the pathogenic bacteria is detected by using a perforation method (see figure 1), and the inventor finds that the screened streptomyces saxadoxorubicin has a good bacteriostatic effect on the 9 fish pathogenic bacteria, the diameter of a bacteriostatic circle is not less than 14 mm, the bacteriostatic effect on abnormal aeromonas saccharophila is optimal, and the diameter of the bacteriostatic circle reaches 23.70 mm. The streptomyces saxadoxorubicin is presumed to be a novel streptomyces saxadoxorubicin strain and is named as Sha A streptomyces S1-6.
Cell morphology characteristics of (II) Sha A streptomyces S1-6
Bacterial colony spores of the strain S1-6 on a Gao' S first plate are in an opaque state and are white, the edges of the spores are neat, the particles are scattered and distributed, the bacterial colony with rich spore growth is tightly combined with a culture medium, and the bacterial colony is not easy to pick up. The bacterial strain is gram-positive bacteria. Under the scanning electron microscope, it was observed that S1-6 mycelia of the cells were developed, elongated, developed, and bent and spirally wound, and also branched (see FIG. 2). As can be seen, the morphological characteristics exhibited by strain S1-6 correspond to those typical of actinomycete colonies and thalli.
(III) identification of 16S rRNA Gene of Sha A Mycomycin Streptomyces S1-6
The S1-6 strain was inoculated into a liquid high-grade medium, cultured at 30 ℃ for 4 days at 120 rpm, the cells were collected, the genomic DNA of the strain S1-6 was extracted using an Ezup column type bacterial genomic DNA extraction kit (purchased from Shanghai Biotechnology, ltd.), and a 16S rRNA gene amplification primer was used.
27F：5′-AGAGTTTGATCCTGGCTCAG-3′；
1492R：5′-ACGGCTACCTTGTTACGACTT-3′。
The 16S rRNA gene sequence was amplified with the primers described above, and the expected length of the sequence was about 1500 bp. Specific parameters used for PCR are shown in tables 1 and 2.
TABLE 1 PCR reaction System
TABLE 2 PCR amplification cycles
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 S1-6 is sequenced to show that the length is 1434 bp, and the amplification sequencing resultA phylogenetic tree of this strain was constructed in the software MEGA X by using the adjacency method by BLAST alignment analysis at NCBI (see FIG. 3). AndStreptomyces zaomyceticus strain QMA47 is closest.
Physicochemical property analysis of (IV) Sha A mycin streptomyces S1-6 bacteriostatic active substance
(1) Thermal stability
Inoculating the strain S1-6 into an Am6 culture medium, carrying out shaking culture at 28 ℃ and 120 rpm for 72 hours, centrifuging at 13000 rpm for 10 minutes, dividing the obtained supernatant into 4 groups, taking 1 ml of each group, respectively putting the groups into a water bath kettle at 20 ℃, 40 ℃, 60 ℃ and 75 ℃ for water bath treatment for 1 hour, recovering to room temperature, setting a control group (the fermentation supernatant is not treated), carrying out an antibacterial experiment by taking aeromonas hydrophila as an indicator bacterium, carrying out upright culture on 24 h at 30 ℃, and observing the antibacterial change of each group. The results show that the bacteriostatic activity of the strain S1-6 is almost the same after being treated at 20 ℃ as that of the control group (no treatment), while the bacteriostatic activity of the fermentation broth after being treated at 40 ℃, 60 ℃, 65 ℃ and 70 ℃ is slightly lower than that of the control group, which may be because the bacteriostatic substances generated by the strain S1-6 during the fermentation culture process are sensitive to high temperature, but generally, the strain S1-6 still has a good bacteriostatic effect after being treated at high temperature (see the test results in FIG. 4).
(2) Acid and base resistance
Inoculating the strain S1-6 into a liquid AM6 culture medium, culturing at 28 ℃ at 120 rpm for 72 hours, respectively taking fermentation liquor, respectively adjusting the pH of the fermentation liquor to 1, 3, 5, 7, 9 and 10, treating for 1 hour, then adjusting the pH of the fermentation supernatant to 7, setting a control group (the fermentation supernatant is not treated), carrying out an antibacterial experiment by taking aeromonas hydrophila as an indicator bacterium, culturing for 24 hours at 30 ℃, and observing the antibacterial change of each group. The results show that the bacteriostatic activity of the fermentation broth of the strain S1-6 is almost the same after the treatment of the group with pH =3 and 5 as compared with the control group (the original pH is 5.8), while the bacteriostatic activity of the fermentation broth after the treatment of the group with pH =1 is the strongest, and the diameter of the bacteriostatic zone reaches 26.50 mm, wherein the bacteriostatic activity of the fermentation broth after the treatment of the group with pH =7 is slightly higher than that of the control group, and when the pH is higher than 7, the bacteriostatic activity of the treated fermentation broth is reduced along with the increase of the pH value, but still can achieve a better bacteriostatic effect (see the test result in FIG. 5).
(3) Resistance to ultraviolet light
Inoculating the strain S1-6 into a liquid AM6 culture medium, culturing at 30 ℃ at 120 rpm for 72 hours, taking a fermentation liquid to irradiate under an ultraviolet lamp for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours and 6 hours respectively, performing a bacteriostasis experiment by taking aeromonas hydrophila as a pathogen indicator and taking the aeromonas hydrophila as an indicator, culturing at the temperature of 30 ℃ for 24 hours, and observing the bacteriostasis change of each group. The results show that the fermentation liquor of the strain S1-6 has almost no difference in bacteriostatic activity compared with the control group (no treatment) after being treated by 6 groups of 1 hour, 2 hours, 3 hours, 4 hours, 5 hours and 6 hours of ultraviolet irradiation, and show that bacteriostatic substances generated by the strain S1-6 in the growth and metabolism process are insensitive to ultraviolet irradiation and have strong ultraviolet stability (the test results are shown in figure 6).
(4) Protease resistance
Inoculating the strain S1-6 into a liquid AM6 culture medium, culturing at 30 ℃ at 120 rpm for 72 hours, treating at 37 ℃ for 1 hour by using protease K and trypsin respectively, performing an antibacterial experiment by using aeromonas hydrophila as an indicator bacterium, culturing at the temperature of 30 ℃ for 24 hours, and observing the antibacterial change of each group. The results show that after the fermentation liquor of the strain S1-6 is treated by the proteinase K56 ℃ water bath for 1 hour and treated by the trypsin 37 ℃ water bath for 1 hour, compared with the control group (no treatment), the bacteriostatic activity is slightly lower than that of the control group, but the bacteriostatic activity is not completely lost (the test results are shown in figure 7).
Therefore, in the research, the test and the determination of the physicochemical properties such as the thermal stability and the like of the fermentation liquor of the strain S1-6 are carried out, and the finding shows that the fermentation liquor of the strain S1-6 has stronger thermal stability and acid resistance, and the fermentation liquor of the strain S1-6 is not influenced by ultraviolet irradiation, so the strain S1-6 meets the requirement of good stability of probiotics.
(V) preparation of Sha A streptomycin S1-6 microecological preparation
(1) Inoculating and activating: transferring Sha A streptomycin S1-6 slant strain to shake flask seed activation culture medium for activation to obtain seed liquid, wherein the seed activation culture medium is Am6 liquid culture medium; the activation culture conditions are as follows: the temperature is 28 ℃, and the rotating speed of the shaking table is 120 r/min;
(2) First amplification culture: inoculating the seed solution obtained in the step (1) into a fermentation tank filled with a fermentation culture medium for amplification culture to obtain a culture solution for the first amplification culture, wherein the inoculation amount of the seed solution inoculated into the fermentation tank is 2% of the volume of the culture medium, and the formula of the culture medium comprises 2 g/L of soluble starch, 1 g/L of glucose and CaCO 3 0.2 g/L, 0.2 g/L yeast extract, 0.5 g/L bacteriological peptone and 50% dissolved oxygen; the temperature of the first amplification culture is 28 ℃; the time of the first amplification culture is 56 hours;
(3) And (3) performing 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 fermentation product, wherein the inoculation amount of the seed liquid in the second amplification culture is 10% of the volume of the culture medium; the formula of the fermentation medium comprises 8 g/L of glucose, 4 g/L of bacteriological peptone, 20 g/L of soluble starch, 5 g/L of yeast extract and CaCO 3 3 g/l;
(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 filtering the microecological liquid microbial inoculum by using a ceramic membrane, collecting filtrate, and spray-drying the filtrate to obtain microecological microbial powder.
Application of (VI) Sha A streptomycin S1-6 in fish culture
a. 5363 colonizing and protecting force of Streptomyces Sha A mycin S1-6 in grass carp body
Selecting healthy grass carps with consistent sizes, randomly dividing the grass carps into 1 control group and 1 experimental group, wherein 10 grass carps are arranged in each group, and repeating the steps. Feeding grass carp for 7 days before experiment, acclimatizing to make grass carp adapt to experiment environment, aerating 48 hr water temperature controlled at about 25 deg.C, feeding grass carp of control group with basic feed, scraping appropriate amount of spore of strain S1-6EGFP with inoculating ring, inoculating to fermentation medium, culturing at 28 deg.C for 72 hr at 120 rpmCentrifugation was carried out at 12000 rpm for 10 minutes to obtain a pellet (belonging to a microecological preparation). The experiment is divided into two groups, wherein each group contains 35 grass carps with weight of about 30 g, mixed feed is obtained by mixing grass carps with bacteria, the grass carps are fed once a day, the feed weight fed each time is 2% of the initial weight of the grass carps, and the grass carps fed with normal feed are used as a control group and fed once a day. The grass carp was placed in a small animal In Vivo Imaging System (IVIS) for observation of fluorescence signals every 2 days (see fig. 8). After 14 days, 100 microliters of grass carp in the test group and the control group was injected at a concentration of 1X 10 6 Colony Forming Unit/milliliter (cfu/mL) A. H mCherry The strain is infected, the death condition and the fluorescent signal condition of the strain are observed every 1 day, and the observation period is 7 days (see figure 9).
In feeding strain S1-6 EGFP The fluorescence signal can be detected after 1 day, and as can be seen from the figure, the fluorescence signal is enhanced along with the prolonging of the feeding time, and after 13 days of continuous observation, the fluorescence is not weakened, and the growth condition of the grass carp is good, which indicates that the strain S1-6 EGFP Can be stably colonized, proliferated and distributed in gill part and intestinal tract of grass carp, and has no toxicity to grass carp. 13 days later with pathogen A. H mCherry Two groups of grass carps are infected, and A, h in the grass carps in the test group can be seen mCherry The fluorescence signal of (A) was significantly weaker than that of the control group, indicating that the strain S1-6 was present EGFP Can inhibit A, h in grass carp body mCherry The resistance of grass carp to pathogenic bacteria is improved (see fig. 8 and 9).
b. Effect of Sha A mycin streptomyces S1-6 on grass carp growth and disease resistance
Grass carp with the weight of about 32 g is selected, and 35 grass carp fishes are divided into one group. Firstly, aerating the water in the culture tank in advance, feeding the grass carp with normal feed for 7 days, and domesticating the grass carp. After the domestication is finished, the grass carps of the control group are continuously fed with normal feed, and the grass carps of the test group are respectively fed with the feed with the concentration of 1 × 10 7 Colony Forming units/gram (cfu/g) and 1X 10 9 cfu/g of S1-6 strain feed, after the experiment is carried out for 30 days, 5 grass carps in each group are randomly selected, and tail fin venous blood of each group is about 1 mlAnd precipitating at-4 deg.C overnight; collecting liver, kidney, spleen and intestinal tissue of test group and control group, and storing at-80 deg.C in refrigerator.
(1) Influence of Sha A streptomycin S1-6 on growth performance of grass carp
The test was carried out with grass carp weighed on day 1 and day 30 of the experiment, respectively. The Weight Gain Ratio (WGR), the feed Factor (FC) and the Survival Rate (SR) were calculated as follows: SR (%) =100% × (final number of grass carps surviving)/(number of grass carps surviving initially); WGR (%) =100% × (Wt-W0)/W0; FC = F/(Wt-W0) (see table 1).
TABLE 3 grass carp growth Performance parameters
Note: i: a control group; II:10 9 cfu/g；Ⅲ：10 7 cfu/g (different superscripts in the same column indicate significant differences, a,p > 0.05；b，p < 0.05）
(2) Sha A streptomyces S1-6 influence on nonspecific immunity index of grass carp serum
The S1-6 strain was added to feed and fed to grass carp for 30 days, and the serum of grass carp was analyzed for alkaline phosphatase (AKP), acid phosphatase (ACP), lysozyme (LZM), catalase (CAT) and superoxide dismutase (SOD) activities (see FIGS. 10-14). The research finds that after the grass carp is fed by the feed containing the S1-6 strain additive, the enzyme activity expressions of 5 enzymes such as grass carp catalase in a test group are all stronger than those in a control group, so that the activity of nonspecific immunity indexes such as SOD and AKP of the grass carp can be improved by feeding the strain S1-6, the immunity level of the grass carp body is improved, the grass carp can better adapt to the external environment, and the feed plays an important role in the immune defense of the grass carp body.
(3) Effect of Sha A mycin Streptomyces S1-6 on immune-related gene expression in grass carp tissues
The S1-6 strain is added into feed to feed grass carp for 30 days, and the expression level of immune related genes in liver, kidney, intestinal tract and spleen of grass carp is analyzed (see FIGS. 15-18). Research hairNow, in the test group, compared with the control groupTNF-αThe expression level of (a) is significantly up-regulated in the spleen;C3is significantly up-regulated in the expression level in the kidney and spleen;IL-8the expression level of (a) is significantly up-regulated in kidney, spleen and intestinal tract;LSZthe expression level of the polypeptide is obviously increased in the liver, the kidney and the spleen;Keap1is significantly up-regulated in the liver and kidney; the up-regulation amplitude is different along with the different proportion of the thalli in the feed. The results show that the antioxidant factors and the immune factors related to various tissues of the grass carp fed by the S1-6 group are improved to different degrees compared with those of a control group by feeding the strain S1-6, and the strain S1-6 can improve the specific immunity capability of the grass carp and improve the immunity of the grass carp.
(4) Sha A streptomyces S1-6 protection test for grass carp
After the S1-6 strain is added into feed and fed to grass carp for 30 days, and after using aeromonas hydrophila to counteract poison, the living state and death condition of each group of grass carp are continuously observed and recorded within 7 days (see table 2). The research shows that the survival rate of the grass carp in the control group fed with the basic feed is 0 at 5 days after challenge, and the addition amount of the S1-6 strain is 1 multiplied by 10 7 cfu/g and 1X 10 9 The grass carp survival rate of the experimental group of cfu/g group is obviously higher than that of the control group, and is respectively 60% and 80%.
TABLE 2 protective experiment of grass carp with S1-6 strains
Claims (10)
1. A Sha A streptomycin strain is characterized in that the strain is classified and named as Sha A streptomycin S1-6 (Streptomyces zaomyceticusS1-6) is preserved in China Center for Type Culture Collection (CCTCC) at 23/6 in 2022 with the preservation number of CCTCC M2022952.
2. Use of streptomyces saxadoxorubicin as claimed in claim 1 as a fish pathogenic bacteria antagonist.
3. A microecological formulation prepared from Streptomyces saxadoxus according to claim 1.
4. The microecological formulation according to claim 3, which is a liquid formulation or a solid formulation; can be used for aquaculture.
5. The microecological formulation according to claim 4, wherein the aquaculture animal is a freshwater fish.
6. A method of making a probiotic according to any of claims 3~5 comprising the steps of:
(1) Inoculating and activating: transferring Sha A streptomycin S1-6 slant strain to shake flask seed activation culture medium for activation to obtain seed liquid;
(2) First amplification culture: inoculating the seed solution obtained in the step (1) into a fermentation tank filled with a fermentation medium for amplification culture to obtain an amplification culture solution I;
(3) And (3) performing second amplification culture: inoculating the expanded culture solution I obtained in the step (2) into a fermentation tank filled with a fermentation culture medium, and culturing again to obtain an expanded culture solution II;
(4) Concentrating and collecting: collecting the enlarged culture solution II obtained in the step (3), and concentrating to obtain a liquid preparation;
the solid preparation is obtained by filtering the liquid preparation with ceramic membrane, collecting filtrate, and spray drying the filtrate.
7. The method for preparing a micro-ecological preparation according to claim 6, wherein in the step (1), the shake flask seed activation medium is an Am6 liquid medium; the activation conditions are as follows: the temperature is 28-30 ℃, and the rotating speed of the shaking table is 100-150 rpm/min.
8. The method for preparing a microecological formulation according to claim 6 or 7,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 solvent of the fermentation medium used for the first amplification culture is water, and the formula is as follows: 1-3 g/L of soluble starch, 0.5-2 g/L of glucose and CaCO 3 0.2 to 0.5 g/L, 0.1 to 0.5 g/L of yeast extract and 0.2 to 0.6 g/L of bacteriological peptone.
9. The method for preparing a microecological preparation according to any one of claims 6~8, wherein in step (2), the dissolved oxygen of the first scale-up culture is from 40% to 50%; the temperature of the first amplification culture is 28-30 ℃; the time of the first amplification culture is 48-72 h; during the culture process, fermentation antifoaming agent is used to avoid foaming.
10. The method for preparing a microecological agent according to any one of claims 6~9, wherein in step (3), the amount of inoculation of expanded culture medium I is 10-15% of the volume of the culture medium; the solvent of the fermentation medium is water, and the formula is as follows: 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 to 5 g/L; the dissolved oxygen of the culture is 40 to 50 percent; the temperature of the culture is 28-30 ℃; the culture time is 72-96 h; during the culture process, a fermentation antifoaming agent is used to avoid foaming.
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| CN108004183A (en) * | 2018-01-04 | 2018-05-08 | 项鹏 | One plant of zaomycin streptomycete for having the anti-soybean cyst nematode Heterodera glycines of inducing soybean and application |
| CN110093295A (en) * | 2019-05-15 | 2019-08-06 | 湖南师范大学 | The Huang Sansu streptomycete of one plant of anti-fish-pathogenic bacteria and its application |
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| CN113621533A (en) * | 2021-07-09 | 2021-11-09 | 湖南师范大学 | Streptomyces rubiginosus Z1-26, microecological preparation and preparation method thereof |
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| CN108004183A (en) * | 2018-01-04 | 2018-05-08 | 项鹏 | One plant of zaomycin streptomycete for having the anti-soybean cyst nematode Heterodera glycines of inducing soybean and application |
| CN110093295A (en) * | 2019-05-15 | 2019-08-06 | 湖南师范大学 | The Huang Sansu streptomycete of one plant of anti-fish-pathogenic bacteria and its application |
| CN110331103A (en) * | 2019-06-04 | 2019-10-15 | 湖南师范大学 | One plant of molten algae streptomycete N1-32, its probiotics and preparation method thereof |
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