CN111849835B - Brown bacillus strain and screening method and application thereof - Google Patents
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Abstract
The invention relates to a brown bacillus strain, and also relates to a screening method and application of the brown bacillus strain, wherein the brown bacillus strain is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with the preservation number of: the strain of the brown bacillus in the invention can effectively inhibit the growth of vibrio, especially inhibit vibrio parahaemolyticus and vibrio harveyi, can obviously reduce the mortality rate of artificially infected vibrio to prawns, and can obviously improve the survival rate of the prawns when being applied to prawn culture. According to the brown characteristic of brown bacteria on 2216E agar medium and 2216E liquid medium, target bacterial colony can be accurately positioned, and further the brown bacteria strain with disease resistance potential can be rapidly screened out through pathogenic bacteria inhibition capability test.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a brown bacillus strain and a screening method and application thereof.
Background
Shrimp culture is one of the most representative industries in the aquaculture industry in China. In recent years, infectious diseases are becoming popular and increasingly severe in shrimp culture, and huge economic losses are brought to shrimp culture industry, so that the healthy development of the industry is severely restricted. In the traditional cultivation mode, the application of antibiotics is taken as a main method for preventing and controlling the occurrence of prawn diseases, but the abuse of antibiotics not only can damage the balance of a cultivation ecological system, but also can cause the problems of environmental pollution, food safety and the like. The probiotics serving as one of main substitutes of antibiotics has the advantages of no toxicity, no side effect, no residue and the like, and is more and more favored in preventing and controlling the shrimp culture diseases. Although the commercial aquatic probiotics are various at present, most of them are separated from terrestrial environment, the effect of these strains in aquaculture is not obvious, and it is difficult to support sustainable development of intensive prawn culture, so development of novel aquatic probiotics is needed.
The prawn intestinal canal is an important resource for screening the probiotics of the prawn, and the probiotics screened from the prawn intestinal canal can better adapt to the intestinal canal environment, thereby ensuring the performance of the probiotics. The rhodobacter family is the intestinal core and dominant group of each growth stage of Litopenaeus vannamei, and a plurality of bacteria of the family produce bioactive substances such as indole derivatives, indigoid, tryptanthrin and tropodithietic acid (TDA) for inhibiting pathogenic bacteria. Among them, TDA is an antibiotic secreted by Phaeobacillus (Phaeobacillus) and the like to kill pathogenic bacteria by destroying the proton motive force of the bacterial cell wall, and has been confirmed to inhibit the growth of various aquatic animal conditional pathogenic bacteria such as Vibrio anguillarum (V. Anguilarum), vibrio parahaemolyticus (V. Parahaeolyticus), vibrio harveyi (V. Harveyi) and the like. Several studies have demonstrated that bacteria of the genus brown bacillus have a great probiotic potential in the disease resistance of the breeding of cod, abalone and litopenaeus vannamei. Therefore, the separation of the brown bacillus strain from the intestinal canal of the prawn has important significance for preventing and controlling diseases of prawn culture.
Disclosure of Invention
The first technical problem to be solved by the present invention is to provide a strain of brown bacillus having an inhibitory effect on the activity of vibrio, especially vibrio parahaemolyticus and vibrio harveyi, against the prior art.
The second technical problem to be solved by the invention is to provide a screening method of the brown bacillus strain aiming at the prior art.
The third technical problem to be solved by the invention is to provide an application of the brown bacillus strain in prawn culture aiming at the prior art.
The technical scheme adopted by the invention for solving the first technical problem is as follows: the brown bacillus strain is named G18 and is preserved in China general microbiological culture Collection center with the preservation number of China Committee for culture Collection of microorganisms: CGMCC No.19893.
Further, the brown bacillus strain is a gram negative bacterium, and is cultured on 2216E agar medium at 30 ℃ for 24 hours, bacterial colony is brown, and is cultured in 2216E liquid medium in a shaking way for 12 hours, and bacterial liquid is brown.
The invention adopts the technical proposal for solving the second technical problem that: a method for screening a strain of brown bacillus as described above, characterized by comprising the steps of:
(1) Isolating and screening potential target strains:
taking fresh shrimp intestinal tracts, grinding, adding sterile phosphate buffer solution, shaking uniformly, and placing in a shaking table at 25-35 ℃ for shaking culture for 30-60 min;
taking a proper amount of cultured bacterial liquid, coating the bacterial liquid on 2216E agar culture medium after gradient dilution, transferring the culture medium into an incubator at 25-35 ℃, and inversely culturing for 3-7 d;
picking brown colonies, repeatedly streaking and inoculating on a new 2216E agar medium for 2 times, picking single colonies, adding into 20% -30% glycerol, and preserving at-80 ℃ to obtain strains to be tested;
(2) Vibrio inhibition capability test:
inoculating the strain to be detected into 2216E liquid culture medium with an inoculum size of 1-5%, culturing for 24h at 25-35 ℃, measuring an OD600 value of the bacterial liquid, and regulating the OD value to 0.6-0.8 for standby by using the sterile 2216E liquid culture medium;
meanwhile, the pathogenic indicator bacteria (vibrio parahaemolyticus or vibrio harveyi) are cultivated for 24 hours by using the same culture medium and culture conditions, the OD600 value of the pathogenic indicator bacteria is adjusted to be 0.1 by using a sterile 2216E liquid culture medium, and a culture medium plate containing the pathogenic bacteria is manufactured after uniform mixing, and the pathogenic bacteria is air-dried for 20-40 minutes in a sterile environment for standby;
dripping the bacteria liquid to be detected on the flat plate containing the pathogenic bacteria, after the bacteria liquid is air-dried, culturing at 30 ℃ for 24 hours, measuring the size of a bacteriostasis zone, and selecting a strain with an inhibition effect on pathogenic indicator bacteria;
(3) Identification of strains:
extracting the DNA of the strain obtained in the step (2), and identifying the DNA.
The invention adopts the technical proposal for solving the third technical problem that: an application of the brown bacillus strain in prawn culture.
The invention adopts the technical proposal for solving the third technical problem that: use of a strain of brown bacillus as described above for combating vibrio pathogen infection in a prawn.
Further, the vibrio pathogenic bacteria is at least one of vibrio parahaemolyticus or vibrio harveyi.
The invention adopts the technical proposal for solving the third technical problem that: an application of the brown bacillus strain in preparing a prawn culture probiotics preparation.
Further, the bacterial preparation is a probiotic feed additive or a culture water additive.
Compared with the prior art, the invention has the advantages that: the brown bacillus strain can effectively inhibit the growth of vibrio, especially inhibit vibrio parahaemolyticus and vibrio harveyi, can obviously reduce the mortality rate of artificially infected vibrio to prawns, and can obviously improve the survival rate of the prawns when being applied to prawn culture. According to the brown characteristic of brown bacteria on 2216E agar medium and 2216E liquid medium, the brown bacteria strain screening method can accurately locate target bacterial colony, and further can rapidly screen brown bacteria strain with disease resistance potential through pathogenic bacteria inhibition capability test.
Drawings
FIG. 1 is a schematic view showing the inhibitory effect of Brown bacillus G18 on Vibrio parahaemolyticus (A) and Vibrio harveyi (B) in example 1 of the present invention;
FIG. 2 is a graph of colony morphology of Brown bacillus G18;
FIG. 3 shows the changes of OD600 (A) and color (B) of the bacterial liquid of Brown bacillus G18 in example 1 of the present invention at different culture times;
FIG. 4 shows the effect of the addition of Brown bacillus G18 on the yield and survival rate of prawns according to example 2 of the invention;
FIG. 5 shows the effect of Brown bacillus G18 on the contents of trypsin, amylase, catalase and glutathione in example 2 of the present invention;
FIG. 6 shows the effect of feeding Brown bacillus G18 to increase the survival rate of prawns after infection of Vibrio parahaemolyticus in example 3 of the invention.
Detailed Description
The invention is described in further detail below with reference to the embodiments of the drawings.
Example 1: screening method of brown bacillus strain
1. Isolation and purification of Brown bacillus strains
10 fresh intestinal tracts of Litopenaeus vannamei (from Ningbo sea microecological technology Co., ltd.) are taken in an ultra clean bench, put in a sterile mortar, ground, added with sterile phosphate buffer (pH 7.4, concentration 10 nM), vigorously shaken for 30s, and put in a shaking table at 28 ℃ and 180rpm for shaking culture for 30min.
Taking 1mL of cultured bacterial liquid, continuously diluting in a gradient increasing by 10 times, and selecting 10 -5 、10 -6 Two dilution gradients of multiple, each gradient was set to 3 replicates, 100. Mu.L each was plated on pre-prepared 2216E agar medium (produced by Qingdao sea Bo Biotechnology Co., ltd.), and the medium was transferred to an incubator at 25℃for inverted culture.
After 3-7 d of culture, picking brown colonies, repeatedly streaking and inoculating on a new 2216E agar culture medium for 2 times, and culturing for 24-48 h at 25 ℃ to obtain purified strains. Single colonies were picked with a sterile inoculating loop, added to 20% glycerol, and stored at-80℃for further use.
2. Bacteriostasis test of brown bacillus strain
Inoculating the strain to be detected into a test tube containing 2216E liquid culture medium with an inoculum size of 1% -5%, culturing for 24 hours at 180rpm and 25 ℃, measuring the OD600 value of the bacterial liquid, and regulating the OD value to 0.6-0.8 for standby by using the sterile 2216E liquid culture medium.
Synchronous with the bacteria to be detected, the pathogenic indicator bacteria are cultivated for 24 hours by using the same culture medium and cultivation conditions, the OD600 value of the pathogenic indicator bacteria is adjusted to 0.1 by using a sterile 2216E liquid culture medium, the pathogenic indicator bacteria and the sterile 2216E liquid culture medium are poured into a disposable culture dish to manufacture a culture medium plate containing the pathogenic bacteria after being uniformly mixed, and the pathogenic indicator bacteria are dried for 30 minutes in a sterile ultra-clean bench for standby. In this example, the pathogenic bacteria are vibrio parahaemolyticus and vibrio harveyi respectively, and the two pathogenic bacteria are parallel experiments, and the experimental method is as described above, and the two pathogenic bacteria are separated from the diseased shrimp and stored in the laboratory.
And (3) dripping 5 mu L of the bacterial liquid to be detected on a plate containing pathogenic bacteria, and after the bacterial liquid is air-dried, culturing at 30 ℃ for 24 hours, and then measuring the size of a bacteriostasis zone to obtain a strain G18 with inhibition effect on vibrio parahaemolyticus and vibrio harveyi, wherein the strain G18 is shown in figure 1.
The strain G18 of the present invention was cultured on 2216E agar medium at 30℃for 24 hours, and the colony was brown, as shown in FIG. 2. When the strain was cultured in 2216E liquid medium for 12 hours with shaking, the OD600 value of the bacterial liquid reached the maximum value (about 1.7), and the bacterial liquid was brown, as shown in FIG. 3.
The 2216E liquid medium (Qingdao sea Bo Biotechnology Co., ltd.) formula was: 5.0g of peptone, 1.0g of yeast powder, 19.45g of sodium chloride, 5.98g of magnesium chloride, 3.24g of sodium sulfate, 1.8g of calcium chloride, 0.55g of potassium chloride, 0.16g of sodium carbonate, 0.1g of ferric citrate, 1000mL of distilled water and pH 7.6+/-0.2; the 2216E agar medium is prepared by adding 15g of agar into the 2216E liquid medium.
3. Identification of Brown bacillus Strain
In a sterile workbench, absorbing 100 mu L of bacterial liquid of the strain G18, centrifuging for 5min at 8000rpm, removing supernatant, adding 100 mu L of sterile phosphate buffer (pH value is 7.4 and concentration is 10 nM) to wash precipitate, repeating the centrifugation and washing operation once, adding 100 mu L of sterile phosphate buffer, mixing uniformly, boiling for 10min, and immediately adopting ice bath cooling to obtain the crude DNA template.
A general primer of 16S rDNA (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 14992R: 5 '-GGTTACCTTGTTACGACTT-3') was used in a 50. Mu.L PCR system (ddH 2 O18. Mu.L, 2 XSanTaq PCR Mix 25. Mu.L, primer F1. Mu.L, primer R1. Mu.L). The PCR conditions were: pre-denaturation at 94℃for 2min; denaturation at 94℃for 30s, renaturation at 60℃for 30s, extension at 72℃for 1min,30 cycles; finally, the extension is carried out for 5min at 72 ℃.
1% agarose gel was prepared, 3. Mu.L of DNA Marker and amplified PCR products were added to each spot well, the voltage was adjusted to 120V, the current was 380mA, and the electrophoresis time was 20min. The PCR products of acceptable concentration and purity were subjected to 16S rDNA sequencing (done by engineering (Shanghai) Co., ltd.). The sequence was submitted to EZBioCloud alignment and found that this strain has a similarity to the model strain Brown-restricted strain (Phaeobacter inhibens) DSM16374 strain of up to 100.0% and was therefore designated Brown bacillus G18.
Example 2:
the experiment is carried out at an aquaculture base of a Xiangxiang town in Ningbo city, and the experiment system comprises 14 simply modified 600L circular aquaculture barrels (with the diameter of 1m and the height of 0.8 m), wherein 6 barrels are used as a brown bacillus G18 adding group, and 8 barrels are used as a sterile adding control group.
Before the experiment, 130 shrimp larvae (purchased from Ningbo city sea microecological technology Co., ltd., body length of 7.95+ -0.53 cm, and weight of 4.16+ -0.76 g) were randomly loaded in each barrel, and the culture density was 325 tails/m 3 . The experimental period was 20 days, 8 per day: 00. 12:00 and 17:00 are respectively fed with prawn compound feed (from Shenzhen Australian group Co., ltd.) and activated brown bacillus G18 bacterial liquid is added every 4 days. Activating and adding a microbial inoculum: inoculating purified brown bacillus G18 into 200mL cone bottle containing 2216E liquid culture medium according to 1% -5% inoculum size, culturing in shaking table (180 rpm) at 30deg.C for 24 hr, and measuring bacterial liquid OD600 value of about 1.6 and concentration of about 1.2X10 9 cfu/ml。
And (3) adding the feed into the cultured brown bacillus liquid, and after the culture is fermented for 10min for a short time, adding the liquid and the feed into a culture barrel together. After the experiment, the yield and the survival rate of the prawns are measured, and the contents of trypsin, amylase, catalase and glutathione are measured. As shown by experimental results, the yield and the survival rate of the brown bacillus G18 added group prawns are obviously improved by 15.31 percent and 13.54 percent respectively compared with the control group after 20 days of culture (shown in figure 4), and trypsin, amylase, catalase and glutathione of the G18 group prawns are obviously higher than those of the control group (shown in figure 5).
Example 3:
after the end of the experiment of example 2, 100 prawns were randomly collected in the brown bacillus G18 added group and the control group of example 2, respectively, for testing the tolerance to the challenge of Vibrio parahaemolyticus.
The vibrio parahaemolyticus challenge experiment was performed in 15 culture glass jars (50L) containing oxygenation devices, the glass jars being randomly divided into 3 groups (brown bacillus G18 added group, control group and PBS control group), each group being repeated 5 times. 10 shrimps were filled into each glass jar before the experiment started. Vibrio toxicity attack is carried out by adopting a reverse clysis mode, 200 mu L of vibrio parahaemolyticus (separated from diseased shrimps and stored in the laboratory) bacterial suspension is respectively injected into the prawns of the G18 addition group and the control group, and the same amount of phosphate buffer solution is injected into the prawns of the PBS group. The number of dead shrimp was recorded after 6, 12, 24, 36, 48, 60, 72 and 84 hours of challenge. Experimental results show that the survival rate (80%) of the brown bacillus G18 added group prawns is significantly higher than that of the control group (48%) after 84h of toxicity attack (as shown in FIG. 6).
A vibrio harveyi challenge experiment is performed according to the method in example 3, which shows that the brown bacillus G18 in the invention can also obviously reduce the mortality rate of artificially infected vibrio harveyi to prawns.
In conclusion, the activity of vibrio pathogenic bacteria in the culture of the brown bacillus G18 prawn has better inhibition effect, in particular to vibrio parahaemolyticus and vibrio harveyi.
Claims (3)
1. A strain of brown bacillus (Phaeobacter inhibens) deposited in the China general microbiological culture Collection center, having accession number: CGMCC No.19893.
2. Use of the brown bacillus strain according to claim 1 for preparing a prawn culture probiotic preparation.
3. Use of a strain of brown bacillus according to claim 2 for the preparation of a probiotic preparation for prawn culture, wherein the preparation is a probiotic feed additive or a culture water additive.
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