CN103834596A - Bacillus subtilis shou003, anti-vibrio protein and preparation method and applications of bacillus subtilis shou003 and anti-vibrio protein - Google Patents
Bacillus subtilis shou003, anti-vibrio protein and preparation method and applications of bacillus subtilis shou003 and anti-vibrio protein Download PDFInfo
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Abstract
The invention provides bacillus subtilis (Bacillussubtilis) shou003, an anti-vibrio protein and a preparation method and applications of the bacillus subtilis shou003 and the anti-vibrio protein. The preparation method of the anti-vibrio protein comprises the following steps: screening out bacillus subtilis shou003 (preserved in China Center for Type Culture Collection with a number of CCTCC No.: M2013571 on November 13, 2013) from intestinal tract of a healthy large yellow croaker; and then extracting the anti-vibrio protein from the fermentation broth of bacillus subtilis shou003, wherein the amino acid sequence of the anti-vibrio protein is shown as SEQ ID No.: 1. The bacillus subtilis shou003 and the anti-vibrio protein show good effects on inhibiting aquatic pathogenic bacteria, in particular pathogenic vibrio; in addition, the bacillus subtilis shou003 shows outstanding tolerance to temperature, NaCl, gastric juice, intestinal juice and cholate and can be widely applied to the prevention of germs during aquaculture; on that basis, the optimal fermentation culture method of the bacillus subtilis shou003, and a preparation method of the anti-vibrio protein are provided.
Description
Technical field
The invention belongs to microbial technology field, relate in particular to a kind of subtilis shou003, anti-vibrios albumen and preparation method thereof and application.
Background technology
Vibriosis (Vibriosis) is bacterial by Vibrio (Vibrio), cultivates generally popular in the aquatic animals such as fish, shrimp, crab and shellfish and endanger maximum bacteriosis all over the world, and the financial loss that culture fishery is caused is inestimable.
At present, the Main Means that is used for controlling vibriosis in culture fishery is microbiotic control, because microbiotic is easy to use, and convenient operation, and also it takes effect than comparatively fast, and curative effect is also relatively good.But due to prolonged application and the abuse of the chemicalses such as microbiotic, cause many bacteriums it to be produced to resistance gradually, thereby increased the difficulty of prevention and cure of disease, and some drug residue is serious, serious harm is to the mankind's health.
Therefore people try to explore to use the new measure such as herbal medicine, immunological reagent, probiotic bacterium to prevent and treat disease, wherein, probiotic bacterium is as a kind of biological control method, not only can effectively control the quantity of pathogenic bacteria, reduce the generation of disease, strengthen the immunizing power of cultivated animals, and can also improve breeding ecological environment, safeguard microecological balance, have broad application prospects.The good probiotic bacterium of screening antibacterial effect, most important for biological control vibriosis.
Summary of the invention
The object of the present invention is to provide a kind of subtilis shou003, be intended to solve existing microbiotic prevents and treats that bacterium that vibriosis causes produces that resistance, difficulty of prevention and cure increase and can harm humans health etc. problem.
A further object of the present invention is to provide the method for above-mentioned subtilis shou003 fermentation culture.
A further object of the present invention is to provide the application of above-mentioned subtilis shou003.
It is a kind of by the above-mentioned subtilis shou003 anti-vibrios albumen obtaining that ferments that a further object of the present invention is to provide, and this albumen has good restraining effect to pathogenic vibrio.
A further object of the present invention is to provide the preparation method of above-mentioned anti-vibrios albumen.
The present invention is achieved in that a kind of subtilis (Bacillussubtilis) shou003, is preserved in Chinese Typical Representative culture collection center on November 13rd, 2013, and deposit number is CCTCCNO:M2013571.
The method that the present invention further provides above-mentioned subtilis shou003 fermentation culture, comprises the following steps:
(1) get described subtilis shou003 bacterial classification and be inoculated in activation medium, 37 ℃ of shaking tables are cultivated after 18～20h, centrifugal 5min under 8000rpm/min condition, and getting precipitation, to wash and be made into concentration with sterilized water be 10
7cFU/ml bacterium liquid;
(2) described bacterium liquid being seeded in fermention medium by the inoculum size of fermention medium 1% volume, is that under 4～7 conditions, shaking table is cultivated 10～16h at 4～40 ℃, pH, obtains subtilis shou003 fermented liquid.
Preferably, in step (1), described activation medium comprises following by the each component of mass parts:
9～10 parts of peptones,
2～3 parts of extracted beef powders,
4～5 parts, sodium-chlor;
In step (2), described fermention medium pH is 5, and described fermention medium comprises following by the each component of mass parts:
1～2 part of maltose,
1～2 part of extractum carnis,
C
acl
20.2～0.3 part.
Preferably, in step (1), described activation medium comprises following by the each component of mass parts:
10 parts of peptones,
3 parts of extracted beef powders,
5 parts, sodium-chlor;
In step (2), described fermention medium comprises following by the each component of mass parts:
2 parts of maltose,
2 parts of extractum carniss,
C
acl
20.3 part.
The present invention further provides the application of above-mentioned subtilis shou003 in aquaculture.
Preferably, described subtilis shou003 is used for suppressing aquatic pathogenic bacterium.
Preferably, described aquatic pathogenic bacterium comprises Vibrio parahaemolyticus (Vibrioparahaemolyticus), vibrio alginolyticus (Vibrioalginolyticus), Aeromonas hydrophila (Aeromonashydrophila), Wdwardsiella tarda (Edwardsiellatarda).
The present invention further provides a kind of anti-vibrios albumen, obtained by the extracellular products of above-mentioned subtilis shou003 is carried out to concentration extraction, the aminoacid sequence of this anti-vibrios albumen is as shown in SEQIDNO:1.
The preparation method who the present invention further provides above-mentioned anti-vibrios albumen, comprises the following steps:
(1) after being activated on nutrient agar medium inclined-plane, subtilis shou003 claimed in claim 1 is inoculated in nutrient broth medium, 28 ℃ of constant-temperature shaking culture 18～20h, centrifugal 20min under 9600rpm/min, 4 ℃ of conditions, get supernatant liquor through 0.22 μ m filtering with microporous membrane, obtain filter liquide;
(2) with ammonium sulfate precipitation method, described filter liquide is extracted, obtain anti-vibrios protein extract.
Preferably, described ammonium sulfate precipitation method comprises the following steps: get genus bacillus filtered liquid 50ml, add solid ammonium sulfate to make solution reach mass percent concentration 70%, at 4 ℃, spend the night, after the centrifugal 20min of 8000rpm/min, obtain throw out, precipitation is dissolved with 0.05MTris-HCl damping fluid, and after dialysis 48h, material in bag taking, is the albumen that this concentration ammonium sulfate precipitation obtains.
The present invention overcomes the deficiencies in the prior art, a kind of subtilis shou003 is provided, anti-vibrios albumen and preparation method thereof and application, be preserved in Chinese Typical Representative culture collection center on November 13rd, 2013 by filter out a bacillus subtilis (Bacillussubtilis) shou003(in the enteron aisle of healthy large yellow croaker, deposit number is CCTCCNO:M2013571), and arrive a kind of anti-vibrios albumen by this subtilis shou003 fermented extracted, the aminoacid sequence of this anti-vibrios albumen is as shown in SEQIDNO:1, wherein, subtilis shou003 and anti-vibrios albumen are to aquatic pathogenic bacterium, especially pathogenic vibrio has good restraining effect, and subtilis shou003 is to gastric juice, intestinal juice and cholate all have good tolerance, under the high temperature of 70 ℃, still have active and can grow, to common antibiotic sensitive, there is no transferable resistance determining factor, and laboratory animal is had no side effect safely, illustrate that this bacterium has the characteristic of probiotic bacterium, can be applied to the bacteria-treating in aquaculture process, not only can effectively control the quantity of pathogenic bacteria, reduce the generation of disease, strengthen the immunizing power of cultivated animals, and can also improve breeding ecological environment, safeguard microecological balance, have broad application prospects.On this basis, the invention provides the extracting method of preparing of the fermentation culture method of above-mentioned subtilis shou003 optimum and anti-vibrios albumen.
Accompanying drawing explanation
Fig. 1 is the tolerance test result of subtilis shou003 to temperature in the embodiment of the present invention 3;
Fig. 2 is the tolerance test result of subtilis shou003 to NaCl in the embodiment of the present invention 3;
Fig. 3 is the tolerance test result of subtilis shou003 to simulated gastric fluid in the embodiment of the present invention 3;
Fig. 4 is the tolerance test result of subtilis shou003 to simulated intestinal fluid in the embodiment of the present invention 3;
Fig. 5 is the tolerance test result of subtilis shou003 to cholate in the embodiment of the present invention 3;
Fig. 6 is the SDS-PAGE electrophoresis structure iron of anti-vibrios albumen in the embodiment of the present invention 7.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
The separation of embodiment 1 subtilis shou003
Subtilis shou003(Bacillussubtilis of the present invention) separate and obtain by the enteron aisle healthy large yellow croaker, separation method is specially:
First carry out body surface sterilization with 75% alcohol; Under aseptic condition, dissect, take out enteron aisle, 75% cotton ball soaked in alcohol wiping intestinal tube outer wall, stroke-physiological saline solution is rinsed repeatedly.While preparing the total bacteria suspension of enteron aisle, (comprise intestines wall and intestinal contents), first with aseptic cotton thread ligation anterior intestine, middle intestines and hindgut position respectively, respectively getting the long tissue of about 0.5cm mixes as sample, after weighing in 1: ratio 10(w/v) add sterile saline mix, fully grind evenly with sterilizing homogenizer, the sample of grinding is made as 10
-1, then with sterile saline to 10
-1sample carry out doubling dilution to 10
-6, each extent of dilution sample is all placed on whirlpool mixed instrument and vibrates evenly.Get respectively each extent of dilution sample 0.1ml and coat manganese nutrition agar, thiosulfuric acid saline citrate cholate sucrose agar (TCBS), eosin methylene blue agar (EMB), seawater agar (2216E), fresh water agar (FWA) and nutrient agar flat board, separately do two parallel controls.Wherein manganese nutrition agar plate is put 60 ℃ and is processed after 2h in 28 ℃ and cultivate 1～2d, and other flat boards are directly put 28 ℃ and cultivated 1～2d.Observe and record the colony characteristics on each flat board.
The fermentation culture of embodiment 2 subtilis shou003
(1) get described subtilis shou003 bacterial classification and be inoculated in activation medium, 37 ℃ of shaking tables are cultivated after 18～20h, centrifugal 5min under 8000rpm/min condition, and getting precipitation, to wash and be made into concentration with sterilized water be 10
7cFU/ml bacterium liquid; Wherein, the preparation process of activation medium is: peptone 9g, and extracted beef powder 2g, sodium-chlor 4g, adding distil water 1000ml, packing flask after heating for dissolving, through 121 ℃ of sterilizing 15min.
(2) described bacterium liquid being seeded in fermention medium by the inoculum size of fermention medium 1% volume, is that under 4 conditions, shaking table is cultivated 10～16h at 4～40 ℃, pH, obtains subtilis shou003 fermented liquid; The preparation process of described fermention medium is: maltose 1g, and extractum carnis 1g, CaCl20.2g, adding distil water 1000ml, packing flask after heating for dissolving, through 121 ℃ of sterilizing 15min.
The tolerance of embodiment 3 subtilis shou003
Be inoculated in activation medium by transfering loop picking subtilis shou003 slant strains, 37 ℃ of shaking tables are cultivated after 18～20h, and 8000rpm/min is centrifugal, and 5min abandons supernatant, with sterilized water washing 3 times, finally make bacterium liquid with sterilized water, utilize Maxwell opacity tube that bacterial concentration is adjusted to 10
7cFU/ml, for the tolerance test to temperature, NaCl, gastric juice, intestinal juice and cholate.
(1) temperature tolerance
Get 12 thick test tubes and be divided into 4 groups, every group 3 are repeated parallel, every adds 10ml fermention medium, after sterilizing, subtilis shou003 is inoculated in respectively in 12 test tubes with 1% inoculum size, then process after 30min respectively at 70 ℃, 80 ℃, 90 ℃ and 100 ℃, at 37 ℃, cultivate 12h after sampling survey the OD value at each group of bacterium liquid 600nm place.
As shown in Figure 1, after 70 ℃ of processing, genus bacillus still can grow test-results, and still, along with the rising for the treatment of temp, OD value reduces gradually; Under the effect of 80 ℃ and 90 ℃ high temperature, the growth of bacterium is affected, but or can grow, and after 100 ℃ of effects, the growth of bacterium is suppressed, and does not substantially grow.
(2) salt tolerance
In fermention medium, add NaCl, make its massfraction be respectively 0 ‰, 5 ‰, 20 ‰, 50 ‰, 70 ‰, 100 ‰, be sub-packed in 250ml Erlenmeyer flask, every bottle of 100ml, genus bacillus bacteria suspension is accessed in above-mentioned substratum with 1% inoculum size, in 37 ℃ of shaking tables, cultivate after 12h, survey the OD value at 600nm place.
Test-results as shown in Figure 2, subtilis shou003 has certain demand to NaCl in process of growth, in 0～35 ‰ scope, bacterial growth is good, in the time of 5 ‰ NaCl concentration, grow best, when concentration exceedes after 5 ‰, along with the rising of concentration, the growth of bacterium is subject to certain inhibition, but salinity is that 50 ‰ bacterial growths are not bad.The average salinity of general seawater, take Pacific Ocean seawater as example, is 35 ‰, so this bacterium all can adapt to seawater and limneticum, subject range is wider.
(3) resistant to gastric juice
The distilled water of sterilizing is diluted with 1mol/LHCl, make the pH of sterilized water be respectively 1.5,2.5 and 3.5, each pH gets respectively in 100ml to 250ml triangular flask, 121 ℃ of sterilizing 30min; After below being cooled to 50 ℃, in each triangular flask, add 0.5g stomach en-to mix; The bacteria suspension of genus bacillus is added in Erlenmeyer flask by 1% the bacterium amount that connects, in 37 ℃ of shaking tables, cultivates, measure respectively each genus bacillus 0,1,2, the OD of 3h
600nm.
As shown in Figure 3, subtilis shou003 has good tolerance to simulated gastric fluid to test-results, is 3.5 o'clock at pH, and the impact that thalline is subject to is less, and bacterial number has reduced approximately 30%; And along with the reduction of pH, acid enhancing, the restraining effect that thalli growth is subject to also strengthens gradually, and under the gastric juice environment that is 1.5 at pH, after effect 2h, thalline quantity is reduced to original 48.9%.
(4) resistance to intestinal juice
Get 0.68g potassium primary phosphate and be dissolved in 100ml water, then with the NaOH of 1mol/L, pH is adjusted to 6.8,121 ℃ of sterilizing 30min, be cooled to 50 ℃ following after, in each Erlenmeyer flask, add 1g trypsinase to mix; The bacteria suspension of genus bacillus is added in Erlenmeyer flask by 1% the bacterium amount that connects, in 37 ℃ of shaking tables, cultivates, measure respectively each genus bacillus 0,1,2, the OD of 3h
600nm.
Test-results as shown in Figure 4, the subtilis shou003 3h that grows under simulated intestinal fluid condition, first 2 hours, the content of bacterium prolongation in time reduces gradually, and after 3h, the content of bacterium raises on the contrary, has only reduced by 9% compared with initial value, and this subtilis has good tolerance to intestinal juice.
(5) bile tolerance
In fermention medium, add cholate, make its massfraction be respectively 0.25%, 0.5%, 1%, 1.5% and 2%, be sub-packed in the Erlenmeyer flask of 250ml, every bottle of 100ml, 121 ℃ of sterilizing 30min; The bacteria suspension of genus bacillus is added in Erlenmeyer flask by 1% the bacterium amount that connects, in 37 ℃ of shaking tables, cultivate, respectively at 0,1,2,3h measures OD
600nm.
Test-results as shown in Figure 5, subtilis is containing the 3h that grows under cholate condition, first 2 hours, bacteria content prolongation in time reduces gradually, and after 3h, bacteria content raises on the contrary, illustrate that this subtilis has good tolerance to cholate, in the time that cholate content is higher, though genus bacillus can not grow, unlikely death.
Embodiment 4 subtilis shou003 are to antibiotic susceptibility
Get 0.1ml genus bacillus bacteria suspension and coat on detection culture medium flat plate, after bacterium liquid is absorbed, stick drug sensitive test paper after 37 ℃ of cultivation 16h, measure antibacterial circle diameter.Test microbiotic used and result as shown in table 1 below:
Table 1 subtilis shou003 is to antibiotic susceptibility
Subtilis shou003 is all more responsive to 18 kinds of conventional feeding antibiotics, is safe for animal and fowl fodder, has the potential quality as probiotic bacterium, meanwhile, should note in use avoiding using with microbiotic simultaneously.
The security of embodiment 5 subtilis shou003
Blood agar: by the bacterial strain streak inoculation to be measured of incubated overnight in nutrient broth medium on sheep blood agar, cultivate 24h for 37 ℃, according to the formation of periphery of bacterial colonies transparent circle, judge the generation of hemolysin, and be so kind as to give with vibrio alginolyticus Va-Y(teacher Fu Lixia of Yangzhou University, at document: " king builds; Qu Xiancheng for Wang Juan; Feng Yonghui, Cai Lisheng, Zhang Qinghua (communication author).From screening and the evaluation of the vibrios Antagonistic Fungi of large yellow croaker enteron aisle, Oceanologia et Limnologia Sinica, 2010,41(5): 707-713. " in open) as positive control.
Mouse: will in nutrient broth, cultivate the bacterial strain of 18h, the centrifugal substratum that goes, with 0.85% physiological saline gradient dilution be 9 × 10
10cFU/ml, 9 × 10
9cFU/ml, 9 × 10
8tri-concentration of CFU/ml.Then abdominal injection infecting mouse, only, 6 every group, physiological saline is as negative control for 1ml/, and vibrio alginolyticus Va-Y is as positive control (9 × 10
9cFU/ml).Feed after one week, observe mouse growth situation.
Hybridized prussian carp: the same mouse experiment of method, abdominal injection infects crucian, 1ml/ tail, every group of 10 tails, physiological saline is as negative control, and vibrio alginolyticus Va-Y is as positive control (9 × 10
9cFU/ml).Feed after one week, observe crucian growing state.
Find by dull and stereotyped hemolytic test, vibrio alginolyticus Va-Y has haemolysis on blood agar, and subtilis shou003 does not observe haemolysis on blood agar, tentatively shows that this bacterial strain does not produce hemolysin, does not have potential pathogenic.
Infect the mouse of vibrio alginolyticus Va-Y, in 2 days, occur intoxicating phenomenon in various degree, and intoxicating phenomenon does not appear in experiment mice one Zhou Houjun, with control group indifference, can judge that this bacterial strain does not have pathogenic to mouse.
All dead in the crucian second day of injection vibrio alginolyticus Va-Y, and all there is not intoxicating phenomenon in experiment mice, with blank indifference, can judge that this bacterial strain does not have pathogenic to crucian.
The preparation of the anti-vibrios albumen of embodiment 6
(1) after being activated on nutrient agar medium inclined-plane, subtilis shou003 claimed in claim 1 is inoculated in nutrient broth medium, 28 ℃ of constant-temperature shaking culture 18～20h, centrifugal 20min under 9600rpm/min, 4 ℃ of conditions, get supernatant liquor through 0.22 μ m filtering with microporous membrane, obtain filter liquide;
(2) with ammonium sulfate precipitation method (reference literature: " and model is bright for Wang Jiazheng, biological chemistry. protein technical manual: Science Press; 2000 " record method operation) described filter liquide is extracted, obtain anti-vibrios protein extract; Wherein, to add the mass concentration after described filter liquide be 70% to ammonium sulfate.
In step (2), described ammonium sulfate precipitation method specifically comprises the following steps: get genus bacillus filtered liquid 50ml, add solid ammonium sulfate to make solution reach mass percent concentration 70%, at 4 ℃, spend the night, after the centrifugal 20min of 8000rpm/min, obtain throw out, precipitation is dissolved with 0.05MTris-HCl damping fluid, and after dialysis 48h, material in bag taking, is the albumen that this concentration ammonium sulfate precipitation obtains.
(3) antagonistic experiment detects and finds that being numbered 7 protein is so kind as to give vibrio alginolyticus Va-Y(teacher Fu Lixia of Yangzhou University, at document: " Wang Juan, Feng Yonghui, Cai Lisheng, king builds, Qu Xiancheng, Zhang Qinghua (communication author).From screening and the evaluation of the vibrios Antagonistic Fungi of large yellow croaker enteron aisle, Oceanologia et Limnologia Sinica, 2010,41(5): 707-713. " in open) growth inhibited.
The detection of the anti-vibrios albumen of embodiment 7
(1) get 4.9ml distilled water, 6ml30% acrylamide, 3.8ml1.5mol/LTris-HCl, 150 μ l10%SDS, 150 μ l10% ammonium persulphates, 6 μ lTEMED mix, encapsulating is after suitable height, add 1ml dehydrated alcohol to seal, after colloid solidification, ethanol is poured out, thoroughly blot ethanol with thieving paper; Fill 5% concentrated glue: get 5.5ml distilled water, 1.3ml30% acrylamide, 1.0ml1.5mol/LTris-HCl, 80 μ l10%SDS, 80 μ l10% ammonium persulphates, 8 μ lTEMED mix, inject separation gel upper strata, after insert immediately comb, after 30min colloid solidification, take out comb, pour 1 × electrophoretic buffer into electrophoresis chamber, and flood short sheet glass.
(2) get 20 μ l protein extracts and mix with 4 μ l sample buffers (5 ×), make protein denaturation in 100 ℃ of heating in water bath 3min, the amount injection point sample hole with liquid-transfering gun with 20 μ l, using sample buffer as negative control.
(3) while carrying out electrophoresis, adopting the voltage in concentrated glue is 80V, and in separation gel, voltage is 120V, powered-down in the time that tetrabromophenol sulfonphthalein forward position is run extremely apart from bottom line 1cm.
(4) after proteins gel electrophoresis finishes, gel is carefully taken off from sheet glass, add 100ml pure water, level shake (30rpm/min) 30min; Pure water is poured out, added staining fluid 100ml, seal, (30rpm/min) 1h is to spending the night in level shake; Staining fluid is poured out, added destainer 100ml, seal, level shake (30rpm/min); When being reduced to acceptable to the background of gel, destainer is poured out, poured into 100ml pure water, scanning gel, as shown in Figure 6, the molecular weight of this anti-vibrios albumen is 59KD to result.
(5) adopt raw work rubber tapping to reclaim test kit (MicroProteinPAGERecoveryKit, BSP062) and carry out protein recovery.
Antagonistic experiment and the order-checking of the anti-vibrios albumen of embodiment 8
(1) will after vibrio alginolyticus Va-Y cultivation 18～20h, wash lower lawn with 0.85% sterile saline, regulating its concentration with reference to Maxwell opacity tube is 10
5cFU/ml, gets 0.1ml and coats on suitable flat board, even with spreading rod coating.
(2) with tweezers, 4 aseptic Oxford cups are put into equably to the plate of horizontal positioned, drawn 0.25ml protein solution in the cup of Oxford, note not overflowing outside the cup of Oxford.Cultivating 24～48h observation for 37 ℃ has or not inhibition zone to occur and records diameter.
(3) the anti-vibrios albumen that has inhibition zone to produce is delivered to the Shanghai biological company limited of raw work and carried out Mass Spectrometric Identification, sequencing result is as shown in SEQIDNO:1.Specific peptide sequence correspondence specific albumen, thereby identifies albumen to be detected according to peptide sequence.In the peptide fingerprinting spectrum of subtilis shou003, have 16 peptide sections and chitinase to match, protein sequence fraction of coverage is about 38%, qualification result demonstration, and protein vibrios to bacteriostatic action is chitinase.
The antimicrobial spectrum of embodiment 9shou003 is measured
With reference to the method (SmithP of Smith etc., DaveyS, 1993.Evidenceforthecompetitive excludingofAeromonassalmonicidafromfishwithstress-induci blefurunculosisby aFluorescentPseudomonad.JournalofFishDisease, 16:521-524) slightly make an amendment.Concrete steps are: by a certain amount of stroke-physiological saline solution (0.85%NaCl), the lawn of Vibrio parahaemolyticus (Vp1.2164), vibrio alginolyticus (Va-Y) after activation, Aeromonas hydrophila (Ah-9), Wdwardsiella tarda (Et-Y) is washed down, drawn respectively 100 μ l and add on nutrient agar flat board and be coated with.Then distinguish scraping experimental strain dibbling on above-mentioned flat board of enlarged culturing 24h with transfering loop, and put on corresponding numbering, 28 ℃ of constant temperature culture, 24～48h carries out observation and the measurement of inhibition zone, and result is as shown in table 2 below:
The antimicrobial spectrum of table 2shou003 bacterial strain to pathogenic bacteria
As can be seen from Table 2, bacterial strain shou003 is stronger to the antagonistic action of two pathogen strain vibrios, and the antibacterial circle diameter of Vibrio parahaemolyticus (Vp1.2164) and vibrio alginolyticus (Va-Y) is reached respectively to 16mm and 9.5mm; And to two kinds of pathogenic bacterias common on aquatic products, the antibacterial circle diameter of Aeromonas hydrophila (Ah-9), Wdwardsiella tarda (Et-Y) reaches respectively: 15.2mm, 11.3mm.
Compare and the shortcoming and defect of prior art, the present invention has following beneficial effect:
(1) subtilis shou003 of the present invention has good restraining effect to pathogenic vibrio, and to salinity, gastric juice, intestinal juice and cholate all have good tolerance, under the high temperature of 70 ℃, still have active and can grow, to common antibiotic sensitive, there is no transferable resistance determining factor, and laboratory animal is had no side effect safely, illustrate that this bacterium has the characteristic of probiotic bacterium, can be applied to the bacteria-treating in aquaculture process, effectively control the quantity of pathogenic bacteria, reduce the generation of disease, strengthen the immunizing power of cultivated animals, have broad application prospects.
(2) method of subtilis shou003 fermentation culture of the present invention is the fermentation process of this subtilis shou003 optimum, can carry out mass-producing fermentation, is suitable for industrialized development.
(3) anti-vibrios albumen of the present invention has good restraining effect to pathogenic vibrio, can be prepared into independent reagent or medicine, has a good application prospect.
(4) preparation method of anti-vibrios albumen of the present invention is simple, is suitable for industrialized development.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. subtilis (Bacillussubtilis) shou003, is preserved in Chinese Typical Representative culture collection center on November 13rd, 2013, and deposit number is CCTCCNO:M2013571.
2. the method for subtilis shou003 fermentation culture claimed in claim 1, is characterized in that comprising the following steps:
(1) get described subtilis shou003 bacterial classification and be inoculated in activation medium, 37 ℃ of shaking tables are cultivated after 18～20h, centrifugal 5min under 8000rpm/min condition, and getting precipitation, to wash and be made into concentration with sterilized water be 10
7cFU/ml bacterium liquid;
(2) described bacterium liquid being seeded in fermention medium by the inoculum size of fermention medium 1% volume, is that under 4～7 conditions, shaking table is cultivated 10～16h at 4～40 ℃, pH, obtains subtilis shou003 fermented liquid.
3. the method for subtilis shou003 fermentation culture as claimed in claim 2, is characterized in that, in step (1), described activation medium comprises following by the each component of mass parts:
9～10 parts of peptones,
2～3 parts of extracted beef powders,
4～5 parts, sodium-chlor;
In step (2), described fermention medium pH is 5, and described fermention medium comprises following by the each component of mass parts:
1～2 part of maltose,
1～2 part of extractum carnis,
C
acl
20.2～0.3 part.
4. the method for subtilis shou003 fermentation culture as claimed in claim 3, is characterized in that, in step (1), described activation medium comprises following by the each component of mass parts:
10 parts of peptones,
3 parts of extracted beef powders,
5 parts, sodium-chlor;
In step (2), described fermention medium comprises following by the each component of mass parts:
2 parts of maltose,
2 parts of extractum carniss,
C
acl
20.3 part.
5. the application of subtilis shou003 claimed in claim 1 in aquaculture.
6. the application of subtilis shou003 as claimed in claim 4 in aquaculture, is characterized in that, described subtilis shou003 is used for suppressing aquatic pathogenic bacterium.
7. the application of subtilis shou003 as claimed in claim 5 in aquaculture, it is characterized in that, described aquatic pathogenic bacterium comprises Vibrio parahaemolyticus (Vibrioparahaemolyticus), vibrio alginolyticus (Vibrioalginolyticus), Aeromonas hydrophila (Aeromonashydrophila), Wdwardsiella tarda (Edwardsiellatarda).
8. an anti-vibrios albumen, is characterized in that, obtains by the extracellular products of subtilis shou003 claimed in claim 1 is carried out to concentration extraction, and the aminoacid sequence of this anti-vibrios albumen is as shown in SEQIDNO:1.
9. the preparation method of anti-vibrios albumen claimed in claim 6, is characterized in that comprising the following steps:
(1) after being activated on nutrient agar medium inclined-plane, subtilis shou003 claimed in claim 1 is inoculated in nutrient broth medium, 28 ℃ of constant-temperature shaking culture 18～20h, centrifugal 20min under 9600rpm/min, 4 ℃ of conditions, get supernatant liquor through 0.22 μ m filtering with microporous membrane, obtain filter liquide;
(2) with ammonium sulfate precipitation method, described filter liquide is extracted, obtain anti-vibrios protein extract.
10. the preparation method of anti-vibrios albumen as claimed in claim 9, it is characterized in that, described ammonium sulfate precipitation method comprises the following steps: get genus bacillus filtered liquid 50ml, add solid ammonium sulfate to make solution reach mass percent concentration 70%, at 4 ℃, spend the night, obtain throw out after the centrifugal 20min of 8000rpm/min, precipitation is dissolved with 0.05MTris-HCl damping fluid, after dialysis 48h, material in bag taking, is the albumen that this concentration ammonium sulfate precipitation obtains.
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