CN103834596B - 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, more particularly, to a kind of bacillus subtilises shou003, anti-vibrio albumen and
Its preparation method and application.
Background technology
Vibriosises (Vibriosis) are bacterial by vibrio (Vibrio), cultivate fish, shrimp, Eriocheir sinensiss all over the world
And generally popular and endanger maximum bacterial disease in the aquatic animal such as shellfish, the economic loss that culture fishery is caused is not
Can estimate.
At present, being used for controlling the Main Means of vibriosises in culture fishery is antibiotic preventing and treating, because antibiotic usage
It is convenient to get up, and is easy to operate, and it takes effect, ratio is very fast, and curative effect is also relatively good.Length yet with chemicalses such as antibiotic
Phase application and abuse, lead to many antibacterials gradually to produce drug resistance to it, thus increased the difficulty of prevention and cure of disease, and some
Drug residue is serious, the health of serious harm to the mankind.
Therefore people try to explore using the New Measure controlling disease such as Chinese herbal medicine, immune formulation, probiotic bacteria, wherein, prebiotic
Bacterium, as a kind of biological control method, can not only effectively control the quantity of pathogen, reduces the generation of disease, strengthens cultivation
The immunity of animal, and breeding ecological environment can also be improved, safeguard microecological balance, have broad application prospects.Screening
The good probiotic bacteria of antibacterial effect, most important for Biological control vibriosises.
Content of the invention
It is an object of the invention to provide a kind of bacillus subtilises shou003 prevents and treats arc it is intended to solve existing antibiotic
The problems such as antibacterial that bacterium disease leads to produces drug resistance, difficulty of prevention and cure increases and can endanger human health.
It is still another object of the present invention to provide the method for above-mentioned bacillus subtilises shou003 fermentation culture.
It is still another object of the present invention to provide the application of above-mentioned bacillus subtilises shou003.
It is still another object of the present invention to provide a kind of anti-arc being obtained by above-mentioned bacillus subtilises shou003 fermentation
Mycoprotein, this albumen has good inhibitory action to pathogenic vibrio.
It is still another object of the present invention to provide the preparation method of above-mentioned anti-vibrio albumen.
The present invention is achieved in that a kind of bacillus subtilises (Bacillus subtilis) shou003, in 2013
On November 13, in is preserved in China typical culture collection center, and deposit number is CCTCC NO：M 2013571.
Invention further provides the method for above-mentioned bacillus subtilises shou003 fermentation culture, comprise the following steps：
(1) described bacillus subtilises shou003 strain is taken to be inoculated in activation medium, 37 DEG C of shaking table cultures 18～
After 20h, under the conditions of 8000r/min, it is centrifuged 5min, taking precipitation with aseptic water washing and being made into concentration is 107CFU/ml bacterium solution；
(2) described bacterium solution is seeded in fermentation medium by the inoculum concentration of fermentation medium 1% volume, 4～40 DEG C,
PH is shaking table culture 10～16h under the conditions of 4～7, obtains bacillus subtilises shou003 fermentation liquid.
Preferably, in step (1), described activation medium includes each component in parts by mass：
9～10 parts of peptone,
2～3 parts of extracted beef powder,
4～5 parts of sodium chloride；
In step (2), described fermentation medium pH be 5, and described fermentation medium include each in parts by mass
Component：
1～2 part of maltose,
1～2 part of Carnis Bovis seu Bubali cream,
CaCl20.2～0.3 part.
Preferably, in step (1), described activation medium includes each component in parts by mass：
10 parts of peptone,
3 parts of extracted beef powder,
5 parts of sodium chloride；
In step (2), described fermentation medium includes each component in parts by mass：
2 parts of maltose,
2 parts of Carnis Bovis seu Bubali cream,
CaCl20.3 part.
Invention further provides application in aquaculture for the above-mentioned bacillus subtilises shou003.
Preferably, described bacillus subtilises shou003 is used for suppressing aquatic pathogenic bacterium.
Preferably, described aquatic pathogenic bacterium includes vibrio parahaemolytious (Vibrio parahaemolyticus), vibrio alginolyticus
(Vibrio alginolyticus), Aeromonas hydrophila (Aeromonas hydrophila), Wdwardsiella tarda
(Edwardsiella tarda).
Invention further provides a kind of anti-vibrio albumen, by extracellular to above-mentioned bacillus subtilises shou003
Product carries out concentration extraction and obtains, the aminoacid sequence such as SEQ ID NO of this anti-vibrio albumen：Shown in 1.
Invention further provides the preparation method of above-mentioned anti-vibrio albumen, comprise the following steps：
(1) it is inoculated in battalion after the bacillus subtilises shou003 described in claim 1 being activated on nutrient agar slopes
Foster broth bouillon, 28 DEG C of constant-temperature shaking culture 18～20h, it is centrifuged 20min under the conditions of 9600r/min, 4 DEG C, take supernatant
Through 0.22 μm of filtering with microporous membrane, obtain filter liquid；
(2) with ammonium sulfate precipitation method, described filter liquid is extracted, obtain anti-vibrio protein extract.
Preferably, described ammonium sulfate precipitation method comprises the following steps：Take bacillus cereuss filtrate 50ml, add solid sulphuric acid
Ammonium makes solution reach mass percent concentration 70%, at 4 DEG C overnight, obtains precipitate, precipitation after 8000r/min centrifugation 20min
With 0.05M Tris-HCl buffer solution, material in bag taking after the 48h that dialyses, it is the obtained egg of this concentration sulphuric acid ammonium precipitation
In vain.
The present invention overcomes the deficiencies in the prior art, provides a kind of bacillus subtilises shou003, anti-vibrio albumen and its system
Preparation Method and application, by filtering out a bacillus subtilis (Bacillus in the intestinal of healthy Carnis Pseudosciaenae
Subtilis) shou003 (is preserved in China typical culture collection center on November 13rd, 2013, deposit number is CCTCC
NO：M 2013571), and by this bacillus subtilises shou003 fermented extracted to a kind of anti-vibrio albumen, this anti-vibrio egg
White aminoacid sequence such as SEQ ID NO：Shown in 1, wherein, bacillus subtilises shou003 and anti-vibrio albumen are to Aquatic product
Pathogen, especially pathogenic vibrio have good inhibitory action, and bacillus subtilises shou003 to gastric juice, intestinal juice with
And cholate all has preferable toleration, still active and can grow under 70 DEG C of high temperature, to common antibiotic sensitive, do not have
There is transferable drug-resistance factor, and laboratory animal is had no side effect safely, illustrate that this bacterium has the characteristic of probiotic bacteria, can apply
Bacteria-treating in aquaculture process, can not only effectively control the quantity of pathogen, reduce the generation of disease, strengthen
The immunity of cultivated animals, and breeding ecological environment can also be improved, safeguard microecological balance, have broad application prospects.
On this basis, the invention provides the optimum fermentation culture method of above-mentioned bacillus subtilises shou003 and anti-vibrio egg
White prepares extracting method.
Brief description
Fig. 1 is the tolerance test result to temperature for the bacillus subtilises shou003 in the embodiment of the present invention 3；
Fig. 2 is the tolerance test result to NaCl for the bacillus subtilises shou003 in the embodiment of the present invention 3；
Fig. 3 is the tolerance test result to simulated gastric fluid for the bacillus subtilises shou003 in the embodiment of the present invention 3；
Fig. 4 is the tolerance test result to simulated intestinal fluid for the bacillus subtilises shou003 in the embodiment of the present invention 3；
Fig. 5 is the tolerance test result to cholate for the bacillus subtilises shou003 in the embodiment of the present invention 3；
Fig. 6 is the SDS-PAGE electrooptical structure figure of anti-vibrio albumen in the embodiment of the present invention 7.
Specific embodiment
In order that the objects, technical solutions and advantages of the present invention become more apparent, below in conjunction with drawings and Examples, right
The present invention is further elaborated.It should be appreciated that specific embodiment described herein is only in order to explain the present invention, and
It is not used in the restriction present invention.
The separation of embodiment 1 bacillus subtilises shou003
Bacillus subtilises shou003 (Bacillus subtilis) of the present invention is passed through in healthy Carnis Pseudosciaenae
Intestinal separates and obtains, and separation method is specially：
Carry out body surface sterilization with 75% ethanol first；Dissect under aseptic condition, take out intestinal, 75% cotton ball soaked in alcohol wipes
Intestinal tube outer wall, physiological saline solution rinses repeatedly.(intestinal wall and intestinal contentses are included), first with aseptic when preparing the total bacteria suspension of intestinal
Cotton thread ligatures anterior intestine, middle intestinal and hindgut position respectively, respectively takes the tissue of about 0.5cm length to be blended together as sample, after weighing
Add sterile saline mixing in the ratio of 1: 10 (w/v), be fully ground uniformly with sterilizing homogenizer, the sample of grinding is set to
10-1, then with sterile saline to 10-1Sample carries out doubling dilution to 10-6, each dilution factor sample is placed in whirlpool mixed instrument
Upper shaken well.Each dilution factor sample 0.1ml is taken to coat manganese nutrition agar, thiosulphate citrate cholate sucrose respectively
Agar (TCBS), eosin methylene blue agar (EMB), sea water agar (2216E), fresh water agar (FWA) and nutrient agar are put down
Plate, separately does two parallel controls.Wherein manganese nutrition agar plate is put 60 DEG C and is processed 2h after 28 DEG C of culture 1～2d, other flat boards
Directly put 28 DEG C of culture 1～2d.Observe and record the colony characteristicses on each flat board.
The fermentation culture of embodiment 2 bacillus subtilises shou003
(1) described bacillus subtilises shou003 strain is taken to be inoculated in activation medium, 37 DEG C of shaking table cultures 18～
After 20h, under the conditions of 8000r/min, it is centrifuged 5min, taking precipitation with aseptic water washing and being made into concentration is 107CFU/ml bacterium solution；
Wherein, the preparation process of activation medium is：Peptone 9g, extracted beef powder 2g, sodium chloride 4g, plus distilled water 1000ml, plus
Subpackage flask after heat of solution, through 121 DEG C of sterilizing 15min.
(2) described bacterium solution is seeded in fermentation medium by the inoculum concentration of fermentation medium 1% volume, 4～40 DEG C,
PH is shaking table culture 10～16h under the conditions of 4, obtains bacillus subtilises shou003 fermentation liquid；The preparation of described fermentation medium
Process is：Maltose 1g, Carnis Bovis seu Bubali cream 1g, CaCl20.2g, plus distilled water 1000ml, subpackage flask after heating for dissolving, through 121 DEG C
Sterilizing 15min.
The toleration of embodiment 3 bacillus subtilises shou003
It is inoculated in activation medium with inoculating loop picking bacillus subtilises shou003 slant strains, 37 DEG C of shaking table trainings
After supporting 18～20h, 8000r/min centrifugation 5min abandons supernatant, with aseptic water washing 3 times, finally makes bacterium solution with sterilized water, utilizes
Bacterial concentration is adjusted to 10 by Maxwell opacity tube7CFU/ml, for the toleration examination to temperature, NaCl, gastric juice, intestinal juice and cholate
Test.
(1) temperature tolerance
12 thick test tubes are taken to be divided into 4 groups, every group 3 are repeated parallel, every addition 10ml fermentation medium, will be withered after sterilizing
Careless bacillus cereuss shou003 is inoculated in 12 test tubes respectively with 1% inoculum concentration, then respectively at 70 DEG C, 80 DEG C, 90 DEG C and
After 100 DEG C process 30min, the OD value at each group bacterium solution 600nm is surveyed in sampling after culture 12h at 37 DEG C.
After result of the test is as shown in figure 1, process through 70 DEG C, bacillus cereuss still are able to grow, but are as treatment temperature
Raise, OD value is gradually lowered；In the presence of 80 DEG C and 90 DEG C of high temperature, the growth of antibacterial is affected, but or can grow,
And after 100 DEG C of effects, the growth of antibacterial is suppressed, and does not substantially grow.
(2) salt tolerance
In the fermentation medium add NaCl so as to mass fraction be respectively 0 ‰, 5 ‰, 20 ‰, 50 ‰, 70 ‰,
100 ‰, it is sub-packed in 250ml conical flask, every bottle of 100ml, bacillus cereuss bacteria suspension is accessed above-mentioned culture with 1% inoculum concentration
In base, after culture 12h in 37 DEG C of shaking tables, survey the OD value at 600nm.
Result of the test as shown in Fig. 2 bacillus subtilises shou003 has certain demand to NaCl in growth course,
In the range of 0～35 ‰, bacterial growth is good, grows best in 5 ‰ NaCl concentration, after concentration is more than 5 ‰, with
The rising of concentration, the growth of bacterium is necessarily suppressed, but salinity is that 50 ‰ bacterial growths are not bad.The average salt of general sea water
Degree, is 35 ‰ taking Pacific Ocean sea water as a example, so, this bacterium all adapts to sea water and limneticum, and subject range is relatively wide.
(3) resistant to gastric juice
With 1mol/L HCl, the distilled water of sterilizing is diluted, makes the pH of sterilized water be respectively 1.5,2.5 and 3.5, often
Individual pH takes in 100ml to 250ml triangular flask respectively, 121 DEG C of sterilizing 30min；After being cooled to less than 50 DEG C, add in each triangular flask
Enter 0.5g pepsin to mix；The bacteria suspension of bacillus cereuss is added in conical flask by 1% bacterium amount that connects, in 37 DEG C of shaking tables
Culture, measure respectively each bacillus cereuss 0,1,2, the OD of 3h600nm.
Result of the test is as shown in figure 3, bacillus subtilises shou003 has preferable toleration to simulated gastric fluid, in pH
During for 3.5, thalline is less by being affected, and bacterial number reduces about 30%；And the reduction with pH, the enhancing of acidity, bacterium
The inhibitory action that bulk-growth is subject to also gradually strengthens, pH be 1.5 gastric juice environment under, effect 2h after, thalline quantity is reduced to
48.9% originally.
(4) resistance to intestinal juice
Take 0.68g potassium dihydrogen phosphate to be dissolved in 100ml water, then with the NaOH of 1mol/L, pH is adjusted to 6.8,121 DEG C
Sterilizing 30min, after being cooled to less than 50 DEG C, adds 1g trypsin to mix in each conical flask；The bacterium of bacillus cereuss is hanged
Liquid is added in conical flask by 1% bacterium amount that connects, cultivate in 37 DEG C of shaking tables, measure respectively each bacillus cereuss 0,1,2,3h
OD600nm.
, as shown in figure 4, bacillus subtilises shou003 grows 3h under the conditions of simulated intestinal fluid, first 2 little for result of the test
When, the prolongation in time of the content of antibacterial is gradually lowered, and after 3h, the content of antibacterial raises on the contrary, only drops compared with initial value
Low by 9%, this bacillus subtilis has good toleration to intestinal juice.
(5) bile tolerance
Add cholate in the fermentation medium so as to mass fraction is respectively 0.25%, 0.5%, 1%, 1.5% and 2%,
It is sub-packed in the conical flask of 250ml, every bottle of 100ml, 121 DEG C of sterilizing 30min；By the bacteria suspension of bacillus cereuss by 1% connect
Bacterium amount adds in conical flask, cultivates in 37 DEG C of shaking tables, respectively at 0,1,2,3h measure OD600nm.
Result of the test is as shown in figure 5, bacillus subtilises grow 3h, front 2 hours, bacterial content under the conditions of containing cholate
Prolongation in time is gradually lowered, and after 3h, bacterial content raises on the contrary, illustrates that this bacillus subtilis has preferably to cholate
Toleration, when cholate content is higher, though bacillus cereuss can not grow, will not be dead.
The sensitivity to antibiotic for the embodiment 4 bacillus subtilises shou003
Take 0.1ml bacillus cereuss bacteria suspension to coat on detection culture medium flat plate, after bacterium solution is absorbed, stick susceptibility paper
Piece, after 37 DEG C of culture 16h, measures antibacterial circle diameter.Antibiotic used by test and result are as shown in table 1 below：
The sensitivity to antibiotic for the table 1 bacillus subtilises shou003
Bacillus subtilises shou003 is all more sensitive to 18 kinds of conventional feeding antibiotics, is safe for animal and fowl fodder,
There is the potential quality as probiotic bacteria, meanwhile, when using it should be noted that avoid using with antibiotic simultaneously.
The safety of embodiment 5 bacillus subtilises shou003
Blood plate：By the test strains streak inoculation of incubated overnight in nutrient broth medium on Sanguis caprae seu ovis flat board, 37 DEG C
Culture 24h, according to the formation of periphery of bacterial colonies transparent circle, to judge the generation of hemolysin, and with vibrio alginolyticus Va-Y (Yangzhou University
Teacher Fu Lixia give, in document：" Wang Juan, Feng Yonghui, Cai Lisheng, Wang Jian, Qu Xiancheng, Zhang Qinghua (communication author).It is derived from
The screening of vibrio Antagonistic Fungi of Carnis Pseudosciaenae intestinal and identification, Oceanologia et Limnologia Sinica, 2010,41 (5)：707-713. " disclosed in) conduct
Positive control.
Mice：The bacterial strain of 18h will be cultivated, culture medium is gone in centrifugation, and the normal saline gradient with 0.85% is dilute in nutrient broth
It is interpreted as 9 × 1010CFU/ml, 9 × 109CFU/ml, 9 × 108Tri- concentration of CFU/ml.Then lumbar injection infecting mouse, 1ml/
Only, every group 6, as negative control, vibrio alginolyticus Va-Y is as positive control (9 × 10 for normal saline9CFU/ml).Feed one
Zhou Hou, observes mouse growth situation.
Carassius aurutus gibelio：The same mouse experiment of method, lumbar injection infects Carassius auratuss, 1ml/ tail, every group of 10 tails, normal saline conduct
Negative control, vibrio alginolyticus Va-Y is as positive control (9 × 109CFU/ml).After feeding one week, observe Carassius auratuss growing state.
Found by flat board hemolytic test, vibrio alginolyticus Va-Y has haemolysises on blood plate, and bacillus subtilises
Shou003 does not observe haemolysises on blood plate, tentatively shows that this bacterial strain does not produce hemolysin, does not have and potentially cause a disease
Property.
, different degrees of intoxicating phenomenon in 2 days in the mice of infection vibrio alginolyticus Va-Y, and experiment mice one Zhou Houjun
Intoxicating phenomenon does not occur, with matched group zero difference it can be determined that this bacterial strain mice is not had pathogenic.
All dead in the Carassius auratuss second day of injection vibrio alginolyticus Va-Y, and all intoxicating phenomenon in experiment mice, with
Blank zero difference it can be determined that this bacterial strain Carassius auratuss are not had pathogenic.
The preparation of the anti-vibrio albumen of embodiment 6
(1) it is inoculated in battalion after the bacillus subtilises shou003 described in claim 1 being activated on nutrient agar slopes
Foster broth bouillon, 28 DEG C of constant-temperature shaking culture 18～20h, it is centrifuged 20min under the conditions of 9600r/min, 4 DEG C, take supernatant
Through 0.22 μm of filtering with microporous membrane, obtain filter liquid；
(2) use ammonium sulfate precipitation method (reference literature：" Wang Jiazheng, biochemistry, Fan Ming. DNA techniques handbook：Science
Publishing house；2000 " record method operation) described filter liquid is extracted, obtain anti-vibrio protein extract；Wherein, ammonium sulfate
The mass concentration after described filter liquid is added to be 70%.
In step (2), described ammonium sulfate precipitation method specifically includes following steps：Take bacillus cereuss filtrate 50ml, plus
Entering solid ammonium sulfate makes solution reach mass percent concentration 70%, at 4 DEG C overnight, must sink after 8000r/min centrifugation 20min
Starch, precipitation 0.05M Tris-HCl buffer solution, material in bag taking after dialysis 48h, it is this concentration sulphuric acid ammonium precipitation
The albumen being obtained.
(3) antagonistic experiment detection find numbering be 7 protein to vibrio alginolyticus Va-Y (Yangzhou University's teacher's Fu Lixia favour
Give, in document：" Wang Juan, Feng Yonghui, Cai Lisheng, Wang Jian, Qu Xiancheng, Zhang Qinghua (communication author).From Carnis Pseudosciaenae intestinal
The screening of vibrio Antagonistic Fungi and identification, Oceanologia et Limnologia Sinica, 2010,41 (5)：Disclosed in 707-713. ") growth have suppression make
With.
The detection of the anti-vibrio albumen of embodiment 7
(1) 4.9ml distilled water, 6ml 30% acrylamide, 3.8ml 1.5mol/L Tris-HCl, 150 μ l 10% are taken
SDS, 150 μ l 10% Ammonium persulfate., 6 μ l TEMED mix homogeneously, encapsulating, to after suitably highly, adds 1ml dehydrated alcohol envelope
Live, after colloid solidification, ethanol is poured out, thoroughly blot ethanol with absorbent paper；Fill 5% concentration glue：Take 5.5ml distilled water,
1.3ml 30% acrylamide, 1.0ml 1.5mol/L Tris-HCl, 80 μ l 10%SDS, 80 μ l 10% Ammonium persulfate., 8 μ l
TEMED mix homogeneously, injects separation gel upper strata, after be immediately inserted into comb, take out comb after 30min colloid solidification, by 1 × electricity
Electrophoresis tank poured into by swimming buffer, and floods short glass plate.
(2) take 20 μ l protein extracts to mix with 4 μ l sample buffer (5 ×), make albumen in 100 DEG C of heating in water bath 3min
Degeneration, injects loading wells with liquid-transfering gun with the amount of 20 μ l, using sample buffer as negative control.
(3) when carrying out electrophoresis, it is 80V using the voltage concentrating in glue, in separation gel, voltage is 120V, when bromophenol blue forward position
Run to away from bottom line 1cm when close power supply.
(4), after proteins gel electrophoresis terminate, gel is carefully taken off from glass plate, plus 100ml pure water, level shake
(30r/min)30min；Pure water is poured out, adds dyeing liquor 100ml, seal, level shake (30r/min) 1h is to overnight；
Dyeing liquor is poured out, adds destaining solution 100ml, seal, level shakes (30r/min)；To the background of gel, be reduced to can
When to accept, destaining solution is poured out, pour 100ml pure water into, scan gel, result is as shown in fig. 6, this anti-vibrio albumen
Molecular weight is 59KD.
(5) carried out using raw work rubber tapping QIAquick Gel Extraction Kit (Micro Protein PAGE Recovery Kit, BSP062)
Protein Recovery.
The antagonistic experiment of the anti-vibrio albumen of embodiment 8 and sequencing
(1) vibrio alginolyticus Va-Y is cultivated and wash lower lawn with 0.85% sterile saline after 18～20h, with reference to Maxwell
It is 10 that opacity tube adjusts its concentration5CFU/ml, takes 0.1ml to coat on suitable flat board, uniform with coating rod coating.
(2) with tweezers, 4 aseptic Oxford cups are equably put in the plate of horizontal positioned, draw 0.25ml albumen molten
Liquid, in Oxford cup, is careful not to overflow outside Oxford cup.37 DEG C of culture 24～48h observations have or not inhibition zone to be occurred and records straight
Footpath.
(3) the anti-vibrio albumen having inhibition zone to produce is delivered to Shanghai raw work biology company limited and carry out Mass Spectrometric Identification, survey
Sequence result such as SEQ ID NO：Shown in 1.Specific peptide sequence correspond to specific albumen, thus identifying according to peptide sequence
Albumen to be detected.16 peptide fragments are had to match with chitinase in the peptide fingerprinting spectrum of bacillus subtilises shou003, albumen sequence
Row coverage rate is about 38%, and qualification result shows, the protein to vibrio with bacteriostasis is chitinase.
The antimicrobial spectrum of embodiment 9 shou003 measures
Method (Smith P, Davey S, 1993.Evidence for the competitive with reference to Smith etc.
excluding of Aeromonas salmonicida from fish with stress-inducible
furunculosis by a Fluorescent Pseudomonad.Journal of Fish Disease,16:521-524)
It is modified slightly.Concretely comprise the following steps：With a certain amount of physiological saline solution (0.85%NaCl) by activate after vibrio parahaemolytious
(Vp1.2164), vibrio alginolyticus (Va-Y), Aeromonas hydrophila (Ah-9), the lawn of Wdwardsiella tarda (Et-Y) are washed down, point
Do not draw and be coated on 100 μ l addition nutrient agar flat boards.Then scrape amplification culture 24h respectively with inoculating loop
Experimental strain dibbling on above-mentioned flat board, and put on corresponding numbering, 28 DEG C of constant temperature culture, 24～48h carries out the sight of inhibition zone
Examine and measure, result is as shown in table 2 below：
The antimicrobial spectrum to pathogen for the table 2 shou003 bacterial strain
From Table 2, it can be seen that bacterial strain shou003 is stronger to the antagonism of two pathogen strain vibrios, to vibrio parahaemolytious
And the antibacterial circle diameter of vibrio alginolyticus (Va-Y) respectively reaches 16mm and 9.5mm (Vp1.2164)；And to common on Aquatic product
Two kinds of pathogen, Aeromonas hydrophila (Ah-9), the antibacterial circle diameter of Wdwardsiella tarda (Et-Y) respectively reach：15.2mm、
11.3mm.
Compare the shortcoming and defect with prior art, the invention has the advantages that：
(1) the bacillus subtilises shou003 of the present invention has good inhibitory action to pathogenic vibrio, and to salt
Degree, gastric juice, intestinal juice and cholate all have preferable toleration, still active and can grow, to common under 70 DEG C of high temperature
Antibiotic sensitive, there is no transferable drug-resistance factor, and laboratory animal had no side effect safely, illustrate that this bacterium has probiotic bacteria
Characteristic, can be applied to the bacteria-treating in aquaculture process, the quantity of effective control pathogen, reduce sending out of disease
Raw, strengthen the immunity of cultivated animals, have broad application prospects.
(2) method of the bacillus subtilises shou003 fermentation culture of the present invention be this bacillus subtilises shou003
Excellent fermentation process, can carry out scale fermentation, be suitable to industrialized development.
(3) the anti-vibrio albumen of the present invention has good inhibitory action to pathogenic vibrio, can be prepared into single examination
Agent or medicine, have a good application prospect.
(4) preparation method of the anti-vibrio albumen of the present invention is simple, is suitable to industrialized development.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all essences in the present invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.
Claims (5)
1. a kind of bacillus subtilises (Bacillus subtilis) shou003, is preserved in Chinese allusion quotation on November 13rd, 2013
Type culture collection, deposit number is CCTCC NO：M 2013571.
2. a kind of method of the bacillus subtilises shou003 described in fermentation culture claim 1 is it is characterised in that include following
Step：
(1) described bacillus subtilises shou003 strain is taken to be inoculated in activation medium, after 37 DEG C of shaking table culture 18～20h,
It is centrifuged 5min under the conditions of 8000r/min, taking precipitation with aseptic water washing and being made into concentration is 107CFU/ml bacterium solution；
(2) described bacterium solution is seeded in fermentation medium by the inoculum concentration of fermentation medium 1% volume, 4～40 DEG C, pH be
Shaking table culture 10～16h under the conditions of 4～7, obtains bacillus subtilises shou003 fermentation liquid；
In step (1), described activation medium includes following component：
Peptone 9～10g/L,
Extracted beef powder 2～3g/L,
Sodium chloride 4～5g/L；
In step (2), described fermentation medium pH is 5, and described fermentation medium includes following component：
Maltose 1～2g/L,
Carnis Bovis seu Bubali cream 1～2g/L,
CaCl20.2～0.3g/L.
3. the method for fermentation culture bacillus subtilises shou003 as claimed in claim 2 is it is characterised in that in step (1)
In, described activation medium includes following component：
Peptone 10g/L,
Extracted beef powder 3g/L,
Sodium chloride 5g/L；
In step (2), described fermentation medium includes following component：
Maltose 2g/L,
Carnis Bovis seu Bubali cream 2g/L,
CaCl20.3g/L.
4. application in preparing aquaculture feed for the bacillus subtilises shou003 described in claim 1.
5. application in preparing aquaculture feed for the bacillus subtilises shou003 as claimed in claim 4, its feature exists
In described bacillus subtilises shou003 is used for suppressing aquatic pathogenic bacterium；
Described aquatic pathogenic bacterium includes vibrio parahaemolytious (Vibrio parahaemolyticus), vibrio alginolyticus (Vibrio
Alginolyticus), Aeromonas hydrophila (Aeromonas hydrophila), Wdwardsiella tarda (Edwardsiella
tarda).
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| CN107937300A (en) * | 2017-11-08 | 2018-04-20 | 青岛农业大学 | A kind of bacillus subtilis and its application in aquaculture |
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