CN108103032A - A kind of vibrio alginolyticus bacteriophage probiotics and its preparation method and application - Google Patents
A kind of vibrio alginolyticus bacteriophage probiotics and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of vibrio alginolyticus bacteriophage probioticses and its preparation method and application, belong to microbial technology field.The preparation method is to be seeded in fermentation culture and carry out fermented and cultured vibrio alginolyticus liquid and vibrio alginolyticus phagocytosis body fluid；When fermented and cultured is carried out to 6~15h, stream plus vibrio alginolyticus liquid into host's zymotic fluid continue fermented and cultured, control the control of entire fermentation period 18~for 24 hours, zymotic fluid carries out inactivation vibrio alginolyticus host, obtains vibrio alginolyticus bacteriophage probiotics.The present invention can realize the potency on the premise of not extending fermentation time, greatly improving vibrio alginolyticus bacteriophage by the way that fermentation flow added-time machine is controlled to carry out 6~15h, stream plus host's liquid for fermented and cultured.
Description
Technical field
The invention belongs to microbial technology fields, and in particular to a kind of vibrio alginolyticus bacteriophage probiotics and its preparation
Methods and applications.
Background technology
At present, the main means for controlling and eliminating pathogenic bacteria are still various methods physically or chemically, including various antibiosis
The use of element.Method physically or chemically all there is it is apparent the drawbacks of.First, elimination of the method for physics to various pathogenic bacteria
Effect can be only applied to a certain link of association area, it is impossible to realize the application of all links in entire field.Secondly, chemical method
Though soda acid processing in can obtain certain control effect, and after harmful bacteria forms biomembrane, the effect of these methods is with regard to pole
It is undesirable.The research and development of microbial ecological preparation are subject to each side to pay close attention to, and the research of particularly bacteriophage is favored be subject to people.
Since bacteriophage is found by Frederick Twort (1915) and F é lix DH é relle (1917), Europe and the former Soviet Union etc.
Country has just just started to treat bacterium infection with bacteriophage.However in the emergence of the antibiotic such as penicillin, streptomysin, with
And preprophage drug failure it is fast the shortcomings of these drugs be eliminated soon.Although antibiotic begins to use effect to show
It writes, but long-time service, it can not only enhance the resistance to the action of a drug of pathogen, but also inhibit profitable strain, cannot finally efficiently control,
Eliminate pathogenic bacteria.Late 1990s, many researchers, which are advocated, re-applies bacteriophage to clinical treatment, phagocytosis
Autogenic therapy is gradually recovered.In addition, bacteriophage has a wide range of applications in itself, such as Bacteria Identification and parting, for diagnosing
With treatment disease, the experimental tool of molecular biology research is also served as.Therefore, bacteriophage is widely applied value promotion bacteriophage
Fermenting and producing have been to be concerned by more and more people.
At present, the fermentation method for producing of bacteriophage has very much, for example, number of patent application is 94114314.7, it is entitled " to bite
The application for a patent for invention of the preparation method of thalline ecological agent " proposes a kind of medium culture method：In 10L aspirator bottles, liquid is filled
Bacteriophage is cultivated using host vibrios 5~7 days, final phage titer is 10 for 5000ml5~108PFU/ml.Number of patent application
200810145709.5 patent of invention uses host vibrios freeze-dried powder, and the potency of bacteriophage is not also high as nutrient source, be 1 ×
107PFU/ml.The life of bacteriophage is carried out in the patent of invention of number of patent application 200810202809.7 using Aeromonas hydrophila
Production, fermentation period is long, is 72~120h, and phage titer is 1 × 108PFU/ml.The hair of bacteriophage disclosed in above-mentioned patent
Fermenting process there are phage titer it is low the problem of.
The content of the invention
In view of this, it is an object of the invention to provide a kind of vibrio alginolyticus bacteriophage probioticses and preparation method thereof
And application, the preparation method ferment to obtain the vibrio alginolyticus bacteriophage of high-titer.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The present invention provides a kind of preparation methods of vibrio alginolyticus bacteriophage probiotics, comprise the following steps：
1) vibrio alginolyticus liquid is accessed in fermentation medium and cultivated, obtain host's zymotic fluid；Vibrio alginolyticus host is being fermented
Initial cell concentration in culture medium is 1010~1016CFU/ml；
2) it is 10 by concentration8~1016The vibrio alginolyticus phagocytosis body fluid of PFU/ml, which is seeded in host's zymotic fluid, to ferment
Culture；The inoculum concentration of vibrio alginolyticus phagocytosis body fluid is 1%~10%；
3) when fermented and cultured carries out 6~12h in the step 2), solubilization is flowed in host's zymotic fluid into the step 2)
Algae vibrios liquid continues fermented and cultured, control entire fermentation period for 18~for 24 hours, obtain zymotic fluid；
4) zymotic fluid obtained in the step 3) is subjected to inactivation vibrio alginolyticus host, it is micro- obtains vibrio alginolyticus bacteriophage
Ecological agent.
Preferably, the stream of vibrio alginolyticus liquid plus volume are the 1%~10% of the volume of vibrio alginolyticus liquid in the step 3)；
The concentration of vibrio alginolyticus liquid is 1010~1014PFU/ml。
Preferably, the volume of stream plus vibrio alginolyticus liquid is the 0.5% of host's fermentating liquid volume per hour in the step 3)
~5%.
Preferably, in the step 3) between the stream added-time of vibrio alginolyticus liquid it is 2~4h.
Preferably, fermentation medium includes following content component in the step 1)：1~20g/L of peptone, beef extract powder
1~20g/L of 1~20g/L and sodium chloride；The pH value of the fermentation medium is 7.0~9.0.
Preferably, the fermentation culture conditions in the step 2) and step 3) are independently as follows：Fermentation temperature is 28~35 DEG C,
The pH value of host's zymotic fluid maintains 6~9；Fermentation carries out under agitation, and speed of agitator is 50~200rpm.
Preferably, the method for inactivation is water-bath in the step 4)；The temperature of water-bath is 45~70 DEG C；The time of water-bath is
2~5h.
The present invention provides a kind of microorganism formulation of vibrio alginolyticus bacteriophage, the potency of vibrio alginolyticus bacteriophage is 1012
~1016PFU/ml。
The present invention provides the vibrio alginolyticus bacteriophage microorganism formulation identify or detect in aquaculture it is molten
Application in algae vibrios.
The present invention provides a kind of preparation method of vibrio alginolyticus bacteriophage probiotics, by vibrio alginolyticus liquid and molten algae
Vibriophage liquid, which is seeded in fermentation culture, carries out fermented and cultured；When fermented and cultured carries out 6~15h, ferment to host
Stream plus vibrio alginolyticus liquid in liquid, continue fermented and cultured, the control of entire fermentation period 18~for 24 hours, obtained zymotic fluid goes out
Vibrio alginolyticus host living, obtains vibrio alginolyticus bacteriophage probiotics.The present invention is avoided after disposably adding vibrio alginolyticus liquid
Make the reduction of fermentation later stage host's bacteria concentration, and then influence the fermenting and producing of bacteriophage, flowed when fermented and cultured is carried out to 6~12h
Add supplement host's liquid, can realize the potency on the premise of not extending fermentation time, greatly improving bacteriophage.By different fermentations
The experimental verification of volume, using preparation method provided by the invention, the potency of the vibrio alginolyticus bacteriophage of fermenting and producing is 1010~
1016Compared with the method for the bacteriophage of prior art production, 2~6 orders of magnitude are improved in potency by PFU/ml.
Meanwhile the inactivation treatment of zymotic fluid has been carried out in preparation method provided by the invention, vibrio alginolyticus host is made to be gone out
It is living, the content of host vibrios in bacteriophage probiotics can be not only eliminated, but also has been avoided that the harm to environment.
Further, preparation method provided by the invention specifically defines the item of fermentation medium component and fermented and cultured
Part prepares vibrio alginolyticus bacteriophage microorganisms microbial inoculum with interflow plus the operation of host's zymotic fluid, can effectively further improve together
The potency of vibrio alginolyticus bacteriophage.
Specific embodiment
The present invention provides a kind of preparation methods of vibrio alginolyticus bacteriophage probiotics, comprise the following steps：
1) vibrio alginolyticus liquid is accessed in fermentation medium and cultivated, obtain host's zymotic fluid；Vibrio alginolyticus host is being fermented
Initial cell concentration in culture medium is 1010~1016CFU/ml；
2) it is 10 by concentration8~1016The vibrio alginolyticus phagocytosis body fluid of PFU/ml, which is seeded in host's zymotic fluid, to ferment
Culture；The inoculum concentration of vibrio alginolyticus phagocytosis body fluid is 1%~10%；
3) when fermented and cultured carries out 6~12h in the step 2), solubilization is flowed in host's zymotic fluid into the step 2)
Algae vibrios liquid continues fermented and cultured, control entire fermentation period for 18~for 24 hours, obtain zymotic fluid；
4) zymotic fluid obtained in the step 3) is subjected to inactivation vibrio alginolyticus host, it is micro- obtains vibrio alginolyticus bacteriophage
Ecological agent.
Vibrio alginolyticus liquid is accessed in fermentation medium and cultivated by the present invention, obtains host's zymotic fluid；Vibrio alginolyticus host exists
Initial cell concentration in fermentation medium is 1010~1016CFU/ml。
The present invention is not particularly limited the method for the access, using access way well-known to those skilled in the art
.
In the present invention, the fermentation medium fermentation medium preferably includes following content component：1~20g/ of peptone
L, 1~20g/L of 1~20g/L of beef extract powder and sodium chloride；More preferably 5~15g/L of peptone, 5~15g/L of beef extract powder and
3~10g/L of sodium chloride；Most preferably peptone 10g/L, beef extract powder 10g/L and sodium chloride 5g/L.The fermentation medium
PH value is preferably 7.0~9.0, and more preferably 7.5~8.5, it is most preferably 8.0.Preparation of the present invention to the fermentation medium
Method is not particularly limited, using the preparation method of culture medium well-known to those skilled in the art.
In the present invention, after in the vibrio alginolyticus liquid access fermentation medium, vibrio alginolyticus host is in fermentation medium
In cell concentration be preferably 1010~1016CFU/ml, more preferably 1012~1015CFU/ml is most preferably 1014CFU/ml。
The present invention is not particularly limited the source of the vibrio alginolyticus host, using vibrio alginolyticus well-known to those skilled in the art
Bacterial strain.In embodiments of the present invention, the vibrio alginolyticus is isolated from Penaeus Vannmei enteron aisle.Leg Shrimp Litopenaeus vannamei Culture Raceway-type Ponds position
In Shandong, Fujian, Guangdong, Hainan and other places, acquisition time 2016.9.Isolated vibrio alginolyticus carries out by conventional method
Expand culture, obtain vibrio alginolyticus liquid.
After obtaining host's zymotic fluid, concentration is 10 by the present invention8~1016The vibrio alginolyticus phagocytosis body fluid of PFU/ml is seeded to
Fermented and cultured in host's zymotic fluid；The inoculum concentration of vibrio alginolyticus phagocytosis body fluid is 1%~10%.
In the present invention, the source of the vibrio alginolyticus bacteriophage is not particularly limited, using those skilled in the art institute
Well known vibrio alginolyticus bacteriophage source.In the embodiment of the present invention, the vibrio alginolyticus bacteriophage is divided from vibrio alginolyticus
From obtaining.The present invention is not particularly limited the separated method, using bacteriophage well-known to those skilled in the art point
From method.
The present invention is not particularly limited the method for the inoculation, using access way well-known to those skilled in the art
.In the present invention, the concentration of vibrio alginolyticus phagocytosis body fluid is preferably 1010~1014PFU/ml, more preferably 1012PFU/
ml.The inoculum concentration of vibrio alginolyticus phagocytosis body fluid is preferably 3%~7%, and more preferably 5%.
In the present invention, the conditional sampling that fermented and cultured is carried out to the bacteriophage is preferably as follows：Fermentation temperature for 28~
35 DEG C, more preferably 30~32 DEG C.The pH value of host's zymotic fluid maintains 7~9, more preferably 7.5~8.5, is most preferably 8.0；
Fermentation carries out under agitation, and speed of agitator is 50~200rpm, more preferable 100~150rpm.
The present invention is when the fermented and cultured carries out 6~15h, stream plus vibrio alginolyticus liquid into host's zymotic fluid, after supervention
Ferment culture, control entire fermentation period for 18~for 24 hours, obtain zymotic fluid.
In the present invention, the stream of the vibrio alginolyticus liquid plus volume are preferably the 1%~10% of host's fermentating liquid volume, more
Preferably 2%~8%, it is most preferably 5%.The concentration of the vibrio alginolyticus liquid is 1010~1014PFU/ml。
In the present invention, the volume of stream plus vibrio alginolyticus liquid is the 0.5%~5% of host's fermentating liquid volume per hour, more
Preferably 1%~4%, it is most preferably 3%.
In the present invention, it is preferably 2~4h between the stream added-time of the vibrio alginolyticus liquid, more preferably 3h.
In the present invention, the condition for continuing fermented and cultured is preferably as follows：Fermentation temperature is 28~35 DEG C, more preferably
30~32 DEG C.The pH value of host's zymotic fluid maintains 6~9, more preferably 7~8；Fermentation carries out under agitation, speed of agitator
For 50~200rpm, more preferable 100~150rpm.
After obtained zymotic fluid, the zymotic fluid is carried out inactivation vibrio alginolyticus host by the present invention, is obtained vibrio alginolyticus and is bitten
Thalline probiotics.
In the present invention, the method for the inactivation is preferably water-bath；The temperature of water-bath is preferably 45~70 DEG C, more preferably
50℃.The time of water-bath is preferably 2~5h, more preferably 3~4h.
The present invention provides a kind of microorganism formulation of vibrio alginolyticus bacteriophage, the potency of vibrio alginolyticus bacteriophage is 1012
~1016PFU/ml。
In the present invention, the titration method of the vibrio alginolyticus bacteriophage, is measured with double-layer agar technique；The bilayer
Flat band method：1mL bacteriophage zymotic fluids are taken, 8000rpm centrifugation 20min take 100 μ L supernatants, add in 900 μ L sterile salines
In shake up, carry out gradient dilution successively.Take 10- 12~-14The dilution 100 μ L and 100 μ L of dilution gradient are in exponential phase
Host strain is added to 8mL and is cooled in 50 DEG C or so of semisolid culturemedium, mixing, and 33 DEG C are inverted culture 12h, and plaque is carried out
It counts.The number of plaque and the calculation formula of potency are as follows：
Plaque number × extension rate × 10 on thalline potency (PFU/m L)=tablet.
Microorganism formulation or the vibrio alginolyticus the present invention provides the vibrio alginolyticus bacteriophage of the method preparation
The application in vibrio alginolyticus in aquaculture is identified or detected to the microorganism formulation of bacteriophage.
A kind of vibrio alginolyticus bacteriophage probiotics provided by the invention and vibrio alginolyticus are bitten with reference to embodiment
Thalline fermentation preparation and application are described in detail, but they cannot be interpreted as the limit to the scope of the present invention
It is fixed.
Embodiment 1
Vibrio alginolyticus is isolated from leg Shrimp Litopenaeus vannamei Culture Raceway-type Ponds.Leg Shrimp Litopenaeus vannamei Culture Raceway-type Ponds are in Shandong, Fujian, Guangdong, Hainan
Etc. ground have.Acquisition time is in September, 2016.Vibrio alginolyticus separates from Penaeus Vannmei enteron aisle, vibrio alginolyticus bacteriophage from
It is separated in culture of Penaeus vannamei water and in seawater.
Separated 3 plants of vibrio alginolyticus bacteriophages expand cultural method, are as follows：First measure the optimal sense of bacteriophage
Dye plural number：When infection multiplicity (multiplicity of infection, MOI) refers to bacteriophage primary infection host strain
The ratio of bacteriophage quantity and host strain quantity, also referred to as infects multiple.In host strain inoculation 100ul to 200ml NB culture solutions,
150r/min 12h are cultivated to logarithmic phase.Then it is 10 to take initial potency9The bacteriophage culture solution of PFU/m L, it is multiple according to infection
Number be respectively 100,10,1,0.1,0.01,0.001,0.0001,0.00001,0.000001 ratios, by bacteriophage VP11 with
PVP11 is added in equal volume in fluid nutrient medium, 37 DEG C of shaking tables, 120r/min culture 10h, 4000r/min centrifugation 15min,
Remove precipitation, 0.22 μm of membrane filtration of supernatant, filter 23 time, with the potency of double-layer agar technique measure bacteriophage, potency highest
Infection multiplicity be optimal multiplicity of infection.The double-layer agar technique：1mL bacteriophage zymotic fluids are taken, 8000rpm centrifugation 20min take
100 μ L supernatants add in 900 μ L sterile salines and shake up, carry out gradient dilution successively.Take 10-11The dilution of dilution gradient
The host strain that liquid 100 μ L and 100 μ L are in exponential phase is added to 8mL and is cooled in 50 DEG C or so of semisolid culturemedium, mixes
Even, 33 DEG C are inverted culture 12h, and plaque is counted.It is inoculated with according to optimal multiplicity of infection, each of the total volume 2%, 33
DEG C, 150rpm cultivates 18h, obtains 3 plants of vibrio alginolyticus phagocytosis body fluid.
Embodiment 2
Stream plus the method for bacteriophage host fermenting and producing bacteriophage, comprise the following steps：
1) fermented using 50L fermentation tanks, the volume of fermentation medium is contained in fermentation tank as 35L, host is accessed
In fermentation medium, it is 10 to make clump count in initial medium10CFU/ml；The fermentative medium formula is as follows：Peptone
10g/L；Beef extract powder 10gL；Sodium chloride 1g/L；PH is 9.0；
2) the 10 of the 1% of first plant of fermentating liquid volume are added8The vibrio alginolyticus phagocytosis body fluid of PFU/ml is into fermentation tank
It is cultivated, fermentation temperature is 28 DEG C, rotating speed 200rpm；PH value is controlled 9.
3) when fermentation carries out 6h, stream plus bacteriophage host into fermentation tank, controlled concentration is 1014Between CFU/ml, stream
It is 3h, flow acceleration 2L/h between added-time, stream adds up the amount of fermentation volume 6%, when fermentation period is 18 small, waits zymotic fluids color
When becoming limpid by muddiness, you can put tank；
4) by the zymotic fluid of gained, the water-bath 5h under the conditions of 45 DEG C, host is to get bacteriophage probiotics for inactivation.
5) the titration method of bacteriophage probiotics uses double-layer agar technique, plaque is counted, specifically
For：1mL bacteriophage zymotic fluids are taken, 8000rpm centrifugation 20min take 100 μ L supernatants, add in 900 μ L sterile salines and shake
It is even, gradient dilution is carried out successively.Take 10-11The dilution 100 μ L and 100 μ L of dilution gradient are in the host strain of exponential phase
It is added to 8mL to be cooled in 50 DEG C or so of semisolid culturemedium, mixing, 33 DEG C are inverted culture 12h, count plaque.
The number of plaque and the calculation formula of potency are as follows：
Plaque number × extension rate × 10 on thalline potency (PFU/m L)=tablet.
Embodiment 3
Stream plus the method for bacteriophage host fermenting and producing bacteriophage, comprise the following steps：
1) fermented using 1000L fermentation tanks, the volume of fermentation medium is contained in fermentation tank as 800L, host is connect
Enter in fermentation medium, the clump count for keeping initial medium is 1015CFU/mL.The fermentative medium formula is as follows：Albumen
Peptone 20g/L；Beef extract powder 10g/L；Sodium chloride 5g/L；PH is 8.0.
2) PFU for adding other one plant of fermentating liquid volume 5% is 1016The phagocytosis body fluid of PFU/ml is carried out into fermentation tank
Culture, fermentation temperature are 35 DEG C, rotating speed 200rpm；PH is controlled 8.0.
3) interior when fermentation progress 8 is small, stream plus bacteriophage host, controlled concentration is 1012CFU/ml is small for 4 between the stream added-time
When, flow acceleration 35L/h, stream adds up the amount of fermentation volume 8%, when fermentation period is 20 small, zymotic fluids color is waited to present dark
During yellow, you can put tank.
4) by the zymotic fluid of gained, the water-bath 2h under the conditions of 70 DEG C, host is to get bacteriophage probiotics for inactivation.
5) the titration method of bacteriophage probiotics uses double-layer agar technique, plaque is counted, specifically
For：1mL bacteriophage zymotic fluids are taken, 8000rpm centrifugation 20min take 100 μ L supernatants, add in 900 μ L sterile salines and shake
It is even, gradient dilution is carried out successively.Take 10-13The dilution 100 μ L and 100 μ L of dilution gradient are in the host strain of exponential phase
It is added to 8mL to be cooled in 50 DEG C or so of semisolid culturemedium, mixing, 33 DEG C are inverted culture 12h, count plaque.
The number of plaque and the calculation formula of potency are as follows：
Plaque number × extension rate × 10 on thalline potency (PFU/m L)=tablet.
Embodiment 4
Stream plus the method for bacteriophage host fermenting and producing bacteriophage, comprise the following steps：
1) fermented using 5000L fermentation tanks, the volume of fermentation medium is contained in fermentation tank as 4000L, by host
It accesses in fermentation medium, the clump count for keeping initial medium is 1016CFU/ml；The fermentative medium formula is as follows：Egg
White peptone 10g/L；Beef extract powder 20g/L；Sodium chloride 8g/L；PH is 7.5.
2) PFU for adding the 3rd plant of fermentating liquid volume 1~10% is 108~1016The phagocytosis body fluid of PFU/ml extremely ferments
It is cultivated in tank, fermentation temperature is 32 DEG C, rotating speed 150rpm；Control is 7.5.
3) interior when fermentation progress 12 is small, stream plus bacteriophage host, controlled concentration is 1015Between CFU/ml, between the stream added-time
For 2 it is small when, flow acceleration 200L/h, stream add up fermentation volume 10% amount, fermentation period for 24 it is small when, wait zymotic fluids color
When becoming limpid by muddiness, you can put tank.
4) by the zymotic fluid of gained, the water-bath 3h under the conditions of 55 DEG C, host is to get bacteriophage probiotics for inactivation.
5) the titration method of bacteriophage probiotics is measured using double-layer agar technique, is specially：Take 1mL bacteriophages
Zymotic fluid, 8000rpm centrifugation 20min, takes 100 μ L supernatants, adds in 900 μ L sterile salines and shake up, carry out ladder successively
Degree dilution.Take 10-12The host strain that 100 μ L of the dilution and 100 μ L of dilution gradient are in exponential phase is added to 8mL and is cooled to
In 50 DEG C or so of semisolid culturemedium, mixing, 33 DEG C are inverted culture 12h, and plaque is counted.
The number of plaque and the calculation formula of potency are as follows：
Plaque number × extension rate × 10 on thalline potency (PFU/m L)=tablet.
Comparative example 1
Scheme according to embodiment 4 is fermented, and difference is as follows for fermentative medium formula：Tryptone 10g/L,
Yeast extract 5g/L, sodium chloride 10g/L.The obtained potency of bacteriophage probiotics of fermenting according to embodiment 4 method into
Row statistics and calculating.
Comparative example 2
Scheme according to embodiment 4 is fermented, be a difference in that will stream plus bacteriophage host's liquid in step 2) together
It adds in fermentation tank, the input constancy of volume of bacteriophage host's liquid.
The bioactivity of the bacteriophage probiotics of embodiment 2~4 and comparative example 1~2 the results are shown in Table 1.
The potency list of the bacteriophage probiotics of 1 embodiment 2~4 of table and comparative example 5
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of preparation method of vibrio alginolyticus bacteriophage probiotics, which is characterized in that comprise the following steps：
1) vibrio alginolyticus liquid is accessed in fermentation medium and cultivated, obtain host's zymotic fluid；Vibrio alginolyticus host is in fermented and cultured
Initial cell concentration is 10 in base10~1016CFU/ml；
2) it is 10 by concentration8~1016The vibrio alginolyticus phagocytosis body fluid of PFU/ml is seeded to fermented and cultured in host's zymotic fluid；
The inoculum concentration of vibrio alginolyticus phagocytosis body fluid is 1%~10%；
3) when fermented and cultured is carried out to 6~12h in the step 2), solubilization algae is flowed in host's zymotic fluid into the step 2)
Vibrios liquid continues fermented and cultured, control entire fermentation period for 18~for 24 hours, obtain zymotic fluid；
4) zymotic fluid obtained in the step 3) is subjected to inactivation vibrio alginolyticus host, obtains vibrio alginolyticus bacteriophage Tiny ecosystem
Preparation.
2. preparation method according to claim 1, which is characterized in that the stream of vibrio alginolyticus liquid adds volume in the step 3)
For the 1%~10% of host's fermentating liquid volume；The concentration of vibrio alginolyticus liquid is 1010~1014PFU/ml。
3. preparation method according to claim 1, which is characterized in that stream adds vibrio alginolyticus liquid per hour in the step 3)
Volume be host's fermentating liquid volume 0.5%~5%.
4. according to the preparation method described in claims 1 to 3 any one, which is characterized in that vibrio alginolyticus in the step 3)
It is 2~4h between the stream added-time of liquid.
5. preparation method according to claim 1, which is characterized in that fermentation medium includes containing below in the step 1)
Measure component：1~20g/L of 1~20g/L of peptone, 1~20g/L of beef extract powder and sodium chloride；The pH value of the fermentation medium is
7.0~9.0.
6. preparation method according to claim 1, which is characterized in that the fermented and cultured item in the step 2) and step 3)
Part is independently as follows：Fermentation temperature is 28~35 DEG C, and the pH value of host's zymotic fluid maintains 6~9；Fermentation carries out under agitation,
Speed of agitator is 50~200rpm.
7. according to the preparation method described in any one of claims 1 to 3,5 and 6, which is characterized in that inactivation in the step 4)
Method be water-bath；The temperature of water-bath is 45~70 DEG C；The time of water-bath is 2~5h.
8. the microorganism formulation of vibrio alginolyticus bacteriophage prepared by claim 1~7 any one the method, feature exist
In the potency of vibrio alginolyticus bacteriophage is 1012~1016PFU/ml。
9. vibrio alginolyticus in aquaculture is identified or detected to the microorganism formulation of the vibrio alginolyticus bacteriophage described in claim 8
In application.
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