CN108018262A - A kind of vibrio parahaemolyticus phage probiotics and its preparation method and application - Google Patents
A kind of vibrio parahaemolyticus phage probiotics and its preparation method and application Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 239000006041 probiotic Substances 0.000 title claims abstract description 25
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 25
- 238000000855 fermentation Methods 0.000 claims abstract description 72
- 230000004151 fermentation Effects 0.000 claims abstract description 72
- 239000007788 liquid Substances 0.000 claims abstract description 58
- 241000607598 Vibrio Species 0.000 claims abstract description 57
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- 230000002779 inactivation Effects 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims description 31
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 18
- 239000000843 powder Substances 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 9
- 239000001888 Peptone Substances 0.000 claims description 8
- 108010080698 Peptones Proteins 0.000 claims description 8
- 235000015278 beef Nutrition 0.000 claims description 8
- 239000000284 extract Substances 0.000 claims description 8
- 235000019319 peptone Nutrition 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 7
- 206010047400 Vibrio infections Diseases 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 206010018910 Haemolysis Diseases 0.000 claims description 6
- 238000009472 formulation Methods 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 6
- 230000008588 hemolysis Effects 0.000 claims description 6
- 244000005700 microbiome Species 0.000 claims description 6
- 239000002054 inoculum Substances 0.000 claims description 5
- 238000013019 agitation Methods 0.000 claims description 3
- 238000009360 aquaculture Methods 0.000 claims description 3
- 244000144974 aquaculture Species 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 230000000813 microbial effect Effects 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 3
- 241001515965 unidentified phage Species 0.000 description 47
- 238000010790 dilution Methods 0.000 description 12
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- 206010057249 Phagocytosis Diseases 0.000 description 11
- 230000008782 phagocytosis Effects 0.000 description 11
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- 229920001817 Agar Polymers 0.000 description 8
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- 241000238553 Litopenaeus vannamei Species 0.000 description 4
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- 238000004448 titration Methods 0.000 description 4
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- 241000607528 Aeromonas hydrophila Species 0.000 description 1
- 101001124039 Banna virus (strain Indonesia/JKT-6423/1980) Non-structural protein 4 Proteins 0.000 description 1
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- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
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Abstract
The present invention provides a kind of vibrio parahaemolyticus phage probiotics and its preparation method and application, belong to microbial technology field.The preparation method is that vibrio parahaemolytious liquid and vibrio parahaemolyticus phage liquid are seeded in fermentation culture to carry out fermented and cultured；When fermented and cultured is carried out to 6~12h, stream plus vibrio parahaemolytious liquid into host's zymotic fluid, continue fermented and cultured, in 18~24h, zymotic fluid carries out inactivation vibrio parahaemolytious host, obtains vibrio parahaemolyticus phage probiotics for whole fermentation period control.The present invention can realize the potency on the premise of not extending fermentation time, greatly improving vibrio parahaemolyticus phage by controlling fermentation flow added-time machine to carry out 6~12h, stream plus vibrio parahaemolytious liquid for fermented and cultured.
Description
Technical field
The invention belongs to microbial technology field, and in particular to a kind of vibrio parahaemolyticus phage probiotics and 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 obvious 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 whole field.Secondly, chemical method
Though soda acid processing in can obtain certain prevention 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.But in the emergence of the antibiotic such as penicillin, streptomysin, with
And preprophage drug failure it is fast the shortcomings of these medicines be eliminated soon.Although antibiotic begins to use effect to show
Write, but long-time service, it can not only strengthen the resistance to the action of a drug of pathogen, but also suppress 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 length 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 parahaemolyticus phage probiotics and its preparation side
Method and application, the preparation method ferment to obtain the vibrio parahaemolyticus phage 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 method of vibrio parahaemolyticus phage probiotics, comprise the following steps：
1) vibrio parahaemolytious liquid is accessed in fermentation medium and cultivated, obtain host's zymotic fluid；Vibrio parahaemolytious host exists
Initial cell concentration in fermentation medium is 1010~1016CFU/ml；
2) it is 10 by concentration8~1016The vibrio parahaemolyticus phage liquid of PFU/ml is seeded in host's zymotic fluid and sends out
Ferment culture；The inoculum concentration of vibrio parahaemolyticus phage liquid is 1%~10%；
3) when fermented and cultured carries out 6~12h in the step 2), stream plus vibrio parahaemolytious into obtained host's zymotic fluid
Liquid, continues fermented and cultured, it is 18~24h to control whole fermentation period, obtains zymotic fluid；
4) zymotic fluid obtained in the step 3) is subjected to inactivation vibrio parahaemolytious host, obtains vibrio parahaemolytious phagocytosis
Body probiotics.
Preferably, the stream of vibrio parahaemolytious liquid plus volume are the 1%~10% of host's fermentating liquid volume in the step 3)；
The concentration of secondary molten vibrios host liquid is 1010~1014PFU/ml。
Preferably, the volume of stream plus vibrio parahaemolytious liquid is host's fermentating liquid volume per hour in the step 3)
0.5%~5%.
Preferably, in the step 3) between the stream added-time of vibrio parahaemolytious 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 45~65 DEG C；The time of water-bath is
2~5h.
The microorganism formulation of the vibrio parahaemolyticus phage prepared the present invention provides the method, vibrio parahaemolytious phagocytosis
The potency of body is 1012~1016PFU/ml。
Microorganism formulation the present invention provides the vibrio parahaemolyticus phage is identified or detected in aquaculture
Application in vibrio parahaemolytious.
The present invention provides a kind of preparation method of vibrio parahaemolyticus phage probiotics, by vibrio parahaemolytious liquid and
Vibrio parahaemolyticus phage liquid, which is seeded in fermentation culture, carries out fermented and cultured；When fermented and cultured carries out 6~15h, to place
Stream plus vibrio parahaemolytious liquid in main fermentation liquid, continue fermented and cultured, whole fermentation period control is in 18~24h, obtained fermentation
Liquid carries out inactivation vibrio parahaemolytious host, obtains vibrio parahaemolyticus phage probiotics.The present invention avoids disposably adding
Make after vibrio parahaemolytious liquid fermentation later stage host's bacteria concentration reduction, and then influence bacteriophage fermenting and producing, fermented and cultured into
Row can realize the effect on the premise of not extending fermentation time, greatly improving bacteriophage to 6~12h, supplement stream plus host's liquid
Valency.By the experimental verification of different fermentations volume, using preparation method provided by the invention, the vibrio parahaemolytious of fermenting and producing bites
The potency of thalline is 1010~1016PFU/ml, compared with the method for the bacteriophage of prior art production, 2~6 numbers are improved in potency
Magnitude.
Meanwhile the inactivation treatment of zymotic fluid has been carried out in preparation method provided by the invention, make vibrio parahaemolytious host's quilt
Inactivation, can not only eliminate the content of host vibrios in bacteriophage probiotics, but also be avoided that the harm to environment.
Further, preparation method provided by the invention, specifically defines the bar of fermentation medium component and fermented and cultured
Part, prepares vibrio parahaemolyticus phage microbial bacterial agent with interflow plus the operation of host's zymotic fluid, effectively can further carry together
The potency of high vibrio parahaemolyticus phage.
Embodiment
The present invention provides a kind of preparation method of vibrio parahaemolyticus phage probiotics, comprise the following steps：
1) vibrio parahaemolytious liquid is accessed in fermentation medium and cultivated, obtain host's zymotic fluid；Vibrio parahaemolytious host exists
Initial cell concentration in fermentation medium is 1010~1016CFU/ml；
2) it is 10 by concentration8~1016The vibrio parahaemolyticus phage liquid of PFU/ml is seeded in host's zymotic fluid and sends out
Ferment culture；The inoculum concentration of vibrio parahaemolyticus phage liquid is 1%~10%；
3) when fermented and cultured carries out 6~12h in the step 2), into the step 2), stream plus pair are molten in host's zymotic fluid
Blood vibrios liquid, continues fermented and cultured, it is 18~24h to control whole fermentation period, obtains zymotic fluid；
4) zymotic fluid obtained in the step 3) is subjected to inactivation vibrio parahaemolytious host, obtains vibrio parahaemolytious phagocytosis
Body probiotics.
Vibrio parahaemolytious liquid is accessed in fermentation medium and cultivated by the present invention, obtains host's zymotic fluid；Vibrio parahaemolytious place
Main initial cell concentration in the 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 preferably includes following content component：1~20g/L of peptone, beef extract powder
1~20g/L of 1~20g/L and sodium chloride；More preferably 5~15g/L of peptone, 5~15g/L of beef extract powder and sodium chloride 3~
10g/L；Most preferably peptone 10g/L, beef extract powder 10g/L and sodium chloride 5g/L.The pH value of the fermentation medium is preferred
It is most preferably 8.0 for 7.0~9.0, more preferably 7.5~8.5.The present invention does not have the preparation method of the fermentation medium
It is specifically limited, using the preparation method of culture medium well-known to those skilled in the art.
In the present invention, the initial cell concentration of vibrio parahaemolytious in the fermentation medium is preferably 1010~1016CFU/
Ml, more preferably 1012~1015CFU/ml, is most preferably 1014CFU/ml.The present invention does not have the source of the vibrio parahaemolytious
It is specifically limited, using vibrio parahaemolytious bacterial strain well-known to those skilled in the art.In embodiments of the present invention, the pair
Hemolysis vibrion is isolated from Penaeus Vannmei enteron aisle.Leg Shrimp Litopenaeus vannamei Culture Raceway-type Ponds are located at the ground such as Shandong, Fujian, Guangdong, Hainan, collection
Time is 2016.9.Isolated vibrio parahaemolytious is enlarged culture by conventional method, obtains vibrio parahaemolytious liquid.
After obtaining host's zymotic fluid, concentration is 10 by the present invention8~1016The vibrio parahaemolyticus phage liquid inoculation of PFU/ml
The fermented and cultured into host's zymotic fluid；The inoculum concentration of vibrio parahaemolyticus phage liquid is 1%~10%.
In the present invention, the source of the vibrio parahaemolyticus phage is not particularly limited, using those skilled in the art
Known vibrio parahaemolyticus phage source.In the embodiment of the present invention, the vibrio parahaemolyticus phage is from secondary haemolysis
It is isolated in vibrios.The present invention is not particularly limited the separated method, and use is well-known to those skilled in the art
Bacteriophage separation method.
The present invention is not particularly limited the method for the inoculation, using access way well-known to those skilled in the art
.The concentration of vibrio parahaemolyticus phage liquid is preferably 1010~1014PFU/ml, more preferably 1012PFU/ml.Secondary haemolysis arc
The inoculum concentration of bacterium phagocytosis body fluid is preferably 3%~7%, and more preferably 5%.
In the present invention, the condition of the vibrio parahaemolyticus phage progress 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 is in stirring bar
Carried out under part, speed of agitator is 50~200rpm, more preferably 100~150rpm.
In the present invention, the present invention is when the fermented and cultured carries out 6~15h, in the host's zymotic fluid obtained to culture
Stream plus vibrio parahaemolytious liquid, continue fermented and cultured, it is 18~24h to control whole fermentation period, obtains zymotic fluid.
In the present invention, the stream of the vibrio parahaemolytious 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 vibrio parahaemolytious liquid is preferably 1010~1014PFU/ml, more preferably
1012~1013PFU/ml。
In the present invention, the volume of stream plus vibrio parahaemolytious 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, between the stream added-time of the vibrio parahaemolytious liquid be preferably 2~
4h, more preferably 3h.
In the present invention, the continuation fermentation culture conditions are 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 7~9, more preferably 7.5~8.5, is most preferably 8.0；Fermentation is under agitation
Carry out, speed of agitator is 50~200rpm, more preferably 100~150rpm.
After obtained zymotic fluid, the zymotic fluid is carried out inactivation vibrio parahaemolytious host by the present invention, obtains secondary haemolysis arc
Bacterium bacteriophage probiotics.
In the present invention, the method for the inactivation is preferably water-bath；The temperature of water-bath is preferably 45~65 DEG C, more preferably
50℃.The time of water-bath is preferably 2~5h, more preferably 3~4h.
The microorganism formulation of the vibrio parahaemolyticus phage prepared the present invention provides such scheme, vibrio parahaemolytious phagocytosis
The potency of body is 1012~1016PFU/ml。
In the present invention, the titration method of the vibrio parahaemolyticus phage, is measured with double-layer agar technique；It is described double
Layer flat band method：1mL bacteriophage zymotic fluids are taken, 8000rpm centrifugation 20min, take 100 μ L of supernatant liquid, add 900 μ L sterile physiological salt
Shaken up in water, carry out gradient dilution successively.Take 10- 11~-13The dilution 100 μ L and 100 μ L of dilution gradient are in exponential phase
Host strain be added to 8mL and be cooled in 50 DEG C or so of semisolid culturemedium, mix, 33 DEG C are inverted culture 12h, to plaque into
Row 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 the present invention provides the vibrio parahaemolyticus phage is identified or detected in aquaculture
Application in vibrio parahaemolytious.
With reference to embodiment to a kind of vibrio parahaemolyticus phage probiotics provided by the invention and its preparation side
Method and application are described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
Vibrio parahaemolytious is isolated from leg Shrimp Litopenaeus vannamei Culture Raceway-type Ponds.Leg Shrimp Litopenaeus vannamei Culture Raceway-type Ponds are in Shandong, Fujian, Guangdong, sea
The ground such as south have.Acquisition time is in September, 2016.Vibrio parahaemolytious separates from Penaeus Vannmei enteron aisle, and vibrio parahaemolytious bites
Thalline is separated from culture of Penaeus vannamei water and in seawater.
Separated 3 plants of vibrio parahaemolyticus phages expand cultural method, comprise the following steps that：First measure the optimal of bacteriophage
Infection multiplicity：Infection multiplicity (multiplicity of infection, MOI) refer to bacteriophage primary infection host strain when
The ratio of bacteriophage quantity and host strain quantity is waited, also referred to as infects multiple.Host strain is inoculated with 100ul vibrio parahaemolytious to 200ml
In NB nutrient solutions, 150r/min 12h are cultivated to logarithmic phase.Then it is 10 to take initial potency9The bacteriophage nutrient solution of PFU/m L,
It is respectively 100,10,1,0.1,0.01,0.001,0.0001,0.00001,0.000001 ratio according to infection multiplicity, will bites
Thalline VP11 and PVP11 is added in fluid nutrient medium with equal volume, 37 DEG C of shaking tables, 120r/min cultures 10h, 4000r/min
15min is centrifuged, removes precipitation, 0.22 μm of membrane filtration of supernatant, filter 23 time, with the effect of double-layer agar technique measure bacteriophage
Valency, the highest infection multiplicity of potency are optimal multiplicity of infection.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 parahaemolyticus phage liquid.
Embodiment 2
Stream plus the method for bacteriophage host fermenting and producing bacteriophage, it is characterised in that comprise the following steps：
1) fermented using 50L fermentation tanks, the volume that fermentation medium is contained in fermentation tank is 35L, and host is accessed
In fermentation medium, it is 10 to make clump count in every milliliter of initial medium10CFU/ml；The fermentative medium formula is as follows：
Peptone 10g/L；Beef extract powder 10g/L；Sodium chloride 1g/L；PH is 8.0；
2) the 10 of the 1% of one plant of fermentating liquid volume are added8The vibrio parahaemolyticus phage liquid of PFU/ml is into fermentation tank
Cultivated, fermentation temperature is 28 DEG C, rotating speed 200rpm；Control is 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 fluid color
When being changed into limpid from muddiness, you can put tank；
4) by the zymotic fluid of gained, the water-bath 5h under the conditions of 50 DEG C, inactivates host, up to bacteriophage probiotics.
5) the titration method of bacteriophage probiotics, is measured with double-layer agar technique；The double-layer agar technique：Take
1mL bacteriophage zymotic fluids, 8000rpm centrifugation 20min, take 100 μ L of supernatant liquid, add in 900 μ L sterile salines and shake up, according to
Secondary carry out gradient dilution.Take 10-10The host strain that the 100 μ L of dilution of dilution gradient are in exponential phase with 100 μ L is added to
8mL is cooled in 50 DEG C or so of semisolid culturemedium, is mixed, and 33 DEG C are inverted culture 12h, and plaque is counted.Phagocytosis
The number of spot 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, it is characterised in that comprise the following steps：
1) fermented using 1000L fermentation tanks, the volume that fermentation medium is contained in fermentation tank is 800L, and 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 7.0.
2) PFU for adding other one plant of fermentating liquid volume 5% is 1012The phagocytosis body fluid of PFU/ml is carried out into fermentation tank
Culture, fermentation temperature are 35 DEG C, rotating speed 200rpm；Control is 7.
3) interior when fermentation progress 8 is small, stream plus bacteriophage host, controlled concentration is 1010CFU/mL is small for 4 between the stream added-time
When, flow acceleration 35L/h, stream add up fermentation volume 8% amount, fermentation period for 20 it is small when, wait zymotic fluid color present by
When muddiness is changed into limpid, you can put tank.
4) by the zymotic fluid of gained, the water-bath 2h under the conditions of 65 DEG C, inactivates host, up to bacteriophage probiotics.
5) the titration method of bacteriophage probiotics, is measured with double-layer agar technique；The double-layer agar technique：Take
1mL bacteriophage zymotic fluids, 8000rpm centrifugation 20min, take 100 μ L of supernatant liquid, add in 900 μ L sterile salines and shake up, according to
Secondary carry out gradient dilution.Take 10-12The host strain that the 100 μ L of dilution of dilution gradient are in exponential phase with 100 μ L is added to
8mL is cooled in 50 DEG C or so of semisolid culturemedium, is mixed, and 33 DEG C are inverted culture 12h, and plaque is counted.Phagocytosis
The number of spot and the calculation formula of potency are as follows：
Plaque number × extension rate × 10 on thalline potency (PFU/mL)=tablet.
Embodiment 4
Stream plus the method for bacteriophage host fermenting and producing bacteriophage, it is characterised in that comprise the following steps：
1) fermented using 5000L fermentation tanks, the volume that fermentation medium is contained in fermentation tank is 4000L, by host
Access in fermentation medium, the clump count for keeping initial medium is 1015CFU/ml；The fermentative medium formula is as follows：Egg
White peptone 10g/L；Beef extract powder 20g/L；Sodium chloride 8g/L；PH is 9.0.
2) PFU for adding the 3rd plant of fermentating liquid volume 8% is 1010The phagocytosis body fluid of PFU/ml is trained into fermentation tank
Support, fermentation temperature is 32 DEG C, rotating speed 150rpm；Control is 9.
3) interior when fermentation progress 12 is small, stream plus bacteriophage host, controlled concentration is 1015Between CFU/m, it is between the stream added-time
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 fluid color by
When muddiness is changed into limpid, you can put tank.
4) by the zymotic fluid of gained, the water-bath 3h under the conditions of 55 DEG C, inactivates host, up to bacteriophage probiotics.
5) the titration method of bacteriophage probiotics, is measured with double-layer agar technique；The double-layer agar technique：Take
1mL bacteriophage zymotic fluids, 8000rpm centrifugation 20min, take 100 μ L of supernatant liquid, add in 900 μ L sterile salines and shake up, according to
Secondary carry out gradient dilution.Take 10-13The host strain that the 100 μ L of dilution of dilution gradient are in exponential phase with 100 μ L is added to
8mL is cooled in 50 DEG C or so of semisolid culturemedium, is mixed, and 33 DEG C are inverted culture 12h, and plaque is counted.Phagocytosis
The number of spot 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
Add 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
| Embodiment | Potency (PFU/ml) |
| Embodiment 2 | 1015 |
| Embodiment 3 | 1012 |
| Embodiment 4 | 1014 |
| Comparative example 1 | 1010 |
| Comparative example 2 | 109 |
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 parahaemolyticus phage probiotics, it is characterised in that comprise the following steps：
1) vibrio parahaemolytious liquid is accessed in fermentation medium and cultivated, obtain host's zymotic fluid；Vibrio parahaemolytious host is being fermented
Initial cell concentration in culture medium is 1010~1016CFU/ml；
2) it is 10 by concentration8~1016The vibrio parahaemolyticus phage liquid of PFU/ml is seeded in host's zymotic fluid training of fermenting
Support；The inoculum concentration of vibrio parahaemolyticus phage liquid is 1%~10%；
3) when fermented and cultured is carried out to 6~12h in the step 2), stream plus vibrio parahaemolytious into obtained host's zymotic fluid
Liquid, continues fermented and cultured, it is 18~24h to control whole fermentation period, obtains zymotic fluid；
4) zymotic fluid obtained in the step 3) is subjected to inactivation vibrio parahaemolytious host, it is micro- obtains vibrio parahaemolyticus phage
Ecological agent.
2. preparation method according to claim 1, it is characterised in that the stream of vibrio parahaemolytious liquid adds body in the step 3)
Product is the 1%~10% of host's fermentating liquid volume；The concentration of vibrio parahaemolytious liquid is 1010~1014PFU/ml。
3. preparation method according to claim 1, it is characterised in that stream adds vibrio parahaemolytious per hour in the step 3)
The volume of liquid is the 0.5%~5% of host's fermentating liquid volume.
4. according to the preparation method described in claims 1 to 3 any one, it is characterised in that secondary haemolysis arc in the step 3)
It is 2~4h between the stream added-time of bacterium solution.
5. preparation method according to claim 1, it is characterised 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, it is characterised in that the fermented and cultured bar 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, it is characterised in that inactivation in the step 4)
Method be water-bath；The temperature of water-bath is 45~65 DEG C；The time of water-bath is 2~5h.
8. the microorganism formulation of vibrio parahaemolyticus phage prepared by claim 1~7 any one the method, its feature exist
In the potency of vibrio parahaemolyticus phage is 1012~1016PFU/ml。
9. secondary haemolysis is identified or detected to the microorganism formulation of the vibrio parahaemolyticus phage described in claim 8 in aquaculture
Application in vibrios.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109652383A (en) * | 2019-01-08 | 2019-04-19 | 集美大学 | A kind of preparation method, probiotics and the application of common eel Edwardsiella bacteriophage |
| CN114015661A (en) * | 2021-12-14 | 2022-02-08 | 青岛润达生物科技有限公司 | Culture medium and method for improving titer of phage |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105331587A (en) * | 2015-12-03 | 2016-02-17 | 江苏省农业科学院 | Vibrio parahaemolyticus phage and preparation method and application thereof |
| CN106995803A (en) * | 2017-01-23 | 2017-08-01 | 集美大学 | One plant is used to prevent and treat the sick bacteriophage of prawn vibrio parahaemolytious and its propagation method |
-
2018
- 2018-01-17 CN CN201810042463.2A patent/CN108018262A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105331587A (en) * | 2015-12-03 | 2016-02-17 | 江苏省农业科学院 | Vibrio parahaemolyticus phage and preparation method and application thereof |
| CN106995803A (en) * | 2017-01-23 | 2017-08-01 | 集美大学 | One plant is used to prevent and treat the sick bacteriophage of prawn vibrio parahaemolytious and its propagation method |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109652383A (en) * | 2019-01-08 | 2019-04-19 | 集美大学 | A kind of preparation method, probiotics and the application of common eel Edwardsiella bacteriophage |
| CN114015661A (en) * | 2021-12-14 | 2022-02-08 | 青岛润达生物科技有限公司 | Culture medium and method for improving titer of phage |
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