CN109652383A - A kind of preparation method, probiotics and the application of common eel Edwardsiella bacteriophage - Google Patents
A kind of preparation method, probiotics and the application of common eel Edwardsiella bacteriophage Download PDFInfo
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- CN109652383A CN109652383A CN201910014847.8A CN201910014847A CN109652383A CN 109652383 A CN109652383 A CN 109652383A CN 201910014847 A CN201910014847 A CN 201910014847A CN 109652383 A CN109652383 A CN 109652383A
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Abstract
The present invention provides preparation method, probiotics and the applications of a kind of common eel tarda bacteriophage, belong to common eel cultural technique field, the preparation method of the common eel tarda bacteriophage, comprising the following steps: 1) common eel tarda is inoculated in fluid nutrient medium and carries out fermented and cultured acquisition zymocyte liquid；2) bacteriophage that the common eel tarda is accessed into zymocyte liquid described in step 1) obtains bacteriophage cultivating system, carries out bacteriophage culture；3) when the bacteriophage is cultivated to 10~20h, start stream plus tarda bacterium solution into bacteriophage cultivating system；Make the bacteria concentration 10 of tarda in the bacteriophage cultivating system8~1014CFU/ml；4) when the bacteriophage cultivates to 18~for 24 hours when terminate to cultivate, obtain common eel tarda bacteriophage.The method is easy to operate, and cultivation cycle is short, and the phage titer of acquisition is high.
Description
Technical field
The invention belongs to common eel cultural technique field more particularly to a kind of preparation sides of common eel tarda bacteriophage
Method, probiotics and application.
Background technique
Common eel is a kind of rivers straddling fish stocks, originates in marine, passes up in fresh water and grows up, after return to marine oviposition.
Annual spring, large quantities of elvers (also referred to as white young, eel line) enter rivers mouth from sea in groups.Common eel fine and tender taste, it is delicious, in common eel
Meat in, protein, vitamin A. D. E, minerals and unsaturated fatty acid DHA/EPA rich in；People can be provided
Class growth, sustain life required nutritional ingredient.
Since common eel has very high nutritive value, common eel aquaculture development is rapid；The common eel aquaculture in China has become
One of the important industry earned foreign exchange for product water outlet.Tarda disease is one of the principal disease of common eel cultivation, main to show
For the enlargement of liver kidney or fester.The traditional therapy of tarda is oral chloramphenicol, but contains in chloramphenicol and inhibit bone
The related NO of marrow hematopoietic function2Group can generate harm to human health, through being prohibited from using.Phage therapy is being pacified
Advantage is had more than antibiotic in terms of full property.
But the preparation method cultivation cycle of existing bacteriophage is long；The phage titer prepared is low.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of preparation method of common eel tarda bacteriophage and answering
With；The method cultivation cycle is short, and the phage titer for cultivating acquisition is high.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical schemes:
A kind of preparation method of common eel tarda bacteriophage, comprising the following steps:
1) common eel tarda is inoculated in fluid nutrient medium and carries out fermented and cultured acquisition zymocyte liquid；
2) bacteriophage that the common eel tarda is accessed in zymocyte liquid described in Xiang Suoshu step 1) obtains phagocytosis
Body cultivating system carries out bacteriophage culture；
3) when the bacteriophage is cultivated to 10~20h, start the continuous flow into bacteriophage cultivating system and add Ai Dehuashi
Bacterium bacterium solution；Make the bacteria concentration 10 of tarda in the bacteriophage cultivating system8~1014CFU/ml；
4) when the bacteriophage cultivates to 18~for 24 hours when terminate to cultivate, obtain common eel tarda bacteriophage fermentation liquid.
Preferably, continuous flow described in step 3) adds the tarda bacterium solution time to be 4~10h；The continuous flow adds love
The total volume of moral Fahrenheit bacterium bacterium solution is the 5~20% of the bacteriophage cultivating system volume.
Preferably, the volume of the bacteriophage of access described in step 2) is the 1~10% of zymocyte liquid volume.
Preferably, the potency of the bacteriophage of access described in step 2) is 108~1012PFU/ml。
Preferably, the temperature of the culture of bacteriophage described in step 2) is 24~32 DEG C, during the bacteriophage culture
With stirring, the revolving speed of the stirring is 100~220rpm.
Preferably, the bacteria concentration of tarda is 10 in zymocyte liquid described in step 1)8~1014CFU/ml。
Preferably, fluid nutrient medium described in step 1) includes following components:
The brain heart soaks 1~20g/L of powder；1~20g/L of peptone；1~10g/L of D-Glucose；1~5g/L of sodium dihydrogen phosphate；Chlorine
Change 1~10g/L of sodium；The pH value of the fluid nutrient medium is 7.0~8.0.
The present invention also provides a kind of common eel tarda bacteriophage probiotics, the preparations of the probiotics
Method, comprising the following steps: the common eel tarda bacteriophage for preparing the preparation method is in 45~65 DEG C of conditions
2~5h of lower heating obtains common eel tarda bacteriophage probiotics.
Preferably, the potency of the common eel tarda bacteriophage probiotics pnagus medius is 1010~
1016PFU/ml。
The present invention also provides common eel tarda bacteriophage probiotics to prevent and treat common eel tarda in preparation
Application in medicine.
Beneficial effects of the present invention: the preparation method of common eel tarda bacteriophage provided by the invention, by biting
Stream plus host's tarda, are continuously replenished fresh host strain, in addition host strain self-reproduction, makes to bite during thallus culture
The viable count of tarda maintains 10 in thallus cultivating system8~1014CFU/ml；It can guarantee in bacteriophage cultivating system
There is sufficient host strain for bacteriophage multiplication, to obtain the bacteriophage of high concentration.
Further, the potency of heretofore described bacteriophage probiotics pnagus medius is up to 1010~1016PFU/
ml；Host strain is inactivated simultaneously, avoids potential environmental hazard.
Further, the medium component of the method for the invention bacteriophage is simple, and a step continuous flow adds tarda
Bacterium solution, easy to operate, cultivation cycle is short.
Specific embodiment
The present invention provides a kind of preparation methods of common eel tarda bacteriophage, comprising the following steps: 1) by common eel
Tarda, which is inoculated in fluid nutrient medium, carries out fermented and cultured acquisition zymocyte liquid；2) to zymocyte liquid described in step 1)
The bacteriophage of the middle access common eel tarda obtains bacteriophage cultivating system, carries out bacteriophage culture；3) it is bitten when described
When thallus culture is to 10~20h, starts the continuous flow into bacteriophage cultivating system and add tarda bacterium solution；Make the bacteriophage
The bacteria concentration of tarda is 10 in cultivating system8~1014CFU/ml；4) when the bacteriophage cultivates to 18~for 24 hours when knot
Beam culture obtains common eel tarda bacteriophage fermentation liquid.
Common eel tarda is inoculated in fluid nutrient medium by the present invention carries out fermented and cultured acquisition zymocyte liquid.This hair
The bright type to the common eel tarda is not particularly limited, to cause common eel to suffer from the pathogenic bacteria of tarda disease i.e.
It can.The present invention is not particularly limited the source of the common eel tarda, is preferably isolated from and suffers from tarda disease
In common eel body, it is also possible to the common eel tarda of commercialization.Heretofore described fluid nutrient medium preferably include with
Lower component: the brain heart soaks 1~20g/L of powder；1~20g/L of peptone；1~10g/L of D-Glucose；1~5g/L of sodium dihydrogen phosphate；Chlorine
Change 1~10g/L of sodium；More preferably are as follows: the brain heart soaks 5~15g/L of powder；5~15g/L of peptone；4~6g/L of D-Glucose；Phosphoric acid
2~4g/L of sodium dihydrogen；3~7g/L of sodium chloride；The pH value of heretofore described fluid nutrient medium is preferably 7.0~8.0.The present invention
Described in the inoculation volume (V/V) of common eel tarda be preferably 1~10%, more preferably 3~8%.It is described in the present invention
The temperature of fermented and cultured is preferably 24~32 DEG C；The revolving speed of the fermented and cultured is preferably 100~220rpm.The present invention is to described
Initial bacteria concentration, the fermented incubation time of common eel tarda are not particularly limited；It is preferred to cultivate to the zymocyte liquid
The bacteria concentration of middle tarda is 108~1014Until CFU/ml.
The present invention accesses biting for the common eel tarda after obtaining the zymocyte liquid in Xiang Suoshu zymocyte liquid
Thallus obtains bacteriophage cultivating system, carries out bacteriophage culture.The common eel tarda of the access in the present invention
The volume of bacteriophage be preferably the 1~10% of zymocyte liquid volume, more preferably 2~8%, more preferably 4~6%.This hair
The potency of the bacteriophage of access described in bright is preferably 108~1012PFU/ml, more preferably 109~1011PFU/ml.The present invention
The source of the bacteriophage is not particularly limited, is common eel tarda specific bacteriophage in above-mentioned steps.This
In invention, the temperature of the bacteriophage culture is preferably 24~32 DEG C, and more preferably 26~30 DEG C；The mistake of the bacteriophage culture
Preferred with stirring in journey, the revolving speed of the stirring is preferably 100~220rpm, more preferably 120~200rpm.
The present invention starts the continuous flow Jia Aide into bacteriophage cultivating system when the bacteriophage is cultivated to 10~20h
Fahrenheit bacterium bacterium solution；Make the bacteria concentration 10 of tarda in the bacteriophage cultivating system8~1014CFU/ml.In the present invention
In, the stream plus the time of tarda bacterium solution of starting is preferably 12~18h.In the present invention, the continuous flow adds Edward
The total time of Salmonella bacterium solution is preferably 4~10h, more preferably 5~8h；The continuous flow adds the total volume of tarda bacterium solution
5~20%, more preferably the 10~18% of the preferably described bacteriophage cultivating system volume.The present invention adds the continuous flow
Speed is not particularly limited, to maintain the bacteria concentration of tarda in bacteriophage cultivating system for 108~1014CFU/ml is
It is quasi-.In 10~20h of bacteriophage culture；The viable count decline of host strain common eel tarda is more, and the present invention is at this
Between continuous flow add the tarda bacterium solution, tarda can be replenished in time, conducive to the rapidly and efficiently breeding of bacteriophage.
Stream of the present invention adds the bacterium in fresh tarda bacterium solution plus the breeding of tarda itself in bacteriophage cultivating system,
The viable count of tarda in bacteriophage cultivating system can be made to maintain 108~1014CFU/ml；Guarantee that bacteriophage cultivates body
There is sufficient host strain for bacteriophage multiplication in system, to obtain the bacteriophage of high concentration.
The present invention cultivates to 18 in the bacteriophage~for 24 hours when terminate to cultivate, obtain common eel tarda bacteriophage hair
Zymotic fluid.In the present invention, the time for terminating culture is preferably 19~23h, more preferably 20~22h；It is heretofore described
Terminate culture time be more preferably the bacteriophage cultivating system color it is clear by xanthochromia when.
Above-mentioned steps of the present invention preferably carry out in round, in specific implementation process of the present invention, preferably exist
It is carried out in fermentor；The present invention does not have particular/special requirement to the specification and brand of the fermentor, using this field normal fermentation tank
, can be the fermentor of 5L, 250L, 1000L scale.
The present invention also provides a kind of common eel tarda bacteriophage probiotics, the preparations of the probiotics
Method, comprising the following steps: the common eel tarda bacteriophage for preparing the preparation method is in 45~65 DEG C of conditions
2~5h of lower heating obtains common eel tarda bacteriophage probiotics.In the present invention, the temperature of the heating is preferred
It is 50~60 DEG C, more preferably 55~58 DEG C；The time of the heating is preferably 3~4h；The mode of the heating is preferably water
Bath heating.The purpose that the common eel tarda bacteriophage is heated in the present invention is by host common eel tarda therein
Inactivation is conducive to later period application, avoids potential environmental hazard.In the present invention, the micro- life of common eel tarda bacteriophage
The potency of state preparation pnagus medius is preferably 1010~1016PFU/ml, more preferably 1014~1015PFU/ml。
The present invention also provides common eel tarda bacteriophage probiotics to prevent and treat common eel tarda in preparation
Application in medicine.In the present invention, the common eel tarda bacteriophage probiotics can be directly as prevention and treatment
The drug of common eel tarda can also be mixed with the medicine of prevention and treatment common eel tarda with other active components or auxiliary material
Object；The present invention is not particularly limited the type and dosage of the other active components or auxiliary material, can be received using bacteriophage
Type and dosage.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Embodiment 1
The preparation method of common eel tarda bacteriophage
Fluid nutrient medium: the brain heart soaks powder 10g/L；Peptone 10g/L；D-Glucose 5g/L；Sodium dihydrogen phosphate 3g/L；Chlorination
Sodium 5g/L；PH value 7.5.The fluid nutrient medium is placed in fermentor and sterilized, cooled down.
1) the common eel tarda being isolated from common eel sick body progress fermented and cultured in fluid nutrient medium is inoculated in obtain
Obtain zymocyte liquid；The viable count of zymocyte liquid is 1013CFU/ml；
2) it is 10 that 5% volume PFU is accessed in Xiang Suoshu zymocyte liquid8~1012The bacteriophage of PFU/ml obtains bacteriophage training
The system of supporting carries out bacteriophage culture；28 DEG C of temperature, stirring rate 150rpm；
3) when the bacteriophage is cultivated to 12h, start stream plus tarda bacterium solution into bacteriophage cultivating system；Make
The bacteria concentration of tarda is 10 in the bacteriophage cultivating system12CFU/ml；Stream plus 6h, stream aggregation fermentation volume 10%
Amount.
Fermentation period is 20h, when waiting fermentation liquids color clear by dark yellow discoloration, can put tank；Harvest bacteriophage, potency
1016PFU/ml。
Embodiment 2
Common eel tarda bacteriophage probiotics
Common eel tarda bacteriophage Tiny ecosystem is obtained by 4h is heated under the conditions of 60 DEG C of bacteriophage obtained in embodiment 1
Preparation, potency 1015PFU/ml。
Embodiment 3
The preparation method of common eel tarda bacteriophage
Fluid nutrient medium: the brain heart soaks powder 12g/L；Peptone 8g/L；D-Glucose 4g/L；Sodium dihydrogen phosphate 4g/L；Chlorination
Sodium 6g/L；PH value 7.2.The fluid nutrient medium is placed in fermentor and sterilized, cooled down.
1) the common eel tarda being isolated from common eel sick body progress fermented and cultured in fluid nutrient medium is inoculated in obtain
Obtain zymocyte liquid；The viable count of zymocyte liquid is 1014CFU/ml；
2) it is 10 that 8% volume PFU is accessed in Xiang Suoshu zymocyte liquid10The bacteriophage of PFU/ml obtains bacteriophage and cultivates body
System carries out bacteriophage culture；25 DEG C of temperature, stirring rate 180rpm；
3) when the bacteriophage is cultivated to 14h, start stream plus tarda bacterium solution into bacteriophage cultivating system；Make
The bacteria concentration of tarda is 10 in the bacteriophage cultivating system12CFU/ml；Stream plus 4h, stream aggregation fermentation volume 15%
Amount.
Fermentation period is 20h, when waiting fermentation liquids color clear by dark yellow discoloration, can put tank；Harvest bacteriophage, potency
1015PFU/ml。
Embodiment 4
Common eel tarda bacteriophage probiotics
Common eel tarda bacteriophage Tiny ecosystem is obtained by 4h is heated under the conditions of 55 DEG C of bacteriophage obtained in embodiment 1
Preparation, potency 1014PFU/ml。
Embodiment 5
The preparation method of common eel tarda bacteriophage
Fluid nutrient medium: the brain heart soaks powder 10g/L；Peptone 10g/L；D-Glucose 5g/L；Sodium dihydrogen phosphate 3g/L；Chlorination
Sodium 5g/L；PH value 7.5.The fluid nutrient medium is placed in fermentor and sterilized, cooled down.
1) the common eel tarda being isolated from common eel sick body progress fermented and cultured in fluid nutrient medium is inoculated in obtain
Obtain zymocyte liquid；The viable count of zymocyte liquid is 1012CFU/ml；
2) it is 10 that 3% volume PFU is accessed in Xiang Suoshu zymocyte liquid11The bacteriophage of PFU/ml obtains bacteriophage and cultivates body
System carries out bacteriophage culture；30 DEG C of temperature, stirring rate 200rpm；
3) when the bacteriophage is cultivated to 10h, start stream plus tarda bacterium solution into bacteriophage cultivating system；Make
The bacteria concentration of tarda is 10 in the bacteriophage cultivating system12CFU/ml；Stream plus 10h, stream aggregation fermentation volume 10%
Amount.
Fermentation period is 22h, when waiting fermentation liquids color clear by dark yellow discoloration, can put tank；Harvest bacteriophage, potency 5.5
×1015PFU/ml。
Embodiment 6
Common eel tarda bacteriophage probiotics
Common eel tarda bacteriophage Tiny ecosystem is obtained by 3h is heated under the conditions of 65 DEG C of bacteriophage obtained in embodiment 1
Preparation, potency 5.5 × 1014PFU/ml。
The above is only a 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 are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of preparation method of common eel tarda bacteriophage, comprising the following steps:
1) common eel tarda is inoculated in fluid nutrient medium and carries out fermented and cultured acquisition zymocyte liquid；
2) bacteriophage that the common eel tarda is accessed into zymocyte liquid described in step 1) obtains bacteriophage culture body
System carries out bacteriophage culture；
3) when the bacteriophage is cultivated to 10~20h, start the continuous flow into bacteriophage cultivating system and add tarda bacterium
Liquid；Make the bacteria concentration 10 of tarda in the bacteriophage cultivating system8~1014CFU/ml；
4) when the bacteriophage cultivates to 18~for 24 hours when terminate to cultivate, obtain common eel tarda bacteriophage fermentation liquid.
2. preparation method according to claim 1, which is characterized in that continuous flow described in step 3) adds tarda bacterium
The total time of liquid is 4~10h；The continuous flow adds the total volume of tarda bacterium solution to be the bacteriophage cultivating system volume
5~20%.
3. preparation method according to claim 2, which is characterized in that the volume of the bacteriophage of access described in step 2) is
The 1~10% of zymocyte liquid volume.
4. preparation method according to claim 3, which is characterized in that the potency of the bacteriophage of access described in step 2) is
108~1012PFU/ml。
5. preparation method according to claim 1, which is characterized in that the temperature of the culture of bacteriophage described in step 2) is 24
~32 DEG C, with stirring during the bacteriophage culture, the revolving speed of the stirring is 100~220rpm.
6. preparation method according to claim 1, which is characterized in that Ai Dehuashi in zymocyte liquid described in step 1)
The bacteria concentration of bacterium is 108~1014CFU/ml。
7. preparation method according to claim 1, which is characterized in that fluid nutrient medium described in step 1) includes following
Component:
The brain heart soaks 1~20g/L of powder；1~20g/L of peptone；1~10g/L of D-Glucose；1~5g/L of sodium dihydrogen phosphate；Sodium chloride
1~10g/L；The pH value of the fluid nutrient medium is 7.0~8.0.
8. a kind of common eel tarda bacteriophage probiotics, which is characterized in that the preparation method of the probiotics,
The following steps are included: the common eel tarda bacteriophage that preparation method described in Claims 1 to 5 any one is prepared
2~5h is heated under the conditions of 45~65 DEG C, obtains common eel tarda bacteriophage probiotics.
9. common eel tarda bacteriophage probiotics according to claim 8, which is characterized in that the common eel love
The potency of moral Fahrenheit bacterium bacteriophage probiotics pnagus medius is 1010~1016PFU/ml。
10. the common eel tarda bacteriophage probiotics of claim 8 or 9 are in preparation prevention and treatment common eel tarda disease
Application in drug.
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| CN114807057A (en) * | 2022-04-01 | 2022-07-29 | 中国科学院大学 | Edwardsiella piscicida phage EPP-1 and application thereof |
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