CN109207401A - The bacterial strain and its screening technique of vibrio parahaemolytious in antagonism litopenaeus vannamei enteron aisle - Google Patents
The bacterial strain and its screening technique of vibrio parahaemolytious in antagonism litopenaeus vannamei enteron aisle Download PDFInfo
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Abstract
The present invention relates to the bacterial strains and its screening technique of vibrio parahaemolytious in a kind of antagonism litopenaeus vannamei enteron aisle, belong to bacterial strain technical field.The screening technique is the following steps are included: sample acquires: choosing litopenaeus vannamei, aseptically dissects litopenaeus vannamei, shred shrimp enteron aisle, be placed in sterilized physiological saline, is homogenized after concussion to get the homogenate of shrimp enteron aisle；Strain culturing: the homogenate of above-mentioned shrimp enteron aisle is added in MRS culture solution and is cultivated, culture stoste is obtained；Strain isolation: it draws above-mentioned culture stoste and is coated on containing CaCO3MRS culture medium on, until grow bacterium colony, the bacterium colony for selecting molten calcium circle is crossed on MRS culture medium for culture, repeats above step until the bacterium colony bacterial strain in the same size to get vibrio parahaemolytious in antagonism litopenaeus vannamei enteron aisle.The strain that the screening technique obtains, tool is relatively resistant to by force the ability of the poor environments and antagonism pathogenic bacteria such as gastric acid, cholate, pancreatin compared with ordinary lactic acid bacteria, has extremely strong inhibiting effect to vibrio parahaemolytious.
Description
Technical field
The present invention relates to bacterial strain technical fields, more particularly to vibrio parahaemolytious in a kind of antagonism litopenaeus vannamei enteron aisle
Bacterial strain and its screening technique.
Background technique
Vibrio parahaemolytious is widely distributed in the marine eco-environment, is the main pathogenic fungi for causing marine organisms vibriosis,
The diseases such as " Deaths syndrome " and the tissue necrosis of fish such as " Acute Hepatic pancreatic necrosis " and lefteye flounder including shrimp aquaculture.
Currently, the effective ways of vibrio parahaemolytious include: using antimicrobials such as lactic norfloxacinis in control aquaculture process
Object is prevented and treated using the Chinese herbal and crude drugs preparations such as Chinese gall and the coptis and sustained release microspheres vaccine etc..
Lactic acid bacteria refers to that fermenting carbohydrate primary product is a kind of general name without gemma, gram-positive bacterium of lactic acid,
It is the common name of a kind of bacterium that a large amount of lactic acid can be generated using fermentable carbohydrate.Lactic acid bacteria can play perhaps in animal body
More physiological functions.Such as: lactic acid bacteria can adjust body gastrointestinal tract flora, keep microecological balance, mention with disease preventing and treating
The advantages that high breeding performonce fo animals.The control and prevention of disease of lactic acid bacteria and its formulation application in aquaculture process has safe, green
Color, environmentally friendly feature.
Lactic acid bacteria has been applied in aquaculture disease control as probiotics, lactic acid bacteria and its formulation application
Control and prevention of disease in aquaculture process has the characteristics that safety, green and environmental protection, and screens from litopenaeus vannamei enteron aisle
There is the lactic acid bacteria of antagonism but to rarely have discovery vibrio parahaemolytious.
Summary of the invention
Based on this, it is necessary in view of the above-mentioned problems, providing a kind of bacterium of vibrio parahaemolytious in antagonism litopenaeus vannamei enteron aisle
Strain and its screening technique, the bacterial strain obtained using the screening technique can have vibrio parahaemolytious in litopenaeus vannamei enteron aisle
Antagonism.
The screening technique of the bacterial strain of vibrio parahaemolytious in a kind of antagonism litopenaeus vannamei enteron aisle, comprising the following steps:
Sample acquisition: choosing litopenaeus vannamei, aseptically dissect litopenaeus vannamei, shred shrimp enteron aisle,
It is placed in sterilized physiological saline, with tissue refiner's concussion homogenate to get the homogenate of shrimp enteron aisle；
Strain culturing: the homogenate of above-mentioned shrimp enteron aisle is added in MRS culture solution and is cultivated, culture stoste is obtained；
Strain isolation: it draws above-mentioned culture stoste and is coated on containing CaCO3MRS culture medium on, culture until grow bacterium colony,
The bacterium colony for selecting molten calcium circle is crossed on MRS culture medium, is repeated above step and is received until bacterium colony is in the same size to get antagonism is all
The bacterial strain of vibrio parahaemolytious in the gut of shrimp of shore.
Above-mentioned screening technique, the strain colonized in enteron aisle screened from gut of shrimp, compared with ordinary lactic acid bacteria
Tool is relatively resistant to by force the ability of the poor environments and antagonism pathogenic bacteria such as gastric acid, cholate, pancreatin, has extremely strong inhibition to make vibrio parahaemolytious
With.
In one of the embodiments, in the sample acquisition step, it is disease-free and not that the litopenaeus vannamei chooses health
Injected the litopenaeus vannamei of drug.There is universality to ensure to screen obtained bacterial strain.
In one of the embodiments, in the strain culturing step, the initial pH of the MRS culture solution is 5-6.It is preferred that
PH is 5.4.With above-mentioned pH value culture, conducive to the growth of lactic acid bacteria.
In one of the embodiments, in the sample acquisition step, according to the ratio of 1g shrimp enteron aisle and 10ml physiological saline
Example be homogenized, in the strain culturing step, according to shrimp enteron aisle homogenate be MRS nutrient solution volume percentage be 1%~2% into
Row inoculation.
In one of the embodiments, in the strain culturing step, 37 ± 2 DEG C of culture 12-24h must cultivate stoste.This
When reach best bacteria concentration.
In one of the embodiments, in the strain isolation step, CaCO in the MRS culture medium3Quality percentage
Content is 0.5-1.5%.The too low molten calcium circle of calcium carbonate content is unobvious, is not easy to observe；Too high levels influence lactobacter growth, on
State CaCO3Content has preferable screening effect.
The invention also discloses the screening technique sieves of the bacterial strain of vibrio parahaemolytious in above-mentioned antagonism litopenaeus vannamei enteron aisle
Select obtained bacterial strain.
Above-mentioned bacterial strains screen to obtain from gut of shrimp, compared with ordinary lactic acid bacteria or other bacterial strains tool relatively by force tolerance gastric acid,
The ability of the poor environments such as cholate, pancreatin and antagonism pathogenic bacteria has extremely strong inhibiting effect to vibrio parahaemolytious.
The bacterial strain is lactobacillus acidophilus in one of the embodiments,.
The invention also discloses a kind of feed for litopenaeus vannamei, above-mentioned bacterial strain is contained in the feed.
The above-mentioned bacterial strain through screening can significantly improve immunity of organisms as additive feeding litopenaeus vannamei, reduce bait
Expect coefficient.
Content of the bacterial strain in the feed is 1 × 10 in one of the embodiments,8~1 × 1010cfu/ml。
It is preferred that 1 × 105Cfu/ml~1 × 109cfu/ml.Litopenaeus vannamei is fed using above-mentioned dosage, there is preferable feeding effect.
Compared with prior art, the invention has the following advantages:
The screening technique of the bacterial strain of vibrio parahaemolytious in a kind of antagonism litopenaeus vannamei enteron aisle of the invention, from gut of shrimp
The strain colonized in enteron aisle screened, tool is relatively resistant to by force the bad rings such as gastric acid, cholate, pancreatin compared with ordinary lactic acid bacteria
The ability in border and antagonism pathogenic bacteria has extremely strong inhibiting effect to vibrio parahaemolytious.And have it is with strong points, batch it is small, manually at
This low advantage.
A kind of feed for litopenaeus vannamei of the invention, containing above-mentioned bacterial strain, machine can be significantly improved by feeding litopenaeus vannamei
Body immunity reduces feed coefficient.
Specific embodiment
To facilitate the understanding of the present invention, below with reference to embodiment to invention is more fully described.But this hair
It is bright to realize in many different forms, however it is not limited to embodiment described herein.On the contrary, providing these embodiments
Purpose be to make the disclosure of the present invention more thorough and comprehensive.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.
Culture medium prescription used in the embodiment of the present invention refers to " manufacture and application of microbiological culture media ", specifically such as
Under:
MRS culture solution (liquid): peptone 10g, MRS culture medium protein peptone 10.0g, beef extract 10.0g, yeast extract
5.0g, diammonium hydrogen citrate [(NH4)2HC6H5O7] 2.0g, glucose (C6H12O6·H2O) 20.0g, Tween 80 1.0m, acetic acid
Sodium (CH3COONa·3H2O) 5.0g, dipotassium hydrogen phosphate (K2HPO4·3H2O) 2.0g, magnesium sulfate (MgSO4·7H2O) 0.58g, sulphur
Sour manganese (MnSO4·H2O 121 DEG C of autoclavings after) 0.25g, distilled water 1000mL dissolution, it is spare to set 4 DEG C of refrigerators.
MRS culture medium (solid): peptone 10g, MRS culture medium protein peptone 10.0g, beef extract 10.0g, yeast extract
5.0g, diammonium hydrogen citrate [(NH4)2HC6H5O7] 2.0g, glucose (C6H12O6·H2O) 20.0g, Tween 80 1.0mL acetic acid
Sodium (CH3COONa·3H2O) 5.0g, dipotassium hydrogen phosphate (K2HPO4·3H2O) 2.0g, magnesium sulfate (MgSO4·7H2O) 0.58g, sulphur
Sour manganese (MnSO4·H2O) 0.25g, 1% calcium carbonate powder, 1 000mL of distilled water dissolution after be added 121 DEG C of height of 18g agar powder
Steam sterilization is pressed, it is spare to set 4 DEG C of refrigerators.
Embodiment 1
The screening technique of the bacterial strain of vibrio parahaemolytious in a kind of antagonism litopenaeus vannamei enteron aisle, comprising the following steps:
(1) sample acquires:
The disease-free litopenaeus vannamei of health is chosen, did not injected antibiotic and other drugs.Aseptically received to all
Prawn is dissected for shore, shreds to obtain 1g shrimp enteron aisle, be placed in 10ml sterile saline, with the mixing of shrimp enteron aisle after fulling shake
Homogenate obtains the homogenate of shrimp enteron aisle.
(2) strain culturing:
The shrimp enteron aisle of step (1) is homogenized and is added in MRS culture solution according to the ratio of volumn concentration 1%~2%,
37 DEG C of culture 12-24h, obtain culture stoste.
(3) strain isolation:
Bacterial strain is screened using the method for coating and scribing line, specifically:
The 0.1ml culture stoste cultivated in aspiration step (2) is coated on containing 1wt%CaCO3MRS culture medium (diameter
On 8.6cm), 37 DEG C of culture 24-48h, until growing bacterium colony.
The bacterium colony for selecting molten calcium circle is crossed on MRS culture medium, repeat above step 3 times or more, until bacterium colony size
Unanimously.
It provokes single colonie and carries out Gram's staining observation thalli morphology, obtain two plants of identical Bacillus acidi lacticis, puncture of test tube 4
DEG C refrigerator saves.
Colony characteristics are as follows: the bacterial strain is gram-positive bacteria, is white, wet, intermediate projections small on MRS plate
Bacterium colony, Gram's staining are the positive, are rod-short or club-shaped under microscopy.
Embodiment 2
The test of Bacillus acidi lactici biochemical reaction.
Biochemical reaction identification is carried out to lactic acid bacteria isolated in above-described embodiment 1, test is reacted using micro biochemical
Pipe, tested bacterial strain are negative in the glucose that turns sour, catalase, benzidine reaction and gelatin liquefaction reaction, but not
Generate H2S, this meets the biochemical reaction characteristic of Bacillus acidi lactici, can primarily determine as Bacillus acidi lactici.
And referring to " Berger bacterial identification manual " and " lactic acid bacteria taxonomic identification and experimental method ", this institute is primarily determined
The bacterial strain being separated to is lactobacillus acidophilus (L.acidophi lus).
Embodiment 3
Embodiment 1 screens obtained lactic acid bacteria strains Oxford cup bacteriostatic experiment.
First draw vibrio parahaemolytious reference culture culture solution 0.1ml be added plain agar culture medium on, with spreading rod into
Row coating.Embodiment 1 is screened to obtained lactic acid bacteria strains culture fermentation liquid 10mL for 24 hours, is centrifuged through 4 DEG C, 8000r/min
10min, Aspirate supernatant obtain lactic acid bacteria cell-free supernatants through 0.45 μm of filter filtering, and the nothing that suction pipe draws strain subject is thin
Born of the same parents' supernatant adds in the Oxford cup being placed on plate, 37 DEG C of overnight incubations, observes fungistatic effect.
3 repetitions are done in experiment, with vernier caliper measurement inhibition zone, take its average value.In this bacteriostatic experiment, acidophilus lactic acid
Bacillus has obvious inhibiting effect to vibrio parahaemolytious, and inhibition zone is 12 ± 1.14mm.
Embodiment 4
Embodiment 1 screens obtained lactic acid bacteria strains Bile salt resistance experiment.
With 50ml MRS fluid nutrient medium culture, 30 DEG C, 180r/min shaking table shake culture for 24 hours.Embodiment 1 is screened
To bacterial strain be configured to 1 × 109Cfu/ml bacteria suspension.It prepares simultaneously and contains 0.1%, 0.2%, 0.4%, 0.6% bovine bile
The MRS culture medium of bovine bile is not added as control in MRS culture medium.
Test strain is coated on culture medium by 1%, culture for 24 hours, calculates survival rate, as a result as shown in the table.
1. Lactobacillus acidophilus of table cultivated for 24 hours under different gallbladder salinities after survival rate
From the above it is found that the Lactobacillus acidophilus remain to deposit in the case where gallbladder salinity is 0.1~0.4 concentration levels
It is living, illustrate that the bacterial strain can survive in high concentration cholate, it is more stronger than ordinary lactic acid bacteria (enterococcus faecalis) cholate tolerance.
Embodiment 5
Embodiment 1 screens the obtained acidproof experiment of lactic acid bacteria strains.
Above-mentioned lactic acid bacteria is made 10 with broth bouillon9Cfu/ml bacteria suspension, and pH value is adjusted to 2.0,3.0,
4.0,5.0 in 37 DEG C of isothermal holdings 1h, 2h, 3h, take each isothermal holding liquid respectively, carry out bacterium colony counting with MRS culture medium, use
The lactic acid bacteria suspension of pH6.0 is control, calculates Survival probability of bacteria, as a result as shown in the table.
Survival rate of 2. Lactobacillus acidophilus of table under different PH condition of culture
As can be seen that pH is in 3-6 in from the above, bacterium when 1 bacterial strain survival rate of embodiment is equal to 2 close to 100%, pH
Strain side is suppressed, and illustrates that the experimental strain acid-fast ability is strong.
Embodiment 6
Embodiment 1 is screened to obtained Lactobacillus acidophilus and feeds litopenaeus vannamei as feed addictive.
6 400ml glass jars are chosen, if 3 groups, it is respectively as follows:
A group: Lactobacillus acidophilus' low concentration group (1 × 105cfu/ml)；
B group: high concentration group (1 × 109cfu/ml)；
C group: blank group.
Every group two parallel, is averaged as experimental result.
Experiment condition are as follows: water salinity is 22 ‰~25 ‰, dissolved oxygen content 6.5mg/l or more, 28 degrees Celsius of left sides of water temperature
The right side, pH value are 7.5~8.2.
Test shrimp weight is 1.2 ± 0.2g, a length of 7 ± 1.2cm of body, chooses 240 test shrimps, is manually felt using feeding method
After contaminating vibrio parahaemolytious, 40 tails is taken to be put into each glass jar at random.Spice is fed 48 days, is fed daily 4 times.Every 16 days measurement shrimps
Weight, rate of body weight gain, feed coefficient, the death rate, shrimp enteron aisle vibrio parahaemolytious quantity detect be averaged for 3 times as experiment knot altogether
Fruit, as a result as shown in the table.
Spice feeds each growth indexes parameter of Penaeus Vannmei under 3. test strain various concentration of table.
From the above it is found that test strain group (A and B group) feed coefficient, the death rate are significantly lower than blank group, and body
Weight, body are long, rate of body weight gain is higher than blank group, while the quantity of vibrio parahaemolytious is obviously controlled.
I.e. experiments prove that: the bacterial strain have promote prawn growth, inhibit the effect of vibrio parahaemolytious.
To sum up, the results showed, embodiment 1, which screens obtained lactobacillus acidophilus, has significantly prawn vibrio parahaemolytious
Inhibiting effect, test strain have the ability of stronger Bile salt resistance and antagonism pathogenic bacteria, have a extensive future, can be used as micro-
Ecological agent or additive can adjust gastrointestinal bacterial flora balance, protect micro-ecological environment, have as probiotics strain
There are preferable potentiality.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. the screening technique of the bacterial strain of vibrio parahaemolytious in a kind of antagonism litopenaeus vannamei enteron aisle, which is characterized in that including following
Step:
Sample acquisition: litopenaeus vannamei is chosen, aseptically litopenaeus vannamei is dissected, shrimp enteron aisle is shredded, is placed in
In sterilized physiological saline, with tissue refiner's concussion homogenate to get the homogenate of shrimp enteron aisle；
Strain culturing: the homogenate of above-mentioned shrimp enteron aisle is added in MRS culture solution and is cultivated, culture stoste is obtained；
Strain isolation: it draws above-mentioned culture stoste and is coated on containing CaCO3MRS culture medium on, culture is selected until grow bacterium colony
There is the bacterium colony of molten calcium circle to cross on MRS culture medium, repeats above step until bacterium colony is in the same size to get antagonism vannamei boone pair
The bacterial strain of vibrio parahaemolytious in shrimp enteron aisle.
2. the screening technique of the bacterial strain of vibrio parahaemolytious in antagonism litopenaeus vannamei enteron aisle according to claim 1, special
Sign is, in the sample acquisition step, the litopenaeus vannamei chooses health vannamei boone pair that is disease-free and not injecting drug
Shrimp.
3. the screening technique of the bacterial strain of vibrio parahaemolytious in antagonism litopenaeus vannamei enteron aisle according to claim 1, special
Sign is, in the strain culturing step, the initial pH of the MRS culture solution is 5-6.
4. the screening technique of the bacterial strain of vibrio parahaemolytious in antagonism litopenaeus vannamei enteron aisle according to claim 1, special
Sign is, in the sample acquisition step, is homogenized according to the ratio of 1g shrimp enteron aisle and 10ml physiological saline, the bacterial strain training
It supports in step, be MRS nutrient solution volume percentage according to the homogenate of shrimp enteron aisle is 1%~2% to be inoculated with.
5. the screening technique of the bacterial strain of vibrio parahaemolytious in antagonism litopenaeus vannamei enteron aisle according to claim 1, special
Sign is, in the strain culturing step, 37 ± 2 DEG C of culture 12-24h must cultivate stoste.
6. the screening technique of the bacterial strain of vibrio parahaemolytious in antagonism litopenaeus vannamei enteron aisle according to claim 1, special
Sign is, in the strain isolation step, CaCO in the MRS culture medium3Mass percentage be 0.5-1.5%.
7. the screening technique sieve of the bacterial strain of vibrio parahaemolytious in the described in any item antagonism litopenaeus vannamei enteron aisles of claim 1-7
Select obtained bacterial strain.
8. bacterial strain according to claim 9, which is characterized in that the bacterial strain is lactobacillus acidophilus.
9. a kind of feed for litopenaeus vannamei, which is characterized in that contain the described in any item bacterium of claim 7-8 in the feed
Strain.
10. feed for litopenaeus vannamei according to claim 9, which is characterized in that the bacterial strain containing in the feed
Amount is 1 × 108~1 × 1010cfu/ml。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111849835A (en) * | 2020-08-07 | 2020-10-30 | 宁波大学 | Bacillus fusobacterium strain and screening method and application thereof |
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