CN106676029A - Bacillus pumilus strain and micro-ecological preparation and feed thereof - Google Patents
Bacillus pumilus strain and micro-ecological preparation and feed thereof Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention belongs to the technical field of biologics, and discloses bacillus pumilus capable of inhibiting streptococcus agalactiae and application thereof. The bacillus pumilus has the advantages that the screened bacillus pumilus metabolite can be used for obviously inhibiting the metabolism and growth of the streptococcus agalactiae, the number of streptococcus agalactiae in the tlapia mossambica culture environment is reduced, and the occurrence of streptococcicosis is inhibited; the bacillus pumilus is subject to multiple times of strain selection and breeding, and can be performed with liquid deep fermenting, so as to prepare water preparation products and powder preparation products; the powder preparation products are prepared into a tlapia mossambica particle feed according to a certain ratio, and then the tlapia mossambica particle feed can be applied into aquaculture.
Description
Technical field
The invention belongs to biological technical field, screens one plant and can suppress tilapia streptococcus
Bacillus pumilus, and carried out fermentation optimization, be prepared into aqua product and powder product,
And can apply in aquatic feeds.
Background technology
Tilapia (Tilapia) is used as one of topmost breed variety of world's marine industry, tool
Have the advantages that fast growth, feeding habits are miscellaneous, meat is good, be of high nutritive value, fertility it is strong
It is distributed widely in the light salt water domain of tropical and subtropical zone.China's Coastal Areas be particularly Hainan,
The provinces such as Guangdong, Fujian, Guangxi cultivation Rofe fish crop account for the world more than half.But
Tilapia cultured area and cultivation density more and more higher were accompanied by the last few years by streptococcus agalactiae
The tilapia streptococcicosises that (Streptococcus agalactiae) infection causes have become obstruction
The principal element of China's tilapia aquaculture healthy and sustainable development.Particularly since two thousand nine
Tilapia " streptococcicosises " presents the trend of whole nation outburst, in annual high temperature season especially
It it is 7-9 months, more than 50%, mortality rate reaches as high as more than 80% to sickness rate
Many at present usually to control the streptococcic generation of tilapia and infection using antibiosis, this is supported
Grow pattern and result in the exceeded problem of abuse of antibiotics and residual in breeding process, it is serious to restrict
The outlet and sale of tilapia finished product, becomes and limits the bottleneck of tilapia industry development and ask
Topic.
The content of the invention
The present invention screens a bacillus pumilus from the breeding environment of tilapia, and this is short
Bacillus pumiluss can suppress to cause the streptococcus agalactiae of tilapia streptococcicosises to grow, can
The generation and infection of significant control tilapia streptococcicosises, reduces antibiotic usage, reduces
Tilapia cultivation risk, improves the culture benefit of tilapia.
First, the present invention provides one plant of short and small spore bar that can suppress tilapia streptococcicosises
Bacterium new strains.
The present invention is separated from tilapia Cultivated water, oriented primary dcreening operation and secondary screening obtain one plant
Bacillus pumilus.Its trophocyte thin rod shape, its spore are oval or long tubular, in being
Raw or secondary end life, sporangiocyst are slightly expanded.Isolate or in short chain, rod end is semicircle.In 12~16h
Interior energy forms bacterium colony, and bacterium colony is circle, or irregularly, edge is hair-like, bacteria colony white,
It is opaque, corrugationless.Gram-positive.
Enter performing PCR identification to the bacterial strain for screening using the universal primer of antibacterial：By antibacterial
DNA extraction kit operating instruction extracts template DNA.Using the conservative sequences of 16S rRNA
5 '-AGAGTT TGA TCC TGG CTC AG-3 ' (SEQ ID No.2) of forward primer of row
Expand with 5 '-GGT TACCTT GTT ACG ACT T-3 ' (SEQ ID No.3) of downstream primer
Increase antibacterial 16s rRNA genetic fragments.The Pseudomonas is obtained in Bacillus pumilus through sequencing
(Bacillus pumilus), the sequencing result of amplified fragments are shown in SEQ ID No.1.
Using LB culture medium:Tryptone 10/L, yeast extract 5g/L, NaCl 10g/L,
Bacillus pumilus CD6 to obtaining has carried out biocidal property, heat resistance, fermentation character reality
Test.In flat board test process, it is found that CD6 can significantly inhibit the growth of streptococcus agalactiae,
Form significantly transparent suppression circle.CD6 spore heat resistance is good, and spore is in 85 DEG C of water-baths
10min, spore survival rate more than 80%.
The Bacillus pumilus for screening (Bacillus pumilus) are named as into CD6, in 2016
On March 9, in is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center,
Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica,
Deposit number is：CGMCC No.12197.
The present invention also provides the high density fermentation culture medium of the Bacillus pumilus, and which contains：
KH2PO40.8~1.5g/L, 3~6g/L of sucrose, 8~12g/L of soybean cake powder, FeSO4
0.25~0.5g/L, CaCO31~3g/L, MgSO42.5~3.5g/L, 4~6g/L of Semen Maydis flour,
6~10g/L of fish flour, defoamer 0.05~0.1%, initial pH7.0~7.5.
Preferably, described high density fermentation culture medium contains KH2PO41.0g/L, sucrose 3
G/L, soybean cake powder 8g/L, FeSO40.30g/L, CaCO31.5g/L, MgSO43.0g/L,
Semen Maydis flour 5g/L fish flour 8g/L, defoamer 0.05%, initial pH7.5.
The present invention also provides the fermentation process in high density of the Bacillus pumilus, it include as
Lower step：Seed liquor is inoculated in described fermentation medium by the inoculum concentration of 3-6%, temperature
30~37 DEG C, 200~400r/min of rotating speed, tank pressure 0.05Mpa, ventilation ratio 1：0.4~0.6,
12~16h of fermentation time, obtains spore production rate more than 90%, and viable count is 109~
1010The Bacillus pumilus fermentation liquid of CFU/ml.
The present invention also provides a kind of microbial ecological agent for suppressing tilapia hammer disease, and which contains short
The fermentation liquid of bacillus pumiluss (Bacillus pumilus) CGMCC No.12197.
In one embodiment of the invention, the described Tiny ecosystem for suppressing tilapia hammer disease
The preparation also NaCl containing final concentration of 12-15%.
The present invention also provides another kind of microbial ecological agent for suppressing tilapia hammer disease, and which contains
The mycopowder of Bacillus pumilus (Bacillus pumilus) CGMCC No.12197, its viable bacteria
Number is 1010~1011CFU/g。
Wherein, described mycopowder preparation method is as follows：High density fermentation liquid is multiple using ceramics
After closing 3 times of membrance concentration, 10% starch of addition is spray-dried, dried strip as carrier
Part is：200~400 DEG C of inlet temperature, 70~85 DEG C of outlet temperature, charging rate 10~
12L/min, is atomized rotary head 20000~30000r/min of speed.
The microbial ecological agent of the exploitation of the present invention, water preparation are directly splashed to Cultivated water, suppress
The growth of streptococcus agalactiae；Powder product is added in tilapia particulate material, improves tilapia
Resistivity to streptococcicosises.
Mycopowder prepared by this product can prepare tilapia particulate material as feed additive, can
Improve resistance of the tilapia to streptococcicosises.
Description of the drawings
Fig. 1：Bacillus pumilus bacillus CD6 inhibition zones.
Specific embodiment
Screening technique of the invention, preparation technology and application are carried out in detail by following examples
State, the explanation is descriptive, is not intended to limit protection scope of the present invention.
The bacteriostatic experiment method of 1 Bacillus pumilus CD6 of embodiment
1) cultural method of streptococcus agalactiae
Fluid medium：Peptone 10.0g/L, is dehydrated little Medulla Bovis seu Bubali and soaks powder 12.5g/L, dehydration
Beef heart infusion 5.0g/L, Sodium Chloride 5.0g/L, glucose 2.0g/L, disodium hydrogen phosphate 2.5
G/L, pH7.5.
Solid medium：Peptone 10.0g/L, is dehydrated little Medulla Bovis seu Bubali and soaks powder 12.5g/L, dehydration
Beef heart infusion 5.0g/L, Sodium Chloride 5.0g/L, glucose 2.0g/L, disodium hydrogen phosphate 2.5
G/L, pH7.5, agar powder 1.5%.
2) screening flat board makes
Streptococcus agalactiae single bacterium colony on flat board is inoculated in into fluid medium, 25 DEG C of culture 16h.
It is cooled to after aseptically pouring cultured fermentation liquid into sterilizing according to 10% ratio
In 40~45 DEG C of solid mediums, mix, in aseptic flat board, 30ml/ flat boards, cooling
Solidification is stand-by.
3) Odontothrips loti screening Bacillus pumilus
Aseptic Oxford cup is placed on the flat board that 2) method is prepared, by CD6LB culture medium
200 μ L of fermentation liquid are added in Oxford cup, 30 DEG C of culture 16h, observe inhibition zone situation.
By above method, it can be seen that inhibitions of the CD6 to streptococcus agalactiae, such as scheme
Shown in 1, there is around the Oxford cup containing CD6 significant transparent suppression circle.
The measure of 2 Bacillus pumilus CD6 fermented supernatant fluid minimum inhibitory concentrations of embodiment
1) preparation method of Bacillus pumilus CD6 fermented liquid supernatants liquid
CD6 is inoculated in the 100ml triangle samples containing 25ml LB culture medium, 37 DEG C
Culture 24h, takes 10ml fermentation liquids, and 5000r/min centrifuges are centrifuged 15min, collect not
Supernatant containing thalline.
2) preparation of variable concentrations CD6 fermented liquid supernatants liquid
5 teat glasses are taken, 0,1,2,3,4, in every test tube are respectively labeled as, plus
Enter the supernatant of not mycetome after 1ml centrifugations, No. 1 test tube adds pure water 1ml, No. 2 examinations
Pipe adds water 2ml, and No. 3 test tubes add pure water 3ml, and No. 4 test tubes add water 4ml.1,2,3,
No. 4 test tube supernatants are diluted 2 times respectively, 3 times, 4 times, 5 times.
3) measure of Bacillus pumilus CD6 fermented supernatant fluids minimum inhibitory concentration
According to the method for embodiment 1, the suppression to streptococcus agalactiae under different diluted concentrations is determined
System, experimental result are as follows.
The measure of table 1CD6 fermented supernatant fluid least concentrations
| Numbering | 0 | 1 | 2 | 3 | 4 |
| Diluted concentration | 1 times | 2 times | 3 times | 4 times | 5 times |
| Fungistatic effect | +++++ | ++++ | ++ | + |
As it can be seen from table 1 the fermented supernatant fluid of CD6 has excellent suppression agalactia hammer
The effect of bacterium, 4 times of dilution still have fungistatic effect.
The preparation of 3 short and small spore bacillus cereuss high density fermentation liquid of embodiment
1) flat board culture rejuvenation：Bacillus pumilus strain is inoculated in into LB plating mediums
On, 24h is cultivated in 30 DEG C, make Bacillus pumilus rejuvenation, and form single bacterium colony, picking
On inoculation medium, 37 DEG C are cultivated 24h to single bacterium colony；
2) preparation of first order seed：By step 1) the Bacillus pumilus strain transfer eggplant cultivated
On sub- bottle LB slant mediums, 37 DEG C of culture 24h make in late log phase, obtain one-level kind
Son；
3) preparation of secondary seed：By step 2) first order seed for preparing made with sterilized water
Bacteria suspension, is inoculated in the 100L seed tanks equipped with 60L LB seed culture mediums, temperature
30 DEG C, rotating speed 200r/min, tank pressure 0.05MPA, ventilating ratio：1：0.6 culture 14h, obtains two
Level seed liquor.
4) preparation of Bacillus pumilus fermentation liquid：By step 3) secondary seed solution for preparing
The 1m of 600L fermentation medium is inoculated into by 5% inoculum concentration3In fermentation tank, 30 DEG C of temperature,
Rotating speed 200r/min, tank pressure 0.05Mpa, ventilating ratio 1：0.5,16h is cultivated, spore life is obtained
Into rate more than 90%, viable count is 5 × 109The Bacillus pumilus fermentation liquid of cfu/ml；
Described fermentation medium is (mass percent)：KH2PO41.0g/L, sucrose 3g/L,
Soybean cake powder 8g/L, FeSO40.30g/L, CaCO3 1.5g/L,MgSO43.0g/L, Semen Maydiss
Face 5g/L fish flour 8g/L, defoamer 0.05%, initial pH7.5.
The preparation of 4 water preparation Tiny ecosystem of embodiment
In fermentation liquid obtained in embodiment 2, the 15% of fermentation liquid weight Sodium Chloride is added,
Shelf-life stable aqua product is can be made into, aqua product is using 1L or 5L PET materials
Packing plastic bottle.
The preparation of 5 powder Tiny ecosystem of embodiment
1) fermentation liquid concentration：The fermentation liquid of production adopts aperture for 1~10 micron of ceramic membrane
Filtering fermentating liquid, realizes the separation of thalline and liquid, makes three times concentrated solution.
2) carrier addition：The volume of concentrate is installed, adds 10% corn starch, stirring is equal
It is even standby.
3) spray drying condition：160~180 DEG C of inlet temperature, 70~80 DEG C of leaving air temp,
20000~30000r/min of atomizer rotating speed, realizes leaving air temp by controlling charging rate
Stablize.
4) prepared by mycopowder：The mixed liquor that 2) method is prepared according to method 3) spray drying condition,
It is prepared into the mycopowder of water content 5%, viable count content 1010CFU/g。
The application of 6 water preparation Tiny ecosystem of embodiment
The water preparation Tiny ecosystem prepared according to embodiment 3 is applied in tilapia cultivating pool, is pressed
According to 1.5 meters of the depth of water, 1 mu/liter dosage is used.The short and small spore in sampling detection finds water body
The quantity of bacillus can reach 103CFU/L, this concentration exceed well over natural bacterium colony in water body
Concentration, forms colony growth advantage, can play the purpose for suppressing streptococcus agalactiae growth.
Table 2 is using one week interior survival number of Bacillus pumilus in water body after product
From Table 2, it can be seen that after applying water preparation Tiny ecosystem of the present invention, within one week,
The Bacillus pumilus viable bacteria of higher level, the 3rd day cell concentration can be detected in pond
Reach highest.
Application of tilapia feed of the embodiment 7 containing CD6 powder in cultivation
The control pool and the test pool are each two mouthfuls, 20 mu of area, 1.5 meters of the depth of water.Tilapia kind
Ji Fu, 3000 tails/mu, 60 days experimental periods.
Control pool feeding basal diet.The test pool is the feedstuff containing CD6 mycopowder, according to reality
The powder product that example 4 is worth is added in the middle of tilapia basal diet according to 0.1% ratio.
CD6 viable counts in obtained tilapia pellet according to the method described above>106CFU/g。
Tilapia feeding method：Once, the control pool and the test pool feed feeding for daily feeding sooner or later
Doses is identical.
Draw a design assay method：Every mouthful of pond draws a design 10, gravimetry, and one was determined per 15 days
It is secondary, and count fish kills situation.
3 tilapia of table weightening statistics
As shown in Table 2, with the feeding tilapia of the feedstuff containing CD6, can improve and cultivate
Weight gain amount in journey, reduces feedstuff-meat ratio, improves food conversion ratio.
In mortality statisticses, after finding fed control feedstuff, the mortality rate of tilapia is more right
Substantially reduce according to group.In the process of the test of 60 days, the mortality rate for testing pool tilapia is more right
10% is reduced according to the pool.
As can be seen here, microbial ecological agent product of the invention is not only safe but also significantly can promote
Enter the individual weight gain of aquatic animal, increase the yield per unit area and total amount, so as to increase Aquatic product
The income of cultivation, increases the benefit.
The above is only the preferred embodiment of the present invention, it is noted that for this technology
For the those of ordinary skill in field, on the premise of without departing from the technology of the present invention principle, also
Some improvements and modifications can be made, these improvements and modifications also should be regarded as the protection of the present invention
Scope.
Claims (10)
1. a bacillus pumilus (Bacillus pumilus) new strains, its preserving number is
CGMCC No.12197。
2. Bacillus pumilus as claimed in claim 1, it is characterised in that its
16sRNA gene orders are as shown in SEQ ID No.1.
3. the high density fermentation culture medium of the Bacillus pumilus described in claim 1, which contains
There is KH2PO40.8~1.5g/L, 3~6g/L of sucrose, 8~12g/L of soybean cake powder, FeSO4
0.25~0.5g/L, CaCO31~3g/L, MgSO42.5~3.5g/L, 4~6g/L of Semen Maydis flour,
6~10g/L of fish flour, defoamer 0.05~0.1%, initial pH7.0~7.5.
4. the fermentation medium of the Bacillus pumilus described in claim 3, it is characterised in that
Containing KH2PO41.0g/L, sucrose 3g/L, soybean cake powder 8g/L, FeSO40.30g/L,
CaCO31.5g/L, MgSO43.0g/L, Semen Maydis flour 5g/L fish flour 8g/L, defoamer
0.05%, initial pH7.5.
5. the fermentation process in high density of Bacillus pumilus described in claim 1, it include as
Lower step：Fermentation of the seed liquor as described in the inoculum concentration of 3-6% is inoculated into claim 3 or 4
In culture medium, 30~37 DEG C of temperature, 200~400r/min of rotating speed, tank pressure 0.05Mpa lead to
Gas compares 1：0.4~0.6,12~16h of fermentation time, obtain spore production rate more than 90%, living
Bacterium number is 109~1010The Bacillus pumilus fermentation liquid of CFU/ml.
6. a kind of microbial ecological agent for suppressing tilapia hammer disease, which contains claim 1 institute
The fermentation liquid of the Bacillus pumilus stated.
7. the microbial ecological agent of tilapia hammer disease is suppressed as claimed in claim 6, and which is special
Levy and be, the NaCl also containing final concentration of 12-15%.
8. a kind of microbial ecological agent for suppressing tilapia hammer disease, which contains claim 1 institute
The mycopowder of the Bacillus pumilus new strains stated, wherein, the work of described Bacillus pumilus
Bacterium number is 1010~1011CFU/g。
9. the microbial ecological agent of tilapia hammer disease is suppressed as claimed in claim 8, and which is special
Levy and be, described mycopowder preparation method is as follows：High density fermentation liquid is utilized into Ceramic Composite
After 3~5 times of membrance concentration, 10~30% starch of addition are spray-dried as carrier, are done
Dry condition is：200~400 DEG C of inlet temperature, 70~85 DEG C of outlet temperature, charging rate
10~12L/min, is atomized rotary head 20000~30000r/min of speed.
10. the tilapia feed containing the Bacillus pumilus mycopowder described in claim 1,
Characterized in that, the addition of the Bacillus pumilus mycopowder is 0.5~5 ‰.
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Address after: 301606 West of Jiuyuan Highway, 3 km south of Linting Kou Town, Baodi District, Tianjin Patentee after: Tianjin DaBeiNong Biotechnology Co.,Ltd. Patentee after: Fujian Dabei nonghuayou Aquatic Technology Group Co.,Ltd. Patentee after: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. Address before: 301606 West of Jiuyuan Highway, 3 km south of Linting Kou Town, Baodi District, Tianjin Patentee before: TIANJIN CHANGNONG SCIENCE & TECHNOLOGY CO.,LTD. Patentee before: FUJIAN DABEINONG FISHERIES SCIENCE & TECHNOLOGY Co.,Ltd. Patentee before: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. |