CN109929784B - One bacillus subtilis and its cultural method and application - Google Patents
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Abstract
The present invention relates to a bacillus subtilis and its cultural method and applications.One bacillus subtilis (Bacillus subtilis) Bsbc01, Chinese microorganism strain collection is preserved on January 25th, 2019, address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, culture presevation number CGMCC No.17239.Present invention firstly discloses a kind of with the bacillus subtilis obtained from the screening of Qinghai-Tibet culture zone, the more existing known bacillus subtilis of the bacterium has significant hypoxemia resistance characteristics, has better application effect as the more existing bacillus subtilis of animal feed additive.
Description
Technical field
The present invention relates to a bacillus subtilis and its cultural method and applications, belong to microorganisms technical field.
Background technique
Bacillus subtilis (Bacillus subtilis) is one kind of bacillus, CAS 68038-70-0.It is single
0.7~0.8 × 2~3 microns of a cell, uniform coloring.Without pod membrane, peritrichous can be moved.Gram-positive bacteria, gemma 0.6
~0.9 × 1.0~1.5 microns, ellipse arrives column, and central or slightly inclined positioned at thallus, thallus does not expand after sporulation.Bacterium colony table
Face is coarse opaque, dirty white or yellowish, and when growing in liquid medium, wrinkle mould is commonly formed.Aerobic bacteria.Using albumen
Matter, a variety of sugar and starch, decompose tryptophan and form indoles.It is widely used in genetics research, to the purine nucleotides of this bacterium
Route of synthesis and its adjustment mechanism research it is clearer.It is widely distributed in soil and the organic matter of corruption, easily soaks juice in withered grass
Middle breeding, it is therefore named.Bacterial strain be alpha-amylase and neutral proteinase important production bacterium；Some bacterial strains have strong degradation core
The enzyme system of thuja acid, therefore often make the parental plant of breeding nucleosides production bacterium or produce the strain of 5'-nucleotidase.
Chinese patent literature CN109161506A (application number 201811098313.X) discloses a bacillus subtilis
And its application, it is related to biological control plant disease field.The bacterial strain is bacillus subtilis (Bacillus subtilis)
Yt1004, deposit number are CGMCC No.14672.The invention also discloses the biological control agent containing above-mentioned bacterial strains and its
Preparation method.The microorganism formulation can be used as biological pesticide or bio-feritlizer prevention and treatment crop root knot nematode disease evil, including cucumber
The one or more such as root-knot nematode, tomato root-knot nematode, great Jiang root-knot nematode, muskmelon root-knot nematode.
Chinese patent literature CN109337841A (application number 201811350457.X) discloses one plant of deposit number
CGMCC NO.14220 has the bacillus subtilis BYS2 of Efficient antibacterial ability；The bacillus subtilis BYS2 can be resistant to
High temperature, acidproof, bile tolerance not only adapt to environment in gastrointestinal tract, but also to Escherichia coli, staphylococcus aureus, production gas pod
Film clostridium has significant inhibiting effect；It is micro- to improve the disease-resistant original of animal body as feed addictive by bacillus subtilis BYS2
Biological infection ability, that is, disease resistance (p < 0.05) remarkably promotes meat chicken growth performance, improves culture efficiency；There is peace simultaneously
Entirely, feature efficiently, prebiotic, has no toxic side effect to animal body.
Bacillus subtilis known to those skilled in the art can realize Reverse transcriptase phase by consumption free oxygen
Pathogenic bacteria are closed, to realize the purpose for killing pathogenic bacteria, therefore, the Hypoxia toleration of bacillus subtilis directly will affect withered grass
The fungistatic effect of bacillus in certain circumstances, therefore, find the high bacillus subtilis of hypoxemia resistance characteristics be improve with
The main path of feed addictive one of of the bacillus subtilis as main antipathogenic composition.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a bacillus subtilis and its cultural method and applications.
Technical solution of the present invention is as follows:
It is micro- to be preserved in China on January 25th, 2019 by one bacillus subtilis (Bacillus subtilis) Bsbc01
Biological inoculum collection, address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, strain
Deposit number CGMCC No.17239.
The cultural method of above-mentioned bacillus subtilis (Bacillus subtilis) Bsbc01, steps are as follows:
(1) by bacillus subtilis (Bacillus subtilis) Bsbc01 streak inoculation in LB solid medium tablets
Activated strains are made in upper 35~38 DEG C of 20~28h of activation culture；
(2) activated strains made from step (1) are inoculated in LB liquid medium, 35~38 DEG C, 180~220rpm vibration
16~20h of culture is swung, seed liquor is made；
(3) seed liquor made from step (2) is inoculated into fluid nutrient medium by the inoculum concentration of percent by volume 5~8%,
Primary fermentation liquid is made in 37 DEG C of culture 8h；
(4) primary fermentation liquid made from step (3) is inoculated into fermented and cultured by the inoculum concentration of percent by volume 5~8%
In base, fermentation liquid is made in 35~38 DEG C of 20~28h of culture.
Preferred according to the present invention, in the step (3), fluid nutrient medium component is as follows:
Corn flour 2%, beancake powder 3%, wheat bran 2%, sodium citrate 0.5%, KH2PO40.03%, K2HPO40.01%,
CaCl20.01%, excess water, pH7.0~7.2.
Preferred according to the present invention, in the step (4), every liter of component of the fermentation medium is as follows:
Corn flour 2%, beancake powder 3%, wheat bran 2%, sodium citrate 0.5%, KH2PO4 0.03%, K2HPO40.01%,
CaCl20.01%, excess water, pH7.0~7.2.
Above-mentioned bacillus subtilis (Bacillus subtilis) Bsbc01 is preparing answering in animal feed additive
With.
A kind of animal feed is mixed above-mentioned fermentation liquid in the ratio of feed total weight 0.05%~0.2% with daily feed
It closes uniform.
Preferred according to the present invention, the cell concentration of the fermentation liquid is (2~8) × 109cfu/ml。
Beneficial effect
Present invention firstly discloses a kind of with screening the bacillus subtilis that obtains from Qinghai-Tibet culture zone, the bacterium compared with
Existing known bacillus subtilis has significant hypoxemia resistance characteristics, as the more existing bacillus subtilis of animal feed additive
Bacterium has better application effect.
Detailed description of the invention
Fig. 1 is the result photo that different strains described in embodiment 1 are grown under low oxygen environment condition；
Fig. 2 is after applying the application bacterial strain and bacillus subtilis CMCC63501 described in embodiment 3 under the same conditions,
Utilize the cultivation results photo of maconkey agar culture medium directional separation farming animals enteron aisle disease bacterium；
In figure: left figure is application bacillus subtilis CMCC63501 result figure；Right figure is to apply herein described withered grass bud
Spore bacillus result figure.
Specific embodiment
Technical solution of the present invention is further elaborated below with reference to embodiment, but institute's protection scope of the present invention is not limited to
This.
Bacillus subtilis (Bacillus subtilis) Bsbc01 as described in the examples was protected on January 25th, 2019
It is hidden in Chinese microorganism strain collection, address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences microorganism is ground
Study carefully institute, culture presevation number CGMCC No.17239；
Embodiment 1
It is derived from yak in Qinghai-tibet free-ranging area soil, sample is subjected to 10 times of gradient dilutions, is respectively coated and is trained in solid LB
It supports and is cultivated on base and solid TSB culture medium, the single colonie grown is forwarded to fluid nutrient medium after 48 hours, continues to cultivate
24 hours.It is applied to solid LB media and carries out pure culture.
It randomly selects 1000 monoclonals and carries out restraining and sterilizing bacteria experiment detection.The pathogenic bacteria kind used are as follows: carrot soft
Rot Erwinia (Erwinia carotovora), carrot pectin bacillus (Pectobacterium carotovorum), copper
Green pseudomonad (Pseudomonas aeruginosa PAO1), rice pathogenic bacteria cloth kirschner bacterium (Burkholderia
Glumae), glume blight of rice plant bacterium (Burkholdria glumae) is solved Erzvinia amylovora (Erwinia amylovora), beautiful
Rice bacterial wilt germ (Pantoea stewartii ssp.stewartii)；Bacterial strain wherein with narrow spectrum bactericidal activity
Totally 39 plants, have 12 plants of bacterial strain of broad spectrum antibacterial activity, there will be the active 1 plant of bacterial strain of most strong restraining and sterilizing bacteria to be sequenced, and name
For bacillus subtilis Bsbc.
By analysis bacillus Bsbc growth characteristics result it is found that in terms of salt tolerance, the salt tolerance of Bsbc is stronger, In
Well-grown under the culture medium condition that the mass percentage content of NaCl is 12%, Bsbc is resistant to low temperature in terms of growth temperature
And hot environment, it can be grown under the conditions of 4 DEG C~45 DEG C；In terms of resistance to acid and alkali, it is adapted to life of the pH 5.5~9.0
Long environment, metal ion are little to the growth effect of Bsbc.The advantage for needing to particularly point out is that Bsbc hypoxemia resistance characteristics are especially
It is prominent, using micro- aerobic productions airbag to Bsbc and before reported bacillus subtilis ATCC 6633 and CMCC 63501 (with
The Yt1004 homology referred in background technique is 100%) to be compared, and carries out (11000Pa, normal atmosphere under low-oxygen environment
The 50% of partial pressure of oxygen), through 48 hours progress viability examinations of LB culture medium culture, as the result is shown existence of the Bsbc under low-oxygen environment
Rate is 3.6 times of control group, and sterilizing ability is 3.3 times of control group.
Ultraviolet accidental ultraviolet mutagenesis then is carried out to Bsbc, anti-low-oxygen environment mutant strain is then screened under low-oxygen environment,
Mutant strain Bsbc01 is obtained, is compared with wild strain, survival rate of the Bsbc01 under low-oxygen environment is wild as the result is shown
2 times of type bacterial strain, sterilizing ability are 1.8 times of wild-type strain.After being cultivated 48 hours under low-oxygen environment, bacterial strain survival ability ratio
It is as shown in Figure 1 compared with result.
Bacillus subtilis (Bacillus subtilis) Bsbc01 was preserved in the micro- life of China on January 25th, 2019
Object Culture Collection Center, address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, strain are protected
Hide number CGMCC No.17239.
Embodiment 2
The cultural method of above-mentioned bacillus subtilis (Bacillus subtilis) Bsbc01, steps are as follows:
(1) by bacillus subtilis (Bacillus subtilis) Bsbc01 streak inoculation in LB solid medium tablets
For 24 hours, activated strains are made in upper 37 DEG C of activation cultures；
(2) activated strains made from step (1) are inoculated in LB liquid medium, 37 DEG C, 180rpm shaken cultivation
Seed liquor is made in 18h；
(3) seed liquor made from step (2) is inoculated into fluid nutrient medium by the inoculum concentration of percent by volume 5%, 37
DEG C culture 8h, be made primary fermentation liquid；
The fluid nutrient medium component is as follows:
Corn flour 2%, beancake powder 3%, wheat bran 2%, sodium citrate 0.5%, KH2PO40.03%, K2HPO40.01%,
CaCl20.01%, excess water, pH7.0；
(4) primary fermentation liquid made from step (3) is inoculated into fermentation medium by the inoculum concentration of percent by volume 8%
In, for 24 hours, fermentation liquid is made in 37 DEG C of cultures；
Every liter of component of the fermentation medium is as follows:
Corn flour 2%, beancake powder 3%, wheat bran 2%, sodium citrate 0.5%, KH2PO40.03%, K2HPO40.01%,
CaCl20.01%, excess water, pH7.0.
Through detecting, cell concentration is 5 × 10 in fermentation liquid9cfu/ml。
Embodiment 3
Fermentation liquid is mixed according to 0.1% ratio with daily feed (ordinary commercial products), as experimental group；Separately
If adding the contrast groups of identical 63501 fermentation liquid of cell concentration bacillus subtilis CMCC, fermentation liquid adding proportion and daily feeding
Expect identical as experimental group；Two groups with dosage by being fed in the usual way.
Through carrying out intestinal flora separation to the farming animals after above-mentioned products applied, and laboratory cultures are carried out, cultivates item
Part is respectively LB culture medium, maconkey agar culture medium, MA culture medium, TSB culture medium, and cultivation temperature is respectively 37 DEG C, and 32 DEG C,
30 DEG C, incubation time is 48 hours, analyzes bacterial diversity.Meanwhile the high-throughput and macro gene order-checking of enteron aisle is carried out, it will be sequenced
Result afterwards is compared, and discovery this product can significantly reduce the ratio of enteron aisle disease bacterium Escherichia coli and pseudomonas aeruginosa
Rate, two kinds of bacterium experimental groups reduce 38.2% and 26.1% compared with abundance of the contrast groups in enteron aisle respectively；Meanwhile it improving beneficial
Bacterium bacillus subtilis, the ratio of enteron aisle lactic acid bacteria, two kinds of bacterium experimental groups are respectively increased compared with abundance of the contrast groups in enteron aisle
12.8% and 22.6%.Wherein, the fungistatic effect of disease Escherichia coli is the most obvious, and experimental group is significantly higher than contrast groups, as a result
As shown in Figure 2.
Claims (7)
1. a bacillus subtilis (Bacillus subtilis) Bsbc01 is preserved in the micro- life of China on January 25th, 2019
Object Culture Collection Center, address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, strain are protected
Hide number CGMCC No.17239.
2. the cultural method of bacillus subtilis (Bacillus subtilis) Bsbc01 as described in claim 1, feature
It is, steps are as follows:
(1) by bacillus subtilis (Bacillus subtilis) Bsbc01 streak inoculation in 35 in LB solid medium tablets
Activated strains are made in~38 DEG C of 20~28h of activation culture；
(2) activated strains made from step (1) are inoculated in LB liquid medium, 35~38 DEG C, 180~220rpm oscillation training
16~20h is supported, seed liquor is made；
(3) seed liquor made from step (2) is inoculated into fluid nutrient medium by the inoculum concentration of percent by volume 5~8%, 37 DEG C
8h is cultivated, primary fermentation liquid is made；
(4) primary fermentation liquid made from step (3) is inoculated into fermentation medium by the inoculum concentration of percent by volume 5~8%,
Fermentation liquid is made in 35~38 DEG C of 20~28h of culture.
3. cultural method as claimed in claim 2, which is characterized in that in the step (3), fluid nutrient medium component is as follows:
Corn flour 2%, beancake powder 3%, wheat bran 2%, sodium citrate 0.5%, KH2PO40.03%, K2HPO40.01%, CaCl2
0.01%, excess water, pH7.0~7.2.
4. cultural method as claimed in claim 2, which is characterized in that in the step (4), every liter of group of the fermentation medium
Divide as follows:
Corn flour 2%, beancake powder 3%, wheat bran 2%, sodium citrate 0.5%, KH2PO40.03%, K2HPO40.01%, CaCl2
0.01%, excess water, pH7.0~7.2.
5. bacillus subtilis described in claim 1 (Bacillus subtilis) Bsbc01 is preparing animal feed additive
In application.
6. a kind of animal feed, which is characterized in that fermentation liquid made from step described in claim 2 (4) is pressed feed total weight
0.05%~0.2% ratio is uniformly mixed with daily feed.
7. animal feed as claimed in claim 6, which is characterized in that the cell concentration of the fermentation liquid be (2~8) ×
109cfu/ml。
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| CN110150478A (en) * | 2019-06-28 | 2019-08-23 | 青岛宝创生物科技有限公司 | A kind of feed addictive and the preparation method and application thereof reducing mastitis for milk cows disease incidence |
| CN111172085B (en) * | 2020-03-15 | 2021-04-02 | 青岛宝创生物科技有限公司 | Application of bacillus subtilis in production of substances inhibiting fungi |
| CN112094770B (en) * | 2020-08-18 | 2022-04-08 | 江苏滋百农生态农业股份有限公司 | Bacillus altitudinis, application of active substance compound solution and microbial inoculum thereof in preventing and treating root knot nematode disease |
| CN113151065B (en) * | 2021-03-30 | 2022-08-05 | 山东蔚蓝生物科技有限公司 | Bacillus subtilis for preventing and treating nematode diseases and application thereof |
| CN113308411B (en) * | 2021-07-09 | 2023-07-04 | 广东海纳川生物科技股份有限公司 | Bacillus subtilis culture method and application thereof |
| CN114958676B (en) * | 2022-06-15 | 2024-02-20 | 扬州大学 | Antibacterial agent based on bacillus fermentation product, and preparation method and application thereof |
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| CN103652508A (en) * | 2013-12-20 | 2014-03-26 | 南京农业大学 | Ensilage combination including barley straws and festuca arundinacea and application thereof |
| CN108576424A (en) * | 2018-05-21 | 2018-09-28 | 侯宝良 | Improve the yak fermented feed of growth performance |
| CN109161509B (en) * | 2018-10-10 | 2021-09-03 | 中国农业科学院兰州兽医研究所 | Bacterial strain capable of preventing and treating bovine and sheep diarrhea |
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