CN105441348B - One bacillus subtilis novel bacterial strain, probiotics and application - Google Patents
One bacillus subtilis novel bacterial strain, probiotics and application Download PDFInfo
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- CN105441348B CN105441348B CN201410460159.1A CN201410460159A CN105441348B CN 105441348 B CN105441348 B CN 105441348B CN 201410460159 A CN201410460159 A CN 201410460159A CN 105441348 B CN105441348 B CN 105441348B
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Abstract
The invention belongs to aquatic products probiotics technical fields, and in particular to a bacillus subtilis novel bacterial strain, probiotics and application.Bacillus subtilis disclosed by the invention (Bacillus subtilis) H8-1 deposit number is CGMCC No.9356, harmful substance such as ammonia nitrogen and nitrite nitrogen in water is reduced in a manner of pollution-free and noresidue, improves cultivation quality, improves economic benefit of aquaculture, and there is promotional value.Bacillus subtilis H8-1 in the present invention is respectively 81.72%, 99.70% to ammonia nitrogen, the nitrite nitrogen degradation rate that concentration is 2.5mg/L.
Description
Technical field
The invention belongs to aquatic products probiotics technical fields, and in particular to bacillus subtilis new strains, Tiny ecosystem system
Agent and application.
Background technique
In recent years, high density, intensive aquaculture gradually replace traditional free-range mode, greatly improve aquaculture
Yield and economic benefit.But people also result in a series of while cultivation density is continuously improved to pursue cultured output
Common problem.Excessive bait throwing in causes the reduction of mixed bait utilization rate, Organic substance in water is caused to increase；Dose increases, and causes
Nitrogen, phosphorus surge in water；The organic pollutions breeding water bodies such as a large amount of excrement；Water transparency decline, water quality deterioration.It is excessive in water body
Organic matter decomposition not only consume the dissolved oxygen in water body, water body is in anaerobic condition, releases many harmful substances, such as ammonia
Nitrogen, nitrite nitrogen and hydrogen sulfide etc., directly or indirectly influence the diversity of organism in water, the cultivation of high-quality aquatic products kind by
The limitation of water quality and food organisms.Control cultivating pool water ecological setting has been the pass that China's pond aquaculture develops in a healthy way
Key problem, the control and purification of water quality of aquaculture pond are extremely urgent.
Currently, domestic and foreign scholars have made intensive studies, and probiotics are pollution-free with its, noresidue and multi-functional
Property the features such as, and sporogenic form exists within producing bacillus subtilis product, to poor environments such as high temperature, low temperature, high salinity
Resistance it is very strong, with significant ground degradation of ammonia nitrogen and nitrite nitrogen ability and inulinase-producing activity.Currently, bacillus conduct
The technical issues of dropping nitrogen bacterium is primarily present in the production phase, and there are also in product practical application effect.Although bacterial strain is very much,
Be bacterial strain effect difference it is big, it is actually active but seldom.
Summary of the invention
To solve the above problems, the present invention provides a bacillus subtilis novel bacterial strain and probiotics.
In the present invention bacillus subtilis (Bacillus subtilis) H8-1, it is applicant from Fujian shrimp feed coefficient
Isolated in water, biological property is as follows: bacterium colony surface is smooth, translucent, and in dirty white, bacterium colony is round, edge at
Zigzag；Colonial morphology be it is rod-shaped, size is 0.8 ~ 1.2 × 1.5 ~ 4.0um, gemma form be it is oval arrive column, be located at thallus
Central or slightly inclined, thallus does not expand after sporulation.Bacillus subtilis and existing bacillus subtilis area in the present invention
Not in can under 0 ‰, 3 ‰, 15 ‰ salt concentration conditions equal well-grown, 10-15 DEG C of low temperature, well-grown can be resistant to.
In the present invention bacillus subtilis (Bacillus subtilis) H8-1, gelatin reacting positive, jellyization ox
Cream restores nitrate, edwardsiella hoshinae, H2S, V-P reacting positive, starch experiment is positive, and glucose fermentation produces acid but do not produce gas, and needs
Oxygen is suitable for 25 ~ 37 DEG C of growths.
In the present invention bacillus subtilis (Bacillus subtilis) H8-1,16S rRNA nucleic acid sequence such as SEQ
Shown in ID No.1.Be put into Genbank and be compared, as the result is shown withBacillus subtilis16S rRNA sequence
Similitude is up to 99%.In conjunction with bacterial morphological characteristic, physio-biochemical characteristics, growth conditions, determine bacillus (Bacillus sp.)
H8-1 is bacillus subtilis.
In the present invention bacillus subtilis (Bacillus subtilis) H8-1, in preservation on June 18 in 2014
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, abbreviation CGMCC, address: the Chaoyang District, Beijing City North Star
The institute 3 of West Road 1, Institute of Microorganism, Academia Sinica, deposit number are CGMCC No. 9356.
The present invention also provides the probiotics for containing the bacterial strain.
Bacterial strain of the invention can be prepared into feed addictive, can also be added in conventional feed addictive, therefore, this
Invention also provides a kind of feed addictive containing the bacterial strain.
The feed addictive of the invention can be added to conventional premix, such as vitamin and minerals premix
In, therefore, the present invention also provides a kind of premixes containing the feed addictive.
The present invention also provides the application of the bacterial strain or the probiotics in improvement breeding water body.
Specifically, the ammonia nitrogen and/or nitrous state of bacterial strain of the present invention or the probiotics for water body of degrading
Nitrogen level.
In the present invention bacillus subtilis (Bacillus subtilis) H8-1 be breeding water body improve probiotics
Provenance is provided, harmful substance such as ammonia nitrogen and nitrite nitrogen in water is reduced in a manner of pollution-free and noresidue, improves cultivation product
Matter improves economic benefit of aquaculture, has promotional value.Bacillus subtilis H8-1 in the present invention is 2.5mg/L's to concentration
Ammonia nitrogen, nitrite nitrogen degradation rate are respectively 81.72%, 99.70%.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..
Embodiment 1
The water sample from Tianjin Wuqing, Fujian, Jiangsu fish pond and the shrimp pool and mud sample 124 are taken, 1mL water sample or mud sample are taken
1g is in the 9mL LB liquid medium of 18ml test tube, 85 DEG C of water bath processing 10min.
It is 30 DEG C that test tube after high-temperature process, which is placed on temperature, under the aerobic condition of 180rpm after constant-temperature shaking culture 16h
Obtain enrichment culture liquid.
Enrichment culture liquid is carried out by gradient dilution using coubling dilution, is coated on LB solid medium, is placed in 30
Culture 20h is carried out in DEG C insulating box, the different single colonies grown on picking plate are crossed, and are purified twice, are lived
Single colonie, altogether obtain 275 plants of bacterium.
Single colonie after purifying twice is placed in 30 DEG C of insulating boxs and is seen with toothpick dibbling on fresh haemolysis plate
Examine for 24 hours, 48h, 72h, if there is haemolysis circle, according to the Capability Categories for making haemocyte agar haemolysis: 1) α haemolysis: grass green is molten
There is the grass green colour circle of 1 ~ 2mm in blood, periphery of bacterial colonies culture medium, are caused by ferrihemoglobin；2) β haemolysis: complete hemolysis, bacterium colony
Surrounding forms a completely Clear & Transparent zone of hemolysis, is caused by bacteriogenic hemolysin is completely dissolved red blood cell；3)γ
Haemolysis: not haemolysis.There to be haemolysis circle bacterial strain to eliminate.
It will cross on Selective solid culture medium without hemolytic strain, well-grown bacterial strain LB Liquid Culture,
30 DEG C of constant temperature expand culture 16h.
Solid selective medium is two kinds: with NH4 +- N is for the ammonia nitrogen selective medium of only nitrogen source and with NO2 --N
For the nitrite nitrogen culture medium of only nitrogen source；
Ammonia nitrogen Selective solid culture medium is every L by (NH4)2SO40.5g, sodium succinate 2.17g, Vickers salting liquid 50
ML, agar 20g and the water of surplus composition, pH7.0-7.2,115 DEG C of high pressure sterilization 20min, the Vickers salting liquid be every L by
K2HPO45.0g, MgSO4·7H2O 2.5g, NaCl 2.5g, FeSO4·7H2O 0.05 g, MnSO4·4H20.05 g of O and remaining
The water of amount forms；
Nitrite nitrogen Selective solid culture medium is every L by NaNO20.5g, sodium succinate 2.17g, Vickers salting liquid 50
ML, agar 20g and the water of surplus composition, pH7.0-7.2,115 DEG C of high pressure sterilization 20min, the Vickers salting liquid be every L by
K2HPO45.0g, MgSO4·7H2O 2.5g, NaCl 2.5g, FeSO4·7H2O 0.05 g, MnSO4·4H20.05 g of O and remaining
The water of amount forms.
It is final to obtain without haemolysis circle and well-grown 63 plants of buds on ammonia nitrogen and nitrite nitrogen Selective solid culture medium
Spore bacillus.
Nitrogen function drops in 2 high throughput assay bacterial strain of embodiment, screens high efficient strain
In super-clean bench, 63 plants of strain to be tested bacterium solutions are accessed in corresponding screening and culturing medium, inoculation final concentration of 5 ×
105CFU/mL, repeat number 3；It is put into shaking table and carries out 30 DEG C, 180 r/min of culture, cultivate 48h；Uniform sampling is to 1.5mL
In centrifuge tube, 12000 rpm are centrifuged 3min, take 200 μ L of supernatant that 96 orifice plates are added；
It when measuring nitrite nitrogen, is added after 200 μ L of test sample supernatant in 96 orifice plates, Ge Lisi examination is successively added in every hole
Each 20 μ L of agent A, B, in 550nm colorimetric estimation；When measuring ammonium nitrogen, 20 μ L of nessler reagent is added in every hole, is surveyed in 450nm colorimetric
It is fixed；
Screening and culturing medium is two kinds: ammonia nitrogen screening and culturing medium and nitrite nitrogen screening and culturing medium；
Ammonia nitrogen screening and culturing medium is every 50mL by (NH4)2SO450 μ l of mother liquor, the commercially available water for crushing shrimp material 0.05g and surplus
Composition, ammonium nitrogen concentration are as follows: 2.5mg/l；
Nitrite nitrogen screening and culturing medium is every 50mL by NaNO250 μ l of mother liquor, the commercially available water for crushing shrimp material 0.05g and surplus
Composition, nitrite nitrogen concentration are as follows: 2.5mg/l.
Ammonium sulfate liquor is to weigh 9 g (NH4)2SO41L is added and simulates shrimp pool water, ammonium nitrogen concentration is about at this time are as follows: 2.5g/
l；
Sodium nitrite mother liquor is to weigh 3.75 g NaNO21L is added and simulates shrimp pool water, nitrite nitrogen concentration is about at this time are as follows:
2.5g/l。
It chooses 10 plant bacterium (number A1-A10) of the ammonia nitrogen degradation rate higher than 30% or cultured water degradation rate is higher than 70%
10 plants of bacterium (number Y1-Y10) carry out replication not to be inoculated with any bacterial strain as negative control (CK).
Measuring method: in super-clean bench, strain to be tested bacterium solution is accessed in corresponding screening and culturing medium, inoculation final concentration of 5
×105CFU/mL, repeat number 3；Be put into shaking table carry out culture 30 DEG C, 180 r/min, culture for 24 hours, 48h；Uniform sampling is arrived
In 1.5mL centrifuge tube, 12000 r/min are centrifuged 3min, take 200 μ L of supernatant that 96 orifice plates are added.
It when measuring nitrite nitrogen, is added after 200 μ L of test sample supernatant in 96 orifice plates, Ge Lisi examination is successively added in every hole
Each 20 μ L of agent A, B, in 550nm colorimetric estimation；It measures every hole when ammonia nitrogen and 20 μ L of nessler reagent is added, in 450nm colorimetric estimation.
Qualitative determination: when measurement nitrite nitrogen, solution, which becomes pink, rose, orange or brown etc., indicates Asia
Nitrate reduction, reacts for the positive, i.e. the bright degradation effect of color more elementary introduction is better；When measuring ammonium nitrogen, solution becomes orange, yellow
Color etc. is positive reaction, is shallowly preferred.
Quantitative determination: nitrite nitrogen concentration (Y is formulated1) and measurement OD value (X1) between reference standard curve and ammonia nitrogen concentration
(Y2) and measurement OD value (X2) between reference standard curve.
Screening and culturing medium includes ammonia nitrogen screening and culturing medium and nitrite nitrogen screening and culturing medium；
Ammonia nitrogen screening and culturing medium: (NH4)2SO450 μ l of mother liquor, shrimp material 0.05g, distilled water 50mL, ammonia nitrogen concentration is about are as follows:
2.5mg/L, 121 DEG C of sterilizings.Ammonium sulfate liquor: 9 g (NH are weighed4)2SO41L distilled water is added, ammonium nitrogen concentration is about at this time are as follows:
2.5g/l。
Nitrite nitrogen screening and culturing medium: NaNO250 μ l of mother liquor, shrimp material 0.05g, distilled water 50mL, nitrous nitrogen concentration is about:
2.5 mg/l, 121 DEG C of sterilizings.Sodium nitrite mother liquor: 3.75 g NaNO are weighed21L distilled water is added, at this time nitrite nitrogen concentration
About are as follows: 2.5g/l.
Ge Lisishi reagent (Griess) A: p-aminobenzene sulfonic acid 0.5g, spirit of vinegar (10% or so) 150mL, brown bottle are kept away
Light, 4 DEG C of preservations.Ge Lisishi reagent B: αnaphthylamine 0.1g, distilled water 20mL, 150 mL of spirit of vinegar (10% or so), brown bottle are kept away
4 DEG C of light preservations.
Nessler reagent: cash purchase, 4 DEG C of preservations.
As a result as shown in Table 1 and Table 2, the average ammonia nitrogen degradation rate of H8-1 is 81.72%, and average nitrous degradation rate is
99.70%, nitrogen ability highest drops.
1 10 plants of preferred strain ammonia nitrogen degradation rate comparative measurements of table (No. A10 is H8-1)
| Bacterial strain number | A1 | A2 | A3 | A4 | A5 | A6 | A7 | A8 | A9 | A10 | ck |
| Ammonia nitrogen for 24 hours | 34.096 | 30.000 | 19.789 | 45.702 | 37.728 | 39.985 | 59.574 | 21.277 | 75.487 | 79.572 | 0.011 |
| 48h ammonia nitrogen | 45.897 | 35.786 | 38.931 | 56.971 | 40.302 | 44.818 | 31.405 | 40.626 | 61.079 | 83.873 | 0.441 |
2 10 plants of preferred strain nitrite nitrogen degradation rate comparative measurements of table (No. Y10 is H8-1)
| Bacterial strain number | Y1 | Y2 | Y3 | Y4 | Y5 | Y6 | Y7 | Y8 | Y9 | Y10 | ck |
| Nitrous for 24 hours | 83.121 | 74.374 | 90.671 | 92.332 | 75.629 | 74.506 | 90.467 | 95.714 | 92.196 | 99.603 | 0.002 |
| 48h nitrous | 83.018 | 74.508 | 90.128 | 93.671 | 75.457 | 70.165 | 92.467 | 96.541 | 94.467 | 99.806 | 2.443 |
The bacillus subtilis H8-1 screened is preserved in Chinese microorganism strain preservation pipe on June 18th, 2014
Reason committee common micro-organisms center, abbreviation CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences
Institute of microbiology, deposit number are CGMCC No. 9356.
H8-1 growing state compares under the different salt concentration conditions of embodiment 3
LB solid medium is prepared using the water that salinity is respectively 0 ‰, 3 ‰, 15 ‰, with the toothpick dibbling H8-1 after sterilizing
It on the plate of different salinity, is placed in 30 DEG C of incubators and cultivates 16h, observe bacterium colony growing state on plate.It is sent out after culture
Existing, bacterium colony growth of the dibbling on the different plate of three kinds of salinity is good, and the bacterium colony size after the completion of growing is more consistent, explanation
H8-1 salt resistant character is strong.
LB culture medium: tryptone: 10g/L；Yeast extract 5g/L；Sodium chloride 10g/L；Agar powder 15g/L.Use salinity
Meter prepares the water that salinity is respectively 0 ‰, 3 ‰, 15 ‰, carries out the preparation of culture medium.
Nitrogen performance measurement drops in H8-1 under the different salt concentration conditions of embodiment 4
It the results are shown in Table 3, it is as shown in the table, under 0 ‰, 3 ‰, 15 ‰ salinity, the drop nitrogen performance of H8-1 difference, but by salt
The influence very little of degree.
Table 3 measures the drop nitrogen performance of H8-1 under different salinity
Ammonia nitrogen screening and culturing medium: (NH4)2SO4Mother liquor 50 μ l, shrimp material 0.05mg use water (0 ‰ salt of different salinity respectively
The water of degree: distilled water；The water of 3 ‰ and 15 ‰ salinity: addition sea salt is measured into distilled water with salinometer, reaches salinity difference
For 3 ‰ and 15 ‰) 50mL, ammonia nitrogen concentration is about are as follows: 2.5mg/L, 121 DEG C of sterilizings.Ammonium sulfate liquor: 9 g (NH are weighed4)2SO4Add
Enter 1L distilled water, ammonium nitrogen concentration is about at this time are as follows: 2.5g/l.
Nitrite nitrogen screening and culturing medium: NaNO2Mother liquor 50 μ l, shrimp material 0.05mg, the water 50mL of different salinity, nitrous nitrogen concentration
About are as follows: 2.5 mg/l, 121 DEG C of sterilizings.Sodium nitrite mother liquor: 3.75 g NaNO are weighed21L distilled water is added, at this time nitrous state
Nitrogen concentration is about are as follows: 2.5g/l.
Measuring method is the same as embodiment 2.
The H8-1 growing state under cryogenic of embodiment 5
With the toothpick dibbling H8-1 single colonie after sterilizing on LB solid plate, be individually positioned in temperature be 10 DEG C, 15 DEG C,
In 30 DEG C of incubators for 24 hours, bacterium colony growing state on plate is observed.It finds after culture, is grown in 10 DEG C and 15 DEG C of incubators
Bacterium colony size is similar on plate, is be grown in 30 DEG C of incubators bacterium colony size on plate 1/2, this illustrates H8-1 in temperature
In lower breeding environment also can normal growth and play drop nitrogen function.
LB solid medium: tryptone: 10g/L；Yeast extract 5g/L；Sodium chloride 10g/L；Agar powder 15g/L.
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, without departing from the technical principles of the invention, several improvements and modifications can also be made, these improvements and modifications
Also it should be regarded as protection scope of the present invention.
Sequence table
<110>BeiJing DaBei farm Science Group Co., Ltd
Fujian Da Bei Nong Aqua Sciences Inc.
The prosperous agriculture science and technology limited Company in Tianjin
<120>one bacillus subtilis novel bacterial strains, probiotics and application
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1424
<212> DNA
<213>bacillus subtilis (Bacillus subtilis)
<400> 1
cttggcggct gctctaaagg ttacctcacc gacttcgggt gttacaaact ctcgtggtgt 60
gacgggcggt gtgtacaagg cccgggaacg tattcaccgc ggcatgctga tccgcgatta 120
ctagcgattc cagcttcacg cagtcgagtt gcagactgcg atccgaactg agaacagatt 180
tgtgggattg gcttaacctc gcggtttcgc tgccctttgt tctgtccatt gtagcacgtg 240
tgtagcccag gtcataaggg gcatgatgat ttgacgtcat ccccaccttc ctccggtttg 300
tcaccggcag tcaccttaga gtgcccaact gaatgctggc aactaagatc aagggttgcg 360
ctcgttgcgg gacttaaccc aacatctcac gacacgagct gacgacaacc atgcaccacc 420
tgtcactctg cccccgaagg ggacgtccta tctctaggat tgtcagagga tgtcaagacc 480
tggtaaggtt cttcgcgttg cttcgaatta aaccacatgc tccaccgctt gtgcgggccc 540
ccgtcaattc ctttgagttt cagtcttgcg accgtactcc ccaggcggag tgcttaatgc 600
gttagctgca gcactaaggg gcggaaaccc cctaacactt agcactcatc gtttacggcg 660
tggactacca gggtatctaa tcctgttcgc tccccacgct ttcgctcctc agcgtcagtt 720
acagaccaga gagtcgcctt cgccactggt gttcctccac atctctacgc atttcaccgc 780
tacacgtgga attccactct cctcttctgc actcaagttc cccagtttcc aatgaccctc 840
cccggttgag ccgggggctt tcacatcaga cttaagaaac cgcctgcgag ccctttacgc 900
ccaataattc cggacaacgc ttgccaccta cgtattaccg cggctgctgg cacgtagtta 960
gccgtggctt tctggttagg taccgtcaag gtaccgccct attcgaacgg tacttgttct 1020
tccctaacaa cagagcttta cgatccgaaa accttcatca ctcacgcggc gttgctccgt 1080
cagactttcg tccattgcgg aagattccct actgctgcct cccgtaggag tctgggccgt 1140
gtctcagtcc cagtgtggcc gatcaccctc tcaggtcggc tacgcatcgt tgccttggtg 1200
agccgttacc tcaccaacta gctaatgcgc cgcgggtcca tctgtaagtg gtagccgaag 1260
ccacctttta tgtttgaacc atgcggttca aacaaccatc cggtattagc cccggtttcc 1320
cggagttatc ccagtcttac aggcaggtta cccacgtgtt actcacccgt ccgccgctaa 1380
catcagggag caagctccca tctgtccgct cgactgcatg atac 1424
Claims (5)
1. a bacillus subtilis (Bacillus subtilis) bacterial strain, which is characterized in that its deposit number is CGMCC
No.9356。
2. the probiotics containing bacterial strain described in claim 1.
3. the feed addictive containing bacterial strain described in claim 1.
4. the premix containing feed addictive described in claim 3.
5. application of the bacterial strain described in claim 1 in improvement breeding water body, which is characterized in that the ammonia nitrogen for water body of degrading
And/or nitrite nitrogen is horizontal.
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| CN105969708B (en) * | 2016-08-01 | 2019-09-03 | 浙江大学 | Has the bacillus subtilis SC228 and application thereof of nitrification function |
| CN108865940B (en) * | 2018-07-17 | 2021-10-08 | 中国海洋大学 | Heterotrophic nitrification-aerobic denitrification bacillus and composite bacterial preparation thereof |
| CN112011486B (en) * | 2020-09-09 | 2022-03-01 | 天津市农业科学院 | Synchronous nitrification and denitrification bacillus subtilis K8 and application thereof |
| CN112553112B (en) * | 2020-12-19 | 2023-01-17 | 武汉水之国环保科技有限公司 | Denitrifying bacillus subtilis and application thereof in strain detoxification |
| CN113249273B (en) * | 2021-06-30 | 2022-03-08 | 泸州老窖股份有限公司 | Salt-tolerant bacillus subtilis and application thereof in high-salt ammonia nitrogen wastewater treatment |
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| CN1603248A (en) * | 2003-02-14 | 2005-04-06 | 中国科学院南京土壤研究所 | Biological denitrification method for ammonium-nitrogen containing waste water and microbes thereof |
| CN103773722A (en) * | 2014-01-16 | 2014-05-07 | 中国药科大学 | Salt-tolerance bacillus subtilis with low-temperature biological deamination function and application of bacillus subtilis |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1603248A (en) * | 2003-02-14 | 2005-04-06 | 中国科学院南京土壤研究所 | Biological denitrification method for ammonium-nitrogen containing waste water and microbes thereof |
| CN103773722A (en) * | 2014-01-16 | 2014-05-07 | 中国药科大学 | Salt-tolerance bacillus subtilis with low-temperature biological deamination function and application of bacillus subtilis |
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| Title |
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| 枯草芽孢杆菌改善水质提高凡纳滨对虾幼体抗逆性的研究;陆家昌 等;《齐鲁渔业》;20101231;第27卷(第3期);摘要部分 * |
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