CN103289933B - Bacillus amyloliquefaciens plant subspecies B-01 and application thereof - Google Patents
Bacillus amyloliquefaciens plant subspecies B-01 and application thereof Download PDFInfo
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Abstract
The invention relates to the field of microbes, and particularly relates to bacillus amyloliquefaciens and application thereof. The bacillus amyloliquefaciens is a bacillus amyloliquefaciens plant subspecies B-01, and the preservation number of the bacillus amyloliquefaciens plant subspecies B-01 is CCTCC: M2012191. The strain disclosed by the invention can be used as a water environment ecological purifying agent for purification and can keep the characteristic of higher enzyme activity under the condition of low temperature and play a better biological role in a majority of national regions with lower environmental temperature.
Description
Technical field
The present invention relates to microorganism field, be specifically related to a kind of bacillus amyloliquefaciens and application thereof.
Background technology
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens, BA) be that the current Ministry of Agriculture makes (2008 No. 1126) zymin class fodder additives allow the probiotic bacterium kind of using, the Dan Gaijun Ministry of Agriculture makes in animal microorganism fodder additives column and not yet listing.Research shows that this bacterium can regulate and control animal digestive tract micro-ecological environment as probiotic bacterium, suppress harmful bacteria, improve organism metabolism function, this type of bacterial classification suitable growth temperature that current national conservation center provides is generally 28-35 ℃ of left and right, under low temperature water body envrionment conditions, because related enzyme systems vigor is lower, suppressed greatly the performance of probiotic bacterium quasi-biology function, this mushroom is mainly used in the production of zymin at present, in purification of water quality, not yet relates to.
Summary of the invention
In order to solve the existing B-01 bacillus amyloliquefaciens lower problem of enzymic activity at low temperatures, separation in the barnyard grass of the present invention from the cold environment of the northern area of China-35 ℃, purifying, cultivation are to strain low temperature high activity bacterial strain a conservation, the relevant bacterial classification existing from current national conservation center has different biological characteristicses, effectively solved the deficiency that present technology exists.
The national preservation mechanism such as Bacillus amyloliquefaciens strain of the present invention and Chinese industrial microbial strains preservation administrative center existing bacterial classification source and biological characteristics are different, have low temperature tolerance characteristics, well-grown under 18 ℃ of conditions.In the preservation of Chinese industrial microbial strains, administrative center carries out bacterium classification evaluation, clearly identify that B-01 is bacillus amyloliquefaciens plant subspecies (Bacillus amyloliquefaciens subsp.plantarum), by the preservation of Chinese industrial microbial strains, administrative center has carried out genetic stability test, proves that this bacterial strain proterties can genetic stability.Its deposit number is CCTCC:M2012191
B-01 bacillus amyloliquefaciens plant subspecies of the present invention can be brought into play higher biologic activity in purification of water quality.This bacterium is well-grown under 18 ℃ of inclined to one side cold condition, secreted amylase, lipase, the proteolytic enzyme of this bacterium all has higher enzyme activity, in the low water temperature environment of eutrophy, larger molecular organics can be degraded to small-molecule substance, on the one hand can back feeding probiotic bacterium, can directly be utilized by aquatic animal, plants on the other hand, contribute to the restoration of the ecosystem of water body environment.This bacterial strain is as water quality cleansing agent, the larger molecular organics (protein, carbohydrate, fat) that can effectively degrade in water body, reduce chemical oxygen demand (COD) (COD), ammonia nitrogen, nitrite,, improve oxygen capacity (DO), improve the indexs such as pH, turbidity, suspension content.
B-01 bacillus amyloliquefaciens can be used as new type water environment probiotic bacterium ecological purificant and the application of novel animal microorganism feed addictive.
Compare with existing bacillus amyloliquefaciens, bacterial strain of the present invention can keep higher enzyme activity characteristic under cold condition, can compared with lowland area, bring into play good biological action in the most of envrionment temperatures of China.Can effectively the degrade organic substance of overnutrition in environment water of bacterial strain of the present invention, is degraded to by larger molecular organics the small-molecule substance that aquatic animals and plants can utilize, and turns waste into wealth, as water surrounding ecological purificant.This,, for alleviating the current water resource environment increasingly worsening, purifies the restoration of the ecosystem of livestock industry aquaculture water and life fluvial-environment, and life, the production water surrounding of building green safety, have positive sociology meaning and economic value.In addition, this bacterial strain also can be used as animal microorganism fodder additives and is applied to livestock breeding industry, as Substitutes For Antibiotic application, can be and alleviates abuse of antibiotics present situation performance significant role.
Accompanying drawing explanation
Different condition bacterial count temporal evolution figure in the orthogonal experimental design of Fig. 1 culture condition.
Fig. 2 probiotic bacterium liquid fermenting viable count temporal evolution figure.
Fig. 3 probiotic bacterium liquid fermenting pH temporal evolution figure.
Fig. 4 probiotic bacterium liquid fermenting dehydrogenase activity temporal evolution figure.
Fig. 5 is bacillus amyloliquefaciens plant subspecies B-01 bacterial strain field emission scanning electron microscope photo of the present invention.20000 times of S4800 field emission scanning electron microscope photos, the long 0.8-3um of bacterium, wide 0.6um.
Fig. 6 is photo under bacillus amyloliquefaciens plant subspecies B-01 bacterial strain oil mirror of the present invention
Note: NA substratum, 37 ℃, cultivate thalli morphology 1 day: shaft-like, 0.5 μ m * 2.0～2.67 μ m, single, paired or paliform is arranged, Gram-positive.
Fig. 7 is that bacillus amyloliquefaciens plant subspecies B-01 bacterial strain agar of the present invention is cultivated bacterium colony photo
Note: NA substratum, 37 ℃, cultivate colonial morphology 1 day: oyster white, irregular shape, surface ruffle, coarse, translucent, edge is irregular.
18 ℃ of amylase of Fig. 8 (AMS) vigor temporal evolution figure.
18 ℃ of lipase of Fig. 9 (LPS) vigor temporal evolution figure.
18 ℃ of proteinase activity temporal evolution figure of Figure 10.
18 ℃ of bacterial count temporal evolution figure of Figure 11.
Figure 12 laboratory probiotic bacterium effects of purification quality figure (before experiment)
Note: the eutrophic water control bottle that does not add probiotic bacterium.
Figure 13 laboratory probiotic bacterium effects of purification quality figure (after experiment)
Note: the eutrophic water assay flask that adds 2%B-01 bacillus amyloliquefaciens.
The a large amount of pond of Figure 14 sewage (210m
3) probiotic bacterium purification of water quality (before experiment).
The a large amount of pond of Figure 15 sewage (210m
3) probiotic bacterium purification of water quality (after experiment).
Figure 16 B-01 bacterial strain is grown tree to the 16SrDNA sequential system of relevant kind.
Figure 17 B-01 and relevant gyrA gene order phylogenetic tree of planting.
Figure 18 B-01 bacterial strain (the 1st batch) and relevant gyrA gene order phylogenetic tree of planting.
Figure 19 B-01 bacterial strain (the 1st batch continuous passage 5 times after bacterial strain) and the gyrA gene order phylogenetic tree of relevant kind.
Bacillus amyloliquefaciens plant subspecies B-01 of the present invention (Bacillus amyloliquefaciens subsp.plantarum B-01), on May 30th, 2012, be preserved in the Chinese Typical Representative culture collection center that is positioned at Wuhan, China university, its preserving number is CCTCC NO:M2012191.
Embodiment
The screening of embodiment mono-bacterial strain
The separation of 1.1 bacterial strains, screening, purge process
Choose each 1 gram, barnyard grass, fiber crops grass, sheep's hay in the cold environment of the northern area of China-35 ℃ and be inoculated in physiological saline and soak 2h, get 2mL soak solution and be inoculated in 100mL and stimulate gemma grown cultures liquid (peptone 10g, yeast extract paste 3g, starch 3g, MgSO
40.1g, KH
2pO41.5g, Na
2hPO
42g distilled water 1000mL, pH7.8,121 ℃ of sterilizing 20min) 18 ℃ be cultured to adularescent mycelia and grow.
Get bacteria suspension and kill not spore production bacteria body in 80 ℃ of water-bath 15min(), choose 10 times of dilution plates of bacteria suspension and coat gemma isolation medium (peptone 5g, glucose 5g, yeast extract paste 5g, K
2hPO
44g, 3.08%MnSO41mL, agar 18g, distilled water 1000mL, pH nature) 18 ℃ be cultured to single bacterium colony and grown.
Select the good bacterium colony of growth conditions under cold condition, dyeing is observed, and bacterium colony cryogenic purincation is cultivated, called after B-01.
2.1 nutrition substrate positive quadraturing design tests
To guarantee optimal culture condition, cultivate.Adopt viable count as the deliberated index of cultivation conditions.
Orthogonal design is in Table 1: inoculation B-01 bacterium liquid measure is 30%+70% nutrient solution (weight percent)
Table 1 orthogonal experimental design scheme
According to the different content of energy, protein and mineral substance, application gold is herded software design and is gone out to fill a prescription as table 2:
Table 2 orthogonal test scheme (9 kinds of optimization processes)
According to the trophic level formula of table design, application gold is herded software design and is gone out composition of raw materials as shown:
Table 3.9 group raw material nutritional formula
Note: all add small powder by table 4 in above-mentioned 9 group of formula
Table 4 adds little material formula
| Each formula group is all added small powder: | MgSO 4 | MnSO 4 | K 2HPO 4 | Add up to |
| Weight percent (%) | 0.15 | 0.01 | 0.11 | 0.27 |
Comparison between orthogonal design different affecting factors, each test combinations colony number is in Table 5.
Table 5 orthogonal experimental design measurement result
Table 6 orthogonal experimental design list factor statistics
From table 6 intuitive analysis, the order of excellence of each level of factor is: A
1>A
2>A
3, B
1>B
2>B
3, C
2>C
3>C
1.
Table 7 orthogonal experimental design the results of analysis of variance
| Soruces of variation | Sum of sguares of deviation from mean | Degree of freedom | All square | F value | P value |
| Energy (A) | 353.018 | 2 | 176.509 | 45.808 | 0.021 |
| Crude protein (B) | 995.704 | 2 | 497.852 | 129.205 | 0.008 |
| Mineral substance (C) | 23.341 | 2 | 11.670 | 3.029 | 0.248 |
| Error | 7.706 | 2 | 3.853 | ? | ? |
| Total variation | 1379.769 | 8 | ? | ? | ? |
From the results of analysis of variance of table 7, crude protein (B) has utmost point significance impact (P < 0.001) to colony number; Energy (A) has a significant impact (P < 0.005) to colony number; Mineral substance is less on colony number impact, and difference is not remarkable, and each trophic factor primary and secondary is sequentially B>A>C, and the best nutritional substrate of selected microbial culture is A
1b
1c
2, that is, and energy 2.0MC/kg, protein 15 %, mineral substance 3.5%(CaHPO
43%, NaCl0.5%)
In conjunction with orthogonal experiments, utilize gold to herd software (VF123-2006 version) and design nutrition substrate formula as table 8:
Table 8 nutrition substrate optimum formula
| Title | Wheat bran | Dregs of beans | Corn | Rice husk | CaHPO 4 | NaCl | Small powder |
| Weight percent (%) | 29.8 | 15.18 | 35.8 | 15.45 | 3.0 | 0.5 | 0.27 |
2.2 culture condition orthogonal tests
Table 9 orthogonal experimental design scheme
By table 9, carry out 4 factor 3 horizontal quadrature design experiments, every 3h measures the viable count of bacterium, amounts to 48h.In the orthogonal experimental design of culture condition, different condition bacterial count temporal evolution figure is shown in Fig. 1. 24h viable count, for bacterial count growth peak point, is therefore usingd as criterion in the known 24h of different condition colony number temporal evolution left and right.
Table 10 orthogonal experimental design the results of analysis of variance
| Soruces of variation | Sum of sguares of deviation from mean | Degree of freedom | All square | F value | P value |
| pH(A) | 3513.849 | 2 | 1756.924 | 184.608 | .000 |
| Temperature (B) | 721.529 | 2 | 360.764 | 37.907 | .000 |
| Inoculum size (C) | 4386.596 | 2 | 2193.298 | 230.460 | .000 |
| Substrate (D) | 264.427 | 2 | 132.213 | 13.892 | .000 |
| Error | 171.307 | 18 | 9.517. | ? | ? |
| Total variation | 9057.707 | 26 | ? | ? | ? |
Orthogonal experimental design the results of analysis of variance is in Table 10, the results of analysis of variance from table 10, each factor of ABCD all has extremely significance impact (P<0.001) to viable count, each factor primary and secondary is sequentially C>A>B>D, and the best nutritional substrate of selected microbial culture is A
2b
2c
3d
2.
Draw the optimal culture condition of B-01: pH7.2,35 ℃ of temperature, inoculum size 15%, concentration of substrate 10%.
2.3B-01 the dynamic research of Bacillus amyloliquefaciens strain liquid fermenting metabolic rule
The suitableeest above-mentioned breeding condition of selecting B-01 bacterial strain, fermentation capacity is 30L, every 3h dynamic measurement viable count, pH value and desaturase total activity, 24h, repeats 4 times altogether.As shown in Fig. 2 .B-01 Bacillus amyloliquefaciens strain liquid fermenting colony number temporal evolution figure, the 1st batch, the 2nd batch, the 3rd batch, the 4th batch colony number expands numerous to 141 * 108cfu/mL by 8 * 108cfu/mL respectively, by 13 * 108cfu/mL, expand numerous to 149 * 108cfu/mL, numerous to 168 * 108cfu/mL by 16 * 108cfu/mL expansion, by 17 * 108cfu/mL, expand numerous to 162 * 108cfu/mL.0h compares with fermentation, and 6-24h colony number is higher, and difference is (P<0.01) extremely significantly; 3h, 6h compare with 9h with fermentation, and 12-24h colony number is higher, and difference is (P<0.01) extremely significantly; 12h, 15h compare with fermentation, and 18-24h colony number is higher, and difference is (P<0.01) extremely significantly; 18h compares with fermentation, and 24h colony number is higher, and difference is (P<0.01) extremely significantly.Constantly cultivation is in rising trend along with bacterium liquid for colony number, beneficial flora increased activity; As Fig. 3 .B-01 Bacillus amyloliquefaciens strain liquid fermenting, pH counts as shown in temporal evolution figure, and the 1st crowd, the 2nd crowd, the 3rd crowd, the 4th crowd pH drops to 6.2 by 6.76 respectively, drops to 6.01 by 6.62, drops to 5.94 by 6.78, by 6.76, drops to 5.99.0h, 3h compare with fermentation, and 6-24h pH reduces, and difference is (P<0.01) extremely significantly; 6h compares with fermentation, and 18-24hpH reduces, and difference is (P<0.01) extremely significantly; Fermentation 9h-21h compares, and 24h pH reduces, and difference is (P<0.01) extremely significantly.PH value temporal evolution and constantly reducing, shows that bacterium is in constantly fermentation propagation; As Fig. 4, shown in B-01 Bacillus amyloliquefaciens strain liquid fermenting TDHA temporal evolution figure, the 1st crowd, the 2nd crowd, the 3rd crowd, the 4th crowd TDHA brings up to 0.694 by 0.005 respectively, by 0.010, brings up to 0.896, bring up to 1.003 by 0.023, by 0.029, bring up to 0.997.0h, 3h compare with fermentation, and 12-24h TDHA is higher, and difference is (P<0.01) extremely significantly; 6-12h compares with fermentation, and 18-24h TDHA is higher, and difference is (P<0.01) extremely significantly; 15-21h compares with fermentation, and 24h TDHA is higher, and difference is (P<0.01) extremely significantly.In 24h fermentation period, TDHA increases trend gradually along with course of fermentation is, and shows that the bacterium enzyme activity of beneficial flora strengthens gradually.
3, low temperature modification high reactivity B-01 bacillus amyloliquefaciens morphology research
Above-mentioned screening obtained strains B-01 at 20,000 times of S4800 field emission scanning electron microscope photos as Fig. 5, the long 0.8-3um of bacterium, wide 0.6um.
By bacterial strain B-01 at NA substratum, 37 ℃, cultivate 1 day, oily Microscopic observation then, photo is as Fig. 6, visible thalli morphology: shaft-like, 0.5 μ m * 2.0～2.67 μ m, single, in pairs or paliform arrange, Gram-positive.
Bacterial strain B-01, at NA substratum, 37 ℃, is cultivated to colonial morphology 1 day: oyster white, irregular shape, surface ruffle, coarse, translucent, edge irregular (Fig. 7).
The evaluation of embodiment bis-bacterial strains
Bacterial strain B-01 send Chinese industrial microbial strains preservation administrative center (CICC) to carry out microorganism identification analysis.
1.BA bacterial strain and phase and phase close the 16S rDNA sequential system of planting and grow tree
Adopt MEGA4.1 software, ortho position connection method shows B-01 and relevant 16S rDNA phylogenetic tree of planting, and carries out the similarity double counting of 1000 times, grows tree node and only show that Bootstrap value is greater than 50% numerical value, (E., Escherichia) in figure.
By Figure 16. the 16S rDNA phylogenetic tree analysis of bacterial strain B-01 shows: a plurality of kinds of Ju Yige phylogeny branches in bacterial strain B-01 and bacillus subtilis flora (Bacillus subtilis group), sequence homology is more than 99.2%.
B-01 bacterial strain DNA sequence dna is as shown in SEQ ID No.1.
2.B-01 and relevant gyrA gene order phylogenetic tree of planting
Adopt MEGA4.1 software, ortho position connection method shows B-01 and relevant gyrA Phylogenetic Tree of planting, and carries out the similarity double counting of 1000 times, grows tree node and only show that Bootstrap value is greater than 50% numerical value in figure.
By Figure 17 .B-01 Bacillus amyloliquefaciens strain, shown to the pheS gene order phylogenetic tree Phylogenetic Analysis of relevant kind: bacterial strain B-01 and bacillus amyloliquefaciens plant subspecies (Bacillusamyloliquefaciens subsp.plantarum) Ju Yige phylogeny branch, sequence homology is 98.7%.This bacterial strain can utilize glycan to produce acid, and raw in gemma, this feature is consistent with bacillus amyloliquefaciens.
By 16S rDNA sequence and gyrA gene order Phylogenetic Analysis, bacterial strain B-01 is accredited as: bacillus amyloliquefaciens plant subspecies (Bacillus amyloliquefaciens subsp.plantarum).3.B-01 bacillus amyloliquefaciens bacterial classification genetic stability is analyzed
Bacterial strain bacillus amyloliquefaciens B-01 send Chinese industrial microbial strains preservation administrative center (CICC) to carry out the analysis of microbial strains genetic stability.Bacterial strain bacillus amyloliquefaciens B-01 (1 crowd) cultivates through continuous passage, and strain morphology is learned feature and do not found visible change, and gyrA gene order is unchanged, and physiological and biochemical property is without considerable change, and bacterial strain proterties can genetic stability.
3.1B-01 bacterial strain physiological and biochemical property (in Table 11)
Table 11B-01 bacterial strain physiological and biochemical property
Nomenclature: "+", the positive; "-", feminine gender; "+w ", the weak positive; ND, does not survey.
3.2 sequential analysis
Sequence alignment: bacillus amyloliquefaciens B-01 (1 crowd)
98.4%Bacillus?amyloliquefaciens?subsp.plantarum?FZB42T(CP000560)
95.1%Bacillus?amyloliquefaciens?supsp.amyloliquefaciens?KCTC1660T(AF272015)
83.6%Bacillus?mojavensis?NRRL?B-14698T(EU138598)
83.4%Bacillus?subtilis?subsp.inaquosorum?NRRL?B-23052T(EU138605)
83.0%Bacillus?subtilis?subsp.spizizenii?NRRL?B-23049T(AF272020)
82.6%Bacillus?subtilis?subsp.subtilis?KCTC3135T(AF272021)
82.4%Bacillus?tequilensis?NRRL?B-41771T(EU138625)
82.3%Bacillus?atrophaeus?KCTC3701T(AF272016)
82.2%Bacillus?vallismortis?NRRL?B-14890T(AF272025)
80.3%Bacillus?sonorensis?NRRL?B-23154T(EU138611)
Bacterial strain bacillus amyloliquefaciens B-01 (1 crowd) and relevant gyrA gene order phylogenetic tree of planting (Figure 18 B-01 Bacillus amyloliquefaciens strain (1 batch) and relevant gyrA gene order phylogenetic tree of planting)
Expert's conclusion: bacillus amyloliquefaciens plant subspecies B.amyloliquefaciens subsp.Plantarum
Sequence alignment: bacillus amyloliquefaciens B-01 (1 crowd continuous passage 5 times after bacterial strain)
98.4%Bacillus?amyloliquefaciens?subsp.plantarum?FZB42T(CP000560)
95.1%Bacillus?amyloliquefaciens?supsp.amyloliquefaciens?KCTC1660T(AF272015)
83.6%Bacillus?mojavensis?NRRL?B-14698T(EU138598)
83.4%Bacillus?subtilis?subsp.inaquosorum?NRRL?B-23052T(EU138605)
83.0%Bacillus?subtilis?subsp.spizizenii?NRRL?B-23049T(AF272020)
82.6%Bacillus?subtilis?subsp.subtilis?KCTC3135T(AF272021)
82.4%Bacillus?tequilensis?NRRL?B-41771T(EU138625)
82.3%Bacillus?atrophaeus?KCTC3701T(AF272016)
82.2%Bacillus?vallismortis?NRRL?B-14890T(AF272025)
80.3%Bacillus?sonorensis?NRRL?B-23154T(EU138611)
Bacterial strain bacillus amyloliquefaciens B-01 (1 crowd continuous passage 5 times after bacterial strain) and the gyrA gene order phylogenetic tree of relevant kind (seeing Figure 19 B-01 Bacillus amyloliquefaciens strain (1 batch continuous passage 5 times after bacterial strain) and the gyrA gene order phylogenetic tree of relevant kind)
Expert's conclusion: bacillus amyloliquefaciens plant subspecies B.amyloliquefaciens subsp.Plantarum.
Embodiment tri-low temperature modification high reactivity B-01 Bacillus amyloliquefaciens strain enzymic activity dynamic studies and dynamic of bacteria counting
Relevant fungus strain enzyme assay is all controlled under 18 ℃ of culture condition to be carried out, and every duplicate samples fermentation capacity is 200Ml, 3 parts of parallel repetition samples.The enzyme assay of existing market application product mushroom, because enzyme activity is poor under cold condition, conventionally measure hot conditionss such as adopting 60 ℃ of amylase activities, 40 ℃, lipase, proteolytic enzyme 40-55 ℃, low temperature modification probiotic products will manifest unique advantage in the lower purification of water quality process of actual environment temperature, can keep higher biologic activity, therefore there is good market application foreground.
18 ℃ are carried out enzyme activity determination (build up with Nanjing lipase, the determination of amylase test kit that Bioengineering Research Institute provides, measure proteinase activity by GB/T1803-1993 method)
Amylase activity unit definition: the AMS in 100ml enzyme liquid, 18 ℃ with substrate-function 30 minutes, hydrolysis 10mg starch is 1 unit
Amylase AMSu/dl=(blank tube absorbancy-mensuration pipe absorbancy) extension rate before/blank tube absorbancy * (0.4*0.5/10) * (30 minutes/7.5 minutes) * (1/0.05) * test sample
=(blank tube absorbancy-mensuration pipe absorbancy)/blank tube absorbancy * 0.16
Lipase activity unit definition: under 18 ℃ of conditions, every person of outstanding talent rise enzyme liquid in this reaction system with substrate reactions 1 minute, every consumption 1umol substrate is an enzyme activity unit.
LPS vigor=(the A1-A2)/extension rate * reaction times (10 minute) of As* standard pipe concentration (umol/l) * sample in reaction system
Proteinase activity definition: 1ml liquid enzymes is at 18 ℃, and under PH7.3 condition, it is an enzyme activity unit (u/ml) that 1min hydrolyzed casein produces 1ug tyrosine
Proteinase activity=A*K*4/10*n
The amylase of 1.B-01 bacterium, lipase, the research of proteinase activity power dynamic rule
Shown in Fig. 8 .B-01 Bacillus amyloliquefaciens strain amylase activity temporal evolution figure, figure is known for 18 ℃ of amylase (AMS) vigor temporal evolution, amylase activity is rising always, amylase activity has significantly growth at 44h and 62h respectively, 62h-74h maintains higher level, and takadiastase vigor is 11.532u/dl.
Shown in Fig. 9 .B-01 Bacillus amyloliquefaciens strain lipase activity temporal evolution figure, figure is known for 18 ℃ of lipase (LPS) vigor temporal evolution, when 44h, lipase activity significantly rises, until reach maximum during 56h, is 1.56411u/l, and lipase activity declines again to some extent subsequently.
Shown in Figure 10 .B-01 Bacillus amyloliquefaciens strain proteinase activity temporal evolution figure, 18 ℃ of proteinase activity temporal evolution figure are known, proteinase activity temporal evolution and constantly raising, until maintain higher level during 62h-74h, wherein when 62h, reaching maximum is 8.836u/mL.
2.B-01 bacterium colony number dynamic rule research in time under low temperature culture condition (colony number temporal evolution figure under Figure 11 .B-01 Bacillus amyloliquefaciens strain cold condition)
After 20h, measure colony number under cold condition, as shown in figure 11,20h-44h bacterial growth is slower, and 44h-68h thalli growth is vigorous, reaches the highest colony number 81 * 10 during 68h
8cfu/mL, bacteria containing amount decreases subsequently.
The research of embodiment tetra-B-01 bacillus amyloliquefaciens purification of water quality characteristics
1. laboratory probiotic bacterium purification of water quality
Under laboratory condition, adopt special eutrophic water (formula: ground pork 50g+ bran powder 50g+ bean cake powder 50g+ Semen Maydis powder 50g+ water 1L, through 100 ℃, boil after 1h, get filtrate and add water to 10L constant volume), by every 100mL nutritive water, add B-01 bacillus amyloliquefaciens bacterium liquid 2mL(viable count of the present invention: 10,000,000,000/mL) mix, the water quality apparent variation of standing observation in the time of 20 days is as Figure 12,13.
Do not add the eutrophic water control bottle water body muddiness of probiotic bacterium and give an offensive smell, adding the eutrophic water assay flask water body of 2%B-01 bacillus amyloliquefaciens limpid bright, free from extraneous odour.
2. a large amount of pond sewage (210m3) probiotic bacterium purification of water qualitys contrasted before and after 20 days
In sewage lagoon, splash 1 ton of bacterium liquid (0.48% bacterium liquid) to the water surface, observe purifying water effect and measure the every water-quality guideline contrasting before and after 20 days, in Table 12.The apparent variation of water quality is as Figure 14,15.
Every water-quality determination index all adopts National Standard Method of Determination:
1. chemical oxygen demand (COD) (COD) adopts potassium permanganate process GB/T15456-2008,
2. oxygen capacity (DO) adopts iodimetry,iodometry GB7489-87
3. ammonia nitrogen Nessler's reagent spectrophotometry GB7479-87
4. nitrite spectrophotometry GBT7493-87
5.pH measures with pH meter
Before and after table 12 employing B-01 bacillus amyloliquefaciens purifies water, correlation water index changes
| Purification of water quality index | Water body before purifying | Water body after purifying |
| Chemical oxygen demand (COD) (COD) | 11.4mg/L | 5.8mg/L |
| Oxygen capacity (DO) | 1.72mg/L | 6.8mg/L |
| : ammonia nitrogen | 2.7mg/L | 1.83mg/L |
| Nitrite | 0.047mg/L | 0.023mg/L |
| pH | 6.4 | 7.2 |
Note: before purifying, after water body muddiness the purification that gives an offensive smell, water quality is limpid, free from extraneous odour.
Claims (3)
1. bacillus amyloliquefaciens plant subspecies (Bacillus amyloliquefaciens subsp.plantarum) B-01 bacterial strain, its preserving number is CCTCC NO:M2012191.
2. bacillus amyloliquefaciens plant subspecies B-01 bacterial strain claimed in claim 1 is as the application of water surrounding ecological purificant.
3. bacillus amyloliquefaciens plant subspecies B-01 bacterial strain claimed in claim 1 is as the application of animal microorganism fodder additives.
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