CN104673730A - Brevibacillus laterosporu with function in quickly decomposing nitrite nitrogen and bacteriostasis function and application thereof - Google Patents

Brevibacillus laterosporu with function in quickly decomposing nitrite nitrogen and bacteriostasis function and application thereof Download PDF

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CN104673730A
CN104673730A CN201510128510.1A CN201510128510A CN104673730A CN 104673730 A CN104673730 A CN 104673730A CN 201510128510 A CN201510128510 A CN 201510128510A CN 104673730 A CN104673730 A CN 104673730A
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徐永平
车鉴
李淑英
李晓宇
王丽丽
王熙涛
张建成
阳桂菊
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Dalian University of Technology
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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/10—Inorganic compounds
    • C02F2101/16—Nitrogen compounds, e.g. ammonia
    • C02F2101/166—Nitrites
    • C—CHEMISTRY; METALLURGY
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Abstract

The invention discloses a Brevibacillus laterosporu with function in quickly decomposing nitrite nitrogen and bacteriostasis function and an application thereof. The screening strain is Brevibacillus laterosporu YB-02, the strain collection number is CGMCC-No. 9757. The strain provided by the invention has a capacity of effectively decomposing nitrite nitrogen in water, has cholate resistance and trypsin resistance, and further has higher inhibiting effects to mariculture pathogenic bacteria like vibrio splendidus, shewanella smarisflavi and edwardsiella tarda. Therefore, the strain provided by the invention can be used for developing probiotics products for mariculture water purification and bacterial inhibition, the antibiotics abuse is avoided, the fishing disease is prevented and treated effectively, the quality and yield of marine food products are improved; the strain has effective, fast and safe water purification effect to acute water pollution, and therefore, the strain is wide in application prospect.

Description

One strain has Brevibacillus laterosporus and the application thereof of fast degradation nitrite nitrogen function and bacteria resistance function
Technical field
The invention belongs to microbial technology field, relate to Brevibacillus laterosporus and application thereof that a strain has fast degradation nitrite nitrogen function and bacteria resistance function.
Background technology
Seawater is the Essential Environment condition that marine cultured animal is depended on for existence, as the saying goes: " breeding fish for three points, seven points of foster water ".The quality of water quality directly has influence on their upgrowth situation and the economic benefit of culturist.Along with growth in the living standard, the demand of people to fishery products increases day by day, and when amount of fishing can not be met the need of market, highdensity intensive aquaculture mode obtains fast development.But, in this aquaculture model, the pollution level of water body is considerably beyond the detergent power of water body self, remaining bait and movement deposit in water body, cause water body self-pollution and even water quality deterioration, show as water body serious eutrophication, Suspended Matter in Water increases, the content of chemical oxygen demand (COD), ammonia nitrogen, nitrite nitrogen, hydrogen sulfide increases, harmful heavy metal content is accumulated, and dissolved oxygen declines, water acidification and the smelly end.Especially the toxicity of nitrite nitrogen is higher than ammonia nitrogen, " Seawater Quality Standards " (GB 3097-1997) specifies that the nitrite nitrogen of marine culture water is lower than 0.1mg/L, otherwise particularly serious to end breath type animal (as sea cucumber, turbot and Fugu rubripes (Temmincket Schlegel) etc.), the lighter damages cultivated animals neural system and impact grows, development and fecundity; Severe one causes epidemic disease to take place frequently and directly death.
The method controlling water nitrite state nitrogen concentration in existing seawater intensive culture process adopts clear pond to fall pond, and circulating filtration, throws in chemical water purification agent and probiotics etc.But ubiquity is wasted time and energy and the shortcoming of high energy consumption high cost; Although worsen for the water body of burst, chemical water purification agent energy rapid reaction transforms objectionable impurities, can cause secondary pollution to water body environment; Probiotics is environmentally friendly water purification scheme, but have that flora cleaning water is low, specificity is poor, Survival Reproduction bad adaptability and the quality guaranteed period viable bacteria problem such as to reduce sharply.
Prebiotic bacterial classification at present for the probiotics of water surrounding mainly contains photosynthetic bacterium, genus bacillus, Bacterium lacticum, bifidus bacillus, nitrobacteria etc.These microbial strainss are single, and decontamination effect improving is limited; Be mostly fresh water bacterial strain, be not suitable for sea farming application; Spore production bacteria agent survival time is not short, to environmental resistance ability; Some growth are slow, and in using, " activation " time is long, causes the problems such as the processing of active bacteria formulation, preservation, transport and use, hinders the research and development of probiotics, production and application.
In No. 2045th, Ministry of Agriculture bulletin " fodder additives kind catalogue (2013) ", allow there are 5 kinds of genus bacillus (Bacillus licheniformis, subtilis, bacillus lentus, bacillus pumilus and Brevibacillus laterosporus) in the 36 kinds of microorganisms used.But these genus bacillus mostly are fresh water bacterial strain, are not suitable for sea farming, for be exclusively used in sea farming, the research of the genus bacillus with highly effective sea-water aquaculture water cleaning action and its microbial preparation is less.
The present invention screens the genus bacillus met in Ministry of Agriculture's " fodder additives kind catalogue (2013) ", and this bacterial strain has the effect of the efficient nitrite nitrogen removed in marine culture water, long quality-guarantee period strong to environmental resistance; Meanwhile, to kind of the pathogenic bacterium of 6 in sea farming, there is certain fungistatic effect, the antibiotic sick epidemic disease and death avoiding water body to worsen causing can be reduced simultaneously in sea farming.
Summary of the invention
In view of above-mentioned deficiency of the prior art, an object of the present invention is to provide the bacterial strain that a strain has nitrite nitrogen function and bacteriostatic action in fast degradation marine culture water.
Bacterial strain provided by the invention is Brevibacillus laterosporus (Brevibacillus laterosporu) YB-02, and its culture presevation is numbered CGMCC-No.9757.
Brevibacillus laterosporus (Brevibacillus laterosporu) YB-02 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (Institute of Microorganism, Academia Sinica on October 11st, 2014, postcode 100101), address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Brevibacillus laterosporus YB-02 (Brevibacillus laterosporu) CGMCC-No.9757 removes the application of nitrite nitrogen in seawater and also belongs to protection scope of the present invention in sea farming.Particularly, Brevibacillus laterosporus YB-02CGMCC-No.9757 of the present invention can be applied to the preparation of marine aquaculture water body water purification agent.Described marine aquaculture is holothruian cultures, turbot cultivation and Fugu rubripes (Temmincket Schlegel) cultivation.
Brevibacillus laterosporus (Brevibacillus laterosporu) CGMCC-No.9757 suppresses the application of sea farming pathogenic bacterium also to belong to protection scope of the present invention in sea farming.Preferably, described sea farming pathogenic bacterium are Vibrio splindidus, Huanghai Sea Shewanella, Edwardsiella tarda and Vibrio parahaemolyticus.
The present invention is separated Brevibacillus laterosporus (Brevibacillus laterosporu) CGMCC-No.9757 obtaining having efficient nitrite nitrogen degradation capability from the bed mud sample of stichopus japonicus seawater-culture pond collection.
Experiment proves, bacterial strain of the present invention has the characteristic of efficient degradation nitrite nitrogen; CGMCC-No.9757 is the suitableeest grows under 26 ~ 28 DEG C of conditions for Brevibacillus laterosporus (Brevibacillus laterosporu), and fermentation pH 7.2 ~ 7.5 reaches late log phase at 14 ~ 16h, and bacterium number reaches 5 × 10 10cfu/ml.Experiment also proves, Brevibacillus laterosporus (Brevibacillus laterosporu) reaches 99.87% to nitrite nitrogen degraded in 24h, does not also have the seawater micro-ecological bacterial of nitrite nitrogen degradation capability so efficiently at present; To pathogenic bacterium common in sea farming, there is certain fungistatic effect simultaneously, wherein for Vibrio splindidus, Huanghai Sea Shewanella, Edwardsiella tarda and Vibrio parahaemolyticus, there is good fungistatic effect comparatively obvious, overcome current seawater pond culture epidemic disease to take place frequently and the problem of abuse of antibiotics, in the cultivation of seawater Harmless, will have broad application prospects.
Accompanying drawing explanation
Fig. 1 is the colony morphology characteristic of Brevibacillus laterosporus YB-02 on 2216E flat board.
Embodiment
Following embodiment is convenient to better understand the present invention, but does not limit the present invention.In addition, in following embodiment, if no special instructions, the experimental technique used is ordinary method, and material therefor, reagent etc. all can be bought from biological or chemical Reagent Company.
Below by example, the present invention is described in further detail.
The screening of embodiment 1 bacterial strain, Isolation and ldentification
1. the preparation of substratum:
Nitrite nitrogen degradation bacteria enrichment isolation liquid nutrient medium: NaNO 21g, Na 2cO 31g, K 2hPO 40.5g, MgSO 47H 2o 0.03g, FeSO 47H 2o 0.4g, Chen Haishui 1L, pH 7.2-7.5.
Nitrite nitrogen degradation bacteria separation and purification solid medium: NaNO 20.5g, Na 2cO 31g, K 2hPO 40.5g, MgSO 47H 2o 0.03g, FeSO 47H 2o 0.4g, Chen Haishui 1L, agar powder 12g, pH 7.2 ~ 7.5.
2216E liquid nutrient medium: peptone 5g, yeast extract paste 1g, FePO 40.1g, Chen Haishui 1L, pH 7.2; Solid medium is add 15g l in liquid nutrient medium -1agar powder.
Nutrient broth (NB) substratum: peptone 10g, extractum carnis powder 3g, sodium-chlor 30g, distilled water 1000ml, pH value 7.2; Solid medium is add 15g l in liquid nutrient medium -1agar powder.
Before above substratum uses, 121 DEG C of sterilizing 20min, room temperature preservation.
2. bacteria selection
Get the internal layer sample 2g of the bed mud sample that stichopus japonicus seawater-culture pond gathers, add the PBS buffered soln of 50ml, vibration 5min, makes sample evenly muddy, the centrifugal 5min of 1000rpm, collects bed mud sample supernatant, for subsequent use.
The isolation and purification of nitrite nitrogen degradation bacteria bacterial strain YB-02:
Get 10ml sea cucumber seawater-culture pond bed mud sample supernatant, join in the Erlenmeyer flask that 200ml ammonia nitrogen degradation bacterium enrichment isolation nutrient solution is housed respectively, 28 DEG C, 150rpm constant-temperature table cultivates 3d, enrichment 3 times.Get 100 μ l after getting the bacteria suspension gradient dilution of cultivation to coat on ammonia nitrogen degradation bacterium separation and purification solid medium, be placed in 28 DEG C of incubators and cultivate.After 72h, picking different shape list bacterium colony is streak culture on 2216E solid medium, repeated isolation purifying 3 times, and the single bacterium colony finally obtained carries out 16S rDNA order-checking, this single bacterium colony called after YB-02.
Sequence in the 16S rDNA sequencing result of YB-02 and GenBank compared, compare with accession number AY_372923.1 (Brevibacillus laterosporu), genetic homology is 99%.According to " the outstanding Bacteria Identification handbook of uncle " (the 8th edition) to colony morphology characteristic and Physiology and biochemistry qualification, determine that YB-02 is Brevibacillus laterosporus (Brevibacillus laterosporu), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preserving number is CGMCC-No.9757.
The sequence length of the 16S rDNA of YB-02 is 1388bp, and sequence is shown in sequence table.
The morphological specificity of embodiment 2 YB-02 and physiological and biochemical property
The morphological specificity of YB-02: as shown in Figure 1, the bacterium colony of YB-02 on 2216E flat board is circular, khaki color, and smooth surface is moistening glossy, and middle slightly projection, opaque, neat in edge, without halo.
The cultural characteristic of table 1. bacterial strain on 2216E substratum
Bacterial strain Physiology and biochemistry detection method according to " the outstanding Bacteria Identification handbook of uncle " (the 8th edition) detects Brevibacillus laterosporus YB-02.
The YB-02 that separation and purification goes out has eurysalinity, and all can grow in 20 ~ 40 ‰ salinity range, the most suitable growth salinity is 28 ~ 32 ‰; Can normal growth 10 ~ 40 DEG C of temperature ranges, optimum growth temperature is 20 ~ 30 DEG C; All can grow in the scope of pH value 7 ~ 9, the most suitable growth pH value is 7.2 ~ 8.5, poor to the tolerance of sour environment; Can normal growth within the scope of dissolved oxygen content 1 ~ 5mg/L, the most suitable growth dissolved oxygen concentration belongs to facultative anaerobe at 4 ~ 5mg/L, YB-02.
The physiological and biochemical property of table 2. Brevibacillus laterosporus YB-02
Note: in table 1, <+> represents positive, <-> represents negative.
The growth characteristics of embodiment 3 Brevibacillus laterosporus YB-02 and culture condition
Brevibacillus laterosporus (Brevibacillus laterosporu) CGMCC-No.9757 is because be separated from sea water culture environment, and therefore have eurysalinity, the most suitable growth salinity is 28 ~ 32 ‰; Optimum growth temperature is at 26 ~ 28 DEG C, and the most suitable growth pH value is 7.2 ~ 8.5.Fermentation 14 ~ 16h reaches late log phase, and bacterium number reaches 5 × 10 10cfu/mL.
It is as follows for cultivating substratum used:
Seed culture medium (nutrient broth (NB) substratum): extractum carnis powder 5g, peptone 10g, sodium-chlor 30g, water 1L, pH 7.2 ~ 7.5.
2216E liquid nutrient medium: peptone 5g, yeast extract paste 1g, FePO4 0.1g, Chen Haishui 1L, pH 7.2 ~ 7.5.
Fermention medium: Semen Maydis powder 150g, soybean cake powder 250g, middle temperature amylase 3.5g, neutral protease 2.5g, corn steep liquor 40g, glucose 25g, magnesium sulfate 10g, Chen Haishui is settled to 5L, pH 7.2 ~ 7.5.Preparation method is: quality according to the rules, takes Semen Maydis powder and soybean cake powder, after adding the old seawater uniform stirring of 3L, goes in fermentor tank, adds neutral protease and middle temperature amylase simultaneously, successively at 50 DEG C, 65 DEG C Water Under solution 40 ~ 60min; Proceed in fermentor tank by the appropriate Chen Haishui adding magnesium sulfate and glucose, finally proceed in fermentor tank by corn steep liquor, Chen Haishui is settled to 5L, adjusts pH 7.2 ~ 7.5,121 DEG C of sterilizing 30min.
Cultivate: by the Brevibacillus laterosporus YB-02 of slant culture, be seeded in seed liquor substratum, at pH 7.2-7.5,26 ~ 28 DEG C of constant-temperature tables are cultivated, and obtain Brevibacillus laterosporus YB-02 probiotics bacterial liquid.Probiotics bacterial liquid cultivate further in NB substratum after for detecting its performance measurement test (cholate and trypsinase resistance test, nitrite nitrogen clearance etc.), or cultivate the zymocyte liquid that the obtains scavenging agent for the preparation of marine culture water in the fermentation medium.Described scavenging agent can adopt described Brevibacillus laterosporus YB-02 zymocyte liquid to prepare separately, also can prepare the scavenging agent of marine culture water with other probiotic bacteriums or its zymocyte liquid mode used in combination according to a certain percentage.
Embodiment 4 cholate and trypsinase resistance test
In order to check the survival performance of Brevibacillus laterosporus YB-02 CGMCC-No.9757 under cholate and trypsinase existence condition, different concns cholate and tryptic tolerance situation are detected.By the Brevibacillus laterosporus YB-02 of slant culture, be seeded in 2216E liquid nutrient medium, at pH 7.2-7.5,26 ~ 28 DEG C of constant-temperature tables are cultivated, and obtain Brevibacillus laterosporus YB-02 probiotics bacterial liquid, bacterial concentration 5 × 10 8cfu/mL, is inoculated in NB liquid nutrient medium by 10% and cultivates, and processes 1h when being respectively 0,1,2,4 and 5g/L by cholate and trypsinase mass concentration respectively at 26 DEG C, observes the upgrowth situation of YB-02.
The impact that table 3. cholate grows Brevibacillus laterosporus YB-02
The impact that table 4. trypsinase grows Brevibacillus laterosporus YB-02
When cholate mass concentration is 5g/L, although YB-02 is survived, number is minimum, also has 5.6 × 10 5cfu/ml, illustrates that YB-02 can tolerate certain density cholate; Be after 5g/L process 1h through trypsinase mass concentration, YB-02 survives number 5.1 × 10 6cfu/ml, shows that the growth effect of trypsinase to YB-02 is less.
Embodiment 5 nitrite nitrogen clearance is tested
Hydrochloride naphthodiamide diazonium azo colorimetry (GB_17378.4-2007) is adopted to detect water nitrite state nitrogen concentration.(mainly salinity 32 ‰, pH 7.5 and temperature 24 ~ 26 under simulated seawater cultivating condition, Rong Xie Yang≤5mg/L), carry out the 24h purification experiment of YB-02 to the nitrite nitrogen initial concentration water body containing 0.5,2,5 and 10mg/L, and calculate nitrite nitrogen clearance, every concentration gradient establishes 3 repetitions, and result is as table 5.The preparation of YB-02: YB-02 probiotic bacterium seed liquor is seeded in fermention medium by the volume ratio of 5%, adjustment tank pressure is 0.1MPa, temperature 28 ~ 30 DEG C, dissolved oxygen >=30%.Be be after 250rpm, 4h be 300rpm after 200rpm, 3h after initial stirring velocity 150rpm, 2h, ferment 10 hours, obtain the zymocyte liquid of YB-02, its concentration is about 5 × 10 10cfu/ml.Get 5ml zymocyte liquid centrifugal after the thalline precipitated joined in the test fluid of 4 concentration nitrite nitrogen initial concentrations of 100ml, carry out the test of 24 hours nitrite nitrogen clearances.
Table 5. Brevibacillus laterosporus YB-0224h nitrite nitrogen clearance is tested
Through 24h process, YB-02 is respectively 99.87% and 99.53% to nitrite nitrogen clearance in the simulated solution of 0.5mg/L and 2mg/L concentration; 5mg/L is the upper limit of marine culture water Central Asia nitrate, and nitrite nitrogen clearance also has 95.02%, can efficient degradation nitrite nitrogen.
Embodiment 6 fungistatic effect is tested
Adopt filter paper lysoplate assay, get 6mm aseptic filter paper sheet immersion YB-02 concentration and be 1.0 × 10 8in cfu/ml nutrient solution.The pathogenic vibrio being cultured to logarithmic phase is diluted to 1.0 × 10 8cfu/ml, gets 100ml and is spread evenly across NB agar plate, and the filter paper of inserting containing YB-02 carries out antagonism test.Cultivate 24h in 28 DEG C and observe bacterial growth situation, measure antibacterial circle diameter, 3 repetitions established by every plate.Result is as shown in table 6, and Brevibacillus laterosporus YB-02 has inhibition to multiple sea farming pathogenic bacterium, wherein has higher inhibition to Vibrio splindidus, Huanghai Sea Shewanella and Edwardsiella tarda.
Table 6. Brevibacillus laterosporus YB-02 is to the inhibition of Main Pathogenic Bacteria
Note: d≤8mm represents to have bacteriostatic activity, and d<8mm represents not have bacteriostatic activity.

Claims (3)

1. a strain has Brevibacillus laterosporus (Brevibacillus laterosporu) YB-02 of degraded nitrite nitrogen function and bacteria resistance function, and its culture presevation number is CGMCC-No.9757.
2. Brevibacillus laterosporus YB-02 as claimed in claim 1 is preparing the application in marine aquaculture water body water purification agent.
3. application according to claim 1, is characterized in that, described marine aquaculture is holothruian cultures, turbot cultivation and Fugu rubripes (Temmincket Schlegel) cultivation.
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CN105969708A (en) * 2016-08-01 2016-09-28 浙江大学 Bacillus subtilis SC228 with nitration function and application thereof
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CN110964676A (en) * 2020-01-04 2020-04-07 山东得和明兴生物科技有限公司 Method for culturing high-bacterial-quantity fermentation liquor of brevibacillus laterosporus
CN112111435A (en) * 2020-10-27 2020-12-22 集美大学 Bacillus NB-1 and culture method and application thereof
CN112625974A (en) * 2021-01-06 2021-04-09 北京波尔莱特饲料有限公司 Brevibacillus laterosporus BL11, fermentation liquid thereof, preparation method and application
CN112625974B (en) * 2021-01-06 2021-12-07 北京波尔莱特饲料有限公司 Brevibacillus laterosporus BL11, fermentation liquid thereof, preparation method and application
CN112940998A (en) * 2021-05-17 2021-06-11 中国科学院烟台海岸带研究所 Bacterial strain for preventing bacterial diseases and application
CN112940998B (en) * 2021-05-17 2021-08-03 中国科学院烟台海岸带研究所 Bacterial strain for preventing bacterial diseases and application
CN114507629A (en) * 2022-04-19 2022-05-17 中国科学院烟台海岸带研究所 Aquaculture probiotics and preparation and application of microbial inoculum thereof

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