CN105062931A - Preparation method of high-concentration bacillus subtilis Cohn and application of high-concentration bacillus subtilisCohn in aquaculture - Google Patents
Preparation method of high-concentration bacillus subtilis Cohn and application of high-concentration bacillus subtilisCohn in aquaculture Download PDFInfo
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- CN105062931A CN105062931A CN201510563339.7A CN201510563339A CN105062931A CN 105062931 A CN105062931 A CN 105062931A CN 201510563339 A CN201510563339 A CN 201510563339A CN 105062931 A CN105062931 A CN 105062931A
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Abstract
The invention relates to a preparation method of high-concentration bacillus subtilis Cohn and an application of high-concentration bacillus subtilis Cohn in aquaculture. The preparation method is characterized in that the high-concentration bacillus subtilis Cohn is prepared by culturing and fermenting original bacillus strains according to a specific proportion, wherein the original bacillus strains are collected in offshore mariculture areas in Rizhao and Qingdao in Shandong province. An appropriate culture medium and culture conditions are selected in the process, so that bacillus is subjected to quick and efficient expanding culture and high-concentration and high-purity bacillus is finally obtained. Through inspection by a test center in Shandong province, the strains in bacillus liquid prepared by the method can total 7.5*10<10>CFU/mL. Being applied to micro-ecological regulation research of pond fish culture, bacillus subtilis Cohn provided by the invention can greatly increase the degradation rates of ammonia nitrogen, phosphate and nitrite in aquaculture water and purifying the aquaculture water, provides use technological guidance for future large-scale application of probiotic preparations and has high scientific research and market application values.
Description
Technical field
The present invention relates to a kind of preparation method of high density subtilis bacterium liquid and the application in aquaculture thereof, belong to field of biological pharmacy.
background of invention
In order to the popular outburst of Control and prevention aquatic products disease, aquatic products Disease management mainly relies on chemicals and the various sterilizing agents such as various agricultural chemicals, microbiotic, quinolones, sulfamido, furans for a long time.These chemicalses and microbiotic blindly use, although can suppress fast or kill pathogenic bacteria to a certain extent, effectively control the generation of disease and spread, and improve the survival rate of aquatic animal.But the drawback that medical treatment exists is also more and more obvious, create a lot of negative effect: the generation of drug-resistant pathogen strains, microbiotic and chemicals usage quantity strengthen gradually, fishery products drug residue jeopardizes food safety, drug-resistant pathogen bacteria strain plasmid is transferred in human pathogen bacterium, increase the degree of difficulty of human disease treatment, bring grave danger to human health.Therefore, extensive style cultivation for many years, Drug abuse cause the food-safety problem of culture fishery.Based on these negative effects, people start to find the control of substitute for aquatic animal disease of green safety microbiotic and chemicals.Improve aquaculture water micro-ecological environment with microbial preparation to be subject to people and more and more to pay close attention to.
Subtilis (BacillussubtilisCohn) is a kind of aerobic gram-positive microorganism.It can consume the organic matter in water body in a large number, is decomposed into small molecular organic acid, amino acid and ammonia, improves water quality, for Micro Algae provides nutrition; Gut pH and ammonia density also can be made to reduce, produce comparatively strong active proteolytic enzyme and amylase, promote the digestion of aquatic animal; Also the immunizing power growing and improve animal of pathogenic bacteria can be suppressed.Given this, subtilis has been widely used in aquaculture.In water body or feed, add bacillus subtilis formulation, effectively can suppress or kill in water body or some harmful pathogenic bacteria in aquaculture organism body, and probiotics group can be strengthened, and reaching the object of control aquatic products disease.
In the laboratory study of present stage and suitability for industrialized production, also exist that fermentation period is long, spore forming rate is low, quantity is few, high in cost of production series of problems.
Summary of the invention:
Object of the present invention is exactly to overcome above-mentioned prior art Problems existing, provides a kind of preparation method preparing high density subtilis bacterium liquid, and this genus bacillus bacterium liquid effect in raising aquaculture products quality is better.
The present invention is separated the subtilis obtaining a strain and purify water in the aquaculture area of From Shandong Rizhao, Qingdao Area, its feature is as follows: surface is thick and coarse, opaque canescence bacterium colony, in nutrient broth, form mycoderm and precipitate on a small quantity, Gram-positive, in shaft-like, the oval gemma of middle life, this bacterium glucose fermentation, produces acid not aerogenesis, nonfermented N.F,USP MANNITOL, lactose, V-P measures the positive, nitrate reduction is positive, and gelatin is positive, and hydrogen peroxide is positive.
A production method for high density subtilis bacterium liquid, comprises the following steps:
The preparation technology of high density subtilis bacterium liquid (BacillussubfilisCohn) involved in the present invention is: the original genus bacillus gathered through the aquaculture area of From Shandong Rizhao, Qingdao Area is seeded in seed culture medium by the ratio in 5%, 12-48h is cultivated at 35-55 DEG C, be extended in fermention medium with the inoculum size of 10% again, at 35-55 DEG C, air flow is 5-10L/min, stirring velocity is 150-300rpm, pH is between 7.2 ~ 7.4, cultivate 72-100h, obtained high density production microbial inoculum.
Preferably, described seed culture based component is as follows: containing yeast extract 8g, Tryptones 10g, sucrose 20g, dipotassium hydrogen phosphate 2.5g in every 1000mL water, potassium primary phosphate 1g, pH7.3.
Preferably, described fermentation medium components is as follows: Semen Maydis powder 8g, urea 5g, yeast extract paste 1g, fermented bean drink 10g, KH
2pO
40.5g, K
2hPO
40.8g, MgSO
47H
2o0.15g, MnSO
4h
2o0.02g, polypropylene glycol 20002.4g, water 1000mL, 121 DEG C of real elimination bacterium 20min, are cooled to room temperature.
Preferably, described culture temperature is 37 DEG C,
Preferably, in described seed culture medium, incubation time is 36h.
Preferably, in described fermention medium, air flow is 5-10L/min, stirring velocity be 150-300rpm, pH between 7.2 ~ 7.4, incubation time is 72-100h.
High density fermentation of bacillus subtilis liquid after having fermented is the microbial preparation prepared.
The present invention has the following advantages:
Subtilis bacterium liquid provided by the invention under the inoculation condition of 5% in 100h bacterial classification sum can reach 7.5 × 10
10cFU/mL (Shandong Forecasting and Analysis Center's inspection), quantity, far above currently available technology, has feature that is efficient and high yield.
Subtilis bacterium liquid provided by the invention is applied in the microecological regulation and control research of aqiuculrue, greatly can improve aquaculture water ammonia nitrogen degradation rate, phosphoric acid salt degradation rate and nitrite degradation rate, purifying aquaculture water quality, large-scale application for probiotics preparation from now on provides operation technique and instructs, and has higher scientific research and market using value.
Accompanying drawing explanation
Fig. 1 is production of the present invention and uses schema.
Fig. 2 is that the Bacillus subtillis bacterium liquid of patent of the present invention production is on the impact of Aquacultural water phosphate concn.
Fig. 3 is that the Bacillus subtillis bacterium liquid of patent of the present invention production is on the impact of Aquacultural water nitrite concentration.
Fig. 4 is that Bacillus subtillis bacterium liquid is on the impact of aquaculture water ammonia nitrogen concentration.
Fig. 5 is that Bacillus subtillis bacterium liquid use-pattern affects aquaculture water nutrient concentration.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.
Following examples for illustration of the present invention, but do not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method; Experiment material used in following embodiment, if no special instructions, is routine biochemistry reagent.
Substratum involved in following examples is:
(1) short bud substratum: containing peptone 10g in every 1000mL water, extractum carnis 5g, NaCl5g, Semen Maydis powder 0.5g, yeast extract paste 0.1g, pH7.2-7.4.
(2) nutrient agar: containing peptone 10g in every 1000mL water, extractum carnis 3g, NaCl5g, agar 15g, pH7.2-7.4.
(3) seed culture medium: containing yeast extract 8g, Tryptones 10g, sucrose 20g, dipotassium hydrogen phosphate 2.5g in every 1000mL water, potassium primary phosphate 1g, pH7.3
(4) fermention medium: Semen Maydis powder 8g, urea 5g, yeast extract paste 1g, fermented bean drink 10g, KH
2pO40.5g, K
2hPO
40.8g, MgSO
47H
2o0.15g, MnSO
4h
2o0.02g, polypropylene glycol 20002.4g, water 1000mL.
Embodiment 1
1. bacterial classification source: the aquaculture area of From Shandong Rizhao, Qingdao Area, concrete separation method is as follows:
(1) enrichment: get 5g culture zone bed mud and put into triangular flask (band bead) containing 50mL sterilized water, concussion 15min, get 5mL bacteria suspension to add 45mL and urge in bud substratum, 75 DEG C-80 DEG C water-bath 20min, be down to 35 DEG C-37 DEG C, 150-200r/min vibrates 24h, and dyeing microscopic examination, repeats 2-3 time.
(2) separation and purification: get enrichment bacterium liquid and be placed in 75 DEG C-80 DEG C water-bath 15-20min, gradient dilution 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, select three suitable gradients to coat respectively on nutrient agar.Each gradient two plates, are inverted flat board, in 35 DEG C-37 DEG C, cultivate 24-48h.Be numbered the single bacterium colony grown, growth selection is fast, bacterium colony is large, surface drying, coarse, opaque bacterium colony is transferred for subsequent use in inclined-plane.The a little lawn smear of picking, does brood cell's dyeing and determines whether genus bacillus.
(3) biochemical reaction: carry out sugar-fermenting, gelatin, V.P, nitrate reduction, gelatin, hydrogen peroxide test with the sporiferous bacterium colony after being separated.
Embodiment 2
The preparation of high density bacillus subtilis microbial agent:
The subtilis of the incubated overnight of separation is seeded in seed culture medium by the ratio in 5%, 36h is cultivated at 37 DEG C, be extended in fermention medium with the inoculum size of 10% again, 37 DEG C, air flow is 5-10L/min, stirring velocity be 150-300rpm, pH between 7.2 ~ 7.4, cultivate 72-100h.
After artificial separation, purifying, optimization, fermentation, through Shandong Forecasting and Analysis Center's inspection, bacterial classification sum reaches 7.5 × 10
10cFU/mL.
Embodiment 3
The application test of high density bacillus subtilis microbial agent in aquaculture:
Subtilis the claims produced, when maintaining aquaculture water 100,000 cell/mL level, uses different number of days, and measure phosphoric acid salt, nitrite and ammonia nitrogen concentration in aquaculture water, acquired results is shown in accompanying drawing 2,3,4.Result shows: when maintaining aquaculture water 100,000 cell/mL subtilis level, use gemma bacillus agent after 2 days, aquaculture water phosphate concn starts to decline, use aquaculture water phosphoric acid salt degradation rate after 3 days to reach 69%, use continuously afterwards and can keep phosphatic degradation efficiency; Use gemma bacillus agent after 4 days cultivation water nitrite concentration significantly decline, afterwards continuously use bacillus more than 67.9% is reached to nitrite degradation rate; Use the aquaculture water ammonia nitrogen concentration decline after 6 days of bacillus microbial inoculum, after this continue to use aquaculture water ammonia nitrogen degradation rate to reach 88.9%.
Arrange and do not add ecological agent group (control group), lasting interpolation group and interval interpolation group, detect the concentration (see accompanying drawing 5) of aqueous systems Middle nutrition salt.Result shows: use probiotics after 6 days continuously, obviously reducing appears in the nutritive salt such as Aquacultural water ammonia nitrogen, nitrite, phosphoric acid salt, thereafter in stopping interpolation genus bacillus 4 days, aquaculture water nutrient concentration still sustaining degradation, illustrate that genus bacillus possibility partial adsorbates is in bed mud, analyzes and also has certain regulating and controlling effect to substrate.To add after genus bacillus the 5th day in stopping, aquaculture water Middle nutrition salts contg starts to raise.Therefore, now should continue to use bacillus preparation to regulate and control.
The subtilis preparation method that the present invention relates to, by reasonably controlling Culture and fermentation conditions, realize genus bacillus quick, efficiently spread cultivation, finally obtain the genus bacillus of high density, purity, through the inspection of test center of Shandong Province, bacterial classification sum can reach 7.5 × 10
10cFU/mL, quantity, far above currently available technology, achieves feature that is efficient and high yield.Be applied in aquaculture water, result show its to water quality environment keep and performance of control better, the preparation method of a kind of high density subtilis that therefore the present invention relates to and its application in aquaculture, instruct for the large-scale application of probiotics preparation from now on provides operation technique.
Although above-mentioned to invention has been detailed description; but be not limited thereto; those skilled in the art can principle according to the present invention modify, and therefore, all various amendments carried out according to principle of the present invention all should be understood to fall into protection scope of the present invention.
Claims (9)
1. purify water a microbial preparation, it is characterized in that, said preparation is high density subtilis (BacillussubfilisCohn) bacterium liquid.
2., as above-mentioned high density subtilis bacterium liquid according to claim 1, it is characterized in that, subtilis bacterial concentration can reach 7.5 × 10
10cFU/mL.
3. the high density subtilis bacterium liquid as described in the claims 1 ~ 2, it is characterized in that, preparation technology is: the original genus bacillus gathered through the aquaculture area of From Shandong Rizhao, Qingdao Area is seeded in seed culture medium by the ratio in 5%, 12-48h is cultivated at 35-55 DEG C, be extended in fermention medium with the inoculum size of 10% again, at 35-55 DEG C, air flow is 5-10L/min, stirring velocity is 150-300rpm, pH is between 7.2 ~ 7.4, cultivate 72-100h, obtained high density production microbial inoculum.
4. as the preparation technology of above-mentioned high density subtilis bacterium liquid according to claim 3, it is characterized in that, described seed culture based component is as follows: containing yeast extract 8g in every 1000mL water, Tryptones 10g, sucrose 20g, dipotassium hydrogen phosphate 2.5g, potassium primary phosphate 1g, pH7.3.
5., as the preparation technology of above-mentioned high density subtilis bacterium liquid according to claim 3, it is characterized in that, described fermentation medium components is as follows: Semen Maydis powder 8g, urea 5g, yeast extract paste 1g, fermented bean drink 10g, KH
2pO
40.5g, K
2hPO
40.8g, MgSO
47H
2o0.15g, MnSO
4h
2o0.02g, polypropylene glycol 20002.4g, water 1000mL, 121 DEG C of real elimination bacterium 20min, are cooled to room temperature.
6., as the preparation technology of above-mentioned high density subtilis bacterium liquid according to claim 3, it is characterized in that, described culture temperature is preferably 37 DEG C.
7., as the preparation technology of above-mentioned high density subtilis bacterium liquid according to claim 3, it is characterized in that, in described seed culture medium, incubation time is preferably 36h.
8. as the preparation technology of above-mentioned high density subtilis bacterium liquid according to claim 3, it is characterized in that, described is 5-10L/min to air flow in fermention medium, and stirring velocity is 150-300rpm, pH is between 7.2 ~ 7.4, and incubation time is 72-100h.
9. as above-mentioned high density subtilis bacterium liquid according to claim 1, it is characterized in that, be applied to and purify water, improve aquaculture products quality.
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| CN109355223A (en) * | 2018-11-08 | 2019-02-19 | 中国科学院南海海洋研究所 | One plant of bacillus subtilis N2 and its application with ammonia nitrogen degradation function |
| CN109704437A (en) * | 2019-03-01 | 2019-05-03 | 黑龙江翰恒环保科技有限公司 | A kind of method of microwave/hydrogen peroxide treatment cattle manure biogas slurry |
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| CN109355223B (en) * | 2018-11-08 | 2020-10-09 | 中国科学院南海海洋研究所 | Bacillus subtilis N2 with ammonia nitrogen degradation function and application thereof |
| CN109704437A (en) * | 2019-03-01 | 2019-05-03 | 黑龙江翰恒环保科技有限公司 | A kind of method of microwave/hydrogen peroxide treatment cattle manure biogas slurry |
| CN109704437B (en) * | 2019-03-01 | 2021-07-27 | 黑龙江翰恒环保科技有限公司 | Method for treating cow dung fermentation biogas slurry by microwave/hydrogen peroxide |
| CN113943685A (en) * | 2021-11-24 | 2022-01-18 | 上海中询生物科技有限公司 | Culture method and application of special bacillus subtilis microbial inoculum for sewage |
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