CN103160453A - Preparation method of microecologics for nitrite degradation, microecologics and use thereof - Google Patents
Preparation method of microecologics for nitrite degradation, microecologics and use thereof Download PDFInfo
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- CN103160453A CN103160453A CN2013100589859A CN201310058985A CN103160453A CN 103160453 A CN103160453 A CN 103160453A CN 2013100589859 A CN2013100589859 A CN 2013100589859A CN 201310058985 A CN201310058985 A CN 201310058985A CN 103160453 A CN103160453 A CN 103160453A
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Abstract
The invention discloses a preparation method of microecologics for nitrite degradation. The method of the microecologics for the nitrite degradation comprises the steps of preparing a seed solution of bacillus subtilis, enabling the seed solution to be inoculated into a liquid fermentation medium of a fermentation tank for fermental cultivation, adopting a two-step temperature control method during the fermental cultivation, adopting different temperatures to carry out fermentation in the earlier stage of the fermentation and in the later stage of the fermentation, after the fermentation is completed, concentrating a bacterium solution, drying thalli, diluting the thalli, detecting the thalli, packaging the thalli, and obtaining the microecologics of the degradation nitrite. Futher disclosed are the microecologics for the nitrite degradation and use of the microecologics for the nitrite degradation in aquaculture water quality improvement. The microecologics for the nitrite degradation is obtained through the method. According to the preparation method of the microecologics for the nitrite degradation, the two-step temperature control method is adopted, a viable count and an endospore rate are improved, and stability of product quality is ensured. Due to addition of zeolite powder in a liquid state fermentation media component, compared with a general immobilization method, the preparation method of the microecologics of the degradation nitrite achieves more effective immobilization, prolongs a quality guarantee period of a product, and an effect of the microecologics for the nitrite degradation is good.
Description
Technical field
The present invention relates to a kind of preparation method of probiotics of degrading nitrite, the invention still further relates to the probiotics that uses the degrading nitrite that the method makes, the present invention has also further related to the purposes of probiotics in the aquaculture water water quality improvement of this degrading nitrite.
Background technology
Along with domestic and international culture fishery fast development, the expanding day of cultivation scale, improving constantly of intensive degree, the amount of transfiniting is put in a suitable place to breed and is concentrated bait throwing in to cause aquaculture water to produce a large amount of residual baits, ight soil and dead animals and plants corpse, these organic depositions are at the bottom of the pond, putrid fermentation under the effect of heterotrophic bacterium, wherein protein and nucleic acid slowly are decomposed, produce a large amount of nitrogenous objectionable impuritiess, aquaculture water water quality is worsened rapidly, cause serious self-pollution, make whole breeding ecological environment all be destroyed when serious.pollute when occuring, ammonium nitrogen in water body, the hazardous and noxious substances such as nitrite produce in a large number, especially the nitrite of high density descends cultivation object blood transport oxygen ability, impel the oxyphorase in blood to be converted into methemoglobin, lose the ability with the oxygen combination, not only directly endanger cultivated animals, while is due to its long-term accumulate poisoning effect, cause fish, the disease resistances such as shrimp reduce, easily cause the invasion and attack of various pathogenic bacterias, therefore be considered to be fish, the pathogenic root of shrimp, the disease of bringing thus makes the raiser suffer heavy losses, the pollution problem of water surrounding nitrite has become one of bottleneck of puzzlement culture fishery development, limited greatly the development of culture fishery.
The method of degrading nitrite mainly contains physics, chemistry and biological method in the market, employing is changed water, absorption, throwing and is executed the physico-chemical processes such as redox agent and come degrading nitrite, having the shortcomings such as consumption is large, the feature of environmental protection is poor, degradation rate is not high, it is short to hold time, easy bounce-back, is the behave of curing the symptoms, not the disease.And adopt the biological method degrading nitrite add probiotics have simple to operate, have no side effect, noresidue, effect is remarkable, cost is low, do not destroy many-sided advantage such as the eubiosis, has become the recent development trend of aquaculture water purification techniques.The outstanding effects of probiotics aspect degrading nitrite such as genus bacillus, photosynthetic bacterium, milk-acid bacteria constantly have research report, and wherein the advantage of subtilis, anti-storage strong with its fecundity, be subject to extensive concern.
Existing probiotics product fermentation manufacturing technique mainly contains liquid-state fermentation technology and solid-state fermentation process, and liquid state fermentation product ubiquity living bacteria count and the low problem of gemma rate cause in the subsequent production treating processes thalline loss serious; The difficult monitoring of processing parameter and control in the solid fermentation products production process, the miscellaneous bacteria rate is high.In addition; after probiotics drops into aquaculture water, thalline is unbound state; tolerance to poor environment in water is low; be difficult to resist competition antagonism and the hydrobiological predation of indigenous bacterium in the pond; and immobilization technology can play to the function yeast in probiotics effective protection; but existing immobilization embedded technological operation is loaded down with trivial details, is difficult to be applied to scale operation.The immobilization adsorption technology is simple to operate, carrier is with low cost, and present main processing mode is to drop into fixation support absorption with fermented liquid is concentrated after fermentation ends again, between thalline and carrier a little less than bonding force, after entry, thalline is easy to analyse and takes off, and does not reach effective immobilized purpose.
Although probiotics has many advantages in the application of degraded aquaculture system nitrite, but owing to there being above-mentioned many-sided defective, development so far, still exist in actual applications effect unstable or use the unfruitful problem of rear milli, substantially also do not have on the market can be really the effective specifics of degrading nitrite, cause nitrite pollution to become making the problem of aquaculture headache.
Summary of the invention
The object of the invention is to overcome the defective that exists in prior art, provide that a kind of production cost is lower, simple to operate, the preparation method of the probiotics of product viable count and the high degrading nitrite of gemma rate.Another object of the present invention is to disclose probiotics and the purposes of this probiotics in the aquaculture water water quality improvement of using the prepared degrading nitrite of the inventive method.
For reaching described purpose, technical scheme of the present invention is as follows:
A kind of preparation method of probiotics of degrading nitrite comprises the steps:
(a) seed liquor of preparation subtilis (Bacillus subtilis).
(b) seed liquor of subtilis (Bacillus subtilis) is seeded in the liquid fermentation medium of fermentor tank and carries out fermentation culture, adopt two step method of temperature-control by in fermentation culture, earlier fermentation uses different temperature to ferment with the later stage, higher at the earlier fermentation leavening temperature, lower in fermentation later stage fermentation temperature;
At earlier fermentation, thalline is in lag phase and logarithmic growth is early stage, higher tank temperature is conducive to accelerate the sprouting of thalline, shorten time lag phase, in the fermentation later stage, thalline enters logarithmic growth vigorous period, and self metabolic heat production is serious, it is too high that local temperature easily appears, suitably reduce control temperature more favourable with the raising of fermentative production bacterium number and the formation of later stage gemma.
(c) after fermentation is completed, concentrated broth, thalline is dry, and dilution detects, and packs, and namely obtains the probiotics of degrading nitrite.
Wherein, in above-mentioned steps (b), the inoculum size of subtilis (Bacillus subtilis) seed liquor is 5～10% of liquid fermentation medium weight; Described fermentation culture conditions is: Initial pH is 7～7.5, and rotating speed is 150～200r/min, and tank pressure is 0.04～0.06MPa, and fermentation time is 24～28h, and 35～38 ℃ of 1～16h temperature controls, 16h is to 28～32 ℃ of fermentation ends temperature controls; Described liquid state fermentation substratum is composed of the following components: remember by weight percentage, Semen Maydis powder 2～3%, molasses 1～2%, soybean cake powder 1～2%, ammonium sulfate 0.2～0.5%, dipotassium hydrogen phosphate 0.1～0.2%, magnesium sulfate heptahydrate 0.03%, manganese sulfate monohydrate 0.01%, zeolite powder 5～10%, surplus are water.
Wherein, in above-mentioned steps (b), the inoculum size of subtilis (Bacillus subtilis) bacterium liquid is preferably 5% of liquid fermentation medium weight; Described fermentation culture conditions is preferably: Initial pH is 7～7.5, and rotating speed is 200r/min, and tank pressure is 0.04～0.06MPa, and fermentation time is 24h, and 37 ℃ of 1～16h temperature controls, 16h is to 32 ℃ of fermentation ends temperature controls; Described each component concentration of liquid state fermentation substratum is preferably: remember by weight percentage, Semen Maydis powder 2%, molasses 1%, soybean cake powder 2%, ammonium sulfate 0.2%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.03%, manganese sulfate monohydrate 0.01%, zeolite powder 10%, surplus are water.
Wherein, in described step (c), the method for concentrated broth is centrifugal rear collection thalline; The method of thalline drying is conventional spray-drying process.
The probiotics of the degrading nitrite that obtains with aforesaid method and the purposes of probiotics in the aquaculture water water quality improvement of described degrading nitrite also belong to protection scope of the present invention.
The present invention compared with prior art has the following advantages:
(1) adopt two step method of temperature-control by at liquid fermentation method, the fermented liquid viable count can reach 10,300,000,000/ml, and the gemma rate has effectively improved viable count and gemma rate more than 99%, has guaranteed the stability of quality product;
(2) add zeolite powder in the liquid state fermentation culture medium prescription, thalline is entered in fermentation culture in the microvoid structure of zeolite powder, realization and carrier combines closely in growing microorganism, forming firmly, microbial film is, realize more firmly keying action than the conventional process for fixation that adds again zeolite powder after fermentation ends, realize effective immobilization, simultaneously, this operation has also extended the quality guaranteed period of probiotics;
(3) effect of gained probiotics degraded water nitrite is fine, and produces, uses simple to operate, with low cost, is easy to apply.
Detailed content of the present invention can obtain by explanation described later and institute's accompanying drawing.
Description of drawings
Fig. 1 is preparation method's the process route chart of the probiotics of degrading nitrite of the present invention.
Fig. 2 is that in differing temps control mode bottom fermentation liquid, the viable count situation of growth compares.
Embodiment
Below in conjunction with drawings and Examples, the specific embodiment of the present invention is further described, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not consisted of any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can modify or replace details and the form of technical solution of the present invention, but these modifications and replacement all fall within the scope of protection of the present invention.
Experiment material
The present invention subtilis used (Bacillus subtilis) is purchased from China Committee for Culture Collection of Microorganisms agricultural microorganism center, and production code member is: ACCC02973.
The preparation of preparation embodiment 1 liquid state fermentation substratum
Preparation liquid state fermentation substratum, it consists of: remembers by weight percentage, Semen Maydis powder 2%, molasses 1%, soybean cake powder 2%, ammonium sulfate 0.2%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.03%, manganese sulfate monohydrate 0.01%, zeolite powder 10%, surplus is water; Take by weight percentage each component and be added to the water, be stirred to evenly.
Preparation embodiment 2 does not contain the preparation of the liquid state fermentation substratum of zeolite powder
Preparation does not contain the liquid state fermentation substratum of zeolite powder, and it consists of: remember by weight percentage, and Semen Maydis powder 2%, molasses 1%, soybean cake powder 2%, ammonium sulfate 0.2%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.03%, manganese sulfate monohydrate 0.01%, surplus is water; Take by weight percentage each component and be added to the water, be stirred to and be uniformly dissolved.
The preparation of the probiotics of embodiment 1 degrading nitrite
1. will buy subtilis (Bacillus subtilis) bacterial strain that comes and activate through the PDA slant medium, and choose bacterium and cultivate to the triangular flask that the LB liquid nutrient medium is housed, culture temperature is 37 ℃, and incubation time is 24h; Again cultured bacterium liquid in triangular flask is seeded to the seeding tank that the LB liquid nutrient medium is housed and enlarges fermentation culture, culture temperature is 37 ℃, and incubation time is 24h, obtains the seed liquor of subtilis (Bacillus subtilis).
2. the seed liquor of step 1 gained is seeded in the liquid state fermentation substratum of fermentor tank and carries out fermentation culture, the inoculum size of seed liquor is 5% of liquid fermentation medium weight, fermentation culture conditions is: Initial pH is 7～7.5, rotating speed is 200r/min, tank pressure is 0.04～0.06MPa, fermentation time is 24h, and 37 ℃ of 1～16h temperature controls, 16h is to 32 ℃ of fermentation ends temperature controls.
3. after fermentation was completed, with the centrifugal rear collection thalline of fermented liquid liquid, the thalline that obtains used conventional spray-drying process to carry out drying, and being diluted to viable count is 21,000,000,000/g, detects, and packs, and namely obtains the probiotics of degrading nitrite.
The preparation of the probiotics of the rear immobilization degrading nitrite of reference examples 1
1. will buy subtilis (Bacillus subtilis) bacterial strain that comes and activate through the PDA slant medium, and choose bacterium and cultivate to the triangular flask that the LB liquid nutrient medium is housed, culture temperature is 37 ℃, and incubation time is 24h; Again cultured bacterium liquid in triangular flask is seeded to the seeding tank that the LB liquid nutrient medium is housed and enlarges fermentation culture, culture temperature is 37 ℃, and incubation time is 24h, obtains the seed liquor of subtilis (Bacillus subtilis).
2. the seed liquor of step 1 gained is seeded to and carries out fermentation culture in fermentor tank, fermentation culture is used the liquid state fermentation substratum that does not contain zeolite powder, the inoculum size of seed liquor is 5% of liquid fermentation medium weight, fermentation culture conditions is: Initial pH is 7～7.5, rotating speed is 200r/min, and tank pressure is 0.04～0.06MPa, and fermentation time is 24h, 37 ℃ of 1～16h temperature controls, 16h is to 32 ℃ of fermentation ends temperature controls.
3. after fermentation is completed, add 10% zeolite powder absorption in fermented liquid, with the centrifugal rear collection thalline of fermented liquid, the thalline that obtains uses conventional spray-drying process to carry out drying, and being diluted to viable count is 21,000,000,000/g, detects, pack, namely obtain the probiotics of rear immobilization degrading nitrite.
The preparation of the probiotics of reference examples 2 not immobilization degrading nitrites
1. will buy subtilis (Bacillus subtilis) bacterial strain that comes and activate through the PDA slant medium, and choose bacterium and cultivate to the triangular flask that the LB liquid nutrient medium is housed, culture temperature is 37 ℃, and incubation time is 24h; Again cultured bacterium liquid in triangular flask is seeded to the seeding tank that the LB liquid nutrient medium is housed and enlarges fermentation culture, culture temperature is 37 ℃, and incubation time is 24h, obtains the seed liquor of subtilis (Bacillus subtilis).
2. the seed liquor of step 1 gained is seeded to and carries out fermentation culture in fermentor tank, fermentation culture is used the liquid state fermentation substratum that does not contain zeolite powder, the inoculum size of seed liquor is 5% of liquid fermentation medium weight, fermentation culture conditions is: Initial pH is 7～7.5, rotating speed is 200r/min, and tank pressure is 0.04～0.06MPa, and fermentation time is 24h, 37 ℃ of 1～16h temperature controls, 16h is to 32 ℃ of fermentation ends temperature controls.
3. after fermentation was completed, with the centrifugal rear collection thalline of fermented liquid, the thalline that obtains used conventional spray-drying process to carry out drying, and being diluted to viable count is 21,000,000,000/g, detects, and packs, and namely obtains the probiotics of not immobilization degrading nitrite.
1 two step of test example method of temperature-control by and step method of temperature-control by ferment effect contrast
1. test method
According to the seed liquor of described step 1 preparation of embodiment 1 subtilis, in step 2, three groups of tests are set carry out the ferment effect contrast, wherein:
Process A: 32 ℃ of omnidistance temperature controls ferment;
Treatments B: 37 ℃ of omnidistance temperature controls ferment;
Process C: adopt two step method of temperature-control by, temperature is carried out segmentation control, 37 ℃ of front 16h temperature controls, after 16h, 32 ℃ of temperature controls.
In fermenting process, every 4h extracts the fermented liquid of every group and detects its viable count, the results are shown in Figure 2; Detect viable count and the gemma rate of every group of fermented liquid during fermentation ends, the results are shown in Table 1.
2. test-results
2.1 in fermenting process, the viable count situation of growth relatively
As can be seen from Figure 2, process the omnidistance temperature control of A at the lesser temps of 32 ℃, early stage, the bacterium number rose slowly, and the later stage enters that increased logarithmic phase bacterium number increases comparatively fast but be highly limited; The omnidistance temperature control of treatments B is at the comparatively high temps of 37 ℃, and early stage, the bacterium number rose soon, but fermentation later stage bacterium is counted rate of rise not as good as processing A and processing C; Process two step of C employing method of temperature-control by segmentation control temperature and have a clear superiority in compared to processing A and treatments B, early stage, the rising of bacterium number was fast, and later stage while can be realized the logarithmic growth of two-forty.
2.2 in differing temps control mode bottom fermentation liquid, viable count and gemma rate are relatively
The comparison of viable count and gemma rate in table 1 differing temps control mode bottom fermentation liquid
| ? | A organizes (32 ℃) | B organizes (37 ℃) | C group (two step temperature controls) |
| Viable count (hundred million/mL) | 71 | 62 | 103 |
| Gemma rate (%) | 98.8 | 98.4 | 99.5 |
Table 1 data presentation adopts that in two step temperature control method fermented liquids, bacterium number and gemma rate reach respectively 10,300,000,000/mL and 99.5%, all obviously is better than the fermented liquid of the temperature control method gained of 32 ℃ of omnidistance fixed temperatures and 37 ℃.The actual purifying water effect of test example 2 different treatment probioticses relatively
Tested on June 16,10 days～2011 June in 2011 and carry out in Wuhan City plant of many Foucaults of Jiangxia District skill farm.Polycultured ponds A, B and the C that 3 nitrite exceed standard, these 3 pond NO are selected in test
- 2-N concentration has all surpassed 0.15mg/L.Pond A is wherein made to throw the probiotics of executing the embodiment of the present invention 1 to be processed; Pond B makes to throw the probiotics of executing reference examples 1 of the present invention and processes; Pond C makes to throw the probiotics of executing reference examples 2 of the present invention and processes; Each pond is thrown the bacterium amount and is 200g/ mu rice.
Nitrite content after throwing on June 10 bacterium in 3 fishponds of continuous monitoring the results are shown in Table 2
Table 2 nitrite exceed standard the pond administer before and after nitrite content change (mg/L)
As can be seen from Table 2, the pond nitrite concentration that three kinds of probioticses have been executed in throwing has decline in various degree, wherein, the effect that embodiment 1 is executed in throwing is more remarkable than reference examples 1 and reference examples 2, its final degradation rate reaches 94.2%, apparently higher than rear immobilized 75.5% and not immobilized 58.2%, explanation is added zeolite powder in the liquid state fermentation culture medium prescription and is carried out front immobilization, than being fixed is not effective, realize more firmly keying action than the conventional process for fixation that adds again zeolite powder after fermentation ends, realize effective immobilization.
The quality guaranteed period of test example 3 different treatment probioticses relatively
Three kinds of probioticses, 25 ℃ of preservations of constant temperature under air-proof condition with embodiment 1, reference examples 1 and reference examples 2 gained regularly detect viable count, the results are shown in Table 3.
In three kinds of probiotics preservation perives of table 3, viable count relatively
Table 3 data presentation, in two years, the probiotics viable count storage rate of embodiment 1 reaches 91.6%, and the probiotics viable count storage rate of reference examples 1 is 43.8%, and the probiotics viable count storage rate of reference examples 2 is 27.3%.Result shows, the present invention adds zeolite powder in the liquid state fermentation culture medium prescription and carries out front immobilized mode, and the survival of subtilis is had provide protection, can effectively extend the preservation period of microbial inoculum.
Test example 4 product of the present invention purifying water effect in actual cultivating pool detects
This is tested on July 20,14 days～2011 July in 2011 and carries out in Wuhan City plant of many Foucaults of Jiangxia District skill farm.Grass carp cultivating pool A, B and the C that 3 nitrite exceed standard serious, these 3 pond NO are selected in test
- 2-N concentration has all surpassed 0.2mg/L, the aging blackout of Chi Shui, and ammonia nitrogen exceeds standard, and has large stretch of blue-green algae floating.Pond A is wherein made to throw the probiotics of executing the embodiment of the present invention 1 process, throwing the bacterium amount is 200g/ mu rice; Pond B does to throw and executes market like product microbial inoculum processing, and throwing the bacterium amount is 200g/ mu rice; Pond C does not throw the bacterium contrast.
Nitrite content after throwing on July 14 bacterium in 3 fishponds of continuous monitoring the results are shown in Table 4:
Table 4 nitrite exceed standard the pond administer before and after nitrite content change (mg/L)
As can be seen from Table 4, thrown in that the nitrite concentration of pond A is obvious downtrending after the probiotics of degrading cultivation water nitrite of the present invention, throw bacterium after 3 days nitrite drop to 0.023mg/L by 0.281mg/L, clearance 91.8%, and at later stage pond nitrite concentration, bounce-back is not arranged.The same period, two other pond nitrite concentration does not obviously descend, and is on the rise on the contrary.
Simultaneously, at duration of test, observe pond A water quality and obviously improve, water colour transfers green feeling well to by deceiving, and ammonia nitrogen is reduced to 0.31mg/L by 0.52mg/L after testing.
Claims (8)
1. the preparation method of the probiotics of a degrading nitrite, comprise the steps:
(a) seed liquor of preparation subtilis (Bacillus subtilis).
(b) seed liquor of subtilis (Bacillus subtilis) is seeded in the liquid fermentation medium of fermentor tank and carries out fermentation culture, adopt two step method of temperature-control by in fermentation culture, earlier fermentation uses different temperature to ferment with the later stage, higher at the earlier fermentation leavening temperature, lower in fermentation later stage fermentation temperature;
(c) after fermentation is completed, concentrated broth, thalline is dry, and dilution detects, and packs, and namely obtains the probiotics of degrading nitrite.
2. it is characterized in that in accordance with the method for claim 1:
In described step (b), the inoculum size of subtilis (Bacillus subtilis) seed liquor is 5～10% of liquid fermentation medium weight;
Described fermentation culture conditions is: Initial pH is 7～7.5, and rotating speed is 150～200r/min, and tank pressure is 0.04～0.06MPa, and fermentation time is 24～28h, and 35～38 ℃ of 1～16h temperature controls, 16h is to 28～32 ℃ of fermentation ends temperature controls.
3. it is characterized in that in accordance with the method for claim 1:
In described step (b), the liquid state fermentation substratum is composed of the following components: remember by weight percentage, Semen Maydis powder 2～3%, molasses 1～2%, soybean cake powder 1～2%, ammonium sulfate 0.2～0.5%, dipotassium hydrogen phosphate 0.1～0.2%, magnesium sulfate heptahydrate 0.03%, manganese sulfate monohydrate 0.01%, zeolite powder 5～10%, surplus are water.
4. it is characterized in that in accordance with the method for claim 1:
In described step (c), the method for concentrated broth is centrifugal rear collection thalline; The method of thalline drying is conventional spray-drying process.
5. it is characterized in that in accordance with the method for claim 2:
In described step (b), the inoculum size of subtilis (Bacillus subtilis) bacterium liquid is 5% of liquid fermentation medium weight;
Described fermentation culture conditions is: Initial pH is 7～7.5, and rotating speed is 200r/min, and tank pressure is 0.04～0.06MPa, and fermentation time is 24h, and 37 ℃ of 1～16h temperature controls, 16h is to 32 ℃ of fermentation ends temperature controls.
6. it is characterized in that in accordance with the method for claim 3:
In described step (b), the liquid state fermentation substratum is composed of the following components: remember by weight percentage, Semen Maydis powder 2%, molasses 1%, soybean cake powder 2%, ammonium sulfate 0.2%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.03%, manganese sulfate monohydrate 0.01%, zeolite powder 10%, surplus are water.
7. the probiotics of the degrading nitrite that obtains according to any one described method of claim 1～6.
8. according to the purposes of probiotics in the aquaculture water water quality improvement of degrading nitrite claimed in claim 7.
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| CN105884040A (en) * | 2016-05-11 | 2016-08-24 | 长沙学院 | Preparation method of water body denitrification material |
| CN114836333A (en) * | 2021-02-01 | 2022-08-02 | 武汉渔庆家电子商务有限公司 | Blue algae purifying agent and preparation and application thereof |
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| CN103160453B (en) | 2014-07-16 |
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