CN103693750A - Holothurian culture water restoration agent capable of effectively preventing occurrence of holothurian disease, and preparation method and application thereof - Google Patents
Holothurian culture water restoration agent capable of effectively preventing occurrence of holothurian disease, and preparation method and application thereof Download PDFInfo
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- CN103693750A CN103693750A CN201310713627.7A CN201310713627A CN103693750A CN 103693750 A CN103693750 A CN 103693750A CN 201310713627 A CN201310713627 A CN 201310713627A CN 103693750 A CN103693750 A CN 103693750A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention discloses a holothurian culture water restoration agent capable of effectively preventing occurrence of holothurian disease, and a preparation method and application thereof, belonging to the technical fields of aquiculture and marine microorganisms. The preparation method comprises the following steps: through a marine microorganism technology, based on physiological habits and culture characteristics of holothurians, separating and screening Bacillus flexus CGMCC No.5115, Bacillus subtilis CGMCC No.5116 and Bacillus cereus CGMCC No.5117 from a holothurian culture environment; selecting corn flour and bean pulp flour as main culture raw materials, and fermenting at 28-37 DEG C for 24-48h; and selecting kieselguhr, attapulgite and rice hull as adsorption and suspension compound carriers, and processing to prepare the holothurian culture water restoration agent capable of effectively preventing occurrence of holothurian disease. The product has high tolerance to the water body environment and has favorable floatability, suspensibility and floating/sinking controllability, thereby being beneficial to three-dimensional stereoscopic purifying effect of a marine culture water body; and the acidification and bottom stinking of the water body can be avoided, and pathogenic microorganisms can be effectively inhibited, thus preventing occurrence of holothurian disease and increasing the culture output and quality of holothurians.
Description
Technical field
The present invention, in aquaculture and Marine Microbial Biotechnology: A Review field, is specifically related to a kind of agent of holothruian cultures Water quality restoration and method for making and purposes of generation of effective prevention sea cucumber disease.
Background technology
Seawater is the Essential Environment condition that the precious marine products such as sea cucumber are depended on for existence, as the saying goes: " within three minutes, breeding fish, seven minutes foster water ".The quality of water quality directly has influence on the upgrowth situation of sea cucumber and raiser's economic benefit.Along with economy and the reach of science, people increase day by day to the demand of sea cucumber, natural, fish for can not meet the need of market in the situation that, holothruian cultures had been converted into the breeding way of present intensive and batch production already by traditional extensive management, Resource Dependence type breeding way, apostichopus japonicus culture has become one of mainstay industry of Chinese Fishery aquaculture.Yet; along with stichopus japonicus is intensive and large-scale cultivation develops rapidly; there are some serious problems, restricted the development of its industry, such as remaining bait and movement and animal dead volume deposit in water body; decompose and generate a lot of objectionable impuritiess; worsened water quality, pathogenic bacteria amount reproduction, causes sea cucumber to produce strong stress reaction; occurred shaking the head do not eat, mouthful swollen, health shrink stiff, arrange the phenomenons such as dirty, ulcer in body surface, last body wall big area is rotted and dead.In order to resist the sick epidemic disease of sea cucumber, long-term and a large amount of abuses of antibiotics and chemical synthetic drug, not only caused the resistance of sea cucumber to the susceptibility of pathogenic micro-organism and bacteria pathogeny microorganism etc., also can make microbiotic detect exceeds standard, and cause the pollution of whole ecotope, had a strong impact on the Sustainable development of saving reduction of discharging, food safety and having hindered nuisanceless apostichopus japonicus culture industry.
Diatomite is a kind of biogenic silicastone, mainly by diatom remains, formed, in diatom have many different shapes, as discoid, needle-like, tubular, pinniform etc., its Chemical Composition is mainly SiO
2, the good characteristics such as voidage is high, specific surface is large, proportion is little owing to having, wear-resisting, acidproof, adsorptivity is strong, heat conductance is low, heat-insulating flame-retardant, heat preservation and soundproof, are widely used in the industries such as diet, building materials, chemical industry, the energy, water treatment.Diatomite has the characteristic that specific surface area is large, and the larger adsorptive capacity of specific surface area is larger; Aperture is larger, and the rate of diffusion of adsorptive capacity in hole is larger, is more conducive to reach adsorption equilibrium.And diatomaceous surface and internal surface of hole be distributed with a large amount of silicon hydroxyls, these silicon hydroxyls dissociation in the aqueous solution goes out H
+thereby, make particle performance go out certain surperficial electronegativity, make it in water treatment procedure, not only can remove the pollution substance of particulate form and colloidal attitude, can also effectively reduce principal pollutant phosphorus and the ammonia nitrogen of water turbidity and removal body eutrophication, and Hg
2+, Pb
2+, Cr
6+, Cd
2+, Cu
2+etc. objectionable impuritiess such as heavy metal ion.
Attapulgite clay is under the jurisdiction of sepiolite group on mineralogy classification, and the magnesia silicate of layer chain for moisture, has unique layer chain-like structure feature, and its desirable chemical molecular formula is: Mg
5si
80
20(OH)
2(OH
2)
24H
2o.Attapulgite is owing to having the crystalline structure that is needle-like, fibrous agrregate, the features such as its specific surface area is large, chemical stability good, high adsorption capacity, it shows good performance at aspects such as Adsorption of Heavy Metal Ions, organic pollutant, radioelement, aspect water treatment, has a good application prospect.
Rice husk is one deck shell of paddy outside, by inner glume and larger coetonium composition.The long 5-10mm of rice husk, 2.5-5mm, thick 23-30 micron, it is golden yellow that its color and luster is generally, and after being pulverized, tap density can reach 384-400kg/m
3, as the auxiliary material of this product, can increase floatability, suspension and the sink-float controllability of product, be conducive to the decontamination effect improving of marine culture water 3 D stereo.
Aquatic products probiotics, it is the active bacteria formulation that utilizes the beneficial microorganism in aquatic animal body or in aquaculture water to form through special processing process, there is the effects such as ammonia oxidation, nitrated, denitrification, phosphorus decomposing, sulphur removal and denitrogenation, effectively reduce the concentration of COD, ammonia nitrogen, nitrous acid nitrogen and sulfide in water, thereby effectively improve water quality.At present, be applied to the microecological water matter conditioning agent of aquaculture, be only limited to simple microbial inoculum or carrier microbial inoculum, in using, there is the inferior positions such as flora Survival Reproduction bad adaptability and quantity minimizing in the former, the latter be substantially major part be sink to pond at the bottom of, centering upper strata water body purification rate is low, cuts pool bottom environment dissolved oxygen low, is unfavorable for the breeding of microecological microbial agent.So, be badly in need of a kind of probiotics and can 3 D stereo is comprehensive can remove holothruian cultures COD in water body, ammonia nitrogen, nitrous acid nitrogen and sulfide, can remove Heavy Metals in Waters ion again, and suppress harmful microbe breeding, reach simultaneously the once Water quality restoration agent of purifying aquatic water completely.
Summary of the invention
The present situations such as in prior art, cultivation water is seriously polluted, disease takes place frequently in order to solve in the present invention, sea cucumber high mortality, sea cucumber heavy metal of body, chemical residual and antibiotic remains exceed standard, and invent a kind of agent of holothruian cultures Water quality restoration and method for making and purposes of generation of effective prevention sea cucumber disease.This effectively prevents the holothruian cultures Water quality restoration agent of the generation of sea cucumber disease mainly by genus bacillus, to be mixed and formed, separated also subtilis (Bacillussubtilis) YB-1CGMCCNo.5116, bacillus cereus (Bacilluscereus) YB-2CGMCCNo.5117 and Bacillus flexus (Bacillusflexus) XH-1CGMCCNo.5115 of screening in holothruian cultures environment, take Semen Maydis powder and bean cake powder as main fermention medium, using diatomite, attapulgite and rice husk as absorption and suspending carrier, be processed into solid powder.The probiotics relating in the present invention, in 2011 08 month No. 05, deliver to China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation (Institute of Microorganism, Academia Sinica, postcode 100101), address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Main aspect of the present invention is: a kind of preparation method of holothruian cultures Water quality restoration agent of generation of effective prevention sea cucumber disease, comprises following operation steps:
1. prepare probiotic bacterium stoste
Will be on LB agar slant culture-medium well-grown subtilis YB-1CGMCCNo.5116, bacillus cereus YB-2CGMCCNo.5117 and Bacillus flexus XH-1CGMCCNo.5115, add respectively 2116E substratum, pH7.0～7.5,12～16h is cultivated in 33～37 ℃ of concussions, with the volume ratio of 1:1:1, mix again, be prepared into probiotic bacterium stoste;
2. liquid state fermentation is cultivated
To add fermented liquid in closed fermentation reactor, and add nutritive substance by 0.1～1% volume ratio, then the probiotic bacterium stoste that 1. step is prepared, press the volume ratio inoculation of 3-5%, ferment, pressure is 0.1～0.25Mpa, 37 ℃ of fermentation 24h, make tunning;
3. spray drying treatment
The above-mentioned steps 2. tunning of gained is sprayed to absorption carrier, drying treatment;
Wherein, step is said seawater LB nutrient agar and 2116E substratum 1., is culture medium prescription in < < molecular cloning experiment guide > >;
Step 2. said nutritive substance by Semen Maydis powder: bean cake powder: seawater is by (1.5～3): (0.5-1.5): 100 weight ratio forms;
Step is consisting of of described absorption carrier 3.: the volume ratio of diatomite in powder, Attapulgite stone flour and rice husk 2:2:1 is mixed.
Under optimal conditions: in above-mentioned preparation method, the step 1. cell concentration of middle bacteria suspension is all about 1 * 10
8cfu/ml.
Under optimal conditions: in above-mentioned preparation method, step is 3. in described absorption carrier, and diatomite in powder and Attapulgite stone flour are 150～200 orders; End product absorption carrier and probiotic composition, the final concentration that carries bacterial content is about 10
8～10
10cfu/g.
Under optimal conditions: in above-mentioned preparation method, the weight ratio that adds absorption carrier in the product 3. making to step is 1:1～1000.
Under optimal conditions: in above-mentioned preparation method, the step method that 3. middle tunning is spray dried into absorption carrier is: fermentation culture is with vehicle weight than being 1:1～4, and be 12～24h time of drying, and product water content is not higher than 5%.
Other one side of the present invention is, utilizes the effectively holothruian cultures Water quality restoration agent product of the generation of prevention sea cucumber disease of above-mentioned method preparation.
Other one side of the present invention is, the application of the holothruian cultures Water quality restoration agent of generation that utilizes above-mentioned effective prevention sea cucumber disease aspect holothruian cultures.
The holothruian cultures Water quality restoration agent environmental resistance of the generation of effective prevention sea cucumber disease of the present invention is strong, floatability, suspension and sink-float controllability are good, decontamination effect improving to the aquaculture water 3 D stereo of sea cucumber is remarkable, especially in this effectively holothruian cultures Water quality restoration agent of the generation of prevention sea cucumber disease of regularly splashing of the nursery stage of sea cucumber and mature stage, can remove fast heavy metal ion and degradation of ammonia nitrogen in aquaculture water, nitrite, the objectionable impuritiess such as hydrogen sulfide, avoid water acidification and the smelly end, effectively suppress pathogenic bacteria, alleviate the stress reaction of environment to sea cucumber, the effectively generation of prevention sea cucumber disease, improve the Quality and yield of holothruian cultures.
Embodiment
Below in conjunction with technical scheme, describe embodiments of the invention in detail.
Raw materials used by ordinary method preparation or obtained by commercial sources in embodiment.
LB agar slant culture-medium: it contains in forming every liter: pancreatin solution peptone 5g, yeast extract paste 1g, sea crystal 33g, high ferric phosphate 0.1g, agar 15g, 1000mL water, pH7.6.Peptone and yeast extract paste are purchased from Britain Oxoid company, and agar powder is purchased from Beijing extensive and profound in meaning star biotechnology limited liability company, and other raw materials are all purchased from Shanghai Sheng Gong biotechnology limited-liability company.
2216E liquid nutrient medium: it contains in forming every liter: pancreatin solution peptone 5g, yeast extract paste 1g, high ferric phosphate 0.1g, Chen Haishui 1000mL, pH7.6.Peptone and yeast extract paste are purchased from Britain Oxoid company, and other raw materials are all purchased from Shanghai Sheng Gong biotechnology limited-liability company.
Semen Maydis powder and bean cake powder, be 100-200 order, is purchased from Wandefu Ind Co., Ltd., Shandong.
Rice husk, is purchased from Dalian Sai Mu group.
Diatomite in powder, is purchased from governor Bai Xian green hill, Jilin source diatomite company limited, 150-200 order.
Attapulgite stone flour, is purchased from Jingyuan County, Gansu Province Hao Di industry and trade limited liability company, 150-200 order.
The probiotics relating in the embodiment of the present invention comprises: in holothruian cultures environment, the genus bacillus of separation screening comprises: subtilis (Bacillussubtilis) YB-1CGMCCNo.5116, bacillus cereus (Bacilluscereus) YB-2CGMCCNo.5117 and Bacillus flexus (Bacillusflexus) XH-1CGMCCNo.5115.
Three strain bacterial strains are preserved in delivers to China Committee for Culture Collection of Microorganisms's common micro-organisms center (Institute of Microorganism, Academia Sinica, postcode 100101) for 2011 08 month No. 05, and address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Below by example, the present invention is described in further detail.
Embodiment 1:
1. prepare probiotic bacterium stoste
Will be on LB agar slant culture-medium well-grown subtilis YB-1CGMCCNo.5116, bacillus cereus YB-2CGMCCNo.5117 and Bacillus flexus XH-1CGMCCNo.5115, add respectively 2116E substratum, pH7.0～7.5,12～16h is cultivated in 37 ℃ of concussions, with the volume ratio of 1:1:1, mix again, be prepared into probiotic bacterium stoste, cell concentration is about 1 * 10
8cfu/ml.
2. liquid state fermentation is cultivated
To add fermented liquid in closed fermentation reactor, and add nutritive substance by 0.1～1% volume ratio, then the probiotic bacterium stoste that 1. step is prepared, press the volume ratio inoculation of 3-5%, ferment, pressure is 0.1～0.25Mpa, 37 ℃ of fermentation 24h, make tunning;
Above-mentioned nutritive substance is by Semen Maydis powder: bean cake powder: seawater is by (1.5～3): (0.5-1.5): 100 weight ratio forms.
3. spray drying treatment
Mixture absorption carrier is 1:1:1 mixed preparing by 150-200 object diatomite in powder, Attapulgite stone flour and rice husk by volume;
The tunning of above-mentioned steps 2 gained is spray dried in above-mentioned absorption carrier, and tunning is with vehicle weight than being 1:4, and be 24h time of drying, and product water content, not higher than 5%, makes the holothruian cultures Water quality restoration agent of the generation of effective prevention sea cucumber disease.
Embodiment 2
1. prepare probiotic bacterium stoste
Will be on LB agar slant culture-medium well-grown subtilis YB-1CGMCCNo.5116, bacillus cereus YB-2CGMCCNo.5117 and Bacillus flexus XH-1CGMCCNo.5115, add respectively 2116E substratum, pH7.0～7.5,12～16h is cultivated in 37 ℃ of concussions, with the volume ratio of 1:1:1, mix again, be prepared into probiotic bacterium stoste, cell concentration is about 1 * 108cfu/ml.
2. liquid state fermentation is cultivated
To add fermented liquid in closed fermentation reactor, and add nutritive substance by 0.1～1% volume ratio, then the probiotic bacterium stoste that 1. step is prepared, press the volume ratio inoculation of 3-5%, ferment, pressure is 0.1～0.25Mpa, 37 ℃ of fermentation 24h, make tunning;
Above-mentioned nutritive substance is by Semen Maydis powder: bean cake powder: seawater is by (1.5～3): (0.5-1.5): 100 weight ratio forms.
3. spray drying treatment
Absorption carrier is 2:2:1 mixed preparing by 150-200 object diatomite in powder, Attapulgite stone flour and rice husk by volume;
The tunning of above-mentioned steps 2 gained is spray dried in above-mentioned absorption carrier, and tunning is with vehicle weight than being 1:4, and be 24h time of drying, and product water content, not higher than 5%, makes the holothruian cultures Water quality restoration agent of the generation of effective prevention sea cucumber disease.
Result of use
Choose the holothruian cultures circle that 3 place's conditions are identical, average depth 1.5-2m, seedling specification 30-50 head/500g, density is at 9-10 head/m
2.
No. 1 circle is control group (not using the effectively holothruian cultures Water quality restoration agent of the generation of prevention sea cucumber disease), and 2, No. 3 circles are experimental group (using the effectively holothruian cultures Water quality restoration agent of the generation of prevention sea cucumber disease); Experimental group is except the holothruian cultures Water quality restoration agent of the generation of the effective prevention sea cucumber disease described in the embodiment 1,2 that regularly splashes, and the mode of feeding is the same with control group with management process.
Experimental period be in September, 2013 to October, about 45 days, experimental group was splashed 1 time every 3 days, the amount of splashing is 5g/m
3.
Experimental session, regularly detects water quality, observes the sea cucumber situation of ingesting, every 15 all over the world water touch spy, measure sea cucumber body weight.Salinity is 35-38 ‰, and water temperature is 16-23 ℃, and pH is 7.6-8.5, and dissolved oxygen is not less than 4mg/L.
1. use after the present embodiment 1,2 the water-quality guideline comparative result of the holothruian cultures circle of the 45th day
2. use the impact of latter 45 days of the present embodiment 1,2 on sea cucumber growth and immunity function
| ? | Number/m2 | Average weight gain amount (g) | Respiratory burst vigor | Phagocytic rate (%) |
| Control group | 7.5 | 4.33 | 0.162±0.013 | 35.35±0.52 |
| Embodiment 1 | 8.5 | 8.75 | 0.474±0.076 | 43.63±0.59 |
| Embodiment 2 | 8.9 | 12.42 | 0.652±0.032 | 49.89±0.62 |
Result shows: the holothruian cultures Water quality restoration agent of the generation of effective prevention sea cucumber disease of the present invention, water quality in holothruian cultures circle is had to obvious decontamination effect improving, can significantly reduce the content of the objectionable impuritiess such as ammonia nitrogen, nitrite, hydrogen sulfide, stablize water body pH, effectively suppress pathogenic bacteria, improve the immunizing power of sea cucumber, the generation of prevention sea cucumber disease, the Quality and yield of raising culturing sea cucumbers.
Claims (8)
1. a preparation method for the holothruian cultures Water quality restoration agent of the generation of effective prevention sea cucumber disease, comprises following operation step
Rapid:
1. prepare probiotic bacterium stoste
Will be on LB agar slant culture-medium well-grown subtilis YB-1CGMCCNo.5116, bacillus cereus YB-2CGMCCNo.5117 and Bacillus flexus XH-1CGMCCNo.5115, add respectively 2116E substratum, pH7.0～7.5,12～16h is cultivated in 33～37 ℃ of concussions, with the volume ratio of 1:1:1, mix again, be prepared into probiotic bacterium stoste;
2. liquid state fermentation is cultivated
To add fermented liquid in closed fermentation reactor, and add nutritive substance by 0.1～1% volume ratio, then the probiotic bacterium stoste that 1. step is prepared, press the volume ratio inoculation of 3-5%, ferment, pressure is 0.1～0.25Mpa, 37 ℃ of fermentation 24h, make tunning;
3. spray drying treatment
The above-mentioned steps 2. tunning of gained is sprayed to absorption and suspension complex carrier, drying treatment;
Wherein, step is said seawater LB nutrient agar and 2116E substratum 1., is culture medium prescription in < < molecular cloning experiment guide > >;
Step 2. said nutritive substance by Semen Maydis powder: bean cake powder: seawater is by (1.5～3): (0.5-1.5): 100 weight ratio forms;
Step is consisting of of described complex carrier 3.: diatomite in powder, Attapulgite stone flour and rice husk are by the volume ratio mixing of 2:2:1.
2. preparation method according to claim 1, is characterized in that, step is described complex carrier 3., and diatomite in powder and Attapulgite stone flour are 150～200 orders.
3. preparation method according to claim 2, is characterized in that, the step 1. cell concentration of middle bacteria suspension is 1 * 10
8cfu/ml.
4. according to the preparation method described in any one in claim 1,2 or 3, characterized by further comprising: in the product 3. making to step, add complex carrier, mix and make the final concentration of bacterial content for being about 10
8-10
10cfu/g.
5. preparation method according to claim 4, is characterized in that, the weight ratio that adds complex carrier in the product 3. making to step is 1:1～1000.
6. according to the preparation method described in any one in claim 1,2 or 5, it is characterized in that, the step method that 3. middle tunning is spray dried into complex carrier is: fermentation culture is with vehicle weight than being 1:1～4, and be 12～24h time of drying, and product water content is not higher than 5%.
7. the holothruian cultures Water quality restoration agent of the generation of effective prevention sea cucumber disease that prepared by the method for claim 1.
8. the application of the holothruian cultures Water quality restoration agent of the generation of effective prevention sea cucumber disease as claimed in claim 7 aspect holothruian cultures.
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| CN106587371A (en) * | 2016-12-11 | 2017-04-26 | 钦州学院 | Water quality purifying agent and preparation method thereof |
| CN106809958A (en) * | 2017-01-19 | 2017-06-09 | 许星星 | A kind of mycotic holothruian cultures Water quality restoration agent of anti-sea cucumber and its preparation method and application |
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| KR20120014813A (en) * | 2010-08-10 | 2012-02-20 | 서원대학교산학협력단 | (bacillus pumilus with capacity of cellulose-decomposition and cultivation method thereof |
| CN102616941A (en) * | 2012-03-26 | 2012-08-01 | 大连理工大学 | Sea cucumber microecological water quality conditioning agent and method for preparing same |
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| CN101323843A (en) * | 2008-07-25 | 2008-12-17 | 浙江大学 | Composite bacillus preparation for improving marine culture water environment and preparation thereof |
| KR20120014813A (en) * | 2010-08-10 | 2012-02-20 | 서원대학교산학협력단 | (bacillus pumilus with capacity of cellulose-decomposition and cultivation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106587371A (en) * | 2016-12-11 | 2017-04-26 | 钦州学院 | Water quality purifying agent and preparation method thereof |
| CN106809958A (en) * | 2017-01-19 | 2017-06-09 | 许星星 | A kind of mycotic holothruian cultures Water quality restoration agent of anti-sea cucumber and its preparation method and application |
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Application publication date: 20140402 |