CN102583777A - Micro-organism preparation for preventing and curing streptococcicosis of Tilapia mossambica as well as preparation method and application thereof - Google Patents
Micro-organism preparation for preventing and curing streptococcicosis of Tilapia mossambica as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a micro-organism preparation for preventing and curing streptococcicosis of Tilapia mossambica as well as a preparation method and an application thereof. The micro-organism preparation method for preventing and curing streptococcicosis of Tilapia mossambica comprises steps of compounding mixed vaccine with red Nocardia ATCC11653 and coral Nocardia ACCC40100, fermenting and cultivating the mixed vaccine with fermentation medium which is compounded of nitrogen source, carbon source and inorganic salt, and processing resultants into solid powder after the fermentation and cultivation. The micro-organism preparation for preventing and curing streptococcicosis of Tilapia mossambica, which provided by the invention, can reduce the content of ammonia nitrogen and nitrate nitrogen in the Tilapia mossambica breeding water body, antagonize the streptococcicosis of the Tilapia mossambica, reduce the accumulation of harmful substances of the middle and later breeding periods, enhance the immunity of the Tilapia mossambica,, achieve the aim of preventing and curing streptococcicosis of Tilapia mossambica, reduce the economic loss caused by diseases, and ensure the breeding output and quality of the Tilapia mossambica. The micro-organism preparation provided by the invention has advantages of convenience in use, small amount of usage and stable effect after use.
Description
Technical field
The present invention relates to a kind of microbial preparation, its preparation method and application that is used to prevent and treat the tilapia streptococcicosis, belong to technical field of aquaculture.
Background technology
Tilapia originates in Africa, is landlocked tropical fish, is subordinate to Perciformes, eel Gyrinocheilidae, tilapia genus.Growth is fast because tilapia has, omnivory, characteristics such as disease is few, disease resistance is strong, wide adaptability, tilapia is become introduced a fine variety breed fingerling the most widely in the world.China is after last century, introduced tilapia the seventies, and the tilapia industry obtains fast development.The cultured output of China tilapia in 2008 reaches 1,200,000 tons, accounts for the world 60%, becomes one of outlet fish species that China mainly earns foreign exchange.Along with the increase of China's tilapia cultivation density, the decline of breeding environment quality, the tilapia disease constantly takes place, and incidence is on the rise.Streptococcicosis is that tilapia is prone to one of predominantly bacteria property disease of suffering from, and its main pathogenic bacterium are Streptococcus iniae and streptococcus agalactiae.The suis that domestic investigator is separated to from ill tilapia is accredited as Streptococcus iniae more, causes tilapia disease incidence in recent years and constantly increases, and the morbidity scope also enlarges gradually.Especially in 2009, the tilapia streptococcicosis that China breaks out the South China on a large scale caused enormous economic loss to China, brought also for China's tilapia industry and seriously influenced.Thereby the tilapia streptococcicosis has become the important factor of restriction tilapia aquaculture industry Sustainable development.
The main method of control aquatic animal disease has at present: chemical prevention, immune protection and bionomic control (probiotics) etc.In the generating process of control aquatic animal disease, certain active effect has been played in the application of microbiotic and chemicals.But life-time service also brings a series of spinoffs, and many pathogenic micro-organisms produce resistance through the generegulation of sudden change and plasmid to microbiotic, cause the increase of antibiotic dosage, cause autogenous infection and superinfection; Destroy and disturbed body normal microflora and breeding ecological balance simultaneously; Make aquatic animal produce immunosuppressive action; Many chemicalses cause drug residue at the aquatic animal cylinder accumulation, and this has not only influenced the trade outlet of quality product and China's fishery products, and directly harm humans is healthy.Though fishing has safe, efficient, pollution-free, noresidue with vaccine and does not produce advantage such as resistance; But in evolution, still be faced with many difficulties, its reach of vaccine is little, with strong points, cost is higher, workload is big etc., and problem has restricted application and the development of fishing with vaccine to a certain extent.In recent years; Along with people's is goed deep into food safety, environmental safety understanding; Aquaculture must develop towards the direction of the environmental friendliness and the strategy of sustainable development, and this just needs to seek the new controlling way of the drug risk of can improving the ecological environment and can reduce, and probiotics itself not only has the immunizing power of optimizing water environment, raising aquaculture organism body and the function that suppresses the pathogenic bacteria growth; And safe, noresidue, advantage such as free from environmental pollution; Therefore prevent and treat in the method numerous, probiotics obtains more and more many concerns as one of measure of integrated control aquatic products disease in culture fishery.But present microbial preparation is many to be main with improvement water quality and enhancing body digestion; The microbial preparation of bacteriostatic and disease prevention of specialization is still rare, develops therefore that a kind of collection purifies water, suppresses cause of disease, improves immunity, the microbial preparation of the control tilapia streptococcicosis of specialization is very necessary safely and effectively.
Summary of the invention
The objective of the invention is to overcome the deficiency that exists in the prior art, a kind of microbial preparation, its preparation method and application that is used to prevent and treat the tilapia streptococcicosis is provided, it utilizes two specific specificity microbial strainss through assembly rationally; After the mixed culture, form probiotics, have the ammonia nitrogen that reduces in the tilapia aquaculture water water quality and the content of nitrous acid nitrogen; Antagonism tilapia streptococcicosis cause of disease; Reduce the accumulation of culturing the middle and later periods objectionable impurities, improve the immunizing power of tilapia body, reach the purpose of biological control tilapia streptococcicosis; Alleviate the financial loss that disease causes, guarantee cultured output and the quality of tilapia.
According to technical scheme provided by the invention: a kind of microbial preparation that is used to prevent and treat the tilapia streptococcicosis is characterized in that: the activeconstituents of said microbial preparation is nocardia rubra ATCC11653 and coral Nocardia bacteria ACCC40100.
A kind of preparation method who is used to prevent and treat the microbial preparation of tilapia streptococcicosis; It is characterized in that: form mixed bacterium by nocardia rubra ATCC11653 and coral Nocardia bacteria ACCC40100; Adopt nitrogenous source, carbon source and inorganic salt to form fermention medium and carry out fermentation culture, after fermentation culture is accomplished, be processed into solid powder; Make and have the ammonia nitrogen that reduces in the aquaculture water and the content of nitrous acid nitrogen; Antagonism tilapia streptococcicosis cause of disease, the immunizing power of raising tilapia body, the solid powder type microbial preparation of control tilapia streptococcicosis effect.
As a further improvement on the present invention, described preparation method specifically may further comprise the steps:
(1) microbial strains:
Bacterial classification A: nocardia rubra ATCC 11653;
Bacterial classification B: coral Nocardia bacteria ACCC 40100;
Mixed bacterium: bacterial classification A and bacterial classification B are mixed, and with bacterial cell dry weight basis proportioning ratio, bacterial classification A: bacterial classification B is 1:0.5～2;
(2) substratum:
Obtain the collective media of big living weight: with inorganic nitrogen-sourced and organic carbon source as nutritive substance; The collective media prescription is: yam 200g/L, glucose 20g/L, Zulkovsky starch 25g/L, ammonium sulfate 5g/L, potassium hydrogenphosphate 0.85g/L; Concrete compound method is: earlier yam is cleaned peeling, take by weighing 200g again and be cut into small pieces, add that poach is mashed (to be boiled 20～30 minutes; Can be poked by glass stick and get final product), with four layers of filtered through gauze, add glucose and Zulkovsky starch in the filtrating; Continue the heated and stirred mixing; Add other composition again after the cooling slightly, and supply moisture to 1000 milliliter, transfer pH7.5;
Detect solid medium that bacterial classification uses and be in above-mentioned collective media, add 2% weight percent agar; It all is to adopt collective media to cultivate that aphalangia is understood under the condition;
3) strain fermentation preparation:
A) shaking table is cultivated: bacterial classification A and bacterial classification B access that the inclined-plane is preserved are equipped with among the 500mL or 1000mL triangular flask of collective media; The collective media amount is 1/3 of a triangular flask capacity; The combined amount of bacterial classification A and bacterial classification B is by the proportioning configuration of mixed bacterium, and inoculum size is chosen completely with the standard inoculation ring and is as the criterion; Shaking table is cultivated 12～18h, 30～35 ℃ of control culture temperature, and pH 7.5～8.5, rotating speed 130～180rpm;
B) seed fermentation: inoculum size 6-20mL/L; 50～200L seed fermentation jar is advanced in inoculation from the triangular flask that shaking table is cultivated, and adopts the collective media fermentation culture, 30～35 ℃ of control leavening temperatures; PH7.5～8.5; Seed fermentation jar mixing speed 130～180rpm, fermentation time 48～120 hours treats that viable count is 5 * 10
9～8 * 10
9Individual/during mL; Tunning directly is prepared into preparation or further carries out ferment tank as seed;
C) ferment tank: the seed fermentation product is inoculated the fermentor tank into 500-2000L as seed, carries out ferment tank, still adopts collective media, and fermentation condition is with the seed ferment tank;
(4) preparation of solid powder: the fermented liquid after the fermentation culture is prepared into the solid powder of certain particle size through drying.
As further improvement of the present invention; In the said step (4), solid powder adopts vacuum drying method to make, and concrete operations are: the fermented liquid after the fermentation culture adds absorption carrier; Fermented liquid: the absorption carrier weight ratio is 1:2～5, carries out drying treatment after the absorption; According to drying temperature, ventilation situation with elaborate thickness, be 8～30 hours time of drying, must adsorb powder; Dried absorption powder carries out dilution process according to absorption carrier of the same type is added in the requirement of viable count, and the absorption powder: the absorption carrier weight ratio is 1:1～10, and viable bacteria content is 1 * 10
8～2 * 10
8Individual/g.
As further improvement of the present invention; In the said step (4); Solid powder adopts low temperature drying method to make, and concrete operations are: the fermented liquid after the fermentation culture adopts whizzer centrifugal 10～15min under 5000～8000rpm, collects the deposition thalline after centrifugal; Carry out low temperature (80 ℃) lyophilize processing after adding protective material, be prepared into lyophilized powder; Lyophilized powder after the lyophilize, carry out dilution process, lyophilized powder according to absorption carrier is added in the requirement of viable count: the absorption carrier weight ratio is 1:1～1000, and viable bacteria content is 1 * 10
8～2 * 10
8Individual/g.
As further improvement of the present invention, said absorption carrier is selected wilkinite, wheat bran, inferior powder or starch for use.
As further improvement of the present invention, said protective material is selected one or more in trehalose, sucrose, bovine serum albumin, the milk for use.
The application of microbial preparation of the present invention aspect control tilapia streptococcicosis, it is used for the tilapia cultivating pool, and consumption is 0.5～1.0Kg/ mu Mi Shuishen.
Among the present invention, all be to adopt collective media to cultivate under aphalangia is understood condition.In the time of need detecting product of the present invention, adopt the collective media that adds 2% weight percent agar, i.e. solid medium.
The action principle of each component is following among the present invention:
1, nocardia rubra: mainly be to purify the tilapia cultivation water, remove ammonia nitrogen and nitrite nitrogen, improve the immunizing power of tilapia.Laboratory result shows: nocardia rubra reaches 10 in the water body
5Individual/as during mL, to act on after 15 days, ammonia nitrogen, nitrite content have reduced by 85.51% and 54.38% in the water; Tilapia immunizing power significantly improves, and its SEAP (AKP), complement C3 vigor improve more than 50%.
2, coral Nocardia bacteria: mainly be the material of secretion antagonism tilapia streptococcicosis cause of disease, suppress pathogenic micro-organism.Laboratory result shows: mainly from its meta-bolites, the antibacterial vigor of antagonism is 8AU/ml to coral Nocardia bacteria to the antagonism of Streptococcus iniae.Preliminary research has been carried out in the physicochemical property of coral Nocardia bacteria antibacterial substance, separation etc., and research shows: antibacterial substance has thermostability preferably, and 100 ℃ keep active completely after handling 30min; To ph stability, the bacteriostatic activity in the pH value is the 4-11 scope changes little, and the pH value is that 11 o'clock bacteriostatic activities still keep 94.08%; Responsive to Proteinase K and trypsinase; To ultraviolet light stabilized; Good water solubility is slightly soluble in ETHYLE ACETATE, propyl carbinol, the sherwood oil, is insoluble to EC; Under uv absorption spectrum, has proteinic characteristic absorbance.Above-mentioned explanation antibacterial substance possibly be a kind of protein matter.The sedimentable matter that coral nocardial filtration sterilization supernatant is handled gained through 40% saturated ammonium sulphate has bacteriostatic activity, and the supernatant after the processing does not have bacteriostatic activity, and further specifying antibacterial substance is a kind of macro-molecular protein.
3, bacterial classification A (nocardia rubra) among the present invention and bacterial classification B (coral Nocardia bacteria); Proportioning ratio (bacterial cell dry weight ratio) is 1:0.5～2; Form the complex microorganism product; Effectively remove ammonia nitrogen, nitrite nitrogen, antagonism tilapia streptococcicosis cause of disease, the immunizing power of raising tilapia body.
Compared with present technology the present invention has the following advantages:
The present invention utilizes two kinds of harmless to aquaculture organisms, useful and harmless to natural aquatic attitude environment promise Ka Shi actinomycetes; After compound proportioning fermentation culture; Utilize its transformation, improve the water body environment quality, and utilize the meta-bolites antagonism of its secreted generation and suppress the cause of disease of tilapia streptococcicosis ammonia nitrogen and nitrous acid nitrogen; Improve the immunizing power of tilapia body, alleviate the generation and the consequent financial loss of tilapia streptococcicosis.
The microbial preparation of control tilapia streptococcicosis of the present invention is easy to use, and usage quantity is little, and the minimum amount of every meter depth of water of every mu of water surface is merely 0.5kg; Use the back effect stability; Can significantly reduce content of harmful such as ammonia nitrogen in the tilapia aquaculture water, nitrite nitrogen, reduce the pathogenic micro-organism quantity of tilapia streptococcicosis, increase the water body dissolved oxygen; Improve the immunizing power of tilapia body; Improve the life condition of tilapia, effectively prevent the generation and the sudden death of tilapia streptococcicosis, improve cultured output and the quality of tilapia.
Embodiment
Below in conjunction with specific embodiment the present invention is described further.
Embodiment 1
(1) bacterial classification A (nocardia rubra ATCC 11653) and bacterial classification B (coral Nocardia bacteria ACCC 40100) are mixed, with bacterial cell dry weight basis proportioning ratio, bacterial classification A: bacterial classification B is 1:1;
(2) substratum:
Obtain the collective media of big living weight: with inorganic nitrogen-sourced and organic carbon source as nutritive substance; The collective media prescription is: yam 200g/L, glucose 20g/L, Zulkovsky starch 25g/L, ammonium sulfate 5g/L, potassium hydrogenphosphate 0.85g/L; Concrete compound method is: earlier yam is cleaned peeling, take by weighing 200g again and be cut into small pieces, add poach and get final product to being poked by glass stick; With four layers of filtered through gauze; Add glucose and Zulkovsky starch in the filtrating, continue the heated and stirred mixing, add other composition again after the cooling slightly; And supply moisture to 1000 milliliter, transfer pH7.5;
3) strain fermentation preparation:
A) shaking table is cultivated: bacterial classification A and bacterial classification B access that the inclined-plane is preserved are equipped with in the 1000mL triangular flask of collective media; The collective media amount is 1/3 of a triangular flask capacity; The combined amount of bacterial classification A and bacterial classification B is by the proportioning configuration of mixed bacterium, and inoculum size is chosen completely with the standard inoculation ring and is as the criterion; Shaking table is cultivated 12h, 35 ℃ of control culture temperature, and pH 8.5, rotating speed 180rpm;
B) seed fermentation: inoculum size 6mL/L, 50L seed fermentation jar is advanced in inoculation from the triangular flask that shaking table is cultivated, and adopts the collective media fermentation culture; 35 ℃ of control leavening temperatures, pH8.5, seed fermentation jar mixing speed 180rpm; Fermentation time 72 hours treats that viable count is 5 * 10
9Individual/during the mL left and right sides; Tunning directly is prepared into preparation;
(4) preparation of solid powder: the fermented liquid after the fermentation culture is prepared into the solid powder of certain particle size through vacuum-drying; Concrete operations are: the fermented liquid after the fermentation culture adds wilkinite as absorption carrier; Fermented liquid: the wilkinite weight ratio is 1:2; Carry out drying treatment after the absorption, be 30 hours time of drying, must adsorb powder; Dried absorption powder carries out dilution process according to absorption carrier of the same type is added in the requirement of viable count, and the absorption powder: the wilkinite weight ratio is 1:2, and viable bacteria content is 1 * 10
8About individual/g.
The practical effect of this microbial preparation is following:
One tilapia cultivating pool uses in Maoming, Guangdong, 15 mu of areas, 1.5 meters of the depth of waters; Summer high temperature is the pond water quality overrich when (6～August), and water colour is bad, and ammonia nitrogen, nitrous acid nitrogen reach 2.20mg/L, 0.45mg/L respectively in the water quality; It is serious to exceed standard, and Streptococcus iniae reaches 5 * 10 in the water body
5Individual/mL, influence the growth of tilapia.A fine morning about 10 microbial preparations with the control tilapia streptococcicosis of above-mentioned prescription be applied in the cultivating pool, consumption is 0.5Kg/ mu Mi Shuishen.Take a turn for the better with back 3 days water colour, transfer oyster to by strong green, transparency increases; Ammonia nitrogen, nitrous acid nitrogen content are reduced to 0.50mg/L and 0.12mg/L in the water body; After 5 days, Streptococcus iniae quantity drops to 8 * 10
3Individual/mL, tilapia ingests normally.
Embodiment 2
(1) bacterial classification A (nocardia rubra ATCC 11653) and bacterial classification B (coral Nocardia bacteria ACCC 40100) are mixed, with bacterial cell dry weight basis proportioning ratio, bacterial classification A: bacterial classification B is 1:2;
(2) substratum:
Obtain the collective media of big living weight: with inorganic nitrogen-sourced and organic carbon source as nutritive substance; The collective media prescription is: yam 200g/L, glucose 20g/L, Zulkovsky starch 25g/L, ammonium sulfate 5g/L, potassium hydrogenphosphate 0.85g/L; Concrete compound method is: earlier yam is cleaned peeling, take by weighing 200g again and be cut into small pieces, add poach and get final product to being poked by glass stick; With four layers of filtered through gauze; Add glucose and Zulkovsky starch in the filtrating, continue the heated and stirred mixing, add other composition again after the cooling slightly; And supply moisture to 1000 milliliter, transfer pH7.5;
3) strain fermentation preparation:
A) shaking table is cultivated: bacterial classification A and bacterial classification B access that the inclined-plane is preserved are equipped with in the 1000mL triangular flask of collective media; The collective media amount is 1/3 of a triangular flask capacity; The combined amount of bacterial classification A and bacterial classification B is by the proportioning configuration of mixed bacterium, and inoculum size is chosen completely with the standard inoculation ring and is as the criterion; Shaking table is cultivated 15h, 32 ℃ of control culture temperature, and pH 8.0, rotating speed 155rpm;
B) seed fermentation: inoculum size 6mL/L, 50L seed fermentation jar is advanced in inoculation from the triangular flask that shaking table is cultivated, and adopts the collective media fermentation culture; 32 ℃ of control leavening temperatures, pH8.0, seed fermentation jar mixing speed 155rpm; Fermentation time 80 hours treats that viable count is 6 * 10
9Individual/during the mL left and right sides; Tunning directly is prepared into preparation;
(4) preparation of solid powder: the fermented liquid after the fermentation culture is prepared into the solid powder of certain particle size through frozen drying; Concrete operations are: the fermented liquid after the fermentation culture adopts whizzer centrifugal 15min under 6000rpm; Collect the deposition thalline after centrifugal; Add trehalose as protective material, carry out low temperature (80 ℃) lyophilize then and handle, be prepared into lyophilized powder; Lyophilized powder after the lyophilize, carry out dilution process, lyophilized powder according to time powder is added in the requirement of viable count: inferior powder weight ratio is 1:500, and viable bacteria content is 1.5 * 10
8About individual/g.
The practical effect of this microbial preparation is following:
Use 20 mu of areas, 1.5 meters of the depth of waters at Guangdong Jiangmen one tilapia cultivating pool; Summer high temperature is the pond water quality overrich when (6～August), and water colour is bad, and ammonia nitrogen, nitrous acid nitrogen reach 2.85mg/L, 0.52mg/L respectively in the water quality; It is serious to exceed standard, and Streptococcus iniae reaches 2 * 10 in the water body
6Individual/mL, influence the growth of tilapia, tilapia has the mortality ratio about 20%.A fine morning about 10 microbial solid pulvis with the control tilapia streptococcicosis of above-mentioned prescription be applied in the cultivating pool, consumption is 1.0Kg/ mu Mi Shuishen, logotype 3 days.Take a turn for the better with back 5 days water colour, transfer oyster to by strong green, transparency increases; Ammonia nitrogen, nitrous acid nitrogen content are reduced to 0.42mg/L and 0.10mg/L in the water body; After 5 days, Streptococcus iniae quantity drops to 3 * 10
4Individual/mL, the tilapia mortality ratio descends 60%, and tilapia ingests and recovers normal.
Embodiment 3
(1) bacterial classification A (nocardia rubra ATCC 11653) and bacterial classification B (coral Nocardia bacteria ACCC 40100) are mixed, with bacterial cell dry weight basis proportioning ratio, bacterial classification A: bacterial classification B is 2:1;
(2) substratum:
Obtain the collective media of big living weight: with inorganic nitrogen-sourced and organic carbon source as nutritive substance; The collective media prescription is: yam 200g/L, glucose 20g/L, Zulkovsky starch 25g/L, ammonium sulfate 5g/L, potassium hydrogenphosphate 0.85g/L; Concrete compound method is: earlier yam is cleaned peeling, take by weighing 200g again and be cut into small pieces, add poach and get final product to being poked by glass stick; With four layers of filtered through gauze; Add glucose and Zulkovsky starch in the filtrating, continue the heated and stirred mixing, add other composition again after the cooling slightly; And supply moisture to 1000 milliliter, transfer pH7.5;
(3) strain fermentation preparation:
A) shaking table is cultivated: bacterial classification A and bacterial classification B that the inclined-plane is preserved insert in the 1000mL triangular flask that collective media is housed (connecing 3 bottles altogether); The collective media amount is 1/3 of a triangular flask capacity; The combined amount of bacterial classification A and bacterial classification B is by the proportioning configuration of mixed bacterium, and inoculum size is chosen completely with the standard inoculation ring and is as the criterion; Shaking table is cultivated 18h, 30 ℃ of control culture temperature, and pH 7.5, rotating speed 130rpm;
B) seed fermentation: inoculum size 10mL/L, 100L seed fermentation jar is advanced in inoculation from the triangular flask that shaking table is cultivated, and adopts the collective media fermentation culture; 30 ℃ of control leavening temperatures, pH7.5, seed fermentation jar mixing speed 130rpm; Fermentation time 48 hours treats that viable count is 8 * 10
9Individual/during the mL left and right sides; Tunning further carries out ferment tank as seed;
C) ferment tank: the seed fermentation product is inoculated the fermentor tank into 2000L as seed, carries out ferment tank, still adopts collective media, and fermentation condition is with the seed ferment tank;
(4) preparation of solid powder: the fermented liquid after the fermentation culture is prepared into the solid powder of certain particle size through vacuum-drying; Concrete operations are: the fermented liquid after the fermentation culture adds wilkinite as absorption carrier; Fermented liquid: the wilkinite weight ratio is 1:5; Carry out drying treatment after the absorption, be 20 hours time of drying, must adsorb powder; Dried absorption powder carries out dilution process according to absorption carrier of the same type is added in the requirement of viable count, and the absorption powder: the wilkinite weight ratio is 1:5, and viable bacteria content is 2 * 10
8About individual/g.
The practical effect of this microbial preparation is following:
One tilapia cultivating pool uses in Hainan, 10 mu of areas, 1.5 meters of the depth of waters; Summer high temperature is the pond water quality overrich when (6～August), and water colour is bad, and ammonia nitrogen, nitrous acid nitrogen reach 3.18mg/L, 0.48mg/L respectively in the water quality; It is serious to exceed standard, and Streptococcus iniae reaches 1 * 10 in the water body
6Individual/mL, influence the growth of tilapia, tilapia has the mortality ratio about 10%.A fine morning about 10 microbial solid pulvis with the control tilapia streptococcicosis of above-mentioned prescription be applied in the cultivating pool, consumption is 0.8Kg/ mu Mi Shuishen.Take a turn for the better with back 2 days water colour, transfer oyster to by strong green, transparency increases; Ammonia nitrogen, nitrous acid nitrogen content are reduced to 0.62mg/L and 0.08mg/L in the water body; After 5 days, Streptococcus iniae quantity drops to 1 * 10
4Individual/mL, the tilapia mortality ratio descends 50%, and tilapia ingests and recovers normal.
In addition, in the foregoing description 1～3, after bacterial classification A and the bacterial classification B proportional mixing fermentation culture, obtain the composite bacterial cell.Under laboratory condition, when dried cell weight concentration is 2.0～3.0 g/L, be that the clearance of nitrite nitrogen of ammonia nitrogen and the 0.6mg/L of 5.0mg/L reaches 62.31%～85.51% and 21.07%～54.38% in 15 days to starting point concentration; To starting point concentration is 10
6The antagonistic rate of the Streptococcus iniae of individual/mL reaches 25.7%～65.3%; Tilapia mortality ratio to being caused by suis can reduce by 37.5%～48.2%.
Claims (8)
1. microbial preparation that is used to prevent and treat the tilapia streptococcicosis, it is characterized in that: the activeconstituents of said microbial preparation is nocardia rubra ATCC11653 and coral Nocardia bacteria ACCC40100.
2. preparation method who is used to prevent and treat the microbial preparation of tilapia streptococcicosis; It is characterized in that: form mixed bacterium by nocardia rubra ATCC11653 and coral Nocardia bacteria ACCC40100; Adopt nitrogenous source, carbon source and inorganic salt to form fermention medium and carry out fermentation culture, after fermentation culture is accomplished, be processed into solid powder; Make and have the ammonia nitrogen that reduces in the aquaculture water and the content of nitrous acid nitrogen; Antagonism tilapia streptococcicosis cause of disease, the immunizing power of raising tilapia body, the solid powder type microbial preparation of control tilapia streptococcicosis effect.
3. the preparation method who is used to prevent and treat the microbial preparation of tilapia streptococcicosis as claimed in claim 2 is characterized in that: specifically may further comprise the steps:
(1) microbial strains:
Bacterial classification A: nocardia rubra ATCC 11653;
Bacterial classification B: coral Nocardia bacteria ACCC 40100;
Mixed bacterium: bacterial classification A and bacterial classification B are mixed, and with bacterial cell dry weight basis proportioning ratio, bacterial classification A: bacterial classification B is 1:0.5～2;
(2) substratum:
Obtain the collective media of big living weight: with inorganic nitrogen-sourced and organic carbon source as nutritive substance; The collective media prescription is: yam 200g/L, glucose 20g/L, Zulkovsky starch 25g/L, ammonium sulfate 5g/L, potassium hydrogenphosphate 0.85g/L, pH7.5;
Detect solid medium that bacterial classification uses and be in above-mentioned collective media, add 2% weight percent agar; It all is to adopt collective media to cultivate that aphalangia is understood under the condition;
(3) strain fermentation preparation:
A) shaking table is cultivated: bacterial classification A and bacterial classification B access that the inclined-plane is preserved are equipped with in the triangular flask of collective media; The collective media amount is 1/3 of a triangular flask capacity; The combined amount of bacterial classification A and bacterial classification B is by the proportioning configuration of mixed bacterium, and inoculum size is chosen completely with the standard inoculation ring and is as the criterion; Shaking table is cultivated 12～18h, 30～35 ℃ of control culture temperature, and pH 7.5～8.5, rotating speed 130～180rpm;
B) seed fermentation: inoculum size 6-20mL/L; 50～200L seed fermentation jar is advanced in inoculation from the triangular flask that shaking table is cultivated, and adopts the collective media fermentation culture, 30～35 ℃ of control leavening temperatures; PH7.5～8.5; Seed fermentation jar mixing speed 130～180rpm, fermentation time 48～120 hours treats that viable count is 5 * 10
9～8 * 10
9Individual/during mL; Tunning directly is prepared into preparation or further carries out ferment tank as seed;
C) ferment tank: the seed fermentation product is inoculated the fermentor tank into 500-2000L as seed, carries out ferment tank, still adopts collective media, and fermentation condition is with the seed ferment tank;
(4) preparation of solid powder: the fermented liquid after the fermentation culture is prepared into the solid powder of certain particle size through drying.
4. the preparation method who is used to prevent and treat the microbial preparation of tilapia streptococcicosis as claimed in claim 3; It is characterized in that: in the said step (4); Solid powder adopts vacuum drying method to make; Concrete operations are: the fermented liquid after the fermentation culture adds absorption carrier, and fermented liquid: the absorption carrier weight ratio is 1:2～5, carries out drying treatment after the absorption; According to drying temperature, ventilation situation with elaborate thickness, be 8～30 hours time of drying, must adsorb powder; Dried absorption powder carries out dilution process according to absorption carrier of the same type is added in the requirement of viable count, and the absorption powder: the absorption carrier weight ratio is 1:1～10, and viable bacteria content is 1 * 10
8～2 * 10
8Individual/g.
5. the preparation method who is used to prevent and treat the microbial preparation of tilapia streptococcicosis as claimed in claim 3; It is characterized in that: in the said step (4), solid powder adopts low temperature drying method to make, and concrete operations are: the fermented liquid after the fermentation culture adopts whizzer centrifugal 10～15min under 5000～8000rpm; Collect the deposition thalline after centrifugal; Carry out the frozen drying processing after adding protective material, be prepared into lyophilized powder, said cryogenic temperature is-80 ℃; Lyophilized powder after the lyophilize, carry out dilution process, lyophilized powder according to absorption carrier is added in the requirement of viable count: the absorption carrier weight ratio is 1:1～1000, and viable bacteria content is 1 * 10
8～2 * 10
8Individual/g.
6. like claim 4 or the 5 described preparing methods that are used to prevent and treat the microbial preparation of tilapia streptococcicosis, it is characterized in that: said absorption carrier is selected wilkinite, wheat bran, inferior powder or starch for use.
7. the preparation method who is used to prevent and treat the microbial preparation of tilapia streptococcicosis as claimed in claim 5, it is characterized in that: said protective material is selected one or more in trehalose, sucrose, bovine serum albumin, the milk for use.
8. press the application of the described microbial preparation of claim 1 aspect control tilapia streptococcicosis for one kind, it is characterized in that: said microbial preparation is used for the tilapia cultivating pool, and consumption is 0.5～1.0Kg/ mu Mi Shuishen.
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| CN106578672A (en) * | 2016-11-30 | 2017-04-26 | 广西南宁市武鸣明山红农业科技开发有限公司 | High yield and high efficiency health culture feed of tilapia and preparation method thereof |
| CN105152362B (en) * | 2015-09-30 | 2017-05-24 | 江苏农林职业技术学院 | Preparation method for aquaculture water bottom environment modifier |
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| CN106721529A (en) * | 2016-11-30 | 2017-05-31 | 广西南宁市武鸣明山红农业科技开发有限公司 | A kind of albumen powder and preparation method and application |
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| CN114409756A (en) * | 2022-01-21 | 2022-04-29 | 中国科学院海洋研究所 | Application of paralichthys olivaceus complement component C3(PoC3) |
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| CN104099271A (en) * | 2014-07-08 | 2014-10-15 | 谢必峰 | Novel culture method of red Nocard's bacilli |
| CN104513803A (en) * | 2015-01-26 | 2015-04-15 | 鹭滨环保科技（上海）有限公司 | Nocardiacoeliaca and application thereof in domestic sewage treatment |
| CN105152362B (en) * | 2015-09-30 | 2017-05-24 | 江苏农林职业技术学院 | Preparation method for aquaculture water bottom environment modifier |
| CN106578672A (en) * | 2016-11-30 | 2017-04-26 | 广西南宁市武鸣明山红农业科技开发有限公司 | High yield and high efficiency health culture feed of tilapia and preparation method thereof |
| CN106721534A (en) * | 2016-11-30 | 2017-05-31 | 广西南宁市武鸣明山红农业科技开发有限公司 | A kind of feed for preventing and treating Tilapia mossambica Streptococcus iniae disease and preparation method thereof |
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| CN106721538A (en) * | 2016-11-30 | 2017-05-31 | 广西南宁市武鸣明山红农业科技开发有限公司 | A kind of Tilapia mossambica phagostimulant and preparation method and application |
| CN106721529A (en) * | 2016-11-30 | 2017-05-31 | 广西南宁市武鸣明山红农业科技开发有限公司 | A kind of albumen powder and preparation method and application |
| CN108496857A (en) * | 2018-04-19 | 2018-09-07 | 广州普麟生物制品有限公司 | A kind of closed zero-emission batch production method for breeding tilapia |
| CN114409756A (en) * | 2022-01-21 | 2022-04-29 | 中国科学院海洋研究所 | Application of paralichthys olivaceus complement component C3(PoC3) |
| CN114409756B (en) * | 2022-01-21 | 2023-06-09 | 中国科学院海洋研究所 | Application of flounder complement component C3 (PoC 3) |
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