CN109055266B - Biological agent capable of inhibiting growth of vibrio in shrimp pond and preparation method and application thereof - Google Patents
Biological agent capable of inhibiting growth of vibrio in shrimp pond and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a leaven of a biological preparation for inhibiting the growth of vibrio in a shrimp pond, which comprises bdellovibrio bacteriovorus, 5-15% of bdellovibrio stailinus, 20-40% of bacillus, 20-40% of lactic acid bacteria and 10-30% of rhodotorula benthicus, wherein the bacteria content of the bdellovibrio bacteriovorus accounts for 5-15% of the total bacteria number. The invention also provides a biological preparation for inhibiting the growth of vibrio in the shrimp pond and a preparation method thereof. The biological preparation for effectively inhibiting the growth of the vibrios in the shrimp pond is prepared by reasonably matching various probiotic fermentation raw materials and utilizing the mutual cooperation of different strains.
Description
Technical Field
The invention relates to the field of aquaculture water quality regulation, and particularly relates to a biological agent capable of inhibiting growth of vibrios in a shrimp pond, and a preparation method and application thereof.
Background
Prawn culture is an important prop industry in the aquaculture industry of China. However, in recent years, with the increase of the aquaculture yield, the aquaculture density is increased, the large-scale development of prawn aquaculture is realized, the aquaculture pollution is aggravated, the aquaculture environment quality is continuously worsened, the pathogenic organism species are increased, the propagation speed is accelerated, the aquaculture diseases are increasingly serious, diseases, particularly bacterial diseases, in prawn aquaculture are frequently generated and seriously influenced, huge economic losses are caused, and the development of the aquaculture industry in China is greatly limited. The problems of breeding diseases and breeding environment become one of bottleneck factors restricting the development of the mariculture industry, seriously troubles the development of the mariculture industry in China and restricts the healthy development of blue agriculture.
Vibriosis is the most common and most harmful bacterial venereal disease in mariculture, and is mainly caused by Vibrio (Vibrio), which belongs to the fields of prokaryotes, bacteroides, gram-facultative anaerobes, Vibrionaceae, (Vibrio, Aeromonas, Plesiomonas, Photobacterium and photobacterium) in Bergey's Manual of bacteria identification, which is universally adopted internationally (also most authoritative worldwide). 91 species have been named so far. Common pathogenic vibrios are Vibrio anguillarum (Vibrio anguillarum), Vibrio harveyi (v. harveyi), Vibrio alginolyticus (v. alginolyticus), Vibrio parahaemolyticus (v. parahaemolyticus), Vibrio vulnificus (v. vulnifficus), and the like. Research shows that almost all marine culture animals, such as prawns, portunus trituberculatus, grouper, red sea bream, large yellow croaker, paralichthys olivaceus, octopus (seriola quinqueradiata) and the like, suffer from vibriosis, can cause blindness, red body disease, gill rot disease, death disease (EMS) and the like of prawns, and can also cause complications and secondary symptoms of various diseases and the like. For example, the vibrio parahaemolyticus is a marine bacterium and is widely distributed in seawater and mariculture products, the detection rate of the vibrio parahaemolyticus in the seawater along the coast in east China is 47.5-66.5%, the average bacteria carrying rate of fish and shrimp in the seawater is 45.6-48.7%, and the bacteria carrying rate in summer can reach more than 90%. The hemolytic toxin has direct hemolytic property, and can cause hemolysis of various cells of prawn, dissolve prawn blood, and kill cells. The heat-resistant direct hemolytic toxin can cause prawn digestive tract tissue injury, ileum erosion, gastric mucositis, even liver, pancreas and digestive dysfunction. It is also believed that vibrio parahaemolyticus produces urease, so that intestinal fluid of prawns accumulates to produce intestinal dropsy, and the phenomenon of white feces may occur in severe cases. The extracellular product of vibrio parahaemolyticus can obviously reduce the immunity of prawns and has slow growth speed, so that other various diseases are easily caused.
At present, the main means for controlling the bacterial diseases of aquaculture animals is to adopt antibacterial drugs for treatment, and a large number of antibiotic drugs are applied to each stage of the culture process. Due to incomplete understanding on aspects of etiology, pharmacology and the like, the phenomena of abuse and indiscriminate use of drugs such as mixed use of a plurality of high-dose drugs exist in the culture process, so that serious problems of drug resistance, drug resistance and antibiotic residue are brought, and the social attention is attracted. Currently, the prawn culture production still mainly adopts a pond culture mode, including an intertidal zone comprehensive ecological culture mode, a common prawn pond semi-intensive culture mode, a mulching film laying high-level pond intensive culture mode and the like. After more than ten years of development, the culture effect is summarized, and the disadvantages of difficult water quality regulation, serious eutrophication, low unit water body culture yield and the like still exist, and particularly, the development space is greatly restricted due to the influence of sudden factors such as temperature and season limitation, storm tide invasion and the like. With the continuous improvement of the culture scale in recent years, the water resource is utilized without restriction, and the pond culture mode faces the pressure of water environment pollution, year-by-year increase of diseases, food safety requirements, coastal industrial land extrusion, national advocated energy conservation and emission reduction and the like.
Biological (mainly microbial) control has been regarded as important because of various disadvantages of the physical and chemical methods commonly used to control water quality. The microecological technology regulates and controls water quality, controls pathogeny, enhances immunity, conditions nutrition, reduces or does not use chemical drugs, has the advantages of low cost, high yield, no pollution and the like, becomes one of the most main development directions of the current prawn culture technology, and is generally adopted and has remarkable benefits in prawn culture. The use of the microorganism can improve the immunity of the prawn culture organism, accelerate the material circulation metabolism, purify the water body and improve the health of the culture water body. The principle of treating the culture wastewater by the microorganisms is as follows: mineralizing soluble protein, carbohydrate and fatty acid into inorganic salts by heterotrophic bacteria, and removing NH by nitrifying bacteria4+、NO2Conversion to low-toxic NO3-，Then uses denitrifying bacteria to remove NO3Denitrogenation to N2. At present, technologies such as biological contact oxidation (2008, etc.), biological rotating disc (Brazil, 2006), biological fluidized bed (Bortone et al,2003) and the like are mostly applied to the biochemical treatment of the aquaculture wastewater, most of biological organisms in the technologies are derived from oligotrophic florae in natural or artificial environments and cannot adapt to the trophic environment of the aquaculture wastewater, and the unit volume treatment load of a microorganism treatment module is actually not high, so that the application of the traditional microorganism treatment technology is greatly influenced.
At present, single or compound microorganism strains are generally adopted at home and abroad to control the water quality, but the microorganisms are greatly influenced by the external environment, the adverse environment impact resistance is poor, and once the system is damaged, the recovery is difficult, so the treatment effect is unstable. There is therefore a need for a scientific proportioning of the species and composition of the microorganisms to improve their efficiency. Wuweiwei et al (2001) identified that if 3 strains were treated as 2: 1: 2, the composite bacterial strain is combined and immobilized, and the conversion effect on ammonia nitrogen and nitrite nitrogen in the aquaculture water body is better.
Therefore, the invention aims to develop a biological preparation for inhibiting the growth of vibrio in a shrimp pond, and the biological preparation mainly comprises bdellovibrio, bacillus, rhodotorula marinus and lactic acid bacteria.
Bdellovibrio bacteriovorus belongs to the family of bdelloviridae, is widely distributed in nature, and is widely distributed in soil, river water, nearby ocean water areas and sewage of sewers, the number of bacteria in the sewage is 10/ml, the number of bacteria in the sewage can be tens of thousands/ml, the number of bacteria in the soil is 10/g, and the bdellovibrio bacteriovorus has all morphological characteristics of the bacteria: single cells, curved or comma-shaped, sometimes in the form of a spiral. The size is 0.3-0.6X 0.8-1.2 microns, or 1/3-1/4 microns in length of bacillus only. There are few more than one terminal flagella, and some have a bundle of cilia at the other end. The flagella of the bdellovibrio aquaticus also have a tunica vaginalis, which is an extension of the cell wall and surrounds flagella filaments, and thus are 3-4 times thicker than the flagella of other bacteria, which is a remarkable feature. Bdellovibrio is active in movement and gram-negative in staining. The protein content of the cells is high, and the protein accounts for 60-70% of the dry weight. DNA content 5%, GC percentage 42-51. It has been reported that the DNA synthesis of Bdellovibrio is carried out after complete lysis of the DNA of the host cell, where 80% of the DNA is incorporated into the parasite. The bacteria do not depend on external sources. Therefore, it can grow even in an ecosystem such as an ocean, a river, a lake, etc., where nutrients are few.
The Bacillus (Bacillus) is different from vegetative cells in structure and chemical composition, has the characteristic of strong resistance, has strong resistance to high temperature, ultraviolet rays, drying and the like, and can resist the extreme environments of oceans such as oxidation, extrusion, high temperature and the like. Meanwhile, the bacillus can generate a plurality of active enzymes, and has the characteristic of improving the growth performance of aquaculture animals. Sugita et al found that Bacillus can provide some nutrients to promote the development of intestinal tract of Paralichthys olivaceus and the expression of digestive enzymes, thereby promoting the absorption of nutrients. The xuhaiyan research shows that the bacillus subtilis generates volatile fatty acids such as acetic acid, propionic acid, butyric acid and the like in the growth and reproduction process, reduces the pH value of intestinal tracts, inhibits the growth of harmful bacteria, provides an acidic condition for the growth of beneficial bacteria, promotes the growth of animals and improves the production performance. The results of researches on the simple biofilter and the microbial preparation on the river sensitivity and the like for the treatment of the seedling water body of the macrobrachium rosenbergii show that the cured biofilter can effectively improve the water quality of the seedling water, reduce the organic load of the water body, reduce the content of ammonia nitrogen and nitrite nitrogen, has an obvious growth promoting effect on the macrobrachium rosenbergii larvae, can reduce the content of ammonia nitrogen in the water body to a certain extent and promote the growth of the macrobrachium rosenbergii larvae.
Rhodotorula benthica (Rhodotorula benthica) is a unicellular eukaryotic organism separated from ocean bottom mud, contains various physiologically active substances such as abundant proteins, saccharides, vitamins, digestive enzymes, growth factors and the like, and is a good starter for economic animals such as fish, shrimp, shellfish, sea cucumber and the like. Practice shows that the rhodotorula benthica can obviously improve the survival rate of aquatic animal seedlings, improve the feed effect and feed reward, enhance the immune function of animals and reduce the using amount of antibiotics, and is an excellent additive for ecological breeding. The aquatic product fed with the rhodotorula benthami feed has the characteristics of strong disease resistance, high survival rate, bright skin and muscle color, good taste and the like.
Lactic acid bacteria (Lactcacic bacteria) are a general term for a group of gram-positive bacteria that are not completely identical in morphology, metabolic performance and physiological characteristics. The mechanism of action of lactobacillus for promoting animal growth and immunity can be divided into two types: the first is direct action, namely the adhesion or degradation of the thalli and active metabolites thereof to harmful substances; the other is indirect effect, namely the lactobacillus plays a role of indirect benefit in the modes of regulating the immunity activity of the organism and the partial enzyme activity of the organism and the like by regulating the intestinal flora and the metabolic enzyme activity thereof. The lactobacillus can produce B vitamins including folic acid, biotin, vitamin B, vitamin K and the like, and can directly provide necessary nutrient substances for a host to promote the growth of animals. In addition, the acidic metabolite of the lactobacillus can enhance intestinal peristalsis and is beneficial to digestion and absorption of nutrients. Can generate antibacterial substances such as organic acid, hydrogen peroxide and the like in the growth and metabolism process of the lactic acid bacteria, and achieves the purpose of inhibiting the growth of pathogenic bacteria and exogenous pathogenic bacteria. Lactic acid bacteria prevent the attachment, colonization and invasion of intestinal cells by pathogenic bacteria on the surface of intestinal epithelial cells, a mechanism known as "adhesion resistance".
Disclosure of Invention
In view of the above, the invention provides a biological preparation capable of inhibiting growth of vibrios in a shrimp pond, and a preparation method and application thereof.
In order to inhibit the growth of vibrios in the shrimp pond, the invention provides a leavening agent of a biological preparation for inhibiting the growth of the vibrios in the shrimp pond, which comprises a compound bacterium A and a compound bacterium B;
wherein the compound bacteria A comprise one or more of Bdellovibrio bacteriovorus and Bdellovibrio stardani; the compound bacteria B comprises one or more of bacillus, lactic acid bacteria and rhodotorula benthica.
In an embodiment of the invention, when the leavening agent of the biological agent for inhibiting the growth of the vibrio in the shrimp pond comprises bdellovibrio bacteriovorus, bdellovibrio staphyli, bacillus, lactobacillus and rhodotorula benthica, the content of the bdellovibrio bacteriovorus accounts for 5-15% of the total bacteria number; the content of the bdellovibrio starling accounts for 5 to 15 percent of the total bacteria number; the content of the bacillus accounts for 20 to 40 percent of the total bacteria number; the content of the lactic acid bacteria accounts for 20 to 40 percent of the total bacteria number; the content of the rhodotorula benthica accounts for 10 to 30 percent of the total bacteria number.
In a preferred embodiment of the present invention, when the leavening agent of the biological agent for inhibiting the growth of vibrio in the shrimp pond comprises bdellovibrio bacteriovorus, bdellovibrio staphylicus, bacillus, lactobacillus and rhodotorula benthica, the content of bdellovibrio bacteriovorus is 10% of the total bacteria number; the content of the bdellovibrio starling accounts for 10 percent of the total bacteria number; the content of the bacillus accounts for 30 percent of the total bacteria number; the content of the lactic acid bacteria accounts for 30 percent of the total bacteria number; the content of the rhodotorula benthica accounts for 20 percent of the total bacteria number.
It can be understood that, according to the strain formula provided by the present invention, a person skilled in the art can add the strain into a corresponding culture medium according to a conventional addition amount (the mass percentage of the strain is 1-2%) to prepare a bacterial liquid or a bacterial powder, and in a specific commercial application, the leavening agent of the biological agent having the function of inhibiting the growth of vibrio in a shrimp pond, which is provided by the present invention, can be the bacterial liquid or the bacterial powder.
In an embodiment of the invention, the invention provides a leavening agent with a biological agent for inhibiting the growth of vibrio in a shrimp pond, and the leavening agent comprises the leavening agent with the biological agent for inhibiting the growth of the vibrio in the shrimp pond provided by the first aspect of the invention, and further comprises a liquid culture medium, a solid culture medium or a powder culture medium.
In a preferred embodiment of the invention, the production medium of the compound bacteria A comprises the following components in parts by weight: 1000 parts of clean tap water, 100 parts of corn flour, 25 parts of soybean meal, 0.03 part of manganese sulfate, 1 part of magnesium sulfate, 1.5 parts of sodium dihydrogen phosphate and disodium hydrogen phosphate3 parts of a mixture; the optimal fermentation process conditions are that the culture temperature is 28 ℃, the initial pH7.0 and the ventilation volume is 2.0m3The pressure in the tank is 0.03MPa, and the fermentation period is 36 h. The bacterial concentration reaches 8.0 multiplied by 1010cfu/mL。
In a preferred embodiment of the present invention, the production medium of the compound bacteria B comprises the following components in parts by weight: 1000 parts of clean tap water, 12 parts of molasses, 5 parts of yeast extract, 1 part of peptone, 4 parts of glucose, 3 parts of corn steep liquor, 5 parts of sodium acetate, 5 parts of sodium chloride, 2.5 parts of dipotassium hydrogen phosphate, 2.5 parts of potassium dihydrogen phosphate, 0.5 part of magnesium sulfate and 0.25 part of manganese sulfate.
The invention provides a biological agent for inhibiting growth of vibrio in a shrimp pond in a second aspect, which is mainly prepared by fermenting raw materials after activating the leavening agent provided by the first aspect of the invention, wherein the raw materials comprise the following components in parts by weight: 1 and bran.
In an embodiment of the present invention, the biological agent includes a compound bacterium a and a compound bacterium B;
wherein the compound bacteria A comprise one or more of Bdellovibrio bacteriovorus and Bdellovibrio stardani; the compound bacteria B comprises one or more of bacillus, lactic acid bacteria and rhodotorula benthica.
In an embodiment of the invention, when the leavening agent of the biological agent for inhibiting the growth of the vibrio in the shrimp pond comprises bdellovibrio bacteriovorus, bdellovibrio staphyli, bacillus, lactobacillus and rhodotorula benthica, the content of the bdellovibrio bacteriovorus accounts for 5-15% of the total bacteria number; the content of the bdellovibrio starling accounts for 5 to 15 percent of the total bacteria number; the content of the bacillus accounts for 20 to 40 percent of the total bacteria number; the content of the lactic acid bacteria accounts for 20 to 40 percent of the total bacteria number; the content of the rhodotorula benthica accounts for 10 to 30 percent of the total bacteria number.
In a preferred embodiment of the invention, when the leavening agent of the biological agent for inhibiting the growth of the vibrio in the shrimp pond comprises bdellovibrio bacteriovorus, bdellovibrio staphyli, bacillus, lactobacillus and rhodotorula benthica, the content of the bdellovibrio bacteriovorus accounts for 10 percent of the total bacteria number; the content of the bdellovibrio starling accounts for 10 percent of the total bacteria number; the content of the bacillus accounts for 30 percent of the total bacteria number; the content of the lactic acid bacteria accounts for 30 percent of the total bacteria number; the content of the rhodotorula benthica accounts for 20 percent of the total bacteria number.
In an embodiment of the present invention, the activation medium for activating the fermentation agent includes the following components in parts by weight: 1000 parts of aquaculture pond water, 20 parts of brown sugar, 0.5 part of ammonium sulfate, 0.05 part of magnesium sulfate heptahydrate, 0.5 part of dipotassium hydrogen phosphate, 14.29 parts of calcium carbonate, 2 parts of sodium chloride, 1 part of brine, 0.02 part of calcium chloride monohydrate, 0.05 part of ferrous sulfate heptahydrate and 0.05 part of copper sulfate pentahydrate.
The third aspect of the invention provides a preparation method of a biological agent for inhibiting the growth of vibrio in a shrimp pond, which comprises the following steps:
(1) pretreatment of fermentation raw materials: mixing the following components in parts by weight: 1, crushing the soybean meal and the bran to obtain a first mixture;
(2) activating a leavening agent: respectively adding the compound bacteria A and the compound bacteria B into an activation culture medium, and culturing and activating to obtain fermentation liquor A and fermentation liquor B;
(3) fermentation: and (3) adding the fermentation liquor A prepared in the step (2) into the first mixture obtained in the step (1) to obtain a second mixture, carrying out aerobic fermentation, adding the fermentation liquor B to obtain a third mixture, and carrying out closed fermentation.
(4) And (4) adding the low-molecular fulvic acid into the third mixture obtained in the step (3), and uniformly stirring to obtain the biological preparation for inhibiting the growth of vibrio in the shrimp pond.
In an embodiment of the present invention, in the step (2), the activation medium includes the following components in parts by weight: 1000 parts of aquaculture pond water, 20 parts of brown sugar, 0.5 part of ammonium sulfate, 0.05 part of magnesium sulfate heptahydrate, 0.5 part of dipotassium hydrogen phosphate, 14.29 parts of calcium carbonate, 2 parts of sodium chloride, 1 part of brine, 0.02 part of calcium chloride monohydrate, 0.05 part of ferrous sulfate heptahydrate and 0.05 part of copper sulfate pentahydrate.
In one embodiment of the present invention, in the step (2), the activation culture temperature of the compound bacteria a is 27-30 ℃, the culture time is 24-48 hours, and the activation is performed by using an aerobic activation method.
In an embodiment of the invention, in the step (2), the activation culture temperature of the compound bacteria B is 27-30 ℃, the culture time is 24-48 hours, and the activation is performed by adopting a sealed activation mode.
In an embodiment of the present invention, in the step (3), a weight ratio of the first mixture to the fermentation liquid a is 1: 2.
in one embodiment of the present invention, in the step (3), the second mixture is aerobically fermented for 2 to 3 days.
In an embodiment of the present invention, in the step (3), the time for the closed fermentation of the third mixture is 5 to 7 days.
In an embodiment of the invention, in the step (4), the addition amount of the low molecular fulvic acid is 1-2% of the third mixture.
In a fourth aspect, the invention provides the application of the leavening agent with the biological preparation for inhibiting the growth of vibrio in the shrimp pond according to the first aspect of the invention, the biological preparation for inhibiting the growth of vibrio in the shrimp pond according to the second aspect of the invention or the preparation method of the biological preparation for inhibiting the growth of vibrio in the shrimp pond according to the third aspect of the invention in the field of aquaculture water quality control.
The invention has the advantages that:
the method has the advantages that various probiotics are reasonably matched, various fermentation raw materials are used, some bioactive factors are generated through fermentation and can be used as effective carbon sources in the environment, the probiotics in the environment are directionally cultured, the synergistic effect among used fermentation strains can be fully embodied, ammonia nitrogen harm is efficiently and safely reduced, the water safety guarantee is improved, various physical and chemical indexes of the water are improved, and the water quality and the bottom quality are improved.
And secondly, a large amount of active substances and a large amount of beneficial live bacteria, such as rich and easily absorbed biological organic matters, balanced amino acids, vitamins, trace elements and the like are introduced, various nutrients are complete, the proportion is reasonable, the content of the active organic matters in the culture water body can be obviously increased after the compound feed is applied to prawn culture, good organic debris is provided for the prawns, and the immunity of the prawn body is improved.
And the integral process is simple, the requirement on equipment is low, the operability is strong, and the method is extremely easy to popularize and use in large-scale farms or aquaculture cooperative.
Detailed Description
While the following is a description of the preferred embodiments of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
The preferred embodiments of the present invention will be described in detail below. The experimental methods in the examples, in which specific conditions are not specified, are generally carried out under conventional conditions. In the examples of the present invention, unless otherwise specified, reagents and consumables used therein are commercially available.
Example 1
1. Preparation of leavening agent
1) Preparation of activation Medium
The activating culture medium used for activating the leavening agent comprises the following components in parts by weight: 1000 parts of aquaculture pond water, 20 parts of brown sugar, 0.5 part of ammonium sulfate, 0.05 part of magnesium sulfate heptahydrate, 0.5 part of dipotassium hydrogen phosphate, 14.29 parts of calcium carbonate, 2 parts of sodium chloride, 1 part of brine, 0.02 part of calcium chloride monohydrate, 0.05 part of ferrous sulfate heptahydrate and 0.05 part of copper sulfate pentahydrate.
2) Activating compound bacteria A:
adding the activated culture medium prepared in the step 1) and adopting an aerobic activation mode, and culturing for 36 hours at the temperature of 30 ℃ for activation to obtain fermentation liquor A.
3) Activation of composite bacteria B:
adding the activated culture medium prepared in the step 1) and adopting a sealing activation mode, and culturing for 36 hours at the temperature of 30 ℃ for activation to obtain fermentation liquor B.
2. Preparation of biological agent for inhibiting growth of vibrio in shrimp pond
1) Pretreatment of fermentation feedstock
Crushing raw materials (50% of each of soybean meal and bran) to be fermented to obtain a first mixture;
2) fermentation of
Adding 2 times of fermentation liquor A into the first mixture obtained in the step 1), uniformly stirring to obtain a second mixture, carrying out aerobic fermentation for 3 days at 30 ℃, then adding fermentation liquor B with the same amount as the fermentation liquor to obtain a third mixture, and carrying out closed fermentation for 7 days.
3) Adding 1.5% of low molecular fulvic acid, stirring and mixing uniformly to obtain concentrated tea color paste aqueous organic liquid, adding fermentation precipitate, stirring to obtain suspension, and having fermentation fragrance, namely the biological preparation for inhibiting the growth of vibrio in the shrimp pond. The obtained biological preparation is diluted 10 times with pond water, and sprayed into the whole pond.
Example 2
1. Preparation of leavening agent
1) Preparation of activation Medium
The activating culture medium used for activating the leavening agent comprises the following components in parts by weight: 1000 parts of aquaculture pond water, 20 parts of brown sugar, 0.5 part of ammonium sulfate, 0.05 part of magnesium sulfate heptahydrate, 0.5 part of dipotassium hydrogen phosphate, 14.29 parts of calcium carbonate, 2 parts of sodium chloride, 1 part of brine, 0.02 part of calcium chloride monohydrate, 0.05 part of ferrous sulfate heptahydrate and 0.05 part of copper sulfate pentahydrate.
2) Activating compound bacteria A:
adding the activated culture medium prepared in the step 1) and adopting an aerobic activation mode, and culturing for 48 hours at 27 ℃ for activation to obtain fermentation liquor A.
3) Activation of composite bacteria B: adding the activated culture medium prepared in the step 1) and adopting a sealing activation mode, and culturing for 48 hours at 27 ℃ for activation to obtain fermentation liquor B.
2. Preparation of biological agent for inhibiting growth of vibrio in shrimp pond
1) Pretreatment of fermentation feedstock
Crushing raw materials (50% of each of soybean meal and bran) to be fermented to obtain a first mixture;
2) fermentation of
Adding 2 times of fermentation liquor A into the first mixture obtained in the step 1), uniformly stirring to obtain a second mixture, carrying out aerobic fermentation for 3 days at 30 ℃, then adding fermentation liquor B with the same amount as the fermentation liquor to obtain a third mixture, and carrying out closed fermentation for 7 days.
3) Adding 2% of low molecular fulvic acid, stirring and mixing uniformly to obtain concentrated tea color paste aqueous organic liquid, adding fermentation precipitate, stirring to obtain suspension, and obtaining the biological preparation with fermentation fragrance for inhibiting the growth of vibrio in the shrimp pond. The obtained biological preparation is diluted 15 times with pond water, and sprinkled in the whole pond.
Example 3
1. Preparation of leavening agent
1) Preparation of activation Medium
The activating culture medium used for activating the leavening agent comprises the following components in parts by weight: 1000 parts of aquaculture pond water, 20 parts of brown sugar, 0.5 part of ammonium sulfate, 0.05 part of magnesium sulfate heptahydrate, 0.5 part of dipotassium hydrogen phosphate, 14.29 parts of calcium carbonate, 2 parts of sodium chloride, 1 part of brine, 0.02 part of calcium chloride monohydrate, 0.05 part of ferrous sulfate heptahydrate and 0.05 part of copper sulfate pentahydrate.
2) Activating compound bacteria A:
adding the activated culture medium prepared in the step 1) and adopting an aerobic activation mode, and culturing for 24 hours at the temperature of 30 ℃ for activation to obtain fermentation liquor A.
3) Activation of composite bacteria B:
adding the activated culture medium prepared in the step 1) and adopting a sealing activation mode, and culturing for 24 hours at the temperature of 30 ℃ for activation to obtain fermentation liquor B.
2. Preparation of biological agent for inhibiting growth of vibrio in shrimp pond
1) Pretreatment of fermentation feedstock
Crushing raw materials (50% of each of soybean meal and bran) to be fermented to obtain a first mixture;
2) fermentation of
Adding 4 times of fermentation liquor A by mass into the first mixture obtained in the step 1), uniformly stirring to obtain a second mixture, carrying out aerobic fermentation for 2 days at 30 ℃, then adding fermentation liquor B with the same amount as the fermentation liquor to obtain a third mixture, and carrying out closed fermentation for 5 days.
3) Adding 1% of low molecular fulvic acid, stirring and uniformly mixing to obtain concentrated tea color paste aqueous organic liquid, adding fermentation precipitate, stirring to obtain suspension, and obtaining the biological preparation with fermentation fragrance for inhibiting the growth of vibrio in the shrimp pond. The obtained biological preparation is diluted 20 times with pond water, and sprinkled in the whole pond.
Example 4
1. Biological agent stability experiment with inhibition of growth of vibrio in shrimp pond:
after the biological preparation is stored for 0 month, 2 months and 4 months in a dark place at room temperature, sampling and activating to detect the content of various bacteria in the microbial inoculum, wherein the unit of the content is (CFU/mL), and the results are as follows:
application examples
Application test of biological agent provided by the invention in prawn culture
1. Test products: the total viable count of the biological preparation of example 1 is more than 10 hundred million/ml.
2. Testing the prawns: the seedlings are thrown to the raised prawns for use in the whole process. Set 3 experimental pools and 3 control pools.
3. Test field: a certain prawn farm in Yangdong of Guangdong province.
4. The test method comprises the following steps: in daily feeding, the biological preparation of the embodiment 1 is uniformly sprinkled in a pool. The feed is used once every five days, the amount of the culture water used in each mu is 1-2L, and the feed is used in double times when the water quality is deteriorated. While the control group used no biological agent.
5. And (3) test results:
the using effect of the biological agent provided by the embodiment 1 is mainly reflected in the aspects of improving water quality, controlling the number of aquatic vibrios, preventing and treating prawn diseases and the like, and specifically comprises the following steps:
1) the water quality is improved, when the water quality is aged, the compound microbial inoculum is splashed, the water color is clear, fresh, tender and alive on the next day, and during the use period, the water color is good, the pH value is stable, the algae grows vigorously, and the algae cannot be poured;
2) the biological preparation provided by the embodiment 1 is used regularly, the vibrio in the water body is maintained at a lower level, and a disinfectant is not used basically in the culture process;
3) in the whole breeding process, the shrimps in 3 experimental ponds using the biological agent provided by the embodiment 1 are basically free from diseases, the shrimps in 3 control ponds are frequently damaged, and more disinfectants are used, so that the shrimps die in a large scale due to serious diseases and are finally discharged. And when the prawns are finally harvested, the yield of the experimental pond is increased by about 1000-1500 kg/mu compared with that of the control pond.
Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the true spirit and scope of the present invention.
Claims (8)
1. The biological preparation capable of inhibiting growth of vibrios in shrimp ponds is characterized by being prepared by fermenting raw materials after activating a leavening agent, wherein the raw materials comprise the following components in parts by weight: 1, the leavening agent comprises a composite bacterium A and a composite bacterium B, the composite bacterium A comprises bdellovibrio bacteriovorus and bdellovibrio stavorus, the composite bacterium B comprises bacillus, lactic acid bacteria and rhodotorula benthica, the content of the bdellovibrio bacteriovorus accounts for 5-15% of the total bacterial number, the content of the bdellovibrio stavorus accounts for 5-15% of the total bacterial number, the content of the bacillus accounts for 20-40% of the total bacterial number, the content of the lactic acid bacteria accounts for 20-40% of the total bacterial number, and the content of the rhodotorula benthica accounts for 10-30% of the total bacterial number.
2. The biological agent according to claim 1, wherein the activating medium for activating the fermentation agent comprises the following components in parts by weight: 800 portions of aquaculture pond water, 10 to 30 portions of brown sugar, 0.1 to 1 portion of ammonium sulfate, 0.01 to 0.1 portion of magnesium sulfate heptahydrate, 0.2 to 0.8 portion of dipotassium phosphate, 10 to 20 portions of calcium carbonate, 1 to 3 portions of sodium chloride, 0.5 to 2 portions of brine, 0.01 to 0.05 portion of calcium chloride monohydrate, 0.01 to 0.1 portion of ferrous sulfate heptahydrate and 0.01 to 0.1 portion of copper sulfate pentahydrate.
3. A preparation method of a biological agent for inhibiting growth of vibrio in a shrimp pond is characterized by comprising the following steps:
(1) pretreatment of fermentation raw materials: mixing the components in a weight ratio of 1: 1, crushing the soybean meal and the bran to obtain a first mixture;
(2) activating a leavening agent: respectively adding an activation culture medium to the compound bacteria A and the compound bacteria B as described in claim 1, and culturing and activating to obtain fermentation liquor A and fermentation liquor B;
(3) fermentation: adding the fermentation liquor A prepared in the step (2) into the first mixture obtained in the step (1) to obtain a second mixture, carrying out aerobic fermentation, adding the fermentation liquor B to obtain a third mixture, and carrying out closed fermentation;
(4) and (4) adding the low-molecular fulvic acid into the third mixture obtained in the step (3), and uniformly stirring to obtain the biological preparation for inhibiting the growth of vibrio in the shrimp pond.
4. The method according to claim 3, wherein the activation culture temperature of the complex bacteria A in the step (2) is 27 to 30 ℃, the culture time is 24 to 48 hours, and the activation is carried out by aerobic activation.
5. The preparation method according to claim 3, wherein in the step (2), the activation culture temperature of the compound bacteria B is 27-30 ℃, the culture time is 24-48 hours, and the activation is carried out in a sealed activation mode.
6. The preparation method according to claim 3, wherein in the step (3), the weight ratio of the first mixture, the fermentation liquid A and the fermentation liquid B is 1: 2: 2.
7. the method of claim 3, wherein in step (3), the second mixture is fermented aerobically for 2-3 days, and the third mixture is fermented closed for 5-7 days.
8. The application of the biological preparation for inhibiting the growth of vibrio in a shrimp pond according to claim 1 or the preparation method of the biological preparation for inhibiting the growth of vibrio in a shrimp pond according to claim 3 in the field of aquaculture water quality regulation.
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