KR101738591B1 - Microorganism agent for crustacea aquaculture and a method for improving productivity of crustacea aquaculture using the same - Google Patents

Abstract

The present invention can be achieved by increasing microorganisms beneficial to crustaceans and decreasing harmful microorganisms by administering the microbial agents comprising at least one of the bio-flak 1 and bio-flak 2 to aquaculture water in a crustacean farm. The present invention improves the quality of aquaculture water and improves the immunity of crustaceans through this right ignition, thereby reducing the mortality of crustaceans caused by infections of white spot syndrome virus and other harmful bacteria. Therefore, according to the method according to the present invention, it is possible to increase the productivity of crustaceans by enabling the annual production of crustacean in Korea.

Description

TECHNICAL FIELD The present invention relates to a microorganism for crustacean production, and a method for enhancing the productivity of crustacea using the microorganism,

The present disclosure relates to a microbial agent for crustacean production for enhancing the productivity of crustaceans and a method for enhancing the productivity of crustaceans using the same.

Shrimp are sensitive to water temperature and are domesticated domestically from May to late September. The shrimp indoor style can be 2-3 shrimp per year, but there is a disadvantage that installation and management cost for house and water temperature maintenance facilities are large, and outdoor style is mainly used. However, shrimp outdoor farming is inexpensive, but can only be farmed once between May and September due to water temperature changes, vulnerability to viral infections, and illness. If you lose one of these opportunities, There is a problem that the form can not be done at all.

Meanwhile, among the viruses that can affect shrimp, white spot syndrome virus is one of the most deadly viruses in shrimp. The white spot syndrome virus (WSSV) has a tail-like attachment on the distal end of the viaon and has a rod-shaped capsid and envelope similar to ovoid bacillus , And whispovirus. White Spot Syndrome Virus (WSSD) is expressed mainly in shrimp, lobster, crabs and other crustaceans. Especially, when white spot syndrome virus is infected, white spot syndrome virus (WSSD) White patches appear in appendage and cuticle, and hepatopancreas become red. Depending on the intensity of the infection, 100 ~ 80% of the dead within 3 ~ 10 days, 20 days is a deadly disease all dead shrimp. In particular, Fenneropenaeus chinensis is very vulnerable to the above-mentioned White Spot Syndrome Disease (WSSD), and production in the domestic market has been greatly reduced.

The crustacean aquaculture industry, which includes shrimp for the past several decades, is one of the fastest growing food production industries in the world as well as in the world, increasing by 16.8% per year. On the other hand, shrimp losses from shrimp disease have been reported to reach $ 3 billion worldwide. Therefore, in order to improve the productivity of shrimp, it is urgent to solve the problem of infection caused by the white spot syndrome virus as described above.

Korean Patent Registration No. 10-0824106 (Apr. 15, 2008)

The present invention provides a microbial agent capable of reducing the virus-induced crustacean mortality and increasing the productivity by administering microorganisms useful in seawater and / or fresh water used in the culture of crustaceans, and a method for increasing the productivity of crustaceans using the microbial agent.

In order to achieve the above object, the present invention provides a microorganism preparation for crustacean production for improving crustacean productivity, comprising Bacillus subtilis, Bacillus coagulans, Bacillus subtilis, at least one strain of Bacillus sp. and Lactobacillus lactis selected from the group consisting of lichenfornis, Bacillus cereus and Bacillus polyfermenticus, Lactobacillus helveticus, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus ruteri, Lactobacillus spp., Lactobacillus spp., Lactobacillus spp. reuteri), Lactobacillus para Kasei (Lactobacillus paracasei) and Lactobacillus Bulgaria Switch (Lactobacillus bulgaricus) pH containing the culture, and of the Lactobacillus genus or more selected one kinds (Lacobacillu sp.) To strain and Saccharomyces (Saccharomyces cerevisiae) in MRS celebrity bicyclic mixed strain mixed culture from the group consisting of 3 to 4 and the number of bacteria of the mixed strain was 1 × 10 ⁸ to 10 ⁹ cfu / ml; Bio-floc 1; And a culture of one photosynthetic strain selected from the group consisting of Rhodobacter capsulatus and Rhodopseudomonas palustris , wherein the pH is 8 to 9 and the number of photosynthetic strains is 1 x 10 < RTI ID = 0.0 > ⁸ to 10 < ⁹ > cfu / ml of Bio-floc 2 as an active ingredient.

Embodiments of the present invention are a method for producing a microorganism agent for crustacean culture, wherein the method for producing biofloat 1 comprises the steps of injecting the mixed strain into a culture medium containing molasses, sugar, rice bran, and fresh water, sea water, step; Culturing the culture solution in which the mixed strain is injected by blocking air at 32 to 38 ° C for 15 to 20 days, wherein the photosynthetic strain is cultured in a culture medium containing maltitol, vitamin B, Into a culture medium containing a mixture thereof; And culturing the cultured medium containing the photosynthetic strain at 28 to 32 ° C for 2 to 4 days.

In addition, embodiments of the present invention include a step of administering the microorganism preparation for crustacean culture repeatedly to the fishes of the crustacean farm in a period of 4 to 6 days, and a step of maintaining the temperature of the fishes at 28 to 32 ° C, And a crustacean culture step to breed crustaceans.

The present invention can be achieved by increasing microorganisms beneficial to crustaceans and decreasing harmful microorganisms by administering microbial agents comprising at least one of the bio-floc 1 and bio-flak 2 to aquaculture water in crustacean shrimp farms . The present invention improves the quality of aquaculture and enhances the immunity of crustaceans through such a right-wing, thereby reducing mortality of crustaceans caused by infection with white spot syndrome virus and other harmful bacteria, and shortening the growth period of crustaceans. Therefore, according to the method according to the present invention, it is possible to increase the productivity of crustaceans by enabling annual cultivation of crustacean in domestic outdoor culture.

FIG. 1 is a graph showing changes in dissolved oxygen (DO) of a feed water treated with a microbial agent according to an embodiment of the present invention, according to the concentration of the microbial agent.
FIG. 2 is a graph showing a change in chemical oxygen demand (COD) of a feed water treated with a microbial agent according to an embodiment of the present invention, according to the concentration of the microbial agent.
FIG. 3 is a graph showing changes in suspended solids (SS) of a feed water administered with a microbial agent according to an embodiment of the present invention, according to the concentration of the microbial agent.
FIG. 4 is a graph showing the total nitrogen (Total-N) change of the feed water treated with the microbial agent according to an embodiment of the present invention, according to the concentration of the microbial agent.
FIG. 5 is a graph showing changes in ammonia nitrogen (NH ₄ ⁺ -N) concentration of a feed water to which a microorganism agent is administered according to an embodiment of the present invention.
FIG. 6 is a graph showing changes in phosphorus phosphate (PO ₄ ^- -P) in the breeding water to which the microbial agent is administered according to the concentration of the microbial agent according to an embodiment of the present invention.
FIG. 7 is a graph showing a change in the number of bacteria in the Bacillus sp. According to the concentration and elapsed time of the microbial agent according to one embodiment of the present invention.
8 is a graph showing changes in the number of bacteria in Lactobacillus sp. According to the concentration and elapsed time of the microbial agent according to one embodiment of the present invention.
FIG. 9 is a graph showing changes in the number of bacterial capsules in accordance with the concentration and elapsed time of the microbial agent according to an embodiment of the present invention.
10 is a graph showing changes in the total number of bacteria according to the concentration and elapsed time of the microbial agent according to one embodiment of the present invention.
FIG. 11 is a graph comparing the weight gain of shrimp according to microorganisms after 45 days and 90 days after administration of the microbial agent according to one embodiment of the present invention to the water for breeding.
FIG. 12 is a graph comparing the increase in the total length of shrimp according to microorganisms after 45 days and 90 days after administration of the microbial agent according to an embodiment of the present invention.
FIG. 13 is a graph showing the expression of proPO (proPhenoloxidase) gene as an immune gene in the liver pancreas of a shrimp after administering the microbial agent according to an embodiment of the present invention to the stock water.
FIG. 14 is a graph showing changes in LYS (Lysozyme) gene expression as an immune gene in the liver pancreas of a shrimp after administering the microbial agent according to an embodiment of the present invention to raising water.
FIG. 15 is a graph showing the expression of SP (Serine Proteinase) gene as an immune gene in the liver pancreas of a shrimp after administering the microbial agent according to an embodiment of the present invention to the stock water.
16 is a graph showing the results of analysis of SOD (superoxide dismutase) activity in liver pancreas of shrimp exposed to a microbial agent according to an embodiment of the present invention.
FIG. 17 is a graph showing the results of analysis of catalase activity in liver pancreas of shrimp exposed to a microbial agent according to an embodiment of the present invention. FIG.
18 is a graph showing the results of analysis of glutathione content in liver pancreas of shrimp exposed to a microbial agent according to an embodiment of the present invention.
FIG. 19 is a graph showing the change in the number of cultures of a culture according to a culture period (0 to 30 days) in the production of biofrac 1 according to an embodiment of the present invention. FIG.
FIG. 20 is a graph showing changes in pH of a culture according to a culture period (0 to 30 days) in the production of bioflak 1 according to an embodiment of the present invention. FIG.
FIG. 21 is a graph showing changes in the number of strains of the culture according to the incubation period (0 to 30 days) and the incubation temperature (20, 25, 30, 35 ° C) in the production of bioflak 1 according to an embodiment of the present invention Fig.

Hereinafter, the present invention will be described in detail.

Embodiments of the present invention include microbial agents for crustacean production to enhance the productivity of crustaceans , such as Bacillus subtilis, Bacillus coagulans, Bacillus lichenfornis, Bacillus cereus Bacillus sp.) And Lactobacillus lactis, Lactobacillus helveticus, Lactobacillus spp., And Lactobacillus spp ., Selected from the group consisting of Bacillus cereus and Bacillus polyfermenticus , Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus paracasei, Lactobacillus rhamnosus, a (Lactobacillus paracasei) Lactobacillus and Bulgaria kusu (Lactobacillus bulgaricus) is At least one member selected from the eojin group Lactobacillus genus (Lacobacillu sp.) Strains and Saccharomyces to a MRS celebrity bicyclic on (Saccharomyces cerevisiae) strain that contains a culture of a mixed mixed culture and, pH is 3-4, and the mixed culture The number of bacteria was 1 x 10 ⁸ to 10 ⁹ cfu / ml Bio-floc 1; And a culture of one photosynthetic strain selected from the group consisting of Rhodobacter capsulatus and Rhodopseudomonas palustris , wherein the pH is 8 to 9 and the number of photosynthetic strains is 1 x 10 < RTI ID = 0.0 > ⁸ to 10 < ⁹ > cfu / ml of Bio-floc 2 as an active ingredient.

Herein, Bio floc refers to a high-quality organic matter entangled with microorganisms, plankton, feed residue, and the like present in the breeding water, and the breeding water is the water which is cultured in crustaceans such as shrimp , Which is the broadest concept that includes all of the water supplied or stored in a farm.

According to embodiments of the present invention, the crustaceans are arthropods living underwater and include at least one member selected from the group consisting of shrimp, crab and crayfish. As an embodiment, since shrimp have non-specific immune responses as invertebrates, vaccines or immunostimulants are effective only for a short period of time against specific pathogens. However, the present invention provides bioprocts 1 and 2 cultured with the useful microorganisms as described above, resulting in an increase in serological immunity and competitive exclusion in the field of shrimp, thereby excellently protecting nonspecific diseases.

According to one embodiment of the present invention, the Bacillus strain of Bioflack 1 secretes various exoenzyme, and it can block the harmful bacteria by being present in the intestine, thereby increasing the survival rate of crustaceans through preemption of useful microorganisms. It can also increase the survival rate of crustaceans by forming clusters in the digestive tract of aquaculture and crustaceans, naturally occurring in the field instead of Vibrio and producing a wide variety of antibiotics. It is able to increase the activity of lipase, protease and amylase in the digestive tract of crustaceans, and it can prevent disease through cell and humoral immune defense activity in crustaceans. It can also act as an immunogen on crustaceans by stimulating the cellular activity of granulocyte leukocytes.

The Lactobacillus strain of Bioflack 1 can reduce harmful bacteria existing in digestive organs of crustaceans, improve host metabolism, decrease serum cholesterol and amines, and can effectively reduce the mortality of crustaceans.

In addition, in the aquaculture, especially in crustacean culture, a large amount of nitrogenous substances such as nitrite nitrogen, nitrate nitrogen, ammonia nitrogen and phosphorus are generated. These phenomena cause amplification of algae and overloading And the like.

Accordingly, the photosynthetic strain of Bioflac 2 according to one embodiment of the present invention is an anaerobic photo-effect organism and plays a role of decomposing organic matter under vacancy or anaerobic conditions. Therefore, it plays a role of improving the quality of water by decomposing nutrients against pollution of organic matter and water in aquaculture environment.

As an embodiment of the present invention, the microbial agent for crustacean culture may contain BioFlag 1 and BioFlag 2 at a volume ratio of 8 to 12: 1, more specifically, at a volume ratio of 10: 1. Also, according to one embodiment, the mixed strain of Bioflack 1 contains the strain of the genus Lactobacillus and the strain of Saccharomyces cerevisiae in a ratio of CFU (Colony Forming Unit) of 1.5-2.5: 0.4-0.7 based on the total volume of the culture liquid can do.

Embodiments of the present invention may include a method of manufacturing bioflavons 1 and 2 described below as a method for producing a microorganism agent for crustacean culture as described above.

Specifically, the method for producing biofract 1 comprises the steps of injecting the mixed strain into a culture solution containing molasses, sugar, rice bran, and fresh water, seawater, or a mixture thereof; Culturing the cultured medium in which the mixed strain is injected by blocking air at 25 to 40 ° C, more specifically 32 to 38 ° C for 15 to 20 days. In addition, the method for producing biofloat 2 comprises the steps of: injecting the photosynthetic strain into a culture solution containing a malate, vitamin B and fresh water, seawater, or a mixture thereof; Culturing the culture solution in which the photosynthetic strain has been injected at 25 to 35 ° C for 2 to 4 days, more specifically at 28 to 32 ° C for 3 days. At this time, the production method of Biofract 2 can slightly aerate the cultured medium in which the photosynthetic strain is injected.

The fresh water used for the cultivation of the bioflakes 1 and 2 means conventional sea water having a salt content of not more than 500 mg / l, and the sea water contains 3.5 to 3.6% of salt, , Sodium chloride, potassium sulfate, magnesium sulfate, etc., nutrients, gases such as oxygen and nitrogen, and the like. In addition, a solution containing seawater and fresh water can be changed to a salinity of 3.5% or less.

According to one embodiment, the culture broth of Bioflack 1 is a mixture of 10 to 50 L of molasses, 1 to 5 kg of sugar, 1 to 10 L of rice bran, and 1000 L of fresh water, seawater or a mixture thereof. In addition, the culture solution of Bioflack 2 may be a mixture of 1 to 20 kg of malate, 0.5 to 5 L of vitamin B and 1000 L of fresh water, seawater or a mixture thereof. More specifically, fresh water, seawater, or a mixture thereof of Bioflakes 1 and 2 may be a mixture of sea water and fresh water at a volume ratio of 3: 1.5 to 2.5.

In addition, in the step of culturing the culture medium in which the photosynthetic strain is injected in the method for producing Biofract 2, the photoperiod may be 1,000 to 30,000 Lx and the wavelength may be 650 to 800 nm.

As another embodiment of the present invention, the present invention provides a microbiological agent-containing step for repeatedly administering the above-mentioned microorganism agent for crustacean culture to the cultured water of a crustacean farm in a period of 3 to 8 days; And a crustacean culture step of raising crustaceans while maintaining the water temperature of the above-mentioned water at 20 to 35 占 폚.

Although the concentration of microorganisms varies depending on the microorganism species, the survival rate is low when the search is too clear, and the survival rate is about 10 ⁶ cfu / ml. The clearness of the search is indicated by the presence of microorganisms, the amount of organic matter, the amount of suspended solids (SS), etc. It takes about 5 days for the concentration of microbes to change from 10 ⁸ to 10 ⁹ to 10 ⁶ to 10 ⁷ do. Therefore, by repeatedly administering the microorganism agent according to the embodiments of the present invention to the rearing water for 4 to 6 days, more specifically, every 5 days, the water quality can be adjusted to be 10 ⁶ or less by the action of the microorganism have.

According to an embodiment of the present invention, the step of administering the microorganism agent may be carried out such that the concentration of the microorganism agent in the feed water is 100 to 140% when the concentration of 1 L of the microorganism agent is 50% And adjusting the amount of the liquid. If the concentration interval is exceeded, the ability of the microbial agent to purify water may be deteriorated, and the growth of microorganisms may be slow.

In addition, the microbial agent dosing step may include one or more of nitrite concentration of 1 to 5 ppm, ammonia concentration of 0.01 to 0.1 ppm, pH of 6 to 9, and chemical oxygen demand (COD) of 3 to 10 ppm And adjusting the condition to satisfy the condition. When the number of breeding water meets the above conditions, the amount of contaminants such as ammonia and nitrite in the breeding water is balanced with the amount of microorganisms to be administered, and the crustacean can be cultured without returning the number of breeding.

Accordingly, the method of enhancing the productivity of crustacean according to the embodiments of the present invention can improve the productivity of the crustacean by indirectly promoting the immunity of the crustacean without using the direct immune enhancing agent such as the white spot syndrome virus antigen by achieving the right- To reduce the rate of crustacean mortality due to white spot virus. As a result, it can ultimately improve the productivity of crustaceans.

Hereinafter, the present invention will be described with reference to the following Production Examples and Experimental Examples. The Examples and Experimental Examples are intended to further illustrate the present invention and are not intended to limit the scope of the present invention.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope of the invention.

[Preparation Example 1] Production of microbial agent (bioflak 1) for shrimp cultivation

Bacillus cereus, Bacillus subtilis, Lactobacillus paracasei, and Saccharomyces cerevisiae strains are 5 × 10 ⁵ to 10 ⁶ cfu / ml, 1 ^{x 10 8 ~ 10 10 cfu /} ml, 1 x 10 6 ~ 10 7 to ^{cfu / ml, 5 x 10 5} ~ 10 6 samples (EM-1, ㈜ Ever Miracle) of 20L included in cfu / ml molasses 20L, The microorganisms were cultured at 35 DEG C for 15 days to obtain a microorganism having a pH of 3-4 and a number of microorganisms of 1 x 10 < ⁸ > to 10 < ⁹ > cfu / mL. To obtain a culture (Preparation Example 1).

[Preparation Example 2] Preparation of microbial agent (bioflak 2) for shrimp cultivation

Rhodobacter la capsule Tuscan (Rhodobactorcapsulatus) (DS-PSB, ( Note) Doosan echo bijeunet) is 1 x 10 ⁶ the end of the sample contained in 0.5L cfu / ml acid 5kg, vitamin B 1L and water 99.4L a mixed culture And cultured under weak aeration at 30 DEG C for 3 days to prepare a microbial culture (Preparation Example 2) having a pH of 8-9 and a number of microorganisms of 1 x 10 ⁸ to 10 ⁹ cfu / mL.

[Preparation Example 3] Preparation of microbial agent for shrimp cultivation

Bioflakes 1 and 2 prepared in Preparation Examples 1 and 2 were mixed at a volume ratio of 10: 1 to prepare a microbial preparation (Preparation Example 3). In the following Experimental Examples, experiments were conducted using the microorganism materials of Preparation Example 3. [

[Experimental Example 1] Analysis of water quality improvement result by administration of a microbial agent

Setting experiment conditions

The experimental shrimp used in the experiments described below were Fenneropenaeuschinensis, which was purchased from the Incheon Fisheries Research Institute. The shrimp were subjected to a week of fermentation, and a total length of about 1.5 cm and an average weight of 0.00575 g were used. The selected specimens were stocked with 180 L of water in a circular water tank of 400 × 400 × 600 mm and filled with about 3 cm of sand. The external temperature for the experiment was kept constant at 24.5 ± 0.5 ℃ and the water quality of the seawater (breeding water) used in the experiment is shown in Table 1 below.

division value Temperature (℃) 24.5 ± 0.5 pH 8.0 ± 0.5 Salinity (‰) 30.5 ± 1.0 DO (mg / L) 7.1 ± 0.3 COD (mg / L) 1.13 ± 0.1 SS (mg / L) 1.22 ± 0.5 ^{_{NO 2 - -N (㎍ / L}} ) 1.3 ± 0.3 ^{_{NO 3 - -N (㎍ / L}} ) 11.48 ± 1.0 ^{_{NH 4 + -N (㎍ / L}} ) 12.5 ± 0.7 PO ₄ ^- -P (μg / L) 1.5 ± 0.5

Prior to the present experiment, when the concentration of the microorganism agent was 0, 60, 80, 100, 120, or 140% when the 1 L of the microorganism agent prepared in Preparation Example 3 was administered to 50 tons of seawater at a concentration of 100% In the seawater.

Water quality analysis

According to the embodiment of the present invention, the change in the water quality of the breeding water was measured 45 days and 90 days after the administration of the microbial agent, and the measurement method was applied to the marine environment process test standard (December 12, 2010 revision) set by the Ministry of Land, Transport and Maritime Affairs I followed.

Specifically, the dissolved oxygen (DO) was obtained by the modification of Strickland and Parsons (1968) by Winkler (1888). Manganese chloride and sodium iodide (NaI) The precipitated iodine corresponding to the amount of dissolved oxygen was titrated with sodium thiosulfate and quantitatively measured.

The chemical oxygen demand (COD) was determined by adding sodium permanganate as an oxidizing agent and then heating for 60 minutes to oxidize chemically oxidizable substances and titration with sodium thiosulfate (Na ₂ S ₂ O ₃ ). At this time, the amount of oxygen consumed was indirectly measured by measuring the amount of oxygen consumed.

The suspended solids (SS) were filtered through a glass fiber filter paper (GF / F filter paper, pore size 0.7 μm) and dried at 105-110 ° C in a constant volume to increase the weight of the filter paper .

In the total nitrogen (N), nitrite nitrogen reacts with a sulfonylamide to form a diazonium ion, and then reacts with naphthylethylenediamine to produce an azo compound. The color-developed sample is analyzed by a spectrophotometer The absorbance was measured at 543 nm. Since nitrate nitrogen is relatively thermodynamically stable in nitrogenous compounds in seawater, it is reduced to nitrite nitrogen by using a cadmium reduction tube treated with copper catalyst and then measured in the principle of nitrite nitrogen measurement. Ammonia nitrogen (NH ₄ ⁺ -N) is oxidized with a basic hypochlorous acid solution to form monochloramine. Phenol and catalysts such as nitroprase and hypochlorous acid are used to form indolephenol. The developed sample is analyzed by spectrophotometer And the absorbance was measured at 640 nm.

Phosphoric acid (PO ₄ ^{- -} P) reacts with ammonium molybdate phosphate and potassium antimony tartrate under acidic conditions to form a phosphodoric acid maldiventric acid complex which is reduced by ascorbic acid to produce a blue solution. The color-developed sample was measured at 885 nm using a spectrophotometer.

The measurement results of the above indices are shown in Figs. 1-6. The amount of Total-N, NH ₄ ⁺ -N and PO ₄ ^- -P showed a tendency to decrease as time passed, ie, 90 days after 45 days compared to the control. However, there was no change in the water quality according to the concentration of the microbial agent of the present invention.

[Experimental Example 2] Analysis of microbial composition in aquaculture water after administration of a microbial agent

The microbial agent according to one embodiment of the present invention was administered to seawater at a concentration of 100% and 150%, and the compositional change of microorganisms included in the microbial agent after 5 days and 10 days was analyzed.

Depending on the composition of microorganisms, water was collected from each tank and diluted 10-fold. The number of colonies was measured at 35 ℃ for 48 hours.

Specifically, total bacterial count (TBC) was prepared by dissolving 23 g of 1% NaCl and NA (Nutrient Agar) in 1 L of distilled water, autoclaving at 121 ° C for 15 minutes under autoclave, And the number of colonies was determined by measuring the concentration of each sample by dilution stepwise.

Lactobacillus spp. Was prepared by dissolving 55 g of MRS broth, 1.7% of Agar and 1% of NaCl in 1 L of distilled water and autoclaving at 121 ° C for 15 minutes in autoclave. After boiling in Petri dishes, The number of colonies was confirmed by dilution. At this time, bacteria were identified through biochemical tests such as API (E & 50CHB & CHL).

Bacillus subtilis was prepared by dissolving 46 g of MYP (Mannitol-Egg Yolk-Polymyxin) agar and 1% NaCl in 1 L of distilled water, autoclaving the autoclave at 121 ° C for 15 minutes, cooling to 45-50 ° C, 50% of Egg Yolk Enrichment and 4.1 ml of Polymyxin were added and mixed well. Then, the Petri dishes were boiled and the samples of each concentration were diluted stepwise to confirm the number of colonies. At this time, bacteria were identified through biochemical tests such as API (E & 50CHB & CHL).

Rhodobacter capsulatum was prepared by dissolving 1.7% agar and 1% NaCl in 1 L of distilled water in Malate basal broth and autoclaving at 121 ° C for 15 minutes. The number of colonies was confirmed by diluting stepwise samples.

As a result, as shown in FIG. 7-10, the concentration of each microorganism decreased with time, and the concentration of the microorganism agent decreased, and the concentration of the microorganism decreased to a smaller extent.

[Experimental Example 3] Analysis of shrimp productivity enhancement by administration of microbial agent

Growth rate analysis of shrimp

In order to analyze the effect of the microbial agent on the degree of growth of the shrimp according to the embodiment of the present invention, the total electric field and the body weight of the shrimp were measured before the experiment, and the microbial agent was applied at 0, 60, 80, 100, And the total length and weight of the shrimp were measured after 45 days and 90 days, respectively. Daily growth rate and daily weight gain were measured as follows.

[Equation 1]

Daily growth gain = (W _f - W _i ) / day

W _f = Final length or weight

W _i = Initial length or weight

The results of measuring the weight growth of shrimp after 45 days and 90 days, respectively, are shown in Fig. After 45 days, the microbial agents showed significant weight gain at 100, 120 and 140%, and the highest growth rate at 120% concentration. The change of total length after 90 days also showed a significant increase when the concentration of microorganism was 120% and 140%, and the highest growth was observed at 120% concentration.

In addition, the change of total length of shrimp after 45 days showed a significant increase at 100, 120 and 140% concentration of microorganism and the highest growth rate at 120% concentration. The change of total length after 90 days also showed a significant increase at the concentrations of 100, 120 and 140% of the microbial agent and the highest growth rate at the 120% concentration.

Immunity analysis of shrimp

The microbial agent according to one embodiment of the present invention was added to the stock water at the concentrations of 0, 60, 80, 100, 120 and 140%, respectively. After 45 days and 90 days, the immunity of the shrimp was analyzed.

The tissues were homogenized using a Trisol teflon-glass homogenizer 099CK4424, Glass-Col, Germany to measure the immunogen gene expression of the liver pancreas of the shrimp. After that, chloroform was added and vortexed. The supernatant was separated by centrifugation at 4 ° C and 12,000 g for 15 minutes. The same amount of isopropanol as the supernatant was added and centrifuged at 12,000 g for 15 minutes at 4 ° C to form pellets. After addition of 80% ethyl alcohol, the cells were washed by centrifugation at 12,000 g for 15 minutes at 4 ° C.

Next, the RNA was diluted with NFW, and then 1 μg / μl was quantified and synthesized as a cDNA using a cDNA synthesis kit (Promega, MADI, USA). The synthesized cDNA was used as a primer according to the following Table 2, and β-actin was used as a house keeping gene. The primer was primer 3 primer 3 program and then primer was supplied from bioneer. Relative mRNA expression was confirmed by real-time PCR (Roche) with TOPrealq PCR 2X Premix (SYBR Green, Enzynomics, Korea). The conditions of the PCR are shown in Table 3 below. The primer sequences of proPO (proPhenoloxiase), LYS (Lysozyme) and SP (Serine Proteinase) as the immunogen used in the experiment are shown in Table 4-6.

Primer β-actin Sequence (5 'to 3') Forward CGA GGT ATC CTC ACC CTGA Reverse CGG AGC TCG TTG TAG AAG G

Temperature (° C) Time cycle Pre-incubation 95 10 min One Denaturation 95 10 sec 45 Annealing 60 15 sec Extension 72 25 sec Melting curve 95, 60, 72 10, 15, 25 sec One

Primer proPhenoloxiase (proPO) Sequence (5 'to 3') Forward GAT ATC CTC GGC GAT GTG T Reverse AGG GTC ATG CGA GAA AGC T

Primer Lysozyme (LYS) Sequence (5 'to 3') Forward GTA ACA AAC GCG ACC TCGA Reverse CCG TGC CAG GCT GTA TAT C

Primer Serine Proteinase (SP) Sequence (5 'to 3') Forward TAT GTG GCG GAT CCC TTA T Reverse GGT GAT AGT CCC CAA GAC G

First, the results of immunoprotein expression of proPO (proPhenoloxidase) in hepatic pancreas of shrimp exposed to a microbial agent according to an embodiment of the present invention are shown in FIG. The expression of proPO was not significantly different in the other groups as compared with the control group, but the concentration of microbial agent was significantly increased at 60, 120 and 140%. The proPO is a gene that plays an important role in the immune function of shrimp, and is converted into PO by the enzyme of SP, and the immune function is activated. Since proPO is increased, it means that the immunity of shrimp is increased.

In addition, the results of immunological gene expression of LYS (Lysozyme) in liver pancreas of shrimp exposed to a microbial agent according to an embodiment of the present invention are shown in FIG. LYS activity was significantly increased with increasing concentrations of microbial agent compared to the control group, and the expression of LYS tended to be increased, and the increase was significant at 120 and 140 intervals. Since LYS is the most important function in the innate immunity of shrimp, the increase of LYS means that the immunity of shrimp is increased.

Finally, the immunogen gene expression results of SP (Serine Proteinase) in liver pancreas of shrimp exposed to the microbial agent according to an embodiment of the present invention are shown in FIG. SP did not show any significant difference as the concentration of microbial agent increased in comparison with the control group, but SP activity tended to increase at the highest concentration range of 140%.

Biochemical analysis of shrimp

The microbial agent according to one embodiment of the present invention was added to the water at the concentrations of 0, 60, 80, 100, 120 and 140%, respectively. After 45 days and 90 days, the enzymatic activity of the shrimp was biochemically analyzed.

The tissue was washed with wash buffer (0.1 M KCl, pH 7.4) to measure the enzymatic activity of the liver pancreas of the shrimp, and treated with Teflon-glass homogenizer (099CK4424, Glass -Col, Germany). The supernatant was used for the experiment by centrifugation at 10000 g for 30 minutes at 4 캜.

First, tissues were homogenized using 1X lysis buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 0.1 mM EDTA) to measure superoxide dismutase (SOD) activity and then centrifuged at 12,000 g for 10 minutes at 4 ° C, All the supernatants were stored at -75 ° C (MDF-U53V, SANYO Electric Co. Ltd., Japan) until the experiment. Protein content of tissues was measured by Bradford (1976) kit (Biorad. , Ltd.). SOD activity was measured by SOD Assay Kit (Cell biolabsInc), which is measured by the inhibitor rate of chromagen reduction. Each supernatant was diluted with 0.1mM PBS in multiples of 5 and then measured at absorbance 490nm using a spectrophotometer. The inhibitor rate was calculated as unit / mg protein. At this time, SOD activity was calculated according to the following equation (2).

&Quot; (2) "

SOD activity (inhibition%) = (OD _blank- OD _sample ) / (OD _blank ) x 100

The results of analysis of SOD activity in liver pancreas of shrimp exposed to the microbial agent of one embodiment of the present invention are shown in FIG. The activity of SOD was decreased from 80% as the concentration of microorganism increased and the activity of SOD was decreased at 120 and 140%, respectively.

Catalase activity was then measured using a catalase assay (Cell biolabsInc), which measures the amount of catalase that reacts with the remaining hydrogen peroxide relative to the concentration of hydrogen peroxide. The absorbance was measured at 520 nm using a spectrophotometer through two reactions. Catalase activity was measured for the OD value using a standard curve of catalase activity assay and expressed as unit / mg protein.

FIG. 17 shows the results of analysis of catalase activity in liver pancreas of shrimp exposed to the microbial agent of one embodiment of the present invention. The activity of catalase was significantly decreased at 120 and 140% of the concentration of the microbial agent compared to the control, and the activity was the lowest at 120%.

Finally, the content of reduced glutathione (GSH) was analyzed by Butler et al. (1963). The supernatant was mixed with metaphosphoric acid (Na ₂ EDTA, NaCl) and centrifuged at 4500 g for 10 minutes. The supernatant was loaded with 0.3M Na ₂ HPO ₄ , developed with 0.5 mM DTNB and absorbance was measured at 412 nm with a spectrophotometer. The content of glutathione was measured using a reduced glutathione standard curve and expressed as nmol GSH / mg protein.

The results of the analysis of the reduced glutathione content in the liver pancreas of shrimp exposed to the microbial agent of one embodiment of the present invention are shown in FIG. Glutathione content was significantly decreased compared to the control group, but there was no significant difference in the concentration of microbial agent.

[Experimental Example 4] Analysis of manufacturing conditions of microbial agent (Bioflak 1) for shrimp cultivation

Bacillus cereus, Bacillus subtilis , Lactobacillus paracasei, and Saccharomyces cerevisiae strains have a concentration of 5 × 10 ⁵ to 10 ⁶ cfu / ml, respectively, (EM-1, EVER MIRACLE CO., LTD.) Containing 1 x 10 ⁸ to 10 ¹⁰ cfu / ml, 1 x 10 ⁶ to 10 ⁷ cfu / ml and 5 x 10 ⁵ to 10 ⁶ cfu / ml, (0 to 30 days), water temperature (20, 25, 30, 35 ° C), and the number of cultures after incubation (0 to 30 days) And pH changes were measured.

As a result, as shown in Figs. 19 and 20, when the incubation period was from 15 days to 20 days, the desired biofactor 1 was effectively produced with a strain number of 10 ⁹ cfu / ml (at a culture temperature of 25 to 40 ° C). As shown in Fig. 21, when the incubation period was 15 to 20 days and the incubation temperature was 30 to 35 ° C, the number of the strains was 10 ⁹ cfu / ml, confirming that the desired biofract 1 was effectively produced.

Claims (10)

Shrimp for Growth Enhancement Shrimp Outdoors As a microbial agent,
( 1 ) selected from the group consisting of Bacillus subtilis, Bacillus coagulans, Bacillus lichenfornis, Bacillus cereus and Bacillus polyfermenticus. A bacterium belonging to the genus Bacillus sp. Or more, Lactobacillus lactis, Lactobacillus helveticus, Lactobacillus rhamnosus, Lactobacillus acidophilus, ( 1 ) selected from the group consisting of Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus paracasei and Lactobacillus bulgaricus. A strain of Lactobacillus sp. Or more, and a strain of Saccharomyces cerevisiae A mixture comprising Saccharomyces cerevisiae strain and a mixture comprising molasses, sugar, rice bran, fresh water and seawater, wherein the pH is 3 to 4 and the number of bacteria of the mixed strain is 1 × 10 ⁸ to 10 ⁹ cfu / ml Bio-floc 1; And
A culture comprising a photosynthetic strain selected from the group consisting of Rhodobacter capsulatus and Rhodopseudomonas palustris , and a mixture comprising malate, vitamin B and seawater, The present invention relates to a shrimp outdoor culture microorganism agent comprising a bio-floc 2 having a pH of 8 to 9 and a bacterial count of 1 × 10 ⁸ to 10 ⁹ cfu / ml as a photosynthetic strain,
The volume ratio of the bioflakes 1 and 2 is 8 to 12: 1,
The volume ratio of the seawater and the fresh water contained in the Biofract 1 is 3: 1.5 to 2.5,
Among the mixed strains of Bioflack 1, the strains of the genus Lactobacillus and Saccharomyces cerevisiae contain the colony forming unit (CFU) ratio of 1.5 to 2.5: 0.4 to 0.7 based on the total volume of the culture,
The CFU ratio of the Lactobacillus sp. Strain and Saccharomyces cerevisiae strain to the Bacillus sp. Strain among the mixed strains of Bioflack 1 is 6.7 to 6670: 1,
The microorganism agent is provided so as to be 120 to 140% when 1 L of the microorganism agent is 50% by weight, and is administered by 1.2 to 1.4 L,
Wherein said microbial agent reduces the mortality of the shrimp by promoting the immunity of the shrimp to the white spot syndrome virus.
Shrimp outdoors to improve growth potential of shrimp. delete delete delete A method of producing a shrimp outdoor cultured microorganism material for improving growth of shrimp according to claim 1,
The method for producing Biofract 1 comprises the steps of: injecting the mixed strain into a culture solution containing a mixture of molasses, sugar, rice bran, fresh water, and seawater; Culturing the cultured medium in which the mixed strain is injected by blocking air at 25 to 40 DEG C for 15 to 20 days,
The method for producing Biofract 2 comprises the steps of: injecting the photosynthetic strain into a culture medium containing a mixture of malate, vitamin B and seawater; Culturing the cultured medium containing the photosynthetic strain at 25 to 35 DEG C for 2 to 4 days.
A method for manufacturing a microbial agent for outdoor cultivation of shrimp for improving the growth of shrimp. 6. The method of claim 5,
The culture broth of Bioflack 1 is a mixture of 10 to 50 L of molasses, 1 to 5 kg of sugar, 1 to 10 L of rice bran, 1000 L of fresh water,
The culture broth of Bioflack 2 is a mixture of 1 to 20 kg of malate, 0.5 to 5 L of vitamin B, and 1000 L of seawater.
A method for manufacturing a microbial agent for outdoor cultivation of shrimp for improving the growth of shrimp. A microbiological agent administration step of repeatedly administering the shrimp outdoor culture microorganism material for improving the growth performance of the shrimp according to claim 1 to the shrimp farm in the shrimp farm every 4 to 6 days; And
The shrimp culture step of raising the shrimp by maintaining the water temperature of the breeding water at 20-35 < 0 > C,
How to Promote Shrimp Growth. delete 8. The method of claim 7,
The step of administering the microorganism agent is adjusted so as to satisfy at least one of a nitrite concentration of 1 to 5 ppm, an ammonia concentration of 0.01 to 0.1 ppm, a pH of 6 to 9, and a chemical oxygen demand (COD) of 3 to 10 ppm in the breeding water prior to administration of the microorganism agent Further comprising:
How to Promote Shrimp Growth. delete

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