KR20020087591A - Preparation and its production method for immunopotentiating, antibacterial activity and growth promotion of aquacultured fish - Google Patents

Abstract

PURPOSE: A process of preparing a formulation having enhanced immune activity on cultured fishes by mixing Paecilomyces japonicus formulations and probiotics is provided. Whereby, the product enhances immunity and growth of the cultured fishes to reduce the death rate and increase the production amount thereof. CONSTITUTION: A mixed formulation contains Paecilomyces japonicus formulations and probiotics having proliferation-inhibiting activity on Edwardsiella tarda, Pseudomonas fluorescens and Vibrio cholerae, wherein the Paecilomyces japonicus formulations are obtained by culturing 6L strains of Paecilomyces japonicus in a medium containing 15 to 25% by weight of starch, 5 to 10% by weight of soybean protein, 5 to 10% by weight of dried yeast, 0.05% by weight of calcium carbonate, 0.025% by weight of K2HPO4 and 0.025% by weight of KH2PO4 at 25deg.C in a pH of 5.0 to 6.0, mixing 2kg starch per 1L cultured liquid and then drying at 50 to 60deg.C for 24hr.

Description

Preparation and its preparation method for immune enhancement and anti-potentiation and increase of aquaculture fish {{Preparation and its production method for immunopotentiating, antibacterial activity and growth promotion of aquacultured fish}

[Industrial use]

The present invention relates to a cordyceps herb preparation, a probiotic and a cordyceps herb preparation containing a probiotic, and a method for preparing the same. And a cultivation process of a cordyceps and a probiotics, which can be used as feed additives for fish farming by establishing a cultivation process containing a mixture of probiotics and cordyceps in a short time at a high concentration and containing a large amount of immune enhancing substances. It relates to a manufacturing method.

[Private Technology]

The use of antibiotics or vaccines is high due to the high mortality caused by high-density farming, but antibiotics are increasingly regulated, and vaccines are difficult to administer and use. Therefore, it is necessary to use a formulation that is easy to use and inexpensive and has excellent anti-medical effect. Cordyceps sinensis in the form of feed additives and mixtures of cordyceps and probiotics can significantly reduce mortality due to increased immunity of fish, enhanced growth and anti-history, and reduction of antibiotic imports due to increased catches and antibiotic abuse suppression and prevention of expression of antibiotic resistant bacteria. Can and is very beneficial to fish consumers.

The purpose of the present invention is to develop a liquid immunity enhancement, anti-cancer, anti-diabetic, gastric ulcer prevention, improvement of exercise ability in the human body as part of the development of preventive immune system for enhancing the immune system in aquaculture in a short time Cordyceps sinensis which reduces the mortality rate and promotes growth and weight gain by formulating probiotics that can be formulated inexpensively by mass harvesting and by forming probiotics that form beneficial bacteria in the intestines of fish To provide a formulation with a probiotic and Cordyceps sinensis and a probiotic.

1 is a flow chart of a formulation method according to the invention

The present invention to achieve the object of the present invention as described above is mixed with mixture which is Cordyceps (Paecilomyces japonicus) is used as a feed additive for fish culture dry product formulation MJ100 and deposited with Accession No. KCCM-10265 microorganism MP100 and MJ100 and MP100 One formulation MJP100 is provided. Hereinafter, the present invention will be described in more detail.

The present invention is a beneficial microorganism capable of growing at 15-25 ° C. separated from low-temperature ripened kimchi in consideration of the breeding environment of Cordyceps mycelium mycelium preparation MJ100 and cultured fish using liquid-cultured special medium for the development of anti-potentiation by the immune activity of fish. Phosphorus mixed microorganisms MP100 and MJP100 mixed with MJ100 and MP100 were developed. Paecilomyces japonicus was selected as the fungus of Cordyceps sinensis, and it was established to establish a culture process technology for rapid mass cultivation of Cordyceps with excellent immunity-improving effect based on the culture conditions and physiological characteristics of the selected strains. The experiment was performed. In addition, the composition and culture conditions of the selective medium excellent immunity enhancing effect by the classification, physiological and chemical properties of the selected Cordyceps sinensis are shown in Table 1 below.

Cordyceps sinensis strains were homogenized by mixing 2Kg of starch per 1L of culture after incubation using a production medium for immuno-enhancement in a 6L fermentation tank (NBS) and then dried at 50 to 60 ° C for 24 hours. The dried culture dry matter was pulverized and ingested in fish, and then examined for immunity. The strength of MJ100 in fish was measured by adding MJ100 to the flounder fry for 4 weeks, and after aseptic isolation of ducin from the flounder annealed with MS222 (tricaine methanesulfonate), Hank's balanced salt solution refrigerated at 4 ℃ Isolate the cells using a mesh that goes into (HBSS). The isolated scalp cells were centrifuged at 400 × g for 30 minutes at 4 ° C. using a 34/51% percoll density gradient, and then separated between 34% and 51% cell layers using a thin Pasteur pipette. Finally, the isolated cells were washed twice with HBSS at 400 × g for 5 minutes. The viability of the cells was analyzed using trypan blue stain, and the cell number of the phagocytes used in the experiment was controlled by 1 × 10 ⁶ cells / ml HBSS. Reactive oxygen intermediates (ROIs) released from phagocytes were measured using an automatic photoluminometer (Bio-orbit 1251, Finland) for chemiluminescent (CL) response analysis. That is, each test cuvette was mixed with 0.7 ml of luminol and 0.4 ml of cell dispersion and incubated at room temperature for 5 minutes, and then measured for 100 minutes by adding 0.3 ml of opsonized zymosan immediately before measurement. MJ100 was added 0.1%, 0.3%, and 0.5% to the feed for 4 weeks, and then the flounders of each experimental group were randomly taken to isolate the phagocytes. After the CL response was measured, the concentration of MJ100 was increased. The CL value was gradually increased, and when 0.5% was added, it showed more than twice the immunity compared to the control.

Fig. 1. CL response effect of flounder head phagocytes with different concentrations of MJ100. In addition, 0.1%, 0.3%, and 0.5% of MJP100 was added to the feed for 4 weeks. As a result of measuring the CL response, it was shown in Figure 2 that the immune activity was improved by 2 times at 0.3% and 3 times at 0.5% when compared to the control group.

Figure 2. CL response effect of flounder head phagocytes with different concentrations of MJP100

(a; MJ100 0.1% + MP100 0.1%, b; MJ100 0.3% + MP100 0.3%, c; MJ100 0.5% + MP100 0.5%)

In order to investigate the inhibition of growth of marine fish pathogens by mixing cultures of MP100 and marine fish pathogens, MP100 produced four inoculated samples containing 10 ⁸ cells per gram of bacteria, and Edward Siella spp. Edwardsiellar tarda are Tryptic soy broth, Pseudomonas fluorescens and Vibrio cholerae containing tryptone and soy flour, and lactose broth containing 2.5% NaCl. After incubation at 25 ° C. for 24 hours using a medium, the solution was inoculated with physiological saline to give 10 ⁶ cells / ml of viable cells. And Vibrio cholera bacteria were inoculated with 1% each of lactose broth medium containing 2.5% NaCl and incubated at 25 ° C. Each mixed culture solution was taken at 6 hour intervals, diluted with sterile saline, and the number of viable cells was measured. The viable cell counts of Edward Siela tada and MP100-containing bacteria were plated on tryptic soy agar medium and MRS medium and incubated at 25 ° C. for 24 hours, and the viable cell counts of each bacterium were measured. Silver NaCl was plated on 2.5% LB plate medium and bromocresol purple (BCP) medium, respectively, and cultured at 25 ° C. for 24 hours, and the viable cell counts of pathogens and MP100-containing bacteria were measured. In order to measure the growth inhibition and antimicrobial activity of MP100 against Vibrio cholera, Vibrio cholera was mixed with Vibrio cholera alone and MP100 for 48 hours using LB medium containing 2.5% NaCl at 25 ° C. In the case of culture alone, the growth was normal, but in the mixed culture, the number of bacteria started to decrease after 12 hours after inoculation, and completely inhibited the growth of Vibrio cholera bacteria after 30 hours.

Figure 3.Inhibitory Effect of Vibrio Cholera by MP100

●; Viable Cell Count of Vibrio Cholera by Vibrio Cholera Culture

■; Number of viable cells of MP100-containing strains when mixed with MP100

▲; Viable Cell Number of Vibrio Cholera in Mixed Culture with MP100

In order to measure the proliferation inhibitory and antimicrobial activity of Edwards siella tada against MP100, the result of cultivation of Edwards siella tada alone and MP100 for 48 hours using tryptic soy broth medium at 25 ° C was shown in Figure 4. In the case of cultivation of ella tada alone, normal growth was observed, but in the case of mixed culture, the number of bacteria started to decrease after 12 hours after inoculation and completely inhibited the growth of Edwardsella elda bacteria after 24 hours.

Figure 4. Growth Inhibition Effect of Edward Siella Tadida by MP100

●; The number of viable bacteria of Edward ciella tada by cultivation of Edward ciella tada alone

■; Number of viable cells of MP100-containing strains when mixed with MP100

▲; The viable cell count of Ed with Siela tada in culture with MP100

In order to determine the growth inhibition and antimicrobial activity of Pseudomonas pullulicense bacteria of MP100 using Pseudomonas pullulicesen alone and MP100 incubated for 48 hours with Lactobacillus ion medium (LB medium) at 25 ℃ as shown in Figure 5 Likewise, when Pseudomonas pullulicense bacteria were cultured alone, normal growth was observed, but in the case of mixed culture, the number of bacteria started to decrease after 12 hours after inoculation and completely inhibited the growth of Pseudomonas pullulicense bacteria after 18 hours.

Figure 5.Inhibitory Effect of Pseudomonas Fluriscens Growth by MP100

●; Viable Counts of Pseudomonas Flurisense Bacteria by Pseudomonas Flurisense Monoculture

■; Number of viable cells of MP100-containing strains when mixed with MP100

▲; Viable Counts of Pseudomonas Fluriscens in Mixed Culture with MP100

EXAMPLE

The present invention describes preferred examples and comparative examples. However, the following examples are merely examples of the present invention showing the constitution and effects of the present invention, and the present invention is not limited to the following examples. MJ100 and MP100 and MJP100 according to the present invention was administered to the flounder fry and the weight gain and mortality of the fish were investigated. In aquaculture experiments, 720 flounder fry flounders were randomly divided into 30 tanks and placed in 24 tanks for 2 weeks. MJ100, MP100 and MJP100 were added 0.1%, 0.3%, 0.5%, 1.0 % Was administered and the control group was administered only pure feed. Each experimental group was subjected to two relapses and analyzed for gain and mortality after administration of the experimental feed for 4 weeks. Fish body weight was determined by randomly taking 10 animals from each experimental group, and the average weight gain of the fish was calculated by repeating this method twice. The mortality was measured in a one-ton tank with each network, and the seaborne fish pathogenic bacteria Edwardsiella tarda was dispersed at 3.4 × 10 ⁷ bacteria per liter (L) of seawater. After the composition, the cumulative mortality of each experimental group was investigated.

The weight gain after MJ100 was added to feed for 4 weeks in flounder fry was 28% and 17% higher in MJ100 and 0.5% and 1.0% than control, respectively. This is shown in Table 2.

The weight gain after MP100 was added to feed for 4 weeks in flounder fry was 4% and 18% increase compared to the control in the group that added 0.3% and 0.5% MP100, respectively. Was observed and is shown in Table 3.

In addition, the increase rate of MJP100 at 0.1%, 0.3%, and 0.5% was increased to 1.6, 2.9, and 2.1 times, respectively, compared to the control group. This is shown in Table 4.

After dispersing the seaweed pathogen Edward Siela tada in the tank and stopping the feeding, the control group examined the anti-histopathy against the MP100 group, and the control group had 80% cumulative mortality rate for 7 days after the seawater pathogen was administered, whereas the MP100 0.3% group The 40%, 0.5% treated group showed 30% and 1.0% treated group showed a mortality rate of 57%, and it was shown that adding 0.5% MP100 to the feed showed the best anti-morbidity.

In addition, when MJP100 was administered, the control group had 80% cumulative mortality rate for 7 days after stopping the feeding of seawater pathogens, whereas 0.1%, 0.3%, 0.5% of MJP100 was administered. The group administered with 0.3% MJP100 at 35%, 10%, and 20% showed a very high survival rate of 8 times or more compared to the control group, which is shown in Table 6.

Cordyceps herb preparations of the present invention and probiotics containing four fungi and preparations mixed with cordyceps herb preparations and probiotics improve the immunity of fish and increase the morbidity, increase catches, and increase the yield by increasing the viability and promoting growth.

Claims (5)

Cordyceps sinensis production medium composition and preparations to increase immune activity, weight gain and reduce mortality for cultured fish MJ100 Mixture of Cordyceps Sinensis and Probiotics to Enhance Immune Activity, Weight Gain and Reduce Mortality in Aquaculture Fish MJP100 Probiotics MP100 Increases Growth Rate and Reduces Mortality in Aquaculture Fish Probiotics MP100 with antiproliferative activity of Edwardsiellar tarda , Pseudomonas fluorescens and Vibrio cholerae Mixed microorganism MP100, deposited with accession No. KCCM-10265, used as feed additive for fish.