KR100831354B1 - Method for exterminating watermold and fish pathogenic bacteria in fishes - Google Patents

Abstract

A method for controlling aquatic fungi and fish pathogenic bacteria of cultivated fish is provided to improve productivity of the cultivated fish by effectively controlling the aquatic fungi and fish pathogenic bacteria using a proper amount of an extract of Opuntia ficus-indica var. saboten Makino and provide safe aquatic products to consumers by using environment-friendly and harmless Opuntia ficus-indica var. saboten Makino extract. To exterminate aquatic fungi of fish eggs, the fish eggs are treated with 50-80 ppm of a hydrolysis fraction of Opuntia ficus-indica var. saboten Makino for 30 minutes every 3 days until the fish eggs become germinated egg. To exterminate aquatic fungi of fish, the fish is treated with 200 ppm of the hydrolysis fraction of Opuntia ficus-indica var. saboten Makino for 2 hours per day one or two times. To treat and prevent fish pathogenic bacteria of cultivated fish, 0.1% hydrolysis fraction of Opuntia ficus-indica var. saboten Makino is absorbed into an expansion pellet and the expansion pellet is administered into the cultivated fish for 4 weeks.

Description

Method for exterminating watermold and fish pathogenic bacteria in fishes

Figure 1 is a photograph showing the experimental bath subjected to the bath treatment of palm cactus extract.

Figure 2 is a graph showing the growth of aquatic bacteria according to the concentration of the palm cactus extract.

Figure 3 is a photograph of the development of aquatic bacteria according to the concentration of the palm cactus extract.

Figure 4 is a photograph showing the eel (control) infected with aquatic bacteria.

Figure 5 is a photograph showing that the treatment by bathing the palm cactus extract in eel infected with aquatic bacteria.

The present invention relates to a method for controlling aquatic bacteria and fish pathogens of farmed fish, and more particularly, to aquatic bacteria infected with farmed fish using palm cactus extract, and to prevent and control fish pathogens causing diseases on farmed flounder. It is about.

Since malachite green (MG), which has been used as a treatment for in vitro parasites of fertilized eggs and larvae in freshwater fish farms such as trout, has recently been recognized as a suspected human harmful substance, the world has restricted or banned its use. have.

In light of this situation, studies on substances to replace malachite green (MG) have been conducted in various countries around the world. In particular, formalin, hydrogen peroxide and sodium chloride have been found to be effective as antibacterial substances. However, the stability of these antimicrobial substances has not been verified, and the proper use of aquaculture fish is not well established.

In addition, in recent years, as the development of environmentally friendly and harmless alternative treatments is continuously required, research on plant herbal medicine extracts such as gojija and baejaja and green tea extracts have been conducted, but their efficacy has not been verified and their practical application It is still incomplete.

An object of the present invention is to rescue aquatic bacteria and fish pathogens infected with farmed fish with environmentally friendly and harmless drugs.

Another object of the present invention is to prevent and treat aquatic bacteria infected with aquaculture fish by bathing, and to prevent bacterial diseases of aquaculture fish by oral administration of drugs.

Still another object of the present invention is to provide a proper use of the drug while at the same time selecting the appropriate drug in the treatment of aquatic bacteria and fish disease bacteria of aquaculture fish.

In order to achieve the above object, the present inventors select a total of seven kinds of drugs used in human body, animal husbandry, and fisheries through extensive literature search together with research data on aquatic bacteria and fish disease bacteria. The effects of bactericidal, propagation inhibition and growth inhibition were investigated. As a result, Opuntia ficus - indica var . Saboten Makino) The stem extract (hereinafter referred to as 'palm cactus extract') replaces malachite green and has been found to be most effective in the sterilization of aquatic bacteria.

In addition, the antimicrobial activity of 18 strains of fish disease bacterial strains of the palm cactus extract was investigated. As a result, the palm cactus extract was found to be superior to the antimicrobial activity.

The palm cactus extract used in this example is a material fractionated and extracted from the stem of the palm cactus by Microbiotech Co., Ltd. The palm cactus extract has a molecular weight of about 120 and freezing point of about 30-33 ℃, boiling point 245-246 ℃, specific gravity 1.134g / ml (25 ℃), pH 2.5 is a liquid substance that is well soluble in water. This substance has been tested for safety through cytotoxicity, toxicity, mutagenicity, and mouse oral toxicity tests.

In the present invention, using the palm cactus extract to control aquatic bacteria of aquaculture organisms, a method of treating the aquatic bacteria is preferably a bath treatment method.

Especially in fish eggs, 50-80ppm palm cactus extract is effective in bathing for 30 minutes at intervals of 3 days until it becomes a bald egg.

In addition, for fish, 200 ppm of palm cactus extract may be treated once or twice a day for 2 hours / day. If it is less than this range, the treatment of aquatic bacteria is insufficient, and even if it exceeds this range, the improvement of the treatment of aquatic bacteria is insignificant.

According to another feature of the present invention, the present invention is to treat and prevent fish pathogens of cultured fish using palm cactus extract.

Particularly in farmed fish, palm cactus extract is preferably administered orally into the body.

At this time, oral administration of cultured fish is preferably administered for 4 weeks by adsorbing 0.1% palm cactus extract to the wound feed.

The present invention improves the productivity of farmed fish by effectively controlling aquatic bacteria and fish pathogens of farmed fish by providing appropriate usage and usage method of palm cactus extract, and safe marine products for consumers by using environmentally friendly and harmless palm cactus extract. There is an advantage to provide.

An embodiment of the present invention is as follows.

<Example 1>

* Identification and culture of aquatic bacteria

Aquatic bacteria used in this example were isolated and cultured as follows.

The isolates of aquatic bacteria were salmon fertilized eggs (Gangwon Yangyang), rainbow trout fertilized eggs (Gangnam Milyang), rainbow trout eosan (Gangwon Pyeongchang) and mountain trout (Gangwon Pyeongchang). The medium used at this time was penicillin & streptomycin added to Sabouraud dextrose agar (SDA, Difco) and GY agar (1% glucose, 0.25% yeast extract, 1.5% agar).

For morphological examination of aquatic bacteria, the fungus isolated from GY agar was made into blocks of 8 mm diameter and transferred to 100 ml of 10 mM PBS (pH 7.0) for 24 hours, followed by culturing supernatant in PBS containing sunflower seeds. Induce and make asexual reproduction by transferring to a plate containing sterile distilled water, or inoculate a purely isolated fungus on Malt agar (Difco) by inoculating a block of 8 mm diameter, and grow the grown mycelium in 20 ml of sterile tap water. ) Was added to the plate, and sterilized tap water every 24 hours, incubated at room temperature for 5-7 days to make a sexual reproduction state, and stained with Latophenol cotton blue (LPCB) staining solution and observed under a microscope.

Amplification of SSU rDNA common to eukaryotes for genetic classification of aquatic bacteria was performed by primers for 18s rRNA (forward, E21f 5'-ATC TGG TTG ATC CTG CCA GT-3 '; revers, E1778r 5'-AAT GAT CCT TCC GCA GGT TC-3 ') was used, and the sequence was commissioned by Bioneer (Korea), and the result was a phylogenetic tree based on GenBank accession.

In this example, the aquatic bacteria infected with domestically grown salmon (fish eggs) were found to be Saprolegnia parasitica, and the aquatic bacteria were secured by continuously passing the aquatic bacteria to GY agar for a certain period of time.

<Example 2>

* Test of inhibitory effect of aquatic bacteria on drug

In the present embodiment, a total of 9 kinds of drugs malachite green, bronopol (15%), palm cactus extract, NaCl, o medium (herbal extract), pneumatic (chlorine dioxide, 5%), formalin (37% formaldehyde), hydrogen peroxide ( 30%, reagent grade) and iodine (reagent grade) were subjected to in vitro screening.

Aquatic bacteria growth suppression experiment was carried out by sterilizing SDA (Sabouraud dextrose Agar, SDA) flat medium or SDB (Sabouraud dextrose broth, SDB) liquid medium, and then pre-cultivating aquatic bacteria in medium prepared by adding nine drugs by concentration. Agar pieces were inoculated and grown at 15 ° C. for a week to examine their growth. The experimental results are shown in Table 1.

According to this embodiment, it can be seen that the palmitary cactus extract next to malachite green has the best effect of inhibiting aquatic bacteria growth.

<Example 3>

* Test of the effectiveness of the drug to treat aquatic bacteria

In this example, a total of seven drug malachite green, palm cactus extract, bronopol, chlorine dioxide (2.5%), hydrogen peroxide, povidin, and formalin were used. And the experimental tank was a PVC tank of 3 tons capacity (1 ton volume) and the flow rate was around 500L per hour (Fig. 1).

Each tank contains 18,000 ~ 25,000 fertilized eggs, and the chlorine dioxide solution and hydrogen peroxide are 5ppm in concentrations of 5ppm once a day and once a day for 30 minutes, while povidin and formalin are 100ppm and 120ppm concentrations. Water baths, malachite green, palm cactus hydrolysate fractions, bronopol and sun salt in 2 to 3 day intervals were bathed for 30 minutes at 3 ppm, 50 ppm, 80 ppm and 10 ppm concentrations at 3 days intervals, and then in control (untreated). Relative initiation rate was evaluated. The experimental results are shown in Table 2.

According to this embodiment, it can be seen that the palmitary cactus extract next to malachite green has the best treatment effect for aquatic bacteria infection.

<Example 4>

* Development and Reproduction Inhibition Test of Aquatic Bacteria from Palm Cactus Extract

Aquatic growth inhibition experiment of palm cactus extract was performed after sterilization of SDA (Sabouraud dextrose Agar, SDA) flat medium or SDB (Sabouraud dextrose broth, SDB) liquid medium, and then added to 50 to 500 ppm concentration. Inoculated with aquatic bacteria agar pieces cultured at 15 ℃, a week incubation was investigated.

Figure 2 is a graph showing the growth of aquatic bacteria according to the concentration of palm cactus extract (MBT). And Figure 3 is a photograph of the development of the three days of aquatic bacteria according to the concentration of palm cactus extract added.

According to this embodiment it can be seen that the concentration of the palm cactus extract should be 50ppm or more to have the effect of inhibiting the growth and reproduction of aquatic bacteria.

<Example 5>

* Treatment of aquatic bacteria infected eel from palm cactus extract

The eel used in this experiment was an eel naturally infected with aquatic bacteria. The experimental tank was a 500L PVC tank, and water was not flowed during the experiment. Ten eel infected with aquatic bacteria were stored in this tank, and then, after bathing once or twice a day at 200 ppm concentration of palm cactus extract, they were compared with the control (non-medicated).

5 is a photograph of the eel of the control, Figure 6 is a photograph of the eel treated by a bath treatment.

According to this embodiment it can be seen that the aquatic bacteria can be treated once or twice a bath for 2 hours at 200ppm concentration of palm cactus extract.

<Example 6>

* Antimicrobial Efficacy Test of Fish Cactus Extract of Palm Cactus Extract

In this embodiment, Gram-negative bacteria ( Edderella) tarda J2-27K and 4 strains; Vibrio spp YT85805 and 4 strains) and Gram-positive bacteria ( Lactococcus) spp. L2 and 4 strains; Streptococcus spp. KCTC3651 and 2 strains). Paper disc for antimicrobial activity was prepared by diluting the stock solution of palm cactus extract, and then absorbing 50 µl each with a paper disc (diameter 8 mm, ADVANTEC D0811606) and drying (50 ° C). 25, 10, 5 and 1 μg.

The antimicrobial activity test was performed by MacFaland No. After adjusting to 0.5, 200 μl of the bacterial solution was evenly spread on a Muller Hinton plate medium, and the prepared experimental paper disc was placed and incubated in a 25 ° C. incubator for 24 to 48 hours, and then the lowland was measured by vernier calipers. As a positive control group, oxytetracycline (BBL, 30㎍ / disc) and a commercially available antibiotic disc with the best sensitivity were used. The experimental results are shown in Table 3.

According to the present embodiment it can be seen that the palm cactus extract 25 ㎍ containing disk exhibits a much better antimicrobial effect than the control antimicrobial agent against the fish disease bacteria.

<Example 7>

* Prophylactic test for fish disease bacteria of palm cactus extract

The flounder used in the experiments were healthy individuals with no apparent signs of disease. The experimental tank was a one-ton capacity FRP circular tank with a return rate of 30 ml per second. Experimental feed with palm cactus extract was prepared by adjusting the concentration to 0.1% by absorbing commercial EP feed. Experimental fish were housed in two repetitions of 120 fish per tank and then fulled every day. It was administered, the breeding water temperature was natural water temperature. Experimental fish were randomly collected a total of 30 animals in each tank at 4, 8 and 12 weeks and used as samples for analysis.

In order to investigate the blood plasma components of the flounder, the Ht, TP, GLU, TCHO, GOT and GPT of the fish were analyzed. For histological examination of the flounder, the gills, liver, spleen, kidney, stomach, intestine, and heart were extracted, and tissue specimens were prepared and examined according to the conventional method. Nonspecific cellular immune function of the flounder was administered aseptically and the CL (Chemiluminescence) response was investigated according to the conventional method. The nonspecific liquid immune function of the flounder was tested according to the conventional method. Serum of fish was determined by lysozyme activity test.

All the data were analyzed by one-way ANOVA test. If there were significant differences, Duncan's multiple range test was used to analyze the significance between the means (P <0.05). In order to investigate the anti-microbial effect of the flounder on the present material, Edwardsiella was tested on each fish. tarda (G3 strain) from 3.72 to 9.28 × 10 ⁵ CFU / mari (10) and Streptcoccus The iniae (2-24 strain) was intraperitoneally injected at a concentration of 1.17∼4.13 × 10 ⁷ CFU / horse (10 mice), and the cumulative mortality was assessed over 2 weeks. The anti-microbial effects of palm cactus-treated flounder by period are shown in Tables 4 and 5.

Growth, total weight, liver weight index, Ht TP, GLU, TCHO, GOT and GPT values of the olive flounder-treated olive flounder were not significantly different from the control group according to the administration period (P> 0.05). Cellular degeneration of the flounder tissues was not confirmed due to poisoning, and no significant difference was observed between the control groups. The CL response of the flounder administered with this material was significantly higher in the 4 week treatment group (P <0.05) than in the control group (p <0.05). . Serum lysozyme activity of the flounder was not significantly different from the control group according to the administration period (P> 0.05). The trend was shown.

As a result of the experiment of the present embodiment, it can be seen that the anti-microbial effect of the olive flounder administered with palm cactus extract was the best at 4 weeks.

As can be seen through the above embodiment, the treatment method of palm cactus extract for aquatic bacteria of aquaculture fish of the present invention ensures the food safety of aquatic products according to the use of therapeutic agents compared to the anti-fungal drugs of malachite green, etc. can do.

In addition, the oral administration of palm cactus extract against bacterial diseases of the flat flounder can minimize the emergence of drug-resistant strains following the use of drugs compared to the conventional oral administration of antibiotics, thus ensuring the stability of farmed fish. to be.

Claims (7)

In exterminating aquatic bacteria of fish eggs, The method of controlling aquatic bacteria of cultured fish, characterized in that the fibrous cactus stem hydrolysis fraction 50 to 80ppm bathed in fish eggs every 30 days for 30 minutes until the eggs become the eggs. In exterminating aquatic bacteria of fish, A method for extinguishing aquatic bacteria of cultured fish, characterized in that the palm cactus stem hydrolysis fraction 200ppm is soaked in the fish once or twice a day for 2 hours / day. In treating and preventing fish pathogens of farmed fish, A method for controlling fish pathogens of cultured fish, characterized in that 0.1% of palm cactus stem hydrolyzate is adsorbed on floating feed and administered to cultured fish for 4 weeks. delete delete delete delete

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