KR20000032630A - Red tide eliminating composition and therapeutic composition for ectoparasitic diseases of fish using alginate polygalacturonite oligomer derivative - Google Patents

Abstract

PURPOSE: A red tide eliminating composition and therapeutic composition for ectoparasitic diseases and ectobacterial diseases of fish containing alginate polygalacturonite oligomer derivative are provided by controlling the physiological properties of alginate. CONSTITUTION: An alginate polygalacturonite oligomer derivative is prepared by hydrolysis of alginic acid extracted from seaweed such as kelp and the methods of hydrolysis can be one of the followings: 1) alginic acid is put into the acidic/alkalic solution of 20-100°C, then stirred for hydrolysis; or 2) alginic acid is hydrolyzed by using alginase at pH 8-10. The alginate polygalacturonite oligomer derivative produced by hydrolyzing alginic acid having average molecular weight of no more than 8000 dalton, can be used as red tide eliminating composition, antibacterial agent against fish pathogen or therapeutic composition for ectoparasitic diseases of fish. The content of the alginate polygalacturonite oligomer derivative in the mixture needs to be no less than 60 %.

Description

Red tide remover and remedy for disease of fishes using alginate polygalacturonite oligomer derivatives

The present invention relates to a red tide annihilating agent and a fish disease treatment agent using an alginic acid oligosaccharide derivative, and more specifically, to the alginate oligosaccharide derivative having a red tide biokilling effect and a fish disease treatment effect prepared by hydrolysis of alginic acid, a natural polymer material, as an active ingredient. It relates to a red tide extinguishing agent and a fish disease treatment agent.

The research trend on alginic acid and alginic acid-oligo manufacturing has been progressed since the production of alginic acid-oligosaccharide by chemical method and alginase enzyme in Alginic acid was known in 1976. The applicability to the antimicrobial effect is suggested.

Thus, standard production methods for the separation and purification of alginic acid and alginic acid oligos have not yet been established, and studies of their functional parts are insufficient. An example of a foreign country that has recently attempted to industrialize with alginic acid is when it is applied to increase the "viscosity of fish feed for young fish" (日本 公開 特許 公報 昭 60-199353, 穉 魚 用 飼料). In other words, the nutrients of fry are embedded with an insoluble material made by reacting cationic and anionic water-soluble high molecular materials to produce granules or pellets with a diameter of 0.1 to 3 mm and are used as feed for fry. The growth rate of eel and flounder fry increased by about 1.4 times that of normal feed, suggesting the potential for livestock feed and human food additives.

Skjermo et al. [Immunostimulation of juvenile turbot (Scophthalmus maximus L.) using an alginate with high mannuronic acid content administered via the live food organism Artemia. Fish and Shellfish Immunology, 5 (7): 531-534: 13ref, 1995] showed an increase in survival when artificial infection of Vibrio anguillarum was performed after long-term oral administration of alginic acid to larvae of fry. This suggests that it triggered the immune system in the fish body, suggesting that the application of antimicrobial activity could be expanded.

In recent years, the north gate, etc., have alginic acid oligosaccharides prepared by reacting polymannurate and polygluronate using enzymes isolated from two types of Vibrio spp. As a result of antibacterial experiments, some common bacteria and intestinal bacteria have been reported to show a slight bacteriostatic action.

However, as the main parasites of the mass mortality of fish, the control method for the infection of Scutika insects includes formalin, hydrogen peroxide, and freshwater treatment (regarding the dynamics and rescue of Scuticociliatids parasitic in South Sea cultured flatfish. Korean Journal of Pathology, 10 (1): 21-29, 1997).

Scuticociliatids are protozoan larvae that parasitic in the epidermis or body of a fish and have been reported around 1985. Since 1988, Scuticociliatids have been known as representative parasites causing massive mortality in fish farms both domestically and internationally.乙 竹, etc., Para ラ メ Paralichthys olivaceus 穉 魚 の ス ク-チ カ 纖毛蟲 (膜 口 類), 養殖 年報, 9: 65-68, 1896).

The Scutica ciliates are 20-45 μm long, rugby-shaped, and the rim edges are actively moving as cilia. When infected, the fish is poorly eaten, the body color is blackening overall, and in severe cases, partial whitening of the epidermis, body surface, fins, redness and corrosion inside the gills, gill anemia, etc. (Fischthal, JH: A peritrichous protozoan, on hatchery book about Prog. Fish-oult, 11: 122-124, 1949).

In addition, they penetrate into the fin body scale scales, connective tissue beneath the dermis, and the brain at high density and are easily observed under a microscope.

And recently, according to Lee et al., The distribution of Scutica ciliforms in fish bodies contaminated with Scutica ciliforms has been extensively induced by these Scutica ciliforms into blood vessels, neural tissues, subcutaneous epidermal tissue and spinal cavity and gill tissues. Histopathological changes in fingerlings of Japaness flounder, Paralichthys olivaceus, with severe Scuticociliatosis. J. Fish Pathol., 7 (1): 151-160, 1994).

Meanwhile, aquatic medicines that appear in marine fish and are used for the prevention and remedy of bacteria, fungi, viruses, and parasites that cause massive mortality include sulfamonomethoxine sodium and sulfadimethoxine sodium. Sulfases, such as sulfisozol and sulfamerazine, and oxytetracycline HCl, tetratracycline, chlorotetracycline HCl, ampicillin trihydrate and amoxyllin antibiotics such as trihydrate, doxycycline hydrate, erythromycine thiocyanate, spiramycine enbonate, kitasamycin, chloramphenicol, and their combined antibacterial agents, oxolinic acid (oxolinic acid), flumequine, piromidic acid, nalidinic acid chemotherapeutic agent, nifurstyrenate sod nitrates such as nitrofuran, bithinol, and trichlorofon.

Using these medicines, a certain amount is administered in a bath or oral manner according to its own properties and needs, and the erosion and glidosis bacterium caused by the Flexibacter maritimus of the flounder, Vibrio disease caused by Vibrio anguillarum, Streptococcosis caused by Streptococcus spp., Edward swelling caused by Edward strain (Edwardsiella tarda) and Edward's disease causing hernia, Intestinal turbidity caused by Vibrio sp., Trichodina, Diseases causing epithelial cell disruption by ciliary insects such as Hymenostoniata and diseases caused by protozoa such as Ichtyophonus hoferi are being treated.

In addition, the same or similar diseases are prevalent in representative farmed fishes such as domes, defenses, jeopbolacs, and perch, resulting in complications and massive deaths, resulting in huge economic losses. Most marine fish and fish remedies use the drugs mentioned above and use extreme methods such as formalin, water temperature control, and freshwater baths for pathogens such as viruses and some parasites. have. Fundamentally, if a drug is misused without precise identification of the pathogen causing the disease, or if a small amount of drug is continuously administered to prevent it, new resistant bacteria will emerge. Persistence issues can pose serious risks to the health of consumers as consumers. In addition, formalin liquid, which is commonly used as the only primary treatment for scuticasis, which causes abundant mortality in aquaculture, should be used in a minimum amount because it is an environmentally regulated substance. Large quantities of farmed water are introduced into the sea, and the possibility of environmental degradation around the farm cannot be ignored.

In addition, what is currently used for red tide control is the method of depositing red soil on land by using the yellow soil spreading method, and when red tide occurs, the red tide is deposited by mixing the yellow soil with water and spraying it into the sea.

However, if we continue to use the loess spreading method, the problem of stockpiling and environmental destruction caused by the development of loess on land and the continuous inflow of loess into the sea may change the bottom quality of the sea and cause ecosystem changes such as transition of benthic organisms. have. Products developed other than loess (bacteria or drugs) for the control of red tide organisms are very insignificant and have not yet been applied to the field.

The present invention is to solve the above problems, to provide a red tide extinction agent that can extinguish the red tide by killing red tide organisms without causing environmental problems such as disturbing the ecosystem by effectively controlling the properties of alginic acid 1 There is a purpose.

In addition, there is a second object to provide an antimicrobial agent against fish disease bacteria by effectively controlling the alginic acid properties, and a third object to provide a fish parasitic agent using the same material.

The present invention for achieving the first object, provides a red tide quenching agent using an alginate polygalacturonite oligomer derivative prepared by hydrolyzing alginic acid as an active ingredient.

The alginic acid may be extracted from seaweeds such as kelp, and the alginic acid may be hydrolyzed by adding alginic acid to an acidic solution or an alkaline solution at a temperature of 20 to 100 ° C., followed by stirring. The method of hydrolyzing alginic acid using arginase enzyme can also be used. It is preferable to hydrolyze the pH at the time of hydrolyzing alginic acid using the said arginase enzyme in the range of 8-10.

In addition, the alginic acid oligosaccharide derivative preferably has a molecular weight distribution chart consisting of 1 to 16 sugars.

Together with the alginic acid oligosaccharide derivative, alginic acid having an average molecular weight of 8,000 or less may be used as a red tide annihilating agent.

At this time, it is preferable that the alginic acid and alginic acid oligosaccharide derivatives have an alginic acid oligosaccharide derivative content of 60% or more in a mixture of both substances.

The present invention for achieving the second object, provides an antimicrobial agent against fish disease bacteria comprising the alginic acid oligosaccharide derivative prepared by hydrolysis of alginic acid as an active ingredient.

In this case, as described above, alginic acid having an average molecular weight of less than 8,000 together with the alginic acid oligosaccharide derivative may be used as an antimicrobial agent against fish disease, and the alginate oligosaccharide derivative content in the mixture of both substances is preferably 60% or more.

The present invention for achieving the third object provides a fish parasite agent comprising an alginic acid oligosaccharide derivative prepared by hydrolysis of alginic acid as an active ingredient.

Like the antimicrobial agent of fish disease bacteria, alginic acid having an average molecular weight of less than 8,000 together with the alginic acid oligosaccharide derivative may be used as a fish parasitic agent, and the alginate oligosaccharide derivative content in the mixture of both substances is preferably 60% or more.

As described above, the alginic acid oligosaccharide derivative according to the present invention kills red tide by killing red tide, and can also be used as a therapeutic agent for fish disease by showing antibacterial effects against fish disease bacteria, and can kill fish parasites.

Hereinafter, the configuration and operation of the present invention will be described in detail through examples and experimental examples.

In the following Examples, the viscosity, which is a decrease in molecular weight, was measured at 100 rpm using a Brookfield Viscometer (USA), and the alginic acid derivative solution used in the measurement was 0.5% (W / W) in a tertiary deionized water solution such that 600 ml was used. After the alginic acid was completely dissolved, bubbles were removed and measured at 25 ° C., and the measuring spindle was measured using Nos. 1 to 4.

In addition, the measurement of total uronic acid content to confirm the purity of the alginate used for the first time, Haug and Larsen (Haug, A. and B. Larsen, Quantitive determination of the uronic acid composition of alginate, Acta. Chem. Scand., 16, 1908-1918, 1962). Molecular weight distribution analysis for the change of molecular weight over time by chemical hydrolysis and enzymatic hydrolysis was performed using gel pemeation chromatography (GPC) analysis system (JASCO, Model LC-90, Jasco Co., Japan). The analysis was carried out under the following conditions.

1. Device

System: JASCO GPC, Model (LCSS-905)

Pump: PU-980

Detector: RI-930

Injector: Rheodyne 7125

Oven: CO-965

2. Conditions

Column: Asahipak GS-220 HQ + GS-320 HQ + GS-520HQ + GS-620HQ (7.6mm ID x 300mm L)

Moble phase: Use 0.1M NaCl solution in deionized water

Flow rate: 1ml / min

Column Temp. : 40 ℃

Injection volumn: 200μL

3. Sample Analysis

MW maker: Pullulan (MW 853000, 95400, 23700, 5800), chitosan-oligosaccharides (MW 322.32, 483, 706, 876)

Alginate oligo derivative: 0.1% (W / V) solution (0.45μL membrane after filtration)

In addition, each pH value was measured by a pH meter when 1g of the prepared graded alginate oligosaccharide derivatives prepared in 100 mL deionized water, stirred, and then sufficiently dissolved, and the pH range of the total alginic acid derivatives ranged from 6.94 to 7.5.

Example 1 Extraction of Alginic Acid

Alginic acid used as a base material to prepare an alginic acid oligo derivative related to the present invention was extracted and purified as follows. That is, the seaweed is usually finely ground and then naturally dried. The dried kelp powder was prepared from alginic acid without iodine and alginic acid without iodine. That is, iodine was extracted with acetone solvent using an Soxlet extractor using an organic solvent, and the remaining kelp powder was extracted with NaHCO3 solution to separate alginic acid possessed by algae. After powder crystallization, lyophilization was performed to obtain a polymer alginate powder from which iodine was removed. The extracted iodine was hydrolyzed by alginic acid and then applied in an appropriate amount again to each example.

However, alginic acid with iodine did not use organic solvent extraction. Enzymes used for the decomposition of macromolecular alginate for the control of physical properties that may exhibit the antimicrobial effect of red trough plankton, fish disease parasites and fish pathogens identified in the present invention are arginase (sigma, Lot No .: A2137). Was used, and the reverse value of the enzyme was 12,500 units / g.

Example 2 Preparation of Alginic Acid Oligosaccharide Derivatives

In a 0.5 L three-necked flask, 0.3 L of 2N-HCl solution was added, and the temperature was heated to 80 to 100 ° C., and 30 g of the polymer alginic acid was added thereto, followed by stirring to hydrolyze. In order to stop the hydrolysis by time, the pH was adjusted to 5 with 10N-NaOH and 3N-HCl solution to terminate the hydrolysis and the reaction, and the alginate, which is an intermediate hydrolyzate in powder form, was separated and the reaction solution was removed. In addition, after the filtered intermediate was added, the mixture was further dispersed to 1% solution (w / v) with normal filtered seawater and tertiary deionized water, and then sufficiently dispersed, and then the pH was adjusted to 7-9.

Finally, low molecular weight alginic acid and alginic acid oligosaccharide derivatives were obtained over time.

Example 3 Preparation of Alginic Acid Oligosaccharide Derivatives

After filling the 3L beaker with 2 L of filtered seawater and tertiary deionized water, the pH was adjusted to 12, and additionally added 20 g of polymer alginic acid at room temperature to completely dissolve. The 0.5L three-necked flask was filled with an alkaline solution adjusted to pH 12 of 0.3L, and then warmed by varying the temperature to 65-85 ° C., respectively. Let's do it. In order to stop the hydrolysis by time, the pH was adjusted to 7-9 with 10N-NaOH and 3N-HCl solution to obtain a hydrolyzed low molecular weight alginic acid. In addition, the pH was adjusted to 7-9 with 1% solution (w / v) with normal filtered seawater and tertiary deionized water. Finally, the solution-derived low molecular weight alginic acid and alginic acid oligo derivatives were obtained over time.

Note) * Oligosaccharide production (content of less than 10 sugars is 70% or more)

Standard deviation of real-time results: mean ± 5 to mean ± 0.5

Example 4 Preparation of Alginic Acid Oligosaccharide Derivatives

2L of tertiary deionized water was added to a 3L beaker, and the temperature was adjusted to 37 ° C. and the pH was adjusted to 9.5. The polymer alginic acid was stirred to a solution of 30 g (1%, w / v), and dissolved in a 0.5L three-necked flask. An amount of 300 ml was aliquoted, and arginase 25, 10, 5, 2.5, 1.5 was added thereto, and hydrolysis was performed.

The reaction was carried out for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, and 24 hours with stirring at 150 rpm at a pH of 9.5 and a temperature of 37 ° C. In order to stop the enzymatic reaction at each time, each three-necked round flask was sterilized at 80 ° C. for 30 minutes to stop the activity of the enzyme, and then the precipitate precipitate was filtered off and finally the low molecular weight of the solution phase obtained by time. Alginic acid and alginic acid oligo derivatives were prepared, and their respective molecular weights were measured by viscosity, and are shown in Table 2 below.

Note) * Oligosaccharide production (content of less than 10 sugars is 70% or more)

Standard deviation of real-time results: mean ± 7 to mean ± 0.5

Example 5 Preparation of Fish Parasites and Alginic Acid Oligosaccharide Derivatives for Assaying Antiparasitic Effects

Isolation of Scuticociliatids to be used in the present invention is to collect 20 individuals of cultured flounder (Paralichthys olivaceus) 5-20 cm in length infected with Scutica ciliform, and isolated and harvested from the wound site of these individuals. Was used for. Scuticociliatids were collected in a medium containing 10% FBS in Eagle's MEM (Gibco) without antibiotics and immediately used as a test material. Alginate derivatives were used to test antiparasitic effects. The alginate derivative having the first physical properties (450 cps) and the physical properties of the alginate oligosaccharide (1-16 sugar) in the range of 8.6 to 13 cps having the characteristics of general alginic acid among the derivatives obtained in Examples 1 to 3 Only those possessing the alginic acid oligomerized physical property at 1.2 cps, which is 70% or more, were selected as shown in Table 3 below.

Table of GPC Analysis of Alginate Oligosaccharides Obtained Antiparasitic Effect by Molecular Weight division Molecular weight range Average molecular weight (Mw) Content of alginic acid / (* alginic acid oligosaccharide) Remarks General Alginic Acid (AOH-0) 1,358,721-72,676 361,381 100 (0) AOH-1 5,326-279 2,227 38.74 (61.25) AOH-2 128,089-122 7,582 39.23 (60.76) AOH-3 16,406-259 1,722 20.84 (79.16)

Note) * Alginate oligosaccharide was composed of 2 to 16 sugars.

Experimental Example 1. Killing Effect of Alginate Oligosaccharide Derivatives of Scuticociliatids

(Experimental method)

Collected Scutica ciliates were adjusted to 3.4 × 10 4 subjects / ml in a tube containing 10 ml of normal seawater, and then housed at room temperature at 17 ° C. Then, the prepared alginic acid oligo derivatives were prepared for each type, and a control group in which the alginate oligo acid derivatives were not added for each concentration of a derivative having physical properties according to molecular weight shown in Table 4 was set. The experimental group was also set up with five experimental groups to which 0.05%, 0.04%, 0.03%, and 0.01% of the same alginic acid derivatives were added, respectively, and before administration of the alginic acid oligo derivative (0 minutes), after administration. From 0.1 to 2, 3, 4, 5, 30 minutes, 1 hour, 3 hours, and 10 hours, 0.1 ml of each hour is collected, and the number of killings is counted three times for the total population under the microscope. At the same time, a CC camera was attached to the microscope to observe the morphology and morphology of Scutica ciliary worms over time.

As a result, as shown in Table 4 below, when AHO-3 was used, the 0.1% alginic acid derivative-added group showed a killing effect of the entire Scutika insects that caused fish tissue invasion and necrosis within 5 minutes after the addition.

And 0.05% treatment group 30-40 minutes, 0.03% 100% killing effect was observed after 2 hours or more.

However, the killing effect was not recognized when the concentration of 0.01% or less was added.

In the case of AHO-1 and AHO-2, antiparasitic effects did not appear.

In order to confirm the effect of alginic acid oligosaccharide derivatives on the killing process, Scutica was directly contacted with alginic acid oligosaccharide derivatives having a concentration of at least 0.03% (v / v). Immediately after contact, the active motility of the Scutica ciliary deteriorated rapidly, and as time passed, the contractile vesicles expanded, changing from streamline to protoplast, and the parasite was completely destroyed after 2 hours.

AOH-3 killing effect test result of Scutica ciliate (expressed as cumulative value) Test group Test conditions (cap tube, 10ml, 17 degrees) Death effect (%) Elapsed time and percent mortality after administration of alginic acid oligo derivatives 0 min 1 minute 2 minutes 3 minutes 4 minutes 5 minutes 30 minutes 1 hours 3 hours 10 hours Comparative example No administration 0 0 0 0 0 0 0 0 0 0 Example 0.1% administration group 0 100 100 100 100 100 100 100 100 100 0.05% administration group 0 100 100 100 100 100 100 100 100 100 0.04% administration group 0 88 90 92 92 95 98 99 100 100 0.01% administration group 0 12 24 32 45 54 67 77 100 100 0.008% administration group 0 0 0 0 0 0 0 10 12 30

Note) 1. Significance review: (P <0.01)

2. Average error range: ± 5 ～ ± 24

Experimental Example 2. Antimicrobial Effect against Fish Bacteria

(Choice of fish strains for antibacterial assay)

Fish strains for the antimicrobial assay were selected as the most common bacterial pathogens in aquaculture. It is characterized by the appearance of fin spots and distinct spots and bleeding spots in the abdomen and anus are observed as the symptoms progress. Edwardsiella tarda Edk-2, which causes bloating and hernia symptoms, Vibrio anguillarum PT-493, a cause of Vibrio disease, which causes ulcers with enlarged scales, fin collapse and defects , Streptococcus sp., Which causes redness or rash of the gill cover and gill soft bone mesentery, and inflammatory reactions of the liver and the digestive tract, or multiple fillings. NG8266, Staphylococcus sp. HR-1 was used. Four strains except Aeromonas hydrophila ATCC7966 strains were identified and isolated from Pukyong National University.

(Preparation of Media for Antimicrobial Assay and Cultivation of Pathogens)

Nutrient broth (NB) was added to 1% (w / v) of NaCl medium (NBN) was autoclaved for 15 minutes at 121 ℃ and then used as a basal medium for the culture of bacteria. Streptococcus sp. In the case of NG8266 and Vibrio anguillarum PT-493, Brain Heart Infusion (BHI) medium was used for species culture. The medium preparation for this culture was prepared by adding 1% solution of alginic acid oligosaccharide derivative adjusted to pH 6.5 and sterilized distilled water to the doubled concentration of NB medium (pH 6.5) autoclaved at 121 ° C. for 15 minutes. The final concentrations were 0.5% (w / v), 0.1% (w / v), 0.02% (w / v) and 0.004% (w / v), and control medium was added with no additive NBN medium. The main culture was inoculated with 0.1% (v / v) of the culture medium incubated overnight, 26 ° C for Vibrio anguillarum, and other strains Aeromonas hydrophila ATCC7966, Edwardsiella tarda Edk-2, Streptococcus sp. NG8266 and Staplylococcus sp. HR-1 was incubated for 24 hours at 37 ℃ for 24 hours at 200rpm. The number of colonies that survived by time was confirmed by colony count appearing on nutrient solid medium and cultured, and converted to percentage based on the administration of the alginate oligosaccharide for the first time.

(Antibacterial activity against fish disease bacteria)

After overnight incubation of the culture medium, the culture medium was inoculated with 105 to 106 bacteria per ml, and at 26 ° C for Vibrio anguillarum PT-493, Aeromonas hydrophila ATCC7966, Edwardsiella tarda Edk-2, Streptococcus sp. NG8266 and Staphylococcus sp. HR-1 was shaken at 37 rpm for 200 rpm. After 12 hours, the susceptibility to growth of microorganisms was confirmed by measuring the susceptibility with a spectrophotometer at a wavelength of 600 nm. Degree differences are shown in Table 5 below as a percentage of control suspension.

Antimicrobial activity against fish disease strains (%, OD measurement 12 hours after preparation) Strain Distilled Water Medium (pH6.5) Seawater Medium (pH6.5) 0.5 0.1 0.02 0.004 0.5 0.1 0.02 0.004 Aeromonas hydrophilaATCC 7966 100 100 100 92 100 100 100 89 Edwardsiella tardaEdk-2 100 100 100 100 100 100 100 98 Streptococcus spNG 8266 100 100 100 100 100 100 100 60 Staphylococcus spHR-1 100 100 100 100 100 100 100 100 Vibrio anguillarumPT-493 100 100 100 100 100 100 100 45

(Antibacterial Activity of Alginate Oligosaccharide Derivatives in Seawater Environment)

Cell cultures obtained by centrifugation of 1 ml of cultures in NBN and BHI medium for each strain were centrifuged at 15,000 rpm for 30 seconds with a microcentrifuge. After diluting the suspension with 104-105 bacteria / ml, the alginate oligosaccharide derivative solution was concentrated to a final concentration of 0.5% (w / v), 0.1% (w / v), 0.02% (w / v) and 0.004% (w / v). ) Was added and mixed. The mixture was rotated at low speed (50rpm) at 22 ℃, which is a general seawater temperature, and then 0.1ml was directly smeared on the appropriate medium after 1 hour, 2 hours, 4 hours, 6 hours and 12 hours, respectively. The reduction rate of viable cells compared to the number of cells grown by smearing 0.1 ml of is shown in Table 6 below. Bacterial killing rate was different by strain, but the number of viable cells decreased rapidly until 1 hour after contact with microorganism and alginate oligosaccharide derivatives, and there was no change in the number of cells. In this case, it could be confirmed that the 3 hour treatment can achieve the purpose at the concentration of administration, and it can be seen that it is necessary to repeat the treatment for a certain period in order to reduce the number of cells under the onset concentration.

Antimicrobial Assay of Alginate Oligosaccharide Derivatives by Concentration and Hours in Fish Pathogens (%) Strain name 0 One 2 4 6 12 Aeromonashydrophila ATCC 7966 0 100 102 107 115 128 136 0.004 100 26 - - 43 100 0.02 100 56 - 100 100 100 0.1 100 100 - - 100 - 0.5 100 100 - - - 100 Edwardsiellatarda Edk-2 0 100 101 107 112 116 165 0.004 100 21 - - 56 98 0.02 100 - 73 - - 100 0.1 100 - 100 100 - 100 0.5 100 100 - - - 100 Streptococcussp. NG 8266 0 100 102 - - - 198 0.004 100 45 0.02 100 61 89 100 - 100 0.1 100 100 - - 100 100 0.5 100 100 - 100 - 100 Staphylococcus sp. HR-1 0 100 106 - - - 202 0.004 100 67 0.02 100 85 - - - 100 0.1 100 98 100 - 100 100 0.5 100 100 - - 100 100 Vibrioanguillarum PT-493 0 100 105 - - - 198 0.004 100 13 - 68 98 100 0.02 100 66 75 88 100 100 0.1 100 88 100 - - 100 0.5 100 96 - 100 - 100

Note) 1. Average deviation of viable cells: ± 8-12%

2.-: No measurement

Experimental Example 3. Killing of Red Tide by Alginate Oligosaccharide Derivatives

Red tide organisms selected for the red tide killing test using the alginic acid oligosaccharide derivatives of the present invention are Cochlodonium polykrikoides and Gymnodinium sanguineum, one of the red tide organisms, which have caused red tide in the coastal area of the South Sea in Korea in recent years. In order to verify the effect of alginic acid oligosaccharide derivatives on the control of red tide organisms, Cochlodonium polykrikoides was grown in the laboratory and Gymnodinium sanguineum and alginic acid oligosaccharide derivatives collected in the field were contacted. Red algae in contact with the alginic acid oligosaccharide derivatives destroyed the cells. Treatment compositions and results of alginic acid oligosaccharide derivatives are summarized in Table 7 below.

Effect of Alginate Oligosaccharide Derivatives on Red Tide Killing Effect Creature Alginate Oligosaccharide Derivative Concentration Kill time Remarks Cochlodiniumpolykrikoides 0.05% 0.025% 0.01% 0.005% 0.0007% Within 20 seconds 2 minutes 10 seconds 2 minutes 40 seconds 4 minutes 30 seconds 14 minutes Gymnodiniumsanguineum 0.05% 0.025% 0.01% 0.005% 0.0007% Within 25 seconds 3 minutes 13 seconds 6 minutes 20 seconds 10 minutes 30 minutes

As described above, the present invention can be used as a red tide annihilating agent or red tide biokilling agent by preparing a derivative of alginic acid, which is a natural polymer material, with excellent red tide extinction or red tide killing effect, antimicrobial effect against fish disease bacteria and killing of parasites. It can be used as an antimicrobial agent against fish disease bacteria and can be used as a fish parasite agent.

Claims (14)

Red algae quenching agent using an alginate polygalacturonite oligomer derivative prepared by hydrolysis of alginic acid as an active ingredient. The red tide quenching agent of claim 1, wherein the alginic acid is extracted from kelp. 2. The red tide annihilating agent according to claim 1, wherein the alginic acid is hydrolyzed by adding alginic acid to an acidic solution or an alkaline solution at a temperature of 20 to 100 ° C, followed by stirring. The red tide quenching agent according to claim 1, wherein the alginic acid is hydrolyzed using an arginase enzyme. The red tide annihilating agent according to claim 4, wherein the alginate is hydrolyzed using the arginase enzyme in a pH range of 8 to 10. The red tide annihilating agent according to claim 1, wherein the alginic acid oligosaccharide derivative has a molecular weight distribution consisting of 1 to 16 sugars. The red tide annihilating agent according to any one of claims 1 to 6, wherein alginic acid having an average molecular weight of 8,000 or less is used as the active ingredient together with the alginic acid oligosaccharide derivative. 8. The red tide quenching agent according to claim 7, wherein the alginic acid and alginic acid oligosaccharide derivative has an alginic acid oligosaccharide derivative content of 60% or more in a mixture of both substances. An antimicrobial agent against fish pathogens comprising alginic acid oligosaccharide derivatives prepared by hydrolysis of alginic acid as an active ingredient. The antimicrobial agent of claim 9, wherein the alginate oligosaccharide derivative is used as an active ingredient with alginic acid having an average molecular weight of 8,000 or less. 10. The antimicrobial agent of claim 9, wherein the alginic acid and alginic acid oligosaccharide derivatives have an alginic acid oligosaccharide derivative content of 60% or more in a mixture of both substances. A fish parasitic agent comprising an alginic acid oligosaccharide derivative prepared by hydrolysis of alginic acid as an active ingredient. The fish parasite preparation according to claim 12, wherein the alginic acid oligosaccharide derivative is used as an active ingredient with alginic acid having an average molecular weight of 8,000 or less. The parasitic insecticide according to claim 12, wherein the alginic acid and alginic acid oligosaccharide derivatives have an alginic acid oligosaccharide derivative content of 60% or more in a mixture of both substances.