WO2011025133A1

WO2011025133A1 - Water soluble free amine chitosan with algicidal activity against harmful algal bloom species induced red tide, anti-red tide agent including the same as an active ingredient, and removal method of red tide using the same

Info

Publication number: WO2011025133A1
Application number: PCT/KR2010/003914
Authority: WO
Inventors: Jae Woon Nah; Yoonkyung Park
Original assignee: DYNAMIC Co Ltd
Current assignee: DYNAMIC Co Ltd
Priority date: 2009-08-31
Filing date: 2010-06-17
Publication date: 2011-03-03
Anticipated expiration: 2012-02-29
Also published as: KR101099075B1; KR20110023307A

Abstract

The present invention relates to a water soluble free amine chitosan with algicidal activity against harmful algal bloom species induced red tide, an anti-red tide agent including the same as an active ingredient, and a removal method of red tide using the same. A water-soluble free amine chitosan according to the present invention is acted on cell membranes of the red tide-causing algae to exhibit excellent anti-red tide activities by inducing the water-soluble free amine chitosan to destroy the cell membranes, and the water-soluble free amine chitosan is optionally acted on the red tide-causing algae only such that the water-soluble free amine chitosan can be usefully used as a safe anti-red tide agent in the marine ecosystem.

Description

WATER SOLUBLE FREE AMINE CHITOSAN WITH ALGICIDAL ACTIVITY AGAINST HARMFUL ALGAL BLOOM SPECIES INDUCED RED TIDE, ANTI-RED TIDE AGENT INCLUDING THE SAME AS AN ACTIVE INGREDIENT, AND REMOVAL METHOD OF RED TIDE USING THE SAME

The present disclosure relates to a water soluble free amine chitosan with algicidal activity against harmful algal bloom species induced red tide, an anti-red tide agent including the same as an active ingredient, and a removal method of red tide using the same.

Red tide means a event that microscopic organisms such as animal or phytoplankton, protozoan and bacteria generally living in the ocean are rapidly increased in large amounts or biologically or physically accumulated to change colors of the seawater and have bad effects on marine organisms.

The red tide is widely generated in all coastal waters all over the world, and is frequently generated particularly in Seto inland sea of Japan, Californian coast of the United States of America, coasts of the Southeast Asia, and coats of the North Sea. Recently, red tide frequently generated in Southern coastal seas, Western coastal seas and Southeast coastal seas of Korea has routinely been generated in all seasons, and particularly red tide organisms are showing tendencies that they are changed from diatoms to flagellated algae and their densities are increased.

In case of our country, Korea, the red tide had been generated in July and August by 1981, and the red tide has been generated even in April after 1981 to show a tendency that the red tide has been generated increasingly quickly. Moreover, the red tide is observed from some waters of certain seas even in December such that the width of the generation period of the red tide is widened. Although the main generating period of the red tide was a high water temperature period of June to September every year, and the duration of the red tide was also about 20 days in 1981, the red tide has been continued usually for about one week to two weeks except long-term red tide of the bay of Masan after 1981. On the other hand, the red tide is generated in coasts of the whole country. Although the red tide is generated very intermittently in the East Sea and Yellow Sea of Korea, the red tide is generated throughout the year in the southern coast of Korea, particularly in the bay of Jinhae. Although the red tide has locally been generated on a small scale in closed back bays of Korea before 1980, an unprecedented red tide was generated on a large scale in the whole region of the bay of Jinhae in 1981. However, a tendency that the red tide has been locally generated again from 1982 is shown and the red tide has been generated from 1982 in conditions similar to those of 1984 although waters in which the red tide is generated may differ slightly by year. Such red tide is frequently being generated in southwestern coasts of Korea, southern coasts of the East Sea, particularly in closed back bays including Jinhae bay, Kwangyang bay, Onsan bay, and others. The red tide has routinely been generated every year particularly in the bay of Masan, and the red tide has considerably frequently been generated also in the western coast of the Jinhae bay including the bay of Dangdong and the bay of Wonmoon. Harmful red tide directly killing fish has been generated in surrounding waters of Saryang Island particularly since 1989.

The generation of such red tide brings about fatal damages to men and fish and shellfish. Namely, since there are many toxic species of flagellatae to cause death of fish and shellfish and to cause poisoning of the man when a man eats the toxified fish and shellfish. The red tide is adhered to gills of fish and shellfish such that the fish and shellfish are suffocated by the red tide. The red tide caused great damage to domestic raising industries since there is lack of oxygen in water due to oxidation decomposition of the red tide to cause death of the fish and shellfish. When the red tide is generated, the fish and shellfish are suffocated by the lack of dissolved oxygen in water, the fish and shellfish are poisoned to death by poisonous substances produced by red tide organisms, or toxic materials such as secondary hydrogen sulfide, methane gas, and ammonia, and the value of fishing ground is lowered. Further, various shellfish poisonings may occur in the men if men eat fish and shellfish which had eaten toxic phytoplankton.

Organisms causing red tide are phytoplankton such as flagellated algae, diatom and protozoan, wherein the flagellated algae moves using a flagellate and also ingests decayed nutrients in addition to photosynthesis. The flagellated algae is reproduced at a slow rate, i.e., a reproduction of the flagellated algae through division takes 2 to 5 days, and the flagellated algae mainly causes direct damages. Further, the diatom does not have a flagellate, and mobility of the diatom is none or weak. The diatom is reproduced at a fast rate, i.e., a reproduction of the diatom through division takes 0.5 to 2 days, and the diatom mainly causes indirect damages. It is known that the protozoan moves using cilia, a moving speed of the protozoan is very high, and the protozoan is generated in a brackish zone primarily influenced by fresh water. Typical red tide-causing organisms generated in Korea may include six main types of organisms such as Cochlodinium polykrikoides, Gymnodinium impudicum, Akashiwo sanguine, Lingulodinium polyedrum, Prorocentrum micans, and Gymnodinium catenatum.

An environmental factor in which red tide-causing organisms proliferate can be divided into three main environmental factors.

A first environmental factor is closed waters which are in the geological form of a back bay, and which are less exchanged with waters in the outer part of the sea. A second environmental factor should be the summer season in which a living water temperature of red tide organisms, 15 to 20 ℃, is capable of being maintained when it is seen from the physical aspect. An additional condition is that the amount of sunshine required for photosynthetic activities of these red tide organisms should be sufficient, or stable water mass should be formed. A third environmental factor is that nutrients, fertilizer constituents required for the growth and propagation of the red tide organisms, are flown in the seawater from the land by rainfall and others such that the nutrients should be dissolved into the seawater. It is particularly known that nitrate and phosphate act as factors of limiting the development of the red tide organisms, and vitamin complex, traces of elements, and others in addition to the nitrate and phosphate should be dissolved into the seawater.

Various methods for preventing red tide or reducing damages due to the red tide have been studied until now. There are methods eliminating the red tide organisms chemically and physically by spraying chemicals as suggested by Steidinger in 1983 and Ryu et al. in 1998, or by using an ultrasonic homogenizer, an ozone generator, and a centrifuge. Further, methods of settling the red tide-causing organisms on the bottom by adsorbing red tide-causing organisms using clay have been developed by Na et al. in 1996 and Choi et al. 1998. However, the above methods may have another bad effect on the ecosystem, and have many problems when using the methods in a wide sea area at which the red tide is generated. Biological methods of removing the red tide-causing organisms are being made currently. The biological methods are methods of removing unwanted organisms using the prey-predator relationship within the ecology system and have been suggested by Ishio et al. in 1989, Sakata et al. and Fukami et al. in 1992, and Part et al. in 1998. However, the above methods may cause other problems by changing the food chain of the ecology system. Methods of killing the red tide organisms by substances secreted by marine bacteria are being actively studied in other fields. Attempts have recently been made to control the red tide organisms using protein synthesis inhibitors, cell wallinhibitors, and antibiotics acting on cell membranes, or low molecular materials may artificially produced through the organic synthesis using these substances as models, wherein the low molecular materials are not decomposed in the sea, but are accumulated in the sea to result in a problem that the low molecular materials are accumulated in the human being through food produced from the marine ecosystem or sea just as problems caused by agricultural chemicals used in the agricultural field.

A chitosan preparation is a preparation capable of being used as an anti-red tide agent which is nature friendly and is harmless to the human being or nature ecosystem when considering such problems. The chitosan preparation can be produced in a large amount since chitosan is derived from chitin forming exoskeletons of sea organisms such as crabs, shells and others, and the chitosan preparation may be nature friendly in the aspect that chitosan is substance extracted from the sea organisms.

Chitosan as β-1,4 linked pyranosa units of glucosamine is a natural polymer in which 5,000 or more of glucosamine residues are linked to one another, and which has a molecular weight of 1,000,000 or more. Such chitosan may be extracted from a marine system including crustacean such as crab shell or shrimp, and squid. The chitosan has a molecular structure similar to that of cellulose, a type of polysaccharide. The chitosan is applied to the pharmaceutical industry since the chitosan is excellent in biocompatibility to prevent rejection from being occurred during the immune reaction. After the certification of chitosan as food has recently been received from the Food and Drug Administration of the USA, chitosan is being applied to material for important biological industry and biomedical uses. The chitosan has widely been used in the a wastewater treatment field including flocculant, heavy metal adsorbent, dyeing wastewater-clarifying agent and others, and an agricultural field including soil ameliorator, insecticide, plant antiviral agent, agricultural chemicals and others as main use examples of the chitosan. Recently, uses of the chitosan have further been expanded into food and beverage application fields, health and hygiene application fields, cosmetics application field, textile-related application field, and pharmaceutical application field. It is expected that the chitosan can be applied to the health food field, food and beverage application fields, cosmetics application field, health and hygiene fields, and pharmaceutical supplies fields since chitosan with a specific molecular weight range of 20,000 to 100,000 is known to have a strong physiological activity function.

However, little has been known about an effect of conventional chitosan on anti-red tide activities. Thus, the present inventors have completed the present invention by finding out that a water-soluble free amine chitosan not only exhibits excellent anti-red tide activities, but also is optionally applied to red tide-causing algae only such that the chitosan can be usefully used as a safe anti-red tide agent in the marine ecosystem while performing research to develop an anti-red tide agent which is nature friendly and harmless to the human being or nature ecosystem.

One object of the present invention is to provide a water-soluble free amine chitosan having anti-red tide activities against red tide-causing algae.

Another object of the present invention is to provide an anti-red tide agent containing the water-soluble free amine chitosan as an active ingredient.

Still another object of the present invention is to provide a method of spraying the anti-red tide agent containing the water-soluble free amine chitosan as the active ingredient in red tide-generating waters to remove red tide.

In order to achieve the objects, the present invention provides a water-soluble free amine chitosan which has anti-red tide activities against red tide-causing algae, and of which a molecular weight is 20,000 to 100,000 daltons.

Furthermore, the present invention provides an anti-red tide agent containing the water-soluble free amine chitosan as an active ingredient.

Furthermore, the present invention provides a method of spraying the anti-red tide agent containing the water-soluble free amine chitosan as the active ingredient in red tide-generating waters to remove red tide.

A water-soluble free amine chitosan according to the present invention is acted on cell membranes of the red tide-causing algae to exhibit excellent anti-red tide activities by inducing the water-soluble free amine chitosan to destroy the cell membranes, and the water-soluble free amine chitosan is optionally acted on the red tide-causing algae only such that the water-soluble free amine chitosan can be usefully used as a safe anti-red tide agent in the marine ecosystem.

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

fig. 1a to fig. 1e are optical microscopic images of algae treated with the water-soluble free amine chitosan to examine anti-red tide activities of a water-soluble free amine chitosan according to the present invention, wherein fig. 1a shows Heterosigma akashiwo as a red tide-causing alga, fig. 1b shows Chattonella sp. as the red tide-causing alga, fig. 1c shows Cochlodinium polykrikoides as the red tide-causing alga, fig. 1d shows Prorocentrum minimum as the red tide-causing alga, and fig. 1e shows Skeletonema costatum as a harmless marine alga;

fig. 2a and 2b are graphs illustrating cell density variations of algae treated with the water-soluble free amine chitosan for measuring an optional annihilation extent of red tide-causing algae of a water-soluble free amine chitosan according to the present invention, wherein fig. 2a is a graph illustrating cell density variations of Skeletonema costatum and Heterosigma akashiwo treated with the water-soluble free amine chitosan, and fig. 2b is a graph illustrating cell density variations of Skeletonema costatum and Prorocentrum minimum treated with the water-soluble free amine chitosan;

fig. 3 is optical microscopic images of Skeletonema costatum and Chattonella sp. treated with the water-soluble free amine chitosan for measuring an optional annihilation extent of red tide-causing algae of a water-soluble free amine chitosan according to the present invention;

fig. 4a and 4b are confocal laser scanning microscopic images of the red tide-causing algae treated with the water-soluble free amine chitosan marked by tetramethylrhodamine for analyzing the action part in red tide-causing algae of a water-soluble free amine chitosan according to the present invention, wherein fig. 4a is a confocal laser scanning microscopic image of Heterosigma akashiwo, and fig. 4b is a confocal laser scanning microscopic image of Prorocentrum minimum; and

fig. 5a and fig. 5b are continuously taken multifocal images using a confocal laser scanning microscope of the red tide-causing algae treated with the water-soluble free amine chitosan marked by tetramethylrhodamine for analyzing the action part in red tide-causing algae of a water-soluble free amine chitosan according to the present invention, wherein fig. 5a is a continuously taken multifocal image using a confocal laser scanning microscope of Heterosigma akashiwo, and fig. 5b is a continuously taken multifocal image using a confocal laser scanning microscope of Prorocentrum minimum.

Features and advantages of the present invention will be more clearly understood by the following detailed description of the present preferred embodiments by reference to the accompanying drawings. It is first noted that terms or words used herein should be construed as meanings or concepts corresponding with the technical sprit of the present invention, based on the principle that the inventor can appropriately define the concepts of the terms to best describe his own invention. Also, it should be understood that detailed descriptions of well-known functions and structures related to the present invention will be omitted so as not to unnecessarily obscure the important point of the present invention.

The present invention provides chitosan having anti-red tide activities in red tide-causing algae.

Hereinafter, the present invention will be described in detail.

A chitosan having anti-red tide activities according to the present invention is a water-soluble free amine chitosan, and it is preferable to use the water-soluble free amine chitosan prepared by a method mentioned in Korean Registration Patent No. 10-0441270 applied for and registered by the present inventors, wherein the water-soluble free amine chitosan preferably has a molecular weight of 20,000 to 100,000 daltons.

The water-soluble free amine chitosan may exhibit anti-red tide activities to red tide-causing marine algae such as Chattonella sp., Prorocentrum minimum, Prorocentrum micans, Heterosigma akashiwo, Cochlodinium polykrikoides, Chattonella marina, and the like.

The water-soluble free amine chitosan may be prepared by a method mentioned in Korean Registration Patent No. 10-0441270 applied for and registered by the present inventors. More specifically, pure water-soluble free amine chitosan may be prepared by the method comprising (1) treating an organic or inorganic acid salt solution of chitosan oligosaccharide with trialkylamine that is a base, (2) adding an organic solvent in the solution to recover the chitosan oligosaccharide in which an organic acid or inorganic acid linked to the chitosan oligosaccharide is removed in the form of a trialkylamine salt and (3) purifying the treated chitosan oligosaccharide solution using an activated carbon/ion exchange resin column after treating the acid-removed chitosan oligosaccharide solution with an inorganic acid. The pure water-soluble free amine chitosan prepared by performing the foregoing method shows physiological activities since the pure water-soluble free amine chitosan has a molecular weight of 1,000 to 100,000 daltons as well as a high solubility with respect to water.

A water-soluble free amine chitosan according to the present invention may be used as a nature friendly anti-red tide agent by exhibiting anti-red tide activities. This is described based on concrete test results.

First, referring to Example 1, Table 1 and fig. 1, it can be seen that a water-soluble free amine chitosan according to the present invention exhibits excellent anti-red tide activities by showing 0.25 mg/ml or less of growth inhibitory concentration of red tide-causing marine algae and showing 0.5 mg/ml or less of 100% growth inhibitory concentration in tests of measuring anti-red tide activities of the water-soluble free amine chitosan.

Furthermore, referring to Example 2, fig. 2 and fig. 3, it can be seen that the water-soluble free amine chitosan is optionally acted on the red tide-causing marine algae only to annihilate the red tide-causing marine algae since the water-soluble free amine chitosan annihilates the cells of the red tide-causing marine algae by decreasing the cell density of the red tide-causing marine algae to almost zero although a water-soluble free amine chitosan according to the present invention hardly influences the density of cells of Skeletonema costatum that is a harmless marine alga in tests of measuring optional annihilation extents of red tide-causing algae.

Furthermore, referring to Example 3, fig. 4 and fig. 5, it can be seen that a water-soluble free amine chitosan according to the present invention is acted on cell membranes of the red tide-causing marine algae in tests of analyzing acted parts of red tide-causing marine algae to exhibit anti-red tide activities by inducing destruction of the cell membranes.

Therefore, a water-soluble free amine chitosan according to the present invention is acted on cell membranes of the red tide-causing algae to exhibit excellent anti-red tide activities by inducing the water-soluble free amine chitosan to destroy the cell membranes, and the water-soluble free amine chitosan is optionally acted on the red tide-causing algae only such that the water-soluble free amine chitosan can be usefully used as a safe anti-red tide agent in the marine ecosystem.

Furthermore, the present invention provides an anti-red tide agent containing as an active ingredient a water-soluble free amine chitosan which has anti-red tide activities against red tide-causing algae, and of which a molecular weight is 20,000 to 100,000 daltons.

Furthermore, the present invention provides a method of spraying the anti-red tide agent containing as an active ingredient the water-soluble free amine chitosan which has anti-red tide activities against red tide-causing algae, and of which a molecular weight is 20,000 to 100,000 daltons in red tide-generating waters to remove red tide.

Hereinafter, the present invention will be described in more detail with reference to the following preparation examples and examples. However, the following preparation examples and examples are provided for illustrative purposes only, and the scope of the present invention should not be limited thereto in any manner.

<Preparation Example 1> Preparation of a water-soluble free amine chitosan having a molecular weight of 20,000 daltons

A 5% chitosan solution was prepared using lactic acid as solvent. 100 ml of the 5% chitosan solution having a pH of 5.0 to 5.5 and chitosanase enzyme derived from Bacillus pumilus BN-262 of a unit 5 were mixed and reacted with each other at 40 ℃ for 36 hours. After finishing the reaction process, the reactant was primarily filtered using a prefilter with a pore size of 1 ㎛, and the primarily-filtered reactant was filtered again using a hollow fiber filter paper with a molecular weight of 20,000 to obtain a filtrate. A chitosan oligosaccharide was prepared by concentrating the filtrate obtained in the above step using a nano-filter system, sterilizing the concentrated filtrate through the sterilizing process, and drying the sterilized filtrate in an air spray dryer.

After dissolving the chitosan oligosaccharide into 1 L of PBS 7.0, 0.52 L of triethylamine was slowly dropped into the dissolved solution, wherein two equivalents of triethylamine were reacted with one equivalent of an amine group in the chitosan oligosaccharide. After reacting the above reactant at room temperature for 2 hours, adding acetonein the reactant, and agitating the reactant and acetone, the mixture was centrifuged. After repeating the foregoing processes 2 to 3 times, the resulting material was air dried and freeze dried, wherein the centrifuging process was performed to 15,000 revolutions per minute at 4 ℃ for 20 minutes by using Supra 30 K.

After adding 30 to 50 ml of 0.001 N hydrochloric acid in a product obtained through the foregoing processes, reacting the product and 0.001 N hydrochloric acid to obtain a reactant, adding acetone in the reactant to agitate the reactant and acetone, the mixture was centrifuged in the same conditions as above. After repeating such processes 2 to 3 times, the resulting material was air dried and freeze dried. After dissolving the dried product into second distilled water and purifying the dissolved solution using an activated carbon/ion exchange resin column to obtain an aqueous solution, the aqueous solution was freeze dried to obtain a white free amine chitosan having a molecular weight of 20,000 daltons.

<Preparation Example 2> Preparation of a water-soluble free amine chitosan having a molecular weight of 100,000 daltons

A 5% chitosan solution was prepared using lactic acid as solvent. 100 ml of the 5% chitosan solution having a pH of 5.0 to 5.5 and chitosanase enzyme derived from Bacillus pumilus BN-262 of a unit 5 were mixed and reacted with each other at 40 ℃ for 36 hours. After finishing the reaction process, the reactant was primarily filtered using a prefilter with a pore size of 1 ㎛, and the primarily-filtered reactant was filtered again using a hollow fiber filter paper with a molecular weight of 100,000 to obtain a filtrate. A chitosan oligosaccharide was prepared by concentrating the filtrate obtained in the above step using a nano-filter system, sterilizing the concentrated filtrate through the sterilizing process, and drying the sterilized filtrate in an air spray dryer.

After adding 30 to 50 ml of 0.001 N hydrochloric acid in a product obtained through the foregoing processes, reacting the product and 0.001 N hydrochloric acid to obtain a reactant, adding acetone in the reactant to agitate the reactant and acetone, the mixture was centrifuged in the same conditions as above. After repeating such processes 2 to 3 times, the resulting material was air dried and freeze dried. After dissolving the dried product into second distilled water and purifying the dissolved solution using an activated carbon/ion exchange resin column to obtain an aqueous solution, the aqueous solution was freeze dried to obtain a white free amine chitosan having a molecular weight of 100,000 daltons.

<Example 1> Measuring anti-red tide activities of water-soluble free amine chitosan

The following tests were performed to measure anti-red tide activities of water-soluble free amine chitosan according to the present invention.

1A : Growth inhibitory concentration of red tide organisms

Anti-red tide activities of a water-soluble free amine chitosan according to the present invention were measured by analyzing the growth inhibitory concentration of the red tide organisms.

Chattonella sp., Prorocentrum minimum, Prorocentrum micans, and Heterosigma akashiwo parceled out from Korea Marine Microalgae Culture Center were used as red tide-causing algae for measuring the anti-red tide activities. Further, Cochlodinium polykrikoides and Chatonella marina directly separated from coastal waters of the southern sea in Korea were used, and Skeletonema costatum that is a harmless marine alga was used.

Each of the respective algae was pre-cultured by constantly maintaining cultivation conditions such as a temperature of 22 ℃, a salt concentration of 34%, an illumination intensity of 3,000 luxes, and a light/dark cycle of 14 hours/10 hours in an F2 culture medium comprising 150 mg of NaNO₃, 8.69 mg of NaH₂PO₄·9H₂O, 10 mg of ferric EDTA, 0.22 mg of MnCl₂, 0.11 mg of CoCl₂, 0.0196 mg of CuSO₄·5H₂O, 0.022 mg of ZnSO₄·7H₂O, 50 mg of Na₂SiO₃·9H₂O, 0.012 mg of Na₂MoO₄·2H₂O, 1 ㎍ of vitamin B₁₂, 1 ㎍ of biotin, and 0.2 ㎍ of thiamine·hydrochloric acid contained in 1 L of filtered seawater. Pre-cultured cells of red tide organisms were diluted to a concentration of 10⁴ cells/ml such that the diluted pre-cultured cells of the red tide organisms were inoculated on 24 well micro titrate plates. Water-soluble free amine chitosans of the preparation example 1 and preparation example 2 were diluted as much as a half time starting from the 6.25 (mg/ml)/well concentration such that the diluted water-soluble free amine chitosans were added in the plates into which strains were inoculated. Further, an F2 culture medium in which the water-soluble free amine chitosans were not contained were added instead of the water-soluble free amine chitosans such that the F2 culture medium could be used as a control group. A 50% growth inhibitory concentration(hereinafter referred to as ‘IC₅₀’) value and a 100% growth inhibitory concentration(hereinafter referred to as ‘IC₁₀₀’) value were determined by measuring the number of red tide organisms every 6 hours using hemocytometer while cultivating the algae for 2 days in the above cultivation conditions, and measurement results were represented in the following Table 1.

Table 1

Marine algae	Growth inhibitory concentration (mg/ml)
	Preparation example 1		Preparation example 2
	IC₅₀	IC₁₀₀	IC₅₀	IC₁₀₀
Heterosigma akashiwo	0.25	0.5	0.25	0.5
Chattonella sp.	0.125	0.25	0.125	0.25
Chattonella marina	0.063	0.125	0.63	0.125
Cochlodinium polykrikoides	0.125	0.25	0.25	0.5
Prorocentrum minimum	0.25	0.5	0.25	0.5
Prorocentrum micans	0.25	0.5	0.25	0.5
Skeletonema costatum	>8	>8	>8	>8

Referring to the Table 1, it can be seen that growths of the red tide-causing algae of the preparation examples 1 and 2 are effectively inhibited since 50% growth inhibitory concentrations of red tide-causing algae including Heterosigma akashiwo, Chattonella sp., Chattonella marina, Cochlodinium polykrikoides, Prorocentrum minimum and Prorocentrum micans are represented to be not more than 0.25 mg/ml, and 100% growth inhibitory concentrations of the red tide-causing algae are represented to be not more than 0.5 mg/ml. On the other hand, it can be seen that the growth of Skeletonema costatum as the harmless marine alga is not inhibited in the preparation examples 1 and 2 since a growth inhibitory concentration of Skeletonema costatum as a harmless marine alga is represented to be not less than 8 mg/ml.

Accordingly, it can be seen that a water-soluble free amine chitosan according to the present invention exhibits excellent anti-red tide activities by effectively inhibiting the growth of the red tide-causing algae.

1B : Visualization of anti-red tide activities

Anti-red tide activities of a water-soluble free amine chitosan according to the present invention were visualized through the optical microscopic analysis.

The inoculated red tide-causing marine algae were cultivated after inoculating the red tide-causing marine algae used in the example 1A in an F2 culture medium comprising 150 mg of NaNO₃, 8.69 mg of NaH₂PO₄·9H₂O, 10 mg of ferric EDTA, 0.22 mg of MnCl₂, 0.11 mg of CoCl₂, 0.0196 mg of CuSO₄·5H₂O, 0.022 mg of ZnSO₄·7H₂O, 50 mg of Na₂SiO₃·9H₂O, 0.012 mg of Na₂MoO₄·2H₂O, 1 ㎍ of vitamin B₁₂, 1 ㎍ of biotin, and 0.2 ㎍ of thiamine·hydrochloric acid contained in 1 L of filtered seawater. After inoculating the cultivated red tide organisms to a concentration of 10⁴cells/ml on 24 well micro titrate plates, water-soluble free amine chitosans of the preparation examples 1 and 2 were added to a concentration of 0.5 mg/ml per well in the plates inoculated with the red tide organisms such that the water-soluble free amine chitosans were cultivated at 20 ℃ for 2 hours. Further, an F2 culture medium in which the water-soluble free amine chitosans were not contained were added instead of the water-soluble free amine chitosans such that the F2 culture medium was used as a control group. After cultivating the water-soluble free amine chitosans, the cultivated water-soluble free amine chitosans were observed through an optical microscope in order to visualize the cultivated water-soluble free amine chitosans, and optical microscopic images were illustrated in fig. 1. More specifically, fig. 1a shows an optical microscopic image of Heterosigma akashiwo as a red tide-causing alga, fig. 1b shows an optical microscopic image of Chattonella sp., and fig. 1c shows an optical microscopic image of Cochlodinium polykrikoides. Furthermore, fig. 1d shows an optical microscopic image of Prorocentrum minimum and fig. 1e shows an optical microscopic image of Skeletonema costatum as a harmless marine alga.

Referring to fig. 1a to 1e, it can be seen that materials or organelles in the organisms are discharged into cells and cell walls and membranes are destroyed in case of treating the water-soluble free amine chitosans of the preparation examples 1 and 2 according to the present invention although the number of organisms is maintained and there is no external changes in the organisms in case of a control group in which the red tide-causing algae are not treated at all. Further, referring to fig. 1e, it can be seen that the water-soluble free amine chitosans of the preparation examples 1 and 2 according to the present invention do not have an effect on the growth of Skeletonema costatum as a harmless marine alga.

<Example 2> Measuring optional annihilation extents of red tide-causing algae of water-soluble free amine chitosan

The following test was performed to measure the optional annihilation extents of red tide-causing algae of a water-soluble free amine chitosan according to the present invention.

The test was divided into test groups of Chattonella sp. and Skeletonema costatum, Prorocentrum minimum and Skeletonema costatum, and Heterosigma akashiwo and Skeletonema costatum in order to see optional growth inhibitory extents of the red tide-causing algae in co-cultivation of red tide-causing algae such as Chattonella sp., Prorocentrum minimum and Heterosigma akashiwo, and a harmless marine alga of Skeletonema costatum. After adding 1 mg/ml of water-soluble free amine chitosans of the preparation examples 1 and 2 in 24 well micro titrate plates and diluting cells of the red tide-causing algae of each test group to a concentration of 2×10⁴cells/ml, the diluted cells of the red tide-causing algae were inoculated into the 24 well micro titrate plates, and all of the water-soluble free amine chitosans of the preparation examples 1 and 2 were not treated as control groups. The measurement results were represented in fig. 2 after measuring the number of cells of the red tide-causing algae in every six hours using a hemocytometer while cultivating the water-soluble free amine chitosans for two days in the same cultivation conditions as those of the example 1. Further, optical microscopic images were illustrated in fig. 3. More specifically, cell densities of Heterosigma akashiwo and Skeletonema costatum were illustrated in fig. 2a, cell densities of Prorocentrum minimum and Skeletonema costatum were illustrated in fig. 2b, and optical microscopic images of Chattonella sp. and Skeletonema costatum were illustrated in fig. 3.

Referring to fig. 2a, although Skeletonema costatum and Heterosigma akashiwo which are not treated are grown well by coexisting with each other, it can be seen that the cell density of Skeletonema costatum as the harmless marine alga is slightly reduced, and Heterosigma akashiwo as the red tide-causing alga is completely annihilated in 30 minutes when treating Skeletonema costatum and Heterosigma akashiwo with the water-soluble free amine chitosans of the preparation examples 1 and 2. Further, referring to fig. 2b, although Skeletonema costatum and Prorocentrum minimum which are not treated are grown well by coexisting with each other, it can be seen that the cell density of Skeletonema costatum as the harmless marine alga is slightly reduced, and Prorocentrum minimum as the red tide-causing alga is almost annihilated after 6 hours when treating Skeletonema costatum and Prorocentrum minimum with the water-soluble free amine chitosans of the preparation examples 1 and 2.

Further, referring to fig. 3, although Skeletonema costatum and Chattonella sp. which are not treated are grown well by coexisting with each other, it can be seen that the cell density of Skeletonema costatum as the harmless marine alga is slightly reduced, and cells of Chattonella sp. as the red tide-causing alga are almost annihilated when treating Skeletonema costatum and Chattonella sp. with the water-soluble free amine chitosans of the preparation examples 1 and 2.

Accordingly, it can be seen that a water-soluble free amine chitosan according to the present invention is optionally acted on red tide-causing marine algae only to annihilate the red tide-causing marine algae.

<Example 3> Analyzing acted parts in red tide-causing algae of a water-soluble free amine chitosan

The following tests were performed to see acted parts in the red tide-causing algae of the water-soluble free amine chitosan according to the present invention.

After dissolving each 10 mg of powder of the preparation examples 1 and 2 into 50 mM sodium borate buffer having a pH of 8.5, the dissolved solutions were mixed with tetramethylrhodamine(TAMRA) as fluorescent substance such that the dissolved solutions were reacted with tetramethylrhodamine at room temperature for 12 hours. After separating water-soluble free amine chitosans of the preparation examples 1 and 2 marked with Sephadex G-25, the separated water-soluble free amine chitosans of the preparation examples 1 and 2 were freeze dried to calculate contents of the water-soluble free amine chitosans. Each of the samples was washed three times using an F2 culture medium in two minutes after treating 0.12 mg/ml of the water-soluble free amine chitosans of the preparation examples 1 and 2 marked with tetramethylrhodamine by the method mentioned in the Example 1. Right after washing the samples, fluorescent light of the samples was observed under a confocal laser scanning microscope, and the observation results were illustrated in fig. 4 and fig. 5.

More specifically, a confocal laser scanning microscopic image of Heterosigma akashiwo treated on the water-soluble free amine chitosan of the preparation example 1 was illustrated in fig. 4a, and a confocal laser scanning microscopic image of Prorocentrum minimum was illustrated in fig. 4b. Further, a continuously taken multifocal image using a confocal laser scanning microscope of Heterosigma akashiwo treated on the water-soluble free amine chitosan of the preparation example 2 was illustrated in fig. 5a, and a continuously taken multifocal image using a confocal laser scanning microscope of Prorocentrum minimum was illustrated in fig. 5b.

Referring to fig 4 and fig. 5, it can be seen that red fluorescent light is congregated around the cell membranes.

Accordingly, a water-soluble free amine chitosan according to the present invention is acted on cell membranes of the red tide-causing algae to exhibit anti-red tide activities by inducing the water-soluble free amine chitosan to destroy the cell membranes.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

A water-soluble free amine chitosan which has anti-red tide activities against red tide-causing algae, and of which a molecular weight is 20,000 to 100,000 daltons.
The water-soluble free amine chitosan as set forth in claim 1, showing anti-red tide activities with respect to one or more red tide-causing marine algae selected from the group consisting of Chattonella sp., Prorocentrum minimum, Prorocentrum micans, Heterosigma akashiwo, Cochlodinium polykrikoides, and Chattonella marina.
An anti-red tide agent containing as an active ingredient the water-soluble free amine chitosan which has anti-red tide activities against red tide-causing algae, and of which a molecular weight is 20,000 to 100,000 daltons of claim 1.
A method of spraying the anti-red tide agent containing as an active ingredient the water-soluble free amine chitosan which has anti-red tide activities against red tide-causing algae, and of which a molecular weight is 20,000 to 100,000 daltons of claim 3 in red tide-generating waters to remove red tide.