KR20110135536A - Method for manufacturing red tide organisms inhibitors by biologically active substances in the sediments of marine bacteria - Google Patents

Abstract

PURPOSE: A method for manufacturing an inhibitor for red tidal organisms using physiologically active materials is provided to degrade cell wall of the red tidal organisms and to prevent secondary contamination. CONSTITUTION: A method for manufacturing an inhibitor for red tidal organisms using physiologically active materials comprises: a step of smearing marine bacteria existing in a marine sediment and culturing; a step of confirming colony; a step of performing pure culture and species identification of several colonies one by one to recognize bacteria characteristic; and a step of centrifuging culture liquid to collect physiologically active materials.

Description

METHODS FOR MANUFACTURING RED TIDE ORGANISMS INHIBITORS BY BIOLOGICALLY ACTIVE SUBSTANCES IN THE SEDIMENTS OF MARINE BACTERIA}

The present invention obtains the bioactive material as a secondary metabolite by extracting and separating and culturing marine bacteria present in the marine sediment, and the biological activity generated by the marine bacteria of the marine sediment killing harmful red tide organisms using the bioactive material. The present invention relates to a method of preparing a red tide inhibitor using a substance, and more particularly, to obtain a high concentration of a bioactive substance by obtaining a secondary metabolite produced by the metabolism of a marine bacterium separated and cultured in a marine sediment, extracting and concentrating a solvent. And injecting the bioactive substance into the red tide, destroying the cell wall of the red tide, causing chloroplasts and nuclei to flow out of the cell, and inducing osmotic perturbation due to concentration differences due to the influx of the bioactive substance. Inhibits red tide organisms due to shell shelling and creates dormant spores The present invention relates to a method for producing red tide inhibitors using bioactive substances produced by marine bacteria in marine sediments that can prevent secondary contamination.

There is a wide variety of microorganisms in the ocean, and in order to adapt to the marine environment, marine microorganisms have produced metabolic substances or developed physiological activities different from terrestrial microorganisms. The higher levels of taxonomy are dominated by marine life worldwide. Species diversity of marine organisms is more potential for microorganisms than for animals and plants. Bacteria can be divided into bacteria that float in water layers and those that inhabit sediments accumulated in lower layers. Typically, 1 million microbes live in 1 ml of seawater, most of which is neither known nor known. In addition, sediments in the ocean floor create different environments with water layers such as nutrients, heavy metals content, ignition loss (IL), total sulfide (TS), redox value (ORP), and chemical oxygen demand (COD). This will form. Therefore, researches using marine bacteria themselves or secondary metabolites are actively underway.

On the other hand, long-term red tide occurs on the coast of Korea every year, and economic damages such as aquaculture farms and coastal ecosystems are being destroyed by red tide. Physical methods, chemical methods using chemical algae, ocher or clay spraying methods, coagulation agents or additives such as magnesium hydroxide, quicklime, clam shell powder and zeolites to effectively remove red tide organisms when such red tide occurs , Sonication, natural application, etc. This will be described in more detail as follows.

First, the physical method is a method for artificially separating organisms by separating the algae in the seawater by the centrifugal force action of the centrifugal separator, there is a problem that the processing capacity is small as 200ℓ / hr, lacking economic efficiency. Chemical methods include the use of chemicals such as copper sulfate or organic compounds with lethality to control red tide, which can affect other organisms due to the use of chemicals. There is a problem that can not be inhibited, the method of using a flocculant is a method of coagulating red tide by using flocculants such as magnesium hydroxide, quicklime, clam shell powder and zeolite mentioned above to induce to sink to deep seabed. There was a problem that can cause secondary pollution by living things. In addition, ultrasonic treatment is a method of inhibiting red tide by generating ultrasonic waves in the ocean when red tide occurs, and may affect not only the red tide but also the ecology and growth of various marine organisms. It is difficult to apply.

Therefore, the most widely applied method of red tide inhibition is the only method of spraying large quantities of ocher or clay. However, in the case of yellow soil spraying, the red tide can temporarily inhibit red tide, but the yellow soil that aggregates and sinks in red tide blocks the oxygen supply of light and benthic species, causing secondary pollution such as killing of benthic species and disturbance of marine ecosystem. (Hallagraeff GM, 1993; Kim H G., 1997; Lee Y. S, Lee SY, 2006; Sun XX, Choi JK, Kim EK, 2004). In addition, the ocher contains a large amount of phosphorus, which may promote the growth of phytoplankton, thereby implying the germination of dormant spores of red tide organisms.

Therefore, the above-described conventional techniques can artificially separate and treat organisms using a centrifugal separator in a physical method, but the processing capacity is small as 200 L / hr, and there is a problem of lack of economic efficiency, and the chemical method has a lethal force. It is possible to kill red tide by administering a chemical to the ocean, but the use of chemicals may affect other marine life except red tide, and there is a problem that it cannot continuously inhibit red tide in large scale occurrences. The use of flocculants can cause red tide to agglomerate and sink to deep sea floors, resulting in secondary contamination by sunken red tide. Ultrasonic treatment can affect the ecology and growth of not only red tide but also various marine organisms. It is possible, the natural use is difficult to apply the actual situation of the natural culture and movement of natural enemies, In the case of yellow soil spraying, the red tide can be temporarily inhibited, but the yellow soil aggregated with the red tide will sink the oxygen supply of light and benthic organisms, causing secondary pollution such as killing of benthic organisms and disturbing marine ecosystems. And, to solve this problem is a problem of the present invention.

Therefore, the present invention after culturing the marine bacteria present in the marine sediment on a plate medium and cultured, to identify the different bacterial colonies formed, to identify the characteristics of bacteria by pure culture and species identification of several colonies individually, Bacteria by centrifugation of the culture medium cultured in a liquid medium (a substance containing secondary metabolites in the growth of marine bacteria using the medium and a substance in which bioactive substances and bacteria coexist, which are secondary metabolites of marine bacteria) With the exception of the residues, the marine metabolites are obtained by obtaining bioactive substances, which are secondary metabolites produced by marine bacteria, extracting high concentrations of bioactive substances by rotary concentration, and controlling red tide organisms using the extracted bioactive substances. It is an object of the present invention to provide a method for preparing red tide inhibitors using bioactive substances produced by marine bacteria in sediments.

The objects of the present invention are not limited to the above-mentioned objects, and other objects and advantages of the present invention, which are not mentioned above, can be understood by the following description, and the objects and advantages of the present invention are shown in the claims and It will be readily appreciated that the combination can be realized.

The present invention for achieving the above object, In the method for producing red tide inhibitors using the bioactive material produced by the marine bacteria of the marine sediment, Marine sediment collecting step of collecting the marine sediment with a collector; A marine sediment suspension step of suspending the marine sediment collected in the marine sediment collection step in saline; Whole marine bacterial smearing and culturing step of smelting and culturing the floating marine sediment in a flat medium after precipitating the marine sediment suspended in the marine sediment suspension step; A primary pure culture step of selecting a specific marine bacterial colony (colony) from the total marine bacteria cultured in the whole marine bacteria smear and culture step to primary pure culture; A secondary pure culture step of culturing the secondary pure to obtain one specific species of marine bacteria cultured primary in the primary pure culture step; A low temperature storage step of culturing the second pure cultured marine bacteria in a flat medium for storage in the second pure culture step and storing the same at a low temperature; Liquid medium inoculation and shaking culture step of inoculating and shaking culture of the marine bacteria stored in the cold storage step; A culture supernatant harvesting step of collecting the culture supernatant which is a physiologically active substance excluding bacterial residues after centrifuging the marine bacteria cultured in the liquid medium inoculation and shaking culture step; A bioactive material and a solvent mixing step of mixing a solvent to obtain a bioactive material concentrated in the culture supernatant collected in the culture supernatant collecting step; Separation step of separating the solvent and water containing the bioactive material by stagnating the mixed material in the bioactive material and solvent mixing step; Concentrating step of concentrating the bioactive material using a rotary concentration of the solvent separated in the separation step; And a concentrated bioactive substance extraction step of extracting by dissolving the bioactive substance concentrated in the concentration step by using a solvent.

As described above, the present invention does not affect other marine organisms other than the red tide because it uses a bioactive material that is a secondary metabolite of marine bacteria by culturing various marine bacteria of marine sediments, and uses physical methods or chemicals. There is no problem of disturbing marine ecosystem, and it does not block light and oxygen like the existing red tide control method, so it can maintain the ecology and growth of the seabed organisms. The dormant spores produced by dormant spontaneously grow and grow again under favorable conditions. Can prevent secondary pollution. In addition, it is possible to kill red tide organisms in a short time in the early stages of red tide generation, thereby reducing the economic and environmental damage caused by red tide, which has the effect of eco-friendly red tide control.

In addition, the present invention, when the marine bacteria present in the marine sediment is plated on the plate medium to isolate and culture, and species identification to find a new species or to obtain the bioactive material of known bacteria, marine bacteria selection medium It is possible to extract useful physiologically active substances that are secreted and produced as the bacteria grow by culturing in the culture. The separated and cultivated bacteria can be stored at low temperature (for example, -72 ° C) and can be cultured at any time if necessary. There is no difficulty in obtaining, it is possible to cultivate a large amount of bacteria by deriving the optimum culture conditions of the bacteria to produce a bioactive material, and also to massify the bioactive material. As such, when the bacteria in the natural environment are used, useful bioactive substances produced by various bacteria can be extracted, and the bioactive substances can be used to inhibit red tide organisms. It also has the effect of preserving marine ecosystems.

1 is a diagram illustrating an embodiment of a method for preparing a red tide organism inhibitor using a bioactive material produced by marine bacteria in a marine sediment according to the present invention.
Figure 2 is an embodiment explanatory diagram for the 10 marine bacteria isolated in the second pure culture step of FIG.
Figure 3a to 3c is an embodiment illustrating the inhibitory effect by the bioactive material prepared by the biotide inhibitor production method using the bioactive material produced by the marine bacteria of the marine sediment according to the present invention.
Figures 4a to 4d is an exemplary explanatory diagram showing the results of red tide inhibition of the bioactive material by the red tide biological inhibitor production method using the bioactive material produced by the marine bacteria of the marine sediment according to the present invention.
5 is Heterocapsa according to an embodiment of the present invention. Triquetra inhibition experiment (5 min stirring after 1% infusion) results.
6 is Akashiwo according to an embodiment of the present invention. Results of sanguinea inhibition experiment (5 min stirring after 1% infusion).
7 is a Prorocentrum in accordance with an embodiment of the present invention. micans Results of inhibition experiments (5 min stirring after 1% injection).
8 is a Photobacterium in accordance with an embodiment of the present invention. sp . Inhibitory experiments by concentration (checked after 5 min stirring).
9A, 9B and 9C show Photobacterium , respectively sp . Gram staining images (a), colony images (b), and bacterial community images (c).
10A and 10B illustrate Heterocapsa according to an embodiment of the present invention. Images showing triquetra inhibition results (a is a pre-treatment image, b is a post-treatment image).
11A and 11B illustrate Akashiwo according to an embodiment of the present invention. Images showing sanguinea inhibition results (a before treatment and b after treatment).
12A and 12B illustrate Prorocentrum according to an embodiment of the present invention. micans Images showing inhibition results (a is a pre-treatment image, b is a post-treatment image).

BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features and advantages of the present invention will become more apparent from the following detailed description of the present invention when taken in conjunction with the accompanying drawings, It can be easily carried out. In the following description, well-known functions or constructions are not described in detail since they would obscure the invention in unnecessary detail. Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 is a diagram illustrating an embodiment of a method for preparing a red tide inhibitor using a bioactive material produced by marine bacteria in a marine sediment according to the present invention.

As shown in Figure 1, the method for producing red tide inhibitors using the bioactive material produced by the marine bacteria of the marine sediment, marine sediment collecting step of first collecting the marine sediment with a collector (for example, grab chaney) (101), the marine sediment suspension step of suspending the marine sediment collected in the marine sediment collecting step 101 in saline (102), the sediment of the marine sediment suspended in the marine sediment suspending step (102) is suspended Whole marine bacteria smear and culture step (103) for smearing and culturing marine bacteria on a plate medium (for example, 30 ° C., 24-48 hours incubation), whole cultured in the whole marine bacteria smear and culture step (103) A primary pure culture step (104) of selecting a specific marine bacteria colonies (colony) of the marine bacteria (for example, 30 ℃, 24 to 48 hours incubation), the primary pure culture step (104) Marine Bacteria Primaryly Cultivated in The secondary pure culture step 105, the secondary pure culture step 105, the secondary pure culture step (105, 24 to 48 hours incubation) to obtain a specific species, the secondary pure culture step 105 Cold storage step 106 of culturing (for example, 30 ℃, 24 hours incubation) for storage of marine bacteria in a flat medium and stored at low temperature (for example, freezer below -72 ℃), the low temperature Liquid medium inoculation and shaking culture step (107), inoculating and shaking culture (for example, 30 ℃, 24 to 48 hours incubation) in the marine medium stored in the storage step 106, the liquid medium inoculation and shaking culture Centrifugation of the marine bacteria cultured in step 107, the culture supernatant harvesting step (108), which collects the culture supernatant which is a physiologically active substance excluding bacterial residues, concentrated in the culture supernatant collected in the culture supernatant collection step (108) Physiological mixing of solvents to obtain bioactive substances Active material and solvent mixing step 109, the step of separating the solvent and water containing the bioactive material by stagnating the mixed material in the bioactive material and solvent mixing step 109, the separation step ( Concentration step (111) for concentrating the bioactive material by using the rotary concentration of the solvent separated in 110), and concentrated bioactive material that is dissolved by extracting the bioactive material concentrated in the concentration step (111) using a solvent Extraction step 112 is included.

The physiologically active substance is a secondary metabolite produced by marine bacteria in marine sediments, and refers to a culture supernatant excluding bacterial residues after centrifugation of marine bacterial culture solution, and is a substance that inhibits red tide organisms.

As described above, the red tide organism inhibitor manufacturing method using the bioactive material of the marine sediment according to the present invention will be described in more detail as follows.

In the marine sediment collecting step 101, which collects marine sediment by using a collector (for example, grab chafing), a rope is connected to a collector such as grab chafing and put into the seawater, and marine sediments of the seabed and the bottom are collected. In addition, marine sediments are collected from the tidal flat using a collector.

In the marine sediment suspension step 102 in which the collected marine sediments are suspended in saline, the above-mentioned marine sediments are put in saline (for example, 0.87% saline), and the marine sediments are shaken to float the microorganisms present in the marine sediments. Suspend in saline.

The whole marine bacterial smear and culturing step 103, in which the suspended marine sediment is precipitated, and the whole floating marine bacteria are plated and cultured on a plate medium (for example, at 30 ° C. for 24 to 48 hours) are suspended. Precipitate the marine sediment, pipet 50 microliters of microorganisms suspended in saline solution, divide into flat media and smear with a triangular loader. Since many bacteria grow in one plate medium, the raw water is incubated by diluting up to 10 ⁴ , and the plated medium is incubated for 24 to 48 hours in a 30 ° C. incubator.

In the first pure culture step (104) of primary marine culture by selecting a specific marine bacterial colony (colony) of the total marine bacteria cultured in the whole marine bacteria smear and culture step (103), the entire cultured marine In order to cultivate marine bacteria of one species, it is separated by the color and form of the entire cultured marine bacterial colonies, checked in a container on a plate medium, and colonized by sterilized platinum. ), One by one to each plate medium and primary incubation for 24 to 48 hours in a 30 ℃ incubator.

The secondary pure culture step 105 of secondary pure culture to obtain a specific species of one of the marine bacteria cultured pure in the primary pure culture step 104 is cultured in each plate medium in the primary pure culture step The marine bacteria are plated one by one again on a plate medium and secondly pure incubated for 24 to 48 hours in a 30 ℃ incubator.

Cold storage step (106) to store at low temperature after incubation for storage of the second pure cultured marine bacteria in the plate medium in the second pure culture step 105 to obtain a specific species of the first marine cultured marine bacteria. In the above), as described above, the secondary pure cultured marine bacteria were plated again for use in stock medium and incubated for 24 hours in a 30 ° C. incubator, and the stock solution was added to the bacteria of the flat medium. After scraping with a triangular loader and inject one by one into the centrifuge tube to be stored at low temperatures (for example, -72 ℃ below freezers) to be incubated whenever necessary.

In the liquid medium inoculation and shaking culture step 107 of inoculating and shaking culture of the marine bacteria stored in the cold storage step 106 in a liquid medium, one of the stored marine bacteria is inoculated into 6 ml of liquid medium at 30 ° C. After culturing for 24 hours, 50 μl of the marine bacteria in 6 ml of liquid medium was inoculated into 50 ml of liquid medium, followed by shaking culture in a 30 ° C. incubator for 48 hours.

After centrifugation of the marine bacteria cultured in the liquid medium inoculation and shaking culture step (107), the culture supernatant harvesting step (108) for collecting the culture supernatant which is a physiologically active substance excluding the bacterial residues, the culture solution of the shaking cultured marine bacteria 13,000 Centrifuge for 20 minutes at rpm to discard the precipitated bacterial debris and collect the culture supernatant, a bioactive substance.

The culture supernatant may be used for inhibiting red tide organisms in the bioactive material and solvent mixing step 109 of mixing the solvent to obtain the bioactive material concentrated in the culture supernatant collected in the culture supernatant collecting step 108. In order to obtain concentrated bioactive substance, ethyl acetate (Ethyl acetate), which is a solvent, and the culture supernatant are injected in a 1: 1 ratio, and shaken for 15-20 minutes to dissolve the bioactive substance in the solvent.

Separation step 110 of separating the solvent and water containing the physiologically active material by stagnating the mixed material in the physiologically active substance and solvent mixing step 109 is opened for 15 minutes by opening the lid of the separatory funnel mentioned above In stagnation, ethyl acetate, which contains a bioactive substance, is separated from water.

The concentration step 111 of concentrating the bioactive material by using the rotary concentration of the solvent separated in the separation step 110 adds sodium sulfate to ethyl acetate containing the bioactive material. Dehydration of the water remaining in the ethyl acetate (Ethyl acetate) containing the bioactive material, and connected to a rotary concentrator, and concentrated only a high concentration of the bioactive material using a vacuum.

In addition, the concentrated bioactive substance extracting step 112, in which the bioactive substance concentrated in the concentration step 111 is dissolved and extracted using a solvent, is a solvent (for example, Methanol) in the highly concentrated bioactive substance mentioned above. Etc.) to dissolve the concentrated bioactive substance, and to recover the highly concentrated bioactive substance, it can be applied to inhibit red tide organisms.

FIG. 2 is a diagram illustrating one embodiment of ten marine bacteria isolated in the second pure culture step of FIG. 1.

As shown in Figure 2, the marine bacteria obtained in the method for producing red tide inhibitor using the bioactive material of the marine sediment according to the present invention Vibrio sp. Includes nine other marine bacteria.

Figure 3a to 3c is an embodiment explanatory diagram showing the inhibitory effect by the bioactive material produced by the red tide inhibitors using the bioactive material of the marine sediment according to the present invention.

Figure 3a is a red algae heterocapsa triquetra using the bioactive material of 10 marine bacteria according to the present invention ( Heterocapsa triquetra ) and Akashiwo sanguinea inhibition results of one embodiment.

Vibrio has the highest inhibitory efficiency against Heterocapsa triquetra . sp. (83%), the lowest being Zooshkella sp. For 64% respectively, and the O Casio California (Akashiwo sanguinea) Marinomonas sp. (82%) showed the highest inhibitory effect, Pseudoalteromonas sp. (26%) showed the lowest inhibitory efficacy.

3 b is a heterocapsa Triquatra using the bioactive material of 10 marine bacteria according to the present invention (Heterocapsa triquetra) And the Casio Zhenia (Akashiwo sanguineaExplanatory diagram of an embodiment showing the time of inhibition experiment.

In order to confirm the inhibitory efficacy time of marine bacterial bioactive substances, 0.01% of each injection was injected to determine life / dead within 8 minutes after the reaction time of 3 minutes.

Figure 3c is a diagram illustrating an embodiment showing the results after 30 minutes reaction by injecting 10 biophysiological substances of marine bacteria according to the present invention in a heterocapsa triquetra ( Heterocapsa triquetra ).

The inhibitory effect within 10 minutes after the 0.01% injection of the bioactive substance into the red tide organisms showed an inhibitory effect of 60-90%, and a high inhibitory effect of more than 95% was confirmed after 20-30 minutes of the administration of the bioactive substance. This was about 30 minutes earlier than the known red tide inhibitor using bacteria, and most of the red tide organisms could break out of the cell wall, causing chloroplasts to escape, and the shells to be shelled.

Figures 4a to 4d is an exemplary explanatory diagram showing the results of red tide inhibition of the bioactive material by the red tide biological inhibitor production method using the bioactive material produced by the marine bacteria of the marine sediment according to the present invention.

Figure 4a is a photograph of living organisms before injecting marine bacteria bioactive material into Heterocapsa triquetra , Heterocapsa triquetra ) is fusiform, and the amortium is rounded to the side and has a round or square end. The inferior angle varies in shape, the traverse is slightly dug, compressed through the center to the right. Species are not clear and do not reach the base. Cells are 16-30 μm in length and 9-18 μm in width (Korean flora and fauna, 1994). It is an acquaintance of the flagella, which moves the flagella and does not form a colony.

Figure 4b is a photograph of a dead organism after injecting marine bacteria bioactive material into Heterocapsa triquetra , the cell wall of the killed individual is bursting, chloroplasts and nuclei are leaked, the cells burst due to osmotic pressure disturbance As a result, it can be seen that the form of the creature is blurred and inconsistent. Heterocapsa triquetra , which is stagnant without the movement of flagella, has been observed to disintegrate cell walls, leak out chloroplasts and nuclei, and shell the envelop surrounding the individual.

Figure 4c is a photograph of living organisms before injecting marine bacteria bioactive material into Akashiwo sanguinea , Akashiwo sanguinea ) The appearance of the cells is elliptical to rounded pentagonal, and the dorsum is flat. The hemispheres are near the center of the cell and are relatively narrow. The boil is not in lettuce. The cell length is 40-80 micrometers.

Figure 4d is a photograph of a living organism that died after injecting the marine bacteria bioactive material into Akashiwo sanguinea , after the injection of the bioactive material, the morphology of the cells decreased and the cell wall was rounded. Loosening, bursting and dying could be seen.

Heterocapsa of Physiologically Active Substances by a Red Tide Inhibitor Production Method Using Bioactive Substances Produced by Marine Bacteria of Marine Sediments According to the Present Invention Triquetra inhibition experiments (5 min stirring after 1% injection) are shown in Table 1 below and FIG. 5.

Species Live Dead efficiency Control 1990 0 One Vibrio sp. 330 390 83.4 2 Zooshikella sp. 710 360 64.3 3 Pseudoalteromonas sp. 510 690 74.4 4 Marinomonas sp. 610 310 69.3 5 Bacillus sp. 680 330 65.8 6 Vibrio sp. 600 310 69.8 7 Bacillus sp. 470 340 76.4 8 Vibrio sp. 790 220 60.3 9 Marinobacterium sp. 690 250 65.3 10 Photobacterium sp. 302 310 84.8

Akashiwo of Physiologically Active Substances by the Method of Manufacturing Red Tide Inhibitors Using Physiologically Active Substances Produced by Marine Bacteria of Marine Sediments According to the Present Invention sanguinea Inhibition experiments (5 min stirring after 1% injection) are shown in Table 2 below and FIG. 6.

Species Live Dead efficiency Control 650 0 One Vibrio sp. 170 140 73.8 2 Zooshikella sp. 220 90 66.2 3 Pseudoalteromonas sp. 250 140 61.5 4 Marinomonas sp. 290 130 55.4 5 Bacillus sp. 200 140 69.2 6 Vibrio sp. 480 130 26.2 7 Bacillus sp. 120 140 81.5 8 Vibrio sp. 160 200 75.4 9 Marinobacterium sp. 370 200 43.1 10 Photobacterium sp. 150 220 76.9

Prorocentrum of physiologically active substances by a method of preparing red tide inhibitors using physiologically active substances produced by marine bacteria of marine sediment according to the present invention The micans inhibition experiment (5 min stirring after 1% infusion) results are shown in Table 3 below and FIG. 7.

Species Live Dead efficiency Control 1940 100 4 Vibrio sp. 670 1510 65.5 21 Zooshikella sp. 1190 730 38.7 32 Pseudoalteromonas sp. 1460 400 24.7 34 Marinomonas sp. 1720 100 11.3 36 Bacillus sp. 1000 1180 48.5 37 Vibrio sp. 520 1150 73.2 38 Bacillus sp. 690 790 64.4 39 Vibrio sp. 810 1100 58.2 40 Marinobacterium sp. 1490 130 23.2 41 Photobacterium sp. 650 720 66.5

Photobacterium of physiologically active substance by the method for preparing red tide organism inhibitor using physiologically active substance produced by marine bacteria of marine sediment according to the present invention sp . Inhibitory experiments for each concentration (checked after 5 minutes of stirring) are shown in Table 4 and FIG. 8.

density(%) efficiency(%) 0.01 36.24454148 0.05 100 0.1 100

10 to 12 shows the results of inhibition of physiologically active substances by the method for preparing red tide inhibitors using physiologically active substances produced by marine bacteria of marine sediments according to FIGS.

10A and 10B illustrate Heterocapsa according to an embodiment of the present invention. An image showing the result of triquetra inhibition (a is a pre-treatment image, b is a post-treatment image).

11A and 11B illustrate Akashiwo according to an embodiment of the present invention. An image showing sanguinea inhibition results (a is a pre-treatment image and b is a post-treatment image).

12A and 12B illustrate Prorocentrum according to an embodiment of the present invention. micans An image showing the inhibition result (a is a pre-treatment image, b is a post-treatment image).

The red tide organisms used in the present invention can be easily obtained from Korea Maritime Microalgae Bank and the like.

The present invention can be used as an environmentally friendly red tide inhibitor.

Claims (1)

Claims [1] A method for producing a red tide inhibitor using a bioactive material produced by marine bacteria in a marine sediment, the method comprising: collecting a marine sediment using a collector;
A marine sediment suspension step of suspending the marine sediment collected in the marine sediment collection step in saline;
Whole marine bacterial smearing and culturing step of smelting and culturing the floating marine sediment in a flat medium after precipitating the marine sediment suspended in the marine sediment suspension step;
A primary pure culture step of selecting a specific marine bacterial colony (colony) from the total marine bacteria cultured in the whole marine bacteria smear and culture step to primary pure culture;
A secondary pure culture step of culturing the secondary pure to obtain one specific species of marine bacteria cultured primary in the primary pure culture step;
A low temperature storage step of culturing the second pure cultured marine bacteria in a flat medium for storage in the second pure culture step and storing the same at a low temperature;
Liquid medium inoculation and shaking culture step of inoculating and shaking culture of the marine bacteria stored in the cold storage step;
A culture supernatant harvesting step of collecting the culture supernatant which is a physiologically active substance excluding bacterial residues after centrifuging the marine bacteria cultured in the liquid medium inoculation and shaking culture step;
A bioactive material and a solvent mixing step of mixing a solvent to obtain a bioactive material concentrated in the culture supernatant collected in the culture supernatant collecting step;
Separation step of separating the solvent and water containing the bioactive material by stagnating the mixed material in the bioactive material and solvent mixing step;
Concentrating step of concentrating the bioactive material using a rotary concentration of the solvent separated in the separation step; And
Concentrated physiologically active substance extraction step of melting and extracting the physiologically active substance concentrated in the concentration step using a solvent
Red tide biological inhibitor production method using a biologically active material of the marine sediment comprising a.

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