JP4469840B2 - Red pigment with an algicidal effect derived from Hahera Chejuensis - Google Patents

Description

  The present invention relates to a red pigment having an algicidal effect derived from Hahera chejuensis, and more specifically, a red pigment (RP10356) is produced by culturing Haella chejuensis strain 96CJ10356 (KCTC 2396). Regarding the method. Furthermore, the present invention is characterized by using a red pigment (RP10356) having an algicidal effect, an algicidal preparation containing the red pigment as an active ingredient, and the strain culture solution or the red pigment, which is prepared by this method. The present invention relates to a method for removing red tide organisms.

  Since the 1980s, harmful and toxic red tides have occurred in the eutrophic waters of South Korea. The red tide caused by some microalgal species has caused enormous damage to the aquaculture every year, especially the cochlodinium polykricoides, which appear every year after August, are significant for coastal aquaculture in Korea. (Park et al., J. Korean Fish. Soc., 31: 767-73, 1998). For this reason, research on the extinction and growth suppression of microalgae species that cause red tide is underway (Yoshinaga et al., Japan Fish Sci., 61: 780-6, 1995).

  In marine ecosystems, microbial communities play an important role in the development and extinction of red tide (Furuka et al., Marine Pollution Bull., 23: 189-93, 1992), and the number of algicidal bacteria is Depending on the host organism in the sea area, the highest concentration is reached when the red tide disappears (Yoshinaga et al., Japan Fish Sci., 61: 780-6, 1995).

  Bacteria having an algicidal ability include Alteromonas, Cytophaga, Flavobacterium, Pseudoalteromonas, Sporospira, Vibrio (Vibrio) Etc. are known. These bacteria have been reported to be directly or indirectly involved in red tide extinction (Lovevey et al., Appl. Environ. Microbiol., 64: 2806-13, 1993). Algae-killing bacteria can kill or dissolve algae of various species, and each bacterium has a species-specific algaecidal range. For example, the algicidal bacterium Cytophoga sp. Has various algicidal effects effective against diatoms (Bacillariaphyceae), Raphydophyceae and Dinophyceae (Imai et al.). Mar. Biol., 116: 527-32, 1993).

  However, no significant progress has been made in studies on the algicidal mechanism of algicidal bacteria, and the rapid decline of red tide organisms is due to the lysis of biological cells (Imai et al., Mar. Biol., 116: 527-32, 1993), Mating stage from asexual reproduction to sexual reproduction, as in the death of organisms by growth inhibition (Imai et al., Mar. Biol., 116: 527-32, 1993) and Alexandrium catanella (Sawayama et al., Nippon Suisan Gakkaishi, 59: 291-4, 1993) and the like.

  The algicidal mechanism of the algicidal bacteria is as follows: (1) a mechanism by contact that the algicidal bacteria adhere to the surface of the red tide organism and dissolve the red tide organism (Imai et al., Mar. Biol., 116: 527-32). 1993), and (2) the mechanism by which the algicidal bacteria secrete the algicidal substance out of the cell to suppress or dissolve the growth of red tide organisms. Most algicidal bacteria are involved in the latter mechanism: antibiotics (Kawano et al., J. Mar. Biotechnol., 5: 225-9, 1997), small oligopeptides (Sawayama et al. , Nippon Suisan Gakkaishi, 59: 291-4, 1993), protein (Baker and Herson, Appl. Environ. Microbiol., 35: 791-6, 1978) and heat labile extracellular material (Mitsutani et al.,). Nippon Suisan Gakkaishi, 58: 2159-69, 1992) has been reported. Such a decrease in microalgae appears hours to days after inoculation with most algicidal bacteria. Such slow reaction time is directly related to the activity of the algicidal substance, and shows various time differences depending on the difference in the algicidal substance production and the inducer.

  For this reason, various studies have been actively conducted to reduce the damage to the aquaculture industry due to red tide by suppressing the growth of microalgae causing red tide using the algicidal substance produced by such algicidal microorganisms. It is being broken. In particular, the coclodinium polyclicoides, which is said to have caused red tide for the first time in 1982, has been causing red tide continuously since 1990. Furthermore, since 1995, high-density red tides of 10,000 cells / mL or more have been caused on a large scale from the south coast of South Korea to the southeastern sea area, causing enormous marine damage. Research on red tide control measures to minimize the risk is underway. However, there have been no reports of attempts to use pigments as algicidal substances.

  The pigments are classified into natural pigments and synthetic pigments and are used in various applications such as food industry, cosmetics, pharmaceuticals and livestock feed additives. Among these, natural pigments using microorganisms can be mass-produced by fermentation as compared with pigments produced by animals and plants, and can produce products having stable quality. Microbial pigments can be obtained in various colors depending on the culture method and medium used, and even in the same strain, pigments of various colors can be obtained depending on the components of the medium, culture temperature, pH, etc. (Carels et al., Can. J. Microbiol., 23: 1360-72, 1977). In particular, pigments are secondary metabolites, which are said to be produced by typical regulatory mechanisms by fermentation of secondary metabolites such as antibiotics, toxins, carbon sources, nitrogen sources, phosphates and trace amounts. It has been reported that the production of dyes is greatly influenced by the elements of (Wong et al., Myclogia, 73: 649-54, 1981).

  Hahera Chejuensis strain 96CJ10356 (KCTC 2396 = IMSNU 11157) is a marine microorganism isolated from seabed sediment collected from the coast of Jeju, South Korea, and is a gram-negative motile bacillus. They also produce large amounts of exopolysaccharides and red pigments (Lee et al., Systemic and Evolutionary Microbiology, 51: 661-6, 2001).

  However, the algicidal effect obtained with pigments derived from Hahera microorganisms including the Hahera Chejuensis 96CJ10356 strain has not been known yet.

  Therefore, as a result of diligent efforts to obtain a novel microorganism having an excellent algicidal effect, the present inventors show that the red pigment (RP10356) obtained from the Hahera Chejuensis strain 96CJ10356 exhibits an excellent algicidal effect. As a result, the present invention has been completed.

  Accordingly, a main object of the present invention is to provide a method for producing a red pigment having an algicidal effect, which is characterized by culturing a Hahera spp. Microorganism.

  Another object of the present invention is to provide a red pigment (RP10356) having an algicidal effect and an algicidal formulation containing this as an active ingredient.

  In order to achieve the above object, the present invention includes a step of culturing a Hahera microorganism and a step of separating and purifying a pigment substance exhibiting an algicidal effect from the culture solution using an organic solvent. A method for producing a coloring substance having

  In the present invention, the microorganism belonging to the genus Hahera is preferably Hahera chejuensis, and in particular, the Hahera chejuensis strain 96CJ10356 (KCTC 2396) is preferred.

In addition, the present invention is produced by the above-described method, and an Rf value of 0.00 is obtained by silica gel thin film chromatography (TLC). 8 8 (petroleum ether: acetone: methanol = 5: 3: 2) shows a, and is characterized by exhibiting a maximum absorbance at about 53 5 nm, to provide a red dye having algicidal effect.

  Furthermore, the present invention provides an algicidal preparation containing the red pigment as an active ingredient.

  In the present invention, the red pigment has an algicidal effect on Cochrodinium polykricoides, Gyrodinium impucumum, or Heterosigma akashiwo. .

  In addition, the present invention provides a method for removing red tide organisms, characterized by using the red pigment, Hahera microorganism culture solution, and an organic solvent extract of the culture solution.

  In the present invention, the red pigment (RP10356) was purely separated by extracting the culture solution of Hahera Chejuensis strain 96CJ10356 with an organic solvent. In order to obtain a red pigment RP10356 having an algicidal effect from Hahera Chejuensis strain 96CJ10356, it is necessary to culture the microorganism in a suitable medium containing nutrient salts, trace amounts of inorganic salts and aged seawater.

In the present invention, the nutrient medium includes 5% glucose as a carbon source, 0.5% peptone as a nitrogen source, 0.083 g / L KH ₂ PO ₄ as a phosphate source, and 0.067 g / L K _2. HPO ₄ , 5 g / L MgSO ₄ , 1 g / L CaCl _{2 and the} like can be used. Further, as a trace amount of inorganic salts, 0.005 g / L FeCl ₂ .6H ₂ O, 0.001 g / L MnCl ₂ .4H ₂ O, 0.001 g / L Na ₂ MoO ₄ , 0 .001 g / L ZnCl ₂ or the like.

The crude pigment extracted from the culture broth and RP10356 purified from the crude pigment are the causative species of red tide, coclodinium polycricoides, gyrodinium impjicum, heterosigma acacio, alexandrium catanella and procent As a result of investigating the algicidal effect on L. mycanthus, it was found that it has an excellent algicidal effect on coclodinium polycricoides, gyrodynium impujim and heterosigma acacio. Red dye RP10356 according to the present invention, the maximum absorbance at acidic ethanol is lipophilic red dye is 486 nm, 53 5 nm.

It is a figure which shows the extraction and isolation | separation process of the red pigment | dye which Hahera Chejuensis strain 96CJ10356 strain (KCTC 2396) produces. It is a figure which shows the algicidal effect of Coclodinium polycricoides by the crude pigment which Hahera Chejuensis 96CJ10356 strain (KCTC 2396) produces. It is a figure which shows the algicidal effect of Gyrodinium impucumum by the crude pigment which Hahera Chejuensis 96CJ10356 strain (KCTC 2396) produces. It is a figure which shows the algicidal effect of Heterosigma akashio by the crude pigment which Hahera Chejuensis 96CJ10356 strain (KCTC 2396) produces. It is a figure which shows the algicidal effect of Cochlodinium polycricoides by RP10356 which Hahera chejuensis 96CJ10356 strain (KCTC 2396) produces. It is a figure which shows the shape change of a test strain in the algicidal action by the crude pigment which Hahera chejuensis 96CJ10356 strain (KCTC 2396) produces (A: Coclodinium polyclicoides, B: Gyrodinium impujicum, C: Heterosigma Acacio, D: Alexandrium Catanella, E: Procentrium Mycanth). It is a figure which shows the production | generation of pH, a microbial cell, and RP10356 by culture | cultivation of Hahera Chejuensis 96CJ10356 strain (KCTC 2396). It is a figure which shows the thin-film chromatographic analysis result of the pigment | dye RP10356 isolate | separated from the crude pigment | dye isolate | separated from the crude pigment | dye produced | generated by Hahera Chejuensis 96CJ10356 strain (KCTC 2396), and crude RP10356. ). It is a figure which shows UV absorbance of RP10356 which Hahera Chejuensis 96CJ10356 strain (KCTC 2396) produces. It is a figure which shows the thin film chromatographic analysis result of each fraction by the silica chromatography of RP10356 which Hahera chejuensis 96CJ10356 strain (KCTC 2396) produces (1: 2-propanol crude pigment, 2: fraction 1, 3, 3: minutes) Fraction 2, 4: Fraction 3, 5: Fraction 4, solvent ratio: petroleum ether: acetone: methanol = 5: 3: 2). It is a figure which shows the high-speed chromatographic analysis result of RP10356 which Hahera Chejuensis 96CJ10356 strain (KCTC 2396) produces. It is a figure which shows the high-speed chromatographic analysis result in 300-550 nm by DAD (Diode Array Detector) detector with respect to RP10356 which Hahera Chejuensis 96CJ10356 strain (KCTC 2396) produces.

Hereinafter, the present invention will be described in detail with reference to examples. However, it will be apparent to those skilled in the art that the following examples are merely illustrative of the invention and that the scope of the invention is not limited by the examples.

<Example 1> Extraction and Separation of RP10356 The strain used in this experiment was Hahera Jejuensis (Systematic and evolutionary Microbiology. 51: 661-666, 2001) isolated from sedimentary soil on the Jeju coast ( Hachela chejuensis gen.nov., Sp.nov.) 96CJ10356 strain (KTCC 2396 = IMSNU 11157). Here, the strain was inoculated into an STN solid medium (Table 1), cultured at 25 ° C. for 48 hours, and then stored in a refrigerated storage at 4 ° C. and transferred to a new medium every two weeks for use.

  In order to obtain the red dye RP10356 from the 96CJ10356 strain, the strain was inoculated into a SzoBell medium (Table 2) serving as a basic medium and cultured at 25 ° C. and 120 rpm for 3 days. Thereafter, the red dye RP10356 was separated from the culture solution.

  A red pigment was extracted from the culture solution of the 96CJ10356 strain by the method of FIG. The 96CJ10356 strain is a microorganism that produces a large amount of extracellular polysaccharide, and in order to remove the polysaccharide from the culture solution and to separate the pigment, 2-isopropanol is added to 1 L of the strain culture solution in a double amount. It was. Then, the polysaccharide was removed from the culture solution by treating for 1 hour using an ultrasonic device (Branson 8210, USA) and leaving it at 4 ° C. for 24 hours. The culture solution from which the polysaccharide has been removed is centrifuged at 12,000 g and 30 minutes, and then filtered using GF / F (Whatman, φ47 mm, USA) to separate the crude pigment from which the cells have been removed. did.

  In order to separate RP10356 from 2-isopropanol dye extract (crude dye) from which polysaccharides and cells were removed, the 2-isopropanol dye extract was vacuum concentrated at 45 ° C. to remove 2-isopropanol. Then, in order to remove the water-soluble components and a large amount of salt from the remaining aqueous layer and extract the fat-soluble fraction, 1-fold petroleum ether was added to the aqueous layer and shaken for 1 hour, followed by a separatory funnel. Was used to separate the petroleum ether layer. In order to remove moisture from the separated petroleum ether layer, the separated petroleum ether layer was dried over anhydrous sodium sulfate and filtered through filter paper (No 5, Whatman, USA), and then further 40 ° C. Concentrated in vacuo to remove petroleum ether (FIG. 1).

  In order to separate the algaecidal pigment from the crude pigment extract, silica chromatography was performed. The crude extract was added to silica gel (silica gel 60, 0.040-0.063 mm, Merck, Germany), and added to a glass column (φ2.5 × 30 cm) containing the same resin with petroleum ether: acetone (7: 3 ) Was passed through the developing solvent to separate the crude dye. As a result, the separated pigments were fractionated into 4 types of Rf 0.98 (light yellow), Rf 0.96 (dark red), Rf 0.88 (light red) and Rf 0.59 (blue), respectively. It was. Among them, since a pigment having an Rf value of 0.88 is the main pigment, this was named “RP10356” and the algicidal effect was examined.

<Example 2> Algicidal effect of RP10356 Algae used in this experiment are shown in Table 3. Microalgae were handed over from the Department of Aquaculture at BKU University and the Korea Ocean Research & Development Institute (KORDI) red tide research team. In addition, microalgae were isolated directly in areas where red tides occurred.

F / 2 medium was used for the culture of microalgae (Table 4). The algal culture was performed in an algal incubator (Je-il Science, Korea) under a light-dark photoperiod condition of a temperature of 22.5 ° C., a light intensity of 150 μEin / m ² / s, and a time of 16 hours: 8 hours. As the algal culture for measuring the algicidal effect, cells at the logarithmic growth stage were used. Algae growth is measured using a fluorimeter (Hitachi, F-2000, Japan) or fixed with 2% Lugol solution, then using an inverted microscope (Zeiss, Axon-1000, Germany) Counting was performed.

(1) Algicidal effect of crude pigment 1 × 10 ³ cells / mL of crude pigment and RP10356 separated in Example 1 at concentrations of 0, 0.1, 1.0, 10, 50 and 100 μL / mL Was added to the culture solution of microalgae (coclodinium / polyclicoides, gyrodium / impzicum, heterosigma / acacio, alexandrium / catanella and procentrium / mycanthus) that caused red tides. After 30 minutes, 1 hour, 2 hours and 24 hours, each treated microalgae was fixed with 2% Lugol solution, and the number of undissolved cells was counted using an inverted microscope (Zeiss, Axon 100, Germany). The number of lysed cells was counted from the initial inoculated cell number, and the algicidal effect was calculated by the following calculation formula 1.

Formula 1

  As a result of investigating the algicidal effect of the crude pigment dissolved in ethanol, when coclodinium polycricoides was treated with a crude pigment having a concentration of 0.1 μL / mL, after 30 minutes and 24 hours, respectively, 3.8% and The algaecidal effect was 72.1%. When treated at a concentration of 1.0 μL / mL, 63.8% and 98.7% of algicidal effects were exhibited after 30 minutes and 24 hours, respectively. When the treatment was performed at a maximum treatment concentration of 100 μL / mL, the algicidal effect was 91.3% after 30 minutes. As a result, it showed an algicidal effect at a concentration of 0.1 μL / mL or more against coclodinium polycricoideun, which caused the greatest red tide damage. ).

  When gyrodinium impjicum was treated with a crude pigment at a concentration of 0.1 μL / mL, it showed 1.4% and 40.8% algicidal effects after 30 minutes and 24 hours, respectively, and 1.0 μL / When treated at mL concentration, the algicidal effect was 28.7% and 93.0% after 30 minutes and 24 hours, respectively. When the treatment was performed at a maximum treatment concentration of 100 μL / mL, an algicidal effect of 60.6% was exhibited after 30 minutes. From this result, it was found that gyrodinium impjicum exhibited a slightly lower algicidal ability than that for coclodinium polycricoides (FIG. 3).

  When heterosigma acacio was treated with crude pigment at a concentration of 0.1 μL / mL, it showed an algicidal effect of 6.5% and 78.8% after 30 minutes and 24 hours, respectively, and reached a concentration of 1.0 μL / mL. And 23.8% and 96.3% of the algaecidal effect, respectively. When the treatment was performed at a maximum treatment concentration of 100 μL / mL, an algicidal effect of 46.4% was exhibited after 30 minutes (FIG. 4).

  In addition, the crude pigment showed a weak algicidal ability against Alexandrium catanella and Procentrium mycanth. As a result, the crude pigment extracted from the culture solution of 96CJ10356 strain showed a different algicidal effect on the strain causing red tide, and of these, an extremely high algicidal effect on coclodinium polycrichoides.

(2) The algaecidal effect of RP10356 Against Coclodinium polycricoides, which caused red tide damage in Korea every year and showed the most excellent algaecidal effect in the algaecidal test with crude pigment The effect was investigated. The algaecidal effect test of the four fractions was carried out in the same manner as the algaecidal effect test of the crude pigment. As a result of comparison with the algicidal effect of the crude pigment, it was found that only RP10356 had an algicidal effect among the 4 pigment fractions. As a result of comparing the algicidal effect on the basis of treatment for 30 minutes, 1 . For 0 [mu] L / m L concentration, the crude pigment while indicating algicidal effect of 63.8%, RP10356 showed algicidal effect of higher 83.5%. In particular, the concentration of RP10356 indicating algicidal ability of 50%, a 30 minute treatment showed a 0.65μL / m L, at 24 hours showed 0.17μL / m L (FIG. 5 and Table 5).

(3) Observation of microalgae Changes in cell shape due to treatment with crude pigment and RP10356 were observed using an inverted microscope. Changes in cell shape due to treatment with RP10356 were observed based on growth inhibition, swelling of cells, lysis of cells, and ecdysis of armor.

96CJ10356 If a strain derived from crude pigment was treated at a concentration of 1.0 [mu] L / m L, Kokurodjiniumu-Porikuri carp Death of crude pigment exhibits algicidal effects, the swelling of the cells over time in the gyro-pyridinium-Inpujikumu and Heterosigma akashiwo And cell lysis was observed (A, B, C in FIG. 6). On the other hand, the detachment of a part of the outer coat was observed in Alexandrium catanella having a low algal killing effect with the crude pigment (FIG. 6D), and the shape change was hardly observed in Procentrium mycanth ( FIG. 6E).

  Further, when microalgae were treated with RP10356, the same shape variation as that in FIG. 6A was observed in coclodinium polyclicoides. From this point, it was confirmed that the algicidal substance was due to the crude pigment RP10356 pigment.

<Example 3> Production of RP10356 To mass-produce RP10356, an algicidal substance, using Hahera Chejuensis 96CJ10356, glucose 5 wt%, 0.1 wt% peptone, 0.42 g / L KH ₂ PO ₄ , 0.34 g / L K ₂ HPO ₄ , 0.5 g / L MgSO ₄ , 2.0 g / L CaCl ₂ , 0.001 g / L CoCl ₂ .6H ₂ O, 0.001 g / L MnCl ₃ , 0.001 g / L ZnSO ₄ , 0.001 g / L NaMoO ₄ , 750 mL of distilled water and aged seawater were added to a final volume of 3,000 mL, and then inoculated with the 96CJ10356 strain. Subsequently, the cells were cultured in a 5 L batch incubator. At this time, the culture temperature was 25 ° C., and the aeration rate at 200 rpm was 1.5 vvm.

Polysaccharide production increased with the passage of culture time, and the maximum value was maintained after 81 hours of culture. The cell growth increased until 48 hours of culture, but the cell growth decreased after 74 hours. In order to measure the concentration of RP10356 produced, 2 mL of cold ethanol was added to 1 mL of the culture solution, followed by treatment with an ultrasonic grinder for 30 minutes, followed by centrifugation (8,000 × g, 15 minutes). The body was removed. Thereafter, the absorbance (A ₅₃₉ ) of the supernatant was measured using a spectrophotometer. As a result, 209 mg / L RP10356 was finally obtained (FIG. 7).

Example 4 Characteristics of RP10356 (1) Thin-layer chromatography (TLC) analysis To purify RP10356 from 2-propanol crude extract, 1 μg of the crude extract was dissolved in 1 mL of ethanol, and then Then, 5 μL was dropped _onto a silica 60F _254S TLC plate (Merck) and developed 10 cm with a mixed solution of petroleum ether: acetone: methanol (5: 3: 2) (FIG. 8). As a result, four types of dyes were obtained: Pf 0.98 (light yellow), Rf 0.96 (dark red), Rf 0.88 (light red), and Rf 0.59 (blue).

(2) Absorption spectrum analysis using an absorptiometer (UV) In order to investigate the absorption type of RP10356, an ethanol solution of RP103561 μg / mL was used at a wavelength of 300 to 700 nm using a spectrophotometer (Shimadzu, UV-VIS2401PC, Japan). The maximum absorption wavelength was investigated in the range (Figure 9). As a result, the maximum absorbance of RP10356 by absorptiometer was 5 3 5 nm.

(3) Mass purification of pigments by silica gel chromatography (Silica gel chromatography) In order to mass-separate and purify pigments extracted with 2-propanol from culture broth, acetone: methanol (9: 1) was used as a developing solvent. Silica chromatography (silica chromatography: silica gel 60, 0.040-0.063 mm, Merck, Germany) was performed to separate the crude pigment (FIG. 10). As a result of separation and purification of the crude pigment by silica chromatography, it was fractionated into four types. This fraction was made in the order of bright yellow, dark red, bright red, and blue in the same manner as the TLC result, and RP10356 that became bright red was separated.

(4) High Performance Liquid Chromatography (HPLC) Analysis The purified dye was analyzed using the HP1050 system (Hewlett Packard, USA). At this time, using a silica column (YMC-Pack SIL, 250 × 4.6 mm, Japan) as the column, 1.0 mL of acetone: methanol (9: 1) solvent was eluted per minute, and DAD-UV ( Hewlett Packard, USA) 53 5 nm using, 486 nm, and at 300 to 800 nm, and analyzed (FIGS. 11 and 12). According to the analysis by HPLC, RP10356 has a retention time of 3.52 minutes under the above conditions, and it was confirmed that a single peak was separated even at 300 to 800 nm.

  As described above, the present invention has an effect of providing a red pigment having an algicidal effect derived from a Hahera chejuensis strain. The red pigment (RP10356) according to the present invention has an excellent tide-killing effect against red tide-causing species such as coclodinium / polyclicoides, gyrodium / impzicum, heterosigma / acassio, etc. It is useful as an active ingredient of the algaecidal preparation to be removed.

Claims (3)

A method for removing red tide organisms, comprising using a culture solution of Hahera Chejuensis strain 96CJ10356 (KCTC 2396) or an organic solvent extract of said culture solution. The method for removing red tide organisms according to claim 1 , wherein the organic solvent is 2-isopropanol. The red tide organisms Kokurodjiniumu-Porikuri carp des (Cochlodinium polykrikoides), gyro pyridinium-Inpujikumu (Gyrodinium impudicum), or Heterosigma akashiwo (Heterosigma akashiwo) red tide organisms removing method of claim 1 is due.

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