KR20160149760A - Method for preventing and curing the disease of fish using bule LED - Google Patents
Method for preventing and curing the disease of fish using bule LED Download PDFInfo
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- KR20160149760A KR20160149760A KR1020150087358A KR20150087358A KR20160149760A KR 20160149760 A KR20160149760 A KR 20160149760A KR 1020150087358 A KR1020150087358 A KR 1020150087358A KR 20150087358 A KR20150087358 A KR 20150087358A KR 20160149760 A KR20160149760 A KR 20160149760A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K63/00—Receptacles for live fish, e.g. aquaria; Terraria
- A01K63/04—Arrangements for treating water specially adapted to receptacles for live fish
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/30—Treatment of water, waste water, or sewage by irradiation
-
- F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
- F21—LIGHTING
- F21K—NON-ELECTRIC LIGHT SOURCES USING LUMINESCENCE; LIGHT SOURCES USING ELECTROCHEMILUMINESCENCE; LIGHT SOURCES USING CHARGES OF COMBUSTIBLE MATERIAL; LIGHT SOURCES USING SEMICONDUCTOR DEVICES AS LIGHT-GENERATING ELEMENTS; LIGHT SOURCES NOT OTHERWISE PROVIDED FOR
- F21K9/00—Light sources using semiconductor devices as light-generating elements, e.g. using light-emitting diodes [LED] or lasers
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2303/00—Specific treatment goals
- C02F2303/04—Disinfection
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Engineering & Computer Science (AREA)
- Marine Sciences & Fisheries (AREA)
- Biodiversity & Conservation Biology (AREA)
- Animal Husbandry (AREA)
- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Physics & Mathematics (AREA)
- Optics & Photonics (AREA)
- Microelectronics & Electronic Packaging (AREA)
- Health & Medical Sciences (AREA)
- Toxicology (AREA)
- Hydrology & Water Resources (AREA)
- Environmental & Geological Engineering (AREA)
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Farming Of Fish And Shellfish (AREA)
Abstract
Description
The present invention relates to a method of sterilizing and bacteriolizing a fish pathogen using a blue light LED. The present invention also relates to a sterilizing or bacteriostatic apparatus for a fish pathogen using the method for sterilizing or bacteriolizing the fish pathogen. The present invention also relates to a method for preventing or treating fish diseases using the method for sterilizing or bacterium of the above-mentioned fish pathogen.
Today, aquaculture industry is one of the fastest growing fisheries industry, and its economic importance is acknowledged globally as a result of the surging demand for aquatic products. In Korea, almost all the coastal areas are being used for aquaculture. Cultured fish are often in an environment that is susceptible to infection such as bacteria and parasites, because of their high immunity due to dense breeding and worsening of water quality.
Currently, several kinds of antibiotics are used alone or in feed for the prevention of disease and infection in Korea. However, safety is more demanded and continuous use of antibiotics causes increase of resistant microorganisms, It can be transmitted to humans, and with the destruction of bacterial flora in the intestines itself, the accumulation of antibiotics may not only hinder the growth of the intestinal flora itself, but may also have adverse effects on food.
LED is an abbreviation of Light Emitting Diode (LED). LED light source is widely used in industry because it can emit more light while consuming less power than conventional fluorescent lamps and incandescent lamps. In addition, this advantage has enabled us to make breakthroughs in photodynamic therapy (PDT). PDT refers to a technique for treating intractable diseases such as cancer without using surgery using a photosensitizing substance. In particular, PDT, which is used for cancer treatment, is a method of inserting a photosensitizing substance capable of generating or absorbing only in specific cancer cells, and then irradiating light to change the oxygen molecule (O 2 ) into active oxygen, Or create new species to selectively kill cancer cells. Such a PDT method can be used not only in cancer cells but also in pathogens producing photosensitizing substances. However, the present PDT treatment is targeted to humans, and there are no examples of application to marine organisms yet, and it is difficult to use the method applied to humans as it is due to the specificity of the species.
The inventors of the present invention discovered that when an LED light source of blue light is irradiated to an underwater environment including a fish pathogen while searching for a method of sterilizing or bacteriating a fish pathogen, the fish pathogens are not environmentally friendly and do not use antibiotics Sterilization or bacteriolysis, and the present invention has been completed.
Accordingly, the present invention provides a method for sterilizing or bacteriolizing a fish pathogen, comprising the step of irradiating an LED light source of blue light to an underwater environment including a fish pathogen.
The present invention also provides a sterilizing or bacteriostatic apparatus for a fish pathogen using the method for sterilizing or bacteriolizing the fish pathogen.
The present invention also provides a method for preventing or treating fish diseases by using a method for sterilizing or bacteriolizing the fish pathogen.
In order to achieve the above object,
The present invention
And irradiating an LED light source of blue light to an underwater environment including a fish pathogen.
In addition, the present invention provides a sterilizing or bacteriostatic apparatus for a fish pathogen using the method for sterilizing or bacteriolizing the fish pathogen.
The present invention also provides a method for preventing or treating fish diseases by using a method for sterilizing or bacteriolizing the fish pathogen.
Hereinafter, the present invention will be described in detail.
The present invention provides a method for sterilizing or bacterium of a fish pathogen, comprising the step of irradiating an LED light source of blue light to an underwater environment including a fish pathogen.
The method for sterilizing or bacterium of a fish pathogen according to the present invention is a method for sterilizing or bacteriolizing only a fish pathogen without any physiological and physical damage to the fish by irradiating the LED light source of a specific wavelength to an underwater environment including a fish pathogen .
The blue light preferably has a wavelength in the range of 380 to 480 nm, and is preferably maintained at a light amount in the range of 50 to 550 mu mol.m -2 s -1 . The wavelength and the amount of light of the blue light LED are used to sterilize or bacteriologically isolate fish pathogens but not to physically damage fish such as inflammation or necrosis and to prevent the expression of stress related factors, . According to one embodiment of the present invention, the condition for 99.9% sterilization of the fish pathogen is proportional to the power of the light amount.
The fish includes all kinds that can be infected with a pathogen, and can be cultured fish or aquarium fish. Specific examples include flounder, carp, cilia, eel, trout, mackerel, mackerel, mackerel, flounder, ten gang, strong legs, rockfish, uroguardia, molasses, codfish, saury, herring, eel, sardine, salmon, tuna, bonito , Anchovy, hawk, red sea bream, early sea, and the like, and it is preferably flounder or carp.
The fish pathogen can be isolated from diseased flounder and selected from TSA (Tryptic Soy Agar), TSB (Tryptic Soy Broth), BHIA (Brain Heart Infusion Agar), BHIB (Brain Heart Infusion Broth) or LB (Luria Bertani) It is preferable to culture them in one or more kinds of medium. It is preferable that the TSA, TSB, BHIA, BHIB or LB can increase the expression of the strain more than the SAPW, TCG, A1BFe-C or A1-C medium and thus it is possible to obtain a large amount of pathogen.
The fish pathogen may be Gram-negative or Gram-positive homogeneous.
The gram-negative bacteria is Vibrio Harvey (V. harveyi), Vibrio aenggwil rarum (V. anguillarum), Vibrio ripening thio yen tri (V. ichthyoenteri), all non-Broglie bridge (V. odrdalii), Vibrio assembly blood kusu (V. vulnificus), V. Let us live monitor (V. salmonicida), Vibrio para Molly tee hee Syracuse (V. parahaemolyticus), Salah photos tumefaciens dam (P. damselae), Flavobacterium five branches pilyum (F. brachiophilum), Playa F. columnare , F. johnsoniae , F. psychrophilum , T. maritiumum , Bacillus subtilis , T. ovolyticum , E. tarda , E. ictaluri , A. salmonicida , eromonas hydrofilas, and the like. A. hydrophila , and the like.
In addition, the Gram-positive bacteria may be selected from the group consisting of Streptococcus iniae , S. parauberis , R. salmoninarum , L. garvieae , N. seriolae , and the like can be used.
The method for sterilizing or bacterium of a fish pathogen according to the present invention is effective for sterilizing and bacterium of both Gram-negative bacteria and Gram-positive bacteria. Gram-positive bacteria have thick peptidoglycan cell walls that consist of numerous individual peptidoglycan layers surrounding the cell membrane. In contrast, Gram-negative bacteria have only a thin layer of peptidoglycan surrounding the cell membrane, which is additionally surrounded by an additional outer membrane. This additional layer allows gram-negative and gram-positive bacteria to be distinguished by the gram method.
According to one embodiment of the present invention, when the Gram-negative bacteria are irradiated with a strong LED light source having a wavelength of 390 to 420 nm, the germ-negative bacteria exhibit more excellent sterilization and bacteriostatic effect than an LED light source having a wavelength of 450 to 480 nm But the Gram-positive bacteria did not show a significant difference between wavelengths. As a result, it is preferable to maintain the LED light source having a wavelength of 390 to 420 nm at 50 to 300 μ mol · m -2 · s -1 for Gram-negative bacteria, and 450 to 480 nm When the wavelength of the LED light source is maintained at a light amount of 300 to 530 μ mol · m -2 s -1 , it is possible to exhibit a more excellent sterilization or bacteriostatic effect without damaging the fish.
Specifically, the V. harveyi strain is a Gram-negative organism and is known to be a luminescence vibrio that infects shrimp, which is an invertebrate animal, which causes many mortalities in flounder. It is known to be one of the most serious diseases that cause 100% mortality due to infection with earfish. According to the present invention, the V. harveyi strain does not exhibit a bactericidal effect for an LED light source having a wavelength of 450 to 480 nm, but when the LED light source having a wavelength of 390 to 420 nm is irradiated for 22 to 26 hours, It was able to sterilize 90 ~ 100% of the strain without any damage. In addition, the Streptococcus iniae strain is a representative marine Gram-positive bacterium, and is a major causative agent of streptococcosis, which causes hemorrhagic septicemia in fishes, particularly in flounder. According to the present invention, the Streptococcus iniae strain has a characteristic that when irradiated with an LED light source having a wavelength of 450 to 480 nm for 5 to 7 hours compared with the case of irradiating a strong LED light source having a wavelength of 390 to 420 nm, It was able to sterilize 90 ~ 100% of the strain without any damage.
The fish pathogen may be included in the water at a concentration of 1 × 10 3 to 1 × 10 8 CFU / mL, but is not limited thereto.
The method for sterilizing or bacterium of a fish pathogen according to the present invention can be carried out by treating a photosensitizer to a fish pathogen and then irradiating an LED light source of blue light. The photosensitizer can be activated by light to change its oxygen molecule (O 2 ) to active oxygen, to create new radicals, or to create new species and destroy pathogens. The photosensitizer material may be indocyanine green, methylene blue, toluidine blue, aminolevulinic acid, phthalocyanine, porphyrin, texapyrine, bacterioclomer, merocyanine, psoralen, benzoporphyrin derivatives, But is not limited thereto.
The underwater environment may be, but not limited to, a fish tank, an aquarium, a farm, a pond, a kennel, or the ocean.
In addition, the present invention provides a sterilizing or bacteriostatic apparatus for a fish pathogen using the method for sterilizing or bacteriolizing the fish pathogen.
Further, the present invention provides a method for preventing or treating fish diseases using the method for sterilizing or bacteriolizing the above-described fish pathogen.
The fish disease is preferably a bacterial disease, and more specifically, it can be a streptococcal disease, a Vibrio disease, a slide bacterial disease, an Edward's disease, a photobacterium disease, and the like. For the prevention or treatment of fish diseases, conventional antibiotics are conventionally administered by oral administration to feedstuffs in combination with feed. However, this method is problematic in that bacterial diseases caused by streptococci occur, and pathogens colonize and proliferate repeatedly Treatment is not good, and the use of antibiotics is likely to produce resistant pathogens. On the other hand. When the method for sterilizing or bacteriolizing a fish pathogen using the blue light LED of the present invention is used, fish diseases can be prevented or treated without any side effects.
The method of sterilizing or bacteriolizing a fish pathogen according to the present invention is performed by irradiating an LED light source of blue light to an underwater environment including a fish pathogen, thereby being eco-friendly and economical, There is an effect.
FIG. 1 is a graph showing the bactericidal effect of a fish pathogen after irradiating an LED light source with a wavelength of 405 nm or 465 nm according to Example 1. FIG.
2 is a graph showing the correlation between the (a) sterilizing effect of an LED light source with a wavelength of 405 nm and the exposure time of a light source according to Example 1 against an Edwardian E. tarda strain .
FIG. 3 is a chart showing relative growth rates of the fish pathogens according to Example 1 for the LED light source (a) at 405 nm and (b) at 465 nm wavelength.
FIG. 4 is a graph showing changes in concentration of fish pathogens in raising water according to irradiation of an LED light source (experimental group) and natural light (control group) at a wavelength of 405 nm or 465 nm according to Example 4-1.
FIG. 5 is a graph showing the results of irradiation of 14 days of LED light source (experimental group) and natural light (control group) of 405 nm or 465 nm wavelength according to Example 4-1, fishes present in head kidneys and spleen FIG.
Fig. 6 is a graph showing the results obtained by irradiating fishes for 0 to 28 days with 405 nm wavelength of (a), 465 nm wavelength of (a), and natural light (control) of LED light source according to Example 5-2, Fig.
FIG. 7 is a graph showing the results of irradiating fishes for 0 to 28 days with a wavelength of 405 nm, (b) 465 nm wavelength and (c) natural light (control group) of the LED light source according to Example 5-2, Fig.
FIG. 8 is a graph showing the fluorescence spectra of (a) HSP70, (b) 11? -HSD2 and (c) HSP70 expressed at a wavelength of 465 nm and (d) 11? -HSD2 expressed at 405 nm wavelength of the LED light source according to Example 5-4 And shows the degree of expression in the head and neck.
Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.
Example 1. Preparation of LED light source and fish pathogen
1-1 Preparation of LED light source
The LED light source was a blue LED light source with wavelengths of 405nm and 465nm provided by Pukyong University marine LED center, and each light quantity was measured with a radio meter (Biospherical Instruments Inc., USA). At this time, the light amount of the 405 nm LED light source was 284 μmol / m² ㆍ s and the light amount of the 465 nm LED light source was 516 μmol / m² ㆍ s.
1-2 Preparation of fish pathogens
The pathogenic strain of the present invention was isolated from diseased flounder by using seven strains known to cause serious damage to fish by causing disease (see Table 1).
(strain code)
( Vibrio harveyi )
( Vibrio anguillarum )
( Photobacterium damselae )
( Edwardsiella tarda )
( Aeromonas salmonicida )
( Streptococcus iniae )
( Streptococcus parauberis )
Of the fish pathogens, gram-negative bacteria is Vibrio Harvey (V. harveyi), Vibrio aenggwil rarum (V. anguillarum), picture tumefaciens dam Cellar (P. damselae), Edward when Ella the tar (E. tarda), Pseudomonas aero A. salmonicida strains were stored in TSA (Tryptic Soy Agar) until use, and then cultured for 24 hours in 1% NaCl medium for use. In addition, Gram-positive bacteria such as S. iniae , S. parauberis strains, BHIA (Brain Heart Infusion Agar), and then cultured in 1% NaCl medium for 24 hours at 28 ° C for use.
After that, V. anguillarum , a strain isolated and preserved in the Korean Collection for Type Culture (KCTC), and E. tarda and Aeromonas salmonii , already identified in previous studies, Except for A. salmonicida strains (see Journal of Fish Diseases , 2006, 29, 601-609, and Journal of Fish Diseases , 2011, 34, 47-55 ) , V. harveyi , P. damselae , S. iniae , and S. parauberis were re-identified by polymerase chain reaction (PCR) using a specific primer.
The specific primers are shown in Table 2.
( Vibrio harveyi )
Tox R: 5`-GGTGAAGACTCATCAGCA-3`
( Photobacterium damselae )
Car 2: 5`-CACCTCGCGGTCTTGCTG -3`
Ure-3`: 5`-CTTGAATATCCATCTCATCTGC-3`
Ure-5`: 5`-TCCGGAATAGGTAAAGCGGG-3`
( Streptococcus iniae )
Lox R: 5'-ATATCTGATTGGGCCGTCTAA-3`
( Streptococcus parauberis )
Spa R: 5`-GCTTCATATATCGCTATACT-3`
The PCR conditions were as follows: (1) V. harveyi strain was pre-denaturated once at 95 ° C for 5 minutes, denatured at 94 ° C for 1 minute, annealed at 55 ° C for 1 minute, And 72 ° C for 1 minute were repeated 30 times, and then the amplification was completed by maintaining 1 time at 72 ° C for 10 minutes (Expected size: 382 bp). (2) P. damselae was subjected to multiflex PCR, denatured at 95 ° C for 1 minute, denatured at 95 ° C for 1 minute, denatured at 95 ° C for 1 minute, denatured at 95 ° C for 1 minute, And a cycle of amplifying at 72 DEG C for 40 seconds was repeated 30 times, and then the amplification was completed at 72 DEG C for 10 minutes to complete the amplification (Expected size: 448, 267 bp). (3) Streptococcus ini . iniae ) was subjected to one cycle of denaturation at 95 ° C for 1 minute, denaturation at 92 ° C for 1 minute, annealing at 55 ° C for 1 minute, and amplification at 72 ° C for 2 minutes. (4) The S. parauberis strain was subjected to a first denaturation at 95 ° C for 1 min, followed by a denaturation at 92 ° C for 1 min, followed by a final denaturation at 95 ° C for 1 min. Annealing for 1 minute at 55 캜, and amplification for 1 minute and 30 seconds at 72 캜 were repeated 25 times, Up was complete the amplification maintained once for 5 minutes (Expected size: 870bp). PCR was performed to identify the strains used in this experiment.
Example 2: Sterilizing effect of fish pathogen
2-1 Sterilization effect of fish pathogen according to wavelength of LED light source
V. anguillarum , V. harveyi , E. edarda , A. salmonicida , A. salmonicida , etc., which are Gram-negative bacteria according to Example 1, sterilization was carried out tests on positive bacteria Streptococcus am a lover (S. iniae), Streptococcus para Ube lease (S. parauberis) - tumefaciens dam Cellar (P. damselae), and gram. First, each of the fish pathogens was suspended in physiological saline at a concentration of about 10 5 CFU / mL, and 10 mL of the fish pathogens were inoculated into a petri dish (60 mm × 15 mm). Thereafter, an LED light source with a wavelength of 405 nm or 465 nm was irradiated, and 0.1 ml of the bacterial solution was inoculated on the medium at 0, 1, 3, 6, 12, 24, and 48 hours, respectively, and viable cell counts were confirmed by the flat plate method.
The bactericidal effect of a fish pathogen after irradiation with an LED light source of wavelength 405nm or 465nm according to Example 1 is shown in Fig. 1 (repeated
As shown in Figure 1, A. salmonicida , S. iniae , and < RTI ID = 0.0 & gt ; The S. parauberis pathogen shows a slight decrease in the number of pathogens over time even in the control group that does not irradiate the LED light source. In addition, the V. harveyi pathogen showed a bactericidal effect at an LED wavelength of 405 nm but was almost not sterilized at an LED wavelength of 465 nm. Gram-negative bacteria such as V. anguillarum , V. harveyi , E. tarda , and P. damselae was completely sterilized at 6 hours, 24 hours, 12 hours, and 6 hours respectively for 405nm LED wavelengths. However, S. iniae , a Gram-positive strain , exhibited a stronger bactericidal effect at a wavelength of 465 nm rather than an LED wavelength of 405 nm, while S. parauberis showed a strong bactericidal effect at 405 nm and 465 nm LEDs But showed almost no significant difference in wavelength.
2-2 Sterilization effect of fish pathogen according to light amount of LED light source
The fish pathogen according to Example 1 A sterilization test was conducted according to the amount of light by using E. edarda strain. First, the E. tarda strain was suspended in physiological saline at a concentration of 10 5 CFU / mL, and 10 mL of the strain was inoculated into a Petri dish (60 mm × 15 mm). Thereafter, the LED light source with a wavelength of 405 nm was irradiated with light quantities of 286, 161, 67, 18, and 9 占 mol m 占 퐉 -2 s -1 , respectively, and after 3, 6, 12, 24, and 48 hours Each broth was plated on TNA medium to confirm viable cell counts, and the results were compared with the control group without light (repeated 3 times).
(A) the sterilizing effect of the LED light source of 405 nm wavelength according to the amount of light and (b) the amount of light and the exposure time of the light source for the Edwardian E. tarda strain according to Example 1, Respectively.
As shown in FIG. 2 (a), as the light amount of the LED light source decreases, the bactericidal effect against the strain decreases. In addition, as shown in Fig. 2 (b), the condition for reducing 99.5% of the E. tarda strain of about 10 5 CFU / mL was found to be proportional to the power of the light amount.
Example 3: Bacteriostatic effect of a fish pathogen
V. anguillarum , V. harveyi , E. edarda , A. salmonicida , A. salmonicida , etc., which are Gram-negative bacteria according to Example 1, bacteriostatic test was performed on positive bacteria Streptococcus am a lover (S. iniae), Streptococcus para Ube lease (S. parauberis) - tumefaciens dam Cellar (P. damselae), and gram. Each of the fish pathogens was suspended in BHIB (Brain Heart Infusion Broth) and 1% NaCl solution at a concentration of 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , and 10 8 CFU // mL, And then inoculated into a well microplate. Thereafter, the relative growth rate of the strain was confirmed by using the absorbance value of 630 nm wavelength in comparison with the control group in which the LED light source of 405 nm or 465 nm was irradiated with the LED light source.
Relative growth rate = (the LED light irradiation section 630 OD / control section of OD 630) * 100, where, where OD is the extinction density (optical density).
The relative growth rates of the LED light sources (a) 405 nm and (b) 465 nm wavelength of the fish pathogen according to Example 1 are shown in FIG. 3, and the results are also shown in Table 3.
As shown in FIG. 3 and Table 3, the susceptibility to the LED light source of 405 nm wavelength was high in A. salmonicida and Edwardian elata tarda , but the LED light of 465 nm wavelength Sensitivity to the light source is determined by the presence of Gram-positive bacteria, S. iniae , Streptococcus parauberis ( S. parauberis ) was relatively high. In addition, the V. harveyi strain showed almost no sensitivity to both 405 nm and 465 nm wavelengths, and P. damselae showed a germicidal effect in the physiological saline solution, No bacteriostatic or bactericidal effect was observed in the medium.
Example 4. Effect of inhibiting horizontal propagation of fish pathogens
4-1. How to measure
The fish pathogen according to Example 1 Edwardian E. tarda strains were used to analyze the horizontal propagation of fish pathogens. First, the E. tarda strain was inoculated into TSA (Trypticase Soy Agar) medium and cultured for 24 hours. Thereafter, the cultured strains were suspended in PBS at a concentration of 3.3 × 10 4 CFU / mL, 0.1 mL of each of them was intraperitoneally injected into each of 405 nm and 465 nm wavelength LED light source test groups and 10 controllable carp of natural light, The tail fin was cut and labeled to distinguish it from the non-group. In addition, each of the 10 carps of the group not injected with the strain was inserted into the 405 nm and 465 nm wavelength LED light sources, and the LED light source was irradiated every 12 hours. Thereafter, the number of breeding water was sampled at 0, 1, 3, 7, and 14 days in order to confirm whether the total strains were changed in the breeding water according to the LED light source irradiation.
In order to confirm the change in the number of total pathogens in the cultivated water according to the LED light source irradiation, 0.1 mL of water was inoculated on TSA medium at 0, 1, 3, 7, and 14 days, Were counted and the number of viable cells was confirmed in the total number of breeding (three times repeated measurement).
After completion of the 14th day of the experiment, the carp of each group of 405 nm and 465 nm wavelengths was dissected and the dorsal and spleen were collected to measure viable cell counts Respectively. Specifically, after the weight of each tissue was measured, 1 mL of PBS was added, and the tissue was ground and cultured on SS (Shalmonella shigella) medium. Then, the number of viable cells growing after 24 hours was counted.
4-2. Measurement result
FIG. 4 shows changes in the concentrations of fish pathogens in the breeding stock according to the irradiation of LED light source (experimental group) and natural light (control group) of 405 nm or 465 nm wavelength according to Example 4-1.
As shown in FIG. 4, the 0th day 405nm or 465nm wavelength LED irradiation group showed a higher total number of bacteria in the breeding water than the control group, but the number of bacteria in the breeding water decreased to 3, 7 and 14 days (P < 0.05).
After 14 days of irradiation with the LED light source (experimental group) and natural light (control group) at the wavelength of 405 nm or 465 nm according to Example 4-1, the number of fish pathogens present in the head kidney and spleen of the breeding water Is shown in Fig. The pelletization rate and the infection rate thereof are shown in Table 4.
As shown in FIG. 5 and Table 4, when the LED light source of 405 nm or 465 nm wavelength was irradiated with respect to the natural light (control group), the number of fish pathogens present in the head kidney and spleen of the breeding water was lower, The infection rate and the mortality rate are also decreased.
Example 5. Assessment of harmfulness to fish
5-1. Species and Experimental Methods
Each 100 carp (cyprinus carpio) (average weight = 2.22 g ± 0.52) was placed in an aerated tap water at 26 ° C for 3 days and then moved again for 2 weeks to make it normal. Of these, 60 carp were divided into an LED light source (experimental group) and natural light (control group) having a wavelength of 405 nm or 465 nm, and then, they were placed in a water tank (35 cm x 50 cm x 45 cm). After that, the light cycle was adjusted to 12 hours every day, and the whole body was returned half by one, and 1% of the body weight was fed daily (commercial fish feed). Three carp were then anesthetized with AQUI-S diluent at intervals of 0, 1, 3, 7, 14, and 28 days, and eye and skin tissues were fixed in buccal fluid, , And stored at -80 ° C.
5-2. Pathological analysis method
The eye and skin tissues fixed to the buoy solution for 24 hours according to Example 5-1 were cut into 0.5-1 cm size and put into the buoy solution and fixed for 24 hours. Then, tissue processing, Leica TP 1020 , Germany) to infiltrate paraffin. Thereafter, it was embedded using a Porsiger machine (Medite TBS 88, Germany) and then discontinued using a pedicle (Leica RM 2125 RTS, Germany). The thickness of the dissected tissue section was made 5 쨉 m, and the cut section was stained with herris hematoxylin-eosin method.
5-3 cDNA synthesis using total RNA isolation and reverse transcription
Total RNA was isolated from the skins stored in the later stage of the RNA according to Example 5-1 using Trizol RNA isolation method, treated with DNase, and 1 μg of total RNA was synthesized according to the cDNA synthesis kit (Bioneer method) .
5-4 Real-time PCR (qPCR) analysis method
Real-time polymerase chain reaction (RT-PCR) was performed to confirm the expression levels of β-actin, HSP70 and 11β-HSD2. The reaction mixture of RT-PCR was analyzed using an Exicycler TM 96 Real-Time Quantitative Thermal Block (Bioneer), and each tube for qPCR analysis contained 12.5 μL of
R: 5`-TTTCTCTTTCGGCTGTGGTGGTG-3`
R: 5`-TTGTCAAAGTCCTCCCCACC-3`
R: 5'-AGATTCAGGGCAGCTTTTGA-3`
5-5 Measurement results
(A) 405 nm wavelength, (b) 465 nm wavelength, and (c) natural light (control group) of the LED light source according to Example 5-2 were irradiated to fish for 0 to 28 days, and then an optical microscope image Is shown in Fig. (A) 405 nm wavelength, (b) 465 nm wavelength, and (c) natural light (control group) of the LED light source according to Example 5-2 were irradiated to fish for 0 to 28 days, A microscope image is shown in Fig.
As shown in FIGS. 6 and 7, in the LED light source of 405 nm wavelength, light of about 284 μmol / m² ㆍ s and the LED light source of 465 nm wavelength of about 516 μmol / m² ㆍ s light amount, It can be seen that it did not cause physical damage.
(A) HSP70, (b) 11? -HSD2, and (c) HSP70 expressed at a wavelength of 465 nm and (d) 11? -HSD2 expressed at 405 nm wavelength of the LED light source according to Example 5-4, Is shown in Fig.
As shown in Fig. 8, at 405 nm and 465 nm wavelength, the highest expression of stress-related factors was induced on
Claims (17)
Wherein the blue light has a wavelength in the range of 380 to 480 nm.
Wherein the blue light is maintained at an amount of light in the range of 50 to 550 mu mol.m- 2 s < -1 & gt ; .
The fishes include halibut, carp, cilia, eel, trout, mackerel, mackerel, mackerel, flounder, ten gang, strong legs, rockfish, uroguardia, mackerel, codfish, saury, herring, eel, sardine, salmon, tuna, bonito , Anchovy, hairtail, red sea bream, and early fish.
The fish pathogens Vibrio Harvey (V. harveyi), Vibrio aenggwil rarum (V. anguillarum), Vibrio ripening thio yen tri (V. ichthyoenteri), all non-Broglie bridge (V. odrdalii), Vibrio assembly blood kusu (V. vulnificus ) , V. salmonicida , V. parahaemolyticus , P. damselae , F. brachiophilum , Flavio F. columnare , F. johnsoniae , F. psychrophilum , T. maritiumum , Tena spp . T. ovolyticum , E. tarda , E. ictaluri , A. salmonicida , and eromonas hydrofilas ( E. coli) A. hydrophila ). Wherein the microorganism is a gram-negative organism of a species or more.
And the LED light source having a wavelength of 390 to 420 nm is maintained at a light amount of 50 to 300 占 mol m 占 퐉 -2? S- 1 for the Gram-negative bacteria.
The fish pathogens include S. iniae , A species selected from the group consisting of S. parauberis , R. salmoninarum , L. garvieae and N. seriolae , which is selected from the group consisting of Staphylococcus aureus , Or more of the Gram-positive bacteria.
And the LED light source having a wavelength of 450 to 480 nm for the Gram-positive bacteria is maintained at a light amount of 300 to 530 占 mol m 占 퐉 -2? S- 1 .
Wherein the Streptococcus iniae strain is sterilized by 90 to 100% of the strain when irradiated with an LED light source having a wavelength of 450 to 480 nm for 5 to 7 hours.
Wherein the fish pathogen is contained at a concentration of 1 x 10 3 to 1 x 10 8 CFU / mL.
Characterized in that the fish pathogen is treated with a photosensitizer and then an LED light source of blue light is irradiated.
Wherein the photosensitizing substance is selected from the group consisting of indocyanine green, methylene blue, toluidine blue, aminolevulinic acid, phthalocyanine, porphyrin, texapyrine, bacterioclomer, merocyanine, psoralen, benzoporphyrin derivatives and sodium formamide And a method for sterilizing or bacterium of a fish pathogen.
Wherein the underwater environment is a fish tank, an aquarium, a farm, a pond, a breeding ground, or a marine.
Wherein said fish disease is bacterial disease of Streptococcus, Vibrio, Slide bacterial, Edward, Photobacterium, or a combination thereof.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107667927A (en) * | 2017-11-21 | 2018-02-09 | 蚌埠市渔歌农业科技有限公司 | A kind of cultural method of high yield high nutrition snakeheaded fish |
| KR20190127223A (en) | 2018-05-04 | 2019-11-13 | 한국해양대학교 산학협력단 | Aquaculture method using a reducing stress and enhancing immunity by irradiation of light emitting diode in the shrimp |
| KR20210041207A (en) | 2019-10-07 | 2021-04-15 | 블루오션영어조합법인 | Fish disease detecting apparatus and method thereof |
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2015
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107667927A (en) * | 2017-11-21 | 2018-02-09 | 蚌埠市渔歌农业科技有限公司 | A kind of cultural method of high yield high nutrition snakeheaded fish |
| KR20190127223A (en) | 2018-05-04 | 2019-11-13 | 한국해양대학교 산학협력단 | Aquaculture method using a reducing stress and enhancing immunity by irradiation of light emitting diode in the shrimp |
| KR20210041207A (en) | 2019-10-07 | 2021-04-15 | 블루오션영어조합법인 | Fish disease detecting apparatus and method thereof |
| KR20210064157A (en) | 2019-10-07 | 2021-06-02 | 블루오션영어조합법인 | Fish disease detecting apparatus |
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