Novel Flavobacteriaceae and fish protection

Abstract

To provide novel microorganisms that protect fish.SOLUTION: In one aspect, the disclosure provides a method for obtaining a microorganism having a fish-protection ability. In another aspect, the disclosure provides a microorganism capable of protecting fish or a biocomponent thereof, and a composition comprising the same. The fish-protecting microorganism may be a strain of Flavobacteriaceae. In one aspect, the disclosure provides a method for protecting fish using a microorganism capable of protecting fish or a biocomponent thereof, and a composition comprising the same.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to microorganisms having the ability to protect fish, methods for obtaining such microorganisms, and uses of such microorganisms. [Background technology]

[0002] Antibiotics are attracting international attention as something that should be reduced due to concerns about the emergence of multidrug-resistant bacteria and environmental pollution. In research into disease prevention in farmed fish, attention has been focused on the development of fish disease vaccines as an alternative to antibiotics (Non-Patent Document 1). Although fish disease vaccines have proven effective, they are only effective against specific pathogens in specific fish species, and the types of vaccines approved for use in the field are limited, making this method insufficient. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Ministry of Agriculture, Forestry and Fisheries, Food Safety and Consumer Affairs Bureau, Livestock and Fisheries Safety Management Division, "Regarding the Use of Fisheries Pharmaceuticals," Report No. 29 (January 31, 2016) Summary of the Invention [Means for solving the problem]

[0004] As a result of extensive research, the present inventors have developed a method for obtaining microorganisms capable of inhibiting harmful microorganisms and have found that the microorganisms thus obtained are useful for protecting fish. Microorganisms that may be useful for protecting fish include bacteria of the Flavobacterium family. These microorganisms have been found to be capable of inhibiting harmful microorganisms. Therefore, the present disclosure provides the microorganisms thus discovered, their derivatives, and uses thereof. The present disclosure also provides a method for obtaining microorganisms capable of protecting fish.

[0005] Thus, the present disclosure provides: (Item 1) A bacterium from the family Flavobacteriaceae that has the ability to protect fish. (Item 2) Any of the above-mentioned Flavobacteria of the family Flavobacteriaceae, which has the ability to function as a probiotic for fish. (Item 3) Any of the above-mentioned Flavobacteriaceae bacteria having the ability to function as a probiotic for the epidermis of fish. (Item 4) 2. The bacterium of the family Flavobacterium according to any one of the preceding items, which has the ability to inhibit at least one harmful microorganism. (Item 5) The Flavobacterium family bacterium according to any of the preceding items, wherein the harmful microorganism has at least one of the following abilities: the ability to cause skin diseases in fish, the ability to infect fish through the skin, the ability to infect fish through wounds, and the ability to infect fish through contact. (Item 6) Any of the Flavobacterium family bacteria listed above, wherein the harmful microorganism is capable of causing edwardsiellosis, vibriosis, furunculosis, atypical Aeromonas salmonicida infection, Aeromonas hydrophila infection, red spot disease, pseudomonad disease in sweetfish, red mouth disease, bacterial gill disease, columnaris disease (chondrosis, tail rot, fin rot, mouth rot), cold water disease, gliding bacteriosis, bacterial kidney disease, mycobacteriosis, nocardiosis, and / or streptococcosis. (Item 7) The harmful microorganisms include Edwardsiella tarda, Edwardsiella ictaluri, Listonella anguillara (Vibrio anguillarum), Vibrio ordalii, Vibrio ichthyoenteri, Vibrio vulnificus, Vibrio salmonicida, Aeromonas salmonicida, atypical Aeromonas salmonicida, Aeromonas hydrophila, Pseudomonas. anguilliseptica, Pseudomonas plecoglossicida, Yersinia ruckeri, Flavobacterium branchiophilum, Flavobacterium columnare, Flavobacterium psychrophilum, Tenacibaculum maritimum, Renibacterium salmoninarum, Mycobacterium marinum, Mycobacterium fortuitum, Mycobacterium chelonei, Nocardia seriolae, Streptococcus iniae, Lactococcus garvieae, Aeromonas caviae, or a combination thereof. (Item 8) The Flavobacterium family bacterium of any of the preceding items, wherein the harmful microorganism comprises Edwardsiella tarda, Edwardsiella ictaluri, or a combination thereof. (Item 9) 2. The Flavobacterium family bacterium according to any one of the preceding items, which has the ability to improve the survival rate of fish. (Item 10) The Flavobacteriaceae bacterium according to any one of the preceding items, wherein the fish are protected by adding the Flavobacteriaceae bacterium to a breeding solution in a breeding environment for the fish. (Item 11) The Flavobacterium family bacterium according to any of the preceding items, wherein the fish is a farmed fish. (Item 12) The Flavobacterium family bacterium according to any of the preceding items, wherein the farmed fish is catfish, eel, sea bass, flounder, turbot, herring, rainbow trout, sweetfish, yellowtail, trout, sea bream, carp, amberjack, tuna, salmon, horse mackerel, tilapia, pufferfish, yellowtail, grouper, mackerel, or saury. (Item 13) The Flavobacterium family bacterium according to any of the preceding items, wherein the farmed fish is catfish, eel, sea bass, flounder, turbot, herring, rainbow trout, or sweetfish. (Item 14) The Flavobacteriaceae bacterium of any of the preceding items, which is a bacterium of the genus Flavobacterium or Chryseobacterium. (Item 15) Any of the Flavobacteriaceae bacteria described above, having a 16S rRNA base sequence that is most homologous or identical to the 16S rRNA base sequence of a type strain of a species selected from the group consisting of Flavobacterium procerum, resistens, micromati, limicola, reichenbachii, tiangeerense, xueshanense, psychrolimnae, algicola, faecale, frigidarium, omnivorum, fryxellicola, degerlachei, gillisiae, frigoris, sinopsychrotolerans, xinjiangense, and urumqiense, among type strains of Flavobacterium bacteria. (Item 16) A Flavobacterium family bacterium according to any of the above items, having a 16S rRNA nucleotide sequence that is most homologous or identical to the 16S rRNA nucleotide sequence of a type strain of a species selected from the group consisting of Chryseobacterium vietnamense, aquifrigidense, culicis, nakagawai, jejuense, bernardetii, rhizosphaerae, kwangjuense, elymi, lathyri, oranimense, contaminans, gallinarum, artocarpi, ureilyticum, oncorhynchi, joostei, viscerum, tructae, lactis, rhizoplanae, sediminis, indologenes, gleum, and arthrosphaerae, among type strains of Chryseobacterium bacteria. (Item 17) A Flavobacterium family bacterium, which is MUCF01 (received number: NITE AP-03323), MUCF02 (received number: NITE AP-03324), or MUCF03 (received number: NITE AP-03325). (Item 18) A fish protection agent comprising any of the above-mentioned Flavobacteriaceae bacteria. (Item 19) A probiotic agent for fish containing any of the above-mentioned Flavobacteriaceae bacteria. (Item 20) The probiotic agent according to any of the preceding items, which is a probiotic for the epidermis of said fish. (Item 21) The fish protectant or probiotic agent of any of the preceding items, which is a protectant or probiotic agent for farmed fish. (Item 22) A fish protectant or probiotic agent according to any of the above items, which is a protectant or probiotic agent for catfish, eel, sea bass, flounder, turbot, herring, rainbow trout, sweetfish, yellowtail, trout, sea bream, carp, amberjack, tuna, salmon, horse mackerel, tilapia, pufferfish, yellowtail, grouper, mackerel or saury. (Item 23) A fish protectant or probiotic agent according to any of the preceding items, which is a protectant or probiotic agent for catfish, eel, sea bass, flounder, turbot, herring, rainbow trout or sweetfish. (Item 24) A method for protecting fish, comprising the step of contacting said fish with any of the Flavobacteriaceae bacteria described in the preceding paragraphs. (Item 25) A method for protecting fish, comprising the step of growing the fish in water in which any of the Flavobacteriaceae bacteria described in the preceding items is present. (Item 26) A strain belonging to the Flavobacterium family, The strain is (i) streaking the strain linearly on NBR2A medium and culturing it at 25°C for 24 to 48 hours; (ii) then streaking the harmful microorganisms vertically from the strain without touching the strain, and culturing the strain at 25°C for 24 to 48 hours; and (ii) is characterized by the presence of a clear zone extending over an area of ​​10 mm or more from the streak of the strain, when tested in a method comprising: Here, the harmful microorganisms are Edwardsiella sp. (NBRC 12716), Edwardsiella sp. (NBRC 12717), and Edwardsiella ictaluri (NBRC 105724). T ) Strains.

[0006] It is contemplated that the present disclosure may provide one or more of the above-described features in combinations other than those explicitly stated. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary. [Effects of the Invention]

[0007] The microorganisms disclosed herein can be used as an alternative to antibiotics in fish farming and other fish breeding, thereby reducing the use of antibiotics and lowering the environmental burden. Furthermore, the method for obtaining microorganisms that protect fish disclosed herein can provide new useful microorganisms, thereby achieving more stable, inexpensive, highly productive, and / or diverse fish breeding. [Brief explanation of the drawings]

[0008] [Figure 1] The results of the cross-streak inhibition test in Example 2 are shown. The results are for MUCF01. The horizontal streaks are for Edwardsiella sp. (NBRC 12716), Edwardsiella ictaluri (NBRC 105724T), and Edwardsiella sp. (NBRC 12717), respectively. [Figure 2] The results of the cross-streak inhibition test in Example 2 are shown. The results are for MUCF02. The horizontal streaks are for Edwardsiella sp. (NBRC 12716), Edwardsiella ictaluri (NBRC 105724T), and Edwardsiella sp. (NBRC 12717), respectively. [Figure 3] The results of the cross-streak inhibition test in Example 2 are shown. The results are for MUCF03. The horizontal streaks are for Edwardsiella sp. (NBRC 12716), Edwardsiella ictaluri (NBRC 105724T), and Edwardsiella sp. (NBRC 12717), respectively. [Figure 4] The results of the cross-streak inhibition test in Example 2 are shown. The results are for strains that do not exhibit Edwardsiella inhibitory activity. The horizontal streaks are for Edwardsiella sp. (NBRC 12716), Edwardsiella ictaluri (NBRC 105724T), and Edwardsiella sp. (NBRC 12717), respectively. [Figure 5]This shows a molecular phylogenetic tree based on the 16S rRNA base sequence of MUCF01 (and MUCF02) from Example 3. The line at the bottom left indicates the scale bar. The numbers at the branching points of the phylogenetic tree indicate bootstrap values. [Figure 6] 1 shows a molecular phylogenetic tree based on the 16S rRNA base sequence of MUCF03 in Example 3. The line at the bottom left indicates the scale bar. The numbers at the branching points of the phylogenetic tree indicate bootstrap values. [Figure 7] This shows a cluster containing MUCF01 (as well as MUCF02) in a molecular phylogenetic tree based on the 16S rRNA base sequences of various type strains of Flavobacterium bacteria in Example 3. The numbers at the branching points of the phylogenetic tree indicate bootstrap values. [Figure 8] 1 shows a cluster including MUCF03 in a molecular phylogenetic tree based on 16S rRNA base sequences with various type strains of Chryseobacterium bacteria in Example 3. Numbers at branching points in the phylogenetic tree indicate bootstrap values. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present disclosure will now be described, illustrating the best mode thereof. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Thus, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, terms used in this specification should be understood to have the meaning commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the event of conflict, the present specification (including definitions) will prevail.

[0010] The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0011] (Definition, etc.) As used herein, "fish" refers to organisms belonging to the subphylum Vertebrata, excluding tetrapods. Fish include, but are not limited to, farmed fish used for food, ornamental fish, and pet fish. In one embodiment, any fish having a skin may be included in the present disclosure.

[0012] As used herein, "microorganisms" refers to organisms including eubacteria and archaea, as well as small (e.g., 1 mm or less, or 0.1 mm or less, or invisible to the naked eye) algae, protozoa, fungi (molds, fungi, etc.), yeast, and animals (protozoa, metazoa, etc.).

[0013] As used herein, the term "harmful microorganisms" refers to microorganisms that deteriorate or may deteriorate the survival, health, flesh quality, recovery from injury, and / or infection state of fish. Harmful microorganisms may also include opportunistic pathogens.

[0014] As used herein, "suppression" of microorganisms (also referred to as "inhibition" in Japanese, but both terms are synonymous in this specification) means a reduction and / or death of microorganisms and / or a slowing down of the growth rate of microorganisms, and also includes slowing down the growth or proliferation of microorganisms and killing microorganisms. Microorganism suppression can be evaluated, for example, based on the transparency of a medium inoculated with microorganisms and the area of ​​the medium that is clear. The ability of a microorganism to suppress or inhibit harmful microorganisms can be measured and identified, for example, by the following tests. Furthermore, if a result showing an inhibitory effect is obtained in any one of the tests, it is understood that this falls within the scope of inhibition in this specification. A test in which a candidate microorganism is added to a medium, and then harmful microorganisms are added to the same medium to check the transparency of the medium and / or the area of ​​the medium that is clear. A test in which candidate microorganisms and harmful microorganisms are grown on the same medium and a growth inhibition zone (clear zone) in which harmful microorganisms cannot grow near the candidate microorganisms is observed. A test to confirm that the growth of harmful microorganisms is reduced by adding the culture supernatant of a candidate microorganism to a liquid medium inoculated with harmful microorganisms. A test in which an impregnated culture medium or culture supernatant of a candidate microorganism is placed on a medium, or the culture medium or culture supernatant of a candidate microorganism is placed in a small bottomless cylinder, and the growth of harmful microorganisms inoculated over the entire medium is confirmed to be suppressed around it. A test in which candidate microorganisms and harmful microorganisms are cultured in the same liquid medium, and then the growth of the two microorganisms is compared to distinguish them, and it is confirmed that the harmful microorganisms are suppressed.

[0015] As used herein, the "skin" of a fish refers to the surface of the body (body surface), eyes, scales, fins, gills, mouth, anus, and genitals of a fish. The skin of the body surface of a fish can be classified into a scale type (scale-type fish) and a mucous type (mucous-type fish).

[0016] As used herein, "protection" of fish refers to improving the survival, health, flesh quality, recovery from injury, and / or infection status (including susceptibility to infection) of fish. The protection of fish can be evaluated, for example, by improving the survival rate of injured fish when they are brought into contact with harmful microorganisms. For example, fish can be protected by adding the Flavobacteriaceae bacteria to the rearing environment of fish (e.g., rearing liquid, rearing water, etc.). Here, the rearing environment refers to any environment in which fish organisms grow, and is not necessarily limited to a rearing liquid, although in the case of fish eggs, the water content may be low. The ability of a microorganism to protect fish can be measured and identified, for example, by the following test. A test in which fish are subjected to stress and harmful microorganisms are added to a control group, and a test in which harmful microorganisms and candidate microorganisms are added to the fish are compared to confirm an improvement in the survival rate of the fish in the treatment group. - A test to confirm the improvement in the survival rate of fish in the treatment group by first raising fish in the presence of candidate microorganisms for a certain period of time and then comparing the results between a treatment group to which harmful microorganisms are added and a control group to which harmful microorganisms are added without the pretreatment.

[0017] As used herein, "probiotics" refers to compositions containing biological components (e.g., microorganisms in their entirety or parts thereof) that have functions such as acting on harmful microorganisms, forming beneficial microbial flora in fish, maintaining or improving the health of fish skin, and / or stimulating the immune system of fish, or the microorganisms or biological components thereof, or compositions containing these. The ability of a microorganism to function as a probiotic can be measured and identified, for example, by the following test. A test to confirm that the survival rate of fish is improved when they are raised in the presence of a candidate microorganism for a certain period of time, compared to when they are raised in the absence of the candidate microorganism. After raising fish in the presence of candidate microorganisms for a certain period of time, tests are conducted to confirm signs of enhanced immune activity in the fish, such as increased cytokine secretion and improved expression of immune-related genes. A test to confirm that the population of harmful microorganisms in the microflora of fish is reduced by raising fish in the presence of candidate microorganisms for a certain period of time. A test in which fish are raised in the presence of candidate microorganisms for a certain period of time as a pretreatment, and then harmful microorganisms are added to the breeding water to be compared with a control group to which harmful microorganisms were added without pretreatment, to confirm that the population of the harmful microorganisms in the microbial flora of the fish in the treatment group is lower than that of the control group.

[0018] As used herein, "colonization" refers to the ability of a microorganism to remain attached to a target and survive (e.g., grow) for a certain period of time. The ability of a microorganism to colonize fish can be measured and identified, for example, by the following test. A test in which fish are raised in water to which candidate microorganisms have been added, and the number or quantity of candidate microorganisms obtained from the raised fish is confirmed. After raising fish in breeding water to which the candidate microorganisms have been added, the fish are observed under a microscope to confirm the presence of the candidate microorganisms on the fish (e.g., on their skin). After raising fish in breeding water to which candidate microorganisms have been added, the microbial flora of the fish (on their skin, etc.) is analyzed to confirm the presence of the candidate microorganisms in the flora.

[0019] As used herein, the term "fish protection agent" refers to a preparation that contains the microorganism of the present disclosure as an active ingredient and that can protect fish.

[0020] As used herein, the term "probiotic agent" refers to a preparation that contains the microorganism of the present disclosure as an active ingredient and is capable of exerting a probiotic function.

[0021] As used herein, a "derivative strain," "analogous strain," or "mutant strain" preferably, but without limitation, comprises a gene (e.g., 16S rDNA) containing a region substantially homologous to the DNA of the microorganism of interest, and in various embodiments, such a strain has a whole genome sequence that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical when compared to the whole genome sequence of the original strain by alignment using computer homology programs known in the art. This refers to a microorganism that has been modified by gene mutation, substitution, deletion, and / or addition, such that the derivative still exhibits the biological functions of the original microorganism, although not necessarily to the same degree. For example, genetic mutations can be introduced using any known mutagen, UV, plasma, genome editing technology, etc. In one embodiment, a "derivative strain," "analogous strain," or "mutant strain" is a strain of the same genus and / or species as the original strain. For example, the biological function of such microorganisms can be examined by suitable available in vitro assays described herein or known in the art.

[0022] As used herein, a "purified" biological factor (e.g., a specific strain of organism or intracellular component) refers to a biological factor from which at least a portion of the factors naturally associated with the biological factor have been removed. Thus, the purity of the biological factor in a purified biological factor is typically higher (i.e., more concentrated) than in the state in which the biological factor normally exists. As used herein, the term "purified" means that preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight of the same type of biological factor (e.g., a specific organism strain) is present.

[0023] As used herein, "homology" of genes or nucleotide sequences refers to the degree of identity between two or more gene sequences. Generally, "homology" refers to a high degree of identity or similarity. Therefore, the higher the homology between two genes, the higher the identity or similarity between their sequences. Whether two genes are homologous can be determined by direct sequence comparison or, in the case of nucleic acids, by hybridization under stringent conditions. When two gene sequences are directly compared, the genes are homologous if the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, and more preferably at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical. As used herein, "similarity" of genes or nucleotide sequences refers to the degree of similarity between two or more gene sequences, and refers to a high degree of similarity between sequences that are identical. "Similarity" is a numerical value that takes into account not only identity but also similar bases, where similar bases refer to partial matches in mixed bases (e.g., R=A+G, M=A+C, W=A+T, S=C+G, Y=C+T, K=G+T, H=A+T+C, B=G+T+C, D=G+A+T, V=A+C+G, N=A+C+G+T).

[0024] Amino acids may be referred to herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted one-letter codes. Herein, comparisons of amino acid and nucleotide sequence similarity, identity, and homology are calculated using the sequence analysis tool BLAST with default parameters. Identity searches can be performed, for example, using NCBI's BLAST 2.7.1 (published October 19, 2017). The "identity" value herein generally refers to the value obtained when aligned using the above-mentioned BLAST under default conditions. However, if a higher value is obtained by changing the parameters, the highest value is used as the identity value. When identity is evaluated in multiple regions, the highest value among them is used as the identity value. "Similarity" is a numerical value that takes into account not only identity but also similar amino acids.

[0025] "Identity" is calculated by the percentage of homologous bases between two or more base sequences, using known methods such as those described above. Specifically, before calculating the percentage, the base sequences of the base sequences to be compared are aligned, and gaps are introduced into the base sequences, if necessary, to maximize the percentage of identical base nucleic acids. Alignment methods, percentage calculation methods, comparison methods, and related computer programs are well known in the art (e.g., BLAST, as described above). Unless otherwise specified, "identity" herein can be expressed as a value measured using NCBI's BLAST. The algorithm for comparing base sequences using BLAST can be Blastp, with default settings. The measurement results are quantified as positives or identities. In this case, when "similarity" is used instead of "identity," the value also takes into account the definition of "similar" "bases" described herein.

[0026] As used herein, the term "biological function," when referring to a microorganism, refers to a specific function that the microorganism may have, such as, but not limited to, protecting fish (e.g., improving fish survival rate). In the present disclosure, examples include, but are not limited to, improving fish survival rate as well as suppressing harmful microorganisms. As used herein, a biological function can be exerted by a corresponding "biological activity." As used herein, "biological activity" refers to an activity that a microorganism may have in a certain environment, and includes activities that exert various functions (e.g., improving fish survival rate). Such biological activity can be measured by techniques well known in the art. Thus, "activity" refers to various measurable indicators that affect a response (i.e., have a measurable effect in response to some exposure or stimulus), and can include, for example, a measure of the amount of an upstream or downstream protein or other similar function of the microorganism of the present disclosure after some stimulus or event.

[0027] As used herein, the "amount" of an analyte in a sample generally refers to an absolute value that reflects the mass of the analyte that can be detected in a volume of the sample. However, the amount also contemplates a relative amount compared to the amount of another analyte. For example, the amount of an analyte in a sample may be an amount that is greater than a control level or normal level of the analyte that is normally present in the sample.

[0028] As used herein, the term "about" refers to the indicated value plus or minus 10%, unless otherwise specified.

[0029] As used herein, the term "kit" refers to a unit in which the parts to be provided (e.g., a composition comprising the microorganism of the present disclosure, additional components, buffer solutions, instructions, etc.) are provided, typically separated into two or more compartments. This kit form is preferred when the purpose is to provide a composition that should not be provided in a mixed state for reasons of stability, etc., but is preferably mixed immediately before use. Such a kit advantageously includes instructions or manuals describing how to use or process the provided parts (e.g., a composition comprising the microorganism, additional components, etc.). When the term "kit" is used herein, the kit typically includes instructions describing how to use the microorganism, composition, etc. of the present disclosure.

[0030] As used herein, "instructions" refers to instructions to the user on how to use the present disclosure. The instructions contain wording that instructs on how to use the present disclosure. If necessary, the instructions are prepared in accordance with a format specified by the regulatory agency of the country in which the present disclosure is implemented (e.g., the Ministry of Health, Labor and Welfare or the Ministry of Agriculture, Forestry and Fisheries in Japan, or the Food and Drug Administration (FDA) or the Department of Agriculture (USDA) in the United States), and it is clearly stated that the instructions have been approved by the regulatory agency. The instructions may be provided in paper form, but are not limited thereto, and may also be provided in the form of electronic media (e.g., a homepage provided on the Internet, email, etc.).

[0031] As used herein, "ingredients for fish farming" refers to any ingredient that is not expected to have the effects of the microorganisms of the present disclosure on the fish to be farmed, but that plays a certain role when combined with the microorganisms of the present disclosure, and examples thereof include carriers (including water), salts, surfactants, carriers, drying protectants, preservatives, antibacterial agents, excipients, fortifiers, antioxidants, stabilizers, diluents, buffers, binders, dispersants, flocculants, other microorganisms, etc.

[0032] As used herein, the term "fish culture liquid" refers to a fluid component of fish culture, and may be a solution or a suspension, or a mixture of a fluid component and a solid component (which may or may not be dissolved). Typically, the fish culture liquid may be a liquid that simulates the environment in which fish live, such as a river, ocean, or lake, or a concentrated solution thereof.

[0033] As used herein, the term "protective culture fluid" refers to a fluid for protecting fish, which contains the microorganisms of the present disclosure and a culture fluid for culturing fish. Typically, fish are raised in the protective culture fluid or a fluid diluted at any dilution factor (e.g., about 2 to 1,000,000 times).

[0034] (Preferred embodiment) Preferred embodiments of the present disclosure will be described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description herein. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.

[0035] (useful microorganisms) In one aspect, the present disclosure provides novel microorganisms capable of protecting fish and / or suppressing harmful microorganisms. In particular, the microorganisms disclosed herein are capable of improving the survival rate of fish, colonizing fish (e.g., their epidermis), and / or suppressing harmful microorganisms. In one embodiment, fish can be protected by adding the microorganisms disclosed herein to the rearing environment (e.g., rearing solution, rearing water, etc.) of fish. The microorganisms disclosed herein are also expected to have effects such as direct action against harmful microorganisms, formation of a microbial flora in fish (e.g., their epidermis), maintenance and / or improvement of the health of fish (e.g., their epidermis), and stimulation of fish immunity. Furthermore, application of the microorganisms disclosed herein to fish eggs and / or fry is expected to improve the hatching rate and / or survival rate of fry.

[0036] In one embodiment, the Flavobacterium family bacterium of the present disclosure is a specific strain that, when tested using a method comprising the steps of (i) streaking the strain linearly on NB agar medium and culturing overnight at 28°C, and (ii) then streaking harmful microorganisms vertically from the strain without touching the strain and culturing overnight at 28°C, can be characterized by the presence of a clear zone, for example, at least 3 mm (e.g., at least 3 mm, at least 4 mm, at least 5 mm, at least 6 mm, at least 7 mm, at least 8 mm, at least 9 mm, at least 10 mm) from the streak of the strain after step (ii), wherein the harmful microorganism comprises at least one species selected from the group consisting of Edwardsiella tarda and Edwardsiella ictaluri.

[0037] In one embodiment, the microorganism of the present disclosure is a bacterium of the family Flavobacteriaceae. Flavobacteriaceae microorganisms are eubacteria and are mostly aerobic. Flavobacterium family includes Aequorivita, Aestuariicola, Aestuariibaculum, Algibacter, Aquibacter, Aquimarina, Arenibacter, Bergeyella, Bizionia, Capnocytophaga, Cellulophaga, Chryseobacterium, Cloacibacterium, Coenonia, Confluentibacter, Costertonia, Crocei bacter, Croceitalea, Croceivirga, Cruoricaptor, Dokdonia, Donghaeana, Elizabethkingia, Empedobacter, Epilithonimonas, Flagellimonas, Flaviramulus, Flavobacterium, Formosa, Gaetbulibacter, Galbibacter, Gelidibacter, Gillisia, Gilvibacter, Grammell a, Joostella, Kaistella, Kordia, Krokinobacter, Leeuwenhoekiella, Lutibacter, Lutimonas, Mangrovimonas, Maribacter, Mariniflexile, Marixanthomonas, Mesonia, Moheibacter, Muricauda, ​​Myroides, Nonlabens, Ornithobacterium, Pibocella, Polaribacter, Ps ychroflexus, Psychroserpens, Riemerella, Robiginitalea, Sabulilitoribacter, Salegentibacter, Salinimicrobium, Sandarakinotalea, Sediminibacter, Sediminicola, Sejongia, Spongiimonas, Stenothermobacter, Subsaxibacter, Subsaximicrobium, Tamlana,The genera include Tenacibaculum, Ulvibacter, Vitellibacter, Wautersiella, Weeksella, Winogradskyella, Yeosuana, Zeaxanthinibacter, Zhouia, Zobellia, and Zunongwangia. In one embodiment, the microorganism of the present disclosure is a bacterium of the genus Flavobacterium or Chryseobacterium.

[0038] Microorganisms of the genus Flavobacterium are Gram-negative, non-motile or motile bacilli. The genus Flavobacterium includes acidificum, aciduliphilum, acidurans, ahnfeltiae, algicola, anatoliense, anhuiense, antarcticum, aquaticum, akiainvivens, aquatile, aquicola, aquidurense, araucananum, arcticum, arsenatis, arsenitoxidans, aureus, banpakuense, beibuense, branchiarum, branchiicola, branchiophilum, breve, brevivitae, buctense, caeni, cauense, ceti, cheniae, cheonanense, cheonhonense, chilense, chungangense, chungbukense, chungnamense, collinsense, collinsii, columnare, compostarboris, crassostreae, croceum, cucumis, cutihirudinis, daejeonense, daemonensis, dankookense, defluvii, degerlache, denitrificans, devorans, dispersum, dongtanense, eburneum, endophyticum, enshiense, faecale, ferrugineum, filum, flaviflagrans, flevense, fluvii, fontis, frigidarium, frigidimaris, Ffrigoris, fryxellicola, fulvum, gelidilacus, gillisiae, ginsengisoli, ginsenosidimutans, glaciei, glycines, granul, halmophilum, haoranii, hauense, hercynium, hibernum, humicola, hydatis, indicum, inkyongense, jejuense, johnsoniae, jumunjinense, koreense, kyungheense, lacunae,lacus, limicola, limnosediminis, lindanitolerans, longum, luticocti, lutivivi, macrobrachii, maotaiense, marinum, maris, micromati, mizutaii, myungsuense, multivorum, nitratereducens, nitrogenifigens, noncentrifugens, notoginsengisoli, oceanosedimentum, omnivorum, oncorhynchi, oceanokoites, orientale, oryzae, palustre, paronense, pectinovorum, pedocola, phragmitis, piscis, plurextorum, ponti, procerum, psychrolimnae, psychrophilum, qiangtangense, rakeshii, reichenbachii, resistens, rivuli, saccharophilum, saliperosum, sasangense, segetis, salegens, seoulense, sinopsychrotolerans, sole, spartansii, shrimps, suaeda, subsaxonicum, succinans, suncheonense, suzhouense, swingsii, tegetincola, earth, terrigena, terriphilus, thermophilus, tiangeerense, tilapia, tistrianus, trout, tyrosinilitus, ummariens e, urocaniciphilum, urumqiense, verecundum, vireti, weaverense, xanthum, xinjiangense, xueshanense, yanchengense, yonginense, etc. are included.The microorganism has a 16S rRNA sequence that is most homologous or identical to the 16S rRNA sequence of a type strain of a species selected from the group consisting of Flavobacterium psychrolimnae, algicola, faecale, frigidarium, omnivorum, fryxellicola, degerlachei, gillisiae, frigoris, sinopsychrotolerans, xinjiangense, and urumqiense. In one embodiment, the microorganism of the present disclosure has a 16S rRNA sequence that is most homologous or identical to the 16S rRNA sequence of a type strain of Flavobacterium procerum or Flavobacterium resistens, among type strains of Flavobacterium bacteria.

[0039] Microorganisms of the genus Chryseobacterium are typically organotrophic Gram-negative bacilli that form yellow-orange colonies. The genus Chryseobacterium includes aahli, angstadtii, antarcticum, anthropi, aquaticum, aquifrigidense, arachidiradicis, arachidis, arthrosphaerae, artocarpi, balustinum, bernardetii, bovis, caeni, camelliae, carnipullorum, carnis, chaponense, cucumeris, contaminans, culicis, daecheongense, daeguense, defluvii, echinoideorum, endophyticum, elymi, flavum, formosense, frigidisoli, frigidum, gallinarum, gambrini, geocarposphaerae, ginsengiterrae, ginsengisoli, ginsenosidimutans, glaciei, gleum, greenlandense, gregarium, gwangjuense, hagamense, haifense, halperniae, hispalense, hispanicum, hominis, humi, hungaricum, indologenes, indoltheticum, jejuense, jeonii, joostei, koreense, kwangjuense, limigenitum, lactis, lathyri, lineare, luteum, marinum, montanum, molle, nakagawai, nematophagum, nepalense, oleae, oncorhynchi, oranimense, pallidum, palustre, piperi, piscicola, piscium, polytrichastri, profundimaris, psychrotolerans, reticulitermitis, rhizoplanae, rhizosphaerae, rigui, salipaludis, sediminis, scophthalmum, shandongense, shigense,Species include soldanellicola, solani, soli, solincola, taeanense, taichungense, taihuense, taiwanense, takakiae, taklimakanense, tenax, treverense, tructae, ureilyticum, vietnamense, viscerum, vrystaatense, wanjuense, xinjiangense, xixisoli, yonginense, and zeae. In one embodiment, the microorganism of the present disclosure has a 16S rRNA nucleotide sequence that is most homologous or identical to the 16S rRNA nucleotide sequence of a type strain of a species of Chryseobacterium selected from the group consisting of vietnamense, aquifrigidense, culicis, nakagawai, jejuense, bernardetii, rhizosphaerae, kwangjuense, elymi, lathyri, oranimense, contaminans, gallinarum, artocarpi, ureilyticum, oncorhynchi, joostei, viscerum, tructae, lactis, rhizoplanae, sediminis, indologenes, gleum, and arthrosphaerae, among type strains of bacteria of the genus Chryseobacterium. In one embodiment, the microorganism of the present disclosure has a 16S rRNA nucleotide sequence that is most homologous or identical to the 16S rRNA nucleotide sequence of a type strain of a species of Chryseobacterium selected from the group consisting of vietnamense, aquifrigidense, culicis, nakagawai, jejuense, bernardetii, rhizosphaerae, and kwangjuense, among type strains of bacteria of the genus Chryseobacterium.

[0040] The inventors discovered new microorganisms belonging to the genus Flavobacterium and Chryseobacterium by examining their ability to inhibit harmful microorganisms, and named them MUCF01, MUCF02, and MUCF03, respectively. These strains were deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation (NITE) and received on November 27, 2020. The accession numbers are NITE AP-03323 (NUCF01), NITE AP-03324 (NUCF02), and NITE AP-03325 (NUCF03), respectively. In addition, accession certificates for NUCF01, NUCF02, and NUCF01 were issued on December 14, 2020, and were assigned the accession numbers NITE P-03323, NITE P-03324, and NITE P-03325, respectively. In one embodiment, the microorganism of the present disclosure is MUCF01 (Accession Number: NITE AP-03323 , Accession number: NITE P-03323 ), MUCF02 (Receipt number: NITE AP-03324 , Accession number: NITE P-03324 ) or MUCF03 (Receipt number: NITE AP-03325 , Accession number: NITE P-03325 ) or a derivative thereof.

[0041] In one embodiment, the microorganism of the present disclosure is MUCF01 (Accession Number: NITE AP-03323 , Accession number: NITE P-03323 ), MUCF02 (Receipt number: NITE AP-03324 , Accession number: NITE P-03324 ) or MUCF03 (Receipt number: NITE AP-03325 , Accession number: NITE P-03325) as a derivative strain. Here, a derivative strain does not necessarily have to be a strain derived from MUCF01, MUCF02, or MUCF03, and refers to a microorganism that exhibits the biological functions of MUCF01, MUCF02, or MUCF03, although not necessarily to the same degree. In one embodiment, a microorganism that is a derivative strain of the present disclosure exhibits a biological function selected from the ability to protect fish, the ability to colonize fish (e.g., the epidermis), and the ability to suppress harmful microorganisms, similar to MUCF01, MUCF02, or MUCF03, but the degree of the biological function may differ from that of MUCF01, MUCF02, or MUCF03. In one embodiment, a microorganism that is a derivative strain of the present disclosure may be a bacterium of the genus Flavobacterium or Chryseobacterium. In one embodiment, the ability of the microorganisms of the present disclosure (including derivatives of MUCF01, MUCF02, or MUCF03) to protect fish, suppress or inhibit harmful microorganisms, colonize fish, and function as probiotics may be confirmed by any of the tests described herein, or modifications thereof, for confirming each of these abilities.

[0042] In one embodiment, the microorganisms of the present disclosure (including derivatives of MUCF01, MUCF02, or MUCF03) have the ability to suppress harmful microorganisms, and this ability can be confirmed by taking a loopful of colonies of the microorganisms of the present disclosure formed on an agar medium (using a φ2.7 mm disposable loop), streaking this in a straight line on NBR2A medium (NB medium + R2A medium), culturing at 25°C for 24 to 28 hours, and then taking a loopful of colonies of the harmful microorganisms formed on the agar medium (using a φ2.7 mm disposable loop), This can be confirmed by observing a clear zone of 0.1 mm or more, 0.2 mm or more, 0.5 mm or more, 0.7 mm or more, 1 mm or more, 2 mm or more, 3 mm or more, 4 mm or more, 5 mm or more, 6 mm or more, 7 mm or more, 8 mm or more, 9 mm or more, 10 mm or more, 12 mm or more, 15 mm or more, 17 mm or more, 20 mm or more, 25 mm or more, 30 mm or more, 35 mm or more, or 40 mm or more when the plate is streaked perpendicularly from the streak without touching the microorganism of the present disclosure and cultured at 25°C for 24 to 28 hours. In one embodiment, the harmful microorganism is a bacterium of the genus Edwardsiella (e.g., Edwardsiella sp. (NBRC 12716), Edwardsiella sp. (NBRC 12717), or Edwardsiella ictaluri (NBRC 105724). T The clear zone can be determined visually by a person skilled in the art.

[0043] Those skilled in the art can test derivatives of MUCF01, MUCF02 or MUCF03 using the above criteria as appropriate to obtain derivatives that have the above biological functions (and their extent).

[0044] In one embodiment, the microorganism of the present disclosure (including derivatives of MUCF01, MUCF02, or MUCF03) is a microorganism obtained by the method for obtaining a microorganism described below.

[0045] In another aspect, the present disclosure provides a bacterial strain belonging to the family Flavobacteriaceae, wherein the bacterial strain is characterized in that, when tested by a method comprising the steps of: (i) streaking the strain linearly on NBR2A medium and culturing the strain at 25°C for 24 to 48 hours; and (ii) thereafter streaking harmful microorganisms vertically from the strain without touching the strain, and culturing the strain at 25°C for 24 to 48 hours, a clear zone is present over a range of 10 mm or more from the streak of the strain after step (ii), wherein the harmful microorganisms are Edwardsiella sp. (NBRC 12716), Edwardsiella sp. (NBRC 12717), and Edwardsiella ictaluri (NBRC 105724). T The conditions (abilities) that these strains should have and the conditions for testing them are provided in the present specification and examples, and those skilled in the art can identify these strains by referring to publicly known information as appropriate.

[0046] (Method for obtaining useful microorganisms) In one aspect, the present disclosure provides a method for obtaining microorganisms capable of protecting fish. This method may include the steps of (a) obtaining a candidate microorganism from the epidermis of fish, (b) adding the candidate microorganism to a culture medium containing harmful microorganisms, (c) confirming the suppression of the harmful microorganisms in the culture medium, and (d) obtaining the candidate microorganism as a microorganism capable of protecting fish if the suppression of the harmful microorganisms in the culture medium is confirmed. Because fish often come into direct contact with microorganisms in water, and therefore frequently suffer from skin infections, targeting microorganisms on the epidermis of fish may be useful for efficiently obtaining microorganisms capable of protecting fish. Using microorganisms originally present on the epidermis of fish may enable safe, simple, and / or environmentally friendly protection of fish. The epidermis of the fish from which the candidate microorganisms are obtained may be any surface of the body, eyes, scales, fins, gills, mouth, anus, and genitals, and in one embodiment, it is the surface of the body.

[0047] In one embodiment, the confirming step (c) can be carried out by confirming the ability of the microorganisms of the present disclosure (including derivatives of MUCF01, MUCF02, or MUCF03) to protect fish, suppress or inhibit harmful microorganisms, colonize fish, and function as probiotics using any of the tests described herein or modifications thereof. In one embodiment, the confirming step (c) can be carried out by confirming the suppression of harmful microorganisms in a culture medium. In one embodiment, the confirming step (c) can be carried out by growing the candidate microorganism and the harmful microorganism in the same culture medium and confirming a growth inhibition zone (clear zone) in which the harmful microorganism cannot grow near the candidate microorganism. In one embodiment, the confirming step (c) can be carried out by confirming the suppression of harmful microorganisms in a liquid culture medium containing the candidate microorganism or its culture supernatant and the harmful microorganism. In one embodiment, the confirming step (c) can be carried out by placing an impregnated product of the culture medium or culture supernatant of the candidate microorganism or a bottomless cylinder containing the culture medium or culture supernatant on an agar medium or equivalent medium (e.g., gellan gum, etc.) that has been inoculated entirely with harmful microorganisms, and confirming the suppression of harmful microorganisms around the impregnated product or cylinder. In one embodiment, the confirming step (c) can be carried out by incubating a liquid medium containing the candidate microorganism and harmful microorganisms, comparing the growth of the candidate microorganism and harmful microorganism, and confirming that the growth of the harmful microorganism is more suppressed. In one embodiment, the confirming step (c) can be carried out by inoculating the candidate microorganism on an agar medium or equivalent medium (e.g., gellan gum, etc.) that has been inoculated entirely with harmful microorganisms, or on an agar medium or equivalent medium (e.g., gellan gum, etc.) that has been completely grown with harmful microorganisms, and confirming that the candidate microorganism forms a colony and that a zone of growth inhibition or a clear zone due to the death of the harmful microorganism is observed around the colony. Different types of microorganisms cultured in the same liquid medium can be distinguished by means of colony shape, color, the use of selective media, etc.For example, methods for distinguishing and examining populations include counting individual colonies by plate culture if the colonies differ in color or shape, directly counting each microorganism under a microscope if different morphologies are observed under a microscope, designing gene primers that can distinguish each microorganism and performing real-time PCR, fluorescent in situ hybridization (FISH), etc. Methods for identifying candidate microorganisms under a microscope include the well-known FISH method and methods that utilize the fluorescence of fluorescent proteins previously introduced into candidate microorganisms.

[0048] In one embodiment, candidate microorganisms can be obtained from colonies formed on a culture medium inoculated with scrapings (e.g., scrapings with a cotton swab) of fish skin (e.g., body surface). One or more microorganisms can be obtained as microorganisms capable of protecting fish. In one embodiment, microorganisms capable of protecting fish can be obtained from the skin of catfish (e.g., Silurus asotus, Ictalurus punctatus). In one embodiment, microorganisms capable of protecting fish can be obtained from the skin of mucus-type fish.

[0049] In one embodiment, a harmful microorganism to be confirmed for inhibition can be selected that is suspected to have at least one of the following abilities: causing skin disease in fish (e.g., farmed fish), transmitting through the skin, infecting through wounds, and transmitting through contact. For example, a person skilled in the art can appropriately select a microorganism that has the ability to cause skin disease in fish (e.g., farmed fish), transmitting through the skin, infecting through wounds, and / or transmitting through contact by referring to "Fish Pathology" (edited by Kodama Hiroshi, Midori Shobo, Tokyo, 2012) and "Infectious and Parasitic Diseases of Fish and Shellfish" (edited by Wakabayashi and Muroga, and written by Ogawa Kazuo, Koseisha Kouseikaku, Tokyo, 2004), etc.

[0050] In one embodiment, the harmful microorganism selected may be a microorganism capable of causing edwardsiellosis, vibriosis, furunculosis, atypical Aeromonas salmonicida infection, Aeromonas hydrophila infection, edwardsiellosis, red spot disease, pseudomonadosis in sweetfish, red mouth disease, bacterial gill disease, columnaris disease (chondrosis, tail rot, fin rot, mouth rot), cold water disease, gliding bacteriosis, bacterial kidney disease, mycobacteriosis, nocardiosis, and / or streptococcosis. The typical causative microorganisms for each fish disease are as follows: Edwardsiellosis (Edwardsiella tarda, Edwardsiella ictaluri); Vibriosis (Listonella anguillara (formerly Vibrio anguillarum), Vibrio ordalii, Vibrio ichthyoenteri, Vibrio vulnificus, Vibrio salmonicida); Furunculosis (Aeromonas salmonicida); Atypical Aeromonas salmonicida infection (Aeromonas salmonicida); Aeromonas hydrophila infection (Aeromonas hydrophila); Red spot disease (Pseudomonas anguilliseptica); Pseudomonas disease in sweetfish (Pseudomonas plecoglossicida); Redmouth disease (Yersinia ruckeri); Bacterial gill disease (Flavobacterium branchiophilum); Columnaris disease (gill rot, tail rot, fin rot) (Flavobacterium columnare); coldwater disease (Flavobacterium psychrophilum); gliding bacteriosis (Tenacibaculum maritimum); bacterial kidney disease (Renibacterium salmoninarum); mycobacteriosis (Mycobacterium marinum, Mycobacterium fortuitum, Mycobacterium chelonei); nocardiosis (Nocardia seriolae); streptococcosis (Streptococcus iniae).Edwardsiellosis is a problem in fish such as perciformes, eels, flatfish (such as flounder), clupeformes (such as sweetfish), and rainbow trout. In one embodiment, the harmful microorganism may include Edwardsiella tarda, Edwardsiella ictaluri, or a combination thereof.

[0051] (Composition containing microorganisms) In one aspect, the present disclosure provides a composition comprising the microorganism of the present disclosure. The microorganism of the present disclosure can be produced by culturing using any suitable method. In one embodiment, the composition is a fish protectant. In one embodiment, the composition is a probiotic agent for fish (e.g., the skin). Use of the fish protectant or probiotic agent of the present disclosure can improve at least one of the survival, health condition, flesh quality, recovery from injury, and infection status (including susceptibility to infection) of fish.

[0052] (Applicable to) In one embodiment, the microorganisms or compositions of the present disclosure can be applied to fish, including, but not limited to, farmed fish and ornamental fish. Farmed fish include, but are not limited to, catfish, eels, sea bass, flounder, turbot, herring, rainbow trout, sweetfish, yellowtail, trout, sea bream, carp, amberjack, tuna, salmon, horse mackerel, tilapia, pufferfish, yellowtail, grouper, mackerel, and saury. The microorganisms of the present disclosure can be obtained from and / or applied to the skin of fish, which can have scale-type or slime-type skin, such as eels and catfish. In one embodiment, the microorganisms or compositions of the present disclosure can be applied to catfish, eels, sea bass, flounder, turbot, herring, rainbow trout, or sweetfish. The microorganisms or compositions of the present disclosure can be used at any stage of fish development, such as eggs, hatchlings, fry, adults, sexually mature fish, etc.

[0053] (Usage form) The form of the microorganism or composition of the present disclosure may be, for example, a liquid state or a solid state. Examples of liquid-state microorganisms or compositions include a microbial culture solution, and a solution in which microorganisms are collected from the culture solution by centrifugation or the like and then re-dispersed in water, a buffer solution, or a culture solution. Examples of solid-state microorganisms or compositions include those dehydrated by centrifugation or pressing, those in a paste or mayonnaise state that is intermediate between a solid and a liquid, and those obtained by drying (e.g., vacuum drying or freeze-drying). Examples of solid forms include powders, granules, tablets, and the like. The composition may also be provided in a state in which the microorganism or culture supernatant is immobilized on a carrier.

[0054] In one embodiment, the microorganism or composition of the disclosure comprises about 1 x 10 8 cells / mL, approximately 1×10 7 cells / mL, approximately 1×10 6 cells / mL, approximately 1×10 5 cells / mL, approximately 1×10 4 cells / mL, approximately 1×10 3 cells / mL, approximately 1×10 2 It can be added to breeding water to give a density of 10 cells / mL or about 10 cells / mL and used for fish.

[0055] (Applicable environment) The microorganisms or compositions of the present disclosure can be used in any suitable environment. In one embodiment, the microorganism or composition of the present disclosure may be used in any temperature environment, such as 0 to 100°C, 5 to 70°C, 10 to 50°C, 15 to 40°C, 20 to 35°C, less than 70°C, less than 60°C, less than 50°C, less than 40°C, less than 30°C, less than 25°C, less than 20°C, less than 15°C, less than 10°C, less than 5°C, less than 0°C, about 70°C, about 60°C, about 50°C, about 40°C, about 30°C, about 25°C, about 15°C, about 10°C, about 5°C, or about 0°C.

[0056] In one embodiment, the microorganism or composition of the present disclosure can be used in any pH environment, such as pH 3 to 13, pH 4 to 12, pH 5 to 11, pH 6 to 10, pH 7 to 9, pH 5.5 to 8.5, about pH 3, about pH 4, about pH 5, about pH 6, about pH 7, about pH 8, about pH 9, about pH 10, about pH 11, about pH 12, or about pH 13.

[0057] In one embodiment, the microorganisms or compositions of the present disclosure may be used in an environment with any dissolved oxygen concentration (DO), such as about 0.05 mg / L, about 0.1 mg / L, about 0.5 mg / L, about 1 mg / L, about 1.5 mg / L, about 2 mg / L, about 3 mg / L, about 5 mg / L, about 8 mg / L, or about 10 mg / L, and in one embodiment, may be used in an environment with a dissolved oxygen concentration (DO) of up to about 8 mg / L, which is close to saturation or suitable for fish farming.

[0058] In one embodiment, the microorganisms or compositions of the present disclosure may be used in an environment with any salt (e.g., sodium chloride) concentration, such as about 0 g / L, about 0.05 g / L, about 0.1 g / L, about 0.5 g / L, about 0.7 g / L, about 1 g / L, about 1.5 g / L, about 2 g / L, about 2.5 g / L, about 3 g / L, about 4 g / L, about 5 g / L, about 7 g / L, or about 10 g / L.

[0059] In one embodiment, the microorganisms or compositions of the present disclosure may be used in the presence of salts, surfactants, light, electric current, agitation, aeration, or any combination thereof.

[0060] In one embodiment, the microorganism or composition of the present disclosure may be used with a carrier capable of immobilizing the microorganism. The use of such a carrier can effectively prevent washout. The material of the carrier is not particularly limited as long as it is capable of immobilizing the microorganism. Examples include carbon fiber (PAN-based, pitch-based, phenolic resin-based, etc.), polyethylene resin, polypropylene resin, polyurethane resin, polystyrene resin, polyvinyl chloride resin, polyvinyl acetate resin, polyvinyl alcohol resin, polyethylene glycol resin, acrylic resin, gelatin, sodium alginate, carrageenan, dextrin, ceramics, silicon, metal, charcoal, activated carbon, minerals (zeolite, diatomaceous earth, etc.), glass, glass waste, and composites thereof. To increase the immobilization rate and the efficiency of microbial activity, it is preferable to use a porous or fibrous carrier. Alternatively, the microorganism may be encapsulated in a gel-like carrier. The shape of the carrier may be, for example, a cube, a rectangular parallelepiped, a cylinder, a sphere, a disk, a sheet, a film, or the like. For information on microbial immobilization technology, see, for example, "Wastewater Treatment by Microbial Immobilization Methods" (edited by Ryuichi Sudo, Industrial Water Research Association) and "Water Treatment by Microbial Immobilization Methods - Carrier Immobilization Method, Entrapment Immobilization Method, Biological Activated Carbon Method (New Water Treatment Series (1))" (written by Kazuhiro Mochizuki, Katsutoshi Hori, and Hideki Tatemoto, NTS Co., Ltd.).

[0061] In one embodiment, the microorganisms or compositions of the present disclosure may be used alone or in combination with other microorganisms without the use of a carrier, allowing the microorganisms to form flocs or granules by themselves. Additionally, nucleating materials or microorganisms that promote the formation of flocs or granules may be used.

[0062] (additional ingredients) In one embodiment, the microorganism or composition of the present disclosure may be used in combination with additional components, which may be added to the composition or may be used separately from the microorganism or composition, and if used separately, may be provided as a kit.

[0063] In one embodiment, the additional components include, but are not limited to, components that enhance the activity of the microorganisms used, surfactants, drying protectants, components for maintaining the microorganisms for a long period of time, preservatives, excipients, strengthening agents, antioxidants, dispersants, flocculants, and other microorganisms, and any suitable components can be used.

[0064] In another embodiment, the additional component may include any component that can be used when contacting or applying to fish, such as a fish culture liquid or a fish culture component. When such an additional component is included, the composition can be used as is when used in fish culture, allowing the fish to be raised while being protected. The fish culture liquid, the fish culture component, etc. may be provided as part of a composition, or may be used separately from the microorganism or composition, or may be provided as a kit.

[0065] (Method using microorganisms) In one aspect, the present disclosure provides a method for protecting fish, comprising contacting the fish with a microorganism or composition of the present disclosure. In one embodiment, the method for protecting fish comprises raising the fish in water containing the microorganism of the present disclosure. The fish to be protected can be any of the fish described herein to which the microorganism or composition of the present disclosure can be applied. In one embodiment, the microorganism or composition of the present disclosure can be applied to the same species of fish as the fish from which the microorganism was obtained from its skin, or to a different species of fish. In one embodiment, the microorganism or composition of the present disclosure can be applied to fish raised in the same environment (e.g., a fish farm) as the fish from which the microorganism was obtained from its skin, or to fish raised in a different environment. The method for protecting fish of the present disclosure can be carried out in any of the environments described herein to which the microorganism or composition of the present disclosure can be applied. The method for protecting fish of the present disclosure can use any of the additional components described herein that can be used in combination with the microorganism or composition of the present disclosure.

[0066] In one embodiment, the disclosed method includes treating or preventing a disease or illness in fish. Alternatively, the disclosed method includes protecting the skin of fish. Targeted fish diseases or illnesses include, for example, skin diseases and infectious diseases, such as Edwardsiellosis, Vibriosis, Furunculosis, Atypical Aeromonas salmonicida infection, Aeromonas hydrophila infection, Red spot disease, Pseudomonas disease in sweetfish, Redmouth disease, Bacterial gill disease, Columnaris disease (gill rot, tail rot, fin rot, mouth rot), Coldwater disease, Gliding bacteriosis, Bacterial kidney disease, Mycobacteriosis, Nocardiosis, and / or Streptococcosis.

[0067] In some embodiments, the microorganism or composition of the present disclosure comprises an effective amount of the Flavobacteriaceae bacteria of the present disclosure, or is provided so as to provide an effective amount upon contact. In one embodiment, the microorganism or composition of the present disclosure is provided in water in which an effective amount of the Flavobacteriaceae bacteria of the present disclosure is present, and fish may be grown therein.

[0068] In this way, by providing an effective amount of the microorganism or composition of the present disclosure, fish are protected from at least one harmful microorganism. The harmful microorganism that can be protected by the method of the present disclosure can be any of those described herein, such as a microorganism that has at least one of the following abilities: causing skin diseases in fish, transmitting infection through the skin, infecting fish through wounds, and infecting fish through contact. Specific examples include harmful microorganisms that can cause edwardsiellosis, vibriosis, furunculosis, atypical Aeromonas salmonicida infection, Aeromonas hydrophila infection, red spot disease, pseudomonas disease in sweetfish, red mouth disease, bacterial gill disease, columnaris disease (gill rot, tail rot, fin rot, mouth rot), coldwater disease, gliding bacteriosis, bacterial kidney disease, mycobacteriosis, nocardiosis, and / or streptococcosis. Such harmful microorganisms include Edwardsiella tarda, Edwardsiella ictaluri, Listonella anguillara (Vibrio anguillarum), Vibrio ordalii, Vibrio ichthyoenteri, Vibrio vulnificus, Vibrio salmonicida, Aeromonas salmonicida, atypical Aeromonas salmonicida, Aeromonas hydrophila, Pseudomonas anguilliseptica, Pseudomonas plecoglossicida, Yersinia ruckeri, Flavobacterium branchiophilum, Flavobacterium columnare, Flavobacterium psychrophilum, Tenacibaculum maritimum, Renibacterium salmoninarum, Mycobacterium marinum, Mycobacterium fortuitum, Mycobacterium chelonei, Nocardia seriolae, Streptococcus iniae, Lactococcus garvieae, Aeromonas caviae, or a combination thereof.

[0069] Therefore, a method for protecting fish, which includes a step of contacting the microorganisms or compositions of the present disclosure with fish or a step of raising fish in water containing the microorganisms of the present disclosure, may include a step of testing the fish for harmful microorganisms before, during, or after contacting the fish with or raising the fish with the microorganisms or compositions. Testing for such harmful microorganisms can be carried out by techniques known in the art, and samples may be collected directly from the fish or from the rearing solution during rearing.

[0070] In a preferred embodiment, once the type of harmful microorganism present is examined and the type of harmful microorganism is identified, preliminary experiments may be conducted to determine the optimal conditions for administering the microorganism or composition of the present disclosure. Such preliminary experiments may include, for example, confirming the effectiveness against the harmful microorganisms present and the effective concentration. The microorganism or composition of the present disclosure to be administered may also include confirming in advance the safety of the microorganism or composition to be administered to the fish species to be protected. Such safety confirmation may include determining the acceptable concentration and other administration conditions, and any method known in the art may be used. Once such conditions, such as the acceptable concentration, are determined, actual contact and rearing may be carried out taking into account the appropriate administration conditions and the appropriate effective concentration.

[0071] In another embodiment, in a method for protecting fish, the method includes contacting fish with a microorganism or composition of the present disclosure or raising fish in water containing the microorganism of the present disclosure, and providing the Flavobacteriaceae bacteria of the present disclosure under conditions sufficient for colonization on the epidermis of the fish. The conditions sufficient for colonization on the epidermis of the fish may be determined in advance, and in such cases, the contact or rearing is carried out under those conditions. Alternatively, a preliminary experiment may be carried out to determine the conditions sufficient for colonization on the epidermis of the fish during contact or rearing, and any method known in the art may be used.

[0072] (General technology) The molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art, and include, for example, Savli, H., Karadenizli, A., Kolayli, F., Gundes, S., Ozbek, U., and Vahaboglu, H. 2003. Expression stability of six housekeeping genes: A proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR. J. Med. Microbiol. 52:403-408; Marie-Ange Teste, Manon Duquenne, Jean M Francois and Jean-Luc Parrou 2009. Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae. BMC Molecular Biology 10:99; Bergey's Manual of Systematic Bacteriology, Bergey's Manual of Systematic of Archaea and Bacteria, etc., which are incorporated herein by reference in relevant parts (possibly in their entirety).

[0073] (Note) In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when it is specified that "within a range" of "two values," the range includes the two values ​​themselves.

[0074] All references cited herein, including scientific literature, patents, patent applications, and the like, are incorporated by reference in their entirety to the same extent as if each were specifically set forth. The present disclosure has been described above with reference to preferred embodiments for ease of understanding.

[0075] The present disclosure will be described below based on examples, but the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present invention. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but is limited only by the claims. [Example]

[0076] Examples are described below. When necessary, the handling of organisms used in the following examples complied with the standards stipulated by Nagoya University, Mie University, regulatory authorities, and the Cartagena Protocol. While the specific reagents used were those listed in the examples, equivalent products from other manufacturers (Sigma-Aldrich, Fujifilm, Wako Pure Chemical Industries, Nakarai, R&D Systems, USCN Life Science Inc., Kanto Chemical, Funakoshi, Tokyo Chemical Industry, Merck, etc.) can also be used.

[0077] Example 1: Isolation of microorganisms from catfish skin Adult catfish (Silurus asotus) were obtained from an aquarium at Mie University (3 individuals) and a catfish farm in Wakayama Prefecture (4 individuals), and American catfish (Ictalurus punctatus) (3 individuals + 1 body part (skin))) were obtained from Lake Kasumigaura.

[0078] The epidermis of these catfish was swabbed with a sterile swab, suspended in sterile saline, and plated on R2A agar medium at 25°C for 24 to 168 hours. [Table 1]

[0079] Thirty-six bacterial strains were isolated from catfish in tanks at Mie University, 78 from catfish at a catfish farm in Wakayama Prefecture, and 20 from American catfish.

[0080] (Example 2: Screening for useful bacteria by the Cross Streak method) Edwardsiella sp. (NBRC 12716) and Edwardsiella ictaluri (NBRC 105724) are pathogenic bacteria. T ), and Edwardsiella sp. (NBRC 12717) were used.

[0081] The cross-streak method was performed with reference to the method of Toth et al. (Erika M. Toth, Andrea K. Borsodi, Tamas Felfoeldi, Balazs Vajna, Rita Sipos, Karoly Marialigeti. 2013. Practical Microbiology: based on the Hungarian practical notes entitled "Mikrobiologiai Laboratoriumi Gyakorlatok". Marialigeti EMTaK, editor. Budapest: Eoetvoes Lorand University.) The candidate microorganisms for each isolate in Example 1 were streaked linearly onto NBR2A medium (NB medium + R2A medium) and cultured at 25°C for 24 to 28 hours. Subsequently, pathogenic bacteria were streaked perpendicular to the candidate microorganism streak without touching the candidate microorganisms, and cultured at 25°C for 24 to 28 hours. Growth inhibition ability was assessed based on the presence of a growth inhibition zone (clear zone) after culture. At this time, one loopful (using a φ2.7 mm disposable loop) was taken from each colony (pathogenic bacteria and candidate microorganisms) on the agar medium and inoculated onto the agar medium for testing. [Table 2]

[0082] As a result, three of the 134 isolates showed growth inhibition against Edwardsiella. These three isolates (named MUCF01, MUCF02, and MUCF03, respectively) had particularly excellent Edwardsiella-suppressing ability (Fig. 1, 2, and 3), which was remarkable compared with the strains that did not show Edwardsiella-suppressing ability (Fig. 4).

[0083] (Example 3: Identification of useful bacteria) Species identification of MUCF01, MUCF02, and MUCF03 by 16S ribosomal RNA (rRNA) was performed as follows. Colonies of MUCF01, MUCF02, and MUCF03 were removed from the agar plates, resuspended in TE buffer, and heated to 96°C for 5 minutes. Aliquots were added to PCR tubes. PCR was performed using 16S rRNA gene primers: 27F-W: 5'-AGRGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 1) and 1492R-W: 5'-GGYTACCTTGTTACGACTT-3' (SEQ ID NO: 2). The cycling conditions were 98°C for 30 seconds (1 cycle), 98°C for 10 seconds, 50°C for 30 seconds, and 72°C for 90 seconds (30 cycles). The PCR reaction contained 2 μL of template solution, EmeraldAmp PCR Master Mix (Takara Bio, Japan), and 0.2 μM 27F-W and 1492R-W primers. The amplified gene fragments were sequenced using standard methods. The sequences obtained by sequencing were compared for homology with other bacterial 16S rRNA gene sequences using online sequence databases available at http: / / www.ncbi.nlm.nih.gov / BLAST / and https: / / www.ezbiocloud.net / identify.

[0084] The database used was EzBioCloud database 2020.05.13.

[0085] The 16S rRNA sequences of MUCF01 and MUCF02 isolated from different individuals of catfish (Silurus asotus) were 100% identical and were determined as follows. [ka]

[0086] Table 3 shows the results of a homology comparison of the 16S rRNA sequences of MUCF01 and MUCF02 with the 16S rRNA gene sequences of other bacteria. MUCF01 and MUCF02 showed 98.05% homology to Flavobacterium plurextorum (CCUG 60112) and were identified as belonging to the genus Flavobacterium. Using a homology of 98.7% or higher as the standard for species identity (Int J Syst Evol Microbiol. 2018 Jan;68(1):461-466.), MUCF01 and MUCF02 may represent new species closely related to Flavobacterium plurextorum. [Table 3]

[0087] The molecular phylogenetic tree based on the 16S rRNA sequences of MUCF01 and MUCF02 is shown in Figure 5. The molecular phylogenetic tree was constructed using only information from the type strains.

[0088] The 16S rRNA sequence of MUCF03 isolated from channel catfish (Ictalurus punctatus) was determined as follows. [ka]

[0089] Table 4 shows the results of a homology comparison of the 16S rRNA sequence of MUCF03 with the 16S rRNA gene sequences of other bacteria. MUCF03 showed 99.78% homology to CP018786_s, which is predicted to be Chryseobacterium vietnamense, and was identified as belonging to the genus Chryseobacterium. MUCF03 is predicted to be Chryseobacterium vietnamense. [Table 4]

[0090] The molecular phylogenetic tree based on the 16S rRNA base sequence of MUCF03 is shown in Figure 6. The molecular phylogenetic tree was constructed using only information from the type strain.

[0091] Furthermore, we selected good-quality sequences from the type strains of the genera Flavobacterium and Chryseobacterium registered in the Ribosomal Database Project (RDP) (Nucleic Acids Res. 1994 Sep;22(17):3485-7.) (RDP Release 11.5). This resulted in data for 148 strains of Flavobacterium and 105 strains of Chryseobacterium. The 16S rRNA sequences of MUCF01 or MUCF02 and MUCF03 were added to the Flavobacterium and Chryseobacterium data, respectively, and a molecular phylogenetic tree was constructed using the neighbor-joining method.

[0092] Enlarged views of the clusters containing MUCF01 (MUCF02) and MUCF03 are shown in Figures 7 and 8. MUCF01 (MUCF02) and MUCF03 formed clusters with the following strains, respectively: MUCF01 (MUCF02) S004473868 Flavobacterium procerum (T) T3 KF857168 S000942420 Flavobacterium resistens (T) BD-b365 EF575563 S000263970 Flavobacterium micromati (T) type strain: LMG 21919 AJ557888 S000121350 Flavobacterium limicola (T) ST-82 AB075230 S000623909 Flavobacterium reichenbachii (T) type strain: WB 3.2-61 AM177616 S000903062 Flavobacterium tiangeerense (T) 0563 EU036219 S002352122 Flavobacterium xueshanense (T) Sr22 HQ436466 S000539443 Flavobacterium psychrolimnae (T) type strain: LMG 22018 AJ585428 S001187274 Flavobacterium algicola (T) TC2 ( NBRC 102673 CIP 109574) AB455265 S004049298 Flavobacterium faecale (T) WV33 KF214259 S000388383 Flavobacterium frigidarium (T) A2i ATCC 700810 NCIMB 13737 AF162266 S000394137 Flavobacterium omnivorum (T) AS 1.2747 JCM 11313 AF433174 S000539636 Flavobacterium fryxellicola (T) type strain: LMG 22022 AJ811961 S000263802 Flavobacterium degerlachei (T) type strain: LMG 21915 AJ557886 S000438605 Flavobacterium gillisiae (T) IC001 U85889 S000264514 Flavobacterium frigoris (T) type strain: LMG 21922 AJ557887 S001329026 Flavobacterium sinopsychrotolerans (T) 0533 FJ654474 S000394136 Flavobacterium xinjiangense (T) AS 1.2749 JCM 11314 AF433173 S002352123 Flavobacterium urumqiense (T) Sr25 HQ436467 ·MUCF03 S002227137 Chryseobacterium vietnamense (T) GIMN1.005 HM212415 S000893188 Chryseobacterium aquifrigidense (T) CW9 EF644913 S001611617 Chryseobacterium culicis (T) type strain: R4-1A FN554975 S003613221 Chryseobacterium nakagawai (T) G41 JX100822 S000871528 Chryseobacterium jejuense (T) JS17-8 EF591303 S003613215 Chryseobacterium bernardetii (T) G229 JX100816 S000721937 Chryseobacterium rhizosphaerae (T) RSB3-1 DQ673670 S000330373 Chryseobacterium kwangjuense (T) KJ1R5 AY514021 S000721938 Chryseobacterium elymi (T) RHA3-1 DQ673671 S000721941 Chryseobacterium lathyri (T) RBA2-6 DQ673674 S000965788 Chryseobacterium oranimense (T) H8 EF204451 S003922571 Chryseobacterium contaminans (T) C-26 KF652079 S004008497 Chryseobacterium gallinarum (T) 100 KC494697 S004056704 Chryseobacterium artocarpi (T) UTM-3 KF751867 S000651736 Chryseobacterium ureilyticum (T) type strain: F-Fue-04IIIaaaa AM232806 S002157006 Chryseobacterium oncorhynchi (T) 701B-08 FN674441 S000134119 Chryseobacterium joostei (T) LMG 18212 AJ271010 S002907085 Chryseobacterium viscerum (T) type strain: 687B-08 FR871426 S002907088 Chryseobacterium tructae (T) type strain: 1084-08 FR871429 S003613220 Chryseobacterium lactis (T) KC1864 JX100821 S004453705 Chryseobacterium rhizoplanae (T) JM-534 KP033261 S004488660 Chryseobacterium sediminis (T) IMT-174 KR349467 S000690525 Chryseobacterium indologenes (T) LMG 8337 AM232813 S000690524 Chryseobacterium gleum (T) CCUG 14555 AM232812 S001575200 Chryseobacterium arthrosphaerae (T) CC-VM-7 FN398101

[0093] Furthermore, MUCF01 (MUCF02) and MUCF03 formed smaller clusters with the following strains, respectively: MUCF01 (MUCF02) S004473868 Flavobacterium procerum (T) T3 KF857168 S000942420 Flavobacterium resistens (T) BD-b365 EF575563 MUCF03 S002227137 Chryseobacterium vietnamense (T) GIMN1.005 HM212415 S000893188 Chryseobacterium aquifrigidense (T) CW9 EF644913 S001611617 Chryseobacterium culicis (T) type strain: R4-1A FN554975 S003613221 Chryseobacterium nakagawai (T) G41 JX100822 S000871528 Chryseobacterium jejuense (T) JS17-8 EF591303 S003613215 Chryseobacterium bernardetii (T) G229 JX100816 S000721937 Chryseobacterium rhizosphaerae (T) RSB3-1 DQ673670 S000330373 Chryseobacterium kwangjuense (T) KJ1R5 AY514021

[0094] Bacteria whose 16S rRNA sequences show high homology to the type strains of these species are likely to exhibit properties similar to MUCF01, MUCF02, or MUCF03.

[0095] Furthermore, MUCF01, MUCF02, and MUCF03 are strains isolated from the epidermis of catfish, and therefore may have the ability to colonize the epidermis of fish.

[0096] Example 4 Obtaining related strains By performing screening as shown in the above examples using catfish living in other places as the isolation source, related strains of MUCF01, MUCF02 and MUCF03 can be obtained.

[0097] Other embodiments Example 5 Tests were conducted to confirm the colonization of the useful microorganisms disclosed in this disclosure in farmed fish and to protect the fish. Experimental zone 1, experimental zone 2, and a control zone were set up in a rearing tank. Ten young catfish (approximately 6 cm in body length) were randomly assigned to each zone and released and reared for two weeks. Experimental zone 1 was then set up in a suspension of Edwardsiella ictaluri (1 x 10 7 In experimental group 2, the cells were immersed in a suspension of MUCF01, MUCF02, or MUCF03 (1x10 cells / mL) for 30 minutes. 7 cells / mL) for 30 min, followed by a suspension of Edwardsiella ictaluri (1x10 7The catfish are then immersed in a solution of 1000 cells / mL for 30 minutes. After that, they are kept at 25°C, and the colonization is confirmed by analyzing the bacterial flora on the surface of the catfish's body, and the mortality rate in each tank is measured.

[0098] (Note) While the present disclosure has been illustrated by preferred embodiments thereof, it is understood that the scope of the present invention is to be construed solely in terms of the claims that follow. It is understood that the patents, patent applications, and other documents cited herein are incorporated by reference into this specification in their entirety as if the contents themselves were specifically set forth herein. [Industrial Applicability]

[0099] The present disclosure provides microorganisms and methods for protecting fish, thereby achieving more stable, inexpensive, highly productive and / or diverse fish breeding, and / or reducing the environmental burden by reducing the use of existing drugs, etc. [Accession number]

[0100] MUCF01 (Receipt number: NITE AP-03323 , Accession number: NITE P-03323 ) MUCF02 (Receipt number: NITE AP-03324 , Accession number: NITE P-03324 ) MUCF03 (Receipt number: NITE AP-03325 , Accession number: NITE P-03325 ) [Sequence List Free Text]

[0101] SEQ ID NO: 1: 16S rRNA gene primer (forward) AGRGTTTGATCMTGGCTCAG SEQ ID NO: 2: 16S rRNA gene primer (reverse) GGYTACCTTGTTACGACTT SEQ ID NO: 3: 16S rRNA sequence of MUCF01 SEQ ID NO: 4: 16S rRNA sequence of MUCF02 SEQ ID NO: 5: 16S rRNA sequence of MUCF03

Claims

1. A fish protective agent against Edwardsiellosis, comprising a bacterium of the Flavobacterium family having the ability to protect fish, wherein the bacterium of the Flavobacterium family is a bacterium of the genus Flavobacterium or Chryseobacterium.

2. 10. The protectant of claim 1 for controlling harmful microorganisms, including Edwardsiella tarda, Edwardsiella ictaluri, or a combination thereof.

3. A protective agent as described in claim 1, which is a protective agent for fish having a mucus-type epidermis.

4. The protective agent according to claim 3, wherein the fish is a catfish.

5. A protective agent as described in claim 1, wherein the Flavobacterium family bacterium is a bacterium of the genus Chryseobacterium.

6. The Flavobacterium family bacterium is (i) streaking the Flavobacteriaceae bacteria on NBR2A medium and culturing the bacteria at 25°C for 24 to 48 hours; (ii) then streaking harmful microorganisms vertically away from the Flavobacteriaceae bacteria without touching them, and culturing at 25°C for 24 to 48 hours; and (ii) having the ability to inhibit microorganisms characterized by the presence of a clear zone extending over an area of ​​10 mm or more from the streak of the Flavobacteriaceae bacteria after step (ii), when tested in a method comprising: wherein the harmful microorganisms are Edwardsiella sp. (NBRC 12716), Edwardsiella sp. (NBRC 12717) and Edwardsiella ictaluri (NBRC 105724 Ｔ ) The protective agent according to claim 1 .

7. The Flavobacteriaceae bacterium has homology or identity to the 16S rRNA base sequence of a type strain of a Flavobacterium bacterium selected from the group consisting of procerum, resistens, micromati, limicola, reichenbachii, tiangeerense, xueshanense, psychrolimnae, algicola, faecalis, frigidarium, omnivorum, fryxellicola, degerlachei, gillisiae, frigoris, sinopsychrotolerans, xinjiangense, and urumqiense of the genus Flavobacterium. The protecting agent according to any one of claims 1 to 4, which is a bacterium of the genus Flavobacterium having a 16S rRNA base sequence that has the highest homology or identity to the rRNA base sequence.

8. The Flavobacteriaceae bacterium has homology or identity to the 16S rRNA base sequence of the type strain of Chryseobacterium genus bacteria, and is selected from the group consisting of vietnamese, aquifrigidense, culicis, nakagawai, jejuense, bernardetii, rhizosphaerae, kwangjuense, elymi, lathyri, oranimense, and contam The protective agent according to any one of claims 1 to 4, wherein the bacterium is a bacterium of the genus Chryseobacterium having a 16S rRNA base sequence that is most homologous or identical to the 16S rRNA base sequence of a type strain of a species selected from the group consisting of Bacillus subtilis, Bacillus gallinarum, Bacillus artocarpi, Bacillus ureilyticum, Bacillus oncorhynchus, Bacillus joostei, Bacillus viscerum, Bacillus tractae, Bacillus lactis, Bacillus rhizoplanae, Bacillus sediminis, Bacillus indologenes, Bacillus gleum, and Bacillus arthrosphaerae.

9. A bacterium belonging to the family Flavobacteriaceae, which is MUCF01 (accession number: NITE P-03323), MUCF02 (accession number: NITE P-03324), or MUCF03 (accession number: NITE P-03325), or a culture thereof.

10. A protective agent for fish against Edwardsiellosis, comprising the Flavobacterium family bacterium according to claim 9.

11. The protective agent according to claim 10, which is a protective agent for catfish, eel, sea bass, flounder, turbot, herring, rainbow trout, sweetfish, yellowtail, trout, sea bream, carp, amberjack, tuna, salmon, horse mackerel, tilapia, pufferfish, yellowtail, grouper, mackerel, or saury.

12. The protective agent according to claim 10, which is a protective agent for catfish, eel, sea bass, flounder, turbot, herring, rainbow trout or sweetfish.

13. The protective agent according to any one of claims 1 to 4 and 10 to 12, for protecting the fish by adding it to a rearing liquid in a rearing environment of the fish.

14. A method for protecting fish from Edwardsiellosis, comprising a step of contacting the fish with the Flavobacterium family bacterium described in claim 9 or the protectant described in any one of claims 1 to 8 and 10 to 13.

15. A method for protecting fish from Edwardsiellosis, comprising a step of growing the fish in water in the presence of the Flavobacterium family bacterium described in claim 9 or the protectant described in any one of claims 1 to 8 and 10 to 13.

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