WO2020246609A1

WO2020246609A1 - Microorganisms useful for fish skin probiotics

Info

Publication number: WO2020246609A1
Application number: PCT/JP2020/022437
Authority: WO
Inventors: 克敏堀
Original assignee: 国立大学法人東海国立大学機構
Priority date: 2019-06-06
Filing date: 2020-06-05
Publication date: 2020-12-10
Also published as: JPWO2020246609A1

Abstract

The present disclosure provides novel microorganisms that protect fish. In one aspect, the present disclosure provides a method for acquiring microorganisms having the capability to protect fish. In one aspect, the present disclosure provides: microorganisms, or the product thereof, having the capability to protect fish; and a composition including the microorganisms or the product thereof. In one embodiment, the microorganisms having the capability to protect fish can be bacteria of the genus pseudomonas. In one aspect, the present disclosure provides: microorganisms, or the product thereof, having the capability to protect fish; and a method for using a composition including the microorganisms or the product thereof to protect the fish.

Description

Microorganisms useful for fish epidermis probiotics

The present disclosure relates to microorganisms capable of protecting fish, methods for obtaining such microorganisms and the use of such microorganisms.

Antibiotics are being watched internationally as something that should be reduced due to concerns about the emergence of multidrug-resistant bacteria and environmental pollution. In research on disease prevention in farmed fish, attention has been paid to the development of vaccines for fish diseases as an alternative means to antibiotics (Non-Patent Document 1). Although vaccines for fish diseases are effective, they are effective only against specific pathogens of specific fish species, cannot cover a wide range of fish species and pathogens, and the types of vaccines approved for use in the field. This method alone is not sufficient in that it is limited.

As a result of diligent research, the present inventors have developed a method for obtaining a microorganism having the ability to protect fish (for example, the ability to function as epidermal probiotics of fish), and thus obtained. We have found that microorganisms are useful for the protection of fish. Microorganisms that could be useful in fish protection included Pseudomonadaceae bacteria. It was also found that this microorganism can colonize fish and / or suppress harmful microorganisms. As such, the present disclosure provides the microorganisms and derivatives thereof found in this way, as well as their use. The disclosure also provides a method for obtaining microorganisms capable of protecting fish.

Accordingly, the present disclosure provides:
(Item 1)
A Pseudomonadaceae bacterium that has the ability to protect fish.
(Item 2)
The Pseudomonadaceae bacterium according to item 1, which has an ability to function as a probiotic of fish.
(Item 3)
The Pseudomonadaceae bacterium according to

item

1 or 2, which has an ability to function as a probiotic of the epidermis of fish.
(Item 4)
The Pseudomonadaceae bacterium according to any one of items 1 to 3, which has an ability to settle on the epidermis of fish.
(Item 5)
The Pseudomonadaceae bacterium according to any one of items 1 to 4, which has an ability to improve the survival rate of the fish.
(Item 6)
The Pseudomonadaceae bacterium according to any one of items 1 to 5, wherein the fish is protected by adding the Pseudomonadaceae bacterium to the breeding environment (for example, breeding solution, breeding water, etc.) of the fish.
(Item 7)
The Pseudomonadaceae bacterium according to any one of items 1 to 6, which has an ability to suppress at least one harmful microorganism.
(Item 8)
The Pseudomonadaceae according to item 7, wherein the harmful microorganism has at least one of the ability to cause skin diseases in fish, the ability to infect fish transdermally, the ability to infect wounds, and the ability to infect contact. Bacteria.
(Item 9)
The harmful microorganisms include vibrio disease, asthma disease, atypical Eromonas salmonicida infection, Eromonas hydrophila infection, edwardsiellosis, red spot disease, Ayu's pseudomonas disease, red mouse disease, bacterial gill disease, and streptococculosis. (Selected, tailed, torn, torn), cold water disease, gliding bacillosis, bacterial kidney disease, mycobacteriosis, nocardiosis, and / or capable of causing streptococculosis, item 7 or 8. The described Pseudomonas family bacteria.
(Item 10)
The harmful microorganisms, Listonella anguillara (Vibrio anguillarum), Vibrio ordalii, Vibrio ichthyoenteri, Vibrio vulnificus, Vibrio salmonicida, Aeromonas salmonicida, atypical Aeromonas salmonicida, Aeromonas hydrophila, Edwardsiella tarda, 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, Pseudomonas family bacteria of claim 7 or 8.
(Item 11)
The harmful microorganisms include Aeromonas hydrophila, Aeromonas caviae, Yersinia ruckeri, Edwardsiella tarda, Flavobacterium colorare, Vibrio angilarum, Vibrio angilarum, Vibrio angilarum, Vibrio angilarum. (Item 12)
The Pseudomonadaceae bacterium according to any one of items 1 to 11, wherein the bacterium is determined to have an ability using zebrafish (Dario rerio) as a standard fish.
(Item 13)
The Pseudomonadaceae bacterium according to any one of items 1 to 12, wherein the fish is a farmed fish.
(Item 14)
Item 13. The Pseudomonas family according to Item 13, wherein the farmed fish is eel, ayu, yellowtail, trout, Thailand, carp, amberjack, tuna, salmon, horse mackerel, horse mackerel, tilapia, blowfish, horse mackerel, hata, mackerel, saury or catfish. Bacteria.
(Item 15)
The Pseudomonadaceae bacterium according to Item 13, wherein the farmed fish belongs to the family Salmonidae.
(Item 16)
The Pseudomonadaceae bacterium according to item 13, wherein the cultured fish is trout.
(Item 17)
The Pseudomonas family bacterium according to any one of items 1 to 16, which is a bacterium of the genus Pseudomonas.
(Item 18)
The Pseudomonas family bacterium according to any one of items 1 to 16, which is a Pseudomonas putida group.
(Item 19)
The Pseudomonas family bacterium according to any one of items 1 to 16, which is Pseudomonas mossellii.
(Item 20)
KH-ZF1 (accession number: NITE BP-20967), a Pseudomonadaceae bacterium.
(Item 21)
A fish protective agent containing the Pseudomonadaceae bacterium according to any one of items 1 to 20.
(Item 22)
A fish probiotic agent comprising the Pseudomonadaceae bacterium according to any one of items 1 to 20.
(Item 23)
The probiotic agent according to item 22, which is the probiotic of the epidermis of the fish.
(Item 24)
The probiotic agent according to item 22 or 23, wherein the Pseudomonadaceae bacterium has an ability to settle in the epidermis of fish.
(Item 25)
The fish protective agent according to item 21, or the probiotic agent according to any one of items 22 to 24, which is a protective agent or probiotic agent for farmed fish.
(Item 26)
Item 21 which is a protective agent or probiotics agent for eel, ayu, yellowtail, trout, tie, carp, amberjack, tuna, salmon, horse mackerel, flatfish, tilapia, puffer, hamachi, hata, mackerel, saury or catfish. The fish protective agent or the probiotics agent according to any one of items 22 to 24.
(Item 27)
The fish protectant according to item 21, or the probiotic agent according to any one of items 22 to 24, which is a salmonid fish protectant or probiotic agent.
(Item 28)
The fish protectant according to item 21, or the probiotic agent according to any one of items 22 to 24, which is a trout protectant or probiotic agent.
(Item 29)
A method for protecting fish, the method comprising contacting the fish with the Pseudomonadaceae bacterium according to any one of items 1 to 20.
(Item 30)
A method for protecting fish, which comprises the step of growing the fish in water in which the Pseudomonadaceae bacterium according to any one of items 1 to 20 is present.
(Item 31)
A method for obtaining microorganisms that have the ability to protect fish.
(A) Step of obtaining candidate microorganisms from the epidermis of fish,
(B) A step of adding the candidate microorganism to a medium containing a harmful microorganism,
(C) A step of confirming the suppression of the harmful microorganism in the medium, and (d) when the suppression of the harmful microorganism in the medium is confirmed, the candidate microorganism is acquired as a microorganism having an ability to protect the fish. Process to do,
Including methods.
(Item 32)
The method according to item 31, wherein in the step (c), the suppression of the harmful microorganisms on the medium is confirmed.
(Item 33)
In the step (c), the candidate microorganism and the harmful microorganism are grown on the same medium (for example, agar medium or an equivalent medium (for example, gellan gum)), and the harmful microorganism is generated in the vicinity of the candidate microorganism. The method according to item 31, wherein the growth inhibition zone where the growth cannot be performed is confirmed.
(Item 34)
The method according to item 31, wherein in the step (c), suppression of the harmful microorganism in the candidate microorganism or the culture supernatant thereof and the liquid medium containing the harmful microorganism is confirmed.
(Item 35)
In the step (c), the culture medium or culture supernatant of the candidate microorganism is contained on a medium in which the harmful microorganism is inoculated as a whole (for example, an agar medium or an equivalent medium (for example, gellan gum)). 31. The method of item 31, wherein a bottomless cylinder containing the invaders or the culture medium or the culture supernatant is placed and the suppression of the harmful microorganisms around the invaders or the cylinder is confirmed.
(Item 36)
In the step (c), after incubating the liquid medium containing the candidate microorganism and the harmful microorganism, the growth of the candidate microorganism and the harmful microorganism is compared, and the growth of the harmful microorganism is further suppressed. The method according to item 31 to be confirmed.
(Item 37)
In the step (c), a medium in which the harmful microorganisms are inoculated entirely (for example, an agar medium or an equivalent medium (for example, gellan gum)) or a medium in which the harmful microorganisms are totally grown (for example, gellan gum). For example, the candidate microorganism is inoculated on an agar medium or an equivalent medium (for example, gellan gum), the candidate microorganism forms a colony, and a growth inhibition circle of the harmful microorganism is observed around the colony. The method according to item 31 for confirming that.
(Item 38)
The method according to any one of items 31 to 37, wherein the candidate microorganism is obtained from a colony formed on a medium inoculated with a scraped product of the epidermis of the fish.
(Item 39)
The method according to any one of items 31 to 38, wherein one or more kinds of microorganisms are acquired as microorganisms having an ability to protect the fish.
(Item 40)
Any of items 31-39, wherein the harmful microorganism has at least one of the ability to cause skin diseases in fish, transdermally infect fish, infect wounds, and infect contact. The method described in paragraph 1.
(Item 41)
The harmful microorganisms include vibrio disease, furunculosis, atypical Eromonas salmonicida infection, Eromonas hydrophila infection, edwardsiellosis, red spot disease, Ayu's pseudomonas disease, red mouse disease, bacterial gill disease, and streptococculosis. (Selected, tailed, torn, torn), cold water disease, gliding bacillosis, bacterial kidney disease, mycobacteriosis, nocardiosis, and / or capable of causing streptococculosis, items 31-39 The method according to any one item.
(Item 42)
The method according to any one of items 31 to 41, wherein a candidate microorganism is obtained from the epidermis of zebrafish (Danio rerio).
(Item 43)
A microorganism obtained by the method according to any one of items 31 to 42.
(Item 44)
The microorganism according to item 43, which belongs to the genus Pseudomonas.
(Item 45)
The microorganism according to item 43, which is a Pseudomonas putida group.
(Item 46)
A method for protecting fish, which comprises a step of bringing a microorganism obtained by the method according to any one of items 31 to 42 into contact with the fish.
(Item 47)
It is a strain belonging to Pseudomonadaceae and
The strain is
(I) A step of linearly drawing the strain on an NB agar medium and culturing at 28 ° C. overnight.
(Ii) After that, a step of drawing harmful microorganisms vertically from the strain so as not to touch the strain and culturing at 28 ° C. overnight.
When tested in a method comprising, after step (ii), the strain is characterized by the presence of a clear zone over a range of 10 mm or more from the stroke.
Here, the harmful microorganisms are Aeromonas hydrophila (ATCC 700183), Aeromonas hydrophila (JCM 1027), Aeromonas caviae (JCM 1043), Flavobacterium columnare (JCM 103), Flavobacterium colorNeria (JCM) 2 Containing at least one selected from the group consisting of
Strain.
(Item XX1)
A Pseudomonadaceae bacterium that has the ability to protect fish.
(Item XX2)
A Pseudomonadaceae bacterium of any of the above items having the ability to function as a fish probiotic.
(Item XX3)
A Pseudomonadaceae bacterium of any of the above items having the ability to function as a probiotic of the epidermis of fish.
(Item XX4)
A Pseudomonadaceae bacterium that has the ability to colonize the epidermis of fish.
(Item XX5)
A Pseudomonadaceae bacterium according to any one of the above items, which has an ability to improve the survival rate of the fish.
(Item XX6)
The Pseudomonadaceae bacterium according to any one of the above items, wherein the Pseudomonadaceae bacterium is protected by adding the Pseudomonadaceae bacterium to the fish breeding environment breeding solution.
(Item XX7)
A Pseudomonadaceae bacterium according to any one of the above items, which has an ability to suppress at least one harmful microorganism.
(Item XX8)
Pseudomonadas according to any one of the above items, wherein the harmful microorganism has at least one ability of causing skin disease of fish, transdermally infecting fish, infecting wounds, and infecting contact. Family bacteria.
(Item XX9)
The harmful microorganisms include vibrio disease, asthma disease, atypical Eromonas salmonicida infection, Eromonas hydrophila infection, edwardsiellosis, red spot disease, Ayu's pseudomonas disease, red mouse disease, bacterial gill disease, and streptococculosis. Any of the above items that are capable of causing cold water disease, gliding bacillosis, bacterial kidney disease, mycobacteriosis, nocardiosis, and / or streptococculosis (selected, tailed, torn, squeezed) Pseudomonas bacterium.
(Item XX10)
The harmful microorganisms, Listonella anguillara (Vibrio anguillarum), Vibrio ordalii, Vibrio ichthyoenteri, Vibrio vulnificus, Vibrio salmonicida, Aeromonas salmonicida, atypical Aeromonas salmonicida, Aeromonas hydrophila, Edwardsiella tarda, 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, or Pseudomonas family bacteria of the item.
(Item XX11)
The harmful microorganisms include Aeromonas hydrophila, Aeromonas caviae, Yersinia ruckeri, Edwardsiella tarda, Flavobacterium colorare, Vibrio angilarum, Vibrio angilarum, Vibrio angilarum.
(Item XX12)
The bacterium is a Pseudomonadaceae bacterium according to any one of the above items, the ability of which is determined by using zebrafish (Dario rerio) as a standard fish.
(Item XX13)
A Pseudomonadaceae bacterium according to any one of the above items, wherein the fish is a farmed fish.
(Item XX14)
Pseudomonas of any of the above items, wherein the farmed fish is eel, ayu, yellowtail, trout, tie, carp, amberjack, tuna, salmon, horse mackerel, flatfish, tilapia, blowfish, hamachi, hata, mackerel, saury or catfish. Family bacteria.
(Item XX15)
A Pseudomonadaceae bacterium according to any of the above items, wherein the farmed fish belongs to the family Salmonidae.
(Item XX16)
A Pseudomonadaceae bacterium according to any of the above items, wherein the cultured fish is trout.
(Item XX17)
A bacterium of the family Pseudomonas, which is a bacterium of the genus Pseudomonas.
(Item XX18)
A Pseudomonas bacterium according to any of the above items, which is a Pseudomonas putida group, a Pseudomonas fluorescens group, or a Pseudomonas koreensis group.
(Item XX19)
Pseudomonas Moseri (Pseudomonas mosselii), Pseudomonas Maruginarisu (Pseudomonas marginalis), a Pseudomonas Korenshisu (Pseudomonas koreensis), Pseudomonas Purotegensu (Pseudomonas protegens) or Pseudomonas Parafuruba (Pseudomonas parafulva), one of the Pseudomonas family of the above-mentioned items Bacteria.
(Item XX20)
KH-ZF1 (accession number: NITE BP-02967), KH-RT1 (receipt number: NITE ABP-03222), KH-RT2 (receipt number: NITE ABP-03223), KH-RT3 (receipt number: NITE ABP-03224) ) Or KH-RT4 (receipt number: NITE ABP-03225), a Pseudomonadaceae bacterium.
(Item XX21)
A fish protectant containing any of the above items, Pseudomonadaceae.
(Item XX22)
A fish probiotic agent containing any of the above items, Pseudomonadaceae.
(Item XX23)
The probiotic agent according to any one of the above items, which is the probiotic of the epidermis of the fish.
(Item XX24)
The probiotic agent according to any one of the above items, wherein the Pseudomonadaceae bacterium has an ability to settle on the epidermis of fish.
(Item XX25)
A fish protective agent or a probiotic agent according to any one of the above items, which is a protective agent or probiotic agent for farmed fish.
(Item XX26)
Any of the above items, which is a protective agent or probiotics agent for eel, ayu, yellowtail, trout, tie, carp, amberjack, tuna, salmon, horse mackerel, flatfish, tilapia, puffer, hamachi, hata, mackerel, saury or catfish. The fish protectant or the probiotics agent of any of the above items.
(Item XX27)
A fish protectant or probiotic agent of any of the above items, which is a salmonid fish protectant or probiotic agent.
(Item XX28)
A fish protectant of any of the above items or a probiotic agent of any of the above items, which is a trout protectant or probiotic agent.
(Item XX29)
A method for protecting fish, the method comprising contacting the fish with a Pseudomonadaceae bacterium of any of the above items.
(Item XX30)
A method for protecting fish, which comprises the step of growing the fish in water in which the Pseudomonadaceae bacterium according to any one of the above items is present.
(Item XX31)
A method for obtaining microorganisms that have the ability to protect fish.
(A) Step of obtaining candidate microorganisms from the epidermis of fish,
(B) A step of adding the candidate microorganism to a medium containing a harmful microorganism,
(C) A step of confirming the suppression of the harmful microorganism in the medium, and (d) when the suppression of the harmful microorganism in the medium is confirmed, the candidate microorganism is acquired as a microorganism having an ability to protect the fish. Process to do,
Including methods.
(Item XX32)
The method according to any one of the above items, which confirms the suppression of the harmful microorganisms on the solid medium in the step (c).
(Item XX33)
In the step (c), any of the above items, in which the candidate microorganism and the harmful microorganism are grown on the same medium, and a growth inhibition zone in which the harmful microorganism cannot grow is confirmed in the vicinity of the candidate microorganism. ..
(Item XX34)
The method according to any one of the above items, wherein in the step (c), suppression of the harmful microorganism in the candidate microorganism or the culture supernatant thereof and the liquid medium containing the harmful microorganism is confirmed.
(Item XX35)
In the step (c), a bottomless cylinder containing the culture solution of the candidate microorganism or an impregnated substance of the culture supernatant or the culture solution or the culture supernatant is placed on a medium in which the harmful microorganism is inoculated as a whole. The method of any of the above items, which is arranged to confirm the suppression of the harmful microorganisms around the impregnated substance or the cylinder.
(Item XX36)
In the step (c), after incubating the liquid medium containing the candidate microorganism and the harmful microorganism, the growth of the candidate microorganism and the harmful microorganism is compared, and the growth of the harmful microorganism is further suppressed. One of the above methods to check.
(Item XX37)
In the step (c), the candidate microorganism is inoculated on the medium in which the harmful microorganism is inoculated as a whole, or on the medium in which the harmful microorganism is grown as a whole, and the candidate microorganism forms a colony. Any of the above-mentioned methods for confirming that a growth-inhibiting circle of the harmful microorganism is observed in the vicinity thereof.
(Item XX38)
The method of any of the above items, wherein the candidate microorganism is obtained from a colony formed on a medium inoculated with a scraped product of the epidermis of the fish.
(Item XX39)
The method of any of the above items, wherein one or more microorganisms are obtained as microorganisms capable of protecting the fish.
(Item XX40)
The method of any of the above items, wherein the harmful microorganism has at least one of the ability to cause skin disease in fish, the ability to infect fish percutaneously, the ability to infect wounds, and the ability to infect contact. ..
(Item XX41)
The harmful microorganisms include vibrio disease, furunculosis, atypical Eromonas salmonicida infection, Eromonas hydrophila infection, edwardsiellosis, red spot disease, Ayu's pseudomonas disease, red mouse disease, bacterial gill disease, and streptococculosis. Any of the above items that are capable of causing cold water disease, gliding bacillosis, bacterial kidney disease, mycobacteriosis, nocardiosis, and / or streptococculosis (selected, tailed, torn, torn) the method of.
(Item XX42)
The method of any of the above items for obtaining candidate microorganisms from zebrafish (Danio rerio) or trout epidermis.
(Item XX43)
Microorganisms obtained by any of the above items.
(Item XX44)
A microorganism belonging to the genus Pseudomonas and any of the above items.
(Item XX45)
A microorganism according to any of the above items, which is a Pseudomonas putida group, a Pseudomonas fluorescens group, or a Pseudomonas putenas group.
(Item XX46)
A method for protecting fish, which comprises a step of bringing a microorganism obtained by any of the above items into contact with the fish.
(Item XX47)
It is a strain belonging to Pseudomonadaceae and
The strain is
(I) A step of linearly drawing the strain on an NB agar medium and culturing at 28 ° C. overnight.
(Ii) After that, a step of drawing harmful microorganisms vertically from the strain so as not to touch the strain and culturing at 28 ° C. overnight.
When tested in a method comprising, after step (ii), the strain is characterized by the presence of a clear zone over a range of 10 mm or more from the stroke.
Here, the harmful microorganisms are Aeromonas hydrophila (ATCC 700183), Aeromonas hydrophila (JCM 1027), Aeromonas caviae (JCM 1043), Flavobacterium columnare (JCM 103), Flavobacterium colorNeria (JCM) 2 Containing at least one selected from the group consisting of
Strain.
(Item XX48)
It is a strain belonging to Pseudomonadaceae and
The strain is
(I) A step of linearly drawing the strain on an NB agar medium and culturing at 28 ° C. overnight.
(Ii) Then, a step of drawing harmful microorganisms in a direction perpendicular to the image of the strain so as to intersect the image of the strain and culturing at 28 ° C. overnight.
In the case of testing by the method including, after the step (ii), the colony by the strain occupies an area of 50% or more in the image portion of the harmful microorganism beyond the portion intersecting the image line of the strain. Characterized by
Here, the harmful microorganism is selected from the group consisting of Yersinia ruckeri (NVH 3758), Aeromonas hydrophila (NRIA14), Vibrio anglilarum (NRIA83) and Vibrio ordali (NRIA90).
Strain.
(Item XX49)
A composition comprising an effective amount of Pseudomonadaceae bacteria in any of the above items.
(Item XX50)
With an effective amount of Pseudomonadaceae bacteria in any of the above items,
A composition comprising a salt, a surfactant, a carrier, a drying protectant, a preservative, an excipient, a strengthening agent, an antioxidant, a dispersant, a flocculant and at least one of other microorganisms.
(Item XX51)
A protective aquaculture solution for fish, which comprises an effective amount of a Pseudomonadaceae bacterium and a fish culture solution for any of the above items.
(Item XX52)
A fish protective agent containing an effective amount of Pseudomonadaceae bacteria according to any of the above items and a component for fish farming.
(Item XX53)
A kit for the protection of fish, which comprises an effective amount of Pseudomonadaceae bacteria in any of the above items and a component for fish farming.
(Item XX54)
A method for protecting fish, comprising contacting the fish with an effective amount of a Pseudomonadaceae bacterium in any of the above items.
(Item XX55)
A method for protecting a fish, which comprises a step of growing the fish in water in which an effective amount of any of the above-mentioned Pseudomonadaceae bacteria is present.
(Item XX56)
The method of any of the above items, wherein the fish is protected from at least one harmful microorganism.
(Item XX57)
The method of any of the above items, wherein the harmful microorganism has at least one of the ability to cause skin disease in fish, the ability to infect fish percutaneously, the ability to infect wounds, and the ability to infect contact. ..
(Item XX58)
The harmful microorganisms include vibrio disease, furunculosis, atypical Eromonas salmonicida infection, Eromonas hydrophila infection, edwardsiellosis, red spot disease, Ayu's pseudomonas disease, red mouse disease, bacterial gill disease, and streptococculosis. Any of the above items that are capable of causing cold water disease, gliding bacillosis, bacterial kidney disease, mycobacteriosis, nocardiosis, and / or streptococculosis (selected, tailed, torn, torn) the method of.
(Item XX59)
The harmful microorganisms, Listonella anguillara (Vibrio anguillarum), Vibrio ordalii, Vibrio ichthyoenteri, Vibrio vulnificus, Vibrio salmonicida, Aeromonas salmonicida, atypical Aeromonas salmonicida, Aeromonas hydrophila, Edwardsiella tarda, 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, the method of any of the preceding items.
(Item XX60)
The harmful microorganisms include Aeromonas hydrophila, Aeromonas caviae, Yersinia ruckeri, Edwardsiella tarda, Flavobacterium colorare, Vibrio angurica, and any combination of Vibrio angilarum, Vibrio angilarum, and Vibrio angilarum.
(Item XX61)
The method according to any one of the above items, wherein the contacting step includes adding the Pseudomonadaceae bacterium to the breeding environment (for example, breeding liquid, breeding water, etc.) of the fish.
(Item XX62)
The method of any of the above items, wherein the contacting step is carried out under conditions sufficient for the Pseudomonadaceae bacteria to colonize the epidermis of the fish.
(Item XX63)
The method of any of the above items, wherein the fish is a farmed fish.
(Item XX64)
The method of any of the above items, wherein the fish is eel, ayu, yellowtail, trout, tie, carp, amberjack, tuna, salmon, horse mackerel, flatfish, tilapia, pufferfish, hamachi, hata, mackerel, saury or catfish.
(Item XX65)
The method of any of the above items, wherein the fish belongs to the family Salmonidae.
(Item XX66)
The method of any of the above items, wherein the fish is trout.
(Item XX67)
The method according to any one of the above items, wherein the Pseudomonada bacterium is a bacterium of the genus Pseudomonas.
(Item XX68)
The method according to any one of the above items, wherein the Pseudomonas family bacterium is a Pseudomonas putida group, a Pseudomonas fluoressence group, or a Pseudomonas koreensis group.
(Item XX69)
The Pseudomonas family bacteria are Pseudomonas mossellii, Pseudomonas marginalis, Pseudomonas korenesis, Pseudomonas protegens Pseudomonas protegens Pseudomonas protegens Pseudomonas. Either way.
(Item XX70)
The Pseudomonadaceae bacteria are KH-ZF1 (accession number: NITE BP-02967), KH-RT1 (receipt number: NITE ABP-03222), KH-RT2 (receipt number: NITE ABP-03223), KH-RT3 (receipt). The method of any of the above items, number: NITE ABP-03224) or KH-RT4 (receipt number: NITE ABP-03225).
(Item XX71)
The method of any of the above items, wherein the protection of the fish comprises protecting the epidermis of the fish.
(Item XX72)
The method of any of the above items, wherein protection of the fish comprises treating or preventing a disease or disease of the fish.

In the present disclosure, it is intended that the above one or more features may be provided in a further combination in addition to the specified combinations. Further embodiments and advantages of the present disclosure will be appreciated by those skilled in the art upon reading and understanding the following detailed description as necessary.

Since the microorganisms disclosed in the present disclosure can be a substitute for antibiotics in fish breeding such as aquaculture, the use of antibiotics can be reduced and the environmental load can be reduced. In addition, the method of obtaining the microorganisms that protect the fish of the present disclosure can provide new useful microorganisms and achieve more stable, cheaper, higher production and / or the growth of diverse fish.

It is a schematic diagram of an exemplary method for obtaining microorganisms from the epidermis of fish. The microflora of the epidermis of fish is scraped with a cotton swab, the cotton swab is immersed in sterile water to prepare a microbial solution, and the microbial solution is added to the medium to isolate the formed colonies. It is a schematic diagram of the inhibition test by the Cross-streak method. The result of the inhibition test by the Cross-streak method of Example 2 is shown. The left is the result of KH-ZF1 and the right is the result of another microbial strain. The horizontal strokes are Aeromonas hydrophila (1) (ATCC 700183), Aeromonas hydrophila (2) (JCM 1027), Aeromonas caviae (JCM 1043), Flavobasinia (JCM 1043), Flavobasinia (Yersinia), Yersinia ruckera 3758), Yersinia ruckeri (2) (DSMZ 18506), and Eschericia colli DH5α. Growth inhibition test on a plate by KH-ZF1 of Example 4. The test microorganisms drawn from the center of the plate are Aeromonas hydrophila (1) (ATCC 700183), Aeromonas hydrophila (2) (JCM 1027), Aeromonas caviae (JCM 1043), Yersiella (JCM 1043), and Yersia (Yersia). rukkeri (2) (DSMZ 18506), Edwardsiella tarda (1) (NRIA44), Edwardsiella tarda (2) (NRIA51), Vibrio anglilarum (NRIA83), Vibrio anglilarum (NRIA83), Vibrio angilarum (NRIA83), Vibrio NicoNico (NRIA83), Vibrio Nico Harmful microorganisms (1) and (2)). The area surrounded by the broken line indicates the clear zone. The molecular phylogenetic tree based on the 16S rRNA partial base sequence of KH-ZF1 of Example 5 is shown. The upper left line shows the scale bar. The number at the branch of the phylogenetic tree indicates the bootstrap value. The T after the strain name of the microorganism indicates that the species is the reference strain. BSL indicates the biosafety level (BSL1 or higher is indicated). The result of the fixing test of Example 6 is shown. The vertical axis shows the colony forming unit (CFU) from one zebrafish. The horizontal axis shows the time from the addition of KH-ZF1 to the time when the body surface of the zebrafish is rubbed with a mentip. It is a schematic diagram of the infection control test of Example 7. The test was carried out by placing 5 zebrafish (Danio rerio) in 200 ml of breeding water. After breeding at 28 ° C. for two days to acclimatize to this breeding condition, zebrafish was exposed to the strain by administering KH-ZF1 into the breeding water (final concentration of bacteria OD ₆₀₀ = 0.01). .. After continuing breeding at 28 ° C. for 24 hours in this state, the muscle at the base of the dorsal fin of the zebrafish was injured with a 1.2 mm needle. Immediately after that, the breeding water was replaced, KH-ZF1 was added again (final bacterial concentration OD ₆₀₀ = 0.01), and the animals were bred for another 24 hours. Then, after lowering the breeding temperature to 20 ° C. and breeding for 24 hours, KH-ZF1 and Yersinia ruckeri (NVH3578) are added so that the bacterial concentration OD ₆₀₀ = 0.01, respectively, and the mixture is left at 20 ° C. for 1 hour. Attempted to infect Yersinia ruckeri with zebrafish. After that, the breeding water was exchanged, KH-ZF1 was added (final bacterial concentration D ₆₀₀ = 0.01), and the progress was observed while continuing the breeding at 20 ° C. (treatment group). The control group to which KH-ZF1 was not added at all was also observed. The survival curve of zebrafish treated with pathogenic microorganisms alone (dotted line) or in combination with KH-ZF1 (solid line) is shown. The vertical axis shows the cumulative survival rate, and the horizontal axis shows the number of days elapsed since the start of observation. RStudio was used as software. It is a schematic diagram of the competitive advantage test by the Cross-streak method. 9 is a microscopic image of a larva on day 3 of KH-ZF1 exposure (top) or control condition (bottom) of Example 9. In the KH-ZF1 exposed group, fluorescence of KH-ZF1 was observed on the body surface of the larvae, indicating colonization of KH-ZF1. The results of the survival test of Example 9 are shown. The vertical axis shows the survival rate. The horizontal axis shows the elapsed time (days), and the 0th, 3rd, 5th, 18th, and 26th days from the start of breeding under the conditions of KH-ZF1 exposure (left) or control condition (right). , 28th day and 31st day are shown.

Hereinafter, the present disclosure will be described while showing the best form. Throughout the specification, it should be understood that the singular representation also includes its plural concept, unless otherwise stated. Therefore, it should be understood that singular articles (eg, "a", "an", "the", etc. in English) also include the concept of their plural, unless otherwise noted. It should also be understood that the terms used herein are used in the meaning commonly used in the art unless otherwise noted. Thus, unless otherwise defined, all terminology and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, this specification (including definitions) takes precedence.

The definitions and / or basic technical contents of terms particularly used in the present specification will be described below as appropriate.

(Definition, etc.)
As used herein, the term "fish" refers to an organism belonging to the subphylum Vertebrata (Vertebrata), excluding quadrupeds. Fish include, but are not limited to, farmed fish used for food, fish for viewing and pets, and the like. In one embodiment, fish may be subject to the present disclosure as long as they have an epidermis.

As used herein, the term "microorganism" refers to small (eg, 1 mm or less, 0.1 mm or less, or invisible to the naked eye) algae, protists, and fungi, as well as eubacteria and archaea. It means organisms that also include molds, fungi, etc.), yeasts, and animals (protists, archaea, etc.).

As used herein, the term "harmful microorganism" means a microorganism that aggravates or may aggravate the survival, health condition, meat quality, injury recovery and / or infectious condition of fish. Harmful microorganisms may also include opportunistic infections.

In the present specification, "suppression" of a microorganism (also referred to as "inhibition" in Japanese, but both are synonymous in the present specification) means reduction and / or death of a microorganism, and / or of a microorganism. It means a decrease in the growth rate, and includes reducing the growth and growth of microorganisms and killing microorganisms. The suppression of microorganisms can be evaluated based on, for example, the transparency of the medium inoculated with the microorganisms and the transparent medium area. The ability of a microorganism to suppress or inhibit harmful microorganisms can be measured and identified, for example, in the following tests. In addition, it is understood that if any one of the tests gives a result showing an inhibitory effect, it falls within the scope of inhibition in the present specification.
-A test in which candidate microorganisms are added on a medium, and then harmful microorganisms are added on the same medium to confirm the transparency and / or the transparent medium area of the medium.
-A test in which a candidate microorganism and a harmful microorganism are grown on the same medium, and a growth inhibition zone in the vicinity of the candidate microorganism is observed in which the harmful microorganism cannot grow.
-A test to confirm that the growth of harmful microorganisms is reduced by adding the culture supernatant of the candidate microorganisms to the liquid medium inoculated with harmful microorganisms.
-Place the culture solution or culture supernatant of the candidate microorganism on the medium, or place the culture solution or culture supernatant of the candidate microorganism in a small bottomless cylinder, and grow harmful microorganisms inoculated on the entire medium. A test to confirm that is suppressed around it.
-A test in which candidate microorganisms and harmful microorganisms are cultured in the same liquid medium, and then both microorganisms are distinguished and the growth of the microorganisms is compared to confirm that the harmful microorganisms are suppressed.

In the present specification, the "epidermis" of a fish means the body (body surface) of the fish, eyes, scales, fins, gills, mouth, anus, and the surface of the genital organs. The epidermis on the body surface of fish can be classified into scale type (scale type fish) and mucosal type (mucosal type fish).

As used herein, "protection" of a fish means improving the survival, health, meat quality, injury recovery and / or infectious condition (including susceptibility) of the fish. Fish protection can be assessed, for example, by improving survival when injured fish are brought into contact with harmful microorganisms. For example, the fish are protected by adding the Pseudomonadaceae bacteria to the fish breeding environment (for example, breeding liquid, breeding water, etc.). Here, the breeding environment refers to an arbitrary growing environment of fish organisms, and is not necessarily limited to that because it is usually a breeding solution but the water content may be low in the case of a roe state. The ability of microorganisms to protect fish can be measured and identified, for example, by the following tests.
-Comparison between the control group that stresses the fish and adds harmful microorganisms to this fish and the treatment group that adds harmful microorganisms and candidate microorganisms to this fish to improve the survival rate of the fish in the treatment group. Exam to confirm.
-Comparison between the treatment group in which harmful microorganisms are added after pretreatment in which fish are bred in the presence of candidate microorganisms for a certain period of time or longer and the control group in which harmful microorganisms are added without the pretreatment. A study to be performed to confirm the improvement in fish survival in the treatment group.

As used herein, "probiotics" are compositions containing biological components (eg, whole or part of a microorganism) that act on harmful microorganisms and form useful microbial flora in fish. Refers to a microorganism or a biological component thereof itself having a function of maintaining or improving the health condition of the epidermis and / or activating the immunity of fish, or a composition containing these. The ability of microorganisms to function as probiotics can be measured and identified, for example, in the following tests.
-A test to confirm that when fish are bred in the presence of candidate microorganisms for a certain period of time or longer, the survival rate of fish is improved as compared with the case where fish are bred in the absence of candidate microorganisms.
-A test to confirm signs of enhanced immune activity of fish, such as increased cytokine secretion and increased expression of immune-related genes after breeding fish for a certain period of time or longer in the presence of candidate microorganisms.
-A test to confirm that the population of harmful microorganisms in the microbial flora of fish is reduced by breeding fish for a certain period of time or longer in the presence of candidate microorganisms.
-A treatment group in which harmful microorganisms are added to the breeding water after pretreatment for breeding fish for a certain period of time or longer in the presence of candidate microorganisms, and a control group in which harmful microorganisms are added without pretreatment. A test to confirm that the population of the harmful microorganisms in the microbial flora of the fish in the treatment group is lower than that in the control group.

As used herein, "fixation" refers to the survival (eg, proliferation) of a microorganism that remains attached to the subject for a period of time. The ability of microorganisms to colonize fish can be measured and identified, for example, by the following tests.
-A test in which fish are bred in breeding water containing candidate microorganisms and the number or amount of candidate microorganisms obtained from the bred fish is confirmed.
-A test to confirm the presence of candidate microorganisms in fish (on the epidermis of fish, etc.) by observing the fish under a microscope after breeding the fish in the breeding water to which the candidate microorganisms have been added.
-A test to confirm the presence of candidate microorganisms in the flora by analyzing the microbial flora of fish (on the epidermis of fish, etc.) after breeding fish in breeding water to which candidate microorganisms have been added.

In the present specification, the "fish protectant" refers to a preparation containing the microorganism of the present disclosure as an active ingredient and capable of protecting fish.

In the present specification, the "probiotic agent" refers to a preparation containing the microorganism of the present disclosure as an active ingredient and capable of exerting a function as a probiotic.

As used herein, the "inducible strain", "similar strain" or "mutant strain" preferably comprises a region substantially homologous to the DNA of the microorganism of interest, although not intended to be limiting. Such strains, including genes (eg, 16S rDNA), are aligned in various embodiments by computer homology programs known in the art and compared to the sequence of the entire genome of the original strain. It has a whole genome sequence that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical. This is a microorganism that has been modified by mutations, substitutions, deletions and / or additions of a gene, the inducing strain of which still exhibits the biological function of the original microorganism, although not necessarily to the same extent. means. For example, gene mutations can be introduced using any known mutagen, UV, plasma, genome editing techniques, and the like. In one embodiment, the "inducible strain", "similar strain" or "mutant strain" is a strain of the same genus and / or species as the original strain. For example, suitable and available in vitro assays described herein or known in the art can be used to study the biological function of such microorganisms.

As used herein, a "purified" biological factor (eg, an organism or intracellular component of a particular strain) is one in which at least a portion of the factors naturally associated with the biological factor have been removed. Say. Therefore, the purity of the biological factor in the purified biological factor is usually higher (ie, enriched) than in the state in which the biological factor is normally present. The term "purified" as used herein is 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. It means that the same type of biological factor (eg, a particular strain) is present.

As used herein, the term "homology" of a gene or base sequence refers to the degree of identity of two or more gene sequences to each other, and generally, "homology" refers to the degree of identity or similarity. It means high. Therefore, the higher the homology of two genes, the higher the identity or similarity of their sequences. Whether or not the two genes are homologous can be examined by direct sequence comparison or, in the case of nucleic acids, hybridization under stringent conditions. When directly comparing two gene sequences, the DNA 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%, the genes are homologous. As used herein, the term "similarity" of a gene or base sequence refers to the degree of similarity between two or more gene sequences to each other, and means that the degree of similarity of other sequences of identity is high. "Similarity" is a numerical value that takes into account similar bases in addition to identity. Here, similar bases are mixed bases (for example, 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).

Amino acids can be referred to herein by either their generally known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides can also be referred to by the generally recognized one-letter code. In the present specification, comparison of amino acid sequence and base sequence similarity, identity and homology is calculated using default parameters using BLAST, a tool for sequence analysis. The identity search can be performed using, for example, NCBI's BLAST 2.7.1 (issued 2017.10.19). The value of "identity" in the present specification usually refers to the value when the above BLAST is used and aligned under the default conditions. However, if a higher value is obtained by changing the parameter, the highest value is set as the identity value. When identity is evaluated in multiple regions, the highest value among them is set as the identity value. "Similarity" is a numerical value that takes into account similar amino acids in addition to identity.

In one embodiment of the present disclosure, "70% or more", which is a numerical value such as identity, is, for example, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more. , 97% or more, 98% or more, 99% or more, or 100% or more, and may be within the range of any two of the numerical values that are the starting points thereof. The above "identity" is calculated by calculating the ratio of the number of bases homologous in a base sequence between two or a plurality of bases according to a known method as described above. Specifically, before calculating the ratio, align the base sequences of the base sequence groups to be compared, and if necessary to maximize the ratio of the same base nucleic acid, leave a gap in a part of the base sequence. Introduce. Methods for alignment, methods for calculating proportions, comparison methods, and related computer programs are well known in the art (eg, BLAST, described above). As used herein, "identity" can be expressed as a value measured by NCBI's BLAST unless otherwise specified. Blastp can be used with default settings for the algorithm when comparing base sequences in BLAST. The measurement results are quantified as Positives or Identities. In this case, the term "similarity" instead of "identity" is a numerical value that also considers those that fall under the definition of "similar" "base" described in the present specification.

As used herein, the term "biological function" refers to a specific function that a microorganism may have when referring to a microorganism, which includes, for example, protection of fish (eg, improvement of survival rate of fish). ) Etc., but are not limited to them. In the present disclosure, for example, improvement of survival rate of fish and suppression of harmful microorganisms can be mentioned, but the present invention is not limited thereto. As used herein, a biological function can be exerted by a corresponding "biological activity". As used herein, the term "biological activity" refers to an activity that a microorganism can have in a certain environment, and includes an activity that exerts various functions (for example, improvement of 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 (ie, have a measurable effect in response to some exposure or stimulus), eg, some stimulus of the microorganisms of the present disclosure. Post-event or post-event upstream or downstream protein levels or other similar functional measures may also be included.

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

In the present specification, the term "about" refers to the indicated value plus or minus 10% unless otherwise defined.

As used herein, a "kit" is usually divided into two or more compartments and the parts to be provided (for example, the composition containing the microorganisms of the present disclosure, additional components, buffers, instructions, etc.) The unit provided. The form of this kit is preferred when the purpose is to provide a composition that should not be mixed and provided for stability and the like, but is preferably mixed and used immediately before use. Such kits preferably include instructions or instructions that describe how to use or treat the parts provided (eg, microbial compositions, additional ingredients), etc. It is advantageous to have instructions. When a kit is used herein, the kit typically includes instructions and the like that describe how to use the microorganisms, compositions, etc. of the present disclosure.

In the present specification, the "instruction" describes the method for using the present disclosure to the user. This instruction contains language that directs how to use this disclosure. If necessary, this instruction may be provided by the regulatory agency of the country in which this disclosure is implemented (eg, Ministry of Health, Labor and Welfare or Ministry of Agriculture, Forestry and Fisheries in Japan, Food and Drug Administration (FDA), Department of Agriculture (USDA) in the United States. ) Etc.), and clearly states that it has been approved by the regulatory agency. Instructions may be provided in paper media, but are not limited to, and may also be provided in the form of, for example, electronic media (eg, homepages provided on the Internet, e-mail).

In the present specification, "components for fish cultivation" are not expected to exert the effect of the microorganisms of the present disclosure on the fish to be cultivated, but have a certain role when combined with the microorganisms of the present disclosure. Any ingredient that fulfills the above, for example, carriers (including water), salts, surfactants, carriers, desiccants, preservatives, antibacterial agents, excipients, enhancers, antioxidants, stabilizers. , Diluents, buffers, binders, dispersants, flocculants, other microorganisms and the like.

In the present specification, the “liquid for fish farming” refers to a fluid component among the components for fish farming, which may be a solution or a suspension, and the fluid component and the solid component (dissolved). It may or may not be mixed with. Typically, the fish farming fluid can be a fluid that mimics the environment in which fish live, such as rivers, oceans, lakes, or a concentrate thereof.

As used herein, the term "protective aquaculture fluid" refers to a fluid for protecting fish, including the microorganisms of the present disclosure and a liquid for fish culture. Fish are typically bred in a protected aquaculture solution or a solution diluted at any magnification (eg, about 2 to 1,000,000 times diluted).

(Preferable embodiment)
The preferred embodiments of the present disclosure will be described below. It is understood that the embodiments provided below are provided for a better understanding of the present disclosure and the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that a person skilled in the art can make appropriate modifications within the scope of the present disclosure in consideration of the description in the present specification. It is also understood that the following embodiments of the present disclosure may be used alone or in combination.

(Useful microorganisms)
In one aspect, the present disclosure provides new microorganisms capable of protecting fish and / or suppressing harmful microorganisms. In particular, the microorganisms of the present disclosure are those that have the ability to improve the survival rate of fish, have the ability to colonize fish (eg, epidermis), and / or have the ability to suppress harmful microorganisms. In one embodiment, the fish can be protected by adding the microorganisms of the present disclosure to the breeding environment (eg, breeding fluid, breeding water, etc.) of the fish. The microorganisms of the present disclosure include direct action on harmful microorganisms, formation of microbial flora in fish (eg, their epidermis), maintenance and / or improvement of the health of fish (eg, their epidermis), activation of fish immunity, etc. It is also expected to be effective. In addition, application of the microorganisms of the present disclosure at the stage of fish eggs and / or fry is expected to improve the hatchability and / or the survival rate of fry.

In one embodiment, the Pseudomonas family bacterium of the present disclosure is a specific strain, which is (i) linearly drawn on NB agar medium and cultured at 28 ° C. overnight. When the test was carried out in a method including (ii) a step of vertically drawing harmful microorganisms from the strain so as not to touch the strain and culturing at 28 ° C. overnight. ), The clear zone is present from the image line of the strain, for example, over a range of 10 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). Here, the harmful microorganisms are Aeromonas hydrophila (ATCC 700183), Aeromonas hydrophila (JCM 1027), Aeromonas caviae (JCM 1043), Flavorbacterium (JCM 1043), Flavorbacterium (JCM 1043), Flavorbacterium (JCM 1043), Flavorbacterium (JCM) Includes at least one selected from the group consisting of Yersina rukkeri (DSMZ 18506).

In one embodiment, the microorganism of the present disclosure is a bacterium of the family Pseudomonadaceae. Microorganisms of the Pseudomonadaceae family are non-fermentable, spore-producing, motile, fluorescent dyes with cytochrome C oxidase, with the Entner-Dudlov pathway by glyceraldehyde hexaphosphate dehydrogenase and aldolase. It may have at least one of the properties of producing Pyobergin. Pseudomonadaceae includes Azomonas, Azomonotarichon, Azotobacter, Azotobacter, Cellvibrio, Chryseomonas, Flavimonas, Mesophyllobacter, Permianibaster, Permianibaster, Pseudomonas, Pseudomonas, Pseudomonas. In one embodiment, the microorganism of the present disclosure is a bacterium of the genus Pseudomonas. Microorganisms of the genus Pseudomonas are bacilli, gram-negative, have one or several flagella that give motility, are aerobic, do not form spores, are positive on the catalase test, on the oxidase test. Positive, no gas formation in Hugh-Leifson test with glucose, beta-hemolytic on blood agar, negative indole test, methyl red, negative in Voges-Proskauer test, citric acid It may have at least one characteristic of being positive for the test. The genus Pseudomonas, abietaniphila, acidovorans, aestusnigri, aeruginosa, agarici, alcaligenes, alcaliphila, aminovorans, amygdali, andropogonis, anguilliseptica, antarctica, antimicrobica, argentinensis, arsenicoxydans, asplenii, asturiensis, aurantiaca, aureofaciens, avellanae, avenae, avenae subsavenae, avenae subscitrulli, avenae subskonjaci, azotifigens, azotoformans, baetica, balearica, bauzanensis, beijerinckii, benzenivorans, beteli, borbori, boreopolis, brassicacearum, brassicacearum subsbrassicacearum, brassicacearum subsneoaurantiaca, brenneri, caeni, cannabina, carboxydohydrogena, caricapapayae, caryophylli, cattleyae, cedrina, cedrina subscedrina, cedrina subsfulgida, cepacia, chengduensis, chloritidismutans, chlororaphis, chlororaphis subsaurantiaca, chlororaphis subsaureofaciens, chlororaphis subschlororaphis, chlororaphis subspiscium, cichorii, cissicola, citronellolis, cocovenenans, composti, congelans, corrugata, costantinii, cremoricolorata, cuatrocienegasensis, deceptionensis, delafieldii, delhiensis, diminuta, doudoroffii, duriflava, echinoides , Elongata, entomophila, extremaustralis, extremorientalis, facilis, ficuserectae, flava, flavescens, flectens, fluorescens, fragi, frederiksbergensis, fulva, fuscovaginae, gelidicola, geniculata, gessardii, gladioli, glathei, glumae, graminis, grimontii, guangdongensis, guariconensis, guguanensis , guineae, halophila, helmanticensis, hibiscicola, hussainii, huttiensis, indica, indigofera, iners, japonica, jessenii, jinjuensis, kilonensis, knackmussii, koreensis, kunmingensis, kuykendallii, lanceolata, lemoignei, libanensis, lini, litoralis, lundensis, lurida, lutea , luteola, mallei, maltophilia, mandelii, marginalis, marina, marincola, mediterranea, meliae, mendocina, mephitica, meridiana, mesophilica, migulae, mixta, mohnii, monteilii, moorei, moraviensis, mosselii, mucidolens, multiresinivorans, nautica, nitroreducens, oleovorans , oleovorans subslubricantis, oleovorans subsoleovorans, orientalis, oryzihabitans, otitidis, pachastrellae, palleroniana, palleronii, panacis, panipatensis, parafulva, paucimobilis, pelagia, peli, perfectomarina, pertucinogena, phenazinium, picketti i, pictorum, plantarii, plecoglossicida, poae, pohangensis, prosekii, protegens, proteolytica, pseudoalcaligenes, pseudoalcaligenes subscitrulli, pseudoalcaligenes subskonjaci, pseudoalcaligenes subspseudoalcaligenes, pseudoflava, pseudomallei, psychrophila, psychrotolerans, punonensis, putida, pyrrocinia, radiora, reinekei, resinovorans, rhizosphaerae , rhodesiae, rhodos, rubrilineans, rubrisubalbicans, sabulinigri, saccharophila, salegens, salomonii, saponiphila, savastanoi, segetis, seleniipraecipitans, simiae, solanacearum, spinosa, stanieri, straminea, stutzeri, synxantha, syringae, syringae subssavastanoi, syringae subssyringae, syzygii, taeanensis include taeniospiralis, taetrolens, taiwanensis, testosteroni, thermotolerans, thivervalensis, tolaasii, toyotomiensis, tremae, trivialis, tuomuerensis, umsongensis, vancouverensis, veronii, vesicularis, viridiflava, vranovensis, woodsii, xanthomarina, xiamenensis, xinjiangensis, species such as zeshuii .. In one embodiment, the microorganisms of the present disclosure can be the Pseudomonas putida group. The Pseudomonas putida group, Pseudomonas mosselii, Pseudomonas fulva, Pseudomonas cremoricolorata, Pseudomonas entomophila, Pseudomonas parafulva, Pseudomonas monteilii, Pseudomonas oryzihabitans, Pseudomonas plecoglossicida, capacity can include but Pseudomonas putida, bacteria included in this group were similar to each other Can be predicted to have (Anzai Y et al., Int J Syst Evol Microbiol. 2000 Jul; 50 Pt 4: 1563-89.). In one embodiment, the microorganisms of the present disclosure can be the Pseudomonas fluoressences group. The Pseudomonas fluorescens group, Pseudomonas antarctica, Pseudomonas azotoformans, Pseudomonas blatchfordae, Pseudomonas brassicacearum, Pseudomonas brenneri, Pseudomonas cedrina, Pseudomonas corrugata, Pseudomonas fluorescens, Pseudomonas gessardii, Pseudomonas libanensis, Pseudomonas mandelii, Pseudomonas marginalis, Pseudomonas mediterranea, Pseudomonas meridiana, Pseudomonas migulae , Pseudomonas mucidolens, Pseudomonas orientalis, Pseudomonas panacis, Pseudomonas protegens, Pseudomonas proteolytica, Pseudomonas rhodesiae, Pseudomonas synxantha, Pseudomonas thivervalensis, Pseudomonas tolaasii, but may include Pseudomonas veronii, bacteria included in this group have the capability similar to each other Can be predicted. In one embodiment, the microorganisms of the present disclosure can be the Pseudomonas korenesis group. The Pseudomonas koreensis group may include Pseudomonas koreensis, Pseudomonas mandelii, Pseudomonas lemonnieri (Andreani et al., Food Microbiol. 2014 May; 39: 116-26. Can be expected to have. Since the Pseudomonas kourensis group may also include part of the Pseudomonas fluorescens, it can be expected to have similar abilities to each other. In one embodiment, the microorganisms of the present disclosure are Pseudomonas mossellii, Pseudomonas marginalis, Pseudomonas koreensis, Pseudomonas paragensis, Pseudomonas protegens, Pseudomonas protegens. The present inventor has found by investigating the ability to suppress harmful microorganisms.

The new microorganism was identified as Pseudomonas Mosery and deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation, which was received on June 3, 2019, and a certificate of acceptance was issued on June 17, 2019. Was done. The receipt number is NITE ABP-02967 and the accession number is NITE BP-02967. Furthermore, the present inventor identified four more useful Pseudomonadaceae microorganisms and deposited them at the Patent Microorganisms Depositary Center of the Product Evaluation Technology Infrastructure Organization, which was received on May 26, 2020. The receipt numbers for these four microorganisms are NITE ABP-03222, NITE ABP-03223, NITE ABP-03224 and NITE ABP-03225, respectively. In one embodiment, the microorganisms of the present disclosure are Pseudomonas bacteria KH-ZF1 (accession number: NITE BP-20967), KH-RT1 (receipt number: NITE ABP-03222), KH-RT2 (receipt number: NITE ABP-). 03223), KH-RT3 (receipt number: NITE ABP-03224) or KH-RT4 (receipt number: NITE ABP-03225), or a derivative thereof.

In one embodiment, the microorganisms of the present disclosure are Pseudomonas bacteria KH-ZF1 (accession number: NITE BP-20967), KH-RT1 (receipt number: NITE ABP-03222), KH-RT2 (receipt number: NITE ABP-). 03223), KH-RT3 (receipt number: NITE ABP-03224) or KH-RT4 (receipt number: NITE ABP-03225). Here, the inducing strain does not need to be a strain obtained based on the pseudomonas bacterium KH-ZF1, KH-RT1, KH-RT2, KH-RT3 or KH-RT4, and the pseudomonas bacterium KH-ZF1, Refers to a microorganism that exhibits the biological function of KH-RT1, KH-RT2, KH-RT3 or KH-RT4, although not necessarily to the same degree. In one embodiment, the microorganisms that are the inducible strains of the present disclosure have the ability to protect fish, such as the Pseudomonas bacteria KH-ZF1, KH-RT1, KH-RT2, KH-RT3 or KH-RT4. , Has the ability to colonize the epidermis, and exhibits a biological function selected from the ability to suppress harmful microorganisms, the degree of which biological function is KH-ZF1, KH-RT1, KH-RT2, KH -May be different from RT3 or KH-RT4. In one embodiment, the inducible strain of the present disclosure is a bacterium of the genus Pseudomonas (Pseudomonas), more specifically Pseudomonas mossellii, Pseudomonas maginalis, Collensis. ), Pseudomonas protegens or Pseudomonas bacterium. In one embodiment, the microorganisms of the present disclosure, including derivatives of KH-ZF1, KH-RT1, KH-RT2, KH-RT3 or KH-RT4, have the ability to protect fish, suppress or inhibit harmful microorganisms. Ability, ability to settle in fish, and ability to function as probiotics can be confirmed by any or a modification of the tests described herein to confirm their respective abilities.

In one embodiment, the microorganisms of the present disclosure, including derivatives of KH-ZF1, KH-RT1, KH-RT2, KH-RT3 or KH-RT4, have the ability to suppress harmful microorganisms and this ability. Places an autoclave sterilized paper disc (4905010; Toyo Filter Paper) in the center of NB agar medium (eg, composition according to Example 1) or equivalent medium (eg, gellan gum) in a 9 cm diameter chalet. This paper disk is impregnated with 20 μl of a cell suspension containing the microorganisms of the present disclosure having an OD600 = 1.0 in sterile water, allowed to stand at 20 ° C. for 2 days, and then agar medium (or an equivalent medium (for example, gellan gum)). Etc.)) While applying a force that does not dent the surface of the medium, mentip (the cotton part is immersed in the culture medium of harmful microorganisms (OD ₆₀₀ = 2 to 3) at an angle of about 20 to 30 ° with respect to the medium surface) By moving the culture medium soaked in the culture medium and wiping off excess water), approximately 100 μL of the culture medium was radially drawn from the vicinity of the paper disc so as not to come into contact with the microorganisms of the present disclosure, and at 20 ° C. When cultured for 2 days, 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 can be confirmed by observing a clear zone. A person skilled in the art can visually determine the clear zone.

In one embodiment, the microorganisms of the present disclosure, including derivatives of KH-ZF1, KH-RT1, KH-RT2, KH-RT3 or KH-RT4, may have a competitive advantage over harmful microorganisms. For example, the competitive advantage over harmful microorganisms is at an angle of about 20-30 ° to the surface of the agar medium (or equivalent medium (eg, gellan gum)) while applying force that does not dent the surface of the medium. Approximately 100 μL of bacterial culture solution by moving Mentip (a cotton part immersed in the culture medium of the microorganisms of the present disclosure (OD ₆₀₀ = 2 to 3) soaked with the culture solution and wiped off excess water). After culturing overnight at 28 ° C., move the mentip in the direction perpendicular to the image so as to intersect the image of the candidate microorganism to draw harmful microorganisms (of the microorganisms of the present disclosure). The same drawing method was used except that the culture medium of the harmful microorganism was used instead of the culture medium), and when the candidate microorganism and the harmful microorganism were mixed and cultured overnight at 28 ° C., the image crossed the image of the candidate microorganism. Colonies by candidate microorganisms may occupy an area of 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more in the image area of harmful microorganisms beyond the portion. It can be confirmed by being observed. By having a competitive advantage over harmful microorganisms, the microorganisms of the present disclosure can exert a particularly high inhibitory ability against harmful microorganisms.

In one embodiment, the microorganisms of the present disclosure (including derivatives of KH-ZF1, KH-RT1, KH-RT2, KH-RT3 or KH-RT4) are attached to fish (eg, zebrafish (Dario rerio)). It has the ability to settle, and this ability is the introduction of 6 fish and a fluorescent protein with OD600 = 0.01 into 100 ml of breeding water (1 L of ultrapure water plus 3 g of instant ocean). Put the disclosed microorganisms, breed at 20 ° C., and after 6 and 24 hours, rub the skin of the fish (eg, body surface) with a mentip, immerse the mentip in 0.5 ml of sterile breeding water and stir. Fluorescent color-based colony forming units (CFU) when the prepared suspension was seeded on Defco ™ Pseudomonas Isolation Agar (Japan Beckton Dickinson, Tokyo) (plate size 9 cm) and incubated at 28 ° C. for 12 hours. Is 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more at 24 hours after 6 hours. , 80% or more, 90% or more, 100% or more, 150% or more, 200% or more, 300% or more, 400% or more, 500% or more, 600% or more, 700% or more, 800% or more, 900% or more, or It can be confirmed that the increase is 1000% or more.

In one embodiment, the microorganisms of the present disclosure, including inducers of KH-ZF1, KH-RT1, KH-RT2, KH-RT3 or KH-RT4, have the ability to improve fish viability. For this ability, 5 fish (for example, zebrafish (Dario relio)) were placed in 200 ml of breeding water (1 L of ultrapure water plus 3 g of instant ocean) and bred at 28 ° C. for 2 days. The breeding water was replaced, the microorganisms of the present disclosure were added so as to have an OD600 = 0.01, and the microorganisms were bred at 28 ° C. for 24 hours, and anesthetic solution (tricaine 400 mg, 1M Tris (pH 9) 2.1 ml, and ultrapure water 97). .. Transfer the fish to 9 ml of undiluted solution (adjusted to pH 7) and add 2 ml to 100 ml of ultrapure water to anesthetize the fish, and apply an injection needle (Termo injection needle, NN-1838R, Scratch with 18G (1.20mm), blade shape RB (blade angle 12 °), needle length 38mm), return the damaged fish to the replaced breeding water, and OD600 = 0 for the microorganisms disclosed. Add to 0.01, keep at 28 ° C for 24 hours, lower the temperature from 28 ° C to 20 ° C for 24 hours, replace the breeding water, and replace the microorganisms and harmful microorganisms of the present disclosure (eg, Yersinia). Luckeri) was added so that OD600 = 0.01, left at 20 ° C for 1 hour, the breeding water was replaced, and the microorganisms of the present disclosure were added so that OD600 = 0.01, and fish at 20 ° C. The survival rate of the treatment group was compared for 8 days between the case where the breeding was continued and the progress was observed (treatment group) and the control group different from the treatment group only when the microorganisms of the present disclosure were not used at all. Can be confirmed to be statistically significantly higher than that of the control group. As a statistical method, the method generally described in biostatistics textbooks may be used, but for example, a long-rank test is used. , If the P value is 0.05 or less or 0.01 or less, it can be said that there is a significant difference.

One of ordinary skill in the art will test inducible strains of KH-ZF1, KH-RT1, KH-RT2, KH-RT3 or KH-RT4 using the above criteria appropriately to test the above biological functions (and their biological functions). Degree) can be obtained.

In one embodiment, the microorganisms of the present disclosure, including derivatives of KH-ZF1, KH-RT1, KH-RT2, KH-RT3 or KH-RT4, are obtained by the methods described below for obtaining microorganisms. It is a microorganism.

In another aspect, the present disclosure is a strain belonging to a Pseudomonas family bacterium, which is (i) a step of linearly drawing the strain on an NB agar medium and culturing at 28 ° C. overnight. After that, when the test was carried out in a method including (ii) a step of vertically drawing harmful microorganisms from the strain so as not to touch the strain and culturing at 28 ° C. overnight. After the step, the strain is characterized by the presence of a clear zone over a range of 10 mm or more from the stroke, where the harmful microorganisms are Aeromonas hydrophila (ATCC 700183), Aeromonas hydrophila (JCM 1027), Aeromonas. A strain comprising at least one strain selected from the group consisting of caviae (JCM 1043), Flavobacterium culture (JCM 21327), Yersinia rukkeri (NVH 3758), and Yersinia rukkeri (DSMZ 18506). The conditions (capacity) that these strains should have and the conditions for the test thereof are provided in the present specification and Examples, and those skilled in the art will identify this strain by referring to publicly known information as appropriate. be able to.

(How to obtain useful microorganisms)
In one aspect, the present disclosure provides a method for obtaining microorganisms capable of protecting fish. This method includes (a) a step of obtaining candidate microorganisms from the epidermis of fish, (b) a step of adding candidate microorganisms to a medium containing harmful microorganisms, (c) a step of confirming suppression of harmful microorganisms in the medium, and a step of confirming suppression of harmful microorganisms in the medium. (D) When suppression of harmful microorganisms in the medium is confirmed, the step of acquiring this candidate microorganism as a microorganism having an ability to protect fish may be included. Since there are many skin infections in fish that have many opportunities to come into direct contact with microorganisms in water, targeting microorganisms in the epidermis of fish is useful for efficient acquisition of microorganisms that have the ability to protect fish. obtain. By using microorganisms originally present in the epidermis of fish, it may be possible to protect fish safely, easily and / or with low environmental impact. The epidermis of the fish from which the candidate microorganism is obtained may be any surface of the body, eyes, scallops, fins, gills, mouth, anus, and genitals, and in one embodiment, the surface of the body.

In one embodiment, the confirming step (c) is the ability of the disclosed microorganisms (including the KH-ZF1 inducible strain) to protect fish, suppress or inhibit harmful microorganisms, and colonize fish. , And by confirming by either the tests described herein or modifications thereof to confirm their ability to function as probiotics. In one embodiment, the confirming step (c) can be performed by confirming the suppression of harmful microorganisms on the medium. In one embodiment, the confirmation step (c) is carried out by growing the candidate microorganism and the harmful microorganism on the same medium and confirming a growth inhibition zone in the vicinity of the candidate microorganism in which the harmful microorganism cannot grow. obtain. In one embodiment, the confirmation step (c) can be performed by confirming the suppression of harmful microorganisms in a liquid medium containing the candidate microorganisms or their culture supernatant and harmful microorganisms. In one embodiment, the confirming step (c) involves impregnating a culture medium or equivalent medium (eg, gellan gum) inoculated with harmful microorganisms with a culture medium or culture supernatant of a candidate microorganism. This can be done by placing a bottomless cylinder containing the material or culture medium or culture supernatant and confirming the suppression of harmful microorganisms around the impregnated material or the cylinder. In one embodiment, the confirming step (c) is to incubate a liquid medium containing the candidate and harmful microorganisms and then compare the growth of the candidate and harmful microorganisms to further suppress the growth of the harmful microorganisms. It can be carried out by confirming that. In one embodiment, the confirmation step of (c) is an agar medium in which harmful microorganisms are inoculated entirely or on an equivalent medium (for example, gellan gum), or an agar medium in which harmful microorganisms are totally grown. Or, inoculate the candidate microorganisms on a medium equivalent to that (for example, gellan gum), and the candidate microorganisms form colonies, and a clear zone due to growth inhibition circle or death of harmful microorganisms is observed around the colonies. It can be carried out by confirming. Different types of microorganisms cultured in the same liquid medium can be distinguished by means such as colony shape, color, use of selective medium and the like. For example, a method of distinguishing and examining population is a method of counting each colony by plate culture when the color or shape of the colonies is different, and a method of directly examining each microorganism under a microscope when different morphologies are observed under a microscope. There are a counting method, a method of designing a gene primer capable of distinguishing each microorganism and performing real-time PCR, a fluorescence in situ hybridization (FISH) method, and the like. Examples of the method for confirming the candidate microorganism under a microscope include a known FISH method and a method using fluorescence of a fluorescent protein previously introduced into the candidate microorganism.

In one embodiment, the candidate microorganism can be obtained from a colony formed on a medium inoculated with a scraped material (eg, scraped with a cotton swab) of a fish epidermis (eg, body surface). One or more microorganisms can be obtained as microorganisms capable of protecting fish. In one embodiment, microorganisms capable of protecting fish from the epidermis of zebrafish (Dario rerio) can be obtained. In one embodiment, microorganisms capable of protecting fish from the epidermis of salmonid fish (eg, trout (eg, rainbow trout mykiss)) can be obtained. In one embodiment, scale-type fish Microorganisms capable of protecting fish from the epidermis can be obtained.

In one embodiment, at least the ability to cause skin disease, transcutaneous infection, wound infection, and contact infection to fish (eg, farmed fish) as harmful microorganisms to confirm suppression. Microorganisms presumed to have one can be selected. For example, microorganisms capable of causing skin diseases to fish (eg, farmed fish), transcutaneously infecting, wound-infecting, and / or contact-transmitting are "fish diseases" (supervised by Kodama). See Yo, Midori Shobo (Tokyo), 2012), and "Fish Infectious Diseases / Parasite Diseases" (Wakabayashi, edited by Muroga, Kazuo Ogawa, Seiseisha Koseikaku (Tokyo), 2004). Can be appropriately selected by those skilled in the art.

In one embodiment, the harmful microorganisms include vibrio disease, furunculosis, atypical Eromonas salmonicida infection, Eromonas hydrophila infection, edwardsiellosis, red spot disease, Ayu's pseudomonas disease, Redmouth disease, bacterial Microorganisms capable of causing gill disease, edwardsiellosis (selected, tailed, torn, squeezed), cold water disease, gliding bacillosis, bacterial kidney disease, mycobacteriosis, nocardiosis, and / or streptococculosis Can be selected. For each fish disease, the typical causative microorganisms are as follows: Vibrio disease (Listonella anguillara (formerly Vibrio anglilarum), Vibrio ordalii, Vibrio ichthyoenteri, Vibrio bacterium Aeromonas, Vibrio bacterium, Vibrio bacterium, Vibrio bacterium). Aeromonas salmonicida; Aeromonas hydrophila; Edwardiella tarda; Pseudomosis red spot disease (Pseudomonas) red spot disease (Pseudomonas) Disease (Yersina rukkeri); Bacterial gill disease (Flavovacterium brunchiophilum); Columnnaris disease (selected, tailed, squeezed) (Flavobacterium columnare); Cold water disease (Flavobacterium) bacteriolysis (Flavobacterium) Disease (Renibacterium salmoninarum); Mycobacterium marinum, Mycobacterium fortuitum, Mycobacterium cellonei; Nocardia seriolace; Streptococcus bacillus. Yersinia ruckeri is the causative agent of redmouth disease known to cause pandemics in salmon and trout. In one embodiment, the harmful microorganisms include Aeromonas hydrophila, Aeromonas caviae, Yersinia ruckeri, Edwardsiella tarda, Flavobacterium colora, Flavobacterium colora, Vibrio varia, and Vibrio varia.

(Composition containing microorganisms)
In one aspect, the present disclosure provides a composition comprising the microorganisms of the present disclosure. The microorganisms of the present disclosure can be produced by culturing by any suitable method. In one embodiment, the composition is a fish protectant. In one embodiment, the composition is a probiotic agent for fish (eg, its epidermis). The use of the fish protectants or probiotics of the present disclosure may improve at least one of fish survival, health, meat quality, injury recovery and infectious conditions (including susceptibility to infection).

(Applicable target)
In one embodiment, fish to which the microorganisms or compositions of the present disclosure are applied include, but are not limited to, farmed fish, ornamental fish, and the like. Cultured fish include, for example, eel, ayu, yellowtail, trout (eg, rainbow trout (Oncorhynchus mykiss)), Thailand, carp, amberjack, tuna, salmon, horse mackerel, flatfish, tilapia, pufferfish, hamachi, hata, mackerel, saury, etc. Examples include, but are not limited to, catfish. The microorganisms of the present disclosure can be obtained from the epidermis of fish and / or can be applied to the epidermis of fish, but the epidermis of fish has scale type and mucus type, for example, eel and catfish as mucus type fish. Can be mentioned. In one embodiment, the fish to which the microorganism or composition of the present disclosure is applied can be a salmonid fish (eg, trout). Salmonidae is an example of scale-type fish. The microorganisms or compositions of the present disclosure can be used at any stage of growth of fish, such as eggs, hatched fish, fry, adult fish, sexually mature fish and the like.

(Usage pattern)
Examples of the form of the microorganism or composition of the present disclosure include a liquid state, a solid state and the like. Examples of the liquid-state microorganism or composition include a culture solution of a microorganism, a culture solution in which microorganisms are collected by centrifugation or the like, and then redispersed in water, a buffer solution, or a culture solution. Examples of the solid state microorganism or composition include those dehydrated by centrifugation, press compression, etc., those in a paste state / mayonnaise state such as between solid and liquid, and dried products that have been dried (for example, vacuum-dried, freeze-dried). Etc. are exemplified. Examples of the solid form include powders, granules, tablets and the like. In addition, the composition may be provided in a state where the microorganism or the culture supernatant is fixed on the carrier.

In one embodiment, the microorganisms or compositions of the present disclosure are about 1 × 10 ⁸ cells / mL, about 1 × 10 ⁷ cells / mL, about 1 × 10 ⁶ cells / mL, about 1 × 10 ⁵ cells / mL. , Approximately 1 x 10 ⁴ cells / mL, approximately 1 x 10 ³ cells / mL, approximately 1 x 10 ² cells / mL or approximately 10 cells / mL, added to breeding water and used in fish obtain.

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

In one embodiment, the microorganisms or compositions of the present disclosure are pH 3-13, pH 4-12, pH 5-11, pH 6-10, pH 7-9, pH 5.5-8.5, about pH 3, about pH 4, about. It can be used in any pH environment such as 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.

In one embodiment, the microorganism or composition of the present disclosure is 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. Can be used in an environment of any dissolved oxygen concentration (DO) such as / L, about 3 mg / L, about 5 mg / L, about 8 mg / L or about 10 mg / L, in one embodiment about 8 mg near saturation. It can be used in environments with dissolved oxygen concentrations (DO) up to / L or suitable for fish breeding.

In one embodiment, the microorganisms or compositions of the present disclosure are 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. Arbitrary such as / 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. Can be used in an environment of salt (eg, sodium chloride) concentration.

In one embodiment, the microorganisms or compositions of the present disclosure may be used in the presence of salts, surfactants, photoelectrons, electric currents, agitation operations, aeration operations, or any combination thereof.

In one embodiment, the microorganism or composition of the present disclosure may be used with a carrier capable of immobilizing the microorganism. Washout can be effectively avoided by using such a carrier. The material of the carrier is not particularly limited as long as it can fix microorganisms, for example, carbon fiber (PAN-based, pitch-based, phenol 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 charcoal, minerals (zeolite, diatomaceous earth, etc.), glass, glass waste, Examples thereof include a complex of these. It is preferable to use a porous or fibrous carrier in order to increase the immobilization rate of microorganisms and the efficiency of action of microorganisms. Further, the gel-like carrier may contain microorganisms. Examples of the shape of the carrier include a cube, a rectangular parallelepiped, a columnar shape, a spherical shape, a disk shape, a sheet shape, and a film shape. Regarding microbial immobilization technology, for example, "Wastewater treatment by microbial immobilization method (edited by Ryuichi Sudo, Industrial Water Research Association)" and "Water treatment by microbial immobilization method-carrier immobilization method, comprehensive immobilization method, bioactive charcoal method" (New water treatment series (1)) (Kazuhiro Mochizuki, Katsutoshi Hori, Hideki Tatemoto (Author), NTS Co., Ltd.) ”.

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 carriers to cause the microorganisms themselves to form flocs or granules. In addition, core materials and microorganisms that promote the formation of flocs and granules may be used.

(Additional ingredient)
In one embodiment, the microorganisms or compositions of the present disclosure may be used in combination with additional ingredients. In one embodiment, the additional ingredients may be added to the composition, may be used separately from the microorganism or composition, and when used separately, are provided as a kit. May be good.

In one embodiment, as additional ingredients, ingredients that enhance the activity of the microorganisms used, surfactants, drying protective agents, ingredients for long-term maintenance of microorganisms, preservatives, excipients, fortifiers, antioxidants. Agents, dispersants, flocculants, and other microorganisms can be used, but are not limited thereto, and any suitable component can be used.

In another embodiment, the additional component may include any component that can be used when contacting or imparting to the fish, such as a fish farming solution, a fish farming component, and the like. When such an additional component is contained, it can be used as it is when used for fish farming, and the fish can be bred while being protected. The fish farming liquid, the fish farming component, and the like may be provided in the composition, may be used separately from the microorganism or the composition, or may be provided as a kit.

(Method using microorganisms)
In one aspect, the disclosure provides a method for protecting a fish, comprising contacting the microorganism or composition of the present disclosure with the fish. In one embodiment, the method for protecting fish comprises the step of raising fish in water in the presence of the microorganisms of the present disclosure. The fish to be protected can be any of the fish described herein to which the microorganisms or compositions of the present disclosure can be applied. In one embodiment, the microorganism or composition of the present disclosure may be applied to the same type of fish as the fish obtained from the epidermis of this microorganism, or may be applied to different types of fish. In one embodiment, the microorganism or composition of the present disclosure may be applied to fish raised in the same environment in which the fish from which the microorganism was obtained from the epidermis was bred (eg, a farm). , May be applied to fish raised in different environments. The methods for protecting the fish of the present disclosure can be carried out in any of the environments described herein to which the microorganisms or compositions of the present disclosure can be applied. The methods for protecting the fish of the present disclosure can use any of the additional ingredients described herein that can be used in combination with the microorganisms or compositions of the present disclosure.

In one embodiment, the methods of the present disclosure include treating or preventing a fish disease or disease. Alternatively, the methods of the present disclosure include protecting the epidermis of fish. Fish diseases or diseases that may be of interest may include, for example, skin diseases, infectious diseases, vibrio disease, asthma, atypical Eromonas salmonicida infection, Eromonas hydrophila infection, Edwazierosis, red spot disease. , Ayu's Pseudomonas disease, Redmouth disease, Bacterial gill disease, Columnnaris disease (selected, tailed, torn, squeezed), cold water disease, gliding bacillosis, bacterial kidney disease, mycobacteriosis, nocardiosis, And / or streptococcal disease can be the subject.

In certain embodiments, the microorganism or composition of the present disclosure comprises an effective amount of the disclosed Pseudomonadaceae bacteria or is provided to be in an effective amount upon contact. In one embodiment, the microorganisms or compositions of the present disclosure are provided in water in the presence of an effective amount of the disclosed Pseudomonadaceae bacteria, in which fish may be grown.

By providing an effective amount of the microorganism or composition of the present disclosure in this way, the fish is protected from at least one harmful microorganism. The harmful bacteria that can be protected by the methods of the present disclosure can be any of those described herein, eg, the ability to cause skin disease in fish, the ability to percutaneously infect fish, the ability to infect wounds. , And can be a bacterium having at least one of the abilities to contact, and specific examples include vibrio disease, asthma, atypical Eromonas salmonicida infection, Eromonas hydrophila infection, edwardsiellosis. Disease, Sekiten-byo, Ayu's Pseudomonas disease, Redmouth disease, Bacterial gill disease, Streptococculosis (selected, tailed, torn, squeezed), cold water disease, gliding bacillosis, bacterial kidney disease, mycobacteria Harmful microorganisms capable of causing disease, nocardiellosis, and / or streptococculosis are exemplified. Such harmful microorganisms, Listonella anguillara (Vibrio anguillarum), Vibrio ordalii, Vibrio ichthyoenteri, Vibrio vulnificus, Vibrio salmonicida, Aeromonas salmonicida, atypical Aeromonas salmonicida, Aeromonas hydrophila, Edwardsiella tarda, 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, may be Aeromonas caviae or combinations thereof.

Therefore, methods for protecting fish, including contacting the microorganisms or compositions of the present disclosure with fish or raising fish in water in the presence of the microorganisms of the present disclosure, are methods of contacting the microorganisms or compositions with fish. It may include a step of inspecting what kind of harmful microorganism the fish is affected by, before or during or after breeding the fish. The test for the morbidity of such harmful microorganisms can be carried out by a method known in the art, and a sample may be collected directly from fish or a sample may be collected from a breeding solution being bred.

In a preferred embodiment, a preliminary test is performed to determine what kind of harmful microorganism is affected, the harmful microorganism is confirmed, and then the optimum conditions for the microorganism or composition of the present disclosure to be administered are determined. Experiments may be performed. Such preliminary experiments include, for example, confirming the effectiveness against existing harmful microorganisms, confirming the effective concentration, and the like. It may also include confirming in advance the safety of the microorganisms or compositions of the present disclosure to be administered to the fish to be protected. Such confirmation of safety may include examining permissible concentrations and other administration conditions, and any method known in the art can be used. When conditions such as such an allowable concentration are found, actual contact and breeding may be carried out in consideration of conditions such as appropriate administration conditions and appropriate effective concentrations.

In another embodiment, in a method for protecting a fish, the epidermis of the fish comprises contacting the microorganism or composition of the present disclosure with the fish or raising the fish in water in the presence of the microorganism of the present disclosure. The Pseudomonadaceae bacteria of the present disclosure are provided under conditions sufficient to colonize. Sufficient conditions for colonization of the epidermis of fish may be investigated in advance, in which case contact or breeding is carried out using the conditions. Alternatively, preliminary experiments may be conducted to determine sufficient conditions for colonization of the epidermis of fish during contact or breeding, and any technique known in the art can be used.

(General technology)
The molecular biological, biochemical, and microbiological techniques used herein are well known and commonly used in the art, and are, 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 their entirety.

(Note)
As used herein, "or" is used when "at least one" of the matters listed in the text can be adopted. The same applies to "or". When "within a range" of "two values" is specified in the present specification, the range also includes the two values themselves.

References such as scientific literature, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety to the same extent as they are specifically described. The present disclosure has been described above with reference to preferred embodiments for ease of understanding.

Hereinafter, the present disclosure will be described based on examples, but the above description and the following examples are provided for purposes of illustration only, and are not provided for the purpose of limiting the present invention. Therefore, the scope of the present invention is not limited to the embodiments and examples specifically described in the present specification, but is limited only by the claims.

Examples are described below. If necessary, the handling of organisms used in the following examples complied with the standards stipulated by Nagoya University, regulatory agencies and the Cartagena Act, if necessary. Specifically, the reagents described in the examples were used, but other manufacturers (Sigma-Aldrich, Fujifilm, Wako Pure Chemical Industries, Nakarai, R & D Systems, USCN Life Science INC, Kanto Chemical, Funakoshi, Tokyo Kasei) , Merck, etc.) can be substituted.

(Example 1: Isolation of microorganisms from zebrafish epidermis)
Adult zebrafish (Dario rerio) were ordered from Mie University and Masuko Waterscape and managed in our laboratory. The fish were managed in a different breeding water tank from the source until the experiment was conducted. The water tank used was CROSS MINI (NWC-341; NISSO). Zebrafish were given tetramine super (17653; Spectrum Brands Japan) twice daily every 12 hours using the Tetra Auto Feeder (AF-3; Spectrum Brands Japan). A Tetra LED Mini Light (73333; Spectrum Brands Japan) was used for lighting, and a digital timer (PT70DW; REVEX) was used to turn on and off the lights every 12 hours. The water temperature was maintained at 28 ° C. using Safecover Heat Navi SH80 (7775; Gex).

Zebrafish was made to swim in an aqueous solution containing 2 ml of trikine solution (4.0 mg / ml trikine, 0.021 M Tris) per 100 ml of ultrapure water to anesthetize, and then mentip (1p1504; Japanese cotton swab, Tokyo) on the body surface. The mucous membrane was scraped off. This mentip was dipped in 1 ml of ultrapure water and vortexed to obtain a suspension. 200 μl each of this suspension and a diluted solution obtained by diluting this suspension with ultrapure water 5 times with Nutrient Broth agar (NB medium), Enhanced Cytophaga agar, Zobel 2216E agar, Tryptone Soya Agar. It was dropped onto (TSA medium) and sprayed. Then, the sprayed agar medium was cultured at 28 ° C. for 2 days (Fig. 1). Each of the 124 colonies obtained was subjected to the Cross Streak method.
Composition of various media

(Example 2: Screening of useful bacteria by the Cross Streak method)
As pathogenic bacteria, Aeromonas hydrophila (1) (ATCC 700183), Aeromonas hydrophila (2) (JCM 1027), Aeromonas caviae (JCM 1043), Flavobacterium Yersinia (JCM 1043), Flavobacterium Yersinia (Yersinia ruck) (2) (DSMZ 18506) was used. Escherichia coli DH5α was used as a representative of non-pathogenic bacteria. These bacteria were pre-cultured overnight and used for screening.

The Cross Streak method is 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". EMTaK, editor. Budapest: Eoetvoes Lorand University.) When evaluating growth inhibition against harmful microorganisms, candidate microorganisms obtained from each colony of Example 1 were linearly placed on an NB agar medium (composition described in Example 1) in a plate having a diameter of 9 cm with a mentip. The image was drawn and cultured at 28 ° C. overnight, and then the harmful microorganisms were imaged vertically from the image of the candidate microorganism with a mental tip so as not to touch the candidate microorganism, and cultured at 28 ° C. overnight. When a clear zone exists over a range of 10 mm or more from the image line of the candidate microorganism, it was judged to have a growth inhibitory ability (Fig. 2).
The growth inhibitory ability of various candidate microorganisms is shown below.

KH-ZF1 had a particularly excellent ability to suppress harmful microorganisms (Fig. 3).

(Example 3: Purification of isolated useful bacteria)
The isolated KH-ZF1 was imaged on NB agar medium (composition described in Example 1) and cultured at 28 ° C. for 2 days. Then, 5 colonies were selected and liquid-cultured in NB medium at 28 ° C. overnight, and the above-mentioned Cross Streak method was performed using the culture medium. One of the colonies that showed growth inhibition by the Cross Streak method was selected, streaked on an agar medium, and cultured at 28 ° C. for 2 days. This operation was repeated 5 times or more.

(Example 4: Ability to inhibit harmful microorganisms of useful microorganisms)
Aeromonas hydrophila (1) (ATCC 700183), Aeromonas hydrophila (2) (JCM 1027), Aeromonas caviae (JCM 1043), Yersinia ruckeri (1) (NVeri) (1) (NVH) 1) (NRIA44), Edwardsiella tarda (2) (NRIA51), Vibrio anguillarum (NRIA83), Vibrio ordalii (NRIA90), Streptococcus iniae (NRIA599) used as a harmful microorganism for the test. In addition, two types of non-toxic microorganisms exemplified as negative controls were similarly tested on the same plate. For these, cells cultured overnight were used.

An autoclave-sterilized paper disc (4905010; Toyo Filter Paper, Tokyo) was placed in the center of NB agar medium in a petri dish with a diameter of 9 cm, and a cell suspension of KH-ZF1 (KH-ZF1 was described in Example 1). After culturing in the same composition), the cells were centrifuged at 5000 × g for 10 minutes, and the precipitated cells were collected and resuspended in sterilized water so that the OD600 became 1.0). It was soaked in a paper disc and allowed to stand at 20 ° C. for 2 days. Then, the above-mentioned test microorganism was mentip-lined radially from the addition site of KH-ZF1 so as not to touch KH-ZF1, and cultured at 20 ° C. for 2 days. Specifically, this drawing was performed as follows. Immerse the cotton part of Mentip in 2 mL of the above-mentioned culture solution of harmful microorganisms (OD ₆₀₀ = 2-3), soak the culture solution in it, wipe off excess water, and apply enough force to prevent the surface of the agar medium from denting. In addition, by moving the mentip at an angle of about 20 to 30 ° with respect to the surface of the medium, about 100 μL of the bacterial culture solution is radially drawn from the vicinity of the paper disk on the dish so as not to touch the grown KH-ZF1. did.
The results are shown in FIG. Selective suppression was observed in harmful microorganisms compared to non-toxic microorganisms.

(Example 5: Identification of useful bacteria)
Species identification of KH-ZF1 by 16S ribosomal RNA (rRNA) was requested to Technosuruga Lab (Shizuoka Prefecture) and carried out as follows.
DNA was extracted from KH-ZF1 cultured using achromopeptidase (Fujifilm Wako Pure Chemical Industries, Osaka). PCR amplification was performed with Tks Gflex DNA Polymerase (Takara Bio, Shiga). A BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) was used for the cycle sequence. Primers of 9F and 1510R were used for PCR amplification, and primers of 9F, 515F, 1099F, 536R, 1242R and 1510R were used for sequencing. The sequence was performed using the ABI PRISM 3130 x1 Genetic Analyzer System (Applied Biosystems, USA). Nucleotide sequence was determined using ChromasPro 2.1 (Technellysium, Australia).

For BLAST homology search, analysis software ENKI (Technosuruga Lab, Shizuoka Prefecture), database DB-BA14.1 (Technosuruga Lab, Shizuoka Prefecture) and international base sequence database (DDBJ / ENA (EMBL) / GenBank) was used. In the molecular phylogenetic analysis, the phylogenetic tree was estimated by the neighbor-joining method, the base substitution model of Kimur-2-parameter, and the reliability of the tree shape was evaluated by the bootstrap method (1000 iterations).

As a result, KH-ZF1 showed 99.8% homology to Pseudomonas mosselii (CIP105259) in the BLAST search result for DB-BA, and Pseudomonas mosselii (PtA1) in the BLAST search result for the international nucleotide sequence database. It showed 99.9% homology and was identified as Pseudomonas mossellii.
The molecular phylogenetic tree based on the 16S rRNA partial base sequence of KH-ZF1 is shown in FIG.

(Example 6: Fish epidermis colonization test)
100 ml of breeding water (1 L of ultrapure water plus 3 g of instant ocean) was placed in an Erlenmeyer flask, and 6 zebrafish (Danio rerio) were placed in each flask. KH-ZF1 in which mCherry was introduced was added to this breeding water so that OD600 = 0.01, and zebrafish were bred at 20 ° C. Six hours and 24 hours after the addition of KH-ZF1, the body surface (both sides) of the zebrafish was rubbed with a mentip, and the mentip was immersed in 0.5 ml of sterile breeding water, stirred and suspended. This suspension was seeded on Difco ™ Pseudomonas Isolation Agar (Becton Dickinson, Tokyo, Tokyo) (plate size 9 cm) and incubated at 28 ° C. for 12 hours. The colony forming unit (CFU) of KH-ZF1 was measured based on the fluorescence color development.

KH-ZF1 into which a fluorescent protein (mCherry) was introduced was prepared as follows.
E. to hold the transposon vector pBSL :: mCherry km ^r Use coli WM6026, gene cultured in the introduced onto Pseudomonas isolation agar medium supplemented with kanamycin to KH-ZF1 by conjugal transfer, mCherry and miles ^r gene has acquired KH-ZF1 that has been introduced into the genome.

The results are shown in Fig. 6. It was observed that KH-ZF1 increased the colonization of zebrafish on the body surface over time.

(Example 7: Infection control test)
The infection control ability of KH-ZF1 was examined. The test procedure is shown below.
200 ml of breeding water (1 L of ultrapure water plus 3 g of instant ocean) was placed in a 500 ml triangular flask, and 5 zebrafish (Danio rerio) were placed in each flask. Zebrafish were bred at 28 ° C for 2 days. The breeding water in the Erlenmeyer flask was replaced, KH-ZF1 was added so that OD600 = 0.01, and the zebrafish was bred at 28 ° C. for 24 hours. Zebrafish was added from an Erlenmeyer flask to 100 ml of ultrapure water (400 mg of trikine, 2.1 ml of 1M Tris (pH 9), and 2 ml of undiluted solution of 97.9 ml of ultrapure water (adjusted to pH 7)). And anesthetized. Scratch the muscle at the base of the dorsal fin of zebrafish with an injection needle (Terumo injection needle, NN-1838R, 18G (1.20 mm), blade shape RB (blade angle 12 °), needle length 38 mm) I attached it. The breeding water in the Erlenmeyer flask was replaced, the damaged zebrafish was added, KH-ZF1 was added so that OD600 = 0.01, and the zebrafish was bred at 28 ° C. for 24 hours. The temperature was lowered from 28 ° C to 20 ° C and the zebrafish were bred for 24 hours. The breeding water in the Erlenmeyer flask was replaced, KH-ZF1 and Yersinia ruckeri (NVH3758) were added so that OD600 = 0.01, respectively, and the zebrafish was allowed to swim at 20 ° C. for 1 hour. The breeding water in the Erlenmeyer flask was replaced, KH-ZF1 was added so that OD600 = 0.01, the zebrafish was continued to be bred at 20 ° C, and the life and death situation was followed up (then the breeding water was replaced. Not done) (Fig. 7). Air was sent to the Erlenmeyer flask with an air pump and covered with a wrap to prevent evaporation of breeding water.
In addition to the above-mentioned treatment group to which KH-ZF1 was administered, the test was also conducted in the control group in which KH-ZF1 was not added at all in the above procedure.

The KH-ZF1 and Yersinia ruckeri (NVH3758) used in the test were prepared as follows.
-Preparation of KH-ZF1 KH-ZF1 cultured on a plate was inoculated into a 15 ml tube containing 2 ml of NB medium, and cultured with shaking at 28 ° C. for 24 hours. 100 μl of this culture solution was inoculated into a 100 ml Erlenmeyer flask containing 10 ml of NB medium, and cultured with shaking at 28 ° C. for 24 hours. Centrifugation was performed at 8000 × g for 5 minutes, and the supernatant was discarded to collect bacteria. The cells were resuspended in breeding water, washed, centrifuged at 8000 × g for 5 minutes, and the supernatant was discarded to collect bacteria (this operation was repeated 3 times). OD600 was measured by resuspending in breeding water.
-Preparation of Yersinia ruckeri (NVH3758) Yersinia ruckeri cultured on a plate was inoculated into a 15 ml tube containing 2 ml of LB medium, and cultured with shaking at 28 ° C. for 24 hours. 100 μl of this culture solution was inoculated into a 100 ml Erlenmeyer flask containing 10 ml of LB medium, and cultured with shaking at 28 ° C. for 12 hours. Centrifugation was performed at 8000 × g for 5 minutes, and the supernatant was discarded to collect bacteria. The cells were resuspended in breeding water, washed, centrifuged at 8000 × g for 5 minutes, and the supernatant was discarded to collect bacteria (this operation was repeated 3 times). OD600 was measured by resuspending in breeding water.

The results are shown in Fig. 8. When the log rank test was performed, P = 0.004 between the treatment group and the control group, and there was a significant difference in survival rate.

(Example 8: Useful microorganisms obtained from rainbow trout epidermis)
The mucous membranes on the body surface of adult rainbow trout (Oncorhynchus mykiss) cultivated at Uren fish farm (Aichi prefecture) and Ishii farm (Gifu prefecture) were scraped off. Colonies were formed on the agar medium in the same manner as in Example 1.

The growth inhibitory activity and competitive advantage of the strains obtained from these colonies were evaluated.

The growth inhibitory activity was evaluated by the same method as in Example 2 and FIG. Yersinia ruckeri (NVH 3758), Aeromonas hydrophila (NRIA14), Vibrio angularum (NRIA83) and Vibrio ordalii (NRIA90) were used as pathogenic bacteria. The method of drawing the image line was the same as in Example 4.

The competitive advantage was evaluated as follows (see also Fig. 9). Yersinia ruckeri, Aeromonas hydrophila, Vibrio anglilarum and Vibrio ordalii were used as pathogenic bacteria (harmful microorganisms) (the same strain as the growth inhibitory activity test), and these bacteria were pre-cultured overnight and used for screening. did. Candidate microorganisms obtained from each colony are linearly drawn on NB agar medium (composition described in Example 1) in a plate having a diameter of 9 cm with a mentip, cultured overnight at 28 ° C., and then candidate microorganisms. The mentip was moved in a direction perpendicular to this image so as to intersect the image of No. 1 to draw harmful microorganisms, and the candidate microorganism and the harmful microorganism were mixed and cultured at 28 ° C. overnight. It was determined that there is a competitive advantage when the candidate microorganism grows preferentially in the portion after crossing the image of the candidate microorganism on the image by the mentip carrying the harmful microorganism. The method of drawing the image line was the same as in Example 4.

As a result, KH-RT1, KH-RT2, KH-RT3 and KH-RT4 had excellent inhibitory ability against harmful microorganisms as shown in the table below.

Next, DNA was extracted from each of the cultured KH-RT1, KH-RT2, KH-RT3 and KH-RT4. Based on these DNAs, a sequence comparison with the V1-V3 region of the 16S ribosomal RNA gene revealed that KH-RT1 was identified as Pseudomonas marginalis, KH-RT2 was identified as Pseudomonas koreensis, and KH-RT3 was identified as Pseudomonas protegens. KH-RT4 is a microorganism of the genus Pseudomonas, and is particularly considered to be Pseudomonas parafulva. Similarly, more useful strains can be obtained.

(Example 9)
The protective effect of useful bacteria on larvae was tested.
KH-ZF1 cultured at 28 ° C. was added to the breeding water so that OD600 = 0.01, and larvae (60 larvae) immediately after hatching of rainbow trout were bred in the breeding water at 15 ° C. with ventilation. Immediately after hatching of 74 rainbow trout larvae were bred under control conditions to which KH-ZF1 was not added. On the 3rd day (after counting the number of survivors), some larvae were collected twice between the 5th and 18th days and observed under a microscope. On the 18th day, some larvae were collected for microscopic observation and exposure test, and breeding water was collected to measure CFU. The breeding water was not changed. Survival rates were confirmed on the 0th, 3rd, 5th, 18th, 26th, 28th, and 31st days (larvae collected for observation were not returned and excluded from the count). ..

The results are shown in FIGS. 10 and 11. On the 3rd day of exposure, KH-ZF1 was observed to be colonized on the body surface of the larvae (Fig. 10). The addition of KH-ZF1 improved the survival rate of the larvae, and in particular, a high improvement in the survival rate was observed after the 26th day. It was confirmed that KH-ZF1 also has a high protective effect on larvae of fish. Similar protective effects can be observed in KH-RT1, KH-RT2, KH-RT3, KH-RT4 or other strains.

Other Embodiment (Example 10)
Harmful microorganisms were administered to both the treatment group to which KH-RT1, KH-RT2, KH-RT3, KH-RT4 or other strains were administered and the control group not to be administered, and the survival rate of rainbow trout in the treatment group. Is higher than the survival rate of the control group.

(Example 11)
Similar to KH-ZF1, KH-RT1, KH-RT2, KH-RT3, KH-RT4 or other strains into which the fluorescent dye gene has been introduced are prepared. These are administered to rainbow trout, the rainbow trout are bred for a while, the microorganisms are regularly scraped off from the epidermis of the rainbow trout with a mentip, and the CFU of the strain based on fluorescence coloring is measured by the same method as in Example 6, and fish Confirm the fixation on the epidermis.

(Example 12)
In a breeding water (treatment group) containing rainbow trout, KH-RT1, KH-RT2, KH-RT3, KH-RT4 or other strains and harmful microorganisms (eg, Yersinia ruckeri), as in Example 7. Raise the rainbow trout that was injured in. It is confirmed that the survival rate is improved as compared with the control group in which KH-RT1, KH-RT2, KH-RT3 or KH-RT4 was not added to the breeding water.

(Note)
As described above, the present disclosure has been illustrated using the preferred embodiments of the present disclosure, but it is understood that the scope of the present invention should be interpreted only by the scope of claims. The patents, patent applications and other documents cited herein should be incorporated herein by reference in their content as they are specifically described herein. Is understood.

(Note)
This application claims the benefit of priority to Japanese Patent Application No. 2019-106436 filed on June 6, 2019, the entire contents of which are incorporated herein by reference.

The present disclosure provides microorganisms that protect fish and methods of protecting fish, thereby achieving more stable, cheaper, higher production and / or diverse fish growth and / or existing drug use, etc. The environmental load can be reduced by reducing the amount of water.

KH-ZF1 (accession number: NITE BP-20967)
KH-RT1 (receipt number: NITE ABP-03222)
KH-RT2 (receipt number: NITE ABP-03223)
KH-RT3 (receipt number: NITE ABP-03224)
KH-RT4 (receipt number: NITE ABP-03225)

Claims

Pseudomonadaceae bacteria that have the ability to protect fish.
The Pseudomonadaceae bacterium according to claim 1, which has the ability to function as a probiotic of fish.
The Pseudomonadaceae bacterium according to claim 1 or 2, which has the ability to function as a probiotic of the epidermis of fish.
The Pseudomonadaceae bacterium according to any one of claims 1 to 3, which has the ability to settle on the epidermis of fish.
The Pseudomonadaceae bacterium according to any one of claims 1 to 4, which has an ability to improve the survival rate of the fish.
The Pseudomonadaceae bacterium according to any one of claims 1 to 5, wherein the Pseudomonadaceae bacterium is protected by adding the Pseudomonadaceae bacterium to the fish breeding environment breeding solution.
The Pseudomonadaceae bacterium according to any one of claims 1 to 6, which has an ability to suppress at least one harmful microorganism.
The Pseudomonadaceae according to claim 7, wherein the harmful microorganism has at least one of the ability to cause skin diseases in fish, the ability to infect fish transdermally, the ability to infect wounds, and the ability to infect contact. Family bacteria.
The harmful microorganisms include vibrio disease, asthma disease, atypical Eromonas salmonicida infection, Eromonas hydrophila infection, edwardsiellosis, red spot disease, Ayu's pseudomonas disease, red mouse disease, bacterial gill disease, and streptococculosis. (Selected, tailed, torn, torn), cold water disease, gliding bacillosis, bacterial kidney disease, mycobacteriosis, nocardiosis, and / or capable of causing streptococculosis, claim 7 or 8 Pseudomonas family bacteria described in.
The harmful microorganisms, Listonella anguillara (Vibrio anguillarum), Vibrio ordalii, Vibrio ichthyoenteri, Vibrio vulnificus, Vibrio salmonicida, Aeromonas salmonicida, atypical Aeromonas salmonicida, Aeromonas hydrophila, Edwardsiella tarda, 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, Pseudomonas family bacterium according to claim 7 or 8 ..
The harmful microorganisms include Aeromonas hydrophila, Aeromonas caviae, Yersinia ruckeri, Edwardsiella tarda, Flavobacterium columnare, Vibrio angilarum, Vibrio angilarum, Vibrio angilarum, Vibrio angilarum.
The Pseudomonadaceae bacterium according to any one of claims 1 to 11, wherein the bacterium is determined to have an ability using zebrafish (Dario rerio) as a standard fish.
The Pseudomonadaceae bacterium according to any one of claims 1 to 12, wherein the fish is a farmed fish.
The pseudomonas according to claim 13, wherein the cultured fish is eel, ayu, yellowtail, trout, tie, carp, amberjack, tuna, salmon, horse mackerel, flatfish, tilapia, blowfish, hamachi, hata, mackerel, saury or catfish. Family bacteria.
The Pseudomonadaceae bacterium according to claim 13, wherein the farmed fish belongs to the family Salmonidae.
The Pseudomonadaceae bacterium according to claim 13, wherein the farmed fish is trout.
The Pseudomonas family bacterium according to any one of claims 1 to 16, which is a bacterium of the genus Pseudomonas.
The Pseudomonas family bacterium according to any one of claims 1 to 16, which is a Pseudomonas putida group, a Pseudomonas fluororescens group, or a Pseudomonas koreensis group.
Pseudomonas mossellii, Pseudomonas marginalis, Pseudomonas korensis (Pseudomonas koreensis), Pseudomonas protegens (Pseudomonas protegens) Pseudomonas protegens (Pseudomonas protegens) The Pseudomonas family bacterium described in item 1.
KH-ZF1 (accession number: NITE BP-02967), KH-RT1 (receipt number: NITE ABP-03222), KH-RT2 (receipt number: NITE ABP-03223), KH-RT3 (receipt number: NITE ABP-03224) ) Or KH-RT4 (receipt number: NITE ABP-03225), a Pseudomonadaceae bacterium.
A fish protective agent containing the Pseudomonadaceae bacterium according to any one of claims 1 to 20.
A fish probiotic agent containing the Pseudomonadaceae bacterium according to any one of claims 1 to 20.
The probiotic agent according to claim 22, which is the probiotic of the epidermis of the fish.
The probiotic agent according to claim 22 or 23, wherein the Pseudomonadaceae bacterium has an ability to settle on the epidermis of fish.
The fish protective agent according to claim 21 or the probiotic agent according to any one of claims 22 to 24, which is a protective agent or probiotic agent for farmed fish.
Eel, ayu, yellowtail, trout, tie, carp, amberjack, tuna, salmon, horse mackerel, flatfish, tilapia, puffer, hamachi, hata, mackerel, saury or catfish protectant or probiotics agent, claim 21. The fish protectant or the probiotics agent according to any one of claims 22 to 24.
The fish protective agent according to claim 21 or the probiotic agent according to any one of claims 22 to 24, which is a salmonid fish protective agent or probiotic agent.
The fish protective agent according to claim 21 or the probiotic agent according to any one of claims 22 to 24, which is a trout protective agent or a probiotic agent.
A method for protecting fish, which comprises a step of bringing the fish into contact with the Pseudomonadaceae bacterium according to any one of claims 1 to 20.
A method for protecting fish, which comprises a step of growing the fish in water in which the Pseudomonadaceae bacterium according to any one of claims 1 to 20 is present.
A method for obtaining microorganisms that have the ability to protect fish.
(A) Step of obtaining candidate microorganisms from the epidermis of fish,
(B) A step of adding the candidate microorganism to a medium containing a harmful microorganism,
(C) A step of confirming the suppression of the harmful microorganism in the medium, and (d) when the suppression of the harmful microorganism in the medium is confirmed, the candidate microorganism is acquired as a microorganism having an ability to protect the fish. Process to do,
Including methods.
The method according to claim 31, wherein in the step (c), the suppression of the harmful microorganism on the solid medium is confirmed.
The method according to claim 31, wherein in the step (c), the candidate microorganism and the harmful microorganism are grown on the same medium, and a growth inhibition zone in which the harmful microorganism cannot grow is confirmed in the vicinity of the candidate microorganism. ..
The method according to claim 31, wherein in the step (c), suppression of the harmful microorganism in the liquid medium containing the candidate microorganism or the culture supernatant thereof and the harmful microorganism is confirmed.
In the step (c), a bottomless cylinder containing the culture solution of the candidate microorganism or an impregnated substance of the culture supernatant or the culture solution or the culture supernatant is placed on a medium in which the harmful microorganism is inoculated as a whole. 31. The method of claim 31, wherein the method is arranged to confirm the suppression of the harmful microorganisms around the impregnated material or cylinder.
In the step (c), after incubating the liquid medium containing the candidate microorganism and the harmful microorganism, the growth of the candidate microorganism and the harmful microorganism is compared, and the growth of the harmful microorganism is further suppressed. 31. The method of claim 31 for confirmation.
In the step (c), the candidate microorganism is inoculated on the medium in which the harmful microorganism is inoculated as a whole, or on the medium in which the harmful microorganism is grown as a whole, and the candidate microorganism forms a colony. The method according to claim 31, wherein the growth-inhibiting circle of the harmful microorganism is observed in the vicinity thereof.
The method according to any one of claims 31 to 37, wherein the candidate microorganism is obtained from a colony formed on a medium inoculated with a scraped product of the epidermis of the fish.
The method according to any one of claims 31 to 38, wherein one or more kinds of microorganisms are acquired as microorganisms having an ability to protect the fish.
Any of claims 31-39, wherein the harmful microorganism has at least one of the ability to cause skin disease in fish, transdermally infect fish, infect wounds, and infect contact. The method described in item 1.
The harmful microorganisms include vibrio disease, furunculosis, atypical Eromonas salmonicida infection, Eromonas hydrophila infection, edwardsiellosis, red spot disease, Ayu's pseudomonas disease, red mouse disease, bacterial gill disease, and streptococculosis. (Selected, tailed, torn, torn), capable of causing cold water disease, gliding bacillosis, bacterial kidney disease, mycobacteriosis, nocardiosis, and / or streptococculosis, claims 31-39. The method according to any one of the above.
The method according to any one of claims 31 to 41, wherein a candidate microorganism is obtained from a zebrafish (Danio relio) or the epidermis of trout.
A microorganism obtained by the method according to any one of claims 31 to 42.
The microorganism according to claim 43, which belongs to the genus Pseudomonas.
The microorganism according to claim 43, which is a Pseudomonas putida group, a Pseudomonas fluororescens group, or a Pseudomonas koreensis group.
A method for protecting fish, which comprises a step of bringing a microorganism obtained by the method according to any one of claims 31 to 42 into contact with the fish.
It is a strain belonging to Pseudomonadaceae and
The strain is
(I) A step of linearly drawing the strain on an NB agar medium and culturing at 28 ° C. overnight.
(Ii) After that, a step of drawing harmful microorganisms vertically from the strain so as not to touch the strain and culturing at 28 ° C. overnight.
When tested in a method comprising, after step (ii), the strain is characterized by the presence of a clear zone over a range of 10 mm or more from the stroke.
Here, the harmful microorganisms are Aeromonas hydrophila (ATCC 700183), Aeromonas hydrophila (JCM 1027), Aeromonas caviae (JCM 1043), Flavobacterium columnare (JCM 103), Flavobacterium colorNeria (JCM) 2 Containing at least one selected from the group consisting of
Strain.
It is a strain belonging to Pseudomonadaceae and
The strain is
(I) A step of linearly drawing the strain on an NB agar medium and culturing at 28 ° C. overnight.
(Ii) Then, a step of drawing harmful microorganisms in a direction perpendicular to the image of the strain so as to intersect the image of the strain and culturing at 28 ° C. overnight.
In the case of testing by the method including, after the step (ii), the colony by the strain occupies an area of 50% or more in the image portion of the harmful microorganism beyond the portion intersecting the image line of the strain. Characterized by
Here, the harmful microorganism is selected from the group consisting of Yersinia ruckeri (NVH 3758), Aeromonas hydrophila (NRIA14), Vibrio anglilarum (NRIA83) and Vibrio ordali (NRIA90).
Strain.