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WO2018155613A1 - Composition pour la prévention et/ou le traitement d'une maladie de nécrose hépatopancréatique aiguë des crevettes comprenant une souche de bacillus amyloliquefaciens et/ou un produit traité de ladite souche - Google Patents

Composition pour la prévention et/ou le traitement d'une maladie de nécrose hépatopancréatique aiguë des crevettes comprenant une souche de bacillus amyloliquefaciens et/ou un produit traité de ladite souche Download PDF

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Publication number
WO2018155613A1
WO2018155613A1 PCT/JP2018/006628 JP2018006628W WO2018155613A1 WO 2018155613 A1 WO2018155613 A1 WO 2018155613A1 JP 2018006628 W JP2018006628 W JP 2018006628W WO 2018155613 A1 WO2018155613 A1 WO 2018155613A1
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strain
shrimp
bacillus amyloliquefaciens
present
composition
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PCT/JP2018/006628
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English (en)
Japanese (ja)
Inventor
元一郎 瀬尾
佐藤　直樹
和夫 福井
育生 廣野
秀裕 近藤
健太郎 今泉
智英 増田
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東亜薬品工業株式会社
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Publication of WO2018155613A1 publication Critical patent/WO2018155613A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the present invention relates to a composition for preventing, controlling, suppressing, reducing, alleviating and / or treating shrimp acute hepatopancreatic necrosis, comprising a Bacillus amyloliquefaciens strain and / or a processed product of the strain, and the composition
  • the present invention relates to a method for treating shrimp eye acute hepatopancreatic necrosis.
  • Patent Document 1 discloses a “Bacillus amyloliquefaciens strain with deposit number NITE ABP-01844”. However, Patent Document 1 does not disclose or suggest that the Bacillus amyloliquefaciens strain is effective for shrimp acute hepatopancreatic necrosis.
  • An object of the present invention is to provide a composition effective for shrimp acute hepatopancreatic necrosis and a method for treating shrimp acute hepatopancreatic necrosis.
  • the Bacillus amyloliquefaciens strain has an effect on Vibrio parahemolyticus which is a causative bacterium of shrimp Acute hepatopancreatic necrosis.
  • the present invention has been completed. That is, the present invention is as follows.
  • a composition for preventing, controlling, suppressing, reducing, alleviating, and / or treating shrimp acute hepatopancreatic necrosis comprising a Bacillus amyloliquefaciens strain and / or a processed product of the strain.
  • the shrimp is a shrimp family, a shrimp, a black tiger, a shrimp, or a white shrimp.
  • the composition according to item 1 or 2 wherein the Bacillus amyloliquefaciens strain is a strain having the deposit number NITE ABP-01844. 4).
  • a marine medicinal product, feed, feed additive, or drinking water comprising the composition according to any one of items 1 to 3. 5).
  • Acute shrimp characterized by administering the aquatic drug, feed, feed additive or drinking water according to item 4 above and / or the composition according to any one of items 1 to 3 above
  • a method for breeding shrimp eyes while preventing, controlling, suppressing, reducing, alleviating, and / or treating hepatopancreatic necrosis. 6).
  • Vibrio parahemolyticus control agent containing a Bacillus amyloliquefaciens strain and / or a processed product of the strain. 7).
  • the pesticide according to item 6, wherein the Bacillus amyloliquefaciens strain is a strain having the deposit number NITE ABP-01844. 8).
  • a shrimp that prevents, controls, suppresses, reduces, alleviates, and / or treats a shrimp acute hepatopancreatic necrosis including a step of administering a Bacillus amyloliquefaciens strain and / or a processed product of the strain to the shrimp How to breed.
  • a method for preventing, controlling, suppressing, reducing, alleviating, and / or treating shrimp acute hepatopancreatic necrosis comprising a step of administering a Bacillus amyloliquefaciens strain and / or a processed product of the strain to the shrimp. 10.
  • Bacillus amyloliquefaciens strain is a strain having the deposit number NITE ABP-01844.
  • a method for controlling Vibrio parahemolyticus comprising a step of adding a Bacillus amyloliquefaciens strain and / or a processed product of the strain to a place where Vibrio parahemolyticus is propagated or propagated. 12 12. The method according to item 11 above, wherein the Bacillus amyloliquefaciens strain is a strain having the deposit number NITE ABP-01844.
  • composition, breeding method or treatment method of the present invention had at least one of the following effects.
  • the comparison result of the organic acid production ability of the strain based on this invention and a well-known strain The EMS / AHPND infection test result 14 days after the start of feed feeding containing the composition of the present invention.
  • the vertical axis indicates the survival rate (%), and the horizontal axis indicates the number of days after the start of infection.
  • ⁇ shows the results of the test plot, and ⁇ shows the results of the control plot.
  • the composition for preventing, controlling, suppressing, reducing, alleviating and / or treating shrimp acute hepatopancreatic necrosis of the present invention is at least Bacillus amyloriki.
  • the method for preventing, controlling, suppressing, reducing, alleviating and / or treating shrimp acute hepatopancreatic necrosis of the present invention preferably uses at least a Bacillus amyloliquefaciens strain and / or a processed product of the strain for shrimp. Is administered orally or sprayed to shrimp farms (eg, culture ponds).
  • Bacillus amyloliquefaciens strain Bacillus amyloliquefaciens according to the present invention (Bacillus amyloliquefaciens, Bacillus amyloliquefaciens) strains, can utilize known strains, particularly preferred strains, deposited as accession number NITEABP-01844 Is TOA5001 stock.
  • the mycological properties of the strain (bacteria) according to the present invention are as follows. Gram-positive, Neisseria gonorrhoeae, aerobic, spore-forming ability, motility. It is low in auxotrophy and can grow on various media such as gravy medium, SCD medium, brain heart infusion medium. On the agar plate medium, the periphery is rough and yellowish white. Furthermore, depending on the type of the medium (Brain Heart Infusion Medium), a raised colony having viscosity inside is formed.
  • the strain according to the present invention is not limited to the strain deposited under the deposit number NITE ABP-01844, as long as it belongs to Bacillus amyloliquefaciens and has an effect on Vibrio parahemolyticus, It can be used as a strain according to the present invention. That is, the Bacillus amyloliquefaciens strain of the present invention is intended to include not only the deposited strain itself but also mutants and progeny that are strains having the above-mentioned mycological properties.
  • the mutant preparation method can be obtained by conventionally known irradiation and chemical substance treatment such as nitrosoguanidine, ethylmethanesulfonic acid, methylmethanesulfonic acid and the like.
  • the strain according to the present invention is not toxic even when administered in vivo (particularly in the body of a shrimp), as shown in the Examples below.
  • the strain according to the present invention is obtained by inoculating a nutrient medium containing a carbon source and a nitrogen source and, if necessary, inorganic salts, followed by culturing under aerobic conditions (for example, shaking culture, aeration and agitation culture), Can be obtained.
  • aerobic conditions for example, shaking culture, aeration and agitation culture
  • a culture solution it has an effect on the causative bacteria of acute hepatopancreatic necrosis disease (for example, Vibrio parahemolyticus) as it is in an unconcentrated culture solution, It has the same activity even after freeze-drying or spray-drying.
  • acute liver can be obtained by using a solid itself or a mixture thereof (for example, a contact or mixture with another solid, or a liquid in which a strain-growing solid according to the present invention is immersed). It can have an effect on the causative bacteria of pancreatic necrosis.
  • the solid may be any material as long as the strain according to the present invention can grow, and means, for example, a plant body that is a raw material for agar medium, silage, natto and the like, and curd represented by yogurt.
  • Any carbon source may be used as long as the strain according to the present invention can be used.
  • Glucose, sucrose, starches, cellulose mixtures, and other carbohydrates are preferably used.
  • the nitrogen source may be any as long as the strain according to the present invention can be used, but preferably peptone, yeast extract, gravy extract and the like are used.
  • the strain according to the present invention can be grown in a liquid medium or a plate culture inoculated on a commercially available medium, for example, tryptosy medium, brain heart infusion medium, or gravy medium.
  • the culture When culturing in a small amount in liquid culture, shaking culture using a test tube or flask is preferred. When culturing in large quantities, it is preferable to culture with aeration and agitation as in the case of other fermentation products.
  • culture When culture is performed in a large tank, it is preferable to first inoculate and culture the strain according to the present invention in a relatively small amount of medium as a preculture, and then transfer the culture to a large production tank for production culture.
  • the composition of the medium used for the preculture and the medium used for the production culture may be the same, or may be changed if necessary.
  • the culture conditions can be appropriately changed within the range in which the strain according to the present invention grows. Usually, the culture is performed at 15 to 45 ° C., preferably 25 to 37 ° C. under aerobic conditions.
  • the culture time varies depending on the culture conditions and the culture volume, but is usually about 1 day to 1 week.
  • the strain according to the present invention can be recovered from the culture solution obtained by the above culture by centrifugation and / or membrane concentration.
  • the collected bacterial cells may be used as they are, or dried bacterial cells that have been subjected to freeze-drying or spray-drying as necessary.
  • the treated product of the strain according to the present invention is various treatments on the cultured strain (washing, far-dehydration, heating, gamma ray or neutron irradiation, spray drying, freeze drying, sterilization, enzyme, surface activity Agent, grinding, grinding, etc.).
  • composition of the present invention includes at least the strain (including cells) of the present invention and / or a processed product of the strain, and prevents, controls, suppresses, reduces, alleviates, and / or shrimps acute hepatopancreatic necrosis. Or have a therapeutic effect, and in some cases, a control, elimination, and / or cure effect.
  • Acute hepatopancreatic necrosis is a cerebral microbial infection caused by Vibrio parahaemolyticus infection, particularly causing serious damage in cultured shrimp.
  • the shrimp of the present invention can be exemplified by, for example, lobster family, vaname shrimp, black tiger (bovine shrimp), tiger shrimp, tiger shrimp and white shrimp.
  • the bacterial cell according to the present invention When the bacterial cell according to the present invention is used, any concentration may be used as long as the desired effect can be exhibited.
  • the bacterial strain according to the present invention when administered to shrimp, is 10 2 to 10 2 per kilogram. It is preferable to feed the feed added to 10 10 cfu (Colony forming unit), preferably 10 4 to 10 8 cfu per kilogram.
  • the administration time, administration method, and dose of the composition of the present invention are not particularly limited, but it may be usually added to a commercial feed or mixed diet.
  • the dose of the composition for acute hepatopancreatic necrosis of the present invention is not particularly limited. For example, it is desirable to add 0.01% to 10%, preferably 0.05% to 5% to a commercial feed and administer it. .
  • composition of this invention is as follows, for example.
  • Composition for prevention, control, suppression, reduction, alleviation, and / or treatment of acute hepatopancreatic necrosis (2)
  • Micromp feed, feed additive, and breeding water Prevention of shrimp acute hepatopancreatic necrosis (particularly acute hepatopancreatic necrosis), characterized in that the feed, feed additive or drinking water and / or the composition of the present invention is administered to the shrimp.
  • methods of breeding shrimp eyes while controlling, controlling, reducing, mitigating, and / or treating.
  • the present invention is also directed to a method for combating Vibrio parahemolyticus.
  • the vibrio parahemolyticus control agent of the present invention contains at least a Bacillus amyloliquefaciens strain and / or a processed product of the strain.
  • the pesticide of the present invention prevents the occurrence of acute hepatopancreatic necrosis by adding it to a place (particularly a breeding facility, aquarium, etc.) where Vibrio parahemolyticus is breeding or expected to breed It is possible to suppress, reduce, etc.
  • the Vibrio parahemolyticus extermination method of the present invention is a place (particularly a breeding facility) where Vibrio parahemolyticus breeds or breeds at least a Bacillus amyloliquefaciens strain and / or a processed product of the strain. , A water tank, etc.).
  • the composition of the present invention includes a wide range of marine medicines, foods and drinks, feed additives, feeds (formulated feeds, mixed feeds), and breeding water having the above-mentioned uses.
  • the composition of the present invention is blended with additives such as starch, potato starch, lactose, soybean protein and the like, excipients, binders, disintegrants, lubricants, stabilizers, suspending agents and the like. These can be formulated into powders, tablets, granules, capsules, ointments, liquids and the like by known methods.
  • the organic acid producing ability of the strain according to the present invention was compared with the known strain organic acid producing ability. Details are as follows.
  • the strain according to the present invention and a known strain, B. subtilis standard strain were cultured under the same culture conditions. Specifically, each strain was inoculated into a separate brain heart infusion liquid medium (manufactured by BBL) and cultured with shaking at 37 ° C. for 24 hours. After the culture, the liquid phase and the solid phase cells were separated from the culture solution by cooling centrifugation (10000 ⁇ g, 10 minutes).
  • the obtained liquid phase part was sterilized and filtered through a 0.45 ⁇ m membrane filter.
  • the filtered liquid was used as a culture supernatant for organic acid measurement by the following HPLC.
  • About 0.2 ml of the culture supernatant of each strain was collected in a 1.5 mL tube, and 50 ⁇ L of 500 mM trans-crotonic acid was added as an internal standard substance. Then, it extracted twice with 0.6 mL of 0.25% ammonia as an extract.
  • To the obtained supernatant 0.3 times the amount of 10% HClO 4 was added to precipitate and remove the protein.
  • the obtained supernatant was filtered through a 0.45 ⁇ m membrane filter to obtain an HPLC sample.
  • HPLC was performed under the following conditions.
  • HPLC system manufactured by Waters Column: Organic Acid Column (7.8 ⁇ 300 mm) and guard column (6.0 ⁇ 50 mm) Column temperature and post-column reaction temperature: 60 ° C Flow rate: 0.8mL / min Mobile phase: 0.08% HClO 4 Reaction phase: 0.2 mM BTB, 5.2 mM NaOH, 15 mM Na 2 HPO 4 Detection wavelength: 445 nm
  • the comparison result of the organic acid production ability computed from the result of HPLC is shown in FIG.
  • the acetic acid producing ability of the strain according to the present invention is at least 2.7 times (48.7 / 4) higher than the acetic acid producing ability of the known Bacillus subtilis reference strain. 17.9). From the above results, the strain according to the present invention has higher organic acid producing ability than known strains. More specifically, since organic acids including acetic acid have antibacterial action, it was confirmed that the strain according to the present invention has higher antibacterial activity than known strains.
  • the strain according to the present invention was cultured. Details are as follows.
  • the strain according to the present invention Bacillus amyloliquefaciens TOA5001 strain, is inoculated into a pre-culture medium in which 2% peptone and yeast extract are added to brain heart infusion medium, and cultured with shaking at 30 ° C. for 16 hours. Thus, a culture solution after pre-culture was obtained.
  • the culture solution after the pre-culture is inoculated into a main culture medium containing 4% glucose, 3% peptone and 2% yeast extract, and cultured with shaking at 30 ° C. to 37 ° C. for 2 to 3 days.
  • a culture solution after the main culture was obtained. From the culture solution after the main culture, cells and a culture supernatant were obtained by centrifugation at 14000 ⁇ g for 20 minutes. The cells were freeze-dried and used in the following examples as a dry powder.
  • EMS / AHPND infection test It was evaluated whether acute hepatopancreatic necrosis (EMS / AHPND) can be controlled by adding and feeding the strain according to the present invention to vaname prawn diet.
  • Vaname shrimp obtained from IMT Engineering was used for the test. The average body weight of shrimp was 1.1 g at the start, 2.25 g after 2 weeks, and 3.3 g after 4 weeks.
  • the breeding aquarium used for the feeding test was a 100 L tank, and the filtration system was a circulation type using TOTTO (Biolaboto Co., Ltd.).
  • the breeding water temperature was controlled by an air conditioner, and the water temperature was 26 degrees.
  • a powder feed obtained by pulverizing a general commercial feed used in Thailand in both the test group and the control group was used.
  • powder feed supplemented with 0.5% of the strain according to the present invention was used as the test plot feed.
  • the powdered feed was prepared by mixing 0.5 parts by weight of the dry powder prepared in Example 2 with 100 parts by weight of commercially available feed, adding an equal amount of distilled water and mixing well.
  • a 50 mL syringe was filled with the kneaded bait and extruded to make a spaghetti shaped bait. This was dried at 60 to 65 ° C. for about 2 hours, and after drying, stored in a refrigerator.
  • the daily feeding amount was about 5% by weight / body weight. Feeding was divided into four times a day and an automatic feeder was used. Feeding continued during the infection test.
  • a bacterial infection test by immersion infection was carried out 2 weeks (14 days) after feeding.
  • the bacterial solution was added to 3 ⁇ 10 4 cells or cfu / mL, and the cells were continuously raised. Survival was assessed 2 weeks after infection.
  • compositions, breeding method or treatment method of the present invention had the following effects.
  • the present invention can provide a composition for preventing and / or treating shrimp acute hepatopancreatic necrosis comprising a Bacillus amyloliquefaciens strain and / or a processed product of the strain.

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Abstract

L'invention concerne une composition ayant un effet contre une maladie de nécrose hépatopancréatique aiguë des crevettes. Il a été découvert qu'une souche de Bacillus amyloliquefaciens a un effet contre Vibrio parahaemolyticus, qui est une bactérie provoquant une maladie de nécrose hépatopancréatique aiguë des crevettes.
PCT/JP2018/006628 2017-02-24 2018-02-23 Composition pour la prévention et/ou le traitement d'une maladie de nécrose hépatopancréatique aiguë des crevettes comprenant une souche de bacillus amyloliquefaciens et/ou un produit traité de ladite souche WO2018155613A1 (fr)

Applications Claiming Priority (2)

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JP2017034084 2017-02-24
JP2017-034084 2017-02-24

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WO2018155613A1 true WO2018155613A1 (fr) 2018-08-30

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020165057A1 (fr) * 2019-02-11 2020-08-20 Evonik Operations Gmbh Compositions contenant du bacillaène produisant des bactéries ou des préparations de celles-ci
CN111763644A (zh) * 2020-07-24 2020-10-13 天津开发区坤禾生物技术有限公司 一种防治鱼类肝胆综合症的复合菌剂及其制备方法和应用
US20220313783A1 (en) * 2019-09-05 2022-10-06 Pebble Labs Inc. Systems, Methods and Compositions for the Inhibition of Bacterial Toxins to Treat Early Mortality Syndrome in Aquatic Animals

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CN104974950A (zh) * 2015-05-11 2015-10-14 山东大学（威海） 一种海洋解淀粉芽孢杆菌及其用途
JP2016116466A (ja) * 2014-12-19 2016-06-30 東亜薬品工業株式会社 バチルスアミロリキファシエンス菌株及び該菌株を含む抗菌組成物

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JP2016116466A (ja) * 2014-12-19 2016-06-30 東亜薬品工業株式会社 バチルスアミロリキファシエンス菌株及び該菌株を含む抗菌組成物
CN104974950A (zh) * 2015-05-11 2015-10-14 山东大学（威海） 一种海洋解淀粉芽孢杆菌及其用途

Non-Patent Citations (3)

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Title
BACILLUS AMYLOLIQUEFACIENS TOA5001, 12 September 2017 (2017-09-12), pages 30 *
SIRIKHARIN, R. ET AL.: "Characterization and PCR Detection Of Binary, Pir-Like Toxins from Vibrio parahaemolyticus Isolates that Cause Acute Hepatopancreatic Necrosis Disease (AHPND) in Shrimp", PLOS ONE, vol. 10, no. 5, 2015, pages e0126987, XP055538338, ISSN: 1932-6203 *
SOTO-RODRIGUEZ, S. A. ET AL.: "Field and Experimental Evidence of Vibrio parahaemolyticus as the Causative Agent of Acute Hepatopancreatic Necrosis Disease of Cultured Shrimp (Litopenaeus vannamei) in Northwestern Mexico", APPL ENVIRON MICROBIOL, vol. 81, no. 5, 29 December 2014 (2014-12-29), pages 1689 - 1699, XP055538335, ISSN: 0090-2240 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020165057A1 (fr) * 2019-02-11 2020-08-20 Evonik Operations Gmbh Compositions contenant du bacillaène produisant des bactéries ou des préparations de celles-ci
US20220313783A1 (en) * 2019-09-05 2022-10-06 Pebble Labs Inc. Systems, Methods and Compositions for the Inhibition of Bacterial Toxins to Treat Early Mortality Syndrome in Aquatic Animals
CN111763644A (zh) * 2020-07-24 2020-10-13 天津开发区坤禾生物技术有限公司 一种防治鱼类肝胆综合症的复合菌剂及其制备方法和应用

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