WO2008023580A1 - Additif pour alimentation animale - Google Patents

Additif pour alimentation animale Download PDF

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Publication number
WO2008023580A1
WO2008023580A1 PCT/JP2007/065638 JP2007065638W WO2008023580A1 WO 2008023580 A1 WO2008023580 A1 WO 2008023580A1 JP 2007065638 W JP2007065638 W JP 2007065638W WO 2008023580 A1 WO2008023580 A1 WO 2008023580A1
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Prior art keywords
aspergillus
strain
feed additive
animal feed
animal
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PCT/JP2007/065638
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English (en)
Japanese (ja)
Inventor
Masami Mochizuki
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Idemitsu Kosan Co., Ltd.
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Application filed by Idemitsu Kosan Co., Ltd. filed Critical Idemitsu Kosan Co., Ltd.
Priority to CN200780031445A priority Critical patent/CN101677595A/zh
Priority to JP2008530855A priority patent/JPWO2008023580A1/ja
Publication of WO2008023580A1 publication Critical patent/WO2008023580A1/fr

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    • 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
    • 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
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • 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
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • 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
    • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
    • 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
    • A23K50/60—Feeding-stuffs specially adapted for particular animals for weanlings
    • 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
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • 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
    • C12N1/14—Fungi; Culture media therefor

Definitions

  • the present invention relates to an animal feed additive containing Aspergillus spp. And Bacillus spp. Having the ability to produce acid enzymes.
  • Feeds such as livestock and pets are processed, such as pulverization, but generally are not heat-treated. There is a problem of poor feed efficiency.
  • inflammatory bowel disorders such as ulcerative colitis and Crohn's disease, which are known as human diseases, have also been reported in animals, causing problems such as diarrhea. It is also known that such inflammatory bowel disorders prevent feed absorption and healthy fattening.
  • pathogenic bacteria that cause intestinal infections in animals include pathogenic E. coli, Salmonella bacteria, Clostridium bacteria, Campylobacter bacteria, and the like.
  • Such pathogens are known to produce toxins (enterotoxin, cytotoxin) when they grow abnormally, damage the intestinal mucosa, and cause loose stool and severe diarrhea.
  • Kokushijimu is a protozoan that parasitizes the intestinal tract of chickens, pigs, cattle, etc., and when infected, causes diarrhea and loss of appetite.
  • There are problems such as the emergence of drug-resistant bacteria where antibiotics are used to prevent and treat such inflammatory bowel disorders.
  • Patent Documents 1 and 2 In recent years, in order to improve the balance of the intestinal flora and suppress the growth of pathogenic bacteria in the intestine, a technique using probiotics has attracted attention (Patent Documents 1 and 2). However, lactic acid bacteria and bifidobacteria are strong against microorganisms among bile acids, except for bacteria collectively called coliforms that die at 0.3% deoxycholic acid concentration or die most at pH 4 or lower. ! /, Unable to survive in the presence of antibacterial deoxycholate! /, Many species. Therefore, there is a demand for strains that do not die in the digestive tract of animals and that have beneficial effects on the host.
  • fermented nutrients are added to the crustacean crusts and mixed, and this is selected from Aspergillus-Niger, Aspenoleguinoles' Orisee, Batchinoles' Zubtilis, Batchinoles' Richenifolemis force, etc.
  • a fermented product which is given to animals.
  • Patent Document 6 Although this method has partially confirmed the effect of promoting the growth of animals, it has not been studied to prevent or treat enteric infections by suppressing the growth of pathogenic bacteria in the intestines of animals. Is also not allowed.
  • mixing two or more microorganisms and administering them to animals has not been studied.
  • Bacillus subtilis has been reported to have antibacterial activity against pathogenic bacteria such as pathogenic E. coli (Patent Documents 7 and 8), and studies on oral administration to animals have been made.
  • Bacillus subtilis DB9011 has aflatoxin degradability and is known to inhibit the growth of fungi, and its addition to feed has been studied (Patent Document 9).
  • Patent Document 9 it has been studied so far that Bacillus subtilis is ingested by animals in combination with other strains of microorganisms!
  • Patent Document 1 Japanese Translation of Special Publication 2005-507670
  • Patent Document 2 Japanese Translation of Special Publication 2004-523241
  • Patent Document 3 Japanese Patent Application Laid-Open No. 2004-141147
  • Patent Document 4 Japanese Patent Publication No. 6-319464
  • Patent Document 5 Japanese Patent Laid-Open No. 11 171674
  • Patent Document 6 Japanese Patent Application Laid-Open No. 2002-238466
  • Patent Document 7 Japanese Patent Laid-Open No. 11 332555
  • Patent Document 8 Japanese Patent Laid-Open No. 11 285378
  • Patent Document 9 Japanese Patent No. 3040234
  • Non-Patent Document 1 George A. Burdock, Madhusudan G. Soni, and Vietnamese G. Carabin (200 1) Regulatory Toxicology and Pharmacology 33, 80-101
  • Non-Patent Document 2 Harry E. Morton, Walter ocholaty, Renate Junowicz- Kocholaty, and Albert elner (1945) J. Bacteriol 50, 579—584
  • An object of the present invention is to provide a safe and simple means for assisting an animal's digestive activity and increasing feed efficiency. Specifically, an object of the present invention is to provide a means for preventing and improving intestinal infections by suppressing the growth of pathogenic bacteria in the intestines of animals and for realizing weight gain of animals.
  • Aspergillus sore Aspenoleguinores' Tamari, Aspenoleguinores' Fötidas, Aspenoleginores' Nigaichi and Aspergillus 'Olyse has excellent ability to produce acid enzymes, especially acid amylase, that these bacteria have antibacterial activity against pathogenic bacteria causing enteric infection and protozoan activity against coccidium, and they are probiotics I found it to work. It was also found that the ability of these bacteria to produce acidic enzymes is extremely excellent when cultured using brown rice as a nutrient source.
  • the present invention is as follows.
  • Aspergillus sojae, Aspengilinos Tamari, Aspengillus foetidus, Aspergillus foetidus, Aspergillus niger and Aspergillus niger Animal feed comprising Aspergillus oryzae, at least one Aspergillus spp., A culture containing acid enzymes produced by these fungi, and Bacillus subtilis Animal feed that is an additive and has a total acid enzyme activity of 20 U or more per lg of feed additive and a Bacillus subtilis concentration of 2.5 X 10 7 to 2 X 10 9 CFU / g Additive.
  • Aspergillus oryzae IK-05074 strain (FERM BP-10622) and / or a mutant strain thereof, characterized by having the same ability to produce an acidic enzyme as the aforementioned strain
  • the animal feed additive according to any one of (1) to (4).
  • a feed comprising the animal feed additive according to any one of (1) to (; 10) from 0.01 to 1.0% by mass.
  • OX 10 1Q A method for producing a feed, comprising a step of containing CFU / kg.
  • the animal feed additive of the present invention is produced by at least one Aspergillus spp. Selected from one of Aspergillus sosa, Aspergillus tamari, Aspergillus foetidas, Aspergillus' Niger and Aspergillus oryzae. It is characterized by containing a culture containing an acidic enzyme, and a Bacillus subtilis bacterium.
  • -Olyse is a bacterium that is classified into the above-mentioned species when classified by a method generally used for identification of gonococcal species in this technical field.
  • a method generally used for identification of gonococcal species in this technical field For the identification of fungal species, for example, “IH. Murakami, The Journal of General and Applied Microbiology, 17, p.281-309, (1971)”, “Miyakami Hideya, Journal of the Japan Brewing Society, Vol. 74, No. 12, p.849-853, (1979) ”,“ Nikkum, et al, The journal of General and Applied Microbiology, 44, p.225-230, (1998) ”.
  • Aspergillus soja is a kind of incomplete filamentous fungus found in soil, koji, etc., and is used for brewing soy sauce and miso.
  • Aspergillus soya AOK 210 (Akita Imano Shoten Co., Ltd.) can be preferably used as such a fungus that may use a commercially available strain.
  • Aspergillus tamari is a type of filamentous imperfect fungi found in soil, koji, foods, etc., and is used for brewing soy sauce and miso.
  • the animal feed additive of the present invention has an ability to produce at least one acidic enzyme described in detail below, preferably acidic amylase. If it is safe, it can be used without particular limitation.
  • Aspergillus tamari AOK 43 strain (Akita Imano Co., Ltd.) can be preferably used as such a bacterium that may use a commercially available strain.
  • Aspergillus' foetidas is a type of filamentous incomplete fungus found in soil, koji, cereal grains, etc., and is used for brewing sake, miso, and soy sauce.
  • the animal feed additive of the present invention has an ability to produce at least one, preferably acidic amylase, among the acidic enzymes described in detail below. If it is safe, it can be used without any particular limitation.
  • Aspergillus. Foetidas AOK N4586 strain (Akita Imano Shoten Co., Ltd.) can be preferably used as such a bacterium that may use a commercially available strain.
  • Aspergillus niger is a kind of filamentous imperfect fungi found in soil, straw, grain residue, etc., and is used in various situations such as liquor production, food processing, sugar production and pharmaceutical production.
  • the animal feed additive of the present invention has at least one of the enzyme enzymes described in detail below, and preferably has the ability to produce acid amylase, and is safe for feeding to animals. Anything can be used without particular limitation.
  • Aspergillus niger AOK B650 strain (Akita Imano Shoten Co., Ltd.) can be preferably used as a fungus such as a commercially available strain.
  • AOK 210 strain, AOK 43 strain, AOK N4586 strain or AOK B650 strain mutant Can also be used. Mutants are the same acidic as AOK 210 strain, AOK 43 strain, AOK N4586 strain or AOK B650 strain from strains obtained by natural mutation of these strains or by mutation treatment with chemical mutagens or ultraviolet rays, etc.
  • the ability to select and obtain a strain having the ability to produce an enzyme In addition to the ability to produce an acidic enzyme, it is also preferable to use a mutant strain of the strain further having at least one of the same antibacterial activity, protozoan activity, bile acid resistance, and acid resistance as the above strain.
  • Aspergillus oryzae is a type of filamentous imperfect fungi found in soil, koji, etc., and is used for brewing soy sauce and miso.
  • the animal feed additive of the present invention has the ability to produce at least one of the acidic enzymes described in detail below, preferably acidic amylase, and is safe for feeding to animals. Can be used without any restrictions.
  • a commercially available strain may be used, and aspergillus oryzae strain IK-05074 can be preferably used as such a bacterium.
  • the IK-05074 strain was isolated from various fermented foods.
  • the size is 50-60mm in diameter, the color changes from yellow to green, and the color changes to brownish over time.
  • the mycelium is inconspicuous and the back is colorless.
  • Conidial pattern thin wall to thick wall, smooth surface to slightly rough surface, spherical shape with a diameter of 20 111 or less, length of 2 mm or less, and a gall of 40 to 50 m, 80 m or less in diameter.
  • Basal intestine Most of the large conidia! / Are present, and the length is less than 12 m.
  • Fear Ride Ampoule, length 8 ⁇ ; 12 111, with short neck.
  • Conidia Spherical shape with a diameter of 5-6 m, color from yellow to green, and smooth to fine rough surface.
  • strain IK 05074 belongs to Aspergillus oryzae.
  • a mutant strain of IK 05074 strain can also be used.
  • the mutant strain of IK 05074 has the ability to produce the same acid enzyme as IK-05074 from a strain obtained by natural mutation of IK 05074 strain or mutation treatment with chemical mutagens or ultraviolet rays. It can be obtained by selecting a strain. It is also preferable to use a mutant of IK-05074 that has at least one of the same antibacterial activity, protocidal activity, bile acid resistance, and acid resistance as IK-05074 in addition to the ability to produce acid enzymes. Yes. In addition, it is also preferable to use a mutant strain having the same mycological properties as those of IK-05074.
  • animal feed additive of the present invention for example, Aspergillus' soya, Aspergillus' Tamari, Aspergillus' foetidas, Aspergillus. Nger, Aspergillus.
  • Aspergillus' soya Aspergillus' Tamari
  • Aspergillus' foetidas Aspergillus. Nger
  • Aspergillus Of these, it is possible to isolate and use strains that have the ability to produce acidic enzymes! /.
  • the ability to produce an acidic enzyme refers to producing an acidic enzyme to the extent that acidic enzyme activity can be detected in a culture obtained by culturing bacterial cells. Measurement of the acidic enzyme activity of the culture can be performed according to a conventional method.
  • the acidic enzyme contained in the animal feed additive of the present invention is preferably a digestive enzyme produced by the bacterium described above, and preferably has activity without being inactivated under acidic conditions in the gastrointestinal tract.
  • those having an optimum pH of 2.5 to 5.5 are preferable.
  • acidic ⁇ -amylase, dalcoamylase, takadiastase, protease, senolase, ribonuclease, nuclease, xylanase, pectinase, linase, etc. can be mentioned, and the animal feed additive of the present invention is one of these. Or it contains two or more.
  • an acid amylase that degrades the starch which is one of the main ingredients of livestock feed.
  • the acidic amylase contained in the animal feed additive of the present invention is not particularly limited as long as it is an acidic enzyme that hydrolyzes starch, and examples thereof include ⁇ -amylase, / 3-amylase, and darcoamylase. .
  • acid-resistant ⁇ -amylase having an optimum pH of around 3 can be preferably mentioned.
  • the animal feed additive of the present invention contains at least one of the acid enzymes produced by Aspergillus' soya, Aspergillus' Tamari, Aspenoleginoles' Fötidas, Aspergillus. Yes, but other species may contain enzymes from other species!
  • the acidic enzyme activity of the animal feed additive of the present invention may be set within the following range.
  • the total acid enzyme activity per lg of animal feed additive is 120 U or more, preferably 170 U or more, more preferably 220 U or more, more preferably 2701; to 5400 U.
  • the total acid enzyme activity is preferably the sum of acid amylase activity, acid protease activity and acid carboxypeptidase activity.
  • the acid amylase activity per lg of animal feed additive is preferably 1 U or more, more preferably 5 U or more, further preferably 10 U or more, more preferably 20 to 400 U.
  • the total of acidic protease activity and acidic carboxypeptidase activity per gram of animal feed additive is preferably 100 U or more, more preferably 150 U or more, more preferably 200 U or more, and further preferably 250 to 5000 U. It is.
  • the activity of acid amylase, acid protease and acid carboxypeptidase is determined by the National Tax Agency's prescribed analysis method (Amendment 3rd Tax Agency Directive No. 1).
  • the measurement may be performed in accordance with the measurement method, acid-resistant ⁇ -amylase activity measurement method, acid protease activity measurement method, and acid carboxypeptidase activity measurement method.
  • Aspergillus' soya, Aspergillus, Tamari, Aspergillus' foetidas, Aspergillus' Niger and Aspergillus-Olyse which are contained in the animal feed additive of the present invention, and these bacteria
  • concentration of the culture containing the acidic enzyme to be produced may be adjusted so that the acidic enzyme activity of the feed additive falls within the above range.
  • the above-mentioned Aspergillus spp. Used in the present invention has antibacterial activity against pathogenic bacteria causing intestinal infections in animals!
  • the above pathogenic bacteria usually belong to the family Enterobacteriaceae. Specific examples include bacteria belonging to pathogenic Escherichia coli, Salmonella, Campylobacter, Clostridium, and Staphylococcus aureus. Examples of pathogenic E. coli include enterotoxin-producing E. coli producing enterotoxin (Enterotoxigenic E. coli (ETEC)), enterohemorrhagic Escherichia coli producing verotoxin such as edema disease and 0157 (Verotoxin-producing E. coli). coli (VTEC), enterohemor rhagic E. coli) and the like. Examples of bacteria belonging to the genus Salmonella include S.
  • Examples of bacteria belonging to the genus Campylobacter include C. jejuni, C. coli, and C. fetus.
  • Examples of bacteria belonging to the genus Clostridium include C. perfringens, C. botulinum, and C. difficile.
  • Aspenoreginores which is included in the animal feed additive according to the present invention, is a bacterium belonging to the genus Salmonella, especially S., Aspeneginoles 'Tamari, Aspenoleginoles' Foretidas, Aspergillus 'Niger, Aspergillus' oryzae. It has high antibacterial activity against enteritidis and Clostridium bacteria, especially C. perfringens, enterobacteria such as edema disease, and S. aureus.
  • Having antibacterial activity against pathogenic bacteria means having the ability to suppress the growth of pathogenic bacteria when inoculated in the same medium as the pathogenic bacteria! Asperginores with antibacterial activity 'Soya, Aspenoleginores' Tamari, Aspenoleginoles' Foretidas, Aspenoleginores' Niger, Aspergillus' Oryzae, for example, Salmonella enteritidis (3 ⁇ 4E, added to agar medium inoculated with pathogenic bacteria such as Clostridium perfringens (CP), Escherichia coli (EG), ⁇ taphylococcus cus aureus (SA), and after incubation, isolate the cells that formed the inhibition circle Since the bacteria obtained in this way have the ability to suppress the growth of the above-mentioned pathogenic bacteria, they can be administered to animals to prevent and treat enteric infections in animals. can do.
  • Asperginores with antibacterial activity 'Soya Aspenoleginores' Tamari, Aspenolegin
  • the growth of pathogenic bacteria that cause intestinal infections in animals is suppressed, for example, by measuring the bacterial cell concentration (viable cell count) in the caecal contents of animals. Ability to persevere.
  • the Aspergillus spp. Used in the present invention preferably has a protozoan activity against coccidium causing intestinal infections in animals.
  • Kokujijumu is a protozoan belonging to the sporeworm (Sporozoasida subclass).
  • E imeria moth Isospora genus
  • Toxoplasma moth Cryptosporidiumfeci 'force
  • struck. E. tenella, E. necatrix, E. acervulina, E. maxima E. mitis, E. zuernii, E. bovis, etc.
  • protozoa belonging to the genus Isospora include I. suis, I. belli, and I. hominis.
  • Examples of protozoa belonging to the genus Toxoplasma include the power S such as ⁇ ⁇ gondii.
  • Examples of protozoa belonging to the genus Cryptosporidium include C. parvum.
  • the animal feed additive of the present invention can be suitably used particularly for infectious diseases caused by ⁇ tenella, E. zuernii.
  • Having a protozoan activity against coccidium has the ability to suppress oocyst germination and proliferation and preferably reduce oocysts when coccidiom oocysts and cultures of bacteria are coexistent It means that Specifically, it means that the cell wall of the oocyst is deformed and dissolved, and has the ability to collapse the oocyst. The collapse of the oocysts and the state of the cell wall can be observed with a microscope.
  • Aspergillus 'soya Aspergillus having a protocidal activity against kokusijuum • Tamari, Aspenoleginoles' Foretidas, Aspenoleginores 'Nigai, Aspenoleginoles' olize can be obtained by the following method, for example. Add the source of soil, straw, etc. to a petri dish containing sterilized water suspended in E. tenella or zu zuernii oocysts, incubate at 37 ° C, and observe for ⁇ 7 days. Isolate the cells from the seeds where oocysts are deformed or dissolved, and add them again to a petri dish containing sterile water in which E.
  • Aspergillus 'soya, Aspergillus. Tamari, Aspenoleginoles' Foretidas, Aspenoleginoles 'Niger, Aspenoleginoles' Olise I contained in the animal feed additive of the present invention is gastric acid and bile Those having resistance to acids are preferred.
  • the bacterial cells produce useful acidic enzymes in the intestines of animals and the function of assisting digestion by the acidic enzymes is maintained.
  • these Aspergillus spp. Have antibacterial activity against the above-mentioned pathogenic bacteria, they suppress the growth of pathogenic bacteria causing intestinal infections in animals and also improve the balance of intestinal flora. Contribute.
  • an acid-resistant strain that can be used for the feed additive of the present invention can be obtained, for example, by selecting a strain having the ability to survive when the separation source is treated at pH 3.5 for about 2 hours. I ’ll do that.
  • the bacterium selected in this way is treated for about 24 hours in the presence of a dexioxic acid concentration of lOg / 1 to select a strain having the ability to survive, whereby the feed additive of the present invention is selected. Strains having resistance to gastric acid and bile acids suitable for use can be obtained. In addition, it can be confirmed that the bacteria have reached the intestine without being killed, for example, by measuring the concentration of bacterial cells in animal excrement.
  • the animal feed additive of the present invention can contain a single strain among the above-mentioned bacterial species, or can contain two or more strains in combination. Among these, it is preferable to contain at least one of Aspergillus soja and Aspergillus oryzae.
  • Aspergillus' soya, Aspergillus. Tamari, Aspergillus' foetidas, Aspergillus' niger and Aspergillus-olise contained in the animal feed additive of the present invention are determined when the feed additive is administered to the animal. In the digestive tract, Usually, it should be within the range where it does not die. Usually, the medium is inoculated with a suitable concentration of bacterial cells to produce the above-mentioned feed additive having acidic enzyme activity, and finally the acidic enzyme activity is measured according to the above value. You just have to adjust it.
  • Aspergillus' soya, Aspergillus' Tamari, Aspergillus' foetidas, Aspergillus' Niger and Aspergillus' oryzae used for the animal feed additive of the present invention produce acidic enzymes by culturing under normal culture conditions To do.
  • the cultivation temperature is a force that can be performed at 25 ° C to 40 ° C.
  • a liquid culture method or a solid culture method such as reciprocating shaking culture or jar mentor culture can be used.
  • the acid enzyme production genes possessed by the cells Since some genes are expressed only in solid media, it is preferred to use the solid culture method for this invention! /.
  • the medium component used for the culture may be either animal or vegetable, but preferably contains a plant nutrient source such as brown rice, bran, rice bran, soybean, barley and the like. It is preferable to contain. Among these, brown rice is particularly preferable as a nutrient source. Thereby, the production efficiency of acidic enzymes such as acidic amylase can be increased.
  • sugars such as glucose, sucrose, and molasses can be added as other carbon sources
  • ammonia salts such as ammonia, ammonium chloride, ammonium chloride, and ammonium nitrate can be added as nitrogen sources.
  • the culture of the genus Aspergillus obtained as described above is preferably dried.
  • it is also preferable to improve the quality stability by performing a process such as further adding an optional component for enhancing the storage stability.
  • the drying method is not particularly limited.
  • ventilation drying is preferably used among the forces that can be performed by ventilation drying, natural drying, spray drying, freeze drying, and the like.
  • freeze-drying can also be used, you may add a protective agent in that case.
  • the type of the protective agent is not particularly limited, but it is preferable to use one or more of skim milk, sodium glutamate and saccharide strength.
  • the type of saccharide is not particularly limited, but glucose or trehalose is preferably used.
  • oxygen scavenger and dehydrating agent to the resulting dried product to make the gas nootropic It is preferable to store it in an aluminum bag and store it at room temperature to low temperature. This makes it possible to preserve the cells while alive for a long time.
  • the Bacillus subtilis used in the animal feed additive of the present invention is the "Bacillus subtilis" in the 9th edition (1994) of Bergey's Manual of Determinative Bacteriology.
  • the bacterium is not particularly limited as long as it is classified as (Bacillus subtilis).
  • Examples of such bacteria include Bacillus subtilis DB9011 strain, NBRC3009 strain, NBRC3025 strain, NBRC3108 strain, and NB RC3336 strain.
  • DB9011 stock is the Institute for Microbiological Technology, Ministry of International Trade and Industry, Ministry of International Trade and Industry (currently, the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center, 1-chome Tsukuba, Ibaraki, Japan 305_8566 No.
  • NBRC3009, NBRC3025, NBRC3108, and NBRC3336 strains are registered in the Biological Genetic Resources Division (NBRC) of the National Institute of Technology and Evaluation.
  • Heart infusion agar medium good growth. No gloss. The surface of the colony is wrinkled, and the color of the colony is white.
  • PDA medium good growth. No gloss. The surface of the colony is wrinkled and the color of the colony is white.
  • the Bacillus subtilis DB9011 strain, NBR C3009 strain, NBRC3025 strain, NBRC3108 strain, NBRC3336 strain may be naturally mutated, or mutated with chemical mutagens, ultraviolet rays, etc. Strains having the same mycological properties as each strain can be selected and used. As a mutant of Bacillus subtilis DB9011, it is preferable to select and use a strain having the above-mentioned mycological properties.
  • the concentration of Bacillus subtilis is 2.5 ⁇ 10 7 to 2 ⁇ 10 9 CFU / g, preferably 5 ⁇ 10 7 to 1 ⁇ 10 9 CFU / g, more preferably 1 X 10 8 to 5 X 10 8 CFU / g.
  • the concentration of Bacillus subtilis is preferably 2.0X10 3 to 5.0X10 6 CFU per animal feed additive exhibiting a total of 1U of acidic enzyme activity.
  • the Bacillus subtilis used in the animal feed additive of the present invention is the Aspergillus It preferably has the ability to grow in the presence of the genus fungus and the acid enzyme produced by the fungus.
  • Bacillus subtilis used in the animal feed additive of the present invention is preferably resistant to bile acids.
  • “Resistant to bile acids” refers to the formation of spores that have the ability to germinate and grow in a medium containing a high concentration of bile acids.
  • Bile acids are tetracyclic steroids that are widely found in the bile of mammals, birds, reptiles, amphibians, and fish, and include cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, and ursodeo. Xicholic acid is included. Normally, in the body of an animal, bile acids are present in the bile as conjugated forms of glycine and taurine with amide bonds, and become sodium salts. In the present specification, the term “bile acid” includes the bile acids and salts thereof and conjugates thereof.
  • the medium containing a high concentration of bile acid is, for example, a medium containing bile powder (Oxgall, manufactured by Difco) obtained by concentrating and drying fresh bile 10 times, and the concentration of bile powder is 0 3% by mass or more, preferably 1% by mass or more, more preferably 3% by mass or more.
  • “having the ability to germinate and proliferate” means that a spore is inoculated into a medium containing a high concentration of bile acid as described above, and conditions other than the bile acid concentration are suitable for culturing Bacillus subtilis. When conditions are met, bacteria germinate, proliferation and division resume, and colonies are formed.
  • Bacillus subtilis that is resistant to bile acids can be obtained, for example, as follows. A source containing Bacillus subtilis is cultured under conditions suitable for sporulation to form spores. The obtained spores are inoculated into the above-mentioned high-concentration bile acid-added medium, cultured, and then the formed colonies are separated. From these colonies, those having the bacteriological properties of Bacillus subtilis are selected.
  • the Bacillus subtilis used for the animal feed additive of the present invention is preferably more acid resistant.
  • the bacteria reach the intestine without being killed even in the stomach.
  • “Acid-resistant” means that when bacteria are administered to animals, they do not die even under conditions inside the stomach (normally pH 3.5 to 6 with food ingestion) and reach the intestines In some cases, the number of bacteria that can grow is maintained. Since Bacillus subtilis spores are usually acid-resistant, there is no particular problem when using spores.
  • the animal feed additive of the present invention may contain a single strain of Bacillus subtilis alone, or may contain a combination of two or more strains. Among these, it is particularly preferable to use the DB9011 strain alone or in combination with other strains.
  • the method for cultivating Bacillus subtilis used for the feed additive of the present invention is not particularly limited, and can be carried out by a conventional method under appropriate conditions according to the properties of the bacteria.
  • the culture temperature is a force that can be performed at 20 to 40 ° C. Usually, it is preferable to culture at 30 to 37 ° C.
  • a culture method a liquid culture method or a solid culture method such as stationary culture, reciprocating shake culture, rotary shake culture, and jar armor culture can be used.
  • the medium components used for the culture are not particularly limited, and carbon sources such as glucose, galactose, ratatose, arabinose, mannose, sucrose, starch, starch corn starch, sugars such as molasses, organic acids such as citrate, glycerin, etc.
  • Alcohol is used as a nitrogen source, ammonia, acid ammonium, ammonium chloride, ammonium nitrate, and other ammonium salts and nitrates, sodium chloride, potassium chloride, phosphoric acid lithium, magnesium sulfate, calcium chloride, calcium nitrate.
  • Use inorganic salts such as manganese chloride and ferrous sulfate, peptone, soybean flour, defatted soybean meal, meat extract, yeast extract, etc.
  • the Bacillus subtilis used for the animal feed additive of the present invention is preferably in the form of a spore from the viewpoint of storage stability and acid resistance.
  • the culture conditions such as the composition of the medium, the pH of the medium, the culture temperature, the culture humidity, and the oxygen concentration during the culture should be adapted to the spore formation conditions during the culture cycle. Adjust it.
  • Such methods include, for example, Schaeffer, P., J. Millet, JP Aubert, “Proceedings of the National Academy of Science”, USA, 1965, Vol. 54. , P. 704-711.
  • the culture and spores of Bacillus subtilis obtained by the method described above are From this point, it is preferable to use a dry powder. Drying is preferably performed such that the water content is 20% by mass or less, for example. The method of drying and storage is the same as that for drying cultures of Aspergillus.
  • the animal feed additive of the present invention includes the above-mentioned Aspergillus sauer and Aspergillus.
  • the animal feed additive of the present invention may further contain optional components.
  • These optional ingredients are recognized as safe as feed ingredients, and can be used without particular limitation as long as they do not kill the aforementioned Aspergillus spp. And Bacillus subtilis and do not inactivate acidic enzymes.
  • immunostimulants such as ⁇ -glucan, dalcomannan, mannan oligosaccharide, seaweed, and organic acids such as darconic acid, aminoaminobutyric acid, citrate, malic acid, fumaric acid, succinic acid, pantothenic acid, and butyric acid are preferable.
  • darconic acid aminoaminobutyric acid
  • citrate malic acid
  • fumaric acid succinic acid
  • pantothenic acid pantothenic acid
  • butyric acid are preferable.
  • the animal feed additive of the present invention promotes an increase in the body weight of the animal when ingested by the animal, it can be used as an animal feed additive for promoting growth.
  • the total acid enzyme activity per lg of feed additive is 270 U or more
  • the acid amylase activity is 20 U or more
  • the sum of acid protease activity and acid carboxypeptidase activity S250 U or more and the concentration of Bacillus subtilis
  • the animal feed additive of the present invention prevents intestinal infections and the like by ingesting the animal, it may be used as an animal feed additive for prevention / amelioration of intestinal infections. it can .
  • the total acid enzyme activity per lg of feed additive is 540 U or more
  • the acid amylase activity is 40 U or more
  • the sum of acid protease activity and acid carboxypeptidase activity is 500 U or more
  • Bacillus subtilis It is preferable to prepare such that the concentration power is S, 2.5 ⁇ 10 8 CFU / g or more.
  • the animal feed additive of the present invention preferably has a low concentration of succinic acid from the viewpoint of safety.
  • concentration of succinic acid be 0.1 mg / 1 or less. More preferably, it is 1 mg / 1 or less.
  • feed of the present invention the animal feed additive of the invention to feed the total amount, 0 in the dry state. 01- 1.0 wt 0/0, preferably 0.5 02-0. 5 mass 0 / 0 , more preferably 0.004 to 0.25% by mass.
  • the total acid enzyme activity per kg of feed of the present invention is 12 U or more, preferably 17 U or more, more preferably 22 U or more, and further preferably 27 to 54000 U.
  • the concentration of batinoles subtilis is 2.5 x 10 6 to 2.
  • the acid amylase activity per kg of feed is preferably 0.1 U or more, more preferably 0.5 U or more, further preferably 1 U or more, more preferably 2 to 4000 U.
  • the total of the acidic prosthetic activity and acidic carboxypeptidase activity per kg of feed is preferably 10 U or more, more preferably 15 U or more, further preferably 20 U or more, and further preferably 25 to 50000 U.
  • the feed of the present invention can be produced by adding the animal feed additive of the present invention to a commonly used feed component.
  • the type and ingredients of the feed are not particularly limited as long as the bacterial cells contained in the animal feed additive of the present invention are not killed and the acid enzyme is not deactivated.
  • feed and pet food for animals animals Used as animal feed, such as supplements for foods, added to and mixed with food.
  • the feed of the present invention may be mixed with the feed additive for animals by adding it to the feed ingredients in a dry state, but is used in a liquid or gel form for easy mixing. You can also.
  • water, vegetable oils such as soybean oil, rapeseed oil and corn oil, liquid animal oils, water-soluble polymer compounds such as polybulal alcohol, polybulurpyrrolidone and polyacrylic acid can be used as the liquid carrier.
  • a water-soluble polysaccharide such as alginic acid, sodium alginate, xanthan gum, casein sodium, gum arabic, guar gum, tamarind seed polysaccharide in order to maintain the uniformity of the bacterial cell concentration in the feed.
  • an organic acid in order to prevent the propagation of various bacteria.
  • the feed of the present invention is a solid medium containing a nutrient source for the growth of Aspergillus spp., Aspenoreginoles.soya, Aspenoleginores 'Tamari, Aspeneginores' Foretida Culturing at least one Aspergillus spp. Selected from the group consisting of Aspergillus, Aspergillus niger and Aspergillus oryzae, and mixing the resulting culture and Bacillus subtilis separately into the feed ingredients, And by adjusting the concentration of Bacillus subtilis within the above range.
  • the feed of the present invention can be used as a feed for promoting the growth of animals.
  • Aspergillus spp. Has an antibacterial activity against pathogenic bacteria that cause intestinal infections in animals and / or a protozoan activity against coccidium, enteric infections caused by pathogenic bacteria and / or coccidium Can be used as feed for prevention and treatment.
  • the type of animal that receives the feed of the present invention includes mammals, birds, reptiles, amphibians, and fish. Among these, it can be suitably used particularly for poultry and livestock. Poultry is suitable for chickens, ducks, quails, turkeys, etc., and livestock is suitable for pigs, cattle, sheep, pupae and the like. The amount of food to be ingested by animals can be adjusted as appropriate according to the type of animal, body weight, age, sex, purpose of use, health condition, ingredients of feed, and the like.
  • the pH of the potato dextrose agar medium was adjusted to 5 and sterilized at 121 ° C for 15 minutes.
  • sodium deoxycholate was added at a concentration of lOg per liter of medium, and the genus Aspergillus stored at Akita Imano Co., Ltd. Aspergillus sojae, Aspergillus tamarii, Aspergillus foetidus, Aspergillus niger force S It grew well on the medium.
  • Aspergillus' soya AOK 210 strain Aspenregils' Tamari AOK 43 strain, Aspergillus' Foretidas AOK N4586 strain, and Aspergillus niger AOK B650 strain grew particularly well.
  • a medium containing sodium deoxycholate was inoculated with many Aspergillus spp. Using various fermented foods as a source of separation, and the strain with the best growth among the strains grown on the medium was selected. did.
  • This strain was named Aspergillus oryzae IK-05074, and was deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary.
  • the mycological properties of the strain IK-05074 are as described above.
  • Aspergillus' soya AOK210 strain, Aspergillus' Tamari AOK43 strain, Aspergillus' Foretidus AOK N4586 strain, Aspergillus' Niger AOK B650 strain and Aspergillus' Orysee IK 05074 strain were cultured using brown rice as solid medium. That is, 100 g of brown rice is soaked in water all day and night to swell, then placed in a 14 cm diameter, 10 cm deep polycarbonate container with a sterile filter on the lid to a thickness of 2 cm, and autoclaved at 121 ° C. Sterilized for 15 minutes. Each bacterial cell was inoculated into this container and cultured at 28 ° C for 5 days to produce an inoculum.
  • the brown rice swelled in the same way as above was laminated to a 30 x 40 x 10 cm stainless steel vat with a thickness of 1.5 cm, and a lid with a vent covered with a 20 x 25 cm filter was applied, They were sterilized at 121 ° C for 25 minutes in a large autoclave. After cooling this vat, the whole inoculum was cultured in advance. The vat was placed in a 28 ° C incubator and cultured for 7 days.
  • the above-mentioned acid enzyme is measured by the National Tax Agency's prescribed analysis method (Amendment 3rd Tax Agency Directive No. 1) solid koji analysis method acid resistance a amylase activity measurement method, acid protease activity measurement method and acid carboxypeptidase This was performed according to the activity measurement method.
  • Salmonella enteritidis (SE)! / Was aerobically cultured at 37 ° C for 24 hours on a standard agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.). Colonies that grew on the plate were scraped off and suspended in sterile physiological saline. Prepare 500 ml of brain heart infusion bouillon “Nissui” in a 1 L Erlenmeyer flask, and after autoclaving, the final concentration of SE will be about 1 ⁇ 0 X 10 4 ⁇ ;! ⁇ 0 X 10 5 CFU / ml Aseptically charged.
  • Aspergillus soaker AOK210 strain and Aspergillus oryzae IK 05074 crushed solid culture were aseptically added to each Erlenmeyer flask, 5 g each, to give Test Examples 1 and 2, and a triangular flask without addition of Neisseria gonorrhoeae culture was used as a control.
  • Each Erlenmeyer flask was cultured in a 37 ° C incubator under aerobic conditions with gentle stirring.
  • Clostridium perfringens (CP)! / Antheropack Kenki (Mitsubishi Gas Chemical Co., Ltd.) is used, and egg yolk-added CW agar medium (Nissui Pharmaceutical Co., Ltd.) is 37 ° Anaerobic culture was performed at C for 24 hours. Colonies that grew on the plate were scraped off and suspended in sterile physiological saline. Prepare 500 ml of Brain Heart Infusion Bouillon “Nissui” in a 1 L Erlenmeyer flask.
  • the final concentration of CP was aseptically added so that the final concentration of CP was about 1 ⁇ OX 10 4 ⁇ ; ⁇ ⁇ OX IO 5 CFU / ml.
  • Aspergillus' soya AOK 210 strain and Aspergillus oryzae IK 05074 strained solid cultures are aseptically added to each Erlenmeyer flask in 5 g portions to give Test Examples 3 and 4, and Erlenmeyer flask without addition of Aspergillus oryzae culture. was used as a control.
  • Each Erlenmeyer flask was cultured in a 37 ° C incubator under gentle anaerobic conditions using an aneropack.
  • the CP viable count method is as follows: The collected culture solution is diluted 10-fold with sterilized physiological saline, and 0.1 ml of each diluted solution is added to the yolk-added CW agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.) And anaerobic culture at 37 ° C for 24 hours using an anero-packed kenki, and the characteristic colonies that developed were counted.
  • Table 3 shows the number of viable SE and Table 4 shows the number of viable CP.
  • Escherichia coli was aerobically cultured at 37 ° C for 24 hours on a standard agar medium (Nissui Pharmaceutical Co., Ltd.). Colonies that grew on the plate were scraped off and suspended in sterile saline. Prepare 500 ml of brain heart infusion bouillon “Nissi” (manufactured by Nissui Pharmaceutical Co., Ltd.) in a 1 L Erlenmeyer flask and after autoclaving, the final EC concentration is about 1.0 X 10 5 ⁇ ;! Aseptically charged to 10 6 CFU / ml.
  • Staphylococcus aureus (SA)! / Was aerobically cultured at 37 ° C for 24 hours on a standard agar medium (Nissui Pharmaceutical Co., Ltd.). Colonies that grew on the plate were scraped off and suspended in sterile saline.
  • the EC viable count method is as follows. After the collected culture solution is diluted 10-fold with sterilized physiological saline, 0.1 ml of each diluted solution is added to the chromo-cultiform foamer “Merck” (Merck) And aerobic culture at 37 ° C for 24 hours, and the characteristic colonies developed were counted.
  • the method for measuring the number of viable bacteria in SA was to dilute the collected culture solution 10 times with sterile physiological saline, and then add 0.1 ml of each diluted solution to the yolk-added mannitol saline medium “Eiken” (Eiken). After application to Equipment (manufactured by Co., Ltd.), aerobic culture was performed at 37 ° C for 48 hours, and the characteristic colonies that had developed were counted.
  • Table 5 shows the number of viable EC and Table 6 shows the number of viable SA.
  • the viable cell count and spore count of DB9011 strain, NBRC3009 strain, NBRC3025 strain, NBRC3108 strain, and NBRC3336 strain were measured.
  • the method for measuring the number of viable bacteria in the above strains is that the collected culture solution is diluted 10-fold with sterilized physiological saline, and 0.1 ml of each diluted solution is added to a standard agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.). After application, the cells were subjected to aerobic culture at 37 ° C for 48 hours to count the characteristic colonies that had developed.
  • the method for measuring the number of spores of the above strain is as follows. First, the collected culture was diluted 10-fold with sterilized physiological saline and heat-treated at 70 ° C for 30 minutes. After cooling, dilute 10-fold with sterile saline, apply 0.1 ml of each diluted solution to a standard agar medium (Nissui Pharmaceutical Co., Ltd.), and perform aerobic culture at 37 ° C for 48 hours. And counted the characteristic colonies that developed
  • Table 7 shows the number of viable bacteria of DB9011 strain
  • Table 8 shows the number of spores.
  • NBRC3025 Viable count (CFU / ml) Test Day 0 Test Day 3 Test Day 7 Test Example 13 Go to NBRC3025 + case. Lucky Luz ⁇ ⁇ ⁇ 210 2. 2 10 4 1. 8X 10 8 4. 2X 10 8 Test Example 14 To NBRC3025 + case. IK-05074 2. 2X 10 4 1. 5X 10 8 4. OX 10 s Control NBRC3025 only 2. 2X 10 4 1. 7X10 8 3. 9X 10 s [0094] [Table 12]
  • NBRC3336 Viable count (CFU / ml) Test Day 0 Test 3 Day Test 7 Day 7 Test Example 17 NBRC3336 + Arusukiru Lus Soya AOK 210 2. 5X 10 4 1. 7X 10 8 4. OX 10 8 Test Example 18 NBRC3336 + Ruki's 'Olyse' IK-05074 2. 5X 10 4 1. 9X 10 8 4. 2X 10 8 Control NBRC3336 only 2. 5X 10 4 1. 3X 10 8 3. 3X 10 8 [0100] [Table 16]
  • Lactobacillus acidophilus (LA)! / Using an anero pack kenki (Mitsubishi Gas Chemical Co., Ltd.), BCP added plate count agar "Nissy” (Nissui Pharmaceutical Co., Ltd.) ) At 37 ° C for 72 hours. The colonies that had grown on the plate were removed with force, and floated in sterile saline. Prepare 500 ml of GAM bouillon (manufactured by Nissui Pharmaceutical Co., Ltd.) in a 1 L Erlenmeyer flask, and after autoclave sterilization, the final concentration of LA is about 1 ⁇ 0 X 10 3 ⁇ ;!
  • Bifidobacterium breve (BB)! / Using Aneropack Kenki (Mitsubishi Gas Chemical Co., Ltd.) and BL agar medium (Nissui Pharmaceutical Co., Ltd.) at 37 ° C, Anaerobic culture was performed for 48 hours. Colonies that grew on the flat plate were scraped and suspended in sterile physiological saline. 500 ml of GAM bouillon (manufactured by Nissui Pharmaceutical Co., Ltd.) is prepared in a 1 L triangular flask and the final concentration of BB after autoclave sterilization is about 1.0 X 10 4 to; 1.0 X 10 5 CFU / ml Aseptically charged.
  • Aspergillus' sor AOK 210 strain and Aspergillus olise IK — 05074 strained solid cultures are aseptically added to each conical flask into Test Examples 21 and 22, with no koji mold culture added. An Erlenmeyer flask was used as a control group. Each triangular hula The sco was cultivated in a 37 ° C incubator under anaerobic conditions using aneropack kenki under anaerobic conditions.
  • Viable counts of LA and BB were measured on days 0, 3, and 7 after the start of the test.
  • the LA viable cell count method is as follows: The collected culture solution is diluted 10-fold with sterilized physiological saline, and 0.1 ml of each diluted solution is added to the BCP-added plate count finger “Nissy” ( After application to Nissui Pharmaceutical Co., Ltd., Anaeropackenki (Mitsubishi Gas Chemical Co., Ltd.) was used for anaerobic culture at 37 ° C. for 72 hours, and the characteristic colonies developed were counted.
  • the number of viable bacteria in BB is determined by 10-fold serial dilution of the collected culture with sterile saline, and then 0.1 ml of each dilution is applied to BL agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.). Subsequently, anaerobac kenki (manufactured by Mitsubishi Gas Chemical Co., Inc.) was used for anaerobic culture at 37 ° C for 48 hours, and the characteristic colonies that had developed were counted.
  • Table 17 shows the number of viable LA and Table 18 shows the number of viable BB.
  • solid cultures of Aspergillus soja AOK210 strain and Aspergillus oryzae IK-05074 strain did not show antibacterial activity against LA.
  • solid cultures of Aspergillus' soya AOK 210 and Aspergillus oryzae IK 05074 did not show antibacterial activity against BB.
  • Bovine diarrheal stool naturally infected with Eimeria zuemii was collected, and the oocysts were separated under a stereomicroscope and washed with physiological saline. 5 ml of physiological saline was added to a petri dish with a diameter of 9 cm, and washed oocysts were added at about 2000 / ml. 50 mg of ground solid cultures of AOK 210 strain, AOK 43 strain, AOK N4586 strain, AOK B650 strain and IK-05074 strain were added at a rate of 50 mg per shear. A petri dish without the koji mold culture was used as a control group. Each petri dish was shaken (150 rpm) at 37 ° C. Seven days later, the number of oocysts was measured and the state of cell wall deformation and dissolution was observed under a stereomicroscope. The decrease rate and dissolution modification rate of oocysts were calculated.
  • Bacillus subtilis DB9011 strain, Bacillus subtilis NBRC3009 strain, Bacillus subtilis NBRC3025 strain, Bacillus subtilis NBRC31 08 strain and Bacillus subtilis NBRC3336 strain were liquid-cultured at 37 ° C for 72 hours using the following sporulation medium .
  • the obtained culture solution was centrifuged to collect bacterial cells.
  • the obtained cells were lyophilized and then pulverized to obtain spore powder.
  • the spore density of DB9011, NBRC3009, NBRC3025, NBRC3108, and NBRC3336 is 1.01 X 10 u CFU / g, 1.21 X 10 respectively.
  • the spore density is determined by diluting the obtained spore powder with sterilized water to an appropriate concentration and heating at 70 ° C for 30 minutes to kill only vegetative cells, then inoculating on a normal agar medium and forming colonies formed. The number was measured by counting.
  • Bacillus subtilis DB9011 strain was registered with FERM BP in the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st, 1st, 1st, Tsukuba, Ibaraki, Japan) Deposited as 3418.
  • the NBRC3009 strain, NBR C3025 strain, NBRC3108 strain, and NBRC3336 strain are registered in the Biological Genetic Resource Department (NBRC) of the National Institute of Technology and Evaluation.
  • Chicken chick's feed (for SD broiler early and late period, manufactured by Nippon Compound Feed Co., Ltd., feed with no antibacterial substances) Production Example;! ⁇ 8 feed additive is 0.1% by mass Mixed as It was.
  • a group of 12 chicken chicks hatched from eggs from broiler chickens (brand: Chunky) was given to each group for 35 days, and a growth test was conducted. Breeding with constant feed and free water. In addition, a growth test was conducted in the same manner, using a feed mixed with 0.1% by mass of lactose alone as a control group. The weight of chicken chicks in each group after 35 days was measured, and the average value of each group was calculated.
  • Production Example 5 (B in which a culture of AOK 210 strain or IK-05074 strain 1.0 mass% or more and DB9011 strain or NBRC3009 strain 1.0 x 10 8 CFU / g or more were combined.
  • -G Production Example 6
  • B-G Production Example 7
  • B-G Production Example 8
  • Total feed weight of piglets (standard feed for pre- and post-stage periods of SD piglet artificial milk, manufactured by Nippon Compound Feed Co., Ltd., feed containing no antibacterial substances), production examples;! ⁇ 8 feed additives 0 It was mixed to 1% by mass.
  • a 3-week-old piglet (brand: large Yorkshire) was grouped, and each group was fed with the above-mentioned feed on a continuous basis for 35 days. Further, a growth test was conducted in the same manner, using a feed mixed with 0.1% by mass of lactose alone as a control group. The weight of piglets in each group after 35 days was measured, and the average value of each group was calculated.
  • Feed supplements of Production Example 1 (EG) and Production Example 2 (EG) containing 15% by mass or more of the culture of Aspergillus 'soya AOK 210 strain or Aspergillus' Oryzae IK 05074 strain The piglets that received the additive showed significant weight gain compared to the control piglets.
  • Production Example 5 (B to G) in which a culture of 1.0% by mass or more of AOK 210 strain or IK-05074 strain and spore of DB9011 strain or NBRC3009 strain is combined at least 1.0 X 10 8 CFU / g,
  • Production Example 6 (B to G)
  • Production Example 7 (B to G)
  • Production Example 8 (B to G) were ingested by animals, a significant effect of weight gain was observed.
  • Aspergillus and Bacillus subtilis it was possible to promote weight gain of piglets at a lower concentration than when each active ingredient was used alone.
  • the feed additives of Production Examples 9 to 12 are 0.1% by mass. So that they were mixed.
  • a group of 12 chicken chicks hatched from eggs from broiler chickens (brand: Chunky) was given to each group for 35 days, and a growth test was conducted. Breeding with constant feed and free water. In addition, a growth test was conducted in the same manner, using a feed mixed with 0.1% by mass of lactose alone as a control group.
  • Table 25 shows the results of measuring the weight of chicken chicks in each group after 35 days and calculating the average value of each group.
  • the ratio of the ground solid culture of Aspergillus soja AOK 210 strain or Aspergillus oryzae IK 05074 strain to DB9011 strain or NBRC3009 strain is 4% by mass: 1X10 8 CFU / g ⁇ ; 8 ?
  • the range of 1; / ⁇ was optimal (Production Example 9 (B to F), Production Example 10 (8 to?), Production Example 11 (8 to?), Production Example 12 (8 to?)). From this, it was found that the optimum concentration of Bacillus subtilis per feed additive exhibiting 1U acid-resistant ⁇ -amylase activity was in the range of 0.5 ⁇ 10 6 to 2.0X10 7 CFU / g. It was also found that the concentration of Bacillus subtilis per feed additive indicating the sum of 1U acidic protease and acidic carboxypeptidase was in the range of 4.5X10 4 to 8.5X10 5 CFU / g.
  • feed mass of piglet (standard feed for pre- and post-stage of SD piglet artificial milk, manufactured by Nippon Compound Feed Co., Ltd., feed containing no antibacterial substances) It mixed so that it might become 1 mass%.
  • a 3-week-old piglet (brand: large Yorkshire) was grouped, and each group was fed with the above-mentioned feed on a continuous basis for 35 days.
  • a growth test was carried out in the same manner, using a feed mixed with 0.1% by mass of lactose alone as a control. After 35 days, the weight of each group of piglets was measured, and the average of each group was calculated.
  • the ratio of the ground solid culture of Aspergillus soja AOK 210 or Aspergillus oryzae IK 05074 to DB9011 or NBRC3009 is 4% by mass: 1 X 10 8 CFU / g to 1% by mass: 4
  • the range of 0 X 10 8 CFU / g was optimal (Production Example 9 (B to F), Production Example 10 (B to F), Production Example 11 (B to F), Production Example 12 (B to F ))
  • the cecal contents lg was diluted 10-fold with sterile phosphate buffered saline and mixed well to obtain a sample stock solution.
  • the sample stock solution was serially diluted 10 times with sterile physiological saline to obtain a serially diluted solution.
  • the sample stock solution and serial dilution were added to the SS agar plate Sy ”(manufactured by Nissui Pharmaceutical Co., Ltd.) and brilliant green agar plate medium (manufactured by Difco Laboratories) are smeared by 0.1 ml each, cultured at 37 ° C for 24 hours, and typical SE grown on each plate medium. The number of colonies was measured.
  • the fungus from the colony is used to inoculate lysine decarboxylation test into SIM agar medium “Nissi” (Nissui Pharmaceutical Co., Ltd.) and TSI agar medium “Nissi” (Nissui Pharmaceutical Co., Ltd.). The culture was confirmed at 37 ° C for 24 hours.
  • the number of viable SE per cecal content lg was calculated by multiplying the number of colonies recognized as SE from this by the dilution factor of the diluent. Based on this result, the infection index and the control index were calculated as follows.
  • the infection index is a value indicating the high infection rate of pathogenic bacteria, and the defense index indicates the ability of each feed to protect against infection by pathogens when compared to the control group. Value.
  • Infection index logarithm average of the number of viable SE in the cecal contents of each individual (average value of log CFU / g)
  • the fungus from the colony was inoculated into LIM agar medium “Nissi” (manufactured by Nissui Pharmaceutical Co., Ltd.), SIM agar medium and TSI agar medium, and cultured at 37 ° C for 24 hours to confirm the properties. .
  • Table 27 shows the infection index
  • Table 28 shows the protection index
  • Table 29 shows the number of SE detected individuals in the total excretory cavity collection culms.
  • chicken chicks were bred in the same manner as described above.
  • 5.0 ⁇ 10 7 CFU of Clostridium perfringens (CP) was orally administered per bird.
  • sputum was collected by wiping the contents of the cecum and the total excretory cavity with a cotton swab.
  • the cecal contents lg was diluted 10-fold with sterile phosphate buffered saline and mixed well to obtain a sample stock solution.
  • the sample stock solution was serially diluted 10 times with sterile physiological saline to obtain a serially diluted solution.
  • the fungi were picked from the colonies, inoculated into egg yolk-added CW agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.), and the properties were confirmed by aerobic and anaerobic culture at 35 ° C for 24 48 hours.
  • the number of viable CP per cecal content lg was calculated by multiplying the number of colonies recognized as CP from the dilution factor of the diluent. Based on this result, the infection index and the control index were calculated in the same manner as described above.
  • CP For sputum collected from the total excretory cavity, the properties of CP were confirmed by qualitative culture for each individual by the following method. Specifically, after suspending wrinkles attached to a cotton swab in 10 ml of sterile phosphate buffered saline to prepare a sample stock solution, 0.1 ml of this was applied to a Clostridia medium (manufactured by Nissui Pharmaceutical Co., Ltd.). It was sprayed and anaerobically cultivated at 35 ° C for 24 hours using an aneropack, and the presence or absence of black colonies grown on each plate medium was determined.
  • Clostridia medium manufactured by Nissui Pharmaceutical Co., Ltd.
  • the fungi were picked from the colonies, inoculated into egg yolk-added CW agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.), and the properties were confirmed by aerobic and anaerobic culture at 35 ° C for 24 48 hours.
  • Table 30 shows the infection index
  • Table 31 shows the protection index
  • Table 32 shows the number of detected CPs in the total excretory cavity.
  • the Asuperugirusu 'Soya AOK210 strain also Asuperugirusu' oryzae IK- 0 5074 strain, the combination of Bacillus subtilis DB9011 strain or NBRC3009 strain, the AOK210 strain also (or IK- 05074 strain 2.5 mass 0/0 or more , DB9011 strain (or NBRC300 9 strain added to feed additive at 2.5 X 10 8 CFU / g or more, AOK 210 strain or IK — 05074 strain alone was added to feed additive at 20% by mass or more The same CP control effect was obtained as when the DB9011 strain or the NBRC3009 strain alone was added to the feed additive at a concentration of 2.0 X 10 9 CFU / g or more (Production Example 5 (C to G ), preparation 6 (C to G), preparation 7 (C to 0), production example 8 (Ji to 0)).
  • the AOK210 strain also IK- 05074 strain 20 mass 0/0 or more
  • DB9011 strain or NBRC3009 strain was added to feed additive at 2.0 X 10 9 CFU / g or more
  • CP could be completely controlled (Production Example 5 (G), Production Example 6 (F, G ) Production Example 7 (G), Production Example 8 (G)).
  • Aspergillus' soya AOK210 strain or Aspergillus oryzae IK 05074 strain with Bacillus subtilis DB9011 strain or NBRC300 9 strain, CP infection at a lower concentration than when each active ingredient is used alone. It can be seen that there is an effect to prevent.
  • Centrifugation was performed at 4 ° C (3,000 rpm, 15 minutes), and the pellet was placed in an enteric capsule.
  • 4.5 X 10 8 CFU of EC per head was administered orally once daily for 3 days using enteric capsules.
  • Saline was placed in an enteric capsule in place of the edema disease fungus and administered by oral gavage in the same manner as a non-inoculated case.
  • the body weight was measured and the average weight gain of each group was calculated.
  • Table 33 shows the weight gain during the test
  • Table 34 shows the stool property score
  • Table 35 shows the periocular edema score.
  • Periocular edema score 0. None, 1. Mild, 2. Moderate, 3. Severe Aspergillus' soya AOK 210 strain or Aspergillus olise IK-05074 strain solid culture of 15% by weight or more by itself When added to the additive, the weight gain was similar to that in the non-inoculated case (Production Example 1 (E to G), Production Example 2 (E to G)). Bacillus subtilis DB9011 strain or NBRC3009 strain alone was 15 x 10 8 CFU / g or more, and when added to a feed additive, the weight gain was similar to that in the non-inoculated case (Production Examples 3 to 0) , Production Example 4-0)).
  • the animals were raised to 7 weeks of age and weighed, and the average weight gain for each group was calculated.
  • the fecal properties were observed every day until the end of the gavage test, and the total fecal property score during the test period was calculated for each individual, and the average score of each group was calculated.
  • Table 36 shows the weight gain during the test period and Table 37 shows the stool property score.
  • Nutrient absorption is promoted by mixing the feed containing an animal feed additive containing Aspergillus spp. And the acidic enzyme produced by the fungus of the present invention and an animal feed additive containing Bacillus subtilis into the feed. , Feed efficiency will increase. Specifically, the acid enzyme produced by Aspergillus spp. Shows antibacterial activity against intestinal pathogens, etc., and Bacillus subtilis activates immunity, resulting in the prevention of intestinal infections in animals And contribute to animal weight gain.
  • the feed of the present invention can be suitably used for breeding livestock such as chickens, pigs and cows.

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Abstract

La présente invention concerne des moyens sûrs et pratiques destinés à soutenir l'activité digestive d'un animal et accroître l'efficacité de l'alimentation animale. L'invention porte plus spécifiquement sur des moyens permettant de prévenir/traiter une maladie infectieuse par inhibition de la prolifération d'une bactérie pathogène ou d'un coccidium présent dans les intestins de l'animal et, par conséquent, augmenter le poids corporel de l'animal. L'une au moins des bactéries Aspergillus sélectionnée parmi l'Aspergillus sojae, l'Aspergillus tamarii, l'Aspergillus foetidus, l'Aspergillus niger et l'Aspergillus oryzae, une culture contenant une enzyme acide produite par ladite bactérie et un Bacillus subtilis sont administrés à un animal en une quantité donnée.
PCT/JP2007/065638 2006-08-24 2007-08-09 Additif pour alimentation animale WO2008023580A1 (fr)

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JP2009232793A (ja) 2008-03-27 2009-10-15 Genichiro Soma 飼料添加剤、飼料、その製造方法、斃死予防剤、及び飼育方法
KR101333732B1 (ko) * 2010-11-02 2013-11-27 건국대학교 산학협력단 항산화효과가 우수한 양파발효액을 이용한 삼계닭 항생제 대체 사료첨가제 및 삼계닭 계육 품질유지제의 제조 방법과 그 용도
WO2020162599A1 (fr) 2019-02-08 2020-08-13 出光興産株式会社 Aliment fonctionnel pour animaux
CN113528375A (zh) * 2021-06-16 2021-10-22 东北农业大学 一种具有抑制多种致病菌微生物生长的民猪源枯草芽孢杆菌
CN114711347A (zh) * 2022-05-16 2022-07-08 华东理工大学 提高海水养殖鱼类抗病能力的组合制剂、其制备方法及应用
CN115181675A (zh) * 2022-05-06 2022-10-14 南京思农生物有机肥研究院有限公司 一种贵州木霉菌促生伴侣溜曲霉及其应用

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KR101980098B1 (ko) * 2018-05-23 2019-05-20 대봉엘에스 주식회사 프로바이오틱스 배양용 배지 조성물 및 이를 이용한 어패류의 양식방법
KR102174589B1 (ko) * 2018-08-03 2020-11-05 부경대학교 산학협력단 뱀장어 사료내 항생제 대체를 위한 신바이오틱스 개발
KR20200107373A (ko) 2019-03-07 2020-09-16 농업회사법인 바이오릭스(주) 사료첨가제조성물 및 상기 조성물을 포함하는 동물용 사료
CN110150487A (zh) * 2019-05-09 2019-08-23 淮安市澳华农牧有限公司 缓解断奶仔猪应激反应的lca饲料及饲喂方法
CN110438015B (zh) * 2019-09-04 2022-09-16 桂林理工大学 产橙皮苷酶的枳实内生真菌及其发酵产橙皮苷酶的方法

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Publication number Priority date Publication date Assignee Title
JP2009232793A (ja) 2008-03-27 2009-10-15 Genichiro Soma 飼料添加剤、飼料、その製造方法、斃死予防剤、及び飼育方法
KR101333732B1 (ko) * 2010-11-02 2013-11-27 건국대학교 산학협력단 항산화효과가 우수한 양파발효액을 이용한 삼계닭 항생제 대체 사료첨가제 및 삼계닭 계육 품질유지제의 제조 방법과 그 용도
WO2020162599A1 (fr) 2019-02-08 2020-08-13 出光興産株式会社 Aliment fonctionnel pour animaux
CN113528375A (zh) * 2021-06-16 2021-10-22 东北农业大学 一种具有抑制多种致病菌微生物生长的民猪源枯草芽孢杆菌
CN113528375B (zh) * 2021-06-16 2022-07-12 东北农业大学 一种具有抑制多种致病菌微生物生长的民猪源枯草芽孢杆菌
CN115181675A (zh) * 2022-05-06 2022-10-14 南京思农生物有机肥研究院有限公司 一种贵州木霉菌促生伴侣溜曲霉及其应用
CN115181675B (zh) * 2022-05-06 2024-06-04 南京思农生物有机肥研究院有限公司 一种贵州木霉菌促生伴侣溜曲霉及其应用
CN114711347A (zh) * 2022-05-16 2022-07-08 华东理工大学 提高海水养殖鱼类抗病能力的组合制剂、其制备方法及应用
CN114711347B (zh) * 2022-05-16 2023-11-07 华东理工大学 提高海水养殖鱼类抗病能力的组合制剂、其制备方法及应用

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