WO2008023580A1 - Animal feed additive - Google Patents

Abstract

Disclosed is a safe and convenient means for supporting the digestive activity in an animal and increasing the feed efficiency. Specifically, disclosed is a means for preventing/treating an infectious disease by inhibiting the proliferation of a pathogenic bacterium or a coccidium in the intestine of the animal and, consequently, increasing the body weight of the animal. At least one Aspergillus bacterium selected from Aspergillus sojae, Aspergillus tamarii, Aspergillus foetidus, Aspergillus niger and Aspergillus oryzae, a culture containing an acidic enzyme produced by the bacterium, and Bacillus subtilis are administered to an animal in specified amounts.

Description

 Specification

 Animal feed additive

 Technical field

 [0001] The present invention relates to an animal feed additive containing Aspergillus spp. And Bacillus spp. Having the ability to produce acid enzymes.

 Background art

 [0002] Feeds such as livestock and pets (hereinafter referred to as “animals! /”) Are processed, such as pulverization, but generally are not heat-treated. There is a problem of poor feed efficiency. Recently, 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. Known 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.

 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.

[0004] On the other hand, animal feed is treated with enzymes and enzyme-producing bacteria at the processing stage to obtain a certain amount of feed. It has been reported that techniques for reducing the burden on the gastrointestinal tract of animals by being understood are useful for the prevention and improvement of diseases of the digestive system (Patent Document 3). In addition, a method is known in which fish meal is fermented with koji mold at low moisture to obtain a fish meal fermented feed for fish containing protease, lipase and the like in high concentrations (Patent Document 4). However, this technique has a problem that it requires a complicated process of increasing the water content in the feed, decomposing the feed components, and then drying to produce a product. Furthermore, feeds with a high water content due to decomposition of components also have problems such as the occurrence of spoilage fungi and mold and the difficulty of maintaining quality immediately.

 In addition, it has been reported that orally administering gonococcal spores to animals by modifying the animal's sputum S, suppresses the growth of toxic bacteria that cause inflammation in the intestines of animals, The use of promoting the increase has not been studied, and it has not been known to administer it to animals in a form in which an acid enzyme is produced by Aspergillus oryzae (Patent Document 5).

 In addition, 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. Alternatively, it is described that two or more species are inoculated to decompose chitin or chitosan to obtain 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. In addition, mixing two or more microorganisms and administering them to animals has not been studied.

 [0005] In addition, 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). However, it has been studied so far that Bacillus subtilis is ingested by animals in combination with other strains of microorganisms!

 [0006] 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 Ioana 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

 Disclosure of the invention

 [0007] 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.

[0008] As a result of diligent research to solve the above-mentioned problems, the present inventors have found that 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. By combining these cells and acid enzymes produced by these cells and ingesting them with feed, they promote digestion, prevent intestinal infections, and contribute to animal weight gain. I found out. Further, it has been found that the growth of Bacillus subtilis is not inhibited even when it coexists with the aforementioned Aspergillus genus bacteria and the acidic enzyme produced by the bacteria. The animal is combined with Aspergillus genus bacteria, the acidic enzyme produced by the bacteria, and Bacillus subtilis. It has been found that a further excellent growth promoting effect and intestinal infection prevention / improvement effect can be obtained by ingesting in the present invention, and the present invention has been completed.

 That is, the present invention is as follows.

(1) 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.

 (2) The animal feed additive according to (1), wherein the acid enzyme contains an acid amylase and has an acid amylase activity S 1U or more per lg of feed additive.

 (3) The animal according to (1) or (2), characterized in that it has antibacterial activity against the pathogenic bacteria causing the intestinal infection of the animal, and / or protozoan activity against coccidium. Feed additive.

 (4) The animal feed additive according to any one of (1) to (3), which is Aspergillus sp. And / or Aspergillus-olise.

 (5) 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 (1 The animal feed additive according to any one of (1) to (4).

 (6) The animal feed additive according to any one of (1) to (5), wherein the culture contains a plant nutrient source.

 (7) The animal feed additive according to (6), wherein the plant nutrient source is brown rice.

 (8) The animal feed additive according to any one of (1) to (7), wherein the Bacillus subtilis is DB9011 strain (FERM BP-3418) and / or a mutant thereof.

 (9) The animal feed additive according to any one of (1) to (8), which is for growth promotion.

(10) Prevention of intestinal infection · For any improvement, as described in any one of (1) to (8) Animal feed additives listed.

 (11) A feed comprising the animal feed additive according to any one of (1) to (; 10) from 0.01 to 1.0% by mass.

(12) A solid medium containing nutrient sources for the growth of Aspergillus spp., Aspergillus.soya, Aspenoleginoles.Tamari, Aspenreginoles 'Foretidas, Aspenreginoles' Niger and Aspergillus Culturing at least one Aspergillus genus selected from the group consisting of a step of containing the resulting culture so that the total acid enzyme activity per kg of feed is 12 U or more, and a concentration of Bacillus subtilis of 2 . 5 X 10 ⁶ to 2. OX 10 ^{1Q A} method for producing a feed, comprising a step of containing CFU / kg.

 (13) An animal breeding method characterized by causing an animal to ingest the feed according to (11). BEST MODE FOR CARRYING OUT THE INVENTION

[0009] 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.

 [0010] Aspergillus' soya, Aspergillus. Tamari, Aspergillus' foetidas, Aspergillus' Niger and Aspergillus contained in the animal feed additive of the present invention

-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. 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) ”.

[0011] Aspergillus soja is a kind of incomplete filamentous fungus found in soil, koji, etc., and is used for brewing soy sauce and miso. In the animal feed additive of the present invention, the ability to produce at least one, preferably acidic amylase, among the acidic enzymes described in detail below. If it has strength and is safe for feeding to animals, it can be used without particular limitation. In the animal feed additive of the present invention, Aspergillus soya AOK 210 (Akita Imano Shoten Co., Ltd.) can be preferably used as such a fungus that may use a commercially available strain.

 [0012] 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. In the animal feed additive of the present invention, Aspergillus tamari AOK 43 strain (Akita Imano Co., Ltd.) can be preferably used as such a bacterium that may use a commercially available strain.

 [0013] 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. In the animal feed additive of the present invention, 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.

[0014] 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. In the animal feed additive of the present invention, Aspergillus niger AOK B650 strain (Akita Imano Shoten Co., Ltd.) can be preferably used as a fungus such as a commercially available strain.

[0015] In addition, 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. Furthermore, it is also preferable to use a mutant strain having the same mycological properties as those of the AOK 210 strain, AOK 43 strain, AOK N4586 strain or AOK B650 strain other than those described above.

 [0016] 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. In the animal feed additive of the present invention, 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. On February 15, 2006, the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (T305-8566, Tsukuba Sakai Higashi, Ibaraki, Japan) 1-chome No. 1 1-Chuo 6) was deposited under the deposit number FERM P-20798, transferred to an international deposit under the Budapest Treaty on June 20, 2006, and given the deposit number FERM BP-10622. Yes.

 [0017] The mycological properties of the strain IK-05074 are as follows.

 (1) Colony properties (Czapek—Dox agar medium (cultured at 25 ° C for 7 days))

 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.

 (2) Form

 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.

[0018] From the above, it was estimated that the strain IK 05074 belongs to Aspergillus oryzae.

 [0019] In the animal feed additive of the present invention, 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.

 [0020] Further, in the animal feed additive of the present invention, for example, 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! /.

 [0021] 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.

[0022] 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. Although not particularly limited, those having an optimum pH of 2.5 to 5.5 are preferable. For example, acidic α-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. Among these, in particular, it is preferable to contain 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 α-amylase, / 3-amylase, and darcoamylase. . Among these, acid-resistant α-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!

[0023] 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. Further, 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 α-amylase activity measurement method, acid protease activity measurement method, and acid carboxypeptidase activity measurement method.

[0024] 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 The 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. Usually, 0.25 to 20% by mass, preferably 0.25 to 20% by mass of the culture obtained by culturing the above-mentioned bacteria for about 7 days at 28 ° C using a sufficient amount of plant nutrients such as brown rice 0.5 to 10% by mass, more preferably 1 to 5% by mass. [0025] Further, 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. pullorum, galiinarum, typhis S. typhimurium, S. enteritidis, S. choleraesuis, and derby dublin. 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.

 [0026] 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 (¾E, 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.

In addition, 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.

 [0027] Further, 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). To be specific, 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. are mentioned as protozoa that prey on E mena moth. Examples of 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 Τ · 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 Εtenella, E. zuernii.

 [0028] 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. tenella and Ε zuernii oocysts are suspended, and incubate at 37 ° C. To check the deformation or dissolution of the oocysts. In this way, the bacteria contained in the petri dish are cultured, and the bacteria belonging to Aspergillus soja, Aspenoleginoles. Tamari, Aspenoleginoles' foetidas, Aspenoleginoles' Niger, Aspergillus' olise 1 As a result, the Aspergillus spp. Used in the present invention can be obtained. By administering the bacteria thus obtained to animals, intestinal coccidiosis in animals can be prevented and treated. [0029] In addition, whether or not the growth of coccidium is suppressed can be confirmed by, for example, observing the oocysts of coccidium in the cecum contents of animals under a microscope.

 [0030] 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. As a result, it is considered that the bacterial cells produce useful acidic enzymes in the intestines of animals and the function of assisting digestion by the acidic enzymes is maintained. In addition, when 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. Being resistant to gastric acid and bile acids means reaching the intestine where the bacteria do not die under normal conditions in the digestive tract. Normally, the animal's stomach can reach a pH of 2 or below when fasting. When food is ingested, the pH of the stomach is higher in the range of 3.5 to 6 than when fasting. Therefore, 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. Furthermore, 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.

 [0031] 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.

[0032] 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.

 [0033] 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. For example, the cultivation temperature is a force that can be performed at 25 ° C to 40 ° C. Usually, it is preferable to culture at 28 to 32 ° C. In addition, as the culture method, a liquid culture method or a solid culture method such as reciprocating shaking culture or jar mentor culture can be used. Among 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! /.

 [0034] 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.

 In addition, sugars such as glucose, sucrose, and molasses can be added as other carbon sources, and ammonia salts such as ammonia, ammonium chloride, ammonium chloride, and ammonium nitrate can be added as nitrogen sources.

 [0035] The culture of the genus Aspergillus obtained as described above is preferably dried. In addition, 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. For example, ventilation drying is preferably used among the forces that can be performed by ventilation drying, natural drying, spray drying, freeze drying, and the like. Moreover, although 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. In addition, the type of saccharide is not particularly limited, but glucose or trehalose is preferably used. Furthermore, after drying, add 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.

 [0036] 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. 6) was deposited as FERM BP-3418 on May 21, 1991. The DB9011 strain was classified as Bacillus licheniformis at the time of deposit, but has been confirmed to belong to Bacillus subtilis. The NBRC3009, NBRC3025, NBRC3108, and NBRC3336 strains are registered in the Biological Genetic Resources Division (NBRC) of the National Institute of Technology and Evaluation.

 [0037] The mycological properties of DB9011 strain are as follows.

 [0038] [Table 1]

Item DB901 1

 Expansion of bacterial cells ―

 7% NaCl +

N0 ₃ → N0 ₂ +

 VP reaction +

 Egg * york

 Indole

 60 ° C ―

 Grams +

 Spore position center

 No gloss

Colony surface wrinkles [0039] Form:

 It is a koji mold with a cell width of 0.7 to 0.8 ^ 111. An elliptical spore exists in the middle and does not expand the cells. It is motile and forms R-type colonies. Does not grow under anaerobic conditions.

 [0040] Growth state in each medium:

 (1) DHL agar medium: Not grown.

 (2) McConkey agar: not growing.

 (3) Mannit salt medium: good growth. There is gloss. The color of the colony that wrinkles the surface of the colony is yellow.

 (4) Ordinary agar medium: good growth. No gloss. The surface of the colony is wrinkled, and the color of the colony is white.

 (5) Heart infusion agar medium: good growth. No gloss. The surface of the colony is wrinkled, and the color of the colony is white.

 (6) Blood agar medium (with 10% sheep blood)

 Good growth. No gloss. The surface of the colony is wrinkled, and the color of the colony is white.

 (7) PDA medium: good growth. No gloss. The surface of the colony is wrinkled and the color of the colony is white.

 [0041] Physiological properties:

 Gram reaction: +

 Gelatin test:

 Growing condition: Full liquefaction

 Gelatin liquefaction; +

 Limasmas Minorek:

 Reaction; acid

 Condition; coagulation

 Nitrate reduction: +

 Denitrification reaction:

MR test: Generation:

 Production of hydrogen sulfide:

 Use of citrate

 Urease:

 Oxidase: +

 Catalers'. +

 Range of growth:

 pH; 4-9

 Temperature; 25-50 ° C

 OF test: Fermentation and gas generation (due to glucose degradation)

[0042] Usability of saccharides

 Gas generation Acid generation

 L-Arabinos

 D—Xylose

 D—Gnore Course

 D—Mannose

 D—fructose

 D—galactose

 Maltose (maltose)

 Sucrose

 Lactose (Lattose)

 Toreno loin

 D- D-

[0043] Degradation of esculin: Use of malonic acid:

 Decomposition of arginine: +

 Lysine decarboxylation: +

 Urea decomposition: soil

 Aflatoxin front angle: +

 Ornitine decarboxylation:

 Core Grace:

 Hemolytic: +

 Resistance of sodium chloride: 10% or less

 Potassium cyanide tolerance: growth possible

 Lecithinase:

 [0044] Further, in the animal feed additive of the present invention, 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.

In the animal feed additive of the present invention, 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.

[0046] Further, in the animal feed additive of the present invention, 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. 1.0X10 ^{4 to} 1.0X10 ⁶ CFU or per animal feed additive showing 1U acid amylase activity 1. OX10 ⁵ to 2.5X10 ⁸ CFU, preferably 5.0 X10 ^{5 to} 5. OX10 ⁷ OX10 ^{3 to} 5.0 X10 ⁶ CFU, preferably 1 ΟΧ 10 ⁴ to 1 OX10 per animal feed additive that is adjusted to be CFU or shows the sum of 1U acidic protease activity and acidic carboxypeptidase activity It can also be made ⁶ CFU.

[0047] 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.

 [0048] The 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.

[0049] 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. In addition, “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.

 [0050] 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.

[0051] The Bacillus subtilis used for the animal feed additive of the present invention is preferably more acid resistant. By using such bacteria, 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.

 [0052] 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.

 [0053] 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. For example, 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. In addition, as 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.

 [0054] 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. In the spore state, since it is resistant to heat and drying, it can be sufficiently dried when producing a feed additive, and storage stability is improved. In order for bacteria to form spores, 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.

[0055] In addition, 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.

[0056] The animal feed additive of the present invention includes the above-mentioned Aspergillus sauer and Aspergillus.

 'Tamari, Aspergillus' Foetidas, Aspergillus' Niger and Aspergillus. All or part of the culture containing at least one Aspergillus sp. And can be obtained by mixing.

 [0057] Further, 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. For example, immunostimulants such as β-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. Can be mentioned.

[0058] Since 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. In this case, in particular, 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 However, it is preferable to prepare such that it becomes 1 × 10 ⁸ CFU / g or more.

[0059] Since 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 . In this case, in particular, 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, and Bacillus subtilis It is preferable to prepare such that the concentration power is S, 2.5 × 10 ⁸ CFU / g or more.

[0060] Further, the animal feed additive of the present invention preferably has a low concentration of succinic acid from the viewpoint of safety. In general, it is preferable that the concentration of succinic acid be 0.1 mg / 1 or less. More preferably, it is 1 mg / 1 or less.

[0061] 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. OX 10 ¹⁰ CFU / kg, preferably 5.0 x 10 ⁶ to 1. OX 10 ¹⁰ CFU / kg, more preferably 1.0 x 10 ^~ 5. it is a 0 X 10 ⁹ CFU / kg. Further, 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. Further, 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.

 [0062] 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. Usually, feed and pet food for animals, animals Used as animal feed, such as supplements for foods, added to and mixed with food.

 In addition, 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. In this case, 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. It is also preferable to add 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. . It is also preferable to add an organic acid in order to prevent the propagation of various bacteria.

[0063] Further, 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.

[0064] The feed of the present invention can be used as a feed for promoting the growth of animals. In addition, if the above-mentioned 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.

 [0065] There are no particular restrictions on the type of animal that receives the feed of the present invention, and examples include 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.

 [0066] As a method of ingesting the feed, a commonly used method can be adopted depending on the kind of the animal.

 Example

 [0067] <1> Culture of Aspergillus

The pH of the potato dextrose agar medium was adjusted to 5 and sterilized at 121 ° C for 15 minutes. When the temperature of this agar medium dropped to 60 ° C, 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. Among them, Aspergillus' soya AOK 210 strain, Aspenregils' Tamari AOK 43 strain, Aspergillus' Foretidas AOK N4586 strain, and Aspergillus niger AOK B650 strain grew particularly well. [0068] In the same manner as above, 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.

 [0069] 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.

 Next, 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.

 After culturing, it was air-dried at 35 ° C and pulverized with a jet mill to obtain a solid culture for each bacterial species.

 [0070] The acid-resistant a amylase activity per lg of each solid culture and the sum of the acid protease activity and the acid carboxypeptidase activity were measured. Table 2 shows the enzyme activity per gram of each culture.

 In addition, 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.

[0071] [Table 2] Table 2

 [0072] Measurement of antibacterial activity of cultures of <2> Aspergillus spp.

 The antibacterial activity of these Aspergillus species was tested by co-culturing the bacteria with Aspergillus soja AOK 210 strain and Aspergillus oryzae strain IK 05074.

[0073] 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.

[0074] 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. After sterilization by 1-clave, the final concentration of CP was aseptically added so that the final concentration of CP was about 1 · OX 10 ⁴ 〜; ί · 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.

 [0075] On the 0th, 3rd and 7th days after the start of the test, viable counts of SE and CP were measured. The number of viable bacteria in SE was measured by diluting the collected culture solution 10-fold with sterile physiological saline, and then applying 0.1 ml of each diluted solution to X-SAL agar medium “Nissui”. Then, aerobic culture was performed at 37 ° C for 24 hours, and the characteristic colonies developed were counted. 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.

 [0076] [Table 3]

 Table 3

 [0077] [Table 4]

 Table 4

[0078] As shown in Test Examples 1 and 2, Aspergillus soja AOK 210 strain and Aspergillus Lus. Oryzae IK—05074 solid culture showed antibacterial activity against SE. In the control group, an increase in the number of bacteria was observed until the 7th day of the test, and 2.9 × 10 ⁸ CFU / ml was shown on the 7th day.

As shown in Test Examples 3 and 4, solid cultures of Aspergillus soja AOK210 strain and Aspergillus oryzae IK 05074 strain showed antibacterial activity against CP. In the control group, the increase in the number of bacteria was observed until the 7th day of the test, and on the 7th day, it showed 1.2 × 10 ⁶ CFU / ml.

[0079] Escherichia coli (EC) 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. Aseptically put 5g each of Aspergillus soja AOK 210 strain and Aspergillus olize IK 05074 strain into Erlenmeyer flasks as test examples 5 and 6, and without adding koji mold culture. The flask was used as a control group. Each Erlenmeyer flask was cultured in a 37 ° C incubator under aerobic conditions with gentle stirring.

[0080] 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. 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 SA concentration is about 1.0 X 10 ⁵ to 1.0 Aseptically charged to 10 ⁶ CFU / ml. Asperian flasks were aseptically charged with 5 g each of Aspergillus soja AOK 210 strain and Aspergillus oryzae IK 05074 strain into each Erlenmeyer flask to give Test Examples 7 and 8, and the Erlenmeyer flask without addition of Neisseria gonorrhoeae culture was the control. It was ward. Each Erlenmeyer flask was cultured in a 37 ° C incubator under gentle agitation under aerobic conditions.

[0081] On the 0th, 3rd, and 7th days after the start of the test, viable counts of EC and SA were performed. 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.

 [Table 5]

 Table 5

 [0083] [Table 6]

 Table 6

[0084] As shown in Test Examples 5 and 6, the solid cultures of Aspergillus soja AOK 210 strain and Aspergillus oryzae IK-05074 strain showed antibacterial activity against EC. In the control group, the highest number of bacteria was observed on the third day of the test, showing 4.3 X 10 ⁸ C FU / ml on the third day and 1.7 X 10 ⁷ CFU / ml on the seventh day. It was.

As shown in Test Examples 7 and 8, the solid cultures of Aspergillus' soya AOK 210 strain and Aspergillus oryzae IK-05074 strain showed antibacterial activity against SA. Na us, in the control group the highest number of bacteria observed in test 3 days, the 4 day ^{2 X 10 8 C FU / ml} , 7 days 3 days 7 illustrates a 2 X 10 ⁷ CFU / ml It was.

[0085] Bacillus subtilis DB9011 strain, NBRC3009 strain, NBRC3025 strain, NBRC3108 strain, The NBRC3336 strain 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 concentration of DB9011, NBRC3009, NBRC3025, NBRC3108, and NBRC3336 Was aseptically added to be about 1.0 × 10 ⁴ to 1.0 × 10 ⁵ CFU / ml. Aseptically add 5g each of Aspergillus' soya AOK 210 strain and Aspergillus oryzae IK-05074 strain solid culture into Erlenmeyer flasks to make test examples 9 to 18; No Erlenmeyer flask was used as a control. Each Erlenmeyer flask was cultured with gentle stirring in a 37 ° C incubator under aerobic conditions.

[0086] On the 0th, 3rd, and 7th days after the start of the test, 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, and Table 8 shows the number of spores.

[0087] [Table 7]

 Table 7

DB9011 Viable count (CFU / ml) Test Day 0 Test Day 3 Test Day 7 Test Example 9 Go to DB9011 + Case. Ruki 'Lus-Sorr AOK 210 2. 7 X 10 ⁴ 1.4 X 10 ⁸ 4. 3 X 10 ⁸ Test Example 10 DB901 1 + Ruki 'Rus'Olise' IK-05074 2. 8 X 1 0 ⁴ 1 .3 X 1 0 ⁸ 4. 5 X 1 0 ⁸ Control DB9011 only 2. 8 X 10 ⁴ 1.4 X 10 ⁸ 3. 2 X 10 ^s [0088] [Table 8]

 Table 8

 [0089] The number of viable bacteria of NBRC3009 strain is shown in Table 9, and the number of spores is shown in Table 10.

[0090] [Table 9]

 Table 9

 [0091] [Table 10]

 Table 10

 [0092] The number of viable bacteria of NBRC3025 strain is shown in Table 11, and the number of spores is shown in Table 12.

[0093] [Table 11]

 Table 11

NBRC3025 Viable count (CFU / ml) Test Day 0 Test Day 3 Test Day 7 Test Example 13 Go to NBRC3025 + case. Lucky Luz · ΑΟΚ ΑΟΚ 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]

 Table 12

 [0095] The viable cell count of NBRC3108 strain is shown in Table 13, and the spore count is shown in Table 14.

[0096] [Table 13]

 Table 13

 [0097] [Table 14]

 Table 14

 [0098] The viable cell count of NBRC3336 strain is shown in Table 15, and the spore count is shown in Table 16.

[0099] [Table 15]

 Table 15

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]

 Table 16

 [0101] As shown in Test Examples 9 to 18, the solid cultures of Aspergillus soja AOK210 strain and Aspergillus oryzae IK-05074 strain were Bacillus subtilis DB9011 strain, NBR C3009 strain, NBRC3025 strain, NBRC3108 strain, NBRC3336 strain No antibacterial activity was shown.

[0102] 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 ~;! · 0 X 10 ⁴ CFU / ml It was aseptically added. Aspergillus sosa AOK210 strain and Aspergillus oryzae in the Erlenmeyer flask: [Assemble 5g of ground solid culture of κ 05074 strain aseptically into test samples 19 and 20, respectively. The flask was used as a control group. Each Erlenmeyer flask was cultured in a 37 ° C incubator under gentle anaerobic conditions using an aneropack.

[0103] 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.

[0104] 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.

[0105] [Table 17]

 Table 17

 [0106] [Table 18]

 Table 18

As shown in Test Examples 19 and 20, solid cultures of Aspergillus soja AOK210 strain and Aspergillus oryzae IK-05074 strain did not show antibacterial activity against LA. As shown in Test Examples 21 and 22, solid cultures of Aspergillus' soya AOK 210 and Aspergillus oryzae IK 05074 did not show antibacterial activity against BB.

 [0108] From the above results, Aspergillus 'soya AOK210 strain and Aspergillus' olize 1 IK 05074 strain were not affected by the pathogenic bacteria Escherichia coli, Salmonella enteritidis, Sta phylococcus aureus Clostridium perfringens. On the other hand, Bacillus subtilis, which is promising for pronotics, and preferred! /, Did not show antibacterial activity against Lactobacillus aci dophilus and Bifidobacterium breve, which are intestinal bacteria. Alternatively, by combining the Aspergillus oryzae IK-05074 strain with B. subtilis, it can be exerted without losing the ability of B. subtilis, and it is not useful as an enteric bacterium. It adversely affects acidophilus and B. breve. It was thought that new feed additives for animals and animal feed additives could be developed.

 [0109] <3> Measurement of protozoan activity in cultures of Aspergillus spp.

 (l) Eimeria tenella control test

 Chicken cages naturally infected with Eimeria tenella were collected, and 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 4000 / ml. 50 mg each of ground solid cultures of AOK 210 strain, AOK 43 strain, AOK N4586 strain, AOK B650 strain and IK-05074 strain were added to each petri dish to obtain Test Examples 23 to 27, respectively. In addition, a petri dish without added 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 lysis was observed under a stereomicroscope. The reduction rate and lysis denaturation rate of oocysts were calculated.

 The results are shown in Table 19.

[0110] [Table 19] Table 19

 1) Percentage of remaining oocysts on day 7

[0111] Solid cultures of ί row, ΑΟΚ 210 strain, ΑΟΚ 43 strain, AOK Ν 4586 strain, AO K B650 strain and IK 05074 strain reduced the number of oocysts in E. tenella. It has been found that it exhibits oocyst dissolution-modifying activity. In particular, by treating E. tenella oocysts with AOK 210 and IK-05074, extremely high oocyst reduction and dissolution denaturation rates were obtained.

 [0112] (2) Eimeria zuemii control test

 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.

The results are shown in Table 20. [0113] [Table 20] Table 20

 1) Percentage of remaining oocysts on day 7

[0114] Tests 28 Rows 28-32 示 す As shown, ί 210, ΑΟΚ 43, Ν 586 4586, AO K B650 and IK 05074 solid cultures reduced the number of oocysts in E. zuemii. And showed a higher oocyst dissolution-modifying activity. In particular, by treating E. zuemii oocysts with AOK 210 strain and IK-050 74 strain, extremely high oocyst reduction rate and dissolution denaturation rate were obtained.

 From the above, it is surmised that the above-mentioned Aspergillus spp. Is effective for controlling kokushijimu.

 [0115] <4> Culture of Bacillus subtilis

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. CFU / g, 1.15 X 10 CFU / g, 1.06 X 10 CFU / g, 1.09 X 10 CFU / g. 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.

 As of May 21, 1991, 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.

[0116] Spore Formation Medium Components "

 Difco nutrient broth 8.0 g

 C1 1.0 g

 MgSO-7H O 0.25 g

 4 2

 MnCl -4H O 0. 002 g

 Adjust pH to 7.0, total volume 1,000 ml

 After autoclave sterilization, sterilized CaCl solution and FeSO solution

 twenty four

Each added to a 5 X 10- ⁴ M and 1 X 10- ⁶ M

 D Schaeffer, P., J. Millet, J. P. Aubert, Proc. Natl. Acad. Sci. USA, 54, 704-711 (1965)

 [0117] <5> Feeding test 1

 (1) Manufacture of feed additives

 Diluted Aspergillus 'soya AOK210 ground solid culture, Aspenolgillus' Oryzae IK 05074 ground solid culture, DB9011 strain and NBRC300 9 strain spore obtained in 1) above with lactose, respectively. Feed additive was prepared (Production Example;! ~ 4). In addition, cultures of AOK 210 strain or IK 05074 strain and spores of DB9011 strain or NBR C3009 strain were combined, respectively, and diluted with lactose to produce feed-contained carotenants of different concentrations (Production Examples 5 to 8). Table 21 shows the amounts of solid culture and batinoles subtilis added to each feed additive.

[0118] [Table 21] Table 21

(2) Chicken chick feeding test

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.

Yes

 The results are shown in Table 22.

[Table 22]

 Table 22

The chicks given the feed additives 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, Significant weight gain was observed compared to the control chicks. Spores of Bacillus' subtilis DB9011 strain or NBRC3009 strain 1. 5 X 10 ⁹ CFU / g or more including Production Example 3 (E to G), chicks fed a feed additive in Production Example 4 (E to G) are A significant increase in body weight was observed compared to the control chicks.

On the other hand, 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) and Production Example 8 (B-G) were ingested into animals, a significant effect of weight gain was observed. As a result, it was found that combining the culture of Aspergillus and Bacillus subtilis can promote weight gain of chicken chicks at a lower concentration than when each active ingredient is used alone.

 [0122] (3) Piglet feeding test

 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.

 The results are shown in Table 23.

 [0123] [Table 23]

 Table 23

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. Bacillus' subtilis DB9011 stock 1.5 5 ⁸ times? Pigs given the feed additive of Production Example 3 (£ ~ 0) containing 11 / _§ or more and Production Example 4 (EG) containing NBRC3009 strain spore more than 1.5 X 10 ⁸ CFU / g Significant weight gain was observed compared to the ward piglets.

On the other hand, 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, When Production Example 6 (B to G), Production Example 7 (B to G) and Production Example 8 (B to G) were ingested by animals, a significant effect of weight gain was observed. As a result, by combining the culture of 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.

 [0125] <6> Feeding test 2

 (1) Manufacture of feed additives

 Combining the culture of AOK 210 or IK-05074 obtained in <1> above with the spores of DB9011 or NBRC3009 and diluting with lactose to prepare feed additives containing each component in different ratios (Production Examples 9 to 12). Table 24 shows the amounts of solid culture and Bacillus subtilis added to each feed additive.

[0126] [Table 24]

Table 24

 [0127] (2) Chicken chick feeding test

 For feed total mass of chicken chicks (for the SD broiler pre- and post-stage, manufactured by Nippon Compound Feed Co., Ltd., feed containing no antibacterial substances), 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.

[0128] [Table 25] Table 25

[0129] 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 α-amylase activity was in the range of 0.5Χ10 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.

 [0130] (2) Piglet feeding test

 For 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 results are shown in Table 26.

[0131] [Table 26] Table 26

[0132] 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 ))

 [0133] <7> Pathogen attack test on chicken chicks

 (1) Salmonella enteritidis attack test

To the total mass of chicken chick feed (SD broiler first term, Nippon Compound Feed Co., Ltd., feed with no antibacterial substances added), production examples;! ~ 8 feed additives should be 0.1% by mass Mixed. A group of 12 chicken chicks hatched from eggs from broiler chickens (brand: Chunky) were fed with the above feed for 14 days with constant water supply and free water supply. A test was conducted in the same manner using a feed containing only 0.1% by mass of lactose as a control group. At 7 days of age, 2.3 × 10 ⁷ CFU of Sal monella enteritidis (SE) was orally administered per bird. At 14 days of age, sputum was collected by wiping the contents of the cecum and the total excretory cavity with a cotton swab.

 Measure the number of viable SE in the cecum contents by the following method, and calculate the infection index and the protection index /

The cecal contents lg was diluted 10-fold with sterile phosphate buffered saline and mixed well to obtain a sample stock solution. Next, 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. Furthermore, 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.

 [0134] 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.

 [0135] Infection index: logarithm average of the number of viable SE in the cecal contents of each individual (average value of log CFU / g)

 Defense index: Infection index in the control group / Infection index in each test group

 [0136] For sputum collected from the total excretion cavity, the properties of SE 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 make a sample stock solution, smear 0.1 ml each on SS agar plate and brilliant green agar plates, After culturing at ° C for 24 hours, the presence or absence of typical SE colonies grown on each plate medium was determined. Furthermore, 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, and Table 29 shows the number of SE detected individuals in the total excretory cavity collection culms.

[0137] [Table 27] Table 27

1) Below detection limit 28]

Table 28

29]

Table 29

Aspergillus' soya AOK 210 strain or Aspergillus olise IK- 05074 crushed solid culture alone, when added to feed additives more than 20% by mass, the SE cell concentration in the caecal contents is low and low The infection index of SE was shown (Production Example 1 (F, G), Production Example 2 (F, G)). When Bacillus subtilis DB9011 strain or NBRC3009 strain alone is added to a feed additive of 2.0 X 10 ⁹ CFU / g or more, the concentration of SE in the cecal content decreases, resulting in a low SE infection index. (Production Example 3 (F, G), Production Example 4 (F, G)). On the other hand, in the combination of Aspergillus' soya AOK210 strain or Aspergillus oryzae IK 05074 strain with Bacillus subtilis DB9011 strain or NBRC3009 strain, the culture of AOK210 strain or IK-05074 strain is 2.5 mass% or more, DB9011 strain Or, when NBRC3009 strain is added to feed additive at 2.5 X 10 ⁸ CFU / g or more, when AOK 210 strain or IK 05074 strain is added alone to feed additive at 20% by weight or DB9 011 strain or The same level of SE control effect was obtained as when NBRC3009 strain alone was added to feed additives at 2.0 X 10 ⁹ CFU / g or more (Production Examples 5 (C to G), Production Example 6 (C ~ G), Production Example 7 (ji ~ 0), Production Example 8 (ji ~ 0)). In addition, 20% by mass or more of AOK210 or IK-05074, and 2.0 × 10 ⁹ CFU / g or more of DB9011 or NBRC3009 When the feed additive was applied, SE could be completely controlled (Production Example 5 (G), Production Example 6 (G), Production Example 7 (G), Production Example 8 (G)). . From this, by combining Aspergillus sau or Aspergillus oryzae and Bacillus subtilis to chicken chicks, SE infection can be prevented at a lower concentration than when each active ingredient is used alone. It turns out that there is an effect.

 [0141] (2) Clostridium perfringens attack test

Using the feed additive prepared above, chicken chicks were bred in the same manner as described above. At 7 days of age, 5.0 × 10 ⁷ CFU of Clostridium perfringens (CP) was orally administered per bird. At 14 days of age, sputum was collected by wiping the contents of the cecum and the total excretory cavity with a cotton swab.

 [0142] The number of viable CP in the cecum contents was measured by the following method, and the infection index and the protection index were calculated /

 The cecal contents lg was diluted 10-fold with sterile phosphate buffered saline and mixed well to obtain a sample stock solution. Next, the sample stock solution was serially diluted 10 times with sterile physiological saline to obtain a serially diluted solution. Apply 0 ml each of the sample stock solution and serial dilutions to a clostridia medium (Nissui Pharmaceutical Co., Ltd.), and anaerobically incubate at 35 ° C for 24 hours using an aneropack. The number of black colonies grown on the plate medium was measured. Furthermore, 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.

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. Furthermore, 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, and Table 32 shows the number of detected CPs in the total excretory cavity.

[0143] [Table 30]

 Table 30

 1) Below detection limit

[0144] [Table 31]

Table 31

32]

Table 32

Aspergillus' soya AOK 210 strain or Aspergillus olise IK- 05074 crushed solid culture alone, when added to feed additives at 20% by mass or more, the concentration of CP in the caecal contents is low and low CP infection index was shown (Production Example 1 (F, G), Production Example 2 (F, G)). When Bacillus subtilis DB9011 strain or NBRC3009 strain alone is added to the feed additive at 2.0 X 10 ⁹ CFU / g or more, the concentration of CP in the caecal contents decreases, resulting in a low CP infection index. (Production Example 3 (F, G), Production Example 4 (F, G)). In contrast, 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)). further, the AOK210 strain also IK- 05074 strain 20 mass _^0/0 or more, When 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)). As a result, combining 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.

 [0147] <8> Oedema disease challenge test on piglets

Production examples produced above for the total weight of the piglet feed (standard feed for the first period of SD piglet artificial milk, Nippon Formula Feed Co., Ltd., feed containing no antibacterial substances); feed additives of 8 to 8 Was mixed so as to be 0.1 mass%. A group of 8 3-week-old piglets (brand: Large Yorkshire) was given the above-mentioned feed constantly and freely for 14 days. The test was conducted in the same manner, using a feed mixed with 0.1% by mass of lactose alone as the control. The porcine edema disease bacterium Escherichia coli (EC) was cultured at 37 ° C for 4 hours in Tribacteria soy broth (Difco Laboratories). Centrifugation was performed at 4 ° C (3,000 rpm, 15 minutes), and the pellet was placed in an enteric capsule. At 4 weeks of age, 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. After rearing to 5 weeks of age, the body weight was measured and the average weight gain of each group was calculated. In addition, from the start of oral gavage to the end of the study, clinical observations of fecal properties and periocular edema are conducted every day, and the total of fecal property scores and periocular edema scores during the study period is calculated for each individual. The average score was calculated.

 Table 33 shows the weight gain during the test, Table 34 shows the stool property score, and Table 35 shows the periocular edema score.

[0148] [Table 33] Table 33

34]

Table 34

Normal fecal score: 0. Normal stool, 1. Soft stool, 2. Mud stool, 3. Water-soluble diarrhea 35]

Table 35

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 same tendency was observed for the fecal property score and the periocular edema score. On the other hand, in the combination of Aspergillus soja AOK 210 strain or Aspergillus oryzae IK-05074 strain and Bacillus subtilis DB9011 strain or NBRC3009 strain, 2.5% by mass or more of AOK210 strain or IK-05074 strain, DB901 1 Strain or NBRC3009 strain added to the feed additive at 2.5 X 10 ⁸ CFU / g or more, the weight gain showed the same value as the non-inoculated case (Production Example 5 (C to G), Production Example) 6 (C to G), Production Example 7 (ji ~ 0), Production Example 8 (ji ~ 0)). The same tendency was observed for the fecal property score and the periocular edema score. As a result, Aspergillus 'sor or Aspergillus' It can be seen that the combination of Zeichi and Bacillus subtilis has the effect of preventing edema disease infection at a lower concentration than when each active ingredient is used alone.

[0152] <9> Salmonella Typhimurium attack test on calves

A group of four 1-week-old male calves (Holstein), and commercially available milk substitute for calves (Promilk, Nippon Formula Feed Co., Ltd.) twice a day, 2 L each, 7 weeks old I paid until. Free water supply was used, and hay and commercial artificial milk (super hoiku morlet, whole farming) were used continuously. The above-prepared production examples;! To 8 feed additives were administered by mixing 25 g per day with milk replacer. Only 25 g of lactose was mixed with the milk substitute to make a control, and the test was conducted in the same manner. At 2 weeks of age, 5.0 × 10 ⁶ CFU of Salmonella Typ himurium (ST) suspended in 50 ml of 10% NaHCO per head was orally administered to all the heads. The animals were raised to 7 weeks of age and weighed, and the average weight gain for each group was calculated. In addition, 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.

[0153] [Table 36]

Table 36

37]

Table 37

Normal stool score: 0. Normal stool, 1. Soft stool, 2. Mud stool, 3. Mucus to watery stool, 4. Blood stool Aspergillus 'soya AOK210 strain or Aspergillus' Oryzae IK 05074 strain alone crushed solid culture 20 By adding more than mass% to the feed additive, the weight gain was similar to that in the non-inoculated case (Production Example 1 (F, G), Production Example 2 (F, G)). Bacillus subtilis DB9011 strain or NBRC3009 strain alone added 2.0 x 10 ⁹ CFU / g or more to the feed additive, and the weight gain was similar to that in the non-inoculated case (Production Example 3 ( F, G), Production Example 4 (F, G)). The same tendency was observed for the stool property score. On the other hand, in the combination of Aspergillus' soya AOK 210 or Aspergillus oryzae IK-0507 4 and Bacillus subtilis DB9011 or NBRC3009, A OK210 or IK-05074 is 2.5 mass ⁰ / More than ₀ , DB9011 strain (or NBRC3009 strain was added to the feed additive at 2.5 X 10 ⁸ CFU / g or more, and the weight gain was similar to that in the non-inoculated case (Production Example 5 (C To G), Production Example 6 (C to G), Production Example 7 (C to G), Production Example 8 (C to G)), and the same tendency was observed with respect to the stool property score. Combining Soya or Aspergillus 'Olysee and Bacillus' subtilis Thus, it can be seen that there is an effect of preventing ST infection at a lower concentration than when each active ingredient is used alone.

Industrial applicability

 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.

Claims

The scope of the claims

[1] Aspergillus sojae, Aspergillus tarn arii, Aspergillus tarn arii, Aspergillus foetidus, Aspergillus niger and Aspergillus niger At least one Aspergillus genus selected from Aspergillus oryzae, culture containing acid enzymes produced by these bacteria, and animal feed additive containing Bacillus subtilis Feed additive for animals with a total power of 20 U or more per lg of feed additive and a concentration of Bacillus subtilis of 2.5 X 10 ⁷ to 2 X 10 ⁹ CFU / g .

 [2] The animal feed additive according to claim 1, wherein the acid enzyme contains an acid amylase, and the acid amylase activity per lg of feed additive is 1 U or more.

 [3] The animal feed addition according to claim 1 or 2, characterized in that it has antibacterial activity against pathogenic bacteria causing intestinal infection of the animal, and / or protozoan activity against coccidium. Agent.

 [4] The animal feed additive according to any one of claims 1 to 3, which is Aspergillus sp., Aspergillus 'soya and / or Aspergillus' oryzae.

 [5] Aspergillus oryzae is an IK-05074 strain (FERM BP-10622) and / or a mutant thereof having the same ability to produce an acidic enzyme as the aforementioned strain. Item: Animal feed additive according to any one of!

 [6] The animal feed additive according to any one of claims;! To 5, wherein the culture contains a plant nutrient source.

 7. The animal feed additive according to claim 6, wherein the plant nutrient source is brown rice.

[8] The animal feed additive according to any one of claims 1 to 7, which is a Bacillus subtilis DB9011 strain (FERM BP-3418) and / or a variant thereof.

Yes

 [9] The animal feed additive according to any one of claims 8 to 8, which is for growth promotion.

[10] The animal feed additive according to any one of claims;! To 8, wherein the animal feed additive is used for prevention / amelioration of intestinal infections.

[11] A feed comprising the animal feed additive according to any one of claims 10 to 10 in an amount of 0.01 to 1.0% by mass.

[12] A solid medium containing nutrients for the growth of Aspergillus spp., Aspergillus sore, Aspenoleginoles tamari, Aspenoleginoles fötidas, Aspenoleginoles niga and aspergillus oryzae Culturing at least one Aspergillus genus selected from the group consisting of a step of containing the resulting culture so that the total acid enzyme activity per kg of feed is 12 U or more, and a concentration of Bacillus subtilis of 2. And 5 X 10 ^{6 to} 2.0 X 10 ¹ ° CFU / kg.

 [13] An animal breeding method, characterized by causing the animal to ingest the feed according to claim 11.