TW200814936A - 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

[Technical Field] The present invention relates to an animal feed additive containing bacteria belonging to the genus AspergUlus and Bacillus which have the ability to produce acidic enzymes. [Prior Art] Although the feed of livestock or pets (hereinafter referred to as animals) is subjected to grinding processing, it is generally because the heat treatment is not performed, and the digestion and the yield are lowered, and the feed efficiency is poor. In addition, it has recently been pointed out that inflammatory bowel disorders such as ulcerative colitis or Crohn's disease, which are known to be human diseases, also occur in animals, causing symptoms such as diarrhea. Furthermore, such inflammatory bowel disorders are known to interfere with the absorption or healthy growth of the feed. Pathogenic bacteria causing intestinal infection in animals ' Known as pathogenic coliform, Salmonella bacterium, Clostridium bacterium, and genus Bacillus. This pathogen is known to produce toxins (enteric toxins, cytotoxins) when abnormally augmented, causing obstruction on the intestinal mucosa, and causing soft stools or severe convulsions. In addition, Coccidium is a protozoan that is parasitic in the intestines of chickens, pigs, and cattle. It can cause squatting and loss of appetite when infected. Antibiotics are used to prevent and treat such inflammatory bowel disorders, but there are problems such as the emergence of drug-resistant bacteria. In recent years, in order to improve the balance of microorganisms in the intestines and to suppress the proliferation of intestinal pathogenic bacteria, techniques using probiotics have been attracting attention (Patent Document 1 and Patent Document 2). However, lactic acid bacteria or Bifidobacterium -4- 200814936, mostly died at a concentration of 0.3% deoxycholic acid or died below pH 値4, in addition to the bacteria of the coliform group, there are still many strains among the bile acids. It is impossible to survive in the presence of deoxycholic acid which has strong antibacterial properties against microorganisms. Therefore, it is still looking for a species that does not die even in the digestive tract of an animal and can bring beneficial effects to the host. On the other hand, it has been reported that the animal feed is treated with an enzyme or an enzyme-producing bacterium at the processing stage, and the technique of reducing the gastrointestinal burden of the animal by considerable decomposition is useful for the prevention or improvement of digestive diseases. (Patent Document 3). In addition, a method of fermenting fish meal with low moisture to produce a fishmeal fermented feed for fish such as a high concentration of proteinase or fatty enzyme is known (Patent Document 4). However, in this technique, after the moisture content in the feed is increased and the components of the feed are decomposed, there is a problem that the complicated steps must be dried and then commercialized. Further, in order to decompose the components and increase the moisture content, the feed is liable to cause spoilage bacteria or molds, and the like, and the quality is difficult to maintain. Furthermore, there are reports mentioning the method of oral administration of the cells of the sputum to the animals to change the quality of the feces of the animals, but it has not reviewed the inhibition of the proliferation of toxic bacteria such as inflammation in the intestinal tract of the animals, and can promote The use of weight gain, and it is also known that a bacterium is administered to an animal in the form of an acidic enzyme (Patent Document 5). Furthermore, it is described that a fermented nutrient source is added to the crustacean ground-shell ground material and mixed, and then the genus Fusarium is inoculated to decompose chitin or chitosan to obtain a fermented product, which is then supplied to the animal (Patent Document 6) ). Although the growth promotion effect of the animal by this method can be partially confirmed, it is not observed that the proliferation of pathogenic bacteria in the intestinal tract of the animal is inhibited, and the intestinal infection is prevented and treated, and these effects are not observed. It has not been confirmed. Furthermore, the mixing of more than two microorganisms and their administration to animals has not actually been reviewed. In addition, it has been reported that the Bacillus subtilis has antibacterial activity against pathogenic bacteria such as pathogenic coliforms (Patent Documents 7 and 8), and the animal has been reviewed for oral administration. Further, Bacillus subtilis DB 901 1 is known to have a decomposability of yellow φ scorpion toxin and inhibit the development of fungi, and is added to feed for review (Patent Document 9). However, until now, no one has combined the bacteria of Bacillus subtilis with other systems to allow animal ingestors to review it. Patent Document 1: JP-A-2005-507670 (Patent Document 2): JP-A-2004-52324 No. JP-A-2004-523 Patent Document 5: Japanese Laid-Open Patent Publication No. Hei No. Hei. No. Hei. No. Hei. No. Hei. No. Hei. No. 2002-238. Patent Document 9: Patent No. 3040234 Non-Patent Document 1 · George A. Burdock, Madhusudan G. Soni, and Ioana G. Carabin (2001) Regulatory Toxicology and Pharmacology 33, 80-101 Non-Patent Document 2: Harry E. Morton, Walter Kocholaty, 200814936

Renate Junowicz. Kocholaty, and Albert Kelner Bacteriol 50, 5 79-5 84 [Disclosure of the Invention] The object of the present invention is to provide a safe and simple method for assisting an animal to improve feed efficiency. Specifically, it provides a method for treating intestinal infection and increasing the body weight of an animal by inhibiting the pathogen or coccidia in the intestinal tract of the animal. The inventors of the present invention have made efforts to solve the above problems, and have found that the strains of soy sauce, sputum, scorpion, black sputum, and acid enzymes, particularly acid amylase, have It is resistant to pathogenic bacteria causing intestinal infection and has a protozoal activity against coccidia; and these bacteria function as a concomitant bacteria. Furthermore, it has been found that the acidity of these bacteria is extremely excellent when cultured with myrtle as a nutrient source. These bacteria and the acidic enzymes produced by the bacteria are combined to make the animals ingest, which can promote digestion, prevention, infection, and increase the weight of the animal, thereby completing the talent even if When Bacillus subtilis coexisted with the above-mentioned genus Fusarium and the enzyme injected by the bacterium, it was found that the proliferation was not hindered. Therefore, the bacteria of the genus Fusarium and the acidic enzymes produced by the bacteria, and the combination of the bacteria, and the animals are found to be more excellent when they are ingested 1 945 ) J.

Digestive activities, the subject of proliferation, in order to achieve the results of the study of rice bran is not good; bacterial activity, can be produced as a probiotic, can be found, and with the improvement of intestinal treatment. The acidity of the re-production is promoted by the growth of the bacterium, and the effect of the prevention and improvement of the intestinal infection is promoted. The present inventors have thus completed the present invention. That is, the present invention is as follows. (1) An animal feed additive comprising: selected from the group consisting of Aspergillus sojae, Aspergillus tamarii, Aspergillus foetidus, Aspergillus niger, and rice bran At least one of (Aspergillus oryzae), a culture of an acidic enzyme produced by the same, and an animal feed additive of Bacillus subtilis; characterized in that the sum of the acid enzyme activity per 1 g of the feed additive is 1 20U or more, and the concentration of Bacillus subtilis is 2.5xl07~2xl09 CFU/g. (2) The animal feed additive according to (1), wherein the acidic enzyme comprises acid amylase, and the feed additive has an activity of 1 U or more per 1 g of the acid amylase. (3) The animal feed additive according to (1) or (2), wherein the strain of the genus Aspergillus has an antibacterial activity against a pathogenic bacteria causing an intestinal infection of the animal and/or for a coccidium Protozoal activity. (4) The animal feed additive according to any one of (1) to (3), wherein the fungus (Aspergillus) is a soy mushroom and/or a rice fungus. (5) The animal feed additive according to any one of (1) to (4), wherein the rice bran strain IK-05074 (BCRC 930092) and/or a variant thereof has the same acidity as the aforementioned strain The animal feed-feeding additive, wherein the culture system contains a vegetable nutrient source, such as the animal feed additive of any one of (5) to (5). (7) The animal feed additive according to (6), wherein the plant-based nutrient source is black rice. (8) The animal feed additive according to any one of (1) to (7), wherein the Bacillus subtilis strain DB9011 (CCRC 910024) and/or a variant thereof. (9) The animal feed additive according to any one of (1) to (8), which is used as a growth promoting user. (1) The animal feed additive according to any one of (1) to (8), which is used as a preventive/improving user of intestinal infection. (1 1 ) A feed characterized by containing 1 to 1.0% by mass of an animal feed additive of any one of (1) to (10). (1 2) A method for producing a feed comprising: a solid medium selected from the group consisting of a nutrient source for proliferation of a genus Fusarium, and a culture selected from the group consisting of soy sauce, sputum, scorpion, and black sputum And at least one of the genus Fusarium genus, and the obtained culture has a total of 1 2 U of activity per 1 kg of acid enzyme; and the concentration of Bacillus subtilis is 2.5 x 〇 6 to 2.0 xl01G CFU/kg steps. (1 3 ) An animal breeding method characterized in that an animal ingests a feed such as (1 1 ). The culture containing the genus Fusarium of the present invention and the acidic enzyme produced by the bacterium, and the animal feed additive containing Bacillus subtilis are mixed in the feed, and the animal can be ingested to promote nutrient absorption and enhance the feed. -9- 200814936 Efficiency. Specifically, the acidic enzyme produced by the genus Trichobacteria has an antibacterial activity against intestinal pathogenic bacteria and the like, and Bacillus subtilis can activate the immunity, thereby preventing and improving the intestinal infection of the animal, and making the animal The weight gains. The feed of the present invention is suitable for use in the breeding of livestock such as chicken, pig, and cattle. [Embodiment] The best form for carrying out the invention The animal feed additive of the present invention characterized by comprising at least one selected from the group consisting of soy sauce, sputum, stag beetle, smut, and rice bran Bacteria, cultures of acidic enzymes produced by the same bacteria, and animal feeds for Bacillus subtilis bacteria. The soy sauce, the sputum fungus, the skunk fungus, the black sputum, and the rice blast fungus contained in the animal feed additive of the present invention, as used in the technical field, the general method used for identifying the sputum species When classified, each line is classified into the above-mentioned species. For the identification of strains, for example, "H. Murakami, The Journal of General and Applied Microbiology, 17, ρ. 281-309, (1971)", "Murao Hideo, Japanese Brewing Association, Vol. 74, No. 12, ρ·849-8 5 3, (1979), "Nikkuni, S., et al, The Journal of General and Applied Microbiology, 44, p. 225-230, (1998)" and the like. Soybean soy sauce is a kind of filamentous incomplete fungus found in soil, earthworms, etc., and is used in the cultivation of soy sauce and miso. In the animal feed additive of the present invention, as long as it has the acidic yeast -10- as described in detail below

At least one of the substances of 200814936, preferably having the production of acid amylase, is suitable for the animal to be safe when ingested, and has no particular use. In the animal feed additive of the present invention, it can be used, but it is preferable to use the soy sauce AOK210 strain (Takjinye store). The sputum bacterium is one of the whole fungi found in the soil, the sputum, the food, and the like, and is used in the animal feed additive of the invention for brewing soy sauce and miso, as long as it has at least one of the sex enzymes, preferably. In order to have the production of acid starch, and the animal can be safe when ingested, there is no special use. In the animal feed additive of the present invention, a strain can be used, but it is preferable to use the sputum AOK43 strain Akita Jinno store. Stinky bacillus, which can be used to find S full-bacteria in soil, earthworms, and sputum, and used in wine, soy sauce, and miso! In the animal feed additive of the present invention, as long as it is at least one of the acidic enzymes of P, it is preferred to have the ability to produce acid hydrazine, and the animal can be safe when ingested, and can be used. The strain which is sold in the animal feed additive of the present invention is preferably a bacillus Α K Co., Ltd. Akita Jinno Store. Black sputum can be used to find a whole strain of S in soil, cockroaches, cockroaches, etc., and is used in various conditions such as alcohol production, food processing, and pharmaceuticals. The ability of the kinetic shirt of the present invention, and the limitation, can be obtained by the commercially available strain of the strain of the autumn strain of the strain. The above-mentioned limitations of the ability of the acid enzymes are all commercially available (the filamentous incomplete strains of the company. There are no special restrictions on the starch enzymes as described below, and the city N4586 strain can be used. In the manufacture of a filamentous candy or a feed additive -11 - 200814936, as long as it has at least one of the acidic enzymes described in detail below, it preferably has the ability to produce acid amylase, and is suitable for the animal when ingested. In the animal feed additive of the present invention, a commercially available strain can be used, but it is preferable to use the skunk bacterium B650 strain (Akita Jinye Co., Ltd.). Store). Alternatively, a mutant strain of AOK2 10 strain, AOK43 strain, ΑΟΚN4586 strain, or ΑΟΚB65 0 strain may be used. The mutant strain may be caused by natural mutation of these strains, or by chemical mutagen or Among the strains obtained by the mutation treatment such as ultraviolet rays, each of them has a strain capable of producing the same acidic enzyme as AOK2 10 strain, AOK43 strain, ΑΟΚN45 86 strain, or ΑΟΚB650 strain. In addition to the ability to produce an acidic enzyme, it is preferred to use at least one of the above-mentioned strains of the same antibacterial activity, protozoal activity, bile acid resistance, and acid resistance as the above-mentioned strains. In addition to the above, it is preferred to use other mutant strains having the same bacteriological properties as AOK210 strain, AOK43 strain, ΑΟΚN45 8 6 strain, or AOK B65 0 strain. Rice bran bacteria can be used in soil and earthworms. A kind of a filamentous incomplete fungus found in the above, and used in the brewing of soy sauce and miso. In the animal feed additive of the present invention, it is preferably at least one of the acidic enzymes described in detail below. In order to have the ability to produce acid amylase, and the animal can be safe when ingested, it can be used without particular limitation. In the animal feed additive of the present invention, a commercially available strain can be used. It is preferred to use the rice bacterium IK-05074 strain. IK-05074 strain is a strain isolated from various fermented foods. On February 15, 2006, -12 - 200814936 registered number FERM P- 20798 is registered in the National Institute of Industrial Technology, the Institute of Industrial Technology and Research, and is licensed to the International Depository Center (the sixth in the center of the 1st, 1st, 1st, 1st, 1st, 1st, 1st, 1st, 1st, It is registered as the registration number FERM BP-10622. In addition, IK-05074 strain was deposited in the Food Industry Development Research Institute on November 15, 1995 with the registration number BCRC 930092. - The bacteriological nature of IK-05074 strain It is as follows: φ ( 1 ) The shape of the colony (CZapek_D〇X vegetable medium (25°C culture 7 days)) The size is 50~60 mm in diameter, the color is yellow to green, and it will change with time. brown. Mycelium is not obvious, and the inside is colorless. (2) Forming sub-handle: thin wall ~ thick wall, smooth surface ~ slightly rough surface, diameter 20μπι or less, length 2 mm or less, top capsule is spherical with diameter 40~50μιη, 8 0μηι or less. The basal stalk is almost under the stalk of the large meriste, and the length is below 12 μm. — phialide: ampoule type, length 8~12μιη, with short neck. Meridian: spherical shape with a diameter of 5~6 μπι, the color is yellow to green, and the surface is smooth surface ~ fine rough surface. Based on the above, the ΙΚ-05 074 strain was presumed to be rice bran. Further, 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 strain can be produced by naturally mutating the strain of -13-200814936 IK-05 074 or by mutation with a chemical mutant or purple, and has the ability to produce 0 5 074 strains. A strain of the same acidic enzyme capacity. In addition, in addition to the ability to produce enzymes, it is used to have mutations in the antibacterial activity, the protozoal activity, the bile acid resistance, and the acid resistance of the 1 Κ - 0 507 strain. The strain is preferred. Further, in addition to the use of other strains of the bacteriological properties and the strains of the 1κ-05074 strain, it is preferred. Further, in the animal feed additive of the present invention, for example, the soy sauce, the sputum fungus, the black sputum fungus, and the rice blast fungus isolated from the soil, the cockroach, the food, the slag residue, etc. It is also possible to produce strains of acid ability. The ability to produce acidic enzymes means that the production of acidic enzymes in the culture of the cells can detect the activity of acidic enzymes. The detection of the acid enzyme activity of the culture can be carried out according to conventional knowledge. The acid enzyme contained in the animal feed additive of the present invention is preferably a digestive enzyme produced by the above-mentioned bacteria, and is not active as long as it is not inactivated under gastrointestinal conditions, and is not particularly It is most preferable to have a pH of 2·5 to 5·5. For example, acidic α-amylase, starch glucoamylase, peak temple, protein enzyme, fiber enzyme, ribonuclease, nucleic acid enzyme glycoprotein, pectinase, lipase, etc., the animal additive of the present invention may contain One or two of the others. Among them, the preferred extra-line and the like are at least one of the same as the acidity of the sputum-produced by the method of the degree of cultivation of the soil bacteria and the odorous enzyme, and are limited by the acid inside, that is, powdery enzymes, poly The wood feed addition contains a salt of -14_200814936, and the starch 3 is a bacterium, and the species in the genus is acidic as a more fermented leaven. The lg is above 400 U of the fermented acid and is acidic with an amylase which can be decomposed. The main ingredient in the composition of livestock feed is starch. The acid powder enzyme contained in the animal feed additive of the present invention is not particularly limited as long as it is an acidic enzyme capable of hydrolyzing starch, for example, α-amylase, S-amylase, glucoamylase, etc. . Among them, it is particularly preferable to have an acid-resistant α-amylase having an optimum pH.

The animal feed additive of the present invention may be at least one type of acid yeast produced by the sputum sputum, the scorpion bacillus, the black sputum, and the rice blast fungus, but may further contain other strains or other The enzyme that comes from birth. The acid enzyme activity in the animal feed additive of the present invention may be within the following range. The total enzyme activity per 1 g of animal feed additive is preferably 120 U or more, more preferably 170 U or more, and 220 U or more is optimal, and 270 U to 5400 U is excellent. The sum of the acid activities is preferably the sum of acid amylase activity, acid protein activity, and acid carboxyl peptide activity. Further, the acid amylase activity of the animal feed additive is preferably 1 U, more preferably 5 U or more, more preferably 10 U or more, and 20% is excellent. Furthermore, the sum of the acid protein activity and the acid carboxyl peptide activity per 1 g of the animal feed additive is preferably 100 U, more preferably 150 U or more, more preferably 200 U or more, and 250 to 5000 U. good. The activity of acid amylase, acid protein enzyme, and acidic carboxy-peptidase can be determined according to the analysis method of the National Tax Agency (Revised 3rd Tax Reward, 200814936, No. 1). The measurement method, the α-amylase activity measurement method, the acid-resistant α-amylase activity measurement method, the acid protein enzyme activity measurement method, and the acidic carboxyl peptide activity measurement method were measured. The culture system comprises: at least one of the fungi belonging to the genus Soybean, the sputum, the scorpion, the sputum, and the rice smut, which are contained in the animal feed additive of the present invention, and the same The acidic enzyme produced, and the concentration of the culture 'as long as the acid enzyme activity of the feed additive is within the above range. In general, a sufficient amount of a plant-based nutrient source such as black rice can be used to culture the culture obtained by the above-mentioned bacteria at 28 C, 7 days or so, and G. 25 to 20% by mass is added, preferably 〇. 5 to 1 ()% by mass, the best is to add 1 to 5 mass%. Further, the above-mentioned genus Fusarium used in the present invention is preferably one having an antibacterial activity against a pathogenic bacterium causing intestinal infection in an animal. The above pathogens generally belong to the intestinal bacteria family. Specifically, there are pathogenic Escherichia coli, Salmonella, Campylobacter bacteria, Clostridium bacteria or Staphylococcus aureus. Wait. Pathogenic coliforms, such as enterotoxin-producing coliforms (toxin-producing coliforms, enterotoxigenic E. coli (ETEC)), edema pathogens, or 0157, etc., verotoxin-producing E. coli (VTEC), enterohemorrhagic E. coli), etc. The bacterium of the genus Salmonella includes, for example, S. p u 11 o r u m, S. g a 11 i n a r u m, S · -16- 200814936 typhi, S. typhimurium, S. enteritidis, S. choleaesuis, S. derby, S. dublin, and the like. Bacteria of the genus Corynebacterium, for example, C. jejuni, C. coli, C. fetus, and the like. A bacterium belonging to the genus Clostridium, for example, C. perfringens, C. botulinum, C. difficile, and the like. The soy sauce, the sputum, the stag beetle, the black sputum, and the rice blast fungus contained in the animal feed additive of the present invention, particularly for the bacterium of the genus Salmonella (especially S. enteritidis) and the shuttle Bacillus genus bacteria (especially C. perfringens), edema pathogens and the like, Escherichia coli, S. aureus, have a high antibacterial activity. The term "antibacterial activity against a pathogenic bacteria" means that it has the ability to inhibit the proliferation of pathogenic bacteria when inoculated in the same medium as the pathogenic bacteria. Antibacterial activity of soy sauce, sputum, scorpion, black sputum, and rice bran, for example, soil, sputum and other separation sources can be added to inoculate Salmonella enteritidis (SE), Clostridium perfringens (CP ), Escherichia coli (EC), Staphylococcus aureus (SA) and other pathogens in the vegetable culture medium, and then isolated and formed to prevent the round of the bacteria can be obtained. The bacterial cells thus obtained have the ability to inhibit the proliferation of the above-mentioned pathogenic bacteria, and can be administered to animals to prevent and treat intestinal infections in animals. Further, the inhibition of the growth of the pathogenic bacteria causing the intestinal infection of the animal can be confirmed, for example, by measuring the concentration of the pathogenic bacteria (the number of bacteria) in the cecal contents of the animal or the feces. Further, the above-mentioned genus Fusarium which is used in the present invention is preferably one which has a protozoal activity against a coccidia causing intestinal infection in an animal. -17- 200814936 The so-called coccidia refers to the protozoa belonging to the genus Sporozoasida. Specifically, for example

Eimeria genus, Isospora genus, Toxoplasma genus, Cryptosporidium genus, and the like. Protozoa of the genus Eimeria, such as E. tenella, E. necatrix, E. acervulina, E. maxima, E. mitis, E. zuernii, E. bovis, and the like. Protozoa of the genus Isospora, such as I. Suis, I. belli, I. Hominis, and the like. Protozoa of Toxoplasma, such as Τ·gondii. Protozoa of the genus Cryptosporidium, for example, C. parvum et al. The animal feed additive of the present invention is particularly suitable for use as an infection caused by E·tenella or E. zuernii. The term "protozoal activity against coccidia" means that when the oocysts (Oocyst) of the coccidia are coexisted with the culture of the bacterium, the germination and proliferation of the oocysts are inhibited, and it is preferred to have the ability to reduce the oocysts. Specifically, it means the ability to deform, dissolve, and destroy the oocysts of the cell wall of the oocyst. The destruction of the oocysts or the state of the cell wall can be observed with a microscope. The soy sauce, the sputum, the sputum, the sputum, and the rice bacterium having the protozoal activity against the coccidia can be obtained, for example, by the following method. A separation source such as soil or sputum was added to a petri dish of suspended sterile water to which E. tenella or E. zuernii oocysts were previously added, and cultured at 37 ° C for 1 to 7 days. The cells were separated from the culture dish in which the oocysts were deformed or dissolved, and they were again added to a petri dish containing the suspension of sterile water of E. tenella or E. zuernii oocysts, respectively, by selecting the respective sauces. The fungus of the genus Fusarium used in the present invention is obtained by the bacteria, the sputum fungus, the scorpion bacterium, the black bacillus, and the fungus of the rice bacterium. By administering the bacteria thus obtained to the -18-200814936, it is possible to prevent or treat intestinal coccidiosis in animals. Further, by adding the above-mentioned bacteria of the genus Fusarium, whether or not the growth of the coccidia is inhibited can be confirmed by observing, for example, the cecal contents of the animal or the coccidia oocysts in the feces under a microscope. The soy sauce, the sputum fungus, the scent bacillus, the black sputum, and the rice blast fungus contained in the animal feed additive of the present invention are preferably those having tolerance to gastric acid or citric acid. Thereby, the bacteria can produce useful acidic enzymes even in the intestines of animals, so that the digestion and auxiliary functions by acidic enzymes are sustainable. Further, when the bacterium belonging to the genus Fusarium has an antibacterial activity against the above-mentioned pathogenic bacteria, it can also promote the proliferation of the pathogenic bacteria which cause intestinal infection in the animal, and the balance of the microorganisms in the intestine. The so-called tolerance to stomach acid or bile acid means that the bacteria do not die under the conditions of the general digestive organs and can reach the intestines. Generally, when the stomach of a human or animal is inside an empty stomach, the pH 値 will reach 2 or less, and in the state of ingesting food, the pH inside the stomach is in the range of 3.5 to 6 and higher than in the fasting. Therefore, the acid-resistant strain which can be used for the feed additive of the present invention can be obtained, for example, by treating the separation source at pH 値3.5 or less for about 2 hours, and selecting a strain having viability. Further, the selected bacteria are treated in the presence of a deoxycholic acid concentration of 10 g/Ι for about 24 hours, and then the viable strain is selected to obtain a feed additive suitable for the present invention, and for gastric acid or sputum Strain with acidity tolerance. Further, the bacteria are not dead before reaching the intestine, and can be confirmed, for example, by measuring the concentration of the cells in the animal excrement or the like. The animal feed additive of the present invention may contain one strain alone or -19-200814936 among the above-mentioned species, or may be a combination of two or more strains. Among them, at least one of soy sauce and rice bran bacteria is preferred. The bacterial concentration of the soy sauce, the sputum, the scorpion, the sputum, and the rice blast fungus contained in the animal feed additive of the present invention is as long as the feed additive is administered to the animal. It is not possible to fall within the range of death in the digestive organs. Generally, for the purpose of producing a feed additive having the above-mentioned acidic enzyme activity, the cells are cultured at an appropriate concentration, and the acid enzyme activity is appropriately adjusted to the end. The above 値 can be. The soy sauce, the sputum fungus, the skunk fungus, the black sputum fungus, and the rice blast fungus used in the animal feed additive of the present invention are cultured under ordinary culture conditions to produce an acidic enzyme. For example, the culture temperature can be carried out at 25 ° C to 40 ° C, but it is generally preferred to culture at 28 ° C to 32 ° C. Further, as the culture method, a liquid culture method or a solid culture method such as reciprocating vibration culture, fermentation tank culture, or the like can be used, but in the acidic enzyme gene of the above-mentioned cells, since there are also genes which are expressed only in the solid medium, Therefore, the present invention is preferably a solid culture method. For the medium component used in the culture, any of animal or sputum properties can be used. It is preferred to use a vegetable-based nutrient source, for example, containing black rice, wheat bran, rice bran, soybean, barley, etc. . Among them, it is better to use black rice as a source of nutrition. Thereby, the production efficiency of acidic enzymes such as acid amylase can be improved. In addition, other carbon sources such as glucose 'sucrose, molasses and the like, or nitrogen sources such as ammonium, ammonium sulfate, ammonium chloride, ammonium nitrate or the like, or nitrates may be added. -20- 200814936 The culture of the genus Fusarium obtained as described above is preferably dried in advance. Further, it is preferable to add a component such as an optional component for improving the preservability, which is preferable in terms of improving the quality stability. The drying method is not particularly limited. For example, it is ventilated and dried, naturally dried, spray-dried, freeze-dried, etc., but it is preferably ventilated and dried. Further, although freeze-drying can also be used, a protective agent can also be added at this time. The type of the protective agent is not particularly limited, and one or more of skim milk, sodium glutamate and sugar may be selected. Further, the type of the saccharide is not particularly limited, but it is preferred to use glucosamine or trehalose. Further, after drying, an oxygen scavenger and a dehydrating agent are added to the obtained dried product, and the mixture is sealed in a gas barrier aluminum bag, and is preferably stored at room temperature to low temperature. This allows the cells to be stored for a long time. The Bacillus subtilis used in the animal feed additive of the present invention is not particularly limited as long as it is classified as "Bacillus subtilis" in Bergey's Manual of Determinative Bacteriology 9th Edition (1994). . Examples of such bacteria include Bacillus subtilis DB9011 strain, NBRC3 009 strain, NBRC3 025 strain, NBRC3108 strain, and NBRC3 3 36 strain. DB901 1 was deposited with the Microbial Industrial Technology Research Institute of the Ministry of Industry and Technology of the Ministry of Industry and Commerce of the Ministry of Industry and Technology of the People's Republic of China on May 21, 1991. 1 in the city of Tsukuba, Ibaraki Prefecture, 1st, 1st, 6th). Also DB 901 1 was classified as -21 - 200814936 at the time of registration.

Bacillus licheniformis, but the subsequent confirmation is attributed to Bacillus subtilis. In addition, DB 901 1 was deposited with the Food Industry Development Research Institute on February 7, 1984, under the registration number CCRC 9 1 0024. In addition, NBRC3009 strain, NBRC3 025 strain, NBRC3108 strain, and NBRC 3 3 3 6 strain are strains deposited in the Bioresource Resources Department (NBRC) of the independent administrative agency product evaluation technology base institution. The bacteriological properties of the DB 9011 strain are as follows. [Table 1] Table 1 Project DB901 1 Expansion of bacteria - 7% NaCl + no3 - no2 + VP reaction + Egg · york - 吲哚- 6 0 °C _ GL+ spore position Central Μ 菌 Colony surface Morphology = Bacterial broad 〇·7~0·8μπι bacillus. The elliptical spores are approximately present in the center, and the cells do not swell. It is sporty and forms R-type colonies. Do not give birth under suffocating conditions. Fertility status in each medium: (1) DHL acacia medium: not fertile. -22 - 200814936 (2) McCakey's acacia medium: not fertile. (3) Mannitol salt culture medium: well developed. Glossy. There is no wrinkle on the surface of the colony, and the color of the colony is yellow. (4) Common aquaculture medium: well developed. Matte. There are wrinkles on the surface of the colony, and the colony color is white. (5) Heart infiltration of acacia medium: well developed. Matte. The colony has wrinkles on the surface and the colony color is white. (6) Blood acacia medium (add 1% sheep blood): well developed. Matte. The surface of the colony has wrinkles and the color of the colony is white. (7) PDA medium: well developed. Matte. The surface of the colony is wrinkled and the color of the colony is white. Physiological properties: Gram reaction β· + gelatin test: fertility status; full liquefaction # gelatin liquefaction; + litmus: reaction; acid state; solidified nitrate reduction: + denitrification reaction: - MR test:吲哚 Formation: Hydrogen sulfide formation: -23- 200814936 Citric acid utilization (Christensen): + Urease: - Oxidase: + Touch enzyme: + Fertility range: pH; 4~9

Temperature; 25~50°C

OF test: Fermentation and gas generation (derived from glucose decomposition) Acid utilization of acid generation L-arabinose - + D - xylose - + D-glucose + + D-mannose - + D-fructose - + D-galactose- + maltose - + sucrose - + lactose - + trehalose - + 〇 sorbitol - + D - mannitol - + inositol - + -24- 200814936 Degradation of escin in glycerol starch + + + soil + + 10%: Utilization of malonic acid: decomposition of arginine: decarboxylation of lysine * urea decomposition: φ decomposition of aflatoxin: avian ammonia Acid decarbonation reaction coagulating enzyme: Hemolysis: Sodium chloride tolerance: Potassium cyanide resistance = Lecithinase = # Further, in the animal feed additive of the present invention, Bacillus subtilis DB9011 strain and NBRC3009 strain may also be used. NBRC3 025 strain, NBRC3 108 strain, NBRC3 3 36 strain naturally mutated, or the strain obtained by mutation of chemical u mutant or ultraviolet light, selected to have the same bacteriological properties as each strain Used as a strain. The mutant strain of Bacillus subtilis DB901 1 strain is preferably a user selected from the bacterial strain having the above bacteriological properties. In the animal feed additive of the present invention, the concentration of Bacillus subtilis is generally 2·5χ107 ～2xl09 CFU/g, preferably 5xl07~lxl09 CFU/g, and most -25-200814936 is ΙχΙΟ8~5xl08 CFU/g. Further, in the animal feed additive of the present invention, the degree of subtilis, which is in the total of the active additive which exhibits 1 U of acidic enzyme activity, is generally 2·0χ 103 to 5.0 x 106 CFU, compared with 104 to 1.0 x l 〇 6CFU; in the display of 1U acid amylase• and animal feed additives, the general system l.OxlO5~2.5x » is preferably 5.0xl05~5.0xl07CFU; and shows 1U φ enzyme and acidity The animal feed for the sum of the activity of the carboxy-peptidominase is generally 2·0χ103 ～5.0×10 6 CFU, preferably 1·0 gt; l〇6 CFU. The herbicide used in the animal feed additive of the present invention is preferably one having the proliferative ability of the above-mentioned bacterium of the genus Fusarium and the acid enzyme produced by the bacterium. The herbicide used in the animal feed additive of the present invention is preferably one which has tolerance to bile acids. # “1 Zhanshi acid tolerance” refers to the so-called “bile acid” which can form germination and proliferative cells in a medium containing a high concentration. It refers to mammals, birds, crawlers and fish. The bicyclic structure, which is widely found in bile, contains bile acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, and bear. In general, in the body of an animal, bile acids are present in the bile or in the form of a combination of taurine and guanamine, and in the specification, if the reference is "bile acid", it includes the salt of the above bile and Wait for the fit. Bacillus concentrate with feed giant 1.0 X activity total 1 0 8 CFU, acidic protein additive c 104~1 · 0 X bacillus, under it, with bacillus, its bile acid thinking insects, two steroids, deoxygenation Cholic acid is sodium glycinate. The present invention is a medium containing a high-concentration bile acid, for example, a medium containing a bile powder (Oxgall, manufactured by Difco) which is concentrated and dried by 10 times of fresh bile. The concentration of the bile powder is generally 0.3% by mass or more, preferably 1% by mass or more, and most preferably 3% by mass or more. In addition, the term "having germination and proliferative ability" refers to a condition in which a cell is inoculated in a medium containing the above-mentioned high concentration of bile acid, and the conditions other than the bile acid concentration are suitable for the culture of Bacillus subtilis, the bacterium It can germinate, then proliferate and divide, and form colonies. Bacillus subtilis having bile acid tolerance can be obtained, for example, as follows. The separation source containing Bacillus subtilis is cultured under conditions suitable for cell formation to form a cell. The obtained cells were inoculated into the above-mentioned high-concentration bile acid addition medium, cultured, and the formed colonies were separated. Among the colonies, those having the bacteriological properties of Bacillus subtilis were selected. The Bacillus subtilis used in the animal feed additive of the present invention is preferably further resistant to acid. By using this bacterium, the bacteria can reach the intestines even if they do not die inside the stomach. The term "acid resistance" means that when the bacteria are administered to an animal, they will not die even under the conditions of the stomach (usually pH 値 3.5 to 6 in the state of ingesting food), and will remain in the intestines when they reach the intestines. The number of bacteria that can be proliferated. The cells of Bacillus subtilis are generally not resistant to acidity and do not pose particular problems when using cells. The animal feed additive of the present invention may contain a strain of Bacillus subtilis alone or a combination of two or more strains. Among them, it is preferred to use DB901 1 alone or in combination with other strains. -27- 200814936 The culture method of the Bacillus subtilis used in the feed additive of the present invention is not particularly limited, and can be cultured in a usual manner under appropriate conditions in accordance with the nature of the bacteria. For example, the culture temperature may be 20 to 40 ° C, but it is usually 30 to 37 ° C. Further, the culture method may be a liquid culture method or a solid culture method using static culture, reciprocating vibrating culture, rotary vibrating culture, fermenting tank culture, or the like. The medium to be used for the culture is not particularly limited, and the carbon source may be glucose, galactose, lactose, arabinose, mannose, sucrose, starch, starch hydrolyzate, molasses or the like, organic acids such as citric acid, glycerin, etc. Alcohols; nitrogen sources such as ammonium, ammonium sulfate, ammonium chloride, ammonium nitrate or other ammonium salts or nitrates, or sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, chlorine Inorganic salts such as manganese and ferrous sulfate, protein glands, soybean powder, defatted soybean meal, meat extract, yeast extract, and the like. The Bacillus subtilis used in the feed additive of the present invention is preferably in the form of a cell based on the viewpoint of maintaining stability and acid resistance. The state of the cells is sufficiently resistant to heat and dryness, and can be sufficiently dried in the production of the feed additive, and the preservation stability is improved. For the purpose of forming the cells in the culture, the culture conditions, the pH of the culture medium, the culture temperature, the culture humidity, and the oxygen concentration during the culture can be adjusted in accordance with the conditions for the formation of the cells. Such methods are described, for example, in Schaeffer, P., J. Millet, J. P. Aubert, "

Proceedings of the National Academy of Science, United States, 1965, Vol. 54, pp. 704-711. Further, it is preferred that the culture of Bacillus subtilis obtained by the above method or the cell -28-200814936 "on the basis of preservability" is prepared by previously preparing a dry powder. Drying, for example, the water content thereof can be adjusted to 20% by mass or less. The method of drying and preserving is the same as the drying of the culture of the genus Fusarium. The animal feed additive of the present invention, which is capable of cultivating at least one bacterium belonging to the genus Soybean, the sputum, the scorpion, the sputum, and the rice bacterium, and the acid enzyme produced by the bacterium All or part of the substance is obtained by mixing with the cells of Bacillus subtilis. Further, the animal feed additive of the present invention may further contain an optional component. Any of these optional ingredients can be used as long as it is safe as a feed ingredient, does not cause the above-mentioned bacterium and Bacillus subtilis to die, and does not inactivate the sex enzyme, and is not particularly limited. For example, preferred are /3-glucan, glucomannan, mannan oligosaccharide, seaweed and other immunostimulating agents, gluconic acid, 7-aminobutyric acid, citric acid, malic acid, fumaric acid , succinic acid, pantothenic acid, butyric acid and other organic acids. The animal feed additive of the present invention can be used as an animal feed additive for growth promotion because it can promote the weight gain of the animal by ingesting the animal. At this time, in particular, the total amount of acid enzyme activity per 1 g of feed additive is above 270 U, the activity of acid amylase is above 2〇υ, and the sum of acid 'amylase activity and acid carboxyl peptide activity is more than 250U. Further, it is preferred that the concentration of Bacillus subtilis is adjusted at ixi 〇 8 CFU/g or more. The animal feed additive of the present invention can prevent enteric infections and the like by ingesting animals, and can be used as an animal feed additive for the prevention and improvement of intestinal infections. At this time, in particular, the total activity of -29-200814936 acidic enzyme activity per ig feed additive is above 540U, the activity of acid amylase is above 40U, the sum of acid protein enzyme activity and acid carboxyl peptide activity is above 500U, and the grass is dry. It is preferred that the concentration of the bacillus is adjusted at 2.5 x 10 CFU/g or more. Further, in the animal feed additive of the present invention, it is preferred that the concentration of citric acid is low based on safety. In general, the concentration of citric acid is preferably 0.1% or less, and preferably 〇1 Ι1 mg/Ι or less. The feed of the present invention is preferably 0.02 to 0.5% by mass, preferably 0.02 to 0.5% by mass, based on the total amount of the feed of the animal feed additive of the present invention. 〇·〇4~〇.25% by mass contains its characteristics. In addition, 'the sum of the acidic enzyme activities per 1 kg of the feed of the invention' is generally 12 U or more, preferably 17 U or more, and the best system is 22 U or more. Excellent is 27~5400 0U. Furthermore, the concentration of Bacillus subtilis is generally 2.5 X 1〇6~2·0 X l〇1GCFU/kg, preferably 5·0 χ 1〇6~1.0 x 101GCFU/kg, and the best is ι·〇 Χι〇7~5 〇xl〇9CFU/kg. In addition, the activity of acid amylase per gram of feed is generally 〇.11; the above is preferably 0.5U or more, the optimum is 1U or more, and the best is 2~4000U. Furthermore, the sum of the acid protein enzyme activity and the acid carboxyl peptide activity per 1 kg of feed is generally 1 〇U or more, preferably 1 5 U or more, the best system is 20 U or more, and the best is 25 to 5 0000U. The feed of the present invention can be produced by adding the animal feed additive of the present invention to a feed ingredient which is generally used. The type or composition of the feed -30-200814936 is not particularly limited as long as it does not cause the bacteria contained in the animal feed additive of the present invention to die, and the acidic enzyme is not inactivated. It can be added to livestock feed, pet food, animal adjuvant, etc., and can be added or mixed with animal feed. Further, the feed of the present invention may be used by directly adding or mixing the animal feed additive in a dry state to the feed component, or may be used in the form of a liquid or a gel based on easy mixing. In this case, a water-soluble polymer compound such as vegetable oil, liquid animal oil, polyvinyl alcohol, polyvinylpyrrolidone or polyacrylic acid such as water, soybean oil, vegetable oil or corn oil can be used as a liquid carrier. In addition, in order to maintain the homogeneity of the bacterial concentration in the feed, it is compatible with arginine, sodium arginate, xanthan gum, casein sodium, gum arabic, guar gum, tamarind seed polysaccharides and the like. Preferred polysaccharides are preferred. Further, in order to prevent the growth of the bacteria, it is preferred to use an organic acid. Furthermore, the feed of the present invention may also be cultured in a solid medium containing a nutrient source for the proliferation of Fusarium, selected from the group consisting of soy sauce, sputum, stag beetle, smut, and rice blast fungus. At least one of the genus Fusarium is produced by mixing the obtained culture and Bacillus subtilis separately into the feed component, and adjusting the acid enzyme activity of the feed and the concentration of Bacillus subtilis within the above range. The feed of the present invention can be used as a feed for the growth of animals. Further, 'the above-mentioned genus bacterium can further prevent or treat pathogenic bacteria and/or coccidia when it has an antibacterial activity against a pathogenic bacteria causing an intestinal infection of an animal and/or a protozoal activity against coccidium. Intestinal infection -31 - 200814936 disease feed. There is no particular limitation on the animal species ingesting the feed of the present invention, such as mammals, birds, reptiles, biogenics, fish, and the like. Among them, it is particularly suitable for poultry and livestock. Poultry such as chickens, ducks, donkeys, turkeys, etc. 'live animals such as pigs, cows, sheep, rabbits, and the like. The amount of the feed to be taken by the animal can be appropriately adjusted depending on the type, body weight, age, sex, purpose of use, health condition, feed ingredient, and the like of the animal. The method of ingesting the animal can be carried out in a usual manner depending on the type of the animal. EXAMPLES <1> Culture of the genus Fusarium The pH of the potato glucoamiana culture medium was adjusted to 5 and sterilized at 1 21 °C for 15 minutes. When the temperature of the agar medium is lowered to 6 (pre-TC', sodium deoxycholate is added in a concentration of 1 μg per 1 L of the medium, and the inoculum is stored in Akita Jinno Co., Ltd. (Aug. 24, Daisuke, Daisen, Akita, Japan) The genus Aspergillus, Aspergillus sojae, Aspergillus tamarii, Aspergillus foetidus, and Aspergillus niger all grow well in the medium. The soy sauce AOK210 strain, the sputum AOK43 strain, the skunkia sputum N45 86 strain, and the black sputum sputum B650 strain are particularly well-produced. In the same medium containing sodium deoxycholate as described above, Various fermented foods were used as a separation source to inoculate a number of strains of the genus Fusarium, and the strains which were produced by the medium - 32-200814936 were selected, and the strain with the best growth was selected. The strain was classified as strain of rice bacterium IK-05074 and deposited. In the franchise of the IK-05 074 strain, the bacteriological nature of the IK-05 074 strain is as described above. Using the black rice as a solid medium, the soy sauce bacterium A OK210 strain, Astragalus strain AOK43 strain, Ailanthus altissima strain N4586 strain, Black fungus AOK B650 strain and Rice bran strain IK-05074 strain were cultured. That is, 100 g of black rice was immersed in water for a day and night to expand, and then added. A lid of a sterile filter having a diameter of 14 cm and a depth of 10 cm was attached to the lid to make it 2 cm thick, and sterilized in a sterilizer at 121 ° C for 15 minutes. The above container was inoculated on the container. Each of the cells was cultured at 28 ° C for 5 days to produce an inoculum. Next, the basal rice which was swollen in the same manner as described above was laminated to a thickness of 1.5 cm in a stainless steel pot of 30 x 40 x 10 cm, and was covered with Cover the vent with a 20x25 cm filter, place it in a large autoclave, and sterilize it at 121 °C for 25 minutes. After cooling the pot, inoculate the above-mentioned inoculum that has been cultured in advance. The pots were placed in an incubator at 28 ° C for 7 曰. After the cultivation, they were air-dried at 35 ° C and pulverized by a wet jet mill, and each of them obtained a solid culture of different strains. Each solid culture was tested for its acid-resistant α-pyrokinase per 1 g. The sum of the activities of the acidic and acidic protein enzymes and the acidic carboxyl peptide enzymes. The above enzyme activity of each culture 1 g is shown in Table 2. In addition, the determination of the above acidic enzymes is based on the analysis of the National Tax Agency -33- The method of measuring the acid-resistant α-amylase activity, the acid protein enzyme measurement method, and the acid carboxylase activity measurement method of the solid oxime analysis method of the 200814936 method (amending the third taxation office order No. 1) is carried out. [Table 2] Table 2 _ strain acid-resistant a-amylase activity (u) Acid protein enzyme acid carboxyl group enzyme activity sum (U) A. s aj ae 25 5 5 469 1 7 A . t am ar i 3 13 50248 A. foetidus 5 86 29660 A. niger 399 2034 1 A . oryzae 4250 5 13 0 1

<2> Measurement of antibacterial activity of the culture of the genus Fusarium The strain of S. sojae ΑΟΚ210 and the strain IK-05074 of rice bran were co-cultured with the bacteria, and the antibacterial activity of the genus Fusarium against bacteria was tested. Salmonella enteritidis (SE), which is cultured in a standard Asian vegetable medium (made by Rishui Pharmaceutical Co., Ltd.), is cultured at 37 °C for 24 hours. The colonies developed on the plates were picked up and suspended in sterile physiological saline. 500 ml of brain water extract "Ri Shui" was prepared in a 1 L-capacity flask, and after sterilization in a sterilizer, SE was placed under aseptic conditions to have a final concentration of about 1.0 x 1.04 to 1.0 x 105 CFU/ml. In a triangular flask, 5 g of each of the ground solid cultures of the soy sauce AOK210 strain and the rice bran strain IK-05074 were used as the test examples 1 and 2, and the uncultured culture was further used. A triangular cone bottle was used as a control area. The respective triangular flasks were slowly stirred and cultured under aerobic conditions in a thermostat at 37 °C. -34- 200814936 Clostridium perfringens (CP), using "AnaeroPack" (Mitsubishi Gas Chemical Co., Ltd.), CW Acacia medium (made by Rishui Pharmaceutical Co., Ltd.) 3 7 °C qi culture for 2 4 hours. The colonies developed on the plates were picked and suspended in sterile physiological saline. In a 1 L volume of a triangular flask, a brain water extract broth "Ri Shui" 500 ml was prepared, and the sterilization was carried out. The CP was placed under aseptic conditions to a final concentration of about 1.0 χΙΟ4 to 1.105 CFU/ml. In a triangular flask, the ground solid cultures of the soy sauce sputum strain 210 strain and the sputum IK-05074 strain were each placed under aseptic conditions as test examples 3 and 4, while the triangular cone of the uncultured culture was used. The bottle is the control area. The respective triangular flasks were slowly stirred and cultured in a 37 ° C thermostat with a gas strip. After the start of the test, the number of bacteria of S E CP was measured on the third day, the third day, and the seventh day. The method for measuring the number of bacteria in the SE is to dilute the culture solution taken from the sterile physiological saline solution 10 times, and then apply 0.1 ml to each of the diluted liquids to the X-SAL agar medium "Ri Shui". In the medium, culture at 37 ° C for 24 hours 'recalculate colonies with developmental characteristics. The method for measuring the growth of cp is to dilute the culture solution to be diluted with sterile physiological saline, and then apply the diluted liquid to 〇.ml in the egg yolk CW agar medium (Nisshui Pharmaceutical Co., Ltd.) In the system, the AnaeroPack was used to culture the anaerobic gas at 37 ° C for 24 hours, and then the colonies of developmental characteristics were counted. The number of bacteria in the SE of Table 3 and the number of bacteria in the CP of Table 4 are listed below. It is used in the factory to make the middle and rear Ox meters 5g and the number of the nutrient 10 -35- 200814936 [Table 3] Table 3 SE bacteria number (CFU/ml) Test Day 3 test Day 3 test 7曰Test Example 1 SE+ soy sauce AOK210 4.8χ104 <1.〇χ102 <1.〇χ102 Test Example 2 SE+ Rice Bacteria 1-05074 4.8χ104 <1·〇χ102 <1·〇χ102 Control Area Only SE 4.8χ104 2.6χ108 2.9χ108 [Table 4] Table 4 CP bacteria number (CFU/ml) Test Day 3 test Day 3 Test No. 7 Test Example 3 CP+ soy sauce AOK210 7.3 χΙΟ4 <1.〇 Χ102 <1·〇χ102 Test Example 4 CP+ Rice Bacteria IK-05074 7.3 χΙΟ4 <1·〇χ102 <1.〇χ102 Control area only CP 7.3 χΙΟ4 5.5χ105 1.2x106

As shown in Test Examples 1 and 2, the solid cultures of the soy sauce Α 2 2 2 2 2 0 strain and the rice blast fungus IK-05 074 strain had antibacterial activity against SE. Further, in the control area, the number of bacteria increased on the 7th day of the test, and on the 7th day, it was 2.9 x 10 CFU/ml. As shown in Test Examples 3 and 4, solid cultures of S. sojae AOK2 10 strain and Rice bran strain IK-0 5 074 strain had antibacterial activity against 0. In the control area, the number of bacteria increased on the 7th day of the test, and on the 7th day, it was 1.2×10 6 CFU/ml. Escherichia coli (EC), which is cultured in a standard amaranth culture medium (manufactured by Sakamoto Pharmaceutical Co., Ltd.), is cultured at 37 ° C for 24 hours. The colonies developed on the plates were taken and suspended in a sterile physiological diet -36-200814936 in saline. 5 00 ml of brain water extract gravy "Ri Shui" was prepared in a 1 L volumetric flask. After sterilization in a sterilizer, the EC was placed under aseptic conditions to a final concentration of about 1.0×xl5~l.〇xl〇6 CFU. /ml. In a triangular flask, the ground solid cultures of the soy sauce AOK210 strain and the rice bran strain IK-05 074 were each put into 5 g as the test cases 5 and 6; and the uncultured culture was added. The triangular cone bottle is used as a control area. The flasks were slowly agitated in a 37 t thermostat under aerobic conditions. Staphylococcus aureus (SA), which is cultured in a standard acacia medium and cultured at 37 ° C for 24 hours. The colonies developed on the plates were picked up and suspended in sterile physiological saline. 500 ml of brain water extract gravy "Daishui" was prepared in a triangular flask of 1 L volume, and after sterilization in a sterilizer, SA was placed under aseptic conditions to have a final concentration of about 1.0×x10 to 1.0×10 6 CFU/ml. In a triangular flask, the ground solid cultures of the soy sauce AOK210 strain and the rice bran strain IK-05074 were each placed in a sterile manner to 5 g as test examples 7 and 8; The triangular cone bottle is used as a control area. The respective triangular flasks were slowly agitated and cultured under agitation conditions in a thermostat at 37 °C. After the start of the test, the number of bacteria in EC and SA was measured on the 0th, 3rd, and 7th day. The method for measuring the number of bacteria in EC is to dilute the culture solution to be diluted with sterile physiological saline, and then apply 1 ml of each diluted liquid to Chromocult-COLIFORM "Merck". After the Merck system, the cells were cultured at 37 ° C for 24 hours, and the colonies with developmental characteristics were counted. The method for measuring the number of bacteria in SA is to dilute the culture solution taken with sterile physiological saline 10 times, and then apply the diluted liquid to -37-200814936 〇·1 ml to the mannose added with egg yolk. The alcohol salt culture medium "Rongshen" (manufactured by Rongji Equipment Co., Ltd.) was cultured at 37 ° C for 24 hours, and the colonies with developmental characteristics were counted. The number of bacteria in EC of Table 5 and the number of bacteria in SA of Table 6 are listed below. [Table 5] Table 5 Number of EC bacteria (CFU/ml) Test Day 3 Test Day 3 Test Day 7 Test Example 5 EC+ Soy Sauce ΑΟΚ 210 1.2 χΙΟ5 <1.〇χ102 <1.0 χΙΟ2 Test Exampleό EC+ rice bran IK-05074 1.2χ105 <1·〇χ102 <1.〇χ102 Control area only EC 1·2χ105 4.3χ108 1·7χ107 [Table 6] Table 6 SA bacteria number (CFU/ml) Test No. 〇曰 test day 3 test day 7 test case 7 SA + soy sauce ΑΟΚ 210 2.7 χ 105 < 1. 〇χ 102 < 1. 〇χ 102 test case 8 SA + rice bacterium IK-05074 2.5 χ 105 < 1. 〇χ 102 <1·〇χ102 Control area only SA 2.5x10s 4.2x108 7.2χ107

As shown in Test Examples 5 and 6, solid cultures of S. sojae AOK2 10 strain and Rice bran strain IK-05 0 74 strain have antibacterial activity against EC. Furthermore, the highest number of bacteria was confirmed in the control area on the third day of the test, which was 4.3 x 10 CFU/ml on the third day and 1.7 x 107 CFU/ml on the seventh day. The solid cultures of the strains of the soy sauce AOK2 10 strain and the rice bran strain IK-05 074 as shown in Test Examples 7 and 8 have antibacterial activity against SA. -38- 200814936 Furthermore, the highest number of bacteria was confirmed in the control area on the third day of the test, with the third 曰 being 4.2xl08 CFU/ml and the seventh day being 7.2xl07 CFU/ml. Bacillus subtilis DB901 1 strain, NBRC3 009 strain, NBRC3 025 strain, NBRC3108 strain, and NBRC3 3 3 6 strain were cultured in standard acacia medium (made by Rishui Pharmaceutical Co., Ltd.), and cultured at 37 °C for 24 hours. It. Extract the colonies developed on the plate and suspend them in sterile physiological saline. ^ In the 1 L volume of the triangular flask, the brain water extract broth "Ri Shui" 5 00 φ ml was prepared, and after sterilization in the sterilizer, DB9 01 1 strain, NBRC3009 strain, NBRC3025 strain, and NBRC3 1 08 strain were placed under aseptic conditions. , NBRC3336 strain, so that the final concentration is about l.OxlO4~1.0xl05 CFU/ml. In a triangular flask, the ground solid cultures of the soy sauce AOK210 strain and the rice bran strain IK-05 074 were each put into 5 g as a test case 9 to 18; and the uncultured culture was added. The triangular cone bottle is used as a control area. The flasks were slowly agitated in a 37 ° C thermostat with good aerobic conditions. • After the start of the test, the number of bacteria and the number of cells of DB9011 strain, NBRC3009 strain, NBRC3025 strain, NBRC3 108 strain, and N B R C 3 3 3 6 strain were measured on the third day, the third day, and the seventh day. The method for measuring the number of bacteria in the above-mentioned strains is to dilute the culture solution taken from the sterile physiological saline solution by 10 times, and apply 0.1 ml of each diluted liquid to the standard acacia medium (Nisshui Pharmaceutical Co., Ltd. ( After the stock system), it was cultured for 48 hours with 37 gas, and then the colonies with developmental characteristics were recalculated. The method for measuring the number of cells of the above strain is as follows. First, the culture solution taken was diluted with a sterile physiological saline solution at a doubling stage, and then heat-treated at 70 ° C for 30 minutes. After cooling, re-39-200814936 was diluted with sterile physiological saline 1 〇, and then 0.1 ml of each dilution stage liquid was applied to standard acacia medium (made by Rishui Pharmaceutical Co., Ltd.) to 3 7 °C was cultured for 48 hours, and then the colonies with developmental characteristics were counted. The number of bacteria of the DB9011 strain of Table 7 and the number of cells of Table 8 are listed as follows. [Table 7] Table 7 DB9011 bacteria number (CRJ/ml) Test No. 3 test Day 3 test Day 9 Test Example 9 DB9011+ Soybean sojae AOK 210 2.7χ104 1.4 xlO8 4·3 χΙΟ8 Test Example 10 DB9011+ rice bran IK-05074 2.8χ104 1.3xl〇8 4.5x10s Control area only DB9011 2·8χ104 1.4χ108 3.2χ108 [Table 8] Table 8 DB9011 number of cells (CFU/ml) Test Day 3 Test 3rd Test 7th day test Example 9 DB9011+ soy sauce AOK 210 2.4x104 1.3x10s 3.8χ10δ Test Example 10 DB9011+ Rice Bacteria IK-05074 2.4x104 1.2χ108 3.5x10s Control area only DB9011 2.1χ104 1·2χ108 3.〇χ108

The number of bacteria in the NBRC 3 009 strain of Table 9 and the number of cells in Table 10 are listed below. -40- 200814936 [Table 9] Table 9 NBRC3009 Bacterial Count (CFU/ml) Test Day 3 Test Day 3 Test No. 7 Test Example 11 NBRC3009+ Soy Sauce AOK 210 2.0 χΙΟ4 l.SxlO8 4.1x10s Test Example 12 NBRC3009+ rice bran IK-05074 2·〇χ104 1.7χ108 4·〇χ108 Control area only NBRC3009 2.〇χ104 1.6χ108 3.5χ108 [表ίο] Table 1 0 NBRC3009 Cell number (CFU/ml) Test Day 1 test Day 3 Test Day 7 Test Example 11 NBRC3009+ Soy Sauce AOK 210 1.9χ104 1.4χ108 3.6x10s Test Example 12 NBRC3009+ Rice Bacteria IK-05074 1.9χ104 1.5x10 s 3.2χ108 Control Area Only NBRC3009 1.9χ104 1.5χ108 3.2χ108

The number of bacteria in the NBRC 3 025 strain in Table 11 and the number of cells in Table 12 are listed below.

Table 11 NBRC3025 bacterial count (CFU/ml) test day 3 test 3rd test 7th test case 13 NBRC3025+ soy sauce AOK 210 2.2x104 1.8χ108 4.2χ108 test case 14 NBRC3025+ rice bacterium IK-05074 2.2χ104 1.5x10s 4.0χ10δ Control area only NBRC3025 2.2χ104 1.7χ108 3.9χ108 -41 - 200814936 [Table 12] Table 12 NBRC3025 Cell number (CFU/ml) Test No. 3 test Day 3 Test Day 13 Test Example 13 NBRC3025+ Soy Sauce Bacteria AOK 210 2.1χ104 1.5x10s 3.6χ108 Test Example 14 NBRC3025+ Rice Bacteria IK-05074 2.0χ104 1.4χ108 3.7χ108 Control area only NBRC3025 2.0χ104 1.4χ108 3.1x10s

The number of bacteria in the NBRC 3 10 8 strain of Table 13 and the number of cells in Table 14 are listed below. [Table 13] Table 13 NBRC3108 Bacterial Count (CFU/ml) Test Day 0 Test Day 3 Test Day 7 Test Example 15 NBRC3108 + Soy Sauce AOK 210 3.1 χΙΟ4 1.1x10s 3.9χ108 Test Example 16 NBRC3108+ Rice Bacteria IK -05074 3.1χ104 1.2x10 s 3.7χ108 Control area only NBRC3108 3.1Χ104 l.lxlO8 3·4χ108 [Table 14] Table 14 NBRC3108 Cell number (CFU/ml) Test Day 3 test Day 3 Test No. 7 Test Example 15 NBRC3108+ soy sauce ΑΟΚ 210 2.9x104 1.0x10s 3.5χ108 mm i6 NBRC3108+ rice bacterium IK-05074 2.9x104 1.0 χΙΟ8 3·2χ108 Control area only NBRC3108 2.9χ104 1.1x10s 2.9χ108 The NBRC3 3 3 6 strain of Table 15 The number and the number of cells in Table 16 are listed below. -42- 200814936

[Table 1 6] Table 1 6

NBRC3336 Cell Count (CFU/ml) Test No. Day 3 Test 7th Test Example 17 NBRC3336 + Soy Sauce AOK 210 2.3 χΙΟ4 1.5χ108 3.7xl〇!_ Test Example 18 NBRC3336+ Rice Bacteria IK-05074 2·4χ104 1.8χ10δ 3.6x1〇!_ Control Area Only NBRC3336 2.3χ104 1.0x10s 2.8x1〇L

[Table 15] Table 15 NBRC3336 bacteria number (CFU/ml) One test Day 3 test Day 3 Test Day 7 Test Example 17 NBRC3336+ Soy Sauce ΑΟΚ 210 2.5 χΙΟ4 1.7χ108 4.〇xl〇!_ Test Example 18 NBRC3336+ rice bran IK-05074 2.5 χΙΟ4 1.9χ108 4.2χ1〇!_ Control area only NBRC3336 2.5χ104 1·3χ108 3.3χ1〇8 As shown in Test Examples 9~18, soy sauce AOK210 strain and rice bran IK The solid culture of strain -05074 did not have antibacterial activity against DB9011 strain, NBRC3009 strain, NBRC3025 strain, NBRC3108 strain, and NBRC3 3 3 6 strain. Lactobacillus acidophilus (LA) of the genus Lactobacillus acidophilus (LA), which is used in the plated counting of acacia medium (made by Risho Pharmaceutical Co., Ltd.) with BCP using "An aero Pack gas" (manufactured by Mitsubishi Gas Chemical Co., Ltd.) 37T: It is incubated for 72 hours. The colonies developed on the plates were removed and suspended in sterile physiological saline. After making 500 ml of GAM gravy (made by Risho Pharmaceutical Co., Ltd.) in a 1 L volumetric flask, the sterilizer was sterilized, and LA was placed under aseptic conditions to a final concentration of about 1. Οχ -43- 200814936 103 ~1.0xl04CFU/mb In the triangular flask, the ground solid culture of the soy sauce AOK210 strain and the rice bran strain IK-05074 strain were each placed under aseptic conditions, 5g, as test examples 19 and 20, but not The triangular flask of the culture of the bacterium was used as a control zone. The flasks were placed in a thermostat, and the "AnaeroPack" was slowly stirred and cultured under anaerobic conditions. Bifidobacterium breve ( BB ), which is cultured at 37 ° C in BL vegetable medium (made by Mitsui Chemicals Co., Ltd.) using "AnaeroPack" (Mitsubishi Gas Chemical Co., Ltd.) 48 hours. The colonies developed on the plates were picked up and suspended in sterile physiological saline. After making 500 ml of GAM gravy (made by Rishui Pharmaceutical Co., Ltd.) in a 1 L volumetric flask, the sterilizer was sterilized, and BB was placed under aseptic conditions to make the final concentration about Ι.ΟχΙΟ4~l.〇xl 〇 5 CFU/ml. In a triangular flask, the ground solid cultures of the soy sauce AOK2 10 strain and the rice bran strain IK-05074 strain were each put into 5 g as the test cases 2 1 and 22, and the uncultured bacteria culture was carried out. The triangular cone bottle is used as a control area. Each of the two-breasted flasks was slowly stirred and cultured under a temper condition using a "AnaeroPack" gas in a thermostat at 37 °C. After the start of the test, the number of bacteria in LA and BB was measured on the third day, the third day, and the seventh day. The method for measuring the number of bacteria in LA is to dilute the culture solution to be diluted with sterile physiological saline, and then apply 0.1 ml of each diluted liquid to the plated counting medium containing BCP (Sunshine) After the pharmaceutical (stock) system, the cells were cultured at 37 ° C for 72 hours, and the colonies with developmental characteristics were counted. The method for measuring the number of bacteria in the BB is to dilute the culture medium to be diluted with sterile physiological saline, and then apply 0.1 ml of the diluted liquid to the 44-200814936 liquid to the BL vegetable medium. After the water pharmacy (stock) system, the "AnaeroPack suffocating gas" was used to culture for 37 hours at 37 lt., and the colonies with developmental characteristics were counted. The number of bacteria in LA of Table 17 and the number of bacteria in bb of 袠Μ are listed as follows. [Table 17] Table 17 _ LA bacteria number (CFU/mi) a Test Day 3 Test No. 7 Test Example 19 LA+ Soy Sauce ΑΟΚ 210 -- 3.6 χ 106 2.6 χ 106 Test Example 20 LA+ Rice Bacteria IK-05074 _U_xl03 3.3χ106 2.2χ106 Control area only LA—---- _Uxl〇3 2.3χ106 1·〇χ106 [Table 18] Table 18 BB bacteria number (CFU/ml) Test Day 0 Test Day 3 Test Day 7 Test Example 21 BB+ soy sauce AOK 210 2.4x1ο4 5.6χ106 8·〇χ106 Test Example 22 BB+ Rice Bacteria IK-05074 2.2xl〇4 5.1χ106 6.7χ106 Control area only BB 2·〇χ1〇4 4.6χ106 4.0χ106 As test The solid cultures of the 'Soybean agaric strain AOK2 10 strain and the rice bran strain IK-05074 strain shown in Examples 19 and 20 did not have antibacterial activity against LA. As shown in Test Examples 21 and 22, the solid cultures of the soy sauce AOK210 strain and the rice bran strain IK-05 074 did not have antibacterial activity against BB. According to the results, the soy sauce A〇K21 strain and the rice strain 1K_05074 -45- 200814936 strain, its pathogenic bacteria Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, Clostridium spores Clostridium perfringens has antibacterial activity, and has no antibacterial activity against Lactobacillus acidophilus and Bifidobacterium, breve, which have the opportunity to become a probiotic Bacillus subtilis or a preferred intestinal bacterium. Therefore, by combining with the soy sauce φ AOK210 strain and/or the rice bacterium IK-05 074 strain and B. subtilis B. subtilis, the function of not losing the ability of B. subtilis is not exhibited, nor is it It is thought to be adversely affected by the Lactobacillus L. acidophilus and the Bifidobacterium B. breve which are useful as intestinal bacteria, and is therefore considered to be a novel animal feed additive. <3 > Determination of the protozoal activity of the culture of the genus Fusarium (1) Eimeriatenella control test φ Collect chicken feces that have been naturally infected with Eimeria tenella, separate the oocysts under a stereoscopic microscope, and wash them with physiological saline. . In a culture of 9 cm in diameter, 5 ml of physiological saline was added to the dish, and the washed oocysts were put into about 4000 cells/ml. A ground solid culture of AOK210 strain, AOK43 strain, ΑΟΚN45 86 strain, AOK B650 strain, and IK-05074 strain was placed at 50 mg per culture dish, and each was used as Test Examples 23 to 27. A petri dish without a sputum culture was also used as a control zone. Each dish was incubated at 37 rpm (150 rpm). After 7 days, the number of oocysts was measured under a stereoscopic microscope, and the state of cell wall deformation or dissolution was observed, and the reduction rate of oocysts -46-200814936 and dissolution denaturation rate were calculated. The results are shown in Table 19. [Table 19] Table 1 9 Reduction rate of oocysts (%) Dissolution denaturation rate n (%) Day 7 Day 7 Test Example 23 4000 40 0 90 25 Test Example 24 45 00 800 82 12 Test Example 25 4400 900 80 11 Test Example 26 410 0 900 78 11 Test Example 27 42 0 0 380 91 29 Control area 4400 4400 0 0 1) Proportion of residual oocysts on day 7

As shown in Test Examples 23 to 27, solid cultures of AOK210 strain, AOK43 strain, AOK N45 86 strain, AOK B65 0 strain, and IK-05074 strain reduced the number of oocysts of E. tenella and showed oocysts. Dissolution denaturation activity. Among them, in particular, by treating the oocysts of E. tenella with AOK210 strain and IK-05074 strain, extremely high oocyst reduction rate and dissolution degeneration rate can be obtained. (2) E i m e r i a z u e r n i i Control test Collect the sputum of the cow that has been naturally infected with Eimeria zuernii, and separate the oocyst under a solid microscope and wash it with physiological saline. 5 ml of physiological saline was added to a culture dish of 9 cm in diameter, and the washed oocysts were put into about 2 000 /ml. A ground solid culture of AOK210 strain, AOK43 strain, AOK N4 5 86 strain, AOK B65 0 strain, and IK-05074 strain was placed at 50 mg per culture dish, and each was used as test examples 28 to 32. A petri dish containing no -47-200814936 sputum culture was used as a control area. Each dish was incubated at 37 ° C with shaking (150 rpm). After 7 days, the number of oocysts was measured under a stereoscopic microscope, and the state of deformation or dissolution of the cell wall was observed, and the reduction rate and dissolution rate of the oocysts were calculated. The results are shown in Table 20. [Table 20] Table 20 Reduction rate of oocysts (%) Dissolution denaturation rate n (%) Day 7 Test Example 2 8 2200 600 73 20 Test Example 2 9 2200 1100 50 9 Test Example 30 2000 1100 45 8 Test Example 3 1 2300 1200 48 8 Test Example 32 2000 5 00 75 3 1 Control area 2300 2 30 0 0 0 1) Proportion of residual oocysts on the 7th day

As shown in Test Examples 28 to 32, a solid culture of AOK210 strain, AOK43 strain, AOK N45 86 strain, ΑΟΚB650 strain, and IK-050 74 strain reduced the number of oocysts of E. zuernii and showed oocyst dissolution. Denaturation activity. Among them, in particular, by treating the oocysts of E. zuernii with the AOK210 strain and the IK-05074 strain, an extremely high oocyst reduction rate and a dissolution denaturation rate can be obtained. According to the above, the above-mentioned genus Fusarium is presumed to be effective for the control of coccidia. <4> Culture of Bacillus subtilis using the cell formation medium of the following composition: Bacillus subtilis-48 - 200814936 DB901 1 strain, Bacillus subtilis NBRC3009 strain, Bacillus subtilis NBRC3025 strain, Bacillus subtilis NBRC3108 strain, Bacillus subtilis NBRC3 3 3 6 strain Each was incubated in liquid at 72 ° C for 72 hours. The obtained culture solution was centrifuged, and the cells were collected. The obtained cells were freeze-dried and ground to obtain a cell powder. The cell densities of DB9011 strain, NBRC3 009 strain, NBRC3 025 strain, NBRC3108 strain, and NBRC3 3 36 strain were 1.01 X 10MCFU/g ^ 1.21 X 10MCFU/g > 1 · 1 5 x 1 0 11 CFU/g, 1.06 x lO^CFU/g, l.ogxloHCFU/g. For the cell density, the obtained cell powder was diluted to a suitable concentration with sterile water, and heated at 7 (rc for 30 minutes, so that only the vegetative cells died, and then inoculated into a common acacia medium, and the number of colonies formed was measured. The Bacillus subtilis DB9011 strain was deposited in the consigned bio-storage center of the Industrial and Commercial Research Institute of the Industrial and Commercial Research Institute of the National Institute of Industrial Science and Technology, Izumi, Izumi, Japan, on May 21, 1991. ), it was classified as Bacillus licheniformis at the time of storage, but it was confirmed that it was attributed to Bacillus subtilis. In addition, NBRC3009 strain, NBRC3 025 strain, NBRC3108 strain, and NBRC 3 3 3 6 strain were deposited in independent administrative legal person product evaluation. Strain of the Biological Genetic Resources Department (NBRC) of the technical base mechanism. Cell formation medium component Difco nutrient solution 8.0g KC1 l.〇g MgS〇4 * 7H2〇〇.25g MnCl2 · 4H20 0.002g -49- 200814936 Adjusting the pH to 7.0 The whole amount is 1,000ml. After sterilization in the sterilization kettle, the sterilized CaCl2 solution and the FeS04 solution are each added to 5 χ1 (Γ4Μ and 1 χ10·6Μ 1) Schaeff Er, P·, J· Millet, J·Ρ· Aubert, Proc. Natl. Acad. Sci. USA, 54, 7 0 4-7 1 1 (1 965) <5> Feeding Test-1 The above <1> The obtained ground solid culture of the soy sauce AOK210 strain, the ground solid culture of the rice bacterium IK-0 5 074 strain, the DB 9011 strain, and the NBRC300 strained cells were each diluted with lactose to prepare Different concentrations of feed additives (Production Examples 1 to 4). Further, cultures of AOK210 strain or IK-05074 strain, and cells of DB9011 strain or NBRC30〇9 strain were each diluted with lactose to prepare different concentrations. Feed additive (Production Examples 5 to 8). Among the respective feed additives, the solid culture and the addition amount of Bacillus subtilis are shown in Table 21. 1 -50- 200814936 imi] Table 21

Production example Addition of Bacillus bacterium culture (% by mass) Bacillus bacteria (xl08CFU/g) Acid-resistant α-amylase activity (U) Acid protein enzyme acid carboxyl group enzyme activity total (U) 1 A AOK210 1.0 - 26 469 B 2.0 - 51 938 C 5.0 128 2346 D 10 - 256 4692 E 15 - 383 7038 F 20 - 511 9383 G 40 - 1022 18767 2 A IK-05074 1.0 - 43 513 B 2.0 - 85 1026 C 5.0 - 213 2565 D 10 - 425 5130 E 15 - 638 7695 F 20 - 850 10260 G 40 - 1700 20520 3 A DB9011 - 1.0 - - B - 2.0 - - C - 5.0 - - D - 10 - - E - 15 - - F - 20 - - G - 40 - - 4 A NBRC3009 - 1.0 - - B - 2.0 - - C - 5.0 - - D - 10 - - E - 15 - - F - 20 - G - 40 - - 5 A AOK210 + DB9011 0.5 0.5 13 235 B 1.0 1.0 26 469 C 2.5 2.5 64 1173 D 5.0 5.0 128 2346 E 7.5 7.5 192 3519 F 10 10 256 4692 G 20 20 511 9383 6 A IK-50074 + DB9011 0.5 0.5 21 257 B 1.0 1.0 43 513 C 2.5 2.5 106 1283 D 5.0 5.0 213 2565 E 7.5 7.5 319 3848 F 10 10 425 5130 G 20 20 850 10260 7 A AOK210 + NBRC3009 0.5 0.5 13 235 B 1.0 1.0 26 469 C 2.5 2.5 64 1173 D 5.0 5.0 128 2346 E 7.5 7.5 192 3519 F 10 10 256 4692 G 20 20 511 9383 8 A IK-05074 + NBRC3009 0.5 0.5 21 257 B 1.0 1.0 43 513 C 2.5 2.5 106 1283 D 5.0 5.0 213 2565 E 7.5 7.5 319 3848 F 10 10 425 5130 G 20 20 850 10260 -51 - 200814936 (2) Chicken feeding test The feed additives of Examples 1 to 8 will be manufactured in comparison with the feed (SD before and after the tender chicken, Japanese compound feed system) The company's system, the feed without added antibacterial substances) is the total mass of 〇. 1% by mass mixed in the feed. Twelve chicks hatched from the chicken breeder (trade name: CHUNKY) were grouped, and the above-mentioned feed was administered to each group for 35 days to carry out a growth test. Feed the bait continuously and feed it in a free way. Further, a feed having only lactose mash and 1% by mass was mixed as a control zone, and a growth test was carried out in the same manner. Determine the body weight of each group of chicks after 3 days, and then calculate the average of each group.

The results are shown in Table 22. [Table 22] Table 22 Average weight of chickens added to the production example (g) ABCDEFG 1 AOK210 1500 1502 1537 1546 1591 1636 1642 2 IK-05074 1507 1505 1524 1536 1594 1633 1646 3 DB9011 1512 1508 1502 1566 1640 1665 1680 4 NBRC3009 1509 1501 1511 1571 1644 1652 1679 5 AOK210+DB9011 1561 1651 1606 1638 1621 1665 1669 6 IK-05074+DB90I1 1589 1636 1649 1629 1641 1660 1664 7 AOK210+NBRC3009 1533 1601 1633 1639 1630 1655 1660 8 IK-05074+NBRC3009 1540 1610 1641 1645 1639 1651 1666 Control area lactose 0.1% 1487 / / / / / /

The feed additive containing the cultures of the soy sauce AOK210 strain or the rice bran strain IK-05 074 is 15% by mass or more of the production examples 1 (E to G) and the production example 2 (E to G), and the chicks are cast. In the case of the chicks of the control zone -52-200814936, the production example 3 (E to G) of the bacterium of the genus DB9011 strain or the NBRC3009 strain having a significant weight gain was confirmed, and the production example 4 (E, for Chicks were administered in comparison with the significant weight gain of the control area. On the other hand, AOK210 strain or IK_ was 1.0% by mass or more, and DB9011 strain or 108 CFU/g or more, and each was combined into Production Example 5 ( (B to G), Production Example 7 (B to G), and the effect of confirming significant weight gain when the product was ingested, and the effect of using the active ingredients in the culture of the genus Fusarium and Bacillus subtilis was lower. Concentration, and promote (3) piglet feeding test will produce the feed additives of Examples 1~8, with the standard feed of the SD piglets before and after artificial milk, the system made by the company, without the addition of antibacterial substances) In the 3 week old piglet (commodity: head group, at 35 During the day, the group continued to experiment with the free water supply. Lactose 〇·1% by mass of the feed was used as the control area. The weight of each group of piglets after 3 days was measured, and the results were not shown. 23. In the case of the feed additive containing 1.5×l09 CFU/g to G), it was confirmed that there were _05074 cultures of NBRC3009 strain 1. Ox: B to G), and Production Example 6 8 (B~ G), make the fruit. By this, it is known that the combination of the individual can increase the weight of the individual chicks.丨 For the feed of piglets (this compound feed type is 0.1% by mass mixed with S: about kexia) 8 The above feed is given to the bait. In addition, only the mixed plots are used for the growth test. -53- 200814936 [Table 23] Table 23 Average weight of piglets added to the manufacturing example (g) ABCDEFG 1 AOK210 22.6 22.3 22.5 23.6 25.0 24.7 24.9 2 IK-05074 22.5 22.7 22.4 23.8 24.8 25.0 25.1 3 DB9011 22.9 22.7 22.7 23.5 24.2 24.4 24.6 4 NBRC3009 22.4 22.7 23.0 23.4 24.5 24.9 24.8 5 AOK210+DB9011 22.8 24.4 24.8 25.0 24.8 25.0 25.3 6 IK-05074+DB9011 23.1 24.6 25.1 24.9 25.1 25.2 25.1 7 AOK210+NBRC3009 23.0 24.5 24.8 24.6 24.9 25.0 25.1 8 IK-05074+ NBRC3009 23.2 24.7 25.0 24.9 25.1 24.8 24.9 Control Zone Lactose 0.1% 22.4 / / / / /

The feed additive containing the cultures of the soy sauce AOK210 strain or the rice bran strain IK-05074 is 15% by mass or more of the production examples 1 (E to G) and the production example 2 (E to G), and is administered to the piglets. Significant weight gain was confirmed in comparison to piglets in the control area. Will contain

The Bacillus subtilis DB9011 strain is a feed additive of Production Example 3 (E to G) of 1.5×10 08 CFU/g or more and Preparation No. 4 (E to G) of a cell of NBRC3009 of 1.5×10 8 CFU/g or more, and is administered to piglets. Significant weight gain was confirmed in comparison to piglets in the control area. On the other hand, the culture of the AOK210 strain or the IK-05074 strain is 1.0% by mass or more, and the cells of the DB9011 strain or the NBRC3 009 strain are 1.0×10 08 CFU/g or more, and are combined into Production Example 5 (B to G). In Examples 6 (B to G), Production Example 7 (B to G), and Production Example 8 (B to G), when an animal was ingested, it was confirmed that there was a significant weight gain effect. Thereby, it is known that by combining the culture of the genus Fusarium and the Bacillus subtilis, the lower concentration of each active ingredient can be used in comparison with -54-200814936, and the weight gain of the piglets is promoted. <6> Feeding test-2 (1) Production of feed additive The culture of AOK210 strain or IK-05074 strain obtained in the above <1>, and the cells of DB9011 strain or NBRC3〇09 strain were combined to obtain milk H The "release" was prepared as a feed additive containing various components in various ratios (Production Examples 9 to 12). The solid culture and the amount of the added barium in the respective feed additives are shown in Table 24.

-55- 200814936

[Table 24] Table 24 Production example Addition of bacillus bacterium valency %) Bacillus bacterium (xl08CFU/g) Acid-resistant α-amylase activity (U) Acid protein enzyme acidic carboxy-peptide activity (U) 9 A AOK210 + DB9011 4.5 0.5 115 2111 B 4.0 1.0 102 1876 C 3.5 1.5 89 1642 D 2.5 2.5 64 1173 E 1.5 3.5 38 704 F 1.0 4.0 26 469 G 0.5 4.5 13 235 10 A IK-05074 + DB9011 4.5 0.5 191 2309 B 4.0 1.0 170 2052 C 3.5 1.5 149 1796 D 2.5 2.5 106 1283 E 1.5 3.5 64 770 F 1.0 4.0 43 513 G 0.5 4.5 21 257 11 A AOK210 + NBRC3009 4.5 0.5 115 2111 B 4.0 1.0 102 1876 C 3.5 1.5 89 1642 D 2.5 2.5 64 1173 E 1.5 3.5 38 704 F 1.0 4.0 26 469 G 0.5 4.5 13 235 12 A IK-05074 + NBRC3009 4.5 0.5 191 2309 B 4.0 1.0 170 2052 C 3.5 1.5 149 1796 D 2.5 2.5 106 1283 E 1.5 3.5 64 770 F 1.0 4.0 43 513 G 0.5 4.5 21 257 -56- 200814936 (2) Feeding test for chicks The feed additives of Examples 9 to 12 will be prepared for the feed of the chickens (for the early and late use of SD tender chicken, Japan Compound Feed Co., Ltd.) System, no added antibacterial substances Fodder) mass of the whole square. 1 mass% in the feed mixture. The chicks hatched from the chicken breeder (trade name: CHUNKY) were planted in groups of 12, and the above-mentioned feed was administered to each group for 35 days to carry out a growth test. Feed the bait continuously and feed it in a free way. In addition, only lactose 〇 will be mixed. 1 mass ° /. The feed was used as a control area and the growth test was performed in the same manner. The body weight of each group of chicks after 3 days was measured, and the average 値 of each group was calculated. The results are shown in Table 25. [Table 25] Table 25 Average weight of chicks added to the production example (g) ABCDEFG 9 AOK210+DB9011 1658 1771 1809 1819 1755 1716 1642 10 IK-05074+DB9011 1650 1787 1812 1803 1769 1723 1651 11 AOK210+NBRC3009 1642 1753 1791 1801 1738 1704 1626 12 IK-05074+NBRC3009 1634 1769 1794 1785 1751 1706 1637 Control area lactose 0.1% 1594 7 / / / / Soybean soya AOK210 strain or rice bran strain IK-05074 strain of ground solid culture, and DB9011 strain The ratio of the NBRC3 009 strain is 4% by mass: lxl08CFU/g to 1% by mass: 4xl08CFU/g is most suitable (manufacturing example 9 (B~F), manufacturing example 10 (B~F), manufacturing example 1 1 -57- 200814936 (B to F), Manufacturing Example 12 (B to F)). From this, it was found that the concentration of Bacillus subtilis for each of the feed additives having the acid-resistant α-amylase activity of ι is most suitable in the range of 〇·5χ106 to 2.0×10 7 CFU/g. Further, for the feed additive having a total of 1 U of acidic protein enzyme and acidic carboxy peptide enzyme, the concentration of Bacillus subtilis is most preferably in the range of 4.5 X 104 8.5 8.5 105 CFU/g. (2) Piglet feeding test The feed additives of Examples 9 to 12 will be produced for the feed of piglets (standard feed for SD piglets before and after artificial milk, manufactured by Japan Compound Feed Co., Ltd., without added antibacterial substance) The mass is 〇. 1% by mass mixed in the feed. A group of 8 piglets of 3 weeks old (trade name: about kexia) was used as a group for the above-mentioned feeds for each group within 35 days, and the growth test was carried out by means of free water supply. Further, a feed in which only lactose 〇·1% by mass was mixed was used as a control zone, and a growth test was carried out in the same manner. The body weight of each group of piglets after 3 days was measured, and the average 値 of each group was calculated. The results are shown in Table 26. [Table 26] Table 2 6 Production Example Addition of average weight of piglets (g) ABCDEFG 9 AOK210+DB9011 23.8 24.5 25.9 26.1 25.6 24.6 23.5 10 IK-05074+DB9011 23.5 24.9 26.0 25.8 25.8 24.8 23.3 11 AOK210+NBRC3009 23.6 24.3 25.6 25.8 25.3 24.4 23.3 12 IK-05074+NBRC3009 23.3 24.7 25.7 25.5 25.5 24.5 23.0 Control area lactose 〇·1% 23.1 / / / 7 / / -58- 200814936 Soybean soya AOK210 strain or rice bacterium IK-05074 strain The ratio of the crushed solid culture and the DB9011. strain or the NBRC3009 strain is 4% by mass: lxl〇8 CFU/g to 1% by mass: 4×10 8 CFU/g is most suitable (manufacturing example 9 (B to F), production) Example 1 0 (B to F), Production Example 11 (B to F), and Production Example 12 (B to F)). <7> Pathogen challenge test for chickens (1) Salmonella enteritidis challenge test for the feed of chicks (pre-SD chicken, Japanese compound feed (stock), feed without antibacterial substance), quality, The feed additives of Production Examples 1 to 8 were mixed in the feed at a concentration of 0.1% by mass. One of the chicks, which was won by the egg from the tender chicken breeder (trade name: CHUNKY), was used as a group, and the above feed was given to the bait without interruption, and the water was freely supplied for 14 days. Further, only lactose mash.; [% by mass of the feed was used as a control zone, and the test was carried out in the same manner. At the time of the 7th, each oral administration of 2.3xl07CFU Salmonella Salmonella enteritidis (SE) was administered. At the age of 14 days, the cecal contents and the total excretory cavity were wiped with a cotton swab to take the feces. The number of S E bacteria in the cecal contents was measured by the following method, and the infection index and the defense index were calculated. The cecal contents lg were added to the sterile phosphate buffer physiological test water, diluted to 1 〇 times, and thoroughly mixed to prepare a sample stock solution. Next, the sample stock solution was diluted to 10 times with sterile physiological test water, and the stage was diluted -59-200814936, and then a stage dilution was made. The sample stock solution and the step-dilution solution were applied to the SS agar medium ("Ri Shui" (manufactured by Rishui Pharmaceutical Co., Ltd.) and Brilliant Green Tablet Culture Laboratories) at a temperature of 37 ° C. After 24 hours, the number of slumps of typical SEs grown on each plate medium was measured. Furthermore, the fungus was picked up by the colony, and it was inoculated into the deacidification test of the amino acid, the SIM vegetable medium "-Sizhui" (made by Rishui Music Co., Ltd.), and the TSI vegetable medium "Ri Shui". (Nissui Pharmaceutical Co., Ltd.) was cultured at 37 ° C for 24 hours, and its properties were confirmed. The number of colonies in which the SE was confirmed was multiplied by the dilution ratio of the diluent, and the number of S E bacteria per gram of the cecal contents was calculated. Based on this result, the infection index and defense index were calculated as shown below. The so-called infection index refers to the degree of infection rate of 75 pathogens; the so-called defense index refers to the ability of defensive pathogens to be detected by their respective feeds when compared to feeds containing no sputum. Xin Infection Index · Logarithmic mean SE of SE bacteria in the contents of the intestines of each body (average log of log CFU/g) > Defense index: infection index of control area / infection index of each test area - About The feces taken from the total excretion chamber were qualitatively cultured in accordance with the following methods to confirm the characteristics of SE. That is, the feces attached to the cotton swab are suspended in 1 〇ml of sterile phosphate buffered saline to prepare a sample solution, which is then applied to s S agar medium and Brilliant green. On the plate culture medium, it was cultured for 24 hours, and then the colony formation of the typical s E was determined on each plate medium. Furthermore, from -60 to 200814936, the bacteria were picked up by the colony, and they were inoculated into the LIM vegetable medium "Ri Shui" (made by Rishui Pharmaceutical Co., Ltd.), SIM Acacia medium and TSI vegetable medium, and cultured at 37 °C. After 24 hours, confirm the traits. The infection index is shown in Table 27, the defense index is shown in Table 28, and the total number of individuals in the total excretory cavity taking feces SE is shown in Table 29. [Table 27] Table 27 Addition of bacterial infection index A Β CDEFG 1 AOK210 6.4 6.4 6.1 4.9 4.1 3.3 3.2 2 IK-05074 6.3 6.2 6.3 4.0 3.2 3.2 3.1 3 DB9011 6.7 6.5 6.4 5.0 4.5 3.9 4.0 4 NBRC3009 6.4 6.3 6.0 4.8 4.3 3.7 3.8 5 AOK210+DB9011 6.2 4.9 3.3 3.0 3.1 2.6 <2i) 6 IK-05074+DB9011 5.8 4.7 3.2 3.1 3.0 2.4 <21) 7 AOK210+NBRC3009 5.6 4.7 3.0 2.9 3.0 2.5 <21) 8 IK- 05074+NBRC3009 5.3 4.5 3.0 3.0 2.9 2.2 <21) Control area lactose 〇·1% 6·4 / / / / / 7

1) Detection limit below -61 - 200814936 [Table 28] Table 28 Manufacturing example added bacteria defense index A Β CDEFG 1 AOK210 1.0 1.0 1.0 1.3 1.6 1.9 2.0 2 IK-05074 1.0 1.0 1.0 1.4 1.6 2.0 2.1 3 DB9011 1.0 1.0 1.0 1.3 1.5 1.7 1.7 4 NBRC3009 1.0 1.0 1.1 1.3 1.5 1.7 1.7 5 AOK210+DB9011 1.1 1.4 2.0 2.2 2.2 2.6 >4 6 IK-05074+DB9011 1.2 1.4 2.1 2.1 2.2 2.8 >4 7 AOK210+NBRC3009 1.1 1.4 2.1 2.2 2.1 2.6 >4 8 IK-05074+NBRC3009 1.2 1.4 2.1 2.1 2.2 2.9 >4 Control area lactose 0.1% 1·0 / / / / / / [Table 29] Table 29 Number of individuals tested for added bacteria SE (only / 12) A Β CDEFG 1 AOK210 12 12 12 12 11 8 6 2 IK-05074 12 12 12 12 11 8 6 3 DB9011 12 12 12 12 12 8 7 4 NBRC3009 12 12 12 12 10 5 5 5 AOK210+DB9011 12 12 6 6 6 3 0 6 IK-05074+DB9011 12 12 7 6 5 2 0 7 AOK210+NBRC3009 12 12 6 5 5 1 0 8 IK-05074+NBRC3009 12 12 6 6 4 2 0 Control area lactose 〇·1% 12 / / / / / /

If the ground solid culture of the soy sauce AOK210 strain or the rice bran strain IK-05074 is separately added to the feed additive by 20% by mass or more, the SE concentration of the cecal contents will be low, and the SE will be low. Sense-62-200814936 dyeing index (manufacturing example i (f, g), manufacturing example 2 (F, G)). In addition, if B. subtilis DB9011 strain or NBRC3009 strain is separately added with 2·〇χ 109 CFU/g or more in the feed additive, the concentration of se in the cecal contents will become lower, and the infection index of low SE will be displayed (manufacturing example) 3 (F, G), Manufacturing Example 4 (F, G)). In contrast, in the combination of the soy sauce AOK210 strain, the rice bran ιΚ_〇5〇74 strain, and the Bacillus subtilis DB9011 strain or the NBRC3009 strain, the culture of the ΑΟΚ210 strain or the IK-05074 strain in the feed additive is used. When the addition of 2.5 mass% or more and the addition of DB9011 strain or NBRC3009 strain to 2.5×10 CFU/g or more, it is possible to add 20% by mass or more of AOK210 strain or IK-05074 strain alone to the feed additive, or DB901 1 When the strain or the NBRC3009 strain was added alone at 2.0×10 CFU/g or more, the same degree of control effect of SE (manufacturing example 5 (c to G), production example 6 (C to G), and production example 7 (C to G), Production Example 8 (C to G)). Furthermore, when a feed additive comprising AOK210 strain or rice bran strain IK-05074 is 20% by mass or more, and DB9011 strain or NBRC3009 strain is 2.0xl09 CFU/g or more, the effect of completely preventing SE can be obtained (manufacture Example 5 (〇), Production Example 6 (G), Production Example 7 (G), and Production Example 8 (G)). Thus, by administering the combination of soy sauce or rice bran and Bacillus subtilis to the chicks, it is possible to prevent SE infection at a lower concentration when the respective active ingredients are used separately. The effect. (2) Assault test of Clostridium perfringens The chicks were bred in the same manner as above using the feed additive prepared above -63-200814936. At the time of 7th, each of the 5.0Xl〇7CFU shuttle Clostridium perfringens (CP) was administered orally with a cotton swab to wipe the cecal contents and the total excretion cavity to control the number of CP bacteria in the cecum. Calculate the infection index and defense index. 1 g of the cecal contents were added to a sterile phosphate buffer to give a 10-fold volume, and the mixture was thoroughly mixed to prepare a sample stock solution. The stock solution is diluted 1 times with sterile physiological test water, and then made into a stage dilution. The sample stock solution and the stage were each smeared on Clostridia (measured by Clostridia), and then cultured in an anero pack for 24 hours, and the number of drops on each plate medium was measured. Further, The bacteria are picked up by the colony, and they are inoculated into CW agar medium (made by Rishui Pharmaceutical Co., Ltd.) for 24 to 48 hours, and cultured in a good gas and suffocating gas, and then the number of colonies confirmed by CP is multiplied by The above dilution was used to calculate the number of CP bacteria per lg of the contents of the calf. The infection index and the defense index were calculated in the same manner as above. Regarding the feces taken from the total excretion chamber, the following methods were used to confirm the culture. The trait of CP. That is, the cotton feces are suspended in 10 ml of sterile phosphate buffered saline solution, and then applied to the fusiform stomach at 0·1 m 1 respectively. ()), Bacillus genus, and take its feces at 14 。. Perform the measurement 'reconstruction test water' dilute, then dilute the solution to 0.1 ml of the nutrient in the sample (daily water! 3 5 °C suspected birth black set with eggs The dilution ratio confirmed by the culturing of the trait at 35 ° C, and the result is, and the method is applied to the individual flower sticks. The sample bacteria are prepared (Clostridia is cultivated at 35 ° C - 64 - 200814936) After 24 hours, it was determined whether or not black colonies were formed on each of the plate mediums. Further, the bacteria were picked up by the colonies, and they were inoculated into CW agar medium (manufactured by Ussui Pharmaceutical Co., Ltd.) supplemented with egg yolk, and 35t: culture 24 ~48 hours, confirm the traits with good and irritated culture. The infection index is shown in Table 30, 'The defense index is shown in Table 31, and the total excretion chamber is the number of individuals tested by feces. In Table 32. [Table 30]

Table 30 Addition bacteria in the manufacturing example_Infection index A Β CDEFG 1 AOK210 6.8 6·8 5.7 5.0 4.2 3.4 3.3 2 IK-05074 6·5 6·7 5.6 4.9 A 7 ^ ο 3 DB9011 6.9 6.9 6.1 4.9 4 7 mJ · J 3 8 3.8 4 NBRC3009 6.6 6.4 5.8 4.7 4 5 3 6 3 6 5 AOK210+DB9011 6.5 5.1 ”3 3.1 3.0 •J •XJ 2.8 mJ · Ky &lt;2^ 6 IK-05074+DB9011 6.0 5.2 3.4 3.2 2 Q <21) <21) 7 AOK210+NBRC3009 5.8 4.9 3.1 3.0 0 Q 9 7 <21) 8 IK-05074+NBRC3009 6.1 5.0 3.2 3.0 2 9 2 5 &lt;2l) Control area lactose 0·1ο/〇6.6 / / / / / 1) Detection limit below -65- 200814936 [Table 3 1 ] Table 31 Manufacturing example added bacteria defense index ABCDEFG 1 AOK210 1.0 1.0 1.2 1.3 1.6 1.9 2.0 2 IK-05074 1.0 1.0 1.2 1.3 1.5 2.0 2.1 3 DB9011 1.0 1.0 1.1 1.3 1.4 1.7 1.7 4 NBRC3009 1.0 1.0 1.1 1.4 1.5 1.8 1.8 5 AOK210+DB9011 1.1 1.3 2.0 2.1 2.2 2.4 &gt;4 6 IK-05074+DB9011 1.1 1.3 1.9 2.1 2.3 &gt;4 &gt;4 7 AOK210+NBRC3009 1.1 13 2.1 2.2 2.3 2.4 &gt;4 8 IK-05074+NBRC3009 1.1 1.3 2.1 2.2 2.3 2.6 &gt;4 Control Area Lactose 〇·!% 1.0 / / / / / / [Table 32] Table 32 Number of individuals tested in the addition of bacteria SE (only / 12) ABCDEFG 1 AOK210 12 12 12 12 11 7 6 2 IK-05074 12 12 12 12 10 8 7 3 DB9011 12 12 12 12 12 8 7 4 NBRC3009 12 12 12 12 10 7 7 5 AOK210+DB9011 12 12 7 8 6 4 0 6 IK-05074+DB9011 12 12 8 7 7 0 0 7 AOK210+NBRC3009 12 12 8 7 7 3 0 8 IK-05074+NBRC3009 12 12 9 8 5 1 0 Control area lactose 0.1% 12 / / / / / /

If the ground solid culture of the soy sauce AOK210 strain or the rice bran strain IK-05074 is separately added to the feed additive by 20% by mass or more, the bacterial concentration of the CP content of the sausage contents becomes low and is displayed. Low CP-66 - 200814936 Infection index (manufacturing example 1 (F, g), manufacturing example 2 (F, G)). In addition, if B. subtilis DB9011 strain or NBrC30〇9 strain is separately added with 2.0x 109 CFU/g or more in the feed additive, the cp factor concentration of the cecal contents becomes low, and the infection index of low CP is displayed (manufacturing example) 3 (F, G), Manufacturing Example 4 (F, 〇)). On the other hand, in the combination of the soy sauce AOK210 strain, the rice bran ιΚ-〇5〇74 strain, and the Bacillus subtilis DB9011 strain or the NBRC3009 strain, for example, in the feed additive, ΑΟΚ210 strain or ΙΚ·05〇74 strain When the culture is added in an amount of 25% by mass or more, when DB901 1 strain or NBRC3 009 strain is added at 2.5×10 CFU/g or more, it is possible to add 20% by mass or more of AOK210 strain or IK-05074 strain separately to the feed additive. Or the DB9011 strain or the NBRC300 strain is separately added by 2.〇xi〇9 CFU/g or more, and is a control effect of CP of the same degree (manufacturing example 5 (c~G), manufacturing example 6 (C~G) Production Example 7 (C to G) and Production Example 8 (C to G)). Further, when a feed additive having a combination of AOK210 strain or rice bran ΪΚ-05074 strain of 20% by mass or more and DB901 1 strain or NBRC3009 strain of 2.0×10 09 CFU/g or more is administered, the effect of completely preventing CP can be obtained ( Production Example 5 (G), Production Example 6 (F, G), Production Example 7 (G), and Production Example 8 (G)). Therefore, by using the combination of the soy sauce AOK210 strain or the rice bran strain IK-05074 strain and the Bacillus subtilis DB90U strain or the NBRC3 009 strain, it is possible to obtain the lower active ingredients when the respective active ingredients are used separately. The effect of Cp infection can be prevented at the concentration. &lt;8&gt; edema disease test for piglets-67- 200814936 For the full quality of feed for piglets (pre-emergence of s D piglets, Japanese compound feed (stock), feed without antibacterial substances), The feed additives of Production Examples 1 to 8 produced were mixed in a feed at a concentration of 〇·1% by mass. Three-week-old piglets (about ketia species), with a group of eight heads, were continuously fed to each group for the above-mentioned feed, and were given free water for 14 days. Further, a feed in which only lactose 〇.1% by mass was mixed was used as a control zone, and the test was carried out in the same manner. Escherichia coli (EC) was cultured in a trypsin gland liquid medium (manufactured by Difco Laboratories) at 37 ° C for 4 hours. After centrifugation (3,000 rpm, 15 minutes) at 4 ° C, the pellet was added to an enteric capsule. At 4 weeks of age, an enteric capsule of 4.5x1 08 CFU of EC was used per head, once a day, for 3 consecutive days for mandatory oral administration. Further, physiological saline was added to the enteric capsule instead of the edema disease, and the same test was carried out by forced oral administration as a no-skin case. When the animals were raised to 5 weeks old, the body weight was measured, and the average weight of the weight gain of each group was calculated. In addition, from the start of forced oral administration to the end of the trial, the daily clinical observation of fecal traits and edema around the eyes 'calculate the total of fecal trait scores and edema around the eyes of each individual during the trial period, and calculate The average number of scores for each group. The weight gains during the test period are shown in Table 3 3, and the fecal trait scores are shown in Table 3 4 ' and the edema around the eyes is shown in Table 35. -68- 200814936 [Table 33] Table 33 Addition of bacteria to increase body weight [g) ABCDEFG 1 AOK210 13 39 65 116 232 230 238 2 IK-05074 11 33 69 110 226 239 237 3 DB9011 22 42 65 90 225 220 223 4 NBRC3009 23 46 68 95 236 231 234 5 AOK210+DB9011 48 96 210 225 224 223 222 6 IK-05074+DB9011 55 105 209 223 225 225 224 7 AOK2104^BRC3009 48 101 221 235 234 233 237 8 IK-05074+NBRC3009 59 110 219 236 236 235 234 Non-inoculated lactose 0.1% 242 / / / Control area lactose 0.1% -5 / / / / [Table 3 4] Table 34 Production example added bacteria feces normal score ABCDEFG 1 AOK210 18.8 18.5 16.2 5.1 1.0 0.7 0.2 2 IK-05074 18.4 18.1 14.7 4.4 0.8 0.5 0.0 3 DB9011 17.4 17.1 14.7 2.8 0.5 0.0 0.0 4 NBRC3009 18.3 18.2 15.4 2.9 0.7 0.2 0.0 5 AOK210+DB9011 16.2 11.9 2.1 0.9 1.0 0.0 0.0 6 IK-05074+DB9011 15.1 10.5 1.8 0.5 0.0 0.0 0.0 7 AOK210+NBRC3009 18.1 12.5 2.2 0.9 0.8 0.0 0.0 8 IK-05074+NBRC3009 17.7 11.0 1.9 0.6 0.0 0.0 0.0 No inoculation lactose 〇·1% 0.0 / / / / / / Control area lactose 0.1% 19.1 / / / / / /

Normal fecal score: 〇. Normal stool, 1. Soft stool, 2. Muddy stool, 3. Water-soluble sputum-69- 200814936 m 35] Table 35 Circumference scores around the added bacteria in the manufacturing case ABCDEFG 1 AOK210 16.0 15.8 13.2 6.9 0.0 0.0 0.0 _ 2 IK-05074 15.9 15.5 12.1 5.7 1 0.0 0.0 0.0 3 DB9011 15.0 14.5 10.5 3.2 0.0 0.0 0.0 ^ 4 NBRC3009 15.2 14.9 11.0 4.5 0.0 0.0 0.0 5 AOK210+DB9011 11.3 7.1 0.0 0.0 0.0 0.0 0.0 6 IK-05074+DB9011 10.5 7.7 0.0 0.0 0.0 0.0 0.0 7 AOK210+NBRC3009 11.9 7.5 0.0 0.0 0.0 0.0 0.0 8 IK-05074+NBRC3009 10.3 7.9 0.0 0.0 0.0 0.0 0.0 No inoculation lactose 0.1% 0.0 / / 7 Control area lactose 0.1% 16.2 / / / Peripheral edema score: 〇·n,1·mild, 2·moderate, 3·severe, such as soy sauce sputum sputum 210 strain or rice blast fungus IK-05074 strain, ground solid culture system separately added 15% by mass or more In the feed additive, the weight gain was the same as that of the no-inoculation example (Production Example 1 (E to G), Production Example 2 (E to G)). When the B. subtilis DB9011 strain or the NBRC3 009 strain was separately added to the feed additive by 15×10 08 CFU/g or more, the weight gain system showed the same degree as the no-inoculation example (Production Example 3 (E to G), Production Example 4) (E~G)). In addition, the score of fecal traits and the edema around the eyes also showed the same tendency. In contrast, in the combination of the soy sauce AOK210 strain or the rice bran strain IK-05 074 strain, and the Bacillus subtilis DB9011 strain or the NBRC3 009 strain, the AOK210 strain or the IK-05074 strain was added by the feed additive by 2.5% by mass. In addition, when the DB901 1 strain or the NBRC3 009 strain was added at 2.5×10 CFU/g or more, the body weight gain was the same as that of the no-inoculation example (Production Example 5 (C to G), Production Example 6). (C to G), Production Example 7 (C to G), and Production Example 8 (C to G)). In addition, the fecal trait score and the edema around the eyes also showed the same tendency. Thus, by combining soy sauce or rice bran and Bacillus subtilis, it is possible to obtain an effect of preventing edema infection at a lower concentration when the respective active ingredients are used alone. &lt;9&gt; Salmonella Typhimurium for the cattle attack test 1 year after birth of the male male cattle (Holstein species in the Netherlands), 4 for a group, the marketed lactating cows with substitute milk (Pro milk, Japanese compound feed ( Share)), in the morning and evening, each time 2L each to 7 weeks. Free water, and hay and commercially available artificial milk (super-care, full-fledged) are constantly feeding. The feed additives of Production Examples 1 to 8 produced above were mixed and administered in a substitute milk at 25 g per day. In addition, only 25 g of lactose was mixed in the substitute milk as a control zone, and the test was carried out in the same manner. At 2 weeks of age, Salmonella Salmonella Typhimurium (ST), 5.0 x 106 CFU/g, was suspended in 10% NaHC03 per head, and all cattle were orally administered. When the animals were raised to 7 weeks old, the body weight was measured, and the average body weight of each group was calculated. Further, from the start of the forced oral administration to the end of the test, the observation of the fecal traits was performed daily to calculate the total of the fecal trait scores of each individual during the test period, and the average 値 of the scores of the respective groups was calculated. The weight gains during the test period are shown in Table 3, and the fecal trait scores are shown in Table 7-7 for the -71 - 200814936. [Table 36] Table 36 Addition of bacteria to increase the body weight j (g) ABCDEFG 1 AOK210 23.6 23.4 23.2 24.0 26.1 34.1 34.4 2 IK-05074 22.9 23.5 23.7 23.4 26.5 33.6 34.8 3 DB9011 22.1 21.8 21.5 22.0 24.5 32.2 33.2 4 NBRC3009 23.2 22.9 22.6 23.1 25.7 33.8 34.9 5 AOK210+DB9011 23.0 25.5 32.0 33.6 32.9 33.4 33.1 6 IK-05074+DB9011 23.5 26.7 32.5 33.9 33.2 33.5 33.8 7 AOK210+NBRC3009 24.2 26.8 33.6 34.5 35.1 34.8 35.2 8 IK-05074+NBRC3009 24.9 28.1 34.1 34.9 35.2 35.5 35.3 Non-inoculated lactose 0.1% 26.! / / / / / Control area lactose 0.1% 21.5 / / / / / / [Table 37] Table 37 Addition of bacterial feces normal score ABCDEFG 1 AOK210 38.3 38.5 31.5 14.4 5.8 0.8 0.3 2 IK-05074 39.0 38.1 30.5 13.8 4.7 1.0 0.1 3 DB9011 36.0 32.5 29.5 12.5 5.3 0.8 0.1 4 NBRC3009 37.8 37.1 31.0 13.1 4.9 0.9 0.2 5 AOK210+DB9011 24.2 13.2 1·5 0.5 0.0 0.0 0.0 6 IK-05074+DB9011 25.4 13.8 1.2 0.3 0.2 0.0 0.0 7 AOK210+NBRC3009 26.7 13.9 1.2 0.3 0.1 0.0 0.0 8 IK-05074+NBRC3009 27.6 14.5 1.8 0.5 0,0' 0.0 0.0 No inoculation · 0.0 billion 1% / / / / / / control zone lactose 0.1% 39.5 / / / / / /

Normal fecal score: 〇·normal stool, 1. soft stool, 2. muddy stool, 3. water-soluble sputum, 4. bloody stool-72- 200814936 such as soy sauce sputum sputum 210 strain or rice bacterium IK_〇 5 〇 74 strain When the ground solid culture is added separately to 20% by mass or more of the feed additive, the weight gain is the same as that of the non-inoculation example (Production Example i (F, G), Production Example 2 (F, G) ). By separately adding B. subtilis, DB9011 strain or NBRC3009 strain to 2.0×10 CFU/g or more in the feed additive, the weight gain system showed the same degree as the no-inoculation case (❿ Manufacturing Example 3 (F, G)- - Production Example 4 (F, G)). In addition, the fecal trait score' also showed the same tendency. In contrast, soy sauce

In the combination of AOK210 strain or rice bran strain IK-05074 strain, and Bacillus subtilis DB90U strain or NBRC3 009 strain, DB9011 will be added by adding 2.5% by mass or more of ΑK K 0 0 strain or IK-05074 strain in the feed additive. When the strain or the NBRC3 009 strain was added in an amount of 2.5×10 〇8 CFU/g or more, the body weight gain was the same as that in the no-inoculation example (Production Example 5 (C to G), Production Example 6 (C to G), and Production Example). 7 (C to G), Manufacturing Example 8 • (C to G)). In addition, the fecal trait score also showed the same tendency. Therefore, by combining soy sauce or rice bran and Bacillus subtilis, it is possible to prevent the effect of S T infection at a lower concentration when the respective active ingredients are used more separately. -73-

Claims (1)

200814936 X. Patent application scope 1 · An animal feed additive containing: selected from Aspergillus sojae, Aspergillus tamarii, Aspergillus foetidus, Aspergillus niger And at least one of the genus Aspergillus oryzae, a culture of an acid enzyme produced by the same species, and an animal feed additive of Bacillus subtilis; The sum of the acid enzyme activities per 1 g of the feed additive is above 20 U, and the concentration of B. subtilis is 2.5 x 107 to 2 x 109 CFU/g. 2. The animal feed additive according to claim 1, wherein the acidic enzyme comprises an acid amylase, and the feed additive has an activity of 1 U or more per 1 g of the acid amylase. 3. The animal feed additive according to claim 1 or 2, wherein the strain of AspergiHus has antibacterial activity against a pathogenic bacteria causing intestinal infection in an animal and/or for coccidia (c〇 Cidium •) kills protozoal activity. 4. The animal feed additive according to any one of claims 1 to 3, wherein the fungus (Aspergillus) is a strain of soy sauce and/or rice fungus. 5. The animal feed additive according to any one of claims 1 to 4, wherein the rice bran strain IK-05074 strain (BCRC 93 0092) and/or a variant thereof has the same strain as the aforementioned strain A strain of acidic enzyme producing ability. The animal feed additive of any one of claims 1 to 5, wherein the culture contains a vegetable nutrient source. 7. The animal feed additive according to claim 6, wherein the plant nutrient source is black rice. The animal feed additive according to any one of claims 1 to 7, wherein the Bacillus subtilis strain DB901 1 (CCRC 91 0024) and a variant thereof. The animal feed roller for animal use according to any one of claims 1 to 8, which is used as a growth promotion user. 10. The animal feed additive according to any one of claims 1 to 8, wherein the animal feed additive is used as a preventive/improving agent for intestinal infection. And a feed additive for animal use according to any one of claims 1 to 10, wherein the amount is 0.01% to 1.0% by mass. 12. A method for producing a feed comprising: a solid medium selected from the group consisting of a nutrient source for the proliferation of a genus Fusarium, and a culture selected from the group consisting of soy sauce, sputum, scorpion, black sputum, and At least one of the genus Fusarium genus, and the resulting culture has a total activity of more than 12 U per 1 kg of acid enzyme activity, and the concentration of Bacillus subtilis is 2·5 χ 106 to 2.0 x 101 ( ) CFU/kg steps. 1 3 · An animal breeding method characterized in that an animal is ingested as in claim 1 of the patent application. -75- 200814936 七无• · Ming said single simple number table is the map of the generation of the map table on behalf of the map: the table of the map, the table, the generation} } one or two fingers c C no eight, the case if there is a chemical formula When revealing the chemical formula that best shows the characteristics of the invention: no-3-