KR20090053927A - Animal feed additive - Google Patents

Abstract

Provides a safe and convenient means to aid the animal's digestive activities and improve feed efficiency. Specifically, the present invention provides a means for preventing and treating infectious diseases by suppressing the growth of pathogens and coccidium in the intestine of animals, thereby realizing weight gain of animals. Aspergillus sojae, Aspergillus tamarii, Aspergillus foetidus, Aspergillus niger and Aspergillus sojae Animals containing at least one Aspergillus species selected from Aspergillus oryzae, cultures containing acidic enzymes produced by these bacteria, and Bacillus subtilis To administer.

Description

Animal feed additives {ANIMAL FEED ADDITIVE}

The present invention relates to an animal feed additive containing Aspergillus bacteria, and Bacillus bacteria, having the ability to produce acidic enzymes.

Feeds such as livestock and pets (hereinafter referred to as animals) are processed such as crushing, but generally do not undergo heat treatment or the like, and thus have a problem of low digestion absorption and poor feed efficiency. In recent years, inflammatory bowel disorders such as ulcerative colitis and Crohn's disease, which are known as human diseases, have also been reported in animals, causing a problem such as diarrhea. It is also known that such inflammatory bowel disorders interfere with the decrease in feed absorption and healthy fattening. As pathogens that cause intestinal infections in animals, pathogenic Escherichia coli, Salmonella bacteria, Clostridium bacteria, Campylobacter bacteria and the like are known. Such pathogens are known to produce toxins (enterotoxins and cytotoxins) when abnormally propagated, cause disorders in the intestinal mucosa, and cause stool or severe diarrhea. In addition, coccidium is a parasitic parasite in the intestines of chickens, pigs, cows, and the like. When infected, it causes diarrhea and loss of appetite. In order to prevent and treat such inflammatory bowel disorders, antimicrobial substances are used, such as the emergence of drug-resistant bacteria.

In recent years, in order to improve the balance of intestinal flora and to suppress the proliferation of pathogens in the intestine, a technique using probiotics has attracted attention (Patent Documents 1 and 2). However, lactic acid bacteria and bifidus bacteria are often killed at a concentration of 0.3% dioxycholic acid, or are killed at pH 4 or lower, and except for bacteria collectively referred to as E. coli group, dioxycholic acid having a strong antibacterial activity against microorganisms in bile acids. There are many species that cannot survive in the presence. Therefore, there is a demand for a bacterium that does not die within the digestive tract of an animal and has a beneficial effect on the host.

On the other hand, it is reported that a technique for reducing the gastrointestinal burden of animals by treating animal feed with enzymes or enzyme producing bacteria at the processing stage and degrading it to some extent is useful for preventing or improving digestive diseases. (Patent Document 3). Moreover, the method of obtaining fish meal fermentation feed for fish containing a high concentration of protease, a lipase, etc. by fermenting fish meal in low moisture using Nuruk bacteria is known (patent document 4). However, in such a technique, there is a problem that a complicated process of increasing the water content in the feed to decompose the feed and then drying and commercializing the feed is necessary. In addition, the feed having a high water content for the decomposition of the ingredients, such as rot bacteria, mold and the like easily occurs, there was also a problem such as difficult to maintain the quality.

In addition, there has been reported a method of modifying the feces of animals by orally administering spores of Nuruk bacteria to the animals. It has not been investigated and it is not known to administer to an animal in the form which produced the acidic enzyme to Nuruk (patent document 5).

Further, fermented nutrients are added to the crustaceans of crustaceans and mixed, and this is selected from Aspergillus niger, Aspergillus aurise, Bacillus subtilis, Bacillus rickenformis. Or inoculating two or more species to decompose chitin or chitosan to obtain a fermented product and to give it to an animal (Patent Document 6). Although the growth promoting effect of the animal by this method has been partially confirmed, it has not been examined to suppress the proliferation of pathogens in the animal intestine and to prevent and treat intestinal infectious diseases, and such effects have not been confirmed. In addition, mixing and administering two or more kinds of microorganisms to animals has not been studied in practice.

Moreover, it is reported that Bacillus subtilis has antibacterial activity against pathogens, such as Escherichia coli (patent document 7, 8), and is examined about oral administration to an animal. In addition, Bacillus subtilis DB9011 has aflatoxin degradability and is known to inhibit the growth of fungi, and it has been studied to add it to feed (Patent Document 9). However, the ingestion of Bacillus subtilis to animals in combination with other microorganisms has not been studied until now.

Patent Document 1: Japanese Patent Publication No. 2005-507670

Patent Document 2: Japanese Patent Application Publication No. 2004-523241

Patent Document 3: Japanese Unexamined Patent Publication No. 2004-141147

Patent Document 4: Japanese Patent Application Laid-open No. Hei 6-319464

Patent Document 5: Japanese Patent Application Laid-Open No. 11-171674

Patent Document 6: Japanese Unexamined Patent Publication No. 2002-238466

Patent Document 7: Japanese Patent Application Laid-Open No. 11-332555

Patent Document 8: Japanese Patent Application Laid-Open No. 11-285378

Patent Document 9: Patent No. 3040234

Non Patent Literature 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, Renate Junowicz-Kocholaty, and Albert Kelner (1945) J. Bacteriol 50, 579-584

Disclosure of Invention

An object of the present invention is to provide a safe and convenient means for assisting the animal's digestive activity and to improve feed efficiency. Specifically, the object of the present invention is to provide a means for preventing and improving intestinal infectious diseases and realizing animal weight gain by inhibiting the growth of pathogens and coccidium in the intestine of animals.

MEANS TO SOLVE THE PROBLEM As a result of earnestly researching in order to solve the said subject, aspergillus elementary, Aspergillus tamari, Aspergillus poetiduth, Aspergillus niger, and Asperium Pergirus-Oriase has excellent ability to produce acidic enzymes, especially acid amylase, these bacteria have antimicrobial activity against pathogens causing intestinal infections and insecticidal activity against coccidium, and they function as probiotics. Figured out. In addition, it was found that the acidic enzyme-producing ability of these bacteria was very excellent when the brown rice was cultured as a nutrient source. In addition, by combining these cells with acidic enzymes produced by the cells and ingesting them with the feed, they were found to promote digestion, prevent and improve intestinal infectious diseases, and contribute to animal weight gain. Moreover, Bacillus subtilis discovered that even if it coexists with the said Aspergillus microbe and the acidic enzyme which the fungus produces, the proliferation is not inhibited. And by ingesting into animals the combination of Aspergillus bacteria, acidic enzymes produced by the bacterium and Bacillus subtilis, it was found that more excellent growth promoting effect and intestinal infectious disease prevention and improvement effect were obtained. The present invention has been completed.

That is, this invention is as follows.

(1) Aspergillus sojae, Aspergillus tamarii, Aspergillus foetidus, Aspergillus niger ) And at least one Aspergillus strain selected from Aspergillus oryzae, cultures containing acidic enzymes produced by these bacteria, and Bacillus subtilis. An animal feed additive to be contained, wherein the total amount of acidic enzyme activity per 1 g of the feed additive is 120 U or more, and the animal feed additive having a Bacillus subtilis concentration of 2.5 × 10 ⁷ to 2 × 10 ⁹ CFU / g.

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

(3) the animal feed additive according to (1) or (2), wherein the genus Aspergillus has antimicrobial activity against pathogens causing intestinal infections in animals and / or insecticidal activity against coccidium. .

(4) The animal feed additive according to any one of (1) to (3), wherein the genus Aspergillus is aspergillus soja and / or aspergillus aurize.

(5) Aspergillus aurise is a strain having IK-05074 strain (FERM BP-10622) and / or a mutant strain thereof, which has the same acidic enzyme-producing ability as that of the strain (1) to (4). Animal feed additive according to any one of).

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

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

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

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

(10) The animal feed additive according to any one of (1) to (8), which is used for prevention and improvement of intestinal infectious diseases.

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

(12) In a solid medium containing a nutrient for propagation of Aspergillus bacteria, Aspergillus spores, Aspergillus tamari, Aspergillus poetidus, Aspergillus A step of culturing at least one Aspergillus genus selected from Niger, Aspergillus and duck, and containing the resulting culture so that the total amount of acidic enzyme activity per kg of feed is 12 U or more, and Bacillus A method for producing a feed comprising a step of containing subtilis such that the concentration is 2.5 × 10 ⁶ to 2.0 × 10 ¹⁰ CFU / kg.

(13) A method of breeding an animal, wherein the animal is ingested with the feed according to (11).

Best Mode for Carrying Out the Invention

The animal feed additive of the present invention is selected from aspergillus, soda, aspergillus tamari, aspergillus poetidus, aspergillus niger, and aspergillus duck. It is characterized by containing at least one species of genus Aspergillus, a culture containing acidic enzymes produced by these bacteria, and Bacillus subtilis bacteria.

Aspergillus demagnet contained in the animal feed additive of the present invention, Aspergillus tamari, Aspergillus poetidus, Aspergillus niger and Aspergillus duck Is a bacterium classified into the above-mentioned species, when classified by the method generally used for identification of the yeast bacterium in the technical field. For identification of the fungus, for example, H. Murakami, The Journal of General and Applied Microbiology, 17, p.281-309, (1971), Hideka Murakami, Nippon Brewing Association, Vol. 74, No. 12, p. 849-853, (1979), '' Nikkuni, S., et al, The Journal of General and Applied Microbiology, 44, p. 225-230, (1998).

Aspergillus and Soja are one of the filamentous imperfect fungi found in soil and yeast, and are used for brewing soy sauce and miso. The animal feed additive of the present invention can be used without particular limitation as long as it has the ability to produce at least one of the acidic enzymes described in detail below, preferably acidic amylase, and is safe for feeding to animals. . In the animal feed additive of the present invention, a commercially available strain may be used, and as such bacteria, AOK 210 strain (Akita Konno Co., Ltd.) can be suitably used for Aspergillus soda.

Aspergillus tamari is a type of filamentous incomplete fungus found in soil, yeast, and food, and is used for brewing soy sauce and miso. The animal feed additive of the present invention can be used without particular limitation as long as it has the ability to produce at least one of the acidic enzymes described in detail below, preferably acidic amylase, and is safe for feeding to animals. . In the animal feed additive of the present invention, a commercially available strain may be used, and as such bacteria, Aspergillus tamari AOK 43 strain (Akita Konno Co., Ltd.) can be preferably used.

Aspergillus and Poetidus are one of the most incomplete fungi found in soil, yeast and grain husks, and are used for brewing sake, miso and soy sauce. The animal feed additive of the present invention can be used without particular limitation as long as it has the ability to produce at least one of the acidic enzymes described in detail below, preferably acidic amylase, and is safe for feeding to animals. . In the animal feed additive of the present invention, commercially available strains may be used, and as such bacteria, Aspergillus poetidutus AOK N4586 (Akita Konno Co., Ltd.) can be preferably used.

Aspergillus niger is one of the most incomplete fungi found in soil, yeast, grain husk, etc., and is used in various places such as liquor production, food processing, sugar production, and pharmaceutical production. The animal feed additive of the present invention can be used without particular limitation as long as it has the ability to produce at least one of the acidic enzymes described in detail below, preferably acidic amylase, and is safe for feeding to animals. . In the animal feed additive of the present invention, a commercially available strain may be used, and as such bacteria, Aspergillus niger AOK B650 (Akita Konno Co., Ltd.) can be preferably used.

A variant strain of AOK 210 strain, AOK 43 strain, AOK N4586 strain, or AOK B650 strain may be used. The mutant strains have the ability to generate acidic enzymes identical to those of AOK 210, AOK 43, AOK N4586 or AOK B650, respectively, from strains obtained by naturally mutating these strains or by mutating them with chemical or ultraviolet or the like. Can be obtained by selecting. In addition to the ability to produce acidic enzymes, it is also preferable to use a mutant strain of the strain further having at least one of the same antimicrobial activity, insecticidal activity, bile acid resistance and acid resistance as the strain. Moreover, it is also preferable to use the same mutant strains as AOK 210 strain, AOK 43 strain, AOK N4586 strain, or AOK B650 strain other than the above.

Aspergillus and duck are one of the most incomplete fungi found in soil, yeast, etc. and are used for brewing soy sauce and miso. In the animal feed additive of the present invention, any one of acidic enzymes described in detail below, preferably having an ability to produce acidic amylase, can be used without particular limitation as long as it is safe for feeding to animals. In the animal feed additive of the present invention, commercially available strains may be used, and Aspergillus aurise IK-05074 strain can be preferably used as such a bacterium. IK-05074 state is state separated from various fermented foods, and, on February 15, 2006, independent administrative corporation industrial technology synthesis institute patent biological deposit center (1 chome Higashi, Tsukuba-shi, Ibaraki, Japan, zip code 305-8566 1 Chuo Die 6) was deposited as Accession No. FERM P-20798 and transferred to the International Deposit under the Budapest Treaty on June 20, 2006, and is assigned Accession No. FERM BP-10622.

The bacteriological properties of IK-05074 are as follows.

(1) The appearance of colonies (Czapek-Dox agar medium (7 days incubation at 25 ℃))

The size is 50-60 mm in diameter, the color is yellow to green, and changes to brownish color with time. Mycelium is inconspicuous and the background is colorless.

(2) form

Conidia bag: thin wall to thick wall, smooth surface to slightly rough surface, diameter of 20 µm or less, length of 2 mm or less, seminal vesicles of 40 to 50 µm in diameter and 80 µm or less.

Basal diameter: present in most of the large conidia bags, the length of which is 12 μm or less.

Filide: Ampoule type, 8-12 micrometers in length, and a short neck part.

Conidia: A spherical particle having a diameter of 5 to 6 µm, a color of yellow to green, and a smooth surface to a fine rough surface.

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

Moreover, in the animal feed additive of this invention, the mutant strain of IK-05074 strain can also be used. Mutant strains of IK-05074 strains can be obtained by selecting strains having the ability to produce acidic enzymes identical to those of IK-05074 strains from strains obtained by naturally mutating the IK-05074 strains or by mutating the chemical with a mutant or ultraviolet light. have. In addition to the ability to produce an acidic enzyme, it is also preferable to use a mutant strain of IK-05074 strain which further has at least one of the same antibacterial activity, insecticidal activity, bile acid resistance and acid resistance as the IK-05074 strain. Moreover, it is also preferable to use the same mutant strain as IK-05074 strain other than the above.

In addition, in the animal feed additive of the present invention, for example, Aspergillus soda isolated from soil, yeast, food, grain husk, Aspergillus tamari, Aspergillus poe You may isolate and use the strain which has the ability to produce an acidic enzyme among Tidus, Aspergillus niger, Aspergillus aurise.

The ability to produce acidic enzymes refers to the production of acidic enzymes to the extent that acidic enzyme activity can be detected in a culture obtained by culturing cells. The measurement of the acidic enzyme activity of the culture can be carried out according to a conventional method.

The acidic enzyme contained in the animal feed additive of the present invention is preferably a digestive enzyme produced by the bacterium, and is not particularly limited as long as it is inactivated and active under acidic conditions in the gastrointestinal tract. It is preferable to have it in -5.5. For example, acidic α-amylase, glucoamylase, tacadiastatase, protease, cellulase, ribonuclease, nuclease, xylanase, pectinase, lipase and the like can be mentioned. The additive contains one or two or more of these. Among these, it is preferable to contain the acidic amylase which decomposes starch which is one of the main components in the feed component of livestock. The acid amylase contained in the animal feed additive of the present invention is not particularly limited as long as it is an acidic enzyme that hydrolyzes starch, and examples thereof include α-amylase, β-amylase, glucoamylase and the like. Among these, acid-resistant alpha-amylase which has an optimum pH around 3 can be mentioned preferably.

The feed additive for animals of the present invention is produced by Aspergillus tamari, Aspergillus tamari, Aspergillus poetidus, Aspergillus niger, Aspergillus arige Although at least 1 type of acidic enzyme should be contained, you may contain the enzyme derived from another species or other species.

What is necessary is just to make the acidic enzyme activity of the animal feed additive of this invention into the following range. The total amount of acidic enzyme activity per gram of animal feed additive is 120 U or more, preferably 170 U or more, more preferably 220 U or more, and more preferably 270 U to 5400 U. The sum of acidic enzyme activity is preferably the sum of acidic amylase activity, acidic protease activity and acidic carboxypeptidase activity. The acidic amylase activity per 1 g of animal feed additive is preferably 1 U or more, more preferably 5 U or more, more preferably 10 U or more, and more preferably 20 to 400 U. The total amount of the acidic protease activity and the acidic carboxypeptidase activity per 1 g of animal feed additive is preferably 100 U or more, more preferably 150 U or more, more preferably 200 U or more, and more preferably 250 to 5000 U.

The activity of acidic amylase, acidic protease and acidic carboxypeptidase is determined by glucoamylase activity assay, α-amylase activity assay, and acid-resistant α-amylase activity assay, which is the assay of solid yeast by the National Tax Service prescribed assay (Revised 3rd Instruction No. 1). What is necessary is just to measure based on the acidic protease activity measuring method and the acidic carboxypeptidase activity measuring method.

Aspergillus demagnet contained in the animal feed additive of the present invention, Aspergillus tamari, Aspergillus poetidus, Aspergillus niger and Aspergillus duck The concentration of at least one Aspergillus genus selected from the above and the culture containing the acidic enzymes produced by these bacteria may be adjusted so that the acidic enzyme activity of the feed additive is in the above range. Usually, a culture obtained by culturing the bacteria for about 7 days at 28 ° C. using a sufficient amount of vegetable nutrients such as brown rice is 0.25 to 20% by mass, preferably 0.5 to 10% by mass, and more preferably 1 to 1 What is necessary is just to add in 5 mass%.

Moreover, it is preferable that the said Aspergillus microorganism used for this invention has antimicrobial activity with respect to the pathogen which causes intestinal infection of an animal.

The pathogen usually belongs to the intestinal bacteria family. Specific examples include bacteria belonging to Escherichia coli, Salmonella, Campylobacter, Clostridium, Staphylococcus aureus, and the like. Pathogenic Escherichia coli includes, for example, enterotoxin-producing E. coli (toxin-producing E. coli (ETEC)), edema pathogens or enterotoxin-producing E. coli (Verotoxin-producing E. coli (e. VTEC), enterohemorrhagic E. coli), and the like. Examples of bacteria belonging to the genus Salmonella include S. pullorum, S. gallinarum, S. typhi, S. typhimurium, S. enteritidis, S. choleraesuis, S. derby, and S. dublin. Bacteria belonging to the genus Campylobacter include C. jejuni, C. coli, C. fetus and the like. Bacteria belonging to the genus Clostridium include C. perfringens, C. botulinum, C. difficile. Aspergillus demagnet contained in the animal feed additive of the present invention, Aspergillus tamari, Aspergillus poetidus, Aspergillus niger, Aspergillus duck In particular, has high antibacterial activity against Salmonella bacteria, S. enteritidis and Clostridium bacteria, E. coli, such as C. perfringens, edema, and S. aureus.

Having antimicrobial activity against a pathogen means that when inoculated into the same medium as the pathogen, it has the ability to suppress the growth of the pathogen. Aspergillus tamari, aspergillus poetidus, aspergillus niger, aspergillus duck agent which have antibacterial activity are soil, for example , Yeast and other isolates were added to agar medium inoculated with pathogens such as Salmonella enteritidis (SE), Clostridium perfringens (CP), Escherichia coli (EC), Staphylococcus aureus (SA), and cultured. It can obtain by isolate | separating the cell which formed viii). The bacterium thus obtained has the ability to inhibit the growth of the above-described pathogens, so that the intestinal infection of the animal can be prevented and treated by administering it to the animal.

In addition, the proliferation of the pathogen causing the intestinal infection of the animal can be confirmed by measuring, for example, the cell concentration (the number of viable cells) of the pathogen in the cecal contents of the animal or in the feces.

Moreover, it is preferable that the said Aspergillus genus used for this invention has an insecticidal activity with respect to the coccidium which causes the intestinal infection of an animal.

Coccidi refers to a protozoan belonging to the spores (Sporozoasida subclass). Specifically, the genera Eimeria, Isospora, Toxoplasma and Cryptosporidium can be cited. Protozoans belonging to the genus Eimeria include E. tenella, E. necatrix, E. acervulina, E. maxima, E. mitis, E. zuernii, and E. bovis. Protozoans belonging to the genus Isospora include I. suis, I. belli and I. hominis. Protozoans belonging to the genus Toxoplasma include T. gondii. Protozoans belonging to the genus Cryptosporidium include C. parvum. The animal feed additive of the present invention can be suitably used particularly for infections caused by E. tenella and E. zuernii.

Having an insecticidal activity against coccidium means that the coexistence of coccidium and a culture of a bacterium coexists, and it has the ability to suppress germination and proliferation of the acist, and preferably to reduce the acist. Specifically, it is said to have the ability to deform and dissolve the cell wall of an oscillator, and to disintegrate the oscillator. The collapse of the oscillator and the state of the cell wall may be observed using a microscope.

Aspergillus demagnetism having an insecticidal activity against coccidium, Aspergillus tamari, Aspergillus poetidus, Aspergillus niger, Aspergillus duck, For example, it can obtain by the following method. Soils, yeasts, and other sources are added to a chalet containing sterile water suspended in E. tenella or E. zuernii, and incubated at 37 ° C for 1 to 7 days. The cells were isolated from the chalets where the alteration or dissolution of the austist was confirmed.These cells were added to a chalet containing sterile water in which the austist of E. tenella or E. zuernii was suspended and incubated at 37 ° C. Check for deformation or dissolution. In this way, the microorganisms contained in the chalet were cultured, and each of Aspergillus and Soja, Aspergillus tamari, Aspergillus poetipidus, Aspergillus niger, and Aspergillus. By selecting the bacteria belonging to the duckling agent, the genus Aspergillus used in the present invention can be obtained. By administering the bacterium obtained in this way to the animal, the intestinal coccidial infection of the animal can be prevented and treated.

In addition, whether or not proliferation of coccidium is suppressed can be confirmed by observing, for example, the microscopic contents of the cecal contents of the animal and the oscillation of coccidium in the feces.

Aspergillus demagnet contained in the animal feed additive of the present invention, Aspergillus tamari, Aspergillus poetidus, Aspergillus niger, Aspergillus duck It is preferable to have resistance to gastric acid and bile acid. Thus, it is considered that even in the intestines of animals, cells produce useful acidic enzymes, and the function of digestive assistance by acidic enzymes is maintained. Moreover, when these Aspergillus microorganisms have antimicrobial activity against the above-mentioned pathogens, they suppress the proliferation of pathogens causing intestinal infections in animals and contribute to improving the balance of intestinal flora. Resistant to gastric acid or bile acid means that the bacteria reach the intestine without dying under conditions in the normal digestive organs. Usually, the stomach inside of an animal may reach pH 2 or less at the time of an empty stomach, but when food is ingested, pH of stomach inside is higher than an empty stomach in the range of 3.5-6. Therefore, a strain having acid resistance that can be used in the feed additive of the present invention can be obtained by selecting a strain having the ability to survive, for example, when the separation source is treated at pH 3.5 for about 2 hours. In addition, by treating the bacteria selected in this way for about 24 hours in the presence of 10 g / ℓ of deoxycholic acid, and selecting a strain having the ability to survive, by selecting a strain having a viability to the stomach acid or bile acid suitable for use in the feed additive of the present invention A strain having resistance can be obtained. In addition, it is possible to confirm that the bacteria reach the intestine without killing by measuring the concentration of the bacteria in the feces of the animal.

The animal feed additive of the present invention may contain one strain alone, or may contain two or more strains in combination. Among these, it is preferable to contain at least 1 sort (s) of an Aspergillus soja and Aspergillus duck.

Aspergillus demagnet contained in the animal feed additive of the present invention, Aspergillus tamari, Aspergillus poetidus, Aspergillus niger and Aspergillus duck The concentration of the cells should be in a range in which the cells do not die in the digestive tract when the feed additive is administered to the animal. Usually, the cells are inoculated with a medium having a concentration suitable for producing a feed additive having the acidic enzyme activity. After culturing, the acidic enzyme activity is finally adjusted to the above value.

Aspergillus saccharin used for the animal feed additive of the present invention, Aspergillus tamari, Aspergillus poetidus, Aspergillus niger, and Aspergillus arige The acidic enzyme is produced by culturing under normal culture conditions. For example, although culture temperature can be performed at 25 degreeC-40 degreeC, it is preferable to culture at 28-32 degreeC normally. The culture method may be a liquid culture method or a solid culture method using a reciprocating shake culture, a fermentation tank culture, or the like. Among the acidic enzyme generating genes of the cells, genes which are expressed only in a solid medium are present. In this case, it is preferable to use a solid culture method.

The medium component to be used for culture may be either animal or vegetable, but preferably contains a nutrient of vegetable origin, for example, brown rice, bran, rice bran, soybean, and soybean. Among these, it is particularly preferable to use brown rice as a nutrient source. Thereby, the production | generation efficiency of acidic enzymes, such as acidic amylase, can be improved.

As other carbon sources, sugars such as glucose, sucrose and molasses, and ammonium salts such as ammonia, ammonium sulfate, ammonium chloride and ammonium nitrate, nitrates and the like can also be added as nitrogen sources.

It is preferable to dry the culture of the genus Aspergillus obtained as described above. Moreover, it is also preferable to further process, such as adding the arbitrary component for improving storage property, and to improve quality stability.

A drying method is not specifically limited, For example, although drying can be performed by ventilation drying, natural drying, spray drying, freeze drying, etc. Among these, ventilation drying is used preferably. In addition, freeze drying may be used, and a protective agent may be added at that time. Although the kind of protective agent is not specifically limited, It is preferable to select and use 1 type (s) or 2 or more types from scheme milk, sodium glutamate, and saccharides. Moreover, when using a saccharide, the kind in particular is not restrict | limited, It is preferable to use glucose or trehalose.

Moreover, after drying, it is preferable to add an oxygen scavenger and a dehydrating agent to the obtained dried thing, to put it into a gas barrier aluminum bag, to seal, and to store it at low temperature rather than room temperature. As a result, the cells can be stored alive for a long time.

Bacillus subtilis to be used in the animal feed additive of the present invention, in the 9th edition (1994) of Bergey's Manual of Determinative Bacteriology, The bacterium classified as "Bacillus subtilis" is not particularly limited. As such bacteria, Bacillus subtilis DB9011 strain, NBRC3009 strain, NBRC3025 strain, NBRC3108 strain, NBRC3336 strain, and the like can be used. DB9011 is usually from Ministry of Trade, Industry and Technology Institute microbial industrial technology research institute (currently, independent administrative corporation industrial technology synthesis research institute patent biological deposit center, postal code 305-8566 Higashi 1, Tsumbashi, Tsukuba-shi, Ibaraki, Japan 1 6 1 chome 1 Chuo Dai 6) 1991 On 21 May, it was deposited as FERM BP-3418. Moreover, although DB9011 strain was classified as Bacillus licheniformis at the time of deposit, it was confirmed that it belongs to Bacillus subtilis after that. In addition, NBRC3009, NBRC3025, NBRC3108, and NBRC3336 are registered states in the Biodiversity Resources Division (NBRC) of the Product Evaluation Technology Infrastructure Organization.

The bacteriological properties of DB9011 strain are as follows.

shape :

It is a bacterium with a cell width of 0.7 to 0.8 µm. Oval spores are slightly centered and do not expand cells. There is motility and forms R-type colonies. It does not grow under anaerobic conditions.

Growth state in each medium:

(1) DHL agar badge: not growing.

(2) McConkie Agar Badge: Not developed.

(3) Mannitol salt medium: Good development. Glossy. There are no wrinkles on the surface of the colony, and the color of the colony is yellow.

(4) Normal agar medium: good development. No gloss. There are wrinkles on the surface of the colony, and the color of the colony is white.

(5) Heart Infusion Agar Badge: Good development. No gloss. There are wrinkles on the surface of the colony, and the color of the colony is white.

(6) blood agar medium (10% cotton blood added)

Good development. No gloss. There are wrinkles on the surface of the colony, and the color of the colony is white.

(7) PDA medium: good development. No gloss. There are wrinkles on the surface of the colony, and the color of the colony is white.

Physiological Properties:

 Gram reaction: +

Gelatin test:

 Growth state; Full Liquefaction

 Gelatin liquefaction; +

Litmus milk:

 reaction ; mountain

 condition ; coagulation

Nitrate Reduction: +

Denitrification:

MR test:

Generation of indole:

Generation of hydrogen sulfide:

Use of citric acid (crisstensen): +

Urease:

Oxidase: +

Catalase: +

Range of growth:

 pH; 4 to 9

 Temperature ; 25-50 ℃

OF test: fermentation and gas generation (by glucose decomposition)

Availability of sugars

                   Gas generating acid generating

L-arabinose-+

D-xylose-+

D-glucose + +

D-mannose － +

D-fractose-+

D-galactose － +

Maltose (maltose)-+

Sucrose (Sucrose) － ＋

Lactose (lactose) － +

Trehalose-+

D-sorbitol-+

D-mannitol-+

Inositol--

Glycerin-+

Starch － ＋

Decomposition of Esculin: +

Use of malonic acid:

Decomposition of arginine: +

Decarboxylation reaction of lysine: +

Element decomposition: ±

Aflatoxin digestion: +

Denitrification of Ornithine:

Coaglase:-

Hemolytic: +

Sodium chloride resistance: 10% or less

Resistance of Potassium Cyanide: Possible to Develop

Recitase:-

In addition, the animal feed additive of the present invention is a strain obtained by naturally varying Bacillus subtilis DB9011 strains, NBRC3009 strains, NBRC3025 strains, NBRC3108 strains, NBRC3336 strains, or by chemical mutations or ultraviolet rays, Strains having the same bacteriological properties as each strain may be selected and used. As a variant strain of Bacillus subtilis DB9011 strain, it is preferable to select and use a strain having the above-mentioned bacteriological properties.

In the animal feed additive of the present invention, the concentration of Bacillus subtilis is 2.5 × 10 ⁷ to 2 × 10 ⁹ CFU / g, preferably 5 × 10 ⁷ to 1 × 10 ⁹ CFU / g, more preferably Preferably 1 × 10 ⁸ to 5 × 10 ⁸ CFU / g.

Moreover, in the animal feed additive of this invention, the concentration of Bacillus subtilis is 2.0 * 10 <^3> -5.0 * 10 <^6> CFU per animal feed additive which shows the sum total of 1U acidic enzyme activity, Preferably it is 1.0. ^{× 10 4 ~ 1.0 × 10 6} CFU is such that, or per feed additives for animals showing the acid amylase activity ^{1U 1.0 × 10 5 ~ 2.5 ×} 10 8 CFU, preferably from ^{5.0 × 10 5 ~ 5.0 × 10} 7 CFU a Or 2.0 × 10 ³ to 5.0 × 10 ⁶ CFU, preferably 1.0 × 10 ⁴ to 1.0 × 10 ⁶ CFU, per animal feed additive representing the sum of 1U acidic protease activity and acidic carboxypeptidase activity. have.

Bacillus subtilis used in the animal feed additive of the present invention preferably has the ability to proliferate in the presence of the above Aspergillus bacteria and acidic enzymes produced by the bacteria.

It is preferable that Bacillus subtilis used for the animal feed additive of this invention is bile acid tolerance.

"Bile acid resistance" means the formation of spores having the ability to germinate and proliferate in a medium containing high concentration of bile acids.

Bile acids refer to steroids of the four-ring structure widely found in bile of mammals, birds, reptiles, amphibians, and fish, and contain cholic acid, kenodeoxycholic acid, dioxycholic acid, lithocholic acid and ursodeoxycholic acid. Usually, in the body of an animal, bile acid exists as a conjugate type which amide couple | bonded with glycine and taurine in bile, and becomes a sodium salt. In the present specification, simply referred to as "bile acid" includes the bile acids, salts thereof, and conjugates thereof.

A medium containing a high concentration of bile acid is, for example, a medium containing bile powder (Oxgall, manufactured by Difco) in which fresh bile is concentrated and dried 10 times. The concentration of bile is 0.3 mass% or more, preferably 1 mass. % Or more, More preferably, it is 3 mass% or more. In the case of "having ability to germinate and propagate", when spores are inoculated into a medium containing a high concentration of bile acids as described above, and conditions other than the bile acid concentration are set to conditions suitable for culturing Bacillus subtilis, Bacteria germinate, resume proliferative division, and colony formation.

Bacillus subtilis that is bile acid resistant can be obtained as follows, for example. A separation source containing Bacillus subtilis is incubated under conditions suitable for sporulation to form spores. The resulting spores are inoculated into the medium of the above-mentioned bile acid addition medium, and after culturing, the formed colonies are separated. Among these colonies, those having bacteriostatic properties of Bacillus subtilis are selected.

It is preferable that Bacillus subtilis used for the animal feed additive of this invention is further acid resistant. By using such bacteria, even within the stomach, the bacteria reach the intestine without being killed. The term "acid resistance" means that when bacteria are administered to animals, the number of germs that can grow in the intestine is maintained even if the intestines do not die under the condition of the stomach (normally pH 3.5 to 6 with food ingested). Say what you are doing. Since spores of Bacillus subtilis are usually acid resistant, there is no particular problem when spores are used.

The animal feed additive of the present invention may contain one strain of Bacillus subtilis alone or may contain two or more strains in combination. Among these, it is especially preferable to use DB9011 strain alone or in combination with other strains.

The method for culturing Bacillus subtilis used in the feed additive of the present invention is not particularly limited and can be carried out by a conventional method under suitable conditions according to the properties of bacteria. For example, although culture temperature can be performed at 20-40 degreeC, it is preferable to culture at 30-37 degreeC normally. As the culture method, a liquid culture method or a solid culture method by standing culture, reciprocating shake culture, rotary shake culture, fermentation tank culture, or the like can be used.

The medium component used for the culture is also not particularly limited, and as a carbon source, glucose, galactose, lactose, arabinose, mannose, sucrose, starch, starch hydrolyzate, sugars such as molasses, organic acids such as citric acid, alcohols such as glycerin Ammonium salts and nitrates such as ammonia, ammonium sulfate, ammonium chloride and ammonium nitrate as the nitrogen source; inorganic salts such as sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride and ferrous sulfate; Peptone, soybean flour, skim soybean white, meat extract, yeast extract, etc. can be used.

It is preferable that the Bacillus subtilis used for the animal feed additive of the present invention is in a spore state from the viewpoint of storage stability and acid resistance. In the spore state, since it is resistant to heat and drying, it can be sufficiently dried when producing a feed additive, and storage stability is improved. In order to form spores in bacteria, in the culture cycle, the culture conditions such as the composition of the medium, the pH of the medium, the culture temperature, the culture humidity, and the oxygen concentration at the time of culture may be adjusted to suit the spore formation conditions. As such a method, for example, Schaeffer, P., J. Millet, J.P. Aubert, "Proceedings of the National Academy of Science," USA, 1965, Volume 54, pp. 704-711.

Moreover, it is preferable to make the culture and spores of Bacillus subtilis obtained by the above-mentioned method into dry powder from a viewpoint of storage property. It is preferable to perform drying so that moisture content may be 20 mass% or less, for example. The method of drying and preservation is the same as that of the culture of the genus Aspergillus.

As for the animal feed additive of this invention, the above-mentioned Aspergillus element, Aspergillus tamari, Aspergillus poetiduth, Aspergillus niger, and Aspergillus It can be obtained by mixing all or part of a culture containing at least one Aspergillus spp. And the acidic enzyme produced by the bacterium with Bacillus subtilis cells.

Moreover, the animal feed additive of this invention can add an arbitrary component further. These optional components can be used without particular limitation as long as they are confirmed as safety as feed ingredients, and do not kill the Aspergillus bacteria and Bacillus subtilis and do not deactivate the acidic enzymes. For example, immune activators such as β-glucan, glucomannan, mannan oligosaccharide, and seaweed, organic acids such as gluconic acid, γ aminobutyric acid, citric acid, malic acid, fumaric acid, succinic acid, pantothenic acid, butyric acid, and the like are preferable.

Since the animal feed additive of the present invention promotes the weight gain of the animal by ingesting the animal, it can be used as an animal feed additive for growth promotion. In this case, in particular, the total amount of acidic enzyme activity per gram of feed additive is 270 U or more, the acid amylase activity is 20 U or more, the total sum of acid protease activity and acid carboxypeptidase activity is 250 U or more, and the concentration of Bacillus subtilis is 1 × 10. It is preferable to prepare so that it may be ⁸ CFU / g or more.

Since the animal feed additive of the present invention prevents intestinal infections and the like by ingesting the animal, it can be used as an animal feed additive for the prevention and improvement of the intestinal infections. In this case, in particular, the total amount of acidic enzyme activity per gram of feed additive is 540U or more, the acid amylase activity is 40U or more, the total sum of acidic protease activity and acidic carboxypeptidase activity is 500U or more, and the concentration of Bacillus subtilis is 2.5 × It is preferable to prepare so that it may be 10 ⁸ CFU / g or more.

In addition, the animal feed additive of the present invention preferably has a low concentration of kojic acid from the viewpoint of safety. Usually, it is preferable that the content concentration of kojic acid is 0.1 mg / L or less, and it is more preferable that it is 0.01 mg / L or less.

The feed of the present invention contains 0.01 to 1.0% by mass, preferably 0.02 to 0.5% by mass, and more preferably 0.04 to 0.25% by mass of the animal feed additive of the present invention with respect to the total amount of the feed. It features.

The total amount of acidic enzyme activity per kilogram of feed of the present invention is 12 U or more, preferably 17 U or more, more preferably 22 U or more, and more preferably 27 to 54000 U. The concentration of Bacillus subtilis is 2.5 × 10 ⁶ to 2.0 × 10 ¹⁰ CFU / kg, preferably 5.0 × 10 ⁶ to 1.0 × 10 ¹⁰ CFU / kg, and more preferably 1.0 × 10 ⁷ to 5.0 × 10 ⁹ CFU / kg. The acid amylase activity per kg of feed is preferably 0.1 U or more, more preferably 0.5 U or more, more preferably 1 U or more, and still more preferably 2 to 4000 U. The total amount of the acidic protease activity and the acidic carboxypeptidase activity per kg of feed is preferably 10 U or more, more preferably 15 U or more, more preferably 20 U or more, and more preferably 25 to 50000 U.

The feed of the present invention can be produced by adding the animal feed additive of the present invention to a component of a feed that is commonly used. The kind and components of the feed are not particularly limited as long as the cells contained in the animal feed additive of the present invention are not killed and the acidic enzyme is inactivated, and is usually used for livestock feed, pet feed, animal supplements, and the like. What is necessary is just to add and mix with what is used as animal feed.

In addition, although the feed of this invention may add and mix animal feed additives to a feed component in a dry state, in order to make mixing easy, you may use it as a liquid form or a gel form. In this case, water, soybean oil, rapeseed oil, vegetable oils such as corn oil, liquid animal oil, water-soluble high molecular compounds such as polyvinyl alcohol, polyvinylpyrrolidone and polyacrylic acid can be used as the liquid carrier. In addition, in order to maintain the uniformity of the cell concentration in the feed, water-soluble polysaccharides such as alginic acid, sodium alginate, xanthan gum, sodium caseinate, gum arabic, guar gum and tamarind seed polysaccharides are preferably blended. Moreover, in order to prevent propagation of various germs, it is also preferable to mix | blend an organic acid.

In addition, the feed of the present invention, aspergillus tamari, aspergillus poetidutus in aspergillus soda in a solid medium containing a nutrient source for propagation of Aspergillus spp. , A culture obtained by culturing at least one species of Aspergillus bacteria selected from Aspergillus niger and Aspergillus aurise, and Bacillus subtilis are separately mixed into the feed ingredients, and It can also manufacture by adjusting the acidic enzyme activity and the concentration of Bacillus subtilis in the above range.

The feed of the present invention can be used as a feed for promoting the growth of an animal. In addition, when the above-mentioned Aspergillus bacteria also have antimicrobial activity against pathogens causing animal intestinal infections and / or insecticidal activity against coccidial, the intestinal infections caused by pathogens and / or coccidial It can be used as a feed for prevention and treatment.

The kind of animal to which the feed of this invention is ingested is not restrict | limited, For example, a mammal, a bird, a reptile, an amphibian, fish, etc. are mentioned. Especially, it can use preferably for poultry and a livestock. Poultry is preferred for chickens, ducks, quails, turkeys, and the like, and livestock is preferred for pigs, cattle, sheep, rabbits, and the like. The amount of feed consumed by the animal can be appropriately adjusted according to the type, weight, age, sex, purpose of use, health condition, and ingredients of the feed.

The method of ingesting a feed can also take the method normally used according to the kind of animal.

<1> culture of Aspergillus spp.

The pH of the potato dextrose agar medium was adjusted to 5 and sterilized for 15 minutes at 121 ° C. When the temperature of the agar medium was lowered to 60 ° C., sodium dioxycholic acid was added at a concentration of 10 g per 1 L of the medium, and the asper was stored in Akita Konno Co., Ltd. (248, Daisen-shi Garibano, Akita, Japan). The genus was inoculated with Aspergillus sojae, Aspergillus tamarii, Aspergillus foetidus and Aspergillus. Aspergillus niger grew well in the medium. Among them, AOK 210 strains, Aspergillus tamari AOK 43 strains, Aspergillus poetidutus AOK N4586 strains, and Aspergillus niger AOK B650 strains are particularly preferred in Aspergillus and Soja. It grew.

In the same manner as above, a variety of strains of Aspergillus were inoculated into the medium containing sodium dioxycholate as the separation source, and the most favorable strains were selected among the strains grown on the medium. Aspergillus aurise IK-05074 was made this week, and it deposited in the patent biological deposit center of the Industrial Technology Research Institute of an independent administrative corporation.

The bacteriological properties of IK-05074 are as described above.

Using brown rice as a solid medium, Aspergillus soda AOK 210 shares, Aspergillus tammari AOK 43 shares, Aspergillus poetidutus AOK N4586 strain, Aspergillus niger AOK B650 Cultures of the strain and Aspergillus aurise IK-05074 strain were performed. That is, 100 g of brown rice was soaked in water all day, and swelled. Then, it was placed in a polycarbonate container having a sterile filter with a sterile filter at a diameter of 2 centimeters and 2 centimeters thick, and sterilized for 15 minutes at 121 ° C. in an autoclave. Each cell was inoculated into this container and incubated at 28 ° C. for 5 days to prepare a seed.

Next, the swollen brown rice was swelled in the same manner as above in a 30 × 40 × 10 centimeter stainless steel batt with a thickness of 1.5 centimeters, and covered in a large autoclave with a lid having a ventilation hole covered with a 20 × 25 centimeter filter. Sterilization for 25 minutes at 121 ℃. After the bat was cooled, the entire amount of the spawned seed was inoculated. The bat was placed in an incubator at 28 ° C and incubated for 7 days.

After incubation, air drying at 35 ° C. and trituration with a jet mill yielded a solid culture for each species.

The acid resistant α-amylase activity, and the sum of acidic protease activity and acidic carboxypeptidase activity, per gram of each solid culture were measured. The enzyme activity is shown in Table 2 per gram of each culture.

In addition, the measurement of the said acidic enzyme was performed based on the acid-resistant alpha-amylase activity measuring method, the acidic protease activity measuring method, and the acidic carboxypeptidase activity measuring method of the solid yeast analysis method of the Internal Revenue Service prescribed | required by the Internal Revenue Service (Revised 3rd Revenue Directive No. 1). .

 <2> Measurement of antimicrobial activity of culture of genus Aspergillus

The antimicrobial activity of these Aspergillus strains was tested by coculturing the bacteria with AOK 210 strain and Aspergillus aurise IK-05074 strain in Aspergillus dendrites.

For Salmonella enteritidis (SE), aerobic culture was performed at 37 ° C. for 24 hours in a standard agar medium (manufactured by Nisui Pharmaceutical Co., Ltd.). Colonies developed on the plate were scraped off and suspended in sterile saline. 500 ml of brain heart infusion bouillon "Nisui" was prepared in a 1 L Erlenmeyer flask, and after autoclave sterilization, aseptically, the final concentration of SE was about 1.0 x 10 ⁴ to 1.0 x 10 ⁵ CFU / ml. Was added. To the Erlenmeyer flask, 5 g of crushed solid cultures of AOK 210 strain and Aspergillus duck, IK-05074 strain, were aseptically added to Aspergillus decanter, respectively. An Erlenmeyer flask without addition was used as a control. Each Erlenmeyer flask was gently incubated in an incubator at 37 ° C. under aerobic conditions.

Regarding Clostridium perfringens (CP), anaerobic kenki (manufactured by Mitsubishi Gas Chemical Co., Ltd.) was used to perform anaerobic culture at 37 ° C for 24 hours in a yolk-added CW agar medium (manufactured by Nisui Pharmaceutical Co., Ltd.). . Colonies developed on the plate were scraped off and suspended in sterile saline. 500 ml of brain heart infusion bouillon "Nisui" was prepared in a 1-liter Erlenmeyer flask, and after autoclave sterilization, it was added aseptically so that the final concentration of CP would be about 1.0 x 10 ⁴ to 1.0 x 10 ⁵ CFU / ml. . To the Erlenmeyer flask, 5 g of crushed solid cultures of AOK 210 strain and Aspergillus duck, IK-05074 strain, were aseptically added to Aspergillus decanter, respectively. An Erlenmeyer flask without addition was used as a control. Each Erlenmeyer flask was gently incubated under anaerobic conditions using aneropakkenki in an incubator at 37 ° C.

The viable cell count of SE and CP was measured on the 0th, 3rd, and 7th day after the start of the test. In the method for measuring the number of viable cells of SE, after diluting the collected culture solution 10 times with sterile physiological saline solution, 0.1 ml of each dilution step solution is applied to X-SAL agar medium `` Nisui '' and breathed at 37 ° C for 24 hours. Cultivation was performed and the characteristic colonies developed were counted. In the method for measuring the viable cell count of CP, after diluting the collected culture solution 10 times with sterile physiological saline solution, 0.1 ml of each dilution step solution is applied to yolk-added CW agar medium (manufactured by Nisui Pharmaceutical Co., Ltd.), Anaeropak kenki was used to carry out anaerobic culture at 37 ° C. for 24 hours, and the developed characteristic colonies were counted.

The number of viable cells of SE is shown in Table 3, and the number of viable cells of CP is shown in Table 4.

As shown in Test Examples 1 and 2, the solid cultures of the AOK 210 strain and the Aspergillus aurise IK-05074 strain on the Aspergillus dendrites showed antimicrobial activity against SE. In addition, in the control group, the increase in the number of bacteria was confirmed until the 7th day of the test, and showed 2.9 x 10 ⁸ CFU / ml on the 7th day.

As shown in Test Examples 3 and 4, the solid cultures of the AOK 210 strain and the Aspergillus aurise IK-05074 strain on the Aspergillus dendrites showed antimicrobial activity against CP. In the control group, the increase in the number of bacteria was confirmed until the 7th day of the test, and was 1.2 × 10 ⁶ CFU / ml on the 7th day.

Escherichia coli (EC) was exhaled for 24 hours at 37 ° C in a standard agar medium (Nisui Pharmaceutical Co., Ltd.). Colonies developed on the plate were scraped off and suspended in sterile saline. In a 1 L volumetric flask, 500 ml of Brain Heart Infusion Bundle "Nisui" (manufactured by Nissui Pharmaceutical Co., Ltd.) was prepared, and after autoclave sterilization, the final concentration of EC was about 1.0 × 10 ⁵ to 1.0 × 10 ⁶ CFU. It was added aseptically to / ml. To the Erlenmeyer flask, 5 g of each of the crushed solid cultures of AOK 210 strain and Aspergillus duckase IK-05074 strain was aseptically added to Aspergillus decanter, respectively, to make Test Examples 5 and 6, and the yeast cell culture. An Erlenmeyer flask without addition was used as a control. Each Erlenmeyer flask was gently incubated in an incubator at 37 ° C. under aerobic conditions.

Staphylococcus aureus (SA) was aerobic culture for 24 hours at 37 ° C in a standard agar medium (manufactured by Nisui Pharmaceutical Co., Ltd.). Colonies developed on the plate were scraped off and suspended in sterile saline. In a 1 L volumetric flask, 500 ml of Brain Heart Infusion Bouillon Nissui (manufactured by Nissui Pharmaceutical Co., Ltd.) was prepared, and after autoclave sterilization, the final concentration of SA was about 1.0 × 10 ⁵ to 1.0 × 10 ⁶ CFU. It was added aseptically to / ml. To the Erlenmeyer flask, 5 g of crushed solid cultures of AOK 210 strain and Aspergillus duckase IK-05074 strain were each aseptically added to Aspergillus decanter, and Test Example 7 and 8 were used as test samples 7 and 8, respectively. An Erlenmeyer flask without addition was used as a control. Each Erlenmeyer flask was gently incubated in an incubator at 37 ° C. under aerobic conditions.

The viable cell count of EC and SA was measured on the 0th, 3rd, and 7th day after the start of the test. The method of measuring the number of viable cells of EC is that after diluting the collected culture solution 10 times with sterile physiological saline solution, 0.1 ml of each dilution step solution is applied to Chromocult Coliform agar `` Merck '' (manufactured by Merck). Aerobic culture was performed for 24 hours at 0 ° C., and the characteristic colonies developed were counted. In the method of measuring the viable cell count of SA, after diluting the collected culture solution 10 times with sterile physiological saline solution, 0.1 ml of each dilution step solution was added with yolk mannitol saline medium `` eiken '' (manufactured by Eiken Corporation). After application to, aerobic culture was performed at 37 ° C. for 48 hours, and developed characteristic colonies were counted.

Table 5 shows the number of viable cells of EC and Table 6 shows the number of viable cells of SA.

As shown in Test Examples 5 and 6, the solid cultures of the AOK 210 strain and the Aspergillus aurise IK-05074 strain in the Aspergillus dendrites showed antimicrobial activity against EC. In the control group, the highest bacterial count was confirmed on the third day of the test, and 4.3 x 10 ⁸ CFU / ml on the third day and 1.7 x 10 ⁷ CFU / ml on the seventh day.

As shown in Test Examples 7 and 8, the solid cultures of the AOK 210 strain and the Aspergillus aurise IK-05074 strain on the Aspergillus dendrites showed antimicrobial activity against SA. In addition, in the control, the highest bacterial count was confirmed on the third day of the test, showing 4.2 × 10 ⁸ CFU / ml on the third day and 7.2 × 10 ⁷ CFU / ml on the seventh day.

Bacillus subtilis DB9011 strain, NBRC3009 strain, NBRC3025 strain, NBRC3108 strain, and NBRC3336 strain were incubated for 24 hours at 37 ° C in a standard agar medium (manufactured by Nisui Pharmaceutical Co., Ltd.). Colonies developed on the plate were scraped off and suspended in sterile saline. In a 1 L volumetric flask, 500 ml of Brain Heart Infusion Bundle "Nisui" (manufactured by Nissui Pharmaceutical Co., Ltd.) was prepared, and after autoclave sterilization, the final DB9011, NBRC3009, NBRC3025, NBRC3108, and NBRC3336 Aseptically, the concentration was about 1.0 × 10 ⁴ to 1.0 × 10 ⁵ CFU / mL. To the Erlenmeyer flask, 5 g of crushed solid cultures of AOK 210 strain and Aspergillus duck, IK-05074 strain, were aseptically added to Aspergillus decanter, respectively, to each of Test Examples 9 to 18. An Erlenmeyer flask without addition was used as a control. Each Erlenmeyer flask was gently incubated in an incubator at 37 ° C. under aerobic conditions.

The viable cell count and spore count of DB9011, NBRC3009, NBRC3025, NBRC3108, and NBRC3336 were measured at 0, 3, and 7 days after the start of the test. In the method for measuring viable cell count in the above state, after diluting the collected culture solution 10 times with sterile physiological saline solution, 0.1 ml of each dilution step solution is applied to standard agar medium (Nisui Pharmaceutical Co., Ltd.), and then 37 ° C. For 48 hours, aerobic culture was performed, and the characteristic colonies developed were counted. The method of measuring the number of spores of the caution is as follows. First, the collected culture solution was diluted 10 times with sterile physiological saline, and heated at 70 ° C. for 30 minutes. After cooling, after diluting 10 times step with sterile physiological saline solution, 0.1 ml of each dilution step solution was applied to standard agar medium (manufactured by Nisui Pharmaceutical Co., Ltd.), followed by aerobic culture at 37 ° C. for 48 hours, Characterized colonies developed were counted.

The viable cell count of DB9011 strain is shown in Table 7, and the spore count is shown in Table 8.

The viable cell count of NBRC3009 strain is shown in Table 9, and the spore count is shown in Table 10.

The viable cell count of NBRC3025 strain is shown in Table 11, and the spore count is shown in Table 12.

The number of viable cells of NBRC3108 strain is shown in Table 13, and the number of spores is shown in Table 14.

The number of viable cells of NBRC3336 strain is shown in Table 15, and the number of spores is shown in Table 16.

As shown in Test Examples 9 to 18, the solid cultures of the AOK 210 strain and the Aspergillus duckase IK-05074 strain in the Aspergillus demagnetization were Bacillus subtilis DB9011 strain, NBRC3009 strain, NBRC3025 strain, and NBRC3108 strain. , Did not show antimicrobial activity against NBRC3336.

For Lactobacillus acidophilus (LA), anaerobic kenki (manufactured by Mitsubishi Gas Chemical Co., Ltd.) was used, and BCP-added plate count agar "Nisui" (manufactured by Nisui Pharmaceutical Co., Ltd.) was subjected to anaerobic treatment at 37 ° C for 72 hours. Incubated. Colonies developed on the plate were scraped off and suspended in sterile saline. 500 ml of GAM bouillon (manufactured by Nisui Pharmaceutical Co., Ltd.) was prepared in a 1 L volumetric flask, and after autoclave sterilization, aseptically, the final concentration of LA was about 1.0 × 10 ³ to 1.0 × 10 ⁴ CFU / ml. Was added. To the Erlenmeyer flask, 5 g of crushed solid cultures of AOK 210 strain and Aspergillus duck, IK-05074 strain, were aseptically added to Aspergillus decanter, respectively, to each of Test Examples 19 and 20. An Erlenmeyer flask without addition was used as a control. Each Erlenmeyer flask was gently incubated under anaerobic conditions using aneropakkenki in an incubator at 37 ° C.

Regarding Bifidobacterium breve (BB), anaerobic kenki (manufactured by Mitsubishi Gas Chemical Co., Ltd.) was used to perform anaerobic culture at 37 ° C. for 48 hours in BL agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.). Colonies developed on the plate were scraped off and suspended in sterile saline. 500 ml of GAM bouillon (manufactured by Nisui Pharmaceutical Co., Ltd.) were prepared in a 1 L volumetric flask, and aseptically, after autoclave sterilization, the final concentration of BB was about 1.0 × 10 ⁴ to 1.0 × 10 ⁵ CFU / mL. Input. To the Erlenmeyer flask, 5 g of crushed solid cultures of AOK 210 strain and Aspergillus duck, IK-05074 strain, were aseptically added to Aspergillus decanter, respectively, to test examples 21 and 22. An Erlenmeyer flask without addition was used as a control. Each Erlenmeyer flask was gently incubated under anaerobic conditions using aneropakkenki in an incubator at 37 ° C.

The viable cell count of LA and BB was measured on the 0th, 3rd, and 7th day after the start of the test. In LA, the method for measuring the number of viable bacteria was diluted 10-fold step with sterile physiological saline solution, and then 0.1 ml of each dilution step solution was added with BCP plate count agar "Nisui" (manufactured by Nisui Pharmaceutical Co., Ltd.) After application to, the anaerobic culture was carried out at 37 ° C. for 72 hours using Aneropakkenki (manufactured by Mitsubishi Gas Chemical Co., Ltd.), and the developed characteristic colonies were counted. In the method for measuring the number of viable bacteria of BB, after diluting the collected culture solution 10 times with sterile physiological saline solution, 0.1 ml of each dilution step solution is applied to BL agar medium (manufactured by Nisui Pharmaceutical Co., Ltd.). Anaerobic culture was carried out for 48 hours at 37 ° C using Lopak Kenki (manufactured by Mitsubishi Gas Chemical Co., Ltd.), and the developed characteristic colonies were counted.

The number of viable cells of LA is shown in Table 17, and the number of viable cells of BB is shown in Table 18.

As shown in Test Examples 19 and 20, the solid cultures of the AOK 210 strain and the Aspergillus aurise IK-05074 strain in the Aspergillus dendrites did not show antibacterial activity against LA.

As shown in Test Examples 21 and 22, the solid cultures of the AOK 210 strain and the Aspergillus aurise IK-05074 strain in the Aspergillus dendrites did not show antibacterial activity against BB.

From the above results, probiotics AOK 210 strain and Aspergillus aurise IK-05074 strains showed as antibacterial activity to Escherichia coli, Salmonella enteritidis, Staphylococcus aureus, Clostridium perfringens, which are pathogenic bacteria. It did not show antimicrobial activity against promising Bacillus subtilis or Lactobacillus acidophilus and Bifidobacterium breve. Thus, by combining AOK 210 strains and / or Aspergillus aurise IK-05074 strains with B. subtilis in Aspergillus soda, the B. subtilis can be exerted without losing its ability. It is thought that new animal feed additives could be developed that do not adversely affect L. acidophilus and B. breve, which are useful as enteric bacteria.

<3> Determination of Insecticidal Activity of Aspergillus Bacteria Culture

(1) Eimeria tenella control test

Feces from chickens naturally infected with Eimeria tenella were collected, and the austist was separated under a stereoscopic microscope and washed with physiological saline. 5 ml of saline solution was added to a 9 cm diameter chalet, and the washed austist was added to about 4000 pieces / ml. Grinded solid cultures of AOK 210 strain, AOK 43 strain, AOK N4586 strain, AOK B650 strain, and IK-05074 strain were charged 50 mg per chalet, respectively, to be Test Examples 23-27. In addition, a chalet to which no yeast culture was added was used as a control. Each chalet was shaken (150 rpm) at 37 ° C. After 7 days, the number of ossists was measured under a stereomicroscope, the deformation and dissolution state of the cell wall were observed, and the reduction rate and dissolution denaturation rate of the astigmats were calculated.

The results are shown in Table 19.

As shown in Test Examples 23 to 27, solid cultures of AOK 210 strain, AOK 43 strain, AOK N4586 strain, AOK B650 strain, and IK-05074 strain reduced the number of ossists in E. tenella or denatured the acist soluble denaturation. It was found to exhibit activity.

In particular, by treating the AOK 210 strains and the IK-05074 strains of E. tenella mainly, very high astigmatism reduction and dissolution denaturation rates were obtained.

(2) Eimeria zuernii control test

Bovine diarrhea spontaneously infected with Eimeria zuernii was collected and the austist was separated under a stereoscopic microscope and washed with physiological saline. 5 ml of physiological saline was added to a 9-cm-diameter chalet, and the rinsed austen was added to about 2000 pieces / ml. Grinded solid cultures of AOK 210 strain, AOK 43 strain, AOK N4586 strain, AOK B650 strain, and IK-05074 strain were charged 50 mg per chalet, respectively, to be Test Examples 28-32. A chalet without the yeast culture was used as a control. Each chalet was shaken (150 rpm) at 37 ° C. After 7 days, the number of ossists was measured under a stereomicroscope, the deformation and dissolution state of the cell wall were observed, and the reduction rate and dissolution denaturation rate of the astigmats were calculated.

The results are shown in Table 20.

As shown in Test Examples 28 to 32, solid cultures of AOK 210 strain, AOK 43 strain, AOK N4586 strain, AOK B650 strain, and IK-05074 strain reduced the number of aches of E. zuernii and resulted in a higher astigmatism. Dissolution denaturing activity was shown. In particular, by treating the austria of AOK 210 strain and IK-05074 strain mainly to E. zuernii, a very high rate of reduction in acest and dissolution modification rate was obtained.

As mentioned above, the said Aspergillus microorganism is estimated to be effective also in the control of coccidium.

<4> Culture of Bacillus subtilis

Bacillus subtilis DB9011 strains, Bacillus subtilis NBRC309 strains, Bacillus subtilis NBRC3025 strains, Bacillus subtilis NBRC3108 strains, and Bacillus subtilis NBRC3336 strains were cultured at 37 ° C. for 72 hours using spore-forming medium having the composition shown below. The obtained culture was centrifuged to collect the cells. The obtained cells were lyophilized and then ground to obtain spore powder. Spore densities of DB9011, NBRC3009, NBRC3025, NBRC3108, and NBRC3336 strains were 1.01 × 10 ¹¹ CFU / g, 1.21 × 10 ¹¹ CFU / g, 1.15 × 10 ¹¹ CFU / g, 1.06 × 10 ¹¹ CFU / g, 1.09 × 10 ¹¹ CFU / g. Spore density was measured by diluting the obtained spore powder with sterile water to an appropriate concentration, killing only feeder cells by heating at 70 ° C. for 30 minutes, and then inoculating the agar medium, and counting the number of colonies formed.

In addition, the Bacillus subtilis DB9011 note, as a FERM BP-3418, was established on May 21, 1991 in the Independent Administrative Institutional Industrial Technology Research Institute Patent Biodeposit Center (Higashi 1 Thome, Ichome 1 Banchi 1 Chuo Dai 6, Ibaraki, Japan). Deposited. In addition, NBRC3009, NBRC3025, NBRC3108, and NBRC3336 are states registered in the Biogenetic Resources Division (NBRC) of the Product Evaluation Technology Infrastructure Organization, respectively.

Spore-forming medium component ¹⁾

Difco nutrient broth 8.0g

KCl 1.0g

MGSO _{4 7} H ₂ O 0.25 g

MnCl ₂ 4H ₂ O 0.002 g

Adjust pH to 7.0, total amount 1,000 ml

After autoclave sterilization, the sterilized CaCl ₂ solution and FeSO ₄ solution were

Add 5 × 10 ^-4 M and 1 × 10 ^-6 M, respectively

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

<5> breeding examination-1

(1) Preparation of feed additives

In the Aspergillus dendrites obtained from the above <1>, the spores of the AOK 210 strain, the ground solid culture of the Aspergillus aurise IK-05074 strain, the spores of the DB9011 strain and the NBRC3009 strain were diluted with lactose, respectively. Feed additives of concentration were prepared (Preparation Examples 1-4). In addition, a culture of AOK 210 strain or IK-05074 strain and spores of DB9011 strain or NBRC3009 strain were respectively combined and diluted with lactose to prepare feed additives having different concentrations (Preparation Examples 5 to 8). The addition amount of the solid culture and Bacillus subtilis in each feed additive is shown in Table 21.

(2) chick breeding test

The feed additives of the preparation examples 1-8 were mixed so that the feed additives of manufacture examples 1-8 may be 0.1 mass% with respect to the whole mass of the chick feed (SD broiler full-duration, Nippon compound feed Co., Ltd. product, antimicrobial substance-free feed). Twelve chicks hatched from broiler chickens (breed: chunky) -derived eggs were used as a group, and each group was fed with the feed for 35 days, and a growth test was conducted. Untied feed (不斷 給 餌), was raised in free water. In addition, a growth test was conducted in the same manner using a feed obtained by mixing only 0.1% by mass of lactose as a control. The body weight of the chicks of each group after 35 days was measured, and the average value of each group was calculated.

The results are shown in Table 22.

Forages of Production Examples 1 (E-G) and Production Examples 2 (E-G) containing 15% by mass or more of AOK 210 strain or Aspergillus duck O. IK-05074 strain culture The chicks given the additive showed a marked weight gain compared to the chicks of the control. The chicks given the feed additives of Preparation Example 3 (E to G) and Preparation Example 4 (E to G) containing spores of Bacillus subtilis DB9011 strain or NBRC3009 strain at least 1.5 × 10 ⁹ CFU / g were the chicks of the control. Significant weight gain was observed.

On the other hand, Production Example 5 (B-G) which combined 1.0 mass% or more of culture | cultivation of AOK 210 strain or IK-05074 strain, and 1.0x10 <^8> CFU / g or more of DB9011 strain or NBRC3009 strain, respectively, Production Example 6 (B- When the animal was ingested G), Production Example 7 (B to G) and Production Example 8 (B to G), the effect of remarkable weight gain was confirmed. As a result, it was found that by combining the culture of genus Aspergillus and Bacillus subtilis, the weight gain of the chick can be promoted at a lower concentration than when each active ingredient is used alone.

(3) piglet breeding test

The feed additives of Production Examples 1 to 8 were mixed so as to be 0.1% by mass with respect to the feed mass of the piglet (standard feed for SD piglet artificial oil, the feed of Nippon Blended Food Co., Ltd., and the feed without an antimicrobial substance). Eight piglets (breed: large Yorkshire) three weeks old were used as a group, and each group was fed with the feed for 35 days in uninterrupted feeding and free watering, and a growth test was conducted. In addition, a growth test was conducted in the same manner using a feed obtained by mixing only 0.1% by mass of lactose as a control. The body weight of piglets in each group after 35 days was measured, and the average value of each group was calculated.

The results are shown in Table 23.

Forages of Production Examples 1 (E-G) and Production Examples 2 (E-G) containing 15% by mass or more of AOK 210 strain or Aspergillus duck O. IK-05074 strain culture Piglets given the additive showed a marked weight gain compared to piglets in the control. Production Example 3 (E-G) containing Bacillus subtilis DB9011 strain 1.5 × 10 ⁸ CFU / g or more and Production Example 4 (E-G) containing NBRC3009 strain spores 1.5 × 10 ⁸ CFU / g or more Piglets given feed additives were found to have marked weight gains compared to piglets in control.

On the other hand, AOK 210 weeks, or manufactured by a IK-05074 Note combining incubated over water 1.0% by weight and DB9011 state or NBRC3009 attention spores 1.0 × 10 ⁸ CFU / g or higher Example 5 (B ~ G), Preparation 6 (B ~ G ), When the animal was ingested in Production Example 7 (B to G) and Production Example 8 (B to G), the effect of remarkable weight gain was confirmed. As a result, it was found that by combining the culture of genus Aspergillus and Bacillus subtilis, the weight gain of the piglets can be promoted at a lower concentration than when each active ingredient is used alone.

<6> breeding examination-2

(1) Preparation of feed additives

A feed additive containing a culture of AOK 210 strain or IK-05074 strain obtained from the above <1> and DB9011 strain or NBRC3009 strain was diluted with lactose and prepared in different proportions. 12). The addition amount of the solid culture and Bacillus subtilis in each feed additive is shown in Table 24.

(2) chick breeding test

The feed additives of Production Examples 9 to 12 were mixed so as to be 0.1% by mass with respect to the total feed mass of the chick (SD broiler late-end, Nippon Blended Feed Co., Ltd. make, antimicrobial-free feed). Twelve chicks hatched from egg breeders derived from broiler chickens (variety: chunky) were used as a group, and each group was fed the feed for 35 days, and a growth test was performed. Untied feed was raised by free water supply. In addition, a growth test was conducted in the same manner using a feed obtained by mixing only 0.1% by mass of lactose as a control. The body weight of the chicks of each group after 35 days was measured, and the average value of each group was calculated.

The results are shown in Table 25.

The ratio of AOK 210 strains or Aspergillus aurise IK-05074 strains to Aspergillus dendrites and DB9011 strains or NBRC3009 strains is 4% by mass: 1 × 10 ⁸ CFU / g to 1 mass. %: The range of 4 * 10 <^8> CFU / g was optimal (Manufacture example 9 (B-F), manufacture example 10 (B-F), manufacture example 11 (B-F), manufacture example 12 (B-F). ). From this, it was found that the concentration of Bacillus subtilis per feed additive exhibiting 1 U acid resistance α-amylase activity was in the range of 0.5 × 10 ⁶ to 2.0 × 10 ⁷ CFU / g. Moreover, it turned out that the range of 4.5x10 <^4> -8.5 * 10 <^5> CFU / g of the concentration of Bacillus subtilis per feed additive which shows the sum total of 1U acidic protease and acidic carboxypeptidase was optimal.

(2) piglet breeding test

The feed additives of Production Examples 9 to 12 were mixed so as to be 0.1 mass% with respect to the feed mass of the piglet (standard feed for SD piglet artificial oil late-end, Nippon Blended Feed Co., Ltd. product, feed without an antimicrobial substance). Eight piglets (breed: large Yorkshire) three weeks old were used as a group, and each group was fed with the feed for 35 days in uninterrupted feeding and free watering, and a growth test was conducted. The growth test was performed similarly using the feed which mixed only 0.1 mass% of lactose as a control. The body weight of piglets in each group after 35 days was measured, and the average value of each group was calculated.

The results are shown in Table 26.

The ratio of AOK 210 strains or Aspergillus aurise IK-05074 strains to Aspergillus dendrites and DB9011 strains or NBRC3009 strains is 4% by mass: 1 × 10 ⁸ CFU / g to 1 mass. %: The range of 4.0x10 <^8> CFU / g was optimal (Manufacture example 9 (B-F), manufacture example 10 (B-F), manufacture example 11 (B-F), manufacture example 12 (B-F) ).

<7> pathogen attack test for chick

(1) Salmonella enteritidis attack test

The feed additives of Production Examples 1 to 8 were mixed so as to be 0.1% by mass with respect to the feed mass of the chick (SD broiler electric, Nippon Blended Feed Co., Ltd. make, antimicrobial-free feed). Twelve chicks hatched from broiler chickens (variety: chunky) -derived eggs were used as a group, and the feed was fed for 14 days by feeding and free feeding. The test was similarly carried out using the feed which mixed only 0.1 mass% of lactose as a control. At 7 days of age, 2.3 × 10 ⁷ CFU of Salmonella enteritidis (SE) was administered orally. At 14 days of age, feces were collected by wiping the cecal contents and total excretory cavity with a cotton swab.

The number of SE viable cells of the cecal contents was measured by the following method, and the infection index and the defense index were calculated.

1 g of cecal contents were diluted 10-fold by the addition of sterile phosphate buffered saline, and mixed sufficiently to prepare a sample stock solution. Next, the sample stock solution was diluted 10-fold using sterile physiological saline to obtain a step-diluted solution. The sample stock solution and the step dilution solution were each coated with 0.1 ml of SS agar flat medium "Nisui" (manufactured by Nisui Pharmaceutical Co., Ltd.) and brilliant green agar flat medium (manufactured by Difco Laboratories), and incubated at 37 ° C for 24 hours. The number of colonies of typical SE grown in the medium was measured. In addition, it is sterilized from colonies, and inoculated into SIM agar medium "Nisui" (manufactured by Nissui Pharmaceutical Co., Ltd.) and TSI agar medium "Nisui" (manufactured by Nisui Pharmaceutical Co., Ltd.) for lysine decarbonization test. After incubation at 37 ° C. for 24 hours, the appearance was confirmed.

The number of SE viable cells per 1 g of cecal contents was calculated by multiplying the number of colonies identified as SE by the dilution ratio of the diluted solution. Based on this result, the infection index and the defense index were computed as follows. The infection index is a value indicating the low infection rate of a pathogen, and the defense index is a value indicating the ability of each feed to protect the infection of the pathogen as compared with the case of administering the feed of the control.

Infection Index: The mean value of the log of the number of SE live bacteria in the caecum contents of each individual (mean value of log CFU / g)

Defense index: Infection index of control / infection index of each test

Regarding the feces collected from the total excretory cavity, the properties of SE were confirmed by qualitatively culturing each individual by the following method. That is, the stool attached to the cotton swab was suspended in 10 ml of sterile phosphate buffered saline solution, and used as a sample stock solution, which was then coated with 0.1 ml of SS agar plate medium and brilliant green agar plate medium, and incubated at 37 ° C for 24 hours. The presence or absence of colony formation of typical SE grown on each flat medium was determined. In addition, the bacteria were inoculated into LIM agar medium "Nisui" (manufactured by Nisui Pharmaceutical Co., Ltd.), SIM agar medium, and TSI agar medium, and cultured at 37 ° C for 24 hours to confirm the appearance.

Infection index is shown in Table 27, a defense index is shown in Table 28, and the detected population of SE of the total excretory river collection feces is shown in Table 29.

AOK 210 strains of Aspergillus or Aspergillus aurise IK-05074 Note When pulverized solid cultures alone were added to feed additives of 20% by mass or more, the cell concentration of SE in the cecum contents was increased. It became low and showed the infection index of low SE (production example 1 (F, G), preparation example 2 (F, G)). When Bacillus subtilis DB9011 strain or NBRC3009 strain alone were added to feed additives of 2.0 × 10 ⁹ CFU / g or more, the cell concentration of SE in the caecum contents decreased and showed a low SE infection index. Example 3 (F, G), Production Example 4 (F, G)). In contrast, in the combination of AOK 210 strain or Aspergillus aurise IK-05074 strain and Bacillus subtilis DB9011 strain or NBRC3009 strain in Aspergillus soybean cultures, AOK 210 strain or IK-05074 strain was cultured. Is added to feed additives of AOK 210 or IK-05074 alone or more than 20% by mass to feed additives, or when DB9011 or DB9011 or NBRC3009 is added to feed additives of 2.5 × 10 ⁸ CFU / g or higher. The same control effect as SE was obtained when the strain or NBRC3009 strain alone was added to a feed additive of 2.0 × 10 ⁹ CFU / g or more (Production Example 5 (C to G), Production Example 6 (C to G). , Production Example 7 (C to G), Production Example 8 (C to G)). In addition, SE was able to be completely controlled when a feed additive obtained by combining AOK 210 strain or IK-05074 strain with 20 mass% or more, DB9011 strain or NBRC3009 strain with 2.0 × 10 ⁹ CFU / g or more was obtained. 5 (G), Production Example 6 (G), Production Example 7 (G), Production Example 8 (G)). From this, aspergillus soja or aspergillus aurise and Bacillus subtilis are given to chicks, so that SE infections are lowered at lower concentrations than when each active ingredient is used alone. It can be seen that there is an effect to prevent.

(2) Clostridium perfringens attack test

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

The number of CP viable cells of the cecal contents was measured by the following method, and the infection index and the defense index were calculated.

1 g of cecal contents were diluted 10-fold by the addition of sterile phosphate buffered saline, and mixed sufficiently to prepare a sample stock solution. Next, the sample stock solution was diluted 10-fold using sterile physiological saline to obtain a step-diluted solution. 0.1 ml each of the sample stock solution and the step dilution solution were respectively coated in Clostridia measurement medium (manufactured by Nisui Pharmaceutical Co., Ltd.), and anaerobic culture was performed at 35 ° C for 24 hours using aneropakkenki, and grown on each flat medium. The number of black colonies was measured. Furthermore, it was sterilized from colonies, inoculated into yolk-added CW agar medium (manufactured by Nisui Pharmaceutical Co., Ltd.), and aerobic and anaerobic culture were carried out at 35 ° C for 24 to 48 hours to confirm the properties.

The number of CP viable cells per 1 g of cecal contents was calculated by multiplying the number of colonies identified as CP by the dilution ratio of the diluent. Based on this result, the infection index and the defense index were computed similarly to the above.

Regarding the stool collected from the total excretory cavity, the properties of CP were confirmed by qualitatively culturing for each individual by the following method. That is, the feces adhering to the cotton swab are suspended in 10 ml of sterile phosphate buffered saline solution, and the sample stock solution is used. Then, 0.1 ml of each is coated on Clostridia measurement medium (made by Nisui Pharmaceutical Co., Ltd.), followed by anero. Anaerobic incubation was carried out at 35 ° C. for 24 hours using Pakkenki, and the presence or absence of black colony growth in each plate medium was determined. Furthermore, it was sterilized from colonies, inoculated into yolk-added CW agar medium (manufactured by Nisui Pharmaceutical Co., Ltd.), and aerobic and anaerobic culture were carried out at 35 ° C for 24 to 48 hours to confirm the properties.

The infection index is shown in Table 30, the defense index is shown in Table 31, and the number of detected individuals of CP of the excretory scavenging stool is shown in Table 32.

AOK 210 strains of Aspergillus or Aspergillus aurise IK-05074 Note When the pulverized solid culture alone was added to a feed additive of 20% by mass or more, the cell concentration of CP in the cecum contents was increased. It was lowered and showed an infection index of low CP (Production Example 1 (F, G), Production Example 2 (F, G)). When Bacillus subtilis DB9011 strain or NBRC3009 strain alone were added to feed additives of 2.0 × 10 ⁹ CFU / g or more, the cell concentration of CP in the cecal contents was lowered and showed a low CP infection index (preparation) Example 3 (F, G), Production Example 4 (F, G)). In contrast, in the combination of AOK 210 strain or Aspergillus aurise IK-05074 strain with Bacillus subtilis DB9011 strain or NBRC3009 strain, the AOK 210 strain or IK-05074 strain was added to Aspergillus soda 2.5. When adding at least 20% by mass, DB9011 or NBRC3009 strains to a feed additive of at least 2.5 × 10 ⁸ CFU / g, when AOK 210 or IK-05074 strains alone were added to at least 20% by mass to the feed additive, or DB9011 The same control effect as CP was obtained when the strain or the NBRC3009 strain alone was added to a feed additive of 2.0 × 10 ⁹ CFU / g or more (Production Example 5 (C to G), Production Example 6 (C to G) , Production Example 7 (C to G), Production Example 8 (C to G)). In addition, CP was completely controlled when AOK 210 strain or IK-05074 strain was added at least 20 mass%, DB9011 strain or NBRC3009 strain was added to a feed additive of 2.0 × 10 ⁹ CFU / g or higher (Production Example 5 (G), Production Example 6 (F, G) Production Example 7 (G), Production Example 8 (G)). From this, each active ingredient is used alone by combining AOK 210 strain or Aspergillus aurise IK-05074 strain with Bacillus subtilis DB9011 strain or NBRC3009 strain to Aspergillus soda. It can be seen that it is effective in preventing CP infection at a lower concentration than that.

<8> edema germ attack test for piglets

0.1 mass% of the feed additives of the preparation examples 1-8 prepared above were added with respect to the feed mass of the piglet (the standard feed for the SD piglet artificial oil, the Nippon compound feed, and the antimicrobial feed). Mix as much as possible. Eight piglets (breed: large Yorkshire) three weeks old were taken as one group, and the feed was given to each group for 14 days in uninterrupted feeding and free watering. The test was similarly carried out using the feed which mixed only 0.1 mass% of lactose as a control. Porcine edema pathogen Escherichia coli (EC) was incubated at 37 ° C for 4 hours in Tryptic Soy Broth (manufactured by Difco Laboratories). Centrifugation (3,000 rpm, 15 minutes) was carried out at 4 degreeC, and the pellet was put into an enteric capsule. At 4 weeks of age, 4.5 × 10 ⁸ CFU of EC was administered orally once daily for three consecutive days using an enteric capsule. Instead of the edema pathogen, physiological saline was placed in an enteric capsule, and forced oral administration in the same manner was used as an example of no inoculation, and the same test was performed. After breeding up to 5 weeks of age, body weight was measured and the average value of the weight gain of each group was calculated. From the start of forced oral administration to the end of the trial, clinical observations of fecal appearance and peri-eye edema are performed daily, and the sum of the fecal constellation score and peri-eye edema score during the trial period is calculated for each individual, and each group The average value of the score of was computed.

The weight gain during the test period is shown in Table 33, the fecal appearance score in Table 34, and the perioperative edema score in Table 35.

AOK 210 strains of Aspergillus or Aspergillus aurise IK-05074 Note The ground solid culture was added to feed additives by 15 mass% or more alone, so that the weight gain was the same as that of the non-inoculated example. The value of the grade was shown (Manufacture example 1 (E-G), manufacture example 2 (E-G)). Bacillus subtilis DB9011 strain or NBRC3009 strain alone added to feed additives of 15 × 10 ⁸ CFU / g or higher, showing the same level of weight gain as that of the non-inoculated example (Preparation Example 3 (E-G ), Production Example 4 (E to G)). The same tendency was also shown with respect to the fecal appearance score and the perioperative edema score. In contrast, in the combination of AOK 210 strain or Aspergillus aurise IK-05074 strain with Bacillus subtilis DB9011 strain or NBRC3009 strain, the AOK 210 strain or IK-05074 strain was added to Aspergillus soda 2.5. By adding at least% by mass, DB9011 strain or NBRC3009 strain to the feed additive of 2.5 × 10 ⁸ CFU / g or more, the weight gain showed the same value as the non-inoculated example (Production Example 5 (C to G), Preparation Example 6 (C-G), manufacture example 7 (C-G), manufacture example 8 (C-G)). The same tendency was also shown with respect to the fecal appearance score and the perioperative edema score. As a result, by combining Aspergillus Soja or Aspergillus aurise with Bacillus subtilis, the effect of preventing edema germ infection at a lower concentration than when each active ingredient is used alone. You can see that there is.

<9> Salmonella Typhimurium attack test on calf

1 week-old male calf (Holstein species) is a group of four, and commercial mammalian calf substitutes (Professional: Promilk, Nippon Blended Food Co., Ltd.) are supplied twice a day, 2 liters each for 7 weeks of age. It was. Free water was used, and hay and commercial artificial oils (Spa Hoiku Moretto and Genno) were taken continuously. 25 g per day per day of the feed additives of Preparation Examples 1 to 8 prepared above were mixed and administered to a substitute oil. Only 25 g of lactose was mixed with the substitute oil as a control, and the same test was performed. At 2 weeks of age, 5.0 × 10 ⁶ CFU of Salmonella Typhimurium (ST) suspended in 50 ml of 10% NaHCO ₃ per animal was orally administered. The animals were reared up to 7 weeks of age, weighed, and the average weight gain of each group was calculated. From the start of forced oral administration to the end of the test, fecal appearances were observed every day, and the sum of the fecal appearance scores during the test period was calculated for each individual, and the average value of the scores of each group was calculated.

Table 36 shows the weight gain during the test period and Table 37 shows the fecal property score.

AOK 210 strains of Aspergillus or Aspergillus aurise IK-05074 Notes The ground solid culture is added to feed additives by 20 mass% or more alone, so that the weight gain is the same as that of the non-inoculated example. The value of (Manufacture example 1 (F, G), manufacture example 2 (F, G)) was shown. Bacillus subtilis DB9011 strain or NBRC3009 strain alone added 2.0 × 10 ⁹ CFU / g or more of the feed additive, and showed the same level of weight gain as the non-inoculated example (Preparation Example 3 (F, G) ), Preparation Example 4 (F, G)). The same tendency was also observed with the fecal constellation score. In contrast, in the combination of AOK 210 strain or Aspergillus aurise IK-05074 strain with Bacillus subtilis DB9011 strain or NBRC3009 strain, the AOK 210 strain or IK-05074 strain was added to Aspergillus soda 2.5. By adding at least% by mass, DB9011 strain or NBRC3009 strain to the feed additive of 2.5 × 10 ⁸ CFU / g or more, the weight gain showed the same value as the non-inoculated example (Production Example 5 (C to G), Preparation Example 6 (C-G), manufacture example 7 (C-G), manufacture example 8 (C-G)). The same tendency was also observed with the fecal constellation score. As a result, by combining Aspergillus sorrea or Aspergillus arige with Bacillus subtilis, the effect of preventing ST infection at a lower concentration than when each active ingredient is used alone is obtained. I can see that there is.

By absorbing aspergillus microorganism of the present invention, the culture containing the acidic enzyme produced by the bacterium, and the animal feed additive containing Bacillus subtilis into the feed and ingesting the animal, the absorption of nutrition is promoted. Feed efficiency rises. Specifically, acidic enzymes produced by the genus Aspergillus show antimicrobial activity against intestinal pathogens and the like, and Bacillus subtilis revives immunity. As a result, the intestinal infections of animals are prevented and improved. Contribute to weight gain. The feed of the present invention can be preferably used for raising livestock such as chickens, pigs, and cattle.

Claims (13)

Aspergillus sojae, Aspergillus tamarii, Aspergillus foetidus, Aspergillus niger and Aspergillus sojae At least one Aspergillus species selected from Aspergillus oryzae, cultures containing acidic enzymes produced by these bacteria, and animals containing Bacillus subtilis A feed additive for animals, wherein the total amount of acidic enzyme activity per 1 g of feed additive is 120 U or more, and the concentration of Bacillus subtilis is 2.5 × 10 ⁷ to 2 × 10 ⁹ CFU / g. The method of claim 1, The animal feed additive, characterized in that the acidic enzyme contains an acidic amylase, and the acidic amylase activity is 1 U or more per 1g of the feed additive. The method according to claim 1 or 2, The animal feed additive, characterized in that the Aspergillus bacteria have an antimicrobial activity against the pathogen causing the intestinal infection of the animal and / or insecticidal activity against coccidial. The method according to any one of claims 1 to 3, The animal feed additive for which Aspergillus spp. Is Aspergillus soja and / or Aspergillus aurize. The method according to any one of claims 1 to 4, Aspergillus aurise is IK-05074 strain (FERM BP-10622) and / or a mutant strain thereof, which is a strain having the same acidic enzyme-producing ability as the strain. The method according to any one of claims 1 to 5, Animal feed additives, wherein the culture contains a plant nutrient. The method of claim 6, Animal feed additives, wherein the plant nutrition source is brown rice. The method according to any one of claims 1 to 7, Bacillus subtilis is a DB9011 strain (FERM BP-3418) and / or a mutant strain thereof. The method according to any one of claims 1 to 8, Animal feed additives for promoting growth. The method according to any one of claims 1 to 8, Animal feed additives for the prevention and improvement of intestinal infectious diseases. The feed containing 0.01-1.0 mass% of animal feed additives of any one of Claims 1-10. In a solid medium containing a nutrient for propagation of Aspergillus bacteria, Aspergillus soda, Aspergillus tamari, Aspergillus poetidus, Aspergillus niger and A step of culturing at least one Aspergillus genus selected from Aspergillus aurise and containing the resulting culture so that the total amount of acidic enzyme activity per kg of feed is 12 U or more, and Bacillus subtilis A method of producing a feed comprising a step of containing a so that the concentration is 2.5 × 10 ⁶ ~ 2.0 × 10 ¹⁰ CFU / kg. An animal breeding method comprising feeding an animal according to claim 11 to an animal.