Compositions for oral administration and uses thereof

Abstract

To provide a novel composition for oral administration useful for preventing or treating mastitis of ruminants.SOLUTION: A composition for oral administration contains Bacillus subtilis TTCC903 strain (accession number: FERM P-21173) as an active ingredient and is used for preventing or treating mastitis of ruminants. A feed additive contains the composition for oral administration. A feed contains the composition for oral administration. A pharmaceutical composition contains the composition for oral administration. A method for preventing mastitis of ruminants includes administering the composition for oral administration to ruminants. A method for treating mastitis of ruminants includes administering the composition for oral administration to ruminants.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a composition for oral administration and its use. Specifically, the present invention relates to a composition for oral administration used for preventing or treating mastitis in ruminants, a feed additive, a feed, and a pharmaceutical composition containing the composition for oral administration, a method for preventing and treating mastitis in ruminants using the composition for oral administration, and a method for improving the rearing environment of ruminants. [Background technology]

[0002] The udder is a collection of mammary gland tissue (cells that produce milk). Mastitis is a disease caused by infection of the udder with pathogenic microorganisms such as Staphylococcus aureus, (non-dairy or environmental) Streptococcus, Escherichia coli, and Mycoplasma. In particular, cows develop mastitis when their resistance is weakened for some reason, or when humans do not properly manage milking or livestock housing, allowing many bacteria to enter through the teat opening.

[0003] Considering the route of infection, mastitis can be classified into "contagious mastitis" and "environmental mastitis." Contagious mastitis is mastitis that spreads from cow to cow via the milker's hands or milking equipment. Staphylococcus aureus and agalactiae streptococcus are the main species that cause contagious mastitis. Staphylococcus aureus in particular is a species that is widely isolated (detected) in Japan. It creates an abscess (a lump the size of a ping-pong ball or baseball) in the udder, and in many cases the bacteria lurk inside it. Even if treatment is performed with antibiotics, the drugs do not reach the inside of the abscess, so developing a technology to eliminate Staphylococcus aureus is considered a major challenge in veterinary medicine. In contrast, environmental mastitis occurs when bacteria in the environment (especially the bedding, etc.) enter the cow through the teat opening and settle in the mammary gland. Bacteria that cause environmental mastitis include Escherichia coli, Klebsiella, and Pseudomonas aeruginosa. Appropriate environmental management is important for controlling environmental mastitis.

[0004] Various therapeutic agents have been developed so far for the prevention or treatment of mastitis in ruminants such as cows. For example, Patent Document 1 discloses a preventive or therapeutic agent for ruminant mastitis, which contains Bacillus subtilis C-3102 strain (international deposit number FERM BP-1096) as an active ingredient. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6415430 Summary of the Invention [Problem to be solved by the invention]

[0006] Dairy cows that develop mastitis experience a decrease in milk production and milk components, and if chemotherapeutic agents such as antibiotics are used, the milk cannot be shipped, resulting in large economic losses for dairy farmers. Therefore, there is a demand for a preventive or therapeutic agent for mastitis in ruminants that is more effective than conventional agents, is safe, and can be taken continuously. In addition, odors from excrement and other materials produced in the raising of ruminants are generally recognized as foul odors and are a cause of complaints. Therefore, there is a demand for a method for reducing the foul odors in the raising environment of ruminants.

[0007] The present invention has been made in view of the above circumstances, and provides a novel composition for oral administration that is effective for preventing or treating mastitis in ruminants, and a method for improving the rearing environment of ruminants using the composition for oral administration. [Means for solving the problem]

[0008] That is, the present invention includes the following aspects. (1) A composition for oral administration used for preventing or treating mastitis in ruminants, comprising Bacillus subtilis TTCC903 strain (accession number FERM P-21173) as an active ingredient. (2) The composition for oral administration described in (1), wherein the ruminant is a cow. (3) A feed additive containing the composition for oral administration described in (1) or (2). (4) A feed containing the composition for oral administration described in (1) or (2). (5) A pharmaceutical composition comprising the composition for oral administration according to (1) or (2). (6) A method for preventing mastitis in ruminants, comprising ingesting the composition for oral administration described in (1) or (2) to a ruminant. (7) A method for treating mastitis in ruminants, comprising ingesting the composition for oral administration described in (1) or (2) to a ruminant. (8) A method for improving the rearing environment of ruminants, comprising having the ruminants ingest a composition for oral administration containing, as an active ingredient, Bacillus subtilis TTCC903 strain (accession number FERM P-21173). Effect of the Invention

[0009] According to the composition for oral administration of the above aspect, it is possible to provide a composition for oral administration that is effective for preventing or treating mastitis in ruminants. According to the method for improving the above aspect, it is possible to effectively improve the rearing environment of ruminants. [Brief description of the drawings]

[0010] [Figure 1] 1 is a graph showing the annual trends in weighted average somatic cell counts at three farms over the past five years (2014 to 2018), 2019, and 2020 in Example 1. [Diagram 2] 1 is a graph showing the percentage of individuals with a somatic cell count linear score of 4.5 or more in early lactation during the two years before and after the start of feeding TTCC903 Natto in Example 1. [Diagram 3] This is a graph showing the annual trends in weighted average milk yields at three farms for the past five years (2014 to 2018), 2019, and 2020 in Example 1. [Figure 4] 1 shows an agarose gel electrophoresis image of colony PCR products in Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] <Composition for oral administration> A composition for oral administration according to one embodiment of the present invention (hereinafter, may be simply abbreviated as "the composition for oral administration of this embodiment") contains Bacillus subtilis TTCC903 strain (accession number FERM P-21173) as an active ingredient, and is used for preventing or treating mastitis in ruminants.

[0012] In the present invention, "containing Bacillus subtilis TTCC903 strain (Accession No. FERM P-21173) as an active ingredient" means containing Bacillus subtilis TTCC903 strain as an ingredient having a preventive or therapeutic effect on mastitis, or containing Bacillus subtilis TTCC903 strain as an ingredient having an effect of improving the rearing environment of ruminants.

[0013] [Mastitis] Mastitis is broadly divided into "clinical mastitis" and "subclinical mastitis" based on its symptoms. Clinical mastitis is when abnormalities can be seen with the naked eye, such as redness and swelling of the breast, fever, pain, or the presence of particles in the milk. Clinical mastitis can be classified as either acute mastitis, when the breast suddenly becomes red and swollen and painful, or peracute mastitis, when these are accompanied by systemic symptoms such as difficulty standing and increased pulse and respiratory rate. In contrast, subclinical mastitis cannot be identified by examining the udder alone, but can be detected by testing the milk for an increased somatic cell count or bacterial infection. The incidence rate of subclinical mastitis is far higher, affecting approximately 25% to 50% of dairy cows. Clinical mastitis is easily cured with antibiotic treatment, but subclinical mastitis is difficult to treat and there is no effective treatment, so the only option is to take measures such as isolating infected animals.

[0014] The composition for oral administration of this embodiment is effective for both clinical mastitis and subclinical mastitis. In particular, the composition for oral administration of this embodiment is effective for subclinical mastitis.

[0015] In the present specification, the term "ruminant" refers to livestock animals such as cows, goats, and sheep. Of these, cows and goats are preferred because they are ruminants that secrete and produce milk. Preferred types of cows include dairy cows, specifically Holstein, Jersey, Guernsey, Ayrshire, Kerry, Dairy Shorthorn, Red Danish, Simmental, and the like. The composition for oral administration of this embodiment can be applied to normal dairy cows, dairy cows at risk of developing mastitis, dairy cows currently developing mastitis, and dairy cows with a history of mastitis, and can be used as a preventive agent for normal dairy cows, dairy cows at risk of developing mastitis, or normal dairy cows with a history of mastitis, or as a therapeutic agent for dairy cows currently developing mastitis.

[0016] [Bacillus subtilis TTCC903 strain] Bacillus subtilis TTCC903 strain (hereinafter, sometimes simply abbreviated as "TTCC903 strain") was deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on February 2, 2007 under the accession number FERM P-21173.

[0017] The TTCC903 strain can be cultured under the culture conditions for Bacillus subtilis commonly known to those skilled in the art, for example, in a medium containing at least a carbon source, a nitrogen source, and inorganic salts, and having a pH of usually 6.0 to 9.0, preferably 6.5 to 8.5, at a temperature of usually 20°C to 45°C, preferably 35°C to 42°C. Media used for culturing the TTCC903 strain include, for example, GSP medium (composition: sodium glutamate·1H2O: 15 g, sucrose: 30 g, Phytone: 15 g, KH2PO4: 2.5 g, Na2HPO4: 1.7 g, NaCl: 0.5 g, MgCl2·7H2O: 0.5 g, biotin: 100 μg, agar: 15 g, distilled water: 1000 mL). Other examples include LB medium, nutrient broth, or modified media thereof. The culture may be a shaking culture, an agitation culture, or a stationary culture, and may also be a batch culture, a semi-batch culture, or a continuous culture. Other culture conditions are not particularly limited as long as they allow the proliferation of Bacillus subtilis natto.

[0018] The TTCC903 strain may be used as a culture thereof as is, or as a dried powder, which can be obtained by separating or concentrating bacterial components by a separation means such as centrifugation or filtration, and then drying the components by a drying method such as freeze drying, vacuum heat drying, or spray drying.

[0019] The TTCC903 strain may be used in the form of a solid, semi-solid or liquid formulation. In the case of a solid, examples of the form include powder, granules, pills, pellets, tablets, capsules, and the like. In the case of a semi-solid, examples of the form include a gel. In the case of liquids, examples include the form of suspension, etc. Specifically, for example, a suspension in which dry powder of the TTCC903 strain is mixed with drinking water for ruminants can be ingested by ruminants.

[0020] As a culture of the TTCC903 strain, for example, natto inoculated with the TTCC903 strain and fermented can be used as it is, as shown in the Examples described below. In addition to the TTCC903 strain, the natto is rich in various nutritional components such as protein, vitamin B6, potassium, magnesium, iron, dietary fiber, vitamin E, calcium, and vitamin B2, and is therefore expected to have health-promoting effects and intestinal regulation effects, and can be preferably ingested by ruminants. In addition, the safety of natto is guaranteed by human dietary experience, and it can be continuously ingested by ruminants.

[0021] An example of a method for producing natto containing the TTCC903 strain will be described below. First, steamed soybeans are inoculated with the TTCC903 strain and fermented. The steamed soybeans can be prepared using a known method. The method for producing the steamed soybeans specifically includes a soaking step and a steaming step. In the soaking step, when the soybeans are whole soybeans, the soaking time can be, for example, 10 hours or more and 22 hours or less, and preferably 14 hours or more and 18 hours or less. When the soybeans are crushed soybeans, the soaking time can be, for example, 2 hours or more and 5 hours or less, and preferably 3 hours or more and 4 hours or less. In addition, the soaking temperature can be, for example, 15°C or more and 30°C or less, and preferably 18°C ​​or more and 22°C or less, in both the whole soybeans and crushed soybeans. In the steaming step, the steaming temperature can be, for example, about 100°C or more and 140°C or less, and the steaming time can be, for example, 5 minutes or more and 90 minutes or less. In addition, the pressure in the steaming step can be 0.02 MPa or more and 0.28 MPa or less. The steamed soybeans used in the fermentation process may be whole soybeans or cracked soybeans. Note that "whole soybeans" refers to soybeans that have not been cracked. In addition, the soybeans used in the fermentation process are not particularly limited, and may be domestically or overseas. In addition, a wide range of particle sizes are applicable, including large to very small, and are not limited. In addition, the soybeans are not limited to yellow soybeans, and colored beans such as black soybeans and blue soybeans are also applicable. In the manufacturing method of this embodiment, the beans used as the raw material for natto are not limited to soybeans (beans belonging to the Glycine max genus), but can be any beans that contain protein or protein and sugar and have stringy properties due to fermentation by Bacillus subtilis natto. Specific examples of such beans include beans belonging to the Phaseolus genus, such as white gold beans, red kidney beans, pinto beans, tiger beans, and white kidney beans.

[0022] As for fermentation conditions, when the steamed soybeans are crushed soybeans, the fermentation temperature can be 37° C. to 47° C., preferably 38° C. to 44° C. The fermentation time can usually be about 5 hours to 15 hours. On the other hand, when the steamed soybeans are whole soybeans, the fermentation temperature can be from 37° C. to 47° C., preferably from 38° C. to 44° C. The fermentation time can usually be from 7 hours to 20 hours. In addition, fermentation can be terminated by lowering the temperature to below 10°C. The additives to be mixed during the production of natto and other production conditions conform to known methods for producing natto.

[0023] A mutant strain of the TTCC903 strain that has a preventive or therapeutic effect on ruminant mastitis can also be used. The mutant strain of the TTCC903 strain can be obtained, for example, by the following method. First, mutations are artificially induced by culturing under irradiation of high-energy rays such as ultraviolet rays or X-rays, or in the presence of mutagens such as nitrosoguanidine, N-ethyl-N-nitrosourea, or nitrosamines, and a mutant colony is isolated. Next, the mutant is tested for being a mutant by, for example, comparing the base sequence of the rDNA (usually used for bacterial classification) of the mutant with the base sequence of the TTCC903 strain. Next, the mutant is fed to ruminants to confirm that it has a preventive or therapeutic effect on mastitis in ruminants.

[0024] [Intake Amount] The intake amount of the composition for oral administration of this embodiment is 2×10 per kg of body weight of a ruminant (particularly, a cow) per day in terms of the amount of the TTCC903 strain cells. 6 pcs / kg or more 2×10 8 pcs / kg or less, preferably 5×10 6 pcs / kg or more 1×10 8 pcs / kg or less, more preferably 9×10 6 pieces / kg or more 9×10 7The composition for oral administration of this embodiment can be ingested by a ruminant in an amount of not more than 1 / kg once or in several doses. The composition for oral administration of this embodiment can be ingested alone, or can be mixed with a drink, feed, or the like so as to obtain the above-mentioned dose. For cows such as dairy cows, roughage such as hay or concentrated feed is often given as feed, and it is preferable to mix the composition for oral administration of this embodiment with such feed. The composition for oral administration of this embodiment can be ingested at a fixed time every day, or can be ingested ad libitum.

[0025] In the composition for oral administration of this embodiment, the content of the TTCC903 strain is, in terms of the amount of bacterial cells, for example, 1 × 10 4 pcs / g or more 1×10 10 It is possible to keep the number of particles to about 1 / g or less.

[0026] According to the composition for oral administration of this embodiment, as shown in the examples described later, it is possible to effectively suppress the onset of mastitis in ruminants (particularly dairy cows). The onset of mastitis in ruminants can be determined, for example, by measuring the number of somatic cells in the milk of ruminants.

[0027] The "somatic cells" referred to here are broadly divided into mammary epithelial cells and white blood cells, and are considered an indicator of mastitis. Mammary epithelial cells are responsible for milk production, but as the number of cows produces milk, they fall off from the mammary tissue and become visible in the milk. They account for about 2% of all somatic cells, and their number does not increase due to mastitis. In contrast, white blood cells are cells that are originally produced in the bone (bone marrow) and arrive at the breast via the blood. They play a role in processing bacteria, and even in normal milk, there are between tens of thousands and 100,000 in 1 mL. Once white blood cells that are naturally present in the breast find bacteria, they send a signal to the blood to call in a large number of white blood cells from the blood. If there are no bacteria, white blood cells are merely sentinels, but in the presence of bacteria, white blood cells become extremely energetic. White blood cells process bacteria to protect the mammary glands, but they can also damage some of the mammary glands and greatly inhibit their function.

[0028] <Feed additives> A feed additive according to one embodiment of the present invention (hereinafter, sometimes simply referred to as the "feed additive of this embodiment") contains the above-mentioned composition for oral administration. The feed additive of this embodiment assists in the prevention or treatment of mastitis in ruminants.

[0029] The feed additive of this embodiment can be produced by mixing the above-mentioned composition for oral administration with any other approved components constituting the feed additive so as to contain an effective amount of the TTCC903 strain for preventing or treating mastitis in ruminants, where the effective amount of the TTCC903 strain is the dose of the TTCC903 strain described above. Specifically, in the feed additive of this embodiment, the composition for oral administration can be blended so that the content of the composition for oral administration is, for example, 1% by mass or more and 99% by mass or less, preferably 5% by mass or more and 95% by mass or less, and more preferably 10% by mass or more and 90% by mass or less, relative to the total mass of the feed additive.

[0030] Generally, a "feed additive" is a substance added to animal feed to impart a desired function. The function imparted by the feed additive of the present embodiment is a function of preventing or treating mastitis in ruminants (particularly, cows), or a function of suppressing the onset of mastitis or alleviating the symptoms.

[0031] The feed additive of this embodiment may further contain an antibiotic for treating mastitis. As the antibiotic, the same ones as those exemplified in the pharmaceutical composition described below can be used.

[0032] The feed additive of this embodiment may contain, in addition to the active ingredient, for example, a carrier (excipient or diluent), additives, etc. for forming the agent.

[0033] The feed additive of this embodiment may be in any form of solid, semi-solid or liquid, preferably in solid form, and specific examples thereof include powder, granules, pills, pellets, tablets, capsules, etc. The form of the composition for oral administration can be selected and blended according to the form of the feed additive of this embodiment.

[0034] Conventionally, mastitis treatment agents listed in the "Livestock Mutual Aid Drug Price Standard Table by Efficacy" (Japan Animal Pharmaceuticals Association) have been used to treat mastitis in cows, etc., and antibiotics have been used as the main treatment agent, but they have not yet been able to prevent recurrence. The above-mentioned composition for oral administration and the feed additive containing it can complement antibiotic treatment.

[0035] <Feed> A feed additive according to one embodiment of the present invention (hereinafter, sometimes simply referred to as "the feed of this embodiment") contains the composition for oral administration. The feed of this embodiment assists in the prevention or treatment of mastitis in ruminants.

[0036] The feed of the present embodiment is different from known feeds in that it contains an effective amount of the TTCC903 strain for preventing or treating mastitis in ruminants, where the effective amount of the TTCC903 strain is the dose of the TTCC903 strain described above.

[0037] The feed of this embodiment can be produced by mixing the above-mentioned oral administration composition with known components that constitute the feed so as to contain an effective amount of the TTCC903 strain for preventing or treating mastitis in ruminants. Specifically, in the feed of this embodiment, the composition for oral administration can be blended so that the content of the composition for oral administration is, for example, 0.03% by mass or more and 0.5% by mass or less, preferably 0.05% by mass or more and 0.4% by mass or less, and more preferably 0.1% by mass or more and 0.3% by mass or less, relative to the total mass of the feed.

[0038] Examples of known components of feed include roughage mainly composed of fiber (e.g., green grass, hay, silage rice straw, etc.); concentrated feed (also called compound feed) containing a lot of starch and protein (e.g., grains such as corn, soybean, rice bran, bran, barley, milo, etc.; vegetable oil cakes such as soybean oil cake, rapeseed oil cake, cottonseed oil cake, etc.). In addition, the feed of this embodiment mainly contains vitamins (vitamin A, D3, E, B1, B2, B6, B12, C, K3, inositol, biotin, pantothenic acid, niacin, choline, folic acid, etc.), amino acids, salt, minerals, etc. As for the components of the feed, for example, those listed in the Japanese Standard Feed Composition Table (edited by the National Agriculture and Food Research Organization, National Institute) can also be used.

[0039] <Pharmaceutical Composition> A pharmaceutical composition according to one embodiment of the present invention (hereinafter sometimes simply abbreviated as "the pharmaceutical composition of this embodiment") contains the above-mentioned composition for oral administration.

[0040] According to the pharmaceutical composition of this embodiment, the onset of mastitis in ruminants can be significantly suppressed, and also, in ruminants currently developing mastitis, mastitis can be treated or symptoms can be alleviated.

[0041] In the pharmaceutical composition of this embodiment, the content of the TTCC903 strain can be formulated to achieve the dose of the TTCC903 strain described above. Specifically, in the pharmaceutical composition of this embodiment, the composition for oral administration can be blended so that the content of the composition for oral administration is, for example, 1% by mass or more and 99% by mass or less, preferably 5% by mass or more and 95% by mass or less, and more preferably 10% by mass or more and 90% by mass or less, relative to the total mass of the pharmaceutical composition.

[0042] [Other ingredients] The pharmaceutical composition of the present embodiment may further contain a mastitis therapeutic agent (antibiotics, etc.) in some cases. Examples of mastitis therapeutic agents include antibiotics that are effective against bacteria such as Staphylococcus aureus, Escherichia coli, Streptococcus, and Pseudomonas aeruginosa, which are known to cause mastitis and are listed in the "Livestock Mutual Aid Drug Price Standard Table by Efficacy 2011 Edition" (Japan Animal Pharmaceutical Association), specifically, antibiotics such as cefam antibiotics, penicillin antibiotics, and macrolide antibiotics, more specifically, erythromycin, oxytetracycline hydrochloride, dicloxacillin sodium, cefazolin, cefuroxime sodium, nafcillin sodium, benzylpenicillin procaine, dihydrostreptomycin sulfate, kanamycin sulfate, cephapirin sodium, monoammonium glycyrrhizinate, and cephalonium. These antibiotics can be used alone or in a mixture of multiple types.

[0043] Furthermore, the pharmaceutical composition of this embodiment may contain, in addition to the TTCC903 strain as an active ingredient, for example, a carrier (e.g., an excipient, a diluent, etc.) and, in some cases, an additive for formulating the composition into a dosage form.

[0044] The additives are not limited to those listed below, but may be, for example, additives generally used in pharmaceutical compositions for ruminants, such as binders, disintegrants, lubricants, surfactants, antioxidants, fungicides, and thickeners.

[0045] In the pharmaceutical composition of this embodiment, the contents of other components can be appropriately determined by those skilled in the art based on the form, etc.

[0046] <Methods for preventing and treating mastitis in ruminants> A method for preventing mastitis in a ruminant according to one embodiment of the present invention (the prevention method of this embodiment) is a method in which a ruminant ingests the above-mentioned composition for oral administration. A method for treating mastitis in a ruminant according to one embodiment of the present invention (the treatment method of this embodiment) is a method in which the above-mentioned composition for oral administration is ingested by a ruminant. Hereinafter, the preventive method of this embodiment and the therapeutic method of this embodiment may be collectively abbreviated as the preventive or therapeutic method of this embodiment.

[0047] The dosage of the composition for oral administration is the same as the dosage exemplified for the TTCC903 strain.

[0048] The method of ingestion is such that the amount of the TTCC903 strain cells is the above-mentioned dose, either once or in several doses per day. The composition for oral administration may be ingested alone, or the composition for oral administration may be mixed with food such as beverages or feed and ingested. For cows such as dairy cows, roughage such as hay or concentrated feed is often ingested as feed, and it is preferable to mix the composition for oral administration with such feed.

[0049] In the prevention or treatment method of this embodiment, when the subject is a ruminant suffering from mastitis, in addition to taking the composition for oral administration, if necessary, a mastitis treatment agent (antibiotics, etc.) that has been conventionally used for treatment can be used in combination, which is expected to increase the rate of complete cure of mastitis or shorten the period until complete cure of mastitis. An antibiotic is administered to a ruminant suffering from mastitis by a known method such as intravenous administration, intramuscular administration, or oral administration, while the composition for oral administration of this embodiment can be ingested by the ruminant before, during, or after administration of the antibiotic.

[0050] <Methods for improving the rearing environment for ruminants> The above-mentioned composition for oral administration can also be used to improve the rearing environment of ruminants. That is, a method for improving the rearing environment of ruminants according to one embodiment of the present invention (the improvement method of this embodiment) is a method for having ruminants ingest a composition for oral administration containing Bacillus subtilis TTCC903 strain (accession number FERM P-21173) as an active ingredient.

[0051] As shown in the examples described below, by continuously feeding the above-mentioned oral administration composition to ruminants, the generation of various odorous substances can be effectively suppressed, thereby improving the rearing environment of ruminants. In addition, the "improvement of the rearing environment" here includes the reduction of the amount of specific odorous substances in the wind of buildings where ruminants are reared, and the reduction of odorous substances derived from ruminant excrement. Examples of specific odorous substances include, but are not limited to, 22 substances (ammonia, methyl mercaptan, hydrogen sulfide, methyl sulfide, methyl disulfide, trimethylamine, acetaldehyde, propionaldehyde, normal butyraldehyde, isobutyraldehyde, normal valeraldehyde, isovaleraldehyde, isobutanol, ethyl acetate, methyl isobutyl ketone, toluene, styrene, xylene, propionic acid, normal butyric acid, normal valeric acid, and isovaleric acid) designated as specific odorous substances in the Offensive Odor Prevention Law and for which regulated standard concentrations are set. In addition, examples of odorous substances derived from ruminant excrement include indole, skatole, methane, phenol, p-cresol, and 4-ethylphenol. The amount of reduction depends on the type of specific malodorous substance or odorous substance, but specifically, the amount of the specific malodorous substance present in a rearing environment in which the above-mentioned oral administration composition is ingested is preferably reduced to 90% or less of the amount of the specific malodorous substance present in a rearing environment in which the above-mentioned oral administration composition is not ingested, more preferably reduced to 70% or less, even more preferably reduced to 50% or less, particularly preferably reduced to 30% or less, and most preferably reduced to 10% or less. That is, the improvement method of the present embodiment can be said to be a method for suppressing or reducing the generation of malodorous substances in the rearing environment of ruminants. The malodorous substances referred to here include the above-mentioned specific malodorous substances and odorous substances derived from the excrement of ruminants.

[0052] The dosage of the composition for oral administration is the same as the dosage exemplified for the TTCC903 strain.

[0053] The method of ingestion is such that the amount of the TTCC903 strain cells is the above-mentioned dose, either once or in several doses per day. The composition for oral administration may be ingested alone, or the composition for oral administration may be mixed with food such as beverages or feed and ingested. For cows such as dairy cows, roughage such as hay or concentrated feed is often ingested as feed, and it is preferable to mix the composition for oral administration with such feed. EXAMPLES

[0054] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0055] [Example 1] 1. Types of cattle used in the study All the cows used in the test were Holsteins. A test of feeding natto to a total of 120 cows was conducted at the following three farms, A to C. Farm A (Kasumigaura City, Ibaraki Prefecture): 60 head B Farm (Ishioka City, Ibaraki Prefecture): 40 head C Farm (Hokota City, Ibaraki Prefecture): 20 head

[0056] In addition, the trends in the average parity of cows (average number of births per cow) (unit: head) at each farm are shown in Table 1 below.

[0057] [Table 1]

[0058] As shown in Table 1, each farm had a rapid replacement rate of dairy cows and there was no significant difference in average calving numbers.

[0059] 2. Manufacturing of natto for feed (TTCC903 natto) Bacillus subtilis TTCC903 strain was inoculated into boiled soybeans. Then, to produce three different masses of natto, 1 kg, 2 kg, and 3 kg, plastic bags of the appropriate size for each mass were prepared as shown below. For 1kg: 360mm x 500mm (no gusset) For 2kg: 460mm x 600mm (without gusset) For 3kg: 650mm x 800mm (no gusset)

[0060] Next, boiled soybeans were placed in each plastic bag and spread evenly so that the thickness of the plastic bag was within 3 cm. Next, small holes were evenly made on the surface of the plastic bag for fermentation, and the bag was placed in an incubator to promote fermentation. After confirming that fermentation was complete, the bag was refrigerated until use.

[0061] 3. Natto feeding method and feeding schedule At three farms, A to C, each cow was given 50 g of TTCC903 strain natto (TTCC903 strain bacterial count in natto: 1 × 10 9 Natto was fed raw (approximately 100 pieces / g). A refrigerator was installed at each farm to prevent deterioration of the quality of the natto. Inventory was managed so that the natto could be fed within 30 days of production. The detailed feeding method and feeding schedule for natto at each farm are shown below.

[0062] (1) A Farm Test start date: February 1, 2019 Natto feeding time: Ad libitum feeding (new feed was given at 4:00 pm in the evening and 6:00 am in the morning) Feeding method: Feed was prepared once a day using a TMR (Total Mixed Ration) mixer and fed to the cattle as TMR. Specifically, when various simple feeds were put into the TMR mixer, 3 kg (0.11% by mass) of TTCC903 natto was added simultaneously for a total mass of 2700 kg of feed and stirred until it was uniformly mixed. In order to confirm that the feed was mixed uniformly, samples were taken from several places after mixing to check the number of natto bacteria and confirm that there was no variation.

[0063] (2)B Ranch Test start date: February 18, 2019 Natto feeding time: once a day at 5pm Feeding method: Each single ingredient was fed to each individual cow. TTCC903 Natto was fed to each individual cow by mixing 2 kg (29% by mass) of TTCC903 Natto with the pelleted additive for a total mass of 5 kg of additive and natto. After feeding, it was confirmed that the cows had eaten all the food.

[0064] (3)C Ranch Test start date: February 18, 2019 Natto feeding time: once a day at 5pm Feeding method: Each animal was fed a single ingredient. TTCC903 Natto was fed by itself. 50g of TTCC903 Natto was placed directly in the feed trough, and after feeding, it was confirmed that the cow had eaten it all.

[0065] 4. Measurement of Somatic Cell Count The number of somatic cells in milk squeezed each month at each farm was measured at the request of the Kanto Raw Milk Sales Agricultural Cooperative Association. Samples were collected by thoroughly stirring the tank (bulk milk) containing the milk of all dairy cows at each farm, and then collecting a portion of the milk to use as a sample for measuring the somatic cell count. Surprise inspections were conducted three times a month. Figure 1 shows the annual trends in the weighted average value of somatic cell counts at the three farms in 2019 and 2020. Figure 1 also shows the annual trends in the weighted average value of somatic cell counts at the three farms for the past five years (from 2014 to 2018) when TTCC903 natto was not fed as a control.

[0066] Figure 1 shows that the somatic cell counts in 2019 and 2020 when TTCC903 natto was fed were lower than those in the past five years when TTCC903 natto was not fed. The decrease in somatic cell counts was particularly noticeable during the high temperature period from June to September. In addition, there was a tendency for the somatic cell count to increase slightly from December 2019 to January to March 2020 compared to the somatic cell count in the past five years. This is thought to be due to the damage to cows caused by rising temperatures. Specifically, the average temperature and the average minimum temperature in August to October 2019 tended to be 1 to 2 degrees Celsius higher than in the past five years. Cows are resistant to the cold, but their thickness is very weak and they are easily damaged. Therefore, it is presumed that the damage suffered during the day could not be recovered when the temperature dropped at night. It is usually known that the effects of these damages become apparent several months after they occur in dairy cows. Therefore, it is thought that the damage caused by the rise in temperature in August to October 2019 became apparent from December 2019 to January to March 2020.

[0067] In addition, in the data on somatic cell counts for each farm, cows with milking days of 45 days or less were considered to be in early lactation, and the percentage of individuals with a somatic cell linear score of 4.5 or more in that early lactation stage was analyzed. A "somatic cell linear score of 4.5 or more" is usually the standard value for determining mastitis. The results are shown in Table 2 and Figure 2 below.

[0068] [Table 2]

[0069] As shown in Table 2 and Figure 2, the percentage of cows with a somatic cell linear score of 4.5 or more in early lactation decreased after the start of feeding TTCC903 natto compared to before the start of feeding TTCC903 natto in all farms. In other words, it was confirmed that feeding TTCC903 natto suppressed the onset of mastitis.

[0070] 5. Measurement of milk yield The amount of milk extracted each month was measured at each farm. Figure 3 shows the annual trends in the weighted average milk yields at the three farms in 2019 and 2020. In Figure 3, the weighted average milk yields at the three farms for each month over the past five years (from 2014 to 2018) are shown as relative values ​​converted to 100.

[0071] Figure 3 shows that milk yields in 2019 and 2020 when TTCC903 natto was fed were significantly higher than those in the past five years when TTCC903 natto was not fed.

[0072] [Example 2] In Example 1, several people commented that the odor (bad odor) in the rearing environment of each of the three farms that fed TTCC903 natto decreased over time. Therefore, a new farm was selected and a measurement test of the odorous substances was conducted to see if there was any change in the amount of odorous substances present.

[0073] 1. Types of cattle used in the study All the cows used in the test were Holsteins. A test of feeding natto to a total of 140 cows was conducted at the four farms D to F shown below. D Farm (Ibaraki Town, Ibaraki Prefecture): 30 head E Farm (Hokota City, Ibaraki Prefecture): 40 head F Farm (Ishioka City, Ibaraki Prefecture): 70 head

[0074] 2. Manufacturing of natto for feed (TTCC903 natto) Bacillus subtilis TTCC903 strain was inoculated into boiled soybeans. Then, to produce three different masses of natto, 1 kg, 2 kg, and 3 kg, plastic bags of the appropriate size for each mass were prepared as shown below. For 1kg: 360mm x 500mm (no gusset) For 2kg: 460mm x 600mm (without gusset) For 3kg: 650mm x 800mm (no gusset)

[0075] Next, boiled soybeans were placed in each plastic bag and spread evenly so that the thickness of the plastic bag was within 3 cm. Next, small holes were evenly made on the surface of the plastic bag for fermentation, and the bag was placed in an incubator to promote fermentation. After confirming that fermentation was complete, the bag was refrigerated until use.

[0076] 3. Natto feeding method and feeding schedule At three farms (D-F), each cow was given 50g of TTCC903 strain natto (TTCC903 strain bacterial count in natto: 1×10 9 Natto was fed raw (approximately 100 pieces / g). A refrigerator was installed at each farm to prevent deterioration of the quality of the natto. Inventory was managed so that the natto could be fed within 30 days of production. The detailed feeding method and feeding schedule for natto at each farm are shown below.

[0077] (1)D Ranch Test start date: April 7, 2020 Natto feeding time: Ad libitum feeding (new feed was given at 4:00 pm in the evening and 6:00 am in the morning) Feeding method: Feed was prepared once a day using a TMR mixer and fed to the cows as TMR. Specifically, when various plain feeds were put into the TMR mixer, 1.7 kg (0.12% by mass) of TTCC903 natto was added at the same time for a total mass of 1400 kg of feed, and the mixture was stirred until it was uniformly mixed.

[0078] (2)E Ranch Test start date: April 7, 2020 Natto feeding time: feeding ad libitum (new feed was given at 5pm in the evening and 7am in the morning) Feeding method: Feed was prepared once a day using a TMR mixer and fed to the cows as TMR. Specifically, when various plain feeds were put into the TMR mixer, 2.5 kg (0.19% by mass) of TTCC903 natto was added at the same time for a total mass of 1300 kg of feed, and the mixture was stirred until it was uniformly mixed.

[0079] (3) F Ranch Test start date: April 11, 2020 Natto feeding time: feeding ad libitum (new feed was given at 5pm in the evening and 7am in the morning) Feeding method: Feed was prepared once a day using a TMR mixer and fed to the cows as TMR. Specifically, when various plain feeds were put into the TMR mixer, 3.5 kg (0.15% by mass) of TTCC903 natto was added at the same time for a total mass of 2400 kg of feed, and the mixture was stirred until it was uniformly mixed.

[0080] 4. Measurement of odorants The odor concentration downwind from the building was measured for 22 types of "specific odorous substances" regulated by the Offensive Odor Prevention Law, based on the measurement method specified in the Environment Agency Notification No. 9 of 1972. The amount of putrefactive matter in the cow dung (amount of phenol, p-cresol, 4-ethylphenol, indole, and skatole) was measured using solvent extraction and GC-MS. In addition, the odor index was measured based on the measurement method specified in the Environment Agency Notification No. 63 of 1995. The odor index measurement was carried out from June. These measurements were carried out once every two months. However, for the above 22 types of "specific odorous substances," items that were not detected multiple times (below the measurement limit) were not measured from the next time onwards. The measurement results are shown in Tables 3 to 5 below. In Tables 3 to 5, "-" indicates that no measurement was carried out, and "ND" indicates that the measurement was below the lower limit of quantification.

[0081] [Table 3]

[0082] [Table 4]

[0083] [Table 5]

[0084] From Tables 3 to 5, it is clear that feeding TTCC903 natto reduces the odor index over time. At Farm F, the odor index increased in August compared to June, but in August, when the temperature and humidity are favorable conditions for the generation of bad odors, the increase in the odor index from June was only 5, and the total was kept to 17, below 20. It is presumed that this is because feeding TTCC903 natto suppressed the generation of bad odors. In addition, compared to the April measurement results, even in June and August, when no changes were observed in the concentration of specific odorous substances in the air or the odor index, it was confirmed that the various odorous substances contained in cow dung were decreasing over time.

[0085] 5. Confirmation of the presence of Bacillus subtilis TTCC903 strain in the environment Next, we confirmed whether the TTCC903 strain was present in the environment at each farm where TTCC903 natto was fed. Specifically, samples taken from the bodies and bedding of cows at each farm were diluted with saline to 1:10 or more. 2 The diluted solution was diluted to about 1:1000 and heated at 72°C for 15 minutes, then applied to a viscous medium and cultured overnight. The viscous medium used was GSP medium (composition: 15g sodium glutamate·1H2O, 30g saccharose, 15g Phytone, 2.5g KH2PO4, 1.7g Na2HPO4, 0.5g NaCl, 0.5g MgCl2·7H2O, 100μg biotin, 15g agar, 1000mL distilled water) (Reference 1: Patent Publication No. 4134254). The next day, one to three colonies that were identified as natto bacteria by visual observation were selected from each sample, and these were further streaked and cultured on a viscous medium. The next day, colony PCR was performed on each colony by a known method. As a positive control, PCR was performed on the genomic DNA of the TTCC903 strain and other natto strains using primers designed from the sequences of those genomic DNAs. Each PCR product was subjected to agarose gel electrophoresis to confirm the bands. The results are shown in Figure 4.

[0086] As shown in Figure 4, the presence of the TTCC903 strain was confirmed in most farms. This suggests that the presence of the TTCC903 strain in the rearing environment suppresses the generation of malodorous substances. [Industrial Applicability]

[0087] According to the composition for oral administration of the present embodiment, it is possible to provide a composition for oral administration that is effective for preventing or treating mastitis in ruminants. According to the improvement method of the above aspect, it is possible to effectively improve the rearing environment of ruminants. [Accession number]

[0088] "Bacillus subtilis TTCC903 strain" was deposited on February 2, 2007 with the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under the accession number FERM P-21173.

Claims

1. A composition for oral administration for preventing or treating mastitis in ruminants, comprising Bacillus subtilis strain TTCC903 (accession number FERM P-21173) as an active ingredient.

2. The composition for oral administration according to claim 1 , wherein the ruminant is a cow.

3. The composition for oral administration according to claim 1 , which is natto.

4. A feed additive for preventing or treating mastitis in ruminants, comprising the composition for oral administration according to any one of claims 1 to 3.

5. A feed for preventing or treating mastitis in ruminants, comprising the composition for oral administration according to any one of claims 1 to 3.

6. A pharmaceutical composition for preventing or treating mastitis in ruminants, comprising the composition for oral administration according to any one of claims 1 to 3.

7. A method for preventing mastitis in ruminants, comprising administering the composition for oral administration according to any one of claims 1 to 3 to a ruminant.

8. A method for treating mastitis in a ruminant, comprising administering to the ruminant the composition for oral administration according to any one of claims 1 to 3.

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