WO2010071222A1 - Methane emission inhibitor for ruminants and feed composition - Google Patents
Methane emission inhibitor for ruminants and feed composition Download PDFInfo
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- WO2010071222A1 WO2010071222A1 PCT/JP2009/071362 JP2009071362W WO2010071222A1 WO 2010071222 A1 WO2010071222 A1 WO 2010071222A1 JP 2009071362 W JP2009071362 W JP 2009071362W WO 2010071222 A1 WO2010071222 A1 WO 2010071222A1
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- ruminant
- methane
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
Definitions
- the present invention relates to a methane production inhibitor, a feed additive and a feed composition for ruminants, and a method for inhibiting ruminant methane production and a method for improving growth, characterized by using these.
- Methane generation from ruminant rumen not only results in a loss of feed energy use efficiency, but methane is a powerful greenhouse gas that contributes to global warming, reducing methane production in ruminant rumen Is extremely important. In the lumen, fermentation by a wide variety of microorganisms is performed, and various metabolites are generated. Methane is one of them, and is thought to be produced by methanogenic bacteria.
- the rumen methane-producing bacteria are hydrogen-utilizing bacteria that use hydrogen to reduce carbon dioxide and produce methane. Therefore, if there is a stronger reduction reaction than this, methane production is inhibited.
- Patent Document 1 As a method for suppressing methane generation in the rumen, a method of feeding ruminants with an ionophore, which is an antibacterial substance such as monensin or ivelin, is known.
- methods that pay attention to the regulation of reducing ability in rumen include a method of feeding cysteine (Patent Document 3), a method of feeding fumaric acid (Patent Documents 2 and 3), and the like.
- Addition of nitrate has also been reported to be effective, but toxic nitrite accumulates in the rumen, which is known to cause ruminant poisoning in ruminants. Studies are also underway on the antibacterial bacteriocin produced by microorganisms.
- Non-patent Document 4 Although there is a report that uses nisin produced by lactic acid bacteria alone (Patent Document 4), it is reported that nisin has no sustained effect because it is easily degraded in rumen, and nisin-resistant bacteria also appear (Non-patent Document). 1). Moreover, although there exists a method of improving feed efficiency by using the preparation of sorbic acid and bacteriocin together (patent document 5), it is not clarified about suppression of methane generation when added as ruminant feed.
- the present invention provides a methane production inhibitor that suppresses methane generation in rumen of ruminants and improves feed efficiency, feed additives and feed compositions containing the same, and feeds these to ruminants to produce methane.
- An object is to provide a method for inhibiting production and thus improving its growth.
- a ruminant characterized by administering the ruminant feed additive and / or ruminant feed composition according to (2) to the ruminant and / or the ruminant feed additive and / or ruminant feed composition according to (2).
- a method for suppressing methane generation from animal rumen (4)
- a ruminant characterized by administering the ruminant feed additive and / or ruminant feed composition according to (2) to the ruminant and / or the ruminant feed additive and / or ruminant feed composition according to (2). Animal growth improvement method.
- methane production in rumen rumen is significantly suppressed, the energy efficiency of the feed is improved, and the development of the ruminant is improved. It can also contribute to environmental problems such as global warming by reducing the production of methane, a greenhouse gas. Confirmation of the effect of suppressing the methane production in the lumen by the administration of the methane production inhibitor of the present invention was confirmed by an artificial rumen system (T. Hino et al., J. Gen. Appl. Microbiol., 39, 35-45 ( 1993)), but it goes without saying that it may be confirmed by oral administration to ruminants in vivo.
- FIG. 1 is an explanatory diagram showing the relationship between the concentration of hydrogen peroxide and the methane production inhibitory activity.
- FIG. 2 is an explanatory diagram showing the relationship between the concentration of hydrogen peroxide and the amount of hydrogen produced.
- the methane production inhibitor of the present invention is a ruminant methane production inhibitor characterized by containing hydrogen peroxide as an active ingredient.
- the ruminant in the present invention refers to a mammal belonging to the order of the artiodactylid ruminant, and refers to one in which the stomach is divided into 3 or 4 chambers and ruins food. Examples include cows and sheep.
- the active substance is hydrogen peroxide, which is highly safe when used at an appropriate concentration, and is preferable from the viewpoint of the health of ruminants. Moreover, since it is rapidly decomposed into water and oxygen, there is no residue in ruminant meat or milk.
- Hydrogen peroxide used as an active ingredient in the methane inhibitor of the present invention can be easily obtained commercially, for example, purchased from Takasugi Pharmaceutical, Naito Shoten, Nippon Peroxide, Wako Pure Chemical, etc. I can do it.
- cultures such as microorganisms that produce hydrogen peroxide, such as lactic acid bacteria can also be used as the hydrogen peroxide source. .
- the timing of administration of the methane production inhibitor is not particularly limited as long as the methane production inhibitory effect of the present invention is exhibited, and can be administered at any time as long as the feed remains in the lumen. However, since it is preferable that a methane formation inhibitor is present in the lumen before and after methane is most actively produced, it is preferable to administer the methane production inhibitor immediately before or simultaneously with the administration of feed.
- the ruminant methane production inhibitor according to the present invention can be used in various forms, and is not particularly limited as long as the methane production inhibitory effect is exerted. For example, an aqueous solution, a capsule, or a feed addition containing these Various forms such as food and feed composition.
- the dosage of the methane production inhibitor of the present invention is such that the hydrogen peroxide concentration in the rumen solution immediately after administration is at least 0.001 to 10 mM, more preferably 0.01 to 5 mM. To do. Since hydrogen peroxide as an active ingredient decomposes rapidly after administration, it may be administered multiple times a day as long as the concentration is within the above range. By administering at such a dose, methane bacteria are preferentially sterilized or bacteriostatic without adversely affecting useful rumen microorganisms such as fibrinolytic bacteria.
- the present invention will be further described by examples. Needless to say, the present invention is not limited to these examples, and can be arbitrarily changed without departing from the gist of the present invention.
- the methane production inhibitory activity of the methane production inhibitor can be confirmed by an activity measurement method described later. Rumen fermentation simplified evaluation method ⁇ Materials and experimental method> Rumen fluid collected from cattle raised mainly in hay before feeding was used. (In this experiment, Holstein cattle were used. However, the type of cattle, the type of feed, and the method for collecting rumen fluid are commonly used in this field. As long as the law does not limit).
- Example 1 Hydrogen peroxide concentration-dependent methane production inhibitory effect A 30-35% aqueous hydrogen peroxide solution (Wako Pure Chemical Industries, Ltd.) whose concentration was accurately analyzed was diluted with physiological saline, and the hydrogen peroxide concentration was reduced to 0. 0. 0. Sample series having 1, 0.3, 0.5, 0.7, 0.9, 1.1, 1.3, 1.5, 3.0, 5.0, 10.0 mM were prepared. These samples were subjected to the rumen fermentation simplified evaluation method. In this study, independent tests were performed three times, and the average value and standard error are shown in FIGS.
- the horizontal axis indicates the hydrogen peroxide sample concentration (mM), and the vertical axis indicates the relative amount of methane generated in each sample administration group when the negative control (hydrogen peroxide sample concentration 0 mM) is 100. Is shown.
- the bar graph shows the average value of three independent experiments, and the error bar shows the standard error obtained from the three experiments.
- the horizontal axis indicates the hydrogen peroxide sample concentration (mM), and the vertical axis indicates the amount of hydrogen generated when the H 2 O 2 concentration is 1.1 mM in each sample administration group. The amount of hydrogen generation is shown as a relative value.
- the bar graph shows the average value of three independent experiments, and the error bar shows the standard error obtained from the three experiments.
- methane which is a powerful greenhouse gas emitted from ruminants. It is extremely useful because it improves growth.
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Abstract
Description
本発明は反芻動物用のメタン生成抑制剤、飼料添加物および飼料組成物、ならびにこれらを使用することを特徴とする反芻動物のメタン生成抑制方法および発育改善方法に関する。 The present invention relates to a methane production inhibitor, a feed additive and a feed composition for ruminants, and a method for inhibiting ruminant methane production and a method for improving growth, characterized by using these.
反芻動物のルーメンからのメタン発生は、飼料のエネルギー利用効率の損失となるのみでなく、メタンは地球温暖化現象に寄与する強力な温室効果ガスであり、反芻動物のルーメンにおけるメタン生成を減らすことは極めて重要である。
ルーメン内では多種多様の微生物による発酵が行われており、種々の代謝産物が生成される。メタンもその１つで、メタン生成細菌により生成すると考えられている。ルーメン内メタン生成細菌は水素資化菌で、水素を利用して二酸化炭素を還元し、メタンを生成する。従って、これより強力な還元反応が存在すれば、メタン生成は阻害される。（特許文献１等）
ルーメン内のメタン発生を抑制する方法としては、反芻動物にモネンシンやアイベリンといった抗菌物質であるイオノフォアを給与する方法が知られている。一方、ルーメンでの還元能の調節に着目した方法として、システインを給与する方法（特許文献３）、フマル酸を給与する方法（特許文献２および３）などが知られている。また、硝酸塩を添加することも効果があると報告されているが、ルーメン内に毒性のある亜硝酸が蓄積するため、反芻動物の亜硝酸中毒を引き起こすことも知られている。
微生物の産出する抗菌物質バクテリオシンについても検討が実施されている。乳酸菌の産出するナイシンを単独で用いる報告もあるが（特許文献４）、ナイシンはルーメン中で容易に分解されるため持続効果がなく、またナイシン耐性菌も出現すると報告されている（非特許文献１）。また、ソルビン酸とバクテリオシンの調製物を併用することで飼料効率を改善する方法もあるが（特許文献５）、反芻動物の飼料として添加した場合のメタン発生抑制については明らかにされていない。
Methane generation from ruminant rumen not only results in a loss of feed energy use efficiency, but methane is a powerful greenhouse gas that contributes to global warming, reducing methane production in ruminant rumen Is extremely important.
In the lumen, fermentation by a wide variety of microorganisms is performed, and various metabolites are generated. Methane is one of them, and is thought to be produced by methanogenic bacteria. The rumen methane-producing bacteria are hydrogen-utilizing bacteria that use hydrogen to reduce carbon dioxide and produce methane. Therefore, if there is a stronger reduction reaction than this, methane production is inhibited. (Patent Document 1 etc.)
As a method for suppressing methane generation in the rumen, a method of feeding ruminants with an ionophore, which is an antibacterial substance such as monensin or ivelin, is known. On the other hand, methods that pay attention to the regulation of reducing ability in rumen include a method of feeding cysteine (Patent Document 3), a method of feeding fumaric acid (
Studies are also underway on the antibacterial bacteriocin produced by microorganisms. Although there is a report that uses nisin produced by lactic acid bacteria alone (Patent Document 4), it is reported that nisin has no sustained effect because it is easily degraded in rumen, and nisin-resistant bacteria also appear (Non-patent Document). 1). Moreover, although there exists a method of improving feed efficiency by using the preparation of sorbic acid and bacteriocin together (patent document 5), it is not clarified about suppression of methane generation when added as ruminant feed.
そこで、本発明は、反芻動物のルーメン内におけるメタン発生を抑制して飼料効率を向上せしめるメタン生成抑制剤、これを配合した飼料添加物や飼料組成物、およびこれらを反芻動物に給与してメタン生成を抑制し、延いてはその発育を改善する方法を提供することを目的とする。 Accordingly, the present invention provides a methane production inhibitor that suppresses methane generation in rumen of ruminants and improves feed efficiency, feed additives and feed compositions containing the same, and feeds these to ruminants to produce methane. An object is to provide a method for inhibiting production and thus improving its growth.
本発明者らは、上記従来技術の問題点に鑑み、鋭意研究を重ねたところ、ルーメンに対して有効成分として過酸化水素を含むメタン生成抑制剤を添加することでルーメンにおけるメタン生成を有意に抑えることを見出した。
本発明はかかる知見に基づいて完成されたものであり、以下の態様を含む。
（１）有効成分として過酸化水素を含有することを特徴とする反芻動物用メタン生成抑制剤。
（２）上記（１）に記載のメタン生成抑制剤を含有することを特徴とする反芻動物用飼料添加物又は反芻動物用飼料組成物。
（３）上記（１）に記載のメタン生成抑制剤及び／又は（２）に記載の反芻動物用飼料添加物及び／又は反芻動物用飼料組成物を反芻動物に投与することを特徴とする反芻動物のルーメン由来のメタン発生を抑制する方法。
（４）上記（１）に記載のメタン生成抑制剤及び／又は（２）に記載の反芻動物用飼料添加物及び／又は反芻動物用飼料組成物を反芻動物に投与することを特徴とする反芻動物の発育改善方法。
In light of the above-mentioned problems of the prior art, the present inventors have conducted extensive research and found that the addition of a methane production inhibitor containing hydrogen peroxide as an active ingredient to rumen significantly improves methane production in the lumen. I found it to be suppressed.
The present invention has been completed based on such findings and includes the following aspects.
(1) A ruminant methane production inhibitor characterized by containing hydrogen peroxide as an active ingredient.
(2) A feed additive for ruminants or a feed composition for ruminants comprising the methane production inhibitor according to (1) above.
(3) A ruminant characterized by administering the ruminant feed additive and / or ruminant feed composition according to (2) to the ruminant and / or the ruminant feed additive and / or ruminant feed composition according to (2). A method for suppressing methane generation from animal rumen.
(4) A ruminant characterized by administering the ruminant feed additive and / or ruminant feed composition according to (2) to the ruminant and / or the ruminant feed additive and / or ruminant feed composition according to (2). Animal growth improvement method.
本発明のメタン生成抑制剤の投与により、反芻動物のルーメンにおけるメタン生成は有意に抑制され、飼料のエネルギー効率が向上し、延いては反芻動物の発育が改善される。また、温室効果ガスであるメタン生成を減らすことによって地球温暖化現象などの環境問題へも貢献できる。
本発明のメタン生成抑制剤の投与によりルーメン内のメタン生成が抑制される効果の確認は、人工ルーメン系（Ｔ．Ｈｉｎｏ ｅｔ ａｌ．，Ｊ．Ｇｅｎ．Ａｐｐｌ．Ｍｉｃｒｏｂｉｏｌ．，３９，３５−４５（１９９３））で確認出来るが、ｉｎ ｖｉｖｏで実際に反芻動物に経口投与して確認してもよいことはいうまでもない。
By the administration of the methane production inhibitor of the present invention, methane production in rumen rumen is significantly suppressed, the energy efficiency of the feed is improved, and the development of the ruminant is improved. It can also contribute to environmental problems such as global warming by reducing the production of methane, a greenhouse gas.
Confirmation of the effect of suppressing the methane production in the lumen by the administration of the methane production inhibitor of the present invention was confirmed by an artificial rumen system (T. Hino et al., J. Gen. Appl. Microbiol., 39, 35-45 ( 1993)), but it goes without saying that it may be confirmed by oral administration to ruminants in vivo.
図１は、過酸化水素の濃度とメタン生成抑制活性の関係を示す説明図である。
図２は、過酸化水素の濃度と水素生成量の関係を示す説明図である。
FIG. 1 is an explanatory diagram showing the relationship between the concentration of hydrogen peroxide and the methane production inhibitory activity.
FIG. 2 is an explanatory diagram showing the relationship between the concentration of hydrogen peroxide and the amount of hydrogen produced.
以下に本発明を詳細に説明する。
本発明のメタン生成抑制剤は、有効成分として過酸化水素を含有することを特徴とする反芻動物用のメタン生成抑制剤である。また、本発明における反芻動物とは、偶蹄目反芻亜目に属する哺乳類のことであり、胃が３または４室に分かれ、食物を反芻するもののことを指す。例えば、牛、羊などが挙げられる。
本発明のメタン生成抑制剤は、有効物質が過酸化水素であり、適正な濃度における使用では安全性が高く、反芻動物の健康面から好ましいものである。また、速やかに水と酸素に分解されるため、反芻動物の肉や乳などへの残存が無い。さらに、同様の理由から、排泄物中への残存も無く、環境保全の面においても非常に優れている。
本発明のメタン抑制剤に有効成分として使用される過酸化水素は、商業的に容易に入手することが出来、例えば、高杉製薬、内藤商店、日本パーオキサイド、和光純薬等から購入することが出来る。また、過酸化水素を生産する微生物、例えば広く乳酸菌（Ａ．Ｉｔｏ ｅｔ ａｌ．，Ｃｕｒｒ．Ｍｉｃｒｏｂｉｏｌ．，４７，２３１−２３６（２００３））などの培養物を過酸化水素源として使用することも出来る。
メタン生成抑制剤の投与時期は、本発明のメタン生成抑制効果の奏される限りは特に制限されるものではなく、飼料がルーメン内に滞留している間であれば何れの時期に投与しても良いが、メタンが最も活発に生成される前後にルーメンにメタン生成抑制剤が存在することが好ましいことから、飼料投与直前或いは同時にメタン生成抑制剤を投与することが好ましい。
本発明に係る反芻動物用メタン生成抑制剤は様々な形態で用いることが可能であり、メタン生成抑制効果の奏される限りは特に制限されず、例えば、水溶液やカプセル或いはこれらを配合した飼料添加物や飼料組成物等の各種の形態が挙げられる。
本発明のメタン生成抑制剤の投与量については、投与直後のルーメン液中の過酸化水素濃度が、少なくとも０．００１~１０ｍＭとなるように、更に好ましくは０．０１~５ｍＭとなるように投与する。有効成分である過酸化水素は投与後速やかに分解するため、上記範囲内の濃度であれば日に複数回投与しても良い。この様な投与量で投与することで、繊維分解菌等の有用なルーメン微生物には悪影響を与えず、メタン菌が優先的に殺菌もしくは静菌される。
The present invention is described in detail below.
The methane production inhibitor of the present invention is a ruminant methane production inhibitor characterized by containing hydrogen peroxide as an active ingredient. In addition, the ruminant in the present invention refers to a mammal belonging to the order of the artiodactylid ruminant, and refers to one in which the stomach is divided into 3 or 4 chambers and ruins food. Examples include cows and sheep.
In the methane production inhibitor of the present invention, the active substance is hydrogen peroxide, which is highly safe when used at an appropriate concentration, and is preferable from the viewpoint of the health of ruminants. Moreover, since it is rapidly decomposed into water and oxygen, there is no residue in ruminant meat or milk. Furthermore, for the same reason, it does not remain in excrement and is very excellent in terms of environmental conservation.
Hydrogen peroxide used as an active ingredient in the methane inhibitor of the present invention can be easily obtained commercially, for example, purchased from Takasugi Pharmaceutical, Naito Shoten, Nippon Peroxide, Wako Pure Chemical, etc. I can do it. In addition, cultures such as microorganisms that produce hydrogen peroxide, such as lactic acid bacteria (A. Ito et al., Curr. Microbiol., 47, 231-236 (2003)) can also be used as the hydrogen peroxide source. .
The timing of administration of the methane production inhibitor is not particularly limited as long as the methane production inhibitory effect of the present invention is exhibited, and can be administered at any time as long as the feed remains in the lumen. However, since it is preferable that a methane formation inhibitor is present in the lumen before and after methane is most actively produced, it is preferable to administer the methane production inhibitor immediately before or simultaneously with the administration of feed.
The ruminant methane production inhibitor according to the present invention can be used in various forms, and is not particularly limited as long as the methane production inhibitory effect is exerted. For example, an aqueous solution, a capsule, or a feed addition containing these Various forms such as food and feed composition.
The dosage of the methane production inhibitor of the present invention is such that the hydrogen peroxide concentration in the rumen solution immediately after administration is at least 0.001 to 10 mM, more preferably 0.01 to 5 mM. To do. Since hydrogen peroxide as an active ingredient decomposes rapidly after administration, it may be administered multiple times a day as long as the concentration is within the above range. By administering at such a dose, methane bacteria are preferentially sterilized or bacteriostatic without adversely affecting useful rumen microorganisms such as fibrinolytic bacteria.
以下、実施例により本発明をさらに説明する。なお、本発明は、これらの実施例のみに限定されるものではなく、本発明の要旨を逸脱しない範囲で任意に変更が可能であることは言うまでも無い。
上記メタン生成抑制剤のメタン生成抑制活性は後に記載の活性測定法にて確認できる。
ルーメン発酵簡易評価法
＜材料および実験方法＞
乾草を中心に飼育された牛から飼料給与前に採取したルーメン液を使用した（本実験ではホルスタイン種の牛を用いたが、牛の種類や飼料の種類、ルーメン液採取方法は本分野における常法に則る限り特に限定しない）。ＭｃＤｏｕｇａｌｌ人工唾液（ＭｃＤｏｕｇａｌｌ，１９４８）７ｍｌおよび上記ルーメン液２ｍｌ、活性測定サンプル１ｍｌの計１０ｍｌを１４ｍｌ容試験管内で混合した（コントロールには活性測定サンプル１ｍｌの代わりに０．９％生理食塩水１ｍｌを用いた）。更に、発酵基質としてクレイングラス乾草粉末（ＤＭ：８７．３３％，ＯＭ：９８．９８％，ＣＰ：１４．０％，ＡＤＦ：３８．８４％，ＮＤＦ：７３．２６％，ＡＤＬ：４．１％，ＧＥ：４．４５Ｍｃａｌ／ｋｇＤＭ）および濃厚飼料粉末（ＤＭ：９４．１３％，ＣＰ：１６．０６％，ＡＤＦ：６．５５％，ＮＤＦ：１９．０６％，ＡＤＬ：１．１４％，ＧＥ：１．７７Ｍｃａｌ／ｋｇＤＭ）を０．０５ｇずつ添加した。シリコン栓にて密栓し、内容物を十分に混合した後、テルモシリンジ（ＴＥＲＵＭＯ）およびテルモ注射針１８Ｇ（ＴＥＲＵＭＯ）をシリコン栓に刺し、試験管内にて発生したガスがシリンジに捕集される様にした。尚、実験操作中ルーメン液は常に３５℃以上４０℃未満に保ち、嫌気条件下で行った（酸素濃度１％以下）。振盪培養機ＢＲ−３０００ＬＦ（ＴＡＩＴＥＣ）を使用し、４０℃、１５０ｒｐｍにて２０~２４ｈ培養を行った。
＜メタンおよび水素の分析方法＞
培養後、シリンジ内に捕集された発生ガスをインジェクター（Ｐｒｅｓｓｕｒｅ−Ｌｏｋ、ＶＩＣＩ）を用いて一部採取し、ガスクロマトグラフィーＧＣ３２０（ＧＬ Ｓｃｉｅｎｃｅｓ）によりメタンと水素および酸素を分析した。カラムはＧＣ３２０ ＪＫ１２（ＧＬ Ｓｃｉｅｎｃｅｓ）を使用し、分析データはインテグレーターＤ−７５００（ＨＩＴＡＣＨＩ）にて出力を行った。ガス分析を行ったサンプル中の酸素濃度から、サンプル採取時に混入した空気量を計算し補正を行うことで発生したガス中のメタン濃度および水素濃度を算出した。
実施例１：過酸化水素濃度依存的なメタン生成抑制効果
濃度を正確に分析した３０~３５％過酸化水素水溶液（和光純薬）を生理食塩水にて希釈し、過酸化水素濃度が０．１，０．３，０．５，０．７，０．９，１．１，１．３，１．５，３．０，５．０，１０．０ｍＭであるサンプル系列を作成した。これらサンプルを上記ルーメン発酵簡易評価法に供した。なお、本検討は独立した試験を３回行い、その平均値および標準誤差を結果として後掲図１および図２に示す。
なお、図１中、横軸は過酸化水素サンプル濃度（ｍＭ）を示し、縦軸はネガティブコントロール（過酸化水素サンプル濃度０ｍＭ）を１００とした場合の各サンプル投与区におけるメタン発生量を相対値にて示している。棒グラフは独立した３回の実験の平均値を示しており、エラーバーはその３回の実験から得られた標準誤差を示している。そして、図２中、横軸は過酸化水素サンプル濃度（ｍＭ）を示し、縦軸はＨ２Ｏ２の濃度１．１ｍＭのときの水素の発生量を１００とした場合の各サンプル投与区における水素発生量を相対値にて示している。棒グラフは独立した３回の実験の平均値を示しており、エラーバーはその３回の実験から得られた標準誤差を示している。
図１の結果から、ルーメン発酵簡易評価法において過酸化水素がメタン生成を抑制するには、少なくとも０．５ｍＭ以上の濃度の過酸化水素の投与が必要であることが明らかとなった。また、図２から、投与した過酸化水素の濃度が０．５~５．０ｍＭでは他の濃度域に比べて水素が多く発生している。このことから、この範囲内においては、メタン生産菌は死滅もしくは静菌されるが、繊維分解菌などの有用なルーメン微生物は活動可能であることが確認できた。
本実施例１のルーメン発酵簡易評価法ではルーメン液を希釈して使用しているため、過酸化水素への感受性がルーメン液の原液よりも高くなっている可能性がある。実際の反芻動物へのメタン生成抑制剤の投与では投与対象となる動物や地域、飼育方法毎に事前に条件検討を行う必要がある。
Hereinafter, the present invention will be further described by examples. Needless to say, the present invention is not limited to these examples, and can be arbitrarily changed without departing from the gist of the present invention.
The methane production inhibitory activity of the methane production inhibitor can be confirmed by an activity measurement method described later.
Rumen fermentation simplified evaluation method <Materials and experimental method>
Rumen fluid collected from cattle raised mainly in hay before feeding was used. (In this experiment, Holstein cattle were used. However, the type of cattle, the type of feed, and the method for collecting rumen fluid are commonly used in this field. As long as the law does not limit). 7 ml of McDoughall artificial saliva (McDougal, 1948) and 2 ml of the above rumen solution and 1 ml of the activity measurement sample were mixed in a 14 ml test tube (1 ml of 0.9% physiological saline was used instead of 1 ml of the activity measurement sample as a control). Using). Furthermore, Kleingrass hay powder (DM: 87.33%, OM: 98.98%, CP: 14.0%, ADF: 38.84%, NDF: 73.26%, ADL: 4.1 as a fermentation substrate. %, GE: 4.45 Mcal / kg DM) and concentrated feed powder (DM: 94.13%, CP: 16.06%, ADF: 6.55%, NDF: 19.06%, ADL: 1.14%, GE: 1.77 Mcal / kg DM) was added in 0.05 g increments. After sealing with a silicon stopper and thoroughly mixing the contents, the Terumo syringe (TERUMO) and the Terumo injection needle 18G (TERUMO) are inserted into the silicon stopper, and the gas generated in the test tube is collected in the syringe. I made it. Note that the rumen solution was always kept at 35 ° C. or higher and lower than 40 ° C. under the anaerobic condition during the experimental operation (oxygen concentration 1% or less). Using a shaking incubator BR-3000LF (TAITEC), culturing was performed at 40 ° C. and 150 rpm for 20 to 24 hours.
<Method for analyzing methane and hydrogen>
After the culture, part of the generated gas collected in the syringe was collected using an injector (Pressure-Lok, VICI), and methane, hydrogen and oxygen were analyzed by gas chromatography GC320 (GL Sciences). The column used was GC320 JK12 (GL Sciences), and the analysis data was output by an integrator D-7500 (HITACHI). From the oxygen concentration in the sample subjected to gas analysis, the amount of air mixed at the time of sampling was calculated and corrected to calculate the methane concentration and hydrogen concentration in the generated gas.
Example 1: Hydrogen peroxide concentration-dependent methane production inhibitory effect A 30-35% aqueous hydrogen peroxide solution (Wako Pure Chemical Industries, Ltd.) whose concentration was accurately analyzed was diluted with physiological saline, and the hydrogen peroxide concentration was reduced to 0. 0. Sample series having 1, 0.3, 0.5, 0.7, 0.9, 1.1, 1.3, 1.5, 3.0, 5.0, 10.0 mM were prepared. These samples were subjected to the rumen fermentation simplified evaluation method. In this study, independent tests were performed three times, and the average value and standard error are shown in FIGS. 1 and 2 as a result.
In FIG. 1, the horizontal axis indicates the hydrogen peroxide sample concentration (mM), and the vertical axis indicates the relative amount of methane generated in each sample administration group when the negative control (hydrogen
From the results of FIG. 1, it was revealed that administration of hydrogen peroxide at a concentration of at least 0.5 mM is necessary for hydrogen peroxide to suppress methane formation in the simple rumen fermentation evaluation method. Further, from FIG. 2, when the concentration of the administered hydrogen peroxide is 0.5 to 5.0 mM, more hydrogen is generated than in other concentration ranges. From this, it was confirmed that within this range, methane-producing bacteria were killed or bacteriostatic, but useful rumen microorganisms such as fiber-degrading bacteria could be active.
Since the rumen liquid is diluted and used in the simple rumen fermentation evaluation method of Example 1, there is a possibility that the sensitivity to hydrogen peroxide is higher than the stock solution of rumen liquid. In the actual administration of methane production inhibitors to ruminants, it is necessary to examine the conditions in advance for each animal, region, and breeding method to be administered.
本発明により、反芻動物から排出される強力な温室効果ガスであるメタンを削減可能であり、さらに、メタンを削減することにより反芻動物の飼料エネルギーの利用効率が向上し、延いては反芻動物の発育が改善するため、極めて有用である。 According to the present invention, it is possible to reduce methane, which is a powerful greenhouse gas emitted from ruminants. It is extremely useful because it improves growth.
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