JP2010131001A - Method for increasing conjugated linoleic acid in milk or meat of rumination cattle - Google Patents
Method for increasing conjugated linoleic acid in milk or meat of rumination cattle Download PDFInfo
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- JP2010131001A JP2010131001A JP2009228231A JP2009228231A JP2010131001A JP 2010131001 A JP2010131001 A JP 2010131001A JP 2009228231 A JP2009228231 A JP 2009228231A JP 2009228231 A JP2009228231 A JP 2009228231A JP 2010131001 A JP2010131001 A JP 2010131001A
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- milk
- ruminant livestock
- feed
- linoleic acid
- meat
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Abstract
Description
本発明は、反芻家畜の乳及び／又は肉中の共役リノール酸の含量を効率的に高める方法に関する。さらに詳しくは、反芻家畜に給与する、乳及び／又は肉中の共役リノール酸含量を効率的に高める栄養素、及びこれを用いた高機能性乳及び／又は高機能性肉の生産方法に関する。 The present invention relates to a method for efficiently increasing the content of conjugated linoleic acid in the milk and / or meat of ruminant livestock. More specifically, the present invention relates to a nutrient that efficiently feeds conjugated linoleic acid content in milk and / or meat to be fed to ruminant livestock, and a method for producing highly functional milk and / or highly functional meat using the nutrient.
共役リノール酸（ＣＬＡ；Ｃｏｎｊｕｇａｔｅｄ ｌｉｎｏｌｅｉｃ ａｃｉｄｓ）は反芻家畜に特徴的な脂肪酸であり、リノール酸（ｃｉｓ−９，ｃｉｓ−１２ ｏｃｔａｄｅｃａｄｉｅｎｏｉｃ ａｃｉｄ）の位置的、幾何学的な異性体であって、共役二重結合を有する、つまり共役ジエン構造を有するものの総称である。二重結合の位置は、９と１１、１０と１２、１１と１３のように異なった位置にある。オクタデカジエン酸のシス−９，トランス−１１異性体：すなわち、ｃｉｓ−９，ｔｒａｎｓ−１１ Ｃ１８：２（ｃｉｓ−９，ｔｒａｎｓ−１１ ｏｃｔａｄｅｃａｄｉｅｎｏｉｃ ａｃｉｄ）は、乳製品中に含まれるＣＬＡの８０％以上を占めている。そして、例えば牛乳中の平均的なＣＬＡ含量は、３ｍｇ／ｇ Ｆａｔ以上、６ｍｇ／ｇ Ｆａｔ未満、すなわち総脂肪量の０．３重量％以上、０．６重量％未満であるが、その変動する幅は大きい。牛乳中のＣＬＡの大部分は、ｃｉｓ−９，ｔｒａｎｓ−１１ ＣＬＡ異性体であるので、この異性体（シス−９，トランス−１１ Ｃ１８：２）をＣＬＡということもある。 Conjugated linoleic acid (CLA) is a fatty acid characteristic of ruminant livestock, and is a positional and geometric isomer of linoleic acid (cis-9, cis-12 octadecadienoic acid). It is a general term for those having a heavy bond, that is, having a conjugated diene structure. The positions of the double bonds are different positions such as 9 and 11, 10 and 12, 11 and 13. The cis-9, trans-11 isomer of octadecadienoic acid: cis-9, trans-11 C18: 2 (cis-9, trans-11 octadecadienoic acid) is 80% of CLA in dairy products It accounts for the above. For example, the average CLA content in milk is 3 mg / g Fat or more and less than 6 mg / g Fat, that is, 0.3% by weight or more and less than 0.6% by weight of the total fat amount, but varies. The width is large. Since most of CLA in milk is a cis-9, trans-11 CLA isomer, this isomer (cis-9, trans-11 C18: 2) is sometimes referred to as CLA.
共役リノール酸（ｃｉｓ−９，ｔｒａｎｓ−１１ ＣＬＡ異性体）の様々な生理機能が明らかにされ、ＣＬＡのヒトヘの給源として、特に牛乳及び牛肉中のＣＬＡが注目されている。ＣＬＡの生理機能としては、抗がん作用、体脂肪低減作用、脂質代謝調節、抗動脈硬化作用、免疫調節作用、ＩＩ型糖尿病抑制作用、抗血小板作用、肥満防止、抗アレルギー作用、骨形成の改善、慢性腎炎の抑制、増体促進、飼料効率の向上などが挙げられる（非特許文献１）。 Various physiological functions of conjugated linoleic acid (cis-9, trans-11 CLA isomer) have been clarified, and CLA in milk and beef has attracted attention as a human source of CLA. Physiological functions of CLA include anti-cancer action, body fat reduction action, lipid metabolism regulation, anti-arteriosclerosis action, immunoregulatory action, type II diabetes suppression action, anti-platelet action, anti-obesity, anti-allergic action, bone formation Examples include improvement, suppression of chronic nephritis, promotion of body gain, and improvement of feed efficiency (Non-Patent Document 1).
反芻家畜の乳や肉中のＣＬＡ含量は、反芻家畜への給餌飼料、季節、年齢（泌乳回数）、個体、ルーメン内微生物叢の変化などの要因で変動する。これらの中でも、給餌飼料の影響が最も大きく寄与していると考えられている。そこで、飼料の成分を変えることで牛乳などのＣＬＡ含量を高める試みが数多く行われており、リノール酸を含むしょうゆ油や醤油粕を飼料に加える方法（特許文献１、２）、トランスバクセン酸、すなわち、オクタデセン酸のトランス−１１異性体（ＴＶＡ；ｔｒａｎｓ−１１ Ｃ１８：１）を直接給与する方法（特許文献３）などが開示されている。
The content of CLA in ruminant livestock milk and meat fluctuates due to factors such as changes in feed, season, age (number of lactations), individuals, and rumen microbiota. Among these, it is thought that the influence of feed feed contributes the most. Therefore, many attempts have been made to increase the content of CLA such as milk by changing the ingredients of the feed, such as a method of adding soy sauce oil or soy sauce cake containing linoleic acid to the feed (
しかし、ＣＬＡやＴＶＡなどの油脂の直接給与は、ＣＬＡのある程度の増加は認められるものの、飼料の嗜好性を低下させ、ルーメンの微生物に悪影響を及ぼし、乳脂率を急激に低下させるなどといった点で全体的にはマイナス作用を有し、好ましくはない。したがって、油脂の直接投与によるＣＬＡ増加には限界がある。 However, the direct supply of fats and oils such as CLA and TVA, although some increase in CLA is recognized, it reduces feed preference, adversely affects rumen microorganisms, and rapidly lowers the percentage of milk fat. Overall it has a negative effect and is not preferred. Therefore, there is a limit to the increase in CLA by direct administration of fats and oils.
なお、反芻家畜におけるＣＬＡは、飼料中のリノール酸やα−リノレン酸などの高度不飽和脂肪酸がルーメン微生物によってＴＶＡに変化し、消化吸収され乳腺組織あるいは脂肪組織に移行し、不飽和化による生合成により生成される。しかし、反芻家畜の生体内でのＴＶＡからＣＬＡへの変換率は約２０％程度でしかなく、決して大きくはない。 CLA in ruminant livestock is a highly unsaturated fatty acid such as linoleic acid or α-linolenic acid in feed that is converted to TVA by rumen microorganisms, digested and absorbed, and transferred to mammary gland tissue or adipose tissue. Generated by synthesis. However, the conversion rate of TVA to CLA in the living body of ruminant livestock is only about 20%, which is never large.
さらに、反芻家畜への醤油粕やしょうゆ油の給与は、乳や肉中のＣＬＡ含量を高めることができるが、同時に生産物中のＴＶＡ濃度も著しく高くなり、生体内でＴＶＡが効率よくＣＬＡに転換するわけではない。 Furthermore, feeding soy sauce and soy sauce oil to ruminant livestock can increase the CLA content in milk and meat, but at the same time, the TVA concentration in the product is significantly increased, and TVA is efficiently converted to CLA in vivo. It doesn't convert.
牛の組織中におけるＴＶＡからＣＬＡへの変換は、不飽和化酵素であるｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅ（ステアロイル−ＣｏＡ不飽和化酵素：ＳＣＤ）によって制御されている。 Conversion of TVA to CLA in bovine tissue is controlled by the desaturase, stearoyl-CoA desaturase (stearoyl-CoA desaturase: SCD).
ｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅは、牛の体脂肪を不飽和化する酵素として知られ、生体内で脂質の合成や分解に重要な役割を果たすｓｔｅａｒｏｙｌ‐ＣｏＡを不飽和化する酵素である。この遺伝子配列及びアミノ酸配列については、既に開示されている（非特許文献２）。しかしながら、この酵素（ＳＣＤ）をコードする遺伝子の発現を高めたり、及び／又は、この酵素の活性を高めたりすることにより、ＴＶＡからＣＬＡへの変換を促進することについては、従来、報告例は見当たらない。ましてや、特定のアミノ酸やビタミン類が、ＳＣＤ遺伝子の発現を高めたり酵素活性を高めたりすることは、全く知られていない。 stearoyl-CoA desaturase is known as an enzyme that desaturates bovine body fat, and is an enzyme that desaturates stearoyl-CoA, which plays an important role in lipid synthesis and degradation in vivo. This gene sequence and amino acid sequence have already been disclosed (Non-patent Document 2). However, there have been reports in the past about promoting the conversion of TVA to CLA by increasing the expression of the gene encoding this enzyme (SCD) and / or increasing the activity of this enzyme. I can't find it. Furthermore, it is not known at all that specific amino acids and vitamins increase the expression of the SCD gene or increase the enzyme activity.
本発明は、食品としての反芻家畜の乳や肉の重要性に鑑み、その付加価値をできるだけ高め、国民の健康に貢献するため、反芻家畜の乳量や乳質、肉質を保ったまま、ＣＬＡの産生能の亢進が可能な飼料成分の提供を目的としてなされたものである。さらに、この飼料成分を用いた効率的なＣＬＡ濃度の高い機能性乳、機能性肉を生産する新規飼養システムの提供も目的としてなされたものである。 In view of the importance of ruminant livestock milk and meat as food, the present invention increases the added value as much as possible and contributes to the health of the nation. Therefore, while maintaining the milk amount, milk quality and meat quality of ruminant livestock, It was made for the purpose of providing a feed ingredient capable of enhancing productivity. Furthermore, the provision of a new rearing system for producing functional milk and functional meat having high CLA concentration efficiently using this feed component was also made.
上記目的を達成するため、本発明者らは各方面から検討の結果、ｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅ（ＳＣＤ）及び該酵素をコードする遺伝子に着目した。そして、反芻家畜の生体内でのＳＣＤをコードする遺伝子の発現亢進や該酵素の活性化ができれば、ＣＬＡの産生能の亢進、生体機能性亢進が期待できると考え、ルーメンの微生物による発酵状態を正常に保ったままで、この遺伝子発現亢進や酵素の活性化をすることが可能な飼料成分の開発が可能であるとの観点にはじめてたった。 In order to achieve the above-mentioned object, the present inventors have focused on stearoyl-CoA desaturase (SCD) and a gene encoding the enzyme as a result of examination from various directions. And, if the expression of the gene encoding SCD and the activation of the enzyme in the ruminant live organism can be increased, it is expected that the production ability of CLA and the enhancement of the biological function can be expected. It was the first time that it was possible to develop a feed ingredient that could enhance the gene expression and activate the enzyme while keeping it normal.
そして更に、本発明者らは鋭意研究の結果、リジン、ロイシン、アルギニン、ビオチン、ビタミンCなどの栄養素を、リノール酸やα−リノレン酸などの高度不飽和脂肪酸を含む家畜給与飼料に添加して給与することにより、反芻家畜の乳腺組織や脂肪組織中のｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅの遺伝子発現や酵素活性を高め、乳量や乳質、肉質を保ったまま、効率的にこれらのＣＬＡ（シス−９，トランス−１１ Ｃ１８：２脂肪酸）濃度を高めることを見出し、本発明に至った。 Furthermore, as a result of earnest research, the present inventors added nutrients such as lysine, leucine, arginine, biotin and vitamin C to livestock feeds containing highly unsaturated fatty acids such as linoleic acid and α-linolenic acid. By feeding, the gene expression and enzyme activity of stearoyl-CoA desaturase in the mammary gland tissue and adipose tissue of ruminant livestock are increased, and these CLA (cis-9, cis-9, It was found that the concentration of trans-11 C18: 2 fatty acid) was increased, and the present invention was achieved.
すなわち、本発明の実施形態は次のとおりである。
（１）反芻家畜に、ｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅの遺伝子発現及び／又は該酵素活性を高める栄養素を給与することにより、反芻家畜の乳及び／又は肉中の共役リノール酸（ＣＬＡ）含量を高める方法。
（２）反芻家畜に、ｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅの遺伝子発現及び／又は酵素活性を高める栄養素を給与することにより、共役リノール酸（ＣＬＡ）含量が高まった高機能性乳及び／又は高機能性肉を生産する方法。
（３）栄養素が、リジン、ロイシン、アルギニン、ビオチン、ビタミンＣから選ばれる少なくとも１つであることを特徴とする（１）又は（２）に記載の方法。
（４）反芻家畜が、乳用牛又は肉用牛であることを特徴とする（１）〜（３）のいずれか１つに記載の方法。
（５）ｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅの遺伝子発現及び／又は酵素活性を高める栄養素を配合した、反芻家畜の乳及び／又は肉中の共役リノール酸含量を高める反芻家畜用飼料添加物。
（６）栄養素が、リジン、ロイシン、アルギニン、ビオチン、ビタミンＣから選ばれる少なくとも１つであることを特徴とする（５）に記載の反芻家畜用飼料添加物。
（７）反芻家畜が、乳用牛又は肉用牛であることを特徴とする（５）又は（６）に記載の反芻家畜用飼料添加物。
（８）（５）〜（７）のいずれか１項に記載の反芻家畜用飼料添加物を含有してなることを特徴とする反芻家畜用飼料。
（９）（８）に記載の飼料を反芻家畜に給与することにより得られた、共役リノール酸含量が総脂肪量の０．６０重量％以上（例えば、総脂肪量の０．６０〜１．８０重量％）であることを特徴とする反芻家畜乳又は反芻家畜肉（特に牛乳又は牛肉）。
That is, the embodiment of the present invention is as follows.
(1) A method for increasing the content of conjugated linoleic acid (CLA) in milk and / or meat of ruminant livestock by feeding ruminant livestock with nutrients that increase the gene expression of stearoyl-CoA desaturase and / or the enzyme activity.
(2) Feeding ruminant livestock with high functional milk and / or high functional meat with increased conjugated linoleic acid (CLA) content by feeding nutrients that enhance the gene expression and / or enzyme activity of stearoyl-CoA desaturase How to produce.
(3) The method according to (1) or (2), wherein the nutrient is at least one selected from lysine, leucine, arginine, biotin, and vitamin C.
(4) The method according to any one of (1) to (3), wherein the ruminant livestock is a dairy cow or a beef cow.
(5) A feed additive for ruminant livestock that increases the content of conjugated linoleic acid in milk and / or meat of ruminant livestock, which contains nutrients that increase gene expression and / or enzyme activity of stearoyl-CoA desaturase.
(6) The feed additive for ruminant livestock according to (5), wherein the nutrient is at least one selected from lysine, leucine, arginine, biotin, and vitamin C.
(7) The feed additive for ruminant livestock according to (5) or (6), wherein the ruminant livestock is dairy cattle or beef cattle.
(8) A ruminant livestock feed comprising the ruminant livestock feed additive according to any one of (5) to (7).
(9) Conjugated linoleic acid content obtained by feeding the ruminant livestock feed described in (8) to 0.60% by weight or more of the total fat amount (for example, 0.60 to 1.% of the total fat amount). 80% by weight) ruminant livestock milk or ruminant livestock meat (especially milk or beef).
また、本発明の他の実施態様としては、特定の栄養素を使用（添加、経口投与）することにより、ＴＶＡからＣＬＡへの変換を触媒する酵素であるＳＣＤの酵素活性を高めたり、ＳＣＤ遺伝子の発現を高めたりする方法、そしてその結果として、反芻家畜の細胞又は乳又は肉中のＣＬＡ含量を高める方法、及び、そのために使用する剤（動物用医薬、飼料、飼料添加物の少なくともひとつ）を挙げることができ、以下に例示される。 In another embodiment of the present invention, the use of a specific nutrient (addition, oral administration) increases the enzymatic activity of SCD, an enzyme that catalyzes the conversion of TVA to CLA, A method for increasing expression and, as a result, a method for increasing CLA content in cells or milk or meat of ruminant livestock, and an agent (at least one of veterinary medicine, feed, feed additive) used therefor Can be mentioned and is exemplified below.
（１）タンパク質代謝又はエネルギー代謝に関与する栄養成分を用いて、反芻家畜の細胞中のｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅ遺伝子発現及び／又はｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅ酵素活性を高める方法。
（２）タンパク質代謝又はエネルギー代謝に関与する栄養成分を用いて、反芻家畜の細胞中のｓｔｅａｒｏｙｌ‐ＣｏＡ ｄｅｓａｔｕｒａｓｅ遺伝子発現及び／又はｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅ酵素活性を高めることで、その細胞中の共役リノール酸含量を高める方法。
（３）栄養成分が、リジン、ロイシン、アルギニン、ビオチン、ビタミンＣから選ばれる少なくとも１つであることを特徴とする（１）又は（２）に記載の方法。
（４）反芻家畜の細胞が、乳腺組織又は脂肪組織より得られたものであることを特徴とする（１）〜（３）のいずれか１項に記載の方法。
（５）反芻家畜の細胞が、ウシの組織より得られたものであることを特徴とする（１）〜（４）のいずれか１項に記載の方法。
（６）タンパク質代謝又はエネルギー代謝に関与する栄養成分を有効成分とする、反芻家畜の細胞中のｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅ遺伝子発現及び／又はｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅ酵素活性の促進剤（増強剤）。
（７）タンパク質代謝又はエネルギー代謝に関与する栄養成分を有効成分とすることを特徴とする反芻家畜の細胞中の共役リノール酸含量を高める剤。
（８）栄養成分が、リジン、ロイシン、アルギニン、ビオチン、ビタミンＣから選ばれる少なくとも１つであることを特徴とする（６）又は（７）に記載の剤。
（９）反芻家畜の細胞が、乳腺組織又は脂肪組織より得られたものであることを特徴とする（６）〜（８）のいずれか１項に記載の剤。
（１０）反芻家畜の細胞が、ウシの組織より得られたものであることを特徴とする（６）〜（９）のいずれか１項に記載の剤。
(1) A method for enhancing stearoyl-CoA desaturase gene expression and / or stearoyl-CoA desaturase enzyme activity in ruminant livestock cells using a nutrient component involved in protein metabolism or energy metabolism.
(2) Conjugated linoleic acid in cells by enhancing stearoyl-CoA desaturase gene expression and / or stearoyl-CoA desaturase enzyme activity in ruminant livestock cells using nutrient components involved in protein metabolism or energy metabolism Method to increase the content.
(3) The method according to (1) or (2), wherein the nutritional component is at least one selected from lysine, leucine, arginine, biotin, and vitamin C.
(4) The method according to any one of (1) to (3), wherein the ruminant livestock cells are obtained from mammary gland tissue or adipose tissue.
(5) The method according to any one of (1) to (4), wherein the ruminant livestock cells are obtained from bovine tissue.
(6) A promoter (enhancer) of stearoyl-CoA desaturase gene expression and / or stearoyl-CoA desaturase enzyme activity in ruminant livestock cells, comprising as an active ingredient a nutrient component involved in protein metabolism or energy metabolism.
(7) An agent for increasing the content of conjugated linoleic acid in cells of ruminant livestock, characterized in that a nutritional component involved in protein metabolism or energy metabolism is used as an active ingredient.
(8) The agent according to (6) or (7), wherein the nutritional component is at least one selected from lysine, leucine, arginine, biotin, and vitamin C.
(9) The agent according to any one of (6) to (8), wherein the ruminant livestock cells are obtained from mammary gland tissue or adipose tissue.
(10) The agent according to any one of (6) to (9), wherein the ruminant livestock cells are obtained from bovine tissue.
本発明によれば、反芻家畜のルーメンの微生物による発酵状態を正常に保ったまま、つまり反芻家畜の乳量や乳質、肉質へ悪い影響を及ぼすことなく、生体内のｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅの遺伝子発現及び酵素活性を高めて、ＣＬＡの産生能の亢進が可能となる。また、これによって効率的なＣＬＡ濃度の高い機能性乳、機能性肉を生産する新規飼養システムを提供することが可能となる。 According to the present invention, the gene expression of stearoyl-CoA desaturase in vivo is maintained while maintaining a normal fermentation state by rumen microorganisms of ruminant livestock, that is, without adversely affecting milk yield, milk quality, and meat quality of ruminant livestock. In addition, the enzyme activity is increased, and the CLA production ability can be enhanced. In addition, this makes it possible to provide a new breeding system that produces functional milk and functional meat with an efficient CLA concentration.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明で栄養素の給与対象としている家畜は、生体内でＴＶＡをＣＬＡへ変換する酵素ｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅが働き、その乳や肉中にＣＬＡを蓄積する反芻家畜（ウシ、ヤギ、ヒツジ、シカ、水牛等）である。特に、牛乳や牛肉の生産のための乳用牛又は肉用牛に対して有効であり、以下乳用牛又は肉用牛を給与対象として例示して説明する。 The livestock subject to feeding nutrients in the present invention is a ruminant livestock (cattle, goat, sheep, deer, etc.) in which the enzyme stearoyl-CoA desaturase that converts TVA to CLA works in vivo and accumulates CLA in its milk and meat. Buffalo etc.). This is particularly effective for dairy cows or beef cows for the production of milk or beef. Hereinafter, dairy cows or beef cows will be exemplified and described as salary targets.
牛は、飼料中の多価不飽和脂肪酸（リノール酸やα−リノレン酸など）をルーメン中の微生物発酵によりＴＶＡ（トランス−１１ Ｃ１８：１）に変換して消化・吸収を行う。そして、吸収されたＴＶＡは、乳腺組織や脂肪組織などに移行し、不飽和化酵素であるｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅによってＣＬＡ（シス−９，トランス−１１ Ｃ１８：２）に変換される。しかし、その変換効率は約２０％程度と非常に低い。本発明では、ルーメンの微生物による発酵状態を正常に保ったままでこの酵素の遺伝子発現及び酵素活性を高めることが可能な栄養素を牛に給与することで、牛乳の乳量や乳質（風味、乳脂肪率等）、牛肉の肉質（風味、食感等）へ悪い影響を及ぼすことなく効率的にＣＬＡ濃度を高めるものである。 Cattle digest and absorb by converting polyunsaturated fatty acids (such as linoleic acid and α-linolenic acid) in feed into TVA (trans-11 C18: 1) by microbial fermentation in rumen. Then, the absorbed TVA is transferred to mammary gland tissue, adipose tissue, etc., and converted to CLA (cis-9, trans-11 C18: 2) by stearoyl-CoA desaturase which is an desaturase. However, the conversion efficiency is as low as about 20%. In the present invention, by supplying a cow with nutrients capable of enhancing the gene expression and enzyme activity of this enzyme while maintaining the fermentation state of rumen microorganisms in a normal state, milk yield and milk quality (flavor, milk fat) The CLA concentration is efficiently increased without adversely affecting the beef quality (flavor, texture, etc.).
ｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅの遺伝子発現及び酵素活性を高めるために牛に給与する栄養素としては、タンパク質代謝、あるいはエネルギー代謝を調節するものが選ばれる。これは、ｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅ遺伝子のプロモーターにエネルギー関連転写因子結合可能領域が認められ、また乳腺組織や脂肪組織ではタンパク質合成が盛んなためである。そこで、各栄養素について鋭意研究した結果、特に、牛の乳腺組織中ではリジン、ロイシン、アルギニン、ビオチン、ビタミンＣが、脂肪組織中ではリジン、ビオチンが非常に効果的に遺伝子発現及び酵素活性を高めることを発見し、本発明を完成させるに至った。これらの栄養素は、乳牛等の飼料への配合実績があり、ルーメンの発酵状態に影響を与えないことが明らかとなっているものである。 Nutrients fed to cows to increase stearoyl-CoA desaturase gene expression and enzyme activity are those that regulate protein metabolism or energy metabolism. This is because an energy-related transcription factor-binding region is recognized in the promoter of the stearoyl-CoA desaturase gene, and protein synthesis is active in mammary gland tissue and adipose tissue. Therefore, as a result of diligent research on each nutrient, lysine, leucine, arginine, biotin and vitamin C are particularly effective in bovine mammary gland tissue, and lysine and biotin enhance gene expression and enzyme activity very effectively in adipose tissue. This has been discovered and the present invention has been completed. These nutrients have a proven track record in feeds such as dairy cows, and it has become clear that they do not affect the fermentation state of rumen.
上記栄養素は、各アミノ酸やビタミンの純品乾燥物が使用できることはもちろんのこと、その粗精製物（ペースト化物、希釈物、乳化物、懸濁物など）も使用可能である。また、デンプンやデキストリン等の賦形剤を加えて顆粒化したり、タブレットにしたりして製剤化したものも使用可能である。さらには、ルーメンで分解されないように油脂等でコーティングしたもの（バイパスアミノ酸等）も使用可能である。 As the nutrients, not only pure products of amino acids and vitamins can be used, but also crudely purified products (pastes, dilutions, emulsions, suspensions, etc.) can be used. In addition, it is possible to use a granulated product by adding an excipient such as starch or dextrin, or a tableted product. Furthermore, what was coated with fats and oils etc. (bypass amino acid etc.) so that it may not be decomposed | disassembled with a lumen | rumen can also be used.
上記栄養素は、これを有効成分として、そのまま、飼料添加物、飼料、飼料組成物、動物医薬、促進剤（増強剤）、その他の剤として使用することができる。また、上記栄養素は、常用される飼料成分を添加、混合、使用して、飼料添加物、飼料、飼料組成物を提供することも可能である。そのうえ、上記栄養素を有効成分とする剤も提供することができる。この場合は、動物医薬製剤の常法にしたがって製剤化すればよい。 The above nutrient can be used as it is as an active ingredient, as it is, as a feed additive, feed, feed composition, animal medicine, promoter (enhancement agent), and other agents. Moreover, the said nutrient can also provide a feed additive, a feed, and a feed composition by adding, mixing and using a commonly used feed component. In addition, an agent containing the above nutrient as an active ingredient can also be provided. In this case, it may be formulated according to a conventional method for animal pharmaceutical preparations.
有効成分としては、上記栄養素の純品乾燥物それ自体及び／又はその粗精製物（濃縮物、ペースト化物、乾燥物、稀釈物、乳化物、懸濁物）の少なくともひとつが使用される。本有効成分は、種々の形態で投与される。その投与形態としては例えば錠剤、カプセル剤、顆粒剤、散剤、シロッブ剤等による経口投与をあげることができる。これらの各種製剤は、常法に従って主薬に賦形剤、結合剤、崩壊剤、滑沢剤、矯味矯臭剤、溶解補助剤、懸濁剤、コーティング剤などの動物医薬の製剤技術分野において通常使用しうる既知の補助剤を用いて製剤化することができる。 As the active ingredient, at least one of the above-mentioned pure dried product of the nutrient itself and / or its crude product (concentrate, pasted product, dried product, diluted product, emulsion product, suspension) is used. The active ingredient is administered in various forms. Examples of the dosage form include oral administration using tablets, capsules, granules, powders, sylobic agents and the like. These various preparations are usually used in the field of veterinary pharmaceutical formulation techniques such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, coating agents, etc. It can be formulated with known adjuvants.
なお、このようにして製剤化してなる動物医薬は、飼料組成物として使用することも充分可能であって、それ自体を飼料として直接家畜に給与することもできるし、飼料添加剤として他の飼料原料に添加、混合して用いることも可能である。 The veterinary medicine thus formulated can also be used as a feed composition, and can be fed directly to livestock as a feed, or other feed as a feed additive. It is also possible to add and mix with the raw material.
以下、本発明を乳牛を例にとって説明するが、本発明は乳牛のみに限定されるものではなく、各種の反芻家畜を広くその対象とするものである。 Hereinafter, although this invention is demonstrated taking a dairy cow as an example, this invention is not limited only to a dairy cow, The various ruminant livestock is made into the object widely.
栄養素の給与量としては特に制限はないが、例えば、栄養素としてビタミンなら１０〜５００ｍｇ／日（好ましくは１００〜３００ｍｇ／日）、アミノ酸なら１〜５０ｇ／日（好ましくは３０〜５０ｇ／日）で、搾乳期間連続して給与するのが好ましい。また、栄養素を配合する飼料には、ＣＬＡ及びＴＶＡの基質となる多価不飽和脂肪酸（リノール酸やα−リノレン酸）が充分含まれていることが好ましい。例えば、糖脂質やリン脂質が含まれる牧草主体の飼料、醤油粕やしょうゆ油を含む飼料、トリグリセリドが多く含まれる濃厚飼料などが好ましい。飼料に使用する油脂類としては、大豆油、綿実油、コーン油、サフラワー油、菜種油、ひまわり油、ごま油、こめ油、亜麻仁油などが好ましい。 There is no particular limitation on the amount of nutrients to be supplied. For example, vitamins are 10 to 500 mg / day (preferably 100 to 300 mg / day) for vitamins, and 1 to 50 g / day (preferably 30 to 50 g / day) for amino acids. It is preferable to feed the milking period continuously. Moreover, it is preferable that the polyunsaturated fatty acid (linoleic acid and (alpha) -linolenic acid) used as the substrate of CLA and TVA is fully contained in the feed which mixes a nutrient. For example, grass-based feeds containing glycolipids and phospholipids, feeds containing soy sauce cake and soy sauce oil, concentrated feeds rich in triglycerides, and the like are preferable. As fats and oils used for feed, soybean oil, cottonseed oil, corn oil, safflower oil, rapeseed oil, sunflower oil, sesame oil, rice bran oil, linseed oil and the like are preferable.
これらの構成をとることにより、乳用牛や肉用牛のルーメンの微生物による発酵状態を正常に保ったまま、生体内のｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅの遺伝子発現及び酵素活性を高めて、ＣＬＡの産生能の亢進が可能となるだけでなく、これによって効率的なＣＬＡ濃度の高い機能性乳、機能性肉を生産する新規飼養システムを提供することができる。 By adopting these constitutions, the gene expression and enzyme activity of stearoyl-CoA desaturase in vivo are increased while maintaining the normal fermentation state by the microorganisms of the rumen of dairy cattle and beef cattle, and the CLA production ability This makes it possible to provide a new breeding system that produces functional milk and functional meat with high CLA concentration.
以下、本発明の実施例について述べるが、本発明はこれらのみに限定されるものではない Examples of the present invention will be described below, but the present invention is not limited to these examples.
ウシ乳腺組織由来の乳腺上皮細胞及び、ウシ脂肪組織由来のｓｔｒｏｍａｌ ｖａｓｃｕｌａｒ細胞（間質血管細胞）におけるＣＬＡ産生に関連するｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅの作用と制御機構についてｉｎ ｖｉｔｒｏ試験を行った。 In vitro tests were conducted on the action and control mechanism of stearoyl-CoA desaturase related to CLA production in bovine mammary tissue-derived mammary epithelial cells and bovine adipose tissue-derived stromal vascular cells (stromal vascular cells).
乳腺上皮細胞は、Ｊ．Ｅｎｄｃｒｉｎｏｌ．，１６９：３８１−３８８（２００１）に記載の方法に従って取得した。すなわち、ウシ乳腺組織より乳腺実質部を取り出し、コラゲナーゼ処理を行った後ナイロンメッシュにより細胞を得て、数個〜数十個の細胞をトリプシン処理して細胞を分散して、ホルモン処理をしてクローニングした分化可能な乳腺上皮細胞を試験に供した。 Mammary epithelial cells are described in J. Endcrilinol. , 169: 381-388 (2001). That is, the mammary gland parenchyma is taken out from bovine mammary gland tissue, and after collagenase treatment, cells are obtained with a nylon mesh, several to several tens of cells are trypsinized to disperse the cells, and then subjected to hormone treatment. The cloned differentiable mammary epithelial cells were subjected to the test.
ｓｔｒｏｍａｌ ｖａｓｃｕｌａｒ細胞は、Ｊ．Ａｎｉｍａｌ Ｓｃｉ．，８２：４２９−４３７（２００４）に記載の方法に従い、ウシの脂肪組織を酵素処理することによって得たｓｔｒｏｍａｌ ｖａｓｃｕｌａｒのｐｒｉｍａｒｙ細胞を試験に供した。 Stromal vascular cells are described in J. Org. Animal Sci. , 82: 429-437 (2004), basal primary cells obtained by enzymatic treatment of bovine adipose tissue were subjected to the test.
これらの乳腺上皮細胞及びｓｔｒｏｍａｌ ｖａｓｃｕｌａｒ細胞を培養皿に播種し、１０％Ｆｅｔａｌ ｂｏｖｉｎｅ ｓｅｒｕｍ（ＦＢＳ）を含むＤＭＥＭ培地にて、５％ＣＯ２条件下で培養した。試験は、コラーゲンｔｙｐｅＩをコートした１２ｗｅｌｌプレートに１×１０４／ｃｍ２の細胞を播種し、ｓｔｒｏｍａｌ ｖａｓｃｕｌａｒ細胞はサブコンフルエントの状態で、乳腺上皮細胞はサブコンフルエントの状態にＬａｃｔｏｇｅｎｉｃ ｈｏｒｍｏｎｅ ｃｏｍｐｌｅｘを添加し、４８時間培養して、分化させた後に試験に供した。分化させない乳腺上皮細胞をｂａｓｉｃ ｃｏｎｔｒｏｌのサンプルとして用いた。 These mammary epithelial cells and stromal vascular cells were seeded on a culture dish and cultured in DMEM medium containing 10% Fetal bovine serum (FBS) under 5% CO 2 conditions. In the test, 1 × 10 4 / cm 2 cells were seeded on a 12-well plate coated with collagen type I, the basal vascular cells were subconfluent, the mammary epithelial cells were subconfluent, and lactogenic harmonic complex was added. The cells were cultured for 48 hours and differentiated before being subjected to the test. Non-differentiated mammary epithelial cells were used as samples for basic control.
（１）ｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅ遺伝子発現に対する各種ホルモンの影響。
ｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅ（ＳＣＤ）遺伝子発現に対する各種ホルモンの影響を見るために、以下の試験を行った。
分化させた乳腺上皮細胞あるいはｓｔｒｏｍａｌ ｖａｓｃｕｌａｒ細胞を無血清培地で１２時間培養した後、インスリン様成長因子−１（ＩＧＦ−１）、インスリン、成長ホルモン（ＧＨ）（各１０−１０００ｎｇ／ｍｌ）およびＬａｃｔｏｇｅｎｉｃ ｈｏｒｍｏｎｅ ｃｏｍｐｌｅｘ（１０μｇ／ｍｌ ｄｅｘａｍｅｔｈａｓｏｎｅ、１０μｇ／ｍｌ インスリン、１０μｇ／ｍｌ ｐｒｏｌａｃｔｉｎ）を無血清のＤＭＥＭ培地に添加し、２４時間培養後、細胞を回収した。ＳＣＤ遺伝子発現応答はリアルタイムＰＣＲで観察した。分化誘導を行わない乳腺上皮細胞をｂａｓｉｃ ｃｏｎｔｒｏｌとした。リボゾームタンパク質Ｓ９（ＲＰＳ９；Ｊ．Ｄａｉｒｙ Ｓｃｉ．，９０：２２４６−２２５２（２００７））をｈｏｕｓｅ ｋｅｅｐｉｎｇ ｇｅｎｅとして使用した。以後、遺伝子発現の結果はすべてＳＣＤ／ＲＰＳ９で示している。
(1) Effects of various hormones on stearoyl-CoA desaturase gene expression.
In order to see the effect of various hormones on stearoyl-CoA desaturase (SCD) gene expression, the following tests were performed.
Differentiated mammary epithelial cells or stromal vascular cells are cultured in serum-free medium for 12 hours, and then insulin-like growth factor-1 (IGF-1), insulin, growth hormone (GH) (each 10-1000 ng / ml) and lactogenic A homone complex (10 μg / ml dexamethasone, 10 μg / ml insulin, 10 μg / ml protease) was added to serum-free DMEM medium, and the cells were collected after 24 hours of culture. The SCD gene expression response was observed by real-time PCR. Mammary epithelial cells that were not induced to differentiate were designated as basic controls. Ribosomal protein S9 (RPS9; J. Dairy Sci., 90: 2246-2252 (2007)) was used as the house keeping gene. Hereinafter, all the results of gene expression are shown as SCD / RPS9.
リアルタイムＰＣＲに使用したプライマーの塩基配列を、配列番号１〜４及び表１に示した。具体的には、ＳＣＤ遺伝子発現解析のためのフォワードプライマーを配列番号１及び表１上段左に、リバースプライマーを配列番号２及び表１上段右に、ＲＰＳ９遺伝子発現解析のためのフォワードプライマーを配列番号３及び表１下段左に、リバースプライマーを配列番号４及び表１下段右に示した。 The base sequences of the primers used for real-time PCR are shown in SEQ ID NOs: 1 to 4 and Table 1. Specifically, the forward primer for SCD gene expression analysis is shown in SEQ ID NO: 1 and the upper left of Table 1, the reverse primer is shown in SEQ ID NO: 2 and upper right of Table 1, and the forward primer for RPS9 gene expression analysis is shown in SEQ ID NO: The reverse primer is shown in SEQ ID NO: 4 and in the bottom right of Table 1 on the left side of 3 and Table 1.
乳腺上皮細胞におけるＩＧＦ−１、インスリン及びＧＨに対するＳＣＤ遺伝子発現の応答を図１に示した。乳腺上皮細胞のＳＣＤ遺伝子発現は、Ｌａｃｔｏｇｅｎｉｃ ｈｏｒｍｏｎｅの添加により約１．６倍に上昇した。一方、インスリン及びＩＧＦ−１を分化誘導後の細胞の培養培地に添加すると、添加量に応じた濃度依存的な遺伝子発現上昇が認められ、インスリンでは１μｇ／ｍｌの添加の濃度においてコントロールに比べ約３倍、ＩＧＦ−１では約４倍に上昇した。一方、ＧＨに対する応答は認められなかった。 The response of SCD gene expression to IGF-1, insulin and GH in mammary epithelial cells is shown in FIG. The expression of SCD gene in mammary epithelial cells was increased about 1.6 times by the addition of lactogenic homone. On the other hand, when insulin and IGF-1 were added to the cell culture medium after differentiation induction, a concentration-dependent increase in gene expression was observed depending on the amount added, and insulin was about 1 μg / ml compared to the control at a concentration of 1 μg / ml. It increased 3 times and about 4 times with IGF-1. On the other hand, no response to GH was observed.
ｓｔｒｏｍａｌ ｖａｓｃｕｌａｒ細胞におけるＩＧＦ−１及びインスリンに対するＳＣＤ遺伝子発現の応答を図２に示した。ｓｔｒｏｍａｌ ｖａｓｃｕｌａｒ細胞のＳＣＤ遺伝子発現も、インスリン及びＩＧＦ−１に応答する傾向が認められ、特にインスリン１μｇ／ｍｌの添加ではコントロールとの比較でｐ＝０．０７であったものの、その発現の上昇は乳腺上皮細胞に比べてやや低いものであった。 The response of SCD gene expression to IGF-1 and insulin in stromal vascular cells is shown in FIG. The SCD gene expression in the stromal vascular cells also showed a tendency to respond to insulin and IGF-1, and in particular, the addition of 1 μg / ml of insulin was p = 0.07 compared to the control, but the increase in the expression was Slightly lower than mammary epithelial cells.
これらの結果から、乳腺上皮細胞及びｓｔｒｏｍａｌ ｖａｓｃｕｌａｒ細胞、特に分化した乳腺上皮細胞では、遺伝子導入により調査されたＳＣＤプロモーター解析の報告と同様に、ＳＣＤ遺伝子発現はインスリンとＩＧＦ−１により亢進されることが明らかとなった。これは、これらの細胞でのＳＣＤ遺伝子発現が、他の組織と同様な転写因子の制御を受けている可能性が高いことを明らかにしている。 From these results, SCD gene expression is enhanced by insulin and IGF-1 in mammary epithelial cells and stromal vascular cells, particularly differentiated mammary epithelial cells, as reported in the SCD promoter analysis investigated by gene transfer. Became clear. This reveals that the SCD gene expression in these cells is likely to be controlled by transcription factors similar to those in other tissues.
（２）ｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅ遺伝子発現に対する各種栄養素添加の影響。
ＳＣＤ遺伝子発現に対する各種栄養素添加の影響を見るために、以下の試験を行った。
試験に用いた栄養素は、（１）の試験結果及びＳＣＤプロモーターにエネルギー関連転写因子結合可能領域が認められること、乳汁分泌及び細胞分化のためのタンパク質合成が盛んなことを考慮して、タンパク質代謝、あるいはエネルギー代謝を調節する栄養素を選出した。
そして、（１）と同様に分化させた乳腺上皮細胞あるいはｓｔｒｏｍａｌ ｖａｓｃｕｌａｒ細胞を無血清培地で１２時間培養した後、アミノ酸（リジン、ロイシン、グルタミン酸、アルギニン、メチオニン）、ミネラル（Ｍｇ、Ｍｎ、Ｃａ）、ウリジル酸、脂肪酸（酢酸、オクタン酸）、ビタミン（ビタミンＡ、ビタミンＣ、ビオチン）、コレステロールを無血清のＤＭＥＭ培地に４００μＭ添加し、２４時間培養して細胞を回収した。ＳＣＤ遺伝子発現応答は（１）と同様にリアルタイムＰＣＲで観察した。
(2) Effect of various nutrients on stearoyl-CoA desaturase gene expression.
In order to see the effect of various nutrient additions on SCD gene expression, the following tests were performed.
Nutrients used in the test were analyzed for protein metabolism in consideration of the test results in (1), the presence of an energy-related transcription factor binding region in the SCD promoter, and active protein synthesis for lactation and cell differentiation. Or, select nutrients that regulate energy metabolism.
Then, after differentiation of mammary epithelial cells or stromal vascular cells in the same manner as in (1) for 12 hours in serum-free medium, amino acids (lysine, leucine, glutamic acid, arginine, methionine), minerals (Mg, Mn, Ca) 400 μM uridylic acid, fatty acids (acetic acid, octanoic acid), vitamins (vitamin A, vitamin C, biotin) and cholesterol were added to serum-free DMEM medium, and the cells were collected by culturing for 24 hours. The SCD gene expression response was observed by real-time PCR as in (1).
乳腺上皮細胞における、各種栄養素に対するＳＣＤ遺伝子発現の応答を図３に示した。乳腺上皮細胞のＳＣＤ遺伝子発現は、リジン、ロイシン、アルギニンの添加により約２倍に上昇した。さらに、ビタミンＣ及びビオチンの添加でも高発現が認められた。 The response of SCD gene expression to various nutrients in mammary epithelial cells is shown in FIG. SCD gene expression in mammary epithelial cells was increased about 2-fold by the addition of lysine, leucine and arginine. Furthermore, high expression was observed even when vitamin C and biotin were added.
ｓｔｒｏｍａｌ ｖａｓｃｕｌａｒ細胞における各種栄養素に対するＳＣＤ遺伝子発現の応答を図４に示した。ｓｔｒｏｍａｌ ｖａｓｃｕｌａｒ細胞のＳＣＤ遺伝子発現は、乳腺上皮細胞と比較して応答性は低かったが、ほぼ同様の傾向を示し、リジン及びビオチン添加時に有意な発現上昇が認められた。これらの結果から、上記栄養素は乳腺上皮細胞あるいはｓｔｒｏｍａｌ ｖａｓｃｕｌａｒ細胞中のｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅ遺伝子発現を高める栄養素であることが明らかとなった。 FIG. 4 shows the response of SCD gene expression to various nutrients in the stromal vascular cells. Although the responsiveness of the SCD gene expression in the stromal vascular cells was lower than that of the mammary epithelial cells, it showed almost the same tendency, and a significant increase in expression was observed when lysine and biotin were added. From these results, it was revealed that the nutrient is a nutrient that enhances the expression of stearoyl-CoA desaturase gene in mammary epithelial cells or stromal vascular cells.
（３）ｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅ遺伝子発現を制御する栄養素添加時のｓｔｅａｒｏｙｌ‐ＣｏＡ ｄｅｓａｔｕｒａｓｅ活性に対する影響。
分化後のウシ乳腺上皮細胞にリジン及びビオチンを５００μＭの濃度で添加し、２４時間培養後の細胞ミクロソームのｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅ活性をフェナシーレブロミドにより脂肪酸を誘導化し、高速液体クロマトグラフィー（ＨＰＬＣ）で定量する方法で測定した。基質はパルミチン酸を用い、活性は１分間に１ｎｍｏｌのパルミトオレイン酸を産生する活性を１Ｕとした。
(3) Influence on the activity of stearoyl-CoA desaturase at the time of addition of nutrients that control stearoyl-CoA desaturase gene expression
Lysine and biotin are added to the differentiated bovine mammary epithelial cells at a concentration of 500 μM, and the stearoyl-CoA desaturase activity of the cell microsomes after 24 hours of culture is derivatized with phenacylrebromide and subjected to high performance liquid chromatography (HPLC). Measured by a quantitative method. Palmitic acid was used as the substrate, and the activity was 1 U for producing 1 nmol of palmitooleic acid per minute.
結果を図５に示した。リジン及びビオチンの添加により、乳腺上皮細胞のｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅ活性は明らかに増加した。これは、リジン及びビオチンによりＳＣＤ遺伝子発現が活性化されるだけでなく、ｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅの酵素活性自体が高められたことによるものと考えられる。これらにより、上記栄養素はＳＣＤ遺伝子発現を活性化するとともに、ｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅの酵素活性を高め、ＴＶＡからＣＬＡへの変換を促進する能力を持つことが明らかとなった。 The results are shown in FIG. The addition of lysine and biotin clearly increased the stearoyl-CoA desaturase activity of mammary epithelial cells. This is considered to be due to the fact that not only SCD gene expression was activated by lysine and biotin but also the enzyme activity of stearoyl-CoA desaturase itself was enhanced. These revealed that the nutrients have the ability to activate SCD gene expression, increase the enzyme activity of stearoyl-CoA desaturase, and promote the conversion from TVA to CLA.
（４）ＳＣＤ遺伝子発現を制御する栄養素添加時の乳腺上皮細胞のＣＬＡ産生能に対する影響
ｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅを活性化する栄養素であるリジン及びビオチンが、乳腺上皮細胞内で、ＴＶＡからのＣＬＡ産生能を亢進することができるかについて試験を行った。培地中に、ＴＶＡ（１００μＭ）と栄養素（５００μＭ）を同時添加し、添加後２４時間における細胞内のＣＬＡ産生量をガスクロマトグラフィーで測定した。
(4) Influence on the CLA production ability of mammary epithelial cells upon addition of nutrients that control SCD gene expression The lysine and biotin that activate stearoyl-CoA desaturase are capable of producing CLA from TVA in the mammary epithelial cells. A test was conducted as to whether or not TVA (100 μM) and nutrients (500 μM) were simultaneously added to the medium, and intracellular CLA production in 24 hours after the addition was measured by gas chromatography.
結果を図６に示した。乳腺上皮細胞内のＴＶＡからＣＬＡの産生能は、ＴＶＡのみを添加した細胞と無添加の細胞をコントロールとした。コントロールでのＣＬＡ量を１とした場合、リジン添加区では１．７０（Ｐ＝０．１１８）、ビオチン添加区では１．８０（Ｐ＝０．０８１）であり、明らかに増加する傾向が認められた（ｎ＝４）。ＳＣＤ遺伝子発現を高め、ＳＣＤ酵素を活性化したリジンとビオチンの乳腺上皮細胞への添加は、ＣＬＡの産生量を増加させた。これらにより、リジンとビオチンが乳腺上皮細胞の細胞内におけるＳＣＤの活性化を介してＴＶＡからＣＬＡの産生を増加させていることが明らかになった。 The results are shown in FIG. The ability to produce CLA from TVA in mammary epithelial cells was controlled by cells added with TVA alone and cells without additive. When the CLA amount in the control was 1, it was 1.70 (P = 0.118) in the lysine-added group and 1.80 (P = 0.081) in the biotin-added group. (N = 4). Addition of lysine and biotin with enhanced SCD gene expression and SCD enzyme activation to mammary epithelial cells increased the production of CLA. These revealed that lysine and biotin increased the production of CLA from TVA through the activation of SCD in the mammary epithelial cells.
各種栄養素による発現応答性を比較検討すると、蛋白質合成に関与するアミノ酸であるリジン、ロイシンと、機能性物質であるＮＯ産生の基質となるアミノ酸であるアルギニンを添加すると、乳腺上皮細胞におけるｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅ遺伝子発現は上昇した。特に、リジンではｓｔｒｏｍａｌ ｖａｓｃｕｌａｒ細胞においても発現を有意に亢進させたことから、ｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅ遺伝子発現に大きく関与していると推察された。さらに、ビタミンＣやビオチンでも有意な発現上昇が認められたが、ビオチンは蛋白質合成にも関与するビタミンであり、一般に乳牛では不足している可能性が示唆されている。また、ビオチンは蹄病の予防にも有効であることが知られており、同時にｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅを活性化し、乳及び肉中のＣＬＡを増加させることができれば、有効な飼料・添加物として活用可能であることが示唆された。 Comparing the expression responsiveness of various nutrients, lysine and leucine, which are amino acids involved in protein synthesis, and arginine, which is a functional substance, a substrate for NO production, add stearoyl-CoA in mammary epithelial cells. desaturase gene expression was elevated. In particular, lysine significantly increased the expression in stromal basal cells, so it was assumed that lysine was greatly involved in stearoyl-CoA desaturase gene expression. Furthermore, a significant increase in expression was also observed with vitamin C and biotin, but biotin is a vitamin involved in protein synthesis, and it is generally suggested that it may be deficient in dairy cows. Biotin is also known to be effective in preventing hoof disease, and at the same time, if stearoyl-CoA desaturase can be activated to increase CLA in milk and meat, it can be used as an effective feed and additive. It was suggested that it was possible.
特に、リジンやビオチンは、乳腺上皮細胞のミクロソーム画分でＳＣＤ活性を有意に上昇させ、乳腺上皮細胞内でＴＶＡからＣＬＡの産生を促進していることも明らかになった。リジンやビオチンは、乳腺細胞や脂肪細胞においてｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅ遺伝子発現を介して、酵素活性を高め、ＴＶＡからＣＬＡの産生を増加させていることから、これらの栄養素は、乳・肉中のＣＬＡを上昇させ、機能性乳や機能性肉を生産させるのに画期的な飼料・添加物として活用可能であることが示唆された。 In particular, lysine and biotin have also been shown to significantly increase SCD activity in the microsomal fraction of mammary epithelial cells and promote production of CLA from TVA in mammary epithelial cells. Since lysine and biotin increase enzyme activity and increase production of CLA from TVA through stearoyl-CoA desaturase gene expression in mammary cells and adipocytes, these nutrients are produced in CLA in milk and meat. It was suggested that it can be used as an innovative feed and additive for producing functional milk and functional meat.
乳腺上皮細胞を用いたｉｎ ｖｉｔｒｏ試験で得られた結果に基づいて、泌乳牛にビオチンあるいはリジン含有飼料を給与した場合の牛乳中のＣＬＡ濃度の挙動確認のため、ｉｎ ｖｉｖｏ試験を行った。 Based on the results obtained in an in vitro test using mammary epithelial cells, an in vivo test was performed to confirm the behavior of CLA concentration in milk when a lactating cow was fed a biotin- or lysine-containing feed.
泌乳牛７頭（平均体重７１８ｋｇ、平均乳量２６ｋｇ／日、平均分娩日数１２８日）を用いて、基礎飼料に脂肪酸Ｃａ１８０ｇ添加した区（対照区）、脂肪酸Ｃａ１８０ｇにビオチンを１日当たり２００ｍｇ添加した区（ビオチン区）、脂肪酸Ｃａ１８０ｇにバイパスリジン（リジンがルーメンで分解されないように脂肪酸でコーティングしたもの）を１日当たり２００ｇ添加した区（リジン区、リジン量として４０ｇ／日）の３つの飼料区で各区１４日間のラテン方格実験配置で試験を行った。用いた基礎飼料は、乾物重量比でスーダングラス乾草１４．１％、トウモロコシサイレージ２０．９％、アルファルファヘイキューブ２０．４％、自家配合飼料（ビートパルプ１、綿実２、ふすま３、くず米２、大豆粕１の割合で混合）１１．６％、乳牛用配合飼料（明治飼糧社製品：ワタナベハイ乳配）３３．１％の組成とした。なお、乳牛用配合飼料のワタナベハイ乳配（明治飼糧社製品）は、粗蛋白質１６．０％以上、粗脂肪２．５％以上、粗繊維７．０％以下、粗灰分７．０％以下、カルシウム０．４０％以上、リン０．３０％以上、ＴＤＮ（可消化養分総量）７６．０％以上が含有成分として保証されている飼料である。
Using 7 lactating cows (average body weight 718 kg, average milk yield 26 kg / day, average number of days of delivery 128 days), a group in which 180 g fatty acid Ca was added to the basic feed (control group), and a group in which 200 mg biotin was added to fatty acid Ca 180 g per day (Biotin group), each feed group of three groups of feed (200g / day lysine group, 40g / day as lysine amount) in which 200g per day of bypass lysine (coated with fatty acid so that lysine is not decomposed by rumen) was added to fatty acid Ca 180g Tests were conducted in a 14-day Latin square experimental setup. The basic feed used was 14.1% Sudangrass hay, 20.9% corn silage, 20.4% alfalfa hay cube, dry feed (
試験は、各区の最終２日間で行い、各区の乳牛の乳量、得られた牛乳の乳成分量（乳糖、乳蛋白質、乳脂肪、無脂乳固形分）、乳脂肪酸組成（トランスバクセン酸、リノール酸、共役リノール酸の脂肪酸メチルエステル（ＦＡＭＥ）１００ｍｇ中の含有量）、共役リノール酸産生量（１日当たり）について測定した。
各区の乳量及び乳成分量の平均値を表２に、乳脂肪酸組成及び共役リノール酸産生量の平均値を表３に示した。乳脂肪は、ビオチンやリジン含有飼料給与区で増加する傾向が見られたほかは、ビオチン及びリジン含有飼料給与による変化は見られなかった。つまり、ビオチン及びリジン含有飼料を給与しても、基本的な乳成分量等の乳品質には影響を与えないことが明らかとなった。
乳脂肪酸組成については、ビオチンやリジン含有飼料の給与によりＣＬＡが有意に増加し、共役リノール酸含量が０．６０ｍｇ／１００ｍｇＦＡＭＥ以上に高まった牛乳が得られた。また、牛乳中に分泌されるＣＬＡの総量も１日当たり８ｇ以上まで増加した。これを総脂肪量当たりのＣＬＡ含有量で比較すると、対照区が総脂肪量の０．５３重量％であるのに対し、ビオチン区が総脂肪量の０．６０重量％、リジン区が総脂肪量の０．６３重量％であった。なお、乳脂肪酸組成のうち、ＴＶＡには変化が見られなかった。さらに、リノール酸は、ビオチンやリジン含有飼料投与によって明らかに減少した。
この結果、ｉｎ ｖｉｖｏにおいて、泌乳牛の飼料にビオチンやリジンを添加することにより、乳中のＣＬＡ濃度を有意に増加させることができるのを確認できた。このような、乳中のＣＬＡ濃度を特定の栄養素給与で高めるという飼養技術の開発は画期的な成果といえ、この飼養技術により乳中のＣＬＡ濃度として総脂肪量の０．６０〜０．７０重量％、あるいは０．６０〜１．００重量％、あるいはそれ以上のＣＬＡ濃度の乳を得ることも可能となった。
The test is conducted in the last two days of each ward, and the milk amount of each dairy cow, the milk content of the obtained milk (lactose, milk protein, milk fat, non-fat milk solids), milk fatty acid composition (transbacsenic acid, Linoleic acid, content of conjugated linoleic acid in 100 mg of fatty acid methyl ester (FAME)) and conjugated linoleic acid production (per day) were measured.
Table 2 shows the average value of milk amount and milk component amount in each section, and Table 3 shows the average value of milk fatty acid composition and conjugated linoleic acid production. Milk fat did not change with feed containing biotin and lysine, except for a tendency to increase in feed groups containing biotin and lysine. In other words, it was clarified that feeding feed containing biotin and lysine does not affect milk quality such as basic milk component amount.
Regarding milk fatty acid composition, CLA was significantly increased by feeding with feed containing biotin or lysine, and milk with a conjugated linoleic acid content increased to 0.60 mg / 100 mg FAME or more was obtained. In addition, the total amount of CLA secreted in milk increased to 8 g or more per day. Comparing this with the CLA content per total fat, the control group is 0.53% by weight of the total fat, whereas the biotin group is 0.60% by weight of the total fat and the lysine group is total fat. 0.63% by weight of the amount. Of the milk fatty acid composition, TVA did not change. Furthermore, linoleic acid was clearly reduced by administration of biotin or lysine-containing feed.
As a result, it was confirmed in vivo that the concentration of CLA in milk can be significantly increased by adding biotin or lysine to the feed of lactating cows. The development of such a breeding technique of increasing the CLA concentration in milk with a specific nutrient supply is an epoch-making result. By this feeding technique, the CLA concentration in milk is 0.60 to 0. It became possible to obtain milk having a CLA concentration of 70% by weight, or 0.60 to 1.00% by weight, or more.
なお、本発明を要約すれば次のとおりである。
すなわち、本発明は、反芻家畜の乳及び肉中に含まれる、機能性物質である共役リノール酸の濃度を効率的に高める栄養学的技術を提供することを課題とする。そして、反芻家畜の乳腺組織や脂肪組織におけるｓｔｅａｒｏｙｌ−ＣｏＡ ｄｅｓａｔｕｒａｓｅの発現と酵素活性をリジン、ロイシン、アルギニン、ビオチン、ビタミンＣなどの栄養素の給与により高めて、乳腺組織や脂肪組織で効率的にトランスバクセン酸を共役リノール酸に変換させ、乳及び肉中の共役リノール酸濃度を高めることにより、上記課題を解決するものである。
The present invention is summarized as follows.
That is, an object of the present invention is to provide a nutritional technique for efficiently increasing the concentration of conjugated linoleic acid, which is a functional substance, contained in milk and meat of ruminant livestock. Then, the expression and enzyme activity of stearoyl-CoA desaturase in the mammary gland tissue and adipose tissue of ruminant livestock are increased by feeding nutrients such as lysine, leucine, arginine, biotin, vitamin C, etc. The above-mentioned problems are solved by converting vaccenic acid to conjugated linoleic acid and increasing the conjugated linoleic acid concentration in milk and meat.
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| US20160205969A1 (en) * | 2013-07-30 | 2016-07-21 | Benemilk Oy | Solid dietary compositions for ruminants and methods of making and using the same |
| JP2018078888A (en) * | 2016-11-05 | 2018-05-24 | Ｆｓｋ三昭株式会社 | Feed, feed production method, and feed production apparatus |
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| CN102210285A (en) * | 2011-07-04 | 2011-10-12 | 广西壮族自治区水牛研究所 | Milk-production buffalo feeding method for improving conjugated linoleic acid content in buffalo milk |
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| US20160205969A1 (en) * | 2013-07-30 | 2016-07-21 | Benemilk Oy | Solid dietary compositions for ruminants and methods of making and using the same |
| JP2018078888A (en) * | 2016-11-05 | 2018-05-24 | Ｆｓｋ三昭株式会社 | Feed, feed production method, and feed production apparatus |
| CN113980890A (en) * | 2021-11-03 | 2022-01-28 | 吉林大学 | Application method of lithium chloride for regulating bovine mammary epithelial cells |
| CN113980890B (en) * | 2021-11-03 | 2024-02-27 | 吉林大学 | Application method of lithium chloride for regulating bovine mammary epithelial cells |
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