JP2009044971A - New yeast which adds probiotic effect on ruminant farm animal to lactobacillus for silage preparation and use thereof - Google Patents
New yeast which adds probiotic effect on ruminant farm animal to lactobacillus for silage preparation and use thereof Download PDFInfo
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本発明は新規酵母および該酵母を利用して動物にプロバイオティック効果を付与する方法に関する。 The present invention relates to a novel yeast and a method for imparting a probiotic effect to an animal using the yeast.
現在、医療現場では抗生物質耐性菌の出現が問題となっている。病原菌が抗生物質耐性を獲得する原因の一つの可能性として、家畜飼料への抗菌性飼料添加物等の添加が指摘されている。EUでは、2006年1月より、家畜の成長促進目的で抗菌性飼料添加物を使用することが禁止された。食に対する消費者の安心・安全志向の高まりと共に、我が国でも抗菌性飼料添加物等の使用量削減が検討されており、抗菌性飼料添加物に頼らない家畜飼養技術の開発が急務となっている。 Currently, the emergence of antibiotic-resistant bacteria is a problem in the medical field. Addition of antibacterial feed additives and the like to livestock feed has been pointed out as one possible cause of pathogens acquiring antibiotic resistance. In January 2006, the EU banned the use of antimicrobial feed additives to promote livestock growth. Along with the increasing consumer safety and safety consciousness towards food, in Japan, the use of antibacterial feed additives, etc. is being studied, and the development of livestock rearing technology that does not rely on antibacterial feed additives is urgently needed. .
近年、家畜飼養の技術として、生菌剤の利用が注目されている。これは、生菌によるプロバイオティック効果、すなわち家畜の発育促進、腸内細菌叢の改善、免疫機能の向上、下痢の防止等を期待できることによる。 In recent years, the use of viable bacteria has attracted attention as a livestock breeding technique. This is because probiotic effects by viable bacteria, that is, growth promotion of livestock, improvement of intestinal bacterial flora, improvement of immune function, prevention of diarrhea and the like can be expected.
しかし現状では、家畜、特に反芻家畜に対して有効なプロバイオティック生菌剤の開発には至っていない。この理由は、反芻家畜の場合、経口摂取された生菌はまず反芻胃に流入するが、そこに共生するバクテリアによる競合的排除や原生動物による捕食によって、投与された生菌の生存性が多大な影響を受けるためと考えられる。したがって、ブタ、ヒト等の単胃動物用に開発された生菌を反芻家畜へそのまま使用しても有効な効果を得ることは困難である。反芻家畜を含む、家畜全般に対して有効なプロバイオティック生菌は、反芻家畜を用いた菌株投与試験によって新たに選抜する必要がある。 However, at present, no probiotic viable agent effective for livestock, particularly ruminant livestock, has been developed. The reason for this is that in the case of ruminant livestock, orally ingested viable bacteria first flow into the rumen, but the viability of the administered viable bacteria is greatly increased by competitive exclusion by bacteria that coexist there and by predation by protozoa. This is thought to be affected by the influence. Therefore, it is difficult to obtain an effective effect even if live bacteria developed for monogastric animals such as pigs and humans are directly used for ruminant livestock. It is necessary to newly select probiotic viable bacteria effective for all livestock including ruminant livestock by a strain administration test using ruminant livestock.
従来のプロバイオティック生菌剤として、例えば、サッカロマイセス（Saccharomyces）属酵母菌株を単独またはエンテロコッカス属乳酸菌と組み合わせてブタに投与し、抗下痢活性等を確認した例がある（特許文献１）。また、ブタ腸内から単離したエンテロコッカス属乳酸菌をビール酵母等とともに栄養組成物として調製し、該栄養組成物をブタに投与し、飼料要求率などを検討した例がある（特許文献２）。他には、ラクトバチルス属乳酸菌とサッカロマイセス（Saccharomyces）属酵母菌等をブタやブロイラーに投与した例（特許文献３）、各種乳酸菌とサッカロマイセス（Saccharomyces）属酵母菌をラット、ブロイラー等に投与した例（特許文献４）があるが、これらいずれの文献も、反芻家畜に対する生菌の効果を全く検討していない。 As a conventional probiotic viable agent, for example, there is an example in which Saccharomyces genus yeast strain is administered alone or in combination with Enterococcus lactic acid bacteria to confirm antidiarrheal activity (Patent Document 1). In addition, there is an example in which Enterococcus lactic acid bacteria isolated from the intestines of pigs are prepared as a nutritional composition together with brewer's yeast and the like, and the nutritional composition is administered to pigs to examine the feed requirement rate (Patent Document 2). Other examples include administration of Lactobacillus lactic acid bacteria and Saccharomyces yeasts to pigs and broilers (Patent Document 3), and administration of various lactic acid bacteria and Saccharomyces yeasts to rats, broilers, etc. Although there is (Patent Document 4), none of these documents considers the effect of viable bacteria on ruminant livestock at all.
反芻家畜は飼料として乳酸菌による発酵飼料であるサイレージを給与され、サイレージに含まれる大量の乳酸菌を日常的に摂取している。乳酸菌はプロバイオティック生菌として汎用されている微生物の一つである。よって反芻動物へのプロバイオティック効果を有するサイレージ調製用乳酸菌を選抜出来れば、その菌株は反芻家畜用生菌剤や反芻家畜用発酵飼料などとして利用可能であり、その反芻家畜に対する有用性は飛躍的に高まると考えられる。 Ruminant livestock are fed silage that is fermented feed by lactic acid bacteria as feed, and ingest large amounts of lactic acid bacteria contained in silage on a daily basis. Lactic acid bacteria are one of the microorganisms widely used as probiotic live bacteria. Therefore, if a lactic acid bacterium for silage preparation having a probiotic effect on ruminants can be selected, the strain can be used as a ruminant livestock bacterium or a ruminant livestock fermented feed. It is thought that it will increase.
これまでに様々な特性を備えたサイレージ調製用乳酸菌株が開発されている。このようなサイレージ調製用乳酸菌株の例として、ペディオコッカス・エスピーLA3株およびLA35株を挙げることができる（特許文献５）。上記菌株は、優れた耐酸性および高温耐性を有し、高品質のサイレージの調製を可能とする。また、ラクトバチルス・プランタルム畜草１号株およびラクトコッカス・ラクティスRO50株は、付着する乳酸菌が少なく茎が中空構造のため不良発酵しやすい飼料用稲を用いて高品質サイレージを調製するために開発された（特許文献６）。しかし、上記乳酸菌株は、あくまでもサイレージ調製用として乳酸発酵能を主な指標として選抜されているため、これら菌株がプロバイオティック効果を有するかどうかは検証されていない。 So far, lactic acid strains for silage preparation with various characteristics have been developed. Examples of such lactic acid strains for silage preparation include Pediococcus sp. Strain LA3 and LA35 (Patent Document 5). The above strain has excellent acid resistance and high temperature resistance, and enables the preparation of high-quality silage. In addition, Lactobacillus plantarum 1 and Lactococcus lactis RO50 were developed to prepare high-quality silage using rice for feed that is susceptible to poor fermentation due to a hollow stalk structure with few lactic acid bacteria. (Patent Document 6). However, since the above lactic acid strains are selected for the silage preparation using lactic acid fermentation ability as the main index, it has not been verified whether these strains have a probiotic effect.
また反芻家畜、特にウシに対し、乳酸菌等の菌株給与試験を行った報告も存在するが（非特許文献１−３）、検討された菌株は、ウシの発育促進、腸内フローラ改善、免疫機能の向上、下痢の防止等に対して明確なプロバイオティック効果を発揮していない。 In addition, although there are reports of lactic acid bacteria and other strain feeding tests on ruminant livestock, especially cattle (Non-patent Documents 1-3), the studied strains are cattle growth promotion, intestinal flora improvement, immune function It does not show a clear probiotic effect for improvement of diarrhea and prevention of diarrhea.
本発明は上記状況を鑑みてなされたものであり、本発明が解決しようとする課題は、乳酸菌と組み合わせることにより、反芻家畜等に対して発育促進、腸内細菌叢改善、免疫機能の向上、下痢の防止等のプロバイオティック効果を発揮する新規微生物の提供である。 The present invention has been made in view of the above situation, and the problem to be solved by the present invention is to promote growth against ruminant livestock and the like, improve intestinal bacterial flora, improve immune function, in combination with lactic acid bacteria, The provision of novel microorganisms that exert probiotic effects such as prevention of diarrhea.
上記課題を解決すべく、本発明者らは鋭意研究を行った。本発明者らはまず、牧草等から分離された様々なサイレージ調製用乳酸菌株をウシに投与してそのプロバイオティック効果を検証する試験を行った。しかし、サイレージ調製用乳酸菌株単独の投与試験では、ウシの発育促進、腸内フローラ改善、免疫機能の向上、下痢の防止等のプロバイオティック効果を有する菌株は選抜されなかった。例えば、本発明者らがラクトバチルス・プランタルム畜草１号株をウシに給与する試験を行ったところ、ラクトバチルス・プランタルム畜草１号株の単独給与ではウシの発育促進、腸内フローラ改善、免疫機能の向上、下痢の防止等のプロバイオティック効果は一切観察されなかった。よって、サイレージ調製用乳酸菌を反芻家畜用プロバイオティック生菌として有効利用するには、その機能を補完するための新たな技術開発が必要となることが判明した。そこで本発明者等は、様々な菌株の培養液を組み合わせてウシに給与し、そのプロバイオティック効果を検証したところ、酵母菌の一種であるCandida sp. (カンジダ・エスピー) CO119株をラクトバチルス・プランタルム畜草１号株と組み合わせた際に顕著なプロバイオティック効果が観察された。具体的には、上記菌株の組み合わせは、ウシの体重増加促進および飼料効率改善、粗飼料に対する消化率向上、腸内細菌叢の改善、下痢・軟便防止、液性および細胞性免疫機能向上の各種効果をもたらした。また、意外なことに、Candida sp. (カンジダ・エスピー) CO119株を単独でウシに投与しても、顕著なプロバイオティック効果は得られなかった。すなわち本発明は、乳酸菌と組み合わせることにより、反芻家畜に対してもプロバイオティック効果を発揮する新規酵母に関し、具体的には下記発明を提供するものである。
（１） 乳酸菌とともに投与することにより前記乳酸菌にプロバイオティック効果を付与する、カンジダ属に属する酵母菌カンジダ・エスピーCO119株（受託番号：NITE P-375）および／またはその処理物、
（２） 前記乳酸菌がサイレージ調製用乳酸菌である、上記（１）に記載の酵母菌および／またはその処理物、
（３） 前記サイレージ調製用乳酸菌がエンテロコッカス属乳酸菌またはラクトバチルス属乳酸菌である、上記（２）記載の酵母菌および／またはその処理物、
（４） 前記乳酸菌がラクトバチルス・プランタルム畜草１号株（FERM P-18930）である、上記（１）から（３）のいずれかに記載の酵母菌および／またはその処理物、
（５） 乳酸菌共存下においてプロバイオティック効果を発揮し、乳酸菌非共存下においてプロバイオティック効果を発揮しない、カンジダ属に属する酵母菌カンジダ・エスピーCO119株（受託番号：NITE P-375）、
（６） 上記（１）から（４）のいずれかに記載の酵母菌および／またはその処理物、あるいは上記（５）記載の酵母菌を含む、飼料添加剤、
（７） 下記（ａ）および（ｂ）を組み合わせてなる、プロバイオティック効果を有する飼料添加剤
（ａ）上記（１）から（４）のいずれかに記載の酵母菌および／またはその処理物、あるいは上記（５）記載の酵母菌
（ｂ）乳酸菌、
（８） 上記（１）から（４）のいずれかに記載の酵母菌および／またはその処理物、あるいは上記（５）記載の酵母菌を含む、飼料、
（９） 下記（ａ）および（ｂ）を組み合わせてなる、プロバイオティック効果を有する飼料。
（ａ）上記（１）から（４）のいずれかに記載の酵母菌および／またはその処理物、あるいは上記（５）記載の酵母菌
（ｂ）乳酸菌、
（１０） 上記（１）から（４）のいずれかに記載の酵母菌および／またはその処理物、あるいは上記（５）記載の酵母菌を含む、下痢治療および予防のいずれかまたは両方のための医薬品、
（１１） 下記（ａ）および（ｂ）を組み合わせてなる、上記（１０）記載の医薬品
（ａ）上記（１）から（４）のいずれかに記載の酵母菌および／またはその処理物、あるいは上記（５）記載の酵母菌
（ｂ）乳酸菌、
（１２） 上記（１）から（４）のいずれかに記載の酵母菌および／またはその処理物、あるいは上記（５）記載の酵母菌を含む、感染症の治療および予防のいずれかまたは両方のための医薬品、
（１３） 下記（ａ）および（ｂ）を組み合わせてなる、上記（１２）に記載の医薬品
（ａ）上記（１）から（４）のいずれかに記載の酵母菌および／またはその処理物、あるいは上記（５）記載の酵母菌
（ｂ）乳酸菌、
（１４） 上記（１）から（４）のいずれかに記載の酵母菌および／またはその処理物、あるいは上記（５）記載の酵母菌を含む、免疫担当細胞活性化剤、
（１５） 上記（１）から（４）のいずれかに記載の酵母菌および／またはその処理物、あるいは上記（５）記載の酵母菌を含む、消化改善剤、
（１６） 上記（１）から（４）のいずれかに記載の酵母菌および／またはその処理物、あるいは上記（５）記載の酵母菌を含む、整腸剤、
（１７） 上記（１）から（４）のいずれかに記載の酵母菌および／またはその処理物、あるいは上記（５）記載の酵母菌を含む、発育促進剤、
（１８） カンジダ・エスピーCO119株（受託番号：NITE P-375）および乳酸菌を含む組成物、
（１９） 下記（ａ）および（ｂ）を、同時にまたは別々に投与することを特徴とする、動物の下痢を治療または予防する方法
（ａ）上記（１）から（４）のいずれかに記載の酵母菌および／またはその処理物、あるいは上記（５）記載の酵母菌
（ｂ）乳酸菌、
（２０） 下記（ａ）および（ｂ）を、同時にまたは別々に投与することを特徴とする、動物の免疫機能を向上させる方法
（ａ）上記（１）から（４）のいずれかに記載の酵母菌および／またはその処理物、あるいは上記（５）記載の酵母菌
（ｂ）乳酸菌、
（２１） 下記（ａ）および（ｂ）を、同時にまたは別々に投与することを特徴とする、動物の感染症を治療および／または予防する方法
（ａ）上記（１）から（４）のいずれかに記載の酵母菌および／またはその処理物、あるいは上記（５）記載の酵母菌
（ｂ）乳酸菌、
（２２） 下記（ａ）および（ｂ）を、同時にまたは別々に投与することを特徴とする、動物の発育を促進する方法
（ａ）上記（１）から（４）のいずれかに記載の酵母菌および／またはその処理物、あるいは上記（５）記載の酵母菌
（ｂ）乳酸菌、
（２３） 下記（ａ）および（ｂ）を、同時にまたは別々に投与することを特徴とする、動物の腸内環境を改善させる方法。
（ａ）上記（１）から（４）のいずれかに記載の酵母菌および／またはその処理物、あるいは上記（５）記載の酵母菌
（ｂ）乳酸菌、
（２４） 上記（８）または（９）に記載の飼料を投与することを特徴とする、動物の下痢を治療または予防する方法、
（２５） 上記（８）または（９）に記載の飼料を投与することを特徴とする、動物の免疫機能を向上させる方法、
（２６） 上記（８）または（９）に記載の飼料を投与することを特徴とする、動物の発育を促進する方法、
（２７） 上記（８）または（９）に記載の飼料を投与することを特徴とする、動物の腸内環境を改善させる方法、
（２８） 上記（８）または（９）に記載の飼料を投与することを特徴とする、動物の感染症を予防および／または治療する方法、
（２９） 前記動物がヒト以外の動物である、上記（１９）から（２８）のいずれかに記載の方法。
In order to solve the above problems, the present inventors have conducted intensive research. The present inventors first conducted a test for verifying the probiotic effect by administering various lactic acid strains for silage preparation isolated from grass and the like to cattle. However, in the administration test of the lactic acid strain for silage preparation alone, no strain having a probiotic effect such as promotion of bovine growth, improvement of intestinal flora, improvement of immune function, prevention of diarrhea, etc. was selected. For example, when the present inventors conducted a test of feeding Lactobacillus plantarum No. 1 strain to cattle, the Lactobacillus plantarum No. 1 strain alone could promote cattle growth, improve intestinal flora, No probiotic effects such as improvement of immune function and prevention of diarrhea were observed. Therefore, it has been found that in order to effectively use the lactic acid bacterium for silage preparation as a live probiotic bacterium for ruminant livestock, it is necessary to develop a new technology to complement its function. Therefore, the inventors of the present invention combined the culture solutions of various strains and fed them to cattle and verified the probiotic effect. As a result, Candida sp. (Candida sp.) CO119 strain, a kind of yeast, was obtained. -A remarkable probiotic effect was observed when combined with Plantarm No. 1 strain. Specifically, the combination of the above strains has various effects of promoting weight gain of cattle and improving feed efficiency, improving digestibility for roughage, improving intestinal bacterial flora, preventing diarrhea and loose stool, improving humoral and cellular immune functions. Brought about. Surprisingly, no significant probiotic effect was obtained even when Candida sp. CO119 strain was administered alone to cattle. That is, this invention relates to the novel yeast which exhibits a probiotic effect also to ruminant livestock by combining with lactic acid bacteria, and provides the following invention specifically.
(1) A yeast Candida sp. CO119 strain (accession number: NITE P-375) belonging to the genus Candida and / or a processed product thereof, which imparts a probiotic effect to the lactic acid bacterium by administration together with the lactic acid bacterium,
(2) The yeast and / or processed product thereof according to (1) above, wherein the lactic acid bacterium is a lactic acid bacterium for silage preparation,
(3) The yeast and / or treated product thereof according to (2) above, wherein the lactic acid bacterium for silage preparation is Enterococcus lactic acid bacterium or Lactobacillus lactic acid bacterium,
(4) The yeast and / or processed product thereof according to any one of (1) to (3), wherein the lactic acid bacterium is Lactobacillus plantarum No. 1 strain (FERM P-18930),
(5) Candida sp. CO119 strain (accession number: NITE P-375) belonging to the genus Candida, which exhibits a probiotic effect in the presence of lactic acid bacteria and does not exhibit a probiotic effect in the absence of lactic acid bacteria,
(6) A feed additive comprising the yeast according to any one of (1) to (4) above and / or a processed product thereof, or the yeast according to (5) above,
(7) A feed additive having a probiotic effect, which is a combination of the following (a) and (b): (a) The yeast according to any one of (1) to (4) above and / or a processed product thereof Or yeast (b) lactic acid bacteria described in (5) above,
(8) A feed comprising the yeast according to any one of (1) to (4) above and / or a processed product thereof, or the yeast according to (5) above,
(9) A feed having a probiotic effect, which is a combination of the following (a) and (b).
(A) The yeast according to any one of (1) to (4) above and / or a processed product thereof, or the yeast according to (5) above (b) lactic acid bacteria,
(10) For either or both of treatment and prevention of diarrhea including the yeast according to any one of (1) to (4) above and / or a processed product thereof, or the yeast according to (5) above Pharmaceuticals,
(11) The pharmaceutical according to (10) above, which is a combination of the following (a) and (b): (a) the yeast and / or processed product thereof according to any of (1) to (4) above, or The yeast (b) lactic acid bacterium according to (5) above,
(12) Either or both of the treatment and prevention of infectious diseases including the yeast according to any one of (1) to (4) above and / or a processed product thereof, or the yeast according to (5) above. Pharmaceuticals for
(13) The pharmaceutical according to (12) above, which is a combination of the following (a) and (b): (a) the yeast and / or processed product thereof according to any one of (1) to (4) above, Or the yeast (b) lactic acid bacteria of the said (5) description,
(14) An immunocompetent cell activator comprising the yeast according to any one of (1) to (4) above and / or a processed product thereof, or the yeast according to (5) above,
(15) A digestion improving agent comprising the yeast according to any one of (1) to (4) above and / or a processed product thereof, or the yeast according to (5) above,
(16) An intestinal regulating agent comprising the yeast according to any one of (1) to (4) above and / or a processed product thereof, or the yeast according to (5) above,
(17) A growth promoter comprising the yeast according to any one of (1) to (4) above and / or a processed product thereof, or the yeast described in (5) above,
(18) A composition comprising Candida sp. CO119 strain (accession number: NITE P-375) and lactic acid bacteria,
(19) A method for treating or preventing diarrhea in animals, characterized by administering the following (a) and (b) simultaneously or separately: (a) The method according to any one of (1) to (4) above Yeast and / or processed product thereof, or yeast (b) lactic acid bacterium according to (5) above,
(20) A method for improving the immune function of an animal, characterized in that the following (a) and (b) are administered simultaneously or separately: (a) The method according to any one of (1) to (4) above Yeast and / or processed product thereof, or yeast (b) lactic acid bacteria described in (5) above,
(21) A method for treating and / or preventing an infectious disease of an animal, characterized by administering the following (a) and (b) simultaneously or separately: (a) any one of (1) to (4) above The yeast described in the above and / or the processed product thereof, or the yeast (b) lactic acid bacterium described in (5) above,
(22) The method according to any one of (1) to (4) above, wherein the following (a) and (b) are administered simultaneously or separately: Fungus and / or processed product thereof, or yeast (b) lactic acid bacterium described in (5) above,
(23) A method for improving the intestinal environment of an animal, which comprises administering the following (a) and (b) simultaneously or separately.
(A) The yeast according to any one of (1) to (4) above and / or a processed product thereof, or the yeast according to (5) above (b) lactic acid bacteria,
(24) A method for treating or preventing animal diarrhea, which comprises administering the feed according to (8) or (9) above,
(25) A method for improving the immune function of an animal, comprising administering the feed according to (8) or (9) above,
(26) A method for promoting the growth of an animal, comprising administering the feed according to (8) or (9) above,
(27) A method for improving the intestinal environment of an animal, comprising administering the feed according to (8) or (9) above,
(28) A method for preventing and / or treating an infectious disease in an animal, comprising administering the feed according to (8) or (9) above,
(29) The method according to any one of (19) to (28), wherein the animal is a non-human animal.
本発明によって、乳酸菌との組み合わせにより、反芻家畜に対しても有用なプロバイオティック効果を発揮する新規酵母菌株CO119株が提供された。本発明の酵母菌株と乳酸菌の組み合わせは、体重増加促進および飼料効率改善、粗飼料に対する消化率向上、腸内細菌叢の改善、下痢・軟便防止、液性および細胞性免疫機能向上など、多岐にわたるプロバイオティック効果をもたらし、幼若および成熟反芻家畜の健全な飼養を可能とするのみならず、結果的に反芻家畜への抗生物質および抗菌性飼料添加物の使用量を削減することができる。さらに、発育促進および粗飼料消化率向上の各効果を有することから、畜産物の品質向上、飼料コストの削減、成長目的で使用される抗菌性飼料添加物の使用量削減が可能となる。また本発明の新規酵母菌株は、反芻動物のみならず広く動物全般に対し、プロバイオティック効果を付与できるものと期待される。 According to the present invention, a novel yeast strain CO119 strain exhibiting a probiotic effect useful for ruminant livestock in combination with lactic acid bacteria has been provided. The combination of the yeast strain and lactic acid bacteria of the present invention has a wide range of pros such as promoting weight gain and improving feed efficiency, improving digestibility for roughage, improving intestinal bacterial flora, preventing diarrhea and loose stool, improving humoral and cellular immune functions. In addition to providing a biotic effect and allowing healthy feeding of young and mature ruminant livestock, the use of antibiotics and antibacterial feed additives to ruminant livestock can be reduced as a result. Furthermore, since it has each effect of promoting growth and improving the digestibility of roughage, it is possible to improve the quality of livestock products, reduce feed costs, and reduce the amount of antibacterial feed additives used for growth purposes. Further, the novel yeast strain of the present invention is expected to be able to impart a probiotic effect not only to ruminants but also to all animals.
本発明は、乳酸菌とともに投与することにより前記乳酸菌にプロバイオティック効果を付与する、カンジダ属に属する新規酵母菌カンジダ・エスピーCO119株（受託番号：NITE P-375）および／またはその処理物、およびその利用に関する。本発明のカンジダ・エスピーCO119株（以下において、「CO119株」とも称す）は、栃木県那須塩原市の畜産草地研究所にて採取されたウシの直腸内容物から単離された分離株の中から、菌学的諸性質、生理的・生化学的性質の検証、およびウシに対する菌株供与試験を実施し、プロバイオティクス効果を指標として選抜された新規酵母菌株である。 The present invention provides a novel yeast Candida sp. CO119 strain belonging to the genus Candida (accession number: NITE P-375) and / or a processed product thereof, which imparts a probiotic effect to the lactic acid bacterium by administration with the lactic acid bacterium, and Regarding its use. The Candida sp. CO119 strain of the present invention (hereinafter also referred to as “CO119 strain”) is one of the isolates isolated from the rectal contents of cattle collected at the Livestock Pasture Research Institute in Nasushiobara City, Tochigi Prefecture. Therefore, it is a novel yeast strain that was selected using the probiotic effect as an index after conducting various mycological properties, physiological and biochemical properties verification, and strain donation tests on cattle.
反芻家畜の腸管下部に存在する細菌は、反芻胃、第四胃（単胃動物の胃に相当する）、腸管上部を死滅せずに通過して腸管下部に到達している。よって上記の方法で採取・選抜した菌株は反芻家畜の反芻胃液、胃酸および胆汁酸に対する耐性を有しており、その経口投与により反芻家畜の腸内に生きたまま到達すると考えられる。 Bacteria present in the lower intestinal tract of ruminant livestock pass through the rumen, the fourth stomach (corresponding to the stomach of a monogastric animal), and the upper intestinal tract without being killed, and reach the lower intestinal tract. Therefore, the strains collected and selected by the above method are resistant to ruminant gastric juice, gastric acid, and bile acids of ruminant livestock, and are considered to reach the intestine of ruminant livestock by oral administration.
本発明のCO119株の菌学的性質は、後述する実施例に記載のとおりである。該菌学的性質および28SリボゾームDNAのD1/D2領域の塩基配列から、CO119株は新規カンジダ属酵母と判断される。また本発明のCO119株は、酵母培養の常法として汎用される一般的な液体培地（例えば、YM Broth(Difco社製)）を用いて培養することができる。あるいは、後述する資化性に基づいて各種栄養成分を含む培地（例えば、廃糖蜜やスキムミルク等を用いた液体培地）を調製し、培養することもできる。 The mycological properties of the CO119 strain of the present invention are as described in the examples described later. From the mycological properties and the nucleotide sequence of the D1 / D2 region of 28S ribosomal DNA, the CO119 strain is judged as a novel Candida yeast. Further, the CO119 strain of the present invention can be cultured using a general liquid medium (for example, YM Broth (manufactured by Difco)) widely used as a conventional method for yeast culture. Alternatively, a medium (for example, a liquid medium using waste molasses, skim milk, or the like) containing various nutritional components can be prepared and cultured based on the assimilation properties described below.
カンジダ・エスピーCO119株は、独立行政法人 製品評価技術基盤機構 特許微生物寄託センターに寄託された。以下に、寄託を特定する情報を記載する。
（i) 寄託機関の名称および住所
名称：独立行政法人 製品評価技術基盤機構 特許微生物寄託センター
住所：日本国千葉県木更津市かずさ鎌足２−５−８ 郵便番号292-0818
（ii) 寄託日：平成１９年（２００７年）６月１３日
（iii) 受託番号：NITE P-375
Candida SP CO119 was deposited at the Patent Microorganism Depositary Center for Product Evaluation Technology. The information specifying the deposit is described below.
(I) Name and address of depositary institution: National Institute of Technology and Evaluation Patent Microorganism Depositary Address Address: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan ZIP Code 292-0818
(Ii) Deposit date: June 13, 2007 (iii) Deposit number: NITE P-375
本発明のCO119株は、生菌の状態で利用することもできるが、本発明の酵母菌株に対して何らかの処理を施した「処理物」として利用することもできる。本発明の処理物の例としては、微生物を培養した培地、微生物を培養した培地の上清画分およびその他の画分、凍結乾燥微生物、微生物破砕物、を挙げることができるが、これらに限られない。 Although the CO119 strain of the present invention can be used in the state of viable bacteria, it can also be used as a “processed product” obtained by subjecting the yeast strain of the present invention to some kind of treatment. Examples of the treated product of the present invention include, but are not limited to, a culture medium in which a microorganism is cultured, a supernatant fraction and other fractions of a culture medium in which a microorganism is cultured, a freeze-dried microorganism, and a microorganism disrupted product. I can't.
本発明のCO119株は、生菌単独でウシに投与した場合には、プロバイオティック効果が確認されなかったが、乳酸菌とともに投与した場合には、体重増加促進および飼料効率改善、粗飼料に対する消化率向上、腸内細菌叢の改善、下痢・軟便防止、液性および細胞性免疫機能向上など、広範囲にわたるプロバイオティック効果が確認された。したがって、本発明のCO119株またはその処理物を動物に投与する際は、動物体内中に乳酸菌とともに存在するように投与することが望ましい。本発明のCO119株と乳酸菌のそれぞれを同時に投与してもよく、またはCO119株と乳酸菌とを混合して投与してもよい。また、動物の体内で同時に共存しうる限り、CO119株の投与と乳酸菌の投与は、必ずしも同時でなくてもよい。 The CO119 strain of the present invention was not confirmed to have a probiotic effect when administered to cattle alone with live bacteria, but when administered with lactic acid bacteria, it promoted weight gain and improved feed efficiency, digestibility against roughage A wide range of probiotic effects were confirmed, including improvement, improvement of intestinal flora, prevention of diarrhea and loose stool, and improvement of humoral and cellular immune functions. Therefore, when administering the CO119 strain of the present invention or a processed product thereof to an animal, it is desirable to administer it so that it is present together with lactic acid bacteria in the animal body. Each of the CO119 strain and lactic acid bacteria of the present invention may be administered simultaneously, or the CO119 strain and lactic acid bacteria may be mixed and administered. Moreover, the CO119 strain and lactic acid bacteria do not necessarily have to be administered at the same time as long as they can coexist simultaneously in the animal body.
あるいは、動物体内にCO119株と共存させる乳酸菌として、サイレージまたは飼料用植物に付着した乳酸菌を利用してもよい。植物には天然の乳酸菌が付着している場合がある。またサイレージは、サイレージの品質向上を目的として、サイレージ調製時に乳酸菌を添加することがある。このようなサイレージや飼料に人為的または天然に存在している乳酸菌を利用して、CO119株を投与してもよい。 Alternatively, lactic acid bacteria attached to silage or feed plants may be used as lactic acid bacteria to coexist with the CO119 strain in the animal body. Natural lactic acid bacteria may adhere to the plant. Silage may add lactic acid bacteria at the time of silage preparation for the purpose of improving the quality of silage. The CO119 strain may be administered using lactic acid bacteria that exist artificially or naturally in such silage or feed.
さらに別の投与形態としては、CO119株を単独で、または乳酸菌とともに植物に混合し、CO119株を含んだサイレージを製造し、該サイレージを飼料として動物に与えることによって、CO119株を動物に投与してもよい。 As yet another dosage form, the CO119 strain is administered to an animal by mixing the CO119 strain alone or with a lactic acid bacterium into a plant, producing a silage containing the CO119 strain, and feeding the silage to the animal as a feed. May be.
CO119株またはその処理物とともに投与する乳酸菌としては、CO119株またはその処理物共存下において動物生体に好ましい効果を発揮する限り、特に限定されないが、好ましくはサイレージの乳酸発酵に寄与しうる植物由来の乳酸菌であり、より好ましくはラクトバチルス属またはエンテロコッカス属のサイレージ調製用乳酸菌である。このような乳酸菌の具体例としては、ラクトバチルス・プランタルム畜草１号株を挙げることができる。ラクトバチルス・プランタルム畜草１号株は、独立行政法人 産業技術総合研究所 特許生物寄託センターに受託番号FERM P-18930として寄託された乳酸菌である。またラクトバチルス・プランタルム畜草１号株は、雪印種苗株式会社（北海道札幌市）よりサイレージ調製用粉末製剤として既に市販されている。 The lactic acid bacteria to be administered together with the CO119 strain or its treated product is not particularly limited as long as it exerts a favorable effect on the animal body in the presence of the CO119 strain or its treated product, but preferably derived from a plant that can contribute to lactic acid fermentation of silage. Lactic acid bacteria, more preferably lactobacillus of Lactobacillus or Enterococcus for silage preparation. Specific examples of such lactic acid bacteria include Lactobacillus plantarum No. 1 strain. Lactobacillus plantarum stock 1 is a lactic acid bacterium deposited under the accession number FERM P-18930 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology. Lactobacillus plantarum stock No. 1 is already commercially available as a powder formulation for silage preparation from Snow Brand Seedling Co., Ltd. (Sapporo, Hokkaido).
本発明の酵母菌株CO119株またはその処理物が乳酸菌とともに発揮するプロバイオティック効果の例としては、体重増加促進および飼料効率改善、粗飼料に対する消化率向上、腸内細菌叢の改善、下痢・軟便防止、液性および細胞性免疫機能向上を挙げることができるが、CO119株またはその処理物が乳酸菌とともに発揮する生体に有益な効果である限り、本発明におけるプロバイオティック効果は上記に限られない。CO119株の特に優れた点の一つは、反芻動物にさえプロバイオティック効果をもたらす点である。反芻動物（ウシ、ヤギ、ヒツジ、シカ等の反芻亜目、ラクダ、ラマ等のラクダ亜目）の場合、経口摂取された生菌はまず反芻胃に流入するが、そこに共生するバクテリアによる競合的排除や原生動物による捕食によって、投与された生菌の生存性が多大な影響を受ける。そのため、単胃動物に対するプロバイオティック効果が確認された微生物であっても、反芻動物に対する有効な効果を示すとは限らない。逆に、反芻動物にプロバイオティック効果をもたらす微生物は、各種単胃動物に対しても、プロバイオティック効果をもたらすと考えることができる。 Examples of the probiotic effect exhibited by the yeast strain CO119 of the present invention or a processed product thereof together with lactic acid bacteria include weight gain promotion and feed efficiency improvement, digestibility improvement for roughage, intestinal bacterial flora improvement, diarrhea and loose stool prevention However, the probiotic effect in the present invention is not limited to the above as long as the CO119 strain or a processed product thereof has a beneficial effect on the living body exhibited together with lactic acid bacteria. One particular advantage of the CO119 strain is that it provides a probiotic effect even for ruminants. In the case of ruminants (bovine, goat, sheep, deer, etc., camels, llama, etc.), live bacteria ingested first flow into the rumen, but they compete with bacteria that live there Survival of the administered viable bacteria is greatly affected by genetic exclusion and protozoan predation. Therefore, even microorganisms that have been confirmed to have a probiotic effect on monogastric animals do not always exhibit an effective effect on ruminants. Conversely, a microorganism that provides a probiotic effect to ruminants can be considered to provide a probiotic effect to various monogastric animals.
上記効果の有無は、当業者に一般的な方法で判断することができる。例えば、飼料効率とは、動物の体重増加量を飼料摂取量にて除した値であり、摂取した飼料の利用性の指標として栄養学で汎用されるものである（例えば、小野寺ら著、家畜栄養学、1989年、川島書店、を参照）。また、反芻動物における消化率は、摂取した飼料が反芻胃内で分解される割合のことであり、採取した反芻胃液を入れた試験管内で飼料を消化させることで簡易的に求めることが出来る（例えば、自給飼料品質評価研究会編、改訂 粗飼料の品質評価ガイドブック、2001年、日本草地畜産種子協会、を参照）。腸内細菌叢の改善とは、反芻家畜の腸内に共存する腸内細菌の菌種構成割合が家畜にとって好ましい方向に変化することを意味し、実施例３に示すように動物の直腸内容物における菌種構成を調べることにより求めることが出来る。その好ましい変化とは、例えば乳酸菌数の増加または大腸菌群数、クロストリジウム属数、バチルス属数の減少などが挙げられる（例えば、光岡知足著、「腸内細菌学」、1990年、朝倉書店、を参照）。また下痢および軟便防止とは、これは実施例６に示すように、糞の形状をその外観から１（正常）、２（やや軟便）、３（軟便）、４（水様）の４段階にスコア評価することで求めることが出来る（Larsonら、J. Dairy Sci., 60, p. 989-991, 1977を参照）。哺乳期および育成期の幼若反芻家畜は下痢等に罹患しやすいため、これら幼若反芻家畜に対する下痢および軟便防止効果は、家畜飼養において重要性が高い。また液性免疫機能の評価は、動物体内に侵入した異物に対する動物の抗体産生能力を測定すればよい。例えば、実施例４に示すように、病原菌ワクチンを投与されたウシの血清と、ワクチンとして用いた病原菌体とを混合し、その凝集反応を観察して病原菌に対する抗体価を測定することにより評価できる。また細胞性免疫機能の評価は、貪食細胞による異物処理能力によって判断できる。例えば実施例３に示すように、ウシの好中球および単球に、蛍光色素で標識した大腸菌を取り込ませ、その蛍光強度を測定することにより評価することができる。液性および細胞性免疫機能の概要については、免疫学に関する一般的な教科書に記載されている（例えば、今西著、免疫学の入門、2004年、金芳堂、を参照）。 The presence or absence of the above effect can be determined by a method common to those skilled in the art. For example, feed efficiency is a value obtained by dividing the amount of weight gain of an animal by the amount of feed intake, and is commonly used in nutrition as an indicator of the availability of ingested feed (eg, Onodera et al., Livestock (See Nutrition, 1989, Kawashima Shoten). In addition, the digestibility in ruminants is the rate at which the ingested feed is degraded in the rumen, and can be easily determined by digesting the feed in a test tube containing the collected ruminant fluid ( (See, for example, the Self-Fedible Feed Quality Evaluation Study, Revised Forage Quality Evaluation Guidebook, 2001, Japan Grassland Livestock Seed Association). The improvement of the intestinal microflora means that the bacterial species composition ratio of the intestinal bacteria coexisting in the intestine of ruminant livestock changes in a favorable direction for the livestock, and as shown in Example 3, the rectal contents of the animal This can be determined by examining the bacterial species composition in Examples of preferable changes include an increase in the number of lactic acid bacteria or a decrease in the number of coliforms, the number of genus Clostridium, the number of the genus Bacillus, and the like (for example, Tomoashi Mitsuoka, “Intestinal Bacteriology”, 1990, Asakura Shoten, reference). In addition, as shown in Example 6, diarrhea and loose stool prevention is classified into four stages of 1 (normal), 2 (slightly loose stool), 3 (soft stool), and 4 (watery) from the appearance of feces. It can be obtained by evaluating the score (see Larson et al., J. Dairy Sci., 60, p. 989-991, 1977). Since young ruminant livestock in the suckling and breeding periods are susceptible to diarrhea and the like, the effects of preventing diarrhea and loose stool on these ruminant livestock are highly important in raising livestock. The evaluation of the humoral immune function may be performed by measuring the animal's ability to produce antibodies against foreign substances that have entered the animal body. For example, as shown in Example 4, it can be evaluated by mixing bovine serum administered with the pathogen vaccine and the pathogen used as the vaccine, observing the aggregation reaction and measuring the antibody titer against the pathogen. . The evaluation of the cellular immune function can be judged by the foreign substance processing ability by the phagocytic cells. For example, as shown in Example 3, bovine neutrophils and monocytes can be evaluated by incorporating E. coli labeled with a fluorescent dye and measuring the fluorescence intensity. An overview of humoral and cellular immune functions can be found in general textbooks on immunology (see, for example, Imanishi, Introduction to Immunology, 2004, Kinyoshido).
本発明のCO119株は、上記プロバイオティック効果を有することから、飼料、サプリメント、健康食品、食品、医薬品、整腸剤、下痢治療および／または予防薬、感染症治療および／または予防薬、消化改善剤、発育促進剤、等として利用可能である。また、CO119株は、プロバイオティック効果を有するサイレージ調製用資材または飼料添加物としても利用できる。本発明において「飼料」とは、動物用食品であるCO119株、CO119株の処理物、および、これらを含む製剤を意味する他、CO119株を添加して調製されたサイレージも、本発明の「飼料」に含まれる。 Since the CO119 strain of the present invention has the above-mentioned probiotic effect, it is a feed, supplement, health food, food, pharmaceutical, intestinal preparation, diarrhea treatment and / or prevention agent, infection treatment and / or prevention agent, digestion improving agent It can be used as a growth promoter, etc. The CO119 strain can also be used as a silage preparation material or a feed additive having a probiotic effect. In the present invention, “feed” means CO119 strain, which is a food for animals, a processed product of CO119 strain, and a preparation containing these, as well as silage prepared by adding CO119 strain. Included in “Feed”.
本発明のCO119株およびその処理物の投与経路は、上記プロバイオティック効果を発揮できる限り特に問わないが、好ましくは経口投与である。本発明のCO119株または処理物は、公知製剤技術を用いて適当な剤型の製剤とすることができる。該剤形は、投与経路にあわせて適宜選択することができる。経口投与であれば、例えば、丸剤、錠剤、顆粒剤、粉剤、シロップ剤、カプセル剤、液剤、等の中から選択できる。該製剤は、必要に応じ、薬学的に許容される賦形剤、保存料、滑沢剤、着色料、安定化剤、結合剤、崩壊剤、等の添加剤を含むことができる。 The route of administration of the CO119 strain of the present invention and its treated product is not particularly limited as long as the above-mentioned probiotic effect can be exhibited, but oral administration is preferred. The CO119 strain or treated product of the present invention can be made into a preparation of an appropriate dosage form using a known preparation technique. The dosage form can be appropriately selected according to the administration route. For oral administration, it can be selected from pills, tablets, granules, powders, syrups, capsules, liquids, and the like. The preparation can contain additives such as pharmaceutically acceptable excipients, preservatives, lubricants, colorants, stabilizers, binders, disintegrants, and the like as necessary.
本発明のCO119株および／またはその処理物は、有効成分として単独で製剤化してもよく、または、乳酸菌と混合して製剤化し、CO119株と乳酸菌を含む一つの製剤としてもよい。あるいは、CO119株および／またはその処理物と乳酸菌とを別個に製剤化して組み合わせ、同時に使用可能なキットとして製品化することもできる。 The CO119 strain and / or processed product thereof of the present invention may be formulated as an active ingredient alone, or may be mixed with lactic acid bacteria to form a single preparation containing the CO119 strain and lactic acid bacteria. Alternatively, the CO119 strain and / or the processed product thereof and lactic acid bacteria can be formulated separately and combined to produce a kit that can be used simultaneously.
本発明のCO119株および／またはその処理物、および乳酸菌の投与量は、投与する動物個体の種類、年齢、体重、投与経路等の各種条件を考慮して、適宜調節することができる。乳酸菌数を動物の体重1 kgあたり108から1010個になるように投与することが好ましく、また、CO119菌株を体重1kgあたり106から108個になるように投与することが好ましい。 The dose of the CO119 strain of the present invention and / or its treated product, and lactic acid bacteria can be appropriately adjusted in consideration of various conditions such as the type, age, weight, route of administration, etc. of the individual animal to be administered. It is preferable to administer the lactic acid bacteria so that the number of lactic acid bacteria is 10 8 to 10 10 per kg body weight of the animal, and it is preferable to administer the CO119 strain so as to be 10 6 to 10 8 per kg body weight.
以下、実施例により本発明をさらに詳細に説明するが、本発明はこれら実施例に制限されるものではない。
［実施例１］新規酵母菌株の選抜
栃木県那須塩原市の畜産草地研究所にて採取されたウシの直腸内容物10 gを無菌的にストマッカ用ビニール袋に入れ、滅菌した生理食塩水90 mlを加えて激しく振とうして10倍から100,000倍の希釈液を作成した。各倍率の希釈液をYM寒天培地（Difco YM Broth（Difco社製））に接種し、30℃で2〜3日間の静置培養を行った。これらの培地から出現した各形状の異なるコロニーを単離して分離株とした。それらの分離株について、菌学的諸性質や生理的・生化学的性質等を調査し、さらにウシに対する菌株給与試験を実施して、酵母菌CO119株を選抜した。
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited to these Examples.
[Example 1] Selection of new yeast strains 10 g of bovine rectal contents collected at the National Institute of Animal Husbandry, Nasushiobara City, Tochigi Prefecture, aseptically placed in a plastic bag for stomacher, and 90 ml of sterile physiological saline And diluted vigorously to make a 10-fold to 100,000-fold dilution. The diluted solution at each magnification was inoculated into YM agar medium (Difco YM Broth (manufactured by Difco)), and static culture was performed at 30 ° C. for 2 to 3 days. Colonies with different shapes that emerged from these media were isolated and used as isolates. The isolates were examined for mycological properties, physiological and biochemical properties, etc., and a strain feeding test was conducted on cattle to select the yeast strain CO119.
本発明の酵母菌CO119株は、以下の菌学的性質を有する。なお、CO119株の菌学的性質に関する試験は、文献１（C. P. Kurtzman and J. W. Fell(ed.),The Yeasts, a Taxonomic Study, 4th revised and enlarged edition, Elsevier, 1997）、文献２（J. A. Barnettら,Yeasts, Characteristics and identification, 3rd edition, Cambridge University Press, 2000）、文献３（厚生労働省監修、食品衛生検査指針微生物編、日本食品衛生協会、2004）の方法に従って遂行した。
（１）形態：楕円（図１）
（２）増殖の形式：出芽
（３）有性胞子形成：なし
（４）炭素源の資化
L-アラビノース：−
D-キシロース：−
D-グルコース：＋
D-マンノース：＋
D-ガラクトース：−
L-ラムノース：−
L-ソルボース：−
マルトース：−
ラクトース：−
メリビオース：−
セロビオース：−
トレハロース：＋
ラフィノース：−
メレジトース：−
白糖：−
α-メチル-D-グルコシド：−
N-アセチルグルコサミン：−
アルブチン：−
エスクリン：−
エリスリトール：−
D-ソルビトール：−
D-グルコン酸塩：−
グリセリン：−
乳酸塩：−
クエン酸塩：−
キシリトール：−
（５）窒素源の資化
硝酸塩：−
尿素：−
L-リジン：−
L-アルギニン：−
L-グルタミン酸：−
上記の菌学的性質はカンジダ属に類似するものであるが、グルコン酸塩、キシリトール、アルギニン、グルタミン酸の資化性が代表的なCandida属の菌株（例えばCandida glabrata(カンジダ・グラブラタ)ATCCMYA-2950株、Candida lusitaniae（カンジダ・ラシタニア）ATCC 34449株、Candida utilis (カンジダ・ユチリス) ATCC 9950株、等）とは異なる。また本発明の新規酵母の28SリボゾームDNAのD1/D2領域の塩基配列を検討したところ、カンジダ属のものと高い相同性を示すことが示された。以上の事から、本発明の新規酵母菌株は、種名までは同定出来ていないもののカンジダ属の一種であると判断し、便宜的にカンジダ・エスピーと表記することとした。
The yeast strain CO119 of the present invention has the following mycological properties. Tests on the mycological properties of the CO119 strain are described in Reference 1 (CP Kurtzman and JW Fell (ed.), The Yeasts, a Taxonomic Study, 4th revised and enlarged edition, Elsevier, 1997), Reference 2 (JA Barnett et al. Yeasts, Characteristics and identification, 3rd edition, Cambridge University Press, 2000), literature 3 (supervised by the Ministry of Health, Labor and Welfare, Food Sanitation Inspection Guidelines for Microorganisms, Japan Food Sanitation Association, 2004).
(1) Form: Ellipse (Figure 1)
(2) Form of growth: budding (3) Sexual spore formation: None (4) Utilization of carbon source
L-arabinose:-
D-xylose:-
D-glucose: +
D-Mannose: +
D-galactose:-
L-rhamnose:-
L-sorbose:-
Maltose:-
Lactose:-
Melibiose:-
Cellobiose:-
Trehalose: +
Raffinose:-
Merezitose:-
Sucrose:-
α-methyl-D-glucoside: −
N-acetylglucosamine:-
Arbutin:-
Esclin:-
Erythritol:-
D-sorbitol:-
D-gluconate:-
Glycerin:-
Lactate:-
Citrate:-
Xylitol:-
(5) Utilization of nitrogen source
Nitrate:-
Urea:-
L-lysine:-
L-Arginine: −
L-glutamic acid:-
Although the above bacteriological properties are similar to those of Candida, strains of the genus Candida (eg, Candida glabrata ATCCMYA-2950, which are representative of assimilability of gluconate, xylitol, arginine, and glutamic acid) And Candida lusitaniae ATCC 34449, Candida utilis ATCC 9950, etc.). Further, when the nucleotide sequence of the D1 / D2 region of the 28S ribosomal DNA of the novel yeast of the present invention was examined, it was shown to show high homology with that of Candida. From the above, the novel yeast strain of the present invention was judged to be a kind of Candida spp. Although it could not be identified up to the species name, and for convenience, it was designated as Candida sp.
［実施例２］培養物の作成
滅菌したYM Broth 液体培地（Difco社製)に酵母菌カンジダ・エスピーCO119株を植え付け、30℃で48時間保温したものを培養物とした。また乳酸菌については、滅菌したMRS Broth液体培地（Difco社製)にラクトバチルス・プランタルム畜草１号株（以下において、「畜産１号株」と称す）を植え付け、30℃で48時間保温したものを培養物とした。これらの培養物には、CO119株は1.4-2.8×108個／ml、畜草１号株は2.7-6.7×109個／mlが含有されていた。
[Example 2] Production of culture A yeast culture of Candida sp. CO119 strain was planted in a sterilized YM Broth liquid medium (Difco) and incubated at 30 ° C for 48 hours to obtain a culture. For lactic acid bacteria, Lactobacillus plantarum 1 stock (hereinafter referred to as “Livestock 1”) was planted in a sterilized MRS Broth liquid medium (Difco) and kept at 30 ° C. for 48 hours. Was used as a culture. These cultures contained the CO119 strain at 1.4-2.8 × 10 8 cells / ml and the No. 1 stock at 2.7-6.7 × 10 9 cells / ml.
［実施例３］菌株投与による体重増加促進、飼料効率改善、腸内細菌叢の改善、細胞性免疫機能向上の各効果
（３−１試験設計）
６ヶ月齢のホルスタイン去勢ウシ８頭を２頭ずつ４試験区に分割した。試験区は菌株を飼料に添加しない区（無添加区）、乳酸菌畜草１号株のみを添加する区（乳酸菌区）、酵母菌CO119株のみを添加する区（酵母区）、畜草１号株とCO119株との両方を添加する区（両菌添加区）とした。実施例２で作成した培養物を、CO119株は9 ml／頭、畜草１号株は100 ml／頭の分量で飼料に添加した。飼料はウシ用市販濃厚飼料と乾草とを乾物比7:3の割合で給与し、１回あたり４週間の菌株添加試験を行った。試験設定はラテン方格法とし、１回の試験が終了する毎に試験区を入れ替え、合計４回の反復試験を行った。
[Example 3] Each effect of weight gain promotion, feed efficiency improvement, intestinal bacterial flora improvement and cellular immune function improvement by strain administration (3-1 study design)
Eight 6-month-old Holstein steers were divided into four test plots, two each. The test group is a group in which the strain is not added to the feed (no added group), a group in which only the lactic acid bacteria livestock No. 1 strain is added (lactic acid bacteria group), a group in which only the yeast CO119 strain is added (yeast group), and livestock No. 1 It was set as a group (both bacteria-added group) to which both the strain and CO119 strain were added. The culture prepared in Example 2 was added to the feed in an amount of 9 ml / head for CO119 strain and 100 ml / head for No. 1 stock. The feed was a commercial concentrated feed for cattle and hay at a dry matter ratio of 7: 3, and the strain addition test was conducted for 4 weeks each time. The test was set to the Latin square method, and the test section was changed every time one test was completed, and a total of four repeated tests were performed.
ここでいうラテン方格法とは、試験群を複数の組に分割し、試験区を反復して試験を行うことで実験誤差を最小限にすることを目的とした実験計画法である（吉田実、畜産を中心とする実験計画法、1983年、養賢堂、を参照）。ウシは多頭数を確保することが困難であり、また個体間の差異が大きいため、ラテン方格法による試験設計を行った。実施例３におけるラテン方格法の実際を以下の表１に示す。 Here, the Latin square method is an experimental design method that aims to minimize experimental errors by dividing the test group into multiple groups and repeating the test section to perform the test (Yoshida In fact, see the experimental design method centered on livestock, 1983, Yokendo.) Cattle were difficult to secure a large number of animals, and because there were large differences between individuals, a test design was conducted using the Latin square method. The actual Latin square method in Example 3 is shown in Table 1 below.
[表１]
（実施例３におけるラテン方格法の実際）
[Table 1]
(Actual of Latin square method in Example 3)
（３−２測定項目・方法）
試験期間中に体重および飼料摂取量を測定して、体重増加量および飼料効率を求めた。また直腸内容物を採取して10倍〜100,000倍に希釈し、各種寒天培地に接種してコロニー数を計測し、腸内微生物菌種構成を求めた。また血液を採取し、蛍光色素で標識した大腸菌と混合して37℃、30分間静置して大腸菌を貪食細胞（好中球および単球）に取り込ませ、細胞を洗浄後にその蛍光強度を測定することで異物を細胞内に取り込む能力、つまり細胞性免疫機能を評価した。
(3-2 Measurement items and methods)
Body weight and feed intake were measured during the study period to determine weight gain and feed efficiency. Rectal contents were collected and diluted 10 to 100,000 times, inoculated on various agar media, the number of colonies was counted, and the intestinal microbial species composition was determined. In addition, blood is collected, mixed with Escherichia coli labeled with a fluorescent dye, and allowed to stand at 37 ° C for 30 minutes to incorporate Escherichia coli into phagocytic cells (neutrophils and monocytes), and the fluorescence intensity is measured after washing the cells. Thus, the ability to take in foreign substances into cells, that is, the cellular immune function was evaluated.
（３−３）結果
体重増加量は、乳酸菌区および酵母区では無添加区と比較して差がなかったが、両菌添加区で向上した（図２Ａ）。また飼料効率も両菌添加区で向上した（図２Ｂ）。また腸内細菌叢の変化では、両菌添加区において腸管内の畜草１号株を除いた乳酸菌数が増加した（図３）。また両菌添加区で好中球および単球の異物取り込みが上昇し、細胞性免疫機能が向上した（図４Ａ、Ｂ）。
以上から、本発明の酵母菌CO119株を乳酸菌畜草１号株と同時に飼料添加することにより、体重増加促進、飼料効率改善、腸内細菌叢の改善および細胞性免疫機能向上が認められた。
(3-3) Results Although there was no difference in the weight gain in the lactic acid bacteria group and the yeast group compared with the non-addition group, it improved in the both bacteria addition group (FIG. 2A). The feed efficiency was also improved in the both bacteria-added section (FIG. 2B). In addition, in the change of the intestinal microflora, the number of lactic acid bacteria in the intestinal tract excluding the No. 1 stock in the intestinal tract increased in both bacteria-added sections (FIG. 3). In addition, the foreign substance uptake of neutrophils and monocytes increased in the both bacteria-added sections, and the cellular immune function was improved (FIGS. 4A and B).
From the above, it was confirmed that by adding the yeast CO119 strain of the present invention at the same time as the lactic acid bacterium livestock No. 1 strain, promotion of weight gain, improvement of feed efficiency, improvement of intestinal bacterial flora and improvement of cellular immune function were observed.
［実施例４］菌株投与による液性免疫機能の向上
（４−１）試験設計
15ヶ月齢のホルスタイン去勢ウシ８頭を４頭ずつ２試験区に分け、無添加区と両菌添加区とを設定した。両菌添加区には実施例２で作成した培養物を、CO119株は9 ml／頭、畜草１号株は100 ml／頭の分量で飼料に添加した。菌添加２週間後に、ウシ用大腸菌ワクチン（商品名ゼンノウ、科学飼料研究所製）を5 ml、全頭に皮下注射した。１ヶ月後に再度、ウシ用大腸菌ワクチンを全頭に皮下注射し、その１ヶ月後に採血して血清を分離した。血清は２倍ずつ段階的に希釈し、そこへワクチンに使用した死菌体の大腸菌を添加して凝集反応を観察し、その時の血清の希釈倍率によりワクチンに対する抗体価を測定した。
[Example 4] Improvement of humoral immune function by strain administration (4-1) Test design
Eight 15-month-old Holstein steers were divided into two test groups of 4 each, and an additive-free group and a group with both bacteria added were set. The culture prepared in Example 2 was added to the feed for both strains in an amount of 9 ml / head for the CO119 strain and 100 ml / head for the No. 1 stock. Two weeks after the addition of the bacteria, 5 ml of bovine Escherichia coli vaccine (trade name: Zennow, manufactured by Scientific Feed Research Institute) was subcutaneously injected into all the heads. One month later, bovine E. coli vaccine was subcutaneously injected into all the heads, and blood was collected one month later to separate the serum. Serum was diluted stepwise by 2 times, and dead E. coli used for the vaccine was added thereto to observe the agglutination reaction, and the antibody titer against the vaccine was measured by the dilution ratio of the serum at that time.
（４−２）結果
無添加区と比較して、両菌添加区でウシ用大腸菌ワクチンに対する抗体価が上昇した（図５）。以上から、本発明の酵母菌CO119株を乳酸菌畜草１号株と同時に飼料添加することにより、液性免疫機能の向上が観察された。
(4-2) Results Compared with the non-addition group, the antibody titer against the bovine Escherichia coli vaccine increased in the both bacteria-added group (FIG. 5). From the above, it was observed that the humoral immunity function was improved by adding the yeast strain CO119 of the present invention at the same time as the feed of the lactic acid bacteria stock No. 1.
［実施例５］菌株投与による粗飼料消化率の向上
（５−１）試験設計・測定項目・方法
黒毛和種反芻胃より採取した反芻胃液（第一胃液）と人工唾液とを1:4の割合で混合した人工反芻胃液20 mlに、トウモロコシ粉末または乾草粉末を0.2 g加えたものを複数作成した。それらを無添加区、乳酸菌区、酵母区、両菌添加区の４試験区に分け、実施例２で作成した培養物を0.01 mlの分量でそれぞれの試験区に合わせて添加した。それらを39℃で24時間静置培養し、終了後のトウモロコシ粉末および乾草粉末の重量を測定してそれぞれの消化率を算出した。
[Example 5] Improvement of digestibility of roughage by administration of strain (5-1) Test design / measurement items / method A ratio of 1: 4 between rumen fluid (ruminal fluid) and artificial saliva collected from Japanese black rumen stomach A plurality of 20 ml of artificial ruminant gas mixed in the above was added with 0.2 g of corn powder or hay powder. They were divided into four test groups, an additive-free group, a lactic acid bacteria group, a yeast group, and both bacteria-added groups, and the culture prepared in Example 2 was added in an amount of 0.01 ml according to each test group. They were incubated at 39 ° C. for 24 hours, and the weights of the corn powder and hay powder after completion were measured to calculate their digestibility.
（５−２）結果
乾草粉末の消化率において、乳酸菌区および酵母区は無添加区と比較して差がなかったが、両菌添加区では向上した（図６）。トウモロコシ粉末の消化率は差がなかった。以上から、本発明の酵母菌CO119株を乳酸菌畜草１号株と同時に飼料添加することにより、粗飼料の反芻胃内消化率の向上が認められた。
(5-2) Results In the digestibility of hay powder, the lactic acid bacteria group and the yeast group were not different from the non-added group, but improved in the both bacteria-added group (FIG. 6). There was no difference in the digestibility of corn powder. From the above, it was confirmed that the rumen digestibility of the roughage was improved by adding the yeast strain CO119 of the present invention at the same time as the lactic acid bacteria No. 1 strain.
［実施例６］菌株投与による下痢および軟便防止効果
（６−１）試験設計・測定項目・方法
6日齢のホルスタイン哺乳子ウシ８頭を４頭ずつ２試験区に分け、無添加区および両菌添加区を設定した。実施例２で作成した培養物を、CO119株は9 ml／頭、畜草１号株は100 ml／頭の分量で両菌添加区のウシ用ミルクに添加し、４週間飼育した。試験期間中に、糞の形状をその外観から１（正常）、２（やや軟便）、３（軟便）、４（水様）の４段階にスコア評価した数値を糞スコアとして１日３回記録し、下痢の判定を行った。
[Example 6] Effect of strain administration to prevent diarrhea and loose stool (6-1) Test design / measurement item / method
Eight 6-day-old Holstein calves were divided into two test groups of 4 each, and an additive-free group and a group with both bacteria added were set. The culture prepared in Example 2 was added to the milk for cows in the both bacteria-added group at a volume of 9 ml / head for the CO119 strain and 100 ml / head for the livestock No. 1 strain and reared for 4 weeks. During the test period, the feces shape was scored three times a day as the fecal score, which was scored in four stages: 1 (normal), 2 (slightly loose stool), 3 (soft stool), and 4 (watery). The diarrhea was determined.
（６−２）結果
無添加区と比較して、両菌添加区で糞スコア数値が低く、下痢の発症が抑制された（図７）。
(6-2) Results Compared with the non-addition group, fecal score values were lower in the both bacteria-added group, and the onset of diarrhea was suppressed (FIG. 7).
本発明により、反芻家畜に対しても有用なプロバイオティック効果を発揮する新規酵母菌株、およびこれらを含有する生菌剤が提供される。その生菌剤は、腸内細菌叢の改善、免疫機能の向上、下痢・軟便の防止の各効果を有することから、幼若および成熟反芻家畜などに投与することでそれらを健全に飼養でき、反芻家畜への抗生物質および抗菌性飼料添加物の使用量削減が可能である。また、その生菌剤は発育促進および粗飼料消化率向上の各効果を有することから、畜産物の品質向上、飼料コストの削減、成長目的で使用される抗菌性飼料添加物の使用量削減が可能となる。 According to the present invention, a novel yeast strain that exhibits a useful probiotic effect even on ruminant livestock, and a viable agent containing these are provided. Because the viable agent has the effects of improving intestinal flora, improving immune function, preventing diarrhea and loose stool, it can be kept healthy by administering it to young and mature ruminants, etc. It is possible to reduce the use of antibiotics and antibacterial feed additives to ruminants. In addition, the viable agent has the effects of promoting growth and improving the digestibility of roughage, so it is possible to improve the quality of livestock products, reduce feed costs, and reduce the amount of antibacterial feed additives used for growth purposes. It becomes.
また、本発明により、通常の反芻家畜用発酵飼料に上記のプロバイオティック効果を付加する為の添加物が提供される。本発明の酵母菌株を添加した反芻家畜用発酵飼料は、発育促進、腸内細菌叢改善、免疫機能の向上、下痢・軟便の防止の各効果を有する機能性飼料として使用可能であり、国内産飼料の高付加価値化に寄与すると考えられる。 Moreover, the additive for adding said probiotic effect to the normal fermented feed for ruminant livestock is provided by this invention. The fermented feed for ruminant livestock added with the yeast strain of the present invention can be used as a functional feed having effects of promoting growth, improving intestinal microflora, improving immune function, preventing diarrhea and loose stool. It is thought that it contributes to high value-added feed.
Claims (29)
（ａ）請求項１から４のいずれかに記載の酵母菌および／またはその処理物、あるいは請求項５記載の酵母菌
（ｂ）乳酸菌 A feed additive having a probiotic effect, comprising a combination of the following (a) and (b).
(A) The yeast according to any one of claims 1 to 4 and / or a processed product thereof, or the yeast according to claim 5 (b) lactic acid bacteria
（ａ）請求項１から４のいずれかに記載の酵母菌および／またはその処理物、あるいは請求項５記載の酵母菌
（ｂ）乳酸菌 A feed having a probiotic effect, comprising a combination of the following (a) and (b).
(A) The yeast according to any one of claims 1 to 4 and / or a processed product thereof, or the yeast according to claim 5 (b) lactic acid bacteria
（ａ）請求項１から４のいずれかに記載の酵母菌および／またはその処理物、あるいは請求項５記載の酵母菌
（ｂ）乳酸菌 The pharmaceutical product according to claim 10, which is a combination of the following (a) and (b).
(A) The yeast according to any one of claims 1 to 4 and / or a processed product thereof, or the yeast according to claim 5 (b) lactic acid bacteria
（ａ）請求項１から４のいずれかに記載の酵母菌および／またはその処理物、あるいは請求項５記載の酵母菌
（ｂ）乳酸菌 The pharmaceutical product according to claim 12, which is a combination of the following (a) and (b).
(A) The yeast according to any one of claims 1 to 4 and / or a processed product thereof, or the yeast according to claim 5 (b) lactic acid bacteria
（ａ）請求項１から４のいずれかに記載の酵母菌および／またはその処理物、あるいは請求項５記載の酵母菌
（ｂ）乳酸菌 A method for treating or preventing diarrhea in an animal, which comprises administering (a) and (b) below or simultaneously:
(A) The yeast according to any one of claims 1 to 4 and / or a processed product thereof, or the yeast according to claim 5 (b) lactic acid bacteria
（ａ）請求項１から４のいずれかに記載の酵母菌および／またはその処理物、あるいは請求項５記載の酵母菌
（ｂ）乳酸菌 A method for improving the immune function of an animal, which comprises administering the following (a) and (b) simultaneously or separately.
(A) The yeast according to any one of claims 1 to 4 and / or a processed product thereof, or the yeast according to claim 5 (b) lactic acid bacteria
（ａ）請求項１から４のいずれかに記載の酵母菌および／またはその処理物、あるいは請求項５記載の酵母菌
（ｂ）乳酸菌 A method for treating and / or preventing an infectious disease of an animal, which comprises administering the following (a) and (b) simultaneously or separately.
(A) The yeast according to any one of claims 1 to 4 and / or a processed product thereof, or the yeast according to claim 5 (b) lactic acid bacteria
（ａ）請求項１から４のいずれかに記載の酵母菌および／またはその処理物、あるいは請求項５記載の酵母菌
（ｂ）乳酸菌 A method for promoting animal growth, which comprises administering (a) and (b) below simultaneously or separately.
(A) The yeast according to any one of claims 1 to 4 and / or a processed product thereof, or the yeast according to claim 5 (b) lactic acid bacteria
（ａ）請求項１から４のいずれかに記載の酵母菌および／またはその処理物、あるいは請求項５記載の酵母菌
（ｂ）乳酸菌 A method for improving the intestinal environment of an animal, wherein the following (a) and (b) are administered simultaneously or separately:
(A) The yeast according to any one of claims 1 to 4 and / or a processed product thereof, or the yeast according to claim 5 (b) lactic acid bacteria
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2014007946A1 (en) * | 2012-06-12 | 2014-01-09 | Pioneer Hi-Bred International, Inc. | Yeast-containing silage inoculants for the enhancement of silage digestion and fermentation in the rumen |
| JP2016505254A (en) * | 2012-12-12 | 2016-02-25 | ルサッフル・エ・コンパニーＬｅｓａｆｆｒｅ Ｅｔ Ｃｏｍｐａｇｎｉｅ | Probiotic strains for treating and / or preventing diarrhea |
| EP3431580A4 (en) * | 2016-03-15 | 2019-11-13 | Snow Brand Seed Co., Ltd. | Lactic acid bacteria for silage preparation and additive for silage preparation |
| CN111903852A (en) * | 2020-07-18 | 2020-11-10 | 黑龙江省农业科学院畜牧研究所 | Feed additive for promoting production performance of ruminants and application thereof |
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2007
- 2007-08-15 JP JP2007211785A patent/JP2009044971A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014007946A1 (en) * | 2012-06-12 | 2014-01-09 | Pioneer Hi-Bred International, Inc. | Yeast-containing silage inoculants for the enhancement of silage digestion and fermentation in the rumen |
| CN104640457A (en) * | 2012-06-12 | 2015-05-20 | 先锋国际良种公司 | Yeast-containing silage inoculants for the enhancement of silage digestion and fermentation in the rumen |
| AU2013287171B2 (en) * | 2012-06-12 | 2017-02-16 | Pioneer Hi-Bred International, Inc. | Yeast-containing silage inoculants for the enhancement of silage digestion and fermentation in the rumen |
| US10945450B2 (en) | 2012-06-12 | 2021-03-16 | Pioneer Hi-Bred International, Inc. | Yeast-containing silage inoculants for the enhancement of silage digestion and fermentation in the rumen |
| JP2016505254A (en) * | 2012-12-12 | 2016-02-25 | ルサッフル・エ・コンパニーＬｅｓａｆｆｒｅ Ｅｔ Ｃｏｍｐａｇｎｉｅ | Probiotic strains for treating and / or preventing diarrhea |
| EP3431580A4 (en) * | 2016-03-15 | 2019-11-13 | Snow Brand Seed Co., Ltd. | Lactic acid bacteria for silage preparation and additive for silage preparation |
| CN111903852A (en) * | 2020-07-18 | 2020-11-10 | 黑龙江省农业科学院畜牧研究所 | Feed additive for promoting production performance of ruminants and application thereof |
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