JP2004357528A - Feed additive and feed containing the same - Google Patents

Feed additive and feed containing the same Download PDF

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JP2004357528A
JP2004357528A JP2003157115A JP2003157115A JP2004357528A JP 2004357528 A JP2004357528 A JP 2004357528A JP 2003157115 A JP2003157115 A JP 2003157115A JP 2003157115 A JP2003157115 A JP 2003157115A JP 2004357528 A JP2004357528 A JP 2004357528A
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lactobacillus
strain
feed
strains
cells
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JP4115338B2 (en
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Yukiko Sumi
有希子 角
Masami Morotomi
正己 諸富
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Yakult Honsha Co Ltd
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Yakult Honsha Co Ltd
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  • Feed For Specific Animals (AREA)
  • Fodder In General (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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Abstract

<P>PROBLEM TO BE SOLVED: To obtain a feed additive which is effective for preventing and improving gastrointestinal disturbances, such as diarrhea, as side effects by the administration of antibiotics, and to provide a feed containing the same. <P>SOLUTION: The feed additive comprises (A) the cells of Lactobacillus equi, and (B) the cells of at least one selected from the group consisting of Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus crispatus, and Lactobacillus johnsonii. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【0001】
【発明の属する技術分野】
本発明は、馬の飼育管理において、抗生物質投与の副作用による消化管障害を予防及び改善する飼料用添加剤、及びそれを含む飼料に関するものである。
【0002】
【従来の技術】
一般に、家畜動物として飼育管理されている代表的なものとしては、牛、豚、馬などが挙げられるが、これらの家畜動物は、飼育環境等の変化によってその発育過程や健康状態に影響を受けやすいため、飼育管理に十分な配慮が必要とされている。例えば、馬の場合には、食事などの飼育環境の変化による種々のストレス状態から誘発される下痢などの身体症状を起こしやすいことが知られている。馬(特に仔馬)は、他の家畜動物に比べて腸内に存在する細菌が少なく、その上、これらの細菌は、飼育環境の変化により極端に減少するため、腸内細菌の生態系を構成する腸内フローラが障害を受け、そのバランスを崩すことにより下痢などの症状を起こす。このような症状は、消化吸収能力の低下、成長阻害及び免疫力の低下等の生理学的機能に悪影響を及ぼすことから、軽種馬生産現場に与える損害は大きい。
【0003】
近年、馬の飼育管理において、乳酸菌やビフィズス菌に代表される微生物を生菌で含有する飼料が広く利用されている(例えば、特許文献1〜4参照)。これらの飼料は、含有される微生物が馬の腸内に付着することにより、腸内フローラを改善し、発育促進、下痢改善等の生理効果を期待するものである。
しかしながら、これらの飼料を使用しても投与した微生物が馬の腸内に付着及び増殖しないため、下痢などの身体症状の改善効果を十分に発揮できない場合が多く、実際には症状に応じて各種抗生物質を投与しているのが現状である。
【0004】
投与される抗生物質は、馬の身体症状を効果的に改善するものであるが、同時に馬の腸内に存在する細菌を死滅させる等の影響を与え、逆に下痢症状の悪化等の副作用を引き起こす原因となることが多い。また、これらの抗生物質は、馬の腸内に存在する細菌だけでなく、飼料等により別途投与される有用微生物も死滅させるため、有用微生物を生菌で含有する飼料を馬に投与した場合であっても、投与した微生物の十分な効果を得ることなく、逆に下痢などの消化管障害を引き起こす身体症状の改善効果も低いものであった。
【0005】
このことから、馬の飼育管理上で使用する微生物含有飼料においては、腸内環境を改善する効果を有する乳酸菌やビフィズス菌等の微生物を単に選択するだけでは十分ではなく、馬の消化管上皮への特異的な付着性と増殖活性を示し、馬の腸内環境を改善する優れた効果を有する微生物を選択すること、さらに、馬の身体症状に応じて随時投与する可能性のある各種抗生物質に対して優れた耐性を有する微生物を選択することが必要である。
【0006】
【特許文献1】
特開昭59−46208号公報
【特許文献2】
特許第3220699号公報
【特許文献3】
特表2001−519144号公報
【特許文献4】
特開2002−58432号公報
【0007】
【発明が解決しようとする課題】
従って、本発明の目的は、馬の消化管上皮への特異的な付着性及び増殖活性を示すだけでなく、抗生物質に対して優れた耐性を有し、抗生物質投与の副作用による下痢等の消化管障害の予防及び改善に有効である飼料用添加剤、及びそれを含む飼料を提供することにある。
【0008】
【課題を解決するための手段】
本発明者らは、鋭意研究の結果、ラクトバチルス属に属する菌種のうち、馬の胃粘膜上皮細胞への優れた付着性及び増殖活性を有する5種類の菌株は、馬(特に仔馬)の下痢予防・改善効果が高く、また、その他の菌に比べ抗生物質に対する耐性に優れていることを見出した。
また、それらのラクトバチルス属に属する5種類の菌株は、優れた抗生物質耐性を示すと共に、同一の属に属するにもかかわらず、それぞれ異なる抗菌スペクトルを有することを知見し、本発明を完成した。
【0009】
すなわち、本発明は、(A)ラクトバチルス・イクイ(Lactobacillus equi)の菌体と、(B)ラクトバチルス・サリバリウス(Lactobacillus salivarius)、ラクトバチルス・ロイテリ(Lactobacillus reuteri)、ラクトバチルス・クリスパータス(Lactobacillus crispatus)及びラクトバチルス・ジョンソニー(Lactobacillus johnsonii)からなる群より選ばれる少なくとも1種の菌体と、を含む飼料用添加剤を提供するものである。
また、本発明は、(C)セフトリアキソンナトリウム、硫酸カナマイシン、ジヒドロストレプトマイシン、スルファモノメトキシン、バクトラミン、ゲンタマイシン、オフロキサシン、セファロチンナトリウム、アンピシリン、オキシテトラサイクリン塩酸塩及びベンジルペニシリンからなる群より選ばれる少なくとも1種の抗生物質を更に含む前記飼料用添加剤を提供するものである。
また、本発明は、前記(C)抗生物質と組合わせて添加するための前記飼料用添加剤を提供するものである。
また、本発明は、前記飼料用添加剤を含む飼料を提供するものである。
また、本発明は、以下の(イ)〜(ニ)からなる群より選ばれる少なくとも1種のラクトバチルス属に属する菌株:(イ)ラクトバチルス・サリバリウス YIT0479株(寄託番号:FERM P−19332号);(ロ)ラクトバチルス・ロイテリ YIT0480株(寄託番号:FERM P−19333号);(ハ)ラクトバチルス・クリスパータス YIT0481株(寄託番号:FERM P−19334号)及び(ニ)ラクトバチルス・ジョンソニー YIT0482株(寄託番号:FERM P−19335号)を提供するものである。
【0010】
【発明の実施の形態】
本発明の飼料用添加剤は、(A)ラクトバチルス・イクイ(Lactobacillus equi)の菌体と、(B)ラクトバチルス・サリバリウス(Lactobacillus salivarius)、ラクトバチルス・ロイテリ(Lactobacillus reuteri)、ラクトバチルス・クリスパータス(Lactobacillus crispatus)及びラクトバチルス・ジョンソニー(Lactobacillus johnsonii)からなる群より選ばれる少なくとも1種の菌体とから構成される。
【0011】
本発明で使用する菌株は、5種類すべてラクトバチルス属に属し、いずれも馬の糞便から単離された菌株である。単離は、ラクトバチルス属の選択培地としてLBS培地を、さらに、非選択培地としてMRS培地を使用し、それぞれ嫌気条件下、約37℃で培養することによって行われる。単離された菌株は、形態的性質、培養的性質、生理学的性質又は化学分類学的性質等によって特徴付けられる。上記菌株は、ラクトバチルス属に属する菌種の中でも特に馬の胃粘膜上皮細胞への優れた付着性及び増殖活性を示し、並びに抗生物質に対する優れた耐性を有する菌株である。
【0012】
(A)ラクトバチルス・イクイの菌株としては、特に、ラクトバチルス・イクイ YIT0483株(独立行政法人産業技術総合研究所に寄託、微生物寄託番号「FERM P−18677号」)が特に付着性及び増殖活性に優れている点で好ましい。
【0013】
また、(B)ラクトバチルス属の4種類の菌株としては、特に、(イ)ラクトバチルス・サリバリウス YIT0479株(独立行政法人産業技術総合研究所に寄託、微生物寄託番号「FERM P−19332号」)、(ロ)ラクトバチルス・ロイテリ YIT0480株(独立行政法人産業技術総合研究所に寄託、微生物寄託番号「FERM P−19333号」)、(ハ)ラクトバチルス・クリスパータス YIT0481株(独立行政法人産業技術総合研究所に寄託、微生物寄託番号「FERM P−19334号」)及び(ニ)ラクトバチルス・ジョンソニー YIT0482株(独立行政法人産業技術総合研究所に寄託、微生物寄託番号「FERM P−19335号」)がいずれも抗生物質に対する優れた耐性を有する点で好ましい。
【0014】
ここで、上記(B)ラクトバチルス属の4種類の菌株である、(イ)ラクトバチルス・サリバリウス YIT0479株、(ロ)ラクトバチルス・ロイテリ YIT0480株、(ハ)ラクトバチルス・クリスパータス YIT0481株及び(ニ)ラクトバチルス・ジョンソニー YIT0482株は、新たに単離された菌株であるので、以下に(a)形態的性質、(b)培養的性質及び(c)化学分類学的性質等を示す。なお、本発明の(イ)〜(ニ)の4菌株は、それぞれの菌株の(a)〜(c)の性質において、有意な差は見られない。
【0015】
(a)形態的性質
本発明の(イ)〜(ニ)の4菌株は、いずれも以下の性質を示す。また、上記(イ)〜(ニ)の4菌株のグラム染色像は、いずれも陽性であった。
(1)細菌の形:桿状
(2)細菌の大きさ:0.6〜0.8×1.3〜3.5μm
(3)細菌の多形性:無
(4)運動性:無
(5)胞子:無
グラム染色像によれば、(イ)L.サリバリウス YIT0479株は、大きさが均一の桿菌であり、(ロ)L.ロイテリ YIT0480株は、大きさが均一の短桿菌であり、(ハ)L.クリスパータス YIT0481株及び(ニ)L.ジョンソニー YIT0482株は、大きさが均一の桿菌(やや細い)であることが示される。
【0016】
(b)培養的性質(生育状態)
(1)本発明の(イ)〜(ニ)の4菌株のMRS寒天平板培養(37℃、2日間)による生育状態について以下に示す。
(イ)L.サリバリウス YIT0479株は、直径1mm程度の白色スムース球形コロニーを生じる。(ロ)L.ロイテリ YIT0480株は、直径2mm程度の白色スムース球形コロニーを生じる。(ハ)L.クリスパータス YIT0481株は、直径2mm程度の灰白色ラフフラットコロニーを生じる。(ニ)L.ジョンソニー YIT0482株は、直径1mm程度の灰白色スムースフラットコロニーを生じる。
(2)本発明の(イ)〜(ニ)の4菌株のMRS液体培養(37℃、24時間)による生育状態については、いずれの菌株にも良好な生育が見られ、静置しておくと沈殿を生じる。
【0017】
(c)化学分類学的性質
(GC含量の測定及びDNA−DNAハイブリダイゼーション)
本発明の(イ)〜(ニ)4菌株のGC含量の測定、及び同4菌株と縁菌種の基準株とのDNA−DNAハイブリダイゼーションは、以下の方法に従って行う。
GC含量の測定方法は、常法の高速液体クロマトグラフィーを用いたEzakiらの方法(文献名:FEMS Microbiol Lett 55,127−130、1990)により行うことができる。測定された本発明の(イ)〜(ニ)4菌株のGC含量は、それぞれ33〜41mol%である。
DNA−DNAハイブリダイゼーションは、常法の蛍光標識マイクロプレート法(文献名:Int J Syst Bacteriol 39,224−229,1989)により行うことができる。以下の20株の基準株(全てラクトバチルス(Lactobacillus)属)を、本発明の(イ)〜(ニ)4菌株と比較するのに使用する。本発明の(イ)〜(ニ)4菌株の各々は、相同性が認められた基準株により同定される。
【0018】
L.acidophilus YIT 0070 (ATTC 4356 )、L.agilis YIT 0253 (JCM 1187 )、L.amylovorus YIT 0211 (JCM 1126 )、L.animalis YIT 0256 (JCM 5670 )、L.brevis YIT 0076 (ATTC 14869 )、L.buchneri YIT 0077 (ATTC 4005 )、L.casei YIT 0180 (ATTC 344 )、L.coryniformis subsp.coryniformis YIT 0237(JCM 1164 )、L.crispatus YIT 0212 (JCM 1185 )、L.fermentum YIT 0081 (ATTC 14931 )、L.gasseri YIT 0192 (DSM 20243 )、L.graminis YIT 0260 (NRIC 1775 )、L.johnsonii YIT 0219 (JCM 2012 )、L.murinus YIT 0239 (JCM 1717 )、L.plantarum YIT 0102 (ATTC 14917 )、L.reuteri YIT 0197 (JCM 1112 )、L.rhamnosus YIT 0105 (ATTC 7469 )、L.ruminis YIT 0221 (JCM 1152 )、L.salivarius subsp.salicinius YIT 0089 (ATTC 11742 ) 及び L.salivarius subsp.salivarius YIT 0104 (ATTC 11741)。
【0019】
本発明の飼料用添加剤に含まれる菌体は各種抗生物質に対して優れた耐性を有するので、馬の健康管理上必要に応じて、各種抗生物質と混合して混合物として飼料に添加してもよい。また、菌体のみで構成される飼料用添加剤を単独で飼料に添加するだけでなく、各種抗生物質と組合わせて使用してもよい。このように飼料用添加剤は、馬の身体症状にあわせて各種抗生物質を適宜選択し如何なる場合にも組合わせることができるので、馬の健康管理、身体症状の改善に有効である。
【0020】
本発明で使用する(C)抗生物質としては、馬の健康管理用に一般的に使用される抗生物質でよく、例えば、仔馬の下痢及び白痢症状等に有用なゲンタマイシン、オキシテトラサイクリン塩酸塩等、肺炎及び関節炎による各種感染症に有用な硫酸カナマイシン、スルファモノメトキシン、バクトラミン、セフトリアキソンナトリウム、セファロチンナトリウム、オフロキサシン、ジヒドロストレプトマイシン等、及び外傷による感染症予防及び輸送熱予防並びに蕁麻疹予防に有用なベンジルペニシリン、アンピシリン等が挙げられる。
【0021】
上記(C)抗生物質のうちいずれを使用するかは、選択された(B)ラクトバチルス属の種類に応じて決定される。例えば、硫酸カナマイシン、スルファモノメトキシン、及びバクトラミン等は、(B)ラクトバチルス属の4種菌株のいずれに対しても使用することができる。また、オフロキサシンは、(B)ラクトバチルス・ロイテリ、ラクトバチルス・クリスパータス及びラクトバチルス・ジョンソニーに対して使用することができる。さらに、セファロチンナトリウム、オキシテトラサイクリン塩酸塩、ベンジルペニシリン及びアンピシリン等は、(B)ラクトバチルス・ロイテリに対して使用することができる。
【0022】
次に、飼料用添加剤の製造方法について示す。
飼料用添加剤は、(A)ラクトバチルス・イクイの菌株と、(B)ラクトバチルス・サリバリウス、ラクトバチルス・ロイテリ、ラクトバチルス・クリスパータス及びラクトバチルス・ジョンソニーからなる群より選ばれる少なくとも1種の菌株とをそれぞれ培養し、培養した菌体を回収し、(A)菌体と(B)菌体とを混合することにより製造される。
上記培養及び回収は通常の方法で行うことができる。使用する培地としては、ラクトバチルス属細菌の増殖可能なMRS broth(Difco Laboratories社製)培地、GAM broth(日水社製)培地などが挙げられる。培養条件はいずれの菌株も37℃で12〜20時間である。菌体の回収には通常使用される遠心分離等の手段を用いることができる。得られた飼料用添加剤は、用途に応じて適当な形態にすればよい。
【0023】
上記製造方法では、菌株によって増殖の程度が異なるため、上記培地以外に、それぞれの菌株に最適な培地を選択して培養することが望ましい。例えば、ラクトバチルス・ロイテリ YIT0480株には、PSYL培地[組成:20%PSM*)5%、ミーストP2G(日本製薬社製)1%、CHCOONa(和光社製)0.3%、(NHSO(和光社製)0.3%、KHPO(和光社製)1%、KHPO(和光社製)0.2%、Met sol**)0.5%]が好ましい。
*):脱脂粉乳(四つ葉社製)200g、オリエンターゼ5N(阪急バイオ社製)4g/1L
**):MgSO・7HO 115g、FeSO・7HO 6.8g、MnSO・7HO 24g(和光社製)/1L
【0024】
また、菌株の増殖性を向上させる手段として、必要に応じて、資化性糖を培地に添加することが望ましい。例えば、ラクトバチルス・イクイ YIT0483株及びラクトバチルス・クリスパータス YIT0481株には、培地に資化性糖としてラクトースを添加することが好ましい。また、ラクトバチルス・サリバリウス YIT0479株及びラクトバチルス・ジョンソニー YIT0482株には、培地に資化性糖としてグルコースを添加することが好ましい。
本発明では、使用する培地は上記に限られるものではなく、一般に使用される培地に含まれる組成を適宜組合わせて各菌株に最適な培地を作製したものを用いてもよい。
【0025】
本発明の飼料用添加剤では、使用する菌体が生菌体であることが望ましく、生菌体であればいかなる状態のものでもよい。例えば、各菌株の培養回収菌体、培養物、凍結乾燥菌体等が挙げられる。なかでも、生菌体の保存性が高い凍結乾燥菌体を使用することが望ましい。なお、死菌体は、馬に投与した時の下痢などの消化管障害の予防及び改善や、早期腸内フローラ形成促進等の生理効果が低いため好ましくない。
【0026】
本発明の飼料用添加剤は、馬の胃粘膜上皮細胞への優れた付着性及び増殖活性を示し、並びに抗生物質に対する優れた耐性を有する。これにより、この飼料用添加剤は、早期の腸内フローラ形成により下痢等の馬の消化管(特に腸管)障害を早期に予防及び改善すると共に、予防及び改善された馬の消化管内環境が、馬の健康管理上必要に応じて使用される上記(C)抗生物質の影響を受けることなく良好に維持され、種々のストレスから誘発される馬の身体症状を効果的に改善することができる。さらに、本発明では、特に(A)ラクトバチルス・イクイの菌体と、(B)ラクトバチルス属の4種類の全ての菌体とを含む飼料用添加剤は、上記のような馬の身体症状の改善効果が顕著であるので好ましい。
【0027】
本発明の飼料用添加剤の投与量は、馬一頭に対して1日あたり上記(A)と(B)との菌体総数として1×10個以上、好ましくは1×1010個〜5×1010個投与することが望ましい。1日の投与量が1×10個より少ない場合には、馬の身体症状に十分な改善効果が得られないため好ましくない。さらに、投与する飼料用添加剤における各種菌株の菌体数割合は、該飼料用添加剤に含有させる菌株の組合わせによって任意に調整することが可能であるが、(A)ラクトバチルス・イクイを菌体総数の5%〜25%、好ましくは10%〜20%の範囲で含有させることにより、馬の身体症状改善に顕著な効果を有する。
【0028】
また、本発明の飼料用添加剤の馬への投与方法は、特に限定されるものではなく、注射器などを用いて直接馬の腸内に投与してもよく、各種糖質、タンパク質、脂質、繊維質、ビタミン類、及びミネラル類などと共に、常法により粉剤、錠剤、顆粒剤、ペレットなどの製剤にして経口投与してもよい。
【0029】
本発明は、これらの飼料用添加剤と、他の成分、例えば脂質や繊維質等とを含有する飼料を提供する。脂質や繊維質としては、飼料成分として一般的に使用可能なものであればいずれでもよく、例えば、大豆粕、アルファルファミール、及びふすま等が挙げられる。本発明の飼料を馬に投与することにより、消化管障害の予防及び改善され、飼料効率を向上させることができるので軽種馬経営上好ましい。
【0030】
【実施例】
以下、実施例を挙げて本発明を更に詳細に説明するが、本発明はこれらに限定されるものではない。
【0031】
<製造例1>
(各菌株の菌体の製造)
下記表1に示す組成の培地を、5Lコルベンに4.5Lずつ作製し、pHを6.8に調整後、高圧加熱殺菌した。次いで、下記表1に示す(A)及び(B)ラクトバチルス属の5種類の菌株の前培養菌体のクレット値を測定し、クレット300に希釈した菌液450mlを前記培地に植菌し、37℃、16時間静置培養して培養液を得た。この培養液を1,500×Gで遠心分離し、5種菌株の菌体をそれぞれ集菌した。各菌株の菌体の4.5Lあたりの集菌量(湿重量)は表1の通りである。
【0032】
【表1】

Figure 2004357528
【0033】
<実施例1>
(増殖活性試験及び馬の胃粘膜上皮細胞に対する菌体の付着性試験)
10%スキムミルク(SM)、0.1%酵母抽出物(Difco社製)+10%スキムミルク(YE)、0.1%ペプトン(Difco社製)+10%スキムミルク(P)及び0.1%酵母抽出物(Difco社製)+0.1%ペプトン(Difco社製)+10%スキムミルク(YP)の4種類の培地を115℃で15分間高圧加熱滅菌した後、それぞれの培地3mlに前記製造例1で得られた(A)及び(B)の5種類の菌株の各菌体培養液を30μl(各菌株の菌体数:約3×10個/ml)ずつ接種し、37℃で24時間及び48時間培養した。増殖活性試験は、増殖した菌体により培養液が完全に凝固しているものを○、菌体は僅かに増殖したが培養液が半流動状態のものを△、菌体が増殖せずに培養液が液状のままのものを×とした判断基準に基づいて、判定を行った。
【0034】
また、前記製造例1で得られた(A)及び(B)の5種類の菌株の各菌体培養液3ml(各菌株の菌体数:約3×10個/ml)に馬から採取した胃粘膜上皮組織片1cmを入れ、37℃で振とう培養した後、この胃粘膜上皮組織片を緩衝液(組成:0.8%NaCl;0.121%KHPO;0.034%KHPO;pH7.2)で数回洗浄して、実体顕微鏡下で胃粘膜上皮組織片から胃粘膜上皮細胞を掻き取り、ギムザ染色を行った。これを用いて顕微鏡下で一つの胃粘膜上皮細胞に付着した各菌株の菌体数を測定し、付着性を判定した。表2にその結果を示す。
【0035】
【表2】
Figure 2004357528
【0036】
表2の結果から、本発明で使用する(A)及び(B)ラクトバチルス属の5種類の菌株の各菌体は、各培地条件において優れた増殖活性を示すと共に、馬の胃粘膜上皮細胞に対しても良好な付着性を示す。また、5種菌株の中でも、(A)L.イクイ YIT0483株が他の(B)の4種菌株に比べて増殖活性及び馬の胃粘膜上皮細胞への付着性に優れていることが認められた。なお、増殖活性試験において、僅かに増殖したが培養液が半流動状態(△判定)のものはなかった。
【0037】
<実施例2>
(抗生物質耐性試験)
下記表3に示された各抗生物質(11種類)を100、10、1、0.1及び0μg/mlの濃度となるようにそれぞれ添加してMRS寒天培地(Difco社製)を作製した。次に、製造例1で得られた(A)及び(B)の5種類の各菌株の菌体培養液を、それぞれの抗生物質含有MRS寒天培地に10μlずつ接種し(各菌株の菌体数:約1×10個/ml)、嫌気条件下で37℃、48時間塗抹培養した。増殖した菌を肉眼で判定し、各抗生物質に対する最小発育濃度(μg/ml)を求めた結果を表3に示す。
【0038】
【表3】
Figure 2004357528
【0039】
表3の結果から、(A)及び(B)ラクトバチルス属の5種類の各菌株は、いずれも各種抗生物質、特に硫酸カナマイシン、スルファモノメトキシン、ジヒドロストレプトマイシン、バクトラミンなどに優れた耐性を示した。特に、L.イクイ株はセフトリアキソンナトリウムに、L.ジョンソニー株はゲンタマイシンに、L.ロイテリ株、L.クリスパータス株及びL.ジョンソニー株はオフロキサシンに、それぞれ特異的に優れた耐性を示していた。このように、用いた5種類の各菌株は、同属に属するにもかかわらず、それぞれ異なる抗菌スペクトルを示した。L.ロイテリ株は5種類の菌株の中でもとりわけ耐性に優れ、特にセファロチンナトリウム、オキシテトラサイクリン塩酸塩及びアンピシリンに対して優れていた。また、このL.ロイテリ株は、他の4種類の菌株よりもベンジルペニシリンに対して耐性が強いので、ベンジルペニシリンを使用する場合に特に有用である。
【0040】
また、(A)L.イクイ YIT0483株と(B)ラクトバチルス属の4種類の菌株から選ばれる1種以上の菌株とを組合わせた菌体培養液を用いて、抗生物質存在下での増殖活性試験を行った。
【0041】
(増殖活性試験1)
抗生物質としてオキシテトラサイクリン塩酸塩を10、5、2及び0μg/mlの濃度となるようにそれぞれ添加したMRS寒天培地(Difco社製)を作製した。次に、製造例1で得られた(A)L.イクイ株単独の菌体培養液と、(A)L.イクイ株及び(B)L.ロイテリ株の混合菌体培養液とを、それぞれの抗生物質含有MRS寒天培地に10μlずつ接種し(各菌体数:約1×10個/ml)、嫌気条件下で37℃、48時間塗抹培養した後、菌の増殖活性を肉眼で判定した。
【0042】
(増殖活性試験2)
抗生物質としてゲンタマイシンを100、50、20及び0μg/mlの濃度となるようにそれぞれ添加したMRS寒天培地(Difco社製)を作製した。次に、製造例1で得られた(A)L.イクイ株単独の菌体培養液と、(A)L.イクイ株及び(B)L.ジョンソニー株の混合菌体培養液とを、それぞれの抗生物質含有MRS寒天培地に10μlずつ接種し(各菌体数:約1×10個/ml)、嫌気条件下で37℃、48時間塗抹培養した後、菌の増殖活性を肉眼で判定した。
【0043】
(増殖活性試験3)
抗生物質としてオフロキサシンを100、50、20及び0μg/mlの濃度となるようにそれぞれ添加したMRS寒天培地(Difco社製)を作製した。製造例1で得られた(A)L.イクイ株単独の菌体培養液と、(A)L.イクイ株及び(B)L.ジョンソニー株又は(A)L.イクイ株及び(B)L.クリスパータス株の混合菌体培養液とを、それぞれの抗生物質含有MRS寒天培地に10μlずつ接種し(各菌体数:約1×10個/ml)、嫌気条件下で37℃、48時間塗抹培養した後、菌の増殖活性を肉眼で判定した。
以上の増殖活性試験1〜3の結果を表4−1〜3に示す。
【0044】
【表4】
Figure 2004357528
【0045】
表4−1〜3の結果から、抗菌スペクトルの異なる(A)ラクトバチルス・イクイ YIT0483株と(B)ラクトバチルス属の4種類の菌株から選ばれる1種以上の菌株とを組合わせた菌体培養液を用いれば、抗生物質存在下でも優れた増殖活性を有することが認められた。
【0046】
<製造例2>
(凍結乾燥菌体の製造)
製造例1により得られた(A)及び(B)5種類の菌株の菌体培養液を、それぞれ10N水酸化ナトリウムでpH6.8〜7.0に調整した。次いで、5,000回転で10分間遠心して上清を除去後、残った菌体に分散媒(組成:スキムミルク20%、及びトレハロース20%水溶液)を8%の割合で添加して均一化した。次いで、−30℃、3時間の予備凍結の後、常法に従い凍結乾燥処理をした。凍結乾燥後の重量を測定し、等量の乾燥澱粉を加えて4℃で保存した。
【0047】
<実施例3>
(早期腸内フローラ形成促進効果)
出生直後の6頭のサラブレッド種仔馬に、製造例2で得られた凍結乾燥菌体5g((A)及び(B)ラクトバチルス属の5種菌株を生菌体総数で3.8×1010個/5g含有する)を5%ブドウ糖50mlに溶解したものを毎日計7回経口投与した。出生後1、2、3、5、7及び14日毎に直腸便を採取し、希釈液を加えて均一化した後、さらに適宜希釈し、LBS寒天培地(Becton Dickinson社製)に接種し、嫌気条件下37℃で4日間培養し、増殖したコロニー数と希釈率から直腸便の乳酸桿菌数を算出した。コントロールとして、凍結乾燥菌体を投与しない6頭の仔馬からも同様に直腸便の乳酸桿菌数を算出した。なお、表5には、(乳酸桿菌が検出された仔馬数)/(調べた全ての仔馬数)×100から算出された検出率(%)を示す。
【0048】
【表5】
Figure 2004357528
【0049】
表5の結果から、投与群では、投与5日目で全ての仔馬の腸内に乳酸桿菌の付着が確認され、コントロール群より2日も早い腸内フローラ形成促進効果が認められた。
【0050】
<実施例4>
(下痢の改善効果 検討1)
上記実施例3と同様の試験を出生直後の仔馬54頭(コントロール群27頭、投与群27頭)において実施し、出生30日目までの下痢の発生を観察した。下痢は、肉眼的に軟便、軽度下痢(下痢便を排泄するが、回数が少なく肛門附近の付着性が僅かなもの)及び重度下痢(悪臭のある水様性の下痢を何度となく排泄し、肛門から臂部にかけて多量の下痢便を付着しているもの)の3段階として、それぞれの発症率(%)を表6に示す。
【0051】
【表6】
Figure 2004357528
【0052】
表6の結果から、軟便の発症率は、3週目に投与群で有意に低い値となった。また、軽度下痢、重度下痢の発症率については、3週目以降に投与群で有意に低い傾向が見られ、特に、重度下痢の発症は、投与群では全く見られず、4週目では、治療処置及び抗生物質の投与もしなくても良いほど改善の効果が見られた。
【0053】
<実施例5>
(下痢の改善効果 検討2)
22頭のサラブレッド種仔馬に、製造例2で得られた凍結乾燥菌体5g((A)及び(B)ラクトバチルス属の5種菌株を生菌体総数で1.4×1010個/5g含有する)を5%ブドウ糖50mlに溶解したものを1日目〜7日目の投与期間に、1日当たり計7回経口投与した。0日目(投与なし)から14日目までの下痢の発生を観察した。コントロールとして、凍結乾燥菌体を投与していない6頭の仔馬についても下痢の発生を観察した。下痢は、肉眼的に軟便、重度下痢(悪臭のある水様性の下痢を何度となく排泄し、肛門から臂部にかけて多量の下痢便を付着しているもの)の2段階とした。軟便の発症率(%)を図1に、重度下痢の発症率(%)を図2にそれぞれ示す。
【0054】
図1及び図2の結果から、軟便の発症において、投与群ではコントロール群と比較して投与6日目で顕著に減少する傾向が見られた。また、下痢の発症において、コントロール群では発症していた下痢症状が、投与群では投与5日目以降全く確認されなかった。
【0055】
【発明の効果】
本発明によれば、馬の消化管上皮への特異的な付着性及び増殖活性を示すだけでなく、抗生物質に対して優れた耐性を有し、抗生物質投与の副作用による下痢等の消化管障害の予防及び改善に有効である飼料用添加剤、及びそれを含む飼料が提供される。
【図面の簡単な説明】
【図1】実施例5の飼料用添加剤の投与群及びコントロール群おける仔馬の軟便の発症率を示した図である。
【図2】実施例5の飼料用添加剤の投与群及びコントロール群おける仔馬の重度下痢の発症率を示した図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a feed additive for preventing and improving gastrointestinal disorders due to side effects of antibiotic administration in horse rearing management, and a feed containing the same.
[0002]
[Prior art]
In general, cattle, pigs, horses, etc. are listed as representative animals that are reared and managed as livestock animals. However, these livestock animals are affected by the development process and health status due to changes in the breeding environment. Because it is easy, careful consideration is required for breeding management. For example, in the case of horses, it is known that physical symptoms such as diarrhea are likely to occur due to various stress conditions due to changes in the breeding environment such as meals. Horses (especially foals) have fewer bacteria in the intestine than other livestock animals, and these bacteria are extremely reduced by changes in the breeding environment. The intestinal flora is damaged, causing symptoms such as diarrhea by breaking the balance. Such a symptom adversely affects physiological functions such as a decrease in digestive and absorption capacity, growth inhibition, and a decrease in immunity.
[0003]
In recent years, in the breeding management of horses, feeds containing microorganisms typified by lactic acid bacteria and bifidobacteria are widely used (see, for example, Patent Documents 1 to 4). These feeds are expected to improve the intestinal flora by adhering the contained microorganisms to the intestine of the horse, and to promote physiological effects such as growth promotion and diarrhea improvement.
However, even if these feeds are used, the administered microorganisms do not adhere to and proliferate in the intestines of horses, and thus often cannot fully improve the physical symptoms such as diarrhea. Currently, antibiotics are being administered.
[0004]
Antibiotics administered effectively improve the physical symptoms of horses, but at the same time have effects such as killing bacteria present in the horse's intestines, and adverse effects such as worsening of diarrhea symptoms. Often causes to cause. In addition, these antibiotics kill not only bacteria present in the intestines of horses, but also useful microorganisms that are separately administered with feed, etc., so when feed containing live microorganisms with useful microorganisms is administered to horses. Even in such cases, the effect of improving the physical symptoms that cause gastrointestinal disorders such as diarrhea was low without obtaining sufficient effects of the administered microorganism.
[0005]
Therefore, it is not sufficient to simply select microorganisms such as lactic acid bacteria and bifidobacteria that have an effect of improving the intestinal environment in the feed containing microorganisms used for the breeding management of horses. Selective of microorganisms that exhibit specific adhesion and proliferative activity and have excellent effects on improving the intestinal environment of horses, and various antibiotics that may be administered as needed depending on the physical symptoms of the horse It is necessary to select microorganisms that have excellent resistance to.
[0006]
[Patent Document 1]
JP 59-46208
[Patent Document 2]
Japanese Patent No. 3220699
[Patent Document 3]
Special table 2001-519144 gazette
[Patent Document 4]
JP 2002-58432 A
[0007]
[Problems to be solved by the invention]
Therefore, the object of the present invention is not only to show specific adhesion and proliferation activity to the digestive tract epithelium of horses, but also has excellent resistance to antibiotics, such as diarrhea due to side effects of antibiotic administration, etc. An object of the present invention is to provide a feed additive effective for prevention and improvement of digestive tract disorders, and a feed containing the same.
[0008]
[Means for Solving the Problems]
As a result of diligent research, the present inventors have found that among the bacterial species belonging to the genus Lactobacillus, five strains having excellent adhesion and proliferation activity to equine gastric mucosal epithelial cells are horses (particularly foals). It was found that diarrhea is highly effective in preventing and improving, and that it is more resistant to antibiotics than other bacteria.
In addition, the five strains belonging to the genus Lactobacillus showed excellent antibiotic resistance, and although they belong to the same genus, they were found to have different antibacterial spectra, thereby completing the present invention. .
[0009]
That is, the present invention includes (A) Lactobacillus equii (B) Lactobacillus salvarius, Lactobacillus reuteri (Lactobacillus reuteri), Lactobacillus reuteri (Lactobacillus reuteri), And at least one cell selected from the group consisting of Lactobacillus johnsonii.
Further, the present invention is selected from the group consisting of (C) ceftriaxone sodium, kanamycin sulfate, dihydrostreptomycin, sulfamonomethoxine, bactramine, gentamicin, ofloxacin, cephalothin sodium, ampicillin, oxytetracycline hydrochloride and benzylpenicillin. The feed additive further comprises at least one antibiotic.
Moreover, this invention provides the said additive for feed for adding in combination with the said (C) antibiotics.
Moreover, this invention provides the feed containing the said additive for feed.
The present invention also relates to at least one strain belonging to the genus Lactobacillus selected from the group consisting of the following (a) to (d): (i) Lactobacillus salivaius YIT0479 strain (deposit number: FERM P-19332) (B) Lactobacillus reuteri YIT0480 strain (Deposit number: FERM P-19333); (C) Lactobacillus chryspatus YIT0481 strain (Deposit number: FERM P-19334) and (d) Lactobacillus johnsonii YIT0482 strain (deposit number: FERM P-19335) is provided.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
The feed additive of the present invention includes (A) Lactobacillus equii (B) Lactobacillus salivarius, Lactobacillus reuteri (Lactobacillus reuteri), Lactobacillus reuteri, Lactobacillus reuteri (Lactobacillus crisis) and Lactobacillus johnsonii (Lactobacillus johnsonii) are comprised from at least 1 sort (s) of microbial cells selected from the group.
[0011]
All five strains used in the present invention belong to the genus Lactobacillus, and all are strains isolated from horse feces. Isolation is performed by culturing at about 37 ° C. under anaerobic conditions using an LBS medium as a selective medium for Lactobacillus and an MRS medium as a non-selective medium. Isolated strains are characterized by morphological, cultural, physiological or chemical taxonomic properties. Among the strains belonging to the genus Lactobacillus, the strain is a strain that exhibits excellent adhesion and proliferation activity particularly to equine gastric mucosal epithelial cells and has excellent resistance to antibiotics.
[0012]
(A) As a strain of Lactobacillus ikui, in particular, Lactobacillus ikui YIT0483 strain (deposited with the National Institute of Advanced Industrial Science and Technology, microbial deposit number “FERM P-18777”) is particularly adherent and proliferative activity It is preferable at the point which is excellent in.
[0013]
In addition, (B) Lactobacillus genus strains include, in particular, (i) Lactobacillus salivarius YIT0479 strain (deposited with the National Institute of Advanced Industrial Science and Technology, microbial deposit number “FERM P-19332”) , (B) Lactobacillus reuteri YIT0480 (deposited with the National Institute of Advanced Industrial Science and Technology, microbial deposit number “FERM P-19333”), (c) Lactobacillus chryspatus YIT0481 (Incorporated administrative agency, National Institute of Advanced Industrial Science and Technology) (Deposited with the institute, microorganism deposit number “FERM P-19334”) and (d) Lactobacillus johnsonii YIT0482 strain (deposited with the National Institute of Advanced Industrial Science and Technology, microorganism deposit number “FERM P-19335”) Are preferable in that they have excellent resistance to antibiotics.
[0014]
Here, (B) Lactobacillus salivarius YIT0479 strain, (b) Lactobacillus reuteri YIT0480 strain, (C) Lactobacillus chryspatus YIT0481 strain, ) Since Lactobacillus johnsonii strain YIT0482 is a newly isolated strain, (a) morphological properties, (b) culture properties, (c) chemical taxonomic properties, etc. are shown below. In addition, the four strains (a) to (d) of the present invention do not show a significant difference in the properties (a) to (c) of the respective strains.
[0015]
(A) Morphological properties
The four strains (i) to (d) of the present invention all exhibit the following properties. In addition, the gram-stained images of the four strains (i) to (d) above were all positive.
(1) Bacteria shape: Spider
(2) Bacterial size: 0.6-0.8 × 1.3-3.5 μm
(3) Bacterial polymorphism: None
(4) Mobility: None
(5) Spore: None
According to the Gram stained image, Sarivarius YIT0479 strain is a uniform gonococcus. Reuteri YIT0480 strain is a short gonococcus having a uniform size. Chris Partus YIT0481 strain and (d) L. John Sony YIT0482 strain is shown to be a uniform gonococcus (slightly thin).
[0016]
(B) Culture properties (growth state)
(1) The growth state of the four strains (i) to (d) of the present invention by MRS agar plate culture (37 ° C., 2 days) is shown below.
(A) L. Sarivarius strain YIT0479 produces white smooth spherical colonies with a diameter of about 1 mm. (B) L. Reuteri YIT0480 strain produces white smooth spherical colonies with a diameter of about 2 mm. (C) L. Crispatus strain YIT0481 produces grayish white rough flat colonies with a diameter of about 2 mm. (D) L. John Sony YIT0482 strain produces grayish white smooth flat colonies with a diameter of about 1 mm.
(2) About the growth state by MRS liquid culture (37 degreeC, 24 hours) of 4 strains of (i)-(d) of this invention, all growths have seen favorable growth and leave still. And precipitate.
[0017]
(C) Chemical taxonomic properties
(Measurement of GC content and DNA-DNA hybridization)
The measurement of the GC content of the four strains (i) to (d) of the present invention and the DNA-DNA hybridization between the four strains and the reference strain of the relative strain are performed according to the following method.
The GC content can be measured by the method of Ezaki et al. (Literature name: FEMS Microbiol Lett 55, 127-130, 1990) using conventional high performance liquid chromatography. The measured GC contents of (i) to (d) 4 strains of the present invention are 33 to 41 mol%, respectively.
DNA-DNA hybridization can be performed by a conventional fluorescent labeling microplate method (literature name: Int J Systact Bacteriol 39, 224-229, 1989). The following 20 reference strains (all belonging to the genus Lactobacillus) are used for comparison with the (i) to (d) 4 strains of the present invention. Each of the four strains (i) to (d) of the present invention is identified by a reference strain in which homology is recognized.
[0018]
L. acidophilus YIT 0070 (ATTC 4356 T ), L. agilis YIT 0253 (JCM 1187 T ), L. amylovorus YIT 0211 (JCM 1126 T ), L. animalis YIT 0256 (JCM 5670 T ), L. brevis YIT 0076 (ATTC 14869 T ), L. buchneri YIT 0077 (ATTC 4005 T ), L. casei YIT 0180 (ATTC 344 T ), L. coriniformis subsp. coriniformis YIT 0237 (JCM 1164 T ), L. crisptus YIT 0212 (JCM 1185 T ), L. fermentum YIT 0081 (ATTC 14931 T ), L. gasseri YIT 0192 (DSM 20243 T ), L. Graminis YIT 0260 (NRIC 1775 T ), L. johnsonii YIT 0219 (JCM 2012 T ), L. Murinus YIT 0239 (JCM 1717 T ), L. plantarum YIT 0102 (ATTC 14917 T ), L. reuteri YIT 0197 (JCM 1112 T ), L. rhamnosus YIT 0105 (ATTC 7469 T ), L. ruminis YIT 0221 (JCM 1152 T ), L. salivarius subsp. salicinius YIT 0089 (ATTC 11742 T ) And L. salivarius subsp. salivarius YIT 0104 (ATTC 11741 T ).
[0019]
Since the bacterial cells contained in the feed additive of the present invention have excellent resistance to various antibiotics, add them to the feed as a mixture by mixing with various antibiotics as needed for horse health management. Also good. Moreover, you may use in combination with various antibiotics not only adding the additive for feed comprised only with a microbial cell to feed independently. As described above, the feed additive is effective for the health management of horses and the improvement of physical symptoms because various antibiotics can be appropriately selected according to the physical symptoms of horses and combined in any case.
[0020]
The antibiotic (C) used in the present invention may be an antibiotic generally used for horse health management, such as gentamicin, oxytetracycline hydrochloride, etc. useful for foal diarrhea and white diarrhea, etc. Kanamycin sulfate, sulfamonomethoxine, bactramine, ceftriaxone sodium, cephalothin sodium, ofloxacin, dihydrostreptomycin, etc. useful for various infections caused by pneumonia and arthritis, and prevention of infection and transport fever and urticaria due to trauma Benzylpenicillin, ampicillin and the like useful in the above.
[0021]
Which of the above (C) antibiotics is used is determined according to the type of (B) Lactobacillus selected. For example, kanamycin sulfate, sulfamonomethoxine, and bactramine can be used for any of the four strains of the genus (B) Lactobacillus. Ofloxacin can also be used against (B) Lactobacillus reuteri, Lactobacillus crispatus and Lactobacillus johnsonii. Furthermore, cephalotin sodium, oxytetracycline hydrochloride, benzylpenicillin, ampicillin and the like can be used for (B) Lactobacillus reuteri.
[0022]
Next, a method for producing a feed additive will be described.
The feed additive is at least one selected from the group consisting of (A) a strain of Lactobacillus ikui, and (B) Lactobacillus salivaius, Lactobacillus reuteri, Lactobacillus crispatus and Lactobacillus johnsonii Each strain is cultured, the cultured cells are collected, and (A) the cells and (B) the cells are mixed.
The culture and recovery can be performed by a usual method. Examples of the medium to be used include MRS broth (manufactured by Difco Laboratories) medium and GAM broth (manufactured by Nissui) medium capable of growing Lactobacillus bacteria. The culture conditions are 12-20 hours at 37 ° C. for all strains. Means such as centrifugation, which are usually used, can be used for collecting the cells. What is necessary is just to make the additive for feed obtained into a suitable form according to a use.
[0023]
In the above production method, since the degree of growth varies depending on the strain, it is desirable to select and culture a medium optimal for each strain in addition to the above medium. For example, Lactobacillus reuteri YIT0480 strain contains PSYL medium [composition: 20% PSM. *) 5%, Mist P2G (Nippon Pharmaceutical Co., Ltd.) 1%, CH 3 COONa (made by Wako) 0.3%, (NH 4 ) 2 SO 4 (Wako) 0.3%, KH 2 PO 4 (Wako) 1%, K 2 HPO 4 (Wako) 0.2%, Met sol **) 0.5%] is preferable.
*) : Skim milk powder (Yotsuba) 200g, orientase 5N (Hankyu Bio) 4g / 1L
**) : MgSO 4 ・ 7H 2 115 g of O, FeSO 4 ・ 7H 2 6.8 g of O, MnSO 4 ・ 7H 2 O 24g (manufactured by Wako) / 1L
[0024]
Moreover, as a means for improving the growth of the strain, it is desirable to add an assimilating sugar to the medium as necessary. For example, it is preferable to add lactose as an assimilating sugar to the culture medium for Lactobacillus iqui YIT0483 strain and Lactobacillus crispartus YIT0481 strain. Moreover, it is preferable to add glucose as an assimilating sugar to the culture medium for Lactobacillus salivaius YIT0479 strain and Lactobacillus johnsonii YIT0482 strain.
In the present invention, the medium to be used is not limited to the above, and a medium in which an optimal medium for each strain is prepared by appropriately combining compositions contained in a commonly used medium may be used.
[0025]
In the feed additive of the present invention, it is desirable that the microbial cell to be used is a live microbial cell, and it may be in any state as long as it is a live microbial cell. For example, the culture | cultivation collection | recovery microbial cell of each strain, a culture, freeze-dried microbial cell, etc. are mentioned. Among them, it is desirable to use freeze-dried cells that have high storage stability of viable cells. Note that dead cells are not preferred because they have low physiological effects such as prevention and improvement of gastrointestinal disorders such as diarrhea when administered to horses and promotion of early intestinal flora formation.
[0026]
The feed additive of the present invention exhibits excellent adhesion to and proliferation activity of equine gastric mucosal epithelial cells, and has excellent resistance to antibiotics. As a result, this feed additive prevents and improves early gastrointestinal (particularly intestinal) disorders such as diarrhea due to early intestinal flora formation, and prevents and improves the digestive tract environment of the horse. It is well maintained without being affected by the antibiotic (C) used as necessary for the health management of horses, and can effectively improve the physical symptoms of horses induced by various stresses. Furthermore, in the present invention, in particular, the feed additive containing (A) Lactobacillus icui cells and (B) all four types of Lactobacillus genus cells is a physical symptom of the horse as described above. Since the improvement effect of is remarkable, it is preferable.
[0027]
The dosage of the feed additive of the present invention is 1 × 10 as the total number of cells of the above (A) and (B) per day per horse. 9 1 or more, preferably 1 × 10 10 Pieces-5 x 10 10 Individual administration is desirable. Daily dose is 1 x 10 9 When the number is less than 1, it is not preferable because a sufficient improvement effect on the physical symptoms of the horse cannot be obtained. Furthermore, the number ratio of various strains in the feed additive to be administered can be arbitrarily adjusted by the combination of strains contained in the feed additive, but (A) Lactobacillus By containing it in the range of 5% to 25%, preferably 10% to 20% of the total number of cells, it has a remarkable effect on improving the physical symptoms of horses.
[0028]
Further, the method for administering the feed additive of the present invention to the horse is not particularly limited, and it may be directly administered into the intestine of the horse using a syringe or the like, and various carbohydrates, proteins, lipids, Along with fiber, vitamins, minerals and the like, they may be orally administered in the form of powders, tablets, granules, pellets, etc. by conventional methods.
[0029]
The present invention provides a feed containing these feed additives and other components such as lipids and fibers. Any lipid or fiber may be used as long as it is generally usable as a feed ingredient, and examples thereof include soybean meal, alfalfa meal, and bran. By administering the feed of the present invention to horses, gastrointestinal tract disorders can be prevented and improved, and feed efficiency can be improved.
[0030]
【Example】
EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these.
[0031]
<Production Example 1>
(Manufacture of cells of each strain)
A culture medium having the composition shown in Table 1 below was prepared in 4.5 L per 5 L Kolben, adjusted to pH 6.8, and then sterilized by high-pressure heating. Next, the Klett value of the precultured cells of 5 strains of (A) and (B) Lactobacillus genus shown in Table 1 below is measured, and 450 ml of the bacterial solution diluted in Klett 300 is inoculated into the medium, The culture solution was obtained by stationary culture at 37 ° C. for 16 hours. This culture solution was centrifuged at 1,500 × G, and the cells of 5 strains were collected. The collected amount (wet weight) per 4.5 L of the bacterial cells of each strain is as shown in Table 1.
[0032]
[Table 1]
Figure 2004357528
[0033]
<Example 1>
(Proliferation activity test and adhesion test of bacterial cells to stomach mucosa epithelial cells of horse)
10% skim milk (SM), 0.1% yeast extract (Difco) + 10% skim milk (YE), 0.1% peptone (Difco) + 10% skim milk (P) and 0.1% yeast extract Four types of media (Difco) + 0.1% peptone (Difco) + 10% skim milk (YP) were sterilized by high-pressure heat at 115 ° C. for 15 minutes, and then 3 ml of each medium was obtained in Production Example 1 above. 30 μl of each cell culture solution of the five strains (A) and (B) (number of cells of each strain: about 3 × 10 8 Per ml) and cultured at 37 ° C. for 24 and 48 hours. Proliferation activity test: ○ when the culture solution is completely coagulated by the proliferated cells, △ when the cells are slightly grown but the culture solution is semi-fluid, cultivate without cell growth Judgment was performed based on a judgment standard in which the liquid remains liquid.
[0034]
In addition, 3 ml of each cell culture solution of the five strains (A) and (B) obtained in Production Example 1 (the number of cells of each strain: about 3 × 10 8 Gastric mucosal epithelial tissue piece 1 cm collected from horses 2 And the gastric mucosal epithelial tissue pieces were buffered (composition: 0.8% NaCl; 0.121% K). 2 HPO 4 ; 0.034% KH 2 PO 4 Washed several times at pH 7.2), and gastric mucosal epithelial cells were scraped from the gastric mucosal epithelial tissue piece under a stereomicroscope, and Giemsa staining was performed. Using this, the number of bacterial cells of each strain adhering to one gastric mucosal epithelial cell was measured under a microscope to determine adhesion. Table 2 shows the results.
[0035]
[Table 2]
Figure 2004357528
[0036]
From the results of Table 2, the cells of the five strains of the genus (A) and (B) used in the present invention show excellent proliferation activity in each medium condition, and equine gastric mucosal epithelial cells. Also shows good adhesion. Among the five strains, (A) L. It was confirmed that Ikuy YIT0483 strain was superior in growth activity and adhesion to equine gastric mucosal epithelial cells as compared with the other four strains of (B). In the growth activity test, there was no growth in which the culture broth was in a semi-fluid state (Δ judgment).
[0037]
<Example 2>
(Antibiotic resistance test)
MRS agar medium (manufactured by Difco) was prepared by adding each antibiotic (11 types) shown in Table 3 below to concentrations of 100, 10, 1, 0.1, and 0 μg / ml. Next, 10 μl of each of the five bacterial strains (A) and (B) obtained in Production Example 1 was inoculated into each antibiotic-containing MRS agar medium (the number of bacterial strains of each strain). : About 1 × 10 8 Cells / ml), and smear culture was performed at 37 ° C. for 48 hours under anaerobic conditions. Table 3 shows the results of determining the grown bacteria with the naked eye and determining the minimum growth concentration (μg / ml) for each antibiotic.
[0038]
[Table 3]
Figure 2004357528
[0039]
From the results of Table 3, each of the five strains of the genus (A) and (B) Lactobacillus has excellent resistance to various antibiotics, particularly kanamycin sulfate, sulfamonomethoxine, dihydrostreptomycin, bactramine and the like. Indicated. In particular, L. The Ikui strain is ceftriaxone sodium, L. John Sony shares gentamicin with L. Reuteri strain, L. Chris Partus strain and L. John Sony's strains showed particularly good resistance to ofloxacin. Thus, each of the five strains used showed different antibacterial spectra despite belonging to the same genus. L. The Reuteri strain was particularly resistant among the five strains, and was particularly superior to cephalothin sodium, oxytetracycline hydrochloride and ampicillin. In addition, the L. Reuteri strains are particularly useful when benzylpenicillin is used because they are more resistant to benzylpenicillin than the other four strains.
[0040]
In addition, (A) L. A growth activity test was carried out in the presence of antibiotics using a cell culture medium in which Ikui YIT0483 strain and (B) one or more strains selected from four strains of the genus Lactobacillus were combined.
[0041]
(Proliferation activity test 1)
MRS agar medium (manufactured by Difco) was prepared by adding oxytetracycline hydrochloride as antibiotics to concentrations of 10, 5, 2, and 0 μg / ml, respectively. Next, (A) L. A cell culture solution of Ikui strain alone, (A) L. Ikui strain and (B) L. 10 μl of each antibiotic-containing MRS agar medium was inoculated with the mixed bacterial cell culture solution of Reuteri strain (the number of each bacterial cell: about 1 × 10 6 After the cells were smeared and cultured at 37 ° C. for 48 hours under anaerobic conditions, the growth activity of the bacteria was determined with the naked eye.
[0042]
(Proliferation activity test 2)
MRS agar medium (manufactured by Difco) was prepared by adding gentamicin as antibiotics to concentrations of 100, 50, 20 and 0 μg / ml, respectively. Next, (A) L. A cell culture solution of Ikui strain alone, (A) L. Ikui strain and (B) L. 10 μl each of the mixed cell culture solution of John Sony strain is inoculated into each antibiotic-containing MRS agar medium (number of each cell: about 1 × 10 6 6 After the cells were smeared and cultured at 37 ° C. for 48 hours under anaerobic conditions, the growth activity of the bacteria was determined with the naked eye.
[0043]
(Proliferation activity test 3)
MRS agar medium (manufactured by Difco) was prepared by adding ofloxacin as an antibiotic to concentrations of 100, 50, 20 and 0 μg / ml, respectively. (A) L. obtained in Production Example 1 A cell culture solution of Ikui strain alone, (A) L. Ikui strain and (B) L. John Sony Corporation or (A) L. Ikui strain and (B) L. 10 μl of each antibiotic-containing MRS agar medium was inoculated with the mixed cell culture solution of the crispartus strain (the number of each cell: about 1 × 10 6 6 After the cells were smeared and cultured at 37 ° C. for 48 hours under anaerobic conditions, the growth activity of the bacteria was determined with the naked eye.
The results of the above proliferation activity tests 1 to 3 are shown in Tables 4-1 to 3-1.
[0044]
[Table 4]
Figure 2004357528
[0045]
From the results shown in Tables 4-1 to 3, microbial cells obtained by combining (A) Lactobacillus icui YIT0483 strain having different antibacterial spectra and (B) one or more strains selected from four strains of the genus Lactobacillus. It was confirmed that the culture solution had excellent growth activity even in the presence of antibiotics.
[0046]
<Production Example 2>
(Production of freeze-dried cells)
The cell culture solutions of (A) and (B) 5 strains obtained in Production Example 1 were adjusted to pH 6.8 to 7.0 with 10N sodium hydroxide, respectively. Subsequently, after centrifugation at 5,000 rpm for 10 minutes to remove the supernatant, a dispersion medium (composition: 20% skim milk and 20% trehalose aqueous solution) was added to the remaining cells at a rate of 8% to make uniform. Then, after pre-freezing at -30 ° C. for 3 hours, lyophilization treatment was performed according to a conventional method. The weight after lyophilization was measured, and an equal amount of dried starch was added and stored at 4 ° C.
[0047]
<Example 3>
(Early intestinal flora formation promotion effect)
The 6 thoroughbred foals immediately after birth were subjected to 5 g of the lyophilized bacterial cells obtained in Production Example 2 ((A) and (B) 5 strains of the genus Lactobacillus in total 3.8 × 10 viable cells). 10 Of 5% glucose) was orally administered daily for a total of 7 times. Rectal stool is collected every 1, 2, 3, 5, 7 and 14 days after birth, and after adding and diluting a diluent, it is further diluted as appropriate and inoculated on LBS agar medium (Becton Dickinson) and anaerobic. The culture was carried out at 37 ° C for 4 days, and the number of lactobacilli in the rectal stool was calculated from the number of colonies grown and the dilution rate. As a control, the number of lactobacilli in rectal stool was calculated in the same manner from 6 foals not administered with lyophilized cells. Table 5 shows the detection rate (%) calculated from (number of foals in which lactobacilli were detected) / (number of all foals examined) × 100.
[0048]
[Table 5]
Figure 2004357528
[0049]
From the results of Table 5, in the administration group, adherence of lactobacilli was confirmed in the intestines of all foals on the 5th day of administration, and an effect of promoting intestinal flora formation as early as 2 days was observed compared to the control group.
[0050]
<Example 4>
(Improvement effect of diarrhea 1)
The same test as in Example 3 was performed on 54 foals immediately after birth (27 in the control group and 27 in the administration group), and the occurrence of diarrhea was observed until the 30th day of birth. Diarrhea is macroscopically loose stool, mild diarrhea (excretion of diarrheal stool, but less frequent and slight adhesion near the anus) and severe diarrhea (smelling watery diarrhea over and over again) Table 6 shows the onset rate (%) for each of the three stages (a large amount of diarrheal stool from the anus to the buttocks).
[0051]
[Table 6]
Figure 2004357528
[0052]
From the results in Table 6, the incidence of loose stool was significantly lower in the administration group at 3 weeks. In addition, the incidence of mild diarrhea and severe diarrhea tended to be significantly lower in the administration group after the 3rd week. In particular, the onset of severe diarrhea was not observed in the administration group at all, and at 4 weeks, The effect of improvement was seen so that treatment treatment and administration of antibiotics were not necessary.
[0053]
<Example 5>
(Improvement effect of diarrhea 2)
In 22 thoroughbred foals, 5 g of the freeze-dried microbial cells obtained in Production Example 2 ((A) and (B) 5 strains of the genus Lactobacillus) were 1.4 × 10 6 in total. 10 Of 5% glucose) was orally administered a total of 7 times per day during the administration period from the first day to the seventh day. The occurrence of diarrhea was observed from day 0 (no administration) to day 14. As a control, the occurrence of diarrhea was also observed in 6 foals not administered with lyophilized cells. Diarrhea was classified into two stages: macroscopic loose stool and severe diarrhea (a odor-like watery diarrhea was excreted several times and a large amount of diarrheal stool was attached from the anus to the buttocks). The incidence (%) of loose stool is shown in FIG. 1, and the incidence (%) of severe diarrhea is shown in FIG.
[0054]
From the results of FIG. 1 and FIG. 2, in the onset of loose stool, the administration group tended to decrease significantly on the 6th day of administration compared to the control group. Further, in the onset of diarrhea, no diarrheal symptoms that had developed in the control group were confirmed in the administration group after the fifth day of administration.
[0055]
【The invention's effect】
According to the present invention, the gastrointestinal tract such as diarrhea caused by side effects of antibiotic administration has not only exhibited specific adhesion and proliferation activity to the digestive tract epithelium of horses, but also excellent resistance to antibiotics. Provided are feed additives that are effective in preventing and improving disorders, and feeds containing the same.
[Brief description of the drawings]
FIG. 1 is a graph showing the incidence of soft stools in foals in a feed additive administration group and a control group in Example 5. FIG.
FIG. 2 is a graph showing the incidence of severe diarrhea in foals in the administration group of feed additive and the control group of Example 5.

Claims (5)

(A)ラクトバチルス・イクイ(Lactobacillus equi)の菌体と、
(B)ラクトバチルス・サリバリウス(Lactobacillus salivarius)、ラクトバチルス・ロイテリ(Lactobacillus reuteri)、ラクトバチルス・クリスパータス(Lactobacillus crispatus)及びラクトバチルス・ジョンソニー(Lactobacillus johnsonii)からなる群より選ばれる少なくとも1種の菌体と、
を含む飼料用添加剤。
(A) Lactobacillus equi (Lactobacillus equi),
(B) Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus crispatus and Lactobacillus johnsons group Lactobacillus Body,
Additive for feed containing.
(C)セフトリアキソンナトリウム、硫酸カナマイシン、ジヒドロストレプトマイシン、スルファモノメトキシン、バクトラミン、ゲンタマイシン、オフロキサシン、セファロチンナトリウム、アンピシリン、オキシテトラサイクリン塩酸塩及びベンジルペニシリンからなる群より選ばれる少なくとも1種の抗生物質を更に含む請求項1記載の飼料用添加剤。(C) at least one antibiotic selected from the group consisting of ceftriaxone sodium, kanamycin sulfate, dihydrostreptomycin, sulfamonomethoxine, bactramine, gentamicin, ofloxacin, cephalothin sodium, ampicillin, oxytetracycline hydrochloride and benzylpenicillin The feed additive according to claim 1, further comprising a substance. 前記(C)抗生物質と組合わせて添加するための請求項1記載の飼料用添加剤。The feed additive according to claim 1, which is added in combination with the (C) antibiotic. 請求項1ないし3のいずれかに記載の飼料用添加剤を含む飼料。A feed comprising the feed additive according to any one of claims 1 to 3. 以下の(イ)〜(ニ)からなる群より選ばれる少なくとも1種のラクトバチルス属に属する菌株:
(イ)ラクトバチルス・サリバリウス YIT0479株(寄託番号:FERMP−19332号);
(ロ)ラクトバチルス・ロイテリ YIT0480株(寄託番号:FERM P−19333号);
(ハ)ラクトバチルス・クリスパータス YIT0481株(寄託番号:FERM P−19334号)及び
(ニ)ラクトバチルス・ジョンソニー YIT0482株(寄託番号:FERMP−19335号)。
At least one strain belonging to the genus Lactobacillus selected from the group consisting of (i) to (d) below:
(I) Lactobacillus saliva varius YIT0479 strain (deposit number: FERMP-19332);
(B) Lactobacillus reuteri YIT0480 strain (deposit number: FERM P-19333);
(C) Lactobacillus crispertus YIT0481 strain (deposit number: FERM P-19334) and (d) Lactobacillus johnsonii YIT0482 strain (deposit number: FERMP-19335).
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JP2008195635A (en) * 2007-02-09 2008-08-28 Crossfield Bio Inc Lactic acid bacteria preparation for horse
JP2008212140A (en) * 2007-02-09 2008-09-18 Crossfield Bio Inc New microorganism of genus lactobacillus and lactic acid bacterium preparation
JP2009100692A (en) * 2007-10-24 2009-05-14 Crossfield Bio Inc New microorganism of genus lactobacillus and lactic acid bacterial preparation for mammal
JP2010041940A (en) * 2008-08-11 2010-02-25 Crossfield Bio Inc Microorganism belonging to new genus sharpea and microorganism pharmaceutical preparation and oral composition
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