JP2004057047A - Lactobacillus growth promoter and method for producing the same - Google Patents
Lactobacillus growth promoter and method for producing the same Download PDFInfo
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- JP2004057047A JP2004057047A JP2002217940A JP2002217940A JP2004057047A JP 2004057047 A JP2004057047 A JP 2004057047A JP 2002217940 A JP2002217940 A JP 2002217940A JP 2002217940 A JP2002217940 A JP 2002217940A JP 2004057047 A JP2004057047 A JP 2004057047A
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Abstract
Description
【０００１】
【発明の属する技術分野】
本発明は、ホエータンパク質をタンパク質分解酵素で処理することにより得られる分解物、及びこの分解物を限外ろ過により分画したリテンテイト画分を有効成分とする乳酸菌生育促進剤に関する。また、本発明はこの乳酸菌生育促進剤を製造する方法に関する。
本発明の乳酸菌生育促進剤は、乳酸菌の生育促進に優れた効果を有する。また、本発明の乳酸菌生育促進剤を添加して得られる乳酸菌培養物は、そのまま乳酸菌飲料や乳酸菌添加食品等の飲食物に直接供することが可能であり、得られる乳酸菌培養物を粉末化して、乳酸菌添加食品等の飲食物に供することができる。
【０００２】
【従来の技術】
乳酸菌は、古来より食品の製造に広く利用されており、チーズ、発酵乳、乳酸菌飲料、発酵バター等の乳製品、キムチや漬物といった食品、発酵ソーセージや発酵サラミなどの畜肉製品を製造する際に用いられている。また、パンのスターターや飼料用サイレージのスターターとしても利用されている。さらに最近では、乳酸菌の有する整腸効果等の生理効果が次々と明らかとなり、乳酸菌の菌体自体や乳酸菌培養物等を健康食品や医薬品等の素材として利用するための開発がなされている。このように、乳酸菌の利用は多岐にわたっており、乳酸菌の生育を促進させて菌体濃度を高めるとともに培養時間を短縮することは、乳酸菌培養物を簡便かつ安価に製造することを可能とし、産業上極めて大きな意義を有する。
【０００３】
しかし、乳酸菌は複雑な栄養要求性を示し、培養する場合には、ブドウ糖や乳糖等の炭素源に、酵母エキス、ビタミン、核酸、アミノ酸類、無機塩類、肉抽出物、金属塩等を添加して培地を作成する必要があり、乳酸菌の培養に使用する培地の調製は極めて煩雑であった。
また、乳酸菌体を取得することを目的として調製する場合には、通常乳酸菌を培養する場合に使用する乳を主体とする培地ではなく、ＭＲＳ培地やＧＡＭ培地等の合成培地を用いることとなる。しかし、これらの合成培地で乳酸菌を培養した場合には、乳を主体とする培地で培養した場合に比べて菌体濃度が高まらないのが一般的であり、合成培地を用いて乳酸菌の培養を行う際には、大量の培地が必要であり、工業化する上では、コストや設備の面でも問題があった。
【０００４】
このような理由から、従来より各種の乳酸菌増殖促進物質が検討されている。蛋白分解酵素処理した酒粕水抽出物を有効成分とする乳酸菌の増殖促進剤（特開平５−１５３６６号公報）、ローヤルゼリー由来の蛋白質の酵素分解物からなる微生物培養培地用ペプトン（特開平７−２６５０６６号公報）や蛋白分解酵素で処理した卵白を乳酸菌の増殖促進物質として用いる（特開２０００−９３１６６号公報）等の提案がされているが、十分な菌数の増加は得られていない。また、ホエー粉等のプロテアーゼ処理物を培地の主成分としたプロピオン酸菌の培養方法が開示されているが（特開平１０−３０４８７１号公報）、プロピオン酸菌が産生する比ビフィズス菌増殖活性を高めるためのプロピオン酸菌高濃度培養方法であり、乳酸菌を対象とはしていない。
【０００５】
【発明が解決しようとする課題】
乳酸菌に対して生育促進効果を有する上記の従来物質は、調製が煩雑であったり、物質自体が高価であったり、また、製品を製造する場合に、特異な味や香りが製品に移行するのを防ぐため、製品への風味に影響しないようにごくわずかの量しか培地に添加できないという問題があった。したがって、本発明は、上記した現状に鑑み、乳酸菌の生育促進に優れた効果を有し、かつ製品の風味に悪影響を与えない乳酸菌生育促進剤を提供することを課題とする。また、本発明は、新規な乳酸菌生育促進剤を製造する方法を提供することを課題とする。
【０００６】
【課題を解決するための手段】
これらの課題を解決するため、本発明者らは、新たに乳酸菌の生育促進に優れた効果を有する物質について鋭意検討をすすめたところ、ホエータンパク質をタンパク質分解酵素処理することにより得られる分解物、及び、その得られた分解物を限外ろ過により分画したときのリテンテイト画分（保持画分）に乳酸菌の生育を著しく促進する効果があることを発見した。さらに、乳酸菌用の合成培地にこのホエータンパク質分解物等を添加することで、乳酸菌の生育を促進できることを見出し、本発明を完成するに至った。微生物は、タンパク質より、タンパク質を分解したペプトンの方が利用しやすいのと同様に、低分子のアミノ酸の方が微生物にとって利用しやすいと一般的に言われてきた。ところが、本発明は意外にも、分子量の大きいホエータンパク質の酵素分解物、および限外ろ過により酵素分解物の低分子画分をさらに除いたリテンテイト画分（保持画分）のような分子量が大きい画分に、乳酸菌の生育を著しく促進する効果があることを見出した。 本発明は、ホエータンパク質をタンパク質分解酵素処理して得られる分解物を有効成分とする、乳酸菌の生育促進に優れた乳酸菌生育促進剤を提供する。また、本発明はホエータンパク質をタンパク質分解酵素処理して得られる分解物を限外ろ過により分画したリテンテイト画分（保持画分）を有効成分とする、乳酸菌の生育促進に優れた乳酸菌生育促進剤を提供する。さらに、本発明はホエータンパク質の水溶液をタンパク質分解酵素で酵素分解し、ついでこの分解物を限外ろ過により分画してリテンテイト画分（保持画分）を得ることを特徴とする乳酸菌生育促進剤の製造法を提供する。
【０００７】
【発明の実施の形態】
本発明におけるホエータンパク質は、牛乳、水牛や山羊などの乳のホエー、その濃縮物、粉末あるいはホエータンパク質精製物をいい、これを酵素反応させるときは水溶液の状態で使用する。これらのホエータンパク質を０．５〜２０重量％含むように水溶液を調製する。ホエータンパク質水溶液を塩酸、クエン酸、乳酸、苛性ソーダ、水酸化カルシウム及びリン酸ソーダ等の溶液を用いてｐＨ６〜１０に調整し、６０〜８０℃程度加熱する。ホエータンパク質にタンパク質分解酵素を作用させる反応条件は、使用する酵素の最適条件に設定することが好ましく、一般には、１０〜９０℃、ｐＨ３〜９に調整して酵素反応を行う。使用する酵素は特に限定されず、トリプシン、ペプシン、パパイン、パンクレアチンなど、植物、動物、微生物由来のタンパク質分解に通常使用するタンパク質分解酵素であれば問題はなく、複数の酵素を組み合わせて利用することもできる。
タンパク質分解酵素処理を終了したホエータンパク質水溶液は、反応液を加熱して酵素を失活させる。酵素の失活は反応液を９０℃以上で１０分間以上の加熱履歴で加熱することにより行うことができ、１１５℃で３〜５秒間加熱して行うこともできる。
【０００８】
ホエータンパク質含有水溶液の酵素分解処理には耐熱性のタンパク質分解酵素も使用し得る。例えば、特開平４−１１２７５３号公報では、ホエータンパク質を耐熱性のタンパク質加水分解酵素で分解処理しており、このような条件にて酵素反応を行うこともできる。以下にその条件を示す。
ホエータンパク質水溶液を、ｐＨ６〜１０に調整し、加熱は６０〜８０℃で行う。耐熱性のタンパク質加水分解酵素を用いる場合には、この温度にして添加するよりもむしろ加熱前から加え、酵素分解を行ったほうが収率の面から好ましい。一般的なプロテアーゼの至適温度は６０℃以下であるが、耐熱性のタンパク質加水分解酵素は７０℃以上であり、このような至適温度を有する耐熱性のタンパク質加水分解酵素として知られているものであれば特に制限はなく使用することができる。このような耐熱性タンパク質加水分解酵素としてパパイン、プロテアーゼＳ（商品名）、プロレザー（商品名）、サモアーゼ（商品名）、アルカラーゼ（商品名）、プロチンＡ（商品名）等を例示することができる。これらは、８０℃で３０分間加熱して残存活性が約１０％あるいはそれ以上になるものが望ましい。また、単独よりも複数の酵素を併用するとより効果的である。反応は３０分〜１０時間行うことが好ましい。酵素の失活は反応液を９０℃以上で１０分間以上加熱することにより行う。
【０００９】
このようにして得られたホエータンパク質分解物は全固形分中に約５０％以上のタンパク質分解物を含有している状態にある。このホエータンパク質分解物はそのままで乳酸菌生育促進剤として使用することができるが、得られた分解物をさらに限外ろ過を通して濃縮し、そのリテンテイト画分（保持画分）を得て、これを乳酸菌生育促進剤として使用することがより好ましい。試験の結果によると、限外ろ過したリテンテイト画分（保持画分）からなる乳酸菌生育促進剤の方が、限外ろ過処理しないで酵素分解処理をしただけの乳酸菌生育促進剤より、菌体濃度が増加した。
本発明において限外ろ過に用いる膜の分画分子量の大きさは、酵素分解の程度により、自由に選択することができる。上記の限外ろ過したパーミエイト画分には低分子の分解物が多く含まれ、本発明において分解酵素として耐熱性酵素を使用した場合、このパーミエイト画分は特開平４−１１２７５３号公報に開示されている低アレルゲン化ホエータンパク質加水分解物に近い組成となり、低アレルゲン化食品の原料として使用することができる。逆に、低アレルゲン化食品原料の取得を主たる目的とする場合には、そのリテンテイト画分を乳酸菌生育促進剤として最適に利用する本発明は、資源の有効利用につながるという多大な利点を持つ。
【００１０】
また、このリテンテイト画分（保持画分）をさらにＤＦ（ｄｉａ−ｆｉｌｔｒａｔｉｏｎ）に供して濃縮し、この濃縮リテンテイト画分を乳酸菌生育促進剤として使用することもできる。試験の結果では、この濃縮リテンテイト画分（保持画分）からなる乳酸菌生育促進剤の方が、限外ろ過処理しないで酵素分解処理をしただけの乳酸菌生育促進剤より菌体濃度が増加したが、試験に供した乳酸菌の種類によっては、限外ろ過処理したリテンテイト画分（保持画分）からなる乳酸菌生育促進剤とは菌体濃度には差が無かった。
【００１１】
このタンパク質分解酵素処理により得られる分解物は、従来より乳酸菌を培養する際に使用されている合成培地に固形換算で０．１〜１５重量％、好ましくは１〜５重量％となるように添加すれば良い。なお、乳酸菌生育促進剤の添加量を増やすことにより、乳酸菌の菌体濃度も増加する。このように本発明の生育促進剤を添加することにより、乳酸菌の菌体濃度を容易に高めることが可能であり、培地の使用量を著しく減少させることができるため、安価に乳酸菌体及び乳酸菌培養物を製造することができる。乳酸菌で、このような高い生育促進効果が認められた物質は、今までに報告されていない。
【００１２】
本発明の乳酸菌生育促進剤は、水溶液のままで、あるいは濃縮して、または、凍結乾燥等の適宜の方法によって粉末とした状態で使用可能である。
本生育促進剤は、乳由来のホエータンパク質を原材料としているため、この生育促進剤を添加して得られた乳酸菌培養物は、そのまま乳酸菌飲料や乳酸菌添加食品等の飲食物に直接供することが可能である。特に、前記の様に、耐熱性酵素により分解して得られた酵素分解物は、分解物に特有の苦味が少なく、食品素材としてそのまま利用できることは大きな利点である。また、得られた乳酸菌培養物を、凍結乾燥、噴霧乾燥等の乾燥手段で粉末化して、乳酸菌添加食品等の飲食物に供することもできる。さらに、得られた乳酸菌培養液から、ろ過や遠心分離等の処理にて乳酸菌体のみを分離し、その乳酸菌体を凍結乾燥、噴霧乾燥等で粉末化処理することにより、取扱の容易な形態の機能性食品原料として供給することも可能である。
【００１３】
次に実施例を示し、本発明を詳細に説明する。なお、以下に記載する実施例は本発明を説明するものであり、酵素やその反応条件などを実施例の記述に限定するものではない（％は重量％をあらわす）。
【００１４】
【実施例１】
ホエープロテインコンセントレイト（Ｌａｃｐｒｏｄａｎ ８０ 、Ａｒｌａ Ｆｏｏｄ 社製）のタンパク質濃度５％溶液を調製し、水酸化カリウムによりｐＨ７．０に調整した。ついで７５℃、２分間加熱後、５０℃まで冷却し、水酸化カルシウムによりｐＨ８．０に調整した。ついで基質に対して０．５％のプロテアーゼＰ６Ｇ（天野エンザイム社製）を添加し５０℃、２時間酵素反応させた。反応終了後、ｐＨを水酸化カルシウムにより８．０に調整し、アルカラーゼ（ノボノルディスクバイオインダストリー社製）を基質に対して２％添加し、６０℃、４時間処理後、１１５℃、３〜５秒間加熱することによりホエータンパク質分解物を得た。得られた分解物の固形分あたりの成分組成比率を表１に示す。
【００１５】
【表１】
【実施例２】
ホエープロテインコンセントレイト（Ｌａｃｐｒｏｄａｎ ８０ 、Ａｒｌａ Ｆｏｏｄ 社製）のタンパク質濃度５％溶液を調製し、水酸化カリウムによりｐＨを７．０に調整した。ついで７５℃、２分間加熱後、５０℃まで冷却し、水酸化カルシウムによりｐＨを８．０に調整した。この基質に対して０．５％のプロテアーゼＰ６Ｇ（天野エンザイム社製）を添加し５０℃、２時間酵素反応させた。反応終了後、ｐＨを水酸化カルシウムにより８．０に調整し、アルカラーゼ（ノボノルディスクバイオインダストリー社製）を基質に対して２％添加し、６０℃、４時間処理後、１１５℃、３〜５秒間加熱することによりホエータンパク質分解物を得た。
得られたホエータンパク質分解物３６ｋｇを限外ろ過（分画分子量１万）により６倍濃縮し、リテンテイト６ｋｇ、パーミエイト３０ｋｇを得た。リテンテイト画分は凍結乾燥し、４℃で保存した。得られたリテンテイト画分の固形分あたりの成分組成比率を表２に示す。
【００１７】
【表２】
【実施例３】
ホエープロテインコンセントレイト（Ｌａｃｐｒｏｄａｎ ８０ 、Ａｒｌａ Ｆｏｏｄ 社製）のタンパク質濃度５％溶液を調製し、水酸化カリウムによりｐＨを７．０に調整した。ついで７５℃、２分間加熱後、５０℃まで冷却し、水酸化カルシウムによりｐＨを８．０に調整した。ついで基質に対して、０．５％のプロテアーゼＰ６Ｇ（天野エンザイム社製）を添加し５０℃、２時間酵素反応させた。反応終了後、ｐＨを水酸化カルシウムにより８．０に調整し、アルカラーゼ（ノボノルディスクバイオインダストリー社製）を基質に対して２％添加し、６０℃、４時間処理後、１１５℃、３〜５秒間加熱することによりホエータンパク質分解物を得た。
得られたホエータンパク質分解物３６ｋｇを限外ろ過（分画分子量１万）により６倍濃縮し、リテンテイト６ｋｇ、パーミエイト３０ｋｇを得た。さらにリテンテイトをＤＦ（ｄｉａ−ｆｉｌｔｒａｔｉｏｎ）に供し２倍濃縮した。リテンテイト画分は凍結乾燥し、４℃で保存した。得られた濃縮リテンテイト画分の固形分あたりの成分組成比率を表３に示す。
【００１９】
【表３】
【試験例１】
本試験には、乳酸菌として、ラクトバチルス・アシドフィラス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ａｃｉｄｏｐｈｉｌｕｓ） ＪＣＭ１１３２、ラクトバチルス・ガセリ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｇａｓｓｅｒｉ） ＪＣＭ１１３１、ラクトバチルス・ガセリ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｇａｓｓｅｒｉ） ＦＥＲＭ Ｐ−１５５３、ラクトバチルス・デルブリッキ・サブスピーシズ・ブルガリカス （Ｌａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ ｓｕｂｓｐ．ｂｕｌｇａｒｉｃｕｓ）ＪＣＭ１００２の４株を供試した。試験培地には、表４に示す組成の合成培地に、実施例１で得られた乳酸菌生育促進剤を試験培地１：無添加、試験培地２：１％添加、試験培地３：３％添加および試験培地４：５％添加し、乳酸菌培養用培地を調製した。まず、ネジ口試験管に各培地を１０ｍｌずつ分注し、１２１℃で１５分間高圧滅菌し試験培地とした。一方、ＭＲＳ液体培地（Ｄｉｆｃｏ社製、ＵＳＡ）で継代培養した各乳酸菌を同培地で３７℃、１６時間培養し乳酸菌前培養物とした。この乳酸菌前培養物を遠心分離し、菌体をリン酸緩衝化生理食塩水（ＰＢＳ）（−）で２回洗浄後、ＰＢＳ（−）に懸濁した。ついで、試験培地１〜４にこの洗浄菌体を３％ずつ接種して３７℃で１６時間培養し、培養終了後、ＭＲＳ寒天培地を用いたプレートカウント法により各試験培地の乳酸菌の生菌数を測定した。その結果を表５に示す。
【００２１】
【表４】
【表５】
【００２３】
【試験例２】
本試験には、乳酸菌として、ラクトバチルス・アシドフィラス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ａｃｉｄｏｐｈｉｌｕｓ） ＪＣＭ１１３２、ラクトバチルス・ガセリ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｇａｓｓｅｒｉ） ＪＣＭ１１３１、ラクトバチルス・ガセリ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｇａｓｓｅｒｉ） ＦＥＲＭ Ｐ−１５５３、ラクトバチルス・デルブリッキ・サブスピーシズ・ブルガリカス （Ｌａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ ｓｕｂｓｐ． ｂｕｌｇａｒｉｃｕｓ）ＪＣＭ１００２の４株を供試した。
試験培地には、表４に示す組成の合成培地に、実施例２で得られた乳酸菌生育促進剤を試験培地１：無添加、試験培地２：１％添加、試験培地３：３％添加および試験培地４：５％添加し、乳酸菌培養用培地を調製した。まず、ネジ口試験管に各培地を１０ｍｌずつ分注し、１２１℃で１５分間高圧滅菌し試験培地とした。一方、ＭＲＳ液体培地（Ｄｉｆｃｏ社製、ＵＳＡ）で継代培養した各乳酸菌を同培地で３７℃、１６時間培養し乳酸菌前培養物とした。この乳酸菌前培養物を遠心分離し菌体をリン酸緩衝化生理食塩水（ＰＢＳ）（−）で２回洗浄後、ＰＢＳ（−）に懸濁した。ついで、試験培地１〜４にこの洗浄菌体を３％接種して３７℃で１６時間培養し、培養終了後、ＭＲＳ寒天培地を用いたプレートカウント法により各試験培地の乳酸菌の生菌数を測定した。その結果を表６に示す。
【００２４】
【表６】
表４に示す合成培地には微生物の窒素源として一般的に使用されるペプトンが配合されているが、乳酸菌生育促進剤を添加していない試験培地１での乳酸菌の生育は比較的良くない。ところが、本発明の乳酸菌生育促進剤を添加するといずれの菌株においても明らかに生育性は改善され、さらに添加する乳酸菌生育促進剤が増加するとともに乳酸菌の生菌数も増加するという結果となった。また、実施例１で得られたタンパク質分解物に比べて、本実施例の限外ろ過によるリテンテイト画分は１．３〜１．５倍程度の菌体濃度の増加があった。
【００２６】
【試験例３】
本試験には、乳酸菌として、ラクトバチルス・アシドフィラス（Ｌａｃｔｏｂａｃｉｌｌｕｓ ａｃｉｄｏｐｈｉｌｕｓ） ＪＣＭ１１３２、ラクトバチルス・ガセリ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｇａｓｓｅｒｉ） ＪＣＭ１１３１、ラクトバチルス・ガセリ（Ｌａｃｔｏｂａｃｉｌｌｕｓ ｇａｓｓｅｒｉ） ＦＥＲＭ Ｐ−１５５３、ラクトバチルス・デルブリッキ・サブスピーシズ・ブルガリカス （Ｌａｃｔｏｂａｃｉｌｌｕｓ ｄｅｌｂｒｕｅｃｋｉｉ ｓｕｂｓｐ． ｂｕｌｇａｒｉｃｕｓ） ＪＣＭ１００２の４株を供試した。
試験培地には、表４に示す組成の合成培地に、実施例３で得られた乳酸菌生育促進剤を試験培地１：無添加、試験培地２：１％添加、試験培地３：３％添加および試験培地４：５％添加し、乳酸菌培養用培地とした。まず、ネジ口試験管に各培地を１０ｍｌずつ分注し、１２１℃で１５分間高圧滅菌し試験培地とした。一方、ＭＲＳ液体培地（Ｄｉｆｃｏ社製、ＵＳＡ）で継代培養した各乳酸菌を同培地で３７℃、１６時間培養し乳酸菌前培養物とした。この乳酸菌前培養物を遠心分離し菌体をリン酸緩衝化生理食塩水（ＰＢＳ）（−）で２回洗浄後、ＰＢＳ（−）に懸濁した。ついで、試験培地１〜４にこの洗浄菌体を３％接種して３７℃で１６時間培養し、培養終了後、ＭＲＳ寒天培地を用いたプレートカウント法により各試験培地の乳酸菌の生菌数を測定した。その結果を表７に示す。
【００２７】
【表７】
本結果によると、乳酸菌生育促進剤の添加によりいずれの菌株においても明らかに生育性は改善され、さらに添加する乳酸菌生育促進剤を増加させるとともに乳酸菌の生菌数も増加する。また、実施例１で得られたタンパク質分解物に比べて、本実施例の濃縮リテンテイト画分は１．３〜１．５倍程度の菌体濃度の増加があった。
【００２９】
【発明の効果】
本発明により新規な乳酸菌培養用生育促進剤が提供される。本生育促進剤を添加することにより得られる乳酸菌及び乳酸菌培養物は、顕著に生育が促進され、到達生菌数が高く、効率的に乳酸菌体を得ることが可能であり、乳酸菌菌体製造及び乳酸菌培養物製造において製造コストを削減することができる。また乳酸菌の高濃度培養の場合にも有用である。[0001]
TECHNICAL FIELD OF THE INVENTION
TECHNICAL FIELD The present invention relates to a degradation product obtained by treating whey protein with a protease, and a lactic acid bacteria growth promoter comprising as an active ingredient a retentate fraction obtained by fractionating the degradation product by ultrafiltration. The present invention also relates to a method for producing the lactic acid bacteria growth promoter.
The lactic acid bacteria growth promoter of the present invention has an excellent effect on promoting the growth of lactic acid bacteria. In addition, the lactic acid bacteria culture obtained by adding the lactic acid bacteria growth promoter of the present invention can be directly provided to foods and drinks such as lactic acid bacteria beverages and foods containing lactic acid bacteria, and the resulting lactic acid bacteria culture is powdered, It can be used in foods and drinks such as foods containing lactic acid bacteria.
[0002]
[Prior art]
Lactic acid bacteria have been widely used in the production of foods since ancient times, and when producing dairy products such as cheese, fermented milk, lactic acid bacteria drinks, fermented butter, foods such as kimchi and pickles, and animal meat products such as fermented sausages and fermented salami. Used. It is also used as a starter for bread and a silage for feed. More recently, physiological effects, such as an intestinal regulation effect, of lactic acid bacteria have been revealed one after another, and developments have been made to utilize lactic acid bacteria cells themselves and lactic acid bacteria cultures as materials for health foods, pharmaceuticals, and the like. As described above, the use of lactic acid bacteria is diversified, and promoting the growth of lactic acid bacteria to increase the cell concentration and shorten the cultivation time makes it possible to produce lactic acid bacteria cultures easily and inexpensively. It has enormous significance.
[0003]
However, lactic acid bacteria show complex nutritional requirements, and when cultured, yeast extract, vitamins, nucleic acids, amino acids, inorganic salts, meat extracts, metal salts, etc. are added to carbon sources such as glucose and lactose. Therefore, the preparation of a medium used for culturing lactic acid bacteria was extremely complicated.
When the lactic acid bacteria are prepared for the purpose of obtaining the lactic acid bacteria, a synthetic medium such as an MRS medium or a GAM medium is used instead of a medium mainly composed of milk used for culturing lactic acid bacteria. However, when lactic acid bacteria are cultured in these synthetic media, the bacterial cell concentration is generally not higher than when cultured in a milk-based medium. When performing the method, a large amount of medium is required, and there are problems in terms of cost and equipment in industrialization.
[0004]
For these reasons, various lactic acid bacteria growth promoting substances have been studied. A lactic acid bacterium growth promoter containing a water extract of sake lees treated with a proteolytic enzyme as an active ingredient (JP-A-5-15366), and a peptone for a microorganism culture medium comprising an enzymatically-decomposed product of royal jelly-derived protein (JP-A-7-265066) Japanese Patent Application Laid-Open No. 2000-93166) and the use of egg white treated with a protease as a growth promoting substance for lactic acid bacteria have been proposed, but a sufficient increase in the number of bacteria has not been obtained. In addition, a method for culturing propionic acid bacteria using a protease-treated product such as whey powder as a main component of a medium has been disclosed (Japanese Patent Application Laid-Open No. 10-304871), but the growth activity of the specific bifidobacteria produced by the propionic acid bacteria has been disclosed. This is a high-concentration culture method for propionibacteria to enhance lactic acid bacteria.
[0005]
[Problems to be solved by the invention]
The above-mentioned conventional substances having a growth promoting effect on lactic acid bacteria are complicated to prepare, the substances themselves are expensive, and when a product is manufactured, a unique taste or aroma is transferred to the product. There is a problem that only a very small amount can be added to the culture medium so as not to affect the flavor of the product in order to prevent the production of the medium. Therefore, an object of the present invention is to provide a lactic acid bacteria growth promoter which has an excellent effect on promoting the growth of lactic acid bacteria and does not adversely affect the flavor of the product, in view of the above-mentioned situation. Another object of the present invention is to provide a method for producing a novel lactic acid bacteria growth promoter.
[0006]
[Means for Solving the Problems]
In order to solve these problems, the present inventors have intensively studied a new substance having an excellent effect on promoting the growth of lactic acid bacteria, a decomposition product obtained by treating whey protein with a protease, In addition, it has been found that a retentate fraction (retention fraction) obtained by fractionating the obtained decomposed product by ultrafiltration has an effect of remarkably promoting the growth of lactic acid bacteria. Furthermore, they have found that the growth of lactic acid bacteria can be promoted by adding the whey protein hydrolyzate or the like to a synthetic medium for lactic acid bacteria, and have completed the present invention. In microorganisms, it has been generally said that low-molecular-weight amino acids are more accessible to microorganisms, just as protein-degraded peptone is more accessible than protein. However, the present invention surprisingly has a large molecular weight such as an enzymatically degraded product of whey protein having a large molecular weight and a retentate fraction (retained fraction) obtained by further removing the low molecular weight fraction of the enzymatically degraded product by ultrafiltration. It was found that the fraction had an effect of significantly promoting the growth of lactic acid bacteria. The present invention provides a lactic acid bacteria growth promoter excellent in promoting the growth of lactic acid bacteria, comprising a degradation product obtained by treating whey protein with a protease, as an active ingredient. The present invention also provides a lactic acid bacterium that promotes the growth of lactic acid bacteria, comprising a retained fraction obtained by ultrafiltration of a degradation product obtained by treating whey protein with proteolytic enzymes, as an active ingredient. Provide the agent. Further, the present invention provides a lactic acid bacterium growth promoting agent characterized in that an aqueous solution of whey protein is enzymatically decomposed with a proteolytic enzyme, and the degraded product is fractionated by ultrafiltration to obtain a retained fraction (retained fraction). And a method for producing the same.
[0007]
BEST MODE FOR CARRYING OUT THE INVENTION
The whey protein in the present invention refers to whey of milk such as milk, buffalo and goat, a concentrate thereof, a powder or a purified whey protein. When the enzyme is subjected to an enzymatic reaction, it is used in the form of an aqueous solution. An aqueous solution is prepared to contain 0.5 to 20% by weight of these whey proteins. The aqueous whey protein solution is adjusted to pH 6 to 10 using a solution of hydrochloric acid, citric acid, lactic acid, caustic soda, calcium hydroxide, sodium phosphate, and the like, and heated at about 60 to 80 ° C. The reaction conditions for causing the protease to act on the whey protein are preferably set to the optimum conditions for the enzyme used. In general, the enzyme reaction is performed at 10 to 90 ° C. and pH 3 to 9 to carry out the enzyme reaction. The enzyme to be used is not particularly limited, and there is no problem as long as it is a proteolytic enzyme normally used for proteolysis derived from plants, animals, and microorganisms, such as trypsin, pepsin, papain, and pancreatin. You can also.
After the proteolytic enzyme treatment, the aqueous whey protein solution heats the reaction solution to deactivate the enzyme. The inactivation of the enzyme can be carried out by heating the reaction solution at 90 ° C. or higher with a heating history of 10 minutes or more, or by heating at 115 ° C. for 3 to 5 seconds.
[0008]
A heat-resistant proteolytic enzyme can also be used in the enzymatic decomposition treatment of the aqueous solution containing whey protein. For example, in JP-A-4-112753, whey protein is decomposed with a heat-resistant protein hydrolase, and an enzymatic reaction can be performed under such conditions. The conditions are shown below.
The pH of the whey protein aqueous solution is adjusted to 6 to 10, and heating is performed at 60 to 80 ° C. When a heat-resistant protein hydrolase is used, it is preferable to add the enzyme before heating and to carry out enzymatic degradation rather than adding at this temperature from the viewpoint of yield. The optimum temperature of a general protease is 60 ° C. or lower, while the temperature of a heat-resistant protein hydrolase is 70 ° C. or higher, and is known as a heat-resistant protein hydrolase having such an optimum temperature. Any material can be used without particular limitation. Examples of such a heat-resistant protein hydrolase include papain, protease S (trade name), proleather (trade name), samoase (trade name), alcalase (trade name), and protin A (trade name). it can. These are desirably those having a residual activity of about 10% or more when heated at 80 ° C. for 30 minutes. It is more effective to use a plurality of enzymes in combination than to use them alone. The reaction is preferably performed for 30 minutes to 10 hours. The enzyme is inactivated by heating the reaction solution at 90 ° C. or higher for 10 minutes or longer.
[0009]
The whey protein hydrolyzate thus obtained is in a state containing about 50% or more of the protein hydrolyzate in the total solid content. The whey protein hydrolyzate can be used as it is as a lactic acid bacteria growth promoter, but the obtained hydrolyzate is further concentrated through ultrafiltration to obtain a retentate fraction (retention fraction), More preferably, it is used as a growth promoter. According to the results of the test, the lactic acid bacteria growth promoter comprising the ultrafiltered retentate fraction (retention fraction) had a higher bacterial concentration than the lactic acid bacteria growth promoter which had been subjected to enzymatic degradation treatment without ultrafiltration. increased.
In the present invention, the size of the molecular weight cut-off of the membrane used for ultrafiltration can be freely selected depending on the degree of enzymatic decomposition. The ultrafiltered permeate fraction contains a large amount of low molecular weight decomposed products. When a thermostable enzyme is used as a decomposing enzyme in the present invention, this permeate fraction is disclosed in JP-A-4-112753. It has a composition close to that of the allergen-reduced whey protein hydrolyzate, and can be used as a raw material for allergen-reduced foods. Conversely, when the main purpose is to obtain an allergen-reduced food material, the present invention in which the retentate fraction is optimally used as a lactic acid bacteria growth promoter has a great advantage in that the resources are effectively used.
[0010]
The retentate fraction (retention fraction) can be further subjected to DF (dia-filtration) to be concentrated, and the concentrated retentate fraction can be used as a lactic acid bacteria growth promoter. According to the test results, the concentration of the lactic acid bacteria growth promoter comprising the concentrated retentate fraction (retention fraction) was higher than that of the lactic acid bacteria growth promoter which was merely subjected to enzymatic decomposition treatment without ultrafiltration. In addition, depending on the type of lactic acid bacteria subjected to the test, there was no difference in the bacterial cell concentration from the lactic acid bacteria growth promoter composed of the ultrafiltration-treated retentate fraction (retention fraction).
[0011]
The degraded product obtained by this proteolytic enzyme treatment is added to a synthetic medium conventionally used for culturing lactic acid bacteria so as to have a solid content of 0.1 to 15% by weight, preferably 1 to 5% by weight. Just do it. By increasing the amount of the lactic acid bacteria growth promoter added, the concentration of the lactic acid bacteria cells also increases. As described above, by adding the growth promoter of the present invention, it is possible to easily increase the cell concentration of lactic acid bacteria, and it is possible to significantly reduce the amount of medium to be used. Things can be manufactured. No substance that has been shown to have such a high growth promoting effect in lactic acid bacteria has been reported so far.
[0012]
The lactic acid bacteria growth promoter of the present invention can be used in the form of an aqueous solution, concentrated, or powdered by an appropriate method such as freeze-drying.
Since the present growth promoter uses whey protein derived from milk as a raw material, the lactic acid bacteria culture obtained by adding the growth promoter can be directly provided to foods and drinks such as lactic acid bacteria beverages and foods containing lactic acid bacteria. It is. In particular, as described above, an enzymatically decomposed product obtained by decomposing with a heat-resistant enzyme has little bitterness peculiar to the decomposed product, and it is a great advantage that it can be used as it is as a food material. Moreover, the obtained lactic acid bacteria culture can be powdered by a drying means such as freeze-drying or spray-drying, and can be provided for foods and drinks such as foods containing lactic acid bacteria. Furthermore, from the obtained lactic acid bacteria culture solution, only lactic acid bacteria are separated by a treatment such as filtration or centrifugation, and the lactic acid bacteria are powdered by freeze-drying, spray-drying, etc., so that the form is easy to handle. It can also be supplied as a functional food ingredient.
[0013]
Next, an example is shown and the present invention is explained in detail. The examples described below illustrate the present invention, and do not limit the enzymes and their reaction conditions to those described in the examples (% represents% by weight).
[0014]
Embodiment 1
A 5% protein solution of whey protein concentrate (Lacprodan 80, manufactured by Arla Food) was prepared and adjusted to pH 7.0 with potassium hydroxide. Then, the mixture was heated at 75 ° C. for 2 minutes, cooled to 50 ° C., and adjusted to pH 8.0 with calcium hydroxide. Then, 0.5% protease P6G (manufactured by Amano Enzyme Co., Ltd.) was added to the substrate, and an enzyme reaction was performed at 50 ° C. for 2 hours. After the reaction, the pH was adjusted to 8.0 with calcium hydroxide, Alcalase (manufactured by Novo Nordisk Bioindustry) was added to the substrate at 2%, and the mixture was treated at 60 ° C for 4 hours. By heating for 5 seconds, a whey protein degradation product was obtained. Table 1 shows the component composition ratio per solid content of the obtained decomposition product.
[0015]
[Table 1]
Embodiment 2
A 5% protein concentration solution of whey protein concentrate (Lacprodan 80, manufactured by Arla Food) was prepared, and the pH was adjusted to 7.0 with potassium hydroxide. Then, after heating at 75 ° C for 2 minutes, the mixture was cooled to 50 ° C, and the pH was adjusted to 8.0 with calcium hydroxide. To this substrate, 0.5% protease P6G (manufactured by Amano Enzyme) was added, and an enzyme reaction was performed at 50 ° C. for 2 hours. After the reaction, the pH was adjusted to 8.0 with calcium hydroxide, Alcalase (manufactured by Novo Nordisk Bioindustry) was added to the substrate at 2%, and the mixture was treated at 60 ° C for 4 hours. By heating for 5 seconds, a whey protein degradation product was obtained.
36 kg of the obtained whey protein hydrolyzate was concentrated 6 times by ultrafiltration (fraction molecular weight: 10,000) to obtain 6 kg of retentate and 30 kg of permeate. The retentate fraction was lyophilized and stored at 4 ° C. Table 2 shows the component composition ratio per solid content of the obtained retentate fraction.
[0017]
[Table 2]
Embodiment 3
A 5% protein concentration solution of whey protein concentrate (Lacprodan 80, manufactured by Arla Food) was prepared, and the pH was adjusted to 7.0 with potassium hydroxide. Then, after heating at 75 ° C for 2 minutes, the mixture was cooled to 50 ° C, and the pH was adjusted to 8.0 with calcium hydroxide. Then, 0.5% protease P6G (manufactured by Amano Enzyme Co., Ltd.) was added to the substrate, and an enzyme reaction was performed at 50 ° C. for 2 hours. After the reaction, the pH was adjusted to 8.0 with calcium hydroxide, Alcalase (manufactured by Novo Nordisk Bioindustry) was added to the substrate at 2%, and the mixture was treated at 60 ° C for 4 hours. By heating for 5 seconds, a whey protein degradation product was obtained.
36 kg of the obtained whey protein hydrolyzate was concentrated 6 times by ultrafiltration (fraction molecular weight: 10,000) to obtain 6 kg of retentate and 30 kg of permeate. Further, the retentate was subjected to DF (dia-filtration) and concentrated twice. The retentate fraction was lyophilized and stored at 4 ° C. Table 3 shows the component composition ratio per solid content of the obtained concentrated retentate fraction.
[0019]
[Table 3]
[Test Example 1]
In this test, as lactic acid bacteria, Lactobacillus acidophilus JCM1132, Lactobacillus gasseri JCM1131, Lactobacillus gasseri subsp. (Lactobacillus delbrueckii subsp. Bulgaricus) 4 strains of JCM1002 were tested. In the test medium, the lactic acid bacteria growth promoter obtained in Example 1 was not added to the synthetic medium having the composition shown in Table 4, test medium 1: no test medium, test medium 2: 1%, test medium 3: 3%, and Test medium 4: 5% was added to prepare a lactic acid bacteria culture medium. First, 10 ml of each medium was dispensed into a screw-mouth test tube, and subjected to high-pressure sterilization at 121 ° C. for 15 minutes to obtain a test medium. On the other hand, each lactic acid bacterium subcultured in an MRS liquid medium (manufactured by Difco, USA) was cultured in the same medium at 37 ° C. for 16 hours to obtain a lactic acid bacterium preculture. The lactic acid bacteria preculture was centrifuged, and the cells were washed twice with phosphate buffered saline (PBS) (-) and suspended in PBS (-). Next, the washed cells were inoculated in 3% of each of the test media 1 to 4 and cultured at 37 ° C. for 16 hours. After completion of the culture, the viable cell count of lactic acid bacteria in each test medium was measured by a plate counting method using MRS agar medium. Was measured. Table 5 shows the results.
[0021]
[Table 4]
[Table 5]
[0023]
[Test Example 2]
In this test, as lactic acid bacteria, Lactobacillus acidophilus JCM1132, Lactobacillus gasseri JCM1131, Lactobacillus gasseri subsp. (Lactobacillus delbrueckii subsp. bulgaricus) JCM1002.
In the test medium, the lactic acid bacteria growth promoter obtained in Example 2 was not added to the synthetic medium having the composition shown in Table 4, test medium 1: no test medium, test medium 2: 1%, test medium 3: 3%, and Test medium 4: 5% was added to prepare a lactic acid bacteria culture medium. First, 10 ml of each medium was dispensed into a screw-mouth test tube, and subjected to high-pressure sterilization at 121 ° C. for 15 minutes to obtain a test medium. On the other hand, each lactic acid bacterium subcultured in an MRS liquid medium (manufactured by Difco, USA) was cultured in the same medium at 37 ° C. for 16 hours to obtain a lactic acid bacterium preculture. The lactic acid bacteria preculture was centrifuged, and the cells were washed twice with phosphate buffered saline (PBS) (-) and suspended in PBS (-). Then, 3% of the washed cells were inoculated into test mediums 1 to 4 and cultured at 37 ° C. for 16 hours. After completion of the culture, the viable cell count of lactic acid bacteria in each test medium was determined by a plate counting method using MRS agar medium. It was measured. Table 6 shows the results.
[0024]
[Table 6]
Although the synthetic medium shown in Table 4 contains peptone, which is generally used as a nitrogen source for microorganisms, the growth of lactic acid bacteria in the test medium 1 to which no lactic acid bacteria growth promoter was added was relatively poor. However, when the lactic acid bacteria growth promoter of the present invention was added, the growth was obviously improved in any of the strains, and the added lactic acid bacteria growth promoter increased and the number of viable lactic acid bacteria increased. In addition, as compared with the protein hydrolyzate obtained in Example 1, the retentate fraction obtained by the ultrafiltration of this example had an increase in the cell concentration of about 1.3 to 1.5 times.
[0026]
[Test Example 3]
In this test, as lactic acid bacteria, Lactobacillus acidophilus JCM1132, Lactobacillus gasseri JCM1131, Lactobacillus gasseri subsp. (Lactobacillus delbrueckii subsp. bulgaricus) Four strains of JCM1002 were tested.
As the test medium, the lactic acid bacteria growth promoter obtained in Example 3 was not added to the synthetic medium having the composition shown in Table 4 in the test medium 1: no test medium, test medium 2: 1%, test medium 3: 3%, and Test medium 4: 5% was added to obtain a lactic acid bacteria culture medium. First, 10 ml of each medium was dispensed into a screw-mouth test tube, and subjected to high-pressure sterilization at 121 ° C. for 15 minutes to obtain a test medium. On the other hand, each lactic acid bacterium subcultured in MRS liquid medium (manufactured by Difco, USA) was cultured in the same medium at 37 ° C. for 16 hours to obtain a lactic acid bacterium preculture. The lactic acid bacteria preculture was centrifuged, and the cells were washed twice with phosphate buffered saline (PBS) (-) and suspended in PBS (-). Then, 3% of the washed cells were inoculated into test mediums 1 to 4 and cultured at 37 ° C. for 16 hours. After completion of the culture, the viable cell count of lactic acid bacteria in each test medium was determined by a plate counting method using MRS agar medium. It was measured. Table 7 shows the results.
[0027]
[Table 7]
According to the present results, the growth of any of the strains was obviously improved by the addition of the lactic acid bacteria growth promoter, and the number of lactic acid bacteria viable cells increased with the increase of the added lactic acid bacteria growth promoter. In addition, as compared with the protein hydrolyzate obtained in Example 1, the concentration of the concentrated retentate fraction of the present example was increased by about 1.3 to 1.5 times.
[0029]
【The invention's effect】
The present invention provides a novel growth promoter for lactic acid bacteria culture. Lactic acid bacteria and lactic acid bacteria cultures obtained by adding the present growth promoter are remarkably promoted in growth, the number of living bacteria reached is high, lactic acid cells can be obtained efficiently, and lactic acid bacteria cell production and Production cost can be reduced in the production of lactic acid bacteria culture. It is also useful for high concentration culture of lactic acid bacteria.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005097972A1 (en) * | 2004-03-31 | 2005-10-20 | Nihon University | Medium for lactic acid bacteria |
| JP2007189914A (en) * | 2006-01-17 | 2007-08-02 | Kitasato Gakuen | Bifidobacteria proliferation-promoting peptide |
| WO2008001497A1 (en) | 2006-06-30 | 2008-01-03 | Snow Brand Milk Products Co., Ltd. | Enhancer of proliferation of lactic acid bacterium, and agent for improvement in survivability of lactic acid bacterium |
| JP2009044983A (en) * | 2007-08-17 | 2009-03-05 | Tohoku Univ | Method for producing gassericin a and food preservative |
| KR101227816B1 (en) | 2011-03-15 | 2013-01-29 | 연세대학교 원주산학협력단 | Composition for promoting growth of lactic acid bacteria containing hydrolyzed whey concentrate fermented by yeast |
| JP2015050993A (en) * | 2013-08-07 | 2015-03-19 | 国立大学法人 岡山大学 | Gastrointestinal tract and feces-derived lactobacilli culture milk or soybean milk-derived composition, culture or fermentation material and lactic fermentation product |
| JPWO2013085009A1 (en) * | 2011-12-06 | 2015-04-27 | 雪印メグミルク株式会社 | Food / beverage products with high survival of gasseri bacteria and method for producing the same |
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2002
- 2002-07-26 JP JP2002217940A patent/JP4307026B2/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005097972A1 (en) * | 2004-03-31 | 2005-10-20 | Nihon University | Medium for lactic acid bacteria |
| JP2007189914A (en) * | 2006-01-17 | 2007-08-02 | Kitasato Gakuen | Bifidobacteria proliferation-promoting peptide |
| JP4726129B2 (en) * | 2006-01-17 | 2011-07-20 | 学校法人北里研究所 | Bifidobacterium growth-promoting peptide |
| WO2008001497A1 (en) | 2006-06-30 | 2008-01-03 | Snow Brand Milk Products Co., Ltd. | Enhancer of proliferation of lactic acid bacterium, and agent for improvement in survivability of lactic acid bacterium |
| JP2009044983A (en) * | 2007-08-17 | 2009-03-05 | Tohoku Univ | Method for producing gassericin a and food preservative |
| KR101227816B1 (en) | 2011-03-15 | 2013-01-29 | 연세대학교 원주산학협력단 | Composition for promoting growth of lactic acid bacteria containing hydrolyzed whey concentrate fermented by yeast |
| JPWO2013085009A1 (en) * | 2011-12-06 | 2015-04-27 | 雪印メグミルク株式会社 | Food / beverage products with high survival of gasseri bacteria and method for producing the same |
| JP2015050993A (en) * | 2013-08-07 | 2015-03-19 | 国立大学法人 岡山大学 | Gastrointestinal tract and feces-derived lactobacilli culture milk or soybean milk-derived composition, culture or fermentation material and lactic fermentation product |
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