JP2019149968A - オフフレーバーのリスクを低減できるラクターゼ調製物 - Google Patents
オフフレーバーのリスクを低減できるラクターゼ調製物 Download PDFInfo
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- JP2019149968A JP2019149968A JP2018037762A JP2018037762A JP2019149968A JP 2019149968 A JP2019149968 A JP 2019149968A JP 2018037762 A JP2018037762 A JP 2018037762A JP 2018037762 A JP2018037762 A JP 2018037762A JP 2019149968 A JP2019149968 A JP 2019149968A
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- lactase
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- glucuronidase
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Abstract
Description
本発明の第1の態様によれば、イソ吉草酸に基づくオフフレーバーリスクを低減できるラクターゼ調製物が提供される。
本発明の第2の態様によれば、ラクターゼ調製物の製造方法が提供される。
第1の実施形態のラクターゼ調製物の製造方法では、β−グルクロニダーゼ活性体を生来的に産生し得るラクターゼ産生微生物からラクターゼ調製物を製造し、その製造方法の中でβ−グルクロニダーゼ活性体の量および/または活性が野生株よりも低減されるようにする処理が実行される。
第2の実施形態のラクターゼ調製物の製造方法では、ラクターゼを産生できる微生物からラクターゼ調製物を製造し、その製造方法の中でラクターゼ調製物のβ−グルクロニダーゼの活性を1回以上測定する。
第3の実施形態のラクターゼ調製物の製造方法では、β−グルクロニダーゼ活性体を生来的に産生し得るラクターゼ産生微生物からラクターゼ調製物を製造し、その製造方法の中でβ−グルクロニダーゼ活性体の量および/または活性が野生株よりも低減されるようにする処理を実行し、およびラクターゼ調製物のβ−グルクロニダーゼ活性体の活性を1回以上測定する。
本発明の第3の態様によれば、原料乳と上述のラクターゼ調製物とを含む混合物が提供される。
本発明の第4の態様によれば、ラクターゼ調製物に含まれるβ−グルクロニダーゼ活性を指標として、ラクターゼ調製物を原料乳に作用させたときのイソ吉草酸の生成量および/またはオフフレーバーのリスクを予測する方法が提供される。
本発明者らは幾つかの因子について、以下に記載する手法によってオフフレーバーが生じるかを確認した。このうちオフフレーバーの発生が確認された因子の1つがK. lactisのβ−グルクロニダーゼ活性を有するβ−グルコシダーゼであった。以下、β−グルクロニダーゼ活性体であるβ−グルコシダーゼをオフフレーバー要因因子の代表例としての手法を具体的に詳述する。
コントロールとして、88gの無脂肪牛乳(小岩井乳業株式会社製)に対して純水12g添加したサンプルを使用した。
精製したβ−グルクロニダーゼ活性体粉末を溶解させてβ−グルクロニダーゼ活性体溶液を調製した。88gの無脂肪牛乳(小岩井乳業株式会社製)に対して、0.22μmフィルターを通じて12gのβ−グルクロニダーゼ活性体溶液(12万U)を添加してサンプルを得た。
K. lactis由来のβ−グルクロニダーゼ活性体の代わりに、市販の大腸菌由来グルクロニダーゼ(メルク株式会社製)を添加した以外はサンプル2と同じ操作を行いサンプルを得た。
β−グルクロニダーゼ活性体(配列番号1の酵素)の構造遺伝子を少なくとも1つ欠損したK. lactisを以下の方法で作製した。
(1):配列番号1のβ−グルクロニダーゼ活性体遺伝子の両端1000bpの二つの塩基配列でマーカー遺伝子と呼ばれる抗生物質などの薬剤耐性遺伝子を挟んだ融合配列を持つ直鎖状二本鎖DNAを、PCR(polymerase chain reaction)法を用いて合成した。
(2):(1)にて作成したDNA配列を宿主生物に形質転換した。
(3):抗生物質などの薬剤を添加した宿主細胞培養用の培地を用いて、染色体中に形質転換用のDNA配列が挿入された形質転換体だけを成育させた。
(4):宿主細胞内の染色体中の標的遺伝子が正確にマーカー遺伝子によって置き換わった、つまり標的遺伝子が破壊されたかどうか、PCR法を用いて確認した。
Claims (8)
- ラクターゼ活性100U/gあたりのβ−グルクロニダーゼ活性が2,400U/g以下であるラクターゼ調製物。
- ラクターゼを産生できる微生物からラクターゼ調製物を製造する方法であって、
微生物がβ−グルクロニダーゼ活性体を生来的に産生する微生物であり、
β−グルクロニダーゼ活性体の量および/または活性が野生株に比べて制限されるような処理を含む、ラクターゼ調製物の製造方法。 - 前記微生物が、配列番号1で表されるアミノ酸配列の酵素もしくはそのファミリー酵素をコードする構造遺伝子をゲノム上に生来的に有するKluyveromyces属に属する微生物であり、
前記処理が、これら構造遺伝子の発現を抑制するように遺伝子の1つ以上を付加、欠損、置換または改変する操作である、
ことを特徴とする請求項2に記載のラクターゼ調製物の製造方法。 - ラクターゼを産生できる微生物からラクターゼ調製物を製造する方法であって、
ラクターゼ調製物のβ−グルクロニダーゼの活性を1回以上測定する、ラクターゼ調製物の製造方法。 - 測定したβ−グルクロニダーゼ活性を所定の基準値と比較すること、
測定したβ−グルクロニダーゼ活性が所定の基準値を超えた場合には所定の基準値以下となるように精製すること、
を含むことを特徴とする請求項4に記載のラクターゼ調製物の製造方法。 - 請求項2〜5のいずれか1項に記載のラクターゼ調製物の製造方法によって製造されたラクターゼ調製物。
- 原料乳と、請求項1または6に記載のラクターゼ調製物と、を含む混合物。
- ラクターゼ調製物に含まれるβ−グルクロニダーゼ活性を指標として、ラクターゼ調製物を原料乳に作用させたときのイソ吉草酸の生成量および/またはオフフレーバーのリスクを予測する方法。
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