JP3628605B2 - Green tea beverage and its production method - Google Patents

Description

この結果から、様々なヘミセルラーゼの中でもβ−キシラナーゼとβ−マンナナーゼに緑茶沈殿抑制効果があることが判明した。しかし、β−キシラナーゼの効果は、β−マンナナーゼよりも弱いものであった。
ヘミセルラーゼには属さないが、植物由来多糖を分解する酵素、例えば、セルラーゼ製剤やペクチナーゼ製剤では緑茶沈殿抑制効果は得られなかった。
【００３５】
実施例３：ペニシリウム・マルチカラー ｍｃｈ１３−２株のβ−マンナナーゼβ−マンナナーゼ生産菌、ペニシリウム・マルチカラー ｍｃｈ１３−２株は、群馬県の土壌より単離した。１５ｍｌの試験管に分注した１０ｇ／リットルのローカスト・ビーン・ガム、１０ｇ／リットルのバクトペプトン（ディフコ社製）、１ｇ／リットルのイースト・エキストラクト（ディフコ社製）、２ｇ／リットルのリン酸二水素カリウム、および０．５ｇ／リットルの硫酸マグネシウム・七水和物からなる液体培地に植菌して振とう培養を行い、β−マンナナーゼ活性を測定し、最も活性の高い菌株としてｍｃｈ１３−２株を得た。本菌株はペニシリウム・マルチカラー（Ｐｅｎｉｃｉｌｌｉｕｍ ｍｕｌｔｉｃｏｌｏｒ）ｍｃｈ１３−２株と命名し、通商産業省工業技術生命工学工業技術研究所に（受託番号）ＦＥＲＭ ＢＰ−６８３１として寄託してある。
なお、β−マンナナーゼの活性測定は次の方法により行った。
１０ｇ／リットルのローカストビーンガム溶液３ｍｌと１５０ｍＭ酢酸ナトリウム緩衝液（ｐＨ５．０）２ｍｌに被験液１ｍｌを加えて４０℃で１０分反応させた。反応液中に生じた還元糖をソモギーネルソン法にて定量した。β−マンナナーゼ活性は被験液１ｍｌが１分間に１μｍｏｌｅのマンノースに相当する還元力を生じる酵素活性を１Ｕｎｉｔ（Ｕ）として示した。なお、ローカストビーンガム（シグマ社製Ｎｏ．Ｇ−０７５３）は、１００℃、３分間攪拌しながら溶解した後、９５００ｒｐｍ、１０分間にて遠心分離した上清を用いた。
【００３６】
実施例４：ペニシリウム・マルチカラー ｍｃｈ１３−２由来のβ−マンナナーゼの精製
３０ｇ／リットルのコプラミール（不二製油社製）、２ｇ／リットルのリン酸二水素カリウム、および０．５ｇ／リットルの硫酸マグネシウム・七水和物を含む培地１．５リットルを、２．５Ｌのミニジャーファーメンターに入れ、１２１℃、４０分滅菌した。
ペニシリウム・マルチカラー ｍｃｈ１３−２株を、上記のようにして調製したミニジャーファーメンターに植菌し、２７℃、５００ｒｐｍ、０．５ｖｖｍにて、七日間培養した。培養液はヌッチェにて菌体を濾過分離し、培養上清約１．３Ｌを得た。なお、以後の操作は全て４℃にて行った。また、β−マンナナーゼ活性測定法は実施例３に記載の方法にて行った。
上記のようにして得られた培養上清を、限外濾過膜（分子量カット１３０００）にて濃縮した後、加水してＵＦ脱塩し、５２ｍｌの濃縮液を得た。
上記のように遠心分離して得られた溶液に最終濃度５０ｍＭとなるようトリス・塩酸緩衝液（ｐＨ８．０）を添加した。その半量ずつを２回に分けて、同緩衝液にて平衡化したイオン交換クロマトグラフィー（カラム：ＴＯＳＯＨ ＴＳＫ−ｇｅｌ ＤＥＡＥ−ＴＯＹＯＰＥＡＲＬ ６５０Ｓ １２０ｍｌ）に通した。カラムを同緩衝液にて洗浄し、引き続き３００ｍｌの０〜０．３Ｍ食塩の線状勾配でβ−マンナナーゼを溶離した。活性画分を限外濾過膜（分子量カット１００００）にて濃縮し、最終濃度５０ｍＭトリス・塩酸（ｐＨ８．０）、１．５Ｍ硫酸アンモニウムとなるように添加し、１０ｍｌの溶液とした。
この溶液を、同緩衝液にて平衡化した疎水クロマトグラフィー（カラム：ＴＯＳＯＨ ＴＳＫ−ｇｅｌ Ｐｈｅｎｙｌ−ＴＯＹＯＰＥＡＲＬ ６５０Ｓ １２０ｍｌ）に通した。カラムを同緩衝液にて洗浄し、次に３００ｍｌの１．５Ｍ〜０Ｍ硫安の線状勾配でβ−マンナナーゼを溶離した。活性画分を限外濾過膜（分子量カット１００００）にて濃縮し、引き続き１０ｍＭトリス・塩酸、０．１５Ｍ ＮａＣｌ緩衝液（ｐＨ７．５）にてＵＦ洗浄、脱塩濃縮し、２ｍｌの溶液とした。
次に濃縮液を同緩衝液で平衡化したゲル濾過クロマトグラフィー（カラム：Ｐｈａｒｍａｃｉａ ＨｉＬｏａｄ １６／６０ Ｓｕｐｅｒｄｅｘ ２００ｐｇ ）にアプライし、同緩衝液にてβ−マンナナーゼを溶出した。活性画分を限外濾過膜（分子量カット１００００）にて濃縮し、２ｍｌの溶液を得た。
最終的にＳＤＳポリアクリルアミドゲル電気泳動、および等電点電気泳動にて単一バンドを示す精製酵素を得た。
各精製ステップにおける総酵素活性、総蛋白量、比活性を以下の第２表に示す。
なお、蛋白量は、ＢＳＡ（牛血清アルブミン）を標準物質として、フォーリン・ローリー法にて測定した。

【００３７】
実施例５：ペニシリウム マルチカラー ｍｃｈ１３−２由来のβ − マンナナーゼの諸性質
実施例４で得られた精製酵素の酵素学的諸性質を測定した。
なお、酵素活性は上記実施例３に記載の方法にて測定した。
（１）作用
β−１，４−Ｄ−マンノピラノシド結合しているローカストビーンガム０．２％溶液に、本発明の酵素を２．０Ｕ／ｍｌの濃度になるように添加し、経時的にサンプルをとり、反応生成物を液体高速クロマトグラフィー（カラムＢｉｏ−ｒａｄ社Ａｍｉｎｅｘ ４２Ａ）にて分析した。その結果、反応開始後６０分後には高分子のローカストビーンガムは消失し、主として中〜低分子の酵素分解物が観察されたが、反応開始後５時間後にはそれらがさらに低分子化していた。反応初期に高分子がすみやかに消失し、中〜低分子の酵素分解物が観察されたことで、本酵素がβ−１，４−Ｄ−マンノピラノシド結合にエンド型で非特異的に作用し、マンノオリゴ糖、マンノースを生成したと考えられる。
（２）基質特異性
ローカストビーンガム（ガラクトマンナン）、グルコマンナン（コンニャク由来）、アラビノキシラン（大麦由来）、キシラン、トラガントガム、セルロース、カルボキシメチルセルロースの各０．２％溶液に本酵素２．０Ｕ／ｍｌになるように混合し、４５℃にて６６時間反応させた。その後、反応を１００℃にて停止後、反応生成物を液体高速クロマトグラフィー（カラムＢｉｏ−ｒａｄ社Ａｍｉｎｅｘ ４２Ａ）にて分析した。その結果、ローカストビーンガム、グルコマンナンでは低分子化が観察されたが、それ以外のアラビノキシラン、キシラン、トラガントガム、セルロース、カルボキシメチルセルロースでは変化は認められなかった。
（３）分子量
分子量の測定はＳＤＳポリアクリルアミドゲル電気泳動により行った。マーカータンパク質として２００，０００、１１６，３００、９７，４００、６６，３００、５５，４００、３６，５００、３１，０００、２１，５００、１４，４００を用いた。その結果、本酵素は糖鎖がついていると考えられ、ＳＤＳポリアクリルアミドゲル電気泳動のバンドはスメアになった。分子量範囲は４２，０００〜４５，０００であり、メインバンドの分子量は４３，０００であった。
（４）等電点
ファーマライトｐＨ２．５〜５．０（ファルマシア社製）３に対してファーマライトｐＨ４．５〜５．４（ファルマシア社製）を１加えた溶液を作成した。さらに、その溶液１に対して１５の割合で純水を加えた液にて膨潤させたアガロースゲルを用いて、等電点電気泳動を行った。その結果、等電点は３．２であった。
（５）安定性
本酵素は５０℃で１時間の処理で安定（１００％の残存活性）であり、６０℃１時間の処理で７０％の残存活性であった。またｐＨ３．０〜１０．０の２０℃で２０時間処理で安定であった。
（６）反応性
本酵素は７０℃付近に反応最適温度、ｐＨ５．５付近に反応最適ｐＨを有する。
（７）各種活性化剤、阻害剤の影響
実施例３のβ−マンナナーゼ活性測定法において、以下の第３表に示す物質を基質と共に添加し、それぞれの場合の活性測定を行い、活性化または阻害の有無を調べた。その結果、マンガンイオン、Ｎ−ブロモコハク酸イミドにより阻害を受けることがわかった。

【００３８】
実施例６：ヘミセルラーゼＧＭ「アマノ」（天野製薬社製）由来のβ−マンナナーゼの精製
ヘミセルラーゼＧＭ「アマノ」は、アスペルギルス属に属する微生物が生産した粗酵素剤である。精製操作は全て４℃にて行った。また、β−マンナナーゼ活性測定法は実施例３に記載の方法にて行った。
ヘミセルラーゼＧＭ「アマノ」１２ｇを、最終濃度５０ｍＭとなるようトリス・塩酸緩衝液（ｐＨ７．０）を添加し、４０ｍｌの溶液とした。この溶液を同緩衝液にて平衡化したイオン交換クロマトグラフィー（カラム：ＴＯＳＯＨ ＴＳＫ−ｇｅｌ ＤＥＡＥ−ＴＯＹＯＰＥＡＲＬ ６５０Ｓ １２０ｍｌ）に通した。カラムを同緩衝液にて洗浄し、引き続き３００ｍｌの０〜０．３Ｍ食塩の線状勾配でβ−マンナナーゼを溶離した。活性画分を限外濾過膜（分子量カット１００００）にて濃縮し、最終濃度１０ｍＭトリス・塩酸（ｐＨ７．５）、１．５Ｍ硫酸アンモニウムとなるよう添加し、１１ｍｌの溶液とした。
この溶液を、同緩衝液にて平衡化した疎水クロマトグラフィー（カラム：ＴＯＳＯＨ ＴＳＫ−ｇｅｌ Ｐｈｅｎｙｌ−ＴＯＹＯＰＥＡＲＬ ６５０Ｓ １２０ｍｌ）に通した。カラムを同緩衝液にて洗浄し、次に３００ｍｌの１．５Ｍ〜０Ｍ硫安の線状勾配でβ−マンナナーゼを溶離した。活性画分を限外濾過膜（分子量カット１００００）にて濃縮し、引き続き１０ｍＭトリス・塩酸、０．１５Ｍ ＮａＣｌ緩衝液（ｐＨ７．５）にてＵＦ洗浄、脱塩濃縮し、２．５ｍｌの溶液とした。
次に濃縮液を同緩衝液で平衡化したゲル濾過クロマトグラフィー（カラム：Ｐｈａｒｍａｃｉａ ＨｉＬｏａｄ １６／６０ Ｓｕｐｅｒｄｅｘ ２００ｐｇ ）にアプライし、同緩衝液にてβ−マンナナーゼを溶出した。活性画分を限外濾過膜（分子量カット１００００）にて濃縮し、１４．４ｍｌの溶液を得た。
最終的にＳＤＳポリアクリルアミドゲル電気泳動、および等電点電気泳動にて単一バンドを示す精製酵素を得た。
各精製ステップにおける総酵素活性、総蛋白量、比活性を以下の第４表に示す。
なお、蛋白量は、ＢＳＡ（牛血清アルブミン）を標準物質として、フォーリン・ローリー法にて測定した。

【００３９】
実施例７：ヘミセルラーゼＧＭ「アマノ」由来のβ − マンナナーゼの諸性質
実施例６で得られた精製酵素の酵素学的諸性質の概略は以下の通りであった。なお、酵素活性は上記実施例３に記載の方法にて測定した。
（１）作用
β−１，４−Ｄ−マンノピラノシド結合しているローカストビーンガムなどにエンド型で作用するガラクトマンナナーゼ活性を有する。
（２）基質特異性
ローカストビーンガム、グルコマンナン、グアーガムに作用し低分子化が観察されたが、アラビノキシラン、キシラン、トラガントガム、セルロース、カルボキシメチルセルロースでは変化は認められなかった。
（３）分子量
分子量の測定はＳＤＳポリアクリルアミドゲル電気泳動により行った。マーカータンパク質として２００，０００、１１６，３００、９７，４００、６６，３００、５５，４００、３６，５００、３１，０００、２１，５００、１４，４００を用いた。その結果、本酵素は糖鎖がついていると考えられ、ＳＤＳポリアクリルアミドゲル電気泳動のバンドはスメアになった。分子量範囲は４１，０００〜４７，０００であった。
（４）反応性
得られた精製酵素は７０℃付近に反応最適温度、ｐＨ４．０〜５．０付近に反応最適ｐＨを有する。
【００４０】
実施例８：セルラーゼＹ−ＮＣ（ヤクルト社製）由来のキシラナーゼの精製
セルラーゼＹ−ＮＣは、アスペルギルス属に属する微生物が生産した酵素である。精製操作は全て４℃にて行った。 なお、本発明のキシラナーゼの活性測定は次の方法により行った。
基質溶液（以下に詳細な調製法を示す）４ｍｌに被験液１ｍｌを加えて４０℃で１０分反応させた。反応液中に生じた還元糖をソモギーネルソン法にて定量した。
基質溶液の調整法
１） 乾物０．６２５ｇのキシラン（Ｏａｔ Ｓｐｅｌｔｓ由来、Ｌｏｔ．ＧＧ０１、東京化成工業製）に３ｍｌ水を加える。
２） ２Ｎ水酸化ナトリウム５ｍｌ加え、さらに１０ｍｌ水を加え、沸騰水浴中で加温し溶解させる。
３） 水約４０ｍｌを加え、室温まで冷やす。
４） １Ｍ酢酸２０ｍｌを１０〜３０分、かけて徐々に加える。
５） １Ｍ酢酸または１Ｎ水酸化ナトリウムを加えてｐＨ４．５にする。
６） 水を加え全量を１００ｍｌにする。
以上の手順に従って調製した溶液をキシラナーゼ活性測定の基質溶液とする。
キシラナーゼ活性は被験液１ｍｌが１分間に１μｍｏｌｅのキシロースに相当する還元力を生じる酵素活性を１Ｕｎｉｔ（Ｕ）として示した。
セルラーゼＹ−ＮＣ５．０ｇを、最終濃度１０ｍＭ酢酸ナトリウム緩衝液（ｐＨ４．０）、１．０Ｍ硫酸アンモニウムとなるよう添加し、５０ｍｌの溶液とした。この溶液を、同緩衝液にて平衡化した疎水クロマトグラフィー（カラム：ＴＯＳＯＨ ＴＳＫ−ｇｅｌ Ｐｈｅｎｙｌ−ＴＯＹＯＰＥＡＲＬ ６５０Ｓ １２０ｍｌ）に通した。カラムを同緩衝液にて洗浄し、次に３００ｍｌの１．０Ｍ〜０Ｍ硫安の線状勾配でキシラナーゼを溶離した。
活性画分を限外濾過膜（分子量カット１００００）にて濃縮し、引き続き２０ｍＭトリス・塩酸緩衝液（ｐＨ６．０）にてＵＦ洗浄、脱塩濃縮し、１５ｍｌの溶液とした。この溶液を同緩衝液にて平衡化したイオン交換クロマトグラフィー（カラム：ＴＯＳＯＨ ＴＳＫ−ｇｅｌ ＤＥＡＥ−ＴＯＹＯＰＥＡＲＬ ６５０Ｓ １２０ｍｌ）に通した。カラムを同緩衝液にて洗浄し、非吸着画分に溶出したキシラナーゼを溶離した。
活性画分を限外濾過膜（分子量カット１００００）にて濃縮し、引き続き５０ｍＭトリス・塩酸、０．２ＭＮａＣｌ緩衝液（ｐＨ６．０）にてＵＦ洗浄、脱塩濃縮し、２．０ｍｌの溶液とした。
次に濃縮液を同緩衝液で平衡化したゲル濾過クロマトグラフィー（カラム：Ｐｈａｒｍａｃｉａ ＨｉＬｏａｄ １６／６０ Ｓｕｐｅｒｄｅｘ ２００ｐｇ ）にアプライし、同緩衝液にてキシラナーゼを溶出した。活性画分を限外濾過膜（分子量カット１００００）にて濃縮し、１７．６ｍｌの溶液を得た。
最終的にＳＤＳポリアクリルアミドゲル電気泳動、および等電点電気泳動にて単一バンドを示す精製酵素を得た。
各精製ステップにおける総酵素活性、総蛋白量、比活性を以下の第５表に示す。
なお、蛋白量は、ＢＳＡ（牛血清アルブミン）を標準物質として、フォーリン・ローリー法にて測定した。

【００４１】
実施例９：セルラーゼＹ−ＮＣ由来のキシラナーゼの諸性質
実施例８で得られた精製酵素の酵素学的諸性質の概略は以下の通りであった。なお、酵素活性は上記実施例８に記載の方法にて測定した。
（１）作用
β−１，４−Ｄ−キシロピラノシド結合しているキシランなどにエンド型で作用するキシラナーゼ活性を有する。
（２）基質特異性
アラビノキシラン、キシランに作用し低分子化が観察されたが、ローカストビーンガム、グルコマンナン、グアーガム、トラガントガムでは変化は認められなかった。
（３）分子量
分子量の測定はＳＤＳポリアクリルアミドゲル電気泳動により行った。マーカータンパク質として２０５，０００、１１６，０００、９７，４００、６９，０００、５５，０００、３６，５００、２９，０００、２０，１００、１４，３００を用いた。その結果、分子量は約２０，０００であった。
（４）反応性
得られた精製酵素は６０℃付近に反応最適温度、ｐＨ４．０〜５．０付近に反応最適ｐＨを有する。
【００４２】
実施例１０：β−マンナナーゼ精製酵素とキシラナーゼ精製酵素の緑茶沈殿抑制効果の比較
実施例４で得られたペニシリウム・マルチカラー ｍｃｈ１３−２株由来の精製β−マンナナーゼ（β−マンナナーゼ１）、実施例６で得られたヘミセルラーゼＧＭ「アマノ」由来の精製β−マンナナーゼ（β−マンナナーゼ２）、実施例８で得られたセルラーゼＹ−ＮＣ由来の精製キシラナーゼの緑茶沈殿抑制効果について評価を行った。その方法について以下に示す。
各酵素を下記の濃度で添加し、４０℃、二週間保存時の各酵素の閾値添加量を求めた。なお、閾値添加量あるいは閾値添加濃度とは、沈殿生成評価において「綿状沈殿なし」となる最小量を意味する。
ヘミセルラーゼ活性を示す統一的な値が無いので両酵素を比較するのに蛋白質量に換算して行った（第６表）。また緑茶沈殿抑制効果の評価については実施例１に記載の方法に従って行った。

以上の結果を第７表に示す。

同様にして４０℃、四週間保存時の結果を第８表に示す。

二週間保存時では、β−マンナナーゼは、キシラナーゼに比べ１／９．２４〜１／５．４５量の添加で緑茶沈殿を抑制しており、すなわちβ−マンナナーゼの方が沈殿抑制効果が約５〜１０倍高いことがわかった。
【００４３】
実施例１１：β−マンナナーゼ粗酵素液の酵素処理時のｐＨ検討
緑茶をβ−マンナナーゼ酵素処理する際のｐＨについて検討した。
１． 炭酸水素ナトリウムを滅菌後にｐＨ６．２５になるように添加し調節後、酵素処理した場合。
実施例１に記載の方法に従って行った。これを、炭酸水素ナトリウム一段添加法とする。
２． 炭酸水素ナトリウムを添加しｐＨを５．０に調節後、酵素処理し、さらに炭酸水素ナトリウムを加えて滅菌後にｐＨ６．２５になるように添加する場合。
これを、炭酸水素ナトリウム二段添加法とする。以下に具体的に示す。
静岡産「やぶきた種」、一番茶中級の緑茶葉（茶葉１）１０ｇを６５℃のイオン交換水３５０ｇに添加し、５分間静置抽出した（茶葉投入直後、２．５分後にそれぞれ１０秒間攪拌）。３０メッシュと１２０メッシュ ステンレスフィルターを用いて茶葉を粗濾過後、約２０℃に冷却した抽出液を遠心分離（３０００回転／分、１０分間）し、さらに２００メッシュ ステンレスフィルターを用いて抽出液を濾過し、清澄液を得た。品質安定化のために、イオン交換水にて３．７倍に希釈後、アスコルビン酸ナトリウムを終濃度０．０４％になるように添加し、その後炭酸水素ナトリウムをｐＨ５．０になるように加え調節した。次に１０ｍｌサイズのキャップ付き耐熱試験管に１０ｍｌずつ分注した。そしてβ−マンナナーゼ活性を主成分とする粗酵素剤として本発明者らの既出願（特願平１１−３３０５５８号）のペニシリウム・マルチカラー ｍｃｈ１３−２由来β−マンナナーゼを使用し、３０℃（水浴）、３０分で酵素処理を行った。酵素処理後、再度炭酸水素ナトリウムを滅菌後にｐＨ６．２５になるように添加し調節した。次いで、１２１℃で１０分の殺菌処理を行った。以上の操作で上記実施例と同様に閾値添加濃度を求めた。
以上二通りの方法で酵素処理を行い、４０℃二週間保存時の閾値添加濃度を第９表にまとめた。

以上の結果から酵素反応をｐＨ５．０から６．２５の範囲で行っても閾値添加量は変わらないことがわかった。そして炭酸水素ナトリウムの添加は一回または二回に分けて行っても同様の効果が認められることがわかった。
【００４４】
実施例１２：各種粗酵素剤の蛋白量と、緑茶沈殿防止作用の閾値添加濃度の評価
実施例３で得られたペニシリウム・マルチカラー ｍｃｈ１３−２株由来の粗酵素液、および以下の第１０表に示す各種粗酵素剤のβ−マンナナーゼ活性とキシラナーゼ活性を実施例３および、８に示す方法にて測定した。
また、フォーリン・ローリー法にて、それぞれの蛋白量を測定した。

ついで、実施例１の方法に従って、１０ｍｌの茶溶液に対する粗酵素剤（１０％溶液として用いた）の添加量を２、５、１０、２０、５０、１００、２００、５００μｌとなるよう実験区を設け酵素処理を行い、４０℃、二週間保存後、綿状沈殿が発生しない閾値添加量の測定を行い、それぞれの粗酵素剤について、β−マンナナーゼ／キシラナーゼ活性比率との関係を評価した。
第１１表に、綿状沈殿抑制の閾値での蛋白添加量と、β−マンナナーゼ／キシラナーゼ活性比率を示した。なお、綿状沈殿抑制効果のなかったものは除いた。

この結果からわかる様に、β−マンナナーゼ／キシラナーゼ活性比率が高い程、閾値での蛋白添加量は少なくて済む事がわかった。すなわち、この現象は精製酵素だけでなく、粗酵素剤でも確認できた。そして、β−マンナナーゼ／キシラナーゼ活性比率は、２０以上で顕著になる事も確認できた。ほとんどがβ−マンナナーゼで構成されるペニシリウム・マルチカラー ｍｃｈ１３−２は、８０６と非常に高い値であると同時に、閾値での蛋白添加量は０．０１３ｍｇ蛋白／ｍｌ茶液と、非常に少ないものであった。
なお、含有するキシラナーゼの量と閾値添加量の値を比較すると、β−マンナナーゼとの相加効果がある傾向が認められた。
【００４５】
実施例１３：β−マンナナーゼ精製酵素とキシラナーゼ精製酵素の緑茶沈殿防止作用における相乗効果の評価
実施例４で得られたペニシリウム・マルチカラー ｍｃｈ１３−２株由来の精製β−マンナナーゼ、および実施例８で得られたセルラーゼＹ−ＮＣ由来の精製キシラナーゼを用いて、緑茶沈殿防止作用における相乗効果の有無の評価を行った。
実施例 １の方法に従って、以下に示す様に、１０ｍｌの茶液に対して、１０段階希釈した各濃度のβ−マンナナーゼを添加して酵素処理を行った実験区系列を基本に、それに精製キシラナーゼでの閾値添加量（実施例１０で０．１２１ｍｇ／ｍｌ＝２．１８Ｕ／ｍｌと求められている）の３．３割、および６．７割にあたる精製キシラナーゼをさらに添加して酵素処理を行った３つの実験区系列を設けた。これを４０℃、二週間保存し、綿状沈殿が発生しない閾値添加濃度の測定を行った。

その結果、いずれの実験区系列においても、精製β−マンナナーゼを０．８Ｕ／ｍｌ以上添加したもので綿状沈殿の解消が認められ、キシラナーゼを閾値添加量の６．７割添加しても、β−マンナナーゼでの綿状沈殿解消効果に影響を与えない事がわかり、相乗効果はないものと考えられた。この結果は、実施例１２の両酵素の相加効果が認められる事と一致していた。
【００４６】
実施例１４：上級茶葉での効果の検証
上級茶葉であるほど二次沈殿を引き起こす多糖が多く含まれることが知られている。また、上級茶葉を用いた場合酵素添加量は上がり、蛋白質由来の沈殿が出やすくなる。そこで、セルラーゼＹ−ＮＣと本発明者ら既出願（特願平１１−３３０５５８号）のペニシリウム・マルチカラー ｍｃｈ１３−２由来のβ−マンナナーゼ粗酵素液を用いて、上述実施例で用いていた静岡産「やぶきた種」、一番茶中級（茶葉１）の緑茶沈殿評価用茶葉よりも上級な静岡産「やぶきた種」一番茶、中級茶葉（これを茶葉２とする）での閾値添加量の評価を行った。また評価試験は、実施例１に記載の方法と同様に１０ｍｌサイズの耐熱試験管で行った。なお、茶葉２は茶葉１に比べ上級であり、沈殿生成量の多い茶葉である。
酵素濃度については第１２表に示したとおりである。

（注）セルラーゼＹ−ＮＣに関してはキシラナーゼ活性で、ｍｃｈ１３−２由来β−マンナナーゼ粗酵素液はβ−マンナナーゼ活性で示している。
４０℃、二週間保存の結果、セルラーゼＹ−ＮＣでは、緑茶沈殿抑制効果は発揮できず、これ以上添加酵素量を上げても蛋白質由来沈殿が生成されるため、上級な茶葉（茶葉２）では効果を得ることができなかった。それに対して、β−マンナナーゼ粗酵素液の場合４１Ｕ／ｍｌで沈殿は完全に抑制できた。従って本β−マンナナーゼ粗酵素液は上級な茶葉（茶葉２）においても緑茶沈殿抑制効果が確認された。蛋白質由来の沈殿が生成されないのは、効果が大きいβ−マンナナーゼを含むこと、および夾雑酵素をほとんど含まないことが考えられる。また本β−マンナナーゼ自体の比活性も高く、添加蛋白量が少ないためと考えられる。以上の結果から、β−マンナナーゼを主成分とするβ−マンナナーゼ粗酵素剤を用いることにより、従来難しいと考えられていた上級茶葉を用いた緑茶飲料の製造も可能になると考えられる。
【００４７】
実施例１５：呈味性の評価
静岡産やぶきた種、一番茶中級緑茶葉（茶葉１）１６０ｇを６５℃のイオン交換水５．６ｋｇに添加し、５分間静置抽出した（茶葉投入直後、２．５分後にそれぞれ１０秒間攪拌）。３０メッシュと１２０メッシュ ステンレスフィルターを用いて茶葉を粗濾過後、約２０℃に冷却した抽出液を遠心分離（３０００回転／分、１０分間）し、さらに２００メッシュ ステンレスフィルターを用いて抽出液を濾過し、清澄液を得た。品質安定化のために、イオン交換水にて３．７倍に希釈後、アスコルビン酸ナトリウムを終濃度０．０４％になるように添加し、その後炭酸水素ナトリウムを滅菌後にｐＨ６．２５になるように添加し調節した。そしてβ−マンナナーゼ活性を主成分とする粗酵素剤として本発明者らの既出願（特願平１１−３３０５５８号）のペニシリウム・マルチカラー ｍｃｈ１３−２由来β−マンナナーゼ粗酵素液または比較例としてセルラーゼＹ−ＮＣを使用し、３０℃（水浴）、３０分で酵素処理を行った。次いで、２００ｍｌサイズ耐熱ガラス瓶に入れ、レトルト殺菌処理を行って、緑茶飲料を得た。
以下の第１３表に示した。

（注）セルラーゼＹ−ＮＣに関してはキシラナーゼ活性で、ｍｃｈ１３−２由来β−マンナナーゼ粗酵素液はβ−マンナナーゼ活性で示している。
上記緑茶抽出液を２５℃および４０℃で四週間保存時の緑茶沈殿抑制評価および、一週間保存時の風味について経験豊富なパネラー３名で官能検査を実施してその品質を比較した。その結果を第１４表に示す。ただし２５℃および４０℃保存では同じ結果が得られた。

以上のように、セルラーゼＹ−ＮＣでは沈殿の生成が抑制されたものの風味には低濃度ですでに酵素由来の土臭が発生していた。一方ｍｃｈ１３−２由来β−マンナナーゼ粗酵素液では、沈殿の抑制ができたと同時にこく味も増し、より茶感が増した。これは明らかに品質向上効果を示すものであった。
【００４８】
【発明の効果】
上述してきたように、本発明によれば、複雑な工程を必要とせず、かつ緑茶抽出液本来の風味・呈味を失うことなく、長期間にわたり、濁りあるいは沈殿のない緑茶飲料、特に透明密閉容器入り緑茶飲料を提供することができる。
また、緑茶飲料を長期に亘って保存しても、従来、保存中に不可避的にみられた二次沈澱の発生を有効に抑制することができると共に、嗜好飲料としての緑茶飲料の茶感（緑茶本来の風味および呈味）が従来製品と比較して一段と向上した高品質の緑茶飲料の製造法およびその製品を提供することができる。
さらに、従来沈殿抑制効果があると言われていたキシラナーゼと比較して、β−マンナナーゼは効果が５〜１０倍程度高く、その結果、β−マンナナーゼを主成分とする酵素剤の場合、閾値添加量が少なくて済むため、酵素由来の「土臭」などの雑味を生じることが無い。また、上級茶葉を用いた場合でも、蛋白質由来の沈殿を生じることなく、完全に二次沈殿（綿状沈殿）を抑制することが可能となった。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to the production of a green tea beverage in which the formation of turbidity or precipitation is suppressed, and more specifically, by treating a green tea extract with β-mannanase or an enzyme agent containing β-mannanase as a main component during storage. A new high-quality green tea that suppresses the generation of secondary precipitates such as flocs caused by hemicellulose and can further improve the original flavor of green tea beverages compared to conventional products The present invention relates to a method for producing a beverage.
[0002]
[Prior art]
In recent years, the market of green tea beverages in sealed containers that are distributed and sold for a long time after being filled in sealed containers such as cans and plastic bottles has been expanding. However, tea beverages, especially green tea beverages, can cause turbidity and precipitation during long-term storage after production. Secondary precipitation such as cotton-like precipitates (floc) caused by hemicellulose and the like has been a major factor that impairs the appearance and significantly lowers the commercial value, particularly in the case of transparent container-packed beverage products.
The turbidity and precipitation of the green tea extract has been reported to be a turbid particle by polymerizing polysaccharides in a complex of catechins (for example, catechin gallate) and caffeine, but polyphenols, caffeine, proteins, It is said that components such as pectin, polysaccharides and calcium ions are involved in a complicated manner.
[0003]
The precipitation formation mechanism is complicated, and since there is no one way, precipitation suppression techniques are numerous. Examples of conventional techniques include a method of physically removing the causative agent for precipitation, a method for preventing precipitation, a method for preventing aggregation precipitation by adding a stabilizer, and a method for preventing precipitation by enzymatic decomposition of the polysaccharide causing the precipitation. For example, the following method is known.
[0004]
In JP-A-4-45744, water-soluble green tea components obtained by extracting green tea or fresh or dried tea leaves are fractionated by ultrafiltration using an ultrafiltration membrane based on organic materials and inorganic materials, It is described that, by removing almost all polymer components having a molecular weight of about 10,000 or more, crystallization of white thread-like and cotton-like solids in green tea beverages is prevented except for polymer compounds that dissolve in green tea beverages. ing.
[0005]
In JP-A-4-31348, green tea is extracted with warm water, preferably about 40 to 100 ° C. for about 1 to 10 minutes, and then the tea shell is removed with a metal net, cloth, etc., and L-ascorbic acid is obtained. And adjusting the pH of the extract to an acidic range of about 4.0 to 5.0, and then rapidly cooling it to room temperature or lower, preferably 20 ° C. or lower, to promote the formation of turbidity and orientation, After various polymer compounds are taken into turbidity and sediment to form particles, turbidity and orientation components including the particles are removed by centrifugation or other arbitrary means, and then a filter aid is added to the supernatant. Add and filter the residue to remove the remaining high molecular compound, and then adjust the pH of the extract to the neutral range of about 5.5 to 7.0, preferably bottles, plastic containers, etc., preferably transparent containers And sterilize by conventional methods. Manufacturing method of the turbidity and sediment of green tea beverages causing no occurrence is described.
[0006]
In JP-A-6-269246, tea is extracted with warm water, and the resulting extract is cooled, then tannic acid is added and left to stand, and then turbidity such as fine tea particles is removed by centrifugation or the like. A method for producing a tea beverage having long-term storage characteristics, characterized in that it is removed and the extract is further clarified by diatomaceous earth filtration, is described.
[0007]
Japanese Patent Application Laid-Open No. 2-100632 describes a method of adding a water-soluble flavonoid or a glucoside of a flavonoid to a green tea extract. Although there is no decrease in taste components, this method is effective in preventing tannin, caffeine, protein and the like from aggregating and precipitating, but is not effective in preventing precipitation by polysaccharides such as hemicellulose. .
[0008]
In JP-A-8-228684, a hot water extract of green tea is clarified, then treated with an enzyme having a hemicellulase activity containing xylanase (xylase) in the presence of ascorbic acid or a salt thereof, and further heated if necessary. A method for producing a green tea beverage that is treated to prevent the occurrence of floc is described. However, if a large amount of enzyme agent is required to eliminate precipitation of polysaccharides derived from tea leaves, there is a problem of causing protein precipitation of the enzyme. In addition, there is also a problem that the taste is significantly reduced by imparting an “earthy odor” derived from the enzyme agent.
[0009]
Thus, conventionally, various methods have been proposed for preventing secondary precipitation of green tea beverages. However, in the method such as filtering at the molecular level as described above, since the precipitation component is removed, the occurrence of secondary precipitation can be suppressed, but at the same time the taste component is also reduced, so it lacks the rich taste and becomes watery. There is a problem that the taste becomes low and the product becomes inferior in flavor.
In addition, in the method such as addition of a stabilizer and an enzyme agent, the suppression of secondary precipitation is not complete, and there is a problem that the flavor is affected if the addition amount is increased in order to obtain the effect.
Therefore, there is a strong demand for the development of a new production method that can easily produce a high-quality product that does not have the above-described drawbacks and has an excellent flavor and taste with respect to green tea beverages as taste beverages. It was in.
[0010]
[Problems to be solved by the invention]
The above-mentioned conventional technology relating to the suppression of the turbidity or precipitation of green tea beverages has a problem that the flavor and taste components in the tea extract are also removed to some extent by the turbidity or precipitation removal operation by physical filtration. In addition, the addition of readily water-soluble flavonoids was not completely effective, and the addition of hemicellulase containing xylanase was not completely effective due to protein-derived precipitation. Especially in high-grade tea leaves, there are many polysaccharides that cause precipitation, so a large amount of enzyme is required, and the above-mentioned protein-derived precipitation and unfavorable “soil odor” are generated, so formulate green tea drinks using high-grade tea leaves It was virtually impossible. The object of the present invention is to provide a green tea beverage that does not require turbidity or precipitation over a long period of time, particularly a green tea beverage in a transparent sealed container, without requiring a complicated process and without losing the original flavor and taste of the green tea extract. There is to do.
[0011]
In addition, the present invention can effectively suppress the occurrence of secondary precipitation, which has been inevitably seen during storage, even when the green tea beverage is stored for a long period of time. It is an object of the present invention to provide a method for producing a high-quality green tea beverage and a product thereof in which the tea feeling (original flavor and taste of green tea) is further improved compared to conventional products.
[0012]
[Means for Solving the Problems]
The present inventors have intensively studied to solve the above problems, and unexpectedly, β-mannanase (Patent No. 3043560, Japanese Patent Application No. 11-330558) described as a coffee precipitation preventing enzyme is used in green tea. Has also been found to show a precipitation-preventing effect.
More specifically, it has been found that β-mannanase is about 5 to 10 times more effective than xylanase, which has been said to have a precipitation-preventing effect. As a result, in the case of an enzyme agent containing β-mannanase as a main component, the amount of the threshold added is small, so that there is no mischief such as “earthy odor” derived from the enzyme. Moreover, even when high-grade tea leaves were used, it was found that secondary precipitation (cotton-like precipitation) was completely suppressed without causing protein-derived precipitation.
The present invention has been completed based on the above findings.
As described above, the present invention relates to the production of a high-quality green tea beverage having good flavor and taste without causing secondary precipitation.
That is, the present invention is a method for producing a green tea beverage with suppressed turbidity or precipitation, characterized by treating a green tea extract with β-mannanase or an enzyme agent containing β-mannanase as a main component.
The present invention is also a use of β-mannanase or an enzyme agent containing β-mannanase activity as a main component for the production of green tea beverages, particularly for the suppression of turbidity or precipitation.
Accordingly, the present invention also relates to a green tea beverage obtained by the above production method or use.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
As described above, the method for producing a green tea beverage according to the present invention is characterized in that the green tea extract is treated with β-mannanase or an enzyme agent containing β-mannanase as a main component.
A basic preferred embodiment of the present invention is to clarify a hot water extract of green tea by ordinary centrifugation or filtration, and to add an antioxidant (for example, L-ascorbic acid or a salt thereof) to the extract. Accordingly, the secondary precipitation is characterized in that the pH is adjusted with a pH adjusting agent (for example, sodium hydrogen carbonate (bicarbonate), etc.) at least once in any step and treated with an enzyme agent containing β-mannanase as a main component. This is a method for producing a green tea beverage that does not cause (cotton-like precipitation). However, the filtration treatment shown here does not indicate filtration at the molecular level as described later, but is filtration treatment that can avoid the removal of the taste component.
[0014]
In the present invention, green tea is the first step in the production of raw tea leaves or tea buds, steaming tea leaves or tea buds with steam or roasting in a kettle to stop enzyme activity in tea leaves or tea buds without causing fermentation. This refers to non-fermented tea made and does not include black tea, which is fermented tea, or oolong tea, which is semi-fermented tea. Specific examples of the green tea in the present invention include sencha, gyokuro, matcha, bancha, roasted tea, steamed tamago green tea, and kama sen tamago green tea. In the present invention, green tea is sometimes referred to as green tea leaves.
[0015]
The hot water extract of green tea used in the present invention is obtained by extracting the above green tea leaves with an aqueous medium (usually hot water) at an appropriate temperature by a conventional method (for example, see Example 1), and by using appropriate means such as centrifugation. The thing which separated the soot etc. may be sufficient. At this time, the type of tea leaves, the concentration of the extract, etc. can be used regardless of the concentration, but it is usual to obtain a concentrated extract with regard to the concentration.
[0016]
As an extraction treatment condition of the concentrated green tea extract, for example, an aqueous medium having a weight of 20 to 50 times, preferably 30 to 40 times that of tea leaves, particularly an aqueous medium at 60 to 70 ° C. is used for 3 to 10 minutes. An extraction treatment with a degree, preferably 1 to several times of stirring can be mentioned. As a method for removing the solid content and insoluble components after the extraction process, after the extraction process is completed, the tea leaves are filtered by net filtration, and then cooled to around 10 to 20 ° C., for example, centrifugation at 3000 rpm for about 10 minutes. A method for removing insoluble components can be exemplified. However, the centrifugation and filtration of the extract means not the separation at the molecular level as in the ultrafiltration described above, but means the removal of insoluble components without removing the taste components.
[0017]
For example, 0.1 to 3% by weight of ascorbic acid is added to the liquid clarified by centrifugation or filtration as described above as an antioxidant for the tea leaves. In general, in the production of green tea beverages, the addition of L-ascorbic acid or sodium ascorbate is carried out for the prevention of oxidation. In the present invention, the addition of ascorbic acid is, for example, L-ascorbic acid or ascorbic acid. What is necessary is just to carry out according to a conventional method using sodium or a thing with the same effect as them.
[0018]
In general, green tea beverages can be produced by bringing green tea into contact with an aqueous medium such as hot water and extracting the tea leaves, removing the solids and insoluble components, and then converting the green tea extract directly into a green tea beverage. Although the method of diluting the obtained green tea extract with formulated water such as ion-exchanged water to make a green tea beverage is known, in terms of industrial efficiency, such as the efficiency of extraction efficiency and ingredient / quality adjustment, A method of diluting a concentrated green tea extract to obtain a green tea beverage is employed. The green tea beverage in which the formation of precipitates according to the present invention is suppressed is characterized in that it is treated with β-mannanase or an enzyme agent containing β-mannanase as a main component as described above. The blending of β-mannanase or an enzyme agent containing β-mannanase as a main component may be an additive blend to an aqueous medium for green tea extract, but is preferably blended with a green tea extract. In the case of addition blending to such green tea extract, β-mannanase or an enzyme agent containing β-mannanase as a main component is added directly to green tea extract or dissolved, or added and dissolved in the preparation water. It may be added to the green tea extract.
[0019]
In the present invention, β-mannanase specifically acts on a substrate having a β-1,4-D-mannopyranoside bond, such as mannan, galactomannan, glucomannan, and non-specifically β-1,4-D-mannopyranoside. It refers to the enzyme β-mannanase that hydrolyzes the bond to produce mannose and mannooligosaccharides. This β-mannanase does not act on arabinoxylan, xylan, tragacanth gum, cellulose, or carboxymethylcellulose.
[0020]
In the present invention, an enzyme agent containing β-mannanase as a main component means a xylanase having a β-mannanase activity per gram of enzyme agent of usually 5,000 U or more, preferably 10,000 U or more, and further contained as a contaminating enzyme. And an activity ratio (β-mannanase / xylanase) is usually 5 or more, preferably 20 or more. The activity measurement method and the definition of activity of β-mannanase and xylanase are as described in detail in the following examples.
As the β-mannanase or the enzyme agent containing this as a main component in the present invention, any agent can be used as long as it has the properties described above. However, the β-mannanase / xylanase activity ratio is as described above. Those of 5 or more or 20 or more are preferred. Specifically, for example, β-mannanase derived from Penicillium multicolor mch13-2 (FERM BP-6831) of an already filed application (Japanese Patent Application No. 11-330558) by the present inventors, cellulosin GM5 ( Β-mannanase derived from Hankyu Bioindustry Co., Ltd., β-mannanase derived from hemicellulase GM “Amano” (manufactured by Amano Pharmaceutical Co., Ltd.), and the like (see Examples below). Among them, the β-mannanase preparation derived from the above-mentioned Penicillium multicolor mch13-2 (FERM BP-6831) by the present inventors has a β-mannanase / xylanase activity ratio of 806, which is the highest among the crude enzyme agents. It can be used as suitable.
[0021]
β-mannanase is, by definition, included in the enzyme group of hemicellulase. This is because hemicellulase does not refer to an enzyme that acts on a single substrate, but generally refers to a group of enzymes that act on a wide variety of polysaccharides extracted from plant tissues with alkali. Because. In a strict definition, it means an enzyme group that acts on polysaccharides (arabinoxylan, xylan, xyloglucan, glucomannan, arabinan, β-glucan) capable of strongly hydrogen bonding to insoluble cellulose (Japanese Patent Laid-Open No. Hei 8). -228684). However, the definition of hemicellulase is too broad, and when the present inventors examined the green tea precipitation inhibitory effect of commercially available hemicellulase preparations, not all hemicellulase preparations had a green tea precipitation inhibitory effect. . For enzyme agents that had no effect, precipitation could not be suppressed even when the concentration was increased, but protein-derived precipitates were formed. Further, although not belonging to hemicellulase, the effects of the present invention were not obtained only with an enzyme that degrades plant-derived polysaccharides, pectinase preparations or cellulase preparations.
[0022]
Specifically, as shown in Example 2, the effect of various hemicellulases on green tea precipitation was examined. The enzymes studied as hemicellulases are β-mannanase, β-xylanase (xylanase), endo-arabinanase, α-L-arabinofuranosidase, endo β-1,4-galactanase, exo-β-glucanase, endo- β-glucanase. Among these, β-mannanase was confirmed to have the highest precipitation inhibitory effect and the highest. Xylanase was also confirmed to have a green tea precipitation inhibitory effect, but not as much as β-mannanase.
[0023]
Therefore, purified β-mannanase and purified xylanase were compared, and the threshold addition amount for green tea precipitation inhibition was compared. Hemicellulase is a generic term for enzymes that act on a wide variety of polysaccharides, and since there is no uniform value indicating their activity, the threshold addition concentrations were compared in terms of protein mass to compare both enzymes. As a result, 0.12 mg / ml (final concentration) of xylanase was required to suppress green tea cotton-like precipitation at the used tea leaf and tea leaf concentration, whereas β-mannanase was 0.013-0.022 mg / ml. ml (final concentration) was required (at 40 ° C. for 2 weeks storage). Therefore, when compared by protein mass, if the precipitation-inhibiting enzyme is xylanase, about 5 to 10 times that of β-mannanase is required. That is, it was found that β-mannanase is the most effective in inhibiting green tea precipitation in the hemicellulase enzyme group.
[0024]
Therefore, when comparing commercially available hemicellulase enzyme agents, the higher the β-mannanase activity ratio, the higher the green tea precipitation inhibitory effect. For this reason, an enzyme agent having a high ratio requires less addition amount for suppressing green tea precipitation. That is, it has been confirmed in Example 12 that will be described later that an enzyme agent having a higher β-mannanase activity ratio requires less protein in the enzyme agent that is an actual embodiment. For example, the β-mannanase derived from Penicillium multicolor mch13-2 strain (FERM BP-6831) of the already filed application (Japanese Patent Application No. 11-330558) by the present inventors is the most β-mannanase among the hemicellulase enzyme agents examined. It was found that the ratio of activity was high and therefore the highest in terms of inhibiting the formation of green tea precipitates. Moreover, it has been found that β-mannanase contained in the present β-mannanase preparation has high specific activity even when compared with other β-mannanases. Therefore, this β-mannanase preparation, which is the most effective for inhibiting green tea precipitation and has a high specific activity β-mannanase activity as a main component, has a smaller amount of addition required for inhibiting green tea precipitation than other β-mannanase preparations. It turned out to be sufficient.
[0025]
In general, it is known that the green tea cotton-like precipitate is more easily generated as the tea leaves become higher grade. This is because the higher the tea leaves, the higher the polysaccharide content in the green tea extract. Therefore, in the case of high-grade tea leaves, the necessary amount of enzyme agent added to suppress precipitation inevitably increases. However, if the amount of the enzyme agent added is increased, the formation of protein-derived precipitate is promoted, and the original role of the enzyme agent cannot be achieved. However, since β-mannanase is about 5 to 10 times more effective in suppressing green tea precipitation than xylanase, if treated with β-mannanase preparation, protein-derived precipitates are produced even when the threshold enzyme amount for higher tea leaves is added. And secondary precipitation (cotton-like precipitation) can be suppressed. In particular, the β-mannanase already filed by the present inventors (Japanese Patent Application No. 11-330558) is more advantageous because it has a high specific activity and requires less protein. This makes it possible to prescribe PET bottle green tea beverages using high-grade tea leaves, which were previously considered difficult to achieve because of the occurrence of flocculent precipitation, which is very useful industrially. It can be said.
[0026]
In recent years, it is said that green tea secondary precipitation is mainly caused by water-soluble polysaccharides having a molecular weight of 20,000 or more. In addition, it has been reported that there are two high molecular weight polysaccharides having a molecular weight of 100,000 or more and an average molecular weight of 10,000 regarding green tea water-soluble polysaccharides. [Teiichi Takeo, “The Journal of Japan Society for Food Science and Technology”, Vol. 45, no. 4, p270-272 (1998)]. The following hypothesis can be considered as a mechanism by which β-mannanase preferentially suppresses secondary precipitation.
-The green tea precipitated polysaccharide contains a single polysaccharide, and both β-mannanase and xylanase can degrade green tea polysaccharide, but β-mannanase shows higher degradability.
-The green tea precipitated polysaccharide contains multiple types of polysaccharides, and the amount of polysaccharides that can be decomposed by β-mannanase is larger. The amount of polysaccharide that can be degraded by xylanase is low in quantity. -Green tea precipitated polysaccharide contains multiple types of polysaccharides, and some polysaccharides are easily degraded by β-mannanase. On the other hand, another polysaccharide has a property that it is difficult to be degraded by xylanase.
・ Polysaccharides that can be degraded by β-mannanase can suppress precipitation by only partially degrading them, whereas polysaccharides that can be decomposed by xylanase cannot suppress precipitation unless they are completely decomposed. Necessary.
[0027]
In the present invention, normal treatment conditions with β-mannanase are as follows.
The optimum pH of β-mannanase is approximately 4.5 to 6.0. However, even if the enzyme treatment is not performed at the optimum pH, the effect is equivalent if the pH is in the range of 5.0 to 6.5. In the production of green tea beverages, ascorbic acid or a salt thereof is usually added as an antioxidant, and then sodium bicarbonate, disodium hydrogen phosphate or the like is appropriately added as a pH adjuster at the time of enzyme treatment. Adjustment is easy. As described above, the pH adjustment of the green tea extract can be performed by adding at least one pH adjuster in any step (enzyme treatment, addition of antioxidant, centrifugation, etc.) in the above production method. preferable.
[0028]
Next, the range of temperature suitable for the action of the β-mannanase preparation is 20 to 70 ° C. The β-mannanase treatment can be performed at a high temperature of 60 to 70 ° C. simultaneously with the green tea extraction by adding an enzyme agent to the aqueous medium for green tea extraction, but the β-mannanase treatment is performed at 20 to 40 ° C. with the green tea extract. It can also be done. Alternatively, both can be performed. In any case, if the time exceeding 40 ° C. is long, the green tea extract may be deteriorated, which is not preferable. If the temperature is too low, the enzyme treatment time becomes long. This is not preferable because it may cause deterioration of the extract. The enzyme treatment time is usually required to be 30 minutes or longer, but may be set as appropriate depending on the amount of enzyme used, the type of tea leaf, the concentration of tea leaf, and the like. However, in order to prevent the deterioration of the extract, it is necessary to adjust appropriately so that the treatment can be performed in as short a time as possible.
[0029]
The amount of the enzyme added is usually 0.05 U to 100 U, preferably 0.8 U to 41 U in terms of β-mannanase activity per 1 ml of green tea extract (when stored at 40 ° C. for 1 month). However, the amount to be added varies depending on the type of tea leaf used, the concentration of the green tea extract, the type, nature, and various conditions of the enzyme used, so it is necessary to set appropriately.
[0030]
The green tea beverage obtained by the above production method has an extraordinary effect that the flavor and taste are further improved as compared with conventional green tea beverage products. This is an enzymatic treatment with an enzyme having β-mannanase activity as described above, whereby the polysaccharides contained in the tea extract are decomposed, and a wide variety of saccharides are decomposed to lower their molecular weight. This is based on the action of further improving the flavor and taste of the green tea beverage as a good flavor component or taste component. Moreover, since only a small amount of enzyme is required, there is an advantage that there is no miscellaneous taste derived from the enzyme agent.
[0031]
After the enzyme treatment, the enzyme reaction is usually stopped simultaneously with the heat sterilization treatment. For these processes, Example 15 can be referred to, for example. In addition, as described above, pH adjustment can be easily performed with an edible alkali such as sodium hydrogencarbonate at least once before and / or after any of the treatment steps in the present invention. The pH for pH adjustment in the method of the present invention is preferably adjusted to pH 6.0 to 6.5 after sterilization for maintaining flavor and improving quality.
[0032]
A typical green tea beverage of the present invention is prepared by diluting a concentrated green tea extract with preparation water such as ion-exchanged water, blending ascorbic acid or a salt thereof, and the like, and adjusting pH with sodium hydrogen carbonate or the like. -After being treated with an enzyme agent containing mannanase or β-mannanase as a main component, it is filled in a sealed container. Moreover, although a sealed container is not specifically limited, A plastic container, a can, a paper container, a glass bottle etc. are illustrated. Filling and sterilization may be performed by a normal processing method and is not particularly limited. For example, in the case of a can, reheating, filling, and high-pressure sterilization (retort sterilization) are performed, and in the case of a PET bottle, an instant sterilization method (130 to 135 ° C., around 30 seconds) is applied. A green tea beverage product in a sealed container is produced (see, for example, Example 15). Since the green tea beverage of the present invention produced in this way is suppressed from producing precipitates, it is advantageous that it can be made into a green tea beverage in a sealed container, particularly a green tea beverage in a transparent sealed container such as a PET bottle.
[0033]
【Example】
EXAMPLES Next, although this invention is demonstrated concretely based on an Example, this invention is not limited at all by the said Example.
Example 1:Green tea precipitation inhibition evaluation method
The green tea precipitation suppression evaluation for determining the enzyme threshold addition amount was performed by the following method.
"Yabukita seeds" from Shizuoka, 10g of green tea leaves of the first tea level (referred to as tea leaves 1) were added to 350g of ion-exchanged water at 65 ° C and extracted by standing for 5 minutes (immediately after adding tea leaves, 2.5 minutes) Each later stirred for 10 seconds). After roughly filtering tea leaves using a 30 mesh and 120 mesh stainless steel filter, the extract cooled to about 20 ° C. is centrifuged (3000 rpm / minute, 10 minutes), and further filtered using a 200 mesh stainless steel filter. A clear liquid was obtained. In order to stabilize the quality, after diluting 3.7 times with ion-exchanged water, sodium ascorbate was added to a final concentration of 0.04%, and then sodium bicarbonate was adjusted to pH 6.25 after sterilization. Added to adjust. Next, 10 ml each was dispensed into a heat-resistant test tube with a cap of 10 ml size. Each enzyme agent was added at each concentration, and the enzyme treatment was performed at 30 ° C. (water bath) for 30 minutes. Next, sterilization treatment was performed at 121 ° C. for 10 minutes and stored at 40 ° C.
[0034]
Example 2:Evaluation of green tea precipitation inhibitory effect of various hemicellulases
The green tea precipitation inhibitory effect of various commercially available purified hemicellulase enzyme agents and crude enzyme agents other than hemicellulase shown in Table 1 below was examined (stored at 40 ° C. for one week). The green tea precipitation inhibition evaluation was performed according to the method described in Example 1.

From these results, it was found that among various hemicellulases, β-xylanase and β-mannanase have a green tea precipitation inhibitory effect. However, the effect of β-xylanase was weaker than β-mannanase.
Although not belonging to hemicellulase, an enzyme that degrades plant-derived polysaccharides, such as cellulase preparations and pectinase preparations, did not provide a green tea precipitation inhibitory effect.
[0035]
Example 3:Β-mannanase of Penicillium multicolor mch13-2 strainA β-mannanase-producing bacterium, Penicillium multicolor mch13-2 strain, was isolated from soil in Gunma Prefecture. 10 g / liter locust bean gum dispensed into a 15 ml test tube, 10 g / liter bactopeptone (Difco), 1 g / liter yeast extract (Difco), 2 g / liter phosphoric acid Inoculated into a liquid medium consisting of potassium dihydrogen and 0.5 g / liter magnesium sulfate heptahydrate, cultured with shaking, β-mannanase activity was measured, and mch13-2 was the most active strain. Got the stock. This strain is designated as Penicillium multicolor mch13-2 strain, and is deposited as FERM BP-6831 at the Ministry of International Trade and Industry, Industrial Technology and Biotechnology Institute.
The β-mannanase activity was measured by the following method.
1 ml of the test solution was added to 3 ml of 10 g / liter locust bean gum solution and 2 ml of 150 mM sodium acetate buffer (pH 5.0) and reacted at 40 ° C. for 10 minutes. The reducing sugar produced in the reaction solution was quantified by the Somogy Nelson method. The β-mannanase activity was expressed as 1 Unit (U), where 1 ml of the test solution produces a reducing power corresponding to 1 μmole of mannose per minute. Locust bean gum (Sigma No. G-0753) was dissolved with stirring at 100 ° C. for 3 minutes and then centrifuged at 9500 rpm for 10 minutes.
[0036]
Example 4:Purification of β-mannanase derived from Penicillium multicolor mch13-2
1.5 L of medium containing 30 g / L of copra meal (manufactured by Fuji Oil Co., Ltd.), 2 g / L of potassium dihydrogen phosphate, and 0.5 g / L of magnesium sulfate heptahydrate was added to 2.5 L. Placed in a mini jar fermenter and sterilized at 121 ° C. for 40 minutes.
Penicillium multicolor mch13-2 strain was inoculated into the mini jar fermenter prepared as described above and cultured at 27 ° C., 500 rpm, 0.5 vvm for 7 days. The culture broth was separated by filtration with Nutsche to obtain about 1.3 L of culture supernatant. All subsequent operations were performed at 4 ° C. The β-mannanase activity was measured by the method described in Example 3.
The culture supernatant obtained as described above was concentrated with an ultrafiltration membrane (molecular weight cut: 13000), then hydrated and desalted with UF to obtain 52 ml of a concentrated solution.
Tris / hydrochloric acid buffer (pH 8.0) was added to the solution obtained by centrifugation as described above to a final concentration of 50 mM. Half of each was divided into two portions and passed through ion exchange chromatography (column: TOSOH TSK-gel DEAE-TOYOPARL 650S 120 ml) equilibrated with the same buffer. The column was washed with the same buffer followed by elution of β-mannanase with a linear gradient of 300 ml of 0-0.3 M saline. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 10000), and added to a final concentration of 50 mM Tris-hydrochloric acid (pH 8.0) and 1.5 M ammonium sulfate to obtain a 10 ml solution.
This solution was passed through hydrophobic chromatography (column: TOSOH TSK-gel Phenyl-TOYOPEARL 650S 120 ml) equilibrated with the same buffer. The column was washed with the same buffer and then β-mannanase was eluted with a linear gradient of 300 ml of 1.5 M to 0 M ammonium sulfate. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 10000), then washed with UF and desalted and concentrated with 10 mM Tris-HCl and 0.15 M NaCl buffer (pH 7.5) to obtain a 2 ml solution. .
Next, the concentrate was applied to gel filtration chromatography (column: Pharmacia HiLoad 16/60 Superdex 200 pg) equilibrated with the same buffer, and β-mannanase was eluted with the same buffer. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 10,000) to obtain 2 ml of a solution.
Finally, a purified enzyme showing a single band was obtained by SDS polyacrylamide gel electrophoresis and isoelectric focusing.
The total enzyme activity, total protein amount, and specific activity in each purification step are shown in Table 2 below.
The amount of protein was measured by the Foreign-Raleigh method using BSA (bovine serum albumin) as a standard substance.

[0037]
Example 5:Β derived from Penicillium multicolor mch13-2 − Properties of mannanase
The enzymatic properties of the purified enzyme obtained in Example 4 were measured.
The enzyme activity was measured by the method described in Example 3 above.
(1) Action
The enzyme of the present invention was added to a 0.2% solution of locust bean gum to which β-1,4-D-mannopyranoside is bound so as to have a concentration of 2.0 U / ml. The product was analyzed by liquid high performance chromatography (column Bio-rad Aminex 42A). As a result, high molecular locust bean gum disappeared 60 minutes after the start of the reaction, and medium to low molecular enzyme degradation products were mainly observed, but they were further reduced in molecular weight 5 hours after the start of the reaction. . In the early stage of the reaction, the polymer disappeared quickly, and an enzyme degradation product of medium to low molecular weight was observed, so that this enzyme acts on the β-1,4-D-mannopyranoside bond non-specifically, It is thought that mannooligosaccharide and mannose were produced.
(2) Substrate specificity
Locust bean gum (galactomannan), glucomannan (derived from konjac), arabinoxylan (derived from barley), xylan, tragacanth gum, cellulose and carboxymethylcellulose are mixed so that the enzyme is 2.0 U / ml. , And reacted at 45 ° C. for 66 hours. Thereafter, the reaction was stopped at 100 ° C., and the reaction product was analyzed by liquid high performance chromatography (column Bio-rad Aminex 42A). As a result, low molecular weight was observed in locust bean gum and glucomannan, but no change was observed in other arabinoxylan, xylan, tragacanth gum, cellulose, and carboxymethylcellulose.
(3) Molecular weight
The molecular weight was measured by SDS polyacrylamide gel electrophoresis. 200,000, 116,300, 97,400, 66,300, 55,400, 36,500, 31,000, 21,500, 14,400 were used as marker proteins. As a result, this enzyme was considered to have a sugar chain, and the band of SDS polyacrylamide gel electrophoresis was smeared. The molecular weight range was 42,000-45,000, and the molecular weight of the main band was 43,000.
(4) Isoelectric point
A solution was prepared by adding 1 of Pharmalite pH 4.5 to 5.4 (Pharmacia) to Pharmalite pH 2.5 to 5.0 (Pharmacia) 3. Furthermore, isoelectric focusing was performed using an agarose gel swollen with a solution obtained by adding pure water at a ratio of 15 to the solution 1. As a result, the isoelectric point was 3.2.
(5) Stability
This enzyme was stable (100% residual activity) after treatment at 50 ° C. for 1 hour, and 70% residual activity after treatment at 60 ° C. for 1 hour. Moreover, it was stable by treatment for 20 hours at 20 ° C. at pH 3.0 to 10.0.
(6) Reactivity
This enzyme has a reaction optimum temperature around 70 ° C. and a reaction optimum pH around pH 5.5.
(7) Effects of various activators and inhibitors
In the β-mannanase activity measurement method of Example 3, the substances shown in Table 3 below were added together with the substrate, the activity was measured in each case, and the presence or absence of activation or inhibition was examined. As a result, it was found that inhibition was caused by manganese ions and N-bromosuccinimide.

[0038]
Example 6:Purification of β-mannanase derived from hemicellulase GM “Amano” (manufactured by Amano Pharmaceutical Co., Ltd.)
Hemicellulase GM “Amano” is a crude enzyme produced by a microorganism belonging to the genus Aspergillus. All purification operations were performed at 4 ° C. The β-mannanase activity was measured by the method described in Example 3.
Tris-hydrochloric acid buffer (pH 7.0) was added to 12 g of hemicellulase GM “Amano” to a final concentration of 50 mM to obtain a 40 ml solution. This solution was passed through ion exchange chromatography (column: TOSOH TSK-gel DEAE-TOYOPEARL 650S 120 ml) equilibrated with the same buffer. The column was washed with the same buffer followed by elution of β-mannanase with a linear gradient of 300 ml of 0-0.3 M saline. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 10000), and added to a final concentration of 10 mM Tris / hydrochloric acid (pH 7.5) and 1.5 M ammonium sulfate to obtain an 11 ml solution.
This solution was passed through hydrophobic chromatography (column: TOSOH TSK-gel Phenyl-TOYOPEARL 650S 120 ml) equilibrated with the same buffer. The column was washed with the same buffer and then β-mannanase was eluted with a linear gradient of 300 ml of 1.5 M to 0 M ammonium sulfate. The active fraction is concentrated with an ultrafiltration membrane (molecular weight cut 10000), followed by UF washing with 10 mM Tris / hydrochloric acid and 0.15M NaCl buffer (pH 7.5), desalting and concentration, and a 2.5 ml solution. It was.
Next, the concentrate was applied to gel filtration chromatography (column: Pharmacia HiLoad 16/60 Superdex 200 pg) equilibrated with the same buffer, and β-mannanase was eluted with the same buffer. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 10,000) to obtain 14.4 ml of a solution.
Finally, a purified enzyme showing a single band was obtained by SDS polyacrylamide gel electrophoresis and isoelectric focusing.
The total enzyme activity, total protein amount, and specific activity in each purification step are shown in Table 4 below.
The amount of protein was measured by the Foreign-Raleigh method using BSA (bovine serum albumin) as a standard substance.

[0039]
Example 7:Β derived from hemicellulase GM "Amano" − Properties of mannanase
The outline of the enzymatic properties of the purified enzyme obtained in Example 6 was as follows. The enzyme activity was measured by the method described in Example 3 above.
(1) Action
It has a galactomannanase activity that acts in an endo-type on locust bean gum or the like that is linked to β-1,4-D-mannopyranoside.
(2) Substrate specificity
Low molecular weight was observed by acting on locust bean gum, glucomannan and guar gum, but no change was observed in arabinoxylan, xylan, tragacanth gum, cellulose and carboxymethylcellulose.
(3) Molecular weight
The molecular weight was measured by SDS polyacrylamide gel electrophoresis. 200,000, 116,300, 97,400, 66,300, 55,400, 36,500, 31,000, 21,500, 14,400 were used as marker proteins. As a result, this enzyme was considered to have a sugar chain, and the band of SDS polyacrylamide gel electrophoresis was smeared. The molecular weight range was 41,000-47,000.
(4) Reactivity
The obtained purified enzyme has a reaction optimum temperature around 70 ° C. and a reaction optimum pH around pH 4.0 to 5.0.
[0040]
Example 8:Purification of xylanase derived from cellulase Y-NC (manufactured by Yakult)
Cellulase Y-NC is an enzyme produced by a microorganism belonging to the genus Aspergillus. All purification operations were performed at 4 ° C. The activity of the xylanase of the present invention was measured by the following method.
1 ml of the test solution was added to 4 ml of the substrate solution (detailed preparation method is shown below) and reacted at 40 ° C. for 10 minutes. The reducing sugar produced in the reaction solution was quantified by the Somogy Nelson method.
Preparation of substrate solution
1) 3 ml of water is added to 0.625 g of dry matter xylan (Oat Sparts origin, Lot. GG01, manufactured by Tokyo Chemical Industry Co., Ltd.).
2) Add 5 ml of 2N sodium hydroxide, add 10 ml of water, and warm to dissolve in a boiling water bath.
3) Add about 40 ml of water and cool to room temperature.
4) Gradually add 20 ml of 1M acetic acid over 10-30 minutes.
5) Add 1M acetic acid or 1N sodium hydroxide to pH 4.5.
6) Add water to bring the total volume to 100 ml.
The solution prepared according to the above procedure is used as a substrate solution for xylanase activity measurement.
Xylanase activity was expressed as 1 Unit (U), where 1 ml of the test solution produces a reducing power corresponding to 1 μmole of xylose per minute.
Cellulase Y-NC (5.0 g) was added to a final concentration of 10 mM sodium acetate buffer (pH 4.0) and 1.0 M ammonium sulfate to obtain a 50 ml solution. This solution was passed through hydrophobic chromatography (column: TOSOH TSK-gel Phenyl-TOYOPEARL 650S 120 ml) equilibrated with the same buffer. The column was washed with the same buffer and then the xylanase was eluted with a 300 ml linear gradient of 1.0 M to 0 M ammonium sulfate.
The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 10000), then washed with UF in 20 mM Tris / HCl buffer (pH 6.0), desalted and concentrated to give a 15 ml solution. This solution was passed through ion exchange chromatography (column: TOSOH TSK-gel DEAE-TOYOPEARL 650S 120 ml) equilibrated with the same buffer. The column was washed with the same buffer to elute xylanase eluted in the non-adsorbed fraction.
The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 10000), then washed with UF in 50 mM Tris-hydrochloric acid, 0.2 M NaCl buffer (pH 6.0), desalted and concentrated, and 2.0 ml of solution was added. did.
Next, the concentrate was applied to gel filtration chromatography (column: Pharmacia HiLoad 16/60 Superdex 200 pg) equilibrated with the same buffer, and xylanase was eluted with the same buffer. The active fraction was concentrated with an ultrafiltration membrane (molecular weight cut 10,000) to obtain 17.6 ml of a solution.
Finally, a purified enzyme showing a single band was obtained by SDS polyacrylamide gel electrophoresis and isoelectric focusing.
The total enzyme activity, total protein amount, and specific activity in each purification step are shown in Table 5 below.
The amount of protein was measured by the Foreign-Raleigh method using BSA (bovine serum albumin) as a standard substance.

[0041]
Example 9:Properties of xylanase derived from cellulase Y-NC
The outline of the enzymatic properties of the purified enzyme obtained in Example 8 was as follows. The enzyme activity was measured by the method described in Example 8 above.
(1) Action
It has a xylanase activity that acts in an endo form on xylan or the like having a β-1,4-D-xylopyranoside bond.
(2) Substrate specificity
Low molecular weight was observed by acting on arabinoxylan and xylan, but no change was observed in locust bean gum, glucomannan, guar gum, and tragacanth gum.
(3) Molecular weight
The molecular weight was measured by SDS polyacrylamide gel electrophoresis. As marker proteins, 205,000, 116,000, 97,400, 69,000, 55,000, 36,500, 29,000, 20,100, 14,300 were used. As a result, the molecular weight was about 20,000.
(4) Reactivity
The obtained purified enzyme has a reaction optimum temperature around 60 ° C. and a reaction optimum pH around pH 4.0 to 5.0.
[0042]
Example 10:Comparison of green tea precipitation inhibitory effect of β-mannanase purified enzyme and xylanase purified enzyme
Purified β-mannanase (β-mannanase 1) derived from Penicillium multicolor mch13-2 strain obtained in Example 4 and purified β-mannanase derived from hemicellulase GM “Amano” obtained in Example 6 (β- Mannanase 2) and the green tea precipitation inhibitory effect of purified xylanase derived from cellulase Y-NC obtained in Example 8 were evaluated. The method is shown below.
Each enzyme was added at the following concentration, and the threshold addition amount of each enzyme when stored at 40 ° C. for 2 weeks was determined. The threshold addition amount or the threshold addition concentration means the minimum amount at which “no flocculent precipitation” is obtained in the precipitation formation evaluation.
Since there is no uniform value indicating hemicellulase activity, the two enzymes were compared in terms of protein mass (Table 6). The evaluation of the green tea precipitation inhibitory effect was performed according to the method described in Example 1.

The results are shown in Table 7.

Similarly, the results of storage at 40 ° C. for 4 weeks are shown in Table 8.

When stored for two weeks, β-mannanase suppresses green tea precipitation by addition of 1 / 9.24 to 1 / 5.45 compared to xylanase, that is, β-mannanase has a precipitation suppression effect of about 5 It was found to be 10 times higher.
[0043]
Example 11:Examination of pH during enzyme treatment of β-mannanase crude enzyme solution
The pH when green tea was treated with β-mannanase enzyme was examined.
1. When sodium bicarbonate is added to adjust the pH to 6.25 after sterilization, and after enzyme treatment.
The procedure described in Example 1 was followed. This is a sodium hydrogen carbonate one-stage addition method.
2. When sodium bicarbonate is added to adjust the pH to 5.0, enzyme treatment is performed, and sodium bicarbonate is further added to sterilize to add pH 6.25.
This is a sodium hydrogen carbonate two-stage addition method. This is specifically shown below.
10g of green tea leaves (tea leaves 1) from Shizuoka "Yabukita Seed", the first tea, were added to 350g of ion-exchanged water at 65 ° C, and extracted by standing for 5 minutes (10 seconds each after the tea leaves were added and 2.5 minutes later) Stirring). After roughly filtering tea leaves using a 30 mesh and 120 mesh stainless steel filter, the extract cooled to about 20 ° C. is centrifuged (3000 rpm / minute, 10 minutes), and further filtered using a 200 mesh stainless steel filter. A clear liquid was obtained. To stabilize quality, dilute 3.7 times with ion-exchanged water, add sodium ascorbate to a final concentration of 0.04%, and then add sodium bicarbonate to pH 5.0. Adjusted. Next, 10 ml each was dispensed into a heat-resistant test tube with a cap of 10 ml size. Then, as a crude enzyme agent having β-mannanase activity as a main component, penicillium multicolor mch13-2 derived β-mannanase of the present applicant's application (Japanese Patent Application No. 11-330558) is used, ), And the enzyme treatment was performed in 30 minutes. After the enzyme treatment, sodium bicarbonate was added again and adjusted to pH 6.25 after sterilization. Next, sterilization treatment was performed at 121 ° C. for 10 minutes. With the above operation, the threshold addition concentration was determined in the same manner as in the above example.
The enzyme treatment was carried out by the two methods described above, and the threshold addition concentrations when stored at 40 ° C. for 2 weeks are summarized in Table 9.

From the above results, it was found that the threshold addition amount did not change even when the enzyme reaction was carried out in the range of pH 5.0 to 6.25. It was also found that the same effect was observed when sodium bicarbonate was added once or twice.
[0044]
Example 12:Evaluation of protein content of various crude enzyme agents and threshold addition concentration of green tea precipitation prevention effect
Examples 3 and 8 show the β-mannanase activity and xylanase activity of the crude enzyme solution derived from the Penicillium multicolor mch13-2 strain obtained in Example 3 and various crude enzyme agents shown in Table 10 below. Measured by the method.
In addition, the amount of each protein was measured by the Foreign-Raleigh method.

Next, according to the method of Example 1, the experimental group was adjusted so that the amount of the crude enzyme agent (used as a 10% solution) added to 10 ml of tea solution was 2, 5, 10, 20, 50, 100, 200, 500 μl An enzyme treatment was performed, and after storage at 40 ° C. for 2 weeks, a threshold addition amount at which no flocculent precipitate was generated was measured, and the relationship with the β-mannanase / xylanase activity ratio was evaluated for each crude enzyme agent.
Table 11 shows the amount of protein added and the β-mannanase / xylanase activity ratio at the threshold for inhibiting the flocculent precipitation. In addition, the thing which did not have the cotton-like precipitation inhibitory effect was excluded.

As can be seen from this result, it was found that the higher the β-mannanase / xylanase activity ratio, the smaller the amount of protein added at the threshold. That is, this phenomenon could be confirmed not only with the purified enzyme but also with the crude enzyme agent. It was also confirmed that the β-mannanase / xylanase activity ratio became prominent at 20 or more. Penicillium multicolor mch13-2, which is mostly composed of β-mannanase, has a very high value of 806, and at the same time, the amount of protein added at the threshold is very small, 0.013 mg protein / ml tea liquid. Met.
In addition, when the amount of xylanase contained and the value of the threshold addition amount were compared, there was a tendency to have an additive effect with β-mannanase.
[0045]
Example 13:Evaluation of synergistic effect of β-mannanase purified enzyme and xylanase purified enzyme on green tea precipitation prevention
Using the purified β-mannanase derived from the Penicillium multicolor mch13-2 strain obtained in Example 4 and the purified xylanase derived from the cellulase Y-NC obtained in Example 8, a synergistic effect on the green tea precipitation preventing action was demonstrated. The presence or absence was evaluated.
In accordance with the method of Example 1, as shown below, purified xylanase was based on a series of experimental plots in which β-mannanase diluted in 10 steps was added to 10 ml of tea liquid and subjected to enzyme treatment. Purified xylanase equivalent to 3.3% and 6.7% of the threshold addition amount in Example 10 (determined as 0.121 mg / ml = 2.18 U / ml in Example 10) was added to perform enzyme treatment. Three experimental section series were established. This was stored at 40 ° C. for 2 weeks, and a threshold addition concentration at which no flocculent precipitation occurred was measured.

As a result, in any experimental section series, the dissolution of flocculent precipitation was observed with the addition of 0.8 U / ml or more of purified β-mannanase, and even when 6.7% of the threshold addition amount was added, It was found that β-mannanase did not affect the effect of eliminating flocculent precipitation, and it was considered that there was no synergistic effect. This result was consistent with the fact that the additive effect of both enzymes of Example 12 was observed.
[0046]
Example 14:Verification of effects in advanced tea leaves
It is known that the higher the tea leaves, the more polysaccharides that cause secondary precipitation. In addition, when high-grade tea leaves are used, the amount of enzyme added increases and protein-derived precipitates are likely to appear. Therefore, using the cellulase Y-NC and the β-mannanase crude enzyme solution derived from the Penicillium multicolor mch13-2 filed by the present inventors (Japanese Patent Application No. 11-330558), Shizuoka used in the above examples. “Yabukita Seed” produced in Shizuoka, which is more advanced than the tea leaves used for the evaluation of green tea precipitation in the first tea intermediate (tea leaf 1) Evaluation was performed. The evaluation test was carried out in a 10 ml size heat resistant test tube in the same manner as in the method described in Example 1. Note that tea leaf 2 is higher than tea leaf 1 and is a tea leaf with a large amount of precipitation.
The enzyme concentration is as shown in Table 12.

(Note) Cellulase Y-NC is represented by xylanase activity, and mch13-2-derived β-mannanase crude enzyme solution is represented by β-mannanase activity.
As a result of storage at 40 ° C. for 2 weeks, cellulase Y-NC cannot exert the effect of suppressing green tea precipitation, and protein-derived precipitates are produced even if the amount of the added enzyme is further increased. Therefore, in advanced tea leaves (tea leaves 2) The effect could not be obtained. In contrast, in the case of the β-mannanase crude enzyme solution, precipitation could be completely suppressed at 41 U / ml. Therefore, this β-mannanase crude enzyme solution was confirmed to have a green tea precipitation inhibitory effect even in advanced tea leaves (tea leaves 2). It is conceivable that the protein-derived precipitate is not produced because it contains a highly effective β-mannanase and hardly contains a contaminating enzyme. Moreover, it is considered that the specific activity of the β-mannanase itself is high and the amount of added protein is small. From the above results, it is considered that by using a β-mannanase crude enzyme agent containing β-mannanase as a main component, it is possible to produce a green tea beverage using high-grade tea leaves that have been considered difficult in the past.
[0047]
Example 15:Evaluation of taste
160g of Shizuoka Yabukita seed, Ichibancha middle-grade green tea leaf (tea leaf 1) was added to 5.6kg of ion-exchanged water at 65 ° C and extracted by standing for 5 minutes. ). After roughly filtering tea leaves using a 30 mesh and 120 mesh stainless steel filter, the extract cooled to about 20 ° C. is centrifuged (3000 rpm / minute, 10 minutes), and further filtered using a 200 mesh stainless steel filter. A clear liquid was obtained. In order to stabilize the quality, after diluting 3.7 times with ion-exchanged water, sodium ascorbate was added to a final concentration of 0.04%, and then sodium bicarbonate was adjusted to pH 6.25 after sterilization. Added to adjust. And as a crude enzyme agent having β-mannanase activity as a main component, a penicillium multicolor mch13-2 derived β-mannanase crude enzyme solution of the present inventors (Japanese Patent Application No. 11-330558) or cellulase as a comparative example. Using Y-NC, the enzyme treatment was performed at 30 ° C. (water bath) for 30 minutes. Then, it put into the 200 ml size heat-resistant glass bottle, and the retort sterilization process was performed, and the green tea drink was obtained.
The results are shown in Table 13 below.

(Note) Cellulase Y-NC is represented by xylanase activity, and mch13-2-derived β-mannanase crude enzyme solution is represented by β-mannanase activity.
The quality of the green tea extract was compared by conducting sensory tests with three experienced panelists on the evaluation of green tea precipitation inhibition when stored at 25 ° C. and 40 ° C. for 4 weeks and the flavor when stored for 1 week. The results are shown in Table 14. However, the same results were obtained when stored at 25 ° C and 40 ° C.

As described above, in cellulase Y-NC, the formation of precipitates was suppressed, but an enzyme-derived earthy odor had already occurred in the flavor at a low concentration. On the other hand, in the mch13-2-derived β-mannanase crude enzyme solution, precipitation could be suppressed and at the same time the rich taste increased and the tea feeling further increased. This clearly showed a quality improvement effect.
[0048]
【The invention's effect】
As described above, according to the present invention, a green tea beverage, in particular, a transparent sealed, that does not require a complicated process and has no turbidity or precipitation over a long period of time without losing the original flavor and taste of the green tea extract. A green tea beverage in a container can be provided.
Moreover, even if the green tea beverage is stored for a long period of time, it is possible to effectively suppress the occurrence of secondary precipitation, which has been inevitably seen during storage, and the tea feeling of the green tea beverage as a favorite beverage ( It is possible to provide a method for producing a high-quality green tea beverage and a product thereof, in which the original flavor and taste of green tea are further improved compared to conventional products.
Furthermore, β-mannanase is about 5 to 10 times more effective than xylanase, which has been said to have a precipitation-preventing effect, and as a result, in the case of an enzyme agent containing β-mannanase as a main component, a threshold value is added. Since the amount is small, there is no occurrence of miscellaneous taste such as “earthy odor” derived from the enzyme. Moreover, even when high-grade tea leaves are used, secondary precipitation (cotton-like precipitation) can be completely suppressed without causing protein-derived precipitation.

Claims (11)

A method for producing a green tea beverage with suppressed turbidity or precipitation, characterized in that the green tea extract is treated with β-mannanase or an enzyme containing β-mannanase as a main component. The method for producing a green tea beverage according to claim 1, wherein the green tea extract is treated with β-mannanase or an enzyme agent containing β-mannanase as a main component and an antioxidant is blended. The manufacturing method of the green tea drink of Claim 1 or 2 which performs an enzyme process at the temperature of 20-70 degreeC. The method for producing a green tea beverage according to any one of claims 1 to 3, wherein the pH adjusting agent is added at least once. It treats with the said enzyme agent whose (beta) -mannanase / xylanase activity ratio is 5 or more, The manufacturing method of the green tea drink of any one of Claims 1-4 characterized by the above-mentioned. The method for producing a green tea beverage according to any one of claims 1 to 5, wherein the treatment is performed with the enzyme agent having a β-mannanase / xylanase activity ratio of 20 or more. The manufacturing method of the green tea drink of any one of Claims 1-6 whose (beta) -mannanase is penicillium multicolor origin (beta) -mannanase. The method for producing a green tea beverage according to claim 7, wherein Penicillium multicolor is Penicillium multicolor mch13-2 strain (FERM BP-6831). Use of β-mannanase or an enzyme agent containing β-mannanase as a main component for producing a green tea beverage. Use according to claim 9, wherein the enzyme agent suppresses the turbidity or precipitation of the green tea beverage. The green tea drink obtained by the manufacturing method of any one of Claims 1-8, or the use of Claim 9 or 10.