JP7144478B2 - タンパク質加水分解物を用いたタンパク質-多糖類二重コーティング乳酸菌の製造方法 - Google Patents
タンパク質加水分解物を用いたタンパク質-多糖類二重コーティング乳酸菌の製造方法 Download PDFInfo
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- JP7144478B2 JP7144478B2 JP2020069218A JP2020069218A JP7144478B2 JP 7144478 B2 JP7144478 B2 JP 7144478B2 JP 2020069218 A JP2020069218 A JP 2020069218A JP 2020069218 A JP2020069218 A JP 2020069218A JP 7144478 B2 JP7144478 B2 JP 7144478B2
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Description
本出願は、2019年4月15日に出願された韓国特許出願第10-2019-0043610号の優先権を主張し、その開示は参照によりその全体が本明細書に組み入れられる。
(a)タンパク質水溶液にタンパク質加水分解酵素を処理して、タンパク質の加水分解率が45%~95%であるタンパク質加水分解水溶液を製造するステップ;
(b)上記製造されたタンパク質加水分解水溶液に乳酸菌培養のための糖成分及び窒素源成分を添加して殺菌した後、乳酸菌を接種して培養するステップ;
(c)上記培養して得た乳酸菌の発酵培養液から乳酸菌の菌体を回収するステップ;
(d)上記回収した乳酸菌の菌体に凍結保護剤及び多糖類水溶液を添加して、混合及び均質化するステップ;及び
(e)上記均質化された乳酸菌菌体水溶液を凍結乾燥させるステップ。
本発明において、乳酸菌のコーティングに使用されるタンパク質は、加水分解処理した後に使用する。
上記製造されたタンパク質加水分解水溶液に乳酸菌培養のための糖成分及び窒素源成分を添加して殺菌した後、乳酸菌を接種して培養する。
乳酸菌を培養した培養液から濾液を最大限多く除去して、固形分である乳酸菌菌体(Cell Mass)の比重が高い状態に濃縮する。乳酸菌菌体を稠密に濃縮するほど凍結乾燥乳酸菌の製造において次のような利点を有する:i)トレイ(Tray)当たりの分注量が減少し、蒸発させるべき水分の量も少なくなり、ii)凍結過程で浸透圧差による水分の流出により凍結速度が速く、iii)氷結晶が小さく均一であり、細胞形態の変化も小さくなり、凍結中の乳酸菌細胞膜の損傷が少なくなり、iv)凍結過程での変性も少なく生じる。
本発明の方法において、乳酸菌を凍結乾燥して乳酸菌の保存安定性を増進させることができる。凍結乾燥処理のために回収及び濃縮された乳酸菌菌体に凍結保護剤を添加して、混合及び均質化する。好ましくは凍結保護剤成分が添加された凍結保護剤水溶液を使用することができる。
本発明において、乳酸菌の凍結乾燥生存率、耐酸性、及び耐胆汁性を向上させるために、濃縮された乳酸菌を多糖類でコーティングすることができる。
上述したように、タンパク質コーティングされた乳酸菌菌体に凍結保護剤及び多糖類成分を添加し、混合及び均質化した乳酸菌菌体を凍結乾燥する。
1-1. 加水分解率の測定方法
乳酸菌二重コーティングに使用されるタンパク質源として脱脂粉乳(skim milk)、分離大豆タンパク(ISP)、脱脂粉乳及び分離大豆タンパク混合物に対して、タンパク質分解酵素による加水分解率を測定した。脱脂粉乳と分離大豆タンパクがそれぞれ異なる濃度で含まれた濃度別タンパク質水溶液を製造した。製造した濃度別タンパク質水溶液を攪拌器が装着された反応容器に入れて、60℃にて100RPMで懸濁及び均質化した後、製造した懸濁液に1N NaOHを添加して、pH8.2±0.2に調整した。下記表1、表2、及び表3に、脱脂粉乳、分離大豆タンパク、及び脱脂粉乳+分離大豆タンパク混合物の懸濁液をそれぞれ異なる濃度で製造した例を示す。
加水分解率S(%)=[ΔOD]/[始点OD]×100%
[ΔOD]=始点OD-終点OD
加水分解率I(%)=[Δppt]/[始点ppt]×100%
[Δppt]=始点ppt-終点ppt
加水分解率S&I(%)=[加水分解率S+加水分解率I]/2
脱脂粉乳の濃度及び酵素の濃度による脱脂粉乳の加水分解率を測定した結果は、下記表4に示す。
脱脂粉乳及び分離大豆タンパクの濃度による加水分解率の測定結果は、下記表6に示す。
* 使用酵素:プロテアーゼ(製品名:Alcalase 2.4L FG、製造社:Novozymes A/S Denmark)0.015%使用
2-1:脱脂粉乳の濃度別加水分解率による培養性実験
実施例1で製造したタンパク質加水分解水溶液に混合乳糖30kg、大豆ペプトン6kg、酵母エキス12kg、第二リン酸カリウム1.2kg、硫酸マグネシウム120g、L-アスコルビン酸600g、L-グルタミン酸240g、ポリソルベート-80 600gを溶解させて、最終液量を1,200Lにして、熱交換器(Alfalaval、スウェーデン)を用いて温度130℃、流量1,850L/hの条件で殺菌し、1.2KL容量の嫌気的発酵管に移送してStreptococcus thermophilus CBT ST3種菌5Lを接種した後、アンモニアでpH6.0を維持しながら13時間発酵させた。発酵後、脱脂粉乳及びタンパク質分解酵素の濃度別加水分解率による乳酸菌の培養性を測定した。乳酸菌培養性の測定は、希釈水9mLに培養液1mLを取ってボルテックス(vortexing)を実施した後、十進希釈法で生菌数を分析した。測定結果は、下記表7に示す。
脱脂粉乳と同様に実施例1で得た加水分解溶解液にブドウ糖24kg、大豆ペプトン6kg、酵母エキス18kg、酢酸ナトリウム1.2kg、クエン酸カリウム1.2kg、硫酸マグネシウム120g、L-システイン塩酸塩1.8kg、L-アスコルビン酸600g、ポリソルベート-80 1.2kgを溶解させて、最終液量を1,200Lにして、熱交換器(Alfalaval、スウェーデン)を用いて温度130℃、流量1,850L/hの条件で殺菌し、1.2KL容量の嫌気的発酵管に移送して Bifidobacterium breve CBT BR3種菌5Lを接種した後、アンモニアでpH6.5を維持しながら14時間発酵させた。発酵後、分離大豆タンパク及びタンパク質分解酵素の濃度別加水分解率による乳酸菌の培養性を測定した。乳酸菌の培養性の測定は、希釈水9mLに培養液1mLを取ってボルテックスを実施した後、十進希釈法で生菌数を分析した。測定結果は表8に示す。
実施例1で確保された脱脂粉乳及び分離大豆タンパク混合物の加水分解溶解液に結晶果糖36kg、酵母エキス36kg、第二リン酸カリウム2.4kg、酢酸ナトリウム6kg、硫酸マグネシウム1.2kg、硫酸マンガン6g、L-システイン塩酸塩1.2kg、L-アスコルビン酸1.2kg、ポリソルベート-80 2.4kg、精製塩7.2kgを溶解させ、最終液量を1,200Lにして、熱交換器(Alfalaval、スウェーデン)を用いて温度130℃、流量1,850L/hの条件で殺菌し、1.2KL容量の嫌気的発酵管に移送してLactobacillus acidophilus CBT LA1種菌5Lを接種した後、アンモニアでpH5.5を維持しながら20時間発酵させた。発酵後、脱脂粉乳、分離大豆タンパクの濃度別加水分解率による乳酸菌の培養性を確認した。乳酸菌の培養性の測定は、希釈水9mLに培養液1mLを取ってボルテックスを実施した後、十進希釈法で生菌数を分析した。測定結果は表9に示す。
3-1:脱脂粉乳の濃度別加水分解率による凍結乾燥生存率実験
実施例2-1で得た発酵液を4.0L/分間の流速で円筒型(Tubular Type)の高速遠心分離機(RPM15,000以上、G-force13,200以上)を用いて、菌体と残存タンパク質成分を沈積させて、コーティングしながら回収した。トレハロース3kg、マルトデキストリン1kg、マンニトール1kg、脱脂粉乳1kgから作製された凍結保護剤水溶液10Lを加圧殺菌して製造し、キサンタンガム(Xantan gum)20g、セルロース(Cellulose)20gを溶解させた多糖類水溶液10Lを加圧殺菌して製造した。次に、回収菌体と、上記製造した凍結保護剤水溶液及び多糖類水溶液をホイッパーが取り付けられた縦型混合機で、200RPMで撹拌して均質化し、-40℃の予備凍結庫で急速凍結した後、凍結乾燥機の棚温度を0℃から2時間単位で10℃ずつ段階的に昇温して、最終的に37℃の条件で凍結乾燥した。タンパク質分解酵素による加水分解の過程で半水溶性の残存タンパク成分は菌体を包括してタンパク質コーティングを形成し、キサンタンガム(Xantan gum)、セルロース(Cellulose)多糖類成分は、菌体間の結合によって非常に緻密な構造を有する菌塊を形成した。このとき、半水溶性のペプチド量が特定の量に比べて高いほど、菌が固まる傾向を示して培養性が低下し、適正量の半水溶性ペプチドは、凍結乾燥の過程で加えられる熱から菌体を保護して、優れた凍結生存率、加速安定性、耐酸性、耐胆汁性を示した。これによる試験結果は、下記表10に示す。
[凍結乾燥後の1g当たりの生菌数×凍結乾燥後の重量(g)]/[凍結乾燥前の1g当たりの生菌数×凍結乾燥前の重量(g)]×100%
実施例3-1で説明された実験方法によって、実施例2-2で得た発酵液を使用して、分離大豆タンパクの濃度別加水分解率による凍結乾燥生存率実験を行った結果を、下記表11に示す。
[凍結乾燥後の1g当たりの生菌数×凍結乾燥後の重量(g)]/[凍結乾燥前の1g当たりの生菌数×凍結乾燥前の重量(g)]×100%
実施例3-1で説明された実験方法によって、上記実施例2-3で得た発酵液を使用して、脱脂粉乳及び分離大豆タンパクの濃度別加水分解率による凍結乾燥生存率実験を行った結果は、下記表12に示す。
[凍結乾燥後の1g当たりの生菌数×凍結乾燥後の重量(g)]/[凍結乾燥前の1g当たりの生菌数×凍結乾燥前の重量(g)]×100%
乳酸菌をコーティングしていない場合、タンパク質コーティングのみ行った場合、二重コーティングした場合に、加速安定性、耐酸性及び耐胆汁性実験を行った。その結果は、下記表13に記載する。
* XG:キサンタンガム
* 加速安定性:希釈水9mLに試料1gを取ってボルテックスを実施した後、十進希釈法で初期生菌数を分析した。さらに試料を40℃で培養した後、1週間単位で4週間まで生菌数分析を行い、初期生菌数に対する生存率を確認した。
* 耐酸性:1M HCl溶液で補正したpH2.1 MRSブロス(broth)溶液9.9mlにそれぞれの試料0.1gを溶解した後、37℃の温度を維持しながら0時間、2時間目の生菌数を分析して、0時間に対する生存率を確認した。
* 耐胆汁性:0.5%オックスゴール(oxgall)が添加されたMRSブロス溶液9.9mlにそれぞれの試料0.1gを溶解した後、37℃の温度を維持しながら0時間、2時間目の生菌数を分析して、0時間に対する生存率を確認した。
Claims (11)
- タンパク質-多糖類コーティングの二重コーティングを有する乳酸菌の製造方法であって、前記方法は、次のステップ:
(a)脱脂粉乳、分離大豆タンパク(ISP)、又は脱脂粉乳及び分離大豆タンパク(ISP)の混合物を含むタンパク質水溶液にタンパク質加水分解酵素を処理して、タンパク質加水分解水溶液を製造するステップであって、
前記脱脂粉乳を含むタンパク質水溶液に対する、前記脱脂粉乳の濃度は2.0重量%~5.0重量%であり、前記タンパク質加水分解酵素の濃度は0.003重量%~0.005重量%であり、
前記分離大豆タンパク(ISP)を含むタンパク質水溶液に対する、前記分離大豆タンパク(ISP)の濃度は0.45重量%~0.75重量%であり、前記タンパク質加水分解酵素の濃度は0.015重量%~0.025重量%であり、又は
前記脱脂粉乳及び分離大豆タンパク(ISP)の混合物を含むタンパク質水溶液に対する、前記脱脂粉乳の濃度は1.5重量%~2.0重量%であり、前記分離大豆タンパク(ISP)の濃度は0.30重量%であり、前記タンパク質加水分解酵素の濃度は0.015重量%であり;
(b)前記製造されたタンパク質加水分解水溶液に乳酸菌培養のための糖成分及び窒素源成分を添加して殺菌した後、乳酸菌を接種して培養するステップ;
(c)前記培養して得た乳酸菌の発酵培養液から乳酸菌の菌体を回収するステップ;
(d)前記回収した乳酸菌の菌体に凍結保護剤水溶液及び多糖類水溶液を添加して、混合及び均質化するステップ;及び
(e)前記均質化された乳酸菌菌体水溶液を凍結乾燥させるステップ、
を含み、
前記ステップ(a)における前記脱脂粉乳の加水分解率は、以下のi)の方法を使用して測定した百分率値であり、
前記ステップ(a)における前記分離大豆タンパク(ISP)の加水分解率は、以下のii)の方法を使用して測定した百分率値であり、
前記ステップ(a)における前記脱脂粉乳及び分離大豆タンパク(ISP)の混合物の加水分解率は、以下のi)及びii)の方法を使用して測定した百分率値の平均値であり、
i)前記タンパク質加水分解酵素を処理する前のタンパク質水溶液の吸光度の測定値(始点OD)と前記タンパク質加水分解酵素を処理した後のタンパク質水溶液の吸光度の測定値(終点OD)との差であるΔOD値を前記始点OD値で割った値の百分率値、及び、
ii)前記タンパク質加水分解酵素を処理する前のタンパク質水溶液を遠心分離して得る沈殿物の重さの測定値(始点ppt)と前記タンパク質加水分解酵素を処理した後のタンパク質水溶液を遠心分離して得る沈殿物の重さの測定値(終点ppt)との差であるΔppt値を前記始点ppt値で割った値の百分率値、
前記ステップ(a)における前記脱脂粉乳の加水分解率は、75%~92%であり、
前記ステップ(a)における前記分離大豆タンパク(ISP)の加水分解率は、61%~80%であり、
前記ステップ(a)における前記脱脂粉乳及び分離大豆タンパク(ISP)の混合物の加水分解率は、76%~85%である
ことを特徴とする、タンパク質-多糖類コーティングの二重コーティング層を有する乳酸菌の製造方法。 - 前記ステップ(a)における前記脱脂粉乳の加水分解率は、80%~90%であることを特徴とする、請求項1に記載のタンパク質-多糖類コーティングの二重コーティング層を有する乳酸菌の製造方法。
- 前記ステップ(a)における前記分離大豆タンパクの加水分解率は、70%~80%であることを特徴とする、請求項1に記載のタンパク質-多糖類コーティングの二重コーティング層を有する乳酸菌の製造方法。
- 前記ステップ(a)における前記脱脂粉乳及び分離大豆タンパクの混合物の加水分解率は、78%~83%であることを特徴とする、請求項1に記載のタンパク質-多糖類コーティングの二重コーティング層を有する乳酸菌の製造方法。
- 前記ステップ(b)の乳酸菌培養のための糖成分は、混合乳糖、果糖、砂糖(Sucrose)、及びブドウ糖からなる群より選択された1種以上であることを特徴とする、請求項1から4のいずれかに記載のタンパク質-多糖類コーティングの二重コーティング層を有する乳酸菌の製造方法。
- 前記ステップ(b)の乳酸菌培養のための窒素源成分は、酵母エキス又は大豆ペプトンであることを特徴とする、請求項1から5のいずれかに記載のタンパク質-多糖類コーティングの二重コーティング層を有する乳酸菌の製造方法。
- 前記ステップ(d)の凍結保護剤は、トレハロース、マルトデキストリン、マンニトール、及び脱脂粉乳からなる群より選択された1種又はこれらの2種以上の混合物であることを特徴とする、請求項1から6のいずれかに記載のタンパク質-多糖類コーティングの二重コーティング層を有する乳酸菌の製造方法。
- 前記ステップ(d)の多糖類は、ガム(gum)類、水溶性食物繊維、難消化性マルトデキストリン、不溶性食物繊維、デンプン、レバン(Levan)、及び抵抗性デンプンからなる群より選択された1種以上であることを特徴とする、請求項1から7のいずれかに記載のタンパク質-多糖類コーティングの二重コーティング層を有する乳酸菌の製造方法。
- 前記乳酸菌は、ストレプトコッカス(Streptococcus)属、ラクトコッカス(Lactococcus)属、エンテロコッカス(Enterococcus)属、ラクトバチルス(Lactobacillus)属、ペディオコッカス(Pediococcus)属、リューコノストック(Leuconostoc)属、ワイセラ(Weissella)属、及びビフィドバクテリウム(Bifidobacterium)属からなる群より選択された一つ以上の菌株であることを特徴とする、請求項1から8のいずれかに記載のタンパク質-多糖類コーティングの二重コーティングを有する乳酸菌の製造方法。
- 請求項1から9のいずれか一項に記載の方法によって製造された、タンパク質-多糖類コーティングの二重コーティングを有する乳酸菌。
- 請求項1から9のいずれか一項に記載の方法によってタンパク質-多糖類コーティングの二重コーティングを有する乳酸菌を製造し、製造されたタンパク質-多糖類コーティングの二重コーティングを有する乳酸菌を食品組成物に添加する、乳酸菌を含む食品組成物の製造方法。
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