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1 Extraction of Trehalose from Saccharomyces cerevisiae by High-Pressure Treatment Miwako KINEFucHI,* Akira YAMAZAKI* and Kouzi YAMAMOTO** * Research Institute, Echigo Seika Co., Ltd. (1-4-5, Gofukumachi, Nagaoka 940, Japan) * * Department of Bioengineering, Nagaoka University of Technology (1603-1, Kamitomioka-cho, Nagaoka , Japan) Commercially available yeasts normally contain approximately 10% dry weight of trehalose. For the purpose of obtaining liquid trehalose economically and safely, without using heat or an organic solvent, we used a high-pressure treatment to extract the trehalose, with inactivating trehalase. Our results showed that trehalase is inactivated by the application of a minimum of 700 MPa and that trehalose is not hydrolyzed even at pressures as high as 1500 MPa. Using high-pressure treatment, therefore, we have succeeded in extracting trehalose from yeast at 12% dry weight when 700 MPa is applied for 10 min at 30 Ž. It has been confirmed that high-pressure treatment is very effective for simultaneously inactivating trehalase and extracting trehalose.
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3 Fig. 1. Relationship between trehalase activity and pressure under different temperatures. A pressure was applied to a trehalase solution (ph 6.8) for 10 min. The trehalase activity decreased with increasing pressure, and trehalase was finally inactivated by high-pressure treatment of 700 MPa.
4 Fig. 2. Hydrolysis of trehalose by high-pressure treat ment. Pressure was applied to a 0.15 M trehalose solution for 10 min. The trehalose was not hydrolyzed even when the pressure was increased to as high as 1500 MPa. Fig. 3. Relationship between yield of trehalose and applied pressure. Yeast was subjected to heat- and pressure-application treatment. The yield of trehalose increased with increasing applied pressure, and attained equilibrium when the applied pressure was 700 MPa. Fig. 4. Extractability of trehalose from yeast after treatment at various pressures. The trehalose weight in the extracted liquid increased with increasing applied pressure and attained equilibrium at 700 MPa.
5 高圧 処理 を利 用 した酵 母 か らの トレハ ロース の抽 出 Fig. 5. Microscopic photograph of pressurized 241 yeasts. A, non-treatment; B, 700 MPa. Each with 0.1% methylene blue dyeing. The pressurized cells changed shape slightly. 処 理 を し た 細 胞 の 顕 微 鏡 観 察 の 結 果 を 示 し た もの で あ る.大 き く割 れ た り 引 き ち ぎ れ た と い う形 の もの は 見 受 け ら れ な か っ た.し り,圧 か し酵 母 は 多 角 形 に 変 型 し て お 力 解 除 さ れ た 後 で も細 胞 が 一 度 強 く圧 縮 さ れ た 痕 跡 を示 し て い る. Fig.6に 示 した よ うに酵 母 は 常 圧 では ポ ロ ポ ロ した 粒 塊 で あ る.し か し圧 力 を 上 昇 さ せ て い く と500MPa 付 近 か ら 流 動 化 し700MPaで 験 で 明 らか に な っ た.圧 液状 とな る こ とが 本実 力 解 除 後 の細 胞 の 大 きさは ほ とん ど変 わ ら な い こ と を 考 え る と,細 胞 液 が 浸 出 し, 空 洞 が あ ち こ ち に で き て い る 細 胞 が 残 っ た と推 定 さ れ た. 3)圧 力 処 理 の有 効 性 生 体 か ら有 用 物 質 を分 離 す る 固 液 抽 出 の 工 程 を 考 え た 場 合,抽 体(生 出 速 度 が 大 き な 課 題 と な る.抽 体)内 部 で の 抽 質(ト 出速 度 は 固 レ ハ ロ ー ス)の 拡 散 と生 体 外 部 の 境 膜 拡 散 と に 分 け て 考 え な け れ ば な ら な い が, 律 速 は 生 体 内 部 の 拡 散 速 度 で あ る と考 え ら れ る. も し生 体 内 が ス ポ ン ジ の よ う に 多 孔 質 で あ れ ば 固 体 Fig. 6. Photograph of non- and pressurized a yeast fluid. A yeast solid changed treatment at 700 MPa. to a yeast fluid yeasts and の 形 状 を平 板 か 球 と して 扱 う こ とが で き る と い わ れ て い る.そ by pressure の 場 合,表 え ら れ る2D. 抽 残 率=1一 細 胞 内 部 の 組 織 が 損 傷 し細 胞 液 が 浸 出 しや す くな っ て い る こ と を うか が わせ る. Fig.5は,生 酵 母 を 脱 気 包 装 し て700MPaの 面 濃 度 は 外 部 液 濃 度 と等 し く一 定 に 保 た れ る と仮 定 す れ ば 抽 出 残 率 と抽 出 率 は 次 式 で 与 才由出 率 =8/π2 Σ1/(2n+1)2exp[-(2n+1)2 π2det/4δ2], 圧 力 (Σ=0か ら )
6 1) G. M. BROWN, D. C. ROHRER and C. A. BEEVERS Acta Cryst, B28, (1972). 3) Y. ODA, K. UNO and S. OHTA : Appl. Environ. Microbiol., (1986). 5) R. P. ADAMS, E. KENDALL and K. K. KARTHA. Bio chem. Sys. Ecol., 18, (1990). 8) J. H. CROWE, and L. M. CROWE : Science, 223, (1984). 21) A. B. NEWMAN : Trans. Am. Inst. Chem. Eng., 27, 310 (1931).
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