十字花科蔬菜中硫醣苷含量與芥子酶特性之研究

Abstract

十字花科蔬菜中普遍具有硫醣苷類（glucosinolates）之植物化合物，硫醣苷在經芥子酶（myrosinase, thioglucoside glucohydrolase；EC 3.2.3.1）水解後會生成包括異硫氰酸酯在內之多種化合物，而異硫氰酸酯是少數目前已被研究證明具有預防癌症發生之生物活性物質。 本試驗之目的為：1. 分析國人日常食用之數種十字花科蔬菜中之硫醣苷含量以及該硫醣苷經添加芥子酶水解後之異硫氰酸酯生成量。2. 探討採後貯藏環境對花椰菜中硫醣苷含量及異硫氰酸酯生成量之影響。3. 瞭解芥子酶之萃取條件及其酵素基本特性。 在從市場購得之蘿蔔、花椰菜、芥菜、青花菜、甘藍、白菜、結球白菜、芥藍、豆瓣菜等九種蔬菜中，硫醣苷含量以豆瓣菜的567.40 μmole / 100g-FW最高，青花菜的188.02 μmole / 100g-FW最低。各蔬菜中之硫醣苷經芥子酶水解後之異硫氰酸酯生成量則以甘藍的96.89 μmole / 100g-FW最高，芥菜的21.57 μmole/100g-FW最低；硫醣苷水解後生成異硫氰酸酯的比例約在5 ％-28 ％不等。不同品種甘藍間之硫醣苷含量無明顯差異；但‘永明’甘藍之異硫氰酸酯生成量明顯高於‘夏峰一號’，而‘夏山’與‘初秋’之異硫氰酸酯生成量相似，介於前兩者中間。花椰菜不同部位之硫醣苷含量無顯著差異；但花蕾部位異硫氰酸酯生成量明顯大於花梗及莖部之異硫氰酸酯生成量。 花椰菜於0 ℃、5 ℃、10 ℃、15 ℃等不同溫度下貯藏7天過程中，貯藏之前3天硫醣苷含量會上升，之後又減少。貯藏於不同溫度下之花椰菜，其硫醣苷含量無顯著差異。整體而言，硫醣苷水解後之異硫氰酸酯生成量，以貯藏前最高，貯藏前3天含量緩慢下降，第3天後又緩慢上升；但此貯藏期間之異硫氰酸酯生成量之變化在統計上無顯著差異。貯藏於高相對濕度能維持較高的硫醣苷含量；相對濕度高低對貯藏前3天之異硫氰酸酯生成量無顯著影響，但至第5天及第7天時，低相對溼度組之異硫氰酸酯生成量，約為高相對溼度組的2倍。 蘿蔔種子之芥子酶粗萃取液，以飽和硫酸銨劃分法進行部份純化，結果以60-90 ％具有最高的比活性（681.11 U g-1）。利用60-90 ％飽和硫酸銨劃分之蘿蔔芥子酶Km值為0.1 mM，而芥菜芥子酶Km值為0.22 mM，蘿蔔種子每克總活性高於芥菜種子；蘿蔔種子及芥菜種子以去離子水24小時浸潤處理後，浸潤後種子之總活性低於乾燥種子。比較蘿蔔及芥菜之芥子酶在不同溫度中反應1小時之活性，以50 ℃中具有最高之芥子酶活性，在60 ℃時活性降為83 %，70 ℃下之活性為零。蘿蔔及芥菜之芥子酶其最適反應酸鹼值（pH）皆為5.0。 於芥子酶之酵素特性反應液中添加不同濃度的抗壞血酸（0.1、0.5、1.0 mM）可以增加芥子酶之Vmax及Km值；利用基質濃度倒數（1/A0）及反應速度倒數（1/V0）作圖，會得到一系列平行的直線，此結果顯示抗壞血酸活化芥子酶模式屬於一種很特別的無競爭型活化類型（uncompetitive activation），與Shikita等人（Biochem. J. （1999）341: 725-732）之觀察結果相同。

Glucosinolates(GS) are a group of phytochemicals that present widely in cruciferous vegetables. The hydrolysis of glucosinolates by myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) results in a number of compounds, including isothiocyanates. Isothiocyanates(ITC) have been recognized in many studies as one of the few bioactive compounds that are associated with a reduced incidence of cancer. The objects of this study include: 1. To determine the total GS contents in crucifer vegetables that are commonly consumed by our people, and the amount of ITC formed after these GS were treated with an exogenous myrosinase. 2. To study the effect of storage environments on total GS contents and ITC formation in cauliflower. 3. To study extraction methods of myrosinase and some characteristics of this enzyme. Nine vegetables, including Chinese radish, cauliflower, Chinese mustard, broccoli, cabbage, Pai-tsai, Chinese cabbage, Chinese kale and water cress, were obtained from local market for analysis. Among them, water cress had the highest GS content of 567.40 μmole/100g-FW, and broccoli had the lowest of 188.02 μmole/100g-FW. With regard to the ITC formed after its GS was hydrolyzed with myrosinase, Cabbage had the highest yield of 96.89 μmole/100g-FW and Chinese mustard had the lowest yield of 21.57 μmole/100g-FW. The conversion ratio of ITC from GS was between 5 – 28%. There were no differences in GS contents among four cultivars of cabbage. However, there were differences among the ITC yields; ‘Yungming’ cabbage was the highest and ‘Siafong No. 1’ was the lowest, ‘Chuchiou’ and ‘Siashan’ cabbages were in the middle with similar values. There were no significant differences among the various parts of cauliflower heads, but the florets had higher ITC yields than that of the pedicels and the stem. Cauliflower heads were stored at 0, 5, 10 and 10℃ for 7 days and their GS contents and ITC yields were determined. The GS contents increase during the first 3 days of storage then declined and there were no differences among temperature treatments. The ITC yields were highest at the beginning, declined for 3 days then went up slightly; statistically, there were no differences among these values. Storage in high RH maintains higher GS contents, relative humidity had no effect on the ITC yield during the first 3 days of storage, but on the 5th and 7th day the ITC yield of low RH treatment was twice of the high RH treatment. Crude extracts of myrosinase from Chinese radish seeds were partial purified with ammonium sulfate precipitation. The highest enzyme activity was found in the 60 – 90% cut with 681.11U g-1 . The Km values of myrosinase from seeds of Chinese radish and Chinese kale was 0.1 mM and 0.22 mM respectively. The total myrosinase activity extracted from the seeds of Chinese radish was higher than that from the seed of Chinese kale. Imbibition of the seeds of both Chinese radish and Chinese kale for 24 hours before extraction resulted in lower extractable myrosinase activity. The optimum temperature for myrosinase activity was determined after incubation of reaction mixture at various temperature for 1 hour. Myrosinases from seeds of both Chinese radish and Chinese kale displayed highest activity at 50℃; the activity declined to 83% at 60℃ and had zero activity at 70℃. The optimum pH for myrosinase from both seeds of Chinese radish and Chinese kale were 5.0. Addition of 0.1, 0.5, 1.0 mM ascorbic acid to myrosinase reaction mixture resulted an increase in both the Vmax and the Km values. A series of parallel lines were obtained when the reciprocal of substrate (sinigrin) concentrations were plotted against the reciprocal of reaction rates at various ascorbic acid concentration. This result indicated that the activation of myrosinase by ascorbic acid follows an unusual model of “uncompetitive activation”, which is the same as reported by Shikita et al. (Biochem. J. 1999. 341:725-732).