創傷與抑制褐化處理對結球萵苣主莖切面褐化與對苯丙氨酸氨基裂解酶活性之影響

Abstract

結球萵苣 (Lactuca sativa L. var. capitata) 為菊科萵苣屬作物，是國內冬季重要外銷蔬菜。結球萵苣採收時由主莖基部切下，此切面於創傷後轉為紅褐色，稱為底部褐化 (butt discoloration)，影響採後外觀品質。已知創傷會引起組織中苯丙胺酸氨基裂解酶 (phenylalanine ammonia lyase, PAL) 活性上升，造成酚類物質累積，進而被多酚氧化酶氧化成醌類，並聚合為褐化物。已有研究指出以熱處理、正丁醇、醋酸及含硫氫基化合物處理，均具抑制萵苣傷口褐化之效果。本研究探討不同處理對萵苣主莖褐化及對PAL活性之影響，以瞭解各種抑制褐化處理之效果與組織中PAL活性變化之關係。結球萵苣主莖切片於5℃中其PAL活性於創傷後2小時即開始上升，至48小時達活性高峰，隨後快速下降；切面片褐化於創傷後48小時出現，至96小時明顯褐化。主莖切片經50℃熱水浴處理90秒能有效抑制褐化發生，切片之PAL活性於熱處理後120小時內均無增加。正丁醇 (1-butanol) 為磷脂酶D (Phospholipase D) 之專一性抑制物，可抑制植物由創傷引起之磷脂訊息傳遞路徑。主莖於創傷後立即以3%正丁醇處理5分鐘，可有效延緩褐化並及抑制PAL活性上升，於創傷後兩小時施用3%正丁醇則無法抑制褐化，亦無法完全抑制PAL活性上升，顯示創傷訊息在2小時內即可導致PAL之生成。主莖在創傷後以1.5%醋酸浸泡1分鐘、2%醋酸浸泡30秒或以3%醋酸浸泡5秒，均可有效維持切片原色澤，亦完全抑制PAL活性之上升。主莖於創傷後12小時施用3%醋酸5秒，仍可有效抑制褐化，並完全抑制PAL活性上升；表示醋酸應作用於PAL酵素本身，使其失去作用。以10%醋酸鈉溶液浸泡5分鐘亦具同樣抑制效果。主莖創傷後以1%、2%及3%之Cysteine (Cys)、L-cysteine hydrochloride (CysH) 及N-acetyl cysteine (NAC) 等含硫氫基化合物處理後，切面之色澤變化與PAL活性出現時間有關。以3% CysH處理之切片其切面色澤不變，其PAL活性完全受到抑制；以2% CysH處理之切片，其切面在創傷後5天變為黃綠色，其PAL活性在創傷後3天開始上升，第4天達到高峰。在創傷後2小時以3% CysH處理切片，亦具有抑制褐化及PAL活性之效果，但是延後至6及12小時處理，無法抑制切面變色，其PAL活性於創傷後3天後開始上升。以Cys處理之切片，1%及2%於創傷後4天變為黃綠色，其PAL活性在第3天後開始上升，3%於創傷後5天變為黃綠色，PAL活性在第4天後開始上升。經NAC處理之切片，1%於創傷後3天開始出現褐化及黃綠色，其PAL活性在切面變色前上升，且達到與對照組相似之最高活性。於創傷後2、6或12小時處理3% NAC，抑制褐化效果隨處理時間延後而遞減，PAL活性開始上升之時間則依序提前。由含硫氫基化合物處理結果可知，此類化合物無法完全抑制結球萵苣主莖切面變色，原因為無法完全抑制PAL活性之上升；而創傷後切面變色的出現時間與組織中PAL活性之增加有關。以硫酸銨沈澱法進一步純化PAL，主要的活性集中在40 - 60%飽和濃度之間，純化程度為粗萃取液的3倍。將純化後PAL與80 mM醋酸反應，可完全抑制PAL活性，且此抑制為不可逆。

Iceberg lettuce (Lactuca sativa L. var. capitata ), of the family Asteraceae, is an important cold-season crop for exportation in Taiwan. Iceberg lettuce is harvested at the bottom of stem and the cut surface turns from white to reddish-brown after wounding. This phenomenon called butt discoloration and it reduces the quality of lettuce appearance during postharvest. Wounding of plant tissues usually induces the activity of phenylalanine ammonialyase (PAL) and results in the accumulation of phenolic compounds, which are oxidized to quinones and eventually condense spontaneously to form brown pigments. Researchs indicated that heat shock, 1-butanol, acetic acid and sulfhydryl compounds treatments can prevent cut surface from browning. Therefore, inhibiting the activity of PAL is considered as the way to alleviate butt discoloration. The objective of this study is to study the influence of different treatments on wounded stem disk browning and PAL activity. When stem disks are storage at 5℃, PAL activity in untreated stem disks started to increase at 2 hours, and finally reached its peak at 48 hours. At the same time, browning of stem disks appeared one day after the rise of PAL activity. Heat shock treatment at 50℃ for 90 seconds could inhibit disks browning effectively by keeping PAL activity at the low level within 120 hours after wounding. 1-Butanol is the specific inhibitor of Phospholipase D which could suppress the wounding signal. Stem disks treated with 3% 1-butanol for 5 minutes immediately after wounding could delay the browning, whereas the treatment at 2 hours after wounding fail to inhibit tissue browning. The result suggested that wounding signal is triggered in 2 hours and lead to formation of PAL. Stem disks soaked with 1.5% acetic acid solution for 1min, 2% for 30 sec, or 3% for 5 sec could keep appearance as white as original and repress PAL activity in the disks. Stem disks soaked with 3% acetic acid solution for 5 sec at 12 hours after wounding could effectively inhibit browning and maintain low PAL activity in the disks. It reveals that acetic acid directly interact with PAL to inhibit enzyme activity. 10% acetic sodium solution treatment is the same effect as acetic acid solution treatment. The stem disks treated with sulfhydryl compounds, including Cysteine (Cys), L-cysteine hydrochloride (CysH), and N-acetyl cysteine (NAC) at the concentration of 1%, 2%, and 3%, the color of wounded stem disks are associated with PAL activity. Treatment of 3% CysH could maintain the appearance of stem disks and keep the PAL activity at low level. In the treatment of 2% CysH, the color of wounded stem disks turned green-yellowish. The activity of PAL increased at 3 days after wounding and reached its peak at 4 day. In the treatment of 3% CysH at 2 hours after wounding, browning and PAL activity were completely repress. However, cut surface browning could not be inhibit in the treatment of 3% CysH at 6 or 12 hours after wounding. Meanwhile, PAL activity increased at 3 days after wounding. In the treatment of 1% and 2% Cys, the color of wounded stem disks turned green-yellowish at 4 days after wounding and PAL activity increased at 3 days after wounding. In the treatment of 3% Cys, the color of wounded stem disks turned green-yellowish at 5 days after wounding and PAL activity increased at 4 days after wounding. The appearance of wounded stem disks treated with 1% NAC are brown and green-yellowish at 3 days after wounding; besides, the PAL activity rises ahead of color alteration and reach to the highest activity similar to control treatment. In the treatment of 3% NAC at 2,6 or 12 hours after wounding, the effect of browning inhibition decreased as the treatment time lengthened, the initiation time of PAL activity increase shifts earlier. The result of sulfhydryl compounds treatments suggest that they only partially inhibited color alteration of iceberg lettuce cut surface because PAL activity can not be completely inhibited. The appearance of color alteration after wounding is correlated with the increase of PAL activity in stem tissuse. The PAL was purificated by 40-60% ammonium sulfate saturation, and its purification is three times than crude extract. Adding 80mM acetic acid to purified PAL can irreversiblely inhibit the activity of PAL.