番石榴採收後生理變化及其果膠甲基酯酶分離純化之探討

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論文名稱: 番石榴採收後生理變化及其果膠甲基酯酶分離純化之探討
研究生姓名: 郭孫憲
指導教授姓名: 江啟銘;吳明昌
出版年: 2007
學校名稱: 國立屏東科技大學
系所名稱: 食品科學系所
關鍵字: 中山月拔;採收後生理變化;果膠甲基酯酶;純化;Tongshan guava;postharvest physiology;pectin methyesterase;purification;
摘要: 番石榴為台灣常見之熱帶水果,種類甚多,其中中山月拔(Tongshan)為果汁工業生產芭樂果汁主要原料之一,除了具濃郁氣味外,果肉的軟化相當快速,因此,適合於加工生產,而軟化之主因可能為果膠甲基酯酶(pectin methyleaterase, PE)作用所致;本研究主要探討番石榴之”中山月拔”品種於採收後之熟成期間生理變化並找出番石榴PE的最適萃取條件及其特性表現。中山月拔於採收後在25±1℃貯藏下,其軟化速率相當快速,一天內硬度、脆度即減少50%,且於此後熟期間果實含水量及pH值方面則無明顯之改變,果皮顏色則由深綠轉變成淡黃色,果皮葉綠素含量則依後熟天數的增加而有逐漸下降之趨勢,果膠酯化度和總果膠含量則於前3天減少速率較為快速,隨後減少速率逐漸趨緩,纖維素酶(cellulase, CE)和聚半乳糖醛酸酶(polygalacturonase, PG)之活性於初期隨後熟天數而增加,而果膠甲基酯酶(PE)則於放置1天後達到最高活性表現,果膠酸解離酶(pectate lyase, PAL)則於第5天時始有活性的出現,隨後一直增加。而對於果膠甲基酯酶抑制劑(pectin methylesterase inhibitor, PEI)方面,則利用PVPP的添加得知番石榴中主要之PEI為多酚類物質(polyphenol)。另外,對於中山月拔PE之研究結果顯示利用0.5M NaCl、7%PVPP與pH 11.5之萃取液,並利用超過濾與離心輔助配合,相較於先前文獻可改善萃取率,所得之粗萃液以CM-Sepharose (pH 8.0)與DEAE-Sepharose (pH 5.0)之條件分別可分離出PE-1與PE-3,接著進一步以HM CL-AIS親和性管柱以及Sephacryl S-100膠過濾管柱層析可得PE-1與PE-3之純質,以SDS-PAGE鑑定其分子量皆為40.0 kDa,回收率分別為2.93 %和1.17 %,純化倍數為99.9及47.0,利用不同之pH緩衝溶液下經CM-Sepharose與DEAE-Sepharose推測出PE-1與PE-3之pI點分別為pH 9.6及3.5,而PE-3於pH 2.0時依然有活性表現,顯示出高耐酸性為其特點。The guava (Psidium guajava Linn.) is the common tropical fruit in Taiwan, there are several kinds of varieties here, the “Tongshan”is usually used for juice processing, for “Tongshan”is rich and distinct flavor, flesh soften quickly, the pectin methylesterase (PE) is the main enzyme for flesh softened, this study is to survey the physiochemical property and the change of soften enzyme during the guava ripening and the characteristic of these enzyme. The harvesting fruits were stored at 25±1℃, the flesh became soft quickly, lost over 50% just one day in firmness and crispness, there were not change on pH and water content during ripening, the color of the fruit peel turned from dark green to yellowish, total chlorophyll content reduced gradually, the degree of esterification on pectin and total pectin content decreased sharphy in the first day, following reduced slowly, the activity of cellulase and polygalacturonase increased gradually during ripening. The pectin methylesterase activity was highest in the first day during ripening, the pectate lyase activity wsa detected in the fifth day increased gradually, the PEI from “Tongshan” may be polyphenolics. The PE of “Tongshan”could be extracted with 0.5M NaCl, 7% PVPP at pH 11.5 and combined with ultrafiltration and centrifugation, the pure PE-1 and PE-3 could be gotten from the crude enzyme by CM-Sepharose (pH 8.0), DEAE-Sepharose (pH 5.0) respectively, and following with HM CL-AIS affinity column, Sephacryl S-100 gel filtration chromatogram, the molecular weight of both enzymes are about 40 kDa by SDS-PAGE, the recovery were 2.93 % and 1.17 %, and the purification fold wrer 99.9 and 47.0 respectively, the pI of PE-1 and PE-3 were 9.6 and 3.5 from CM-Sepharose and DEAE-Sepharose respectively, the PE-3 was acid resistant on pH 2.0.
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