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標題: 麴菌蛋白酶之特性分析及其應用於牛肉熟成之探討
Characterization of protease from Aspergillus oryzae and its application on tenderization in beef
作者: 石嘉雯
Shih, Chia-Wen
Contributors: 劉登城
動物科學系所
關鍵字: 蛋白酶熟成嫩化
Aspergillus oryzae;protease;tenderization
日期: 2012
Issue Date: 2013-11-18 14:58:52 (UTC+8)
Publisher: 動物科學系所
摘要: 麴菌 (Aspergillus oryzae) 為傳統上釀造醬油、醬菜或酒類之菌元,其在發酵過程中會產生澱粉酶、蛋白酶等酵素,這些酵素近年來廣泛地應用於食品、藥品及其他產業中;而肉品加工業中,牛肉之嫩度為一重要指標,許多研究指出牛後腿肉具有使消費者難以接受之硬度,因此需另外施以物理性或化學性之嫩化方式來提升其嫩度,而先前文獻指出於肉品中添加外源性酵素可有效地促使肌原纖維與膠原蛋白降解,進而提升肉品嫩度並產生風味物質。故本篇試驗將萃取麴菌之蛋白酶且分析其特性,後於牛後腿肉中添加衍自麴菌之不同酵素活性 (20與40 U) 之中性蛋白酶,再將其放置於10 ℃與15 ℃下進行熟成,之後探討牛後腿肉在熟成期間其嫩度與品質之變化。
結果顯示:麴菌接種至固態基質中進行發酵,於發酵第72小時產生之蛋白質濃度、蛋白酶活性及蛋白酶比活性最高,且其蛋白酶於酸鹼值 pH 7 與溫度50 ℃之作用環境下具有最佳活性;另此蛋白酶在酸性 (pH 5) 與中溫 (30 與 40 ℃) 環境下穩定度佳,而以 SDS-PAGE 分析麴菌粗酵素液之分子量大小可發現,其在35 kDa 有明顯之環帶產生;另外,在牛後腿肉中添加麴菌粗酵素液之分析試驗中可發現,對照組及各處理組之滴液損失率、烹煮失重、總生菌數、乳酸菌數、 MFI 及 VBN 值皆隨熟成時間增加而提升,40 U 與 20 U 處理組之 VBN 值與 MFI 於各時間點皆較對照組高,且在15 ℃熟成之處理組上升趨勢較10 ℃之處理組快,另於 pH 值試驗中,VBN 值雖逐漸上升,但由於乳酸菌之生長,因此使對照組與蛋白酶處理組於熟成期間 pH 值皆小於 pH 6,而於截切值、截切能量測定與感官品評試驗中可發現,添加麴菌粗酵素液處理組至熟成後期其截切值與截切能量顯著較對照組低,而感官品評嫩度評分則顯著高於對照組,且在熟成溫度15 ℃時有較明顯之效果,另對照組與各處理組在其他感官品評之測試項目中皆無顯著差異,而於 SDS-PAGE 試驗中可明顯觀察到 40 U 處理組在熟成後期 (於10 ℃與15 ℃下分別熟成14天與7天) 牛肉之肌原纖維與肌漿蛋白質被降解之現象,且以掃描式電子顯微鏡觀察肌纖維之顯微結構可發現,添加蛋白酶之處理組相較於對照組其肌纖維有明顯斷裂之情形。綜觀上述,麴菌所產生之蛋白酶應用於牛後腿進行熟成嫩化,可快速且有效地降解肌原纖維與肌漿蛋白質,促使牛肉嫩度提升,且對肉品無不良影響,因此麴菌粗酵素液具有應用於肉品嫩化之潛力。
Aspergillus oryzae are wildly utilized in the production of protease and amylase in addition to the production of traditional fermented foods such as soy sauce and beverages. Tenderness is an important driver of beef consumer satisfaction and as such has been extensively investigated. Many reports indicated a high level of unacceptably tough retail cuts originating from beef round. There are several means for tenderizing meat, both chemically and physically. Treatment with proteolytic enzymes is one of the popular methods for meat tenderization, and exogenous enzymes added to meat causing degradation to both myofibrillar and collagen proteins. The aim of this study was to investigate the characterization of protease from A. oryzae and evaluate the tenderizing effect of beef rounds.
The results showed that the maximum protein concentration, protease activity and specific amount protease of A. oryzae was obtained at 72 hours of cultivation. The protease from A. oryzae had an optimum pH of around 7 and its optimum temperature was approximately 50 ℃. In addition, the protease were quite stable at the acidic condition and around 30-40 ℃. SDS-PAGE of protease showed the molecular mass was 35 kDa. In the drip loss, cooking loss, total plate count, lactic acid bacteria count, MFI and VBN value determination of the beef, all treatments were increased with aging time at 10 and 15 ℃. In addition, the MFI and VBN value of the treatments with protease from A. oryzae (20 U and 40 U) were higher than control during aging. On the other hand, the VBN value of all treatments increasing gradually, but due to the growth of lactic acid bacteria, thus the pH value of all treatments were less than pH 6 during aging period. In the analysis of peak force, shearing energy and sensory evaluation, a significant reduction in peak force values and shearing energy values were observed in all protease-treated samples compare to control, and the protease-treated samples also received better scores for tenderness. SDS-PAGE of the myofibrillar protein and sarcoplasmic protein also revealed extensive proteolysis and reduction in number of protein bands in the treatments with protease from A. oryzae (20 U and 40 U). The microstructure of muscle fibers showed loss of muscle fiber structure only in protease-treated samples. In conclusion, the protease from A. oryzae degraded the myofibrillar protein and sarcoplasmic protein of meat, and it is the main factor contributing to the development of tenderness. Therefore, the protease from A. oryzae could be of potential to improve tenderness of meat.
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