- 學位論文
轉麩胺酸醯胺基酶處理對河粉膠體物性影響之研究
Modification in physical properties of rice gel by microbial transglutaminase
摘要
微生物轉麩胺酸醯胺基酶（Microbial transglutaminase； MTGase）催化蛋白質分子間或分子內形成ε-（γ-glutamyl）lysine共價鍵，可用來轉變分子結構以改善食品中蛋白質性質。本研究直接採用市售MTGase粗酵素粉末，經過加水萃取，此粗酵素液先用50~80%硫酸銨沈澱，可得最大酵素活性；再經過CM-Sepharose CL-6B膠濾層析，以及Blue Sepharose 6 Fast Flow 親和性管柱分離，經過這幾個步驟，以SDS-PAGE證明，可得到純化的MTGase。 米穀粉添加不同比例MTGase，以動態流變儀（Rheometer）測其黏彈性，顯示出其彈性模式（G′）與黏性模式（G〞）均隨MTGase添加而增加；至於質地組織分析（texture analysis），其質地參數（硬度、膠性）也是隨MTGase添加而增加；在熱性質測試，添加MTGase因其作用蛋白質分子，交叉鍵結形成聚合分子，有降低焓值的現象（enthalpy）。但值得注意的是添加過多的MTGase（0.3U/mg，以米蛋白質為基重）反而降低其膠體強度與TPA各種物性，此可能會由於太多ε-(γ-glutamyl)lysine的鍵結會抑制蛋白質網狀結構的發展而降低其彈性，以添加MTGase 0.1U/mg與0.2U/mg rice protein可得較佳效果，所以此為建議添加量。SDS-PAGE profile證明MTGase會鍵結米蛋白質形成較大分子量的聚合物（HMW）。由電子顯微掃瞄（SEM）可觀察到，MTGase添加量愈多，米中蛋白質結構愈細緻，孔洞愈小，而且排列愈呈組織化。 以另一種方式探討MTGase影響秈米中蛋白質學性質，將秈米的蛋白質依不同溶劑中溶解性不同來區分，其萃出可分為四大類：水溶（albumin）、鹽溶（globulin）、鹼溶（glutelin）以及醇溶（prolamin），含量多寡依序為鹼溶蛋白＞鹽溶蛋白＞水溶蛋白＞醇溶蛋白。醇溶蛋白，經SDS-PAGE分析只有一個次單位（subunit）。 從動態流變性質測知，白米中水溶蛋白(albumin)對增進河粉膠體強度較無貢獻，所以建議米食加工時可以去除。米蛋白中，穀蛋白的半胱胺酸含量多，對產品的影響較大，由SDS-PAGE圖可觀察到添加MTGase的交聯效果。
並列摘要
Microbial transglutaminase (MTGase) may catalyze the cross-linking between a peptide-bound glutaminyl residue and an ε-amino group of lysine residue in protein. MTGase has been used to modify food protein for improving their functionality. Not pure MTGase could be supplied, so making it by myself. The commercial microbial transglutaminase was obtained from Ajinomoto Co.（Japan）. Added to water and followed by centrifugation. The supernatant was filtered to obtain the crude enzyme solution. The solution was precipitated with ammonium sulfate. In the obtained precipitates, 50~80% showed the better enzyme activity. Then the supernatant was purified by gel filtration through a CM- Sepharose CL-6B column and a Blue Sepharose Fast Flow column. Used SDS-PAGE profile could prove to get pure MTGase. Rice flour was treated with different concentrations of MTGase. The rheological properties, elastic (G') and viscous (G") moduli of the rice gel increased at the addition of MTGase. However, high amount of the addition of MTGase at 0.3U/mg may reduce textural parameters. Therefore, rheological properties and textural parameters might be affected by the existing threshold number of connections for cross-links in rice protein. Differential scanning calorimetry (DSC) showed that MTGase treatment led to decrease the denaturation enthalpy. Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that rice proteins were polymerized through the MTGase reaction. Scanning electron microscope (SEM) figure showed the more concentrations of MTGase added to rice flour, the better texture of rice flour gel. Therefore, those SEM photographs proved the added MTGase improved the rheological properties and textural parameters (hardness, and gumminess). Here another method was used to investigate the effects of MTGase to properties of rice protein. Rice protein was treated with different solvents and there were four kinds of extractions were obtained: water soluble (albumin), salt soluble (globulin), alkali soluble (glutelin) and alcohol soluble (prolamin) fractions. The amount obtained were in the order of alkali soluble protein＞salt soluble protein＞water soluble protein＞alcohol soluble protein. After SDS-PAGE analysis, alcohol soluble protein only one subunit. From Rheology test, water soluble protein (albumin) was proved has no contribution in strength of rice gel. Therefore it is suggested to remove it at the rice processing. Rice protein contain much cysteine, and affects the properties of the products. The rice contains much glutelin, from SDS-PAGE profile, the effects of MTGase addition to the linkages were observed.