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Purification and Characteristics of Transglutaminase and Its Application
Low-salt restructured pork
|摘要:||本研究旨在比較禽畜血液之特性、純化豬血漿之麩醯基轉移(Transglutaminase，TGase)和探討其特性，並應用TGase於低鹽肉製品。首先比較豬血、雞血和鴨血之TGase活性及探討其經自然凝固和不同加熱處理(80℃, 30或60分鐘及90℃, 30或60分鐘)後之血塊的物性和顯微構造變化。其次進行純化豬血漿TGase，並探討TGase於不同溫度下(4, 25 和37℃)對淘汰蛋雞胸肌肌球蛋白重鏈(Myosin heavy chain )和肌動蛋白(Actin)之影響。而且，也探討pH值( pH 4, 5, 6, 7, 8和9)、溫度 (5, 15, 25, 35, 45,55和65℃)、金屬離子(Ca2+, Mg2+, Na2, K+, Zn2+, Fe2+, Co2+和Cu2+)和抑制劑(iodoacetate, IAA; N-ethylmaleimide, NEM; phenylmethylsulfonylfluoride, PMSF)等因素對TGase之影響。進而探討TGase對模擬低鹽重組肉之影響，最後實際應用TGase在低鹽雞肉貢丸和低鹽重組鴨排之製作，並分析製品之製成率、色澤、凝膠強度、觀察顯微構造變化和官能品評，以瞭解所純化之TGase的實用價值性。結果顯示如下:
1.鴨血之酵素比活性及自然凝固血塊之破裂強度(Breaking strength)為最高，雞血最低(P<0.05);不同加熱處理後血塊之破裂強度，仍以鴨血塊最高，且雞血塊和鴨血塊之破裂強度皆隨加熱溫度及時間增加而有增加趨勢，但豬血塊則於加熱90℃,60分鐘後有下降現象。顯微構造觀察顯示鴨血自然凝固之血纖維蛋白凝塊(Fibrin clot)和加熱後之血塊構造皆較緻密，雞血塊則有最大之孔洞。
6.低鹽重組鴨排之油煎失重以0.5%之豬血漿粗TGase配合添加單離大豆蛋白(isolated soy protein, TISP)者最低（P<0.05），凝膠強度則以0.5%之豬血漿粗TGase配合添加0.5%之酪蛋白鈉(sodium caseinate, TSC)最高（P<0.05）；但在色澤方面，各處理間皆無顯著差異。在官能品評之外觀、質地、多汁性和總接受性方面，TSC、TISP和0.5%之豬血漿粗TGase配合添加乳清蛋白(whey protein, TWP)者皆顯著高於（P<0.05）對照組。|
The purpose of this research was to compare the characteristics of pig, chicken and duck blood, to purify transglutaminase (TGase) derived from pig plasma and examine its characteristics, and to apply TGase to process low-salt meat products. The first, to compare the activity of TGase in pig, chicken and duck blood, and also to study the rheological properties and microstructure changes in the natural blood clots and curds under different cooking temperatures (80℃, for 30 and 60 minutes and 90℃, for 30 and 60 minutes). Secondarily, to purify pig plasma TGase and examine its effects on myosin heavy chain and actin of the breast muscles from spent hens at different temperatures(4, 25 and 37℃). Moreover, to examine the effect of pig plasma TGase at different factors as pH values ( pH 4, 5, 6, 7, 8 and 9), temperatures (5, 15, 25, 35, 45, 55 and 65℃), metal ions (Ca2+, Mg2+, Na+, K+, Zn2+, Fe2+, Co2+ and Cu2+) and inhibitors (iodoacetate, IAA; N-ethylmaleimide, NEM; phenylmethylsulfonylfluoride, PMSF) conditions. Furthermore, to examine the effect of TGase on the quality of simulated low-salt restructured pork. Finally, to practically apply TGase to make low-salt chicken meat-balls and restructured duck steak. Yield, color, gel strength, microstructure and sensory evaluation were measured. The results were shown as follows: 1. TGase specific activity of duck plasma and the breaking strength of the natural blood duck clot were the highest and that of the chicken blood was the lowest (P<0.05). Having tested different cooking temperatures and times, the breaking strength of duck blood curd proved to be the highest among the three treatments (P<0.05). The breaking strength of chicken and duck blood curds gained with increasing cooking temperature and time. However, the breaking strength of pig blood curd decreased at longer cooking time (60 minutes) at 90℃. Observing the microstructure, the duck blood clot was found to be more dense. Chicken blood curd had the largest holes in the microstructure among all blood curd 2. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that plasma TGase was composed of molecular weights of approximately 75,000 and 80,000. TGase added to the myofibrillar proteins solution indicated that the intensity of myosin heavy chain and actin decreased when incubated at 4℃ for 48 hr. However, the intensity of myosin heavy chain and actin obviously decreased when incubated at 25℃ for 2 hr. Moreover, the relative intensity analyzed by scanning densitometry after SDS-PAGE indicated that myosin heavy chain and actin decreased to 45 and 64% respectively. In addition, the relative intensity of myosin heavy chain and actin declined to 7 and 63% at 37℃ for 5 min. Furthermore, the relative intensity of myosin heavy chain and actin decreased with reaction time when incubated at 25 and 37℃. 3. Pig plasma TGase had the highest relative activity at pH 7-8, 35-45℃ and in the present Ca2+ . However, the activity of this enzyme inhibited by IAA, NEM and PMSF. 4. Low-salt restructured pork with TGase had higher (P<0.05) gel strength and L-value, but lower (P<0.05) a-value than the control group. Scanning electron microscopy (SEM) photomicrographs showed that the low-salt restructured pork had a rather regular gel network structure when TGase was added. 5. Both the yield and gel strength of low-salt chicken meat-balls increased when TGase supplement were increased (P<0.05). However, color did not differ among treatments. Observation of scanning electron microscopy (SEM), a rather firm and regular gel network structure of meat-balls were obtained when more TGase was added. Texture, juiciness and overall acceptability of sensory evaluation were not differed statistically until the level of TGase was up to 1﹪ (P<0.05). 6. Low-salt restructured duck steak supplemented with 0.5% crude pig plasma TGase mixed with 0.5% isolated soy protein (TISP) had the lowest frying loss (P<0.05) while 0.5% crude pig plasma TGase mixed with 0.5% sodium caseinate (TSC) had the highest gel strength (P<0.05). Color of products was found to be similar among lots. Regarding the sensory evaluation, the scores of appearance, texture, juiciness and overall acceptability of duck steak from TSC, TISP and 0.5% crude pig plasma TGase mixed with 0.5% whey protein (TWP) were higher than those of the control (P<0.05).
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