转谷氨酰胺酶对草鱼糜凝胶性的影响
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摘要
鱼糜制品在水产品加工业中占有重要的地位。鱼糜及其制品的加工原料多为海产品类,但受海洋捕捞和海水养殖生产不同程度的停滞,海产品的供应趋势明显减少,满足不了鱼糜及鱼糜制品的市场需求,因此开发淡水鱼鱼糜及鱼糜制品是今后鱼糜加工业的重要课题。但是淡水鱼糜的凝胶能力普遍较差,尤其是经过冷冻储藏之后,研究发现转谷氨酰胺酶(Transglutaminase,简称TGase)可以提高鱼糜的凝胶强度。本文以草鱼为研究对象,研究鱼中内源性转谷氨酰胺酶(FishTransglutaminase,简称FTG)的活性,内源性及微生物转谷氨酰胺酶对草鱼糜中蛋白质的交联性的影响,及微生物转谷氨酰胺酶(Microbial Transglutaminase,简称MTG)对草鱼糜凝胶特性的影响。
草鱼糜中的内源性转谷氨酰胺酶的活性会受到PH、温度、离子强度、激活剂、抑制剂及鱼糜冻藏时间等因素的影响。研究表明,草鱼糜的内源性转谷氨酰胺酶的最适pH为8-9,最适温度为45℃,需要钙离子(1 mmol/L)激活,DTT在低浓度(0-1 mM)范围内可以保护酶的活性基团,从而提高酶的活性,Cu2+Ba2+、Zn2+、Mn2+、Fe2+、Pb2+、Fe3+、Li+、Mg2+、K+、Na+等金属离子及一些酶活抑制剂会抑制酶的活性,甚至导致酶失活,酶活也受鱼糜冻藏时间的影响,冻藏时间越长,酶活也越低。
在加热过程中,加入TGase的溶液中,测得溶液中自然肌动球蛋白(Natural actomyosin,简称NAM)的含量比不加酶时要低,说明加入TGase可以增强NAM的交联程度。比较加入相同酶活单位的MTG和FTG后,发现MTG对NAM交联程度的影响比草鱼糜中内源性转谷氨酰胺酶要好。溶液中NAM含量受温度影响很大。低温条件(25-35℃),溶液中NAM的含量随着温度的上升略有上升;在40-45℃时,溶液NAM含量降到最低,说明这个温度下,NAM的交联程度最大；随着温度的进一步升高,NAM的交联程度大大降低,可能是因为组织蛋白酶在较高温度下被激活的原因。
在加酶量为1.5%、自然PH值下,于42℃保温0.5h,草鱼糜的凝胶强度达到最大。MTG作用条件对白度、持水性、蒸煮损失的研究表明,鱼糜凝胶的白度与凝胶强度及凝胶含水量有一定的关系,持水性及蒸煮损失一般与鱼糜凝胶强度成正相关。钙离子浓度在20-50mmol/kg时可以增强草鱼糜凝胶强度,草鱼糜中加入EDTA或NH4C1,鱼糜凝胶变差。
在MTG优化条件下,添加马铃薯淀粉、小麦淀粉、玉米淀粉的鱼糜凝胶品质较好,研究马铃薯淀粉添加量的影响表明,淀粉添加量在100%-15%的鱼糜品质较好,水分添加量在15%-25%时,鱼糜凝胶的品质较好。
Surimi products occupies an important position in processing industry of aquatic products. The raw material of surimi and surimi products mostly are seafood. But the supply of seafood is reducing and it can't meet the market demands of surimi and surimi products, because marine fishing and aqua culture production is stagnated to varying degrees. Therefore the development of freshwater surimi products is an important issue in the future surimi processing industry. However the gelation of freshwater surimi is generally poor, especially after frozen storage. It has been found that transglutaminase can enhance surimi gel strength. In this thesis which focuced on grass carp, we studied the characterization of transglutaminase (TGase) from grass carp under different conditions, effects of FTG and MTG on cross-linking of natural actomyosin, and effects of MTG on gel strength of grass carp.
The activity of TGase from grass carp can be affected by PH, setting temperature, ionic strength, activator, inhibitor, frozen storage time and other factors. The results showed that optimal pH and optimal temperature of transglutaminase from grass carp were 8-9 and 25-45℃, respectively. It could be activated by calcium. Dithiothreitol (DTT) at low concentrations (0～1 mM)can protect the active groups of enzyme, so as to increase the enzyme activity. Cu2+、Ba2+、Zn2+、Mn2+、Fe2+、Pb2+、Fe3+、Li+ Mg2+、K+、Na+ and other metal ions can inhibit the activity of TGase, and even lead to enzyme inactivation. Enzyme activity can also be affected by frozen storage time of surimi, enzyme activity decreases with frozen storage time.
It was measured that the concentration of natural actomyosin (NAM) in solution which was added TGase was lower than which wasn't added in the heating process. It indicated adding TGase in solution can enhance the craoss-linking of NAM. Cross-linking of NAM in solution was more improved by MTG when MTG and FTG were added to solution both at 3 U TGase/ml. The concentration of NAM was greatly affected by temperature. It reduced to a minimum in 40～45℃. It indicated the cross-linking of NAM was best in this temperature. As the temperature increased further, the cross-linking of NAM significantly reduced, probably because of cathepsin is activated in higher temperatures.
Surimi gel strength of grass carp was biggest under sunch conditions as 1～1.5% enzyme dosage, setting 0.5h at 42℃with natural PH value. Studies on whiteness, water holding capacity, cooking-loss of surimi gels under different conditions have shown whiteness of surimi gels had a certain relationship to gel strength and water content of surimi gels, and water holding capacity and cooking-loss of surimi gels is generally positively related to the gel strength of surimi. Calcium could improve the gel strength of surimi in concentration of 20-50mmol/kg. The gelation of surimi became bad by adding EDTA or NHUCl.
Under optimized conditions, surimis adding potato starch, wheat starch or corn starch would form good gels. The adding amount of potato starch in 10～15%, surimi gels quality was better. The adding amount of water in 15～25%, surimi gels quality was better.
草鱼糜中的内源性转谷氨酰胺酶的活性会受到PH、温度、离子强度、激活剂、抑制剂及鱼糜冻藏时间等因素的影响。研究表明,草鱼糜的内源性转谷氨酰胺酶的最适pH为8-9,最适温度为45℃,需要钙离子(1 mmol/L)激活,DTT在低浓度(0-1 mM)范围内可以保护酶的活性基团,从而提高酶的活性,Cu2+Ba2+、Zn2+、Mn2+、Fe2+、Pb2+、Fe3+、Li+、Mg2+、K+、Na+等金属离子及一些酶活抑制剂会抑制酶的活性,甚至导致酶失活,酶活也受鱼糜冻藏时间的影响,冻藏时间越长,酶活也越低。
在加热过程中,加入TGase的溶液中,测得溶液中自然肌动球蛋白(Natural actomyosin,简称NAM)的含量比不加酶时要低,说明加入TGase可以增强NAM的交联程度。比较加入相同酶活单位的MTG和FTG后,发现MTG对NAM交联程度的影响比草鱼糜中内源性转谷氨酰胺酶要好。溶液中NAM含量受温度影响很大。低温条件(25-35℃),溶液中NAM的含量随着温度的上升略有上升;在40-45℃时,溶液NAM含量降到最低,说明这个温度下,NAM的交联程度最大；随着温度的进一步升高,NAM的交联程度大大降低,可能是因为组织蛋白酶在较高温度下被激活的原因。
在加酶量为1.5%、自然PH值下,于42℃保温0.5h,草鱼糜的凝胶强度达到最大。MTG作用条件对白度、持水性、蒸煮损失的研究表明,鱼糜凝胶的白度与凝胶强度及凝胶含水量有一定的关系,持水性及蒸煮损失一般与鱼糜凝胶强度成正相关。钙离子浓度在20-50mmol/kg时可以增强草鱼糜凝胶强度,草鱼糜中加入EDTA或NH4C1,鱼糜凝胶变差。
在MTG优化条件下,添加马铃薯淀粉、小麦淀粉、玉米淀粉的鱼糜凝胶品质较好,研究马铃薯淀粉添加量的影响表明,淀粉添加量在100%-15%的鱼糜品质较好,水分添加量在15%-25%时,鱼糜凝胶的品质较好。
Surimi products occupies an important position in processing industry of aquatic products. The raw material of surimi and surimi products mostly are seafood. But the supply of seafood is reducing and it can't meet the market demands of surimi and surimi products, because marine fishing and aqua culture production is stagnated to varying degrees. Therefore the development of freshwater surimi products is an important issue in the future surimi processing industry. However the gelation of freshwater surimi is generally poor, especially after frozen storage. It has been found that transglutaminase can enhance surimi gel strength. In this thesis which focuced on grass carp, we studied the characterization of transglutaminase (TGase) from grass carp under different conditions, effects of FTG and MTG on cross-linking of natural actomyosin, and effects of MTG on gel strength of grass carp.
The activity of TGase from grass carp can be affected by PH, setting temperature, ionic strength, activator, inhibitor, frozen storage time and other factors. The results showed that optimal pH and optimal temperature of transglutaminase from grass carp were 8-9 and 25-45℃, respectively. It could be activated by calcium. Dithiothreitol (DTT) at low concentrations (0～1 mM)can protect the active groups of enzyme, so as to increase the enzyme activity. Cu2+、Ba2+、Zn2+、Mn2+、Fe2+、Pb2+、Fe3+、Li+ Mg2+、K+、Na+ and other metal ions can inhibit the activity of TGase, and even lead to enzyme inactivation. Enzyme activity can also be affected by frozen storage time of surimi, enzyme activity decreases with frozen storage time.
It was measured that the concentration of natural actomyosin (NAM) in solution which was added TGase was lower than which wasn't added in the heating process. It indicated adding TGase in solution can enhance the craoss-linking of NAM. Cross-linking of NAM in solution was more improved by MTG when MTG and FTG were added to solution both at 3 U TGase/ml. The concentration of NAM was greatly affected by temperature. It reduced to a minimum in 40～45℃. It indicated the cross-linking of NAM was best in this temperature. As the temperature increased further, the cross-linking of NAM significantly reduced, probably because of cathepsin is activated in higher temperatures.
Surimi gel strength of grass carp was biggest under sunch conditions as 1～1.5% enzyme dosage, setting 0.5h at 42℃with natural PH value. Studies on whiteness, water holding capacity, cooking-loss of surimi gels under different conditions have shown whiteness of surimi gels had a certain relationship to gel strength and water content of surimi gels, and water holding capacity and cooking-loss of surimi gels is generally positively related to the gel strength of surimi. Calcium could improve the gel strength of surimi in concentration of 20-50mmol/kg. The gelation of surimi became bad by adding EDTA or NHUCl.
Under optimized conditions, surimis adding potato starch, wheat starch or corn starch would form good gels. The adding amount of potato starch in 10～15%, surimi gels quality was better. The adding amount of water in 15～25%, surimi gels quality was better.
引文
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