漂洗对蓝圆鲹鱼糜凝胶特性的影响

目的 研究漂洗对蓝圆鲹鱼糜凝胶特性的影响。方法以白度、持水性、pH、水分分布、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、 微观结构和粘弹性等为评价指标,分析了不同漂洗方法对蓝圆鲹鱼糜凝胶特性的影响。结果 娃哈哈纯净水和盐水漂洗均可提高蓝圆鲹鱼糜凝胶强度;经一次纯净水娃哈哈纯净水漂洗和两次0.15%盐水漂洗处理后,凝胶白度、肌原纤维蛋白含量相对、不易流动水含量M21 ,持水性均增加,凝胶强度增强至7581.51 g·mm（P<0.05）,形成致密且平滑的凝胶网状结构;动态流变学结果显示,储能模量G′最大,此时凝胶以弹性为主要特征。结论 经过一次娃哈哈纯净水漂洗和两次0.15% (m/m)盐水漂洗,得到的蓝圆鲹鱼糜凝胶特性最佳。     

电子束辐照对带鱼鱼糜内源性蛋白酶活性及其构象单元的影响

鱼类内源性蛋白酶是引起鱼糜凝胶劣化的重要因素之一。以不同剂量电子束辐照带鱼鱼糜,测定鱼糜内源性肌原纤维结合型丝氨酸蛋白酶（myofibril-bound serine proteinase,MBSP）和组织蛋白酶L（cathepsin L,Cat-L）活力及其最适反应温度,结合圆二色光谱分析其构象单元变化,探讨电子束辐照对鱼糜内源性MBSP和Cat-L的影响。结果表明：随着辐照剂量的增加,鱼糜MBSP和Cat-L活力降低,当辐照剂量为9 kGy时Cat-L活力下降更为明显;对照组和辐照组鱼糜MBSP的最适温度均为55 ℃,但3 kGy及9 kGy辐照对Cat-L的最适温度产生影响,使其由55 ℃降低至45 ℃;辐照引起鱼糜MBSP和Cat-L分子中的二级结构单元互相转化,导致其构象变化,削弱其降解肌原纤维蛋白的能力。结论：电子束辐照能够影响带鱼鱼糜MBSP和Cat-L的二级结构及活力,减轻二者对肌原纤维蛋白的降解作用,适宜剂量的电子束辐照能有效抑制鱼糜内源性蛋白酶活性,防止凝胶劣化,有利于鱼糜凝胶的形成。     

蓝圆鲹肌肉中丝氨酸蛋白酶的分离纯化及性质研究

鱼类死后肌肉容易发生软化现象。研究表明,这与肌肉中的丝氨酸蛋白酶有着密切的关系。本研究通过硫酸铵盐析、DEAE-Sephacel、Q-Sepharose及Capto Q等柱层析相结合的方法,从蓝圆鲹肌肉中纯化得到一种具有分解明胶能力的丝氨酸蛋白酶,SDS-PAGE结果显示其分子量约为60ku,该酶最适温度及最适pH分别为40℃和9.0。丝氨酸蛋白酶抑制剂Pefabloc SC、Benzamidine、MBTI、PMSF和LBTI均能明显的抑制该酶的活性,而其他蛋白酶抑制剂对其活性没有明显的影响。底物特异性表明其能有效的降解丝氨酸蛋白酶荧光底物Boc-Leu-Lys-Arg-MCA,但进一步研究发现,该酶对I型胶原蛋白及明胶有明显的分解能力,同时对肌球蛋白重链也有一定的分解作用,说明该酶可能参与鱼肉保鲜中肌肉软化的过程。     

戊糖片球菌产蛋白酶对发酵鲢鱼鱼糜凝胶性能的影响

对选用的戊糖片球菌产生的蛋白酶进行分离纯化,利用葡萄糖酸内酯(GDL)和抗生素建立模拟发酵体系,以鱼糜蛋白质降解、肌动球蛋白聚集和鱼糜凝胶强度为指标研究了戊糖片球菌产蛋白酶对发酵鱼糜凝胶性能的影响。结果表明:戊糖片球菌产蛋白酶的添加导致了鱼糜蛋白质非蛋白氮含量和TCA-溶解肽含量增加,两种pH下肌动球蛋白浊度降低,粒径变小,且在pH 4.5条件下更为显著。同时,加酶组鱼糜样品的凝胶强度明显小于空白组鱼糜样品的凝胶强度。综上,戊糖片球菌产蛋白酶对发酵鱼糜中蛋白质有降解作用,影响了肌动球蛋白的聚集性,对凝胶性能产生劣化影响。     

NaCl对添加丝氨酸蛋白酶的肌原纤维蛋白凝胶特性的影响

以养殖大黄鱼作为研究对象,探究NaCl对添加了丝氨酸蛋白酶的肌原纤维蛋白凝胶特性的影响。首先对养殖大黄鱼肉经过提取分离纯化得到的丝氨酸蛋白酶进行研究,探讨其最适温度、最适pH以及NaCl对丝氨酸蛋白酶活性的影响。并进一步研究NaCl对含有丝氨酸蛋白酶的肌原纤维蛋白凝胶的质构特性、持水性、白度、拉曼光谱、荧光分析、微观结构等特性的影响。结果发现,丝氨酸蛋白酶的最适温度为50℃,最适pH值为7.0。NaCl的添加使肌原纤维蛋白凝胶的二级结构发生改变,蛋白凝胶结构越来越紧密稳定,但荧光强度相对有所下降。在NaCl质量浓度为0~20 g/L时,肌原纤维蛋白凝胶的胶黏性、咀嚼性不断上升,且白度增大。在NaCl添加量为40 g/L时,肌原纤维蛋白凝胶的持水性最强。因此可得出结论,添加NaCl后,丝氨酸蛋白酶的活性受到抑制,使其对肌原纤维蛋白凝胶的破坏减弱,从而改善鱼糜凝胶特性。     

茶多酚延缓冷藏大黄鱼肌原纤维蛋白变性降解机理研究

研究经过0.3%茶多酚浸泡的养殖大黄鱼鱼片和对照组在-2℃冷藏条件下25 d内肌原纤维蛋白生化特性以及鱼糜凝胶特性变化规律。定期分析处理组和对照组大黄鱼肌原纤维蛋白生化指标(钙酶活性、巯基含量和表面疏水性)和鱼糜凝胶特性指标(凝胶弹性、凝胶强度和凝胶持水性)。试验结果表明,茶多酚处理组和对照组的鱼片肌原纤维蛋白的生化特性具有显著差异性,储藏期间鱼片中肌原纤维蛋白的SH含量下降,表面疏水性增大,Ca2+-ATPase活性逐渐下降。同时显示,冷藏过程中茶多酚处理组和对照组鱼糜凝胶弹性、凝胶强度及凝胶持水性随着贮藏时间的延长而明显下降。SDS-PAGE电泳结果显示,肌原纤维蛋白发生一定程度的降解。此外,肌纤维蛋白质的SH含量和表面疏水性与贮藏时间呈显著线性相关,可以作为蛋白质变性程度的指示指标。茶多酚能延缓肌原纤维蛋白变性和降解程度,延缓鱼片和鱼糜氧化变质程度。     

表没食子儿茶素没食子酸酯对冷冻罗非鱼鱼糜抗冻作用机制

通过肌原纤维蛋白理化指标、鱼糜凝胶特性的测定及十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE）,研究表没食子儿茶素没食子酸酯（epigallocatechin gallate,EGCG）对冷冻罗非鱼鱼糜的抗冻效果并探讨其作用机制。结果表明,罗非鱼鱼糜中添加EGCG具有一定的抗冻效果,对比空白组具有显著差异性。冻藏期间空白组鱼糜中盐溶性蛋白含量、总巯基含量分别下降了68.2%和62.9%,而添加0.01% EGCG后二者的降幅分别为54.5%和49.2%,且EGCG还明显延缓了肌原纤维蛋白的氧化羰基化作用。此外,冻藏过程中,EGCG组和空白组鱼糜凝胶强度和持水性随冻藏时间延长逐渐降低,且EGCG添加量较大时,反而加快凝胶劣化。SDS-PAGE显示,EGCG能够有效抑制肌原纤维蛋白降解,其中质量分数为0.01%的EGCG效果最佳。EGCG能延缓肌原纤维蛋白变性和降解程度,延缓鱼糜氧化变质程度,有望成为一种新型的鱼糜抗冻剂。     

凡纳滨对虾肌肉中组织蛋白酶L提取工艺优化及分离纯化

优化凡纳滨对虾肌肉中组织蛋白酶L提取工艺,分离纯化组织蛋白酶L并验证其对肌原纤维蛋白的降解作用。以凡纳滨对虾肌肉为原料,采用单因素试验、Plackett-Burman试验和双因素等重复试验对肌肉中组织蛋白酶L的提取工艺进行了优化。采用Tris-HCl缓冲液浸提、硫酸铵沉淀、Q-Seharose F.F阴离子交换层析、Sephacryl S-100凝胶过滤层析和SDS-PAGA等方法对组织蛋白酶L粗酶液进行分离纯化,并验证提纯后的组织蛋白酶L对肌原纤维蛋白的降解作用。最终确定对虾肌肉中组织蛋白酶L最佳提取工艺为:缓冲液体系(Tris-HCl缓冲液浓度40 mmol/L,半胱氨酸浓度6 mmol/L,苯甲基磺酰氟浓度0.4 mmol/L),料液比1∶8(g∶mL),pH 6.0。优化后组织蛋白酶L总酶活力值升至378.36 U,酶活力得率达到150.1 U/g,纯化后的组织蛋白酶L的比活力从0.34 U/mg提高到了101.15 U/mg,纯化倍数达到298.28倍,得率为16.50%,分子质量为46.4 kDa,并进一步验证了提纯后的组织蛋白酶L对肌球蛋白重链和肌动蛋白有降解作用。发现对...     

鲢鱼加工副产物酶解产物对冻融鱼糜肌原纤维蛋白性质的影响

分析了鲢鱼加工副产物酶解产物对冻融鱼糜肌原纤维蛋白性质的影响。采用复合蛋白酶制备酶解产物,分别添加2%,6%的酶解产物(FPH-2%、FPH-6%)于鱼糜中,设置空白对照,冻融循环6次后,提取鱼糜中的肌原纤维蛋白并测定其性质。结果表明:两种添加量的酶解产物对冻融鱼糜中肌原纤维蛋白均具有良好的保护作用,其中,添加6%酶解产物的鱼糜冻融循环6次后,肌原纤维蛋白浓度和巯基含量降低最少且羰基含量较低,DSC和SDS-PAGE分析表明:6%酶解产物添加组中肌原纤维蛋白变性程度较低。本研究有助于明确该酶解产物对于鱼糜结构的作用,为鲢鱼加工业提供理论依据;同时能有效利用蛋白资源,提高水产加工产业的附加值。     

蓝圆鲹骨骼肌脯氨酸内肽酶的分离纯化及其对胶原肽的作用

以蓝圆鲹为研究对象,探讨脯氨酸内肽酶(Prolyl endopeptidase,PEP)对鱼类肌肉胶原蛋白的作用及其机理。通过硫酸铵分级盐析,DEAE-Sephacel阴离子交换,Phenyl-Sepharose疏水层析和Q-Sepharose阴离子交换,从蓝圆鲹骨骼肌中分离纯化得到一种脯氨酸内肽酶。SDS-PAGE结果显示,PEP的分子量为82 ku,肽质量指纹图谱分析得到16个肽片段,共169个氨基酸残基。片段序列与墨西哥鲷鱼(Neolamprologus brichardi)PEP的同源性达98.8%,证明纯化得到的酶是PEP。PEP的最适温度为35℃,但热稳定性较差;最适p H为6.0,在p H 5.0~7.5之间有较好的稳定性。将PEP与合成的鱼胶原蛋白小肽反应,利用反相高效液相色谱对产物进行分离,电喷雾质谱分析结果显示PEP的水解位点在脯氨酸残基的羧基端。以上结果表明,鱼类肌肉中的PEP能够协同金属蛋白酶,通过切割脯氨酸残基进一步降解胶原小肽从而参与到鱼肌肉胶原蛋白的新陈代谢中,是鱼死后参与胶原蛋白降解的重要酶类。     

Purification and characterisation of cathepsin L from the skeletal muscle of blue scad (Decapterus maruadsi) and comparison of its role with myofibril-bound serine proteinase in the degradation of myofibrillar proteins

The modori phenomenon during surimi production is caused by endogenous proteinases, especially cathepsin L and myofibril-bound serine proteinase (MBSP). Cathepsin L from the skeletal muscle of blue scad (Decapterus maruadsi) was purified to homogeneity by ammonium sulphate fractionation and a series of column chromatographies and revealed a single band with molecular mass of 30 kDa on SDS–PAGE. Peptide mass fingerprinting (PMF) obtained three fragments with 48 amino acid residues, which were highly identical to cathepsin L from other fish species. Its optimal pH and temperature were 5.5 and 55 °C, respectively. Meanwhile, MBSP was purified from the skeletal muscle of blue scad, and the roles of cathepsin L and MBSP in the degradation of myofibrillar proteins were compared. The results indicated that MBSP is more effective than cathepsin L in promoting the degradation of myofibrillar proteins, especially myosin heavy chain (MHC), suggesting that MBSP plays a more significant role.     

Myosin heavy chain degradation during post mortem storage of Atlantic cod (Gadus morhua L.)

Post mortem proteolytic degradation of fish fillets leads to textural changes like muscle softening and gaping. In this study proteolytic degradation of myosin heavy chain (MHC) was monitored during storage of muscle and of isolated myofibrils at different temperatures and pH-values by the use of MHC-specific antibodies. The ability of cathepsin D to associate to myofibrillar proteins was also studied. Muscle stored at 6 °C and isolated myofibrils stored at 0 °C, 6 °C and 20 °C were degraded at pH 6.3 or lower. Cathepsin D could be found associated with extensively washed myofibrils. Inhibition of cathepsin D during storage affected the observed MHC-degradation at pH 5.5, but not at pH 6.3. This indicates that cathepsin D to a less extend than formerly believed, is responsible post mortem degradation of MHC.     

Purification and Characterization of Cathepsin B from the Skeletal Muscle of Fresh Water Fish, Tilapia mossambica

Cathepsin B from the skeletal muscle of a fresh water fish Tilapia mossambica was purified 4280-fold with 9% recovery. The electrophoretic homogeneity of the preparation was established both under native and denatured conditions. The molecular weight of cathepsin B on the basis of its gel filtration profile was 23,500 daltons. The enzyme, an endopeptidase, hydrolysed Z-arg-arg-NNap and Bz-arg-NNap, with Km values of 0.57 and 3.23 mM, respectively. Cathepsin B did not display aminopeptidase activity, but cleaved Bz-arg-NH₂, exhibiting the specificity of a carboxypeptidase. Among protein substrates tested, only azocoll was hydrolyzed at lower pH values. Leu-peptin, antipain and thiol blockers abolished the enzyme activity completely. The Kcat set^-1 value of fish cathepsin B seemed to be lower than that of mammalian enzyme.     

凡纳滨对虾组织蛋白酶L性质分析及其对肌肉蛋白的降解

本研究从凡纳滨对虾肝胰腺中纯化获得一种组织蛋白酶L,其分子质量约为31 k D,肽质量指纹图谱分析得到8个片段共112个氨基酸残基,与报道的凡纳滨对虾组织蛋白酶L序列完全一致。该酶的最适温度和最适pH值分别为35℃和5.5,且在0~40℃以及pH 5.5~6.5之间酶活力稳定。该酶仅对底物Z-Phe-Arg-MCA特异分解。半胱氨酸蛋白酶抑制剂E-64和Leupeptin对其有明显的抑制作用,而金属蛋白酶抑制剂乙二胺四乙酸和乙二醇二乙醚二胺四乙酸对其有少量的激活作用。扫描电子显微镜观察结果显示,随着低温贮藏时间的延长,对虾肌肉纤维的断裂程度不断增加。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析显示,组织蛋白酶L可使肌肉蛋白发生分解,推测其可能参与对虾低温贮藏过程中肌肉的降解。     

Broad‐spectrum inhibition of proteolytic enzymes by allicin and application in mitigating textural deterioration of ice‐stored grass carp (Ctenopharyngodon idella) fillets

The inhibitory effect of allicin on proteolytic enzymes and textural deterioration of ice‐stored grass carp (Ctenopharyngodon idella) fillets was investigated. The results of in vitro study showed that allicin inhibited the activity of cathepsin B, L and D, calpain and collagenase in crude extract of grass carp muscle. Among endogenous enzymes, cathepsin B, L and collagenase were the most susceptible to allicin. Proteolysis of myofibrillar proteins by either crude enzyme or cathepsin B and L was almost prevented by allicin when employed at a concentration higher than 100 mm . After storage of 21 days, shear force of fillets treated with allicin at 10–100 mm was 39–51% higher than that of control. Myofibrillar proteins of fillets during storage were well protected against degradation when allicin concentration increased to 100 mm , as evidenced by SDS‐PAGE. Therefore, allicin could be a potential broad‐spectrum inhibitor to retard softening of fish fillets via mitigating myofibrillar proteolysis by endogenous enzymes especially cathepsin B and L during ice‐storage.     

Isolation of cathepsin B from the muscle of silver carp (Hypophthalmichthys molitrix) and comparison of cathepsins B and L actions on surimi gel softening

Cathepsin B from silver carp muscle was purified to 263-fold by acid treatment, ammonium sulfate fractionation, followed by a series of chromatographic separations. The molecular mass of the purified enzyme was 29 kDa as determined by SDS-PAGE and immunoblotting. The purified enzyme was activated by dithiothreitol and cysteine while it was substantially inhibited by E-64, suggesting the purified enzyme belongs to the cysteine proteinase family. Optimal pH and temperature were 5.5 and 35 °C, respectively. The enzyme catalyzed the hydrolysis of Z-Arg-Arg-MCA with a parameter of K_m (90 μM) and K_cat (20.3 s⁻¹), but hardly hydrolyzed Arg-MCA. Analysis of surimi gel strength and microstructure showed that cathepsins B and L were capable of destroying the network structure of silver carp surimi gels, consequently causing gel softening. Cathepsin L might play an important role in the modori effect.     

Degradation of myofibrils from rabbit, chicken and beef by cathepsin l and lysosomal lysates

The degradation of rabbit, chicken and beef myofibrils by cathepsin L or lysosomal lysates was studied by SDS-polyacrylamide-gel electrophoresis and electron microscopy (EM). Similar degradation patterns were observed for each myofibrillar preparation incubated with cathepsin L, except that myosin heavy chain and tropomyosin of beef were more susceptible than those of rabbit and chicken. Otherwise, troponin T, troponin in I and C-protein were rapidly degraded with slower degradation of titin, nebulin, myosin heavy chain, α-actinin, α-tropomyosin, actin and myosin light chains, LC1 and LC2. However, the component of 30 000 Mr was found to be further degraded to smaller peptides. Degradation at pH 5·5 (approximate post-mortem limit value) was faster than at pH 6·0 but slower than at pH 5·0. A number of new protein bands were identified (130 000, 120 000, 90 000, 85 000, 80 000, 31 000 and 30 000 Mr). The degradation patterns of rabbit myofibrils by rabbit muscle lysosomal lysates were similar to that of myofibrils incubated with purified cathepsin L except for the retention of the 30 000 Mr component and reduced degradation of actin, due presumably to the reduced amount or stability of cathepsin L in the crude enzyme preparations. Electron micrographs revealed that myofibrillar degradation by cathepsin L occurred preferentially at the Z-lines leading to removal of the Z-line proteins and fracturing of the myofibrils at these sites. Catheptic damage was seen to be most rapid in chicken myofibrils and least rapid in beef myofibrils consistent with the more rapid conditioning process in chicken.     

Cathepsin Degradation of Pacific Whiting Surimi Proteins

Cathepsin B was the most active cysteine protease in Pacific whiting fish fillets; cathepsin L was predominant in surimi. Cathepsin L showed highest activity at 55°C in both fish fillets and surimi, indicating its function in myosin degradation during conventional heating of fillets and surimi, gels. Washing during surimi processing removed cathepsin B and H but not cathepsin L. Myosin heavy chain was the primary substrate during autolysis of surimi paste and actin and myosin light chain showed limited hydrolysis during 2 hr incubation. Purified Pacific whiting cathepsin L hydrolyzed myofibrils, myosin and native and heat-denatured collagen. The degradation pattern of myofibrils by the protease was the same as the autolytic pattern of surimi.     

CATHEPSIN D AND ITS EFFECTS ON MYOFIBRILLAR PROTEINS: A REVIEW1

The molecular and enzymatic properties of the extensively studied enzyme cathepsin D are reviewed and additional information concerning its activity presented. Cathepsin D at pH 5.5 (37°C) degraded several myofibrillar proteins. The most rapidly hydrolyzed included titin and perhaps nebulin, myosin heavy chain, and M and C-proteins. The effects of cathepsin D on myofibrillar structure under these conditions included reduction in A band width, cleared central region in the A band, and dislocation of the Z line. Temperature was found to exert a strong influence on activity of cathepsin D and maximum activity was observed at 45°C with both muscle and hemoglobin substrates. Activity was evident at even higher temperatures and approximately 49% remained at 55°C (hemoglobin assay). Low temperature (i.e., < 15°C) however, has been observed to result in almost complete inactivity of the enzyme. The implications of this information for involvement of cathepsin D in postmortem proteolysis and tenderization were discussed.     

鱿鱼肝脏蛋白酶的鉴定及组织蛋白酶L的分离纯化

鱿鱼肝脏含有丰富的蛋白酶,为利用其内源蛋白酶进行可控的酶解,本研究以鲤鱼肌原纤维蛋白为底物对鱿鱼肝脏内源蛋白酶的种类和性质进行了研究。反应体系中添加E-64、1,10-菲啰啉和苯甲基磺酰氟(phenylmethylsulfonyl?fluoride,PMSF)后,肌球蛋白重链(myosin?heavy?chain,MHC)的降解得到了显著抑制,确定了鱿鱼肝脏含有金属类、半胱氨酸类、丝氨酸类3类蛋白酶。半胱氨酸类蛋白酶热稳定性最好,在50℃以上仍然具有较大活性,可将肌原纤维蛋白酶解成小分子质量的降解产物。利用特异性底物对半胱氨酸蛋白酶种类进行鉴定发现,该酶只酶解Z-Phe-Arg-MCA,添加亮抑酶肽后相对酶活性为0%,添加E-64相对酶活性仅存0.6%,初步确定鱿鱼肝脏中的半胱氨酸蛋白酶主要为组织蛋白酶L。最后,通过硫酸铵沉淀、离子交换层析、凝胶过滤对组织蛋白酶L进行分离纯化,在电泳上得到了分子质量约为25?kD单一条带。