Enhanced expression of chicken cystatin as a thioredoxin fusion form in Escherichia coli AD494(DE3)pLysS and its effect on the prevention of surimi gel softening

The DNA encoding chicken lung cystatin was ligated into a thioredoxin-pET 23a+ expression vector and transformed into Escherichia coli AD494(DE3)pLysS. A high level of soluble recombinant thioredoxin-cystatin (trx-cystatin) was expressed in the cytoplasm of the E. coli transformant. As compared with recombinant cystatin (trx-free), a 38.7% increase of inhibitory activity in the soluble fraction was achieved by introducing the trx fusion protein. Trx-cystatin was purified to electrophoretical homogeneity by 3 min of heating at 90 degrees C and Sephacryl S-100 chromatography. The molecular mass of trx-cystatin was 29 kDa, which was the expected size based on its composition of recombinant trx (16 kDa) and chicken cystatin (13 kDa). The purified trx-cystatin behaved as a thermally stable and papain-like proteinase inhibitor comparable to either recombinant or natural chicken cystatins. The inhibitor could inhibit the gel softening of mackerel surimi.     

Effect of glycosylation modification (N-Q-(108)I --> N-Q-(108)T) on the freezing stability of recombinant chicken Cystatin overexpressed in Pichia pastoris X-33

The cDNAs encoding chicken cystatin and its N-glycosylation-modified mutant (Asn(106)-Ile(108)-->Asn(106)-Thr(108)) were cloned into the pGAPZ alpha C expression vector, using the GAP as promoter and Zeocin as resistant agent, and transformed into Pichia pastoris X-33 expression host. The effect of N-glycosylation on the stability of recombinant chicken cystatin was investigated. A large quantity of recombinant chicken cystatin and the Asn(106)-glycosylated cystatins were expressed and secreted into broth using alpha-factor preprosequence. The K(i) of the recombinant chicken cystatin (0.08 nM) was similar to that of wild-type chicken cystatin (0.05 nM). They acted as a competitive inhibition reaction against papain. According to the K(i), the inhibition ability of Asn(106)-glycosylated mutant cystatin (K(i) = 9.5 nM) was weaker than that of the wild-type one. However, N-glycosylation at Asn(106) substantially enhanced the freezing stability of recombinant chicken cystatin overexpressed in P. pastoris.     

High-level production of recombinant chicken cystatin by Pichia pastoris and its application in mackerel surimi

A high level of the secreted form of recombinant chicken cystatin was expressed in Pichia pastoris X-33 by chromosomal integration of multiple copies of an expression cassette containing chicken cystatin under the control of glyceraldehyde-3-phosphate dehydrogenase promoter. The inhibition ability of the recombinant for papain-like proteinase was found to correspond to those of natural chicken cystatin. The recombinant cystatin substantially inhibited the proteolysis of myosin and gel softening, which consequently improved the gel properties of mackerel surimi.     

Overexpression of the soluble form of chicken cystatin in Escherichia coli and its purification

A cDNA encoding chicken cystatin was cloned into the pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3)pLysS expression host. An active soluble form of cystatin was expressed in the cytoplasm of E. coli induced by isopropyl beta-D-thiogalactopyranoside. The recombinant chicken cystatin was purified to electrophoretic homogeneity by a simple and rapid method involving heat treatment and Sephacryl S-100 gel filtration chromatography. The recombinant cystatin behaved as a thermal-stable protein and exhibited papain-like protease inhibition activity comparable to the natural chicken cystatin.     

Cloning, functional expression, and characterization of cystatin in sesame seed

A cDNA fragment encoding cystatin, a cysteine protease inhibitor, was obtained from maturing sesame seeds. The clone was constructed in a nonfusion or fusion vector and then overexpressed in Escherichia coli. The recombinant cystatins were found in the soluble fraction of cell extract and were demonstrated to be functionally active in a reverse zymographic assay. The corresponding endogenous 22 kDa cystatin of low abundance in mature seeds was purified to homogeneity via a papain-coupling affinity column and confirmed by western blotting with antibodies against the recombinant cystatin. Both endogenous and recombinant cystatin proteins showed effective inhibitory activities against papain with K(i) values of 7.89 x 10(-8) M and 2.77 x 10(-8) M, respectively. Immunodetection indicated that cystatin was specifically expressed in maturing seeds and rapidly degraded in germination. Accordingly, zymographic and inhibition analyses showed that sesame cystatin could not inhibit the de novo synthesized proteases in germinating seeds. It is suggested that sesame cystatin may play a role in the regulation of endogenous cysteine proteases during seed maturation and germination.     

Expression of soluble form carp (Cyprinus carpio) ovarian cystatin in Escherichia coli and its purification

A DNA encoding thioredoxin-mature carp ovarian cystatin (trx-cystatin) fusion protein was ligated into a pET-23a(+) expression vector and then transformed into Escherichia coli AD494(DE3) expression host. After induction by isopropyl beta-D-thiogalactopyranoside, a high level of the soluble form of recombinant trx-cystatin was expressed in the cytoplasm of E. coli. The recombinant trx-cystatin could be purified by Ni(2+)-NTA agarose affinity chromatography. The molecular mass (M) of the recombinant trx-cystatin was approximately 28 kDa composed of recombinant thioredoxin (16 kDa) and recombinant mature carp ovarian cystatin (12 kDa). Both recombinant trx-fused and mature carp ovarian cystatins were stable at pH 6-11. No obvious decrease in activity was observed even after 5 min of incubation at 60 degrees C. They exhibited papain-like protease inhibition activity comparable to that of the mature carp ovarian cystatin, which could inhibit papain and mackerel cathepsins L and L-like, but not cathepsin B.     

Interactive effects of microbial transglutaminase and recombinant cystatin on the mackerel and hairtail muscle protein

Interactive effects of microbial transglutaminase (MTGase) and recombinant cystatin on the mackerel and hairtail water soluble protein (WSP), salt soluble protein (SSP), and muscle protein (MP) were investigated. According to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzymic activity analyses, cross-linking of mackerel and hairtail myosin heavy chain and low molecular mass compounds and formation of epsilon-(gamma-glutamyl)lysine cross-links were observed on samples with MTGase, while the recombinant cystatin could effectively inhibit the cathepsins and subsequently prevent degradation of proteins during setting. The cathepsins and MTGase activities in WSP, SSP, and MP solutions decreased, but the recombinant cystatin activity increased during setting at 45 degrees C.     

Incorporation and stabilization of omega-3 fatty acids in surimi made from cod, Gadus morhua

Surimi containing omega-3 fatty acids from algal oil was prepared by the addition of oil-in-water emulsions or bulk oil. Emulsion and bulk oil were added separately to surimi to provide approximately 500 mg of omega-3 fatty acids per serving of surimi (85 g). Addition of the emulsion had no effect on surimi gel strength, whereas bulk oil decreased gel strength an average of 31%. All surimi treatments containing algal oil increased in Hunter b values due to the presence of carotenoids in the oil. Among cryoprotectants, sodium tripolyphosphate was the major surimi additive responsible for retarding the formation of lipid hydroperoxides and thiobarbituric acid reactive substances (TBARS). Lipid hydroperoxide and TBARS formation was lower in surimi containing bulk oil compared to surimi with emulsified oil. Both EDTA and lipid soluble antioxidants were able to decrease lipid oxidation in surimi fortified with omega-3 fatty acids. This suggests that surimi containing nutritionally beneficial omega-3 fatty acids could be developed with good oxidative stability and gel strength.     

用低场核磁共振研究盐溶液漂洗对带鱼鱼糜凝胶品质的影响

为了比较单一成分盐溶液(质量分数为0.30%Na Cl、0.06%Ca Cl2或0.50%柠檬酸钠)和复合盐溶液(0.15%Na Cl、0.04%Ca Cl2、0.35%柠檬酸钠)漂洗对带鱼鱼糜凝胶水分分布及其凝胶特性的影响,该文运用低场核磁技术测定鱼糜凝胶的水分弛豫时间和水分质子密度,并结合鱼糜凝胶含水率、持水性、凝胶强度及其电镜扫描图,对盐溶液漂洗效果进行探究。鱼肉经单一成分0.30%Na Cl、0.06%Ca Cl2或0.50%柠檬酸钠溶液漂洗,柠檬酸钠组鱼糜凝胶中的氢质子束缚力最大,水分难以迁移,自由水的相对含量最低;与单一成分漂洗相比,经过复合盐溶液漂洗后,鱼糜凝胶水分迁移力相对较低,自由水含量较少,而水分质子密度较高,水分分布均匀;盐溶液漂洗后鱼糜凝胶的持水性、含水率、凝胶强度、凝胶显微结构与鱼糜凝胶中的水分分布相关,利用复合盐溶液漂洗带鱼鱼肉,能更好地改善带鱼鱼糜凝胶品质。研究结果为改善鱼肉漂洗工艺、提高鱼糜品质提供参考。     

不同亲水胶体对带鱼鱼糜凝胶品质的影响

为确定合适的亲水胶体种类及其添加量,以带鱼鱼糜为原料,通过测定凝胶强度、质构(硬度、粘聚性、弹性和咀嚼性)、白度、持水性和凝胶溶解度等指标,分析瓜尔胶、魔芋胶和沙蒿胶3种亲水胶体对带鱼鱼糜凝胶品质的影响。结果表明,瓜尔胶、魔芋胶和沙蒿胶均能提高带鱼鱼糜凝胶强度,但相同添加量下魔芋胶的改善效果最好,而瓜尔胶对鱼糜凝胶强度的影响不显著。魔芋胶能显著改进带鱼鱼糜凝胶的质构特性;瓜尔胶添加量为2.0%时鱼糜凝胶硬度、弹性、咀嚼性、粘聚性比对照组低;沙蒿胶对鱼糜凝胶的粘聚性影响显著,当添加量为1.5%或2.0%时,带鱼鱼糜凝胶的咀嚼性、硬度增加,弹性下降。瓜尔胶、魔芋胶和沙蒿胶均能提高带鱼鱼糜持水性,降低鱼糜凝胶白度和溶解度,但对鱼糜凝胶色泽的影响不显著。扫描电镜结果显示,1.5%魔芋胶处理组形成的鱼糜凝胶表面规则有序、网络结构致密均一。综合比较,添加1.5%魔芋胶能有效改善带鱼鱼糜的凝胶品质。本研究结果为提高带鱼鱼糜制品品质及生产研发提供了一定的理论依据。     

鱿鱼与白鲢鱼混合比例对鱼糜制品凝胶特性的影响研究

为了改善鱿鱼与白鲢鱼混合鱼糜制品的凝胶特性,本研究通过鱼糜制品的感官评价、凝胶强度、持水性、白度、水分分布、肌原纤维蛋白凝胶电泳和光学显微镜观察等指标,分析鱿鱼与白鲢鱼混合比例对鱼糜制品凝胶特性的影响。结果表明,当鱿鱼与白鲢鱼混合比例为3∶4时,凝胶特性最好,其中鱼糜凝胶的凝胶强度及持水性较混合比例为5∶2时分别提高了42.74%和2.93个百分点;感官评价总分、白度值显著高于其他混合比例（P<0.05）,分别为76.3和72.3,不易流动水相对含量显著高于其他混合比例（P<0.05）,且该比例下鱼糜凝胶网络结构致密均一,对水分的束缚能力强。肌原纤维蛋白电泳结果显示,混合比例为3∶4时,肌球蛋白重链交联程度高,形成了大分子聚集体。综上,当鱿鱼与白鲢鱼混合比例为3∶4时,可明显改善混合鱼糜制品的凝胶品质。本研究结果为提高鱿鱼鱼糜制品的凝胶品质及生产研发提供了一定的理论依据。     

金鲳鱼肉和罗非鱼皮明胶改善罗非鱼碎肉鱼糜品质

为了改善罗非鱼碎肉鱼糜制品的品质,研究添加金鲳鱼肉和罗非鱼皮明胶对罗非鱼碎肉鱼糜制品品质的影响。将少量金鲳鱼肉与罗非鱼碎肉混合,制作成鱼糜(金鲳鱼肉质量分别占复合鱼糜总质量的0%、5%、10%、15%、20%),研究鱼糜制品品质的变化规律。通过检测样品的质构、凝胶强度、出水率及颜色发现,随着复合鱼糜中金鲳鱼肉含量的上升,复合鱼糜制品的硬度、胶着性、咀嚼性、弹性、内聚性、凝胶强度、白度均呈现上升趋势,但出水率无显著变化。进一步研究,将罗非鱼鱼肉和金鲳鱼鱼肉以一定比例混合,制作成复合鱼糜,然后加入不同量的明胶,研究复合鱼糜制品品质的变化规律发现,将明胶添加到复合鱼糜制品中会使鱼糜制品的硬度、胶着性、咀嚼性、凝胶强度、白度均上升,出水率下降,但鱼糜的弹性、内聚性、破断距离等差异不显著。试验结果表明,金鲳鱼肉和罗非鱼皮明胶可以用于罗非鱼碎肉鱼糜制品品质的改良。研究结果为改善罗非鱼碎肉鱼糜制品的品质提供了参考。     

高密度CO2处理虾肌球蛋白形成凝胶的临界浓度与凝胶强度

为了探讨高密度CO_2(dense phase carbon dioxide,DPCD)诱导蛋白质形成凝胶的机制,以凡纳滨对虾肌球蛋白为研究对象,研究了DPCD处理压强、温度和时间对虾肌球蛋白形成凝胶的临界浓度和对虾肉糜凝胶强度的影响。研究结果表明:DPCD处理压强和温度对虾肌球蛋白溶液形成凝胶的临界浓度有显著影响,处理时间对肌球蛋白溶液形成凝胶的临界浓度无显著影响,但增加处理时间,可以形成更加紧实的凝胶。在40℃和5~30 MPa时虾肌球蛋白溶液形成凝胶的临界质量浓度为14 mg/mL,在50℃和5、10 MPa时虾肌球蛋白溶液形成凝胶的临界质量浓度为12 mg/mL,在50℃和15~30 MPa时虾肌球蛋白溶液形成凝胶的临界质量浓度为11 mg/mL,在60℃和5~30 MPa时虾肌球蛋白溶液形成凝胶的临界质量浓度为10 mg/mL。DPCD处理压强和温度对虾肉糜的凝胶强度也具有显著影响(P0.05),且随着压强增加和温度升高,虾肉糜凝胶强度呈增加趋势(P0.05);在50℃和25 MPa下处理虾肉糜20 min,形成的凝胶强度较好,达到了(14.28±0.57)N·mm。DPCD处理温度越高,虾肌球蛋白形成凝胶的临界浓度就越低,而虾肉糜形成凝胶的强度越高;DPCD处理压强越高,虽然对虾肌球蛋白形成凝胶的临界浓度影响较小,但能使虾肌球蛋白和虾肉糜形成凝胶的强度增加。从分析中还可以推断,DPCD低压(5~10 MPa)诱导虾肉糜形成凝胶主要是热效应的作用,DPCD较高压强(10 MPa)诱导虾肉糜形成凝胶是热和CO_2分子效应的共同作用。研究结果为进一步阐明DPCD诱导蛋白质形成凝胶的机制提供了基础数据。     

乙酰化二淀粉磷酸酯对再加热鱼糜凝胶冻藏特性的影响

为探究乙酰化二淀粉磷酸酯（ADSP）添加量对再加热鱼糜凝胶冻藏品质的影响,本研究以冷冻阿拉斯加狭鳕鱼糜为原料,改变二段加热的凝胶化过程,对鱼糜进行低温预凝胶-冷冻贮藏-高温再加热处理,分析冻藏期间鱼糜凝胶的微观结构、失水率、水分分布状态、质构特性和动态粘弹性。结果表明,随着冻藏时间的延长,鱼糜凝胶的孔隙变大,失水率增加,不易流动水减少,自由水增加,硬度和胶粘性先下降后上升,粘附性增大,内聚性无变化,动态粘弹性下降。在同一冻藏周期下,随着ADSP添加量的增加,鱼糜凝胶的孔隙变小,失水率降低,水分分布状态较稳定,其中5%和7%添加量的效果较好;质构特性结果显示,ADSP添加量的增加有利于提高再加热鱼糜凝胶品质,虽然添加量为5%和7%的鱼糜凝胶硬度、内聚性和胶粘性基本一致,但5%添加量的鱼糜凝胶的粘附性在冻藏期间开始增加的时间较晚且变化幅度较小;动态粘弹性结果显示,高温加热后期5%添加量的鱼糜凝胶的储能模量最高,其次是7%添加量;在损耗模量中,7%添加量的鱼糜凝胶粘性最高,其次是5%添加量。综合分析认为,添加5%ADSP能有效降低再加热鱼糜凝胶在冻藏期间的品质变化。本研究结果为冷冻鱼糜制品的...     

Plant seed cystatins and their target enzymes of endogenous and exogenous origin

Cystatins are protein inhibitors of cysteine proteinases of the papain family, and those of animal origin have long been studied from medical and physiological aspects. In the meantime, oryzacystatin cloned from rice seeds in 1987 was recognized as the first well-defined cystatin of plant origin. Cloning studies followed to disclose various plant cytstatins including those of corn and soybean origin, their similarities to and differences from animal cystatins being analyzed in detail. Plant seed cystatins are now understood as factors controlling germination by inhibition of endogenous cysteine proteinases. They can also recognize insect midgut proteinases as exogenous target enzymes to control. This paper discusses chemical and phytophysiological relationships between cystatins and their targets.     

Characterization of the proteases involved in gel weakening of beef heart surimi.

The goal of this study was to elucidate the nature and characteristics of the proteases involved in gel weakening of beef heart surimi. Acidic (E1) and neutral (E2) protease extracts were prepared from the surimi. The major active components in E1 were found to be cathepsins B and L. E1 exhibited optimum activity to hydrolyze substrates specific to cathepsins B and B+L at 50 degrees C and pH 5.5. At pH 6.0, it retained approximately 50% of its maximum activity. The catheptic activity of E1 was inhibited almost completely by E-64 and leupeptin. The active component in E2 was unidentified and was not inhibited by cysteine or serine protease inhibitors. However, beef plasma powder effectively inhibited the hydrolysis of FITC-casein and myosin heavy chain by E2.     

Tyrosinase-aided protein cross-linking: effects on gel formation of chicken breast myofibrils and texture and water-holding of chicken breast meat homogenate gels

The effects of Trichoderma reesei tyrosinase-catalyzed cross-linking of isolated chicken breast myofibril proteins as a simplified model system were studied with special emphasis on the thermal stability and gel formation of myofibrillar proteins. In addition, tyrosinase-catalyzed cross-linking was utilized to modify the firmness, water-holding capacity (WHC), and microstructure of cooked chicken breast meat homogenate gels. According to SDS-PAGE, the myosin heavy chain (MHC) and troponin T were the most sensitive proteins to the action of tyrosinase, whereas actin was not affected to the same extent. Calorimetric enthalpy (DeltaH) of the major thermal transition associated with myosin denaturation was reduced and with actin denaturation increased in the presence of tyrosinase. Low-amplitude viscoelastic measurements at constant temperatures of 25 degrees C and 40 degrees C showed that tyrosinase substantially increased the storage modulus (G') of the 4% myofibrillar protein suspension in the 0.35 M NaCl concentration. The effect was the most pronounced with high-enzyme dosages and at 40 degrees C. Without tyrosinase, the G' increase was low. Tyrosinase increased the firmness of the cooked phosphate-free and low-meat chicken breast meat homogenate gels compared to the corresponding controls. Tyrosinase maintained gel firmness at the control level of the low-salt homogenate gel and weakened it when both salt and phosphate levels were low. Tyrosinase improved the WHC of the low-meat and low-salt homogenate gels and maintained it at the level of the corresponding controls of phosphate-free and low-salt/low-phosphate homogenate gels. Microstructural characterization showed that a collagen network was formed in the presence of tyrosinase. Keywords: Chicken myofibrillar proteins; protein modification; cross-linking; tyrosinase; gelation; thermal stability; texture; water-holding capacity; microstructure.     

鱼糜凝胶脆性的力学性能表征与模型建立

为了用客观准确的方法评价鱼糜凝胶的脆性,以求为鱼糜凝胶的质构调控等相关研究奠定基础,该研究采用感官评价和单轴压缩、三点弯曲、穿刺等物性分析方法,对不同交联度的鱼糜凝胶感官脆性和力学指标进行测定,研究鱼糜凝胶感官脆性与交联度和力学指标的关系并建立鱼糜凝胶脆性的表征模型。结果表明,不同交联度的鱼糜凝胶表现出不同的口感,鱼糜凝胶的感官脆性评分随着交联度的增加而增大。当鱼糜凝胶的交联度小于30%时,咀嚼时鱼糜凝胶不具备脆性。当交联度超过30%后,鱼糜凝胶开始出现脆性,且随着交联度的增加其脆性显著增加。而当交联度超过75%时,感官脆性不再明显增加。在交联度30%~75.5%范围内,对鱼糜凝胶脆性进行力学表征并进行多元回归分析,建立了基于破断力、脆裂功、断裂应力、初始切割系数和压缩常数5个参数的鱼糜凝胶的脆性表征方程(R~2=0.981 9),可较好地表征鱼糜凝胶脆性。经验证,该模型预测值与实测值无差异(P0.05),具有较好的准确性和精确度,可客观表征鱼糜凝胶脆性,为开发不同口感鱼糜制品提供理论基础。     

漂洗和斩拌对海鲈鱼肌球蛋白理化特性的影响

为探究漂洗和斩拌对海鲈鱼肌球蛋白理化特性的影响,分别提取原料、漂洗鱼糜、斩拌鱼糜的肌球蛋白,通过测定总巯基、活性巯基、表面疏水性、浊度等基本性质,并结合红外光谱和原子力显微镜(atomic force microscope,AFM)技术对肌球蛋白二级结构和表面形貌进行研究。结果表明:相比原料,漂洗鱼糜肌球蛋白总巯基含量降低19.5%,活性巯基增大63.9%,而斩拌鱼糜肌球蛋白总巯基和活性巯基的质量分数相比漂洗鱼糜分别降低22.6%和66.8%,漂洗鱼糜的肌球蛋白变性程度最大;肌球蛋白浊度和表面疏水性在经过漂洗和斩拌均增大,漂洗对表面疏水性影响更大,斩拌对浊度影响更大;红外光谱研究结果显示,漂洗对二级结构影响更明显,原料经过漂洗后,α-螺旋相对含量降低了33.16%,无规则卷曲相对含量增加了79.42%,β-折叠和β-转角分别增加1.11%和10.38%,斩拌后,鱼糜肌球蛋白二级结构变化率较低;漂洗和斩拌都可改变肌球蛋白的表面形貌,使肌球蛋白聚集簇明显减小,聚集高度增加。研究结果证明,漂洗和斩拌对肌球蛋白理化特性有很大的影响,是鱼糜具有更好的凝胶性能的重要步骤。     

Structural changes in natural actomyosin and surimi from ling cod (Ophiodon elongatus) during frozen storage in the absence or presence of cryoprotectants

Surimi and natural actomyosin (NAM) from ling cod (Ophiodon elongatus) were subjected to frozen storage in the absence or presence of cryoprotectants (sorbitol, sucrose, lactitol, and Litesse, either individually or in combination). Effects of frozen storage were studied for NAM frozen at -10 degrees C for 10 days and for surimi after eight freeze-thaw cycles. A commercial blend cryoprotectant (4% sucrose and 4% sorbitol), individual cryoprotectants at 8%, and optimal blends at 4, 5.5, 6, and 8%, were effective in maintaining the gel strength of surimi and NAM gels. Surimi or NAM frozen in the absence of cryoprotectants or with only 4% individual cryoprotectants, showed increased percent alpha-helical content by Raman analysis. Increased disulfide content was also observed in the treatment without cryoprotectants by the Raman SS stretching band and by chemical determination. Tyrosine residues were in a buried environment before and after freezing for all treatments, and surface hydrophobicity measured by 1-anilinonaphthalene-8-sulfonate decreased after frozen storage in the absence of cryoprotectants.