Dissociation of natural actomyosin from kuruma prawn muscle induced by pyrophosphate

Effect of pyrophosphate (PP) on the dissociation and stability of natural actomyosin (NAM) from kuruma prawn muscle was studied in comparison with adenosine 5′-triphosphate (ATP). In the presence of PP up to 5 mM, NAM exhibited lower Mg²⁺-ATPase activity (P < 0.05), while no marked change was observed in NAM treated with ATP at all concentrations tested (0.25–10 mM) (P > 0.05). Ca²⁺-ATPase activity of NAM treated with 5 mM PP decreased markedly when incubated at temperatures greater than 30 °C, suggesting lowered thermal stability of the liberated myosin molecule. Nevertheless, Ca²⁺-ATPase activity of ATP-treated NAM was similar to the control NAM. In the presence of 5–10 mM MgCl₂, NAM treated with 5 mM PP underwent dissociation effectively, as evidenced by a greater decrease in Mg²⁺-ATPase activity as well as an increased band intensity of actin released. Therefore, addition of PP in combination with MgCl₂ was more effective than was ATP in dissociating the actomyosin complex of prawn muscle.     

Participation of cysteine protease cathepsin L in the gel disintegration of red bulleye (Priacanthus macracanthus) surimi gel paste

BACKGROUND: Endogenous proteases, among them cysteine‐type proteases, are reported to contribute to gel disintegration, resulting in kamaboko of poor quality. Severe gel disintegration occurs in red bulleye surimi gel paste. The objective of this study was to clarify the participation of cysteine protease cathepsin L in the gel disintegration of red bulleye surimi. The surimi was made into kamaboko with and without cathepsin L inhibitors. To confirm its hydrolysis action, crude cathepsin L was also extracted and added to the surimi to make kamaboko. RESULTS: The gel strength of kamaboko obtained by both one‐step (50 °C, 2 h) and two‐step (50 °C, 2 h + 80 °C, 20 min) heating was very low in the absence of inhibitors. Protease inhibitors E‐64 and leupeptin were found to enhance the gel strength considerably. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the hydrolysis of kamaboko was promoted by crude cathepsin L and inhibited by E‐64 and leupeptin. The gel strength of two‐step heated kamaboko was increased from 12 to 110 and 130 g cm^?2 by E‐64 and leupeptin respectively at a concentration of 0.2 g kg^?1 surimi. CONCLUSION: Endogenous cathepsin L of red bulleye surimi participates in gel disintegration during kamaboko processing. It does so by degrading the myosin heavy chain of actomyosin and consequently hindering the gelation of red bulleye surimi. Copyright © 2009 Society of Chemical Industry     

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.     

Oxidation desensitizes actomyosin to magnesium pyrophosphate-induced dissociation

This study aimed to establish the influence of protein oxidation on the ability of magnesium pyrophosphate (PP) to dissociate actomyosin. Actomyosin isolated from pork muscle then suspended in 0.1 M NaCl at pH 6.2 was oxidatively stressed with 10 μM FeCl₃/0.1 mM ascorbate/1 mM H₂O₂ for 6 or 12 h at 4 °C. Protein oxidation was evidenced by the loss of myosin and actin, the concomitant formation of disulphide-cross-linked polymers, and elevated myosin ATPase activity. The intrinsic viscosity of oxidized actomyosin had a weaker response to PP-Mg²⁺ than that of non-oxidized actomyosin, indicating the suppression of actomyosin dissociation. Moreover, oxidized actomyosin solutions were devoid of small particles (<10 nm) and the stressed actomyosin exhibited weaker binding of PP-Mg²⁺ than non-oxidized, which further suggested a reduced myosin–PP interaction and subsequent dissociation of the actomyosin complexes.     

Thermal Gel Degradation (Modori) in Sturgeon (Acipenseridae) Surimi Gels

Sturgeon meat has been found to be suitable as surimi raw materials. The present study determined the modori phenomenon in sturgeon surimi gels and identified its relationship with cathepsins. In all heat‐treated gels (25 to 90 °C, at 5 °C intervals), the 40 °C‐incubated sturgeon surimi gel showed the weakest gel properties and water‐holding capacity (P < 0.05), a rough protein gel network under SEM, and the highest protein solubility and trichloroacetic acid‐soluble peptides content (P < 0.05). SDS‐PAGE indicated that the myosin heavy chain band of sturgeon surimi gels was almost completely degraded at 40 °C. Moreover, the highest cathepsin L activity was observed in 40 °C‐treated sturgeon surimi gels (P < 0.05). Our results suggested that the modori phenomenon in sturgeon surimi gels occurred at 40 °C, which was partially attributed to cathepsin L, thereby allowing for the better exploitation and utilization of sturgeon surimi.     

Modification of volatile profiles of silver carp surimi gel by immersion treatment with hydrogen peroxide (H2O2)

The effect of immersion treatment with different concentration (0, 1.0, 5.0, 20.0 and 100.0 mm ) of hydrogen peroxide (H₂O₂) on volatile profiles and gel properties of silver carp surimi gel were investigated. The results showed that the immersion treatment with H₂O₂ obviously changed flavour characteristic of surimi gel depending on H₂O₂ concentration and different layers (outside, middle and inner). The contents of typical fishy off-flavour substances such as 1-octen-3-ol, heptanal, nonanal, octanal and decanal were largely reduced in the outside layer of surimi gel after H₂O₂ treatment, with no obvious change in the inner portion. The gel strength and whiteness were increased after H₂O₂ treatment with concentration higher than 20 mm . The content of carbonyl compounds was positively correlated with the concentration of H₂O₂, suggesting that the volatiles variation of surimi gel probably be highly related to the protein oxidation. In conclusion, immersion treatment with H₂O₂ provided a promising method to remove fishy odour of surimi gel with improved gel properties.     

Trypsin Inhibitory Activity and Gel‐Enhancing Effect of Sarcoplasmic Proteins from Common Carp

Abstract: Proteinase inhibitory activity of sarcoplasmic protein (SP) extracted from common carp (Cyprinus carpio) muscle and its gel‐improving ability were investigated. SPs displayed 89% and 54% inhibitory activity toward trypsin at 40 and 65 °C, respectively. Protein bands with molecular mass of 69, 50, 44, 41, and 35 kDa appeared on trypsin inhibitory activity staining under nonreducing condition when incubated at 40 °C, while 2 protein bands at 54 and 35 kDa were observed at 65 °C. Addition of SP at 0.18 g protein/100 g increased textural properties of threadfin bream surimi gel. However, when SP was added in combination with various CaCl₂ concentrations (0.1% to 0.5%) it did not further improve textural properties as compared to the addition of SP alone. Retention of myosin heavy chain of threadfin bream surimi was greater with the addition of SP. These results indicated that the gel‐enhancing effect of common carp SP was due to the inhibitory activity toward endogenous trypsin‐like proteinases in threadfin bream surimi. Practical Application: Sarcoplasmic protein from common carp muscle could be used as a functional protein ingredient that minimizes muscle proteolysis and improves textural properties of surimi containing trypsin‐like endogenous proteinases.     

不同辅料对草鱼鱼糜品质的影响

为了研究不同辅料对鱼糜品质的影响,以新鲜草鱼鱼糜为原料,分别加入糖类辅料和蛋白类辅料,对鱼糜的组织蛋白酶活性、pH值、凝胶强度、折叠柔韧性、持水性能和白度值进行测定。结果表明：不同辅料对草鱼鱼糜组织蛋白酶B、L产生不同抑制效果,对凝胶强度提升最大的辅料为蔗糖＋山梨醇;马铃薯淀粉加入草鱼鱼糜后,鱼糜凝胶的持水性最强;除紫山药和魔芋精粉外,其余辅料对鱼糜白度值的影响无显著性差异;马铃薯淀粉、蔗糖＋山梨醇、魔芋精粉和胶原蛋白能明显增强草鱼鱼糜折叠等级。     

辅料改善草鱼鱼糜凝胶性能的机理

以草鱼为原料,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、质构分析和化学作用力测定等方法,研究辅料改善草鱼鱼糜凝胶性能的机理。结果表明：草鱼鱼糜蛋白自溶最适温度和pH值分别为65 ℃和6.5;马铃薯淀粉对鱼糜蛋白交联结构的填充能力强于胶原蛋白,而胶原蛋白对鱼糜组织蛋白酶的抑制效果好于马铃薯淀粉;辅料加入后,改变了鱼糜蛋白间的离子键、氢键、疏水性相互作用和二硫键等化学作用力,从而达到改善鱼糜品质的目的。     

Transglutaminase from Streptoverticillium ladakanum and application to minced fish product

The transglutaminase (TGase) from Streptoverticillium ladakanum was purified to electrophoretic homogeneity after ammonium sulfate fractionation and Blue Sepharose Fast Flow chromatography. The molecular weight of the purified TGase was 30.5 kDa estimated by Superdex 75HR gel filtration, and 37.5 kDa by SDS-PAGE. This enzyme, with optima at pH at 6.0 and 50°C was very stable at pH 5.0–7.0. It was strongly inhibited by PCMB, PMSF, Pb²⁺, Zn²⁺ and Cu²⁺, but not affected by EDTA and Ca²⁺. This suggested that the purified TGase was calcium-independent and its active center contained cysteine. It catalyzed the crosslinking of fish myosin heavy chain and substantially increased the gel strength of mackerel surimi.     

含冷冻带鱼糜的复合鱼糜凝胶特性的研究

通过质构测定和SDS-PAGE方法,分析了冷冻带鱼糜加入到白鲢鱼糜后带鱼糜对复合鱼糜凝胶特性的影响。结果表明,非热处理带鱼糜可显著降低复合鱼糜的凝胶强度,而凝胶劣化特性则被显著提高;带鱼糜经热处理后使复合鱼糜的凝胶强度和凝胶劣化指数都得到明显改善;加入带鱼糜的蛋白酶提取液经55℃反应240min后肌球蛋白重链（Myosin Heavy Chains,MHC）全部消失。证明冷冻带鱼糜中含有蛋白酶能够降低复合鱼糜凝胶特性。      

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.     

Color improvement by titanium dioxide and its effect on gelation and texture of proteins recovered from whole fish using isoelectric solubilization/precipitation

Whiteness is a critical attribute for restructured fish products such as surimi seafood. However, the whiteness of gels made from proteins recovered from fish processing by-products or whole fish using isoelectric solubilization/precipitation is poor. The by-products and whole fish contain bones, scales, skin, etc. that affect gel color. Therefore, whiteness needs to be improved if marketable products are to be developed from recovered proteins. The objectives of this study were to determine effects of titanium dioxide (TiO₂) on: (1) color; (2) texture; and (3) viscoelasticity (G′) of gels made from isolated carp proteins and Alaska pollock surimi. Carp proteins were recovered with isoelectric solubilization/precipitation. TiO₂ was added to carp proteins at 0-0.5 g/100 g. TiO₂ was not added to surimi. Due to much higher (P < 0.05) yellowness (b^∗) and lower (P < 0.05) lightness (L^∗), the whiteness of carp gels without TiO₂ was lower (P < 0.05) than surimi gels. TiO₂ at ≥ 0.2 g/100 g resulted in better (P < 0.05) whiteness of carp gels than surimi gels without chalky and artificially white appearance. TiO₂ did not affect texture or viscoelasticity. This research demonstrates that whiteness of restructured fish products based on proteins recovered from whole fish via isoelectric solubilization/precipitation can be similar to the whiteness of surimi seafood.     

Quality Improvement of Mackerel Surimi with NADPH-Sulfite Reductase from Escherichia coli

The application of NADPH‐sulfite reductase, produced from Escherichia coli, on mackerel surimi was investigated. The activity of this reductase was very stable. There was about 70%, 80%, and 90% activity left even after 8–wk storage at 25 °C and 12–wk storage at 4 or ‐30 °C, respectively. Increase in mackerel‐surimi gel properties containing reductase was consistent with the amounts of reactive sulfhydryl groups detected. SDS‐PAGE indicated that NADPH‐sulfite reductase could reconstitute myosin heavy‐chain and increase soluble actomyosin. From the data obtained, E. coli NADPH‐sulfite reductase effectively improved the gel properties of mackerel surimi.     

Participation of cathepsin L in modori phenomenon in carp (Cyprinus carpio) surimi gel

Cathepsin L (Cat L) in carp (Cyprinus carpio) dorsal muscles was purified and its molecular weight determined by SDS polyacrylamide gel electrophoresis (SDS–PAGE) was 36 kDa. Its optimal temperature and pH were 50 °C and 5.5, respectively. The results of the effects of specific substrates, activators and inhibitors on the enzymatic activity showed that Cat L belongs to the family of cysteine proteinases containing thiol. Compared to the control, the gel strength of surimi with the addition of purified Cat L decreased significantly by 24.33% while that of surimi with both purified Cat L and inhibitors increased by 13.7% and 21.6%, respectively, suggesting the participation of Cat L in the modori phenomenon occurring in carp surimi. Both the SDS–PAGE electrophoretic pattern and microstructure figure revealed that Cat L could hydrolyse the main protein in carp surimi and was one of the enzymes involved in the modori phenomenon.     

Proteinase in Pacific Whiting Surimi Wash Water: Identification and Characterization

A proteinase in Pacific whiting surimi wash water (SWW) was characterized to be cathepsin L based on molecular mass (M_r), substrate specificity, and SDS-substrate gel electrophoresis. The proteinase was highly active on Z-Phe-Arg-NMec, and the native M_r was 27,400 based on size exclusion (SEC)-HPLC. Acidification of the SEC-HPLC fractions showed a two-fold increase in activity on Z-Phe-Arg-NMec. SWW proteolytic activity was found at M_r 54,200 on SDS-substrate gel. However, acidification shifted the activity zone to M_r 39,500 corresponding to cathepsin L. No evidence of activity by calpain or cathepsin B or H was found in Pacific whiting SWW.     

鲢鱼与金线鱼混合鱼糜的凝胶特性

为研究鲢鱼与金线鱼混合鱼糜的凝胶特性,本实验对混合鱼糜凝胶的凝胶强度、持水性、蒸煮损失、白度值、横向弛豫时间、微观结构和肌原纤维蛋白进行分析。结果表明：混合鱼糜的白度值较纯金线鱼鱼糜显著提高（P＜0.05）,且鲢鱼鱼糜与金线鱼鱼糜质量比5∶1混合后的凝胶白度值高于纯鲢鱼鱼糜;此配比下的混合鱼糜凝胶性能最好,其中凝胶破断力、凹陷距离、凝胶强度和持水性较鲢鱼鱼糜分别提高了18.33%、27.29%、51.01%和5.65%,较金线鱼鱼糜分别提高了5.84%、19.50%、26.49%和3.45%;该配比下的混合鱼糜凝胶的蒸煮损失较鲢鱼鱼糜和金线鱼鱼糜分别降低了15.88%和7.48%。低场核磁共振分析显示鲢鱼鱼糜与金线鱼鱼糜质量比5∶1混合后的鱼糜凝胶的横向弛豫时间T22比鲢鱼鱼糜凝胶T22缩短了10.34 ms。通过扫描电镜观察发现鲢鱼鱼糜与金线鱼鱼糜质量比5∶1混合时可形成高度均匀、致密的空间凝胶网络结构。肌原纤维蛋白凝胶电泳图显示该配比下肌球蛋白重链（myosin heavy china,MHC）发生交联,大分子聚集体形成,进入凝胶中MHC更少,条带更细窄。     

Gel properties,physicochemical properties,and sensory attributes of white leg shrimp (Litopenaeus vannamei) surimi gel treated with sodium chloride (NaCl) substitutes

This study investigated the effects of different NaCl substitutes on the gel properties, physicochemical properties, and sensory attributes of shrimp surimi gel to produce high-quality reduced-salt shrimp surimi gel. The results showed that CaCl₂, calcium ascorbate (Vc-Ca), and L-arginine (L-Arg) could significantly improve the gel strength and texture of shrimp surimi gel compared to NaCl. The addition of CaCl₂, Vc-Ca, and L-Arg significantly increased the number of disulphide bonds and the content of β-sheet structures compared to NaCl. The electrophoretic analysis revealed that CaCl₂, Vc-Ca, and L-Arg had protective effects on the myosin heavy chain during thermal gelation. Additionally, CaCl₂ and L-Arg promoted the cross-linking of myofibrillar proteins to form the denser and less porous microstructures, and thus improved the gel properties of shrimp surimi gel. In general, the introduction of L-Arg as a substitute for NaCl acquired the best gel properties and sensory attributes of shrimp surimi gels, followed by CaCl₂ and Vc-Ca.     

Protein cross-linking ability of sarcoplasmic proteins extracted from threadfin bream

Sarcoplasmic proteins extracted from threadfin bream (TB, Nemipterus sp.) contained transglutaminase (TGase) activity exhibiting a pH optima at 7.5. Activity increased with CaCl₂ and reached the maximum at 5 mmol/L, showing the Ca²⁺-dependent characteristic. TGase activity staining on native-polyacrylamide gel electrophoresis (native-PAGE) showed two fluorescent bands catalyzing incorporation of monodansylcadavarine (MDC) into di-methylated casein (DMC). However, both fluorescent bands showed only one major protein band with a molecular weight of about 66 kDa on sodium dodecylsulfate-PAGE (SDS-PAGE), suggesting the presence of isozymes. The TB sarcoplasmic protein (TBSP) concentrated using a 30-kDa ultrafiltration membrane induced cross-linkings of bovine serum albumin when incubated at 25 °C for 6 h. Addition of the concentrated TBSP at 1.6 g protein/100 g along with 0.1 g CaCl₂/100 g resulted in a 1.8-fold increase in the breaking force of the proteinase-laden lizardfish surimi pre-incubated at 37 °C for 20 min. TBSP effectively minimized proteolysis of lizardfish surimi at 37 °C. TBSP could be a promising protein additive that improves textural properties of proteinase-laden surimi.     

鱼种和亲水胶体对鱼糜制品凝胶性质的影响

考察了添加复合亲水胶体对鲢鱼、带鱼、金线鱼等鱼糜制品凝胶特性的影响。结果发现,添加亲水胶体后,带鱼鱼糜制品凝胶强度从58.38 g·cm提高至107.27 g·cm,金线鱼鱼糜制品从328.68 g·cm下降至137.55 g·cm,但鲢鱼鱼糜制品没有明显变化。另一方面,添加亲水胶体后鱼糜制品的硬度、粘结性、咀嚼性等发生下降,但水分含量、持水性、蒸煮吸水率均有显著提高。而且,添加亲水胶体后带鱼鱼糜制品中肌球蛋白重链的降解受到一定的抑制。SEM结果发现亲水胶体可以填充到带鱼和鲢鱼鱼糜制品中,但在金线鱼鱼糜凝胶结构中容易形成胶体块状,导致凝胶强度下降。