Contribution of sarcoplasmic proteins to myofibrillar proteins gelation

Surimi, a refined protein extract, is produced by solubilizing myofibrillar proteins during the comminuting and salting stages of manufacturing. The resulting paste gels on heating to produce kamaboko or a range of analog shellfish such as crab claw, filament sticks, fish mushroom, and so on. The myosin molecule is the major myofibrillar protein in gelation. It is believed that washing steps during the traditional surimi process play an important role in enhancing the gel properties of the resultant kamaboko by removing water-soluble (sarcoplasmic, Sp-P) proteins. By contrast, some researchers claim that retaining Sp-P or adding it into the surimi gel network not only does not interfere with the action of myofibrillar proteins during the sol-gel transition step but also improves the gel characteristics of the resultant kamaboko. It seems that retention of Sp-P or their addition into raw surimi does enhance the textural properties of kamaboko gel perhaps by functioning as a proteinase inhibitor, particularly against trypsin and trypsin-like proteinases but this depends on the type of applied surimi process. Among different types of Sp-P, it has been claimed that some proteins such as endogenous transglutaminase (TGase) play a more important role than other Sp-P in bond formation, by catalyzing the cross-linking of myosin heavy chain (MHC) molecules during low-temperature setting of surimi, resulting a more elastic kamaboko gel.     

The effect of soybean trypsin inhibitor on the degradation of myofibrillar proteins by an endogenous serine proteinase of crucian carp

The effect of soybean trypsin inhibitor (STI) on the degradation of crucian carp myofibrillar proteins caused by an endogenous myofibril-bound serine proteinase (MBSP) was studied. Soybean trypsin inhibitor was purified to high homogeneity and mixed with myofibrils and its inhibitory effect on myofibrillar protein degradation was investigated. In the absence of STI, proteolysis of myofibrillar proteins including myosin heavy chain, α-actinin, actin and tropomyosin could be identified after incubation at 55 °C for 5–20 min depending on the kind of the protein. In contrast, in the presence of STI, with final concentration of 0.75 μg/ml, proteolysis of these proteins was greatly suppressed even after incubation for 1 h, suggesting STI is an effective inhibitor in preventing myofibrillar protein degradation caused by a serine proteinase that is quite possibly MBSP. Though STI has disadvantages for food digestion, as a native food grade inhibitor, it is safe as a food additive, especially at low concentration. Because in surimi production, the decrease of elasticity is always accompanied with the degradation of myofibrillar proteins, thus, the present result suggested the possibility that STI could be applicable in surimi production in order to enhance the elasticity that is the quality of the final products.     

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.     

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     

IDENTIFICATION OF A MYOFIBRIL-BOUND SERINE PROTEINASE IN THE SKELETAL MUSCLE OF SILVER CARP

Myofibril‐bound serine proteinase (MBSP) in the skeletal muscle of silver carp was characterized. Myosin heavy chain (MHC) degraded markedly when silver carp myofibril was incubated at 55–60C as shown by SDS‐PAGE. Prolonged incubation of myofibrils also caused the degradation of other myofibrillar proteins such as α‐actinin, actin and tropomyosin to some degree. The results suggest the existence of an endogenous myofibril associated proteinase. Serine proteinase inhibitors (Pefabloc SC and Lima bean trypsin inhibitor) greatly suppressed the degradation of myosin heavy chain, while inhibitors for cysteine, metallo, and aspartic proteinases did not show any effect, indicating that the endogeneous proteinase is a myofibril‐bound serine proteinase.     

Degradation of myofibrillar proteins by a myofibril-bound serine proteinase in the skeletal muscle of crucian carp (Carasius auratus)

A myofibril-bound serine proteinase (MBSP) in the skeletal muscle of crucian carp (Carasius auratus) was identified. Hydrolysis of myofibrillar proteins by the endogenous MBSP was studied. Myosin heavy chain (MHC) was degraded markedly when crucian carp myofibril was incubated at 55 °C, as shown by SDS-PAGE. Prolonged incubation of myofibril at 55 °C also caused the obvious degradation of tropomyosin, while the decomposition of other myofibrillar proteins, such as α-actinin and actin, was slight, as detected by Western blotting. The results suggest the existence of an endogenous myofibril-associated proteinase in crucian carp myofibril, which efficiently cleaves MHC and tropomyosin. Serine proteinase inhibitors (Lima bean trypsin inhibitor, PMSF and benzamidine) greatly suppressed the degradation of MHC, caused by the enzyme, while inhibitors for cysteine, metallo-, and aspartic proteinases showed only partial or incomplete inhibitory effects, indicating that the endogenous proteinase is a serine proteinase. Substrate specificity analysis, using partially purified crucian carp MBSP, suggested that the enzyme is a trypsin-like serine proteinase.     

Cross-Linking of Myosin Heavy Chains from Cod, Herring and Silver Hake During Thermal Setting

Cross-linking of myofibrillar proteins extracted from cod (Gadus morhua), herring (Clupea harengus) and silver hake (Merluccius bilinearis) was studied in 0.6M NaCl, pH 6.5 at 40°C and evaluated turbidimetrically and by SDS polyacrylamide gel electrophoresis coupled with l-ethyl-3-(3-dimethylaminopropyl) carbodiimide as a zero-length crosslinker. Turbidities of heat-treated cod and silver hake myofibril/myosin solutions were significantly higher than those of herring. Electrophoretic results showed that the myosin heavy chain (MHC) was the principal myofibrillar protein cross-linked to form a polymerized complex during the heat treatment. Cross-linking ability of MHC from the three fish species was different; herring MHC formed only small polymers (n≦3) but cod and silver hake MHC formed both small and large polymers (n≦6).     

Effect of preheating temperature on the microstructure of walleye pollack surimi gels under the inhibition of the polymerisation and degradation of myosin heavy chain

BACKGROUND: The physical attribute of heat‐induced gel texture is highly dependent on the microstructure of the gel. In this study the microstructures of walleye pollack surimi gels preheated at various temperatures with and without inhibitors (ethylenediamine‐N,N,N′,N′‐tetraacetic acid, iodoacetamide and leupeptin) were observed with a natural scanning electron microscope. RESULTS: Without inhibitors, gels preheated at 30 °C showed a fine and uniform network structure together with the highest polymerisation of myosin heavy chain (MHC) and the highest gel strength. At 60 °C, gels exhibited a broken, disrupted and loose cluster‐like structure together with the highest degradation of MHC and the lowest gel strength. Under the inhibition of polymerisation and degradation of MHC a fine network was observed up to 40 °C during preheating. However, after a second step of heating at 80 °C the microstructures were disrupted and resembled each other regardless of the preheating temperature. CONCLUSION: Heat‐induced gel formation is related to the polymerisation and degradation of MHC and the microstructure of the gel during preheating. Gelation occurred during setting even under the inhibitory condition, and the formation of covalent bonding by transglutaminase is not essential to the formation of a three‐dimensional network during setting but is essential to the gel strength enhancement effect of setting by subsequent heating at 80 °C. Copyright © 2010 Society of Chemical Industry     

The effect of acidification on myofibrillar proteins

Isolated myofibrillar proteins of mutton, beef and chicken were treated with an acidulent to give various pH values and stored at 5°C for 20 h before analysing the proteins using sodium dodecylsulphate polyacrylamide gel electrophoresis. It was found that protein degradation occurred below pH 4·5, with a decrease in band intensity of all major myofibrillar proteins, particularly myosin heavy chain, and the appearance of new bands at approximately 140 and 70 kd. The degradation had an optimum around pH 3·0 and was inhibited by a high temperature pre-treatment or the presence of the endopeptidase inhibitors pepstatin A and leupeptin. Results are discussed in terms of the action of acid proteinase enzymes.     

Heat-activated proteolysis in lizardfish (Saurida tumbil) muscle

Autolysis of lizardfish mince and washed mince during heating at elevated temperatures was studied. Higher degradation of myosin heavy chain (MHC) was generally observed in mince, compared to washed mince. The highest autolysis was observed at 65 and 60 °C for mince and washed mince, respectively. Autolysis was extended as the incubation time increased by the result of the decrease in MHC band intensity on SDS-PAGE and the increase in TCA-soluble peptides. Autolysis of washed mince was markedly inhibited by E-64 and soybean trypsin inhibitor, suggesting that myofibril-associated proteinases were both cysteine and serine proteinases. Sarcoplasmic proteinase was characterized to be heat-activated alkaline proteinase, which had the optimal pH and temperature of 8.0 and 65 °C, respectively. The preteinase was inhibited by E-64 and activated by reducing agents, which was one of the characteristics of cysteine proteinase. Therefore, endogenous sarcoplasmic and myofibril-associated proteinases play an important role in degradation of myofibrillar proteins of lizardfish during heat-induced gelation, which results in gel weakening.     

Effect of medium temperature setting on gelling characteristics of surimi from some tropical fish

Effects of setting at 25 °C on textural properties and cross-linking of myofibrillar proteins in surimi produced from threadfin bream (Nemipterus bleekeri), bigeye snapper (Priacanthus tayenus), barracuda (Sphyraena jello) and bigeye croaker (Pennahai macrophthalmus) were investigated. Increase in setting time (0–8 h) resulted in a higher breaking force and deformation for all surimi gels tested (P<0.05). Increased gel strength was associated with increase in non-disulfide bond formation and decreased heavy chain myosin. Proteins underwent degradation during setting; however polymerization occurred to a much higher extent, leading to a strengthened gel matrix. Therefore, setting at 25 °C, for an appropriate time, should be a promising means to improve gelling properties of surimi produced from tropical fish.     

Effects of Thermal Sensitivity of Fish Proteins from Various Species on Rheological Properties of Gels

ABSTRACT: Thermal sensitivity of myofibrillar proteins from various fish species were compared at various preincubation (setting) treatments and chopping temperatures. There was a significant species effect on gel texture by both pre‐incubation and chopping temperatures. Whereas Alaska pollock had the highest shear stress values at 5 °C or lower temperatures, big eye, lizardfish, and threadfin bream had higher fracture shear stress values at 25 °C or higher temperatures. Decreased intensity of myosin heavy chain (MHC) for warm‐water species set at 40 °C clearly revealed higher thermal stability of these particular species.     

Proteinase inhibitory activity of sarcoplasmic proteins from threadfin bream (Nemipterus spp.)

BACKGROUND: Thailand is the second largest surimi producer in the world and 50% of surimi is produced from threadfin bream. During surimi processing, sarcoplasmic proteins are removed through water washing and discarded in the waste stream. This study was aimed at investigating the proteinase inhibitory activity of sarcoplasmic proteins. RESULTS: Sarcoplasmic proteins from threadfin bream (TBSP) exhibited inhibitory activity toward trypsin but did not inhibit papain and chymotrypsin. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis under non‐reducing condition stained by trypsin inhibitory activity revealed three protein bands of molecular mass of 95, 41 and 37 kDa. Inhibitory activity of TBSP reached a maximum when subjected to 45 °C and completely disappeared at 60 °C. The breaking force and deformation of lizardfish surimi gel with added TBSP and pre‐incubated at 37° for 20 min increased with additional levels of TBSP (P < 0.05). Trichloroacetic acid–oligopeptide content of lizardfish surimi gel with added TBSP decreased with the addition of 4 g kg^?1 TBSP (P < 0.05). Retention of myosin heavy chain (MHC) increased when TBSP concentration was increased. TBSP effectively protected MHC from proteolysis at 37 °C to a similar extent as egg white powder, but efficacy of TBSP was not observed at 65 °C. CONCLUSION: TBSP could be applied to reduce proteolytic degradation of lizardfish surimi or other surimi associated with trypsin‐like proteinase, rendering an improvement in surimi gelation set at 37–40 °C. Copyright © 2009 Society of Chemical Industry     

Effects of Postmortem Storage and Temperature on Muscle Protein Degradation: Analysis by SDS Gel Electrophoresis

The proteolytic breakdown of the major contractile proteins of bovine longissimus muscle was examined during postmortem storage by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Samples of muscle stored at 4°C for 14 days exhibited little proteolysis of the major contractile proteins; however, samples stored at 37°C for 1 day showed significant degradation of myosin heavy chains and almost complete proteolysis of this protein by day 14. Major degradation products of the myosin heavy chains included a series of polypeptides having molecular weights between 145,000 and 125,000. These experiments demonstrate that substantial degradation of the myosin heavy chain and other muscle proteins can occur during the storage of meat, and this phenomenon was highly temperature dependent.     

Degradation Kinetics of Myosin Heavy Chain of Pacific Whiting Surimi

Pacific whiting surimi paste was ohmically heated to investigate degradation of myosin heavy chain (MHC) caused by endogenous proteinase over a range of 40–85°C and 0.5–35 min. Degradation was best described with an apparent reaction order of 1.4. Changes of degradation rate increased with temperature and reached a maximum at 57°C. Then, rate of MHC degradation decreased with higher temperature and reached a minimum at 75°C. E_a values of activation and inactivation zone were 142.3 and 83.1 kJ/mol, respectively. Generally, failure shear stress and shear strain increased linearly with MHC content. Proteolytic degradation of actin exhibited the same trend as that of MHC but at a slower rate. The synergistic effect of actin in the gelation of whiting surimi was predominant at ≥75°C.     

Effect of heating temperature and duration on the texture and protein composition of Bighead Carp (Aristichthys nobilis) muscle

The effect of heating temperature and duration on the texture and protein composition of bighead carp (Aristichthys nobilis) muscle was investigated. Samples were heated at 90°C, 100°C, 110°C, and 120°C for different times. The results showed that cooking loss increased significantly with increasing temperature and heating time, and a fractional conversion model was used to describe the increase. Hardness, chewiness, and gumminess exhibited a similar four-phase change with two peaks at 90°C, 100°C, and 110°C, and the trend was not observed at 120°C. Analysis of sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) revealed that contents of myosin heavy chain decreased entirely to disappear and contents of actin significantly decreased. Increasing the intensity of the heavy molecular weight bands indicated the aggregation of myosin. Content of water-soluble proteins presented an increasing trend, which reflected the protein degradation and the release of low-molecular-weight compounds.     

Proteolysis Affects Thermal Gelation of Squid Mantle Muscle

Thermal gelation of squid mantle muscle was studied by means of gel strength measurement and SDS-polyacrylamide gel electrophoresis. The gel strength of thermally induced squid meat gel decreased when squid meat paste was heated in two steps (35°C for 30 min followed by 90°C for 30 min). This decrease in gel strength was effectively depressed by the addition of protease inhibitors such as ethylenediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), and soybean trypsin inhibitor, indicating that me-tallo- and/or serine-proteases were mainly involved in the deterioration of squid meat gel prepared by two-step heating. SDS-PAGE analysis demonstrated that EDTA or PMSF suppressed the degradation of myosin heavy chain.     

Original article: Gel properties of red tilapia surimi: effects of setting condition,fish freshness and frozen storage

This study aimed to determine effects of setting condition, fish freshness and storage time of frozen surimi on properties of red tilapia surimi gel. To investigate the effect of setting condition, a combination of eight setting temperatures (35–70 °C) and four setting times (30–120 min) was used. Maximum breaking force, deformation and gel strength were obtained after the gel had been set at 40 °C for 90 or 120 min. Setting at 65 °C resulted in the lowest obtained gel strength, because of proteolytic degradation of myosin heavy chain. Increasing storage time of raw fish material in ice caused a significant decrease in gel strength of the resultant surimi gel (P < 0.05). Gels produced from surimi kept in frozen storage for up to 9 months also exhibited reduced gel strength, with a concomitant increase in the expressible drip, with increasing storage time (P < 0.05).     

Identification of myofibrillar substrates for μ-calpain

To identify myofibrillar substrates of μ-calpain under post-mortem conditions, a combination of SDS–PAGE, two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) was used. Purified myofibrils were incubated with μ-calpain under post-mortem-simulated conditions for two or four days at 4 °C. The resulting protein changes were analyzed by SDS–PAGE and 2DE. The μ-calpain-mediated protein changes were identified by peptide-mass mapping using MALDI-TOF MS and revealed that desmin, actin, myosin heavy chain, myosin light chain I, troponin T, tropomyosin 1, tropomyosin 4, thioredoxin and CapZ are all degraded in vitro by μ-calpain. The findings that actin and myosin heavy chain are substrates of μ-calpain were rather surprising, as it has previously been reported that these proteins are resistant to μ-calpain degradation. However, both actin and myosin heavy chain are poor substrates compared with desmin.     

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。