Gelation of mixed myofibrillar/wheat gluten proteins treated with microbial transglutaminase

Gluten (Glu) and an acid-extracted protein fraction (AF) from wheat flour were mixed (1:1 ratio) with myofibrillar protein (MP) and treated with microbial transglutaminase (MTGase) to observe the effect on heat-induced gelation. Dynamic rheological properties and thermal denaturation patterns of treated samples were measured, respectively, with an oscillatory rheometer and a differential scanning calorimeter. The storage modulus (G′) of control MP sample (no MTGase), with a value of 533 Pa at end of heating (77 °C), was not affected (P > 0.05) by Glu nor by AF. However, mixed protein samples after the MTGase treatment produced higher gel elasticity values that differed (P < 0.05) between samples (1355, 1700 and 1875 Pa at 77 °C for MP, MP/AF, and MP/Glu, respectively). The MP sample underwent three endothermic transitions (peaking at 61.5, 68.0, and 78.5 °C) during thermal scan. The treatment with MTGase and/or addition of Glu or AF tended to lower the temperature for the first transition but raised the temperature for the third transition, suggesting possible interactions of the muscle with non-muscle proteins.     

Jumbo squid (Dosidicus gigas) myofibrillar protein concentrate for edible packaging films and storage stability

Properties and storage stability of two different jumbo squid myofibrillar protein-based films were investigated. Myofibrillar proteins were extracted by isoelectric precipitation after acidic and alkaline solubilization, obtaining the same extraction yield. During extraction the edible fraction, which was discarded during mantle skinning, was recovered, and the fishy flavour produced by nitrogen and other undesirable compounds was removed, although some sarcoplasmic proteins were lost simultaneously. In alkaline-Concentrate (C), myosin unfolding led to water resistant films, less water vapour permeable and more mechanically resistant than acidic–Films (F); whereas acidic-C protein hydrolysis resulted in more transparent and soluble films, with higher protein release (∼80 g/L). During 4 months of storage some structure reorganization occurred, and both films incremented their yellowish tendency, especially the acidic-F, being this attributed to a Maillard reaction with plasticizers. After storage time, water solubility increased in C-films. While acidic-F aggregation led to a protein release reduction and tensile strength improvement, alkali-F became weak and brittle, loosing transparency. C-films offered different filmogenic properties, proving to be promising biodegradable packaging materials.     

Emulsion properties of pork myofibrillar protein in combination with microbial transglutaminase and calcium alginate under various pH conditions

In this study, the effects of microbial transglutaminase (MTG) and calcium alginate (CA) systems in combination with soybean oil on the emulsion properties of porcine myofibrillar protein (MP) were evaluated under various pH conditions. MTG was shown to improve emulsifying capacity and creaming stability, which increased with increasing pH values up to 6.5. The CA did not influence emulsifying capacity, but it improved the creaming stability of the MP-stabilized emulsions. Both MTG and CA enhanced the rheological properties, but their effects on the physical characteristics of the protein evidenced an opposite trend in relation to pH, i.e., the MTG system improved both the emulsion and gelling properties with increasing pH, whereas the CA system was effective when the pH was lowered. By combining the two MP gelling systems, a stable and pH-insensible emulsion could be produced.     

Analysis of texture and dynamics of water binding in enzymatically modified myofibrillar preparation obtained from washed mechanically recovered poultry meat

Experiments were conducted on a myofibrillar preparation, obtained from washed mechanically recovered poultry meat. An enzymatic preparation containing microbial transglutaminase (MTG) was added to samples of the myofibril isolate. The binding of water contained in the protein preparation with added MTG was assessed using low field nuclear magnetic resonance (NMR). Moreover, texture was analyzed in myofibril samples with the addition of transglutaminase pre-incubated for 0.5, 1.5, 3, 4.5 and 6 h. All measurements were taken at 7 ± 0.2 °C. Samples with added transglutaminase exhibited improved mechanical failure strength and better water binding capacity. The most dynamic increase of texture parameter values was observed in the interval from 1.5 to 3 h pre-incubation of the preparation with the added enzyme. Based on NMR (T ₁) testing it was established that the highest amount of water was bound by protein in the period from approximately 1 to 1.5 h pre-incubation. After that time free water content in the sample was again found to increase. This means that water was displaced from the system as a result of protein–protein interactions dominating over protein–water interactions. The above suggests that the enzymatic modification of the protein preparation contributed to the intensification of cross-linking between proteins in the preparation.     

Effects of microbial transglutaminase and sodium alginate on cold-set gelation of porcine myofibrillar protein with various salt levels

The effects of microbial transglutaminase (TG) and sodium alginate (SA) systems on cold-set gelation of myofibrillar protein (MP) at various salt levels were investigated. The gelation kinetics data showed that both TG and SA had optimal but different salt levels to form an acceptable cold-set MP gel. Although their gelling characteristics were altered with different salt levels, the combination of TG and SA showed a rapid cold-set MP gel formation, regardless of salt levels. The effect of TG and SA on gel strength was reversed by increasing the salt level, while the cold-set MP gel strength of the TG and SA combination was not affected by salt levels. Furthermore, the SA system contributed to improved water-binding ability and cold-set MP gel formation, while the TG system enhanced the gel strength of the MP after cooking at low-salt levels. In addition, TG's effects on gel strength of MP increased at higher salt levels, the SA system prevented the moisture loss induced by the TG reaction. There was no evidence of any interaction between SA and MP in the thermogram and gel electrophoresis data. Results from this study suggested that a system combining TG with SA had the potential advantage of improving the water-binding ability and producing cold-set MP gelation at an even lower salt level than TG alone.     

Skeletal muscle fiber type and myofibrillar proteins in relation to meat quality

Although numerous studies have reported the relationships among muscle fiber characteristics, lean meat content and meat quality, controversial perspectives still remain. Conventional histochemical classifications may be involved in a high level of error, subjectivity and it could not clearly explain variety of myofibrillar protein isoforms. Therefore, more information is needed on how different factors, such as species, breeds, gender, nutrient conditions, physiological state of animals, and environment factors, affect ultimate meat quality in order to evaluate these uncertainness. Unfortunately, there is little information that completely covers with relationship among the muscle fiber types, myofibrillar proteins and enzymatic proteolysis. In addition to the perspective of postmortem metabolism, protein quality control in skeletal muscle and proteolytic degradation of muscle proteins during postmortem period could help to clarify this relationship. Therefore, the present review will focus on muscle fiber types, typing methods, muscle proteins and meat quality, and will summarize aspects of enzymatic view of proteasome.     

Influence of muscle type on rheological properties of porcine myofibrillar protein during heat-induced gelation

The gelation characteristics of myofibrillar proteins are indicative of meat product texture. Defining the performance of myofibrillar proteins during gelation is beneficial in maintaining quality and developing processed meat products and processes. This study investigates the impact of pH on viscoelastic properties of porcine myofibrillar proteins prepared from different muscles (semimembranosus (SM), longissimus dorsi (LD) and psoas major (PM)) during heat-induced gelation. Dynamic rheological properties were measured while heating at 1 °C/min from 20 to 85 °C, followed by a holding phase at 85 °C for 3 min and a cooling phase from 85 to 5 °C at a rate of 5 °C/min. Storage modulus (G′, the elastic response of the gelling material) increased as gel formation occurred, but decreased after reaching the temperature of myosin denaturation (52 °C) until approximately 60 °C when the gel strength increased again. This resulted in a peak and depression in the thermogram. Following 60 °C, the treatments maintained observed trends in gel strength, showing SM myofibrils produced the strongest gels. Myofibrillar protein from SM and PM formed stronger gels at pH 6.0 than at pH 6.5. Differences may be attributed to subtle variations in their protein profile related to muscle type or postmortem metabolism. Significant correlations were determined between G′ at 57, 72, 85 and 5 °C, indicating that changes affecting gel strength took effect prior to 57 °C. Muscle type was found to influence water-holding capacity to a greater degree than pH.     

Study on the Functional Properties of Soybean Protein Isolate Cross-Linked with Gelatin by Microbial Transglutaminase

A microbial transglutaminase was used to cross-link soybean protein isolates in the presence of gelatin to prepare a cross-linked composite soybean protein-gelatin. Cross-linking conditions, such as total proteins, the ratio of soybean protein isolates to gelatin, and original pH, were fixed at 5% (w/v), 4:1 (w/w), and 7.5, respectively. The 4-hydroxyproline content in the modified product was measured and used as an index to select suitable transglutaminase addition, reaction temperature, and time by single factor experiments, which were found to be 20 U/g proteins, 40°C, and 2 h, respectively. Under the identified conditions, a modified product with 4-hydroxyproline content of about 36 mg/g proteins was prepared, and its functional properties were evaluated. SDS-PAGE analysis showed that several protein polymers existed in the modified product. Compared to the original soybean protein isolates or a cross-linked soybean protein isolates, the modified product had a better emulsifying stability and water holding capacity but a poor enzymatic digestibility in vitro, emulsifying activity and oil binding capacity; meanwhile, the dispersions formed by the modified product exhibited the highest apparent viscosity, storage modulus, and viscous modulus.     

Modification of functional,rheological and structural characteristics of myofibrillar proteins by high-intensity ultrasonic and papain treatment

The effects of sonication duration (15 and 30 min) and papain enzyme treatments on the conformational, physicochemical and functional traits of myofibrillar proteins (MPs) were addressed in this study. As the ultrasound duration was increased, the water holding capacity (WHC), solubility, foaming and emulsifying properties were improved as a result of the changes in the particles size distribution and zeta potential. Our results indicated that the turbulence force caused increments in the surface hydrophobicity and significant changes in the secondary structure. Also, the rheological properties were influenced by both cavitation force and papain treatment. According to the observations, the samples treated with sonication (for 15 and 30 min) and enzyme were more elastic with higher viscosity, as compared to the control. Compared with the control, enzymolyzed samples indicated better functionality. Moreover, papain treatment led to the increase of the hydrophobicity groups on the surface of proteins and the decrease of the amount of α-helix and β-sheet structures. It was noteworthy that the most changes in the structure and techno-functionality of myofibrillar proteins were observed for the sample affected by the simultaneous application of papain treatment and 15 min sonication. The highest values in the surface hydrophobicity, specific surface area, net charge, storage modulus, solubility and WHC belonged to this sample. However, enzyme-modified samples exposed to the longer sonication time (30 min) demonstrated reductions in solubility, WHC and surface hydrophobicity, as well as increases in the particle size distribution and protein turbidity.     

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.     

The effect of active caspase-3 on degradation of chicken myofibrillar proteins and structure of myofibrils

The objective of this study was to investigate the potential contribution of caspase-3 to meat postmortem tenderisation by examining the role of caspase-3 in the degradation of myofibrillar proteins and disruption of myofibril structure in vitro. Myofibrillar protein prepared from chicken muscle was incubated with EDTA or EDTA plus caspase-3 at 25 °C for 16 h and used for detecting muscle protein degradation and ultrastructure of myofibril. Results revealed that caspase-3 reproduced the degradation patterns of titin, nebulin and α-actinin during postmortem storage of meat, but caused little proteolysis of desmin and no appearance of 28–30 kDa peptides. Meanwhile, caspase-3 also induced the weakening in the I band adjacent to Z-lines, which occurred during meat postmortem ageing. Therefore, caspase-3 could account only for a part of the myofibrillar protein degradation observed in naturally aged meat and is likely involved in postmortem tenderisation of meat together with other endogenous proteases.     

Dependence of microbial transglutaminase on meat type in myofibrillar proteins cross-linking

The objectives of this study were to determine the factors that cause differences in the improvements of gel strength and ε(γ-glutamyl)lysine (G-L) content in chicken and beef (Japanese black cattle) myofibrillar proteins after adding microbial transglutaminase (MTG). As the amount of MTG added increased, the breaking strength increased progressively (p < 0.01) in chicken and beef samples, with the exception of chicken samples treated at 40 °C. The values of elasticity in the chicken samples were lower than those of the beef samples (p < 0.01). Surprisingly, the elasticity level, ε(γ-glutamyl)lysine contents and myosin heavy chain (MHC) band sizes of chicken and beef at all levels of MTG were significantly different (p < 0.01). The results of this study suggest that MTG activity was affected by MTG inhibitors; that MTG develops the texture of myofibrils differently in different species. However, the activity is limited and inconstant among meat proteins, as suggested by the data collected from the chicken samples. As a result, when the transferable amino acid residues are depleted (cross-linked) by MTG activity, the function of MTG will be insignificant. The correlation between MTG and different sources of meat protein is quite unstable but it is strong, which was observed when chicken and beef responded differently to MTG because their chemical and physiological properties were different. The remarkable rate of formation of cross-linked proteins and the discrepancy between the expected and observed amount of dipeptide raises the possibility that there are enzymes capable of reversing the reaction induced by transglutaminase in chicken and beef myofibrils. In summary, our results suggest that access of MTG to chicken and beef myofibrils is different because it depends on physiological (muscles and their fibre types), biological (substrates) and biochemical (inhibitors and amino acids) variables.     

食品酶制剂对冷冻面团流变学和微结构的影响

应用动态流变仪,Brabender拉伸仪,扫描电子显微镜(SEM)研究了葡萄糖氧化酶和谷氨酰胺转胺酶对冷冻面团粘弹模性量粘弹模量,抗拉伸阻力R5及微结构影响。空白面团(未加添加剂),含有葡萄糖氧化酶面团和含有谷氨酰胺转胺酶面团于-18℃冷冻贮藏7,21,35d,随冷冻贮藏时间延长,面团弹性模量(G′)降低。在同一冷冻贮藏时期内空白面团弹性模量最小,添加葡萄糖氧化酶面团弹性模量最大;含有葡萄糖氧化酶和含有谷氨酰胺转胺酶面团抗拉伸阻力R5大于空白面团。葡萄糖氧化酶和谷氨酰胺转胺酶使新鲜面团(未冷冻面团)面筋网络增强,淀粉颗粒镶嵌于交错的面筋网络之间,在-18℃经过35d冷冻贮藏,空白面团面筋网络不再连续,支离破碎,并与淀粉颗粒分离,而且面筋膜变薄。含有葡萄糖氧化酶和含有谷氨酰胺转胺酶面团依然有大量连续面筋网络存在。葡萄糖氧化酶和谷氨酰胺转胺酶抑制了面团弹性模量和抗拉伸阻力R5的恶化,而且抑制冰晶对面团中面筋三维网络结构的破坏。     

Effect of enzymatic cross-linking of milk proteins on functional properties of set-style yoghurt

This paper presents research on the effect of enzymatic cross-linking of milk proteins on the properties of yoghurt. Whole milk was incubated with transglutaminase (TG) prior to fermentation (2 h, 40°C, E/S ratio 1/2000). Enzyme action was stopped by heating (1 min, 80°C). Skim-milk was treated by simultaneous use of TG and thermophilic yoghurt starter culture without inactivation of the enzyme. A TG treatment of milk prior to fermentation led to prolonged fermentation, while the concomitant use of TG and culture had no influence on fermentation time. Post acidification of yoghurt during storage was lower for products from enzyme-treated milk. This applies both for products cross-linked prior to fermentation with enzyme inactivation, and for simultaneous use of culture and TG without inactivation of the enzyme. Scanning electron microscopic studies revealed that a TG treatment of milk led to reduced mesh sizes of the protein network, and a more regular distribution of the proteins in the yoghurt gel. As a result, yoghurt products from enzyme-treated milk showed increased gel strength and less syneresis, especially when the enzyme was not inactivated. Sensory studies revealed that odour and consistency properties of products from TG-treated milk were assessed as less 'yoghurt specific'. On the other hand, products from enzyme-treated milk were described as being more creamy, indicating that a TG treatment may simulate fat in fermented milk products.     

Solubilization of myofibrillar proteins in water or low ionic strength media: Classical techniques,basic principles,and novel functionalities

The qualitative characteristics of meat products are closely related to the functionality of muscle proteins. Myofibrillar proteins (MPs), comprising approximately 50% of total muscle proteins, are generally considered to be insoluble in solutions of low ionic strength (< 0.2 M), requiring high concentrations of salt (> 0.3 M) for solubilization. These soluble proteins are the ones which determine many functional properties of meat products, including emulsification and thermal gelation. In order to increase the utilization of meat and meat products, many studies have investigated the solubilization of MPs in water or low ionic strength media and determining their functionality. However, there still remains a lack of systematic information on the functional properties of MPs solubilized in this manner. Hence, this review will explore some typical techniques that have been used. The main procedures used for their solubilization, the fundamental principles and their functionalities in water (low ionic strength medium) are comprehensively discussed. In addition, advantages and disadvantages of each technique are summarized. Finally, future considerations are presented to facilitate progress in this new area and to enable water soluble muscle MPs to be utilized as novel meat ingredients in the food industry.     

Characterisation and quantification of the reaction(s) catalysed by transglutaminase using the o‐phthaldialdehyde reagent

The potential application of the o‐phthaldialdehyde (OPA) reagent for quantification of the type and extent of the reaction(s) catalysed by transglutaminase (TGase) during incubation with sodium caseinate (NaCN) was investigated. Initial studies were performed to ensure that NH₃, a by‐product of TGase activity, could be determined with the OPA reagent in trichloroacetic acid (TCA) supernatants of NaCN solutions. The detectable concentration of exogenously added NH₃ (at NH₃ concentrations > 10 mM ) was found to decrease during extended incubation at 23, 37 and 50°C and at either pH 7.0 or 8.0 in 4% w/v NaCN solutions, even when taking into account the evaporation of water from the sample. The recovery of NH₃ from 12% w/v TCA supernatants of NaCN solutions spiked with 5 mM NH₃ at 23°C and pH 7.0 was found to be 88%. The release of NH₃ and the decrease in ε‐amino groups on incubating NaCN with TGase was subsequently quantified using the OPA reagent. Incubation of NaCN (4% w/v) with TGase at 23°C resulted in progressive increases and decreases, respectively, in NH₃ and ε‐amino group concentration with increased incubation time. These changes were dependent on TGase : NaCN. It was estimated that approximately 20% of the available Lys residues in NaCN were involved in TGase‐catalysed cross‐links. However, the observed decrease in ε‐amino group concentration was higher than expected. This may be due to concealment of noncross‐linked amino groups in polymerised NaCN, making them unavailable for reaction with the OPA reagent.     

Preliminary study on the effect of caspase-6 and calpain inhibitors on postmortem proteolysis of myofibrillar proteins in chicken breast muscle

The objective was to determine the effect of three different protease inhibitors, caspase-6 specific inhibitor VEID-CHO (N-Acetyl-Val-Glu-Ile-Asp-al), calpain inhibitor leupeptin or calpain inhibitor EGTA on protein degradation, ultrastructure of myofibrils and calpain activity during postmortem (PM) aging of chicken muscle. Results showed that proteolysis of nebulin, troponin-T and desmin during 14-days postmortem storage were inhibited significantly by leupeptin. Inhibitive effects of VEID-CHO and EGTA on these protein degradations were significant only during 1-day postmortem storage. The activities of calpains were inhibited noticeably by leupeptin and EGTA, but not by VEID-CHO. Samples treated with VEID-CHO, leupeptin and EGTA retarded structural disruption of chicken muscle fibers. These results demonstrate that calpain is a major contributor to PM tenderization; while caspase-6 plays, if any, a minimal role in the conversion of chicken muscle to meat.     

Role of disulphide linkages between protein-coated lipid droplets and the protein matrix in the rheological properties of porcine myofibrillar protein-peanut oil emulsion composite gels

The objective of the study was to establish disulphide interaction between protein-coated oil droplets and the surrounding protein matrix in myofibrillar protein (MP)-emulsion composite gels. An MP-stabilized peanut oil emulsion was treated with 0, 1, 3, 5 and 10 mM N-ethylmaleimide (NEM, a sulphydryl-blocking agent) and subsequently incorporated into a bulk MP sol to produce 5%-lipid, 2%-protein composites at pH 6.2. About 69% of sulphydryls in the emulsion (1% protein) were blocked by 1 mM NEM, and almost all were bound at ≥3 mM NEM. The loss of free sulphydryls resulted in a significant drop in the storage modulus (G') and rupture force of the composite gels. Microstructural examination revealed pores and oil leakage from emulsion droplets by NEM treatments, corresponding to declining rheological properties of the MP-emulsion composites. The results supported the hypothesis that disulphide cross-linking between MP-coated oil droplets and protein matrix contributed to the stabilization and reinforcement of protein-emulsion composite gels formed in comminuted muscle foods.     

Analysis of fish and squid myofibrillar proteins by capillary sodium dodecyl sulfate gel electrophoresis: actin and myosin quantification

 A capillary electrophoretic method for the separation and quantification of fish and squid myofibrillar proteins was developed. The method uses sodium dodecyl sulfate and β-mercaptoethanol for solubilization and analysis of myofibrillar protein subunits. The separation of the different polypeptides is achieved by the sieving effect of the gel inside the capillary. A calibration curve for myosin heavy chain and actin UV absorbance quantification of these proteins was developed. Received: 2 November 1999 / Revised version: 16 February 2000