Microbial Transglutaminase and ε-(γ-Glutamyl)lysine Crosslink Effects on Elastic Properties of Kamaboko Gels

Kamaboko gels from Alaska pollock surimi (SA? and 2nd? grades) were prepared by setting at 10 or 45°C with Microbial transglutaminase (MTGase) and its effect on gel properties was investigated. At 10 and 45°C, gels from 2nd? grade surimi paste showed increases in breaking strength, without decline in deformation. Gel from SA? graded surimi paste showed an increase in breaking strength with no changes in deformation in 45°C setting, up to 0.03% MTGase. Crosslinking of myosin heavy chains through ε-(γ-glutamyl)lysine bonds was observed and a possible correlation was shown between ε-(γ-glutamyl)lysine content and gel strength (breaking strength X strain). ε-(γ-Glutamyl)lysine content up to 3 μmol/100g or MTGase 0.03% or higher improved gel properties.     

NONDISULFIDE COVALENT CROSS-LINKING OF MYOSIN HEAVY CHAIN IN “SETTING” OF ALASKA POLLOCK AND ATLANTIC CROAKER SURIMI1

ABSTRACT Myosin heavy chain (MHC) content of cooked gels of pollock and croaker surimi decreased during preincubation (“setting”) at temperatures ranging from 4–50C. Decreases in MHC content were attributed to either nondisulfide covalent cross-linking or proteolysis. Depending upon which process dominated at a given temperature, formation of stronger or weaker gels occurred, respectively. Maximum production of cross-linked polymers occurred at the optimum setting temperatures, i.e., at 25C for pollock surimi and 40C for croaker surimi. Subsequent cooking of these set gels at 90C decreased the amount of cross-linked polymers formed at the optimum setting temperature. Addition of free lysine-HCl inhibited formation of cross-linked polymers of MHC during setting and the increase in cooked gel strength for both species. This supports published evidence that cross-linking of MHC during setting may be of the ε-amino-(γ-glutamyl) lysine I type, mediated by a transglutaminase enzyme.     

Transglutaminase-mediated setting in bigeye snapper Surimi

Effect of setting induced by endogenous transglutaminase (TGase) in two species of bigeye snapper, Priacanthus tayenus and Priacanthus macracanthus, on gel properties and protein cross-linking was investigated. Setting at either 25 or 40 °C, prior to heating at 90 °C resulted in the increase in both breaking force and deformation of surimi from both species, particularly when setting time increased (P<0.05). A decrease in solubility of surimi gels in a mixture of sodium dodecyl-sulfate, urea and β-mercaptoethanol suggested increased formation of non-disulfide covalent bonding which coincided with increased gel strength and the decrease in myosin heavy chain (MHC) polypeptide. The optimum conditions for setting of surimi sol was found to be 40 °C for 2 h for P. tayenus and 25 °C for 3 h for P. macracanthus. Assayed by monodancylcadaverine (MDC)-incorporation method, TGase from P. tayenus and P. macracanthus exhibited an optimum temperature at 40 and 25 °C, respectively. In addition, the breaking force and deformation of surimi from both species increased markedly with the addition of calcium chloride, while they decreased considerably in the presence of EDTA, N-methylmaleimide and ammonium chloride. The results confirmed that endogenous transglutaminase played an important role in gel enhancement of surimi from both species of bigeye snapper.     

Effect of soy protein isolate on gel properties of Alaska pollock and common carp surimi at different setting conditions

The effects of soy protein isolate (SPI) on the gel properties of different grade Alaska pollock and common carp surimi at different setting conditions were evaluated and compared. Breaking force and distance of gels decreased with increasing SPI concentrations in direct cook (85 °C for 30 min) and in cook after setting at 30 °C for 60 min conditions. The effect of SPI on gel strength of common carp surimi was less than in Alaska pollock surimi. The breaking force obtained for addition of 10% SPI to Alaska pollock surimi was higher than for surimi alone when cooked after incubation at 50 °C for 60 min. Addition of SPI decreased the whiteness and increased the yellowness of the gel. The gel structure showed that the addition of SPI modified the microstructure of the fish protein gel, thus resulting in surimi with different gelling properties. Copyright © 2004 Society of Chemical Industry     

Transglutaminase Effects on Low Temperature Gelation of Fish Protein Sols

Myosin polymerization and formation of ?-(γ-glutamyl)lysine linkages were quantified in Alaska pollock surimi gels which contained no additive (control), or a commercial microbial transglutaminase (MTGase). As preincubation (“setting”) time at 25°C was increased, the gel strength of control and 0.2% MTGase-added samples increased, with greater increases at higher MTGase levels. SDS-PAGE and HPLC analyses showed increasing nondisulfide polymerization and ?-(γ-glutamyl)lysine dipeptide content, with increasing setting time and/or added MTGase. Content of ?-(γ-glutamyl)lysine dipeptide correlated with gel strength (shear stress) and shear modulus at failure (G_f) for these gels. Higher stresses were measured in samples containing 0.2% MTGase than in controls at corresponding levels of ?-(γ-glutamyl)lysine dipeptide, indicating that rate of myosin polymerization may affect ultimate gel strength.     

High Pressure Effects on Gelation of Surimi and Turkey Breast Muscle Enhanced by Microbial Transglutaminase

High pressure effects on the strength (stress) and elasticity/deformability (strain) of surimi and turkey breast meat gels containing microbial transglutaminase (TGase) were evaluated. Pressurization of muscle proteins at 4°C prior to incubation at 25°C or 40°C (setting) increased gel strength 2–3 fold in uncooked surimi gels, but not in uncooked turkey gels. However, pressurization at 40°C or 50°C prior to setting increased the strength of turkey gels. Similar effects of prior pressurization, but of lesser magnitude, occurred in gels formed by directly or subsequently (following setting) cooking at 90°C. SDS-PAGE confirmed that myosin crosslinking occurred due to TGase activity during the setting treatment, which had survived prior pressure treatment. High pressure rendered protein substrates more accessible to TGase thereby enhancing intermolecular cross-link formation and gel strength.     

Gel Strength Enhancement by Addition of Microbial Transglutaminase during Onshore Surimi Manufacture

Surimi from Alaska pollock flesh was manufactured onshore with Microbial transglutaminase (MTGase). Effect of MTGase was investigated by evaluating breaking strength and deformation of gels from MTGase-treated surimi with and without setting at 30°C. Quantitative analysis of ε-(γ-glutamyl)lysine (GL) crosslink was also carried out to monitor the MTGase reaction. In set gels, breaking strength and GL crosslink increased, and myosin heavy chain decreased correspondingly with MTGase concentration. These changes were smaller in gels prepared without setting. Results suggest that surimi gel could be improved through the formation of GL crosslinks by added MTGase in surimi.     

淡水鱼肉蛋白质组成及其在鱼糜制品加工中的变化

本文通过鱼肉蛋白质组成、鱼糜凝胶特性的测定及SDS-PAGE凝胶电泳,研究了鲢、鳙、草、鲫四种淡水鱼鱼糜凝胶特性及其凝胶形成过程中蛋白质组成的变化。四种淡水鱼肉粗蛋白质含量基本相同,但凝胶特性存在明显差异,其原因是四种淡水鱼肉的盐溶性蛋白含量不同。在鱼糜制品加工过程中,盐溶性蛋白质、水溶性蛋白的比例逐渐下降,而不溶性蛋白比例逐渐上升。弹性凝胶体的形成始于盐擂阶段,主要形成于低温凝胶化阶段。SDS-PAGE凝胶电泳图谱显示,肌球蛋白重链的量在鱼糜凝胶形成过程中明显减少,而肌球蛋白轻链和肌动蛋白的量几乎未发生变化。肌球蛋白重链在内源转谷氨酰胺酶作用下,发生分子间或分子内的ε-(γ-Glu)-Lys共价交联,形成了网络状的不溶性蛋白。     

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     

Characterization of the Effect of Solutes on the Water-Binding and Gel Strength Properties of Carrageenan

The water activity of carrageenan gels with incorporated solutes was determined by cryoscopic osmometry. Kappa (k), kappalambda (kλ) and iota (κ) carrageenan were utilized at concentrations of from 0.1 2% solids producing a sol, viscous sol, and gel. The solutes used were sucrose, NaCl, Na₂SO₄, KCl, NH₄Cl and urea at concentrations ranging from 0.05–1.0 kinetic units per kg H₂O. All the solutes were found to give a_w values as measured cryoscopically withhin 0.0005 units of literature values. The water activity of pure carrageenans at 0.1–2% solids was greater than 0.999. Addition of some solutes increased the water binding of the carrageenan-solute-water system as was found by a lower a_w than expected. This interaction effect increased with increasing concentration of solute and carrageenan and was greater for solutes with lower activity coefficients. The interaction effect increased in the following order: Na₂SO₄, NaCl, KCl, and NH₄ Cl. The force required to rupture the gel was measured using the Instron Universal Testing Machine. Solutes were found to influence the gel strength of k and kλ carrageenans, but not ι carrageenan. The solutes increased kλ carrageenan's gel strength in increasing order: urea, sucrose, Na₂SO₄, NaCl, NH₄Cl, and KCl and k carrageenan's gel strength in increasing order: sucrose, Na₂SO₄ and NaCl. There appears to be no simple relationship between gel strength and water-binding when solutes are added to carrageenan.     

CHITOSAN AFFECTS TRANSGLUTAMINASE-INDUCED SURIMI GELATION

Effect of chitosan on barred garfish (Hemiramphus far) surimi gel was studied in the presence of EDTA and microbial transglutaminase (MTGase). An increase in breaking force of surimi gels added with 1.0% prawn shell chitosan indicated the gel enhancing effect of chitosan on the heat‐induced gelation of fish myofibrillar proteins. However, gel‐forming ability of surimi containing chitosan was inhibited in the presence of EDTA, especially at higher concentration. Therefore, the enhancing effect of chitosan was possibly mediated through the action of endogenous transglutaminase (TGase) during setting, resulting in the formation of protein‐protein and protein‐chitosan conjugates. In general, addition of MTGase remarkably increased both breaking force and deformation of surimi gel (P<0.05). However, enhancing effect of MTGase was retarded in the presence of chitosan, resulting in lower magnitude of breaking force and deformation (P<0.05). Scanning electron microscopy showed that chitosan particles were uniformly dispersed in the gel matrix. A tightly associated gel network was formed in surimi containing MTGase, whereas a large number of voids were noted in gels with EDTA. These results suggest that chitosan acted as a surimi gel enhancer in combination with endogenous TGase in fish muscle, but hindered gel formation in the presence of MTGase.     

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.     

Cross-linking activity of sarcoplasmic fraction from bigeye snapper (Priacanthus tayenus) muscle

Addition of sarcoplasmic fraction from bigeye snapper (Priacanthus tayenus) into natural actomyosin in combination with setting at 40°C resulted in the cross-linking of myosin heavy chain (MHC). Higher amount of sarcoplasmic fraction and extended setting time resulted in a higher cross-linking, indicating the presence of endogenous transglutaminase (TGase) in bigeye snapper muscle. TGase activity was activated by calcium ion and reducing agents (β-mercaptoethanol and dithiotreitol), but was inhibited by N-ethylmaleimide (NEM), NH₄Cl and EDTA. TGase in the sarcoplasmic fraction was not stable when heated at temperature above 40°C, particularly with an increasing heating time. TGase was stable at pH ranging from 5.0 to 7.0, in which more than 70% activity was retained. Therefore, sarcoplasmic fraction possessed a cross-linking activity caused by TGase and its recovery for further uses should be considered.     

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).     

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     

Temperature and pH Affect Transglutaminase-Catalyzed "Setting" of Crude Fish Actomyosin

Dynamic rheological properties of actomyosin from two species (Alaska pollock, Atlantic croaker) were monitored during preincubation (setting) at 25°C and 37°C. followed bv nronrammed (l°C/min) cookinn to 77°C. Added guinea pig liver transgluianase enhanced gelation, as indicated by increases in both storage modulus (G′) and percentage of polymerized myosin heavy chain (MHC). In the presence of added transglutaminase maximum G′ and MHC polymerization occurred at the same conditions of pH and temperature which were optimum for setting of surimi pastes. This suggested that the transglutaminase-mediated setting reaction in surimi was constrained more by the conformation of the substrate (i.e., myosin) than by that of the enzyme.     

Mackerel Cathepsins B and L Effects on Thermal Degradation of Surimi

During surimi processing, cathepsins B and L activities in minced, leached and NaCl-ground meats were 6.02, 5.23, and 4.07 units/g, respectively. About 80% activity remained in surimi after 8 wk storage at ?40°C suggesting that these proteinases were stable and difficult to remove. At 40°～ 55°C, pH 6.5 ～ 7.5, cathepsins B and L and purified cathepsin B had high hydrolytic activity on myosin heavy chain (MHC). The strength of surimi gel with cathepsins B and L or with purified B decreased (p < 0.05) after 2 hr incubation at 55°C. This suggested that the residual cathepsins B and L had MHC-degrading activity and consequently caused gel softening.     

低温交联时间对酸诱导罗非鱼碎肉鱼糜凝胶特性的影响

本实验以罗非鱼(Oreochromis niloticus)碎肉为试验对象,研究2.4%葡萄糖酸内酯(GDL)介导下的蛋白在低温(4 ℃)交联过程中,鱼糜凝胶的化学作用力和质构特性随交联时间(0~24 h)的变化规律。结果表明,随着交联时间的延长,低温酸诱导过程中γ-羧酰胺基和伯胺产生的交联键、二硫键和疏水相互作用使得碎肉蛋白之间发生交联从而形成了高强度的凝胶。鱼糜凝胶在交联过程中,蛋白凝胶的持水性随着交联时间的延长而下降,但凝胶特性随着交联时间的延长而显著增加(P<0.05)。交联时间为20~24 h时,鱼糜凝胶强度均显著高于5~15 h(P<0.05);硬度在20 h达到最高,而咀嚼度在15~20 h时达到最高(P<0.05)。综上所述,罗非鱼碎肉蛋白在低温酸诱导鱼糜凝胶形成过程中,20 h为最佳交联时间。     

Oxidative characteristics and gel properties of porcine myofibrillar proteins affected by l-lysine and l-histidine in a dose-dependent manner at a low and high salt concentration

This study investigated the effects of l -lysine (Lys) and l -histidine (His) on the oxidative characteristics and gel properties of porcine myofibrillar proteins (MP). Results showed that Lys and His had a strong ferrous ion-chelating ability and hydroxyl radical-scavenging activity. Moreover, Lys and His inhibited the protein carbonyl formation and MP aggregation at 0.2 M and 0.6 M NaCl, respectively, in a dose-dependent manner. Furthermore, 2 and 4 mg mL⁻¹ Lys and His decreased the oxidation-induced loss of the tertiary structure of MP accompanied by the lower surface hydrophobicity. The water-holding capacity and gel strength of MP gels increased with increasing Lys and His concentrations due to more regular and lamellar structures with smaller and homogeneous pores at 0.6 M NaCl and more orderly crosslinking via fibrous filament at 0.2 M NaCl. In summary, Lys and His chelated the ferrous ions and scavenged hydroxyl radicals, decreased the oxidation-induced physicochemical changes, thus preventing oxidative damage during the formation of a three-dimensional gel network, which resulted in better gel quality.     

葡萄糖酸内酯酸化处理对未漂洗鲟鱼糜凝胶特性的影响

本研究中,以未漂洗鲟鱼糜为研究对象,探究了不同添加量（0、1%、2%、3%、4%）的葡萄糖酸内酯（GDL）对低温交联24 h后的鱼糜凝胶特性的影响,并结合化学作用力和SDS-PAGE凝胶电泳对相关作用机理进行分析。结果表明,与对照组相比,GDL可以提高鱼糜凝胶白度和持水性;添加GDL后,凝胶强度、硬度、咀嚼性和胶黏性随着添加量的增加逐渐增大,并在添加量为3%时达到最大。鱼糜凝胶蛋白分子间氢键和离子键呈下降趋势,疏水相互作用呈现先增加后略微下降的趋势。SDS-PAGE凝胶电泳结果显示,MHC条带随着GDL添加量的增加逐渐减弱,说明添加GDL可以提高MHC的交联,从而改善凝胶特性。综合分析,添加GDL可改善未漂洗鲟鱼糜凝胶特性,且添加量为3%时较为适宜,可获得品质较好的鱼糜凝胶。