Aggregation and structural changes of silver carp actomyosin as affected by mild acidification with d-gluconic acid δ-lactone

The structural changes and aggregation properties of silver carp actomyosin acidified with d-gluconic acid-δ-lactone (GDL) were investigated. Results showed that silver carp actomyosin underwent aggregation and formation of precipitate as indicated by turbidity and centrifugation coupled electrophoresis analysis. Circular dichroism indicated that myosin rod unfolded during acidification, resulting in a gradual decrease in α-helical content. The changes in tertiary structure of actomyosin under acidic conditions were demonstrated by second-derivative UV spectra and intrinsic fluorescence. Tyrosine residues were exposed to the surface of proteins when pH was decreased to 5.5, and were buried inside the protein aggregates with further reduction in pH. In contrast, more tryptophan residues were exposed to the polar environment with decreasing pH. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinking experiments showed that the intensity of myosin heavy chain (MHC) bands decreased sharply with decreasing pH and the actin bands decreased more slowly, suggesting that MHC is the major protein component involved in the non-covalent cross-linking and formation of aggregates during acidification of silver carp actomyosin.     

Acid-induced gelation of natural actomyosin from Atlantic cod (Gadus morhua) and burbot (Lota lota)

The acid-induced gelation of natural actomyosin (NAM) from burbot (Lota lota) and Atlantic cod (Gardus morhua) added with d-gluconic acid-δ-lactone (GDL) during incubation at room temperature (22–23 °C) for 48 h was investigated. During acidification, pH values of both NAMs reached 4.6 within 48 h. Both NAMs underwent aggregation during acidification as evidenced by increases in turbidity and particle size, especially after 6 h of incubation. The decreases in Ca²⁺-ATPase activity and salt solubility of both NAMs were observed during incubation. Decreases in total sulphydryl content with the concomitant increases in disulphide bond content of NAM from both species were also noticeable. Additionally, surface hydrophobicity of NAM increased, suggesting the conformational changes in NAM induced by acidification. The storage modulus (G′) values increased with increasing incubation time and G′ development was greater in Atlantic cod NAM, compared with burbot NAM. Differential scanning calorimetry (DSC) revealed that T_max and enthalpy of myosin peak shifted to the lower values and endothermic peak of actin completely disappeared. In general, gel development was more pronounced in Atlantic cod NAM, compared with the burbot counterpart. As visualised by transmission electron microscopy, network strands of aggregates from Atlantic cod were finer and more uniform than those of the burbot counterpart. Acid-induced gelation of NAM from both fish species therefore involved both denaturation and aggregation processes. However, gelation varied with fish species and had an impact on the resulting gels.     

Effect of glucono-δ-lactone acidification and heat treatment on the physicochemical properties of silver carp mince

The physicochemical and rheological properties of silver carp mince with different levels of glucono-δ-lactone (GDL) with or without heat treatment at 90 °C for 20 min were investigated. Breaking force, deformation, and whiteness of GDL-induced gels increased with increasing GDL concentration. Heat treatment of GDL-induced gels led to a significant increase in breaking force and whiteness, but decreased deformation. Disulphide bond formation was observed in GDL-induced gels, which increased after heating of the acidified gels. Solubility profiles of gels in different solutions suggested that gel networks were mainly due to hydrophobic interactions, although other chemical interactions may also occur. SDS-PAGE showed that acidification resulted in a large decrease in myosin heavy chain (MHC) band intensity, suggesting the extensive formation of covalent cross-links among silver carp myosins during GDL-induced gelation.     

Effect of concentration and incubation temperature on the acid induced aggregation of soymilk

The molecular details of the aggregation of soy protein particles in soymilk during acid-induced gelation using glucono-delta-lactone (GDL) were investigated. Soymilk samples were prepared from different water-to-bean ratios and contained approximately 4% and 7% protein. The effects of protein concentration and incubation temperature (30 or 7 °C) on soymilk gelation were observed using rheology and diffusing wave spectroscopy at two different GDL concentrations. During acidification, there was a decrease in electrostatic repulsion between particles which well correlated with the pH of aggregation determined by dynamic light scattering. Gelation of soymilk occurred at about pH 5.8, and neither the rate of acidification nor the protein concentration affected the gelation pH. Gel stiffness was affected by protein concentration. A detailed study of the soluble fraction during the preceding stages of aggregation clearly demonstrated that glycinin components were the first to destabilize during acidification, followed by the β-conglycinin subunits. Decreasing the incubation temperature from 30 °C to 7 °C increased the pH of gelation and the gel modulus (G′) measured by rheology. It was concluded that short range interactions play a major role in the formation of the protein network in soymilk curd.     

Effect of frozen storage on thermal stability of sarcoplasmic protein and myofibrillar protein from common carp (Cyprinus carpio) muscle

Thermal stability of sarcoplasmic protein and myofibrillar protein extracted from fresh and frozen common carp was comparatively studied. Total sulphydryl content (SH) in sarcoplasmic protein solution from 5‐month frozen carp decreased by 19.43% compared with fresh sample. The SDS‐PAGE patterns showed that all the bands of sarcoplasmic protein from frozen‐stored samples were almost invisible at 80 °C. Myofibrillar protein from fresh sample exhibited lower turbidity and surface hydrophobicity and higher Ca²⁺‐ATPase activity and SH content than frozen‐stored sample when heated from 20 to 80 °C. The Ca²⁺‐ATPase activity from fresh (M0), 2 (M2)‐ and 5 (M5)‐month frozen‐stored carp was completely lost at 48, 46 and 46 °C, respectively. When heated to 80 °C, the SH content of myofibrillar solutions in M0, M2 and M5 decreased by 26%, 60% and 70%, respectively. Sarcoplasmic and myofibrillar proteins from frozen carp were more susceptible to aggregate during heating treatment.     

Changes in Physicochemical Properties of Bighead Carp (Aristichthys mobilis) Actomyosin by Thermal Treatment

Physicochemical properties of actomyosin from bighead carp during heat-treatment were investigated. Turbidity, solubility, and surface hydrophobicity showed significant changes from 40–55°C (P < 0.05). Heat-treatment before 45°C could make actomyosin unfold, and the disulfide bonds were formed at temperatures above 45°C. Three enthalpy transitions at 41.2, 61.7, and 63.4°C were found in differential scanning calorimetry thermogram of bighead carp actomyosin. Storage modulus (G′) increased gradually and reached the first peak at 36.5°C and the second peak at 50°C. Myosin suffered conformation changes and lead to aggregation during the heating period.     

Protein molecular interactions involved in the gel network formation of fermented silver carp mince inoculated with Pediococcus pentosaceus

The types of interactions and the proteins involved in the formation of gel network of fermented silver carp mince were investigated. Results showed that the amount of each type of protein interactions varied significantly depending on fermentation time. The content of sulphydryl groups decreased continually with a concomitant increase in disulphide bonds during fermentation. The higher gel strength and more compact fibrous network of fermented silver carp mince corresponded to a higher percentage of hydrophobic interactions, disulphide bonds and non-disulphide covalent bonds. SDS–PAGE showed that the major myofibrillar proteins, particularly myosin heavy chain were involved in the formation of aggregates through hydrophobic interactions and disulphide bonds during fermentation. Furthermore, low molecular weight proteins probably produced by proteolysis were also involved in the formation of gel matrix.     

Impact of high-power sonication on yield,molecular structure,and functional properties of soy protein isolate

This study reports the influence of high-power sonication (HPS) pre-treatment on protein yield, molecular structure, and functional properties. HPS energy densities (0–720 J/mL) were applied to soybean flour and flakes prior to protein extraction at pH 8.5 for 15 or 30 min at 60⁰C, and 5 min at room temperature. The isolates were prepared by precipitating at pH 4.5 and had >90% protein. HPS of 720 J/mL to flakes substrates extracted 80% protein without any incubation and 85% with 15 min incubation when no-sonication control showed an extraction of 71%. FTIR showed some aggregation and intrinsic fluorescence and free SH content showed pronounced effects on isolates from flour. HPS increased the surface hydrophobicity, reduced solubility, and enhanced the emulsification and foaming properties for a few treatments. Therefore, incubation duration changed structural and functional properties more prominently than did HPS in the isolates from flour than flakes.     

Gel properties of surimi from silver carp (Hypophthalmichthys molitrix) as affected by heat treatment and soy protein isolate

The effects of setting conditions and soy protein isolate (SPI) on textural properties of surimi produced from silver carp were investigated. Effects of setting temperature, setting time and protein concentration on the gel strength were evaluated and compared utilizing response surface methodology. Models for breaking force and breaking distance of silver carp surimi were established. The total protein content was 13.4% in all experimental samples. Setting temperature and protein concentration were the major factors affecting the gel strength. In the range of the additive SPI protein (10–40%), breaking force and distance of silver carp surimi gels decreased when the protein ratio of SPI was increased in the total protein at 30 and 40 °C for 60 min setting and heating at 85 °C for 30 min, but the breaking force obtained for 90% surimi protein plus 10% SPI protein was higher than surimi alone at 50 °C for 60 min incubation and heating at 85 °C for 30 min.     

Carp Natural Actomyosin: Thermal Denaturation Mechanism

Structural changes of actomyosin, the major protein of muscle, on heating have been estimated on ATPase activity. We investigated carp actomyosin molecule changes on heating based on biophysical and biochemical techniques. Actomyosin molecules began to unfold at ～30°C. Hydrophobic amino acid residues and SH groups, which had been inside the molecule, emerged to the surface. Because of hydrophobic interactions and disulfide bonds, actomyosin molecules formed aggregates. At > 40°C, a part of myosin molecules was dissociated from actin filaments. Thus, dissociated myosin and the myosin-lacking molecules co-existed. In addition, fragmentation of actin filaments was observed, which was associated with the dissociation of myosin molecules. At ≥ 60 °C actomyosin molecules formed larger aggregates, in which no filamentous shape was observed. This aggregation occurred mainly by formation of SS bonds.     

Comparative study on acid-induced gelation of myosin from Atlantic cod (Gardus morhua) and burbot (Lota lota)

Physicochemical and rheological properties of myosin from Atlantic cod and burbot during acid-induced gelation at room temperature (22–23 °C) by d-gluconic acid-δ-lactone (GDL) were monitored. Turbidity and particle size of both myosins increased and salt soluble content decreased when pH decreased, suggesting the formation of protein aggregates caused by acidification. The formation of disulphide bonds in myosin gelation was induced by acid. Ca²⁺-ATPase activity of myosin decreased (p < 0.05), while surface hydrophobicity increased during acidification (p < 0.05). Furthermore, the decreases in maximum transition temperature (T_max) and the denaturation enthalpies (ΔH) were found in both myosins. During acidification, the increases in storage modulus (G′) and loss modulus (G″) of myosin were observed (p < 0.05), revealing the formation of elastic gel matrix. Thus, gelation of myosin from Atlantic cod and burbot could take place under acidic pH via denaturation and aggregation. However, myosin from Atlantic cod was generally more favourable to gelation than was burbot myosin.     

Physical and chemical changes of silver carp sausages during fermentation with Pediococcus pentosaceus

Changes in protein composition and physicochemical properties of silver carp sausages during fermentation were investigated. As fermentation progressed, the amount of salt-soluble and water-soluble proteins decreased gradually with a concomitant increase in insoluble proteins and non-protein constituents. The rapid reduction in pH to 4.5 within 48 h of fermentation coincided with a progressive increase in titratable acidity. The increasing content of TCA-soluble peptides during fermentation indicated intensive degradation of muscle proteins. SDS–PAGE showed that muscle proteins tended to aggregate into large polymers through disulphide bonds and non-disulphide covalent bonds during fermentation, which may be responsible for the formation of superior textural properties of silver carp sausages.     

Effect of high pressure treatment on the physicochemical and functional properties of egg yolk

The effects of high-pressure (HP) treatment at 100–500 MPa on some physicochemical and functional properties of egg yolk (EY) were investigated. Protein solubility, viscosity, surface hydrophobicity (H₀), free sulfhydryl (SH) content, differential scanning calorimetry characteristics, emulsifying activities and emulsifying stability were evaluated. HP-treatment resulted in protein aggregation, as evidenced by gradual decrease in protein solubility and significantly increased in viscosity. HP-treatment at 100–500 MPa induced a gradual decrease in H₀ and SH content, possibly due to protein unfolding and subsequent aggregation/re-association of unfolded proteins. Emulsifying activity index (EAI) was slightly decreased between 100 and 300 MPa and when the pressure is above 400 MPa, EAI was significantly (P < 0.05) decreased relative to the untreated EY. HP-treatment at 100 MPa significantly (P < 0.05) increased the ESI values of EY, while a significant (P < 0.05) decrease was observed when the pressure was above 200 MPa. It was also investigated that there are significant correlations between physicochemical properties of EY, and the differences in the modification of EY protein by HP treatment at different pressure levels may be attributed to the differences in aggregation and unfolding/refolding extents of proteins.     

Effects of the Acid- and Alkali-Aided Processes on Bighead Carp (Aristichthys nobilis) Muscle Proteins

The objective of this study was to investigate the effects of the acid- and alkali-aided processes on bighead carp (Aristichthys nobilis) muscle protein as compared with the conventional washing method. Acid- and alkali-aided processes resulted in higher protein recoveries than conventional washing method. In textural profile analyses, the highest hardness and cohesiveness were observed in the gels prepared by conventional washing and alkali-aided processing (p < 0.05). No endothermic peak was found in the differential scanning calorimetry thermogram of protein isolates after acid- and alkali-aided processes. The hydrophobic interactions and disulfide bonds were the main chemical bonds to maintain the stability of the gel matrix, while the sulfhydryl group in bighead carp muscle was apt to be activated during alkali-aided processing. The α-helical content of actomyosin decreased remarkably after the acid- and alkali-aided processes (p < 0.05). Overall, there is a potential to apply the alkali-aided processing to extract proteins from bighead carp muscle for the production of surimi-based seafoods.     

Changes in the physical properties,solubility, and heat stability of milk protein concentrates prepared from partially acidified milk

A limiting factor in using milk protein concentrates (MPC) as a high-quality protein source for different food applications is their poor reconstitutability. Solubilization of colloidal calcium phosphate (CCP) from casein micelles during membrane filtration (e.g., through acidification) may affect the structural organization of these protein particles and consequently the rehydration and functional properties of the resulting MPC powder. The main objective of this study was to investigate the effects of acidification of milk by glucono-δ-lactone (GDL) before ultrafiltration (UF) on the composition, physical properties, solubility, and thermal stability (after reconstitution) of MPC powders. The MPC samples were manufactured in duplicate, either by UF (65% protein, MPC65) or by UF followed by diafiltration (80% protein, MPC80), using pasteurized skim milk, at either the native milk pH (~pH 6.6) or at pH 6.0 after addition of GDL, followed by spray drying. Samples of different treatments were reconstituted at 5% (wt/wt) protein to compare their solubility and thermal stability. Powders were tested in duplicate for basic composition, calcium content, reconstitutability, particle size, particle density, and microstructure. Acidification of milk did not have any significant effect on the proximate composition, particle size, particle density, or surface morphology of the MPC powders; however, the total calcium content of MPC80 decreased significantly with acidification (from 1.84 ± 0.03 to 1.59 ± 0.03 g/100 g of powder). Calcium-depleted MPC80 powders were also more soluble than the control powders. Diafiltered dispersions were significantly less heat stable (at 120°C) than UF samples when dissolved at 5% solids. The present work contributes to a better understanding of the differences in MPC commonly observed during processing.     

Effects of composition and oxidation of proteins on their solubility,aggregation and proteolytic susceptibility during processing of Cantonese sausage

Cantonese sausage proteins were classified into five fractions based on solubility. The alkali-soluble protein was the major protein component of Cantonese sausage and increased during the process. The salt- (myofibrillar proteins) and water-soluble protein fractions (sarcoplasmic proteins), which decreased throughout the process, played an important role in the proteolysis. The total carbonyl content was found to significantly increase (p < 0.05) in sarcoplasmic, myofibrillar and alkali-soluble proteins. The SH group level decreased, whereas S–S group level increased gradually. The protein solubility determinations suggested that hydrogen bonds, hydrophobic interactions, disulphide bonds and ionic bonds gave rise to a three-dimensional network structure of Cantonese sausage proteins. Average particle size and specific surface area of Cantonese sausage proteins were affected by the changes in composition and oxidation of proteins. Results of proteolysis suggested that proteolytic susceptibility was influenced by the degree of protein oxidation.     

盐添加量和热处理对内酯鱼糜凝胶品质的影响

采用葡萄糖酸内酯(GDL)诱导鲢鱼糜形成凝胶,考察了食盐添加量和热处理对内酯鱼糜凝胶品质的影响。结果表明,食盐添加量对内酯鱼糜凝胶的持水性和质构特性具有显著影响,鱼糜凝胶的失水率和蒸煮损失均随盐含量的增加而显著降低,凝胶强度和弹性在0.5%～2%的食盐添加范围内随盐添加量的增加而提高,当食盐添加量为1.5%～2%时,内酯鱼糜凝胶强度和弹性显著提高。随食盐添加量的增加,鱼糜凝胶的白度略有下降。蒸煮处理导致内酯鱼糜破断强度和硬度显著增加,弹性略有下降。      

Inhibition of frozen storage‐induced oxidation and structural changes in myofibril of common carp (Cyprinus carpio) surimi by cryoprotectant and hydrolysed whey protein addition

The objective of the present study was to evaluate the efficacy of combined cryoprotectants (sucrose + sorbitol) and whey protein isolate (WPI) hydrolysates to inhibit protein oxidation and quality loss in common carp (Cyprinus carpio) surimi during frozen storage at ?25 °C. With increasing storage time, the carbonyl content of myofibrillar proteins increased from 4.02 nmolmg^‐1 protein (0 day) to 7.25, 6.31, 5.26 and 4.83 nmol mg^?1 protein (180 days; P < 0.05) for the control and samples with cryoprotectants, with cryoprotectants + WPI hydrolysates and with cryoprotectants + propyl gallate, respectively; protein surface hydrophobicity and turbidity increased in a similar trend, while sulfhydryl content, Ca‐ATPase activity, protein solubility and protein thermal stability decreased (P < 0.05). These results suggest that treatments with combined cryoprotectants and antioxidative WPI hydrolysates offer an effective approach to reducing the extent of protein oxidation in common carp surimi, thereby limiting protein structural changes known to impair texture of surimi products.     

Chemical cross-linking and molecular aggregation of soybean protein during extrusion cooking at low and high moisture content

Soybean protein isolate (SPI) was extruded using a co-rotating twin-screw extruder at low (28%) and high (60%) moisture content. Dead-stop operation was employed to obtain samples from different zones of the extruder. The protein solubility of extrudate was analyzed based on 8 different combinations of chemical bond-breaking solutions, which contained only phosphate buffer (P); two reagents, i.e. sodium dodecyl sulfate (SDS) in P (P + S), urea in P (P + U) and 2-mercaptoethanol (2-ME) in P (P + M); three reagents, i.e. (P + S + U), (P + U + M) and (P + M + S); four reagents, i.e. (P + S + U + M). Based on the protein solubility in the combined extraction solutions, each specific chemical bond and their interactions responsible for supporting the extrudate structure were further analyzed. The aggregation of protein subunits in extruded sample was investigated by using SDS-PAGE electrophoresis. The results showed that hydrophobic interactions, hydrogen bonds, disulfide bonds and their interactions collectively hold the structure of extrudate; and the importance of non-covalent bonds outweighs covalent bonds. Increasing feed moisture content could increase the interactions between disulfide bonds and hydrogen bonds and between disulfide bonds and hydrophobic interactions (p < 0.05), reduce the degree of aggregation and the difference in protein-protein interactions and protein subunits among different zones within the extruder.     

Low‐salt restructured fish products using microbial transglutaminase as binding agent

Low‐salt restructured silver carp products were obtained using mechanically deboned fish meat from filleting wastes of silver carp (Hypophthalmichthys molitrix). The additives used were NaCl at three levels (0 (control), 10 and 20 g kg^?1) and microbial transglutaminase (MTGase) also at three levels (0 (control), 3 and 6 g kg^?1). The fish meat was massaged with the additives at <15 °C for 1 h. The massaged fish paste was then packed into steel stainless tubes and cooked at 40 °C for 30 min followed by 90 °C for 15 min. Changes in mechanical properties (texture profile analysis and punch test), solubility, electrophoretic profile and expressible water were evaluated. Hardness was in the range from 26.3 to 52.4 N, cohesiveness varied from 0.185 to 0.318 and springiness varied from 0.418 to 0.768. Increasing the amount of both additives improved the mechanical and functional properties of the restructured silver carp products. MTGase activity was associated with a decrease in protein solubility and a decrease in the myosin band (SDS‐PAGE). Increasing NaCl decreased the amount of expressible water. The results indicated that it is feasible to obtain low‐salt restructured silver carp products with improved mechanical and good functional properties using 3 g kg^?1 MTGase and 10 g kg^?1 NaCl. © 2002 Society of Chemical Industry