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Suppression mechanism of l -arginine in the heat-induced aggregation of bighead carp (Aristichthys nobilis) myosin: The significance of ionic linkage effects and hydrogen bond effects
Food Hydrocolloids 102 (2020) 105596
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Suppression mechanism of L-arginine in the heat-induced aggregation of bighead carp (Aristichthys nobilis) myosin: The significance of ionic linkage effects and hydrogen bond effects Tong Shi a, Zhiyu Xiong a, Wengang Jin b, Li Yuan a, Quancai Sun a, Yuhao Zhang c, Xiuting Li d, **, Ruichang Gao a, b, * a
School of Food and Biological Engineering, Jiangsu University, Zhenjiang, Jiangsu Province, 212013, China Bio-resources Key Laboratory of Shaanxi Province, School of Biological Science and Engineering, Sha’anxi University of Technology, Hanzhong, 723001, China c College of Food Science, Southwest University, Chongqing, 400716, China d Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University, Beijing, 100048, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Arg Myosin Heat-induced aggregation Ionic interaction effects Hydrogen bond effect
The suppression mechanism of L-arginine (Arg) in fish myosin heat-induced aggregation was investigated in this work. The hydrodynamic radius determined by dynamic light scattering (DLS) and the turbidity of a myosin solution decreased in a dose-dependently manner with Arg after heating, especially in a solution without pH modification (P < 0.05). The surface hydrophobicity of myosin exhibited the same trend as that for hydrody namic radius and turbidity (P < 0.05). The myosin secondary structures clearly changed with Arg concentration during heating (P < 0.05). These results demonstrate that positively-charged Arg could form ionic interactions with negatively charged myosin, as well as promote repulsion between myosin molecules induced by higher pH values. Furthermore, the resultant hydrogen bonding observed indicated that, in addition to Arg having ionic linkage effects, the specific structure of Arg played an important role in the suppression of myosin aggregation. Essentially, Arg markedly disturbed the hydrogen bonds of the myosin backbone and formed new hydrogen bonds with myosin molecules via two amino groups (-NH2), which are part of the guanidinium moiety of Arg, preferentially hydrogen bonding with the backbone carbonyl oxygen atoms of myosin molecules. This work provides support for using Arg as an additive to modify the texture of fish products to produce liquid and semiliquid food suitable for the elderly consumers in an aging society.
1. Introduction Myosin, one component of the myofibrillar protein matrix, is a dominant gelling protein, with applications in achieving desired texture and fat-and-water stabilization in the processing of meat products (Zhou et al., 2014a,b). The thermal gelation of myosin from surimi, a source of protein with high quality, has been studied in detail (Lanier, Carvajal, Yongsawatdigul, & Park, 2005; Liu et al., 2010). Thermal gelation re sults from protein denaturation that leads to the formation of intermo lecular covalent bonds and noncovalent interactions in an ordered fashion to generate a continuous network structure (Chen et al., 2014). Attempts have been made to improve the network structure and gel strength to make more chewable and flexible surimi products (Jia, You,
Hu, Liu, & Xiong, 2015; Jian et al., 2016; Liu, Xu, Zhang, Zhao, & Ding, 2016; Zhuang et al., 2018). However, research is lacking in ways to modify the texture and color of fish products to obtain liquid and semiliquid food in consideration of the aging population. The application of amino acids in the meat industry has recently attracted considerable interest (Hao, Peng, Zhou, & Wang, 2015; Wachirasiri, Wanlapa, Uttapap, & Rungsardthong, 2016; Zhou, Li, & Tan, 2014). Lysine (Lys) and Arg have been reported as phosphate al ternatives to improve the quality of frozen white shrimp (Wachirasiri et al., 2016). Arg has been found to affect the thermal stability and gelation of salt-soluble meat proteins from breast muscle (Hao et al., 2015). The physicochemical properties of pork sausage have also been linked to the intake of Lys (Zhou et al., 2014a,b). In addition, various
* Corresponding author. School of Food and Biological Engineering, Jiangsu University, No. 301, Xuefu Road, Zhenjiang, Jiangsu Province, 212013, China. ** Corresponding author. E-mail addresses: [email protected] (X. Li), [email protected] (R. Gao). https://doi.org/10.1016/j.foodhyd.2019.105596 Received 14 September 2019; Received in revised form 3 December 2019; Accepted 16 December 2019 Available online 18 December 2019 0268-005X/© 2019 Elsevier Ltd. All rights reserved.
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studies have investigated the effects of amino acids on the solubility of myosin at low salt levels (Guo, Peng, Zhang, Liu, & Cui, 2015; Hay akawa, Ito, Wakamatsu, Nishimura, & Hattori, 2009; Chen et al., 2016). Furthermore, the role of Lys, Arg and L-histidine in suppressing heat-induced aggregation of bighead carp myosin was revealed in the authors’ previous studies (Gao et al., 2019; Gao, Wang, Mu, Shi, & Yuan, 2018; Shi, Yuan, Wang, & Gao, 2018). These results indicate that there may be an interaction between amino acids and myosin, which serves to protect the myosin from aggregating during heating. The guanidinium group, the functional group of Arg, has been speculated to have a potential role in the suppression of protein aggre gation by Arg (Gao et al., 2019; Hamada, Takahashi, Noguchi, & Shiraki, 2008; Matsuoka, Hamada, Matsumoto, & Shiraki, 2010). One study showed that the guanidinium group alone was insufficient in myosin solubilization (Takai, Yoshizawa, Ejima, Arakawa, & Shiraki, 2013). Recent research shows that guanidinium ions from guanidine hydro chloride may replace water molecules that form cages around exposed hydrophobic residues during the unfolding of hen egg-white lysozyme (Raskar, Yeow, Niebling, Kini, & Hosur, 2019). However, to our knowledge, no investigation to date has clearly detailed the molecular mechanism and intermolecular interactions related to suppression of the heat-induced aggregation of fish myosin by Arg, a process that is critical in modifying of the texture and color of fish products. The objective of this study was to investigate the suppression mechanism of Arg in the heat-induced aggregation of fish myosin, which was carried out using a particular experimental design and various instrumental techniques. For this purpose, it is feasible to broaden the versatile use of Arg for application as an additive to modify in the texture and color of fish products to obtain liquid and semiliquid food for elderly consumers, or as chemical denaturants in food applications.
carried out and samples were then stored in a refrigerator at 4 � C prior to the hydrogen bond determination; a native myosin-Arg solution was used as a control group. 2.5. Dynamic light scattering (DLS) Particle diameter was estimated by DLS measurement using a Lite sizer™ 500 (Anton Paar Instruments Ltd, Austria) equipped with a 658 nm laser source. The myosin-Arg and myosin-guanidinium suspensions (1.0 mg/mL) were placed in a 1.0-cm path-length glass cuvette with a thermal insulation cover and subjected to DLS measurement with a detection angle of 175� under Particle Size Series Mode (from 25 � C to 90 � C at 1 � C/min). Particle diameters of myosin aggregates were measured every 5 � C during the programmed heating process and monitored by the cumulants model, which is based on a single expo nential fit of the autocorrelation function to calculate the mean particle size. All samples were measured in triplicate. 2.6. Turbidity Turbidity was determined by modification of a previously reported procedure (Shi, Yuan, Wang, & Gao, 2018). Myosin-Arg and myosin-guanidinium suspensions (2.0 mg/mL) were placed in test tubes and heated from 25 � C to 90 � C at 1 � C/min with a programmable water bath. The absorbance values at 340 nm were measured every 5 � C using a UV/visible spectrophotometer (UV 1600, Rayleigh Analytical In struments, Beijing, China). All samples were measured in triplicate. 2.7. Surface hydrophobicity To determine the surface hydrophobicity (ANS-S0) of myosin-Arg sols, 8-anilino-1-naphthalene sulfonic acid (ANS) was used as a hydro phobic fluorescence probe (Gao et al., 2018; Guerrieri, Alberti, Lavelli, & Cerletti, 1996). The myosin-Arg sols were diluted with 0.5 M NaCl-20 mM Tris-HCl (pH 7.0) to a final volume of 4 mL with a resulting myosin concentration ranging from 0.0625 to 0.5 mg/mL. Subsequently, 20 μL of 8.0 mM ANS solution (dissolved in 0.5 M NaCl-20 mM Tris-HCl, pH 7.0) was added to the diluted sols, which were then incubated in the dark for 10 min. The relative fluorescence at different concentrations was detected in a Spectramax microplate reader (Spectramax M2, Mo lecular Devices, Sunnyvale, CA, USA) at excitation and emission wave lengths of 370 nm and 426 nm, respectively. The initial slope of the fluorescence intensity against the myosin concentration was computed as the index of ANS-S0. All samples were measured in triplicate.
2. Materials and methods 2.1. Materials Live bighead carp (Aristichthys nobilis) was obtained from the Auchan supermarket (Zhenjiang, China). Adenosine-triphosphate (ATP), Arg, guanidinoacetic acid, and guanidinium sulfate were purchased from Aladdin Industrial Corporation (Ontario, CA). All other chemicals of analytical grade were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2.2. Extraction of myosin Myosin was extracted from the dorsal muscle of bighead carp as previously reported (Yuan et al., 2017). The protein concentration of protein was measured by the Biuret method (Dustin, 1950). Analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a gel imaging system (Universal Hood II, Bio-Rad Co., USA) showed that the purity of the extracted myosin was over 90%.
2.8. Secondary structures Far-UV circular dichroism (CD) spectra of native myosin and myosinArg sols at 0.1 mg/mL were recorded from 205 to 240 nm using a Jasco J-815 spectropolarimeter (Jasco Co. Ltd., Tokyo, Japan) (Shi, Yuan, Mu, & Gao, 2019). The temperature was regulated with a Peltier controller from 25 � C to 85 � C. Spectra were obtained every 10 � C using a 1.0-cm path-length quartz cell at a scan rate of 100 nm/min and a bandwidth of 5 nm. Subsequently, the percentages of α-helix, β-sheet, β-turn and random coil structures were calculated using the protein secondary structure estimation program provided with the Jasco spec tropolarimeter. All spectra were corrected for the buffered solvent, and all samples were measured in triplicate.
2.3. Preparation of myosin-Arg sols A1: Arg was added to the myosin solution at various concentrations (0, 1, 10, 40 and 100 mM). A2: Arg was added to the myosin solution at various concentrations (0, 1, 10, 40 and 100 mM) with a final pH value of 7.0. The myosin-Arg sols were heated at 40 � C for 60 min in a water bath, and then heated at 90 � C for 30 min (two-step heating) prior to surface hydrophobicity measurements. Additionally, a native myosinArg solution was used as a control group.
2.9. Hydrogen bonds Hydrogen bonds of myosin-guanidinium mixtures were analyzed using a centrifugal method (Tan, Lai, & Kuochiang, 2010; Zhao et al., 2016). Two grams of samples prepared as described in 2.4 was ho mogenized at 5000 rpm with 10 mL of S1 (0.6 M sodium chloride) for 5 min. The resulting homogenate was stirred at 4 � C for 1 h and then
2.4. Preparation of myosin-guanidinium sols Each guanidinium material (Arg, guanidinoacetic acid and guanidi nium sulfate) was separately added to the myosin solution with a final concentration of 40 mM and pH value of 7.0. Two-step heating was 2
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Fig. 1. Effect of Arg on the hydrodynamic radius and turbidity of myosin during heating. (A & a: A1, pH values were not adjusted; B & b: A2, pH values were adjusted to 7.0).
centrifuged at 18, 600�g for 25 min. The obtained pellet was homoge nized with 10 mL of S2 (1.5 M urea þ0.6 M sodium chloride) and centrifuged by the same process. To prevent interference in protein determination, supernatants were treated with an equal volume (5 mL) of 20% trichloroacetic acid for protein precipitation, and the pellet was recovered in dissolved in 0.5 mL of 1 M NaOH. The protein concentra tion was determined based on the Lowry method (Rao et al., 2017), and the contribution of hydrogen bonding interactions was expressed as the percentage of the concentration retained after centrifugation compared to the initial concentration.
Arakawa, & Shiraki, 2015). As shown in Fig. 1A and Fig. 1B, the hy drodynamic radius of myosin aggregates in the control group (myosin solution without Arg), representing the size of myosin aggregates induced from heating, began to increase sharply from 35 � C and reached the first peak value at 50 � C before a moderate decline, after which it increased again with the increasing temperature and reached its highest point at 80 � C. Simultaneously, as Fig. 1a and b show, the turbidity of the myosin solution sharply increased from 40 � C and reached its first peak value at 70 � C before a slight decline, followed by an increase with increasing temperature. Presumably, the myosin molecules unfold when the temperature is above the thermal denaturation temperature point and subsequently aggregate. The observed increase in hydrodynamic radius and turbidity indicates that the size of myosin aggregates formed was large enough to scatter light strongly (Brenner, Johannsson, & Nicolai, 2009; Yarnpakdee, Benjakul, Visessanguan, & Kijroongrojana, 2009). This phenomenon was consistent with that observed in our pre vious work (Gao et al., 2019). Additionally, the two pivotal temperature ranges of 35–50 � C and 65–80 � C observed in the DLS measurements were theoretically attributed to the initial association of the myosin heads and the subsequent cross-linking of myosin rod regions, respec tively (Hayakawa et al., 2015; Samejima, Ishioroshi, & Yasui, 2010; Sharp & Offer, 2010). Moreover, the slight decrease in turbidity at 70 � C might be due to the settlement of myosin particles as a result of rapid and intense aggregation. The turbidity significantly decreased when the temperature was above 80 � C due to the aggregates being too large to settle; consequently, the supernatant became clear.
2.10. Statistical analysis The analyses of variances, means, and standard errors were carried out using Excel 2003 (Microsoft Office Excel 2003 for Windows). All the figures were drawn in Origin 2018. A significance level of P < 0.05 was used to determine the differences between the samples by Tukey tests with SPSS Statistics 17.0 (SPSS Inc., Chicago, IL, USA). 3. Results and discussion 3.1. DLS and turbidity DLS is an effective technology used to determine myosin filament dissociation or monomer aggregation by measuring the hydrodynamic radius of particles in solution (Chen et al., 2016; Shimada, Takai, Ejima, 3
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two abovementioned factors are herein called ionic linkage effects. Last but not least, specific interactions between moieties of Arg and myosin molecules were implicated, as shown by the dose-dependent decrease in the hydrodynamic radius and turbidity, just as the amount of net charges on the myosin molecules remained invariant in the presence of Arg with the modification of pH 7.0 (Fig. 1B and b). 3.2. Surface hydrophobicity The formation of intermolecular hydrophobic interactions, which results from the thermodynamic response of a protein surface exposed to the water in which it is dispersed or solubilized, is presently thought to be a primary mechanism for surimi gel formation resulting from heating or high pressure (�300 MPa) (Gilleland, Lanier, & Hamann, 2010; Lanier et al., 2005). As shown in Fig. 2, the surface hydrophobicity of natural myosin was not affected by Arg regardless of pH modification. The lack of an influence of surface hydrophobicity by Arg in the un heated treatment with 0.5 M NaCl was consistent with a previous report on myosin (Gao et al., 2018). After two-step heating, the surface hy drophobicity of all samples increased significantly (P < 0.05), which can be attributed to the unfolding of the protein molecules leading to exposure of some of the buried nonpolar amino acids during heating (Gao et al., 2018). Moreover, the increase in surface hydrophobicity of the myosin-Arg sols without pH modification (A1) was smaller than that of the control, and the higher the concentration of Arg was, the less the increase in surface hydrophobicity that was observed (P < 0.05). However, the surface hydrophobicity of myosin-Arg sols with pH modification (A2) increased as high as that of the control at 1 and 10 mM. In addition, the surface hydrophobicity of myosin-Arg with pH modification at 40 and 100 mM was higher than that of myosin-Arg without pH modification. Accordingly, this result indicates that Arg altered the tertiary structure of myosin during heating, which could be ascribed to the limited exposure of hydrophobic groups located and buried along the axis of the myosin molecule. Furthermore, the ionic linkage effects mentioned before were indicated, as A1 showed a more apparent suppressing effect than that of A2. As reported (Lanier et al., 2005), if, during unfolding of the protein and exposure of the hydrophobic core, the attraction and structuring of water resulting from the exposure of the backbone carbonyl and amide groups is not sufficiently strong, then exposure of the hydrophobic groups, leading to aggregation and gelation of neighboring proteins, would not take place. Based on this theory, it is reasonable to hypoth esize that Arg showed effects on the exposure of the backbone carbonyl and amide groups, a phenomenon called the “hydrogen bond effect” in this paper (see later discussion). The increasing pH value illustrated the capacity to promote this hydrogen bond effect. Furthermore, the gua nidinium ion, the main moiety of Arg, might replace water molecules that form cages around exposed hydrophobic residues (Raskar et al., 2019) so that the hydrophobic interaction required for myosin heat-induced aggregation would be reduced.
Fig. 2. Effect of Arg on the surface hydrophobicity of myosin during heating. Numbers represent mean � S.E. (n ¼ 3). Different lowercase letters indicate significant difference (P < 0.05).
The hydrodynamic radius of myosin versus temperature was obvi ously altered by Arg during heating (Fig. 1A and B), regardless of pH modification. The hydrodynamic radius of myosin with Arg without pH modification (A1) showed only one peak near 40 � C, corresponding to the association of myosin heads. Simultaneously, the peak value decreased in a dose-dependently manner with Arg concentration (Fig. 1A). The decrease of hydrodynamic radius in the presence of Arg was consistent with the previous work on myosin (Li, Zheng, Xu, Zhu, & Zhou, 2018). Furthermore, the peak corresponding to the cross-linking of myosin rods did not appear at any concentration. These results indi cated that the aggregation of the rod region of myosin molecules, which plays an important role in the formation of cross-linking with gelation, was significantly suppressed. The association of myosin heads was also clearly suppressed. Fig. 1B shows that the aggregation of myosin with Arg at pH 7.0 (A2) was also significantly suppressed, though the pH was modified the same as that for the control group. Furthermore, the effect was dose-dependent with Arg concentration. However, compared to Arg without pH modification (A1), Arg with pH modification (A2) showed a relatively weak ability to suppress the aggregation of myosin. In the group with pH modification, the cross-linking of myosin rods was completely suppressed over an Arg concentration of 10 mM, while the suppression of cross-linking occurred at less than 1 mM Arg in the group without pH modification. The turbidity of the myosin-Arg sols (Fig. 1a) was lower in a dose-dependently manner than that of the control at the same temperature during heating, which was consistent with the results of DLS (Fig. 1A). As a cationic amino acid, Arg exhibited a higher pH value in A1 than in A2 at the same concentration, which in turn led to a smaller hydrodynamic radius and lower turbidity during heating (Fig. 1A and a). This outcome was consistent with the theory that it was difficult to form filaments if the periodic charges were imbalanced, resulting from the changed conformation of the myosin rod (Hayakawa et al., 2009). These results indicated that the Arg-induced suppression of the heat-induced aggregation of myosin correlates not only to the pH but also to the structure of Arg. On the one hand, at the pH used in this experiment, the carboxyl groups (COO ) of glutamic acid and aspartic acid, two amino acids on the myosin chain, are negatively charged (Lanier et al., 2005), while Arg is net positively charged. It is expected that an ionic attraction will be presented between these groups, preventing filament formation. On the other hand, the total amount of net negative charge on the myosin molecules increases with the change in pH values induced by Arg. This effect more repulsion between myosin molecules, which helps stabilize them against aggregation compared with the case of the control. These
3.3. Secondary structures The secondary structure of myosin was significantly affected by the introduction of Arg (Fig. 3). It was observed that the α-helix content of myosin decreased remarkably, followed by an increase in the content of other secondary structure elements, particularly random coils (Fig. 3A), which can be attributed to the unfolding of the protein molecules during heating (Ota, Ikeguchi, Tanaka, & Hamada, 2016). The decreased α-helix content during heating was consistent with the study of myofi brillar proteins observed by Raman spectroscopy (Xu, Han, Fei, & Zhou, 2011). At a temperature equal to that for the control, the α-helix content of both A1 and A2 decreased proportionally with an increasing amount of Arg below 10 mM (Fig. 3A, B, 3b, 3C and 3c), and then remained at a stable level when higher concentrations (�40 mM, Fig. 3D, d, 3E and 3e) of Arg were added. For A1, Arg promoted myosin molecule unfolding in 4
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Fig. 3. Effect of Arg on the secondary structures of myosin during heating. Capital letters (A–E) indicate the A1 group, in which pH values were not adjusted. Lower case letters (b–e) indicate the A2 group, in which pH values were adjusted to 7.0. Letters on the same line represent the same addition, and the concentration of Arg from A to E are 0, 1 mM, 10 mM, 40 mM and 100 mM, respectively.
a concentration-dependent manner. The moderate concentration of Arg (�40 mM) was conducive to strong unfolding of the myosin molecule even if at the initial heating temperature of 25 � C (Fig. 3D and E). For A2, the random coil content of myosin containing 1 mM, 10 mM, 40 mM and
100 mM Arg reached the highest level at temperatures of 65 � C, 65 � C, 75 � C and 85 � C, respectively, indicating that the structure of Arg showed the potential to enhance the thermal stability of myosin molecules. 5
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Fig. 3. (continued).
As the rods of myosin molecules were composed of a large number of helical structures (Lanier et al., 2005), it was speculated that the α-helix losses of myosin induced by Arg were ascribed to conformational changes of the rod region, which was consistent with the results of DLS (Fig. 1A and B). The main causes of these synergistic effects appeared to be both pH (ionic linkage effects) and the structure of Arg, which were shown by the lower increase in random coil content of A2 with the same addition of Arg compared to that of A1. It is well known that the α-helix structure of myosin is mainly stabilized by hydrogen bonds between the carbonyl oxygen (-CO) and amino hydrogen (NH-) groups of the poly peptide chain (Cao & Xiong, 2015; Liu, Zhao, Xiong, Xie, & Qin, 2008). Therefore, the presence of Arg may have disturbed the hydrogen bonds in the polypeptide chain. It was theoretically hypothesized that two amino groups (-NH2) of the Arg molecule, one of which is located in the guanidinium group, replaced amino groups of the backbone in terms of forming new hydrogen bonds with the backbone carbonyl oxygens atoms (-CO). This phenomenon was similar to the effect of adding guanidine hydrochloride added to hen egg-white lysozyme, and the authors suggest that guanidinium ions preferentially form hydrogen bonds with the backbone carbonyl oxygen atoms (Raskar et al., 2019). Thus, the influence described in this study on the suppression of heat-induced aggregation of myosin caused by the structure of Arg was referred to as a hydrogen bond effect.
Fig. 4. Effect of guanidinium on the hydrodynamic radius and turbidity of myosin during heating.
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Fig. 5. Effect of guanidinium on the secondary structures of myosin during heating (pH 7.0). (A: control; B: 40 mM Arg; C: 40 mM guanidineacetic acid; D: 40 mM guanidinium sulfate).
3.4. Role of hydrogen bonds
3.4.2. Secondary structures Fig. 5 shows that all three additives changed the secondary structure of the myosin molecule, especially the α-helix structure, indicating the conformational changes of the rod region induced by the guanidinium ion (Lanier et al., 2005). It was observed that the α-helix content of myosin remained at a stable level in the presence of 40 mM Arg (Fig. 5B), suggesting that among the additives, Arg promoted a higher unfolding of the myosin molecule even if at the initial heating temperature of 25 � C. However, guanidinoacetic acid did not promote a high unfolding of myosin at the same concentration (Fig. 5C), which might be due to its strong interference in the determination of CD spectra (data not shown). The effect of guanidinium sulfate on the myosin secondary structure was the weakest among the three myosin-guanidinium mixtures (Fig. 5D). These results suggest that the ability to effectively influence the sec ondary structures increased in the following order: (1) myosin-guanidinium sulfate, (2) myosin-guanidinoacetic acid, and (3) myosin-Arg. It was hypothesized that the guanidinium ion of the three materials might have disturbed the hydrogen bonds that stabilize the α-helix structures of the myosin backbone. Furthermore, new hydrogen bonds may form between the amino hydrogen (NH-) of guanidinium and backbone carbonyl oxygens atoms (-CO) of myosin molecules (Raskar et al., 2019). However, it is also reasonable to conclude that guanidi nium is a critical factor, but not the only decisive factor, in the sup pression of the heat-induced aggregation behavior of myosin by Arg. The reason is because differences were displayed among the three additives,
3.4.1. DLS To verify the existence of a hydrogen bond effect, especially the hydrogen bonds between guanidinium ions and myosin molecules, the effects of Arg, guanidinoacetic acid, and guanidinium sulfate on the hydrodynamic radius, secondary structures, and hydrogen bond number of myosin were investigated at a concentration of 40 mM at pH 7.0. As shown in Fig. 4, the distribution of hydrodynamic radii indicated that myosin without additives tended to aggregate into particles and clusters as a result of head-head aggregation and rod-rod cross-linking (Chen et al., 2016). However, the hydrodynamic radius of myosin molecules in the presence of any one of the three additives significantly decreased during the whole programmed heating process. Furthermore, it was equally of interest to observe that the hydrodynamic radius of myosin in the presence of Arg, guanidinoacetic acid and guanidinium sulfate was maintained between 180 nm and 400 nm in a temperature-independent manner. This synergistic observation illus trated that soluble myosin species such as oligomers, filaments, and their complexes, rather than large particles or clusters, coexisted in the myosin-guanidinium sols during heating. As a result, it was reasonable to conclude that the guanidinium ion, the common group of three compounds, was the prime structural element involved in inhibiting the heat-induced aggregation behavior of myosin and possessed the ability to maintain myosin in a soluble state during heating.
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hydrophobicity during heating. Additionally, compared Arg without pH modification, Arg with pH modification showed a relatively weak ability to suppress myosin aggregation, indicating that the suppression induced by Arg in the heat-induced aggregation of myosin correlates not only to the pH, but also to the structure of Arg. CD studies indicated that the interaction of Arg with myosin caused significant conformational changes in the secondary structures of the protein. Furthermore, gua nidinium was a critical factor, but not the only decisive factor, in the suppression of the heat-induced aggregation behavior of myosin by Arg. In the present study, the suppression mechanism of Arg in the heatinduced myosin aggregation process was illustrated from two aspects. First, ionic attraction between positively charged Arg and negatively charged myosin occurred, and the repulsion between myosin molecules was promoted, induced by the higher pH values in the presence of Arg. Second, the two amino groups of Arg preferentially form hydrogen bonds with the backbone carbonyl oxygen atoms of the myosin mole cule. These results may be of interest to further broaden the versatile use of Arg as an additive for modifying of the texture of fish products to obtain liquid and semiliquid food for the elderly consumers in an increasingly aging society. Declaration of competing interest
Fig. 6. Effect of guanidinium on the hydrogen bonds of myosin (40 mM, pH 7.0). Different lowercase letters indicate significant difference (P < 0.05).
which contained the same concentration of guanidinium at the same pH value.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
3.5. Hydrogen bonds
Acknowledgments
As shown in Fig. 6, the number of hydrogen bonds of the control increased after two-step heating treatment. This transition could be ascribed to the gel form of myosin without Arg since the samples with high protein concentrations were stored at 4 � C immediately after twostep heating. This phenomenon was consistent with the theory that hydrogen bonds between proteins are more numerous when the gel is colder, producing a firmer gel structure (Lanier et al., 2005). All of the three additives containing guanidinium ions increased the number of hydrogen bonds of myosin in the unheated treatment, but the number decreased after two-step heating. This result indicated that the hydrogen bonds were first structurally integrated between each material and the myosin molecule but then were broken during heating. However, the number of hydrogen bonds in the myosin-guanidinium mixtures was lower than that of the control after two-step heating. This result showed that the hydrogen bonds formed between each guanidinium material and the myosin molecules were more easily broken than those formed within the myosin molecule backbone. The number of hydrogen bonds of natural myosin with Arg was the greatest among the three myosin-guanidinium mixtures, which was nearly twice as much as that of myosin with guanidinoacetic acid and guanidinium sulfate. This discrepancy might be due to the unequal number and particular structure of amino groups (-NH2) in these three materials. All of the additives possess a –NH2 group from the guanidi nium group. Essentially, Arg and guanidinium sulfate possess another –NH2 group, while the latter is joined to the sulfate group. This obser vation suggested that not only the guanidinium group, but also the other amino group in the Arg molecule was of prime importance in sup pressing the heat-induced aggregation of myosin, fundamentally via forming hydrogen bonds with carbonyl oxygen atoms of the myosin backbone (as shown before in the secondary structure analysis).
This work was supported by the National Natural Science Foundation of China (No. 31671882), the Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University (BTBU). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.foodhyd.2019.105596. Author contribution statement Tong Shi: Conceptualization, Experimentation, Data treatment, Interpretation, Writing Original Draft. Zhiyu Xiong: Methodology, Investigation. Wengang Jin: Conceptualization, Supervision. Li Yuan: Data treatment, Interpretation, Validation. Quancai Sun: Reviewing Draft. Yuhao Zhang: Data treatment, Writing Original Draft. Xiuting Li: Data treatment, Interpretation, Supervision, Validation. Ruichang Gao: Conceptualization, Data treatment, Interpretation, Validation, Writing, Reviewing, Editing, Supervision. References Brenner, T., Johannsson, R., & Nicolai, T. (2009). Characterization of fish myosin aggregates using static and dynamic light scattering. Food Hydrocolloids, 23(2), 296–305. Cao, Y. G., & Xiong, Y. L. (2015). Chlorogenic acid-mediated gel formation of oxidatively stressed myofibrillar protein. Food Chemistry, 180, 235–243. Chen, X., Li, P. J., Nishiumi, T., Takumi, H., Suzuki, A., & Chen, C. G. (2014). Effects of high-pressure processing on the cooking loss and gel strength of chicken breast actomyosin containing sodium alginate. Food and Bioprocess Technology, 7(12), 3608–3617. Chen, X., Zou, Y. F., Han, M. Y., Pan, L. H., Xing, T., Xu, X. L., et al. (2016). Solubilisation of myosin in a solution of low ionic strength l-histidine: Significance of the imidazole ring. Food Chemistry, 196(3), 42–49. Dustin, J. P. (1950). Determination of proteins by the biuret reaction. Bulletin de la Societe de Chimie Biologique, 32(9–10), 696.
4. Conclusion Arg was demonstrated to suppress the heat-induced aggregation of fish myosin regardless of pH modification, which was shown by a dosedependent decrease in hydrodynamic radius, turbidity and surface 8
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Gao, R. C., Shi, T., Sun, Q. C., Li, X. T., McClements, D. J., & Yuan, L. (2019). Effects of Larginine and L-histidine on heat-induced aggregation of fish myosin: Bighead carp (Aristichthys nobilis). Food Chemistry, 295, 320–326. Gao, R. C., Wang, Y. M., Mu, J. L., Shi, T., & Yuan, L. (2018). Effect of L-histidine on the heat-induced aggregation of bighead carp (Aristichthys nobilis) myosin in low/high ionic strength solution. Food Hydrocolloids, 75, 174–181. Gilleland, G. M., Lanier, T. C., & Hamann, D. D. (2010). Covalent bonding in pressureinduced fish protein gels. Journal of Food Science, 62(4), 713–733. Guerrieri, N., Alberti, E., Lavelli, V., & Cerletti, P. (1996). Use of spectroscopic and fluorescence techniques to assess heat-induced molecular modifications of gluten. Cereal Chemistry, 73(3), 368–374. Guo, X. Y., Peng, Z. Q., Zhang, Y. W., Liu, B., & Cui, Y. Q. (2015). The solubility and conformational characteristics of porcine myosin as affected by the presence of Llysine and L-histidine. Food Chemistry, 170, 212–217. Hamada, H., Takahashi, R., Noguchi, T., & Shiraki, K. (2008). Differences in the effects of solution additives on heat- and refolding-induced aggregation. Biotechnology Progress, 24(2), 436–443. Hao, Q., Peng, X., Zhou, C. L., & Wang, Y. J. (2015). Effects of l-Arginine on water holding capacity and texture of heat-induced gel of salt-soluble proteins from breast muscle. Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 63 (2), 912–918. Hayakawa, T., Ito, T., Wakamatsu, J., Nishimura, T., & Hattori, A. (2009). Myosin is solubilized in a neutral and low ionic strength solution containing L-histidine. Meat Science, 82(2), 151–154. Hayakawa, T., Yoshida, Y., Yasui, M., Ito, T., Wakamatsu, J.-i., Hattori, A., et al. (2015). Role of heavy meromyosin in heat-induced gelation in low ionic strength solution containing L-histidine. Journal of Food Science, 80(8), C1641–C1645. Jian, W. J., Wu, H. Y., Wu, L. L., Wu, Y. H., Jia, L. N., Pang, J., et al. (2016). Effect of molecular characteristics of Konjac glucomannan on gelling and rheological properties of Tilapia myofibrillar protein. Carbohydrate Polymers, 150, 21–31. Jia, D., You, J., Hu, Y., Liu, R., & Xiong, S. B. (2015). Effect of CaCl2 on denaturation and aggregation of silver carp myosin during setting. Food Chemistry, 185, 212–218. Lanier, T. C., Carvajal, P., Yongsawatdigul, J., & Park, J. W. (2005). Surimi gelation chemistry. Liu, J. H., Xu, Q. H., Zhang, J. J., Zhao, P. C., & Ding, Y. T. (2016). Characterization of silver carp ( Hypophthalmichthys molitrix ) myosin protein glycated with konjac oligo-glucomannan. Food Hydrocolloids, 57, 114–121. Liu, R., Zhao, S. M., Liu, Y. M., Yang, H., Xiong, S. B., Xie, B. J., et al. (2010). Effect of pH on the gel properties and secondary structure of fish myosin. Food Chemistry, 121(1), 196–202. Liu, R., Zhao, S. M., Xiong, S. B., Xie, B. J., & Qin, L. H. (2008). Role of secondary structures in the gelation of porcine myosin at different pH values. Meat Science, 80 (3), 632–639. Li, S. Y., Zheng, Y. D., Xu, P., Zhu, X. X., & Zhou, C. L. (2018). L-Lysine and L-arginine inhibit myosin aggregation and interact with acidic amino acid residues of myosin: The role in increasing myosin solubility. Food Chemistry, 242, 22–28. Matsuoka, T., Hamada, H., Matsumoto, K., & Shiraki, K. (2010). Indispensable structure of solution additives to prevent inactivation of lysozyme for heating and refolding. Biotechnology Progress, 25(5), 1515–1524. Ota, C., Ikeguchi, M., Tanaka, A., & Hamada, D. (2016). Residual structures in the unfolded state of starch-binding domain of glucoamylase revealed by near-UV circular dichroism and protein engineering techniques. BBA - Proteins and Proteomics, 1864(10), 1464–1472.
Rao, W. L., Li, X., Wang, Z. Y., Yang, Y., Qu, Y. L., Gao, Y., et al. (2017). Dense phase carbon dioxide combined with mild heating induced myosin denaturation, texture improvement and gel properties of sausage. Journal of Food Process Engineering, 40 (2), 1–10. Raskar, T., Yeow, C., Niebling, S., Kini, R. M., & Hosur, M. V. (2019). X-ray crystallographic analysis of time-dependent binding of guanidine hydrochloride to HEWL: First steps during protein unfolding. International Journal of Biological Macromolecules, 122, 903–913. Samejima, K., Ishioroshi, M., & Yasui, T. (2010). Relative roles of the head and tail portions of the molecule in heat-induced gelation of myosin. Journal of Food Science, 46(5), 1412–1418. Sharp, A., & Offer, G. (2010). The mechanism of formation of gels from myosin molecules. Journal of the Science of Food and Agriculture, 58(1), 63–73. Shi, T., Yuan, L., Wang, Y. M., & Gao, R. C. (2018). Influence of L-lysine on heat-induced aggregation of bighead carp myosin. Food Science, 39(8), 1–5. Shimada, M., Takai, E., Ejima, D., Arakawa, T., & Shiraki, K. (2015). Heat-induced formation of myosin oligomer-soluble filament complex in high-salt solution. International Journal of Biological Macromolecules, 73, 17–22. Shi, T., Yuan, L., Mu, J. L., & Gao, R. C. (2019). The effect of Arginine, Lysine and Histidine in the myosin secondary structure by circular dichroism and Raman spectroscopy. CyTA - Journal of Food, 17(1), 656–660. Takai, E., Yoshizawa, S., Ejima, D., Arakawa, T., & Shiraki, K. (2013). Synergistic solubilization of porcine myosin in physiological salt solution by arginine. International Journal of Biological Macromolecules, 62(11), 647–651. Tan, F. J., Lai, K. M., & Kuochiang, H. (2010). A comparative study on physical properties and chemical interactions of gels from tilapia meat pastes induced by heat and pressure. Journal of Texture Studies, 41(2), 153–170. Wachirasiri, K., Wanlapa, S., Uttapap, D., & Rungsardthong, V. (2016). Use of amino acids as a phosphate alternative and their effects on quality of frozen white shrimps ( Penaeus vanamei ). Lebensmittel-Wissenschaft und -Technologie- Food Science and Technology, 69, 303–311. Xu, X. L., Han, M. Y., Fei, Y., & Zhou, G. H. (2011). Raman spectroscopic study of heatinduced gelation of pork myofibrillar proteins and its relationship with textural characteristic. Meat Science, 87(3), 159–164. Yarnpakdee, S., Benjakul, S., Visessanguan, W., & Kijroongrojana, K. (2009). Thermal properties and heat-induced aggregation of natural actomyosin extracted from goatfish (Mulloidichthys martinicus) muscle as influenced by iced storage. Food Hydrocolloids, 23(7), 1779–1784. Yuan, L., Liu, Y. A., Ge, J., Feng, X. P., & Gao, R. C. (2017). Effects of heat treatment at two temperatures on the myosin cluster of bighead carp for gel formation. CyTA Journal of Food, 15(12), 574–581. Zhao, Y., Chen, Z. Y., Li, J. K., Xu, M. S., Shao, Y. Y., & Tu, Y. G. (2016). Changes of microstructure characteristics and intermolecular interactions of preserved egg white gel during pickling. Food Chemistry, 203, 323–330. Zhou, Y. Z., Chen, C. G., Chen, X., Li, P. J., Ma, F., & Lu, Q. H. (2014). Contribution of three ionic types of polysaccharides to the thermal gelling properties of chicken breast myosin. Journal of Agricultural and Food Chemistry, 62, 2655–2662. Zhou, C. L., Li, J., & Tan, S. J. (2014). Effect of l-lysine on the physicochemical properties of pork sausage. Food Science & Biotechnology, 23(3), 775–780. Zhuang, X. B., Han, M. Y., Bai, Y., Liu, Y. F., Xing, L. J., Xu, X. L., et al. (2018). Insight into the mechanism of myofibrillar protein gel improved by insoluble dietary fiber. Food Hydrocolloids, 74, 219–226.
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Food Hydrocolloids journal homepage: http://www.elsevier.com/locate/foodhyd
Suppression mechanism of L-arginine in the heat-induced aggregation of bighead carp (Aristichthys nobilis) myosin: The significance of ionic linkage effects and hydrogen bond effects Tong Shi a, Zhiyu Xiong a, Wengang Jin b, Li Yuan a, Quancai Sun a, Yuhao Zhang c, Xiuting Li d, **, Ruichang Gao a, b, * a
School of Food and Biological Engineering, Jiangsu University, Zhenjiang, Jiangsu Province, 212013, China Bio-resources Key Laboratory of Shaanxi Province, School of Biological Science and Engineering, Sha’anxi University of Technology, Hanzhong, 723001, China c College of Food Science, Southwest University, Chongqing, 400716, China d Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University, Beijing, 100048, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Arg Myosin Heat-induced aggregation Ionic interaction effects Hydrogen bond effect
The suppression mechanism of L-arginine (Arg) in fish myosin heat-induced aggregation was investigated in this work. The hydrodynamic radius determined by dynamic light scattering (DLS) and the turbidity of a myosin solution decreased in a dose-dependently manner with Arg after heating, especially in a solution without pH modification (P < 0.05). The surface hydrophobicity of myosin exhibited the same trend as that for hydrody namic radius and turbidity (P < 0.05). The myosin secondary structures clearly changed with Arg concentration during heating (P < 0.05). These results demonstrate that positively-charged Arg could form ionic interactions with negatively charged myosin, as well as promote repulsion between myosin molecules induced by higher pH values. Furthermore, the resultant hydrogen bonding observed indicated that, in addition to Arg having ionic linkage effects, the specific structure of Arg played an important role in the suppression of myosin aggregation. Essentially, Arg markedly disturbed the hydrogen bonds of the myosin backbone and formed new hydrogen bonds with myosin molecules via two amino groups (-NH2), which are part of the guanidinium moiety of Arg, preferentially hydrogen bonding with the backbone carbonyl oxygen atoms of myosin molecules. This work provides support for using Arg as an additive to modify the texture of fish products to produce liquid and semiliquid food suitable for the elderly consumers in an aging society.
1. Introduction Myosin, one component of the myofibrillar protein matrix, is a dominant gelling protein, with applications in achieving desired texture and fat-and-water stabilization in the processing of meat products (Zhou et al., 2014a,b). The thermal gelation of myosin from surimi, a source of protein with high quality, has been studied in detail (Lanier, Carvajal, Yongsawatdigul, & Park, 2005; Liu et al., 2010). Thermal gelation re sults from protein denaturation that leads to the formation of intermo lecular covalent bonds and noncovalent interactions in an ordered fashion to generate a continuous network structure (Chen et al., 2014). Attempts have been made to improve the network structure and gel strength to make more chewable and flexible surimi products (Jia, You,
Hu, Liu, & Xiong, 2015; Jian et al., 2016; Liu, Xu, Zhang, Zhao, & Ding, 2016; Zhuang et al., 2018). However, research is lacking in ways to modify the texture and color of fish products to obtain liquid and semiliquid food in consideration of the aging population. The application of amino acids in the meat industry has recently attracted considerable interest (Hao, Peng, Zhou, & Wang, 2015; Wachirasiri, Wanlapa, Uttapap, & Rungsardthong, 2016; Zhou, Li, & Tan, 2014). Lysine (Lys) and Arg have been reported as phosphate al ternatives to improve the quality of frozen white shrimp (Wachirasiri et al., 2016). Arg has been found to affect the thermal stability and gelation of salt-soluble meat proteins from breast muscle (Hao et al., 2015). The physicochemical properties of pork sausage have also been linked to the intake of Lys (Zhou et al., 2014a,b). In addition, various
* Corresponding author. School of Food and Biological Engineering, Jiangsu University, No. 301, Xuefu Road, Zhenjiang, Jiangsu Province, 212013, China. ** Corresponding author. E-mail addresses: [email protected] (X. Li), [email protected] (R. Gao). https://doi.org/10.1016/j.foodhyd.2019.105596 Received 14 September 2019; Received in revised form 3 December 2019; Accepted 16 December 2019 Available online 18 December 2019 0268-005X/© 2019 Elsevier Ltd. All rights reserved.
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studies have investigated the effects of amino acids on the solubility of myosin at low salt levels (Guo, Peng, Zhang, Liu, & Cui, 2015; Hay akawa, Ito, Wakamatsu, Nishimura, & Hattori, 2009; Chen et al., 2016). Furthermore, the role of Lys, Arg and L-histidine in suppressing heat-induced aggregation of bighead carp myosin was revealed in the authors’ previous studies (Gao et al., 2019; Gao, Wang, Mu, Shi, & Yuan, 2018; Shi, Yuan, Wang, & Gao, 2018). These results indicate that there may be an interaction between amino acids and myosin, which serves to protect the myosin from aggregating during heating. The guanidinium group, the functional group of Arg, has been speculated to have a potential role in the suppression of protein aggre gation by Arg (Gao et al., 2019; Hamada, Takahashi, Noguchi, & Shiraki, 2008; Matsuoka, Hamada, Matsumoto, & Shiraki, 2010). One study showed that the guanidinium group alone was insufficient in myosin solubilization (Takai, Yoshizawa, Ejima, Arakawa, & Shiraki, 2013). Recent research shows that guanidinium ions from guanidine hydro chloride may replace water molecules that form cages around exposed hydrophobic residues during the unfolding of hen egg-white lysozyme (Raskar, Yeow, Niebling, Kini, & Hosur, 2019). However, to our knowledge, no investigation to date has clearly detailed the molecular mechanism and intermolecular interactions related to suppression of the heat-induced aggregation of fish myosin by Arg, a process that is critical in modifying of the texture and color of fish products. The objective of this study was to investigate the suppression mechanism of Arg in the heat-induced aggregation of fish myosin, which was carried out using a particular experimental design and various instrumental techniques. For this purpose, it is feasible to broaden the versatile use of Arg for application as an additive to modify in the texture and color of fish products to obtain liquid and semiliquid food for elderly consumers, or as chemical denaturants in food applications.
carried out and samples were then stored in a refrigerator at 4 � C prior to the hydrogen bond determination; a native myosin-Arg solution was used as a control group. 2.5. Dynamic light scattering (DLS) Particle diameter was estimated by DLS measurement using a Lite sizer™ 500 (Anton Paar Instruments Ltd, Austria) equipped with a 658 nm laser source. The myosin-Arg and myosin-guanidinium suspensions (1.0 mg/mL) were placed in a 1.0-cm path-length glass cuvette with a thermal insulation cover and subjected to DLS measurement with a detection angle of 175� under Particle Size Series Mode (from 25 � C to 90 � C at 1 � C/min). Particle diameters of myosin aggregates were measured every 5 � C during the programmed heating process and monitored by the cumulants model, which is based on a single expo nential fit of the autocorrelation function to calculate the mean particle size. All samples were measured in triplicate. 2.6. Turbidity Turbidity was determined by modification of a previously reported procedure (Shi, Yuan, Wang, & Gao, 2018). Myosin-Arg and myosin-guanidinium suspensions (2.0 mg/mL) were placed in test tubes and heated from 25 � C to 90 � C at 1 � C/min with a programmable water bath. The absorbance values at 340 nm were measured every 5 � C using a UV/visible spectrophotometer (UV 1600, Rayleigh Analytical In struments, Beijing, China). All samples were measured in triplicate. 2.7. Surface hydrophobicity To determine the surface hydrophobicity (ANS-S0) of myosin-Arg sols, 8-anilino-1-naphthalene sulfonic acid (ANS) was used as a hydro phobic fluorescence probe (Gao et al., 2018; Guerrieri, Alberti, Lavelli, & Cerletti, 1996). The myosin-Arg sols were diluted with 0.5 M NaCl-20 mM Tris-HCl (pH 7.0) to a final volume of 4 mL with a resulting myosin concentration ranging from 0.0625 to 0.5 mg/mL. Subsequently, 20 μL of 8.0 mM ANS solution (dissolved in 0.5 M NaCl-20 mM Tris-HCl, pH 7.0) was added to the diluted sols, which were then incubated in the dark for 10 min. The relative fluorescence at different concentrations was detected in a Spectramax microplate reader (Spectramax M2, Mo lecular Devices, Sunnyvale, CA, USA) at excitation and emission wave lengths of 370 nm and 426 nm, respectively. The initial slope of the fluorescence intensity against the myosin concentration was computed as the index of ANS-S0. All samples were measured in triplicate.
2. Materials and methods 2.1. Materials Live bighead carp (Aristichthys nobilis) was obtained from the Auchan supermarket (Zhenjiang, China). Adenosine-triphosphate (ATP), Arg, guanidinoacetic acid, and guanidinium sulfate were purchased from Aladdin Industrial Corporation (Ontario, CA). All other chemicals of analytical grade were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2.2. Extraction of myosin Myosin was extracted from the dorsal muscle of bighead carp as previously reported (Yuan et al., 2017). The protein concentration of protein was measured by the Biuret method (Dustin, 1950). Analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a gel imaging system (Universal Hood II, Bio-Rad Co., USA) showed that the purity of the extracted myosin was over 90%.
2.8. Secondary structures Far-UV circular dichroism (CD) spectra of native myosin and myosinArg sols at 0.1 mg/mL were recorded from 205 to 240 nm using a Jasco J-815 spectropolarimeter (Jasco Co. Ltd., Tokyo, Japan) (Shi, Yuan, Mu, & Gao, 2019). The temperature was regulated with a Peltier controller from 25 � C to 85 � C. Spectra were obtained every 10 � C using a 1.0-cm path-length quartz cell at a scan rate of 100 nm/min and a bandwidth of 5 nm. Subsequently, the percentages of α-helix, β-sheet, β-turn and random coil structures were calculated using the protein secondary structure estimation program provided with the Jasco spec tropolarimeter. All spectra were corrected for the buffered solvent, and all samples were measured in triplicate.
2.3. Preparation of myosin-Arg sols A1: Arg was added to the myosin solution at various concentrations (0, 1, 10, 40 and 100 mM). A2: Arg was added to the myosin solution at various concentrations (0, 1, 10, 40 and 100 mM) with a final pH value of 7.0. The myosin-Arg sols were heated at 40 � C for 60 min in a water bath, and then heated at 90 � C for 30 min (two-step heating) prior to surface hydrophobicity measurements. Additionally, a native myosinArg solution was used as a control group.
2.9. Hydrogen bonds Hydrogen bonds of myosin-guanidinium mixtures were analyzed using a centrifugal method (Tan, Lai, & Kuochiang, 2010; Zhao et al., 2016). Two grams of samples prepared as described in 2.4 was ho mogenized at 5000 rpm with 10 mL of S1 (0.6 M sodium chloride) for 5 min. The resulting homogenate was stirred at 4 � C for 1 h and then
2.4. Preparation of myosin-guanidinium sols Each guanidinium material (Arg, guanidinoacetic acid and guanidi nium sulfate) was separately added to the myosin solution with a final concentration of 40 mM and pH value of 7.0. Two-step heating was 2
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Fig. 1. Effect of Arg on the hydrodynamic radius and turbidity of myosin during heating. (A & a: A1, pH values were not adjusted; B & b: A2, pH values were adjusted to 7.0).
centrifuged at 18, 600�g for 25 min. The obtained pellet was homoge nized with 10 mL of S2 (1.5 M urea þ0.6 M sodium chloride) and centrifuged by the same process. To prevent interference in protein determination, supernatants were treated with an equal volume (5 mL) of 20% trichloroacetic acid for protein precipitation, and the pellet was recovered in dissolved in 0.5 mL of 1 M NaOH. The protein concentra tion was determined based on the Lowry method (Rao et al., 2017), and the contribution of hydrogen bonding interactions was expressed as the percentage of the concentration retained after centrifugation compared to the initial concentration.
Arakawa, & Shiraki, 2015). As shown in Fig. 1A and Fig. 1B, the hy drodynamic radius of myosin aggregates in the control group (myosin solution without Arg), representing the size of myosin aggregates induced from heating, began to increase sharply from 35 � C and reached the first peak value at 50 � C before a moderate decline, after which it increased again with the increasing temperature and reached its highest point at 80 � C. Simultaneously, as Fig. 1a and b show, the turbidity of the myosin solution sharply increased from 40 � C and reached its first peak value at 70 � C before a slight decline, followed by an increase with increasing temperature. Presumably, the myosin molecules unfold when the temperature is above the thermal denaturation temperature point and subsequently aggregate. The observed increase in hydrodynamic radius and turbidity indicates that the size of myosin aggregates formed was large enough to scatter light strongly (Brenner, Johannsson, & Nicolai, 2009; Yarnpakdee, Benjakul, Visessanguan, & Kijroongrojana, 2009). This phenomenon was consistent with that observed in our pre vious work (Gao et al., 2019). Additionally, the two pivotal temperature ranges of 35–50 � C and 65–80 � C observed in the DLS measurements were theoretically attributed to the initial association of the myosin heads and the subsequent cross-linking of myosin rod regions, respec tively (Hayakawa et al., 2015; Samejima, Ishioroshi, & Yasui, 2010; Sharp & Offer, 2010). Moreover, the slight decrease in turbidity at 70 � C might be due to the settlement of myosin particles as a result of rapid and intense aggregation. The turbidity significantly decreased when the temperature was above 80 � C due to the aggregates being too large to settle; consequently, the supernatant became clear.
2.10. Statistical analysis The analyses of variances, means, and standard errors were carried out using Excel 2003 (Microsoft Office Excel 2003 for Windows). All the figures were drawn in Origin 2018. A significance level of P < 0.05 was used to determine the differences between the samples by Tukey tests with SPSS Statistics 17.0 (SPSS Inc., Chicago, IL, USA). 3. Results and discussion 3.1. DLS and turbidity DLS is an effective technology used to determine myosin filament dissociation or monomer aggregation by measuring the hydrodynamic radius of particles in solution (Chen et al., 2016; Shimada, Takai, Ejima, 3
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two abovementioned factors are herein called ionic linkage effects. Last but not least, specific interactions between moieties of Arg and myosin molecules were implicated, as shown by the dose-dependent decrease in the hydrodynamic radius and turbidity, just as the amount of net charges on the myosin molecules remained invariant in the presence of Arg with the modification of pH 7.0 (Fig. 1B and b). 3.2. Surface hydrophobicity The formation of intermolecular hydrophobic interactions, which results from the thermodynamic response of a protein surface exposed to the water in which it is dispersed or solubilized, is presently thought to be a primary mechanism for surimi gel formation resulting from heating or high pressure (�300 MPa) (Gilleland, Lanier, & Hamann, 2010; Lanier et al., 2005). As shown in Fig. 2, the surface hydrophobicity of natural myosin was not affected by Arg regardless of pH modification. The lack of an influence of surface hydrophobicity by Arg in the un heated treatment with 0.5 M NaCl was consistent with a previous report on myosin (Gao et al., 2018). After two-step heating, the surface hy drophobicity of all samples increased significantly (P < 0.05), which can be attributed to the unfolding of the protein molecules leading to exposure of some of the buried nonpolar amino acids during heating (Gao et al., 2018). Moreover, the increase in surface hydrophobicity of the myosin-Arg sols without pH modification (A1) was smaller than that of the control, and the higher the concentration of Arg was, the less the increase in surface hydrophobicity that was observed (P < 0.05). However, the surface hydrophobicity of myosin-Arg sols with pH modification (A2) increased as high as that of the control at 1 and 10 mM. In addition, the surface hydrophobicity of myosin-Arg with pH modification at 40 and 100 mM was higher than that of myosin-Arg without pH modification. Accordingly, this result indicates that Arg altered the tertiary structure of myosin during heating, which could be ascribed to the limited exposure of hydrophobic groups located and buried along the axis of the myosin molecule. Furthermore, the ionic linkage effects mentioned before were indicated, as A1 showed a more apparent suppressing effect than that of A2. As reported (Lanier et al., 2005), if, during unfolding of the protein and exposure of the hydrophobic core, the attraction and structuring of water resulting from the exposure of the backbone carbonyl and amide groups is not sufficiently strong, then exposure of the hydrophobic groups, leading to aggregation and gelation of neighboring proteins, would not take place. Based on this theory, it is reasonable to hypoth esize that Arg showed effects on the exposure of the backbone carbonyl and amide groups, a phenomenon called the “hydrogen bond effect” in this paper (see later discussion). The increasing pH value illustrated the capacity to promote this hydrogen bond effect. Furthermore, the gua nidinium ion, the main moiety of Arg, might replace water molecules that form cages around exposed hydrophobic residues (Raskar et al., 2019) so that the hydrophobic interaction required for myosin heat-induced aggregation would be reduced.
Fig. 2. Effect of Arg on the surface hydrophobicity of myosin during heating. Numbers represent mean � S.E. (n ¼ 3). Different lowercase letters indicate significant difference (P < 0.05).
The hydrodynamic radius of myosin versus temperature was obvi ously altered by Arg during heating (Fig. 1A and B), regardless of pH modification. The hydrodynamic radius of myosin with Arg without pH modification (A1) showed only one peak near 40 � C, corresponding to the association of myosin heads. Simultaneously, the peak value decreased in a dose-dependently manner with Arg concentration (Fig. 1A). The decrease of hydrodynamic radius in the presence of Arg was consistent with the previous work on myosin (Li, Zheng, Xu, Zhu, & Zhou, 2018). Furthermore, the peak corresponding to the cross-linking of myosin rods did not appear at any concentration. These results indi cated that the aggregation of the rod region of myosin molecules, which plays an important role in the formation of cross-linking with gelation, was significantly suppressed. The association of myosin heads was also clearly suppressed. Fig. 1B shows that the aggregation of myosin with Arg at pH 7.0 (A2) was also significantly suppressed, though the pH was modified the same as that for the control group. Furthermore, the effect was dose-dependent with Arg concentration. However, compared to Arg without pH modification (A1), Arg with pH modification (A2) showed a relatively weak ability to suppress the aggregation of myosin. In the group with pH modification, the cross-linking of myosin rods was completely suppressed over an Arg concentration of 10 mM, while the suppression of cross-linking occurred at less than 1 mM Arg in the group without pH modification. The turbidity of the myosin-Arg sols (Fig. 1a) was lower in a dose-dependently manner than that of the control at the same temperature during heating, which was consistent with the results of DLS (Fig. 1A). As a cationic amino acid, Arg exhibited a higher pH value in A1 than in A2 at the same concentration, which in turn led to a smaller hydrodynamic radius and lower turbidity during heating (Fig. 1A and a). This outcome was consistent with the theory that it was difficult to form filaments if the periodic charges were imbalanced, resulting from the changed conformation of the myosin rod (Hayakawa et al., 2009). These results indicated that the Arg-induced suppression of the heat-induced aggregation of myosin correlates not only to the pH but also to the structure of Arg. On the one hand, at the pH used in this experiment, the carboxyl groups (COO ) of glutamic acid and aspartic acid, two amino acids on the myosin chain, are negatively charged (Lanier et al., 2005), while Arg is net positively charged. It is expected that an ionic attraction will be presented between these groups, preventing filament formation. On the other hand, the total amount of net negative charge on the myosin molecules increases with the change in pH values induced by Arg. This effect more repulsion between myosin molecules, which helps stabilize them against aggregation compared with the case of the control. These
3.3. Secondary structures The secondary structure of myosin was significantly affected by the introduction of Arg (Fig. 3). It was observed that the α-helix content of myosin decreased remarkably, followed by an increase in the content of other secondary structure elements, particularly random coils (Fig. 3A), which can be attributed to the unfolding of the protein molecules during heating (Ota, Ikeguchi, Tanaka, & Hamada, 2016). The decreased α-helix content during heating was consistent with the study of myofi brillar proteins observed by Raman spectroscopy (Xu, Han, Fei, & Zhou, 2011). At a temperature equal to that for the control, the α-helix content of both A1 and A2 decreased proportionally with an increasing amount of Arg below 10 mM (Fig. 3A, B, 3b, 3C and 3c), and then remained at a stable level when higher concentrations (�40 mM, Fig. 3D, d, 3E and 3e) of Arg were added. For A1, Arg promoted myosin molecule unfolding in 4
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Fig. 3. Effect of Arg on the secondary structures of myosin during heating. Capital letters (A–E) indicate the A1 group, in which pH values were not adjusted. Lower case letters (b–e) indicate the A2 group, in which pH values were adjusted to 7.0. Letters on the same line represent the same addition, and the concentration of Arg from A to E are 0, 1 mM, 10 mM, 40 mM and 100 mM, respectively.
a concentration-dependent manner. The moderate concentration of Arg (�40 mM) was conducive to strong unfolding of the myosin molecule even if at the initial heating temperature of 25 � C (Fig. 3D and E). For A2, the random coil content of myosin containing 1 mM, 10 mM, 40 mM and
100 mM Arg reached the highest level at temperatures of 65 � C, 65 � C, 75 � C and 85 � C, respectively, indicating that the structure of Arg showed the potential to enhance the thermal stability of myosin molecules. 5
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Fig. 3. (continued).
As the rods of myosin molecules were composed of a large number of helical structures (Lanier et al., 2005), it was speculated that the α-helix losses of myosin induced by Arg were ascribed to conformational changes of the rod region, which was consistent with the results of DLS (Fig. 1A and B). The main causes of these synergistic effects appeared to be both pH (ionic linkage effects) and the structure of Arg, which were shown by the lower increase in random coil content of A2 with the same addition of Arg compared to that of A1. It is well known that the α-helix structure of myosin is mainly stabilized by hydrogen bonds between the carbonyl oxygen (-CO) and amino hydrogen (NH-) groups of the poly peptide chain (Cao & Xiong, 2015; Liu, Zhao, Xiong, Xie, & Qin, 2008). Therefore, the presence of Arg may have disturbed the hydrogen bonds in the polypeptide chain. It was theoretically hypothesized that two amino groups (-NH2) of the Arg molecule, one of which is located in the guanidinium group, replaced amino groups of the backbone in terms of forming new hydrogen bonds with the backbone carbonyl oxygens atoms (-CO). This phenomenon was similar to the effect of adding guanidine hydrochloride added to hen egg-white lysozyme, and the authors suggest that guanidinium ions preferentially form hydrogen bonds with the backbone carbonyl oxygen atoms (Raskar et al., 2019). Thus, the influence described in this study on the suppression of heat-induced aggregation of myosin caused by the structure of Arg was referred to as a hydrogen bond effect.
Fig. 4. Effect of guanidinium on the hydrodynamic radius and turbidity of myosin during heating.
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Fig. 5. Effect of guanidinium on the secondary structures of myosin during heating (pH 7.0). (A: control; B: 40 mM Arg; C: 40 mM guanidineacetic acid; D: 40 mM guanidinium sulfate).
3.4. Role of hydrogen bonds
3.4.2. Secondary structures Fig. 5 shows that all three additives changed the secondary structure of the myosin molecule, especially the α-helix structure, indicating the conformational changes of the rod region induced by the guanidinium ion (Lanier et al., 2005). It was observed that the α-helix content of myosin remained at a stable level in the presence of 40 mM Arg (Fig. 5B), suggesting that among the additives, Arg promoted a higher unfolding of the myosin molecule even if at the initial heating temperature of 25 � C. However, guanidinoacetic acid did not promote a high unfolding of myosin at the same concentration (Fig. 5C), which might be due to its strong interference in the determination of CD spectra (data not shown). The effect of guanidinium sulfate on the myosin secondary structure was the weakest among the three myosin-guanidinium mixtures (Fig. 5D). These results suggest that the ability to effectively influence the sec ondary structures increased in the following order: (1) myosin-guanidinium sulfate, (2) myosin-guanidinoacetic acid, and (3) myosin-Arg. It was hypothesized that the guanidinium ion of the three materials might have disturbed the hydrogen bonds that stabilize the α-helix structures of the myosin backbone. Furthermore, new hydrogen bonds may form between the amino hydrogen (NH-) of guanidinium and backbone carbonyl oxygens atoms (-CO) of myosin molecules (Raskar et al., 2019). However, it is also reasonable to conclude that guanidi nium is a critical factor, but not the only decisive factor, in the sup pression of the heat-induced aggregation behavior of myosin by Arg. The reason is because differences were displayed among the three additives,
3.4.1. DLS To verify the existence of a hydrogen bond effect, especially the hydrogen bonds between guanidinium ions and myosin molecules, the effects of Arg, guanidinoacetic acid, and guanidinium sulfate on the hydrodynamic radius, secondary structures, and hydrogen bond number of myosin were investigated at a concentration of 40 mM at pH 7.0. As shown in Fig. 4, the distribution of hydrodynamic radii indicated that myosin without additives tended to aggregate into particles and clusters as a result of head-head aggregation and rod-rod cross-linking (Chen et al., 2016). However, the hydrodynamic radius of myosin molecules in the presence of any one of the three additives significantly decreased during the whole programmed heating process. Furthermore, it was equally of interest to observe that the hydrodynamic radius of myosin in the presence of Arg, guanidinoacetic acid and guanidinium sulfate was maintained between 180 nm and 400 nm in a temperature-independent manner. This synergistic observation illus trated that soluble myosin species such as oligomers, filaments, and their complexes, rather than large particles or clusters, coexisted in the myosin-guanidinium sols during heating. As a result, it was reasonable to conclude that the guanidinium ion, the common group of three compounds, was the prime structural element involved in inhibiting the heat-induced aggregation behavior of myosin and possessed the ability to maintain myosin in a soluble state during heating.
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hydrophobicity during heating. Additionally, compared Arg without pH modification, Arg with pH modification showed a relatively weak ability to suppress myosin aggregation, indicating that the suppression induced by Arg in the heat-induced aggregation of myosin correlates not only to the pH, but also to the structure of Arg. CD studies indicated that the interaction of Arg with myosin caused significant conformational changes in the secondary structures of the protein. Furthermore, gua nidinium was a critical factor, but not the only decisive factor, in the suppression of the heat-induced aggregation behavior of myosin by Arg. In the present study, the suppression mechanism of Arg in the heatinduced myosin aggregation process was illustrated from two aspects. First, ionic attraction between positively charged Arg and negatively charged myosin occurred, and the repulsion between myosin molecules was promoted, induced by the higher pH values in the presence of Arg. Second, the two amino groups of Arg preferentially form hydrogen bonds with the backbone carbonyl oxygen atoms of the myosin mole cule. These results may be of interest to further broaden the versatile use of Arg as an additive for modifying of the texture of fish products to obtain liquid and semiliquid food for the elderly consumers in an increasingly aging society. Declaration of competing interest
Fig. 6. Effect of guanidinium on the hydrogen bonds of myosin (40 mM, pH 7.0). Different lowercase letters indicate significant difference (P < 0.05).
which contained the same concentration of guanidinium at the same pH value.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
3.5. Hydrogen bonds
Acknowledgments
As shown in Fig. 6, the number of hydrogen bonds of the control increased after two-step heating treatment. This transition could be ascribed to the gel form of myosin without Arg since the samples with high protein concentrations were stored at 4 � C immediately after twostep heating. This phenomenon was consistent with the theory that hydrogen bonds between proteins are more numerous when the gel is colder, producing a firmer gel structure (Lanier et al., 2005). All of the three additives containing guanidinium ions increased the number of hydrogen bonds of myosin in the unheated treatment, but the number decreased after two-step heating. This result indicated that the hydrogen bonds were first structurally integrated between each material and the myosin molecule but then were broken during heating. However, the number of hydrogen bonds in the myosin-guanidinium mixtures was lower than that of the control after two-step heating. This result showed that the hydrogen bonds formed between each guanidinium material and the myosin molecules were more easily broken than those formed within the myosin molecule backbone. The number of hydrogen bonds of natural myosin with Arg was the greatest among the three myosin-guanidinium mixtures, which was nearly twice as much as that of myosin with guanidinoacetic acid and guanidinium sulfate. This discrepancy might be due to the unequal number and particular structure of amino groups (-NH2) in these three materials. All of the additives possess a –NH2 group from the guanidi nium group. Essentially, Arg and guanidinium sulfate possess another –NH2 group, while the latter is joined to the sulfate group. This obser vation suggested that not only the guanidinium group, but also the other amino group in the Arg molecule was of prime importance in sup pressing the heat-induced aggregation of myosin, fundamentally via forming hydrogen bonds with carbonyl oxygen atoms of the myosin backbone (as shown before in the secondary structure analysis).
This work was supported by the National Natural Science Foundation of China (No. 31671882), the Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University (BTBU). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.foodhyd.2019.105596. Author contribution statement Tong Shi: Conceptualization, Experimentation, Data treatment, Interpretation, Writing Original Draft. Zhiyu Xiong: Methodology, Investigation. Wengang Jin: Conceptualization, Supervision. Li Yuan: Data treatment, Interpretation, Validation. Quancai Sun: Reviewing Draft. Yuhao Zhang: Data treatment, Writing Original Draft. Xiuting Li: Data treatment, Interpretation, Supervision, Validation. Ruichang Gao: Conceptualization, Data treatment, Interpretation, Validation, Writing, Reviewing, Editing, Supervision. References Brenner, T., Johannsson, R., & Nicolai, T. (2009). Characterization of fish myosin aggregates using static and dynamic light scattering. Food Hydrocolloids, 23(2), 296–305. Cao, Y. G., & Xiong, Y. L. (2015). Chlorogenic acid-mediated gel formation of oxidatively stressed myofibrillar protein. Food Chemistry, 180, 235–243. Chen, X., Li, P. J., Nishiumi, T., Takumi, H., Suzuki, A., & Chen, C. G. (2014). Effects of high-pressure processing on the cooking loss and gel strength of chicken breast actomyosin containing sodium alginate. Food and Bioprocess Technology, 7(12), 3608–3617. Chen, X., Zou, Y. F., Han, M. Y., Pan, L. H., Xing, T., Xu, X. L., et al. (2016). Solubilisation of myosin in a solution of low ionic strength l-histidine: Significance of the imidazole ring. Food Chemistry, 196(3), 42–49. Dustin, J. P. (1950). Determination of proteins by the biuret reaction. Bulletin de la Societe de Chimie Biologique, 32(9–10), 696.
4. Conclusion Arg was demonstrated to suppress the heat-induced aggregation of fish myosin regardless of pH modification, which was shown by a dosedependent decrease in hydrodynamic radius, turbidity and surface 8
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