2020 年 26 巻 6 号 p. 759-769
The optimal conditions for the preparation of maltotriose-conjugated chicken myofibrillar proteins (Mfs), exhibiting high solubility in low ionic strength medium, through the Maillard reaction were investigated using random-centroid optimization. Parameters of temperature, relative humidity (RH), reaction time, and maltotriose to chicken Mfs mixing ratio were examined, resulting in 13 vertices. Evaluations were carried out related to each individual vertex, and the optimal preparatory conditions resulting in the highest solubility were determined as follows: temperature of 53 °C, RH of 45%, reaction time of 38.5 h, and maltotriose to chicken Mfs mixing ratio of 4.24 (w/w), presenting 54.9 ± 1.9% solubility in low ionic strength medium. Its hydroxyl radical averting capacity showed 8.1 ± 0.4 µmol of gallic acid equivalent per gram of protein. The thermal gel-forming ability of chicken Mfs was retained by the maltotriose-conjugation.
Chicken myofibrillar proteins (Mfs) have been investigated in various studies for alterations in their functional properties through the process of protein glycation (mainly conjugation with glycosyl units), namely the Maillard reaction (Nishimura et al., 2011a; Nishimura et al., 2011b; Isono et al., 2012; Nishimura et al., 2015; Nishimura and Saeki, 2016; Nishimura and Saeki, 2018a; Nishimura and Saeki, 2018b; Nishimura et al., 2019). We previously reported that maltose-conjugated chicken Mfs acquired various alterations in their functional properties, such as solubility in low ionic strength medium, antioxidant capacity, and thermal stability (Nishimura et al., 2011a; Nishimura et al., 2011b). The antioxidant capacity has been attributed to the primary structure or specific peptide sequences of chicken Mfs, irrespective of the protein shape (Isono et al., 2012). Moreover, the maltose-conjugated chicken Mfs were observed to retain their thermal gel forming ability (Nishimura et al., 2015). Further, the optimal preparative conditions were searched using random-centroid optimization (RCO) (Nishimura et al., 1998; Nishimura et al., 2001; Nakai et al., 2009; Yan and Wang, 2009; Nishimura and Saeki, 2016; Nishimura and Saeki, 2018a; Nishimura and Saeki, 2018b; Nishimura et al., 2019) in order to obtain maltose-conjugated chicken Mfs possessing improved solubility in low ionic strength medium and strong antioxidative capacity against hydroxyl radical (•OH). Consequently, the searched conditions included a temperature of 57 °C, 37% relative humidity (RH), 37.2 h of reaction time, and maltose mixing ratio of 5.43 (w/w) for chicken Mfs. The hydroxyl radical antioxidant capacity (HORAC) was measured as 7.8 ± 1.0 µmol of gallic acid (GA) equivalent /g of protein; however, the HORAC of this thermal gel was reduced to 4.4 ± 1.7 µmol of GA equivalent /g of protein (Nishimura and Saeki, 2016). Likewise, investigations have been carried out for monosaccharaides, including ribose and glucose, possessing five and six-member ring structures, respectively, rather than maltose. The ribose-conjugated chicken Mfs have been reported to be prepared in a very short reaction time (4.55 h) and exhibited a HORAC of 5.1 ± 1.3 µmol of GA equivalent /g of protein, however, this resulted in the loss of its thermal gel-forming activity (Nishimura and Saeki, 2018b). The glycated chicken Mfs obtained after glucose conjugation have been observed to possess improved solubility in low ionic strength medium, strong antioxidative capacity (HORAC of 9.7 ± 0.7 µmol of GA equivalent /g of protein), and retention of thermal gel-forming capacity (Nishimura, et al., 2019). The HORAC of this thermal gel was 23.2 ± 0.8 µmol of GA equivalent /g of protein, which was 2.4 times more prior to heating. Similar functional alterations, i.e., water-solubilization and gain of anti-oxidative activity, have been observed in chicken Mfs with the use of glucose and maltose as monosaccharide and disaccharide, respectively, irrespective of the difference in their molecular weights. Therefore, the present study aimed to investigate the effect of the number of glucose units on functional alterations in chicken Mfs.
This study is an attempt to improve the food functionality of chicken Mfs by conjugation with maltotriose consisting of three glucose units. The optimal preparative conditions were searched using RCO in order to prepare maltotriose-conjugated chicken Mfs exhibiting improved solubility in low ionic strength medium, and the obtained Mfs-maltotriose conjugate was subjected to anti-oxidative assay. Further, the acquired functional alterations were analyzed with respect to those of previous reports.
Materials and chemicals Chicken breast meat was purchased from a local poultry farm immediately after slaughter. Hydroxyl radical measurement was performed using a commercial kit (Radical Catch) purchased from Hitachi, Ltd. (Tokyo, Japan). GA was obtained from ChromaDex, Inc. (Irvine, CA, USA). All other chemicals were reagent grade and obtained from either Nacalai Tesque, Inc. (Kyoto, Japan) or Wako Pure Chemicals Industries, Ltd. (Osaka, Japan).
Preparation of Mfs Chicken Mfs were prepared according to the protocol described previously (Nishimura and Saeki, 2018b). Briefly, 30 g of chicken breast meat was finely cut and resuspended in 10 volumes of 0.1 M sodium phosphate buffer at pH 7.5. The suspension was allowed to settle; then, the supernatant was decanted and the process was repeated five times. Thereafter, the meat was homogenized using an AM-9 homogenizer (Nissei Co., Ltd., Tokyo, Japan) for 0.5 min at 10 000 rpm by adding 10 volumes of 0.1 M sodium phosphate buffer (pH 7.5), based upon the initial muscle weight, and this step was repeated five times. Subsequently, the meat homogenate was passed through cotton gauze, and a solution of 20% Triton X-100 was added to the filtrate to reach a final concentration of 0.5% Triton, followed by centrifugation at 7 000 × g for 10 min to collect the chicken Mfs. The precipitate was resuspended in 50 mM NaCl and centrifuged again at 7 000 × g for 10 min. This procedure was repeated five times. Approximately 35 g of precipitate was obtained as prepared chicken Mfs. All the steps were carried out on ice. The prepared chicken Mfs were regarded as native chicken Mfs.
RCO RCO (Nishimura et al., 1998; Nishimura et al., 2001; Nakai et al., 2009; Yan and Wang, 2009; Nishimura and Saeki, 2016; Nishimura and Saeki, 2018a; Nishimura and Saeki, 2018b; Nishimura et al., 2019) was used to determine the optimal conditions for the preparation of glycated chicken Mfs exhibiting the highest solubility in a low ionic strength medium. Briefly, glycated chicken Mfs were prepared in relation to 13 vertices, each representative of specific experimental conditions defined by 4 parameters, and assessed independently in triplicate. The four experimental parameters were defined as follows: temperature range of 40–70 °C, RH range of 30–50%, reaction time range of 10–60 h, and mixing ratio of maltotriose to chicken Mfs was within 3–8 (w/w).
Glycation of chicken Mfs The chicken Mfs were glycated following a previously described protocol (Nishimura and Saeki, 2018b). Prepared chicken Mfs were resuspended in 50 mM NaCl and mixed with maltotriose at ratios predetermined by the RCO vertex. After adjustment of the final protein concentration to 6.0 mg/mL, 5 mL of each chicken Mfs-maltotriose mixture was transferred to a test tube (16 mm in diameter), frozen at −80 °C, and immediately lyophilized using a freeze-dryer (FDU-1110; Tokyo Rikakikai Co., Ltd., Tokyo, Japan). This process led to lysis of the sarcomeres and exposure of the chicken Mfs. Lyophilization was terminated when the temperature of the samples reached 15–18 °C. Each lyophilized protein powder was immediately stored at −40 °C and used within 30 days of preparation. The Maillard reaction between chicken Mfs and maltotriose proceeded by incubating the lyophilized powders under the 13 experimental conditions as defined by the RCO. An incubator/humidity cabinet (KCL-2000A; Tokyo Rikakikai Co., Ltd. Tokyo, Japan) was used to control temperature and RH. The chicken Mfs without sugar were incubated under the optimal conditions of temperature, reaction time, and RH, and subsequently, the unmodified chicken Mfs were prepared.
Solubility of glycated chicken Mfs The solubility of glycated chicken Mfs was measured according to a previously described protocol (Nishimura and Saeki, 2018b). After glycation, the protein powder was immediately mixed with 15 mM sodium phosphate buffer containing 0.1 or 0.5 M NaCl (pH 7.5) at a final concentration of 1.5 mg/mL. After incubation at 8–12 °C overnight, the Mfs were then dispersed using a high-speed blender (T-10 basic Ultra-Turrax, IKA-Labotechnik, Staufen, Germany) at 13 500 rpm for 0.5 min, and this step was repeated. The homogenate was centrifuged at 32 000 × g for 30 min at 4 °C, and the protein concentrations of the homogenate and the supernatant before and after centrifugation were determined using the Kjeldahl method (AOAC, 1990). Total soluble chicken Mfs were expressed as the percentage of protein content in the supernatant in relation to total protein content prior to centrifugation. These values were used to evaluate RCO.
Preparation of samples for HORAC measurement The samples were incubated to allow the glycation reaction to proceed. Each protein powder was immediately mixed with 0.1 M NaCl-15 mM sodium phosphate buffer (pH 7.5), and then dialyzed in the same buffer solution using the membrane with 40–50 Å pore size at 8–12 °C for five days to remove any unreacted maltotriose. Following centrifugation at 32 000 × g for 30 min at 4 °C, the supernatant containing 7–9 mg/mL protein was used as a working solution.
HORAC measurement The HORAC of each protein sample was measured using an antioxidant potential measurement kit (Radical Catch; Aloka Co., Ltd., Tokyo, Japan) and a chemiluminescence reader (AccuFLEX Lumi400; Aloka Co., Ltd., Tokyo, Japan) based on the Fenton reaction, whereby •OH reacts with luminol, resulting in light emission (Yildiz and Demiryürek, 1998; Parejo et al., 2000). Aliquots of the samples were harvested to measure their respective antioxidant capacity. Initially, 50 µL each of cobalt solution and luminol solution were mixed with 20 µL of a diluted sample solution adjusted to a final concentration of 0.59 mg protein/mL, and incubated for 8 min at 37 °C. •OH generation was initiated by the addition of 50 µL of H2O2 solution. Light emission was measured for 120 s at a wavelength of 430 nm immediately after initiation, and the light emissions recorded between 80 to 120 s were integrated. The control sample contained 0.1 M NaCl and 15 mM sodium phosphate buffer (pH 7.5). The residual ratio of •OH was calculated for the glycated chicken Mfs having the highest solubility in low ionic strength medium and their thermal gels. Antioxidant activity was measured as the relative light units (RLU) of a sample / RLU of the control × 100.
Determination of available lysine and fructosamine Quantification of the available lysine and fructosamine was carried out to monitor the progress of the Maillard reaction between the chicken Mfs and maltotriose. Optimally glycated chicken Mfs were dissolved in 0.5 M NaCl-15 mM sodium phosphate buffer (pH 7.5) using a T 10 basic ULTRA-TURRAX high-speed blender (IKA-Labotechnik, Staufen, Germany), and then precipitated with 7.5% trichloroacetic acid in ice water for 30 min. The sodium phosphate buffer, unreacted sugars, and trichloroacetic acid were removed through centrifugation at 1 000 × g for 30 min at 4 °C, and the precipitated protein was reconstituted in 50 mM sodium phosphate buffer (pH 9.5) containing 2% sodium dodecyl sulfate (SDS) (Saeki, 1997). The available lysine content was determined through spectrophotometric analysis using o-phthalaldehyde and N-acetyl-L-cysteine (Hernandez and Alvarez-Coque, 1992).
The optimally glycated chicken Mfs dissolved in 0.5 M NaCl-15 mM sodium phosphate buffer (pH 7.5) were dialyzed against the same buffer at 8–12 °C for 5 days to remove any unreacted maltotriose. Fructosamine, regarded as a ketoamine product of protein glycation, was assayed following the method of Johnson et al. (1983). Glycosylated human serum was used as a standard for the determination of fructosamine content. Assays for the determination of available lysine and fructosamine concentrations were performed just after preparation of samples.
Thermal stability Analysis of thermal stability was followed by monitoring changes in solubility of the protein solution during heating according to a modified method of Fujiwara et al. (1998). Murphy et al. (1998) reported that thermal denaturation of chicken Mfs occurs at 53 °C; therefore, the incubation temperature was set at 50 °C to examine the effect of temperature on solubility. Optimally glycated chicken Mfs, at a final protein concentration of 1.5 mg/mL, were immediately mixed with 15 mM sodium phosphate buffer (pH 7.5) containing 0.1 or 0.5 M NaCl using a T-10 basic ULTRA-TURRAX high-speed blender (IKA-Labotechnik, Staufen, Germany) at 13 500 rpm for 0.5 min twice (total of 1 min) and dialyzed against the same NaCl solution at 4 °C for 5 days. Unreacted maltotriose was removed during the dialysis step. The dialyzed sample was suspended using a T-10 basic ULTRA-TURRAX high-speed blender. The supernatant was incubated at 50 °C for 2 h, and then centrifuged at 32 000 × g for 30 min at 4 °C. Thermal stability was expressed as the percentage of protein concentration in the supernatant with respect to that of the dialyzate prior to 2 h incubation at 50 °C. The protein content of the dissolved chicken Mfs was determined following the Kjeldahl method (AOAC, 1990).
Thermal gel formation and biochemical analysis The optimally glycated chicken Mfs were concentrated up to 15 mg protein/mL, from a starting stock concentration of 7–9 mg protein/mL, in 15 mM sodium phosphate buffer (pH 7.5) containing 0.1 M NaCl. Briefly, sample tubes were dialyzed in solid polyethylene glycol particles at 4 °C. Then, 0.5 mL of the concentrated solutions were placed in test tubes (16 mm in diameter), sealed with polyvinylidene chloride film, and heated in a water bath at 90 °C for 4 h. The samples were observed with a cryo-scanning electron microscope (SEM) and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and HORAC assay. As a control, chicken Mfs without glycation (termed unmodified chicken Mfs) were dissolved in 15 mM sodium phosphate buffer (pH 7.5) containing 0.5 M NaCl and examined in a similar manner.
SDS-PAGE analysis of thermal gels The heated solutions were mixed with an equal volume of 125 mM Tris-HCl (pH 6.8) containing 8 M urea, 4% SDS, and 40 mM N-ethylmaleimide. The mixtures were allowed to stand overnight at room temperature. 2-Mercaptoethanol (2-ME) was added to each mixture to a final concentration of 10% (v/v), and the mixture was boiled for 3 min. Electrophoresis was carried out on a 7.5% acrylamide slab gel according to Laemmli's method (1970), followed by staining with Coomassie Brilliant Blue R-250.
Microscopic observation The microstructures of the 15, 30, 60, and 120 min thermal gels derived from the optimally glycated chicken Mfs were examined under a cryo-SEM (SU8230; High-Technologies Co., Tokyo, Japan). For cryo-SEM observation, the thermal gels were frozen in liquid nitrogen, fractured using a scalpel, and transferred to the cold stage of the preparation chamber. Each sample was then sequentially exposed to approximately −105 °C, −95 °C, and −85 °C for 10 min each under a pressure of less than 7 × 10−3 Pa. Cryo-SEM observation was performed using SEM operated at 1.5 kV at −125 °C.
Protein content determination The protein content of dissolved chicken Mfs was determined following the Kjeldahl method (AOAC, 1990). The glycated chicken Mfs, when measuring the amount of available lysine, and the prepared chicken Mfs were determined as described by Lowry (1951). The biuret method (Gornall et al., 1949) was followed for the other assays, using bovine serum albumin as a protein standard.
Statistical analysis Statistical analysis was performed on three independent samples of glycated chicken Mfs. The results are represented as the mean of three replicate values ± standard deviation. Statistical analysis was performed using Microsoft Excel Ver. 2016 with Ekuseru-Toukei 2010 (Social Survey Research Information Co., Tokyo, Japan). A p value < 0.05 was considered as statistically significant.
Determination of optimal preparative method for glycation of chicken Mfs with maltotriose As shown in Table 1, RCO was used to determine the optimal conditions for the production of maltotriose-conjugated chicken Mfs exhibiting the highest solubility in a low ionic strength medium on the basis of varying temperature, RH, reaction time, and maltotriose to chicken Mfs mixing ratio (w/w). The parameters respective to each experiment were calculated using the RCO program (Nakai et al., 2009) and were then implemented for different preparations of glycated chicken Mfs. All data were mapped as shown in Fig. 1, aiding the visualization of the experimental response surface and indicating data trends (Nakai et al., 2009). RCO is usually repeated until an adequate response is achieved. Therefore, in the present study, the approximate position of the optimal conditions was made clear by conducting a first cycle of the RCO program. Optimal results were obtained with a temperature of 53 °C, 45% RH, reaction time of 38.5 h, and maltotriose to chicken Mfs mixing ratio (w/w) of 4.24, as shown in Fig. 1. In previous studies, we reported that the optimal conditions for glucose (Nishimura et al., 2019) and maltose (Nishimura and Saeki, 2016) were 52 and 57 °C, 38 and 37% RH, 6.79 and 37.2 h reaction time, and a sugar to protein mixing ratio of 11.7 and 5.43 (w/w), respectively. Although the solubility of maltotriose-conjugated chicken Mfs in a low ionic strength medium is not over 60%, instead 55%, these results suggest that the glycation reaction with maltotriose occurs at a slower rate as compared to glucose, a monosaccharide, and is similar to that of maltose, a disaccharide that consists of two glucose units. In addition, a reaction time of more than 38.5 h did not raise the solubility in a low strength medium (data not shown). This result may suggest that the protective effect on the denaturation of chicken Mfs was weak compared to using glucose and maltose. That is, before the introduction of a sufficient amount of sugar chains improves the solubility of Mfs, insolubilization may have progressed due to thermal denaturation of the protein.
| Vertex No. |
Temperature (°C) |
RH (%) |
Reaction Time (h) |
Maltotriose mixing ratio to Mfs (w/w) |
Evaluation (Solubility)a) (%) |
|---|---|---|---|---|---|
| 1 | 53 | 45 | 38.5 | 4.24 | 54.5 |
| 2 | 44 | 50 | 59.6 | 5.15 | 49.8 |
| 3 | 63 | 45 | 33.2 | 7.42 | 34.3 |
| 4 | 65 | 40 | 25.6 | 6.92 | 51.7 |
| 5 | 47 | 35 | 21.9 | 4.75 | 6.3 |
| 6 | 63 | 39 | 33.1 | 4.93 | 34.4 |
| 7 | 66 | 38 | 49.4 | 3.06 | 25.1 |
| 8 | 44 | 49 | 13.6 | 5.00 | 9.2 |
| 9 | 50 | 42 | 34.3 | 5.52 | 43.6 |
| 10b) | 53 | 44 | 39.5 | 5.46 | 50.5 |
| 11b) | 55 | 42 | 38.1 | 5.63 | 49.4 |
| 12b) | 52 | 44 | 41.4 | 4.96 | 53.2 |
| 13b) | 58 | 41 | 32.9 | 5.40 | 51.6 |
Mapping of the solubility in a low ionic strength medium (evaluation) associated with each individual parameter tested during RCO
The evaluation of the vertex was estimated to be equal to the solubility in a low ionic strength medium. The vertex that was associated with the largest evaluation was sought. The effect of variations in (A) temperature, (B) relative humidity, (C) reaction time, and (D) maltotriose to chicken Mfs mixing ratio (w/w) was investigated. The symbol (●) represents one vertex. Lines indicate probable trends. The arrow indicated on each graph shows the best result.
HORAC measurement of optimally glycated chicken Mfs HORAC of the water-soluble glycated Mfs, determined by the data in Fig. 1, was examined; the inhibition curve of HORAC values associated with the optimally glycated chicken Mfs is shown in Fig. 2. Native chicken Mfs hardly demonstrated HORAC within the range of 0.35–1.06 mg of protein/mL. In contrast, the optimally glycated chicken Mfs reduced •OH production with increasing protein concentration, and the reduction ratio reached 44.2 ± 1.6% (n = 3) at a protein concentration of 0.94 mg /mL. The IC50 value of the optimally glycated chicken Mfs was equal to 0.79 ± 0.04 mg of protein / mL, as calculated previously and shown in Fig. 2.
Effect of chicken Mfs concentration on HORAC
Using the optimally glycated chicken Mfs resuspended in 0.1 M NaCl solution (pH 7.5), the antioxidative activity against •OH was independently measured three times using the fluorescence method. The glycated chicken Mfs were prepared under the optimal conditions determined in Fig. 1. The symbol (●) and (○) represent the residual ratio of •OH (%) for optimally glycated and native chicken Mfs, respectively, as measured against chicken Mfs concentration (mg/mL) through the fluorescence method. Data are represented as mean ± standard deviation (n = 3).
The HORAC of the optimally glycated chicken was converted into GA equivalent activity (µmol/mL) based on the IC50 value of GA (6.4 ± 0.1 nmol/mL); hence, a value of 8.1 ± 0.4 µmol of GA equivalent/g of protein (n = 3) was obtained. The HORACs of glucose- (Nishimura et al., 2019) and maltose-conjugated chicken Mfs (Nishimura and Saeki, 2016) were 9.7 ± 0.7 and 7.8 ± 1.0 µmol of GA equivalent/g of protein (n = 3), respectively, values which were significantly different (p < 0.05). However, no significant differences were observed between the HORAC of maltotriose-conjugated chicken Mfs and glucose- and maltose-conjugated chicken Mfs, respectively. These data indicate that the optimal preparative conditions used for maltotriose-conjugated chicken Mfs obtained in the present study resulted in an antioxidant activity that was similar to those of glucose and maltose-conjugated chicken Mfs. Therefore, it is apparent that conjugation with glycosyl units can effectively improve the antioxidant activity in chicken Mfs, regardless of glucose number in the molecule.
Time-dependent changes in solubility, available lysine, and fructosamine in chicken Mfs during glycation Lyophilized chicken Mfs-maltotriose mixtures (chicken Mfs: maltotriose = 1:4.24) were heated at 53 °C and 45% RH, in accordance with the optimal preparative conditions determined for the functional glycated chicken Mfs. The solubility was examined using 15 mM sodium phosphate buffer (pH 7.5) containing 0.1 or 0.5 M NaCl for 38.5 h (Fig. 3A). The solubility in 0.5 M NaCl (pH 7.5) increased from 62.1 ± 15.4% (n = 3) to 54.0 ± 4.3% (n = 3) after 3 h, and to 62.3 ± 4.6% (n = 3) after 38.5 h. Furthermore, the solubility in 0.1 M NaCl (pH 7.5) was initiated at about 10.9 ± 1.1% (n = 3) and increased somewhat with increasing reaction time to 54.5 ± 5.1% (n = 3) after 24 h, and almost reached full solubility in 0.5 M NaCl (pH 7.5). Furthermore, no significant increase in solubility in 0.1 M NaCl occurred after 24 h of reaction, and also no loss in solubility was observed during the glycation period. This result indicates that the introduction of maltotriose abrogated the salt-concentration dependence of the solubility of chicken Mfs and resulted in water-solubility.
Changes in solubility and amounts of available lysine and fructosamine in chicken Mfs during glycation
Mixture of lyophilized chicken Mfs and maltotriose at a ratio of 1:4.24 (w/w) were heated at 53 °C and 45% RH. The solubility (A) in 0.1 M (●) or 0.5 M (○) NaCl at pH 7.5 was measured during the Maillard reaction with maltotriose. The available lysine (B) and fructosamine (C) contents were simultaneously measured. Data are represented as mean ± standard deviation (n = 3).
Figures 3B and 3C show the changes in the amount of available lysine and fructosamine contents in the chicken Mfs upon introducing maltotriose through the Maillard reaction. The amount of available lysine decreased somewhat during the reaction and reached 44.7 ± 14.3% (n = 3) as the Maillard reaction progressed (Fig. 3B). Conversely, the quantity of fructosamine increased with the reaction time to reach 278.0 ± 13.7 µmol/g of protein (n = 3) after 38.5 h (Fig. 3C). Under optimal preparative conditions, the available lysine in glucose and maltose-conjugated chicken Mfs amounted to 33.5 ± 10.3% (n = 4) and 37.5 ± 3.2% (n = 3) µmol/g of protein, while fructosamine amounted to 427 ± 115 (n = 4) and 326 ± 32 (n = 3) µmol/g of protein, respectively (Nishimura et al., 2019; Nishimura and Saeki, 2016). There were no significant differences in these values among groups, suggesting that the respective optimal conditions resulted in a similar early stage Maillard reaction. This could explain the similar HORAC levels in the three kinds of sugar-conjugated chicken Mfs.
The results of Fig. 3 indicated that the improved solubility in 0.1 M NaCl (pH 7.5) was enhanced with the progress of fructosamine production, which is an indicator of the early stage of the Maillard reaction. The fact that 0.5 M NaCl solubility was not compromised clearly demonstrates that maltotriose-conjugated chicken Mfs achieve a high salt-independent solubility without significant protein denaturation at the early stage of the Maillard reaction.
Myosin, the major protein in chicken Mfs, forms water-insoluble filaments under physiological conditions (Huxley, 1963). The non-enzymatic glycation of lysine residues inhibited filament formation by increasing the negative charge repulsion among myosin heavy chains (MHC) and by physically interfering with the self-assembly regardless of the size of the glycosyl units (Katayama et al., 2004). An inhibition effect occurred depending on the molecular size of the introduced sugar units, and maltotriose showed a greater inhibitory influence on myosin self-assembly compared to glucose and maltose (Katayama et al., 2004). Therefore, the water-solubilization of chicken Mfs with the maltotriose modification could have been caused by a harmonic effect induced by the increase in hydrophilicity, negative charge repulsion in MHC, and inhibitory myosin self-assembly at the early stage of the Maillard reaction.
Thermal stability Native chicken Mfs and the optimally glycated chicken Mfs were respectively dissolved in 0.1 and 0.5 M NaCl solutions (pH 7.5), and then heated at 50 °C for 2 h. Figure 4 shows the solubility of both chicken Mfs before (a) and after (b) heating. The solubility of native chicken Mfs in 0.5 M NaCl was significantly decreased from 59.6 ± 9.5% (n = 3) to 16.2 ± 4.2% (n = 3) during heat treatment (p < 0.01). However, no significant decrease in solubility was apparent in the maltotriose-conjugated chicken Mfs in 0.5 M NaCl at the start or end of the 2 h heating. While the solubility of the optimally glycated chicken Mfs in 0.1 M NaCl was 53.3 ± 1.2% (n = 3) at the start, and reached 44.2 ± 4.6% (n = 3) at the end of 2 h heating, showing a significant difference (p < 0.01), the amount of solubility decrease was very small compared to the native chicken Mfs. These results indicate that the thermal stability of chicken Mfs was improved upon conjugation with maltotriose. Although this improvement in the thermal stability of the glycated chicken Mfs has also been observed in our previous studies using maltose (Nishimura et al., 2011a and b), this might be attributed to the restrained thermal aggregation of myosin due to the attachment of sugars, and the increased hydration with water due to the presence of a sufficiently large amount of hydroxyl groups. Increased hydration due to the presence of a sufficient amount of hydroxyl groups may also be involved in this functional improvement.
Effects of glycation with maltotriose on the thermal stability of chicken Mfs
The native and optimally glycated Mfs suspended in 0.1 M or 0.5 M NaCl (a) were heated at 50 °C for 2h (b), and the solubility change due to the heat-treatment was examined to investigate the thermal stabilization of Mfs occurred by attaching maltotriose. Values are mean ± standard deviation (n = 3). **: significant differences between values (p < 0.01).
Thermal gel-forming abilities of optimally glycated chicken Mfs The unmodified and optimally glycated chicken Mfs dissolved in 15 mM sodium phosphate buffer (pH 7.5) containing 0.1 or 0.5 M NaCl were heated at 90 °C for up to 240 min to investigate thermal gel formation (Fig. 5). The turbidity of both chicken Mfs solutions increased with heating; however, the unmodified chicken Mfs solution remained as a suspension, with no change in color and no gel formation even with heating for 240 min (Fig. 5A). On the contrary, the optimally glycated chicken Mfs became turbid upon heating, did not flow upon tilting the test tube, and formed a thermal gel upon heating for 15 to 60 min (Fig. 5B). However, further heating collapsed the thermal gel, and the color changed to light yellow following a 60 min heating period. These results indicate that conjugation with maltotriose improved the gel-forming ability of chicken Mfs, while excessive Maillard reaction inhibited gel formation. In previous investigations (Nishimura and Saeki, 2016; Nishimura et al., 2019), we showed that the conjugation of chicken Mfs with maltose and glucose could promote gel formation, while the beneficial effect was reduced as the Maillard reaction progressed further. Based upon the results of the present and previous studies, we propose that the Maillard products at the advanced stage may inhibit the gel formation of chicken Mfs, leading to the collapse of the thermal gel. Use of maltotriose rather than mono- and disaccharides could not suppress the negative effects of the Maillard products for thermal gelation.
Thermal gel formation in Mfs preparations
Unmodified chicken Mfs (A) and optimally glycated chicken Mfs (B) were dissolved in 0.5 M NaCl and 0.1 M NaCl at pH 7.5, respectively. 0.5 mL of each Mfs solution at 15 mg/mL of protein concentration was placed in a ø16 mm-test tube and heated at 90 °C for 240 min. Thermal gel-formation of both Mfs was examined by tilting the test tubes at an angle.
The thermal gel of the maltotriose-modified chicken Mfs was subjected to HORAC assay and the result was compared with those of the glucose and maltose conjugated chicken Mfs previously reported. The thermal gels of optimally saccharified with glucose and maltose-bound chicken Mfs showed HORAC values of 23.2 ± 0.8 and 4.4 ± 1.7 µmol GA equivalent /g of protein, respectively (Nishimura and Saeki, 2016; Nishimura et al., 2019), whereas the maltotriose-modified chicken Mfs after gel-formation showed no antioxidant activity against •OH. Therefore, the antioxidant activity of glycated chicken Mfs after thermal gel-formation might be inversely proportional to the number of glycosyl units.
Effects of glycation on cross-linking of protein subunits of chicken Mfs during thermal gel-formation The patterns of unmodified and glycated chicken Mfs under the reducing conditions of SDS-PAGE are shown in Figs. 6A and 6B, respectively. Figure 6A shows that the decrease in MHC and appearance of the smaller molecule component under MHC occurred during the incubation of chicken Mfs, under the optimal condition without sugar, suggesting the decomposition of MHC during incubation. The amounts of these components decreased with a lapse in heating time, and a marked decrease in MHC, observed beyond the 60 min-heating period, corresponded to the production of strong covalent bonds among the proteins. However, such cross-linking of proteins did not contribute to the gel formation of chicken Mfs, as shown in Fig. 5A, and no change in the small protein components under actin was observed during heating.
Monitoring of protein subunit contents in unmodified and optimally glycated chicken Mfs during gel-formation
Unmodified (A) and glycated Mfs (B) solutions were gel-formed as described in Fig. 5, and then subjected to SDS-PAGE analysis in the presence of 10% 2-ME. MHC: myosin heavy chain.
On the other hand, the decrease in the mobility of MHC and actin and the loss of protein of lower molecular weight than actin (presumed to be tropomyosin) occurred (Lane 0 min in Fig. 6B), indicating that the conjugation of maltotriose with chicken Mfs proceeded during incubation under optimal conditions. Furthermore, the decrease of MHC and the formation of higher polymer components were observed along with the heating, and almost all the protein subunits disappeared after heating periods of 15 min and 60 min.
These results indicated that glycation promoted the interaction among chicken Mfs and rapidly formed a cross-linked structure, resulting in the formation of the gel observed in Fig. 5B. However, as shown in Fig. 5B, the thermal gel of the glycated chicken Mfs disintegrated when it was heated for more than 120 min. Therefore, the results shown in Figs. 5B and 6B indicate that excessive cross-linking, except for the non-covalent and disulfide bonds among proteins, disrupted the gel structure, although the non-covalent and disulfide bonds induced during the early stage of heating could play a major role in the gel formation of glycated chicken Mfs.
Observation of thermal gel microstructure of optimally glycated chicken Mfs Thermal gels were prepared from the optimally glycated chicken Mfs heated at 90 °C for 15, 30, 60, and 120 min and their microstructures were observed using cryo-SEM (magnification × 5 000) (Fig. 7). A fine network structure, which showed the three-dimensional expanse, contributing to gel formation was observed in the thermal gel obtained after heating for 15, 30, and 60 min (Figs. 7A, B, and C). However, a microstructure that was crude and not observed as a three-dimensional network occurred in the gel after a 120 min heating period (Fig. 7D). This phenomenon is consistent with the state of the heat-induced gels shown in Fig. 5B, demonstrating that continued heating may disrupt the gel microstructure, as demonstrated in Fig. 5B. A similar disruption of thermal gels was previously described in maltose-and glucose-conjugated chicken Mfs (Nishimura and Saeki, 2016; Nishimura et al., 2019). Although we had predicted the presence of Maillard reaction intermediate products that could lead to a collapse of the microstructure, such intermediate products are likely to promote disruption of the gel structure.
Microstructures of optimally glycated chicken Mfs after 15, 30, 60- and 120-min heating at 90 °C
The optimally glycated chicken Mfs solutions obtained from the experiments described in Fig. 5 were heated at 90 °C for 15 (A), 30 (B), 60 (C) and 120 min (D). The microstructure of each respective Mfs was observed under cryo-SEM. Magnification, ×5 000; Scale bar = 5.0 µm.
Acknowledgements We are grateful to Ms. Yuko Nambu, a technical staff member at the Graduate School of Agriculture, Kyoto University, for her skillful technical assistance with the microscopic observation. This work was supported by a Grant-in-Aid for Scientific Research (C) (No. 18K02195) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
gallic acid
HORAChydroxyl radical antioxidant capacity
IC50half-maximal inhibitory concentration
2-ME2-mercaptoethanol
Mfsmyofibrillar proteins
MHCmyosin heavy chain
•OHhydroxyl radical
PAGEpoly-acrylamide gel electrophoresis
RCOrandom-centroid optimization
RHrelative humidity
RLUrelative light unit
SDSsodium dodecyl sulfate
SEMscanning electron microscope
SSdisulfide
Uunit
