Development of glucose-conjugated chicken myofibrillar protein with the strongest superoxide anion radical scavenging activity using random-centroid optimization and maltotriose-conjugated ones

Abstract

The superoxide anion radical scavenging activities of glucose-conjugated chicken myofibrillar proteins (Mfs), which were soluble in low-ionic-strength medium and exhibited maximum antioxidant capacity, and maltotriose-conjugated chicken Mfs, which were maximally soluble in low-ionic-strength medium, were 264 ± 45 (n = 3) and 740 ± 346 units of superoxide dismutase/g of protein (n = 4), respectively. The droplet diameters of the highest peak of o/w emulsions prepared using each conjugate as emulsifier were 8.1 ± 1.3 (n = 3) and 4.7 ± 1.9 µm (n = 3), respectively, a significant difference at p < 0.05. The emulsions prepared with glucose- and maltotriose-conjugated chicken Mfs did not show strong stability. Maltotriose-conjugate was added to an emulsion containing methyl linoleate and Tween 20 at a concentration of 0.3% and stored at 50 °C for 7 days, resulting in significantly suppressed oil oxidation at day 5 only (p < 0.05). These results indicate that the conjugate can act as an antioxidant to inhibit oil oxidation.

Introduction

The authors previously observed that maltose (consisting of 2 glycosyl units)-conjugated chicken myofibrillar proteins (Mfs) obtained using the Maillard reaction (Fayle and Gerrard, 2002) resulted in functional alternations, such as increased solubility in low-ionic-strength medium and improved thermal stability (Nishimura et al., 2011a). Furthermore, we found that this conjugate also acquired antioxidant capacity without losing solubility in low-ionic-strength medium when the reaction time was extended (Nishimura et al., 2011b). In general, the ORAC method (Guohua et al., 1993) is used as a simple method to measure antioxidant capacity. However, the accuracy of this method remains a concern because it measures antioxidant capacity indirectly. Therefore, we selected the hydroxyl radical and super oxide anion radical (O⁻₂) as reactiveoxygen species and directly measured their scavenging ability as antioxidant capacity in the following series of studies. The antioxidant capacity of chicken Mfs has been attributed to the primary structure or specific peptide sequences, irrespective of the protein shape (Isono et al., 2012). Furthermore, maltose-conjugated chicken Mfs were observed to retain their thermal gel forming ability (Nishimura et al., 2015), and the resulting gel was found to maintain its antioxidant properties (Nishimura and Saeki, 2016). These results suggest the possibility of Maillard-type glycation in developing new antioxidant food materials, without the need for large amounts of salt when dissolving Mfs to produce sausage-like paste products. Investigation of the preparative conditions for maltose-conjugated chicken Mfs with hightest antioxidant capacity using random-centroid optimization (RCO) (Nakai et al., 2009) identified the following optimal conditions: temperature, 61 °C; relative humidity (RH), 38%; reaction time, 33.9 h; and maltose weight ratio, 5.59 (w/w) (Nishimura and Saeki, 2018). Superoxide anion radical scavenging activity (SOSA) of this conjugate reached a maximum of 274 ± 86 units (U) of superoxide dismutase (SOD)/g of protein (n = 3).

In this study, glucose and maltotriose, in which the glycosyl units differ from those of maltose, were selected as sugars, and glycated chicken Mfs were prepared and examined for antioxidant capacity. First, for glucose, the RCO program was used to obtain glucose-conjugated chicken Mfs with the highest SOSA value while maintaining a solubility of > 60% in low-ionic-strength medium. On the other hand, SOSA values of maltotriose-conjugated chicken Mfs exhibiting the highest solubility in low-ionic-strength medium were examined, as maltotriose-conjugated chicken Mfs could acquire only 60% or less solubility in low-ionic-strength medium by glycation (Nishimura et al., 2020). Furthermore, the emulsification properties of both glycated chicken Mfs were evaluated. From these investigations, we attempted to clarify the characteristics of both conjugates.

Materials and Methods

Materials and chemicals    Chicken breast meat was purchased from a local poultry farm immediately after slaughter. Biochemical grade SOD from bovine erythrocytes was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). 5,5-Dimethyl-1-pyrroline-N-oxide (99.0%) was obtained from Labotec (Tokyo, Japan), and xanthine oxidase (XOD) was purchased from Oriental Yeast Co. (Tokyo, Japan). Corn oil (reagent grade) was obtained from Nacalai Tesque (Kyoto, Japan). All other chemicals (reagent grade) were purchased from Nacalai Tesque (Kyoto, Japan) or Wako Pure Chemicals Industries (Osaka, Japan). Distilled water used in the electron spin resonance (ESR) spectroscopy was pretreated with a Chelex-100 resin (100–200 mesh; Bio-Rad, Hercules, CA, USA).

Preparation of Mfs    Chicken Mfs were prepared according to a previously described protocol (Nishimura and Saeki, 2018). Finally, approximately 35 g of chicken Mfs was obtained from 30 g of chicken breast because of swelling during preparation, and this was used as the native chicken Mfs.

RCO    To determine the optimal preparative conditions for glucose-conjugated chicken Mfs, which exhibit highest antioxidant capacity while maintaining > 60% solubility in low-ionic-strength medium, RCO (Nakai et al., 2009) was performed according to the previous method (Nishimura and Saeki, 2018), using glucose instead of maltose and ribose. A search was conducted under the experimental ranges of each factor (temperature, 30–70 °C; RH, 30-45%; reaction time, 2–10 h; and glucose mixing ratio (w/w), 6–14), and 24 vertices were obtained. Each vertex was assessed independently in triplicate or more.

Preparation of glucose-conjugated chicken Mfs    Chicken Mfs were glycated using the previously described protocol (Nishimura and Saeki, 2018). Briefly, chicken Mfs suspended in 50 mM NaCl were mixed with glucose at weight ratios determined using the RCO vertex and each lyophilized protein powder was prepared. The Maillard reaction was conducted by incubating the lyophilized powders under the 24 conditions defined by the RCO to determine the optimal conditions. The chicken Mfs incubated without glucose under optimal conditions were designated as glucose-unmodified chicken Mfs.

Preparation of optimal maltotriose-conjugated chicken Mfs    Optimal maltotriose-conjugated chicken Mfs were prepared according to the conditions of maximum solubility in a low-ionic-strength medium (temperature, 53 °C; RH, 45%; reaction time, 38.5 h; and maltotriose to chicken Mfs mixing ratio, 4.24 (w/w)), which were determined using the RCO in the previous study (Nishimura et al., 2020). The chicken Mfs incubated without maltotriose under optimal conditions were designated as maltotriose-unmodified chicken Mfs.

Solubility of glucose-conjugated chicken Mfs    The solubility of glucose-conjugated chicken Mfs was measured as previously described (Nishimura and Saeki, 2018).

ESR Spectroscopy    ESR spectroscopy was performed at room temperature using a 0.4-mm flat cell (ES-LC 12; Jeol Ltd., Tokyo, Japan) using an X-band ESR spectrometer (JES-FA 100; Jeol) with manganese as the internal standard, following a previously published method (Nishimura and Saeki, 2018). The evaluation of antioxidant capacity also used the comparison of the signal intensity of O⁻₂ induced by the hypoxanthine-XOD system in the presence of glucose-conjugated chicken Mfs, as in the previous study (Nishimura and Saeki, 2018). Again, the results suggested that glycated chicken Mfs has no inhibitory effect on XOD.

SOSA evaluation of SOD and optimal glucose- and maltotriose-conjugated chicken Mfs    The SOSAs of SOD and optimal glucose- and maltotriose-conjugated chicken Mfs were calculated according to a previously described protocol (Nishimura and Saeki, 2018). That is, from the relationship between the comparative ESR signal strength and the concentration of the optimal glucose- or maltotriose-conjugated proteins or SOD, each half-maximal inhibitory concentration (IC₅₀) value (mg/mL) was calculated and converted to SOSA (U/g of protein) based on SOD IC₅₀ values (1.0 U/mL), respectively.

Determination of available lysine and fructosamine contents    The available lysine and fructosamine contents were quantified to monitor the progress of the Maillard reaction between chicken Mfs and glucose during the preparation of the optimal glucose-conjugated chicken Mfs, following a previously published method (Nishimura and Saeki, 2018). Briefly, impurities were removed by Saeki's method (Saeki, 1997), proteins were collected, and their available lysine contents were determined according to the method of Hernandez and Alvarez-Coque (1992).

Fructosamine contents in each glucose-conjugated chicken Mfs were assayed according to Johnson's method (Johnson et al., 1983), as in the previous study (Nishimura and Saeki, 2018).

Emulsifying properties of native, optimal glucose- and maltotriose-conjugated chicken Mfs    The emulsifying properties of native, optimal glucose- and maltotriose-conjugated chicken Mfs were measured using the procedure reported by Matsumura et al. (2003) with slight modifications. An o/w emulsion was prepared using a 5 wt.% oil phase and 95 wt.% aqueous phase.

Optimal glucose- and maltotriose-conjugated chicken Mfs dissolved in 0.1 M NaCl-15 mM sodium phosphate buffer (pH 7.5) were dialyzed against the same buffer at 8–12 °C overnight and for 5 days to remove unreacted glucose and maltotriose, respectively. The supernatant containing 3 mg/mL protein and 0.05% sodium azide, obtained after centrifugation at 32 000 × g for 30 min at 4 °C, was used as the aqueous phase. The oil phase was composed of corn oil. The oil and aqueous phases were mixed at a 1:19 weight ratio and homogenized for 3 min in a high-speed blender (Microtec Co., Ltd., Chiba, Japan) operated at 15 000 rpm. The average droplet diameter was further reduced using an ultrasonic homogenizer (WakenBtech Co., Ltd., Kyoto, Japan) operated at maximum power for 5 min.

The emulsifying properties of the protein samples were analyzed by measuring the particle size distribution and calculating the droplet diameter in the highest peak of the emulsion samples using a laser diffraction particle size analyzer (SALD-2200; Shimadzu Co., Ltd., Kyoto, Japan). The refractive index was set to 1.40-0.05i. The stability of emulsions was analyzed by sealing the vials (35-mm diameter) containing the emulsions, maintaining them at 4 °C without agitation, and observing them 5 min and 1 h after emulsification.

The emulsifying properties of native and unmodified chicken Mfs, obtained by incubation in the absence of sugar under both optimal preparative conditions, were also investigated after dissolving them in 0.5 M NaCl-15 mM sodium phosphate buffer (pH 7.5) solution.

Thiobarbituric acid (TBA) test    O/W emulsions were prepared using a 5 wt.% oil phase and 95 wt.% aqueous phase. The aqueous phase was a surfactant solution (2 wt.% Tween 20 in 0.1 M NaCl-15 mM sodium phosphate buffer, pH 7.5). Methyl linoleate was used as the oil phase. The oil and aqueous phases were mixed and homogenized in a high-speed blender (Microtec) at 15 000 rpm for 3 min. The average droplet diameter was further reduced using an ultrasonic homogenizer (WakenBtech) at maximum power for 3 min.

Moreover, to test lipid oxidation inhibition, o/w emulsions prepared as described above were diluted 16.7 times with 0.1 M NaCl-15 mM sodium phosphate buffer, pH 7.5, with or without 0.3% optimal maltotriose-conjugated chicken Mfs. Sodium azide was added at a concentration of 0.05% to these diluted solutions. Dilution is very convenient for performing subsequent TBA tests. In addition, dilution is necessary to minimize oil droplet interactions while evaluating the action of FeSO₄ on the oil droplets. The oil phase concentration in the diluted emulsions for the TBA test was about 0.3% (w/w). The addition of 5 µM FeSO₄ initiated the oxidation of methyl linoleate in the emulsions. Sample emulsions were divided into small portions (2 mL per tube) and stored at 50 °C for 7 days. Samples were removed at predetermined intervals, and the oxidation of methyl linoleate was measured using the TBA test.

Measurement of TBA-reactive substances (TBRS)    TBA values were measured using the method of Buege and Aust (1978). An aliquot (0.5 mL) of oxidized emulsion was mixed with 2.0 mL of TBA solution (0.375% TBA, 15% trichloroacetic acid, and 0.04% butylated hydroxytoluene in 0.25 N HCl) and 0.5 mL of 0.1 M NaCl-15 mM sodium phosphate buffer at pH 7.5. The mixture was heated to 95 °C for 15 min. After cooling, the mixture was centrifuged at 1 700 × g for 15 min. The absorbance of the supernatant at 532 nm was measured using a recording spectrometer. The results were expressed as the amount of TBA-reactive substances (TBARS). The values were calculated as malondialdehyde (MDA) equivalents based on the molecular absorbance of MDA, i.e., 1.56 × 10⁵.

Protein determination    The protein concentrations of dissolved and purified chicken Mfs were determined using the Kjeldahl (AOAC, 1990) and Lowry (Lowry et al., 1951) methods, respectively, with ovalbumin as a standard. The biuret method (Gornall et al., 1949), which uses bovine serum albumin as a standard, was used in other assays.

Statistical analysis    Each experiment was performed on three different lots of glycated Mfs. The results are reported as mean values of at least three determinations, with the error bars indicating the standard deviation. Statistical analysis was performed using Microsoft Excel (Microsoft 365) with Ekuseru-Tokei (Social Survey Research Information Co., Ltd., Tokyo, Japan). Statistical significance was set at p < 0.05.

Results and Discussion

Determination of the optimal preparative method for glucose-conjugated chicken Mfs    As outlined in Table 1, RCO was used to determine the optimal conditions for production of glucose-conjugated chicken Mfs with the highest antioxidant capacity and > 60% solubility in a low-ionic-strength medium on the basis of varying temperature, RH, reaction time, and the glucose mixing ratio to Mfs (w/w) (Table 1). The parameters respective to each experiment were calculated using the RCO program (Nakai et al., 2009) and then implemented for different preparations of glycated chicken Mfs. All data were mapped as shown in Figure 1, aiding in the visualization of the experimental responses and indicating data trends. As RCO is usually repeated until an adequate response is achieved, in the present study, the approximate position of the optimal conditions was made clear by conducting a second cycle of the RCO program. The best result was obtained at Vertex 7 with a temperature of 51 °C, RH of 41%, reaction time of 7.51 h, and glucose mixing ratio to Mfs (w/w) of 11.4, as shown in Fig. 1.

Table 1. Summary data for random-centroid optimization of SOSA using glucose.

Vertex No.	Temperature (°C)	RH (%)	Reaction Time (h)	Glucose Mixing Ratio to Mfs (w/w)	Evaluation (Residual ratio of O−2)a) (%)
1	52	42	3.44	9.6	100.0
2	42	38	5.44	13.0	100.0
3	32	39	5.32	8.8	100.0
4	53	32	7.80	7.6	80.1
5	70	39	8.36	7.0	100.0
6	40	43	8.79	8.8	100.0
7	51	41	7.51	11.4	54.2
8	36	31	3.35	13.6	100.0
9	50	30	5.69	8.9	100.0
10b)	47	33	6.09	10.4	100.0
11b)	48	36	7.45	9.2	100.0
12b)	45	37	6.86	10.3	100.0
13b)	44	36	6.34	10.7	100.0
14	64	32	6.12	6.1	100.0
15	57	35	7.22	9.0	66.2
16	57	37	5.79	8.1	73.2
17	52	36	7.13	6.5	66.3
18	47	44	6.05	11.4	100.0
19	57	34	6.36	12.8	74.2
20	46	39	8.36	12.9	100.0
21c)	54	37	6.91	8.8	82.9
22c)	54	36	7.05	9.9	85.7
23c)	55	37	6.72	10.3	83.1
24c)	54	37	6.70	9.7	81.9

^a)  When solubility of glucose conjugated Mfs did not exceed 60 %, the evaluation (residual ratio of O⁻₂ of this vertex was regarded as 100%.

^b)  Re-centroid points of first cycle.

^c)  Re-centroid points of second cycle.

Fig. 1

Mapping results of experiments subjected to RCO for scavenging O⁻₂ using glucose.

The comparative signal strength of O⁻₂ induced by the hypoxanthine-XOD system in the presence of glucose-conjugated chicken Mfs was used to evaluate the antioxidant capability, and the vertex that provided the smallest evaluation was sought. The ESR signal strength of O⁻₂ induced by the hypoxanthine-XOD system was regarded as 100%. When the solubility of glycated chicken Mfs with glucose did not surpass 60%, the evaluation (residual ratio of O⁻₂ of this vertex was regarded as 100%. (A) Reaction temperature. (B) Relative humidity. (C) Reaction time. (D) Glucose mixing ratio to Mfs (w/w). (●). The curves and lines in the figure are drawn by the software. In this case, they represent approximately in which direction the lowest values exist. The arrow in each graph shows the best result.

When hydroxyl radical scavenging activity was used as an indicator of antioxidant capacity, the optimal conditions were a temperature 52 °C, RH of 38%, reaction time of 6.79 h, and glucose mixing ratio to Mfs (w/w) of 11.7 (Nishimura et al., 2019). However, in this study, only the reaction time of the four preparative condition parameters was slightly longer. This significant difference regarding the reaction time, even though the others were similar, might have affected the production yields of the intermediate and final Maillard reaction products with specific scavenging activity for O⁻₂ or hydroxyl radicals.

Time-dependent changes in solubility and available lysine and fructosamine contents in chicken Mfs during glycation with glucose    A lyophilized chicken Mfs-glucose mixture (chicken Mfs:glucose ratio = 1:11.4) was heated at 51 °C and 41% RH in accordance with the optimal preparative conditions determined as explained above. The solubility was examined for 7.51 h using 15 mM sodium phosphate buffer (pH 7.5) containing 0.1 M or 0.5 M NaCl (Fig. 2A). The solubility in 0.5 M NaCl (pH 7.5) gradually increased from 53.6 ± 8.0% (n = 3) to reach 78.0 ± 3.6% (n = 3) after 7.51 h. On the other hand, the solubility in 0.1 M NaCl (pH 7.5) was 6.0 ± 1.9% (n = 3), and no significant change was observed until 2 h later. The solubility began to increase after 2 h and reached 67.9 ± 2.4% (n = 3) after 7.51 h, although maltotriose-conjugated chicken Mfs did not exceed 60% solubility under any condition (Nishimura et al., 2020). The solubility before the reaction differed according to the NaCl concentration. However, the introduction of glucose resulted in a loss of salt dependence and an increase in the water solubility of the Mfs.

Fig. 2.

Changes in solubility and amounts of available lysine and fructosamine in chicken Mfs during glycation.

Mixture of lyophilized chicken Mfs and glucose at a ratio of 1:11.4 (w/w) were heated at 51 °C and 41% RH. The solubility (A) in 0.1 M (●) or 0.5 M (○) NaCl at pH 7.5 was measured during the Maillard reaction with glucose. The available lysine (B) and fructosamine (C) contents were simultaneously measured. Data represent mean ± standard deviation (n = 3).

Figures 2B and 2C show the changes in the contents of available lysine and fructosamine in the chicken Mfs upon glucose introduction through the Maillard reaction. The content of available lysine decreased mildly during the reaction and reached 48.2 ± 1.1% (n = 3) as the reaction progressed (Fig. 2B). Conversely, the quantity of fructosamine increased with the reaction time, reaching 329.1 ± 32.3 µmol/g of protein (n = 3) after 7.51 h (Fig. 2C). A similar trend was observed in maltotriose-conjugated chicken Mfs (Nishimura et al., 2020).

The results shown in Fig. 2 indicated that the 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 the solubility in 0.5 M NaCl was not compromised clearly demonstrates that glucose-conjugated chicken Mfs exhibit high salt-independent solubility without significant protein denaturation at the early stage of the Maillard reaction, as described previously (Nishimura et al., 2019; Nishimura et al., 2020; Nishimura and Saeki, 2021).

SOSA measurement of optimal glucose- and maltotriose-conjugated chicken Mfs    The inhibition curves of SOSA values associated with optimal glucose- and maltotriose-conjugated chicken Mfs are shown in Fig. 3. Native chicken Mfs hardly demonstrated SOSA within the range of 2.0-6.0 mg of protein/mL (Nishimura and Saeki, 2018). In contrast, both conjugates reduced O⁻₂ production with an increase in protein concentration. The reduction ratios of the optimal glucose- and maltotriose-conjugated chicken Mfs reached 44.6 ± 3.7% (n = 3) and 20.6 ± 8.6% (n = 4) at a protein concentration of 5.0 and 4.0 mg/mL, respectively. The SOSAs derived from the IC₅₀ values based on the SOD IC₅₀ value (1.0 U/mL) were 264 ± 45 (n = 3) and 740 ± 346 U/g of protein (n = 4). In a previous study, the SOSA values of optimal ribose- and maltose-conjugated chicken Mfs were 368 ± 120 (n = 3) and 274 ± 86 U/g of protein (n = 3), respectively (Nishimura and Saeki, 2018). However, there was no significant difference between these four values, suggesting that regardless of the type of sugar, the amounts of intermediate products with antioxidant capacity in the early stage of the Maillard reaction are similar. These results indicate that there is no significant difference in the antioxidant capacity of each conjugate. This trend was also observed when hydroxyl radical scavenging activity was used as an indicator of antioxidant capacity (Nishimura and Saeki, 2021).

Fig. 3.

Antioxidant properties of optimal glucose- and maltotriose-conjugated chicken Mfs

The antioxidant capacity of optimal glucose-conjugated chicken Mfs (●) and optimal maltotriose-conjugated chicken Mfs (■) against O⁻₂ was measured with ESR. The strength of O⁻₂ generated by the hypoxanthine-XOD system was converted to a signal intensity relative to that of manganese, which was regarded as 100%. Values are mean ± standard deviation (n ≥ 3).

Emulsifying properties    O/W emulsions were prepared using five types of chicken Mfs solutions and corn oil, as described in the Materials and Methods, and their particle sizes were measured (Fig. 4). The droplet diameter in the highest peak of the emulsion prepared using native chicken Mfs was 9.6 ± 1.8 µm (n = 3) (Fig. 4A and B), whereas those of emulsions prepared with optimal glucose-conjugated and glucose-unmodified chicken Mfs were 8.1 ± 1.3 µm (n = 3) and 8.5 ± 0.2 µm (n = 3), respectively (Fig. 4A). There was no significant difference among the particle sizes of the three types of chicken Mfs. The droplet diameters in the highest peaks of emulsions prepared with optimal maltotriose-conjugated and maltotriose-unmodified chicken Mfs were 4.7 ± 1.9 µm (n = 3) and 8.6 ± 0.2 µm (n = 3), respectively (Fig. 4B), indicating that the emulsions prepared with optimal maltotriose-conjugated chicken Mfs were significantly smaller than the native emulsions (p < 0.05).

Fig. 4.

Particle size distributions of emulsions of several types of chicken Mfs.

The emulsifying ability of the chicken Mfs was analyzed by measuring the particle size distribution and calculating the droplet diameter in the highest peak of the emulsion samples using a light scattering instrument. Panel A shows the particle size distributions of the emulsion prepared using native chicken Mfs (×), optimal glucose-conjugated chicken Mfs (●) and glucose-unmodified chicken Mfs (○), respectively. Panel B shows the particle size distributions of the emulsion prepared using native chicken Mfs (×), optimal maltotriose-conjugated chicken Mfs (■) and maltotriose-unmodified chicken Mfs (□), respectively. Values are the mean ± SD of three independent experiments.

Emulsions are highly dynamic systems in which the droplets continuously move around and frequently collide with each other. Droplet-droplet collisions are particularly rapid during homogenization because of the intense mechanical agitation of the emulsion. If droplets are not protected by a sufficiently strong emulsifier membrane, they tend to coalesce with one another during a collision (Walstra, 1993). Maltotriose consisting of 3 glycosyl units naturally has more OH groups than glucose, and hence is more hydrophilic. The impartation of the chemical property of maltotriose could be involved in the enhancement of the emulsifying activity of Mfs. Higher hydrophilicity may be responsible for the formation of emulsifier membranes stronger than those formed when using the other samples, leading to a significantly smaller particle size in the peaks of emulsions.

It is generally believed that the smaller the average particle size, the more stable the emulsion (Wang et al., 2019). Therefore, it was considered that the emulsion stability of optimal maltotriose-conjugated chicken Mfs was superior to that of the native chicken Mfs.

The stabilities of the emulsions prepared with five types of chicken Mfs were measured. Emulsions were poured into vials (35.0 mm × 81.5 mm) and sealed. Each state was observed at 4 °C for up to 1 h (Fig. 5). All emulsions except for the one prepared with glucose-unmodified chicken Mfs dispersed and stabilized 5 min after emulsification. However, the emulsions of optimal glucose-conjugated and maltotriose-unmodified chicken Mfs completely separated into water and cream phases after 1 h. The phase separations of native and optimal maltotriose-conjugated chicken Mf emulsions were not observed clearly after 1 h, but both started to separate after 3 h (data not shown), indicating that optimal maltotriose-conjugated chicken Mfs had weak emulsion stability compared to that of native Mfs. Accordingly, this indicates that the optimal maltotriose-conjugated chicken Mfs cannot be used as an emulsifier.

Fig. 5.

Emulsion stability of several types of chicken Mfs.

The stability of emulsions was analyzed by sealing the vials containing the emulsions, maintaining them at 4 °C without agitation, and observing them 5 min and 1 h after emulsification. The emulsion stabilities of native chicken Mfs, optimal glucose-conjugated chicken Mfs, glucose-unmodified chicken Mfs, optimal maltotriose-conjugated chicken Mfs, and maltotriose-unmodified chicken Mfs were determined in three independent experiments. The average emulsion stability of each chicken Mfs sample is shown.

When Tween 20, a typical emulsifier for aqueous phases, was used to prepare o/w emulsions, the average particle size of the emulsions was about 2.3 µm (data not shown). This value is about half that for optimal maltotriose-conjugated chicken Mfs (4.7 ± 1.9 µm (n = 3)). This small particle size resulted in faster diffusion and adsorption of Tween 20 on the oil droplet surface during emulsification.

Effects of maltotriose-conjugated chicken Mfs on lipid oxidation in emulsions    The weak emulsion stability of the optimal maltotriose-conjugated chicken Mfs prevents its use as an emulsifier. However, this conjugate exhibits antioxidant capacity. Accordingly, as an antioxidant, its ability to protect against oil oxidation was considered. In a subsequent experiment, this phenomenon was investigated (Fig. 6). Tween 20 was used for the emulsification. When the addition of FeSO₄ initiated oxidation, the concentrations of TBARS in the absence and presence of optimal maltotriose-conjugated chicken Mfs gradually increased up to 3 days to 7.8 ± 1.6 (n = 6) and 5.2 ± 0.9 µM (n = 3), respectively. Thereafter, both values remained largely unchanged and were 5.1 ± 0.7 (n = 6) and 6.7 ± 1.0 µM (n = 3) after 7 days. There was almost no significant difference between the TBARS values of the two samples. However, a significant difference (p<0.05) was observed on day 5, with the values for the emulsions in the absence and presence of optimal maltotriose-conjugated chicken Mfs being 7.5 ± 1.2 (n = 6) and 4.9 ± 1.1 µM (n = 3), respectively. This indicates that maltotriose-conjugated chicken Mfs may act as an antioxidant to inhibit oil oxidation.

Fig. 6.

Inhibitory effects of maltotriose-conjugated chicken Mfs when used as antioxidants for lipid oxidation.

Emulsions were prepared using Tween 20 and diluted 16.7 times with 0.1M NaCl-15 mM sodium phosphate buffer (pH 7.5) (□), and 0.3% optimal maltotriose-conjugated chicken Mfs (○). All emulsions were stored at 50 °C for 7 days. The extent of oxidation was evaluated using the TBA tests. The TBA test value is expressed as the equivalent of MDA ( TBARS ). Values are the mean ± SD of three independent experiments.

* represents significant differences between values (p < 0.05).

The authors have already pointed out that this antioxidant capacity is attributed to the primary structure or specific peptide sequences of the chicken Mfs, irrespective of the protein shape (Isono et al., 2012). Therefore, it is thought that the antioxidant capacity of maltotriose-conjugated chicken Mfs weakened or disappeared after 7 days, attributed to amino acid sequence masking caused by denaturation over time. We consider that this is the reason why oil oxidation progressed, although there was a significant difference at day 5, and the significant difference between the two disappeared at day 7. However, the details of this point require further investigation.

Conclusion

Glucose-conjugated chicken Mfs, with maximum antioxidant capacity as determined by measuring SOSA and solubility in low-ionic-strength medium using the RCO method, and maltotriose-conjugated chicken Mfs, with maximal solubility in low-ionic-strength medium and antioxidant capacity, were prepared. Droplet diameters of the o/w emulsions prepared using maltotriose-conjugated chicken Mfs as an emulsifier tended to be significantly smaller than those prepared using glucose-conjugated chicken Mfs, but the sugar conjugations did not improve the stability of the chicken Mf emulsions. However, when the maltotriose-chicken Mf conjugates were stored at 50 °C for 7 days, the oxidation of methyl linoleate in the emulsion was markedly suppressed only on day 5. These results indicate that maltotriose-conjugated chicken Mfs may act as an antioxidant to inhibit oil oxidation.

Acknowledgements    The authors appreciate the contributions of Ms. Haruka Aoyama, Ms. Chihiro Sato and Ms. Yuri Ushio of Doshisha Women's College of Liberal Arts in this study. 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.

Conflict of interest    There are no conflicts of interest to declare.

Abbreviations

ESR

electron spin resonance

IC₅₀

half-maximal inhibitory concentration

MDA

malondialdehyde

Mfs

myofibrillar proteins

O⁻₂

superoxide anion radical

RCO

random-centroid optimization

RH

relative humidity

SOD

superoxide dismutase

SOSA

superoxide anion radical scavenging activity

TBA

thiobarbituric acid

TBARS

TBA-reactive substances

U

unit

XOD

xanthine oxidase

References

• AOAC International (1990). Official Methods of Analysis of the Association of Analytical Chemists, 15th edition. Washington DC, USA, pp. 66-88.
•  Buege,  J.A., and  Aust,  S.D. (1978). Microsomal lipid peroxidation. Methods Enzymol., 52, 302-310.
•  Fayle,  S.E. and  Gerrard,  J.A. (2002). “The Maillard Reaction.” The Royal Society of Chemistry, Cambridge.
•  Gornall,  A.G.,  Bardwill,  C.J., and  David,  M.M. (1949). Determination of serum proteins by means of the Biuret reaction. J. Biol. Chem., 177, 751-766.
•  Guohua,  C.,  Helaine,  M. A., and  Richard,  G. C. (1993). Oxygen-radical absorbance capacity assay for antioxidants. Free Radic. Biol. Med., 14, 303-311.
•  Hernandez,  M.J.M. and  Alvarez-Coque,  M.C.G. (1992). Available lysine in protein, assay using o-phthalaldehyde/N-acetyl-L-cysteine spectrophotometric method. J. Food Sci., 57, 503-505.
•  Isono,  M.,  Saeki,  H., and  Nishimura,  K. (2012). Properties of glycated chicken myofibrillar proteins with enhanced antioxidant abilities. J. Home Econ. Jpn., 63, 461-467.
•  Johnson,  R.N.,  Metcalf,  P.A., and  Baker,  J.R. (1983). Fructosamine: A new approach to the estimation of serum glycosylprotein. An index of diabetic control. Clin. Chim. Acta, 127, 87-95.
•  Lowry,  O.H.,  Rosebrough,  N.J.,  Farr,  A.L., and  Randall,  R.J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193, 265-275.
•  Matsumura,  Y.,  Egami,  M.,  Satake,  C.,  Maeda,  Y.,  Takahashi,  T.,  Nakamura,  A., and  Mori,  T. (2003). Inhibitory effects of peptide-bound polysaccharides on lipid oxidation in emulsions. Food Chem., 83, 107-119.
•  Nakai,  S.,  Horimoto,  Y.,  Dou,  J., and  Verdini,  R.A. (2009). Optimization in Food Engineering. In “Random-Centroid Optimization.” ed. by  F.  Erdoğdu. CRC Press, New York, pp. 141-152.
•  Nishimura,  K.,  Murakoshi,  M.,  Katayama,  S., and  Saeki,  H. (2011a). Changes in solubility and thermal stability of chicken myofibrillar protein by glycosylation. Food Sci. Technol. Res., 17, 69-75.
•  Nishimura,  K.,  Murakoshi,  M.,  Katayama,  S., and  Saeki,  H. (2011b). Antioxidative ability of chicken myofibrillar protein developed by glycosylation and changes in the solubility and thermal stability. Biosci. Biotechnol. Biochem., 75, 247-254.
•  Nishimura,  K.,  Isono,  M., and  Saeki,  H. (2015). Gel-forming ability of glycated chicken myofibrillar proteins with enhanced antioxidant properties. J. Home Econ. Jpn., 66, 213-221.
•  Nishimura,  K. and  Saeki,  H. (2016). Preparation of chicken myofibrillar protein-maltose conjugate having multi-food functions using random-centroid optimization. Food Sci. Technol. Res., 22, 801-810.
•  Nishimura,  K. and  Saeki,  H. (2018). Random-centroid optimization reveals the strongest superoxide anion radical scavenging activity of maltose- and ribose-conjugated chicken myofibrillar protein. Food Sci. Technol. Res., 24, 551-557.
•  Nishimura,  K.,  Suzuki,  M., and  Saeki,  H. (2019). Glucose-conjugated chicken myofibrillar proteins derived from random-centroid optimization present potent hydroxyl radical scavenging activity. Biosci. Biotechnol. Biochem., 83, 2307-2317.
•  Nishimura,  K.,  Suzuki,  M., and  Saeki,  H. (2020). Maltotriose-conjugated chicken myofibrillar proteins derived from random-centroid optimization present potent solubility in low ionic strength medium. Food Sci. Technol. Res., 26, 759-769.
•  Nishimura,  K. and  Saeki,  H. (2021). Dextran-glycation improves the solubility of chicken myofibrillar proteins in low ionic strength medium, allowing the proteins to exhibit antioxidant activity. J. Home Econ. Jpn., 72, 377-386.
•  Saeki,  H. (1997). Preparation of neoglycoprotein from carp myofibrillar protein by Maillard reaction with glucose: Biochemical properties and emulsifying properties. J. Agric. Food Chem., 45, 680-684.
•  Walstra,  P. (1993). Principles of emulsion formation. Chem. Eng. Sci., 48, 333-349.
•  Wang,  Y.,  Yu,  J.,  Xu,  N.,  Wang,  G., and  Wang,  X. (2019). Influence of protein hydrolysis on the freeze-thaw stability of emulsions prepared with soy protein - dextran conjugates. J. Oleo Sci., 68, 959-965.