Combined effects of high-intensity ultrasound treatment and hydrogen peroxide addition on the thermal stabilities of myofibrillar protein emulsions at low ionic strengths

Graphical abstract

Highlights

• •H₂O₂ inhibited the disulfide bonds, suppressing the myosin cross-linking.
• •HIU destroyed the filamentous structures, inhibiting the rod region aggregation.
• •HIU increased the exposure of –SH groups, promoting blockage effects of H₂O₂.
• •Combination of HIU and H₂O₂ enhanced the thermal stability of MP emulsions.

Abstract

In this study, the effects of high-intensity ultrasound (HIU) treatment combined with hydrogen peroxide (H₂O₂) addition on the thermal stability of myofibrillar protein (MP)–stabilized emulsions in low-salt conditions were investigated. Results showed that compared to using either HIU or H₂O₂ treatment alone, HIU treatment combined with H₂O₂ was most effective in enhancing the physical stability of emulsions. Moreover, the emulsion stabilized by MPs co-treated with HIU and H₂O₂ exhibited the most uniform distribution, highest absolute zeta potential, and optimal rheological properties upon heating. This combination effect during heating was caused by the inhibition of disulfide bond cross-linking of myosin heads by H₂O₂ and the dissociation of filamentous myosin structures using the HIU treatment. In addition, the results of oxidative stability analysis indicated that the addition of H₂O₂ increased the content of oxidation products; however, the overall influence on the oxidative stability of emulsions was not significant. In conclusion, the combination of HIU and H₂O₂ treatment is a promising approach to suppress heat-induced MP aggregation and improve the thermal stability of corresponding emulsions.

1. Introduction

Oil-in-water (O/W) emulsions are multiphase systems in which immiscible liquids are uniformly dispersed as droplets in a continuous phase [1]. Recently, O/W emulsions have been widely used in the food industry, including fluid foods, creams, functional beverages, and emulsified meat products [2]. Emulsions are thermodynamically unstable systems and undergo phase separation over time; therefore, emulsifiers play a key role in reducing interfacial tension at the O/W interface and maintain emulsion stability. Amphiphilic proteins are generally used as emulsifiers to coat oil droplets and provide additional nutrients [3]. Nowadays, most natural protein emulsifiers used in the food industry are plant and milk proteins, such as soy, pea, and whey protein isolate [4], [5], [6]. Although meat proteins have not been used as widely as these proteins, they have garnered increasing attention owing to their emulsifying activities and rich nutritional benefits.

Myofibrillar proteins (MPs) account for ∼ 60 %–65 % of the total meat proteins, which are mainly composed of myosin, actin, tropomyosin, and troponin in a 10:4:1:1 ratio [7]. MPs are high-quality proteins because they can provide essential amino acids, particularly a high lysine content. These essential amino acids are highly digestible, which is beneficial to human health [8]. The emulsifying activities of MPs have also been studied. Wu et al., [9] reported that when MPs adsorb at the O/W interface, they undergo structural changes. The hydrophobic groups embedded in the oil phase are exposed, and the hydrophilic groups in the disperse phase are rearranged. This process enables interfacial proteins to interact with neighboring proteins or the oil phase, forming a viscoelastic film resistant to mechanical stress and providing electrostatic repulsion and steric hindrance to prevent the flocculation or agglomeration of oil droplets. Owing to these virtues, MPs can be considered as promising emulsifiers, and MP emulsions have the potential to be used in functional beverages and fluid foods. However, the utilization of MPs in stabilizing emulsions is still limited by certain factors, and thus the application of MP emulsions poses challenges.

Myosin, a major component in MPs, tends to pack and coil in low-salt media and form insoluble filamentous polymers, exhibiting poor solubility and stability. Thus, MPs cannot be used as effective emulsifiers to stabilize emulsions under low-ionic environments. Our prior investigations on the potential mechanisms for myosin molecular assembly indicate that this self-assembly behavior is possibly driven by the electrostatic attraction between regularly positioned charge clusters in the myosin tail [10]. MPs have high solubility in high-salt solutions (0.47–0.68 M NaCl); however, salt ions cause charge shielding, thereby negatively affecting the stability of emulsion systems. Furthermore, a high-salt diet is harmful to the body [11]. Therefore, the solubility of MPs in low-salt conditions must be enhanced to improve the stability of MP emulsions.

High-intensity ultrasound (HIU) is an environmentally friendly and efficient approach that uses acoustic cavitation to enhance the functional properties of proteins [12]. During ultrasound transmission, cavitation bubbles collapse when they reach a critical size in the medium and generate strong physical forces such as shear force and turbulence around the liquid, modifying protein structures [13]. HIU treatment induced the depolymerization of filamentous myosin polymers and suppressed the subsequent self-assembly behaviors of myosin rods [8], [14]. The as-obtained myosin monomers can exhibit high solubility in low-salt conditions and efficiently coat oil droplets, ensuring the stable performance of emulsion systems.

Another issue that limits the application of MP emulsions is the poor thermal stability of MPs. In food industry, many emulsion products are inevitably subjected to strict low-temperature thermal treatment to ensure food safety and shelf life such as pasteurization (<90℃). As MPs are prone to intermolecular cross-linking, aggregation, and gel formation after heating, the stability and functional properties of emulsions stabilized by MPs can be adversely affected [15]. Intermolecular covalent cross-linking and hydrophobic interactions are the key drivers of MP aggregation behaviors. Specifically, under heat treatment, the initial aggregation of myosin heads is caused by sulfhydryl (–SH)-disulfide (S–S) interchange in the myosin subfragment-1 region and the subsequent aggregation of myosin rods is caused by hydrophobic and hydrogen bonds [16]. These theoretical foundations indicate that the blockage of reactive –SH groups and inhibition of hydrophobic interactions are key to arresting the thermal aggregation of MPs.

Hydrogen peroxide (H₂O₂), an oxidizing compound, is an FDA-permitted food additive that is generally recognized as safe (GRAS); it has been widely used in food industry for various purposes [17]. Sutariya et al., [18] revealed that H₂O₂ could block –SH groups, inhibit the formation of disulfide bonds, and suppress the thermal aggregation of MPs. The addition of H₂O₂ oxidizes the –SH groups to sulfinic or sulfonic acids with high water solubility, which replace the stable disulfide bond structure, thereby interfering with the heat-induced protein aggregation [19]. Thus, the issue of intermolecular cross-linking between myosin heads seems to be addressed. Our previous works illustrated that the dissociation of myosin filaments induced by HIU treatment can increase the long-range intermolecular electrostatic repulsion, which may resist the intermolecular hydrophobic attraction and enhance the thermal stability of MPs [8], [14].

In fact, we previously revealed that the resistance caused by HIU treatment to hydrophobic interactions, combined with the disruptions of H₂O₂ to sulfhydryl-disulfide interchange, inhibited the heat-induced aggregation of MPs in low-ionic-strength media [20]. However, the performance of co-treated MPs in emulsion systems remains unclear·H₂O₂, a pro-oxidant, may induce free radical chain reactions in the presence of oils. However, the effects of H₂O₂-modified proteins on the oxidative stability of corresponding emulsions have not been reported yet. Therefore, emulsions were prepared herein using MPs treated only with H₂O₂ and HIU and co-treated with H₂O₂ and HIU. These emulsions were heat-treated under the same conditions, and their physical stability, rheological properties, and droplet characterization were analyzed before and after heating. Moreover, their oxidative stability was also monitored to investigate the negative influence of H₂O₂ addition. The primary aim of this study is to develop an emulsion system with strong resistance to heat and broaden the application of MP emulsions in the food industry.

2. Materials and methods

2.1. Materials

Porcine longissimus muscle within 24 h of slaughter was purchased from a local market (Harbin, Heilongjiang, China). Soybean oil was obtained from Jinlongyu Co., Ltd. (Beijing, China). 30 % H₂O₂ was provided by Novozymes (Bagsvaerd, Denmark). All other chemicals and reagents were of analytical grade.

2.2. Extraction of MPs

MPs were extracted following the methodology described by Liu et al., [20]. Minced meat was added into 10 volumes of sodium phosphate buffer (20 mM, pH 7.0, 25-mM NaCl, and 5.0-mM EDTA) before a 2-min homogenization, and the homogenate was centrifuged (10000 g, 15 min) at 4 ℃. Then, the obtained precipitate was washed thrice with 20-mM sodium phosphate buffer (pH 7.0, 0.1-M NaCl, 5.0-mM EDTA), followed by the removal of connective tissue and external fat using four layers of coarse mesh gauze. The resulting sediment was suspended in 5-mM sodium phosphate buffer (pH 7.0, 1-mM EDTA) and centrifuged twice to obtain the MP samples. The extracted MPs were resuspended in 5.0-mM sodium phosphate buffer (pH 7.0, 1-mM EDTA) to prepare a specific concentration (10 mg/mL) for use.

2.3. HIU and H₂O₂ treatment

The MP suspension (10 mg/mL) was placed in a double-walled beaker (50 mL) and treated in a Scientz-Ⅱ D ultrasound generator (Scientz Biotechnology Co., Ltd., Ningbo, China). The settings for the ultrasound treatment were an ultrasonic power of 450 W and a time of 10 min (pulse durations of 2.0 s on and 3.0 s off) [8]. Throughout the treatment, an ice-water mixture was circulated around the double-walled beaker to avoid protein denaturation, and a temperature probe was integrated for real-time monitoring. After the HIU treatment [20], H₂O₂ with the concentration of 200-μmol/g protein was added to the MP suspensions (10 mg/mL) at 4 ℃, followed by vortex mixing for 30 s to ensure a complete reaction. The untreated MP suspension was named “Native-MP,” and the HIU-treated MP suspension without H₂O₂ treatment was named “HIU-MP.” The untreated MPs after adding H₂O₂ was named “Native-MP + H₂O₂,” and the HIU-treated MPs after adding H₂O₂ was named “HIU-MP + H₂O₂.”.

2.4. Emulsion preparation

Each of MP suspensions (45 mL), i.e., Native-MP, Native-MP + H₂O₂, HIU-MP, and HIU-MP + H₂O₂ was used as disperse phase, and soybean oil (5 mL) was used as the oil phase. To form coarse emulsions, the disperse and oil phases were blended using an Ultra-Turrax homogenizer (IKA T20 Basic, IKA-Werke GmbH and Co. KG, Staufen, Germany) for 3 min at a speed of 10,000 rpm. Then, to obtain fine emulsions, the coarse emulsions were further homogenized through a high-pressure homogenizer (ATS Engineer, Inc., Shanghai, China) at 60 MPa for two cycles, with a 5-min rest between cycles.

2.5. Heat treatment

Most current food protein products are subjected to low-temperature heat sterilization treatment (≤90 ℃); therefore, the heat-treatment temperature was set as 90 ℃ herein. The emulsion samples were placed in glass vials with sealing films and heated in a water bath at 90 ℃ for 10 min. After heating, the samples were cooled in an ice-water bath for 20 min and used immediately for subsequent experiments. Additionally, a set of the same emulsion samples was prepared using the aforementioned method and stored in an incubator (37 ℃) for measuring the oxidative stability. To prevent microbial growth during storage, sodium azide (3.0 mM in final emulsions) was added to each emulsion sample.

2.6. Physical stability analysis

The physical stability of emulsions stabilized by Native-MP, HIU-MP, Native-MP + H₂O₂, and HIU-MP + H₂O₂ was evaluated via centrifugal sedimentation analysis using the LUMiSizer stability analyzer (LUM GmbH, Berlin, Germany) [21]. Specifically, the LUMiSizer was set at 25 ℃; rotational speed, 4000 rpm; scanning rate, once every 20 s for 6000 s; the number of scanning times, 300. During testing, the continuous centrifugal sedimentation was applied to accelerate instability processes. Simultaneously, the parallel near-infrared (IR) light was passed through the sample cell, collecting the intensity of transmitted light. Finally, the overall transmittance change curve of the samples with respect to time was linearly regressed to obtain a value for the slope of the curve.

2.7. Measurement of droplet size distribution and zeta potential of emulsions

The droplet size distribution and zeta potential of emulsion samples before and after heating were determined using a static light scattering instrument (Mastersizer 2000, Malvern Instruments Ltd., Worcestershire, UK) and a zeta potential analyzer (Zeta Plus, Malvern, UK), respectively. To avoid multiple scattering effects, each of the samples was diluted 100-fold with 0.01-M phosphate buffer (pH 7.0) before measurements.

2.8. Confocal laser scanning microscopy (CLSM)

A Leica TCS SP5 confocal laser scanning microscope (Leica, Heidelberg, Germany) was used to observe the micromorphology of MP emulsions with different treatments. By modifying the previously proposed method [22], 25 μL of Nile blue (0.1 %, w/v) and 20 μL of Nile red (0.1 %, w/v) were mixed with 1 mL of emulsion samples in the dark for 30 min to stain protein and oil, respectively. Each stained sample (5 μL) was then placed on a concave microscope slide with a cover glass. The protein and oil phases of emulsions were observed at excitation wavelengths of 633 and 488 nm, respectively, using a 40 × HCPL APO/20 × oil-immersion objective.

2.9. Rheological behavior analysis

2.9.1. Dynamic rheology

The dynamic rheological behavior of emulsions was analyzed using a model CVO rheometer (Malvern Instruments, Westborough, MA) based on the methodology reported by Sutariya et al., [18]. The emulsion samples were placed between two parallel plates, followed by heating from 25 ℃ to 90 ℃ at a rate of 4 ℃/min and maintaining at 90 ℃ for 10 min. Then, the samples were cooled to 25 ℃ at a rate of 4 ℃/min and maintained for 15 min. The temperature sweeps were measured at a maximum shear strain of 2.0 % and frequency of 2.0 Hz. The storage modulus (G′) and loss modulus (G′′) were recorded every 30 s.

2.9.2. Apparent viscosity

The samples were subjected to shear rate scanning before and after heating using a model CVO rheometer (Malvern Instruments, Westborough, MA), with a parallel plate diameter of 50 mm and a gap of 0.5 mm [23]. Then, the variation curves of apparent viscosity (η) with respect to shear rate (γ) were recorded in the range of 1–1000 s⁻¹.

2.10. Oxidative stability analysis

The oxidative stability of emulsion samples placed in a thermostatic incubator (37 °C) was evaluated by measuring the peroxide value (POV) and malondialdehyde or thiobarbituric acid reactive substance (TBARS) concentration on days 0, 1, 4, 7, and 14. The trend of POV was determined using the AOCS standard procedure with sodium thiosulphate (Na₂S₂O₃) titration [24]. TBARS was determined using the method reported by Li et al., [25] with some modifications. Briefly, 1 mL of the oxidated emulsions were mixed with 2 mL of a thiobarbituric acid (TBA) solution and vortexed for 1 min. The mixtures were placed in a boiling water bath for 10 min and cooled to room temperature (∼22 °C), followed by centrifuging at 4000 g for 30 min. Then, the absorbance of the supernatant was determined at 532 nm using a spectrophotometer. The TBARS concentration was calculated using the molar extinction coefficient of malondialdehyde (1.56 × 10⁵ L/mol cm⁻¹).

2.11. Statistical analysis

Each measurement was conducted in triplicate, and the values were reported as mean ± standard deviation (SD). The Statistix 8.1 software package (Analytical Software, St Paul, MN, USA) was used to determine the significant differences between means (P < 0.05) via one-way ANOVA with Tukey’s multiple comparisons.

3. Results and discussion

3.1. Physical stability analysis

The creaming degree of emulsion samples before and after heating was quantified by plotting the overall transmittance versus time. In general, the slope of the resulting curve is negatively correlated with emulsion stability, i.e., the larger slope, the poorer the stability of emulsions [26]. As shown in Fig. 1, the slope of curves is expressed in terms of delamination rate (%/h). The emulsion stabilized by Native-MP exhibited relatively high delamination rate, indicating its poor stability. This phenomenon is related to the properties of myosin, which is the main salt-soluble protein in MP. In a low-salt environment, myosin monomers tend to pack and coil regularly through electrostatic interactions at the tails (rods) to form ordered self-assembled filamentous structures [27]. Therefore, myosin could not adsorb well around oil droplets and form complete interfacial films in the emulsion system, leading to poor emulsion stability. Upon heating, the delamination rate of Native-MP emulsion further increased (P < 0.05) due to the formation of strand-like structures induced by the interfilamental cross-linking of myosin heads [28]. Regardless, Native-MP is unsuitable as an emulsifier to stabilize O/W emulsion systems because the low physical stability can induce phase separation of Native-MP emulsion. Thus, the final products present unstable inhomogeneous undesirable conditions, which are not acceptable to consumers [29].

Fig. 1.

Effect of heating on the physical stability of emulsion samples with different pretreatment conditions (stability was expressed as the slope of the integrated transmission (%)-time (h) curve, as measured by the LUMiSizer). Uppercase (A − E) letters indicate the significant differences between emulsion samples (P < 0.05).

As shown in Fig. 1, after the addition of H₂O₂, the Native-MP + H₂O₂ emulsion stability did not change considerably before heating compared with Native-MP emulsion (P > 0.05). Interestingly, the Native-MP + H₂O₂ emulsion after heat treatment showed considerably reduced delamination rate (P < 0.05). This is because H₂O₂ irreversibly oxidized -SH to stable sulfonic acid or sulfinic acid, which could not form disulfide bonds, thereby disrupting the exchange process of sulfhydryl–disulfide bonds. Consequently, the cross-linking of myosin heads was inhibited [30]. In this case, Native-MP + H₂O₂ was adsorbed at the interface and provided physical protection for oil droplets under heating. However, the denaturation of the myosin subfragment-2 (S2) and tail domains still occurred during heat treatment, which induced the formation of MP aggregates and the instability of emulsions [20].

Fig. 1 shows that the emulsions stabilized by HIU-MP and HIU-MP + H₂O₂ exhibited excellent stability before the heat treatment. Han et al., [2] reported that HIU treatment can suppress and disassemble the formation of myosin filament by dissociating the double chain helix of myosin tails. The resulting myosin monomers were uniformly distributed in the continuous phase and adsorbed at the O/W interfaces to form a stable and dense interfacial film, which substantially improved the stability of emulsions before heating. Nevertheless, the stability of HIU-MP emulsion considerably decreased after heating (P < 0.05) and the delamination rate reached 124.87 %/h, indicating that the emulsion could not resist the aggregation behavior induced by the cross-linking of myosin heads when subjected to heating (Fig. 1). The delamination rate remained low after heating for the emulsion stabilized by HIU-MP + H₂O₂, exhibiting the highest thermal stability among the samples. Compared with HIU-MP emulsion, HIU-MP + H₂O₂ emulsion exhibited higher thermal stability after the addition of H₂O₂. This is because H₂O₂ interfered with the exchange process of sulfhydryl–disulfide bonds and thus inhibit the protein aggregation. Compared with Native-MP + H₂O₂ emulsion, the HIU treatment exposed more -SH sites trapped by H₂O₂ and more effectively inhibited the cross-linking of myosin heads during heating [17]; this confirmed the advantage of using HIU and H₂O₂ in combination. Therefore, HIU-MP + H₂O₂ was well adsorbed at the O/W interface and provided strong resistance against heat treatment.

3.2. Droplet characteristics of emulsions

3.2.1. Droplet size distribution

To further explore the potential mechanism of the combination of HIU and H₂O₂ for enhancing the thermal stability of MP emulsions, the droplet size distributions of emulsions before and after heating were determined. The droplet size distribution of emulsion samples before heating is shown in Fig. 2A. It can be seen that Native-MP emulsion showed relatively large droplet size and a slightly raised peak appeared at 100 μm due to myosin filaments at the disperse phase and O/W interfaces. Compared with Native-MP emulsion, the emulsion stabilized by HIU-MP exhibited smaller droplet size with a significantly higher and narrower peak. This phenomenon indicates that the HIU-MP was more effective to stabilize emulsions with a uniform droplet distribution (Fig. 2A). This is because HIU treatment destroyed the insoluble myosin filaments and improved the emulsifying ability of MPs [2]. After the addition of H₂O₂, the emulsion droplet size did not change considerably, suggesting that H₂O₂ did not adversely affect the emulsion.

Fig. 2.

Particle size distribution of emulsion samples with different pretreatments before (A) and after (B) heating and the appearance (C) after heating.

As depicted in Fig. 2B, the peaks of all samples shifted to the right to varying degrees upon heating. In addition, the peak width grew larger, indicating that the droplets of samples tended to be non-uniform after heating. The Native-MP emulsion shows a clear bimodal droplet size distribution, where the larger size distribution peak is attributed to the separated oil droplets, and aggregates formed by interfilamental cross-linking between myosin heads [30]. Moreover, a severe phase separation occurred in the Native-MP emulsion during heating, showing an irregular gel form (Fig. 2C). A new droplet size distribution peak at ∼ 1 μm was observed for the heated Native-MP emulsion. Fu et al., [31] noted that heating induced the unfolding of the exposed head region of myosin filaments and released a large number of cysteine residues, which subsequently caused the interfilamental cross-linking by converting the cysteine–disulfide bonds in head-head interactions. Further, continued heating caused the denaturation of the S2 region of myosin and enhanced irreversible interfilamental interactions, during which the myosin filaments became loosely structured and monomeric proteins and fragmented structures escaped [32]. Therefore, the distribution peak at ∼ 1 μm was possibly due to the free monomers and fragmented structures, indicating that the dissociation of myosin filaments and interfilamental cross-linking occurred simultaneously during heating. Similarly, the emulsion stabilized by HIU-MP shows an evident bimodal droplet size distribution after heating (Fig. 2B). As most MPs were in the form of monomers or fragmented structures at this point, the large volume distribution peak was likely related to the aggregation of these proteins and the separation of oil droplets. For the heated emulsions stabilized by Native-MP + H₂O₂ and HIU-MP + H₂O₂, the droplet size decreased significantly compared with the emulsions without H₂O₂. In addition, no obvious delamination phenomenon was observed in their appearance, particularly the HIU-MP + H₂O₂ emulsion, which was in a homogeneous fluid state. These results suggest that H₂O₂ might irreversibly oxidize -SH to form sulfinic acid or sulfonic acid. In general, sulfinic and sulfonic acids are stable and do not participate in the formation of disulfide bonds. Thus, the heat-induced aggregation behaviors of MPs were suppressed, which accordingly increased the thermal stability of emulsions [30]. HIU-MP + H₂O₂ emulsion exhibited the highest thermal stability because of the mechanical dissociation of myosin filaments by HIU treatment and the crossing-linking inhibition by H₂O₂ addition. The combined effects provided strong resistance for the emulsion system against the heat treatment [21]. The resistance to the droplet and protein aggregation is beneficial to the strong interactions between oil droplets, thereby providing the emulsion products (such as functional beverages and fluid foods) with high stability against external environment [33].

3.2.2. Zeta potential

To investigate the effects of different pretreatment conditions on the interaction between oil droplets in emulsions, the zeta potential of the emulsion samples was determined (Fig. 3). The emulsion stabilized by Native-MP exhibited low net-negative charge (−7.2 mV) before heating mainly due to the closed structure of filamentous myosin polymers covering the polar sites [34]. However, the absolute zeta potential considerably increased for the emulsion stabilized by Native-MP + H₂O₂ (P < 0.05). The irreversible oxidation of -SH groups by H₂O₂ led to the formation of sulfinic and sulfonic acids, whose deprotonation could shift the isoelectric point toward the acidic side and increase the overall charge density. Accordingly, the emulsion net charge increased after the addition of H₂O₂ [35]. The high net charge of HIU-MP emulsion is attributed to the large number of polar sites released by myosin filaments after HIU treatment. Yu et al., [36], who treated ovalbumin with HIU, reported a similar increase in zeta potential. After H₂O₂ addition, the zeta potential increased significantly (Fig. 3), indicating higher amounts of sulfonic and sulfinic acids in the emulsion system; extensive deprotonation effects increased the overall net charge strength.

Fig. 3.

Zeta potential of emulsion samples with different pretreatment conditions before and after heating. Uppercase (A − E) letters indicate the significant differences between emulsion samples (P < 0.05).

The net charge of all samples decreased to varying degrees after the heat treatment than that before the treatment, indicating the thermal aggregation of MPs (Fig. 3). Although heating could induce the unfolding of protein structures and increase the surface charge [37], excessive unfolding of proteins at higher temperatures would result in strong thermal aggregation behaviors. Subsequently, the formation of aggregates covered the polar sites, thereby decreasing the net charge [38]. For emulsion systems, the decrease in net charge implies a weakened interaction between oil droplets [39]. Therefore, emulsion systems stabilized by thermally intolerant proteins exhibit markedly unstable behavior after heating [40]. As displayed in Fig. 3, the addition of H₂O₂ to HIU-MP significantly considerably retarded the decrease in surface charge of the emulsion system upon heating (P < 0.05). However, this phenomenon was not obvious in the emulsion stabilized by Native-MP + H₂O₂ mainly because the exogenous H₂O₂ could not reach the -SH groups located inside the polymer structure. Thus, the intermolecular covalent cross-linking was unavoidable during the heating process [31].

As expected, the emulsion stabilized by HIU-MP + H₂O₂ maintained a high net charge after heating (Fig. 3), suggesting that the thermal stability of the emulsion was significantly improved using the combination of HIU and H₂O₂. As H₂O₂ trapped more -SH groups exposed by HIU, the number of S-S bonds in the heated HIU-MP + H₂O₂ was lower and most proteins remained free as monomers. This effect allowed the emulsion to maintain an intact interfacial structure after heating and exhibited high resistance to high temperatures. On the other hand, H₂O₂-mediated -SH sulfonation increased the system charge density and thus enhanced the electrostatic repulsion between oil droplets and between protein molecules [41]. Thus, the droplet aggregation and protein flocculation were inhibited, which improved the thermal stability of the emulsion.

3.2.3. Microscopic morphology of droplets in emulsions

CLSM images were captured to observe the micromorphology of emulsions stabilized by MP under different treatment conditions after heating. The red and green fluorescence in Fig. 4 represent oil droplets and MPs, respectively. According to Fig. 4A, the green fluorescence (Native-MP) appeared to be widely aggregated, indicating that extensive gelation occurred in Native-MP upon heating. In addition, the oil droplets exhibited non-uniform distribution. It can be supposed that Native-MP formed a three-dimensional gel network structure via the interfilamental cross-linking of myosin and bound most of the oil droplets within it [42]. After the addition of H₂O₂, the gel behavior of Native-MP + H₂O₂ was suppressed due to the interference of head–head interactions between MP filaments by H₂O₂, while extensive aggregation still occurred (Fig. 4B). Fig. 4C shows that gelation did not occur in the emulsion stabilized by HIU-MP after heating. Moreover, the degree of heat-induced aggregation of HIU-MP was milder than those of Native-MP and Native-MP + H₂O₂ due to the mechanical forces exerted by HIU, dissociating the aggregation structure of filamentous myosin and inhibiting its assembly in low-ionic-strength media [27]. For HIU-MP + H₂O₂ emulsion, the droplets remained uniformly distributed after heating, indicating significant improvement in its thermal stability (Fig. 4D). Although there was a tendency of aggregation, it was acceptable for a heated emulsified system. These results further confirm our above analysis, that is, HIU combined with H₂O₂ treatment can considerably enhance the heat resistance of low-ionic-MP emulsions.

Fig. 4.

CLSM image of the emulsion samples stabilized by MPs under different pretreatment conditions after heating. (A) Native-MP; (B) Native-MP + H₂O₂; (C) HIU-MP; and (D) HIU-MP + H₂O₂.

3.3. Rheological behavior analysis

3.3.1. Dynamic rheology

The viscoelasticity is an important indicator to evaluate the organoleptic quality of emulsion drink products. Therefore, it is necessary to determine the viscoelastic behavior of MP emulsions in our study [43]. The changes in the viscoelastic behavior of emulsions stabilized by Native-MP, Native-MP + H₂O₂, HIU-MP, and HIU-MP + H₂O₂ during heating were analyzed via variable-temperature rheology measurements (Fig. 5). The storage modulus (G') measured during heating is shown in Fig. 5A, which reflects the elastic behavior of the emulsion samples. It can be seen that the G' of Native-MP emulsion gradually increased upon heating, indicating an enhancing elastic behavior. A dramatic increase in G' was observed at ∼ 43 ℃, which is attributed to the interfilamental cross-linking caused by myosin head–head interactions [31]. Another increase in G' was observed at 90 ℃, suggesting that the S2 region of myosin was denatured at this point. Thus, the irreversible interactions between the molecules enhanced, causing interfilamental self-association and a pronounced elastic behavior [17]. The great increase in G' and the gelation occurrence during heating could inevitably reduce the fluidity of emulsions, thereby inducing the deterioration of sensory properties of emulsion beverages [33]. Additionally, the denaturation of MPs would decrease nutritional values of final products. Interestingly, the addition of H₂O₂ could address the issue to certain degree. As shown in Fig. 5A, the G' of Native-MP + H₂O₂ emulsion considerably reduced and did not increase until ∼ 43 ℃. This phenomenon indicates that H₂O₂ inhibited myosin the head–head covalent cross-linking, thereby suppressing the formation of strand-type aggregates. However, G' slightly increased during cooling and then remained almost constant as the temperature decreased to 25 ℃. This is possibly due to the aggregation of monomeric and fragmented structures released by the dissociated myosin filaments during heating [44]. In this case, the aggregates mainly existed as clusters and retained abilities to interact with the oil. Therefore, Native-MP + H₂O₂ emulsion still maintained its fluidity with an unobtrusive elastic behavior. The emulsion stabilized by HIU-MP exhibited a different phenomenon. As shown in Fig. 5A, the G' of HIU-MP emulsion was significantly lower than that of Native-MP emulsion because the HIU treatment disrupted the highly ordered filamentous myosin structure. This increased the emulsifying activities of MPs and decreased the elastic behavior of the emulsion system [27]. After the temperature increased to 90 ℃, G' increased sharply and then declined rapidly. Liu et al., [20] reported that although HIU treatment inhibited the formation of myosin filaments, the intermolecular cross-linking induced by disulfide bonds still occurred during heating. Therefore, herein, the interfacial MPs were subjected to thermal aggregation during the heat treatment, thereby increasing G' value. The subsequent decrease in G' may be due to the improved system fluidity resulting from the interactions between the aggregates and the oil. After the addition of H₂O₂, the G' of HIU-MP + H₂O₂ emulsion remained stable throughout the heating process, with no significant increase in any heating interval. This reveals that H₂O₂ effectively hampered the heat-induced protein aggregation, and the emulsion formed by HIU-MP + H₂O₂ maintained high stability and fluidity upon heating. Chen et al., [17] also reported that high-pressure homogenization combined with H₂O₂ suppressed the aggregation of myofibrillar protein in chicken breast and retarded the increase in G'.

Fig. 5.

Changes in storage modulus (G') (A) and loss modulus (G'') (B) of emulsion samples stabilized by MPs with different pretreatment conditions during heating.

Loss modulus (G''), also known as the viscous modulus, reflects the viscosity of materials [43]. As displayed in Fig. 5B, the G'' of all samples exhibited similar trends to G'. The G'' of Native-MP emulsion was relatively high and showed large alteration at 43 ℃ and 90 ℃, indicating that the emulsion had certain viscoelastic properties. The G'' of emulsions stabilized by Native-MP + H₂O₂, HIU-MP, and HIU-MP + H₂O₂ reduced to varying degrees compared with that of the Native-MP emulsion (Fig. 5B). These results indicate that H₂O₂ significantly inhibited the thermal aggregation behavior of MPs in the emulsion systems. Moreover, the effects were more remarkable under HIU combined with H₂O₂ treatment.

3.3.2. Apparent viscosity

The fluidity of functional drink products considerably impacts the subjective consumer preference. In addition to changes in viscoelasticity during heating, the final viscosity and flow behavior are critical to the product quality [45]. Thus, the apparent viscosities of emulsion samples stabilized by different pretreated MPs before and after heating were measured; the results are shown in Fig. 6. It can be observed that the apparent viscosity of emulsions stabilized by Native-MP and Native-MP + H₂O₂ gradually decreased as the shear rate increased, exhibiting a shear-thinning behavior [46]. As the aggregates formed by filamentous myosin were prone to cause large steric hindrance and internal friction effects, the emulsion system exhibited high initial viscosity [47]. As the shear rate increases, the connection between myosin filaments weakened, which subsequently reduced the intermolecular steric hindrance and friction effects. Accordingly, the fluidity of the emulsion system was enhanced, showing a shear-thinning behavior [9]. With the addition of H₂O₂, the apparent viscosity of emulsion decreased due to the H₂O₂-induced sulfonation of cysteines in the myosin head that loosened the myosin filamentous structures. Similar results can be found in the reports of Zhang et al., [48], who treated porcine myofibrillar proteins with H₂O₂ of different concentrations. They demonstrated that appropriate oxidation was beneficial to weaken the interaction between protein fibrillar. Interestingly, the emulsion stabilized by HIU-MP exhibited relatively low apparent viscosity with typical Newtonian fluid properties. This phenomenon is attributed to the mechanical fragmentation of myosin filaments by HIU, which significantly improved the interfacial behavior of MP in low-ionic emulsion systems and reduced the emulsion droplet size. Wang et al., [49] noted that the viscosity of emulsions is generally positively correlated with the oil droplet radius. Therefore, a decrease in the droplet size increased the fluidity of HIU-MP emulsion. After adding H₂O₂, the emulsion viscosity further decreased due to the ability of H₂O₂ in loosening the myosin filamentous structures.

Fig. 6.

Apparent viscosity of emulsion samples stabilized with different pretreated MPs before (A) and after (B) heating.

According to Fig. 6B, after heating, the apparent viscosity of all emulsions increased significantly, indicating the occurrence of heat-induced interfilamental cross-linking and protein aggregation. Notably, the heated HIU-MP + H₂O₂ emulsion had the lowest apparent viscosity, close to the apparent viscosity of unheated HIU-MP emulsion. This suggests that the MPs co-treated with HIU and H₂O₂ can form emulsions with high resistance to high temperatures, which further supports the analysis of the viscoelastic properties discussed in section 3.3.1. Although higher apparent viscosity contributed to emulsion stability according to the Stoke’s law [50], all emulsion samples in our study exhibited apparent viscosity below 0.1 Pa. s. Consequently, the influence of the apparent viscosity on the emulsion stability was negligible. In this case, it cannot be concluded that HIU-MP + H₂O₂ emulsion with lower viscosity was less stable.

3.4. Oxidative stability analysis

Oxidative stability plays a crucial role in the quality of emulsion systems [51]. Li et al., [52] reported that oxidation generally occurs in the oil phase in emulsion systems. In addition, as a pro-oxidant, H₂O₂ may induce free radical chain reactions in the presence of oils. Therefore, the oxidative stability of emulsions stabilized by H₂O₂-treated MPs was determined by monitoring the POV and TBARS value throughout the storage process.

As shown in Fig. 7, the POV and TBARS value of all samples were relatively low at day 0 and increased to different degrees as the storage time increased (P < 0.05). Compared with Native-MP emulsion, the POV and TBARS value of HIU-MP emulsion were considerably lower during storage. This is because HIU treatment disrupted the original highly ordered structures of MP and exposed the internal groups with antioxidant capacity, thereby increasing the antioxidant properties. On the other hand, the dense interfacial film formed by HIU-MPs prevented the oxidation of oil droplets; thus, HIU-MP emulsion exhibited high oxidative stability [53]. Fig. 7 shows that the POV and TBARS value of Native-MP + H₂O₂ emulsion were higher than that of Native-MP emulsion. Likewise, the POV and TBARS value of HIU-MP emulsion slightly increased after adding H₂O₂. These phenomena indicate that the introduction of H₂O₂ promoted the emulsion oxidation. Interestingly, the increase in POV and TBARS value of HIU-MP + H₂O₂ emulsion was much slighter than that of Native-MP + H₂O₂. This is because HIU treatment released more –SH groups to react with H₂O₂. Since more H₂O₂ involved in the reaction, its effects on oxidative stability of emulsions were considerably reduced. Although the addition of H₂O₂ declined the oxidative stability of emulsions, the impact was relatively small. Therefore, the combination of HIU and H₂O₂ is a feasible approach to modify MPs and improve the thermal stability of MP emulsions.

Fig. 7.

Oxidative stability of emulsion samples with different pretreatment conditions during storage.

3.5. Thermal stability mechanism of MP emulsions affected by HIU pretreatment combined with H₂O₂

Fig. 8 shows the mechanism of HIU pretreatment combined with H₂O₂ for improving the thermal stability of MP emulsions. In low-salt settings, myosin in the MPs was prone to pack and coil via electrostatic interactions in the rod region to form filamentous polymers. In this case, the MPs cannot form a firm and complete interface layer, leading to the poor stability of emulsions. After heating, the interfilamental cross-linking of the myosin heads and the hydrophobic attractions in the myosin rods induced the formation of strand-like aggregates in the disperse phase or at the O/W interface. This exacerbated the emulsion instability. After treating MPs with H₂O₂, its emulsion thermal stability was somewhat improved. Fig. 8 shows that the addition of H₂O₂ can oxidize -SH groups to sulfonic acid or sulfinic acid and then inhibit the formation of disulfide bonds. Therefore, during heating, the thermal aggregation of myosin heads was suppressed, providing the emulsion with higher thermal stability than Native-MP emulsion. However, most -SH groups were not exposed on the surface of MPs. These -SH groups would participate in the formation of disulfide bonds, rather than being captured by H₂O₂. Consequently, aggregates still appeared in Native-MP + H₂O₂ emulsion after heating. In addition, the aggregation of myosin rods occurred through hydrophobic interactions during heating. These are the reasons for the existence of aggregates in the emulsion system.

Fig. 8.

Thermal stability mechanism of MP emulsions affected by HIU pretreatment combined with H₂O₂.

HIU-MPs firmly adsorbed at the O/W interface and exhibited excellent performance before heating due to the mechanical forces that destroyed the filamentous polymers. After heat treatment, the aggregates appeared in the emulsion system due to the formation of disulfide bonds. The aggregation of HIU-MPs was relatively feeble, and the emulsion depicted stronger thermal stability compared to Native-MP emulsion. As shown in Fig. 8, any further addition of H₂O₂ based on HIU treatment could substantially enhance the emulsion thermal stability. This is attributed to the combination effect of HIU and H₂O₂, i.e., the suppression of hydrophobic interactions by HIU treatment and the interference of disulfide bond formation by H₂O₂. On the other hand, HIU treatment could lead to the exposure of more -SH groups, increasing the reaction between -SH and H₂O₂. Thus, HIU-MP + H₂O₂ adsorbed well on the oil droplet surface and inhibited the heat-induced aggregation of oil droplets, allowing the emulsion to have the highest thermal stability.

4. Conclusions

In this study, MPs co-treated with HIU and H₂O₂ were used to prepare emulsions with excellent thermal stability. The emulsion stabilized by HIU-MP + H₂O₂ depicted the highest physical stability, most uniform oil droplets, and optimal rheological properties after heating than the emulsions stabilized by native MPs and single treated MPs. The thermal stability enhanced because HIU pretreatment destroyed the filamentous myosin polymer by mechanical force and induced the release of –SH groups. On the other hand, H₂O₂ oxidized the –SH groups to stable sulfonic acid or sulfinic acid and interfered with the exchange process of sulfhydryl-disulfide bonds, thereby inhibiting the cross-linking of myosin heads during heating. Consequently, the combination effects endow the emulsion with high thermal stability. The oxidative stability analysis results indicated that the addition of H₂O₂ had little adverse effect on the improvement of lipid oxidation. In summary, the combination treatment of HIU and H₂O₂ can be a promising physicochemical-modulating method to improve the thermal stability of MP emulsions.

CRediT authorship contribution statement

Siqi Zhao: Writing – original draft, Methodology, Investigation, Conceptualization. Yubo Zhao: Software, Investigation. Haotian Liu: Writing – review & editing, Supervision, Funding acquisition. Qian Chen: Visualization, Resources. Hongbo Sun: Project administration, Methodology, Conceptualization. Baohua Kong: Writing – review & editing, Supervision, Investigation, Funding acquisition, Conceptualization.

Declaration of competing interest

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.