A novel strategy for improving the stability of myofibrillar protein emulsions at low ionic strength using high-intensity ultrasound combined with non-enzymatic glycation

Graphical abstract

Highlights

• •Glycation of MPs with DX was accelerated by HIU pretreatment.
• •Conjugates pretreated by HIU possessed the higher adsorption at oil–water interfaces.
• •HIU pretreatment combined with glycation enhanced the emulsion stability.
• •Higher electrostatic repulsion and smaller size enhanced the emulsion stability.

Abstract

Poor emulsification of myofibrillar proteins (MPs) limits the production of meat protein emulsion-type products, and it is related to the myosin self-assembles in low-salt settings. The effect of high-intensity ultrasound (HIU) pretreatment combined with non-enzymatic glycation on MP-stabilized emulsions in low-salt settings was investigated in this study, and the potential mechanism was revealed. The results indicated that, compared to using either HIU or glycation treatment alone, HIU pretreatment in combination with glycation significantly improves the physical stability of emulsions while increasing the distribution uniformity and reducing the droplet particle size from 18.05 μm to 2.54 μm (P < 0.05). Correspondingly, the emulsion prepared using this approach exhibited a relatively high absolute zeta potential (−23.58 mV) and a high interfacial protein content (38.78 %) (P < 0.05), promoting molecular rearrangement and forming a continuous and stable interfacial layer. HIU pretreatment combined with glycation could offer reinforced electrostatic repulsion and steric hindrance to depolymerize self-assembled filamentous polymers, thus enhancing the stability of droplets. Additionally, the thermal sensitivity of the glycated MPs pretreated by HIU was remarkably reduced, thus improving the thermal stability of the corresponding emulsions.

1. Introduction

Oil-in-water (O/W) emulsions are heterogeneous systems consisted of oil phase dispersed in the water phase [1]. It is frequently used in the food industry for items including creams, beverages, and emulsified meat products [2]. However, O/W emulsions tend to undergo phase separate over time owing to its immiscibility in both phases, which eventually causes a decline in emulsifying performance and emulsion stability [3]. Recently, meat researchers have shown considerable interest in the development of protein-stabilized emulsions because of their potential application in novel functional formula foods, such as the delivery of high nutritional and bioactive ingredients into food matrices [4]. As amphiphilic macromolecules, proteins can adhere to the oil-droplet surface and lower interfacial tension to form a uniformly dispersed O/W emulsion system [5]. Some natural proteins, especially milk and soy proteins, have been well used as potent emulsifiers in food processing [4].

Myofibrillar proteins (MPs) are the major protein components in muscle, containing premium nutrients and essential amino acids, with excellent digestion and absorption capacity [6]. Furthermore, the excellent surface activity of MPs makes suitable emulsifiers for stabilizing emulsions [7]. However, despite these virtues, the development of natural meat proteins in emulsion systems is strictly restricted owing to the poor aqueous stability of MPs in low-salt settings. MPs that are salt-soluble can dissolve well in high-salt solutions (0.47–0.68 M NaCl), which is not ideal for a healthy promoting diet [6]. Studies have reported that the low solubility of MPs in aqueous conditions will further prevent the formation of interfacial protein films, limiting its application in low-salt meat protein-based emulsion-type products [8]. It is thus essential to explore methods of enhancing the stability of emulsions stabilize by MPs in low-salt settings and develop novel meat protein-based O/W beverages.

Currently, non-enzymatic glycation is an effective strategy to improving the emulsifying properties of protein, which is performed under mild and safe environments without foreign chemicals. [9]. Non-enzymatic glycation under aqueous conditions has the outstanding advantage of short heating time and is suitable for heat-sensitive muscle proteins [10]. Glycation refers to the covalent binding between the carbonyl group of reducing sugar and free amino group, mainly ε-amino group of the lysine and arginine in proteins [11]. Protein–polysaccharide conjugates can efficiently enhance the emulsifying properties of emulsions by strongly adsorbing and arranging at the interface through the hydrophobic protein groups and then extending into the water phase through the hydrophilic polysaccharide groups [12]. Hydrophilic polysaccharides could be grafted into oil droplets to enhance electrostatic repulsion and steric stabilization forces, maintaining the subsequent stability of emulsions [10]. Droplets with thick interfacial protein layers are also more stable to environmental stresses, including pH, temperature, and ionic strength variations [13].

High-intensity ultrasound (HIU) is an effective physical approach used to alter the structure characteristics of proteins, particularly plant proteins [14], [15], egg proteins [16], [17], and dairy proteins [18]. Liu et al., [2] revealed that HIU treatment could dissociate myosin filaments by unwinding the superhelix of myosin rods. The obtained myosin oligomer, subunit, or monomer could evenly disperse and rapidly cover the oil droplets without the limitations of myosin self-assembly. Moreover, our previous research established that the partial unfolding of protein structures caused by HIU pretreatment could expose the internal reactive groups and glycation sites, facilitating subsequent glycation reactions [19]. This work unlocked an effective method for producing dissolved and stably dispersed MP aqueous solutions and also provided more ideas for improving the functional characteristics of MPs in low-salt settings. Therefore, studies on the emulsification performance of MP conjugates pretreated with HIU are worth exploring.

Based on our previous work and the actual needs of protein–polysaccharide conjugates in formulated foods, the aim of this work is to investigate the effect of HIU pretreatment and glycation on low-salt emulsion stabilized using porcine MPs. The property improvement of the MP-stabilized emulsions using this combined treatment was investigated systemically. The findings will help guide the formulation of new muscle protein-based emulsion-type products with excellent physical and thermal stability.

2. Materials and methods

2.1. Materials

Longissimus muscle from pork meat and soybean oil (Jinlongyu) were obtained from a supermarket in Harbin (Heilongjiang, China). Dextran (DX, 70 kDa) was obtained from Yuanye Biotechnology Co., Ltd. (Shanghai, China). All other chemicals were of analytical grade.

2.2. Preparation of MPs

The MPs were extracted using a previously reported method [20]. The extracted protein was suspended in 50 mM sodium phosphate buffer (pH 7.5), and the final protein concentration was adjusted to 20 mg/ml. The protein content was calculated by the Biuret method.

2.3. HIU pretreatment combined with glycation

MP-DX conjugate was prepared according to our previously reported method [19]. The MP suspension (20 mg/mL) was transferred into beakers and treated using Scientz-II D ultrasound generator with a 6.0 mm diameter probe (Scientz Biotechnology Co., Ltd., Ningbo, China). The volume of the MP suspension in reaction vessel was 100 mL. The parameters of the ultrasonic treatment were ultrasonic power 400 W (20 kHz), duration 15 min, and pulse mode (5 s on and 3 s off). The actual ultrasonic power into the medium was 4.65 W and the ultrasonic intensity was 16.45 W/cm², as measured by a calorimetric method according to Jambrak et al., [21]. Throughout the entire procedure, the beakers were immersed in iced water. After HIU treatment, the MPs (20 mg/mL) and DX (20 mg/mL) were stirred and blended in 5.0 mM sodium phosphate buffer. Subsequently, the mixtures were continuously shaken while reacting at 37 °C for 8 h. The reaction was instantly stopped by cooling in an ice-water bath. To remove the unreacted DX, the resulting glycated samples were dialyzed (dialysis membrane: 70 kDa molecular weight cut-off) at 4 °C for 24 h with 50 mM sodium phosphate buffer (pH 7.5). The native (untreated) MP suspensions were labeled “MP”, the HIU-treated MP samples without glycation were labeled “UMP”, the glycated MP samples without HIU pretreatment were labeled “MP-DX”, and the glycated MP samples pretreated using HIU were labeled “UMP-DX”.

2.4. Amino acid composition

The samples were hydrolyzed in hydrochloric acid (6 M), blown with nitrogen, and stored in an oven for 22 h at 110 °C. After complete hydrolysis, the samples were transferred and diluted in 50 mL of ultrapure water, after which 1 mL of the sample was withdrawn and lyophilized at 40–50 °C. The amino acid contents were then determined by the L-8800 automatic amino acid analyzer (Tokyo, Japan) [22].

2.5. Emulsion preparation

Each of the aqueous MP, UMP, MP-DX, and UMP-DX suspensions (45 mL) was combined with 5 mL of soybean oil before being pre-emulsified at 10,000 rpm for 3 min with an Ultra Turrax T20 homogenizer (IKA Labortechnik, Staufen, Germany). A 5-minute break was taken after every two cycles of the high-pressure homogenizer (Mini DeBee, USA), which homogenized each coarse emulsion twice at 60 MPa [2]. As an antibacterial agent, sodium azide (0.02 % w/v) was used in the MP emulsions.

2.6. Backscattering (BS) light and turbiscan stability index (TSI)

A Turbiscan Tower instrument (Turbiscan LAB Expert, Formulation, France) was used for determining the BS and TSI [23]. The emulsion samples (25 mL; 10 mg/mL) were scanned using near-infrared light (880 nm) at 25 °C for 24 h, during which the macroscopic BS fingerprint pattern and the TSI were obtained.

2.7. Droplet size and zeta potential

Zeta potential, particle size and distribution of MP emulsions were evaluated by a laser light scattering instrument (Microtrac S3500, Florida, USA) and a Malvern Zetasizer instrument (Nano-ZS90, UK), as described by Xu et al., [9]. Each sample was injected onto a quartz colorimetric dish after being diluted to 0.1 mg/mL with 5.0 mM of phosphate buffer for analysis.

2.8. Super-resolution microscopy (SRM)

The microgram of samples was observed using SRM (Deltavision OMX SR, General Electric Company, US). Briefly, 25 μL of Nile blue (0.1 %, w/v) and 25 μL of Nile red (0.1 %, w/v) were mixed with the MP emulsions (1.0 mL) and left in the dark for 20 min. Thereafter, the stained samples were then put into a microscope slide at a volume of 5 L for observation. The protein phase's excitation wavelength was adjusted to 640 nm.

2.9. Cryogenic scanning electron microscopy (Cryo-SEM)

The microgram of the different emulsions (10 mg/mL) was assessed using a cryo-SEM (SU8100, Hitachi, Japan) as reported by Cao et al., [24]. The emulsions were sublimated at 90 °C for 10 min after being frozen in liquid nitrogen. Each sample was then platinum-coated and placed in a freezer for observation.

2.10. Interfacial protein characteristics

The percentage and composition of adsorbed and non-adsorbed proteins were determined using centrifugation (10,000 g; 30 min; 4 °C), as described by Jiang et al., [25]. After centrifuging the different emulsions, a syringe was used to obtain the aqueous solution to determine the content of non-adsorbed proteins. Thereafter, the differences between the protein content in the initial emulsion in the water layer was used to determine the adsorbed protein content.

To examine the non-adsorbed protein composition, the aqueous phase was subjected to SDS–PAGE, while the upper emulsified layer was re-dissolved in sodium phosphate buffer (5.0 mM) and centrifuged at 10,000 g for 30 min at 4 ℃. The aforementioned steps were replicated three times, and the upper emulsions were filtered using Whatman #1 filter paper to obtain more purified protein samples. Subsequently, the proteins were dissolved with sodium phosphate buffer (5.0 mM) and subjected to SDS–PAGE to examine the adsorbed protein composition. A gradient gel of 4 %–20 % Bis–Tris was used for SDS–PAGE, as described in previous study [20].

2.11. Dynamic rheology measurement

The rheological behavior tests of all the samples were conducted by a MARS 40 rheometer (Thermo Scientific, USA) fitted with a parallel plate (40 mm diameter), as described by Du et al., [26]. The viscoelastic properties of the emulsions were evaluated using temperature sweeps. The temperature sweeps were performed at a 1.0 % strain and 0.1 Hz frequency. Each sample was heated from 25 °C to 80 °C at a rate of 2 °C /min and subsequently cooled to 25 °C at a rate of 4 °C/min.

2.12. Confocal laser scanning microscopy (CLSM)

The morphology of the MP emulsions with different treatments were investigated by CLSM (Zeiss, LSM710, Germany), according to our previously described method [27]. Briefly, 25 μL of Nile blue (0.1 %, w/v) and 25 μL of Nile red (0.1 %, w/v) were mixed with the MP emulsions (1.0 mL) and vortexed for 2 min before incubating for 20 min in the dark. Subsequently, each stained sample (5 μL) was placed on a 1.5 mm-thick microscope slide with a cover glass. The CLSM microgram was collected by a 40 × magnification objective.

2.13. Statistical analysis

Each measurement weas conducted in triplicate, and the data were reported as mean values ± standard deviations (SD). One-way ANOVA was performed to analyze the data using the Statistix 8.1 software program (Analytical Software, USA). The Tukey multiple comparison test was used to identify the mean differences at the P < 0.05 level.

3. Results and discussion

3.1. Analysis of amino acids

As shown in Table 1, the relative lysine and arginine content in the native MPs and UMP samples were significantly higher than those of the MP-DX and UMP-DX conjugates (P < 0.05), confirming that the MPs were successfully conjugated with DX. Notably, UMP-DX consumed more lysine and arginine than MP-DX (P < 0.05), indicating that HIU pretreatment promoted subsequent glycation and improved grafting of DX. This is in agreement with the grafting degree results measured in our previous work [19]. This could be due to the protein structure, which tended to unfold under the cavitation effect of ultrasound, exposing more free amino groups that are conducive to the subsequent glycation reactions [28].

Table 1.

Amino acid composition (%) of MPs treated with different treatments.

Amino acid	MP	UMP	MP-DX	UMP-DX
Asp	9.77 ± 1.13	9.86 ± 0.94	9.90 ± 1.56	10.08 ± 0.89
Thr	5.31 ± 0.56	5.26 ± 0.83	5.33 ± 0.43	5.97 ± 0.67
Ser	3.76 ± 0.36	3.81 ± 0.41	4.16 ± 0.75	4.12 ± 0.64
Glu	16.61 ± 0.93	16.66 ± 0.26	17.26 ± 0.22	17.64 ± 0.18
Gly	3.06 ± 0.34	3.07 ± 0.56	3.19 ± 0.47	3.15 ± 0.73
Ala	5.75 ± 0.25	5.89 ± 0.63	6.03 ± 0.47	6.08 ± 0.56
Cys	1.49 ± 0.33	1.78 ± 0.38	1.00 ± 0.26	0.91 ± 0.45
Val	4.86 ± 0.45	4.98 ± 0.31	5.13 ± 0.76	5.25 ± 0.59
Met	2.28 ± 0.78	2.57 ± 0.41	2.65 ± 0.36	2.76 ± 0.47
IIe	4.85 ± 0.31	4.86 ± 0.68	5.2 ± 0.33	5.19 ± 0.62
Leu	9.36 ± 0.35	9.52 ± 0.57	9.61 ± 0.41	9.83 ± 0.43
Tyr	3.65 ± 0.17	3.76 ± 0.34	3.95 ± 0.23	3.98 ± 0.42
Phe	4.40 ± 0.43	4.09 ± 0.62	4.69 ± 0.69	4.96 ± 0.35
Lys	10.36 ± 0.47a	10.57 ± 0.64a	8.95 ± 0.53b	7.77 ± 0.41c
His	3.00 ± 0.53	2.86 ± 0.48	2.83 ± 0.67	2.88 ± 0.39
Arg	7.51 ± 0.68a	7.63 ± 0.47a	6.35 ± 0.43b	5.61 ± 0.31c
Pro	4.61 ± 0.39	3.56 ± 0.71	4.16 ± 0.45	4.04 ± 0.96

Note: Different lowercase letters mean significant difference in the same row (P < 0.05).

3.2. Multiple light scattering

The BS variation (ΔBS) reflects conditions of clarification, flocculation/ coalescence, and creaming/precipitation of emulsions during storage [29]. Fig. 1a–d shows the 24-hour ΔBS results of the MP emulsions. For the four examined emulsions, during the scan, the ΔBS decreased at the bottom of the sample (left of the graph) and increased at the top (right), yielding negative and positive peaks, respectively. This observation indicated that the creaming layer at the top and the serum phase at the bottom appeared as a result of droplet migration and aggregation [3]. Furthermore, with the increase of the phase separation position, the scanning line are shifted, and the distance among each line gradually becomes shorter.

Fig. 1.

Changes in the backscattering light intensity (a) and TSI (b) of emulsions prepared using MPs following different treatments at low ionic strength.

Ultrasonicated emulsions had a reduced transversal range of negative peaks, when they are compared to the native MP emulsions. This means that the serum layer was reduced, indicating an improvement in the physical stability of the UMP emulsions. Additionally, the UMP emulsions had a narrower spacing among vertical lines than native MP emulsions, especially at the bottom of the bottle, suggesting that the phase separation rate was decreased. It is plausible that HIU may improve the physical stability by suppressing and disassembling myosin filament formation in water, thereby releasing myosin monomers and their dimers and oligomers while improving the interfacial adsorption ability [2]. Similarly, the addition of DX further reduced the serum height and droplet migration velocity, with slowest the creaming velocity and the lowest serum layer observed in the UMP-DX emulsions. The hydrophilicity of the MP conjugates was significantly improved because of the addition of more hydroxyl groups into MP molecules [13]. Therefore, the hydrophilic polysaccharides extended into the water phase, and the hydrophobic region of the proteins extended into the oil phase. This facilitates the formation of a continuous interfacial film of MP-DX conjugates on the oil droplet surfaces as well as the reduction of the interfacial tension. Additionally, complex long DX chains entangled at the oil–water interfaces may restrict the migrations of droplets, thereby inhibiting the phase separation of emulsions [30]. It is worth noting that UMP-DX emulsions possessed better physical stability than the other emulsions, suggesting a positive synergy between HIU and glycation.

The above conclusion was further corroborated by the TSI results (Fig. 1e), which were negatively correlated to the physical stability of the emulsions [31]. As expected, the native MP emulsions have the highest TSI values, followed by the UMP, MP-DX, and UMP-DX emulsions. This indicated that the UMP-DX emulsions had the highest physical stability, followed by MP-DX and UMP, while the native MP emulsions had the lowest stability.

3.3. Droplet size distribution and droplet size

As showen in Fig. 2, all fresh emulsions showed a homogeneous white appearance across the samples, and the fresh emulsions stabilized using native MPs had a wider droplet distribution (Fig. 2a–d) and a larger droplet size (Fig. 2e). During the MP treatment with either HIU or glycation, the droplet distribution peak of the emulsions was narrow, and the droplet size significantly decreased (P < 0.05), indicating an improvement in the uniformity of the formed emulsion particles. For the UMP-DX emulsions, the droplet size was significantly decreased, and the droplet dispersion was the most to the left and more uniform than other samples (P < 0.05). These results suggest that the strong mechanical effects generated by HIU caused the filamentous polymer to depolymerize into small fragments, resulting in a small droplet size in the emulsions and promoting adsorption of MPs on the oil droplet surface [2]. Moreover, the MP conjugates had a flexible conformation, which facilitated the rapid adsorption of MPs on the surface of oil droplets [32]. HIU pretreatment promoted covalent grafting between DX and the MPs and enhanced the hydrophilicity of the glycoconjugates by introducing more hydroxyl groups (Table 1), which, together with the hydrophobic groups, enhanced the amphiphilicity of the glycated MPs, thus forming a stable emulsion system with high distribution uniformity and small droplet size [13].

Fig. 2.

Droplet size distribution of the emulsions prepared using native MP (a), UMP (b), MP-DX (c), and UMP-DX (d). (The insets represent typical appearance photographs for 0 d and 7 d). Average droplet size (e) before (green) and after (orange) storage at 4 °C for 7 d. The different lowercase and uppercase letters denote the significant difference (P < 0.05). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

After 7 d of storage, the native MPs, UMP, and MP-DX emulsions exhibited obvious stratification, while the UMP-DX emulsions exhibited a relatively uniform white appearance. The distribution curves of the emulsion was clearly shifted to the right after 7 d of storage, indicating the convergence of the emulsion droplets and the increasing of droplet size, especially in the native MPs and UMP emulsions. Notablely, the non-glycated MP samples showed higher numbers of particles from 100 to 700 μm, while the MP-DX conjugates showed relatively minor size particle increases in the 30 to 250 μm region. The result further confirmed that glycation stabilized the MP emulsions, making them resistant to flocculation and coalescence. The UMP-DX emulsions (Fig. 2d) showed no new peaks of large particle sizes after storage; moreover, the distance between the droplet distribution peak and the change in average particle size before and after storage decreased, suggesting that the emulsion stabilized using the HIU and glycation-treated MPs had better storage stability. This finding is consistent with that of a previous work [13], which reported that the increase in the grafting degree of glycation contributed to better emulsion stability. Droplet coalescence and flocculation in the UMP-DX emulsions were possibly inhibited by higher steric repulsions between the MP molecules as a result of the introduction of larger amounts of DX as molecular spacers on its surface [33].

3.4. Microscopic morphology of droplets

The green and red fluorescence in the SRM images represent the oil droplet and MPs, respectively. As exhibited in Fig. 3, the actual distribution of MP emulsions was consistent with the aforementioned droplet size result (Fig. 2e). The emulsion stabilized using native MPs exhibited a larger size than other emulsions, and the emulsion droplets were aggregated, further confirming the instability of the emulsions. This might be due to the tendency of the interfacial proteins to assemble into fibrous aggregates at low ionic strength [2]. As a result, few proteins adsorbed on the oil droplet surface of the native MP emulsions, and adjacent droplets fused (Fig. 3).

Fig. 3.

SRM micrographs of the emulsions prepared using native MP, UMP, MP-DX, and UMP-DX. Green color represents oil phase, and red color represents the distribution of proteins. Select typical areas to observe the coating of interface proteins. The scale bar corresponds to 15 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Notably, the droplet size reduced along with more uniform droplet distribution in MP emulsions treated with HIU or glycation alone, and the smallest droplet size with the most uniform size distribution was shown in the UMP-DX emulsions (Fig. 3). Furthermore, the typical region images showed that the aggregated state gradually dissipated and MPs were uniformly wrapped around the oil droplets of the UMP-DX emulsions, forming a red fluorescent ring. These results suggested that HIU pretreatment disrupted the filamentous polymerization structure, thereby promoting effective adsorption of proteins at the interface and reducing droplet aggregation. Additionally, the HIU pretreatment could expose more amino groups and glycation sites buried inside the protein molecules, facilitating subsequent glycation reactions [19]. In this case, the grafting of additional macromolecule DX contributed to form a dense barrier at the oil–water interfaces, thereby maintaining the separation distance between the droplets. Moreover, the enhanced steric repulsion was expected to resist Van der Waals gravity among the droplets [34].

Furthermore, the distribution characteristics of the emulsion droplets were acquired using Cryo-SEM. As shown in Fig. 4a, the native MPs were present in the form of self-assembled filamentous polymers, and a few particles adsorbed at the interface. A possible explanation is that the rigidly structured myosin filaments could not be covered quickly and accurately at the oil droplet surfaces. After HIU treatment, the protein aggregates disintegrated into numerous small units, dispersed in the continuous phase, and covered on oil droplet surface (Fig. 4b). However, the interfacial films of the UMP emulsions were not continuous, and the oil droplets were not completely wrapped. Obvious coalescence of adjacent droplets was showed in Fig. 4b, which is related to the discontinuous interfacial films [35]. Notably, the emulsion droplets stabilized using conjugates showed obvious network structure around them (Fig. 4c and d), especially in the UMP-DX emulsions. The polysaccharide chains tightly adhered to the film surface and randomly linked together with the polysaccharide chains adsorbed on the adjacent droplet surface to form a thick and dense three-dimensional (3D) network structure. Therefore, it is reasonable to speculate that the dense network layer might strengthen the stabilization of the interfacial film, acting as a spatial barrier to inhibit the flocculation and convergence of adjacent droplets during storage. He et al., [36] also reported that the stabilization of emulsions was achieved via a 3D network formed by the intramolecular interactions between sugar chains and the interfacial protein.

Fig. 4.

Cryo-SEM images and schematic diagrams of the emulsions prepared using MPs following different treatments at low ionic strength. Pictures a-d are emulsions stabilized by native MP (a), UMP (b), MP-DX (c), and UMP-DX (d), respectively. The scale bar corresponds to 3 μm.

3.5. Zeta potential

Electrostatic repulsion is tightly related to the stability and aggregation of emulsion droplets [37]. As exhibited in Fig. 5a, the zeta potential of all emulsions was negative, indicating the presence of more amino acids with negative charges on the protein surface [13]. Notably, the net negative charge of emulsions stabilized using MPs treated using either HIU or glycation alone increased significantly compared to that of the native MP emulsions (P < 0.05). This result could be due to the exposure of the internal polar groups caused by the depolymerization of filamentous polymers and the expansion of the MP structures via HIU or glycation treatment [19], [20]. The increased net negative charge generally represents the enhanced electrostatic repulsion and the increased separation between emulsion droplets [2].

Fig. 5.

The zeta-potential (a), percentages of adsorbed proteins and non-adsorbed proteins (b), and the SDS-PAGE profiles (c) of the emulsions prepared using MPs following different treatments. The different lowercase and uppercase letters denote the significant difference (P < 0.05). Lanes: M, markers; 1–4 and 5–8 indicate adsorbed proteins and non-adsorbed proteins of emulsion stabilized using native MP, UMP, MP-DX, and UMP-DX, respectively. MHC: myosin heavy chain; MLC: myosin light chain. The letters A–D and a–d indicate the significant differences (P < 0.05) in the non-adsorbed proteins and adsorbed proteins.

The UMP-DX emulsions exhibited the highest charge value (−23.58 mV), and this is in agreatment with the date on physical stability (Fig. 1) and droplet size (Fig. 2e). This may be due to the fact that HIU pretreatment exposed more specific amino groups, which facilitated the consumption of positively charged lysine and arginine in subsequent glycation reaction (Table 1) as well as the grafting of negatively charged hydroxyl groups. Therefore, the reinforced electrostatic repulsion between the emulsion droplets prepared with UMP-DX was expected to improve the uniformity and stability of droplet dispersion and limit unstable behaviors during storage.

3.6. Interfacial protein characteristics

The stability of the emulsions is tightly related to the interfacial protein ratio [38]. As exhibited in Fig. 5b, the level of adsorbed proteins in the emulsion stabilized using MPs treated with either HIU or glycation alone was significantly higher compared to the native MP emulsions (P < 0.05), and the level of adsorbed proteins was highest in UMP-DX emulsions (P < 0.05). Additionally, the level of non-adsorbed proteins showed an opposite changing pattern in the water phase.

The small particle size of protein treated by HIU and glycation, which increased the likelihood of contact among the MPs and oil droplets during homogenization, is responsible for the increase in the amount of adsorbed protein [39]. This was also supported by the study of Li et al., [40], which reported that the decrease in the particle size of chicken MPs increased the adsorbed protein ratio in the emulsion. It is reasonable to conclude that the proteins with small particle sizes could anchor more quickly and easily on the oil droplet surfaces to increase the thickness and density of the interfacial protein film (Fig. 2). This implies that the coalescence barrier was strengthened, making droplets more difficult to aggregate, and ultimately improving the stability of the emulsions (Fig. 1).

SDS–PAGE was used to further identify the composition of the adsorbed and non-adsorbed proteins. As exhibited in Fig. 5c, the adsorbed proteins were composed of myosin heavy chain (MHC), actin, and three faint light chain bands (MLC 1, MLC 2, and MLC 3), indicating that MHC and actin were the primary proteins involved in the creation of the interfacial film. The native MP emulsions were found to have a relatively low band intensity of adsorbed proteins, further confirming their weak adsorption ability [2]. It is worth noting that a portion of actin was still adsorbed on oil droplets in the control sample. Hegarty et al., [41] reported that actin possessed an excellent adsorption capacity on the oil droplet surfaces at low ionic strength. Similar result was also found by Ma et al., [42], who proposed that the main component of interfacial proteins was cod actin. In this study, however, we found that both MHC and actin played significant roles in stabilizing the emulsions. Notably, the HIU and glycation treatments promoted the adsorption of MHC and actin in the cream layer (Fig. 5c). As expected, the highest expression level of adsorbed proteins bands was observed in the UMP-DX emulsions, and this is in agreement with the date of adsorbed protein content (Fig. 5b). These results illustrate that HIU combined with glycation treatment facilitated MP adsorption on the oil–water interface.

3.7. Thermal stability of the emulsions

3.7.1. Confocal laser scanning microscopy, particle size, and zeta potential

Emulsion-based products should have good thermal sterilization stability to maintain the quality during hot processing. To further understand how heating affects the stability of MP emulsions, freshly prepared emulsions were heated at 80 °C for 30 min. As shown in Fig. 6a, the emulsion had a homogeneous white appearance before heat treatment. Upon heating, the emulsions prepared with the MP and UMP underwent flocculation, indicating thermal aggregation. In contrast, the conjugate-stabilized emulsions, particularly the UMP-DX emulsions, had fewer changes after heating, with no significant delamination. Additionally, the native MP emulsions significantly aggregated after heating (Fig. 6b) probably because of the weak electrostatic repulsion (Fig. 6c), resulting in a marked increase in droplet size (Fig. 2e and 6d). In general, the protein denaturation induced by high temperature could affect the interface protein distribution [43]. Interface protein denaturation occurred during heating, increasing protein–protein interactions and droplet aggregation caused by disulfide connections between interface molecules [4]. Notably, the UMP-DX emulsions maintained good dispersibility after heating, confirming their excellent thermal stability (Fig. 6b). This behavior is attributable to the following: (i) strong electrostatic repulsion between droplets, which keeps them separate (Fig. 6c), as evidenced by the smaller droplet size (Fig. 6d); (ii) the grafted hydrophilic DX adsorbs in the aqueous phase, weakening the interactions among adsorbed and non-absorbed proteins, thereby inhibiting heat-induced aggregation [44]; (iii) a thick interface layer formed by the conjugated MP, which reduces the sensitivity of the MPs to heat treatment and contributes to the thermal stability of the oil droplets [45].

Fig. 6.

The visual observations (a), CLSM micrographs (b), zeta-potential (c), and average droplet size (d) of the emulsions prepared using MPs following different treatments upon heating. The different uppercase letters denote the significant differences (P < 0.05).

3.7.2. Rheological properties

Temperature rheological scanning was used to further assess the susceptibility of the MPs emulsions to temperature. The changes in the storage modulus (G′) of each emulsion showed different changing trends during the temperature increase from 25 °C to 80 °C (Fig. 7). The native MP emulsions displayed a typical rheological transition that sharply increased from 41 °C to a peak at approximately 53 °C. Thereafter, the G′ sharply decreased to a minimum value at 64 °C, followed by a continuous increase to the endpoint. The first increase in G′ was attributable to the myosin head cross-linking, while the subsequent drop and increase were probably caused by denaturation and unfolding of the myosin LMM and association of LMM through tail-to-tail interactions, respectively [46]. See (Fig. 8).

Fig. 7.

Changes in the storage modulus (G′) of the emulsions prepared using MPs following different treatments under temperature sweep mode.

Fig. 8.

Schematic illustration of the mechanism of HIU pretreatment combined with glycation to promote MP emulsion stability.

The G′ value of the UMP and MP-DX emulsions significantly decreased compared to the native MP emulsions (Fig. 7), suggesting that the intermolecular cross-linking of the MPs in the emulsions was hampered. Moreover, the MP-DX emulsions achieved typical peak at a relatively high temperature. This implied that a large amount of DX grafted by glycation reduced the thermal sensitivity of adsorbed proteins, protecting the MPs from unfolding at lower temperatures [47]. In turn, this would result in less denaturation of the adsorbed proteins after heating, improving the thermal stability of MP emulsions. Hrynets et al., [33] reported that glycation treatment increased thermodynamic stability by shifting the maximum gelation temperatures of natural actomyosin to higher values. For the UMP-DX emulsions, the transition peak was essentially lost, and the G′ value remained at a low level during the heating and cooling process. These results indicate that higher steric repulsions between protein molecules caused by the grafting of additional DX may have prevented the denaturation and aggregation of adsorbed proteins, enhancing the stability of the interfacial protein film [33]. Therefore, the UMP-DX emulsions can resist thermal stress in emulsion-based products.

3.8. Stability mechanism of MP emulsions affected by high-intensity ultrasound pretreatment combined with glycation

In low-salt settings, myosin in the MPs tended to self assemble to form filamentous polymers [48]. This specific self-assembly behavior made it difficult to persistently adsorb at the oil–water interfaces after emulsification, leading to the fusion of adjacent droplets (Fig. 3, Fig. 4a). Upon heating, protein denaturation caused by high temperature could increase the protein–protein interactions and droplet aggregation (Fig. 6b). HIU treatment results in unfolding of MPs and exposure of the internal polar groups on the MP surface (Fig. 5a). The strong electrostatic repulsion can disintegrate the filamentous polymers into myosin monomers or subunits, promoting the formation of the interfacial films. However, the interfacial films of the UMP emulsions were not continuous, which resulted in the coalescence of adjacent droplets (Fig. 3, Fig. 4b). Moreover, the interfacial proteins of the UMP emulsions exhibited an aggregation behavior after heating, causing heat instability in the UMP emulsions (Fig. 6b). For the UMP-DX emulsions, the reinforced electrostatic repulsion among droplets can improve the uniformity and stability of emulsion droplet dispersion (Fig. 5a). Additionally, grafting more macromolecular DX can provide greater steric resistance, thereby preventing subsequent aggregation of interfacial proteins (Fig. 4d). Therefore, UMP-DX conjugate adsorbed well on the oil droplet surface and formed thicker and denser interfacial films. The thick interface layer can reduce the sensitivity of the MPs to heat treatment and inhibit heat-induced aggregation of oil droplets (Fig. 6b and 7).

4. Conclusions

In this study, the HIU combined with glycation treatment was proven to effectively improve the physical stability of the emulsions at low ionic strength, as evidenced by the reduced droplet size, uniform droplet distribution, and enhanced inter-droplet interactions. Moreover, the emulsions prepared with this combination had a lower tendency to phase separate during the storage period. The excellent stability of UMP-DX emulsions was attributed to its high protein adsorption on the surface of oil droplets, and its high steric hindrance against flocculation and coalescence. Additionally, the UMP-DX-stabilized emulsions exhibited noticeable stability against coalescence under heating. In conclusion, these findings have important implications for the development of low-salt meat protein emulsion-type products.

CRediT authorship contribution statement

Ge Han: Data curation, Writing – original draft. Siqi Zhao: Formal analysis, Investigation. Fangda Sun: Data curation. Xiufang Xia: Software, Resources. Haotian Liu: Conceptualization, Visualization, Supervision. Baohua Kong: Conceptualization, Writing – review & editing, Methodology, Funding acquisition.

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.

Data availability

The data that has been used is confidential.