Improvement of physicochemical properties, microstructure and stability of lotus root starch/xanthan gum stabilized emulsion by multi-frequency power ultrasound

Highlight

• •Lotus root starch/xanthan gum nanoparticles emulsion was prepared by ultrasound.
• •Ultrasound improved the physiochemical and structural properties of the emulsion.
• •Ultrasound enhanced the adsorption of LS/XG-NP nanoparticles at oil–water interface.
• •The emulsions showed good resistance to acid, alkali and salt ions at 30 d storage.
• •The prepared emulsions can effectively inhibit the volatilization of fishy odor.

Abstract

Multi-frequency power ultrasound was applied as an environmentally friendly technique to control the nanoparticles (LS/XG-NPs) embedded with lotus root starch/xanthan gum, with the aim of enhancing the stability of Pickering emulsions. The present investigation was centered on evaluating the impact of ultrasound technology on various aspects of the emulsions, encompassing their mean particle size, particle size distribution, zeta potential, microstructure, rheological characteristics, and environmental stability. The findings of this study indicate that ultrasonic treatment enhanced the adsorption of LS/XG-NP onto oil droplets surface, resulting in a reduction in their size. Additionally, ultrasonic treatment decreased the viscosity and Brownian motion rate of the emulsion stabilized by LS/XG-NP, leading to increased fluidity. Furthermore, the emulsion's thermal stability and resistance to environmental oxidation were significantly enhanced through ultrasonic treatment. The Pickering emulsions that were prepared using ultrasound demonstrated excellent resistance to acid, alkali (pH 2–8) and salt ions (50–300 mM NaCl) for a period of 30 days during storage. It was worth anticipating that ultrasound-assisted LS/XG-NPs could efficiently retard the volatilization of fishy odor components within fish oil. Taken together, the present research has evinced the efficacy of ultrasound in enhancing the stability of Pickering emulsions coated with LS/XG-NPs. These findings offer significant novel insights into the advancement of ultrasound-assisted Pickering emulsions that are stabilized with starch-based or biopolymeric materials.

1. Introduction

Pickering emulsion is a particular type of emulsion that is fortified by solid particles, including but not limited to nanoparticles, microparticles, colloidal particles, or a blend of these particles [1]. The enhanced stability of Pickering emulsions can be attributed to the irreversible adsorption of particles at the interface of oil and water. This mechanism effectively prevents droplet aggregations, flocculation, and Ostwald ripening phenomena, thereby making them more stable than conventional emulsions. The utilization of natural macromolecules, including polysaccharides, proteins, fats, and polyphenols, for stabilising Pickering emulsions has gained significant attention in light of the green cleaning concept [2]. Moreover, these particles are relatively cheap, widely available, biodegradable and non-toxic. Exploring the potential of novel-assembly of food-grade encapsulants has become a vibrant field of research.

Starch, as a natural and abundant biological macromolecule, is widely extracted from corn, wheat, peas, potatoes, bananas, and other plants and has been aroused as a promising candidate for stabilizing emulsified systems [3]. However, due to their large particle size and high affinity for the aqueous phase, natural starch granules are not appropriate for directly stabilizing Pickering emulsions [4], [5]. It has been documented that the structure of starch granules may be modified to increase the Pickering emulsion’s stability by nanoprecipitation, acid hydrolysis, enzymatic hydrolysis, and recrystallization, among other efficient approaches ([6]. In our prior work, we have successfully developed a new series of nanoparticles composed of modified lotus root starch and xanthan gum (LS/XG-NPs) that have proven to be efficacious in stabilizing Pickering emulsions. This study revealed that utilizing the autoclave-cooling cycle method was successful in decreasing the droplet size of starch-coated nanoparticles. Additionally, the addition of XG resulted in improved wettability of the starch nanoparticles. As the combination of starch and XG was confirmed to be dependent on hydrogen bonding and electrostatic interaction, which directly determined the structure and morphology of LS/XG-NPs closely related to emulsion stability [7]. Therefore, further research needs to explore novel methods to change the intermolecular forces to construct ideal LS/XG-NPs that can more effectively stabilize Pickering emulsions.

Ultrasound is a green and eco-friendly food processing strategy that has found widespread use in the preparation of emulsions [8]. Ultrasound propagation in a liquid system results in a distinctive cavitation phenomenon that includes nucleation, bubble growth, and rupture. When bubbles break apart, they release energy that can result in various effects such as liquid jet, high pressure, and temperature [9], [10]. These physical effects can indeed have an impact on the dispersed phase droplets within emulsion systems [8]. They can lead to a reduction in droplet size, an increase in the adsorption of emulsifiers per unit area, and consequently, an enhancement in emulsion stability. However, traditional ultrasonic treatment of emulsions typically relies on a single frequency. Therefore, additional research is necessary to explore the potential benefits of other frequencies and operating modes, such as simultaneous processing. This further investigation will provide a clearer understanding of the possibilities and advantages that can be achieved in emulsion treatment. Multi-frequency power ultrasound, which combines the beneficial effects of multiple ultrasound frequencies, demonstrates significant promise in enhancing interfacial properties and improving the performance of emulsions. Additionally, it plays a crucial role in the formation of diverse interfacial structures within emulsions. Ouyang et al. [11] revealed that emulsions subjected to dual-frequency ultrasound at 28/68 kHz demonstrated remarkable improvements, including a reduction in particle size to 2.05 μm and an increase in zeta potential to 40 mV. These enhancements played a pivotal role in significantly enhancing the stability of the walnut emulsion. The findings provide clear evidence that the utilization of multi-frequency ultrasound presents notable advantages in effectively controlling conventional emulsions. Currently, there is limited research on the use of multi-frequency power ultrasound to stabilize Pickering emulsions using starch-based nanoparticles. Only a few studies have been reported, indicating that further investigation is needed to fully understand the potential of U-LS/XG-NPs [12]. Therefore, our research team hypothesized that multi-frequency power ultrasound treatment could significantly enhance the stability of produced Pickering emulsions by altering the structure and intermolecular interactions of LS/XG-NPs.

The main objective of the study was to assess the impact of multi-frequency power ultrasound treatment on the physical characteristics and microstructures of Pickering emulsions stabilized by LS/XG-NPs. Various physical properties such as particle size distribution, average droplet size, zeta potential, thermal stability, pH stability, ionic strength stability, and oxidative stability were measured to evaluate these changes. Additionally, the study aimed: (1) to investigate the rheological properties, micro-rheological behavior, and microstructural features of Pickering emulsions coated with LS/XG-NPs. (2) to stabilize fish oil using a Pickering emulsion prepared with ultrasonically-treated LS/XG-NPs (U-LS/XG-NPs). The study's findings may offer a novel perspective for designing starch nanoparticles-stabilized Pickering emulsions. Moreover, this technology offers a promising opportunity to broaden the utilization of Pickering emulsions in functional foods. The ultimate goal is to develop functional emulsions that can be labeled as health-promoting, thereby opening up new possibilities for incorporating beneficial properties into food products.

2. Materials and methods

2.1. Materials

An industrial-grade LS, boasting a starch content of 85.23 %, was procured from Fuzhou Jiaxinzheng Foods Co., Ltd. located in Fuzhou, China. The chemical compounds XG, Nile red, and Nile blue were acquired from Aladdin Biochemical Technology Co., Ltd. located in Shanghai, China. First-grade oil derived from commercially grown soybeans, was gifted by Jiusan Huikang Food Co., Ltd. (Harbin, China). All additional reagents utilized in this investigation were of analytical quality.

2.2. Fabrication of LS/XG composite nanoparticles (LS/XG-NPs)

The fabrication of LS/XG-NPs was carried out in accordance with the methodologies established in prior research [7]. Briefly, LS was weighed precisely and then mixed with distilled water at a proportion of 1:4 (starch: water, w/v). After that, the resulting suspension was then placed in a boiling water bath for 5 min while being constantly stirred to ensure that it was evenly dispersed. The LS solution was autoclaved for 20 min at 121 °C. To produce the LSNPs suspension, the starch paste that had undergone autoclaving was subjected to cooling at ambient temperature, followed by refrigeration at 4 °C for a duration of 9 h. Finally, LSNPs were mixed with an equal volume of 1.2 % XG solution (dissolved in water), and the mixture was magnetically agitated for a period of two hours to produce a suspension of LS/XG-NPs.

2.3. Preparation of ultrasonic-assisted Pickering emulsions stabilized with LS/XG-NPs (U-LS/XG-NPs emulsion)

A course emulsion was produced by homogenizing the fresh LS/XG-NPs suspension with 25 % (v/v) soybean oil in a T18 homogenizer (IKA, Germany) at 12000 rpm for 2 min. The course emulsion was treated by our team using an apparatus known as energy-gathered multi-frequency power ultrasound, which was created by our research group and produced by Five Pines Biotechnology Co., Ltd. (Jiangda Town, Zhenjiang City, Jiangsu Province, China. The ultrasonic equipment is equipped with a pair of ultrasonic probes operating at frequencies of 20 kHz and 40 kHz. It offers the flexibility to process emulsions in both single-frequency and dual-frequency synchronous operating modes. During the process, the emulsion samples are placed in a container with a jacket, which facilitates temperature control by exchanging heat with cooling water in the jacket [13]. It was worth noting that when the frequency is optimized, the power is 400 W and the time is 6 min. When optimizing power, the frequency is 20/40 kHz and the time is 6 min; when optimizing the time, the frequency is 20/40 kHz and the power is 600 W. A water bath with a constant flow of water was utilized to keep the sample's temperature at 25 °C during the ultrasonic operation. The traditional method of preparing Pickering emulsion embedded with LSNPs was modified by replacing ultrasound with an electromagnetic stirrer while keeping all other conditions constant.

2.4. Measurement of mean droplet size and zeta potential of Pickering emulsions

An apparatus called the Mastersizer 3000 (Malvern Instruments Ltd, Malvern, UK) was employed to determine the mean droplet size of created emulsions. The refractive index of the sample was 1.47, while that of the solvent water was 1.33. The Zetasizer Nano ZS90 instrument (Malvern Instruments, UK) was used to measure the zeta potential of the emulsion sample. To mitigate the occurrence of multiple scattering phenomena, the zeta potential of the samples was assessed through a process of dilution with ultrapure water at a ratio of 1:100, subsequent to the preparation of the emulsions.

2.5. Microstructure analysis

First, the emulsions' morphology was studied with the aid of an optical microscope (BM2000, Jiangnan Novel, China). A 20 μL droplet of the emulsion was deposited onto a microscope slide, which was subsequently covered with a coverslip, and the specimen was seen at a magnification of 10 × 4. The structure of Pickering emulsion was accessed using Confocal laser scanning microscopy (CLSM; Leica TCS SP5, Germany) to observe its microscopic features. A microscope slide containing 10 μL of emulsion was coated with a coverslip and examined at 25 °C using a 100 × oil immersion objective lens. Each image was captured at a scanning frequency of 200 Hz at a resolution of 1024x1024.

2.6. Rheological property of Pickering emulsion

The rheological properties of fresh developed Pickering emulsions were evaluated using a 40 mm (diameter) parallel plate rheometer with a 1 mm gap height. For the steady-state flow measurements, the sample was subjected to continuous shearing ranging from 1 to 100 s − 1 [14]. Furthermore, the flow behavior of the sample was examined using Power law Eq. (1) models.

$$\tau =K·{\gamma }^n$$ (1)

where τ is the shear stress (Pa), K is the consistency index (Pa⋅sⁿ), γ is the shear rate (s⁻¹), and n is the fluidity index.

2.7. Micro-rheological characteristics of Pickering emulsion

The micro-rheological characteristics of emulsion samples were carried out at 25℃ for 7 h by a diffusion spectrometer (Rheolaser Master, Formula, France). The viscoelastic properties of particles, along with the elastic index (EI), macroscopic viscosity index (MVI), fluidity index (FI), and solid–liquid equilibrium (SLB), were determined by measuring the displacement of the particles in the sample caused by Brownian motion.

2.8. Rapid analysis of emulsion stability

2.8.1. Emulsion thermal stability by Turbiscan^TM

The stability of emulsions with respect to creaming and sedimentation was evaluated using a Turbiscan TOWER stability analyzer manufactured by Formulaction Inc. in France. For this experiment, a 20 mL emulsion contained within a cylindrical glass bottle was subjected to scanning every 10 min for a duration of 6 h at two distinct temperatures (25 °C and 65 °C). The Turbisoft 2.0 software was utilized to compute the backscattering intensity (BS) and Turbiscan stability index (TSI).

2.8.2. pH stability

After adjusting the pH of the emulsion samples to 2, 4, 6, and 8 using 0.5 M HCl and NaOH, the emulsion was kept at 25 °C for 30 days. The macroscopic and microscopic changes of the emulsion were monitored by direct photography and microscopic optical microscopy on days 1, 7 and 30.

2.8.3. Ionic strength stability

The NaCl concentrations of the emulsion samples were adjusted to 50 mM, 100 mM, 200 mM, and 300 mM by direct addition of NaCl. The macroscopic and microscopic changes of the emulsion were monitored by direct photography and microscopic optical microscopy on days 1, 7 and 30.

2.8.4. Oxidative stability

For oxidation analysis, freshly prepared emulsion samples were placed in a glass tank that was tightly closed and kept at 25 °C for 4 weeks. Peroxide value (POV) and malondialdehyde content (MDA) were determined in order to determine the amount of lipid hydrogen peroxide and secondary oxidation products present in the emulsions, respectively. [15], [16]. Standard curves for the determination of POV and MDA were plotted using cumene hydroperoxide and 1,1,3,3-tetraethoxypropane, respectively.

2.9. Determination of volatile substances

The fish oil was used to replace the soybean oil in Section 2.3 to study the impact of ultrasound-assisted LS/XG-NPs nanoparticles on the fishy smell of fish oil. T80 stabilized fish oil Pickering emulsion was prepared by replacing LS/XG-NPs nanoparticles with an equal amount of T80 as a control.

2.9.1. Solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC/MS)

The SPME-GC/MS method was used to determine the volatile substances in the samples. The 5 mL samples were freshly prepared and subjected to equilibration at a temperature of 60 °C for a duration of 10 min within a 15 mL headspace vial. Subsequently, the specimens were subjected to adsorption at a temperature of 60 °C for a duration of 30 min, utilizing the extraction head that had undergone aging. The sample was injected with GC and then desorbed at 250 °C for 3 min. The GC conditions used were as follows: the inlet temperature was set at 250 °C, with spitless injection. The temperature program was initiated at 50 °C for a duration of 5 min, followed by an increase to 280 °C at a rate of 8 °C/min. Subsequently, the temperature was elevated to 320 °C with a heating rate of 30 °C/min and sustained at this level for a duration of 3 min. The working conditions for MS were set as follows: ion source temperature at 230 °C, interface temperature at 250 °C, electron energy at 70 eV, and scanning range from 45 to 550 m/z. The components were subjected to qualitative analysis by searching the NIST database. Only the identification results with a positive and negative matching degree greater than 80, with a maximum value of 100, were considered. The purpose of the quantitative analysis was to figure out the amount of each volatile component in the emulsion using the area normalization method.

2.9.2. Electronic nose

The volatile substances of the emulsion were detected by an electronic nose (AIRSENSE, PEN 3, Germany), and the corresponding sensing substances of each detector are shown in Table 2. After 10 mL of emulsion was placed in a sealed 30 mL glass sample bottle for 15 min, the probe was inserted to absorb air to detect volatile substances. The working parameters were set as follows: injection flow, 400 mL/min; injection time, 120 s; self-cleaning purge flow, 600 mL/min; purge time, 130 s. During the measurement process, the sensor response value gradually increased and tended to be gentle after 60 s. The 70th second signal was used as the time point for sensor signal analysis. Each sample was repeated 10 times.

Table 2.

Electronic nose sensor and its performance description.

Channels	characteristic
W1C	aromatic
W5S	broadrange
W3C	aromatic
W6S	hydrogen
W5C	arom-aliph
W1S	broad-methane
W1W	sulphur-organic
W2S	broad-alcohol
W2W	sulph-chlor
W3S	methane-aliph

2.10. Statistical analysis

Each analysis was conducted a minimum of three times. The experimental results were presented in the format of means ± standard deviations. The mean difference between groups was analyzed using Duncan's multiple range test and variance analysis. A significance level of p < 0.05 was employed. The statistical assessment of the data was conducted using SPSS 17.0 software developed by SPSS Inc. in Chicago, IL, USA.

3. Results and discussion

3.1. The droplet size and zeta potential of Pickering emulsion

Emulsion droplet size and zeta potential are powerful features that determine its physiochemical properties. The principal mechanism underlying the destabilization of starch in emulsions is the creation of a robust surface layer surrounding the droplets. The prevention of condensation is achieved by binding irreversibly to the oil–water interface. Reducing the droplet size of an emulsion makes it more difficult for the oil phase to separate from the emulsion. The zeta potential is generally employed to measure the electrical characteristics of emulsion droplets, and smaller droplet size and higher absolute zeta potential can contribute to improved emulsion stability. The data presented in Fig. 1 displays the mean droplet size and zeta potential of LS/XG-NPs stabilized emulsions, which were prepared under varying ultrasonic conditions.

Fig. 1.

Effects of ultrasonic frequency (A and B), power (C and D) and time (E and F) on the average particle size, particle size distribution and |zeta potential| of Pickering emulsions, respectively.

The mean particle size and zeta potential of LS/XG-NPs-coated emulsions equipped with ultrasonic frequencies of 20, 40, and 20/40 kHz are shown in Fig. 1A and B. The emulsion's optimal ultrasound frequency for the indexes was found to be 20/40 kHz. The application of ultrasound at this frequency led to a noteworthy reduction of 47.0 % in the average particle size, along with a 15 % rise in the zeta potential when compared to the control sample that was not subjected to ultrasound treatment. According to the emulsion particle size distribution results, it was observed that the maximum particle size reduced and the particle size distribution became narrower due to the application of ultrasound treatment, particularly the 20/40 kHz dual-frequency ultrasound. As a result, the emulsion's average particle size was reduced. Thus, it can be figured out that dual-frequency synchronous ultrasonic treatment is more beneficial for stabilizing the emulsion of LS/XG-NPs compared to single-frequency ultrasonic treatment. In Ouyang's research [11], it was noted that the application of multi-frequency ultrasonic treatment led to a decrease in particle size and an elevation in zeta potential within the emulsion. These combined effects ultimately resulted in a significant improvement in the emulsion's stability. Numerous studies have also found that dual-frequency acoustic chemical reactors are more cost-efficient than single-frequency ones. This could be attributed to the instantaneous power intensity of the system, the acoustic flow phenomenon, and the interaction between frequency modes. [17], [18], [19]. When compared to single frequency operation, double frequency operation increases the cavitation activity in the reactor, leading to a higher overall cavitation yield. This is due to the presence of more cavities and stronger interactions between bubble–bubble and bubble-sound fields, which can cause droplets to decompose into smaller, uniformly sized droplets in the emulsion [19]. Moreover, the interaction between two frequencies can result in mutual interference or superposition. This interaction can lead to increased resonance with various substances in the emulsion system [20]. This resonance can be advantageous in breaking down the structure of the LS/XG-NPs and increasing the electrostatic repulsion among the nanoparticles. This might eventually make the emulsion more stable.

Fig. 1C and D demonstrate the impact of varying ultrasound power (200, 400, 600, 800, and 1000 W) on the particle size and zeta potential of LS/XG-NPs stabilized emulsions. As the ultrasonic power increases, the average particle size of the emulsion gradually decreases. The reason for this can be attributed to the rise in bubble collapse pressure, active volume of cavitation, and lifespan. The findings obtained from this study were in accordance with previous research indicating that ultrasonic treatment can decrease the droplet size of protein-stabilized emulsions [21]. As the ultrasonic power was increased, the zeta potential of the emulsion initially increased but then decreased, which was different from the trend observed in particle size. Our findings depicted that the ultrasound treatment dynamically changed the electrostatic repulsion on the surface of nanoparticles, which might be attributed to the modification of nanoparticles structure by ultrasound. In fact, it has been proven that ultrasound can alter the morphology and attributes of starch. This is achieved through the generation of strong shear force, free radicals, and high temperature in a localized area. The extent of this change is dependent on various factors such as ultrasonic power, frequency, temperature, and other conditions [22]. Recently, Saleh et al. (2017) reported that specific conditions of ultrasound could degrade the xanthan gum polymer into smaller segments, thereby facilitating the exposure of the action bond and contributing to changes in zeta potential [23]. In order to maintain stability against electrostatic aggregation, it is necessary for particles to possess a zeta potential absolute value exceeding 30. The influence of particle size and zeta potential on the stability of emulsion was considered, and an ultrasonic power of 600 W was selected as the optimal condition. The resulting emulsion had a particle size of 12.03 ± 0.45 μm and a zeta potential of 32.58 ± 0.33 mV, which showed a decrease of 47 % and an increase of 26 %, respectively, compared to the control.

The droplet size and zeta potential of LS/XG-NPs stabilized emulsions subjected to ultrasound treatment for 0–10 min are depicted in Fig. 1E and F. The impact of ultrasonic duration on the emulsion's particle size and zeta potential was comparable to that of ultrasonic intensity. Short-term ultrasound treatment remarkably improved (p < 0.05) the zeta potential of the emulsion, with a maximum value of 35.38 ± 0.62 mV observed at 4 min. Increasing the duration of ultrasonic treatment to 6–10 min led to a noteworthy decrease in the zeta potential. Nevertheless, the observed decrease failed to attain statistical significance (p ≥ 0.05) relative to the control group. The findings indicate that the zeta potential of emulsions is influenced by ultrasound duration, likely due to alterations in nanoparticle structure and intermolecular force resulting from the ultrasound [24]. It is possible that the application of ultrasound treatment for a very short period could induce structural relaxation in LS/XG-NPs, thereby increasing the exposure of intermolecular bonds and promoting intermolecular electrostatic repulsion and hydrophobic forces. With an increase in ultrasound exposure time, there is a possibility that the modified intermolecular forces could lead to the aggregation of nanoparticles, ultimately causing a reduction in the zeta potential. The most suitable ultrasonic parameters were established through a systematic optimization process, resulting in the selection of an ultrasonic frequency of 20/40 kHz, power of 600 W, and a duration of 4 min. The present study further investigated the mechanism of enhancing emulsion stability through ultrasonic treatment under the specified conditions.

3.2. Microstructure observation

LS/XG-NPs stabilized emulsion's morphology and microstructure were examined using optical and CLSM microscopes for exploring the impact of ultrasound treatment, as illustrated in Fig. 2. The images of sonicated emulsions were captured using microscopic techniques, which revealed that the droplets were evenly distributed throughout the continuous phase. The distribution of nanoparticles and oil droplets was observed via CLSM, utilizing fluorescent dyes Nile blue A (red) and Nile red (green) for visualization purposes. Compared with the LS/XG-NPs emulsion (Fig. 2A, C), the droplet size of the U-LS/XG-NPs emulsion (Fig. 2B, D) was noteworthy reduced. Additionally, the oil droplets were observed to be more uniformly dispersed throughout the continuous phase. According to the study's findings, applying ultrasonic treatment significantly reduced the Pickering emulsion's particle size and made it easier for droplets to be distributed more uniformly. The observed outcome is consistent with the findings presented in Fig. 1 with respect to the influence of ultrasound on the particle size of the emulsion. The impact of nanoparticle distribution at the interface on the stability of the emulsion interface layer is closely associated with the stability of the Pickering emulsion [25].

Fig. 2.

Optical microscopy images and CLSM images of crude Pickering emulsion stabilized by LS/XG-NPs (A, C, E, G) and ultrasonic emulsified Pickering emulsion stabilized by LS/XG-NPs (B, D, F, H).

The results depicted in Fig. 2E and G clearly indicate that some LS/XG-NPs could be attached to the droplets and form the ring structure for the Pickering emulsion, confirming the ability of LS/XG-NPs to stabilize the emulsion to some extent. After ultrasonic treatment, the color of the red ring deepened and the number of rings also increased, as portrayed in Fig. 2F. This suggests that ultrasonic treatment enhanced the interfacial adsorption capability of LS/XG-NPs, leading to a greater number of LS/XG-NPs being adsorbed onto the interface between oil and water, consequently resulting in the formation of a compact interfacial film. Simultaneously, it can be observed that a framework for networks consisting of LS/XG-NPs was formed in the continuous phase to hinder the amalgamation and agglomeration of oil droplets (as shown in Fig. 2H). It is therefore apparent that the ultrasonic treatment facilitated the entrapment of oil droplets more efficiently within the gel network of LS/XG-NPs. On the one hand, one possible explanation for the observed rise in adsorbed nanoparticles is the occurrence of cavitation induced by ultrasonic treatment, which could have led to a reduction in droplet size and an increment in droplet surface area [26]. Conversely, ultrasonic treatment might cause changes in the structure and molecular spatial arrangement of LS/XG-NPs. Additionally, the reduced size of nanoparticles facilitates their rapid dispersion towards the exterior of oil droplets, thereby augmenting the quantity of adsorbed nanoparticles.

3.3. Rheological property of Pickering emulsion

The rheological characteristics of ultrasonically-stabilized LS/XG-NPs emulsions can potentially be evaluated through steady shear rheological experiments. The Power Law model is capable of accurately representing the flow curve of each emulsion within a specific range of shear rate (R² > 0.98) [27]. In line of this, Table 1 presents the Power Law rheological model parameters of the prepared emulsion. It was observed that all ultrasonically-equipped emulsions exhibited non-Newtonian pseudoplastic properties, with a flow behavior index (n) of less than 1. The results shown in Fig. 3A and B demonstrate a gradual reduction in both the apparent viscosity and shear stress with an increase in shear rate. The utilization of ultrasound therapy led to a decrease in the apparent viscosity of the emulsion that was stabilized by LS/XG-NPs. For instance, Abker and his colleagues have exhibited that the implementation of high intensity ultrasonic treatment led to a decrease in viscosity within egg white protein emulsion. The observed outcome was attained via a reduction in particle dimensions, alteration of microstructure, and modulation of interfacial tensions [21]. Similarly, the results of another study indicate that the emulsion subjected to ultrasonic treatment demonstrated a statistically significant reduction in the consistency coefficient (K) value and a consequent rise in the flow behavior index (n) value, when compared to the control group. These findings propose that the use of ultrasound treatment enhanced the fluidity of the LS/XG-NPs stabilized emulsion. The direct correlation between the K value and n value of an emulsion determines the relationship between starch molecules and the viscosity of the continuous phase [27]. The results of CLSM in Section 3.2 showed ultrasound enhanced connection between nanoparticles. Therefore, it could be speculated that the main contribution to the above results comes from the reduction of the viscosity of the XG caused by ultrasound. Recently, Wang and his co-workers also reported the reduced viscosity of the soy protein isolate-pectin emulsion to the fact that ultrasound affects the viscosity by reducing the molecular weight of the pectin [25].

Table 1.

Rheological parameters of Pickering emulsions.

Samples	n	K	R2
Control	0.207 ± 0.005	8.882 ± 0.167	0.989
UT	0.314 ± 0.004	3.213 ± 0.048	0.997

*Different lowercase letters in the same index indicate significant differences between groups (p < 0.05).

Fig. 3.

Viscosity (A) and shear stress (B) of Pickering emulsions as a function of shear rate, MSD of crude Pickering emulsion (C) and ultrasonic emulsified Pickering emulsion (D) stabilized by LS/XG-NPs, EI (E), FI (F), MVI (G) and SLB (H) of Pickering emulsions.

3.4. Micro-rheological properties of the Pickering emulsion

3.4.1. Mean square displacement (MSD)

Micro-rheology is a commonly employed rheological technique that enables the investigation of the microrheological characteristics of emulsions and facilitates the anticipation of their physical stability [28]. The method utilized in this study is based on the Diffusing Wave Spectroscopy (DWS) theory, which involves the conversion of the velocity of Brownian motion of the dispersed phase into an MSD curve. This approach enables the acquisition of microscopic viscoelasticity information of the sample [29]. The micro-rheological properties of Pickering emulsions emulsified with starch-based nanoparticles have not been extensively studied. Fig. 3C and D depict the decorrelation time curves of the Pickering emulsions based on LS/XG-NPs that were made using various emulsification techniques at different times. The Pickering emulsions displayed viscoelastic behavior and typical shear thinning behavior, as evidenced by their nonlinear curves. The present observation aligns with the results depicted in Fig. 3A, indicating that the perceived viscosity exhibited fluctuations upon increasing of the shear rate. The MSD curve was divided into three regions, a linear region on the left, a platform region in the middle, and a linear region on the right. The curve of the linear region on the right reflected the viscosity of the sample, and the slope was positively correlated with the viscosity. The platform region of the MSD curve partially reflected the elasticity of nanoparticles. The lower the MSD value of the platform, the stronger the elasticity of the interfacial film on the particle surface; conversely, the weaker the elasticity of the particles. The curves of the linear regions of the emulsions stabilized by the two samples were similar. In comparison to the control group, it was observed that the MSD value of the platform region of the red curve of the U-LS/XG-NPs stabilized emulsion presented a declining trend which suggests a reduction in the viscosity of the emulsion that was prepared through ultrasonic treatment. Yue and his coworkers obtained comparable outcomes, anticipating that ultrasound treatment decreased the viscosity of Pickering emulsion that was stabilized by chitosan self-assembled nanoparticles [33]. Through the extraction of pertinent data from the MSD diagram of the emulsion, it is possible to derive the EI, MVI, FI, and SLB values, which provide a more comprehensive understanding of the emulsion's viscosity and elasticity.

3.4.2. The EI, MVI, FI and SLB

The EI can be defined as the reciprocal of the plateau value of the mean squared displacement [30]. It could be seen from the Fig. 3E that at the beginning of the test, the EI value of LS/XG-NPs stabilized emulsion was found to be higher as compared to U-LS/XG-NPs stabilized emulsion, which mean that the strong interaction between the original LS/XG-NPs particles was broken by ultrasound, and the mutual collision between the particles was reduced, resulting in a lower elasticity of U-LS/XG-NPs stabilized emulsion. The macroscopic viscosity of an emulsion can be characterized by calculating the MVI, which is derived from the slope of the final segment of the MSD [31]. The calculation of FI is based on the characteristic time of particle movement and it has a negative correlation with MVI. Fig. 3F and G demonstrate that the MVI value of the LS/XG-NPs-coated Pickering emulsion was greater compared to the U-LS/XG-NPs stabilized emulsion. Conversely, the FI value was lower for the LS/XG-NPs stabilized emulsion compared to the U-LS/XG-NPs stabilized emulsion.

The findings indicate that the droplet velocity was enhanced and the time taken for droplet displacement was reduced when subjected to ultrasonic treatment, as opposed to the conventional emulsion. It was assumed that, ultrasonic treatment appears to have a restricting impact on Brownian motion. This effect may lead to some degree of fracture in the XG network, resulting in increased fluidity and decreased viscosity of the emulsion [32]. The ratio of the sample's solid-to-liquid behavior, represented by the slope of the MSD platform, can be used to calculate the solid–liquid equilibrium. The measurement of the SLB involves the determination of the slope of the MSD plateau, which is indicative of the proportion of solid-like and liquid-like characteristics exhibited by the sample. According to the data, an SLB value of 0.5 signifies a balanced ratio of liquid and solid constituents. When the SLB value is less than 0.5, it indicates that the droplet movement is primarily influenced by solid behavior, which is commonly referred to as gel behavior. On the other hand, SLB values between 0.5 and 1 indicate a higher proportion of liquid or viscous behavior. Fig. 3H showed the spectrum of the SLB value of the two samples. The observations suggest that the initial SLB values of all samples were observed to be higher than 0.5. As the storage time increased, the SLB value exhibited a gradual decline and eventually stabilized below 0.5. This phenomenon is indicative of a transformation in the emulsion's rheological properties from a more fluidic nature to a more solid-like behavior within a short time. Careful observation showed that the SLB value of LS/XG-NPs stabilized emulsion were higher than that of U-LS/XG-NPs stabilized emulsion, indicating the liquid behavior of emulsions obtained by ultrasonic treatment was more pronounced. In conclusion, the application of ultrasound has the potential to decrease the viscosity of emulsions and enhance their fluidity, which could be attributed to the stronger effect of ultrasound on the network structure within the emulsion [33].

3.5. Assessment of emulsion stability

3.5.1. Thermal stability

The Turbiscan stability analyzer was utilized to rapidly assess the Pickering emulsion's thermal stability. By utilizing the principle of multiple light scattering, it is possible to obtain the variation of BS (ΔBS) of an emulsion by continuously scanning the sample. The measurement has the potential to yield significant insights into the directional patterns of the particles and anticipate the emulsion's thermal stability. The measurement time and height of the sample cell are important factors that can change the physio-chemical characteristics of Pickering emulsion [34]. Fig. 4 depicts the impact of ultrasonic treatment on the durability of the Pickering emulsion under varying temperatures of 25 °C and 65 °C. Since 25 °C and 65 °C are the commonly used room temperature and pasteurization temperature respectively, the selection of these two temperatures could reflect the stability of the emulsion in the storage and sterilization environment to a certain extent. The horizontal coordinate, left longitudinal axis and right longitudinal axis represent the height of the sample, the variation in the percentage of BS relative to the initial stage and the test scanning time, respectively. It could be observed that an increase in the duration of measurement led to a minor reduction in the intensity of ΔBS at the lower part of the emulsion in the control group (Fig. 4A), indicating that the bottom of the emulsion was slightly clarified at 25 °C. While the ΔBS intensity at the bottom of the emulsion in the ultrasonic treatment group was almost unchanged (Fig. 4C). For the emulsion with a height of 2 mm-40 mm, the ΔBS intensity almost stabilized with time, indicating that the middle layer of the two emulsions showed good stability. It was worth noting that the floating of dispersed particles and the stratification at the top of the emulsion were confirmed by the rise in droplet concentration at the emulsion's surface, which tracked the rising trend of ΔBS. Following ultrasound treatment, the magnitude of ΔBS at the apex of the emulsion experienced a significant reduction from 15 % to below 5 %. The observed phenomena suggest that ultrasonic treatment has a notable impact on the migration rate of LS/XG-NPs, resulting in improved emulsion stability at a temperature of 25 °C. Evidently, the ΔBS curves depicted in Fig. 4B and D display an obvious resemblance to those in Fig. 4A and C. Over time, the ΔBS curve of the emulsion exhibited a discernible degree of deviation. However, it is noteworthy that this variation was more pronounced at 65 °C as opposed to 25 °C, thereby implying that lower temperatures are more conducive to maintaining the stability of the emulsion. The reason for this phenomenon may be that high viscosity at low temperatures limited the migration and movement of particle size, and high temperatures might increase the possibility of collisions of oil molecules, thus accelerating the destruction process [35]. Compared with Fig. 4B, the shape of ΔBS curve at the top of the emulsion in Fig. 4D changed, and the strength of ΔBS decreased, indicating that application of ultrasonic waves could enhance the emulsions stability at 65 °C. Careful comparison showed that the influence of ultrasonic treatment on the top ΔBS curve of the emulsion stored at 65 °C was less than that at 25 °C. This suggests that ultrasonic treatment is more favorable for enhancing the stability of the emulsion at lower temperatures.

Fig. 4.

ΔBS of crude Pickering emulsion (A), ultrasonic emulsified Pickering emulsion (C) and TSI of Pickering emulsions (E) at 25℃; ΔBS of crude Pickering emulsion (B), ultrasonic emulsified Pickering emulsion (D) and TSI of Pickering emulsions (F) at 65℃.

The Turbiscan stability analyzer can provide an intuitive indicator called TSI, which measures the Pickering emulsion’s stability. As the TSI value increases, the emulsion becomes more unstable. Fig. 4E and F demonstrate that the emulsion assisted via ultrasonic treatment at 25 °C and 65 °C had higher TSI values compared to the control group. Moreover, the difference between the two increased with the storage time. The results confirmed that the emulsion prepared through ultrasonic treatment exhibited stability during storage. The influence of ultrasound emulsification on the TSI was in line with that of ΔBS. Yue and his colleagues (2022) have documented that ultrasonic treatment can decelerate the migration rate of constituents and enhance the storage stability of emulsions [33].

3.5.2. pH and ionic strength stability

Environmental stability is a prerequisite for the application of many emulsions in various fields. Fig. 5A and B depict the impact of pH and ionic strength stability on the visual appearance and optical microscopy images of LS/XG-NPs stabilized emulsions at varying storage times (1, 7, and 30 days). After a storage period of 30 days, it was observed that all emulsions remained stable and showed no significant changes in their appearance. This indicates that LS/XG-NPs are highly effective as emulsion stabilizers and possess a certain level of resistance towards acid, alkali (pH 2–8), and salt ions (50–300 mM of NaCl). For example, it has been observed that Xanthan gum with the appropriate viscosity can successfully stop the aggregation of oil droplets and the demulsification of emulsions. This property is beneficial for creating a stable and uniform emulsion system [36]. The good thickening properties of XG are attributed to its long-chain polymer properties, which allow it to easily dissolve in cold water at a pH range of 3–8.5. According to Xuran and his colleagues, while acidic and neutral pH levels may alter the conformation of XG, the modified XG chain has a low tendency to generate hydrogen bonds with water molecules. As a result, any changes in the XG chain's conformation have minimal impact on surface tension [27]. Therefore, the coordination between XG/LS-NPs could enhance the stability of the emulsion over a prolonged period within the pH range of 2–8. The loading of NaCl can alter the interaction among polysaccharide molecules, leading to changes in the droplet size and zeta potential of the nanoparticles. However, certain polysaccharides can withstand the flocculation precipitation caused by a specific concentration of NaCl. The emulsion, which had 1 % enzymatically hydrolyzed Enteromorpha prolifra polysaccharide added to it, demonstrated stability within the range of 0–400 mM NaCl [37]. Cheng et al also reported that XG-stabilized emulsions remained fairly stable over the entire test NaCl concentration range (0–200 mM) [40]. The optical microscopic image of the LS/XG-NPs emulsion provides further confirmation of its exceptional stability against acid-base and salt ions. It could be seen that the droplets were evenly distributed, there was a certain space between the droplets, and there was almost no agglomeration. While the overall stability of the emulsions did not exhibit significant variation in comparison to their visual appearance, microscopic analysis revealed that the droplets in the ultrasonic treatment group exhibited a reduction in size and a more uniform distribution instead of control sample. The outcome was in accordance with the reduction in droplet size and the augmentation of zeta potential instigated by ultrasound, as demonstrated in Fig. 1.

Fig. 5.

Macro and micro-monitoring of Pickering emulsions at different pH (A) and ionic strength (B) during storage for 30 days; dynamic values of POV and MDA of Pickering emulsion (C).

3.5.3. Oxidative stability

The principal product (POV) and secondary product (MDA) of lipid oxidation during natural oxidation were measured in order to explore the impact of ultrasound on the oxidation stability of the Pickering emulsion stabilized by LS/XG-NPs. The POV and MDA values of the freshly made U-LS/XG-NPs stabilized emulsion were greater than those of the freshly made LS/XG-NPs stabilized emulsion, as shown in Fig. 5C. It was attributed to the chemical effects of ultrasound, which could result in free radical production to encourage oil oxidation during ultrasonic propagation in liquid media. According to a study, ultrasonic treatment enhanced the peroxide value of emulsified skim milk powder, XG, monoglyceride, and olive oil from 9.17 meq O₂/kg oil to 11.50 meq O₂/kg oil [12]. The level of autoxidation in the oil emulsion exhibited a positive correlation with the duration of storage. This effect was particularly noticeable in the LS/XG-NPs stabilized emulsion, which had a significantly higher rate of autoxidation compared to the U-LS/XG-NPs stabilized emulsion. After 30 days of autoxidation, the POV and MDA values of LS/XG-NPs and U-LS/XG-NPs emulsions increased by 178.75 and 40.41 μmol/Kg oil, and 110.70 and 13.03 μmol/Kg oil, respectively. The obtained result suggests that the application of ultrasound treatment is effective in delaying the auto-oxidation of the emulsion while it is being stored. The emulsions stabilized by LS/XG-NPs have an antioxidant mechanism wherein the LS/XG-NPs act as an effective emulsifier by adsorbing and attaching themselves to the oil–water interface. This inhibits the oil droplets from adsorbing pro-oxidants present in the aqueous continuous phase. The more composite nanoparticles adsorbed on the interface, the denser the interface layer formed, the better the phase separation between the oil phase and the continuous phase, which helps to improve the oxidation resistance [39]. Therefore, it was speculated that ultrasonic treatment increased the adsorption of LS/XG-NPs on the interface and enlarged the thickness of the interface layer. The above speculation was confirmed by CLSM image analysis as depicted in Fig. 2.

3.6. Volatile component analysis

3.6.1. SPME-GC/MS

The consumption of fish oil, abundant in unsaturated fatty acids, is known to possess significant nutritional and health-promoting benefits. However, the fishy smell of fish oil seriously limits its acceptance by consumers and affects the application of fish oil products and the development of the industry. The results of volatile components of emulsion samples and fish oil detected by SPME-GC/MS are shown in Fig. 6A. The main volatile components in natural fish oil were 13 aldehydes, 13 ketones, 8 alcohols, 25 hydrocarbons, 42 esters, and 8 other substances; the total content of aldehydes and ketones accounted for 19.14 %. In fact, fish oil itself contains a large amount of aldehydes, and ketones formed by lipid oxidation are affected by the experimental environment; these two substances are one of the main sources of fishy smell, and the olfactory threshold is low in the total volatiles [40]. Compared with the original fish oil, the flavor components of T80 and U-LS/XG-NPs stabilized fish oil changed to varying degrees; the total content of aldehydes and ketones accounted for 19.54 % and 14.87 %, respectively. The above results showed that U-LS/XG-NPs could effectively encapsulate aldehydes and ketones in fish oil and inhibit fishy smells to a certain extent.

Fig. 6.

Percentage of volatile substances in emulsions (A), PCA of electronic nose sensor data of emulsions(B), Electronic nose radar profiles of emulsions(C).

3.6.2. Electronic nose

Electronic nose can use electrochemical sensing systems to analyze, identify, and detect odor and volatile substances [41]. By using multiple sensors sensitive to different odors to detect volatile substances and convert them into data signals, the odor analysis of the sample to be tested is realized. The analysis results of the electronic nose on fish oil samples are portrayed in the Fig. 6B and C. The data for natural fish oil, T80, and U-LS/XG-NPs stabilized fish oil were collected by electronic nose sensors and made into a radar map. It could be seen from the Fig. 6B that different sensors showed different signal intensities in response to flavor substances in natural fish oil. The response values of natural fish oil on W5S, W1W and W2W sensors were larger than those on other sensors. Since these three sensors were sensitive to nitrogen oxides, inorganic sulfides, and sulfur-containing organics, the odor detected by these three sensors was the characteristic fishy smell of fish oil [42]. Compared with the original fish oil, the response values of the above three sensors to T80 and U-LS/XG-NPs stabilized fish oil were significantly reduced, and the order was T80 emulsion > U-LS/XG-NPs emulsion, indicating that the strategy of preparing emulsion could reduce the fishy smell of fish oil, and U-LS/XG-NPs emulsion has the better effect on inhibiting the volatilization of fishy smell. Principal component analysis, also referred to as PCA, is a statistical technique that reduces the dimensionality of data and turns numerous variables into two or three key variables [43]. The PCA analysis results of the data information obtained by all sensors of the electronic nose showed two-dimensional scatter points that could form clusters, indicating that the detection results of the electronic nose are stable and reliable. As mentioned in Fig. 6C, the total contribution rate of the first principal component (88.7 %) plus the second principal component (8.8 %) was 97.5 %, far more than 85 %, indicating that these two principal components could well describe the main characteristics of the sample flavor [44]. Based on the PCA findings, it could be inferred that the first principal component should be the characteristic fishy odor of fish oil. The order on the first principal component is natural fish oil < T80 emulsion < U-LS/XG-NPs emulsion, indicating that U-LS/XG-NPs have superior embedding performance for fishy oil odor. The results of the electronic nose of fish oil Pickering emulsions and the results of their SPME-GC/MS were mutually confirmed.

4. Conclusion

The utilization of ultrasonic emulsification as an environmentally friendly processing strategy has the potential to yield significant advancements in the improvement of the physical characteristics and microstructures of LS/XG-NP Pickering emulsions. The present work exhibited that ultrasonic parameters significantly affected the particle size distribution, mean droplet size and zeta potential of LS/XG-NP embedded emulsions. The utilization of CLSM and optical microscopy revealed that the application of ultrasound resulted in a decrease in the dimensions of oil droplets and an increment in the adsorption of LS/XG-NP onto the oil droplet surface. The emulsion stabilized by LS/XG-NPs displayed characteristics of non-Newtonian pseudoplasticity, viscoelasticity, and the expected shear thinning behavior. Ultrasonic treatment decreased the viscosity of LS/XG-NP stabilized emulsion, reduced the Brownian motion rate and increased the fluidity, which was demonstrated via rheological and microscopic rheological properties analysis. In addition, ultrasonic treatment could improve the thermal stability of the emulsion, and its effect on the emulsion at 25 °C was better than that at 65 °C. After 30 days of self-oxidation, the increased POV and MDA values of Pickering emulsion prepared by ultrasound were significantly lower than that prepared by traditional conditions, and the Pickering emulsion prepared by ultrasound had stronger resistance to self-oxidation during storage. It was worth mentioning that the results of SPME-GC/MS and electronic nose showed that U-LS/XG-NPs can retard the volatilization of fishy odor components in fish oil to some extent. This study successfully applied ultrasonic technology to the Pickering emulsion stabilized by starch-based/polysaccharide composites, which provided a new idea for the development of Pickering emulsion with excellent stability. Additionally, this technology has the potential to broaden the application of Pickering emulsions in functional foods, offering more preparation possibilities for various complex emulsion systems.

Declarations of interest

There are no conflicts of interest to declare. This article does not contain any studies involving human or animal subjects.

CRediT authorship contribution statement

Qiufang Liang: Investigation, Conceptualization, Writing – review & editing, Funding acquisition. Chengwei Zhou: Methodology, Formal analysis, Writing – original draft, Data curation. Abdur Rehman: Formal analysis, Writing – review & editing. Abdul Qayum: Formal analysis, Writing – review & editing. Yuxuan Liu: Data curation, Formal analysis. Xiaofeng Ren: Supervision, Conceptualization, Investigation, Writing – review & editing, 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

Data will be made available on request.