Effect of ultrasonic treatment on rheological and emulsifying properties of sugar beet pectin

Abstract The effects of ultrasonic treatment on rheological and emulsifying properties of sugar beet pectin were studied. Results indicated that intrinsic viscosity ([η]) and viscosity average molecular weight ([M v]) decreased with the increased time from 0 to 30 min but increased when the duration prolonged to 45 min. The change of apparent viscosity with shear rate of all pectin solutions could be well described by Sisko model (R 2 ≥ .996) and the infinite‐rate viscosity (η ∞) and the consistency coefficient (k s) values decreased after ultrasonic treatment. Ultrasonic treatment could have an effect on dynamic moduli and activation energy of sugar beet pectin solutions. Particle size of pectin emulsions decreased and absolute zeta potential increased with increased time from 0 to 20 min. Excessive ultrasonic duration (30 and 45 min) could result in the aggregation of oil droplets in pectin emulsion and decrease in emulsifying stability. It could be concluded that ultrasonic treatment could affect the rheological and emulsifying properties of sugar beet pectin. The results have important implications for understanding the ultrasonic modification of sugar beet pectin.

as excessive time consumption, environmental pollutions, expensive cost, and complex procedures (Zhang, Zhang, Liu, Ding, & Ye, 2015). Alternative sustainable techniques should be applied in the modification of pectin.
Ultrasound is the applied science and technology of sound waves with frequency above human hearing ability ranged from 20 kHz to 10 MHz (Ma, Yang, Zhao, & Guo, 2018;Sattar et al., 2019). During processing, ultrasonication can create localized high temperature and pressure spots which could be affected by factors, such as ultrasound frequency, power intensity, temperature, and treatment time . As an emerging and green technology, ultrasound could be used for extraction and modification of products in food industry with relatively easy, cheap, and energy saving (Awad, Moharram, Shaltout, Asker, & Youssef, 2012). Recently, ultrasound is a promising alternative method to apply in assisted extraction of pectin from different sources compared with conventional extraction process (Bayar et al., 2017;Chen, Fu, & Luo, 2015;Maran & Priya, 2015). High efficiency of ultrasonic assisted extraction could contribute to achieving in less processing time, lower extraction temperature, and reduced energy consumption. Meanwhile, ultrasonic treatment can influence the physicochemical property, antioxidant activity, and structure of pectin in an aqueous system. It has been demonstrated that ultrasound decreased average molecular weight, changed the methylation degree, and degraded the neutral sugar side chains of citrus pectin (Zhang, Ye, Xue, et al., 2013).

Intermolecular and intramolecular hydrogen bonds of citrus pectin
were destructed during ultrasonic processing (Qiu, Cai, Wang, & Yan, 2019). Ultrasound also changed the rheological property and structure of apple pectin, which suggested that ultrasonic processing could be a feasible alternative method for pectin modification (Zhang, Ye, Ding, et al., 2013). Emulsifying capacities of citrus pectin had been significantly improved by ultrasound treatment (Wang et al., 2020). Modified pectin showed improved properties after ultrasonic processing. However, there has been no research on influence of ultrasonic treatment on sugar beet pectin characteristics.
In the current study, the effects of ultrasonic treatment on the rheological and emulsifying properties of sugar beet pectin under different time (0-45 min) were investigated. Rheological properties were characterized by viscosity, molecular weight, and dynamic moduli. Emulsifying properties of sugar beet pectin emulsions were measured via particle size, zeta potential, emulsifying activity, and physical stability. In addition, confocal laser scanning microscope images were also obtained to get a better understanding of ultrasonic effect on sugar beet pectin.

| Ultrasonic treatment
The stock solution of sugar beet pectin (20.0 g/L) was obtained by dissolving 2.0 g pectin in volume of 100 ml deionized water under constant stirring at ambient temperature for 12 hr. Ultrasonic treatment was conducted by a JY92-IIN Ultrasonic Homogenizer (Ningbo Scientz Biotechnology Co.) equipped with a 10 mm (diameter) probe.
The probe diameter, operating frequency, and output power were 10 mm, 20 kHz, and 650 W (1%-99%), respectively. The pectin solutions in 150 ml glass beaker were placed in the noise isolating chamber, and the ultrasonic probe was installed at the fixed depth of 20 mm below the liquid surface. The pectin solutions were then sonicated for 0, 5, 10, 20, 30, and 45 min (2 s on and 1 s off period) at the power ratio of 99% and then placed in refrigerator 4°C for further analyses.

| Rheological property of ultrasonic sugar beet pectin
Rheological property of different pectin solutions (20.0 g/L) was determined by an AR 2000 ex rheometer (TA instruments). The aluminum cone plate geometry with 1° angle, 40 mm diameter, and 27 μm gap was used and each step was performed separately.

| Apparent viscosity measurement
The apparent viscosity of sugar beet pectin was determined using the steady-state flow step with the shear rate ranged from 0.1 to 100 s −1 at 25°C. The apparent viscosity (η) as a function of shear rate (γ) can be fitted by the Sisko model (Mothé & Rao, 1999): where η ∞ is the infinite-rate viscosity (Pa·s), k s is the consistency coefficient of Sisko model (Pa·s n ), and n is the flow behavior index (dimensionless).

| Frequency sweep test
The frequency sweep tests of ultrasonic sugar beet pectin were conducted with the angular frequency ranged from 0.6283 to 62.83 rad/s at the temperature of 25°C and strain of 0.5%. The frequency (ω) dependence of the G′ and G″ could be described by the following Power Law equations: where K′ and K″ are constants (Pa·s n ), n′ and n″ are frequency exponents dimensionless (Zhu, Li, & Wang, 2019).

| Temperature ramp measurement
The apparent viscosity as a function of temperature can be used to characterize the activation energy (E a ) (Wang, Wang, Li, Xue, & Mao, 2009). Temperature ramp sweep was performed with the temperature ranged from 10 to 60°C at the heating rate of 10°C/min and angular velocity of 0.1 rad/s. The E a could be determined according to the Arrhenius equation: where η a and T are the apparent viscosity (Pa·s) and absolute temperature (K), η ∞ and R are the frequency factor (dimensionless) and ideal gas constant (8.3145 J/mol·K).

| Pectin emulsions preparation
The 5.0 g of corn oil (density of 840 g/L) and 100 ml of ultrasonic treated pectin solutions (concentration of 20.0 g/L) were mixed and subjected to prehomogenization process by a digital Ultra-Turrax Homogenizer (T25, IKA) at the speed of 12,000 rpm for 2 min. The mixtures were homogenized by an AH-100 D homogenizer (ATS Engineering Inc.) at the pressure of 50 MPa for three passes. The prepared emulsions were placed in the refrigerator at 4°C for the following analyses.

| Particle size and zeta potential
Particle size and zeta potential of sugar beet pectin emulsions were determined by a Zetasizer Nano ZS (Malvern Instruments). To avoid multiple scattering effects, different sugar beet pectin emulsions were diluted with deionized water for 900 times (30 times each) before measurement and then injected into clear disposable zeta cell.
Refractive indices of oil droplet and solvent were 1.45 and 1.33, respectively. All measurements were conducted at 25°C for at least in triplicate.

| Emulsifying activity
The prepared sugar beet pectin emulsions were diluted 900 times with 1.0 g/L sodium dodecyl sulfate (SDS) and then tested the absorbance at 500 nm using a UV spectrophotometer, and the SDS solution (1.0 g/L) was used as the blank control. The turbidity (T) and emulsifying activity index (EAI) were calculated by the equations below (Wang, Wang, Li, Adhikari, & Shi, 2011): where A, V, and I are absorbance, dilution factor, and path length (0.01 m), respectively.
where Ø and c represent oil volume fraction and pectin concentration in the emulsion, respectively.

| Confocal laser scanning microscopy (CLSM) test
The morphological characteristics of emulsion droplets were deter- To avoid the fluorescence quenching, the stained emulsion was kept in darkened before the CLSM measurements. The excitation and emission wavelengths of Nile red are 488 nm and 600 to 700 nm, respectively. The CLSM image resolution was 1,024 × 1,024 pixels, which was corresponded to viewing filed of 200 μm × 200 μm.

| Physical stability
Physical stability of sugar beet pectin emulsions was determined using an analytical centrifuge (LUMiFuge, LUM GmbH). Emulsions (420 μl) were transferred into polycarbonate rectangular synthetic cell (2 × 8 mm) and analyzed by an emitting light beam at a wavelength of 865 nm. The samples were centrifuged at a speed of 400 rpm at 25°C with a rate of 30 s interval for 2 hr.

| Statistical analysis
Each test was conducted at least three replicates in this experiment.
At least three replicates were tested for all experiments. Data analysis was performed using the statistical software SPSS 22.0 (SPSS Inc.). Duncan's multiple comparison tests were applied to determine the significance (p < .05).

| Intrinsic viscosity ([η]) and viscosity average molecular weight ([M v ]) analysis
The [η] could directly reflect polymer solution behaviors in many applications, which is one of the most important parameters of hydrodynamic volume of given polymer mass (Rushing & Hester, 2003). As shown in Figure 1,

| Steady-state flow analysis
The  Table 1. The infinite-rate viscosity (η ∞ ) and the consistency coefficient (k s ) values decreased after ultrasonic treatment but revealed no significant difference with the increasing time from 5 to 45 min (p ˂ .5). The flow behavior index (n) of Sisko model decreased from 0 to 30 min but increased from 30 to 45 min.

| Frequency sweep analysis
The storage modulus (G′) and loss modulus (

| Activation energy analysis
The sensitivity of viscosity to temperature can be characterized by activation energy (E a ) (Pongsawatmanit, Temsiripong, Ikeda, & Nishinari, 2006). The E a values of sugar beet pectin solutions under different ultrasonic time are displayed in Figure 4. The E a value decreased from 21.7 to 11.7 kJ/mol with the increment time from 0 to 30 min but increased from 30 to 45 min, which was consistent with trends in the [η] and [M v ] as a function of ultrasonic time. The untreated sample (0 min) had the highest E a values and temperature sensitivity in viscosity, indicating that ultrasonic treatment weakened the intermolecular interactions between pectin molecules as well as intramolecular interactions between pectin polymer chains (Li, Li, Geng, Song, & Wu, 2017;Lopes Da Silva, Gonçalves, & Rao, 1994).

| Particle size distribution, zeta potential, and emulsifying activity analysis
The particle size distribution of different sugar beet pectin emulsions is shown in Figure 5. The unimodal distribution was observed in pectin emulsions with ultrasonic time from 0 to 20 min, and bimodal phenomena were detected in 30 and 45 min samples. It could be observed in Table 3 that particle size of pectin emulsions decreased as the ultrasonic time increased from 0 to 20 min and increased from 30 to 45 min, indicating that the reduction of molecular weight could promote the accessibility of surface-active groups but too short entangled polymer chains resulted in aggregation of emulsion droplets (Alba & Kontogiorgos, 2017).
The zeta potential is a measure of the surface charge density which could characterize the potential stability of emulsion system and larger absolute value represents a more stable system with stronger electrostatic repulsive force (Dickinson, 2009). The zeta potential and emulsifying activity index (EAI) are presented in Table 3. It could be observed from the table that absolute zeta potential and EAI values increased first and then decreased. The 20min treated emulsion showed the greatest absolute zeta potential and EAI values, which meant the highest energy barrier between emulsion droplets (Sui et al., 2017). As ultrasonic time continued to increase to 30 and 45 min, the decrease in absolute values of zeta potential and EAI values was related to the aggregations of emulsion droplets, leading to a decrease in the stability and activity of different pectin emulsion. The results were in accordance with the flax seed oil emulsion (Shanmugam & Ashokkumar, 2014) and myofibrillar protein-xanthan gum emulsion (Xiong et al., 2019).  aggregations of oil droplets, which further verified the particle size results in Figure 5 and Table 3.

| Physical stability analysis
Macro photographs of ultrasonic pectin emulsions after centrifugation were shown in Figure 7. The abscissa is the position (mm) and ordinate is the transmission (%). Red profiles at the bottom and green ones at the top were obtained in first and last scanning. The recorded spectrum could be used to estimate the emulsion stability, and greater change of light transmittance indicates worse emulsion stability (Sobisch & Lerche, 2008;Yuan, Xu, Qi, Zhao, & Gao, 2013).
It could be seen from Figure

| CON CLUS IONS
The

ACK N OWLED G M ENT
This research was financially supported by Basal Scientific Research (No. 2019JK035) from Chinese Academy of Inspection and Quarantine.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L A PPROVA L
The authors declare that this study did not involve human or animal subjects, and human and animal testing are unnecessary in this study.