Effect of ultrasonication on the stability and storage of a soy protein isolate-phosphatidylcholine nanoemulsions

Phosphatidylcholine-soybean protein isolate (PC-SPI) nanoemulsions were prepared by ultrasonication. The effects of preparation conditions (SPI and PC addition, ultrasonic power and time) on the structural properties of the nanoemulsions and their storage stability were investigated. The results showed that the most optimal adsorption capacity and adsorption tightness at the oil–water interface under optimal conditions (1.5% SPI, 0.20% PC, 500 W ultrasonic power and 9 min ultrasonic time) were exhibited by the SPI-PC conjugate, which demonstrated that this nanoemulsions can be categorized as a high-quality emulsion suitable for research. To test its stability, and the high-quality nanoemulsion of β-carotene was stored. After degradation of the nanoemulsions during storage, β-carotene was released. The β-carotene retention rate of the high-quality emulsion was maintained above 86% at different temperatures in the absence of light for up to 30 days. This study provides new information for the development of transport and stability systems for nanoemulsions.

Nanoemulsions are carriers with strong kinetic stability and bioavailability that are used to transport lipophilic substances 1 . Compared with conventional emulsions, nanoemulsions with particle sizes of mainly 100-500 nm have higher stability, solubility, bioavailability, and transport efficiency 2 . Nanoemulsions can increase the availability of biologically active substances in specific parts via controlled release 3 . Nanoemulsions are usually prepared using a high-energy (mechanically based) or low-energy (chemically based) method. High energy technologies such as homogenization, microfluidization and ultrasound technology have been used to prepare nanoemulsions 4 . However, microfluidic and homogenization technologies were not readily available in industry due to high costs and scalability issues 5 . Ultrasonic processing has the advantages of easy operation of the system, low production cost, low requirements for instruments and high energy efficiency 6 . Hydraulic shear and cavitation are produced by ultrasonication to break droplets to nanodroplets 7,8 .
Soybean protein isolate (SPI) is widely used in food formulations due to its nutritional value and functional characteristics 9 . SPI as an emulsifier plays a vital role in the formation of small oil droplets. The introduction of external emulsifiers (such as lecithin) can enhance the emulsification properties of SPI in producing nanoemulsions 10 . Phosphatidylcholine (PC) is the main phospholipid in lecithin 11 . Lecithin contains a lipophilic group in the form of a fatty acid group and a hydrophilic group in the form of a phosphate. The amphiphilic molecular structure is responsible for the excellent emulsification ability of PC, and the physical stability of an SPI nanoemulsion can be increased by adding lecithin 12,13 . Emulsifiability increases when PC interacts with SPI through a hydrophobic group. Li et al. 14 proved that a binding site can be a hydrophobic region of the α-helix of a protein.
β-Carotene (C40H56) is a natural pigment that is found in various fruits, grains, and oils. It is a lipophilic substance and a highly conjugated long-chain isoprenoid micronutrient 15 . β-Carotene has been widely used in food, feed, and other nutraceuticals as an antioxidant 16 . Some functionalities of β-carotene are limited because its lower chemical stability of carotenoids, which is easily degraded by heat, oxygen, and light. Because the solubility
Particle size, ζ-potential, and polydispersity index (PDI) analysis. The particle size, ζ-potential and PDI of nanoemuslions were measured using a dynamic light-scattering device (Zetasizer Nano-ZS 90 light scattering particle size analyzer). Nanoemulsions were diluted (1:1,000) with a phosphate buffer before measurement to prevent multiple scattering 21 . Aqueous dispersion and the refractive index medium were set at 1.33 and 1.45, respectively. The fresh nanoemulsions and phosphate buffer were diluted in proportion (1:100, v/v) during ζ-potential analysis. Turbidity analysis. Turbidity values were obtained using a previously published method with slight modification 22 . The nanoemulsions were diluted 40 times with a phosphate buffer solution. The absorbance was measured at 600 nm with a spectrophotometer (LW-1600FC UV, Shanghai, China). Phosphate buffer solution was used as a blank. Turbidity was calculated using the formula: where A = the absorbance of diluted nanoemulsions at 600 nm, I = optical path extent (0.01 m), and V = dilution (40).
Emulsification yield analysis. The emulsification yield of the nanoemulsions was determined using a previously published method 11 . The β-carotene in 1 mL of the nanoemulsions was extracted using an organic phase (ethanol: n-hexane = 1:1, 95%, 5 mL) and was quantitated using an Agilent 1,100 HPLC (Agilent Technologies Inc., Santa Clara, CA, USA) system with a UV-Vis diode array absorption detector. A reversed phase analytical column C30 (YMC Carotenoid, 250 × 4.6 mm i.d., 5 μm, YMC, Inc., Wilmington, NC, USA) was used with a flow rate of 1.0 mL/min by mobile phase. A 20-μL sample was analyzed using a diode array detector (DAD) at 450 nm. Standard curves were obtained using different concentrations of β-carotene standard samples (assuming a purity of 100%). The amount of β-carotene in the nanoemulsions was determined after processing the data with Dionex Chromeleon software. The emulsion yield (%) of the nanoemulsions was calculated by the following formula:

Raman microscopic observation of nanoemulsion morphology. Raman microscopic observation
was used as an interface structural analysis of the nanoemulsions 20 . A 5 mW laser was focused on a slide with droplets in the center, and the droplets were observed. The scanning distance was set in the direction of the two axes (x-axis, y-axis) according to the required scanning range. Spectral analysis was performed using Spec 32 software, which was supplied with the spectrometer. The moving distance of the spot was 2 μm, which enabled automatic and efficient scanning of the nanoemulsion sample to be tested. The 3D imaging portion observed by the microscope was based on 2D imaging. The laser focusing depth (i.e., z-axis) was adjusted so that layer-bylayer scanning of the sample could be manually conducted.
Nanoemulsion storage stability. The β-carotene nanoemulsions were prepared with 1.5% SPI, 0.17% PC, ultrasonic power 500 W, and ultrasonic time 9 min. The storage stability of the β-carotene nanoemulsions was evaluated by adding 1 mL of the emulsion to a brown tube (1.5 mL) that was stored at 4, 25, or 55°C 24 . Next, 1 mL sample was extracted with 5 mL of n-hexane: ethanol = 3:2, and the hexane phase was removed.
After repeating twice, their absorbances were measured using a spectrophotometer at 450 nm. A standard curve of β-carotene was prepared under similar conditions, and the β-carotene concentration was measured. The β-carotene retention rate was calculated using the formula: where R β-carotene denotes β-carotene retention, C N indicates the concentration of β-carotene in the nanoemulsions, and C I represents the initial concentration of β-carotene.
Statistical analysis. All measurements were analyzed by t-test via SPSS 16 software, and the results are expressed as the mean ± standard deviation (SD). Analysis of variance (ANOVA) was used to determine differences between groups.

Results
Effect of SPI addition. The effect of SPI addition on the characteristics and stability of nanoemulsions is shown in Table 1. The average particle size, PDI, turbidity, and TSI of the nanoemulsions were drastically decreased with the SPI content increased from 0.5% to 1.5%. The particle size value, turbidity, TSI and PDI value were minimized when the SPI addition was 1.5%. Hebishy found that when the whey protein content was low, the prepared nanoemulsions had larger droplets, which was consistent with the current results 25 . SPI as an emulsifier plays a vital role in the formation of small oil droplets by reducing the tension at the oil-water interface. When SPI is adsorbed on the surface of oil droplets, it prevents the oil droplets from accumulating and coalescing 26 .The thickness of the interfacial layer gradually increased and the interfacial tension at the oil-water Table 1. Effect of the soybean protein content on the characteristics and stability of nanoemulsions. Data represent mean ± standard deviations. Different letters in the same column represent a significant difference between samples (p < 0.05). Arrange all the averages in descending order, and use the letter "a" on the maximum average. www.nature.com/scientificreports/ interface decreased with SPI concentration, which increased the stability of the emulsion. High interfacial tension caused the emulsion droplets to fuse and aggregate with each other 27 .The maximum concentration of SPI adsorped at the interface is the critical micelle concentration. If the amount of SPI adsorption at the interface exceeded a critical value, the adsorption capacity of the protein molecule was weakened at the interface of the continuous phase. The stability of the nanoemulsion was positively correlated with its ζ-potential 11 . Table 1 showed that the absolute value of the ζ-potential was greater than 30 mV between 1.5% SPI and 2.5% SPI. It was observed that the ζ-potential values had a maximal value (-33.4 mV) at 1.5% SPI. The nanoemulsions exhibited satisfactory stability through electrostatic repulsion above 30 mV of the ζ-potential value 28 . It could be deduced that the nanoemuslions stabilized by SPI-PC complex had a high stability. With increasing addition of protein, the unabsorbed soluble protein formed aggregates under the action of ultrasonic cavitation and thermal effects due to enhanced hydrophobic interactions 20 . The protein at the oil-water interface was replaced by aggregates, leading to a decrease in emulsion stability 29 . Ghosh 5 found that water molecules were difficult to penetrate the protein molecules under highly protein concentration. Protein molecules were aggregated, which decreased the stability of the nanoemulsions. Therefore, the emulsion yield value and the TSI value of the nanoemulsions were reduced.
Effect of PC addition. PC is a surfactant with excellent biocompatibility and amphiphilicity, and it can increase the load capacity and reduce the interfacial tension of the dispersed phase by forming an embedded structure at the water/oil interface 30 . PC interacts with soy protein through electrostatic and hydrophobic interactions, and significantly affects the functional properties of soy protein 31 . The hydrophobic region of SPI can bind to the hydrophobic group of PC, and the binding force will maintain the interaction between SPI and PC 14,32 . Thus, The particle size, PDI value, TSI and turbidity of SPI-PC nanoemuslions were increased with the addition of PC from 0.13% to 0.17% as shown in Table 2. The smaller droplets were formed when the PC was increased due to sufficient protein isolate and insufficient interfacial tension to maintain newly formed droplets 7 and thus, the stability of SPI-PC nanoemuslion was enhanced by adding more PC. This mixture system lowered the interfacial tension and also prevented coalescence, and thus improved the stability of a protein-stabilized oil-water emulsion 33 . Moreover, the soy protein molecule was negatively charged at neutral pH, while PC was a zwitterion with both positive and negative charges 34 . The addition of PC could increase the interface adsorption by enhancing the electrostatic interaction between hydrophobic SPI and PC. In the case of the emulsification interface, the blank palce found in soy protein was subsequently filled with PC.
This mixture system lowers the interfacial tension and also prevents coalescence, and thus improves the stability of a protein-stabilized oil-water emulsion 33 . Therefore, after PC addition (from 0.13 to 0.19%), the ζ-potential value and the stability of the nanoemulsions were increased. Excessive PC addition increased the PC content in the aqueous phase of the emulsion. Based on the principle of displacement solubilisation, the oil-water interface protein was replaced by PC and entered the aqueous phase, which reduced the interfacial protein content and increased the particle size of the emulsion 35-37 . Effect of ultrasonic power. Ultrasonication is more economical and practical than microfluidization when taking into consideration production costs, maintenance, and aseptic production. Ultrasonication has become an excellent and superior tool in the emulsification process 38 . As shown in Table 3, an increase in ultrasonic power (from 100 to 400 W) led to an increase in average particle size, PDI, and TSI, but a decrease in turbidity value, which in turn increased ζ-potential and emulsion production. The cavitation effect produced by ultrasonication can reduce the particle size of SPI and affect the interfacial layer of the emulsion 39 . As ultrasonic power increased, the distribution became more uniform. These results suggest that 500 W resulted in the best values. After the ultrasonic power is increased to 500 W, the formation and collapse of cavitation bubbles can generate strong shock waves and jets. This strong force causes a significant increase in particle size and PDI that results in the tendency of the emulsion to re-agglomerate. Research has shown that the hydrophobicity of milk proteins was reported to have increased after ultrasonication 40,41 . The hydrophobic interaction forces aggregated droplets and increased the PDI, turbidity, and particle size of the emulsion. The increase in the of ultrasonic power increasesd the frequency of collisions between emulsion droplets, which subsequently leads to a higher probability of coalescence of droplets 28 . The increasing turbidity values may be related to the aggregation of emulsion Table 2. Effect of the lecithin content on the characteristics and stability of nanoemulsions. Data represent mean ± standard deviations. Different letters in the same column represent a significant difference between samples (p < 0.05). Arrange all the averages in descending order, and use the letter "a" on the maximum average.

PC addition
Particle size (nm) ζ-potential (mV) PDI Emulsification yield (%) Turbidity TSI Effect of ultrasonication time. The effect of ultrasonic processing time on the properties of nanoemulsions is shown in Table 4. PDI, turbidity, and the particle size of the nanoemulsions decreased and the emulsification yield increased when the ultrasound time increased from 6 to 9 min. Properly increasing the ultrasonic time can improve the dispersion state and emulsion stability of nanoemulsion. When the ultrasound time was continuously increased (between 9 and 10 min), the particle size increased from 287.9 to 468.5 nm. The particle size of the nanoemulsions increased by longer sonication treatment. The effect of long-term sonication on particle size is related referred to in the literature as "over-processing". Ultrasonication generated a large amount of energy, increasing the aggregation of droplets 34 . Lago et al. observed a similar trend with ultrasonic treatment of emulsion particle size 42 . The greater turbulence and heating of the system increased the frequency of collisions between the oil balls and caused emulsion instability 19 . Over-processing also caused the ζ-potential value to decrease 28,39,43. Additionally, previous researchers found that long-term ultrasonication decreased the emulsion stability due to a decrease in interfacial tension and viscosity, while smaller interfacial tension values caused emulsion interface instability 40,44 . Therefore, nanoemulsions exhibited satisfactory emulsion characteristics when the ultrasonic time was 9 min.
Ultrasonic nanoemulsion droplet structure. The 3D images help to observe the spatial structural morphology. In this study, the 3D confocal Raman imaging was used to observe the structure of SPI-PC nanoemulsion. The protein in emulsion exhibited significant Raman absorption at 1,660 cm −1 , and the feature regions were used for extraction and computational imaging. A microscopic image of a single drop in the yellow borderline region was analyzed in Fig. 1A. A Raman imaging structure of six nanoemulsion droplets (Fig. 1B) was formed as the observed position gradually probed into the center of the oil phase. The figure shows that a large number of green markers (SPI components) were more widely distributed in the oil-water interface of the emulsion droplets. It can be seen that soy protein were evenly distributed at the interface of nanoemulsion droplets according to our processing conditions. Figure 2 shows the Raman characteristic absorption peak image of the emulsion. The symmetric stretching vibration and the anti-symmetric stretching vibration characteristic peak of the C-H 2 group of PC were 2,840 cm −1 and 2,880 cm −1 , respectively. The characteristic peak of amide I of SPI located at 1665 cm −1 . The results indicated that PC and SPI were distributed at the oil-water interface of the emulsion droplets, and our results were consistent with those of a previous study 20 . The total Raman intensity decreased when the observed position gradually moved toward the center of the oil droplet, and this effect be related to the reduction of the soy protein content. However, the characteristic peaks of SPI and PC have not disappeared in the figure, which proved that SPI and PC were interacted and uniformly absorbed at the interface of the nanoemulsions. Table 3. Effect of ultrasonic power on the characteristics and stability of nanoemulsions. Data represent mean ± standard deviations. Different letters in the same column represent a significant difference between samples (p < 0.05). Arrange all the averages in descending order, and use the letter "a" on the maximum average.  Table 4. Effect of ultrasonic power on the characteristics and stability of nanoemulsions. Data represent mean ± standard deviations. Different letters in the same column represent a significant difference between samples (p < 0.05). Arrange all the averages in descending order, and use the letter "a" on the maximum average. www.nature.com/scientificreports/ The 3D images help to observe the spatial structural morphology. In this study, the 3D confocal Raman imaging was used to observe the structure of SPI-PC nanoemulsion. The protein in emulsion exhibited significant Raman absorption at 1,660 cm −1 , and the feature regions were used for extraction and computational imaging. A microscopic image of a single drop in the yellow borderline region was analyzed in Fig. 1A. A Raman imaging structure of six nanoemulsion droplets (Fig. 1B) was formed as the observed position gradually probed into the center of the oil phase. The figure shows that a large number of green markers (SPI components) were more widely distributed in the oil-water interface of the emulsion droplets. It can be seen that soy protein were evenly distributed at the interface of nanoemulsion droplets according to our processing conditions. Figure 2 shows the Raman characteristic absorption peak image of the emulsion. The symmetric stretching vibration and the anti-symmetric stretching vibration characteristic peak of the C-H 2 group of PC were 2,840 cm −1 and 2,880 cm −1 , respectively. The characteristic peak of amide I of SPI located at 1665 cm −1 . The results indicated that PC and SPI were distributed at the oil-water interface of the emulsion droplets, and our results were consistent with those of a previous study 20 . The total Raman intensity decreased when the observed position gradually moved toward the center of the oil droplet, and this effect be related to the reduction of the soy protein content. However, the characteristic peaks of SPI and PC have not disappeared in the figure, which proved that SPI and PC were interacted and uniformly absorbed at the interface of the nanoemulsions.

Ultrasonic time (min) Particle size (nm) ζ-potential (mV) PDI
Storage stability of nanoemulsions. The stability of the product is essential for nanoemulsion-based delivery systems in most practical applications. Temperature, pH, storage time, processing method, and ionic strength can influence the stability of emulsions 20 . Storage temperature and time are main factors influencing the stability of emulsions. The effects of different storage temperatures and storage times on nanoemulsion stability is shown in Table 5, When the storage temperature increased, the particle size increased, and the β-carotene retention and ζ-potential rate decreased with both storage temperature and time. The average particle size of the emulsion was maintained at 100-500 nm of nanodroplets, and the ζ-potential of the nanoemulsion was reduced to 35-30 mV during storage. The rate of liquid phase separation of nanoemulsions during storage also depends on the frequency of contact collisions between droplets, because Brownian motion increases as temperature storage increases. The droplet collision frequency affected the mass transfer kinetics of surfactant and oil mol-  www.nature.com/scientificreports/ ecules between the water and oil phases, resulting in an increase of particle size 45 . The ζ-potential value was an important indicator for the stability of the nanoemulsions. After storage at 4 °C, 25 °C, and 55 °C for 30 days, the absolute ζ-potential values of emulsions gradually decreased to 31.5, 30.1, and 29.6, respectively. This may be due to the aging of austenite and the change in protein conformation over time, leading to the formation of hydrophobic bonds and hydrogen bonds between adjacent proteins at the interface 46 . Nanoemulsions had a lower electrostatic interaction while they were stored at high temperatures or during long-term storage, which caused the conformational change of the biopolymer of the surfactant molecules [47][48][49] .The stability nanoemulsion is related to the storage environment(time, temperature). The nanoemulsions gradually oxidized during storage, and the degree of oxidation increased with the increase in storage time and temperature. Lipid oxidation might change the interfacial composition of the nanoemulsion, and cause the emulsifier to rearrange and desorb at the interface and reduce the stability of the emulsion system. Eventually, the nanoemulsions gradually broke up, which caused β-carotene to be released from the nanoemulsions. Although the retention rate of β-carotene in the emulsion gradually decreased, 86% of β-carotene was remained even storaging for a long time at a higher storage temperature (Table 5), exhibiting excellent resistance to droplet coalescence during storage. This may be due to the antioxidant activities of SPI and PC. Borba et al. 50 demonstrated that Tween 20 β-carotene nanoemulsions prepared by a high-pressure homogenizer had a retention rate of 70-80% during storage, and an increased ability to encapsulate β-carotene as compared to conventional emulsions. Early studies have found that protein emulsifiers inhibit lipid oxidation better than small-molecule surfactants 51,52 . This provides a method to increase the potential use of carotenoids in the food industry, including applications of β-carotene.

Conclusions
In this research, the SPI-PC complex was used as the aqueous phase and sunflower oil was used as the oil phase, and the β-carotene nanoemulsion was prepared using ultrasonic technology. The results show that when SPI is 1.5%, PC is 0.22%, ultrasonic power is 500 W, ultrasonic time is 9 min, nanoemulsion has the best stability. 3D Raman imaging shows that ultrasonic treatment can make SPI and PC evenly distributed on the water-oil interface of nanoemulsion droplets. During storage, the ζ-potential and β-carotene retention rate were higher than 30 mV and 86%, respectively. The optimal preparation process provides a method and theoretical basis for the ultrasonic preparation of nanoemulsions, and may lead to further food design, including the design of β-carotene.
Received: 8 May 2020; Accepted: 23 July 2020 Table 5. Storage stability of nanoemulsion. Data represent mean ± standard deviations. Different letters in the same column represent a significant difference between samples (p < 0.05). Arrange all the averages in descending order, and use the letter "a" and "A" on the minimum average.