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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 results25. 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 coalescing26.The thickness of the interfacial layer gradually increased and the interfacial tension at the oil–water 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 other27.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.

Table 1 Effect of the soybean protein content on the characteristics and stability of nanoemulsions.

The stability of the nanoemulsion was positively correlated with its ζ-potential11. 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 value28. 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 interactions20. The protein at the oil–water interface was replaced by aggregates, leading to a decrease in emulsion stability29. Ghosh5 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 interface30. PC interacts with soy protein through electrostatic and hydrophobic interactions, and significantly affects the functional properties of soy protein31. The hydrophobic region of SPI can bind to the hydrophobic group of PC, and the binding force will maintain the interaction between SPI and PC14,32. 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 droplets7 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 emulsion33. Moreover, the soy protein molecule was negatively charged at neutral pH, while PC was a zwitterion with both positive and negative charges34. 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.

Table 2 Effect of the lecithin content on the characteristics and stability of nanoemulsions.

This mixture system lowers the interfacial tension and also prevents coalescence, and thus improves the stability of a protein-stabilized oil–water emulsion33. 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 emulsion35,36,37.

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 process38. 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 emulsion39. 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 ultrasonication40,41. 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 droplets28. The increasing turbidity values may be related to the aggregation of emulsion molecules. The emulsification interface was unstable state under the condition of high ultrasonic power (600 W) output. At this time, the absolute value of the ζ-potential of the nanoemulsion was significantly reduced.

Table 3 Effect of ultrasonic power on the characteristics and stability of nanoemulsions.

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 droplets34. Lago et al. observed a similar trend with ultrasonic treatment of emulsion particle size42. The greater turbulence and heating of the system increased the frequency of collisions between the oil balls and caused emulsion instability19. Over-processing also caused the ζ-potential value to decrease28,39,43. 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 instability40,44. Therefore, nanoemulsions exhibited satisfactory emulsion characteristics when the ultrasonic time was 9 min.

Table 4 Effect of ultrasonic power on the characteristics and stability of nanoemulsions.

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 1
figure1

3D-Raman microscopic image of ultrasonic preparation of nanoemulsions. In the figure, (A) a single nanoemulsions droplet was selected for analysis, and (B) 6 Raman imaging plots of the single nanoemulsions droplet were formed as the observational displacement gradually probed into the oil phase.

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–H2 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 study20. 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.

Figure 2
figure2

Raman image of the ultrasonic preparation of nanoemulsions. (U-1, U-2, U-3, U-4, U-5 and U-6 represent six observation positions from top to bottom, respectively).

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-H2 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 study20. 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.

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 emulsions20. 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 molecules between the water and oil phases, resulting in an increase of particle size45. 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 interface46. 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 molecules47,48,49.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.

Table 5 Storage stability of nanoemulsion.

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 surfactants51,52. This provides a method to increase the potential use of carotenoids in the food industry, including applications of β-carotene.

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