Food additives SiO${}_2$ and TiO${}_2$ particles were purchased from Evonik Industries AG (Essen, Germany) and Tioxide Europe S.R.L (Varese, Italy), respectively. TX-114, casein, humic acid (HA, sodium salt), Si standard solution, and Ti standard solution were provided by Sigma-Aldrich (St. Louis, MO, USA). Nitric acid (HNO${}_3$), hydrogen peroxide (H${}_2$O${}_2$), hydrofluoric acid (HF), sodium chloride (NaCl), sodium hydroxide (NaOH), and ethyl alcohol were supplied by Samchun Pure Chemical Co., Ltd. (Pyeongtaek, Gyeonggi-do, Republic of Korea). Powdered sugar was purchased from Samyang Co. (Seoul, Republic of Korea). Conical-bottom glass centrifuge tubes (15 mL) were obtained from Daeyoung Science (Seoul, Republic of Korea). Commercial products containing SiO${}_2$ (candy, powder, coffee mix, milk tea, and snacks) or TiO${}_2$ (candy, powder, sauces, chocolate, and snacks) as food additives were purchased from supermarkets located in Seoul, Republic of Korea in 2022.

Particle size and shape of SiO${}_2$ or TiO${}_2$ were analyzed by field emission transmission electron microscope (FE-TEM; JEM-2100F, JEOL, Tokyo, Japan) equipped with energy-dispersive X-ray spectroscopy (EDS). The particles were dispersed in distilled and deionized water (DDW) and the suspensions (0.1 mg/mL) were prepared in ethyl alcohol solution and sonicated for 15 min (160 W, Bransonic 5800, Branson Ultrasonics, Danbury, CT, USA). The suspensions (5 $\mu$L) were dropped on a carbon-coated copper grid (200 mesh, PELCO® TEM Grids, Ted Pella Inc., Redding, CA, USA). After drying at room temperature, TEM images were acquired at an accelerating voltage of 200 kV. The average particle sizes and size distributions of SiO${}_2$ or TiO${}_2$ particles were determined using ImageJ software (version 1.53k, National Institutes of Health, Bethesda, MD, USA).

Crystal structure of SiO${}_2$ or TiO${}_2$ was determined by powder X-ray diffraction (XRD) patterns using X-ray diffractometer (SmartLab, Rigaku Co., Tokyo, Japan) with Ni-filtered CuK$\alpha$ radiation ($\lambda$ = 1.5418Å, a voltage of 40 kV, a current of 40 mA, a scan range of 5–80° with a step size of 0.02°, and a scanning rate of 3°/min).

Hydrodynamic diameters and zeta potentials of SiO${}_2$ or TiO${}_2$ were measured by dynamic light scattering (DLS) and electrophoretic light scattering (ELS), respectively, using a Zetasizer Nano System (Malvern Instruments, Worcestershire, UK). SiO${}_2$ or TiO${}_2$ suspension (0.1 mg/mL) was prepared in DDW or humic acid (HA) solution (10 $\mu$g/mL) and stirred for 30 min. After further sonication for 15 min, the suspensions (1 mL) were immediately put in plastic cuvettes and the measurements were carried out at room temperature.

CPE method for SiO${}_2$ and TiO${}_2$ was optimized with SiO${}_2$ or TiO${}_2$ dispersions (0.1 mg/mL) in DDW or HA solution (10 $\mu$g/mL), respectively. After stirring for 30 min and sonication for 15 min at room temperature, the suspensions (7 mL) were transferred to bottom glass centrifuge tubes (15 mL) and the pH was adjusted to 3.0 and 4.0 for SiO${}_2$ and TiO${}_2$, respectively, with NaOH or HNO${}_3$ solution. Next, TX-114 (0.5 mL of 5% (w/v)) and NaCl (0.75 mL of 0.2 M) solutions were added in the suspensions and diluted to 10 mL with DDW. The mixed solutions were incubated for 30 min at 45 °C to promote phase separation and centrifuged at 2500$\times$g for 5 min at 25 °C. The precipitates and the supernatants where particles and ions were present, respectively, were digested for inductively coupled plasma-atomic emission spectroscopy (ICP-AES) analysis (JY2000 Ultrace, HORIBA Jobin Yvon, Longjumeau, France) as described in “2.7. Digestion of Organic Materials, Microwave digestion, and ICP-AES Analysis”.

The CPE developed for SiO${}_2$ or TiO${}_2$ was applied by spiking SiO${}_2$ or TiO${}_2$ into representative food matrices, powdered sugar or casein. The contents of SiO${}_2$ and TiO${}_2$ were adjusted to be 2% (w/w) and 1% (w/w) based on FDA recommendations, respectively. SiO${}_2$ suspension (0.1 mg/mL) was spiked into 0.1 g of powdered sugar or casein at the concentration of 2% (w/w) and dispersed in DDW (7 mL). TiO${}_2$ (0.1 mg/mL) was spiked into 0.1 g of food matrices at the concentration of 1% (w/w) and dispersed in HA solution (7 mL). After stirring for 30 min and sonication for 15 min, the same procedure was applied as described in “2.3. Optimizations of CPE for SiO${}_2$ and TiO${}_2$”.

Commercial foods (10 g), such as candy, powder, coffee mix, milk tea, chocolate, sauce, and snacks, containing SiO${}_2$ or TiO${}_2$ as a food additive on product labeling were homogenized in an agate mortar. Homogenized commercial products were dispersed in 7 mL of DDW and 7 mL of HA solution for the determination of SiO${}_2$ and TiO${}_2$, respectively. After stirring for 30 min and sonication for 15 min, the same procedure was applied as described in “2.3. Optimizations of CPE for SiO${}_2$ and TiO${}_2$”.

Dissolution properties of SiO${}_2$ and TiO${}_2$ in food matrices were evaluated in powdered sugar or casein, spiked with SiO${}_2$ (2% (w/w)) or TiO${}_2$ (1% (w/w)). Commercial foods (10 g) containing SiO${}_2$ or TiO${}_2$ as a food additive indicated on product labeling were homogenized in an agate mortar. Food matrices (0.1 g) or homogenized commercial products (0.1 g) spiked with SiO${}_2$ and TiO${}_2$ were dispersed in 10 mL of DDW and HA, respectively, and stirred for 30 min, followed by sonication for 15 min at room temperature. Further incubation was carried out for 30 min at 45 °C, as described in “2.3. Optimizations of CPE for SiO${}_2$ and TiO${}_2$”. The suspensions were then centrifuged at 16,000$\times$g for 15 min to collect the supernatants containing dissolved Si or Ti. The Si and Ti concentrations in the supernatants were determined by ICP-AES analysis after pre-digestion with HNO${}_3$ and H${}_2$O${}_2$ as described in “2.7. Digestion of Organic Materials, Microwave digestion, and ICP-AES Analysis”.

Si or Ti concentrations were quantified by measuring total Si or Ti contents using ICP-AES analysis, with Si or Ti standard solutions of different concentrations based on our previous reports [15, 30]. It was reported that the amount of SiO${}_2$ and TiO${}_2$ in commercial foods are ranged from 1.5 to 7 mg/g and 0.2 to 10 mg/g, which are enough to be detected by ICP-AES [15, 31, 32]. Organic materials in the supernatants obtained after dissolution or CPE experiments were digested with 10 mL of ultrapure HNO${}_3$ and 1 mL of H${}_2$O${}_2$ at 180 °C until the solution was colorless and entirely evaporated. The precipitates containing SiO${}_2$ or TiO${}_2$ particles after CPE application were digested in perfluoroalkoxy microwave digestion vessels using a microwave system (ETHOS EASY, Milestone Srl, Sorisole, Italy). Briefly, SiO${}_2$ particles were digested with 6 mL of 70% HNO${}_3$ and 1 mL of 40% HF. TiO${}_2$ particles were digested with 6 mL of 70% HNO${}_3$ and 2 mL of 40% HF [33, 34]. The samples were digested for 55 min at 1600 W by irradiation at 120, 160, and 210 °C for 15, 10, and 30 min, respectively, followed by holding for 1 min. After digestion, all samples were diluted to suitable volumes with DDW, shaken up and down, and analyzed by ICP-AES. Method blanks were determined by performing the same procedure in the absence of Si or Ti samples. Instrument operating conditions are described in Supplementary Table 1.

Results were presented as means $\pm$ standard deviations. One-way analysis of variance with Tukey’s test was performed using the SAS Ver.9.4 (SAS Institute Inc., Cary, NC, USA) to determine the significances of intergroup differences. Statistical significance was accepted for p values of 0.05.

To optimize TX-114-based CPE methods for SiO${}_2$ and TiO${}_2$, commercially available food-grade SiO${}_2$ and TiO${}_2$ were purchased and the characterization of each particle was carried out. Supplementary Fig. 1A,B show that the constituent particle sizes of SiO${}_2$ and TiO${}_2$ by TEM analysis were 14 $\pm$ 4 nm and 109 $\pm$ 35 nm, respectively. Zeta potential values of SiO${}_2$ and TiO${}_2$ under different pH conditions are presented in Supplementary Fig. 1C, showing isoelectric points (IEP) of the former and the latter were pH 1.9 and 3.1, respectively. It was reported that the electrostatic repulsion of particles is minimized at pH close to the IEP due to reduction of Brownian motion, and thus, the pH where zeta potentials are close to zero is optimal for the formation of NPs captured in TX-114-based micelles [35, 36]. But, the addition of NaCl during the CPE process elevated zeta potential values of NPs. Hence, the pH values for the CPEs were set at higher pHs than IEP values, 3.0 and 4.0 for SiO${}_2$ and TiO${}_2$, respectively. As a consequence, particles can be captured in TX-114-based micells as precipitates, whereas ionized forms are present in supernatants after phase separation using CPE followed by centrifugation.

Table 1 demonstrates the changes in hydrodynamic diameters and zeta potentials before and after CPE application. The hydrodynamic diameters of SiO${}_2$ suspension in DDW were statistically same to those obtained by CPE, whereas SiO${}_2$ in HA solution had increased hydrodynamic diameters after CPE application (Table 1). In case of TiO${}_2$, no increase in hydrodynamic diameters after CPE was found when it was dispersed in HA solution. It is worth noting that particles captured in TX-114 micelle by CPE must have the same particle size distribution compared with pristine particles without aggregation or dissolution, which is critical for fate determination as intact forms. Hence, SiO${}_2$ and TiO${}_2$ were dispersed in DDW and HA solution, respectively, for CPE application. This result also suggests that the CPE process can capture SiO${}_2$ and TiO${}_2$ as intact forms without aggregate formation or degradation. On the other hand, Table 1 shows that the zeta potential values of both SiO${}_2$ and TiO${}_2$ changed to less negative charges after CPE, probably resulted from NaCl addition during the CPE process.

Total recoveries of SiO${}_2$ and TiO${}_2$ were about 92.8% and 97.5%, respectively, and only 0.6% and 0.1% of SiO${}_2$ and TiO${}_2$ were detected as Si and Ti ionic forms, respectively, after CPE application (Fig. 1A,B). Meanwhile, the solubility of pristine SiO${}_2$ and TiO${}_2$ was assessed under the CPE conditions, showing 0.5% and 0.2% solubilities for the former and the latter, respectively, without significant differences between solubility and ionic fates (Fig. 1B). These results clearly indicate that SiO${}_2$ and TiO${}_2$ particles can be obtained as intact particle forms by CPE approaches.

Fig. 1.

Particle or ionic fates and total recoveries of pristine food additive (A) SiO${}_{\mathrm{2}}$ and (B) TiO${}_{\mathrm{2}}$ by CPE.

Representative food matrices such as powdered sugar and casein were spiked with SiO${}_2$ or TiO${}_2$, and the CPE methods optimized were applied. Powedered sugar and casein were chosen as food matrices due to their frequent usage in confectionary. Fig. 2A shows that there were no statistical changes in hydrodynamic diameters between pristine SiO${}_2$ or TiO${}_2$ and particles recovered from particles-spiked powdered sugar or casein by CPE application, suggesting that the CPE methods can recover SiO${}_2$ and TiO${}_2$ particles from food matrices as intact forms without aggregation or dissolution. The recoveries of SiO${}_2$ and TiO${}_2$ in powdered sugar or casein as particle forms were ranged from 93.2% and 102.6% (Fig. 2B,C). Only ~0.7% and 0.1% of SiO${}_2$ and TiO${}_2$ were detected as ionic forms, respectively, regardless of matrix type. Total recoveries of both particles and ions were 93.3%–103.3% for all cases, implying reliability of the results. All the results suggest that SiO${}_2$ and TiO${}_2$ added in food matrices can be separated as intact particle forms by applying the CPE.

Fig. 2.

Hydrodynamic diameters, fates, and solubility of SiO${}_{\mathrm{2}}$ and TiO${}_{\mathrm{2}}$ particles in food matrices. (A) Hydrodynamic diameters of SiO${}_2$ and TiO${}_2$ in food matrices before (pristine) and after CPE. No significant differences between before and after CPE were found (p $\mathrm{>}$ 0.05). Particle or ionic fates and total recoveries of pristine food additive SiO${}_2$ and TiO${}_2$ in (B) powdered sugar and (C) casein by CPE. (D) Dissolution properties of food additive SiO${}_2$ and TiO${}_2$ in food matrices.

On the other hand, the solubility of SiO${}_2$ and TiO${}_2$ was also evaluated because particles can dissolve to some extent in food matrices. As shown in Fig. 2D, the solubilities of SiO${}_2$ and TiO${}_2$ were ~0.9% and 0.1%, respectively, without significant differences compared with ionic forms (%) obtained by CPE (Fig. 2B,C). This result clearly supports that the CPE methods can separate particles from ionic forms in food matrices.

Quantitative analytical methods using acid digestion in a microwave system were validated by calculating recovery (%), linearity (coefficient of determination values, R${}^2$), accuracy (relative error, RE), precision (coefficient of variation, CV), limit of detection (LOD), and limit of quantification (LOQ). Table 2 shows that the recoveries of pristine SiO${}_2$ and TiO${}_2$ or SiO${}_2$- and TiO${}_2$-spiked powdered sugar or casein were range from 90.18% to 108.32%. The CV and RE values for all the cases were 0.40%–9.69% and –9.82% to 8.32%, respectively. Fig. 3 demonstrates a good linearity for pristine particles and particles-spiked in food matrices. All the results indicate reliable analytical parameters for SiO${}_2$ and TiO${}_2$ in food matrices, which is consistent with other previous reports [15, 30, 37, 38].

Fig. 3.

Quantitative analysis of SiO${}_{\mathrm{2}}$ and TiO${}_{\mathrm{2}}$. Standard curves for (A) pristine food additive SiO${}_2$ and TiO${}_2$, (B) powdered sugar spiked with SiO${}_2$ or TiO${}_2$, and (C) casein spiked with SiO${}_2$ or TiO${}_2$ obtained by acid digestion in a microwave system, followed by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) analysis.

On the other hand, the LOD and LOQ values were 0.03–0.10 $\mu$g/mL and 0.10–0.30 $\mu$g/mL for SiO${}_2$, and 0.01–0.13 $\mu$g/mL and 0.03–0.38 $\mu$g/mL for TiO${}_2$, respectively. These are similar or lower values compared with those obtained by other results, showing that the LOD and LOQ were 0.07 $\mu$g/g and 0.20 $\mu$g/g for SiO${}_2$, and 0.03–0.34 $\mu$g/mL and 0.09–1.04 $\mu$g/mL for TiO${}_2$, respectively [15, 31]. Sensitively low LOQ and LOD values were obtained, suggesting that the analytical methods by acid digestion in a microwave system are effective and reliable.

The CPE methods were applied to eight commercial products available on the market indicating SiO${}_2$ or TiO${}_2$ as a food additive on product labelling. Fig. 4 shows that 90.9%–101.8% of SiO${}_2$ were present as particle forms, only 0.02%–7.3% ionized forms were detected. When total recovery percentage (91.3%–108.3%) of both SiO${}_2$ particles and Si ions from products was considered, about 93.5%–99.9% of added SiO${}_2$ were found as intact particles. In case of TiO${}_2$, particle forms were detected in the range of 92.4% to 105.9%, whereas ionic forms were only found at 0.1%–0.9% (Fig. 5). Based on total recovery (93.3%–106.3%) of both TiO${}_2$ particles and Ti ions, about 99.1%–99.9% of added TiO${}_2$ were detected as intact particle forms. The results demonstrate that most of food additive SiO${}_2$ and TiO${}_2$ are not degraded and present as particles, and these two additives have low solubility in food matrices. When detected ionic forms were compared, higher Si ions than Ti ions were found, probably indicating high solubility of the former than the latter. Indeed, it was reported that the solubility of SiO${}_2$ was higher than TiO${}_2$ [4, 39, 40], which is in good agreement with our results.

Fig. 4.

Particle or ionic fates and total recoveries of SiO${}_{\mathrm{2}}$ in commercial foods by CPE. Particle or ionic fates and total recoveries of SiO${}_2$ in commercial (A) candy 1, (B) candy 2, (C) powder 1, (D) powder 2, (E) powder 3, (F) powder 4, (G) coffee mix, and (H) milk tea by CPE.

Fig. 5.

Particle or ionic fates and total recoveries of TiO${}_{\mathrm{2}}$ in commercial foods by CPE. Particle or ionic fates and total recoveries of TiO${}_2$ in commercial (A) candy 1, (B) candy 2, (C) powder, (D) snack, (E) chocolate 1, (F) chocolate 2, (G) sauce 1, and (H) sauce 2 by CPE.

Dissolution property of SiO${}_2$ and TiO${}_2$ was further evaluated by spiking known amount of pristine SiO${}_2$ and TiO${}_2$ in commercial food matrices, the same products where the CPEs were applied (Figs. 4,5). As shown in Fig. 6A,B, the solubilities of SiO${}_2$ were ranged from 0.1% to 5.5%, whereas TiO${}_2$ had 0.1%–0.9% solubilities depending on the matrix type. Overall results indicate that the solubility of two additives was low in commercial complex food matrices, but SiO${}_2$ has high solubility than TiO${}_2$, which is in good agreement with previous report [4, 5]. It is worth noting that no significant differences between solubility (Fig. 6) and ionic fate (Figs. 4,5) were found in the same products (p $>$ 0.05), indicating that the solubility critically affects the fates of two additives in foods. The results support reliable fate determination of SiO${}_2$ and TiO${}_2$ in commercial foods by CPE, and also suggest that major fates of SiO${}_2$ and TiO${}_2$ are particle forms in commercial processed products. Relatively high solubility of SiO${}_2$ in powder 1, powder 2, and milk tea seems to be related to its matrix interactions because their pH values were neutral (pH ~7.0). Indeed, the interactions between food additive SiO${}_2$ and food matrices were demonstrated, although the interactions seem to be not strong [3, 6, 13]. Quantitative analysis revealed that saccharides, proteins, fatty acids, and minerals can be adsorbed on the surface of SiO${}_2$ [13]. Hence, the adsorption of food matrices on SiO${}_2$ may increase its solubility in commercial food products. Meanwhile, the interaction between TiO${}_2$ and food matrices is likely to be negligible based on its extremely low solubility (Fig. 6B) and minor ionic fates (Fig. 5).

Fig. 6.

Dissolution properties of (A) SiO${}_{\mathrm{2}}$ and (B) TiO${}_{\mathrm{2}}$ in commercial foods.

Physico-chemical characterization of separated and recovered SiO${}_2$ and TiO${}_2$ from commercial food products by CPE was further carried out. The constituent particle sizes and size distributions were analyzed by TEM. Fig. 7A,B and Fig. 8A,B show that the constituent particle sizes of separated SiO${}_2$ and TiO${}_2$ were ranged from 12 $\pm$ 3 nm to 21 $\pm$ 4 nm and from 112 $\pm$ 30 nm to 132 $\pm$ 37 nm, respectively, without significant differences from pristine SiO${}_2$ (14 $\pm$ 4 nm) and TiO${}_2$ (109 $\pm$ 35 nm) (p $>$ 0.05). No remarkable aggregate formation or decomposed form was observed. The TEM-EDS analysis clearly reveals the presence of Si or Ti ions in the particles observed (Figs. 7C,8C). The results confirm that food additive SiO${}_2$ and TiO${}_2$ are primarily present as particles without degradation or aggregation.

Fig. 7.

Characterization of separated SiO${}_{\mathrm{2}}$ from commercial foods. (A) Transmission electron microscopy (TEM) images, (B) size distribution obtained by the TEM images, and (C) energy dispersive X-ray spectroscopy (EDS) of separated SiO${}_2$ from commercial foods.

Fig. 8.

Characterization of separated TiO${}_{\mathrm{2}}$ from commercial foods. (A) Transmission electron microscopy (TEM) images, (B) size distribution obtained by the TEM images, and (C) energy dispersive X-ray spectroscopy (EDS) of separated TiO${}_2$ from commercial foods.

The crystalline phases of separated SiO${}_2$ and TiO${}_2$ from commercial foods were analyzed by XRD patterns. The results indicate that pristine SiO${}_2$ and TiO${}_2$ have typical amorphous and anatase phases, respectively, and the same crystalline phases were observed in separated SiO${}_2$ and TiO${}_2$ particles from commercial foods (Figs. 9,10). It should be noted that amorphous SiO${}_2$ and anatase TiO${}_2$ are authorized as food additives [3, 23]. All the results imply that food additive SiO${}_2$ and TiO${}_2$ are present as intact particle forms without changes in crystalline phase and constituent particle size.

Fig. 9.

X-ray diffraction (XRD) patterns of pristine food additive SiO${}_{\mathrm{2}}$ and separated SiO${}_{\mathrm{2}}$ from commercial foods.

Fig. 10.

X-ray diffraction (XRD) patterns of pristine TiO${}_{\mathrm{2}}$ particles and separated TiO${}_{\mathrm{2}}$ from commercial foods.