Charge-switchable magnetic separation and characterization of food additive titanium dioxide nanoparticles from commercial food

Journal of Hazardous Materials 393 (2020) 122483

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Charge-switchable magnetic separation and characterization of food additive titanium dioxide nanoparticles from commercial food

T

Ke Luoa, Hyein Parka, Hazzel Joy Adraa, Jian Ryua, Jun-Hee Leea, Jin Yub, Soo-Jin Choib, Young-Rok Kima,* a b

Graduate School of Biotechnology & Department of Food Science and Biotechnology, College of Life Sciences, Kyung Hee University, Yongin, 17104, South Korea Department of Applied Food System, Major of Food Science & Technology, Seoul Women’s University, Seoul, 01797, South Korea

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO

A B S T R A C T

Editor: Navid B Saleh

Growing concerns about the potential health effects of nanoscale titanium dioxide (TiO2) have necessitated the need for monitoring the size distribution and physicochemical properties of food additive TiO2 that are present in commercial food. Acid digestion is by far the most widely used method to remove interfering food matrices, but the highly corrosive nature of the reaction could alter the physicochemical properties of the TiO2, which may give a skewed information about the materials. Here, we report an effective approach to extract intact form of food additive TiO2 nanoparticles from processed food through charge-charge interaction between TiO2 particles and charge-switchable starch magnetic beads (PL@SMBs), of which the captured TiO2 is readily harvested by switching the surface charge of PL@SMBs to neutral. The size and surface property of extracted TiO2 were shown to be well maintained due to the mild nature of the reaction. The extracted TiO2 particles from 10 commercial processed food showed a size distribution from 40 to 250 nm with a mean diameter of 115 nm, of which 22 % of them were less than 100 nm. The extracted TiO2 did not exhibit short-term cytotoxicity, but induced cellular oxidative stress at high concentration.

Keywords: Titanium dioxide Food additives Nanoparticles Magnetic separation Cytotoxicity

⁎

Corresponding author. E-mail address: [email protected] (Y.-R. Kim).

https://doi.org/10.1016/j.jhazmat.2020.122483 Received 3 February 2020; Received in revised form 4 March 2020; Accepted 5 March 2020 Available online 06 March 2020 0304-3894/ © 2020 Elsevier B.V. All rights reserved.

Journal of Hazardous Materials 393 (2020) 122483

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1. Introduction

matrices are based on wet digestion of the sample with a strong acid, such as nitric acid (HNO3), perchloric acid (HClO4), sulfuric acid (H2SO4), hydrofluoric acid (HF), etc. The rate of acid digestion could be accelerated by elevating temperature. For instance, microwave-assisted digestion with strong acids and oxidants, such as HNO3 and H2O2, respectively, can effectively oxidize organic matrices in foods, converting them to carbon dioxide or other volatile gases (Wang et al., 2004). But, such processes require corrosive acids and other reactive reagents that could have a negative impacts on human health and environment, thus utmost caution must be taken when used. Moreover, the size or morphology of some inorganic nanomaterials could be affected by harsh acid treatment. For instance, E171 TiO2 is known to be ionized by H2SO4 and HF, of which its physical properties, such as size and shape, could be altered over the course of acid treatment. Alternatively, physical separation techniques (e.g. centrifugation or filtration) can be employed to separate inorganic food additives from the food matrix based upon their size, density and fluid properties. But their performance is also limited by the fact that the fraction of inorganic food additives is small and there are number of other food substances that are comparable to the inorganic food additives in size and density. Also, the viscous nature of food products derived from various proteins and polysaccharides is an additional barrier that has to be taken into account when centrifugation and filtration are employed for the extraction. Magnetic separation is a robust technique for rapid separation and concentration of analytes of interest from complex sample matrices without affecting their physical properties and morphological features (Luo et al., 2019a, a; Luo et al., 2018b). By using an appropriate affinity towards the target analytes, magnetic separation could offer high efficiency and specificity when compared with conventional centrifugation or filtration methods. Recently, magnetic materials coupled with charged ligand have been used to separate oppositely charged biomolecules, such as DNA (Esser et al., 2006), peptide (Bu et al., 2015), polysaccharide (Liu et al., 2014), and microorganism (Luo et al., 2018a). Herein, we present a fairly simple and effective approach for the extraction and concentration of food additive TiO2 from commercial foods by utilizing charge-switchable magnetic material, poly-L-lysine coated starch magnetic bead (PL@SMBs), of which its surface charge could be modulated from strong positive to neutral depending on the pH of surrounding environment. Food additive TiO2 having negative surface charge could be captured and released by switching the surface charge of magnetic particles. The morphology and size distribution of the magnetically extracted TiO2 from commercial processed foods were investigated by electron microscopy, and the titanium contents in food samples were analyzed by ICP-AES. In addition, the potential cytotoxicity of commercial E171 in pristine form and food additive TiO2 extracted from processed food were examined in vitro.

Titanium dioxide (TiO2) is a naturally occurring oxide of titanium, exhibiting three crystalline phases known as anatase, rutile, and brookite (Oveisi et al., 2010). TiO2 is one of the most commonly used pigments with a range of applications in food, cosmetics, paint, paper, plastic, rubber, and so on (Weir et al., 2012). The use of TiO2 as a white pigment has consistently increased since 1916, and the global production of TiO2 exceeded 7 million metric tons in 2018 (Loosli et al., 2019). Expanding use of TiO2 as part of a food additive in modern processed food would substantially increase its oral intake regardless of the age and gender of consumers. Food-grade TiO2 or E171 have been used as a colorant to enhance and brighten the color of food and personal care products, such as candies, chewing gum, toothpaste, drug capsule, and tablet type dietary supplements. The US food and drug administration (FDA) approved the use of TiO2 in food with an allowing level up to 1% (w/w), and the annual consumption of TiO2 as a part of food is estimated to be 2780 tons (Yang et al., 2014). Moreover, a Monte Carlo analysis of human exposure associated with TiO2 in food revealed that children under the age of 10 show the highest exposure level (1−3 mg TiO2/kg of body weight/day) compared to adults among American and British because TiO2 content in sweets is higher than other types of food products (Weir et al., 2012). E171 has been authorized without any established limitation in daily intake due to its low intestinal absorption, solubility, and toxicity (Authority, 2005). However, excess use of E171 has raised concerns on public health since significant levels of dietary intake is associated with the nanoscale TiO2, having one or more external dimensions in the size range of 1–100 nm. Although food additive TiO2 is not regarded as nanomaterials and industrial producers are not required to report it as a nanomaterial, food-grade TiO2 (E171) has a very broad size distribution (30−400 nm) with up to 36 % of total particles falling below 100 nm in at least one dimension (Weir et al., 2012). A number of toxicological studies have shown that TiO2 nanoparticles (NPs) could cause adverse health effects associated with inflammation, tissue necrosis, renal apoptosis, and immune response (Grande and Tucci, 2016; Bettini et al., 2017; Gui et al., 2013). Moreover, the International Agency for Research on Cancer has classified TiO2 NPs as “possibly carcinogenic to humans” on the basis of the experimental evidence from animal inhalation studies (Baan et al., 2006). In response to the emerging health concern towards oral consumption of nanoscale TiO2, France government announced a ban for the use of food products containing TiO2 from 2020 based on the opinion proposed by the French food safety agency (Audran, 2019). As the toxicity of the nanoparticles on human depends on its size, surface property, and lattice structure, it is important to monitor and characterize the food grade E171 TiO2 in commercial food in order to fully assess its potential impact on human health. Several tools, such as inductively coupled plasma mass spectrometry (ICP-MS), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction analysis (XRD), and dynamic light scattering (DLS), etc., are regarded as robust and effective tools to detect, characterize, and quantify food additive TiO2 in their raw or pristine states (Yang et al., 2014). In reality, however, there are significant analytical challenges in the characterization of TiO2 in food, because they are present at low levels and often tightly complexed with various food matrices. The commercial processed food containing E171 typically take the form of dry solids like powders or cereals, and partially hydrated or semi-fluidic materials like gums, syrups, and oils, which would adversely affect the analytical power of the instruments (Singh et al., 2014). Those issues associated with the complex interaction of TiO2 with food matrices and diverse form of TiO2-containing food could bring about analytical challenges, which should be resolved prior to the analysis. Over the past decades, the most widely used methods for the extraction/separation of inorganic nanomaterials from the complex

2. Materials and methods 2.1. Materials Pullulanase, ferrous chloride tetrahydrate (FeCl2·4H2O), poly-L-lysine (Mw 150,000–300,000 Da), dextran (Mw 9,000–11,000), crystal violet, ferric chloride hexahydrate (FeCl3·6H2O), ammonium hydroxide was purchased from Sigma-Aldrich (St. Louis, MO, USA). Waxy maize starch was obtained from Samyang Co. (Seoul, Korea). Hydrogen peroxide, nitric acid, and sulfuric acid were purchased from Samchun Pure Chemical Co., Ltd., (Pyeongtaek, Gyeonggi-do, Korea). Human intestinal epithelial Caco-2 cells were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea). Minimum essential medium (MEM) and streptomycin were purchased from Welgene (Gyeongsan, Korea). Water-soluble tetrazolium salt (WST-1) solution was obtained from Roche (Mannheim, Germany). Carboxy-2′,7′-dichlorofluorescein diacetate (H2DCFDA) were purchased from Molecular Probes Inc. (Eugene, OR, USA). 2

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2.2. Preparation of poly-L-lysine-coated starch magnetic beads (PL@SMBs)

after magnetic separation.

Starch magnetic beads (SMBs) were prepared by the self-assembly process of short chain glucans (SCG) generated by enzymatic debranching of amylopectins in waxy maize starch, while dextran-coated iron oxide (Fe3O4) nanoparticles (Dex@IONPs, ∼130 nm in diameter) prepared by the coprecipitation process using dextran and iron chloride (see the supporting information) were spontaneously incorporated into the growing particle during the self-assembly reaction (Luo et al., 2018a, c). Debranching of amylopectins in waxy maize starch was carried out as described elsewhere (Luo et al., 2019b, c). Briefly, two grams of waxy maize starch was dissolved in 50 ml of deionized distilled water (DW) and boiled at 100 °C for 30 min for gelatinization. After cooling to 60 °C, pullulanase was added to the gelatinized starch solution to a final concentration of 100 ASPU/ml and incubated at 60 °C for 24 h. After the incubation, the reaction solution was centrifuged at 15,000 xg for 20 min, and the supernatant was transferred to a fresh tube, followed by addition of Dex@IONPs to a final concentration of 5 mg/ml. The mixture was then incubated at 4 °C for 24 h to induce the self-assembly reaction to form SMBs. The synthesized SMBs were washed three times with DW and its morphology and composition was analyzed by scanning electron microscopy (SEM, TM 3000, Hitachi, Tokyo, Japan) and field emission transmission electron microscopy (FETEM, JEM-2100 F, JEOL, USA) equipped with EDS elemental mapping of iron, carbon and oxygen. Magnetic properties of SMBs were measured using physical property measurement system (16 T PPMS Dynacool, Quantum Design, USA) at room temperature from −12,000 to 12,000 Oe. To prepare poly-L-lysine-coated SMBs (PL@SMBs), 10 mg of SMBs was introduced to 1 ml of poly-L-lysine (0.1 % w/v) solution, and incubated at room temperature for 30 min with gentle rotation. The sample was washed with DW 3 times to remove residual poly-L-lysine. The surface charge of the prepared PL@SMBs was measured by using dynamic light scattering (DLS, Zetasizer Nano ZS90, Malvern Instruments). The final product was stored at 4 °C until use.

%RE=1-

2.4. Quantitative analysis The content of TiO2 particles was measured as described elsewhere with modification (Hwang et al., 2019). Briefly, food sample was suspended in 10 ml DW to a final concentration of 100 mg/ml. 10 ml of sulfuric acid was added to each sample and heated at 200 °C for 2 h to dissolve Ti from TiO2 particles and heated at 380 °C until the remaining solution was removed. The digested products were resuspended in 5 ml DW and total Ti levels were analyzed by ICP-AES (JY2000 Ultrace). 2.5. Separation of TiO2 particles from simulated food by acid digestion and centrifugation Simulated chewing gum was prepared by coating the surface of 1.0 g of gum base with 0.02 g of food additive TiO2 (T5) which was subsequently covered with a thin layer of caramelized sugar and solidified at room temperature. For preparation of simulated candy, 0.1 g of TiO2 powder (T5) was thoroughly mixed with 4.9 g of ground sugar (Samyang Co., Seoul, Korea). The mixture in glass vial was melted on hot plate, poured into a mold to a final mass of 1 g and solidified at room temperature. Separation of TiO2 particles from the simulated food by acid digestion in combination with centrifugation was conducted as described elsewhere with modification (Lim et al., 2018). Briefly, 1 g of test food samples was transferred to a glass vial and dissolved in 5 ml of H2O2/HNO3 (10:0.1, v/v), followed by boiling at 100 °C for 10 min. The samples were then transferred to a clean 15 ml tubes and centrifuged at 7000 xg for 10 min. The pellet was resuspended in 2 ml of nitric acid and boiled for 20 min, followed by centrifugation at 7000 xg for 10 min. The harvested particles as a pellet were washed three time with DW and acetone, respectively. The extracted samples were dispersed in ethanol and stored at 4 °C until use. The cytotoxicity assay of extracted TiO2 particles was carried out as described in supporting information.

Five representative commercial food additive TiO2 (T1-T5) were dispersed in DW to a final concentration of 1 mg/ml. PL@SMBs were introduced to the sample suspension to a final concentration of 5 mg/ml and incubated at room temperature for 10 min with gentle rotation. After that, the eppendorf (EP) tube containing the mixture was placed close to a neodymium magnet (50 mm × 5 mm×25.4 mm) for 3 min to collect TiO2 particles bound to the PL@SMBs to the side of tube, and the supernatant containing unbound TiO2 was transferred to a fresh EP tube. The harvested TiO2 particles-PL@SMBs were resuspended in 25 mM NH4OH solution and subjected to bath sonication for 5 min to elute TiO2 particles from the magnetic beads. PL@SMBs were subsequently removed by magnet, and the solution containing eluted TiO2 particles was transferred to a fresh EP tube. The amount of captured and eluted TiO2 particles was measured by using inductively coupled plasmaatomic emission spectroscopy (ICP-AES, JY2000 Ultrace, HORIBA Jobin Yvon, longjumeau, France). The mass of TiO2 was calculated by the following equation, Eq. (1):

2.6. Characterization of TiO2 Food additive TiO2 recovered from food samples were characterized by Fourier transform infrared (FT-IR), X-ray diffraction (XRD), TEM, and SEM analysis. FT-IR spectra for the TiO2 samples were recorded using a Perkin-Elmer Spectrum One System spectrometer (Foster City, CA, USA) with KBr pellets in the range of 1500 to 4000 cm−1 (Luo et al., 2018c). The crystal structure of TiO2 particle was analyzed from 4° to 80° (2θ) using Cu-Kα radiation on a Bruker D8 Advance X-ray diffractometer (Bruker, Karlsruhe, Germany). All samples were fully dehydrated in a vacuum desiccator before XRD analysis. The morphology and composition of the recovered TiO2 samples were analyzed by TEM (JEM-2100 F) and high-resolution scanning electron microscope (HR-SEM, MERLIN Carl Zeiss, Oberkochen, Germany) equipped with EDS elemental mapping of titanium (Ti) and oxygen (O). The particle size distribution TiO2 was estimated from the HR-SEM images by measuring the diameter of at least 100 particles. Hydrodynamic

(1)

where MTiO2 is the mass of TiO2 in test sample, and MTi is the mass of titanium measured by ICP-AES. The relative capture efficiency (%CE), Eq. (2), and recovery efficiency (%RE), Eq. (3), of the PL@SMBs for TiO2 particles were calculated by the following equation:

%CE=

Ninitial − Nunbound ×100 Ninitial

(3)

where Ninitial is the initial concentration of TiO2 in test sample prior to magnetic separation, and Nreleased is the concentration of released TiO2 from PL@SMBs. To separate and quantify TiO2 particles from real food, ten different commercial food (mentos-candy, drug capsule, vitamin pill, chocolate, gum, M&M (milk chocolate), M&M (peanut chocolate), skittles-candy, syrup, jelly manufactured by multinational food company were purchased from market. One gram of food samples was resuspended in 10 ml DW, followed by addition of PL@SMBs to a final concentration of 5 mg/ml. The rest procedures for the separation of TiO2 particles from the food were same as described above.

2.3. Magnetic separation of TiO2 by PL@SMBs

MTiO2 = MTi × 1.668

Ninitial − Nreleased ×100 Ninitial

(2)

where Ninitial is the initial concentration of TiO2 in test sample prior to magnetic separation, and Nunbound is the concentration of unbound TiO2 3

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Fig. 1. (A) Schematics illustrating the fabrication of Starch magnetic beads (SMBs) through co-crystallization of iron oxide nanoparticles (IONPs) and short-chain glucan (SCG) obtained by enzymatic hydrolysis of waxy maize starch using pullulanase. (B) SEM image of SMBs. (C) Magnetization curve for PL@SMBs. The insert shows magnetic separation of SMBs suspended in DW. (D) TEM image of SMBs combined with elemental mapping images representing iron (Fe), oxygen (O), and carbon (C).

gradually neutralized in the presence of TiO2, suggesting that the surface charge of PL@SMBs was passivated by oppositely charged TiO2 particles in concentration dependent manner (Bastakoti et al., 2014) (Fig. 2B). Fig. 2C shows a magnetic separation of TiO2 using PL@SMBs. The TiO2 particles dispersed in DW were effectively captured by PL@ SMBs through electrostatic interaction and collected to the side of tube by using a neodymium magnet. The captured TiO2 was readily released from the PL@SMBs by switching the pH of solution to basic with 1 vol% NH4OH. Upon the elution of TiO2 particles, PL@SMBs were selectively removed from the mixture by applying external magnetic field. The eluted TiO2 was quantified by ICP-AES in order to evaluate the capture and recovery efficiency of PL@SMBs. The PL@SMBs showed an excellent capture and recovery efficiency (over 85 %) for all TiO2 samples regardless of the brand (Fig. 2D). It should be noted that both TiO2 and PL@SMBs maintained their original surface charge after recovery, indicating that poly-L-lysine was not eluted along with TiO2 from the surface of SMBs (Fig. 2E). Subsequently, the SMBs were further washed with 20 % ethanol and DW to remove the remaining ammonium. The capture efficiency as well as recovery efficiency of PL@SMBs for food additive TiO2 (T5) particles was not deteriorated over the course of three consecutive recycling processes (Fig. S3). Moreover, the charge density of PL@SMBs was not much affected by the recycling steps, demonstrating the excellent structural and functional stability of the magnetic particles.

diameter and zeta-potential of the TiO2 were measured by using dynamic light scattering (DLS, Zetasizer Nano ZS90, Malvern Instruments). 3. Results and discussion 3.1. Preparation of PL@SMBs for magnetic separation of food additive TiO2 from food Starch magnetic beads (SMBs) were synthesized through co-crystallization of iron oxide nanoparticles (IONPs) and short-chain glucan (SCG) obtained by enzymatic hydrolysis of waxy maize starch using pullulanase (Fig. 1A) (Luo et al., 2018a). SEM image revealed a welldefined spherical SMBs with a uniform size distribution centered at 2.2 μm (Figs. 1B and S1). The synthesized SMBs were shown to have a strong superparamagnetic behavior with a saturated magnetization value of 20 emu g−1 (Fig. 1C). Since starch particles produced by recrystallization of SCG have a B-type crystalline structure that is very stable in acid and neutral environment (Luo et al., 2018d), the SMB was very stable and maintained its structural integrity throughout the course of magnetic separation. TEM analysis and elemental mapping showed the presence of well-distributed Fe within the microstructure along with C and O, which were considered as skeletal materials of SMBs (Fig. 1D). The surface of SMBs was functionalized with poly-L-lysine by electrostatic interactions between negatively charged SMBs and positively charged poly-L-lysine, which conferred a highly positive surface charge on the PL@SMBs (Fig. 2A). The Zeta-potential of PL@SMBs in DW was found to maintain over 35 mV even after three consecutive washing with DW, suggesting the strong adhesion of poly-L-lysine on the surface of magnetic microbeads. Owing to the strong electrostatic repulsive forces, PL@SMBs or TiO2 alone was well-dispersed in DW as a homogeneous suspension. But, upon mixing with E171 TiO2, PL@SMBs instantly formed large aggregates due to the strong electrostatic attraction between the oppositely charged species (Bastakoti et al., 2015) (Fig. S2). In addition, we observed that the surface charge of PL@SMBs was

3.2. Characterization of commercial food additive TiO2 The physical properties of commercial food additive TiO2 (E171) from five major manufacturers were analyzed prior to the investigation of the TiO2 present in commercial processed food. SEM analysis revealed that all the E171 samples from five different manufacturers were spherical shaped with a mean primary particle size of 150.3 ± 26.5 nm (T1), 153.1 ± 15.6 nm (T2), 169.8 ± 21.9 nm (T3), 120.5 ± 21.1 nm (T4), and 132.8 ± 23.7 nm (T5) with a size distribution ranging from 50 to 300 nm (Figs. 3A and S4). The majority of TiO2 particles (∼82 %) were between 100−200 nm in diameter, whereas those below 100 nm and over 200 nm accounted for around 13 % and 6%, respectively 4

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Fig. 2. (A) Illustration of poly-L-lysine-coated starch magnetic beads (PL@SMBs). (B) Surface charge of PL@SMBs (5 mg/ml) in the presence of varying concentration of commercial E171. Transmission light microscopy images of PL@SMBs (i), mixture of PL@SMBs and E171 (ii), and E171 (iii) in aqueous solution were shown above the graph. (C) Collection of the TiO2 bound to PL@SMBs by magnetic field, followed by elution of the captured TiO2 from the magnetic beads with NH4OH in ultrasonic (US) bath for 5 min. (D) Capture and recovery efficiency of PL@SMBs for the five commercial E171 (T1-T5). (E) Surface charge of PL@SMBs and TiO2 after elution step.

materials from complicated food matrices would be plausible.

(Fig. 3B). XRD analysis was carried out to determine the crystal structure of food additive E171. All the five E171 samples showed distinctive diffraction peaks at 2θ values of 25.3°, 37.8°, 38.5°, 48.1°, 53.9°, 55.1°, 62.7°, 68.9°, 70.3°, 75.1° and 76.1° corresponding to the crystal planes of (101), (004), (112), (200), (105), (211), (204), (116), (220), (215) and (301), respectively (Fig. 3C). This result indicated that anatase-type TiO2 are dominating in all tested commercial food additive E171 samples. It has been reported that the toxicity of anatase-type TiO2 is greater than that of rutile-type in nanoparticulate form but reactive oxygen species (ROS) generation by anatase-type TiO2 is not significant under ambient light condition (Fadeel and Garcia-Bennett, 2010). We subsequently investigated the surface charge of E171 in a range of pH in order to validate the applicability of charge-based separation of TiO2 from processed food. As shown in Fig. 3D, the surface charges of all five samples were negative at pH value higher than its isoelectric points, which is approximately 3.5. The Zeta-potential of E171 in aqueous solution reached higher than −20 mV at the pH above 5. The highly negative surface charge of the E171 in neutral and basic environments indicated that charge-based capture and separation of the inorganic

3.3. The effect of separation methods on the size and morphology of food additive TiO2 The methods or procedures that are employed to extract inorganic food additives from food should have a minimal effect on the physical morphology of extracted materials. Any alteration in chemical and physical properties of extracted TiO2, which could take place during the separation procedure, would give a false information about the food additives in processed food. Considering that acid digestion of food matrices using strong acid is a conventional method that is the most widely used to liberate inorganic particles from the food, we investigate the effect of magnetic separation method on size and morphology of TiO2 in comparison with that of acid digestion with HNO3 and H2O2. In order to investigate the effect of separation methods on the size and morphology of food additive TiO2, we prepared simulated gum and candy samples spiked with 2 % (w/w) of commercial food additive E171 (T5). The extracted TiO2 particles from the simulated food using magnetic separation and acid digestion were characterized in terms of

Fig. 3. SEM image (A), histogram of primary particle size distribution (B), and XRD patterns (C) of five commercial food additive TiO2, which are named as T1 (i), T2 (ii), T3 (iii), T4 (iv), and T5 (v). The particle size distribution of each TiO2 sample was estimated by measuring the diameter of at least 100 particles from the SEM images. (D) Zeta-potential of five commercial food additive TiO2 (T1-T5) as a function of pH. 5

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Fig. 4. TEM (A), SEM (B), particle size distribution (C), FT-IR (D), and XRD (E) analysis of pristine TiO2 (i), TiO2 recovered from simulated gum (ii) and simulated candy (iii) samples by magnetic separation, and TiO2 recovered from simulated gum (iv) and simulated candy (v) samples by acid digestion. The estimated mean particle size of each sample was labeled on top of corresponding peak. The fraction (%) of particles smaller than 100 nm was indicated in each histogram.

physical properties of TiO2 in particle diameter, which resulted in an decrease in surface charge density of the particles (Suttiponparnit et al., 2010). XRD pattern demonstrated that the pristine TiO2 has an average crystallite sizes of around 89.23 nm that is comparable to the size observed with TEM. It was found that the crystal characteristics of the TiO2 particles were also not affected by both methods. Moreover, XRD patterns of the recovered TiO2 exhibited clear diffraction peaks that are characteristic to anatase phase, which are identical to the original E171 (Fig. 4E). This finding indicates that although there were no changes in crystalline structure, acid digestion affected the surface charge and size of TiO2 particles. Since surface charge and particles size are regarded as the most important factors affecting cellular uptake and cytotoxicity of TiO2 particles in human cells study (Kim et al., 2013), the magnetic separation method employing PL@SMBs would provide an effective means of extracting intact form of food additive TiO2 from food matrices for the accurate evaluation of its potential health risks.

chemical composition, crystallinity, surface charge, particle size and morphology. The pristine E171 (T5) without any treatment was also examined as a control. TEM analysis revealed that there is a negligible change in the morphology of TiO2 recovered by magnetic separation in comparison with that of pristine E171 TiO2 (control) (Fig. 4A). On the other hand, overall size of TiO2 particles recovered by acid digestion was found to be smaller than the original E171. Furthermore, a number of fragmented particles smaller than 10 nm were observed in recovered sample. Although TiO2 has been reported to be resistant to nitric acid (Singh et al., 2014), the harsh treatment of the sample with strong acid in high temperature would have affected the integrity of the inorganic particles, leading to the alteration in their sizes and morphologies. The effect of these two methods on the size of TiO2 particles was further investigated by SEM analysis and particle size distribution measurements (Fig. 4B and C). The size distribution analysis revealed that the mean particle size of TiO2 recovered by magnetic separation from simulated gum and candy samples were 131.8 ± 25.4 nm and 132.5 ± 19.2 nm, respectively, which were not much different from that of control E171 before separation (133.2 ± 24.7 nm) (Fig. 4C). On the other hand, a significant shift in size distribution was observed when acid digestion was employed for the extraction of TiO2 from simulated foods. The mean particle sizes of TiO2 recovered from simulated gum and candy by acid digestion were 94.3 ± 26.1 nm and 84.1 ± 22.9 nm, respectively, which were notably smaller than the pristine E171 before extraction. The fraction of TiO2 particles smaller than 100 nm increased from 21 % to 76 % after the acid digestion. In addition, the TiO2 particles recovered by acid digestion and magnetic separation were subjected to FT-IR and XRD analysis in order to investigate if there is any alteration in chemical and physical properties of TiO2 during the extraction procedure. FT-IR data suggested that the surface of TiO2 particles were free from any chemical debris originated from food components regardless of extraction methods (Fig. 4D). The absorption band between 400–800 cm−1 was derived from the Ti-O stretching and Ti-O-Ti bridging stretching vibrations (Jiang et al., 2013). This result suggests that both acid digestion and magnetic separation effectively recovered TiO2 particles from food samples without any notable adsorption of food matrices on the surface of particle. However, the surface charge of TiO2 particles (Gum/AD and Candy/ AD) significantly decreased to almost a half after extracting the particles by acid digestion, whereas magnetic separation brought about negligible changes on the surface charge of TiO2 particles (Gum/MS and Candy/MS) (Fig. S5). Even though there is no sign of debris remaining on the surface of TiO2 particles, acid treatment affected

3.4. Extraction and analysis of TiO2 from commercial food The PL@SMBs developed in this study were applied to extract the food additive E171 from real processed food. Ten different types of commercial food products that were manufactured by major multinational food companies were selected to investigate the morphology and size of TiO2 added to the food products as a food additive. All the selected food products were labeled as containing TiO2 in varying content. The food samples were first dissolved in DW, followed by centrifugation to remove soluble food matrices, such as protein, lipid and polysaccharide. The PL@SMBs showed an excellent capture efficiency that separated over 80 % of TiO2 from all the tested food samples except Mentos® candy showing about 60 % capture efficiency (Fig. S6). The final recovery efficiency of TiO2 from food by magnetic separation was from 30 % to 83 % depending on the types of food (Fig. S7). The decrease in the recovery efficiency might be due to the adsorption of food matrix, such as protein and polysaccharide, on both TiO2 particles and PL@SMBs, leading to cross-linking of these two particles through nonspecific hydrophobic interactions. That is, TiO2 particles could be captured by PL@SMBs through both electrostatic and hydrophobic interactions, of which the physically adsorbed TiO2 particles was not fully eluted by elevating pH with NH4OH. The extracted E171 TiO2 from commercial food were characterized by electron microscopy in combination with element mapping of titanium (Ti) and oxygen (O). SEM images show that food additive TiO2 were effectively extracted in 6

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Fig. 5. (A) SEM images of TiO2 before (upper panel) and after (lower panel) magnetic separation from the ten commercial food. (B) EDX elemental mapping of the corresponding SEM image of the recovered TiO2 showing Ti element (upper panel) and O element (lower panel). The size distributions of TiO2 particles measured after magnetic separation were presented below the corresponding SEM images. The mean particle sizes and the fraction (%) of TiO2 particles smaller than 100 nm were labeled in each histogram. S1, mentos-candy; S2, M&M-peanut chocolate; S3, chocolate; S4, vitamin pill; S5, skittles candy; S6, jelly; S7, syrup; S8, drug capsule; S9, gum; S10, M&M (milk chocolate). The scale bar represents 1 μm.

derived from the lipid crystal. TiO2 particles were heavily covered with food-origin bulk organic materials in most samples except chewing gum. In case of chewing gum, most of the food additive TiO2 (> 90 %) are known to be present in water soluble outer shell (Weir et al., 2012), and insoluble gum base can be easily removed from the suspension, making it relatively simple to recover TiO2 particles by washing and centrifugation. However, most of food products contain various bulk organic substances, such as carbohydrates, proteins and fats, which are typically complexed with the food additive TiO2. Therefore, selective capture and separation of TiO2 particles as well as removing all the food matrices would be essential to recover intact TiO2 particles from food products. In this regard, magnetic separation using PL@SMBs was found to be effective for the extraction of intact food additive TiO2 in highly pure form.

highly pure form from the commercial food (Fig. 5A). The size and morphology of the extracted TiO2 particles were comparable to those of pristine E171 that were widely used in food industry. The magnetic particles that were employed to capture and separate the TiO2 were not observed in extracted samples, suggesting that the magnetic particles were effectively removed from the sample by external magnetic field. Furthermore, elemental mapping analysis confirmed that the extracted particles were mainly composed of Ti and O (Fig. 5B). The food samples without magnetic separation were also examined by electron microscopy. Food samples were thoroughly resuspended in water and subjected to centrifugation to remove soluble food matrices. The SEM image revealed that the major components of the pellet were organic substances that were precipitated during the centrifugation (Fig. 5A). The crystal structures observed in chocolate sample would be 7

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Fig. 6. (A) Histogram of primary particle size distribution of food additive TiO2 extracted from 10 commercial processed food. Inset shows the Gaussian curve fitted to the corresponding size distribution. The estimated mean particle sizes of the extracted TiO2 were labeled on top of the Gaussian peak. The fraction (%) of particles below 100 nm was indicated in the histogram. (B) Titanium contents in 10 commercial food measured by ICP-AES. Fig. 7. Effect of food additive TiO2 on Caco-2 cell proliferation (A) and intracellular ROS generation (B) after incubation for 24 h. Abbreviation: DW, deionized water as a control; TiO2, pristine TiO2; A-TiO2, commercial E171 TiO2 recovered from water by acid digestion; M-TiO2, commercial E171 TiO2 recovered from water by magnetic separation; Agum, TiO2 recovered from gum by acid digestion; M-gum, TiO2 recovered from gum by magnetic separation; A-cho, TiO2 recovered from chocolate by acid digestion; M-gum, TiO2 recovered from chocolate by magnetic separation. Different lower-case letters (a and b) indicate significant differences from controls (DW) (p < 0.05).

Fig. 8. Cytotoxic effect of varying concentrations of food additive TiO2 on colony-forming ability after incubation for 14 days. Representative images (left) of cell colony formation assay and (right) histogram of colony number. Abbreviation: DW, control; pristine TiO2; A-TiO2, pristine TiO2 recovered from water by acidic digestion; M-TiO2, pristine TiO2 recovered from water by magnetic separation; A-gum, TiO2 recovered from gum by acidic digestion; M-gum, TiO2 recovered from gum by magnetic separation; A-cho, TiO2 recovered from chocolate by acidic digestion; M-gum, TiO2 recovered from chocolate by magnetic separation. Different lower-case letters (a and b) indicate significant differences from untreated controls (DW) (p < 0.05).

commercial food products, thus the direct comparison of the TiO2 particles extracted from the product with the original ones was not performed in this study. Considering that magnetic separation by using PL@SMBs did not affect the size of TiO2 particles (Fig. 4), a slight degradation of E171 during the food processing would be a possible reason for the relatively smaller size of TiO2 in commercial food in comparison to that of pristine E171 in market. Furthermore, the Ti contents of 10 commercial foods were determined by ICP-AES measurement, of which the total mass of TiO2 was calculated on the basis of the ratio of titanium and oxygen. The content of TiO2 in food products varied from 0.01–10 mg per gram of food, where mentos® candy was found to contain the highest amount of TiO2 with a concentration of 8.7

In addition, the primary particle size distribution of TiO2 as well as the Ti contents in all the 10 commercial food were determined by SEM analysis and ICP-AES, respectively. The size distribution histogram showed that the extracted TiO2 particles from 10 commercial food have a broad size distribution (40−250 nm) with a mean particle size of 114.7 ± 38.7 nm (Fig. 6A). It should be noted that around 21.8 % of the particles extracted from 10 commercial food fell below 100 nm in diameter. The ratio of TiO2 particles below 100 nm varied from 6 % to 38 % depending on the type of food products, which were found to be higher than that of the most widely distributed commercial E171 (approximately 12.8 %) (Fig. 3B). We were unable to identify the original source of the materials (E171) that were actually added to the

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understanding the nature of food additive TiO2 in commercial food. We applied the PL@SMBs developed in this study to extract TiO2 particles from 10 commercial processed food manufactured by multinational food companies. The extracted TiO2 particles from 10 commercial food showed a broad size distribution (40−250 nm) with a mean particle diameter of 115 nm, of which around 22 % of them fell below 100 nm. According to the in vitro cytotoxicity analysis, the TiO2 particles extracted from food exhibited insignificant toxicity to human intestinal epithelial cells but was shown to induce cellular oxidative stress at high concentration. In this regard, the charge-switchable magnetic separation proposed in this study would provide a more accurate means of understanding the nature of TiO2 that are present in commercial food and evaluating their potential toxicity by further study in near future.

mg/g (Fig. 6B). It is interesting to note that vitamin pill and dietary supplement capsule contain a higher amount of TiO2 in comparison to other sweets products, such as chewing gums, chocolate, and jelly, which have been reported to have higher TiO2 contents. As a limited information about the food additive TiO2 is listed on the product packaging, the method proposed in this study would provide a useful means of monitoring and understanding the nature of TiO2 present in food. This method could also be applied to monitor various Ti-O nanostructures, such as nanotubes (Kiatkittipong and Assabumrungrat, 2017) and nanoribbons (Eiamsa-ard and Kiatkittipong, 2019) in a range of consumer products and environment. 3.5. Cytotoxicity of food additive TiO2

Credit author statement

To investigate the cytotoxic effect of food additive TiO2 on cell viability and proliferation, human intestinal epithelial Caco-2 cells cultured in 96-well plates were treated with varying concentration of pristine commercial E171 (T5) and TiO2 particles extracted from water and food products, including chewing gum and chocolate, by magnetic separation and acid digestion. Regardless of the recovery methods, no significant viability variations were observed over 24 h of treatment with food additive TiO2 recovered from food samples with a concentration ranging from 0 to 1000 μg/ml (Fig. 7A). The H2DCF-DA assay was carried out to evaluate intracellular ROS generation in Caco2 cells treated with varying concentrations of TiO2. Oxidative stress was estimated based on the oxidation of DCFDA to a fluorescent product, 2′,7′-dichlorofluorescein (DCF) in the presence of ROS. Exposure of the cells to TiO2 led to different degrees of ROS generation, in which a significant (p < 0.05) increase of ROS level was observed at TiO2 concentration over 125 μg/ml in a dose dependent manner. On the other hand, we did not observe significant differences in ROS generation between 7 different types of test sample (Fig. 7B). The effect of food additive TiO2 on long-term cell growth was further investigated by clonogenic survival assay. As shown in Fig. 8 treatment with 250 μg/ml of TiO2 significantly inhibited (p < 0.05) the colony formation, whereas no significant differences in growth inhibition were observed between test samples. From the results, it is clear that the dose-dependent cytotoxicity of TiO2 was predominant at high concentration in terms of cellular oxidative stress and inhibition of cell growth. Its cytotoxicity observed in the present study was comparable to those reported by other study obtained by TiO2 nanoparticles (Hwang et al., 2019). The results also show that the extraction methods, both magnetic separation and acid digestion, did not affect the cytotoxicity of TiO2, even though acid digestion was found to cause a decrease in particle size of TiO2 (Fig. 4). As we have developed an effective method for extracting intact form of food additive TiO2 from commercial food, we expect that more accurate evaluation on the potential toxicity of food additive TiO2 particles that are present in a wide range of commercial food would be possible through animal studies in near future.

I certify that all authors have seen and approved the final version of the manuscript being submitted. They warrant that the article is the authors' original work, hasn’t received prior publication and isn't under consideration for publication elsewhere. Declaration of Competing Interest The authors declare that there are no conflicts of interest. Acknowledgement This research was supported by a grant (18163MFDS011) from Ministry of Food and Drug Safety in 2018. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jhazmat.2020.122483. References Audran, X., 2019. France Bans Titanium Dioxide in Food Products by January 2020. United States Department of Agriculture. Authority, E.F.S., 2005. Opinion of the Scientific Panel on food additives, flavourings, processing aids and materials in contact with food (AFC) on Titanium dioxide. EFSA J. 3, 163. Baan, R., Straif, K., Grosse, Y., Secretan, B., El Ghissassi, F., Cogliano, V., 2006. Carcinogenicity of carbon black, titanium dioxide, and talc. Lancet Oncol. 7, 295–296. Bastakoti, B.P., Ishihara, S., Leo, S.-Y., Ariga, K., Wu, K.C.W., Yamauchi, Y., 2014. Polymeric micelle assembly for preparation of large-sized mesoporous metal oxides with various compositions. Langmuir 30, 651–659. Bastakoti, B.P., Li, Y., Imura, M., Miyamoto, N., Nakato, T., Sasaki, T., Yamauchi, Y., 2015. Polymeric micelle assembly with inorganic nanosheets for construction of mesoporous architectures with crystallized walls. Angew. Chem. 54, 4222–4225. Bettini, S., Boutet-Robinet, E., Cartier, C., Coméra, C., Gaultier, E., Dupuy, J., Naud, N., Taché, S., Grysan, P., Reguer, S., Thieriet, N., Réfrégiers, M., Thiaudière, D., Cravedi, J.-P., Carrière, M., Audinot, J.-N., Pierre, F.H., Guzylack-Piriou, L., Houdeau, E., 2017. Food-grade TiO2 impairs intestinal and systemic immune homeostasis, initiates preneoplastic lesions and promotes aberrant crypt development in the rat colon. Sci. Rep. 7, 40373. Bu, T., Zako, T., Zeltner, M., Sörgjerd, K.M., Schumacher, C.M., Hofer, C.J., Stark, W.J., Maeda, M., 2015. Adsorption and separation of amyloid beta aggregates using ferromagnetic nanoparticles coated with charged polymer brushes. J. Mater. Chem. B 3, 3351–3357. Eiamsa-ard, S., Kiatkittipong, K., 2019. Thermohydraulics of TiO2/Water nanofluid in a round tube with twisted tape inserts. Int. J. Thermophys. 40, 28. Esser, K.-H., Marx, W.H., Lisowsky, T., 2006. maxXbond: first regeneration system for DNA binding silica matrices. Nat. Methods 3, i–ii. Fadeel, B., Garcia-Bennett, A.E., 2010. Better safe than sorry: understanding the toxicological properties of inorganic nanoparticles manufactured for biomedical applications. Adv. Drug Del. Rev. 62, 362–374. Grande, F., Tucci, P., 2016. Titanium dioxide nanoparticles: a risk for human health? Mini Rev. Med. Chem. 16, 762–769. Gui, S., Sang, X., Zheng, L., Ze, Y., Zhao, X., Sheng, L., Sun, Q., Cheng, Z., Cheng, J., Hu, R., Wang, L., Hong, F., Tang, M., 2013. Retracted article: Intragastric exposureto titanium dioxide nanoparticles induced nephrotoxicity in mice, assessed byphysiological and gene expression modifications. Part. Fibre Toxicol. 10, 4.

4. Conclusions Here, we report a facile and effective approach to extract intact form of food additive TiO2 from commercial processed food by utilizing charge-switchable starch magnetic beads (PL@SMBs). The strong electrostatic interaction between negatively charged food additive TiO2 and positively charged PL@SMBs was the key for the capture and separation of TiO2 particles with a capture efficiency over 80 % in most processed food, of which the captured TiO2 particles were readily harvested from PL@SMBs by switching its charge to neutral. Significant alterations in size and surface charge were observed when the most widely used acid digestion was employed for the extraction of food additive TiO2 from food. On the other hand, magnetic extraction of TiO2 from food using PL@SMBs maintained their original size and morphology, which provides more accurate means of monitoring and 9

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