Cloud point extraction and characterization of zinc oxide nanoparticles isolated from infant milk formulas (original) (raw)

Abstract

The increasing presence of nanoparticles in food products, especially in those consumed by sensitive populations like infants, raises justified health concerns. The presence of zinc oxide nanoparticles (ZnO-NPs) in three different commercial infant milk formulas were analyzed. In addition, one maternal food supplement was included in this study. Notably, existing regulations lack specificity regarding the size distribution of nanoparticles (NPs) and the maximum permissible concentrations in commercial infant products. Except in one sample, the total zinc content exceeded the reported amount in the nutritional label, which varied from 34 to 119 µg/g. This work validated the cloud point extraction (CPE) technique for the effective isolation of ZnO-NPs from the selected products. CPE was then used to evaluate the ZnO-NPs concentrations in commercially available infant formulas and maternal supplements. Using inductively coupled plasma optical emission spectrometry (ICP-OES), the ZnO-NPs and total Zn concentrations were determined. The ZnO-NPs concentration ranged from 16 to 39 µg/g, representing a considerable portion of the total zinc content. Transmission electron microscopy (TEM) analysis indicated the presence of nanoparticles with an average diameter of 6.3 nm. The NPs size could determine their cell internalization, and thus, the potential cytotoxic effects are discussed. These findings underscore the need for rigorous isolation and quantification of nanoparticles from infant milk formulas, and as an inevitable first step for in vitro and in vivo toxicity studies to address the potential health impact of nanoparticles in food products.

Introduction

Nanotechnological progress is revolutionizing food quality, safety, and functionality [[1](#ref-CR1 "C. Chellaram, G. Murugaboopathi, A.A. John, R. Sivakumar, S. Ganesan, S. Krithika, G. Priya, Significance of nanotechnology in food industry. APCBEE Procedia. 8, 109–113 (2014). https://doi.org/10.1016/j.apcbee.2014.03.010

            "),[2](#ref-CR2 "S. Jacobson, Chemical food preservation. Published by Bibliotex, Canada. (2022). Website www.bibliotex.co. ISBN: 9781984665973"),[3](/article/10.1007/s11694-024-02881-4#ref-CR3 "J. Chen, Y. Guo, X. Zhang, J. Liu, P. Gong, Z. Su, L. Fan, G. Li, Emerging nanoparticles in food: sources, application, and safety. J. Agricult Food Chem. 71, 3564–3582 (2023). 
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            ")\]. Recent research has explored the nanoparticles application in food packaging, food fortification, and nutrient delivery \[[4](#ref-CR4 "D.J. McClements, H. Xiao, Is nano safe in foods? Establishing the factors impacting the gastrointestinal fate and toxicity of organic and inorganic food-grade nanoparticles. Npj Sci. Food. 1, 6 (2017). 
              https://doi.org/10.1038/s41538-017-0005-1
              
            "),[5](#ref-CR5 "S.H. Nile, V. Baskar, D. Selvaraj, A. Nile, J. Xiao, G. Kai, Nanotechnologies in food science: applications, recent trends, and future perspectives. Nano-Micro Lett. 12, 1–34 (2020). 
              https://doi.org/10.1007/s40820-020-0383-9
              
            "),[6](#ref-CR6 "M. Zare, K. Namratha, S. Ilyas, A. Sultana, A. Hezam, R.A. Surmenev, M.B. Nayan, S. Ramakrishna, S. Mathur, K. Byrappa, Emerging trends for ZnO nanoparticles and their applications in food packaging. ACS Food Sci. Technol. 2, 763–781 (2022). 
              https://doi.org/10.1021/acsfoodscitech.2c00043
              
            "),[7](/article/10.1007/s11694-024-02881-4#ref-CR7 "S. Wang, H. Alenius, H. El-Nezami, P. Karisola, A new look at the effects of engineered ZnO and TiO2 nanoparticles: evidence from transcriptomics studies. Nanomaterials. 12, 1247 (2022). 
              https://doi.org/10.3390/nano12081247
              
            ")\]. Moreover, these nanoparticles exhibit precise control over physical attributes, potentially modifying food products texture and sensory experience and ultimately enhancing consumer satisfaction \[[8](/article/10.1007/s11694-024-02881-4#ref-CR8 "Y.H. Zhou, A.S. Mujumdar, S.K. Vidyarthi, M. Zielinska, H. Liu, L.Z. Deng, H.W. Xiao, Nanotechnology for food safety and security: a comprehensive review. Food Rev. Int. 39, 3858–3878 (2023). hppts://doi.org/1080/87559129.2021.2013872")\]. However, it is unavoidable to acknowledge the imperative for comprehensive safety assessments of nanoparticles employed as food additives. The distinct characteristics of nanoparticles may entail potential health implications, mainly when consumed in substantial quantities over prolonged periods \[[7](/article/10.1007/s11694-024-02881-4#ref-CR7 "S. Wang, H. Alenius, H. El-Nezami, P. Karisola, A new look at the effects of engineered ZnO and TiO2 nanoparticles: evidence from transcriptomics studies. Nanomaterials. 12, 1247 (2022). 
              https://doi.org/10.3390/nano12081247
              
            "), [9](/article/10.1007/s11694-024-02881-4#ref-CR9 "M. Moradi, R. Razavi, A.K. Omer, A. Farhangfar, D.J. McClements, Interactions between nanoparticle-based food additives and other food ingredients: a review of current knowledge. Trends Food Sci. Technol. 120, 75–87 (2022). 
              https://doi.org/10.1016/j.tifs.2022.01.012
              
            ")\]. Vigilant scrutiny is essential to ascertain the safety profile of nanoparticles in food, ensuring they do not pose any undue risks to human health. Through rigorous evaluation and cautious application, nanoparticles stand poised to reshape the landscape of food additives, offering a pathway to elevated quality and functionality in food products \[[4](/article/10.1007/s11694-024-02881-4#ref-CR4 "D.J. McClements, H. Xiao, Is nano safe in foods? Establishing the factors impacting the gastrointestinal fate and toxicity of organic and inorganic food-grade nanoparticles. Npj Sci. Food. 1, 6 (2017). 
              https://doi.org/10.1038/s41538-017-0005-1
              
            "), [10](/article/10.1007/s11694-024-02881-4#ref-CR10 "Z. Ali, C. Zhang, J. Zhu, G. Jin, Z. Wang, Y. Wu, M.A. Khan, J. Dai, Y. Tang, The role of nanotechnology in food safety: current status and future perspective. J. Nanosci. Nanotechnol. 18, 7983–8002 (2018). 
              https://doi.org/10.1166/jnn.2018.16395
              
            ")\].

In particular, ZnO-NPs are considered convenient for their high surface area, UV-blocking, electrical, and antimicrobial properties. ZnO-NPs in food preparation may offer anti-inflammatory and antioxidant benefits [[4](/article/10.1007/s11694-024-02881-4#ref-CR4 "D.J. McClements, H. Xiao, Is nano safe in foods? Establishing the factors impacting the gastrointestinal fate and toxicity of organic and inorganic food-grade nanoparticles. Npj Sci. Food. 1, 6 (2017). https://doi.org/10.1038/s41538-017-0005-1

            "), [6](/article/10.1007/s11694-024-02881-4#ref-CR6 "M. Zare, K. Namratha, S. Ilyas, A. Sultana, A. Hezam, R.A. Surmenev, M.B. Nayan, S. Ramakrishna, S. Mathur, K. Byrappa, Emerging trends for ZnO nanoparticles and their applications in food packaging. ACS Food Sci. Technol. 2, 763–781 (2022). 
              https://doi.org/10.1021/acsfoodscitech.2c00043
              
            ")\]. Moreover, ZnO-NPs as food additive enhance also the quality, safety, and functionality of products. However, it is increasingly relevant to study the health issues that metal oxide compounds, including ZnO-NPs, may cause to human body due when exposed to substantial consumption over prolonged periods of time \[[7](/article/10.1007/s11694-024-02881-4#ref-CR7 "S. Wang, H. Alenius, H. El-Nezami, P. Karisola, A new look at the effects of engineered ZnO and TiO2 nanoparticles: evidence from transcriptomics studies. Nanomaterials. 12, 1247 (2022). 
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There is a growing awareness of the critical necessity to thoroughly comprehend the safety implications of nanoparticles, particularly when they constitute integral components of crucial dietary sources like infant formulas [[11](#ref-CR11 "K. Radad, M. Al-Shraim, R. Moldzio, W.D. Rausch, Recent advances in benefits and hazards of engineered nanoparticles. Environ. Toxicol. Pharmacol. 34, 661–672 (2012). https://doi.org/10.1016/j.etap.2012.07.011

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            ")\]. Infants, characterized by their developing physiological systems and nascent metabolic capacities, are notably susceptible to potential adverse effects from dietary sources. Comprehensive assessments of bioavailability, health risks, and physicochemical interactions of NPs are essential for safe and effective infant formula formulation and regulation \[[5](/article/10.1007/s11694-024-02881-4#ref-CR5 "S.H. Nile, V. Baskar, D. Selvaraj, A. Nile, J. Xiao, G. Kai, Nanotechnologies in food science: applications, recent trends, and future perspectives. Nano-Micro Lett. 12, 1–34 (2020). 
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            "), [14](/article/10.1007/s11694-024-02881-4#ref-CR14 "L. Tran, Q. Chaudhry, Engineered nanoparticles and food: an assessment of exposure and hazard. In Nanotechnologies in food (Eds. Q. Chaudhry, E. Castle, R. Watkins) Ch. 8, pp. 120–133 (2010). RCS- Thomas Graham House, Cambridge UK. ISBN 978-0-85404-169-5"), [15](/article/10.1007/s11694-024-02881-4#ref-CR15 "N. Durán, P.D. Marcato, Nanobiotechnology perspectives. Role of nanotechnology in the food industry: a review. Int. J. Food Sci. Technol. 48, 1127–1134 (2013). 
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            ")\].

The main challenge of toxicity studies lies in accurately determining whether metal oxide NPs are actually present in a food item. For this purpose, several techniques and analytical procedures have been developed. This work focuses on studying metal oxide NPs using the Cloud Point Extraction (CPE) methodology. Several techniques and analytical approaches can be employed to determine whether the food we consume contains metal oxide NPs. Among them, the Cloud Point Extraction (CPE) methodology is a highly valuable and versatile technique for isolating NPs from complex mixtures, offering selective separation and reducing interference from impurities (Fig. 1). Since its development in the 1970s, CPE has evolved to a pivotal sample preparation method in analytical chemistry, enhancing the sensitivity of nanoparticle detection and remaining accessible to researchers without the need for specialized equipment [[16](/article/10.1007/s11694-024-02881-4#ref-CR16 "F.H. Quina, W.L. Hinze, Surfactant-mediated cloud point extractions: an environmentally benign alternative separation approach. Ind. Eng. Chem. Res. 38, 4150–4168 (1999). https://doi.org/10.1021/ie980389n

            ")\]. CPE has attracted attention for its alignment with “green chemistry” principles, as it uses dilute surfactant solutions of a low toxicity, instead of the harmful classical organic solvents traditionally employed in liquid-liquid extraction \[[17](/article/10.1007/s11694-024-02881-4#ref-CR17 "N. Hasić, E. Horozić, Cloud point extraction as a method for preconcentration of metal ions. J. Eng. Proces Manag. 12, 44–49 (2020). 
              https://doi.org/10.7251/JEPM2002044H
              
            ")\]. The advantages of CPE include low cost, stability, simplicity, and a high efficiency, in obtaining highly preconcentrated samples from small initial sample volumes. The cloud point phenomenon, which is fundamental to the CPE process, occurs when a solution containing nonionic or zwitterionic surfactants separates into two phases upon heating to an appropriate temperature. This phase separation requires the surfactant concentration to exceed its critical micellar concentration (CMC). The NPs extraction mechanism in CPE involves specific interactions, such as hydrogen bonding, between functional groups on chelating reagents and the nonionic surfactant, resulting in the chelate being extracted into the hydrophobic portion of the micelles. Extraction efficiency can be influenced by the ionic strength of the solution, where an increase in ionic strength does not significantly alter extraction efficiency but facilitates phase separation.

Fig. 1

Fig. 1

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Schematic illustration of the Triton X-100 (TX-100) based cloud point extraction (CPE) approach for the fate determination of ZnO-NPs in infant milk formula. (A) Infant milk formula; (B) Addition of surfactant and binding of metal to the interior of the micelle; (C) Incubation and formation of ZnO + Triton TX-100 micelles; (D) Phase separation after heating and centrifugation, supernatant containing free Zn ions; (E) Surfactant rich phase and separated from the dilute aqueous phase.

This work has developed and validated a CPE procedure for ZnO-NPs isolation and characterization as an analytical methodology. The ZnO-NPs concentration, and nanoparticle size and morphology from different commercial infant formulas, commonly called “baby food” and form one maternal supplement, were determined.

Materials and methods

Reagents

Commercial foods used for the experiment were as follows: Three different infant milk formulas from the world’s most important trademarks and that cover most of the US market [18, [19](/article/10.1007/s11694-024-02881-4#ref-CR19 "G. Kent, WIC’s promotion of infant formula in the United States, Int. Breastfeed. J. 1, 1–14 (2006). https://doi.org/10.1186/1746-4358-1-8

            ")\] were used and named A, B and C, and one maternal food supplement (named D) was also analyzed. All products were purchased from local supermarkets. The names of commercial products, as well the can batch numbers, are available after justified request. Surfactant Triton TX-100, humic acid, nitric acid, hydrogen peroxide, and ZnO-NPs were obtained from Sigma Aldrich (St. Louis, MO).

Cloud point extraction

To isolate ZnO-NPs from infant milk formula, to fully analyze and characterize these additives, a modified cloud point extraction (CPE) procedure reported by Tani and collaborators [[20](/article/10.1007/s11694-024-02881-4#ref-CR20 "H. Tani, T. Kamidate, H. Watanabe, Micelle-mediated extraction. J. Chromatogr. A 780, 229–241 (1997). https://doi.org/10.1016/S0021-9673(97)00345-2

            ")\] was performed. First, 0.1 g of powdered infant formula was weighed, dispersed in 7 mL of distilled water, and then 70 µL of humic acid solution (10 µg/mL) were added. Th solution was stirred for 30 min, and then left to incubate at 60 °C for 30 min. Thereafter, the mixture was ultrasonicated for 15 min in the 9.9 s on and 9.9 s off program. Subsequently, the pH was adjusted to 10 with NaOH solution, and Triton TX-100 surfactant was then added to reach 5% (w/w) in the suspension. The mixture was incubated for one hour at 60 °C. Finally, it was centrifuged for 10 min at 2,500 _g_ and 25 °C from which the pellet and the supernatant were obtained.

Inductively coupled plasma (ICP) analysis

The Total Zn concentration in the pellet, supernatant, and total sample was determined by Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) [21]. After ZnO-NPs were isolated by the CPE method, each sample was digested with 10 mL of ultrapure nitric acid (HNO3) and 1 mL of 30% hydrogen peroxide (H2O2), and heated at 160 °C until the samples were colorless. Then, the total Zn concentrations were determined, using ZnCl2 as standard, in an ICP optical emission spectrometer (Varian, Vista-MPX CCD Simultaneous).

TEM analysis

The size and morphology of isolated nanoparticles were determined with transmission emission microscopy (TEM, JEOL-2010, JEOL Ltd., Peabody, MA, USA) operated at 200 kV. After the CPE isolation from the infant formula, 10 µL of the pellet resuspended in deionized water at a concentration of 0.1 mg/mL were deposited on the copper grids with carbon and Formvar coating, then left for 1 min, and the excess was removed. Images were processed using Gatan software.

Thermogravimetric analysis

Water and organic matter in isolated ZnO-NPs were estimated by thermogravimetric analysis (TGA) in Thermogravimetric analyzer (SDT-Q600, TA Instruments, New Castle, DE). The heating program started at 300 °C and the temperature was raised to 800 °C under a N2 atmosphere. ZnO-NPs samples (10 mg) from the infant formula were deposited on the thermogravimetric analyzer (SDT-Q600, TA Instruments, New Castle, DE).

Statistical analysis

All determinations were performed by at least three independent replicates. The average and standard deviations were obtained by the Excel software.

Results and discussion

Validation of the cloud point extraction technique

To validate the Cloud Point Extraction procedure, pristine commercial ZnO nanoparticles were first characterized. Figure 2a and b show the TEM micrographs depicting a predominant polygonal sheet shape with smooth surfaces for most nanoparticles. The average diameter of the ZnO-NPs was estimated from over 20 measurements of particles in random fields of the TEM micrograph, revealing an average size of 100 nm (Fig. 2c).

Fig. 2

Fig. 2

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Morphology and size distribution of ZnO NPs. A and B are images of pristine ZnO-NPs from Sigma-Aldrich by transmission electron microscopy (TEM). C) The size distribution was obtained by measuring NPs from TEM images, which showed an average size of 100 nm

The suspension of the commercial sample of ZnO-NPs was isolated by CPE and then analyzed. Figure 3a shows the spectra obtained from the sample of ZnO-NPs at a concentration of 1 mg/mL, which was previously dispersed in deionized water by ultrasonication for 15 min with two vortex agitation intervals. Figure 3A shows an intense absorption band at 375 nm in the pellet after centrifugation and resuspended, containing ZnO-NPs. The obtained spectrum coincides with that reported by Alarifi et al. [[22](/article/10.1007/s11694-024-02881-4#ref-CR22 "S. Alarifi, D. Ali, S. Alkahtani, A. Verma, M. Ahamed, M. Ahmed, H.A. Alhadlaq, Induction of oxidative stress, DNA damage, and apoptosis in a malignant human skin melanoma cell line after exposure to zinc oxide nanoparticles. Int. J. Nanomed. 983–993 (2013). https://doi.org/10.2147/IJN.S42028

            ")\] where the maximum absorption band was reported at 375 nm, and with that reported by Pudukudy and Yaakob \[[23](/article/10.1007/s11694-024-02881-4#ref-CR23 "M. Pudukudy, Z. Yaakob, Facile synthesis of quasi spherical ZnO nanoparticles with excellent photocatalytic activity. J. Clust Sci. 26, 1187–1201 (2014). 
              https://doi.org/10.1007/s10876-014-0806-1
              
            ")\] at 378 nm.

The ZnO-NPs dispersion was also analyzed by Dispersion Light Scattering (DLS). Figure 3B shows the hydrodynamic diameter of 299.1 nm with a polydispersity index of 0.261. The zeta potential of ZnO-NPs was determined to be -28.6 mV (Fig. 3C).

Fig. 3

Fig. 3

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(A) UV-vis absorption spectra obtained of commercial ZnO-NPs sample (Sigma-Aldrich) isolated by the CPE method. An intense absorption band is evident in the pellet (375 nm) and absent in the supernatant. (B) Size distribution of ZnO-NPs by dynamic light scattering (DLS). (C) Zeta potential in ZnO-NPs: -28.6 mV 299.1 nm.

The CPE method validation involved quantifying Zn in the different fractions by inductively coupled plasma-optical emission spectroscopy (ICP-OES). Table 1 shows the amounts of Zn found in each fraction. An 85.2% of initial Zn content in the sample was recovered after CPE extraction, from which 93.3% was found in the NPs fraction. A substantial recovery of ZnO-NPs at 64 °C in the presence of humic acids was obtained in the precipitate after CPE, confirming the effectiveness of CPE in isolating ZnO-NPs in particulate form. Minimal amounts (less than 6%) of ZnO-NPs were detected in the supernatants attributed to Zn ions, confirming that only a small quantity of Zn ions was released from the ZnO-NPs during CPE. This observation aligns with the findings of Jeon et al. [[24](/article/10.1007/s11694-024-02881-4#ref-CR24 "Y.R. Jeon, J. Yu, S.J. Choi, Fate determination of ZnO in commercial foods and human intestinal cells. Int. J. Mol. Sci. 21, 433 (2020). https://doi.org/10.3390/ijms21020433

            ")\], who reported a 93.4% recovery of pristine ZnO-NPs in particulate form after employing CPE with humic acid as a dispersing agent, according to the procedure reported by Majedi et al. \[[25](/article/10.1007/s11694-024-02881-4#ref-CR25 "S.M. Majedi, H.K. Lee, B.C. Kelly, Chemometric analytical approach for the cloud point extraction and inductively coupled plasma mass spectrometric determination of zinc oxide nanoparticles in water samples. Anal. Chem. 84, 6546–6552 (2012). 
              https://doi.org/10.1021/ac300833t
              
            ")\], and Triton TX-114 as surfactant. Thus, this established condition consequently was employed to elucidate the fate of ZnO in the commercial infant formula and maternal food supplements used for the present study.

In addition, the hydrodynamic diameters and primary particle sizes of ZnO-NPs obtained by CPE were found to be statistically indistinguishable from those of pristine nanoparticles, indicating that the CPE procedure did not impact the integrity, particle size or size distribution. Thus, the CPE was demonstrated to be an efficient method for isolating ZnO-NPs.

Table 1 Concentrations of Zn before and after Cloud Point extraction of ZnO-NPs from Sigma-Aldrich, where 100% corresponds to the total zn in the initial sample

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In general, nanoparticles have been isolated and quantified by a variety of methods that include capillary electrophoresis [[26](/article/10.1007/s11694-024-02881-4#ref-CR26 "G. Vicente, L.A. Colón, Separation of bioconjugated quantum dots using capillary electrophoresis. Anal. Chem. 80, 1988–1994 (2008). https://doi.org/10.1021/ac702062u

            "), [27](/article/10.1007/s11694-024-02881-4#ref-CR27 "M. López-Pastor, A. Domínguez-Vidal, M.J. Ayora-Cañada, B.M. Simonet, B. Lendl, M. Valcárcel, Separation of single-walled carbon nanotubes by use of ionic liquid-aided capillary electrophoresis. Anal. Chem. 80, 2672–2679 (2008). 
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            ")\], field flow fractionation \[[28](/article/10.1007/s11694-024-02881-4#ref-CR28 "M. Hassellöv, B. Lyvén, C. Haraldsson, W. Sirinawin, Determination of continuous size and trace element distribution of colloidal material in natural water by on-line coupling of flow field-flow fractionation with ICPMS. Anal. Chem. 71, 3497–3502 (1999). 
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            "), [29](/article/10.1007/s11694-024-02881-4#ref-CR29 "W.J. Lee, B.-R. Min, M.H. Moon, Improvement in particle separation by hollow fiber flow field-flow fractionation and the potential use in obtaining particle size distribution. Anal. Chem. 71, 3446–3452 (1999). 
              https://doi.org/10.1021/ac981204p
              
            ")\], hydrodynamic chromatography \[[30](/article/10.1007/s11694-024-02881-4#ref-CR30 "E. Chmela, R. Tijssen, M.T. Blom, H.J.G.E. Gardeniers, A. van den Berg, A chip system for size separation of macromolecules and particles by hydrodynamic chromatography. Anal. Chem. 74, 3470–3475 (2002). 
              https://doi.org/10.1021/ac0256078
              
            ")\], ionic liquid extraction \[[31](/article/10.1007/s11694-024-02881-4#ref-CR31 "H.-L. Huang, H.P. Wang, G.-T. Wei, I.-W. Sun, J.-F. Huang, Y.W. Yang, Extraction of nanosized copper pollutants with an ionic liquid. Environ. Sci. Technol. 40, 4761–4764 (2006). 
              https://doi.org/10.1021/es060034s
              
            ")\], size-exclusion chromatography \[[32](/article/10.1007/s11694-024-02881-4#ref-CR32 "X. Huang, R.S. Mclean, M. Zheng, High-resolution length sorting and purification of DNA-wrapped carbon nanotubes by size-exclusion chromatography. Anal. Chem. 77, 6225–6228 (2005). 
              https://doi.org/10.1021/ac0508954
              
            "), [33](/article/10.1007/s11694-024-02881-4#ref-CR33 "K.M. Krueger, A.M. Al-Somali, J.C. Falkner, V.L. Colvin, Characterization of nanocrystalline CdSe by size exclusion chromatography. Anal. Chem. 77, 3511–3515 (2005). 
              https://doi.org/10.1021/ac0481912
              
            ")\], and solid-phase extraction \[[34](/article/10.1007/s11694-024-02881-4#ref-CR34 "Z. Chen, P. Westerhoff, P. Herckes, Quantification of C60 fullerene concentrations in water. Environ. Toxicol. Chem. 27, 1852–1859 (2008)")\]. However, extraction of trace nanoparticles from complex matrixes such as food, biological, or environmental samples is a challenge and represents a bottleneck obstructing the quantitative determination and characterization. Scarce information is available on the nanoparticle isolation from complex mixtures. A simple and accurate method is urgently needed to isolate the nanoparticles present in complex mixtures. Due to the extremely low concentration of nanoparticles in these complex mixtures, the isolation procedure should include a preconcentration step and preserve the size and shape of nanoparticles. Cloud Point Extraction (CPE) \[[35](/article/10.1007/s11694-024-02881-4#ref-CR35 "J.-F. Liu, J.-B. Chao, R. Liu, Z.-Q. Tan, Y.-G. Yin, Y. Wu, G.-B. Jiang, Cloud point extraction as an advantageous preconcentration approach for analysis of trace silver nanoparticles in environmental waters. Anal. Chem. 81, 6496–6502 (2009). 
              https://doi.org/10.1021/ac900918e
              
            ")\] has been demonstrated to be applicable for the concentration and separation of nanoparticles from aqueous dispersions, in which the size and shape of nanoparticles are preserved during phase transfer and storage in a surfactant-rich phase. The efficiency and advances of nanoparticles purification by cloud point extraction have been recently reviewed \[[36](/article/10.1007/s11694-024-02881-4#ref-CR36 "K.B. Gavazov, I. Hagarová, R. Halko, V. Andruch, Recent advances in the application of nanoparticles in cloud point extraction. J. Mol. Liquids. 281, 93–99 (2019). 
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            ")\].

To enable future studies aimed at unraveling the impact of oxide metal NPs in humans, it is essential first to understand which methodologies can be applied to isolate and analyze the metal oxide NPs, such as ZnO-NPs in food products, especially in infant milk formula. Here, the successful isolation of ZnO-NPs by the CPE technique used in this work is an important step to characterize ZnO-NPs from the precipitated Triton TX-100 rich phase. Substantial recovery of ZnO-NPs (85%) at 64 °C in the presence of humic acids was obtained in the pellet after CPE, underscoring the effectiveness of CPE in isolating ZnO-NPs in particulate form.

Analysis of three different commercial infant milk formulas and one maternal food supplement

After the CPE method validation for ZnO-NPs isolation, the ZnO-NPs content in three commercial infant formulas (A, B, y C) and one maternal food supplement (D) were determined. The selected infant milk formulas are from the world’s most important trademarks, which cover most of the US market [18, [19](/article/10.1007/s11694-024-02881-4#ref-CR19 "G. Kent, WIC’s promotion of infant formula in the United States, Int. Breastfeed. J. 1, 1–14 (2006). https://doi.org/10.1186/1746-4358-1-8

            ")\]. All products were purchased from local supermarkets and named A, B, C, and D. The names of commercial products and the can batch numbers are available after justified request. The Zn content in both pellet (containing ZnO-NPs) and supernatant (containing ionic zinc) was determined by ICP-OES analysis.

All products analyzed showed the presence of ZnO-NPs with values from 16 to 40 mg/g of formula (Table 2). The percentage of Zn content determined as ZnO-NPs varied from 18% in the product C to 60% in the product B. Except for infant formula A, all commercial products contained larger amounts of total zinc than those indicated on the product nutritional label.

Table 2 Zinc concentration as ZnO-NPs and soluble ions in the three infant formulas and one maternal supplement a

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TEM analyses confirmed the presence of ZnO-NPs in the Triton TX-100 rich fraction precipitated from all tested products (Fig. 4). The ZnO-NPs are observed as black dots with an average diameter of 6.3 nm (Fig. 4A and B). The TEM images also show an intricate network with bifurcations, demonstrating an encapsulation of nanoparticles by assembled micelles or corona, in the infant formulas. This encapsulation is characterized by a dark gray color and an irregular shape (see red circle in Fig. 4B). Notably, Liu and coworkers [[37](/article/10.1007/s11694-024-02881-4#ref-CR37 "J.F. Liu, R. Liu, Y.G. Yin, G.B. Jiang, Triton X-114 based cloud point extraction: a thermoreversible approach for separation/concentration and dispersion of nanomaterials in the aqueous phase. Chem. Commun. 2009, 1514–1516 (2009). https://doi.org/10.1039/B821124H

            ")\] reported similar micelle assembly in the CPE extraction of Au-NPs using Triton TX-114 as a surfactant. Particle size determined by DLS revealed consistent hydrodynamic diameters of ZnO-NPs (Fig. [4](/article/10.1007/s11694-024-02881-4#Fig4)C).

Fig. 4

Fig. 4

The alternative text for this image may have been generated using AI.

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Characterizations of ZnO-NPs by transmission electron microscopy (TEM). (a) NPs generated with the Cloud Point Extraction method with 100 nm scale. (b) Same ZnO-NPs but with 50 nm scale. (c) Size distribution plot of ZnO-NPs from the infant formula with an average size of 6.3 nm

The organic material content in the isolated nanoparticles from the infant formulas, forming a corona, was determined through thermogravimetric analysis (TGA), as depicted in Table 3.

Table 3 Organic matter content on the isolated nanoparticles determined by thermogravimetric analysis (TGA)

Full size table

The use of zinc oxide nanoparticles (ZnO-NPs) in the food industry offers multifaceted advantages, prominently characterized by their antimicrobial properties, UV protection capabilities, and potential for nanoencapsulation. Their antimicrobial qualities extend the shelf life of food products by inhibiting microbial growth [[38](/article/10.1007/s11694-024-02881-4#ref-CR38 "S.L. Chia, D.T. Leong, Reducing ZnO nanoparticles toxicity through silica coating. Heliyon. 2, e00177 (2016). https://doi.org/10.1016/j.heliyon.2016.e00177

            "), [39](/article/10.1007/s11694-024-02881-4#ref-CR39 "A. Grasso, M. Ferrante, A. Moreda-Pineiro, G. Arena, R. Magarini, G.O. Conti, A. Cristaldi, C. Copat, Dietary exposure of zinc oxide nanoparticles (ZnO-NPs) from canned seafood by single particle ICP-MS: Balancing of risks and benefits for human health. Ecotoxicol. Environ. Saf. 231, 113217 (2022). 
              https://doi.org/10.1016/j.ecoenv.2022.113217
              
            ")\]. Additionally, when integrated into food packaging, these nanoparticles safeguard light-sensitive compounds, such as vitamins, helping to maintain the nutritional value and product quality \[[40](/article/10.1007/s11694-024-02881-4#ref-CR40 "J. Fujihara, N. Nishimoto, Review of zinc oxide nanoparticles: toxicokinetics, tissue distribution for various exposure routes, toxicological effects, toxicity mechanism in mammals, and an approach for toxicity reduction. Biol. Trace Elem. Res. 202, 9–23 (2024). 
              https://doi.org/10.1007/s12011-023-03644-w
              
            "), [41](/article/10.1007/s11694-024-02881-4#ref-CR41 "P. Chaudhary, F. Fatima, A. Kumar, Relevance of nanomaterials in food packaging and its advanced future prospects. J. Inorg. Organomet. Polym. Mater. 30, 5180–5192 (2020). 
              https://doi.org/10.1007/s10904-020-01674-8
              
            ")\]. Nanoencapsulation into ZnO-NPs aids in improving the stability and bioavailability of certain nutrients or bioactive ingredients, preventing degradation during storage and processing \[[42](/article/10.1007/s11694-024-02881-4#ref-CR42 "P.J.P. Espitia, N.D.F.F. Soares, J.S.D.R. Coimbra, N.J. de Andrade, R.S. Cruz, E.A.A. Medeiros, Zinc oxide nanoparticles: synthesis, antimicrobial activity and food packaging applications. Food Bioproc. Technol. 5, 1447–1464 (2012). 
              https://doi.org/10.1007/s11947-012-0797-6
              
            "), [43](/article/10.1007/s11694-024-02881-4#ref-CR43 "I.I. Muhamad, D.N.A. Zaidel, Z. Hashim, N.A. Mohammad, N.F.A. Bakar, Improving the delivery system and bioavailability of beverages through nanoencapsulation. In Nanoengineering in the beverage industry (Eds. A.M. Grumezescu, A.M. Holban, Academic Press, 2020, Pages 301–332. 
              https://doi.org/10.1016/B978-0-12-816677-2.00010-7
              
            ")\]. Moreover, incorporating zinc oxide nanoparticles can enhance the texture and appearance of specific food items. For instance, they may act as whitening agents in confectionery and dairy products. Furthermore, ZnO-NPs may be utilized for nutrient delivery, particularly for essential micronutrients like zinc itself. Their reported antioxidant properties also protect food products from oxidative damage, preserving flavors, colors, and nutritional content. While these benefits are promising, it is crucial to acknowledge the regulatory considerations surrounding the use of nanoparticles in food. Safety concerns and potential health effects require careful examination, and adherence to local regulations and guidelines is essential to ensure the responsible and safe incorporation of ZnO-NPs or similar nanomaterials in food applications \[[44](#ref-CR44 "M. Wang, S. Li, Z. Chen, J. Zhu, W. Hao, G. Jia, W. Chen, Y. Zheng, W. Qu, Y. Liu, Safety assessment of nanoparticles in food: current status and prospective. Nano Today. 39, 101169 (2021). 
              https://doi.org/10.1016/j.nantod.2021.101169
              
            "),[45](#ref-CR45 "M.S. Sheteiwy, H. Shaghaleh, Y.A. Hamoud, P. Holford, H. Shao, W. Qi, M. Zaffar Hashmi, T. Wu, Zinc oxide nanoparticles: potential effects on soil properties, crop production, food processing, and food quality. Environ. Sci. Pollut Res. 28, 36942–36966 (2021). 
              https://doi.org/10.1007/s11356-021-14542-w
              
            "),[46](/article/10.1007/s11694-024-02881-4#ref-CR46 "S. Smaoui, I. Chérif, H.B. Hlima, M.U. Khan, M. Rebezov, M. Thiruvengadam, T. Sarkar, M. Ali Shariati, J.M. Lorenzo, Zinc oxide nanoparticles in meat packaging: a systematic review of recent literature. Food Packaging Shelf Life. 36, 101045 (2023). 
              https://doi.org/10.1016/j.fpsl.2023.101045
              
            ")\].

Zinc is an essential element for life, but its toxicity becomes apparent at elevated levels. Numerous studies have indicated that nanosized particles, such as zinc oxide nanoparticles (ZnO-NPs), exhibit more severe toxicity than their bulk counterparts. The toxicological impact of ZnO-NPs has been evaluated through various exposure routes, including oral, inhalation, and intratracheal administration. Studies on mice exposed to ZnO-NPs via digestive tract revealed damage to primary organs, with the spleen and brain cells remaining normal. The severity of pathological changes induced by ZnO-NPs was found to be both size and dose-dependent [[40](/article/10.1007/s11694-024-02881-4#ref-CR40 "J. Fujihara, N. Nishimoto, Review of zinc oxide nanoparticles: toxicokinetics, tissue distribution for various exposure routes, toxicological effects, toxicity mechanism in mammals, and an approach for toxicity reduction. Biol. Trace Elem. Res. 202, 9–23 (2024). https://doi.org/10.1007/s12011-023-03644-w

            "), [47](/article/10.1007/s11694-024-02881-4#ref-CR47 "B. Wang, W. Feng, M. Wang, T. Wang, Y. Gu, M. Zhu, H. Ouyang, J. Shi, F. Zhang, Y. Zhao, H. Wang, J. Wang, Acute toxicological impact of nano-and submicro-scaled zinc oxide powder on healthy adult mice. J. Nanopart. Res. 10, 263–276 (2008). 
              https://doi.org/10.1007/s11051-007-9245-3
              
            ")\]. In cell culture studies, ZnO-NPs have been implicated in cytotoxicity, causing oxidative stress in human colon cancer cells. The time and dose-dependent decrease in cell number compared to untreated cells suggests potential risks associated with ZnO NPs exposure \[[48](/article/10.1007/s11694-024-02881-4#ref-CR48 "M. Horie, K. Nishi, K. Fujita, S. Endoh, A. Miyauchi, Y. Saito, H. Iwahashi, K. Yamamoto, H. Murayama, H. Nakano, N. Nanashima, E. Niki, Y. Yoshida, Protein adsorption of ultrafine metal oxide and its influence on cytotoxicity toward cultured cells. Chem. Res. Toxicol. 22, 543–553 (2009). 
              https://doi.org/10.1021/tx800289z
              
            "), [49](/article/10.1007/s11694-024-02881-4#ref-CR49 "B. De Berardis, G. Civitelli, M. Condello, P. Lista, R. Pozzi, G. Arancia, S. Meschini, Exposure to ZnO nanoparticles induces oxidative stress and cytotoxicity in human colon carcinoma cells. Toxicol. Appl. Pharmacol. 246, 116–127 (2010). 
              https://doi.org/10.1016/j.taap.2010.04.012
              
            ")\]. Additionally, it has been observed that smaller nanoparticles are more toxic to aquatic organisms compared to bulk preparations. The gastrointestinal tract plays a crucial role in the fate of ingested nanoparticles, where acidic conditions can dissolve metal and metal oxide nanoparticles into ions that can penetrate cells \[[50](/article/10.1007/s11694-024-02881-4#ref-CR50 "J.J. Powell, N. Faria, E. Thomas-McKay, L.C. Pele, Origin and fate of dietary nanoparticles and microparticles in the gastrointestinal tract. J. Autoimm. 34, J226–J233 (2010). 
              https://doi.org/10.1016/j.jaut.2009.11.006
              
            "), [51](/article/10.1007/s11694-024-02881-4#ref-CR51 "L.J. Du, K. Xiang, J.H. Liu, Z.M. Song, Y. Liu, A. Cao, H. Wang, Intestinal injury alters tissue distribution and toxicity of ZnO nanoparticles in mice. Toxicol. Lett. 295, 74–85 (2018). 
              https://doi.org/10.1016/j.toxlet.2018.05.038
              
            ")\].

As the global demand for infant milk formula rises, driven by population growth and changing dietary habits, understanding the potential impact of ZnO-NPs in infant nutrition becomes extremely important. Infant milk formula, designed as a substitute for breast milk, aims to mimic the nutritional profile of human milk, emphasizing the need for comprehensive research on the safety and toxicity of nanoparticles in such formulations [[52](/article/10.1007/s11694-024-02881-4#ref-CR52 "C.R. Martin, P.R. Ling, G.L. Blackburn, Review of infant feeding: key features of breast milk and infant formula. Nutrients. 8, 279 (2016). https://doi.org/10.3390/nu8050279

            "), [53](/article/10.1007/s11694-024-02881-4#ref-CR53 "A. Ali, A.R. Phull, M. Zia, Elemental zinc to zinc nanoparticles: is ZnO-NPs crucial for life? Synthesis, toxicological, and environmental concerns. Nanotechnol Rev. 7, 413–441 (2018). 
              https://doi.org/10.1515/ntrev-2018-0067
              
            ")\]. Some potential health risks associated with the use of zinc oxide nanoparticles in infants include: (i) Toxicity: ZnO nanoparticles may exhibit toxicity, and their small size allows them to penetrate biological barriers, raising concerns about potential adverse effects on developing organs and systems in infants. (ii) Inflammatory responses: The immune system of infants is still developing, and exposure to nanoparticles may trigger inflammatory responses. This could potentially lead to respiratory or other health issues. (iii) Bioaccumulation: There is a concern that nanoparticles may accumulate in organs and tissues over time, and the long-term effects of such bioaccumulation, especially in infants, are not fully elucidated. (iv) Potential for developmental effects: The impact of ZnO nanoparticles on infant development, including the central nervous system, is an area of concern and ongoing research. (v) Gastrointestinal absorption: If ingested, there is a possibility that ZnO nanoparticles may be absorbed through the gastrointestinal tract, raising questions about their effects on the digestive system and nutrient absorption \[[4](/article/10.1007/s11694-024-02881-4#ref-CR4 "D.J. McClements, H. Xiao, Is nano safe in foods? Establishing the factors impacting the gastrointestinal fate and toxicity of organic and inorganic food-grade nanoparticles. Npj Sci. Food. 1, 6 (2017). 
              https://doi.org/10.1038/s41538-017-0005-1
              
            "), [54](#ref-CR54 "S. Tang, M. Wang, K.E. Germ, H. Du, W.J. Su, W.M. Gao, G.D. Mayer, Health implications of engineered nanoparticles in infants and children. World J. Pediat. 11, 197–206 (2015). 
              https://doi.org/10.1007/s12519-015-0028-0
              
            "),[55](#ref-CR55 "I. Martín-Carrasco, P. Carbonero-Aguilar, B. Dahiri, I.M. Moreno, M. Hinojosa, Comparison between pollutants found in breast milk and infant formula in the last decade: a review. Sci. Total Environ. 875, 162461 (2023). 
              https://doi.org/10.1016/j.scitotenv.2023.162461
              
            "),[56](#ref-CR56 "W.S. Cho, B.C. Kang, J.K. Lee, J. Jeong, J.-H. Che, S.H. Seok, Comparative absorption, distribution, and excretion of titanium dioxide and zinc oxide nanoparticles after repeated oral administration. Part. Fibre Toxicol. 10, 9 (2013). 
              https://doi.org/10.1186/1743-8977-10-9
              
            "),[57](/article/10.1007/s11694-024-02881-4#ref-CR57 "A. Beegam, P. Prasad, J. Jose, M. Oliveira, F.G. Costa, A.M.V.M. Soares, P.P. Gonçalves, T. Trinidade, N. Kalarikkal, S. Thomas, M.L. Pereira, Environmental fate of zinc oxide nanoparticles: risks and benefits. In Toxicology-New Aspects to This Scientific Conundrum (Eds. S. Soloneski, M.L. Larramendy), Ch. 5, pp. 81–112 (2016) IntechOpen. 
              https://doi.org/10.5772/65266
              
            ")\]. It is important to point out that research on the safety of zinc oxide nanoparticles, particularly in the context of infant exposure, is continually evolving. The susceptibility of infancy to metabolic programming underscores the significance of providing appropriate nutrition to mitigate the risk of future diseases. Consequently, there is a need to precisely determine the composition of infant formulas to assess the potential exposure and ingestion of ZnO-NPs by infants \[[52](/article/10.1007/s11694-024-02881-4#ref-CR52 "C.R. Martin, P.R. Ling, G.L. Blackburn, Review of infant feeding: key features of breast milk and infant formula. Nutrients. 8, 279 (2016). 
              https://doi.org/10.3390/nu8050279
              
            "), [58](/article/10.1007/s11694-024-02881-4#ref-CR58 "R.P. Happe, L. Gambelli, Infant formula. In: Specialty oils and fats in food and nutrition (Ed. G. Talbot) pp. 285–315 (2015). Woodhead Publishing. 
              https://doi.org/10.1016/B978-1-78242-376-8.00012-0
              
            ")\]. A thorough quantitative analysis facilitates a more accurate understanding of the concentration levels of ZnO-NPs in these formulas, contributing to the assessment of their safety and potential health consequences for infants. The method described in this work holds critical importance for regulatory offices, research laboratories, and industrial manufacturers, enabling the establishment of guidelines, conducting thorough risk assessments, and ensuring the alignment of infant milk formulations with essential safety standards.

Conclusions

Cloud Point Extraction (CPE) has proven to be an effective technique for isolating and quantifying ZnO-NPs from infant formulas. ICP-OES characterization reveals concentrations in commercial formulas of ZnO-NPs from 16 to 40 µg/g, and from 12 to 95 µg/g of soluble Zn+ 2 ions. The total Zn content, encompassing both Zn particles and Zn ions, was higher in some samples than the Zn content shown on the nutritional product label, while in another case, this amount was significantly lower than the content shown on the product label. Thermogravimetric analysis indicates the formation of an organic corona covering the nanoparticles, which can be removed by simple washing. Thus, the isolation method of nanoparticles is essential for nanotoxicological studies due to the potential cell internalization of ZnO-NPs into mammalian cells. Further in vitro and ex vivo studies are imperative to examine the impact of the determined concentrations of ZnO-NPs isolated by CPE from infant milk formulas.

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Acknowledgements

We thank Dr. Oscar Gonzalez Davis and Itandehui Betanzo for their technical assistence, and Isabel Pérez Monfort for her editorial work. This work was funded by UNAM (PAPIIT-UNAM Grant IA204223).

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Centro de Nanociencias y Nanotecnología, Universidad Nacional Autónoma de México, Km 107 carretera Tijuana-Ensenada, Ensenada, 22860, Baja California, México
Gloria Salinas-Lucero & Rafael Vazquez-Duhalt
Centro de Física Aplicada y Tecnología Avanzada, Universidad Nacional Autónoma de México, Boulevard Juriquilla 3001, Juriquilla, 76230, Querétaro, México
Karla Juarez-Moreno
Centro de Investigaciones Científicas y de Estudios Superiores de Ensenada, Carretera Ensenada- Tijuana No.3918, Zona Playitas, Ensenada, CP.22860, B.C, México
Gloria Salinas-Lucero

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Gloria Salinas-Lucero
Karla Juarez-Moreno
Rafael Vazquez-Duhalt

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Correspondence toRafael Vazquez-Duhalt.

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The authors declare no competing interests.

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The authors declare to be fair and objective making the commercial products analysis. The information on products trademarks and the can batch numbers are availables after justified request.

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Salinas-Lucero, G., Juarez-Moreno, K. & Vazquez-Duhalt, R. Cloud point extraction and characterization of zinc oxide nanoparticles isolated from infant milk formulas.Food Measure 18, 9330–9340 (2024). https://doi.org/10.1007/s11694-024-02881-4

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Received: 04 April 2024
Accepted: 10 September 2024
Published: 28 September 2024
Version of record: 28 September 2024
Issue date: November 2024
DOI: https://doi.org/10.1007/s11694-024-02881-4

Cloud point extraction and characterization of zinc oxide nanoparticles isolated from infant milk formulas (original) (raw)

Abstract

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Introduction

Materials and methods

Reagents

Cloud point extraction

Inductively coupled plasma (ICP) analysis

TEM analysis

Thermogravimetric analysis

Statistical analysis

Results and discussion

Validation of the cloud point extraction technique

Analysis of three different commercial infant milk formulas and one maternal food supplement

Conclusions

References

Acknowledgements

Author information

Authors and Affiliations

Corresponding author

Ethics declarations

Ethics approval

Competing interests

Ethical Statement

Additional information

Publisher’s note

Rights and permissions

About this article

Cite this article

Keywords

Cloud point extraction and characterization of zinc oxide nanoparticles isolated from infant milk formulas (original) (raw)

Abstract

Similar content being viewed by others

Introduction

Materials and methods

Reagents

Cloud point extraction

Inductively coupled plasma (ICP) analysis

TEM analysis

Thermogravimetric analysis

Statistical analysis

Results and discussion

Validation of the cloud point extraction technique

Analysis of three different commercial infant milk formulas and one maternal food supplement

Conclusions

References

Acknowledgements

Author information

Authors and Affiliations

Corresponding author

Ethics declarations

Ethics approval

Consent for publication

Competing interests

Ethical Statement

Additional information

Publisher’s note

Rights and permissions

About this article

Cite this article

Keywords