**1. Introduction**

E171 (food grade titanium dioxide (TiO2)) is one of the most widely used food additives containing nanoparticles [1]. Pigment grade TiO2 with its primary size ranging from 200–350 nm shows a high refractive index (2.6–2.9), negligible absorption in visible range of light spectrum, high scattering, and excellent masking power [2]. In addition, it is stable across a wide range of measurements of pH, temperature, and humidity, and it does not react with matrix components, making it a desired material for food and pharmaceutical products. E171 is copiously incorporated as a white pigment and a masking agen<sup>t</sup> in over 900 commonly consumed food products such as dairy products and analogues, edible ices, confectionaries, surimi and similar products, food supplements, and seasonings and sauces [3,4].

TiO2 is generally recognized as safe (GRAS) according to the U.S. Food and Drug Administration (FDA) and is permitted to be used in food up to 1% without being declared on labels. Recent studies point towards potential adverse health outcomes from oral administration of TiO2 nanoparticles [2,5]. E171 particles are not designed to be nanoparticles,

**Citation:** Ngew, E.; Phue, W.H.; Liu, Z.; George, S. Composite of Layered Double Hydroxide with Casein and Carboxymethylcellulose as a White Pigment for Food Application. *Foods* **2022**, *11*, 1120. https://doi.org/10.3390/ foods11081120

Academic Editor: Beatriz Gandul-Rojas

Received: 17 February 2022 Accepted: 8 April 2022 Published: 13 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

but pristine and recovered E171 particles from several food products showed a substantial proportion of nanoparticles (below 100 nm) [4,6]. The presence of nanoparticles in E171 has triggered concerns regarding the undesirable impacts of TiO2 nanoparticles on human health. In addition, recent publications from our group have reported the negative impacts of TiO2 nanoparticles on the functional integrity of intestinal epithelial cells and potential allergenicity of food allergens [7,8]. Pinget et al. also found that food grade TiO2 could impair the homeostasis in gu<sup>t</sup> microbiota-host interactions [3]. The European food safety authority (EFSA) recently deemed that E171 is "not safe" as a food additive after evaluating the outcomes from recent health risk assessments [9]. Consequently, the European Union moved to ban the application of TiO2 as a food additive under Regulation (EC) No. 2022/63 in the Official Journal of the European Union, with a transition period until 7 August 2022. This and the growing consumer demand for natural and organic foods have resulted in food manufacturers using white pigment alternatives like rice starch (RS), silicon dioxide (E551), and calcium phosphate.

Layered double hydroxides (LDH) have recently fascinated researchers for their wide range of applications in various fields due to their biocompatibility, cost and resource effective methods of synthesis, and suitability for modification [10–12]. LDH is composed of positively charged two-dimensional (2D) metal hydroxide layers which stack alternatively with interlayers of anions, forming a three-dimensional (3D) "lasagna" structure. Structurally, the 2D "lasagna sheets" are composed of divalent cations octahedrally surrounded by OH–ions where the octahedra share edges forming an infinite 2D layer. The positively charged brucite-like (M2+ (OH)2) lasagna sheets are separated by an interlayer region containing charge compensating anions and solvation molecules, forming a 3D structure held together via electrostatic interactions and hydrogen bonds [13]. Anionic organic species like peptides, amino acids, and proteins could be intercalated in between the (M2+ (OH)2) layers through anion exchange chemistry [14]. LDH-composites thus generated have shown promising applications in medicine and energy conversion as well as storage and environmental remediation [15].

In this article, we report the generation of composite materials where Mg-Al LDH was intercalated with casein through anion exchange, and the surface was further modified with carboxymethylcellulose (CMC). LDH and its composites were characterized and compared to currently used white pigments for their physicochemical properties, masking power and safety. We report the superior performance, cost, and safety features of LDH composite in comparison to generally used E171 alternatives for its potential applications in products intended for human consumption.

### **2. Materials and Methodologies**

Food additive silicon dioxide particles E551 (SIPERNAT 22) were obtained from Evonik Corporation (Parsippany, NJ, USA) and food additive titanium dioxide particles (E171, cat # 13463-67-7) were obtained from Minerals-Water, UK. All particles were used as received.

Commercial bovine milk casein (cat # 5890), magnesium oxide (MgO), aluminum oxide (Al2O3), sodium hydroxide (NaOH), rice starch (RS), and carboxymethyl cellulose (CMC) with 0.55–1.0 degree of substitution (DS) were purchased from Sigma Chemical Co. (St-Louis, MO, USA).

Stock solutions of particles (E171, E551) and rice starch were prepared by dispersing 10 mg/mL in deionized (DI) water obtained from a Milli-Q water system (Millipore Sigma, Milford, MA, USA).

### *2.1. Synthesis of Mg-Al LDH*

Mg-based LDH was chosen because it is white color [16]; and Al3+ was chosen as the "guest" metal cation due to its ability to stabilize the α- form of hydroxides during LDH formation [17]. LDH was synthesized using a hydrothermal method as detailed elsewhere [18,19]. We chose the hydrothermal method as it improves the crystallinity of LDH which is desirable for the scattering of light within the LDH structure [20].

Accordingly, we mixed MgO: Al2O3 in a molar ratio of 2 in 50 mL of deionized water using ultrasonication for 30 m in a clean glass bottle. A Mg/Al ratio of 2 was chosen to form a clear hydrotalcite phase and higher basal spacing which is preferred for the anionic exchange reaction in the following steps [21,22]. One molar NaOH was added dropwise into the bottle under vigorous stirring until the pH of the solution reached ~10. The obtained mixture was stirred at 110 ◦C for 12 h in the tightly closed bottle, followed by ageing in the oven at 110 ◦C for 10 days for a dissociation-deposition-diffusion mechanism to mediate the formation of LDH [19,23]. The synthesized LDH was then washed with DI water twice at 12,500 rpm and the precipitate was freeze-dried overnight to obtain dry powder of LDH.

### *2.2. Synthesis of Mg-Al-Casein LDH (CLDH)*

CLDH was synthesized by rehydration of LDH in the presence of casein. For this, 1.5 g LDH and 2.5 g casein were added to 100 mL DI water under vigorous stirring, followed by 30 min bath sonication to fully disperse the particles. NaOH (1 M) was added dropwise to the suspension under vigorous stirring. The suspension (pH~10) was further stirred in a tightly sealed bottle for 10 h. At this relatively high pH way above the isoelectric point of casein (pI = 4.6), casein was intercalated into the LDH interlayer region between the [Mg2Al (OH)6]+ "lasagna sheets". Subsequently, the white suspension was collected by centrifugation at 12,500 rpm and washed with DI water twice to remove excess casein. The white precipitate from final wash was then freeze dried overnight to obtain CLDH.

### *2.3. Synthesis of Carboxymethyl Cellulose (CMC) Modified CLDH (CCLDH)*

CMC (1.0 g) was dissolved in DI water (500 mL) with vigorous stirring at room temperature for 5 h for it to be fully dispersed. CLDH suspension in water (8 mg/mL) was added dropwise into the CMC solution such that the mass ratio of CMC to CLDH is 9 into a glass bottle. After 6 h, the CCLDH suspension was washed with DI water thrice, and was dried overnight in a freeze dryer.
