*2.4. Materials Characterization*

X-Ray Diffraction (XRD) analysis was performed to observe and compare crystal structure of LDH, CLDH, and CCLDH composite and other particles used in this study. For this, the powdered particles were placed on the XRD specimen holder and pressed with a glass plate to fill the holder. The XRD patterns were recorded using a diffractometer (Bruker D8 Discover diffractometer with VANTEC–2000 detector system and Cu source) and analyzed with a diffraction angle range between 0 and 50◦ (2 theta). The voltage, current and pass time used were 40 Kv, 40 mA, and 1 s, respectively.

Attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR, Bruker, Billerica, MA, USA) was used to identify the functional groups of the particles. For this, the dried powder samples were placed on the ATR probe and pressed. The FTIR spectra were taken in the range of 400–4000 cm<sup>−</sup>1, with a resolution of 4 cm<sup>−</sup><sup>1</sup> and 24 scans. Spectra were deconvoluted and analyzed using OMNIC software.

Transmission electron microscope (TEM) images were obtained using transmission electron microscope (Field Electron and Ion Company (FEI), Hillsboro, OR, USA) operating at 120 kV. For this, 1 mg/mL of particles (LDH, CLDH, CCLDH) were dispersed in ethanol and bath sonicated for 15 min. The suspension was further diluted in ethanol to 100 μg/mL. Five μL of samples were dropped on the TEM grid, air dried for 30 min, and imaged. Size distribution analysis for E171 was performed on at least 20 particles identified on the grid using ImageJ software (NIH, Bethesda, MD, USA).

The average hydrodynamic diameter, polydispersity index (PdI) and zeta potential of the particles were determined by dynamic light scattering (DLS) on a Zetasizer nano-ZS (Malvern Instruments, Malvern, UK) at 25 ◦C. The particles were dispersed and diluted to 50 μg/mL in deionized water and were measured twice. Size results are given as intensity distribution by the mean diameter with its standard deviation.

### *2.5. Transmittance and Opacity Measurements*

The UV-Visible absorbance spectra could be used to associate with opacity [24]. The total reflectance spectra of the synthesized particles were measured and compared with E171, E551, and RS. For this, equal quantities of dry powders of particles were pressed onto the wall of the quartz cuvette of a UV–VIS diffuse reflectance spectrophotometer (Lambda 750, PerkinElmer Life and Analytical Sciences, Shelton, CT, USA) operated by UV WinLab™ software (Version 4.0, PerkinElmer Life and Analytical Sciences, Shelton, CT, USA), and % reflectance was measured in the range of 200–800 nm with 1 nm interval.

Opacity is defined as the proficiency of the pigment to mask the features of the underlying substrate [25]. Opacities of the synthesized particles were compared with that of E171 by measuring % transmittance. For transmittance measurement, 100 μL of particle suspensions (1 mg/mL) were dispensed into wells of 96 well plate and % transmittance at 550 nm was measured using a microplate reader (Spectra Max i3x, Molecular Devices, San Jose, CA, USA). Masking power comparisons between E171 and synthesized particles were conducted visually with optimized concentrations of hydroxy propyl methylcellulose (HPMC) (85% *w*/*v*) and plasticizer (Glycerol 10% *w/v*) for preparation of a film layer. Firstly, dry particles of E171, E551, and RS were suspended (1 mg/mL) in DI water, while the synthesized particles in dry powder form were suspended (1 mg/mL) at pH ~10 adjusted solution (with 0.1 M NaOH). These suspensions were individually mixed with slurry containing 85% hydroxy propyl methylcellulose (HPMC), 10% glycerol, and 5% solution. The resulting thick pastes (1 mL) of particles were spread evenly on a glass plate using a paint brush and left to dry overnight at room temperature. The glass plate was imaged against a worded background for visual comparison of masking power, recorded using digital camera.

### *2.6. Characterization of Stability of Particle Suspension to pH and Temperature*

Stabilities of aqueous suspensions of LDH, CLDH, CCLDH, and E171, E551 (1 mg/mL) were evaluated as a function of time and under different pH and temperature conditions. For this, the particle suspensions were probe sonicated for three intervals of 15 s to evenly disperse the particles in DI water.

### 2.6.1. Effect of pH

Stability of particle suspensions as a function of pH was performed by adjusting the pH of the suspension using 0.1 M NaOH and 1 M HNO3 to obtain pH levels of 3 and 9. These two pH levels were chosen as they represent the acidic and alkaline pH typically encountered in food processing. The suspension of particles with pH adjusted were left undisturbed for 30 min at room temperature, and the % transmittance (550 nm) was measured to assess the extent of particle sedimentation. The experiment was repeated in triplicates, and the mean value was plotted as % transmittance vs. pH.

### 2.6.2. Effect of Temperature

Stability of particle suspension as a function of temperature was determined by heating 1 mg/mL samples in Eppendorf tubes in a thermostat water bath to 25, 50, 75, and 100 ◦C. After holding samples at the stipulated temperatures for 5 min, suspensions were cooled in ice bath to room temperature, and 100 μL of each suspension was aliquoted into a well of 96 wells plate. The % transmittance at 550 nm was determined using a microplate reader. The results of the three replicates were averaged and a graph of % transmittance vs. temperature was plotted with mean values and standard deviations.

### *2.7. Casein Antigenicity Study*

One of the potential challenges for the application of the CCLDH composite would be the allergenicity of casein. Therefore, we compared the antigenicity of casein before and after conjugating with LDH using enzyme-linked immunosorbent assay (ELISA) as reported [8]. The ELISA plate was prepared by adding 100 μL of antigen solution (casein, CLDH and CCLDH (100 ng/mL) in coating buffer (0.1 M Na2HPO4 in DI water- pH 9.5)) to individual wells of a 96-well plate (Nunc Maxisorp 96-well ELISA plate). The plate was kept overnight at 4 ◦C for adsorption of antigen (casein, CLDH or CCLDH) to well surface. The uncoated antigen was discarded, and the residual free binding sites were blocked with blocking buffer (2% BSA in 1× TBS containing 50 mM Tris-HCl and 150 mM NaCl) for 1 h at 37 ◦C. After discarding the blocking buffer, primary antibody (1: 2000) (anti-casein rabbit antibody- cat # ab166596) was added in the blocking buffer and incubated for 1 h at room temperature. Excess of primary antibody (remaining in solution) was discarded, and the wells were washed four times with washing buffer (0.05% tween −20 in 1× TBS). The wells were then incubated with secondary antibody (1: 10,000) (anti-rabbit antibody- cat # 6721) in blocking buffer for 1 h at RT. Subsequently, the excess unbound secondary antibody was discarded, and the wells were washed four times with washing buffer. These wells were further added with 100 μL of substrate solution (5 mM of tetramethylbenzidine-TMB) and incubated for 3 min at RT for the enzymatic color development, followed by the addition of stop solution (100 μL of 2 M sulphuric acid) to terminate the reaction. Absorbance at a wavelength of 450 nm was determined by using a microplate reader. The amount of primary antibody binding onto antigen (casein) was determined using the standard curve, and the antigenicity is expressed as casein antigen response (ng/mL). The result presented is an average of values from triplicate experiments.
