In this study, hybrid particles of AgNPs-loaded eggshell calcium carbonate (AgNPs/eCaCO₃) were prepared in situ by precipitating the eggshell in the presence of freshly prepared AgNPs with a particle size of 10-30 nm. The hybrid particles were comparatively precipitated in situ at 25 °C and 35 °C using poly (sodium 4-styrenesulphonate) as a polyelectrolyte. The AgNPs/eCaCO₃ particles prepared at 25 °C had a spherical morphology with a mean diameter of 3.56 mm, and BET surface area of 85.08 m²/g. On the other hand, the particles prepared at 35 °C had a broader size distribution with a mean diameter of 3.19 mm, and a BET surface area of 79.25 m²/g. AgNPs-loaded commercial calcium carbonate particles (AgNPs/e=CaCO₃) comparatively prepared at 35 °C were perfectly spherical with a mean diameter of 5.61 mm. At preparing temperature of 25 °C, the hybrid particles contain AgNPs of 0.78 wt% for AgNPs/eCaCO₃ and 3.20 wt% for AgNPs/CaCO₃. The AgNPs/eCaCO₃ and AgNPs/CaCO₃ particles exhibited the same efficiency against bacteria extracted from beef with an average inhibition zone diameter of 7–10 mm according to the modified Kirby–Bauer disc diffusion assay depending on their concentration and beef source. Freshly prepared silver colloids showed comparatively poorer antimicrobial efficiency with an inhibition zone diameter around 7 mm. 

I. Particle diameter

Field Emission Scanning Electron Microscopy (FESEM) was carried out on Carl Zeiss, Auriga to observe the particle morphology and particle size of AgNPs, eCaCO₃, AgNPs/eCaCO₃, and AgNPs/CaCO₃ using an accelerating voltage of 3.0 kV. Prior to the observation, each sample was coated using a gold sputter coater (Neo Coater) for 2 min. The diameter of the particles was measured from the SEM micrographs using an analysis software (Image J) and calculated by randomly selecting 200 particles. MATLAB R2022a was used to obtain the mean diameter, size distribution, and respective histogram.

II. Inhibition zone diameters 

The digital images of the Inhibition zone diameters against beef-extracted bacteria obtained from AgNPs/eCaCO₃ and AgNPs/CaCO₃ were measured three times from a disc using ImageJ and an average made from three replica. The antimicrobial test was performed in triplicate and the average diameter of the inhibition zone with standard deviation was calculated.

Excel and image-related program.