Four foodborne pathogenic strains were provided by the Microbiology Laboratory of the National Research Centre (Egypt), which included E. coli, S. aureus, Rhizopus, A. niger. Microbial cultures were maintained in a suitable agar medium inclined at an angle of at 4°C (slant culture), which served as the stock cultures. The pathogens were grown in Mueller–Hinton agar (MHA) or Mueller–Hinton broth (MHB).

In this study, CaCO₃@Cur-FA microparticles were prepared by a method described previously. Briefly, water: acetone (3:1) solution was mixed with Na₂CO₃ (0.5 M) and CaCl₂ (0.5 M) with Cur-FA1 (0.1 M), Cur-FA2 (0.2 M), or Cur-FA3 (0.3 M) to prepare the stock solutions. The CaCl₂ solution was mixed with Cur-FA (Cur-FA1, Cur-FA2, or Cur-FA3) in a 50-mL beaker, followed by 10 min of stirring with the 85-1 constant temperature magnetic stirrer (Shanghai Zhiwei Electric Appliance Co., Ltd) to prepare CaCO₃@Cur-FA microparticles. After the rapid addition of Na₂CO₃ solution to the aforementioned mixture, the obtained solution was subjected to 5 min of stirring at 500 rpm at 40°C, followed by centrifugation to collect the products. CaCO₃ microparticles were synthesized using a similar method without adding Cur-FA solution.

The Zetasizer Nano-Zeta potentiometer (Nano ZS90, Malvern, UK) was used to measure zeta-potential and particle size of the CaCO₃@Cur-FA microparticles. The scanning electron microscope (SEM, Model S-4800 II FESEM, Hitachi, High-Technologies Co., Ltd., Japan) was used to observe the particle morphology.

To assess the pH sensitivity of CaCO₃@Cur-FA microparticles in a food microenvironment, the CaCO₃@Cur-FA samples (0.2 mg, 2 mL) were dissolved in phosphate buffered saline (PBS) of various pH (5.5, 6.8, and 7.4) and added into dialysis tubes (MWCO 3500). Thereafter, these tubes were soaked in 10 mL of PBS with corresponding pH (within the centrifuge tube) by constant shaking below 37°C. We took out 1 mL dialysate at pre-determined time intervals to measure the absorption, thereby assessing the amount of released Cur-FA. Then, 1 mL fresh PBS was added to the centrifuge tube.

The antioxidant activity of CaCO₃@Cur-FA was assessed by measuring its ability to scavenge 2,2′-azino-bis (3-ethylbenzothiazoline-6 sulfonic acid; ABTS⁺) and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radicals. In brief, 0.2 g CaCO₃@Cur-FA was dissolved in 2 mL distilled water before the test. Further, 2 mL of the CaCO₃@Cur-FA supernatant was added into ~2 mL of DPPH methanol solution (0.1 mM) to react for 30 min in dark. Subsequently, the ultraviolet spectrophotometer (Shimadzu, UV-2007, Japan) was used to measure the absorbance (optical density; OD) at 517 nm. The test tube was shaken for ensuring solution uniformity prior to measurements. For the composite film, its ability to scavenge ABTS⁺ free radicals were determined using the modified approach.

The ABTS⁺ or DPPH scavenging ability [R (%)] was determined through (Eq. 1)

A0 is the OD value of DPPH (ABTS⁺/PBS) in distilled water;

A1 is the OD value of CaCO₃@Cur-FA solution in distilled water mixed with the DPPH methanol solution (ABTS+/PBS mixed solution), and

A2 indicates the OD value of DPPH methanol solution (ABTS⁺/PBS mixed solution).

In this study, we adopted agar–well diffusion approach to perform antimicrobial studies. In brief, 1 mL of active strain culture (10⁵ cells /mL) was added to 20 mL MHB (Becton Dickinson, USA) and poured into petri dishes containing MHA. When the agar solidified, wells (diameter, 5 mm) were cut using a sterile borer. Further, 50 μL of the nano-microemulsion or bulk extract was added to each well. The plates were incubated for 2 h under ambient temperature, so that the solutions in the wells diffused into the agar. Moreover, the plates were incubated for 24 h at 37°C. Afterwards, we determined the microbial growth inhibition rate and inhibition zone diameters. Every sample was measured thrice (penicillin G, bulk extract, and nano-microemulsion), and the experiment was performed in triplicates.

Discs containing penicillin G (10 U) and ethanol were used as the positive and negative controls, respectively.

The differences among the samples were analyzed using ANOVA. Statistical analysis was performed using SPSS software. The significance of difference (P < 0.05) was compared by Duncan's multiple range tests.

This study successfully prepared CaCO₃ microparticles using the mineralisation approach (Figure 1A). As shown in Figure 1B, the SEM data of CaCO₃ and CaCO₃@Cur-FA microparticles clearly revealed the spherical morphology as well as the almost even size distribution. The sample particle size is given in Figure 1C. Table 1 displays the sample zeta potential. The average hydrodynamic sizes of the microparticles of CaCO₃, CaCO₃@Cur-FA1, CaCO₃@Cur-FA2, and CaCO₃@Cur-FA3 were 1070.1 ± 76 nm, 1303.5 ± 66 nm, 1464.8 ± 58 nm, and 1460.7 ±72 nm, respectively. Thus, the optimal concentration of Cur-FA required for preparing uniform-sized CaCO₃@Cur-FA microparticles was 0.2 M. The mean size of CaCO₃@Cur-FA mildly increased relative to that of the unmodified CaCO₃ microparticles, indicating the successful encapsulation of Cur-FA into the microemulsion. In addition, no droplet aggregation was observed, which indicated that CaCO₃@Cur-FA maintained its identity in the process of drying. The image of CaCO₃@Cur-FA microparticles is shown in Figure 1D.

The pH sensitivity of CaCO₃@Cur-FA2 microparticles was evaluated in PBS of pH 7.4, 6.8, and 5.5. As shown in Figure 2, CaCO₃@Cur-FA2 released only 24.5% of loaded Cur-FA2 at pH 7.4 after 128 h. On the contrary, 70.1 and 45.6% of Cur-FA2 was released from CaCO₃@Cur-FA2 at pH 5.5 and 6.8, respectively, which was associated with CaCO₃ decomposition under the acidic condition. The results demonstrated that CaCO₃@Cur-FA could be used as a food additive with a visible pH sensitivity.

Antioxidant activity plays a vital role in the food industry. It is the ability of inhibiting or delaying additional molecular oxidation (14). Previous results have demonstrated that Cur (15) and FA (16) have very high antioxidant activity because of phenolic hydroxyl groups in their molecular structure. In our previous study, the antioxidant activity of Cur ester derivatives was shown to be significantly increased. A higher Cur-FA level resulted in the higher antioxidant activity of the Cur-FA film. The DPPH scavenging activity of CaCO₃@Cur-FA1 microparticles was increased three times compared with that of CaCO₃ microparticles (Figure 3A). The ABTS⁺ scavenging ability of CaCO3@Cur-FA was similar to the DPPH scavenging ability (Figure 3B). The free radical scavenging ability of CaCO₃@Cur-FA microparticles was increased by 40% compared with that of CaCO₃ microparticles. Thus, CaCO₃@Cur-FA exhibited good antioxidant ability.

CaCO₃@Cur-FA exhibited antimicrobial activity, which is significant for its application as a food additive (16). In the present work, we adopted the agar–well diffusion method to detect the ability of CaCO₃@Cur-FA to inhibit Rhizopus, A. niger, E. coli, and S. aureus, and the inhibition zones were found to be 5.6 ± 0.3, 13.6 ± 0.3, 14.6 ± 0.2, and 9.3 ± 0.4 mm, respectively (Table 2). Thus, CaCO₃@Cur-FA inhibited the growth of these pathogens. CaCO₃@Cur-FA microparticles demonstrated superior antimicrobial activity than CaCO₃ microparticles, indicating that adding CaCO₃ promoted the antimicrobial activity of Cur-FA.