**2. Results**

#### *2.1. Synthesis and Characterization of Ag-CaGP Nanocomposites*

UV-Vis absorption spectroscopy (UV-Vis) showed that Ag-CaGP nanocomposites presented silver in nanosized dimensions in all of the nanocomposites synthesized, regardless of the reducing agen<sup>t</sup> used. It was demonstrated by the presence of an intense absorption peak, denominated plasmonic band, which occurred between 420 and 450 nm (Figure 1a, Figure S1). It characterizes noble metal

nanoparticles, with strong absorption band being observed in the visible region [27]. The CaGP did not exhibit absorption peak in the visible region of the electromagnetic spectrum.

X-ray diffraction (XRD) pattern indicated that all of the Ag-CaGP nanocomposites were composed of AgNP and CaGP for confirming the presence of silver in Ag-CaGP nanocomposites through comparison of the nanoparticles and CaGP. The typical powder XRD pattern of the prepared CaGP showed diffraction peaks at 2θ = 6.30◦, 12.3◦, 26.4◦, 41.1◦, and 44.2◦ (Figure 1b, Figure S2), and the corresponding crystallographic form (PDF No. 1-17) [28]. The typical powder XRD pattern of the silver nanoparticles showed (Figure 1b) diffraction peaks at 2θ = 38.2◦, 44.4◦, 64.6◦, 77.5◦, and 81.7◦, which can be indexed to (111), (200), (220), (311), and (222) planes of pure silver with face-centered cubic system (PDF No. 04-0783).

**Figure 1.** (**a**) UV-Vis (**b**) XRD pattern of Ag-CaGP (B4 group) nanocomposite, silver nanoparticles, and CaGP.

Nanostructured materials that exhibit a pattern of small nanoparticles scattered on a larger surface, similar to glass bead embellishments on a Christmas tree, are generally classified as a decorated material. The scanning electron microscopy (SEM) images of Figure 2 show this typical pattern, with spherical silver nanoparticles (indicated by arrows) decorating the surface of the CaGP microparticles in all synthesized nanocomposites containing 10% Ag (B4; B8; C4). In addition, transmission electron microscopy (TEM) was performed for the nanocomposite B4 (Figure S3).

**Figure 2.** SEM images of the Ag-CaGP nanocomposites: B4 (**<sup>a</sup>**,**b**), C4 (**<sup>c</sup>**,**d**), and B8 (**<sup>e</sup>**,**f**) at different magnifications. The arrows indicate silver nanoparticles on the surface of CaGP in B4 and in the bulk of CaGP in C4 and B8.

The energy-dispersive X-ray sprectroscopy (EDS) clearly showed the outline of Ag-CaGP nanocomposites in all micrographs. Also, Figure 3 (B4), Figure 4 (C4) and Figures S4–S6, the two-dimensional (2D) images were constructed by analyzing the energy released from the issuance Si Kα,OK<sup>α</sup>,PKα, Ca Kα, and Ag Kα, indicating the distribution of these elements on the demarcated area in the micrograph.

**Figure 3.** SEM and EDS mapped in 2D elements issuance Si Kα,OK<sup>α</sup>,PKα, Ca Kα, and Ag Kα false color. Analysis of the distribution of silver nanoparticles on the Ag-CaGP for sample B4: (**a**) SEM image; (**b**) chemical mapping of silicon element present in the substrate, where the electron beam was focused directly on the substrate and is showed in green color, and the dark regions the beam was focused in Ag-CaGP nanocomposite B4; (**<sup>c</sup>**–**f**) oxygen, calcium, phosphorus, and silver, respectively, demonstrating they are constituents of the Ag-CaGP.

**Figure 4.** SEM and EDS mapped in 2D elements issuance Si Kα,OK<sup>α</sup>,PKα, Ca Kα, and Ag Kα false color. Analysis of the distribution of silver nanoparticles on the Ag-CaGP for sample C4: (**a**) SEM image; (**b**) chemical mapping of silicon element present in the substrate, where the electron beam was focused directly on the substrate and is showed in green color, and the dark regions the beam was focused in Ag-CaGP nanocomposite C4; and, (**<sup>c</sup>**–**f**) oxygen, calcium, phosphorus, and silver, respectively, demonstrating they are constituents of the Ag-CaGP.

#### *2.2. Minimum Inhibitory Concentration*

The results showed that the MIC values were related to the synthesis process and the Ag concentration used (Table 1). Nanocomposites that were obtained using Na3C6H5O7 as reducing agen<sup>t</sup> showed the most effective antimicrobial activity against *C. albicans* and *S. mutans*. In these composites, the lowest MIC values were observed for those containing 10% of Ag (C3 and C4), being between 19.05 and 39.05 μg/mL for *C. albicans* and 156.2 and 625 μg/mL for *S. mutans*. The nanocomposites that were synthesized using NaBH4 as reducing agen<sup>t</sup> and isopropanol as solvent showed fungicidal effect varying between 100 and 1600 μg/mL, whilst no effect against *S. mutans* was observed. While the nanocomposites synthesized using the same reducing agen<sup>t</sup> and deionized water as solvent did not show any effect against both microorganisms. In addition to the MICs found for the synthesized compounds, it was carried out the microdilution assay to find the MIC values for the solutions containing only AgNP or CaGP diluted in deionized water, besides the other compounds used in the synthesis reaction as reducing and surfactant agents. These data are showed in Table 2.

**Table 1.** Values of minimum inhibitory concentration (MIC) of the nanocompounds based on μg of AgCaGP mL−<sup>1</sup> and on μg of Ag mL−<sup>1</sup> in each ones, synthesized using sodium borohydride (Group B) and sodium citrate (Group C), and silver ions concentration (μg Ag+/mL) in all nanocompounds tested.


**Table 2.** Values of minimum inhibitory concentrations (MIC) (μg/mL) and silver ions (Ag+) (μg Ag+/mL) of the control solutions: AgNP reduced by sodium citrate (Na3C6H5O7), and by sodium borohydride (NaBH4); silver nitrate (AgNO3); nanoparticulated CaGP (CaGP-nano), and CaGP in commercial form (CaGP-commercial); sodium citrate and surfactant (Na3C6H5O7+NH-PM), and sodium borohydride and surfactant (NaBH4+NH-PM).


#### *2.3. Determination of Ag+ Concentration*

The Ag+ concentration of all the nanocomposites containing Ag (AgNP and Ag-CaGP) is showed in Table 1. For samples that were obtained through NaBH4 route (B1–B8), a reduction of ionic silver higher than 98% was observed, when considering the total amount of ionic silver added to the reaction was 500 μg Ag+/mL for B1, B2, B5, B6, and 5000 μg Ag+/mL for B3, B4, B7, and B8. While for the compounds that were synthesized using Na3C6H5O7 as reducing agent, the ionic silver remaining was higher and reached about 10% in those samples that were produced using initially 5000 μg Ag+/mL in the reaction process (C3 and C4). C1 and C2 presented 61.1% and 33.3%, respectively, of ionic silver in samples as the total Ag+ added in the reaction was 500 μg Ag+/mL. For AgNP with no CaGP added to the reaction (Table 2) obtained by Na3C6H5O7 route (nanoAg(Na3C6H5O7)) the Ag+ concentration was 107.25 μg/mL, whereas for AgNP produced through NaBH4 (nanoAg(NaBH4)) the Ag+ concentration was 576.19 μg/mL.
