Nanocomposite Egg Shell Powder with in situ Generated Silver Nanoparticles Using Inherent Collagen as Reducing Agent

Due to the unique properties such as higher surface to volume ratio and antibacterial activity, metal nanoparticles (MNPs) have many applications in nanomedicine. Of all the MNPs, silver nanoparticles (AgNPs) are important due to their outstanding antibacterial activity. Though there are several methods of synthesizing the MNPs, bio-reduction approach gets attention due to its environmentally friendly nature and simplicity. In this direction, many workers synthesized the AgNPs using aqueous leaf and fruit extracts as reducing agents. For instance in some studies, the AgNPs were synthesized by using the leaf extracts of Ocimum sanctum, Berberis vulgaris, Psidium guajav, banana peel, Azadirachta indica, Taxus baccata, some Indian medicinal plants etc. (Singhal et al., 2011; Banerjee et al., 2014; Sriram and Pandidurai, 2014; Ibrahim, 2015; Ahmed et al., 2016; Asadi et al., 2018; Behravan et al., 2019). In order to widen their applications, nanocomposite cellulose films were prepared with in situ generated AgNPs by using the leaf extracts of O. sanctum, Cassia alata, A. indica, Terminalia cattapp etc. (Sadanand et al., 2017; Kishanji et al., 2017; Muthulakshmi et al., 2017; Sivaranjana et al., 2017). However, in order to in situ generate the AgNPs in polymer matrices by single step; recently, thermal assisted methods exploiting the inherent reducing agents presented in the polymer matrices have been adopted. For example, Sadanand et al. (2017) in situ generated the AgNPs in cotton fabrics by one pot hydrothermal method in which the OH groups presented in the matrix acted as the reducing agents. Similarly, Pusphalatha et al. (2019) in situ generated the AgNPs in polyester fabrics by one pot hydrothermal method.

Due to the increasing environmental awareness, the trend is now shifting towards utilizing natural materials, kitchen and agro waste materials as fillers in the preparation of polymer green composites. Jawaid and Siengchin (2019) stressed the importance of natural materials in the preparation of hybrid composites. Pan et al. (2016) suggested the usage of bio-based polymers for packaging materials. Lin et al. (2017) used lignin and hemicelluloses for facial synthesis of gold, platinum and palladium nanoparticles. Egg shell powder (ESP) is a poultry waste which poses environmental problems if properly not disposed. In this direction, some researchers aimed to add values for this poultry and kitchen waste. Chen et al. (2016) developed decolorants based on poultry keratin for the treatment of waste water in dyeing industries. Feng et al. (2014) prepared the composites of polypropylene carbonate by adding the ESP as a filler. Similarly, Ashok et al. (2014) used the ESP in polylactic acid matrix and prepared their composites. In another study, the ESP was utilized to immobilize cadmium and lead in a contaminated soil (Ok et al., 2011). Haroon et al. (2015) suggested the usage of the ESP as a cleaning powder for house ware. In order to impart antibacterial activity to the ESP, in the present study, the nanocomposite egg shell powder (NCESP) with in situ generated AgNPs was prepared by one pot thermal assisted method. The ESP has 95 wt% of CaCO₃ and 5 wt% of X-collagen (the binding protein) as its components (Ly et al., 2016). Further, the main constituents of X-collagen present in ESP include the three amino acids—glycine, proline and hydroxy proline (Haroon et al., 2015; Ly et al., 2016). In the present study, the authors exploited the functional groups of X-collagen as reducing agents in the in situ generation of AgNPs in ESP. The NCESP was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD), thermogravimetric analysis (TGA) and antibacterial tests. This work aims to make antibacterial ESP for applications such as antibacterial cleaning powder for house ware and also as antibacterial low-cost filler in the preparation of hybrid nanocomposites.

Disposed egg shells were collected from the local restaurants. Silver nitrate (Sigma-Aldrich Chemicals Ltd., Mumbai, India) was used as received. Deionized water was used in all the preparation.

The collected chicken egg shells were cleaned thoroughly with deionized water and dried in the open at ambient conditions. The dry egg shells were ground into fine powder in kitchen mixer grinder. The obtained ESP was sieved and the powder with a size < 25 m was stored in desiccators till used.

A quantity of 3 g of sieved ESP was added to 100 mL of water solution of AgNO₃ with a concentration of 5 mmol/L. The AgNO₃ solution and the mixture maintained at 80 ℃ was stirred at a rotating speed of 300 r/min on magnetic stirrer for 24 h. The NCESP with in situ generated AgNPs was filtered, washed thoroughly with eionized water several times and dried. The dried NCESP was stored in the desiccators till tested.

The SEM images of the NCESP were recorded on Zeiss EVO scanning electron microscope operated at 10 kV. The specimens were gold coated prior to capturing the images. For elemental analysis, the energy dispersive X-ray (EDX) spectra were recorded by using the attachment of the same SEM.

To probe the interactions between the generated AgNPs and the ESP in the NCESP, the FT-IR spectra of the ESP and the NCESP were recorded by using RXI Perkin Elmer FT-IR spectrometer in the wavenumber region of 4000–500 cm^–1 at a resolution of 4 cm^–1.

In order to probe the state of the generated AgNPs in the NCESP, the X-ray diffractograms of the ESP and NCESP were recorded by using Rigaku Ultimo Ⅳ X-ray diffractometer operated at 40 kV and 30 mA. The diffractograms were recorded in the range of 2θ= 10°–80° at a scanning rate of 2 °/min.

In order to study the effect of generated AgNPs on the thermal stability of the NCESP, the primary thermograms of both ESP and NCESP were recorded by using Perkin Elmer TGA-7 instrument in the temperature range of 100–700 ℃ at a heating rate of 10 ℃/min under nitrogen atmosphere.

The AgNPs are known to possess antibacterial activity. In order to probe whether the NCESP with in situ generated AgNPs also exhibits antibacterial activity or not, the antibacterial tested by standard disk method was used (Ashok et al., 2018). The test was conducted against both Gram negative (Escherichia coli and Pseudomonas aeruginosa) and Gram positive (Staphylococcus aureus and Bacillus licheniformis) bacteria. The clear zones formed indicated that the inhibitions to the growth of bacteria were photographedand in each case the zone diameter was measured.

In order to visualize the appearance of the ESP and NCESP, their digital images were photographed and are presented in Fig. 1b and Fig. 1c respectively. The photograph of the egg shells is presented in Fig. 1a. From Fig. 1b and Fig. 1c, it is found that the ESP was white in color whereas the NCESP with in situ generated AgNPs was dark brown. The change in color preliminarily indicated the in situ generation of the AgNPs in NCESP. In order to confirm this further, the SEM analysis was carried out.

Figure 1.  Digital images of egg shells (a); egg shell powder (ESP) (b) and nanocomposite egg shell powder (NCESP) with in situ generated silver nanoparticles (AgNPs) (c).

The SEM image, EDX spectrum and histogram showing the particle size distribution of in situ generated AgNPs in the NCESP are presented in Fig. 2a, Fig. 2b and Fig. 2c, respectively. From Fig. 2a, we can see the presence of tiny spherical AgNPs on the surface of the ESP particle. Along with individual tiny AgNPs, we can also observe the agglomerated AgNPs. From Fig. 2b, the peaks corresponding to silver and calcium elements can be found besides the usual carbon and oxygen. As the ESP had 95% CaCO₃, the presence of calcium element in the NCESP was justified. The presence of peak corresponding to silver element confirmed that the nanoparticles generated were those of silver. From Fig. 2c, we can find that the size of in situ generated AgNPs in the NCESP varied between 50 nm and 120 nm with most of them in the range of 81–90 nm. Further, the average size of the AgNPs generated was 88 nm. Thus the SEM analysis confirmed the in situ generation of spherical AgNPs in the NCESP.

Figure 2.  The SEM image (a); EDX spectrum (b) and histogram indicating particle size distribution (c) of NCESP. Some of the generated AgNPs are shown by arrows.

In order to probe the possible interactions between the generated AgNPs and ESP in the NCESP, the FT-IR analysis was carried. The FT-IR spectra of the ESP and NCESP in the conventional wavenumber range of 4000–600 cm^–1 are presented in Fig. 3. The spectra of the ESP and NCESP are similar with common peaks. The band at 1743 cm^–1 arose due to the C==O vibrations from carbonate ion (CO₃^2–) of the ESP (Ashok et al., 2014; Feng et al., 2014) and also the C==O of the amideⅠ of collagen component of the ESP (Belbachir et al., 2009). The band observed at 1654 cm^–1 arose due to the OH bending vibrations of adsorbed water (Chen et al., 2008) and also due to the absorption by AmideⅠ groups of collagen (Camacho et al., 2001). The broad band at 1418 cm^–1 (ranging from 1580 cm^–1 to 1150 cm^–1) was due to the CO₃^2– moiety of calcium carbonate (Meejoo et al., 2006). The other prominent peaks at 875 cm^–1 and 715 cm^–1 also belong to calcium carbonate of the ESP (Iram, 2019). In addition, the other bands of collagen corresponding to CH₂, CH₃, C—N and N—H vibrations are expected to be at 1454, 1403, 1340 cm^–1 and 1282–1205 cm^–1 respectively (Belbachir et al., 2009). These peaks could not be observed separately in Fig. 3 as they were obscured by the intense peak centered at 1418 cm^–1 that covers the wide range of 1580–1150 cm^–1. A close observation of the spectra of ESP and NCESP indicated that the intensity of the peak centered at 1418 cm^–1 and 1654 cm^–1 for the NCESP is lower than that of the ESP, indicating the involvement of the functional groups of collagen component in the generation of the AgNPs.

Figure 3.  The FT-IR spectra of NCESP and ESP in spectral range of 4000–600 cm^–1.

In order to examine state of the generated silver nanoparticles in the NCESP, the X-ray analysis was carried out. The X-ray diffractograms of the ESP and NCESP are presented in Fig. 4a. There are several sharp peaks in both the diffractograms of the ESP and NCESP. These sharp peaks correspond to the reflections from the planes of calcite (calcium carbonate) present in the ESP (Wang et al., 2006; Galván-Ruiz et al., 2009). From Fig. 4a, it is also found that the intensity of the peaks of the ESP was higher than that of the NCESP. As the NCESP contained calcite as the major component when compared with the minute quantity of generated AgNPs, the peaks of the minor components could not be identified from Fig. 4a as the strong peaks of calcite obscured them. In order to observe the obscured peaks of the AgNPs, the diffractogram of the NCESP was expanded in the range of 2θ= 30°–80° and is presented separately in Fig. 4b. Fig. 4b shows that there were some very low-intensity peaks (which were not present in the diffractogram of the ESP) identified among which the peaks observed at 2θ= 38°, 45.8° and 78° arose due to the reflections from (111), (200) and (311) planes respectively of the AgNPs (Sivaranjana et al., 2007). Similarly, some faint peaks observed at 2θ= 32.1°, 54.7° and 69.2° were due to the reflections from the planes of (111), (220) and (222) respectively of the AgO nanoparticles (AgONPs) (Sadanand et al., 2018). Hence, the NCESP had both AgNPs and AgONPs nanoparticles.

Figure 4.  The X-ray diffractograms of NCESP and ESP (a) and NCESP expanded in range of 2θ= 30°–80° (b).

Collagen protein in egg shell contains mainly three amino acids—glycine, proline and hydroxy proline (Ly et al., 2016). The possible mechanism involving proline in reducing Ag⁺ to Ag is presented in Fig. 5. The amino acid proline contains two hydroxyl groups (Rajesh Kumar et al., 2016; Birusanti et al., 2019) which can form an intermediate silver-amino acid complex. This further rearrangement forms AgNPs and AgONPs as described in Fig. 5a and Fig. 5b, respectively. The lower intensity of the two peaks at 1654 and 1481 cm^–1 of the NCEP over that of the ESP further supported the role of the protein component of the ESP in the generation of the AgNPs and AgONPs.

Figure 5.  Mechanism of generation of AgNPs and silver oxide nanoparticles (AgONPs).

In order to examine the influence of generated AgNPs on the thermal stability of the NCESP, the thermogravimetric analysis was carried out. The primary thermograms of the ESP and NCESP with in situ generated AgNPs are shown in Fig. 6. The thermal stability of the NCESP was higher than that of the ESP. Therefore, the in situ generated AgNPs enhanced the thermal stability of the NCESP. In other words, the generated AgNPs retarded the thermal degradation of the NCESP. Similar observation was made by Makvandi et al. (2015) in the case of polymethyl methacrylate nanocomposites with in situ generated AgNPs. The rigid nature of the generated AgNPs might be responsible for the enhanced thermal stability of the NCESP.

Figure 6.  Primary thermograms of ESP and NCESP.

It is an established fact that AgNPs exhibit antibacterial property (Karunagaran et al., 2014). In order to verify whether the NCESP having in situ generated AgNPs also possessed antibacterial activity or not, the antibacterial test by disk method was carried against both Gram negative (E.coi and P.auroginosa) and Gram positive (S.aureus and B.licheniformis) bacteria. The zones of clearance indicating the inhibition of bacteria were photographed and the images are shown in Fig. 7. For comparison the test was also conducted for the ESP.

Figure 7.  Zones of clearance formed for ESP (O) and NCESP (X) against Escherichia coli (a); Escherichia coli (b); Staphylococcus aureus (c) and Bacillus licheniformis (d) bacteria.

From Fig. 7, it is found that the ESP did not form any zone of clearance indicating its inability to inhibit the growth of the bacteria. However, the NCESP having in situ generated AgNPs formed prominent zones of clearance exhibiting excellent antibacterial activity. Using the images in Fig. 7, the diameters of zones of clearance formed by the NCESP against each bacterium were measured and the values are summarized in Table 1. The diameters of the zones of clearance formed by the NCESP varied between 33 mm and 43 mm. It indicated that the NCESP possessed excellent antibacterial activity and hence can be used as filler in different polymer matrices to make them antibacterial hybrid nanocomposites and also as low-cost antibacterial cleaning powder for house ware.

The NCESP with in situ generated AgNPs was prepared by one step thermal assisted (hydrothermal) method. The in situ generation of the AgNPs in the NCESP was proved by SEM coupled (EDX spectrum and X-ray analysis). The thermal stability of the NCESP was found to be higher than that of ESP. The NCESP exhibited excellent antibacterial activity against both Gram negative and Gram positive bacteria. The NCESP with in situ generated silver based nanoparticles can be utilized as low-cost antibacterial cleaning powder for house ware and also as low-cost antibacterial filler in polymer matrices to make antibacterial nanocomposites.

There are no conflicts to declare.

This research was supported by Natural Composite Research Group (NCR), Department of Mechanical, Process Engineering (MEPE), TGGS, King Mongkut's University of Technology North Bangkok (KMUTNB), Thailand and the Thailand Research Fund through the Royal Golden Jubilee PhD Program (No. PHD/0109/2560 to K.Y. and S.C.)