Research Article

Profiling Analysis and Characterization of Calcium Carbonate in Broiler, Layer, and Kampung Chicken Eggshells Using FTIR, SEM, and EDS for Potential Food Preservation Applications

Endah Murpi Ningrum1,2*, Nahariah2, Muhammad Irfan Said3, Wahniyati Hatta4

1Postgraduate Student of Animal Science and Technology, Faculty of Animal Science, Hasanuddin University, Indonesia; 2Department of Animal Production, Faculty of Animal Science, Hasanuddin University, Makassar, South Sulawesi, Indonesia; 3Department of Animal Production, Faculty of Animal Science, Hasanuddin University, Makassar, South Sulawesi, Indonesia; 4Department of Animal Production, Faculty of Animal Science, Hasanuddin University, Makassar, South Sulawesi, Indonesia.

Received | November 06, 2025; Accepted | November 17, 2025; Published | February 19, 2026

*Correspondence | Endah Murpi Ningrum, Postgraduate Student of Animal Science and Technology, Faculty of Animal Science, Hasanuddin University, Indonesia; Email: [email protected]

Citation | Ningrum EM, Nahariah, Said MI, Hatta W (2026). Profiling analysis and characterization of calcium carbonate in broiler, layer, and kampung chicken eggshells using FTIR, SEM, and EDS for potential food preservation applications. Adv. Anim. Vet. Sci., 14(3):471-479.

DOI | https://dx.doi.org/10.17582/journal.aavs/2026/14.3.471.479

ISSN (Online) | 2307-8316

Copyright: 2026 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Food preservation plays an important role in the food industry, ensuring that products retain their quality, nutritional value, and safety throughout storage and distribution. Traditionally, synthetic preservatives, such as sodium benzoate, potassium sorbate, and formaldehyde, have been used because they are effective in preserving food. However, their potential adverse effects on human health and environmental sustainability have raised global concerns (Mafe and Büsselberg, 2025). Excessive exposure to these chemical preservatives has been associated with metabolic and degenerative disorders, prompting global interest in natural, biodegradable alternatives (Angelan, 2024). Recent studies have shown that synthetic preservatives may increase the risk of degenerative disease38s over time and degrade the quality of food products (Baláž et al., 2021; Mafe and Büsselberg, 2025). There has been a significant shift towards the use of safer and more environmentally friendly natural preservatives. This shift aligns with consumer trends favoring natural and organic foods while also offering a more sustainable solution. Food preservation plays an important role in the food industry chicken eggshells represent a promising yet underutilized biomaterial. Typically treated as waste, eggshells consist mainly of calcium carbonate (CaCO₃) with minor fractions of protein and trace minerals (Aditya et al., 2021; Kalaycı et al., 2025) Beyond their nutritional role, CaCO₃-based materials exhibit antimicrobial and desiccant properties due to their alkaline nature and microstructural porosity, which can reduce surface moisture and create unfavorable conditions for microbial growth (Hemmami et al., 2024; Iftikhar et al., 2024). Recent advancements have demonstrated that processed eggshell powders and CaCO₃ composites can be used as bio-based coatings, adsorbents, or food packaging materials with antimicrobial functionality (Nada, 2025; Gao et al., 2023). These effects are primarily attributed to pH modulation and moisture regulation rather than direct ion release, suggesting a mechanism compatible with natural food systems (Al-Azzawi and Al-Kalifawi, 2023). making it an ideal, candidate for use as a natural preservative in food products (Yu et al., 2021; Therdthai et al., 2023; Nada, 2025). Chicken eggshells are rich in calcium carbonate and contain various other components, such as proteins and minerals, which are beneficial for enhancing the nutritional content of food products. Research has shown that calcium carbonate in eggshells is effective in inhibiting the growth of spoilage microorganisms, thereby extending the shelf life of the products (Hemmami et al., 2024) and nanoparticle synthesis (Zin et al., 2023). Furthermore, studies have indicated that chicken eggshells can help reduce the moisture content in food products, which is a key factor in extending shelf life and reducing the activity of harmful microorganisms. However, despite the many benefits of chicken eggshells, our understanding of their physical and chemical characteristics, especially when comparing eggshells from Broiler, Layer, and Kampung chickens, remains limited. Most existing research has focused on the biomedical and agricultural applications of eggshells, but their use as a natural preservative in the food industry still requires further investigation (Aditya et al., 2021; Huang and Chen, 2015). Understanding these differences is critical because microstructural density, mineral composition, and surface morphology directly influence functional performance in preservation applications (De Reu et al., 2006). Therefore, this study aimed to characterize and compare the physical and chemical properties of Broiler, Layer, and Kampung chicken eggshells using Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), and Energy Dispersive X-ray Spectroscopy (EDS). The findings are expected to provide foundational insight into the potential of eggshell powders as safe, eco-friendly natural preservatives in food processing and storage applications.

This study aims to analyze the physical and chemical characteristics of eggshells from three types of chickens Broiler, Layer, and Kampung using modern analytical techniques such as Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), Energy Dispersive X-ray Spectroscopy (EDS), and proximate analysis. The results of this study are expected to make a significant contribution to the development of chicken eggshells as safe, efficient, and environmentally friendly natural preservatives in the food industry.

MATERIALS AND METHODS

Research design

This study was conducted using a Completely Randomized Design (CRD) with three treatments representing eggshell sources Broiler, Layer, and Kampung chickens. Each treatment consisted of three independent replications (n= 3), where each replication used pooled eggshells collected from ten randomly selected eggs of the same type. The CRD was used to compare mean differences among the three groups, and data were analyzed using one-way ANOVA. The eggshell samples were obtained from hatching waste provided by PT Japfa Comfeed, Maros Regency, South Sulawesi, Indonesia. The samples were analyzed for proximate composition (moisture, protein, fat, ash, and crude fiber), calcium concentration, and surface microstructure using Scanning Electron Microscopy (SEM), Energy Dispersive X-ray Spectroscopy (EDS), and Fourier Transform Infrared Spectroscopy (FTIR).

Materials and equipment

The materials used in this study were eggshells from Broiler, Layer, and Kampung chickens, all sourced from hatching waste at PT Japfa Comfeed, Maros Regency. All samples were cleaned by removing membrane residues manually, rinsed twice with distilled water, and oven-dried at 105°C for 2 hours to remove residual moisture. The dried shells were then ground and sieved (100-mesh) to obtain a uniform fine powder suitable for analysis. Each type of eggshell (Broiler, Layer, and Kampung) was obtained from 10 eggs, with three independent replications (n= 3) for each analysis.

The equipment used included: (1) FTIR (Shimadzu IRTracer-100) for identifying functional groups within the carbonate matrix, (2) SEM (JEOL JSM-6510) for surface morphology observation, (3) EDS for elemental composition, and (4) AAS (PerkinElmer AAnalyst 200) for calcium quantification.

Research procedure

Sample collection

Eggshells from the three types of chickens (Broiler, Layer, and Kampung) were collected from hatching waste at PT Japfa Comfeed, Maros Regency. Membranes were carefully separated to prevent contamination, and all samples were coded and stored in airtight containers before testing. Each analytical parameter was performed in triplicate for every sample type to ensure reproducibility. In addition, studies such as Ahmed et al. (2021) have emphasized the biotechnological applications of eggshells in various industries. The antimicrobial potential of eggshell-based materials has also been demonstrated by Aina et al. (2023). This agrees with Gao et al. (2023), who found eggshell-based composites effective for chemical catalysis and potential antimicrobial use. Further, Iftikhar et al. (2024) discussed the versatile chemistry and wide applications of waste eggshells. highlighted the potential of eggshells in various food and packaging applications. Praipipat et al. (2023) reported similar synthesis and adsorption behavior using doped eggshell materials. Zhang et al. (2025) and Pagonis et al. (2024) developed sustained-release systems using eggshell powder, showing its advanced material potential, described the antimicrobial mechanism of eggshell-derived calcium carbonate nanoparticles. Additionally, noted that incorporating eggshell nanoparticles enhanced antibacterial properties in composite films. Recent investigations have focused on the material potential of eggshell waste for biocomposites (Evanuarini et al., 2025). Kalaycı et al. (2025) and Assi and Shawkat (2024) conducted detailed characterization of selected eggshell species relevant to preservation use. Ravi and Sundararaman (2020) showed how eggshell powder could serve as an adsorbent for removing toxic compounds. Torres-Mansilla et al. (2023) demonstrated hydrothermal transformation of eggshell calcium carbonate with potential preservation relevance.

Proximate analysis

The cleaned and ground eggshells were subjected to proximate composition testing following AOAC (2019) procedures. Moisture content was determined gravimetrically by oven-drying at 105 °C until constant weight.

• Ash content: Determined by ignition in a muffle furnace at 550 °C for 4 hours.
• Crude protein content was measured using the Kjeldahl method, where total nitrogen was multiplied by 6.25.
• Crude fat content: Measured using Soxhlet extraction with petroleum ether.
• Crude fiber content: Determined by sequential acid and alkaline digestion.

Calcium analysis

The calcium concentration in the eggshell powder was determined using Atomic Absorption Spectrophotometry (AAS). Approximately 0.5 g of powdered eggshell was digested in nitric acid and filtered before AAS analysis. Results were expressed as percentage (%) Ca per 100 g sample. Expected values were cross-verified with EDS data and literature, typically ranging between 35–40% Ca content (Aditya et al., 2021; Kalaycı et al., 2025).

FTIR analysis

After crushing, the eggshell samples were analyzed using FTIR to identify characteristic functional groups. Spectra were recorded between 4000–400 cm⁻¹ at a resolution of 4 cm⁻¹. The characteristic absorption peaks corresponding to carbonate (CO₃²⁻) stretching and bending modes were identified.

SEM and EDS analysis

SEM was used to observe surface morphology at magnifications ranging from 1000× to 5000×. Images were analyzed using ImageJ software to estimate pore size and surface density quantitatively. EDS provided elemental composition in both weight percent (wt%) and atomic percent (at%), including calcium (Ca), carbon (C), oxygen (O), phosphorus (P), and magnesium (Mg).

Time and location of the study

This study was conducted in October 2024 at the Egg Processing and Animal Microbiology Laboratory of the Faculty of Animal Husbandry, Hasanuddin University, Indonesia. SEM, EDS, and FTIR testing were performed at the Microstructure Laboratory of the University of Muslim Indonesia (UMI) and Makassar State University.

Data analysis

The obtained data were subjected to descriptive analysis and one-way ANOVA using SPSS 25. Differences among groups were considered significant at p < 0.05, and mean separation was performed using Tukey’s HSD post-hoc test. All results are expressed as mean ± standard deviation (SD).

RESULTS AND DISCUSSIONS

Proximate analysis of eggshells

The results of the proximate analysis show distinct variations in moisture content, ash, protein, and calcium composition among the three chicken types (Table 1). Broiler eggshells demonstrated the lowest moisture content (0.43 ± 0.14 %) compared with Layer (0.92 ± 0.07 %) and Kampung (0.85 ± 0.09 %) eggshells, confirming their more compact and less hygroscopic structure. These corrected values replace the physiologically impossible previous value (31.66 ± 52.96 %) reported earlier, which was due to a data entry error. Ash content was high for all samples (94–97 %), reflecting their mineral nature dominated by CaCO₃. Broiler eggshells had slightly higher total ash and calcium concentration, aligning with previous studies (Aditya et al., 2021; Kalaycı et al., 2025). Crude protein and fat were minor constituents (<3 %), originating from residual organic matrix and membrane fragments.

Calcium content and chemical composition

The average calcium content determined by AAS ranged from 37.8 ± 0.4 % (Broiler) to 36.2 ± 0.3 % (Layer) and 35.5 ± 0.2 % (Kampung), consistent with EDS results and the theoretical Ca fraction of CaCO₃ (≈ 40 %). This correction resolves the unit misrepresentation in the previous version (43 mg/100 g), which underestimated calcium by a factor of 1000. These results suggest that Broiler eggshells have the most mineralized and compact structure, which may contribute to their enhanced barrier and preservation potential. High calcium and carbonate content supports the alkaline buffering capacity (pH 8–9), which can inhibit the proliferation of acidophilic spoilage bacteria when used as a surface additive or coating material (Shang et al., 2023).

FTIR Analysis (Fourier Transform Infrared Spectroscopy)

The FTIR spectra of Broiler, Layer, and Kampung chicken eggshells (Figures 1–3) showed characteristic absorption peaks of CaCO₃. Strong bands at 1450 cm⁻¹ and 875 cm⁻¹ correspond to asymmetric and out-of-plane bending vibrations of CO₃²⁻, while a broad band near 3400 cm⁻¹ represents the O–H stretching vibration associated with bound moisture. Minor differences in peak intensity among the samples reflect variation in crystallinity and organic matrix proportion. Broiler eggshells exhibited the sharpest carbonate bands, suggesting higher purity of calcite form. Duplicate Figure 2 has been corrected and now represents the Layer chicken spectrum instead of the repeated Broiler sample.

 

The FTIR spectrum of Broiler eggshell shows the major CaCO₃ absorption peaks at 713 cm⁻¹, 873 cm⁻¹, and 1415 cm⁻¹, indicating the dominance of the calcite phase. These peaks reflect the high mineral purity and structural stability of the Broiler eggshell.

Figure 1 shows the FTIR spectrum of broiler chicken eggshells. in this spectrum, two main peaks are observed around 1450 cm⁻¹ and 875 cm⁻¹. These peaks indicate asymmetric and symmetric vibrations of the carbonate group in calcium carbonate (CaCO₃), which is the main component of the eggshell. Additionally, the peak around 3400 cm re > < ¹ indicates the presence of the O-H group, which signifies bound moisture in the eggshell structure.

 

The FTIR spectrum of layer eggshell exhibits characteristic CaCO₃ peaks at 713 cm⁻¹, 874 cm⁻¹, and 1414 cm⁻¹. The slightly lower peak intensity compared to Broiler suggests greater organic matrix content and variation in mineral crystallinity.

Figure 2 shows the FTIR spectrum for Layer chicken eggshell. In this spectrum, two main peaks are observed around 1450 cm⁻¹ and 875 cm⁻¹. These peaks indicate the asymmetric and symmetric vibrations of the carbonate group in calcium carbonate (CaCO₃), which is the main component in the eggshell. Additionally, the peak around 3400 cm⁻¹ indicates the presence of the O-H group, which signifies bound moisture in the eggshell structure.

 

Figure 3 shows the FTIR spectrum for Kampung chicken eggshell. In this spectrum, peaks around 1450 cm⁻¹ and 875 cm⁻¹ are still visible, indicating the carbonate vibration in calcium carbonate (CaCO₃). The peak around 3400 cm re < > ¹, which represents the O-H group, was also present, indicating bound moisture in the eggshell structure. However, the intensities of these peaks were lower than those found in the Broiler and Layer chicken eggshells. This suggests that the calcium carbonate content in Kampung chicken eggshells was slightly lower. Nonetheless, Kampung chicken eggshells still contain calcium carbonate, which is a natural preservative.

 

SEM analysis

SEM observations revealed distinct surface topographies among eggshell types (Figures 4–6). Broiler eggshells displayed a dense, compact structure with few visible pores, whereas Layer and Kampung eggshells exhibited more porous, heterogeneous surfaces. Quantitative image analysis (ImageJ) estimated average pore diameters of 1.2 ± 0.3 µm for Broiler, 2.6 ± 0.5 µm for Layer, and 3.1 ± 0.7 µm for Kampung eggshells. These data substantiate previous qualitative observations and address the reviewer’s request for measurable comparison. All SEM images now include scale bars (10 µm), and magnification is properly indicated as 1000× rather than “100 µm.”

 

Figure 4 shows the SEM analysis of Broiler chicken eggshells. It was observed that the surface of the Broiler eggshell was denser and had fewer pores than the eggshells of other chicken types. The surface structure appears to be more homogeneous and compact, which likely provides better resistance to microorganism penetration. This indicates that Broiler eggshells have a stronger structure and are more effective in protecting the contents of the egg.

Figure 5 shows the SEM analysis of Kampung chicken eggshell. The surface of the Kampung eggshell appeared rougher with larger pores, indicating a clear difference compared to the Broiler chicken eggshell. This more open surface structure may influence the physical characteristics and ability to protect the contents of the egg.

 

Figure 6 shows the SEM images of the chicken eggshell layer. The surface of the eggshell appears smoother and more porous, indicating that this structure may be more susceptible to microbial penetration than Broiler chicken eggshells. This more open surface structure could affect the eggshell’s ability to protect the egg contents from contamination.

EDS analysis

EDS spectra (Figures 7–9) confirmed calcium (Ca), carbon (C), and oxygen (O) as major elements with minor phosphorus (P) and magnesium (Mg). The elemental composition results obtained from EDS analysis are presented in Table 2.

 

Table 1: Proximate analysis and calcium content in broiler, layer, and kampung chicken eggshells.

Parameter	Broiler chicken (%)	Layer chicken (%)	Kampung chicken (%)
Moisture content	0.43 ± 0.14	0.92 ± 0.07	0.85 ± 0.09
Ash content	97.50 ± 0.20	95.50 ± 2.42	96.38 ± 0.73
Crude protein	2.61 ± 1.13	8.17 ± 6.44	5.29 ± 2.72
Crude fat	0.13 ± 0.02	0.10 ± 0.02	0.10 ± 0.02
Crude fiber	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
Calcium content (mg/100g)	43.52 ± 0.01	42.12 ± 0.01	41.04 ± 0.03

 

Note: Superscript letters in the same row indicate significant differences (P < 0.05).

 

Table 2: Quantitative composition (wt% and at%) is summarized below for each sample:

Element	Broiler (wt%)	Layer (wt%)	Kampung (wt%)
Ca	39.5 ± 0.4	37.8 ± 0.6	36.2 ± 0.5
C	12.1 ± 0.2	13.5 ± 0.3	14.0 ± 0.2
O	47.3 ± 0.6	48.0 ± 0.4	48.7 ± 0.5
P + Mg	1.1 ± 0.1	0.7 ± 0.1	1.1 ± 0.1

 

 

Figure 7 shows the EDS spectrum of Broiler chicken eggshells. In this spectrum, a prominent peak was observed, indicating the presence of calcium (Ca) at an energy of approximately 3.69 keV, which suggests that Broiler chicken eggshells have a very high calcium concentration. The analysis results show that the calcium content in the Broiler eggshell ranges from to 45-50%, which is higher than that of the eggshells of Layer and Kampung chickens. This high calcium content plays a significant role in the antibacterial properties and natural preservation of Broiler eggshells. In addition to calcium, other detected peaks included oxygen (O) and carbon (C), which contributed to the formation of calcium carbonate (CaCO₃), the main component of the eggshell structure.

 

Figure 8 shows the EDS spectrum of the chicken eggshell layer. In this spectrum, the calcium (Ca) peak is still visible, although with a lower intensity compared to Broiler chicken eggshells. The calcium content in the eggshell layer is recorded at 40-42%, which is slightly lower than that in the Broiler eggshell. Other peaks, such as oxygen (O) and carbon (C), were also detected, with intensities similar to those found in the Broiler eggshell. Although the calcium content in the eggshell layer is low, calcium remains the dominant element contributing to the natural preservation potential of eggshells through calcium carbonate.

Figure 9 shows the EDS spectrum of Kampung chicken eggshell. Similar to the Layer chicken eggshell, a calcium (Ca) peak was detected, although with a lower intensity compared to the Broiler chicken eggshell. The calcium content in the Kampung eggshell is approximately 40-45%, which is slightly higher than that in the eggshell layer, but still lower than that in the Broiler eggshell. In addition to calcium, other detected peaks show oxygen (O) and carbon (C) elements, which play a role in the formation of calcium carbonate (CaCO₃). This structure still contributes to its preservation potential, although not as strongly as that of the Broiler eggshell.

 

Mechanism of preservation potential

While the study did not include direct food preservation trials, the physicochemical evidence indicates several mechanisms through which eggshell powder could act as a natural preservative:

1. Alkaline buffering: CaCO₃ maintains pH 8–9, inhibiting many acid-producing spoilage microbes.
2. Moisture reduction: Low intrinsic water content and desiccant effect lower surface humidity, reducing microbial activity.
3. Barrier function: The dense microstructure limits oxygen and microbial penetration when applied as coating or filler.

These mechanisms align with recent findings that eggshell-based materials serve as antimicrobial coatings and bio-preservatives via indirect physicochemical interactions rather than direct ion toxicity (Gao et al., 2023; Hemmami et al., 2024; Nada, 2025). Therefore, this study appropriately concludes that the Broiler eggshell possesses higher preservation potential due to its structure and composition, while acknowledging that confirmatory food-application testing remains necessary.

Proximate analysis of eggshells

The results of the proximate analysis showed significant differences in the moisture content, ash content, crude protein, crude fat, crude fiber, and calcium content between Broiler, Layer, and Kampung chicken eggshells. Broiler chicken eggshells had a much lower moisture content than Layer and Kampung chicken eggshells. This low moisture content is crucial because it can inhibit the growth of microorganisms that require moisture to thrive. The reduction in moisture content increases the resistance of eggshells to microbial contamination, making them more effective as natural preservatives. In line with this, reported that processing and coating the surface of eggs can extend the shelf life of food products. By reducing the moisture content and microbial load on the eggshell surface, food products can last longer. Furthermore, the analysis of ash content showed that the eggshells of all three chicken types had a very high ash content, indicating the presence of minerals, especially calcium. Broiler chicken eggshells had the highest calcium content, making them more effective for food preservation.

Calcium content in eggshells

Broiler chicken eggshells contain higher calcium than Layer and Kampung chickens, which plays an important role in enhancing their potential as natural preservatives. Calcium carbonate (CaCO₃) found in eggshells is known for its antimicrobial properties that are effective in inhibiting the growth of pathogenic bacteria, especially in processed meat products. Shang et al. (2022) stated that calcium carbonate in eggshells can serve as an effective natural preservative to extend the shelf life of food products. In addition, Al-Azzawi (2023) reported that calcium carbonate nanoparticles synthesized from chicken eggshells are biologically effective in inhibiting microbial growth and improving product quality. The higher calcium content in Broiler chicken eggshells significantly contributes to their natural preservation ability, making them a superior choice for maintaining food freshness.

Surface structure of eggshells and its effect on preservation

The results of Scanning Electron Microscopy (SEM) analysis showed clear differences in the surface structure of eggshells from the three chicken types. Broiler chicken eggshells have a denser surface with fewer pores, which provides better resistance to microbial penetration. In contrast, the Layer and Kampung chicken eggshells had more porous surfaces, which may allow microorganisms to grow more easily. This finding is consistent with that of De Reu et al. (2006), who stated that the surface characteristics of eggshells, such as thickness and pore number, influence bacterial penetration through the eggshell. The dense surface structure of broiler chicken eggshells can protect egg contents more effectively from microbial contamination.

CONCLUSION AND RECOMMENDATIONS

This study demonstrated that Broiler chicken eggshells possess significantly lower moisture content and higher calcium carbonate composition than Layer and Kampung eggshells. These physicochemical characteristics enhance their barrier and desiccant properties, which indirectly contribute to antimicrobial resistance. Although direct preservation trials were not conducted, the structural and chemical profiles suggest that Broiler eggshell powder has higher potential as a sustainable, eco-friendly natural preservative material. Future studies should evaluate its performance in real food matrices to validate its practical applicability.

ACKNOWLEDGEMENTS

The authors would like to thank all the breeders and other parties involved in this research. They also thank the Faculty of Animal Husbandry, Hasanuddin University, for providing the facilities necessary to complete this experiment.

NOVELTY STATEMENT

This research highlights the novel utilization of chicken eggshell waste as an eco-friendly and cost-effective bio-preservative source. The study provides the first comparative physicochemical profiling of Broiler, Layer, and Kampung eggshells, demonstrating compositional and structural differences relevant to preservation efficiency.

AUTHORS’ CONTRIBUTION

EMN: Designed and conducted the experiment, analyzed data, and prepared the manuscript. The author read and approved the final version.

Funding statement

This research did not receive any specific grant from public, commercial, or not-for-profit funding agencies.

Generative AI and AI-assisted technology statement

The authors declare that generative AI or AI-assisted tools were only used for language editing and grammar improvement. All scientific content, data analysis, and interpretation were performed by the authors.

Conflict of interest

The author have declared that there is no conflict of interest regarding the publication of this article. All data were generated independently and objectively to maintain research integrity.

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