This study did not require approval from an Institutional Animal Care and Use Committee because no live animals underwent experimental procedures. Rumen fluid was collected from apparently healthy goats immediately after routine commercial slaughter at a licensed local slaughterhouse. Sample collection was performed postmortem without any additional animal handling or intervention for research purposes. All procedures involving sample collection, transportation, microbial cultivation, and laboratory analyses complied with institutional biosafety and laboratory hygiene regulations. Laboratory procedures involving microorganisms were conducted under biosafety level 2 conditions to ensure personnel safety and prevent environmental contamination.

The study was conducted from June to December 2025 at the Feed Technology Laboratory, Faculty of Animal Science, Universitas Andalas, Padang, West Sumatra, Indonesia. Production of postbiotic propionic acid, calcium propionate synthesis, and physicochemical characterization were performed at the same institution.

This experimental laboratory study evaluated the synthesis of calcium propionate from bio-derived propionic acid produced through microbial co-culture fermentation. Four alternative calcium sources were investigated: eggshell flour (P1), shellfish flour (P2), bone meal (P3), and indigenous limestone flour (P4). Calcium propionate synthesized from each calcium source was comparatively characterized using SEM–EDS, FTIR, XRD, and thermogravimetric analysis (TGA) to evaluate differences in morphology, elemental composition, functional groups, crystalline structure, and thermal stability.

The microbial strains used in this study were S. harbinensis and P. kudriavzevii, obtained from the Feed Technology Laboratory culture collection, Universitas Andalas, Padang, Indonesia. These microorganisms were selected because of their reported synergistic interactions in co-culture systems that enhance substrate utilization efficiency and organic acid production [4, 5].

Each strain was activated separately in an appropriate growth medium under controlled conditions before co-cultivation. Stock cultures were preserved at −80°C in their respective media supplemented with 20% (v/v) glycerol. Before experimentation, S. harbinensis was revived in De Man, Rogosa, and Sharpe (Merck, Darmstadt, Germany) broth and incubated anaerobically at 37°C for 24–48 h until the mid-logarithmic growth phase (OD₆₀₀ ≈ 0.8). P. kudriavzevii was cultured in Yeast extract–peptone–dextrose (Sigma-Aldrich, Merck, Darmstadt, Germany) broth at 35°C–37°C for 24–48 h until the exponential growth phase was reached. The inoculum density of each microorganism was standardized to ensure reproducibility and optimal fermentation performance, as recommended for mixed-culture fermentation systems [6].

Bio-derived propionic acid used in this study was obtained from a previously optimized co-culture fermentation of S. harbinensis and P. kudriavzevii, as described by Marlida et al. [4]. This study focused on using the resulting postbiotic propionic acid for calcium propionate synthesis and physicochemical characterization.

Propionic acid production was carried out using a previously optimized co-culture fermentation system. The fermentation medium composition, pH, incubation temperature, and inoculum ratio were adopted from previously optimized conditions because these variables significantly influence organic acid production [4]. Fermentation was performed under anaerobic or microaerophilic conditions favorable for propionic acid biosynthesis [7]. Samples were collected periodically to monitor propionic acid concentration, substrate utilization, and microbial growth throughout fermentation.

Based on the previous optimization study, fermentation was conducted under low carbon availability (0.1 g/L), high nitrogen concentration (15 g/L), and a 72 h incubation period. A 1:1 inoculum ratio with a total inoculum level of 4%, an initial pH of 7.0, and a forage-to-concentrate substrate ratio of 60:40 were used [4, 6]. These optimized conditions were selected because they critically affect microbial metabolic activity, fermentation efficiency, propionic acid production, and overall process productivity [8].

Raw materials: The calcium sources evaluated in this study were eggshell flour, shellfish flour, bone meal, and indigenous limestone flour. These materials were selected for their high calcium content, low cost, wide availability, and potential as sustainable alternatives to commercially refined calcium sources.

Before synthesis, eggshells and shellfish shells were thoroughly washed with distilled water to remove adhering organic matter, air-dried, oven-dried to constant weight, ground into fine powder, and passed through a 200-mesh sieve. Bone meal and indigenous limestone were similarly dried, milled, and sieved to obtain a uniform particle size before use as calcium precursors.

Experimental treatments: Four calcium source treatments were evaluated: eggshell flour (P1), shellfish flour (P2), bone meal (P3), and indigenous limestone flour (P4). The synthesized calcium propionate products were comparatively characterized using SEM–EDS, FTIR, XRD, and TGA to evaluate differences in morphology, elemental composition, functional groups, crystalline structure, and thermal stability.

Synthesis procedure: Calcium propionate was synthesized by reacting bio-derived propionic acid with each calcium source under optimized reaction conditions reported previously [9]. The synthesis was performed using a particle size of 200 mesh, a propionic acid concentration of 15%, a solid-to-liquid ratio of 0.14:1, a reaction temperature of 80°C, and a reaction time of 2.5 h.

After the reaction was complete, the resulting slurry was filtered and washed thoroughly with distilled water to remove residual reactants. The recovered product was subsequently dried before physicochemical characterization.

The synthesized calcium propionate was characterized using complementary analytical techniques to evaluate its physicochemical properties.

FTIR analysis was performed using an IRTracer-100 spectrometer (Shimadzu Corporation, Kyoto, Japan) to identify functional groups and confirm the chemical structure of the synthesized products [10]. XRD analysis was conducted using a D8 Advance diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) to determine crystalline structure and phase composition [11]. SEM–EDS analysis was performed using a JSM-6510LV scanning electron microscope (JEOL Ltd., Tokyo, Japan) to evaluate surface morphology and elemental composition [12]. TGA was performed using a DTG-60H thermal analyzer (Shimadzu Corporation, Kyoto, Japan) to assess thermal stability and compositional characteristics of the synthesized products [13]. These complementary analytical techniques are widely employed for comprehensive physicochemical characterization and quality assessment of calcium-based materials.

This study primarily involved qualitative physicochemical characterization of calcium propionate synthesized from four different calcium sources. Surface morphology, elemental composition, functional groups, crystalline structure, and thermal stability were comparatively evaluated using SEM–EDS, FTIR, XRD, and TGA. Because the study did not generate replicated quantitative response variables suitable for statistical hypothesis testing, inferential statistical analyses were not performed. Instead, comparative interpretation of the analytical findings was undertaken to identify the calcium source that produced calcium propionate with the most favorable physicochemical characteristics.

SEM revealed distinct differences in the surface morphology of calcium propionate synthesized from the four calcium sources (Figure 1). Calcium propionate synthesized from shellfish meal (Figure 1A) consisted of relatively large plate-like particles with smooth surfaces and compact aggregates. In contrast, the bone meal-derived product (Figure 1B) exhibited densely packed irregular granules with rough, heterogeneous surfaces, indicating a less uniform particle distribution. The eggshell flour-derived product (Figure 1C) displayed a highly porous and irregular microstructure characterized by interconnected cavities and loosely aggregated particles, suggesting a substantially greater apparent surface area than the other calcium sources. Meanwhile, calcium propionate synthesized from indigenous limestone flour (Figure 1D) exhibited layered, fractured particles with a relatively compact morphology and lower apparent porosity than the eggshell-derived product.

Among the evaluated calcium sources, eggshell flour produced calcium propionate with the most porous and heterogeneous morphology. The interconnected pore network and rough surface texture indicate a greater specific surface area, which may facilitate enhanced contact with aqueous media and improve dissolution characteristics. Such morphological properties are advantageous for feed additive applications because they can increase the release and bioavailability of calcium and propionate ions following ingestion. Similar observations have been reported for porous calcium-based materials, which exhibit improved physicochemical characteristics and faster dissolution than dense crystalline structures [14].

The marked differences in particle morphology demonstrate that the calcium precursor strongly influenced the microstructure of the synthesized calcium propionate. Eggshell flour consists predominantly of highly purified calcite (approximately 94%–97% calcium carbonate) with only minor quantities of organic matrix and trace minerals. This relatively homogeneous composition promotes a more complete neutralization reaction with propionic acid, resulting in fewer residual mineral phases and a more open microstructure [15]. In contrast, shellfish meal and bone meal contain appreciable amounts of phosphate, magnesium, silica, and other inorganic constituents that may interfere with crystal formation and promote heterogeneous particle aggregation. Mahmood et al. [14] similarly reported that calcium propionate synthesized from high-purity calcium carbonate exhibited greater structural uniformity and fewer morphological defects than products derived from less purified calcium sources.

The porous morphology of the eggshell-derived product may also provide important functional advantages in livestock nutrition. Materials with higher surface roughness and interconnected pores generally possess greater specific surface area, facilitating rapid hydration and dissolution. Improved dissolution enhances the availability of calcium and propionate ions, which contribute to calcium homeostasis and provide glucogenic substrates during periods of increased metabolic demand in ruminants [16]. Similar relationships between particle morphology, surface area, dissolution kinetics, and bioavailability have been widely documented for calcium carbonate biomaterials derived from natural waste resources [14, 17]. Therefore, the porous microstructure of eggshell-derived calcium propionate is expected to improve its physicochemical performance following incorporation into animal feed.

The SEM findings were corroborated by subsequent FTIR and XRD analyses, which confirmed successful calcium propionate synthesis and showed that the eggshell-derived product had greater chemical homogeneity and crystallinity than products synthesized from the other calcium sources. The agreement among morphological, structural, and crystallographic analyses indicates that eggshell flour is the most suitable calcium precursor for producing high-quality calcium propionate. Moreover, using eggshell waste provides an environmentally sustainable strategy for converting an abundant agro-industrial by-product into a value-added feed additive while supporting resource recycling and circular bioeconomy principles [14, 18].

EDS was performed to determine the elemental composition of calcium propionate synthesized from the four calcium sources (Figure 2). The spectra confirmed that calcium, carbon, and oxygen were the predominant elements in all synthesized products, consistent with the expected composition of calcium propionate. However, noticeable differences in elemental purity and the presence of minor inorganic constituents were observed among the treatments, reflecting differences in the mineral composition of the precursor materials.

The eggshell flour-derived product exhibited the most homogeneous elemental composition, with calcium, carbon, and oxygen accounting for most detected elements and relatively few detectable impurities. This finding is consistent with the high calcium carbonate content of eggshells and supports the formation of a chemically uniform product. In contrast, products synthesized from shellfish meal, bone meal, and indigenous limestone contained additional minor elements, including phosphorus, magnesium, silicon, and other inorganic constituents originating from the respective raw materials. The persistence of these elements indicates incomplete removal of naturally occurring mineral impurities during synthesis and is consistent with previous observations for alternative calcium sources [14].

The higher elemental purity observed in the eggshell-derived calcium propionate supports the SEM findings, which demonstrated a more porous and homogeneous microstructure. Together, these results indicate that the relatively pure calcite composition of eggshell flour promoted a more efficient neutralization reaction with bio-derived propionic acid, thereby minimizing the incorporation of secondary mineral phases into the final product. Mahmood et al. [14] similarly reported that calcium propionate synthesized from high-purity calcium carbonate sources exhibited greater chemical uniformity and fewer structural impurities than products derived from less refined calcium precursors.

From a functional perspective, the combination of higher elemental purity and porous morphology may improve dissolution efficiency and the subsequent release of calcium and propionate ions following ingestion. Enhanced dissolution characteristics are desirable for livestock feed additives because they improve mineral availability and facilitate the rapid utilization of propionate as a glucogenic precursor during periods of increased metabolic demand [19–21].

The EDS findings complement the SEM, FTIR, and XRD analyses, collectively demonstrating that eggshell flour provides the most suitable calcium precursor for synthesizing high-quality calcium propionate. In addition to improving product quality, using eggshell waste offers an environmentally sustainable approach that converts an abundant agro-industrial by-product into a value-added feed additive while reducing dependence on commercially refined calcium sources and supporting circular bioeconomy strategies [22].

XRD analysis showed that the crystalline characteristics of the synthesized calcium propionate varied with the calcium precursor used during synthesis (Figure 4). All diffraction patterns exhibited the characteristic reflections of calcium-containing crystalline phases, confirming successful conversion of bio-derived propionic acid into calcium propionate. However, the four treatments showed distinct differences in peak sharpness, intensity, and phase complexity. Calcium propionate synthesized from eggshell flour (Figure 4C) exhibited sharp, well-resolved diffraction peaks with relatively low background intensity, indicating a more ordered crystalline structure and greater phase homogeneity. In contrast, products synthesized from bone meal (Figure 4B) and indigenous limestone flour (Figure 4D) displayed broader, more complex diffraction patterns with additional reflections attributable to phosphate-, magnesium-, and silica-containing mineral phases, reflecting the heterogeneous mineral composition of these calcium sources. The shellfish meal-derived product (Figure 4A) exhibited an intermediate diffraction pattern, with greater crystallinity than bone meal but lower structural uniformity than the eggshell-derived product.

The superior crystallinity of the eggshell-derived calcium propionate is closely associated with the mineralogical composition of eggshells, which consist predominantly of highly crystalline calcite (CaCO₃) with only minor quantities of organic matrix and trace minerals [22]. The high purity of calcite provides readily available calcium that reacts efficiently with propionic acid, thereby minimizing the formation of residual secondary mineral phases during synthesis. Conversely, bone meal naturally contains hydroxyapatite [Ca₁₀(PO₄)₆(OH)₂], whereas indigenous limestone commonly contains dolomite, silica, magnesium carbonate, and other accessory minerals that contribute to more complex diffraction profiles and indicate incomplete conversion of the precursor minerals. Mahmood et al. [14] similarly demonstrated that calcium propionate synthesized from high-purity calcium carbonate exhibited sharper diffraction peaks and fewer crystalline impurities than products prepared from heterogeneous calcium sources. Likewise, recent investigations of waste-derived calcium materials have shown that precursor purity is a major determinant of crystallinity, crystal growth, and structural homogeneity in synthesized calcium salts [26].

The greater crystallinity observed in the eggshell-derived product is likely to enhance its physicochemical stability during storage and processing. Well-ordered crystalline materials generally exhibit greater structural uniformity and reduced structural defects, contributing to improved product consistency and handling characteristics. Although excessive crystallinity may reduce dissolution in some mineral systems, the present findings indicate that the highly porous morphology observed by SEM compensated for this effect by providing an extensive surface area for hydration. Consequently, the eggshell-derived calcium propionate combined favorable crystallinity with a porous microstructure, characteristics desirable for feed additive applications requiring both storage stability and efficient dissolution after ingestion.

The XRD findings complement the SEM, EDS, and FTIR results, collectively demonstrating that eggshell flour produced calcium propionate with the most favorable morphological, elemental, chemical, and crystallographic characteristics among the evaluated calcium sources. The close agreement among these complementary analytical techniques provides strong evidence that eggshell flour is the most suitable alternative calcium precursor for synthesizing high-quality calcium propionate from bio-derived propionic acid. Furthermore, valorization of eggshell waste provides an environmentally sustainable strategy for converting an abundant agro-industrial by-product into a value-added feed additive while reducing reliance on commercially refined calcium sources and supporting circular bioeconomy principles [18, 22, 27].

The present study demonstrates that calcium propionate synthesized from bio-derived propionic acid possesses physicochemical characteristics that support its potential application as a livestock feed additive. Complementary findings from SEM, EDS, FTIR, and XRD confirmed successful synthesis of calcium propionate and showed that the calcium precursor substantially influenced product quality. Among the evaluated calcium sources, eggshell flour consistently produced calcium propionate with the most favorable combination of porous morphology, relatively homogeneous elemental composition, well-defined functional groups, and highly ordered crystalline structure. Together, these characteristics are expected to improve product stability while facilitating rapid hydration and dissolution, thereby enhancing the release and availability of calcium and propionate ions within the gastrointestinal tract. Previous studies have shown that calcium propionate readily dissociates into calcium and propionate, enabling simultaneous mineral supplementation and provision of glucogenic substrates in ruminants [2, 28].

From a nutritional perspective, propionate is the principal precursor for hepatic gluconeogenesis in ruminants and contributes substantially to endogenous glucose production, particularly during periods of increased metabolic demand such as early lactation [6]. Adequate glucose availability is essential for maintaining milk production, supporting immune competence, and minimizing metabolic disorders associated with NEB. Dietary supplementation with calcium propionate has been shown to improve dry matter intake, feed efficiency, milk yield, and metabolic status while reducing the incidence of hypocalcemia, ketosis, and other transition-related disorders in dairy animals [1, 2, 28]. Although the present investigation focused exclusively on physicochemical characterization, the favorable structural properties of the eggshell-derived product suggest considerable potential for comparable biological performance. Nevertheless, controlled in vivo feeding trials are required to verify its bioavailability, safety, digestibility, and productive responses under commercial livestock production systems.

Beyond its nutritional advantages, utilizing eggshell waste as the primary calcium source provides substantial environmental and economic benefits. Eggshells are generated in large quantities by the food processing industry and are frequently discarded despite containing approximately 94%–97% calcium carbonate, making them an abundant and renewable calcium resource [22]. Converting this agro-industrial by-product into calcium propionate represents an effective waste valorization strategy that improves resource efficiency while reducing dependence on commercially refined calcium carbonate. Integration of bio-derived propionic acid production with eggshell recycling therefore represents a sustainable manufacturing approach that supports circular bioeconomy principles and offers a promising pathway for producing environmentally friendly, value-added feed additives for future livestock production systems [18].