Gelatin, a protein derived from the partial hydrolysis of collagen found in animal tissues such as porcine and bovine skins, is a common ingredient in the food, pharmaceutical, and cosmetic industries due to its functional properties, including gelling, emulsifying, and stabilizing capabilities (Dille et al., 2021; Schrieber and Gareis, 2007). Sold predominantly in powdered form, its low water activity (<0.6) inhibits microbial growth and enzymatic degradation, extending shelf life while simplifying handling, storage, and precise dosing in formulations (Beuchat et al., 2011).

The manufacturing process of gelatin includes raw material pretreatment (acid or alkaline hydrolysis), thermal extraction, filtration, vacuum evaporation, concentration, sterilization, tunnel drying, and grinding (Alipal et al., 2021; Schrieber and Gareis, 2007). Despite the use of harsh chemical and thermal treatments throughout production, final gelatin products are still frequently contaminated with a variety of microorganisms, including Enterobacteriaceae, Staphylococcus spp., and spore-forming bacteria such as B. cereus, B. licheniformis, B. subtilis, B. pumilus, G. stearothermophilus, Anoxybacillus flavithermus, and C. sporogenes (De Clerck et al., 2004; de Clerck and de Vos, 2002; Heckler et al., 2024; Sharma et al., 2006a; Sharma et al., 2006b; Sharma et al., 2003).

This contamination is often attributed to the high microbial load of animal-derived raw materials, such as skins and bones, which can harbor up to 106–108 CFU/g of bacteria prior to processing (Antic et al., 2010; Bacon et al., 2000; Ilboudo et al., 2016). Biofilm formation on equipment surfaces, particularly in hard-to-clean areas, along with inadequate sanitation procedures, and prolonged exposure to critical temperatures (20–60 °C), contribute to increased microbial contamination risks during processing (Karaca and Coleri Cihan, 2020).

Among the most persistent contaminants in gelatins are spore-forming bacteria, particularly those from the Bacillus and Clostridium genera. These microorganisms produce spores, which are metabolically dormant structures protected by layers of calcium dipicolinate, a cortex, and protein coats. These spores exhibit remarkable resistance to heat, chemical agents, and desiccation, enabling them to survive even after rigorous sanitation protocols. (ICMSF, 1996; Setlow, 2006). This resilience is amplified in gelatin matrices, where reduced moisture during drying may create protective microenvironments that further shield spores from thermal inactivation (De Clerck et al., 2004). Surviving spores in pharmaceutical gelatin (e.g., capsule shells) or nutritional products for immunocompromised populations can lead to severe health risks, as the gelatin in powder form can provide a suitable environment for spore germination when rehydrated, particularly under conditions of moisture and temperature that support bacterial growth. This germination can result in the production of vegetative cells that are capable of causing infections, especially in individuals with weakened immune systems (Emmanuel et al., 2015).

Thermal processing steps, such as concentration (55 °C for up to 6 h) and tunnel drying (20–80 °C for 4 h), aim to reduce water activity and ensure product stability. Concentration occurs in tanks, where chemical adjustments are made to the gelatin solution. While drying is conducted in tunnels that employ a temperature gradient to prevent structural collapse of the gelatin gel, starting with air at 20–30 °C and gradually increasing to 60–80 °C (Schrieber and Gareis, 2007; Silva et al., 2001). Although these processing temperatures, which are moderate compared to sterilization conditions (> 100 °C), are effective for preserving functional properties and inactivating vegetative cells, this thermal profile may fail to eliminate spores, particularly of thermophiles like G. stearothermophilus, which exhibit heat resistance above 120 °C (Durand et al., 2015b; Wells-Bennik et al., 2016, Wells-Bennik et al., 2019). Moreover, the thermal inactivation behavior in viscous gelatin solutions may be influenced by spore aggregation or interactions between matrix proteins and spore structures, which can delay heat diffusion and create heterogeneous thermal gradients (Periago et al., 2004; van Asselt and Zwietering, 2006).

To advance understanding of spore behavior during gelatin processing, this study aimed to: (1) evaluate the inactivation of spores from twelve spore-forming bacterial strains (isolated from gelatin processing stages) during concentration (55 °C for 3.5 and 6 h) and drying (20–80 °C for 4 h); (2) analyze microorganism, biological, and experimental variability in spore inactivation under these conditions; and (3) determine thermal inactivation kinetics and parameters (D-values, z-values) for B. cereus (105–115 °C) and G. stearothermophilus (120–140 °C) spores under isothermal conditions in capillary tubes.