Enumeration and identification of bacterial spores in cocoa powders.

Presence of bacterial spores in cocoa powders is inevitable due to the cocoa bean fermentation process, during which members of the genus Bacillus and Geobacillus are typically present. Spores are a concern in heat treated foods when they survive heat treatments and the finished product supports germination, growth and potentially toxin production. In this study, available methods for the enumeration of total mesophilic and thermophilic spores (TMS and TTS) were evaluated, leading to the recommendation of one global method specifically for cocoa powders. The proposed method was validated during a ring test on seven selected cocoa powders and applied during routine analyses on commercial powders. The method includes dilution of cocoa powder using Buffered Peptone Water, heating at 80 C for 10 min for TMS and TTS count and heating at 100 C for 30 min for heat resistant (HR) spore count. Tryptic Soy Agar is used as a recovery medium with a maximal concentration of cocoa powder of 2.5 mg/mL (to prevent growth inhibition) and a non-nutrient agar overlay to prevent swarming of bacteria. Plates are incubated for at least 72 hours at 30 C for recovery of mesophilic bacteria and 55 C for thermophilic bacteria. Suitable alternatives to specific method parameters are provided.Median values of total spore concentrations are low (<400 CFU/g for TMS and <75 CFU/g for TTS) and concentrations of HR spores are very low (<5 CFU/g). Importantly, the relation between concentrations of (HR) spores in cocoa powder and incidence of spoilage of heat treated beverages containing cocoa is currently unclear. In the powders included in this study, Bacillus subtilis and Bacillus licheniformis were the predominant spore forming species identified (49% and 39%, respectively). Both species are known for high variability in spore heat resistance. Development of reliable and sensitive molecular methods is therefore required to assess the risk of spoilage caused by spores present in cocoa powders.