In addition, specifications for maximum spore concentrations in cocoa powder for quality control purposes are determined by customer demands and often vary among companies. The application of different methods influences the analytical outcome of spore counts in food ingredients and complicates interpretations of results in relation to risk assessments for the quality and stability of finished products ( 8, 17).

The methods mainly differ in the volumes of diluent used to dilute a sample, the heating parameters applied, the plating media used, and the incubation parameters applied for enumeration of mesophilic spore-forming species (e.g., ranging from 30 to 37вЊ and 48 to 72 h). An inventory of methods (as performed by the authors of this article) showed that over 35 different methods are applied within the market to enumerate total and HR spores in cocoa powders, including published standard methods for the microbiological examination of foods in general ( 15) and cocoa specifically ( 10, 12). Routine microbial quality assessment of cocoa powders is generally performed using classical plating methods. A previous study by Lima and coworkers ( 19) showed the presence of HRMS in 9 of 25 cocoa samples (ranging from 1.0 to 1.8 log CFU/g) and the presence of HRTS in 3 of 25 cocoa samples (ranging from <1 to 1.4 log CFU/g), as determined by a most-probable-number approach. The concentrations of HR spores in cocoa powders are often very low, <10 CFU/g, which is below the detection limit of classical plating techniques. The specification used in the cocoa industry to assess the overall microbial quality of powders is a total microbial count of 5,000 CFU/g, and no spore-specific specifications are currently in place. Generally, the total concentrations of spores in cocoa powders range from below detection level to a maximum of 3.7 log CFU/g for TMS and a maximum of 3.1 log CFU/g for TTS ( 19). Concentrations of spores with a high heat resistance are determined by applying a heat treatment of 30 min for 100вЊ, followed by incubation at 30 to 37вЊ for heat-resistant (HR) mesophilic spore (HRMS) formers and at 55вЊ for HR thermophilic spore (HRTS) formers. Generally, total spore concentrations are established by applying a heat treatment of 10 min at 80вЊ and plating on a suitable recovery medium, followed by incubation at 30 to 37вЊ for total mesophilic spores (TMS) or at 55вЊ for (facultative) total thermophilic spores (TTS). Spore-forming bacteria can be grouped according to the heat resistance of their spores and the range of growth temperatures of the organism.

The development of reliable and sensitive molecular methods is therefore required to assess the risk of spoilage caused by spores present in cocoa powders. Both species are known for high variability in spore heat resistance. In the powders included in this study, Bacillus subtilis and Bacillus licheniformis were the predominant spore-forming species identified (49 and 39%, respectively). Importantly, the relation between concentrations of HR spores in cocoa powder and incidence of spoilage of heat-treated beverages containing cocoa is currently unclear. 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). Suitable alternatives to specific method parameters are provided. Plates are incubated for at least 72 h at 30вЊ for recovery of mesophilic bacteria and 55вЊ for thermophilic bacteria. 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 nonnutrient agar overlay to prevent swarming of bacteria. The method includes dilution of cocoa powder using buffered peptone water, heating at 80вЊ for 10 min for TMS and TTS counts, and heating at 100вЊ for 30 min for a heat-resistant (HR) spore count. The proposed method was validated during a ring test on seven selected cocoa powders and applied during routine analyses on commercial powders. In this study, available methods for the enumeration of total mesophilic and thermophilic spores (TMS and TTS, respectively) were evaluated, leading to the recommendation of one global method specifically for cocoa powders.

Spores are a concern in heat-treated foods when they survive heat treatments and the finished product supports germination, growth, and potentially toxin production. The presence of bacterial spores in cocoa powders is inevitable due to the cocoa bean fermentation process, during which members of the genera Bacillus and Geobacillus are typically present.