A standard set of testing methods reliably enumerates spores across commercial milk powders

Contamination of dairy powders with sporeforming bacteria is a concern for dairy processors who wish to penetrate markets with stringent spore count specifications (e.g., infant powders). Despite instituted specifications, no standard methodology is used for spore testing across the dairy industry. Instead, a variety of spore enumeration methods are in use, varying primarily by heat-shock treatments, plating method, recovery medium, and incubation temperature. Importantly, testing the same product using different methodologies leads to differences in spore count outcomes, which is a major issue for those required to meet specifications. As such, we set out to identify method(s) to recommend for standardized milk powder spore testing. To this end, 10 commercial milk powders were evaluated using methods varying by (1) heat treatment (e.g., 80°C/12 min), (2) plating method (e.g., spread plating), (3) medium type (e.g., plate count milk agar), and (4) incubation time and temperature combinations (e.g., 32°C for 48 h). The resulting data set included a total of 48 methods. With this data set, we used a stepwise process to identify optimal method(s) that would explain a high proportion of variance in spore count outcomes and would be practical to implement across the dairy industry. Ultimately, spore pasteurized mesophilic spore count (80°C/12 min, incubated at 32°C for 48 h), highly heat resistant thermophilic spore count (100°C/30 min, incubated at 55°C for 48 h), and specially thermoresistant spore enumeration (106°C/30 min, incubated at 55°C for 48 h) spread plating on plate count milk agar were identified as the optimal method set for reliable enumeration of spores in milk powders. Subsequently, we assessed different powder sampling strategies as a way to reduce variation in powder spore testing outcomes using our recommended method set. Results indicated that 33-g composite sampling may reduce variation in spore testing outcomes for highly heat resistant thermophilic spore count over 11-g and 33-g discrete sampling, whereas there was no significant difference across sampling strategies for specially thermoresistant spore enumeration or spore pasteurized mesophilic spore count. Finally, an interlaboratory study using our recommended method set and a modified method set (using tryptic soy agar with 1% starch) among both university and industry laboratories showed increased variation in spore count outcomes within milk powders, which not only was due to natural variation in powders but also was hypothesized to be due to technical errors, highlighting the need for specialized training for technicians who perform spore testing on milk powders. Overall, this study addresses challenges to milk powder spore testing and recommends a method set for standardized spore testing for implementation across the dairy industry.