Category Archives: Spores

Laboratory Pasteurization Count (LPC) enumerates thermoduric bacteria and is one parameter used to assess raw milk quality. While there is currently no regulatory limit for LPC, LPC data are used by some dairy processors and cooperatives to designate raw milk quality premiums paid to farmers and may also be used for troubleshooting bacterial contamination issues. Despite occasionally being used as a proxy for levels of bacterial spores in raw milk, there is limited knowledge of the types of organisms that are enumerated by LPC in contemporary raw milk supplies. While historical studies have reported that thermoduric bacteria quantified by LPC may predominantly represent Gram-positive cocci, updated knowledge on microbial populations enumerated by LPC in contemporary organic raw milk supplies is needed. To address this gap, organic raw milk samples from across the United States (n = 94) were assessed using LPC, and bacterial isolates were characterized. LPC ranged from below detection (<0.70 log cfu/mL) to 4.07 log cfu/mL, with a geometric mean of 1.48 log cfu/mL. Among 380 isolates characterized by 16S rDNA sequencing, 52.6%, 44.5%, and 2.4% were identified as Gram-positive sporeformers, Gram-positive non-sporeformers, and Gram-negatives, respectively, and 0.5% that could not be categorized into those groups because they could only be assigned a higher level of taxonomy. Isolates identified as Gram-positive sporeformers were predominantly Bacillus (168/200) and Gram-positive non-sporeformers were predominately Brachybacterium (56/169) and Kocuria (47/169). To elucidate if the LPC level can be an indicator of the type of thermoduric (e.g., sporeforming bacteria) present in raw milk, we evaluated the proportion of sporeformers in raw milk samples with LPC of ≤100 cfu/mL, 100 to 200 cfu/mL, and ≥200 cfu/mL (51%, 67%, and 35%), showing a trend for sporeformers to represent a smaller proportion of the total thermoduric population when LPC increases, although overall linear regression showed no significant association between the proportion of sporeformers and the LPC concentration. Hence, LPC level alone provides no insight into the makeup of the thermoduric population in raw milk and further characterization is needed to elucidate the bacterial drivers of elevated LPC in raw milk. We therefore further characterized the isolates from this study using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), a rapid microbial identification tool that is more readily available to dairy producers than 16S rDNA PCR and sequencing. While our data indicated agreement between 16S and MALDI-TOF MS for 66.6% of isolates at the genus level, 24.2% and 9.2% could not be reliably identified or were mischaracterized using MALDI-TOF, respectively. This suggests that further optimization of this method is needed to allow for accurate characterization of thermoduric organisms commonly found in raw milk. Ultimately, our study provides a contemporary perspective on thermoduric bacteria selected by the LPC method and establishes that the LPC alone is not sufficient for identifying the bacterial drivers of LPC levels. Further development of rapid characterization methods that are accessible to producers, cooperatives, and processors will support milk quality troubleshooting efforts and ultimately improve outcomes for dairy industry community members.