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Identification of Spoilage Bacteria Present in Laboratory Heat- Treated and Commercially Pasteurized Milk: A Case Study Involving Milk Production Chain in Nebraska

Maricarmen Estrada-Anzueto, Bismarck Martinez, Jayne Stratton and Andreia Bianchini

Dairy processing facilities need to control spoilage microorganisms in fluid milk in order to reach markets and increase their profits. The major contributors for early spoilage on fluid milk are sporeforming bacteria capable of growing under refrigeration temperature and Post- Pasteurization Contamination (PPC) organism. Since these two groups of organisms have different survival characteristics, strategies to control them may require different approaches. Therefore, identification of bacteria contributing to spoilage becomes essential. The objective of this research was to identify the spoilage bacteria, mainly sporeformers, associated with laboratory heat-treated milk (80°C for 12 min) and commercially-pasteurized fluid milk, throughout a production chain in Nebraska. Identification was done using the rpoB gene and/or partial 16S rDNA sequences. Raw and pasteurized milk samples were collected from different locations (farm, trucks, processing plant, and packaged products) during three periods (Spring-2012, Fall- 2012 and Spring-2013). All raw and some pasteurized milk samples (Spring-2013) were heat-treated in the laboratory (80°C, 12 minutes) to eliminate vegetative cells allowing the exclusive survival of spores. All milk samples were stored at refrigeration conditions (5 ± 1°C) during 21 days. Bacterial isolates were collected at different shelf-life time points and identified using rpoB and/or 16S rRNA sequencing. A total number of 207 bacterial isolates were identified. The isolates obtained from laboratory heat-treated raw milk samples included Bacillus spp. (84%), Paenibacillus spp. (11%) and other sporeforming bacteria (5%); while the ones from commercially pasteurized milk samples were identified as Bacillus spp. (54%), PPC (29%), Paenibacillus spp. (12%), and other sporeforming bacteria (5%). Although most of the isolates were identified as sporeforming bacteria, PPC is still a concern in commercially pasteurized milk, especially during the filling stage. Psychrotrophic sporeformers were detected in samples from different locations throughout the production chain, thus strategies to control them would need to encompass the entire production chain.