Molecular Subtyping and Characterization of Psychrotolerant Endospore-Forming Bacteria in Two New York State Fluid Milk Processing Systems

Abstract

Psychrotolerant endospore-forming bacteria Bacillus and Paenibacillus spp. are important spoilage organisms in fluid milk. A recently developed rpoB subtyping method was applied to characterize the diversity and phylogenetic relationships among Bacillus and related sporeformers associated with milk processing systems. Milk samples representing the processing continuum from raw milk to pasteurized products were collected from two fluid milk processing plants, held at 6°C uptothe code date that had been established by each processing plant (i.e., either 18 or 21 days), and plated for bacterial enumeration throughout storage. Bacterial colonies selected to represent the visible diversity in colony morphology on enumeration plates were examined further. Among 385 bacterial isolates characterized, 35% were Bacillus spp., and 65% were Paenibacillus spp. A total of 92 rpoB allelic types were identified among these isolates, indicating considerable diversity among endospore-forming spoilage organisms present in fluid milk systems. Of the 92 allelic types identified, 19 were isolated from samples collected from both processing plants. The same rpoB allelic types were frequently identified in paired raw milk and packaged product samples, indicating that Bacillus and Paenibacillus spp. can enter dairy processing systems through raw milk. Certain subtypes were found exclusively in pasteurized samples, including those that were temporally independent, suggesting the possibility of in-plant sources for these spoilage organisms, including through the persistence of selected subtypes in processing plants. Development of effective control strategies for the diverse array of psychrotolerant endospore-forming organisms that currently limit the shelf lives of high-temperature short-time fluid milk products will require comprehensive, integrated efforts along the entire milk processing continuum.