Sources, transmission, and tracking of sporeforming bacterial contaminants in dairy systems.

Bacterial endospores, or simply spores, are formed by a diverse group of members within the phylum Bacillota and include notable genera such as Bacillus, Paenibacillus, and Clostridium. Spores are distributed ubiquitously in natural environments, with soil being an important primary reservoir for these microbes. As such, spores are present throughout the dairy farm environment, and transmission into raw milk occurs through several pathways that coalesce at the point of milk harvest. Despite the very low spore concentrations typically found in bulk tank raw milk, the impact of spores on dairy product quality, safety, and product conformance is widely documented. Processed dairy products affected by the presence of sporeforming bacteria include milk, cheese, dairy powders, ice cream mix, and more. Although raw milk is a major source of spores leading to quality, safety, and conformance issues in dairy products, the impact of other sources should not be discounted and may include ingredients (e.g., cocoa powder), contamination originating from biofilms in processing equipment, and even cross-contamination from the processing environment itself. Addressing spore contamination in the dairy system is complicated by this widespread distribution and by the diversity of these organisms, and successful source tracking often requires discriminatory molecular subtyping tools. Here, we review the key sources of sporeforming bacteria in the dairy system, the factors leading to the transmission of this diverse group of microbes into processed dairy products, and methods employed to enumerate and track spore contaminants.

Invited review: Redefining raw milk quality-Evaluation of raw milk microbiological parameters to ensure high-quality processed dairy products.

Raw milk typically has little bacterial contamination as it leaves the udder of the animal; however, through a variety of pathways, it can become contaminated with bacteria originating from environmental sources, the cow herself, and contact with contaminated equipment. Although the types of bacteria found in raw milk are very diverse, select groups are particularly important from the perspective of finished product quality. In particular, psychrophilic and psychrotolerant bacteria that grow quickly at low temperatures (e.g., species in the genus Pseudomonas and the family Enterobacteriaceae) and produce heat-stable enzymes, and sporeforming bacteria that survive processing hurdles in spore form, are the 2 primary groups of bacteria related to effects on processed dairy products. Understanding factors leading to the presence of these important bacterial groups in raw milk is key to reducing their influence on processed dairy product quality. Here we examine the raw milk microbiological parameters used in the contemporary dairy industry for their utility in identifying raw milk supplies that will perform well in processed dairy products. We further recommend the use of a single microbiological indicator of raw milk quality, namely the total bacteria count, and call for the development of a whole-farm approach to raw milk quality that will use data-driven, risk-based tools integrated across the continuum from production to processing and shelf-life to ensure continuous improvement in dairy product quality.

Bedding and bedding management practices are associated with mesophilic and thermophilic spore levels in bulk tank raw milk.

Mesophilic and thermophilic spore-forming bacteria represent a challenge to the dairy industry, as these bacteria are capable of surviving adverse conditions associated with processing and sanitation and eventually spoil dairy products. The dairy farm environment, including soil, manure, silage, and bedding, has been implicated as a source for spores in raw milk. High levels of spores have previously been isolated from bedding, and different bedding materials have been associated with spore levels in bulk tank (BT) raw milk; however, the effect of different bedding types, bedding management practices, and bedding spore levels on the variance of spore levels in BT raw milk has not been investigated. To this end, farm and bedding management surveys were administered and unused bedding, used bedding, and BT raw milk samples were collected from dairy farms (1 or 2 times per farm) across the United States over 1 yr; the final data set included 182 dairy farms in 18 states. Bedding suspensions and BT raw milk were spore pasteurized (80°C for 12 min), and mesophilic and thermophilic spores were enumerated. Piecewise structural equation modeling analysis was used to determine direct and indirect pathways of association among farm and bedding practices, levels of spores in unused and used bedding, and levels of spores in BT raw milk. Separate models were constructed for mesophilic and thermophilic spore levels. The analyses showed that bedding material had a direct influence on levels of spores in unused and used bedding as well as an indirect association with spore levels in BT raw milk through used bedding spore levels. Specific bedding and farm management practices as well as cow hygiene in the housing area were associated with mesophilic and thermophilic spore levels in unused bedding, used bedding, and BT raw milk. Notably, levels of spores in used bedding were positively related to those in unused bedding, and used bedding spore levels were positively related to those in BT raw milk. The results of this study increase the understanding of the levels and ecology of mesophilic and thermophilic spores in raw milk, emphasize the possible role of bedding as a source of spores on-farm, and present opportunities for dairy producers to reduce spore levels in BT raw milk.

Internal transcribed spacer (ITS) sequence-based characterization of fungal isolates from multiple yogurt facilities-A case study.

Fungal spoilage remains a significant issue in dairy product quality, especially for cultured dairy products such as yogurt formulated without preservatives such as potassium sorbate. Fungal contamination can occur throughout the processing continuum, from the dairy farm environment to the finished product processing environment. As molecular characterization of fungal isolates is used more frequently, we obtained fungal isolates obtained in 2 yogurt processing facilities as part of routine fungal testing of raw materials (e.g., fruit preparations, added ingredients), in-process product samples, environmental samples (e.g., air plates, equipment surfaces such as valves, face plates, air nozzles), and finished product samples, to determine whether internal transcribed spacer (ITS) barcoding data would be helpful to support source tracking of fungal contamination issues. Internal transcribed spacer PCR amplification and sequencing allowed us to classify the 852 isolates from these 2 facilities into 200 unique ITS allelic types (AT), representing the phyla Ascomycota (743 isolates), Basidiomycota (97 isolates), and Mucoromycota (12 isolates). Thirty ITS AT were isolated from both facilities; 62 and 108 ITS AT were isolated from only facility A or only facility B, respectively. Nine ITS AT were each represented by more than 20 isolates; these AT comprised 53% of the 852 isolates. The considerable diversity of fungal isolates even within a single facility illustrates the challenge associated with controlling fungal contamination of dairy products. The ITS barcoding technique, however, did show promise for facilitating the source tracking of fungal contamination, particularly for ITS AT over-represented in a given facility. For example, we found evidence for equipment-specific reservoirs for 2 AT (14 and 219) in facility B. Our data suggest that despite its limited discriminatory power, ITS sequencing can provide initial information that can help trace fungal contamination along the processing continuum. However, development and implementation of discriminatory subtyping methods will be needed to further improve the ability to identify sources of fungal contamination in dairy facilities. Developing and implementing sampling plans that comprehensively capture yeast and mold diversity in a given processing facility remain a considerable challenge.

Internal transcribed spacer (ITS) sequencing reveals considerable fungal diversity in dairy products.

Fungi are important spoilage organisms in dairy products. However, little is known about the diversity of naturally occurring spoilage fungi in raw milk and processed dairy products, due at least in part to the fact that classical fungal identification methods require considerable expertise. To gain further insight into the fungal diversity in the dairy system, we isolated fungi from raw milk, raw and pasteurized milk cheese, and yogurt using the selective dichloran rose bengal chloramphenicol agar. In total, 361 fungal isolates were obtained and further characterized by DNA sequencing of the internal transcribed spacer (ITS) region and the nuclear ribosomal large subunit (LSU) rRNA gene if needed. We conducted BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) searches of the ITS region sequences against the UNITE Database (https://unite.ut.ee/analysis.php), and selected other databases if needed, which allowed identification to the species level of 183 isolates and to the genus level of 107 of the 346 isolates that were successfully ITS sequenced. The isolates characterized represented 3 phyla and 19 genera; the most common genera isolated were Penicillium (25% of isolates), Debaryomyces (18%), and Candida (9%). This study not only provides, by using modern molecular tools, a baseline understanding of the types of fungi in dairy products, but also confirms that ITS sequencing is a useful approach for identification of fungal organisms found in the dairy food chain.