Composition, Succession, and Source Tracking of Microbial Communities throughout the Traditional Production of a Farmstead Cheese

ABSTRACT Prior to the advent of milk pasteurization and the use of defined-strain starter cultures, the production and ripening of cheese relied on the introduction and growth of adventitious microbes from the environment. This study characterized microbial community structures throughout a traditional farmstead cheese production continuum and evaluated the role of the environment in microbial transfer. In total, 118 samples (e.g., raw milk, cheese, and environmental surfaces) were collected from milk harvesting through cheese ripening. Microbial communities were characterized based on amplicon sequencing of bacterial 16S rRNA and fungal internal transcribed spacer genes using the Illumina MiSeq platform. Results indicated that the environment in each processing room harbored unique microbial ecosystems and consistently contributed microbes to milk, curd, and cheese. The diverse microbial composition of milk was initially attributed to milker hands and cow teats and then changed substantially following overnight ripening in a wooden vat to one dominated by lactic acid bacteria, including Lactococcus lactis, Lactobacillus, and Leuconostoc, as well as fungi such as Exophiala, Kluyveromyces, and Candida. Additional microbial contributions were attributed to processing tools, but the composition of the cheese paste remained relatively stable over 60 days of ripening. In contrast, rind communities that were largely influenced by direct contact with bamboo aging mats showed a distinct succession pattern compared to the interior sections. Overall, these findings highlight the critical role of traditional tools and practices in shaping the microbial composition of cheese and broaden our understanding of processing environments as important sources of microbes in food. IMPORTANCE Throughout the 20th century, especially in the United States, sanitation practices, pasteurization of milk, and the use of commercial defined-strain starter cultures have enhanced the safety and consistency of cheese. However, these practices can reduce cheese microbial diversity. The rapid growth of the artisanal cheese industry in the United States has renewed interest in recapturing the diversity of dairy products and the microbes involved in their production. Here, we demonstrate the essential role of the environment, including the use of wooden tools and cheesemaking equipment, as sources of dominant microbes that shape the fermentation and ripening processes of a traditional farmstead cheese produced without the addition of starter cultures or direct inoculation of any other bacteria or fungi. These data enrich our understanding of the microbial interactions between products and the environment and identify taxa that contribute to the microbial diversity of cheese and cheese production.