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

Abstract

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.

Cheese yeasts

“…hansenii , and Pichia sp. are among the more frequently occurring yeast species in raw milk (Boutrou & Gueguen, ; Büchl & Seiler, ; Buehler, Evanowski, Martin, Boor, & Wiedmann, ; Irlinger, Layec, Hélinck, & Dugat‐Bony, ). The same species have also been isolated from autochthonal whey and milk starters for cheese production (Binetti, Carrasco, Reinheimer, & Suarez, ).…”

Evaluation of biopreservatives in Greek yogurt to inhibit yeast and mold spoilage and development of a yogurt spoilage predictive model

“…In this model, all of the initial mold contamination in the yogurt was assumed to be the mold P. commune. Previous studies of fungal diversity have found that P. commune is frequently isolated from the yogurt production facility (Gougouli et al, 2011;Buehler et al, 2017). Growth of P. commune was adjusted for changes in storage temperature over time by assuming instantaneous adaptation of growth rate when temperature changed; lag phase was cumulative over storage time, similar to previous approaches (Koutsoumanis, 2001;Gougouli and Koutsoumanis, 2010).…”

“…Based on the isolates characterized in Buehler et al (2017) and information listed in Food Microbe Tracker (www .foodmicrobetracker .com; Vangay et al, 2013), 6 isolates were selected to represent a broad range of dairy-relevant fungal spoilage organisms. The selected isolates represent 5 yeast organisms and 1 mold organism (Table 1).…”

“…The selected isolates represent 5 yeast organisms and 1 mold organism (Table 1). Identification of yeast isolates to the species level and the mold isolate to the genus level was completed in a previous study and was based on internal transcribed spacer (ITS) sequencing (Buehler et al, 2017). The mold isolate was identified to species level by phenotypic classification according to Frisvad and Samson (2004).…”

“…Fungal contamination can occur throughout the dairy processing continuum, from the dairy farm environment to the finished product-processing environment (Kure et al, 2001;Temelli et al, 2006;Vacheyrou et al, 2011). Although it has been reported that raw milk is a source for natural fungal contaminants (Lavoie et al, 2012;Atanassova et al, 2016;Buehler et al, 2017), these organisms are typically not heat resistant and thus not the main route for fungal contamination of dairy products (Jacques and Casaregola, 2008;Garnier et al, 2017b). The processing environment, specifically after pasteurization, represents the most common source for fungal contamination of dairy products.…”