Fast detection and quantification of four dairy propionic acid bacteria in milk samples using real-time quantitative polymerase chain reaction Meral Turgay * , Walter Schaeren, Daniel Wechsler, Ueli Bütikofer, Hans Ulrich Graber Agroscope, Institute for Food Sciences IFS, CH-3003 Bern, Switzerland article info Article history: Received 18 November 2015 Received in revised form 2 March 2016 Accepted 31 March 2016 Available online 13 April 2016 abstract Propionibacterium freudenreichii is added to vat milk to create the characteristic eyes and typical nutty flavour of Emmentaler Protected Designation of Origin (PDO) cheese, but leads to serious quality defects in other raw milk cheeses from Switzerland. To trace propionic acid bacteria (PAB) in raw milk, we developed and validated a fast quantitative polymerase chain reaction (qPCR)-based method for P. freudenreichii, Propionibacterium thoenii, Propionibacterium jensenii, and Propionibacterium acid- ipropionici. qPCR-standard curves were linear over five log units down to 10 1 copies per reaction (R  0.997); efficiencies ranged from 0.83 to 0.97. In spiking experiments, the lower limits of quantifi- cation were 10 1 e10 2 cfu mL 1 raw milk. Fifty one vat milk samples were analysed with plate count method and qPCR in parallel. Compared with the classic plate count method, the newly developed qPCR method gave faster and species specific determination of four dairy PAB in milk and yielded comparable quantitative results. © 2016 Elsevier Ltd. All rights reserved. 1. Introduction Raw milk cheeses are considered to be rich in taste and are part of the cultural heritage of many countries (Montel et al., 2014). In Switzerland, for example, various traditional cheese varieties with Protected Designation of Origin (PDO), such as Emmentaler PDO, Gruy ere PDO, L'Etivaz PDO, Sbrinz PDO, T^ ete de Moine PDO, Berner Alp- and Hobelk€ ase PDO, Formaggio d'Alpe Ticinese PDO, and Raclette du Valais PDO, are still produced entirely from raw milk. To achieve good cheese quality, outstanding raw milk quality is required. The raw milk microflora contributes in an important way to the sensory characteristics of raw milk cheeses. However, it may contain undesirable bacteria that lead to serious cheese defects during the prolonged ripening of semi-hard, hard, and extra-hard cheeses. Important economic losses due to insufficient raw milk quality can only be prevented through regular microbial control of the vat milk and the bulk tank milk of individual farmers. Apart from the defect of late blowing, which is usually due to the anaerobic fermentation of lactate to butyrate, CO 2 , and H 2 by Clostridium tyrobutyricum, several other important cheese defects, such as the formation of reddish-brown spots in the curd, an atypical sweet flavour, opening defects, and poor storage quality, are caused by the undesired growth of propionic acid bacteria (Baer & Ryba, 1992; Carcano, Todesco, Lodi, & Brasca, 1995; Decarvalho, Guezenec, Gautier, & Grimont, 1995; Fessler, Casey, & Puhan, 1999a; Turgay et al., 2011). For the production of raw milk cheeses without propionic acid fermentation, a concentration of propionic acid bacteria of <30 cfu mL 1 in the milk is recommended (Bachmann et al., 2011). Even in Swiss-type cheeses made from raw milk, such as Emmentaler PDO, where adjuncts of Propionibacterium freu- denreichii are added to the vat milk, naturally occurring contami- nations of propionic acid bacteria (PAB) in raw milk should be avoided since the growth of PAB wild-type strains with a high aspartase activity can reduce the storage quality of the cheeses (Turgay et al., 2011). Contaminations of bulk tank milk with PAB result mostly from contaminations in the milking plant (Fessler, Casey, & Puhan, 1999b). Moreover, insufficiently cleaned equip- ment at the farm (e.g., milk cans, bulk tanks) and in cheese dairies (e.g., seals in valves and pipelines) are important contamination sources (Fessler, 1997). The classical term “dairy PAB” comprises the four species of P. freudenreichii, Propionibacterium thoenii, Propionibacterium * Corresponding author. Tel.: þ41 58 463 82 62. E-mail address: meral.turgay@agroscope.admin.ch (M. Turgay). Contents lists available at ScienceDirect International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj http://dx.doi.org/10.1016/j.idairyj.2016.03.014 0958-6946/© 2016 Elsevier Ltd. All rights reserved. International Dairy Journal 61 (2016) 37e43