Food Microbiology 34 (2013) 137e144 Contents lists available at SciVerse ScienceDirect Food Microbiology journal homepage: www.elsevier.com/locate/fm High content of biogenic amines in Pecorino cheeses Maria Schirone, Rosanna Tofalo, Giuseppe Fasoli, Giorgia Perpetuini, Aldo Corsetti, Anna Chiara Manetta, Aurora Ciarrocchi, Giovanna Suzzi* Dipartimento di Scienze degli Alimenti, Università degli Studi di Teramo, Via C.R. Lerici 1, 64023 Mosciano Sant’Angelo, Teramo, Italy article info abstract Article history: Pecorino refers to Italian cheeses made exclusively from raw or pasteurized ewes’ milk, characterized by Received 6 August 2012 a high content of fat matter and it is mainly produced in the Middle and South of Italy by traditional Received in revised form procedures. The autochthonous microbiota plays an important role in the organoleptic traits of Pecorino 19 November 2012 cheese and it can influence biogenic amines (BA) content. Accepted 20 November 2012 The aim of this study was to characterize from microbiological and chemical point of view 12 Available online 10 December 2012 randomly purchased commercial cheeses produced in Abruzzo region. Moreover, the BA content and the bacteria showing a decarboxylating activity were detected. For this purpose, a real-time quantitative PCR Keywords: Abruzzo ewe cheese (qPCR) was applied to evaluate histamine and tyramine-producers. Biogenic amines The samples were well differentiated for microbial groups composition, such as aerobic mesophilic Quantitative PCR bacteria, Enterobacteriaceae, coagulase-negative staphylococci, yeasts, enterococci, mesophilic and thermophilic lactobacilli. Pathogens such as Salmonella spp., Listeria monocytogenes and Escherichia coli O157:H7 were absent in all samples. In most samples the content of BA resulted to be high, with prevalence of histamine and tyramine. In particular, total BA content reached 5861 mg/kg in Pecorino di Fossa cheese. The qPCR method resulted to be very useful to understand the role of autochthonous Pecorino cheese microbiota on BA accumu- lation in many different products. In fact, since the ability of microorganisms to decarboxylate amino- acids is highly variable being in most cases strain-specific, the detection of bacteria possessing this activity is important to estimate the risk of BA cheese content. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction responsible for the accumulation of undesirable substances, such as biogenic amines (BA). The presence of these compounds is often Cheesemaking is one of the major industries worldwide, and unavoidable in fermented foods (Ladero et al., 2010a; Suzzi and great part is still practiced on relatively small scale which accounts Gardini, 2003). Nevertheless, their presence can cause several for the rich diversity of cheese available (Fox and McSweeney, problems for susceptible consumers, such as nausea, respiratory 2004). The growing interest in artisanal cheeses is partly due to distress, hot flushes, sweating, heart palpitation, headache, bright the uniqueness of such products, in which specialized microor- red rash, oral burning, hyper- or hypo-tension, whose intensity is ganisms can grow and contribute to their organoleptic and quali- dependent on quantitative and qualitative differences (Ladero et al., tative characteristics. However, these dairy products are often 2010a; Stratton et al., 1991). Cheese is among the most commonly manufactured under poor or uncontrolled hygiene conditions; in implicated foods associated with histamine poisoning and tyra- addition, they are produced following different protocols, which mine toxicity (EFSA Panel on Biological Hazards-BIOHAZ, 2011). In can vary from one cheesemaker to another. Many cheesemakers the Netherlands, United States and France several cases of BA use raw milk considering it essential for good and stronger flavor poisoning have been described. Outbreaks involved Swiss cheese than pasteurized milk, primarily due to greater proteolysis and containing more than 100 mg histamine/100 g cheese. Low levels of lipolysis by the raw milk microbiota in the cheese (Buffa et al., BA could have negative health effects in adults with histamine 2001). This microbiota plays a major role in the development of intolerance or those being administered monoamine oxidase the organoleptic characteristics of cheeses but it can also be inhibitors (MAOI) (indirect sympathomimetic drugs). Moreover, cheeses with high content of BA could be related to pediatric and adolescent migraine (Komprda et al., 2007; Millichap and Yee, * Corresponding author. Tel.: þ39 0861 266938; fax: þ39 0861 266940. 2003). In particular, close relation between consumption of E-mail address: [email protected] (G. Suzzi). tyramine-rich foods, such as cheese, migraine crisis has been found, 0740-0020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fm.2012.11.022 138 M. Schirone et al. / Food Microbiology 34 (2013) 137e144 and the toxic effects have been named as cheese-reaction (Coutts 2. Materials and methods et al., 1986; Hanington, 1967). In cheese the most important BA are histamine, tyramine, 2.1. Samples of Abruzzo cheeses putrescine and cadaverine, that originate from specific aminoacid decarboxylase activities of cheese microbiota. In fact, many Twelve randomly purchased commercial cheeses produced in different genera and species of Gram negative and positive bacteria Abruzzo region (two from each dairy farm), were analyzed possess the ability to produce BA (Linares et al., 2012). (Table 1). These cheeses are produced in the local farms of Abruzzo In addition, the BA accumulation is related not only to the pres- region according to the traditional cheese making procedures, ence of BA-producing microorganisms, but also to several variables which did not include the use of starter cultures. such as their proteolytic and decarboxylase activity, starter cultures, pasteurization, physico-chemical factors, ripening time, ripening 2.2. Microbiological analyses and storage temperature, etc. (Buñková et al., 2010; Burdychova and Komprda, 2007; Fernández et al., 2007; Gardini et al., 2001; Ladero Samples (10 g) of cheese were diluted in 90 ml of a sodium citrate et al., 2010a; La Gioia et al., 2011; Naila et al., 2010; Martuscelli et al., (2% w/v) solution and homogenized with a Stomacher Lab-Blender 2005; Wolken et al., 2006). It is well known that the decarboxylase 400 (Steward Medical, London, UK) for 1 min. Serial dilutions in activity of microorganisms is highly variable, being in most cases sterile peptone water (0.1% w/v) were plated in duplicate on selective strain-specific, so the detection of these bacteria is important not media to enumerate the following microorganisms: aerobic meso- only to estimate the risk of BA food content but also to prevent their philic bacteria on Plate Count Agar (PCA; Oxoid, Milan, Italy) at 30 C accumulation in fermented foods. In this context the application of for 48 h; mesophilic and thermophilic lactobacilli on MRS agar quantitative real-time PCR (qPCR) could be useful because it allows (Oxoid), acidified to pH 5.4 with acetic acid, at 30 C and 45 C, the rapid detection of producers and the quantification of genes and respectively for 48 h in anaerobic conditions using the Gas-Pack gene expression (Martinez et al., 2011; Postollec et al., 2011). anaerobic system (AnaeroGen; Oxoid); coagulase-negative staphy- Different qPCR assays have been developed to detect and quantify lococci (CNS) on Baird-Parker agar with egg yolk tellurite enrichment Gram positive bacteria able to produce BA in different food matrices (Oxoid) at 37 C for 24 h; enterococci on Slanetz-Bartley agar (Oxoid) (Fernández et al., 2006; Ladero et al., 2010b; Lucas et al., 2008; at 37 C for 48 h; Enterobacteriaceae on Violet Red Bile Glucose Nannelli et al., 2008; Torriani et al., 2008). Recently, this method has Agar (VRBGA; Oxoid) at 37 C for 24 h. Yeasts were grown on Yeast been applied to detect histamine producing Gram negative bacteria Peptone Dextrose Agar (YPD; 1% w/v yeast extract, 2% w/v peptone, in fish (Bjornsdottir-Butler et al., 2011; Martinez et al., 2011). 2% w/v glucose and 2% w/v agar; Oxoid) supplemented with chlor- Due to the impact of BA on human health and food safety, amphenicol (150 mg/l) at 25 Cfor3e5 days. The presence of monitoring their levels in foodstuffs is still gaining importance Salmonella spp., Listeria monocytogenes and Escherichia coli O157:H7 (Önal, 2007). In Italy there is a large production of Pecorino cheeses was determined according to standard methods (Association (w62,000 t of production in 2010) of different varieties, which have Française de Normalisation, 1997; International Organization for typical characters and originate from a delimited geographical area Standardization, 1998, 2002). (Coda et al., 2006; Pirisi et al., 2011). The name Pecorino is commonly given to Italian cheeses made exclusively from pure 2.3. Chemical analyses ewes’ milk and it has in most cases a Protected Designation of Origin or PDO status. Generally this type of cheese is produced in A radial slice of each cheese was taken at random, and used for the Middle and South of Italy by a traditional procedure, charac- chemical assays. The rind of each slice was carefully removed, and terized by a different ripening time ranging from 8 to 12 months (Di the rindless material was fully shredded. The determinations of ash Cagno et al., 2003). and total protein were carried out according to standard methods Pecorino di Farindola and Pecorino Abruzzese are traditional (AOAC, 2000a,b). Moisture was evaluated using an oven from Wtb varieties of cheese made from ewes’ milk, produced in the Abruzzo region, on the east side of the Gran Sasso mountain (National Park of Table 1 Gran Sasso, Italy). These two Pecorino cheeses show unique organ- Main characteristics of cheese samples used in the study. oleptic characteristics deriving from the environmental conditions and manufacturing practices already existing in the production area Product Sample Type of milk Ripening code (Chaves-López et al., 2006; Martuscelli et al., 2005; Schirone et al., Months Temperatures 2011; Serio et al., 2007).